Arsenic-induced Cell Research Articles

Aberrant Cell Proliferation by Enhanced Mitochondrial Biogenesis via mtTFA in Arsenical Skin Cancers

Arsenic-induced Bowen's disease (As-BD), a cutaneous carcinoma in situ, is thought to arise from gene mutation and uncontrolled proliferation. However, how mitochondria regulate the arsenic-induced cell proliferation remains unclear. The aim of this study was to clarify whether arsenic interfered with mitochondrial biogenesis and function, leading to aberrant cell proliferation in As-BD. Skin biopsy samples from patients with As-BD and controls were stained for cytochrome c oxidase (Complex IV), measured for mitochondrial DNA (mtDNA) copy number and the expression levels of mitochondrial biogenesis-related genes, including peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α), nuclear respiratory factor 1 (NRF-1), and mitochondrial transcription factor A (mtTFA). The results showed that expression of cytochrome c oxidase, mtTFA, NRF-1, and PGC-1α was increased in As-BD compared with in healthy subjects. Treatment of primary keratinocytes with arsenic at concentrations lower than 1.0 μmol/L induced cell proliferation, along with enhanced mitochondrial biogenesis. Furthermore, we observed that the mitochondrial oxygen consumption rate and intracellular ATP level were increased in arsenic-treated keratinocytes. Blocking of mitochondrial function by oligomycin A (Complex V inhibitor) or knockdown of mtTFA by RNA interference abrogated arsenic-induced cell proliferation without affecting cyclin D1 expression. We concluded that mtTFA up-regulation, augmented mitochondrial biogenesis, and enhanced mitochondrial functions may contribute to arsenic-induced cell proliferation. Targeting mitochondrial biogenesis may help treat arsenical cancers at the stage of cell proliferation.

Roles of ROS, Akt, ERK signaling in arsenic‐induced cell transformation and tumor growth

Arsenic is strongly associated with lung cancer. But mechanism of arsenic in carcinogenesis still remains to be elucidated. This study sets to understand roles of reactive oxygen species (ROS) and signaling molecules in arsenic-inducing cell transformation and tumor growth using lung bronchial epithelial (BEAS-2B) cells. Acute arsenic treatment and arsenic-transformed (As-T) cells have much higher ROS generation. Arsenic activated Akt and ERK signaling which was abated with ROS inhibition via catalase treatment. As-T cells increased cell proliferation and colony formation in soft agar. Interruption of ROS generation, PI3K/Akt, and MEK/ERK signaling significantly decreased proliferation and colony formation in As-T cells. As-T cells formed a greater number of primary tumors with larger tumor volume in immune-deficient mice when compared to untreated control cells. Tumor weight was significantly reduced with catalase treatment. Tumor formation of As-T cells was completely ablated with Akt interruption. We conclude that arsenic-induced cell transformation and tumor growth requires ROS generation and Akt/ERK signaling. Funding was provided by National Institutes of Health and American Cancer Society.

Activation and up‐regulation of translation initiation factor 4B contribute to arsenic‐induced transformation

Arsenic is a known human carcinogen. However, the mechanism of how arsenic induces cell transformation remains unclear. In this study, we demonstrated that long-term exposure to sodium arsenite at low-dose (2 µM) increases cell proliferation and neoplastic transformation in a mouse epidermal cell model, JB6 promotion-susceptible cells. The phosphorylation of AKT and its downstream targets, 70-kDa ribosomal protein S6 kinase (p70S6K) and translation initiation factor 4B (eIF4B), are increased in the arsenite treated cells, indicating that long-term arsenite treatment activates AKT-p70S6K signaling pathway. In addition, long-term exposure to arsenite up-regulates eIF4B expression and increases the rate of translation. Knockdown of eIF4B expression resulted in inhibition of arsenic-induced cell proliferation, transformation, and translation. Moreover, the expression of c-Myc which is up-regulated by long-term arsenite treatment is inhibited by eIF4B knockdown. Taken together, these results indicate that activation and up-regulation of eIF4B contributes to arsenic-induced transformation in JB6 cells.

