Abstract  DNA double strand breaks (DSBs), induced by gamma-irradiation in Chinese hamster ovary cells, were used to examine whether antimony compounds affect the repair of DNA damage. The cells were first incubated with antimony trichloride or antimony potassium tartrate (both Sb(III)) for 2 h, and then irradiated with gamma-rays at a dose of 40 Gy. The DNA DSB was quantified with pulsed field gel electrophoresis immediately after irradiation (non-repair group) as well as at 30 min post-irradiation (repair group). The degree of repair inhibition was determined by the differences in the amount of DNA DSB between non-repair and repair groups. Both antimony compounds inhibited repair of DNA DSB in a dose dependent manner. In trichloride, 0.2 mM antimony significantly inhibited the rejoining of DSB, while 0.4 mM was necessary in potassium antimony tartrate. The mean lethal doses, D(0), for the treatment with antimony trichloride and antimony potassium tartrate, were approximately 0.21 and 0.12 mM, respectively. This indicates that the repair inhibition by antimony trichloride occurred in the dose range near D(0), but the antimony potassium tartrate inhibited the repair at doses where most cells lost their proliferating ability. This is the first report to indicate that antimony compounds may inhibit the repair of radiation-induced DNA DSB.

Abstract  The U. S. Environmental Protection Agency’s (EPA) Office of Pesticide Programs (OPP), Office of Water (OW), and Office of Research and Development (ORD) coordinated the development of two scientific documents that address the carcinogenicity of Dimethylarsinic Acid (DMAV) and inorganic arsenic (iAs). In response to an Agency request, the Science Advisory Board (SAB) convened an expert panel to review and comment on key scientific issues presented in these two documents, including: (a) the metabolism and toxic responses of arsenic species; (b) mode(s) of carcinogenic action; (c) data selection for dose-response assessment; and (d) approaches and methods for low-dose extrapolation for DMAV and iAs. The SAB Panel supported the Agency’s conclusion that on the basis of available data, human exposure to DMAV appears to result in a narrower spectrum of active metabolites than those expected in the metabolic profile associated with exposure to iAs. Therefore, the Panel agreed with EPA that, in the absence of human data on DMAV, the bladder tumor data from DMAV rat bioassays is better suited for DMAV cancer risk assessment than is epidemiology data from iAs exposure. The Panel, however, noted that there remain significant uncertainties associated with the use of animal data for DMAV cancer risk assessment due to the observed metabolic differences between rats and humans. The Panel agreed with the Agency’s conclusion that DMAV-induced bladder cancer in rats, at high dose, is mediated by a cytotoxic mode of action, and that this MOA should be considered relevant to humans. However, the Panel concluded there are not sufficient data to support a reactive oxygenated species-mediated mode of direct genetic action for DMAV. The Panel supported the nonlinear approach for low dose extrapolation of DMAV and the use of uncertainty factors to account for interspecies differences and human variability for sensitive human populations, and concluded that presently there is no arsenic-specific information that can inform the choice of specific values. This means that, at least for now, such choices must be based on more general considerations, including EPA’s science policy judgment of the degree of precaution that it deems appropriate. EPA concluded that the mechanisms by which inorganic arsenic induces bladder cancer in humans are not yet known, but they are likely to be mediated by multiple modes of action. The Agency used a linear default approach for low dose extrapolation because it lacked a full understanding of the iAs modes of carcinogenic action. The Panel agreed that available human and animal data do not fully describe the shape of the iAs carcinogenic dose-response curve at low doses. Given the considerable uncertainties regarding low dose extrapolation, the Panel supported the use of a linear cancer risk model for iAs as recommended by the National Research Council in its 2001 report. The Panel also supported the use of the epidemiologic data on the Taiwanese population for estimating human cancer risk for iAs especially to identify the potential range of responses of human populations. However, the Panel recognized limitations to these data, and that there is some evidence on iAs from animal toxicology, pharmacokinetics, and pharmacodynamics research, that suggests other than a linear bladder cancer dose-response. The Panel urged the Agency to consider other epidemiologic studies from the U.S. and other countries, utilizing a uniformset of evaluative criteria. The Panel also recommended sensitivity analyses be conducted to account for human variability in drinking water consumption rates, dietary intake of iAs from food, and certain other assumptions currently used in EPA’s assessment. The Panel made several suggestions for improvements in the currently applied risk model’s programming and documentation conventions. Finally, the Panel believes there is a critical need for a continued research effort to strengthen EPA’s cancer risk assessment for DMAV and iAs. The scientific bases for the Panel’s conclusions and research recommendations are detailed throughout this report.

