DNA adducts in human placenta as biomarkers for environmental pollution, analysed by the 32P-HPLC method

During pregnancy, mothers are exposed to complex chemical mixtures, such as air pollution and smoke from incomplete combustion. In this study DNA adducts were measured in human placentas from 29 mothers. Environmental exposure and several possible biomarkers in relation to levels of DNA adducts were measured. Placental aromatic and bulky DNA adducts were measured with the ³²     

Capillary zone electrophoresis with on-line blotting for separation and detection of 32P-postlabelled DNA adducts

A new technique for the detection of ³²P-postlabelled DNA adducts separated by capillary electrophoresis was developed. By direct transfer from the capillary outlet to a positively charged moving filter paper, eluted radioactive peaks can be quantified using a phosphor imaging detector. With this method it is possible to separate DNA adducts from different carcinogens after ³²P-postlabelling of the modified and unmodified nucleotides with high sensitivity approaching 1 adduct per 10⁹ nucleotides.     

Capillary zone electrophoresis with on-line blotting for separation and detection of 32P-postlabelled DNA adducts

A new technique for the detection of ³²P-postlabelled DNA adducts separated by capillary electrophoresis was developed. By direct transfer from the capillary outlet to a positively charged moving filter paper, eluted radioactive peaks can be quantified using a phosphor imaging detector. With this method it is possible to separate DNA adducts from different carcinogens after ³²P-postlabelling of the modified and unmodified nucleotides with high sensitivity approaching 1 adduct per 10⁹ nucleotides.     

Postlabelling HPLC analysis of lipophilic DNA adducts from human lung

A new modification of the 32P postlabelling method was described for the analysis of lipophilic DNA in human tissues. To isolate these DNA adducts the method applied nuclease P1 enrichment before labelling and butanol extraction after labelling, followed by high performance liquid chromatography HPLC separation and flow through radioactivity detection. These enrichment methods are known to work for many adducts of polycyclic aromatic hydrocarbons PAHs . In human peripheral lung tissue fro m smokers the apparent level of the DNA adducts observed was 25-244 adducts per 108 nucelotides; in two alleged non smokers the level of adducts was 17 and 109 adducts per 108 nucleotides. When the same samples were analysed by thin layer chromatography TLC , as in the conventional postlabelling assay, the recovery was 5 of that of the HPLC method. Nevertheless, the results from the two methods correlated. In TLC the adducts were lost because they did not remain in the origin in D1 of the TLC development. There was no large difference in recovery between the two techniques for the PAH-DNA adduct standards used. The present results are underestimates of the true adduct levels because there is no way to correct for labelling efficiency and recovery of unknown adducts. Yet they give a lower estimate of the level of lipophilic DNA adducts in human lung tissue.     

Postlabelling HPLC analysis of lipophilic DNA adducts from human lung

A new modification of the 32P postlabelling method was described for the analysis of lipophilic DNA in human tissues. To isolate these DNA adducts the method applied nuclease P1 enrichment before labelling and butanol extraction after labelling, followed by high performance liquid chromatography HPLC separation and flow through radioactivity detection. These enrichment methods are known to work for many adducts of polycyclic aromatic hydrocarbons PAHs. In human peripheral lung tissue fro m smokers the apparent level of the DNA adducts observed was 25-244 adducts per 108 nucelotides; in two alleged non smokers the level of adducts was 17 and 109 adducts per 108 nucleotides. When the same samples were analysed by thin layer chromatography TLC, as in the conventional postlabelling assay, the recovery was 5 of that of the HPLC method. Nevertheless, the results from the two methods correlated. In TLC the adducts were lost because they did not remain in the origin in D1 of the TLC development. There was no large difference in recovery between the two techniques for the PAH-DNA adduct standards used. The present results are underestimates of the true adduct levels because there is no way to correct for labelling efficiency and recovery of unknown adducts. Yet they give a lower estimate of the level of lipophilic DNA adducts in human lung tissue.     

