Role of the Fancg gene in protecting cells from particulate chromate-induced chromosome instability

Particulate hexavalent chromium (Cr(VI)) is a known human lung carcinogen. Cr(VI)-induced tumors exhibit chromosome instability (CIN), but the mechanisms underlying these effects are unknown. We investigated a possible role for the Fanconi anemia (FA) pathway in particulate Cr(VI)-induced chromosomal damage by focusing on the Fancg gene, which plays an important role in cellular resistance to DNA interstrand crosslinks. We used the isogenic Chinese hamster ovary (CHO) KO40 fancg mutant compared with parental and gene-complemented cells. We found that fancg cells treated with lead chromate had lower intracellular Cr ion levels than control cell lines. Accounting for differences of Cr ion levels between cell lines, we discovered that fancg cells treated with lead chromate had increased cytotoxicity and chromosomal aberrations, which was not observed after restoring the Fancg gene. Chromosomal damage was manifest as increased total chromosome damage and percent metaphases with damage, specifically an increase in chromatid and isochromatid breaks. We conclude that Fancg protects cells from particulate Cr(VI)-induced cytotoxicity and chromosome damage, which is consistent with the known sensitivity of fancg cells to crosslinking damage and the ability of Cr(VI) to produce crosslinks.     

The role of intracellular zinc in chromium(VI)-induced oxidative stress, DNA damage and apoptosis

Several studies have demonstrated that zinc is required for the optimal functioning of the skin. Changes in intracellular zinc concentrations have been associated with both improved protection of skin cells against various noxious factors as well as with increased susceptibility to external stress. Still, little is known about the role of intracellular zinc in hexavalent chromium (Cr(VI))-induced skin injury. To address this question, the effects of zinc deficiency or supplementation on Cr(VI)-induced cytotoxicity, oxidative stress, DNA injury and cell death were investigated in human diploid dermal fibroblasts during 48 h. Zinc levels in fibroblasts were manipulated by pretreatment of cells with 100 microM ZnSO4 and 4 or 25 microM zinc chelator TPEN. Cr(VI) (50, 10 and 1 microM) was found to produce time- and dose-dependent cytotoxicity resulting in oxidative stress, suppression of antioxidant systems and activation of p53-dependent apoptosis which is reported for the first time in this model in relation to environmental Cr(VI). Increased intracellular zinc partially attenuated Cr(VI)-induced cytotoxicity, oxidative stress and apoptosis by enhancing cellular antioxidant systems while inhibiting Cr(VI)-dependent apoptosis by preventing the activation of caspase-3. Decreased intracellular zinc enhanced cytotoxic effects of all the tested Cr(VI) concentrations, leading to rapid loss of cell membrane integrity and nuclear dispersion--hallmarks of necrosis. These new findings suggest that Cr(VI) as a model environmental toxin may damage in deeper regions residing skin fibroblasts whose susceptibility to such toxin depends among others on their intracellular Zn levels. Further investigation of the impact of Zn status on skin cells as well as any other cell populations exposed to Cr(VI) or other heavy metals is warranted.     

1-bromoalkanes as new potent nontoxic glutathione depletors in isolated rat hepatocytes

The effect of 1-bromlalkanes on intracellular glutathione (GSH) was studied in freshly isolated rat hepatocytes. Treatment of cells with bromoalkanes depleted cellular GSH levels without causing cytotoxicity. The extent of GSH depletion was directly proportional to the concentration and increasing chain length of 1-bromoalkanes (C2-C7). Bromoheptane (100 microM) depleted GSH by 87% in 30 mins which remained depleted for the 4 hr study period without causing cytotoxicity. A 30 fold higher concentration of bromoheptane was required before cytotoxicity ensued. Bromoheptane would therefore be particularly useful for studying the role of GSH in modulating xenobiotic cytotoxicity.     

