How Tobacco Smoke Causes Disease the Biology and Behavioral Basis for Smoking-Attributable Disease

How Tobacco Smoke Causes Disease The Biology and Behavioral Basis for Smoking-Attributable Disease

A Report of the Surgeon General

U.S. Department of Health and Human Services

DiseaseCover2.indd 1 4/22/2010 2:50:59 PM How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease

A Report of the Surgeon General

2010

U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES Public Health Service Office of the Surgeon General Rockville, MD National Library of Medicine Cataloging in Publication

How tobacco smoke causes disease: the biology and behavioral basis for smoking-attributable disease : a report of the Surgeon General. – Rockville, MD : Dept. of Health and Human Services, Public Health Service, Office of Surgeon General, 2010. p. 706 Includes bibliographical references.

1. Tobacco – adverse effects. 2. Smoking – adverse effects. 3. Disease – etiology. 4. Tobacco Use Disorder – complications. 5. Tobacco Smoke Pollution – adverse effects. I. United States. Public Health Service. Office of the Surgeon General. II. United States. Office on Smoking and Health.

QV 137 H847 2010

U.S. Department of Health and Human Services Centers for Disease Control and Prevention National Center for Chronic Disease Prevention and Health Promotion Office on Smoking and Health

This publication is available on the World Wide Web at http://www.surgeongeneral.gov/library

Suggested Citation U.S. Department of Health and Human Services. How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease: A Report of the Surgeon General. Atlanta, GA: U.S. Department of Health and Human Services, Centers for Disease Control and Prevention, National Center for Chronic Disease Prevention and Health Promotion, Office on Smoking and Health, 2010.

For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, DC 20402. ISBN 978-0-16-084078-4

Use of trade names is for identification only and does not constitute endorsement by the U.S. Department of Health and Human Services. Any recommendations expressed by nongovernmental individuals or organizations do not necessarily represent the views or opinions of the U.S. Department of Health and Human Services. Message from Kathleen Sebelius Secretary of Health and Human Services

The enormous public health and financial impact on this nation from tobacco use is completely avoidable. Until we end tobacco use, more people will become addicted, more people will become sick, more families will be devastated by the loss of loved ones, and the nation will continue to incur damaging medical and lost productivity costs. Now is the time to fully implement the proven and effective interventions that reduce tobacco-caused death and disease and to help end this public health epidemic once and for all. Cigarettes are responsible for approximately 443,000 deaths each year in the United States, which is one in every five deaths. The chronic diseases caused by tobacco use are the leading causes of death and disability in the United States and are an unnecessary drain on our health care system. The economic burden of cigarette use includes more than $193 billion annually in health care costs and loss of productivity. We can prevent the staggering toll that tobacco takes on the individual, our families, and our communities. This new Surgeon General’s report focuses on cigarettes and cigarette smoke to provide further evidence on how cigarettes cause addiction and death and will further add to the robust evidence base on effective interventions for tobacco control and prevention. The report identifies key pathways of disease production and, through knowledge of these pathways, points the way to finding better approaches to cessation and prevention and should bring new directions to lowering the still too high burden of smoking-caused disease. Twenty years of successful state efforts show that the more states invest in tobacco control programs, the greater the reductions in smoking; and the longer states maintain such programs, the greater and faster the impact. The largest impacts come when we increase tobacco prices, ban smoking in public places, offer affordable and accessible cessation treatments and services, and combine media campaigns with other initiatives. We have outlined a level of state investment in comprehensive tobacco control and prevention efforts that, if implemented, would result in an estimated five million fewer smokers over the next five years. As a result, hundreds of thousands of premature deaths caused by tobacco use would be prevented, and many fewer of the nations’ children would be robbed of their aunts, uncles, parents, and grandparents. Importantly, in 2009 the U.S. Food and Drug Administration received statutory authority to regulate tobacco products. This has the potential to greatly accelerate progress in reducing morbidity and mortality from tobacco use. Tobacco prevention and control efforts need to be commensurate with the harm caused by tobacco use, or tobacco use will remain the largest cause of preventable illness and death in our nation for decades, even though we possess the knowledge and the tools to largely eliminate it. When we help Americans quit tobacco use and prevent our youth from ever starting, we all benefit. Now is the time for comprehensive public health and regulatory approaches to tobacco control. If we seize this moment, we will make a difference in all of our communities and in the lives of generations to come.

Foreword

In 1964, the first Surgeon General’s report on the effects of smoking on health was released. In the nearly 50 years since, extensive data from thousands of studies have consistently substantiated the devastating effects of smoking on the lives of millions of Americans. Yet today in the United States, tobacco use remains the single largest preventable cause of death and disease for both men and women. Now, this 2010 report of the Surgeon General explains beyond a shadow of a doubt how tobacco smoke causes disease, validates earlier findings, and expands and strengthens the science base. Armed with this irrefutable data, the time has come to mount a full-scale assault on the tobacco epidemic. More than 1,000 people are killed every day by cigarettes, and one-half of all long-term smokers are killed by smoking-related diseases. A large proportion of these deaths are from early heart attacks, chronic lung diseases, and cancers. For every person who dies from tobacco use, another 20 Americans continue to suffer with at least one serious tobacco-related illness. But the harmful effects of smoking do not end with the smoker. Every year, thousands of nonsmokers die from heart disease and lung cancer, and hundreds of thousands of children suffer from respiratory infections because of exposure to secondhand smoke. There is no risk-free level of exposure to tobacco smoke, and there is no safe tobacco product. This new Surgeon General’s report describes in detail the ways tobacco smoke damages every organ in the body and causes disease and death. We must build on our successes and more effectively educate people about the health risks of tobacco use, prevent youth from ever using tobacco products, expand access to proven cessation treatments and services, and reduce exposure to secondhand smoke. Putting laws and other restrictions in place, including making tobacco products progressively less affordable, will ultimately lead to our goal of a healthier America by reducing the devastating effects of smoking. The Centers for Disease Control and Prevention (CDC), the U.S. Food and Drug Administration (FDA), and other federal agencies are diligently working toward this goal by implementing and supporting policies and regulations that strengthen our resolve to end the tobacco epidemic. CDC has incorporated the World Health Organization’s MPOWER approach into its actions at the local, state, and national levels. MPOWER consists of six key interventions proven to reduce tobacco use that can prevent millions of deaths. CDC, along with federal, state, and local partners, is committed to monitoring the tobacco epidemic and prevention policies; protecting people from secondhand smoke where they live, work, and play; offering quit assistance to current smokers; warning about the dangers of tobacco; enforcing comprehensive restrictions on tobacco advertising, promotion, and sponsorship; and raising taxes and prices on tobacco products. In 2009, the Family Smoking Prevention and Tobacco Control Act was enacted, giving FDA explicit regulatory authority over tobacco products to protect and promote the health of the American public. Among other things, this historic legislation gave the agency the authority to require compa- nies to reveal all of the ingredients in tobacco products—including the amount of nicotine—and to prohibit the sale of tobacco products labeled as “light,” “mild,” or “low.” Further, with this new regulatory mandate, FDA will regulate tobacco advertising and require manufacturers to use more effective warning labels, as well as restrict the access of young people to their products. FDA will also assess and regulate modified risk products, taking into account the impact their availability and marketing has on initiation and cessation of tobacco use. Reducing the tremendous toll of disease, disability, and death caused by tobacco use in the United States is an urgent need and a shared responsibility. All public health agencies need to partner together to develop common strategies to combat the dangers of smoking and tobacco use and defeat this epidemic for good.

i This 2010 Surgeon General’s report represents another important step in the developing recognition, both in this nation and around the world, that tobacco use is devastating to public health. Past investments in research and in comprehensive tobacco control programs—combined with the findings presented by this new report—provide the foundation, evidence, and impetus to increase the urgency of our actions to end the epidemic of tobacco use.

Thomas R. Frieden, M.D., M.P.H. Margaret A. Hamburg, M.D. Director Commissioner of Food and Drugs Centers for Disease Control and Prevention U.S. Food and Drug Administration and Administrator Agency for Toxic Substances and Disease Registry

ii Preface from the Surgeon General, United States Public Health Service

In 1964, the Surgeon General released a landmark report on the dangers of smoking. During the intervening 45 years, 29 Surgeon General’s reports have documented the overwhelming and conclu- sive biologic, epidemiologic, behavioral, and pharmacologic evidence that tobacco use is deadly. Our newest report, How Tobacco Smoke Causes Disease, is a comprehensive, scientific discussion of how main¬stream and secondhand smoke exposures damage the human body. Decades of research have enabled scientists to identify the specific mechanisms of smoking-related diseases and to characterize them in great detail. Those biologic processes of cigarette smoke and disease are the focus of this report. One-third of people who have ever tried smoking become daily smokers. This report investi¬gates how and why smokers become addicted and documents how nicotine compares with heroin and cocaine in its hold on users and its effects on the brain. The way tobacco is grown, mixed, and processed today has made cigarettes more addictive than ever before. Because of this, the majority of smokers who try to quit on their own typically require many attempts. It is imperative that we use this information to prevent initiation, make tobacco products less addictive, and provide access to treatments and services to help smokers quit successfully. This new report also substantiates the evidence that there is no safe level of exposure to cigarette smoke. When individuals inhale cigarette smoke, either directly or secondhand, they are inhaling more than 7,000 chemicals: hundreds of these are hazardous, and at least 69 are known to cause cancer. The chemicals are rapidly absorbed by cells in the body and produce disease-causing cellular changes. This report explains those changes and identifies the mechanisms by which the major classes of the chemi¬cals in cigarette smoke contribute to specific disease processes. In addition, the report discusses how chemicals in cigarette smoke impair the immune system and cause the kind of cellular damage that leads to cancer and other diseases. Insight is provided as to why smokers are far more likely to suffer from chronic disease than are nonsmokers. By learning how tobacco smoke causes disease, we learn more about how chemicals harm cells, how genes may make us susceptible, and how tobacco users become addicted to nicotine. The answers to these questions will help us to more effectively prevent tobacco addiction and treat tobacco-caused disease. Understanding the complexity of genetic, biochemical, and other influences discussed in this report offers the promise of reducing the disease burden from tobacco use through earlier detection and better treatment; however, even with all of the science presented here, it currently remains true that the only proven strategies to reduce the risks of tobacco-caused disease are preventing initiation, facilitating cessation, and eliminating exposure to secondhand smoke. My priority as Surgeon General is the health of the American people. Although we have made great strides in tobacco control, more than 440,000 deaths each year are caused by smoking and expo¬sure to secondhand smoke. The cost to the nation is tremendous: a staggering amount is spent on medical care and lost productivity. But most important is the immeasurable cost in human suffering and loss. In 1964, Surgeon General Luther Terry called for “appropriate remedial actions” to address the adverse effects of smoking. With this report, the devastating effects of smoking have been character¬ized in great detail and the need for appropriate remedial action is even more apparent. The harmful effects of tobacco smoke do not end with the users of tobacco. There is no safe level of exposure to tobacco smoke. Every inhalation of tobacco smoke exposes our children, our families, and our loved ones to dangerous chemicals that can damage their bodies and result in life-threatening diseases such as cancer and heart disease. And, although not a focus of this report, we know that smokeless tobacco causes cancer and has other adverse health effects. The science is now clear that “appropriate remedial actions” include protecting everyone in the country from having to breathe secondhand smoke; mak¬ing all tobacco products progressively less affordable; expanding access to proven cessation treatments and services;

iii taking actions at the federal, state, and local levels to counteract the influence of tobacco advertising, promotions, and sponsorship; and ensuring that all adults and children clearly understand that the result of tobacco use is addiction, suffering, reduced quality of life, and all too often, early death. Forty- five years after Surgeon General Terry called on this nation to act, I say, if not now, when? The health of our nation depends on it.

Regina Benjamin, M.D., M.B.A. Surgeon General

iv How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease

Acknowledgments

This report was prepared by the U.S. Department of Health Samuel F. Posner, Ph.D., Associate Director for Science and Human Services under the general direction of the (Acting), National Center for Chronic Disease Prevention Centers for Disease Control and Prevention, National Cen- and Health Promotion, Centers for Disease Control and ter for Chronic Disease Prevention and Health Promotion, Prevention, Atlanta, Georgia. Office on Smoking and Health. Matthew T. McKenna, M.D., M.P.H., Director, Office on Regina Benjamin, M.D., M.B.A., Surgeon General, U.S. Smoking and Health, National Center for Chronic Disease Public Health Service, Office of Public Health and Science, Prevention and Health Promotion, Centers for Disease Office of the Surgeon General, Office of the Secretary, U.S. Control and Prevention, Atlanta, Georgia. Department of Health and Human Services, Washington, D.C. Terry F. Pechacek, Ph.D., Associate Director for Sci- ence, Office on Smoking and Health, National Center for Rear Admiral (Retired) Steven K. Galson, M.D., M.P.H., Chronic Disease Prevention and Health Promotion, Cen- former Acting Surgeon General, U.S. Public Health Ser- ters for Disease Control and Prevention, Atlanta, Georgia. vice, Office of the Surgeon General, Office of Public Health and Science, Office of the Secretary, U.S. Department of Health and Human Services, Washington, D.C. The editors of the report were David Sidransky, M.D., Senior Scientific Editor, Professor, Rear Admiral Robert C. Williams, P.E., D.E.E., Acting Dep- Otolaryngology-Head & Neck Surgery, Oncology, Pathol- uty Surgeon General, U.S. Public Health Service, Office ogy, Urology, Cellular and Molecular Medicine, and Direc- of the Surgeon General, Office of Public Health and Sci- tor, Head and Neck Cancer Research, The Johns Hopkins ence, Office of the Secretary, U.S. Department of Health University School of Medicine, Johns Hopkins Medical and Human Services, Washington, D.C. Institutions, Baltimore, Maryland. Rear Admiral Carol A. Romano, Ph.D., R.N., F.A.A.N., Act- Leslie A. Norman, Managing Editor, Office on Smoking ing Chief of Staff, U.S. Public Health Service, Office of the and Health, National Center for Chronic Disease Preven- Surgeon General, Office of Public Health and Science, tion and Health Promotion, Centers for Disease Control Office of the Secretary, U.S. Department of Health and and Prevention, Atlanta, Georgia. Human Services, Washington, D.C. Anne McCarthy, Technical Editor, Northrop Grumman Mary Beth Bigley, Dr.P.H., M.S.N., A.N.P., Acting Director supporting National Center for Health Marketing, Divi- of the Office of Science and Communication, U.S. Public sion of Creative Services, Centers for Disease Control and Health Service, Office of the Surgeon General, Office of Prevention, Atlanta, Georgia. Public Health and Science, Office of the Secretary, U.S. Department of Health and Human Services, Washington, Peter L. Taylor, M.B.A., Technical Editor, Senior Editor, D.C. Palladian Partners, Silver Spring, Maryland. Rear Admiral (Retired) Kenneth P. Moritsugu, M.D., M.P.H., former Acting Surgeon General, U.S. Public Contributing editors were Health Service, Office of the Surgeon General, Office of Public Health and Science, Office of the Secretary, U.S. David L. Ashley, Ph.D., Chief, Emergency Response and Department of Health and Human Services, Washington, Air Toxicants Branch, Division of Laboratory Sciences, D.C. National Center for Environmental Health, Centers for Disease Control and Prevention, Atlanta, Georgia. Thomas R. Frieden, M.D., M.P.H., Director, Centers for Disease Control and Prevention, Atlanta, Georgia. Neal L. Benowitz, M.D., Professor of Medicine and Bio- pharmaceutical Sciences and Chief, Division of Clinical Janet Collins, Ph.D., Director, National Center for Chronic Pharmacology and Experimental Therapeutics, University Disease Prevention and Health Promotion, Centers for of California, San Francisco, California. Disease Control and Prevention, Atlanta, Georgia.

v Surgeon General’s Report

Dorothy Hatsukami, Ph.D., Forster Family Professor in Tomoku Betsuyaku, M.D., Ph.D., Associate Professor, First Cancer Prevention, Professor of Psychiatry, University of Department of Medicine, Hokkaido University School of Minnesota Tobacco Use Research Center, University of Medicine, Sapporo, Japan. Minnesota, Minneapolis, Minnesota. Ann M. Bode, Ph.D., Research Professor and Associate Stephen S. Hecht, Ph.D., Wallin Professor of Cancer Pre- Director, The Hormel Institute, University of Minnesota, vention, Masonic Cancer Center, University of Minnesota, Austin, Minnesota. Minneapolis, Minnesota. Anne Burke, M.D., Assistant Professor of Medicine, Gas- Jack E. Henningfield, Ph.D., Vice President Research and troenterology Division, University of Pennsylvania School Health Policy, Pinney Associates, Bethesda, Maryland; and of Medicine, Philadelphia, Pennsylvania. Professor, Adjunct, Department of Psychiatry and Behav- ioral Sciences, The Johns Hopkins University School of David M. Burns, M.D., Professor Emeritus, Department of Medicine, Baltimore, Maryland. Family and Preventive Medicine, School of Medicine, Uni- versity of California, San Diego, California. Patricia Richter, Ph.D., DABT, Toxicologist, Office on Smoking and Health, National Center for Chronic Disease Rebecca Buus, Ph.D., Health Scientist, Maternal and Infant Prevention and Health Promotion, Centers for Disease Health Branch, Division of Reproductive Health, National Control and Prevention, Atlanta, Georgia. Center for Chronic Disease Prevention and Health Promo- tion, Centers for Disease Control and Prevention, Atlanta, Jonathan M. Samet, M.D., M.S., Professor and Chairman, Georgia. Department of Epidemiology, Bloomberg School of Public Health, The Johns Hopkins University, Baltimore, Mary- Laurie Chassin, Ph.D., Regents Professor of Psychology, land (1994–2008); and Director, USC Institute for Global Psychology Department, Arizona State University, Tempe, Health, and Chairman, Department of Preventive Medi- Arizona. cine, Keck School of Medicine, University of Southern California, Los Angeles, California (2008–present). David Christiani, M.D., M.P.H., Professor, Departments of Environmental Health and Epidemiology, Harvard School Gayle C. Windham, Ph.D., M.S.P.H., Research Scientist of Public Health, Harvard University, Boston, Massachu- Supervisor, Chief, Environmental Surveillance Unit, Divi- setts. sion of Environmental and Occupational Disease Control, California Department of Public Health, Richmond, Cali- Pamela I. Clark, M.S.P.H., Ph.D., Research Professor, fornia. School of Public Health, University of Maryland, College Park, Maryland.

Contributing authors were John P. Cooke, M.D., Ph.D., Professor of Medicine, Stan- ford University, Stanford, California. David L. Ashley, Ph.D., Chief, Emergency Response and Air Toxicants Branch, Division of Laboratory Sciences, Adolfo Correa, M.D., Ph.D., M.P.H., Medical Officer, Divi- National Center for Environmental Health, Centers for sion of Birth Defects and Developmental Disabilities, Disease Control and Prevention, Atlanta, Georgia. National Center on Birth Defects and Developmental Disabilities, Centers for Disease Control and Prevention, Timothy B. Baker, Ph.D., Professor of Medicine, University Atlanta, Georgia. of Wisconsin School of Medicine and Public Health, Madi- son, Wisconsin. Johannes Czernin, M.D., Professor, Molecular and Medical Pharmacology, and Chief, Ahmanson Biological Imaging Steve A. Belinsky, Ph.D., Director, Lung Cancer Program, Division, David Geffen School of Medicine, University of Senior Scientist, Lovelace Respiratory Research Institute, California, Los Angeles, California. Albuquerque, New Mexico. Gerald N. DeLorenze, Ph.D., Research Scientist, Division Neal L. Benowitz M.D., Professor of Medicine and Bio- of Research, Kaiser Permanente Northern California, Oak- pharmaceutical Sciences and Chief, Division of Clinical land, California. Pharmacology and Experimental Therapeutics, University of California, San Francisco, California.

vi How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease

David M. DeMarini, Ph.D., Genetic Toxicologist, Integrated Dorothy Hatsukami, Ph.D., Forster Family Professor in Systems Toxicology Division, U.S. Environmental Protec- Cancer Prevention, Professor of Psychiatry, University of tion Agency, Research Triangle Park, North Carolina. Minnesota Tobacco Use Research Center, University of Minnesota, Minneapolis, Minnesota. Delia Dempsey, M.D., M.S., Assistant Professor, Pediatrics, Medicine and Clinical Pharmacology, Division of Clinical Stephen S. Hecht, Ph.D., Wallin Professor of Cancer Pre- Pharmacology, University of California, San Francisco, vention, Masonic Cancer Center, University of Minnesota, San Francisco, California. Minneapolis, Minnesota.

Phillip A. Dennis, M.D., Ph.D., Senior Investigator, Medical Marc K. Hellerstein, M.D., Ph.D., Professor (DH Calloway Oncology Branch, Center for Cancer Research, National Chair in Human Nutrition), Department of Nutritional Cancer Institute, National Institutes of Health, Bethesda, Sciences, University of California, Berkeley, California; Maryland. and Professor of Endocrinology, Metabolism and Nutri- tion, Department of Medicine, University of California, Yan Shirley Ding, Ph.D., Senior Service Fellow, Emer- San Francisco, California. gency Response and Air Toxicants Branch, Division of Laboratory Sciences, National Center for Environmen- Jack E. Henningfield, Ph.D., Vice President Research and tal Health, Centers for Disease Control and Prevention, Health Policy, Pinney Associates, Bethesda, Maryland; and Atlanta, Georgia. Professor, Adjunct, Department of Psychiatry and Behav- ioral Sciences, The Johns Hopkins University School of Mirjana V. Djordjevic, Ph.D., Program Director, Tobacco Medicine, Baltimore, Maryland. Control Research Branch, Behavioral Research Program, Division of Cancer Control and Population Sciences, James C. Hogg, M.D., Ph.D., Emeritus Professor of National Cancer Institute, National Institutes of Health, Pathology and Lab Medicine, iCAPTURE Centre for Car- Bethesda, Maryland. diovascular and Pulmonary Research, University of Brit- ish Columbia and St. Paul’s Hospital, Vancouver, British Zigang Dong, M.D., M.S., Dr.P.H., Professor, The Hormel Columbia, Canada. Institute, University of Minnesota, Austin, Minnesota. Anne M. Joseph, M.D., M.P.H., Wexler Professor of Medi- Björn Eliasson, M.D., Ph.D., Associate Professor, Depart- cine, and Director, Applied Clinical Research Program, ment of Internal Medicine, Sahlgrenska University Hospi- University of Minnesota Medical School, Minneapolis, tal, Göteborg, Sweden. Minnesota.

Lucinda England, M.D., M.S.P.H., Medical Epidemiologist, Caryn Lerman, Ph.D., Mary W. Calkins Professor, Depart- Maternal and Infant Health Branch, Division of Reproduc- ment of Psychiatry and Annenberg Public Policy Center; tive Health, National Center for Chronic Disease Preven- and Director, Tobacco Use Research Center, Scientific tion and Health Promotion, Centers for Disease Control Director, Abramson Cancer Center, University of Pennsyl- and Prevention, Atlanta, Georgia. vania, Philadelphia, Pennsylvania.

Reginald Fant, Ph.D., Senior Scientist and Director, Pin- William MacNee, Professor, ELEGI Colt Research Labs, ney Associates, Bethesda, Maryland. MRC Centre for Inflammation Research, The Queen’s Medical Research Institute, University of Edinburgh, Garret A. FitzGerald, M.D., Chair, Department of Pharma- Edinburgh, Scotland, United Kingdom. cology, and Director, Institute for Translational Medicine and Therapeutics, University of Pennsylvania School of Athina Markou, Ph.D., Professor, Department of Psy- Medicine, Philadelphia, Pennsylvania. chiatry, School of Medicine, University of California, San Diego, La Jolla, California. Mari S. Golub, Ph.D., Staff Toxicologist, Office of Environ- mental Health Hazard Assessment, California Environ- Robert Merritt, M.A., Branch Chief, Epidemiology and mental Protection Agency, Sacramento, California. Surveillance Branch, Division of Heart Disease and Stroke Prevention, National Center for Chronic Disease Preven- tion and Health Promotion, Centers for Disease Control and Prevention, Atlanta, Georgia.

vii Surgeon General’s Report

Dennis L. Molfese, Ph.D., Distinguished University Scholar Halit Pinar, M.D., Director, Division of Perinatal and Pedi- and Professor, Birth Defects Center, University of Louis- atric Pathology, Women and Infants Hospital of Rhode ville, Louisville, Kentucky. Island; and Professor, Department of Pathology and Labo- ratory Medicine, Brown University Alpert School of Medi- Masaaki Moriya, Ph.D., Research Professor, Laboratory of cine, Providence, Rhode Island. Chemical Biology, Department of Pharmacological Sci- ences, University at Stony Brook, The State University of Gregory Polzin, Ph.D., Research Chemist, Emergency New York, Stony Brook, New York. Response and Air Toxicants Branch, Division of Laboratory Sciences, National Center for Environmental Health, Cen- Marcus Munafò, M.A., M.Sc., Ph.D., Reader in Biological ters for Disease Control and Prevention, Atlanta, Georgia. Psychology, Department of Experimental Psychology, Uni- versity of Bristol, Bristol, United Kingdom. Patricia Richter, Ph.D., DABT, Toxicologist, Office on Smoking and Health, National Center for Chronic Disease Sharon E. Murphy, Ph.D., Professor, Biochemistry, Molec- Prevention and Health Promotion, Centers for Disease ular Biology and BioPhysics, Masonic Cancer Center, Uni- Control and Prevention, Atlanta, Georgia. versity of Minnesota, Minneapolis, Minnesota. Nancy Rigotti, M.D., Professor of Medicine, Harvard Medi- Raymond Niaura, Ph.D., Professor, Department of Psy- cal School; and Director, Tobacco Research and Treatment chiatry and Human Behavior, The Warren Alpert Center, Massachusetts General Hospital, Boston, Massa- Medical School of Brown University; and Director, Trans- chusetts. disciplinary Research, Butler Hospital, Providence, Rhode Island. Wendie A. Robbins, Ph.D., Associate Professor, UCLA Schools of Nursing and Public Health, University of Cali- James Pankow, Ph.D., Professor, Department of Chem- fornia, Los Angeles, California. istry, and Department of Civil and Environmental Engi- neering, Portland State University, Portland, Oregon. Andrew G. Salmon, M.A., D.Phil., Senior Toxicologist, Office of Environmental Health Hazard Assessment, Cali- Steve Pappas, Ph.D., Research Chemist, Emergency fornia Department of Health Services, Oakland, California. Response and Air Toxicants Branch, Division of Laboratory Sciences, National Center for Environmental Health, Cen- Jonathan M. Samet, M.D., M.S., Professor and Chairman, ters for Disease Control and Prevention, Atlanta, Georgia. Department of Epidemiology, Bloomberg School of Public Health, The Johns Hopkins University, Baltimore, Mary- Terry F. Pechacek, Ph.D., Associate Director for Science, land (1994–2008); and Director, USC Institute for Global Office on Smoking and Health, National Center for Health, and Chairman, Department of Preventive Medi- Chronic Disease Prevention and Health Promotion, Cen- cine, Keck School of Medicine, University of Southern ters for Disease Control and Prevention, Atlanta, Georgia. California, Los Angeles, California (2008–present).

Kenneth A. Perkins, Ph.D., Professor of Psychiatry, West- Robert M. Senior, M.D., Professor of Pulmonary Medicine, ern Psychiatric Institute and Clinic, University of Pitts- Cell Biology & Physiology, Division of Pulmonary and burgh School of Medicine, Pittsburgh, Pennsylvania. Critical Care, Washington University School of Medicine, St. Louis, Missouri. Lisa A. Peterson, Ph.D., Professor, Masonic Cancer Center and the Division of Environmental Health Sciences, Uni- Edwin K. Silverman, M.D., Ph.D., Associate Professor of versity of Minnesota, Minneapolis, Minnesota. Medicine, Harvard Medical School, Harvard University; and Associate Physician, Brigham and Women’s Hospital, Gerd P. Pfeifer, Ph.D., Professor and Chair, Division of Biol- Boston, Massachusetts. ogy, Beckman Research Institute, City of Hope National Medical Center, Duarte, California. Margaret R. Spitz, M.D., M.P.H., Professor and Chair, Department of Epidemiology, The University of Texas, M. D. Anderson Cancer Center, Houston, Texas.

viii How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease

Stephen B. Stanfill, M.S., Chemist, Emergency Response Mary Beth Bigley, Dr.P.H., M.S.N., A.N.P., Acting Direc- and Air Toxicants Branch, National Center for Environ- tor of the Office of Science and Communication, Office mental Health, Centers for Disease Control and Preven- of the Surgeon General, Office of Public Health and Sci- tion, Atlanta, Georgia. ence, Office of the Secretary, U.S. Department of Health and Human Services, Washington, D.C. Prudence Talbot, Ph.D., Professor of Cell Biology, Depart- ment of Cell Biology and Neuroscience, University of Cali- Michele Bloch, M.D., Ph.D., Medical Officer, Tobacco Con- fornia, Riverside, California. trol Research Branch, Behavioral Research Program, Divi- sion of Cancer Control and Population Sciences, National Anne Thorndike, M.D., M.P.H., Instructor in Medicine, Cancer Institute, National Institutes of Health, Bethesda, General Medicine Unit, Massachusetts General Hospital, Maryland. Boston, Massachusetts. Naomi Breslau, Ph.D., Professor, Department of Epidemi- Clifford H. Watson, Ph.D., Lead Research Chemist, Emer- ology, Michigan State University, East Lansing, Michigan. gency Response and Air Toxicants Branch, National Cen- ter for Environmental Health, Centers for Disease Control Klaus D. Brunnemann, M.S., Research Associate, New and Prevention, Atlanta, Georgia. York Medical College, Valhalla, New York.

Gayle C. Windham, Ph.D., M.S.P.H., Research Scientist David M. Burns, M.D., Professor Emeritus, Department of Supervisor, Chief, Environmental Surveillance Unit, Divi- Family and Preventive Medicine, School of Medicine, Uni- sion of Environmental and Occupational Disease Control, versity of California, San Diego, California. California Department of Public Health, Richmond, Cali- fornia. Pamela I. Clark, M.S.P.H., Ph.D., Research Professor, School of Public Health, University of Maryland, College Weija (William) Wu, Ph.D., Senior Service Fellow, Emer- Park, Maryland. gency Response and Air Toxicants Branch, Division of Laboratory Sciences, National Center for Environmen- Gregory N. Connolly, D.M.D., M.P.H., Professor, Depart- tal Health, Centers for Disease Control and Prevention, ment of Society, Human Development, and Health, Har- Atlanta, Georgia. vard School of Public Health, Harvard University, Boston, Massachusetts. Mitchell Zeller, J.D., Vice President for Policy and Strate- gic Communications, Pinney Associates, Bethesda, Mary- William A. Corrigall, Ph.D., Professor of Psychiatry, Uni- land. versity of Minnesota; and Senior Scientist, Minneapolis Medical Research Foundation, Minneapolis, Minnesota.

Reviewers were James D. Crapo, M.D., Professor of Medicine, National Jewish Health, Denver, Colorado. Erik M. Augustson, Ph.D., M.P.H., Behavioral Scientist/ Psychologist, Tobacco Control Research Branch, Behav- Michael Criqui, M.D., M.P.H., Professor and Chief, Divi- ioral Research Program, Division of Cancer Control and sion of Preventive Medicine, Department of Family and Population Sciences, National Cancer Institute, National Preventive Medicine; and Professor, Department of Medi- Institutes of Health, Bethesda, Maryland. cine, School of Medicine, University of California, San Diego, La Jolla, California. Cathy L. Backinger, Ph.D., M.P.H., Chief, Tobacco Control Research Branch, Behavioral Research Program, Divi- K. Michael Cummings, Ph.D., M.P.H., Senior Research sion of Cancer Control and Population Sciences, National Scientist, Chair, Department of Health Behavior, Division Cancer Institute, National Institutes of Health, Bethesda, of Cancer Prevention and Population Sciences, Roswell Maryland. Park Cancer Institute, Buffalo, New York. Neal L. Benowitz M.D., Professor of Medicine and Bio- pharmaceutical Sciences and Chief, Division of Clinical Pharmacology and Experimental Therapeutics, University of California, San Francisco, California.

ix Surgeon General’s Report

Mirjana V. Djordjevic, Ph.D., Program Director, Tobacco John E. Hokanson, Ph.D., Associate Professor and Chair, Control Research Branch, Behavioral Research Program, Department of Epidemiology, Colorado School of Public Division of Cancer Control and Population Sciences, Health, University of Colorado, Denver, Aurora, Colorado. National Cancer Institute, National Institutes of Health, Bethesda, Maryland. John Hughes, M.D., Professor, Department of Psychiatry, University of Vermont, Burlington, Vermont. Erik Dybing, M.D., Ph.D., Division Director and Professor (retired), Division of Environmental Health, Norwegian Richard D. Hurt, M.D., Professor of Medicine, Mayo Clinic Institute of Public Health, Oslo, Norway. College of Medicine, Rochester, Minnesota.

Michael Eriksen, Sc.D., Professor, and Director, Institute Jared B. Jobe, Ph.D., FABMR, Program Director, Clinical of Public Health, Georgia State University, Atlanta, Geor- Applications and Prevention Branch, Division of Preven- gia. tion and Population Sciences, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Jefferson Fowles, Ph.D., Toxicologist, LyondellBasell Maryland. Industries, Rotterdam, The Netherlands. Rachel Kaufmann, Ph.D., M.P.H., Deputy Associate Direc- Adi F. Gazdar, M.D., Professor, Department of Pathology, tor for Science, Office on Smoking and Health, National University of Texas Southwestern Medical Center, Dallas, Center for Chronic Disease Prevention and Health Promo- Texas. tion, Centers for Disease Control and Prevention, Atlanta, Georgia. Gary A. Giovino, Ph.D., M.S., Professor and Chair, Depart- ment of Health Behavior, School of Public Health and Karl T. Kelsey, M.D., M.O.H., Professor, Community Health Professions, University at Buffalo, The State Uni- Health and Pathology and Laboratory Medicine, Director, versity of New York, Buffalo, New York. Center for Environmental Health and Technology, Brown University, Providence, Rhode Island. Stanton Glantz, Ph.D., Professor of Medicine, Division of Cardiology, University of California, San Francisco, Cali- Juliette Kendrick, M.D., Medical Officer, Division of fornia. Reproductive Health, National Center for Chronic Disease Prevention and Health Promotion, Centers for Disease Michael R. Guerin, Ph.D., Associate Division Director Control and Prevention, Atlanta, Georgia. (retired), Oak Ridge National Laboratory, Knoxville, Ten- nessee. Martin Kharrazi, Ph.D., Chief, Program Research and Demonstration Section, Genetic Disease Screening Pro- Dorothy Hatsukami, Ph.D., Forster Family Professor in gram, California Department of Public Health, Richmond, Cancer Prevention, Professor of Psychiatry, University of California. Minnesota Tobacco Use Research Center, University of Minnesota, Minneapolis, Minnesota. Taline V. Khroyan, Ph.D., Behavioral Pharmacologist, SRI International, Center for Health Sciences, Menlo Park, Jack E. Henningfield, Ph.D., Vice President Research and California. Health Policy, Pinney Associates, Bethesda, Maryland; and Professor, Adjunct, Department of Psychiatry and Behav- Walther N.M. Klerx, Food and Consumer Product Safety ioral Sciences, The Johns Hopkins University School of Authority, Eindhoven, The Netherlands. Medicine, Baltimore, Maryland. Philip Lazarus, Ph.D., Professor of Pharmacology, College Allison Chausmer Hoffman, Ph.D., Program Director, of Medicine, Pennsylvania State University, Milton S. Her- National Institute on Drug Abuse, National Institutes of shey Medical Center, Hershey, Pennsylvania. Health, Bethesda, Maryland. Scott Leischow, Ph.D., Associate Director for Behavioral John R. Hoidal, M.D., Professor and Chairman of Medi- and Social Sciences Research, Arizona Cancer Center, The cine, School of Medicine, University of Utah Health Sci- University of Arizona, Tucson, Arizona. ences Center, Salt Lake City, Utah.

x How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease

Christina N. Lessov-Schlaggar, Ph.D., Research Assistant Scott Rogers, M.P.H., Public Health Advisor, Epidemiology Professor, Department of Psychiatry, Washington Univer- and Genetics Research Program, Division of Cancer Con- sity School of Medicine, St. Louis, Missouri. trol and Population Sciences, National Cancer Institute, National Institutes of Health, Bethesda, Maryland. Catherine Lorraine, Director, Policy Development and Coordination Staff, Food and Drug Administration, Silver Dale P. Sandler, Ph.D., Chief, Epidemiology Branch, Spring, Maryland. National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, Cathy Melvin, Ph.D., M.P.H., Director, Child Health Ser- North Carolina. vices Program, Cecil G. Sheps Center for Health Services Research, University of North Carolina, Chapel Hill, North David A. Scott, Ph.D., Associate Professor, Oral Health and Carolina. Systemic Disease Research Group, University of Louis- ville, Louisville, Kentucky. John D. Minna, M.D., Director, Hamon Center for Thera- peutic Oncology Research, University of Texas Southwest- Harold E. Seifried, Ph.D., DABT, Chemist, Toxicologist, ern Medical Center, Dallas, Texas. Industrial Hygienist (retired), Program Director, Nutri- tional Sciences Research Group, Division of Cancer Pre- Joshua E. Muscat, Ph.D., Professor, Department of Public vention, National Cancer Institute, National Institutes of Health Sciences, College of Medicine, Pennsylvania State Health, Bethesda, Maryland. University, Milton S. Hershey Medical Center, Hershey, Pennsylvania. Peter G. Shields, M.D., Professor of Medicine and Oncol- ogy, Deputy Director, Lombardi Comprehensive Cancer Mark Parascandola, Ph.D., M.P.H., Epidemiologist, Center, Georgetown University Medical Center, Washing- Tobacco Control Research Branch, Behavioral Research ton, D.C. Program, Division of Cancer Control and Population Sci- ences, National Cancer Institute, National Institutes of Saul Shiffman, Ph.D., Research Professor, Psychology and Health, Bethesda, Maryland. Psychiatry, University of Pittsburgh; and Senior Scientific Advisor, Pinney Associates, Pittsburgh, Pennsylvania. Wallace Pickworth, Ph.D., Health Research Leader, Bat- telle Centers for Public Health Research and Evaluation, Donald R. Shopland, Sr., U.S. Public Health Service Baltimore, Maryland. (retired), Ringgold, Georgia.

John M. Pinney, President, Pinney Associates, Bethesda, Stephen Sidney, M.D., M.P.H., Associate Director for Clini- Maryland. cal Research, Division of Research, Kaiser Permanente, Oakland, California. Britt C. Reid, D.D.S., Ph.D., Chief, Modifiable Risk Factors Branch, Epidemiology and Genetics Research Program, Gary D. Stoner, Ph.D., Professor Emeritus, Department of Division of Cancer Control and Population Sciences, Internal Medicine, The Ohio State University, Columbus, National Cancer Institute, National Institutes of Health, Ohio. Bethesda, Maryland. Gary E. Swan, Ph.D., Director, Center for Health Sciences, John E. Repine, M.D., Director, Webb-Waring Center, War- SRI International Fellow, SRI International, Center for ing Professor of Medicine and Pediatrics, School of Medi- Health Sciences, Menlo Park, California. cine, University of Colorado, Denver, Colorado. Michael J. Thun, M.D., M.S., Vice President Emeritus, Epi- William Rickert, Ph.D., Chief Executive Officer, Labstat demiology and Surveillance Research, American Cancer International ULC, Kitchener, Ontario, Canada. Society, Atlanta, Georgia.

David J. Riley, M.D., Professor, Department of Medicine, Robert B. Wallace, M.D., M.Sc., Irene Ensminger Stecher University of Medicine and Dentistry of New Jersey, Robert Professor of Epidemiology and Internal Medicine, Depart- Wood Johnson Medical School, Piscataway, New Jersey. ment of Epidemiology, University of Iowa College of Pub- lic Health, Iowa City, Iowa.

xi Surgeon General’s Report

Deborah M. Winn, Ph.D., Deputy Director, Epidemiology Marta Gwinn, M.D., M.P.H., Medical Officer, Office of Pub- and Genetics Research Program, Division of Cancer Con- lic Health Genomics, National Center for Chronic Disease trol and Population Sciences, National Cancer Institute, Prevention and Health Promotion, Centers for Disease National Institutes of Health, Bethesda, Maryland. Control and Prevention, Atlanta, Georgia.

Nancy Leonard, Administrative Coordinator to Dr. Jona- Other contributors were than M. Samet, Department of Epidemiology, Bloomberg School of Public Health, The Johns Hopkins University, Charlotte Gerczak, M.L.A., Communications Associate, Baltimore, Maryland. Department of Epidemiology, Bloomberg School of Public Health, The Johns Hopkins University, Baltimore, Mary- Deborah Williams, Desktop Publishing Specialist to Dr. land. Jonathan M. Samet, Department of Epidemiology, Bloom- berg School of Public Health, The Johns Hopkins Univer- Roberta B. Gray, Administrative Supervisor to Dr. Jona- sity, Baltimore, Maryland. than M. Samet, Department of Epidemiology, Bloomberg School of Public Health, The Johns Hopkins University, Baltimore, Maryland.

xii How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease

Chapter 5. Cancer 221 Introduction 225 Carcinogen Exposure, Metabolism, and DNA Adducts 227 DNA Repair and Conversion of Adducts to Mutations 252 Gene Mutations in Tobacco-Induced Cancer 274 Loss of Mechanisms for Growth Control 284 Other Aspects 296 Evidence Summary 302 Conclusions 304 References 305

Chapter 6. Cardiovascular Diseases 351 Introduction 355 Tobacco Use and Cardiovascular Disease 355 Secondhand Tobacco Smoke and Cardiovascular Disease 363 Pathophysiology 363 Hemodynamic Effects 368 Smoking and the Endothelium 370 Thrombogenic Effects 374 Inflammation 382 Smoking and Diabetes 383 Lipid Abnormalities 387 Cardiovascular Biomarkers 391 Smoking Cessation and Cardiovascular Disease 394 Methods to Reduce Exposure 402 Evidence Summary 407 Conclusions 408 References 409

List of Abbreviations 671

List of Tables and Figures 675

Definitions and Alternative Nomenclature of Genetic Symbols Used in This Report 679

Index 689

xiii

Chapter 5 Cancer

Introduction 225

Carcinogen Exposure, Metabolism, and DNA Adducts 227 Carcinogens in Cigarette Smoke 227 Biomarkers of Carcinogens in Smokers 230 Urinary Biomarkers 230 Breath and Blood Biomarkers 234 Summary 234 Metabolic Activation and Detoxification of Carcinogens 235 Enzymology of Carcinogen Metabolism 237 Introduction 237 Cytochrome P-450 Enzymes 237 Epoxide Hydrolases 238 Glutathione-S-Transferases 239 Uridine-5’-Diphosphate-Glucuronosyltransferases 240 N-Acetyltransferases 241 DNA Adducts and Biomarkers 242 Introduction 242 Characterized Adducts in the Human Lung 242 Uncharacterized Adducts in Human Lung Tissue 244 Adducts in Other Tissues 244 Protein Adducts as Surrogates for DNA Adducts 245 Summary 245 Molecular Epidemiology of Polymorphisms in Carcinogen-Metabolizing Genes 245 Introduction 245 CYP1A1 Gene 246 CYP2E1 Gene 247 CYP2A13 Gene 247 GSTM1 Gene 248 CYP1A1 and GSTM1 in Combination 248 GSTP1 Gene 249 GSTT1 Gene 249 NAT2 Gene 249 Microsomal Epoxide Hydrolase 250 Genes in the Pathway for Metabolism of Reactive Oxygen Species 251 Summary 251

DNA Repair and Conversion of Adducts to Mutations 252 Repair of DNA Adducts 252 Introduction 252 O6-Alkylguanine–DNA Alkyltransferase 252 Base Excision Repair 253 Nucleotide Excision Repair 254

221 Mismatch Repair 258 Double-Strand Break Repair 259 Molecular Epidemiology of DNA Repair 259 Functional Assays of DNA Damage and Repair to Tobacco Carcinogens 261 8-Oxoguanine DNA Glycosylase Activity Assay 263 Mutagen Sensitivity Assays 263 Comet Assay 263 Polymorphisms in O6-Alkylguanine–DNA Alkyltransferase 264 Polymorphisms in the Pathway for Repair of Base Excision 264 Polymorphisms in the Pathway for Nucleotide Excision Repair 266 Genotype-Phenotype Correlations 267 Polymorphisms in the Pathway for Double-Strand Break Repair 268 Summary 268 Conversion of DNA Adducts to Mutations 268 Molecular Analysis of Conversion to Mutations 269 Translesion Synthesis in Mammalian Cells 269 Factors in Outcome of Translesion Synthesis 271 Conversion of Cigarette-Smoke-Induced DNA Adducts to Mutations 272 Assessment of Genotoxicity of DNA Adducts 273

Gene Mutations in Tobacco-Induced Cancer 274 Chromosome Instability and Loss 274 Lung Cancer 274 Identification of Tumor-Suppressor Genes 274 Tumor-Suppressor Genes Inactivated in Lung Cancer 275 Activation of Oncogenes in Lung Cancer 275 Oncogene Activation, Tumor-Suppressor Gene Inactivation, and Lung Cancer Survival 277 Relationship of TP53 Mutations to Smoking and Carcinogens 278 TP53 Mutations in Smoking-Associated Lung Cancers 278 G→T Transversions in Lung Cancer 279 TP53 Gene Mutations in Other Smoking-Associated Cancers 283 Limitations to the Study of TP53 Mutations and Smoking-Induced Cancer 283

Loss of Mechanisms for Growth Control 284 Signal Transduction 284 Introduction 284 Apoptosis 284 Key Apoptotic Regulators 284 Regulation of Tumor Suppressors and Proapoptotic Proteins 284 Regulation of Antiapoptotic Proteins and Effects 286 Summary 288 Cigarette Smoke and Activation of Cell-Surface Receptors in Cancer 288 Airway Epithelial Cells 288 Activation of Cytoplasmic Kinase by Tobacco Smoke 290 Downstream Targets of Signaling Cascades Mediated by Tobacco Smoke 292 Gene Promoter Hypermethylation in Cancer Induced by Tobacco Smoke 292 Alternative to Mutation 292 Inactivation of the P16 Gene in Lung Cancer 292 Critical Pathways Inactivated in Non-Small-Cell and Small-Cell Lung Cancer 292 Gene Silencing in Lung Cancer 293 Gene Promoter Hypermethylation, Prognosis, and Clinical Risk Factors 293 Other Tobacco-Related Cancers 294

222 Molecular Epidemiology of Cell-Cycle Control and Tobacco-Induced Cancer 294 Introduction 294 CCND1 Gene 294 P21 Protein 295 TP53 Gene 295 P73 Gene 296

Other Aspects 296 Carcinogenic Effects of Whole Mixture and Fractions of Tobacco Smoke 296 Synergistic Interactions in Tobacco Carcinogenesis 297 Alcohol 297 Asbestos 298 Carcinogens as Causes of Specific Cancers 298 Tobacco Carcinogens, Immune System, and Cancer 300 Epidemiology of Family History and Lung Cancer 300

Evidence Summary 302

Conclusions 304

References 305

223

How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease

Introduction

The 2004 Surgeon General’s report, The Health P-450s may be a critical aspect of cancer susceptibility in Consequences of Smoking: A Report of the Surgeon Gen- smokers. P-450s 1A2, 2A6, 2A13, and 2E1 are also eral (U.S. Department of Health and Human Services important in the activation of cigarette smoke carcino- [USDHHS] 2004), concluded that the evidence is suffi- gens. Competing with the activation process is metabolic cient to infer a causal relationship between smoking and detoxification, which excretes carcinogen metabolites in cancers of the lung, larynx, oral cavity, pharynx, esopha- generally harmless forms and is catalyzed by a variety of gus, pancreas, bladder, kidney, cervix, and stomach, and enzymes, including glutathione-S-transferases (GSTs), acute myeloid leukemia. In addition, the report found that uridine-5’-disphosphate-glucuronosyltransferases (UGTs), evidence suggests a causal relationship between smoking epoxide hydrolases, and sulfatases. The balance between and colorectal and liver cancers. This chapter examines metabolic activation and detoxification of carcinogens the mechanisms by which cigarette smoking induces varies among persons and likely affects cancer susceptibil- cancer. Literature citations for this section’s discussion ity. Persons with a higher activation and lower detoxifica- appear in subsequent sections of this chapter, as appropri- tion capacity are at the highest risk for smoking-related ate. A schematic overview of the pertinent mechanisms cancers. This finding is supported by considerable evi- discussed in this chapter is presented in Figure 5.1. The dence from molecular epidemiologic studies of the poly- figure depicts the major established pathways of cancer morphisms (variants) in these enzymes. causation by cigarette smoking: (1) the exposure to car- The metabolic activation of carcinogens results cinogens (cancer-causing substances), (2) the formation in the formation of DNA adducts, which are absolutely of covalent bonds between the carcinogens and DNA (DNA central to the carcinogenic process. However, some car- adduct formation), and (3) the resulting accumulation cinogens can directly form DNA adducts without meta- of permanent somatic mutations in critical genes (genes bolic activation. Since the mid-1980s, extensive studies appear in italics). Somatic mutations lead to clonal out- have examined the presence of DNA adducts in human growth and, through accumulation of additional muta- tissues. Studies that used nonspecific methods, such as tions, to development of cancer. 32P-postlabeling and immunoassays, to measure adducts Each puff of each cigarette contains a mixture of concluded that adduct levels in the lung and in other tis- thousands of compounds, including more than 60 well- sues are higher in smokers than in nonsmokers. Some established carcinogens. The carcinogens in cigarette epidemiologic data link higher adduct levels with a higher smoke belong to multiple chemical classes, including probability of developing cancer. polycyclic aromatic hydrocarbons (PAHs), N-nitrosamines, There are ample cellular repair systems that can aromatic amines, aldehydes, volatile organic hydrocar- remove DNA adducts and maintain a normal DNA struc- bons, and metals. In addition to these well-established car- ture. These systems include direct repair of DNA bases by cinogens, others have been less thoroughly investigated. alkyltransferases, the excision of DNA damage by base and These include alkylated PAHs, oxidants, free radicals, and nucleotide excision repair, mismatch repair, and double- ethylating agents. Considerable evidence indicates that strand break repair. If repair enzymes are overwhelmed in human cancers caused by cigarette smoking, PAHs, by DNA damage or for other reasons cannot function N-nitrosamines, aromatic amines, and certain volatile efficiently, DNA adducts may persist and increase the organic agents play a major role. Extensive data in the likelihood of developing somatic mutations. Inherited literature demonstrate the uptake of these carcinogens polymorphic variants in some DNA repair enzymes are by smokers. The data confirm the expected presence of also associated with decreased DNA repair activity and a metabolites of these substances in the urine of smokers at potentially higher probability of developing cancer. higher levels than those in nonsmokers. Persistent DNA adducts can cause miscoding (e.g., Most carcinogens in cigarette smoke require a insertion of the wrong base) during replication of DNA metabolic activation process, generally catalyzed by cyto- when DNA polymerase enzymes process the adducts chrome P-450 enzymes (P-450s), to convert the carcino- incorrectly. Considerable specificity exists in the relation- gens to forms that can covalently bind to DNA and form ship between specific DNA adducts caused by carcinogens DNA adducts. P-450s 1A1 and 1B1, which are inducible in cigarette smoke and the types of observed somatic by cigarette smoke through interactions with the aryl mutations; for example, an O6-methylguanine adduct hydrocarbon receptor, are particularly important in the causes G→A transitions. These types of mutations are metabolic activation of PAHs. The inducibility of these frequently observed in the KRAS oncogene in lung

Cancer 225 Surgeon General’s Report

Figure 5.1 Link between cigarette smoking and cancer through carcinogens in tobacco smoke

Protein Receptor kinase A and B binding activation and other changes

Mutations in Initiation of Regular oncogenes Loss of cigarette cigarette Uptake of Metabolic DNA Persistence normal growth and tumor- Cancer smoking/ smoking carcinogens activation adducts miscoding control nicotine suppressor mechanisms addiction genes Metabolic Repair detoxiﬁcation Apoptosis Excretion Normal DNA

Uptake of cocarcinogens Tumor-suppressor and tumor promoters gene inactivation Gene promoter hypermethylation and other changes

cancer and in the TP53 gene in a variety of cancers However, the top and bottom tracks of Figure 5.1 indicate induced by cigarette smoke. The KRAS and TP53 muta- that other pathways also contribute to carcinogenesis. tions observed in lung cancer in smokers appear to reflect Nicotine and tobacco-specific nitrosamines bind to nico- DNA damage caused by metabolically activated PAHs. How- tinic receptors and other cellular receptors. This binding ever, a number of other carcinogens or toxicants, such as then leads to the activation of protein kinase B (AKT, also N-nitrosamines and aldehydes, as well as oxidative dam- known as PKB), protein kinase A (PKA), and other key age, are also likely to be involved. Animal studies have biologic pathways for cytogenetic changes. Cigarette firmly established the cancer-causing role of mutations in smoke activates EGFR and COX-2, both known to be these genes. important in cell proliferation and transformation. Fur- Gene mutations can cause the loss of normal func- thermore, the occurrence of cocarcinogens and tumor tions in control of cellular growth, ultimately resulting promoters in cigarette smoke is well established. Although in cellular proliferation and cancer. Studies have strongly these compounds are not carcinogenic, they clearly linked chromosome damage in cells throughout the enhance the carcinogenicity of cigarette smoke carcino- aerodigestive tract to exposure to cigarette smoke. The gens through mechanisms that usually lead to stimula- protective process of programmed cell death (apoptosis) tion of cell proliferation. The reversibility of cancer risk can counterbalance these mutational events by removing after smoking cessation supports the role of tumor pro- cells with DNA damage. The balance between mechanisms moters and other epigenetic factors in tobacco carcino- leading to apoptosis and those suppressing apoptosis has genesis. However, the specifics of these effects have not a major impact on tumor growth. In addition, research- been fully elucidated. An important epigenetic pathway is ers have observed numerous cytogenetic changes in the enzymatic hypermethylation of promoter regions of lung cancer. genes, which can result in gene silencing. If this occurs The central track of Figure 5.1 that proceeds through in tumor-suppressor genes, the result can be unregulated genetic damage is clearly established as a major pathway cellular proliferation. by which carcinogens in cigarette smoke can cause cancer.

226 Chapter 5 How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease

Carcinogen Exposure, Metabolism, and DNA Adducts

Carcinogens in Cigarette Smoke rats and respiratory tract tumors in mice and hamsters (Hecht 1998). Carcinogens in cigarette smoke that were evalu- Aromatic amines in cigarette smoke are combustion ated by the International Agency for Research on Cancer products that include the well-known human bladder car- (IARC 2004) are listed in Table 5.1. All are carcinogenic cinogens 2-naphthylamine and 4-aminobiphenyl (4-ABP), in laboratory animals, and 15 are rated as carcinogenic in which were first characterized as human carcinogens humans (group 1 carcinogens). Similar evaluations have attributable to industrial exposures in the dye industry been published by the USDHHS (2005). The total exposure (Luch 2005). Heterocyclic aromatic amines are also com- of smokers to these compounds is approximately 1.4 to 2.2 bustion products and are best known for their occurrence milligrams (mg) per cigarette (Table 5.1). This estimate in broiled foods (Sugimura 1995), but they also occur in is based on machine measurements and may underesti- cigarette smoke. mate actual exposure. Some of the strongest of these car- Aldehydes such as formaldehyde and acetaldehyde cinogens are PAHs, N-nitrosamines, and aromatic amines, occur widely in the human environment and are endo- which occur in the lowest amounts, and some of the genous metabolites found in human blood (IARC 1995c, weaker carcinogens, such as acetaldehyde and isoprene, 1999; Gao et al. 2002). The phenolic compounds cat- occur in the highest amounts. Thus, a simple addition of echol and caffeic acid are common dietary constituents. the amounts of carcinogenic agents could be misleading. High doses of catechol cause glandular stomach tumors For other carcinogens in cigarette smoke that IARC has when administered in the diet. Catechol can also act as a not evaluated (e.g., broad spectra of PAHs and aromatic cocarcinogen, enhancing the activity of carcinogens such amines), data on frequency of occurrence, levels, and car- as B[a]P (IARC 1999). Dietary caffeic acid caused renal cinogenic activities are incomplete (IARC 1986). cell tumors in female mice (IARC 1993). The volatile PAHs are incomplete combustion products first hydrocarbons include 1,3-butadiene, a powerful multior- identified as carcinogenic constituents of coal tar (Phil- gan carcinogen in mice that was shown to have weaker lips 1983). These products occur as mixtures in tar, soot, activity in rats, and benzene, a known human leukemogen broiled foods, automobile engine exhaust, and other (IARC 1982, 1999). 1,3-butadiene and benzene are argu- materials generated by incomplete combustion (IARC ably the two most prevalent potent carcinogens in ciga- 1983). Generally, PAHs are carcinogens that act locally. rette smoke, on the basis of toxicologic criteria (Fowles Some PAHs, such as benzo[a]pyrene (B[a]P), have power- and Dybing 2003). ful carcinogenic activity. Studies have typically evaluated Other carcinogenic organic compounds in cigarette PAH carcinogenicity by application to mouse skin, but smoke include the human carcinogens vinyl chloride in PAHs also induce tumors of the lung, trachea, and mam- low amounts and ethylene oxide in substantial quantities mary gland, depending on the route of administration and (IARC 1979). Ethylene oxide is associated with malignan- the animal model used (Dipple et al. 1984). cies of the lymphatic and hematopoietic systems in both Heterocyclic compounds include analogs of PAHs humans and laboratory animals (IARC 1994). Diverse met- containing nitrogen, as well as simpler compounds such als such as the human carcinogen cadmium are also pres- as furan, which is a liver carcinogen. N-nitrosamines are ent in cigarette smoke, as is the radioisotope polonium a large class of carcinogens with demonstrated activity in 210, which is carcinogenic to humans. at least 30 animal species (Preussmann and Stewart 1984). Cigarette smoke also contains oxidants such as They are potent and systemic carcinogens that affect dif- nitric oxide (about 600 micrograms [µg] per cigarette) ferent tissues depending on their structure. Two of the and related species (Hecht 1999). Free radicals have been most important N-nitrosamines in cigarette smoke are the detected by electron spin resonance and spin trapping tobacco-specific 4-(methylnitrosamino)-1-(3-pyridyl)-1- (Hecht 1999). Researchers postulate that the major spe- butanone (NNK) and N’-nitrosonornicotine (NNN) (Hecht cies of free radicals are a quinone-hydroquinone complex. and Hoffmann 1988). NNK caused lung tumors in all spe- Other compounds may also be involved in the oxidative cies tested, and activity in rats was particularly high. Stud- damage produced by cigarette smoke. In addition, several ies using animal models have demonstrated that NNK also studies demonstrate the presence in cigarette smoke of an induces tumors of the pancreas, nasal cavity, and liver. In uncharacterized ethylating agent, which ethylates both addition, NNN produces esophageal and nasal tumors in DNA and hemoglobin (Hb) (Carmella et al. 2002a; Singh et al. 2005).

Cancer 227 Surgeon General’s Report

Table 5.1 IARC evaluations of carcinogens in mainstream cigarette smoke

IARC evaluations of evidence of carcinogenicity in humans Quantity IARC Monographc Carcinogena (per cigarette) In animals In humans IARC groupb (volume, year)

Polycyclic aromatic hydrocarbons Benz[a]anthracene 20–70 ng Sufficient 2A 32, 1983; S7, 1987 Benzo[b]fluoranthene 4–22 ng Sufficient 2B 32, 1983; S7, 1987 Benzo[j]fluoranthene 6–21 ng Sufficient 2B 32, 1983; S7, 1987 Benzo[k]fluoranthene 6–12 ng Sufficient 2B 32, 1983; S7, 1987 Benzo[a]pyrene 8.5–17.6 ng Sufficient Limited 1 32, 1983; S7, 1987; 92, in press Dibenz[a,h]anthracene 4 ng Sufficient 2A 32, 1983; S7, 1987 Dibenzo[a,i]pyrene 1.7–3.2 ng Sufficient 2B 32, 1983; S7, 1987 Dibenzo[a,e]pyrene Present Sufficient 2B 32, 1983; S7, 1987 Indeno[1,2,3-cd]pyrene 4–20 ng Sufficient 2B 32, 1983; S7, 1987 5-methylchrysene ND–0.6 ng Sufficient 2B 32, 1983; S7, 1987

Heterocyclic compounds Furan 20–40 µg Sufficient 2B 63, 1995a Dibenz[a,h]acridine ND–0.1 ng Sufficient 2B 32, 1983; S7, 1987 Dibenz[a,j]acridine ND–10 ng Sufficient 2B 32, 1983; S7, 1987 Dibenzo[c,g]carbazole ND–0.7 ng Sufficient 2B 32, 1983; S7, 1987 Benzo[b]furan Present Sufficient 2B 63, 1995a

N-nitrosamines N-nitrosodimethylamine 0.1–180 ng Sufficient 2A 17, 1978; S7, 1987 N-nitrosoethylmethylamine ND–13 ng Sufficient 2B 17, 1978; S7, 1987 N-nitrosodiethylamine ND–25 ng Sufficient 2A 17, 1978; S7, 1987 N-nitrosopyrrolidine 1.5–110 ng Sufficient 2B 17, 1978; S7, 1987 N-nitrosopiperidine ND–9 ng Sufficient 2B 17, 1978; S7, 1987 N-nitrosodiethanolamine ND–36 ng Sufficient 2B 17, 1978; 77, 2000 N’-nitrosonornicotine 154–196 ng Sufficient Limited 1 37, 1985; S7, 1987; 89, in press 4-(methylnitrosamino)-1-(3-pyridyl)- 110–133 ng Sufficient Limited 1 37, 1985; S7, 1987; 1-butanone 89, in press

Aromatic amines 2-toluidine 30–200 ng Sufficient Limited 2A S7, 1987; 77, 2000 2,6-dimethylaniline 4–50 ng Sufficient 2B 57, 1993 2-naphthylamine 1–22 ng Sufficient Sufficient 1 4, 1974; S7, 1987 4-aminobiphenyl 2–5 ng Sufficient Sufficient 1 1, 1972; S7, 1987

Heterocyclic aromatic amines 2-amino-9H-pyrido[2,3-b]indole 25–260 ng Sufficient 2B 40, 1986; S7, 1987 2-amino-3-methyl-9H-pyrido[2,3-b]indole 2–37 ng Sufficient 2B 40, 1986; S7, 1987 2-amino-3-methylimidazo[4,5-f]quinoline 0.3 ng Sufficient 2A S7, 1987; 56, 1993 3-amino-1,4-dimethyl-5H-pyrido 0.3–0.5 ng Sufficient 2B 31, 1983; S7, 1987 [4,3-b]indole 3-amino-1-methyl-5H-pyrido[4,3-b]indole 0.8–1.1 ng Sufficient 2B 31, 1983; S7, 1987 2-amino-6-methylpyrido[1,2-a:3’, 0.37–0.89 ng Sufficient 2B 40, 1986; S7, 1987 2’-d]imidazole 2-aminodipyrido[1,2-a:3’,2’-d]imidazole 0.25–0.88 ng Sufficient 2B 40, 1986; S7, 1987 2-amino-1-methyl-6-phenylimidazo 11–23 ng Sufficient 2B 56, 1993 [4,5-b]pyridine

228 Chapter 5 How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease

Table 5.1 Continued

IARC evaluations of evidence of carcinogenicity in humans Quantity IARC Monographc Carcinogena (per cigarette) In animals In humans IARC groupb (volume, year)

Aldehydes Formaldehyde 10.3–25 µg Sufficient Sufficient 1 S7, 1987; 62, 1995b Acetaldehyde 770–864 µg Sufficient 2B S7, 1987; 71, 1999

Phenolic compounds Catechol 59–81 µg Sufficient 2B S7, 1987; 71, 1999 Caffeic acid <3 µg Sufficient 2B 56, 1993

Volatile hydrocarbons 1,3-butadiene 20–40 µg Sufficient Limited 2A S7, 1987; 71, 1999 Isoprene 450–1,000 µg Sufficient 2B 60, 1994; 71, 1999 Benzene 12–50 µg Sufficient Sufficient 1 29, 1982; S7, 1987

Nitrohydrocarbons Nitromethane 0.5–0.6 µg Sufficient 2B 77, 2000 2-nitropropane 0.7–1.2 ng Sufficient 2B S7, 1987; 71, 1999 Nitrobenzene 25 µg Sufficient 2B 65, 1996

Miscellaneous organic compounds Acetamide 38–56 µg Sufficient 2B S7, 1987; 71, 1999 Acrylamide Present Sufficient 2A S7, 1987; 60, 1994 Acrylonitrile 3–15 µg Sufficient 2B S7, 1987; 71, 1999 Vinyl chloride 11–15 ng Sufficient Sufficient 1 19, 1979; S7, 1987 1,1-dimethylhydrazine Present Sufficient 2B 4, 1974; 71, 1999 Ethylene oxide 7 µg Sufficient Limited 1 60, 1994; S7, 1987 Propylene oxide 0–100 ng Sufficient 2B 60, 1994; S7, 1987 Urethane 20–38 ng Sufficient 2B 7, 1974; S7, 1987

Metals and inorganic compounds Arsenic 40–120 ng Sufficient Sufficient 1 84, 2004 Beryllium 0.5 ng Sufficient Sufficient 1 S7, 1987; 58, 1993 Nickel ND–600 ng Sufficient Sufficient 1 S7, 1987; 49, 1990 Chromium (hexavalent) 4–70 ng Sufficient Sufficient 1 S7, 1987; 49, 1990 Cadmium 41–62 ng Sufficient Sufficient 1 S7, 1987; 58, 1993 Cobalt 0.13–0.20 ng Sufficient 2B 52, 1991 Lead (inorganic) 34–85 ng Sufficient Limited 2A 23, 1980; S7, 1987; 87, in press Hydrazine 24–43 ng Sufficient 2B S7, 1987; 71, 1999 Radioisotope polonium-210 0.03–1.0 Sufficient 1 78, 2001 picocurie Source: Adapted from Hoffmann et al. 2001 and International Agency for Research on Cancer 2004 with permission from American Chemical Society, © 2001 and International Agency for Research on Cancer, © 2004. Note: IARC = International Agency for Research on Cancer; ND = not detected; ng = nanograms; S7 = Supplement 7; µg = micrograms. aVirtually all these compounds are known carcinogens in experimental animals, and IARC found sufficient evidence for carcinogenicity in animals for all the compounds. bUsing data on cancer in humans and, in some cases, other data, IARC established classifications for compounds as group 1 (carcinogenic to humans), group 2A (probably carcinogenic to humans), and group 2B (possibly carcinogenic to humans). cIf more than two IARC evaluations were performed, only the two most recent monographs are listed.

Cancer 229 Surgeon General’s Report

In summary, cigarette smoke contains diverse car- al. 1997). One metabolite of phenanthrene, trans, anti- cinogens. PAH, N-nitrosamines, aromatic amines, 1,3- PheT, results from the diol epoxide metabolic activation butadiene, benzene, aldehydes, and ethylene oxide are pathway common to many carcinogenic PAHs. This me- probably the most important carcinogens because of their tabolite is a promising new biomarker for PAH uptake and carcinogenic potency and levels in cigarette smoke. metabolic activation and can be readily quantified by gas chromatography (GC)-negative ion chemical ionization– mass spectrometry (MS) (Hecht et al. 2003). Levels of Biomarkers of Carcinogens trans, anti-PheT are higher in smokers than in nonsmokers (Hecht et al. 2003). in Smokers 1-hydroxypyrene. Pyrene is a noncarcinogenic component in all PAH mixtures; levels in mainstream Measurements of carcinogens or their metabolites cigarette smoke were 50 to 270 ng per cigarette (IARC in urine, blood, and breath can provide convenient and 1986). The major metabolite of pyrene is 1-hydroxypyrene reliable quantitative information on human exposure to (1-HOP) glucuronide, which can be measured in urine carcinogens. The information provided by these measure- (Jongeneelen et al. 1985). To quantify 1-HOP in urine, ments, which are biomarkers of exposure, is critical to enzymatic hydrolysis is used to release 1-HOP, which is objective evaluation of carcinogen doses in smokers. then enriched by reverse-phase chromatography and quantified by high-performance liquid chromatography (HPLC) Urinary Biomarkers with fluorescence detection. Studies have described varia- Urinary biomarkers are the most widely applied tions of this method (Carmella et al. 2004b). Hundreds of biomarkers of carcinogen exposure in smokers (Hecht studies of occupational and environmental PAH exposure 2002b). Urine is relatively simple to obtain in large quan- have measured 1-HOP as a surrogate marker for total PAH tities, and obtaining study participants’ consent and speci- exposure. In reviews of the data on the effects of smok- mens for testing is almost never a difficulty. Carcinogens ing (Jongeneelen 1994, 2001; Van Rooij et al. 1994; Levin in cigarette smoke and/or their metabolites are frequently 1995; Heudorf and Angerer 2001; Hecht 2002b), most of present in substantial quantities in urine. Therefore, reli- the studies noted that 1-HOP levels in the urine of smok- able quantitation is generally feasible. This section pro- ers were about twice as high as those in the urine of non- vides an overview of some of the urinary biomarkers most smokers, although some studies have reported greater commonly used to estimate carcinogen doses in smokers. differences. Levels of 1-HOP may be influenced by genetic The chemical structures of all compounds discussed in polymorphisms in carcinogen-metabolizing enzymes this section are illustrated in Figure 5.2. (Alexandrie et al. 2000; Nerurkar et al. 2000; Nan et al. 2001; van Delft et al. 2001). Polycyclic Aromatic Hydrocarbons Other metabolites of polycyclic aromatic hydrocarbons. Studies examining urine biomarkers have Phenanthrene metabolites. Phenanthrene is the measured phenolic metabolites of naphthalene and a simplest PAH with a bay region (the region of a molecule variety of PAHs, which show promise as urinary biomark- between positions 4 and 5), a feature closely associated ers of PAH uptake from cigarette smoke (Hecht 2002b; with the carcinogenic activity of PAHs (Figure 5.2). Phen- Smith et al. 2002a,b; Serdar et al. 2003). Studies have anthrene, however, is inactive as a carcinogen (LaVoie quantified B[a]P metabolites in urine, but the levels are and Rice 1988). Concentrations of phenanthrene in main- generally low, limiting their routine application in large stream smoke range from 85 to 620 nanograms (ng) studies (Hecht 2002b). per cigarette (IARC 1986). Studies have quantified the phenanthrene metabolites phenanthrols, phenanthrene Aromatic Amines and Heterocyclic dihydrodiols, and r-1,t-2,3,c-4-tetrahydroxy-1,2,3,4-tet- Aromatic Amines rahydrophenanthrene (trans, anti-PheT) in human urine (Hecht 2002b). Levels of phenanthrols in human urine Researchers have quantified aromatic amines, but differed between smokers and nonsmokers in some stud- not their metabolites, in human urine. In one study, levels ies but not in others (reviewed in Carmella et al. 2004a). of 2-toluidine excreted by smokers were 6.3 ± 3.7 (stan- There are sources of phenanthrene exposure other than dard deviation [SD]) µg/24 hours and levels excreted by cigarette smoke, and all people have phenanthrene nonsmokers were 4.1 ± 3.2 (SD) µg/24 hours. The differ- metabolites in their urine. This finding is well documented ence was not significant (El-Bayoumy et al. 1986). Another in environmental and occupational settings with high investigation reported urine levels of 2-toluidine that were exposures to PAH (Grimmer et al. 1993, 1997; Angerer et higher in smokers than in nonsmokers (Riffelmann et al.

230 Chapter 5 How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease

Figure 5.2 Chemical structures of biomarkers of carcinogen exposure 3 OH OH 4 2 HO OH 5 1 6 OH OH 7 10 8 9 Phenanthrene 1-hydroxyphenanthrene Trans, anti-PheT 1-HOP OH NH2 N O OH N O N CH3 N NH CH 2 3 CH3

N O+ N 4-aminobiphenyl NNAL 2-toluidine Gluc NNAL-N-Gluc COOH

HO O

HO HO O N O N

N N N O N N CH3 O+ N O Gluc N O N N N NNAL-O-Gluc NNN NNN-N-Gluc NATB S H C CH COOH 3 2 COOH COOH N 2 N N N 4 N O N O N O N O N NAB NPRO NSAR NTCA O O HO O H3C N N OH N CH CH 3 OH N 3 S H S H

O O OH N O OH Iso-NNAC 1,3-butadiene MHBMA O O OH O N O NH O HO N CH 3 HO N CH3 HO N S H N NH2 OH S H O O O OH O OH DHBMA tt-MA S-PMA OH 8-oxodeoxyguanosine O N N H N N OH N N NH2

N HO O N HO O N HO N N N O O O

N N CH CH OH OH OH 2 3 5-hydroxymethyldeoxyuridine 3,N4-ethenodeoxycytidine 1,N6-ethenodeoxycytidine 3-ethyladenine Note: 1-HOP = 1-hydroxypyrene; DHBMA = dihydroxybutylmercapturic acid; iso-NNAC = 4-(methylnitrosamino)-4-(3-pyridyl)butyric acid; MHBMA = monohydroxybutenylmercapturic acid; NAB = N’-nitrosoanabasine; NATB = N’-nitrosoanatabine; NNAL = 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol; NNAL-N-Gluc = 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol-N-glucuronide; NNAL-O-Gluc = 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol-O-glucuronide; NNN = N’-nitrosonornicotine; NNN-N-Gluc = N’-nitrosonornicotine-N-glucuronide; NPRO = N-nitrosoproline; NSAR = N-nitrososarcosine; NTCA = N-nitrosothiazolidine 4-carboxylic acid; S-PMA = S-phenylmercapturic acid; trans, anti-PheT = r-1,t-2,3,c-4-tetrahydroxy-1,2,3,4-tetrahydrophenanthrene; tt-MA = trans,trans- muconic acid.

Cancer 231 Surgeon General’s Report

1995). There appear to be significant sources of human smoke, the origin of NNAL and NNAL glucuronides found uptake of 2-toluidine in addition to cigarette smoke. in urine is the metabolism of NNK. Most investigations Although these sources are not fully characterized, diet demonstrate a correlation between NNAL plus NNAL gluc- is one likely source. Amounts of 4-ABP excreted by smok- uronides and cotinine in urine. This finding indicates that ers (78.6 ± 85.2 [SD] ng/24 hours) were similar to those NNAL and NNAL glucuronides are a biomarker of uptake excreted by nonsmokers (68.1 ± 91.5 ng/24 hours), and of the lung carcinogen NNK and that cotinine is a bio- amounts of 2-naphthylamine excreted by smokers (84.5 marker of nicotine uptake. Ratios of NNAL glucuronides ± 102.7 ng/24 hours) were similar to those excreted by to NNAL vary at least 10-fold in smokers. This ratio could nonsmokers (120.8 ± 279.2 ng/24 hours) (Grimmer et al. be a potential indicator of cancer risk, because NNAL 2000). In another study, Hb adducts appeared to be better glucuronides are detoxification products, whereas NNAL biomarkers of exposure to aromatic amines from tobacco is carcinogenic (Carmella et al. 1995; Richie et al. 1997). smoke than were urinary levels of metabolites (Skipper In human urine, (S)-NNAL-O-glucuronide is the pre- and Tannenbaum 1990). dominant diastereomer of NNAL-O-glucuronide, and the Researchers have measured urinary biomarkers of level of (S)-NNAL is slightly higher than that of (R)-NNAL heterocyclic aromatic amines mainly in studies of dietary (Carmella et al. 1999). (S)-NNAL is the more tumorigenic exposure. Little information is available on the contribu- enantiomer of NNAL in the A/J mouse lung (Upadhyaya tions of cigarette smoke to urinary levels of heterocyclic et al. 1999). NNAL and NNAL glucuronides are released aromatic amines (Hecht 2002b). slowly from the human body only after smoking cessation. This finding has been linked to a particularly strong N-Nitrosamines retention of (S)-NNAL, possibly at a receptor site (Hecht et al. 1999; Zimmerman et al. 2004). Recent studies indicate 4-(methylnitrosamino)-1-(3-pyridyl)-1-bu- that levels of NNAL plus NNAL-glucuronides are not only tanol and its glucuronides. In rodents and humans, biomarkers of NNK exposure but also are biomarkers of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) risk for lung cancer in smokers (Church et al. 2009; Yuan and its glucuronides are quantitatively significant et al. 2009) metabolites of NNK (Hecht 1998). Both NNAL and NNK N’-nitrosonornicotine, N’-nitrosoanatabine, are pulmonary carcinogens with particularly strong activ- N’-nitrosoanabasine, and their pyridine-N-glucuro- ity in rats; NNAL also induces pancreatic tumors (Hecht nides. Researchers developed a method to analyze NNN, 1998). Glucuronidation of NNAL at the pyridine nitrogen N’-nitrosoanatabine (NATB), N’-nitrosoanabasine (NAB), gives NNAL-N-glucuronide, and conjugation at the carbi- and their pyridine N-glucuronides (e.g., NNN-N-glucuro- nol oxygen yields NNAL-O-glucuronide. Both NNAL-N- nide) in human urine. NATB and NAB are tobacco-specific glucuronide and NNAL-O-glucuronide exist as a mixture nitrosamines that like NNN and NNK are formed by the of two diastereomers, and each diastereomer is a mixture nitrosation of tobacco alkaloids (Hecht and Hoffmann of E- and Z-rotamers (Upadhyaya et al. 2001). The NNAL- 1988). Studies show that NATB is not carcinogenic but N-glucuronide and NNAL-O-glucuronide isomers are that NAB is a weak esophageal carcinogen in rats (Hecht collectively referred to as NNAL glucuronides. (R)-NNAL- 1998). Mean levels of total NNN, NATB, and NAB in the O-glucuronide does not induce tumors in mice (Upad- urine of 14 smokers were 0.18 ± 0.22 SD, 0.19 ± 0.20, and hyaya et al. 1999). The (S) isomer has not been tested, 0.040 ± 0.039 picomoles/mg of creatinine, respectively. but glucuronidation generally deactivates a carcinogenic These compounds have not been detected in the urine of metabolite in any event. nonsmokers with no exposure to secondhand smoke. NNAL and NNAL glucuronides can be readily deter- Nitrosamino acids. Researchers have used the mined in urine by using GC with nitrosamine-selective N-nitrosoproline (NPRO) test to compare endogenous detection (Carmella et al. 1993, 1995; Hecht et al. 1999) nitrosation in smokers and nonsmokers (Bartsch et al. and by MS methods (Carmella et al. 1993, 1999; Parsons et 1989). The results of clinical studies indicate that the al. 1998; Lackmann et al. 1999; Hecht et al. 2001; Byrd and frequency of endogenous formation of NPRO is higher Ogden 2003). Typical levels are about 1 nanomole (nmol) in smokers than in nonsmokers and that it may be of NNAL in 24 hours and 2.2 nmol of NNAL glucuronides enhanced by thiocyanate catalysis (Bartsch et al. 1989; in 24 hours, with no detection of unchanged NNK. NNAL Tsuda and Kurashima 1991; Tricker 1997). However, and NNAL glucuronides are absolutely specific to expo- some population-based studies document similar levels of sure to tobacco and have not been detected in the urine NPRO in smokers and nonsmokers, because this precur- of nontobacco users unless they were exposed to second- sor biomarker for nitrosamine formation is primarily from hand smoke. Because NNAL is not present in cigarette dietary sources (Tricker 1997).

