Protective Effect of Azadirachta indica and Vitamin E Against Arsenic Acid-Induced Genotoxicity and Apoptosis in Rats.

Sodium arsenite (NaAsO2) is one of the major environmental toxicants with severe toxicological consequences in some developing and developed countries. Rats in Group A received normal saline. Genotoxicity and apoptosis were induced by single intraperitoneal injection of 10 mg/kg sodium arsenite to rats in Groups B-F. Rats in Groups C and D had earlier been pretreated with Azadirachta indica (100 and 200 mg/kg) or E and F with vitamin E (50 and 100 mg/kg), respectively. Markers of oxidative stress, inflammation, hepatic damage, genotoxicity, and apoptosis were assessed. Pretreatment of rats with either Azadirachta indica or vitamin E led to a significant (p <.05) increase in the activities of glutathione-S-transferase (GST), catalase (CAT), superoxide dismutase (SOD), and reduced glutathione (GSH) in the liver compared to the group that received NaAsO2 alone. Markers of oxidative stress and inflammation, malondialdehyde (MDA), hydrogen peroxide (H2O2) generation, nitric oxide (NO), and myeloperoxidase (MPO), were significantly (p <.05) lowered in rats pretreated with Azadirachta indica or vitamin E. The frequency of micronucleated polychromatic erythrocytes (MNPCEs) and expression of caspase-3 were significantly (p <.05) reduced in rats pretreated with either Azadirachta indica or vitamin E compared to rats intoxicated with arsenite. Histopathology of the liver showed areas of infiltration of inflammatory cells with deaths of numerous hepatocytes in NaAsO2-intoxicated rats, and these were reversed by Azadirachta indica. Together, we report for the first time the genoprotective and antiapoptotic effect of Azadirachta indica by a significant reduction in the frequency of micronuclei-induced apoptosis and oxidative stress by arsenic intoxication.

Linking arsenite- and cadmium-generated oxidative stress to microsatellite instability in vitro and in vivo.

Mismatch repair (MMR) corrects replicative errors and minimizes DNA damage that occurs frequently in microsatellites. MMR deficiency is manifested as microsatellite instability (MSI), which contributes to hypermutability and cancer pathogenesis. Genomic instability, including MSI and chromosomal instability, appears to be responsible for the carcinogenesis of arsenic and cadmium, common contaminants in our environment. However, few studies have addressed arsenic- or cadmium-induced MSI, especially its potential link with arsenic- or cadmium-generated oxidative stress, due to the lack of quantifiable MSI assays and cost-effective animal models. Here, using a dual-fluorescent reporter, we demonstrate that sub-lethal doses of cadmium or arsenite, but not arsenate, increased the MSI frequency in human colorectal cancer cells. Arsenite- and cadmium-induced MSI occurred concomitantly with increased levels of reactive species and oxidative DNA damage, and with decreased levels of MMR proteins. However, N-acetyl-l-cysteine (NAC) suppressed arsenite- and cadmium-induced MSI and oxidative stress while restoring the levels of MMR proteins in the cells. Similarly, MSI was induced separately by arsenite and cadmium, and suppressed by NAC, in zebrafish in a fluorescinated PCR-based assay with newly-developed microsatellite markers and inter-segmental comparisons. Of five selected antioxidants examined, differential effects were exerted on the MSI induction and cytotoxicity of both arsenite and cadmium. Compared to MMR-proficient cells, MMR-deficient cells were more resistant to arsenic-mediated and cadmium-mediated cytotoxicity. Our findings demonstrate a novel linkage between arsenite-generated and cadmium-generated oxidative stress and MSI induction. Our findings also caution that antioxidants must be individually validated before being used for preventing arsenite- and cadmium-induced MSI that is associated with cancer development.

Dual role of resveratrol in modulation of genotoxicity induced by sodium arsenite via oxidative stress and apoptosis.