Abstract  Chronic arsenic exposure is associated with cardiovascular abnormalities. Prolongation of the QT (time between initial deflection of QRS complex to the end of T wave) interval and profound repolarization changes on electrocardiogram (ECG) have been reported in patients with acute promyelocytic leukemia treated with arsenic trioxide. This acquired form of long QT syndrome can result in life-threatening arrhythmias.

The objective of this study was to assess the cardiac effects of arsenic by investigating QT interval alterations in a human population chronically exposed to arsenic.

Residents in Ba Men, Inner Mongolia, have been chronically exposed to arsenic via consumption of water from artesian wells. A total of 313 Ba Men residents with the mean arsenic exposure of 15 years were divided into three arsenic exposure groups: low (< or = 21 microg/L), medium (100-300 microg/L), and high (430-690 microg/L). ECGs were obtained on all study subjects. The normal range for QTc (corrected QT) interval is 0.33-0.44 sec, and QTc > or = 0.45 sec was considered to be prolonged.

The prevalence rates of QT prolongation and water arsenic concentrations showed a dose-dependent relationship (p = 0.001). The prevalence rates of QTc prolongation were 3.9, 11.1, 20.6% for low, medium, and high arsenic exposure, respectively. QTc prolongation was also associated with sex (p < 0.0001) but not age (p = 0.486) or smoking (p = 0.1018). Females were more susceptible to QT prolongation than males.

We found significant association between chronic arsenic exposure and QT interval prolongation in a human population. QT interval may potentially be useful in the detection of early cardiac arsenic toxicity.

Abstract  To gain insight into plant responses to arsenic, the effect of arsenic exposure on maize (Zea mays L.) root proteome has been examined. Maize seedlings were fed hydroponically with 300 microM sodium arsenate or 250 microM sodium arsenite for 24 h, and changes in differentially displayed proteins were studied by two-dimensional electrophoresis and digital image analysis. About 10% of total detected maize root proteins (67 out of 700) were up- or down-regulated by arsenic, among which 20 were selected as being quite reproducibly affected by the metalloid. These were analyzed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and 11 of them could be identified by comparing their peptide mass fingerprints against protein- and expressed sequence tag-databases. The set of identified maize root proteins highly responsive to arsenic exposure included a major and functionally homogeneous group of seven enzymes involved in cellular homeostasis for redox perturbation (e.g., three superoxide dismutases, two glutathione peroxidases, one peroxiredoxin, and one p-benzoquinone reductase) besides four additional, functionally heterogeneous, proteins (e.g., ATP synthase, succinyl-CoA synthetase, cytochrome P450 and guanine nucleotide-binding protein beta subunit). These findings strongly suggest that the induction of oxidative stress is a main process underlying arsenic toxicity in plants.

Abstract  Resistance to chemotherapy is one of the major problems in treatment responses of lung cancer. This study explored the mechanism underlying the arsenic resistance of lung cancer. Four lung cancer cells with different proliferation activity were characterized for cytotoxicity, arsenic influx/efflux, and arsenic effects on intracellular glutathione and 8-hydroxy-2'-deoxyguanosine (8-OHdG) production. Our data revealed that relative proliferation potency of these cells was H1299>A549>CL3>H1355. Moreover, A549, H1299, and H1355 were markedly resistant to As(2)O(3) with IC50 approximately 100 microM, whereas CL3 was sensitive to As(2)O(3) with IC50 approximately 11.8 microM. After treatment with the respective As(2)O(3) at IC50, arsenic influx/efflux activity in CL3 was comparable to those in the other three arsenic-resistant cells. However, differences in glutathione levels and 8-OHdG production were also detected either before or after arsenic treatment, indicating that a certain degree of variation in anti-oxidative systems and/or 8-OHdG repair activity existed in these cell lines. By transfection of an aquaglyceroporin 9 (AQP9) gene, we showed that increased AQP9 expression significantly enhanced arsenic uptake and disrupted arsenic resistance of A549. The present study strongly suggests that membrane transporters responsible for arsenic uptake, such as AQP9, may play a critical role in development of arsenic resistance in human lung cancer cells.

