Evidence of prooxidant and antioxidant action of melatonin on human liver cell line HepG2

The aim of this study was to evaluate melatonin cytotoxicity by measuring its effects on various cellular targets. Cell viability, intracellular reduced glutathione (GSH) level, and reactive oxygen species (ROS) production were assessed in the human liver cell line (HepG2), after incubation with increasing melatonin concentrations (0.1-10,000 microM). The incubation times tested were 24, 72, and 96 h for cell viability and intracellular GSH level, and 15 and 45 minutes for ROS production. Cellular target evaluations were possible in living cells by means of a new microplate cytofluorimeter. This technology was suitable for the assessment of cell viability, GSH level, and ROS overproduction with, respectively, neutral red, monochlorobimane (mBCl), and 2',7'-dichlorofluorescin diacetate (DCFH-DA) fluorescent probes. At the lowest melatonin concentrations (0.1-10 microM) and for a relatively short incubation time (24 h), the antioxidant effect of melatonin was revealed by an increased intracellular GSH level, associated to cell viability improvement. In contrast, after longer incubation (96 h), cell viability significantly decreased with these lowest melatonin concentrations (0.1-10 microM). Moreover, high melatonin concentrations (1,000-10,000 microM) induced GSH depletion. This oxidative stress is associated with ROS overproduction from 10 microM after only 15 minutes of incubation. This dual effect is strong evidence that, in vitro, melatonin can be both antioxidant and prooxidant on the human liver cell line, depending on the concentration and incubation time.     

alpha-tocopherol increases the intracellular glutathione level in HaCaT keratinocytes

-Tocopherol is a lipophilic vitamin that exhibits an antioxidative activity. The purpose of this study was to clarify the roles of -tocopherol in the regulation of intracellular glutathione (GSH) levels in HaCaT keratinocytes. When HaCaT keratinocytes were cultivated with -tocopherol for 24 h, the intracellular GSH was increased at every concentration of -tocopherol tested. Furthermore, the HaCaT keratinocytes cultured with -tocopherol at 50 μM for 24 h exhibited resistance against H 2 O 2 . However, a short exposure of HaCaT keratinocytes to -tocopherol for 1 h did not influence either the GSH level or the resistance to H 2 O 2 . These findings suggest that GSH, which is inductively synthesized by -tocopherol, effectively reduces exogenous oxidative stress. To evaluate the effect of -tocopherol on the GSH level, BSO, which is a typical inhibitor of γ-glutamylcysteine synthetase ( γ-GCS), was used. When BSO was added to HaCaT keratinocytes, no action of -tocopherol on the GSH level was observed. On the other hand, -tocopherol resulted in the up-regulation of γ-GCS-HS (heavy subunit) mRNA. In addition, water soluble -tocopherol derivatives ( -tocopherol phosphate and trolox) caused no changes in GSH level. From these results, it was concluded that -tocopherol increases the intracellular GSH level of HaCaT keratinocytes through the up-regulation of γ-GCS-HS mRNA.     

Acetaminophen decreases intracellular glutathione levels and modulates cytokine production in human alveolar macrophages and type II pneumocytes in vitro

Recent epidemiological observations suggest that acetaminophen (paracetamol) may contribute to asthma morbidity. Impaired endogenous antioxidant defences may have a role in the pathogenesis of a number of inflammatory pulmonary diseases, including asthma. We studied the effect of acetaminophen on the intracellular level of reduced glutathione (GSH) with and without inhibitors of cytochrome P450 or prostaglandin H synthetase, and TNF-alpha, IL-6 and IL-8 protein production in human alveolar macrophages and type II pneumocytes in vitro. Following a 20 h incubation with acetaminophen, cytotoxicity was apparent from > or = 5 and > or = 10 mM in macrophages and type II pneumocytes, respectively. A time- and concentration-dependent decrease of intracellular GSH occurred after acetaminophen (0.05-1 mM) exposure (1-4 h) in pulmonary macrophages (up to 53%) and type II pneumocytes (up to 34%). Diethyldithiocarbamic acid, potassium ethyl xanthate, and indomethacin decreased significantly acetaminophen-induced GSH depletion in the two cell types tested, suggesting the involvement of cytochrome P450 (mainly CYP2E1) and/or prostaglandin H synthetase. In macrophages, acetaminophen decreased the secretion of TNF-alpha (at 4 and 24 h, concentration-related) and IL-6 (at 24 h, at 0.1 mM), and did not affect significantly IL-8 production. These in vitro observations demonstrate that clinically relevant concentrations of acetaminophen decreased: (i) intracellular GSH in human pulmonary macrophages and type II pneumocytes and (ii) the secretion of TNF-alpha and possibly IL-6 by human pulmonary macrophages. These findings provide experimental plausibility to the challenging observations that frequent use of APAP may be a risk factor for asthma morbidity.     