Prolonged exposure to particulate Cr(VI) is cytotoxic and genotoxic to fin whale cells

BackgroundHexavalent chromium [Cr(VI)] is a human lung carcinogen and global marine pollutant. High Cr concentrations, resembling the ones observed in occupationally exposed workers, have been observed in fin whales (Balaenoptera physalus) in the Gulf of Maine. This outcome suggests Cr might be disrupting the health of fin whale populations. Indeed, Cr in acute (24 h) exposure does cause toxicity in fin whale cells. However, human cell culture data indicate prolonged exposures (120 h) induce a higher amount of toxicity compared to 24 h exposure due to an inhibition of homologous recombination repair. However, whether prolonged exposure causes similar outcomes in fin whale cells is unknown.ObjectiveDue to the importance of assessing prolonged exposure toxicity, this study focuses on characterizing acute and prolonged exposure of Cr(VI) in male and female fin whale cells.MethodsCytotoxicity was measured by the clonogenic assay, also known as colony forming assay, which measures the ability of cells to proliferate and form colonies after the treatment. DNA double strand breaks were analyzed by neutral comet assay. Clastogenicity was measured using the chromosome aberration assay. Intracellular Cr levels were measured with Graphite Furnace Atomic Absorption Spectrometry (GFAAS) with Syngistix Software.ResultsIn this study, we demonstrate that particulate Cr(VI) induces cytotoxicity and genotoxicity in a treatment-dependent manner after 24 h and 120 h exposures. Cytotoxicity levels were generally low with relative survival above 64 %. DNA double strand break data and chromosome aberration data were elevated after a 24 h exposure, but decreased after a 120 h exposure. While cytotoxicity was similar after 24 h and 120 h exposures, less DNA double strand breaks and chromosomal instability occurred with prolonged exposure.ConclusionParticulate Cr(VI) is cytotoxic and genotoxic to fin whale cells after acute and prolonged exposures. The reduction of genotoxicity we have observed after 120 h exposure may be partly explained by lower intracellular Cr levels after 120 h. However, the decrease in intracellular levels is not reflected by a similar decrease in chromosome aberrations suggesting other mechanisms may be at play. Male fin whale cells appear to be more susceptible to the genotoxic effects of particulate Cr(VI) while female cells are less susceptible possibly due to increased cell death of damaged cells, but more work is needed to clarify if this outcome reflects a sex difference or interindividual variability. Overall, the study shows particulate Cr(VI) does induce toxicity at both acute and prolonged exposures in fin whales cells indicating Cr(VI) exposure is a health risk for this species.     

In vivo nephrotoxicity induced in mice by chromium(VI)

The role of glutathione (GSH) and chromium (V) in chromium (VI)-induced nephrotoxicity in mice was investigated at 24 h after K2Cr(VI)2O7 ip injection. Nephrotoxicity was assessed by measurements of relative kidney weight and serum urea nitrogen. Cr(VI) nephrotoxicity was accompanied by decreased renal GSH and glutathione reductase (GSSG-R) levels. Pretreatment with buthionine sulfoximine, an inhibitor of GSH biosynthesis, enhanced Cr(VI)-induced nephrotoxicity, and remarkably diminished kidney GSH and GSSG-R levels. In contrast, pretreatment with glutathione methyl ester, a GSH-supplying agent, prevented Cr(VI) from exerting a harmful effect on mouse kidney and restored kidney GSH level. Administration of a Cr(V) compound, K3Cr(V)O8, induced much higher toxicity in mouse kidney than Cr(VI), but it failed to diminish renal GSH level. Another Cr(V) compound, Cr(V)-GSH complex, and Cr(III) nitrate did not cause a nephrotoxic effect in mice. The mechanism of Cr(VI)-induced nephrotoxicity was explained using GSH and Cr(V).     

Cometabolism of Cr(VI) by Shewanella oneidensis MR-1 produces cell-associated reduced chromium and inhibits growth

