32P-Postlabelling analysis of the covalent binding of benzo[ghi]perylene to DNA in vivo and in vitro

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Abstract
Benzo[ghi]perylene (B[ghi]P) is a polycyclic aromatic hydrocarbon (PAH), present in complex combustion products, and evidence for its carcinogenic activity in experimental animals is equivocal, and yet it has demonstrable mutagenic activity in vitro. In order to investigate the possible DNA binding properties of B[ghi]P, groups of male Parkes mice were treated topically with 1.0 μmol of B[ghi]P. Mice were killed up to 3 months after treatment, DNA was isolated from the treated areas of skin and analysed for adducts by 32P-postlabelling. Maximum levels of binding (0.57 fmol/μg DNA) were detected 2 days after treatment and adducts were found to persist for at least 12 weeks after treatment, a property of many PAHs whh known tumor-initiating activity. B[ghi]P also became bound to DNA in vitro in the presence of 3-methylcholanthrene-induced rat liver microsomal preparations. When chromatographed on PEI–cellulose, the major adducts formed by B[ghi]P in vivo and in vitro appeared to be identical. However, they were found to be different when compared by reversed-phase HPLC. These differences might explain, in part, the differences in the biological activity of B[ghi]P in vivo and in vitro,. The behaviour of B[ghi]P when present in a mixture was also examined. B[ghi]P was applied topically to mouse skin with six other PAHs at a dose level of 0.25 μmol/PAH/mouse. DNA isolated 24 h after treatment was analysed for adducts by 32P-postlabelllng. Whilst the total level of binding was 30% lower than expected from the sum of the binding levels that resulted when the hydrocarbons were applied singly, the formation of B[ghi]P–DNA adducts did not appear to be inhibited. The results have demonstrated that B[ghi]P has significant DNA binding ability in vivo and in vitro and on the basis of its DNA binding ability in mouse skin it would be predicted to be at least a weak tumour initiator. The formation of DNA adducts by B[ghi]P when present in an artificial mixture of PAHs suggests that B[ghi]P may contribute to the DNA binding activity of more complex carcinogenic mixtures.

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