Zinc binding ligands and cellular zinc trafficking: apo-metallothionein, glutathione, TPEN, proteomic zinc, and Zn-Sp1

Many cell types contain metal-ion unsaturated metallothionein (MT). Considering the Zn(2+) binding affinity of metallothionein, the existence of this species in the intracellular environment constitutes a substantial "thermodynamic sink". Indeed, the mM concentration of glutathione may be thought of in the same way. In order to understand how apo-MT and the rest of the Zn-proteome manage to co-exist, experiments examined the in vitro reactivity of Zn-proteome with apo-MT, glutathione (GSH), and a series of common Zn(2+) chelating agents including N,N,N',N'-(2-pyridylethyl)ethylenediammine (TPEN), EDTA, and [(2,2'-oxyproplylene-dinitrilo]tetraacetic acid (EGTA). Less than 10% of Zn-proteome from U87mg cells reacted with apo-MT or GSH. In contrast, each of the synthetic chelators was 2-3 times more reactive. TPEN, a cell permeant reagent, also reacted rapidly with both Zn-proteome and Zn-MT in LLC-PK(1) cells. Taking a specific zinc finger protein for further study, apo-MT, GSH, and TPEN inhibited the binding of Zn(3)-Sp1 with its cognate DNA site (GC-1) in the sodium-glucose co-transporter promoter of mouse kidney. In contrast, preformation of Zn(3)-Sp1-(GC-1) prevented reaction with apo-MT and GSH; TPEN remained active but at a higher concentration. Whereas, Zn(3)-Sp1 is active in cells containing apo-MT and GSH, exposure of LLC-PK(1) cells to TPEN for 24h largely inactivated its DNA binding activity. The results help to rationalize the steady state presence of cellular apo-MT in the midst of the many, diverse members of the Zn-proteome. They also show that TPEN is a robust intracellular chelator of proteomic Zn(2+).     

Effects of cyclophosphamide and buthionine sulfoximine on ovarian glutathione and apoptosis

Treatment with the anticancer drug cyclophosphamide (CPA) destroys ovarian follicles. The active metabolites of CPA are detoxified by conjugation with glutathione (GSH). We tested the hypotheses that CPA causes apoptosis in ovarian follicles and that suppression of ovarian GSH synthesis before CPA administration enhances CPA-induced apoptosis. Proestrous rats were given two injections, 2 h apart, with (1) saline, then saline; (2) saline, then 50 mg/kg CPA; (3) saline, then 300 mg/kg CPA; or (4) 5 mmol/kg buthionine sulfoximine (BSO) to inhibit glutamate cysteine ligase (GCL), the rate-limiting enzyme in GSH synthesis, and then 50 mg/kg CPA. Statistically significantly increased DNA fragmentation by agarose gel electrophoresis and granulosa cell apoptosis by TUNEL were observed in the CPA-treated ovaries 24 h after the second injection, but BSO did not enhance the effect of 50 mg/kg CPA. We next tested the hypothesis that CPA depresses ovarian GSH concentration and expression of the rate-limiting enzyme in GSH synthesis, GCL. Proestrous rats were injected with 300 or 50 mg/kg CPA or vehicle and were sacrificed 8 or 24 h later. After CPA treatment, ovarian and hepatic GSH levels decreased significantly, and ovarian GCL subunit mRNA levels increased significantly. There were no significant changes in GCL subunit protein levels. Finally, we tested the hypothesis that GSH depletion causes apoptosis in ovarian follicles. Proestrous or estrous rats were injected with 5 mmol/kg BSO or saline at 0700 and 1900 h. There was a significant increase in the percentage of histologically atretic follicles and a nonsignificant increase in the percentage of apoptotic, TUNEL-positive follicles 24 h after onset of BSO treatment. Our results demonstrate that CPA destroys ovarian follicles by inducing granulosa cell apoptosis and that CPA treatment causes a decline in ovarian GSH levels. More pronounced GSH suppression achieved after BSO treatment did not cause a statistically significant increase in follicular apoptosis. Thus, GSH depletion does not seem to be the mechanism by which CPA causes follicular apoptosis.     

