3-Nitrobenzanthrone (3-nitro-7H-benz [de]anthracen-7-one, 3-NBA) is a potent mutagen and suspected human carcinogen identified in diesel exhaust and air pollution. We compared the ability of human hepatic cytosolic samples to catalyze DNA adduct formation by 3-NBA. Using the 32 P-postlabeling method, we found that 12/12 hepatic cytosols activated 3-NBA to form multiple DNA adducts similar to those formed in vivo in rodents. By comparing 3-NBA-DNA adduct formation in the presence of cofactors of NAD(P)H:quinone oxidoreductase (NQO1) and xanthine oxidase, most of the reductive activation of 3-NBA in human hepatic cytosols was attributed to NQO1. Inhibition of adduct formation by dicoumarol, an NQO1 inhibitor, supported this finding and was confirmed with human recombinant NQO1. When cofactors of N,Oacetyltransferases (NAT) and sulfotransferases (SULT) were added to cytosolic samples, 3-NBA-DNA adduct formation increased 10-to 35-fold. Using human recombinant NQO1 and NATs or SULTs, we found that mainly NAT2, followed by SULT1A2, NAT1, and, to a lesser extent, SULT1A1 activate 3-NBA. We also evaluated the role of hepatic NADPH:cytochrome P450 oxidoreductase (POR) in the activation of 3-NBA in vivo by treating hepatic POR-null mice and wild-type littermates i.p. with 0.2 or 2 mg/kg body weight of 3-NBA. No difference in DNA binding was found in any tissue examined (liver, lung, kidney, bladder, and colon) between null and wild-type mice, indicating that 3-NBA is predominantly activated by cytosolic nitroreductases rather than microsomal POR. Collectively, these results show the role of human hepatic NQO1 to reduce 3-NBA to species being further activated by NATs and SULTs. (Cancer Res 2005; 65(7): 2644-52)

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Abstract3‐Nitrobenzanthrone (3‐NBA) is a potent mutagen and potential human carcinogen identified in diesel exhaust and ambient air particulate matter. Previously, we detected the formation of 3‐NBA‐derived DNA adducts in rodent tissues by 32P‐postlabeling, all of which are derived from reductive metabolites of 3‐NBA bound to purine bases, but structural identification of these adducts has not yet been reported. We have now prepared 3‐NBA‐derived DNA adduct standards for 32P‐postlabeling by reacting N‐acetoxy‐3‐aminobenzanthrone (N‐Aco‐ABA) with purine nucleotides. Three deoxyguanosine (dG) adducts have been characterised as N‐(2′‐deoxyguanosin‐8‐yl)‐3‐aminobenzanthrone‐3′‐phosphate (dG3′p‐C8‐N‐ABA), 2‐(2′‐deoxyguanosin‐N2‐yl)‐3‐aminobenzanthrone‐3′‐phosphate (dG3′p‐N2‐ABA) and 2‐(2′‐deoxyguanosin‐8‐yl)‐3‐aminobenzanthrone‐3′‐phosphate (dG3′p‐C8‐C2‐ABA), and a deoxyadenosine (dA) adduct was characterised as 2‐(2′‐deoxyadenosin‐N6‐yl)‐3‐aminobenzanthrone‐3′‐phosphate (dA3′p‐N6‐ABA). 3‐NBA‐derived DNA adducts formed experimentally in vivo and in vitro were compared with the chemically synthesised adducts. The major 3‐NBA‐derived DNA adduct formed in rat lung cochromatographed with dG3′p‐N2‐ABA in two independent systems (thin layer and high‐performance liquid chromatography). This is also the major adduct formed in tissue of rats or mice treated with 3‐aminobenzanthrone (3‐ABA), the major human metabolite of 3‐NBA. Similarly, dG3′p‐C8‐N‐ABA and dA3′p‐N6‐ABA cochromatographed with two other adducts formed in various organs of rats or mice treated either with 3‐NBA or 3‐ABA, whereas dG3′p‐C8‐C2‐ABA did not cochromatograph with any of the adducts found in vivo. Utilizing different enzymatic systems in vitro, including human hepatic microsomes and cytosols, and purified and recombinant enzymes, we found that a variety of enzymes [NAD(P)H:quinone oxidoreductase, xanthine oxidase, NADPH:cytochrome P450 oxidoreductase, cytochrome P450s 1A1 and 1A2, N,O‐acetyltransferases 1 and 2, sulfotransferases 1A1 and 1A2, and myeloperoxidase] are able to catalyse the formation of 2‐(2′‐deoxyguanosin‐N2‐yl)‐3‐aminobenzanthrone, N‐(2′‐deoxyguanosin‐8‐yl)‐3‐aminobenzanthrone and 2‐(2′‐deoxyadenosin‐N6‐yl)‐3‐aminobenzanthrone in DNA, after incubation with 3‐NBA and/or 3‐ABA. © 2005 Wiley‐Liss, Inc.

The reaction of trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BP-diolepoxide) with deoxyguanosine has been studied. In addition to the expected N2-guanine derivative minor products resulting from reaction at the O6 and 7-positions have been identified. Reaction of racemic, (+) or (-) BP-diolepoxide with [14C] and [3H]purine labelled DNA allowed these same products to be identified and their yields estimated. It was found that the O6 and 7-guanine products were derived mainly from reaction of the (-)isomer. The 7-substituted guanine derivative in DNA was unstable, undergoing either spontaneous release of the substituted guanine or imidazole ring opening.

Alkylation of DNA by the nitrogen mustard bis(2-chloroethyl)methylamine (mechlorethamine; HN2) gave four principal products, derived by mono-alkylation of guanine at N-7 and adenine at N-3 and by cross-linking of guanine to guanine or guanine to adenine at these positions. These products were isolated by hydrolysis from DNA at neutral pH, followed by ion-exchange chromatography on SP-Sephadex and reversed phase chromatography on ODS. They were characterized by identification with products from the reaction of nitrogen mustard with adenine or deoxyguanylic acid, and by their UV, mass, and proton magnetic resonance spectra.