Selenite Downregulates STAT3 Expression and Provokes Lymphocytosis in the Liver of Chronically Exposed Syrian Golden Hamsters.

Arsenic is considered a worldwide pollutant that can be present in drinking water. Arsenic exposure is associated with various diseases, including cancer. Antioxidants as selenite and α-tocopherol-succinate have been shown to modulate arsenic toxic effects. Since changes in STAT3 and PSMD10 gene expression have been associated with carcinogenesis, the aim of this study was to evaluate the effect of arsenic exposure and co-treatments with selenite or α-tocopherol-succinate on the expression of these genes, in the livers of chronically exposed Syrian golden hamsters. Animals were divided into six groups: (i) control, (ii) chronically treated with 100 ppm arsenic, (iii) treated with 6 ppm α-tocopherol-succinate (α-TOS), (iv) treated with 8.5 ppm selenite, (v) treated with arsenic + α-TOS, and (vi) treated with arsenic + selenite. Urine samples and livers were collected after 20 weeks of continuous exposure. The urine samples were analyzed for arsenic species by atomic absorption spectrophotometry, and real-time RT-qPCR analysis was performed for gene expression evaluation. A reduction in STAT3 expression was observed in the selenite-treated group. No differences in PSMD10 expression were found among groups. Histopathological analysis revealed hepatic lymphocytosis in selenite-treated animals. As a conclusion, long-term exposure to arsenic does not significantly alter the expression of STAT3 and PSMD10 oncogenes in the livers of hamsters; however, selenite down-regulates STAT3 expression and provokes lymphocytosis.

Ogg1 genetic background determines the genotoxic potential of environmentally relevant arsenic exposures.

Inorganic arsenic (i-As) is a well-established human carcinogen to which millions of people are exposed worldwide. It is generally accepted that the genotoxic effects of i-As after an acute exposure are partially linked to the i-As-induced production of reactive oxygen species, but it is necessary to better determine whether chronic sub-toxic i-As doses are able to induce biologically significant levels of oxidative DNA damage (ODD). To fill in this gap, we have tested the genotoxic and oxidative effects of environmentally relevant arsenic exposures using mouse embryonic fibroblast MEF mutant Ogg1 cells and their wild-type counterparts. Effects were examined by using the comet assay complemented with the use of FPG enzyme. Our findings indicate that MEF Ogg1-/- cells are more sensitive to arsenite-induced acute toxicity, genotoxicity and ODD. Long-term exposure to sub-toxic doses of arsenite generates a detectable increase in ODD and genotoxic DNA damage only in MEF Ogg1-deficient cells. Altogether, the data presented here point out the relevance of ODD and Ogg1 genetic background on the genotoxic risk of i-As at environmentally plausible doses. The persistent accumulation of DNA 8-OH-dG lesions in Ogg1-/- cells during the complete course of the exposure suggests a relevant role in arsenic-associated carcinogenic risk in turn.

Inhibition of hepatocyte nuclear factor 1 and 4 alpha (HNF1α and HNF4α) as a mechanism of arsenic carcinogenesis.

Inorganic arsenic (i-As) is a naturally occurring toxic metalloid affecting millions of people worldwide. It is known to be carcinogen, liver being a potential target, and related to the prevalence of diabetes in arseniasis-endemic areas. Hepatocyte nuclear factor 1 and 4 alpha (HNF1α and HNF4α) are key members of a transcriptional network essential for normal liver architecture. Changes in HNF1α and HNF4α expression are clearly associated with the development of liver malignancies and diabetes in humans. In this work, hepatic HepG2 cells and golden Syrian hamsters were exposed to sub-toxic, environmentally relevant doses of sodium arsenite (SA; up to 10 µM in vitro, 15 mg/L in vivo) in order to evaluate whether arsenic is able to compromise the expression of hepatocyte nuclear factors. Also, liver histopathological examination was carried out, and several markers of hepatocyte differentiation and glucose metabolism status were determined as a measure of i-As-induced effects. Results show a consistent down-regulation of HNF1α and HNF4α under a scenario of exposure where HepG2 cells (1) gained resistance to arsenic-induced toxicity/apoptosis, (2) attained loss of tissue-specific features (as shown by the observed down-regulation of ALDOB, PEPCK and CYP1A2, triggering of the epithelial-to-mesenchymal transition program and the hypersecretion of matrix metalloproteinase-2 and 9), (3) failed to maintain balanced expression of the "stemness" genes C-MYC, OCT3/4, LIN28 and NOTCH2 and (4) showed glucose metabolism impairment. We conclude that the i-As-induced down-regulation of HNF1α and HNF4α under chronic settings may play a central role in the features of disease and cancer observed both in vivo and in vitro.

