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We sought to establish whether heme-thiolate monooxygenases oxidize, alpha,beta-unsaturated aldehydes generated during lipid peroxidation. Several recombinant P450s co-expressed with NADPH:P450 oxidoreductase were surveyed for aldehyde oxidation activity with anthracene-9-carboxaldehyde and 4-hydroxy-trans-2-nonenal (HNE). Murine P4502c29, human P4503A4, human P4502B6, and rabbit P4502B4 were good catalysts of aldehyde oxidation to carboxylic acids. Other P450s (e.g., P4501A2, 2E1, and 2J2) did not oxidize these aldehydes. P4502c29 and P4503A4 displayed K(m)/S(0.5) values of approx. 1-20microM. The product measured by HPLC that co-migrates with authentic 4-hydroxynonenoic acid (HNA) had a mass spectrum identical to the standard. Using P4502c29, HNE was a mixed-competitive inhibitor of anthracene-9-carboxaldehyde oxidation, suggesting that both aldehydes are substrates for P4502c29. Specific inhibitors of aldehyde dehydrogenases and P450 were used to assess their role in the metabolism of HNE in primary rat hepatocytes. Inhibitors of aldehyde dehydrogenase (cyanamide) inhibited HNA formation by 60% and together cyanamide and miconazole (P450) caused over 85% inhibition of HNA formation. P450s are significant participants in metabolism of endogenous and exogenous unsaturated aldehydes in primary rat hepatocytes.