Buthionine Sulfoximine Pretreatment Research Articles

Abstract 5485: The combined effects of polyamine and GSH inhibition on the viability of human cancer cell lines

Abstract Polyamines play essential roles in DNA stability and cell proliferation, making polyamine metabolism a potential target for cancer therapeutics. The spermine analog N1,N11- Diethylnorspermine (DenSpm) reduces levels of ODC, the enzyme responsible for polyamine biosynthesis, and also induces the enzyme SSAT, responsible for the catabolism of polyamines. Polyamines have been shown to inactivate reactive oxygen species (ROS), including peroxides, protecting mammalian cells from oxidative damage to nucleic acids. The catabolism of polyamines by SSAT, in contrast, leads to the production of ROS, which results in cellular death and limits the progression of cancer. Buthionine sulfoximine (BSO) selectively depletes stores of intracellular glutathione GSH, an antioxidant that neutralizes ROS. We hypothesized that treating cancer cell lines with both DenSpm and BSO will have a synergistic cytotoxic effect on the cells, due to the depletion of ROS-scavenging polyamines, the generation of ROS caused by DenSpm and the absence of GSH. Treatment of the four cell lines, MCF-7, MDA-MD-231, Caco-2 and PANC-1, with DenSpm resulted in moderate reductions in cell viability as measured using the Cell Titer Blue assay (Promega, Inc.). As has been widely reported in other mammalian systems, DenSpm (10 µM) reduced intracellular polyamine concentrations (HPLC of benzoylated polyamine derivatives) and induced SSAT (Western Blots). Treatment of cells with BSO (500 µM) caused a significant decrease in intracellular GSH levels. Similar findings have been reported in previous studies with human neuroblastoma lines (Anderson, et al. 1999). The combination of BSO and DenSpm had effects markedly greater than either compound alone. Pretreatment of cells with BSO (500 µM) for 24 hours with subsequent removal of BSO before DenSpm (10, 100, 1000 µM) addition resulted in significantly reduced viability in all cell lines. DenSpm was found to be most effective in the continued presence of BSO as compared to a 24-hour BSO pretreatment. The compounds used in combination also enhanced ROS production, further demonstrating their synergistic effects. Citation Format: John Grady, Davey Holzer, Kaci Keleher, Emma Meetz, Russ Feirer. The combined effects of polyamine and GSH inhibition on the viability of human cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5485. doi:10.1158/1538-7445.AM2017-5485

Radiation-induced surge of macrophage foam cell formation, oxidative damage, and cytokine release is attenuated by a nanoformulation of curcumin

Purpose: We examined the potential of a dendrosomal nanoformulation of curcumin (DNC) for intervention of ionizing radiation (IR)-induced damage (particularly leading to atherosclerosis), employing an irradiated THP-1 macrophage model.Materials and methods: Differentiated THP-1 macrophages were irradiated and treated with curcumin or DNC nanoformulation (and oxidized low density lipoprotein, ox-LDL, to promote foam cells). Chemical, biochemical, and genetics tools including viability and apoptosis, multiple ELISA, real-time PCR, Western blotting, enzyme activity, and fluorimetry assays were employed to illustrate IR damage as well as the DNC intervention potential.Results: DNC per se at 10 μM exerted no cytotoxic effects on macrophages. However, it caused apoptosis in 2 Gy-irradiated macrophages which were treated with ox-LDL, chiefly through a caspase-dependent pathway involving caspase-3. Concurrently, 10 μM DNC prevented the IR-induced rise in lipid accumulation (72% decrease compared to IR control, p < .0001), dil-oxLDL uptake (78% decrease, p < .005), protein and mRNA expression of cholesterol influx genes, CD36 and SR-A, NF-κB activation (81% less binding activity, p < .001; and lower nuclear presence of p65), cytokine (monocyte chemoattractant protein-1 and interleukin-1β) release, reactive oxygen species (ROS), and oxidative damage to DNA (37% decrease in 8-OHdG, p < .05) and lipids (62% decrease in 8-isoprostane, p < .005). DNC facilitated the uptake of curcumin in irradiated macrophages, increased glutathione peroxidase expression and activity, restored glutathione (GSH) level, and upregulated the expression of a cholesterol efflux gene, ABCA1. Two other antioxidants, resveratrol and N-acetyl cycteine (NAC), could simulate some of the beneficial effects of DNC against IR-induced CD36 expression and lipid accumulation, which were obviated by buthionine sulfoximine (BSO) pre-treatment of macrophages. However, some modulatory effects of DNC, particularly on lipid accumulation and the expression of SR-A and ABCA1 genes, seemed to be independent of its antioxidant effect, since they were still observed in BSO-pretreated macrophages, depleted of GSH.Conclusions: DNC treatment suppresses IR-induced oxidative damage, inflammation, and foam cell formation in macrophages through multiple mechanisms.

