Glutathione Sulfhydryl Research Articles

Glutathione and bicarbonate nanoparticles improve mucociliary transport in cystic fibrosis epithelia.

Cystic fibrosis (CF) airway disease is characterized by thick mucus and impaired mucociliary transport (MCT). Loss of functional cystic fibrosis transmembrane receptor (CFTR) leads to acidification and oxidation of airway surface mucus. Replacing bicarbonate (HCO3 -) topically fails due to rapid reabsorption and neutralization, while the scavenging antioxidant, glutathione sulfhydryl (GSH), is also rapidly degraded. The objective of this study is to investigate GSH/NaHCO3 nanoparticles as novel strategy for CF airway disease. GSH/NaHCO3 poly (lactic-co-glycolic acid) nanoparticles were tested on primary CF (F508del/F508del) epithelial cultures to evaluate dose-release curves, surface pH, toxicity, and MCT indices using micro-optical coherence tomography. In vivo tests were performed in three rabbits to assess safety and toxicity. After 1week of daily injections, histopathology, computed tomography (CT), and blood chemistries were performed and compared to three controls. Fluorescent nanoparticles were injected into a rabbit with maxillary sinusitis and explants visualized with confocal microscopy. Sustained release of GSH and HCO3 - with no cellular toxicity was observed over 2weeks. Apical surface pH gradually increased from 6.54±0.13 (baseline) to 7.07±0.10 (24h) (p<0.001) and 6.87 ± 0.05 at 14 days (p<0.001). MCT, ciliary beat frequency, and periciliary liquid were significantly increased. When injected into the maxillary sinuses of rabbits, there were no changes to histology, CT, or blood chemistries. Nanoparticles penetrated rabbit sinusitis mucus on confocal microscopy. Findings suggest that GSH/NaHCO3 - nanoparticles are a promising treatment option for viscous mucus in CF and other respiratory diseases of mucus obstruction such as chronic rhinosinusitis.

Marjoram oil attenuates oxidative stress and improves colonic epithelial barrier function in dextran sulfate sodium-induced ulcerative colitis in Balb/c mice

In this study, we explored the effects of marjoram oil (MO) on dextran sulfate sodium (DSS)-induced colitis in Balb/c mice. Marjoram oil was found to significantly reduce the severity of DSS-induced colonic inflammation, as reflected by improved disease activity index, prevented colon length shortening, lower histopathological score, decreased myeloperoxidase activity, and reduced interlukin 6 (IL-6) levels. Moreover, marjoram oil pretreatment enhanced the colonic epithelial integrity by decreasing paracellular permeability. Marjoram oil was found to clearly reduce the colonic levels of thiobarbituric acid reactive substances assay and enhance the activity of superoxide dismutase, catalase, and glutathione sulfhydryl content. Marjoram oil could exert a protective effect on ulcerative colitis through its anti-inflammatory and antioxidant properties.

Corn embryo ameliorates cognitive dysfunction and anxiety-like behaviors in D-galactose-induced aging rats via attenuating oxidative stress, apoptosis and up-regulating neurotrophic factors

