Reactive oxygen species can give rise to numerous modifications of DNA. We have investigated the formation of such modifications using the nuclease P1 digestion method of the 32P-postlabelling procedure for the detection of DNA damage. Analysis of DNA that had been treated with a Fenton-type system of copper (or iron) ions and H2O2 resulted in the detection of up to ten discrete 32P-labelled spots, displaying chromatographic characteristics similar to aromatic adducts, on PEI-cellulose TLC. Maximum total levels equivalent to 28 adducts/10(8) nucleotides were achieved after 15 min of treatment with Cu2+/H2O2. The formation of adducts was 1.5 times greater if single-stranded rather than double-stranded DNA was employed, suggesting an intrastrand effect. Experiments with 3'-deoxyribonucleotides demonstrated that the adducts detected did not represent base modifications such as 8-hydroxydeoxyguanosine or thymidine glycols. However, treatment of specific dinucleotides (dApdG and dApdA) was found to produce two major adducts that were chromatographically identical by TLC and HPLC to the two major adducts formed in DNA. It is proposed that these species with aromatic adduct-like characteristics are the result of the intrastrand linking of specific adjacent bases in DNA.

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Dibenz[a,h]anthracene (DB[a,h]A) and the related 3,4-diol and anti- and syn-3,4-diol 1,2-oxides were applied to the shaved dorsal skin of groups of four C57Bl/CB1 mice. Twenty-four hours later the mice were killed, DNA isolated from the treated skin, hydrolysed and examined for the presence of aromatic adducts using the nuclease P1 modification of the 32P-postlabelling technique. Autoradiography of the maps obtained by chromatography on polyethyleneimine-cellulose plates showed that six DNA adduct spots that were derived from DB[a,h]A were also present in the DNA of skin treated with the DBA 3,4-diol and that, whilst four of these adduct spots were also seen in maps prepared from the DNA of skin treated with the anti-3,4-diol-1,2-oxide, they were not present in DNA from skin to which the syn-isomer had been applied. The identity of these adduct spots was confirmed by their coincidence when mixtures of different DNA hydrolysates were chromatographed together. Quantitatively, the highest levels of mouse skin modification were obtained with the diol-epoxides and the lowest with DB[a,h]A. The results suggest that most of the DNA adducts formed in DB[a,h]A-treated mouse skin arise through metabolism of the hydrocarbon to the related 3,4-diol and that some may be formed following the conversion of this diol to the bay-region anti-3,4-diol-1,2-oxide.

Male and female CDF1 mice were administered a single oral dose of 3 mumol of the food mutagens 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) or 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) and killed 24 h later. DNA was isolated from the livers, lungs, kidneys, colon and forestomach and analysed by 32P-postlabelling for the presence of IQ and MeIQ adducts. Several adduct-enrichment procedures were investigated, including ATP-deficient labelling conditions, butanol extraction and nuclease P1 digestion, and only the ATP-deficient procedure was found to produce the same adduct pattern on polyethyleneimine--cellulose TLC as the standard procedure. Up to nine adduct spots were detected in liver DNA from IQ-treated mice, two of which were not detected in other tissues. The levels of binding in both male and female mice were in the order liver greater than kidney greater than colon greater than forestomach greater than lung. Analysis of DNA from MeIQ-treated mice revealed the presence of up to seven adducts, one of which was detected in liver but not in other tissues. The relative order of DNA binding was kidney greater than liver greater than or equal to colon greater than forestomach greater than lung. As dietary feeding of IQ induces liver, lung and forestomach tumours, and MeIQ induces liver and forestomach tumours in this mouse strain, these binding levels do not correlate with the susceptibility of the organs to carcinogenesis induced by these compounds; the results may indicate the importance of additional factors in determining organ specificity of carcinogenicity.

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