Human DNA adduct measurements: state of the art.

Human DNA adduct formation (covalent modification of DNA with chemical carcinogens) is a promising biomarker for elucidating the molecular epidemiology of cancer. Classes of compounds for which human DNA adducts have been observed include polycyclic aromatic hydrocarbons (PAHs), nitrosamines, mycotoxins, aromatic amines, heterocyclic amines, ultraviolet light, and alkylating cancer chemotherapeutic agents. Most human DNA adduct exposure monitoring has been performed with either 32P-postlabeling or immunoassays, neither of which is able to chemically characterize specific DNA adducts. Recently developed combinations of methods with chemical and physical end points have allowed identification of specific adducts in human tissues. Studies are presented that demonstrate that high ambient levels of benzo[a]pyrene are associated with high levels of DNA adducts in human blood cell DNA and that the same DNA adduct levels drop when the ambient PAH levels decrease significantly. DNA adduct dosimetry, which has been achieved with some dietary carcinogens and cancer chemotherapeutic agents, is described, as well as studies correlating DNA adducts with other biomarkers. It is likely that some toxic, noncarcinogenic compounds may have genotoxic effects, including oxidative damage, and that adverse health outcomes other than cancer may be correlated with DNA adduct formation. The studies presented here may serve as useful prototypes for exploration of other toxicological end points.

Methods that have been used for sensitive detection of carcinogen-DNA adducts in humans include immunoassays (5), immunohistochemistry (6,7), 32p_ postlabeling, (8,9), fluorescence and phosphorescence spectroscopy (10), gas chromatography-mass spectrometry (GC-MS) (11), atomic absorbance spectrometry (AAS) (12,13) and electrochemical conductance (ECC) (14). Typically, the techniques that are used without preparative procedures are not absolutely quantitative or able to chemically characterize a specific adduct, but they are highly effective screening tools. Recent advances combining preparative methods [immunoaffinity chromatography (IAC), high performance liquid chromatography (HPLC) or other chromatography] with immunoassays, 32P-postlabeling, synchronous fluorescence spectrometry (SFS), and GC-MS have allowed identification and quantitation of specific DNA adducts in human tissues potentially resulting in more precise exposure documentation.
This paper is an overview of methodologies and their application in exposure biomonitoring, focusing on human blood cell PAH-DNA adduct measurements obtained concomitantly with ambient PAH monitoring. DNA adduct dosimetry and correlation of DNA adducts with other biomarkers are considered. aCompetitive repair assay, analogous to immunoassay.  (15)(16)(17) have been widely used to quantify and localize xenobiotic-induced DNA damage (18)(19)(20)(21) and to measure DNA adduct formation in human tissues (22,23). Competitive radioimmunoassays (RIAs) and enzyme-linked immunosorbent assays (ELISAs), able to detect human DNA damage with sensitivity in the range of one adduct in 108 unmodified nucleotides, have been established with these antisera. Immunoassays are reliable, inexpensive, and allow for the analysis of more than 20 samples per day. Disadvantages include the requirement for relatively large amounts of DNA (200 pg) and a lack of absolute specificity because of antibody cross-reactivity. Cross-reactivity with unmodified DNA, unmodified nucleotides, or carcinogen alone rarely occurs (24), but there may be recognition of other adducts of the same carcinogen or adducts of other chemically related compounds (25). Therefore, unless prior separation of adducts is used, the values obtained for human samples may reflect measurement of multiple, chemically similar DNA adducts. In addition to use with quantitative immunoassays, anticarcinogen DNA adduct antisera have been used for immunohistochemical staining of human tissues (21,23,26). In general, immunohistochemistry is less sensitive than ELISA, but the approach may be relevant for identification of susceptible cell types in complex tissues.
Xenobiotic exposures that have been examined by immunoassay in samples from human subjects (

32p-posdabeling
The 32P-postiabeling technique is widely used for human DNA adduct detection (9,20,23), largely because of its high sensitivity (routinely one adduct in 109 nucleotides) and application to small quantities of DNA (2-10 pg). The method is based on the radiolabeling of adducts with high specific activity 32p from y32P-ATP by T4 polynucleotide kinase. DNA isolated from a tissue of interest is digested to 3'-monophosphates and phosphorylated to form 5'-32P-3'-bisphosphates. Adducts in the labeled mixture are separated by thin layer chromatography (TLC) in multiple (most often four) directions. Relative quantitation has been approached by scraping materials from the TLC plates and subjecting them to radiochemical analysis; however, recent development of a sensitive radiomatic apparatus for chromatogram scanning facilitates the quantitation and provides a measure of protection for laboratory personnel. Advantages and disadvantages of this method have been discussed in detail elsewhere (23,49).
