Division of Environmental Health Science, Joseph L.Mailman School of Public Health at Columbia University, and Herbert Irving Comprehensive Cancer Center, Columbia University, New York, NY 10032, USA
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Abstract |
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Abbreviations: AFB1, aflatoxin B1; IGF-1, insulin-like growth factor-1; PAH, polycyclic aromatic hydrocarbon.
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Introduction |
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Molecular epidemiology has as its ultimate goal the prevention of cancer, a disease that in the USA claims over 500 000 lives annually, with more than 1 000 000 new cases diagnosed each year (9). Even more clearly than in 1982, various lines of evidence indicate that the great majority of cancer is, in principle, preventable because the factors that determine cancer incidence are largely exogenous (10). This evidence comes mainly from epidemiologic studies and includes: (i) time trends in cancer incidence and mortality; (ii) geographic variations and the effects of migration; (iii) the identification of specific causative factors such as cigarette smoking, occupational and environmental chemicals, radiation, dietary factors and viruses; and (iv) the observation that the majority of human cancers do not show simple patterns of inheritance. Genetic factors are clearly important in terms of influencing individual susceptibility to carcinogens; and in certain rare forms of human cancer, hereditary factors play a decisive role. However, external factors represent the greatest opportunity for primary prevention. This is an optimistic message because it means that the development of cancer is not an inherent consequence of the aging process per se, and the human species is not inevitably destined to suffer a high incidence of cancer. This awareness has lent greater urgency to the search for more powerful tools for primary preventionfor early warning systems to identify causal environmental agents and flag risks well before the malignant process is entrenched.
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Overview of recent progress in molecular epidemiology |
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Although molecular epidemiology has the advantage of being directly relevant to human risk, unlike animal or other experimental models that require extrapolation to humans, it is also subject to many of the general limitations of epidemiologic studies, such as vulnerability to confounding factors. In addition, many of the biomarkers used in molecular epidemiologic studies require further validation; as discussed in detail elsewhere (5,11). Furthermore, without adequate precautions the use of these biomarkers carries the potential for serious social harm (reviewed in refs 12 and 13). At the present time, many biomarkers can be useful in assessing exposure, early deleterious molecular and biologic effects and potential risk for a group or population; most are not, however, sufficiently characterized or validated for routine use in screening, diagnosis or quantitative estimation of individual risk of developing cancer.
As we proposed in 1982 (1), biomarkers have generally been classified into four major categories: (i) internal dose; (ii) biologically effective dose; (iii) preclinical biologic effects; and (iv) susceptibility, with some overlap between them (14) (Figure 1). Of the four categories, internal dosimeters have been most widely used, particularly in the workplace, to determine exposure to carcinogens and other toxicants. Because of their precision, reliability, and relevance to individual risk, biomarkers of internal dose have been used to great advantage in conjunction with more traditional approaches: monitoring of ambient or workplace concentrations of the selected agent(s), and analysis of workers' records or questionnaires, including information on smoking, food consumption and other environmental exposures.
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Although markers of internal dose are a valuable supplement to conventional methods of assessing exposure, they do not indicate the extent to which a given compound has interacted with critical cellular targets. Therefore, assays have been developed to measure the `biologically effective dose' of a compound, i.e. the amount that has reacted with critical cellular macromolecules, usually DNA, or a surrogate, such as specific proteins in the blood. The rationale for measuring carcinogenDNA adducts derives from the fact that a number of chemical carcinogens or their metabolites exert their biological effects by binding covalently to cellular DNA, thereby inducing mutations in critical cellular genes. Measures of specific carcinogenDNA adducts reflect not only individual differences in absorption and distribution but also differences in metabolism (activation versus detoxification) of the chemical, as well as the extent of repair of carcinogenDNA adducts that may have taken place subsequent to carcinogen exposure (2,1618). For many studies in humans, DNA from the target tissue is not readily accessible and thus surrogate cells or tissues are often used (e.g. peripheral blood cells, buccal cells or placenta). The relationships between the types and levels of adducts in the more readily available surrogate samples and those in the target tissue have not been well characterized in humans, but have been established for certain carcinogens in experimental animals (19). Another limitation is that, because of DNA repair and tissue renewal, levels of carcinogenDNA adducts generally reflect exposure during the previous several months, rather than exposure in the distant past (20).