Abstract  Arsenic toxicity and distribution are highly dependent on animal species and its chemical species. Recently, thioarsenical has been recognized in highly toxic arsenic metabolites, which was commonly found in human and animal urine. In the present study, we revealed the mechanism underlying the distribution and metabolism of non-thiolated and thiolated dimethylarsenic compounds such as dimethylarsinic acid (DMAV), dimethylarsinous acid (DMAIII), dimethylmonothioarsinic acid (DMMTAV), and dimethyldithioarsinic acid (DMDTAV) after the administration of them into femoral vein of hamsters. DMAV and DMDTAV distributed in organs and body fluids were in their unmodified form, while DMAIII and DMMTAV were bound to proteins and transformed to DMAV in organs. On the other hand, DMAV and DMDTAV were mostly excreted into urine as their intact form 1 h after post-injection, and more than 70% of the doses were recovered in urine as their intact form. By contrast, less than 8–14% of doses were recovered in urine as DMAV, while more than 60% of doses were distributed in muscles and target organs (liver, kidney, and lung) of hamsters after the injection of DMMTAV and DMAIII. However, in red blood cells (RBCs), only a small amount of the arsenicals was distributed (less than 4% of the doses) after the injection of DMAIII and DMMTAV, suggesting that the DMAIII and DMMTAV were hardly accumulated in hamster RBCs. Based on these observations, we suggest that although DMMTAV and DMDTAV are thioarsenicals, DMMTAV is taken up efficiently by organs, in a manner different from that of DMDTAV. In addition, the distribution and metabolism of DMMTAV are like in manner similar to DMAIII in hamsters, while DMDTAV is in a manner similar to DMAV.

Abstract  Arsenic is a human skin, lung, and urinary bladder carcinogen, and may act as a cocarcinogen in the skin and urinary bladder. Possible modes of action of arsenic carcinogenesis/cocarcinogenesis include oxidative stress induction and inhibition of DNA damage repair. We investigated the effects of arsenic in drinking water on DNA damage repair in urinary bladder transitional cells and on micronucleus formation in bone marrow. F344 rats were given 100 ppm arsenate [As(V)] or dimethylarsinic acid [DMA(V)] in drinking water for 1 week. The in vivo repair of cyclophosphamide (CP)-induced DNA damage resulting from a single oral gavage of CP, and the in vitro repair of hydrogen peroxide (H(2)O(2))- or formaldehyde-induced DNA damage, resulting from adding H(2)O(2) or formaldehyde into cell medium, were measured by the Comet assay. DMA(V) effects were not observed on either CP-induced DNA damage induction or on DNA repair. Neither DMA(V) nor As(V) increased the H(2)O(2)- or formaldehyde-induced DNA damage, and neither inhibited the repair of H(2)O(2)-induced DNA damage. Neither DMA(V) nor As(V) increased the micronucleus frequency, nor did they elevate micronucleus frequency resulting from CP treatment above the level observed by the treatment with CP alone. These results suggest that arsenic carcinogenesis/cocarcinogenesis in the urinary bladder may not be via DNA damage repair inhibition. To our knowledge this is the first report of arsenic effects on DNA damage repair in the urinary bladder.

Abstract  Arsenic trioxide is a potent chemotherapeutic agent by virtue of its ability to selectively trigger apoptosis in tumor cells. Previous studies have demonstrated that arsenicals cause direct damage to mitochondria, but it is not clear that these effects initiate apoptosis. Here we used Bak-/- mouse liver mitochondria and virally immortalized Bax-/- Bak-/- mouse embryonic fibroblasts (MEFs) to investigate whether or not multidomain proapoptotic BCL-2 family proteins were required for arsenic-induced mitochondrial damage and cell death. At clinically achievable concentrations, arsenic stimulated cytochrome c release and apoptosis via a Bax/Bak-dependent mechanism. At higher concentrations (125 microM-1 mM), cells died via a Bax/Bak-independent mechanism mediated by oxidative stress that resulted in necrosis. Consistent with previous reports, arsenic directly inhibited complex I of the mitochondrial electron transport chain, which resulted in mitochondrial permeability transition (MPT), accompanying generation of reactive oxygen species (ROS), and thiol oxidation. However, these effects only occurred at concentrations of arsenic trioxide of 50 microM and higher, and the oxidative stress associated with these effects blocked caspase activation. Our data demonstrate for the first time that the cytochrome c release which initiates apoptosis in cells exposed to this classic mitochondrial poison occurs indirectly via the activation of Bax/Bak rather than via direct mitochondrial damage. Furthermore, the results implicate reactive oxygen species in a concentration-dependent mechanistic switch between apoptosis and necrosis.