Effects of glucocorticoids on maturation of pig oocytes and their subsequent fertilizing capacity in vitro

The aim of this study was to assess the possible role of glucocorticoids in the maturation of pig oocytes and their subsequent fertilizing capacity in vitro. Pig cumulus-enclosed oocytes collected from prepubertal gilts were cultured in Waymouth MB752/1 medium supplemented with sodium pyruvate (50 microg/ml), LH (0.5 microg/ml), FSH (0.5 microg/ml), and estradiol-17beta (1 microg/ml) in the presence or absence of cortisol or dexamethasone (DEX) for 24 h; they then were cultured without hormonal supplements in the presence or absence of cortisol or DEX for an additional 16-24 h. Treatment of cumulus-enclosed or denuded oocytes with increasing concentrations of cortisol or DEX for 48 h resulted in a dose-response inhibition of germinal vesicle breakdown (GVB). Increasing duration (12-48 h) of treatment with DEX (10 microg/ml) led to a time-dependent inhibition of GVB, which achieved statistical significance by 12 h. The addition of DEX (10 microg/ml) to maturation medium immediately after culture or at 12 h, 24 h, or 36 h after culture also decreased the percentage of oocytes with GVB. When oocytes were exposed to DEX for 48 h, the maturation rate was reduced. The degree of this reduction was dependent on DEX, and a concentration of DEX higher than 0.1 microg/ml was needed. The inhibitory effect of DEX on the maturation of oocytes was prevented by the glucocorticoid receptor antagonist RU-486. Exposure of oocytes to DEX for 40 h did not prevent sperm penetration, affect the incidence of polyspermy, or decrease the ability of oocytes to form a male pronucleus. The intracellular concentration of glutathione (GSH) in cumulus-enclosed oocytes was 4.4 mM per oocyte. Exposure of oocytes to DEX (0.01-10 microg/ml) had no effect on GSH concentration. These results demonstrate that glucocorticoids directly inhibit the meiotic but not cytoplasmic maturation of pig oocytes in vitro. This inhibitory effect is not mediated through a decrease in the level of intracellular GSH.     

Improvement in bovine embryo production in vitro by treatment with green tea polyphenols during in vitro maturation of oocytes

The present study examined the effect of green tea polyphenols (GTP) during in vitro maturation (IVM) of bovine oocytes on in vitro fertilization (IVF) parameters, intracellular glutathione (GSH) concentration and subsequent embryo development. Cumulus-oocyte complexes were aspirated from the ovaries derived from slaughterhouse and cultured in modified synthetic oviduct fluid (m-SOF) supplemented with 0-25 microM GTP for 24h. After IVM, cumulus-free oocytes were coincubated with frozen-thawed spermatozoa for 15-18 h. Putative embryos were transferred to m-SOF and cultured for 8 days (Experiment 1). In comparison with the absence of GTP, treatment with GTP at a concentration of 15 microM showed a significant increase in the proportion of pronuclear (PN) formation after sperm penetration (65% versus 80%, P<0.05). No significant differences in the rates of sperm penetration and polyspermic fertilization were found among treatments. The cleavage rate at 48 h of in vitro insemination showed no difference in oocytes matured with or without GTP. However, compared to no addition (23.5%), the presence of 15 and 20 microM GTP during IVM significantly (P<0.05) increased the proportion of blastocysts (38.1% and 36.4%) on day 9 of in vitro insemination. A further increase from 20 to 25 microM GTP reduced (P<0.05) the proportion of blastocysts. In Experiment 2, after IVM, oocytes were fixed to analyze the GSH concentration. Compared to no addition, a higher (P<0.05) level of GSH was found in oocytes matured with 15 microM GTP and compared with 15 microM GTP, GSH was low (P<0.05) at 20 and 25 microM GTP. The results suggest that at certain concentrations of GTP (15 microM) in IVM medium has beneficial effects on subsequent embryo development, and is correlated with intracellular GSH level in bovine oocytes.     