232 Chapter 5 How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease

The major nitrosamino acids present in human S-phenylmercapturic acid (S-PMA) is formed by urine are N-nitrososarcosine, N-nitrosothiazolidine the metabolism of the glutathione conjugate of benzene 4-carboxylic acid (NTCA), and trans- and cis-isomers of oxide and has the potential to be specific for benzene N-nitroso-2-methylthiazolidine 4-carboxylic acid (NMTCA) uptake (Stommel et al. 1989; van Sittert et al. 1993; (Bartsch et al. 1989; Tsuda and Kurashima 1991). NTCA Boogaard and van Sittert 1995, 1996; Qu et al. 2000). and NMTCA are formed by reactions of formaldehyde or In one study, S-PMA levels were significantly higher in acetaldehyde with cysteine, followed by nitrosation. Some smokers (1.71 micromoles [μmol]/mole of creatinine) studies demonstrate increased levels of urinary NTCA than those in nonsmokers (0.94 μmol/mole of creatinine), and NMTCA in smokers (Tsuda and Kurashima 1991). whereas tt-MA levels were not significantly different Although some studies show a correlation between (Boogaard and van Sittert 1996). Researchers believe that total nitrosamino acids and urinary nicotine plus cotinine S-PMA and tt-MA are the most sensitive biomarkers for among smokers (Malaveille et al. 1989), other studies low levels of exposure to benzene (Qu et al. 2000, 2003). show mixed results (Tricker 1997). Collectively, the avail- Phenol, hydroquinone, catechol, and 1,2,4-tri- able data support the concept that nitrosamines can be hydroxybenzene are also urinary metabolites of benzene. formed endogenously in smokers under some conditions. Studies relating urinary levels of these metabolites to Studies suggest that 4-(methylnitrosamino)-4-(3- occupational exposure to benzene have mixed results, pyridyl)butyric acid is a potential monitor of endogenous because background levels of the metabolites are high nitrosation of nicotine (Djordjevic et al. 1991). However, (Inoue et al. 1988, 1989; Ong et al. 1995, 1996; Qu et al. researchers could not find any evidence for its formation 2000). Urinary catechol levels did not differ significantly after oral administration of nicotine or cotinine to persons between smokers and nonsmokers (Carmella et al. 1982), abstaining from smoking (Tricker et al. 1993). and diet has been shown to be a major source of urinary 1,3-butadiene. The major urinary metabolites catechol (Carmella et al. 1982). of 1,3-butadiene are monohydroxybutenyl-mercapturic Products of oxidative damage. The presence of acids (MHBMAs) and dihydroxybutyl-mercapturic acid free radicals and oxidants in cigarette smoke can lead to (DHBMA). Levels of MHBMA were 86.4 ± 14.0 (SD) µg/24 oxidative DNA damage and the subsequent formation of hours in smokers and 12.5 ± 1.0 µg/24 hours in nonsmok- products such as 8-oxodeoxyguanosine, thymine glycol, ers—a significant difference (Urban et al. 2003).- Cor thymidine glycol, and 5-hydroxymethyluracil. Repair of responding levels of DHBMA were 644 ± 90 (SD) µg/24 these modified DNA constituents ultimately leads to their hours in smokers and 459 ± 72 µg/24 hours in nonsmok- excretion in urine. Researchers have frequently quantified ers, which were not significantly different (Urban et al. 8-oxodeoxyguanosine in urine of smokers and nonsmok- 2003). DHBMA does not appear to be specific to exposure ers (Loft and Poulsen 1998; Prieme et al. 1998; Renner to 1,3-butadiene and is probably not a useful biomarker. et al. 2000). Cigarette smoking usually results in levels of Hb adducts have also proven useful as markers of long- 8-oxodeoxyguanosine in urine that modestly increase to term exposure to 1,3-butadiene. The long half-lives of 16 to 50 percent higher than those in nonsmokers, but these adducts result in an average measurement that is studies have also reported negative results (Nia et al. 2001; more time weighted than that for some other metabolites Harman et al. 2003; Mukherjee et al. 2004). Smoking ces- (e.g., urinary meta-bolites) (Swenberg et al. 2001; Boysen sation caused a 21-percent decrease in the excretion of et al. 2007). 8-oxodeoxyguanosine (Prieme et al. 1998). Longitudinal Benzene. One path of benzene metabolism pro- studies have not shown convincing increases in urinary ceeds by ring oxidation, ultimately by ring cleavage to 8-oxodeoxyguanosine that were attributable to smoking, trans,trans-muconaldehyde, and finally to trans,trans- and a complex pattern of factors may affect background muconic acid (tt-MA), a metabolite widely used as a bio- levels of this biomarker in urine (Kasai et al. 2001; Pilger marker of benzene uptake (Scherer et al. 1998). Most et al. 2001; Mukherjee et al. 2004). Studies on the effects studies have found significantly elevated levels of tt-MA of smoking on urinary levels of 5-hydroxymethyluracil in the urine of smokers (Scherer et al. 1998; Cocco et or 5-hydroxymethyldeoxyuridine have obtained mixed al. 2003; Lee et al. 2005). Levels of tt-MA were 1.4 to 4.8 results (Pourcelot et al. 1999; Harman et al. 2003). One times higher in smokers than in nonsmokers, and the study showed a correlation between smoking and urinary additional amount of tt-MA excreted by smokers ranged excretion of 3,N4-ethenodeoxycytidine, which may result from 0.022 to 0.20 mg/gram of creatinine (Scherer et al. from endogenous lipid peroxidation (Chen et al. 2004a). 1998). However, sorbic acid, a food constituent that can be Studies have also detected 1,N6-ethenodeoxyadenosine in transformed metabolically into tt-MA, can contribute to human urine, but no differences were observed between urinary levels of tt-MA and thereby decrease its specificity levels in smokers and those in nonsmokers (Hillestrøm as a biomarker for benzene uptake (Scherer et al. 1998; et al. 2004). Pezzagno et al. 1999).

Cancer 233 Surgeon General’s Report

Products of alkylating agents. The reaction of associated with the number of cigarettes smoked (IARC alkylating agents with DNA forms alkyladenines, alkylgua- 2004). Cadmium levels were also higher in the blood of nines, and other products (Singer and Grunberger 1983). smokers. Measurements of NNAL in blood demonstrated a Alkylation at the 3-position of deoxyadenosine or at the mean level of 42 femtomoles/milliliter of plasma in smok- 7-position of deoxyguanosine results in products with ers; NNAL was not detected in nonsmokers (Carmella et an unstable glycosidic bond. These products are readily al. 2005). Cigarette smoke induces oxidative damage as removed from DNA, either spontaneously or by glycosyl- determined by elevated blood protein carbonyls (Reznick ases, which results in the urinary excretion of 3-alkylade- et al. 1992) and blood protein-bound glutathione (Muscat nines and 7-alkylguanines. Studies have more extensively et al. 2004). F2-isoprostane levels, which are biomarkers investigated 3-alkyladenines as biomarkers of exposure to of oxidative damage, were higher in the plasma of smok- alkylating agents, because researchers expected the back- ers than in the plasma of nonsmokers and decreased with ground levels of 3-alkyladenines in urine to be lower than vitamin C treatments (Morrow et al. 1995; Dietrich et al. those of 7-alkylguanines. However, substantial amounts 2002). Hb adducts and DNA adducts in white blood cells of 3-methyladenine occur in the diet (Prevost et al. 1993; are discussed in the next section. Fay et al. 1997). Nevertheless, two controlled studies demonstrated an increase in the urinary excretion of 3-meth- Summary yladenine among smokers (Kopplin et al. 1995; Prevost Quantitative analysis of carcinogens or their metab- and Shuker 1996). Another study found lower background olites in urine, breath, and blood provides a convenient and levels of 3-ethyladenine than those of 3-methyladenine reliable method of comparing carcinogen exposure among (Prevost et al. 1993). Two studies demonstrated convinc- smokers and between smokers and nonsmokers. The most ing increases in urinary levels of 3-ethyladenine in smok- extensive measurements have been made in urine. Uri- ers, indicating the presence in cigarette smoke of an nary biomarkers of several major types of carcinogens unidentified ethylating agent (Kopplin et al. 1995; Prevost in cigarette smoke are reliable indicators of exposure. and Shuker 1996). There was no effect from smoking on These biomarkers include trans, anti-PheT and 1-HOP urinary levels of 3-(2-hydroxyethyl)adenine (Prevost and for PAH; total NNAL (NNAL plus NNAL glucuronides) for Shuker 1996). A population-based study found higher NNK; MHBMA for 1,3-butadiene; and tt-MA and S-PMA levels of both 3-methyladenine and 7-methylguanine in for benzene. The measurements provide good estimates smokers than in nonsmokers, and a second study found of minimum doses of relevant carcinogens in smokers and no difference in the 3-methyladenine levels (Shuker et al. allow comparisons with those in nonsmokers. The total 1991; Stillwell et al. 1991). carcinogen dose is generally difficult to calculate because Metals. Studies of urinary cadmium have most the extent of conversion of a given carcinogen to the mea- consistently demonstrated differences between smok- sured metabolite is usually unknown and can vary widely ers and nonsmokers. Large studies in Germany and the among individuals. Nevertheless, the results of these stud- United States showed increases in urinary cadmium lev- ies are illuminating. They show, for example, that levels els with age and smoking (IARC 2004). These results were of metabolites of benzene (about 1,100 nmol/24 hours of consistent with those of other studies. tt-MA and 8 nmol/24 hours of S-PMA) and 1,3-butadiene (about 340 nmol/24 hours of MHBMA) exceed levels of Breath and Blood Biomarkers other biomarkers (e.g., about 3 nmol/24 hours of NNAL Benzene, 1,3-butadiene, and a variety of volatile plus NNAL glucuronides and 2 nmol/24 hours of 1-HOP). organic compounds including xylenes, styrene, isoprene, These results are consistent with the levels of benzene and 2,5-dimethylfuran, ethane, and octane were measured in 1,3-butadiene in cigarette smoke, which were higher than expired air; levels were generally higher in smokers than those of NNK and PAH. in nonsmokers (Gordon et al. 2002; Perbellini et al. 2003; However, metabolites of benzene and a metabolite IARC 2004). Levels of benzene and 1,3-butadiene in the of 1,3-butadiene (DHBMA) are also found in nonsmokers breath of smokers were 360 and 522 µg/cubic meter (m3), in considerable quantities. Comparisons of smokers and respectively (Gordon et al. 2002). In another study, mean nonsmokers demonstrate that total NNAL is the most benzene levels ranged from 58.1 to 81.3 µg/m3, depending discriminatory carcinogen biomarker because the only on the cigarette brand (IARC 2004). source of the parent carcinogen NNK is tobacco products. Studies have quantified volatile organic compounds, Total NNAL is not detected in nonsmokers unless they including benzene and styrene, in the blood of smokers; have been exposed to secondhand tobacco smoke. There- levels were generally higher than those in the blood of fore, this biomarker is particularly useful for compar- nonsmokers. Benzene levels in blood were significantly ing carcinogen uptake in smokers who, for example, use

234 Chapter 5 How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease different tobacco products, because the measurements are of metabolic activation for a number of PAHs is the forma- not confounded by other exposures such as diet, occupation of diol epoxides in which the epoxide ring is in the bay tion, or the general environment. region of the PAH molecule, similar to that in BPDE (Con- ney 1982; Thakker et al. 1985; Baird and Ralston 1997). Competing with B[a]P metabolic activation processes are Metabolic Activation and detoxification pathways leading to (1) phenols through direct hydroxylation or rearrangement of initially formed Detoxification of Carcinogens epoxides, (2) dihydrodiols through hydration of epoxides catalyzed by epoxide hydrolase, and (3) formation of gluta- Most of the carcinogens listed in Table 5.1 require thione, glucuronide, and sulfate conjugates. Researchers metabolic activation to become intermediate agents, gen- have also observed the formation of quinone metabolites erally electrophiles, which react with nucleophilic sites in from initial hydroxylation at the 6-position, followed by DNA to form DNA adducts. All PAHs, heterocyclic com- further oxidation (Cooper et al. 1983). pounds, N-nitrosamines, aromatic amines, and heterocy- Metabolic activation of NDMA occurs by α-hydro- clic aromatic amines in cigarette smoke require metabolic xylation and leads to an unstable α-hydroxymethyl metab- activation. Other compounds in Table 5.1 that require olite. This compound spontaneously loses formaldehyde metabolic activation are 1,3-butadiene, isoprene, benzene, and forms methanediazohydroxide, the same intermedi- nitromethane, 2-nitropropane, nitrobenzene, acrylamide, ate agent produced in the α-methylene hydroxylation of vinyl chloride, and urethane. Detoxification reactions in NNK. Researchers also observed the consequent forma- most cases compete with metabolic activation and also tion of methyl DNA adducts such as 7-methylguanine, affect the disposition of compounds that do not require O6-methylguanine, and O4-methylthymidine. Denitrosa- metabolic activation, such as ethylene oxide. tion produces nitrite and methylamine and is a detoxifica- An overview of the metabolism of six carcinogens tion pathway (Preussmann and Stewart 1984; Hecht and in tobacco smoke that are implicated in the forma- Samet 2007). The metabolism of NNK and NDMA forms tion of DNA adducts identified in human tissues is pre- aldehydes, whose roles in carcinogenesis are unclear, but sented in Figure 5.3. The six carcinogens are B[a]P, NNK, studies show that formaldehyde reacts with DNA and pro- N-nitrosodimethylamine (NDMA), NNN, ethylene oxide, tein to form cross-links and other products (Chaw et al. and 4-ABP. 1980; Beland et al. 1984; Hecht and Samet 2007). The major metabolic activation pathway of B[a]P α-hydroxylation of NNN adjacent to the pyridine that results in DNA adducts identified in human tissues ring produces the same intermediate agent formed by is the conversion to the highly mutagenic B[a]P-7,8-diol- methyl hydroxylation of NNK, which leads to pyridyloxo- 9,10-epoxides (BPDEs). The formation of BPDE occurs in butyl (POB)-DNA adducts (Hecht 1998). α-hydroxylation three steps: the metabolism of B[a]P to B[a]P-7,8-epoxide; distal from the pyridine ring also produces a reactive hydration of B[a]P-7,8-epoxide to give the dihydrodiol diazohydroxide, but its reactions with DNA have not been B[a]P-7,8-diol; and further epoxidation to produce BPDE. fully characterized. The acetate esters of the α-hydroxy- One of the four enantiomers is strongly carcinogenic and NNN metabolites are mutagenic (Hecht 1998; Hecht and 2 reacts with DNA to form adducts at N of deoxyguanosine Samet 2007). -hydroxylation of NNN, a minor pathway, 2 β (BPDE-N -deoxyguanosine) (Cooper et al. 1983; IARC and pyridine-N-oxidation are detoxification reactions. 1983; Thakker et al. 1985). This adduct was also observed NNN is also detoxified by denitrosation and oxidation to in animals treated with B[a]P. produce norcotinine, and by glucuronidation of the pyri- Two other proposed metabolic activation pathways dine ring (Hecht 1998; Stepanov and Hecht 2005; Hecht of B[a]P exist, but the evidence for their involvement in and Samet 2007). DNA adduct formation in laboratory animals and humans Ethylene oxide reacts directly with DNA to form is not as strong as that for BPDE. One pathway involves 7-(2-hydroxyethyl)guanine and other adducts (IARC 1994; the conversion of B[a]P-7,8-diol to the corresponding cat- Hecht and Samet 2007). Competing detoxification path- echol metabolite catalyzed by dihydrodiol dehydrogenase. ways involve glutathione conjugation and excretion of The catechol can undergo redox cycling to produce a qui- mercapturic acids (IARC 1994). none reactive with DNA, and the redox cycling process can 4-ABP is metabolically activated by N-hydroxylation produce oxidative damage to DNA (Penning et al. 1999; Yu (Kadlubar and Beland 1985; Hecht and Samet 2007). et al. 2002). Another metabolic activation process occurs Conjugation of the resulting hydroxylamine with acetate when one electron oxidation of B[a]P produces unstable or other groups, such as sulfate, ultimately produces depurinating DNA adducts that can lead to apurinic sites nitrenium ions, which react with DNA and form adducts and miscoding (Casale et al. 2001). A common mechanism

Cancer 235 Surgeon General’s Report 3 H a C O - N N ]py NDMA a C 3 H 2 = National NH =O NNAL- N -oxide 2 3 2 NNAL-Glucs = benzo[ P-450s CH CH NO ]P NIH shift a B[ = 4-(methylnitrosamino)- P-450s NNK NNAL α -hydroxy NNALs α -hydroxy NDMA Norcotinine β -hydroxy NNNs NNN- N -oxide 3 H -nitrosodimethylamine; O C N = = adenosine diphosphate; N N ADP O NDMA a =O 2 P-450s P-450s N N a -diphosphate-glucuronosyltransferases. NNK ’ + CH = acetyl; + aldehydes NNN O Diazonium ions Diazonium ions Diazonium ions α -hydroxy NNNs Ac α -hydroxy NNKs N N = uridine-5 -acetyltransferases; N UGTs = glycol = 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol; Ethylene NATs ADP adducts NNK- N -oxide = 4-aminobiphenyl; 2 NNAL H C a DNA adducts X 4-ABP O a O oxide H Ethylene C N 2 -transferases; S H B[ a ]P c A Quinones Sulfates 2 3 Glucuronides H O 2 N GSTs 4 UGTs = cytochrome P-450 enzymes; 1 CO 5 = glutathione- NATs P-450s c 12 P-450s acids H A GSTs 6 N O Mercapturic 11 Phenols NATs 7 H 10 NATs H O 8 9 N shift Glutathione conjugates P-450s = glucuronides; -nitrosonornicotine; ’ c NIH N A H = Glucs P-450s N GSTs NNN P-450s 2 Phenols P-450s EH H N diols Metabolism of six carcinogens in tobacco smoke that produce DNA adducts identified the lungs smokers Epoxides Diol epoxides UGTs = epoxide hydrolase; a Adapted from Cooper et al. 1983; Preussmann and Stewart 1984; Kadlubar Beland 1985; International Agency for Research on C ancer 1994; Hecht 1998, 1999. EH In 4-ABP scheme, X represents conjugates such as glucuronide or sulfate. Figure 5.3 Tetraols 4-ABP Carcinogens in tobacco smoke. Source: Note: rene; Institutes of Health phenomenon hydroxylation-induced intramolecular migration; 1-(3-pyridyl)-1-butanone; a Sulfates Glucuronides 236 Chapter 5 How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease mainly at C-8 of guanine. Other aromatic amines, as well are critical in forming DNA adducts include diol epoxides as heterocyclic aromatic amines, are predominantly acti- of PAH, diazonium ions generated by α-hydroxylation vated metabolically in similar ways. Acetylation of 4-ABP of nitrosamines, nitrenium ions formed from esters of can be a detoxification pathway if it is not followed by N-hydroxylated aromatic amines, and epoxides such as N-hydroxylation. Ring hydroxylation and conjugation of ethylene oxide. Glutathione and glucuronide conjugation the phenols result in detoxification. play major roles in the detoxification of carcinogens in Two other important carcinogens from cigarette cigarette smoke. smoke that require metabolic activation are benzene and 1,3-butadiene. DNA adducts of these compounds have not been detected in human samples. However, there is con- Enzymology of Carcinogen siderable information on their conversion to intermediate agents that react with DNA. Metabolism Benzene is metabolized to benzene epoxide, which is in equilibrium with its 7-member ring tautomer oxepin Introduction (Scherer et al. 2001; Hecht and Samet 2007). Researchers A number of enzyme families are important in have observed the reaction of benzene epoxide with DNA both the activation and detoxification of carcinogens in to produce 7-phenylguanine. Further metabolism of ben- cigarette smoke, including P-450s, GSTs, UGTs, N-acetyl- zene epoxide-oxepin can occur in a variety of ways. One transferases (NATs), epoxide hydrolases, and sulfotrans- way is nonenzymatic rearrangement to phenol, which ferases. The importance of each enzyme to the activation can be further hydroxylated to hydroquinone, catechol, or detoxification of a particular carcinogen depends on and 1,2,4-trihydroxybenzene. These metabolites can then characteristics of both the carcinogen (size, polarity, and be conjugated as glucuronides or sulfates. Hydroquinone lipophilicity) and the enzyme (structure, tissue distribu- can be further oxidized to benzoquinone, which can bind tion, and regulation of expression). The large number of to DNA, or hydration catalyzed by epoxide hydrolase can carcinogens in cigarette smoke and the wide variety of produce benzene dihydrodiol, which can then be con- enzymes involved in metabolizing these carcinogens pre- verted to catechol or tt-MA. Another possibility involves clude a comprehensive discussion of current understand- conjugation with glutathione that ultimately produces ing of the contribution of each enzyme to every pathway. S-PMA. Other pathways of benzene metabolism result in Therefore, the goals of this presentation are to introduce the formation of biphenyl and benzene dioxetane, which the families of enzymes involved and to highlight some can also lead to tt-MA (Scherer et al. 2001; Hecht and of the activation and detoxification reactions for specific Samet 2007). Studies have detected nitrobenzene, nitro- enzymes and carcinogens. biphenyl, and nitrophenol isomers in the bone marrow of mice treated with benzene; these isomers presumably Cytochrome P-450 Enzymes formed from reactions of benzene with endogenously generated nitric oxide (Chen et al. 2004b; Hecht and P-450s, encoded by CYP genes, are microsomal Samet 2007). enzymes that catalyze the oxidation of myriad chemicals, 1,3-butadiene is metabolically activated by epoxida- including many of the carcinogens in cigarette smoke. tion to give a monoepoxide that can be further metabo- Sequencing the human genome has identified 57 CYP lized to a diepoxide and a dihydrodiol epoxide (van Sittert genes, about 15 of which are considered important in et al. 2000; Hecht and Samet 2007), which all form DNA the metabolism of xenobiotics (Nelson 2003; Guengerich adducts. The dihydrodiol epoxide also produces cross-links 2004). Among the P-450s encoded by these genes, a rea- in DNA and may be the most important of these interme- sonable argument can be made for the role of six (1A1, diate agents (Park and Tretyakova 2004; Hecht and Samet 1B1, 1A2, 2A6, 2A13, and 2E1) as important catalysts for 2007). The epoxides can be hydrated to dihydrodiols and the metabolic activation of carcinogens in cigarette smoke. conjugated by reactions with glutathione. 1,3-butadiene PAHs are metabolized by P-450s 1A1 and 1B1 (Shimada and metabolism can also lead to epoxidation and forma- Fujii-Kuriyama 2004), aromatic amines by P-450 1A2 tion of N-terminal Hb adducts, providing a longer-term, (Kim and Guengerich 2005), and N-nitrosamines by “time-weighted” measurement of exposure (Swenberg et P-450s 2A6, 2A13, and 2E1 (Yoo et al. 1988; Guengerich al. 2001). et al. 1991; Yamazaki et al. 1992; Jalas et al. 2005; Wong Although details remain to be determined, the et al. 2005a). P-450 2E1 also catalyzes the epoxidation of major pathways of metabolic activation and detoxification benzene and 1,3-butadiene (Guengerich et al. 1991; Bolt of some of the principal carcinogens in cigarette smoke et al. 2003). are well established. Reactive intermediate agents that

Cancer 237 Surgeon General’s Report

P-450s 1A1 and 1B1 are expressed in a wide range whether the responsible mechanism is common to both of extrahepatic tissues and catalyze both the activation P-450s 1A1 and 1B1. and detoxification reactions of PAH metabolism (Shimada Studies have characterized P-450 1A2 as the best and Fujii-Kuriyama 2004). In addition, both enzymes are catalyst for aromatic amine N-oxidation, which is the first inducible by the PAHs in cigarette smoke (Nebert et step in the activation of these bladder carcinogens (Butler al. 2004). Induction of these two enzymes is generally et al. 1989; Landi et al. 1999; Kim and Guengerich 2005). mediated by the aryl hydrocarbon receptor, but differ- P-450 1A2 is both constitutively expressed and inducible ences may exist in the mode of induction for each enzyme. in the liver. The induction of P-450 1A2 is mediated by the Historically, researchers believed that P-450 1A1 was the aryl hydrocarbon receptor, and hepatic levels vary more predominant P-450 catalyst for the metabolism of PAHs, than 60-fold from person to person (Nebert et al. 2004). particularly in the lung. However, the discovery of P-450 Cigarette smoking induces the levels of this enzyme in the 1B1 (Sutter et al. 1994) clarified the equal or more pre- liver. Researchers have also reported that P-450s 1A1 and dominant role P-450 1B1 may play in the activation of 1B1 metabolically activate a number of aromatic amines, PAHs compared with that of P-450 1A1 (Shimada et al. including 4-ABP, and may play a role in extrahepatic 1996). Studies show that P-450 1B1, which is heterolo- metabolism (Shimada et al. 1996). gously expressed, activates the proximate carcinogen of Although hepatic P-450 2A6 catalyzes the metabolic many PAHs and that in several cases, P-450 1B1 was more activation of NNK (Yamazaki et al. 1992; Jalas et al. 2005), efficient than P-450 1A1 (Shimada et al. 1996). For exam- P-450 2A6 is not a particularly efficient catalyst. The extra- ple, (+)-B[a]P-7,8-diol was activated to a genotoxic species hepatic P-450 2A13 might be a more important catalyst of to a greater extent by P-450 1B1 than by P-450 1A1 (Shi- the activation of this carcinogen (Jalas et al. 2005). P-450 mada et al. 1996). In addition, the ratio of the maximum 2A13 is expressed in the lung (Su et al. 2000) and catalyzes velocity (Vmax) of an enzyme-catalyzed reaction to the the α-hydroxylation of NNK significantly more efficiently concentration of a substrate that leads to half-maximal than does P-450 2A6. P-450 2A13 is an excellent catalyst velocity (Km) for the formation of B[a]P-7,8-diol was 3.5- of NNK α-hydroxylation, with a low Km and a high Vmax. fold greater for P-450 1B1 than for P-450 1A1 (Shimada P-450 2A13 is the sole catalyst of NNK α-hydroxylation in et al. 1999). (The Vmax to Km ratio measures an enzyme’s human fetal nasal tissue and is considered equally impor- efficiency.) In contrast, P-450 1A1 was a better catalyst of tant in the lung (Wong et al. 2005b). P-450 2E1 has also B[a]P 3-hydroxylation, which is a detoxification pathway catalyzed the activation of both NNN and NNK (Yamazaki (Shimada et al. 1997). Taken together, these data indicate et al. 1992). However, the catalytic efficiencies of these that P-450 1B1 activity, but not P-450 1A1 activity, may reactions are poor (Hecht 1998; Jalas et al. 2005). Studies contribute to individual susceptibility to B[a]P-induced have identified P-450 2E1 as the best catalyst of NDMA carcinogenesis. However, cigarette smoke has many dif- metabolism (Yoo et al. 1988; Guengerich et al. 1991) and ferent PAH carcinogens, and either P-450 1B1 or 1A1 as an excellent catalyst of the epoxidation and activation of individually or together may be important in their meta- benzene and 1,3-butadiene (Guengerich et al. 1991; Bolt bolic activation. et al. 2003). As noted previously (see “Cytochrome P-450 Enzymes” earlier in this chapter), both P-450 1A1 and 1B1 Epoxide Hydrolases are inducible by PAHs. Studies have reported that levels Several carcinogens of tobacco smoke, including of messenger RNA (mRNA) and protein of both P-450s PAH, 1,3-butadiene, and benzene, are metabolized to were higher in the lungs of smokers than in the lungs of epoxides. These epoxide metabolites are substrates for lifetime nonsmokers (Willey et al. 1997; Kim et al. 2004a; MEH (also known as EPHX1), an enzyme that catalyzes Port et al. 2004). The levels of P-450 1A1 and 1B1 proteins their hydrolysis (Wood et al. 1976; Snyder et al. 1993; were correlated in lung microsomes from all participants Krause and Elfarra 1997; Fretland and Omiecinski 2000). who smoked. However, the absolute amount of P-450 1A1 In mammals, at least five epoxide hydrolases were identi- in each person was, on average, more than 10-fold greater fied. However, four of these predominantly or exclusively than the amount of P-450 1B1 (Kim et al. 2004a). Despite catalyze the hydrolysis of endogenous substrates (Fretland the ability of P-450 1B1 to more efficiently mediate the and Omiecinski 2000). The fifth, MEH, plays a role in both activation of some PAHs, the higher P-450 1A1 levels may the detoxification and activation of xenobiotics. Specifi- result in each enzyme contributing similarly to the total cally, MEH is involved in the formation of the reactive diol metabolism of PAHs. An equally important factor in deter- epoxide metabolites of PAHs, and its activity is therefore mining the role of these P-450s in the activation of PAHs critical to the carcinogenicity of these compounds (Con- in cigarette smoke is the variability in the induction of ney 1982). For example, MEH catalyzes the hydrolysis of P-450s across individuals. Researchers do not know

238 Chapter 5 How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease

B[a]P-7,8-epoxide to B[a]P-7,8-diol, which is then oxi- these enzymes vary with the diol epoxide substrate. For dized to the ultimate carcinogen BPDE (Levin et al. 1976; example, GSTM1-1 is a more efficient catalyst of gluta- Gautier et al. 1996). The importance of this enzyme to thione conjugation of (+)-anti-BPDE than is either PAH carcinogenicity is supported by the observation that GSTA1-1 or GSTP1-1 (Sundberg et al. 1997). The GSTA1-1 MEH-null mice are highly resistant to carcinogenesis and GSTP1-1 enzymes have overall Kcat/Km values for induced by 7,12-dimethylbenz[a]anthracene (Miyata et catalytic rate or turnover number that are about 3-fold al. 1999). lower than the value for GSTM1-1, but GSTM1-1 is almost In contrast to its role in the activation of PAHs, 30-fold better as a catalyst for the conjugation of (-)-anti- MEH detoxifies the epoxides of 1,3-butadiene (Krause BPDE (Sundberg et al. 1997). The contribution of each GST and Elfarra 1997; Wickliffe et al. 2003). Studies have enzyme to the detoxification of PAH diol epoxides- var reported that several polymorphisms in MEH result in an ies with the substrate and across different tissues on the increased sensitivity to the genotoxic effects of 1,3-buta- basis of their expression levels. In lung tissue from smok- diene (Abdel-Rahman et al. 2003, 2005). Benzene oxide is ers, levels of (+)-anti-BPDE–DNA adducts were depen- also a substrate for MEH (Snyder et al. 1993). However, dent on the GSTM1 genotype (Alexandrov et al. 2002). male mice deficient in MEH are not susceptible to toxic Persons with the GSTM1 null genotype had significantly effects induced by benzene (Bauer et al. 2003). The role of higher adduct levels than did those with the GST wild-type benzene oxide in carcinogenesis is unclear. genotype. These data support the importance of GSTM1-1 activity in BPDE detoxification in the lung, but they do Glutathione-S-Transferases not exclude a role for GSTA1-1 and GSTP1-1 in the detoxification of this or other PAHs. Another mechanism that may detoxify carcinogenic GSTM1-1 and GSTT1-1 enzymes play a key role in epoxides is conjugation with glutathione. This reaction the conjugation of two 1,3-butadiene epoxide metabolites: can be catalyzed by cytosolic GSTs (Sheehan et al. 2001; 3,4-epoxybutene (EB) and diepoxybutane (DEB) (Norppa Hayes et al. 2005), which are dimeric. Seven classes (alpha, et al. 1995; Wiencke et al. 1995; Thier et al. 1996; Ber- mu, pi, sigma, theta, omega, and zeta) exist in mammalian nardini et al. 1998; Landi 2000; Fustinoni et al. 2002; species (Sheehan et al. 2001), and at least 16 GST sub- Schlade-Bartusiak et al. 2004). The direct measurement units exist in humans. However, only four homodimeric of either GSTM1-1 or GSTT1-1 activity with these epoxide enzymes to date have been characterized as catalysts of substrates has not been reported. However, several stud- glutathione conjugation of tobacco smoke carcinogens ies of sister chromatid exchange (SCE) in human lympho- (Cheng et al. 1995; Norppa et al. 1995; Wiencke et al. 1995; cyte cultures from persons with the GSTT1 null genotype Jernstrom et al. 1996; Sundberg et al. 1998, 2002; Landi support the role of GSTT1-1 in the detoxification of DEB 2000; Verdina et al. 2001; Fustinoni et al. 2002; Sørensen (Norppa et al. 1995; Wiencke et al. 1995; Bernardini et et al. 2004a; Hayes et al. 2005). These enzymes are mem- al. 1998; Landi 2000; Schlade-Bartusiak et al. 2004). In bers of four GST classes: alpha (GSTA1-1), mu (GSTM1-1), conflict with these data, one study reports the increased pi (GSTP1-1), and theta (GSTT1-1). The protein levels of mutagenicity of DEB in Salmonella typhimurium TA1535 each GST vary significantly from person to person, as well expressing GSTT1-1, suggesting that the conjugation of as across tissues within an individual (Rowe et al. 1997; this diepoxide is an activation pathway (Thier et al. 1996). Sherratt et al. 1997; Mulder et al. 1999). Researchers have The role of both GSTM1-1 and GSTT1-1 in the detoxifica- identified several polymorphisms in the genes encoding tion of EB is supported by a higher induction of SCE by these subunits (Hayes et al. 2005). Of particular note with EB in lymphocyte cultures from persons with either the regard to cancer risk in smokers are the *NULL alleles GSTM1-1 or the GSTT1-1 null genotype (Uusküla et al. for GSTM1 and GSTT1, which have decreased detoxifica- 1995; Bernardini et al. 1998). Although GSTs play a role tion capacity and elevated DNA damage. GSTA1, GSTM1, in the metabolism of 1,3-butadiene, it remains unclear and GSTT1 are expressed in the liver of persons who are whether polymorphisms in GSTs modulate the carcino- not homozygous for either null phenotype; little GSTP1 is genic effects of 1,3-butadiene in humans (Fustinoni et present in the liver (Rowe et al. 1997; Sherratt et al. 1997; al. 2002). Mulder et al. 1999). In contrast, the lung expresses higher One major excreted metabolite of benzene is levels of GSTP1 than those expressed by the other three S-PMA, which is formed from the glutathione conjugate of subunits (Rowe et al. 1997; Sherratt et al. 1997). benzene oxide (Snyder and Hedli 1996). This glutathione GSTA1-1, GSTM1-1, and GSTP1-1 each catalyze the conjugate may be generated both enzymatically and non- glutathione conjugation of a number of PAH diol epox- enzymatically, and it is not clear which pathway predomi- ides (Jernstrom et al. 1996; Sundberg et al. 1998, 2002). nates. However, a number of studies on benzene exposure However, the efficiencies and stereoselectivity of each of and toxicity have suggested a role for either GSTM1-1 or

Cancer 239 Surgeon General’s Report

GSTT1-1 in the conjugation of benzene oxide (Hsieh et Aromatic amines and their N-hydroxy metabolites al. 1999; Verdina et al. 2001; Wan et al. 2002; Kim et al. are glucuronidated to facilitate excretion (Bock 1991; 2004b). Researchers have not directly measured which Tukey and Strassburg 2000; Zenser et al. 2002). Glucuron- enzyme is the better catalyst of glutathione conjugation idation is a detoxification reaction. Therefore, variations in of benzene oxide. The in vivo role of GSTT1-1 in benzene the expression and catalytic efficiency of the enzymes that oxide detoxification is supported by a report that S-PMA catalyze this reaction may influence the carcinogenicity of levels excreted by persons exposed to benzene who carried particular aromatic amines. In general, researchers have the wild-type GSTT1* allele were higher than those of per- suggested that members of the UGT1A family contribute sons homozygous for the GSTT1* NULL allele (Sørensen to the glucuronidation of these carcinogens (Orzechowski et al. 2004b). et al. 1994; Green and Tephly 1998; Tukey and Strassburg Ethylene oxide is also detoxified by glutathione 2000; Zenser et al. 2002). However, UGT2B7 also catalyzes conjugation (Brown et al. 1996). Although studies have their glucuronidation (Zenser et al. 2002). In most cases, not directly evaluated the role of specific human GSTs, data support UGT1A9 as the best catalyst. For the tobacco evidence supports the role of GSTT1-1 as a catalyst of smoke carcinogen 4-ABP, the relative catalytic efficiency this reaction (Hallier et al. 1993; Fennell et al. 2000). On of N-glucuronidation is UGT1A9>UGT1A4>UGT1A7>UGT exposure to ethylene oxide, lymphocytes from persons 2B7>UGT1A6, but the catalytic efficiency of all these pro- with the GSTT1-1* NULL allele had higher levels of SCE teins is approximately equal to that of UGT1A1 (Zenser et than did those from persons with the wild-type allele (Hall- al. 2002). ier et al. 1993). In addition, levels of 2-hydroxyethylvaline The phenol and diol metabolites of PAHs are pri- Hb adducts were higher in smokers than in nonsmok- marily eliminated as glucuronide conjugates. Researchers ers, because of exposure to ethylene and ethylene oxide have studied the role of specific UGTs in the metabolism in cigarette smoke, and were higher in smokers with the of B[a]P (Bock 1991; Guillemette et al. 2000; Fang et al. GSTT1* NULL allele than in those with the wild-type allele 2002; Dellinger et al. 2006). Studies with UGT1A-deficient (Fennell et al. 2000). rats have implicated UGT1A enzymes in the detoxification of B[a]P (Wells et al. 2004). The glucuronidation of B[a]P- Uridine-5’-Diphosphate-Glucuronosyltransferases 7,8-diol and 3-hydroxy-, 7-hydroxy-, and 9-hydroxy-B[a]P by heterologously expressed human UGTs has been char- Conjugation with glucuronic acid is an important acterized for a number of UGT1A and UGT2B enzymes metabolic pathway for a number of carcinogens in tobacco (Fang et al. 2002; Dellinger et al. 2006). Among the phe- smoke (Bock 1991; Hecht 2002a; Nagar and Remmel 2006). nols, UGT1A10 was the most efficient UGT1A catalyst of (Conjugation is the addition of a polar moiety to a metab- glucuronidation. UGTs 2B7, 2B15, and 2B17 all catalyzed olite to facilitate excretion.) The microsomal enzymes, conjugation of the three B[a]P phenols. However, the K UGTs, catalyze these conjugation reactions. Researchers m of the reaction for UGT2B enzymes was 2- to 250-fold have identified 18 human UGTs that are members of two higher than that for UGT1A10 (Dellinger et al. 2006). For families (UGT1 and UGT2) (Tukey and Strassburg 2000; the carcinogenic (-)-B[a]P-7,8-diol, UGT1A10 was a bet- Burchell 2003; Nagar and Remmel 2006). The UGT1A pro- ter catalyst of glucuronidation than was UGT1A9, and teins are encoded by a single gene cluster, and expression UGT2B7 did not catalyze detectable levels of glucuronida- of the nine members of this subfamily occurs through exon tion (Fang et al. 2002), but UGT2B7 did catalyze the gluc- sharing. Exon 1 is unique for each UGT1A, whereas exon 2 uronidation of (+)-B[a]P-7,8-diol. to exon 5 are shared by all UGT1As (Tukey and Strassburg In smokers, glucuronidation also plays an impor- 2000). Thus, all UGT1A proteins are identical in the 245 tant role in the excretion of the NNK metabolite NNAL amino acids of the carboxyl terminus encoded by exon 2 (Carmella et al. 2002b; Hecht 2002a). Both O-linked and to exon 5 (Tukey and Strassburg 2000; Finel et al. 2005). N-linked NNAL glucuronide conjugates are formed (Car- In contrast, proteins from the UGT2 family are all unique mella et al. 2002b). In addition, the direct detoxifica- gene products (Riedy et al. 2000; Tukey and Strassburg tion of the hydroxymethyl metabolite of NNK occurs by 2000). The expression of UGTs is tissue specific, and there glucuronidation in rats (Murphy et al. 1995). However, are large differences in expression among tissues (Gregory the contribution of this pathway to NNK detoxification et al. 2000, 2004; Tukey and Strassburg 2000; Wells et al. in smokers has not been identified. In vitro studies with 2004). For example, UGTs 1A1, 1A3, 1A4, 1A6, and 1A9 are fibroblasts both from UGT1A-deficient and control rats highly expressed in the liver; UGTs 1A7, 1A8, and 1A10 have confirmed a role for UGT1A enzymes in the protec- are mainly expressed in extrahepatic tissues (Tukey and tion of these cells from NNK-induced micronuclei forma- Strassburg 2000; Gregory et al. 2004; Wells et al. 2004). tion (Kim and Wells 1996). Human UGT1A9, UGT2B7,

240 Chapter 5 How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease and UGT2B17 catalyze NNAL-O-glucuronidation, with Studies with recombinant human NAT1 and NAT2 UGT2B17 being the most active, and UGT1A4 catalyzes have described differences in the N-acetylation of 4-ABP. NNAL-N-glucuronidation (Ren et al. 2000; Wiener et al. The apparent affinity of 4-ABP for NAT2 is significantly 2004b; Lazarus et al. 2005). The rate of NNAL O- and greater than that for NAT1, and ratios of NAT1 activity to N-glucuronidation by human liver microsomes varies sig- NAT2 activity and clearance calculations support a greater nificantly among persons; researchers have suggested that role for NAT2 than for NAT1 in the N-acetylation of aryl- polymorphisms in UGT2B7 and UGT1A4 contribute to this amines (Hein et al. 1993). The characterization of NAT1 variability (Wiener et al. 2004a). as the key catalyst of the O-acetylation (i.e., activation) Glucuronidation may also contribute to the detoxi- of aromatic amines is more speculative and is primarily fication of benzene (Bock 1991). In hepatocytes from rats driven by the tissue distribution of NAT1 (see the discus- treated with 3-methylcholanthrene to induce UGTs, phe- sion below). No data in the literature report differences nol glucuronidation increases compared with sulfation. between the efficiencies of NAT1- and NAT2-catalyzed Glucuronide conjugates are more stable than the cor- O-acetylation of aromatic amines. However, more recent responding sulfates, and researchers have suggested the studies that engineered S. typhimurium strains to over- glucuronidation of phenol as a detoxification pathway express either NAT1 or NAT2 reported that NAT1, but not (Bock 1991). However, to date, the role of glucuronidation NAT2, catalyzed the genotoxic activation of N-hydroxy- in benzene-induced carcinogenesis has not been charac- 4-ABP (Oda 2004). These data provide support for NAT1 terized and is poorly understood. as an important catalyst in the activation of this aromatic amine. N-Acetyltransferases The organ and tissue distribution of NAT1 and NAT2 differ markedly (Dupret and Rodrigues-Lima 2005). The NATs are cytosolic enzymes that catalyze the trans- NAT2 protein is mainly expressed in the gut and liver; the fer of the acetyl group from acetylcoenzyme A to an NAT1 protein is expressed in the liver and a number of acceptor molecule (Hein et al. 2000b). This transfer other tissues, including the colon and bladder. Research- occurs through an enzyme intermediate in which cysteine ers believe that aromatic amines in tobacco smoke con- 68 is acetylated and then deacetylated during the course tribute to smoking-related bladder cancer. Therefore, the of the reaction. Humans express two unique enzymes, potential activation of these compounds in the bladder is NAT1 and NAT2, which catalyze both N- and O-acetylation important in understanding the etiology of bladder can- reactions. Researchers have recognized the polymorphic cer. Researchers have detected NAT1 activity, but not NAT2 nature of NAT2 for more than 40 years and, more recently, activity, in samples of bladder tissue from smokers (Bad- have identified more than 35 alleles (Hein et al. 2000b; wawi et al. 1995). In addition, DNA adduct levels measured Hein 2002). NAT1 is less well studied but is also poly- by 32P-postlabeling correlated with NAT1 activity. These morphic, and more than 25 alleles have been identified data are thus consistent with a role for NAT1 in the activa- (Hein 2002; University of Louisville School of Medicine tion of arylamines in tobacco smoke. 2006). Researchers suggest that polymorphisms in both Epidemiologic studies that demonstrate a modest NAT1 and NAT2 influence the activation and detoxifica- increase in risk of bladder cancer in persons phenotypi- tion of carcinogenic aromatic amines in tobacco smoke cally and genotypically identified as having slow acetyla- (Hein 2002). tion catalyzed by NAT2 further support the role of NAT2 The N-acetylation of aromatic amines, such as in the detoxification of aromatic amines (Green et al. 4-ABP, is a detoxification reaction (Hein 2002). In con- 2000; Hein et al. 2000a; Gu et al. 2005) (see “Molecular trast, O-acetylation of the N-hydroxy metabolites of Epidemiology of Polymorphisms in Carcinogen-Metabo- arylamines generated by P-450 (e.g., N-hydroxy-4-ABP) is lizing Genes” later in this chapter). A number of the NAT2 an activation reaction leading to DNA adduct formation variant alleles identified in persons with slow acetyla- (Hein et al. 1993, 1995; Hein 2002). NAT1 and NAT2 both tion were expressed heterologously and demonstrated a catalyze each of these reactions (Hein et al. 1993). How- decrease in activity for both the N-acetylation of 4-ABP and ever, NAT2 is generally considered the more important O-acetylation of N-hydroxy-4-ABP, primarily because of catalyst of detoxification, and NAT1 is the more - impor the instability of the variant enzymes (Hein et al. 1995; tant catalyst of activation (Badawi et al. 1995; Hein 2002). Zhu et al. 2002). Both activation and detoxification would This assumption is based on differences in the catalytic be diminished in persons expressing variant NAT2 activity, efficiency of the enzymes and their tissue distribution in but NAT1 activity would be maintained. Studies that have humans as well as on studies with animal models (Hein et characterized NAT1 proteins from a number of variants of al. 1993; Hein 2002). this gene have also reported a decrease in enzyme activity (Fretland et al. 2002).

Cancer 241 Surgeon General’s Report

DNA Adducts and Biomarkers and the KRAS oncogene in cells (Tang et al. 1999; Feng et al. 2002). Introduction Several studies have quantified 7-methyldeoxyguanosine in human lung tissue. The source of this adduct in Although formation of carcinogen-DNA adducts is smokers could be NDMA, NNK, or perhaps other methyl- a well-characterized phenomenon in laboratory animals, ating agents. Studies have reported mixed results: some there were no reports of analyses of DNA adducts in smok- show higher adduct levels in smokers than in nonsmokers before the mid-1980s. In the past 20 years, a large ers (Hecht and Tricker 1999; Lewis et al. 2004). One study body of literature on DNA adducts in human tissues has examined O6-methyldeoxyguanosine in human lung tis- emerged with the development of sensitive methods such sue but was too small to draw conclusions about the effect as HPLC fluorescence, GC–MS, liquid chromatography of smoking (Wilson et al. 1989). 32 (LC)–MS, electrochemical detection, P-postlabeling, Three small studies provided evidence for ethyl DNA and immunoassay. Researchers have applied all of these adducts in human lung tissue (Wilson et al. 1989; Blömeke methods to analyze DNA adducts, producing data on these et al. 1996; Godschalk et al. 2002). Levels of both O6-ethyl- biomarkers in molecular epidemiologic studies of can- deoxyguanosine and O4-ethylthymidine were higher in cer susceptibility. Thus, a discussion of DNA adducts in smokers than in nonsmokers. Although one source of human tissues also includes biomarkers of DNA adduct these adducts could be N-nitrosodiethylamine, its level in formation in smokers. cigarette smoke is low. As discussed previously, cigarette smoke contains a direct-acting, but chemically uncharac- Characterized Adducts in the Human Lung terized, ethylating agent that may be responsible for the Available data on characterized DNA adducts in presence of these adducts (see “Carcinogens in Cigarette human lung tissue, the tissue most extensively inves- Smoke” earlier in this chapter). tigated to date, are summarized in Table 5.2. The small NNK and NNN are metabolically activated to inter- number of studies reflects several difficulties in this mediate agents that pyridyloxobutylate DNA. The result- research. First, DNA from human lung tissue is difficult to ing POB-DNA adducts can be hydrolyzed with acid to obtain. The amounts of DNA available from routine proce- yield 4-hydroxy-1-(3-pyridyl)-1-butanone, which can be dures, such as bronchoscopy, are generally too small for detected by GC–MS. Application of this method demon- analysis of specific DNA adducts. Second, the levels of DNA strated higher levels of POB-DNA adducts in lung tissue adducts are generally low: between 1 in 10 million and 1 of smokers than in that of nonsmokers in a small study in 100 million normal DNA bases. Analyzing such small (Foiles et al. 1991). One study detected 7-(2-hydroxyethyl) amounts of material is challenging. Nevertheless, meth- deoxyguanosine in human lung tissue (Zhao et al. 1999), ods such as those listed previously and in Table 5.2 were and ethylene oxide is the likely source of this adduct. Stud- successfully applied. However, because of the limitations ies have detected 4-ABP–DNA adducts in human lungs but noted, the number of participants in most of the studies show no clear effect of smoking on adduct levels (Wilson is small. et al. 1989; Lin et al. 1994). 6 The major DNA adduct of B[a]P observed in labora- Researchers have quantified N1, -ethenodeoxy- 4 tory animals is BPDE-N2-deoxyguanosine. Acid hydrolysis adenosine and 3,N -ethenodeoxycytidine in human lungs 32 of DNA containing this adduct releases B[a]P-7,8,9,10- by P-postlabeling (Godschalk et al. 2002). These adducts tetraol, which can be analyzed by HPLC with fluorescence may result from lipid peroxidation or from metabolic acti- detection (Rojas et al. 1998). Other BPDE-derived DNA vation of vinyl chloride or ethyl carbamate. No differences adducts may be hydrolyzed simultaneously. This assay were reported between smokers and nonsmokers. Studies has been applied to lung tissue obtained during surgery of the oxidative-damage product 8-oxodeoxyguanosine in (Alexandrov et al. 2002; Boysen and Hecht 2003). Com- the human lung obtained mixed results regarding a rela- pared with nonsmokers, smokers with the GSTM1 null tionship between detection of this product and smoking genotype displayed higher levels of BPDE-DNA adducts status (Asami et al. 1997; Lee et al. 1999a). in lung tissue, although this finding is based on a small In summary, data on the quantitation of specific number of cases (Rojas et al. 1998, 2004). BPDE-DNA DNA adducts in the human lung are limited. However, adducts were detectable in 40 percent of the smokers with some studies document clear evidence for elevated lev- whole lung analyses (Boysen and Hecht 2003) and in all els of adducts resulting from exposure to specific car- samples with analyses of bronchial epithelial cells (Rojas cinogens such as B[a]P, NNK, or NNN. Several methods et al. 2004). When the adduct localization in genes was used in these studies—HPLC fluorescence, GC–MS, 32 determined by in vitro studies, one target was seen to be LC–MS, and P-postlabeling with modifications for specific at mutational hot spots in the P53 tumor-suppressor gene adducts—have the potential for application to molecular

242 Chapter 5 How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease

Table 5.2 DNA adducts in human lung tissue

Type of Study Carcinogen DNA base Adduct structuresa evidenceb

Rojas et al. 1998, 2004 Benzo[a]pyrene dG 1 (N2) Boysen and Hecht 2003 3 HO

Wilson et al. 1989 N-nitrosodimethylamine dG 7—CH3 2 Mustonen et al. 1993 NNK Kato et al. 1995 Others Blömeke et al. 1996 6 Petruzzelli et al. 1996 O —CH3 2

Wilson et al. 1989 N-nitrosodiethylamine dG 7—CH3CH2 2 6 Blömeke et al. 1996 Others O —CH3CH2 2 4 Godschalk et al. 2002 T O —CH3CH2 2

Foiles et al. 1991 NNK dG, T, dC O 1 N’-nitrosonornicotine (7-dG O6-dG 2 N O -T O2-dC)

Eide et al. 1999 Ethylene oxide dG 7—HOCH2CH2 2 Wilson et al. 1989 4-aminobiphenyl dG 2 Lin et al. 1994 NH(C-8)

Godschalk et al. 2002 Vinyl chloride Deoxyadenosine N 2 Ethyl carbamate N Oxidants N

N N dR dC N 2

O N dR

Asami et al. 1997 Oxidants dG 8—oxo 3 Lee et al. 1999a Note: dC = deoxycytidine; dG = deoxyguanosine; NNK = 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; T = thymidine. aAdduct structures show position of attachment to the base (e.g., N2-, O6-, or 7- of dG) and the organic moiety derived from the carcinogen. b1 = detection of a released adducted moiety by a specific method; 2 = detection of a nucleoside or base by a relatively nonspecific method (e.g., 32P-postlabeling or immunoassay); 3 = detection of a nucleoside or base by a specific method (e.g., mass spectrometry, high-performance liquid chromatography [HPLC]-fluorescence, or HPLC-electrochemical detection).

Cancer 243 Surgeon General’s Report epidemiologic studies that relate specific DNA adduct lev- Levels of 7-alkyl-deoxyguanosines determined by els to tobacco exposure and cancer risk. 32P-postlabeling in laryngeal DNA were higher in smokers than in nonsmokers (Szyfter et al. 1996), and they Uncharacterized Adducts in Human Lung Tissue correlated with the DRZ in these samples. Studies used immunoassay also to detect 4-ABP–DNA adducts in laryn- Studies have extensively applied two main nonspe- geal tissue (Flamini et al. 1998). Other studies examined cific methods—32P-postlabeling and immunoassay—to the DRZ by 32P-postlabeling (IARC 2004). analyze DNA adducts in human lung tissue, as well as in Researchers have detected 1,N2-propanodeoxy- other tissues. Researchers have discussed the advantages guanosine (PdG) adducts derived from acrolein and cro- and disadvantages of these methods (Kriek et al. 1998; tonaldehyde in the DNA of gingival tissue of smokers and Wild and Pisani 1998; Poirier et al. 2000; Phillips 2002). nonsmokers; adduct levels were higher in smokers (Nath Major advantages include high sensitivity for analyzing et al. 1998). Adducts detected by 32P-postlabeling in oral small amounts of DNA, simplicity of analysis, and no need and nasal tissue were also higher in smokers than in non- for extremely expensive equipment. Disadvantages include smokers. Use of immunoassay techniques revealed that a lack of chemical specificity, particularly in32 P-postlabel- levels of BPDE-DNA, 4-ABP–DNA, and malondialdehyde- ing analyses, and difficulty in quantitation. Studies have DNA adducts in human oral mucosal cells of smokers were extensively reviewed the application of these methods to higher than those for nonsmokers (IARC 2004). tissues obtained from smokers (Phillips 2002; Wiencke Using 32P-postlabeling, researchers found 4-ABP– 2002; IARC 2004). DNA (C-8 deoxyguanosine) adducts in exfoliated uro- The output of assays using 32P-postlabeling is thelial cells and bladder biopsy samples (IARC 2004). In often a “diagonal radioactive zone” (DRZ), which con- studies using antibodies to 4-ABP–DNA, levels detected sists of uncharacterized radioactive components referred in biopsy specimens from the bladder of smokers were to in the literature as hydrophobic or aromatic DNA higher than those for nonsmokers (IARC 2004). Studies adducts. In most cases, little if any evidence supports the using 32P-postlabeling of bladder DNA from smokers and true chemical characteristics of these adducts. Neverthe- nonsmokers yielded mixed results; some studies showed less, the intensity of the DRZ is consistently elevated in higher adduct levels in smokers (IARC 2004). samples from smokers. Immunoassays have used various Using GC–MS, Melikian and colleagues (1999) docu- methods of detection, including the fluorescent staining mented that BPDE-DNA adducts were higher in cervical of tissue specimens that allows for the location of adducts. epithelial cells of smokers than in those of nonsmokers. An Cross-reactivity is a common problem of immunoassays. immunohistochemical analysis using antibodies to BPDE- For example, antibodies raised against protein conjugates DNA adduct in human cervical cells also showed higher of B[a]P–DNA adducts cross-react with adducts generated adduct levels in smokers than in nonsmokers (Mancini from other PAHs. et al. 1999). 32P-postlabeling consistently showed higher Many studies using 32P-postlabeling methods adduct levels in cervical tissues of smokers than in those examined DNA adduct levels in the peripheral lung, bron- of nonsmokers (IARC 2004). chial epithelium, or cells obtained by bronchial lavage of The 32P-postlabeling of DNA from breast tissue smokers. Most of the studies found that adduct levels were yields the characteristic DRZ from smokers. Researchers higher in smokers compared with nonsmokers (Gy rffy ő also investigated adduct levels by using antibodies against et al. 2004; IARC 2004). Investigations that attempted BPDE-DNA; results were generally mixed with respect to to draw quantitative relationships between the extent smoking status (IARC 2004). Studies that used 32P-post- of smoking and adduct levels had inconsistent results labeling to measure adduct levels in pancreatic and stom- (IARC 2004). ach tissues reported a correlation with smoking status (IARC 2004). Adducts in Other Tissues Some studies indicate the presence of smoking- Numerous studies have evaluated DNA adduct related DNA adducts in human placenta, but the overall formation in fetuses and in various tissues and fluids of relationship of placental DNA adducts to smoking is weak smokers, including samples from the larynx, oral and (IARC 2004). Analyses of sperm DNA also reported mixed nasal mucosa, bladder, cervix, breast, pancreas, stomach, results with respect to smoking status (IARC 2004). placenta, and cardiovascular system, and samples of spu- Many studies have examined DNA adducts in blood tum, sperm, and blood cells. Researchers have compre- cells (IARC 2004). The common use of blood cells in these hensively reviewed these studies (Phillips 2002; Weincke studies is obviously related to the ease of clinically obtain- 2002; IARC 2004), most of which used 32P-postlabeling ing these samples. From this viewpoint, blood cell DNA and immunoassay techniques. is advantageous for biomarker studies. A disadvantage of

244 Chapter 5 How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease using blood cells is that adduct levels in blood cells are Summary not necessarily directly related to levels of DNA adducts in Overwhelming evidence indicates that DNA adduct the tissues in which smoking-related cancers occur. The levels are higher in most tissues of smokers than in cor- collective results of the studies are somewhat inconsistent responding tissues of nonsmokers. This observation pro- with respect to the effects of smoking on levels of DNA vides bedrock support for the major pathway of cancer adducts. This inconsistency probably results in part from induction in smokers that proceeds through DNA adduct competing sources of adduct formation such as diet, occu- formation and genetic damage. DNA adducts studied can pation, and the general environment. Another factor is the generally be divided into two classes: nonspecific adducts, lifetime of the blood cells investigated; longer-lived cells which are detected by 32P-postlabeling and immunoassay, appear to provide more consistent results with respect to and specific adducts, which are detected by structure- smoking (IARC 2004). Studies comparing levels of blood specific methods. Studies of nonspecific DNA adducts are cell–DNA adducts in smokers with or without cancer had far more common than studies of specific DNA adducts, mixed results (IARC 2004). which are still scarce and are limited mainly to human A meta-analysis of the relationship of DNA adduct lung tissue. Strong evidence exists for the presence of levels in smokers to cancer, determined by 32P-postlabel- a variety of specific adducts in the human lung, and in ing, used data from case-control studies of lung cancer several cases, adduct levels are higher in smokers than (five studies), oral cancer (one study), and bladder cancer in nonsmokers. Measuring levels of Hb adducts by MS (one study). Six studies measured adducts in white blood provides a simple and perhaps more practical approach cells, and one study used normal lung tissue. Among cur- for assessing carcinogen exposure of the cell. In several rent smokers, adduct levels for case patients were signifi- instances, levels of specific adducts are substantially cantly higher than those for control participants (Veglia higher in smokers than in nonsmokers. Collectively, the et al. 2003). results of these biomarker studies demonstrate the potential for genetic damage in smokers from the persistence Protein Adducts as Surrogates for DNA Adducts of DNA adducts. The propagation of this genetic damage Researchers have proposed that levels of carcino- during clonal outgrowth is consistent with the accumu- gen-Hb adducts and carcinogen-albumin adducts be used lation of multiple genetic changes observed in lung can- as surrogates for the measurements of DNA adducts dis- cer progression. cussed in the preceding section (Osterman-Golkar et al. 1976; Ehrenberg and Osterman-Golkar 1980). Although these proteins are not considered targets for carcinogen- Molecular Epidemiology of esis, all carcinogens that react with DNA are also thought to react with protein to some extent. Advantages of Hb Polymorphisms in Carcinogen- adducts as surrogates include the ready availability of Metabolizing Genes Hb in blood and the long lifetime of the erythrocyte in humans (approximately 120 days), which provides an Introduction opportunity for adducts to accumulate. Other research- Genetic polymorphisms may play a role in tobacco- ers have comprehensively reviewed studies on protein related neoplasms. Researchers have established cigarette adducts in smokers (Phillips 2002; IARC 2004). smoking as a major cause of lung cancer: more than 85 The Hb adducts of aromatic amines have emerged percent of lung cancers are attributable to smoking (Ries as highly informative carcinogen biomarkers. Levels of et al. 2004). However, not all smokers develop lung cancer, these adducts are consistently higher in smokers than in and lung cancer can arise in lifetime nonsmokers. This nonsmokers, particularly for 3-ABP–Hb and 4-ABP–Hb variation in disease has stimulated interest in molecular adducts. Adduct levels decrease with smoking cessa- epidemiologic investigations of genetic polymorphisms, tion and are related to the number of cigarettes smoked including carcinogen-metabolizing enzymes that may (Maclure et al. 1990; Skipper and Tannenbaum 1990; lead to variations in susceptibility to the carcinogens in Castelao et al. 2001). Adducts that form with the amino ter- tobacco smoke (Table 5.3). Considerable data exist on minal valine of Hb are also informative. Important examples genetic polymorphisms in cancers other than lung can- include adducts derived from ethylene oxide, butadiene, cer, but the discussion here focuses only on lung cancer acrylonitrile, and acrylamide (Bergmark 1997; Fennell and bladder cancer, two of the most heavily investi- et al. 2000; Swenberg et al. 2001). Ethylated N-terminal gated cancers. valine of Hb is also higher in smokers than in nonsmokers (Carmella et al. 2002a).

Cancer 245 Surgeon General’s Report

Table 5.3 Selected gene polymorphisms evaluated by molecular epidemiology investigations for relationship to lung cancer through variation in susceptibility to carcinogens in tobacco smoke

Metabolic genes Nucleotide change Amino acid change Enzymatic activity

CYP1A1 T→C (MSPI) NA Increased A→G Ile462Val Increased

CYP2E1 T→A (DRAI) NA Increased G→C (RSAI) NA Increased

CYP2A13 C→T Arg257Cys Decreased

GSTM1 Deletion NA None

GSTP1 A→G Ile105Val Decreased

GSTT1 Deletion NA None

NAT2 T→C Ile114Thr Decreased C→T Lys161Lys Decreased A→G Lys268Arg Decreased G→A Arg197Gln Decreased C→T Tyr94Tyr Decreased G→A Gly286Glu Decreased

MEH T→C Tyr113His Decreased A→G His139Arg Increased Note: NA = not applicable.

Studies have identified polymorphisms in phase I review summarizes the effects of genetic polymorphisms and II enzymes. Phase I enzymes, such as P-450s, gen- on lung cancer (Schwartz et al. 2007), and specific exam- erally add an oxygen atom to a carcinogen, and phase II ples are discussed here. enzymes, such as GSTs or UGTs, modify the carcinogen by making it highly water soluble for more facile excretion. CYP1A1 Gene These enzymes are involved in the activation and detoxi- Researchers hypothesize that interindividual varia- fication of carcinogens and may be associated with a dif- tions in the ability to activate carcinogens such as PAH ferential ability to process carcinogens. Researchers have through the CYP1A1 gene may lead to differential carci- hypothesized that the accumulation of active carcinogen nogenic effects. Studies describe at least two variant poly- metabolites and hence increased DNA adduct formation morphisms in the CYP1A1 gene. The first is a T3801C base add to lung cancer risk. Studies of cases with autopsy of change in intron 6, which results in a new MSPI restric- cancer-free lung tissue indicate that polymorphisms in tion site (Kawajiri et al. 1990). (A restriction site is a site in CYP1A1 and GSTM1 genes may be associated with higher the gene that is cleaved by a specific restriction enzyme.) DNA adduct levels, suggesting that variations in meta- The second polymorphism is an A2455G base change in bolic pathways can play a role in individual response to exon 7, which results in an Ile to Val amino acid change carcinogen exposure (Kato et al. 1995). Numerous studies (Hayashi et al. 1991). Although these polymorphisms ap- have extended this line of analysis to investigate whether pear to be linked, study results are inconsistent, and wide this differential ability to metabolize carcinogens leads to disparities exist among populations. differential lung cancer risk. Overall, data from the study Studies of Japanese and Chinese populations of these polymorphisms have generated inconsistent associate both of the CYP1A1 variant polymorphisms with results. These inconsistencies may be explained in part by an increase in lung cancer risk. Nakachi and colleagues the combination of a small sample size and variable fre- (1991) were the first to report an association of the*MSPI quencies of the polymorphic alleles within different ethnic polymorphism with lung cancer risk. For patients with populations. A summary of some of the specific gene poly- lung cancer, the frequency of harboring the homozygous morphisms investigated is provided in Table 5.3. A recent

246 Chapter 5 How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease variant genotype was more than two times higher than comprised approximately one-half of the case-control that for control participants. Among patients with squa- studies published at that time. Researchers found an mous cell carcinoma (SCC), the homozygous variant association in Whites between the CYP1A1 homozygous genotype was associated with an increased risk of develop- *MSPI variant and lung cancer risk after adjustment ing lung cancer, especially in those with a lower cumula- of values for age and gender (OR = 2.36; 95 percent CI, tive dose of cigarette smoke. At low levels of exposure to 1.16–4.81). The association held for both SCC and adeno- cigarette smoke, the odds ratio (OR) for developing lung carcinomas (Vineis et al. 2003). However, this association cancer among persons with the homozygous variant gen- failed to reach statistical significance among Asians in otype was 7.31 (95 percent confidence interval [CI], 2.13– this analysis. Moreover, studies such as the research con- 25.12). This increased risk was persistent, but of a lesser ducted by Nakachi and colleagues (1991, 1993) discussed magnitude, at higher levels of exposure to cigarette smoke previously in this section were not included, making the (Nakachi et al. 1991). Okada and colleagues (1994) Asian data difficult to interpret. reported similar findings. Studies have also associated the ILE462VAL poly- CYP2E1 Gene morphism of CYP1A1 with lung cancer risk in Japanese The CYP2E1 gene is involved in the metabolic and Chinese populations. Again, the homozygous variant activation of NDMA, as well as several other tobacco *VAL/*VAL genotype was associated with lung cancer at smoke carcinogens. Le Marchand and colleagues (1998) lower cumulative doses of cigarette smoke (Nakachi et al. performed a population-based, case-control study with 1993; Yang et al. 2004; Ng et al. 2005). One explanation 341 lung cancer cases and 456 controls. These researchers posited for this relationship with the dose level in smok- found that CYP2E1 polymorphisms were associated with a ers has been that the relevant enzyme is saturated at high decrease in risk of lung adenocarcinoma. A Chinese study doses but not at low doses of cigarette smoke (Vineis et (Wang et al. 2003c) confirmed this finding. However, the al. 1997). The effects of genetic variability and differential presence of at least one variant CYP1A1 *MSPI allele was enzymatic activity are more apparent at low doses, when associated with an increased risk of SCC, both alone (2.4- saturation has not been reached. fold increase in risk) and in combination with GSTM1 de- Results have been inconsistent outside Asian popu- letion (3.1-fold increase in risk) (Le Marchand et al. 1998). lations. Individual studies often lack statistical power to These researchers suggest that the associations between detect an association (Shields and Harris 2000). Also, CYP1A1 and CYP2E1 polymorphisms and subsets of lung CYP1A1 polymorphisms are common in Asian populations cancer indicate a specificity of PAHs to induce SCC and of (30 percent of the population) (Nakachi et al. 1993), but nitrosamines to induce adenocarcinomas. are far less common among Europeans and North Ameri- cans (<10 percent of the population) (Warren and Shields CYP2A13 Gene 1997). A study of African Americans and Mexican Ameri- cans showed a twofold increase in the risk of lung cancer The CYP2A13 gene is expressed primarily in the among light smokers with the *MSPI variant genotype respiratory tract and participates in the metabolic activa- (Ishibe et al. 1997). However, a Brazilian study showed tion of N-nitrosamines such as NNK. Researchers have an increase in risk with the *ILE/*VAL polymorphism but identified a polymorphism in CYP2A13 in which a C→T not with the MSPI polymorphism (Hamada et al. 1995). A transition leads to an Arg→Cys substitution at position more recent study suggested that in Latinos, the *MSPI 257. The variant 257CYS protein, the product of this gene, variant genotype was associated with an overall inverse has one-half to one-third the capacity of the 257ARG pro- OR of 0.51 (95 percent CI, 0.32–0.81), which reflected the tein to activate NNK (Su et al. 2000; Zhang et al. 2002). In inverse interaction with smoking (Wrensch et al. 2005). a study of 724 lung cancer patients and 791 control par- Reports from Finland, Norway, and Sweden show a lack of ticipants, Wang and colleagues (2003a) demonstrated that association between either of the CYP1A1 polymorphisms the variant CYP2A13 genotype (*C/*T or *T/*T) was asso- and lung cancer risk (Tefre et al. 1991; Hirvonen et al. ciated with a reduced risk for lung cancer, particularly for 1992; Alexandrie et al. 2004). A meta-analysis provides adenocarcinomas (OR = 0.41; 95 percent CI, 0.23–0.71). little support for this association (Houlston 2000). The reduction in risk did not reach statistical significance Because of the small sample sizes in these studies, for SCC or other histologies of lung cancer. The reduced Vineis and colleagues (2003) conducted an analysis of risk for adenocarcinomas was apparent only in smokers, pooled data from the International Collaborative Study and in light smokers rather than in heavy smokers (Wang on Genetic Susceptibility to Environmental Carcinogens, et al. 2003a). This finding again indicates that genetic which included raw data from 22 case-control studies polymorphisms may play a greater role when the carcino- totaling 2,451 cases and 3,358 controls. This data set thus gen dose is low and does not saturate enzymatic capacity.

Cancer 247 Surgeon General’s Report

GSTM1 Gene GSTM1 null genotype who consumed cruciferous vegetables (London et al. 2000; Spitz et al. 2000). One hypoth- Study reports have noted large variations in enzy- esis is that these participants were less able to eliminate matic activity for several GSTs. About 50 percent of the protective isothiocyanates by conjugation with glutathi- White population is homozygous for a deletion in the one. In a case-control study, Cheng and colleagues (1999) GSTM1 gene that leads to null expression (Seidegard et analyzed data from 162 patients with SCC of the head and al. 1988). The GSTM1 enzyme is important in detoxify- neck and 315 healthy control participants. They found ing carcinogens, and numerous studies have investigated that 53.1 percent of the case patients and 42.9 percent of the possible association of the GSTM1 null genotype with the control participants were null for GSTM1 (p <0.05), lung cancer risk. whereas 32.7 percent of case patients and 17.5 percent of Some studies have found an association between control participants were null for GSTT1 (p <0.001). the GSTM1 null mutation and lung cancer across many Thus, the effect of GSTM1 alone may not be dra- populations. In a Japanese population, the GSTM1 null matic. However, it appears to be magnified by gene- genotype was positively correlated with SCC of the lung environment and gene-diet interactions, and the effects but not with adenocarcinomas (Kihara et al. 1993). A sim- were significantly greater as exposure to cigarette smoke ilar analysis in a Finnish population also correlated the increased. In addition, the high frequency of GSTM1 poly- GSTM1 null genotype with SCC (Hirvonen et al. 1993). morphisms observed across all ethnicities may contribute Analyses of Scottish (Zhong et al. 1991), Norwegian to the importance of this variant as a risk factor for devel- (Ryberg et al. 1997), and Turkish populations (Pinarbasi et oping lung cancer (Brennan et al. 2005). al. 2003) had similar findings. A U.S. study also suggested that the GSTM1 null genotype was associated with a mod- CYP1A1 and GSTM1 in Combination est elevation in lung cancer risk, which increased among heavy smokers (Nazar-Stewart et al. 2003). However, some Studies of the effect of combined CYP1A1 and GSTM1 studies have not shown a significant association between variant genotypes hypothesized that increased PAH activa- the GSTM1 null genotype and lung cancer risk for SCC tion and decreased PAH detoxification in tobacco smokers or overall for lung cancer (London et al. 1995; Rebbeck might lead to an increase in lung cancer risk. Numerous 1997). A meta-analysis of data from 12 case-control stud- studies have explored this association. Perhaps the studies comprising 1,593 cases and 2,135 controls showed a ies with the strongest support for this association come moderate increase in the risk of lung cancer across all from Japan, although they are generally limited by small histologies with the GSTM1 null genotype (OR = 1.41; 95 sample sizes. percent CI, 1.23–1.61) (McWilliams et al. 1995). A more Combination of the CYP1A1 variant genotype and recent meta-analysis of 43 studies including more than the GSTM1 null genotype enhanced the risk of smoking- 18,000 persons showed a smaller but statistically signifi- related lung cancers in a Japanese population. Hayashi cant OR of 1.17 (95 percent CI, 1.07–1.27) (Benhamou et and colleagues (1992) demonstrated this finding with the al. 2002). *ILE/*VAL polymorphism. These investigators found an Kihara and colleagues (1994) analyzed data on 178 increased frequency of the homozygous *VAL/*VAL gen- Japanese patients with lung cancer and 201 healthy con- otype combined with the GSTM1 null genotype in lung trol participants and found that the GSTM1 null genotype cancer patients compared with control participants (8.5 was associated with an overall increase in lung cancer risk percent versus 2.2 percent, respectively). Nakachi and (OR = 1.87; 95 percent CI, 1.21–2.87). The strongest asso- colleagues (1991) reported similar results with both the ciation was for SCC (OR = 2.13; 95 percent CI, 1.11–4.07). *MSPI and *ILE/*VAL polymorphisms and the GSTM1 With stratification by the amount of smoking, the propor- null genotype. The case-control study found that for light tion of GSTM1 null genotype increased progressively in smokers, either of the two CYP1A1 susceptible genotypes the SCC group from 50 percent in light smokers to 72 combined synergistically with the deficient GSTM1 geno- percent in heavy smokers (Kihara et al. 1994). One study type to create a high risk for lung cancer (OR = 16; 95 suggested that higher intakes of cruciferous vegetables percent CI, 3.76–68.02 for *MSPI, and OR = 41; 95 percent reduced lung cancer risk among persons with the GSTM1 CI, 8.68–193.61 for *ILE/*VAL). Eighty-seven percent genotype (highest versus lowest tertile for amount of of the light smokers who developed lung cancer had at smoking; OR = 0.61; 95 percent CI, 0.39–0.95) but not least one of the three homozygous variant genotypes. The among persons with the GSTM1 null genotype (highest investigators suggested that particularly when the ciga- versus lowest tertile; OR = 1.15; 95 percent CI, 0.78–1.68) rette dose is low, CYP1A1 and GSTM1 may be an impor- (Wang et al. 2004b). However, several other studies have tant determinant of susceptibility to lung cancer (Nakachi shown a greater protective effect in persons with the et al. 1991).

