Inhibition of glutathione efflux from isolated rat hepatocytes by methionine

A substantial inhibition (50-70%) of GSH efflux by methionine was demonstrated in hepatocytes isolated from fed rats. Concurrent measurements of intracellular GSH revealed maintenance of a higher concentration in methionine-supplemented cells over the 1-h incubation. Analysis of total GSH suggested that maintenance of higher intracellular GSH by methionine could be quantitatively accounted for by inhibition of GSH efflux rather than by net GSH synthesis. This conclusion was supported by studies with propargylglycine, a potent inhibitor of cysteine synthesis from methionine. Identical results were obtained in incubations containing either propargylglycine and methionine or methionine alone, thereby suggesting that net synthesis of GSH from methionine was minimal under the assay conditions. Similar decreases (40-60%) in the rate of extracellular accumulation of GSH were observed with ethionine and buthionine, two higher homologs of methionine, but not with a wide range of other naturally occurring and synthetic amino acids. The inhibition of GSH efflux by methionine was not dependent on the presence of sodium in the medium and did not correlate with metabolic consumption of ATP.     

Metabolic activation and hepatotoxicity. Effect of cysteine, N-acetylcysteine, and methionine on glutathione biosynthesis and bromobenzene toxicity in isolated rat hepatocytes.

Freshly isolated rat hepatocytes contained a high level (30–40 nmol/10⁶ cells) of reduced glutathione (GSH) which decreased steadily upon incubation in an amino acid containing medium lacking cysteine and methionine. This decrease in GSH level was prevented, and turned into a slight increase, when either cysteine, N-acetylcysteine, or methionine was also present in the medium. The amino acid uptake into hepatocytes was more rapid with cysteine than with methionine. Cystine was not taken up, or taken up very slowly, by the cells and could not be used to prevent the decrease in GSH level which occurred in the absence of cysteine and methionine. The level of GSH in hepatocytes freshly isolated from rats pretreated with diethylmaleate was markedly decreased (to ~5 nmol/10⁶ cells) but increased rapidly upon incubation of the cells in a medium containing amino acids including either cysteine, N-acetylcysteine, or methionine. Again, cysteine was taken up into the cells more rapidly than methionine. The rate of uptake of cysteine was moderately enhanced in hepatocytes with a lowered level of intracellular GSH as compared to cells with normal GSH concentration. Exclusion of glutamate and/or glycine from the medium did not markedly affect the rate of resynthesis of GSH by hepatocytes incubated in the presence of exogenously added cysteine or methionine. Incubation of hepatocytes with bromobenzene in an amino acid-containing medium lacking cysteine and methionine resulted in accelerated cell damage. Addition of either cysteine, N-acetylcysteine, or methionine to the medium caused a decrease in bromobenzene toxicity. The protective effect was dependent, however, on the time of addition of the amino acid to the incubate; e.g., the effect on bromobenzene toxicity was greatly reduced when either cysteine or methionine was added after 1 h of preincubation of the hepatocytes with bromobenzene as compared to addition at zero time. This decrease in protective effect in bromobenzene-exposed cells was related to a similar decrease in the rate of uptake of cysteine and methionine into hepatocytes preincubated with bromobenzene. The rate of uptake, and incorporation into cellular protein, of leucine was also markedly inhibited in hepatocytes preincubated with bromobenzene. In contrast, there was no measurable change in the rate of release of leucine from cellular protein as a result of incubation of hepatocytes with bromobenzene. It is concluded that the presence of cysteine, N-acetylcysteine, or methionine in the medium protects hepatocytes from bromobenzene toxicity by providing intracellular cysteine for GSH biosynthesis and suggested that an inhibitory effect on amino acid uptake may contribute to the cytotoxicity of bromobenzene in hepatocytes.     