Individuals receiving many different types of potentially genotoxic exposures have been demonstrated to have potential adduct spots by 32P-postlabeling ( these are termed "aromatic adducts." The chemical identification of such adducts has rarely been achieved, but recent sophisticated modifications to the basic methodology have allowed tentative identification of some specific adducts (30,50,51). Overall, correlations of adduct levels with documented human exposure appear to be good (52)(53)(54)(55)(56), but the quantitative data should be interpreted with caution because the adducts are unidentified and the efficiency of phosphorylation is often unknown and uncontrolled (51,(57)(58)(59)(60).

Luminescence and Phosphorescence Spectroscopy
Luminescence spectroscopy is possible only with carcinogens having intrinsic fluorescence, such as PAHs and aflatoxins. In SFS, both the excitation and emission monochromators are driven simultaneously at a distance equal to the Stoke's shift so that a fluorescence signal is observed when the last excitation maximum and first emission maxima are met. This approach simplifies the spectrum and allows for the generation of more complete fluorescence excitation-emission matrices (61)(62)(63). Conventional SFS detection of (7R)-N2-110-[r-7,t-8,t-9trihydroxy-7,8,9,10tetrahydrobenzo [a] pyrene]yl}-deoxyguanos ine (BPdG) in human tissues rec hydrolysis (0.1 N HCI, 90°C, 3 DNA and isolation of the B[a]P i r-7, t-8, t-9, c-I 0-tetrahydroxytetrahydrobenzo [a] pyrene, (BPtetrol). A detection limit of one 106 to 107 unmodified nucleotide achieved for 100 pg of DNA (64-A recently developed approac the use of analytical solid mat phorescence spectroscopy to d 7,10/8,9-tetrol. The method us matrix composed of hydrophobic trins to adsorb PAH residues fo laser excitation (67), and tht detection is 20 to 50 times l i conventional SFS.
The major limitations to t fluorescence spectroscopy for the of carcinogen DNA damage in h lack of prior knowledge of addi istry, a requirement that the a fluorescent, and a requirement tively large quantities of sam (100-1000 pg). After the initial equipment, assays can be p rapidly and inexpensively. To d; 2), fluorescence studies have been to detection of DNA adducts co pyrene fluorophore (49,68), de exfoliated aflatoxin adducts in u 0' 0 and N7-methyl-deoxyguanosine (N7me-IR dG) and 06methyl-deoxyguanosine </ (06me-dG) adducts in liver in a case of acute poisoning (70).

OCH3
Gas Chromatography-Mass Spectrometry Gas chromatography-mass spectrometry (49,71) is highly specific and has had widespread application in measurement of both carcinogen-protein adducts and car-,CH3 cinogen-DNA adducts (72)(73)(74)(75)(76). Mass s<Ns spectrometry requires derivitization of the compound of interest to increase mass and N volatility; vaporization of the sample; ion-RlsosE ization, which can be achieved in a number d RIBOSE of ways (electron impact, fast atom bombardment, chemical ionization, and laser desorption); collimation of the charged particles; and acceleration into the mass analyzer. The spectrum of ions detected comprises the molecular ion plus the fragment ions including the base peak. The base peak, the most intense signal, is most 0-tetrahydro-commonly used for quantitative sample ianosin-(8-y1)-analysis by single-ion monitoring when the [E) aflatoxin mass spectrum of a compound of interest is xyguanosine. already known. Methods using GC-MS have been developed for several different exposures luires acid (Table 2). Alkyl purine adducts in human hr) of the urine have been the most extensively studresidues as ied (11,72,73,(77)(78)(79)(80). These investigations 7,8,9,10-have examined the exfoliation of adducts -7,10/8,9-by seeking the presence of N7me-dG, adduct in N7et-dG, N7-hydroxethyldeoxyguas has been nosine (N7-OHet-dG), 3-methyladenine -66).