Regio and stereospecific DNA adduct formation in mouse lung at N6 and N7 position of adenine and guanine after 1,3 butadiene inhalation exposure

Butadiene monoepoxide (BMO) alkylated guanine N7 and adenine N 6 adducts were prepared and enriched by solid phase extraction and HPLC. The purified adducts were analysed by a modified 32P-postlabelling assay, which utilized one dimensional TLC chromatography and a subsequent HPLC analysis with UV and radioactivity detectors. In vitro with Ct-DNA the formation of N7-dGMP and N 6-dAMP adducts were linear at a concentration range of 44 to 870 nmol of BMO per mg DNA at physiological pH. N7- dGMP and N 6-dAMP adducts were formed in a ratio of 200:1. In dGMP and in dAMP 48 % and 86 % of adducts were covalently bound to the C-2 carbon of BMO. CD-1 mice were inhalation exposed to butadiene for 5 days and 6 h per day. The N7-dGMP adduct level in lung samples of animals exposed to 200, 500 and 1300 ppm was 2.8 +/- 0.9 fmol, 11 +/- 2.0 fmol and 30 +/- 6.7 fmol in 10 mug DNA, respectively. The level of N 6-dAMP adducts in lung samples after 500 ppm and 1300 ppm exposure was 0.09 +/- 0.06 fmol and 0.11 +/- 0.05 fmol in 10 mug DNA. At 200 ppm the adduct level was below the detection limit. A sub-group of animals exposed to 1300 ppm was killed 3 weeks after the last exposure. N7-dGMP adducts were not detected but the level of N 6-dAMP adducts was not affected. N7-dGMP adducts were formed in a clear stereospecific manner in vivo . S -BMO adducts were the main product and represented 77 % ( n = 4, SD = 2%) of total BMO adducts. No clear conclusion can be drawn about the enantiospecific DNA binding at the N 6 position of dAMP, because of the poor separation of the enantiomers. However, we could separate regioisomeric adducts which indicated that C-2 adducts represented 69 +/- 3 % of the total N 6 adducts formed in mice lung DNA. This observation is supported by the data derived from in vitro DNA experiments but is different to our previously published data, which indicates the 2:1 (C-1:C-2) ratio in regioisomer formation in nucleotides or nucleosides. We suggest that the data presented in this communication indicate a different mechanism between nucleotides and DNA in BMO-derived adduct formation- Dimroth rearrangement dominates in nucleotides, but in double stranded DNA a direct alkylation is probably the major mechanism of adduct formation.     

Regio and stereospecific DNA adduct formation in mouse lung at N6 and N7 position of adenine and guanine after 1,3 butadiene inhalation exposure

