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.

Histological and histometrical study of the protective role of α-tocopherol against sodium arsenite toxicity in rat ovaries.

This study examines histometrical changes induced by sodium arsenite (SA), as an environmental pollutant, and investigates the protective effect of α-tocopherol on ovaries of SA-treated rats during the prenatal stage until sexual maturity. Rats were classified into groups: control, SA (8 ppm/day), α-tocopherol (100 ppm/day), and SA+α-tocopherol. Treatment was performed from pregnancy until maturation when the rats and ovaries were weighed. The Cavalieri method was used to estimate volume of the ovaries, cortex, medulla, and corpus luteum. The mean diameter of oocytes, granulosa cells, and nuclei were measured and volume was estimated using the Nucleator method. The number of oocytes and thickness of the zona pellucida (ZP) were determined using an optical dissector and orthogonal intercept method, respectively. SA reduced the body and ovary weight, the number of secondary, antral and Graafian oocytes, volume of the ovaries, cortex, medulla and corpus luteum, mean diameter and volume of oocytes in primordial and primary follicles, mean diameter and volume of oocyte nuclei in all types of follicles, and mean thickness of the ZP in secondary and antral follicles. Also, the mean diameter and volume of granulosa cells and their nuclei in antral and Graafian follicles decreased significantly. Vacuolization and vascular congestion in the corpus luteum and an increase in the number of atretic oocytes were seen in the SA group. Most of these parameters were unchanged from the control level in the SA+α-tocopherol group. It was concluded that α-tocopherol supplementation reduced the toxic effects of SA exposure on ovarian tissue in rats.

Biogenic synthesis of selenium nanoparticles and their effect on As(III)-induced toxicity on human lymphocytes.

A bioreductive capacity of a plant, Terminalia arjuna leaf extract, was utilized for preparation of selenium nanoparticles. The leaf extract worked as good capping as well as stabilizing agent and facilitated the formation of stable colloidal nanoparticles. Resulting nanoparticles were characterized using UV-Vis spectrophotometer, transmission electron microscopy (TEM), energy dispersive X-ray analysis (EDAX), Fourier transform infrared spectroscopy (FT-IR), and X-ray diffraction analysis (XRD), respectively. The colloidal solution showed the absorption maximum at 390 nm while TEM and selected area electron diffraction (SAED) indicated the formation of polydispersed, crystalline selenium nanoparticles of size raging from 10 to 80 nm. FT-IR analysis suggested the involvement of O-H, N-H, C=O, and C-O functional group of the leaf extract in particle formation while EDAX analysis indicated the presence of selenium in synthesized nanoparticles. The effect of nanoparticles on human lymphocytes treated with arsenite, As(III), has been studied. Studies on cell viability using MTT assay and DNA damage using comet assay revealed that synthesized selenium nanoparticles showed protective effect against As(III)-induced cell death and DNA damage. Chronic ingestion of arsenic infested groundwater, and prevalence of arsenicosis is a serious public health issue. The synthesized benign nanoselenium can be a promising agent to check the chronic toxicity caused due to arsenic exposure.

Sodium arsenite-induced alteration in hepatocyte function of rat with special emphasis on superoxide dismutase expression pathway and its prevention by mushroom lectin.

This study was accomplished to exemplify the possible protective role of ascorbic acid and mushroom lectin against arsenic-induced cytotoxicity and impairment of superoxide dismutase (SOD) production pathway in hepatocytes of rat. Hepatocytes were isolated from rat and treated with sodium arsenite (AS), arsenic plus ascorbic acid (AS + AA) and arsenic plus mushroom lectin (AS + ML). A placebo control was also included. Arsenic treatment resulted in the depletion of cell proliferation, phagocytic activity (nitro blue tetrazolium index) and superoxide dismutase (SOD) activity, relative mRNA expression of superoxide dismutase 2 (SOD(2)) and enhanced production of nitric oxide (NO). Ascorbic acid, a standard antioxidant, could normalize cellular perturbation and SOD production pathway relating to gene expression, whereas partially purified Pleurotus florida lectin (PFL), an edible mushroom containing protein complex, maintained cellular activity and prevented stress by normalizing phagocytic (NBT index) and SOD activities vis-à-vis relative gene expression. It could further defend NO production of hepatocytes. Mushroom lectin strongly prevented sodium arsenite-induced damage of SOD production pathway in hepatocytes, and its effect was also comparable to a standard antioxidant, i.e. ascorbic acid.