Abstract  Arsenite is a human multisite carcinogen, but its mechanism of action is not known. We recently found that extremely low concentrations (

Abstract  Apoptosis or programmed cell death (PCD) is a genetically regulated cellular, physiological and biochemical suicidal mechanism that plays a crucial role in the development and defense of homeostasis, in which the cell participates in its own demise via a cascade of molecular interactions. PCD can be modulated by various stimuli including infectious agents or drugs. Arsenic is one among inducible toxic agent that triggers apoptosis via free radical generation. Since the generation of free radicals during the metabolism of arsenic is thought to be involved in arsenic toxicosis, understanding the deleterious effects caused by the ROS that attack the vital molecules like DNA has become important. The present work was conducted to evaluate the regulatory effect exerted by Vitamin C and Vitamin E upon the apoptotic process, which can be assessed by the presence of cells with apoptosis associated DNA breaks and characterize the role of TNF-alpha and caspase-3 in rats intoxicated with arsenic. Male albino rats of wistar strain (120-150 g) were used in this study and are further divided into seven groups. We observed that ascorbate and alpha-tocopherol selectively altered the extent of DNA damage by reducing TNF-alpha level and inhibiting the activation of caspase cascade, from these observations it is strongly believed that the present vitamins supplementation perspective, though observed in animal model, will have sustainable curative value among the already afflicted populations, neutralizing impact on freshly emerging arsenicosis scenario and possible proactive protection to those potentially susceptible to arsenicals exposure.

Abstract  Previous work has suggested that arsenic exposure contributes to skin carcinogenesis by preserving the proliferative potential of human epidermal keratinocytes, thereby slowing the exit of putative target stem cells into the differentiation pathway. To find a molecular basis for this action, present work has explored the influence of arsenite on keratinocyte responses to epidermal growth factor (EGF). The ability of cultured keratinocytes to found colonies upon passaging several days after confluence was preserved by arsenite and EGF in an additive fashion, but neither was effective when the receptor tyrosine kinase activity was inhibited. Arsenite prevented the loss of EGF receptor protein and phosphorylation of tyrosine 1173, preserving its capability to signal. The level of nuclear beta-catenin was higher in cells treated with arsenite and EGF in parallel to elevated colony forming ability, and expression of a dominant negative beta-catenin suppressed the increase in both colony forming ability and yield of putative stem cells induced by arsenite and EGF. As judged by expression of three genes regulated by beta-catenin, this transcription factor had substantially higher activity in the arsenite/EGF-treated cells. Trivalent antimony exhibited the same effects as arsenite. A novel finding is that insulin in the medium induced the loss of EGF receptor protein, which was largely prevented by arsenite exposure.

Abstract  Arsenic is a well-known carcinogen for human skin, but its mechanism of action and proximal macromolecular targets remain to be elucidated. In the present study, low micromolar concentrations of sodium arsenite maintained the proliferative potential of epidermal keratinocytes, decreasing their exit from the germinative compartment under conditions that promote differentiation of untreated cells. This effect was observed in suspension and in post-confluent surface cultures as measured by colony-forming ability and by proportion of rapidly adhering colony-forming cells. Arsenite-treated cultures exhibited elevated levels of beta1-integrin and beta-catenin, two proteins enriched in cells with high proliferative potential. Levels of phosphorylated (inactive) glycogen synthase kinase 3beta were higher in the treated cultures, likely accounting for the increased levels of transcriptionally available beta-catenin. These findings suggest that arsenic could have co-carcinogenic and tumor co-promoting activities in the epidermis as a result of increasing the population and persistence of germinative cells targeted by tumor initiators and promoters. These findings also identify a critical signal transduction pathway meriting further exploration in pursuit of this phenomenon.

Abstract  There is a wide variation in susceptibility to health effects of arsenic, which, in part, may be due to differences in arsenic metabolism. Arsenic is metabolized by reduction and methylation reactions, catalyzed by reductases and methyltransferases.