Arsenite exposure in human lymphoblastoid cell lines induces autophagy and coordinated induction of lysosomal genes

Chronic exposure to inorganic arsenic is associated with diverse, complex diseases, making the identification of the mechanism underlying arsenic-induced toxicity a challenge. An increasing body of literature from epidemiological and in vitro studies has demonstrated that arsenic is an immunotoxicant, but the mechanism driving arsenic-induced immunotoxicity is not well established. We have previously demonstrated that in human lymphoblastoid cell lines (LCLs), arsenic-induced cell death is strongly associated with the induction of autophagy. In this study we utilized genome-wide gene expression analysis and functional assays to characterize arsenic-induced effects in seven LCLs that were exposed to an environmentally relevant, minimally cytotoxic, concentration of arsenite (0.75 μM) over an eight-day time course. Arsenic exposure resulted in inhibition of cellular growth and induction of autophagy (measured by expansion of acidic vesicles) over the eight-day exposure duration. Gene expression analysis revealed that arsenic exposure increased global lysosomal gene expression, which was associated with increased functional activity of the lysosome protease, cathepsin D. The arsenic-induced expansion of the lysosomal compartment in LCL represents a novel target that may offer insight into the immunotoxic effects of arsenic.

The NRF2-mediated oxidative stress response pathway is associated with tumor cell resistance to arsenic trioxide across the NCI-60 panel

BackgroundDrinking water contaminated with inorganic arsenic is associated with increased risk for different types of cancer. Paradoxically, arsenic trioxide can also be used to induce remission in patients with acute promyelocytic leukemia (APL) with a success rate of approximately 80%. A comprehensive study examining the mechanisms and potential signaling pathways contributing to the anti-tumor properties of arsenic trioxide has not been carried out.MethodsHere we applied a systems biology approach to identify gene biomarkers that underlie tumor cell responses to arsenic-induced cytotoxicity. The baseline gene expression levels of 14,500 well characterized human genes were associated with the GI50 data of the NCI-60 tumor cell line panel from the developmental therapeutics program (DTP) database. Selected biomarkers were tested in vitro for the ability to influence tumor susceptibility to arsenic trioxide.ResultsA significant association was found between the baseline expression levels of 209 human genes and the sensitivity of the tumor cell line panel upon exposure to arsenic trioxide. These genes were overlayed onto protein-protein network maps to identify transcriptional networks that modulate tumor cell responses to arsenic trioxide. The analysis revealed a significant enrichment for the oxidative stress response pathway mediated by nuclear factor erythroid 2-related factor 2 (NRF2) with high expression in arsenic resistant tumor cell lines. The role of the NRF2 pathway in protecting cells against arsenic-induced cell killing was validated in tumor cells using shRNA-mediated knock-down.ConclusionsIn this study, we show that the expression level of genes in the NRF2 pathway serve as potential gene biomarkers of tumor cell responses to arsenic trioxide. Importantly, we demonstrate that tumor cells that are deficient for NRF2 display increased sensitivity to arsenic trioxide. The results of our study will be useful in understanding the mechanism of arsenic-induced cytotoxicity in cells, as well as the increased applicability of arsenic trioxide as a chemotherapeutic agent in cancer treatment.

Arsenic-induced cell proliferation is associated with enhanced ROS generation, Erk signaling and CyclinA expression

Arsenic is a well-established human carcinogen; however molecular mechanisms to arsenic-induced carcinogenesis are complex and elusive. The present study identifies a potential biomarker of arsenic exposure, and redefines arsenic-induced signaling in stimulation of cell proliferation. The effect of arsenic exposure on gene expression was evaluated in PBMC of arsenic-exposed individuals selected from a severely affected district of West Bengal, India. A novel, un-documented biomarker of arsenic exposure, CyclinA was identified by microarray analysis from the study. Non-transformed cell lines HaCat and Int407 when exposed to clinically achievable arsenic concentration showed significant increase of CyclinA substantiating the clinical data. An associated increase in S phase population of cells in cell cycle, indicative of enhanced proliferation was also noticed. On further investigation of the pathway to arsenic-induced proliferation, we observed that arsenic resulted: ROS generation; activated Erk signaling; stimulated AP-1 activity, including immediate early genes, c-Jun and c-Fos. N-Acetyl- l-cysteine, a ROS quencher, blocked the arsenic-induced effects. Our study underlines a previously undefined mechanism by which arsenic imparts its toxicity and results in uncontrolled cell proliferation.

