Oxidation of pyrazole to 4-hydroxypyrazole by intact rat hepatocytes

4-Hydroxypyrazole has been identified as a major metabolite found in the urine of rats and mice after in vivo administration of pyrazole, a potent inhibitor of alcohol dehydrogenase and of ethanol metabolism. The locus and the enzyme systems responsible for the oxidation of pyrazole have not been identified. In the current report, isolated hepatocytes from fed rats were shown to oxidize pyrazole to 4-hydroxypyrazole. An HPLC procedure employing UV and electrochemical detection was utilized to separate and quantify the 4-hydroxypyrazole. The apparent Km for pyrazole by intact hepatocytes was about 2 mM, whereas the apparent Vmax was about 0.06 nmol 4-hydroxypyrazole per min per mg liver cell protein. The production of 4-hydroxypyrazole was inhibited by carbon monoxide and metyrapone, as well as by competitive drug substrates such as aniline or aminopyrine. These results implicate a role for cytochrome P-450 in the oxidation of pyrazole by the hepatocytes. Ethanol was an effective inhibitor of pyrazole oxidation. Hepatocytes were also isolated from rats treated with acetone and 4-methylpyrazole, to attempt to evaluate whether pyrazole oxidation is induced. The rate of 4-hydroxypyrazole production by hepatocytes after acetone and 4-methylpyrazole treatment was actually lower than that of controls. Kinetic assays suggested the presence of an endogenous inhibitor (perhaps the inducer itself) in the induced hepatocytes. In contrast, hepatocytes isolated from rats fasted for 48 hr showed a 2-fold increase in the oxidation of pyrazole to 4-hydroxypyrazole. The Km for pyrazole was the same in hepatocytes from fasted and fed rats, whereas Vmax was increased after fasting. The locus and enzyme system responsible for the oxidation of pyrazole to 4-hydroxypyrazole, and the site of sensitivity to ethanol, appears to be the cytochrome P-450 system of the hepatocyte.