Carbon black nanoparticles promote endothelial activation and lipid accumulation in macrophages independently of intracellular ROS production.

Exposure to nanoparticles (NPs) may cause vascular effects including endothelial dysfunction and foam cell formation, with oxidative stress and inflammation as supposed central mechanisms. We investigated oxidative stress, endothelial dysfunction and lipid accumulation caused by nano-sized carbon black (CB) exposure in cultured human umbilical vein endothelial cells (HUVECs), THP-1 (monocytes) and THP-1 derived macrophages (THP-1a). The proliferation of HUVECs or co-cultures of HUVECs and THP-1 cells were unaffected by CB exposure, whereas there was increased cytotoxicity, assessed by the LDH and WST-1 assays, especially in THP-1 and THP-1a cells. The CB exposure decreased the glutathione (GSH) content in THP-1 and THP-1a cells, whereas GSH was increased in HUVECs. The reactive oxygen species (ROS) production was increased in all cell types after CB exposure. A reduction of the intracellular GSH concentration by buthionine sulfoximine (BSO) pre-treatment further increased the CB-induced ROS production in THP-1 cells and HUVECs. The expression of adhesion molecules ICAM-1 and VCAM-1, but not adhesion of THP-1 to HUVECs or culture dishes, was elevated by CB exposure, whereas these effects were unaffected by BSO pre-treatment. qRT-PCR showed increased VCAM1 expression, but no change in GCLM and HMOX1 expression in CB-exposed HUVECs. Pre-exposure to CB induced lipid accumulation in THP-1a cells, which was not affected by the presence of the antioxidant N-acetylcysteine. In addition, the concentrations of CB to induce lipid accumulation were lower than the concentrations to promote intracellular ROS production in THP-1a cells. In conclusion, exposure to nano-sized CB induced endothelial dysfunction and foam cell formation, which was not dependent on intracellular ROS production.

Pretreatment with buthionine sulfoximine enhanced uptake and retention of BSH in brain tumor

To determine the influence of buthionine sulfoximine (BSO) on boron biodistribution after sulfhydryl borane (BSH) administration for boron neutron capture therapy, the effectiveness of the combination of BSO with sulfhydril- (BSH) and non-sulfhydril (B12H12 and BNH3) boron compounds, and the interval between BSO and BSH administration, the retention of boron in tissues have been evaluated using a 9L rat tumor model. Simultaneous administration of BSH and BSO showed significantly higher boron accumulation compared to that without BSO, however there was no difference in tissue boron level between B12H12 and BNH3 administration with BSO or without BSO. The longer interval (6h) between BSH and BSO administration related to the highest boron concentration in the brain and subcutaneous tumors compared to shorter intervals (0.5, 3h). Boron concentration in subcutaneous and brain tumors was maintained for 6 and 12h after the administration of BSH following BSO pretreatment.