Since 1982, when the in vivo occurrence of carcinogenDNA adducts was first reported in a human population (21), many different types of carcinogenDNA adducts have been detected in human cells and tissues (17,18,2224). [There was one prior report of methylated purine in two samples of human liver as a result of intentional poisoning (25).] Carcinogenprotein adducts, especially adducts formed with hemoglobin or albumin, have also been widely measured as surrogates for DNA damage (26,27). The use of longer-lived adducts with histone and collagen molecules is now being explored (26). The carcinogens whose derivatives have been found to covalently bind to DNA or protein include such diverse agents as ethylene oxide, 4-aminobiphenyl, PAH, AFB1, nitrosamines and cisplatin (17,18,22,2628). Not surprisingly, in many different populations, the adduct data show a general doseresponse relationship for the genotoxic carcinogens studied, with no apparent threshold. Adducts have gained relevance as a potential risk marker from the recent finding that patients with lung cancer had markedly higher PAHDNA adduct levels in their peripheral white blood cells than individuals without cancer, after taking into account differences in the amount of smoking and other potential confounders (17,29). Other molecular epidemiologic studies have prospectively linked DNA damage from AFB1 to liver cancer risk in Chinese and Taiwanese populations (3032). In virtually every study, the levels of DNA damage or protein adducts have been found to vary considerably between persons with apparently similar exposure (20,33,34). As has been shown with PAHDNA adducts, although the observed variability reflects a combination of true biologic variability, unaccounted for differences in exposure, within-person variability, and laboratory variation, a significant fraction is apparently contributed by biologic factors (35). This aspect is discussed later under the topic of biomarkers of susceptibility and geneenvironment interactions.
Although chemical-specific damage to DNA by exogenous agents remains a major mechanism in carcinogenesis, there is increasing recognition of the importance of other types of damage to DNA by both endogenous and exogenous factors, as a driving force in carcinogenesis. The types of non-specific DNA damage include deaminations, alkylations, base losses and oxidations. The magnitude of these effects can be influenced by inflammatory processes and co-exposure to dietary and environmental agents. Therefore, biomarkers that assess the extent of these types of DNA damage will play an increasingly important role in molecular epidemiologic studies. An example is the development of antibodies that recognize 8-hydroxyguanosine, one of the major DNA adducts produced by oxygen radicals (36).
The next category of biomarkers, which reflects subsequent events in the multi-step sequence of carcinogenesis, includes markers of early biologic effects resulting from exposure. Like the preceding categories of markers, these effects can be measured directly in target tissues or in a surrogate source of cells, such as peripheral white blood cells. Most of the available biomarkers assess various types of genotoxicity, including chromosomal aberrations, small deletions [loss of heterozygosity (LOH)], and point mutations.
The powerful PCR method and variations thereof have greatly facilitated the analysis of somatic mutations in molecular epidemiologic studies (37). It is now possible to detect such mutations in tissues of exposed individuals at a very early stage in the carcinogenic process; that is, even before cells carrying mutated genes have undergone clonal expansion. Characteristic patterns of mutations in tumors have provided valuable, albeit circumstantial, molecular evidence of the importance of specific environmental carcinogens. For example, because mutations of the p53 tumor suppressor gene and the ras oncogene are common events in human cancer, they can be useful reporters of carcinogenic events involved in the development of cancer. In a number of cases, the patterns of p53 and ras mutations in human tumors have been consistent with the types of DNA adducts implicated in their causation and with the types of mutations induced experimentally by the specific environmental carcinogens under investigation (38,39). Thus, the mutation profile or spectrum found in certain genes may, in some instances, serve as a fingerprint of the causative agent. In recent studies, PCR-based methods have demonstrated that histologically `normal' areas of the bronchial epithelium of cigarette smokers can display specific chromosomal areas of LOH that are often found in lung cancers (40). LOH can be considered a biomarker of biologic effect that may be useful in identifying individuals who do not yet have cancer but are at high risk.
Other types of biomarkers may be indicative of the dose and effects of agents that enhance the carcinogenic process without forming covalent adducts with cellular DNA or proteins, and without directly inducing gene mutations. These agents appear to act by disrupting gene expression, cell growth, apoptosis and/or differentiation. They include certain tumor promoters (12-O-tetradecanoylphorbol-13-acetate and phenobarbitol), xenobiotics (such as 2,3,7,8-terachlorodibenzo-p-dioxin, PCBs and various pesticides) and natural and synthetic estrogens and androgens. Assays for the biologic effects of these agents are only now being developed, and include occupancy of specific high affinity receptors that mediate the action of these compounds, levels of specific mediators (growth factors, growth factor receptors, second messengers like cAMP and diacylglycerol, protein kinases and the phosphorylation of specific proteins), and the expression of genes related to tumor promotion, cell proliferation, apoptosis and differentiation. Examples of markers related to cell proliferation are proliferating cell nuclear antigen (a nuclear antigen associated with cell division) and cyclins (proteins associated with cell-cycle regulation) (41). In the latter category, cyclin D1 may prove to be a very useful biomarker since it is induced by various mitogens and is frequently overexpressed in a wide variety of human cancers (42). Recently, overexpression of cyclin D1 in plasma has been associated with increased risk of breast cancer (D.L.Tang, A.Rundle, Q.W.Chen, J.Z.Zhou and P.Brandt-Rauf, manuscript submitted).
