Oxidation of tolualdehydes to toluic acids catalyzed by cytochrome P450-dependent aldehyde oxygenase in the mouse liver.
Mouse hepatic microsomal enzymes catalyzed the oxidation of o-, m-, and p-tolualdehydes, intermediate metabolites of xylene, to the corresponding toluic acids. Cofactor requirement for the catalytic activity indicates that the microsomes contain NAD- and NADPH-dependent enzymes for this reaction. GC/MS analyses of the carboxylic acids formed by incubation under oxygen-18 gas indicate that the mechanism for this oxidation is an oxygenation and a dehydrogenation for the NADPH- and NAD-dependent reaction. Vmax/Km (nmol/min/mg protein) ratios indicate that the NADPH-dependent activity is more pronounced than the NAD-dependent activity. These results suggest that the NADPH-dependent reaction is mainly responsible for the microsomal oxidation of tolualdehydes. The NADPH-dependent activity was significantly inhibited by SKF 525-A, disulfiram and menadione, inhibitors of cytochrome P450 (P450), suggesting the involvement of P450 in the reaction. In a reconstituted system, P450 MUT-2 (CYP2C29) purified from mouse hepatic microsomes catalyzed the oxidation of o-, m-, and p-tolualdehydes to the carboxylic acids, and the specific activities (nmol/min/nmol P450) were 1.44, 2.81, and 2.32, respectively. Rabbit antibody raised against P450 MUT-2 significantly inhibited the NADPH-dependent oxidation of tolualdehydes to toluic acids by 88% (o-), 63% (m-), and 62% (p-) using mouse hepatic microsomes. The present study demonstrated that a mouse hepatic microsomal aldehyde oxygenase, P450 MUT-2, catalyzed the most of oxidative activity of tolualdehydes to toluic acids in the microsomes.
Watanabe
K
K
Department of Hygienic Chemistry, Faculty of Pharmaceutical Sciences, Hokuriku University, Kanazawa, Japan.
Matsunaga
T
T
Yamamoto
I
I
Yashimura
H
H
D000445:Aldehyde Oxidoreductases / Q000378:metabolism*; D000818:Animals; D001547:Benzaldehydes / Q000378:metabolism*; D001565:Benzoates / Q000378:metabolism*; D002384:Catalysis; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D013058:Mass Spectrometry; D051379:Mice; D008815:Mice, Inbred Strains; D008862:Microsomes, Liver / Q000201:enzymology* / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction
D001547:Benzaldehydes; D001565:Benzoates; C020627:4-methylbenzaldehyde; C009503:3-toluic acid; D009249:NADP; D003577:Cytochrome P-450 Enzyme System; C009502:2-toluic acid; C009504:4-toluic acid; D000445:Aldehyde Oxidoreductases; C093383:3-methylbenzaldehyde; C093384:2-methylbenzaldehyde