Increase In Intracellular GSH Levels Research Articles

CHAC1 exacerbates arsenite cytotoxicity by lowering intracellular glutathione levels.

We here examined whether CHAC1 is implicated in arsenite (As(III))-induced cytotoxicity in HaCaT cells. We found that HaCaT cells in which the intracellular GSH levels were elevated by transfection with CHAC1 siRNA showed decreased sensitivity to As(III) compared to the control cells. Treatment with BSO (an inhibitor of GSH biosynthesis) abolished the decrease in sensitivity to As(III), suggesting that an increase in intracellular GSH levels was involved in the decrease in sensitivity to As(III) due to the decrease in the levels of CHAC1 expression. When we examined the expression of CHAC1 after exposure of HaCaT cells to As(III), the levels of CHAC1 were increased. Since CHAC1 is a proapoptotic factor, we examined appearance of apoptotic cells and cleavage of caspase-3 after exposure to As(III) to determine whether As(III)-induced CHAC1 up-regulation was involved in apoptosis induction. The results showed that induction of apoptosis by As(III) exposure was not detected in CHAC1 siRNA-transfected cells. Together, our findings indicate that CHAC1 is involved in the sensitivity of HaCaT cells to As(III) by regulating the intracellular GSH levels, and in particular, CHAC1 is involved in As(III)-induced apoptosis.

Bromosulfophthalein suppresses inflammatory effects in lipopolysaccharide-stimulated RAW264.7 macrophages

Objective It has been reported that glutathione (GSH), the most abundant cellular antioxidant, can inhibit production of pro-inflammatory cytokines by activated macrophages. Bromosulfophthalein (BSP) has been recognized as an inhibitor of the efflux of reduced GSH from cells, leading to an increase in the intracellular GSH level. In this study, we evaluated, for the first time, whether BSP possessed anti-inflammatory effects in lipopolysaccharide (LPS)-stimulated macrophages. Materials and methods RAW 264.7 cells were treated with BSP and the levels of proinflammatory cytokines, GSH, and nitrite were assessed. Gene expression of inducible nitric oxide synthase (iNOS), tumor necrosis factor alpha (TNF α), interleukin-1beta (IL-1β), and interleukin-6 (IL-6) was analyzed via quantitative RT-PCR. We also examined various inflammatory signaling pathways including Akt/forkhead box protein O1 (FoxO1)/toll-like receptor 4 (TLR4), mitogen-activated protein kinases (MAPKs), and Fas protein by Western blot and flow cytometry analysis. Results Our study demonstrated that BSP induced an increase in intracellular GSH level in LPS-stimulated macrophages. BSP inhibited production of nitric oxide and proinflammatory cytokines. BSP increased phosphorylation of Akt and nuclear exclusion of FoxO1 and suppressed TLR4 expression. Additionally, BSP decreased MAPKs activation and Fas expression. Discussion and conclusion Taken together, these data suggest that BSP can attenuate inflammation through multiple signaling pathways. These findings highlight the potential of BSP as a new anti-inflammatory agent.

Aqueous-Methanol Extracts of Orange-Fleshed Sweet Potato (Ipomoeabatatas) Ameliorate Oxidative Stress and Modulate Type 2 Diabetes Associated Genes in Insulin Resistant C2C12 Cells.

Edible plants such as sweet potato are sources of natural antioxidants that can be exploited in the management and treatment of insulin resistance. This present study investigated the effects of the extracts of an orange-fleshed sweet potato on oxidative stress biomarkers (glutathione status and lipid peroxidation) and activities of antioxidant enzymes (catalase, CAT and glutathione peroxidase, GPx) in palmitate-induced insulin resistant C2C12 cells. The intracellular antioxidant status of the cells was also measured using Ferric reducing antioxidant power (FRAP) and Trolox equivalent antioxidant capacity (TEAC) assays. Furthermore, this study determined the effect of the extracts on the regulation of some type 2 diabetes associated genes; glucose transporter 4 (glut4), Nuclear respiratory factor 1 (nrf1), Myocyte enhanced factor 2A (mef2a), Carnitine palmitoyltransferase 1 (cpt1) and Acetyl-CoA carboxylase 2 (acc2). The results showed a significant (p < 0.05) increase in intracellular GSH level, a significant reduction in the level of malonaldehyde and a significant improvement in the intracellular antioxidant status upon treatment of the insulin resistant cells with the extracts. The extracts were also able to positively modulate the expression levels of the type 2 diabetes associated genes. On the other hand, HPLC-MS analysis of the extracts showed the presence of polyphenols which could have contributed to the bioactivity of the extracts through their antioxidant effects.

