Nitric oxide-loaded chitosan nanoparticles as an innovative antileishmanial platform.

Leishmaniasis is a neglected tropical disease that demands for new therapeutic strategies due to adverse side effects and resistance development promoted by current drugs. Nitric oxide (NO)-donors show potential to kill Leishmania spp. but their use is limited because of their instability. In this work, we synthesize, characterize, and encapsulate S-nitroso-mercaptosuccinic acid into chitosan nanoparticles (NONPs) and investigate their activity on promastigotes and intracellular amastigotes of Leishmania (Leishmania) amazonensis. Cytotoxicity on macrophages was also evaluated. We verified that NONPs reduced both forms of the parasite in a single treatment. We also noticed reduction of parasitophorous vacuoles as an evidence of inhibition of parasite growth and resolution of infection. No substantial cytotoxicity was detected on macrophages. NONPs were able to provide a sustained parasite killing for both L. (L.) amazonensis infective stages with no toxicity on macrophages, representing a promising nanoplatform for cutaneous leishmaniasis.

Studies of the inhibitory activities of tiopronin and mercaptosuccinic acid on glutathione peroxidase and their cytotoxic and antioxidant properties.

Glutathione peroxidase (GPx), an important antioxidative enzmye, can be inhibited by various thiols, including of tiopronin and mercaptosuccinic acid (MSA). Recently, there has been discussion regarding the combination of tiopronin in anticancer therapy to overcome acquired resistance to anticancer drugs. However, thiols are also known to act as antioxidants, which can be contraindicated in cancer chemotherapy. This article focuses on the inhibitory effects of tiopronin and MSA on bovine and human glutathione peroxidase activities, and their effects on the redox status of cancer cells. IC50 values for the inhibition for the bovine erythrocyte enzyme were 356 and 24.7 µM for tiopronin and MSA, respectively, with the corresponding Ki values of 343 µM and 14.6 µM, respectively at pH 7.4 and 25 °C. MSA inhibited human GPx activity in human cancer cell lysates at its IC50 while tiopronin did not. Both compounds were cytotoxic to human cancer cell lines GUMBUS and HL-60, with IC50 values between 42.7 and 149.4 µM. Neither had an effect on cell cycle. Only MSA induced apoptosis in HL-60 cells but not in GUMBUS cells, while tiopronin resulted in no apoptosis in either cell line. Combination studies of the MSA with hydrogen peroxide in living cells enhanced the production of reactive oxygen species in GUMBUS cells while tiopronin acted as antioxidant in HL-60 cells. MSA and tiopronin antagonized the cytotoxic effect of cisplatin, doxorubicin and methotrexate in combination studies. Our findings indicate that the antioxidant properties of both thiols prevail over their GPx inhibitory activity in human cancer cell lines.

Evaluation of antibacterial activity of nitric oxide-releasing polymeric particles against Staphylococcus aureus and Escherichia coli from bovine mastitis.

Bovine mastitis is a serious veterinary disease that causes great loss to the dairy industry worldwide. It is a major infectious disease and is difficult to manage and control. Furthermore, emerging multidrug resistant bacteria that cause mastitis have complicated such management. The free radical nitric oxide (NO) is a potent antimicrobial agent. Thus, the aims of this study were to prepare and evaluate the antibacterial activity of nitric oxide-releasing polymeric particles against Staphylococcus aureus (MBSA) and Escherichia coli (MBEC), which were isolated from bovine mastitis. Fifteen MBSA isolates and fifteen MBEC were collected from subclinical and clinical bovine mastitis. Biocompatible polymeric particles composed of alginate/chitosan or chitosan/sodium tripolyphosphate (TPP) were prepared and used to encapsulate mercaptosuccinic acid (MSA), which is a thiol-containing molecule. Nitrosation of thiol groups of MSA-containing particles formed S-nitroso-MSA particles, which are NO donors. The NO release kinetics from the S-nitroso-MSA particles showed sustained and controlled NO release over several hours. The antibacterial activity of NO-releasing particles was evaluated by incubating the particles with an MBSA multi-resistant strain, which is responsible for bovine mastitis. The minimum inhibitory concentration for S-nitroso-MSA-alginate/chitosan particles against MBSA ranged from 125 µg/mL to 250 µg/mL. The results indicate that NO-releasing polymeric particles are an interesting approach to combating bacteria resistance in bovine mastitis treatment and prevention.

