Antioxidant protection mechanism of chick hepatic mitochondria exposed to lanthanum chloride & neodymium chloride treatment

Acute lanthanum chloride (250 mg/kg body wt) and neodymium chloride (200 mg/kg body wt) administrations resulted in significant enhancement of glutathione level in chick hepatic mitochondria. However, glutathione-s-transferase activity was depressed. There was no alteration in the activity of glutathione reductase. Activity of glucose-6-phosphate dehydrogenase was not altered under lanthanum and neodymium treatment. There was a significant enhancement of intramitochondrial glutathione peroxidase and superoxide dismutase. Lipid peroxidation remains the same as control group of animals.     

Influence of Methionine on Toxicity of Fluoride in the Liver of Rats

Oxidative stress is a common mechanism by which chemical toxicity can occur in the liver. The aim of the studies conducted has been to determine what influence the administration of methionine during intoxication with sodium fluoride may have upon the selected enzymes of the antioxidative system in rat liver. The experiment was carried out on Wistar FL rats (adult females) that, for 35 days, were administered distilled water, NaF, or NaF with methionine (doses: 10 mg NaF/kg bw/day, 10 mg Met/kg bw/day). The influence of administered NaF and Met was examined by analyzing the activity of the antioxidative enzymes: superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, and glutathione transferase in the liver. The results suggest that fluoride reduces the efficiency of the enzymatic antioxidative system in the liver. Administration of methionine during intoxication with sodium fluoride does not have an advantageous influence upon the activity of superoxide dismutase, catalase, reductase, and glutathione transferase in the liver. The slight increase of the activity of glutathione peroxidase after administration of methionine may indicate its protective influence upon that enzyme.     

Responses of antioxidant systems to lanthanum nitrate treatments in tomato plants during drought stress

Abstract

Lanthanum is one of the most abundant elements in rare earths enriched fertilizers and is supposed to be one of the main responsible of the effects of such fertilizers on crops. In this work, the effect of lanthanum nitrate on H₂O₂ production, lipid peroxidation, ascorbate and glutathione content, and on the activity of cytosolic ascorbate peroxidase, dehydroascorbate reductase, monodehydroascorbate reductase, and glutathione reductase in Lycopersicon esculentum L. cv. Marmande during drought stress was evaluated. The results confirmed that treatments of tomato plants with lanthanum nitrate affect the antioxidant cellular defences and that lanthanum toxicity is dependent on the way of treatment. The stimulation of antioxidant systems did not induce any improvement in drought stress responses in tomato but seemed to be only a consequence of the unbalance in cell metabolism due to the treatment with lanthanum nitrate.     

Gamma-glutamyltransferase of rat kidney. Simultaneous assay of the hydrolysis and transfer reactions with (glutamate-14C)glutathione.

1. The hydrolytic and transfer reactions catalysed by rat kidney-gamma-glutamyltransferase (EC 2.3.2.2) were studied in vitro with substrates [U-14C]glutamic acid-labelled glutathione and methionine. Initial-velocity patterns, isotope-exchange and binding studies were consistent with a branched non-sequential mechanism in which a gamma-glutamyl-enzyme intermediate may react either with water (hydrolysis) or with methionine (gamma-glutamyl transfer). 2. The Michaelis constant for glutathione in hydrolysis was 13.9 +/- 1.4 mum, for glutathione in transfer it was 113 +/- 15 muM and for methionine as substrate it was 4.7 +/- 0.7 mM. At substrate concentrations in the ranges of their respective Michaelis constants, the rate of transfer was about ten times higher than that of hydrolysis, but at concentrations of methionine approximating to the physiological (64 muM in rat plasma) the transfer is negligible. 3. The enzyme is reported to lie on the luminal surface of the proximal straight kidney tubule. In this situation, if the kinetic results obtained with the detergent-solubilized enzyme are relevant to the behavior of the enzyme in vivo, it appears likely that the main function of renal gamma-glutamyltransferase is not in amino acid transport, but rather to hydrolyse glutathione in the renal filtrate.     