248 Chapter 5 How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease

Kihara and colleagues (1994) also demonstrated a were 2.9 times more likely to develop lung cancer than synergistic effect. Persons with these variant genotypes were African Americans without a *G allele (95 percent in both CYP1A1 and GSTM1 had a much higher risk of CI, 1.29–6.20). African Americans with either one or two lung cancer than did those with the variant CYP1A1 and genotypes that carry risk at the GSTM1 and GSTP1 loci wild-type GSTM1 (OR = 21.9; 95 percent CI, 4.68–112.7 were at higher risk of developing lung cancer than were versus OR = 3.2; 95 percent CI, 0.37–24.0). Studies in African Americans who had fully functional GSTM1 and Scandinavian populations (Alexandrie et al. 1994; Anttila GSTP1 genes (OR = 2.8; 95 percent CI, 1.1–7.2 for GSTM1, et al. 1994), as well as U.S. populations (García-Closas et and OR = 4.0; 95 percent CI, 1.3–12.2 for GSTP1). No sig- al. 1997), support an increase in the risk of lung cancer nificant single-gene associations were observed between with the combination of variant CYP1A1 and GSTM1 gen- GSTM1, GSTT1, or GSTP1 and early-onset lung cancer in otypes. Using data from the International Collaborative Whites (Cote et al. 2005). Study on Genetic Susceptibility to Environmental Car- cinogens database, Vineis and colleagues (2004) found a GSTT1 Gene statistically significant effect of the*MSPI variant on lung Previous results have not supported an association cancer risk in Whites (OR = 2.6; 95 percent CI, 1.2–5.7) of the GSTT1 gene with lung cancer risk (To-Figueras et with evidence for an interaction between the MSPI and al. 1997; Malats et al. 2000; Stücker et al. 2002; Ruano- GSTM1 null genotypes (OR = 2.8; 95 percent CI, 0.9–8.4). Ravina et al. 2003; Wang et al. 2003b). In a study of Chi- nese living in Hong Kong, the GSTT1 null genotype was GSTP1 Gene associated with a higher risk of lung cancer than was the Studies have reported polymorphisms in the GSTP1 functional GSTT1 genotype (AOR = 1.69; 95 percent CI, gene family of phase II enzymes with high expression in 1.12–2.56) only in nonsmokers (Chan-Yeung et al. 2004). the lung. One GSTP1 polymorphism includes an A→G A study from Denmark also suggested that the GSTT1 null base change that leads to an isoleucine→valine substi- genotype is associated with a higher risk of lung cancer tution, which results in lower enzymatic activity toward (Sørensen et al. 2004a). 1-chloro-2,4-dinitrobenzene (Watson et al. 1998) but higher activity toward PAH diol epoxides (Sundberg et al. NAT2 Gene 1998). Several studies showed no statistically significant Several widely studied polymorphisms for the NAT2 association between GSTP1 polymorphisms and lung can- gene are associated with decreased activity or reduced cer risk (Harris et al. 1998; Katoh et al. 1999; To-Figueras stability of the enzyme. Phenotypically, these polymor- et al. 1999; Nazar-Stewart et al. 2003). However, in the phisms result in slow or fast acetylation. Study results on study with the largest sample size of 1,042 cases and 1,161 the association of the NAT2 gene with lung cancer risk controls, the GSTP1 homozygous variant genotype was are conflicting. Most studies report no overall increase associated with a higher lung cancer risk at any level of in risk with the genotype for either slow or fast acetyla- exposure to smoke than was the wild-type genotype (Miller tion (Philip et al. 1988; Martinez et al. 1995; Bouchardy et al. 2003). et al. 1998; Saarikoski et al. 2000). However, a few studies The combination of the GSTM1 null genotype and report an increase in risk with the genotype for either slow the GSTP1 *G/*G genotype may increase lung cancer acetylation (Oyama et al. 1997; Seow et al. 1999) or fast risk (Ryberg et al. 1997; Kihara et al. 1999; Perera et al. acetylation (Cascorbi et al. 1996). In the largest study, of 2002). In a study of 1,694 cases and 1,694 controls, double 1,115 lung cancer patients and 1,250 control participants, variants in GSTM1 and GSTP1, as well as in GSTP1 and no association between the NAT2 genotype and lung can- TP53, were associated with an increase in lung cancer cer risk was observed. However, the study noted a signifi- risk among persons aged 55 years or younger (adjusted cant interaction with smoking. Among nonsmokers, the OR [AOR] = 4.03; 95 percent CI, 1.47–11.1 for the M1- genotype for rapid acetylation decreased lung cancer risk P1 double variant, and AOR = 5.10; 95 percent CI, 1.42– more than did the genotype for slow acetylation. This 18.30 for the P1-P53 double variant) (Miller et al. 2002). relationship was reversed among smokers, and persons with Another study included 350 persons younger than age 50 the genotype for rapid acetylation had a higher risk. The years with a diagnosis of lung cancer who were identified authors hypothesized that for nonsmokers, the NAT2 pro- from the metropolitan Detroit Surveillance, Epidemiol- tein may provide a means for N-acetylation, thereby detoxi- ogy, and End Results program. The study compared these fying aromatic amines and protecting a person against patients with 410 control participants matched by age, cancer. However, cigarette smoke markedly induces CYP race, and gender. The results indicated that African Amer- oxidation and could increase the production of reactive icans carrying at least one *G allele at the GSTP1 locus

Cancer 249 Surgeon General’s Report intermediate agents in smokers. In this setting, NAT2 may also suggested that the NAT2 genotype for slow acetyla- instead O-acetylate these metabolites and thereby produce tion was associated with an increased risk of bladder can- more reactive metabolites, thus augmenting the cancer cer, especially with the joint effects of cigarette smoking risk (Zhou et al. 2002b). A study from Denmark confirmed and occupational exposure to aromatic amines (Hung et the associations of the NAT2 gene with smoking status al. 2004; Gu et al. 2005). In a case-control study of blad- (Sørensen et al. 2005). However, a study from Taiwan der cancer in females, exclusive use of permanent hair suggested that the NAT2 genotype for fast acetylation is dye was associated with a 2.9-fold increased risk of blad- associated with an increased risk of lung cancer among der cancer among persons with the NAT2 genotype and women who were lifetime nonsmokers (Chiou et al. 2005). slow acetylation but not in those with the NAT2 genotype The NAT2 protein plays an important role in the bio- and rapid acetylation (Gago-Dominguez et al. 2003). All activation and detoxification of the aromatic amines asso- of these results confirmed that the genotype for NAT2 ciated with bladder cancer induced by cigarette smoke. In slow acetylation is a risk factor for bladder cancer through the phenotypic studies, persons with slow acetylation had interaction with smoking or occupational exposure. How- increased risk of bladder cancer, particularly when they ever, several studies that included populations of Chinese had occupational exposure to arylamines or were cigarette (Ma et al. 2004), northern Indians (Mittal et al. 2004), and smokers (Green et al. 2000; Johns and Houlston 2000). In Poles (Jaskula-Sztul et al. 2001) reported no association the genotype analysis, more than 20 independent studies, between the NAT2 genotype and bladder cancer risk. many with small sample size have assessed the association of NAT2 polymorphisms with the risk of bladder cancer. A Microsomal Epoxide Hydrolase meta-analysis of published case-control studies conducted Like NAT2, MEH can act as both an activator and a in the general population (22 studies, 2,496 cases, and detoxifier of carcinogens. As a detoxifier, MEH catalyzes the 3,340 controls) examined the relationship of acetylation hydrolysis of highly reactive epoxide intermediate agents status (phenotype and genotype) to bladder cancer risk. to less reactive dihydrodiols that are excretable. As an Persons with slow acetylation had a 40-percent increase in activator, MEH is involved in further metabolism of PAH risk compared with risk for persons with rapid acetylation epoxides. Several identified polymorphisms include a T→C (OR = 1.4; 95 percent CI, 1.2–1.6) (Marcus et al. 2000b). base change in exon 3 leading to a tyrosine→histidine However, studies conducted in Asia generated a summary substitution at residue 113, which is associated with a OR of 2.1 (95 percent CI, 1.2–3.8), studies in Europe gen- decrease in enzymatic activity, and an A→G base change erated a summary OR of 1.4 (95 percent CI, 1.2–1.6), and in exon 4, leading to a histidine→arginine substitution studies in the United States generated a summary OR of at residue 139, which leads to an increase in enzymatic 0.9 (95 percent CI, 0.7–1.3). activity (Hassett et al. 1994). Several reports of studies In addition, a case series meta-analysis of data from have noted an increased risk of lung cancer among persons a case series of 16 studies of bladder cancer, conducted in carrying polymorphisms associated with an increase in the general population and involving 1,999 cases, showed enzymatic activity. A study of Mexican Americans and Afri- a weak interaction between smoking status and NAT2 slow can Americans found a greater risk of lung cancer among acetylation (OR = 1.3; 95 percent CI, 1.0–1.6). The interac- young Mexican Americans with the exon 4 polymorphism, tion was stronger when analyses were restricted to studies but not among those with the exon 3 polymorphism. No conducted in Europe (OR = 1.5; 95 percent CI, 1.1–1.9) association was observed among African Americans (Wu (Marcus et al. 2000a). In a pooled analysis of data from et al. 2001). The homozygous variant genotype at exon 4 1,530 cases and 731 controls from four case-control stud- confers increased enzymatic activity and was again asso- ies plus two case series conducted in Whites in European ciated with an increase in lung cancer risk in a study in countries, a significant association was reported between Texas (Cajas-Salazar et al. 2003). A study from Austria sug- NAT2 slow acetylation and bladder cancer (OR = 1.42; 95 gested an association between the exon 3 polymorphism percent CI, 1.14–1.77) (Vineis et al. 2001). The risk of can- of the MEH gene and a significantly decreased risk of lung cer was elevated in smokers and in persons with occupa- cancer (Gsur et al. 2003). The combination of exon 3 and tional exposure to cigarette smoke, and the highest risk exon 4 polymorphisms that conferred high enzymatic was for persons with slow acetylation (Vineis et al. 2001). activity also significantly increased the risk (Cajas-Salazar In a hospital-based, case-control study of 201 men et al. 2003; Park et al. 2005). In a Chinese population in in northern Italy and a case-control study with 507 White Taiwan, high MEH activity, defined by the corresponding patients with bladder cancer in the United States, findings

250 Chapter 5 How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease combination of exon 3 and exon 4 polymorphisms, was enzyme transport into mitochondria. The *VAL/*VAL gen- associated with an increased risk for SCC (Lin et al. 2000). otype is associated with risk of lung cancer higher than In a French population, high MEH activity was similarly that for the wild-type genotype (AOR = 1.67; 95 percent associated with lung cancer risk (Benhamou et al. 1998). CI, 1.27–2.20) (Wang et al. 2001a). Other studies also asso- A study of 974 White patients with lung cancer and ciate the heterozygous variant genotype with an increased 1,142 control participants found no relationship between risk of lung cancer (AOR = 1.34; 95 percent CI, 1.05–1.70) MEH polymorphisms and lung cancer risk overall. How- (Wang et al. 2001a, 2004c). Studies have identified a →G A ever, evidence of gene-environment interactions was polymorphism in the promoter region of myeloperoxidase observed. Low-activity MEH genotypes were a risk factor (MPO) that decreases *A allele transcription. A study of for lung cancer among nonsmokers (OR = 1.89; 95 percent bronchoalveolar lavage fluid and cells from 106 White CI, 1.08–3.28) but were protective among heavy smokers smokers who had lung cancer showed an association of (OR = 0.65; 95 percent CI, 0.42–1.00) (Zhou et al. 2001b). the variant genotypes with reduced MPO activity in the This effect was stronger in SCC than in adenocarcinoma. fluid and reduced levels of smoking-related DNA adducts The researchers hypothesized that this difference may be in bronchoalveolar cells (Van Schooten et al. 2004). The explained by the dual actions of MEH. In nonsmokers, association was stronger in persons having two variant the presence of low MEH activity may lead to a decreased alleles (homozygous variants) than it was in persons ability to detoxify environmental pollutants, thus increas- having one normal and one variant allele (heterozyg- ing lung cancer risk. In smokers, MEH may participate in ous variants). activating the PAHs in cigarette smoke. Therefore, low Findings on lung cancer risk are conflicting. Most activity is protective for heavy smokers. Similar results studies performed since 1999 suggested that the variant were reported in a Slovak study (Habalová et al. 2004). MPO genotypes are associated with a decreased risk of In a meta-analysis of data from seven published lung cancer (Le Marchand et al. 2000; Dally et al. 2002; studies that included 2,078 case patients with lung can- Feyler et al. 2002; Kantarci et al. 2002; Lu et al. 2002; cer and 3,081 control participants, investigators found Schabath et al. 2002). In contrast, two studies found no no consistent overall association for either the exon 3 or association between either the heterozygous or homo- exon 4 polymorphisms with lung cancer risk (Lee et al. zygous variant genotypes and lung cancer risk (Xu et al. 2002c). However, in an analysis of pooled data from eight 2002; Chevrier et al. 2003). Another study suggested that studies (four published and four unpublished at that time) the MPO-G463A polymorphism associated with a novel with 986 case patients and 1,633 control participants, estrogen-receptor-binding site modifies the association researchers observed a significant decrease in lung cancer between the SOD ALA16VAL polymorphism and risk of risk (OR = 0.70; 95 percent CI, 0.51–0.96) for the exon 3 non-small-cell lung cancer (NSCLC) differently by gender. *HIS/*HIS genotype. The protective effect of the exon 3 For women carrying MPO variant genotypes, the AOR of polymorphism seems stronger for adenocarcinomas of the the SOD polymorphism (*VAL/*VAL versus *ALA/*ALA) lung than for other histologic types. Researchers found no was 3.26 (95 percent CI, 1.55–6.83). No associations were overall association between MEH activity and lung cancer found in men or women who carried the MPO *G/*G wild- risk and no consistent modification of the carcinogenic type genotype (Liu et al. 2004). effect of smoking according to the MEH polymorphism. However, the risk of lung cancer decreased among lifetime Summary nonsmokers with high MEH activity and among heavy Studies to date suggest a role for genetic polymor- smokers with the exon 3 *HIS/*HIS genotype (Lee et phisms in the risk of lung and bladder cancer in smokers al. 2002c). and support a possible association between specific genes and smoking status. Investigations continue on the role Genes in the Pathway for Metabolism of multiple genetic variants that occur simultaneously of Reactive Oxygen Species and the interactions between metabolic gene variants and Studies have identified an alanine→valine substi- other kinds of heritable variations, such as DNA repair, tution at codon 16 of manganese superoxide dismutase cell-cycle control, tumor-suppressor genes, and onco- (SOD), which may be associated with a less efficient gene activity.

Cancer 251 Surgeon General’s Report

DNA Repair and Conversion of Adducts to Mutations

Repair of DNA Adducts and Pegg 2002). Therefore, the steric constraints of an active AGT site determine whether it can efficiently repair 6 Introduction a bulky O -alkylguanine adduct such as those more commonly resulting from exposure to smoke. Tobacco products and smoke contain many chemicals that can damage DNA. Multiple repair pathways Protecting Against Mutagenicity of Tobacco protect a human cell against the mutagenic and carcino- Carcinogens genic activities of these DNA-damaging agents. Pathways involved in the repair of tobacco-related DNA damage Tobacco smoke contains a number of alkylating include direct base repair by alkyltransferases, excision of agents, such as tobacco-specific nitrosamines, which are 6 DNA damage by base excision repair (BER), or nucleotide capable of forming O -alkylguanine adducts (Wang et al. excision repair (NER), mismatch repair (MMR), and dou- 1997; Hecht 1998). AGT protects against the mutagenic ble-strand break repair (DSBR). The inadequate removal and carcinogenic properties of alkylating agents. of DNA damage results in increased rates of mutagenesis In vitro studies. Increased expression of AGT pro- 6 and, as a consequence, the increased likelihood of a person tects against the mutagenic effects of O -alkylguanine developing cancer. (Ellison et al. 1989) and alkylating agents (Kaina et al. 1991; Wu et al. 1992; Ferrezuelo et al. 1998a,b). Consis- O6-Alkylguanine–DNA Alkyltransferase tently, alkylating agents are more toxic and mutagenic when coadministered with AGT inactivators such as 6 Overview O -benzylguanine or related compounds (Dolan et al. 1990, 1991; Bronstein et al. 1992). The mutagenic activ- 6 O -alkylguanine adducts are repaired by the repair ity of O6-methylguanine or O6-[4-oxo-4-(3-pyridyl)bu- 6 protein O -alkylguanine–DNA alkyltransferase (AGT) in tyl]guanine is enhanced when cells are pretreated with a reaction involving transfer of the methyl group from O6-benzylguanine (Pauly et al. 1995, 2002). This finding 6 the O position of guanine to a cysteinyl residue on the indicated that AGT is important in protecting against protein (Pegg 2000). This transfer reaction results in an the mutagenic activity of these adducts derived from error-proof repair as it regenerates an unmodified gua- tobacco constituents. nine residue in DNA. However, the repair protein is inacti- In vivo studies. AGT is depleted in tissues from vated as the alkylated protein undergoes a conformational NNK-treated rats, and AGT levels are depleted in Clara change (Daniels et al. 2000) and is degraded (Srivenugo- cells (Belinsky et al. 1988). NNK also reduces AGT levels pal et al. 1996; Xu-Welliver and Pegg 2002). As a conse- in the lungs and liver of A/J mice (Peterson et al. 2001). quence of this repair mechanism, the constitutive levels Although the liver function recovers to control values of AGT determine the initial repair capacity of a cell by within 96 hours after exposure, AGT activity remains 6 this mechanism. Overall capacity of the O -alkylguanine depressed in the lung. Consistently, O6-methylguanine is repair is determined by the rate of protein synthesis and efficiently repaired in the liver of NNK-treated mice, but 6 the amount of alkylation at the O position of guanine. it persists for at least two weeks in lung DNA (Peterson and Hecht 1991; Peterson et al. 2001). Notably, levels of Substrate Specificity O6-methylguanine in lung DNA are highly correlated with Mammalian AGT specifically repairs O6-alkylgua- pulmonary tumorigenic activity in A/J mice (Peterson nine adducts, and it repairs the larger O6-alkylguanine and Hecht 1991; Peterson et al. 2001). These observations 6 residues more readily than does the bacterial protein. In strongly suggest that the inefficient repair of O -meth- rodents, AGT repairs O6-methylguanine, O6-butylguanine, ylguanine, presumably by AGT, is linked to the tumori- and O6-[4-oxo-4-(3‑pyridyl)butyl]guanine at comparable genic activity of NNK. This conclusion is supported by the rates, whereas human AGT repairs the bulky adducts more observation from another study that high AGT levels pro- slowly (Mijal et al. 2004). This ability of the rodent protein tect against NNK-induced lung tumorigenesis and that to accommodate such large structural differences likely NNK is a less potent lung carcinogen in transgenic mice results from the additional amino acid residue (Gly166) containing the human AGT transgene (Liu et al. 1999). in the binding pocket of the rodent proteins (Loktionova

252 Chapter 5 How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease

Base Excision Repair but sometimes overlapping substrate specificities that are subgrouped into type I and type II glycosylases, depending Overview on their mode of action (Christmann et al. 2003). Type I glycosylases catalyze the cleavage of the N-glycosidic BER is a major pathway for the repair of small DNA bond, leaving an AP site. Type II glycosylases remove damage, primarily to alkylated and oxidized DNA bases, as the damaged base in a similar manner. They contain 3’- well as the repair of apurinic/apyrimidinic (AP) sites and endonuclease activity that cleaves the AP site, which gen- single-stranded breaks (Fortini et al. 2003; Fromme et al. erates a single-strand break with a 3’-terminal deoxyribose 2004). BER is initiated by a recognition of the damaged phosphate. Spontaneous hydrolysis of a glycosidic bond DNA by specific DNA glycosylases. Studies have charac- can also directly generate AP sites. AP sites are substrates terized 12 human glycosylases (Table 5.4) (Christmann et for DNA AP endonuclease, which cuts the phosphodiester al. 2003; Fortini et al. 2003). Each enzyme has different

Table 5.4 Human DNA glycosylases

Study Glycosylase Specificity Subgroup

Chakravarti et al. 1991 Alkylpurine DNA glycosylase 3-methyladenine, 7-methylguanine, Type I O’Connor and Laval 1991 or methylpurine DNA 3-methylguanine, ethenoadenine, Samson et al. 1991 glycosylase hypoxanthine

Hendrich and Bird 1998 Methyl-CpG binding endonuclease 1 U or T opposite G, preferentially in Type I Hendrich et al. 1999 CpG sites

Slupska et al. 1996, 1999 Adenine DNA glycosylase A opposite 8-oxoguanine Type I Fortini et al. 2003

Hazra et al. 2002a,b Nei-like DNA glycosylase 1 Formamidopyrimidines, oxidized Type II pyrimidines, 8-oxoguanine opposite C, G, or T

Hazra et al. 2002a,b Nei-like DNA glycosylase 2 5-hydroxyuracil, 5-hydroxycytosine Type II

Takao et al. 2002 Nei-like DNA glycosylase 3 Fragmented and oxidized pyrimidines NR

Aspinwall et al. 1997 Thymine glycol DNA glycosylase 1 Ring-saturated, oxidized, and Type II Hilbert et al. 1997 fragmented pyrimidines Miyabe et al. 2002 Fortini et al. 2003

Bjørås et al. 1997 8-oxoguanine DNA glycosylase 1 8-oxoguanine opposite C, T, or G Type II Radicella et al. 1997 Rosenquist et al. 1997 Fortini et al. 2003

Hazra et al. 1998 8-oxoguanine DNA glycosylase 2 8-oxoguanine opposite A or G NR

Haushalter et al. 1999 Mismatch-specific uracil DNA Uracil opposite G Type I Nilsen et al. 2001 glycosylase 1

Neddermann and Jiricny Thymidine DNA glycosylase Uracil, T, or ethenoC opposite G; Type I 1993, 1994 T opposite G, C, or T Neddermann et al. 1996

Olsen et al. 1989 Uracil DNA glycosylase Uracil Type I Muller and Caradonna 1991 Fortini et al. 2003 Source: Adapted from Christmann et al. 2003 with permission from Elsevier, © 2003. Note: NR = data not reported.

Cancer 253 Surgeon General’s Report

bond and causes the formation of a strand break with a of the various glycosylases and repair pathways. However, 5’-terminal deoxyribose phosphate (Barzilay et al. 1995). mutagenicity of methyl methanesulfonate (MMS) in lym- Once the phosphodiester bond is cleaved, BER can phocytes from alkylpurine-DNA-N-glycosylase–null mice proceed through two pathways: short-patch or long-patch was three to four times higher than that in lymphocytes (Figure 5.4). The balance of the two pathways can depend from wild-type control mice (Elder et al. 1998). Most of on tissue type (Sancar et al. 2004). In general, short-patch the mutations were AT→TA transversions. In addition, BER dominates when BER is initiated by glycosylases. 8-oxoguanine DNA glycosylase 1 (OGG1) knockout mice Long-patch BER is the preferred pathway when BER is that are aging eventually develop lung cancer (Sakumi initiated with the formation of AP sites through spontane- et al. 2003). However, in another study, knockout of pro- ous hydrolysis or oxidative base loss (Sancar et al. 2004). teins involved in the steps after removal of the base caused In short-patch BER, DNA polymerase β (polβ) the knockout mice to die at a very young age (Fortini et inserts a new nucleotide at the lesion site and catalyzes al. 2003). the release of 5’-terminal deoxyribose phosphates by Imbalances in the proteins involved in this pathway β-elimination (Matsumoto and Kim 1995; Sobol et al. could have negative consequences. Chinese hamster ovary 1996; Prasad et al. 1998). This step is followed by ligation cells that overexpress alkylpurine DNA glycosylase are of the remaining break by the ligase III x-ray repair cross- more sensitive to both the toxic and mutagenic effects of complementation group 1 (XRCC1) complex (Kubota et MMS and have higher numbers of mutations at AT base al. 1996). pairs than do normal Chinese hamster ovary cells (Cal- Long-patch BER occurs in oxidized or reduced AP léja et al. 1999). This result suggests that an enhanced sites, 3’-unsaturated aldehydes, or 3’-phosphates, because repair of 3-methyladenine leads to an accumulation of these modifications are resistant toβ -elimination by polβ. unprocessed AP sites that are also mutagenic. Findings Therefore, this damage is further processed by long-patch in another study indicate that imbalances and/or poly- repair dependent on the proliferating cell nuclear antigen morphisms in the proteins involved in BER may cause an (PCNA) after the insertion of a nucleotide at the lesion increase in cancer susceptibility (Fortini et al. 2003). site by polβ (Christmann et al. 2003; Fortini et al. 2003). This mechanism displaces the damaged strand, which is Nucleotide Excision Repair followed by DNA synthesis of an oligonucleotide (up to 10 nucleotides) by pold or pole in concert with PCNA and Overview replication factor C (Stucki et al. 1998). The flap endo- NER repairs a wide class of helix-distorting lesions nuclease 1 (FEN1) recognizes and cleaves off the damaged that interfere with base pairing, blocking transcription oligonucleotide flap structure (Klungland and Lindahl and normal replication (Petit and Sancar 1999; Sancar et 1997). Ligase I catalyzes the final ligation step (Prasad et al. 2004). NER is the primary repair mechanism for bulky al. 1996; Srivastava et al. 1998). DNA damage caused by chemicals or ultraviolet (UV) radiation or as a result of protein-DNA cross-links (Sancar Substrate Specificity et al. 2004). Two NER pathways exist (Figure 5.5): global Tobacco smoke is rich in reactive oxygen species genomic NER (GGR), which surveys the whole genome that can oxidize DNA bases. BER is an important pathway for DNA damage, and transcription-coupled repair (TCR), for the repair of oxidized DNA bases, such as 8-oxogua- which primarily repairs damage that interferes with tran- nine and oxidized pyrimidines, and for the repair of single- scription. Both pathways involve recognition of DNA dam- strand breaks. Tobacco smoke contains N-nitrosamines age and excision of the damaged DNA, followed by the capable of generating small alkylguanine damage that is synthesis and ligation of new DNA. repaired by this pathway. The small chemical alterations Global genomic nucleotide excision repair. frequently miscode if they are not repaired by BER. There- Researchers think that GGR is largely transcription inde- fore, this pathway is particularly important in preventing pendent, occurring throughout the genome because no mutagenesis. gene or strand preference for this repair pathway exists (Hanawalt et al. 2003). Proteins involved in this pathway Protecting Against Mutagenicity and are presented in Table 5.5. The initial step involves recog- Carcinogenicity of Tobacco Carcinogens nition of DNA damage (Figure 5.5). The complex of repair factors in the xeroderma pigmentosum group C (XPC) Single-gene knockouts of glycosylases in mice and the homologous recombinational repair group 23B are well tolerated, with only modest increases in rates (HR23B) can directly recognize some DNA damage (Hey of spontaneous mutagenesis (Fortini et al. 2003). This et al. 2002). However, in some cases, DNA-binding pro- observation likely results from an overlapping specificity

254 Chapter 5 How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease

Figure 5.4 Mechanism of base excision repair

5’ 3’ 3’ 5’

Glycosylase

Formation of apurinic site

or AP- endonuclease Glycosylase AP- lyase activity AP- lyase activity

OH P 5’dRP Polymerase Strand displacement activity pol ß Synthesis of ﬁrst nucleotide

5’dRP in P 5’dRP 5’dRP in hemiacetal form 3’α,β nonsaturated aldehyde form

 P Polymerase pol ß P dRPase activity RF-C FEN1 pol δ/ε PCNA activity

PCNA  Lig III FEN1 5’dRP ﬂap XRCC1 dRPase activity P

Short-patch repair PCNA Lig I

Long-patch repair

Source: Adapted from Christmann et al. 2003 with permission from Elsevier, © 2003. Note: 5’dRP = 5’-deoxyribose phosphate; AP = apurinic/apyrimidinic; dRPase = DNA deoxyribophosphodiesterase; FEN1 = flap endonuclease 1;Lig = ligase; OH = hydroxide; P = phosphate; PCNA = proliferating cell nuclear antigen; pol = polymerase; RF-C = replication factor C; XRCC1 = x-ray repair cross-complementation group 1.

Cancer 255 Surgeon General’s Report

Figure 5.5 Mechanism of nucleotide excision repair: (A) global genomic repair; (B) transcription-coupled repair

A B Lesion Lesion 5’ 3’ 5’ 3’ 3’ 5’ 3’ 5’ XPE DDB1 DDB2 XPC -HR23B mRNA TFIIH RNAPII

TFIIH XPB XPD XPA -RPA RNAPII blocked CSB CSA

TFIIS TFIIH RNAPII XPG

Degradation or removal of RNAPII Incision XPG Incision ERCC1-XPF XPF CSB RNAPII

Excision Excision 27–29 nucleotides

RNAPII Polymerization pol δ , pol ε Ligation PCNA, DNA-Ligase I Polymerization Ligation Restart of transcription

TFIIH RNAPII

Source: Adapted from Christmann et al. 2003 with permission from Elsevier, © 2003. Note: CSA = Cockayne syndrome complementation group A; CSB = Cockayne syndrome complementation group B; DDB = DNA binding protein; ERCC1 = excision repair cross-complementation group 1; HR23B = homologous recombinational repair group 23B; mRNA = messenger RNA; PCNA = proliferating cell nuclear antigen; pol = polymerase; RNAPII = RNA polymerase II; RPA = replication protein A; TFIIH = transcription initiation factor IIH; TFIIS = transcription initiation factor IIS; XP = xeroderma pigmentosum (groups A–G).

256 Chapter 5 How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease

Table 5.5 Factors involved in nucleotide excision repair activity in humans

Factor Proteins Factor activity Role in repair

XPA XPA DNA binding Damage recognition

RPA P70 XPA binding Damage recognition P34 DNA binding NR P11 NR NR

TFIIH XPB DNA-dependent ATPase Formation of preincision complexes XPD Helicase NR P62 GTP NR P52 NR NR P44 CAK NR CDK7 NR NR CYCH NR NR P34 NR NR

XPC XPC DNA binding Molecular matchmaker HR23B NR Stabilization of preincision complex 1

XPG XPG NR 3’ incision

XPF XPF NR 5’ incision ERCC1 NR NR

Source: Adapted from Petit and Sancar 1999 with permission from Elsevier, © 1999. Note: ATPase = adenosine triphosphatase; CAK = cyclin-dependent kinase-activating kinase; CDK7 = cyclin-dependent kinase 7; CYCH = cytochrome c-type biogenesis protein; ERCC1 = excision repair cross-complementation group 1; GTP = guanosine triphosphate; HR23B = homologous recombinational repair group 23; NR = data not reported; RPA = replication protein A; TFIIH = transcription initiation factor IIH; XP = xeroderma pigmentosum (groups A–G).

tein is required to enhance the DNA distortion before the associated proteins (Aboussekhra et al. 1995; Mu et al. recruitment of XPC-HR23B (Tan and Chu 2002; Hanawalt 1995; Araújo et al. 2000). Findings from in vivo studies et al. 2003). Repair factors XPA and replication protein suggest that the NER machinery is assembled in a step- A are then recruited to the complex to verify that the wise manner from the individual components at the lesion alteration in the DNA structure results from a lesion, as site. After the repair of a DNA lesion, the entire complex is opposed to a natural variation in DNA structure (Hanawalt believed by researchers to disassemble (Houtsmuller et al. et al. 2003). 1999; Hoeijmakers 2001). The DNA is then unwound at the site of the lesion. GGR appears to be inducible in humans (Hanawalt This step occurs on recruitment of the basal transcrip- et al. 2003). The proteins that recognize DNA damage in tion initiation factor IIH (TFIIH) multiprotein complex GGR are maintained at low levels under normal physio- (Christmann et al. 2003). This complex is likely involved logical conditions. However, as a result of genomic stress, in a further verification of damage and detection of the the efficiency of GGR is increased through the activation damaged strand. The helicase subunits of TFIIH, XPB, and of the P53 tumor-suppressor gene (Hanawalt et al. 2003). XPD then unwind the DNA around the lesion. Consequently, the levels of XPC and XPE increase. Repair Once the DNA has unwound, the lesion is excised of PAH adducts depends on the presence of the P53 pro- at defined sites on either side of the damage (Evans et al. tein (Hanawalt et al. 2003). These adducts are not repaired 1997). The 3’-incision is catalyzed by XPG (Habraken et al. in human fibroblasts that lack a functional P53 protein or 1994; O’Donovan et al. 1994), and the 5’-incision is cata- other gene product (Lloyd and Hanawalt 2000, 2002; Wani lyzed by the excision repair cross-complementation group et al. 2000). 1 (ERCC1) XPF complex (Sijbers et al. 1996). The result- Transcription-coupled repair. TCR occurs when ing DNA gap is filled in by the PCNA-dependent polymer- DNA damage blocks elongating RNA polymerases (Tor- ases: pold and pole (Aboussekhra et al. 1995; Araújo et al. naletti and Hanawalt 1999). The transcription-coupled, 2000). The final ligation is performed by DNA ligase I and repair-specific factors belong to two Cockayne syndrome

Cancer 257 Surgeon General’s Report

complementation groups (A and B) and are involved in Figure 5.6 Mechanism of mismatch repair the displacement of the stalled polymerase (Christmann et al. 2003). At this point, TFIIH is recruited to the lesion, 5’ G 3’ and the subsequent steps in TCR proceed in a manner 3’ T 5’ apparently similar to the steps for GGR. Because of the HMSH2 HMSH6 mechanism of DNA damage recognition, TCR is specific to a DNA strand. G SSB T Substrate Specificity HMLH1 HPMS2 The substrate specificity of NER ranges from small G to large distortions in DNA structure. The bulky DNA T damage generated by PAHs and aromatic amines in Translocation ATPase activity tobacco smoke is repaired primarily by NER (Friedberg along the DNA of MSH2 and/or MSH6 2001). The finding that human NER repairs 8-oxoguanine G in vitro more efficiently than thymine dimers or thymine T glycol (Reardon et al. 1997) suggests that this pathway may be important for the repair of this mutagenic adduct. Exonuclease I In addition, human NER repairs O6-methylguanine and N6-methyladenine, as well as A:G and G:G mismatches G (Huang et al. 1994). T Polymerase δ Protecting Against Mutagenicity and Ligase Carcinogenicity of Tobacco Carcinogens G C Consistent with the primary role of NER in the repair of bulky DNA damage from PAHs and aromatic Source: Adapted from Christmann et al. 2003 with permission amines, cells deficient in NER are more susceptible to the from Elsevier, © 2003. mutagenic and toxic effects of PAHs (Quan et al. 1994, Note: For two steps dependent on adenosine triphosphatase 1995; Lloyd and Hanawalt 2000, 2002; Wani et al. 2000). (ATPase), see text. C = cytosine; G = guanine; HMLH1 = human mutL homolog 1 protein; HMSH2 = human mutS homolog 2 Mismatch Repair protein; HMSH6 = human mutS homolog 6 protein; HPMS2 = human postmeiotic segregation increased 2 protein; Overview SSB = single-stranded DNA binding protein; T = thymine. MMR corrects replication errors (base-base or insertion-deletion mismatches) resulting from DNA polymerase errors. This repair pathway is also involved in synthesized DNA (Christmann et al. 2003). A second het- the repair of alkylation DNA damage, such as repair of erodimer (mutLα) composed of mutL homologs MLH1 O6-methylguanine (Duckett et al. 1996), cisplatin-derived and PMS2 then binds to the mutSα-DNA complex in an- 1,2-intrastrand cross-links (Duckett et al. 1996), and other ATP-dependent step (Nicolaides et al. 1994; Papado- adducts derived from B[a]P (Wu et al. 1999), 2-aminofluo- poulos et al. 1994; Li and Modrich 1995; Stojic et al. 2004). rene, and N-acetyl-2-aminofluorene (Li et al. 1996). This Exonuclease I then excises the DNA strand containing the pathway can also repair 8-oxoguanine (Colussi et al. 2002). mispaired base (Genschel et al. 2002), followed by the An overview of MMR is displayed in Figure 5.6. Rec- resynthesis of new DNA by pold (Longley et al. 1997). ognition of DNA damage occurs primarily by the mutSα MSH2 can also complex with MSH3 to form mutSβ complex (Christmann et al. 2003). This complex is com- (Acharya et al. 1996; Palombo et al. 1996). The substrate posed of the mutS homologous proteins MSH2 (Fishel specificity of this alternate complex is different from that et al. 1993; Leach et al. 1993) and MSH6 (Palombo et al. of mutSα (Christmann et al. 2003). The mutSα complex 1995). Once the heterodimer is bound to the mismatch, recognizes base-base mismatches, as well as insertion- it undergoes an adenosine-triphosphate (ATP)-dependent deletion mismatches (Umar et al. 1994), whereas mutSβ conformational change (Stojic et al. 2004). This com- binds only to insertion-deletion mismatches (Palombo et plex is involved in determining which strand is the newly al. 1996; Genschel et al. 1998).

258 Chapter 5 How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease

Protecting Against Mutagenicity and RAD51D, as well as XRCC2 and XRCC3 (Christmann et al. Carcinogenicity of Tobacco Carcinogens 2003). RAD51 is able to exchange the single strand with the same sequence from the sister chromatid DNA. This Persistent adducts that escape repair by AGT, BER, double-stranded copy is then used as a template to cor- or NER may be processed by MMR. Because MMR occurs rectly repair with DNA synthesis machinery. The resulting after DNA replication (Stojic et al. 2004), this is the last Holliday structures are subsequently resolved to generate opportunity for DNA damage repair before cell division. the repaired DNA (Constantinou et al. 2001). Consequently, a defective MMR results in an increase in mutagenesis (Schofield and Hsieh 2003). The overall Nonhomologous End-Joining effect of a defective MMR is the likelihood that cells with persistent DNA damage survive with a miscoded DNA. Nonhomologous end-joining merely links the ends This combination results in a higher cancer risk. of a DSB together in the absence of a template (Figure The role of MMR in the toxicity and mutagenicity 5.8). The break is initially recognized by a heterodimer of alkylating agents is well documented for methylating consisting of the proteins KU70 (Reeves and Sthoeger agents (Karran 2001; Stojic et al. 2004). Cells lacking 1989) and KU80 (Jeggo et al. 1992). This binding protects functional MMR are more resistant to the toxic effects of the DNA from digestion and associates it with the protein methylating agents (Koi et al. 1994; Risinger et al. 1995; kinase catalytic subunit DNA-PKcs, which is dependent on Umar et al. 1997; de Wind et al. 1999; Karran 2001; Sto- DNA. This complex, in turn, activates XRCC4-ligase IV, jic et al. 2004). However, these cells are sensitive to the which connects the broken DNA pieces together once the mutagenic effects of these compounds (Umar et al. 1997; MRE11-RAD50-NBS1 complex has processed the break Zhu et al. 1998). These effects are linked to the ineffective (Maser et al. 1997; Nelms et al. 1998). Researchers think MMR of O6-methylguanine. As a result, a defective MMR is that FEN1 and Artemis also play a role in the processing associated with increases in mutagenesis and carcinogen- of DSBs (Christmann et al. 2003). esis mediated by O6-methylguanine (Hickman and Sam- son 1999; Pauly and Moschel 2001). Similar effects were Protecting Against Mutagenicity and reported for ethylating agents (Claij et al. 2003). Carcinogenicity of Tobacco Carcinogens Scientists think that DSBs trigger large chromo- Double-Strand Break Repair somal aberrations such as chromosomal breaks and exchanges (Pfeiffer et al. 2000). Studies link an increase in Overview chromosomal aberrations to tobacco exposure (DeMarini Two pathways exist for DSBRs: homologous recom- 2004). These aberrations are more common in pulmonary bination and nonhomologous end-joining. Homologous lung tumors from smokers than in those from nonsmok- recombination occurs during DNA replication in the S ers (Sanchez-Cespedes et al. 2001). DSBs are also impor- and G2 phases, whereas nonhomologous end-joining tant in triggering cell death (Rich et al. 2000; Lips and occurs during G0 and G1 phases. Homologous recombina- Kaina 2001). tion uses the sister chromatid as the template for aligning the breaks in the proper orientation and is consequently error free (Hoeijmakers 2001). However, nonhomologous Molecular Epidemiology end-joining does not require sequence homology between the two breaks to ligate them and is therefore prone to of DNA Repair errors (Hoeijmakers 2001). Studies support the substantial interindividual vari- Homologous Recombination ations in DNA repair capacity (DRC). Researchers hypothesize that common variants in the genes that regulate The meiotic recombination 11 (MRE11)-RAD50- these protein expressions may modulate repair and influ- NBS1 protein complex initiates DSBR by catalyzing the ence susceptibility to tobacco carcinogenesis. Two com- degradation of the DNA in the 5’ to 3’ direction, generating plementary approaches to studying DNA repair as a risk 3’ single-stranded DNA (Figure 5.7). This single-stranded factor for tobacco carcinogenesis are applying functional DNA is protected from degradation by a heptameric ring assays and genotyping variants in gene pathways for DNA complex of RAD52 proteins (Stasiak et al. 2000). Replica- repair. Table 5.6 summarizes some relevant genes and tion factor A facilitates the assembly of a RAD51 nucleo- their variants in pathways involved in repairing tobacco- protein filament, which consists of RAD51B, RAD51C, and induced DNA damage.

Cancer 259 Surgeon General’s Report

Figure 5.7 Proposed mechanism of homologous recombination

5’ 3’ 5’ 3’ 3’ 5’ 3’ 5’ MRE11 RAD 50 Processing of DNA ends NBS 5’ - 3’ resection

RAD52

RAD51B / RAD51C / RAD51D XRCC2 / XRCC3 Strand exchange

RPA

RAD51

DNA synthesis

DNA ligation, branch migration, and resolution of Holliday junctions

Source: Adapted from Christmann et al. 2003 with permission from Elsevier, © 2003. Note: MRE11 = meiotic recombination 11; RAD = S. cerevisiae DNA damage recognition and repair protein; RPA = replication protein A; XRCC2/XRCC3 = x-ray repair cross-complementation groups 2 and 3.

260 Chapter 5 How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease

Functional Assays of DNA Damage and Figure 5.8 Proposed mechanism of nonhomologous Repair to Tobacco Carcinogens end-joining Berwick and Vineis (2000) extensively reviewed the types of functional assays. The review included assays that 5’ 3’ 5’ 3’ use a chemical or physical mutagen challenge such as 3’ 5’ 3’ 5’ mutagen sensitivity, single-cell microgel electrophoresis KU70 (comet assay), and assays of induced adducts and unsched- KU80 uled DNA synthesis. The review also included the host-cell DNA- PKcs reactivation (HCR) assay, which measures cellular ability to remove adducts from plasmids transfected into lymphocyte cultures in vitro by the expression of damaged FEN-1 reporter genes. MRE11 RAD 50 Artemis NBS Processing of DNA ends Host-Cell Reactivation Assay The HCR assay measures the expression level of damaged reporter genes, which are involved in reacti- XRCC4 vation of the host cell. The assay uses undamaged host lymphocytes, is fast, and objectively measures intrinsic XRCC4 cellular repair (Athas et al. 1991). In the assay, lympho- Ligase IV cytes are transfected with a damaged, nonreplicating recombinant plasmid harboring a chloramphenicol acetyltransferase (CAT) reporter gene (PCMVCAT). To study XRCC4 tobacco-related cancers, the mutagen challenge is B[a]P (Gelboin 1980). Experimental conditions produce at least one BPDE-DNA adduct per plasmid, completely blocking transcription of the PCMVCAT gene without inducing conformational changes in the DNA. This finding is important because conformational change of the plasmid Source: Adapted from Christmann et al. 2003 with permission could reduce the transfection rate. Because even a single from Elsevier, © 2003. unrepaired DNA adduct can effectively block PCMVCAT Note: FEN1 = flap endonuclease 1;MRE11 = meiotic recombination 11; PK = protein kinase catalytic subunit; transcription (Koch et al. 1993), any measurable PCMV- cs XRCC4 = x-ray repair cross-complementation group 4. CAT activity reflects the ability of the transfected cells to remove BPDE-induced adducts from the plasmids (Athas et al. 1991). Both lymphocytes and skin fibroblasts from patients Such an adaptation would be consistent with a baseline with basal cell carcinoma, but not with XP, were found DRC that can be mobilized on an increased demand for to have lower excision repair rates than those from per- repair (Eller et al. 1997; Cheng et al. 1998). This adapta- sons without cancer (Wei et al. 1993). Consequently, the tion of DRC to smoking, if it exists, appears to be long repair capacity of lymphocytes may reflect the overall term rather than transient, because the effect was still repair capacity of a person. Spitz and colleagues (2003) present in former smokers but was not stronger in those showed that case patients with lung cancer had a sig- who had smoked in the 24 hours before the blood sample nificantly lower DRC than did control participants and was drawn (Wei et al. 2000). The finding that long-term that case patients aged 63 years or younger and lifetime heavy smokers with lung cancer had an efficient DRC may nonsmokers had a lower DRC than that of matched con- also indicate that heavy exposure overwhelms even rela- trol participants. DRC appears to be highest among case tively resistant phenotypes. patients and control participants who were current smokers than among those who were former smokers and Luciferase Host-Cell Reactivation Assay lifetime nonsmokers. Heavy smokers among both case patients and control participants tended to have more pro- Researchers have modified the HCR assay by ficient DRC than did light smokers. This finding indicated replacing CAT with luciferase (LUC). The cell-extraction that cigarette smoking may stimulate DRC in response procedure is far more simplified for the LUC assay. A lumi- to the DNA damage caused by carcinogens in tobacco. nometer measures LUC optical density, and the laboratory

Cancer 261 Surgeon General’s Report

Table 5.6 Select candidate genes and polymorphisms implicated in repair of tobacco-induced DNA damage

Gene Nucleotide position Nucleotide change Amino acid change Allele frequency

Alkyltransferases

AGT, MGMT Ile143Val 0.05–0.2

Base excision repair

MBD4 13390 T/C Ser342Pro 0.05 13402 G/A Glu346Lys 0.07 TDG 27090 G/A Gly199Ser 0.09 MUTYH 18556 G/C Gly335His 0.44 OGG1 18069 C/G Ser326Cys 0.25 APEX1 11865 T/G Glu148Asp 0.25 XRCC1 32584 C/T Arg194Trp 0.08 33746 G/A Arg280His 0.05 34432 G/A Arg399Gln 0.31 ADPRT 75787 T/C Val761Ala 0.38 77525 C/T Pro882Leu 0.20 POLD1 24569 G/A Arg19His 0.12 27479 A/G His119Arg 0.16 27715 A/G Ser173Asn 0.06

Nucleotide excision repair

XPC 30197 C/T Ala499Val 0.24 42635 A/C Lys939Gln 0.38 XPD 16232 G/A Asp312Asn 0.40 28572 A/C Lys751Gln 0.33 XPF 34637 T/C Ser662Pro 0.06 RAD23B 48769 C/T Ala249Val 0.18 CCNH 23563 C/T Val270Ala 0.10 XPG 39583 G/C His1104Asp 0.32 LIG1 29008 A/C Ala170Ala 0.39 61130 C/T Asp802Asp 0.21 62525 C/G Ala814Ala 0.30

Double-strand break repair

Homologous recombination RAD51 692 (cDNA) G/T Ala143Ser 0.33 XRCC3 26044 T/C Thr241Met 0.22 RAD52 45137 C/A Ser346Ter 0.05 RAD54L 1222 (cDNA) G/C Arg374Ser 0.11 BRCA2 27113 T/G His372Asn 0.23 93268 G/A Ile3412Val 0.05 RAD50 3374 (cDNA) G/T Arg1111Ile 0.25 NBS1 137331 C/G Gln185Glu 0.46 Nonhomologous end-joining KU70 336 (cDNA) G/A Glu107Lys 0.06 454 (cDNA) T/C Val146Ala 0.11 1825 (cDNA) T/C Leu603Pro 0.10 KU80 1487 (cDNA) C/T Pro485Leu 0.14 LIG4 8 (cDNA) C/T Ala3Val 0.07 27 (cDNA) C/T Thr9Ile 0.14 XRCC4 21965 G/T Ala247Ser 0.08 Note: cDNA = complementary DNA.

262 Chapter 5 How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease procedures are shorter. The results for the DRC phenotype 2003; Strom et al. 1995; Wu et al. 1995, 1998a; Zheng et from the independent CAT and LUC assays in parallel are al. 2003). The risks associated with mutagen sensitivity, highly correlated, with a correlation coefficient of 0.65 stratified by smoking status, are elevated in all smoking (p <0.0001) (Qiao et al. 2002a). This finding suggests that strata but are highest in current smokers and in the heavi- these two assays are comparable. est smokers. For studying tobacco-related cancers, a more appropriate mutagen to trigger DNA damage is BPDE; 8-Oxoguanine DNA Glycosylase Activity Assay BPDE-induced sensitivity is a risk factor for lung cancer (Wei et al. 1996; Wu et al. 1998a,b). The OGG test is another functional repair assay that measures the specific activity of the enzyme 8-oxoguanine Comet Assay DNA N-glycosylase in protein extracts prepared from peripheral blood mononuclear cells (Paz-Elizur et al. The comet assay (single-cell microgel electrophore- 2003). The OGG1 enzyme removes 8-oxoguanine from sis) is applicable to any cell line or tissue from which a DNA and leaves behind an abasic site that is rapidly cleaved single-cell suspension can be obtained (Ross et al. 1995). by the AP lyase of OGG1. OGG activity (in units per µg of Singh and associates (1988) have described the methods in protein extract) is the amount of fragment cleaved by 1 µg detail. For this assay, a cell culture is mixed with agarose of extract in one hour under standard reaction condi- gel and attached to a microscope slide. The cells are lysed tions (Paz-Elizur et al. 2003). In a pilot case-control study by submerging the slides in freshly prepared lysis buffer of NSCLC, the mean value of OGG activity in the case to remove all cellular proteins. To allow for DNA denatur- patients was significantly lower than that in the control ation, unwinding, and expression of the alkali-labile sites, participants and was independent of tumor histology, the slides are then placed in an alkali buffer (pH = 12.0). gender, and smoking status (Paz-Elizur et al. 2003). In To separate the damaged DNA from the intact nuclei, a addition, OGG activity in protein extracts from peripheral constant electric current is applied, and the slides are neu- blood mononuclear cells correlated well with that from tralized, fixed, and stored in the dark at room temperature lung tissue in the same patients (p = 0.003). until ready for analysis. During electrophoresis, damaged DNA migrates from the nucleus toward the anode Mutagen Sensitivity Assays as a result of the constant electric current, which forms the typical “comet” cell. A predetermined number of cells Researchers use cytogenetic assays extensively to are manually selected, and comet cells are automatically measure human exposure and response to genotoxic quantified with appropriate imaging software. agents. These assays are based on the unproven hypoth- Case patients with lung and bladder cancer had esis that the extent of genetic damage in the lymphocytes significantly higher levels of induced DNA damage- af may reflect critical events in carcinogenesis in the affected ter exposure to both BPDE and g-radiation, which were tissues. The assays can only indirectly indicate DRC from assessed by the mean “tail moment” in lymphocytes. (The the cellular damage that remains after mutagenic expo- tail moment is the product of lymphocyte tail length and sure and recovery and therefore probably reflect nonspe- the fraction of the total DNA in the tail.) Higher levels of cific impairment of the DNA repair machinery (Berwick DNA damage were positively associated with increased and Vineis 2000). risk of bladder and lung cancer (Schabath et al. 2003; Wu Hsu and colleagues (1989) developed the in vitro et al. 2005). Schmezer and colleagues (2001) showed that mutagen challenge assay to demonstrate interindividual case patients with lung cancer were significantly more differences in susceptibility to carcinogenic agents. This sensitive to bleomycin and had a reduced DRC (68 percent assay counts the frequency of bleomycin-induced breaks in case patients and 81 percent in control participants in short-term lymphocyte cultures, as a measure of cancer [p <0.001]). Rajee-Behbahani and colleagues (2001) susceptibility. Bleomycin is a clastogenic agent that mim- reported a similar DRC finding of 67 percent in 160 case ics the effects of radiation by generating free oxygen radi- patients with lung cancer and 79.3 percent in 180 con- cals capable of producing DNA single-strand breaks and trol participants. When the data from the cases and con- DSBs after forming a complex with DNA, ferrous ions, and trols were considered together, only 18 percent of the case oxygen that releases oxygen radicals (Burger et al. 1981). patients were below the median level of sensitivity to bleo- Most of the breaks are repaired by BER. Repair is rapid, mycin for all control participants, and 82 percent were in with a half-life of only a few minutes (López-Larraza et the hypersensitive range. al. 1990). Mohrenweiser and Jones (1998) have pointed out Mutagen sensitivity is an independent risk factor for several lines of evidence documenting that differences lung cancer that has a dose-response relationship with the in DRC reflect genetic differences. DRC in lymphocyte number of induced chromosomal breaks (Spitz et al. 1995,

Cancer 263 Surgeon General’s Report subpopulations from an individual exhibited similar may account for some of the interindividual variations in repair capacities. Furthermore, intraindividual variations expression levels. Eight allelic variants in this region occur in repair capacity among subpopulations of lymphocytes at a frequency of at least 0.01. One-half of these variants are significantly smaller than are interindividual varia- have a prevalence of 0.1 to 0.6, and this finding suggests tions (Crompton and Ozsahin 1997). The phenotype of a contribution to the variability in AGT expression within reduced repair capacity in the NER pathway is indepen- populations (Krzesniak et al. 2004). The prevalence of the dent of the phenotype for DSBR (Wu et al. 1998a). best-studied enhancer polymorphism, C1099T, is 0.09 Researchers have performed extensive resequenc- to 0.12 and is associated with an increase in promoter- ing of DNA repair genes to identify variations that may be enhancer activity in the LUC assay (Krzesniak et al. 2004). associated with a reduced function of their encoded pro- However, heterozygotes for this enhancer polymorphism teins rather than an absence of function. Such polymor- do not appear to be at a markedly lower risk of developing phisms could explain interindividual differences in DRC lung cancer (OR = 0.82; 95 percent CI, 0.41–1.67) (Krz- (Spitz et al. 2001; Qiao et al. 2002b; Wu et al. 2003). esniak et al. 2004). None of the other enhancer-promoter Although the variant alleles are likely to be associated polymorphisms have been examined for their relationship with only a modest cancer risk, because they exist at a with AGT or cancer risk. polymorphic frequency, the attributable risks can become Mutations at or near the cysteine acceptor site can substantial. As Berwick and Vineis (2000) noted, stud- affect AGT activity (Crone et al. 1994). Two polymorphisms ies that compare genetic polymorphisms with results of have been identified in this region of the protein: codon functional assays will likely be the most valuable type of 143 *ILE/*VAL and codon 160 *GLY/*ARG. The variant investigations to clarify the role of a defect in DRC with isoform codon 160 *GLY/*ARG has exhibited activity to the development of cancer. repair bulky alkylated DNA adducts that is significantly less than that of the GLY wild-type phenotype (Edara et al. Polymorphisms in O6-Alkylguanine–DNA 1996; Mijal et al. 2004). The estimated prevalence of the Alkyltransferase 143 variant is 0.07 in Whites, 0.03 in African Americans (Kaur et al. 2000), and as high as 0.24 in Swedish popu- The ability of a cell to withstand alkyl damage is lations (Ma et al. 2003). The codon 160 variant allele is related to the number of AGT molecules in the cell and likely a mutant, because it was found at a frequency less the rate of de novo synthesis of AGT. AGT levels differed than 0.01 (Kaur et al. 2000). These researchers reported a among persons, and protein levels in the liver, colon, and marginally significant increase in the risk of lung cancer peripheral blood varied 4-, 10-, and 20-fold, respectively associated with the codon 143 *ILE/*VAL genotype (OR = (Myrnes et al. 1983; Strauss et al. 1989; Povey et al. 2000). 2.1; 95 percent CI, 1.01–4.7), and no interaction of geno- Environmental and genetic factors, but not age, affect type and smoking dose was seen. More recently, Cohet and expression levels of AGT (Margison et al. 2003). AGT lev- colleagues (2004) reported an OR of 2.05 (95 percent CI, els are higher in normal lung, colorectal, and placental 1.03–4.07) for lung cancer among lifetime carriers of the tissues from smokers than in those from nonsmokers, codon 143 and 160 variant alleles who were nonsmokers. although these findings are controversial (Slupphaug et The authors suggested that the strongest risk was associ- al. 1992; Drin et al. 1994; Povey et al. 2000). Exposure ated with exposure to secondhand tobacco smoke. to AGT inhibitory aldehydes, such as formaldehyde from occupational exposure, can deplete AGT activity (Hayes et Polymorphisms in the Pathway al. 1997). Expression of AGT is higher in NSCLC tumor tis- for Repair of Base Excision sues from smokers than in those from nonsmokers (Mat- tern et al. 1998). However, small-scale studies examining Cross-Complementation AGT activity in peripheral lymphocytes have not observed Group 1 for Excision Repair significant differences between smokers and nonsmokers (Vähäkangas et al. 1991; Oesch and Klein 1992; Hall et al. The XRCC1 gene encodes a protein that functions 1993) or between case patients with cancer and control in BER and involves the excision of the damaged region, participants (Boffetta et al. 2002). It is not clear how well followed by repair synthesis that uses the opposite strand AGT activity in lymphocytes predicts levels in lung tissue. as the template. The XRCC1 protein forms scaffolding The AGT gene contains multiple polymorphisms in with DNA ligase III, polβ, and poly (adenosine diphos- the 5’ upstream region, which comprises the promoter phate–ribose) polymerase (PARP) to rejoin DNA strand and enhancer regions, as well as exon 1 (Egyházi et al. breaks and repair gaps left during BER. Of the estimated 2002; Krzesniak et al. 2004). These genetic variations 17 variants, 1 is a polymorphism at the XRCC1 28152 site

264 Chapter 5 How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease

(G→A transition) of codon 399 in exon 10 that results in a healthy and were homozygous for the 399*GLN allele nonconservative amino acid substitution of arginine than among those with the wild-type genotype (15.6 ver- for glutamine. Goode and colleagues (2002) extensively sus 6.78, p = 0.007). Wang and colleagues (2003a) showed reviewed the conflicting results for lung cancer studies. that persons with the variant 194*TRP allele had fewer For example, Divine and colleagues (2001) reported a bleomycin- and BPDE-induced breaks per cell than did more than twofold risk for lung adenocarcinoma associ- those with the wild-type genotype. The XRCC1 codon 399 ated with the *GLN/*GLN genotype. Zhou and colleagues is within the BRCT domain (amino acids 301 to 402) that (2003) found an association that was largely restricted to interacts with PARP and is in many proteins with activity nonsmokers and light smokers. Some studies failed to find involving response to DNA damage and cell-cycle check- any association between the *GLN/*GLN genotype and points. This region also has homology with yeast RAD4 lung cancer risk (Butkiewicz et al. 2001; Ratnasinghe et repair-related genes. Because the role of XRCC1 in BER al. 2001; Popanda et al. 2004; Zhang et al. 2005b). How- brings together DNA polβ, DNA ligase III, and PARP at the ever, Matullo and colleagues (2001a) did find an associa- site of DNA damage, repair activity of the exon 10 vari- tion for bladder cancer. David-Beabes and London (2001) ant may be altered. The codon 194 polymorphism is in reported a decreased risk of lung cancer for both African the linker region of the XRCC1 N-terminal domain sepa- American and White patients. Other studies also report rating the helix 3 and polβ involved in binding a single- conflicting findings regarding an increased risk associated nucleotide gap DNA substrate (Marintchev et al. 1999). with the exon 10 *ARG/*ARG genotype (Lee et al. 2001; Therefore, this polymorphism is less likely to cause a sig- Stern et al. 2001). nificant change in repair function. Another polymorphism at the XRCC1 26304 site (C→T transversion) of codon 194 in exon 6 results in a OGG1 Gene nonconservative amino acid substitution of arginine. Most The product of the OGG1 gene catalyzes the excision studies suggest a reduced risk of cancer associated with of a modified base, 8-oxoguanine, which may be formed by the 399*ARG variant allele (Goode et al. 2002). Studies exposure to reactive oxygen species. The reduced ability to of lung, bladder, head and neck, and gastric cancers all excise 8-oxoguanine may lead to an accumulation of oxi- showed inverse associations with the variant allele; some dation-induced mutations. Studies have identified several studies included evidence of an interaction with smoking polymorphisms at the OGG1 locus. The most frequently (Sturgis et al. 1999; David-Beabes and London 2001; Rat- studied polymorphism is a common C→G transversion in nasinghe et al. 2001; Stern et al. 2001). Two studies noted exon 7 that results in an amino acid alteration at codon no association for esophageal and lung cancers (Butkie- 326 (Ser→Cys). The HOGG1 protein encoded by the wild- wicz et al. 2001; Lee et al. 2001). type 326*SER allele exhibited substantially higher DNA A few earlier studies evaluated the functional sig- repair activity than did the 326*CYS variant in an in vitro nificance of these polymorphisms. Lunn and colleagues Escherichia coli complementation activity assay (Kohno (1999) noted that persons with the 399*GLN allele were et al. 1998). at a significantly higher risk (OR = 2.4) for exhibiting Researchers have observed fairly consistent detectable aflatoxin B adducts and a higher frequency 1 increased risks of OGG1 polymorphisms (Goode et al. of the glycophorin A variant than were carriers of the 2002). The largest study was a U.S.-population-based, 399*ARG/*ARG allele. This study also reported a dose- multiethnic study of lung cancer that identified a -sig response relationship between smoking status and pres- nificantly increased risk associated with the *CYS/*CYS ence of the polymorphism for detecting the adducts and genotype (*CYS/*CYS versus *SER/*SER; OR = 2.1; 95 the glycophorin A variant (Lunn et al. 1999). However, percent CI, 1.2–3.7) (Le Marchand et al. 2002). A small, no significant effects were noted for other XRCC1 poly- hospital-based study of lung cancer in Japan supported morphisms. Duell and colleagues (2000) found elevated these results (Sugimura et al. 1999). Findings in a third SCE frequencies and polyphenol DNA adducts with study of lung cancer that also suggested an increased risk 399*GLN/*GLN homozygous genotypes. Abdel-Rahman had similar findings for comparison of the two homozy- and El-Zein (2000) noted that persons carrying the gote groups (OR = 2.2; 95 percent CI, 0.4–11.8) (Wikman *GLN allele had significantly higher numbers of SCEs in et al. 2000). Analyses of esophageal cancer also showed an response to NNK treatment than did *ARG/*ARG geno- increased risk associated with the *CYS/*CYS genotype type carriers. No differences were detected in persons with (OR = 1.9; 95 percent CI, 1.3–2.6) (Xing et al. 2001). How- the codon 194 genotype. ever, overall findings regarding an interaction with smok- Matullo and colleagues (2001b) found higher DNA ing were inconsistent. adduct levels among lifetime nonsmokers who were

Cancer 265 Surgeon General’s Report

Polymorphisms in the Pathway for Cross-Complementation Group 1 Nucleotide Excision Repair for Excision Repair The XRCC1 gene codes for a 5’ incision subunit of Xeroderma Pigmentosum A the NER complex (Mohrenweiser et al. 1989). The XRCC1 XPA is an essential DNA-binding protein in the and XPF proteins form a stable complex in vivo and in NER pathway that aids in correctly positioning the repair vitro (de Laat et al. 1999). Although studies have reported machinery around the damaged areas and in maintaining no defect in the human XRCC1 gene, cells from XRCC1- contact with core repair factors during the repair process. deficient mice have an increase in genomic instability and XPA interacts with other proteins, such as replication a repair-deficient phenotype (Melton et al. 1998). Five protein A, TFIIH, and XRCC1/XPF (Volker et al. 2001). known polymorphisms of the XRCC1 gene do not cause an Studies have identified an→ A G transversion variant amino acid change and are validated in the SNP500Can- in the 5’ noncoding region (Butkiewicz et al. 2000). cer Database of the National Cancer Institute. How- Researchers investigating this polymorphism in lung can- ever, researchers think that a polymorphism with A→C cer reported similar results in two studies. Wu and col- transversion at nucleotide 8092 in the 3’ untranslated leagues (2003) reported that the presence of one or two region affects mRNA stability (Shen et al. 1998). Studies copies of the *G allele instead of the *A allele was associ- have implicated the XRCC1 polymorphism in the risk of ated with a reduced lung cancer risk for all ethnic groups. adult-onset glioma (Chen et al. 2000) but not in head and Furthermore, control participants with one or two copies neck cancer (Sturgis et al. 2002). Zhou and colleagues of the *G allele demonstrated more efficient DRC, as mea- (2005) found no overall effect on lung cancer risk, but sured by the HCR assay, than did control participants with they did find a lower lung cancer risk in heavy smokers the homozygous A genotype. In a study in Korea, Park and and a significantly higher risk in lifetime nonsmokers colleagues (2002) reported that the *G/*G genotype was (OR = 2.11; 95 percent CI, 1.03–4.31). also associated with a significantly decreased lung cancer risk when the combined *A/*A and *A/*G genotype was Xeroderma Pigmentosum used as the reference group. Butkiewicz and colleagues Complementation Group D (2004) reported similarly increased risks that had border- XPD (XRCC2) is one of the seven genetic comple- line statistical significance for the *A/*A genotype in all mentation groups that encode for proteins in the NER participants and for SCC and adenocarcinomas. For heavy pathway. The XPD protein has a role in both NER and smokers, the risk estimate was 2.52 (95 percent CI, 1.2– basal transcription. XPD functions as an evolutionary 5.4). Popanda and colleagues (2004) reported a nonsignifi- conserved ATP-dependent helicase within the multisub- cant risk of 1.53 for the *A/*A genotype compared with the unit transcription repair factor complex TFIIH. Of the *G/*G genotype. Thus, all studies confirm the protective three polymorphisms identified in XPD, two are in exons effect of the *G/*G genotype and the enhanced risk for the and the third is silent. The G→A transition in exon 10 at *A/*A genotype. codon 312 results in an amino acid change (Asp→Asn). The transition A→C at codon 751 in exon 23 produces a Xeroderma Pigmentosum C Lys→Gln change. The amino acid substitution Lys751Gln XPC is the step-limiting factor in NER. Researchers in exon 23 does not reside in a known helicase/adenos- have found two single nucleotide polymorphisms (SNPs) ine triphosphatase domain, but it is an amino acid resi- in the coding region. ALA499VAL is a single *C/*T nucleo- due identical in human, mouse, hamster, and fish XPD. tide polymorphism that codes for an amino acid substitu- This finding suggests a functional relevance for such tion (Ala/Val) at codon 499. Another SNP, LYS939GLN, is a highly evolutionary conserved sequence (Shen et al. a single *A/*C nucleotide polymorphism that codes for an 1998). Goode and colleagues (2002) extensively reviewed amino acid substitution (Lys/Gln) at codon 939. This vari- numerous reports from case-control studies of lung ant allele is associated with an increased risk of bladder cancer that have conflicting results. The largest study cancer (Sanyal et al. 2004). There is also a bi-allelic poly included 1,092 lung cancer case patients and 1,240 con- AT insertion/deletion polymorphism (PAT) in intron 9 of trol participants who were spouses or friends (Zhou et al. XPC. The *PAT allele has been associated with risk of head 2002a). The overall AOR was 1.47 (95 percent CI, 1.1–2.0) and neck cancer (Shen et al. 2001). for the ASP312ASN polymorphism (*ASN/*ASN versus

266 Chapter 5 How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease

*ASP/*ASP), but there was no association for the LYS- XPF/ERCC1 Gene Complex 751GLN polymorphism (*GLN/*GLN versus *LYS/*LYS). ERCC1 forms a complex with XPF when it makes a Analyses of the interactions between genes and smoking dual incision at the single-strand to double-strand transi- revealed that the adjusted ORs for each of the two polymor- tion at the 5’ end of the damaged DNA strand (Shen et phisms decreased significantly as pack-years1 increased. al. 1998). This complex is required to repair interstrand The interaction between the ASP312ASN polymorphism cross-links. A T→C transition at codon 662 results in a and smoking status was stronger than that between the serine→proline substitution. Fan and colleagues (1999) LYS751GLN polymorphism and smoking. The researchers reported six SNPs, five in coding regions. Three of the concluded that cumulative cigarette smoking modified SNPs resulted in nonconserved amino acid differences. the association between XPD polymorphisms and lung cancer risk. Spitz and colleagues (2001) reported AORs XPG/ERCC5 Gene Complex for the variant LYS751GLN and ASP312ASN genotypes of 1.36 and 1.51, respectively, although neither estimate This complex shows homology with yeast RAD2 was statistically significant. For persons homozygous for and carries out incision at the 3’ end of the lesion in the variant genotype at either locus, the AOR was 1.84 (95 the DNA strand (Harada et al. 1995; Hyytinen et al. percent CI, 1.11–3.04; p = 0.018 for trend). 1999). Only two of the seven validated SNPs appear A recent review and meta-analysis of nine interna- with significant frequency. In one polymorphism, a tional case-control studies that included 2,886 lung can- single nucleotide substitution (G→C) causes an amino cer cases and 3,085 controls for codon 312 (LYS751GLN) acid change (His1104Asp) at codon 1104. In the other and 3,374 lung cancer cases and 3,880 controls for codon polymorphism, a C→G substitution produces an amino 751 (ASP312ASN) did not demonstrate significant associa- acid change from cysteine to serine at codon 529. San- tions with either variant genotype (Benhamou and Sarasin yal and colleagues (2004) showed that the variant *C/*C 2005). However, for U.S. studies alone, both variants were genotype was significantly less frequent in cases of blad- associated with a significantly increased risk for lung can- der cancer than in controls. Jeon and colleagues (2003) cer (ORs = 1.43 and 1.25, respectively). Hu and colleagues reported similar findings for 310 lung cancer cases, in (2004) conducted another meta-analysis. The combined which the frequency of the variant genotype was less than case-control studies reported a 21-percent higher risk that for the other two genotypes combined (AOR = 0.54; for the 751*C/*C genotype (OR = 1.21; 95 percent CI, 95 percent CI, 0.37–0.80). This protective effect was at- 1.02–1.43) and a 27-percent higher risk for the tenuated in heavy smokers. Jeon and colleagues (2003) XPD 312*A/*A genotype (OR = 1.27; 95 percent CI, pointed out that because this SNP is in the C-terminus, it 1.04–1.56) among cases than among controls. Among might alter binding to other proteins in the incision com- studies of persons of Asian versus White descent, only plex, thereby affecting DRC. the studies of Whites found a significantly higher risk for the XPD 751*C/*C genotype (OR = 1.23; 95 percent CI, RAD23B Gene 1.03–1.47). For the XPD 312*A/*A genotype, the risk The RAD23B gene is an evolutionary, well-conserved among Whites compared with other races had border- gene with 10 exons. The protein complexes with XPC to line statistical significance (OR = 1.22; 95 percent CI, bind to different types of lesions and recruit the necessary 0.99–1.49) among cases compared with controls. TFIIH factors for NER. Of the six validated SNPs, four are seen transcriptional activity may be tolerant to amino acid with considerable frequency. One of the most frequently changes in the XPD protein, and mutations may destroy observed polymorphisms is a substitution (C→T) result- or alter the repair function without affecting transcrip- ing in an amino acid change at codon 249 (Ala249Val). tional activity. As Lunn and colleagues (2000) suggested, the effects of the *LYS allele may differ in different repair Genotype-Phenotype Correlations pathways, as assessed by different repair assays. The overall effect of conservative mutations in XPD may be subtle, Amino acid differences, especially at conserved sites because they do not alter XPB and XPD helicase activity, in these enzymes, could result in changes in repair profi- and multiple alterations might be needed before any effect ciency. The next logical step is the challenging task of eval- is noted. uating the functional relevance of these polymorphisms. A variety of factors that modulate the path from genotype to

1Pack-years = the number of years of smoking multiplied by the number of packs of cigarettes smoked per day.

Cancer 267 Surgeon General’s Report phenotype include protein-protein interaction, posttrans- DSB/REC. Shen and colleagues (1998) identified a →C T lational modification, gene silencing, epigenetic regula- substitution in exon 7 at position 18067 of XRCC3, a poly- tion, and environmental factors. Furthermore, proteins morphism that results in a threonine→methionine amino involved in DNA repair pathways are often multifunc- acid substitution at codon 241. David-Beabes and col- tional, resulting in a variety of phenotypes. leagues (2001) found no significant association between Both of the common genotypes *LYS/*LYS 751 and the XRCC3241 polymorphism and lung cancer. This find- 312 *ASP/*ASP XPD were associated with a DRC more ing was consistent with a smaller study of NSCLC that efficient than that for heterozygotes and with a signifi- also found no association after adjustments for age and cantly higher DRC than that for the homozygote mutants smoking (Butkiewicz et al. 2001). Wang and colleagues (Spitz et al. 2001). These results were confirmed in a differ- (2003c) reported an elevated but not statistically signifi- ent study population with a different mutagen challenge: a cant risk of lung cancer associated with polymorphisms UV exposure of 800 joules per square meter that like BPDE of the XRCC3 *T allele in African Americans and Mexican invoked NER (Qiao et al. 2002b). Additional validation Americans, which was evident largely in heavy smokers. came from a correlative study that used the comet assay to Other studies have associated this XRCC3 polymorphism assess DNA damage and repair (Schabath et al. 2003). These with an increased risk for melanoma skin cancer (Winsey data are consistent with some of the published small-scale et al. 2000) and bladder carcinoma (Matullo et al. 2001a). studies of these types of genotype-phenotype correlations. The THR241MET genetic variant may also contribute to Hou and colleagues (2002) noted a significant increase increases in DNA adducts and bladder cancer risk (Stern in DNA adduct levels, as measured by 32P-postlabeling, et al. 2002). with an increased number of variant alleles in exon 10 (p = 0.02) and exon 23 (p = 0.001). In addition, persons Summary with the combined exon 10 *A/*A and exon 23 *C/*C The association between common variants in DNA genotype showed significantly higher levels of adducts repair genes and the risk for tobacco-induced cancers is than those for persons carrying any of the other geno- the focus of considerable interest, but the results to date types (p = 0.02). Lunn and colleagues (2000) reported that are inconsistent. Complementary functional studies are possessing the common XPD genotype, *LYS/*LYS 751, likely to be valuable in addressing these inconsistencies. was associated with an increased risk of suboptimal DRC, Molecular epidemiologists now have better access to high- which was reflected in the number of x-ray–induced lym- throughput genotyping platforms and an enhanced ability phocyte chromatid aberrations. No association with the to focus on analyses based on pathways. Haplotype analy- *ASN312 allele was found. ses also increase the power to detect relevant associations. However, Møller and colleagues (1998) reported no In addition, computational algorithms such as PolyPhen relationship between the LYS751GLN polymorphism and and Scale-Invariant Feature Transform correlate with risk DRC, as measured by HCR or comet assay in 80 partici- estimates, and new analytic tools are being developed. pants, including 20 healthy persons. Another study with a small sample of 76 healthy persons found no association between either SCE frequencies or the presence of DNA adducts by LYS751GLN genotype (Duell et al. 2000). Conversion of DNA Adducts For a complex disease such as cancer, multiple to Mutations genes—each with a small effect—probably act independently or interact with other genes to influence the dis- DNA replication plays a major role in inducing point ease phenotype. Although these data suggest that the mutations—substitutions of one base pair for another and polymorphisms have a functional relevance, biochemical small mutations due to insertion or deletion of bases. DNA and biologic characterizations of the variants are needed adducts per se are not mutations and can be removed by to validate these findings. various DNA repair mechanisms in cells (Friedberg et al. 1995). When repair is not completed before a replication Polymorphisms in the Pathway for complex encounters the DNA adducts or other lesions, Double-Strand Break Repair various events are induced, which are sensed by cell-cycle The XRCC3 gene encodes a protein that acts in checkpoint mechanisms that halt cell-cycle progression the pathway for DSB/homologous recombination repair (Sancar et al. 2004). When the lesion is a strand break, (DSB/REC repair) and repairs chromosomal damage such replication causes a DSB that is repaired by homologous as breaks, translocations, and deletions. XRCC3 is a pro- recombination or by the erroneous nonhomologous end- tein related to RAD51, which is a critical component of joining mechanism. When the lesion is an interstrand

268 Chapter 5 How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease cross-link, the stall of a replication complex triggers the nucleotide terminus is most efficiently extended opposite unhooking of the cross-link by endonucleolytic inci- a lesion. These experiments characterize the efficiency sions on both sides of the cross-link in one strand. When and fidelity of translesion synthesis by a given polymerase. the lesion is a modified base or the loss of a base, a DNA The results of the in vitro experiments may be reflected polymerase often inserts a nucleotide, either correctly or in translesion synthesis in cells if the polymerase is incorrectly, opposite the lesion and extends the DNA involved in the synthesis. The involvement of a candidate strand beyond the site. The modification of a template nu- polymerase is examined by studying translesion synthesis cleotide generally impairs its ability to serve as a template in a host cell lacking the polymerase of interest. Finally, in efficiency and fidelity. Therefore, DNA synthesis slows the involvement is confirmed by a complementation down or is blocked at the site of the adducted template. experiment in mutant cells that express the exogenous Translesion DNA synthesis occurs when a DNA gene for the polymerase of interest. Thus, translesion syn- polymerase succeeds in DNA synthesis over the modified thesis across a given DNA lesion is studied in detail. One template. The synthesis reaction sometimes results in the limitation of this site-specific experiment is the inability insertion of an incorrect nucleotide opposite a lesion. This to study the effects on chromosomal aberrations. In addi- insertion leads to base-substitution mutations, the skip- tion, the results may not apply to other sequence contexts. ping of the lesion nucleotide template, or the realignment Nevertheless, this approach has provided a tremendous of a growing primer strand on the template strand at the amount of information on the mechanism of DNA adduct adducted region, which produces frameshift mutations. conversion to mutations. This step results in the introduction of mutations, and the subsequent replication of the mutated strand estab- Translesion Synthesis in Mammalian Cells lishes the mutation in the genome. This section describes DNA polymerases are key players in mutation induc- the mechanism of mutation induction by translesion tion. They introduce mutations during replication and DNA synthesis. determine the types of mutations generated. Great progress has been made in DNA polymerase studies in the last Molecular Analysis of Conversion to Mutations several years. Many novel DNA polymerases were discov- The strategy for studying the conversion of adducts ered in prokaryotes and eukaryotes (Hübscher et al. 2002). to mutations is to incorporate a chemically characterized These DNA polymerases play important roles in various DNA adduct or lesion into a specific sequence (Basu and aspects of DNA metabolism. The Y family polymerases Essigmann 1988). A DNA adduct can be incorporated into (Ohmori et al. 2001) include eukaryotic polh, polk, and an oligonucleotide sequence by total chemical synthesis. polτ, as well as REV1 and E. coli polIV and polV. In addi- However, a modified oligonucleotide may be prepared by tion, polz is a member of the B family. Researchers think direct reaction with a mutagen, followed by HPLC and/or these Y family polymerases are specialized for translesion gel electrophoresis purification. The modified oligonucle- synthesis (Prakash and Prakash 2002). These discoveries otide is then used as a substrate for in vitro and in vivo have led to the general idea that these polymerases are studies of translesion DNA synthesis, repair, and struc- responsible for overcoming the blocking effects of DNA ture. This experimental approach generally demonstrates adducts and constitute an important mechanism for tol- a clear relationship between cause and effect. The advan- erating unrepaired DNA lesions. tage of this approach is the ability to analyze in detail The role of the polh polymerase is most clearly events in translesion synthesis such as (1) quantification understood. This polymerase is coded by the XPV gene, of the effects of blocking DNA synthesis, (2) miscoding fre- which is defective in persons with XP variant cells (John- quency, and (3) miscoding specificity. son et al. 1999; Masutani et al. 1999). Although these cells In vitro studies of translesion synthesis that use possess NER capability, they carry a predisposition to skin purified DNA polymerases complement the in vivo studies. cancer on exposure to sunlight. The discovery that polh As described in the following section, cells have various is able to bypass the cis-syn thymine-thymine dimer effi- types of DNA polymerases and some of them are responsible ciently and accurately (Masutani et al. 1999) indicates that for translesion synthesis. Therefore, the characterization this polymerase plays a very important role in protection of in vitro translesion synthesis could help to identify the from the deleterious effects of unrepaired UV photoprod- polymerase responsible for translesion synthesis in cells. ucts. In its absence, the unrepaired lesions are bypassed by In vitro studies can be divided into two phases: insertion one or more other polymerases in an error-prone manner and extension steps. The insertion step determines which leading to skin cancer (Gibbs et al. 1998, 2000). X-ray crys- nucleotide is most efficiently inserted opposite a lesion by tallographic studies reveal that Y family polymerases have a given polymerase. The extension step determines which loose catalytic pockets enabling them to accommodate

Cancer 269 Surgeon General’s Report

Table 5.7 Translesion-specialized DNA polymerases (pol) and activities on various DNA lesions

DNA adduct polh polk polz polh + polz poli + polz pold + polz REV1 + polz

cis-syn TT + (a) – (b) – (c) – (c)

(6-4) TT – (a) – (b) – (c) + (d) + (c)

Abasic site + (a) + (b) – (c) + (e) + (c) + (f) + (f)

Acetylaminofluorene + (a) + (b) C8-dG adduct

Cisplatin intrastrand + (a) – (b) dG-dG adduct

1,N6- + (g) + (g) ethenodeoxyadenosine

8-oxodeoxyguanosine + (h) + (i)

(+) BPDE-N2-dG + (j) + (i)

(–) BPDE-N2-dG + (i)

Note: (a) Masutani et al. 2000; (b) Ohashi et al. 2000; (c) Johnson et al. 2000; (d) Johnson et al. 2001b; (e) Yuan et al. 2000; (f) Haracska et al. 2001; (g) Levine et al. 2001; (h) Haracska et al. 2000; (i) Zhang et al. 2000a; (j) Zhang et al. 2000b. BPDE-N2-dG = trans-anti-benzo[a]pyrene-N2-deoxyguanosine; dG = deoxyguanosine; TT = thymine-thymine dimer.

