Abstract
Drug-induced hepatotoxicity is mainly caused by hepatic glutathione (GSH) depletion. In general, the activity of rodent glutathione S-transferase is 10 to 20 times higher than that of humans, which could make the prediction of drug-induced hepatotoxicity in human more difficult. Gamma-glutamylcysteine synthetase (gamma-GCS) mainly regulates de novo synthesis of GSH in mammalian cells and plays a central role in the antioxidant capacity of cells. In this study, we constructed a GSH-depletion experimental rat model for the prediction of human hepatotoxicity. An adenovirus vector with short hairpin RNA against rat gamma-GCS heavy chain subunit (GCSh) (AdGCSh-shRNA) was constructed and used to knock down the GCSh. In in vitro study in H4IIE cells, a rat hepatoma cell line, GCSh mRNA and protein were significantly decreased by 80% and GSH was significantly decreased by 50% 3 days after AdGCSh-shRNA infection. In the in vivo study in rat, the hepatic GSH level was decreased by 80% 14 days after a single dose of AdGCSh-shRNA (2 x 10(11) pfu/ml/body), and this depletion continued for at least 2 weeks. Using this GSH knockdown rat model, acetaminophen-induced hepatotoxicity was shown to be significantly potentiated compared with normal rats. This is the first report of a GSH knockdown rat model, which could be useful for highly sensitive tests of acute and subacute toxicity for drug candidates in preclinical drug development.
Highlights
Glutathione (5-L-glutamyl-L-cysteinylglycine, GSH)2 is one of the most abundant tripeptides, consisting of glycine, glutamic acid, and cysteine
GSH synthetase couples glycine to ␥-glutamylcysteine to form GSH. ␥-GCS is a rate-limiting step in GSH biosynthesis, and GSH is a feedback inhibitor of ␥-GCS activity. ␥-GCS is a heterodimeric enzyme composed of a catalytic subunit [4] and a modulatory subunit [5]
Changes of GCS heavy chain (GCSh) mRNA Expression and GSH Level in Various Hepatoma Cell Lines—To investigate the knockdown effect on the cells, various hepatoma cells were infected with AdGCSh-short hairpin RNA (shRNA) or AdLuc-shRNA at a multiplicity of infection (m.o.i.) of 20 for 3 days
Summary
Glutathione (5-L-glutamyl-L-cysteinylglycine, GSH) is one of the most abundant tripeptides, consisting of glycine, glutamic acid, and cysteine. Knockdown Effects of ␥-Glutamylcysteine Synthetase in Rat active metabolites produced in vivo in rat would be immediately detoxified by GSH conjugation, which would make the prediction of drug-induced hepatotoxicity in human more difficult. From this perspective, the AdGCSh-shRNA-mediated GSH depletion rat model could be useful for predicting the hepatotoxicity caused by unknown active metabolites of drug candidates produced by Phase I enzymes
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