STUDIES ON THE PHARMACOGENETIC ASPECT#OF BRONCHOGENIC CARCINOMA FfESMOKERS by RIAD AYESH A thesis submitted for the degree of Doctor of Philosophy University of London ProQuest Number: 10610830 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a com plete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest ProQuest 10610830 Published by ProQuest LLC(2017). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States C ode Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106- 1346 This work is dedicated to the memory of my parents. "No character owes its existence to inheritance alone or to environmental factors alone. No character is either innate or acquired : all are both, but in varying degrees because the degree of penetrance that genes show can be very variable." Sir Gavin De Beer (1966) Page 1 Contents Page ABSTRACT 2 ACKNOWLEDGEMENTS 3 LIST OF TABLES 4 LIST OF FIGURES 8 CHAPTER ONE Introduction 10 CHAPTER TWO Pharmacogenetics, cytochrome P-450 genes and genetic susceptibility to bronchogenic carcinoma in smokers. 56 CHAPTER THREE Debrisoquine oxidation phenotypes as markers for susceptibility to bronchogenic carcinoma in smokers. 88 CHAPTER FOUR Genetic polymorphism of mcphenytoin 4-hydroxylasc in lung cancer patients. A comparative study with debrisoquine 4-hydroxylase. 150 CHAPTER FIVE Comparison of phenotypes for metabolism of antipyrine and debrisoquine as indicators of lung cancer risk in smokers. 172 CHAPTER SIX General discussion and concluding remarks. 203 BIBLIOGRAPHY 210 APPENDICES 277 Page 2 ABSTRACT Bronchial carcinoma is not an inevitable consequence of smoking cigarettes. This thesis seeks to determine whether any one or more of the well established oxidative polymorphisms might directly control the biological response to cigarette smoking and/or environmental agents. 1. In the first part of the study, two groups (I and II) of European Caucasian patients were investigated with debrisoquine. Patients with lung cancer (n=245) and controls with airflow limitation (n=234) were similar in age (66.5 ± 7.4 (mean + SD) and 67.2 + 3.2 respectively), sex ratio ((M/F) 1.82, 1.89) and smoking history (60.3 + 24.0 (mean + SD), 59.4 + 21.1 pack year). Debrisoquine 4-hydroxylation showed major differences between lung cancer and control series. The metabolic ratios (MR) for smoking controls included 21 recessives and were distributed across the full range of values. On the other hand, the metabolic ratios for cancer patients contained only 4 recessives and were aggregated towards the left end of the spectrum of metabolic ratios. The relative risk for lung cancer for the extensive metabolisers of debrisoquine (MR<1) is 13 (95% Cl : 4.0-43.3). Within this group, the risk for lung cancer increases further with possible and likely exposure to both asbestos and polycyclic aromatic hydrocarbons. The increased risk among this group with likely asbestos or polycyclic aromatic hydrocarbon exposure, is 35-fold and 17-fold respectively compared to non exposed poor and intermediate phenotypes. The debrisoquine hydroxylation status associated with lung cancer risk does not appear to be altered in any way by cigarette smoke or by the direct influence of the tumour mass. 2. A study was undertaken to eliminate the possibility of an enhanced, oxidative metabolism in lung cancer patients being an effect of the disease rather than associated with its cause. Two groups of patients (lung cancer (n=24) and controls (n=27) were investigated with both debrisoquine and mephenytoin. The % mephenytoin recovery as 4-hydroxymephenytoin was variable in both groups but statistically insignificant, 19 ± 11.8 mean ± SD 21.6 + 8.1 for cancer and controls respectively. The data showed no correlation between the debrisoquine metabolic ratios and the % mephenytoin recovery (rs=0.21 P>0.10). 3. A third study was undertaken to investigate the hepatic oxidation rates in lung cancer patients (n=30) and controls (n=29) as measured by antipyrine metabolism and to investigate the ability to metabolise both debrisoquine and antipyrine in both groups. The antipyrine clearance was almost identical in the two groups with mean + SD 2.9 + 0.46 L/h, 3.0 + 0.5 in cases and controls respectively. The results also showed that there is no difference in the pharmacokinetic parameters of antipyrine between both extensive and poor metabolisers for debrisoquine hydroxylation. 4. The results of this thesis suggest a) that cigarette smokers who arc extensive metabolisers of debrisoquine are at an elevated risk of developing lung cancer, b) additive risk between the ability to extensively metabolise debrisoquine and occupational exposure to lung carcinogens in male smokers, c) the metabolic oxidation phenotypes may serve with other genetic markers for assessing susceptibility to lung cancer. 