Low-dose Arsenite Research Articles

Effect of Long-Term Low-Dose Arsenic Exposure on DNA Methylation and Gene Expression in Human Liver Cells.

Millions of people around the world are exposed to elevated levels of arsenic through food or drinking water. Epidemiological studies have linked chronic arsenic exposure to an increased risk of several cancers, cardiovascular disease, central nervous system neuropathies, and genotoxic as well as immunotoxic effects. In addition to the induction of oxidative stress and inhibition of DNA repair processes, epigenetic effects, including altered DNA methylation patterns resulting in aberrant gene expression, may contribute to carcinogenicity. However, the underlying mechanisms by which chronic micromolar concentrations of arsenite affect the methylation status of DNA are not fully understood. In this study, human HepG2 hepatocarcinoma cells were treated with 0.5-10 μM sodium arsenite for 24 h, 10, or 20 days. During these periods, the effects on global DNA methylation, cell cycle phase distribution, and gene expression were investigated. While no impact on DNA methylation was seen after short-term exposure, global hypomethylation was observed at both long-term exposure periods, with concomitant induction of the DNA methyltransferase genes DNMT1 and DNMT3B, while DNMT3A was slightly down-regulated. Pronounced time- and concentration-dependent effects were also seen in the case of genes involved in DNA damage response and repair, inflammation, oxidative stress response, and metal homeostasis. These results suggest that chronic low-dose arsenite exposure can lead to global hypomethylation. As an underlying mechanism, the consistent down-regulation of DNA methyltransferase genes could be excluded; alternatively, interactions at the protein level could play an important role.

Low-dose arsenite causes overexpression of EGF, TGFα, and HSP90 through Trx1-TXNIP-NLRP3 axis mediated signaling pathways in the human bladder epithelial cells

Epidemiological studies have demonstrated an increased incidence of bladder cancer in arseniasis- endemic areas; however, the precise molecular mechanisms remain unknown. Our previous results have shown that the protein levels of EGF, TGFα, and HSP90 in arsenite-treated bladder uroepithelial cells increased markedly and contributed to hyperactivation of EGF receptors. The aim of this study was to further explore the regulatory ways underlying overexpression of EGF, TGFα, and HSP90 in these cells. The present results showed that both Trx and GSH systems were stimulated in arsenite-treated cells, and ROS levels in 2 μM arsenite-treated cells did not changed obviously; however, ROS levels in 4 μM arsenite-treated cells increased significantly. By using the antioxidant and specific inhibitors, we found that in 2 μM arsenite-treated cells, JNK/NF-κB signaling pathway was involved in overexpression of EGF and TGFα, and ERK/NF-κB signaling pathway contributed to HSP90 overexpression, however in 4 μM arsenite-treated cells, both ERK/ and JNK/NF-κB signaling pathways were involved in overexpression of EGF, TGFα, and HSP90, and PI3K/AKT/NF-κB signaling pathway contributed to overexpression of EGF and TGFα. Furthermore, our results also showed that the Trx1-TXNIP-NLRP3 axis was activated in arsenite-treated cells, and played a pivotal role in activation of the signaling pathways involved in overexpression of EGF, TGFα, and HSP90. In conclusion, the Trx1-TXNIP-NLRP3 axis might be activated by arsenite-induced redox imbalance in bladder uroepithelial cells, and mediate the activation of signaling pathways involved in overexpression of EGF, TGFα, and HSP90.

Akt Regulated Phosphorylation of GSK-3β/Cyclin D1, p21 and p27 Contributes to Cell Proliferation Through Cell Cycle Progression From G1 to S/G2M Phase in Low-Dose Arsenite Exposed HaCat Cells.

Arsenic is a toxic environmental contaminant. Long-term exposure to arsenic through drinking water induces human cancers. However, it is as yet uncertain about the mechanisms of arsenic induced carcinogenesis. Although the effects of low-dose arsenicals on proliferation and cell cycle have been revealed by short time exposure, the evidences for long-term exposure were seldom reported. The detailed mechanism has been unclear and supplemented constantly. In the present study, we used normal human keratinocytes (HaCat) to study the effects of long-term, low-dose sodium arsenite (NaAsO2) exposure on cell proliferation with emphasis on the Akt regulated cell cycle signaling pathways. Treatment of NaAsO2 resulted in increased cell proliferation and promotion of cell cycle progression from G1 to S/G2M phase, both of which could be attenuated by MK2206, a highly selective inhibitor of Akt. Along with the increased expression of phospho-Akt (p-Akt, Ser 473), increased expression of p-GSK-3β (Ser 9), p-p21 (Thr 145), p-p27 (Thr 157) and total cyclin D1, and decreased expression of p-cyclin D1 (Thr 286), p21 and p27 were also found in the NaAsO2 exposed cells. Treatment of MK2206 markedly reversed the expression of all of the above proteins. Our findings indicated that the phosphorylated activation of Akt played a role in the proliferation of HaCat cells upon long-term, low-dose NaAsO2 exposure through the phosphorylative regulation of its downstream cell cycle regulating factors of GSK-3β/cyclin D1, p21 and p27, which could induce the promotion of cell cycle progression from G1 to S/G2M phase.