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.

Abstract 88: Targeting Bcl-XL enhances arsenic-induced cell death in ovarian cancer cells

Abstract Autophagy is an evolutionary conserved process in which long-lived proteins and damaged organelles are digested under physiological conditions including starvation. Autophagy has also been shown to act as a programmed cell death mechanism (type II PCD) and considered as caspase independent cell death. Several studies showed that autophagy has been triggered by anti-cancer treatments, such as chemo and hormonal therapy and radiotherapy and growth factor deprivation, function either as a protective survival pathway or cellular death mechanism. Arsenic trioxide (ATO), which is a FDA approved drug and used as a treatment in acute promyelocytic leukemia, has been reported to induce autophagic and/or apoptotic cell death in leukemia, glioblastoma and also ovarian cancer cells, we have previously found that ATO induces autophagy in ovarian cancer cells. Here, we investigated the role of antiapoptotic protein Bcl-Xl, which inhibits autophagy by binding beclin-1, an essential autophagy promoting protein, in arsenic induced apoptosis and determined the effects of ATO/Bcl-XL RNA interference combination therapy in ovarian cancer. We found that ATO induces dose dependant growth inhibition in MTS proliferation assay. ATO treatment (2-4 uM) induced apoptosis in A2780 (40-50 %) and SKOV3 (20-25 %) cells, detected by annexin V staining and flow cytometric (FACS) analysis and by caspase 9 and PARP cleavage (Western blotting). ATO induced marked autophagy in A2780 (60-70 %) and in SKOV3 (15-20 %) as indicated by LC3-II, a marker for autophagy, formation autophagic vacuoles detected by acridine orange staining and FACS and by transmission electron microscopy. ATO induced Bcl-XL protein expression in ovarian cancer cells. We also found expression of high levels of Bcl-XL in all ovarian cancer cell lines including drug (i.e., cisplatin and paclitaxel) resistant cells. Inhibition of Bcl-XL expression by Bcl-XL siRNA induced growth inhibition (40-60 %), apoptosis (40-60 %) and autophagy (50-60% in A2780 and 15-25 % in SKOV3 ovarian cancer cell line). Furthermore, combination of ATO with Bcl-XL siRNA led to 90 % cell death and 70 % apoptosis in both A2780 and SKOV3 cells. Overall, our results suggest that combination of ATO and Bcl-XL induced predominantly apoptosis, although each of them induced autophagy. In conclusion we provided first evidence that inhibition of Bcl-XL induces autophagy in ovarian cancer cells and sensitizes them to ATO that may be a promising treatment regimen for ovarian cancer patients. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 88.

Glutathione Depletion Enhances Arsenic Trioxide-Induced Apoptosis in Lymphoma Cells through Mitochondrial and Caspase-Independent Mechanisms.