Inhibition of glutathione peroxidase mediates the collateral sensitivity of multidrug-resistant cells to tiopronin.

Multidrug resistance (MDR) is a major obstacle to the successful chemotherapy of cancer. MDR is often the result of overexpression of ATP-binding cassette transporters following chemotherapy. A common ATP-binding cassette transporter that is overexpressed in MDR cancer cells is P-glycoprotein, which actively effluxes drugs against a concentration gradient, producing an MDR phenotype. Collateral sensitivity (CS), a phenomenon of drug hypersensitivity, is defined as the ability of certain compounds to selectively target MDR cells, but not the drug-sensitive parent cells from which they were derived. The drug tiopronin has been previously shown to elicit CS. However, unlike other CS agents, the mechanism of action was not dependent on the expression of P-glycoprotein in MDR cells. We have determined that the CS activity of tiopronin is mediated by the generation of reactive oxygen species (ROS) and that CS can be reversed by a variety of ROS-scavenging compounds. Specifically, selective toxicity of tiopronin toward MDR cells is achieved by inhibition of glutathione peroxidase (GPx), and the mode of inhibition of GPx1 by tiopronin is shown in this report. Why MDR cells are particularly sensitive to ROS is discussed, as is the difficulty in exploiting this hypersensitivity to tiopronin in the clinic.

Differential expression and anti-oxidant function of glutathione peroxidase 3 in mouse uterus during decidualization.

Glutathione peroxidase 3 (GPX3) is an important member of antioxidant enzymes for reducing reactive oxygen species and maintaining the oxygen balance. Gpx3 mRNA is strongly expressed in decidual cells from days 5 to 8 of pregnancy. After pregnant mice are treated with GPX inhibitor for 3 days, pregnancy rate is significantly reduced. Progesterone stimulates Gpx3 expression through PR/HIF1α in mouse endometrial stromal cells. In the decidua, the high level of GPX3 expression is closely associated with the reduction of hydrogen peroxide (H2O2). Based on our data, GPX3 may play a major role in reducing H2O2 during decidualization.

Biofunctionalized quantum dots for live monitoring of stem cells: applications in regenerative medicine.

AIM: This study aimed to live monitor the degree of endothelial progenitor cell (EPC) integration onto tissue-engineering scaffolds by conjugating relevant antibodies to quantum dots (QDs). MATERIALS & METHODS: Biocompatible mercaptosuccinic acid-coated QDs were functionalized with two different antibodies to EPC (CD133 with QDs of 640 nm wavelength [λ] and later-stage mature EPCs; and von Willebrand factor with QDs of λ595 and λ555 nm) using conventional carbomide and N-hydroxysuccinimide chemistry. Biofunctionalization was characterized with Fourier-transform infrared spectroscopy. Cell viability assays and gross morphology observations confirmed cytocompatibility and normal patterns of celluar growth. The antigens corresponding to each state of cell maturation were determined using a single excitation at λ488 nm. RESULTS: The optimal concentrations of antibody-QD conjugates were biocompatible, hemocompatible and determined the state of EPC transformation to endothelial cells. CONCLUSION: Antibody-functionalized QDs suggest new applications in tissue engineering of polymer-based implants where cell integration can potentially be monitored without requiring the sacrifice of implants.

Expression of tyrosine hydroxylase increases the resistance of human neuroblastoma cells to oxidative insults.

In this study, we demonstrate that human neuroblastoma SH-SY5Y cells transfected with human tyrosine hydroxylase isoform 1 (SH + TH cells) were substantially more resistant to cell death induced by hydrogen peroxide and 6-hydroxydopamine when compared to wild-type SH-SY5Y cells (SH cells). SH + TH cells exhibit increased levels of dopamine (DA) compared to SH cells. Incubation with hydrogen peroxide or 6-hydroxydopamine (10-100microM) for 24 h caused a significant reduction in cell viability and increased apoptosis in both cell types. However, these effects were significantly reduced in the SH + TH cells when compared to the SH cells. The SH + TH cells showed an improved ability to detoxify peroxide, which correlated with an increase in glutathione peroxidase and glutathione reductase activities, while catalase activity was unchanged. Our data suggest that a preconditioning-like mechanism linked to higher DA levels increased the resistance of SH + TH cells against oxidative insults, which is at least in part related to an augmentation in the activity of glutathione-related antioxidant enzymes.