Low dose arsenite confers resistance to UV induced apoptosis via p53-MDM2 pathway in ketatinocytes

Chronic arsenite and ultraviolet (UV) exposure are associated with skin tumor. To investigate the details by low concentrations of arsenite and UV induced carcinogenesis in skin, hTERT-immortalized human keratinocytes were used as a cellular model with exposure to low concentrations of sodium arsenite and UV. The effect of NaAsO2 on UV treatment-induced apoptosis was measured by flow cytometry and Hoechst staining. We found that the cell apoptosis induced by UV exposure was significantly attenuated after exposure to low-dose arsenite, and knockdown of p53 could block UV-induced apoptosis indicating that this phenomenon depended on p53. Interestingly, the expression of murine double minute 2 (MDM2), including its protein and transcriptional levels, was remarkably high after exposure to low-dose arsenite. Moreover, low-dose arsenite treatment dramatically decreased the MDM2 gene promoter activity, suggesting that this effect has been mediated through transcription. In addition, treatment of PD98059 reversed low-dose arsenite-induced MDM2 expression, and the inhibition of ERK2 expression could significantly block MDM2 expression as a consequence, and p53 expression automatically was increased. To validate the role of p53 in exposure to low-dose arsenite, the expression of p53 was examined by immunohistochemistry in the skin of Sprague−Dawley rats model by chronic arsenite exposure for 6 months and in patients with arsenic keratosis, and the results showed that the expression of p53 was decreased in those samples. Taken together, our results demonstrated that low-dose arsenite-induced resistance to apoptosis through p53 mediated by MDM2 in keratinocytes.

Deficiencies in mitochondrial dynamics sensitize Caenorhabditis elegans to arsenite and other mitochondrial toxicants by reducing mitochondrial adaptability.

Mitochondrial fission, fusion, and mitophagy are interlinked processes that regulate mitochondrial shape, number, and size, as well as metabolic activity and stress response. The fundamental importance of these processes is evident in the fact that mutations in fission (DRP1), fusion (MFN2, OPA1), and mitophagy (PINK1, PARK2) genes can cause human disease (collectively >1/10,000). Interestingly, however, the age of onset and severity of clinical manifestations varies greatly between patients with these diseases (even those harboring identical mutations), suggesting a role for environmental factors in the development and progression of certain mitochondrial diseases. Using the model organism Caenorhabditis elegans, we screened ten mitochondrial toxicants (2, 4-dinitrophenol, acetaldehyde, acrolein, aflatoxin B1, arsenite, cadmium, cisplatin, doxycycline, paraquat, rotenone) for increased or decreased toxicity in fusion (fzo-1, eat-3)-, fission (drp-1)-, and mitophagy (pdr-1, pink-1)-deficient nematodes using a larval growth assay. In general, fusion-deficient nematodes were the most sensitive to toxicants, including aflatoxin B1, arsenite, cisplatin, paraquat, and rotenone. Because arsenite was particularly potent in fission- and fusion-deficient nematodes, and hundreds of millions of people are chronically exposed to arsenic, we investigated the effects of these genetic deficiencies on arsenic toxicity in more depth. We found that deficiencies in fission and fusion sensitized nematodes to arsenite-induced lethality throughout aging. Furthermore, low-dose arsenite, which acted in a "mitohormetic" fashion by increasing mitochondrial function (in particular, basal and maximal oxygen consumption) in wild-type nematodes by a wide range of measures, exacerbated mitochondrial dysfunction in fusion-deficient nematodes. Analysis of multiple mechanistic changes suggested that disruption of pyruvate metabolism and Krebs cycle activity underlie the observed arsenite-induced mitochondrial deficits, and these disruptions are exacerbated in the absence of mitochondrial fusion. This research demonstrates the importance of mitochondrial dynamics in limiting arsenite toxicity by permitting mitochondrial adaptability. It also suggests that individuals suffering from deficiencies in mitodynamic processes may be more susceptible to the mitochondrial toxicity of arsenic and other toxicants.

Arsenite Effects on Mitochondrial Bioenergetics in Human and Mouse Primary Hepatocytes Follow a Nonlinear Dose Response.