Hepatic glutathione concentrations linked to ethionine toxicity in rats

1. The effect of injected ethionine on liver GSH concentrations was studied in male and female rats. 2. Liver GSH concentrations were markedly and consistently decreased in 5hr. and elevated within 24-44hr. By 72hr. the amount of liver GSH of ethionine-poisoned rats had the same values as saline-injected control rats. At no time was erythrocyte GSH concentration affected by ethionine. 3. The concentrations of non-protein thiol compounds, glycogen and ATP were also significantly decreased when the rats were killed 5hr. after ethionine injection. 4. ATP, adenine or methionine did not prevent the decrease of liver GSH produced by ethionine. From these and other observations we conclude that ethionine is not an antagonist of methionine with respect to GSH metabolism. 5. The metabolic relationship between ethionine toxicity and liver GSH concentration is briefly discussed.     

Upregulation of Glyoxalase I fails to Normalize Methylglyoxal Levels: A Possible Mechanism for Biochemical Changes in Diabetic Mouse Lenses

Glyoxalase I is the first enzyme in a two-enzyme glyoxalase system that metabolizes physiological methylglyoxal (MGO). MGO reacts with proteins to form irreversible adducts that may lead to crosslinking and aggregation of lens proteins in diabetes. This study examined the effect of hyperglycemia on glyoxalase I activity and its mRNA content in mouse lens epithelial cells (mLE cells) and in diabetic mouse lenses and investigated the relationship between GSH and MGO in organ cultured lenses. mLE cells cultured with 25 mM D-glucose (high glucose) showed an upregulation of glyoxalase I activity and a higher content of glyoxalase I mRNA when compared with either cells cultured with 5 mM glucose (control) or with 20 mM L-glucose + 5 mM D-glucose. MGO concentration was significantly elevated in cells cultured with high D-glucose, but not in L-glucose. GSH levels were lower in cells incubated with high glucose compared to control cells. Glyoxalase I activity and mRNA levels were elevated in diabetic lenses compared to non-diabetic control mouse lenses. MGO levels in diabetic lenses were higher than in control lenses. Incubation of lenses with buthionine sulfoximine (BSO) resulted in a dramatic decline in GSH but the MGO levels were similar to lenses incubated without BSO. Our data suggest that in mouse lenses MGO accumulation may occur independent of GSH concentration and in diabetes there is an upregulation of glyoxalase I, but this upregulation is inadequate to normalize MGO levels, which could lead to MGO retention and chemical modification of proteins.     

Maintenance of glutathione content is isolated hepatocyctes.

1. During the standard procedure for the preparation of rat hepatocytes, about half of the cellular GSH (reduced glutathione) is lost. 2. This loss is prevented by the addition of 0.1 mM-EGTA (but no EDTA) to the perfusion medium. 3. On incubation with and without EGTA, isolated hepatocytes prepared in the presence of EGTA lose GSH. This loss is prevented by near-physiological concentrations of methionine or homocysteine, but not of cysteine. 4. Cysteine, at concentrations above 0.2 mM, causes a loss of GSH probably by non-enzymic formation of a mixed disulphide. 5. Serine together with methionine or homocystein increases GSH above the value in cells from starved rats in vivo. This is taken to suggest that cystathionine may be a cysteine donor in the synthesis of gamma-glutamylcysteine, the precursor of GSH.     

晶状体生化的研究:正常和亚硒酸钠诱发白内障大鼠晶状体中与谷胱甘肽代谢有关的三种酶活性的测定

测定了用亚硒酸钠诱发的大鼠白内障晶状体中谷胱甘肽过氧化物酶(GSH-Px)、谷胱甘肽还原酶(GSSG-R)和谷胱甘肽硫转移酶(GSH-S)的活性,并与正常晶休中这三种酶的活性作了比较。结果表明,核浊浑期晶状体中GSH-Px的活性比正常晶状体的高一倍,但在整个晶状体浑浊时降低,GSSG-R的活性变化与GSH-PX相似,这两种酶在代谢上是相关的。GSH-S的活性在核浑浊期不改变,但在完全浑浊后降低。     