Involvement of the cystathionine pathway in the biosynthesis of glutathione by isolated rat hepatocytes

The ability of l-methionine to support glutathione biosynthesis has been investigated in isolated rat hepatocytes under conditions of normal and depleted glutathione status. The addition of l-[³⁵S]methionine or [l-[³⁵S]homocysteine to incubation media containing hepatocytes results in the incorporation of ³⁵S into intracellular glutathione. Additionally both l-methionine and l-homocysteine are capable of supporting the resynthesis of glutathione in isolated hepatocytes after prior depletion with diethyl maleate. The inclusion in the incubation medium of 1 mm propargylglycine, which is an irreversible inhibitor of the terminal enzyme of the cystathionine pathway, substantially blocks the incorporation of ³⁵S from methionine and l-homocysteine into cellular glutathione. Propargylglycine treatment of hepatocytes in the presence of [³⁵S]methionine is shown to result in the intracellular accumulation of [³⁵S]cystathionine. These results strongly support the conclusion that in rat hepatocytes the cystathionine pathway enables methionine to provide a significant source of l-cysteine for the support of glutathione biosynthesis, under both normal and glutathione-depleted conditions.     

Decrease in glutathione levels of kidney and liver after injection of methionine sulfoximine into rats

After rats were injected with the convulsant methionine sulfoximine, there was a rapid decrease in the glutathione concentrations of the kidney and liver, but there was no measurable effect (within 5 hours) on brain glutathione. The maximum decreases in the glutathione concentrations of kidney and liver were observed 1 hr after injection and were about 60 and 40%, respectively, of the control levels. The findings suggest that there may be at least two pools of tissue glutathione. Studies in which other amino acids were injected, and earlier $\text{in vitro}$ studies, are consistent with the conclusion that methionine sulfoximine affects glutathione synthesis $\text{in vivo}$ by inhibiting γ-glutamylcysteine synthetase. Injection of glycylglycine also decreased glutathione levels, an effect probably mediated by γ-glutamyltranspeptidase.     

Postacute Effects of Kisspeptin‐10 on Neuronal Injury Induced by l‐Methionine in Rats

Apart from its effect on the regulation of reproductive function, recent studies indicate that kisspeptin may play roles in the antioxidant defense system. The antioxidant defense system and oxidative stress contribute to the etiology and pathogenesis of neuronal cell death after brain injury. We have investigated the postacute effect of kisspeptin‐10 on brain injury induced by l ‐methionine. DNA fragmentation, malondialdehyde (MDA), reduced glutathione levels, and superoxide dismutase (SOD) activities were analyzed. Our results showed that methionine treatment increases apoptotic cell death. Kisspeptin alone showed no side effect on apoptotic cell death. However, kisspeptin treatment reversed the proapoptotic effect of methionine associated with reduced MDA and increased glutathione levels. Furthermore, SOD activity was completely depleted in methionine‐treated animals. In conclusion, our results revealed that delayed kisspeptin‐10 treatment reduces neuronal cell death by activation of SOD activity.     

IN VIVO INHIBITION OF γ-GLUTAMYLCYSTEINE SYNTHETASE BY l-METHIONINE-RS-SULFOXIMINE; INFLUENCE ON INTERMEDIATES OF THE γ-GLUTAMYL CYCLE

—The inhibition of γ-glutamylcysteine synthetase and its influence on the concentration of intermediates associated with the metabolism of glutathione was studied in mice receiving methionine sulfoximine, a convulsant agent. The activity of the enzyme decreased significantly in the liver and kidney 1-4 h after administration of methionine sulfoximine; the activity of the enzyme in the brain was unchanged after 1 and 2 h but decreased significantly after 4 h. There was a rapid and sharp decrease in the concentration of glutathione in the kidney and a slower decrease in the liver. Brain glutathione concentrations were unaffected. Methionine sulfoximine in vivo, inhibited the synthesis of l -γ-glutamyl-l -α-aminobutyrate after administration of l -α-aminobutyrate, a reaction catalyzed by γ-glutamylcysteine synthetase. The inhibitor also lowered the concentration of pyrrolidone carboxylate in mouse tissues and prevented the accumulation of this intermediate after administration of l -α-aminobutyrate. The results show that methionine sulfoximine in vivo affects the metabolism of glutathione and that this action may contribute to its convulsive properties.     

