Purification and characterization of heterologously expressed mouse CYP2A5 and CYP2G1: role in metabolic activation of acetaminophen and 2,6-dichlorobenzonitrile in mouse olfactory mucosal microsomes.
The metabolic activation of two known olfactory mucosal (OM) toxicants, acetaminophen (AP) and 2,6-dichlorobenzonitrile (DCBN), was examined with mouse liver and OM microsomes and purified, heterologously expressed mouse CYP2A5 and CYP2G1. In reconstituted systems, both isoforms were active in metabolizing DCBN and AP to metabolites that formed protein adducts. The formation of DCBN- or AP-protein adducts and other AP metabolites, including 3-hydroxy-AP and, in the presence of glutathione, AP-glutathione conjugate, was also detected in OM microsomal reactions and to a much greater extent than in liver microsomes. Evidence was obtained that CYP2A5 and CYP2G1 play major roles in mouse OM microsomal metabolic activation of DCBN and AP. Immunoblot analysis indicated that CYP2A5 and CYP2G1 are abundant P450 isoforms in OM microsomes. OM microsomal AP and DCBN metabolic activation was inhibited by 5- and 8-methoxsalen, which inhibit both CYP2A5 and CYP2G1, and by an inhibitory anti-CYP2A5 antibody that also inhibits CYP2G1. In addition, the roles of CYP1A2 and CYP2E1 in the OM bioactivation of AP and DCBN were ruled out by comparing activities of acetone-treated mice or Cyp1a2(-/-) mice with those of control mice. Thus, CYP2A5 and CYP2G1 may both contribute to the known OM-selective toxicity of AP and DCBN. Further analysis of the kinetics of AP and DCBN metabolism by the purified P450s suggested that CYP2A5 may play a greater role in OM microsomal metabolism of AP, whereas their relative roles in DCBN metabolism may be dose dependent, with CYP2G1 playing more important roles at low substrate concentrations.
Gu
J
J
Wadsworth Center, New York State Department of Health, Albany, New York, USA.
Zhang
Q Y
QY
Genter
M B
MB
Lipinskas
T W
TW
Negishi
M
M
Nebert
D W
DW
Ding
X
X
D000082:Acetaminophen / Q000378:metabolism*; D018712:Analgesics, Non-Narcotic / Q000378:metabolism*; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D065637:Cytochrome P-450 CYP2A6; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D000072467:Cytochrome P450 Family 2; D005260:Female; D051379:Mice; D008861:Microsomes / Q000201:enzymology* / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D009570:Nitriles / Q000378:metabolism*; D009831:Olfactory Mucosa / Q000201:enzymology* / Q000378:metabolism; D011817:Rabbits; D013250:Steroid Hydroxylases / Q000378:metabolism*
D018712:Analgesics, Non-Narcotic; D009570:Nitriles; D000082:Acetaminophen; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; C585520:Cyp2a5 protein, mouse; D065637:Cytochrome P-450 CYP2A6; D000072467:Cytochrome P450 Family 2; C105920:cytochrome P-450 CYP2G1; C055869:dichlobanil