Received for publication May 17, 2005.
Revised September 17, 2005.
Accepted for publication September 20, 2005.

SATURATION TOXICOKINETICS OF THIOACETAMIDE: ROLE IN INITIATION OF LIVER INJURY

Abstract

Thioacetamide (TA), a potent centrilobular hepatotoxicant, undergoes a two-step bioactivation mediated by microsomal CYP2E1 to TA sulfoxide (TASO), and further to TA-S, S-dioxide (TASO₂), a reactive metabolite that initiates cellular necrosis. Our earlier studies showed that bioactivation-mediated liver injury of TA is not dose-proportional. The objective of this study was to examine whether increasing doses of TA lead to enzyme saturation, thereby resulting in lack of dose-response for injury: bioactivation of TA TASO TASO₂ may follow zero-order kinetics. A 12-fold dose range of TA (50, 300 and 600 mg/kg, i.p.) was injected to male SD rats. TA and TASO were quantified in plasma, liver and urine by HPLC. With increasing doses, the apparent elimination half-lives of TA and TASO increased linearly, indicating that TA bioactivation exhibits saturation kinetics. Increasing TA dose resulted in greater-than-proportional increases in plasma TA and TASO levels. The TASO/TA ratio was inversely proportional to the dose of TA. Covalent binding of ¹⁴C-TA-derived radiolabel to liver macromolecules showed a less-than-dose-proportionate increase with a 12-fold higher dose. Less than dose-proportional covalent binding was confirmed in liver microsomal incubations with ¹⁴C-TA. Three-fold higher excretion of TASO was seen in urine at the highest dose (600 mg/kg) compared to the lowest dose (50 mg TA /kg). Incubation of TA with rat liver microsomes and purified baculovirus-expressed rat and human CYP2E1 "Supersomes^®" over a concentration range of 0.01 to 10 mM, revealed saturation of TA conversion to TASO at and above 0.05 mM TA concentration, comparable to in vivo plasma and liver levels achieved upon administration of higher doses. Calculated Km values for TA (0.1 mM) and TASO (0.6 mM) suggest that the second step of TA bioactivation is 6-fold less efficient. Collectively, the findings indicate saturation of CYP2E1 at the first (TA to TASO) and second steps (TASO to TASO₂) of TA bioactivation.

Key words: bioactivation, hepatotoxicity, reactive intermediate, toxicokinetics