The capacity of the chemical carcinogen ethyl carbamate (EC, urethane) and its metabolites vinyl carbamate (VC) and vinyl carbamate epoxide (VCO) to form ethenobases was studied in liver and lung DNA of 12-day-old and adult CD-1, B6C3F1, C3H/HeJ and C57BL/6J mice. Following single and multiple doses of EC, VC or VCO, the formation of 1,N6-ethenodeoxyadenosine (epsilon dA) and 3,N4-ethenodeoxycytidine (epsilon dC) was quantified by an immunoaffinity chromatography/32P-postlabelling technique. Both etheno adducts were detected in untreated control DNA samples from liver and lung in the range of 2-15 adducts/10(9) parent nucleotides. Following five repeated injections of 250 or 280 nmol/g body wt VC to adult mice, 51, 57 and 78 epsilon dA/10(9) dA and 28, 42 and 42 epsilon dC/10(9) dC (means of duplicate analyses) were detected in liver DNA of CD-1, C3H/HeJ and C57BL/6J mice respectively. In lung DNA of these VC-treated mice, the levels were 87, 49 and 58 (epsilon dA/10(9) dA) and 64, 39 and 43 (epsilon dC/10(9) dC) respectively. Under similar dose regimens, lower levels of etheno adducts were detected in B6C3F1 mice. Etheno-DNA adducts were also formed in liver and lung upon treatment with EC in adult mice, but at 3-fold lower levels as compared with VC. In 12-day-old C3H/HeJ and C57BL/6J mice, 2- to 3-fold higher etheno adduct levels were detected in liver DNA, when compared with adults, upon a single treatment with 250 nmol/g body wt VC, suggesting that young animals are more susceptible to adduct formation. Combined analysis of adduct formation in adult CD-1, C3H/HeJ and C57BL/6J mice at the higher dose showed a statistically significant increase in etheno adduct formation in the order EC > VC. The results demonstrate that EC and its activated intermediates bind to liver and lung DNA to form epsilon dA and epsilon dC, and the differences in DNA binding further support the hypothesis that metabolic activation of EC to VC is involved. Preliminary data also suggest that background levels of epsilon dA and epsilon dC in DNA are affected by the type of diet given to the animals.
Analysis of aristolochic acid I (AAI)-DNA adducts in exfoliated cells in urine, urothelium and entire urinary bladder were studied after oral administration of five daily doses (10 mg/kg body wt) AAI for 3 months to rats. The two major adducts excreted in urine are presumably identical to the two main adducts formed in vitro and in vivo in different organs in the rat, which have previously been characterized in vitro as 7(-deoxyguanosin-N2-yl)-aristolactam I and 7(-deoxyadenosin-N6-yl)-aristolactam I. Urine samples were collected on dry-ice, subsequently pooled and purified according to the protocol of Kadlubar and co-workers. DNA was isolated, digested and AAI-DNA adducts of exfoliated cells in urine and urothelium of rats were detected and quantitated by enhancement methods of the 32P-postlabeling assay, namely nuclease P1 enrichment or butanol extraction. Autoradiograms indicated that adduct patterns in DNA derived from exfoliated cells in urine were very similar to those obtained from DNA isolated from tissues. Quantitative analysis of adducts revealed adduct levels declining for both adducts from DNA isolated from urothelium to DNA isolated from the entire urinary bladder to DNA isolated from exfoliated cells in urine. In general, count rates of two predominant AAI adducts were enhanced by butanol extraction approximately 3- to 8-fold when compared with the nuclease P1 digestion technique. The identity of the two major adducts was confirmed by co-chromatography with eluted spots from in vivo adducts by comparing mobilities on poly-(ethyleneimine)-cellulose plates. Microbiological investigations of the urine revealed no gross contamination with bacteria, so that the isolated DNA supposedly originated from exfoliated urothelial cells. This study indicates that 32P-postlabeling analysis can be used to monitor non-invasively the formation of carcinogen-DNA adducts in animals or humans exposed to carcinogens.
In this study the development of aristolochic acid (AA) induced tumors in rats with and without diallyl sulfide (DAS) was studied. Experiments were also conducted to establish the effects of DAS administration on AA-derived DNA single-stranded regions and DNA adduct formation in the forestomach of such animals. Forestomach, urinary bladder and thymus tumors were induced in male BD-6 rats after oral treatment for 12 weeks with AA (2 x 10 mg/kg/week). Administration of 150 mg/kg DAS intragastrically 4 h prior to AA treatment reduced significantly the number of rats that developed forestomach tumors (6-9 months after the start of experiment). The incidence of AA-induced forestomach tumors was 10% (two out of 20 rats) after co-administration of DAS and 60% (12 out of 20 animals) when AA was administered alone. The high dose of DAS (2 x 150 mg/kg) markedly inhibited the formation of squamous cell carcinomas in the forestomach. However, the thioether did not prevent the formation of forestomach and urinary bladder papillomatosis. Additionally, DAS co-administration decreased the accumulation of single-stranded regions in rat forestomach DNA. Using the nuclease P1 enhancement method of the 32P-postlabeling assay, a decrease in the level of AA-derived adducts was also detected after co-administration of DAS. We conclude that the decrease of DNA damage after DAS co-administration is associated with the delay in conversion of papillomas to malignant forestomach tumors.
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