Interplay between cellular methyl metabolism and adaptive efflux during oncogenic transformation from chronic arsenic exposure in human cells

After protracted low level arsenic exposure, the normal human prostate epithelial cell line RWPE-1 acquires a malignant phenotype with DNA hypomethylation, indicative of disrupted methyl metabolism, and shows arsenic adaptation involving glutathione overproduction and enhanced arsenic efflux. Thus, the interplay between methyl and glutathione metabolism during this progressive arsenic adaptation was studied. Arsenic-treated cells showed a time-dependent increase in LC50 and a marked increase in homocysteine (Hcy) levels. A marked suppression of S-adenosylmethionine (SAM) levels occurred with decreased methionine adenosyltransferase 2A (converts methionine to SAM) expression and increased negative regulator methionine adenosyltransferase B, suggesting reduced conversion of Hcy to SAM. Consistent with Hcy overproduction, activity and expression of S-adenosylhomocysteine hydrolase (converts S-adenosylhomocysteine to Hcy) were both increased. Expression of cystathionine beta-synthase, a key gene in the transsulfuration pathway, and various glutathione production genes were increased, resulting in a 5-fold increase in glutathione. Arsenic efflux increased along with expression of ATP-binding cassette protein C1, which effluxes arsenic as a glutathione conjugate. Evidence of genomic DNA hypomethylation was observed during early arsenic exposure, indicating that the disruption in methyl metabolism had a potential impact related to oncogenesis. Thus, cellular arsenic adaptation is a dynamic, progressive process that involves decreased SAM recycling and concurrent accumulation of Hcy, which is channeled via transsulfuration to increase glutathione and enhance arsenic efflux but may also impact the carcinogenic process.     

High level expression and purification of peptide methionine sulfoxide reductase in Escherichia coli.

The enzyme peptide methionine sulfoxide reductase catalyzes the conversion of methionine sulfoxide residues in proteins to methionine. The 636 nucleotide coding region of the peptide methionine sulfoxide reductase gene has been amplified from a genomic clone using the polymerase chain reaction and the product was subcloned into plasmid pGEX-2T downstream of the glutathione S-transferase gene under control of the tac promoter. Escherichia coli XL1-Blue cells transformed with this plasmid and induced with isopropylthio-beta-galactoside expressed high levels of the fusion protein. The protein was soluble and was purified to homogeneity by affinity binding to a glutathione-agarose resin followed by cleavage of the fusion protein with thrombin. Both the fusion protein and the purified peptide methionine sulfoxide reductase protein showed high peptide methionine sulfoxide reductase activity.     

The effects of dietary supplementation of methionine on genomic stability and p53 gene promoter methylation in rats

Methionine is a component of one-carbon metabolism and a precursor of S-adenosylmethionine (SAM), the methyl donor for DNA methylation. When methionine intake is high, an increase of S-adenosylmethionine (SAM) is expected. DNA methyltransferases convert SAM to S-adenosylhomocysteine (SAH). A high intracellular SAH concentration could inhibit the activity of DNA methyltransferases. Therefore, high methionine ingestion could induce DNA damage and change the methylation pattern of tumor suppressor genes. This study investigated the genotoxicity of a methionine-supplemented diet. It also investigated the diet's effects on glutathione levels, SAM and SAH concentrations and the gene methylation pattern of p53. Wistar rats received either a methionine-supplemented diet (2% methionine) or a control diet (0.3% methionine) for six weeks. The methionine-supplemented diet was neither genotoxic nor antigenotoxic to kidney cells, as assessed by the comet assay. However, the methionine-supplemented diet restored the renal glutathione depletion induced by doxorubicin. This fact may be explained by the transsulfuration pathway, which converts methionine to glutathione in the kidney. Methionine supplementation increased the renal concentration of SAH without changing the SAM/SAH ratio. This unchanged profile was also observed for DNA methylation at the promoter region of the p53 gene. Further studies are necessary to elucidate this diet's effects on genomic stability and DNA methylation.     

