Effect of N,N-diallyl-2,2-dichloroacetamide (R-25788) on efflux and synthesis of glutathione in tobacco suspension cultures

The low level of glutathione synthesis observed in tobacco suspension cultures grown under heterotrophic conditions was stimulated by the addition of t     

Glutathione and glutathione metabolizing enzymes in yeasts

Total glutathione content, glutathione peroxidase, glutathione transferase and glutathione reductase activities have been measured in 12 species of yeasts. All the strains tested contained glutathione, though in different amounts, as well as the above mentioned enzymes. To discriminate between the selenium-dependent and the selenium-independent form, glutathione peroxidase activity has been measured with both H₂O₂ and cumene hydroperoxide. Rhodotorula glutinis appeared to be the only strain in which the selenium-dependent form was not found, but this yeast exhibited the highest level of selenium-independent glutathione peroxidase activity as compared to the other strains.     

A selenium-induced peroxidation of glutathione in algae

Two unicellular marine algae cultured in media containing sodium selenite were examined for glutathione peroxidase activity. The 400 g supernatant from disrupted cells of both the green alga Dunaliella primolecta and the red alga Porphyridium cruentum were able to enhance both the H₂O₂ and the tert-butyl hydroperoxide dependent oxidation of glutathione. The glutathione peroxidation activity of D. primolecta was reduced only slightly by heating the 400 g supernatant, a 30% decrease in the rate with H₂O₂ and 10% decrease in the rate with t-BuOOH being observed. Heating caused the H₂O₂ dependent activity in P. cruentum to be reduced by only 30%, but the activity with t-BuOOH was reduced by 90%. Freezing decreased the t-BuOOH dependent activity of P. cruentum by 90%, but did not lower the t-BuOOH dependent activity of D. primolecta or the H₂O₂ dependent activity of either alga. It was concluded that the heat and cold stable, glutathione peroxidation was non-enzymatic in nature. A variety of small molecules (ascorbate, Cu(NO₃)₂, selenocystine, dimethyldiselenide and selenomethionine) were shown to be able to enhance the hydroperoxide dependent oxidation of glutathione in the assay system employed in this study. Such compounds could be responsible for the activity observed in algae. The heat and cold labile t-BuOOH reductase activity of P. cruentumwas possibly enzymatic, but was not attributable to the presence of glutathione-S-transferase. Both algae, when cultured in the presence of added selenite, displayed an approximate doubling of the non-enzymatic H₂O₂ and t-BuOOH dependent glutathione oxidase activities. The heat and cold labile t-BuOOH reductase activity of P. cruentum was unaltered when the alga was grown in the presence of added selenite. These observations are consistent with the hypothesis that selenium compounds present in the algae are responsible for the selenium induced glutathione peroxidation.     

亚硒酸钠对小麦幼苗中谷胱甘肽过氧化物酶和谷胱甘肽转硫酶活性以及谷胱甘肽含量的影响

用1.0 mg·L-1的亚硒酸钠根施小麦幼苗,测定亚硒酸钠对谷胱甘肽过氧化物酶和谷胱甘肽转硫酶活性以及还原性谷胱甘肽含量的结果表明,外源亚硒酸钠对麦苗地上部的谷胱甘肽过氧化物酶和谷胱甘肽转硫酶活性均有诱导作用,使麦苗体内的谷胱甘肽含量水平增加.     

Development of Mechanisms of Protection Against Oxidative Stress in Doxorubicin-Resistant Rat Tumoral Cells in Culture

We have compared some mechanisms involved in the defense against doxorubicin-induced free radical damage in rat hepatoma and glioblastoma cell lines and their doxorubicin-resistant variants presenting an overexpression of the multidrug resistance gene.

Immediate in vivo production of malondialdehyde was minor and was not different in sensitive and resistant cells. Alpha-tocopherol was undetectable in all cell lines. Glutathione levels were not different in sensitive and resistant cells and these levels did not vary upon doxorubicin treatment. Resistant cells exhibited either a 50% decrease (hepatoma) or a 25% increase (glioblastoma) of glutathione-S-transferase activity. Glutathione reductase presented no important change upon acquisition of resistance. In contrast, selenium-dependent glutathione peroxidase activity was consistently 2-6-fold increased in the resistant cells, which suggests a magnification of protection mechanisms against hydroxyle radical formation from H₂O₂ in resistant cells. Depletion of glutathione levels by buthionine sulfoximine sensitized hepatoma resistant cells to doxorubicin, but had no effect on doxorubicin cytotoxicity to glioblastoma cells.     

