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In order to evaluate the potential of biological markers in epidemiology and risk assessment of complex exposures, we review recent studies of macromolecular adducts and oncogene activation in human populations. Results are discussed in terms of the strengths and weaknesses of various study designs in order to identify the most promising approaches for future research.

To assess the utility of DNA adducts as biomarkers of exposure to carcinogens in an industrial population, a pilot study of roofers occupationally exposed to a mixture of polycyclic aromatic hydrocarbons was conducted. DNA was isolated from peripheral white blood cells of roofers and non-occupationally exposed subjects matched for age, sex and smoking status. Occupational exposures to anthracene, fluoranthene, pyrene, benzanthracene, benzo[a]pyrene, benzo[b]fluoranthene, benzo[g,h,i]perylene and benzo[k]fluoranthene were assessed by personal breathing zone air sampling and skin wipes. Exposures to benzo[a]pyrene in air of exposed subjects ranged from 0.60 microgram/m3 to 1.39 micrograms/m3, and exposures to total polycyclic aromatic hydrocarbon (the sum of eight hydrocarbons) ranged from 6.0 micrograms/m3 to 13.8 micrograms/m3 on the day before blood collection. In the biomarker studies 10 of 12 roofers, but only 2 of 12 comparison subjects, had detectable levels of aromatic DNA adducts by 32P-postlabelling assay (p less than 0.01). The two non-roofers with detectable adducts had levels at or near the detection limit of 2 adducts per 10(9) nucleotides. In two roofer samples which were studied in a mixing experiment, the major adduct spots did not co-migrate with the guanosine N2 adduct of benzo[a]pyrene diol epoxide. These results suggest that the 32P-postlabelling assay may be useful for monitoring exposures to complex mixtures of aromatic hydrocarbons in industrial populations.

Sensitive immunological methods for the detection of carcinogen-DNA adducts have recently been developed. These techniques are particularly useful for screening human populations for exposure to environmental carcinogens. Measurement of the biologically effective dose in humans may be useful in detecting carcinogenic hazards and carrying out risk estimates. We have developed monoclonal antibodies to several carcinogen-DNA adducts. These have included DNA modified by a benzo[a]pyrene diol epoxide (BPDE-I), 1-aminopyrene (1-AP) and 8-methoxypsoralen (8-MOP). BALB/cCr mice were immunized with the modified DNAs complexed electrostatically to methylated bovine serum albumin. Several stable clones have been isolated for each of the modified DNAs and characterized by enzyme-linked immunosorbent assay (ELISA). All antibodies are highly specific for the appropriate modified DNA and do not cross-react with nonmodified DNA. The antibodies to BPDE-I-DNA have significant cross-reactivity with DNAs modified by similar antitrans diol epoxides of benz[a]anthracene and chrysene. These DNAs all contain N-2 of guanine adducts. The antibody probably recognizes a shared determinant encompassing the guanine base and the hydrocarbon ring containing the hydroxide groups. The antibodies cross-react with BPDE-I-dG, the monoadduct isolated from DNA, but with lower sensitivity than for the intact modified DNA. They do not react with acetylaminofluorene (AAF) or 1-AP modified DNA, both of which contain C-8 of guanosine adducts. The antibodies to 1-AP-modified DNA demonstrate cross-reactivity with 8-nitro-1-aminopyrene- and 6-nitro-1-aminopyrene-modified DNA, as well as some slight cross-reactivity with BPDE-I-DNA and AAF-DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Should federal agencies use separate, less stringent guidelines for regulating epigenetic or nongenotoxic carcinogens on the assumption that thresholds are likely to exist for these agents? This article reviews recent initiatives by the Environmental Protection Agency that either propose or informally adopt this approach in light of responses from the scientific community and a review of the recent literature. Relevant background is provided by current research concerning the role of chromosomal damage and oncogene activation in carcinogenesis along with findings that classical promoters or "epigenetic" agents can induce both DNA damage and chromosomal rearrangements. The conclusion is that such a revision of cancer policy is not now supported by available scientific data concerning chemical carcinogenesis.

