Short communication

Importance of the glutathione redox cycle for the resistance of lung epithelial cells against a polymorphonuclear leukocyte-mediated oxidant attack

An imbalance between oxidants and antioxidants in the circulation is blamed to cause preeclampsia and eclampsia. In this study plasma ascorbic acid level was analysed in 13 eclamptic, 14 mild preeclamptic, 12 severe preeclamptic and 20 uncomplicated pregnancies to see whether there is any correlation with blood pressure, proteinuria, serum triglyceride level, erythrocyte fragility and leukocyte count. Plasma ascorbic acid level was normal and had no significant difference among the groups. Fasting serum triglyceride level was significantly higher in the study group than in the control group but it did not differ among the three study groups. Erythrocyte fragility was found to be increased in all three study groups. Blood leukocyte count was increased in the study groups, especially in the eclampsia group. However, plasma ascorbic acid level and erythrocyte fragility were found to have no significant correlation with blood pressure and proteinuria. It was concluded that though the ascorbic acid levels were normal in both the study and the control groups, erythrocyte fragility increased probably due to an elevation in peroxide and free radical levels in preeclampsia and eclampsia groups, but without any correlation with the severity of the clinical picture.

Murine leukemia L1210 cells rendered deficient in glutathione peroxidase (GPX) and phospholipid hydroperoxide glutathione peroxidase (PHGPX) by Se deprivation (L · Se(−) cells) were found to be more sensitive to tert-butyl hydroperoxide (t-BuOOH) cytotoxicity than Se-replete controls (L · Se(+) cells). Human K562 cells, which express PHGPX, but not GPX, were also more sensitive to t-BuOOH in the Se-deficient (K · Se(−)) than Se-satisfied (K · Se(+)) condition. In examining the metabolic basis for selenoperoxidase-dependent resistance, we found that glucose-replete Se(−) cells reduce t-BuOOH to t-butanol far more slowly than Se(+) cells, the ratio of the first-order rate constants approximating that of the GPX activities (L1210 cells) or PHGPX activities (K562 cells). Monitoring peroxide-induced changes in GSH and GSSG gave consistent results; e.g., glucose-depleted L · Se(+) cells exhibited a first order loss of GSH that was substantially faster than that of glucose-depleted L · Se(−) cells. Under the conditions used, peroxide-induced conversion of GSH to GSSG could be stoichiometrically reversed by resupplying D-glucose, indicating that no significant lysis or GSSG efflux and/or interchange had taken place. The apparent first-order rate constant for GSH decay increased progressively for L1210 cells expressing a range of GPX activities from ∼ 5% to 100%, demonstrating that peroxide detoxification is strictly dependent on enzyme content. The initial rate of ¹⁴CO₂ release from d-[1-¹⁴C]glucose supplied in the medium was much greater for L · Se(+) or K · Se(+) cells than for their respective Se(−) counterparts, consistent with greater hexose monophosphate shunt activity in the former. These results highlight the importance of selenoperoxidase action in the glutathione cycle as a means by which tumor cells cope with hydroperoxide stress.

Murine leukemia L1210 cells grown for 5–7 d in the presence of 1% serum without added selenium [Se(−) cells] expressed < 5% of the glutathione peroxidase (GPX) activity of selenium-supplemented controls [Se(+) cells]. Clonogenic survival assays indicated that t-butyl hydroperoxide (t-BuOOH) is much more toxic to Se(−) cells (LC₅₀ ∼ 10 μM) than to Se(+) or selenium-repleted [Se(−/+)] cells (LC₅₀ ∼ 250 μM). Hypersensitivity of Se(−) cells to t-BuOOH was partially reversed treating them with Ebselen, a selenoperoxidase mimetic; thus, selenoperoxidase insufficiency was probably the most serious defect of Se deprivation. Cytotoxicity of t-BuOOH was inhibited by desferrioxamine and by α-tocopherol, indicating that redox iron and free radical intermediates are involved. Elevated sensitivity of Se(−) cells to t-BuOOH was accompanied by an increased susceptibility to free radical lipid peroxidation, which became even more pronounced in cells that had been grown in arachidonate (20:4, n − 6) supplement media. The glutathione (GSH) is required for cytoprotection was established by showing that Se(+) cells are less resistant to t-BuOOH after exposure to buthionine sulfoximine (BSO), an inhibitor of GSH synthesis, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), an inhibitor of glutathione reductase. Coupled enzymatic assays indicated the Se(+) or Se(−/+) cells metabolize t-BuOOH 20–50 times more rapidly than Se(−), consistent with the measured difference in GPX activities of these cells. Correspondingly, when challenged the with t-BuOOH, Se(+) cells showed an initial loss of GSH and elevation of GSSG that exceeded that of Se(−) cells. It was further shown that like Se(−) cells, BSO-or BCNU-treated Se(+) cells metabolized t-BuOOH more slowly than nontreated controls. These results clearly indicate that selenoperoxidase action in the glutathione cycle is a vital element in cellular defense against toxic hydroperoxides.

