Baculovirus-mediated expression and characterization of rat CYP2A3 and human CYP2a6: role in metabolic activation of nasal toxicants.
Cytochrome P450 2A3 (CYP2A3) was previously identified in rat lung by cDNA cloning and recently found to be expressed at a high level in the olfactory mucosa. In the current study, CYP2A3 was expressed in insect cells lacking endogenous cytochrome P450 (P450) activity, and the substrate specificity of the recombinant cytochrome was characterized and compared with that of CYP2A6, a human ortholog of rat CYP2A3, which has been detected in human olfactory mucosa as well as in liver. The CYP2A3 and CYP2A6 cDNAs were cloned into baculovirus, and recombinant viruses were used to produce active enzymes in Spodoptera frugiperta (SF9) cells. The metabolic activities of S. frugiperta cell microsomal fractions containing CYP2A3 or CYP2A6 were studied in a reconstituted system with purified rabbit NADPH-P450 reductase. CYP2A3 was found to be active toward testosterone, producing 15 alpha-hydroxytestosterone and several other metabolites, but it had only low activity toward coumarin. On the other hand, CYP2A6 was active toward coumarin but not toward testosterone. However, both enzymes were active in the metabolic activation of hexamethylphosphoramide, a nasal procarcinogen, and 2,6-dichlorobenzonitrile (DCBN), a herbicide known to cause tissue-specific toxicity in the olfactory mucosa of rodents at very low doses. In addition, both enzymes were active toward 4-nitrophenol, a preferred substrate for CYP2E1. Consistent with CYP2A3 being a major catalyst in microsomal metabolism of DCBN, the activities of both CYP2A3 and rat olfactory microsomes in DCBN metabolism were inhibited strongly by metyrapone and methoxsalen (ID50 < 1 microM, with DCBN at 30 microM), but only marginally by 4-methylpyrazole, an inhibitor of CYP2E1. In contrast, the activity of CYP2A6 was only weakly inhibited by metyrapone or methoxsalen (ID50 > 50 microM). Thus, rat CYP2A3 and human CYP2A6 have differences in substrate specificity as well as tissue distributor. These findings should be taken into account when assessing the risk of exposure to potential nasal toxicants in humans.
Liu
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Laboratory of Human Toxicology and Molecular Epidermiology, Wadsworth Center, New York State Department of Health, New York, USA.
Zhuo
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Gonzalez
F J
FJ
Ding
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D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D016367:Baculoviridae / Q000235:genetics / Q000378:metabolism; D001711:Biotransformation; D003374:Coumarins / Q000378:metabolism; D065637:Cytochrome P-450 CYP2A6; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D000077604:Fomepizole; D006492:Hempa / Q000493:pharmacokinetics* / Q000633:toxicity*; D006801:Humans; D015151:Immunoblotting; D008297:Male; D008730:Methoxsalen / Q000494:pharmacology; D008797:Metyrapone / Q000494:pharmacology; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D009570:Nitriles / Q000493:pharmacokinetics* / Q000633:toxicity*; D009596:Nitrophenols / Q000378:metabolism; D009831:Olfactory Mucosa / Q000187:drug effects* / Q000201:enzymology; D011720:Pyrazoles / Q000494:pharmacology; D011817:Rabbits; D051381:Rats; D017208:Rats, Wistar; D018411:Spodoptera / Q000821:virology; D013379:Substrate Specificity; D013739:Testosterone / Q000378:metabolism
D003374:Coumarins; D065607:Cytochrome P-450 Enzyme Inhibitors; D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D009570:Nitriles; D009596:Nitrophenols; D011720:Pyrazoles; D013739:Testosterone; D000077604:Fomepizole; D003577:Cytochrome P-450 Enzyme System; C030123:coumarin; D006899:Mixed Function Oxygenases; C585519:Cyp2a3 protein, rat; D001189:Aryl Hydrocarbon Hydroxylases; C585710:CYP2A6 protein, human; D065637:Cytochrome P-450 CYP2A6; D006492:Hempa; C055869:dichlobanil; D008730:Methoxsalen; C024836:4-nitrophenol; D008797:Metyrapone