Blood samples were obtained from volunteers working in a Finnish iron foundry who were occupationally exposed to polycyclic aromatic hydrocarbons (PAH) and from control subjects not known to be occupationally exposed to this class of chemical carcinogens. Foundry workers were classified as belonging to high, medium or low exposure groups according to their exposure to airborne benzo[a]pyrene: high, greater than 0.2: medium, 0.05-0.2: low, less than 0.05 micrograms benzo[a]pyrene/m3 air). Aromatic adducts were found to be present in white blood cell DNA from most of the exposed workers using the enzyme-linked immunosorbent assay (ELISA) to detect aromatic DNA adducts and the 32P-postlabelling technique. There was a dose-response relationship between the estimated exposure and adduct levels by both methods, and a reasonable correlation between the results of the immunoassay and postlabelling carried out in two laboratories. The levels of adducts found in the samples from the high and medium exposure groups by ELISA ranged up to five adducts in 10(7) nucleotides: the aromatic adducts detected by the postlabeling assay were at a level of two adducts/10(8) nucleotides in the high and medium exposure categories. No effect due to age, sex or the smoking habits of the subjects was observed. The results indicate that DNA extracted from white blood cells of highly exposed workers is more likely to contain aromatic DNA adducts than that from workers without occupational exposure to PAH, but large interindividual variations were evident. This study suggests that the antibody and 32P-postlabelling assays may be useful in monitoring human exposure to known and previously unidentified environmental genotoxic agents.

A new tool for the study of occupational carcinogenesis is "molecular dosimetry" or biomonitoring to establish the biologically effective dose of carcinogens in workers. Human monitoring of biologically effective dose and preclinical response has the potential to flag the need for protective measures and/or surveillance. Comparable biologically effective dose and preclinical response data in humans and laboratory animals for whom tumor incidence is known can also enhance risk extrapolation between species. This paper will provide a brief overview of biomonitoring methods now under development, including advantages, limitations, applications to date, and research needs. Application to the monitoring of worker populations requires careful thought about the use to which monitoring data will be put.

We studied the level of knowledge that a group of general practitioners (N = 40), selected by means of conglomerate sampling, had about dementias. To perform this study we used a five-question open questionnaire. Our conclusions were: 1) In our sample, 80% of the GP's were not familiar with the initial symptoms of dementia; 2) The full clinical picture of dementia was well recognized; 3) There was little knowledge about the management of a possible case of dementia, the most usual procedure being to remit it to a specialist; 4) In general, they did not know what the possible causes for dementia are, arteriosclerosis being the most frequently mentioned; 5) A considerable number of doctors (40%) believed dementia to be curable.

Iron foundry workers, exposed to high levels of polycyclic aromatic hydrocarbons (PAHs), silica, and metal fumes and dusts, are at elevated risk of lung cancer. Benzo(a)pyrene and a number of structurally related PAHs are metabolically activated to diol epoxides (e.g., 7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a) pyrene) which are mutagenic, carcinogenic in experimental animals, and form covalent adducts with DNA. The levels of these adducts were measured in an enzyme-linked immunosorbent assay using a polyclonal anti-benzo(a)pyrene diol epoxide-I-DNA antibody which cross-reacts with DNA modified by diol epoxides of structurally related PAHs. DNA was analyzed from peripheral blood cells of 35 Finnish foundry workers and 10 controls. Workers were classified as having low (less than 0.05 micrograms/m3), medium (0.05-0.2 micrograms/m3), or high (greater than 0.2 micrograms/m3) exposure to benzo(a)pyrene (as an indicator of PAH). When adjustment was made for cigarette smoking and time since vacation, benzo(a)pyrene exposure was significantly related to adduct levels (P = 0.0001). Each of the three exposure groups had significantly elevated adduct levels compared to controls. Among the exposed workers, the low group differed significantly from the high and medium categories. This study supports the usefulness of monitoring adduct formation in a population occupationally exposed to carcinogens.

Molecular epidemiology is a promising new tool in the study of environmental carcinogenesis and, particularly, in cancer prevention. Genetic damage and mutation are believed to play a critical role in chemical carcinogenesis. By incorporating biologic markers of dose or response to carcinogens (such as mutagenicity of body fluids, carcinogen-DNA adducts, chromosomal abnormalities, and somatic cell mutation) into human bio-monitoring or molecular epidemiologic studies, one can detect potential hazards early and increase the power of studies to determine causal relationships. Such markers can also improve extrapolation of risks from experimental animals to humans or from one human population to another. During the past 5 years, there has been considerable progress in developing markers and applying them in human (largely pilot) studies. A review of this experience--with particular emphasis on carcinogen-DNA adducts--affords a better awareness both of the significance of biologic markers and the research needed to fill gaps in understanding. Criteria for marker validation and sound study design are presented that should greatly enhance future research.