Aging is the primary cause of neurodegenerative diseases, which are mainly characterized by cognitive decline and neuropsychiatric symptoms. Corn embryo, an important component of corn kernels, contains plenty of essential nutrients and bioactive compounds. However, corn embryo is often removed in the process of refining corn. To reveal potential biological benefits of corn embryo, the present study investigated the intervention effects of corn embryo on age-related cognitive decline and neuropsychiatric symptoms. Ninety male Wistar rats were randomly divided into six groups: Control, Corn embryo, Aging model, Low-, Medium- and High-dose intervention group. Aging models induced by an intraperitoneal injection of 60 mg/kg D-galactose plus a gavage of 200 mg/kg aluminum chloride were intervened with a gavage of 0.3, 0.6 or 1 g/kg corn embryo while the Control and Corn embryo groups received saline and 0.6 g/kg corn embryo respectively. Morris water maze and open field test were performed to assess cognitive abilities and anxiety-like behaviors. Brain biochemical parameters including the malondialdehyde, glutathione, glutathione sulfhydryl transferase and γ-glutamylcysteine synthetase were detected to evaluate oxidative stress levels. The mRNA expression of brain-derived neurotrophic factor was determined to estimate neurotrophic factor levels. Besides, histopathological alterations were visualized by hematoxylin-eosin staining and neuronal apoptosis levels were measured by the immunohistochemical staining of Bax and Bcl-2. Ultimately, the mimetic aging rats showed significant cognitive impairment (n = 15, P < 0.01) and anxiety-like behaviors (n = 15, P < 0.01), increased oxidative stress (n = 5, P < 0.001), neurodegeneration (n = 5, P < 0.001) and apoptosis (n = 5, P < 0.01) and reduced neurotrophic factors (n = 5, P = 0.074) in the brain. However, corn embryo effectively prevented the above undesirable neurobehavioral alterations via attenuating oxidative stress (n = 5, P < 0.01), neurodegeneration (n = 5, P < 0.001) and apoptosis (n = 5, P < 0.01) and increasing the levels of neurotrophic factors (n = 5, P < 0.001), suggesting its strong neuroprotective effects.

The Influence of Oxidative Stress on Neurological Outcomes in Spontaneous Intracerebral Hemorrhage.

Spontaneous intracerebral hemorrhage (ICH) causes, besides the primary brain injury, a secondary brain injury (SBI), which is induced, amongst other things, by oxidative stress (OS) and inflammation, determining the patient’s outcome. This study aims to assess the impact of OS in plasma and cerebrospinal fluid (CSF) on clinical outcomes in patients with ICH. A total of 19 ICH (volume > 30 cc) patients and 29 control patients were included. From day one until seven, blood and CSF samples were obtained, and ICH volume was calculated. OS markers, like malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), glutathione-sulfhydryl (GSH), and the total antioxidant status (TAS) were measured. Clinical data on treatment and outcome were determined. Patients with mRS ≤ 4 showed significantly elevated SOD and GSH-Px levels in plasma compared to patients with poor CO (p = 0.004; p = 0.002). Initial increased TAS in plasma and increased MDA in CSF were linked to an unfavorable outcome after six months (p = 0.06, r = 0.45; p = 0.05, r = 0.44). A higher ICH volume was associated with a worse outcome at week six (p = 0.04, r = 0.47). OS plays a significant role in SBI. Larger ICHs, elevated MDA in CSF, and TAS in plasma were associated with a detrimental outcome, whereas higher plasma-SOD and -GSH-Px were associated with a favorable outcome.

Association between Malondialdehyde, GSH/GSSG Ratio and Bone Mineral Density in Postmenopausal Women

Background: Osteoporosis is one of chronic degenerative diseases especially in postmenopausal women, characterized by a decreased bone mass due to imbalance activity between osteoblasts and osteoclasts. Recently, oxidative stress is believed to play an important role in osteoporosis pathogenesis. Oxidative stress is commonly considered as the consequence of an imbalance between pro and antioxidants species, which results in damage in the affected tissue. Malondialdehyde (MDA) is frequently used as a biomarker of oxidative stress in many health problems since MDA is produced at high levels during lipid peroxidation. Meanwhile, glutathione is well known as one of antioxidant which against oxidative stress by preserving its homeostasis in the reduced form of glutathione sulfhydryl (GSH) and the oxidized form of glutathione disulphide (GSSG). This study was aimed to determine the association between MDA, GSH/GSSG ratio and bone mineral density (BMD) in postmenopausal women.Materials and method: We conducted a cross-sectional study in 40 postmenopausal women. MDA and GSH/GSSG ratio were assessed by enzyme-linked immunosorbent assay (ELISA). Bone mineral density (BMD) was obtained from secondary data. The statistical analysis was conducted using Spearman rho’s correlation test.Results: Based on the test, we didn’t found significant correlation between MDA and BMD (r=-0.054, p=0.741), but we found significant moderate correlation between GSH/GSSG ratio (r=0.436, p=0.005) and BMD in postmenopausal women. Conclusion: There was no correlation between MDA and BMD in postmenopausal women. However, there was significant moderate correlation between GSH/GSSG ratio and BMD in postmenopausal women.Keywords: MDA, GSH/GSSG ratio, BMD, osteoporosis