DNA strand breaking capacity of acrylamide and glycidamide in mammalian cells

We compared the DNA damaging potency of acrylamide (AA) and its metabolite glycidamide (GA) in the comet assay in cell systems differing with respect to species origin and cytochrome P450-depended monooxygenase (CYP2E1) expression (V79, Caco-2, primary rat hepatocytes). Only after 24 h incubation in the highest concentration of AA (6 mM) a slight but significant increase in DNA damage was observed in V79 and Caco-2 cells. In primary rat hepatocytes, however, expressing substantial amounts of CYP2E1, no induction of DNA strand breaks was found. At the end of the incubation time period (24 h), still 67+/-19% of the CYP2E1 protein was detected by Western blotting. Direct treatment with GA resulted in a significant increase in DNA damage in V79 cells and primary rat hepatocytes at concentrations > or =100 microM (24 h). Caco-2 cells were found to be less sensitive, exhibiting an increase in DNA strand breaks at concentrations > or 300 microM GA. These data confirm the higher genotoxic potential of GA compared to AA but also indicate that high expression of CYP2E1 per se is not necessarily associated with increased genotoxicity of AA. We, therefore, investigated whether the intracellular glutathione (GSH) level might be a critical determinant for the genotoxicity of AA in cells with different CYP2E1 status. Depletion of intracellular GSH by dl-buthionine-[S,R]-sulfoxime (BSO) in rat hepatocytes and V79 cells resulted in a significant induction of DNA strand breaks after incubation with 1 mM AA. However, at higher concentrations (> or =1.25 mM) a strong increase in cytotoxicity, resulting in a severe loss of viability, was observed. In summary, the DNA strand breaking effect of AA appeared not to be directly correlated with the CYP2E1 status of the cells. Depletion of GSH is associated with an increase in AA genotoxicity but seems also to lead to a substantial enhancement of cytotoxicity.     

Vitamin C inhibits diethylmaleate-induced L-cystine transport in human vascular smooth muscle cells

Adaptive increases in intracellular glutathione (GSH) in response to oxidative stress are mediated by induction of L-cystine uptake via the anionic amino acid transport system x(c)(-). The recently cloned transporter xCT forms a heteromultimeric complex with the heavy chain of 4F2 cell surface antigen (4F2hc/CD98). Depletion of GSH by the electrophile diethylmaleate (DEM) induces the activity and expression of xCT in peritoneal macrophages. We here examine the effects of vitamin C on induction of xCT by DEM in human umbilical artery smooth muscle cells. DEM caused time- (3-24 h) and concentration- (25-100 microM) dependent increases in L-cystine transport, with GSH depleted by 50% after 6 h and restored to basal values after 24 h. xCT mRNA levels increased after 4 h DEM treatment with negligible changes detected for 4F2hc mRNA. DEM caused a rapid (5-30 min) phosphorylation of p38(MAPK). Inhibition of p38(MAPK) by SB203580 (10 microM) enhanced DEM-induced increases in L-cystine transport and GSH, whereas inhibition of p42/p44(MAPK) (PD98059, 10 microM) had no effect. Pretreatment of cells with vitamin C (100 microM, 24 h) attenuated DEM-induced adaptive increases in L-cystine transport and GSH levels. Inhibition of p38(MAPK), but not p42/p44(MAPK), reduced the cytoprotective action of vitamin C. Our findings suggest that DEM induces activation of xCT via intracellular signaling pathways involving p38(MAPK), and that vitamin C, in addition to its antioxidant properties, may modulate this signaling pathway to protect smooth muscle cells from injury.     

Early and delayed glutamate effects in rat primary cortical neurons. Changes in the subcellular distribution of protein kinase C isoforms and in intracellular calcium concentration