Abstract 2708Poster Board II-684 Introduction:Arsenic trioxide (ATO) induces apoptosis in cancer through several mechanisms including modulation of the redox system and targeting the BCL-2 family and mitochondria, resulting in cytochrome c release and activation of caspases. However, the mechanisms of arsenic-induced cell death in lymphoma remain to be elucidated, while methods to enhance ATO-related apoptosis have not been fully explored. In the present study, we evaluated the cytotoxic activity and pathways of cell death induced by ATO alone and in combination with a glutathione (GSH)-depleting agent in non-Hodgkin lymphoma (NHL) cell lines and primary cells from patients with lymphoma. We hypothesized that depletion of GSH would enhance ATO induced apoptosis through mitochondrial and caspase-mediated pathways. Methods:We treated Ramos (Burkitt lymphoma), HF1 (follicular lymphoma), and SUDHL4 (diffuse large B-cell lymphoma) NHL cell lines, and primary follicular lymphoma and CLL/SLL cells with increasing concentrations (10–100μM) of ATO for 24–72 hours (hrs). Apoptosis was determined by Annexin V-Propidium iodide staining followed by flow cytometry and Z-VAD-FMK was used as a pan-caspase inhibitor. ROS was measured by H2DCFDA staining followed by flow cytometry. We further studied the mechanism of apoptosis by measuring alteration in mitochondrial membrane potential (MMP), Bax translocation, and caspase and PARP activation. In addition, we treated wild type and Bax−/−/Bak−/− double knockout mouse endothelial fibroblasts (MEFS) with ATO or ATO/BSO and determined apoptosis and mitochondrial membrane potential compared with MEF wild type (MEF-wt). Results:Treatment of cells with ATO resulted in dose- and time-dependent apoptosis, although high concentrations were necessary for effect (ED50 >10–20μM). ATO treatment redistributed Bax from the cytosol to mitochondria and there were significant changes in MMP, as well as increased PARP, cleaved caspse-3 and -9, while BID was downregulated. Furthermore, ATO-induced apoptosis was caspase-dependent. Addition of the GSH-depleting agent, buthionine sulfoximine (BSO), to ATO induced strong synergistic cell death (combination indices <0.1 in all cell lines), resulting in apoptosis that was ROS-related, but mitochondrial- and caspase-independent. Using immortalized wild-type MEFS and Bax−/−Bak−/− double knockouts, ATO-induced alteration in MMP and apoptosis that was Bax-dependent (see figure below), while targeted disruption of the Bax/Bak genes had no enhancing effects on ATO/BSO-induced MMP or apoptosis. Interestingly, ATO alone induced appreciable apoptosis in primary CLL/SLL and follicular lymphoma cells, while BSO had minimal additive effects in these fresh cells. This appeared due to markedly lower intracellular GSH content within primary cells compared with cell lines (p<0.001). [Display omitted] Conclusion:Altogether, our findings establish that ATO has anti-lymphoma activity and approaches to enhance its cytotoxic activity are feasible via engagement of distinct cell death mechanisms. Continued investigation of arsenic-based therapy, including new arsenical compounds and methods of tissue delivery that are able to overcome mechanisms of cellular resistance, for the treatment of lymphoma is warranted. Disclosures:Gordon:Cure Tech: Membership on an entity's Board of Directors or advisory committees.

Taurine prevents arsenic-induced cardiac oxidative stress and apoptotic damage: Role of NF-κB, p38 and JNK MAPK pathway

Cardiac dysfunction is a major cause of morbidity and mortality worldwide due to its complex pathogenesis. However, little is known about the mechanism of arsenic-induced cardiac abnormalities and the use of antioxidants as the possible protective agents in this pathophysiology. Conditionally essential amino acid, taurine, accounts for 25% to 50% of the amino acid pool in myocardium and possesses antioxidant properties. The present study has, therefore, been carried out to investigate the underlying mechanism of the beneficial role of taurine in arsenic-induced cardiac oxidative damage and cell death. Arsenic reduced cardiomyocyte viability, increased reactive oxygen species (ROS) production and intracellular calcium overload, and induced apoptotic cell death by mitochondrial dependent caspase-3 activation and poly-ADP ribose polymerase (PARP) cleavage. These changes due to arsenic exposure were found to be associated with increased IKK and NF-κB (p65) phosphorylation. Pre-exposure of myocytes to an IKK inhibitor (PS-1145) prevented As-induced caspase-3 and PARP cleavage. Arsenic also markedly increased the activity of p38 and JNK MAPKs, but not ERK to that extent. Pre-treatment with SP600125 (JNK inhibitor) and SB203580 (p38 MAPK inhibitor) attenuated NF-κB and IKK phosphorylation indicating that p38 and JNK MAPKs are mainly involved in arsenic-induced NF-κB activation. Taurine treatment suppressed these apoptotic actions, suggesting that its protective role in arsenic-induced cardiomyocyte apoptosis is mediated by attenuation of p38 and JNK MAPK signaling pathways. Similarly, arsenic intoxication altered a number of biomarkers related to cardiac oxidative stress and other apoptotic indices in vivo and taurine supplementation could reduce it. Results suggest that taurine prevented arsenic-induced myocardial pathophysiology, attenuated NF-κB activation via IKK, p38 and JNK MAPK signaling pathways and could possibly provide a protection against As-induced cardiovascular burden.