The potential benefits of resveratrol as an anticancer (proapoptosis) and antioxidant (pro-survival) compound have been studied extensively. However, the role of resveratrol in modulation of the toxicity induced by sodium arsenite (NaAsO2) is still unclear. In the present study, we examined the effects of resveratrol on NaAsO2-induced cytotoxicity, DNA and chromosomal damage, cell cycle progression, apoptosis and oxidative stress in human lung adenocarcinoma epithelial (A549) cell line at concentrations from 1 to 20 µM after 24h exposure. Our results revealed that at 1 and 5 µM, resveratrol was found to exert benefit effects, promoting cell viability and proliferation over 24h NaAsO2 exposure, whereas, resveratrol was showed to inhibit cell survival under the same condition at 20 µM. Corresponding to the opposing effect of resveratrol at low vs. high concentrations, DNA and chromosomal damage, cell apoptotic rate and level of oxidative stress were also alleviated by lower concentrations (1, 5 µM) of resveratrol, but exacerbated by higher concentration (20 µM) resveratrol. Our study implicates that resveratrol is the most beneficial to cells at 1 and 5 µM and caution should be taken in applying resveratrol as an anticancer therapeutic agent or nutraceutical supplement due to its concentration dependent effect.

Efflux of glutathione and glutathione complexes from human erythrocytes in response to inorganic arsenic exposure.

The objective of the present study was to investigate if arsenic exposure results in glutathione efflux from human erythrocytes. Arsenite significantly depleted intracellular nonprotein thiol level in a time- and concentration-dependent manner. The intracellular nonprotein thiol level was decreased to 0.767 ± 0.0017 µmol/ml erythrocyte following exposure to 10 mM of arsenite for 4 h. Extracellular nonprotein thiol level was increased concomitantly with the intracellular decrease and reached to 0.481 ± 0.0005 µmol/ml erythrocyte in 4 h. In parallel with the change in extracellular nonprotein thiol levels, significant increases in extracellular glutathione levels were detected. Extracellular glutathione levels reached to 0.122 ± 0.0013, 0.226 ± 0.003, and 0.274 ± 0.004 µmol/ml erythrocyte with 1, 5, and 10 mM of arsenite, respectively. Dimercaptosuccinic acid treatment of supernatants significantly increased the glutathione levels measured in the extracellular media. Utilization of MK571 and verapamil, multidrug resistance-associated protein 1 and Pgp inhibitors, decreased the rate of glutathione efflux from erythrocytes suggesting a role for these membrane transporters in the process. The results of the present study indicate that human erythrocytes efflux glutathione in reduced free form and in conjugated form or forms that can be recovered with dimercaptosuccinic acid when exposed to arsenite.

All-trans retinoic acid protects against arsenic-induced uterine toxicity in female Sprague-Dawley rats.

BACKGROUND AND PURPOSE: Arsenic exposure frequently leads to reproductive failures by disrupting the rat uterine histology, hormonal integrity and estrogen signaling components of the rat uterus, possibly by generating reactive oxygen species. All-trans retinoic acid (ATRA) was assessed as a prospective therapeutic agent for reversing reproductive disorders. EXPERIMENTAL APPROACH: Rats exposed to arsenic for 28 days were allowed to either recover naturally or were treated simultaneously with ATRA for 28 days or treatment continued up to 56 days. Hematoxylin-eosin double staining was used to evaluate changes in the uterine histology. Serum gonadotropins and estradiol were assayed by ELISA. Expression of the estrogen receptor (ERα), an estrogen responsive gene vascular endothelial growth factor (VEGF), and cell cycle regulatory proteins, cyclin D1 and CDK4, was assessed by RT-PCR, immunohistochemistry and western blot analysis. KEY RESULTS: ATRA ameliorated sodium arsenite-induced decrease in circulating estradiol and gonadotropin levels in a dose- and time-dependent manner, along with recovery of luminal epithelial cells and endometrial glands. Concomitant up regulation of ERα, VEGF, cyclin D1, CDK4 and Ki-67 was also observed to be more prominent for ATRA-treated rats as compared to the rats that were allowed to recover naturally for 56 days. CONCLUSIONS AND IMPLICATIONS: Collectively, the results reveal that ATRA reverses arsenic-induced disruption of the circulating levels of gonadotropins and estradiol, and degeneration of luminal epithelial cells and endometrial glands of the rat uterus, indicating resumption of their functional status. Since structural and functional maintenance of the pubertal uterus is under the influence of estradiol, ATRA consequently up regulated the estrogen receptor and resumed cellular proliferation, possibly by an antioxidant therapeutic approach against arsenic toxicity.

