Covalent Modification of Lipids and Proteins in Rat Hepatocytes, and In Vitro, by Thioacetamide Metabolites

This document is the Accepted Manuscript version of a Published Work that appeared in final form in Chemical Research in Toxicology, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see http://pubs.acs.org/doi/abs/10.1021/tx3001658Thioacetamide (TA) is a well-known hepatotoxin in rats. Acute doses cause centrilobular necrosis and hyperbilirubinemia while chronic administration leads to biliary hyperplasia and cholangiocarcinoma. Its acute toxicity requires its oxidation to a stable S-oxide (TASO) that is oxidized further to a highly reactive S,S-dioxide (TASO2). To explore possible parallels between the metabolism, covalent binding and toxicity of TA and thiobenzamide (TB) we exposed freshly isolated rat hepatocytes to [14C]-TASO or [13C2D3]-TASO. TLC analysis of the cellular lipids showed a single major spot of radioactivity that mass spectral analysis showed to consist of N-acetimidoyl PE lipids having the same side chain composition as the PE fraction from untreated cells; no carbons or hydrogens from TASO were incorporated into the fatty acyl chains. Many cellular proteins contained N-acetyl- or N-acetimidoyl lysine residues in a 3:1 ratio (details to be reported separately). We also oxidized TASO with hydrogen peroxide in the presence of dipalmitoyl phosphatidylenthanolamine (DPPE) or lysozyme. Lysozyme was covalently modified at five of its six lysine side chains; only acetamide-type adducts were formed. DPPE in liposomes also gave only amide-type adducts, even when the reaction was carried out in tetrahydrofuran with only 10% water added. The exclusive formation of N-acetimidoyl PE in hepatocytes means that the concentration or activity of water must be extremely low in the region where TASO2 is formed, whereas at least some of the TASO2 can hydrolyze to acetylsulfinic acid before it reacts with cellular proteins. The requirement for two sequential oxidations to produce a reactive metabolite is unusual, but it is even more unusual that a reactive metabolite would react with water to form a new compound that retains a high degree of chemical reactivity toward biological nucleophiles. The possible contribution of lipid modification to the hepatotoxicity of TA/TASO remains to be determined

Metabolism and Toxicity of Thioacetamide and Thioacetamide SOxide in Rat Hepatocytes

“This document is the Accepted Manuscript version of a Published Work that appeared in final form in Chemical Research in Toxicology, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see http://pubs.acs.org/doi/abs/10.1021/tx3002719The hepatotoxicity of thioacetamide (TA) has been known since 1948. In rats, single doses cause centrilobular necrosis accompanied by increases in plasma transaminases and bilirubin. To elicit these effects TA requires oxidative bioactivation leading first to its S-oxide (TASO) and then to its chemically reactive S,S-dioxide (TASO2) which ultimately modifies amine-lipids and proteins. To generate a suite of liver proteins adducted by TA metabolites for proteomic analysis, and to reduce the need for both animals and labeled compounds, we treated isolated hepatocytes directly with TA. Surprisingly, TA was not toxic at concentrations up to 50 mM for 40 hr. On the other hand, TASO was highly toxic to isolated hepatocytes as indicated by LDH release, cellular morphology and vital staining with Hoechst 33342/propidium iodide. TASO toxicity was partially blocked by the CYP2E1 inhibitors diallyl sulfide and 4-methylpyrazole, and was strongly inhibited by TA. Significantly, we found that hepatocytes produce TA from TASO relatively efficiently by back-reduction. The covalent binding of [14C]-TASO is inhibited by unlabeled TA which acts as a “cold-trap” for [14C]-TA and prevents its re-oxidation to [14C]-TASO. This in turn increases the net consumption of [14C]-TASO despite the fact that its oxidation to TASO2 is inhibited. The potent inhibition of TASO oxidation by TA, coupled with the back-reduction of TASO and its futile redox cycling with TA may help explain phenomena previously interpreted as “saturation toxicokinetics” in the in vivo metabolism and toxicity of TA and TASO. The improved understanding of the metabolism and covalent binding of TA and TASO facilitates the use of hepatocytes to prepare protein adducts for target protein identification