58 EFFECT OF LYSOPHOSPHATIDIC ACID ON PORCINE OOCYTE IN VITRO MATURATION AND SUBSEQUENT EMBRYONIC DEVELOPMENT AFTER PARTHENOGENETIC ACTIVATION AND IVF

Lysophosphatidic acid (LPA) is a signalling molecule derived from phospholipid, known to have biological activities such as stimulating cell proliferation, differentiation, and migration. The purpose of this study was to investigate the effect of LPA on in vitro maturation (IVM) of porcine oocytes and subsequent embryonic development after in vitro fertilization (IVF) and parthenogenetic activation (PA). We examined nuclear maturation, intracellular glutathione (GSH), and reactive oxygen species (ROS) levels and subsequent embryonic development after IVF in porcine oocytes matured in TCM-199 media containing either 0, 10, 30, or 60 μM LPA (No. of cultured oocytes 225, 225, 230, and 226, respectively). All data were compared for all groups using one-way ANOVA, followed by Duncan’s multiple range test. After 44 h of IVM, no significant difference was observed for nuclear maturation from the different (0, 10, 30, and 60 μM) LPA treatment groups (86.61, 87.48, 92.97, and 88.69%, respectively) compared with the control (P &lt; 0.05). The 30 μM LPA treated group exhibited a significant (P &lt; 0.05) increase in intracellular GSH levels and decrease in intracellular ROS levels compared with other LPA-treated groups. Oocytes matured with 30 μM LPA during IVM had significantly (P &lt; 0.05) higher cleavage rates after PA (88.50%) than other LPA-treated groups (79.42, 83.87, and 83.65%, respectively). Blastocyst formation rates following PA increased up to 30 μM LPA, but the rates decreased in the 60 μM LPA-treated group (49.64, 53.28, 57.00, and 51.09%, respectively). However, a statistical difference was not observed (P &lt; 0.05). Upon evaluation of embryonic competence following IVF, treatment with 30 μM LPA during IVM significantly increased cleavage rates (70.44%; P &lt; 0.05) compared with 0 and 60 μM LPA-treated groups (61.51 and 65.27%, respectively). Blastocyst formation rates improved with increasing concentrations of LPA up to 30 µM; however, the rates decreased in the 60 μM LPA-treated group (26.34, 30.10, 33.26, and 27.77%, respectively). However, like the preceding PA blastocyst formation rates, a statistical difference was not observed (P &lt; 0.05). In conclusion, treatment with 30 μM LPA during IVM improved the developmental potential of PA and IVF porcine embryos by increasing the intracellular GSH level, thereby decreasing the intracellular ROS level during oocyte maturation. This work was supported, in part, by a grant from the Cooperative Research Program for Agriculture Science &amp; Technology Development (Project No. PJ011077, PJ011288), Rural Development Administration, and the National Research Foundation of Korea Grant funded by the Korean Government (NRF-2013R1A2A2A04008751). These are the results of a study from the Leaders INdustry-University Cooperation Project 2015-B-0013–010118, supported by the Ministry of Education, Republic of Korea.

Epalrestat increases glutathione, thioredoxin, and heme oxygenase-1 by stimulating Nrf2 pathway in endothelial cells

Epalrestat (EPS) is the only aldose reductase inhibitor that is currently available for the treatment of diabetic neuropathy. Recently, we found that EPS at near-plasma concentration increases the intracellular levels of glutathione (GSH) in rat Schwann cells. GSH plays a crucial role in protecting endothelial cells from oxidative stress, thereby preventing vascular diseases. Here we show that EPS increases GSH levels in not only Schwann cells but also endothelial cells. Treatment of bovine aortic endothelial cells (BAECs), an in vitro model of the vascular endothelium, with EPS caused a dramatic increase in intracellular GSH levels. This was concomitant with the up-regulation of glutamate cysteine ligase, an enzyme catalyzing the first and rate-limiting step in de novo GSH synthesis. Moreover, EPS stimulated the expression of thioredoxin and heme oxygenase-1, which have important redox regulatory functions in endothelial cells. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a key transcription factor that regulates the expression of antioxidant genes. EPS increased nuclear Nrf2 levels in BAECs. Nrf2 knockdown by siRNA suppressed the EPS-induced glutamate cysteine ligase, thioredoxin-1, and heme oxygenase-1 expression. Interestingly, LY294002, an inhibitor of phosphatidylinositol 3-kinase, abolished the EPS-stimulated GSH synthesis, suggesting that the kinase is associated with Nrf2 activation induced by EPS. Furthermore, EPS reduced the cytotoxicity induced by H2O2 and tert-butylhydroperoxide, indicating that EPS plays a role in protecting cells from oxidative stress. Taken together, the results provide evidence that EPS exerts new beneficial effects on endothelial cells by increasing GSH, thioredoxin, and heme oxygenase-1 levels through the activation of Nrf2. We suggest that EPS has the potential to prevent several vascular diseases caused by oxidative stress.