Development of a Novel Pharmaceutical Formula of Nanoparticle Lipid Carriers of Gentamicin/α-Tocopherol and In Vivo Assessment of the Antioxidant Protective Effect of α-Tocopherol in Gentamicin-Induced Nephrotoxicity

Gentamicin is a potent antibiotic with a nephrotoxicity drawback which limits its use. D-α-tocopherol polyethylene glycol succinate (α-tocopherol) is widely used as a surfactant and have potent antioxidant properties. This study aimed to assess the protective effect of α-tocopherol on gentamicin-induced nephrotoxicity by loading gentamicin on nanostructured lipid carriers (NLC). In vivo, the product was administered intravenously to three groups of rabbits (control, gentamicin and gentamicin/α-tocopherol NLC) for 10 consecutive days. Blood was collected on days 1, 5 and 10 to assess renal function. A significant difference in all plasma parameters related to kidney function were observed in the gentamicin group compared to the control by day 5 and 10, confirming the nephrotoxicity effect. On the other hand, the same parameter levels of the NLC group were significantly different compared to the gentamicin group, confirming the protective effect on kidney function. Gentamicin also caused significant decreases in plasma levels of glutathione sulfhydryl (GSH) and superoxide dismutase (SOD) activity. However, gentamicin-α-tocopherol NLC significantly elevates both plasma levels of GSH as well as SOD activity. The present work indicates that, loading of gentamicin on NLC by using α-tocopherol, is an innovative strategy to protect against aminoglycoside-induced nephrotoxicity due to its antioxidant activity.

The Effects of Allopurinol on Glutathione Sulfhydryl (GSH) Serum Level, Six Minute Walking Test, and CAT Score of COPD Patients

Background: Stress oxidative in COPD impairs striated muscle thus worsening COPD symptoms. Allopurinol as antioxidant reduces stress oxidative so it can improve exercise capacity and clinical symptoms. The aims of this study were to analyze the effects of allopurinol on GSH serum level, six minute walking test (6MWT), and COPD assessment test (CAT) score of COPD patients.&#x0D; Methods: This study was a pre and post test clinical trial held in pulmonology outpatient clinic in Dr. Moewardi General Hospital Surakarta from January to February 2018 using purposive sampling. The COPD group C and D patients were cathegorized as intervention group which received allopurinol 300 mg/day for 4 weeks and control group which did not receive allopurinol. Glutathione sulfhydryl serum level, 6MWT, and CAT score were measured at baseline and after 4 weeks in both groups.&#x0D; Results: A total of 37 stable COPD group C and D patients were included in this study. The intervention group showed decreased GSH level (52.58±38.39) µg/ml and CAT score (10.37±4.46) which were statistically significant compared to control group (P

Neuroprotection by glucose-6-phosphate dehydrogenase and the pentose phosphate pathway.

Glucose-6-phosphate dehydrogenase (G6PD), the rate limiting enzyme that channels glucose catabolism from glycolysis into the pentose phosphate pathway (PPP), is vital for the production of reduced nicotinamide adenine dinucleotide phosphate (NADPH) in cells. NADPH is in turn a substrate for glutathione reductase, which reduces oxidized glutathione disulfide to sulfhydryl glutathione. Best known for inherited deficiencies underlying acute hemolytic anemia due to elevated oxidative stress by food or medication, G6PD, and PPP activation have been associated with neuroprotection. Recent works have now provided more definitive evidence for G6PD's protective role in ischemic brain injury and strengthened its links to neurodegeneration. In Drosophila models, improved proteostasis and lifespan extension result from an increased PPP flux due to G6PD induction, which is phenocopied by transgenic overexpression of G6PD in neurons. Moderate transgenic expression of G6PD was also shown to improve healthspan in mouse. Here, the deciphered and implicated roles of G6PD and PPP in protection against brain injury, neurodegenerative diseases, and in healthspan/lifespan extensions are discussed together with an important caveat, namely NADPH oxidase (NOX) activity and the oxidative stress generated by the latter. Activation of G6PD with selective inhibition of NOX activity could be a viable neuroprotective strategy for brain injury, disease, and aging.

