2-DIMENSIONAL ELECTROPHORETIC ANALYSIS OF COMPARTMENT-SPECIFIC HEPATIC PROTEIN CHARGE MODIFICATION INDUCED BY THIOACETAMIDE EXPOSURE IN RATS

Thioacetamide (TA) is a well-known hepatotoxicant. It has been reporte d that an obligate intermediate of TA binds to proteins with the forma tion of acetylimidolysine derivatives that are responsible for TA-indu ced 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 deat h. Male Sprague-Dawley rats (200-225 g, 7-8 weeks old) were divided in to 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. Usin g whole liver extracts, alterations in the hepatic protein pattern fol lowing treatment with the 12-fold dose range of TA were studied using high-resolution, two-dimensional polyacrylamide gel electrophoresis an d computerized image analysis. The results indicate that charge modifi cation 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, calre ticulin, grp78, and ER60 exhibited acidic charge variants. The effect of TA became more prominent with dose and time. Generally the elevatio n 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 i n the lower two doses of 50 and 150 mg TA/kg, it continued to remain e levated 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 c ell. death observed with a similar dose range of TA, where lower doses (50 and 150 mg TA/kg) predominantly cause cell death via apoptosis wh ile higher doses cause cell death via necrosis. Delayed charge modific ation was observed with the cytosolic hsc70s with the 300 and 600 mg T A/kg treatments, indicating that the reactive metabolite(s) slowly lea k out into the cytosol from the endoplasmic reticulum. There were no a lterations in the mitochondrial proteins hsp60 and grp75, suggesting t hat TA has no effect on the mitochondrion, its effects primarily being confined to the endoplasmic reticulum. The concept of looking at thes e proteins as biomarkers of tissue injury has validity. These changes may be indicators of bioactivation and adduct formation and also may b e signaling events in the regulation of the mode of cell death. (C) 19 96 Society of Toxicology.