Mechanism of Protection by S-(1,2-Dicarboxyethyl) glutathione Triester against Acetaminophen-Induced Hepatotoxicity in Rat Hepatocytes.

Abstract

2-dicarboxyethyl)glutathione (DCE-GS, Fig. 1) was isolated from calf lenses and its chemical structure was determined by comparison with the synthetic compound.2) By HPLC after reaction with 2,4-dinitrofluorobenzene we previously determined the content of this peptide in the lenses of various vertebrates, 3) in various tissues of rat, and in the subcellular fraction of rat liver. 4) We also reported that DCE-GS was enzymatically synthesized using glutathione (GSH) and L-malate, and that the enzyme catalyzing this reaction was purified from the rat liver cytosolic fraction.4) Successively, we found that DCE-GS had potent inhibitory effects on blood coagulation and platelet aggregation, 5,6) and an enhancing effect on epidermal growth factorstimulated DNA synthesis in primary cultures of rat hepatocytes. 7) We also determined the levels of GSH and DCE-GS in rat liver during the regeneration of rat liver with time after partial hepatectomy. The GSH and DCE-GS levels increased to a great extent in regenerating rat liver.8) Recently we showed that oral administration of DCE-GS triester, S-(1,2-diethoxycarbonyl)glutathione isopropyl ester, to rats inhibited the acetaminophen (APAP)-induced hepatotoxicity via an increased hepatic GSH content. 9)However, it remains unclear how DCE-GS triester is transported into hepatocytes and how DCE-GS triester transported into hepatocytes increases the hepatic GSH content. Therefore, in order to clarify the mechanism of protection by DCE-GS triester against APAP-induced hepatotoxicity, we examined the mechanism as to the enhancement of the hepatocellular GSH level on DCE-GS triester treatment using isolated rat hepatocytes. MATERIALS AND METHODS AnimalsMale Wistar rats aged 6 weeks (150-180 g) were used in this study. The animals were obtained from Shizuoka Laboratory Animal (Shizuoka, Japan). Water and food (MF; Oriental Yeast) were provided ad libitum for at least 1 week before use. ChemicalsThe structures of DCE-GS and its esters are shown in Fig. 1, and S-(1,2-diethoxycarbonylethyl)glutathione isopropyl ester (triester) were supplied by Senju Pharmaceutical Co., Ltd. (Osaka, Japan). Cell culture reagents were purchased from Dainippon Pharmaceutical Co., Ltd. (Osaka, Japan). Other compounds were from Wako Pure Chemical Ind., Ltd. (Osaka, Japan).Preparation and Primary Culture of Isolated Hepatocytes Hepatocytes, exhibiting more than 85% initial viability, as measured as trypan blue exclusion, were isolated from rats by means of the in situ perfusion technique with collagenase.10) The isolated hepatocytes were suspended in William's medium E supplemented with 5% fetal bovine serum, 1 mg/ml insulin, 1 mM dexamethasone and 100 mg/ml kanamycine, inoculated at a cell density of 1ϫ10 5 cells/0.2 ml/cm 2 onto 16-or 35-mm diameter Corning plastic dishes, which had been coated with rat tail collagen, and then cultured under a humidified atmosphere of 5% CO 2 : 95% air at 37°C. After an attachment period of 2 h, the medium was replaced by a serum-free medium containing 100 mg/ml kanamycin...

Glutathione Homeostasis and Functions: Potential Targets for Medical Interventions

“…This is usually achieved by supplementation with GSH monoesters and diesters [247–249], GSH precursors such N -acetyl cysteine (NAC); [250–252] or α -mercaptopropionylglycine [87, 253]. Importantly, cysteine is not usually utilized as a precursor presumably due to its toxicity at high concentrations [254].…”