title: Two-dimensional electrophoretic analysis of compartment-specific hepatic protein charge modification induced by thioacetamide exposure in rats.
abstract: Thioacetamide (TA) is a well-known hepatotoxicant. It has been reported that an obligate intermediate of TA binds to proteins with the formation of acetylimidolysine derivatives that are responsible for TA-induced hepatotoxic effects. TA has also been reported to cause chemically induced cell death via both apoptosis and necrosis. The objective of this study was 2-fold: first, to investigate the effect of TA exposure on protein charge modifications in the rat liver and second, to study the role of these molecular correlates in the regulation of cell death. Male Sprague-Dawley rats (200-225 g, 7-8 weeks old) were divided into four major groups and treated intraperitoneally with a 12-fold dose range of TA (50, 150, 300, and 600 mg TA/kg) dissolved in water. Using whole liver extracts, alterations in the hepatic protein pattern following treatment with the 12-fold dose range of TA were studied using high-resolution, two-dimensional polyacrylamide gel electrophoresis and computerized image analysis. The results indicate that charge modification was clearly evident as early as 2 hr with the lowest dose of 50 mg TA/kg. At this dose and time endoplasmic reticulum proteins, calreticulin, grp78, and ER6O exhibited acidic charge variants. The effect of TA became more prominent with dose and time. Generally the elevation of charge modification indices (CMI) by TA appeared to reach a peak between 4 and 6 hr and then while CMI either leveled off or declined in the lower two doses of 50 and 150 mg TA/kg, it continued to remain elevated with the higher doses of 300 and 600 mg TA/kg. This dichotomy in the elevation of CMI is in close correspondence to the pattern of cell death observed with a similar dose range of TA, where lower doses (50 and 150 mg TA/kg) predominantly cause cell death via apoptosis while higher doses cause cell death via necrosis. Delayed charge modification was observed with the cytosolic hsc70s with the 300 and 600 mg TA/kg treatments, indicating that the reactive metabolite(s) slowly leak out into the cytosol from the endoplasmic reticulum. There were no alterations in the mitochondrial proteins hsp60 and grp75, suggesting that TA has no effect on the mitochondrion, its effects primarily being confined to the endoplasmic reticulum. The concept of looking at these proteins as biomarkers of tissue injury has validity. These changes may be indicators of bioactivation and adduct formation and also may be signaling events in the regulation of the mode of cell death.
authors.affiliation: Molecular Anatomy Laboratory, Department of Biology, Indiana University, Columbus 47203-1769, USA.
authors.forename: F A
authors.initials: FA
authors.lastname: Witzmann
authors.forename: C D
authors.initials: CD
authors.lastname: Fultz
authors.forename: R S
authors.initials: RS
authors.lastname: Mangipudy
authors.forename: H M
authors.initials: HM
authors.lastname: Mehendale
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002273:Carcinogens / Q000633:toxicity*; D002455:Cell Division / Q000187:drug effects; D056486:Chemical and Drug Induced Liver Injury / Q000378:metabolism / Q000473:pathology; D015180:Electrophoresis, Gel, Two-Dimensional; D006360:Heat-Shock Proteins / Q000737:chemistry* / Q000187:drug effects*; D007091:Image Processing, Computer-Assisted; D008099:Liver / Q000737:chemistry* / Q000187:drug effects* / Q000473:pathology; D008297:Male; D018832:Molecular Chaperones / Q000737:chemistry; D011522:Protons; D051381:Rats; D017207:Rats, Sprague-Dawley; D013853:Thioacetamide / Q000633:toxicity*
chemical_list: D002273:Carcinogens; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D011522:Protons; D013853:Thioacetamide