Identification of differentially expressed genes in the livers of chronically i-As-treated hamsters.

Inorganic arsenic (i-As) is a human carcinogen causing skin, lung, urinary bladder, liver and kidney tumors. Chronic exposure to this naturally occurring contaminant, mainly via drinking water, is a significant worldwide environmental health concern. To explore the molecular mechanisms of arsenic hepatic injury, a differential display polymerase chain reaction (DD-PCR) screening was undertaken to identify genes with distinct expression patterns between the liver of low i-As-exposed and control animals. Golden Syrian hamsters (5-6 weeks of age) received drinking water containing 15 mg i-As/L as sodium arsenite, or unaltered water for 18 weeks. The in vivo MN test was carried out, and the frequency of micronucleated reticulocytes (MN-RETs) was scored as a measure of exposure and As-related genotoxic/carcinogenic risk. A total of 68 differentially expressed bands were identified in our initial screen, 41 of which could be assigned to specific genes. Differential level of expression of a selected number of genes was verified using real-time RT-PCR with gene-specific primers. Arsenic-altered gene expression included genes related to stress response, cellular metabolism, cell cycle regulation, telomere maintenance, cell-cell communication and signal transduction. Significant differences of MN-RET were found between treated (8.70 ± 0.02 MN/1000RETs) and control (2.5 ± 0.70 MN/1000RETs) groups (P<0.001), demonstrating both the exposure and the i-As genotoxic/carcinogenic risk. Overall, this paper reveals some possible networks involved in hepatic arsenic-related genotoxicity, carcinogenesis and diabetogenesis. Additional studies to explore further the potential implications of each candidate gene are of especial interest. The present work opens the door to new prospects for the study of i-As mechanisms taking place in the liver under chronic settings.

Effects of cerium oxide nanoparticles on differentiated/undifferentiated human intestinal Caco-2 cells.

Since ingestion constitute one of the main routes of nanoparticles (NPs) exposure, intestinal cells seems to be a suitable choice to evaluate their potential harmful effects. Caco-2 cells, derived from a human colon adenocarcinoma, have the ability to differentiate forming consistent cell monolayer structures. For these reasons Caco-2 cells, both in their undifferentiated or differentiated state, are extendedly used. We have used well-structured monolayers of differentiated Caco-2 cells, as a model of intestinal barrier, to evaluate potential harmful effects associated to CeO2NPs exposure via ingestion. Different parameters such as cell toxicity, monolayer integrity and permeability, cell internalization, translocation through the monolayer, and induction of DNA damage were evaluated. No toxic effects of CeO2NPs were observed, independently of the differentiated state of the Caco-2 cells. In the same way, no effects on the monolayer integrity/permeability were observed. Although important cell uptake was demonstrated in undifferentiated cells (by using confocal microscopy), CeO2NPs remained mostly attached to the apical membrane in the differentiated cells. In spite of this apparent lack of uptake in differentiated cells, translocation of CeO2NPs to the basolateral chamber was observed by using confocal microscopy. Finally no genotoxic effects were observed when the comet assay was used, although decreases in the levels of oxidized bases were observed, supporting the antioxidant role of CeO2NPs.

Activity of intracellular phospholipase A1 and A2 in Giardia lamblia.

