Glutamate Dehydrogenase Covalently Binds to a Reactive Metabolite of Acetaminophen

Abstract

The mechanism of the hepatotoxicity of the analgesic acetaminophen is believed to be mediated by covalent binding to protein; however, critical targets which effect the toxicity are unknown. It has been shown that mitochondrial respiration in vivo is inhibited in mice as early as 1 h following a hepatotoxic dose of acetaminophen, and it is postulated that covalent binding to critical mitochondrial proteins may be important. A time course of mitochondrial proteins stained with anti-acetaminophen in an immunoblot detected two major adducts of 50 and 67 kDa as early as 30 min after a hepatotoxic dose of acetaminophen in mice. To further understand the role of covalent binding to mitochondrial proteins and acetaminophen hepatotoxicity, we have purified and identified a 50 kDa mitochondrial protein which becomes covalently bound to a reactive metabolite of acetaminophen. An N-terminal sequence of the 50 kDa adduct was 100% homologous with the deduced amino acid sequence of glutamate dehydrogenase. In addition, the purified protein was immunochemically reactive with rat liver anti-glutamate dehydrogenase. Enzyme activity of glutamate dehydrogenase was significantly decreased in mice 1 h following hepatotoxic treatment with acetaminophen. These data suggest that acetaminophen hepatotoxicity may in part be mediated by covalent binding to glutamate dehydrogenase.

Inhibition of Carbamyl Phosphate Synthetase-I and Glutamine Synthetase by Hepatotoxic Doses of Acetaminophen in Mice

“…Although not discussed, the Western analyses presented in the publications by these groups often show an immunoreactive band in the region of 130 kDa, though these studies employed microsomal or cytosolic fractions. The profile of immunoreactive proteins from mitochondrial fractions reported by Halmes et al (1996) offers more direct evidence for formation of an alkylated species migrating at 130 kDa, which supports the hypothesis of alkylation of CPS-I more strongly. Although the covalent binding of acetaminophen metabolites is observed at greatest levels in the centrilobular hepatocytes (Jollow et al, 1973;Roberts et al, 1991), the alkylation indicated by the immunohistochemical studies reported by Roberts et al was reported to extend through the midzonal hepatocytes.…”

“…The present data show that 2 hr after 400 mg/kg of acetaminophen, glutamine synthetase activities are decreased by 50%, while CPS-I activities are not decreased significantly (Figs. 9 and 10), suggesting that the hyperammonemia observed in mice 2 hr after acetaminophen is more likely to be due to the decrease in glutamine synthetase activities than to decreases in CPS-I activities, although the relative contributions of inhibition of glutamate dehydrogenase inhibition reported by Halmes et al (1996) were not evaluated in the animals we studied. Halmes et al (1996) report 20% inhibition of hepatic glutamate dehydrogenase activities 1 hr after 400 mg/kg of acetaminophen and 30% inhibition after 600 mg/kg.…”

“…9 and 10), suggesting that the hyperammonemia observed in mice 2 hr after acetaminophen is more likely to be due to the decrease in glutamine synthetase activities than to decreases in CPS-I activities, although the relative contributions of inhibition of glutamate dehydrogenase inhibition reported by Halmes et al (1996) were not evaluated in the animals we studied. Halmes et al (1996) report 20% inhibition of hepatic glutamate dehydrogenase activities 1 hr after 400 mg/kg of acetaminophen and 30% inhibition after 600 mg/kg. However, the possible contributions of decreased CPS-I activities to the hyperammonemia we observed are not disproved by the data presently available.…”

“…5-7). In addition, Halmes et al (1996) have shown that acetaminophen metabolites bind to glutamate dehydrogenase, which also would be expected to compromise a mechanism of ammonia clearance. Hepatic glutamine synthetase is located in perivenous hepatocytes, in layers one to three cells thick surrounding terminal venules (Haussinger, 1983;Gebhardt and Mecke, 1983;Gebhardt and Reichen, 1994) and is isolated with the microsomal fraction by differential centrifugation (Bulera et al, 1995).…”

Systems Biology Investigation to Discover Metabolic Biomarkers of Acetaminophen-Induced Hepatic Injury Using Integrated Transcriptomics and Metabolomics

“…Increases in urine levels of α-ketoglutarate have been reported as a mitochondrial malfunction biomarker in transaldolase deficiency patients [27]. Halmes et al [28] also reported that enzyme activity of glutamate dehydrogenase, catalyzing the reversible oxidative deamination of glutamate to α-ketoglutarate in the energy metabolism, is significantly decreased in mice 1 h post-dosing with APAP. As such, α-ketoglutarate levels can reflect energy fluctuation, which can be influenced by mitochondrial damage in hepatic cells as well as hepatocyte recovery.…”