Diethyl maleate inhibits MCA+TPA transformed cell growth via modulation of GSH, MAPK, and cancer pathways

Murine or human cancer cells have high glutathione levels. Depletion of the elevated GSH inhibits proliferation of cancer cells. Molecular basis for this observation is little understood. In an attempt to find out the underlying mechanism, we reproduced these effects in transformed C3H10T1/2 and BALB/c 3T3 cells using diethyl maleate and studied cytogenomic changes in the whole mouse genome using spotted 8×60K arrays. Transformed cells revealed an increase in GSH levels. GSH depletion by DEM inhibited the growth of transformed cells. The non-cytotoxic dose of DEM (0.25mM) resulted in GSH depletion, ROS generation, cell cycle arrest, apoptosis, decrease in anchorage independent growth, gene expression changes and activation of all three members of the MAPK family. Increase in intracellular GSH levels by GSHe countered the effect of DEM. These results support the physiological importance of GSH in regulation of gene expression for transformed cell growth restraint. This study is of interest in not only understanding the molecular biology of the transformed cells, but also in identifying new targets for development of gene therapy together with the chemotherapy.

154 THE EFFECT OF ZINC ON PORCINE IN VITRO MATURATION AND SUBSEQUENT EMBRYONIC DEVELOPMENT AFTER IN VITRO FERTILIZATION

Zinc (Zn) is one of the abundant transition metals in biology and is an essential component of most cells. However, there are few reports about the effect of Zn in porcine oocytes. The objective was to investigate the effects of supplementary Zn during in vitro maturation (IVM) of porcine oocytes. We investigated nuclear maturation, intracellular glutathione (GSH) levels, reactive oxygen species (ROS) levels, and subsequent embryonic development after IVF. Before the experiment, Zn concentrations in IVM medium and body fluids were measured using inductively coupled plasma spectrophotometer (sensitivity: 1 μM) and treatment concentrations were determined. Zinc concentration was 12.6 μM in porcine plasma and 12.9 μM in porcine follicular fluid. We confirmed that Zn was not detected in IVM medium. A total of 541 cumulus–oocyte complexes (COC) were used for the evaluation of nuclear maturation. The COC were matured in TCM-199 medium supplemented with various concentrations of Zn (0, 6, 12, 18, and 24 μM). After 44 h of IVM, no significant difference was observed in all groups (metaphase II rate: 85.7, 88.7, 90.4, 90.3, and 87.2%, respectively). A total of 100 matured oocytes were examined for the effects of different Zn concentrations (0, 6, 12, 18, and 24 μM) on porcine oocyte intracellular GSH and ROS levels, which were measured through fluorescent staining and image analysis program. The groups of 12, 18, and 24 μM showed a significant (P &lt; 0.05) increase in intracellular GSH levels (1.45, 1.67, and 1.78, respectively) compared with the control and 6 μM group (1.00 and 1.08, respectively). The intracellular ROS level of oocytes matured with 12, 18, and 24 μM (0.82, 0.68, and 0.55) were significantly (P &lt; 0.05) decreased compared with the control and 6 μM groups (1.00 and 1.03, respectively). Finally, the developmental competence of oocytes matured with different concentrations of Zn (0, 6, 12, 18, and 24 μM) was evaluated after IVF. There were no significantly different in cleavage rates. However, cleavage patterns and blastocyst (BL) formation were different. Fragmented embryo ratio of the 12 μM group (14.9%) was significantly lower than that of the other groups (control, 6, 18, and 24 μM: 26.4, 17.8, 18.4, and 18.0%, respectively). Oocytes treated with 12 μM Zn during IVM had a significantly higher BL formation rate (28.2%) after IVF compared with the control (19.8%). In conclusion, these results indicate that Zn treatment as body fluid concentration during IVM improved the developmental potential of IVF in porcine embryos by increasing the intracellular GSH concentration and decreasing the ROS level. This work was supported, in part, by a grant from the Next-Generation Bio Green 21 Program (No. PJ00956901), Rural Development Administration, and the National Research Foundation of Korea Grant funded by the Korean Government (NRF-2012R1A1A4A01004885, NRF-2013R1A2A2A04008751), Republic of Korea.