Oxidative stress and phthalate-induced down-regulation of steroidogenesis in MA-10 Leydig cells

Previous studies have shown that phthalate exposure can suppress steroidogenesis. However, the affected components of the steroidogenic pathway, and the mechanisms involved, remain uncertain. We show that incubating MA-10 Leydig cells with mono-(2-ethylhexyl) phthalate (MEHP) resulted in reductions in luteinizing hormone (LH)-stimulated cAMP and progesterone productions. cAMP did not decrease in response to MEHP when the cells were incubated with cholera toxin or forskolin. Incubation of MEHP-treated cells with dibutyryl-cAMP, 22-hydroxycholesterol or pregnenolone inhibited the reductions in progesterone. Increased levels of reactive oxygen species (ROS) occurred in response to MEHP. In cells in which intracellular glutathione was depleted by buthionine sulfoximine pretreatment, the increases in ROS and decreases in progesterone in response to MEHP treatment were exacerbated. These results indicate that MEHP inhibits MA-10 Leydig cell steroidogenesis by targeting LH-stimulated cAMP production and cholesterol transport, and that a likely mechanism by which MEHP acts is through increased oxidative stress.

Neurotoxicity of “ecstasy” and its metabolites in human dopaminergic differentiated SH-SY5Y cells

"Ecstasy" (3,4-methylenedioxymethamphetamine or MDMA) is a widely abused recreational drug, reported to produce neurotoxic effects, both in laboratory animals and in humans. MDMA metabolites can be major contributors for MDMA neurotoxicity. This work studied the neurotoxicity of MDMA and its catechol metabolites, α-methyldopamine (α-MeDA) and N-methyl-α-methyldopamine (N-Me-α-MeDA) in human dopaminergic SH-SY5Y cells differentiated with retinoic acid and 12-O-tetradecanoyl-phorbol-13-acetate. Differentiation led to SH-SY5Y neurons with higher ability to accumulate dopamine and higher resistance towards dopamine neurotoxicity. MDMA catechol metabolites were neurotoxic to SH-SY5Y neurons, leading to caspase 3-independent cell death in a concentration- and time-dependent manner. MDMA did not show a concentration- and time-dependent death. Pre-treatment with the antioxidant and glutathione precursor, N-acetylcysteine (NAC), resulted in strong protection against the MDMA metabolites' neurotoxicity. Neither the superoxide radical scavenger, tiron, nor the inhibitor of the dopamine (DA) transporter, GBR 12909, prevented the metabolites' toxicity. Cells exposed to α-MeDA showed an increase in intracellular glutathione (GSH) levels, which, at the 48 h time-point, was not dependent in the activity increase of γ-glutamylcysteine synthetase (γ-GCS), revealing a possible transient effect. Importantly, pre-treatment with buthionine sulfoximine (BSO), an inhibitor of γ-GCS, prevented α-MeDA induced increase in GSH levels, but did not augment this metabolite cytotoxicity. Even so, BSO pre-treatment abolished NAC protective effects against α-MeDA neurotoxicity, which were, at least partially, due to GSH de novo synthesis. Inversely, pre-treatment of cells with BSO augmented N-Me-α-MeDA-induced neurotoxicity, but only slightly affected NAC neuroprotection. In conclusion, MDMA catechol metabolites promote differential toxic effects to differentiated dopaminergic human SH-SY5Y cells.

Cadmium is a catalytic inhibitor of DNA topoisomerase II

Cadmium (Cd(2+)) is a highly toxic and carcinogenic metal that is an environmental and occupational hazard. DNA topoisomerase II is an essential nuclear enzyme and its inhibition can result in the formation of genotoxic and recombinogenic DNA double strand breaks. In this study we showed that cadmium chloride strongly inhibited the DNA decatenation activity of human topoisomerase IIα in the low micromolar concentration range and that its inhibitory effects were reduced by glutathione. Because the activity of topoisomerase II is strongly inhibited by thiol-reactive compounds this result suggested that cadmium may be binding to critical topoisomerase II cysteine thiols. Cadmium, however, did not stabilize DNA-topoisomerase II covalent complexes, as measured by the lack of formation of DNA double strand breaks. Hence, it is not likely to be a topoisomerase II poison. Consistent with the idea that cadmium cytotoxicity may be modulated by glutathione levels, buthionine sulfoximine pretreatment to decrease glutathione levels resulted in a greatly increased cadmium-induced cytotoxicity in K562 cells. The results of this study suggest that cadmium may exert some of its cell growth inhibitory, and possibly its toxicity and carcinogenicity, by inhibiting topoisomerase IIα through reaction with critical cysteine thiols.