Methylmercury neurotoxicity is associated with inhibition of the antioxidant enzyme glutathione peroxidase.

In this study, we investigated the involvement of glutathione peroxidase-GPx in methylmercury (MeHg)-induced toxicity using three models: (a) in mouse brain after treatment with MeHg (40 mg/L in drinking water), (b) in mouse brain mitochondrial-enriched fractions isolated from MeHg-treated animals, and (c) in cultured human neuroblastoma SH-SY5Y cells. First, adult male Swiss mice exposed to MeHg for 21 days showed a significant decrease in GPx activity in the brain and an increase in poly(ADP-ribose) polymerase cleavage, an index of apoptosis. Second, in mitochondrial-enriched fractions isolated from MeHg-treated mice, there was a significant reduction in GPx activity and a concomitant decrease in mitochondrial activity and increases in ROS formation and lipid peroxidation. Incubation of mitochondrial-enriched fractions with mercaptosuccinic acid, a GPx inhibitor, significantly augmented the toxic effects of MeHg administered in vivo. Incubation of mitochondrial-enriched fractions with exogenous GPx completely blocked MeHg-induced mitochondrial lipid peroxidation. Third, SH-SY5Y cells treated for 24 h with MeHg showed a significant reduction in GPx activity. There was a concomitant significant decrease in cell viability and increase in apoptosis. Inhibition of GPx substantially enhanced MeHg toxicity in the SH-SY5Y cells. These results suggest that GPx is an important target for MeHg-induced neurotoxicity, presumably because this enzyme is essential for counteracting the pro-oxidative effects of MeHg both in vitro and in vivo.

[Role of glycolysis and antioxidant enzymes in the toxicity of amyloid beta peptide Abeta25-35 to erythrocytes].

The role of glycolysis and antioxidant enzymes in amyloid beta peptide Abeta(25-35) toxicity to human and rat erythrocytes was studied. The erythrotoxicity of Abeta(25-35) was shown to increase two- to fourfold both in the absence of glucose in the incubation medium and upon the addition of sodium fluoride, an enolase inhibitor. Potassium cyanide, a Cu,Zn-superoxide dismutase inhibitor, abolishes the toxic effect of Abeta(25-35) to erythrocytes, whereas mercaptosuccinate, a glutathione peroxidase inhibitor, and ouabain, a Na+,K+-ATPase inhibitor, promote it. Sodium azide, a catalase inhibitor, did not affect the cell lysis under the action of Abeta(25-35) . The results support the hypothesis that H2O2, Cu,Zn superoxide dismutase, and glutathione peroxidase are involved in the toxicity mechanism rather than superoxide radical. Glycolysis and Na+,K+-ATPase play a substantial protective role. Fullerene C(60) nanoparticles are toxic to erythrocytes of both types; their toxicity is not related to enhanced oxidative stress and the mechanism of toxicity differs from that of Abeta(25-35) .

Critical role of exposure time to endogenous oxidative stress in hepatocyte apoptosis.

We have previously shown that inhibition of catalase and glutathione peroxidase activities in rat primary hepatocytes by 3-amino-1,2,4-triazole (ATZ) and mercaptosuccinic acid (MS) results in sustained oxidative stress, followed by apoptosis. To examine the effects of duration of oxidative stress, ATZ and MS were removed from culture medium at 3, 6 and 9 h after treatment with both inhibitors. Oxidative stress was induced for periods of time by ATZ and MS exposures in primary hepatocytes. Treatment with ATZ and MS reduced catalase (CAT) and glutathione peroxidase (GPx) activities, and decreased CAT and GPx activities recovered to normal values upon withdrawal. Although oxidative stress of up to 6 h duration did not cause cell death, sustained oxidative stress (over 9 h) induced apoptosis. The increase in the glutathione disulfide/reduced glutathione ratio under oxidative stress up to 6 h was transient and reversible, while that due to sustained oxidative stress was irreversible. These results suggest that irreversible redox shifts resulting from sustained oxidative stress play a critical role in the induction of hepatocyte apoptosis in this experimental system.

Endogenous hydrogen peroxide regulates the excitability of midbrain dopamine neurons via ATP-sensitive potassium channels.

