Hepatic ATP-Depletion Precedes Thioacetamide-Induced Liver Damage

Introduction: Acute thioacetamide (TAA; CH3-CS-NH2) administration is widely used in animal models to investigate mechanisms of toxin-induced liver injury, regeneration and fibrosis. TAA is metabolized by micosomal CYP2E1 to TAA-S-oxide, which in turn is oxidized by CYP2E1 to TAA-S,S-dioxide. This highly reactive metabolite covalently modifies macromolecules in all cell compartments. It has been shown that TAA-bioactivation is subject to saturation kinetics and CYP2E1 has less specificity for TAA-S-oxide than for TAA. However, the exact cause leading to cell death and whether covalent binding to macromolecules is responsible for hepatic necrosis still remains unclear. Aim: Cellular energy metabolism in mouse liver after TAA-administration was followed to elucidate a mechanism that may pre-dispose liver cells to die from necrosis. Methods: BALB/c mice were injected with a non-lethal hepatotoxic dose of TAA (200 mg/kg, ip) and sacrificed 1-6, 12, 18 and 24hrs after TAA-injection. Standard assays were used for serum-ALT/AST and caspase-3 determinations. ATP/ADP/AMP and GSH/GSSG were analyzed by HPLC. NMR analysis: Mice were injected with [U-13C]glucose (500 mg/kg, ip) 45 min prior to sacrifice. Ex vivo 13C-NMR-spectra of water soluble metabolites from liver-extracts were recorded. Flux of 13C through pyruvate carboxylase (PC) and pyruvate dehydrogenase (PDH) was followed up by 13C-isotopomer analysis. Results: One hour after TAA-injection the hepatic energy charge ([ATP]+[ADP]/2)/([ATP]+[ADP]+[AMP]) dropped to 68±8%control (P<0.001) as indicated by decreased ATP (63±13%control, P<0.005) and increased ADP and AMP (130±4, P<0.01 and 214±28, P<0.001; %controls, respectively). Neither the energy charge nor the ATP-levels recovered at any time-point up to 24hrs. In fact, ATP further declined to 27±4%control (P<0.001) at 24hrs. Analysis of 13C-NMR spectra suggests diminished flux trough PC (65±17%control) and decreased glycolytic activity (fractional enrichments in [3-13C]lactate and [3-13C]alanine: 72±3 and 35±4%controls, respectively) at 1hr, while PDH-flux was at control-level. GSH-levels were unchanged up to 12hrs and decreased at 18 and 24 hrs (37±9 and 28±7%controls, P