The fourth category of biomarkers includes those that relate to inherited or acquired variations in host susceptibility. This category is a burgeoning area of research and, among all the categories of biomarkers, generates the greatest concern about ethical issues (8,4345). The basic principles of multistage carcinogenesis and of the molecular biology of cancer predict that a number of factors, in addition to exposure to specific causative agents, influence the likelihood that tumors will develop in a given individual. These individual susceptibility factors can be acquired or inherited. Examples include interindividual differences in the metabolism (activation and detoxification) of carcinogenic chemicals, DNA repair, and the functions of protooncogenes or tumor suppressor genes, as well as interindividual differences in nutritional, hormonal and immunologic factors.
With respect to genetic factors, rare, highly penetrant dominant mutations in genes, such as p53 in the LiFraumeni syndrome and Rb in familial bilateral retinoblastoma confer high individual risk, but account for a small percent of all cancer (12,4648). In contrast, relatively common genetic traits that regulate metabolism and detoxification of carcinogens can have a major impact on the population attributable risk of cancer, even though their individual risk is low. For example, cytochrome P450 `phase I' enzymes can produce highly reactive DNA-damaging intermediates during the normal process of converting chemical carcinogens to excretable forms. CYP1A1, a cytochrome P450 enzyme that metabolizes PAH, is normally inducible. Thus, its level can vary 20-fold in the human liver and 50-fold in the human lung (49,50). Inducibility also varies between individuals, and high CYP1A1 inducibility has been correlated in a number of studies with risk of lung cancer (51,52). Polymorphic variations in the sequences of CYP1A1, CYP1A2, CYP1B1 and other P450s exist in the human population and specific forms have been associated with increased cancer risk in various populations (43).
Interindividual variation in `Phase II' detoxifying enzymes, such as GSTM1, can also contribute to individual susceptibility. About 40% of the population have a deletion at this locus which has been linked to increased risk of bladder and lung cancers (5356). Another common genetic factor related to increased cancer risk is the inheritance of a homozygous recessive mutation in the N-acetyltransferase gene resulting in the inability to efficiently detoxify aromatic amines via N-acetylation. The mutation is carried by ~50% of the population. It has been demonstrated that slow acetylators are at increased risk of bladder cancer, especially those occupationally exposed to aromatic amine bladder carcinogens (57). Furthermore, in a study of blond and black tobacco smokers, slow acetylators had higher levels of 4-aminobiphenyl-hemoglobin (4-AB-Hb) adducts in their red blood cells than fast acetylators who smoked the same type and quantity of cigarettes (58,59). In contrast to its protective effect in bladder cancer, the fast acetylator phenotype is associated with an increased risk of colon cancer (60,61), demonstrating the complexity of geneenvironment interactions with respect to cancer susceptibility. Moreover, combinations of metabolic polymorphisms are increasingly being linked to increased cancer risk (8).
Acquired or inherited variations in the efficiency or fidelity of DNA repair can also influence individual susceptibility to cancer. This principle is illustrated by the rare autosomal recessive disease xeroderma pigmentosum, in which defects in the excision of pyrimidine dimers and other bulky DNA lesions lead to a marked increase in susceptibility to skin cancers induced by sunlight (62). Indeed, studies of human lymphocytes have identified a ~5-fold interindividual variation in unscheduled DNA synthesis induced by UV exposure; and DNA repair deficiency (measured phenotypically) has been linked to increased cancer risk (63,64). In one study, compared with healthy controls, patients with lung cancer were five times more likely to have reduced ability to repair damage induced by the PAH metabolite, benzo[a]pyrene diol epoxide (65). Recent studies suggest that polymorphisms in the XPD nucleotide excision repair gene influence the risk of basal cell carcinomas in patients with psoriasis (66). In addition, studies of Taiwanese suggest that polymorphisms in another nucleotide excision repair gene, XRCC1, result in deficient DNA repair (67).
Rapid progress has been made in identifying the numerous genes that play a role in DNA repair pathways in mammalian cells, including direct damage reversal (via the enzyme O6-methylguanine-DNA methyltransferase), base excision repair, nucleotide excision repair, mismatch repair and double-strand break repair (68). It will be of interest to determine to what extent heritable polymorphic variations in some of these enzymes influence individual susceptibility to specific types of human cancer.