The mouse liver content of carbonic anhydrase III and glutathione S-tranferases A3 and P1 depend on dietary supply of methionine and cysteine

The contents of glutathione S-transferase (GST) subunits, carbonic anhydrase III (CAIII), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and a 230 kDa protein are affected by protein deprivation in mouse liver. In order to know if particular amino acids control these contents, the effects of feeding for 5 days with diets containing different amino acids were examined. After an exploration using SDS-PAGE analysis, the action of selected diets was further examined by distinct techniques. The 230 kDa protein was identified as fatty acid synthase (FAS) by both mass spectrometry and amino acid sequence analyses. Dietary tests showed that: (1) a protein-free diet (PFD) increased the content of glutathione S-transferases P1 and M1, and glyceraldehyde-3-phosphate dehydrogenase, while the content of glutathione S-transferase A3, fatty acid synthase and carbonic anhydrase III decreased; (2) a protein-free diet having either methionine or cysteine preserved the normal contents of glutathione S-transferases P1, A3, M1 and carbonic anydrase III; (3) a protein-free diet having threonine preserved partially the normal contents of glutathione S-transferases P1, A3, M1 and carbonic anhydrase III; (4) a protein-free diet having methionine, threonine and cysteine prevented in part the loss of fatty acid synthase; and (5) the glyceraldehyde-3-phosphate dehydrogenase content was controlled by increased carbohydrate level and/or by lower amino acid content of diets, but not by any specific amino acid. These data indicate that methionine and cysteine exert a main role on the control of liver glutathione S-transferases A3 and P1, and carbonic anhydrase III. Thus, they emerge necessary to prevent unsafe alterations of liver metabolism caused by protein deprivation.     

Zinc toxicity in various lung cell lines is mediated by glutathione and GSSG reductase activity

In a previous work, it was shown that in cells after a decrease of cellular glutathione content, toxic zinc effects, such as protein synthesis inhibition or GSSG (glutathione, oxidized form) increases, were enhanced. In this study, zinc toxicity was determined by detection of methionine incorporation as a parameter of protein synthesis and GSSG increase in various lung cell lines (A549, L2, 11Lu, 16Lu), dependent on enhanced GSSG reductase activities and changed glutathione contents. After pretreatment of cells with dl-buthionine-[R,S]-sulfoximine (BSO) for 72 h, cellular glutathione contents were decreased to 15–40% and GSSG reductase activity was increased to 120–135% in a concentration-dependent manner. In BSO pretreated cells, the IC₅₀ values of zinc for methionine incorporation inhibition were unchanged as compared to cells not pretreated. The GSSG increase in BSO pretreated cells by zinc was enhanced in L2, 11Lu, and 16Lu cells, whereas in A549 cells, the GSSG increase by zinc was enhanced only after pretreatment with the highest BSO concentration. Inhibition of GSSG reductase in alveolar epithelial cells was observed at lower zinc concentrations than needed for methionine incorporation inhibition, whereas in fibroblastlike cells, inhibition of GSSG reductase occurred at markedly higher zinc concentrations as compared to methionine incorporation inhibition. These results demonstrate that GSSG reductase is an important factor in cellular zinc susceptibility. We conclude that reduction of GSSG is reduced in zinc-exposed cells. Therefore, protection of GSH oxidation by various antioxidants as well as enhancement of GSH content are expected to be mechanisms of diminishing toxic cellular effects after exposure to zinc.     

Biosynthesis of thiazole from thiamine in Escherichia coli]

The incorporation into the thiazole moiety of thiamine of several labeled compounds has been studied on short time incubations of washed-cells suspensions. No incorporation of radioactivity from [G-14C] methionine was found in a mutant auxotrophic for methionine. No radioactivity was incorporated from [U-14C] aspartate or from [U-14C] serine. The incorporation of 35S from sulphate was lowered by cysteine or glutathione but was unaffected by methionine or homocystine. Although the synthesis of thiazole is dependent on methionine, neither the sulphur atom nor the carbon chain of thiazole originate from methonine in E. coli. No carbon originates from cysteine which is the likely direct donor of sulphur.     