Specific Contribution of Methionine and Choline in Nutritional Nonalcoholic Steatohepatitis: IMPACT ON MITOCHONDRIAL S-ADENOSYL-l-METHIONINE AND GLUTATHIONE*

The pathogenesis and treatment of nonalcoholic steatohepatitis (NASH) are not well established. Feeding a diet deficient in both methionine and choline (MCD) is one of the most common models of NASH, which is characterized by steatosis, mitochondrial dysfunction, hepatocellular injury, oxidative stress, inflammation, and fibrosis. However, the individual contribution of the lack of methionine and choline in liver steatosis, advanced pathology and impact on mitochondrial S-adenosyl-l-methionine (SAM) and glutathione (GSH), known regulators of disease progression, has not been specifically addressed. Here, we examined the regulation of mitochondrial SAM and GSH and signs of disease in mice fed a MCD, methionine-deficient (MD), or choline-deficient (CD) diet. The MD diet reproduced most of the deleterious effects of MCD feeding, including weight loss, hepatocellular injury, oxidative stress, inflammation, and fibrosis, whereas CD feeding was mainly responsible for steatosis, characterized by triglycerides and free fatty acids accumulation. These findings were preceded by MCD- or MD-mediated SAM and GSH depletion in mitochondria due to decreased mitochondrial membrane fluidity associated with a lower phosphatidylcholine/phosphatidylethanolamine ratio. MCD and MD but not CD feeding resulted in increased ceramide levels by acid sphingomyelinase. Moreover, GSH ethyl ester or SAM therapy restored mitochondrial GSH and ameliorated hepatocellular injury in mice fed a MCD or MD diet. Thus, the depletion of SAM and GSH in mitochondria is an early event in the MCD model of NASH, which is determined by the lack of methionine. Moreover, therapy using permeable GSH prodrugs may be of relevance in NASH.     

Glutathione content during the rinsing and rewarming process of rat hepatocytes preserved in University of Wisconsin solution

The addition of glutathione (GSH) to University of Wisconsin (UW) solution increases the intracellular content of GSH and decreases the release of lactate dehydrogenase used here as a measure of cell viability. However, we found a depletion of GSH when the cells were transferred from UW solution to the rewarming solution. This could sensitize the cells to various forms of oxidative injury. In this study we examined how different compositions of rinsing and rewarming solutions affected the GSH content and the viability of hepatocytes after 72 h of cold storage. For both the rinsing and the rewarming steps we used a Krebs-Henseleit solution with the addition of GSH, methionine, or both GSH and methionine. We found no loss of GSH when the hepatocytes were rinsed in the presence of 3 mM GSH. During the rewarming step we observed a loss of GSH in all of the study groups, but the cells that were incubated with 1 mM methionine showed a lesser depletion of GSH and improved viability. This finding may have valuable applications in hepatocellular transplantation and in the development of bioartificial liver support devices.     

Lens methionine sulfoxide reductase

Investigation of human and bovine lenses has demonstrated the presence of a methionine sulfoxide (Met(O)) peptide reductase activity. The reductase can use either dithiothreitol or thioredoxin but not glutathione as a reducing agent. The enzyme is present primarily in the water soluble fraction. The highest specific activity is in the outer epithelial layer with decreasing activity in the inner layers of the tissue. The known high level of methionine sulfoxide residues in cataractous lens protein is not due to a decreased level of Met (O)-peptide reductase itself since a comparison of normal and cataractous human lenses showed no statistically significant decrease in reductase activity in the cataract population. However, it is not known whether the reducing system for Met (O)-peptide reductase (probably the thioredoxin system) is deficient in cataractous lenses.     

Methionine-mediated lethality in yeast cells at elevated temperature.