Using combinations of biomarkers, molecular epidemiology has reinforced prior evidence that risk from carcinogenic exposures can vary significantly with ethnicity, age or stage of development, gender, pre-existing health impairment and nutritional factors (for review see ref. 8). Biologically based interindividual variation in only a few susceptibility factors can lead to a significant increase in population risk over that expected based on an assumption of uniform susceptibility, possibly by an order of magnitude or more (69). Based on biomarker data, Hattis et al. have calculated that the variability in metabolic activity, detoxification and DNA repair among 95% of the US population could be as high as 85500-fold (69) with correspondingly high variability in cancer risk.
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Future directions and research needs in the field of molecular epidemiology |
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However, as knowledge of mechanisms in carcinogenesis has evolved, the available armamentarium of biomarkers is no longer sufficient. As discussed above, the majority of the available biomarkers used in molecular epidemiologic studies relate to agents that cause DNA damage and are mutagenic. At the same time, we know that a large number of chemicals (hormones, various tumor promoters, pesticides, retinoids, etc.) can enhance or inhibit the carcinogenic process through indirect genotoxic or epigenetic mechanisms. They appear to act by altering gene expression, cell proliferation, differentiation and/or apoptosis (programmed cell death). Therefore, a current challenge is to develop more biomarkers for this category of agents and to incorporate them into molecular epidemiology studies.
For example, there is increasing evidence that in addition to mutations due to damage to DNA, the carcinogenic process is often associated with aberrant hypermethylation of CpG islands in the promoter regions of various genes, leading to their transcriptional inactivation. These genes encode various proteins including the cell-cycle inhibitory protein p16ink4, the DNA mismatch repair protein hMLH1, and the detoxification enzyme glutathione S-transferase (70). Since these changes can enhance the process of tumor progression, they might provide informative biomarkers in cell samples obtained from individuals at risk of developing cancer.
Additional assays are needed to study oxygen radicals and oxidant stress, the metabolism of nitrogen oxide and nitrites, alterations in DNA methylation, the activities of specific protein kinases, activation of various cytoplasmic and nuclear receptors and transcription factors, cyclins and other cell-cycle control proteins, and aberrant cell proliferation, differentiation, apoptosis and angiogenesis. During the multistage process of carcinogenesis, mutations and/or epigenetic disorders in the expression of numerous genes can occur, and this can lead to marked changes in the overall circuitry of signal transduction and cell cycle control (for review see ref. 71). Recent advances in genomics, microarray technology and informatics have made it possible to analyze small numbers of cells for their profiles of expression of many different genes. As these techniques become more available they can be applied to exfoliated cells and small biopsies of normal tissues, precursor lesions and tumors.
As mentioned earlier, polymorphic variation in several types of genes may influence cancer susceptibility at the population level. In addition to the metabolism of xenobiotics, these polymorphisms can affect the metabolism of various dietary factors, the endogenous synthesis, metabolism and action of hormones, DNA repair, immune and inflammatory processes, oxidant stress, signal transduction and cell-cycle control. Therefore, recently launched nationwide efforts to assemble and characterize a large panel of such polymorphic genes through the Human Genome and Environmental Genome Project (72,73) should provide powerful new biomarkers to the field of molecular epidemiology. Automated DNA-chip technology will greatly facilitate the large scale application of this new technology.
An extremely important area for future application of the concepts and methods of molecular epidemiology is the role of nutrition and diet in cancer. Although epidemiologic studies suggest that nutritional factors play a causative role in >30% of human cancers, the precise roles of specific dietary factors are uncertain and often controversial. Current issues include: (i) the identification of specific cancer-preventive chemicals in fruits, vegetables and various phytochemicals, including antioxidants; (ii) the influence of genetic factors on individual cancer risk from dietary factors; (iii) the carcinogenic role of heterocyclic amines that are generated by cooking meat at high temperatures; and (iv) the contribution of specific dietary fats versus caloric excess per se to increased cancer risk. New types of biomarkers need to be developed to analyze these questions in specific human populations.