Arsenite is a known carcinogen and its exposure has been implicated in a variety of noncarcinogenic health concerns. Increased oxidative stress is thought to be the primary cause of arsenite toxicity and the toxic effect is thought to be linear with detrimental effects reported at all concentrations of arsenite. But the paradigm of linear dose response in arsenite toxicity is shifting. In the present study we demonstrate that arsenite effects on mitochondrial respiration in primary hepatocytes follow a nonlinear dose response. In vitro exposure of primary hepatocytes to an environmentally relevant, moderate level of arsenite results in increased oxidant production that appears to arise from changes in the expression and activity of respiratory Complex I of the mitochondrial proton circuit. In primary hepatocytes the excess oxidant production appears to elicit adaptive responses that promote resistance to oxidative stress and a propensity to increased proliferation. Taken together, these results suggest a nonlinear dose-response characteristic of arsenite with low-dose arsenite promoting adaptive responses in a process known as mitohormesis, with transient increase in ROS levels acting as transducers of arsenite-induced mitohormesis.

Continuous activation of Nrf2 and its target antioxidant enzymes leads to arsenite-induced malignant transformation of human bronchial epithelial cells

Long-term exposure to arsenite leads to human lung cancer, but the underlying mechanisms of carcinogenesis remain obscure. The transcription factor of nuclear factor-erythroid-2 p45-related factor (Nrf2)-mediated antioxidant response represents a critical cellular defense mechanism and protection against various diseases. Paradoxically, emerging data suggest that the constitutive activation of Nrf2 is associated with cancer development, progression and chemotherapy resistance. However, the role of Nrf2 in the occurrence of cancer induced by long-term arsenite exposure remains to be fully understood. By establishing transformed human bronchial epithelial (HBE) cells via chronic low-dose arsenite treatment, we showed that, in acquiring this malignant phenotype, continuous low level of ROS and sustained enhancement of Nrf2 and its target antioxidant enzyme levels were observed in the later-stage of arsenite-induced cell transformation. The downregulation of Keap1 level may be responsible for the over-activation of Nrf2 and its target enzymes. To validate these observations, Nrf2 was knocked down in arsenite-transformed HBE cells by SiRNA transfection, and the levels of Nrf2 and its target antioxidant enzymes, ROS, cell proliferation, migration, and colony formation were determined following these treatments. Results showed that blocked Nrf2 expression significantly reduced Nrf2 and its target antioxidant enzyme levels, restored ROS levels, and eventually suppressed cell proliferation, migration, and colony formation of the transformed cells. In summary, the results of the study strongly suggested that the continuous activation of Nrf2 and its target antioxidant enzymes led to the over-depletion of intracellular ROS levels, which contributed to arsenite-induced HBE cell transformation.

Mitochondrial hormesis links low‐dose arsenite exposure to lifespan extension

Arsenite is one of the most toxic chemical substances known and is assumed to exert detrimental effects on viability even at lowest concentrations. By contrast and unlike higher concentrations, we here find that exposure to low-dose arsenite promotes growth of cultured mammalian cells. In the nematode C. elegans, low-dose arsenite promotes resistance against thermal and chemical stressors and extends lifespan of this metazoan, whereas higher concentrations reduce longevity. While arsenite causes a transient increase in reactive oxygen species (ROS) levels in C. elegans, co-exposure to ROS scavengers prevents the lifespan-extending capabilities of arsenite, indicating that transiently increased ROS levels act as transducers of arsenite effects on lifespan, a process known as mitohormesis. This requires two transcription factors, namely DAF-16 and SKN-1, which employ the metallothionein MTL-2 as well as the mitochondrial transporter TIN-9.1 to extend lifespan. Taken together, low-dose arsenite extends lifespan, providing evidence for nonlinear dose-response characteristics of toxin-mediated stress resistance and longevity in a multicellular organism.

Abstract 1329: Up-regulation of FOXM1b promotes low doses of arsenite-induced lung carcinogenesis