Microbial reduction is a promising strategy for chromium remediation, but the effects of competing electron acceptors are still poorly understood. We investigated chromate (Cr(VI)) reduction in batch cultures of Shewanella oneidensis MR-1 under aerobic and denitrifying conditions and in the absence of an additional electron acceptor. Growth and Cr(VI) removal patterns suggested a cometabolic reduction; in the absence of nitrate or oxygen, MR-1 reduced Cr(VI), but without any increase in viable cell counts and rates gradually decreased when cells were respiked. Only a small fraction (1.6%) of the electrons from lactate were transferred to Cr(VI). The 48-h transformation capacity (Tc) was 0.78 mg (15 micromoles) Cr(VI) reduced. [mg protein](-1) for high levels of Cr(VI) added as a single spike. For low levels of Cr(VI) added sequentially, Tc increased to 3.33 mg (64 micromoles) Cr(VI) reduced. [mg protein](-1), indicating that it is limited by toxicity at higher concentrations. During denitrification and aerobic growth, MR-1 reduced Cr(VI), with much faster rates under denitrifying conditions. Cr(VI) had no effect on nitrate reduction at 6 microM, was strongly inhibitory at 45 microM, and stopped nitrate reduction above 200 microM. Cr(VI) had no effect on aerobic growth at 60 microM, but severely inhibited growth above 150 microM. A factor that likely plays a role in Cr(VI) toxicity is intracellular reduced chromium. Transmission electron microscopy (TEM) and electron energy loss spectroscopy (EELS) of denitrifying cells exposed to Cr(VI) showed reduced chromium precipitates both extracellularly on the cell surface and, for the first time, as electron-dense round globules inside cells.     

Mitochondrial poisons cause depletion of reduced glutathione in isolated hepatocytes.

Antimycin A, KCN, and 1-methyl-4-phenylpyridinium ion (MPP+) all produced a marked depletion of cellular GSH levels in freshly isolated hepatocytes. This effect was consistently observed before the onset of cytotoxicity and seemed to be correlated with the loss of cellular ATP induced by these mitochondrial poisons. Concentrations of GSSG remained unchanged both intracellularly and extracellularly, indicating that oxidation was not involved in the events leading to GSH depletion. Approximately 40% of the decrease of intracellular GSH was accounted for by efflux of this tripeptide, assessed by increased formation of cysteinyl-glutathione when hepatocytes were incubated in the presence of 0.2 mM cystine. Therefore, an overall loss of glutathione was observed during incubations with all three inhibitors of mitochondrial function. Addition of 10 mM fructose to the incubation media substantially protected against GSH depletion caused by antimycin A, KCN, and MPP+. These results indicate that energy-dependent mechanisms are involved in the maintenance of intracellular GSH levels, and suggest that GSH depletion may be a general phenomenon associated with impairment of mitochondrial function.     

Metabolism of Cr(VI) by ascorbate but not glutathione is a low oxidant-generating process

Genotoxic activity of hexavalent chromium (chromate) results from its reductive activation inside the cell. Cr(VI) metabolism in vivo is primarily driven by ascorbate (Asc) but in cultured cells by glutathione (GSH). Given the common use of cultured cells for mechanistic studies, it is important to establish whether Cr(VI) activated by Asc and GSH displays the same genotoxic properties. Using 2',7' dichlorofluorescin (DCFH) as a redox sensitive probe, we found that Asc-dependent reduction of Cr(VI) in vitro under physiological conditions generated 25-80 times lower yields of oxidants compared to GSH. When both reducers were present, Asc dominated Cr(VI) metabolism and inhibited DCFH oxidation. Consistent with the findings in defined chemical reactions, restoration of physiological levels of Asc in human lung H460 cells led to the loss of their hypersensitivity to clonogenic killing by Cr(VI) in the presence of methoxyamine, which inhibits base excision repair of oxidative DNA damage. Despite suppressed oxidative damage, Asc-containing cells formed a large number of DNA double-strand breaks after exposure to a dose of Cr(VI) corresponding to the drinking water standard of 100 ppb. Our results indicate that Asc-driven metabolism of Cr(VI) shifts its genotoxicity toward nonoxidative mechanisms.     

Sialoglycoprotein and carbohydrate complexes in chromium toxicity

Chromium(VI) compounds are amongst the most widely encountered industrial carcinogens and are of increasing concern with respect to environmental exposure. Sialoglycoproteins and carbohydrates play a crucial role in stabilizing oxoCr(V) intermediates, which are produced by extracellular and intracellular reduction of chromium(VI). Recent research has addressed the molecular characterization of oxoCr(V)-sialoglycoprotein and -carbohydrate complexes and the roles that these species may play in Cr(VI) metabolism and carcinogenesis. Particular highlights include the role of oxoCr(V) complexes of extracellular sialoglycoproteins, intracellular D-glucose, and related species and their potential roles in Cr(VI)-induced genotoxicity.     