Glutamate-induced changes in the subcellular distribution of protein kinase C isoforms and in the intracellular calcium concentration were investigated in rat primary cortical neurons. Western blot analysis of protein kinase C isoforms (alpha, beta1, beta2, gamma, delta, epsilon, zeta and theta), performed 30 min after a 10 min treatment with 30 microM glutamate, revealed a decrease in the total beta1 (-24%) and beta2 (-40%) isoform levels, without any significant change in any of the other isozymes. All conventional isoforms translocated to the membrane compartment, while delta, epsilon, zeta and theta; maintained their initial subcellular distribution. Twenty-four hours after glutamate treatment, the total protein kinase C labelling had increased, particularly the epsilon isoform, which accounted for 34% of the total densitometric signal. At this time, protein kinase C beta1, delta, epsilon and zeta isoforms were mainly detected in the membrane compartment, while gamma and theta; signals were displayed almost solely in the cytosol. Basal intracellular calcium concentration (FURA 2 assay) was concentration-dependently increased (maximum effect +77%) 30 min, but not 24h after a 10 min glutamate (10-100 microM) treatment, while the net increase induced by electrical stimulation (10 Hz, 10s) was consistently reduced (maximum effect -64%). The N-methyl-d-aspartate receptor antagonist, MK-801, 1 microM, prevented glutamate action both 30 min and 24 h after treatment, while non-selective protein kinase C inhibitors, ineffective at 30 min, potentiated it at 24 h. These findings show that protein kinase C isoforms are differently activated and involved in the early and delayed glutamate actions, and that the prevailing effect of their activation is neuroprotective.     

The role of glutathione in the aerobic radioresponse. I. Sensitization and recovery in the absence of intracellular glutathione

The effect of changes in both the intracellular glutathione (GSH) concentration and the concentration of extracellular reducing equivalents on the aerobic radiosensitization was studied in three cell lines: CHO-10B4, V79, and A549. Intracellular GSH was metabolically depleted after the inhibition of GSH synthesis by buthionine sulfoximine (BSO), while the extracellular environment was controlled through the replacement of growth medium with a thiol-free salt solution and in some experiments by the exogenous addition of either GSH or GSSG. Each of the cell lines examined exhibited an enhanced aerobic radioresponse when the intracellular GSH was extensively depleted (GSH less than 1 nmol GSH/10(6) cells after 1.0 mM BSO/24 h treatment) and the complexity of the extracellular milieu decreased. Although the addition of oxidized glutathione (5 mM GSSG/30 min) to cells prior to irradiation was without effect, much or all of the induced radiosensitivity was overcome by the addition of reduced glutathione (5 mM GSH/15 min). However, the observation that the exogenous GSH addition restores the control radioresponse without increasing the intracellular GSH concentration was entirely unexpected. These results suggest that a number of factors exert an influence on the extent of GSH depletion and determine the extent of aerobic radiosensitization. Furthermore, the interaction of exogenous GSH with--but without penetrating--the cell membrane is sufficient to result in radiorecovery.     

Flavonoid-induced glutathione depletion: potential implications for cancer treatment

The ability of a number of flavonoids to induce glutathione (GSH) depletion was measured in lung (A549), myeloid (HL-60), and prostate (PC-3) human tumor cells. The hydroxychalcone (2'-HC) and the dihydroxychalcones (2',2-, 2',3-, 2',4-, and 2',5'-DHC) were the most effective in A549 and HL-60 cells, depleting more than 50% of intracellular GSH within 4 h of exposure at 25 microM. In contrast, the flavones chrysin and apigenin were the most effective in PC-3 cells, depleting 50-70% of intracellular GSH within 24 h of exposure at 25 microM. In general, these flavonoids were more effective than three classical substrates of multidrug resistance protein 1 (MK-571, indomethacin, and verapamil). Prototypic flavonoids (2',5'-DHC and chrysin) were subsequently tested for their abilities to potentiate the toxicities of prooxidants (etoposide, rotenone, 2-methoxyestradiol, and curcumin). In A549 cells, 2',5'-DHC potentiated the cytotoxicities of rotenone, 2-methoxyestradiol, and curcumin, but not etoposide. In HL-60 and PC-3 cells, chrysin potentiated the cytotoxicity of curcumin, cytotoxicity that was attenuated by the catalytic antioxidant manganese(III) meso-tetrakis(N-ethylpyridinium-2-yl)porphyrin (MnTE-2-PyP). Assessments of mitochondrial GSH levels mitochondrial membrane potential and cytochrome c release showed that the potentiation effects induced by 2',5'-DHC and chrysin involve mitochondrial dysfunction.     