Effects of Combined Exposure to Chronic High-Fat Diet and Arsenic on Thyroid Function and Lipid Profile in Male Mouse.

The thyroid is one of the major endocrine glands that contribute to body and fat metabolism. The present study evaluated the effects of combined exposure to chronic high-fat diet (HFD) and arsenic on thyroid function and lipid profile. In this experimental study, 72 male Naval Medical Research Institute mice were divided into six groups and fed HFD or low-fat diet (LFD) while being exposed to 25 or 50ppm of arsenic in drinking water for 20weeks. After 24h of the last experimental day, blood samples were collected for hormonal and biochemical measurements. The data indicated that exposure to HFD alone increased the levels of triiodothyronine (T3), thyroid-stimulating hormone (TSH), leptin, lipid profile, reactive oxygen species (ROS), and malondialdehyde (MDA) and decreased the levels of high-density lipoprotein, albumin, adiponectin, and glutathione sulfhydryl reductase (GSH), whereas exposure to arsenic alone decreased the levels of T3 and GSH and increased the levels of TSH, leptin, ROS, MDA, and T4/T3 ratio compared to those in the control LFD group. Furthermore, concomitant administration of HFD and arsenic decreased the lipid profile and levels of T4, albumin, total protein, T3, and GSH and increased the levels of TSH, adiponectin, leptin, ROS, MDA, and T4/T3 ratio compared to those in the control LFD or HFD group. In conclusion, combined exposure to HFD and arsenic induced hypothyroidism via reduction of thyroid hormones and enhancement of plasma TSH and T3 uptake levels concomitant with hypolipidemia, hyperleptinemia, hyperadiponectinemia, induction of oxidative stress, and reduction of GSH levels.

Pretreatment of Gymnema sylvestre revealed the protection against acetic acid-induced ulcerative colitis in rats

BackgroundOverproduction of free radicals and decreased antioxidant capacity are well-known risk factors for inflammatory bowel diseases. Gymnema sylvestre (GS) leaves extract is distinguished for its anti-diabetic, antioxidant and anti-inflammatory properties. Present study is designed to evaluate the preventative activities of GS against acetic acid (AA)-induced ulcerative colitis in Wistar rats.MethodsExperimentally ulcerative colitis (UC) was induced by AA in animals pretreated with three different doses of GS leaves extract (50, 100, 200 mg/kg/day) and a single dose of mesalazine (MES, 300 mg/kg/day) for seven days. Twenty four hours later, animals were sacrificed and the colonic tissues were collected. Colonic mucus content was determined using Alcian blue dye binding technique. Levels of thiobarbituric acid reactive substances (TBARS), total glutathione sulfhydryl group (T-GSH) and non-protein sulfhydryl group (NPSH) as well as the activity of the antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) were estimated in colon tissues. Colonic nucleic acids (DNA and RNA) and total protein (TP) concentrations were also determined. Levels of pro-inflammatory cytokines including interleukin-1 beta (IL-1β), tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) as well as prostaglandin E2 (PGE2) and nitric oxide (NO) were estimated in colonic tissues. The histopathological changes of the colonic tissues were also observed.ResultsIn AA administered group TBARS levels were increased, while colonic mucus content, T-GSH and NP-SH, SOD and CAT were reduced in colon. Pretreatment with GS inhibited TBARS elevation as well as mucus content, T-GSH and NP-SH reduction. Enzymatic activities of SOD and CAT were brought back to their normal levels in GS pretreated group. A significant reduction in DNA, RNA and TP levels was seen following AA administration and this inhibition was significantly eliminated by GS treatment. GS pretreatment also inhibited AA-induced elevation of pro-inflammatory cytokines, PGE2 and NO levels in colon. The apparent UC protection was further confirmed by the histopathological screening.ConclusionThe GS leaves extract showed significant amelioration of experimentally induced colitis, which may be attributed to its anti-inflammatory and antioxidant property.