Epalrestat increases intracellular glutathione levels in Schwann cells through transcription regulation

Epalrestat (EPS), approved in Japan, is the only aldose reductase inhibitor that is currently available for the treatment of diabetic neuropathy. Here we report that EPS at near-plasma concentration increases the intracellular levels of glutathione (GSH), which is important for protection against oxidative injury, through transcription regulation. Treatment of Schwann cells with EPS caused a dramatic increase in intracellular GSH levels. EPS increased the mRNA levels of γ-glutamylcysteine synthetase (γ-GCS), the enzyme catalyzing the first and rate-limiting step in de novo GSH synthesis. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a key transcription factor that plays a central role in regulating the expression of γ-GCS. ELISA revealed that EPS increased nuclear Nrf2 levels. Knockdown of Nrf2 by siRNA suppressed the EPS-induced GSH biosynthesis. Furthermore, pretreatment with EPS reduced the cytotoxicity induced by H2O2, tert-butylhydroperoxide, 2,2'-azobis (2-amidinopropane) dihydrochloride, and menadione, indicating that EPS plays a role in protecting against oxidative stress. This is the first study to show that EPS induces GSH biosynthesis via the activation of Nrf2. We suggest that EPS has new beneficial properties that may prevent the development and progression of disorders caused by oxidative stress.

The effects of resveratrol on porcine oocyte in vitro maturation and subsequent embryonic development after parthenogenetic activation and in vitro fertilization

We investigated the effects of resveratrol, a phytoalexin with various pharmacologic activities, on in vitro maturation (IVM) of porcine oocytes. We investigated intracellular glutathione (GSH) and reactive oxygen species (ROS) levels, as well as gene expression in mature oocytes, cumulus cells, and in vitro fertilization (IVF)-derived blastocysts, and subsequent embryonic development after parthenogenetic activation (PA) and IVF. After 44 h of IVM, no significant difference was observed in maturation of the 0.1, 0.5, and 2.0 μM resveratrol groups (83.0%, 84.1%, and 88.3%, respectively) compared with the control (84.1%), but the 10.0 μM resveratrol group showed significantly decreased nuclear maturation (75.0%) (P < 0.05). The 0.5- and 2.0-μm groups showed a significant (P < 0.05) increase in intracellular GSH levels compared with the control and 10.0 μM group. Intracellular ROS levels in oocytes matured with 2.0 μM resveratrol decreased significantly (P < 0.05) compared with those in the other groups. Oocytes treated with 2.0 μM resveratrol during IVM had significantly higher blastocyst formation rates and total cell numbers after PA (62.1% and 49.1 vs. 48.8%, and 41.4, respectively) and IVF (20.5% and 54.0 vs. 11.0% and 43.4, respectively) than the control group. Cumulus-oocytes complex treated with 2.0 μM resveratrol showed lower expression of apoptosis-related genes compared with mature oocytes and cumulus cells. Cumulus cells treated with 2.0 μM resveratrol showed higher (P < 0.05) expression of proliferating cell nuclear antigen than the control group. IVF-derived blastocysts derived from 2.0 μM resveratrol-treated oocytes also had less (P < 0.05) Bak expression than control IVF-derived blastocysts. In conclusion, 2.0 μM resveratrol supplementation during IVM improved the developmental potential of PA and IVF porcine embryos by increasing the intracellular GSH level, decreasing ROS level, and regulating gene expression during oocyte maturation.