Status of reduced glutathione in primary cultures of rat hepatocytes and the effect on conjugation of benzo[a]pyrene-7,8-dihydrodiol-9,10-oxide

The intracellular level of reduced glutathione (GSH) and GSH conjugation have been investigated in primary cell cultures of hepatocytes isolated from control rats, phenobarbitone (PB) and 3-methylcholanthrene (MC) treated rats. The data demonstrate that in all cell cultures the GSH concentrations show a triphasic pattern: (i) within 1 h of culture an initial marked decrease to 50% of the levels found in fresh hepatocytes; (ii) recovery of GSH concentrations to above the levels observed in fresh cells. This occurs after 6 h in culture with control cells and after 10-24 h with cells from either PB or MC treated rats and was most prominent in cells from PB-treated rats. (iii) A slow decline to between 30 and 40 nmol GSH/mg protein from 24 to 96 h in culture. Synthesis of GSH was slower in cultured cells from PB treated rats and this was confirmed by the resynthesis rates when diethylmaleate (DEM) was used to deplete GSH. The formation of GSH conjugates with racemic 7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) was measured in control cells in suspension and after 3 and 24 h in culture. Despite the decrease in GSH concentrations observed between 1 and 4 h after culture, the conjugation rates were not decreased.     

Allicin inhibits cell growth and induces apoptosis through the mitochondrial pathway in HL60 and U937 cells

In this article, the effects of allicin, a biological active compound of garlic, on HL60 and U937 cell lines were examined. Allicin induced growth inhibition and elicited apoptotic events such as blebbing, mitochondrial membrane depolarization, cytochrome c release into the cytosol, activation of caspase 9 and caspase 3 and DNA fragmentation. Pretreatment of HL60 cells with cyclosporine A, an inhibitor of the mitochondrial permeability transition pore (mPTP), inhibited allicin-treated cell death. HL60 cell survival after 1 h pretreatment with cyclosporine A, followed by 16 h in presence of allicin (5 microM) was approximately 80% compared to allicin treatment alone (approximately 50%). Also N-acetyl cysteine, a reduced glutathione (GSH) precursor, prevented cell death. The effects of cyclosporine A and N-acetyl cysteine suggest the involvement of mPTP and intracellular GSH level in the cytotoxicity. Indeed, allicin depleted GSH in the cytosol and mitochondria, and buthionine sulfoximine, a specific inhibitor of GSH synthesis, significantly augmented allicin-induced apoptosis. In HL60 cells treated with allicin (5 microM, 30 min) the redox state for 2GSH/oxidized glutathione shifted from EGSH -240 to -170 mV. The same shift was observed in U937 cells treated with allicin at a higher concentration for a longer period of incubation (20 microM, 2 h). The apoptotic events induced by various concentrations of allicin correlate to intracellular GSH levels in the two cell types tested (HL60: 3.7 nmol/10(6) cells; U937: 7.7 nmol/10(6) cells). The emerging mechanistic basis for the antiproliferative function of allicin, therefore, involves the activation of the mitochondrial apoptotic pathway by GSH depletion and by changes in the intracellular redox status.     

Modulation of intracellular glutathione concentrations alters lymphocyte activation and proliferation

Glutathione (GSH) has been implicated in lymphocyte activation and differentiation, as well as in protection from radiation damage. Since [3H]thymidine ([3H]TdR) at high concentrations in the nucleus causes radiation damage to the cells, it is important to rule out the possibility that changes in [3H]TdR uptake by mitogen-activated lymphocytes are not caused by 3H-induced cell injury following alterations in intracellular GSH concentration. In this study, flow-cytometric analysis of cell cycle was used to measure lymphocyte activation. Intracellular GSH levels were enhanced using 2-L-oxothiazolidine-4-carboxylate (OTC) and 2-mercaptoethanol (2ME), which deliver cysteine intracellularly, and suppressed by buthionine sulfoximine (BSO) which inhibits gamma-glutamylcysteine synthetase. Enhancement of intracellular GSH concentrations in lymphocytes with 2-oxothiazolidine-4-carboxylate or 2-mercaptoethanol augments mitogen-induced lymphocyte activation, and proliferation, while suppression of intracellular GSH levels by buthionine sulfoximine inhibits the progression of cellular proliferation--but not activation, as measured by flow cytometry. There was a linear relationship between intracellular GSH concentration and conA-activated cells by flow cytometry and between GSH concentration and [3H]TdR incorporation as measured at 24 h. We conclude that alterations of intracellular GSH concentrations may be one way to modulate lymphocyte activation and differentiation.     