(3me-Ade), 3-ethyladenine (3et-Ade), 3--h involves hydroxyethyladenine (3OHet-Ade), and 3trix phos-benzyladenine (3bz-Ade) in human urine. Ietect BP-Investigations using single-ion monitoring ses a solid GC-MS have been extended to the meacyclodexsurement of 4-(N-nitrosomethylamino)-1ollowed by (3-pyridyl)-1-butanone (NNK) adducts in e limit of human lung and tracheal DNA of smokers wer than and nonsmokers (81). In addition, the presence of BPdG has been demonstrated the use of in placental tissues of smokers and nondetection smokers (82). Samples of human urinary iumans are bladder and lung DNA, digested and subuct chemjected to negative ion GC-MS, were shown adduct be to contain N-deoxyguanosin-(8-yl)-4t for relaaminobiphenyl (dG-C8-4-ABP) at levels ple DNA that compared well with 32P-postlabeling cost of the analysis of the same samples using approerformed priate standards (83). Similarly, the C-8 ate (Table DNA adduct  Atomic Absorbance Spectrometry Atomic absorbance spectrometry is an analytical method for determining elements based on the absorbance of radiation by free atoms released by high-temperature combustion. It is most valuable in the detection of metal ions and has been successfully applied, for human dosimetry, in the monitoring of DNA from cancer patients treated with the platinum drugs, cisplatin and diamminecyclobutane-dicarboxylatoplatinum (II) (carboplatin) ( Table  2). These drugs have been shown by AAS (12) and other methods to bind covalently to DNA (45, [85][86][87]. For measurement of platinum bound to DNA in tissues of human cancer patients, unknown samples are quantified by comparison to a standard curve, and sufficient sensitivity is only obtained using an AAS with Zeeman background correction (86,88).

Electochemical Conductance
Electrochemical conductance requires the application of a voltage across two electrodes that are immersed in an electrolytic solution. An electric current flows, allowing the measurement of electrical conductance, which is proportional to the analyte concentration (89). This technique has the advantage of specificity because it can be directly applied to individual HPLC fractions (14). However, factors related to the chromatographic conditions (gradient elution, temperature, and pressure) may cause problems with detector performance (90,91). Electrochemical conductance has been used to monitor oxidative damage in DNA ( Table 2). This use of the method is complicated by the fact that oxidation of DNA may occur during the extraction procedure (92); however, 8-hydroxydeoxyguanosine (80H-dG) is detectable, whereas unmodified deoxyguanosine is not. The approach is sufficiently specific and sensitive for human biomonitoring, but investigations so far have been limited to determining 80H-dG in urine, peripheral white blood cells, or bronchial alveolar macrophages (93,94).

Combinations of Methods
The most frequently applied detection methods, immunoassays, 32P-postlabeling, and fluorescence spectroscopy, used in the absence of micropreparative techniques, provide a broad screen and indicate exposure. However, recently devised combinations of methods produce more specific and readily comprehensible data. Such approaches are necessarily more labor intensive, time consuming, expensive, and demanding when applied to large numbers of specimens, but the information they provide is invaluable for human exposure assessment.
Most combinations of methods to improve the specificity of DNA adduct detection use either conventional chromatographic separation or immunoaffinity chromatography as a first step. When a human DNA sample is digested and subjected to HPLC, even though the adducts cannot be observed by conventional monitoring, the fractions known to contain specific adducts can be analyzed by immunoassay, 32P-postlabeling, or GC-MS. For example, the sensitivity and specificity of ELISAs for human DNA adduct monitoring have been enhanced by combination with prior HPLC. This approach has been applied to human gastric mucosa (32) and liver samples (33) using antisera specific for alkyl-modified nucleosides. Chromatographic separation by HPLC has also been combined with 32P-postlabeling; a recent review (59) covers the subject. A highly successful line of experimentation has combined two chromatographic steps with 32p_ postlabeling to detect specific o6and N7-alkyl-dG adducts in human lung and lymphocytes (51,57,95). The development of this method has facilitated the use of internal and cochromatography standards. In an another approach, Stillwell et al. (79) employed two chromatographic steps prior to the GC-MS determination of 3me-dA and 7me-dG adducts in the urine of smokers. Finally, in a novel set of experiments, HPLC was used as the first step of a procedure combining 32P-postlabeling with immunoprecipitation (termed PREPI) for the detection of 06me-dG, 04me-dG, and 04et-dG in human liver and leukocyte DNA samples (50).