Effects of 2(RS)-n-propylthiazolidine-4(R)-carboxylic acid on 4-hydroxy-2-nonenal-induced apoptotic T cell death

4-Hydroxy-2-nonenal (HNE), the aldehydic product of lipid peroxidation, is associated with multiple immune dysfunctions, such as HIV and hepatitis C virus infection. HNE-induced immunosuppression could be due to a decrease in CD4+ T lymphocyte activation or proliferation. Glutathione (GSH) is the most abundant endogenous antioxidant in cells, and an adduct between HNE and GSH has been suggested to be a marker of oxidative stress. Our earlier studies showed that HNE induced cytotoxicity and Akt inactivation, which led to the enhancement of FasL expression and concomitantly decreased cellular FLICE-like inhibitory protein (c-FLIP(S)) levels. In this study, we found that HNE caused intracellular GSH depletion in Jurkat T cells, and we further investigated the role of 2(RS)-n-propylthiazolidine-4(R)-carboxylic acid (PTCA), a GSH prodrug, in attenuating HNE-induced cytotoxicity in CD4+ T lymphocytes. The results show that PTCA protected against HNE-induced apoptosis and depletion of intracellular GSH. PTCA also suppressed FasL expression through increasing levels of Akt kinase as well as antiapoptotic c-FLIP(S) and decreasing the activation of type 2 protein serine/threonine phosphatase. Taken together, these data demonstrate a novel correlation between GSH levels and Akt activation in T lymphocyte survival, which involves FasL down-regulation and c-FLIP(S) expression through increasing intracellular GSH levels. This suggests that PTCA could potentially be used in the treatment of oxidative stress-induced immunosuppressive diseases.     

Multidrug resistance-associated protein 1 as a major mediator of basal and apoptotic glutathione release

The proteins responsible for reduced glutathione (GSH) export under both basal conditions and in cells undergoing apoptosis have not yet been identified, although recent studies implicate some members of the multidrug resistance-associated protein family (MRP/ABCC) in this process. To examine the role of MRP1 in GSH release, the present study measured basal and apoptotic GSH efflux in HEK293 cells stably transfected with human MRP1. MRP1-overexpressing cells had lower intracellular GSH levels and higher levels of GSH release, under both basal conditions and after apoptosis was induced with either Fas antibody or staurosporine. Despite the enhanced GSH efflux in MRP1-overexpressing cells, intracellular GSH levels were not further depleted when cells were treated with Fas antibody or staurosporine, suggesting an increase in GSH synthesis. MRP1-overexpressing cells were also less susceptible to apoptosis, suggesting that the stable intracellular GSH levels may have protected cells from death. Overall, these results demonstrate that basal and apoptotic GSH release are markedly enhanced in cells overexpressing MRP1, suggesting that MRP1 plays a key role in these processes. The enhanced GSH release, with a concurrent decrease of intracellular GSH, appears to be necessary for the progression of apoptosis.     

Role of cellular calcium homeostasis in toxic liver injury induced by the pyrrolizidine alkaloid senecionine and the alkenal trans-4-OH-2-hexenal

The pyrrolizidine alkaloid senecionine has been shown to be hepatotoxic, genotoxic, and cytotoxic. However, the biochemical mechanism by which senecionine produces hepatocellular toxicity remains to be elucidated. The role of calcium homeostasis in toxic liver injury was examined in isolated rat hepatocytes treated with senecionine and trans-4-OH-2-hexenal (t-4HH), a microsomal metabolite of senecionine, and appropriate cofactors. Hepatocytes treated with senecionine and t-4HH demonstrated greater cytotoxicity (leakage of lactate dehydrogenase) when incubated in the absence of extracellular Ca²⁺ than in its presence. Both compounds elicited an increase in cytosolic Ca²⁺ levels of isolated hepatocytes in the presence of extracellular Ca²⁺ In the following study, senecionine and t-4HH depleted intracellular glutathione levels and induced lipid peroxidation and cytotoxicity in isolated hepatocytes. Pretreatment with the thiolgroup reducing agent dithiothreitol prevented depletion of intracellular glutathione and protected hepatocytes against senecionine and t-4HH-induced lipid peroxidation and cytotoxicity. Both compounds also depleted intracellular ATP and NADPH levels. These results suggest that hepatotoxocity induced by senecionine and t-4HH is not dependent on the influx of extracellular Ca²⁺; however, alterations in intracellular Ca²⁺, possibly associated with depletion of intracellular glutathione, NADPH, and ATP, may play a critical role.     