UVC Radiation-induced Effect on Human Primary Thyroid Cell Proliferation and HLA-DR Expression

The aim of this study was to examine how UVC irradiation will affect normal human thyroid cell proliferation and HLA-DR expression. Primary human thyroid cells were exposed to UVC (254 nm wavelength) irradiation. In some experiments 0.5 mM buthionine sulfoximine (BSO) was added. Apoptosis was detected measuring annexin V, proteins involved in apoptotic process (p53, Bax, Bcl-2, caspase 3, and 9) by immunoblot analysis and HLA-DR expression by FACS. UVC induced a cell cycle arrest in G0/G1 phase in the first 24 h, accumulation of cells in the S phase 72 h after treatment, and an increase of apoptotic cells. BSO pretreatment showed an earlier appearance and a higher percentage of apoptosis. p53, caspase 3 and 9 were increased, while Bax and Bcl-2 were decreased. We also observed a transient significant increase in HLA-DR expression. UVC inhibited cell proliferation and induced apoptosis in normal human primary thyroid cells. An inhibitor of glutathione synthesis induced an earlier appearance and higher percentage of apoptosis suggesting that oxidative stress may play a role. Apoptotis involved components of the intrinsic mitochondrial pathway. A transient increase in HLA-DR expression after UVC irradiation could play a role in inducing AITD.

Critical role of GSH in Sulfur Mustard-induced Oxidative Stress and Cytotoxicity in Human Skin Fibroblast Cell Line

In this study the role of glutathione (GSH) in sulfur mustard -induced oxidative stress and cytotoxicity, in human skin fibroblast cell line (HF2FF) was evaluated. Sulfur mustard-induced superoxide radical and hydrogen peroxide formation were evaluated by determination of superoxide dismutase and catalase activity in cell lysate. The cytotoxicity of sulfur mustard was estimated by lactate dehydrogenase leakage. The intracellular GSH content was modulated by N-acetylcysteine (NAC), a GSH precursor, and buthionine sulfoximine (BSO), a specific GSH synthesis inhibitor. It was found that sulfur mustard exposure led to a dose-and time-dependent decrease in GSH content in HF2FF cells. NAC increased intracellular GSH level and protected the cells against sulfur mustard-induced reactive oxygen species formation and lactate dehydrogenase leakage. In contrast, buthionine sulfoximine pretreatment depleted cellular GSH and enhanced the susceptibility of HF2FF to the cytotoxic effects of sulfur mustard. These results indicated that GSH plays a critical role in protecting HF2FF cell line against sulfur mustar-induced cell injury, most probably through its antioxidant activity.

Influence of glutathione on the induction of chromosome aberrations, delay in cell cycle kinetics and cell cycle regulator proteins in irradiated mouse bone marrow cells

Purpose: Reduced glutathione (GSH) is an endogenous thiol and has long been thought to affect the sensitivity of cells to radiation. The aim was to see the influence of GSH on: (i) the production of all types of radiation-induced chromosome aberrations (CA), and (ii) the radiation-induced delay in cell cycle and the levels of cell cycle regulator proteins.Materials and Methods: Cell cycle kinetics were determined by scoring the mitotic index (MI). CA and MI were scored in γ-irradiated buthionine sulfoximine (BSO) (10 h) or GSH (1 h) pretreated and untreated mouse bone marrow cells (BMC). The expression of p53 and p21 proteins after 2 and 6 h of irradiation and for the B-cell lymphoma 2 (Bcl-2) associated X-protein (Bax) after 24 h of irradiation with or without BSO or GSH treatment was analyzed by immunoblotting.Results: Radiation delays mouse BMC in their passage through the cell cycle and induces CA. Exogenous addition of GSH protected CA uniformly at lower doses of radiation but differentially at higher doses, whereas GSH-depletion by BSO increased the frequency of radiation-induced CA. Both GSH and BSO-pretreated cells reduced the delay in cell kinetics after irradiation. Levels of both p53 and p21 were enhanced after irradiation to BSO-pretreated cells. However, in GSH-pretreated cells the level of these proteins was reduced.Conclusion: Data indicate that the induction of CA and delay in cell kinetics by radiation may not always be interlinked and that the level of endogenous GSH exerts its influence on these parameters. Both GSH and BSO pretreatment reduce delays in cell kinetics of irradiated cells which may die apoptotically, since they have either a higher frequency of exchange aberrations or CA, respectively.

