Levels Of DNA Adducts In Liver Research Articles

Hepatic genotoxicity and toxicogenomic responses in Muta™Mouse males treated with dibenz[a,h]anthracene

Dibenz[a,h]anthracene (DB[a,h]A) is a polycyclic aromatic hydrocarbon that is a by-product of combustion and a potent carcinogen. Few studies have investigated the effects of DB[a,h]A on mRNA and microRNA expression to dissect the mechanisms involved in carcinogenesis. In this study, mature male mice (Muta(™)Mouse) were exposed to 6.25, 12.5 and 25mg/kg/day DB[a,h]A by oral gavage for 28 consecutive days. Results were compared with mice similarly exposed to benzo[a]pyrene (B[a]P) in our previous work. Liver DNA adduct levels and lacZ mutant frequency increased dose dependently for both chemicals. Aryl hydrocarbon receptor (AhR) potency was greater for DB[a,h]A than B[a]P using the chemical-activated luciferase expression assay. Microarray analysis revealed 19 up-regulated and 22 down-regulated genes (false discovery rate-adjusted P ≤ 0.05; fold change ≥ 1.5) following treatment with 6.25 mg/kg/day DB[a,h]A. Thirteen transcripts were up-regulated and 32 down-regulated in the 12.5mg/kg/day group. The 25mg/kg/day dose had major effects on mRNA expression with 135 up-regulated and 104 down-regulated genes. Overall, perturbations were greater for DB[a,h]A than for B[a]P; in vitro chemical-activated luciferase expression supports that this may be driven by the AhR. Many of the DB[a,h]A-affected genes are implicated in cancer and are essential in vital biological functions including circadian rhythm, glucose metabolism, lipid metabolism, immune response, cell cycle and apoptosis. Although a number of functional groups were similarly affected by B[a]P and DB[a,h]A, in general the responses generated by each chemical were quite distinct. Commonalities included a DNA damage response leading to induction of cell cycle arrest and apoptosis in both Tp53-dependent and Tp53-independent manners. MicroRNA expression was identical for both chemicals, with only miR-34a showing a dose-dependent increase in treated mice.

Exposure to benzo[a]pyrene of Hepatic Cytochrome P450 Reductase Null (HRN) and P450 Reductase Conditional Null (RCN) mice: Detection of benzo[a]pyrene diol epoxide-DNA adducts by immunohistochemistry and 32P-postlabelling

Benzo[a]pyrene (BaP) is a widespread environmental carcinogen activated by cytochrome P450 (P450) enzymes. In Hepatic P450 Reductase Null (HRN) and Reductase Conditional Null (RCN) mice, P450 oxidoreductase (Por) is deleted specifically in hepatocytes, resulting in the loss of essentially all hepatic P450 function. Treatment of HRN mice with a single i.p. or oral dose of BaP (12.5 or 125mg/kgbody weight) resulted in higher DNA adduct levels in liver (up to 10-fold) than in wild-type (WT) mice, indicating that hepatic P450s appear to be more important for BaP detoxification in vivo. Similar results were obtained in RCN mice. We tested whether differences between hepatocytes and non-hepatocytes in P450 activity may underlie the increased liver BaP-DNA binding in HRN mice. Cellular localisation by immunohistochemistry of BaP-DNA adducts showed that HRN mice have ample capacity for formation of BaP-DNA adducts in liver, indicating that the metabolic process does not result in the generation of a reactive species different from that formed in WT mice. However, increased protein expression of cytochrome b5 in hepatic microsomes of HRN relative to WT mice suggests that cytochrome b5 may modulate the P450-mediated bioactivation of BaP in HRN mice, partially substituting the function of Por.

A comparison of covalent binding of ethanol metabolites to DNA according to Aldh2 genotype

In order to clarify the effects of ALDH2 polymorphism on the carcinogenicity and organ damage caused by ethanol consumption, labeled ethanol was administered to wild-type (C57BL/6, Aldh2+/+) and Aldh2 knock-out ( Aldh2−/−) mice, and DNA adduct levels of organs were compared according to Aldh2 genotype. Aldh2−/− mice, which have the same genetic background as C57BL/6 mice except in the Aldh2 gene, were used as a model of lack of ALDH2 activity in humans. The DNA adduct levels in liver, stomach, and kidney and radioactivity in liver, stomach, kidney, and serum were measured by liquid scintillation counting 6, 12, and 24 h after administration. Though radioactivity levels in all organs decreased over time, there were no significant differences in radioactivity between Aldh2+/+ and Aldh2−/− mice. On the other hand, the DNA radioactivity in each organ tested differed significantly between Aldh2+/+ and Aldh2−/− mice 24 h after administration. These findings show that ethanol consumption affects DNA in Aldh2−/− mice much more strongly than in Aldh2+/+ mice. According to the IARC document, ethanol consumption is carcinogenic to humans (Group 1). Moreover, several studies have shown that ALDH2-deficient humans who habitually consume ethanol have higher rates of cancer than humans with ALDH2. Our results support these findings of epidemiological studies.

