UNIT 2. BIOMARKERS: PRACTICAL ASPECTS CHAPTER 4. Physical/chemical/immunologic analytical methods Jia-Sheng Wang and John D. Groopman UNIT 2 CHAPTER 4 CHAPTER Summary Introduction Biomarkers can be used to measure Over the past 25 years, the validated biomarkers to traditionally the presence of a wide variety of development, validation and descriptive epidemiological studies parent compounds and metabolites application of molecular helps to: delineate the continuum in body fluids and excreta, and biomarkers that reflect events from of events between an exposure and serve as biomarkers of internal environmental exposure to the resulting disease; identify smaller dose. Chemical-macromolecular formation of clinical disease (e.g. exposures to specific xenobiotics; adducts formed in blood and cancer) has rapidly expanded our indicate earlier events in the natural tissue or excreted in urine serve knowledge of the mechanisms history of diseases and reduce as biomarkers of exposure as well, of pathogenesis and provided misclassification of dependent and and in many instances reflect both opportunities for devising improved independent variables; enhance exposure and additional relevant tools for disease treatment and individual and group risk monitoring biological processes. An assortment prevention. Molecular epidemiology and assessments; and reveal of analytical techniques have been and its evolving paradigm refers toxicologic mechanisms by which developed to identify and measure to the use of biomarkers in an exposure and a disease are parent compounds, metabolites, epidemiological research (i.e. related (5,6). A unique feature of chemical-DNA and protein incorporation of molecular, molecular epidemiologic studies is adducts. This chapter will discuss cellular and other biochemical the interdisciplinary collaboration many analytical techniques that measurements into epidemiological between population and field measure biomarkers in molecular studies of the etiology, prevention, scientists and laboratory scientists epidemiologic studies, including and control of health risks faced from various disciplines, such as biological, physical, chemical and by human populations)(1–4) epidemiology, toxicology, molecular immunological methods. (Figure 4.1). The application of biology, genetics, immunology, Unit 2 • Chapter 4. Physical/chemical/immunologic analytical methods 43 Figure 4.1. Molecular epidemiology paradigm biochemistry, pathology and clinical disease (Figure 4.1). Any step or an altered state recognized as analytical chemistry. The analytic in this process may be modified by impairment or disease. A biomarker measurement of biomarkers is host-susceptibility factors including of susceptibility is an indicator or a critical to molecular epidemiologic genetic traits and effect modifiers (e.g. metric of an inherent or acquired studies and requires special diet or environmental exposures). ability of an organism to respond attention to the collection, handling Therefore, biomarkers are indicators to the challenge of exposure to a and storage of biologic specimens, of events for physiologic, cellular, specific xenobiotic substance or as well as development and subcellular and molecular alterations other toxicant. Such a biomarker may validation of analytical methods (7). in the multistage development of be the unusual presence or absence specific diseases (9). of an endogenous component, or Biomarker paradigm Molecular biomarkers are an abnormal functional response and validation strategies typically used as indicators of to an administered challenge exposure, effect or susceptibility. (9). Molecular epidemiology and As adapted from Perera and A biomarker of exposure refers molecular dosimetry thus have Weinstein (1) and the Committee to measurement of the specific great utility in addressing the on Biological Markers of the agent of interest, its metabolite(s), relationships between exposure National Research Council (8), the or its specific interactive products to environmental agents and development of disease as a result in a body compartment or fluid, development of clinical diseases, of exposure to an environmental which indicates the presence and in identifying those individuals agent or other toxicant is multistage: (and magnitude) of current and at high risk for the diseases (2,6,10). it starts with exposure and past exposure. A biomarker of Collectively, these data also help to progresses to internal dose (e.g. effect indicates the presence inform the risk assessment process, deposited body dose), biologically (and magnitude) of a biological where regulations can be tested effective dose (e.g. dose at the response to exposure to an against biological measurements of site of toxic action), early biological environmental agent. Such a exposure to determine the efficacy effect (e.g. at the subcellular level), biomarker may be an endogenous of policies. altered structure or function (e.g. component, a measure of the The development and subclinical changes) and finally to functional capacity of the system, application of molecular biomarkers 44 for environmental chemical agents Extensive efforts have been made and experimental samples. should be based upon specific to identify these high-risk individuals Conceptually, as shown in Figure knowledge of their metabolism, using various genetic and metabolic 4.2, an appropriate animal model is interactive product formation and susceptibility markers (e.g. used to determine the associative general mechanisms of action measurement of polymorphism of or causal role of the biomarker (11,12). Examples in the field are genotype and phenotype of various in the disease or effect pathway, studies on the relationships between enzymes involved in transformative and to establish relations between tobacco smoking and lung cancer metabolic reactions of certain dose and response. The putative (13–16) and between aflatoxin known carcinogens and the DNA biomarker can then be validated exposure and liver cancer (17,18). repair process)(20–23). Although in pilot human studies, where A specific application of biomarker this strategy has not yet proven to sensitivity, specificity, accuracy technology to human cancer is the be broadly applicable to many other and reliability parameters can be study of the variation in response human diseases, progress is being established. Data obtained in these among individuals following made for many types of cancers studies can then be used to assess UNIT 2 exposures to tobacco. For example, (24). intraindividual or interindividual even in heavy tobacco smokers, less The validation of any biomarker- variability, background levels, 4 CHAPTER than 15% of these exposed people effect link requires sequential relationship of the biomarker to develop lung cancer (19); thus, or parallel experimental and external dose or to disease status, intrinsic susceptibility factors must human studies (12). Following the as well as feasibility for use in larger affect the time course of disease development of the hypothesis of population-based studies. It is development and eventual outcome. an exposure-disease linkage, there important to establish a connection The identification of those at highest is the need to develop the analytical between the biomarker and risk for developing cancers should methodology necessary to measure exposure, effect or susceptibility. be facilitated by biomarker studies. these biological markers in human To fully interpret the information that Figure 4.2. Validation scheme for molecular biomarker research Unit 2 • Chapter 4. Physical/chemical/immunologic analytical methods 45 the biomarker provides, prospective toxicity can vary by both charge interindividual variation in dose– epidemiological studies may be state (e.g. tri- and penta-valent state response of environmental necessary to demonstrate the role of arsenic) (30) and methylation (e.g. agents, the central focus in risk that the biomarker plays in the methyl mercury) (31). Measurement assessment. While biomarkers overall pathogenesis of the disease of parent organic compounds in have been most widely applied in or effect. To date, few biomarkers biological samples, although still in the area of clinical pharmacology, have been rigorously validated practice, is less favoured because they are increasingly being used to using this entire process. most organic toxic/carcinogenic document interindividual variation compounds undergo metabolism and in the context of the much lower Techniques and strategies for exert their toxicologic/carcinogenic exposures found in the environment measuring parent compounds effects through metabolic activation (32,33). There are many examples and metabolites (22). Collectively, the problem of variation in the pharmacologic faced in these investigations is the action of drugs in people; in general, Many analytical techniques, including relationship between exposure the response varies about 10-fold biological, physical, chemical, and and dose. Unlike most organic in the general population for most immunological methods, have compounds, metals’ measurements pharmaceutics. At issue with the been developed and standardized often reflect both exposure and extrapolation of these findings to by regulating agencies (e.g. US dose. Figure 4.3 illustrates a environmentally occurring toxicants Environmental Protection Agency) nomograph relating the increase is the recognition that a limited for biohazard identification and risk in analytical sensitivity by various variance in