Cadmium-induced non-apoptotic cell death mediated by oxidative stress under the condition of sulfhydryl deficiency

Cadmium (Cd), which accumulates primarily in the liver and the kidney, induces apoptosis and also causes necrotic cell death in certain pathophysiologic situations. Previously, we have shown that Cd activated mitogen-activated protein kinases and that sulfur amino acid deficiency potentiated Cd-induced cytotoxicity via activation of mitogen-activated protein kinases. In the present study, we established the mechanistic basis of apoptotic and non-apoptotic cell death induced by Cd in H4IIE cells a rat-derived hepatocyte cell line. Cd at 0.3-10 microM decreased viability of cells in a concentration-dependent manner. Cd-induced cytotoxicity was enhanced by pretreatment with buthionine sulfoximine (BSO). Cd at 0.3 microM induced translocation of Bad to mitochondria, decreased the level of mitochondrial BcL(XL) with the release of cytochrome c, and induced procaspase-9 activation and poly(ADP-ribose) polymerase (PARP) cleavage. Sulfhydryl deficiency by BSO, however, blocked PARP cleavage in spite of the decrease in procaspase-9. Cytochrome c release, procaspase-9 activation and PARP cleavage were all increased by 1 microM Cd irrespective of BSO pretreatment. We also used H(2)O(2) (10-100 microM) as a source of oxidative stress. Cd (0.3-1 microM) + H(2)O(2) (70 microM) resulted in greater extents of cytochrome c release, procaspase-9 activation and PARP cleavage in H4IIE cells than Cd alone. Flow cytometric analysis confirmed apoptotic and non-apoptotic cell death by Cd depending on cellular glutathione (GSH) content. These results provide evidence that Cd at the physiologically obtainable concentration causes non-apoptotic cell death under the condition of sufhydryl deficiency, whereas Cd at the micromolar level induces apoptosis. The cell death mechanism involves cytochrome c release from mitochondria and decrease in the level of procaspase-9, but not PARP cleavage, implying that alterations in cellular sulfhydryls may be the major determining factor for the path of cell death in response to low level of Cd.

The role of thiol reduction in hydroquinone-induced apoptosis in HEK293 cells

Hydroquinone (HQ) is a chemical used as a reducing agent, antioxidant, polymerization inhibitor, and chemical intermediate. It has a minor use as a bleaching agent in dermatologic preparations. HQ also occurs as a main metabolite of benzene. In the present study, HQ-induced apoptosis was evaluated by cell morphology changes, determination of phosphatidylserine (PS) externalization and analysis of sub-G1 cells. The effect of HQ on intracellular thiol concentration, including glutathione and protein thiol, and the effect of N-acetylcysteine (NAC) and buthionine sulfoximine (BSO) pretreatment on HQ-induced apoptosis were investigated. The results showed that HQ was able to induce typical apoptosis in HEK293 cells (human embryonic kidney cells) in a dose-dependent manner. Intracellular thiol, including glutathione and protein thiol, was decreased following treatment with HQ. NAC, a precursor of intracellular GSH synthesis, significantly inhibited HQ-induced apoptosis. However, BSO, a specific inhibitor of intracellular GSH synthesis, enhanced HQ-induced apoptosis significantly. Taken together, the present study demonstrates that HQ is able to induce apoptosis in HEK293 cells, most probably through depletion of intracellular thiol. The results also suggest that, at least in HEK293 cells, the control of intracellular redox homeostasis has a central role in the regulation of cell death induced by HQ.