Cytotoxicity of Benzyl Isothiocyanate in Normal Renal Proximal Tubular Cells and Its Modulation by Glutathione

In the present study, we examined the toxicity of benzyl ITC (BITC) and its urinary mercapturic acid metabolite (BITC-NAC), using a normal renal proximal tubular cell line, pig LLC-PK1. BITC increased cell death with an IC(50) value of about 7 μM, whereas the cytotoxic effect of BITC-NAC was five times weaker than that of BITC. We observed a significant necrosis of the compounds on LLC-PK1 cells with oxidative stress. In the presence of 5 mM glutathione (GSH), comparable to physiological levels, the cytotoxicity of BITC-NAC as well as BITC was significantly reduced. Furthermore, the increase in intracellular GSH levels by pretreatment with NAC before the BITC treatment resulted in inhibition of the BITC-induced necrotic events as well as intracellular oxidative stress. These results suggest that GSH is a determinant of cellular resistance against the BITC-mediated and oxidative stress-dependent cytotoxicity in renal proximal tubular cells.

189 THE EFFECTS OF RESVERATROL ON PORCINE OOCYTES IN VITRO MATURATION AND SUBSEQUENT DEVELOPMENT AFTER PARTHENOGENETIC ACTIVATION AND IN VITRO FERTILIZATION

The present study investigated the effects of resveratrol (a phytoalexin with various pharmacological activities) during in vitro maturation (IVM) of porcine oocytes on nuclear maturation, intracellular glutathione (GSH) and reactive oxygen species (ROS) levels, gene expression in matured oocytes and subsequent embryonic development after parthenogenetic activation (PA) and IVF. Data were analyzed with SPSS 17.0 using Duncan's multiple range test. In experiment 1, a total of 1146 cumulus–oocyte complexes (COC) were divided into 5 groups (0, 0.1, 0.5, 2.0 and 10.0 μM resveratrol). In the nuclear maturation after 44-h IVM, the groups of 0.1, 0.5 and 2.0 μM (83.0, 84.1 and 88.3%, respectively) had no significant difference compared to the control group (84.1%). The group of 10.0 μM decreased the nuclear maturation (75.0%) significantly (P &lt; 0.05). In experiment 2, a total of 300 matured oocytes were examined for the effects of different resveratrol concentrations (0, 0.5, 2.0 and 10.0 μM) on porcine oocyte intracellular GSH and ROS levels. The groups of 0.5 and 2.0 μM showed a significant (P &lt; 0.05) increase in intracellular GSH levels (1.3 and 1.3, respectively) compared with the control and 10.0 μM groups (1.0 and 1.0, respectively). The intracellular ROS level of oocytes matured with 2.0 μM resveratrol (0.4) was significantly (P &lt; 0.05) decreased compared to other groups (control: 1.0; 0.5 μM: 0.6; and 10.0 μM: 0.7). In experiment 3, lower expression of apoptosis-related genes (Bax, Caspase-3 and Bak) was observed in matured oocytes treated with 2.0 μM resveratrol when compared with that of the control (P &lt; 0.05). In experiment 4, a total of 728 oocytes were divided into 4 groups (control, 0.5, 2.0 and 10.0 μM) and examined subsequent to embryonic development after PA. Oocytes treated with 2.0 μM resveratrol during IVM had a significantly higher cleavage (CL) rate, blastocyst (BL) formation rate and total cell numbers (TCN) after PA compared with those of the control (2.0 μM: 96.6%, 62.1% and 49.1 vs control: 88.3%, 48.8% and 41.4, respectively) and the 10.0 μM groups (87.3%, 41.4% and 40.9, respectively). Oocytes treated with 0.5 μM resveratrol (87.2%, 50.5% and 48.6, respectively) during IVM had significantly higher TCN, but there were no differences in CL and BL formation rates. In experiment 5, a total of 935 oocytes in 3 groups (control, 2.0 and 10.0 μM resveratrol) were conducted in IVF. The BL formation rate and TCN were significantly higher in the group of 2.0 μM resveratrol (20.5% and 54.0, respectively) than the control (11.0% and 43.4, respectively) and 10.0 μM group (11.7% and 45.0, respectively), but there was no significant difference in CL rate. In conclusion, 2.0 μM resveratrol supplementation during IVM improved the developmental potential of PA and IVF in porcine embryos by increasing the intracellular GSH concentration, decreasing the ROS level and decreasing apoptosis-related gene expression during oocyte maturation. This work was supported by a grant from the Next-Generation BioGreen 21 Program (No. PJ008121), Rural Development Administration, Republic of Korea.