Figure 5.9 Model of mechanism for mammalian translesion synthesis

REV1 d k

i h d N

z h k

d d N N

Note: Replicative polymerase d encounters DNA lesion (open circle) in template, progression of DNA synthesis is blocked, and d temporarily disengages. Y family polymerases (h, k, i, and REV1) are recruited to the sites, and 1 or more polymerases catalyze the insertion of a nucleotide opposite a lesion and the extension from the newly generated terminus. With some DNA lesions, the Y family polymerases can insert a nucleotide (N), but further extension is inhibited. Then, the second translesion polymerase, z, catalyzes the extension step. After translesion synthesis, d resumes replication.

270 Chapter 5 How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease unusual base pairs (Trincao et al. 2001). This structural Factors in Outcome of Translesion Synthesis feature could explain the low fidelity of DNA synthesis on Many studies reveal that the efficiency and fidelity a normal DNA template. of translesion synthesis depend on the host (Moriya et al. In vitro experiments on translesion synthesis using 1994, 1996). Some DNA adducts miscode in one host (hu- purified polymerases reveal that each polymerase cata- man cells) but not in another (E. coli), and the reverse also lyzes bypass synthesis across various lesions with a differ- occurs (Moriya et al. 1994; Pandya and Moriya 1996). This ent efficiency and fidelity (Table 5.7). The current model finding underscores the importance of evaluating transle- of the mechanism for mammalian translesion synthesis sion events in the appropriate host: human cells. The dis- is illustrated in Figure 5.9. When a replicative DNA poly- crepancy most likely reflects the difference in the activity merase is inhibited by a lesion, translesion synthesis can of the translesion polymerases involved. be completed by the action of one polymerase or by the Sequence context also plays an important role in cooperation of two polymerases (Prakash and Prakash determining the outcome of translesion synthesis. Gen- 2002). Among these polymerases, polz is unique because it erally, a DNA adduct in iterated sequences, such as has low ability to insert a nucleotide opposite a lesion but monotonous repeats (e.g., GGGGG) and dinucleotide re- is efficient at extending from unmatched terminal pairs peats (e.g., GCGCGC), tends to cause frameshift mutations (Johnson et al. 2000). Therefore, researchers think that because these sequences misalign easily (Benamira et al. this polymerase plays a role mainly in extending from a 1992). However, the same adduct induces base-substitu- terminus opposite a lesion where another polymerase has tion mutations in a different sequence context (Moriya et inserted a nucleotide and the further extension is blocked.

Table 5.8 Mutational specificity of selected DNA adducts derived from tobacco smoke

Study DNA adduct Mutation specificity

Loechler et al. 1984 O6-methyldeoxyguanosine G→A Dosanjh et al. 1991 Pauly and Moschel 2001

Pauly et al. 2002 O6-[4-oxo-4-(3-pyridyl)butyl]-deoxyguanosine G→A

Dosanjh et al. 1991 O4-methylthymidine T→C Pauly and Moschel 2001

Wood et al. 1990 8-oxodeoxyguanosine G→T Moriya 1993

Kanuri et al. 2002 1,N2-propanodeoxyguanosine from acrolein G→T Yang et al. 2002

Moriya et al. 1994 3,N4-ethenodeoxycytidine C→A, T

Pandya and Moriya 1996 1,N6-ethenodeoxyadenosine A→G, T

Lawrence et al. 1990 Apurinic/apyrimidinic sites AP→T, A, G Cabral Neto et al. 1994 Gibbs and Lawrence 1995

Moriya et al. 1996 Benzo[a]pyrene-7,8-diol-9,10-epoxide-N2-deoxyguanosine G→T, A, C Page et al. 1998

Page et al. 1999 Benzo[a]pyrene-7,8-diol-9,10-epoxide-N6-deoxyadenosine A→T

Verghis et al. 1997 4-aminobiphenyl-C8-deoxyguanosine G→C

Cancer 271 Surgeon General’s Report al. 1994). Sequence context influences base-substitution mutation after exposure to these agents (Pauly and Mos- events (Moriya et al. 1996; Page et al. 1998) and trans- chel 2001). lesion efficiency (Latham et al. 1993) (see “Benzo[a] pyrene-7,8-Diol-9,10-Epoxide-N2-Deoxyguanosine stereo- 8-Oxodeoxyguanosine isomers” later in this chapter). 8-oxodeoxyguanosine is a representative adduct Thus, translesion events are determined by the formed by oxidative damage to DNA, and researchers have interplay between a DNA adduct, its sequence environ- extensively studied its mutagenic properties and repair ment, and the DNA polymerase involved. This finding mechanisms (Grollman and Moriya 1993). The miscod- underscores the importance of conducting experiments ing property of this adduct derives from its propensity to with use of a proper sequence context and host. assume a syn conformation and to pair easily with deoxyadenosine (anti), which leads to G→T transversions. To Conversion of Cigarette-Smoke-Induced avoid this mutation induction, cells have developed an DNA Adducts to Mutations elaborate postreplication BER mechanism that specifi- Conversion of DNA adducts induced by cigarette cally removes misinserted deoxyadenosine by the action smoke to mutations is summarized in Table 5.8. The dis- of the DNA glycosylase, adenine-DNA-glycosylase (Parker cussion that follows provides additional details. and Eshleman 2003). Subsequently, when deoxycytidine monophosphat) is inserted opposite the adduct, 8-oxo- O6-Pyridyloxobutyl-Deoxyguanosine deoxyguanosine is removed by another BER initiated by OGG1, and a G:C pair is restored. O6-POB-deoxyguanosine is formed by a pyridyloxo- This adduct is also formed in the nucleotide pool. butylating metabolite of the tobacco-specific N-nitrosa- When 8-oxodeoxyguanosine-triphosphate is inserted mines NNK and NNN and is removed by AGT. Therefore, opposite a deoxyadenosine template, the misinsertion in the presence of this repair enzyme, the adduct induced leads to an A→C transition. To avoid this event, the cellu- only a moderate miscoding frequency. The resulting lar enzyme MTH1 converts 8-oxodeoxyguanosine triphos- mutations were G→A transitions. In the absence of AGT, phate to 8-oxodeoxyguanosine monophosphate, which is the miscoding frequency markedly increased to more than no longer a substrate for DNA synthesis. Thus, cells have 90 percent (Pauly et al. 2002). The results were similar in developed several layers of defense mechanisms against E. coli and human cells. These results indicate that the 8-oxodeoxyguanosine. Therefore, the apparent frequency frequency of miscoding for this adduct is high. The DNA of mutation induction by this adduct is low in normal polymerase involved almost exclusively inserts deoxythy- cells. However, when MYH is inactivated, the frequency midine monophosphate opposite the adduct, leading to of G→T transversions increases drastically (Moriya and G→A transitions. Thus, repair by the alkyltransferase is Grollman 1993; Hashimoto and Moriya, unpublished data) extremely critical to the avoidance of mutation induction and mutations in this gene lead to a high incidence of by this adduct. The biologic characteristics of this adduct spontaneous human colon cancer (Al-Tassan et al. 2002). are similar to those of O6-methyldeoxyguanosine (Pauly and Moschel 2001). The DNA polymerase that catalyzes 1,N2-Propanodeoxyguanosine the translesion synthesis and the bypass efficiency of this synthesis remain to be determined. Various unsaturated α,β-aldehydes, such as acrolein and crotonaldehyde, produce the DNA adduct PdG. Ac- O6-Methyldeoxyguanosine rolein produces two positional isomers: 8-(g-) and 6-(α-) and O4-Methylthymidine xy hydroxyl PdG. When positioned in double-stranded DNA, the α adduct is more genotoxic than the g adduct. The O6-methyldeoxyguanosine and O4-methyl- The α adduct has significantly more blocking effects, and thymidine adducts induce mutations by stable pairing the g adduct, but not the α adduct, miscodes with G→T to thymidine (Dosanjh et al. 1993) and deoxyguanosine transversions at a frequency of approximately 10 percent (Toorchen and Topal 1983), respectively. Accordingly, (Yang et al. 2002). Most of the miscoding events were their miscoding potentials are high (Dosanjh et al. 1991; induced by polh (Yang et al. 2003). Structural studies Pauly and Moschel 2001) and are similar to those of O6- reveal that the exocyclic ring of the g adduct, but not the POB-deoxyguanosine. MMR acts on base pairs containing α adduct, opens in a manner similar to that of the malo- O6-methyldeoxyguanosine after replication (Branch et al. ndialdehyde-induced deoxyguanosine adduct (Mao et al. 1993) and leads to cell death as a result of a futile MMR. 1999) when paired to deoxycytidine (de los Santos et al. Therefore, MMR mutants are more resistant to methyl- 2001). This finding may account for the weaker blocking ating agents (Branch et al. 1993) and are more prone to effect and the lack of miscoding, because the ring-opened

272 Chapter 5 How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease g adduct pairs nicely to deoxycytidine with the Watson- 4-Aminobiphenyl-C8-Deoxyguanosine Crick type of (anti-anti) conformation. When the g The adduct 4-ABP-C8-deoxyguanosine barely mis- adduct is inserted in single-stranded DNA and replicates codes in E. coli by inducing G→C transversions (Verghis in mammalian cells, the resulting structure miscodes by et al. 1997). Because researchers observed G→T, G→A, inducing G→T transversions (Kanuri et al. 2002). In addi- and G→C mutations in an experiment that used a ran- tion, the ring-opened deoxyguanosine adduct forms inter- domly modified single-strand DNA (Verghis et al. 1997), strand G-G cross-links in the sequence 5’CpG (Kozekov et the possibilities of the effects from the sequence context al. 2003), which may also contribute to the genotoxicity and the involvement of other deoxyguanosine adducts, of acrolein. such as those at N2, remain to be explored. Furthermore, it appears that a 4-ABP-deoxyadenosine adduct induces Exocyclic Etheno Adducts A→T transversions (Lasko et al. 1988; Hatcher and Swam- Although the etheno adduct 1,N6-ethenodeoxy- inathan 1995). adenosine miscodes efficiently in simian kidney cells, it does not miscode in E. coli (Pandya and Moriya 1996). This Assessment of Genotoxicity of DNA Adducts finding emphasizes the importance of the host. The find- Genotoxic properties of a DNA lesion can be char- ing is probably attributable to the difference in the fidelity acterized by using chemically defined substrates. The of the DNA polymerase involved in translesion synthesis. genotoxicity of a DNA lesion is determined by factors The etheno adduct 3,N4-ethenodeoxycytidine miscodes such as the efficiency and fidelity of translesion synthesis efficiently in both hosts (Moriya et al. 1994). and repair. For point mutations, however, the “genotoxic potency” of a DNA adduct can be determined by assessing Apurinic/Apyrimidinic Sites the bypass efficiency and the miscoding potency. Accord- AP sites are generated by the cleavage of a glyco- ing to this formula, a DNA lesion that is easily bypassed sidic bond between a base and a sugar in DNA for various with a high frequency of miscoding events is defined as reasons such as (1) the action of a DNA glycosylase and a highly genotoxic DNA adduct. Furthermore, when the (2) modifications to a base that destabilize the glycosidic genotoxicity of a DNA lesion is assessed, the information bond. These sites do not convey any coding information. on its abundance in the genome, which reflects the bal- Deoxyadenosine is often inserted opposite these sites in ance between formation and removal, should also be con- E. coli (Lawrence et al. 1990), which is known as “the A sidered. Therefore, conceptually, the total genotoxicity of rule” (Strauss 1991). However, this rule does not appear a DNA adduct could be estimated by determining its geno- to be applicable in mammalian cells: various bases are toxic potency and its abundance in DNA. inserted opposite these sites in those cells (Cabral Neto et According to these criteria, the genotoxicity of the al. 1994; Gibbs and Lawrence 1995). 8-oxodeoxyguanosine adduct, which is a unique case, would be high because it exists in high levels in genomic Benzo[a]pyrene-7,8-Diol-9,10-Epoxide-N2- DNA and is easily bypassed by a DNA polymerase with a Deoxyguanosine Stereoisomers high miscoding frequency. However, the apparent genotoxicity is low because of the postreplication repair that Studies have extensively characterized the geno- is catalyzed by MYH. Therefore, when the postreplication toxicity of different stereoisomers of BPDE-N2-deoxy- repair is inactivated, this adduct can become a significant guanosine (Moriya et al. 1996; Fernandes et al. 1998). genotoxic adduct (Al-Tassan et al. 2002). A prominent feature is that both the surrounding DNA Data for the genotoxic effects of DNA lesions derived sequence and the host markedly influence miscoding fre- from tobacco smoke are scarce, and a systematic study quency (Moriya et al. 1996; Fernandes et al. 1998; Page et is needed. Together with information on the abundance al. 1998) and miscoding specificity (Kozack et al. 2000). of each lesion, the genotoxicity of tobacco-related DNA The major adduct, (+)-BPDE-N2-deoxyguanosine, induces adducts might be ranked by using site-specific modified mainly G→T and G→A transversions in 5’-TGC and plasmids, introducing them with the use of host cells, and 5’-AGA sequence contexts, respectively, which research- subsequently recovering them for sequence analysis. ers hypothesize is attributable to differences in adduct conformations in different sequence contexts (Kozack et al. 2000). The deoxyadenosine adduct (BPDE-N6-deoxyadenosine) also miscodes with A→T transversions (Page et al. 1999).

Cancer 273 Surgeon General’s Report

Gene Mutations in Tobacco-Induced Cancer

Chromosome Instability and Loss genome FRA3B maps in the FHIT gene and may contribute to the susceptibility of this locus to gene rearrangement Lung Cancer induced by carcinogens in cigarette smoke. Researchers have observed a loss of the FHIT protein in 50 percent of The detection of numerous cytogenetic changes lung cancers, but somatic mutations are uncommon in provided the first link to the molecular pathogenesis of the FHIT gene. The epigenetic inactivation by methyla- lung cancer. Mapping chromosomal sites for rearrange- tion of the 5’CpG island located in the promoter region ment, breakpoints, and losses revealed both common of FHIT represents another mechanism for inactivating and distinct changes in both SCLC and NSCLC. In SCLC, this gene in lung cancer (see “Gene Promoter Hypermeth- breakpoints are commonly seen in chromosomes 1, 3, 5, ylation in Cancer Induced by Tobacco Smoke” later in and 17, although researchers have observed losses of the this chapter). short arm (p) of chromosomes 3 and 17 and of the long The importance of the inactivation of tumor- arm (q) of chromosome 5 (Balsara and Testa 2002). Sub- suppressor genes FHIT, RB, and TP53 in lung cancer is sequent studies using comparative genomic hybridization evident from their functions. The binding of hypophos- showed that deletions on chromosomes 3p, 4q, 5q, 10q, phorylated RB to cyclin-dependent kinase (CDK) 4 or 6 13q, and 17p were common in SCLC (Petersen et al. 1997). blocks transit of the RB protein through the G1/S bound- In NSCLC, multiple numeric and structural changes were ary of the cell cycle. Inactivating mutations result in the seen across many chromosomes. The most frequent sites loss of a functional hypophosphorylated protein associ- (60 to 80 percent) for chromosome loss were found on ated with a shortening of the G1 phase of the cell cycle chromosomes 3p, 6q, 8p, 9p, 9q, 17p, 18q, 19p, 21q, and and the enhancement of cell proliferation, a hallmark of 22q (Balsara and Testa 2002). Some of the most common the cancer cell (Nevins 1992). The TP53 gene is central to sites for chromosome loss included 3p, 9p, 13q, and 17p. several critical processes needed to control the response These sites were also detected in nonmalignant bron- of the cell to exogenous stress from exposure to ciga- chial epithelium of current and former smokers and were rette smoke. This gene functions as a transcription factor absent in lifetime nonsmokers (Mao et al. 1997; Witsuba et within several pathways and as a sensor of DNA damage al. 1997). These findings strongly link tobacco exposure to (Robles et al. 2002). Thus, the TP53 gene has an important the development of chromosome damage throughout the role in cell-cycle checkpoints, DNA repair, apoptosis, and aerodigestive tract. senescence. A loss of TP53 function is also an early event

Identification of Tumor-Suppressor Genes The commonality for specific regions in the genome Table 5.9 Frequency of mutation or deletion of to lose alleles suggested the presence of tumor-suppres- tumor-suppressor genes in lung cancer sor genes within these loci. The RB gene was the first tumor-suppressor gene linked to lung cancer (Harbour et Frequency (%) al. 1988). A loss of function of this gene through either Non-small- deletion or point mutation occurs in 90 percent of SCLCs, Chromosomal Small-cell cell lung whereas few NSCLCs harbor changes in this tumor- Gene location lung cancer cancer suppressor gene (Table 5.9) (Shimizu et al. 1994). The RB 13q14 90 15 most frequently inactivated tumor-suppressor gene in lung cancer is TP53. TP53 mutations are found in 70 TP53 17p13 70 50 percent of SCLCs, 65 percent of SCCs, and 33 percent of adenocarcinomas (Greenblatt et al. 1994). (For discus- CHFR 12q24 ND 6 sion of specific mutations and their potential relation- MYO18B 22q12 ND 13 ship to carcinogens in cigarette smoke, see “Relationship of TP53 Mutations to Smoking and Carcinogens” later in PTEN 10q23 9 17 this chapter.) LKB1/STK11 19p13 ND 35 A frequent deletion within chromosome 3p14 led to the identification of the FHIT gene (Zabarovsky et Note: ND = not determined. al. 2002). The most common fragile site of the human

274 Chapter 5 How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease in the genesis of SCC that occurs in bronchial dysplasia Two additional genes with poorly characterized (Sozzi et al. 1992; Bennett et al. 1993). Studies have also functions and localized to chromosome 3p are altered detected TP53 mutations in peripheral lung tissue from in lung cancer through deletion or mutation. A specific patients with lung cancer, a finding that supports a role ATG→AGG mutation in codon 50 of the ARP gene was for this gene in the early development of adenocarcinomas seen in 8 of 20 lung cancers. In addition, researchers (Hussain et al. 2001). The FHIT gene induces apoptosis observed either exon deletion or intron insertion in the mediated by CASPASE-8 and independent of mitochon- DLC1 gene in 11 of 30 NSCLCs (Zabarovsky et al. 2002). drial mediators and inhibits cell growth through interac- Frequent deletion involving the short arm of chromosome tions with the SRC protein kinase (Pekarsky et al. 2004; 19 occurs in lung adenocarcinomas (Sanchez-Cespedes Roz et al. 2004). A loss of function of the FHIT, RB, and et al. 2001). One gene mapped to this chromosome TP53, genes leads to the immortalization of bronchial region is STK11, in which germline mutations are causal epithelial cells, a key step in neoplastic transformation for Peutz-Jeghers syndrome. This syndrome is character- (Reddel et al. 1988) (see “Signal Transduction” later in ized by a series of anomalies and increased risk for gas- this chapter). trointestinal and extraintestinal malignancies (Giardiello et al. 1987). Inactivating mutations and/or deletion of the Tumor-Suppressor Genes LKB1/STK11 protein were described in about one-third of Inactivated in Lung Cancer primary adenocarcinomas (Sanchez-Cespedes et al. 2002; Ji et al. 2007), and these abnormalities were closely asso- The search for tumor-suppressor genes inactivated ciated with mutation of the KRAS oncogene in the same through the two-hit mechanism of the loss of one allele tumors (Ji et al. 2007; Matsumoto et al. 2007). The STK11 and the mutation of the remaining allele has not recently gene may function as a growth-inhibiting gene that is identified any genes with a frequency of inactivation activated through phosphorylation by the ATM gene, approaching that seen for the RB and TP53 genes. The which senses DNA damage (Sapkota et al. 2002), and acts discussion that follows describes the involvement of sev- through pathways dependent or independent of the P53 eral genes and their functions in subsets of lung cancer protein to suppress invasion and metastasis (Karuman et (Table 5.9). The mitotic checkpoint gene CHFR, which al. 2001; Upadhyay et al. 2006; Ji et al. 2007). In addition functions in early prophase to regulate chromosome to inactivating by mutation, epigenetic silencing by pro- condensation, was mutated in 3 of 53 lung carcino- moter hypermethylation has emerged as a major mecha- mas (Mariatos et al. 2003). Studies found three somatic nism for inactivating many genes in lung cancer, some of mutations in the proapoptotic gene CASPASE-8 in 2 of 30 which are described here (e.g., MYO18B). (For a detailed lung tumors (Hosomi et al. 2003). MYO18B is a candidate discussion, see “Gene Promoter Hypermethylation in tumor-suppressor gene at chromosome 22q12. Of 46 pri- Cancer Induced by Tobacco Smoke” later in this chapter.) mary NSCLCs, 6 contained somatic mutations within this gene. Restoring MYO18B function in cell lines inhibited Activation of Oncogenes in Lung Cancer anchorage-independent growth, thus supporting its function as a tumor-suppressor gene in lung cancer (Nishioka Oncogenes encode proteins that influence cell et al. 2002). cycling and promote cancer. They are usually “gain-of- The PTEN gene is located on chromosome 10. Its function” mutations of normal genes. Researchers see gene product is phosphatidylinositol 3’-phosphatase, a KRAS gene mutations in approximately 30 to 40 percent protein tyrosine phosphatase that uses the phosphoinosit- of adenocarcinomas but rarely in SCCs, SCLCs, or lung ide second messenger, phosphatidylinositol 3,4,5-triphos- tumors from nonsmokers (Slebos et al. 1990; Westra et phate (PIP3), as a physiological substrate (Maehama et al. al. 1996; Ahrendt et al. 2003). Mutations are localized to 2001). Researchers have identified point mutations of the codons 12, 13, and 61. More than 85 percent occur within PTEN gene in cell lines from 3 of 35 SCLCs and 3 of 18 codon 12. Nearly 70 percent of the mutations are G→T NSCLCs; there were two homozygous deletions in primary transversions within codon 12 that change a glycine SCLCs (Forgacs et al. 1998). Mutations that impair PTEN codon (GGT) to valine (GTT) or cysteine (TGT). Mouse function result in a marked increase in PIP3 levels and in lung tumors induced by B[a]P and other PAHs show exclu- the constitutive activation of AKT survival, thus signaling sively G→T transversions in codon 12 of the Kras gene. pathways that in turn promote hyperplasia and tumor for- These findings support the hypothesis that activation of mation. Thus, although it is not common in lung cancer, this oncogene in lung tumors results from DNA damage a PTEN mutation or deletion profoundly affects an impor- leading to base mispairing of these deoxyguanosines. In tant signaling pathway in the cell. vitro studies have demonstrated that DNA adducts formed

Cancer 275 Surgeon General’s Report from the metabolism of B[a]P, NNK, and reactive oxygen Thus, a RAS oncogene mutation leads to the disruption of species can all lead to G→T transversions (Table 5.8) (You many cellular pathways and provides a strong oncogenic et al. 1989; Belinsky et al. 1992). Thus, the activation of signal for neoplastic transformation (see “Signal Trans- carcinogens in tobacco smoke and the pulmonary inflam- duction” later in this chapter). mation that ensues from exposure to particulate matter The MYC family of genes (C-MYC, N-MYC, and together can lead to activation of the KRAS oncogene. L-MYC) plays a prominent role in the growth of the Studies detected KRAS gene mutations in 39 percent developing and mature adult lung. Extensive studies have of atypical alveolar hyperplasia, a putative precursor to evaluated the expression and amplification of these genes adenocarcinoma (Slebos et al. 1996). A similarity in the in NSCLC and SCLC (Jänne and Johnson 2000). Most percentage of precursor lesions and tumors containing lung cancers express one or more of the MYC family of KRAS mutations supports the importance of this gene in genes, whereas gene amplification is seen in a minority tumor progression in a subset of adenocarcinomas. of primary tumors (Table 5.10). Gene rearrangements The sequence of events leading to activation of the involving different exons are associated with amplifica- RAS signal transduction pathway is well characterized tion detected in cell lines but are uncommon in primary (Lechner and Fugaro 2000). When the RAS protein is tumors (Kinzler et al. 1986; Mäkelä et al. 1991; Sekido activated through mutations in codon 12, 13, or 61, it et al. 1992). Mechanisms responsible for the increased binds irreversibly to guanosine triphosphate (GTP) in expression of the MYC genes in the absence of gene am- the cell, which initiates a cascade of protein activations, plification are not well understood. Increased expression beginning with v-raf-murine leukemia viral oncogene 1 could occur through increased activity in the RAS signal- (RAF-1), that transmits a signal from the cell membrane ing pathway through either KRAS oncogene mutations or to the nuclear transcription machinery. Ultimately, these effects on the activity of genes in this pathway, such as the signals culminate in the activation of transcription factors activity of mitogen-activated protein kinase (MAPK) (Jull including MYC, FOS, and JUN, which in turn influence et al. 2001). many cellular activities such as transcription, translation, Increased gene expression is common in lung can- cytoskeletal organization, and cell-cell interactions. This cer but often is not associated with gene amplification. signal remains active until GTPase (guanosine triphos- Two genes studied extensively are EGFR and NEU (HER-2/ phatase) dephosphorylates GTP to guanosine diphosphate. NEU [ERBB2]). EGFR is the receptor for the epidermal

Table 5.10 Frequency of gene amplification and increased expression of genes in lung cancer

Frequency (%) Gene Tumor histology Amplification Expression

C-MYC Small-cell lung cancer 5 25

N-MYC Small-cell lung cancer 7 3

L-MYC Small-cell lung cancer 12 33

EGFR Small-cell lung cancer 0 0

HER-2/NEU Small-cell lung cancer <1 0–7

C-MYC Non-small-cell lung cancer 8 33

N-MYC Non-small-cell lung cancer 0 ND

L- MYC Non-small-cell lung cancer 3 ND

EGFR Non-small-cell lung cancer 9–25 34–62

HER-2/NEU Non-small-cell lung cancer 2–4 23–58

Note: ND = not determined.

276 Chapter 5 How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease growth factor and the HER-2/NEU protein, and the bind- phosphorylation of BRAF by AKT (Guan et al. 2000). The ing of these growth factors to this receptor is associated disruption of AKT-induced BRAF inhibition could contrib- with increased DNA synthesis, cell proliferation, and dif- ute to malignant transformation. ferentiation. An increased expression of the EGFR gene was not seen in SCLCs but occurred in 34 to 62 percent Oncogene Activation, Tumor-Suppressor Gene of NSCLCs (Hirsch et al. 2003b; Suzuki et al. 2005). In Inactivation, and Lung Cancer Survival addition, an increased expression of this gene was more Researchers have studied the prognostic impact of common in SCC than in adenocarcinoma—82 versus 44 commonly altered genes in lung cancer. The effect of an percent (Hirsch et al. 2003b). In contrast, gene amplifica- activated KRAS oncogene on survival was assessed in 69 tion was detected in 9 to 25 percent of tumors (Hirsch patients, including 48 with stage I adenocarcinoma that et al. 2003b; Suzuki et al. 2005). Expression of the HER- was completely resected (Slebos et al. 1990). Twelve of 19 2/NEU gene was seen in 23 to 58 percent of NSCLCs and patients with a KRAS mutation died within the follow- in 0 to 7 percent of SCLCs (Shi et al. 1992; Junker et al. up period (median, 47 months) compared with 22 of 50 2005; Pelosi et al. 2005). Similar to EGFR, HER-2/NEU patients with a tumor negative for the KRAS oncogene. was more commonly expressed in SCC than in adenocar- This significant difference in survival was observed even cinoma, and gene amplification was rare in all tumors though patients with a KRAS mutation had a less advanced (<5 percent). disease than those with no mutation. All seven patients Observation of EGFR expression in 34 to 62 percent with stage III disease were negative for mutations. Rosell of NSCLCs led to the development of small molecule and colleagues (1993) conducted a similar study of largely inhibitors of the tyrosine kinase domain of the wild-type stage I resected adenocarcinomas that again revealed a EGFR protein (Fukuoka et al. 2003; Herbst and Bunn 2003; reduced survival rate independent of lymph node status Lynch et al. 2004; Amann et al. 2005; Baselga and Arteaga for patients whose tumor contained a mutated KRAS 2005). The clinical response of approximately 10 percent gene. In contrast, a larger study of 127 adenocarcinomas of European patients and 30 percent of patients from found no difference in survival by KRAS mutation status Japan to treatment with the EGFR inhibitors gefitinib or (Keohavong et al. 1996). Overall, data are conflicting with erlotinib led to a search for the mechanism responsible respect to KRAS mutations as prognostic factors and fur- (Kris et al. 2003; Pérez-Soler et al. 2004). The outcome of ther research is needed (Aviel-Ronen et al. 2006). these studies was the identification of somatic mutations Studies have examined the effect of TP53 gene in the tyrosine kinase domain of the EGFR gene in most mutations on prognosis in both early- and late-stage lung patients who had demonstrated a clinical response to the cancers. After four years of follow-up, the hazard ratio for drugs (Lynch et al. 2004; Amann et al. 2005). In addition, 106 patients with stage I resected NSCLC with a TP53 recent studies suggest that the EGFR copy number and mutation was 2.8 for death, compared with patients who KRAS mutation may also be involved in determining a had a wild-type gene (Ahrendt et al. 2003). Four years response to gefitinib and erlotinib. Subsequent studies after surgery, 78 percent of patients with no TP53 muta- have sequenced the EGFR gene in thousands of NSCLCs tion and 52 percent with a TP53 mutation were alive. A from patients in Asia, Europe, and the United States. These previous study by Tomizawa and colleagues (1999) found studies found that most mutations were due to either a a similar survival benefit for patients with stage I NSCLC deletion involving exon 19 or a missense mutation in exon and no TP53 mutations, which also confer a poor clinical 21. In addition, mutations were two to three times more outcome for those with advanced NSCLC. Independent of likely in women than in men and three to five times more chemotherapy or supportive care, median survival dura- likely in nonsmokers than in current or former smok- tion for patients with stage III or IV NSCLC with or with- ers (Johnson and Jänne 2005). Finally, the prevalence of out a TP53 mutation was 17 versus 39 weeks, respectively mutations was 10 percent in tumors of patients from (Murakami et al. 2000). Recently, a study of 420 patients Europe and the United States compared with 30 percent with primary head and neck cancer (Poeta et al. 2007) in tumors from persons of Asian background residing in showed that disruptive TP53 mutations in tumor DNA are Japan and Taiwan. associated with reduced survival after surgical treatment The BRAF gene encodes a RAS-regulated kinase that of SCC of the head and neck (hazard ratio, 1.7; 95 percent can mediate cell growth. BRAF mutations were found in CI, 1.2–2.4; p = 0.003). 5 of 179 NSCLCs and are almost exclusively confined to Together, it is apparent that the inactivation of the adenocarcinomas (Brose et al. 2002; Naoki et al. 2002). TP53 tumor-suppressor gene and the activation of the Although the mutation is relatively uncommon in lung KRAS oncogene in NSCLCs and other tumors are cor- cancer, its location in either exon 11 or exon 15 altered the related with exposure to cigarette smoke and contribute

Cancer 277 Surgeon General’s Report to a phenotype that reduces survival in both early and 1994; Hernandez-Boussard and Hainaut 1998; Pfeifer et advanced stages of the disease. al. 2002). One study shows that the relative risk of having a TP53 mutation in lung cancer was up to 13 times higher in lifetime heavy smokers than in lifetime nonsmokers Relationship of TP53 Mutations (Le Calvez et al. 2005). G→T transversions are commonly observed in smoking-associated lung cancers (Greenb- to Smoking and Carcinogens latt et al. 1994; Hainaut and Hollstein 2000; Hainaut and Pfeifer 2001). The frequency of G→T transversions in lung TP53 Mutations in Smoking-Associated cancers from smokers is higher than that for lung cancers Lung Cancers and most other cancers in nonsmokers (Greenblatt et al. TP53 gene mutations are found in approximately 40 1994; Husgafvel-Pursiainen and Kannio 1996; Hernandez- percent of human lung cancers; TP53 is the most com- Boussard and Hainaut 1998; Bennett et al. 1999; Hainaut monly mutated tumor-suppressor gene in lung cancer and Pfeifer 2001). Mutational patterns for lung cancers (see “Identification of Tumor-Suppressor Genes” earlier in from smokers and nonsmokers are shown in Figure 5.10. this chapter). These mutations are generally more com- The difference between 28.9 percent G→T transversion mon in smokers than in nonsmokers (Greenblatt et al. mutations in “designated smokers” (i.e., smoking status

Figure 5.10 Patterns of TP53 gene mutations and percentage of G→T transversion mutations in human lung cancers All persons with lung cancers minus Lung cancer in nonsmokers nonsmokers (N = 1,580) (N = 186)

13.4%

28.6%

Lung cancer in smokers Breast, brain, and colorectal cancers (N = 529) (N = 5,379) A:T C:G A:T G:C A:T T:A 28.9% 8.8% G:C A:T G:C A:T at CpG G:C C:G G:C T:A Insertion/deletion/complex

Source: Data are from the R9 version (July 2004) of the International Agency for Research on Cancer TP53 mutation database (IARC 2006). Note: Cell lines and metastatic cancers were excluded, as well as cancers with defined exposures other than tobacco (e.g., asbestos, radon, mustard gas, and air pollution) (see database Web site for specifications of exposure data). Nonsmokers included a series of 21 mutations (Le Calvez et al. 2005) not included in the database, in addition to 165 database entries. Data from Gao et al. 1997 were excluded (see Hainaut and Pfeifer 2001 for detailed selection criteria). N = total number of mutations.

278 Chapter 5 How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease indicated in literature) and 13.4 percent G→T mutations bronchial epithelial cells treated with BPDE. Hot spots in nonsmokers has high statistical significance (p <0.001, of G→T mutations in cancers of the brain, breast, and χ2 test). The frequency of G→T transversions is higher in colon differ from those in lung cancers (Pfeifer et al. 2002). lung cancer tumors than in other tumors, except for liver The codons containing mutation hot spots are important cancers associated with geographic areas with evidence because they may allow determination of which carcino- of food contamination from aflatoxins (Greenblatt et gen caused the mutation. However, hot spot codons may al. 1994). exist solely as a consequence of phenotypic selection in In most internal cancers not strongly linked to tumors. To address this issue, studies have compared the smoking, such as breast, brain, and colorectal, the fre- mutational events in different types of cancers at a num- quency of G→T mutations is 8 to 10 percent (Figure ber of common hot spot codons. The major lung cancer 5.10). Nonsmokers have a higher percentage of G→A tran- mutation hot spots at codons 158, 245, 248, and 273 are sitions (42.5 percent) than do smokers (27.9 percent), a commonly G→T transversions in lung cancer but are difference that is also statistically significant. Figure 5.10 generally other mutation types (almost exclusively G→A) includes categories of both designated smokers and “all in other internal tumors not associated with smoking lung cancer cases minus nonsmokers.” This category (Pfeifer et al. 2002). is based on the knowledge that, overall, 90 percent of these lung cancers occur in smokers (Proctor 2001). The G→T Transversions in Lung Cancer proportion of G→T transversions, as well as the overall The major product of the diol epoxide BPDE reac- mutation pattern for all persons with lung cancers, tion with DNA is BPDE-N2-deoxyguanosine, which except nonsmokers, is similar to observations of research- induces mainly G→T transversions, depending on the ers for designated smokers (Figure 5.10). The difference sequence context, after a DNA polymerase carries out in G→T transversions in smokers versus nonsmokers error-prone translesion synthesis past this adduct (Eisen- may be attributable to bias in the database used, which stadt et al. 1982; Chen et al. 1990; Ruggeri et al. 1993; pools data from studies that differ in aims, size, and meth- Yoon et al. 2001) (see “Conversion of DNA Adducts to ods for ascertaining smoking status. However, in a more Mutations” earlier in this chapter). Using the UvrABC recent study of a series of 21 mutations that was designed incision method in combination with a ligation-mediated to address this possibility, TP53 gene mutations were polymerase chain reaction (LMPCR), scientists mapped found in 27.5 percent of current smokers, 15.8 percent of the distribution of BPDE and other PAH diol epoxide former smokers, and 4.8 percent of lifetime nonsmokers adducts at the nucleotide level along exons of the TP53 (Le Calvez et al. 2005). These observations suggest that gene in normal human bronchial epithelial cells treated the difference in G→T transversions in smokers versus with diol epoxide (Denissenko et al. 1996; Smith et al. nonsmokers may be larger than that indicated in the data- 2000). Frequent adduct formation occurred at gua- base, perhaps due to the misclassification in the database nine positions in codons 156, 157, 158, 245, 248, and of long-term former smokers as nonsmokers. 273. These positions of preferential formation of PAH To address the issue of whether the major histologic adducts are major mutational hot spots in human lung types of lung cancer show differences in TP53 mutational cancers (Figure 5.12). The only exception is codon 156, patterns, researchers analyzed the IARC TP53 mutation where G→T substitution commonly results in a pheno- database separately for these tumors (Figure 5.11). The fre- typically silent mutation and is therefore not selected dur- quencies of G→T transversions in the TP53 database were ing tumorigenesis. 31.4 percent in adenocarcinomas, 27.1 percent in SCCs, Researchers analyzed the distribution of BPDE-N2- 27.5 percent in SCLCs, and 34.7 percent in large-cell car- deoxyguanosine within TP53 exons by using stable isotope cinomas. Furthermore, the global mutation patterns were labeling LC-electrospray ionization tandem MS (Tretya- similar in the two main histologic types: adenocarcinoma kova et al. 2002; Matter et al. 2004). In this approach, and SCC. Thus, the different types of lung cancer in smok- specific guanine nucleobases withinTP53 gene sequences ers all show an excess of G→T transversions compared were labeled with 15N so the BPDE adducts originating with cancers unrelated to exposure to tobacco smoke. from these positions could be distinguished from the TP53 mutations do not occur at random along the lesions formed at other sites. Researchers observed an coding sequence. They are typically clustered at mutation excellent agreement with the data from the UvrABC- “hot spots,” which are within the DNA binding domain LMPCR method (Denissenko et al. 1996). All four dia- of the TP53 protein and span codons 120 to 300. Figure stereomers of BPDE-N2-deoxyguanosine were formed 5.12 shows the concordance of codon distribution of G→T preferentially at the frequently mutated TP53 codons 157, transversions (upper panel) along the TP53 gene in lung 158, 245, 248, and 273. The contributions of individual cancer with the distribution of adducts in this gene in

Cancer 279 Surgeon General’s Report

Figure 5.11 Patterns of TP53 gene mutations and percentage of G→T transversion mutations in different histologic types of lung cancer

Lung ADC (N = 392) Lung SCC (N = 527)

31.4% 27.1%

Lung LCC (N = 98) Lung SCLC (N = 138) A:T C:G A:T G:C A:T T:A 34.7% 27.5% G:C A:T G:C A:T at CpG G:C C:G G:C T:A Insertion/deletion/complex

Source: Data are from the R9 version (July 2004) of the International Agency for Research on Cancer TP53 mutation database (IARC 2006). Note: Cancers were classified according toInternational Classification of Diseases, Tenth Revision (ICD-10), World Health Organiza- tion 1994. The data set excluded lung cancers from nonsmokers. Cell lines and cancers metastatic to the lung were excluded, as well as all cancers with defined exposures other than tobacco (e.g., asbestos, radon, mustard gas, and air pollution).ADC = adenocarcinoma (ICD C34-8140/3); LCC = large-cell carcinoma (ICD C34-8012/3); N = total number of mutations; SCC = squamous cell carcinoma (ICD C34-8070/3); SCLC = small-cell lung carcinoma (ICD C34-8041/3).

diastereomers to the total adducts at a given site varied 2001). Methylation at CpG sites may increase the binding but were highest (70.8 to 92.9 percent) for (+)-trans- of planar carcinogenic compounds at the intercalation step BPDE-N2-deoxyguanosine (Matter et al. 2004). through the hydrophobic effect of the methyl group that A mechanistic basis for the selectivity of forma- can stabilize intercalated adduct conformations (Zhang tion of diol epoxide–DNA adducts in the TP53 gene is the et al. 2005a). However, the precise mechanism by which enhancement of adduct formation by 5-methylcytosine cytosine methylation at CpG sites enhances carcinogen bases present at CpG dinucleotide sequences (Denissenko binding and mutagenesis still needs to be determined. In et al. 1997; Chen et al. 1998; Weisenberger and Romano contrast, the presence of 5’-neighboring 5-methylcytosine 1999; Tretyakova et al. 2002; Matter et al. 2004). All CpG inhibited formation of guanine adducts by NNK metabo- sequences in TP53 coding exon 5 through exon 9 were lites (Rajesh et al. 2005). completely methylated in all of the tissues examined, Studies show that the preferential formation of including the lung (Tornaletti and Pfeifer 1995). In the BPDE adducts at methylated CpG sites is reflected in TP53 gene of lung cancers, the five major→ G T muta- the strongly enhanced mutagenesis at CpG sequences tional hot spots at codons 157, 158, 245, 248, and 273 after cells were treated with BPDE. This finding was dem- (Figure 5.12) consisted of methylated CpGs (Yoon et al. onstrated with three different mutated reporter genes,

280 Chapter 5 How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease including two chromosomal genes with methylated CpG adduction profile of acrolein in the P53 gene was similar sequences (Yoon et al. 2001). to that of BPDE and other PAH diol epoxides, indicating Methylated CpG sites are preferentially modified that this α,β-unsaturated aldehyde reacts at methylated by several carcinogens, including aromatic amines and CpG sites and, because of its high concentration in ciga- aflatoxins (Chen et al. 1998). However, the exact range of rette smoke compared with that of PAHs, could contrib- compounds that target methylated CpGs is not known. ute to the TP53 mutations observed in lung tumors from In one study, researchers did not observe a preferential smokers (Feng et al. 2006). mutagenesis at methylated CpGs by the aromatic amine Hussain and colleagues (2001) have shown that 4-ABP (Besaratinia et al. 2002). G→T transversions exposing bronchial epithelial cells to BPDE produces G→T resulting from 8-oxodeoxyguanosine are not specifi- transversions in the TP53 gene at lung cancer hot spot cally targeted to methylated CpG sequences (Lee et al. codons 157, 248, and 249. Nontumorous lung tissues from 2002a). A more recent study demonstrated that the DNA smokers with lung cancer carried a high TP53 mutational

Figure 5.12 Concordance between codon distribution of G→T transversions along TP53 gene in lung cancers (top) and distribution of adducts of benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE)–DNA adducts in bronchial epithelial cells (bottom)

G→T Transversions

10 157 245 273 8 158 249

6 248

% of all mutations 4

0 0.2 0.4 166 245 0.6 158 0.8 1.0 157 156 248 273 Relative adduct frequency BPDE Adducts Relative adduct frequency

Source: Adduct data were quantitated from Denissenko et al. 1996 and Smith et al. 2000. Note: Distribution of G→T mutations is shown along the TP53 coding sequence, and “hot spot” codons for major mutations are indicated. Mutation data from the International Agency for Research on Cancer TP53 mutation database were used. Cell lines and cancers metastatic to the lung were excluded, as well as all cancers with defined exposures other than tobacco (e.g., asbestos, radon, mustard gas, and air pollution). Length of bars indicates relative adduct frequency at major hot spots for adducts. For adducts of BPDE, the strongest binding site has a value of 1. Sites with values less than 0.2 are not shown. Numbers correspond to TP53 codon numbers.

Cancer 281 Surgeon General’s Report

Figure 5.13 Patterns of TP53 gene mutations and percentage of G→T transversions in smoking-associated cancers other than lung cancer

Oral (N = 929) A:T C:G A:T G:C A:T T:A G:C A:T 16.5% G:C A:T at CpG G:C C:G G:C T:A Insertion/deletion/complex

Esophageal SCC (N = 1,051) Esophageal ADC (N = 228)

7.9%

19.6%

Laryngeal cancer (N = 316) Bladder cancer (N = 836)

11.1%

25.9%

Source: Data are from R9 version (July 2004) of International Agency for Research on Cancer TP53 mutation database (IARC 2006). Note: Oral cancers include cancers of oropharynx, hypopharynx, gum, palate, floor of mouth, and tongue. Cases with defined exposures other than tobacco (e.g., asbestos, radon, mustard gas, and air pollution) were excluded. ADC = adenocarcinoma; N = total number of mutations; SCC = squamous cell carcinoma.

282 Chapter 5 How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease load at these codons, even when another TP53 mutation heavy smokers compared with nonsmokers (Brennan et was present in the tumor itself. DeMarini and colleagues al. 1995). In one study of oral and esophageal SCC, how- (2001) studied TP53 and KRAS mutations in lung tumors ever, the frequency of G→T transversions is only slightly from Chinese women who were nonsmokers and whose higher (16.5 and 19.6 percent, respectively) than those in tumors were associated with exposure to smoky coal cancers not strongly related to exposure to tobacco smoke containing high levels of PAHs and probably other com- (e.g., breast, colorectal, and brain cancers). The patterns pounds such as acrolein. The tumors showed a high per- of mutations in both oral and esophageal SCC are similar, centage of mutations that were G→T transversions in perhaps reflecting the importance of common risk factors, the KRAS oncogene (86 percent) or the TP53 gene (76 such as the combined use of tobacco and alcohol, infec- percent). In the TP53 gene, the mutations clustered at tions by human papilloma virus (Gillison and Shah 2003), the CpG-rich codons 153 through 158 and at codons 249 and various lifestyle behaviors such as tobacco chewing and 273. or consuming scalding hot beverages, as well as similar The site specificity of mutagenesis by PAH diol epox- histology in oral and esophageal tissues. In contrast, the ides implies that targeted adduct formation, in addition to mutation pattern is different in esophageal adenocarcino- phenotypic selection, is responsible for shaping the TP53 mas, with a high prevalence of G→T transversions at CpG mutational spectrum in lung tumors. According to the sites (Figure 5.13) and a type of mutation that could be IARC TP53 mutation database, more than 80 percent of associated with the overproduction of reactive nitrogen G→T transversions in lung cancers are targeted to gua- species due to inflammation (Ambs et al. 1999). nines on the nontranscribed DNA strand. This observation For bladder cancer, the mutation pattern shows an suggests that a preferential repair of DNA lesions occurs unusually high prevalence of G→A transitions at non-CpG on the transcribed strand. DNA repair experiments ana- sites. These mutations are not distributed at random, and lyzing BPDE adducts in the TP53 gene showed that the bladder-specific mutation hot spots can be seen at codons nontranscribed strand is repaired more slowly than is the 280 and 285, according to the IARC TP53 database. Both transcribed strand (Denissenko et al. 1998). These find- codons occur within the same primary sequence context ings support the proposal that both the initial DNA adduct (5’AGAG), which raises the possibility that this sequence levels and a bias in repair of DNA strands may contribute may be a preferential target site for a carcinogen involved to the mutational spectrum of the human TP53 gene in in bladder carcinogenesis. However, aromatic amines, lung cancer. a potent class of bladder carcinogens in tobacco smoke, produce mainly G→T mutations (Besaratinia et al. 2002). TP53 Gene Mutations in Other Smoking-Associated Cancers Limitations to the Study of TP53 Mutations and Smoking-Induced Cancer Of four cancer types analyzed, only SCC of the larynx showed a strong similarity with lung cancers. Preva- Although the study of mutations in the TP53 gene lence of G→T transversions was high (25.9 percent), and provides potentially useful leads for understanding mech- many occurred at PAH-target codons 157 and 245. A gra- anisms of tobacco carcinogenesis, this approach also has dient in the upper respiratory tract reflects the prevalence limitations. As already mentioned, various carcinogen- of TP53 G→T transversions in cancers of smokers. This DNA adducts can produce G→T transversions and even prevalence ranges from low in the oral cavity, to interme- similar spectra of mutations. In addition, most of this diate in the larynx, and high in various histologic types of research is not population based, and the studies may lung cancers. The gradient may reflect the existence of an be biased with respect to the stage of lung cancer repre- underlying, parallel gradient in the extent of exposure of sented. Finally, lack of a mutation in the TP53 gene does respiratory tract cells to carcinogens in tobacco smoke. not necessarily mean that the tumor is not related to In oral cancers, studies show that the TP53 mutation load smoking, because other uncharacterized changes could is proportional to the extent of smoking, with an almost have occurred. fourfold increase in the prevalence of mutations among

Cancer 283 Surgeon General’s Report

Loss of Mechanisms for Growth Control

Signal Transduction the survival of lung cancer cells, which is the focus of this section. Introduction Key Apoptotic Regulators Normally, cell signaling is very tightly regulated and begins with the transduction of the signal through a mem- One or more pathways may lead to apoptosis. Stress brane receptor. The signal is conveyed through a series of signals stimulate a pathway that activates proteins to intracellular proteins, and the result is the regulation of respond to DNA damage. These proteins subsequently cellular processes including proliferation and apoptosis. phosphorylate, activate, and stabilize the P53 protein. The In lung cancer cells, the processes governing these events activated P53 protein drives the transcription of genes are frequently deregulated by DNA-damaging mutations associated with cell-cycle arrest, DNA repair, and apop- induced by cigarette smoke or other alterations in the tosis. These genes include the BCL-2 family of proteins, molecules of numerous signaling pathways. The balance which consists of both proapoptotic and antiapoptotic between mechanisms leading to apoptosis (proapoptotic) members. The BCL-2 family of proteins interacts with the and those suppressing apoptosis (antiapoptotic) or sup- outer mitochondrial membrane to regulate the release of pressing increased proliferation will have a major impact cytochrome c, which results in the activation of aspartyl on lung tumor growth. Identifying and targeting signaling and cysteine proteases (caspases) (Igney and Krammer pathways that lead to therapeutic resistance could help to 2002). The caspases are crucial executioners of apoptosis neutralize a patient’s resistance to standard therapies. (Meier et al. 2000; Reed 2000). Once stimulated, the caspases activate endonucleases that subsequently cleave the Apoptosis DNA of the targeted cell into nucleosome-sized fragments, which is a common characteristic of apoptosis. Apoptosis was first described in 1972 (Kerr et al. A multitude of signaling molecules mediate the 1972). The term “apoptosis” is from the Greek word for mechanisms that govern apoptosis. An imbalance in pro- “falling off.” Apoptosis is a natural process that consists of apoptotic and antiapoptotic signaling events contributes a well-orchestrated cascade of distinct biologic and histo- to the development and progression of lung cancer. The logic events (Kerr et al. 1972). These events are critical for mechanisms for the deregulation of apoptosis can be eliminating injured or genomically unstable cells while categorized into (1) the decrease of signaling associated minimizing damage to surrounding normal cells (Martin directly with the induction of apoptosis and (2) the 2002). The induction of apoptosis prevents the malignant increase of signaling leading to the suppression of apop- growth of cancer cells (Rich et al. 2000). The deregulation tosis. This decrease may include mutations induced by of the mechanisms governing apoptosis is a distinctive cigarette smoke or other smoke-related mechanisms that characteristic of most cancer cells (Hanahan and Wein- activate oncogenes or inactivate tumor-suppressor pro- berg 2000). teins or other proapoptotic proteins. The increase may Apoptosis is characterized by morphologic features include mutations induced by cigarette smoke, certain including membrane blebbing, cell shrinking, and chro- kinases, other antiapoptotic proteins or transcription mosomal condensation. Apoptosis is generally believed factors, or overexpression or constitutive activation of to occur through two “effector” mechanisms: extrinsic growth factors. The end result of this deregulation usually (death receptor mediated) and intrinsic (mitochondrial includes a profound resistance to apoptosis. mediated) (Hengartner 2000). The extrinsic pathway is regulated by binding a “death receptor molecule” to the Regulation of Tumor Suppressors and cancer cell’s membrane receptor (i.e., death receptor). Proapoptotic Proteins The intrinsic pathway is mediated by rendering the mitochondrial membrane permeable, a phenomenon directly Decrease of important proapoptotic proteins of the influenced by the ratio of the interaction of proapoptotic BCL-2 family and tumor suppressors such as the P53 and and antiapoptotic proteins. In general, researchers believe RB proteins is a characteristic in many types of cancers, that the inactivation of apoptosis through the intrinsic including lung cancer. This decrease provides lung cancer pathway is the primary mechanism through which DNA- cells with a strong ability to resist apoptosis, which leads damaging agents from tobacco smoke act to enhance to a distinct advantage for cell survival (Figure 5.14).

284 Chapter 5 How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease

Figure 5.14 Tobacco-associated suppression of protease-9 (CASPASE-9), which is required to form the proapoptotic proteins and tumor- “apoptosome” complex that initiates apoptosis. The apop- suppressor proteins totic response is critically dependent on the ratio of the expression of proapoptotic and antiapoptotic BCL-2 mem- DNA-damaging bers (Zha et al. 1997; Korsmeyer 1999; Kroemer 1999; cigarette smoke components Reed 1999; Huang and Strasser 2000; Lutz 2000; Cheng et al. 2001; Ruvolo et al. 2001). A lack of BAX (Zhang et al. 2000a; Schmitt and Lowe 2002) or an increase of BCL-2 BAX or BCL-XL (Schott et al. 1995; Walczak et al. 2000; Chipuk et al. 2001) suppresses apoptosis, whereas a decrease of Wild-type TP53 BCL-XL or BCL-2 enhances apoptosis (Hayward et al. 2003). Dimers containing BAX and BCL-2 inactivate BAX and therefore inhibit apoptosis. In addition, phosphoryla- apoptosome tion of the BAD protein results in its inactivation, because Cytochrome c only the nonphosphorylated form of BAD can antagonize APAF1 the antiapoptotic BCL-2 or BCL-XL at the mitochondrial CASPASE-9 membrane (Hermeking 2003). Cell-cycle arrest DNA repair Nicotine suppresses the death of lung cancer cells Apoptosis by phosphorylation mediated by the extracellular signal- regulated kinase (ERK) of BCL-2 (Heusch and Maneckjee Apoptosis 1998; Mai et al. 2003). Conversely, NNK inactivates BAD through β-adrenergic receptors and protein kinase C Note: Tobacco-associated suppression of proapoptotic proteins (PKC), which promotes survival of NSCLC cells (Lahn et and tumor-suppressor proteins increases cell proliferation and al. 2004; Jin et al. 2005). Nicotine also stimulates cell sur- resistance to apoptosis. Two major signaling pathways that are vival through the phosphorylation and inhibition of BAD downregulated by DNA-damaging tobacco agents are the TP53 activated by β-adrenergic-receptor–mediated AKT-, PKA-, protein and the proapoptotic family of BCL-2 proteins. APAF1 = apoptotic-releasing factor 1; BAX = BCL-2 associated and/or ERK-dependent pathways (Jin et al. 2004a). These X protein; CASPASE-9 = cysteine-aspartic acid protease-9. studies show that BCL-2 family members are critical effec- tors of signaling pathways that promote cancer cell survival in response to components of cigarette smoke—in these cases, through direct receptor binding rather than BCL-2 Family Proteins DNA damage. In normal cells, stresses initiate apoptosis through the mitochrondrial or intrinsic pathway, and the BCL-2 P53 Protein family proteins are important mediators of the apoptotic The P53 pathway is clearly involved in cellular life response. These proteins are characterized by the pres- or death. The P53 tumor-suppressor protein can induce ence of one to four conserved BCL-2 homology (BH) the expression of BAX and additional proapoptotic mem- domains. The BCL-2 family can be divided into antiapop- bers of the BCL-2 family (Miyashita and Reed 1995; Yin et totic members: BCL-2, BCL-XL, and myeloid cell leuke- al. 1997; Oda et al. 2000a,b; Nakano and Vousden 2001). mia-1. The proapoptotic BCL-2 proteins are subdivided In addition to having direct effects on BCL-2 family pro- into two groups: the multidomain BAX subfamily (BAK, teins, the P53 protein also increases activity of the APAF1 BAX, and BOK) and the BH3-only proteins (BAD, BID, gene (Robles et al. 2001), which as indicated earlier, is and BIM) (Korsmeyer 1995; Hale et al. 1996; Adams and a member of the apoptosome complex and is critical for Cory 1998; Huang and Strasser 2000; Cory et al. 2003). the activation of CASPASE-9 to initiate apoptosis (Soen- The BCL-2 family of proteins appears to directly influ- gas et al. 1999) (see “BCL-2 Family Proteins” earlier in ence the permeability of the mitochondrial membrane to this chapter). Although it is primarily a nuclear protein, regulate apoptosis. P53 may function outside the nucleus by translocating The interaction of BAX with the mitochrondrial to the mitochondria, where it interacts directly with an- membrane causes the release of cytochrome c into the tiapoptotic proteins such as BCL-2 and BCL-XL to induce cytosol, where it binds to apoptotic-releasing factor 1. apoptosis (Mihara et al. 2003). The aberrant inactivation The binding of cytochrome c and apoptotic-releasing fac- of P53 leads to a deregulation of cell-cycle control and a tor 1 results in the activation of cysteine-aspartic acid suppression of many crucial proapoptotic pathways (Ford

Cancer 285 Surgeon General’s Report and Hanawalt 1995; Wang et al. 1995a,b; Offer et al. 1999; The inhibition of MDM2 leads to elevated P53 levels and Vogelstein et al. 2000; Zhou et al. 2001a). The loss of P53 apoptosis. The E2F protein can also activate proapoptotic function markedly decreases the sensitivity of lung cancer BCL-2 family members and caspases (Nahle et al. 2002; cells to apoptosis induced by exposure to tobacco smoke Hershko and Ginsberg 2004). or other stresses (Lowe et al. 1994). Regulation of Antiapoptotic Proteins and Effects Retinoblastoma Protein Studies document that many genes and signaling Inactivation of the RB protein results in the release proteins are overexpressed or display gain-of-function and activation of the transcription factor E2F (Flemington mutations in lung cancers. These include the EGFR et al. 1993; Helin et al. 1993). Some E2F family members gene, signal transduction and activator of transcription, induce expression of the genes important in apoptosis, PKC, RAS/MAPK, phosphatidylinositol-3 kinase (PI-3K)/ such as the P14ARF gene (DeGregori et al. 1997; Bates et AKT, PTEN, nuclear factor-kappa B (NF-kB), and COX al. 1998). The P14ARF protein is a negative regulator of (Figure 5.15). murine double minute 2 (MDM2), a P53 binding protein.

Figure 5.15 Protein-signaling pathways deregulated in lung cancer

DNA-damaging BCL-2 BAX cigarette smoke components Survival proliferation

PTEN STAT3 KRAS PI-3K PKC RAF AKT Antiapoptotic NF-κB IκKα proteins BAD or CASPASE-9 BCL-XL SURVIVIN MEK MDM2 MCL-1 RSK2 Antiapoptotic proteins P53 ERK BCL-XL BCL-2 C-MYC Survival COX-2 AP-1 proliferation Survival proliferation

Note: Many protein-signaling pathways deregulated in lung cancer represent a dense interactive network with a range of potential survival-enhancing effects. Tobacco- or cigarette smoke-associated activation of antiapoptotic proteins provides lung cancer cells with a distinct growth advantage. Major protein survival-signaling pathways activated by tobacco carcinogens are illustrated. KRAS and P53 are boxed to emphasize that KRAS and TP53 are the most commonly mutated genes. Mutationally activated KRAS is locked in its active form, resistant to the inactivating effects of GTPase-activating proteins, and cannot hydrolyze guanosine triphosphate to guanosine diphosphate. Similarly, mutated P53 cannot carry out many of its normal protective functions with respect to cell cycle control and apoptosis. AKT = protein kinase B; AP-1 = activator protein-1; BAD = BCL-associated death protein; BAX = BCL-2 associated X protein; CASPASE-9 = cysteine-aspartic acid protease-9; ERK = extracellular signal-regulated kinase; IkKa = I kappa-B kinase alpha; MDM2 = murine double minute 2 protein; MEK = mitogen-activated protein kinase kinase; NF-kB = nuclear factor- kappa beta; PI-3K = phosphatidylinositol 3-kinase; PKC = protein kinase C signaling pathway; PTEN = phosphatase and tensin homolog; RAF = v-raf murine leukemia viral oncogene; RSK2 = P90 ribosomal protein S6 kinase; STAT3 = signal transducer and activator of transcription 3.

286 Chapter 5 How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease

Epidermal Growth Factor Receptor motility migration in numerous cell types (Carpenter and Cantley 1996; Khwaja 1999; Rameh and Cantley 1999; Exposure of oral cells to cigarette smoke caused Blume-Jensen and Hunter 2001; Roymans and Slegers an increase in EGFR tyrosine kinase activity (Moraitis et 2001). Cell survival and oncogenic transformation require al. 2005). Signaling through EGFR can lead to survival PI-3K activation (Datta et al. 1999; Stambolic et al. 1999). signals that suppress dependent (downstream) apoptotic PI-3K–dependent kinases include 3-phosphoinositide– pathways or stimulate cell proliferation. Some evidence dependent protein kinase-1 (PDK1) and AKT (PKB). The suggests that EGFR signaling can influence the levels and PI-3K pathway can also be activated by the EGFR protein activities of antiapoptotic BCL-2 family members (Kari et and by an activated RAS protein (Rodriguez-Viciana et al. 2003). The EGFR gene is overexpressed and thus con- al. 1997). One of the first steps in PI-3K signaling is the stitutively activated in lung cancer cells (Sridhar et al. activation of PDK1, which phosphorylates and activates 2003) and bronchial preneoplastic lesions (Rusch et al. AKT (Coffer et al. 1998; Belham et al. 1999). AKT phos- 1995; Kurie et al. 1996; Piyathilake et al. 2002). In addi- phorylates and inactivates several proapoptotic proteins, tion, a truncated form of the EGFR protein (EGFRvIII) is including BAD and CASPASE-9. Other targets of AKT constitutively active in NSCLC cells (Okamoto et al. 2003). important in the regulation of apoptosis include glycogen- The expression of this mutant form of EGFR is associ- synthase-kinase-3 (Pap and Cooper 1998), the Forkhead ated with an increase in cell transformation and with the transcription factor FKHRL1 (Brunet et al. 1999), and the constitutive activation of important downstream signal- mammalian target of rapamycin/p70S6 kinase (McCor- ing pathways for survival, including the PI-3K/AKT path- mick 2004). Furthermore, AKT inactivates P53 by phos- way (Antonyak et al. 1998; Moscatello et al. 1998; Tang et phorylating MDM2, which increases the ability of MDM2 al. 2000). to bind to and promote P53 degradation (Ogawara et al. 2002). AKT also suppresses apoptosis by activating NF-kB RAS/Mitogen-Activated Protein Kinase through AKT phosphorylation of I kappa-B kinase alpha Activation of the RAS pathway sends a strong anti- (Ozes et al. 1999; Romashkova and Makarov 1999). apoptotic signal, and the constitutive activation of RAS Most NSCLC cells display an increase in PI-3K can transform normal cells. Oncogenic RAS protein has a activity that results in highly active AKT and other down- primary role in the development of lung cancer (Johnson stream mediators (Moore et al. 1998; Brognard et al. et al. 2001a). RAS activates several pathways, including 2001). AKT is important in the survival of lung cancer RAF/mitogen-activated protein kinase kinase (MEK)/ERK, cells and is constitutively activated in most NSCLC cell PKC, PI-3K/AKT, and NF-kB (Kauffmann-Zeh et al. 1997; lines to promote the survival of NSCLC cells under stress- Kennedy et al. 1997; Peeper et al. 1997; Baldwin 2001). ful conditions (Brognard et al. 2001). Studies have also These pathways are commonly deregulated by RAS in lung found AKT expression in SCLC tumor samples (Lee et al. cancers (Adjei 2001a,b). RAF/MEK/ERK pathway activa- 2002b; Mukohara et al. 2003), SCLC cell lines (Moore et tion can lead to changes in downstream gene expression al. 1998), SCLC tumors (Blackhall et al. 2003), mouse through the activation of activator protein-1 (AP-1). AP-1 tumors induced by tobacco carcinogens (West et al. 2003), is a well-characterized transcription factor composed of and human bronchial dysplastic lesions (Tsao et al. 2003). homodimers and/or heterodimers of the JUN and FOS (For additional details on AKT activation by components gene families (Angel and Karin 1991). AP-1 regulates the of cigarette smoke through receptor interactions, see transcription of various genes. Many stimuli, including “Activation of Cytoplasmic Kinase by Tobacco Smoke” tumor promoters, mediate AP-1 binding to the DNA of later in this chapter.) genes that govern cellular processes such as inflammation, proliferation, and apoptosis (Angel and Karin 1991). Nuclear Factor-Kappa B NF-kB is a rapidly induced transcription factor Phosphatidylinositol-3 Kinase, responsive to stress that functions to intensify the tran- Phosphatidylinositol 3’-Phosphatase, scription of a variety of genes, including those encoding and Protein Kinase B cytokines, growth factors, and acute response proteins PI-3K consists of a family of heterodimeric com- (Baldwin 1996). Nicotinic activation of nicotinic acetylcho- plexes, each composed of a p110 catalytic subunit and line receptors (nAChRs) stimulates NF-kB activity down- a regulatory subunit that exists primarily as a p85 form stream of ERK and AKT, which promotes tumor growth (Tolias et al. 1995; Vanhaesebroeck et al. 1997; Wymann and angiogenesis through the vascular endothelial growth and Pirola 1998). This family of proteins is involved in factor (VEGF) in vivo (Heeschen et al. 2001, 2002). More- the regulation of proliferation, viability, adhesion, and over, NF-kB activation by exposure to cigarette smoke in

Cancer 287 Surgeon General’s Report lung cancer cells induces the expression of COX-2 (Anto antiapoptotic proteins and/or suppression of proapoptotic et al. 2002; Shishodia and Aggarwal 2004). Recent results and tumor-suppressor proteins. suggest that nicotine, but not NNK, activates NF-kB– dependent survival of lung cancer cells in addition to their proliferation. These studies illustrate that the activation Cigarette Smoke and Activation of of NF-kB by nicotine or by cigarette smoke in its entirety through receptor binding can promote tumorigenesis in Cell-Surface Receptors in Cancer the lung through many mechanisms, including increased levels of VEGF and COX-2. Airway Epithelial Cells

Cyclooxygenase Nicotinic Acetylcholine Receptors COX-1 and COX-2 were shown to catalyze synthe- Neuronal nAChRs are large membrane-associated sis of prostaglandins from arachidonic acid. Research- proteins that are the first line of contact between cells and ers observed that COX-1 was constitutively expressed in components of cigarette smoke such as nicotine and NNK. most tissues, whereas COX-2 was inducible and found at These proteins were originally described as receptors for elevated levels in various cancers (Koki et al. 2002; Dan- acetylcholine (ACh). Their function in the brain has been nenberg and Subbaramaiah 2003; Dubinett et al. 2003). In studied in detail because of their ability to mediate the lung cancers, researchers have found COX-2 expression at addictive effects of nicotine. Each receptor is made up of most stages of tumor progression (Hida et al. 1998; Huang five subunits arranged in a barrel-like structure, creating et al. 1998; Wolff et al. 1998; Hosomi et al. 2000; Ander- a pore that allows calcium to enter the cell in response son et al. 2002; Fang et al. 2003). Others reported high to ligand binding. Nine alpha subunits (α2 through α10) levels of COX-2 in NSCLC and premalignant lesions, but and three beta subunits (β2 through β4) combine with COX-2 expression is less consistent in SCLC (Wolff et al. each other to form heteropentamers (combinations of α2 1998; Hosomi et al. 2000). Studies show that NNK induces through α6 with β2 through β4) or homopentamers (α7 a high expression of COX-2 in rats (El-Bayoumy et al. through α10). Each nAChR consists of 5 subunits, and 1999). Levels of COX-2 mRNA are about four times higher researchers have identified at least 12 subunits; thus, in the oral mucosa of smokers than in that of lifetime non- many functional nAChRs exist. Different ligands, includ- smokers (Moraitis et al. 2005). Researchers believe that ing nicotine, NNK, and ACh, have varying affinities for at least one role of COX-2 in cancer is associated with different nAChRs. Despite this complexity, the primary cell resistance to apoptosis and an increase in metastatic receptors that mediate the addictive effects of nicotine potential (Gupta and Dubois 2001). The supporting evi- are α4β2 nAChRs, whereas α7 nAChRs are high-affinity dence shows that COX-2 overexpression coincides with an receptors for NNK (Lindstrom 1997, 2003). Moreover, the increased BCL-2 expression (Tsujii and DuBois 1995) and an discovery that mutations in the α4 nAChR subunit lower increased stabilization of the antiapoptotic protein sur- the threshold for addiction raises the possibility that vivin (Li et al. 1998a; Krysan et al. 2004). Lung cancer genetic variations in these receptors could increase sus- cells that were induced to express COX-2 demonstrated ceptibility to nicotine dependence and exposure to car- an increase in survival time (Lin et al. 2001b), and COX-2 cinogens through smoking (Tapper et al. 2004). inhibitors stimulated apoptosis in lung carcinoma cells Although nAChRs were originally thought to be lim- (Hida et al. 2000; Yao et al. 2000; Chang and Weng 2001). ited to neuronal cells, studies have identified functional nAChRs in tissues outside the nervous system. This find- Summary ing raises the possibility that these receptors may mediate some of the systemic effects of smoking. In lung tissues, Apoptosis is commonly suppressed in lung cancer, researchers have discovered nAChRs in human bronchial which correlates with increases in cancer cell survival epithelial cells, vascular endothelial cells, pulmonary neu- and proliferation. Deregulation of the many pathways for roendocrine cells, neuroepithelial bodies, NSCLC cells, growth control (Figure 5.15) in lung cancer is attributable and SCLC cells (Tarroni et al. 1992; Maneckjee and Minna partly to interactions of carcinogens in cigarette smoke 1994; Macklin et al. 1998; Maus et al. 1998; Schuller and with the KRAS oncogene, the P53 tumor-suppressor Orloff 1998; Wang et al. 2001b; Fu et al. 2003; Schuller et gene, and other genes. These pathways represent a dense al. 2003; Song et al. 2003a,b; Tsurutani et al. 2005). interactive network with a range of potential effects on The stimulation of nAChRs by components of ciga- cell survival. Mechanisms associated with cigarette smoke rette smoke has biologic effects on cells that are important that increase resistance to apoptosis include activation of for the initiation, progression, and maintenance of cancer.