Butadiene monoepoxide (BMO) alkylated guanine N7 and adenine N 6 adducts were prepared and enriched by solid phase extraction and HPLC. The purified adducts were analysed by a modified 32P-postlabelling assay, which utilized one dimensional TLC chromatography and a subsequent HPLC analysis with UV and radioactivity detectors. In vitro with Ct-DNA the formation of N7-dGMP and N 6-dAMP adducts were linear at a concentration range of 44 to 870 nmol of BMO per mg DNA at physiological pH. N7- dGMP and N 6-dAMP adducts were formed in a ratio of 200:1. In dGMP and in dAMP 48 % and 86 % of adducts were covalently bound to the C-2 carbon of BMO. CD-1 mice were inhalation exposed to butadiene for 5 days and 6 h per day. The N7-dGMP adduct level in lung samples of animals exposed to 200, 500 and 1300 ppm was 2.8 +/- 0.9 fmol, 11 +/- 2.0 fmol and 30 +/- 6.7 fmol in 10 mug DNA, respectively. The level of N 6-dAMP adducts in lung samples after 500 ppm and 1300 ppm exposure was 0.09 +/- 0.06 fmol and 0.11 +/- 0.05 fmol in 10 mug DNA. At 200 ppm the adduct level was below the detection limit. A sub-group of animals exposed to 1300 ppm was killed 3 weeks after the last exposure. N7-dGMP adducts were not detected but the level of N 6-dAMP adducts was not affected. N7-dGMP adducts were formed in a clear stereospecific manner in vivo. S -BMO adducts were the main product and represented 77 % (n = 4, SD = 2%) of total BMO adducts. No clear conclusion can be drawn about the enantiospecific DNA binding at the N 6 position of dAMP, because of the poor separation of the enantiomers. However, we could separate regioisomeric adducts which indicated that C-2 adducts represented 69 +/- 3 % of the total N 6 adducts formed in mice lung DNA. This observation is supported by the data derived from in vitro DNA experiments but is different to our previously published data, which indicates the 2:1 (C-1:C-2) ratio in regioisomer formation in nucleotides or nucleosides. We suggest that the data presented in this communication indicate a different mechanism between nucleotides and DNA in BMO-derived adduct formation- Dimroth rearrangement dominates in nucleotides, but in double stranded DNA a direct alkylation is probably the major mechanism of adduct formation.     

DNA adducts in marine and freshwater fish as biomarkers of environmental contamination

The analysis of DNA modifications in aquatic animals may serve as a sensitive marker of exposure to genotoxic contaminants. This is of importance in assessing water quality regarding pollution with genotoxic compounds, food safety analysing edible aquatic animals and in terms of ecotoxicology. Covalent modification of DNA is considered a crucial event in chemical carcinogenesis and thus may be considered a biomarker of an early genotoxic effect. Measuring DNA adducts is unique in that these lesions may be considered a biomarker of both exposure and effect. A number of studies have described the analysis of the DNA isolated from the liver of both freshwater and marine fish. Considerable levels of DNA adducts have been observed in some animals from contaminated lakes or rivers. Low levels were observed in DNA from the liver of marine fish. The background levels of DNA adducts have to be determined in animals from appropriate uncontaminated control sites that are matched for species, gender, age and season of the year. It is of crucial importance to consider the species analysed since there have been reports of the non responsiveness of some species.     

DNA adducts in marine and freshwater fish as biomarkers of environmental contamination

The analysis of DNA modifications in aquatic animals may serve as a sensitive marker of exposure to genotoxic contaminants. This is of importance in assessing water quality regarding pollution with genotoxic compounds, food safety analysing edible aquatic animals and in terms of ecotoxicology. Covalent modification of DNA is considered a crucial event in chemical carcinogenesis and thus may be considered a biomarker of an early genotoxic effect. Measuring DNA adducts is unique in that these lesions may be considered a biomarker of both exposure and effect. A number of studies have described the analysis of the DNA isolated from the liver of both freshwater and marine fish. Considerable levels of DNA adducts have been observed in some animals from contaminated lakes or rivers. Low levels were observed in DNA from the liver of marine fish. The background levels of DNA adducts have to be determined in animals from appropriate uncontaminated control sites that are matched for species, gender, age and season of the year. It is of crucial importance to consider the species analysed since there have been reports of the non responsiveness of some species.     

Conversion of 1 nitropyrene by Brown trout Salmo trutta and turbot Scophthalamus maximus to DNA adducts detected by 32P postlabelling

