Abstract
4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent tobacco-specific nitrosamine in animals and has been suggested to play a role in human tobacco-related cancers. Our previous study demonstrated that cytochrome P450 (P450) 1A2 catalyzes the formation of 4-hydroxy-1-(3-pyridyl)-1-butanone (keto alcohol) (an alpha-hydroxylation product) from NNK in human liver microsomes. Phenethyl isothiocyanate (PEITC) inhibits NNK tumorigenesis by blocking the activation of NNK. The purpose of the present study was to elucidate the mechanism of inhibition of P450 1A2-catalyzed NNK activation by PEITC. Human P450 1A2 was expressed in Escherichia coli and purified to homogeneity. In a reconstituted system, P450 1A2 catalyzed the formation of keto alcohol and 4-oxo-1-(3-pyridyl)-1-butanone (keto aldehyde) from NNK, with the keto alcohol being the major metabolite. The apparent Km and Vmax values for keto alcohol formation was 380 microM and 1.7 nmol/min/nmol P450, respectively. For the tobacco-specific nitrosamine N-nitrosonornicotine (NNN), P450 1A2 catalyzed the formation of the derived 4-hydroxy-4-(3-pyridyl)butyric acid (hydroxy acid),4-oxo-4-(3-pyridyl)butyric acid (keto acid) and keto alcohol. In comparison to NNK, NNN had a lower rate of oxidation with P450 1A2. PEITC decreased the formation of the NNK-derived keto alcohol in a concentration-dependent manner, with an IC50 value of 0.14 microM. PEITC was a competitive inhibitor of P450 1A2, exhibiting a Ki value of 0.18 microM. Preincubation of PEITC with NADPH in the reconstituted system resulted in a further decrease (25%) in the catalytic activity of P450 1A2, suggesting that there is a slow metabolism-dependent inhibition of P450 1A2 by PEITC. The formation of keto aldehyde and keto alcohol was inhibited by PEITC in human liver microsomes with IC50 values of 9.5 and 4.6 microM respectively. Methoxyresorufin O-dealkylase activity, a marker for P450 1A2, was decreased by PEITC in a concentration-dependent manner, with an IC50 of 0.34 microM. The results suggest that PEITC itself is a potent inhibitor of P450 1A2 and that a metabolite(s) of PEITC can also inhibit P450 1A2. We conclude that PEITC may be an effective inhibitor of the carcinogenicity or toxicity of chemicals that are activated by P450 1A2.
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