Significance of alterations in the metabolomics of sulfur-containing amino acids during liver regeneration.

It has been known that liver regeneration is accompanied with a profound change in the metabolomics of sulfur-containing substances in liver. However, its physiological significance in the liver regenerative process is still unclear. Our previous work showed that buthioninesulfoximine and phorone, both widely used to deplete intracellular glutathione (GSH) in biological experiments, induced contrasting changes in the sulfur-containing amino acid metabolism in liver. In this study we employed these GSH-depleting agents to evaluate the role of sulfur-containing substances in the early phase of liver regeneration. Male rats treated with buthioninesulfoximine or phorone were subjected to two-thirds partial hepatectomy (PHx). At the doses used, the magnitude of GSH depletion after PHx was comparable, but buthioninesulfoximine administration inhibited the progression of liver regeneration as determined by liver weight increase, elevation of serum alanine aminotransferase activity, and cyclin D1 and proliferating cell nuclear antigen (PCNA) protein expressions, whereas liver recovery was significantly accelerated in the phorone-treated rats, suggesting that the role of GSH in this process is minimal. Hepatic concentrations of methionine, S-adenosylmethionine, cysteine, taurine and GSH were all elevated by PHx. Methionine adenosyltransferase activity was also induced in the remnant liver. Buthioninesulfoximine administration depressed the elevation of S-adenosylmethionine, but increased the catabolism of cysteine to taurine. In contrast, S-adenosylmethionine elevation was augmented whereas cysteine, hypotaurine and taurine were decreased in the phorone-treated rats. PHx elevated hepatic putrescine and spermidine, but lowered spermine concentrations. Buthioninesulfoximine administration increased putrescine further, but decreased spermidine and spermine concentrations. On the contrary, both spermidine and spermine concentrations were elevated in the rats treated with phorone. The results suggest that the availability of S-adenosylmethionine plays a critical role in the progression of liver regeneration via enhancement of polyamine synthesis. These findings raise the possibility that regulating hepatic transsulfuration reactions may be capable of modifying the recovery process after liver injury.

Metabolomic analysis of sulfur-containing substances and polyamines in regenerating rat liver.

We studied the significance of alterations in the metabolomics of sulfur-containing substances in rapidly regenerating rat livers. Male rats were subjected to two-thirds partial hepatectomy (PHx), and the changes in hepatic levels of major sulfur-containing amino acids and related substances were monitored for 2 weeks. Liver weight began to increase from 24 h after the surgery, and appeared to recover fully in 2 weeks. Serum alanine aminotransferase and aspartate aminotransferase activities were elevated immediately after the surgery and returned slowly to normal levels in 2 weeks. Methionine, S-adenosylmethionine (SAM), cystathionine and cysteine were increased rapidly and remained elevated for longer than 1 week. Hepatic glutathione concentration was increased gradually for 24 h, and then decreased thereafter, whereas hypotaurine was elevated drastically right after the surgery. Hepatic concentrations of polyamines were altered significantly by PHx. In the hepatectomized livers putrescine concentration was elevated rapidly, reaching a level 40- to 50-fold greater than normal in 6-12 h. Ornithine, the metabolic substrate for putrescine synthesis, was also elevated markedly. Spermidine was increased significantly, whereas spermine was depressed below normal, which appeared to be due to the increased consumption of decarboxylated SAM for spermidine biosynthesis. The results show that the metabolomics of sulfur-containing amino acids and related substances is altered profoundly in regenerating rat livers until the original weight is recovered. Hepatic concentrations of polyamines after PHx are closely associated with the alteration in the metabolomics of sulfur-containing substances. The implication of these changes in the progression of liver regeneration is discussed.

Protective effect of quercetin against paraquat-induced lung injury in rats.