Measurement of sulfur-containing compounds involved in the metabolism and transport of cysteamine and cystamine. Regional differences in cerebral metabolism

An HPLC method with coulometric detection is presented for the quantitation of cysteamine, cystamine, thialysine, glutathione, glutathione disulfide and an oxidized metabolite of thialysine [S-(2-aminoethyl)-l-cysteine ketimine decarboxylated dimer (AECK-DD)]. The advantage of coulometric detection is that derivatization is unnecessary if the analyte is redox sensitive. The method was used to quantitate several sulfur-containing compounds in plasma and brain following gavage feeding of cysteamine to rats. Cysteamine, cystamine, thialysine and AECK-DD were detected in the brains of these animals. Interestingly, cysteamine treatment resulted in greatly elevated levels of cerebral methionine, despite the fact that cysteamine is not a precursor of methionine.     

高蛋氨酸饲料喂养的大鼠高同型半胱氨酸血症模型的实验研究

探讨高蛋氨酸饲料诱导高同型半胱氨酸血症的适应剂量及其对相关代谢的影响.选择质量分数1%、2%和3%蛋氨酸饲料喂饲大鼠,高压液相色谱法测定血清同型半胱氨酸、半胱氨酸和还原形谷胱甘肽含量.结果表明饲料中1%蛋氨酸可以使血清同型半胱氨酸水平升高且无生长抑制等毒副作用,2%和3%蛋氨酸饲料喂养大鼠后出现摄食量减少和生长抑制等毒...     

Metabolism of extracellular glutathione in rat small-intestinal mucosa

Metabolism of exogenous glutathione was investigated in suspensions of freshly isolated rat small-intestinal mucosal cells. The cells catalyzed the oxidation of reduced glutathione (GSH) to glutathione disulfide (GSSG). Neither serine . borate nor methionine significantly influenced this reaction. Formed GSSG was further metabolized as indicated by its disappearance from the medium. Degradation of GSSG was stimulated by methionine and inhibited by serine . borate. Separation and identification of GSSG metabolites were achieved by high performance liquid chromatography. The results indicate that the preferred route for GSSG metabolism to the constituent amino acids in small intestine, is by hydrolytic removal of the two gamma-glutamyl groups of GSSG to yield cystinyl-bisglycine which is subsequently hydrolyzed to cystine. gamma-Glutamyltransferase activity was compared in isolated intestinal, kidney and liver cells using gamma-glutamyl-p-nitrocarboxyanilide as substrate. Kidney cells were approximately 5-fold and 150-fold more active than intestinal and liver cells, respectively. Serine . borate markedly inhibited, and glycyl-glycine stimulated, hydrolysis of gamma-glutamyl-p-nitrocarboxyanilide in all cell types confirming the involvement of gamma-glutamyltransferase in the reaction. The hydrolysis of gamma-glutamyl-p-nitrocarboxyanilide was inhibited to approximately the same extent by either GSH or GSSG suggesting that both compounds interact at the donor site of gamma-glutamyltransferase. Comparison of the rates of glutathione metabolism by isolated intestinal and kidney cells suggests that the intestinal contribution to the degradation of extracellular glutathione may be physiologically more important than has previously been assumed.     

A study of the sulphur amino acids of rat tissues

1. In a study of the metabolism of l-[(35)S]methionine in vivo, the labelled sulphur compounds of rat liver and brain were separated first by ion-exchange chromatography into two fractions containing (i) free sulphur amino acids such as methionine, cystathionine, cyst(e)ine and homocyst(e)ine and (ii) glutathione. 2. Two-dimensional paper chromatography with butan-1-ol-acetic acid or propionic acid-water in the first direction and 80% acetone or acetone-ethyl methyl ketone-water in the second direction was found superior to other solvent systems for separating the sulphur amino acids. 3. At 10min. after injection of [(35)S]methionine only a small part of the (35)S was found combined in free methionine or other free sulphur amino acids. 4. Evidence was obtained of the presence of adenosyl[(35)S]methionine and adenosyl[(35)S]homocysteine in perchloric acid extracts of rat liver and brain. 5. The trans-sulphuration pathway was active in brain as well as in liver.     

