Serine alleviates oxidative stress via supporting glutathione synthesis and methionine cycle in mice.

SCOPE: Serine lies at the central node linking biosynthetic flux from glycolysis to glutathione synthesis and one-carbon metabolic cycle which are closely related to antioxidant capacity. The present study was conducted to determine the effects of serine supplementation on oxidative stress and its relative mechanisms. METHODS AND RESULTS: Diquat treatment was performed to induce oxidative stress in mice and primary hepatocytes. The results showed that hepatic glutathione anti-oxidant systems were impaired and reactive oxygen species and homocysteine were increased in diquat-induced mice and hepatocytes, while such disadvantageous changes were diminished by serine supplementation both in vivo and in vitro. However, when cystathionine ß-synthase expression was inhibited by interference RNA in hepatocytes, the effects of serine supplementation on the improvement of glutathione synthesis and the alleviation of oxidative stress were diminished. Moreover, when hepatocytes were treated with cycloleucine, an inhibitor of methionine adenosyltransferase, the effects of serine supplementation on the improvement of methionine cycle and the alleviation of DNA hypomethylation and oxidative stress were also diminished. CONCLUSION: Our results indicated that serine supplementation alleviated oxidative stress via supporting glutathione synthesis and methionine cycle, mostly by condensing with homocysteine to synthesize cysteine and providing one-carbon units for homocysteine remethylation.

L-Glutamate Enhances Barrier and Antioxidative Functions in Intestinal Porcine Epithelial Cells.

BACKGROUND: L-Glutamate (Glu) is a major amino acid in milk and postweaning diets for mammals (including pigs and human infants). However, effects of Glu on intestinal mucosal barrier and antioxidative functions are unknown. OBJECTIVE: This study tested the hypothesis that Glu may enhance the barrier function of intestinal porcine epithelial cell line 1 (IPEC-1) cells by upregulating the expression of tight junction proteins. METHODS: IPEC-1 cells were cultured with or without Glu in the presence or absence of 1 mmol/L diquat (an oxidant) for indicated time points. Cell numbers, transepithelial electrical resistance (TEER), mRNA, and protein abundance of glutamate transporter, the release of lactate dehydrogenase (LDH), and the abundance of tight junction proteins were determined. RESULTS: Compared with 0 mmol/L Glu, 0.5-, 1-, and 2 mmol/L Glu stimulated (P < 0.05) cell growth by 13-37% at 24 h and 12-34% at 48 h, respectively. In addition, 0.5 mmol/L Glu increased (P < 0.05) TEER (by 58% at 24 h and by 98% at 48 h, respectively). These effects of Glu were associated with increased mRNA abundance of Glu transporter solute carrier family 1 member 1 (SLC1A1) by 30-130% and protein abundance of excitatory amino acid transporter 3 (encoded by SLC1A1) by 19-34%, respectively. In a cell model of oxidative stress induced by 1 mmol/L diquat, 0.5 mmol/L Glu enhanced cell viability, TEER, and membrane integrity (as indicated by the reduced release of LDH) in IPEC-1 cells by increasing the abundance of the tight junction proteins occludin, claudin-3, zonula occludens (ZO)-2, and ZO-3. CONCLUSION: These findings indicate that Glu plays an important role in mucosal barrier function by enhancing cell growth and maintaining membrane integrity in response to oxidative stress.

Melatonin reduces mortality and oxidatively mediated hepatic and renal damage due to diquat treatment.

The bipyridyl herbicide, diquat, is a potent prooxidant that generates superoxide anions through redox cycling in vivo. Exposure to elevated levels of this compound causes acute hepatic and renal toxicity as well as death in rodents. In the present study, we investigated whether melatonin, a free radical scavenger and antioxidant, could protect against diquat-induced hepatic and renal damage and whether the indole would improve survival of Kunming mice given a lethal dose of diquat. When mice were intraperitoneally (i.p.) given a single dose of diquat (50 mg/kg body weight), liver and kidney injuries were observed at 6 hr as indicated by elevated serum levels of both alanine aminotransferase (ALT) activity and blood urea nitrogen (BUN). In addition, lipid peroxidation levels in both liver and kidney showed significant increases as shown by elevated concentrations of F(2)-isoprostanes. The administration of melatonin (20 mg/kg) 30 min before the diquat injection resulted in a significant reduction in serum levels of ALT and BUN as well as hepatic and renal F(2)-isoprostanes levels. For the survival study, 75 mg/kg diquat was administered i.p. into mice to induce acute death. Without melatonin treatment, 10 of 23 (43.5%) mice died within 24 hr after diquat injection. Pretreatment with melatonin (20 mg/kg) 30 min prior to the injection of diquat and thereafter at 4-hr intervals until the end of the observation period (24 hr), reduced the death rate to two of 22 (9.1%) mice. Chi-squared test revealed a significant difference with P < or = 0.05. In conclusion, melatonin, a broad spectrum antioxidant, reduces hepatic and renal damage and lowers the death rate in diquat-treated mice.