For example, it is well known that calorie restriction can markedly inhibit tumor formation in mice. A variety of mechanisms have been invoked. The level of insulin-like growth factor-1 (IGF-1) may play a critical role since the serum level of this protein is reduced in calorically restricted mice and continuous infusion of IGF-1 into these mice enhances tumor formation (74). A variety of other experimental studies implicate IGF-1 and signalling through its receptor (IGF-1r) in carcinogenesis. Futhermore, several types of human tumors, including breast, colon and lung carcinomas, overexpress either IGF-1 or IGF-1r (for review see ref. 75). High serum levels of IGF1 have been implicated in human cancer of the prostate (76) and breast (77). It is known that diets high in fat and high glycemic index carbohydrates can lead to a state of insulin resistance, with hyperglycemia, impaired glucose tolerance and hyperinsulinemia. One hypothesis is that this process can enhance colon carcinogenesis because of the mitogenic effects of insulin and related factors (for review see ref. 78). Therefore, assays for serum levels of IGF-1, insulin and other polypeptide growth factors may provide informative biomarkers in molecular epidemiologic studies on nutrition and cancer. A related issue is whether biomarkers can be developed to better assess the putative role of exercise as a protective factor for breast and other cancers.
Recent studies suggest that individuals who have a methyl-deficient diet are at higher risk of colon cancer if they carry a defective polymorphic form of the enzyme methylene tetrahydrofolate reductase (79). Moreover, a decreased level of PAHDNA adducts in the peripheral white blood cells of cigarette smokers has been reported in persons with high plasma levels of -tocopherol, but only in those individuals who lack the GSTM1 detoxifying gene, which is detected in 3050% of the Caucasian population (80,81). Studies are needed also on polymorphic variations in genes involved in energy metabolism or in the metabolism of lipids.
Specific viruses or bacteria may be important cofactors in the causation of certain types of human cancer, acting in concert with environmental chemical agents, dietary factors or hereditary susceptibility factors. Examples include hepatitis B and C viruses in the causation of liver cancer, EpsteinBarr virus (EBV) in Burkitt's lymphoma and nasopharyngeal carcinoma, HPV in cervical cancer, human Herpes virus in Kaposi's sarcoma and Helicobacter pylori in gastric cancer (82). Therefore, it is important to include biomarkers for various microorganisms in molecular epidemiologic studies on specific types of human cancer.
A recent trend that brings together cancer researchers interested in cancer epidemiology, chemoprevention and therapy is the increasing recognition that biomarkers developed in the field of molecular epidemiology may also be useful as early or intermediate endpoints in studies on cancer prevention by identifying `at risk' populations and then assessing the efficacy of various types of intervention. For example, in interventions to prevent first or second malignancies, biomarkers can help identify populations or individuals at high risk of cancer resulting from specific environmentgene interactions. Measures could then be taken to reduce exposure (i.e. through clean-up of the workplace, modification of the diet, tighter air quality regulations, etc.), to carry out early cancer detection screening, or to initiate chemoprevention when appropriate. In some situations a combination of these measures could be used. Appropriate biomarkers could then be used to provide feedback during these interventions (8385).
The field of molecular epidemiology is especially relevant to the very promising and rapidly expanding field of cancer chemoprevention, i.e. the use of specific, synthetic or naturally occurring compounds to inhibit the carcinogenic process before the development of malignant tumors (86). Both approaches emphasize the multistage nature of carcinogenesis. Specific agents used in cancer chemoprevention appear to act by inhibiting carcinogenic damage to DNA, mutagenesis, tumor promotion and/or tumor progression. In addition to identifying appropriate candidates for cancer chemoprevention studies, many of the biomarkers used in molecular epidemiology are useful as early or intermediate endpoints, to assess the efficacy of chemopreventive agents in humans. These biomarkers include assays for DNA adducts, mutations in specific genes, abnormalities in cell proliferation and alterations in gene expression. Thus, new bridges are being built between the fields of basic cancer research, epidemiology, prevention and therapy (71).
Finally, with respect to prevention, the use of biomarkers to quantify interindividual variability in response to exposure has significant implications for carcinogenic risk assessment and associated regulatory actions (8). The assumption underlying current risk assessment models, that all humans respond homogeneously to a specific carcinogen or mixture of carcinogens, is belied by the large interindividual variation observed within human populations exposed to similar levels of diverse carcinogens. These observations suggest the need for more stringent regulation of exposures to certain ambient or workplace pollutants or environmental tobacco smoke to protect the more sensitive subpopulations. Further molecular epidemiologic research on the relationship between exposure and susceptibility should also provide a better estimate of the percentage of cancer cases resulting from specific environmental exposures. Such estimates of attributable risk can be used to guide public policy toward reduction or elimination of the most significant causative factors and toward interventions targeted at specific subpopulations or individuals.
In conclusion, we believe that the potential benefits of biomarkers and molecular epidemiology in cancer prevention justify a major commitment to the further development and use of this approach and to addressing the ethical concerns involved in its application to cancer prevention. We are hopeful that this new discipline will continue to provide new insights into the causation of specific human cancers and will help us arrive at more effective strategies for cancer prevention.
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Notes |
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Acknowledgments |
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References |
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