Chem. Res. Toxicol. 1993,6, 764-770
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Molecular Biomarkers for Human Chemical Carcinogen Exposures John D. Groopman' and Thomas W. Kensler Department of Environmental Health Sciences, School of Hygiene and Public Health, The Johns Hopkim University, 615 North Wolfe Street, Baltimore, Maryland 21205 Received July 30, 1993
I. Introduction Increased understanding of the mechanistic basis of chemically-induceddiseases provides opportunities for the identification of molecular biological markers reflecting events from exposure through clinical disease. These molecular biological markers can be classified into three major categories: markers of exposure reflecting either internal or biologically effective dose of toxic agents, markers of effect indicating a biological response to an exposure, and markers of susceptibility that characterize the inherent sensitivity of an individual to the toxic agent (1). By definition, some of these markers are chemical agent-specific, such as carcinogen-DNA or -protein adducts, while others are biological process-specific, such as the altered expression of a gene. While there are many literature reports on biomarkers, a recent two-volume collection of papers has been published featuring a comprehensive spectrum of molecular biomarker studies applied to human cancer (2, 3). In the future, the information obtained from studies of molecular biomarkers in humans can be used for a range of public health applications from primary and secondary prevention to the design of clinical therapies. This article highlights some of the recent strategies used for the development and validation of molecular biomarkers of chemical agents found in the environment. While it is well beyond the scope of this article to comprehensively review this field, we have chosen to emphasize the very recent literature reports in this area to permit as broad-based an overview of this research as possible. The studies cited are generally focused upon exposures to specific environmental toxicants, but it is important to recognize that the multistage nature of carcinogenesismeans that humans are exposed to multiple chemical agents and often in combination with viral and physical agents. Thus, in only a few instances, when high exposures have occurred, will a single chemical agent be an overwhelming risk factor for cancer development. However, it is hoped that the availability of an array of molecular biomarkers will permit the dissection of the role of chronic low-level exposures to multiple chemical agents in the pathogenesis of human diseases. The development of putative molecular biomarkers for environmental chemicals should be based upon specific knowledge of metabolism, macromolecular adduct formation, and general mechanisms of action (4). The validation of any biomarker-cancer link requires parallel experimental animal and human studies. Ideally, an appropriate animal model is used to determine the associative or causal role of the marker in the disease pathway and to establish relationships between dose and response. The putative marker can then be validated in
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pilot human studies where sensitivity, specificity,accuracy, and reliability parameters are established (5). Data obtained in these studies can be used to assess intra- or interindividual variability, background levels, and relationship of the marker to external dose or to disease status, as well as feasibility for use in larger population-based studies. It is important to establish a connection, in humans, between the biological marker and the exposure or the outcome of interest. To fully interpret the information that the marker can provide, prospective epidemiological studies may be necessary to demonstrate the role that the marker plays in the overall pathogenesis of the disease. To date, few chemically specific exposure markers have been rigorously validated using this entire process. 11. Monitoring Ambient Exposures to Chemical
Agents Human exposure to environmental chemical agents occurs as a result of contaminated air, water, soil, and food. The measurement of toxicants in these media is accomplished by a wide array of analytical methodologies. Approaches to make assessments of exposure include questionnaires, personal external monitors, and measurements of chemicals in the ambient environment. Questionnaires have been extensively used to determine broad dietary exposures to compounds, smoking histories, and genetic backgrounds. While questionnaire data have proven useful in some circumstances, for example, in assessing current smoking status, this approach is very imprecise for measuring other exposures, such as those occurringthrough diet where the knowledgebase of specific dietary chemical agents is still limited. A complexity arising from the use of ambient measurements to determine exposure status of individuals is the heterogeneous nature of most environmental contaminations. It is rare for chemicals to be evenly distributed in environmental media. One illustration of this problem is the measurement of aflatoxin levels in grains. The distribution of aflatoxins in grains is very uneven due to variable patterns of mold growth, such that the sampling procedure used to determine aflatoxins in grains often results in a greater than 100% coefficient of variation (6). This extensive variation makes it very difficult to extrapolate data on grain contamination to an individual's exposure. Given these problems, the goal for the development of specific biomarkers to assess exposure is multifold, including an ability to integrate multiple portals of entry, to integrate fluctuating exposures relating time of exposure to dose, and to examine mechanistically important biological targets. This goal assumes increased relevance when it is recognized that safety regulations are often set on the basis of ambient exposure determinations.