AIMS: Paraquat (PQ) is known to induce pulmonary injury via a redox cyclic reaction. The present study was aimed to determine the protective effects of quercetin against PQ-induced pulmonary injury in association with its antioxidant activity. MAIN METHODS: Male rats were challenged acutely by PQ (50 mg/kg, i.p.) with or without quercetin post-treatment. Pulmonary heme oxygenase-1 (HO-1) expression, malondialdehyde (MDA) level, and the total oxyradical scavenging capacity (TOSC) toward hydroxyl, peroxyl radicals and peroxynitrite were measured 24 h after PQ treatment. Different groups of rats were instilled with PQ (0.5 mg/kg) directly into the right lung. Quercetin was administered to the rats daily for 14 days after PQ instillation. Serum NO, pulmonary glutathione (GSH) and 4-hydroxyproline (4-HP) concentrations were quantified in conjunction with histopathological examination to determine the fibrotic changes in lung. KEY FINDINGS: Pulmonary MDA level and HO-1 expression were elevated and the TOSC was reduced rapidly by an intraperitoneal dose of PQ. These changes were inhibited by quercetin post-treatment. In rat lungs instilled with PQ 14 days before, NO, MDA and 4-HP were elevated, and GSH was reduced, which were all inhibited significantly by daily quercetin treatment. Histopathological examination also revealed that quercetin ameliorated the increase in fibroblast distribution and collagen deposition in the lungs instilled with PQ. SIGNIFICANCE: The present results demonstrate that quercetin administration to rats effectively inhibits the development of PQ-induced pulmonary injury most probably via its antioxidant activity.

Alteration in metabolism and toxicity of acetaminophen upon repeated administration in rats.

Our previous studies showed that administration of a subtoxic dose of acetaminophen (APAP) to female rats increased generation of carbon monoxide from dichloromethane, a metabolic reaction catalyzed mainly by cytochrome P450 (CYP) 2E1. In this study we examined the changes in metabolism and toxicity of APAP upon repeated administration. An intraperitoneal dose of APAP (500 mg/kg) alone did not increase aspartate aminotransferase, alanine aminotransferase, or sorbitol dehydrogenase activity in serum, but was significantly hepatotoxic when the rats had been pretreated with an identical dose of APAP 18 h earlier. The concentrations and disappearance of APAP and its metabolites in plasma were monitored for 8 h after the treatment. APAP pretreatment reduced the elevation of APAP-sulfate, but increased APAP-cysteine concentrations in plasma. APAP or APAP-glucuronide concentrations were not altered. Administration of a single dose of APAP 18 h before sacrifice increased microsomal CYP activities measured with p-nitrophenol, p-nitroanisole, and aminopyrine as probes. Expression of CYP2E1, CYP3A, and CYP1A proteins in the liver was also elevated significantly. The results suggest that administration of APAP at a subtoxic dose may result in an induction of hepatic CYP enzymes, thereby altering metabolism and toxicological consequences of various chemical substances that are substrates for the same enzyme system.

Ethanol-induced liver injury and changes in sulfur amino acid metabolomics in glutathione peroxidase and catalase double knockout mice.

BACKGROUND/AIMS: Oxidative stress via generation of reactive oxygen species is suggested to be the major mechanism of alcohol-induced liver injury. We investigated the effects of glutathione peroxidase-1 and catalase double deficiency (Gpx-1(-/-)/Cat(-/-)) on liver injury and changes in the sulfur amino acid metabolism induced by binge ethanol administration. METHODS: Ethanol (5 g/kg) was administered orally to the wild-type and the Gpx-1(-/-)/Cat(-/-) mice every 12 h for a total of three doses. Mice were sacrificed 6 h after the final dose. RESULTS: The Gpx-1/Cat deficiency alone increased malondialdehyde levels in liver significantly. Hepatic methionine adenosyltransferase (MAT) activity and S-adenosylmethionine levels were decreased, however, glutathione contents were not changed. Ethanol administration to the Gpx-1(-/-)/Cat(-/-) mice increased the elevation of serum alanine aminotransferase activity, plasma homocysteine levels, hepatic fat accumulation and lipid peroxidation compared with the wild-type animals challenged with ethanol. Also the reduction of MAT activity and S-adenosylmethionine levels was enhanced, but MATI/III expression was increased significantly. CONCLUSIONS: The results indicate that Gpx-1 and Cat have critical roles in the protection of liver against binge ethanol exposure. Augmentation of ethanol-induced oxidative stress may be responsible for the impairment of the transsulfuration reactions and the aggravation of acute liver injury in the Gpx-1(-/-)/Cat(-/-) mice.

