Covalent binding of isomeric benzo[a]pyrene diol-epoxides to DNA

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Abstract
We have compared the abilities of two diol-epoxide derivatives of benzo[a]pyrene (B[a]P) to bind covalently to DNA in a simple in vitro system. Purified DNA in aqueous solution was allowed to react with (±)-7α, 8β-dihydroxy-9β, 10β-oxy-7,8,9,10-tetrahydroB[a]P (BPDE) or with (±)-9α, 10β-dihydroxy-7α,8α-oxy-7,8,9,10-tetrahydroB[a]P (reverse BPDE) to completion. After repurification of the DNA, binding was detected by fluorescence spectroscopy or by absorbance spectroscopy. Both BPDE and reverse BPDE but not their hydrolysis products exhibited binding which increased linearly with increasing diol epoxide concentration. When DNA modified by reverse BPDE was enzymatically hydrolysed, two major fluorescent deoxyribonucleoside-adducts were detected by reverse phase h.p.l.c. These were separable from the major adduct obtained from BPDE-modified DNA and from the major products obtained by hydrolysis of reverse BPDE in the absence of DNA. Absorbance and fluorescence spectroscopy of modified native DNA suggested that the pyrene nucleus of reverse BPDE but not of BPDE was intercalated in the DNA double helix. This suggestion was supported by fluorescence-quenching studies. In the presence of increasing DNA concentrations, covalent binding of both diol epoxides increased towards an apparent maximum. Double reciprocal analysis of the data indicated a maximum binding level of ∼5% of the total dose for BPDE and 4% for reverse BPDE. This suggests that for both diol epoxides the ratio of the rate constants for covalent binding and for DNA-enhanced hydrolysis are nearly the same. Covalent binding of reverse BPDE to DNA was effectively blocked by low concentrations of Mg 2+ , suggesting that formation of a non-covalent intercalation complex may be a prerequisite for covalent reaction.