Metabolic activation of the environmental contaminant 3-nitrobenzanthrone by human acetyltransferases and sulfotransferase

Abstract

3-Nitrobenzanthrone (3-NBA) an extremely potent mutagen and suspected human carcinogen identified in diesel exhaust and in airborne particulate matter was shown to form multiple DNA adducts in vitro and in vivo in rats. In order to investigate whether human N,O-acetyltransferases (NATs) and sulfotransferases (SULTs) contribute to the metabolic activation of 3-NBA we used a panel of newly constructed Chinese hamster lung fibroblast V79MZ derived cell lines expressing human NAT1, human NAT2 or human SULT1A1, as well as TA1538-derived Salmonella typhimurium strains expressing human NAT1 (DJ400) or human NAT2 (DJ460) and determined DNA binding and mutagenicity. The formation of 3-NBA-derived DNA adducts was analysed by (32)P-postlabelling after exposing V79 cells to 0.01 micro M 3-NBA or 0.1 micro M N-acetyl-N-hydroxy-3-aminobenzanthrone (N-Ac-N-OH-ABA), a potential metabolite of 3-NBA. Similarly up to four major and two minor adducts were detectable for both compounds, the major ones being identical to those detected previously in DNA from rats treated with 3-NBA. Comparison of DNA binding between different V79MZ derived cells revealed that human NAT2 and, to a lesser extent, human NAT1 and human SULT1A1, contribute to the genotoxic potential of 3-NBA and N-Ac-N-OH-ABA to form DNA adducts. However, the extent of DNA binding by 3-NBA was higher in almost all V79 cells at a 10-fold lower concentration than by N-Ac-N-OH-ABA, suggesting that N-Ac-N-OH-ABA is not a major intermediate in the formation of 3-NBA-derived adducts. 3-NBA showed a 3.8-fold and 16.8-fold higher mutagenic activity in Salmonella strains expressing human NAT1 and human NAT2, respectively, than in the acetyltransferase-deficient strain, whereas N-Ac-N-OH-ABA was only clearly (but weakly) mutagenic in Salmonella DJ460 expressing human NAT2. This finding suggests that N-Ac-N-OH-ABA is not a major reactive metabolite responsible for the high mutagenic potency of 3-NBA in Salmonella. Collectively our results indicate that O-acetylation and O-sulfonation by human NATs and SULTs may contribute significantly to the high mutagenic and genotoxic potential of 3-NBA. Moreover, the yet-unidentified four major 3-NBA-derived adducts may be DNA adducts without an N-acetyl group.

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

“…30,32 To resolve which human enzyme systems are able to catalyse the formation of dA-N 6 -ABA, dG-N 2 -ABA and dG-C8-N-ABA from 3-NBA and its main human metabolite 3-ABA, we used different experimental approaches to study the enzymology as described previously. 20,27,[29][30][31][32]36,37 As outlined in Figure 1, POR and NQO1 are mainly responsible for nitroreduction of 3-NBA to form N-OH-ABA. 29,30,32 Recent data indicate that in vivo 3-NBA is predominantly activated by cytosolic nitroreductases rather than microsomal POR.…”

“…27 Enrichment by butanol extraction has been shown to yield more adduct spots and a better recovery of 3-NBAderived DNA adducts than using enrichment by nuclease P1 digestion. For resolution of DNA adduct spots, the following chromatographic conditions were used: D1, 1.0 M sodium phosphate, pH 6.0; D3, 4 M lithium formate, 7 M urea, pH 3.5 and D4, 0.8 lithium chloride, 0.5 M Tris, 8.5 M urea, pH 8.0.…”

“…DNA adduct spots were numbered as reported. 26,27,32,36 DNA adduct levels (RAL, relative adduct labeling) were calculated from the adduct cpm, the specific activity of [g-32 P]ATP and the amount of DNA (pmol of DNA-P) used.…”

“…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.…”

“…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.…”