Effect of N-glucuronidation on urinary bladder genotoxicity of 4-aminobiphenyl in male and female mice

Urinary bladder cancer is the fourth most commonly diagnosed malignancy in men and the tenth most commonly diagnosed malignancy in women in the US. Arylamines have long been associated with bladder cancer and several studies documented that men exposed to arylamines (cigarette smokers, hairdressers, and workers of dye and textile industries) have several times increased risk compared to women. N-glucuronidation is an important phase II conjugation reaction that delivers the active metabolites of arylamines from the liver to the urinary bladder. In the current study, we found that male mice are more active in 4-ABP N-glucuronidation than female mice and this difference was statistically significant. In the in vivo experiments, male and female mice (strain C57BL/6) were treated with 4-ABP after modulating their 4-ABP N-glucuronidation using the plant steroid, hecogenin. The distribution of 4-ABP adducts in liver and bladder was then determined. In animals treated with 4-ABP only, males had statistically significant higher levels of DNA adducts in the bladder ( p-value 0.0004) while females had statistically significant higher levels in the liver ( p-value < 0.0001). Hecogenin co-treatment increased levels of DNA adducts in the liver in both males and females but this increase was statistically significant only in males ( p-value 0.0024). There was a slight decrease in levels of DNA adducts in the bladder in both males and females co-treated with hecogenin, but this decrease was statistically insignificant. Using two-way ANOVA, we found that gender and hecogenin treatment both had a statistically significant effect on liver DNA adduct levels, whereas only gender had statistically significant effect on bladder adduct levels where males have about 2.2-fold higher DNA adducts than females. The current data suggests that N-glucuronidation of 4-ABP may have an important impact on the organ distribution of DNA damage. The fact that there was only a slight decrease in bladder adduct levels compared to the significant increase in the liver in groups co-treated with hecogenin indicates that besides N-glucuronidation, conjugation and metabolic activation by other enzymes may also contribute to the transport of the proximate metabolites of 4-ABP to the bladder.

Dose-dependent benzo(a)pyrene [B(a)P]–DNA adduct levels and persistence in F-344 rats following subchronic dietary exposure to B(a)P

In order to investigate the relationship between BaP–DNA adduct formation and long-term exposure to benzo(a)pyrene (BaP), DNA adduct levels in liver and lung tissues of male and female F-344 rats subchronically exposed to BaP were determined. Doses of 0, 5, 50, and 100 mg/kg BaP, representing control, low, intermediate, and high doses, respectively, were administered in the animal diet over a 90-day period. After dosing, animals were sacrificed, liver and lung tissues were removed, DNA was isolated and analyzed for BaP-induced DNA adducts by the 32P-postlabeling method using a four-directional thin-layer chromatography system. At low and intermediate BaP doses, DNA adduct levels in the tissues were significantly correlated with exposure. However, at high BaP doses, the dose-DNA adduct relationship became non-linear. Similarly, the relative DNA adducts persistence at intermediate and high doses were significantly higher than that measured at low dose. The low and intermediate dose linearity and high dose non-linearity may be due to saturation of metabolic activation and detoxification enzymes, and DNA repair processes.

