Synthesis and structure determination of 6-methylbenzo[a]pyrene-deoxyribonucleoside adducts and their identification and quantitation in vitro and in mouse skin
Introduction
Polycyclic aromatic hydrocarbons (PAH) undergo two main pathways of metabolic activation related to tumor initiation: one-electron oxidation to give radical cations and monooxygenation with formation of bay-region diol epoxides [1], [2], [3]. Activation of PAH can proceed through either of these pathways, or both, to form stable and depurinating DNA adducts. The stable adducts remain covalently bound to DNA, whereas depurinating adducts are eliminated from DNA by cleavage of the glycosyl bond. Determination of the structure of DNA adducts provides information on the mechanism of activation, the type of DNA damage, and the biological significance in terms of tumor initiation. A relationship has been established between Harvey-ras mutations in mouse skin papillomas and apurinic sites generated by loss of depurinating PAH-DNA adducts [4].
To elucidate mechanisms of tumor initiation, the profile of DNA adducts formed by benzo[a]pyrene (BP) in vitro and in vivo has been determined [5], [6]. In mouse skin, 71% of the total adducts are depurinating adducts. Of these, 66% are formed from BP radical cation, with specific reaction at C-6 [6]. Some DNA adducts of 6-fluoroBP and 6-methylbenzo[a]pyrene (6- CH3BP) have been synthesized by electrochemical oxidation [7], and the stable adducts formed by these two PAH in vitro have been investigated [8]. Tumorigenicity studies of BP, 6-fluoroBP, 6-CH3BP, 6-chloroBP, and 6-bromoBP show that 6-fluoroBP and 6-CH3BP are active [9], [11], but less potent than the parent compound, whereas 6-chloroBP and 6-bromoBP are virtually inactive [9], [10].
6-CH3BP contains a methyl substituent at the critical C-6, the preferred position in which the BP radical cation binds to nucleophiles. Charge localization of the 6-CH3BP radical cation at C-6 renders the methyl group reactive with nucleophiles. This has been observed in the oxidation of 6-CH3BP by manganic acetate, as well as in electrochemical oxidation of 6-CH3BP in the presence of dGuo [7], [12]. In these reactions, nucleophilic attack at the methyl group of the 6-CH3BP radical cation competes with attack at the positions of second highest reactivity, C-1 and C-3. This suggests that the reactivity of the methyl group in 6-CH3BP radical cation is less than that of C-6 in BP radical cation, in which no adducts are obtained at C-1 or C-3 [12], [13], [14], [15].
In this paper, we report the synthesis of standard DNA adducts substituted at the 6-methyl group, and at C-1 and C-3, obtained by iodine oxidation of 6-CH3BP in the presence of deoxyribonucleosides and nucleobases. This method yields adducts that are not obtained by electrochemical oxidation [7]. We also report the identification of biologically formed 6-CH3BP-DNA depurinating adducts by comparison with the synthesized standard adducts. Total stable adducts were quantitated by the 32P-postlabeling method [16]. Knowledge of the DNA adducts formed by 6-CH3BP is necessary for understanding the biological significance of the adducts in producing the mutations leading to cancer.
Section snippets
Methods and materials
Caution. The chemicals 6-CH3BP and BP are hazardous and should be handled carefully in accordance with NIH guidelines [17].
Synthesis and structure determination of adducts formed by iodine oxidation of 6-CH3BP in the presence of deoxyribonucleosides or nucleobases
Some adducts were previously synthesized by electrochemical oxidation of 6-CH3BP in the presence of dAdo or dGuo [7]. The adducts previously identified as BP-6-CH2-N3Ade and BP-6-CH2-C8dGuo [7] are, in fact, BP-6-CH2-N7Ade and BP-6-CH2-N2dGuo, respectively. These errors were rectified by the use of 500 MHz NMR [15], [22].
One-electron oxidation of 6-CH3BP produces a radical cation with charge mainly localized at the C-6 position adjacent to the methyl group. Loss of the acidic methyl proton
Conclusions
Reaction of the 6-CH3BP radical cation in chemical and biological systems occurs competitively at the 6-CH3 group and at C-1 and C-3, the positions of second highest charge density in its radical cation. Depurinating adducts of 6-CH3BP are formed in vitro and in mouse skin only at guanine bases. Furthermore, for the first time, a PAH radical cation was found to produce an adduct with thymidine, the stable adduct BP-6-CH2-N3dThd.
6-CH3BP is much less carcinogenic than BP [9], [11]. In biological
Acknowledgements
We gratefully thank Dr. Ronald Cerny at the Nebraska Center for Mass Spectrometry at the University of Nebraska-Lincoln for the valuable mass spectral data. We extend our deepest gratitude to Paula Mailander and Dr. Dhruba Chakravarti for help in isolating DNA from 6-CH3BP-7,8-dihydrodiol-treated mouse skin. We would also like to thank Sheila Higginbotham for her help with the mouse skin experiments. This research was supported by a US PHS grant from the National Cancer Institute (P01 CA49210).
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