Efficacy of Glutathione in Ameliorating Sulfur Mustard Analog-Induced Toxicity in Cultured Skin Epidermal Cells and in SKH-1 Mouse Skin In Vivo

Exposure to chemical warfare agent sulfur mustard (HD) is reported to cause GSH depletion, which plays an important role in HD-linked oxidative stress and skin injury. Using the HD analog 2-chloroethyl ethyl sulfide (CEES), we evaluated the role of GSH and its efficacy in ameliorating CEES-caused skin injury. Using mouse JB6 and human HaCaT epidermal keratinocytes, we observed both protective and therapeutic effects of exogenous GSH (1 or 10 mM) in attenuating a CEES-caused decrease in cell viability and DNA synthesis, as well as S and G(2)M phase arrest in cell cycle progression. However, the protective effect of GSH was stronger than its ability to reverse CEES-induced cytotoxic effect. The observed effect of GSH could be associated with an increase in intracellular GSH levels after its treatment before or after CEES exposure, which strongly depleted cellular GSH levels. N-Acetyl cysteine, a GSH precursor, also showed both protective and therapeutic effects against CEES-caused cytotoxicity. Buthionine sulfoximine, which reduces cellular GSH levels, caused an increased CEES cytotoxicity in both JB6 and HaCaT cells. In further studies translating GSH effects in cell culture, pretreatment of mice with 300 mg/kg GSH via oral gavage 1 h before topical application of CEES resulted in significant protection against CEES-caused increase in skin bifold and epidermal thickness, apoptotic cell death, and myeloperoxidase activity, which could be associated with increased skin GSH levels. Together, these results highlight GSH efficacy in ameliorating CEES-caused skin injury and further support the need for effective antioxidant countermeasures against skin injury by HD exposure.

Transient Glutathione Depletion Determines Terminal Differentiation in HL-60 Cells

To better define the role of glutathione (GSH) in cell differentiation, the present study measured GSH concentrations during terminal HL-60 cell differentiation, in the presence and absence of differentiation-inducing agents, and in the presence and absence of GSH altering agents. Interestingly, there was a small transient increase in intracellular GSH levels during dimethyl sulfoxide (DMSO) or 1alpha,25-dihydroxyvitamin D3 (VD3) induced differentiation. This increase coincided with an increase in nitroblue tetrazolium (NBT) reduction capacity, a measure of superoxide anion production, but there was no apparent change in the GSH/glutathione disulfide (GSSG) ratio. Surprisingly, treatment of cells with low doses of 1-chloro-2,4-dinitrobenzene (CDNB; 5 microM) or diethylmaleate (DEM; 0.5 mM), which transiently deplete GSH levels to about 40% of control levels, resulted in enhanced differentiation of HL-60 cells exposed to VD3 or all-trans-retinoic acid (ATRA), as well as under un-induced conditions (i.e., spontaneous differentiation). Enhanced differentiation occurred when cells were treated with the GSH-depleting agents 4 hours after treatment with differentiation inducers. These findings indicate that intracellular GSH levels are regulated in a complex fashion during HL-60 cell differentiation, and that transient GSH depletion using low doses of CDNB and DEM enhances the differentiation process.

Benzo[ a]pyrene-induced elevation of GSH level protects against oxidative stress and enhances xenobiotic detoxification in human HepG 2 cells

Glutathione (GSH) is one of the most important antioxidants in mammalian cells. It also plays an important role in chemical detoxification. Some evidence showed that polycyclic aromatic hydrocarbons, such as benzo[ a]pyrene (B[ a]P [50-32-8]), could increase GSH content as a defense mechanism against oxidative stress as well as to promote its detoxification. However, there has been very little study on clarifying the role GSH plays in antioxidation and detoxification actions. Therefore, the present study aims to analyze intracellular glutathione metabolism in the human hepatoma cells (HepG2) upon exposure to B[ a]P. Exposure of the cells to B[ a]P (1–100 μM) for 24 h did not cause significant cell death in this cell line. By selecting the sublethal concentration of 10 μM, B[ a]P caused a significant increase in GSH and a small (13%) but significant decrease in glutathione reductase activity. However, there was no change in the activity of glutathione peroxidase, and no detectable increase in reactive oxygen species (ROS) production. Treatment with B[ a]P caused up to 1.5 folds increase in γ-glutamylcysteine synthatase (γ-GCS) activity over control. Buthioneine sulfoximine (BSO), an inhibitor of γ-GCS, could suppress GSH increase in a dose-dependent manner. Assessment of the oxidative state of the cells indicated that the increase in GSH caused the cells to become more reduced. Thus, the results concluded that cells were not suffering from oxidative stress at 24 h after treatment with 10 μM B[ a]P. Upon analyzing the activities of detoxification enzymes, there was an increase in the activity of CYP1A subfamily monooxygenases and glutathione S-transferase. Both changes occurred prior to the changes in γ-GCS activity and the increase in GSH. In summary, results of the present study demonstrate that B[ a]P caused an activation of detoxification enzymes. The increase in intracellular GSH level was due to activation of γ-GCS activities. Oxidative stress may not be an important risk factor for B[ a]P (at 10 μM of up to 24 h) induced injury.