The novel role of fenofibrate in preventing nicotine- and sodium arsenite-induced vascular endothelial dysfunction in the rat.

The present study investigated the effect of fenofibrate, an agonist of PPAR-alpha, in nicotine- and sodium arsenite-induced vascular endothelial dysfunction (VED) in rats. Nicotine (2 mg/kg/day, i.p., 4 weeks) and sodium arsenite (1.5 mg/kg/day, i.p., 2 weeks) were administered to produce VED in rats. The scanning electron microscopy study in thoracic aorta revealed that administration of nicotine or sodium arsenite impaired the integrity of vascular endothelium. Further, administration of nicotine or sodium arsenite significantly decreased serum and aortic concentrations of nitrite/nitrate and subsequently reduced acetylcholine-induced endothelium-dependent relaxation. Moreover, nicotine or sodium arsenite produced oxidative stress by increasing serum thiobarbituric acid reactive substances (TBARS) and aortic superoxide generation. However, treatment with fenofibrate (30 mg/kg/day, p.o.) or atorvastatin (30 mg/kg/day p.o., a standard agent) significantly prevented nicotine- and sodium arsenite-induced VED and oxidative stress by improving the integrity of vascular endothelium, increasing the concentrations of serum and aortic nitrite/nitrate, enhancing the acetylcholine-induced endothelium-dependent relaxation and decreasing serum TBARS and aortic superoxide anion generation. Conversely, co-administration of L-NAME (25 mg/kg/day, i.p.), an inhibitor of nitric oxide synthase, markedly attenuated these vascular protective effects of fenofibrate. The administration of nicotine or sodium arsenite altered the lipid profile by increasing serum cholesterol and triglycerides and consequently decreasing high-density lipoprotein levels, which were significantly prevented by treatment with fenofibrate or atorvastatin. It may be concluded that fenofibrate improves the integrity and function of vascular endothelium, and the vascular protecting potential of fenofibrate in preventing the development of nicotine- and sodium arsenite-induced VED may be attributed to its additional properties (other than lipid lowering effect) such as activation of eNOS and generation of NO and consequent reduction in oxidative stress.

Arsenic induces apoptosis of human umbilical vein endothelial cells through mitochondrial pathways.

To clarify the molecular mechanisms through which arsenic causes injuries to blood vessels, we analyzed the effects of sodium arsenite (NaAsO(2)) on the apoptosis of human umbilical vein endothelial cells (HUVECs), mitochondrial membrane potential (Delta Psi m), intracellular reactive oxygen species (ROS), and the expression of the related genes. HUVECs apoptosis increased and Delta Psi m decreased in a dose-dependent manner following arsenic treatment. Intracellular ROS showed 2 phase alterations: a slight decrease with low levels of arsenic (5 and 10 microM) treatment; but a sharp increase at higher concentrations (>or=20 microM). The arsenic-induced cell apoptosis and intracellular ROS were blocked by the addition of the antioxidant N-acetyl-L-cysteine (NAC). The mRNAs of superoxide dismutase 2 (SOD2) and NAD(P)H:quinone oxidoreductase 1 (NQO1) increased strikingly when cells were treated with a low concentration of NaAsO(2) (5 microM) and the level of induction was decreased with higher concentrations of arsenic treatment. Based on the results, we suggest that the decrease of Delta Psi m caused by arsenic and the resulting cell apoptosis may contribute to the injuries of blood vessel in arsenism. The decrease in intracellular ROS and the increase in SOD2 and NQO1 expressions observed when HUVECs were treated with low concentration of NaAsO(2), suggest the role of the two enzymes in protecting HUVECs from injuries of arsenic exposure.