ATP-sensitive K+ (K(ATP)) channels link metabolic state to cell excitability. Here, we examined regulation of K(ATP) channels in substantia nigra dopamine neurons by hydrogen peroxide (H2O2), which is produced in all cells during aerobic metabolism. Blockade of K(ATP) channels by glibenclamide (100 nM) or depletion of intracellular H2O2 by including catalase, a peroxidase enzyme, in the patch pipette increased the spontaneous firing rate of all dopamine neurons tested in guinea pig midbrain slices. Using fluorescence imaging with dichlorofluorescein to visualize intracellular H2O2, we found that moderate increases in H2O2 during partial inhibition of glutathione (GSH) peroxidase by mercaptosuccinate (0.1-0.3 mM) had no effect on dopamine neuron firing rate. However, with greater GSH inhibition (1 mM mercaptosuccinate) or application of exogenous H2O2, 50% of recorded cells showed K(ATP) channel-dependent hyperpolarization. Responsive cells also hyperpolarized with diazoxide, a selective opener for K(ATP) channels containing sulfonylurea receptor SUR1 subunits, but not with cromakalim, a selective opener for SUR2-based channels, indicating that SUR1-based K(ATP) channels conveyed enhanced sensitivity to elevated H2O2. In contrast, when endogenous H2O2 levels were increased after inhibition of catalase, the predominant peroxidase in the substantia nigra, with 3-amino-1,2,4-triazole (1 mM), all dopamine neurons responded with glibenclamide-reversible hyperpolarization. Fluorescence imaging of H2O2 indicated that catalase inhibition rapidly amplified intracellular H2O2, whereas inhibition of GSH peroxidase, a predominantly glial enzyme, caused a slower, smaller increase, especially in nonresponsive cells. Thus, endogenous H2O2 modulates neuronal activity via K(ATP) channel opening, thereby enhancing the reciprocal relationship between metabolism and excitability.

Effects of arsenic-, platinum-, and gold-containing drugs on the disposition of exogenous selenium in rats.

Having found that the electrophilic model compound sulfobromophthalein markedly altered the fate of exogenous selenium in the body by reacting in vivo with nucleophilic selenium metabolites, the effects of metal-containing drugs with expected selenium reactivity were tested on biliary, urinary, and pulmonary excretion. Tissue distribution of selenium in selenite-injected rats was also examined. Coadministration with [(75)Se]selenite (10 micromol/kg, iv) of the trypanosomicid arsenicals (100 micromol/kg, iv) trimelarsan (TMA) or melarsoprol (MAP), the antitumor cisplatin (25 micromol/kg, iv), or the antirheumatic gold sodium thiomalate (25 or 50 micromol/kg, iv) significantly altered the disposition of (75)Se, whereas carboplatin (100 micromol/kg, iv) did not produce such an effect. The most dramatic alterations included the approximately 20-fold increase in the biliary excretion rate of selenium in response to TMA and MAP, the almost complete cessation of the exhalation of selenium as dimethyl selenide after administration of the arsenic- and gold-containing drugs, and the manifold accumulation of selenium in the blood plasma following gold injection. Direct chemical reaction of the drugs with nucleophilic selenite metabolites in the body may underlie these alterations. The tight coordination in time and extent observed between the biliary excretion of arsenic and selenium in rats receiving either of the arsenicals and selenite supports this hypothesis. However, attempts to detect selenium-containing biliary metabolites of TMA and MAP have failed, possibly owing to their instability. In summary, the arsenic-, platinum- and gold-containing drugs significantly influence the fate of exogenous selenium, whereby they may adversely affect the availability of this essential element for synthesis of selenoenzymes. Furthermore, the capability of TMA and MAP to enhance the biliary and total excretion of selenium renders these drugs significant candidates for antidotes in selenium intoxication.

Glutathione oxidation and PTPase inhibition by hydrogen peroxide in Caco-2 cell monolayer.

