The glutathione precursor l-2-oxothiazolidine-4-carboxylic acid protects against liver injury due to chronic enteral ethanol exposure in the rat

Abstract

L-2-oxothiazolidine-4-carboxylic acid (OTC) is a cysteine prodrug that maintains glutathione in tissues. Here, its effect on alcohol-induced liver injury in an enteral alcohol feeding model was investigated. Male Wistar rats were given control high-fat or ethanol containing diets enterally for 4 weeks. Treated rats received 500 mg/kg/d of dietary OTC. Ethanol delivery, weight gain, and the cyclic pattern of ethanol in the urine were not different between the OTC-ethanol and ethanol groups. After 4 weeks, serum aspartate transaminase (AST), necrosis and inflammation were elevated significantly by ethanol compared with appropriate high-fat controls, effects blocked by OTC. Moreover, ethanol elevated hepatic tumor necrosis factor ␣ (TNF-␣) messenger RNA (mRNA) and the nuclear transcription factor nuclear factor B (NFB) 2-3 fold. NFB in isolated Kupffer cells was also increased by ethanol. These effects were all blocked by OTC treatment. Additionally, superoxide production was higher in Kupffer cells isolated from ethanol-treated rats, an effect blunted by OTC. OTC also increased circulating glutathione (GSH) levels about 2-fold; however, GSH levels were not affected by ethanol or OTC in livers from the groups studied. Surprisingly, GSH was elevated by ethanol and OTC treatment in isolated Kupffer cells about 2-fold. Moreover, GSH (Ki-10 mol/L) and cysteinylglycine, but not oxidized glutathione (GSSG) or OTC, blunted the LPS-induced increase in calcium in isolated Kupffer cells, possibly by activating a glycine-gated chloride channel due to their structural similarity with glycine. Collectively, it is concluded that GSH is protective, in part, by increasing circulating GSH, which blunts activation of Kupffer cells via the glycine-gated chloride channel. (HEPATOLOGY 2000;31:391-398.)Kupffer cells release numerous mediators that participate in metabolic regulation, inflammation, and immune function. 1,2 Recent work from this laboratory has shown a central role for Kupffer cells in mechanisms of alcohol-induced liver injury. For example, when Kupffer cells were destroyed using gadolinium chloride (GdCl 3 ), injury caused by chronic alcohol administration in the Tsukamoto-French enteral alcohol feeding model was blocked. 3 Moreover, Kupffer cells are activated by bacterial endotoxin, and elimination of endotoxin with antibiotics 4 or Lactobacillus feeding 5 also diminished injury to the liver caused by alcohol. This concept was further strengthened by the observation that an antibody to tumor necrosis factor ␣ (TNF-␣) and a mouse lacking the TNF-receptor 1 6 exhibited diminished injury due to chronic ethanol 7 (for review see Thurman 8 ).Using the same enteral feeding model, Hirano et al. 9 showed that ethanol decreased hepatic glutathione (GSH) levels. Decreased glutathione occurs in many disease states including ischemia-reperfusion associated with heart disease. 10 Interestingly, they also showed that ethanol had a more pronounced effect on mitochondrial GSH content, and suggested that it could play an important role...

Role of free radicals in liver diseases

“…In the intragastric infusion of ethanol, liver damage was associated with enhanced lipid peroxidation, formation of 1-hydroxyethyl radical, decreased formation of protein carbonyl in GSH, and formation of lipid radicals [6][7][8][9][10]. Replacement of polyunsaturated lipids (necessary for peroxidation of lipids to occur) with mediumchain triglycerides or saturated lipids in the diets fed to rats intragastrically prevented or lowered the peroxidation of lipids and the alcohol-induced hepatic damage [9,11].…”

“…Iron is known both to produce • OH and to induce liver damage; importantly, addition of iron to these diets exacerbated hepatic damage [12]. Interestingly, administration of antioxidants, such as ebselen, vitamin E, superoxide dismutase (SOD), and precursors of GSH prevented alcohol-induced hepatic damage in rats [8]. Since ethanol-induced liver injury was linked to oxidative stress, Cederbaum and co-workers [13,14] investigated the effect of a compromised antioxidant defense system, copper-zinc SOD1 knockout mice, in an alcohol-induced hepatic damage model.…”

CYP2E1 and oxidative liver injury by alcohol

“…The most convincing data that oxidative stress contributes to alcohol-induced liver injury comes from the studies using the intragastric infusion model of alcohol administration. In these studies, alcoholinduced liver injury was associated with enhanced lipid peroxidation, protein carbonyl formation, formation of the 1-hydroxyethyl radical, formation of lipid radicals, decreases in hepatic antioxidant defense, especially GSH (26)(27)(28)(29)(30). Replacement of polyunsaturated fat (required for lipid peroxidation to occur) with saturated fat or medium chain triglycerides in the diets fed to rats intragastrically, lowered or prevented the lipid peroxidation, and the alcohol-induced liver injury (29,30).…”

“…Addition of iron, known to generate OH and promote oxidative stress, to these diets exacerbated the liver injury (31). Importantly, addition of antioxidants such as vitamin E, ebselen, superoxide dismutase (SOD), GSH precursors, prevented the alcoholinduced liver injury (28). Because alcohol-induced liver injury has been linked to oxidative stress, we investigated the effect of a compromised antioxidant defense system, copper-zinc superoxide dismutase (SOD1) deficiency on alcohol-induced liver injury (32,33).…”

Effects of N-acetylcysteine on ethanol-induced hepatotoxicity in rats fed via total enteral nutrition

“…Similarly, in vivo, feeding with the glutathione precursor L-oxothioazolidine-4-carboxylic acid and with dietary antioxidant extracts from cocoa and green tea, as well as gene therapy resulting in hepatic overexpression of either Cu/Zn or Mn superoxide dismutase has been shown to be protective against ALD [4,[12][13][14][15][16]. It is noteworthy that these later observations were performed in rat intragastric ethanol-infusion models such as that originally developed by Tsukamoto et al [5] where pathology is accompanied by endotoxemia and Kupffer cell activation as measured by increased production of oxidants via NADPH oxidase and increased expression of the endotoxin receptor CD14 [4,[12][13][14][15][16][17][18]. We have developed a total enteral nutrition (TEN) model in which ethanol is also infused intragastrically, but in which ethanol substitututes for carbohydrate as part of an isocaloric diet.…”