The ability of the common aquatic contaminant 1-nitropyrene to form DNA adducts in fish was investigated in vitro and in vivo using Brown trout (Salmo trutta) and turbot (Scophthalmus maximus)in comparison to the Wistar rat. In vitro studies used Brown trout (control and induced (50 mg kg-1-naphthoflavone (NF), i.p. 3 day pre-treatment single injection)) and induced rat (PB; 0 1% w/v for 7 days in drinking water, NF; 80 mg kg-1, single injection 2 days prior to sacrifice). Hepatic 9000 g supernatant (S9 fractions) were incubated for 2 hours (at 25 C for fish and 37 C for rat) with calf thymus DNA (1mg) and 1-NP (100 M). With all S9 fractions the presence of three distinct 1-NP-related DNA adducts was detected using the butanol enrichment procedure of the 32Ppostlabelling assay. A greater level of DNA adducts was observed with the uninduced compared to the induced trout S9 (37, 12 and 8 fold greaterfor adducts in chromatograph areas 1-3 respectively) suggesting the enhancement of detoxification pathways with respect to bulky adducts following NF pre-treatment. DNA adduct levels in the induced rat consistently demonstrated approximately two-fold higher levels as compared to the induced fish, reflecting the lower protein levels in the S9 fraction of Brown trout (42 and 22 mg ml-1 for rat and fish respectively). Turbot, rat and Brown trout (uninduced and induced (NF; 50 mg kg-1; i.p. single injection 3 days prior)) were dosed with 100 mg kg-1 1-NP (i.p. single injection, 24 hours). Liver DNA from both turbot and rat exhibited a 1- NP related adduct spot which was similar in position to that of area 1 in the incubations with S9 from rat and Brown trout. However, in contrast to the in vitro studies no 1-NPrelated adducts were found in liver DNA from induced and uninduced Brown trout. This study highlights the potential, in a marine and a freshwater fish, for 1-NP metabolism to reactive of binding to DNA. However, activation of 1-NP was more optimal in the S9-mediated system, possibly reflecting the influence of detoxification systems.     

Effect of genetic polymorphism for metabolic enzymes on the relationship between smoking dose and DNA adducts in lymphocytes

The genetic polymorphism of metabolic enzymes on the relationship between smoking dose and DNA adduct levels in lymphocytes were evaluated in 51 smokers. The genetic polymorphisms of cytochrome P4501A1 (CYP1A1) and glutathione S-transferase M1 (GSTM1) were analysed by a PCR method. Lymphocyte DNA adducts were measured by two analytical versions of a 32P-postlabelling method; nuclease P1 digested method and butanol extracted method. Mean adduct levels obtained with the nuclease P1 method (1.21 +/- 0.74 per 108 nucleotides) were higher than those obtained with the butanol extracted method (0.82 +/- 0.47, p < 0.01). There was a significant correlation between adduct levels by the nuclease P1 method and those by the butanol extracted method (r = 0.49, p < 0.01). A significant correlation was not found between smoking dose and DNA adduct levels obtained using both methods in lymphocytes of all subjects. When subjects were divided into two groups by CYP1A1 genotypes, significant correlations between smoking indices, such as number of cigarettes per day years or tar intake per day years, and DNA adduct levels measured by the butanol extracted method was found in heterozygous or miner homozygous for CYP1A1 exon 7 polymorphism. We could not get a significant effect of GSTM1 on the relationship between smoking dose and DNA adducts.     

The relationship between aryl hydrocarbon hydroxylase activity and DNA adducts measured by 32P-postlabelling assay in lymphocytes of lung cancer patients

We have investigated the correlation between DNA adduct levels and aryl hydrocarbon hydroxylase (AHH) activity in peripheral lymphocyte samples obtained from 42 lung cancer patients. DNA adducts and AHH activity were determined by the ³²P-postlabelling technique and the fluorometric method, respectively. The mean +/- SD of DNA adduct level was 0.88 +/- 0.37 (ranged from 0.22 to 1.90) per 10⁸ nucleotides. The geometric means of non-induced and 3-methylcholanthrene (MC)-induced AHH activity, as well as AHH inducibility (MC-induced AHH activity/non-induced AHH activity) were 0.029, 0.228 pmol min^-1 10^-6 cells, and 7.776, respectively. There was no statistically significant correlation between DNA adduct levels and non-induced or MC-induced AHH activity. A tendency of positive correlation was found between DNA adduct levels and AHH inducibility for the all subjects (n = 42, r = 0.25, p = 0.11). Such a positive correlation reached statistical significance in the subjects with squamous cell carcinoma (n = 13, r = 0.70, p < 0.01). In addition, similar correlation of DNA adducts with AHH inducibility was also observed in the GSTM1 present genotype (n = 17, r = 0.44, p = 0.07) and GSTP1-AA genotype (n = 29, r = 0.37, p = 0.05) individuals. These findings suggest that DNA adduct levels are mediated by CYP1A1 enzyme, and AHH inducibility may be a more relevant indicator than specific AHH activity for explaining the variation of DNA adduct levels in lymphocytes.     