The role of H(2)O(2) and protein thiol oxidation in oxidative stress-induced epithelial paracellular permeability was investigated in Caco-2 cell monolayers. Treatment with a H(2)O(2) generating system (xanthine oxidase + xanthine) or H(2)O(2) (20 microM) increased the paracellular permeability. Xanthine oxidase-induced permeability was potentiated by superoxide dismutase and prevented by catalase. H(2)O(2)-induced permeability was prevented by ferrous sulfate and potentiated by deferoxamine and 1,10-phenanthroline. GSH, N-acetyl-L-cysteine, dithiothreitol, mercaptosuccinate, and diethylmaleate inhibited H(2)O(2)-induced permeability, but it was potentiated by 1,3-bis(2-chloroethyl)-1-nitrosourea. H(2)O(2) reduced cellular GSH and protein thiols and increased GSSG. H(2)O(2)-mediated reduction of GSH-to-GSSG ratio was prevented by ferrous sulfate, GSH, N-acetyl-L-cysteine, diethylmaleate, and mercaptosuccinate and potentiated by 1,10-phenanthroline and 1, 3-bis(2-chloroethyl)-1-nitrosourea. Incubation of soluble fraction of cells with GSSG reduced protein tyrosine phosphatase (PTPase) activity, which was prevented by coincubation with GSH. PTPase activity was also lower in H(2)O(2)-treated cells. This study indicates that H(2)O(2), but not O(2)(-). or.OH, increases paracellular permeability of Caco-2 cell monolayer by a mechanism that involves oxidation of GSH and inhibition of PTPases.

DNA damage in arsenite- and cadmium-treated bovine aortic endothelial cells.

Reactive oxygen species have been shown to be involved in the mutagenicity, clastogenicity, and apoptosis of mammalian cells treated with arsenic or cadmium. As these endpoints require several hours of cellular processing, it is not clear that reactive oxygen species damage DNA directly or interfere with DNA replication and repair. Using single-cell alkaline electrophoresis, we have detected DNA strand breaks (DSBs) in bovine aortic endothelial cells by a 4-h treatment with sodium arsenite (As) and cadmium chloride (Cd) in sublethal concentrations. As-induced DSBs could be decreased by nitric oxide (NO) synthase inhibitors, superoxide scavengers, and peroxynitrite scavengers and could be increased by superoxide generators and NO generators. Treatment with As also increased nitrite production. These results suggest that As-increased NO may react with O2*- to produce peroxynitrite and cause DNA damage. The results showing that Cd increased cellular H2O2 levels and that Cd-induced DSBs could be modulated by various oxidant modulators suggest that Cd may induce DSBs via O2*-, H2O2, and *OH. Nevertheless, the DSBs in both As- and Cd-treated cells seem to come from the excision of oxidized bases such as formamidopyrimidine and 8-oxoguanine, as the Escherichia coli enzyme formamidopyrimidine-DNA glycosylase (Fpg) increased DSBs in cells treated with As, 3-morpholinosydnonimine (a peroxynitrite-generating agent), Cd, or H2O2.

Arsenic trioxide selectively induces acute promyelocytic leukemia cell apoptosis via a hydrogen peroxide-dependent pathway.

Low concentrations of As(2)O(3) (

The synergism of hydrogen peroxide with plasma S-nitrosothiols in the inhibition of platelet activation.

Earlier studies have shown that inhibition of aggregation of washed platelets (WP) by NO was enhanced almost 100-fold by H2O2. In the present study, the interactions of H2O2 with nitrosothiols, the influence of the presence of plasma and the mechanism of the synergism were investigated. H2O2 strongly enhanced the inhibitory effects of S-nitrosoglutathione (GSNO) on thrombin-induced aggregation of WP. S-Nitrosoalbumin also inhibited platelets, and this was similarly enhanced by H2O2. The synergism with H2O2 was demonstrable for both exogenous GSNO and NO in the presence of plasma when platelets were stimulated with collagen. The inhibition of platelets by GSNO and H2O2 was completely inhibited by guanylate cyclase inhibitors. Synergism was also observed whether the H2O2 was added simultaneously or 1 min before or after the GSNO (or NO). This suggests that the action of H2O2 follows the occupation by NO of haem sites in guanylate cyclase and that a prior reaction between NO and H2O2 was not required. In the absence of exogenous GSNO or NO, H2O2 inhibited activation of platelets in plasma, an effect abolished by guanylate cyclase inhibitors. This suggested that endogenous NO donors in plasma or NO synthesized in platelets may interact with H2O2. Addition of NG-nitro-L-arginine methyl ester (hydrochloride) (L-NAME) decreased the effects of the H2O2 by 25%, indicating that the major endogenous source of NO in platelet-rich plasma was not derived from platelet synthesis of NO but from NO donors in plasma, such as nitrosothiols. Inhibition by H2O2 was also enhanced by beta-mercaptosuccinate, a glutathione peroxidase inhibitor that protects the H2O2. These results suggest a potent synergism of H2O2 with endogenous plasma nitrosothiols that inhibit platelet function through an intracellular mechanism involving guanylate cyclase.