Our goal in this study was to elucidate the influence of various demographic and genetic factors on the metabolism of arsenic.

We studied 415 individuals from Hungary, Romania, and Slovakia by measuring arsenic metabolites in urine using liquid chromatography with hydride generation and inductively coupled plasma mass spectrometry (HPLC-HG-ICPMS). We performed genotyping of arsenic (+III) methyltransferase (AS3MT), glutathione S-transferase omega 1 (GSTO1), and methylene-tetrahydrofolate reductase (MTHFR).

The results show that the M287T (T-->C) polymorphism in the AS3MT gene, the A222V (C-->T) polymorphism in the MTHFR gene, body mass index, and sex are major factors that influence arsenic metabolism in this population, with a median of 8.0 microg/L arsenic in urine. Females < 60 years of age had, in general, higher methylation efficiency than males, indicating an influence of sex steroids. That might also explain the observed better methylation in overweight or obese women, compared with normal weight men. The influence of the M287T (T-->C) polymorphism in the AS3MT gene on the methylation capacity was much more pronounced in men than in women.

The factors investigated explained almost 20% of the variation seen in the metabolism of arsenic among men and only around 4% of the variation among women. The rest of the variation is probably explained by other methyltransferases backing up the methylation of arsenic.

Abstract  Prior research has shown that arsenic exposure induces changes that coincide with most of the developmental, biochemical, pathologic, and clinical features of Alzheimer disease (AD) and associated disorders. On the basis of this literature, we propose the Arsenic Exposure Hypothesis for AD that is inclusive of and cooperative with the existing hypotheses. Arsenic toxicity induces hyperphosphorylation of protein tau and overtranscription of the amyloid precursor protein, which are involved in the formation of neurofibrillary tangles and brain amyloid plaques, consistent with the amyloid hypothesis of AD. Arsenic exposure has been associated with cardiovascular diseases and associated risk factors, which is in agreement with the vascular hypothesis of AD. Arsenic exposure invokes brain inflammatory responses, which resonates with the inflammatory hypotheses of AD. Arsenic exposure has been linked to reduced memory and intellectual abilities in children and adolescents, which provides a biologic basis for the developmental origin of health and disease hypothesis for AD. Arsenic and its metabolites generate free radicals causing oxidative stress and neuronal death, which fits the existing oxidative stress hypothesis. Taken together, the arsenic exposure hypothesis for AD provides a parsimonious testable hypothesis for the development and progression of this devastating disease at least for some subsets of individuals.

Abstract  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.

Abstract  Arsenic (As) is a toxic semi-metal of wide distribution in nature. People living in regions where drinking water contains large quantities of arsenic, have an unusually high likelihood of developing blood-vessel diseases, but little is known about the mechanisms involved, i.e. the blood rheologic alterations that would contribute to the circulatory obstruction. Erythrocytes are the main target cells for arsenic compounds systemically absorbed and their cell membrane is the first place against the toxic. In this paper we have examined the in vitro effect of arsenic (As(V)) on the rheologic properties of human erythrocytes in relation with membrane fluidity and internal microviscosity. According to our present results, As(V) treatment produces oxidative degradation of membrane lipids and alteration of internal microviscosity. These red blood cells (RBCs) membrane and cytoplasmic structural damage consequently alters RBCs rheologic properties: an alteration of the RBCs discoid shape to stomatocytes, a diminution of erythrocyte deformability and an enhancement of osmotic fragility and cell aggregability. These effects impaired blood fluid behaviour that contribute to obstruct peripheral circulation and provides anemia, both clinic evidences typical of arsenic cronic intoxication.

Abstract  Arsenic (As) exposure during pregnancy induces oxidative stress and increases the risk of fetal loss and low birth weight.

In this study we aimed to elucidate the effects of As exposure on immune markers in the placenta and cord blood, and the involvement of oxidative stress.

Pregnant women were enrolled around gestational week (GW) 8 in our longitudinal, population-based, mother-child cohort in Matlab, an area in rural Bangladesh with large variations in As concentrations in well water. Women (n = 130) delivering at local clinics were included in the present study. We collected maternal urine twice during pregnancy (GW8 and GW30) for measurements of As, and placenta and cord blood at delivery for assessment of immune and inflammatory markers. Placental markers were measured by immunohistochemistry, and cord blood cytokines by multiplex cytokine assay.