Mechanism of sulfite cytotoxicity in isolated rat hepatocytes

Sulfite (SO(3)(2-)) has been widely used as preservative and antimicrobial in preventing browning of foods and beverages. SO(2), a common air pollutant, also is capable of producing sulfite and bisulfite depending on the pH of solutions. A molybdenum-dependent mitochondrial enzyme, sulfite oxidase, oxidizes sulfite to inorganic sulfate and prevents its toxic effects. In the present study, sulfite toxicity towards isolated rat hepatocytes was markedly increased by partial inhibition of cytochrome a/a(3) by cyanide or by putting rats on a high-tungsten/low-molybdenum diet, which result in inactivation of sulfite oxidase. Sulfite cytotoxicity was accompanied by a rapid disappearance of GSSG followed by a slow depletion of reduced glutathione (GSH). Depleting hepatocyte GSH beforehand increased cytotoxicity of sulfite. On the other hand, dithiothreitol (DTT), a thiol reductant, added even 1h after the addition of sulfite to hepatocytes, prevented cell death and restored hepatocyte GSH levels. Sulfite cytotoxicity was also accompanied by an increase of oxygen uptake, reactive oxygen species (ROS) formation and lipid peroxidation. Cytochrome P450 inhibitors, metyrapone and piperonyl butoxide also prevented sulfite-induced cytotoxicity and lipid peroxidation. Desferroxamine and antioxidants also protected the cells against sulfite toxicity. These findings suggest that cytotoxicity of sulfite is mediated by free radicals as ROS formation increases by sulfite and antioxidants prevent its toxicity. Reaction of sulfite or its free radical metabolite with disulfide bonds of GSSG and GSH results in the compromise of GSH/GSSG antioxidant system leaving the cell susceptible to oxidative stress. Restoring GSH content of the cell or protein-SH groups by DTT can prevent sulfite cytotoxicity.     

Interleukin-6 protects PC12 cells from 4-hydroxynonenal-induced cytotoxicity by increasing intracellular glutathione levels

Oxidative stress plays an important role in neuronal cell death associated with many different neurodegenerative conditions, and it is reported that 4-hydroxynonenal (HNE), an aldehydic product of membrane lipid peroxidation, is a key mediator of neuronal cell death induced by oxidative stress. Previously, we have demonstrated that interleukin-6 (IL-6) protects PC12 cells from serum deprivation and 6-hydroxydopamine-induced toxicity. Therefore, in the present study, we examined the effects of interleukins on HNE toxicity in PC12 cells. Exposure of PC12 cells to HNE resulted in a decrease in levels of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction, which was due to necrotic and apoptotic cell death. Addition of IL-6 24 h before HNE treatment provided a concentration-dependent protection against HNE toxicity, whereas neither IL-1β nor IL-2 had any effect. Addition of glutathione (GSH)-ethyl ester, but not superoxide dismutase or catalase, before HNE treatment to the culture medium protected PC12 cells from HNE toxicity. We found that IL-6 increases intracellular GSH levels and the activity of γ-glutamylcysteine synthetase (γ-GCS) in PC12 cells. Buthionine sulfoximine (BSO), an inhibitor of γ-GCS, reversed the protective effect of IL-6 against HNE toxicity. These results suggest that IL-6 protects PC12 cells from HNE-induced cytotoxicity by increasing intracellular levels of GSH.     

On the mechanism of the chromate reduction by glutathione: ESR evidence for the glutathionyl radical and an isolable Cr(V) intermediate

With a view of elucidating the role of glutathione (GSH) in the biochemical pathways of the chromate-exposure related carcinogenesis, we carried out electron spin resonance (ESR) spectroscopic investigations of the chromate-GSH redox reactions. The ESR measurements, employing spin-traps, provide evidence for the involvement of the glutathione (GS) radical, as well as an isolable Cr(V)-glutathione intermediate. These results indicate a new mechanism for the reduction of chromate by GSH in in vitro cellular environment and help understand the (unexpected) increase in Cr(VI)-induced DNA strand breaks at elevated GSH levels.     