288 Chapter 5 How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease

The activation of nAChRs in lung epithelial cells by nico- Other Receptors tine or NNK promotes the survival and proliferation of The ERBB family is another group of EGFRs that human mesothelioma and lung cancer cells (Maneckjee indirectly mediate signal transduction associated with cig- and Minna 1994; Schuller and Orloff 1998; West et al. arette smoke. The four types of ERBB receptors are EGFR 2002; Schuller et al. 2003; Trombino et al. 2004; Tsurutani (HER-1), HER-2, HER-3, and HER-4. These receptors act et al. 2005). In normal cells, nicotine can stimulate prop- in pairs to stimulate downstream signaling pathways that erties consistent with cell transformation and the early mediate the survival and proliferation of both normal stages of cancer formation, such as increased cell prolif- cells and cancer cells. Ligands that bind to ERBB family eration, decreased cellular dependence on the extracellu- members include the epidermal growth factor TGFα and lar matrix for survival, and decreased contact inhibition, amphiregulin. In addition, receptors can be activated in which is the natural process of arresting cell growth when the absence of a ligand through overexpression of the two or more cells come in contact with each other (West receptors themselves. Both of these mechanisms play et al. 2003). Furthermore, nicotine stimulation of endo- a role in activation of these receptors mediated by ciga- thelial nAChRs promotes angiogenesis, another property rette smoke. of cancer (Heeschen et al. 2001, 2002; Zhu et al. 2003). The hypothesis that ERBB receptors mediate the Thus, the induced activation of nAChRs in lung tissues effects of cigarette smoke on airway epithelial cells by components of cigarette smoke can promote processes emerged from correlative clinical data and mechanisms required for development of cancer. defined in vitro. Clinical data include many reports of In addition to stimulating nAChRs directly, compo- EGFR and HER-2 overexpression in lung cancer (Hen- nents of cigarette smoke can indirectly stimulate nAChRs dler and Ozanne 1984; Cerny et al. 1986; Veale et al. 1987; by promoting the growth of tobacco-related cancers that Hirsch et al. 2003a; Tan et al. 2003). In addition, some express and secrete ACh, the endogenous ligand for these studies have shown that EGFR overexpression and activa- receptors. SCLC and NSCLC cells synthesize, transport, tion in human lung cancers correlate with shorter sur- and release ACh in vitro, which stimulates proliferation of vival times, suggesting that they play an important role in cancer cells through the autocrine activation of nAChRs development of cancer (Kern et al. 1990; Kanematsu et al. (Song et al. 2003a; Proskocil et al. 2004). This finding 2003; Selvaggi et al. 2004). suggests that there are many mechanisms for activation Clinical data also support the hypothesis that ERBB of nAChRs in lung cancer and further emphasizes the expression and activation change with exposure to ciga- importance of these receptors in the biology of tobacco- rette smoke or its components. Studies have demon- related cancer. strated the overexpression of EGFR and ERBB3 in the bronchial epithelium of smokers (Yoneda 1994; O’Donnell ß-Adrenergic Receptors et al. 2004). Results of mechanistic in vitro studies, such The β-adrenergic receptors are neuronal recep- as the demonstration that NNK-induced transformation tors that may play a role in mediating effects of cigarette of lung epithelial cells is associated with an increase in smoke related to signal transduction. NNK is structur- EGFR expression, support these observations (Lonardo ally similar to epinephrine, the endogenous ligand for et al. 2002). Moreover, exposure to nicotine alone can the β-adrenergic receptor, suggesting that in addition to increase the expression of EGFR in cervical cancer cell binding nAChRs, NNK may bind to these receptors. Once lines (Mathur et al. 2000). Studies also demonstrate that bound to β-adrenergic receptors, NNK can stimulate the exposure to tobacco smoke increases the activity of EGFR, release of arachidonic acid (Schuller et al. 1999; Weddle and metabolites of B[a]P induce activation of EGFR and et al. 2001). The enzyme COX-2 converts arachidonic acid downstream signaling pathways that promote prolifera- to prostaglandin E2, which mediates inflammation and tion (Burdick et al. 2003; Moraitis et al. 2005). These stud- promotes cell survival and proliferation in cancer. This ies support the idea that components of cigarette smoke finding is important because cell lines from human lung modulate the expression and activation of the ERBB fam- cancer overexpress the β-adrenergic receptor (Schuller et ily of receptors. al. 2001), and several studies suggest that the presence or In addition to increasing the expression of ERBB expression of arachidonic acid is a risk factor for pulmo- family members, components of tobacco smoke stimu- nary adenocarcinomas (Alavanja et al. 1993, 2001). Thus, late cells to produce ligands that activate the receptors. In these studies indicate that the β-adrenergic receptor may clinical specimens, studies have described the coexpres- be an important mediator of signal transduction pathways sion of EGFR and its ligand TGFα in human NSCLC. In associated with exposure to cigarette smoke. one study, both EGFR and TGFα were expressed in 38

Cancer 289 Surgeon General’s Report percent of the cases of NSCLC examined (Rusch et al. Activation of Cytoplasmic Kinase 1993). In a second study, 72 percent of SCCs and 34 by Tobacco Smoke percent of adenocarcinomas expressed both EGFR and Activation of cell-surface receptors by components TGFα (Hsieh et al. 2000). This finding may be clinically of tobacco smoke stimulates downstream kinases that important because a retrospective analysis showed that the mediate cancer cell survival, proliferation, and resistance coexpression of EGFR and TGFα is an indicator of a to chemotherapy. The best-described kinases activated poor prognosis (Tateishi et al. 1990). These studies sug- by smoking are AKT, ERK, PKC, and PKA. All of these gest that the stimulation of ERBB ligands induced by kinases can be activated by cigarette smoke components cigarette smoke may be an important mechanism of sig- through nAChRs, but ERBB family members also mediate nal transduction. AKT and ERK activation by cigarette smoke components. Consistent with the clinical data, in vitro studies In addition, β-adrenergic receptor activation by cigarette show that condensate from cigarette smoke stimulates smoke components can activate PKA and PKC. Thus, the release of amphiregulin and TGFα from the cell mem- these proteins can be activated by tobacco smoke compo- brane, which leads to the autocrine activation of EGFR nents through multiple receptor-mediated mechanisms, and cell proliferation (Richter et al. 2002; Lemjabbar et al. suggesting that the proteins are important mediators of 2003; Moraitis et al. 2005). Several studies demonstrate smoking-induced signal transduction. that cigarette smoke condensate activates matrix metalloproteinases (MMPs), which are enzymes on the extracel- Protein Kinase B lular surface of cells that cleave these ligands from the extracellular matrix. Support for these in vitro observa- The serine/threonine kinase AKT may be the critical tions comes from the demonstration that MMP activity is effector of signaling induced by cigarette smoke, because higher in lung tissues from smokers than in those from AKT is stimulated in response to the activation of nAChRs, nonsmokers (Kang et al. 2003; Kangavari et al. 2004). β-adrenergic receptors, and the ERBB family of receptors. In addition to stimulating downstream kinases, Moreover, AKT controls many cellular processes that EGFR activation by cigarette smoke may provide a mecha- promote cell survival, proliferation, and the resistance of nistic link to the increased inflammation characteristic cancer cells to chemotherapy. Clinical data also suggest of smokers by increasing COX-2 activity (see “Activation that AKT activation indicates a poor prognosis in many of Cytoplasmic Kinase by Tobacco Smoke” later in this tobacco-related cancers. Thus, activation of this kinase chapter). In vitro data suggest that autocrine activation by components of tobacco smoke can affect many cellular of EGFR, by the expression of the TGFα and AREG genes processes important for the initiation, growth, and pro- induced by tobacco smoke, stimulates COX-2 expression gression of tumors. (Moraitis et al. 2005). Cigarette smoke also increases AKT might be important for the initiation as well as COX-2 expression by lung fibroblasts in vitro, and B[a]P the maintenance of tobacco-related cancers. Nicotine and increases COX-2 expression by oral epithelial cells (Kelley NNK cause rapid AKT activation through different nAChRs et al. 1997; Martey et al. 2004). Thus, many in vitro stud- (West et al. 2003; Tsurutani et al. 2005). B[a]P metabo- ies demonstrate that EGFR activation by components of lites activate AKT in breast epithelial cells, although the cigarette smoke can contribute to inflammation through cellular receptor responsible for the effect has not been the increased expression and activation of COX-2. identified (Burdick et al. 2003). Furthermore, nicotine- Clinical data support the validity of these in vitro induced AKT activation in normal human bronchial cells observations. For example, studies document increased or in small airway epithelial cells promotes cell survival, levels of COX-2 in the oral mucosa of smokers (Moraitis proliferation, and anchorage-independent growth, all et al. 2005) and in urothelial tissues from smokers with of which are properties of transformed cells (West et al. bladder cancer (Badawi et al. 2002). Moreover, COX-2 is 2003). These studies are important because they suggest expressed only in neoplastic epithelial cells, not in nor- that AKT activation by tobacco smoke components may mal bronchial epithelial cells (Hastürk et al. 2002). COX-2 precede the formation of DNA mutations that cause can- overexpression in lung cancer is associated with tumor cer. Thus, AKT activation could serve as a biochemical angiogenesis and survival and proliferation of tumor cells gatekeeper for lung carcinogenesis by promoting the sur- (Riedl et al. 2004) and with a poor prognosis in NSCLC vival of cells that would normally die from DNA damage. (Achiwa et al. 1999; Yuan et al. 2005). Thus, the stimu- In addition to promoting AKT-dependent growth lation of EGFR that leads to COX-2 activity by exposure and survival of normal epithelial cells, tobacco smoke to cigarette smoke is another mechanism mediated by a components have similar effects on cells throughout the growth factor receptor to promote cell survival and prolif- phenotypic spectrum of transformation. In a mouse model eration in carcinogenesis. of NNK-induced lung tumorigenesis, an increase in AKT

290 Chapter 5 How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease activation was associated with an increase in the progres- resistance to cell death (Zajac-Kaye 2001). Thus, tobacco sion of NNK-induced lung lesions (West et al. 2004b). In smoke components stimulate ERK, which promotes cell human lung cancer cells, nicotine or NNK activated the proliferation and contributes to the poor prognosis of AKT pathway and stimulated AKT-dependent prolifera- lung cancer patients with this biochemical alteration. tion through nAChRs (Tsurutani et al. 2005). Moreover, these researchers showed that nicotinic activation of AKT Protein Kinase C increased survival of lung cancer cells after treatment with The PKC kinases also mediate cellular responses chemotherapeutic agents or radiation (Tsurutani et al. to exposure to tobacco smoke. Several isoforms of PKC 2005). The fact that tobacco smoke components activate can promote cell survival, most notably PKCα. Nico- AKT and promote the survival of cancer cells is important, tine- and NNK-induced activation of PKCα through the and it is supported by the finding that cancer patients who β-adrenergic receptor promotes the survival of lung continue to smoke during chemotherapy have a worse cancer cells (Schuller et al. 2003). In addition, nicotinic prognosis than those who stop smoking (Johnston-Early activation of nAChRs activates PKC in human bronchial et al. 1980; Browman et al. 1993; Videtic et al. 2003). epithelial cells, as well as in lung cancer cells (Maneck- Clinical data and preclinical models support the jee and Minna 1994; Carlisle et al. 2004). In SCLC, NNK- hypothesis that AKT activation is an early event in carci- induced activation of nAChRs causes PKC activation nogenesis. AKT is activated in preneoplastic lung lesions associated with cell proliferation (Jull et al. 2001). induced by exposure to NNK (West et al. 2004a) and in dys- Another PKC isoform, PKCd, seems to act atypically in plastic lung lesions from smokers (Massion et al. 2004). In NSCLC cells. Activation of PKCd in NSCLC promotes cell addition, AKT activation is associated with poor survival survival and resistance to chemotherapeutic agents (Clark in patients with tobacco-related cancers, including lung et al. 2003), and nicotine can prevent chemotherapy from cancer and pancreatic cancer (David et al. 2004; Hirami inhibiting PKC (Heusch and Maneckjee 1998). Like AKT, et al. 2004; Yamamoto et al. 2004; Tsurutani et al. 2005). nicotinic activation of PKC has ramifications for smok- Together, these clinical studies support the idea that AKT ers by contributing to chemotherapeutic resistance. This plays an important role in the formation and maintenance finding is consistent with the finding that patients with of tobacco-related cancers. lung cancer who continue to smoke during chemotherapy have a worse prognosis than those who stop smoking. Extracellular Signal-Regulated Kinases A clinical study also demonstrates the importance In addition to AKT, ERK may play an important role of PKC in tobacco-related cancers. Lahn and colleagues in smoking-related cancers because it can be activated (2004) found that PKCα is overexpressed in a subset of in response to components of tobacco smoke through NSCLC. Collectively, the results suggest that the activa- both nAChR and ERBB receptors. In normal cells, ERK tion of prosurvival PKC isoforms by cigarette smoke is an is activated in response to many extracellular signals and important mechanism of cell proliferation mediated by stimulates cell proliferation. In SCLC and pulmonary neu- nAChRs and β-adrenergic receptors in carcinogenesis. roendocrine cells, NNK-induced activation of nAChR leads to the activation of RAF-1 and its downstream effector ERK Protein Kinase A (Jull et al. 2001; Schuller et al. 2003). In addition, B[a]P Another cytoplasmic kinase activated by compo- metabolites activate ERK (Burdick et al. 2003), and nico- nents of tobacco smoke is PKA. Under normal physi- tine activates ERK and promotes cell survival (Heusch and ological conditions, PKA is stimulated through the Maneckjee 1998). Thus, like AKT, ERK can be activated as production of cyclic adenosine monophosphate by activated an acute response to tobacco smoke components. Because G protein–coupled receptors. Nicotinic activation of PKA ERK and AKT can promote cell survival and proliferation, occurs through both nAChRs and β-adrenergic receptors early activation of both kinases may contribute to the ini- (Dajas-Bailador et al. 2002; Jin et al. 2004a). The primary tiation, promotion, and progression of cancer. effect of nAChR-mediated PKA activation was an increase Researchers have also described ERK activation in cell proliferation. Nicotinic activation of PKA through in tobacco-related cancers, thus validating the mecha- β-adrenergic receptors, however, promoted cell survival. nisms defined in vitro. ERK activation is associated with Although the data on PKA are limited, they suggest that poor survival in SCLC, which occurs almost exclusively PKA might be an important mediator of signal transduc- in smokers (Blackhall et al. 2003). The overexpression of tion, mediating cell survival and proliferation in response C-MYC, an oncogene activated by ERK, has been described to activation by nAChRs and β-adrenergic receptors. in lung cancer and promotes proliferation as well as

Cancer 291 Surgeon General’s Report

Downstream Targets of Signaling Cascades This regulation is lost if either the P16 or the RB gene is Mediated by Tobacco Smoke inactivated. The reciprocal relationship between RB alterations in SCLC and P16 alterations in NSCLC sup- Activation of cell-surface receptors induced by com- ports the premise that dysfunction within the RB pathway ponents of tobacco smoke and the subsequent activation is a major target in research on the genesis of lung cancer of cytoplasmic kinases stimulate other proteins that dic- (Swafford et al. 1997). tate cellular responses, such as cell survival and proliferation. Although activated kinases have many downstream Critical Pathways Inactivated in Non-Small-Cell targets, the two most studied are the transcription fac- and Small-Cell Lung Cancer tor NF-kB and proteins in the BCL-2 family. Activation of these proteins by tobacco smoke components through More than 50 genes are inactivated by gene pro- signaling cascades promotes processes involved in initia- moter hypermethylation in lung cancer, and new genes tion, progression, and maintenance of cancers (see “Sig- are still being identified through genomewide screening nal Transduction” earlier in this chapter). approaches (Suzuki et al. 2002; Palmisano et al. 2003). The pathways and genes involved are summarized in Table 5.11. Gene Promoter Hypermethylation Of particular importance is the DNA repair gene AGT, which protects cells from the carcinogenic effects in Cancer Induced by Tobacco of alkylating agents by removing adducts from the O6 Smoke position of deoxyguanosine (see “Repair of DNA Adducts” earlier in this chapter). Failure to repair this DNA adduct Alternative to Mutation could lead to mutations in genes such as KRAS and TP53. AGT is inactivated by gene promoter methylation in 24 to Gene promoter hypermethylation is an epigenetic 48 percent of adenocarcinomas (Esteller et al. 1999; Zöch- change of a gene involving extensive methylation at the 5’ baur-Müller et al. 2001; Pulling et al. 2003). SCLC studies position of C in CpG islands within the promoter region conducted for methylation of this gene are limited. Stud- and often extending into exon 1 of regulatory genes (Jones ies have reported an association between AGT promoter and Baylin 2002; Herman and Baylin 2003). “Epigenetic” hypermethylation and a G→A transition mutation at CpG refers to alteration in gene expression resulting from sites within the TP53 gene in NSCLC (Wolf et al. 2001). changes other than DNA sequence. The end result of this In contrast, no association was found between AGT gene process can be loss of gene transcription and therefore the methylation and a transition mutation in codon 12 of the silencing of gene function. KRAS gene from adenocarcinomas (Pulling et al. 2003). The RAS superfamily of GTP-binding proteins Inactivation of the P16 Gene in Lung Cancer plays an important role in signal transduction pathways One region on chromosome 9p contains the that control cell proliferation, differentiation, and death CDKN2A (P16) tumor-suppressor gene (Kamb et al. 1994; (Campbell et al. 1998; Downward 2001) (see “Activation Merlo et al. 1994). Mutations within the P16 coding of Oncogenes in Lung Cancer” earlier in this chapter). sequence are uncommon in lung cancer (Kamb et al. Researchers identified a new family of genes that encode 1994). In contrast, this gene is inactivated by hypermeth- RAS-binding proteins. One of these genes, RASSF1A, is ylation at prevalences up to 60 percent and 70 percent in located at chromosome 3p21 and inactivated in 30 per- adenocarcinomas and SCC of the lung, respectively (Merlo cent of NSCLCs and in 100 percent of SCLCs (Dammann et al. 1995; Belinsky et al. 1998; Kim et al. 2001; Zöchbaur- et al. 2000; Burbee et al. 2001). Attempts to determine Müller et al. 2001; Divine et al. 2005). This discovery of the function of this gene are continuing (Agathanggelou inactivation of a tumor-suppressor gene by hypermeth- et al. 2003). RASSF1A protein forms a heterodimer with ylation in lung cancer and identification of such inacti- NORE1A, which allows it to bind with the proapoptotic vation of the P16 gene launched an area of research to protein MST1 (Khokhlatchev et al. 2002). Binding RAS uncover other genes inactivated by this mechanism. The to this complex may mediate RAS-dependent apopto- targeting of P16 for inactivation is likely attributable to sis. NORE1A is also silenced by methylation in NSCLC the critical function of this gene in the cell, which is to but not in SCLC (Hesson et al. 2003). Therefore, silenc- inhibit CDKs that bind cyclin D1 and phosphorylate the ing either RASSF1A or NORE1A could effectively block RB gene product (Lukas et al. 1995; Weinberg 1995). apoptosis mediated by RAS activation. Two other RASSF

292 Chapter 5 How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease

Table 5.11 Pathways altered through gene silencing Gene Silencing in Lung Cancer by promoter methylation The most extensively studied gene with respect to Methylation prevalence (%) timing of methylation in NSCLC is P16. Examination of biopsy specimens from premalignant lesions obtained Non-small- from people without SCLC or from different airways or cell lung Small-cell at different bronchial generations revealed a progressive Pathway Gene cancer lung cancer increase in the prevalence of P16 methylation as the dis- Cell cycle P16 26–70 0 ease developed. The frequency of P16 methylation was PAX5a 64–74 ND 17 percent in basal cell hyperplasia, and the frequency PAX5ß 52–61 ND increased incrementally over the histologic stages to 60 CHFR 10–19 ND percent in SCCs (Belinsky et al. 1998). Further studies examined bronchial epithelial cells obtained by bronchos- DNA repair AGT 27–47 0–19 copy from cancer-free smokers and found that inactiva- Apoptosis DAPK 24–48 33 tion of the P16 gene is likely one of the earliest events in CASPASE-8 0 35–52 lung cancer (Belinsky et al. 2002). Researchers detected FAS ND 40 P16 methylation in specimens from 25 of 137 biopsy pro- TRAIL-R1 ND 40 cedures (18 percent) classified as histologically normal, FHIT 38–45 ND metaplasia, or mild dysplasia. In contrast, no P16 meth- RAS RASSF1A 30 100 ylation was found in biopsy specimens obtained from life- signaling RASSF4 20 20 time nonsmokers. Researchers used an animal model to NORE1A 24 0 determine the timing of P16 methylation in adenocarcinomas. In rats, 94 percent of adenocarcinomas induced Invasion E-CADHERIN 16–19 ND by NNK were hypermethylated at the P16 promoter; this H-CADHERIN 43 ND change was frequently detected in precursor lesions to the TIMP3 19–24 ND tumors: adenomas and hyperplastic lesions (Belinsky et LAMA3 27–58 65 LAMB3 20–32 77 al. 1998). LAMC2 13–32 58 Inactivation of the AGT gene appears to be a later MYO18B 31 45 event in lung cancer than is the inactivation of P16. Only 3 of 40 biopsy specimens (8 percent) from heavy smokers Source: Belinsky 2004. Reprinted with permission from with histologies including normal, hyperplasia, metapla- Macmillan Publishers Ltd., © 2004. sia, and dysplasia showed methylation of the AGT gene Note: ND = not determined. (Pulling et al. 2003). In addition, the prevalence of AGT methylation increased between stage I adenocarcinoma and stages II to IV. Finally, of the 137 bronchial biopsy specimens studied, 3 percent of the DAPK gene and none family members appear to be involved in lung cancer of the RASSF1A genes showed methylation, which sug- development. Studies show that RASSF2 binds directly to gests that the silencing of these genes likely occurs after the KRAS gene in a GTP-dependent manner and appears P16 inactivation in SCC (Pulling et al. 2003). In con- to promote both cell-cycle arrest and apoptosis through trast, the inactivation of DAPK by methylation in alveolar this interaction (Vos et al. 2003). The expression of this hyperplasias in a murine model of lung adenocarcinomas gene is markedly reduced in some lung cancer cell lines, suggests a role for this gene in the early development of thus suggesting silencing by gene promoter hypermethyl- adenocarcinomas (Pulling et al. 2004). ation. There is support for this mechanism of inactivation in studies on colon cancer that document hypermethyl- Gene Promoter Hypermethylation, Prognosis, ation of this gene in 70 percent of tumors (Hesson et al. and Clinical Risk Factors 2005). A third member of this gene family, RASSF4, shares 25 percent homology with RASSF1A and 40 percent with Numerous studies have evaluated relationships RASSF2 and is methylated in approximately 20 percent of between gene promoter methylation and established clini- NSCLCs and SCLCs (Eckfeld et al. 2004). Thus, the loss cal risk factors such as smoking dose and tumor stage. of function among members of the RASSF gene family is In addition, researchers have examined in detail the effect important to the development of lung cancer. of gene-specific methylation on the survival of patients

Cancer 293 Surgeon General’s Report with a diagnosis of early-stage lung cancer. Results from Cigarette smoking was also associated with an increased investigations of the most commonly studied genes in risk of promoter methylation of the P16 gene in bladder lung cancer are highlighted here. cancer (Marsit et al. 2006). P16 methylation was significantly associated with pack-years of smoking and with an independent risk factor that predicts a shorter survival for patients who had resec- Molecular Epidemiology of tion of a stage I adenocarcinoma (Kim et al. 2001). Several other studies also support P16 methylation as a prognostic Cell-Cycle Control and Tobacco- factor for survival of patients who had resection of a stage Induced Cancer I adenocarcinoma (Suzuki et al. 2002; Wang et al. 2004a). In contrast, RASSF1A methylation in stage III NSCLC was Introduction a stronger predictor of poor survival than was P16 meth- Cell-cycle checkpoints delay cell-cycle progression, ylation (Wang et al. 2004a). These two genes may differ thereby affording adequate time for DNA repair to occur. in that the silencing of P16 is an early event involved in Such checkpoint signaling also activates pathways lead- initiation of tumorigenesis, whereas RASSF1A methyla- ing to apoptosis if the damage cannot be repaired. The tion is a later event more likely involved in progression introduction of new techniques of profiling gene expres- of tumorigenesis. Thus, the methylation of RASSF1A may sion has enabled researchers to comprehensively evaluate lead to a more aggressive tumor phenotype. This hypoth- activity of proteins in regulating the cell cycle (Singhal esis is supported by the more frequent involvement of et al. 2003). A hallmark of the neoplastic cell is the abil- RASSF1A methylation in tumors with a vascular invasion, ity to disrupt the tightly regulated cell-cycle control and pleural involvement, and a poorly differentiated histol- enable the cell to bypass checkpoints, especially at the ogy (Tomizawa et al. 2002). Persons who started smoking G /S and G /M boundaries (Hanahan and Weinberg 2000). before 19 years of age were 4.2 times more likely to have 1 2 Persons with defects in cell-cycle checkpoints (acquired or methylation of the RASSF1A gene than were those who inherited) could therefore exhibit chromosome damage, started smoking after 19 years of age (Kim et al. 2003). genomic instability, and increased susceptibility to This research also suggests that for patients with stage I tobacco carcinogenesis. or stage II NSCLC at diagnosis, methylation of this gene is In vitro studies show an association between expo- associated with a poorer prognosis (Kim et al. 2003). sure to tobacco carcinogens and the disruption of cell- cycle control (Khan et al. 1999). Furthermore, Jin and Other Tobacco-Related Cancers colleagues (2004b) provide data showing that NNK pro- In addition to the studies on lung cancer described motes cell survival and proliferation through phosphory- here, other studies have shown association of cigarette lation of the proteins BCL-2 and C-MYC. Studies implicate smoking with gene promoter hypermethylation in other tobacco carcinogens in genetic alterations in the P16-RB tobacco-related cancers, such as the head and neck and and P14ARF-P53 pathways, mainly through the forma- bladder. Aberrant promoter methylation is common in tion of DNA adducts. Variations in cell-cycle checkpoints head and neck cancer and has been detected by using might also be attributed to functional polymorphisms saliva samples (Rosas et al. 2001). Promoter methylation in cell-cycle control genes. The SNP500Cancer Database of the P16, DAPK, E-CADHERIN, and RASSF1A genes was reports that 27 genes related to the cell cycle are polymor- associated with smoking and commonly found in head phic, and these include genes that control checkpoints for and neck cancer (Hasegawa et al. 2002). P16 promoter both the G1/S and G2 phases of the cell cycle. However, hypermethylation and the loss of P16 protein expression only a few genes, including CCND1, TP53, P21, and P73, were detected in head and neck SCC; loss of expression have been studied in tobacco-related cancers. correlated significantly with a history of alcohol- con sumption or tobacco use (Ai et al. 2003). The prevalence CCND1 Gene of P15 methylation in the healthy epithelium of patients The CCND1 gene, together with CDK4, P16, and with head and neck SCC who had long-term smoking the tumor-suppressor gene RB, comprise a linked system and drinking behaviors was significantly higher than that governing the passage of the cell through the cell cycle in nonsmokers (Wong et al. 2003). Another study sug- (Betticher et al. 1997). A common finding in a variety of gested that P15 gene methylation could be induced by cancers is the amplification or overexpression of CCND1, chronic smoking and drinking and could play a role in which contributes to tumor initiation, progression, the early stages of head and neck SCC (Chang et al. 2004).

294 Chapter 5 How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease and outcome, such as death. A G→A polymorphism at TP53 Gene codon 242 in the conserved splice donor region of exon Studies have reported 14 polymorphisms in the 4 increases alternate splicing (Betticher et al. 1995). TP53 gene, 3 of which have been widely studied: a G→C The alternate transcript appears to encode for a protein- polymorphism at codon 72 (proline/arginine), a 16bp missing sequence involved in protein turnover, and there- insertion in intron 3, and a G→A transition in intron 6. fore, the encoded protein may have a longer half-life. This Polymorphisms in codons 21, 36, and 213 are silent. The extended half-life, in turn, would facilitate passage of dam- polymorphism in codon 47 involves a rare allele with a fre- aged cells through the checkpoint for the G /S phase and 1 quency less than 5 percent. The codon 72 polymorphism promote proliferation rather than apoptosis. Researchers on exon 4 produces variant proteins with an arginine have studied the association between the CCND1 genotype (CGC) or proline (CCC) at the site. Thomas and colleagues and cancer risk in several tobacco-related cancers. Qiul- (1999) reported differences between the two variants in ing and colleagues (2003) reported that the CCND1 *A/*A their ability to interact with basic elements of the tran- genotype was associated with a significantly increased scriptional machinery and to induce apoptosis. Weston risk of lung cancer (OR = 1.87; 95 percent CI, 1.01–3.45) and colleagues (1992) reported an increased frequency compared with that for the *G/*G genotype. The risk was of the proline allele in lung adenocarcinoma, which was even higher in young persons and men. A similar finding consistent with findings in a Japanese study of lung cancer was reported in cancer of the head and neck (Zheng et (Kawajiri et al. 1993). Jin and colleagues (1995) reported al. 2001). These investigators demonstrated that carriers significantly higher risks for the *PRO/*PRO genotype of the *A/*A genotype, on average, had diagnoses of can- among patients with lung cancer who were younger than cer 3.5 years earlier than did carriers of the*G/*G geno- 55 years of age and among patients reporting fewer than type. Wang and colleagues (2002) reported that the *A/*A 30 pack-years of smoking. In a study of NSCLC, Nelson genotype was associated with a significantly higher risk and coworkers (2005) found that mutation on the *PRO of transitional cell carcinoma of the bladder than that for allele was associated with a significantly worse outcome the *A/*G plus *G/*G genotypes (OR = 1.76; 95 percent than that for patients with no mutation or with mutation CI, 1.09–2.84). However, neither Cortessis and colleagues on the *ARG allele. (2003) nor Yu and colleagues (2003) reported significant Mutations in intron sequences may initiate aberrant associations with either bladder cancer or esophageal SCC, pre-mRNA splicing that results in a defective protein (Hil- respectively. Spitz and colleagues (2005) demonstrated an lebrandt et al. 1997) or that may influence mutations in increased risk for lung cancer associated with this poly- the coding region. Either result would increase the likeli- morphism. The risk estimate was 1.35 (95 percent CI, hood of a deleterious phenotype (Malkinson and You 1994). 1.05–1.73) for the *A/*A and *A/*G genotypes compared Biroš and colleagues (2001) reported a higher percentage with the *G/*G genotypes. of the intron 6 variant allele in patients with lung cancer than in control participants. However, Birgander and col- P21 Protein leagues (1995) found no association of the allele with lung Cell-cycle inhibitor protein P21 (WAF1/CIP1) acts as cancer. Several studies estimated the pairwise haplotype a checkpoint regulator for the G1/S and G2/M phases. Mar- frequencies for the polymorphisms in exon 4 and introns wick and colleagues (2002) showed a significant increase 3 and 6. The researchers proposed that the P53 haplotypes in P21 mRNA expression in alveolar epithelial cells after were associated with a higher risk for lung cancer (Birgan- exposure to condensate from cigarette smoke and con- der et al. 1995; Biroš et al. 2001). In one study of 635 pairs cluded that oxidative stress induced by cigarette smoke of lung cancer patients and control participants, variant modulates the expression of P21. alleles of TP53 exon 4, introns 3 and 6, and their variant Three studies of lung cancer have examined the haplotypes were associated with an increased risk of lung association of cancer risk with a polymorphism of P21 at cancer (Wu et al. 2002). In a meta-analysis of TP53 poly- codon 31 (SER31ARG), but the findings were inconsistent. morphisms and lung cancer risk that included data from Själander and colleagues (1996) reported an increased fre- 16 case-control studies, Matakidou and colleagues (2003) quency of the variant allele (*ARG) among patients with concluded that persons with the P53 exon 4 *PRO/*PRO lung cancer (p <0.004). Two other studies failed to repli- genotype had a 1.18-fold increase in lung cancer risk cate this finding (Shih et al. 2000; Su et al. 2003). How- (OR = 1.18; 95 percent CI, 0.99–1.41). Other researchers ever, Chen and colleagues (2002) reported that the variant have observed a similar association with polymorphisms allele (*ARG) was associated with increased risk of blad- of P53 introns 3 and 6. However, evidence of these associa- der cancer. tions has not been consistent (Wang et al. 1999; Mabrouk et al. 2003).

Cancer 295 Surgeon General’s Report

P73 Gene significant effect observed was among male smokers (OR = 1.87; 95 percent CI, 1.25–2.80) with SCLC, suggest- The P73 gene activates the promoters of several ing that this P73 polymorphism may have an impact on genes that are responsive to the TP53 gene and participate the repair of tobacco-associated DNA damage. A study of in cell-cycle control, DNA repair, and apoptosis and inhibit 1,054 patients with lung cancer and 1,139 control partici- cell growth in a P53-like manner by inducing apoptosis or pants found a dose-response relationship between the fre- cell-cycle arrest in the G phase (Nomoto et al. 1998; Cai 1 quency of heterozygous or homozygous variant alleles and et al. 2000). Loss of heterozygosity at the P73 locus is rela- risk of lung cancer (trend test, p <0.001). ORs were 1.32 tively common. Studies have identified an estimated 17 (95 percent CI, 1.10–1.59) for the frequency of heterozy- polymorphisms. Two common SNPs at positions 4 (G→A) gous alleles and 1.54 (95 percent CI, 1.05–2.26) for the and 14 (C→T) in the uncoding region of exon 2 of the frequency of homozygous alleles (Li et al. 2004b). The risk P73 gene are in complete linkage disequilibrium and may of lung cancer was more pronounced in persons younger affect P73 function by altering the efficiency of translation than 50 years of age, men, light smokers, and patients with initiation (Kaghad et al. 1997). SCLC. The variant genotypes were also associated with an Studies have reported the role of the P73 increased risk for SCCs of the head and neck that was sta- G4C14→A4T14 polymorphism in the risk of smoking- tistically significant (OR = 1.33) and an even higher risk related cancer (Ryan et al. 2001; Hamajima et al. 2002; among current smokers (OR = 1.77) (Li et al. 2004a). Hiraki et al. 2003; Huang et al. 2003). In NSCLC, the most

Other Aspects

Carcinogenic Effects of Whole Extensive fractionation studies were conducted with cigarette smoke condensate (Hoffmann et al. 1978). Mixture and Fractions of Bioassays of the resulting fractions applied to the skin of Tobacco Smoke mice demonstrated that the neutral portion of the condensate has carcinogenic activity and the acidic portion Researchers have conducted inhalation studies of has tumor-promoting and cocarcinogenic activity. The cigarette smoke in hamsters, rats, mice, rabbits, dogs, and recombined neutral and acidic portions accounted for nonhuman primates. The model systems used in these about 80 percent of the carcinogenic activity of the con- studies had various problems, including the inability of densate. Subfractionation of the neutral portion revealed any study to accurately duplicate human smoking behav- that certain PAHs were the major tumor initiators in this iors. Nevertheless, comprehensive reviews of these studies fraction. However, these PAHs alone, in the levels at which have found a large amount of useful information (IARC they occur, were insufficient to induce tumors. Moreover, 1986, 2004; Coggins 1998; Witschi 2000). Researchers when these PAHs were added to the condensate, the tumor observed the most consistent results on cancer induction yield was higher than with the condensate alone. These in Syrian golden hamsters; whole cigarette smoke and its results indicate that the combination of PAHs acting as particulate phase induced malignant tumors and other tumor initiators, together with cocarcinogens in the con- lesions in the larynx. Tumors were not induced by the gas densate, accounted for the tumorigenicity of the conden- phase of cigarette smoke. sate on mouse skin. Researchers identified catechol and Findings of studies that induced malignant tumors alkyl catechols as major cocarcinogens in the condensate, by inhalation of cigarette smoke and its particulate phase and the weakly acidic portion of the condensate demon- are consistent with those in the substantial amount of strated tumor-promoting activity (Van Duuren and Gold- literature demonstrating that condensate from cigarette schmidt 1976; Hecht et al. 1981). Other cocarcinogens in smoke, which lacks volatile constituents of the gas phase, cigarette smoke include undecane, pyrene, fluoranthene, causes benign and malignant tumors when applied to and B[a]P (Van Duuren and Goldschmidt 1976). The mouse skin and rabbit ears or instilled in rat lungs by identity of tumor promoters in cigarette smoke is largely intrapulmonary administration (Hoffmann et al. 1978; unknown, although simple phenols may contribute IARC 1986, 2004). Collectively, these results clearly show weakly (Hecht et al. 1975). Researchers have also observed that major carcinogenic fractions of cigarette smoke tumor-promoting activity of cigarette smoke in inhalation reside in the particulate phase. experiments with hamsters (IARC 1986). PAH-enriched

296 Chapter 5 How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease fractions of cigarette smoke condensate instilled in the rat esophagus (IARC 2004). No single mechanism clearly lung also resulted in tumor formation (IARC 1986). explains these observations, but several have been pro- These results clearly demonstrate the carcinogenic, posed and there is reasonable support for some. The most tumor-promoting, and cocarcinogenic activity of the par- consistent body of evidence relates to the effects of alcohol ticulate phase of cigarette smoke. However, some data on the distribution of carcinogenic nitrosamines. Swann indicate that constituents of the gas phase also contribute and colleagues (1984) demonstrated that alcohol could to tumor induction. Early studies in Snell’s mice dem- inhibit the hepatic metabolism and clearance of NDMA, onstrated an increase in pulmonary adenocarcinomas in a carcinogen in tobacco smoke. This inhibition occurs animals exposed to the gas phase alone (IARC 2004). In an because ethanol competitively inhibits hepatic cyto- exposure model using 89 percent sidestream smoke and 11 chrome P-450 2E1, the main hepatic enzyme responsible percent mainstream smoke, increased multiplicity of lung for metabolism of NDMA. Consequently, more NDMA adenomas was consistently observed in A/J mice exposed reaches extrahepatic tissues where it can be metabolically to the smoke for five months, followed by a four-month activated and has the potential to cause cancer. Ander- resting period. Tumor response in this model was clearly son and coworkers (1992, 1996) demonstrated that coad- attributable to the gas phase, because filtration had no ministration of ethanol and NDMA to A/J mice resulted effect on multiplicity of lung adenomas (Witschi 2000; in an incidence of lung tumors higher than that in mice IARC 2004). The results of these studies indicate that a treated with NDMA alone and that this increase was a con- volatile carcinogen in cigarette smoke—possibly 1,3-bu- sequence of inhibition of hepatic metabolism and not of tadiene—produced a tumorigenic response in the A/J tumor promotion. Furthermore, administration of etha- mouse lung. nol to patas monkeys before they received NDMA resulted Two other studies not included in the reviews previ- in a 14.6-fold increase in O6-methylguanine in esophageal ously cited here demonstrate convincingly that cigarette DNA and other extrahepatic tissues (Anderson et al. 1996). smoke administered to rats or mice by whole-body expo- Another potential mechanism also involves the sure for extended periods induces benign and malignant effects of ethanol on P-450 2E1, but as an inducer of this tumors of the respiratory tract (Mauderly et al. 2004; enzyme. Chronic ethanol consumption is known to induce Hutt et al. 2005). When male and female F-344 rats were production of hepatic P-450 2E1, and researchers hypothe- exposed to smoke from 1R3 research cigarettes or to clean sized that this induction could lead to increased metabolic air for six hours per day, five days per week for up to 30 activation of carcinogens in tobacco smoke (McCoy et al. months, the exposure significantly increased the -inci 1979). Some N-nitrosamines in tobacco smoke—NDMA, dence of nonneoplastic and neoplastic proliferative lung N-nitrosodiethylamine, and N-nitrosopyrrolidine—are all lesions in females. The combined incidence of bronchoal- substrates for P-450 2E1. McCoy and colleagues (1981) veolar adenomas and carcinomas was 14 percent in the demonstrated that chronic ethanol consumption in ham- high-exposure group (250 mg of particulates per m3 of sters increased the metabolism of N-nitrosopyrrolidine air), 6 percent in the low-exposure group (100 mg/m3), in hepatic and target tissue (e.g., trachea), as well as the and none in the controls. Mutations in codon 12 of the carcinogenicity of this nitrosamine, which increases the KRAS gene occurred in 4 of 23 tumors. Both males and occurrence of tumors in the nasal cavity and trachea. The females had significant increases in neoplasia of the nasal carcinogenicity of N-nitrosodiethylamine in the rat esoph- cavity (Mauderly et al. 2004). Female B6C3F1 mice were agus was also increased by simultaneous administration exposed to smoke for 6 hours per day, 5 days per week, of ethanol (Gibel 1967). Overall, however, the effects of for 925 days (250 mg/m3) or were sham exposed. The ethanol consumption on N-nitrosamine carcinogen- incidence of lung adenoma (28 percent) and lung adeno- esis have been mixed, and they appear to depend on the carcinoma (20 percent) in the mice exposed to smoke N-nitrosamine studied and the protocol used. For exam- were significant (Hutt et al. 2005). ple, long-term ethanol consumption had no effect on the carcinogenicity of NNN in the hamster and only modest or no effect in the rat (McCoy et al. 1981; Trushin et al. 1984). Synergistic Interactions in Other mechanisms for the enhancing effect of alcohol consumption on tobacco carcinogenesis have been Tobacco Carcinogenesis discussed (Pöschl and Seitz 2004). Persons who abuse alcohol generally have nutritional deficiencies, which Alcohol could exacerbate the effects of smoking. They commonly There is persuasive epidemiologic evidence that have folate deficiency that could contribute to an inhibi- alcohol consumption and smoking synergistically increase tion of transmethylation, which is important in gene reg- the risk for cancers of the oral cavity, pharynx, larynx, and ulation. Zinc deficiency is known to result in enhanced

Cancer 297 Surgeon General’s Report carcinogenesis in the rat esophagus (Fong et al. 2001). be particularly potent in the rat. Significant incidence of Reduced serum and hepatic levels of vitamin A in persons lung tumors was induced by total doses as low as 6 mg/ with long-term alcohol abuse may affect carcinogenesis. kilogram (kg) of body weight or by 1.8 mg/kg as part of Alcohol could also act as a solvent, increasing absorption a dose-response trend. These doses are comparable to an of tobacco carcinogens (Squier et al. 1986). estimated NNK dose of 1.1 mg/kg in persons who have smoked for 40 years (Hecht 1998). DNA adducts derived Asbestos from NNK or from the related tobacco-specific nitrosamine, NNN, are present in lung tissue from smokers, Smoking and exposure to asbestos interact synergis- and metabolites of NNK are found in the urine of smok- tically to increase the risk for lung cancer (IARC 2004). ers (Hecht 2002b). Epidemiologic data indicate that a The mechanism for this synergy is unknown. Research- systemic carcinogen causes lung cancer in cigar smokers ers have investigated a number of possibilities, however, who do not inhale the smoke; this finding is consistent and these have been summarized (Nelson and Kelsey with the tumorigenic properties of NNK (Boffetta et al. 2002). It has been proposed that asbestos fibers serve as a 1999; Shapiro et al. 2000). vehicle to deliver tobacco carcinogens to the cell nucleus. The changing histology of lung cancer is also con- Surfactant phospholipids may help to solubilize carcino- sistent with the role of NNK: adenocarcinoma has now genic PAH, increasing their concentrations in the lung overtaken SCC as the most common lung cancer type. epithelium. Studies have also demonstrated that asbestos This nitrosamine in tobacco smoke produces primarily fibers can induce chromosomal aberrations and extensive adenocarcinomas in rodents. However, this outcome has deletions, potentially adding to the DNA damage produced also been attributed to differing inhalation patterns of by carcinogens in tobacco smoke. Furthermore, asbestos current cigarette smokers (Travis et al. 1995; Hecht 1998). may cause oxidative damage that could be related to in- As nitrate concentrations in tobacco increased from 1959 flammation and cell death related to pulmonary fibrosis to 1997, NNK concentrations in mainstream smoke associated with exposure to asbestos. It seems likely that increased and those of B[a]P decreased. Researchers asbestos fibers could cause proliferation that may increase attributed these changes to tobacco blends with higher the probability of mutations attributable to DNA damage levels of air-cured tobacco, the use of reconstituted by tobacco smoke carcinogens. tobacco, and other factors (Hoffmann et al. 2001). Other compounds that could be involved in lung cancer include 1,3-butadiene, isoprene, ethylene oxide, ethyl carbamate, Carcinogens as Causes of aldehydes, benzene, metals, and oxidants, but the collec- Specific Cancers tive evidence for each of these substances is not as strong as the evidence for PAHs and NNK (Hecht 1999). Data from carcinogenicity studies, product analyses, The particulate phase of cigarette smoke causes and findings from studies using biochemistry and molecu- tumors of the larynx in hamsters, which could be attrib- lar biology support a significant role for certain carcino- uted to PAHs (IARC 1986). TP53 gene mutations identified gens in tobacco-induced cancer (Table 5.12). in tumors of the human larynx support a role for PAHs Considerable evidence favors PAHs and NNK as in the development of this cancer (Pfeifer et al. 2002). major factors in development of lung cancer. PAHs are N-nitrosamines, as well as acetaldehyde and formalde- strong carcinogens acting locally; thus, fractions of hyde, induce nasal tumors in rodents and are likely can- tobacco smoke enriched in these compounds are carcino- didates for causing nasal tumors associated with smoking genic (Hoffmann et al. 1978; Deutsch-Wenzel et al. 1983; (Preussmann and Stewart 1984; IARC 1995c, 1999). On IARC 1983) (see “Carcinogens in Cigarette Smoke” ear- the basis of animal studies, PAH, NNK, and NNN are the lier in this chapter). Researchers have detected PAH-DNA most likely causes of oral cancer in smokers (Hoffmann adducts in human lungs, and the spectrum of mutations and Hecht 1990). N-nitrosamines are the most effective in the TP53 gene isolated from lung tumors was similar to esophageal carcinogens known. NNN causes tumors of the pattern of DNA damage produced in vitro by PAH diol the esophagus in rats and is the most prevalent N-nitrosa- epoxide metabolites and in cell cultures by B[a]P (Pfeifer mine carcinogen in cigarette smoke (Hecht and Hoffmann et al. 2002; Phillips 2002; Boysen and Hecht 2003; Liu et 1989; Lijinsky 1992). al. 2005) (see “DNA Adducts and Biomarkers” earlier in NNK and several other N-nitrosamines and furan this chapter). in cigarette smoke are effective hepatocarcinogens in rats NNK is a strong systemic carcinogen in lungs of (Preussmann and Stewart 1984; IARC 1995b). NNK and rodents that induces lung tumors independent of the its major metabolite NNAL are the only known pancreatic route of administration (Hecht 1998). NNK was found to carcinogens in tobacco products. Biochemical data from

298 Chapter 5 How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease

Table 5.12 Carcinogens and tobacco-induced cancers

Study Cancer type Likely carcinogen involvementa

Hoffmann and Hecht 1990 Lung PAHs, NNK (major), 1,3-butadiene, isoprene, ethylene oxide, Hecht et al. 1994 ethyl carbamate, aldehydes, benzene, metals Törnqvist and Ehrenberg 1994 Hecht 1999 Hoffmann et al. 2001 Pfeifer et al. 2002

IARC 1986 Larynx PAHs Hoffmann et al. 2001 Pfeifer et al. 2002

Preussmann and Stewart 1984 Nasal NNK, NNN, other nitrosamines, aldehydes IARC 1995c, 1999 Hecht 1998

Hecht et al. 1986 Oral cavity PAHs, NNK, NNN Hoffmann et al. 1987, 1995, 2001 Hecht and Hoffmann 1988, 1989 Hoffmann and Hecht 1990 Hecht 1998 Vainio and Weiderpass 2003

Hecht and Hoffmann 1989 Esophagus NNN, other nitrosamines Lijinsky 1992 Hecht 1998 Hoffmann et al. 2001

Preussmann and Stewart 1984 Liver NNK, other nitrosamines, furan IARC 1995a Hecht 1998

Rivenson et al. 1988 Pancreas NNK, NNAL Hecht 1998 Hoffmann et al. 2001 Prokopczyk et al. 2002

Melikian et al. 1999 Cervix PAHs, NNK Prokopczyk et al. 2001 Phillips 2002

IARC 1974 Bladder 4-aminobiphenyl, other aromatic amines Hoffmann and Hecht 1990 Skipper and Tannenbaum 1990 Skipper et al. 1994 Landi et al. 1996 Probst-Hensch et al. 2000 Hoffmann et al. 2001

IARC 1982 Leukemia Benzene Source: Adapted from Hecht 2003 with permission. Note: IARC = International Agency for Research on Cancer; NNAL = 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol; NNK = 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; NNN = N’-nitrosonornicotine; PAHs = polycyclic aromatic hydrocarbons. aBased on carcinogenicity studies in laboratory animals, biochemical evidence from human tissues and fluids, and epidemiologic data when available.

Cancer 299 Surgeon General’s Report studies of human tissue provide some support for the smoke. Other studies demonstrated that nicotine is role of these carcinogens in smoking-related pancreatic immunosuppressive and thus might be responsible for cancer, although the studies did not detect DNA adducts some of the effects of cigarette smoke (Sopori 2002). (Rivenson et al. 1988; Prokopczyk et al. 2002, 2005). Bio- chemical studies demonstrate that both NNK and PAHs can reach the cervix in humans and are metabolically Epidemiology of Family History activated in these tissues (Melikian et al. 1999; Prokopc- zyk et al. 2001). Researchers have detected DNA adducts and Lung Cancer derived from B[a]P and other hydrophobic compounds in cervical tissue from smokers (Melikian et al. 1999; Phillips Studies of familial aggregation of lung cancer pro- 2002). Therefore, in combination with the human papil- vide indirect evidence supporting the possibility of an loma virus, these compounds may contribute to develop- inherited component to tobacco carcinogenesis. A num- ment of cervical cancer in smokers (IARC 1995a). 4-ABP ber of published studies showed that significantly more and 2-naphthylamine are known human bladder carcino- lung cancers were reported in first-degree relatives of pro- gens, and considerable data from human studies support bands with lung cancer than were reported in first-degree the role of aromatic amines as the major cause of bladder relatives of healthy control participants. Assuming that cancer in smokers (IARC 1974; Skipper and Tannenbaum the family structure does not differ between cases and 1990; Skipper et al. 1994; Landi et al. 1996; Probst-Hensch controls, this pattern could be explained by shared genes et al. 2000; Castelao et al. 2001). The most probable cause among the family members, shared smoking patterns, or of leukemia in smokers is exposure to benzene, which a combination of both factors. By incorporating smoking occurs in large quantities in cigarette smoke and is a histories of the probands and the first-degree relatives known cause of acute myelogenous leukemia in humans into a study of familial aggregation, researchers can begin (IARC 1982). to assess the level of familial risk of cancer while adjusting Cigarette smoke causes oxidative damage probably for tobacco use. However, few studies of familial aggrega- because it contains free radicals, such as nitric oxide, and tion incorporate history of involuntary exposure of fam- contains mixtures of hydroquinones, semiquinones, and ily members to tobacco smoke. Estimates of the overall quinones that can induce reduction and oxidation (redox proportion of patients with lung cancer who have fam- cycling) (Pryor et al. 1998; Hecht 1999). Smokers have ily history of lung cancer in a first-degree relative range lower levels of ascorbic acid in plasma and, sometimes, from 6 (Li and Hemminki 2004) to 16 percent (Sellers et higher levels of oxidized DNA bases in white blood cells al. 1992). than do nonsmokers. However, the role of oxidative dam- Forty years ago, Tokuhata and Lilienfeld (1963) age as a cause of specific tobacco-induced cancers remains observed that the number of deaths from lung cancer unclear (Hecht 1999). was higher among relatives of lung cancer case patients than it was among relatives of control participants. These researchers also reported a fourfold excess of lung cancer mortality in nonsmoking relatives of 270 lung cancer pro- Tobacco Carcinogens, Immune bands. The effect among relatives who smoked was less System, and Cancer pronounced (twofold). These findings suggest that the risk was not solely attributable to shared smoking patterns Cigarette smoke alters a range of immunologi- in the relatives. Other studies have since demonstrated a cal functions including innate and adaptive immune familial component of risk for lung cancer. The ORs asso- responses (Sopori 2002). These effects, acting as tumor- ciated with family history ranged from 1.3 to 7.2 (Ooi et al. promoting or cocarcinogenic stimuli, could affect 1986; Samet et al. 1986; Wu et al. 1988, 1996; Osann 1991; tobacco-related carcinogenesis. Cigarette smoking Shaw et al. 1991; Schwartz et al. 1996; Mayne et al. 1999). increases the number of alveolar macrophages in the For example, in a comparison of 336 lung cancer pro- lung, possibly leading to higher levels of oxygen radicals bands with relatives of control spouses, Ooi and colleagues and MPO activity, which are hypothesized to be impor- (1986) reported an association between a family history tant in tumor promotion. Investigators examined the of lung cancer and a threefold excess risk (OR = 3.09; 95 effects of smoking on the function of natural killer (NK) percent CI, 1.9–5.0). Shaw and associates (1991) found an cells—a lymphoid cell type involved in surveillance of OR of 2.8 (95 percent CI, 1.2–6.6) for risk of lung cancer tumor growth (Lu et al. 2007). They obtained strong evi- among two or more relatives of case patients. Brownson dence that suppression of NK cell activation was related and colleagues (1997) reported a trend for increasing risk to increased lung metastases in mice exposed to cigarette associated with the number of first-degree relatives with

300 Chapter 5 How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease lung cancer. The risk was more than twofold for persons evidence from a study in Poland for aggregation of cancers with five affected family members. among 757 patients with lung cancer who were younger One approach that evaluates familial aggregation than 50 years of age. while controlling for the impact of smoking focused on On the other hand, Etzel and colleagues (2003) lifetime nonsmokers. Wu and colleagues (2004) evalu- observed the familial aggregation of lung cancer and ated 216 lung cancer probands who were female nonsmoking-related cancers in late-onset lung cancers in smokers and reported that family history of lung cancer persons older than 55 years of age, but not in early-onset was associated with a 5.7-fold (OR = 5.7; 95 percent CI, lung cancers. An advantage of this study was the ability to 1.9–16.9) increase in lung cancer risk. Wu and colleagues adjust for the smoking status of the relatives. The study (1988) noted, after adjustment for exposure to second- noted an excess of cancer among relatives of probands hand smoke, a 30-percent increase in risk that was not who were current smokers but not among relatives of life- statistically significant for history of cancers of the time nonsmokers. More recently, Li and Hemminki (2004) respiratory tract. This association was especially evident evaluated familial risks by using data from the Swedish in mothers and sisters. The risk was also slightly elevated Family Cancer Database and demonstrated that the his- for lung cancer (OR = 1.3; 95 percent CI, 1.0–1.6). Mayne tologic type of lung cancer in relatives was generally ran- and colleagues (1999) also focused on familial risk in a dom. These researchers also estimated that 25 percent of population-based, case-control study of 437 lifetime familial lung cancers were diagnosed before 50 years of nonsmokers and former smokers who had lung cancer age, which represented about 1.6 percent of all lung can- and 437 matched control participants. The investigators cers before 68 years of age. observed increased risk of cancers of the aerodigestive However, a cohort study of lung cancer mortality tract among parents of case patients (OR = 2.78; 95 percent among male twins showed no role for genetic predisposi- CI, 1.30–5.95) and increased risk of lung cancer among tion (Braun et al. 1994). Li and colleagues (1998b) used siblings and offspring of case patients (OR = 4.14; 95 per- a parametric likelihood approach and adjustment for cent CI, 0.88–19.46; p = 0.07). They also reported approxi- shared covariates to study familial association in the age at mately twofold increases in risk of breast cancer among onset that they hypothesized to be attributable to genetic mothers (OR = 2.52; 95 percent CI, 1.21–5.24) and sisters factors. The analysis indicated that a history of smoking, (OR = 2.07; 95 percent CI, 0.99–4.31) of lung cancer exposure to secondhand smoke, and chronic obstructive patients who were nonsmokers. On the other hand, airway disease were all associated with lung cancer risk. Kreuzer and colleagues (2002) reported no evidence of After adjustments were made for these factors, there was familial risk in 234 lung cancer probands who were little evidence of familial aggregation. female nonsmokers. In a recent series with high-risk multiplex fami- Other investigators have reported familial aggrega- lies, Bailey-Wilson and colleagues (2004) mapped a major tion of lung cancer among relatives of case patients who susceptibility locus of lung cancer through a genome- were nonsmokers with early-onset of lung cancer (at wide linkage analysis to chromosome 6q23–25 near the ≤60 years of age). Schwartz and colleagues (1996) noted PARKIN gene, which carries predisposition for a signifi- that family members of these case patients (aged 40 to 59 cantly increased hereditary risk of lung cancer. A study years) had a sixfold increase in risk of lung cancer after of this locus also indicated presence of gene-environment adjustments for the age, gender, and race of each relative. interaction, and even light smoking by carriers of this In a subsequent study involving 118 population-based pro- gene significantly increased the risk for lung cancer com- bands, Schwartz and colleagues (1999) also showed that pared with that among heavy smokers. Carriers who did family members of case patients younger than 40 years of not smoke had much lower risk, comparable to risk for age who had lung cancer were at increased risk for other noncarriers. This finding indicated existence of a -sensi cancers. Kreuzer and colleagues (1998) concluded that tive group of persons for whom any amount of smoking lung cancer in a first-degree relative was associated with a is deleterious. 2.6-fold increase in risk of lung cancer among young case Studies have also presented evidence of Mendelian patients younger than 46 years of age. Elevated risk was inheritance in lung cancer. Sellers and colleagues (1990) not detected in older case patients. A study in Germany of found that the pattern of occurrence of lung cancer was 945 lung cancer cases and 983 controls reported increased compatible with the Mendelian codominant inheritance risk of lung cancer among first-degree relatives (RR = 1.7; of a rare and major autosomal gene. However, a similar 95 percent CI, 1.1–2.5) and a 4.75-fold increase in risk study of families with lung cancer probands who did not among relatives of probands younger than 50 years of age smoke revealed no evidence for a major gene model and who had a diagnosis of lung cancer (Bromen et al. 2000). reported that an environmental model best explained Radzikowska and colleagues (2001) also noted stronger the segregation pattern in the data (Yang et al. 1997).

Cancer 301 Surgeon General’s Report

Gauderman and Morrison (2000) determined that when cancer according to the gender, race, ethnicity, and age the same data were analyzed but missing data for smok- of the respondents. These differences were mostly due to ing behaviors were produced from modeling techniques, a underreporting with respect to these covariates. Zio- single autosomal dominant locus provided a slightly bet- gas and Anton-Culver (2003) found that family histories ter fit than the codominant model suggested by Sellers of cancer reported by probands were more accurate for and colleagues (1990). In addition to possible etiologic first-degree relatives and that probands referred through heterogeneity, the inconsistency of these findings may clinics had lower false-positive rates for reporting of fam- be partly due to the insufficient power for the statistical ily history than did population-based probands. Bondy analysis of limited sample sizes. A reanalysis by Yang and and colleagues (1994) evaluated the accuracy of cancer colleagues (1999) found evidence of a major gene with the diagnosis reported by probands among family members Mendelian codominant model in the families of probands by comparing reported cancer information with docu- of nonsmokers younger than 60 years of age. This analysis mentation available through medical records and death rejected both the codominant and environmental models certificates. The study noted high levels of accuracy for and suggested that multiple genetic and/or environmen- cancers of first-degree relatives, as evidenced by agreement tal factors contribute to the age at onset of lung cancer. between reporting by probands and information in Therefore, researchers need more complex genetic models records. Thus, these findings of familial aggregation sug- for the distribution of age at onset. gest a role for the inherited susceptibility of lung cancer Xu and colleagues (2005) completed a segrega- beyond that associated with familial clustering of smoking tion analysis on 14,378 persons from 1,561 case families behaviors, taking into account family size and structure. with aggregation of lung cancer. In their modeling, these A genomewide association study in 2008 identified researchers adjusted for the effects of smoking, gender, a region of strong linkage disequilibrium on the long arm and age. This work provided evidence for a model involv- of chromosome 15 as a susceptibility locus for lung can- ing multiple gene loci and interactions that contribute to cer (Amos et al. 2008). Studies replicating this association the age at onset of lung cancer. have focused attention on the most likely candidate genes One caveat that applies to all of these studies is in this region, CHRNA3 and CHRNA5, which encode sub- the validity of data on family history, an issue indirectly units of the nAChR (Hung et al. 2008). Le Marchand and addressed in an evaluation of family histories of can- colleagues (2008) found that carriers of the lung-cancer- cer in participants in the Prostate, Lung, Colorectal and associated variants in these genes extract more nicotine Ovarian Cancer Screening Trial (Pinsky et al. 2003). The and are thus exposed to a higher internal dose of carci- data showed that in the ratios of reported-to-expected nogenic nicotine-derived nitrosamines. SNPs in the same rates of cancer in family members, there were impor- region have also been associated with nicotine dependence tant differences in rates of reporting family history of and smoking intensity (Caporaso et al. 2009).

Evidence Summary

Although cigarette smoke contains diverse carcino- Familial predisposition and genetic polymorphisms gens, PAH, N-nitrosamines, aromatic amines, 1,3-butadi- may play a role in tobacco-related neoplasms. Research- ene, benzene, aldehydes, and ethylene oxide are among ers have established cigarette smoking as a major cause the most important carcinogens because of their carcino- of lung cancer; more than 85 percent of lung cancers are genic potency and levels in cigarette smoke. Moreover, the attributable to smoking. However, not all smokers develop major pathways of metabolic activation and detoxification lung cancer, and lung cancer can arise in lifetime non- of some of the principal carcinogens in cigarette smoke are smokers. This variation in disease has stimulated inter- well established. Reactive intermediate agents critical in est in molecular epidemiology of genetic polymorphisms, forming DNA adducts include diol epoxides of PAH, diazo- including genes that regulate the cell cycle and genes nium ions generated by α-hydroxylation of nitrosamines, for carcinogen-metabolizing enzymes that may lead to nitrenium ions formed from esters of N-hydroxylated aro- variations in susceptibility to the carcinogens in tobacco matic amines, and epoxides such as ethylene oxide. Glu- smoke. Studies to date suggest a role for these genetic tathione and glucuronide conjugation play major roles in polymorphisms in the risk of lung and bladder cancer in detoxification of carcinogens in cigarette smoke. smokers, and they support the possibility of interactions between genes and smoking status.

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Quantitative analysis of carcinogens or their metab- adduct formation, in addition to phenotypic selection, is olites in urine, breath, and blood provides a convenient responsible for shaping the TP53 mutational spectrum and reliable method of comparing exposure to carcino- in lung tumors. Propagation of these genetic alterations gens among smokers and between smokers and non- during clonal outgrowth is consistent with accumulation smokers. Urinary biomarkers of several major types of of multiple genetic changes observed in progression of carcinogens in cigarette smoke are reliable indicators of lung cancer. exposure, and the measurements provide good estimates Gene promoter hypermethylation is an epigenetic of minimum doses of relevant carcinogens in smokers change involving extensive methylation at the 5-position and allow comparisons with nonsmokers. The total car- of C in CpG islands within the promoter region and often cinogen dose is generally difficult to calculate because the extending into exon 1 of regulatory genes. The end result extent of conversion of a given carcinogen to the measured of this process can be loss of gene transcription and there- metabolite is usually unknown. However, relative carcino- fore the silencing of gene function. Promoter methylation gen levels in cigarettes generally correlate with metabolite of several genes including P16 occurs early in tumor for- levels in urine. Comparisons of smokers and nonsmokers mation. P16 methylation was significantly associated with demonstrate that total NNAL is the most discriminatory pack-years of smoking and was an independent risk fac- biomarker in that tobacco products are the only source of tor for shorter survival in patients with early resectable the parent carcinogen NNK. adenocarcinomas. Other genes such as RASSF1A may be Evidence is overwhelming that DNA adduct levels more frequently methylated in various tumor types from are higher in most tissues of smokers than in correspond- smokers. Methylation of genes, such as AGT promoter ing tissues of nonsmokers. This observation provides hypermethylation, may increase G→A transition muta- bedrock support for the major pathway of cancer induc- tions at CpG sites within the TP53 gene in NSCLC. tion in smokers that proceeds through formation of DNA The activation of nAChRs in lung epithelial cells adducts and genetic damage. Studies of specific adducts by nicotine or NNK promotes survival and proliferation are still scarce and are limited mainly to human lung tis- of cancer cells and also leads to increased angiogenesis. sue. Strong evidence supports the presence of a variety of Activation of cell-surface receptors induced by components specific adducts in the human lung, and in several studies, of tobacco smoke and subsequent activation of cytoplas- adduct levels are higher in smokers than in nonsmokers. mic kinases stimulate other proteins that dictate cellular Collectively, the results of these biomarker studies clearly responses, such as cell survival and proliferation. Although demonstrate the potential for genetic damage in smokers activated kinases have many downstream targets, the two from the persistence of DNA adducts. most studied are the transcription factor NF-kB and pro- Adducts lead to mutations that drive the process teins in the BCL-2 family. Activation of key intracellular of tumor formation and progression through additional proteins by tobacco smoke components through signaling genetic alterations. Chromosomal losses are more com- cascades promotes processes that are important for initia- mon in tumors from smokers. Furthermore, inactivating tion, progression, and maintenance of cancer. Apoptosis, mutations of the TP53 tumor-suppressor gene and activat- the normal mechanism of endogenous cell elimination, is ing mutations of the KRAS oncogene in NSCLCs and other also commonly suppressed in lung cancer by these com- tumors are correlated with exposure to cigarette smoke, ponents. Thus, key genetic and epigenetic events that lead and they contribute to a phenotype that reduces survival to cancer causation, as well as critical cellular pathways time in both early and advanced stages of the disease. Dif- that further growth and development of transformed cells, ferent types of lung cancer in smokers all show an excess are directly targeted by components of cigarette smoke of G→T transversions compared with cancers that are not individually and in combination as a potent carcino- related to exposure to tobacco smoke. The site specificity genic mixture. of mutagenesis by PAH diol epoxides implies that targeted

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Conclusions

1. The doses of cigarette smoke carcinogens resulting 6. Exposure to cigarette smoke carcinogens leads to from inhalation of tobacco smoke are reflected in lev- DNA damage and subsequent mutations in TP53 and els of these carcinogens or their metabolites in the KRAS in lung cancer. urine of smokers. Certain biomarkers are associated with exposure to specific cigarette smoke carcinogens, 7. There is consistent evidence that smoking leads to the such as urinary metabolites of the tobacco-specific presence of promoter methylation of key tumor sup- nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1- pressor genes such as P16 in lung cancer and other butanone and hemoglobin adducts of aro- smoking-caused cancers. matic amines. 8. There is consistent evidence that smoke constituents 2. The metabolic activation of cigarette smoke carcino- such as nicotine and 4-(methylnitrosamino)-1-(3- gens by cytochrome P-450 enzymes has a direct effect pyridyl)-1-butanone can activate signal transduction on the formation of DNA adducts. pathways directly through receptor-mediated events, allowing the survival of damaged epithelial cells that 3. There is consistent evidence that a combination of would normally die. polymorphisms in the CYP1A1 and GSTM1 genes leads to higher DNA adduct levels in smokers and 9. There is consistent evidence for an inherited sus- higher relative risks for lung cancer than in those ceptibility of lung cancer with some less common smokers without this genetic profile. genotypes unrelated to a familial clustering of smoking behaviors. 4. Carcinogen exposure and resulting DNA damage observed in smokers results directly in the numerous 10. Smoking cessation remains the only proven strategy cytogenetic changes present in lung cancer. for reducing the pathogenic processes leading to cancer in that the specific contribution of many tobacco 5. Smoking increases the frequency of DNA adducts of carcinogens, alone or in combination, to the develop- cigarette smoke carcinogens such as benzo[a]pyrene ment of cancer has not been identified. and tobacco-specific nitrosamines in the lung and other organs.

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Yoneda K. Distribution of proliferating-cell nuclear anti- Zenser TV, Lakshmi VM, Hsu FF, Davis BB. Metabolism gen and epidermal growth factor receptor in intraepi- of N-acetylbenzidine and initiation of bladder cancer. thelial squamous cell lesions of human bronchus. Mutation Research 2002;506–507:29–40. Modern Pathology 1994;7(4):480–6. Zha J, Harada H, Osipov K, Jockel J, Waksman G, Kors- Yoo JS, Guengerich FP, Yang CS. Metabolism of N-nitroso- meyer SJ. BH3 domain of BAD is required for het- dialkylamines by human liver microsomes. Cancer erodimerization with BCL-XL and pro-apoptotic Research 1988;48(6):1499–504. activity. Journal of Biological Chemistry 1997;272(39): Yoon J-H, Smith LE, Feng Z, Tang M-S, Lee C-S, Pfeifer 24101–4. GP. Methylated CpG dinucleotides are the prefer- Zhang L, Yu J, Park BH, Kinzler KW, Vogelstein B. Role ential targets for G-to-T transversion mutations of BAX in the apoptotic response to anticancer agents. induced by benzo[a]pyrene diol epoxide in mammalian Science 2000a;290(5493):989–92. cells: similarities with the p53 mutation spectrum in Zhang N, Lin C, Huang X, Kolbanovskiy A, Hingerty BE, smoking-associated lung cancers. Cancer Research Amin S, Broyde S, Geacintov NE, Patel DJ. Methylation 2001;61(19):7110–7. of cytosine at C5 in a CpG sequence context causes a You M, Candrian U, Maronpot RR, Stoner GD, Anderson conformational switch of a benzo[a]pyrene diol epox- MW. Activation of the Ki-ras protooncogene in sponta- ide-N2-guanine adduct in DNA from a minor groove neously occurring and chemically induced lung tumors alignment to intercalation with base displacement. of the strain A mouse. Proceedings of the National Journal of Molecular Biology 2005a;346(4):951–65. Academy of Sciences of the United States of America Zhang X, Miao X, Liang G, Hao B, Wang Y, Tan W, Li Y, 1989;86(9):3070–4. Guo Y, He F, Wei Q, et al. Polymorphisms in DNA base Yu C, Lu W, Tan W, Xing D, Liang G, Miao X, Lin D. Lack excision repair genes ADPRT and XRCC1 and risk of of association between CCND1 G870A polymorphism lung cancer. Cancer Research 2005b;65(3):722–6. and risk of esophageal squamous cell carcinoma. Can- Zhang X, Su T, Zhang Q-Y, Gu J, Caggana M, Li H, Ding X. cer Epidemiology, Biomarkers & Prevention 2003; Genetic polymorphisms of the human CYP2A13 gene: 12(2):176. identification of single-nucleotide polymorphisms and Yu D, Berlin JA, Penning TM, Field J. Reactive oxygen functional characterization of the Arg257Cys variant. species generated by PAH o-quinones cause change-in- Journal of Pharmacology and Experimental Thera- function mutations in p53. Chemical Research in Toxi- peutics 2002;302(2):416–23. cology 2002;15(6):832–42. Zhang Y, Yuan F, Wu X, Rechkoblit O, Taylor J-S, Geacin- Yuan A, Yu C-J, Shun C-T, Luh K-T, Kuo S-H, Lee Y-C, tov NE, Wang Z. Error-prone lesion bypass by human Yang P-C. Total cyclooxygenase-2 mRNA levels cor- DNA polymerase h. Nucleic Acids Research 2000b; relate with vascular endothelial growth factor mRNA 28(23):4717–24. levels, tumor angiogenesis and prognosis in non-small Zhao C, Tyndyk M, Eide I, Hemminki K. Endogenous cell lung cancer patients. International Journal of Can- and background DNA adducts by methylating and cer 2005;115(4):545–55. 2-hydroxyethylating agents. Mutation Research 1999; Yuan F, Zhang Y, Rajpal DK, Wu X, Guo D, Wang M, Taylor 424(1–2):117–25. J-S, Wang Z. Specificity of DNA lesion bypass by the Zheng Y, Shen H, Sturgis EM, Wang L-E, Eicher SA, yeast DNA polymerase h. Journal of Biological Chem- Strom SS, Frazier ML, Spitz MR, Wei Q. Cyclin D1 istry 2000;275(11):8233–9. polymorphism and risk for squamous cell carcinoma Yuan JM, Koh WP, Murphy SE, Fan Y, Wang R, Carmella of the head and neck: a case-control study. Carcinogen- SG, Han S, Wickham K, Gao YT, Yu MC, Hecht SS. Uri- esis 2001;22(8):1195–9. nary levels of tobacco-specific nitrosamine metabolites Zheng Y-L, Loffredo CA, Yu Z, Jones RT, Krasna MJ, in relation to lung cancer development in two prospec- Alberg AJ, Yung R, Perlmutter D, Enewold L, Harris tive cohorts of cigarette smokers. Cancer Research CC, et al. Bleomycin-induced chromosome breaks as 2009;69(7):2990–5. a risk marker for lung cancer: a case-control study Zabarovsky ER, Lerman MI, Minna JD. Tumor suppressor with population and hospital controls. Carcinogenesis genes on chromosome 3p involved in the pathogen- 2003;24(2):269–74. esis of lung and other cancers. Oncogene 2002;21(45): Zhong S, Howie AF, Ketterer B, Taylor J, Hayes JD, Beck- 6915–35. ett GJ, Wathen CG, Wolf CR, Spurr NK. Glutathione Zajac-Kaye M. Myc oncogene: a key component in cell S-transferase mu locus: use of genotyping and pheno- cycle regulation and its implication for lung cancer. typing assays to assess association with lung cancer Lung Cancer 2001;34(Suppl 2):S43–S46. susceptibility. Carcinogenesis 1991;12(9):1533–7.

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350 Chapter 5 Chapter 6 Cardiovascular Diseases

Introduction 355

Tobacco Use and Cardiovascular Disease 355 Estimation of Risk 355 Coronary Heart Disease 356 Cigarettes Smoked per Day 356 Duration of Smoking 359 Smoking Cessation 359 Women 360 Race and Ethnicity 361 Sudden Death 361 Stroke 361 Aortic Aneurysm 362 Peripheral Arterial Disease 362 Pipes, Cigars, and Low-Tar Cigarettes 362 Summary 362

Secondhand Tobacco Smoke and Cardiovascular Disease 363

Pathophysiology 363 Cigarette Smoke Constituents and Cardiovascular Disease 363 Mechanisms 367

Hemodynamic Effects 368 Blood Pressure and Heart Rate 368 Coronary Blood Flow 368 Summary 370

Smoking and the Endothelium 370 Endothelial Injury or Dysfunction 370 Regeneration of Endothelium 370 Endothelial Dysfunctions 371 Nitric Oxide and Vascular Homeostasis 371 Endothelium-Dependent Vasodilation 372 Impairment of Endothelium-Dependent Vasodilation 372 Pathologic Angiogenesis 373 Summary 374

351 Thrombogenic Effects 374 Alterations in Blood 375 Platelets 375 Red Blood Cells 377 Leukocytes 378 Circulating Proteins 378 Alterations in Blood Vessels 380 Nitric Oxide 380 Prostacyclin Production 380 von Willebrand Factor 380 Tissue Factor 380 Tissue Plasminogen Activator 380 Summary 381 Exposure to Secondhand Tobacco Smoke 381 Nicotine and Thrombosis 381 Summary 382

Inflammation 382

Smoking and Diabetes 383 Risk 383 Metabolic Control 384 Insulin Resistance 384 Microvascular Complications 385 Nephropathy 385 Retinopathy 386 Neuropathy 386 Macrovascular Complications 386 Pathophysiological Mechanisms 387 Summary 387

Lipid Abnormalities 387 Epidemiologic Observations 387 Lipoprotein Composition and Apolipoprotein Levels 388 Plasma- and Lipoprotein-Associated Lipid Transfer Enzymes 388 Oxidized Lipoproteins 388 Postprandial Lipid Changes 388 Metabolism of Free Fatty Acids 389 Reverse Cholesterol Transport 390 Effects of Smoking Cessation 390 Therapeutic Implications of Pathogenic Mechanisms 390 Summary 391

Cardiovascular Biomarkers 391

352 Smoking Cessation and Cardiovascular Disease 394 Methods 395 Interventions 395 Counseling 398 Nicotine Replacement Therapy 399 Bupropion 400 Other Pharmacotherapy 401 Summary 402

Methods to Reduce Exposure 402 Reduced Smoking 403 Nicotine Replacement Therapy 406 Summary 406 Implications 407

Evidence Summary 407

Conclusions 408

References 409

353

How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease

Introduction

This chapter reviews the epidemiology of smoking- secondhand smoke and CVD has been reviewed in the 2006 induced cardiovascular disease (CVD) and the mecha- report of the Surgeon General, The Health Consequences nisms by which tobacco smoke is thought to cause CVD. of Involuntary Exposure to Tobacco Smoke (U.S. Depart- The discussion includes use of biomarkers to diagnose ment of Health and Human Services [USDHHS] 2006), so smoking-induced CVD and treatment implications of discussion of secondhand smoke in this report is limited. the pathophysiology of the disease. The link between

Tobacco Use and Cardiovascular Disease

Cigarette smoking is a major cause of CVD, and smoking results in approximately eight times the risk (2 × past reports of the Surgeon General extensively reviewed 2 × 2) of persons with no risk factors. Cigarette smoking the relevant evidence (U.S. Department of Health, Edu- also is a cause of peripheral arterial disease (PAD), aor- cation, and Welfare [USDHEW] 1971, 1979; USDHHS tic aneurysm, CHD, and cerebrovascular disease, but the 1983, 2001, 2004). Cigarette smoking has been respon- relative risk (RR) of disease varies with the vascular bed sible for approximately 140,000 premature deaths annu- (USDHEW 1971, 1979; USDHHS 1983, 2001, 2004). The ally from CVD (USDHHS 2004). More than 1 in 10 deaths highest RRs are observed for diseases of peripheral arter- worldwide from CVD in 2000 were attributed to smoking ies in the lower extremities, and the lowest are for stroke; (Ezzati et al. 2005). In the United States, smoking RRs are intermediate for CHD and aortic aneurysm. accounted for 33 percent of all deaths from CVD and 20 The general mechanisms by which smoking results percent of deaths from ischemic heart disease in persons in cardiovascular events include development of athero- older than 35 years of age (Centers for Disease Control and sclerotic changes with narrowing of the vascular lumen Prevention 2008). Cigarette smoking also influences other and induction of a hypercoagulable state, which create cardiovascular risk factors, such as glucose intolerance risk of acute thrombosis (USDHHS 1983, 2004). The rapid and low serum levels of high-density lipoprotein cholester- decline in risk of a recurrent myocardial infarction (MI) ol (HDLc). However, studies have reported that smoking after smoking cessation (USDHHS 1990) supports the role increases the risk of CVD beyond the effects of smoking on of smoking in thrombosis. In addition, abundant evidence other risk factors. In other words, the risk attributable to demonstrates that smoking contributes to development smoking persisted even when adjustments were made for of atherosclerotic plaque (Strong and Richards 1976; differences between persons who smoke and nonsmokers Auerbach and Garfinkel 1980; Solberg and Strong 1983; in levels of these other risk factors (Friedman et al. 1979; USDHHS 1983, 2004). USDHHS 1983, 2001, 2004; Shaper et al. 1985; Criqui et al. 1987; Ragland and Brand 1988; Shaten et al. 1991; Nea- ton and Wentworth 1992; Freund et al. 1993; Cremer et Estimation of Risk al. 1997; Gartside et al. 1998; Wannamethee et al. 1998; Jacobs et al. 1999a). For example, in one study, the effect The risk of CHD from cigarette smoking can be of cigarette smoking on the risk of coronary heart disease described in terms of RR and excess risk (Thun et al. (CHD) was evident even among persons with low serum 1997). The RR is the ratio of CHD rates for populations levels of cholesterol (Blanco-Cedres et al. 2002). of smokers to rates for lifetime nonsmokers. Excess risk Beyond its status as an independent risk factor, is the difference between the rates of disease for smokers smoking appears to have a multiplicative interaction with and nonsmokers. the other major risk factors for CHD—high serum levels These two estimates of risk can lead to conflicting of lipids, untreated hypertension, and diabetes mellitus impressions of the changes in smoking-related CHD risks (USDHHS 1983). For instance, if the presence of smoking with advancing age. The RRs and excess death rates for alone doubles the level of risk, the simultaneous presence CHD are shown by age group in data from Cancer Preven- of another major risk factor is estimated to quadruple the tion Study II (CPS-II) (Thun et al. 1997), sponsored by the risk (2 × 2). The presence of two other risk factors with American Cancer Society (see Figure 6.1 for data on men).

