3-aminobenzanthrone, a human metabolite of the environmental pollutant 3-nitrobenzanthrone, forms DNA adducts after metabolic activation by human and rat liver microsomes: evidence for activation by cytochrome P450 1A1 and P450 1A2.
3-Nitrobenzanthrone (3-NBA) is a suspected human carcinogen found in diesel exhaust and ambient air pollution. The main metabolite of 3-NBA, 3-aminobenzanthrone (3-ABA), was recently detected in the urine of salt mining workers occupationally exposed to diesel emissions. Determining the capability of humans to metabolize 3-ABA and understanding which human enzymes are involved in its activation are important in the assessment of individual susceptibility. We compared the ability of eight human hepatic microsomal samples to catalyze DNA adduct formation by 3-ABA. Using the (32)P-postlabeling method, we found that all hepatic microsomes were competent to activate 3-ABA. DNA adduct patterns with multiple adducts, qualitatively similar to those formed in vivo in rats treated with 3-ABA, were observed. These patterns were also similar to those formed by the nitroaromatic counterpart 3-NBA and which derive from reductive metabolites of 3-NBA bound to purine bases in DNA. The role of specific cytochrome P450s (P450s) in the human hepatic microsomal samples in 3-ABA activation was investigated by correlating the P450-linked catalytic activities in each microsomal sample with the level of DNA adducts formed by the same microsomes. On the basis of this analysis, most of the hepatic microsomal activation of 3-ABA was attributable to P450 1A1 and 1A2 enzyme activity. Inhibition of DNA adduct formation in human liver microsomes by alpha-naphthoflavone and furafylline, inhibitors of P450 1A1 and 1A2, and P450 1A2 alone, respectively, supported this finding. Using recombinant human P450 1A1 and 1A2 expressed in Chinese hamster V79 cells and microsomes of baculovirus-transfected insect cells (Supersomes), we confirmed the participation of these enzymes in the formation of 3-ABA-derived DNA adducts. Moreover, essentially the same DNA adduct pattern found in microsomes was detected in metabolically competent human lymphoblastoid MCL-5 cells expressing P450 1A1 and 1A2. Using rat hepatic microsomes, we showed that both human and rat microsomes lead to the same 3-ABA-derived DNA adducts. Pretreatment of rats with beta-naphthoflavone or Sudan I, inducers of P450 1A1 and 1A2, and P450 1A1 alone, respectively, significantly stimulated the levels of 3-ABA-derived DNA adducts formed by rat liver microsomes. Utilizing purified rat recombinant P450 1A1, the participation of this enzyme in DNA adduct formation by 3-ABA was corroborated. In summary, our results strongly suggest a genotoxic potential of 3-ABA for humans. Moreover, 3-ABA is not only a suitable biomarker of exposure to 3-NBA but may also directly contribute to the high genotoxic potential of 3-NBA.
Arlt
Volker M
VM
Section of Molecular Carcinogenesis, Institute of Cancer Research, Brookes Lawley Building, Cotswold Road, Sutton, Surrey SM2 5NG, United Kingdom. volker.arlt@icr.ac.uk
Hewer
Alan
A
Sorg
Bernd L
BL
Schmeiser
Heinz H
HH
Phillips
David H
DH
Stiborova
Marie
M
D000393:Air Pollutants / Q000378:metabolism*; D000818:Animals; D001551:Benz(a)Anthracenes / Q000378:metabolism*; D002461:Cell Line, Transformed; D019363:Cytochrome P-450 CYP1A1 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D019388:Cytochrome P-450 CYP1A2 / Q000235:genetics / Q000378:metabolism*; D065609:Cytochrome P-450 CYP1A2 Inhibitors; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism; D018736:DNA Adducts / Q000378:metabolism*; D006801:Humans; D008862:Microsomes, Liver / Q000201:enzymology*; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
C475568:3-aminobenzanthrone; D000393:Air Pollutants; D001551:Benz(a)Anthracenes; D065609:Cytochrome P-450 CYP1A2 Inhibitors; D018736:DNA Adducts; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; C104464:CYP3A protein, human; D019363:Cytochrome P-450 CYP1A1; D019388:Cytochrome P-450 CYP1A2; D051544:Cytochrome P-450 CYP3A; C117220:3-nitrobenzanthrone