Dietary selenium fortification: a potential solution to chronic arsenic toxicity

Selenium (Se), an important micronutrient and antioxidant, also acts as an antagonist of arsenic (As). Se supplementation of diet was investigated in mitigating chronic As toxicity in mammals. Experiments were conducted to determine whether Se supplementation in As-exposed rats might (i) decrease As-induced lipid peroxidation in liver, (ii) increase blood antioxidant status, and (iii) reverse suppression of the secondary antibody response. Male Wistar rats were exposed to As (40 and 80 ppm) in drinking water and received challenge diets with three different levels of Se (deficient: <0.01 ppm, adequate: 0.15 ppm, and fortified: 0.6 ppm) for 16 weeks. Clinical variables including behavior, body weight, and food and water consumption were recorded weekly, and blood sample was collected monthly. Antioxidant status was assessed through glutathione sulfhydryl (GSH) levels in whole blood. Lipid peroxidation in the liver was evaluated using the malondialdehyde (MDA) assay. The antibody response was measured using keyhole limpet hemocyanin as an immunogen. Se deficiency significantly increased hepatic lipid peroxidation and suppressed antibody production relative to the Se-adequate and -fortified groups, confirming that Se deficiency exacerbates the damage produced by As exposure. Se fortification markedly elevated the blood GSH level in both As-exposed groups, indicating protective effects. At adequate Se levels, rats showed signs of counteracting As-mediated toxicity. However, Se fortification produced more pronounced benefits against As-induced toxicity, a pattern that was particularly notable in the 40 ppm As group.

Contribution of oxidative stress to pulmonary hypertension induced by chronic hypoxia

Chronic hypoxia causes pulmonary hypertension and right ventricular hypertrophy associated with media wall thickening of pulmonary arteries in rats. Platelet-activating factor plays an important role in the pulmonary vascular remodeling induced by chronic hypoxia, and reactive oxygen species are involved in tissue injury induced by platelet activating factor. We therefore hypothesized that reactive oxygen species contribute to the pulmonary hypertension induced by chronic hypoxia, and examined the effect of N-acetyl-L-cysteine (NAC), a free radical scavenger and the precursor of glutathione sulfhydryl (GSH), on hypoxia-induced pulmonary hypertension. We used a chemiluminescence-HPLC assay to measure the levels of phosphatidylcholine hydroperoxide (PCOOH) in the rat lungs exposed to hypoxia. Three weeks of normobaric hypoxia (FiO2 = 0.1) with NAC significantly reduced pulmonary hypertension, right ventricular hypertrophy, and media wall thickening of the pulmonary arteries. NAC had no effect on the hematocrit of normoxic or chronically normobaric hypoxic rats. Lung PCOOH levels were significantly higher in the hypoxic rats than in the control rats, and those in the NAC-treated rats were significantly lower than those in the hypoxic rats that were not given NAC. Lung PCOOH levels were significantly higher in the hypoxic rats than in the control rats, and those in the NAC-treated rats were significantly lower than those in the hypoxic rats that were not given NAC. These results indicate that hypoxia induces oxidative stress in the lung tissue, and that oxidative stress may have a role in the development of pulmonary hypertension induced by chronic hypoxia in rats.