Biological Responses to Arsenic Compounds

Arsenic is a metalloid that generates various biological effects on cells and tissues. Depending on the specific tissue exposed and the time and degree of exposure, diverse responses can be observed. In humans, prolonged and/or high dose exposure to arsenic can have a variety of outcomes, including the development of malignancies, severe gastrointestinal toxicities, diabetes, cardiac arrhythmias, and death. On the other hand, one arsenic derivative, arsenic trioxide (As(2)O(3)), has important antitumor properties. This agent is a potent inducer of antileukemic responses, and it is now approved by the Food and Drug Administration for the treatment of acute promyelocytic leukemia in humans. The promise and therapeutic potential of arsenic and its various derivatives have been exploited for hundreds of years. Remarkably, research focused on the potential use of arsenic compounds in the treatment of human diseases remains highly promising, and it is an area of active investigation. An emerging approach of interest and therapeutic potential involves efforts to target and block cellular pathways activated in a negative feedback manner during treatment of cells with As(2)O(3). Such an approach may ultimately provide the means to selectively enhance the suppressive effects of this agent on malignant cells and render normally resistant tumors sensitive to its antineoplastic properties.

Arsenic induces mitochondria-dependent apoptosis by reactive oxygen species generation rather than glutathione depletion in Chang human hepatocytes

This study was conducted to evaluate the possible involvement of mitochondrial pathway in NaAsO2-induced apoptosis and the role of reactive oxygen species (ROS) and reduced glutathione (GSH) in the apoptotic effect in Chang human hepatocytes. The MTT assay demonstrated that sodium arsenite (NaAsO2) treatment for 24 h caused a dose-dependent decrease of cell viability. NaAsO2 treatment (0-30 microM) was also found to induce phosphatidylserine externalization, a hallmark of apoptosis; to disrupt the mitochondrial membrane potential (Deltapsi ( m )); to cause the release of cytochrome c into the cytosol, and to trigger cleavage of caspase-3 and poly (ADP-ribose) polymerase (PARP) in a dose-dependent manner. All these changes were accompanied with the enhanced generation of intracellular ROS and malondialdehyde (MDA). Increase of intracellular GSH also coincided unexpectedly. Moreover, the extracellular addition of N-acetyl-L-cysteine (NAC, 5 mM) effectively reduced the generation of ROS and MDA, and rescued the cells from NaAsO2 induced apoptosis and related alteration of mitochondria. These data suggest that the arsenic-induced cell apoptosis occurs though the mitochondrial pathway, and is mostly dependent on generation of ROS rather than GSH depletion in Chang human hepatocytes.

Gene expression profiling analysis reveals arsenic-induced cell cycle arrest and apoptosis in p53-proficient and p53-deficient cells through differential gene pathways