Lymphocytes,DNA adducts and genetic polymorphism for metabolic enzymes in low dose cigarette smokers

The aim of this study was to investigate the relationship between genetic polymorphism of metabolic enzymes and DNA adduct levels in lymphocytes of low dose cigarette smokers (less than 20 cigarettes per day). We previously reported the effects of cytochrome P4501A1 (CYP1A1) and glutathione S-transferase M1 (GSTM1) on lymphocyte DNA adducts. This time we considered not only CYP1A1 and GSTM1 but also cytochrome P4502E1 (CYP2E1) and glutathione S-transferase T1 (GSTT1). DNA adducts in lymphocytes obtained from low dose cigarette smokers (n = 41) and nonsmokers (n = 56) were measured by the 32P-postlabelling method. The adduct levels were compared regarding smoking status and polymorphic genotypes of these four enzymes. The mean SD of DNA adduct levels in all low dose cigarette smokers and non-smokers was 1 05 0 83 per 108 nucleotidesand 0 85 0 35 per 108 nucleotides, respectively. In low dose cigarette smokers, adduct levels were higher in the rare homozygous (MM) for CYP1A1-exon 7 polymorphism compared with the other types such as common homozygous (WW) and heterozygous (WM). CYP1A1-WM, MM in combination with GSTM1 null showed highest adduct levelamong low smokers. The low smokers with rare homozygous for CYP2E1 Dra1 polymorphism tended to have lower adduct levels than wild types. Low dose cigarette smokers with combined GSTM1 null and T1 null had a higher tendency for adduct levels than others. However none of the differences reached statistical significance.     

Short Communication: The effect of CYP1A1 induction on the formation of benzo a pyrene adducts in liver and lung DNA and plasma albumin in rats exposed to benzo a pyrene:adduct quantitation by immunoassay and an HPLC method

Induction of cytochrome P450 enzymes by exposure to polycyclic aromatic hydrocarbons (PAH) can result in both decreased or increased PAH adduct levels. The lung is a main target site for PAH-carcinogenesis. By HPLC determination of B [a]P-r-7, t-8-dihydrodiol, t-9, 10-epoxide (BPDE-I)-DNA adducts in rat, the level of the ultimate carcinogenic B[a]P-metabolite was higher in lungs than in liver. However, measured by immunoassay, the total benzo[a]pyrene (B[a]P)-DNA adduct levels were higher in liver than in lungs. Induction of CYP1A1 in vivo in rat by repeated i.p. doses of methylcholanthrene (MC) prior to a single dose of B[a]P resulted in a 2.4 times increase in CYP1A1 activity in liver tissue and 1.5 times higher levelsof total B[a]P-DNA adducts in lung and liver compared with controls which only received B[a]P. Increased levels of BPDE-I-DNA adducts were significantly correlated to increased CYP1A1 activity in induced lung tissue but not in liver. The times to reach maximum adduct levels were similar for both controls and MC-induced rats in both lung and liver,and plasma albumin. The BPDE-I-albumin adducts reached a maximum level around 1 day after B[a]P exposure and could not be used as a reliable marker of the short term PAH exposure in this study.     