Immunoaffinity chromatography provides a valuable purification step that has been widely used to improve the specificity of other methods. Antibodies elicited against DNA adducts or carcinogenmodified DNA samples can be covalently bound to a matrix and the resulting material used in columns that bind and elute specific adducts in a DNA digest. Because most antisera have cross-reactivity for families of structurally similar DNA adducts, IAC concentrates structurally similar DNA adducts (96)(97)(98). Further separation of adducts by HPLC is frequently required before specific adduct determination is possible.
For PAH exposure, IAC and HPLC of human lung and placenta have been combined with different end points, including SFS (82,98,99), GC-MS (82), and 32P-postlabeling (99,100), to provide evidence of BPdG formation. Using this methodology, it is possible to detect one BPdG adduct in 108 nucleotides.
Immunoaffinity chromatography and GC-MS have been combined to detect exfoliated 3-alkyl-adenines in human urine (11,80). Recently, Bianchini et al. (101) reported the combination of IAC, HPLC, and ECC to measure N7me-dG in human pancreas. Aflatoxin exposure dosimetry has been accomplished by combining IAC with HPLC and UV absorbance (A362) to detect adducts in human urine and tissues (28,102,103). In other studies IAC has been combined with 32P-postlabeling and TLC to detect 06 me-dG (104).

Human DNA Adduct Formation as an Exposure Dosimeter Comparison ofAmbient B[a]P Levels with DNA Adduct Formation
A number of investigatiors have attempted to monitor ambient air for B[a]P and other PAHs while simultaneously measuring blood cell DNA adducts in occupationally or environmentally exposed individuals. Studies focused only on smoking have been excluded here because the dosimetry is necessarily crude, and PAH-DNA adduct levels in blood-cell DNA are not consistently higher in smokers. In fact, the results suggest that smoking is only one of many factors that contribute to adduct formation in blood cell DNA. This analysis focuses on adduct measurements in human bloodcell DNA because human tissue biopsies are not practical for routine analysis.
A summary of investigations in which ambient B[a]P levels were compared to PAH-DNA adduct analyses in human blood cells is shown in Table 3. Overall, the data suggest that increased ambient pollution levels (using B[a]P as an indicator) are associated with higher levels of blood-cell PAH-DNA adducts, and that measures taken to remove B[a]P (and presumably other PAHs) from the environment yield dividends in terms of lowered biologically effective dose. Two studies in a population of Finnish foundry workers, performed several years apart, indicate that decreasing the B[a]P levels from 12-200 ng/m3 to < 5-60 ng/m3 significantly reduced the PAH-DNA adduct levels measured by the anti-BPdG-DNA-ELISA (37,41). In addition, lower Environmental Health Perspectives -Vol 104, Supplement 5 * October 1996  PAH-DNA adduct levels were measured in the same workers after time spent on vacation (105).
In a study of U.S. Army soldiers, military personnel were monitored before, during, and after a tour of duty in Kuwait (106). It was expected that higher exposure to PAHs would result from oil-well fires burning at that time. However, the DNA adduct and air sampling data indicate that these soldiers went from a clean environment in Kuwait in August to significantly higher pollution levels in Germany in October, and DNA adducts, assayed by both BPdG-DNA dissociation-enhanced lanhtanide fluoroimmunoassay and 32ppostlabeling, increased significantly (106).
Another example of pollution modulation and concomitant reduction in DNA adduct levels occurred in the Silesian region of Poland in the summertime; the air was approximately 5-fold cleaner than in the winter, and the levels of adducts in lymphocytes was approximately 5-fold lower (107). One should note, however, that the adduct dosimetry shown here to reflect ambient PAH levels is not consistent with the much higher levels of ambient B[a]P and the disproportionately low adduct levels observed in other studies (Table 3).