5. An agenda for subsequent investigations is proposed. Page 3 ACKNOWLEDGEMENTS I would like to thank Professor R.L. Smith for allowing me to work in his Department and for his tremendous help and advice. I would like to thank Dr. M. Hetzel for his unstinted help and kindness and endless encouragement throughout the course of this work. I am deeply grateful to Professor J.R. Idle for his thoughtful and enthusiastic help, guidance and supervision throughout this study. My special thanks to Dr. N.S. Oates for his help with computer graphics and data analysis. My thanks also to Dr. J. Ritchie, Mr. M. Crothcrs for their help in analysing the urine and plasma samples. My thanks also to both Mr. L.A. Wakile and Mr. J. O’Gorman for their support throughout my work. Finally, I am most grateful to Mrs. A. Stolinska for typing this thesis. Page 4 LIST OF TABLES Page Table 1.1 Histological classification of lung tumours. 19 Table 1.2 Histological classification of bronchogenic carcinoma. 20 Table 1.3 Relative risk of lung cancer in large cohort studies among men smoking cigarette and other types of tobacco. 27 Table 1.4 Toxic agents in the gaseous phase of tobacco smoke. 35 Table 1.5 Carcinogenic agents in the particulate phase of tobacco smoke. 36 Table 1.6 Some of the co-carcinogenic agents in the particulate phase of tobacco smoke. - 37 Table 1.7 Known suspected agents of occupational cancer of the lung and their relation to various histological types. 41 Table 1.8 Death rate per 100,000/population in various countries due to lung cancer. 51 Table 1.9 Death rate per 100,000/population in the UK due to lung cancer. 52 Table 1.10 The incidence and mortality rate due to the most common type of cancer in England and Wales. 53 Table 2.1 Examples of known genetic polymorphisms in human and their mode of inheritance. 62 Table 2.2 Associations of some diseases of unknown etiology with human genetic polymorphisms. 63 Page 5 Page Table 2.3 Polymorphisms of human drug metabolising pathways. 74 Table 2.4 Main causes of cancer in the U.K. 77 Table 2.5 Geographical distribution of relative risk of lung cancer by smoking category. 85 Table 3.1 Host factors and human cancer. 90 Table 3.2 The frequency of deficient trait of debrisoquine 4-hydroxylation in various populations. 97 Table 3.3 General characteristics of both cases and controls (Study number one). 110 Table 3.4 Selected characteristics of lung cancer patients and airflow limitation controls. 119 Table 3.5 The distribution of study group members by age, sex and disease. 119 Table 3.6 The measured parameters for both blood chemistry and haematology of the study group patients. 120 Table 3.7 The number of individuals in both cases and controls which have measured routine haematology and clinical chemistry parameters above or/below the clinical range for each parameter. 122 Table 3.8 The distribution of debrisoquine metabolic ratios in association with histology subtypes, sex, % debrisoquine recovery and smoking (PY). 126 Table 3.9 The number of individuals on each drug used by both groups of patients. 127 Page 6 Page Table 3.10 Relative risk of developing lung cancer in smokers by debrisoquine metaboliser status. 129 Table 3.11 The relative risk for lung cancer among extensive metabolisers of debrisoquine by sex and cell type. 130 Table 3.12 The relative risk of lung cancer in maleby occupational exposure to lung carcinogens. 131 Table 3.13 The relative risk (RR) of lung cancer in males by occupational exposure to lung carcinogens and by debrisoquine metabolic ratios. 132 Table 3.14 The influence of cigarette smoking on the debrisoquine metabolic ratios. 133 Table 3.15 The range of clinical syndromes as a consequence of hormones production by bronchogenic carcinoma. 138 Table 3.16 Ectopic hormones produced by bronchogenic carcinoma and other tumours. 139 Table 3.17 Drugs of which one or more metabolic pathways have been reported to co-segregatc with debrisoquine polymorphism as assessed in in vitro and or in vivo studies. 145 Table 3.18 Syggested inherited factors in lung cancer development. 148 Table 4.1 Factors causing interindividual differences in the rate of drug metabolism. 152 Table 4.2 Inter-ethnic differences in pooor metaboliser incidence of mephenytoin oxidation. 157 Page 7 Page Table 4.3 Substrates with inhibiting potency towards mephenytoin 4-hydroxylation in vitro. 159 Table 4.4 General characteristics of lung cancer patients and controls (Mephenytoin Study). 164 Table 4.5 Selected characteristics of lung cancer cases and control (Mephenytoin Study).