The main goals of this research project are to identify human liver cytochromes P-450 involved in the activation of acetaminophen to a reactive intermediate. Studies in this proposal will focus on modulators of cytochrome P-450 catalyzed acetaminophen activation. We will examine the mechanisms involved in ethanol induction, 7,8 benzoflavone (ANF) stimulation and cimetidine inhibition. Ethanol potentiation of acetaminophen hepatotoxicity has been documented in both human and animal studies, suggesting that modulation by induction of the ethanol enzyme is the mechanism by which potentiation of toxicity occurs. In addition, modulators may be used as a means of identifying isozymes of cytochrome P-450. The use of ANF as a stimulator of acetaminophen activation has allowed us to identify the presence of at least two cytochromes P-450 involved in this reaction in human liver. In addition, we propose to purify the human liver cytochromes and prepare monoclonal antibodies which may be employed in defining which population of individuals may be more susceptible to hepatotoxicity from large doses of acetaminophen. The high degree of specificity exhibited by these antibodies will allow them to be used as specific inhibitors in the acetaminophen assay to verify the role of the cytochromes in the metabolism of acetaminophen. Using needle biopsy samples from individuals exposed to various quantities of ethanol for different lengths of time and subjecting the samples to immunoblots developed with the antibody to the human ethanol P-450, should provide information about induction and de-induction of the enzyme. Finally, we will examine whether human kidney microsomes contribute to acetaminophen activation and identify, by use of the monoclonal antibodies prepared to the liver cytochromes, the cytochromes P-450 which may be involved. Thus, these studies provide a two-fold purpose. First, in identifying human liver cytochromes P-450 that may produce activation of acetaminophen and mechanisms of modulating their metabolism to prevent hepatotoxicity and second, to determine the quantity of ethanol and duration of exposure required for induction of human liver ethanol P-450.

Agency
National Institute of Health (NIH)
Institute
National Institute on Alcohol Abuse and Alcoholism (NIAAA)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
5R29AA008139-04
Application #
3452824
Study Section
Biochemistry, Physiology and Medicine Subcommittee (ALCB)
Project Start
1988-09-01
Project End
1993-08-31
Budget Start
1991-09-01
Budget End
1992-08-31
Support Year
4
Fiscal Year
1991
Total Cost
Indirect Cost
Name
University of New Mexico
Department
Type
Schools of Pharmacy
DUNS #
829868723
City
Albuquerque
State
NM
Country
United States
Zip Code
87131
Goldstein, J A; Faletto, M B; Romkes-Sparks, M et al. (1994) Evidence that CYP2C19 is the major (S)-mephenytoin 4'-hydroxylase in humans. Biochemistry 33:1743-52
Raucy, J L; Carpenter, S J (1993) The expression of xenobiotic-metabolizing cytochromes P450 in fetal tissues. J Pharmacol Toxicol Methods 29:121-8
Raucy, J L; Kraner, J C; Lasker, J M (1993) Bioactivation of halogenated hydrocarbons by cytochrome P4502E1. Crit Rev Toxicol 23:1-20
Kraner, J C; Lasker, J M; Corcoran, G B et al. (1993) Induction of P4502E1 by acetone in isolated rabbit hepatocytes. Role of increased protein and mRNA synthesis. Biochem Pharmacol 45:1483-92
Bornheim, L M; Lasker, J M; Raucy, J L (1992) Human hepatic microsomal metabolism of delta 1-tetrahydrocannabinol. Drug Metab Dispos 20:241-6
Raucy, J L; Lasker, J M; Kraner, J C et al. (1991) Induction of cytochrome P450IIE1 in the obese overfed rat. Mol Pharmacol 39:275-80
Raucy, J L; Lasker, J M (1991) Isolation of P450 enzymes from human liver. Methods Enzymol 206:577-87
Ray, S D; Sorge, C L; Tavacoli, A et al. (1991) Extensive alteration of genomic DNA and rise in nuclear Ca2+ in vivo early after hepatotoxic acetaminophen overdose in mice. Adv Exp Med Biol 283:699-705
Wrighton, S A; Brian, W R; Sari, M A et al. (1990) Studies on the expression and metabolic capabilities of human liver cytochrome P450IIIA5 (HLp3). Mol Pharmacol 38:207-13
Ray, S D; Sorge, C L; Raucy, J L et al. (1990) Early loss of large genomic DNA in vivo with accumulation of Ca2+ in the nucleus during acetaminophen-induced liver injury. Toxicol Appl Pharmacol 106:346-51

Showing the most recent 10 out of 11 publications