Drinking water contaminated with arsenic, a human carcinogen, is a worldwide health issue. An understanding of cellular signaling events in response to arsenic exposure and rational designing of strategies to reduce arsenic damages by modulating signaling events are important to fight against arsenic-induced diseases. Previously, we reported that activation of the Nrf2-mediated cellular defense pathway confers protection against toxic effects induced by sodium arsenite [As(III)] or monomethylarsonous acid [MMA(III)]. Paradoxically, arsenic has been reported to induce the Nrf2-dependent signaling pathway. Here, we report the unique mechanism of Nrf2 induction by arsenic. Similar to tert-butylhydroquinone (tBHQ) or sulforaphane (SF), arsenic induced the Nrf2-dependent response through enhancing Nrf2 protein levels by inhibiting Nrf2 ubiquitination and degradation. However, the detailed action of arsenic in Nrf2 induction is different from that of tBHQ or SF. Arsenic markedly enhanced the interaction between Keap1 and Cul3, subunits of the E3 ubiquitin ligase for Nrf2, which led to impaired dynamic assembly/disassembly of the E3 ubiquitin ligase and thus decreased its ligase activity. Furthermore, induction of Nrf2 by arsenic is independent of the previously identified C151 residue in Keap1 that is required for Nrf2 activation by tBHQ or SF. Distinct mechanisms of Nrf2 activation by seemingly harmful and beneficial reagents provide a molecular basis to design Nrf2-activating agents for therapeutic intervention.
Arsenicals are known to induce ROS, which can lead to DNA damage, oxidative stress, and carcinogenesis. A human urothelial cell line,, UROtsa, was used to study the effects of arsenicals on the human bladder. Arsenite [As(III)] and monomethylarsonous acid [MMA(III)] induce oxidative stress in UROtsa cells after exposure to concentrations as low as 1 μM and 50 nM, respectively. Previous research has implicated ROS as signaling molecules in the MAPK signaling pathway. As(III) and MMA(III) have been shown to increase phosphorylation of key proteins in the MAPK signaling cascade downstream of ErbB2. Both Src phosphorylation (p-Src) and cyclooxygenase-2 (COX-2) are induced after exposure to 50 nM MMA(III) and 1 μM As(III). These data suggest that ROS production is a plausible mechanism for the signaling alterations seen in UROtsa cells after acute arsenical exposure. To determine importance of ROS in the MAPK cascade and its downstream induction of p-Src and COX-2, specific ROS antioxidants (both enzymatic and non-enzymatic) were used concomitantly with arsenicals. COX-2 protein and mRNA was shown to be much more influenced by altering the levels of ROS in cells, particularly after MMA(III) treatment. The antioxidant enzyme superoxide dismutase (SOD) effectively blocked both As(III)-and MMA(III)-associated COX-2 induction. The generation of ROS and subsequent altered signaling did lead to changes in protein levels of SOD, which were detected after treatment with either 1 μM As(III) or 50 nM MMA(III). These data suggest that the generation of ROS by arsenicals may be a mechanism leading to the altered cellular signaling seen after low-level arsenical exposure.
arsenite [As(III)]; monomethylarsonous acid [MMA(III)]; ROS; ROS scavengers; MAPK
Bladder cancer has been associated with chronic arsenic exposure. Monomethylarsonous acid [MMA(III)] is a metabolite of inorganic arsenic and has been shown to transform an immortalized urothelial cell line (UROtsa) at concentrations 20-fold less than arsenite. MMA(III) was used as a model arsenical to examine the mechanisms of arsenical-induced transformation of urothelium. A microarray analysis was performed to assess the transcriptional changes in UROtsa during the critical window of chronic 50 nM MMA(III) exposure that leads to transformation at three months of exposure. The analysis revealed only minor changes in gene expression at one and two months of exposure, contrasting with substantial changes observed at three months of exposure. The gene expression changes at three months were analyzed showing distinct alterations in biological processes and pathways such as a response to oxidative stress, enhanced cell proliferation, anti-apoptosis, MAPK signaling, as well as inflammation. Twelve genes selected as markers of these particular biological processes were used to validate the microarray and these genes showed a time-dependent changes at one and two months of exposure, with the most substantial changes occurring at three months of exposure. These results indicate that there is a strong association between the acquired phenotypic changes that occur with chronic MMA(III) exposure and the observed gene expression patterns that are indicative of a malignant transformation. Although the substantial changes that occur at three months of exposure may be a consequence of transformation, there are common occurrences of altered biological processes between the first two months of exposure and the third, which may be pivotal in driving transformation.
