The objective of this project is to obtain information on the identities of reactive hepatotoxic metabolites of certain therapeutic agents and to investigate their interaction with proteins. The drugs to be studied include acetaminophen, isoniazid, iproniazid and valproic acid. The experimental approach to be adopted in this study is novel in that it takes advantage of the irreversible binding of reactive drug metabolites to protein in order to """"""""trap"""""""" these short-lived toxic species. Both direct and indirect methods will be used to characterize the structures of the resulting covalent adducts which, in turn, will reveal the identities of the reactive intermediates themselves. Modern chromatographic and mass spectrometric techniques will play a central role in the proposed studies and the development of improved analytical methodology for the isolation and identification of drug-protein and drug-amino acid conjugates will be a key feature of this investigation. Such methods are likely to find widespread use in the field of biochemical toxicology and related disciplines.
The specific aims of this project are: (1) to identify the covalent adducts formed in vitro between proteins and both hepatotoxic and non-hepatotoxic agents, (2) to compare the types of covalent adduct formed in vivo with those produced in vitro, (3) to study the different types of interaction which take place between proteins and both toxic and non-toxic metabolites, (4) to investigate species differences in the protective effect of ascorbate on acetaminophen-induced liver injury, (5) to identify hepatotoxic metabolites of valproic acid, and (6) to develop versatile and sensitive analytical methodology for the identification of trace amounts of drug-protein adducts in liver tissue and in hemoglobin. Information of the type sought in this project is necessary for the construction of a sound experimental basis from which to develop rational therapeutic and prophylactic treatments of drug-induced hepatitis and, ultimately, from which to predict the potential toxicity of new therapeutic agents.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK030699-05
Application #
3229600
Study Section
Pharmacology A Study Section (PHRA)
Project Start
1981-08-01
Project End
1989-05-31
Budget Start
1987-06-01
Budget End
1988-05-31
Support Year
5
Fiscal Year
1987
Total Cost
Indirect Cost
Name
University of Washington
Department
Type
Schools of Pharmacy
DUNS #
135646524
City
Seattle
State
WA
Country
United States
Zip Code
98195
Baillie, T A (1992) Metabolism of valproate to hepatotoxic intermediates. Pharm Weekbl Sci 14:122-5
Pearson, P G; Slatter, J G; Rashed, M S et al. (1991) Carbamoylation of peptides and proteins in vitro by S-(N-methylcarbamoyl)glutathione and S-(N-methylcarbamoyl)cysteine, two electrophilic S-linked conjugates of methyl isocyanate. Chem Res Toxicol 4:436-44
Slatter, J G; Rashed, M S; Pearson, P G et al. (1991) Biotransformation of methyl isocyanate in the rat. Evidence for glutathione conjugation as a major pathway of metabolism and implications for isocyanate-mediated toxicities. Chem Res Toxicol 4:157-61
Hoffmann, K J; Axworthy, D B; Baillie, T A (1990) Mechanistic studies on the metabolic activation of acetaminophen in vivo. Chem Res Toxicol 3:204-11
Han, D H; Pearson, P G; Baillie, T A et al. (1990) Chemical synthesis and cytotoxic properties of N-alkylcarbamic acid thioesters, metabolites of hepatotoxic formamides. Chem Res Toxicol 3:118-24
Pearson, P G; Slatter, J G; Rashed, M S et al. (1990) S-(N-methylcarbamoyl)glutathione: a reactive S-linked metabolite of methyl isocyanate. Biochem Biophys Res Commun 166:245-50
Decker, C J; Rashed, M S; Baillie, T A et al. (1989) Oxidative metabolism of spironolactone: evidence for the involvement of electrophilic thiosteroid species in drug-mediated destruction of rat hepatic cytochrome P450. Biochemistry 28:5128-36
Rashed, M S; Pearson, P G; Han, D H et al. (1989) Application of liquid chromatography/thermospray mass spectrometry to studies on the formation of glutathione and cysteine conjugates from monomethylcarbamate metabolites of bambuterol. Rapid Commun Mass Spectrom 3:360-3
Porubek, D J; Grillo, M P; Baillie, T A (1989) The covalent binding to protein of valproic acid and its hepatotoxic metabolite, 2-n-propyl-4-pentenoic acid, in rats and in isolated rat hepatocytes. Drug Metab Dispos 17:123-30
Tirmenstein, M A; Nelson, S D (1989) Subcellular binding and effects on calcium homeostasis produced by acetaminophen and a nonhepatotoxic regioisomer, 3'-hydroxyacetanilide, in mouse liver. J Biol Chem 264:9814-9

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