In multivariable adjusted models, maternal urinary As (U-As) exposure both at GW8 and at GW30 was significantly positively associated with placental markers of 8-oxoguanine (8-oxoG) and interleukin-1β (IL-1β); U-As at GW8, with tumor necrosis factor-α (TNFα) and interferon-γ (IFNγ); and U-As at GW30, with leptin; U-As at GW8 was inversely associated with CD3+ T cells in the placenta. Cord blood cytokines (IL-1β, IL-8, IFNγ, TNFα) showed a U-shaped association with U-As at GW30. Placental 8-oxoG was significantly positively associated with placental proinflammatory cytokines. Multivariable adjusted analyses suggested that enhanced placental cytokine expression (TNFα and IFNγ) was primarily influenced by oxidative stress, whereas leptin expression appeared to be mostly mediated by As, and IL-1β appeared to be influenced by both oxidative stress and As.

As exposure during pregnancy appeared to enhance placental inflammatory responses (in part by increasing oxidative stress), reduce placental T cells, and alter cord blood cytokines. These findings suggest that effects of As on immune function may contribute to impaired fetal and infant health.

Abstract  Arsenite is an environmental pollutant. Exposure to inorganic arsenic in drinking water is associated with elevated cancer risk, especially in skin. Arsenite alone does not cause skin cancer in animals, but arsenite can enhance the carcinogenicity of solar UV. Arsenite is not a significant mutagen at non-toxic concentrations, but it enhances the mutagenicity of other carcinogens. The tumor suppressor protein P53 and nuclear enzyme PARP-1 are both key players in DNA damage response. This laboratory demonstrated earlier that in cells treated with arsenite, the P53-dependent increase in p21(WAF1/CIP1) expression, normally a block to cell cycle progression after DNA damage, is deficient. Here we show that although long-term exposure of human keratinocytes (HaCaT) to a nontoxic concentration (0.1 microM) of arsenite decreases the level of global protein poly(ADP-ribosyl)ation, it increases poly(ADP-ribosyl)ation of P53 protein and PARP-1 protein abundance. We also demonstrate that exposure to 0.1 microM arsenite depresses the constitutive expression of p21 mRNA and P21 protein in HaCaT cells. Poly(ADP-ribosyl)ation of P53 is reported to block its activation, DNA binding and its functioning as a transcription factor. Our results suggest that arsenite's interference with activation of P53 via poly(ADP-ribosyl)ation may play a role in the comutagenic and cocarcinogenic effects of arsenite.

Abstract  Sodium arsenite (arsenite)-induced neurotoxicity and its interaction with ferrous citrate (iron) was investigated in rat brain. In vitro data showed that arsenite (1-10 micromol/L) concentration dependently increased lipid peroxidation and the potency of arsenite was less than that of iron. The oxidative activity of arsenite, sodium arsenate (arsenate), monomethylarsonic acid (MMA), and dimethylarsinic acid (DMA) were evaluated by inducing lipid peroxidation in cortical homogenates, and the potency for this effect was as follows: arsenite > arsenate > MMA and DMA. Several well-known antioxidants, including glutathione, melatonin, and beta-estradiol inhibited arsenite-induced lipid peroxidation in a concentration-dependent manner. Our in vivo study employed intranigral infusion of arsenite (5 nmol) in the substantia nigra (SN) of anesthetized rats. Four hours to 7 days after infusion, lipid peroxidation was elevated while glutathione was depleted in the infused SN. The dopamine content in the striatum ipsilateral to arsenite-infused SN was first elevated 24 h and then decreased 7 days after intranigral infusion of arsenite. Using pretreatment of l-buthionine-[S,R]-sulfoximine (l-BSO, i.c.v.) to reduce glutathione content in rat brain, arsenite-induced oxidative injury was augmented. Low doses of arsenite (1.5 nmol) and iron (3 nmol) alone induced minimal oxidative injury; however, co-infusion of arsenite and iron augmented neurotoxicity, including elevated lipid peroxidation and reduced striatal dopamine content. Moreover, expression of heme oxygenase-1, alpha-synuclein aggregation, and DNA fragmentation were significantly enhanced in SN co-infused with low doses of arsenite and iron. Taken together, our study demonstrates that arsenite was less potent than iron in inducing oxidative stress. Furthermore, concomitant arsenite and iron potentiated oxidative injury in the nigrostriatal dopaminergic system, indicating that interaction of metals plays a more clinically-relevant role in pathophysiology of central nervous system neurodegeneration.