Cardiovascular Diseases 355 Surgeon General’s Report

Figure 6.1 Relative risk and excess death rate for coronary heart disease among men, by age group

7 900

800 6 Relative risk 700 Excess death rate 5 600

4 500

3 400 Relative risk

300 2 Death rate per 100,000 200 1 100

0 0 35–39 40–44 45–49 50–54 55–59 60–64 65–69 70–74 75–79 80+ Age group (years)

The RRs were highest at younger ages (35 to 54 years) and deaths from COPD matched and those from lung cancer declined steeply with advancing age. This finding leaves exceeded the excess death rates attributable to CHD. The the false impression that the disease burden of CHD from RR for death from a cerebrovascular disease among smok- smoking declined with age or was low among older smokers was substantially elevated among younger smokers ers. However, excess CHD death rates for smokers by age (RR = 4 to 5; data not shown). However, the absolute rate group presented different evidence. The high RR for CHD of stroke at these younger ages was low, and this finding at younger ages can be explained in part because mortal- resulted in a low excess mortality rate. At older ages, the ity rates are low for death from CHD at those ages and death rate from stroke in the general population increased because coronary events in young people occur primarily and the RR among smokers declined, thus moderating the among smokers. Even though RR declined with increas- excess death rate attributable to smoking. ing age, because the absolute rate of deaths from CHD increased markedly, the magnitude of the CHD burden produced by smoking increased with advancing age. Coronary Heart Disease The age at onset of substantial excess risk differs by disease. For smokers, age-specific excess death rates Cigarettes Smoked per Day attributable to CHD, lung cancer, cerebrovascular disease, and chronic obstructive pulmonary disease (COPD) are Studies showed increased risk of having CHD at all illustrated by data from CPS-II (Figure 6.2 shows data for levels of cigarette smoking, and increased risks were evi- men) (Thun et al. 1997). For persons younger than age 45 dent even for persons who smoked fewer than five ciga- years, CHD was the dominant cause of increased mortality rettes per day (Rosengren et al. 1992; Prescott et al. 2002; attributable to cigarette smoking. Excess rates for death Bjartveit and Tverdal 2005). Prospective mortality studies from lung cancer increased steeply after age 50 years, and conducted in the 1960s and 1970s showed a clear increase excess death rates from COPD were largely confined to in CHD mortality with an increase in the number of ciga- the seventh and eighth decades of life. Late in life, excess rettes smoked per day, regardless of the actual number

356 Chapter 6 How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease

(Doll and Peto 1976; USDHHS 1983). Other studies sug- secondhand smoke. By using serum levels of cotin- gested that risk increased up to at least 40 cigarettes per ine (a metabolite of nicotine) as biomarkers of expo- day (Miettinen et al. 1976; Willett et al. 1987). However, sure, Whincup and colleagues (2004) explored the dose more recent data appeared to show an increase in CHD response relationship between exposure to cigarette risk with more cigarettes smoked per day only up to about smoke and CHD in persons involuntarily exposed to 25 cigarettes; the risk increased relatively little even with cigarette smoke. More than 2,000 men who said they further increases in cigarette consumption (Neaton and did not smoke had blood levels of cotinine measured in Wentworth 1992; Rosengren et al. 1992; Thun et al. 1997). 1978–1980 and then had follow-up for 20 years. Nico- Law and Wald (2003), who conducted a meta- tine exposure was examined by quartiles of blood coti- analysis of five large studies of smoking and CHD, dem- nine as follows: less than or equal to 0.7 nanograms per onstrated a nonlinear dose-response relationship between milliliter (ng/mL), 0 to 1.4, 1.5 to 2.7, and 2.8 to 14.0. the number of cigarettes smoked per day and the RR of Hazard ratios for CHD, which included deaths and non- disease (Figure 6.3). The researchers suggested that the fatal MIs, were significantly increased at all upper quar- effect of cigarette smoking on risk of CHD may have a low tiles (hazard ratios, 1.43 to 1.57) compared with the lowest threshold and that the dose-response characteristics of exposure quartile, after adjustment for established CHD the risk relationship are less steep at higher doses. This risk factors. Hazard ratios were also higher at the first hypothesis was used to explain the seeming anomaly of a and second five-year follow-ups (3.73 to 10.58 and 1.95 high RR of CHD associated with relatively low exposure to to 2.48, respectively) than those at later follow-ups. The

Figure 6.2 Age-specific excess death rates among male smokers for coronary heart disease, lung cancer, chronic obstructive pulmonary disease (COPD), and cerebrovascular disease

1,000

900

800

700

600

500

400

300 Excess death rate per 100,000 200

100

0 35–39 40–44 45–49 50–54 55–59 60–64 65–69 70–74 75–79 80+ Age group (years) Lung cancer Coronary heart disease COPD Cerebrovascular disease

Cardiovascular Diseases 357 Surgeon General’s Report

Figure 6.3 Dose-response relationship between number of cigarettes smoked per day and relative risk of ischemic heart disease

2.0

1.5 Cause and effect: mechanisms other than platelet aggregation (linear dosimetry)

1.3 Cause and effect: platelet aggregation heart disease event (maximal effect at low dose) Relative risk of ischemic

1.0 Confounding 0 5 10 15 20 25 30 Number of cigarettes smoked per day

Source: Law and Wald 2003. Reprinted with permission from Elsevier, © 2003. Note: The dose-response relationship between exposure to tobacco smoke and ischemic heart disease events is compartmentalized into separate associations attributable to confounding (difference between smokers and nonsmokers in blood pressure, body weight, blood lipids, and diet), cause and effect maximal at low dose, and cause and effect with linear dosimetry.

substantial cardiovascular risk attributable to involuntary Smokers titrate cigarette smoke to achieve a consistent exposure to cigarette smoke (USDHHS 2006) and the intake of nicotine by altering the number of cigarettes practice in most CVD studies of not excluding from the smoked per day or by changing the puffing pattern— control group persons who had secondhand smoke expo- that is, by taking deeper, faster, more, or longer puffs sure have resulted in underestimation, in many research (National Cancer Institute [NCI] 2001). When these smoking reports, of the effects of active smoking compared with no behaviors fail to restore the level of nicotine intake, as they exposure to cigarette smoke. may with cigarettes that have very low machine-measured The data on secondhand smoke and CHD risk indi- yields, smokers may increase the number of cigarettes per cate that the dose-response relationship between exposure day to maintain the same level of nicotine intake. to smoke and cardiovascular effects is nonlinear. Another These observations suggest that the number of cig- consideration is that the number of cigarettes smoked arettes smoked per day may have become a less precise per day may not provide a linear measure of exposure to measure of exposure to tobacco smoke with the introduc- tobacco smoke. When carboxyhemoglobin or serum coti- tion of cigarettes with low machine-measured yields of nine levels were used as measures of the smoke taken in, tar and nicotine. This diminished precision of cigarettes persons who smoked more cigarettes per day had higher smoked per day as a measure of exposure may account levels of these biologic substances (Benowitz 1996). Even for some of the discordance between studies that define so, the carboxyhemoglobin and cotinine levels were sub- increased risk by an increase of more than one pack per stantially lower than those predicted by linear extrapola- day in the number of cigarettes smoked. By using serum tion from data on persons who reported smoking 1 to 20 cotinine as an indicator of nicotine intake and exposure cigarettes per day (Law et al. 1997). Among smokers of 40 to tobacco smoke in the population demonstrates a linear or more cigarettes per day, the levels of these biomark- increase for 10 to 15 cigarettes per day. However, as use ers were 35 percent lower than those predicted by linear exceeds 10 to 15 cigarettes per day, a progressively smaller extrapolation from data on persons who reported smoking increment in serum cotinine for each increment in the fewer than 20 cigarettes per day. number of cigarettes smoked per day is observed (Carabal- At the same reported number of cigarettes per day, lo et al. 1998; O’Connor et al. 2006). This flattening of the cotinine levels varied substantially (Benowitz 1996). relationship between exposure and cigarettes smoked per

358 Chapter 6 How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease day was similar to flattening of the relationship between CPS-I, investigators calculated the risk of developing CHD the RR of CHD and the number of cigarettes smoked per by age and duration of smoking (see Table 6.1 for data on day. Thus, researchers should be cautious about defin- White men) (Burns et al. 1997). For almost all age groups ing the absence of a continuing increase in risk among younger than age 70 years, RRs increased with increas- smokers of more than 20 cigarettes per day as evidence ing duration of smoking. Data from CPS-II on men (Thun that increases in actual exposure are not accompanied by et al. 1997) also demonstrated a pattern of increasing RR increases in risk. with age-specific mortality due to CHD and increasing duration of smoking for each level of cigarettes smoked Duration of Smoking per day (Table 6.2). Even though data in these analyses were not adjusted for potential differences in other car- Researchers have not always demonstrated a sig- diovascular risk factors, the findings presented a convinc- nificant relationship between duration of cigarette smoking picture of increasing risk of CHD with longer duration ing and CHD risk when adjustment was made for other of smoking. risk factors and the number of cigarettes smoked per day (Kuller et al. 1991; Tverdal 1999). Variation in the number Smoking Cessation of cigarettes smoked per day and in the products smoked during the lifetime of a smoker is often substantial, but The risks of MI and death from CHD are lower this variable is not well captured in epidemiologic studies. among former smokers than among continuing smokers Age is colinear with duration of smoking, because in many studies, including those with data adjusted for the two variables grow in tandem after a person starts to levels of other risk factors (Gordon et al. 1974; Åberg et smoke and the RRs for smoking and CHD decline with al. 1983; USDHHS 1990; Kuller et al. 1991; Frost et al. advancing age. Furthermore, most smokers begin to 1996). The risk fell rapidly, decreasing about one-half in smoke during adolescence, which promotes the colinear- one year (Lightwood and Glantz 1997). Risks appear to ity. These realities make it difficult to estimate the inde- remain slightly elevated for more than a decade after per- pendent contributions of age and duration of smoking sons stopped smoking, but in some studies this increased to risk of CHD in multivariate models. However, the two risk was not statistically significant (Dagenais et al. 1990; studies of the American Cancer Society are a good source Omenn et al. 1990; Kawachi et al. 1993a, 1994; Jacobs et of data, because each study consists of more than 1 mil- al. 1999a; Qiao et al. 2000). Among smokers who had MI or lion men and women (Burns et al. 1997; Thun et al. 1997). angiographically documented CHD, persons who stopped Analyses of these data stratified by age and the number smoking had a substantially lower rate of reinfarction than of cigarettes smoked per day showed steady increases in did those who continued to smoke. Reduction in risk was CHD mortality rates with increasing duration of smoking evident within the first year after MI. Risk continued to for persons younger than age 70 years. Using data from be lower among former smokers than among continuing

Table 6.1 Rate ratios for coronary heart disease among White men, by age and duration of cigarette smokinga

Duration of smoking (years) Age (years) 20–24 25–29 30–34 35–39 40–44 45–49 50–54 55–59

40–44 1.95 2.47 4.23 NR NR NR NR NR 45–49 1.79 2.5 2.66 2.64 4.39 NR NR NR 50–54 2.06 2.25 2.22 2.74 2.82 3.4 NR NR 55–59 1.71 1.66 2.13 2.03 2.43 2.99 2.17 NR 60–64 1.83 1.44 1.86 1.75 1.92 2.12 2.45 4.06 65–69 1.34 1.56 1.52 1.61 1.49 1.6 2.09 2.25 70–74 1.17 1.14 1.23 1.08 1.55 1.26 1.53 1.78 75–79 1.09 1.2 1.31 1.12 1.55 1.46 0.94 1.36 Source: Adapted from Burns et al. 1997. Note: NR = data not reported. aFrom Cancer Prevention Study I, American Cancer Society.

Cardiovascular Diseases 359 Surgeon General’s Report

Table 6.2 Death rates and rate ratios for death from coronary heart disease among men, by age and duration of smoking by number of cigarettes smoked per daya

Death rates (per 100,000)b Rate ratiosc

Number of years of smoking Number of years of smoking Number of Age Age cigarettes/day (years) 20–29 30–39 40–49 ≥50 (years) 20–29 30–39 40–49 ≥50

50–59 241.1 276 277.9 NR 50–59 2.7 3.1 3.2 NR 1–19 60–69 340.6 560.3 643.5 866.5 60–69 1.1 1.8 2.1 2.8

50–59 213.9 272.6 493.4 956.4 50–59 2.4 3.1 5.6 NR 20 60–69 299.9 509 729.5 1,088.9 60–69 1 1.6 2.4 3.5

50–59 195.8 237.4 367.2 NR 50–59 2.2 2.7 4.2 NR 21–39 60–69 232.2 350.8 607.2 1,113.4 60–69 NR 1.1 2 3.6

50–59 144.9 281.6 321.4 664.5 50–59 1.6 3.2 3.7 NR 40 60–69 470.8 458.3 607.6 988.3 60–69 1.5 1.5 2 3.2 Source: Adapted from Thun et al. 1997. Note: NR = data not reported. aFrom Cancer Prevention Study II, American Cancer Society (CPS-II). bCPS-II data, Appendix Table 10, Thun et al. 1997. cCPS-II data, Appendix Table 12, Thun et al. 1997.

smokers for prolonged periods after the first MI (Daly et al. angiotensin-converting-enzyme (ACE) inhibiting drugs, 1983; Omenn et al. 1990). Studies also demonstrated rapid b-blockers, or spironolactone, which are mainstays for reduction in risk after persons stopped smoking among the treatment of heart failure. populations at high risk for CHD (Ockene et al. 1990) and among women (Kawachi et al. 1993a, 1994). Women Patients with angiographically documented CHD Women have lower absolute rates of CHD than do who stopped smoking at the diagnosis of CHD (Vlietstra et men. However, cigarette smoking has been associated al. 1986) or before diagnosis (Hermanson et al. 1988) had with higher RR of MI (Njølstad et al. 1996) and higher lower death rates from MI or CHD than did continuing CHD mortality (Kawachi et al. 1994; Thun et al. 1997) smokers. In addition, the benefit of stopping smoking did among women than among men. The absolute increase in not decline with advancing age. risk of CHD from smoking is similar for men and women In the 16-year follow-up of the Multiple Risk Factor (USDHHS 1983, 2001). Intervention Trial Research Group (1990, 1996), mortality A prospective evaluation of fatal and nonfatal CVD from CHD was 11.4 percent lower in the “special interven- events among women in the Nurses’ Health Study (Willett tion” group than in the “usual care” group. This result et al. 1987) found that smoking was an independent cause may illustrate the benefit of stopping smoking, because of CVD. Age-adjusted risks of disease increased progres- one of the interventions targeted smoking cessation. A sively with more cigarettes smoked per day up to 45 or trial of advice to civil servants in London, England, to stop more per day. Even when the combined risks of fatal CHD smoking demonstrated an 18-percent reduction in mortal- and nonfatal MI were adjusted for levels of other risk fac- ity from CHD in the intervention group versus the control tors, risks increased with increasing numbers of cigarettes group after 10 years of follow-up (Rose et al. 1982). Suskin per day. and colleagues (2001) reported that in addition to benefits Researchers have demonstrated a rapid decline in for CHD, stopping smoking also reduces morbidity and excess risk of CHD in women after they stopped smok- mortality in patients with left ventricular dysfunction. In ing cigarettes. Even so, 10 to 14 years of nonsmoking are this study, the benefits of stopping smoking on mortality required before risks approach those of lifetime nonsmok- and recurrent congestive heart failure requiring hospital- ers (Kawachi et al. 1993a, 1994). ization were similar to the benefits from treatments with

360 Chapter 6 How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease

Race and Ethnicity dysfunction after MI. Finally, the risk of recurrent cardiac arrest among smokers surviving out-of-hospital cardiac In 2004, heart disease mortality was higher among arrest was lower among persons who then stopped smok- African Americans than among Whites (National Heart, ing than among those who continued to smoke (Hall- Lung, and Blood Institute [NHLBI] 2007). From 1999 strom et al. 1986). through 2004, the prevalence of acute MI was higher for African Americans than for Whites aged 35 through 54 years; however, for ages 55 years and older, the prevalence of acute MI was higher among Whites (NHLBI 2007). Stroke The INTERHEART study is a case-control investigation of acute MI in 52 countries in Africa, Asia, Australia, After adjustment of data for other risk factors, the Middle East Crescent, and North and South Ameri- cigarette smokers have higher risk of stroke and higher ca (Teo et al. 2006). The odds ratio (OR) for acute MI in mortality from cerebrovascular disease than do lifetime smokers was 2.95 for this large multiethnic population nonsmokers, and a dose-response relationship is evident compared with lifetime nonsmokers. In addition, the risk (USDHHS 1983, 2001, 2004; Neaton et al. 1984; Colditz of MI was higher among persons who smoked bidis than et al. 1988; Wolf et al. 1988; Kannel and Higgins 1990; among nonsmokers in countries where use of this form of Kuller et al. 1991; Freund et al. 1993; Hames et al. 1993; tobacco is common. Håheim et al. 1996; Tanne et al. 1998; Jacobs et al. 1999a; Researchers also identified cigarette smoking as Sharrett et al. 1999; Djoussé et al. 2002). In addition, in a significant risk factor for CHD among Hispanic popu- the 20-year follow-up of a prospective study of mortal- lations (Mendelson et al. 1998) and Asian populations ity that controlled for other cardiovascular risk factors, (Kiyohara et al. 1990; Miyake et al. 2000; Lam et al. 2002). cigarette smoking increased the risk of death from stroke and mortality rates grew the number of cigarettes smoked increased (Hart et al. 1999). Sudden Death In a meta-analysis of data from 32 studies, the overall RR for stroke associated with cigarette smoking was 1.5 (95 percent confidence interval [CI], 1.4–1.6) (Shin- Most sudden death is due to CVD. In many epidemi- ton and Beevers 1989). The RRs varied with the stroke ologic studies, RRs for sudden cardiac death were higher subtypes: 1.9 for cerebral infarction, 0.7 for cerebral than RRs for CHD or MI among persons who smoked. The hemorrhage, and 2.9 for subarachnoid hemorrhage. The RRs for sudden death among current smokers, compared researchers reported a dose-response relationship with lifetime nonsmokers, often exceeded 3.0 (USDHEW between the number of cigarettes smoked per day and the 1971, 1979; Dawber 1980; Kannel and Thomas 1982; RR. The data suggested a sustained higher risk of stroke USDHHS 1983; Wannamethee et al. 1995; Sexton et al. among former smokers younger than age 75 years than 1997). In multivariate analyses of the combined data from the risk for nonsmokers in the same age group. For all the Framingham Heart Study and the Albany Study, which ages combined, RR for former smokers was 1.2. examined sudden cardiac death in men aged 45 through During the 26-year follow-up of the cohort in the 64 years, cigarette smoking was the risk factor with the Framingham Heart Study, cigarette smoking was a sig- highest statistical significance (Kannel et al. 1975). In a nificant risk factor for stroke (Wolf et al. 1988). The risk study of data from the 1986 National Mortality Followback declined, however, among smokers who had stopped Survey among persons with no history of CHD, cigarette smoking for two years and was similar to that of lifetime smoking was the only modifiable risk factor associated nonsmokers after five years of abstinence from smok- with sudden coronary death and it was one factor associ- ing. In the 12-year follow-up of the Nurses’ Health Study ated with increased risk of sudden coronary death among (Kawachi et al. 1993b), RR for stroke among current persons with known CHD (Escobedo and Zack 1996; Esc- smokers was 2.58 compared with nonsmokers, but it was obedo and Caspersen 1997). Cigarette smoking was also 1.34 among former smokers compared with nonsmok- associated with risk of sudden cardiac death in the 18-year ers. Once those who stopped smoking had abstained for follow-up of the Honolulu Heart Program (Kagan et al. two to four years, their risk for stroke could not be dis- 1989) and the 28-year follow-up of the Framingham Heart tinguished from that of lifetime nonsmokers. In addition, Study (Cupples et al. 1992). the pattern of decline in total risk for stroke after stopping Peters and colleagues (1995) found an associa- smoking remained the same after adjustments for other tion between smoking cessation and reduction in death risk factors. from cardiac arrhythmia for patients with left ventricular

Cardiovascular Diseases 361 Surgeon General’s Report

Aortic Aneurysm Pipes, Cigars, and Low-Tar Cigarettes Mortality studies consistently demonstrated higher risk of death from abdominal aortic aneurysm among cig- “Low-tar” or “light” cigarettes were designed to arette smokers than among nonsmokers (Hammond and produce low machine-measured yields of tar and nicotine Horn 1958; Weir and Dunn 1970; USDHHS 1983, 2004; (NCI 2001). Design characteristics of low-tar cigarettes Strachan 1991; Nilsson et al. 2001). In addition, the risk include increased ventilation and more rapid cigarette rose with an increasing number of cigarettes smoked per burn rate. By changing the way they smoke or the number day (Kahn 1966; Hammond and Garfinkel 1969; Burns et of cigarettes smoked, persons who smoke these products al. 1997; Blanchard et al. 2000; Vardulaki et al. 2000). can obtain as much nicotine as from “regular” or “full- Studies have demonstrated an association of ciga- flavored” cigarettes, thereby satisfying their addiction rette smoking with prevalence of aortic aneurysm or aor- (USDHEW 1979; NCI 2001). Comprehensive reviews of tic dilation, as determined by ultrasonography in cohorts this issue concluded that use of low-tar cigarettes has not of men and women, even after adjustment for a large resulted in meaningful reduction in the risk of CVD (NCI number of known risk factors (Alcorn et al. 1996; Lee et 2001; Stratton et al. 2001; Scientific Advisory Committee al. 1997; Wilmink et al. 1999; Jamrozik et al. 2000; Led- on Tobacco Product Regulation 2002; USDHHS 2004). erle et al. 2001). The U.S. Preventive Services Task Force Compared with persons who smoke cigarettes, (2005) recommended a one-time screening by ultrasonog- smokers who exclusively smoke pipes or cigars have lower raphy for abdominal aortic aneurysm among men aged 65 risk for many smoking-related diseases (NCI 1998). Smoke to 75 years who had ever smoked. Cigarette smoking has from pipes and cigars contains the same toxic substances been associated with increased growth of abdominal aortic as cigarette smoke, but those who use a pipe or cigar usu- aneurysms (Brady et al. 2004). This finding suggests that ally smoke at lower intensity; observation indicates that more frequent monitoring of smokers for this condition they tend not to inhale the smoke, thus reducing their is necessary. With increasing duration of abstinence from exposure to its toxic substances (USDHEW 1979; NCI smoking, the risk of developing an abdominal aneurysm 1998; Shanks et al. 1998). Most current cigar users are appears to slowly decline (Wilmink et al. 1999). young males who often smoke less than one cigar daily (NCI 1998); no data on risk for this population are available. For older adults who regularly use cigars, particular- Peripheral Arterial Disease ly those who smoke more than one cigar per day or inhale the smoke, risk of CHD is modestly higher than that for Cigarette smoking and diabetes are well established nonsmokers (NCI 1998; Iribarren et al. 1999; Jacobs et al. as the major risk factors for PAD, and a strong dose- 1999b; Baker et al. 2000). Studies have reported similar response relationship for smoking was observed even after increases in risks for CHD and cerebrovascular disease for adjustment for other CVD risk factors (Weiss 1972; Kan- persons who smoke a pipe exclusively (Henley et al. 2004). nel and Shurtleff 1973; USDHHS 1983; Wilt et al. 1996; Price et al. 1999; Meijer et al. 2000; Ness et al. 2000). Data from the Framingham Heart Study demonstrated Summary increased risk of PAD among both young and older male and female cigarette smokers after adjustment for other Cigarette smoking and involuntary exposure to cardiovascular risk factors. In addition, this risk increased cigarette smoke are major causes of CHD, stroke, aortic with the increase in the number of cigarettes smoked per aneurysm, and PAD. The risk is seen both as an increased day, and this result was statistically significant (Freund risk of acute thrombosis of narrowed vessels and as an et al. 1993). The Framingham Offspring Study reported a increased degree of atherosclerosis in the blood vessels similar finding (Murabito et al. 2002). Finally, researchers involved. The cardiovascular risks attributable to cigarette have observed a significantly higher rate of late arterial smoking increase with the number of cigarettes smoked occlusion in patients who continued to smoke after and with the duration of smoking. However, risk is sub- peripheral vascular surgery than in those who stopped stantially increased even by exposure to low levels of cig- smoking (Wray et al. 1971; Ameli et al. 1989; Wiseman et arette smoke as with exposure to secondhand smoke or al. 1989). Among smokers with claudication, progression smoking a few cigarettes per day. Risks are not reduced to critical limb ischemia is reduced in those who stopped by smoking cigarettes with lower machine-measured smoking (Jonason and Bergström 1987).

362 Chapter 6 How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease yields of tar and nicotine. Smokers of only pipes or experience the lower CVD risk of persons who primarily cigars seem to have lower risks of CVD than do cigarette smoke a pipe or cigar. Stopping cigarette smoking and smokers. However, cigarette smokers who switch to pipes eliminating exposure to secondhand smoke rapidly and or cigars often inhale the tobacco smoke and may not substantially reduce risks of various CVDs.

Secondhand Tobacco Smoke and Cardiovascular Disease

The 2006 Surgeon General’s report on involun- for MI after smoke-free laws were put in place (Dinno and tary exposure to tobacco smoke (USDHHS 2006) and Glantz 2007; Lightwood and Glantz 2009; Meyers et al. Barnoya and Glantz (2005) extensively reviewed risks of 2009). As for stroke, the evidence was insufficient to infer CVD among nonsmokers exposed to secondhand tobacco a causal relationship between increased risk of CHD mor- smoke. They found a causal relationship among both men bidity and mortality and exposure to secondhand smoke. and women between exposure to secondhand smoke and Studies of the effects of secondhand smoke on subclini- increased risks of CHD morbidity and mortality. Pooled cal vascular disease, particularly thickening of the walls RRs from meta-analyses indicated a 25- to 30-percent in- of the carotid arteries, also suggest a causal relationship crease in risk of CHD from exposure to secondhand smoke. between exposure to secondhand smoke and atheroscle- The study by Whincup and associates (2004), which was rosis. As mentioned previously, the substantial CVD risk based on blood levels of cotinine in men, suggested a associated with involuntary exposure to cigarette smoke 50- to 60-percent increase in risk of CHD from exposure indicates that the risks estimated in most studies of active to secondhand smoke. The risk of acute MI appeared to smoking are biased downward because the control groups decline rapidly after cessation of exposure to secondhand generally included large numbers of persons with expo- smoke, as evidenced by a decline in hospital admissions sure to secondhand smoke.

Pathophysiology

This section on pathophysiology focuses primarily plasma nicotine levels peaked sharply after each cigarette, on mechanisms by which cigarette smoking may increase trough values also rose during the first six to eight hours risk of CVD. of regular smoking during the day (Benowitz et al. 1982a). This accumulation pattern was consistent with an elimination half-life for nicotine of two hours (Benowitz et al. Cigarette Smoke Constituents and 1982a). In persons who smoke regularly, venous plasma levels of nicotine reached a plateau in early afternoon and Cardiovascular Disease remained at that level until bedtime (Figure 6.4). Signifi- cant levels of nicotine were in the smoker’s venous blood Three constituents of cigarette smoke have received even on waking in the morning. Thus, these findings the greatest attention as potential contributors to CVD: indicate that the regular smoker is exposed to significant nicotine, carbon monoxide (CO), and oxidant gases. Some levels of nicotine 24 hours per day. research also investigated the contributions of polycyclic Nicotine is a sympathomimetic drug that releases aromatic hydrocarbons (PAHs), particulate matter, and catecholamines both locally from neurons and systemi- other constituents of tobacco smoke to the pathophysi- cally from the adrenal gland. In studies of the pharmaco- ology of CVD including atherogenesis (Brook et al. 2004; dynamics of nicotine, the intensity of its maximal effect Vermylen et al. 2005; Bhatnagar 2006). was greater with more rapid delivery (Porchet et al. 1987). Nicotine, which is absorbed rapidly from cigarette Pharmacodynamic studies also indicated that although smoke, was found in arterial blood levels of 40 to 100 ng/ tolerance to the effects of nicotine developed rapidly, tol- mL after each cigarette was smoked (Henningfield et al. erance was incomplete (Porchet et al. 1987). In one study, 1993). The typical dose of nicotine systematically absorbed a constant intravenous infusion of nicotine increased the from each cigarette is 1 to 2 milligrams (mg). Although

Cardiovascular Diseases 363 Surgeon General’s Report

Figure 6.4 Plasma nicotine and carboxyhemoglobin concentrations throughout a day of cigarette smoking

X X X 40 XXX X X X 30 X

(ng/mL) 20 X

10 X Blood nicotine concentrations X X

0 8:00 12:00 4:00 8:00 12:00 4:00 8:00 a.m. p.m. p.m. p.m. a.m. a.m. a.m. Clock time 12

8 X X X X X 6 X X X X Carboxyhemoglobin (%) 4 X X

2 8:00 12:00 4:00 8:00 12:00 4:00 8:00 a.m. p.m. p.m. p.m. a.m. a.m. a.m. Clock time

Source: Benowitz 2003. Adapted from Benowitz et al. 1982b with permission from Elsevier, © 2003. Note: Mean (± standard error of measurement) blood nicotine and carboxyhemoglobin concentrations in cigarette smokers. Participants smoked cigarettes every half-hour from 8:30 a.m. to 11:00 p.m., for a total of 30 cigarettes per day. ng/mL = nanograms per milliliter.

heart rate even though nicotine levels in the blood were In another study, heart rate measured by ambulatory relatively low. As the infusion continued, the heart rate monitoring was higher throughout the day when persons reached a plateau despite a progressive rise in blood levels were smoking than when they were not smoking (Benow- of nicotine (Benowitz et al. 1982a). The same phenome- itz et al. 1984). The extent of elevation was independent of non was observed in comparisons of acceleration of heart the blood level of nicotine absorbed from the cigarettes. rate with level of blood nicotine during regular cigarette The researchers concluded that the elevated heart rate smoking throughout the day (Benowitz et al. 1984). reflected persistent stimulation of the sympathetic

364 Chapter 6 How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease nervous system, a possible contributing factor to CVD. Acrolein, a reactive aldehyde produced by endog- Nicotine may also contribute to endothelial dysfunc- enous lipid peroxidation, is present at high levels in cig- tion, lipid abnormalities, and insulin resistance (Beno arette smoke. Acrolein binds covalently to form protein witz 2003). adducts, and acrolein-induced modification of proteins CO is a major constituent of cigarette smoke. In has been implicated in atherogenesis. Acrolein modifies regular smokers, carboxyhemoglobin levels average apolipoprotein A-I (APO A-I), the major protein in HDL about 5 percent, compared with 10 percent or higher (Shao et al. 2005). HDL protects against atherosclerosis. in heavy smokers (Benowitz et al. 1982b). These values Acrolein-protein adducts co-localize with APO A-I in mac- compare with levels of 0.5 to 2 percent in nonsmokers, rophages in the intima of human atheromatous blood ves- depending on exposure to automobile exhaust. Like nico- sels (Szadkowski and Myers 2008). tine levels, elevated carboxyhemoglobin levels persist for Acrolein also oxidized thioredoxins 1 and 2 in endo- 24 hours a day in smokers (Figure 6.4). thelial cells. Thioredoxins are prominent antioxidant CO exposure can aggravate ischemia and worsen proteins that regulate the oxidation-reduction balance symptoms in persons with vascular disease, although it is critical for normal cell function. These results suggest not clear that CO contributes directly to atherosclerosis that oxidation of thioredoxins can result in dysfunction (Benowitz 2003). CO binds avidly to hemoglobin, reduc- and death of endothelial cells, contributing to athero- ing the amount of hemoglobin available to carry oxygen sclerosis. In addition, acrolein induces production of the and impeding release of oxygen by hemoglobin. In some enzyme cyclooxygenase-2 (COX-2) in human endothelial studies, inhalation of CO at levels comparable to those in cells in vitro (Park et al. 2007). This finding is relevant cigarette smokers reduced exercise tolerance in patients because COX-2 is expressed in atherosclerotic lesions and with angina pectoris, intermittent claudication, or COPD may participate in atherogenesis. Acrolein may contribute (Calverley et al. 1981; Allred et al. 1989). Another study to thrombogenicity in smokers by inhibiting antithrom- reported that CO exposure in persons with obstructive bin activity (Gugliucci 2007). Finally, acrolein induces coronary disease resulted in a greater degree of exercise- hypercontraction in isolated human arteries and could induced ventricular dysfunction and an increase in the contribute to smoking-induced coronary vasospasm number and complexity of ventricular arrhythmias dur- (Conklin et al. 2006). ing exercise (Sheps et al. 1990). Inhaling CO reduced the Cigarette smoke contains a number of metals, in- threshold for ventricular fibrillation in animals (DeBias et cluding aluminum, cadmium, copper, lead, mercury, al. 1976). nickel, and zinc. Metals in cigarette smoke catalyze the Long-term CO exposure in smokers resulted in oxidation of cellular proteins (Bernhard et al. 2005). This greater red blood cell mass and reduced the oxygen- reaction may lead to structural damage, endothelial dys- carrying capacity of red blood cells, resulting in relative function, and detachment of endothelial cells from the hypoxemia (Benowitz 2003). In response to hypoxemia, walls of blood vessels. Mixtures of metals and oxidants red blood cell masses increased to maintain the amount of may be particularly damaging to endothelial cells. Cadmi- oxygen needed by organs in the body. The increase in red um levels are higher in serum of smokers, and cadmium blood cell mass increased blood viscosity and may contrib- accumulates in the aortic walls of smokers (Abu-Hayyeh ute to hypercoagulation in smokers. et al. 2001). Epidemiologic evidence indicates an associa- Cigarette smoke delivers a high level of oxidizing tion between serum levels of cadmium and lead and CVD, chemicals to smokers, including oxides of nitrogen and including hypertension and MI (Abu-Hayyeh et al. 2001). many free radicals from both the gas and tar phases of PAHs found in the tar fraction of cigarette smoke cigarette smoke (Church and Pryor 1985). Exposure to reportedly accelerated atherosclerosis in experimen- oxidant chemicals in smoke was associated with depletion tal animals. Weekly injections of benzo[a]pyrene and of endogenous levels of antioxidants, manifested as lower 7,12-dimethylbenz[a]anthracene, at doses below those blood levels of vitamin C in smokers than in nonsmok- that produce tumors, increased development of athero- ers (Lykkesfeldt et al. 2000). Cigarette smoking also was sclerotic plaque in the aortas of cockerels (Penn and Sny- reported to increase levels of lipid peroxidation products der 1988). Similarly, inhaled butadiene, a component of in the plasma and urine of smokers (Morrow et al. 1995). the vapor phase of cigarette smoke, increased the amount Study results also indicated that oxidant stress contrib- of atherosclerotic plaque in the same animal model (Penn utes to several potential mechanisms of CVD, including and Snyder 1996). The researchers speculated that one inflammation, endothelial dysfunction, lipid abnormali- mechanism of atherogenesis is a mutation, followed by ties such as oxidation of low-density lipoprotein (LDL), hyperproliferation of smooth muscle or other cells that and platelet activation (Burke and FitzGerald 2003). may contribute to growth of atherosclerotic plaque.

Cardiovascular Diseases 365 Surgeon General’s Report

Figure 6.5 Overview of mechanisms by which cigarette smoking causes an acute cardiovascular event

Oxidant chemicals Carbon Nicotine Particulates monoxide Other combustion products

Sympathetic Reduced nervous system Inﬂammation oxygen activation availability

Platelet Increased heart rate activation/ Endothelial Coronary Increased blood pressure thrombosis dysfunction vasoconstriction Increased myocardial contractility

Reduced myocardial Increased blood, oxygen, and myocardial nutrient supply demand for oxygen Coronary occlusion and nutrients

Myocardial ischemia Myocardial infarction

Sudden death

Studies of the cardiovascular effects of smoke- products contain a large array of chemicals, including nic- less tobacco may be informative for understanding the otine, nitrosamines, nitrosamine acids, PAHs, aldehydes, pathophysiology of smoking-induced CVD. Oral and nasal and metals (IARC 2007). A recent systematic review smokeless tobacco products have been used for centuries reported that studies from both the United States and around the world (International Agency for Research on Sweden showed an increased risk of death from MI and Cancer [IARC] 2007). Traditional smokeless tobacco prod- stroke related to the frequency and duration of use of ucts vary widely among countries; however, similar to smokeless tobacco products (Boffetta and Straif 2009). Sweden, forms of oral snuff are the most common types This review relied heavily (85–89 percent of the weight) of products used in the United States (Substance Abuse on results of a large U.S. cohort study conducted in and Mental Health Services Administration 2009). These two waves between 1959–1971 and 1982–1988 and may

366 Chapter 6 How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease not represent risk associated with products currently Mechanisms marketed in the United States and Europe. As in cigarettes, nicotine is the principal alkaloid in smokeless Cigarette smoking produces acute myocardial isch- tobacco products, and the concentrations of nicotine (mg/ emia by adversely affecting the balance of demand for gram [g] tobacco) are similar between cigarettes and the myocardial oxygen and nutrients with myocardial blood types of oral snuff sold in the United States (Djordjevic supply (Figure 6.5). The increase in demand for oxygen and Doran 2009). An analysis comparing the effects of in the myocardium is a consequence of nicotine stimu- using oral snuff with those of smoking cigarettes pro- lation of the sympathetic nervous system and the heart. vided insights into the role of nicotine versus the effects Cigarette smoking acutely increases levels of plasma of other toxins from tobacco smoke on CVD and car- norepinephrine and epinephrine and enhanced 24-hour diovascular risk factors (Benowitz et al. 1988, 1989). In urinary excretion of these catecholamines (review by addition clinical trials of nicotine patches in patients with Benowitz and Gourlay 1997). Regular smoking increases known CVD have not shown that transdermal nicotine the heart rate both in the short term (up to 20 beats per increased cardiovascular risk (Working Group for the minute) and throughout the day (average increase, 7 beats Study of Transdermal Nicotine in Patients with Coronary per minute), as measured during ambulatory monitoring. Artery Disease 1994; Joseph et al. 1996). In the study Nicotine also increases heart rate, blood pressure, and of 3,094 middle-aged smokers with chronic obstruc- myocardial contractility. These hemodynamic changes tive lung disease, the U.S. Lung Health Study found no result in increases in myocardial work that in turn require evidence of increased cardiovascular risk in subjects who increased myocardial blood flow. quit smoking by using nicotine gum versus those who In healthy persons, cigarette smoking increases cor- quit without use of nicotine gum (Murray et al. 1996). onary blood flow in response to increases in myocardial These studies and related evidence suggest that chemi- work. In smokers, the response in coronary blood flow to cals other than nicotine may contribute to the elevated increased myocardial demand was impaired (i.e., reduced risk of death from MI and stroke. In the INTERHEART coronary vasodilatory reserve) (Czernin and Waldherr study, the OR of acute MI was 2.23 among those who 2003). Cigarette smoking played a direct role by constrict- used only smokeless tobacco compared with those who ing coronary arteries through nicotine-mediated action used no tobacco. The OR was comparable to that of cur- on a-adrenergic receptors and by induction of endothelial rent cigarette smokers (OR = 2.95) compared with those dysfunction by nicotine and oxidizing chemicals (Nicod who used no tobacco (Teo et al. 2006). In addition, the et al. 1984; Puranik and Celermajer 2003). In addition, risk of acute MI among smokers who also used smokeless oxidant chemicals contribute to platelet activation and tobacco was the highest risk related to tobacco use (OR = thrombogenesis (Burke and FitzGerald 2003). 4.09), suggesting that some of the toxicants involved in Exposure to CO may also contribute to the adverse the elevated cardiovascular risk could be contained in both hemodynamic effects of cigarette smoking. By producing tobacco smoke and smokeless products. Smokeless functional anemia, CO increases the need for coro- tobacco products have been found to have significant nary blood flow, especially during physical exertion. An amounts of numerous other toxicants and carcinogens, in-adequate vasodilatory flow reserve produced by -ciga particularly tobacco-specific nitrosamines as well as rette smoking, in the face of need for increased coronary volatile aldehydes and PAHs (Stepanov et al. 2008). Addi- blood flow mediated by carbon dioxide, could contribute tional research on these and other toxicants in smokeless to myocardial ischemia with exercise in smokers. tobacco, such as heavy metals like cadmium, is needed to In addition to the mechanisms described in Fig- understand the observed cardiovascular risks among users ure 6.5, cigarette smoking has effects on inflammation, of smokeless tobacco products. insulin sensitivity, and lipid abnormalities that most likely contribute to smoking-induced CVD.

Cardiovascular Diseases 367 Surgeon General’s Report

Hemodynamic Effects

Blood Pressure and Heart Rate Intravenous nicotine, nicotine nasal spray, and nicotine chewing gum all increased the heart rate up to 10 to 15 In 1907, Erich Hesse published “The Influence of beats per minute and raised systolic blood pressure by up to Smoking on the Circulation,” which documents his obser- 5 to 10 millimeters of mercury (mm Hg), responses similar vations on the effects of smoking on heart rate and blood to the effects of cigarette smoking (Gourlay and Benowitz pressure (Hesse 1907). In most study participants, both 1997). Nicotine increased cardiac output by increasing heart rate and blood pressure increased immediately after both heart rate and myocardial contractility. Different vas- smoking. Hesse observed a greater response in blood pres- cular beds express different types and ratios of adrenergic sure after smoking in persons who smoked than in non- receptors. Therefore, not all vascular responses to nico- smokers. Speculating that the increases in blood pressure tine or tobacco smoke are the same. For example, nicotine and heart rate reflected stimulation of the heart or ner- constricts some vascular beds, such as the skin, and cuta- vous system, he instituted a rule prohibiting patients with neous vasoconstriction explains the reduced temperature a “heart weakness” from smoking, to avoid unnecessary of the fingertip observed with administration of nicotine strain and stress for the heart muscle. Many investigators (Benowitz et al. 1982a). Conversely, nicotine appears to confirmed Hesse’s observations on the hemodynamic dilate other vascular beds, such as skeletal muscle (Diana effects of cigarette smoking (Deanfield et al. 1986; Czernin et al. 1990). Vasodilation of skeletal muscle may partly et al. 1995; Barutcu et al. 2004). result from the increase in cardiac output, although the The positive chronotropic, inotropic, and blood release of epinephrine from nerve terminals may also pressure effects of smoking are explained by nicotine- contribute. The net result of increases in heart rate, blood induced activation of the sympathetic nervous system pressure, and myocardial contractility is an increase (review by Benowitz 2003). Nicotine promotes the release in myocardial work, followed by increased myocardial of epinephrine and norepinephrine from the adrenal blood flow. medulla and terminal nerve endings, resulting in increased heart rate and greater contractility through stimulation of myocardial β1 receptors. Peripheral vas- Coronary Blood Flow cular resistance increases through α-receptor mediated vasoconstriction that in turn increases blood pressure. An important hemodynamic consequence of ciga- Coronary β2 and α2 receptors are also stimulated: stimu- rette smoking is its effect on blood flow in the coronary lation of β2 receptors promoted vasodilation, and stimula- arteries. Cigarette smoking acutely increased coronary tion of α2 receptors promoted vasoconstriction (Cryer et blood flow by up to 40 percent, apparently a response to al. 1976; Benowitz 2003). the increase in myocardial work (review by Czernin and Cryer and colleagues (1976) elucidated the mecha- Waldherr 2003). nisms behind observed hemodynamic changes during In anesthetized dogs, coronary blood flow showed smoking. They observed more than a 150-percent increase a biphasic response to nicotine. Initially, researchers in plasma epinephrine levels (from 44 to 113 picograms hypothesized that increases in coronary blood flow— [pg]/mL) at 10 minutes after participants started to smoke in the large coronary vessels as well as the smaller a cigarette. Norepinephrine values increased to a smaller vessels—resulted from an increase in myocardial meta- degree (from 227 to 315 pg/mL) at 12.5 minutes after the bolic demand. start of smoking. These increases were associated with Cigarette smoking impairs the response of coro- significant increases in heart rate and blood pressure. nary blood flow to an increase in myocardial demand for Pretreatment with α-receptor blockers and β-receptor oxygen; that is, it reduces the coronary vasodilatory flow blockers had little effect on the increase in plasma levels of reserve. Thus, the increase in coronary blood flow based catecholamines, but increases in blood pressure and heart on the level of myocardial work is less than would be rate were eliminated. This study confirmed that smoking- expected in the absence of exposure to tobacco smoke. induced increases in blood pressure and heart rate are Considerable evidence indicates that cigarette smok- attributable to adrenergic mechanisms. ing causes dysfunction of the coronary arterial endothe- The hemodynamic effects of cigarette smoking lium (see “Endothelial Injury or Dysfunction” later in are mediated primarily by nicotine, although oxidizing this chapter). chemicals in tobacco smoke also affect vascular function.

368 Chapter 6 How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease

Cigarette smoking may also be associated with In one study, after pretreatment with calcium-channel- coronary vasoconstriction. Although cigarette smoking blocking agents or nitroglycerin, cigarette smoking increases coronary blood flow in a person who does not increased coronary blood flow in patients with CHD who have CHD, it may decrease coronary blood flow in the had manifested no increase after cigarette smoking alone presence of coronary disease. Regan and colleagues (1960) (Winniford et al. 1987). This finding that coronary vaso- measured coronary sinus blood flow in seven male volun- dilator drugs, which block chemically mediated vasocon- teers with documented CHD before and after they smoked striction, permit the usual increase in coronary blood flow two cigarettes during a period of about 25 minutes; car- in response to increased myocardial work supports the diac work increased by about 30 percent during smoking. hypothesis that cigarette smoking directly produces coro- Even so, in response to smoking, coronary blood flow fell nary vasoconstriction. In another study, chewing 4 mg of in three patients, did not change in three patients, and nicotine gum by healthy nonsmokers blunted the increase increased in one patient. Similar paradoxical responses in coronary blood flow that occurs with increased heart were observed after long-term smokers were exposed to rate produced by cardiac pacing (Kaijser and Berglund cold by testing with cold pressors (Campisi et al. 1998). 1985). This result confirmed that even low doses of nico- In the testing, the hand is immersed in ice water for one tine can directly constrict coronary arteries in humans. to two minutes. This painful procedure evokes a mixed Another study found that nicotine worsened myo- adrenergic response involving coronary receptors α1 and cardial dysfunction in “regionally stunned” ischemic α2 (vasoconstriction), β2 (vasodilation), and myocardial myocardium of anesthetized dogs (Przyklenk 1994). In β1 (indirect coronary vasodilation). Endothelial α2 recep- a placebo-controlled experiment, transient ischemia was tors that promote indirect coronary vasodilation may also induced in dogs by clamping the left anterior descending be involved. In healthy volunteers, cold-induced increases coronary artery for 15 minutes. Segmental shortening of about 30 percent in the product of heart rate and blood of the myocardium recovered to only 29 percent of the pressure were associated with appropriate and similar preischemic baseline values in dogs pretreated with nico- increases in blood flow. In contrast, smokers showed no tine, compared with 54 percent in saline-treated control measurable increases in blood flow in response to the dogs. The doses of nicotine administered to the animals cold. Campisi and colleagues (1998) ascribed this observa- did not alter heart rate, blood pressure, or blood flow or tion to coronary endothelial dysfunction. cause myocyte necrosis. Kaufmann and colleagues (2000) provided addi- In patients with vasospastic angina, cigarette smok- tional evidence for the association between smoking and ing is associated with increased occurrence of the condi- endothelial dysfunction. Using quantitative positron emis- tion and a poorer response to medication compared with sion tomography, they found that coronary microvascular the response in nonsmokers (Caralis et al. 1992). The function was abnormal in smokers but could be restored researchers observed that cigarette smoking during coro- by infusion of vitamin C. Earlier, Zeiher and colleagues nary angiography (cardiac catheterization) produced an (1995) used quantitative coronary angiography to mea- acute coronary vasospasm. sure the effects of long-term smoking on the diameter of Schelbert and colleagues (1979) extensively stud- the coronary artery at study entry, during flow-mediated ied the relationship of coronary vasomotion, endothelial coronary vasodilation, and after intracoronary administra- function, and myocardial blood flow to potential revers- tion of nitroglycerin. The study population consisted of ibility of coronary vasomotor abnormalities. They adopted patients with or without mild disease of the left anterior a noninvasive approach by measuring blood flow with descending coronary artery and no other cardiac prob- positron emission tomography using 13N ammonia: (1) lems. Flow-mediated vasodilation was markedly blunted at rest during testing with cold pressors to probe endo- in long-term smokers. In a study of smokers with CHD, thelium-dependent coronary vasomotion and (2) during cigarette smoking increased coronary vascular resistance, dipyridamole-induced hyperemia to assess endothelium- an effect that can be blocked by a-adrenergic blockers. independent coronary vasomotion. Using this protocol, This finding indicates that the mechanism of increased Czernin and associates (1995) investigated the effects of coronary artery resistance is at least partly due to stimula- short- and long-term smoking on myocardial blood flow tion of the sympathetic nervous system by nicotine. and flow reserve in smokers. The investigators sought to Intracoronary measurements by Doppler ultraso- determine whether abnormalities in coronary vasomo- nography demonstrated that cigarette smoking constricts tion in response to cold could be reversed in response to epicardial arteries and increases total coronary vascular l-arginine infusion (Campisi et al. 1998, 1999). The find- resistance. This result indicates that impairment of coro- ings indicated that short-term and long-term smokers nary blood flow by cigarette smoking results from con- had normal coronary vasodilatory capacity. Short-term striction of both epicardial and resistance blood vessels. smoking, however, reduced flow reserve in both short- and

Cardiovascular Diseases 369 Surgeon General’s Report long-term smokers. Also, exposure of long-term smokers Summary to cold resulted in abnormal blood flow. The smoking- associated abnormalities in vasomotion were restored by Cigarette smoking impairs the vascular endothelial intravenous l-arginine, and this result further implicates function and activates the sympathetic nervous system. the endothelium as the target of toxic substances con- These effects can result in inappropriate reduction in or tained in cigarette smoke. Campisi and colleagues (1999) failure to increase coronary blood flow in response to quantified myocardial blood flow during exposure to cold increases in myocardial demand. Together with long-term while l-arginine, the substrate of ENOS, was infused atherosclerotic damage from smoking, these effects con- intravenously for 45 minutes at a dose of 30 g as 10 per- tribute to ischemic cardiac events. Coronary endothelial cent arginine hydrochloride. This infusion produced sig- dysfunction clearly increases the risk for cardiovascular nificant improvement in responses of myocardial blood events. The smoking-induced alterations in vasomotor flow to cold. In addition to active smoking, exposure to function appear to be substantially reversible, which secondhand smoke for 30 minutes abruptly reduces cor- underscores the importance of smoking cessation pro- onary blood flow velocity in nonsmokers, as assessed by grams and policies to promote a smoke-free environment. echocardiography (Otsuka et al. 2001).

Smoking and the Endothelium

Endothelial Injury or Dysfunction generally resists adherence of platelets and infiltration of immune cells. Endothelial injury and dysfunction are thought to contribute to the initiation of atherogenesis and to have a major role in acute cardiovascular events. Cigarette smok- Regeneration of Endothelium ing produces endothelial injury and dysfunction in both peripheral and coronary arteries. Other cardiovascular With aging, the normal functioning of the endo- risk factors such as hypercholesterolemia, diabetes, and thelium requires replacement of apoptotic or injured hypertension also produce endothelial dysfunction. cells. Normally, the turnover of endothelial cells is low, The healthy endothelium is a diaphanous film of tis- on the order of 0.1 percent of the cells undergoing sue that invests the luminal surface of all blood and lym- mitosis at any time (Wright 1972). The rate of endothelial phatic vessels. In larger-conduit vessels, the endothelium turnover increases, however, in areas of disturbed flow forms a monolayer between the circulating blood and the (bends, branches, or bifurcations of blood vessels). The vessel wall. The tissue capillaries, which are the small- length of chromosome telomeres documents that endo- est conduits for blood and lymphatic flow, are composed thelial aging occurs more rapidly in these areas (Chang exclusively of endothelial cells. Because of the ubiquity and Harley 1995). Furthermore, the accelerated aging in of endothelial cells, the surface area of the endothelium these areas may lead to focal senescence, which is demon- in a human weighing 70 kilograms (kg) is 1,000 to 4,000 strated by impaired endothelium-dependent vasodilation square meters—equivalent to two to four tennis courts— (McLenachan et al. 1990). with a weight of approximately 1 kg (Wolinsky 1980). The Persons who smoke may have impaired ability to endothelium produces a variety of paracrine factors that regenerate the endothelium. The endothelial monolayer is regulate vascular homeostasis, including proteins, lip- regenerated in part from circulating endothelial progeni- ids, and small molecules that (1) can relax or activate the tor cells derived from bone marrow, and the supply of these underlying vascular smooth muscle, (2) regulate the inter- cells may be a key determinant of endothelial health. The action of the vessel wall with circulating blood elements, number of circulating endothelial progenitor cells, which and (3) modulate vessel structure (Aird 2005). In healthy is estimated by ex vivo colony counts or by analysis using persons, the endothelium primarily exerts a vasodila- fluorescence-activated cell sorting, is directly associated tor influence that reduces vascular resistance and main- with the ability of the endothelium to induce vasodila- tains blood flow. The endothelium maintains the blood’s tion (Hibbert et al. 2003; Hill et al. 2003). Smokers have fluidity by elaborating anticoagulant substances and reduced numbers of circulating endothelial progenitor

370 Chapter 6 How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease cells and impaired endothelium-dependent vasodilation (PAI-1) (Simpson et al. 1997). By interfering with the (Vasa et al. 2001, Hill et al. 2003). In addition, smoking function of tPA, PAI-1 reduces thrombolytic capacity cessation was associated with a rebound in the number (MacCallum 2005). Imbalance in thrombolytic capacity of circulating endothelial progenitor cells and improve- attributable to higher PAI-1 values or reduction in tPA ment in endothelium-dependent vasodilation (Moreno et levels is associated with occurrence of adverse cardiovas- al. 1998; Kondo et al. 2004). cular events. The healthy endothelium elaborates vasodilator substances such as nitric oxide (NO), prostacyclin, atrial Endothelial Dysfunctions natriuretic peptide, endothelium-derived hyperpolariz- ing factor, and adrenomedullin (Chen and Burnett 1998; A variety of endothelial dysfunctions may contribute Busse and Fleming 2003; Brain and Grant 2004). In to disorders of vessel tone and structure that precede clin- doing so, the healthy endothelium increases the diameter ical vascular disease. Cardiovascular risk factors such as of the blood vessels and reduces resistance to blood flow. hypercholesterolemia, hypertension, diabetes, and use of When the endothelium becomes diseased, synthesis and tobacco cause endothelial aberrations long before clinical bioactivity of the vasodilators are reduced, and the balance vascular disease becomes evident. Endothelial dysfunc- tips in favor of endothelium-derived vasoconstrictors such tion is the first step in vascular disease, because it leads to as endothelin and thromboxane (Vanhoutte et al. 2005). vascular inflammation, cell proliferation, and thrombosis, This derangement in endothelial function has clinical which contribute to progression of vascular disease. consequences. Because vasodilator function is impaired, Endothelial generation of adhesion molecules coronary vascular resistance increases, and ischemia can increases in smokers, as evidenced by higher plasma result. Furthermore, endothelial vasodilator dysfunction levels of soluble adhesion molecules (Blann et al. 1997, in the coronary arteries of humans is associated with 1998). These molecules include soluble forms of the vas- reversible myocardial perfusion defects, which are cular cell adhesion molecule (sVCAM) and the intercel- associated with other vascular abnormalities (Hasdai et al. lular adhesion molecule (sICAM). The soluble adhesion 1997b). These abnormalities include expression of adhe- molecules, which are shed from the endothelium, reflect sion molecules, adherence and infiltration of leukocytes, the increased endothelial production of these adhesion and proliferation of smooth muscle cells. molecules in the context of vascular inflammation. Endo- Most of the endothelium-derived vasodilators also thelial adhesion molecules are required for adherence to oppose key processes involved in atherogenesis (cell adhe- blood leukocytes and their infiltration into the vessel wall sion, proliferation, and inflammation) (Cooke and Dzau (Gimbrone 1995). The increased elaboration of adhe- 1997a,b). Thus, by reducing the generation or bioactiv- sion molecules is an endothelial dysfunction that pro- ity of endothelial vasodilators, exposure to tobacco can motes leukocyte infiltration, vascular inflammation, and accelerate atherosclerosis. This mechanistic explanation progression of atherosclerosis. Studies have associated for tobacco-related CVD is supported by the finding that elevated levels of either sVCAM or sICAM with increased dysfunction of endothelial vasodilators is an independent risk of cardiovascular events (Blankenberg et al. 2001). predictor of vascular events (Schächinger et al. 2000; Smoking also impairs the ability of the endothe- Suwaidi et al. 2000; Gokce et al. 2003). The role of these lium to resist thrombosis. Compared with nonsmokers, mechanisms involving NO is vascular protection, which is smokers have higher levels of von Willebrand factor pro- impaired by exposure to tobacco. tein (MacCallum 2005) and tissue factor (Matetzky et al. 2000; Sambola et al. 2003), which may be generated by the endothelium. Tissue factor activates the coagulation Nitric Oxide and Vascular cascade, and von Willebrand factor protein mediates Homeostasis adherence of platelets to the vessel wall (MacCallum 2005). Furthermore, study findings indicate that smoking NO induces vasodilation by stimulating soluble gua- impairs capacity to lyse the thrombus that is formed. Plas- nylate cyclase to produce cyclic guanosine monophos- ma levels of tissue plasminogen activator (tPA), a throm- phate (Ignarro et al. 1984). NO, which has a short half-life, bolytic protein produced by the endothelium, are reduced avidly interacts with sulfhydryl-containing proteins, heme in smokers (Newby et al. 2001). In contrast, smoking proteins, and oxygen-derived free radicals. By virtue of its increases levels of plasminogen activator inhibitor-1

Cardiovascular Diseases 371 Surgeon General’s Report ability to nitrosylate proteins, NO may change their activ- Endothelium-Dependent ity or behavior (Hess et al. 2005). The significant increase in vascular resistance induced in animals and humans Vasodilation exposed to pharmacologic antagonists of ENOS reflects The effect of exposure to tobacco on endothelium- the physiological importance of this endothelium-derived dependent vasodilation in humans was assessed by observ- vasodilator (Rees et al. 1989; Vallance et al. 1989). ing its effect on flow-mediated vasodilation. As blood flow Endothelium-derived NO also inhibits adherence through a vessel is increased, the vessel relaxes. In animal of platelets and leukocytes to the vessel wall (Kubes et models, this flow-mediated vasodilation was abolished al. 1991; Tsao et al. 1994). This effect is mediated partly by removing the endothelium (Pohl et al. 1986). When by activation of cyclic guanosine monophosphate and a pharmacologic antagonist of ENOS was used, flow- phosphorylation of intracellular signaling proteins such mediated vasodilation in the rabbit iliac artery depended as vasodilator-stimulated phosphoprotein (Smolenski et on the endothelial release of NO (Cooke et al. 1991). Cel- al. 1998). In addition, NO suppresses expression of adhe- ermajer and colleagues (1992) used duplex ultrasonogra- sion molecules and chemokines that regulate endothelial phy to record flow-mediated vasodilation of the brachial interactions with circulating blood elements (Tsao et al. artery in response to hyperemic vasodilation of the fore- 1996, 1997). These observations suggest that NO is an arm. The investigators induced vasodilation by using endogenous antiatherogenic molecule. Impairment of a blood pressure cuff inflated to suprasystolic pressures ENOS contributes to the pathologic alterations in vas- to transiently occlude blood flow in the forearm. Joan- cular reactivity and structure observed in atherosclerosis nides and colleagues (1995) extended this finding by (Cooke and Dzau 1997a,b). The pharmacologic inhibi- showing that flow-mediated vasodilation of the brachial tion or genetic deficiency of ENOS inhibits endothelium- artery could be abolished by pharmacologic antagonism dependent vasodilation, impairs blood flow in tissues, and of ENOS. Subsequently, numerous studies used this raises blood pressure (Huang et al. 1995; Kielstein et al. approach to document impairment of flow-mediated, 2004). Furthermore, NO deficiency promotes adherence endothelium-dependent vasodilation in smokers and in to and intimal accumulation of mononuclear cells and persons exposed to secondhand smoke (Celermajer et al. accelerates formation of lesions in animal models of 1993, 1996; Barua et al. 2001). Researchers also observed atherosclerosis (Kuhlencordt et al. 2001). In contrast, that tobacco use impaired endothelium-dependent vaso- enhancing production of NO in the vessel wall slows or dilation in the coronary microcirculation. Intracoronary even reverses atherogenesis and restenosis (Cooke et al. infusion of acetylcholine induced vasodilation that was 1992; von der Leyen et al. 1995; Candipan et al. 1996). partly attributable to release of NO, and this response was NO is a survival factor for endothelial cells, and it induces blunted in persons who smoked (Kugiyama et al. 1996). apoptosis of macrophages and proliferation of vascular smooth muscle cells (Wang et al. 1999). Certain polymorphisms of the ENOS gene predict development of CHD (Ichihara et al. 1998; Tsukada et al. Impairment of Endothelium- 1998; Yoshimura et al. 1998). The ENOS gene GLU298ASP Dependent Vasodilation polymorphism is more prevalent in patients with variant angina, essential hypertension, and acute MI (Hibi Many factors contribute to the ability of tobacco to et al. 1998; Miyamoto et al. 1998; Shimasaki et al. 1998; impair endothelial function. Tobacco use adversely affects Yoshimura et al. 1998). Intriguingly, this polymorphism is the ENOS pathway. Exposure of cultured endothelial cells associated with greater sensitivity to the effects of smok- derived from human coronary arteries or umbilical veins ing on endothelial vasodilator function. Young men who to sera from smokers reduced expression and activity of are carriers of the ENOS *ASP298 allele have increased ENOS (Barua et al. 2001, 2003). The researchers attrib- susceptibility to smoking-associated reduction in endo- uted this effect partly to the oxygen-derived free radicals thelial function (Leeson et al. 2002). Similarly, a qua- in tobacco smoke. In addition, the half-life of NO was druple repeat of a sequence of 27 base pairs in *intron 4 markedly shortened by oxidative stress (Rubanyi and Van- of the ENOS gene (allele *a) is associated with increased houtte 1986). risk of CHD and acute MI (Ichihara et al. 1998). Smokers Superoxide anion reacts avidly with NO to form a who are homozygous for the ENOS allele *a are at risk for peroxynitrite (ONOO−) anion, which itself is a highly more severe CHD than are those who are not homozygous reactive free radical (Beckman and Koppenol 1996). Oth- (Wang et al. 1996). er sources of free radicals that may inactivate NO and

372 Chapter 6 How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease impair endothelial vasodilator function include activated and inhibited production of transforming growth factor leukocytes, xanthine oxidase, the mitochondrial electron- β1 (Villablanca 1998; Cucina et al. 1999). Nicotine also transport chain, and uncoupling of ENOS itself (Heitzer increased DNA synthesis, mitogenic activity, and endo- et al. 2000; Barua et al. 2003; Kayyali et al. 2003; Sydow thelial proliferation. Nicotine has been shown to induce and Münzel 2003; Guthikonda et al. 2003). Investigators endothelial dysfunction (Chalon et al. 2000; Sarabi and postulated that this uncoupling occurs when amounts of Lind 2000; Neunteufl et al. 2002). the NOS cofactor tetrahydrobiopterin or the NO precursor l-arginine are insufficient. These conditions involve transfer of electrons from ENOS to oxygen and thus formation Pathologic Angiogenesis of a superoxide anion. Oxidative stress also impairs the NOS pathway by Another endothelial function influenced by exposure increasing accumulation of asymmetric dimethylarginine to tobacco is development of new blood vessels (angiogen- (ADMA), an endogenous competitive inhibitor of ENOS esis). Angiogenesis requires activation of endothelial cells produced by all cells during degradation of methylated by an angiogenic cytokine, followed by endothelial cell nuclear proteins (Vallance et al. 1992; Tran et al. 2003). proliferation and migration and the formation of tubes. Most of the ADMA produced is degraded by the enzyme Exposure to secondhand smoke promotes tumor angio- dimethylarginine dimethylaminohydrolase. Oxidative genesis and growth (Zhu et al. 2003). Human lung cancer stress impairs the activity of this enzyme and leads to cells implanted in a subcutaneous or orthotopic location accumulation of ADMA and suppression of ENOS grew more rapidly in mice when they were exposed to sec- (Cooke 2004). ondhand tobacco smoke. Furthermore, these mice had Blood levels of antioxidant vitamins are lower than higher plasma levels of vascular endothelial growth factor, normal in smokers, reflecting endogenous consumption and capillary density in their tumor nodules was greater of these vitamins in response to ongoing oxidant stress than that in control mice. Mecamylamine, an antagonist (Lykkesfeldt et al. 2000). Administration of vitamin C of the nicotinic acetylcholine receptor (nAChR), abol- reverses the impairment of endothelium-mediated ished the effects of exposure to secondhand smoke. These vasodilation in smokers, a finding consistent with an observations indicate that secondhand smoke increases oxidant mechanism of endothelial dysfunction (Heitzer et tumor angiogenesis and growth, at least partly through a al. 1996). nicotine-mediated mechanism. Nicotine itself may injure endothelial cells. Stud- Researchers observed the effect of nicotine in pro- ies showed that in levels similar to those found in the moting pathologic angiogenesis in numerous murine blood of cigarette smokers, nicotine altered structural and models of tobacco-related diseases, including lung can- functional characteristics of cultured vascular smooth cer, atherosclerosis, and retinopathy (Heeschen et al. muscle and endothelial cells (Csonka et al. 1985; Thyberg 2001, 2002; Natori et al. 2003; Shin et al. 2004; Suñer 1986). In one study, oral nicotine administered to rats to et al. 2004). Tumor angiogenesis was required for tumor achieve blood levels comparable to those in human smok- growth, and correspondingly, promotion of tumor ers produced myointimal thickening of the aorta after an angiogenesis accelerated tumor growth (Folkman 2003). experimental injury (denudation of the endothelium with Conversely, antiangiogenic agents inhibit progression a balloon catheter) (Krupski et al. 1987). The excessive of cancer and are now approved as treatment for some myointimal thickening in nicotine-treated animals is con- advanced human malignant diseases (Jain 2005). The sistent with persistent injury to endothelial cells. Stud- effect of nicotine on promotion of tumor angiogenesis ies reported increased numbers of circulating endothelial may be attributable to a direct effect on endothelial cells. cells in the venous blood, reflecting endothelial injury, In clinically relevant levels, nicotine promoted endothe- and decreased platelet aggregate ratios, reflecting platelet liaprocesses that may be involved in tumor angiogenesis. aggregation in persons who had never smoked but who, At these doses, nicotine promoted survival, proliferation, for experimental purposes, smoked cigarettes contain- and migration of endothelial cells (Heeschen et al. 2001, ing tobacco (Davis et al. 1985). These results were not 2002). Nicotine also induced elaboration and release of observed in nonsmokers who smoked cigarettes that did angiogenic factors, including NO, prostacyclin, vascular not contain tobacco. These findings further support a role endothelial growth factor, and fibroblast growth factor for nicotine in injury to endothelial cells. (Carty et al. 1996; Heeschen et al. 2001a; Lane et al. 2005). Nicotine may influence endothelial function in These effects of nicotine were mediated by nAChRs on the other ways. In studies of cultured endothelial cells, nico- endothelium (Heeschen et al. 2002). Conversely, phar- tine enhanced release of the basic fibroblast growth factor macologic inhibition or genetic deficiency of endothelial

Cardiovascular Diseases 373 Surgeon General’s Report nAChRs reduced angiogenic response to nicotine. A vari- before induction of ischemia obliterated angiogenic ety of human lung cancer cells synthesized acetylcholine response to nicotine (Konishi et al. 2010). or expressed nAChRs. Nicotine increased growth of these The relevance of animal models for research on cells in vitro, but this effect was inhibited by antagonists nicotine and angiogenesis to human smokers is not clear. of the nAChRs (Schuller 2002). It is important to differentiate studies that show effects The importance of pathologic angiogenesis in of pure nicotine from those in which the exposure is to growth of tumors is well known. Less widely recognized, tobacco smoke. In mice, effects of nicotine on angiogen- however, is the role of neovascularization in progression esis depended on release of NO, but the net effect of smok- of atherosclerotic plaque. Large atherosclerotic plaques ing in humans seems to be impaired release of NO. Also, in human coronary arteries are well vascularized by most studies on angiogenesis involve short-term admin- microvessels originating from the vasa vasorum (Barger istration of nicotine, although tolerance to the effects of et al. 1984). In mice with hypercholesterolemia, growth nicotine may develop with long-term use. of atheroma can be inhibited by antiangiogenic agents, Inhibition of ACE normalized impaired bradykinin- and in the same murine models, nicotine promoted neo- mediated, endothelium-dependent venodilation in smok- vascularization of plaque and its progression in the aorta ers (Chalon et al. 1999). Furthermore, coronary vasomotor (Heeschen et al. 2001; Moulton et al. 2003). The effect of responses to acetylcholine in patients with CHD nicotine in increasing neovascularization and progression improved in response to the ACE inhibitor quinapril to of plaque may partially explain increased risk of athero- a much greater extent in smokers than in nonsmokers sclerotic disease in persons who smoke. (Schlaifer et al. 1999). ACE inhibitors have an antioxidant Similarly, in a model of age-related macular degen- activity, which could contribute to the clinical benefit eration in mice, nicotine stimulated retinal neovascular- in smokers. ization (Suñer et al. 2004). This effect was antagonized by hexamethonium, another antagonist of nAChRs. In a clinical study, the most virulent form of age-related macu- Summary lopathy is associated with retinal neovascularization that contributes to visual deterioration, and tobacco smokers The endothelium, a delicate monolayer of cells that are at greater risk of age-related macular degeneration invests all blood vessels, is a major regulator of vascular than are nonsmokers (Christen et al. 1996; Seddon et al. reactivity and structure. Healthy endothelium maintains 1996). Thus, a variety of tobacco-related diseases are char- vascular homeostasis by promoting fluidity of the blood, acterized by pathologic neovascularization, an effect that vasodilation, and relaxation of the underlying vascu- may be promoted by nicotine. lar smooth muscle. Endothelial dysfunction regularly Notwithstanding nicotine’s effect as a promoter of accompanies and promotes vascular disease. Endothelial neovascularization, long-term exposure to tobacco may vasodilator dysfunction is an independent risk factor for impair therapeutic angiogenesis. In a murine model major adverse cardiovascular events and mortality. Active of hindlimb ischemia, short-term exposure to nicotine smoking and involuntary exposure to cigarette smoke paradoxically increased capillary density and improved injure endothelial cells and impair endothelial vasodila- regional blood flow in the ischemic hindlimb (Heeschen et tion. Thus, each type of exposure to tobacco or tobacco al. 2001, 2003). However, long-term exposure to nicotine smoke contributes to the development of CVD. for 16 weeks (about one-third of the life span of a mouse)

Thrombogenic Effects

This section on thrombogenic effects reviews the Epidemiologic evidence indicates that cigarette state of knowledge of mechanisms by which smoking smoking increases risk of acute MI and sudden death or secondhand smoke exposure may predispose a per- more than it increases risk of angina pectoris. Researchers son to thrombosis, a pathologic reaction that commonly hypothesize that risk of acute MI and sudden death is results in smoking-related MI or stroke. Smoking-mediat- mediated by thrombosis, whereas angina is mediated ed thrombosis appears to be a major factor in the patho- primarily by hemodynamic factors. Successful revas- genesis of acute cardiovascular events. cularization in patients with MI after treatment with

374 Chapter 6 How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease thrombolysis is more likely in smokers than in non- number of newly formed reticulated platelets, rather than smokers (Bowers et al. 1996). At the time of MI, smokers absolute platelet count, to incidence of thrombotic events are younger and have fewer cardiac risk factors and less (Rinder et al. 1998). severe underlying coronary disease than do nonsmokers Urinary excretion of thromboxane metabolites (Metz and Waters 2003). Enhanced thrombosis superim- (TxMs), such as 2,3-dinor thromboxane B2 (TxB2) and posed on less severely stenotic arteries best explains these 11-dehydro TxB2, which are markers of platelet activation observations. In men who died suddenly, a history of ciga- in vivo, increases in smokers in a dose-dependent manner rette smoking was significantly more likely when patho- (Murray et al. 1985). Studies demonstrated increases in logic examination showed acute thrombosis (75 percent of levels of urinary TxM among smokers who were monozy- cases) than when the finding was plaque with no throm- gotic twins but divergent for smoking behaviors (Lassila bosis (41 percent) (Burke et al. 1997). Conversely, stable et al. 1988). These increases were also observed in young plaque with no thrombosis was the more common finding Swedish army recruits who smoked but had no apparent in nonsmokers. vascular disease (Wennmalm et al. 1991). Studies also Thrombosis occurs when fibrinogen is converted showed that mainstream and sidestream smoke directly to fibrin, a process involving interaction of platelets, promoted platelet activation and enhanced activation blood-borne proteins, endothelial cells, and subendo- induced by shear stress (Rubenstein et al. 2004). thelial vascular tissue. An endogenous antithrombotic Elevated levels of TxM observed in persons who mechanism involves these same components. Imbalance smoke decreased substantially within days of smoking in these pathways results in predisposition to thrombosis. cessation, although they did not reach the levels found in Virchow’s triad, first described in 1856 and modified by nonsmokers (Rangemark et al. 1993; Saareks et al. 2001). Aschoff in 1924, provides a framework for risk factors for In an ex vivo study, platelets from smokers showed greater thrombosis that is still valid. These authorities described aggregation than those from nonsmokers (Takajo et al. the cardinal risk factors as “alterations in blood coagula- 2001). A decline in platelet aggregation during 14 days of tion,” “alterations in blood flow,” and “alterations in the abstinence from smoking was reversed rapidly after smok- blood vessel wall.” ing was resumed (Morita et al. 2005). Ex vivo aggregability, however, is a crude index of in vivo platelet activation and aggregation. Some studies reported diminished ex Alterations in Blood vivo aggregability in smokers. This finding suggests that partial activation in vivo resulted in the harvest of a less Blood contains platelets, red blood cells, and leuko- responsive subset of platelets for study ex vivo. Increased cytes suspended in plasma. Plasma in turn contains a va- levels of urinary TxMs in smokers may be attributable to riety of coagulation proteins and lipids that also contrib- activation of both platelets and macrophages. Low-dose ute to the clotting process. Smokers tend to develop MI aspirin, which inhibits COX activity in platelets, substan- at a lower burden of atheroma than do nonsmokers. This tially depressed the increment of TxM in smokers, lead- finding suggests a greater role for formed elements of ing investigators to hypothesize that TxM was principally blood or for cardiac electrical instability in cardiovascular derived from the activity of platelet COX-1 rather than events in smokers. macrophage COX-2 (Nowak et al. 1987; McAdam et al. 2005). Platelets Generation of Nitric Oxide Platelets, although only a minor component of the solid phase of blood, are critical to the coagulation process Platelets constitutively express ENOS. Although and are important mediators of the impact of smoking on platelet-derived NO is a modest inhibitor of platelet acti- cardiovascular outcomes (Figure 6.6). vation in vitro, it may be critical to inhibit recruitment of platelets to a growing thrombus (Freedman et al. Turnover and Activation 1997). Aggregating platelets obtained from patients with acute coronary syndromes produced less NO than did Studies have reported that sudden cardiac death those from patients with stable angina (Freedman et al. is 2.5 times higher in smokers than in nonsmokers 1998). Such findings must be interpreted with caution, (Kannel et al. 1984; Goldenberg et al. 2003). Research however, because results reflect in vivo platelet activity findings broadly implicated activation of platelets and (see “Turnover and Activation” earlier in this chapter). subsequent focal ischemia in sudden cardiac death among In one study, levels of platelet-derived NO were lower in smokers. The turnover of platelets is accelerated in ciga- smokers than in nonsmokers (Takajo et al. 2001). This rette smokers (Fuster et al. 1981). Researchers related the

Cardiovascular Diseases 375 Surgeon General’s Report

Figure 6.6 Potential sites of actions and mechanisms of effects of smoking on platelets

Oxidative processes

Isoprostane Glycoprotein 1b levels

NO synthesis

Activation of thromboxane NO levels receptor

Sensitivity to Sensitivity to NO shear stress

NO inhibition of platelet activation Platelet activation

Note: Smoking decreases NO-mediated inhibition of platelet activation and increases platelet activation through oxidative stress and other mechanisms. NO = nitric oxide.

finding was associated with lower levels of intraplatelet- which are markers of oxidative stress, were also depressed reduced glutathione—a marker of oxidative stress and after smoking cessation. One study showed that supple- increased platelet aggregation. In another study, both the mentation with vitamin C restored NO levels and platelet platelet-derived release of NO and the glutathione levels aggregation in current smokers to levels observed in non- recovered in a time-dependent manner after smoking ces- smokers (Takajo et al. 2001). Another study demonstrated sation, but they rapidly decreased again when smoking was that the normal morning increase in platelet sensitivity resumed (Morita et al. 2005). Abstinence from smoking to NO ex vivo was lost in smokers, leaving platelets poten- was also associated with decreased agonist-induced platelet tially more susceptible to activation during early morning aggregation ex vivo. Furthermore, levels of intraplatelet hours, when MIs are most common (Sawada et al. 2002). nitrotyrosine and urinary 8-hydroxy-2’-deoxyguanosine, In yet another study, platelets from smokers were less

376 Chapter 6 How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease sensitive to administration of nitroglycerin, a documented however, isoprostane alone did not induce platelet activa- NO donor (Haramaki et al. 2001). tion and partially blocked the proaggregatory effects of TP agonists and high-dose collagen (Cranshaw et al. 2001). Oxidative Stress and Platelet Function Also, isoprostane iPF2α-III was reported to decrease the antiplatelet activity of NO (Minuz et al. 1998). Some Cigarette smoke has been shown to be an abundant researchers speculated that this isoprostane has a role in source of free radicals (Church and Pryor 1985). Levels the resistance to low-dose aspirin observed in patients of isoprostanes—quantitative indices of in vivo oxidative with CVD (Csiszar et al. 2002). It is not known, however, stress—are higher in smokers than in nonsmokers (Reilly how these effects, which are demonstrable in vitro, relate et al. 1996; Chehne et al. 2001; Dietrich et al. 2002), and to endogenous levels of isoprostanes attained locally in they decrease with smoking cessation (Reilly et al. 1996; vivo under conditions of oxidative stress. Praticò et al. 1997). In addition to serving as biomarkers of oxidative stress, isoprostanes may serve as secondary mes- Summary sengers that exert biologic effects, at least in vitro. Studies demonstrated elevated production of iso- Thus, platelets from smokers demonstrate a dose- prostanes and decreased levels of reduced glutathione in dependent increase in activity and adhesiveness that rap- the platelets of smokers (Takajo et al. 2001). Intraplatelet idly decreases with smoking abstinence. Findings suggest levels of nitrotyrosine, which is a marker of modification that the inhibitory NO pathway is impaired and respon- of proteins induced by oxidative stress, decreased with siveness to other agonists is increased at least partially smoking abstinence but increased rapidly when smoking through the mediation of TP. was resumed, together with return of increased sensitivity to agonist-induced platelet aggregation ex vivo (Morita et Red Blood Cells al. 2005). In another study, products from activated platelets induced oxidative stress in vascular smooth muscle Hematocrit in Adults cells, which is associated with increased expression of tis- Smoking is associated with an increase in hemato- sue factor, a highly thrombogenic protein (Görlach et al. crit or red blood cell mass attributable to increased lev- 2000). Other investigators showed that administration of els of CO and carboxyhemoglobin. Hematocrit decreases antioxidants to persons with diabetes, just as to smokers, with smoking cessation, but increased blood viscosity and decreased production of isoprostane and urinary TxM deformability of red blood cells may persist (Haustein et (Davì et al. 1999) and decreased platelet aggregation ex al. 2004). Increases in hematocrit and blood viscosity are vivo (Salonen et al. 1991). associated with increasing risk of CVD. It is unclear, however, whether these risk factors are independent of smok- Isoprostanes and Platelet Function ing and other conventional risk factors, particularly in In one study, platelets oxidized ex vivo demonstrated women (Lowe et al. 1997; Irace et al. 2003; Woodward et increased aggregation induced by shear stress, an effect al. 2003). When data are adjusted for smoking, hyperten- that is only partly inhibited by administration of aspirin sion, and high cholesterol, viscosity remains a significant (Chung et al. 2002). In another study, oxidation of plate- risk factor for stroke and for PAD but not for ischemic let membranes was associated with reduced expression of heart disease. This finding suggests that the effect of high glycoprotein Ib, a receptor for the von Willebrand factor viscosity may be an independent risk factor for stroke or that is critical to platelet activation and aggregation under PAD (Lee et al. 1996; Lowe et al. 1997). conditions of shear stress (Escolar and White 2000). The researchers postulated that decreased expression of gly- Hematocrit in Neonates coprotein Ib indicates a highly reactive status of platelets. Researchers showed that the effects of smoking on Some evidence indicates that isoprostanes may act hematocrit were transmitted to the fetus during preg- as platelet and vascular agonists through ligation of the nancy. Some studies reported a dose-dependent increase thromboxane A receptor (TP) (Audoly et al. 2000). To 2 in hemoglobin levels in infants of mothers who smoked date, no molecular evidence exists for a distinct isopros- (al-Alawi and Jenkins 2000; Habek et al. 2002). Further- tane receptor (Praticò et al. 1996). One study reported that more, infants born to mothers who smoked more than 20 infusion of isoprostanes elevated blood pressure and acti- cigarettes per day had higher rates of fetal hypoxia, poly- vated platelets—effects that are lost in mice lacking TP. cythemia, and neurological complications than did infants Binding of isoprostane iPF -III to TP promoted change 2α of nonsmoking mothers. in platelet shape and facilitated response to other proaggregatory stimuli (Praticò et al. 1996). In another study,

Cardiovascular Diseases 377 Surgeon General’s Report

Leukocytes vitamin C (Weber et al. 1996). In one study, isoprostane iPF2α-III inhibited adhesion of monocytes to cultured der- Polymorphonuclear Leukocytes mal cells or renal endothelial cells in rats but paradoxically increased adhesion of monocytes to human endo- In one study, persons who smoked had higher num- thelial cells in the umbilical vein (Leitinger et al. 2001; bers of circulating polymorphonuclear leukocytes than Kumar et al. 2005). Adhesion of monocytes to endothelial did nonsmokers (Sela et al. 2002). In other research, cells may increase their access into the subendothelium, neutrophils from smokers had higher levels of myeloper- where they differentiate into macrophages and promote oxidase (Bridges et al. 1985) and increased expression of atherogenesis (Ross 1999). Differentiation of monocytes integrins CD11b, CD15, and CD63, which are markers into macrophages depends on production of intracellular of leukocyte activation (Gustafsson et al. 2000). Further- reactive oxygen species induced by nicotinamide adenine more, when stimulated, these leukocytes released super- dinucleotide phosphate, but no evidence exists for a role oxide at a faster rate than did leukocytes from nonsmokers of cigarette smoking in this differentiation (Barbieri et (Sela et al. 2002), which further increased local oxidative al. 2003). stress. Studies documented a greater variety of circulating cellular adhesion molecules, including ICAM-1, VCAM, Summary P-selectin, and E-selectin, in smokers than in nonsmokers (Mazzone et al. 2001; Bermudez et al. 2002). An increase In summary, smoking induced changes in the in these cellular adhesion molecules (monocytes) may numbers and activity of polymorphonuclear leukocytes facilitate recruitment of inflammatory cells to sites of vas- and monocytes. In addition, it promoted expression of cular injury (Figure 6.7). chemoattractant and adhesion molecules and integrins, which would be expected to increase recruitment of Monocytes activated leukocytes to areas of oxidative stress, including sites of platelet deposition after vascular injury. Monocytes from smokers demonstrated increased expression of the integrins CD11b and CD18, which aug- Circulating Proteins ment adhesiveness of monocytes to endothelial cells, at least in vitro (Weber et al. 1996). This process is thought In addition to its effects on the cellular elements to be mediated by activation of protein kinase C (Kalra of blood, smoking alters the proteins involved in the et al. 1994) and is attenuated by supplementation with coagulation pathway by changing procoagulant factors in

Figure 6.7 Potential sites of effects of smoking on thrombosis through oxidative stress and other mechanisms

Vessel wall Foam cell ( Endothelial cells

Vessel lumen

Neutrophil Plasmin 3 Reactive oxygen 1 LDL species cholesterol 2

Note: 1. Increased numbers and activation of polymorphonuclear leukocytes; increased production of superoxide radicals; and increased expression of integrins and adhesion molecules on leukocytes and endothelial cells. 2. Increased oxidation of LDL cholesterol; oxidized LDL cholesterol taken up more easily into macrophages to produce foam cells; and increased adhesiveness of monocytes to endothelial cells. 3. Increased levels of fibrinogen; increased nitration of tyrosine residues on fibrinogen, rendering it more thrombogenic; impaired activity of plasmin; and decreased thrombolysis. LDL = low-density lipoprotein.

