Metabolism and toxicity of thioacetamide and thioacetamide S-oxide in rat hepatocytes

Abstract

The hepatotoxicity of thioacetamide (TA) has been known since 1948. In rats, single doses cause centrolobular 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 (TASO(2)), 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 h. 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 [(14)C]-TASO is inhibited by unlabeled TA, which acts as a "cold-trap" for [(14)C]-TA and prevents its reoxidation to [(14)C]-TASO. This in turn increases the net consumption of [(14)C]-TASO despite the fact that its oxidation to TASO(2) 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.

Figure 1

Cytotoxicity of TA toward isolated hepatocytes shown as the time dependence of cytosolic LDH release in response to the indicated concentrations of TA. Cells only refers to untreated (control) cells.

Figure 2

Cytotoxicity of TASO to isolated hepatocytes. The top panel shows the time dependence of cytosolic LDH release in response to the indicated concentrations of TASO. The bottom panel shows the effect of TASO on cell viability as indicated by H/PI staining.

Figure 3

Protective effect of diallyl sulfide (DAS, top panel) and 4-methylpyrazole (4MP, bottom panel) against TASO cytotoxicity.

Figure 4

Protective effect of TA against TASO cytotoxicity as reflected in inhibition of cytosolic LDH release (top panel) and maintenance of cell viability by H/PI staining (bottom panel).

Figure 5

Quantitation of CYP2E1 in Rat Hepatocytes. Rat hepatocytes were isolated, cultured and treated as indicated on the figure. Equal amounts of microsomal protein were resolved using 10% SDS-PAGE.

Figure 6

Biotransformation of [¹⁴C]-TA (100 mM, top panel) and [¹⁴C]-TASO (10 mM, bottom panel) by isolated rat hepatocytes. Incubations were conducted using 7.5 × 10⁶ cells suspended in 1 mL medium containing the indicated initial substrate concentration. At the indicated times aliquots (0.1 mL) were withdrawn and quenched with strong detergent prior to analysis by TLC. The 6% conversion of TA to TASO (top panel) results in a TASO concentration of 6 mM, corresponding to an average oxidation rate of 178 nmol/hr/mg total cellular protein.

Figure 7

Effect of unlabeled TA (10 mM) on metabolism and covalent binding of [¹⁴C]-TASO (10 mM) by isolated hepatocytes (7.5 × 10⁶ cells/mL).

Figure 8

Cytotoxicity of TASO and protective effects of TA on hepatocytes in culture. Cells were allowed to attach in standard culture medium for 3 hr prior to the start of a 18 hr exposure period, and they were photographed at 10 min intervals throughout the exposure period. The six photos shown are the last frame of each of the six time-lapse movies that document the behavior of the cells throughout the exposure period. The complete movies are available as Supporting Information. Panel A01 shows untreated control cells; they are normal in appearance. Panels A02 and A03 show cells after 18 hr exposure to 1.5 mM TASO or 4.5 mM TASO, respectively; these cells have all died. Panel B01 shows cells exposed to 20 mM TA in medium; no toxic effects are apparent. Panels B02 and B03 show the protective effects of 20 mM TA against the cytotoxicity of 1.5 or 4.5 mM TASO, respectively.

Figure 9

Metabolic pathway diagrams showing the effect of unlabeled TA on the metabolism of [¹⁴C]-TASO in isolated hepatocytes. The numerical values are from Figure 7. In both A and B, the initial concentration of TASO was 10 mM; in B only the initial concentration of TA was 10 mM.

Scheme 1

Formation and reactivity of TA metabolites. In rat hepatocytes, thioacetamide (1) undergoes reversible S-oxidation to TASO (2). Further oxidation generates the highly reactive species TASO₂ (represented by tautomers 3 and 4). TASO₂ can directly imidoylate amine groups (R-NH₂) on cellular proteins or PE phospholipids (7). It can also react with water to form the stable metabolite acetamide (6), or the reactive derivative acetyl sulfinic acid (5) which reacts with protein amine groups to form amide derivatives (8). The latter do not form via hydrolysis of the stable amidine adducts (7). For further details see ref. .