Synthesis and structure determination of 6-methylbenzo[a]pyrene-deoxyribonucleoside adducts and their identification and quantitation in vitro and in mouse skin

Abstract

Activation of the moderate carcinogen 6-methylbenzo[a]pyrene (6-CH₃BP) by one-electron oxidation to form DNA adducts was studied. Iodine oxidation of 6-CH₃BP in the presence of dGuo produces BP-6-CH₂-N²dGuo, BP-6-CH₂-N7Gua and a mixture of 6-CH₃BP-(1&3)-N7Gua, whereas in the presence of Ade the adducts BP-6-CH₂-N1Ade, BP-6-CH₂-N3Ade, BP-6-CH₂-N7Ade and 6-CH₃BP-(1&3)-N1Ade are obtained. Furthermore, for the first time an aromatic hydrocarbon radical cation afforded an adduct with dThd, the stable adduct BP-6-CH₂-N3dThd. Formation of these adducts indicates that the 6-CH₃BP radical cation has charge localized at the 6, 1 and 3 position. When 6-CH₃BP was activated by horseradish peroxidase in the presence of DNA, two depurinating adducts were identified, BP-6-CH₂-N7Gua (48%) and 6-CH₃BP-(1&3)-N7Gua (23%), with 29% unidentified stable adducts. In the binding of 6-CH₃BP catalyzed by rat liver microsomes, the same two depurinating adducts, BP-6-CH₂-N7Gua (22%) and 6-CH₃BP-(1&3)-N7Gua (10%), were identified, with 68% unidentified stable adducts. In 6-CH₃BP-treated mouse skin, the two depurinating adducts, BP-6-CH₂-N7Gua and 6-CH₃BP-(1&3)-N7Gua, were identified. Although quantitation of these two adducts was not possible due to coelution of metabolites on HPLC, they appeared to be the major adducts found in mouse skin. These results show that 6-CH₃BP forms depurinating adducts only with the guanine base of DNA, both in vitro and in mouse skin. The weaker reactivity of 6-CH₃BP radical cation vs. BP radical cation could account for the weaker tumor-initiating activity of 6-CH₃BP in comparison to that of BP.

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.