Causes of DNA single-strand breaks during reduction of chromate by glutathione in vitro and in cells

Carcinogenic chromates induce DNA single-strand breaks (SSB) that are detectable by conventional alkali-based assays. However, the extent of direct breakage has been uncertain because excision repair and hydrolysis of Cr-DNA adducts at alkaline pH also generate SSB. We examined mechanisms of SSB production during chromate reduction by glutathione (GSH) and assessed the significance of these lesions in cells using genetic approaches. Cr(VI) reduction was biphasic and the formation of SSB occurred exclusively during the slow reaction phase. Catalase or iron chelators completely blocked DNA breakage, as did the use of GSH purified by a modified Chelex procedure. Thus, the direct intermediates of GSH-chromate reactions were unable to cause SSB unless activated by H2O2. SSB repair-deficient XRCC1(-/-) and proficient XRCC1+ EM9 cells had identical survival at doses causing up to 60% clonogenic death and accumulation of 1 mM Cr(VI). However, XRCC1(-/-) cells displayed higher lethality in the more toxic range and the depletion of GSH made them hypersensitive even to moderate doses. Elevation of cellular catalase or GSH levels eliminated survival differences between XRCC1(-/-) and XRCC1+ cells. In summary, formation of toxic SSB in cells occurs at relatively high chromate doses, requires H2O2, and is suppressed by high GSH concentrations.     

Roles of glutathione and glutathione peroxidase in the protection against endothelial cell injury induced by 15-hydroperoxyeicosatetraenoic acid.

We investigated the role of the glutathione redox cycle in endothelial cell injury induced by 15(S)-hydroperoxyeicosatetraenoic acid (15-HPETE), an arachidonate lipoxygenase product. Pretreatment of endothelial monolayers with reduced glutathione (GSH) markedly suppressed 15-HPETE-induced cellular injury, which was determined by the 51Cr-release assay. 15-HPETE-induced cytotoxicity was modified by several GSH-modulating agents such as buthionine sulfoximine and 2-oxothiazolidine-4-carboxylate, indicating that this cyto-protective action of GSH was correlated with the intracellular GSH level. These GSH-modulating agents also modified the conversion of 15-HPETE to 15(S)-hydroxyeicosatetraenoic acid by endothelial cells. On the other hand, the exposure of endothelial cell monolayers to 15-HPETE did not deplete intracellular GSH levels but decreased GSH peroxidase activity. In addition, sodium selenite and ebselen, a stimulator and mimic of GSH peroxidase activity, respectively, displayed remarkable protective effects against 15-HPETE-induced cytotoxicity. These results suggest that intracellular GSH plays a pivotal role in the protection against 15-HPETE-induced endothelial cell injury, and that the decreased activity of GSH peroxidase activity is involved in 15-HPETE-induced cytotoxicity.     

The role of oxidative stress in the ochratoxin A-mediated toxicity in proximal tubular cells

Balkan endemic nephropathy (BEN), a disease characterized by progressive renal fibrosis in human patients, has been associated with exposure to ochratoxin A (OTA). This mycotoxin is a frequent contaminant of human and animal food products, and is toxic to all animal species tested. OTA predominantly affects the kidney and is known to accumulate in the proximal tubule (PT). The induction of oxidative stress is implicated in the toxicity of this mycotoxin.In the present study, primary rat PT cells and LLC-PK(1) cells, which express characteristics of the PT, were used to investigate the OTA-mediated oxidative stress response. OTA exposure of these cells resulted in a concentration-dependent elevation of reactive oxygen species (ROS) levels, depletion of cellular glutathione (GSH) levels and an increase in the formation of 8-oxoguanine.The OTA-induced ROS response was significantly reduced following treatment with alpha-tocopherol (TOCO). However, this chain-braking anti-oxidant did not reduce the cytotoxicity of OTA and was unable to prevent the depletion of total GSH levels in OTA-exposed cells. In contrast, pre-incubation of the cell with N-acetyl-L-cysteine (NAC) completely prevented the OTA-induced increase in ROS levels as well as the formation of 8-oxoguanine and completely protected against the cytotoxicity of OTA. In addition, NAC treatment also limited the GSH depletion in OTA-exposed PT- and LLC-PK(1) cells.From these data, we conclude that oxidative stress contributes to the tubular toxicity of OTA. Subsequently, cellular GSH levels play a pivotal role in limiting the short-term toxicity of this mycotoxin in renal tubular cells.