Arsenic; Monomethylarsonous Acid; Bladder Cancer; UROtsa; Gene Expression
Arsenic compounds are classified as toxicants and human carcinogens. Environmental exposure to arsenic imposes a big health issue worldwide. Arsenic elicits its toxic efforts through many mechanisms, including generation of reactive oxygen species (ROS). Nrf2 is the primary transcription factor that controls expression of a main cellular antioxidant response, which is required for neutralizing ROS and thus defending cells from exogenous insults. Previously, we demonstrated a protective role of Nrf2 against arsenic-induced toxicity using a cell culture model. In this report, we present evidence that Nrf2 protects against liver and bladder injury in response to six-weeks of arsenic exposure in a mouse model. Nrf2−/− mice displayed more severe pathological changes in the liver and bladder, compared to Nrf2+/+ mice. Furthermore, Nrf2−/− mice were more sensitive to arsenic-induced DNA hypomethylation, oxidative DNA damage, and apoptotic cell death. These results indicate a protective role of Nrf2 against arsenic toxicity in vivo. Hence, this work demonstrates the feasibility of using dietary compounds that target activation of the Nrf2 signaling pathway to alleviate arsenic-induced damage.
Arsenic is present in the environment and has become a worldwide health concern due to its toxicity and carcinogenicity. However, the specific mechanism(s) by which arsenic elicits its toxic effects has yet to be fully elucidated. The transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2) has been recognized as the master regulator of a cellular defense mechanism against toxic insults. This review highlights studies demonstrating that arsenic activates the Nrf2-Keap1 antioxidant pathway by a distinct mechanism from that of natural compounds such as sulforaphane (SF) found in broccoli sprouts or tert-butylhyrdoquinone (tBHQ), a natural antioxidant commonly used as a food preservative. Evidence also suggests that arsenic prolongs Nrf2 activation and may mimic constitutive activation of Nrf2, which has been found in several human cancers due to disruption of the Nrf2-Keap1 axis. The current literature strongly suggests that activation of Nrf2 by arsenic potentially contributes to, rather than protects against, arsenic toxicity and carcinogenicity. The mechanism(s) by which known Nrf2 activators, such as the natural chemopreventive compounds SF and lipoic acid, protect against the deleterious effects caused by arsenic will also be discussed. These findings will provide insight to further understand how arsenic promotes a prolonged Nrf2 response, which will lead to the identification of novel molecular markers and development of rational therapies for the prevention or intervention of arsenic-induced diseases. The National Institute of Environmental Health Science (NIEHS) Outstanding New Environmental Scientist (ONES) award has provided the opportunity to review the progress both in the fields of arsenic toxicology and Nrf2 biology. Much of the funding has led to (1) the novel discovery that arsenic activates the Nrf2 pathway by a mechanism different to that of other Nrf2 activators, such as sulforaphane and tert-butylhydroquinone, (2) activation of Nrf2 by chemopreventive compounds protects against arsenic toxicity and carcinogenicity both in vitro and in vivo, (3) constitutive activation of Nrf2 by disrupting Keap1-mediated negative regulation contributes to cancer and chemoresistance, (4) p62-mediated sequestration of Keap1 activates the Nrf2 pathway, and (5) arsenic-mediated Nrf2 activation may be through a p62-dependent mechanism. All of these findings have been published and are discussed in this review. This award has laid the foundation for my laboratory to further investigate the molecular mechanism(s) that regulate the Nrf2 pathway and how it may play an integral role in arsenic toxicity. Moreover, understanding the biology behind arsenic toxicity and carcinogenicity will help in the discovery of potential strategies to prevent or control arsenic-mediated adverse effects.