Protective effect of Corchorus olitorius leaves on sodium arsenite-induced toxicity in experimental rats.

The present study was undertaken to evaluate the protective effect of aqueous extract of Corchorus olitorius leaves (AECO) against sodium arsenite-induced toxicity in experimental rats. The animals exposed to sodium arsenite at a dose of 10mg/kg body weight p.o. for 10days exhibited a significant inhibition (p<0.01) of hepatic and renal antioxidant enzymes namely superoxide dismutase, catalase, glutathione-S-transferase, glutathione peroxidase and glutathione reductase. In addition, arsenic intoxication significantly decreased (p<0.01) the level of reduced glutathione and increased (p<0.01) the levels of oxidized glutathione and thiobarbituric acid reactive substances in selected tissues. Treatment with AECO at doses of 50 and 100mg/kg body weight p.o. for 15days prior to arsenic intoxication significantly improved hepatic and renal antioxidant markers in a dose dependant manner. AECO treatment also significantly reduced the arsenic-induced DNA fragmentation of hepatic and renal tissues. Histological studies on the ultrastructural changes of liver and kidney supported the protective activity of the AECO. The results concluded that the treatment with AECO prior to arsenic intoxication has significant role in protecting animals from arsenic-induced hepatic and renal toxicity.

In vitro and in vivo reduction of sodium arsenite induced toxicity by aqueous garlic extract.

BACKGROUND: Arsenic is ubiquitous in the environment, and chronic or acute exposure through food and water as well as occupational sources can contribute to a well-defined spectrum of disease. Despite arsenic being a health hazard and a well-documented human carcinogen, a safe, effective and specific preventive or therapeutic measure for treating arsenic induced toxicity still eludes us. OBJECTIVE: This study was undertaken to evaluate the therapeutic efficacy of aqueous garlic (Allium sativum L.) extract (AGE) in terms of normalization of altered biochemical parameters particularly indicative of oxidative stress following sodium arsenite (NaAsO(2)) exposure and depletion of inorganic arsenic burden, in vitro and in vivo. RESULTS: AGE (2mg/ml) co-administered with 10 microM NaAsO(2) attenuated arsenite induced cytotoxicity, reduced intracellular reactive oxygen species (ROS) level in human malignant melanoma cells (A375), human keratinocyte cells (HaCaT) and in cultured human normal dermal fibroblast cells. Moreover, AGE application in NaAsO(2) intoxicated Sprague-Dawley rats resulted in a marked inhibition of tissue lipid peroxide generation; enhanced level of total tissue sulfhydryl groups and glutathione; and also increased the activities of antioxidant enzymes, superoxide dismutase and catalase to near normal. An increase in blood ROS level and myeloperoxidase activity in arsenic-intoxicated rats was effectively prevented by AGE administration. AGE was also able to counter arsenic mediated incongruity in blood hematological variables and glucose level. CONCLUSIONS: The restorative property of AGE was attributed to its antioxidant activity, chelating efficacy, and/or oxidizing capability of trivalent arsenic to its less toxic pentavalent form. Taken together, evidences indicate that AGE can be a potential protective regimen for arsenic mediated toxicity.

S-adenosyl-L-methionine counteracts mitotic disturbances and cytostatic effects induced by sodium arsenite in HeLa cells.