Neither phospholipase A1 (PLA A1) nor phospholipase A2 (PLA A2), nor their respective genes, have been identified in Giardia lamblia, even though they are essential for lipid metabolism in this parasite. A method to identify, isolate, and characterize these enzymes is needed. The activities of PLA A1 and PLA A2 were analyzed in a total extract (TE) and in vesicular (P30) and soluble (S30) subcellular fractions of G. lamblia trophozoites; the effects of several chemical and physicochemical factors on their activities were investigated. The assays were performed using substrate labeled with 14C, and the mass of the 14C-product was quantified. PLA A1 and PLA A2 activity was present in the TE and the P30 and S30 fractions, and it was dependent on pH and the concentrations of protein and Ca2+. In all trophozoite preparations, PLA A1 and PLA A2 activities were inhibited by ethylenediaminetetraacetic acid and Rosenthal's inhibitor. These results suggest that G. lamblia possesses several PLA A1 and PLA A2 isoforms that may be soluble or associated with membranes. In addition to participating in G. lamblia phospholipid metabolism, PLA A1 and PLA A2 could play important roles in the cytopathogenicity of this parasite.

Selenite restores Pax6 expression in neuronal cells of chronically arsenic-exposed Golden Syrian hamsters.

Arsenic is a worldwide environmental pollutant that generates public health concerns. Various types of cancers and other diseases, including neurological disorders, have been associated with human consumption of arsenic in drinking water. At the molecular level, arsenic and its metabolites have the capacity to provoke genome instability, causing altered expression of genes. One such target of arsenic is the Pax6 gene that encodes a transcription factor in neuronal cells. The aim of this study was to evaluate the effect of two antioxidants, α-tocopheryl succinate (α-TOS) and sodium selenite, on Pax6 gene expression levels in the forebrain and cerebellum of Golden Syrian hamsters chronically exposed to arsenic in drinking water. Animals were divided into six groups. Using quantitative real-time reverse transcriptase (RT)-PCR analysis, we confirmed that arsenic downregulates Pax6 expression in nervous tissues by 53 ± 21% and 32 ± 7% in the forebrain and cerebellum, respectively. In the presence of arsenic, treatment with α-TOS did not modify Pax6 expression in nervous tissues; however, sodium selenite completely restored Pax6 expression in the arsenic-exposed hamster forebrain, but not the cerebellum. Although our results suggest the use of selenite to restore the expression of a neuronal gene in arsenic-exposed animals, its use and efficacy in the human population require further studies.

Tocopherol and selenite modulate the transplacental effects induced by sodium arsenite in hamsters.

Human studies suggest that in utero exposure to arsenic results in adverse pregnancy outcomes. The use of dietary supplements, such as sodium selenite (SS) or α-tocopherol succinate (α-TOS), is a reasonable approach to ameliorate such health effects. Sodium arsenite at 100ppm was administered via drinking water to female hamsters from gestational days 1 or 8 to the time of delivery. Viable fetuses, fetal resorptions and non-viable fetuses were recorded during and after pregnancy and total arsenic and its metabolites were characterized in pregnant animals, placentas and fetuses. Arsenic was found to accumulate in the placenta and fetus, increasing fetal mortality, non-viable fetuses and resorptions. Co-administration of SS and α-TOS significantly reduced the observed teratogenic effects. SS influenced arsenic biotransformation by reducing the MMA/InAs index and increasing the DMA/MMA, whereas α-TOS more likely exerts its protective effect through its potent antioxidant activity.

Tocopherol esters inhibit human glutathione S-transferase omega.