Attenuation of expression of gamma-glutamylcysteine synthetase by ribozyme transfection enhance insulin secretion by pancreatic beta cell line, MIN6

Low levels of intracellular antioxidant enzyme activities as well as glutathione (GSH) concentrations have been described in pancreatic beta cells. We examined the effects of intracellular GSH depletion on insulin secretion and the role of intracellular GSH in signal transduction in beta cell line, MIN6 cells. Anti-gamma-glutamylcysteine synthetase (gamma-GCS) heavy subunit ribozyme was stably transfected to MIN6 cells to reduce intracellular GSH concentration. In the presence of 10 mM glucose, ribozyme-transfected cells (RTC) increased insulin secretion from 0.58 microg/10(6) cells/h in control cells (CC) to 1.48 microg/10(6) cells/h. This was associated with increased intracellular Ca(2+) concentration in RTC, detected by fluo-3 staining. Our results demonstrated that intracellular GSH concentration might influence insulin secretion by MIN6 cells, and suggest that enhanced insulin secretion by beta cells conditioned by chronic depletion of GSH is mediated by increased intracellular Ca(2+) concentration.     

Effect of gonadotropins during in vitro maturation of feline oocytes on oocyte-cumulus cells functional coupling and intracellular concentration of glutathione

Information about the mechanisms of meiotic arrest and resumption of meiosis in feline oocytes is still limited. The aim of this study was to investigate the effect of the presence of gonadotropins during IVM, on meiotic progression in relation to the status of gap junction mediated communications between oocyte and cumulus cells, to the cAMP intracellular content, and to the intra-oocyte concentration of glutathione (GSH) in feline oocytes. Our results indicated that about 50% of cumulus-oocyte complexes (COCs) showed functionally open communications at the time of collection, while the remainder were partially or totally closed. After 3h of culture, the percentage of COCs with functional gap junctions was significantly greater in the group matured in the presence of gonadotropins than in those matured without them, where an interruption of communications was observed. Moreover, this precocious uncoupling was associated with a moderate increase of cAMP concentration in the oocyte, lower than in the group exposed to gonadotropins. Intra-oocyte glutathione levels decreased significantly after 24h of IVM, whether gonadotropins were present or absent during the culturing process. The presence of thiol compounds in the IVM medium induced an intra-oocyte GSH concentration significantly higher than that found in oocytes cultured without these compounds, and similar to the GSH content of immature oocytes. Moreover, the intracellular GSH concentration increased as meiosis progressed. The present study suggests that in feline oocytes, gonadotropins affect the dynamic changes in communications between oocyte and cumulus cells during IVM. However, the intracellular concentration of GSH is not influenced by the gonadotropin stimulation. Moreover, the presence of gonadotropins and thiol compounds results in an increase of GSH levels along with meiotic progression of the oocytes.     

Toxic effects of acute glutathione depletion by buthionine sulfoximine and dimethylfumarate on murine mammary carcinoma cells

Glutathione (GSH) depletion to approximately equal to 5% of control for 48 h or longer by 0.05 mM L-buthionine sulfoximine (BSO) led to appreciable toxicity for the 66 murine mammary carcinoma cells growing in vitro [L.A. Dethlefsen et al., Int. J. Radiat. Oncol. Biol. Phys. 12, 1157-1160 (1986)]. Such toxicity in normal, proliferating cells in vivo would be undesirable. Thus the toxic effects after acute GSH depletion to approximately equal to 5% of control by BSO plus dimethylfumarate (DMF) were evaluated in these same 66 cells to determine if this anti-proliferative effect could be minimized. Two hours of 0.025 mM DMF reduced GSH to 45% of control, while 6 h of 0.05 mM BSO reduced it to 16%. However, BSO (6 h) plus DMF (2 h) and BSO (24 h) plus DMF (2 h) reduced GSH to 4 and 2%, respectively. The incorporation (15-min pulses) of radioactive precursors into protein and RNA were unaffected by these treatment protocols. In contrast, cell growth was only modestly affected, but the incorporation of [3H]thymidine into DNA was reduced to 64% of control by the BSO (24 h) plus DMF (2 h) protocol even though it was unaffected by the BSO (6 h) plus DMF (2 h) treatment. The cellular plating efficiencies from both protocols were reduced to approximately equal to 75% of control cells. However, the aerobic radiation response, as measured by cell survival, was not modified at doses of either 4.0 or 8.0 Gy. The growth rates of treated cultures, after drug removal, quickly returned to control rates and the resynthesis of GSH in cells from both protocols was also rapid. The GSH levels after either protocol were slightly above control by 12 h after drug removal, dramatically over control (approximately equal to 200%) by 24 h, and back to normal by 48 h. Thus even a relatively short treatment with BSO and DMF resulting in a GSH depletion to 2-5% of control had a marked effect on DNA synthesis and plating efficiency and a modest effect on cellular growth. One cannot rule out a direct effect of the drugs, but presumably the antiproliferative effects are due to a depletion of nuclear GSH with the subsequent inhibition of the GSH/glutaredoxin-mediated conversion of ribonucleotides to deoxyribonucleotides. However, even after extended treatment, upon drug removal, GSH was rapidly resynthesized and cellular DNA synthesis and growth quickly resumed.     