Aneuploidy represents a serious problem for human health. Toxicological data have shown that aneuploidy can be caused by exposure to chemical agents known as mitotic spindle poisons, since they arrest cell cycle in mitosis through their interaction with tubulin. Among these agents is arsenic. In previous reports, we demonstrated that the aneugenic events induced by sodium arsenite can be abolished by the exogenous addition of S-adenosyl-l-methionine (SAM). Nevertheless, the mechanisms involved are still unknown. The aim of the present work was to study the influence of SAM on the mitotic disturbances caused by sodium arsenite. To achieve this goal, we analyzed microtubule (MT) polymerization by immunolocalization and live cell microscopy of mitotic cells. Our findings indicate that sodium arsenite alters the dynamics of MT polymerization, induces centrosome amplification and delays mitosis. Furthermore, SAM reduces the alterations on MT dynamics, as well as centrosome amplification, and therefore diminishes the formation of multipolar spindles in treated HeLa cells. In addition, SAM decreases the progression time through mitosis. Taking these data together, we consider that the mechanism by which SAM reduces the frequency of aneuploid cells must be related to the modulation of the dynamics and organization of MT, suggesting a role of SAM on chromosome segregation, which should be further investigated in primary cells.

Vitamin E supplementation protects oxidative stress during arsenic and fluoride antagonism in male mice.

Arsenic and fluoride are common environmental contaminants. Coexposure to these elements can occur through groundwater. We investigated the effects of sodium meta arsenite (50 mg/L in drinking water) and sodium fluoride (50 mg/L in drinking water) individually and in combination. Biochemical parameters suggestive of alterations in heme synthesis pathway, oxidative stress in liver and kidneys, and concentration of essential metals in blood and soft tissues were studied in Swiss albino male mice given the chemicals for 3 weeks. The possible beneficial effect of vitamin E administration (25 mg/kg, oral, alternate days after arsenic/fluoride exposure) on the above variables was investigated. Exposure to arsenic or fluoride caused a significant depletion in blood delta-aminolevulinic acid dehydratase (ALAD) activity, platelet counts (PLT), and glutathione (GSH) level. Blood white blood cell (WBC) counts also decreased. These changes were accompanied by increased reactive oxygen species (ROS) levels. Arsenic and fluoride exposure led to a significant depletion of super oxide dismutase (SOD) activity with no effect on catalase and glutathione peroxidase (GPx) activities. Combined exposure to these toxicants had no synergistic effect on blood ALAD activity and WBC counts, and the effects seen appeared to result predominantly from arsenic. Hepatic catalase activity, on the other hand, increased significantly on exposure to arsenic and fluoride. There was only moderate antagonistic effect on arsenic and fluoride concentration in blood and liver, and kidney arsenic concentration was less pronounced during coexposure compared with arsenic alone. Interestingly, fluoride concentration showed less pronounced uptake during concomitant exposure compared with fluoride exposure alone. Vitamin E supplementation during coexposure to arsenic and fluoride provided only moderate recovery in the altered antioxidant enzymes and in depleting ROS level, but the altered essential metal concentration, particularly calcium level, responded more favorably to vitamin E administration. It can be concluded from the current study that (i) coadministration of arsenic and fluoride was less toxic to the animals compared with individual toxic effects of these toxicants, and (ii) vitamin E supplementation during coexposure had only limited additional beneficial effects in restoring altered biochemical variables, maintaining pro-oxidant/antioxidant balance, and reducing body arsenic store but plays a significant role in maintaining essential metal balance.

Pro-angiogenesis action of arsenic and its reversal by selenium-derived compounds.