High levels of dietary vitamin E do not replace cellular glutathione peroxidase in protecting mice from acute oxidative stress.

Our objective was to determine whether high levels of dietary vitamin E replaced the protection of the Se-dependent cellular glutathione peroxidase (GPX1) against paraquat- or diquat-induced acute oxidative stress in mice. Two experiments were conducted using GPX1 knockout [GPX1(-/-)] mice and wild-type (WT) mice (n = 78/group). In Experiment 1, mice were fed torula yeast-based, Se-adequate (0.4 mg/kg as sodium selenite) diets + 0, 75, 750 or 7,500 mg all-rac-alpha-tocopheryl acetate for 5 wk before an intraperitoneal injection of 50 mg paraquat/kg body weight. In Experiment 2, mice were fed the diet + 0 or 750 mg all-rac-alpha-tocopheryl acetate for 5 wk and were killed 1 or 3 h after an injection of diquat at 12, 24 or 48 mg/kg. In Experiment 1, all mice died of the injection and there were 8- to 15-fold differences (P < 0.001) in survival times between the GPX1(-/-) and the WT mice. Although increasing tocopheryl acetate from 0 to 750 mg/kg extended the survival time of the GPX1(-/-) mice for 2 h (P = 0.06), the highest tocopheryl acetate level resulted in a decrease (P < 0.05) in survival time in the WT mice. The vitamin E-deficient GPX1(-/-) mice had the highest concentration of hepatic thiobarbituric acid reacting substances. In Experiment 2, the diquat-induced formation of hepatic F(2)-isoprostanes was accelerated (P < 0.05) by vitamin E deficiency and was also affected by the GPX1 knockout. Diquat produced much greater (P < 0.01) dose-dependent increases in plasma alanine transaminase (ALT) activities in the GPX1(-/-) than in the WT mice. Hepatic phospholipid hydroperoxide GPX activities were decreased (P < 0.05) by the diquat injection only in the vitamin E-deficient GPX1(-/-) mice. Despite a potent inhibition of hepatic lipid peroxidation, high levels of dietary vitamin E do not replace the protection of GPX1 against the paraquat-induced lethality or the diquat-induced plasma ALT activity increase in mice.

Antioxidant-dependent inhibition of diquat-induced toxicity in vivo.

The abilities of two experimental antioxidants (U-74006F and U-78517G), as well as the model antioxidant, diphenyl-p-phenylenediamine (DPPD), to protect against diquat-induced toxicity in male Fischer-344 rats were examined. Both experimental compounds afforded near complete protection against diquat-induced hepatotoxicity, as measured by clinical chemistry and histopathological indices. When observed, diquat-induced nephrotoxicity was also inhibited. Minimal protection was afforded by the model compound, DPPD. In follow-up studies with U-78517G, no effect on diquat-induced biliary excretion of oxidized glutathione was observed, suggesting that a shift in the thiol:disulfide ratio is not responsible for diquat-induced hepatotoxicity. These data are consistent with those from previous in vitro studies in our laboratory and are in agreement with studies by others which suggest that lipid peroxidation is an important event in diquat-induced hepatotoxicity in vivo. The antioxidant effects were largely route-independent as either oral pre-treatment alone (200 mg/kg, 24 h before diquat), intravenous pre-treatment alone (6 mg/kg, 5 min before diquat) or the combination of both treatments produced a similar degree of protection. While pre-treatment with antioxidants was quite effective, no significant U-78517G-dependent inhibition of toxicity was observed when administration was delayed by as little as 10 min post diquat. These latter data suggest that initiation of diquat-induced hepatotoxicity is rapid and that these compounds would therefore be unlikely to have clinical utility in the treatment of diquat intoxication.