Of all the studies summarized in Table  3, the highest reported ambient B[a]P levels were 2283 ng/m3 for Chinese women breathing coal smoke (108), 7800 ng/m3 for Dutch coke oven workers (109), and 50,000 ng/m3 for Polish coke oven workers (110). Of these, neither Dutch coke oven workers nor Chinese women had proportionately higher DNA adduct levels. The Chinese women, who have a high lung cancer risk, were exposed to coal smoke while cooking meals (108), but the Dutch workers experienced 100 to 7800 ng/m3 during a 40-hr week (109). Therefore, in analyzing these data, a number of confounding factors must be recognized. The use of ambient B[a]P measurements provide an indicator of the pollution levels, but the actual hydrocarbon components vary and are not always measured. The BPdG-DNA immunoassays used actually measure BPdG as well as a broad spectrum of PAH-DNA adducts, since the antiserum used in many studies (96,111,112) recognizes multiple PAH-DNA adducts (98). In addition, cohorts are grouped according to the highest exposure documented, but the range of exposures for one job at one work site can vary considerably. Overall, the data suggest that DNA adduct levels vary with pollution, and that B[a]P may not be the compound responsible for producing the majority of PAH-DNA adducts observed by immunoassay and 32P-postlabeling in human blood cells. DNA Adduct Dosimetry As discussed above, because much of the available human DNA adduct dosimetry for occupational and environmental exposures depends on ambient biomonitoring, precise dose-response relationships have not been possible. However, in some studies with dietary carcinogen exposure and others involving DNA-damaging cancer chemotherapeutic agents, dosimetry has been demonstrated.
In one study involving California firefighters (113), blood samples were taken before the summer firefighting season began and 8 weeks later. PAH-DNA adducts were measured by BPdG-DNA ELISA and did not correlate with the extent of firefighting. Comparison of questionnaire results with DNA adduct values showed that individuals (n= 19) consuming charbroiled food one to two times in the previous week had a mean PAH-DNA adduct value of 1.6 adducts in 108 nucleotides. Twenty-three individuals who reported consumption of these foods three to five times in the previous week and five individuals reporting consumption more than five times in the previous week had mean adduct values of 3.0 and 6.7 adducts/ 108 nucleotides, respectively. The largest single source responsible for PAH-DNA adduct formation was consumption of charbroiled food.
For dosimetry of aflatoxin exposure, 42 individuals in the Guangxi region of China were studied (103). Samples of the food consumed were assayed for aflatoxin content, which was correlated with the urinary excretion of aflatoxin-N7-guanine (AFB1-N7-G) by both males and females. Immunoaffinity chromatography and HPLC were used to isolate metabolites and adducts from urine (28,114) and fluorescence emission spectra were used to identify the AFB1-N7-G. The dosimetry data showed an excellent correlation between dietary aflatoxin intake and urinary adduct excretion (69). Cancer chemotherapy has presented unique opportunities to demonstrate DNA adduct dosimetry because the doses of drug are precisely known. Analysis of blood-cell DNA from 77 previously untreated ovarian and testicular cancer patients receiving platinum drug-based therapy showed strong DNA adduct dosimetry in patients with measurable DNA adduct levels (115,116). In studies with procarbazine and dacarbazine, which were used to treat patients with Hodgkin's lymphoma, an excellent correlation was shown between cumulative drug dose and blood-cell DNA levels of the O6me-dG adduct (39,117). In general, the presence of high levels of DNA adducts appears to correlate positively with favorable clinical outcome (118), and therefore such analyses may become important clinical dosimeters. In addition, they serve to validate the assays commonly used for human DNA adduct measurements, since precise dosimetry is rarely possible in a clinical setting.

Correlation of DNA Adducts with Other Biomarkers
The classes of biomarkers most likely to correlate with PAH-DNA adduct measurements are urinary metabolites (biomarkers of exposure), hemoglobin adducts (surrogates for DNA adducts), mutagenesis (biomarkers of effect), and polymorphisms for enzymes involved in PAH metabolism (biomarkers of susceptibility). Studies currently available in this area have been performed with small numbers of subjects, and conflicting correlations have sometimes been observed. However, as methodological advances improve the specificity of biomarker analysis, there is the promise that batteries of such assays may be usefully employed in future risk assessments.
Metabolic polymorphisms are considered to influence all the other classes of biomarkers. The enzyme complex responsible for the initial metabolism of PAHs, aryl hydrocarbon hydroxylase (AHH), consists of a battery of enzymes that include cytochrome P4501A1. The extent of AHH activity in human lung microsomes has been compared to DNA adduct levels determined in the same lung samples by either HPLC and fluorescence (specific for BPdG) (119) or 32P-postlabeling (53).
Both DNA adduct detection methods yielded results that correlated positively with the extent of AHH activity. Correlations of blood-cell DNA adduct levels with CYPlAl have been examined in chimney sweeps (155) and in California firefighters (156) with ambivalent results. The lack of agreement may be due to differences in experimental approaches to determining polymorphism and to detecting DNA adducts, but it is also possible that neither study had sufficient statistical power to demonstrate subtle effects.