Impact of cyclopentenone-oxylipins on the proteome of Arabidopsis thaliana

Both enzymatically and non-enzymatically generated oxylipins fulfill signalling functions in plant responses to biotic and oxidative stress on the cellular level. We studied the impact of two different exogenously applied cyclopentenone-oxylipins on the proteome of Arabidopsis thaliana leaves: the enzymatically formed 12-oxo-phytodienoic-acid, a member of the jasmonate family of mediators, and A 1-phytoprostane generated by a free-radical mechanism upon biotic and oxidative stress. Infiltration of leaves with these oxylipins led to induction of classical stress proteins like chaperones as well as enzymes connected to the cellular redox and detoxification systems. A large proportion of the regulated proteins are localized in chloroplasts where these oxylipins are formed. Furthermore, we show that cyclopentenone-oxylipins spontaneously react with several proteins and glutathione in vitro and in vivo. Conjugation to the glutathione sulfhydryl group is a reversible process that is also catalysed by glutathione-S-transferases. In vitro, an oxidative stress inducible glutathione-S-transferase, GST6, localized both in plastids and the cytosol can be covalently modified and partially inactivated by several cyclopentenone-oxylipins.

Models of protein modification in Tris–glycine and neutral pH Bis–Tris gels during electrophoresis: Effect of gel pH

The pH of conventional Tris–glycine SDS–PAGE gels during a run is determined to be 9.5, in contrast to Bis–Tris–Mes gels where the pH is 7.2. Concentrations of free acrylamide are determined to be less than 10 mM in commercial gels of both types, and it is found that of the major components in these gels, only glycine and protein amine or sulfhydryl functions are likely to react with residual acrylamide during the time frame of typical separations. The addition of acrylamide to sulfhydryl groups on proteins is modeled using glutathione and cysteine at acrylamide concentrations found in the commercial gels. Rate constants are determined for these reactions as well as for reaction with glycine at the pH that proteins will encounter in these gel types. The half-life for glutathione sulfhydryl at 10 mM acrylamide and pH 7.2 is more than 4 h at room temperature. Rates are significantly lower in Bis–Tris–Mes gels than in Tris–glycine gels, reducing the risk of adventitious protein modification. Commercial Bis–Tris–Mes gels provide a sample reduction buffer at pH 8.5 versus the conventional pH 6.8 of Tris–glycine gels. It is shown that significantly less protein degradation occurs during sample preparation at the higher pH used with Bis–Tris gels.

Amperometric Biosensor for Glutathione Based on Osmium-Polyvinylpyridine Gel Polymer and Glutathione Sulfhydryl Oxidase

A new amperometric biosensor based on glutathione sulfhydryl oxidase (GSH-SOx) and osmium-polyvinylpyridine gel polymer (Os-gel-HRP) bilayer film modified glassy carbon (GC) electrode was demonstrated for glutathione (GSH) and glutathione disulfide (GSSG). Os-gel-HRP was applied at glassy carbon (GC) electrode with a surface coverage of 7.1 µL/cm2 to sense hydrogen peroxide based on horseradish peroxide (HRP) catalytic reaction mediated by osmium. GSH-SOx was immobilized at Os-gel-HRP cast coated GC electrode with an approximate surface coverage of 7 U/cm2 by cross-linking with BAS-GSH-SOx in glutaraldehyde vapor for 5 min. The resultant bienzyme-based sensor was tested toward GSH and GSSG with techniques of cyclic voltammetry (CV), flow cell amperometry and flow injection analysis (FIA). The sensor was polarized at 0.0 V (versus Ag/AgCl, 3M KCl) electrode for the detection of GSH and GSSG in a flow system at a flow rate of 6 µL/min. Linear response to GSH and GSSG in a concentration range from 1 µM to 200 µM and 2 µM to 120 µM was obtained at the sensor with a sensitivity of 1.195 nA/µM and 0.60 nA/µM for GSH and GSSG, respectively. The dependence of current response on pH value of the buffer and operating potential was also tested and optimized.