Vascular Oxidant Stress and Hepatic Ischemia/Reperfusion Injury

The objective of this study was to test the hypothesis that the extracellular oxidation of glutathione (GSH) may represent an important mechanism to limit hepatic ischemia/reperfusion injury in male Fischer rats in vivo. Basal plasma levels of glutatione disulfide (GSSG: 1.5 ± 0.2μM GSH-equivalents), glutathione (GSH: 6.2 ± 0.4 μM) and alanine aminotransferase activities (ALT 12 ± 2U/I) were significantly increased during the l h reperfusion period following l h of partial hepatic no-flow ischemia (GSSG: 19.7 ± 2.2μM; GSH 36.9 ± 7.4μM; ALT: 2260 ± 355 U/l). Pretreatment with 1,3-bis-(2-chloroethyl)-I-nitrosourea (40mg BCNU/kg), which inhibited glutathione reductase activity in the liver by 60%. did not affect any of these parameters. Biliary GSSG and GSH efflux rates were reduced and the GSSG-to-GSH ratio was not altered in controls and BCNU-treated rats at any time during ischemia and reperfusion. A 90% depletion of the hepatic glutathione content by phorone treatment (300 mg/kg) reduced the increase of plasma GSSG levels by 54%, totally suppressed the rise of plasma GSH concentrations and increased plasma ALT to 4290 ± 755 U/I during reperfusion. The data suggest that hepatic glutathione serves to limit ischemialreperfusion injury as a source of extracellular glutathione, not as a cofactor for the intracellular enzymatic detoxification of reactive oxygen species.     

Development of Mechanisms of Protection Against Oxidative Stress in Doxorubicin-Resistant Rat Tumoral Cells in Culture

We have compared some mechanisms involved in the defense against doxorubicin-induced free radical damage in rat hepatoma and glioblastoma cell lines and their doxorubicin-resistant variants presenting an overexpression of the multidrug resistance gene.

Immediate in vivo production of malondialdehyde was minor and was not different in sensitive and resistant cells. Alpha-tocopherol was undetectable in all cell lines. Glutathione levels were not different in sensitive and resistant cells and these levels did not vary upon doxorubicin treatment. Resistant cells exhibited either a 50% decrease (hepatoma) or a 25% increase (glioblastoma) of glutathione-S-transferase activity. Glutathione reductase presented no important change upon acquisition of resistance. In contrast, selenium-dependent glutathione peroxidase activity was consistently 2-6-fold increased in the resistant cells, which suggests a magnification of protection mechanisms against hydroxyle radical formation from H₂O₂ in resistant cells. Depletion of glutathione levels by buthionine sulfoximine sensitized hepatoma resistant cells to doxorubicin, but had no effect on doxorubicin cytotoxicity to glioblastoma cells.     

Elevation of glutathione levels and glutathione S-transferase activity in arsenic-resistant chinese hamster ovary cells

Summary Arsenic-resistant Chinese hamster ovary (CHO) cells were established by progressively increasing the concentration of sodium arsenite in culture medium. One of the resistant clones, SA7, was also cross-resistant to As(V), Zn, Fe(II), Co, and Hg. The susceptibilities to sodium arsenite in parental CHO cells, revertant SA7N cells, and resistant SA7 cells were correlated with their intracellular glutathione (GSH) levels and glutathione S-transferase (GST) activity. The resistance in SA7 cells was diminished by depletion of GSH in cells after treatment with buthionine sulfoximine. Furthermore, after reexposure of revertant SA7N cells to sodium arsenite, the intracellular GSH levels, GST activity, and resistance to sodium arsenite were raised to the same levels as SA7 cells. These data indicate that the elevation of intracellular GSH levels and GST activity in SA7 cells may be responsible for the resistance to arsenite. A p25 protein, which could be a monomer subunit of GST, accumulated in SA7 cells. In addition, an outward transport inhibitor, verapamil, indiscriminately increased the arsenite toxicity in resistant and parental cells. This work was supported in part by grant NSC77-0201-B001-31 from the National Science Council, Republic of China.     

谷胱甘肽的解毒作用与毒性代谢物

谷胱甘肽是细胞内重要的含SH基的非蛋白分子,它对氧自由基、有机氢过氧化物以及亲电子剂的灭活起着极重要的作用.但近年来的研究表明,一些连位二卤烷烃、卤烯烃、含酮结构的化合物以及一些异氰酸酯、异硫氰酸酯、醛、α,β－不饱和醛等与谷胱甘肽轭合后可导致毒性代谢物的形成.     

Overexpression of enzymes involved in glutathione synthesis enhances tolerance to organic pollutants in Brassica juncea

Transgenic Indian mustard (Brassica juncea) overexpressing y-glutamylcysteine synthetase (ECS) or glutathione synthetase (GS) were shown previously to have two-fold higher levels of glutathione and total nonprotein thiols, as well as enhanced cadmium tolerance and accumulation. Here, the hypothesis was tested that these transgenics have enhanced tolerance to organic pollutants, based on the reasoning that many organic xenobiotics are detoxified via conjugation to glutathione. Both the ECS and GS transgenics showed enhanced tolerance to atrazine: while root growth of wildtype seedlings was inhibited 50% by 100 mg L(-1) atrazine, ECS and GS root growth was inhibited 20-30% (P < 0.05). The tolerance of the transgenics to CDNB (1-chloro-2,4-dinitrobenzene). metolachlor, and phenanthrene was also somewhat higher than wild type, but these differences were not as pronounced. Each of the organics treatments significantly enhanced total nonprotein thiol levels in all plant types (2 to 12-fold). Overall, these results suggest that GSH biosynthesis is limiting for atrazine detoxification in Indian mustard and that overexpression of enzymes involved in GSH biosynthesis offers a promising approach to create plants with the enhanced capacity to tolerate not only heavy metals, but also certain organics.