A romatic DNA adduct levels in human peripheral blood lymphocytes and total white blood cells by 32P postlabelling: need for validation

Long lived lymphocytes tend to have higher 32P postlabelling measured levels of adducts than short lived granulocytes in environmental and life style associated i.e. smoking exposures. With the aim of investigating this issue for occupational exposure to PAH and contributing to further validation of some technical aspects of the 32P postlabelling assay, two Italian laboratories analysed PAH-DNA adducts from lymphocytes and total white blood cells WBC . Seventy seven blood samples from coke oven workers employed at a steel plant located in Taranto, Southern Italy, and 14 samples from control subjects were collected. At the University of Padua, DNA was purified from peripheral blood lymphocytes PBL . Two years later, at the University of Bari, white blood cells WBC were isolated from replicate blood samples stored at- 80 C and DNA purified by the same method. In both cases, the nuclease P1 modified postlabelling assay was used to determine aromatic DNA adduct levels. The mean adduct levels were 5.13 3.37 Padua and 2.48 1.27 Bari per 108 nucleotides. Both laboratories observed large inter individual variations of adduct levels ranging from 0.09 to 18.93 per 108 nucleotides. Both the correlation and the agreement of the two sets of data were assessed. Slight correlation r = 0.39; p 0.01 and a poor level of agreement were found, the intra class correlation coefficient being equal to 0.05. Better correlation coefficient r = 0.54, p 0.01 and intra class correlation coefficient r = 0.50 were observed comparing only the adduct levels determined on the diagonal zone DRZ . Our findings seem to confirm the same divergence reported in the literature on DNA adduct levels between lymphocytes and granulocytes.     

The choice of controls in a case-control study on WBC-DNA adducts and metabolic polymorphisms

The choice of the control group is a key issue in case-control studies, particularly in studies of molecular epidemiology. We discuss the potential bias introduced by different options. To exemplify the consequences of different choices, we have analysed two sets of controls in the context of a case-control study on bladder cancer: 55 were patients with urological conditions (cystitis, prostate hypertrophy), while 49 had a miscellany of medical or surgical conditions. We measured DNA adducts in white blood cells (WBC) by ³²P-postlabelling and a series of metabolic polymorphisms (GSTM1, GSTT1, GSTP1, NAT2, NQO1). While no statistically significant differences were found for metabolic polymorphisms, the two series of controls showed different concentrations of DNA adducts, suggesting that conditions related to bladder cancer or intermediate steps leading to bladder cancer, such as chronic cystitis, may be associated with higher adduct levels. An association between DNA adduct levels and infection has been noted before in experimental animals: both in lung and in the skin, an inflammatory response increased the biologically effective doses of polycyclic aromatic hydrocarbons. An alternative explanation is confounding; in fact, after adjustment for the level of consumption of fruit and vegetables (but not for smoking) the difference between the two control groups was no longer statistically significant. In conclusion, the choice of controls in studies of molecular epidemiology has subtle methodological implications, including confounding of metabolic/molecular measurements by complex exposures such as diet.     

Measurement of 7 methyl and 7 2 hydroxyethyl guanine DNA adducts in white blood cells of smokers and non smokers

The goal of the present study was to measure the levels of 7-methylguanine and 7-(2- hydroxyethyl)guanine DNA adducts in human white blood cells in relation to smoking. DNA was isolated from samples of 11 smokers and eight non-smokers. The 32P-postlabelled 7-methylguanine and 7-(2-hydroxyethyl)guanine adducts were analysed by thin-layer chromatography (TLC) combined with a high pressure liquid chromatography (HPLC) assay. In smokers the mean 7-methylguanine and 7-(2-hydroxyethyl)guanine levels were 32.3 +/- 7.1 and 6.6 +/- 2.3 adducts per 108 nucleotides respectively. The corresponding values in non-smokers were 25.0 +/- 7.0 and 3.7 +/- 2.4 adducts per 108 nucleotides. There were significantly higher levels of 7-methylguanine and 7-(2-hydroxyethyl)guanine adducts in WBC in smokers than in non-smokers (p = 0.041; p = 0.018), respectively. A positive correlation between 7-methylguanine and 7-(2-hydroxyethyl)guanine levels was observed.     

