Environmental Pollutant and Potent Mutagen 3-Nitrobenzanthrone Forms DNA Adducts after Reduction by NAD(P)H:Quinone Oxidoreductase and Conjugation by Acetyltransferases and Sulfotransferases in Human Hepatic Cytosols

Abstract

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)

Identification of three major DNA adducts formed by the carcinogenic air pollutant 3‐nitrobenzanthrone in rat lung at the C8 and N2 position of guanine and at the N6 position of adenine

“…Previously, we detected the formation of 3-NBAderived DNA adducts in rodent tissues by 32 P-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 -ABA in two independent systems (thin layer and high-performance liquid chromatography).…”

“…9 Its genotoxicity has been further documented by the detection of specific DNA adducts formed in various chemical and enzymatic in-vitro assays, in cells and in vivo in rodents treated with 3-NBA. [22][23][24][25][26][27][28][29][30][31][32][33][34][35] The main metabolite of 3-NBA, 3-aminobenzanthrone (3-ABA, Fig. 1), has been found in urine samples of underground salt mine workers occupationally exposed to diesel emissions, 10 and this metabolite forms the same DNA adducts as those formed by its nitroaromatic counterpart 3-NBA.…”

“…1) is catalysed primarily by cytosolic reductases, in particular NAD(P)H:quinone oxidoreductase (NQO1) and xanthine oxidase (XO), although microsomal NADPH:cytochrome P450 oxidoreductase (POR) can also be involved in the reduction of 3-NBA. 25,26,[30][31][32] N-OH-ABA formed by nitroreduction can be further activated by phase II enzymes, such as N,O-acetyltransferases (NATs) and sulfotransferases (SULTs), leading to the formation of reactive N-acetoxyor sulfooxy esters capable of reacting with DNA to form adducts. 27,29,32 It has been established that the multiple DNA adducts formed in vivo are derived from purine bases reacted with reductive metabolites of 3-NBA, which do not posses an N-acetyl group.…”

“…25,26,[30][31][32] N-OH-ABA formed by nitroreduction can be further activated by phase II enzymes, such as N,O-acetyltransferases (NATs) and sulfotransferases (SULTs), leading to the formation of reactive N-acetoxyor sulfooxy esters capable of reacting with DNA to form adducts. 27,29,32 It has been established that the multiple DNA adducts formed in vivo are derived from purine bases reacted with reductive metabolites of 3-NBA, which do not posses an N-acetyl group. 21,26,28,32,33 Although analyses by 32 P-postlabeling of in-vitro and in-vivo 3-NBA-derived DNA adducts have shown that they contain deoxyadenosine (dA) and deoxyguanosine (dG), 26,30 the definitive structural characterisation of these adducts has not been reported.…”

The Role of Biotransformation Enzymes in the Development of Renal Injury and Urothelial Cancer Caused by Aristolochic Acid: Urgent Questions and Difficult Answers

“…In this study, the authors used the HRN (Hepatic Cytochrome P450 Reductase Null) mouse model, previously shown to be suitable for examining hepatic versus extrahepatic xenobiotic metabolism in vivo [48][49][50][51] . For this reason , we have advocated this animal model for elucidating AA metabolism 25 .…”