Effect of gold sodium thiomalate and its thiomalate component on the in vitro expression of endothelial cell adhesion molecules.

Endothelial adhesion molecules play an important role in the tissue recruitment of leukocytes in inflammatory conditions such as rheumatoid arthritis. We have investigated the effect of the antirheumatic drug gold sodium thiomalate on adhesion molecule protein and mRNA expression in cultured human endothelial cells. Gold sodium thiomalate inhibited cytokine (TNF, IL-1, IL-4)-stimulated expression of vascular cell adhesion molecule-1 and E-selectin but not intercellular adhesion molecule-1 on endothelial cells. Gold sodium thiomalate also suppressed TNF-stimulated increases in vascular cell adhesion molecule-1 and E-selectin mRNA levels but had no effect on intercellular adhesion molecule-1 mRNA. Thiomalate (mercaptosuccinate), but not gold thioglucose or D-penicillamine, mimics the effect of gold sodium thiomalate at equimolar concentrations. We propose that the inhibition of vascular cell adhesion molecule-1 and E-selectin expression by gold sodium thiomalate is due to its thiomalate and not its gold component. Gold sodium thiomalate has a direct effect on endothelial adhesion molecule expression, and this may contribute to its antiinflammatory activity.

Endogenously generated active oxygen species and cellular glutathione levels in relation to BHK-21 cell proliferation.

In BHK-21 cells (baby hamster kidney fibroblasts) cellularly generated active oxygen species such as hydrogen peroxide and superoxide appear to be important growth regulatory signals as judged from the growth inhibitory effects of catalase, superoxide dismutase and superoxide dismutase mimics. These active oxygen species may contribute a novel redox system of regulatory control superimposed upon established growth signal pathways. This may be achieved by direct oxidative modification of cell regulatory proteins such as transcription factors or protein kinases or indirectly through, for example alterations in levels of glutathione (GSH). This latter possibility is suggested from observations that catalase, or superoxide dismutase treatment of BHK-21 cells brings about increased cellular levels of GSH. However during the normal growth phase cellular levels of GSH actually decline although this effect can be partly reversed by N-acetylcysteine and by mercaptosuccinate which also impair proliferation of these cells.

Thiols, gold-thiols, zinc-thiols and the redox state of hemoglobin.

The beta subunit of human hemoglobin can be oxidized site-specifically through beta-Cys-93 by Cu(II)(His)2. A series of thiol ligands, gold thiols and zinc(II) inhibit this oxidation. The thiol inhibitors formed a transient ternary intermediate involving Cu(I) with consequent inhibition of electron transfer from the Fe(II)-heme. The intermediate led to the formation of a disulfide at the beta-Cys-93 site. The most effective thiols achieved maximum inhibition at one equivalent per beta heme. Gold thiols and zinc complexes inhibited heme oxidation by competing with the Cu(II)(His)2 for the beta-Cys-93 site.

Quantitative analysis of cellular glutathione in early preimplantation mouse embryos developing in vivo and in vitro.

The relative levels of reduced glutathione (GSH) have been measured fluorimetrically in individual eggs and early embryos from two mouse strains, one of which shows developmental arrest in vitro. GSH levels fell by approximately 20-25% at fertilization and by approximately 45% by the late 2-cell and early 4-cell stages. No differences were observed between strains or between embryos cultured in vitro or in vivo. Addition of exogenous H2O2 or diethylmaleate depleted GSH. GSH levels were not affected significantly after inhibition of GSH-peroxidase by mercaptosuccinate nor of catalase by aminotriazole. Mercaptosuccinate did not inhibit development but catalase inhibition caused arrest at the 2-cell stage. Addition of exogenous GSH or thioredoxin did not promote development of 'blocking' embryos through the 2-cell block. It is concluded that early embryos lack a mercaptosuccinate sensitive peroxidase activity for removing H2O2, which may be removed by catalase or the glutathione-S-transferase system. It is suggested that GSH may have a role in detoxifying peroxidated lipids. The results are consistent with a role for reactive oxygen species in the 2-cell block.