Toxicokinetics of acrylamide and glycidamide in Fischer 344 rats

Acrylamide (AA) is a widely studied industrial chemical that is neurotoxic, mutagenic to somatic and germ cells, and carcinogenic in rodents. The recent discovery of AA at ppm levels in a wide variety of commonly consumed foods has energized research efforts worldwide to define toxic mechanisms, particularly toxicokinetics and bioavailability. This study compares the toxicokinetics of AA and its epoxide metabolite, glycidamide (GA), in serum and tissues of male and female F344 rats following acute exposure by intravenous, gavage, and dietary routes at 0.1 mg/kg AA or intravenous and gavage routes with an equimolar amount of GA. AA was rapidly absorbed after oral dosing, was widely distributed to tissues, was efficiently converted to GA, and produced increased levels of GA-DNA adducts in liver. GA was also rapidly absorbed, widely distributed to tissues, and produced increased liver DNA adduct levels. AA bioavailability after aqueous gavage was 60–98% and from the diet was 32–44%; however, first-pass metabolism or other kinetic change resulted in much higher internal exposures to GA (2- to 7-fold) when compared to the intravenous route. A similar effect on metabolism to GA following oral administration was previously observed under an identical exposure paradigm in mice. Furthermore, DNA adduct formation in rat liver showed the same proportionality with the respective GA AUC value as did mice in the previous study. These findings suggest that as the AA content in food is reduced, species-differences in GA formation and subsequent DNA adduct formation may be minimized. These findings provide additional information needed to assess genotoxic risks from the low levels of AA that are pervasive in the food supply.

Toxicokinetics of acrylamide and glycidamide in B6C3F 1 mice

Acrylamide (AA) is a widely studied industrial chemical that is neurotoxic, mutagenic to somatic and germ cells, and carcinogenic in rodents. The recent discovery of AA at ppm levels in a wide variety of commonly consumed foods has energized research efforts worldwide to define toxic mechanisms, particularly toxicokinetics and bioavailability. This study compares the toxicokinetics of AA and its epoxide metabolite glycidamide (GA) in serum and tissues of male and female B6C3F1 mice following acute dosing by intravenous, gavage, and dietary routes at 0.1 mg/kg AA or intravenous and gavage dosing with an equimolar amount of GA. AA was rapidly absorbed from oral dosing, was widely distributed to tissues, was efficiently converted to GA, and increased levels of GA-DNA adducts were observed in liver after complete elimination from serum. GA dosing also resulted in rapid absorption, wide distribution to tissues, and produced liver DNA adduct levels that were approximately 40% higher than those from an equimolar dose of AA. While oral administration was found to attenuate AA bioavailability to 23% from the diet and to 32–52% from aqueous gavage, a first-pass effect or other kinetic change resulted in higher relative internal exposure to GA when compared to the intravenous route. A similar effect on relative GA exposure was also evident as the administered dose was reduced, which suggests that as dosing rate decreases, the conversion of AA to GA is more efficient. These findings are critical to the assessment of genotoxicity of AA at low doses in the food supply, which appears to depend on total exposure to GA.

Effect of CYP1A2 deficiency on heterocyclic amine DNA adduct levels in mice

The contribution of CYP1A2 to the formation of DNA adducts of the cooked meat-derived heterocyclic amines (HCAs) 2-amino-3-methylimidazo[4,5- f]quinoline (IQ) and 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (PhIP) was examined in CYP1A2-null (knock-out, KO) and wild-type (WT) mice. IQ (25 mg and 75 mg/kg) and PhIP (150 mg/kg) were administered by gavage to mice and DNA adduct levels in liver, kidney, mammary gland and colon were examined by the 32P-postlabeling assay. Three hours after either dose of IQ, adducts levels in liver and kidney of KO mice were 20–30% of the levels in WT mice, a difference that was statistically significant (Student's t-test, P<0.05). In the colon, adduct levels in KO mice were significantly lower than in the WT mice only at the lowest dose of IQ (1.6±0.6 vs 4.6±0.7, respectively, relative adduct labeling (RAL)×10 8, mean±S.E.M., n=3–5 mice). In the mammary gland, however, there was no difference in IQ–DNA adduct levels in KO and WT mice at either dose of IQ. Three hours after dosing with PhIP, PhIP–DNA adduct levels were statistically significantly lower in KO mice than in WT mice in all tissues examined. PhIP–DNA adducts in liver and kidney of WT mice were 9.9±1.1 and 22.5±6.9, respectively, whereas no PhIP–DNA adducts were detected in either organ of KO mice (limit of detection, 1.4–2.8×10 9). PhIP–DNA adduct levels in mammary gland and colon of WT mice were 47.1±9.5 and 58.0±21.7, respectively, but accordingly only 3.8±0.7 and 5.4±0.9 in KO mice. The findings indicate that CYP1A2, responsible for IQ and PhIP N-hydroxylation, the first step in the metabolic action, significantly effects DNA adduct formation in vivo. However, the data raise the possibility that other cytochromes P450 as well as other pathways of activation potentially contribute to DNA adduct formation in specific organs, depending on the HCA substrate.

Oral administration of naturally occurring coumarins leads to altered phase I and II enzyme activities and reduced DNA adduct formation by polycyclic aromatic hydrocarbons in various tissues of SENCAR mice.