Abstract  We investigated the effects and modes of action of the nutritional factor folate on arsenic-induced toxicity in Chang human hepatocytes. Cells were cultured in folate-deficient medium, normal folate medium or folate-supplemented medium for 1h and then co-treated with or without 20-microM sodium arsenite (NaAsO(2)) for 24h. The results showed that folate deficiency significantly aggravated the NaAsO(2)-induced apoptotic progression [evidenced by phosphatidylserine externalization, cleavage of caspase-3 and poly (ADP-ribose) polymerase (PARP), collapse of mitochondrial potential, and release of cytochrome c from the mitochondria] and decrease of cell viability. Folate supplementation significantly attenuated all the above mentioned NaAsO(2)-induced effects except phosphatidylserine externalization. The NaAsO(2)-induced generation of intracellular reactive oxygen species and malondialdehyde was aggravated, to some extent, by folate deficiency, but these phenomena were significantly suppressed by folate supplementation. In contrast, NaAsO(2)-induced elevation of reduced glutathione levels was significantly suppressed by folate deficiency, but significantly enhanced by folate supplementation. In addition, folate deficiency significantly decreased the arsenic methylation capacity of the hepatocytes, but had no effects on cellular retention of arsenic. Folate supplementation had no significant effect on cellular retention or methylation of arsenic. These results indicate that folate deficiency aggravates arsenic-induced toxicity and apoptosis, while folate supplementation attenuates these effects. Folate, which plays a role in arsenic metabolism, also exerts its effect on arsenic toxicity at least partly because of its antioxidant property.

Abstract  Differences in arsenic metabolism are known to play a role in individual variability in arsenic-induced disease susceptibility. Genetic variants in genes relevant to arsenic metabolism are considered to be partially responsible for the variation in arsenic metabolism. Specifically, variants in arsenic (3+ oxidation state) methyltransferase (AS3MT), the key gene in the metabolism of arsenic, have been associated with increased arsenic methylation efficiency. Of particular interest is the fact that different studies have reported that several of the AS3MT single nucleotide polymorphisms (SNPs) are in strong linkage-disequilibrium (LD), which also extends to a nearby gene, CYP17A1. In an effort to characterize the extent of the region in LD, we genotyped 46 SNPs in a 347,000 base region of chromosome 10 that included AS3MT in arsenic-exposed subjects from Mexico. Pairwise LD analysis showed strong LD for these polymorphisms, represented by a mean r(2) of 0.82, spanning a region that includes five genes. Genetic association analysis with arsenic metabolism confirmed the previously observed association between AS3MT variants, including this large cluster of linked polymorphisms, and arsenic methylation efficiency. The existence of a large genomic region sharing strong LD with polymorphisms associated with arsenic metabolism presents a predicament because the observed phenotype cannot be unequivocally assigned to a single SNP or even a single gene. The results reported here should be carefully considered for future genomic association studies involving AS3MT and arsenic metabolism.

Abstract  To determine whether arsenic inhibits transcriptional activation of the liver X receptor (LXR)/retinoid X receptor (RXR) heterodimers, thereby impairing cholesterol efflux from macrophages and potentially contributing to a proatherogenic phenotype.

Arsenic is an important environmental contaminant and has been linked to an increased incidence of atherosclerosis. Previous findings showed that arsenic inhibits transcriptional activation of type 2 nuclear receptors, known to heterodimerize with RXR. Environmentally relevant arsenic doses decrease the LXR/RXR ligand-induced expression of the LXR target genes (ABCA1 and SREBP-1c). Arsenic failed to decrease cAMP-induced ABCA1 expression, suggesting a selective LXR/RXR effect. This selectivity correlated with the ability of arsenic to decrease LXR/RXR ligand-induced, but not cAMP-induced, cholesterol efflux. By using chromatin immunoprecipitation assays, we found that arsenic inhibits the ability of LXR/RXR ligands to induce activation markers on the ABCA1 and SREBP-1c promoters and blocks ligand-induced release of the nuclear receptor coexpressor (NCoR) from the promoter. Arsenic did not alter the ability of LXR to transrepress inflammatory gene transcription, further supporting our hypothesis that RXR is the target for arsenic inhibition.