Induction of c-fos expression by nicotine in human periodontal ligament fibroblasts is related to cellular thiol levels.

Cigarette smoking is associated with increased incidence of periodontal disease and poor response to periodontal therapy. Several studies have shown the detrimental effects of nicotine on periodontal tissue. To investigate the molecular toxicological implications of cigarette smoking on periodontal tissue, expression of c-fos early stress response gene was examined in human periodontal ligament fibroblasts (PDLFs) after exposure to nicotine. The exposure of quiescent human PDLFs to nicotine resulted in the induction of c-fos mRNA expression. The levels of the c-fos mRNAs increased about 2.5 and 4.8-fold after exposure to 2.5 mm and 10 mm nicotine for 2 h, respectively. Moreover, the peak of c-fos mRNA levels induced by nicotine was 5 mm at 2-h incubation period. Kinetic investigations of c-fos mRNA expression in nicotine-treated cells revealed a rapid accumulation of the transcript, a significant signal first detectable after 30 min of exposure. This increase was transient and the level of c-fos mRNAs returned rapidly to that of control cells by 8 h. To determine whether thiol levels were important in induction of c-fos by nicotine, we pretreated cells with the glutathione (GSH) precursor, 2-oxothiazolidine-4-carboxylic acid (OTZ), to boost thiol levels, or buthionine sulfoximine (BSO) to deplete GSH. Our results demonstrate that OTZ pretreatment decreased in c-fos mRNA level and BSO pretreatment enhanced in c-fos mRNA level after exposure to nicotine. In addition, nicotine significantly depleted intracellular GSH in a dose-dependent manner (P < 0.05). At a concentration of 5 mm and 20 mm, nicotine depleted about 22.2% and 56% of GSH, respectively. Taken together, c-fos gene expression might be one signal transduction pathway linked to the induction of early response genes by cigarette smoking. These results suggest that the nicotine-dependent stress-specific expression of the c-fos gene correlates with cellular thiol levels in human PDLFs.

Prior induction of heme oxygenase-1 with glutathione depletor ameliorates the renal ischemia and reperfusion injury in the rat

Heme oxygenase (HO)-1 catalyzes the rate-limiting step in heme degradation releasing iron, carbon monoxide, and biliverdin. Induction of HO-1 occurs as an adaptive and protective response to oxidative stress. Ischemia and reperfusion (IR) injury seems to be mainly caused by the oxidative stress. In this study, we have examined whether prior induction of HO-1 with buthionine sulfoximine (BSO), a glutathione (GSH) depletor, affects the subsequent renal IR injury. BSO (2 mmol/kg body weight) was administered intraperitoneally into rats, the levels of HO-1 protein increased within 4 h after the injection. When BSO was administered into rats at 5 h prior to the renal 45 min of ischemia, the renal IR injury was assessed by determining the levels of blood urea nitrogen and serum creatinine, markers for renal injury, after 24 h of reperfusion. The renal injury was significantly improved as compared to the rats treated with IR alone. Administration of zinc-protoporphyrin IX, an inhibitor of HO activity, reduced the efficacy of BSO pretreatment on the renal IR injury. Our findings suggest that the prior induction of HO-1 ameliorates the subsequent renal IR injury.

Renal toxicity caused by cisplatinum in glutathione-depleted metallothionein-null mice