378 Chapter 6 How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease the circulation and anticoagulation factors derived from C-Reactive Protein the endothelium. Chronic, low-level inflammation—reflected by elevated levels of C-reactive protein (CRP) and other bio- Fibrinogen markers—is an important risk factor for atherosclerosis Study findings indicate that circulating levels of (Koenig et al. 1999). Investigators reported that levels of fibrinogen increase in smokers and decrease with smok- CRP, which likely contributes to both oxidative stress and ing cessation (Thomas et al. 1995; Hunter et al. 2001; Tuut mitogenic and fibrogenic characteristics of atheroscle- and Hense 2001). Also, research suggests that elevated rotic plaque, are higher in smokers than in nonsmokers fibrinogen values are an independent risk factor for CHD in a dose-dependent manner (Bakhru and Erlinger 2005). (Paramo et al. 2004) and deep-vein thrombosis (Vayá et al. This increase persisted even after adjustment for diabetes, 2002). For CVD, the predictive effect was reportedly simi- lipid profile, and CVD, as well as age, gender, and race. lar and additive to traditional cardiovascular risk factors More important, five years after smoking cessation, CRP (Woodward et al. 1998). The effect of fibrinogen on CVD is levels were decreased to levels similar to those in lifetime partly attributable to smoking and seems to be mediated nonsmokers. This finding suggests vascular healing. The through alterations in rates of synthesis by the liver. Use timeframe is consistent with that observed in the multi- of snuff is not associated with increased fibrinogen levels national monitoring of trends and determinants of CVD (Eliasson et al. 1995). (MONICA) (Dobson et al. 1991) and in the Northwick Park Nitration of tyrosine residues, a marker of Heart studies (Meade et al. 1987). In those studies, cardio- NO-dependent damage, is increased in smokers (Petru- vascular risk was reduced at two to five years after a person zzelli et al. 1997). Presence of these residues depends stopped smoking. strictly on availability of nitrogen dioxide radicals that are in turn derived from ONOO− (Kirsch et al. 2002). Oxidized Low-Density Lipoprotein Tyrosine nitration modifies a variety of proteins, including Cholesterol fibrinogen (Petruzzelli et al. 1997; Pignatelli et al. 2001). In vitro research on oxidative modification of LDL Nitrogenated fibrinogen is more reactive and thrombo- cholesterol (LDLc) by extracts from cigarette smoke genic than is native fibrinogen (Gole et al. 2000), a fact showed a significant increase in atherogenicity (Chisolm that seems attributable to accelerated formation of clots and Steinberg 2000). In one study, LDL isolated after par- without modification in plasmin-induced thrombolysis ticipants smoked six or seven cigarettes was more suscep- (Vadseth et al. 2004). The antioxidants glutathione and tible to ex vivo oxidation than was LDL isolated after 24 vitamin C protect against formation of nitrogenated hours of abstinence from smoking (Harats et al. 1989). In fibrinogen by interfering with interaction of nitrate radi- another study, oxidizability of LDL ex vivo decreased with cals with tyrosine (Kirsch and de Groot 2000; Kirsch et al. smoking cessation (Sasaki et al. 1997). Oxidized LDLc, 2001). ONOO− is likely to derive from interaction of NO but not native LDL, interacted with scavenger recep- from cigarette smoke with superoxide radicals from pul- tors on lipid-laden lung cells (foam cells) and was readily monary macrophages (Deliconstantinos et al. 1994). In incorporated into atherosclerotic plaque. Findings in oth- studies of both animal models and human volunteers, er studies suggest that oxidized LDL prompts migration even brief exposure to tobacco smoke induced prolonged and degranulation of neutrophils (Sedgwick et al. 2003) production (>30 minutes) of ONOO−, apparently from and increases expression of the Toll-like receptor, a trans- pulmonary macrophages (Deliconstantinos et al. 1994). membrane protein in macrophages, thus promoting their activation (Xu et al. 2001). Plasmin Clinical studies produced conflicting data on the Circulating plasminogen is activated to plasmin by ability of cigarette smoke to oxidize LDLc and on the role fibrin, thrombin, and tPA. Plasminogen has fibrinolytic of cigarette smoke in the process in vivo. Findings in sev- and collagenase activities. In vitro studies showed that eral studies (Scheffler et al. 1992; Mahfouz et al. 1995; levels of ONOO− increased in smokers and that ONOO− Kagota et al. 1996; Gouaźe et al. 1998; Yamaguchi et al. induced nitration of plasminogen in a concentration- 2005), but not all studies (Princen et al. 1992; Siekmeier dependent manner and reduced proteolytic activ- et al. 1996; Marangon et al. 1997; van den Berkmortel et al. ity of plasmin (Nowak et al. 2004). This effect is partially 2000), suggest that smoking increases oxidation of LDLc. reduced by glutathione. Studies of an animal model suggest a similar oxidative

Cardiovascular Diseases 379 Surgeon General’s Report effect (Yamaguchi et al. 2002, 2004). In one study, modi- biosynthesis of prostacyclin is likely to reflect a compen- fication of LDLc by cigarette smoke was diminished in a satory reaction by the vascular endothelium. dose-dependent manner by administration of fluvastatin (Yamaguchi et al. 2002; Franzoni et al. 2003). Fluvastatin von Willebrand Factor is a potent scavenger of the peroxyl radical. A similar Studies reported higher circulating levels of von clinical study showed no decrease in oxidized LDL with Willebrand factor in smokers than in nonsmokers (Blann administration of atorvastatin, despite improvement in and McCollum 1993; Smith et al. 1993), and findings endothelial function (Beckman et al. 2004). It is unclear indicated that these levels may precede clinically overt whether failure to demonstrate an association between atherosclerosis (Prisco et al. 1999). The von Willebrand improved endothelial function and oxidative stress in factor is essential for initial adhesion of activated plate- the study of atorvastatin reflects a true independence of lets to the vessel wall and for expansion of thrombi. It is these effects, a distinction from fluvastatin, or limitations unclear to what degree high levels of von Willebrand fac- attributable to the small sample size. tor might contribute to the increased thrombosis and ath- Thus, cigarette smoking may induce changes in erogenesis observed in smokers. both coagulation and fibrinolytic pathways to promote a prothrombotic state. In addition to its effects on inflam- Tissue Factor matory cells, smoking promotes production of inflammatory markers and acute-phase reactants. Tissue factor is a prothrombotic protein made by numerous cells in the blood vessel wall, predominantly macrophages but also vascular smooth muscle and endo- Alterations in Blood Vessels thelial cells. Tissue factor is released when the endothelium is injured and can start the clotting cascade. Studies Nitric Oxide assessing the effects of smoking on tissue factor yielded mixed results (Barua et al. 2002; Sambola et al. 2003). The Cigarette smoking has injurious effects on the vas- immunoreactivity of tissue factor was higher in specimens cular endothelium (see “Smoking and the Endothelium” of plaque obtained during endarterectomy from smokers earlier in this chapter). Abnormalities in the release of than in those from nonsmokers (Matetzky et al. 2000). In chemical mediators occur as a consequence of endothelial mice with APO E deficiency that were fed high-cholesterol dysfunction and are likely to contribute to the prothrom- diets, exposure to cigarette smoke resulted in higher lev- botic condition of smokers. Examples include decreases in els of tissue factor and VCAM-1 and higher macrophage NO-mediated inhibition of interactions between platelets counts in atherosclerotic plaques than in those of unex- and the blood vessel wall, in platelet-induced NO, and in posed mice (Lykkesfeldt et al. 2000). Aspirin treatment inhibition of platelet activation (see “Platelets” earlier in of cigarette smokers and of mice exposed to smoke was this chapter). Blood vessel tone is more sensitive to low associated with lower levels of tissue factor in plaque. This NO levels than is platelet function (Loscalzo 2001). The finding indicates that aspirin may have a protective role importance of NO deficiency mediated by oxidative stress for smokers. in thrombosis is suggested by familial childhood stroke resulting from deficiency in glutathione peroxidase. This Tissue Plasminogen Activator condition decreases NO levels in association with both increased expression of P-selectin in platelets and platelet Vascular endothelial cells and other tissues secrete aggregation and activation (Kenet et al. 1999). tPA. This protein has a central role in fibrinolysis, which limits expansion of clots during thrombosis and eventu- Prostacyclin Production ally dissolves the clot during thrombolysis. Evidence from the Physicians’ Health Study indicates that high levels In vitro studies showed impaired production of of tPA are an independent risk factor for stroke and sug- prostacyclin by vascular cells exposed to cigarette smoke gests that activation of the endogenous fibrinolytic system extracts. In vivo studies, however, showed increased bio- occurs years before arterial vessels become occluded (Rid- synthesis of prostacyclin that is presumed to be reactive to ker et al. 1994). PAI-1, which is also secreted by vascular accelerated interactions of platelets and neutrophils with endothelial cells, opposes the actions of tPA. vessel walls in smokers (Murray et al. 1990; Lassila and Data on the effects of smoking on tPA and PAI- Laustiola 1992). Consistent with this concept, levels of 1 are conflicting (Blann et al. 2000; Enderle et al. 2000; markers of platelet and leukocyte activation were greater Matetzky et al. 2000; Newby et al. 2001). Using an in vitro in smokers than in nonsmokers. Thus, the augmented model, researchers showed that serum from smokers

380 Chapter 6 How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease impaired tPA production by human umbilical vein endo- research, exposure to secondhand smoke decreased sensi- thelial cells but that PAI-1 production was unchanged tivity of platelets to the inhibitory effects of prostacyclin in (Barua et al. 2002). In an in vivo model, bradykinin (Newby vitro (Burghuber et al. 1986). et al. 1999; Pretorius et al. 2002) and substance P (Newby Thus, many of the effects from active smoking can et al. 1999; Pretorius et al. 2002) stimulated the produc- be observed in persons involuntarily exposed to cigarette tion of tPA, which had decreased in smokers, but there smoke. The magnitude of the effect of secondhand smoke was no effect on the release of methacholine-induced tPA. is relatively large considering the low systemic exposure Vitamin C failed to ameliorate this decrease (Pellegrini et to tobacco smoke for nonsmokers compared with that for al. 2004). Studies showed that levels of PAI-1 may decrease active smokers and supports the finding of high cardiovas- after smoking cessation (Simpson et al. 1997). cular risk at low levels of exposure to smoke. Thus, the evidence suggests that smoking decreases the production of tPA and perhaps also increases the amount of PAI-1 produced. These changes would Nicotine and Thrombosis be expected to impair fibrinolytic activity. Alternatively, a population-based study demonstrated increased lev- Nicotine replacement therapy (NRT) does not seem els of fibrinogen, but fibrinolytic activity in smokers did to increase the acute risk of thrombosis, even in patients not differ from that in nonsmokers (Eliasson et al. 1995). with established cardiac disease (Joseph et al. 1996). Another study suggested that differences between smok- However, investigators have not fully studied cardiovascu- ers and nonsmokers in thrombolysis may be evident only lar effects of extended administration of nicotine, which in older adults (Ikarugi et al. 2003). might be used as an adjunct for persons trying to stop smoking (Stratton et al. 2001). The high urinary excretion Summary of TxM in smokers declines rapidly after smoking cessa- A variety of abnormalities can be observed in tion. In one study, this decline did not occur in smokers endothelial cell function among smokers compared with who were using NRT, suggesting that nicotine may be nonsmokers. These abnormalities affect the ability of the contributing to platelet activation (Saareks et al. 2001). endothelium to modulate vascular tone, platelet func- However, other studies in which smokers switched to nic- tion, thrombogenesis, and thrombolysis. Administra- otine patches found a decline in eicosanoid excretion and tion of antioxidants mitigates some but not all of these that long-term use of smokeless tobacco, which results abnormalities. The relative importance of these abnormal- in nicotine exposure similar to that of cigarette smokers, ities and their interactions in clinical settings remains to does not increase urinary excretion of TxM (Wennmalm be elucidated. et al. 1991; Benowitz et al. 1993). These findings suggest that nicotine per se does not activate platelets. When nicotine or cotinine was added to the platelet-rich plasma of nonsmokers, platelet-dependent formation of thrombin Exposure to Secondhand increased (Hioki et al. 2001). The magnitude of the effect Tobacco Smoke was similar to that observed in smokers, even though the basal nicotine levels in smokers were higher than those In one study, levels of fibrinogen and coagulation in nonsmokers. When cultured endothelial cells from the factor VII were higher in nonsmoking adolescent offspring human brain were exposed to nicotine, tPA levels were of smokers than in nonsmoking adolescent offspring of unchanged (Zidovetzki et al. 1999). Rather, PAI-1 mes- nonsmokers (Stavroulakis et al. 2000). This finding is in senger RNA and protein expression increased, favoring keeping with a prothrombotic state in smokers. In addi- a prothrombotic state. Alternatively, when transdermal tion, levels of PAI-1 were lower in nonsmoking offspring of nicotine was administered to nonsmokers, the release smokers. This result suggests decreased fibrinolysis, but of tPA induced by substance P was greater than that in there was no difference in tPA levels. Levels of thrombo- nonsmokers who had received placebo patches (Pellegrini modulin, which is produced by the vascular endothelium et al. 2001). This finding suggests a more favorable effect and has anticoagulant effects, were higher in nonsmoking in vivo. offspring of smokers, but levels of von Willebrand factor A study of cardiovascular biomarkers indicates that were unchanged. Another study demonstrated that TxM smokeless tobacco produced neither the inflammatory levels increased in one exposure to secondhand smoke; reaction found in smokers nor endothelial dysfunc- after six hours of exposure, levels approached those tion, activation of platelets, or evidence of oxidant stress observed in smokers (Schmid et al. 1996). In other (Axelsson et al. 2001). Leukocyte counts; levels of CRP,

Cardiovascular Diseases 381 Surgeon General’s Report

fibrinogen, and antioxidant vitamins; and lipid profiles cardiovascular risk among smokers will emerge and were similar in users of smokeless tobacco and in persons which genetic variants might particularly influence these who did not use tobacco. risks in smokers. The implications of the hypercoagulable state are observed both in the epidemiology of active and involuntary smoking-related cardiovascular events and in Summary the rapid rate of decline in the major component of excess risk for those events after smoking cessation. A hyperco- Multiple factors produced in the blood and released agulable state can result in acute MI in persons who have from the vasculature determine the likelihood of a clini- less severe underlying coronary disease, so smokers who cally significant thrombosis. Cigarette smoke and com- stop smoking have a better prognosis than do nonsmok- ponents of the smoke stimulate formation or activity ers after MI. A more gradual decline of residual risk may of factors that favor the development of thrombosis. It reflect resolution of smoking-induced vascular injury, remains to be seen whether biomarkers of individual which in turn stimulates platelet activation.

Inflammation

Studies demonstrate that cigarette smoking results al. 2001). The study findings suggest that smoking-related in a chronic inflammatory state, evidenced by increased impairment of NO release is an important determinant of counts of circulating leukocytes, CRP, and acute-phase increased adhesion of monocytes to endothelial cells. reactants such as fibrinogen (Tracy et al. 1997; Jensen et Nicotine may contribute to inflammation by acting al. 1998; Tuut and Hense 2001). Cigarette smoking also as a chemotactic agent for migration of neutrophils (Nicod activates monocytes and enhances recruitment and adhe- et al. 1984). One study indicates that nicotine enhanced sion of leukocytes to blood vessel walls, an integral step leukocyte-endothelium interactions, resulting in greater in vascular inflammation (Lehr et al. 1994). Research leukocyte rolling and adhesion in the cerebral microcir- indicates that inflammation contributes to atherogenesis, culation of mice (Nitenberg et al. 1993). Nicotine report- because high leukocyte counts and high levels of CRP and edly acts on human monocyte-derived dendritic cells to fibrinogen are all powerful predictors of future cardiovas- stimulate an inflammatory response (Nowak et al. 1987). cular events (Libby et al. 2002). Dendritic cells, which were detected in the walls of arter- However, the mechanisms by which cigarette smok- ies and in atherosclerotic lesions, present antigens and are ing promotes inflammation are not completely elucidated. thus required for the start of adaptive immunity. Studies As discussed previously, oxidant stress appears to be a crit- showed that nicotine is a potent inducer of expression of ical factor; oxidized LDL is a proinflammatory stimulus a variety of co-stimulatory molecules and that it increases (see “Cigarette Smoke Constituents and Cardiovascular secretion of the proinflammatory cytokine interleukin-12 Disease” earlier in this chapter). Studies also show that in cultured dendritic cells (Aicher et al. 2003). Nicotine the products of lipid peroxidation are proinflammatory, augmented the capacity of dendritic cells to stimulate acting in part on the receptor for platelet-activating factor proliferation of T cells and cytokines. Finally, intravenous (PAF). In hamsters, the antioxidant vitamin C prevented injection of nicotine increased the movement of dendritic adhesion of leukocytes to the endothelium and leukocyte- cells into atherosclerotic lesions in vivo in mice deficient platelet aggregation (Lehr et al. 1994). In the same animal in APO E. This line of research suggests that nicotine model, adhesion of leukocytes and formation of leukocyte- could contribute to adaptive immunity, which may have platelet aggregates were mediated by PAF-like agonists a role in atherogenesis. However, switching from smoking (Lehr et al. 1997). This PAF-like factor was derived from to transdermal nicotine resulted in a significant decline oxidative modification of phospholipids and was distinct in the leukocyte count (Benowitz et al. 1993). In addition, from biosynthetic PAF. Treatment with vitamin C inhibited use of smokeless tobacco did not produce higher leuko- generation of PAF-like lipids. In contrast, oral l-arginine cyte counts or higher CRP levels than are seen in persons but not vitamin C reversed the effect of sera from smok- who do not use tobacco. These observations suggest that ers by promoting monocyte-endothelial cell adhesion, nicotine is not the main determinant of the inflammatory which is associated with higher levels of ICAM (Newby et response in smokers.

382 Chapter 6 How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease

Smoking and Diabetes

Cigarette smoking is widely known to increase the activity, alcohol intake, social class, undiagnosed CHD, risks of CVD. Even so, this knowledge does not appear to and treatment for hypertension (Wannamethee et al. influence smoking behaviors among patients with diabe- 2001). Mean follow-up was almost 17 years. The increased tes, who bear a higher risk of cardiovascular morbidity and risk of diabetes was lower 5 years after smoking cessation, mortality than those who do not have diabetes (Haffner et and the risk normalized 20 years later. A Japanese study al. 1998). Surveys found that smoking patterns were simi- found similar results and also reported a positive correla- lar in patients with diabetes and comparable populations tion between use of tobacco products and risk of diabetes without that disorder (Ford et al. 1994; Gill et al. 1996). (Uchimoto et al. 1999). Numerous experimental studies demonstrated Almost identical results were presented in the Physi- that smoking had negative effects on the metabolism of cians’ Health Study (Manson et al. 2000). During 12 years glucose and lipids in persons with or without diabetes. (255,830 person-years1) of follow-up, the risk of diabetes Investigators reported that cigarette smoking in patients in men who smoked more than 20 cigarettes per day was with diabetes was associated with deterioration of meta- 70 percent higher than that in nonsmokers, after adjust- bolic control (Madsbad et al. 1980; Bott et al. 1994) and ment for multiple variables. The results also showed increased risk of microvascular and macrovascular com- a significant positive association between the risk of plications and death (Chase et al. 1991; Morrish et al. developing diabetes and higher consumption of cigarettes 1991). Furthermore, cigarette smoking increases risk of (Manson et al. 2000). In addition, the Insulin Resistance type 2 diabetes in the general population (Will et al. 2001). Atherosclerosis Study monitored a cohort of 906 study This risk may be mediated through direct metabolic participants for five years with equal representation of effects alone or in combination with a metabolically unfa- African Americans, Hispanics, and Whites (Foy et al. vorable lifestyle. 2005). For all persons studied, current smoking was associated with development of diabetes: the adjusted OR was 2.66 (95 percent CI, 1.49–4.77). Among participants who Risk had normal glucose tolerance at baseline, the OR was 5.27 (95 percent CI, 2.11–13.16). In several prospective studies, cigarette smoking At least three other studies of men confirmed the was associated with increased risk of type 2 diabetes in main results of these prospective studies (Feskens and both men and women (Willi et al. 2007). Generally, these Kromhout 1989; Kawakami et al. 1997; Ko et al. 2001). prospective studies were large and population based. Most There have been fewer studies with women, but two major of the information was collected by mailing participants prospective surveys yielded similar results. In the Nurses’ a questionnaire, which in some cases, was supplemented Health Study (114,247 women; 1,277,589 person-years with information from medical records. Most of these of follow-up), RR for diabetes in heavy smokers was 1.42, studies included follow-up of more than 10 years. Results after adjustments for other risk factors (Rimm et al. 1993). were generally presented after adjustments for possible In an analysis of data from the Nurses’ Health Study covariates. after 16 years of follow-up, Hu and colleagues (2001) In the Health Professionals Follow-Up Study, the showed that the strongest predictors of diabetes were RR of developing diabetes among men who smoked 25 being overweight or obese. In addition, poor diet, smok- or more cigarettes per day was 1.94 (95 percent CI, 1.25– ing, abstinence from alcohol, and low levels of physi- 3.03) when nonsmokers were the reference group (Rimm cal activity were all independently associated with the et al. 1995). In a smaller British study, the risk of diabetes risk of developing diabetes. Adjusted RR for developing was 50 percent higher among smokers, but this finding diabetes was approximately 1.4 for smokers compared was not independent of other risk factors, such as obe- with nonsmokers. sity and low levels of physical activity (Perry et al. 1995). Data from CPS-I, a prospective cohort study con- Another British study demonstrated that RR for diabetes ducted between 1959 and 1972, were used to analyze the in men who smoked was approximately 1.7, after adjust- correlation between tobacco use and risk of diabetes in ments for the effects of age, body mass index, physical both men and women (Will et al. 2001). In comparison

1Person-year = the sum of the number of years that each member of a population has been smoking.

Cardiovascular Diseases 383 Surgeon General’s Report

with the risk of developing diabetes for nonsmokers, the consistent determinant of HbA1c levels in relatively young risks were higher for men who smoked more than one articipants treated with insulin (Bott et al. 1994). The pack of cigarettes per day (RR = 1.19) or two packs per investigators performed a three-year follow-up on 697 day (RR = 1.45). Risks were also higher for women who patients with diabetes who had no debilitating late com- smoked more than one pack per day (RR = 1.21) or two plications. HbA1c levels were higher throughout the study packs per day (RR = 1.74). After smoking cessation, the in smokers but eventually improved to levels similar to risk returned to normal after 5 years for women and after those in nonsmokers, presumably because of the educa- 10 years for men (Will et al. 2001). tional program. Only a few studies, all from the 1970s or 1980s, failed to demonstrate a positive association between smoking and diabetes, likely due to inadequate study design or lack Insulin Resistance of power in the study to test this hypothesis (Medalie et al. 1975; Keen et al. 1982; Wilson et al. 1986). The metabolic effects of smoking have been gener- It is generally accepted that risk increases more for ally studied in persons who did not have diabetes. Insulin type 2 diabetes than for type 1, because type 1 diabetes is sensitivity was usually determined by using the euglyce- relatively rare among the age groups in the studies. Risk mic hyperinsulinemic clamp technique (DeFronzo et al. for type 2 diabetes is also consistent with the adverse met- 1979). This technique or a slightly modified version of it abolic effects of smoking (see “Insulin Resistance” later in is considered to be the gold standard in metabolic studies. this chapter). Type 1 diabetes is insulin deficiency caused In 1993, Attvall and colleagues showed that short- by autoimmune destruction of pancreatic beta cells; term smoking caused impaired insulin sensitivity in in type 2 diabetes, insulin resistance is combined with healthy young men. Separately, two cross-sectional stud- impaired secretion of insulin (Reaven 1988; Kahn 2001). ies of men compared uptake of insulin-mediated glucose (insulin sensitivity) in smokers and nonsmokers (Fac- chini et al. 1992; Eliasson et al. 1997a). Insulin sensitivity Metabolic Control was significantly lower (by 10 to 40 percent) in smokers. The degree of insulin resistance was positively correlated Some studies examined the effects of cigarette with tobacco use, and in long-term users of nicotine gum, smoking on the body’s requirement for insulin and meta- with serum cotinine values (Eliasson et al. 1994, 1996). bolic control in patients with diabetes. In a cross-sectional Because cotinine is a metabolite of nicotine, serum and study, Madsbad and colleagues (1980) investigated the urine levels of cotinine reflect the amount of nicotine use. relationship between insulin doses and related variables Insulin resistance in smokers normalized eight weeks in patients treated with injections of insulin. Insulin after smoking cessation, despite a weight gain of 2.7 kg doses and serum levels of triglycerides were significantly (Eliasson et al. 1997a). higher in the 114 persons who smoked than in the 163 Smokers in these two cross-sectional studies had who did not smoke, and these values increased in a dose- signs of insulin-resistance syndrome, such as signifi- dependent manner in relation to the number of cigarettes cantly high serum levels of free fatty acids (FFAs) and smoked. Hemoglobin A1c (HbA1c)—a marker of long-term triglycerides and low levels of HDLc (Facchini et al. 1992; glucose elevation—was not measured in this study, but Eliasson et al. 1997b). In the study by Eliasson and col- blood and urine levels of glucose did not differ between leagues (1997b), smokers had a high proportion of ath- the two groups. This finding suggests that in patients who erogenic small and dense LDL particles, high fibrinogen smoke, a larger insulin dose is needed to achieve meta- levels, and high PAI-1 activity compared with those of bolic control similar to that in patients who do not smoke. nonsmokers. PAI-1 activity among long-term users of In a cross-sectional study of 192 patients with type nicotine gum was similar, but effects on lipids were not 1 diabetes, smoking was more common in those with as pronounced as those in smokers (Eliasson et al. 1996). higher HbA1c values (Lundman et al. 1990). Other dif- One aspect of insulin-resistance syndrome that ferences between the smokers and nonsmokers were in attracted attention was postprandial hypertriglyceridemia, attitudes toward diabetes, psychological well-being, and a phenomenon associated with CVD and insulin resistance similar factors. (Patsch et al. 1992; Jeppesen et al. 1995). This phenom- In a relatively large prospective study that exam- enon is also observed in smokers (Axelsen et al. 1995; Eli- ined the effects of intensified insulin treatment and asson et al. 1997b), but its cause is unknown. One possible of an educational program, smoking was the most

384 Chapter 6 How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease explanation is the inability of smokers to adequately clear particularly adverse effects in persons susceptible to dia- triglyceride-rich chylomicrons and their remnants from betes but not in those who are healthy (insulin sensitive). the body (Mero et al. 1997). Other studies that did not use exact measurements of insulin sensitivity reported changes in glucose metabo- Microvascular Complications lism in smokers compared with nonsmokers. Compared with nonsmokers, smokers were hyperinsulinemic and Microvascular complications in diabetes (retinopa- relatively glucose intolerant (Eliasson et al. 1991; Zava- thy, nephropathy, and neuropathy) are linked to metabolic roni et al. 1994; Frati et al. 1996; Ronnemaa et al. 1996). control in both type 1 and type 2 disease (New England A large cross-sectional study showed that after adjust- Journal of Medicine 1993; Lancet 1998). The mechanisms ments for confounding factors, smoking behaviors were for development of microvascular complications are not clearly correlated with HbA1c values in persons who did fully understood, although several pathogenetic pathways not have diabetes (Sargeant et al. 2001), but researchers have been suggested (Brownlee et al. 1984; Tomlinson have debated the importance of HbA1c in persons who do 1999; Cai and Boulton 2002). Hyperglycemia has a central not have diabetes. Even so, these findings add support to role as a trigger for subsequent events, such as conversion the hypothesis that use of tobacco exerts adverse effects on of glucose to sorbitol by aldose reductase; nonenzymatic glucose homeostasis. glycosylation of proteins and receptors in susceptible tis- Results in a few studies did not support these find- sues; increased exposure to oxidative stress; and activation ings. Godsland and Walton (1992) found no differences in of protein kinase C and mitogen-activated protein kinases. insulin sensitivity between women smokers and nonsmok- Researchers have suggested that these pathogenetic path- ers, but this result may be attributable to lower levels of ways lead to the disturbances in morphology and function tobacco use. In addition, the results were from analysis of found in diabetic nephropathy, retinopathy, and neuropa- data obtained to test a different hypothesis. In a study of thy (Brownlee et al. 1984; Tomlinson 1999; Cai and Boul- metabolic changes in patients with or without hyperten- ton 2002). sion, no differences in insulin sensitivity between smokers and nonsmokers were detected (Nilsson et al. 1995). How- Nephropathy ever, the study design likely did not enable discrimination between metabolic changes caused by hypertension and Some studies showed that smoking increased risk of those caused by smoking. microvascular complications in diabetes. Several studies In patients with type 1 diabetes, Helve and colleagues of patients with type 1 diabetes reported negative effects (1986) examined cross-sectional and short-term effects of of tobacco use on albuminuria and renal function. Chase smoking on insulin sensitivity. Despite elevated levels of and colleagues (1991), for example, showed that the albu- circulating epinephrine, cortisol, growth hormone, and min excretion rate was 2.8 times higher in smokers than glucagon after smoking, no effect of smoking on insulin in nonsmokers, after statistical corrections for glycemic sensitivity was observed. The investigators concluded that control, duration of diabetes, age, gender, and blood pres- fluctuations in blood glucose and metabolic control dis- sure. In addition, albuminuria progressed at a more rapid guised the influence of smoking in these patients with dia- rate in smokers than in nonsmokers. betes. In a study of 28 smokers and 12 nonsmokers with Smoking promoted progression of renal disease type 2 diabetes, the researchers measured insulin sensi- in persons with type 2 diabetes (Biesenbach et al. 1997; tivity by using euglycemic clamps (Targher et al. 1997). Chuahirun and Wesson 2002; Chuahirun et al. 2003). Bie- Smokers had higher insulin resistance and glucose intol- senbach and colleagues (1997) studied only 36 patients, erance than did nonsmokers. The researchers concluded but follow-up lasted 13 years. At study entry, smokers and that smoking markedly and in a dose-dependent manner nonsmokers had similar clinical and laboratory character- aggravated insulin resistance observed in patients with istics, but progression of nephropathy and development type 2 diabetes (Targher et al. 1997). of atherosclerotic disease progressed more rapidly in the Axelsson and colleagues (2001) reported that nico- smokers than in the nonsmokers. Multiple regression tine administered intravenously to nonsmokers caused a analysis showed that only tobacco use and blood pres- marked reduction (about 30 percent) in insulin sensitivity sure levels were independently associated with impair- in those with type 2 diabetes but not in healthy control ment in renal function. This finding underscored the participants. These results suggest that nicotine and pos- roles of smoking and vascular disease in susceptibility to sibly tobacco use or other environmental factors may have renal disease (Biesenbach et al. 1997). In two prospective

Cardiovascular Diseases 385 Surgeon General’s Report studies by Chuahirun and colleagues (2002, 2003), the Macrovascular Complications effects of cigarette smoking on acceleration of nephropathy in patients with type 2 diabetes were confirmed even in The multiple effects of smoking on the vascular and those who had optimal therapy for hypertension. Research hemostatic systems and on inflammation are reviewed presented further evidence of functional and structural elsewhere in this chapter (see “Hemodynamic Effects,” changes in the glomeruli of patients with type 2 diabetes “Smoking and the Endothelium,” “Nicotine and Throm- who smoke (Baggio et al. 2002). In a study of 96 patients bosis,” and “Inflammation” earlier in this chapter). Dia- who had biopsy of the kidney, electron and light micros- betes patients are particularly susceptible to some effects copy demonstrated significant changes in glomeruli and of smoking, because their risk of cardiovascular morbidity basal membranes that corresponded to impaired glomeru- and mortality is elevated (Jarrett et al. 1982; Manson et al. lar filtration rates in the smokers. 1991; Morrish et al. 1991). In a study cohort in London, England, in the pro- Retinopathy spective Multinational Study of Vascular Disease in Diabe- Generally, investigators have not considered smok- tes, sponsored by the World Health Organization, smokers ing to be a substantial risk factor for diabetic retinopathy with type 1 or type 2 diabetes had significantly increased (Porta and Bandello 2002). Findings in fairly large stud- risk of CHD, but not stroke, during the eight-year follow- ies with mixed populations showed no strong support for up (Morrish et al. 1991). In the Diabetes Control and Com- such an association, except in older adults with certain plications Trial (New England Journal of Medicine 1993), conditions (Walker et al. 1985; Moss et al. 1991). At least designed to study the role of intensive insulin treatment two studies of patients with type 1 diabetes, however, sug- and optimized glycemic control in type 1 diabetes, smok- gest that smoking does predispose these patients to reti- ing was not a significant risk factor for macrovascular nopathy (Mulhauser et al. 1986, 1996). In addition, Chase complications. Because the participants were relatively and colleagues (1991) showed that retinopathy was more young, this trial was not optimally designed to study the common in patients with type 1 diabetes who smoked than role of tobacco use. Other studies with slightly older par- in those who did not smoke, but after adjustments for ticipants who had type 1 diabetes reported that smoking covariates, differences were not statistically significant. increased risk of CHD (Moy et al. 1990; Sinha et al. 1997). The study also reported accelerated progression of reti- Among patients with type 2 diabetes in the United nopathy in patients who smoked. Thus, smoking may Kingdom Prospective Diabetes Study, cigarette smoking be a risk factor for diabetic retinopathy, but only in cer- was a significant and independent risk factor for CHD tain subgroups. (Turner et al. 1998), stroke (Kothari et al. 2002), and PAD (Adler et al. 2002). Also, an analysis of data from Neuropathy the Nurses’ Health Study demonstrated that for women with type 2 diabetes, a dose-effect relationship existed The role of tobacco in the development of diabetic between smoking behaviors and mortality (Al-Delaimy et neuropathy is relatively difficult to examine because of al. 2001). Compared with nonsmokers, risk of mortality methodologic problems and the frequent prevalence of from all causes was 1.64 for women who smoked 15 to 34 confounding factors (Westerman et al. 1992). Diabetic cigarettes per day and 2.19 for women who smoked more neuropathy usually develops during a long period, and it than 34 cigarettes per day. Ten years after smoking cessa- may affect different sensory, motor, and autonomic nerve tion, risk of mortality had normalized. Researchers pub- fibers in varying degrees in individuals. This variation lished similar data on smoking and CHD risk in the same makes it difficult to standardize study methods. One case- cohort (Al-Delaimy et al. 2002). control study reported that risk of neuropathy was three A relatively large prospective study that analyzed times higher in patients with type 1 diabetes who smoked the effects of smoking cessation on cardiovascular risk in than in those who did not smoke (Mitchell et al. 1990). persons with diabetes compared mortality risk for former Smoking was not related to neuropathy in patients with smokers with that for lifetime nonsmokers (Chaturvedi et type 2 diabetes. In a study of young patients treated with al. 1997). Compared with mortality risk for lifetime non- insulin, independent risk factors for progression of distal smokers, risk of death from all causes was approximately sensory neuropathy, apart from poor glycemic control, 50 percent higher for patients who had stopped smoking were cigarette smoking, greater height, and female gender during the past one to nine years and 25 percent higher (Christen et al. 1999). Other studies in patients with type for those who had not smoked for more than nine years. 1 diabetes confirmed the roles of glycemic control and Smoking cessation reduced mortality risk among per- smoking behaviors in development of clinical neuropathy sons with diabetes, but risks remained high several years (Maser et al. 1989; Reichard 1992).

386 Chapter 6 How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease after smoking cessation and were highly dependent on the sensitivity directly or indirectly through these and possi- duration of smoking. bly other mechanisms. An additional negative factor for insulin-mediated glucose uptake is high circulating levels of FFAs, secondary to increased lipolysis (Bergman and Pathophysiological Mechanisms Ader 2000). Research has shown that smoking acutely elevates circulating FFA levels (Kershbaum and Bellet 1966). Having diabetes, even for nonsmokers, is associ- Researchers have proposed, but not fully elucidated, ated with long-term exposure to oxidative stress, the potential role of endothelial dysfunction or inflam- impaired endothelial function, and dyslipidemia (Brown- mation in development of insulin resistance and type lee et al. 1984; Turner et al. 1998; Dogra et al. 2001; Cai 2 diabetes. and Boulton 2002; Komatsu et al. 2002). The causes of type 2 diabetes are still not fully understood, although the main metabolic aberrations are well characterized. Summary Research showed that type 2 diabetes is caused by insulin resistance in combination with relative impairment of Many clinical and experimental studies have found insulin secretion (Reaven 1988; Kahn 2001). Published significant associations between cigarette smoking and studies have not demonstrated a significant impairment development of diabetes, impaired glycemic control, and in insulin secretion among cigarette smokers (Epifano et diabetic complications (microvascular and macrovascu- al. 1992; Facchini et al. 1992; Persson et al. 2000), but lar). A different lifestyle of smokers, in contrast to that several studies documented a negative effect of smoking maintained by nonsmokers, may also contribute to these on insulin sensitivity (Facchini et al. 1992; Eliasson et effects. Most of the reviewed studies, however, either al. 1997a,b). attempted to statistically adjust for confounding factors Cigarette smoking and intake of nicotine increase or were designed to examine short-term effects of tobacco the circulating levels of insulin-antagonistic hormones and nicotine. (i.e., catecholamines, cortisol, and growth hormone) The development of type 2 diabetes is another harm- (Kershbaum and Bellet 1966; Cryer et al. 1976; Wilkins ful consequence of cigarette smoking, one that adds to the et al. 1982; Kirschbaum et al. 1992). Smoking also acti- heightened risks of CVD. In diabetes care, smoking cessa- vates the sympathetic nervous system (Niedermaier et al. tion is crucial to facilitating glycemic control and limiting 1993; Lucini et al. 1996). Nicotine likely impairs insulin development of complications.

Lipid Abnormalities

Cigarette smoking is associated with an atherogenic between the number of cigarettes smoked per day and lipid profile likely to contribute to risk of CVD. plasma lipid levels (Muscat et al. 1991). In contrast, plasma lipid and lipoprotein levels in former smokers typically are similar to those in nonsmokers. Epidemiologic Observations The ratio of LDLc to HDLc, which is used as a measure of atherogenic risk, is higher in smokers than in Several observations are central to the relationship nonsmokers. Cigarette smoking is thought to raise the between cigarette smoking and lipids. Compared with LDLc to HDLc ratio by 15 to 20 percent. Increased lev- nonsmokers, smokers have higher levels of triglycerides els of plasma triglycerides are associated with lower HDLc associated with very-low-density lipoprotein (VLDL), levels, but reduction in HDLc from cigarette smoking per- total triglycerides, and APO B, in addition to modest in- sists even after corrections for levels of total triglycerides. creases in LDLc and lower levels of plasma HDLc and APO Early epidemiologic studies, such as the Lipid Research A-I (Billimoria et al. 1975; Criqui et al. 1980; Wilson et Clinics Program Prevalence Study and the Framingham al. 1983; Craig et al. 1989; Muscat et al. 1991; Freeman Heart Study (Criqui et al. 1980; Muscat et al. 1991; Free- and Packard 1995; Villablanca et al. 2000). These findings man and Packard 1995), emphasized lower HDLc values as are robust and are reported in numerous survey studies. the primary effect of cigarette smoking. Researchers observed a dose-response relationship

Cardiovascular Diseases 387 Surgeon General’s Report

Researchers have estimated, however, that these Plasma- and Lipoprotein- effects of cigarette smoking on plasma lipids and lipoproteins account for only 10 percent of the observed 70 per- Associated Lipid Transfer Enzymes cent increase in risk of vascular disease associated with Several enzymes in plasma—either free or associ- cigarette smoking (Craig et al. 1989). In the Edinburgh ated with lipoproteins—are involved in transport and Artery Study, for example, adjusting for known CHD risk use of lipids. Lipoprotein lipase (LPL) activity is involved factors reduced the RR of CHD in heavy smokers from in clearance of total triglycerides from triglyceride-rich 3.94 to 2.72 and the RR in moderate smokers from 2.72 to lipoproteins (TGRLs), particularly the chylomicra formed 1.70 (Price et al. 1999). However, cigarette smoking still in persons after a meal containing fat. Cigarette smoking accounted for 75 percent of the risk of developing PAD, reportedly reduced plasma LPL activity after a mixed meal after adjustment for other known risk factors, such as (Freeman et al. 1998). Reduced LPL activity may contrib- hyperlipidemia and type 2 diabetes (Lu and Creager 2004). ute to the reduced clearance of TGRLs reported for total Other researchers reported similar findings (Cullen et al. triglycerides, APO B, and retinyl-ester components of 1998). Cigarette smoking thus appears to have atherogen- TGRLs (Mero et al. 1998). In another study, the cholesterol ic effects that are not explained by traditional CHD risk ester transfer protein (CETP) received considerable atten- factors, including abnormal levels of blood lipids. tion as a therapeutic target for raising HDLc levels (Brous- Analyses of mechanisms related to lipid and lipopro- seau et al. 2004; Ruggeri 2005). CETP mediates transfer tein metabolism may be required for understanding the of cholesterol esters between HDL and other lipoproteins atherogenicity of cigarette smoking. Such mechanisms (VLDL and LDL). However, controversy exists as to wheth- include lipid oxidation; changes in composition of lipo- er CETP activity is beneficial or deleterious to the process proteins; alterations in plasma- and lipoprotein-associated of RCT. Moreover, the proatherogenic or antiatherogenic lipid transfer enzymes; changes in metabolism of fatty consequences of changing HDLc by the CETP mechanism acids; effects on levels of postprandial lipids; and chang- remain uncertain (Ruggeri 2005). Studies have reported es in cholesterol fluxes, particularly reverse cholesterol both increases and decreases in plasma CETP activity in transport (RCT). The following discussion reviews the smokers (Dullaart et al. 1994; Zaratin et al. 2004). effects of cigarette smoking on these potential underly- Some studies measured other enzymes in smokers. ing mechanisms. For example, cigarette smoking did not appear to markedly alter lecithin cholesterol acyltransferase activity (McCall et al. 1994). Lipoprotein Composition and Apolipoprotein Levels Oxidized Lipoproteins Cigarette smoking clearly reduces APO A-I and the ratio of A-I to A-II (Mero et al. 1998). APO A‑I is a major Many investigators hypothesized that oxidized LDL component of HDL particles. The reduction in A-I levels is highly atherogenic (Dullaart et al. 1994; Zaratin et al. observed in smokers is similar to, although perhaps some- 2004), and studies reported increased oxidative damage what lower than, the reduction in HDLc levels seen in this to LDL in smokers (Ambrose and Barua 2004). However, population. For example, Mero and colleagues (1998) doc- attribution of a precise atherogenic contribution from umented that levels of plasma APO A-I were 4 to 6 percent oxidative damage to LDL remains speculative. The inad- lower and HDLc values were 6 to 9 percent lower in mod- equacy of the metrics of pro-oxidative and antioxidative erate-to-heavy smokers. The effects of cigarette smoking status in vivo needs to be resolved before the role of oxida- on APO B and other APOs are also well documented (Bil- tive damage to LDL can be adequately evaluated. limoria et al. 1975; Craig et al. 1989; Muscat et al. 1991; Villablanca et al. 2000). Researchers have associated abnormalities in different subfractions of HDL with different risks of CHD. Postprandial Lipid Changes Cigarette smoking reduced different HDL subfractions in Traditionally, plasma lipid and lipoprotein mea- different studies (Billimoria et al. 1975; Craig et al. 1989; surements used to evaluate CHD risks have, for largely Muscat et al. 1991). Even so, the true atherogenicity of dif- technical reasons, been performed in the fasting (posta- ferent HDL subfractions remains controversial. The role bsorptive) state. Plasma levels of metabolites are more of altered HDL subfractions in arterial disease associated easily characterized in steady-state conditions than in a with cigarette smoking requires further study. nonsteady state. From a pathophysiological perspective,

388 Chapter 6 How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease however, events in the postprandial state may be critical 1975; Perkins et al. 1989; Hellerstein et al. 1994; Neese et in atherogenesis (Zilversmit 1979). Some studies explored al. 1994). However, most of the FFAs released in response effects of cigarette smoking on postprandial lipid metabo- to cigarette smoking are not oxidized but taken up and lism (Mero et al. 1998). For example, total triglycerides reesterified to triglycerides in tissues, particularly the increased to higher levels after a mixed meal in smokers liver. This conclusion was partly based on kinetic studies than in nonsmokers. These researchers observed increases that compared the rates at which plasma FFA appeared in plasma APO B level and reductions in levels of APO A-I, with whole-body rates of fat oxidation (Hellerstein et al. lipoprotein A-I, HDLc, and LDL-APO B after a meal. Other 1994; Neese et al. 1994). These studies demonstrated that investigators postulated that the mechanisms underlying the rate of FFA influx into plasma in response to- ciga altered postprandial lipid changes in smokers include rette smoking greatly exceeded changes in whole-body lower LPL activity (Freeman et al. 1998), but higher fat oxidation. endogenous production of VLDL-total triglycerides by the Accordingly, the catabolic effects of cigarette smok- liver has not been excluded. The postprandial effects of ing on total adipose triglycerides do not directly promote cigarette smoking in particular and their role in athero- oxidation of body fat (weight loss); instead, the primary genesis in general are not completely understood. result is overproduction of VLDL-total triglycerides (Hellerstein et al. 1994; Neese et al. 1994). This “futile cycle,” a substrate cycle in which adipose triglycerides Metabolism of Free Fatty Acids are converted to hepatic VLDL triglycerides, is modestly wasteful of energy. It accounts for about 5 percent of the Changes in FFAs (nonesterified fatty acids) are thermogenic effects of long-term cigarette smoking—for attributed to an increase in adipocyte lipolysis, and they example, fewer than 10 kilocalories (kcal)/day if cigarette represent the most well-characterized mechanistic action smoking increases total energy expenditure by 200 kcal/ of cigarette smoking in the context of alterations in lipids day. This cycle, however, may be the central driving force and lipoproteins. Many studies reported higher plasma lev- behind the atherogenic dyslipidemia associated with ciga- els of FFAs in smokers than in nonsmokers (Kershbaum et rette smoking. Overproduction of VLDL-total triglycerides al. 1963; Bizzi et al. 1972; Walsh et al. 1977; Hellerstein et typically results in elevated plasma levels of VLDL-total al. 1994; Neese et al. 1994). triglycerides and APO B, as well as increased numbers of Using stable (nonradioactive) isotopes to measure LDL particles (Sniderman et al. 2001). Furthermore, high FFA kinetics, researchers demonstrated that cigarette VLDL-total triglycerides contribute to lowering of HDLc smoking immediately and markedly increased influx of through CETP-mediated transfer of cholesterol-ester from FFAs into the bloodstream and thereby raised plasma HDL to VLDL particles (Brousseau et al. 2004; Ruggeri et levels of FFA (Hellerstein et al. 1994; Neese et al. 1994). al. 2005). Influx of FFAs into the liver for reesterification Plasma FFAs are primarily derived from adipose tissue and secretion of total triglycerides is most likely a major by lipolytic breakdown of stored triglycerides. Catechol- reason for the low HDLc levels observed in smokers, but amines stimulate hormone-sensitive lipase activity in perhaps it does not represent the entire effect of smoking adipose tissue and oppose various antilipolytic actions of on HDLc (Criqui et al. 1980; Muscat et al. 1991; Freeman insulin. Increases in FFA levels and flux induced by smok- and Packard 1995). ing were temporally correlated with increases in plasma If nicotine-stimulated release of catecholamine is epinephrine levels (Watts 1960; Bizzi et al. 1972; Arcavi et responsible for the hypertriglyceridemia and low HDLc al. 1994; Hellerstein et al. 1994; Neese et al. 1994). These levels observed in smokers, NRT as an adjunct to smok- increases are prevented by β-adrenergic blockers. Nico- ing cessation should logically prevent improvements in tine increases the adrenal medullary release of epineph- plasma lipids and lipoproteins after smoking cessation. rine in persons with nontolerance of nicotine (Arcavi et Moffatt and colleagues (2000) reported that nicotine- al. 1994). Therefore, the model implicated as the cause of patch therapy prevented the normalization of HDLc lev- increases in plasma FFA levels induced by cigarette smok- els observed with smoking cessation in the absence of the ing seems clear: cigarette smoking→nicotine→increased nicotine patch. The patch also prevented weight gain after plasma epinephrine→increased lipolysis in adipose smoking cessation (Allen et al. 2005), a finding consis- tissue→increased release of FFAs into plasma→increased tent with the hypothesis that shared catecholamines are plasma levels of FFA. the basis for two important effects of cigarette smoking: The fate of FFAs released into the bloodstream in weight reduction and dyslipidemia. Other studies, how- response to cigarette smoking is also relevant. Cigarette ever, did not confirm that use of the nicotine patch as an smoking increases expenditure of energy through the agent for smoking cessation prevents improvements in activity of nicotine and catecholamines (Ilebekk et al. HDL levels (Allen et al. 1994). In addition, lipid profiles

Cardiovascular Diseases 389 Surgeon General’s Report are similar in persons who use smokeless tobacco and in Rabkin 1984; Stamford et al. 1986; Critchley and Capewell those who do not use any form of tobacco. To the extent 2004). Many studies documented the return of normal that dyslipidemia contributes to vascular disease associ- levels of plasma lipids and lipoproteins after cessation of ated with cigarette smoking, it is important to determine cigarette smoking. the full range of effects of NRT on lipid and lipoprotein metabolism. Therapeutic Implications of Pathogenic Mechanisms Reverse Cholesterol Transport If stimulation of lipolysis underlies the atherogenic RCT refers to the pathway by which cholesterol is dyslipidemia associated with cigarette smoking, inhibi- mobilized from tissues, carried through the blood, and tion of lipolysis might be an effective therapeutic strat- excreted from the body. HDL and its associated membrane egy to improve blood lipid profiles in smokers or persons receptors (e.g., SR-B1, ABC-A1, and ABC-G1), plasma receiving NRT. This strategy is an attractive approach in enzymes (e.g., CETP and phospholipid transfer protein), one sense because inhibition of lipolysis does not block APOs (e.g., APO A-I), and hepatobiliary enzymes (e.g., cho- the thermogenic actions of nicotine. The cycle of lipoly- lesterol 7α-hydroxylase) constitute a system that mediates sis and reesterification accounts for less than 5 percent the complex process of RCT through pathways that are of the increase in energy expenditure observed in ciga- increasingly well characterized in molecular terms (Neese rette smokers (Hellerstein et al. 1994; Neese et al. 1994). et al. 1994; Tall 1998). RCT is generally accepted as the Another consideration is that if FFAs released by lipolysis leading explanation for the cardioprotective activity of are involved in the insulin resistance reportedly associat- HDL, although other actions of HDL (e.g., antioxidative ed with cigarette smoking (Facchini et al. 1992), lipolysis and anti-inflammatory) may also be involved. inhibitors may have an additional therapeutic use. Flux through the RCT pathway and, thus, antiath- Niacin, a hypolipidemic agent, is thought to act erogenic activity cannot be predicted simply from plasma at least partly by inhibiting total triglyceride lipolysis in levels of HDL or APO A-I (Tall 1998). Thus, changes in lev- adipose tissue (Meyers et al. 2004). The use of niacin in els of HDLc, VLDL-total triglycerides, and other lipopro- smokers who are at high risk for CHD has not been fully teins may not fully capture the effects of cigarette smoking investigated. The side effects of niacin, including cutane- on pro-atherogenic or antiatherogenic fluxes such as RCT. ous flushing and worsening of insulin resistance in some Until more recently, however, there were no viable tech- persons, perhaps from effects on pancreatic islet function, niques for measuring RCT fluxes. Therefore, this question may discourage its clinical use. The impact of niacin and could be addressed only indirectly—for example, through its analogs on lipolysis is complex. They induce a rebound changes in lipid transfer proteins in plasma that may overshoot of lipolysis after initial inhibition, but niacin influence the efficiency of RCT. Studies reported decreas- does reduce production of VLDL-total triglycerides (Wang es in activity of CETP and phospholipid transfer protein et al. 2001). in smokers after they had smoked a cigarette (Zaratin et Other strategies for use of lipolysis inhibitors to al. 2004). prevent CHD related to cigarette smoking may require Reduced capacity for remodeling HDL particles in development of specific antilipolytic agents that are well the vascular compartment could alter these fluxes in a tolerated. One possibility is the thiazolidinedione class manner not reflected by HDLc levels. This possibility will of insulin-sensitizing drugs. In one study, such drugs remain speculative, however, until RCT fluxes are -mea reduced lipolysis in adipose tissue, perhaps by activating sured in humans. The ability to directly measure effects of glyceroneogenesis and thereby promoting intra-adipocyt- cigarette smoking on the cardioprotective process of RCT ic reesterification of FFAs (Chen et al. 2005). No studies could provide a major tool for advancing understanding are known to have tested the efficacy of thiazolidinediones of the role of lipids in causing vascular disease associated in smokers to determine effects on lipid abnormalities or with cigarette smoking. sensitivity to insulin. However, recent research indicates that rosiglitazone (a thiazolidinedione) increases CHD risk, although pioglitazone, another drug in the same Effects of Smoking Cessation class, does not increase this risk (Lincoff et al. 2007; Nis- sen and Wolski 2007). Thus, it is unclear whether the Research on smoking cessation largely confirmed possible benefits of this class of drugs in smokers will the associations observed in smokers (Gordon et al. 1975; be pursued.

390 Chapter 6 How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease

Summary of FFAs, and reesterification of FFAs by the liver.- How ever, the predicted effect from changes in standard lipid Effects of cigarette smoking on standard mea- risk factors for vascular disease associated with cigarette sures of blood lipids and lipoproteins are well character- smoking appears to be modest. Future research will reveal ized. The most important effects are to lower levels of whether this estimation of a modest effect is a true esti- HDLc and increase levels of total triglycerides. The meta- mate of the pathogenic importance of smoking-induced bolic mechanisms underlying these changes are known changes in blood lipid levels or an inability to measure the to some extent, particularly the catecholamine-mediated full effects of cigarette smoking on atherogenesis. increase in adipocyte lipolysis, changes in plasma levels

Cardiovascular Biomarkers

Biomarkers of smoking-related CVD risk are useful the plasma and urine (Nowak et al. 1987). Other markers for stratifying individual risk and, perhaps, for assessing of oxidative stress in smokers included higher plasma lev- product risk. Biomarkers for CVD risk can be divided into els of oxidized LDL and oxidized fibrinogen, higher urine three categories: (1) constituents of cigarette smoke that levels of substances reactive with thiobarbituric acid, and contribute to CVD, (2) physiological changes involving reduced plasma levels of antioxidant vitamins such as E, potential mechanisms of CVD, and (3) chemical biomark- C, and beta-carotene. ers of cardiovascular dysfunction and disease (Table 6.3). The hemodynamic effects of cigarette smoking can Studies showed that cigarette smoking altered many of the be observed while a person smokes a cigarette. These CVD biomarkers, as evidenced by comparisons of smokers effects include elevation in heart rate, blood pressure, with nonsmokers and former smokers. However, fewer and cardiac output. Coronary blood flow, as assessed by studies prospectively examined reversal of such changes coronary perfusion studies, may increase or decrease with after smoking cessation. More important, to date, there smoking, depending on underlying atherosclerosis and are no data on how changes in smoking-related biomark- endothelial function (Czernin and Waldherr 2003). ers predict risk of disease. Researchers have proposed numerous biomarkers Three constituents of cigarette smoke received the for measuring endothelial dysfunction, and many of these greatest attention as potential contributors to CVD: CO biomarkers are affected by cigarette smoking. The func- measured as exhaled CO or as blood carboxyhemoglobin, tional assessment most widely used is flow-mediated arte- nicotine, and oxidant chemicals (Benowitz 2003). These rial vasodilation (Puranik and Celermajer 2003), a test that constituents are used as general biomarkers of exposure measures the diameter of the brachial artery in response to tobacco or tobacco smoke. Apparently, no direct mea- to changes in forearm blood flow. The brachial artery is sures of levels of oxidizing chemicals in the body have imaged by using Doppler ultrasonography before and been developed, but numerous measures of the biologic after release of a blood pressure cuff that is inflated over consequences of exposure to oxidizing chemicals exist. the forearm to occlude arterial blood flow. With release of Exposure to particulate matter in cigarette smoke is like- the cuff, the increase in blood flow triggers an increase ly to contribute to CVD in smokers (Brook et al. 2004; in the diameter of the brachial artery that is mediated by Vermylen et al. 2005; Bhatnagar 2006), but no direct bio- release of NO and prostacyclin by endothelial cells. Many markers of particulate exposure are available. Particulate researchers demonstrated impairment of flow-mediated matter appears to affect oxidative stress, coagulability, and dilation in populations of active smokers and persons inflammation, for which biomarkers are available. A lesser exposed involuntarily to cigarette smoke, but estimates of body of research suggests that PAHs and other constitu- impairment in persons with no exposure to smoke over- ents of tobacco smoke may also contribute to atherogen- lapped considerably with those for the other two groups. esis (Penn and Snyder 1988, 1996). Urine levels of PAH Other potential markers of endothelial dysfunction that metabolites can also be measured in smokers; 1-hydroxy- can be measured in the blood include ADMA, von Will- pyrene is most widely used for this purpose. ebrand factor, tPA, E-selectin, and P-selectin. Prostacyclin Cigarette smoke exposes the smoker to high levels metabolites can be measured in the urine (Cooke 2000). of potentially oxidizing chemicals (Burke and FitzGerald Selectins are adhesion molecules released by both endo- 2003). In one study, cigarette smoking increased levels of thelial cells and platelets (Ley 2003). lipid peroxidation products, such as F2-isoprostanes, in

Cardiovascular Diseases 391 Surgeon General’s Report

Table 6.3 Biomarkers of risk for cardiovascular disease from exposure to cigarette smoke

Study

Smokers vs. Change with Dose-response Change with Biomarker Measurement of: nonsmokers smoking cessation relationship reduced smoking

Chemical biomarkers

Carbon monoxide Delivery of potential SRNT SRNT Benowitz and Hecht et al. 2004 chemical toxins Subcommittee Subcommittee Jacob 1984 on Biochemical on Biochemical Verification 2002 Verification 2002

Nicotine and Delivery of potential SRNT SRNT Benowitz and Benowitz et al. cotinine chemical toxins Subcommittee Subcommittee Jacob 1984 1983 on Biochemical on Biochemical Verification 2002 Verification 2002

Physiological and biochemical markers

Blood pressure Hemodynamic Benowitz et al. Benowitz et al. 2002 effects 2002

C-reactive protein Inflammation Bazzano et al. Bazzano et al. 2003 Bazzano et al. 2003 2003

Carotid and femoral Atherosclerosis Wallenfeldt et al. artery intima-media 2001 thickness

Circulating Endothelial function Kondo et al. 2004 Kondo et al. 2004 Kondo et al. endothelial 2004 precursor cells

E-selectin Endothelial function Bazzano et al. Bazzano et al. 2003 2003

Fibrinogen Hypercoagulable Bazzano et al. Bazzano et al. 2003 Bazzano et al. state 2003 2003

Flow-mediated Endothelial function Czernin and Czernin and Czernin and dilation Waldherr 2003 Waldherr 2003 Waldherr 2003

Glucose-clamping Insulin resistance Eliasson et al. Eliasson et al. 1997a Eliasson et al. studies 1997a 1997a

HDL cholesterol Lipid marker Stubbe et al. Stubbe et al. 1982 1982

Heart rate Hemodynamic Benowitz et al. Benowitz et al. 1984 effects 1984

Hemoglobin A1c Insulin resistance Sargeant et al. 2001

Homocysteine Hypercoagulable Bazzano et al. Bazzano et al. 2003 Bazzano et al. state 2003 2003

392 Chapter 6 How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease

Table 6.3 Continued

Study

Smokers vs. Change with Dose-response Change with Biomarker Measurement of: nonsmokers smoking cessation relationship reduced smoking

Insulin/glucose Insulin resistance Zavaroni et al. ratio 1994

Interleukin-6 Inflammation Bermudez et al. Bermudez et al. 2002 2002

Nuclear coronary Hemodynamic Czernin and Mahmarian et al. perfusion studies effects Waldherr 2003 1997

Oxidized LDL Oxidative stress/lipid Panagiotakos et Panagiotakos et al. cholesterol marker al. 2004 2004

P-selectin Endothelial function Bazzano et al. Bazzano et al. 2003 2003

Red blood cell mass Hypercoagulable Blann et al. 1997 Blann et al. 1997 state

Serum Oxidative stress Lykkesfeldt et al. concentrations of 2000 vitamin C

Serum triglycerides Lipid marker Axelsen et al. 1995

Soluble Inflammation Scott et al. 2000 Scott et al. 2000 Scott et al. intercellular 2000 adhesion molecule-1

Thiobarbituric acid Oxidative stress reactive substances

Tissue plasminogen Hypercoagulable Simpson et al. activator state 1997

Urine F2- Oxidative stress Morrow et al. Pilz et al. 2000 isoprostanes 1995

Urine thromboxane Hypercoagulable Nowak et al. 1987 Saareks et al. 2001 A2 metabolite state von Willebrand Endothelial function Blann et al. 1997 Blann et al. 1997 factor

White blood cell Inflammation Jensen et al. 1998 Jensen et al. 1998 Jensen et al. count 1998 Note: HDL = high-density lipoprotein; LDL = low-density lipoprotein; SRNT = Society for Research on Nicotine and Tobacco.

Cardiovascular Diseases 393 Surgeon General’s Report

Markers of the hypercoagulable state include carotid and femoral arteries by ultrasonography, which increased urine levels of thromboxane A2 metabolites. provides a direct measure of early atherosclerotic changes Thromboxane A2 is released when platelets aggregate in in blood vessels (de Groot et al. 2004). vivo, and its metabolites in urine are a useful noninvasive Numerous cardiovascular biomarkers that might be measure of the point of activation (Nowak et al. 1987). used to assess the effects of cigarette smoking and invol- Other relevant biomarkers of a hypercoagulable state untary smoking and that are expected to increase the risk include fibrinogen, red blood cell mass, blood viscosity, of CVD are discussed here. Many biomarkers, however, tPA, PAI-1, homocysteine, and P-selectin (Benowitz 2003). do not reflect causal pathways related to development of Biomarkers used to assess an inflammatory state CVD. Instead, they reflect the pathophysiological effects include total leukocyte and neutrophil counts and levels of the constituents of cigarette smoke. In addition, many of CRP, fibrinogen, and interleukin-6 (Pearson et al. 2003). biomarkers are influenced by processes and risk factors In addition, the counts of several cell-surface adhesion that are independent of cigarette smoking. Many of the molecules increased in inflammatory states. These mol- same abnormalities produced by smoking are also pro- ecules included ICAM, sVCAM-1, and monocyte chemoat- duced by diabetes, hypercholesterolemia, and hyper- tractant protein-1. tension. Thus, it is unclear which biomarkers are most Another study found several markers to be useful for specific to cigarette smoking. It is also unclear which bio- assessing insulin resistance (Eliasson 2003). For example, markers best predict the risk of CVD attributable to ciga- in persons with insulin resistance, levels of plasma glu- rette smoke. Also, a given biomarker profile can indicate cose were likely to be elevated in fasting status and two any of several marked differences in a person’s susceptibil- hours after eating. HbA1c levels, which reflect plasma glu- ity to CVD. cose levels throughout the day, were elevated in persons The potential exists to develop improved biomarkers in a hyperglycemic state. The ratio of insulin to glucose for CVD by using advances in high-throughput genom- after glucose loading was useful as an index of insulin ics and by examining the relationships of gene polymor- sensitivity. The most definitive investigations were glu- phisms or alterations in protein expression or activity to cose-clamping studies, in which insulin levels were mea- smoking-induced disease (Zhang et al. 2001). Emerging sured when the glucose level was constant or vice versa. genomic and proteomic technologies may cast light on Numerous standard markers of lipids may be altered the signaling pathways activated by smoking and the con- in cigarette smokers. These markers include HDLc, LDLc, stituents of tobacco smoke that culminate in cardiovascu- the ratio of total cholesterol to HDL, and serum triglycer- lar dysfunction. Such approaches may also contribute to ide levels. an understanding of individual differences in susceptibil- Nuclear coronary perfusion studies with or without ity to the cardiovascular complications of smoking. physical exercise are among several functional studies for Numerous studies of clinical genetics examined dif- diagnosing cardiovascular dysfunction or disease. They ferences in susceptibility to smoking-induced CVD as a indicate that cigarette smoking reduces cardiac perfusion function of different genetic variants (Wang et al. 2003). in patients with coronary disease (Czernin and Waldherr Such studies, combined with genomic and proteomic 2003). Reserve in endothelial function can be assessed approaches, may provide mechanistic information on by studying flow-mediated dilation (see “Endothelium- pathogenesis and, in combination with other biomark- Dependent Vasodilation” earlier in this chapter). Find- ers, may result in better predictions of cardiovascular risk ings in another study indicate that vascular disease can in smokers. be assessed by measuring intima-media thickness of the

Smoking Cessation and Cardiovascular Disease

Smoking cessation reduces the risk of cardiovascular does not cause hypertension, smoking is associated with morbidity and mortality for smokers with or without CVD higher blood pressure in persons with hypertension and (USDHHS 1990). Smoking directly accelerates atherogen- enhances the likelihood of complications, including pro- esis, causes acute cardiovascular events, and contributes gression of renal disease in patients with hypertension to and acts synergistically with other risk factors, such (Green et al. 1986; Mann et al. 1991; McNagny et al. 1997; as hyperlipidemia and diabetes (see “Smoking and Diabe- Regalado et al. 2000). One study demonstrated that ciga- tes” earlier in this chapter). Although cigarette smoking rette smoking was a substantial contributor to morbidity

394 Chapter 6 How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease and mortality in patients with left ventricular dysfunction contribute to tobacco use. Evidence from randomized (Suskin et al. 2001). In such patients, the benefit of reduc- controlled clinical trials of cessation methods has been ing the likelihood of death by smoking cessation is equal summarized in meta-analyses conducted independently to or greater than the benefit of therapy with inhibitors of by two groups—the U.S. Public Health Service (PHS) ACE, β-blockers, or spironolactone. Smoking cessation is and the Cochrane Database of Systematic Reviews (the particularly important in patients with diabetes. For these Cochrane Library). These reviews document the efficacy of patients, smoking markedly increases cardiovascular risks, both psychosocial counseling and pharmacologic agents including the risk that diabetic nephropathy will progress. for cessation (Fiore et al. 2000, 2008). Combination of Smoking also increases insulin resistance and increases the two methods is the most effective strategy. Interven- the difficulty of controlling diabetes. For these and other tions in psychosocial counseling range from brief coun- reasons, smoking cessation in patients with CVD is an seling by the physician to intensive, cognitive-behavioral essential therapeutic intervention. counseling interventions during several weeks. There is a The 1990 Surgeon General’s report on smoking ces- dose-response relationship between behavioral treatment sation (USDHHS 1990) outlines the evidence that stop- and smoking cessation; that is, the efficacy of counsel- ping smoking helps to prevent CVD, and subsequent ing interventions increases with increased intensity and research has reinforced this concept (Hasdai et al. 1997a; duration of the program (Fiore et al. 2000, 2008; USDHHS van Domburg et al. 2000; Wilson et al. 2000). Estimates 2000). The U.S. Food and Drug Administration (FDA) in case-control and cohort studies indicate that most risk has approved pharmacotherapy for tobacco dependence. reduction for mortality occurred in the first one to three The pharmacotherapy includes five types of NRT (gum, years after smoking cessation, and approximately one- transdermal patch, nasal spray, vapor inhaler, and loz- half of the risk of smokers for a nonfatal MI was elimi- enge), sustained-release bupropion, and varenicline. PHS nated in the first year after cessation. It takes about three designated these medications as first-line therapies for to five years of abstinence from smoking for most of the smoking cessation in Treating Tobacco Use and Depen- excess CVD risk to be gone (USDHHS 1990; Lightwood dence: Clinical Practice Guidelines (Fiore et al. 2000). Two and Glantz 1997). other drugs—nortriptyline and clonidine—were effica- Smoking cessation after MI reduces the risk of car- cious in randomized controlled trials and were shown to be diovascular morbidity and mortality by 36 to 50 percent effective in the Cochrane review and in meta-analyses con- (USDHHS 1990; Kumanan et al. 2000; Wilson et al. 2000; ducted for development of the PHS guidelines (Fiore et Rea et al. 2002; Critchley and Capewell 2003). Smoking al. 2000, 2008; Gourlay et al. 2004; Hughes et al. 2004b). cessation is highly cost-effective (Krumholz et al. 1993; These drugs have not been approved by FDA for use in Lightwood 2003) and is recommended in professional smoking cessation, and in the PHS report, they are desig- guidelines for prevention of recurrent cardiovascular nated as second-line interventions. There is no evidence to events in persons with known CVD (Smith et al. 2001). support use of alternative therapies such as acupuncture Evidence supports the central roles of smoking cessation and hypnosis for smoking cessation (Abbot et al. 1998; and eliminating exposure to secondhand smoke in pre- Fiore et al. 2000, 2008; White et al. 2002). venting development and progression of CVD (USDHHS 1990, 2006; Benowitz and Gourlay 1997; Hasdai et al. 1997a; van Domburg et al. 2000; Wilson et al. 2000; Gold- Interventions enberg et al. 2003). Multiple randomized controlled clinical trials demonstrated the benefits of counseling patients with CVD Methods on smoking cessation (Table 6.4) (Thomson and Rigotti 2003). In contrast, relatively few clinical trials tested the Tobacco use and dependence are determined by safety or efficacy of pharmacotherapy for treating smokers complex physiological and psychological factors. Use of with CVD (Table 6.5). Researchers raised concerns about nicotine causes tolerance, physical dependence, and a the safety of NRT and sustained-release bupropion in withdrawal syndrome when smoking is stopped (USDHHS patients with CVD, because both agents can have sym- 1988). Use of tobacco is a learned behavior that becomes pathomimetic activity and can theoretically increase part of the daily routine of a smoker and is often used to myocardial work, and NRT might also reduce the myocar- cope with stress, anxiety, anger, and depression (USDHHS dial oxygen supply through coronary vasoconstriction by 1988; Rigotti 2002). aggravating endothelial dysfunction (Benowitz and Gour- Interventions to achieve smoking cessation tar- lay 1997). get both the physiological and psychological factors that

Cardiovascular Diseases 395 Surgeon General’s Report

Table 6.4 Randomized controlled trials of counseling for smokers hospitalized with cardiovascular disease

In-hospital Smoking RR or OR counselor, duration Postdischarge cessation rates (95% CI) Study Population of counseling counseling (%) p value

Taylor et al. Acute MI Nurse TC: 1, 2, 3 weeks, 12 months 1.9 (1.3, 2.8) 1990 I vs. C: 86 vs. 87 Average duration then every I vs. C: 61 vs. 32 p <0.001 5 hospitals 3.5 hours for baseline month x 4 San Francisco Bay, and postdischarge CV for relapse after California counseling smoking cessation

Ockene et al. Coronary angiogram Health educator TC: 1, 3 weeks; 12 months 1.5 (0.9, 2.4) 1992 I vs. C: 135 vs. 132 30 minutes 3 months for relapse SR: 57 vs. 48 p = 0.06 3 hospitals after smoking I vs. C: 35 vs. 28 1.4 (0.8, 2.4) Massachusetts cessation; 2 and 4 3-vessel CAD p = 0.19 months for relapse I vs. C: 65 vs. 41 13.4 (3.1, 58.0) after smoking cessation

DeBusk et al. Acute MI Nurse TC: 2 days, 1 week, 12 months 1994 I vs. C: 131 vs. 121 Duration not stated monthly x 6 I vs. C: 70 vs. 53 p = 0.03 5 hospitals San Francisco Bay, California

Rigotti et al. Coronary artery Nurse TC: once per 12 months 1994 bypass graft 60 minutes week x 3 I vs. C: 61 vs. 54 p >0.52 I vs. C: 44 vs. 43 Massachusetts General Hospital, Boston

Dornelas et Acute MI Psychologist TC: <1, 4, 8, 12, 16, 6 months al. 2000 I vs. C: 54 vs. 46 Duration not stated 20, and 26 weeks SR: 67 vs. 43 p <0.05 Hartford Hospital 12 months Hartford, SR: 55 vs. 34 p = 0.04 Connecticut FV: 66 vs. 37 p <0.05

Hajek et al. Acute MI or cardiac Nurse None 12 months 2002 bypass surgery 20–30 minutes I vs. C: 37 vs. 41 p = 0.40 I vs. C: 267 vs. 273 17 hospitals England

Quist- Acute MI, unstable Cardiac nurses TC: 2 days, 1 week, 3 12 months Absolute risk Paulsen and angina, or cardiac (no training) weeks, 3 months, 5 I vs. C: 50 vs. 37 reduction: 35% Gallefoss bypass surgery Duration not stated months (0%, 26%) 2003 I vs. C: 100 vs. 118 CV: 6 weeks 1 hospital Norway Note: CAD = coronary artery disease; CI = confidence interval;CV = clinic visit; FV = validated by family; I vs. C = intervention versus control; MI = myocardial infarction; OR = odds ratio; RR = relative risk; SR = self-reported; TC = telephone call.

396 Chapter 6 How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease

Table 6.5 Randomized controlled trials of pharmacologic interventions for smoking cessation in patients with cardiovascular disease

Smoking RR or OR Counseling cessation (95% CI), Adverse events Study Population Treatment (I & C) ratesa (%) p value (I vs. C)

Transdermal nicotine

Working Group Stable outpatients 14 mg could be Group 5 weeks No differences for the Study with CVD increased to counseling I vs. C: 36 p <0.05 in episodes of of Transdermal I vs. C: 77 vs. 79 21 mg after weekly x 5 vs. 22 angina or change Nicotine in General medical 1 week in blood pressure, Patients with and cardiology 5 weeks heart rate, or Coronary Artery clinics EKG readings Disease 1994 Media advertising 4 centers United States

Joseph et al. Stable outpatient 21 mg x 6 weeks Behavioral 14 weeks No difference 1996 veterans with CVD 14 mg x 2 weeks counseling I vs. C: 21 p = 0.001 in primary Joseph and I vs. C: 288 vs. 287 7 mg x 2 weeks 15 minutes at vs. 9 endpoints Antonnucio 1999 10 Veterans Affairs study entry 6 months (death, MI, medical centers 10 minutes at I vs. C: 14 p = 0.67 cardiac arrest, United States 1 and 6 weeks vs. 11 or admission 12 months to hospital) I vs. C: 10 p = 0.41 or secondary vs. 12 endpoints (other CVD)

Tzivoni et al. Stable outpatients 21 mg (>20 Smoking 2 weeks No differences 1998 with CAD in cigarettes/day) cessation I vs. C: 73 p <0.05b in EKG changes, smoking cessation or 14 mg (<20 program vs. 52 exercise testing, program cigarettes/day) Group meetings or heart rate and I vs. C: 52 vs. 54 2 weeks weekly blood pressure 2 centers Israel

Bupropion-sustained release

Tonstad et al. Stable outpatients 150 mg BID TC: 1 day 12 months Cardiovascular 2003 with CVD 7 weeks before smoking I vs. C: 27 p <0.001 events I vs. C: 313 vs. 313 Target date cessation vs. 12 I vs. C: 24 vs. 14 28 centers in for smoking 3 days afterward Continuous (p = NS) 10 European cessation 7–14 Monthly x 12 abstinence countries days after CV: brief weeks 4–52 starting drug counseling at 3, I vs. C: 22 p <0.001 6, and 12 months vs. 9

Cardiovascular Diseases 397 Surgeon General’s Report

Table 6.5 Continued

Smoking RR or OR Counseling cessation (95% CI), Adverse events Study Population Treatment (I & C) ratesa (%) p value (I vs. C)

Rigotti et al. Acute MI, unstable 150 mg BID Nurse 12 weeks 1.61 (0.74, No difference in 2006 angina, or other 12 weeks counseling for I vs. C: 37 2.76) CVD mortality CVD hospital Started during 40 minutes in vs. 27 p = 0.08 (0% vs. 2%) or admission hospitalization hospital 12 months 1.23 (0.68, in CVD events at I vs. C: 124 vs. 124 TC: 48 hours, I vs. C: 25 2.23) 12 weeks (16% 5 academic medical 1, 3, 8, and 12 vs. 21 p = 0.49 vs. 14%, IRR 1.22 centers weeks after [0.61–2.48]) or New England discharge 1 year (26% vs. 18%, IRR 1.56 [0.88, 2.82]) Note: BID = twice a day; CAD = coronary artery disease; CI = confidence interval;CV = clinic visit; CVD = cardiovascular disease; EKG = electrocardiogram; I vs. C = intervention versus control; IRR = incidence rate ratio; mg = milligrams; MI = myocardial infarction; NS = not significant;OR = odds ratio; RR = relative risk; TC = telephone call. aAbstinence measured by point prevalence (no smoking in previous seven days) unless otherwise noted. bp value calculated with c2 test.