Exposure to arsenic enhances the risk of atherosclerosis. We present data that arsenic inhibits the transcriptional activity of the liver X receptor, resulting in decreased cholesterol-induced gene expression and efflux from macrophages. Therefore, arsenic may promote an athersclerotic environment by decreasing the ability of macrophages to efflux excess cholesterol, thereby favoring increased plaque formation.

Abstract  Biomethylation is the major pathway for the metabolism of inorganic arsenic (iAs) in many mammalian species, including the human. However, significant interspecies differences have been reported in the rate of in vivo metabolism of iAs and in yields of iAs metabolites found in urine. Liver is considered the primary site for the methylation of iAs and arsenic (+3 oxidation state) methyltransferase (As3mt) is the key enzyme in this pathway. Thus, the As3mt-catalyzed methylation of iAs in the liver determines in part the rate and the pattern of iAs metabolism in various species. We examined kinetics and concentration-response patterns for iAs methylation by cultured primary hepatocytes derived from human, rat, mice, dog, rabbit, and rhesus monkey. Hepatocytes were exposed to [(73)As]arsenite (iAs(III); 0.3, 0.9, 3.0, 9.0 or 30 nmol As/mg protein) for 24 h and radiolabeled metabolites were analyzed in cells and culture media. Hepatocytes from all six species methylated iAs(III) to methylarsenic (MAs) and dimethylarsenic (DMAs). Notably, dog, rat and monkey hepatocytes were considerably more efficient methylators of iAs(III) than mouse, rabbit or human hepatocytes. The low efficiency of mouse, rabbit and human hepatocytes to methylate iAs(III) was associated with inhibition of DMAs production by moderate concentrations of iAs(III) and with retention of iAs and MAs in cells. No significant correlations were found between the rate of iAs methylation and the thioredoxin reductase activity or glutathione concentration, two factors that modulate the activity of recombinant As3mt. No associations between the rates of iAs methylation and As3mt protein structures were found for the six species examined. Immunoblot analyses indicate that the superior arsenic methylation capacities of dog, rat and monkey hepatocytes examined in this study may be associated with a higher As3mt expression. However, factors other than As3mt expression may also contribute to the interspecies differences in the hepatocyte capacity to methylate iAs.

Abstract  Human exposure to inorganic arsenic (iAs), a potent oxidative stressor, causes various dermal disorders, including hyperkeratosis and skin cancer. Nuclear factor-erythroid 2-related factor 1 (NRF1, also called NFE2L1) plays a critical role in regulating the expression of many antioxidant response element (ARE)-dependent genes.

We investigated the role of NRF1 in arsenic-induced antioxidant response and cytotoxicity in human keratinocytes.

In cultured human keratinocyte HaCaT cells, inorganic arsenite (iAs3+) enhanced the protein accumulation of long isoforms (120-140 kDa) of NRF1 in a dose- and time-dependent fashion. These isoforms accumulated mainly in the nuclei of HaCaT cells. Selective deficiency of NRF1 by lentiviral short-hairpin RNAs in HaCaT cells [NRF1-knockdown (KD)] led to decreased expression of γ-glutamate cysteine ligase catalytic subunit (GCLC) and regulatory subunit (GCLM) and a reduced level of intracellular glutathione. In response to acute iAs3+ exposure, induction of some ARE-dependent genes, including NAD(P)H:quinone oxidoreductase 1 (NQO1), GCLC, and GCLM, was significantly attenuated in NRF1-KD cells. However, the iAs3-induced expression of heme oxygenase 1 (HMOX-1) was unaltered by silencing NRF1, suggesting that HMOX-1 is not regulated by NRF1. In addition, the lack of NRF1 in HaCaT cells did not disturb iAs3+-induced NRF2 accumulation but noticeably decreased Kelch-like ECH-associated protein 1 (KEAP1) levels under basal and iAs3+-exposed conditions, suggesting a potential interaction between NRF1 and KEAP1. Consistent with the critical role of NRF1 in the transcriptional regulation of some ARE-bearing genes, knockdown of NRF1 significantly increased iAs3+-induced cytotoxicity and apoptosis.

Here, we demonstrate for the first time that long isoforms of NRF1 contribute to arsenic-induced antioxidant response in human keratinocytes and protect the cells from acute arsenic cytotoxicity.