To elucidate the protective role of metallothionein (MT) and glutathione (GSH) in renal toxicity caused by cisplatinum (cis-DDP), we examined the sensitivity of GSH-depleted MT-null mice to the renal toxicity of cis-DDP. Blood urea nitrogen and creatinine values in the serum, and histopathological change in the kidney were utilized as indicators of nephrotoxicity caused by cis-DDP. Although cis-DDP exerted renal toxicity in MT-null mice and wild-type mice, the toxicity was more conspicuous in the MT-null mice than in the wild-type mice. Moreover, renal toxicity caused by cis-DDP was enhanced significantly by a decrease in the renal GSH level by buthionine sulfoximine (BSO) pretreatment in both kinds of mice. The cis-DDP-caused nephrotoxicity that was enhanced by BSO-mediated GSH depletion was much more severe in the MT-null mice than in the wild-type mice. However, preadministration of zinc sulfate cancelled the BSO-enhanced, cis-DDP-dependent renal toxicity in the wild-type mice, but not in the MT-null mice. In the present study, we found that MT and GSH play an important, cooperative role in detoxification of severe kidney damage caused by cis-DDP. Moreover, the renal MT preinduced by zinc could protect mice from cis-DDP nephrotoxicity enhanced by GSH depletion.

Enhanced hydroxychavicol-induced cytotoxic effects in glutathione-depleted HepG2 cells

Hydroxychavicol (HC) is the major safrole urinary metabolite in rats and humans. The cytotoxic potential of HC in metabolically competent cells has yet to be studied. HC alone was slightly toxic to HepG2 cells. However, the cytotoxicity increased significantly (P<0.05) when HepG2 cells were pretreated with buthionine sulfoximine (BSO), suggesting that endogenous glutathione participates in HC-induced cytotoxicity. Addition of catalase or N-acetylcysteine prevented the BSO plus HC-mediated cytotoxicity. HC also increased 8-hydroxy-2'-deoxyguanosine formation and apoptosis in BSO-pretreated HepG2 cells and this increase could also be suppressed by catalase. These data suggest that BSO pretreatment enhanced HC-induced cytotoxic effects in HepG2 cells, which are related to oxidative DNA damage.

Ebselen Induces Apoptosis in HepG2 Cells through Rapid Depletion of Intracellular Thiols

Ebselen, 2-phenyl-1,2-benzisoselenazol-3(2H)-one, is a synthetic seleno-organic compound with antioxidant capability. In the present study, we systematically examined the ability of ebselen to induce apoptosis in a human hepatoma cell line, HepG(2). Ebselen-induced apoptosis was evaluated by (i) TdT-mediated dUTP nick end labeling assay; (ii) analysis of sub-G1 cells; (iii) cell morphology, including cell size and granularity examination; and (iv) DNA gel electrophoresis. The results showed that ebselen was able to induce typical apoptosis in HepG(2) cells in a dose- and time-dependent manner. In order to explore the possible mechanisms involved in ebselen-induced apoptosis, the effect of ebselen on intracellular thiol concentrations including reduced glutathione (GSH) and protein thiols and the effect of N-acetylcysteine (NAC) and buthionine sulfoximine (BSO) pretreatment on ebselen-induced apoptosis were investigated. It was found that (i) ebselen rapidly depleted intracellular GSH and protein thiols, moreover, the depletion preceded the occurrence of apoptosis; (ii) NAC, a precursor of intracellular GSH synthesis, significantly alleviated ebselen-induced apoptosis; and (iii) BSO, a specific inhibitor of intracellular GSH synthesis, augmented ebselen-induced apoptosis significantly. Taken together, the present study demonstrates that ebselen is able to induce apoptosis in HepG(2) cells, most probably through rapid depletion of intracellular thiols.

Critical role of GSH in silica-induced oxidative stress, cytotoxicity, and genotoxicity in alveolar macrophages.

The main objective of this study was to evaluate the critical role of glutathione (GSH) in silica-induced oxidative stress, cytotoxicity, and genotoxicity in rat alveolar macrophages (AMs). Silica-induced superoxide radical and hydrogen peroxide formation were determined with lucigenin-dependent chemiluminescence and 2', 7'-dichlorofluorescin diacetate fluorescence test, respectively. The cytotoxicity of silica was estimated by lactate dehydrogenase leakage, and a comet assay was used for examining silica-induced DNA damage in AMs. The intracellular GSH content was modulated by N-acetylcysteine, a GSH precursor, and buthionine sulfoximine, a specific GSH synthesis inhibitor. It was found that silica led to a dose- and time-dependent decrease in GSH content in AMs. N-acetylcysteine increased intracellular GSH level and protected against silica-induced reactive oxygen species formation, lactate dehydrogenase leakage, and DNA strand breaks in AMs. In contrast, buthionine sulfoximine pretreatment depleted cellular GSH and enhanced the susceptibility of AMs to the cytotoxic and genotoxic effects of silica. It thus appears that GSH plays a critical role in protecting against silica-induced cell injury, most probably through its antioxidant activity.

