Two-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 beenreported that an obligate intermediate of TA binds to proteinswith the formation of acetylimidolysine derivatives that areresponsible for TA-induced hepatotoxic effects. TA has alsobeen reported to cause chemically induced cell death via bothapoptosis and necrosis. The objective of this study was 2-fold:first, to investigate the effect of TA exposure on protein chargemodifications in the rat liver and second, to study the roleof 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 intraperitoneallywith a 12-fold dose range of TA (50, 150, 300, and 600 mg TA/kg)dissolved in water. Using whole liver extracts, alterationsin the hepatic protein pattern following treatment with the12-fold dose range of TA were studied using high-resolution,two-dimensional polyacrylamide gel electrophoresis and computerizedimage analysis. The results indicate that charge modificationwas clearly evident as early as 2 hr with the lowest dose of50 mg TA/kg. At this dose and time endoplasmic reticulum proteins,calreticulin, grp78, and ER60 exhibited acidic charge variants.The effect of TA became more prominent with dose and time. Generallythe elevation of charge modification indices (CMI) by TA appearedto reach a peak between 4 and 6 hr and then while CMI eitherleveled off or declined in the lower two doses of 50 and 150mg TA/kg, it continued to remain elevated with the higher dosesof 300 and 600 mg TA/kg. This dichotomy in the elevation ofCMI is in close correspondence to the pattern of cell deathobserved with a similar dose range of TA, where lower doses(50 and 150 mg TA/kg) predominantly cause cell death via apoptosiswhile higher doses cause cell death via necrosis. Delayed chargemodification was observed with the cytosolic hsc70s with the300 and 600 mg TA/kg treatments, indicating that the reactivemetabolite(s) slowly leak out into the cytosol from the endoplasmicreticulum. There were no alterations in the mitochondrial proteinshsp60 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 tissueinjury has validity. These changes may be indicators of bioactivationand adduct formation and also may be signaling events in theregulation of the mode of cell death.