Another metabolic enzyme class, the glutathione S-transferases (GSTs), is involved in PAH detoxification. An 8-kb deletion, including the entire coding region of the GSTMI gene, renders the null genotype in approximately 45% of people who are homozygotes. This genotype has been implicated in increased aflatoxin-DNA adduct formation. To investigate PAH exposure levels in lung tissue, Shields et al. (100) showed that measurable BPdG adduct levels, by SFS and IAC/32P-postlabeling, were present in six of seven people with the null genotype. Among individuals who were negative for these adducts, only 12 of 31 individuals had the null genotype. However, studies with blood-cell DNA adduct measurements did not show a similar correlation. For example, PAH-DNA adduct levels, measured by ELISA in blood cells from heavily smoking males with the null genotype (32/63) were similar to those found in matched individuals with homozygous or heterozygous normal genotypes (31/63) (120). In addition, in California firefighters who had significant increases in blood-cell PAH-DNA adducts due to eating charbroiled food, the presence or absence of the null genotype was not associated with consistent fluctuations in adduct levels (156). It is not likely that a single metabolic polymorphism will be the major determinant of cancer risk, but the results from a metabolic profile may be useful for future elucidation of susceptibility.
Urinary metabolites, hemoglobin adducts, and HPRTmutagenesis are being analyzed in conjunction with DNA adducts in human biomonitoring studies. Although few of these multiple biomarker studies are published, a number of promising new methodologies make this an important direction for the future. Urinary metabolites are considered to provide direct evidence of exposure and reflect the activity of the enzymes discussed above. The metabolite measured most frequently is 1-OH-pyrene (121), and the extent and timing of I-OH-pyrene excretion have been correlated with occupational PAH exposures (122). In one study, PAH-DNA adducts and 1-OH-pyrene were measured in Finnish Foundry workers at three levels of ambient B[a]P exposure (41), and whereas adducts did correlate with exposure, the urinary metabolite did not. Subsequently, analytical procedures have been developed for the BP-7,10/8,9-tetrol (123) and the 1-OH-pyrene glucuronide (124), and these are now being measured in conjunction with DNA adducts (106). A number of methodological approaches for PAH analysis of human hemoglobin have demonstrated the presence of adducts derived from B[a]P and chrysene (66,125,126). Hemoglobin adducts of B[a]P were measured concomitantly with airborne PAH concentration in one study (127), and a weak correlation was demonstrated with exposure, but DNA adducts were not measured. Attempts have been made to correlate HPRT mutagenesis with exposure and DNA adduct formation in Finnish foundry workers (128,129). There was a significant increase in HPRT mutagenesis and DNA adduct formation with exposure, but the numbers of individuals in the mutagenesis portion of the study were small (128) and the correlation requires more substantial validation.
Taken together, these results demonstrate that multiple biomarker correlations are not necessarily straightforward. The tissues studied, the nature of the parameters being measured, and the capabilities of the assays used should all be carefully scrutinized. The numbers of subjects available may be insufficient for solid statistics, and the validity of a particular correlation may not be established until after several independent investigations are performed. However, the present status of development of several biomarkers suggests that good correlation studies with PAH-DNA adduct measurements can now be designed.

Conclusion
Human DNA adduct formation is a promising biomarker for molecular cancer epidemiology. The recent development of methodologies capable of measuring classes of adducts and specific adducts in human blood and tissue samples is likely to lead to improved risk assessment for groups of exposed individuals and may indicate opportunities for chemoprevention. This is true not only for cancer but for other toxic biological end points. Human DNA adduct measurements have been most widely used for exposure documentation. The studies presented here demonstrate that high ambient levels of B[a]P are associated with high levels of DNA adducts in human blood cell DNA and that DNA adduct levels drop when the ambient PAH levels decrease significantly. Precise human DNA adduct dosimetry has been difficult to achieve with PAHs because of the complex nature of the exposures involved; however, correlating DNA adducts with other exposure biomarkers such as urinary metabolites and hemoglobin adducts may substantiate ambient B[a]P measurements and approach dosimetry. Correlations with human mutagenesis and metabolic polymorphisms also suggest the importance of a battery of biomarkers in elucidating toxic mechanisms. It is likely that some toxic, noncarcinogenic compounds may have genotoxic effects and that adverse health outcomes other than cancer may be correlated with DNA adduct formation. Therefore, the studies presented here may serve as useful prototypes for exploration of other toxic end points.