0893-228~/93/2706-0764$04.00/0 0 1993 American Chemical Society
Forum: Frontiers in Molecular Toxicology
Chem. Res. Toxicol., Vol. 6, No. 6,1993 765
exposures; however, they do not provide evidence that toxicologic damage has occurred, possibly resulting in cancer or other diseases. Among the various possible Given the problems described above for extrapolating biomarkers reflective of these disease end points, the ambient measurements to specific individual exposures, measurement of carcinogen-DNA and -protein adducts it has been well recognized that measures of internal dose is of significant interest because they are direct products of a specific agent provide a clearer demonstration that of (or surrogate markers for) damage to critical macroa toxicant has been absorbed and possibly distributed in molecular targets. Many different types of analytical the body. Many direct measurements of toxic chemicals techniques have been devised to measure chemicalor their metabolites in body fluids and excretia (e.g., blood, macromolecular adducts, and these have been recently urine, feces, milk, amniotic fluid, sweat, hair, nails, saliva, reviewed (11). These techniques have been used to breath) have been done. One example of direct measuremeasure composite and specific DNA adducts in cellular ment is human breath analysis to determine specific DNA isolated from peripheral lymphocytes, bladder, and solvent vapors in exhaled air (7). In this type of analysis colonic tissues, as well as excreted DNA adducts in urine the subject exhales through a carbon-containing tube of humans exposed to environmental toxicants. In adconnected to a respirometer with subsequent measurement dition, these types of techniques have been applied to the by gas chromatography. This method has been used to clinical setting to examine DNA adducts of people determine styrene exposure, particularly in workers in the undergoing chemotherapy with alkylating agents in an shipbuilding industry. attempt to associate adduct levels with clinical outcome Smoking status in people has been extensively examined (12,13). by using urinary and blood nicotine or cotinine levels (8). One recent study has examined a variety of molecular Data derived from these studies have shown that, qualbiomarkers to assess human exposure to complex mixtures itatively, cigarette smoking can be assessed; however, the of environmental pollution in Poland (14).Measurement quantitative level of numbers of cigarettes smoked is of genotoxic damage in peripheral blood samples from difficult to determine by these methods. In a more recent residents of high-exposure regions indicated that envistudy, specific metabolites of one of the tobacco-specific ronmental pollution is associated with significant increases nitrosamines, 4-(methylnitrosamino)-l-(3-pyridyl)-l-b~- in carcinogen-DNA adducts (polynuclear aromatic hytanone (NNK),’ a potent chemical carcinogen, were drocarbon-DNA and other aromatic adducts), sister quantified in the urine of smokers (9). These metabolites chromatid exchanges, chromosomal aberrations, and frewere not detected in urine of nonsmokers, and this study quency of increased ras oncogene expression. Perera and provides the first evidence for metabolites of tobaccocolleagues found that the presence of aromatic adducts on specificnitrosamines in human urine. This is an important DNA was significantly correlated with chromosomal finding because these tobacco-specific nitrosamines may mutation, providing a possible link between environmental be causally related to both active and passive smokingexposure and genetic alterations relevant to disease (14). induced cancers, and variations in the levels of these Another example of exposure to complex mixtures occurs markers might be related to disease risk for exposed with cigarette smoking. Polycyclic aromatic hydrocarbonindividuals. DNA (PAH-DNA) adduct levels have been detected in Another example of internal dose determination is the specific subsets of white blood cells. Santella et al. (15) measurement of fat or tissue concentrations of compounds have found that DNA combined from lymphocyte and such as polychlorinated biphenyls (PCBs),polybrominated monocyte fractions of smokers exhibited detectable levels biphenyls (PBBs),organochlorine pesticides, and dioxins of DNA adducts with a mean of 4.38 f 4.29 adducts/l0(8) (TCDD). One recent study reported the relation between nucleotides, while the corresponding values were 1.35 f exposure to PCBs and to DDE [l,l-dichloro-2,
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