Saccharomyces cerevisiae cells grown at 30 degrees C in minimal medium containing methionine lose viability upon transfer to 45 degrees C, whereas cells grown in the absence of methionine survive. Cellular levels of two intermediates in the sulfate assimilation pathway, adenosine 5'-phosphosulfate (APS) and adenosine 5'-phosphosulfate 3'-phosphate, are increased by a posttranslational mechanism after sudden elevation of temperature in yeast cultures grown in the absence of methionine. Yeast cells unable to synthesize APS because of repression by methionine or mutation of the MET3 gene do not survive the temperature shift. Thus, methionine-mediated lethality at elevated temperature is linked to the inability to synthesize APS. The results demonstrate that APS plays an important role in thermotolerance.     

Unique characteristics of rat spleen lymphocyte, L1210 lymphoma and HeLa cells in glutathione biosynthesis from sulfur-containing amino acids

Suspensions of rat spleen lymphocyte, murine L1210 lymphoma and HeLa cells were partially depleted of glutathione (GSH) with diethyl maleate and allowed to utilize either [35S]methionine, [35S]cystine or [35S]-cysteine for GSH synthesis. Lymphocytes preferentially utilized cysteine, compared to cystine, at a ratio of about 30 to 1, which was not related to differences in the extent of amino acid uptake. Only HeLa cells displayed a slight utilization of methionine via the cystathionine pathway for cysteine and GSH biosynthesis. HeLa and L1210 cells readily utilized either cystine or cysteine for GSH synthesis. The three cell types accumulated detectable levels of intracellular cysteine glutathione mixed disulfide when incubated in a medium containing a high concentration of cystine. Various enzyme activities were measured including gamma-glutamyl transpeptidase, GSH S-transferase and gamma-cystathionase. These results support the concept of a dynamic interorgan relationship of GSH to plasma cyst(e)ine that may have importance for growth of various cell types in vivo.     

The effects of dietary methionine and glycine on lead toxicity in choline-deficient chicks

A 2×2×2 factorial experiment was conducted to study the effects of dietary methionine, glycine, and lead (Pb) in cholinedeficient chicks. The variables were: adequate or deficient methionine; adequate or excess glycine; and 0 or 1000 ppm lead (as Pb acetate 3H₂O). Methionine stimulated growth when added to the methionine-deficient diets, but the response was greater when supplemental glycine was present. Addition of glycine to the glycine-adequate diets stimulated growth in the presence of adequate but not deficient methionine. The patterns of response to methionine were the same at both 0 and 1000 ppm dietary Pb. Added Pb depressed growth with all diets, but the depression was greater in methionine-deficient than in methionine-adequate diets. Hepatic nonprotein sulfhydryl (NPSH) and glutathione (GSH) concentrations were increased by both supplemental methionine and lead, and the effects were additive. Glycine levels did not significantly alter NPSH and GSH concentrations. Both methionine and glycine lowered Pb concentrations in kidney, and the effects were additive. The results are consistent with previous observations that added methionine ameliorates Pb-induced growth depression with choline-adequate diets, however, this effect is not as pronounced with choline-deficient diets. The results suggest (1) that glycine is limiting for growth in choline-deficient, methionine-adequate diets, and (2) that methionine and glycine may enhance Pb detoxification by different mechanisms.

Paper No. 10213 of the Journal Series of the NC Agricultural Research Service, Raleigh, NC 27695-7601. The use of trade names implies neither endorsement of the product named nor criticism of similar products not mentioned by the NCARS.     

The effect of Glurenorm (gliquidone) on lenses and skin in experimental diabetes.