Human glutathione S-transferase omega 1-1 (hGSTO1-1) is a newly identified member of the glutathione S-transferase (GST) family of genes, which also contains alpha, mu, pi, sigma, theta, and zeta members. hGSTO1-1 catalyzes the reduction of arsenate, monomethylarsenate (MMA(V)), and dimethylarsenate (DMA(V)) and exhibits thioltransferase and dehydroascorbate reductase activities. Recent evidence has show that cytokine release inhibitory drugs, which specifically inhibit interleukin-1b (IL-1b), directly target hGSTO1-1. We found that (+)-alpha-tocopherol phosphate and (+)-alpha-tocopherol succinate inhibit hGSTO1-1 in a concentration-dependent manner with IC50 values of 2 microM and 4 microM, respectively. A Lineweaver-Burk plot demonstrated the uncompetitive nature of this inhibition. The molecular mechanism behind the inhibition of hGSTO1-1 by alpha-tocopherol esters (vitamin E) is important for understanding neurodegenerative diseases, which are also influenced by vitamin E.

Arsenic induces DNA damage in environmentally exposed Mexican children and adults. Influence of GSTO1 and AS3MT polymorphisms.

Inorganic arsenic (i-As) is an environmental carcinogen to which millions of people are chronically exposed mainly via drinking water. In this study, we used the comet assay to evaluate DNA damage in i-As-exposed inhabitants of the north of Mexico. The environmental monitoring and the exposure assessment were done by measuring both drinking water arsenic (As) content and total urinary As. In addition, the studied population was genetically characterized for four different glutathione S-transferase omega1 (GSTO1) polymorphisms (Ala140Asp, Glu155del, Glu208Lys, and Ala236Val) and the As (+3 oxidation state) methyltransferase (AS3MT) Met287Thr polymorphism to determine whether such variants influence As-related genotoxicity. As content in the drinking water of the population was found to range between 1 and 187 microg/l, with a mean concentration value of 16 microg/l. The total urinary As content of the exposed individuals was found to be correlated with the As content in drinking water, and subjects were classified as low (< 30 microg As/g creatinine), medium (31-60 microg As/g creatinine), and highly exposed (> 61 microg As/g creatinine). A positive association was found between the level of exposure and the genetic damage measured as percentage of DNA in tail (p < 0.001), and AS3MT Met287Thr was found to significantly influence the effect (p < 0.034) among children carrying the 287Thr variant allele. Altogether, our results evidenced that people living in As-contaminated areas are at risk and that AS3MT genetic variation may play an important role modulating such risk in northern Mexico, especially among children.

High arsenic metabolic efficiency in AS3MT287Thr allele carriers.

OBJECTIVES: Epidemiological data indicate the existence of wide interindividual differences in arsenic metabolism. It has recently been shown that arsenic(III)methyltransferase (AS3MT) enzyme catalyses the methylation of arsenite and monomethylarsonous acid (MMA). Thus, genetic variations in the AS3MT gene could explain, at least partly, the interindividual variation in the response to arsenic exposure. In an earlier study, we have demonstrated that the AS3MT Met(287)Thr (C/T) polymorphism affected the urinary arsenic profile in a Chilean group of men (n=50) occupationally exposed to arsenic. METHODS: To confirm, the influence of the Met(287)Thr polymorphism in the metabolism of arsenic, a total of 207 Chilean men working at the copper industry were genotyped and their urinary profiles determined. RESULTS: The results confirm that Met(287)Thr polymorphism does influence arsenic metabolism in this population. Those carriers of the variant ((287)Thr) had a higher methylation efficiency, excreting 4.63% more MMA in urine (P=0.0007) and presenting a 2.98 times higher odd of excreting levels of MMA over the standard (P=0.011) than the participants homozygous for the normal allele. CONCLUSION: We can conclude that individuals with the (287)Thr variant display increased arsenic methylation; thus, those participants might be at increased risk for the toxic and genotoxic effects of arsenic exposure.

Effect of clofibric acid on desmin and vimentin contents in rat myocardiocytes.

The aim of this experimental study was to analyze in vitro effects of clofibric acid on vimentin and desmin contents in rat myocardiocytes, which was carried out in primary myocardiocyte cells that were treated only with clofibric acid at 0.1 mM. The measurement of vimentin and desmin were done by Western blotting and densitometry. This study showed that myocardiocytes exposed to clofibric acid exhibit a 26.3% decrease in vimentin and a 42.1% decrease in desmin. Considering the role that these intermediate filaments play in the anchorage and cellular organization of myocardiocytes, the decrease of desmin and vimentin observed in cells treated with clofibric acid may be partially responsible for the adverse effects observed in patients. In conclusion, the alteration of cytoskeletal proteins may be a cause of cardiopathy in patients treated with these compounds.