Nitric Oxide Inhibits c-Jun DNA Binding by Specifically TargetedS-Glutathionylation

This study addresses potential molecular mechanisms underlying the inhibition of the transcription factor c-Jun by nitric oxide. We show that in the presence of the physiological sulfhydryl glutathione nitric oxide modifies the two cysteine residues contained in the DNA binding module of c-Jun in a selective and distinct way. Although nitric oxide induced the formation of an intermolecular disulfide bridge between cysteine residues in the leucine zipper site of c-Jun monomers, this same radical directed the covalent incorporation of stoichiometric amounts of glutathione to a single conserved cysteine residue in the DNA-binding site of the protein. We found that covalent dimerization of c-Jun apparently did not affect its DNA binding activity, whereas the formation of a mixed disulfide with glutathione correlated well with the inhibition of transcription factor binding to DNA. Furthermore, we provide experimental evidence that nitric oxide-induced S-glutathionylation and inhibition of c-Jun involves the formation of S-nitrosoglutathione. In conclusion, our results support the reversible formation of a mixed disulfide between glutathione and c-Jun as a potential mechanism by which nitrosative stress may be transduced into a functional response at the level of transcription.

Effect of Glutathione Depletion and Metallothionein Gene Expression on Arsenic-Induced Cytotoxicity and c-myc Expression in Vitro

Arsenic exposure is clearly linked to human cancer. In rodent cells, arsenic has been reported to induce aberrant gene expression, including activation of the proto-oncogene c-myc. Abnormal or altered expression of such oncogenes can be involved in the acquisition of a malignant phenotype. Although its mechanism of action is unclear, arsenic is known to exert at least some of its toxic effects through interaction with sulfhydryl groups, and the nonprotein sulfhydryl glutathione (GSH) appears to play an important role in detoxication of arsenic. Similarly, metallothionein (MT), a metal-binding protein with high sulfhydryl content, often functions in defense against metal-induced or oxidative cellular injury. Therefore, we examined the relationship among GSH, MT gene expression, and arsenic-induced toxicity or c-myc expression in cultured rat myoblast (L6) cells. In initial toxicity studies, arsenic was used in both the trivalent (arsenite) and pentavalent (arsenate) forms. The role of GSH was studied by pretreating cells with L-buthionine sulfoximine (BSO), which induces a marked depletion of GSH. In vitro exposure of L6 cells to BSO (1 to 25 μM) resulted in dose-dependent decreases in GSH. GSH depletion sensitized cells to both arsenite and arsenate. Zinc pretreatment, at levels which highly activated MT expression, had no effect on arsenite-induced cytotoxicity. Arsenite (1 μM) alone modestly increased c-myc expression from 1 to 4 h after treatment (maximum of 2.0-fold over control). After GSH depletion cells responded to arsenite exposure with much larger increases in c-myc transcription (3.2-fold over control). Zinc pretreatment had no reductive effect on arsenite-induced c-myc expression despite markedly activating the MT gene. Thus, it appears that the cellular levels of GSH, but not MT gene expression, play an important role in resistance to arsenic toxicity and aberrant gene activation. Moreover, depletion of GSH enhances arsenic-induced proto-on-cogene activation, which might contribute to subsequent transformation.

Direct Reaction of H2O2with Sulfhydryl Groups in HL-60 Cells: Zinc-Metallothionein and Other Sites