The aim of this study was to investigate the effect of administering Glurenorm (gliquidone, 10 mg/kg) on the lenses and skins of streptozotocin-induced diabetic rats. The drug was given to both diabetic and control rats daily, until the end of the experiment, at day 42. The drug was administered to one diabetic and one control group from day 0 and for the other diabetic and control groups from day 14. On day 42, cardiac blood samples, skin samples, and lenses were taken from each rat. Blood glucose (BG) was measured by the o-toluidine method. The total protein, nonenzymatic glycosylation of proteins (NEG), lipid peroxidation (LPO), and glutathione (GSH) levels in the lens and skin homogenates were determined by the Lowry, thiobarbituric acid, Ledwozwy, and Ellman methods, respectively. Laemmli SDS polyacrylamide gel electrophoresis was also carried out on the lens or skin homogenates. After 42 d, Glurenorm given to the diabetic rats produced (i) significant reductions in BG, NEG, and total protein in the lenses; (ii) significant increases in GSH levels in the lenses; (iii) and no significant results in the skin. The body weights of the drug group dropped relative to day 0, but not significantly. SDS polyacrylamide gel electrophoresis revealed no significant differences in any of the protein bands between any of the groups. In the lenses, the gains in turns of reduced NEG and increased GSH may have been offset by the reduction in protein.     

Production of intracellular 35S-glutathione by rat and human hepatocytes for the quantification of xenobiotic reactive intermediates

The quantification and identification of xenobiotic reactive intermediates is difficult in the absence of highly radiolabeled drug. We have developed a method for identifying these intermediates by measuring the formation of adducts to intracellularly generated radiolabeled glutathione (GSH). Freshly isolated adherent rat and human hepatocytes were incubated overnight in methionine and cystine-free ('thio-free') medium. They were then exposed to 100 microM methionine and 10 microCi 35S-labeled methionine in otherwise thio-free medium to replete cellular GSH pools with intracellularly generated 35S-labeled GSH. After 3 h, acetaminophen was added as a test compound and the cells were incubated for an additional 24 h. Intracellular GSH and its specific activity were quantified after reaction with monobromobimane followed by HPLC analysis with fluorescence and radiochemical detection. Radiolabeled GSH was detectable at 3 h and maintained high specific activity and physiological concentrations for up to 24 h. Incubation medium from acetaminophen treated and nontreated hepatocytes were analyzed for radiolabeled peaks by HPLC using radiochemical detection. Radiolabeled peaks not present in nontreated hepatocytes were identified as acetaminophen GSH adducts by LC-MS. Formation of acetaminophen 35S-GSH adducts by rat hepatocytes containing endogenously synthesized 35S-GSH was increased with acetaminophen concentrations ranging from 500 to 2 mM.     

Lack of methionine biosynthesis de novo in Butyrivibrio fibrisolvens strain E14

Butyrivibrio fibrisolvens strain E14 has an absolute requirement for methionine. Metabolism of L-[ β-¹⁴C]-serine to methionine occurred in the methionine-independent B. fibrisolvens strain H17c but not in strain E14. The absolute requirement for methionine in strain E14 could be met by addition of S-adenosylmethionine to the medium, but incorporation was not due to the presence of free methionine in the S-adenosylmethionine preparation. The results show that B. fibrisolvens strain E14 is unable to synthesize methionine de novo , probably due to a lack of methionine synthase. Butyrivibrio fibrisolvens may also possess an alternative pathway of methionine biosynthesis from S-adenosylmethionine.     

Cysteine regulates expression of cysteine dioxygenase and gamma-glutamylcysteine synthetase in cultured rat hepatocytes

Rat hepatocytes cultured for 3 days in basal medium expressed low levels of cysteine dioxygenase (CDO) and high levels of gamma-glutamylcysteine synthetase (GCS). When the medium was supplemented with 2 mmol/l methionine or cysteine, CDO activity and CDO protein increased by >10-fold and CDO mRNA increased by 1.5- or 3.2-fold. In contrast, GCS activity decreased to 51 or 29% of basal, GCS heavy subunit (GCS-HS) protein decreased to 89 or 58% of basal, and GCS mRNA decreased to 79 or 37% of basal for methionine or cysteine supplementation, respectively. Supplementation with cysteine consistently yielded responses of greater magnitude than did supplementation with an equimolar amount of methionine. Addition of propargylglycine to inhibit cystathionine gamma-lyase activity and, hence, cysteine formation from methionine prevented the effects of methionine, but not those of cysteine, on CDO and GCS expression. Addition of buthionine sulfoximine to inhibit GCS, and thus block glutathione synthesis from cysteine, did not alter the ability of methionine or cysteine to increase CDO. GSH concentration was not correlated with changes in either CDO or GCS-HS expression. The effectiveness of cysteine was equivalent to or greater than that of its precursors (S-adenosylmethionine, cystathionine, homocysteine) or metabolites (taurine, sulfate). Taken together, these results suggest that cysteine itself is an important cellular signal for upregulation of CDO and downregulation of GCS.     