Arsenic (As) is a well-known environmental toxicant and carcinogen as well as an effective chemotherapeutic agent. The underlying mechanism of this dual capability, however, is not fully understood. Tumor suppressor gene p53, a pivotal cell cycle checkpoint signaling protein, has been hypothesized to play a possible role in mediating As-induced toxicity and therapeutic efficiency. In this study, we found that arsenite (As 3+) induced apoptosis and cell cycle arrest in a dose-dependent manner in both p53 +/+ and p53 −/− mouse embryonic fibroblasts (MEFs). There was, however, a distinction between genotypes in the apoptotic response, with a more prominent induction of caspase-3 in the p53 −/− cells than in the p53 +/+ cells. To examine this difference further, a systems-based genomic analysis was conducted comparing the critical molecular mechanisms between the p53 genotypes in response to As 3+. A significant alteration in the Nrf2-mediated oxidative stress response pathway was found in both genotypes. In p53 +/+ MEFs, As 3+ induced p53-dependent gene expression alterations in DNA damage and cell cycle regulation genes. However, in the p53 −/− MEFs, As 3+ induced a significant up-regulation of pro-apoptotic genes (Noxa) and down-regulation of genes in immune modulation. Our findings demonstrate that As-induced cell death occurs through a p53-independent pathway in p53 deficient cells while apoptosis induction occurs through p53-dependent pathway in normal tissue. This difference in the mechanism of apoptotic responses between the genotypes provides important information regarding the apparent dichotomy of arsenic's dual mechanisms, and potentially leads to further advancement of its utility as a chemotherapeutic agent.

Neoplastic Transformation of Human Small Airway Epithelial Cells Induced by Arsenic

Human small airway epithelial cells (SAECs) previously immortalized with human telomerase reverse transcriptase (h-TERT) were continuously treated with sodium arsenite at a dose of 0.5 microg/mL in culture for up to 6 months. Arsenic-treated cells progressively displayed an increase in transformed phenotype including enhanced growth saturation density, plating efficiency, and anchorage-independent growth and invasion capability compared with their nontreated control cells. To determine whether arsenic-induced cell transformation was associated with genomic instability, treated and control cells were also analyzed for micronuclei formation. A 4.8-fold increase in micronuclei incidence in arsenic-treated cells was detected in conjunction with increased N-phosphonacetyl-l-aspartate (PALA)-resistant characteristics. In addition, arsenic-treated cells showed an increase in c-H-ras, c-myc, and c-fos protein expression relative to controls. The change in oncoprotein expression correlated with a decrease in wild-type p53 expression and hyperphosphorylated retinoblastoma. Taken together, these results strongly suggest that h-TERT immortalized human small airway epithelial cells underwent step-wise transformation after inorganic arsenic treatment.

The nitric oxide prodrug, V-PYRRO/NO, mitigates arsenic-induced liver cell toxicity and apoptosis

Arsenite is an important cancer chemotherapeutic. The liver is a major target tissue of arsenic toxicity and hepatotoxicity may limit its chemotherapeutic efficacy. O 2-vinyl 1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate (V-PYRRO/NO) is a liver-selective nitric oxide (NO)-producing prodrug metabolized by hepatic P450 enzymes to release NO locally. V-PYRRO/NO protects against various organic or inorganic hepatotoxicants but any role in arsenic hepatotoxicity is undefined. Thus, we studied the effects of V-PYRRO/NO (0–1000 μM) pretreatment on inorganic arsenic-induced toxicity in cultured rat liver (TRL 1215) cells. These cells metabolized the prodrug to release NO, producing extracellular nitrite levels to 41.7-fold above control levels (7.50 ± 0.38 μM) after 24 h V-PYRRO/NO (1000 μM) exposure. The effect of pretreatment with V-PYRRO/NO (24 h) on the cytolethality of arsenic (as NaAsO 2) exposure (24 h) was assessed. Arsenic was markedly less toxic in V-PYRRO/NO pretreated cells (LC 50 = 30.3 μM) compared to control (LC 50 = 20.1 μM) and the increases in LC 50 showed a direct relationship to the level of NO produced (measured as nitrite). Consistent with the cytolethality data, V-PYRRO/NO pretreatment markedly reduced arsenic-induced apoptosis as assessed by DNA fragmentation. Activation of the c-Jun N-terminal kinase (JNK) pathway can be critical to apoptosis and pretreatment with V-PYRRO/NO suppressed arsenic-induced JNK activation. V-PYRRO/NO pretreatment modestly increased metallothionein (MT), a metal-binding protein, but greatly enhanced arsenic induction of MT. Thus, V-PYRRO/NO pretreatment directly mitigates arsenic toxicity in cultured liver cells, reducing cytolethality, apoptosis and related JNK pathway activation, apparently through generation of NO. The role of NO in reducing the hepatotoxicity of arsenical chemotherapeutics in vivo deserves additional study.

