Human enzymes involved in the metabolic activation of the environmental contaminant 3-nitrobenzanthrone: evidence for reductive activation by human NADPH:cytochrome p450 reductase.
Determining the capability of humans to metabolize the suspected carcinogen 3-nitrobenzanthrone (3-NBA) and understanding which human enzymes are involved in its activation are important in the assessment of individual susceptibility to this environmental contaminant found in diesel exhaust and ambient air pollution. We compared the ability of eight human hepatic microsomal samples to catalyze DNA adduct formation by 3-NBA. Using two enrichment procedures of the (32)P-postlabeling method, nuclease P1 digestion and butanol extraction, we found that all hepatic microsomes were competent to activate 3-NBA. DNA adduct patterns with multiple adducts, qualitatively similar to those found recently in vivo in rats, were observed. Additionally one major DNA adduct generated by human microsomes was detected. The role of specific cytochromes p450 (p450) and NADPH:p450 reductase in the human hepatic microsomal samples in 3-NBA activation was investigated by correlating the p450- and NADPH: p450 reductase-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-NBA was attributed to NADPH: p450 reductase. Inhibition of DNA adduct formation in human liver microsomes by alpha-lipoic acid, an inhibitor of NADPH: p450 reductase, supported this finding. Using the purified rabbit enzyme and recombinant human NADPH: p450 reductase expressed in Chinese hamster V79 cells, we confirmed the participation of this enzyme in the formation of 3-NBA-derived DNA adducts. Moreover, essentially the same DNA adduct pattern found in microsomes was detected in metabolically competent human lymphoblastoid MCL-5 cells. The role of individual human recombinant p450s 1A1, 1A2, 1B1, 2A6, 2B6, 2D6, 2C9, 2E1, and 3A4 and of NADPH: p450 reductase in the metabolic activation of 3-NBA, catalyzing DNA adduct formation, was also examined using microsomes of baculovirus-transfected insect cells containing the recombinant enzymes (Supersomes). DNA adducts were observed in all Supersomes preparations, essentially similar to those found with human hepatic microsomes and in human cells. Of all of the recombinant human p450s, p450 2B6 and -2D6 were the most efficient to activate 3-NBA, followed by p450 1A1 and -1A2. These results demonstrate for the first time the potential of human NADPH: p450 reductase and recombinant p450s to contribute to the metabolic activation of 3-NBA by nitroreduction.
Arlt
Volker M
VM
Section of Molecular Carcinogenesis, Institute of Cancer Research, Sutton, Surrey SM2 5NG, United Kingdom. v.arlt@icr.ac.uk
Stiborova
Marie
M
Hewer
Alan
A
Schmeiser
Heinz H
HH
Phillips
David H
DH
D000818:Animals; D001551:Benz(a)Anthracenes / Q000378:metabolism / Q000493:pharmacokinetics*; D001711:Biotransformation; D002478:Cells, Cultured; D006224:Cricetinae; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D018736:DNA Adducts / Q000096:biosynthesis; D004785:Environmental Pollutants / Q000378:metabolism / Q000493:pharmacokinetics*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005347:Fibroblasts / Q000201:enzymology; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism; D008214:Lymphocytes / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology; D009251:NADPH-Ferrihemoprotein Reductase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010084:Oxidation-Reduction; D011817:Rabbits; D051381:Rats; D017208:Rats, Wistar; D011994:Recombinant Proteins / Q000378:metabolism
D001551:Benz(a)Anthracenes; D065607:Cytochrome P-450 Enzyme Inhibitors; D018736:DNA Adducts; D004785:Environmental Pollutants; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; D009251:NADPH-Ferrihemoprotein Reductase; C117220:3-nitrobenzanthrone