Dexamethasone-resistant human Pre-B leukemia 697 cell line evolving elevation of intracellular glutathione level: an additional resistance mechanism.

Glucocorticoids remain among the most important drugs in the treatment of acute lymphoblastic leukemia (ALL). Although the mechanisms of glucocorticoid resistance have been studied in some T‐cell leukemic cell lines, less work has been done with B‐cell lines. We established a dexamethasone (DEX)‐resistant human pre‐B lineage leukemia cell line (697/DEX) and investigated the mechanism of resistance. 697/DEX was over 430–fold more resistant to DEX compared with the parental cells (697/Neo). Overexpression of Bcl–2 protein was not observed in 697/DEX, different from the mechanism of resistance in Bcl–2–virus‐infected cells (697/Bcl–2). Although the expression of p‐glycoprotein (Pgp) in 697/DEX was positive, its functional activity was not detected. The numbers of glucocorticoid receptors (GR) in 697/DEX and 697/Bcl–2 were significantly lower than those in 697/Neo. In addition, 697/DEX and 697/Bcl–2 had higher levels of glutathione (GSH) than 697/Neo. In the presence of l‐buthionine‐(S, R)‐sulfoximine (BSO), an inhibitor of GSH synthesis, both 697/DEX and 697/Bcl–2 recovered their sensitivity to DEX. Interestingly, cell death by the depletion of GSH did not involve caspase–3/7 activation in 697/Bcl–2 and 697/DEX, different from 697/Neo, suggesting a death mechanism through caspase‐independent programmed cell death or necrosis. In conclusion, DEX‐resistance in 697/DEX was related not only to a GR decrease, but also to an increase in intracellular GSH level in the DEX‐resistant B‐cell leukemia cell line. Circumvention of DEX‐resistance with BSO may offer an approach to overcoming resistance to chemotherapy in B‐cell lineage ALL.

Mechanism of resistance to oxidative stress in doxorubicin resistant cells.

Doxorubicin (DOX) is an anthracycline drug widely used in chemotherapy for cancer patients, but it often gives rise to multidrug resistance in cancer cells. The purpose of this work was to study the effect of hydrogen peroxide in DOX-sensitive mouse P388/S leukemia cells and in the DOX-resistant cell line. Hydrogen peroxide induced a significant increase in dose- and time-response cell death in cultured P388/S cells. The degree of cell death in P388/DOX cells induced by hydrogen peroxide was less than that in P388/S cells treated with hydrogen peroxide. Parent cells exposed to 3 mM of hydrogen peroxide showed a loss of mitochondrial membrane potential correlated with cell death. Hydrogen peroxide at a concentration greater than 0.3 mM increased the intracellular Ca2+ of P388/S cells dose-dependently; however, no change following addition of hydrogen peroxide (0.3-1 mM) was observed in the resistant cells. Hydrogen peroxide (0.1 and 1 mM) treatment also induced the production of intracellular ROS in P388/S cells, while no such increase was produced by this substance in P388/DOX cells. Resistant cells also showed a significant level of glutathione (GSH) compared with the parent cells. In addition, N-acetyl-L-cysteine and reduced GSH antioxidants abolished death of P388/S cells caused by hydrogen peroxide. Therefore, it is believed that the reduced effect of oxidative stress towards the resistant cells may be related to an increase in intracellular GSH level.