Several naturally occurring coumarins, to which humans are routinely exposed in the diet, were previously found to inhibit P450-mediated metabolism of benzo[a]pyrene (B[a]P) and 7,12-dimethylbenz[a]anthracene (DMBA) in vitro, block DNA adduct formation in mouse epidermis and inhibit skin tumor initiation by B[a]P and/or DMBA when applied topically to mice. The present study was designed to investigate the effects of two of these compounds, of the linear furanocoumarin type, when given orally (70 mg/kg per os, four successive daily doses), on P450 and glutathione S-transferase (GST) activities and DNA adduct formation by B[a]P and DMBA in various mouse tissues. Imperatorin and isopimpinellin significantly blocked ethoxyresorufin O-deethylase (EROD) and pentoxyresorufin O:-dealkylase (PROD) activities in epidermis at 1 and 24 h after oral dosing. Imperatorin and isopimpinellin modestly inhibited EROD activities in lung and forestomach at 1 h and significantly inhibited PROD activities in lung and forestomach at 1 h after the final oral dose. Twenty-four hours after the final oral dose of imperatorin or isopimpinellin EROD and PROD activities remained inhibited in epidermis and lung. However, forestomach P450 activity had returned to control levels. Interestingly, imperatorin and isopimpinellin treatment inhibited liver EROD activity at 1 h, had no effect on PROD activity at this time point, but elevated both these enzyme activities at 24 h. Elevated EROD and PROD activities coincided with elevated hepatic P450 content. Imperatorin and isopimpinellin treatment also increased liver cytosolic GST activity at both 1 and 24 h after the final oral dose by 1.6-fold compared with corn oil controls. Oral administration of imperatorin and isopimpinellin also had a protective effect against DNA adduct formation by B[a]P and DMBA. Imperatorin pretreatment decreased formation of DNA adducts by DMBA in forestomach. Pretreatment with isopimpinellin led to reduced DNA adduct levels in liver (B[a]P), lung (B[a]P) and mammary epithelial cells (DMBA). These results suggest that imperatorin and isopimpinellin may have potential chemopreventive effects when administered in the diet.

Tissue distribution and macromolecular binding of extremely low doses of [14C]-benzene in B6C3F1 mice.

The tissue distribution and macromolecular binding of benzene was studied over a dose range spanning nine-orders of magnitude to determine the nature of the dose-response and to establish benzene's internal dosimetry at doses encompassing human environmental exposures. [14C]-Benzene was administered to B6C3F1 male mice at doses ranging between 700 pg/kg and 500 mg/kg body wt. Tissues, DNA and protein were analyzed for [14C]-benzene content between 0 and 48 h post-exposure (625 Ng/kg and 5 microg/kg dose) by accelerator mass spectrometry (AMS). [14C]-Benzene levels were highest in the liver and peaked within 0.5 h of exposure. Liver DNA adduct levels peaked at 0.5 h, in contrast to bone marrow DNA adduct levels, which peaked at 12-24 h. Dose-response assessments at 1 h showed that adducts and tissue available doses increased linearly with administered dose up to doses of 16 mg/kg body wt. Tissue available doses and liver protein adducts plateau above the 16 mg/kg dose. Furthermore, a larger percentage of the available dose in bone marrow bound to DNA relative to liver. Protein adduct levels were 9- to 43-fold greater than DNA adduct levels. These data show that benzene is bioavailable at human-relevant doses and that DNA and protein adduct formation is linear with dose over a dose range spanning eight orders of magnitude. Finally, these data show that the dose of bioactive metabolites is greater to the bone marrow than the liver and suggests that protein adducts may contribute to benzene's hematoxicity.

Biomarkers of marine pollution observed in species of mullet living in two eastern Mediterranean harbours

The activities of enzymes associated with xenobiotic metabolism and or oxidative processes, and the levels of aromatic DNA adducts, have been determined in the livers of grey mullet Oedalechilus labeo and Lisa ramada living in two eastern Mediterranean harbours. Glutathione peroxidase GSH P activity was 2.5 times higher 9 IU g-1 liver and glutathione reductase GSSG R activity was twice as high 2.5 IU g-1 liver in fish from the more polluted harbour at Mersin than in the harbour near Erdemli. Superoxide dismutase SOD activity was 25 lower 4.3 IU g-1 liver in the more polluted harbour. The concentrations of glutathione and malondialdehyde varied both with species and environment by a factor of 2.5-3. DNA adducts in liver were determined by 32P postlabelling. In Oedalechilus labeo in the more polluted harbour, adduct levels were 258 21 adducts per 108 nucleotides mean SE; two groups of Lisa ramada were distinguished having 261 48 and 30 6 adducts per 108 nucleotides, respectively. The average adduct level in a group of mullet of mixed species in the less polluted harbour was 3.3 2.3 adducts per 108 nucleotides. The results illuminate the ability of mullet to live in contaminated marine environments, and show that enzyme activities and liver DNA adduct levels can serve as indicators of marine pollution.