Inorganic arsenic (arsenite and arsenate) in drinking water has been associated with skin cancers and increased incidence of cardiovascular diseases. Additionally, studies have demonstrated the pro-angiogenic effect of arsenite and its potential promotion of tumor angiogenesis and tumor progression. Furthermore, recent reports demonstrated reversal of skin co-carcinogenesis by an organoselenium compound. The present study was undertaken to determine the effect and mechanism on angiogenesis of arsenite at low level and its potential reversal by various selenium-derived compounds. The pro-angiogenesis effects and mechanisms of sodium arsenite were determined using the chick chorioallantoic membrane (CAM) model over 3 days and compared with standard pro-angiogenesis factors, such as basic fibroblast growth factor (b-FGF). Additionally, the potential effect of various selenium-derived compounds--such as dimethyl selenone, diphenyl selenone, sodium selenite or Se-methyl selenocysteine--in reversing the pro-angiogenesis effect of arsenite or b-FGF was also determined in the CAM model. The pro-angiogenesis effect of arsenite or b-FGF was significantly (P < 0.01) blocked by dimethyl selenone, diphenyl selenone, sodium selenite or Se-methyl selenocysteine. The pro-angiogenesis effect of either sodium arsenite at 33 nM or b-FGF was blocked (P < 0.01) by the extracellular signal-regulated kinases 1 and 2 (ERK1/2) activation inhibitor, PD 98059. Additionally, the pro-angiogenic effect of arsenic or b-FGF was blocked as well (P < 0.01) by the alphavbeta3 antagonist, XT199. These data suggest that the pro-angiogenesis effect of arsenic is initiated at the plasma membrane integrin alphavbeta3, involves activation of the ERK1/2 pathway and is effectively reversed by various selenium-derived compounds.

Arsenite induces endothelial cytotoxicity by down-regulation of vascular endothelial nitric oxide synthase.

Epidemiological studies have demonstrated a high association of inorganic arsenic exposure with vascular diseases. Recent research has also linked this vascular damage to impairment of endothelial nitric oxide synthase (eNOS) function by arsenic exposure. However, the role of eNOS in regulating the arsenite-induced vascular dysfunction still remains to be clarified. In our present study, we investigated the effect of arsenite on Akt1 and eNOS and its involvement in cytotoxicity of vascular endothelial cells. Our study demonstrated that arsenite decreased the protein levels of both Akt1 and eNOS accompanied with increased levels of ubiquitination of total cell lysates. We found that inhibition of the ubiquitin-proteasome pathway by MG-132 could partially protect Akt1 and eNOS from degradation by arsenite together with a proportional protection from the arsenite-induced cytoxicity. Moreover, up-regulation of eNOS protein expression significantly attenuated the arsenite-induced cytotoxicity and eNOS activity could be significantly inhibited after incubation with arsenite for 24 h in a cell-free system. Our study indicated that endothelial eNOS activity could be attenuated by arsenite via the ubiquitin-proteasome-mediated degradation of Akt1/eNOS as well as via direct inhibition of eNOS activity. Our study also demonstrated that eNOS actually played a protective role in arsenite-induced cytoxicity. These observations supported the hypothesis that the impairment of eNOS function by arsenite is one of the mechanisms leading to vascular changes and diseases.

Amelioration of sodium arsenite-induced clastogenicity by tea extracts in Chinese hamster v79 cells.

Since the early 1980s, an alarming problem of groundwater arsenic (As) contamination has devastated many districts of West Bengal in India. People drinking As-contaminated water have been suffering severe health problems such as hyperkeratosis, blackfoot disease, neuropathy, and cancer of various sites. DNA damage and genetic instability induced by the inorganic arsenicals present in water are thought to be prerequisites for the initiation of carcinogenesis. Many natural polyphenols, which are consumed through our daily diet, possess excellent cancer chemopreventive properties. Tea, a popular beverage worldwide and rich in polyphenols, has exhibited many health benefits. The present study was conducted to examine the anticlastogenic action of tea extracts (both green and black) against the As-induced chromosomal aberrations. We also evaluated the role of tea in inducing antioxidant enzymes such as superoxide dismutase and catalase to provide protection against the oxidative stress induced by As. Our results demonstrated that tea extracts, particularly Darjeeling tea extract, are effective in counteracting the clastogenicity (chromatid breaks, in particular) of the most potent form of As, sodium arsenite. The antioxidant function of tea in reducing clastogenicity may be partly due to the induction of phase II detoxification enymes, such as superoxide dismutase and catalase. Our results suggest that the use of tea may be an effective approach in combating the health crisis generated by As.

Protective effect of N-acetylcysteine against arsenic-induced depletion in vivo of carbohydrate.