Abstract Long-term exposure to low doses of arsenic from environmental sources has been associated with increased incidence of skin, lung, liver, bladder, and prostate cancers. A paradox is that arsenic has not been shown conclusively to be an initiator or a promoter of carcinogenesis in animal models. After E6/E7/hTERT immortalized human airway epithelial cell line NuLi-1 was exposed at 1μM arsenite continuously for 24 weeks, the cells exhibited malignant transformation in the anchorage independent growth assay. Because this cell line is null for p53 and Rb, these data indicated that inactivation of p53 and pRb pathways alone was not sufficient to induce malignant transformation, the addition of long-term exposure of low dose arsenite resulted in malignant transformation. Using a high-throughput method, we detected up-regulated expressions of CDC6, CDC25A and Cyclin D1 (which are critical for cell cycle regulation at G1 and S) when the cells were exposed to 0.1μM arsenite for 24∼72 hours CDC6, CDC25A and Cyclin D1 were expressed over time until cell transformation occurred. Arsenite steadily increased the expression levels of CDC6, CDC25A and Cyclin D1 with time at both transcription and protein levels. Interestingly, the expression of a proliferation-specific transcription factor FOXM1b correlated with increased expression of CDC6, CDC25A and Cyclin D1. FOXM1b is a key regulator of both G1/S and G2/M cell cycle transitions, and mitotic spindle integrity and plays an important role in lung carcinogenesis. Ectopic upregulation of FOXM1b in NuLi-1 cells significantly promoted arsenite-induced malignant transformation; in reverse, knockdown of FOXM1b in the transformed NuLi-1 cells and lung adenocarcinoma cell line A549 significantly decreased malignant transformation. Collectively, the study indicated that upergulation of FoxM1b, of the absence of p53 and pRb in immortalized human airway epithelial cells, promotes low dose arsenite-induced malignant transformation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1329. doi:10.1158/1538-7445.AM2011-1329

Angiogenin-induced tRNA-derived Stress-induced RNAs Promote Stress-induced Stress Granule Assembly

Angiogenin (ANG) is a secreted ribonuclease that cleaves tRNA to initiate a stress-response program in mammalian cells. Here we show that ANG inhibits protein synthesis and promotes arsenite- and pateamine A-induced assembly of stress granules (SGs). These effects are abrogated in cells transfected with the ANG inhibitor RNH1. Transfection of natural or synthetic 5'- but not 3'-tRNA fragments (tRNA-derived stress-induced RNAs; tiRNAs) induces the phospho-eukaryotic translation initiation factor 2alpha-independent assembly of SGs. Natural 5'-tiRNAs but not 3'-tiRNAs are capped with a 5'-monophosphate that is required for optimal SG assembly. These findings reveal that SG assembly is a component of the ANG- and tiRNA-induced stress response program.

C-Jun/AP-1 pathway-mediated cyclin D1 expression participates in low dose arsenite-induced transformation in mouse epidermal JB6 Cl41 cells

Arsenic is a well-documented human carcinogen associated with skin carcinogenesis. Our previous work reveals that arsenite exposure is able to induce cell transformation in mouse epidermal cell JB6 Cl41 through the activation of ERK, rather than JNK pathway. Our current studies further evaluate downstream pathway in low dose arsenite-induced cell transformation in JB6 Cl41 cells. Our results showed that treatment of cells with low dose arsenite induced activation of c-Jun/AP-1 pathway, and ectopic expression of dominant negative mutant of c-Jun (TAM67) blocked arsenite-induced transformation. Furthermore, our data indicated that cyclin D1 was an important downstream molecule involved in c-Jun/AP-1-mediated cell transformation upon low dose arsenite exposure, because inhibition of cyclin D1 expression by its specific siRNA in the JB6 Cl41 cells resulted in impairment of anchorage-independent growth of cells induced by low dose arsenite. Collectively, our results demonstrate that c-Jun/AP-1-mediated cyclin D1 expression is at least one of the key events implicated in cell transformation upon low dose arsenite exposure.

Identification of Genes Responding to Low-Dose Arsenite Using HiCEP

ABSTRACTChronic ingestion of arsenic in polluted food and water can cause various human disorders including skin and lung cancers. Sensitive biomarkers from human tissue/cells could help to prevent chronic intoxication with low-dose arsenite. Using High-Coverage Expression Profiling (HiCEP), an Amplified Fragment Length Polymorphism (AFLP)-based gene expression profiling technique, we analyzed the expression of approximately 11,000 genes in human lung fibroblasts (HFLIII) and compared the profiles between cells, treated and untreated with 1 μM sodium arsenite (NaAsO2). Hundreds of genes appeared upregulated and downregulated more than two-fold, 2 h after the treatment. Marked induction was found (>4.4-fold) in a few genes including HMOX1, INHBA, and ANKRD11. Induction of the HMOX1 was detected with a dose of arsenite at as low as 0.3 μM (0.04 ppm) and reached its maximum at 4 h after the treatment. The arsenite-induced HMOX1 expression was attenuated by the promoted glutathione (GSH) synthesis by N-acetyl-L-cysteine (NAC). However, it was not affected by pretreating the cells with general radical scavengers, consistent with the fact that ionizing radiation at either high-or low-doses has never induced HMOX1 in the same assay system. Thus, induction of HMOX1 gene is highly sensitive and also selective against arsenite in the cells. The present process could provide a useful strategy for exploring biomarkers that might help in assessing the known and unknown risks of any natural and artificial toxic reagents.