The reaction of the sulfhydryl groups in metallothionein with hydrogen peroxide was examined in HL-60 cells. Partial purification of cell cytosol using Sephadex G-75 chromatography showed that zinc-metallothionein (Zn-MT) was induced by 24-h treatment with 100 μMZnCl2, but the cellular glutathione content and glutathione peroxidase and catalase activities were unaffected. The ratio of H2O2concentrations needed to reduce cell survival 50% in Zn-induced cells compared to normal cells was 1.65 to 1. According to alkaline elution experiments, the average ratio of single-strand breaks caused by H2O2at 37°C in Zn-induced vs normal cells was 0.5 to 1. A similar reduction in strand breakage was seen in nuclei from Zn-treated cells exposed to H2O2; however, at 4°C protection against DNA strand breakage by Zn pretreatment was not seen. Incubation of Zn-pretreated cells with H2O2at 37°C but not 4°C was accompanied by loss of Zn bound to MT and a reduction in the number of MT sulfhydryl groups. In the absence or presence of Zn-MT, sulfhydryl groups from glutathione and protein fractions were also reduced by exposure of cells to H2O2. However, thiolate groups in the MT fraction were preferentially lost compared to the other pools of sulfhydryl residues. Zn-MT also spared glutathione sulfhydryl groupsin vitrofrom oxidation by H2O2. Protection against strand breakage correlated with the ability of Zn-MT to reactin vitrowith H2O2at 37°C, but not at 4°C. The reaction was slow and was not inhibited by the presence of an hydroxyl radical scavenger, dimethyl sulfoxide. Similarly, in cells dimethyl sulfoxide did not prevent the loss of sulfhydryl groups from glutathione or protein. Incubation of MT or higher molecular weight fractions from cells exposed to H2O2with either 2-mercaptoethanol or dithiothreitol in the presence of Cd failed to regenerate any detectable, reduced MT, suggesting that MT sulfhydryl groups were oxidized by H2O2beyond the disulfide oxidation state.

GLUTATHIONE SULFHYDRYL TRANSFERASE-PI EXPRESSION IN TESTICULAR GERM-CELL TUMORS - AN IMMUNOHISTOCHEMICAL STUDY OF 30 CASES

Glutathione sulfhydryl transferase (GST) is believed to play a major role in the detoxification of chemotherapeutic agents and therefore is thought to be important in chemoresistance. Thirty primary testicular germ cell tumors were stained immunohistochemically for GST pi using a rabbit polyclonal antibody and formalin-fixed paraffin-embedded tissue. There were 12 pure seminomas and 18 mixed germ cell tumors (GCTs). Immunostaining of each element present was graded as negative (-), weakly positive (+), or strongly positive (++). The results were as follows: seminoma (8/17 +, 9/17 ++); embryonal carcinoma (1/13 -, 7/13 +, 5/13 ++); mature teratoma (9/9 ++); immature teratoma (7/7 +); endodermal sinus tumor (7/8 +, 1/8 ++); and choriocarcinoma (1/1 -). These results show that variability exists in the expression of GST pi between different tumor types and between different examples of the same tumor type. Strongest expression was seen in seminomas and mature teratomas while other germ cell elements tended to show weaker staining. The staining patterns showed no apparent correlation with stage or response to therapy.

Gene expression during phorbol ester-induced differentiation of cultured human megakaryoblastic cells

Platelet protein makeup is determined during transformation of megakaryoblasts to mature megakaryocytes, the immediate precursor of circulating platelets. To better understand the molecular mechanisms of megakaryocyte formation, gene expression was characterized by Northern analysis and RNA fingerprinting of cultured human CHRF-288 megakaryoblastic cells undergoing phorbol ester-stimulated megakaryocytic differentiation or serum-stimulated megakaryoblast proliferation. Protooncogenes c-fos and c-jun were coordinately upregulated in both proliferating and differentiating cells, whereas c-myc transcripts were upregulated during proliferation only. In contrast, mRNAs for transforming growth factor-beta 1 (TGF-beta 1) and thromboxane receptors were coordinately upregulated during differentiation but differentially regulated during proliferation. RNA fingerprinting revealed multiple transcripts specific to either proliferating or differentiated cells. Three of these were identified by homology to known DNA sequence: CDw44 adhesion molecule (upregulated during differentiation), glutathione sulfhydryl peroxidase (downregulated during differentiation), and plectin cytoskeletal protein (upregulated during differentiation). Thus, although megakaryoblast proliferation and megakaryocyte differentiation both involve DNA and protein synthesis, each growth response is characterized by a distinct pattern of gene expression.