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.

Aqueous and ethanolic leaf extracts of Ocimum basilicum (sweet basil) protect against sodium arsenite-induced hepatotoxicity in Wistar rats.

We evaluated the effects of aqueous and ethanolic leaf extracts of Ocimum basilicum (sweet basil) on sodium arsenite-induced hepatotoxicity in Wistar rats. We observed that treatment of the animals with the extracts before or just after sodium arsenite administration significantly (p < 0.05) reduced mean liver and serum γ-Glutamyl transferase (γGT), and serum alkaline phosphatase (ALP) activities when compared with the group administered the toxin alone. In addition, treatments of the animals with aqueous or ethanolic extract of O. basilicum before the administration of sodium arsenite resulted in the attenuation of the sodium arsenite-induced aspartate and alanine aminotransferase activities: ALT (from 282.6% to 167.7% and 157.8%), AST (from 325.1% to 173.5% and 164.2%) for the group administered sodium arsenite alone, the aqueous extracts plus sodium arsenite, and ethanolic extracts plus sodium arsenite respectively, expressed as percentage of the negative control. These findings support the presence of hepatoprotective activity in the O.basilicum extracts.

Protective effect of dietary phytochemicals against arsenite induced genotoxicity in mammalian V79 cells.

Chronic arsenic exposure causes skin diseases, gastrointestinal and neurological disorders, diabetes and cancer in various organs. Oxidative stress associated with arsenic exposure cause genetic instabilities and may initiate carcinogenesis. Phytochemicals present in vegetables, fruits, spices, tea, and medicinal plants, have shown to suppress experimental carcinogenesis in various organs. The aim of the present study was to elucidate the protective effect of some of the phytochemicals against the arsenite induced DNA damage in normal mammalian V79 cells. Comet assay was used for assessment of DNA damage and 2', 7'-dichlorofluorescein dihydroacetate for estimation of ROS generated by arsenite. The effect of the phytochemicals was observed during simultaneous treatment with arsenic, before arsenite exposure and during repair experiments. Of all the phytochemicals tested against arsenic, curcumin gave better protection during simultaneous treatment and resveratrol during pre treatment, which was evident both from comet assay and ROS generation experiments. During pre treatment a longer duration of treatment with lower dose of phytochemicals proved fruitful in reducing the genotoxicity. During repair experiments the phytochemicals enhanced recovery of DNA damage and ellagic acid gave promising results. The results indicated that natural phytochemicals may have the efficacy in reducing arsenic induced genotoxicity, in scavenging ROS and in enhancing the process of DNA repair in V79 cells.

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.

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.

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.

Inhibition of arsenite-induced apoptosis and AP-1 activity by epigallocatechin-3-gallate and theaflavins.

Arsenite-induced apoptosis appears to be important in its toxicity and its role in carcinogenesis. Green tea has been used as a traditional Chinese remedy for detoxification of arsenite-caused toxicity. In the present work, we found that tea polyphenols, EGCG and theaflavins, effectively blocked arsenite-induced apoptosis of JB6 cells and inhibited arsenite-induced AP-1 transcription activity and AP-1 DNA binding activity. EGCG and theaflavins potently inhibited arsenite-induced Erks activity, but not p38 kinase activity. PD 98059, an inhibitor of Erks, and DNM-JNK1 blocked arsenite-induced apoptosis, while SB202190, an inhibitor of p38 kinases, or DNM-p38 kinase did not. We conclude that Erks and JNKs may be involved in arsenite-induced apoptosis, and the inhibition of arsenite-induced apoptosis by EGCG and theaflavins may be mediated by a decreased phosphorylation of Erks and JNKs. Furthermore, these results provide a possible mechanism for the detoxification effect of tea on arsenite-induced toxicity.

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.