N-acetylcysteine (NAC), a synthetic aminothiol, possesses antioxidative and cytoprotective properties. The present study evaluates the effect of NAC supplementation on arsenic-induced depletion in vivo of carbohydrates. Arsenic (as sodium arsenite) treatment (i.p.) of male Wistar rats (120-140 g b.w.) at a dose of 5.55 mg/kg body weight (35% of LD50) per day for a period of 30 days produced a significant decrease in blood glucose level (hypoglycemia) and a fall in liver glycogen and pyruvic acid contents. The free amino acid nitrogen content of liver increased while that of kidney decreased after arsenic treatment. Arsenic also enhanced the liver lactate dehydrogenase activity whereas glucose 6-phosphatase activity in both liver and kidney decreased significantly following arsenic treatment. Transaminase activities in liver and kidney were not significantly altered except the glutamate-pyruvate transaminase activity that was reduced in kidney after arsenic treatment. Oral administration of NAC (163.2 mg/kg/day) for last 7 days of treatment prevented the arsenic-induced hypoglycemia and glycogenolytic effects to an appreciable extent. There was also recovery of liver pyruvic acid as well as liver and kidney free amino acid nitrogen content after NAC supplementation. Arsenic-induced alteration of glucose 6-phosphatase activity in both liver and kidney was also counteracted by NAC. It is suggested that carbohydrate depletion in vivo due to exposure to arsenic can be counteracted by NAC supplementation.

Folic acid protects SWV/Fnn embryo fibroblasts against arsenic toxicity.

It has been proposed that arsenic exerts its toxic effects, in part, by perturbing cellular methyl metabolism. Based on the hypothesis that folic acid treatment will attenuate the cytotoxic and growth inhibitory effects of arsenic, SWV/Fnn embryo fibroblasts were cultured in media supplemented with various concentrations of folic acid during treatment with sodium arsenite or dimethylarsinic acid (DMA). It was found that folic acid protects SWV/Fnn embryo fibroblasts from sodium arsenite and DMA cytotoxicity in a dose-dependent manner. In contrast, folic acid supplementation has no effect on toxicity resulting from treatment with ethanol or staurosporine, suggesting that folic acid is not generally protective against necrosis and apoptosis. Although folic acid protects against acute arsenic toxicity, this agent shows a modest and delayed ability to attenuate the growth inhibitory effect of arsenic on these cells. These results support a model in which perturbations of methyl metabolism contribute to the acute cytotoxicity of arsenic.

Cyclosporin A inhibits chromium(VI)-induced apoptosis and mitochondrial cytochrome c release and restores clonogenic survival in CHO cells.

A variety of key events in the molecular apoptotic pathway involve the mitochondria. Cyclosporin A (csA) affects the mitochondria by inhibiting the mitochondrial permeability transition (MPT), thereby preventing disruption of the transmembrane potential. The role of the MPT in apoptosis is not fully understood, but inhibition of the MPT may prevent the release of mitochondrial caspase activators, such as cytochrome c (cyt c), into the cytosol. Certain hexavalent chromium [Cr(VI)] compounds are known occupational respiratory tract toxins and carcinogens. We have previously shown that these compounds induce apoptosis as a predominant mode of cell death and that this effect can be observed in cell culture using soluble Cr(VI). We show here that Cr(VI)-induced apoptosis in Chinese hamster ovary (CHO) cells involves disruption of mitochondrial stability. Using a cyt c-specific monoclonal antibody, we observed a dose-dependent release of mitochondrial cyt c in cytosolic extracts of CHO cells exposed to apoptogenic doses of sodium chromate. Co-treatment of these cells with csA inhibited the release of cyt c and abrogated Cr(VI)-induced apoptosis as determined by a reduction in internucleosomal DNA fragmentation. Co-treatment with csA also markedly increased clonogenic survival of Cr(VI)-treated CHO cells. In contrast, the general caspase inhibitor Z-VAD-FMK markedly inhibited most of the morphological and biochemical parameters of apoptosis but did not prevent cyt c release and did not increase clonogenic survival. These results suggest that the MPT plays an important role in the regulation of mitochondrial cyt c release and that this may be a critical point in the apoptotic pathway in which cells are irreversibly committed to death.