Effect of vitamin E on the degradation of hydrogen peroxide in cultured human umbilical vein endothelial cells

Vitamin E reacts with radicals such as lipid peroxyl radical (LOO·) and singlet oxygen ( 1O 2), and plays a role in inhibiting lipid peroxidation in cell membranes and preventing the oxidation of low-density lipoproteins (LDL). However, only a few studies have investigated the effect of vitamin E on the degradation of hydrogen peroxide (H 2O 2). Therefore, we examined the effect of vitamin E on glutathione redox cycle-dependent H 2O 2 degradation activity in human umbilical vein endothelial cells (HUVEC). Confluent HUVEC were cultured for seven days in media containing various concentrations of vitamin E (α-tocopherol). The level of glutathione redox cycle-dependent H 2O 2 degradation activity and the intracellular glutathione level were determined. HUVEC that had been cultured in the presence of higher concentrations of vitamin E had a higher level of H 2O 2 degradation activity and a higher intracellular content of the reduced form of glutathione (GSH). Therefore, it is suggested that the vitamin E-induced increase in H 2O 2 degradation activity in HUVEC results from an increase in intracellular GSH level.

Glycine facilitates γ-glutamylcysteinylethyl ester-mediated increase in liver glutathione level

γ-glutamylcysteinylethyl ester ( γ-GCE) increases reduced glutathione (GSH) levels in GSH-depleted rat hepatocytes. Because glycine, a constituent of GSH, exists at 0.3 to 0.4 mM in rat plasma, we examined the influence of glycine added to the medium on the action of γ-GCE to increase GSH levels in the rat hepatocytes. Glycine (0.2-0.8 mM) dose-dependently enhanced γ-GCE-mediated increase in intracellular GSH levels with an increase in intracellular γ-GCE levels. These results indicate that exogenous glycine facilitates γ-GCE-mediated increase in intracellular GSH levels in rat hepatocytes possibly by enhancing the uptake of γ-GCE into the cells.

4-Hydroxy-2-nonenal Cytotoxicity in Renal Proximal Tubular Cells: Protein Modification and Redox Alteration

4-Hydroxy-2-nonenal (HNE), one of the major products of membrane lipid peroxidation, has been shown recently to be present in a form covalently attached to proteins in the renal proximal tubules of rats treated with a renal carcinogen, ferric nitrilotriacetate (Toyokuni, S.,et al.(1994)Proc. Natl. Acad. Sci. USA91, 2616–2620; Uchida, K.,et al.(1995)Arch. Biochem. Biophys.317, 405–411). In the present study, the mechanism of HNE cytotoxicity was studied using the renal tubular epithelial cells (LLC-PK1), focusing on the protein modification and alteration of cellular redox status induced by HNE. Upon treatment with HNE for 2 h, the LLC-PK1cells were found to be resistant to the low concentration (10 μM) of HNE, while HNE at higher concentrations (≥50 μM) mediated cell death. The cytotoxicity of HNE appeared to be correlated with the HNE modification of cellular proteins. Among a number of proteins modified by HNE, a glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase was detected as one of the major targets of HNE in the cells. On the other hand, exposure of LLC-PK1cells to HNE resulted in rapid reduction of cellular glutathione (GSH) levels, suggesting that HNE influenced primarily the redox status of the cells. Depletion of GSH with buthionine sulfoximine, a potent suppressor of GSH biosynthesis, before HNE treatment caused the cells to be sensitive to HNE cytotoxicity and to HNE modification of cellular proteins, whereas the increase in intracellular GSH levels by treatment withN-acetylcysteine before HNE treatment resulted in a dose-dependent inhibition of HNE-mediated protein modification. These results suggest that intracellular GSH is a determinant on cellular resistance against the HNE-mediated cytotoxicity.

Mechanisms of use of extracellular glutathione by lung epithelial cells and pulmonary artery endothelial cells.

Cells in most culture media use cystine as the primary source of the cysteine precursor needed for glutathione (GSH) synthesis. As a result, GSH levels in many cultured cells may be limited by the rate of uptake of cystine into cells. We have shown that incubation with extracellular GSH can result in the reaction of GSH with cystine to generate cysteine, and that bovine pulmonary artery endothelial cells and lung type II epithelial cells transported cysteine more efficiently than cysteine. Cysteine transport was not affected by the presence of GSH. In cells incubated with GSH in RPMI-1640 there was a cystine-dependent increase in intracellular GSH levels. The increases in GSH were not prevented by the presence of acivicin, an inhibitor of the gamma-glutamyl transpeptidase reaction. Incubation with oxidized glutathione (GSSG) did not result in significant increases in intracellular GSH levels. We conclude that a primary mechanism by which extracellular GSH may increase intracellular GSH levels in cultured cells is by reducing cystine to cysteine, which is then rapidly transported and used as a substrate for intracellular GSH synthesis.