Prabhakar D. Devanesan

Radical cations as precursors in the metabolic formation of quinones from benzo[a]pyrene and 6-fluorobenzo[a]pyrene: Fluoro substitution as a probe for one-electron oxidation in aromatic substrates

Three classes of products are formed when benzo[a]pyrene (BP) is metabolized by cytochrome P-450: dihydrodiols, phenols and the quinones, BP 1,6-, 3,6- and 6,12-dione. These products have been thought to arise from attack of a catalytically-activated electrophilic oxygen atom. In this paper we report chemical and biochemical experiments which demonstrate that BP quinones arise from an initial one-electron oxidation of BP to form its radical cation. BP, 6-fluorobenzo[a]pyrene (6-FBP), 6-chlorobenzo[a]pyrene (6-ClBP), and 6-bromobenzo[a]pyrene (6-BrBP) were metabolized by uninduced and 3-methylcholanthrene-induced rat liver microsomes in the presence of NADPH or cumene hydroperoxide (CHP) as cofactor. BP and 6-FBP produced similar metabolic profiles with induced microsomes in the presence of NADPH or 2 mM CHP. With NADPH both compounds produced dihydrodiols, phenols and quinones, whereas with CHP, they yielded only quinones. Metabolism of BP and 6-FBP was also similar with uninduced microsomes and 2 mM CHP, yielding the same BP quinones. With uninduced microsomes in the presence of NADPH, BP produced all three classes of metabolites, whereas 6-FBP afforded only quinones. At a low concentration of CHP (0.10 mM), BP was metabolized to phenols and quinones, whereas 6-FBP gave only quinones. 6-ClBP and 6-BrBP were poor substrates, forming metabolites only with induced microsomes and NADPH. One-electron oxidation of BP by Mn(OAc)3 occurred exclusively at C-6 with predominant formation of 6-acetoxyBP and small amounts of BP quinones. In the one-electron oxidation of 6-FBP by Mn(OAc)3, the major products obtained were 6-acetoxyBP, a mixture of 1,6- and 3,6-diacetoxyBP, and BP quinones. Reaction of BP and 6-FBP radical cation perchlorates with water produced the same BP quinones. Conversely, electrophilic substitution of 6-FBP with bromine or deuterium ion afforded C-1 and/or C-3 derivatives with retention of the fluoro substituent at C-6. These results indicate that metabolic formation of BP quinones from BP and 6-FBP can only derive from their intermediate radical cation.

Radical cations in the horseradish peroxidase and prostaglandin H synthase mediated metabolism and binding of benzo[a]pyrene to deoxyribonucleic acid

Metabolism and DNA binding studies are used to investigate mechanisms of activation for carcinogens. In this paper we describe metabolism of benzo[a]pyrene (BP) and 6-fluorobenzo[a]pyrene (6-FBP) by two peroxidases, horseradish peroxidase (HRP) and prostaglandin H synthase (PHS), which are known to catalyze one-electron oxidation. In addition, binding of BP and BP quinones to DNA was compared in the two enzyme systems. The only metabolites formed from BP or 6-FBP by either enzyme were the quinones, BP 1,6-, 3,6- and 6,12-dione. HRP metabolized BP and 6-FBP to the same extent and produced the same proportion of each dione from both compounds, approximately 40% each of BP 1,6- and 3,6-dione and 20% BP 6,12-dione. PHS formed twice as much quinones from BP as from 6-FBP and produced relatively more BP 3,6-dione from 6-FBP (46%) compared to BP (30%) and relatively less BP 6,12-dione from 6-FBP (16%) compared to BP (33%). Removal of the fluoro substituent in the metabolism of 6-FBP is consistent only with an initial one-electron oxidation of the substrate. Since BP quinones were the only products formed in HRP- and PHS-catalyzed activation of BP, their possible binding to DNA was compared to that of BP. No significant binding of BP quinones to DNA occurred with either HRP or PHS. These results, coupled with those from other chemical and biochemical experiments, demonstrate that HRP- and PHS-catalyzed one-electron oxidation of BP to its radical cation is the mechanism of formation of quinones and binding of BP to DNA.

Effect of 7-Hydroxybenzo[a]pyrene on Benzo[a]pyrene Metabolism and Formation of DNA Adducts

Abstract Two major pathways are responsible for the metabolic activation of benzo[a]pyrene (BP) and other aromatic hydrocarbons, one leading to radical cations and the other to diol epoxides. It has been suggested that 7-hydroxyBP (7-OHBP) might inhibit formation of diol epoxides. We have examined the effect of various concentrations of 7-OHBP on the cytochrome P450-catalyzed metabolism of BP, total DNA binding and formation of BP-DNA adducts. Metabolism of BP was not significantly affected by 7-OHBP. Both total BP-DNA binding and stable DNA adducts, as measured by the 32P-postlabeling method, were inhibited in a concentration-dependent manner, ranging from 25% to 72% inhibition for binding and 23% to 85% for adducts at 7-OHBP:BP ratios of 0.1 to 1.0. Formation of the depurination adducts of BP was inhibited 67% to 100% at a molar ratio of 1:0.25 (BP:7-OHBP), whereas none of the depurination adducts were detected at a molar ratio of 1:0.5 (BP:7-OHBP). These results suggest that 7-OHBP is not a selective i...

Covalent Binding of Benzo[a]pyrene to Free and Membrane-Bound DNA in Isolated Liver Nuclei from 3-Methylcholanthrene-Induced Rats

Abstract Benzo[a]pyrene (BP) is bound to DNA by two major pathways: one-electron oxidation and monooxygenation. One-electron oxidation requires that the highly reactive radical cation be formed in close proximity to the DNA. Thus, in intact cell nuclei, binding of BP radical cation would be expected to occur predominantly in DNA associated with the membrane. To examine this, nuclei from the livers of 3-methylcholanthrene-induced male MRC Wistar rats were incubated with [3H]BP and NADPH. After the incubation, the “free” DNA was separated from “membrane-bound” DNA by extraction in low-Mg buffer. Binding of BP to membrane-bound DNA was 9.5 ± 2.0 μmol/mol DNA-P, whereas binding to free DNA was 2.2 ± 0.7 μmol/mol DNA-P. BP adducts formed by one-electron oxidation and lost from the nuclear DNA by depurination were also examined. The three depurination adducts previously found with rat liver microsomes and in mouse skin were also obtained from nuclei. These results suggest that binding of BP to DNA in intact nuc...

Metabolism of 1- and 3-Fluorobenzo[a]pyrene by Cytochrome p450 and Horseradish Peroxidase

Abstract Benzo[a]pyrene (BP) is a good model for elucidating the mechanism of oxygen transfer for substrates that are considered good electron donors. Fluoro substitution of BP represents a suitable probe for studying mechanisms of oxygen transfer in the metabolic formation of BP quinones and BP phenols. By using this strategy with 6-fluoroBP (6-FBP), we have previously demonstrated that the BP quinones are formed metabolically via an initial one-electron oxidation of BP catalyzed by cytochrome P450. Now we have synthesized 1-FBP and 3-FBP with the purpose of elucidating the mechanism of phenol formation. If formation of 3-hydroxyBP (3-OHBP) (major metabolite of BP) and 1-OHBP (minor metabolite of BP) occurs via an initial electron transfer from BP to cytochrome P450, when we use 3-FBP and 1-FBP as substrates, one of the metabolites formed should be BP-3,6-dione and BP-1,6-dione, respectively, with displacement of fluorine. Metabolism of 1-FBP and 3-FBP by rat liver microsomes and by horseradish peroxidas...