Apoptosis and P53 induction in human lung fibroblasts exposed to chromium (VI): effect of ascorbate and tocopherol.

Some forms of hexavalent chromium [Cr(VI)] are known to cause damage to respiratory tract tissue, and are thought to be human lung carcinogens. Because Cr(VI) is mutagenic and carcinogenic at doses that evoke cell toxicity, the objective of these experiments was to examine the effect of Cr(VI) on the growth, survival, and mode of cell death in normal human lung fibroblasts (HLF cells). DNA adduct formation was monitored as a marker for bioavailability of genotoxic chromium. We also examined the modulation of these endpoints by vitamins C and E. Long-term Cr(VI) exposures were employed, which decreased clonogenic cell survival by 25% to 95% in a dose-dependent manner. The predominant cellular response to Cr(VI) was growth arrest. We found that Cr(VI) caused up to 20% of HLF cells to undergo apoptosis, and documented apoptotic morphology and the phagocytosis of apoptotic bodies by neighboring cells. P53 levels increased 4- to 6-fold in chromium-treated cells. In contrast with previous studies using CHO cells, the present study using HLFs found that pretreatment with either vitamin C or E did not exhibit a significant effect on Cr-induced apoptosis or clonogenic survival. In addition, pretreatment with vitamin C did not affect the p53 induction observed after chromium treatment. Neither vitamin had any effect on Cr-DNA adduct formation. These data indicate that although pretreatment with vitamin C or E alters the spectrum of cellular and/or genetic lesions induced by chromium(VI), neither vitamin altered the initiation or progression of apoptosis in diploid human lung cells.

Prevention of cytotoxic effects of arsenic by short-term dietary supplementation with selenium in mice in vivo.

Interaction between selenium and arsenic has been used to protect against the genotoxic effects of sodium arsenite through dietary intervention by an equivalent amount (1/10 LD50) of sodium selenite. The two salts were administered by gavaging to laboratory bred Swiss albino mice sequentially and in combination. Cytogenetic endpoints, including chromosomal aberrations (CA) and damaged cells (DC) were recorded 24 h after exposure from chromosome spreads in bone marrow cells. Administration of sodium selenite 1 h before sodium arsenite reduced the clastogenic effects of the latter significantly. The protection was less when the salts were given together and negative when arsenite was given before selenite. Histological changes were recorded. Such reduction of arsenic toxicity through dietary intervention by selenium is of significance in protecting against the widespread toxicity observed in human populations exposed to arsenic through drinking water from contaminated deep tubewells in West Bengal and Bangladesh.

Mustard oil and garlic extract as inhibitors of sodium arsenite-induced chromosomal breaks in vivo.

Arsenic, a well-known human carcinogen present as a contaminant in ground water poses a serious threat to public health in various countries. The anticlastogenic properties of two dietary supplements, garlic and mustard oil, were screened against the clastogenic activity of sodium arsenite, since diet may contain factors which affect the process of mutagenesis and carcinogenesis. Aqueous extract of garlic (100 mg/kg b.w.) and mustard oil (0.643 mg/kg b.w.) were fed to Mus musculus for 30 consecutive days either singly or simultaneously. Sodium arsenite (0.1 mg/kg b.w.) was injected subcutaneously on days 7, 14, 21 and 30 of the experiment, singly and together with the dietary supplements. The animals were sacrificed 24 h after the last exposure to sodium arsenite and clastogenic effects were observed in the bone marrow cells. The degree of modulation of sodium arsenite-induced chromosomal aberrations was more pronounced in mustard oil than in garlic extract and simultaneous administration of both the dietary supplements reduced the clastogenic effects of sodium arsenite closer to the level of the negative control. The greater efficacy could be due to the interaction of the two dietary supplements and its radical scavenging property.