Turnover and functions of glutathione studied with isolated hepatic and renal cells

Suspensions of freshly isolated rat hepatocytes and renal tubular cells contain high levels of reduced glutathione (GSH), which exhibits half-lives of 3-5 and 0.7-1 h, respectively. In both cells types the availability of intracellular cysteine is rate limiting for GSH biosynthesis. In hepatocytes, methionine is actively converted to cysteine via the cystathionine pathway, and hepatic glutathione biosynthesis is stimulated by the presence of methionine in the medium. In contrast, extracellular cystine can support renal glutathione synthesis; several disulfides, including cystine, are rapidly taken up by renal cells (but not by hepatocytes) and are reduced to the corresponding thiols via a GSH-linked reaction sequence catalyzed by thiol transferase and glutathione reductase (NAD(P)H). During incubation, hepatocytes release both GSH and glutathione disulfide (GSSG) into the medium; the rate of GSSG efflux is markedly enhanced during hydroperoxide metabolism by glutathione peroxidase. This may lead to GSH depletion and cell injury; the latter seems to be initiated by a perturbation of cellular calcium homeostasis occurring in the glutathione-depleted state. In contrast to hepatocytes, renal cells metabolize extracellular glutathione and glutathione S-conjugates formed during drug biotransformation to the component amino acids and N-acetyl-cysteine S-conjugates, respectively. In addition, renal cells contain a thiol oxidase acting on extracellular GSH and several other thiols. In conclusion, our findings with isolated cells mimic the physiological situation characterized by hepatic synthesis and renal degradation of plasma glutathione and glutathione S-conjugates, and elucidate some of the underlying biochemical mechanisms.     

Effects of free and dipeptide forms of methionine supplementation on oxidative metabolism of broilers under high temperature

Our previous studies have shown that methionine supplementation could help to attenuate the effects of heat stress on the metabolism of broiler chickens. Here we investigated for the first time the effects of methionine supplementation in the form of DL-methionyl-DL-methionine on broilers subjected to heat stress during the growth phase. Broilers were divided into two groups; one group was reared under thermoneutral conditions and the other under continuous heat stress (30 ± 1 °C, 60% relative humidity). Both groups were subdivided into three dietary treatments: a methionine-deficient (MD) diet, a diet supplemented with free methionine (DL-M), and a diet supplemented with methionine dipeptide (DL-MM). Broilers raised under chronic heat stress had lower feed intake and weight gain than broilers raised under thermoneutral conditions (P < 0.05). There were no differences in animal performance between methionine-supplemented diets (DL-M and DL-MM). Heat-stressed birds had significantly higher heterophil/lymphocyte (H/L) ratio than thermoneutral birds. Under heat stress, broilers fed DL-M and DL-MM diets had lower H/L ratio than birds fed the MD diet. Higher concentrations of carbonylated proteins and lower concentration of reduced glutathione were observed in broilers raised under heat stress. In comparing heat-stressed broilers, we found that birds fed the DL-M diet had lower concentrations of thiobarbituric acid-reactive substances and carbonylated proteins than those fed the MD diet (P < 0.05). Higher expression of glutathione peroxidase (GPX) and glutathione synthetase (GSS) genes was observed in heat-stressed broilers (P < 0.05). Under heat stress, the MD diet increased GPX expression compared with other diets. Under thermoneutral conditions, the DL-M diet resulted in the highest GSS expression. There was a negative correlation between DNA methylation and GPX and GSS expression. Our results showed that supplementation of broiler diets with free methionine or methionine dipeptide may help attenuate the effects of heat stress through enhanced activation of genes related to the glutathione antioxidant system. Methionine effects were found for gene regulation, gene expression, and post-translational processing.