Arsenate reductase II. Purine nucleoside phosphorylase in the presence of dihydrolipoic acid is a route for reduction of arsenate to arsenite in mammalian systems.

An arsenate reductase has been partially purified from human liver using ion exchange, molecular exclusion, hydroxyapatite chromatography, preparative isoelectric focusing, and electrophoresis. When SDS-beta-mercaptoethanol-PAGE was performed on the most purified fraction, two bands were obtained. One of these bands was a 34 kDa protein. Each band was excised from the gel and sequenced by LC-MS/MS, and sequest analyses were performed against the OWL database SWISS-PROT with PIR. Mass spectra analysis matched the 34 kDa protein of interest with human purine nucleoside phosphorylase (PNP). The peptide fragments equal to 40.1% of the total protein were 100% identical to the corresponding regions of the human purine nucleoside phosphorylase. Reduction of arsenate in the purine nucleoside arsenolysis reaction required both PNP and dihydrolipoic acid (DHLP). The PNP rate of reduction of arsenate with the reducing agents GSH or ascorbic acid was negligible compared to that with the naturally occurring dithiol DHLP and synthetic dithiols such as BAL (British anti-lewisite), DMPS (2,3-dimercapto-1-propanesulfonate), or DTT (alpha-dithiothreitol). The arsenite production reaction of thymidine phosphorylase had approximately 5% of such PNP activity. Phosphorylase b was inactive. Monomethylarsonate (MMAV) was not reduced by PNP. The experimental results indicate PNP is an important route for the reduction of arsenate to arsenite in mammalian systems.

A review of the enzymology of arsenic metabolism and a new potential role of hydrogen peroxide in the detoxication of the trivalent arsenic species.

This laboratory has studied the enzymology involved in the biotransformation of inorganic arsenic to dimethylarsinous acid (DMA(III)) and in human studies established that monomethylarsonous acid (MMA(III)) and DMA(III) appear in urine of people chronically exposed to arsenic. It appears that only two proteins are required for inorganic arsenic biotransformation in the human, namely, monomethylarsonic acid (MMA(V)) reductase and arsenic methyltransferase. MMA(V) reductase and the unique glutathione transferase omega (hGST-O) are identical proteins. Arsenicals with a +3 oxidation state are more toxic than the +5 species. While methylation of arsenite, MMA(III), and DMA(III) produces less toxic +5 oxidation arsenic species containing an additional methyl group such as MMA(V), dimethylarsinic acid (DMA(V)), and TMAO, a new mechanism involving hydrogen peroxide for detoxifying arsenite, MMA(III), and DMA(III) is proposed based on in vitro experiments.

Evaluation of the mutagenic and cytotoxic effects of mercurous chloride by the micronuclei technique in golden Syrian hamsters.

The aims of this study were to evaluate the mutagenic and cytotoxic activity of mercurous chloride by the micronucleus technique in vivo on the bone marrow of golden Syrian hamsters after a single i.p. drug administration. Forty male golden Syrian hamsters were classified into eight groups: negative control, positive control and six groups treated with different doses of mercurous chloride (1.25, 2.5, 5, 10, 20 and 40 mg/kg). The negative control was injected with physiological saline i.p. and the positive control with cyclophosphamide at a dose of 80 mg/kg i.p. With respect to mutagenic effect, the average number of micronucleated polychromatic erythrocytes (MPE) in hamsters treated with different doses of mercurous chloride was not significant compared with the negative control. With respect to cytotoxic effect, the average polychromatic erythrocyte/red blood cell ratio showed a significant decrease when the doses were higher than the 2.5 mg/kg dose compared with the negative control. In conclusion, this preliminary study shows a cytotoxic effect but not a mutagenic effect of calomel in vivo at one time point (24 h).