Argument for the motion that endocrine disruptors pose a major risk to human health

The contribution of CYP1A2 to the formation of DNA adducts of the cooked meat-derived heterocyclic amines (HCAs) 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) was examined in CYP1A2-null (knock-out, KO) and wild-type (WT) mice. IQ (25 mg and 75 mg/kg) and PhIP (150 mg/kg) were administered by gavage to mice and DNA adduct levels in liver, kidney, mammary gland and colon were examined by the 32P-postlabeling assay. Three hours after either dose of IQ, adducts levels in liver and kidney of KO mice were 20–30% of the levels in WT mice, a difference that was statistically significant (Student's t-test, P<0.05). In the colon, adduct levels in KO mice were significantly lower than in the WT mice only at the lowest dose of IQ (1.6±0.6 vs 4.6±0.7, respectively, relative adduct labeling (RAL)×108, mean±S.E.M., n=3–5 mice). In the mammary gland, however, there was no difference in IQ–DNA adduct levels in KO and WT mice at either dose of IQ. Three hours after dosing with PhIP, PhIP–DNA adduct levels were statistically significantly lower in KO mice than in WT mice in all tissues examined. PhIP–DNA adducts in liver and kidney of WT mice were 9.9±1.1 and 22.5±6.9, respectively, whereas no PhIP–DNA adducts were detected in either organ of KO mice (limit of detection, 1.4–2.8×109). PhIP–DNA adduct levels in mammary gland and colon of WT mice were 47.1±9.5 and 58.0±21.7, respectively, but accordingly only 3.8±0.7 and 5.4±0.9 in KO mice. The findings indicate that CYP1A2, responsible for IQ and PhIP N-hydroxylation, the first step in the metabolic action, significantly effects DNA adduct formation in vivo. However, the data raise the possibility that other cytochromes P450 as well as other pathways of activation potentially contribute to DNA adduct formation in specific organs, depending on the HCA substrate.

Argument for the motion that endocrine disruptors pose a major risk to human health

The contribution of CYP1A2 to the formation of DNA adducts of the cooked meat-derived heterocyclic amines (HCAs) 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) was examined in CYP1A2-null (knock-out, KO) and wild-type (WT) mice. IQ (25 mg and 75 mg/kg) and PhIP (150 mg/kg) were administered by gavage to mice and DNA adduct levels in liver, kidney, mammary gland and colon were examined by the 32P-postlabeling assay. Three hours after either dose of IQ, adducts levels in liver and kidney of KO mice were 20–30% of the levels in WT mice, a difference that was statistically significant (Student's t-test, P<0.05). In the colon, adduct levels in KO mice were significantly lower than in the WT mice only at the lowest dose of IQ (1.6±0.6 vs 4.6±0.7, respectively, relative adduct labeling (RAL)×108, mean±S.E.M., n=3–5 mice). In the mammary gland, however, there was no difference in IQ–DNA adduct levels in KO and WT mice at either dose of IQ. Three hours after dosing with PhIP, PhIP–DNA adduct levels were statistically significantly lower in KO mice than in WT mice in all tissues examined. PhIP–DNA adducts in liver and kidney of WT mice were 9.9±1.1 and 22.5±6.9, respectively, whereas no PhIP–DNA adducts were detected in either organ of KO mice (limit of detection, 1.4–2.8×109). PhIP–DNA adduct levels in mammary gland and colon of WT mice were 47.1±9.5 and 58.0±21.7, respectively, but accordingly only 3.8±0.7 and 5.4±0.9 in KO mice. The findings indicate that CYP1A2, responsible for IQ and PhIP N-hydroxylation, the first step in the metabolic action, significantly effects DNA adduct formation in vivo. However, the data raise the possibility that other cytochromes P450 as well as other pathways of activation potentially contribute to DNA adduct formation in specific organs, depending on the HCA substrate.