A Critical Role for IκB Kinase β in Metallothionein-1 Expression and Protection against Arsenic Toxicity

Arsenic is a widespread environmental toxic agent that has been shown to cause diverse tissue and cell damage and at the same time to be an effective anti-cancer therapeutic agent. The objective of this study is to explore the signaling mechanisms involved in arsenic toxicity. We show that the IkappaB kinase beta (IKKbeta) plays a crucial role in protecting cells from arsenic toxicity. Ikkbeta(-)(/)(-) mouse 3T3 fibroblasts have decreased expression of antioxidant genes, such as metallothionein 1 (Mt1). In contrast to wild type and IKKbeta-reconstituted Ikkbeta(-)(/)(-) cells, IKKbeta-null cells display a marked increase in arsenic-induced reactive oxygen species (ROS) accumulation, which leads to activation of the MKK4-c-Jun NH(2)-terminal kinase (JNK) pathway, c-Jun phosphorylation, and apoptosis. Pretreatment with the antioxidant N-acetylcysteine (NAC) and expression of MT1 in the Ikkbeta(-)(/)(-) cells prevented JNK activation; moreover, NAC pretreatment, MT1 expression, MKK4 ablation, and JNK inhibition all protected cells from death induced by arsenic. Our data show that two signaling pathways appear to be important for modulating arsenic toxicity. First, the IKK-NF-kappaB pathway is crucial for maintaining cellular metallothionein-1 levels to counteract ROS accumulation, and second, when this pathway fails, excessive ROS leads to activation of the MKK4-JNK pathway, resulting in apoptosis.

Induction of Germline Cell Cycle Arrest and Apoptosis by Sodium Arsenite in Caenorhabditis elegans

The nematode Caenorhabditis elegans has been shown to be a model organism in studying aquatic toxicity. Although epidemiological studies have shown that arsenic is teratogenic and carcinogenic to humans, the lethality assay indicated that C. elegans is less sensitive to inorganic arsenic than any other organisms that have been tested thus far. In the present study, we used the more malleable germline of C. elegans as an in vivo system to investigate the genotoxic effects of arsenite. After animals were exposed to sodium arsenite at concentrations ranging from 1 microM to 0.5 mM, mitotic germ cells and germline apoptosis were scored after DAPI staining and acridine orange vital staining, respectively. DMSO rescue experiments were performed by exposing C. elegans to 0.01 mM arsenite in the presence of DMSO (0.1%) for 24 h, and reactive oxygen species (ROS) were semiquantified by CM-H(2)DCFDA vital staining. The results indicated that arsenic exposure reduced the brood size of C. elegans and caused mitotic cell cycle arrest and germline apoptosis, which, to some extent, exhibited a concentration- and time-dependent manner. The addition of 0.1% DMSO completely rescued arsenic-induced cell cycle arrest and partially suppressed germline apoptosis. Furthermore, treatment of animals with arsenite at a dose of 0.01 mM significantly increased ROS production in the intestine, which could be reduced by DMSO treatment. The present study also indicated that C. elegans might be used as an in vivo model system to study the mechanisms of arsenic-induced genotoxic effects.

