Pyrazole and 4-methylprazole (4-MP) are potent inhibitors of alcohol dehydrogenase and block the metabolic actions of ethanol. These compounds have been used in the treatment of methanol and ethylene glycol poisoning and to ameliorate the disulfiram-ethanol reaction and acetaldehyde toxicity in """"""""flushers"""""""". Biochemical and pharmacological properties of pyrazole and 4-MP and enzymatic loci for their metabolism, have not been investigated in detail. Pyrazole and 4-MP were found to induce the same isozyme of cytochrome P-450 as does ethanol, P-450 IIE.1. The pyrazole- induced P-450 has been purified, characterized, and a polyclonal antibody produced Pyrazole and 4-MP produce Type II binding spectra with microsomes and cytochrome P-450, and inhibit the oxidation of drugs, including ethanol, by microsome. Pyrazole was shown to be a good substrate for P-450 IIE.1 and was oxidized to 4-hydroxy-pyrazole. Experiments are planned to continue to characterize the interactions of pyrazole and 4-MP with the microsomal mixed-function oxidase system. We have developed a new method to detect 4-hydroxymethylpyrazole. The ability of microsomes, P-450, hepatocytes and oxygen radicals to oxidize 4-MP to this metabolite, and the effect of inducers of P-450 IIE.1 will be evaluated. Studies to understand at the molecular level mechanisms responsible for the induction of P-450 IIE.1 by pyrazole, 4-MP and ethanol will involve effect of inhibitors of transcription and translation, detailed time and dose response curves comparing changes in P-450 IIE.1 mRNA levels and protein levels and measures of the turnover of P-450 IIE.1 in the absence and presence of the inducer. The acinar localization and induction of P-450 IIE.1. and activation of hepatotoxins in periportal and perivenous hepatocytes will be determined. The ability of glycerol (a possible physiological relevant inducer) to induce P-450 IIE.1 and to interact with and be metabolized by microsomes and P-450 will be investigated. Additional studies will employ intact hepatocytes and human livers, characterize metabolic actions of the metabolites 4- hydroxypyrazole and 4-hydroxymethylpyrazole, and extend the substrate preference of P-450 IIE.1 to toxicologically important agents such as nitriles, e.g., cyanamide and glycols, e.g., anti-freeze. It is hoped that these studies will provide important information and continue to characterize the metabolic, biochemical and pharmacological properties of these valuable compounds.

National Institute of Health (NIH)
National Institute on Alcohol Abuse and Alcoholism (NIAAA)
Method to Extend Research in Time (MERIT) Award (R37)
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Mount Sinai School of Medicine
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Wu, Defeng; Cederbaum, Arthur (2008) Cytochrome P4502E1 sensitizes to tumor necrosis factor alpha-induced liver injury through activation of mitogen-activated protein kinases in mice. Hepatology 47:1005-17
Lu, Yongke; Gong, Pengfei; Cederbaum, Arthur I (2008) Pyrazole induced oxidative liver injury independent of CYP2E1/2A5 induction due to Nrf2 deficiency. Toxicology 252:9-16
Lu, Yongke; Zhuge, Jian; Wang, Xiaodong et al. (2008) Cytochrome P450 2E1 contributes to ethanol-induced fatty liver in mice. Hepatology 47:1483-94
Caro, Andres A; Cederbaum, Arthur I (2007) Role of intracellular calcium and phospholipase A2 in arachidonic acid-induced toxicity in liver cells overexpressing CYP2E1. Arch Biochem Biophys 457:252-63
Kessova, Irina G; Cederbaum, Arthur I (2007) Mitochondrial alterations in livers of Sod1-/- mice fed alcohol. Free Radic Biol Med 42:1470-80
Dey, Aparajita; Cederbaum, Arthur I (2007) Induction of cytochrome P450 2E1 [corrected] promotes liver injury in ob/ob mice. Hepatology 45:1355-65
Zhuge, Jian; Cederbaum, Arthur I (2006) Increased toxicity by transforming growth factor-beta 1 in liver cells overexpressing CYP2E1. Free Radic Biol Med 41:1100-12
Dey, Aparajita; Kessova, Irina G; Cederbaum, Arthur I (2006) Decreased protein and mRNA expression of ER stress proteins GRP78 and GRP94 in HepG2 cells over-expressing CYP2E1. Arch Biochem Biophys 447:155-66
Wu, Defeng; Cederbaum, Arthur (2006) Nitric oxide donors prevent while the nitric oxide synthase inhibitor L-NAME increases arachidonic acid plus CYP2E1-dependent toxicity. Toxicol Appl Pharmacol 216:282-92
Wu, Defeng; Cederbaum, Arthur I (2006) Opposite action of S-adenosyl methionine and its metabolites on CYP2E1-mediated toxicity in pyrazole-induced rat hepatocytes and HepG2 E47 cells. Am J Physiol Gastrointest Liver Physiol 290:G674-84

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