Protein Targets of Thioacetamide Metabolites in Rat Hepatocytes

Abstract

Thioacetamide (TA) has long been known as a hepatotoxicant whose bioactivation requires S-oxidation to thioacetamide S-oxide (TASO) and then to the very reactive S,S-dioxide (TASO2). The latter can tautomerize to form acylating species capable of covalently modifying cellular nucleophiles including phosphatidylethanolamine (PE) lipids and protein lysine side chains. Isolated hepatocytes efficiently oxidize TA to TASO but experience little covalent binding or cytotoxicity because TA is a very potent inhibitor of the oxidation of TASO to TASO2. On the other hand hepatocytes treated with TASO show extensive covalent binding to both lipids and proteins accompanied by extensive cytotoxicity. In this work, we treated rat hepatocytes with [14C]-TASO and submitted the mitochondrial, microsomal and cytosolic fractions to 2DGE which revealed a total of 321 radioactive protein spots. To facilitate the identification of target proteins and adducted peptides we also treated cells with a mixture of TASO/[13C2D3]-TASO. Using a combination of 1DGE- and 2DGE-based proteomic approaches, we identified 187 modified peptides (174 acetylated, 50 acetimidoylated and 37 in both forms) from a total of 88 non-redundant target proteins. Among the latter, 57 are also known targets of at least one other hepatotoxin. The formation of both amide- and amidine-type adducts to protein lysine side chains is in contrast to the exclusive formation of amidine-type adducts with PE phospholipids. Thiobenzamide (TB) undergoes the same two-step oxidative bioactivation as TA, and it also gives rise to both amide and amidine adducts on protein lysine side chains but only amidine adducts to PE lipids. Despite their similarity in functional group chemical reactivity, only 38 of 62 known TB target proteins are found among the 88 known targets of TASO. The potential roles of protein modification by TASO in triggering cytotoxicity are discussed in terms of enzyme inhibition, protein folding and chaperone function, and the emerging role of protein acetylation in intracellular signaling and the regulation of biochemical pathways.

Action of Melatonin on Severe Acute Liver Failure in Rats

“…This experimental model mimics the physiopathogenic and pathophysiological aspects of the disease in humans and helps in the understanding of its progress and the possibility of therapeutic intervention [16,[27][28][29].…”

Potential Ameliorative Effects of Alpha Lipoic Acid and Silymarin on Thioacetamide-Induced Hepatic Damage in Rats

“…Thioacetamide (TAA) is a well-recognized hepatocarcinogen that induces oxidative damage in hepatocytes through generation of reactive oxygen species (ROS), and subsequent liver cirrhosis and liver cell tumors in rodents (Uskoković-Marković et al, 2007). Thus, it is widely used to induce acute toxic hepatic damage through oxidative stress, membrane damage and accumulation of lipid droplets in the hepatocyte cytoplasm (Koen et al, 2013). Alpha lipoic acid (ALA), also named as thiotic acid, is a natural cofactor that is synthesized inside the plant and animal mitochondria (Thaakur and Himabindhu, 2009).…”

Oxidative stress caused by a SOD1 deficiency ameliorates thioacetamide-triggered cell death via CYP2E1 inhibition but stimulates liver steatosis

“…In addition to the direct damage caused by these cytotoxic products, reactive oxygen species (ROS) that are produced by CYP enzymes also have harmful influences on the liver (Yasui et al 2005). Target molecules of the oxygenized metabolites and ROS have been extensively investigated, in attempt to rationalize the molecular mechanisms responsible for the hepatocyte death caused by the TAA treatment (Low et al 2004;Hajovsky et al 2012;Sarma et al 2012;Koen et al 2013). Analyses of the TAA-treated cells indicate that thioacetamide S-oxide (TAA-SO) and thioacetamide S,Sdioxide (TAA-SO 2 ) are produced as reactive metabolites ) and that phosphatidylethanolamine and some proteins act as target molecules, which mainly accounts for the cytotoxicity of TAA (Sarma et al 2012;Koen et al 2013).…”