Protection against arsenic trioxide-induced autophagic cell death in U118 human glioma cells by use of lipoic acid

Arsenic is an environmental toxicant found naturally in ground water. Epidemiological studies have suggested a correlation between chronic arsenic exposure and potential brain tissue damage in clinical case and animal experiments. Lipoic acid (LA) is a thiol-compound naturally occurring in plants and animals, which is thought to be a strong antioxidant and possess neuroprotective effects. The objective of this study was to determine if the AS 2O 3-induced glial cell toxicity could be prevented by LA. The human malignant glioma cell (U118) was selected as a research model. By using acridine orange staining and flow cytometry analysis, we found that autophagic, but not apoptotic, cell death was significantly induced by AS 2O 3 in U118 cells, and that AS 2O 3-mediated autophagic cell death was nearly completely attenuated by LA. Down-regulation of p53 and Bax proteins and the up-regulation of Bcl-2 and HSP-70 proteins were observed by western blot in AS 2O 3-mediated autophagic cell death. Our results implied that LA completely inhibited U118 cells autophagic cell death induced by AS 2O 3. We suggested that LA may emerge as a useful protective agent against arsenic-induced glial cell toxicity and reversing arsenic-induced damage in human brain.

Involvement of Substance P and Neurogenic Inflammation in Arsenic-Induced Early Vascular Dysfunction

Vascular-related diseases, including Blackfoot Disease and atherosclerosis, are prominent clinical findings among populations residing in arseniasis areas. While oxidative stress provided a general but nonspecific mechanistic base for arsenic-induced endothelial cell damage in vitro, more specific mechanism is needed to explain the highly targeted vascular lesions induced by arsenic in vivo. Based on our previous studies, we hypothesized that arsenic exerted its action on blood vessels via the neurogenic inflammation process involving release of a neuropeptide (substance P) and activation of endothelial Neurokinin 1 (NK-1) receptor in vivo. Indeed, our present study demonstrated a significantly higher substance P levels in arsenic-treated tissues when compared to saline-treated controls indicating a rapid release of substance P under the influence of arsenic. Furthermore, the arsenic-induced vascular leakage could be significantly reduced when the neurogenic inflammation process was interrupted (via either disruption on the release of substance P, interference on the action of substance P, or blockage of endothelial NK-1 receptor) showing that the neurogenic inflammation process was indeed involved. Histamine release was not found to play a significant role in arsenic-induced vascular permeability change. Our present study affirmed a de novo concept that a pathophysiological mechanism involving the neurogenic release of substance P and activation of endothelial NK-1 receptor underlies the arsenic-induced vascular injury and dysfunction in vivo. This pathophysiological process constituted a two-tiered biological interaction between the nervous system and vascular system and therefore was not readily unveiled by traditional in vitro studies in the past. Our present finding unveiled an important de novo concept on arsenic vascular toxicity in vivo.

Depletion of Securin Increases Arsenite-Induced Chromosome Instability and Apoptosis via a p53-Independent Pathway

Arsenic is a pathologic factor of cardiovascular diseases and cancers; nevertheless, it also acts as an anticancer agent effective on acute promyelocytic leukemia and multiple myeloma. Securin, a proposed proto-oncogene, regulates cell proliferation and tumorigenesis. However, roles of securin on the arsenic-induced cell cycle arrest and apoptosis remain unknown. In this study, the effects of sodium arsenite on the expression of securin in two tissue types of cell lines, the vascular endothelial and colorectal epithelial cells, were investigated. Arsenite (8-16 microM, 24 h) increased the cytotoxicity, apoptosis, and growth inhibition in both endothelial and epithelial cells. The levels of phospho-CDC2 (threonine-161), CDC2, and cyclin B1 proteins were decreased, and the G2/M fractions were increased by arsenite. Concomitantly, arsenite markedly diminished the securin protein expression and induced the abnormal sister chromatid separation. The depletion of securin proteins increased the induction of mitotic arrest, aberrant chromosome segregation, and apoptosis after arsenite treatment. p53, a tumor suppressor protein, balances the cell survival and apoptosis. Arsenite raised the levels of phospho-p53 (serine-15) and p53 (DO-1) proteins in both the securin-wild-type and -null cells. The p53-functional cells were more susceptible than the p53-mutational cells to arsenite on the cytotoxicity and apoptosis. Besides, arsenite decreased the levels of securin proteins to a similar degree in both the p53-functional and -mutational cells. Together, it is the first time to demonstrate that the inhibition of securin expression induced by arsenite increases the chromosomal instability and apoptosis via a p53-independent pathway.