Peroxide-metabolizing systems of the crystalline lens

The ability of transparent and cataractous human, rabbit and mice lenses to metabolize hydrogen peroxide in the surrounding medium was evaluated. Using a chemiluminescence method in a system of luminol-horseradish peroxidase and a photometric technique, the temperature-dependent kinetics of H₂O₂ decomposition by lenses were measured. The ability of opaque human lenses to catalyze the decomposition of 10^?4 M H₂O₂ was significantly decreased. However, this was reserved by the addition of GSH to the incubation medium. Incubation of the mice lenses with the initial concentration H₂O₂ 10^?4 M led to partial depletion of GSH in normal and cataractous lenses. Human cataractous lenses showed decreased activities of glutathione reductase, glutathione peroxidase (catalyzing reduction of organic hydroperoxides including hydroperoxides of lipids), superoxide dismutase, but no signs of depletion in activities of catalase or glutathione peroxidase (utilizing H₂O₂). The findings indicated an impairment in peroxide metabolism of the mature cataractous lenses compared to normal lenses to be resulted from a deficiency of GSH. An oxidative stress induced by accumulation of lipid peroxidation products in the lens membranes during cataract progression could be considered as a primary cause of GSH deficiency and disturbance of the redox balance in the lens.     

Influence of Methionine Pool Composition on the Formation of Methyl-Deficient Transfer Ribonucleic Acid in Saccharomyces cerevisiae

Methionine auxotrophs of Saccharomyces cerevisiae continue to synthesize ribonucleic acid (RNA) after methionine withdrawal. The newly synthesized transfer RNA (tRNA) is methyl-deficient in some strains, but not in all. Whether such tRNA will accumulate depends on the position of the block in the methionine pathway that is carried by the mutant strain. Free methionine rapidly decreases in the intracellular pool of all strains after its removal from the medium. Certain metabolites derived from methionine are removed from the pool relatively slowly after methionine withdrawal. Notable among these is S-adenosylhomocysteine, which is depleted less rapidly from those strains that accumulate methyl-deficient tRNA than from others. S-adenosylhomocysteine is a potent inhibitor of tRNA-methylating enzymes in vitro.     

Elevation of glutathione levels by ammonium ions in primary cultures of rat astrocytes

It is well established that ammonia is detoxified in the brain to form glutamine and that astrocytes play a major role in this process. The synthesis of glutamine requires glutamate and ATP. Since glutamate and ATP are also required for the synthesis of glutathione (GSH), we examined the effect of pathophysiological concentrations of ammonia on levels of GSH in primary cultures of astrocytes. GSH content in the medium increased in a dose- and time-dependent manner in the presence of ammonia. After an initial decrease, cellular GSH content increased in a similar manner. The levels of glutathione disulfide (GSSG) were also increased. A linear relationship was observed between ammonia concentration and the increase in GSH levels. An increase in the efflux of GSH from cells into medium was also observed under these conditions. Buthionine sulfoximine and acivicin, but not methionine sulfoximine, blocked the ammonia induced increase in GSH levels. No, or minor, changes in the activities of enzymes (gamma-glutamyl transpeptidase, GSH reductase and GSH-peroxidase) that might influence GSH levels were identified and thus could not account for the ammonia induced increase in GSH levels in astrocytes. These findings indicate that pathophysiological concentrations of ammonium ions result in increased astroglial levels of GSH which may affect the metabolism and function of astrocytes.