32P-postlabeling analysis of aromatic DNA-adducts in liver and brain of wild brook trout ( Salvelinus fontinalis)

Several studies have reported the absence of DNA-adducts in fish collected from unpolluted areas. We determined DNA-adduct levels in liver and brain tissue of wild brook trout ( Salvelinus fontinalis) from remote areas of Newfoundland and sub-arctic Labrador, that have no known input of anthropogenic contamination. Varying levels of adducts were detected in both liver and brain, at all locations. Hepatic adduct levels were generally higher than those in brain tissue. Tissue-specific differences in adduct levels and the possible effects of long range transport of air pollutants (LRTAP) are discussed.

Liver DNA adducts in methyl-deficient rats administered a single dose of aflatoxin B1.

Using an 8 week Solt-Farber protocol with selection pressure (2-acetylaminofluorene/partial hepatectomy) applied during weeks 6 and 7, we have observed that a single oral administration of aflatoxin B1 (AFB1) to Fischer 344 rats on day 1 of the study, followed by a 3 week feeding regimen of either a methyl-deficient (CMD) or a basal (CMS) diet, results in a relative increase in hepatic preneoplastic lesions in CMD diet fed rats. It has previously been shown that a multiple dosing regimen with AFB1, started after 3 weeks of CMD diet, enhances tumor incidence. In the present study, the role of metabolic activation in the induction of preneoplastic lesions, and liver DNA adduct levels after the first dose of AFB1 in the tumorigenesis model have been investigated. AFB1-DNA adducts were determined at 2-168 h following a single non-necrogenic (100 micrograms/kg body wt) or necrogenic (600 micrograms/kg body wt) dose of AFB1 on day 1 or day 21 of a 3 week treatment with a complete basal or CMD diet. In all rats irrespective of dose, dietary treatment or time of AFB1 dosing, the patterns of adduct formation and repair did not change. In rats receiving AFB1 on day 1, total DNA adduct levels between the diet or dose groups were not significantly different, and quantitatively did not correlate with the observed increase in preneoplastic lesions, suggesting a contribution by additional factors in the initiation of these lesions. Administration of AFB1 on day 21, however, resulted in significantly reduced levels of total adducts at both dose levels in CMD diet fed rats compared to controls. Serum biochemistry data suggest that a prolonged exposure to CMD diet may cause pathological and/or biochemical alterations in hepatocytes with a resultant decrease in metabolic activation of AFB1, thus making it difficult to evaluate whether DNA damage is directly related to tumorigenesis.

32P-analysis of DNA adducts in somatic and reproductive tissues of rats treated with the anticancer antibiotic, mitomycin C

Mitomycin C (MMC) is clinically used drug with mutagenic and antitumor activities, presumably elicited through its covalent binding to DNA, however, little is known about MMC binding to DNA in vivo. A 32P-postlabeling method that does not require radiolabeled test compounds was employed here to study the formation of DNA adducts in somatic and reproductive tissues of rats 24 h after an i.p. dose of 9 mg/kg MMC. Among 14 tissues studied in female rats, MMC-DNA adduct levels were within a 2-fold range in 11 tissues, i.e. bladder, colon, esophagus, heart, kidney, liver, lung, ovary, pancreas, small intestine and stomach (minimum levels of 9.6–21.9 adducts per 10 7). Three other tissues, i.e. brain, spleen and thymus, exhibited lower adduct levels (0.2, 5.4 and 1.4 adducts, respectively, per 10 7 N). Liver DNA adduct levels were 32% lower in male than in female rats. Testicular DNA contained 2.5 adducts per 10 7 N, i.e. 5.3. times less than ovarian DNA. 32P-labeled adduct patterns were qualitatively similar among the different tissues and consisted of 10 adducts, one of which comprised 71 (±5)% of the total. All these adducts were chromatographically identical to adducts formed by the reaction of chemically reduced MMC with DNA in vitro, demonstrating that metabolic activation of MMC occured via reduction. Using homopolydeoxyribonucleotides modified with MMC, in vivo adducts were shown to be mostly (>90%) gaunine derivatives and small amounts of adenine, cytosine and thymine products. Most of the adducts appeared to be monofunctional derivatives of DNA nucleotides. Dose-dependent MMC-DNA adduct formation was determined in rat liver over an 82-fold range of MMC administered (0.11–9.0 mg/kg). The lowest dose level studied was 4.5 times lower than the recommended single dose for human cancer chemotherapy (20 mg/m 2). Thus, these results predict that 32P-postlabeling methodology is suitable to monitor and quantify DNA adducts in tissue biopsies of patients receiving MMC chemotherapy.