In a molecular epidemiological study of lung cancer cases (n = 81) and noncancer controls (n = 67), polycyclic aromatic hydrocarbon (PAH)-DNA adducts were evaluated in peripheral blood leukocytes from all subjects and in a smaller number of lung tissue specimens collected prior to or at surgery. Sister chromatid exchanges (SCE) in lymphocytes were also studied in a subset of cases and controls. Questionnaire, medical record, or tumor registry data provided a family history of cancer, as well as information on cigarette smoking, dietary and occupational exposure to PAHs, and other factors related to SCEs. In both cases and controls PAH-DNA adducts in leukocytes measured by an enzyme-linked immunosorbent assay were not significantly related to age, sex, ethnicity, amount of cigarette smoking, passive smoking, dietary charcoal, or caffeine consumption. Nor did family history of cancer or histological type of cancer significantly affect adduct levels. However, when subjects were stratified by smoking status (current, former, and nonsmoker), lung cancer cases who were current smokers had significantly higher levels of covalent adducts than current smoker controls. A seasonal variation was observed in PAH-DNA binding, with a peak in adduct levels during July-October. This peak corresponds to that seen in a prior study of aryl hydrocarbon hydroxylase inducibility by other investigators. The finding of significant levels of PAH-DNA adducts in former smokers and non-smokers supports an earlier observation that this marker is not smoking specific but reflects a pervasive and variable "background" exposure to PAH. These results are consistent with a genetically determined enhancement of PAH-DNA adduct formation in leukocytes of lung cancer cases which is evident in current smokers. The results in lung tissue are limited by the small number of samples. Adduct levels were not significantly increased in lung tissue of smokers compared with nonsmokers. An inverse linear correlation was seen between adduct values in lung tissue and age of the donors. SCEs were significantly related to pack years of smoking. However, there was no difference in the frequency of SCE between cases and controls; nor were SCE and DNA adducts significantly correlated in this small sample.

In 1987, investigators (Ames et al.) concluded that the risks of man-made industrial carcinogens and pesticides (outside of the workplace) are trivial compared with the risks of naturally occurring carcinogens found mostly in the diet. They used a ranking system based on human exposure and rodent potency (HERP) data to arrive at this conclusion. As a result, they recommend that regulatory agencies, such as the Environmental Protection Agency and the Food and Drug Administration, base their priorities in this area on their HERP system. We analyzed the assumptions and data set upon which the HERPs were based, concluding that such a simplified approach to set public health policy is inappropriate given the underlying uncertainties. However, we note that when comparisons are consistently based on estimates of average daily exposure to common carcinogens, the HERP scores of many man-made pollutants are comparable to those of naturally occurring carcinogens in the diet.

Benzo(a)pyrene(BaP) originating from fossil fuel and other organic combustion processes is largely adsorbed on fine particulate and hence is a widespread atmospheric pollutant. Available emissions and air quality data are based on the total weight of particulate matter without reference to size and give little information on trends and concentrations of fine particulate BaP. Greater reliance on coal, synfuels and diesel fuel for energy production and transportation will significantly increase ambient levels of BaP. Because of the particulate size, BaP is substantially deposited in the lower lung and readily eluted into surrounding tissue. After elution in the lung, BaP is metabolically activated to its electrophilic, carcinogenic from by a complex enzyme system whose activity is increased by prior exposure to air pollutants, cigarette smoke and certain drugs. The resultant diol epoxide metabolite has been shown to bind covalently with the DNA of the lung. In experimental animals, BaP is a potent initiating carcinogen whose action is enhanced by sulfur dioxide, promoting agents and carrier fine particles. The effect of small, divided doses of BaP has been shown to be greater than that of a single high dose; no threshold has been established. Epidemiological studies show that mixtures containing BaP (such as urban air, industrial emissions and cigarette smoke) are carcinogenic and may interact synergistically. Occupational studies indicate that the action of BaP-containing mixtures is enhanced in the presence of SO2. However, quantitative risk assessment for BaP is precluded by problems in extrapolating to the general population from small-scale animal studies; uncertainties in findings of epidemiology; and imprecise exposure data. Existing stationary and mobile controls preferentially remove coarse particulate matter and are inefficient collectors of the particulate BaP. In the current absence of health and environmental standards for BaP, there is little incentive to control BaP emissions. BaP meets the criteria for regulation under the Clean Air Act; however, no such BaP standards have yet been proposed.

An interlaboratory comparison of immunoassays using antisera elicited against benzo[a]pyrene-diol-epoxide-modified DNA (BPDE-I-DNA) was carried out resulting in standardization of antisera, competitors and assay conditions. The assays used included competitive enzyme-linked immunosorbent assays (ELISA) with color and fluorescence endpoint detection and an ultrasensitive enzyme radioimmunoassay (USERIA) with a radioactive endpoint. Three different antisera were compared, two of which were obtained from different rabbits immunized with the same BPDE-I-DNA and a third from an animal immunized with another BPDE-I-DNA sample. Samples of standardized BPDE-I-DNA with high (36 pmol adduct/microgram DNA; 1.2 adducts/10(2) nucleotides) and low (4.5 fmol/microgram DNA; 1.5 adducts/10(6) nucleotides) modification levels were prepared and used in each laboratory. The antisera were all elicited against DNAs modified to a high extent, and it was therefore not surprising that they detected adducts in a slightly modified DNA sample with lower efficiency than those in highly modified DNA samples. The discrepancy of antibody recognition between the highly and slightly modified samples varied between 1.4- and 11.2-fold depending on the antiserum and assay. To ascertain the quantitative capability of the immunoassays, the modification level of DNA isolated from mouse keratinocytes treated with [3H]benzo[a]pyrene was determined by radioactivity and immunoassay. These results indicated that when a biological sample is assayed against a BPDE-I-DNA standard modified in the same range as the biological samples (4.5 fmol/microgram), quantitative recovery of adducts is achieved by immunoassay. These studies resulted in the realization that interlaboratory differences in immunoassay procedure can have significant consequences for data comparison and that where possible it is preferable for laboratories to use the same antisera and modified DNA standards.