DNA strand breaking capacity of acrylamide and glycidamide in mammalian cells

We compared the DNA damaging potency of acrylamide (AA) and its metabolite glycidamide (GA) in the comet assay in cell systems differing with respect to species origin and cytochrome P450-depended monooxygenase (CYP2E1) expression (V79, Caco-2, primary rat hepatocytes). Only after 24 h incubation in the highest concentration of AA (6 mM) a slight but significant increase in DNA damage was observed in V79 and Caco-2 cells. In primary rat hepatocytes, however, expressing substantial amounts of CYP2E1, no induction of DNA strand breaks was found. At the end of the incubation time period (24 h), still 67 ± 19% of the CYP2E1 protein was detected by Western blotting. Direct treatment with GA resulted in a significant increase in DNA damage in V79 cells and primary rat hepatocytes at concentrations ≥100 μM (24 h). Caco-2 cells were found to be less sensitive, exhibiting an increase in DNA strand breaks at concentrations ≥300 μM GA. These data confirm the higher genotoxic potential of GA compared to AA but also indicate that high expression of CYP2E1 per se is not necessarily associated with increased genotoxicity of AA. We, therefore, investigated whether the intracellular glutathione (GSH) level might be a critical determinant for the genotoxicity of AA in cells with different CYP2E1 status. Depletion of intracellular GSH by DL-buthionine-[S,R]-sulfoxime (BSO) in rat hepatocytes and V79 cells resulted in a significant induction of DNA strand breaks after incubation with 1 mM AA. However, at higher concentrations (≥1.25 mM) a strong increase in cytotoxicity, resulting in a severe loss of viability, was observed. In summary, the DNA strand breaking effect of AA appeared not to be directly correlated with the CYP2E1 status of the cells. Depletion of GSH is associated with an increase in AA genotoxicity but seems also to lead to a substantial enhancement of cytotoxicity.     

Response to copper and cadmium stress in wild-type and copper tolerant strains of the lichen alga <Emphasis Type="Italic">Trebouxia erici</Emphasis>: metal accumulation,toxicity and non-protein thiols

Cysteine, glutathione (GSH) and phytochelatins were determined in the cells of both wild and copper tolerant strains of the lichen alga Trebouxia erici following short-term (24 h) exposure to copper and cadmium and long-term (4 weeks) exposure to copper. Both metals caused concentration dependent synthesis of phytochelatins (PC₂–PC₅), but cadmium was a more potent activator of phytochelatin synthesis, even inducing synthesis of PC₅. The copper-tolerant strain did not reveal a higher degree of phytochelatin synthesis than the wild strain, and at 5 μM Cu production of phytochelatins was in fact significantly lower. Lower levels of phytochelatin correlated with significantly decreased intracellular copper content in the copper-tolerant strain. Both strains maintained high GSH levels even at a high copper concentration of 5 μM, and only the highest copper concentration (10 μM) was toxic for both strains, causing a decrease of GSH and PC content in algal cells. Cadmium had less effect on GSH in the cells of both tested strains. In the long term experiments, only relatively small amounts of PC₂ were detected in both strains, but the copper-tolerant strain retained significantly higher levels of reduced glutathione, probably due to the lesser degree of oxidative stress caused by Cu. The significant increase of cysteine synthesis in the copper-tolerant strain found in the present study may be related to copper tolerance in T. erici, while decreased intracellular Cu uptake, detoxification by PCs and increased free proline levels for protection of chloroplast membranes may also be implicated.