Intracochlear infusion of buthionine sulfoximine potentiates carboplatin ototoxicity in the chinchilla

The aim of this experiment was to determine if buthionine sulfoximine (BSO), an inhibitor of glutathione (GSH) synthesis, enhances the ototoxicity of carboplatin. Osmotic pumps were used to infuse BSO into the right cochleas of 12 adult chinchillas for 14 days. The left cochleas served as controls. Animals were assigned to three groups: a drug control group that did not receive carboplatin, a group that received a single dose of carboplatin (25 mg/kg i.p.), and a group that received a double dose of carboplatin (25 mg/kg i.p. x 2), with 4 days between injections. Carboplatin was administered after three days of BSO pre-treatment. Ototoxicity was assessed with evoked potentials recorded from electrodes implanted in the inferior colliculi (ICPs), distortion product otoacoustic emissions (DPOAEs), and cochleograms. BSO infusion itself caused no long-term functional or morphological changes. One of four animals treated with it single dose of carboplatin showed a significant loss of inner hair cells (IHCs), with greater loss in the BSO-treated ear. All animals in the double-dose carboplatin group showed marked differences between BSO-treated and control ears. Average IHC losses were 59% in BSO-treated ears vs. 18% in control ears. Moreover, BSO-treated ears sustained significantly greater outer hair cell (OHC) losses than control ears (37% vs. 2%, respectively). ICP and DPOAE response amplitudes were reduced slightly in BSO-treated ears relative to control ears, consistent with their greater hair cell loss. The results clearly show that BSO can enhance carboplatin ototoxicity in the chinchilla, supporting a role of GSH and reactive oxygen species in platinum ototoxicity.

Inhibition of tumor necrosis factor-alpha- and interleukin-1-induced endothelial E-selectin expression by thiol-modifying agents.

The expression of endothelial-leukocyte adhesion molecules has been postulated to be regulated by redox-sensitive events. Tumor necrosis factor-alpha (TNF-alpha)- and interleukin-1 (IL-1)-induced E-selectin expression was analyzed after pretreating human umbilical vein endothelial cells with different thiol-modifying agents, ie, diamide, phenylarsine oxide, N-ethylmaleimide, and diethyl maleate. E-selectin protein expression was quantified by indirect immunofluorescence. All compounds suppressed the cytokine-induced E-selectin expression in a concentration-dependent manner, whereas the antioxidant N-acetylcysteine showed no effect. The inhibitory effect of diamide (100 micromol/L, 1 hour) was reversible within 6 hours when the cells were allowed to recover before application of cytokines. Reversibility was strongly delayed when cells were deprived of glutathione by buthionine sulfoximine pretreatment. Glutathione depletion alone did not influence cytokine-induced E-selectin expression. Analysis of cellular glutathione status showed a 3-fold increase in oxidized glutathione after diamide treatment. Monochlorobimane labeling also revealed a decrease in total cellular thiols. During recovery, the glutathione status was restored within 1 hour, whereas total thiol content and E-selectin expression needed at least 6 hours to return to baseline. Complete inhibition of E-selectin expression by the vicinal thiol blocker phenylarsine oxide (0.5 micromol/L) was reversed by dithiols like dithiothreitol or dimercaptopropanol, but not by the monothiol 2-mercaptoethanol. These data suggest that proteins with essential thiols, most probably vicinal thiols. are involved in the IL-1- and TNF-alpha-mediated induction of E-selectin. These thiols must be in the reduced state; oxidation or other modification thereof attenuates or abolishes the cells' response to the cytokines.