In 1987 researchers from Berkeley (CA), proposed an index (HERP) for ranking human carcinogens. The index was derived from human exposure data and rodent carcinogen potency. They concluded that the risks for the US population from man-made industrial carcinogens are trivial compared with the risks from naturally occurring carcinogens in the diet. Analysis of the assumptions and data upon which the HERPs were based revealed major limitations and we concluded that such a simplified approach is inappropriate given the underlying uncertainties. However, when we made a comparison consistently based on estimates of average daily exposure to common carcinogens, the HERP scores of man-made pollutants were comparable to those of naturally occurring carcinogens.

In order to validate markers of internal dose and biologically effective dose of carcinogens, a battery of measurements was made on blood samples from 22 smokers and 24 nonsmokers. The markers included immunoreactivity in an enzyme-linked immunosorbent assay (ELISA) quantified in white blood cells with the use of a polyclonal anti-benzo[a]pyrene diol epoxide-I-DNA antibody, 4-aminobiphenyl hemoglobin (4-ABP-Hb) adducts measured by negative chemical ionization mass spectrometry, sister chromatid exchange (SCE) in cultured lymphocytes, and cotinine in plasma measured by radioimmunoassay. Several blood samples were drawn from each subject. In blood samples 1 and 3 having detectable levels of DNA adducts, mean femtomole-per-microgram levels were consistently higher among smokers compared to nonsmokers. The borderline significance of this difference may be attributable to the small numbers of subjects. Consistently higher adduct levels were seen in females compared to males. In sample 3, adduct levels were significantly correlated with measurements of active smoking in smokers and with passive smoking in nonsmokers. By contrast to the ELISA data, which may reflect cumulative exposure from multiple background sources, the 4-ABP-Hb assay was able to distinguish clearly between smokers and nonsmokers. SCEs were significantly elevated in the smokers compared to nonsmokers. Also observed were significant correlations between 4-ABP-Hb and both cotinine and SCEs, as well as a positive correlation between the 4-ABP-Hb and DNA adduct levels (sample 3) that was highly significant. The correlation between DNA and 4-ABP-Hb adducts was significant in smokers but not nonsmokers (sample 3). These results support the need for batteries of markers to detect and to quantify the carcinogenic dose to humans resulting from both specific and "background" environmental exposures.

Methods are needed to improve the ability of biomonitoring and epidemiological studies to identify potential carcinogenic hazards and to quantify human risk. The limitations of pharmacokinetic models can be mitigated by the direct measurement of molecular markers of biologically effective dose of carcinogen. Parallel animal and human studies are recommended as a means of validating these markers.

Lung cancer is now the major cause of cancer deaths in the USA and is an increasingly significant cancer worldwide. Biological markers could be used to prevent lung cancer by allowing more timely and precise understanding of the role of environmental factors. So far, biological markers that can serve as carcinogen dosimeters have been investigated in only a small number of pilot studies of populations with current or past exposure to lung carcinogens. We describe several of our collaborative studies involving smokers, various worker populations, lung cancer cases and controls in order to illustrate the advantages and the limitations of 'molecular epidemiology'. The enzyme-linked immunosorbent assay (ELISA) with antibodies to polycyclic aromatic hydrocarbon (PAH)-DNA adducts has been used in conjunction with one or more of the following: physicochemical techniques to monitor carcinogen adducts on haemoglobin, cytogenetic methods to quantify sister chromatid exchange (SCE) and chromosomal aberrations, and Southern and western blot analyses of oncogene activation. Increased levels of markers of biologically effective doses have generally been seen in exposed and high-risk groups when compared to controls. We have also observed significant background levels of such markers and interindividual variation in levels of certain biological markers resulting from exposures to carcinogens. Thus, these methods may be particularly useful in identifying segments of the population that have received a significant effective dose and hence can be considered to be at elevated risk of cancer. Such studies are necessary to validate laboratory methods and lay the groundwork for more definitive molecular epidemiological investigations of lung cancer.