Merocyanine 540 (MC540) is a lipophilic photosensitizing dye of biomedical interest in connection with its ability to preferentially inactivate leukemia cells in bone marrow grafts and enveloped viruses in blood products. Evidence that iron plays a role in dye-mediated photo-killing is presented in this report. When sensitized with MC540 and irradiated with visible light, cultured murine leukemia L1210 cells underwent lipid peroxidation (accumulation of iodometrically detectable lipid hydroperoxides) and photokilling (loss of clonogenic capacity). Selenium-deficient [Se(-)] cells, which expressed minimal selenoperoxidase activity, were found to be more sensitive to photoperoxidation and photokilling than selenium-replete [Se(+)] controls. Since redox active iron in the presence of electron donors has been shown to exacerbate photoperoxidative damage in isolated membrane systems, it was of interest to examine the possible role of iron in MC54Oflight-induced cytotoxicity. Involvement of iron was established by showing (i) that desferrioxamine (a high-affinity chelator and redox inhibitor of Fe³⁺) acted protectively on Se(-) and Se(+) cells and (ii) that treating these cells with sublethal concentrations of the lipophilic chelate ferric 8-hydroxyquin-oline [Fe(HQ)₂] made them much more sensitive to photokilling and thiobarbituric acid-detectable lipid peroxidation. Lethal damage induced by t-butyl hydroperoxide was also amplified by Fe(HQ)₂. Fe(HQ)₂-enhanced photoperoxidation and photokilling were suppressed by α-tocopherol, suggesting that iron-catalyzed free radical reactions were involved. A mechanism based on iron-mediated one-electron reduction of nascent photoperoxides is proposed. We believe that under the conditions used, toxic one-electron chemistry overwhelms two-electron detoxification catalyzed by GSH-dependent selenoperoxidase(s).

The purpose of this study was to determine if in vivo ozone exposure results in elevations in the levels of glutathione and glutathione-dependent enzymes in cells derived from bronchoalveolar lavage fluid (BALF). Our hypothesis was that, as part of a defense mechanism against oxygen toxicity, such cells would have increased levels of glutathione (GSH) in response to an oxidant stress. Female F344/N rats were exposed to 0.8 ppm ozone, 6 hr/day, for 1, 3, or 7 days, after which cells were collected by lung lavage. The GSH and GSH-peroxidase activity per milligram of protein in the cellular fraction, both necessary for reducing cellular peroxides, were elevated after 3 days of ozone exposure. After 7 days of exposure, cellular GSH had returned to control values, but the activity of glutathione reductase, the enzyme that reduces oxidized glutathione to GSH, was increased. Extracellular GSH concentration and glutathione reductase activity in BALF were also increased after 7 days of exposure. The totalglutathione equivalents (GSH and GSSG, both cellular and extracellular) in BALF increased throughout the 7-day exposure, with GSH increasing first in the cells, and then in the extracellular fluid. This study demonstrated that the glutathione anti-oxidant system of BALF cells is stimulated by exposure to ozone. This response may serve to protect cells from the toxic effects of oxidant stress.

The effect of selenium deprivation on the viability of murine L1210 cells exposed to various exogenous lipid hydroperoxides has been investigated. Selenoperoxidase activities of cells grown for longer than 1 week in 1% serum with no added selenium [Se(−) cells] were <10% of the activities of selenium-satisfied controls [Se(+) cells] or selenium-repleted counterparts [Se(−/+) cells]. The enzymes measured were classical glutathione peroxidase (GPX) and phospholipid hydroperoxide glutathione peroxidase (PHGPX). Se(−) cells exhibited a compensatory increase in catalase activity. Dye exclusion and clonal survival assays indicated that Se(−) and Se(+) cells were relatively insensitive to photochemically generated phospholipid hydroperoxides in liposomal form. However, both cell types were sensitive to liposomal cholesterol hydroperoxides, e.g., 7-hydroperoxycholesterol (7-OOH), Se(−) being much more so (LD₅₀ ~10 μm) than Se(+) (LD₅₀ ~75 μm). By contrast, 7-hydroxycholesterol over a comparable concentration range was minimally toxic to Se(−) and Se(+) cells. Cell killing by 7-OOH was inhibited by desferrioxamine and by butylated hydroxytoluene, suggesting that iron-mediated free radical reactions are involved. The involvement of glutathione in cytoprotection was confirmed by showing that Se(+) cells were more sensitive to 7-OOH after treating with buthionine sulfoximine, an inhibitor of GSH synthesis. Cellular detoxification of 7-OOH is provisionally attributed to PHGPX rather than GPX, since 7-OOH and other cholesterol hydroperoxides were found to be good substrates for PHGPX in a cell free system, but were unreactive with GPX.