Counseling months reduced smoking rates at one year (50 versus 37 percent, absolute risk reduction, 13 percentage points [95 Several randomized controlled clinical trials demon- percent CI, 0 to 26 percentage points]) (Quist-Paulsen and strated the efficacy of counseling for patients hospitalized Gallefoss 2003). with CVD (Table 6.4). The evidence for efficacy is strongest Other studies that lacked the same intensity of fol- for patients who had acute MI (Pozen et al. 1977; Taylor et low-up after hospital discharge produced less impressive al. 1990; Ockene et al. 1992; DeBusk et al. 1994; Dornelas results. One study examined a multicomponent behav- et al. 2000). In one study, an intensive, nurse-managed ioral smoking intervention delivered to 267 patients hav- intervention to achieve smoking cessation for 173 smoking coronary angiography (Ockene et al. 1992). Compared ers hospitalized with an acute MI doubled the cessation with patients who did not receive the intervention, those rates at one year (61 versus 32 percent, p <0.001) (Taylor et with angiography had higher rates of validated smoking al. 1990). At the bedside, the nurse delivered a 30-minute abstinence at 6 months (45 versus 34 percent) and 12 cognitive-behavioral counseling session focused on self- months (35 versus 28 percent), but these differences were efficacy and prevention of relapse. Additional counseling not statistically significant. Two randomized trials (Rigotti was delivered by telephone at one, two, and three weeks et al. 1994; Hajek et al. 2002) and one partially randomized after discharge and then every month for four months. trial (Bolman et al. 2002) examined the effects of inpatient A second study of the same patients expanded the nurse- counseling with minimal follow-up in cardiac patients. delivered counseling model to target multiple cardiac risk These studies showed no improvement in abstinence rates factors (DeBusk et al. 1994). This study also reported rates with the counseling intervention versus usual care. of smoking abstinence higher than those in the first study The most successful counseling interventions (Taylor et al. 1990). A third trial assigned 100 consecu- for cardiac inpatients include high-intensity baseline tive smokers admitted with MI to either minimal care or counseling with sustained contacts after discharge for bedside counseling with seven follow-up telephone calls prevention of relapse. However, even with the most suc- (Dornelas et al. 2000). Smoking cessation rates at one year cessful counseling interventions, at least 40 percent of were higher in the intervention group than in the mini- smokers who have cardiac disease resume smoking within mal care group (55 versus 34 percent, p <0.05). A more re- one year. Guidelines for smoking cessation recommend cent study of 240 smokers admitted to the hospital for MI, addition of pharmacotherapy to counseling (Fiore et unstable angina, or cardiac bypass surgery demonstrated al. 2000, 2008). Pharmacotherapy has the potential to that counseling by cardiac nurses untrained in counsel- improve smoking cessation rates in smokers with CVD ing and then follow-up counseling during the next five

398 Chapter 6 How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease

(see “Nicotine Replacement Therapy” and “Bupropion” with stable CVD. The first study enrolled 156 patients later in this chapter). with CHD and randomly assigned them to receive either 14-mg nicotine patches or a placebo for five weeks (Work- Nicotine Replacement Therapy ing Group for the Study of Transdermal Nicotine in Patients with Coronary Artery Disease 1994). The dose NRT helps smokers stop smoking and also reduces was increased to 21 mg if smoking persisted. Smoking nicotine withdrawal symptoms, which begin a few hours abstinence was achieved at five weeks by 36 percent in the after the last cigarette is smoked and can last up to four patch group and 22 percent in the placebo group (p <0.05). weeks (Hughes et al. 1992). The typical withdrawal syn- Patients recorded all episodes of angina, palpitations, and drome is characterized by agitation, anxiety, depressed other cardiac symptoms in daily diaries and had a 12-lead mood, difficulty concentrating, increased appetite, insom- electrocardiogram at three time points. The nicotine nia, irritability, restlessness, and an intense craving to patch did not affect the frequency of angina, arrhythmias, smoke. Most smokers who stop smoking relapse to smok- or depression of the ST segment (isoelectric period) on ing within the first week, when withdrawal symptoms electrocardiograms during the five weeks of treatment, are strongest. even in patients who smoked intermittently. In multiple clinical trials, all the NRT products A second randomized trial of transdermal nicotine approximately doubled rates of smoking abstinence com- included 584 outpatients with CVD from 10 Veterans pared with rates for participants receiving a placebo (Fiore Affairs hospitals (Joseph et al. 1996). The participants were et al. 2000; Silagy et al. 2004). Meta-analyses conducted randomly assigned to receive 21-mg patches or a placebo for PHS demonstrated ORs of 1.9 (95 percent CI, 1.7–2.2) for 10 weeks. Primary cardiovascular endpoints during 14 for the nicotine patch and 1.5 (95 percent CI, 1.3–1.8) for weeks of follow-up included MI, cardiac arrest, death, and nicotine gum. Meta-analyses from the Cochrane Library hospital admission for angina, dysrhythmia, or congestive found similar results; ORs were 1.74 (95 percent CI, heart failure. The two groups did not differ in the propor- 1.57–1.93) for the patch and 1.66 (95 percent CI, 1.52– tion of patients who reached at least one cardiovascular 1.81) for the gum (Silagy et al. 2004). For smokers with endpoint (5.4 versus 7.9 percent, p = 0.23). Concomitant greater dependence on nicotine, the 4-mg dose of nicotine use of the nicotine patch and smoking was not associated gum was more effective than the 2-mg dose. Maximum with an increase in adverse events. Although use of the effectiveness depended on correct chewing techniques. patch was safe in this population, no improvement was Similar ORs were reported after meta-analyses of data on observed in rates of short-term or long-term smoking the nicotine inhaler and nicotine nasal spray (Fiore et al. abstinence in comparisons with the placebo group (Joseph 2000). Only one study compared the efficacy of four forms et al. 1996; Joseph and Antonuccio 1999). of NRT (patch, gum, inhaler, and nasal spray); they dem- A third trial tested use of the nicotine patch in 106 onstrated similar efficacy rates after 12 weeks of follow-up smokers with CHD (Tzivoni et al. 1998). Patients were (Hajek et al. 1999). randomly assigned to receive nicotine patches or placebo Nicotine directly affects the cardiovascular system by patches for two weeks. All patients had ambulatory elec- multiple mechanisms (see “Cigarette Smoke Constituents trocardiogram monitoring and exercise testing at study and Cardiovascular Disease” earlier in this chapter). The entry, after the first application of the patch, and at two various effects lead to increased heart rate, blood pressure, weeks. No difference was observed at any of the three time and myocardial contractility, and reduced coronary blood points, between the patch and placebo groups, in resting flow. Nicotine may also contribute to insulin resistance heart rate, blood pressure, the number or duration of isch- and development of a more atherogenic lipid profile. The emic episodes, frequency of arrhythmias, exercise dura- nicotine dose in NRT products is usually lower than the tion, or time to 1-mm depression of the ST segment on an dose from smoking, but there have been concerns about electrocardiogram. In a randomized study of 234 patients the safety of NRT in patients with CVD. Case reports in with both cardiovascular and respiratory diseases, no the medical literature described atrial fibrillation, MI, and increase in adverse events was observed in patients stroke in patients receiving NRT (Joseph and Fu 2003). It assigned to the nicotine patch or the placebo (Campbell is difficult to assess the cardiovascular risk from NRT on et al. 1996). the basis of these reports, because of the inability to con- In a case-control study, Kimmel and colleagues trol for individual risk factors for these events, especially (2001) found no increased risk of a first MI with use of the that these persons were smokers (Benowitz and Gourlay nicotine patch in persons who stopped smoking or those 1997; Joseph and Fu 2003). who continued to smoke. Using a computerized database To date, three randomized controlled trials of trans- for general practice in the United Kingdom, Hubbard and dermal use of nicotine have been conducted in patients

Cardiovascular Diseases 399 Surgeon General’s Report colleagues (2005) studied the relative incidence of MI and to smoke but at reduced levels. The researchers concluded stroke in four two-week periods before and after the first that use of the patch, even with concomitant smoking and prescription for NRT. They found a progressive increase in higher plasma levels of nicotine, resulted in reduction of risk in the 56 days after the first NRT prescription but no exercise-induced ischemia in a comparison with baseline evidence of increased cardiovascular events or mortality values. This finding suggested that components of tobacco in the 56 days after the NRT prescription. smoke other than nicotine are responsible for impaired Meine and colleagues (2005) addressed the safety coronary blood flow. The second study investigated the of transdermal nicotine in the setting of acute CHD. effect of nicotine gum on coronary perfusion in former These investigators analyzed data in the Duke Univer- cigarette smokers having angiography (Nitenberg and sity Cardiovascular Databank for 9,991 smokers who Antony 1999). The findings demonstrated that the gum had cardiac catheterization after hospital admission for did not reduce the surface area of normal or diseased seg- unstable angina or non-ST-segment MI. This retrospective ments of the coronary artery. Other studies of the effects of observational study compared outcomes of patients who smoking cessation on lipids and thrombosis reported did or did not receive transdermal nicotine during hospi- improvements in these markers, even among smokers talization. The study identified 194 patients who had been using NRT (Allen et al. 1994; Lúdvíksdóttir et al. 1999; treated with transdermal nicotine during the hospital Eliasson et al. 2001; Haustein et al. 2002). stay. The investigators used a “propensity score analysis” In summary, despite anecdotal reports of cardiovas- to compare patients receiving transdermal nicotine with cular events attributable to use of NRT, data from multiple matched patients from the database who did not receive clinical trials of smokers with or without CVD show no the medication. Because patients were not randomly evidence for increased cardiovascular risk when NRT assigned to receive transdermal nicotine, selection bias is used to treat tobacco dependence. However, the safe- could have confounded these results. In an attempt to ty of NRT has not been tested in a more acute setting, reduce this bias, patients who did or did not receive trans- such as during hospitalization for a cardiovascular event. dermal nicotine were matched on demographic charac- Observational data suggest that use of the nicotine patch teristics, diagnosis, cardiac risk factors, and mean cardiac in patients with unstable cardiac disease is probably ejection fraction. Rates of cardiac outcomes in the two safe (Meine et al. 2005), but randomized trials are need- groups were compared. No differences in 7-day, 30-day, or ed to confirm these findings. Current PHS guidelines one-year mortality rates were observed. Patients receiving recommend that NRT be used with caution in smokers transdermal nicotine were not more likely to have coro- with unstable angina, MI in the past two weeks, or serious nary artery bypass grafting or percutaneous transluminal arrhythmia (Fiore et al. 2000). coronary angioplasty during hospitalization. It was not possible to control for the dose of medication received, Bupropion the amount of smoking before hospital admission, or Bupropion is an aminoketone approved by FDA in relapse to smoking after hospitalization. These results 1989 for treatment of depression and in 1997 for smoking provide some evidence that NRT was safe in the setting of cessation. The drug is included in national guidelines as acute CVD, but randomized controlled trials are needed to first-line therapy for smoking cessation (Fiore et al. 2000, establish the safety of NRT in patients who have unstable 2008). Its mechanism of action is not fully understood, but cardiac disease. researchers think it acts by inhibiting neuronal uptake of Other studies examined markers of exposure to norepinephrine and dopamine. Bupropion may also block tobacco smoke related to cardiovascular risk as surro- activity of nAChRs. The mechanism of action for smoking gate endpoints for cardiovascular events in clinical trials. cessation appears to be unrelated to the antidepressant There was no evidence that NRT raised blood pressure in effects of bupropion. A preparation of the drug for sus- any of the efficacy trials, but these trials typically excluded tained release provides a better safety profile and more patients with poor control of hypertension. In one small convenient dosing than does the immediate-release form. trial of 30 smokers with or without hypertension, NRT Evidence from several randomized controlled tri- increased mean arterial pressure in smokers with normal als shows that bupropion doubled the smoking cessation blood pressure but not in smokers who had hypertension rates obtained with a placebo. Meta-analyses of data on (Tanus-Santos et al. 2001). bupropion for smoking cessation conducted by PHS and To date, two studies have tested the effect of NRT the Cochrane Library yielded ORs of 2.1 (95 percent CI, on coronary circulation in smokers with CHD. One study 1.5–3.0) and 2.06 (95 percent CI, 1.77–2.40), respectively examined the size of defects in myocardial perfusion in 36 (Fiore et al. 2000; Hughes et al. 2004b). One trial com- patients with baseline CHD who were treated with nicotine pared use of bupropion, a nicotine patch, bupropion plus patches (Mahmarian et al. 1997). Participants continued

400 Chapter 6 How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease a patch, and a placebo among 893 participants (Jorenby deaths) and in the placebo group (two deaths) was not et al. 1999). Smoking abstinence rates at one year were statistically significant. During the 12 weeks of drug treat- 15.6 percent in the placebo group, 16.4 percent in the ment, the difference between the number of cardiovascular nicotine-patch group, 30.3 percent in the bupropion events in the bupropion group (20 events) and the placebo group (p <0.0001 versus the placebo group), and 35.5 group (17 events) was also not significant. Cardiovascular percent in the bupropion-plus-patch group (p <0.0001 events included death, nonfatal MI, unstable angina, con- versus the placebo group; p = 0.06 versus the bupropion- gestive heart failure, stroke, and coronary revasculariza- alone group). tion procedure. At the one-year follow-up, the number of The major risk of bupropion is that it lowers a cardiovascular events in the bupropion group (32 events, person’s seizure threshold. The risk of seizure from the 26 percent) exceeded the number in the placebo group sustained-release formulation is 0.1 percent, which is no (22 events, 18 percent), but this difference was not signifi- different from that for other antidepressants (Hughes et cant. In addition, at one year, the difference between the al. 1999; Rigotti 2002). No seizures were reported in any of smoking abstinence rates for the bupropion group (25 the clinical trials that tested sustained-release bupropion percent) versus the placebo group (21 percent) was not for smoking cessation. significant. However, the results after 12 weeks of drug As with NRT, early case reports of serious cardio- treatment suggested that bupropion had short-term effi- vascular events with sustained-release bupropion raised cacy (37 versus 27 percent, p = 0.08). questions about the safety of this agent in patients with In summary, sustained-release bupropion is effective CVD. These reports, which were mostly in Canada and and safe for treating smokers with stable CVD. The drug England, included cardiac deaths, chest pain, MI, and appears to be less efficacious in smokers hospitalized with myocarditis (Joseph and Fu 2003). Assessment of the con- acute CVD than in other groups of patients. Bupropion is tribution of bupropion to these events is difficult because the only medication for treating tobacco dependence that evaluation of other cardiac risk factors in these patients was has been tested in patients with acute CVD, and it appears not possible. to be safe for those with either stable or acute disease. To date, none of the efficacy trials of bupropion for smoking cessation has reported a significant increase Other Pharmacotherapy in cardiovascular events. Two randomized controlled Varenicline, a partial agonist of the α4β2 nAChR, trials enrolled only smokers with CVD. The first trial has been marketed for the treatment of tobacco depen- enrolled 629 outpatients with stable CVD—that is, MI or an dence but its use in smokers with CVD has not yet been interventional cardiac procedure more than three months studied (Coe et al. 2005). The drug produces approximate- earlier and stable angina pectoris, PAD, or congestive ly 50 percent of the receptor stimulation provided by nico- heart failure (Tonstad et al. 2003). Patients were randomly tine, but it blocks the effects of any nicotine taken in from assigned to receive bupropion or a placebo for seven weeks. cigarette smoking. Clinical trials have found it superior This study found no differences in the number of deaths to bupropion in promoting smoking cessation, and pro- in the two groups—two in the bupropion group and two longed administration has been shown to reduce relapse in the placebo group. Overall, 38 participants (6 percent) in smokers who had been abstinent 12 weeks after initial reported a single adverse cardiovascular event—24 in the therapy (Gonzalez et al. 2006; Jorenby et al. 2006; Tonstad bupropion group and 14 in the placebo group. The most et al. 2006). Two other medications have been demonstrat- common cardiovascular events were angina pectoris, ed to be effective for smoking cessation: nortriptyline, a hypertension, and palpitations; 13 events occurred in the tricyclic antidepressant, and clonidine, a central α-agonist bupropion group versus 8 events in the placebo group. antihypertension agent. However, neither drug has been No statistical tests were performed on the rates of adverse approved by FDA for smoking cessation. Both agents have events. Patients who took bupropion were more likely to potential cardiovascular side effects, and the safety profile have stopped smoking at one year than were patients who of these drugs should be considered carefully before use in took the placebo (27 versus 12 percent, p <0.001). smokers with CVD. A second trial enrolled 248 smokers hospitalized Meta-analyses of data on use of clonidine for treat- with acute CVD, including acute MI, unstable angina, ing smokers resulted in ORs for smoking abstinence of or other cardiovascular conditions (Rigotti et al. 2006). 2.1 (95 percent CI, 1.4–3.2) (Fiore et al. 2000) and 1.89 Patients were randomly assigned to receive sustained- (95 percent CI, 1.30–2.74) (Gourlay et al. 2004). To date, release bupropion or a placebo for 12 weeks, and all no safety data for patients with CVD are available. How- patients received intensive counseling during hospital- ever, clonidine is known to cause orthostatic hypotension, ization and follow-up. At the one-year follow-up, the dif- rebound hypertension from abrupt cessation of the drug, ference between death rates in the bupropion group (no

Cardiovascular Diseases 401 Surgeon General’s Report and rarely, atrioventricular nodal blockade. Nortriptyline smoking cessation therapy has an important impact on is also effective in promoting smoking cessation; meta- CVD, and the cost per life saved is lower than that of many analyses yielded ORs of 3.2 (95 percent CI, 1.8–5.7) (Fiore other therapeutic interventions for CHD that are consid- et al. 2000) and 2.79 (95 percent CI, 1.70–4.59) (Hughes ered to be the standard (such as treatment of hypertension et al. 2004b). Nortriptyline was designated a second-line and hyperlipidemia) (Lightwood 2003). drug for smoking cessation in PHS clinical guidelines because of a smaller evidence base of support and greater side effects than those of other medications for smoking Summary cessation. In general, tricyclic antidepressants are avoided in patients with CVD because of concerns about increased Smoking cessation is a key element in both primary risks for arrhythmias and depression of myocardial con- and secondary prevention of CVD. Guidelines from PHS tractility (Joseph and Fu 2003). recommend counseling, NRT, sustained-release bupro- Although the focus of the preceding section (“Meth- pion, and varenicline as first-line treatments to achieve ods”) was on clinical interventions to reduce smoking, it smoking cessation. Studies show that NRT and bupropion is important to recognize that policy-based interventions, are effective in patients with CVD, although not all trials such as smoke-free environments and community and demonstrated efficacy. Several studies have demonstrated statewide tobacco control programs, are also important the safety of NRT in patients with stable CVD, but ran- elements in a strategy to improve cardiovascular health. domized trials are needed to establish the safety of this For example, smoke-free workplaces are a highly cost- treatment for patients hospitalized with acute disease. effective approach to promoting smoking cessation with Bupropion appears to be safe in patients with stable or an impact on cardiovascular health (Ong and Glantz 2004). unstable CVD, but it is less effective in patients with acute Decreases in admissions to hospitals have been observed disease. Varenicline is a partial agonist of the α4β2 nAChR after smoke-free laws have gone into effect (Dinno and that is effective in treating tobacco dependence, but it has Glantz 2007). The California Tobacco Control Program not yet been studied in smokers with CVD. The develop- substantially accelerated the decline in the heart disease ment of more effective pharmacotherapies to aid smok- death rate in the state (Fichtenberg and Glantz 2000). ing cessation that are safe in persons with CVD is a high It should be noted that although long-term smok- research priority. ing quit rates after various interventions in cardiovascular patients may appear to be low (most less than 30 percent),

Methods to Reduce Exposure

Evidence-based interventions for treating smok- These strategies are often termed “harm reduction” inter- ers include behavioral and pharmacological treatments, ventions, although data are limited as to whether harm is which significantly increase rates for long-term absti- really lessened with reduced exposure to tobacco. To date, nence from smoking. Even so, absolute rates of abstinence the effect of methods for reducing exposure on risk factors are modest; they range from 8 to 25 percent, depending for CVD and on development of CVD has been evaluated on the study population and the treatment. In addition, in a limited number of clinical trials, prospective cohort only a small proportion of smokers are interested in treat- studies, and epidemiologic studies. ment at any given time. Interest in smoking cessation and Endpoints with respect to CVD that have been mea- success in achieving long-term abstinence are greater sured in studies of reduced exposure include measures of among patients with CVD than in the general population exposure to tobacco constituents (e.g., nicotine and CO); of smokers (Thomson and Rigotti 2003). Nevertheless, biomarkers of inflammation (e.g., CRP, leukocyte counts, abstinence rates remain disappointingly low, particu- and fibrinogen); thrombosis (e.g., fibrinogen and PAI-1); larly in light of the important health benefits for this lipid abnormalities (e.g., levels of total cholesterol, HDLc, population when they do stop smoking (USDHHS 1990; LDLc, triglycerides, APOs A-I and B, and HDLc to LDLc Burns 2003). ratio); oxidative stress that reflects and may contribute Suboptimal treatment outcomes prompted inter- to cardiovascular risk (e.g., F2-isoprostanes); and clinical est in testing interventions that might decrease the risk outcomes (blood pressure, heart rate, angina, exercise tol- of smoking among those who continue to use tobacco. erance, MI, other adverse events, and death) (Table 6.3).

402 Chapter 6 How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease

Reduced Smoking the intervention, but 16 percent of those who continued to smoke daily smoked 1 to 24 percent fewer cigarettes Important methodologic considerations in evalu- than at baseline; another 27 percent reduced smoking by ating studies of reduced smoking include the extent and 25 to 49 percent; 19 percent, by 50 to 74 percent; and 11 duration of smoking reduction, use of nicotine replace- percent, by 75 to 99 percent. The mean reduction in ciga- ment products, doses, and timing of endpoint measure- rettes smoked was 29 percent (from 32 to 22 cigarettes ments (Table 6.6). In addition, some studies report per day), and the mean reduction in CO levels was 24 per- outcome data on the basis of intention to treat all par- cent (from 34 to 26 ppm). Thus, more than 80 percent of ticipants, regardless of whether treatment was success- those who did not stop smoking achieved some level of ful. Others report only on subgroups who achieve specific reduction. However, the reduction in CO was not as large goals for smoking reduction. as the reduction in cigarettes per day. This finding again Eliasson and colleagues (2001) tested the effect of suggests compensatory smoking. nicotine nasal spray on achieving smoking reduction and Hatsukami and colleagues (2005) examined the abstinence among 58 healthy adult smokers in an open- effect of smoking reduction on cardiovascular risk fac- label cohort study. The primary goal for the first eight weeks tors among 151 cigarette smokers interested in stopping of the study was to reduce daily smoking by 50 percent; smoking but not in reducing their smoking. Nicotine participants were asked to stop smoking after eight weeks. patches and gum were used to assist with reduction of Cardiovascular risk factors were evaluated at baseline, at smoking. The cardiovascular risk factors (CO and choles- eight weeks, and after eight weeks of abstinence. The 33 terol levels, leukocyte counts, blood pressure, and heart study participants provided data at all three time points. rate) were measured for 12 weeks after study entry. Bio- After participants completed eight weeks of smoking markers did not change among persons who continued to reduction, mean cigarette use decreased by 50.2 percent, smoke ad libitum, but the 61 persons who reduced smok- expired CO dropped 17 percent, and plasma thiocyanate ing achieved significant improvements. Smokers who decreased by 20.1 percent. Significant improvements reduced the self-reported number of cigarettes smoked per included fibrinogen levels (from 2.9 g/liter [L] to 2.65 day by 40 percent or more had significantly reduced leu- 9 g/L, p = 0.011); hemoglobin values (from 13.8 to 13.3 kocyte counts (from 7.39 to 6.98 x 10 /L, p <0.001); high- g/L, p <0.001); leukocyte counts (from 7.0 to 6.2 x 109/L, er HDLc levels (from 50.3 to 52.8 mg per deciliter [dL], p = 0.005); and HDLc to LDLc ratio (from 0.33 to 0.37, p <0.0167); improved HDL to LDL ratios (from 0.47 to p <0.005). Eight weeks of abstinence from smoking was 0.49, p <0.0167); lower APO B levels (from 103.7 to 103.0 associated with further improvements in hemoglobin lev- mg/dL, p <0.0167); lower systolic blood pressure (from els, leukocyte counts, and HDL and LDL levels, and sig- 123.0 to 120.3 mm Hg, p <0.0167); and lower heart rate nificant reduction in PAI-1 activity. These researchers did (from 75.7 to 70.2 beats per minute, p <0.001). Although not observe improvements in HDLc and LDLc levels with some of these changes were statistically significant, they reduction in smoking. are modest, and the clinical importance is undetermined. Hurt and colleagues (2000) conducted a small, open- Levels of triglycerides, total cholesterol, APO A-I, and label cohort study to test the effect of a nicotine inhaler diastolic blood pressure did not change, and LDLc levels on biomarkers of exposure to cigarette smoke among 23 increased from 122.1 to 124.4 mg/dL (p <0.0167). heavy smokers. Levels of blood thiocyanate, several urine To date, only one randomized controlled trial of an carcinogens, and expired CO were measured at study entry intervention for smoking reduction among persons with and at 4, 8, 12, and 24 weeks. Despite an average reduction known CVD has been conducted (Joseph et al. 2005). Treat- among participants from 40 or more to 10 cigarettes per ment included behavioral counseling and NRT with patch- day, expired CO decreased only from 30.4 to 26.0 parts per es and gum. The goal of this study of 152 participants was million (ppm), reflecting compensatory smoking or mis- at least a 50-percent reduction in cigarettes smoked per reporting of reduction in smoking. This change was not day; usual care was the standard for comparison. At study statistically significant. entry, participants smoked an average of 27.4 cigarettes In the U.S. Lung Health Study, 5,887 male and per day. At six months, the intervention group had reduced female smokers were randomly assigned to one of three cigarette use by 39 percent, versus a decline of 25 percent groups: intervention for smoking cessation, including in the usual care group, but this difference was not statis- nicotine gum, plus bronchodilator therapy; intervention tically significant. Biomarkers for carcinogenesis and CVD plus a placebo; or usual care (Hughes et al. 2004a). Among were measured, as were clinical outcomes. No significant 3,923 participants in the intervention at the one-year differences between the treatment groups were observed follow-up, 1,722 continued to smoke daily. Reduction in for levels of CO, fibrinogen, 2 F -isoprostanes, or CRP, or the number of cigarettes smoked was not an objective of for leukocyte counts. The groups did not differ in clinical

Cardiovascular Diseases 403 Surgeon General’s Report

Table 6.6 Smoking reduction and cardiovascular disease endpoints: biomarkers and clinical outcomes

Reduction from baseline

Study Design n Follow-up NRT CPD Nicotine CO (ppm)

Hurt et al. Open-label 23 24 weeks Inhaler ≥40 to 10** -a NS 2000 cohort

Eliasson et al. Open-label 33 9 weeks Nasal spray 21.5 to 10.8**; - - 2001 cohort 50.2%

Godtfredsen et Prospective 643c Mean 13.8 NA ≥50%d Significant 13.2 vs. 8.7*** al. 2003 cohort study years

Hughes et al. Reduction 1,722 1 year Gum 32 to 22; 29% - NS 2004a cohort in RCT

Hatsukami et Reduction 61 12 weeks Patch and 24.16 to 6.93*; -f 19.9 to al. 2005 cohort in RCT gum >40% 6.77***

Joseph et al.h RCT 152 6 months Patch and 27.7 to 16.8* NS NS 2005 gum

Note: APO A-I = apolipoprotein A-I; APO B = apolipoprotein B; BP = blood pressure; CCSC = Canadian Cardiovascular Society classification;CO (ppm) = carbon monoxide (parts per million); CPD = cigarettes/day; Fibr = fibrinogen concentrations; g/L = grams per liter; Hb = hemoglobin; HDL = high-density lipoprotein; hs-CRP = high-sensitivity C-reactive protein; LDL = low-density lipoprotein; mg/dL = milligrams per deciliter; MI = myocardial infarction; n = number in sample (or study) population; NA = not applicable; NRT = nicotine replacement therapy; NS = not significant;PAI-1 = plasminogen activator inhibitor-1; Plts = platelets; RCT = reverse cholesterol transport; SAEs = serious adverse events; Trigly = triglycerides; WBC = white blood cells.

outcomes, including body weight, distance completed in Godtfredsen and colleagues (2003) conducted a pro- a six-minute walking test, the proportion of participants spective cohort study in Denmark to examine changes completing that test, the prevalence and frequency of in the incidence of MI after spontaneous reductions in angina, the need for urgent cardiac care, and other serious cigarette use. This study included 10,956 men and 8,467 adverse events. Because no significant differences between women who provided detailed information on smoking treatment groups were observed and because both groups behavior during two examinations. Mortality registers and achieved significant reductions in cigarette use from base- hospital registers were searched for an incident of hospital line, results for the entire cohort at six months were com- admission or a death attributable to MI. A sample consist- pared with the baseline data. There were no significant ing of pooled data from three population studies yielded differences in biomarkers of cardiovascular risk, including 643 participants who were heavy smokers at study entry leukocyte counts and levels of F2-isoprostanes or CRP, but and were evaluated for the effects of reduced smoking. CO had decreased by 6.0 ppm (p = 0.007). These persons reported unassisted reductions in tobacco use by at least 50 percent and were compared

404 Chapter 6 How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease

Reduction from baseline

Hb (g/L) WBC x 109/L Fibr (g/L) LDL (mg/dL) HDL (mg/dL) Other biomarkers Clinical outcomes

------

13.8 to 13.3** 7.0 to 6.2** 2.9 to 2.6* NSb NS Plts NS - PAI-1 NS

- - - -e NS - Systolic BP NS Diastolic BP 80.7 vs. 78.8*** Risk of MI NS

------

14.49 to 7.39 to - 122.14 to 50.27 to 52.82* Trigly NS Systolic BP 122.97 14.40* 6.98*** 124.36*g APO A-I NS to 122.29* APO B 103.74 to Diastolic BP NS 103.0* Heart rate 75.7 to 70.18***

NS NS NS - - hs-CRP NS 6-minute walk, F2-isoprostanes NS angina presences/ frequency, CCSC classification, cardiac or other SAEs NS aThiocyanate concentrations increased from 189 to 198 micromoles/L (p <0.05). bHDL to LDL ratio increased from 0.33 to 0.37, p <0.005. cTotal cohort = 19,423. d22.2 grams tobacco per day in continued heavy smokers versus 10.5 grams per day in reducers (p <0.001). eTotal cholesterol NS. fAnatabine decreased from 4.69 to 3.02 nanograms per milligram of creatinine (statistically significant). gHDL to LDL ratio increased from 0.47 to 0.49, p <0.0167. hReduction in CPD reported for smoking reduction intervention group, significance tests reported for smoking reduction versus control group. *p <0.05, **p <0.01, ***p <0.001.

with 1,379 persons who reported abstinence from smok- They speculated that an approximate 50-percent reducing. Outcomes were adjusted for baseline cardiovascu- tion (from 20 g to 10 g of tobacco smoked per day) was lar risk factors. Smoking cessation was associated with not sufficient to improve cardiovascular risk. These epi- a hazard ratio for MI of 0.71 (95 percent CI, 0.59–0.85), demiologic data make an important contribution because but smoking reduction was not associated with a statis- they are population based and come from a large cohort tically significant reduction in risk of MI (hazard ratio, of smokers who reduced their smoking during a period 1.15 [95 percent CI, 0.94–1.40]). A subgroup analysis longer than the usual timeframe for clinical trials. demonstrated significant reductions in levels of expired In summary, these studies show that a significant CO among persons who reduced cigarette smoking. The reduction in cigarette use, even to levels as low as 10 ciga- investigators concluded that the results were consis- rettes per day, results in reduction of exposure to CO from tent with a short-term thrombogenic effect of tobacco tobacco smoke that is smaller than expected. This find- exposure rather than with a cumulative effect of exposure. ing most likely reflects compensatory smoking. Some but

Cardiovascular Diseases 405 Surgeon General’s Report not all studies showed reduced exposure to nicotine, as Mahmarian and colleagues (1997) used single pho- well as improvements in values for hemoglobin, leukocyte ton emission computed tomography to measure the com- counts, and fibrinogen and cholesterol levels. However, bined effects of smoking and use of nicotine patches on the improvements in values are relatively minor compared myocardial perfusion. In 36 patients with known CHD who with those observed with abstinence from smoking. Many were treated with nicotine patches, the amount of heart of these improvements occurred in study participants who muscle deprived of normal blood flow (size of perfusion were using NRT (see “Nicotine Replacement Therapy” defects) decreased despite increased serum nicotine lev- below). None of the studies showed improvements in clin- els. The baseline size of defects and changes in CO levels, ical outcomes of heart disease, consistent with evidence but not in nicotine levels, predicted the size of perfusion presented earlier that low levels of smoke exposure trig- defects. The researchers concluded that the reduction in ger many of the adverse cardiovascular effects of smoke the size of defects resulted from reduction in smoking that (see “Exposure to Secondhand Tobacco Smoke” earlier in was facilitated by NRT and from decreased exposure to CO. this chapter). They also concluded that nicotine patches were safe to use in smokers with heart disease. Nitenberg and Antony (1999) used angiography to Nicotine Replacement Therapy examine short-term effects of use of nicotine gum on perfusion of the coronary arteries in former cigarette smok- In addition to providing data on smoking reduction, ers at study entry, after a test with immersion of the hand the Lung Health Study of 3,094 persons offered a unique in cold water (cold pressor test) before and after adminis- opportunity to examine the natural history and safety of tration of the gum. The gum did not augment the result prolonged use of nicotine gum among thousands of peo- of the cold pressor test, which constricted normal and ple during a five-year follow-up (Murray et al. 1996). Per- diseased segments of the coronary artery, reducing their sistent smoking, but not use of nicotine gum, predicted cross-sectional area, and it did not reduce the surface area fatal and nonfatal cardiovascular events and elevation of of the arterial segments at rest or under conditions of diastolic blood pressure. sympathetic stimulation. In general, trials of smoking cessation and smoking These results suggest that reduction of exposure to reduction showed improvements in lipid profiles, even tobacco smoke through use of NRT or with abstinence with NRT. Eliasson and colleagues (2001) observed that from smoking is associated with improvements in bio- use of nicotine nasal spray for smoking reduction or ces- markers of cardiovascular risk. sation yielded significant improvements in HDLc to LDLc ratios at 9 weeks, with further improvements if smokers abstained from smoking for 17 weeks (see “Reduced Summary Smoking” earlier in this chapter). In addition, significant improvements in fibrinogen levels and leukocyte counts Epidemiologic studies demonstrate a strong dose- were observed. A clinical trial of smoking cessation also response relationship between the number of cigarettes reported significant decreases in hemoglobin values, leu- smoked per day and cardiovascular risk. The relationship kocyte counts, and total and LDLc concentrations (Lúd- is not linear, however, and even low levels of exposure to víksdóttir et al. 1999). In addition, HDLc to LDLc ratios tobacco, such as a few cigarettes per day, occasional smok- improved among participants who abstained from smoking, or exposure to secondhand tobacco smoke are suffi- ing after three months of treatment with a nicotine nasal cient to substantially increase risk of cardiac events. Some spray. Allen and colleagues (1994) also observed signifi- interventions have accomplished significant reductions in cantly increased HDLc levels in participants treated with the number of cigarettes smoked per day, but the reduc- the nicotine patch. tions in levels of biomarkers of exposure and biomarkers of Investigators have noted improvements in markers cardiovascular risk factors are not proportional, probably of thrombogenesis among persons who abstained from because of compensatory smoking by study participants smoking and were using medicinal nicotine. In one study, and the nonlinear dose-response relationship. The limited plasma fibrinogen levels were reduced among 164 men data on clinical outcome do not confirm reduction in car- using a combination of a nicotine patch and gum for 12 diovascular events due to reduced smoking. Other meth- weeks to stop smoking (Haustein et al. 2002). In another ods for reducing exposure, including NRT with abstinence study, use of transdermal nicotine appeared to activate from smoking, are associated with more improvement in platelet aggregation less than smoking did (Benowitz et risk factors for CVD than is smoking reduction with or al. 1993).

406 Chapter 6 How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease without pharmacologic support. Accordingly, these meth- would be difficult to accomplish. Reducing exposure by ods hold more potential for reducing risk. reducing smoking, therefore, appears to have limited promise for improving cardiac risk unless this method contributes to eventual smoking cessation (Hughes 2000). Implications Because smoking cessation is associated with marked improvements in the risk of MI, sudden death, and stroke, These findings suggest that to lower cardiac risk, it should be stressed as the goal for interventions deal- interventions would have to reduce exposure to tobacco ing with dependence on tobacco. The safety and efficacy of smoke to extremely low levels or eliminate the exposure. long-term NRT use to reduce cardiovascular risk by main- Studies of smoking reduction to date suggest that goals taining smoking cessation have not been established.

Evidence Summary

Exposure to tobacco smoke is associated with accel- blood pressure and constricting coronary arteries. Nico- erated atherosclerosis and an increased risk of acute MI, tine may also contribute to endothelial dysfunction, insu- stroke, PAD, aortic aneurysm, and sudden death. Smok- lin resistance, and lipid abnormalities. However, interna- ing appears to have both causal relationships and mul- tional epidemiologic evidence and data from clinical trials tiplicative interactions with other major risk factors for of nicotine patches suggest that chemicals other than nic- CHD, including hyperlipidemia, hypertension, and diabe- otine contribute to an elevated risk of death from MI and tes mellitus. stroke. CO reduces the delivery of oxygen to the heart and The cardiovascular risk attributable to cigarette other tissues and can aggravate angina pectoris or PAD smoking increases sharply at low levels of cigarette con- and can lower the threshold for arrhythmias in the pres- sumption and with exposure to secondhand smoke. The ence of CHD. Exposure to particulates is associated with risk then tends to plateau at higher levels of smoking. This oxidant stress and cardiovascular autonomic disturbances finding indicates a low threshold for effect and a nonlinear that potentially contribute to acute cardiovascular events. dose-response relationship. Some of the nonlinearity of Cigarette smoking causes acute cardiovascular the relationship between the number of cigarettes smoked events such as MI and sudden death by adversely affect- per day and CVD risk may be due to impreciseness of this ing the balance of myocardial demand for oxygen and measure of actual exposure to smoke. However, the data nutrients and coronary blood flow. Smoking results in on risk associated with exposure to secondhand smoke increased myocardial work, reduced coronary blood flow, indicate a true nonlinear relationship between exposure and enhanced thrombogenesis. Enhancement of throm- and CVD risk. Cardiovascular risk is not reduced by smok- bogenesis appears to be particularly important in that ing cigarettes of lower machine-delivered yields of nico- smokers with acute MI have less severe underlying cor- tine or tar. onary artery disease than do nonsmokers with MI, but The constituents of tobacco smoke believed to be smokers have a greater burden of thrombus. responsible for cardiovascular disease include oxidiz- Several potential mechanisms appear to contribute ing chemicals, nicotine, CO, and particulate matter. to the effects of smoking in accelerating atherosclerosis. Oxidizing chemicals, including oxides of nitrogen and These mechanisms include inflammation, endothelial many free radicals, increase lipid peroxidation and con- dysfunction, impaired insulin sensitivity, and lipid abnor- tribute to several potential mechanisms of CVD, including malities. Cigarette smoking is a risk factor for diabetes inflammation, endothelial dysfunction, oxidation of LDL, and aggravates insulin resistance in persons with diabetes. and platelet activation. The mechanism appears to involve both the effects of oxi- Nicotine is a sympathomimetic drug that increases dizing chemicals in the smoke and the sympathomimetic heart rate and cardiac contractility, transiently increasing effects of nicotine.

Cardiovascular Diseases 407 Surgeon General’s Report

Conclusions

1. There is a nonlinear dose response between expo- 5. Cigarette smoking produces an atherogenic lipid pro- sure to tobacco smoke and cardiovascular risk, with file, primarily due to an increase in triglycerides and a a sharp increase at low levels of exposure (including decrease in high-density lipoprotein cholesterol. exposures from secondhand smoke or infrequent cigarette smoking) and a shallower dose-response 6. Smoking cessation reduces the risk of cardiovascular relationship as the number of cigarettes smoked per morbidity and mortality for smokers with or without day increases. coronary heart disease.

2. Cigarette smoking leads to endothelial injury 7. The use of nicotine or other medications to facilitate and dysfunction in both coronary and peripheral smoking cessation in people with known cardiovas- arteries. There is consistent evidence that oxidizing cular disease produces far less risk than the risk of chemicals and nicotine are responsible for endothe- continued smoking. lial dysfunction. 8. The evidence to date does not establish that a reduc- 3. Tobacco smoke exposure leads to an increased risk tion of cigarette consumption (that is, smoking fewer of thrombosis, a major factor in the pathogenesis of cigarettes per day) reduces the risks of cardiovascu- smoking-induced cardiovascular events. lar disease.

4. Cigarette smoking produces a chronic inflamma- 9. Cigarette smoking produces insulin resistance and tory state that contributes to the atherogenic disease chronic inflammation, which can accelerate macro- processes and elevates levels of biomarkers of inflam- vascular and microvascular complications, including mation, known powerful predictors of cardiovascu- nephropathy. lar events.

408 Chapter 6 How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease

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432 Chapter 6 How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease von der Leyen HE, Gibbons GH, Morishita R, Lewis NP, Weber C, Erl W, Weber K, Weber PC. Increased adhesive- Zhang L, Nakajima M, Kaneda Y, Cooke JP, Dzau VJ. ness of isolated monocytes to endothelium is prevent- Gene therapy inhibiting neointimal vascular lesion: in ed by vitamin C intake in smokers. Circulation 1996; vivo transfer of endothelial cell nitric oxide synthase 93(8):1488–92. gene. Proceedings of the National Academy of Sciences Weir JM, Dunn JE Jr. Smoking and mortality: a prospec- of the United States of America 1995;92(4):1137–41. tive study. Cancer 1970;25(1):105–12. Walker JM, Cove DH, Beevers DG, Dodson PM, Leath- Weiss NS. The value of roentgenographic abdominal aor- erdale BA, Fletcher RF, Wright AD. Cigarette smok- tic calcification in predicting site of occlusion in arte- ing, blood pressure and the control of blood glucose riosclerosis obliterans. 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434 Chapter 6 How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease

List of Abbreviations

1-HOP 1-hydroxypyrene CDK cyclin-dependent kinase 4-ABP 4-aminobiphenyl CETP cholesterol ester transfer protein 4-HNE 4-hydroxy-2-nonenal CHD coronary heart disease 8-oxo-dG 7-hydroxy-8-oxo-2’-deoxyguanosine CI confidence interval 8-OH-dG 8-hydroxy-2’-deoxyguanosine CNS central nervous system 8-OH-DPAT 8-hydroxy-2-dipropylaminotetralin CO carbon monoxide

AAT α1-antitrypsin CO2 carbon dioxide ACE angiotensin-converting enzyme COPD chronic obstructive pulmonary disease ACh acetylcholine CPS-I Cancer Prevention Study I ACTH adrenocorticotropic hormone CPS-II Cancer Prevention Study II ADMA asymmetric dimethylarginine CREB cyclic adenosine monophosphate–response AGT O6-alkylguanine–DNA alkyltransferase element binding protein AH aryl hydrocarbon CRP C-reactive protein AHH aryl hydrocarbon hydroxylase CS conditioned stimulus AKT protein kinase B CSE cigarette smoke extract AOR adjusted odds ratio CT computed tomography AP apurinic/apyrimidinic CUZNSOD copper zinc superoxide dismutase AP-1 activator protein-1 CVD cardiovascular disease APA American Psychiatric Association DEB diepoxybutane APO apolipoprotein DHBMA dihydroxybutyl mercapturic acid APO A-I apolipoprotein A-I DHβE [3H]dihydro-beta-erythroidine APO B apolipoprotein B dL deciliter APO E apolipoprotein E DMBA 7,12-dimethybenz[a]anthracene ATP adenosine triphosphate DRC DNA repair capacity B[a]P benzo[a]pyrene DRZ diagonal radioactive zone B[b]F benzo[b]fluoranthene DSB double-strand break B[k]F benzo[k]fluoranthene DSBR double-strand break repair BAL bronchoalveolar lavage DSM-III-R Diagnostic and Statistical Manual of Mental BER base excision repair Disorders, 3rd ed. (rev) BH BCL-2 homology DSM-IV Diagnostic and Statistical Manual of Mental BMI body mass index Disorders, 4th ed. BPDE benzo[a]pyrene-7,8-diol-9,10-epoxide EB 3,4-epoxybutene BSID Bayley Scales of Infant Development ECSOD extracellular superoxide dismutase Ca2+ calcium ion EF etiologic fraction Cal/EPA California Environmental Protection Agency ELF epithelial lining fluid CAM chorioallantoic membrane ENOS endothelial nitric oxide synthase CaMKII Ca2+ calmodulin-dependent protein kinase II EPA U.S. Environmental Protection Agency CAN Canadian Intense EPC endothelial progenitor cell CAT chloramphenicol acetyltransferase EPO eosinophil-specific peroxidase

CB1 cannabinoid subtype 1 ERK extracellular signal-regulated kinase CDC Centers for Disease Control and Prevention F-344 Fischer-344

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FDA U.S. Food and Drug Administration HPG hypothalamic-pituitary-gonadal Fe2+ ferrous ion HPLC high-performance liquid chromatography Fe3+ ferric ion HR23B homologous recombinational repair group

FEV1 forced expiratory volume in one second 23B FFA free fatty acid IARC International Agency for Research on Cancer FR fecundability ratio IBF intervillous blood flow FSH follicle-stimulating hormone ICAM intercellular adhesion molecule FTC U.S. Federal Trade Commission ICD-10 International Statistical Classification of FTND Fagerström Test for Nicotine Dependence Diseases and Related Health Problems, Tenth FTQ Fagerström Tolerance Questionnaire Revision FVC forced vital capacity Ig immunoglobulin g gram IgM immunoglobulin M GABA γ-aminobutyric acid IL interleukin GC gas chromatography INOS inducible nitric oxide synthase GCL glutamate cysteine ligase IOM Institute of Medicine GGR global genomic nucleotide excision repair ISO International Organization for GLu-P-1 2-amino-6-methyldipyrido[1,2-a:3’,2’-d] Standardization imidazole IUGR intrauterine growth restriction GOLD Global Initiative for Chronic Obstructive IVF in vitro fertilization Lung Disease kcal kilocalorie GPX glutathione peroxidase kg kilogram GRX glutathione reductase L liter GSH glutathione LBW low birth weight GSSG oxidized glutathione LC liquid chromotography GST glutathione-S-transferase LDL low-density lipoprotein GTP guanosine triphosphate LDLc low-density lipoprotein cholesterol H/R hypoxia/reperfusion LH luteinizing hormone

H2O water LMPCR ligation-mediated polymerase chain reaction H2O2 hydrogen peroxide LOD logarithm of the odds H2S hydrogen sulfide LPL lipoprotein lipase HAT histone acetyltransferase LUC luciferase Hb hemoglobin m3 cubic meter

HbA1c hemoglobin A1c MAO monoamine oxidase HBEC human bronchial epithelial cell MAPK mitogen-activated protein kinase HCA heterocyclic amine MDM2 murine double minute 2 hCG human chorionic gonadotropin MDPH Massachusetts Department of Public Health HCN hydrogen cyanide MEK mitogen-activated protein kinase kinase HCR host cell reactivation mg milligram HDAC histone deacetylase mGluR2/3 metabotropic glutamate receptor subtype 2/3 HDL high-density lipoprotein mGluR5 metabotropic glutamate receptor subtype 5 HDLc high-density lipoprotein cholesterol MHBMA monohydroxybutenyl mercapturic acid Hg mercury MI myocardial infarction HIV human immunodeficiency virus mL milliliter HONC Hooked on Nicotine Checklist mm millimeter HOX hypohalous acids MMP matrix metalloproteinase

672 How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease

MMR mismatch repair NST nonstress test MMS methyl methanesulfonate NTCA N-nitrosothiazolidine 4-carboxylic acid

MNSOD manganese superoxide dismutase O2 oxygen − MPEP 2-methyl-6-(phenylethynyl)pyridine O2• superoxide anion MPO myeloperoxidase OH hydroxyl radical mRNA messenger RNA ONOO− peroxynitrite MRP multidrug-resistance protein ONOOH peroxynitrous acid MS mass spectrometry OR odds ratio MSCA McCarthy Scales of Children’s Abilities p short arm of chromosome MT1 membrane-type 1 PAD peripheral arterial disease MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltet- PAF platelet-activating factor razolium bromide PAH polycyclic aromatic hydrocarbon μg microgram PAI-1 plasminogen activator inhibitor-1 μm micrometer PARP poly (adenosine diphosphate–ribose) μmol micromole polymerase

N2 nitrogen PAT poly AT insertion/deletion polymorphism NAB N’-nitrosoanabasine PCASRM Practice Committee of the American Society nAChR nicotinic acetylcholine receptor for Reproductive Medicine NADPH reduced nicotinamide adenine dinucleotide PCNA proliferating cell nuclear antigen phosphate p-CREB phosphorylated CREB NAT N-acetyltransferase PdG 1,N2-propanodeoxyguanosine NATB N’-nitrosoanatabine PDK1 3-phosphoinositide–dependent protein NCI National Cancer Institute kinase-1 NDMA N-nitrosodimethylamine PET positron emission tomography NER nucleotide excision repair pg picogram

NF-κB nuclear factor-kappa B pHeff effective pH ng nanogram PhIP 2-amino-1-methyl-6-phenylimidazo[4,5-b] NHLBI National Heart, Lung, and Blood Institute pyridine NIH National Institutes of Health PHS U.S. Public Health Service NK natural killer PI protease inhibitor nmol nanomole PI-3K phosphatidylinositol-3 kinase NMTCA N-nitroso-2-methylthiazolidine 4-carboxylic PIP3 phosphatidylinositol 3,4,5-triphosphate acid PKA protein kinase A NNAL 4-(methylnitrosamino)-1-(3-pyridyl)-1-buta- PKB protein kinase B (also known as AKT) nol PKC protein kinase C

NNK 4-(methylnitrosamino)-1-(3-pyridyl)-1-buta- PKcs protein kinase catalytic subunit none PlGF placental growth factor NNN N’-nitrosonornicotine PMN polymorphonuclear neutrophil NO nitric oxide pmol picomole

NO2 nitrogen dioxide p-MPPI 4-(2’-methoxy-phenyl)-1-[2’-(n-2’’-pyridinyl)- NOS nitric oxide synthase p-iodobenzamido]ethyl-piperazine NPRO N-nitrosoproline POB pyridyloxobutyl NRC National Research Council pol polymerase NRT nicotine replacement therapy ppm parts per million NSCLC non-small-cell lung cancer PPROM preterm premature rupture of membranes

673 Surgeon General’s Report

PREP potential reduced-exposure product Th1 helper T-cell subset 1 PRX peroxiredoxin Th2 helper T-cell subset 2 q long arm of chromosome TIMP tissue inhibitor of metalloproteinase − Q• semiquinone radical Tmax time to reach maximum blood concentrations RCT reverse cholesterol transport of nicotine REC homologous recombination TNFα tumor necrosis factor-alpha RI resistance index tPA tissue plasminogen activator RNS reactive nitrogen species TPM total particulate matter ROS reactive oxygen species trans, anti-PheT r-1,t-2,3,c-4-tetrahydroxy-1,2,3,4-tetrahydro- RR relative risk phenanthrene S/D systolic/diastolic Trp-P-1 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole SAB spontaneous abortion Trp-P-2 3-amino-1-methyl-5H-pyrido[4,3-b]indole SARS severe acute respiratory syndrome TRX thioredoxin SCC squamous cell carcinoma TSNA tobacco-specific nitrosamine SCE sister chromatid exchange tt-MA trans,trans-muconic acid

SCENIHR Scientific Commission on Emerging and TxB2 thromboxane B2 Newly Identified Health Risks TxM thromboxane metabolite SCLC small-cell lung cancer UCSF University of California at San Francisco SD standard deviation UGT uridine-5’-diphosphate-glucuronosyltransfer- SES socioeconomic status ase sFlt-1 soluble vascular endothelial growth factor US unconditioned stimulus receptor Flt-1 USDHEW U.S. Department of Health, Education, and SGA small for gestational age Welfare sICAM soluble intercellular adhesion molecule USDHHS U.S. Department of Health and Human SIDS sudden infant death syndrome Services SNO S-nitrosothiol USPSTF U.S. Preventive Services Task Force SNP single nucleotide polymorphism UV ultraviolet SOD superoxide dismutase VCAM vascular cell adhesion molecule S-PMA S-phenylmercapturic acid VEGF vascular endothelial growth factor STR short tandem repeat VEGFR vascular endothelial growth factor receptor sVCAM soluble vascular cell adhesion molecule VLDL very-low-density lipoprotein

TBARS thiobarbituric acid reactive substances Vmax maximum velocity TCR transcription-coupled repair VOC volatile organic compound TFIIH transcription initiation factor IIH VTA ventral tegmental area TGFβ transforming growth factor-beta WHO World Health Organization TGRL triglyceride-rich lipoprotein XP (A–G) xeroderma pigmentosum (groups A–G) γGT γ-glutamyltranspeptidase

674 How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease

List of Tables and Figures

Chapter 5 Cancer Table 5.12 Carcinogens and tobacco-induced cancers 299 Link between cigarette smoking and cancer Table 5.1 IARC evaluations of carcinogens in mainstream Figure 5.1 cigarette smoke 228 through carcinogens in tobacco smoke 226

Figure 5.2 Chemical structures of biomarkers of carcinogen Table 5.2 DNA adducts in human lung tissue 243 exposure 231

Table 5.3 Selected gene polymorphisms evaluated by Figure 5.3 Metabolism of six carcinogens in tobacco smoke molecular epidemiology investigations for rela- that produce DNA adducts identified in the lungs tionship to lung cancer through variation in sus- of smokers 236 ceptibility to carcinogens in tobacco smoke 246 Figure 5.4 Mechanism of base excision repair 255 Mechanism of nucleotide excision repair: (A) Table 5.4 Human DNA glycosylases 253 Figure 5.5 global genomic repair; (B) transcription-coupled repair 256 Table 5.5 Factors involved in nucleotide excision repair activity in humans 257 Figure 5.6 Mechanism of mismatch repair 258 Proposed mechanism of homologous recombina- Table 5.6 Select candidate genes and polymorphisms impli- Figure 5.7 cated in repair of tobacco-induced DNA dam- tion 260 age 262 Figure 5.8 Proposed mechanism of nonhomologous end- joining 261 Translesion-specialized DNA polymerases (pol) Table 5.7 Figure 5.9 Model of mechanism for mammalian translesion and activities on various DNA lesions 270 synthesis 270 Mutational specificity of selected DNA adducts Patterns of TP53 gene mutations and percentage Table 5.8 Figure 5.10 derived from tobacco smoke 271 of G→T transversion mutations in human lung cancers 278 Table 5.9 Frequency of mutation or deletion of tumor-suppressor genes in lung cancer 274

Frequency of gene amplification and increased Table 5.10 Figure 5.11 Patterns of TP53 gene mutations and percentage expression of genes in lung cancer 276 of G→T transversion mutations in different histologic types of lung cancer 280 Table 5.11 Pathways altered through gene silencing by promoter methylation 293 Figure 5.12 Concordance between codon distribution of G→T transversions along TP53 gene in lung cancers (top) and distribution of adducts of benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE)– DNA adducts in bronchial epithelial cells (bottom) 281 Figure 5.13 Patterns of TP53 gene mutations and percentage of G→T transversions in smoking-associated cancers other than lung cancer 212 Figure 5.14 Tobacco-associated suppression of proapoptotic proteins and tumor-suppressor proteins 285

Figure 5.15 Protein-signaling pathways deregulated in lung cancer 286

675 How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease

Chapter 6 Cardiovascular Diseases

Table 6.1 Rate ratios for coronary heart disease among White men, by age and duration of cigarette smoking 359

Table 6.2 Death rates and rate ratios for death from coronary heart disease among men, by age and duration of smoking by number of cigarettes smoked per day 360

Table 6.3 Biomarkers of risk for cardiovascular disease from exposure to cigarette smoke 392

Table 6.4 Randomized controlled trials of counseling for smokers hospitalized with cardiovascular disease 396

Table 6.5 Randomized controlled trials of pharmacologic interventions for smoking cessation in patients with cardiovascular disease 397

Table 6.6 Smoking reduction and cardiovascular disease endpoints: biomarkers and clinical outcomes 404

Figure 6.1 Relative and excess death rate for coronary heart disease among men, by age group 356

Figure 6.2 Age-specific excess death rates among male smokers for coronary heart disease, lung cancer, chronic obstructive pulmonary disease (COPD), and cerebrovascular disease 357

Figure 6.3 Dose-response relationship between number of cigarettes smoked per day and relative risk of ischemic heart disease 358

Figure 6.4 Plasma nicotine and carboxyhemoglobin concentrations throughout a day of cigarette smoking 364

Figure 6.5 Overview of mechanisms by which cigarette smoking causes an acute cardiovascular event 366

Figure 6.6 Potential sites of actions and mechanisms of effects of smoking on platelets 375

Figure 6.7 Potential sites of effects of smoking on thrombosis through oxidative stress and other mechanisms 378

677 How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease

Definitions and Alternative Nomenclature of Genetic Symbols Used in This Report Throughout this report, genes are represented by their abbreviations in italics. In many cases, proteins and enzymes related to these genes have the same abbreviation, presented in roman type. Definitions, alternative genetic symbols, related proteins and enzymes, and polymorphisms and variant genotypes are listed alphabetically by gene abbreviation.

Gene symbol used in this Alternative Polymorphism/variant report Definition gene symbol Related protein/enzyme genotype

5HTT serotonin transporter SLC6A4, 5HTT, SERT, SLC6A4 LPR; VNTR; 5HTTLPR 5HTTLPR

ADPRT poly (ADP-ribose) polymerase PARP1 ADPRT; PARP1 VAL762ALA; PRO882LEU; family, member 1 CYS908TYR

AGT O6-alkylguanine–DNA AGT ILE143VAL; GLY160ARG alkyltransferase

ANKK1 ankyrin repeat and kinase ANKK1, protein kinase PKK2 GLU713LYS (caused by domain containing 1 DRD2 *TAQ1A system)

APAF1 apoptotic peptidase activating APAF1 factor 1

APEX1 APEX nuclease APEX1 GLU148ASP (T11865G) (multifunctional DNA repair enzyme) 1

AREG amphiregulin (schwannoma- SDGF AREG, SDGF, CRDGF derived growth factor) (colorectum cell-derived growth factor)

ARP arginine-rich protein ARMET ARP, ARMET (arginine- rich mutated in early stage tumors)

ATM ataxia telangiectasia mutated TEL1 ATM; TEL1 (telomere maintenance 1)

BAX BCL2-associated X protein BAX

BRAF v-raf murine sarcoma viral BRAF, B-RAF proto-oncogene onco homolog B1 serine/threonine-protein kinase (p94)

BRCA2 breast cancer 2, early onset BRCA2, breast cancer HIS372ASN (T27113G), susceptibility protein ILE3412VAL (G93268A)

C-FOS v-fos FBJ murine osteosarcoma FOS FOS, C-FOS viral oncogene homolog

C4A complement component 4A C4A haplotype (Rodgers blood group)

C4B complement component 4B C4B haplotype (Childo blood group)

CASPASE-3 cysteine-aspartic acid CASPASE-3 protease-3

CASPASE-8 cysteine-aspartic acid CASPASE-8 protease-8

679 Surgeon General’s Report

Gene symbol used in this Alternative Polymorphism/variant report Definition gene symbol Related protein/enzyme genotype

CCK cholecystokinin CCK C-45T promoter polymorphism in SP1 binding region of CCK gene

CCKAR cholecystokinin A receptor CCK1-R CCKAR, CCK1-R T779C, VAL365ILE (cholecystokinin-1 receptor)

CCND1 G1/S-specific cyclin D1 CCND1 G870A, *A/*A, *G/*A, *G/*G

CCNH cyclin H CCNH, cyclin-dependent VAL270ALA kinase-activating kinase

CD14 CD14 molecule CD14 molecule; CD14 C-159T (promoter antigen polymorphism)

CDK4 cyclin-dependent kinase 4 CDK4, cell division kinase 4

CDKN2A cyclin-dependent kinase CDKN2A, P16 inhibitor 2A

C-HA-RAS v-Ha-ras Harvey rat sarcoma C-HRAS, C-HRAS, HRAS1 viral oncogene homolog HRAS proto-oncoprotein

CHFR checkpoint with forkhead and CHFR, mitotic checkpoint ring finger domains protein

CHRNA3 cholinergic receptor, nicotinic, CHRNA3 a3

CHRNA4 cholinergic receptor, nicotinic, CHRNA4, α4 nAChR, SNP RS3746372 (*1); a4 nicotinic acetylcholine haplotype receptor α4 subunit

CHRNA5 cholinergic receptor, nicotinic, CHRNA5, nicotinic SNP RS16969968 a5 acetylcholine receptor α5 (CHRNA5); SNPs subunit, α5 nAChR RS8023462 and RS1948 (CHRNA5/A3/B4 cluster)

CHRNA7 cholinergic receptor, nicotinic, CHRNA7, nicotinic D15S1360 (a microsatellite a7 acetylcholine receptor α7 polymorphic marker in subunit, α7 nAChR INTRON 2) exhibited seven different dinucleotide repeat lengths (99, 109, 111, 113, 115, 117, and 125 bp), the major alleles are *113 and *115 (*113/*113, *113/*115, *115/*115)

CHRNB2 cholinergic receptor, nicotinic, CHRNB2, nicotinic multiple SNPs; haplotype b2 (neuronal) acetylcholine receptor β2 subunit, β2 nAChR

CHRNB3 cholinergic receptor, nicotinic, CHRNB3 b3

680 How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease

Gene symbol used in this Alternative Polymorphism/variant report Definition gene symbol Related protein/enzyme genotype

CHRNB4 cholinergic receptor, nicotinic, CHRNB4 b4

C-MYC avian myelocytomatosis viral MYC C-MYC, myc proto-oncogene oncogene homolog protein

COMT catechol-O-methyltransferase COMT A1947G, *A, *G, VAL158MET

COX-1 cyclooxygenase-1 COX-1

COX-2 cyclooxygenase-2 COX-2

CYP17A1 cytochrome P-450, family 17, CYP17A1 subfamily A, polypeptide 1

CYP1A1 cytochrome P-450, family 1, CYP1A1, MSPI *MSPI (*A/*a, *a/*a), subfamily A, polypeptide 1 ILE462VAL

CYP2A13 cytochrome P-450, family 2, CYP2A13 ARG257CYS (C3375T, subfamily A, polypeptide 13 *C/*T, *T/*T)

CYP2A6 cytochrome P-450, family 2, CYP2A6 *1 (wild type), *1A, *1B, subfamily A, polypeptide 6 *2, *3, *4, *4A, *4B, *4C, *4D, *5, *6, *7, *8, *9, *10, *12, *16, *NULL, *DEL

CYP2B6 cytochrome P-450, family 2, CYP2B6 *5 (C1459T=ARG487CYS); subfamily B, polypeptide 6 *6 (G516T=GLN172HIS); *2 (C64T=ARG22CYS); *3 (C777A=SER259ARG); *4 (A785G=LYS262ARG)

CYP2D6 cytochrome P-450, family 2, CYP2D6 *3, *4A, *5 subfamily D, polypeptide 6

CYP2E1 cytochrome P-450, family 2, CYP2E1 *1C, *1D, *DRA1, *RSA1 subfamily E, polypeptide 1

DAPK1 death-associated protein DAPK DAPK1, DAPK kinase 1

DAT dopamine transporter DAT1 DAT, DAT1 VNTR

DDC dopa decarboxylase (aromatic AADC DDC, AADC haplotype l-amino acid decarboxylase)

DLC1 deleted in liver cancer 1 DLC1

DRD1 dopamine receptor D1 DRD1 *DDE1

DRD2 dopamine receptor D2 DRD2 *TAQ1A (*A), *TAQ1B, -141C *INS/*DEL, *FOK1, *INTRON 2, *MBO1

681 Surgeon General’s Report

Gene symbol used in this Alternative Polymorphism/variant report Definition gene symbol Related protein/enzyme genotype

DRD4 dopamine receptor D4 DRD4 VNTR

DRD5 dopamine receptor D5 DRD5 haplotype

DßH dopamine beta hydroxylase DβH G1368A, C1021T, *A allele (G444A)

E-CADHERIN cadherin 1, type 1, E-cadherin E-CADHERIN, calcium- (epithelial) dependent adhesion protein, epithelial

EGFR epidermal growth factor EGFR receptor (erythroblastic leukemia viral (v-erb-b) onco homolog, avian)

ELA2 elastase, neutrophil expressed ELANE ELA2

EN2 engrailed homeobox 2 EN-2 EN2

ENOS endothelial nitric oxide NOS3 ENOS, NOS3 *A allele (a quadruple synthase repeat of a 27 base-pair sequence in INTRON 4), GLU298ASP (*ASP298)

EPHX1 gene of epoxide hydrolase 1, MEH EPHX1, MEH microsomal EXON 3 SNP, EXON 4 microsomal (xenobiotic) epoxide hydrolase SNP, EXONs 3-4 SNP haplotypes, (T → C) TYR113HIS (*H), (A → G) HIS139ARG

ERCC1 excision repair cross- ERCC1 complementing rodent repair deficiency, complementation group 1 (includes overlapping antisense sequence)

ESR1 estrogen receptor 1 estrogen receptor α *XBAL (c.454-351A → G), *PVULL (c.454-397T → C)

FAS Fas (TNF receptor superfamily, FAS member 6)

FHIT fragile histidine triad FHIT, FRA3B

FMS colony stimulating factor 1 CSF1R, CSF1R, C-FMS, CD115, receptor C-FMS, FIM2, CSFR CD115, FIM2, CSFR

GABARAP GABA receptor-associated GABARAP protein

GABAB2 γ-aminobutyric acid type B GABAB2 haplotype receptor 2

682 How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease

Gene symbol used in this Alternative Polymorphism/variant report Definition gene symbol Related protein/enzyme genotype

GC group-specific component GC *1S (416GLU, 420THR), (vitamin D binding protein) *1F (416ASP, 420THR), *1S/*1F, *2 (416ASP, 420LYS), haplotype

GPX glutathione peroxidase GPX

GSTA1 glutathione-S-transferase 1a GSTA1

GSTM1 glutathione-S-transferase µ1 GSTM1 *NULL, *DEL

GSTP1 glutathione-S-transferase p1 GSTP1 ILE105VAL, *ILE/*ILE, *ILE/*VAL, ALA114VAL, *ALA/*ALA, *ALA/*VAL

GSTT1 glutathione-S-transferase q1 GSTT1 *NULL, *DEL H-CADHERIN cadherin 13, H-cadherin CDH13 H-CADHERIN, CDH13 (heart)

HER-2/NEU v-erb-b2 erythroblastic ERBB2 HER-2/NEU, erb B2 c-erb B2/ leukemia viral oncogene neu protein, HER-2(human homolog 2, neuro/glioblastoma epidermal growth factor derived oncogene homolog receptor 2) (avian)

HPRT hypoxanthine HPRT phosphoribosyltransferase

IL-1ß interleukin-1b IL-1β C-31T (TATA Box), *-31C, *-31T

JUN jun oncogene JUN

KRAS v-Ki-ras2 Kirsten rat sarcoma KRAS viral oncogene homolog

LAMA3 laminin, α3 LAMA3

LAMB3 laminin, β3 LAMB3

LAMC2 laminin, γ2 LAMC2

LIG1 ligase I, DNA, ATP-dependent LIG1 ALA170ALA (A → C), ASP802ASP (C → T), ALA814ALA (C → G)

LIG4 ligase IV, DNA, ATP-dependent LIG4 ALA3ALA (C8T, cDNA), THR9ILE (C27T, cDNA)

LKB1/STK11 serine/threonine kinase 11 LKB1, STK11

LMYC v-myc myelocytomatosis viral L-MYC, L-MYC, LMYC, MYCL1 onco gene homolog 1, lung MYCL1 carcinoma derived

MAOA monoamine oxidase A MAOA silent C1460T (EXON 14), VNTR

683 Surgeon General’s Report

Gene symbol used in this Alternative Polymorphism/variant report Definition gene symbol Related protein/enzyme genotype

MAOB monoamine oxidase B MAOB A644G (*A,*G)

MBD4 methyl-CpG binding domain MBD4 SER342PRO (T → C), protein 4 GLU346LYS (G → A)

MGMT O6-methylguanine-DNA MGMT LEU84PHE, ILE143VAL methyltransferase

MMP-1 matrix metallopeptidase 1 MMP-1 (interstitial collagenase)

MMP-12 matrix metallopeptidase 12 MMP-12 (macrophage elastase)

MPO myeloperoxidase MPO

MSX1 msh homeobox 1 MSX1 haplotype (intronic CA repeat), *X1.3/*X1.3, *X2.4/*X2.4, *2 (rare allele)

MUC5AC mucin 5AC, oligomeric MUC5AC mucus/gel-forming

MUTYH mutY homolog (E. coli) MUTYH, mutY DNA GLN335HIS (G → C) glycosylase, A/G-specific adenine DNA glycosylase

MYO18B myosin XVIIIB MYO18B

NAT1 N-acetyltransferase 1 NAT1, arylamine 1088 *A/*A (T1088A), N-acetyltransferase 1 1095 *A/*A (C1095A)

NAT2 N-acetyltransferase 2 NAT2, arylamine ILE114THR (T → C), N-acetyltransferase 2 LYS161LYS (C → T), LYS268ARG (A → G), ARG197GLN (G → A), TYR94TYR (C → T), GLY286GLU (G → A), *4/*4 (wild type)

NBS1 Nijmegen breakage syndrome 1 NBN NBS1, NBN, nibrin GLN185GLU (C → G)

N-MYC v-myc myelocytomatosis NMYC, MYCN N-MYC, NMYC, MYCN, viral related oncogene, N-myc proto-oncogene neuroblastoma derived (avian) protein

NORE1A RAS association (RALGDS/AF- RASFF5 NORE1A, RASFF5 6) domain family member 5

NRXN1 neurexin 1 NRXN1

NRXN3 neurexin 3 NRXN3

NTRK2 neurotrophic tyrosine kinase, NTRK2 receptor, type 2

684 How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease

Gene symbol used in this Alternative Polymorphism/variant report Definition gene symbol Related protein/enzyme genotype

OGG1 8-oxoguanine DNA glycosylase OGG1 SER326CYS (C → G), 326*CYS/*CYS, 326*SER/*SER

OPRM1 opioid receptor, μ1 OPRM1 ASN40ASP (A118G), *ASP40, *ASN40

P14ARF cyclin-dependent kinase P14 P14, P14ARF (the alternate inhibitor 2A (CDKN2A) reading frame products of CDKN2A gene)

P16 cyclin-dependent kinase P16INK4A P16, P16INK4A (the major inhibitor 2A (CDKN2A) product of CDKN2A gene)

P21 cyclin-dependent kinase P21, CDNK1A, P21, CDNK1A, CIP1, WAF1, SER31ARG inhibitor 1A CIP1, WAF1, SDI1 SDI1

P53 tumor suppressor P53 ARG72PRO (G → C), 72*ARG/*PRO, 72*PRO/*PRO, *MSPI (INTRON 6), 16 bp insertion (INTRON 3)

P73 tumor protein gene 73 TP73 P73, TP73 *GC/*GC, *AT/*AT, *GC/*AT (G4A, C14T in EXON 2)

PARKIN Parkinson disease (autosomal PARK2 PARKIN (ligase) recessive, juvenile) 2, parkin

PAX5 α paired box gene 5 BSAP (B-cell PAX5 α (one of the products specific of PAX5 gene), BSAP (B-cell activator) α specific activator protein)α

PAX5 ß paired box gene 5 BSAP (B-cell PAX β (one of the products specific of PAX5 gene), BSAP (B-cell activator) β specific activator protein)β

PCMVCAT a plasmid construct PCMVCAT, chloramphenicol containing the reporter acetyltransferase as a reporter gene CAT (chloramphenicol acetyltransferase) driven by CMV (cytomegalovirus) promoter

PLAP placental alkaline phosphatase ALPP PLAP, ALPP alkaline *1/*1, *2/*2 phosphatase, placental (Regan isozyme)

POLD1 polymerase (DNA directed), POLD1 ARG19HIS (G → A), delta 1, catalytic subunit HIS119ARG (A → G), 125kDa SER173ASN (A → G)

685 Surgeon General’s Report

Gene symbol used in this Alternative Polymorphism/variant report Definition gene symbol Related protein/enzyme genotype

PTEN phosphatase and tensin PTEN, deleted on this gene may be deleted homolog chromosome 10 on chromosome 10 (10q23) with the development of cancers

RAD23B RAD23 homolog B RAD23B, UV excision repair ALA249VAL (C → T) (S. cerevisiae) protein RAD23 homolog B, XP-C repair complementing protein

RAD4 RAD4 gene of S. cerevisiae RAD4, S. cerevisiae DNA damage recognition and repair protein

RAD50 RAD50 gene homolog RAD50 (human) ARG1111ILE (G3374T, (S. cerevisiae) cDNA)

RAD51 RAD51 homolog (S. cerevisiae) RAD51

RAD52 RAD52 homolog (S. cerevisiae) RAD52

RAD54L RAD54 homolog (S. cerevisiae) S. cerevisiae RAD54L ARG374SER (G1222C, RAD50 gene cDNA) homolog

RASSF1A Ras association (RalGDS/AF-6) RASSF1A domain family member 1 (A isoform)

RASSF2 Ras association (RalGDS/AF-6) RASSF2 domain family member 2

RASSF4 Ras association (RalGDS/AF-6) RASSF4, tumor suppressor domain family member 4 protein

RB retinoblastoma 1 tumor RB1 RB, RB1 suppressor

SERPINA1 serpin peptidase inhibitor, SERPINA1, AAT protein *M, *S, *Z, *NULL clade A (a-1 antiproteinase, antitrypsin), member 1

SERPINA3 serpin peptidase inhibitor, SERPINA3, ACT protein PRO229ALA, LEU55PRO, clade A (a-1 antiproteinase, MET389VAL, ALA- antitrypsin), member 3 15THR (signal peptide), 1258DELAA

SFTPB surfactant protein B SFTPB; surfactant, A-18C (promoter), pulmonary-associated A1013C, C1580T, A9306G, protein B INTRON 4 variants, THR131ILE

STK11 serine/threonine kinase 11 STK11

TDG thymine-DNA glycosylase TDG

TGFa transforming growth factor a TGFa

686 How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease

Gene symbol used in this Alternative Polymorphism/variant report Definition gene symbol Related protein/enzyme genotype

TGFß1 transforming growth factor β1 TGFβ1 SNP RS1800469 (C-509T, promoter), SNP RS2241712 (G-10807A, promoter), SNP RS6957 (3’UTR), SNP RS224178 (3’UTR), SNP RS1982073 (T29C, LEU10PRO, EXON 1)

TGFß3 transforming growth factor β3 TGFβ3

TH tyrosine hydroxylase TH VNTR

TIMP-3 tissue inhibitor of TIMP-3 metalloproteinase 3

TNFα tumor necrosis factor (TNF TNF TNFα, TNF TNF2 ( a promoter SNP, superfamily, member 2) G-308A), SNPs G-376A and G-238A (promoter), G489A (INTRON), SNP RS3091257 (3’UTR), SNP RS769178 (3’UTR)

TP thymidine phosphorylase TP

TPH tryptophan hydroxylase TPH1 TPH, TPH1 C218A, A779C

TP53 tumor protein TP53

TRAIL-R1 TNF-related apoptosis inducing TNFRSF10A TRAIL-R1, TNFRSF10A, ligand receptor 1 death receptor 4

UGT1A UDP glucuronosyltransferase 1 UGT1A family, polypeptide A1

XPC xeroderma pigmentosum, XPCC, XP3 XPC, XPCC, XP3 ALA499VAL (C → T), complementation group C LYS939GLN (A → C), *PAT (poly AT ins/del polymorphism)

XPD xeroderma pigmentosum, ERCC2 XPD, ERCC2 ASP312ASN (G → A, 312 complementation group D *G/*A), LYS751GLN (A → C, 751 *A/*C)

XPF xeroderma pigmentosum, ERCC4 XPF, ERCC4 SER662PRO (T → C), complementation group F ARG415GLN (G1244A, EXON 8 SNP RS1800067), SER835SER (T2505C, EXON 11 SNP RS1799801)

XPG xeroderma pigmentosum, ERCC5 XPG, ERCC5 HIS1104ASP (G3507C, complementation group G SNP RS17655), HIS46HIS (T335C, SNP RS1047768), CYS529SER (SNP RS2227869)

687 Surgeon General’s Report

Gene symbol used in this Alternative Polymorphism/variant report Definition gene symbol Related protein/enzyme genotype

XPV polymerase (DNA directed) eta POLH XPV POLH, human DNA polymerase h

XRCC1 x-ray repair complementing XRCC1 ARG194TRP (C → T), defective repair in Chinese ARG280HIS (G → A), hamster cells 1 ARG399GLN (G → A)

XRCC2 x-ray repair complementing XRCC2 defective repair in Chinese hamster cells 2

XRCC3 x-ray repair complementing XRCC3 THR241MET (T → C) defective repair in Chinese hamster cells 3

XRCC4 x-ray repair complementing XRCC4 ALA247SER (G → T) defective repair in Chinese hamster cells 4

XRCC5 x-ray repair complementing KU80 XRCC5, KU80 defective repair in Chinese hamster cells 5 (double-strand- break rejoining)

XRCC6 x-ray repair complementing KU70 XRCC6, KU70 defective repair in Chinese hamster cells 6

688