A romatic DNA adduct levels in human peripheral blood lymphocytes and total white blood cells by 32P postlabelling: need for validation

Long lived lymphocytes tend to have higher 32P postlabelling measured levels of adducts than short lived granulocytes in environmental and life style associated i.e. smoking exposures. With the aim of investigating this issue for occupational exposure to PAH and contributing to further validation of some technical aspects of the 32P postlabelling assay, two Italian laboratories analysed PAH-DNA adducts from lymphocytes and total white blood cells WBC. Seventy seven blood samples from coke oven workers employed at a steel plant located in Taranto, Southern Italy, and 14 samples from control subjects were collected. At the University of Padua, DNA was purified from peripheral blood lymphocytes PBL. Two years later, at the University of Bari, white blood cells WBC were isolated from replicate blood samples stored at- 80 C and DNA purified by the same method. In both cases, the nuclease P1 modified postlabelling assay was used to determine aromatic DNA adduct levels. The mean adduct levels were 5.13 3.37 Padua and 2.48 1.27 Bari per 108 nucleotides. Both laboratories observed large inter individual variations of adduct levels ranging from 0.09 to 18.93 per 108 nucleotides. Both the correlation and the agreement of the two sets of data were assessed. Slight correlation r = 0.39; p 0.01 and a poor level of agreement were found, the intra class correlation coefficient being equal to 0.05. Better correlation coefficient r = 0.54, p 0.01 and intra class correlation coefficient r = 0.50 were observed comparing only the adduct levels determined on the diagonal zone DRZ. Our findings seem to confirm the same divergence reported in the literature on DNA adduct levels between lymphocytes and granulocytes.     

32P-postlabelling analysis of DNA adducts from Fischer-344 rats administered 2,4-diaminotoluene.

Using 32P-postlabelling and thin layer chromatography, DNA adduct formation by the potent animal carcinogen 2,4-diaminotoluene in Fischer-344 rats was investigated. DNA from four different organs, liver, mammary gland, kidney and lung, were examined for adducts following single administration of this compound. DNA binding was detected in all four organs, with each producing one major and two minor adduct spots on autoradiograms. The adducts induced were qualitatively identical among the different organs, but quantitative differences were observed. The two target organs of 2,4-diaminotoluene induced carcinogenesis, the liver and mammary gland produced higher adduct yields, with levels up to 30-times higher than those for the two non-target organs. Since the liver is the principal target for 2,4-diaminotoluene induced carcinogenesis, we further examined DNA adducts from this site for the effects of different doses and time points. DNA binding in liver was detected following doses as low as 4.1 mumol/kg. At the highest concentration examined (2046 mumol/kg), the level of the major adduct was 29.2 adducted nucleotides per 10(7) total nucleotides. The yields for the two minor adducts were approximately one-tenth that for the major adduct. Following a 410 mumol/kg dose, DNA adduct removal over time was examined. DNA adduct removal exhibited biphasic kinetics, with a rapid initial phase followed by a slower rate of elimination. Up to 60% of maximum adduct levels persisted after 2 weeks. DNA binding by 2,4-diaminotoluene was also compared to that by its weakly carcinogenic analog, 2,4-dinitrotoluene. The two compounds produced identical adduct patterns, suggesting that they share common metabolites and adducts. Adduct yields from 2,4-dinitrotoluene, however, were lower. The results of our studies suggest that the differences in carcinogenic potency between 2,4-diaminotoluene and 2,4-dinitrotoluene, as well as the organotropic effects of 2,4-diaminotoluene may be explained, in part, by quantitative differences in the extent of DNA adduct formation.