Impaired sulfur-amino acid metabolism and oxidative stress in nonalcoholic fatty liver are alleviated by betaine supplementation in rats.

Nonalcoholic fatty liver is involved in the development of nonalcoholic steatohepatitis and chronic liver injury. Impairment of hepatic transsulfuration reactions is suggested to be critically linked with alcoholic liver injury, but its role in nonalcoholic fatty liver remains unknown. We examined the early changes in sulfur-amino acid metabolism and their implication in nonalcoholic fatty liver disease (NAFLD). Male rats were provided with a standard liquid diet or a high-fat liquid diet (HF) for 3 wk. An additional group of rats received the HF diet supplemented with betaine (1%). HF diet intake elevated hepatic triglyceride and serum tumor necrosis factor-alpha (TNFalpha) concentrations. Antioxidant capacity of liver cytosol against hydroxyl and peroxyl radicals was reduced significantly. Hepatic S-adenosylmethionine (SAM) and glutathione (GSH) decreased, but hypotaurine and taurine concentrations increased. Methionine adenosyltransferase (MAT) activity, not its concentration, was depressed, whereas both activity and concentration of cysteine dioxygenase and GSH S-transferase were elevated. Betaine supplementation of the HF diet inhibited hepatic fat accumulation and serum TNFalpha elevation. The decrease in cytosolic antioxidant capacity was also prevented. MAT activity and its concentration were induced significantly. Hepatic SAM and GSH increased and elevation of hypotaurine and taurine was depressed. The results indicate that the metabolism of S-containing substances is significantly disturbed by the HF diet, suggesting a causal role of impairment of hepatic transsulfuration reactions in NAFLD. Betaine supplementation protects the liver from nonalcoholic steatosis and oxidative stress most probably via its effects on the transsulfuration reactions.

Comparison of the effects of buthioninesulfoximine and phorone on the metabolism of sulfur-containing amino acids in rat liver.

Alterations in hepatic transsulfuration reactions were determined in rats treated with a glutathione-depleting agent. A dose of l-buthionine-(S,R)-sulfoximine decreased hepatic methionine, cysteine, S-adenosylmethionine, and glutathione levels rapidly. Methionine adenosyltransferase and gamma-glutamylcysteine lygase activities were decreased transiently, but significantly. The activity of cysteine dioxygenase was increased, resulting in an elevation of hypotaurine and taurine concentrations. Administration of phorone reduced hepatic glutathione and cysteine similarly, but S-adenosylmethionine concentrations were elevated for as long as 72h. Hepatic methionine adenosyltransferase, cystathionine beta-synthase, cystathionine gamma-lyase, and gamma-glutamylcysteine lygase activities were all increased but cysteine dioxygenase activity and taurine generation were markedly depressed. The results show that a decrease in hepatic GSH induces profound changes in sulfur amino acid metabolomics, which would subsequently influence various cellular processes. It is suggested that the change in hepatic levels of sulfur-containing substances and its physiological significance should be considered when a glutathione-depleting agent is utilized in biological experiments.

Alleviation of acute ethanol-induced liver injury and impaired metabolomics of S-containing substances by betaine supplementation.

Oxidative stress is suggested to play a key role in the development of alcoholic liver injury. We investigated the induction of oxidative damage in association with changes in hepatic concentrations of sulfur-containing substances in mice challenged with binge-like ethanol administration. Also the protective effect of dietary betaine against ethanol-induced liver injury was determined. Serum alanine aminotransferase activity, TNFalpha level, and hepatic malondialdehyde level were increased significantly by ethanol administration. Hepatic Cyp2e1 was induced to 250% of control. Ethanol administration decreased hepatic S-adenosylmethionine, cysteine, and glutathione, but elevated hypotaurine and taurine levels. Betaine supplied in drinking water for 2 weeks attenuated the induction of alcoholic liver injury and Cyp2e1 significantly. Reduction of hepatic S-adenosylmethionine and glutathione was alleviated, and elevation of hypotaurine and taurine was depressed. The results suggest that betaine may protect the liver against ethanol-induced oxidative injury most probably via its effects on the sulfur-amino acid metabolism.