title: Limitations of hepatocytes and liver homogenates in modelling in vivo formation of acyl glucuronide-derived drug-protein adducts.
abstract: The covalent binding of drugs or their metabolites to proteins is of increasing interest in the investigation of the toxicity of these compounds. Recent attention on biological consequences of protein adduct formation with carboxylate drugs, derived via their reactive acyl glucuronide metabolites, has focussed on liver tissue. Although the intact animal represents undisturbed hepatic physiology, other hepatic models can offer advantages, e.g., multiple experiments from a single liver. In this study we set out to compare the patterns of covalent binding of zomepirac (ZP) to proteins in the livers of intact rats, isolated rat hepatocytes (in culture or suspension), and in rat liver homogenates. Rats were dosed i.v. with 25 mg ZP/kg, and their livers were removed 3 h later. Isolated hepatocytes or liver homogenates were exposed to ZP at 100 microg/mL for 3 h at 37 degrees C. Liver homogenates were exposed to ZP and also zomepirac acyl glucuronide (ZAG) at 100 microg ZP equivalents/mL for 3 h at 37 degrees C. Covalent binding of ZP species was examined by SDS-PAGE and Western blotting with a polyclonal ZP antiserum. In livers from dosed animals, the strongest staining appeared at about 110120, 140, and 200 kDa. Few similarities existed with the results from isolated hepatocytes and, not surprisingly, liver homogenates. Only the 200-kDa band was common to all treatments. Many proteins seemed to be modified, at least to some extent. The differences in major bands are most likely caused by the loss of liver and hepatocyte architecture. The variability across different model systems in respect to covalent binding to hepatic proteins emphasizes the need for care in interpretation of results.
authors.affiliation: Department of Medicine, Royal Brisbane Hospital, University of Queensland, Australia.
authors.forename: M J
authors.initials: MJ
authors.lastname: Bailey
authors.forename: R G
authors.initials: RG
authors.lastname: Dickinson
mesh_terms: D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000378:metabolism* / Q000494:pharmacology; D001667:Binding, Competitive; D015153:Blotting, Western; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D005965:Glucuronates / Q000378:metabolism*; D006031:Glycosylation; D008099:Liver / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D011485:Protein Binding; D051381:Rats; D017208:Rats, Wistar; D014046:Tolmetin / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology
chemical_list: D000894:Anti-Inflammatory Agents, Non-Steroidal; D005965:Glucuronates; C027368:zomepirac glucuronide; C020549:zomepirac; D014046:Tolmetin