Nrf2; Arsenic; Keap1; Oxidative stress; p62; Autophagy; Chemoprevention
The enzymatic methylation of inorganic As (iAs) is catalyzed by As(+3 oxidation state)-methyltransferase (AS3MT). AS3MT is expressed in rat liver and in human hepatocytes. However, AS3MT is not expressed in UROtsa, human urothelial cells that do not methylate iAs. Thus, UROtsa cells are an ideal null background in which the role of iAs methylation in modulation of toxic and cancer-promoting effects of this metalloid can be examined. A retroviral gene delivery system was used in this study to create a clonal UROtsa cell line (UROtsa/F35) that expresses rat AS3MT. Here, we characterize the metabolism and cytotoxicity of arsenite (iAsIII) and methylated trivalent arsenicals in parental cells and clonal cells expressing AS3MT. In contrast to parental cells, UROtsa/F35 cells effectively methylated iAsIII, yielding methylarsenic (MAs) and dimethylarsenic (DMAs) containing either AsIII or AsV. When exposed to MAsIII, UROtsa/F35 cells produced DMAsIII and DMAsV. MAsIII and DMAsIII were more cytotoxic than iAsIII in UROtsa and UROtsa/F35 cells. The greater cytotoxicity of MAsIII or DMAsIII than of iAsIII was associated with greater cellular uptake and retention of each methylated trivalent arsenical. Notably, UROtsa/F35 cells were more sensitive than parental cells to the cytotoxic effects of iAsIII but were more resistant to cytotoxicity of MAsIII. The increased sensitivity of UROtsa/F35 cells to iAsIII was associated with inhibition of DMAs production and intracellular accumulation of MAs. The resistance of UROtsa/F35 cells to moderate concentrations of MAsIII was linked to its rapid conversion to DMAs and efflux of DMAs. However, concentrations of MAsIII that inhibited DMAs production by UROtsa/F35 cells were equally toxic for parental and clonal cell lines. Thus, the production and accumulation of MAsIII is a key factor contributing to the toxicity of acute iAs exposures in methylating cells.
As; Methyltransferase; AS3MT; Metabolism; Toxicity; Urinary bladder; Human
Groundwater contaminated with arsenic imposes a big challenge to human health worldwide. Using natural compounds to subvert the detrimental effects of arsenic represents an attractive strategy. The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) is a critical regulator of the cellular antioxidant response and xenobiotic metabolism. Recently, activation of the Nrf2 signaling pathway has been reported to confer protection against arsenic-induced toxicity in a cell culture model.
The goal of the present work was to identify a potent Nrf2 activator from plants as a chemopreventive compound and to demonstrate the efficacy of the compound in battling arsenic-induced toxicity.
Oridonin activated the Nrf2 signaling pathway at a low subtoxic dose and was able to stabilize Nrf2 by blocking Nrf2 ubiquitination and degradation, leading to accumulation of the Nrf2 protein and activation of the Nrf2-dependent cytoprotective response. Pretreatment of UROtsa cells with 1.4 μM oridonin significantly enhanced the cellular redox capacity, reduced formation of reactive oxygen species (ROS), and improved cell survival after arsenic challenge.
We identified oridonin as representing a novel class of Nrf2 activators and illustrated the mechanism by which the Nrf2 pathway is activated. Furthermore, we demonstrated the feasibility of using natural compounds targeting Nrf2 as a therapeutic approach to protect humans from various environmental insults that may occur daily.
antioxidant responsive element; antitumor; ARE; arsenic; chemoprevention; diterpenoid; Keap1; Nrf2; oridonin; oxidative stress; rubescensin
Arsenic is a human bladder carcinogen. Arsenic is methylated to both monomethyl and dimethyl metabolites which have been detected in human urine. The trivalent methylated arsenicals are more toxic than inorganic arsenic. It is unknown if these trivalent methylated metabolites can directly cause malignant transformation in human cells. The goal of this study is determine if monomethylarsonous acid (MMAIII) can induce malignant transformation in a human bladder urothelial cell line. To address this goal, a non-tumorigenic human urothelial cell line (UROtsa) was continuously exposed to 0.05 μM MMAIII for 52 weeks. Hyperproliferation was the first phenotypic change observed in exposed UROtsa (URO-MSC). After 12 weeks of exposure, doubling time had decreased from 42 h in unexposed control cells to 27 h in URO-MSC. Hyperproliferation continued to be a quality possessed by the URO-MSC cells after both 24 and 52 weeks of exposure to MMAIII, which had a 40–50% reduction in doubling time. Throughout the 52-week exposure, URO-MSC cells retained an epithelial morphology with subtle morphological differences from control cells. 24 weeks of MMAIII exposure was required to induce anchorage-independent growth as detected by colony formation in soft agar, a characteristic not found in UROtsa cells. To further substantiate that malignant transformation had occurred, URO-MSC cells were tested after 24 and 52 weeks of exposure to MMAIII for the ability to form tumors in SCID mice. Enhanced tumorigenicity in SCID mouse xenografts was observed after 52 weeks of treatment with MMAIII. These observations are the first demonstration of MMAIII-induced malignant transformation in a human bladder urothelial cell line and provide important evidence that MMAIII may be carcinogenic in human tissues.
Arsenic methylation; Monomethylarsonous acid; Bladder cancer; Cell culture; UROtsa
The loading of macrophages with oxidized low density lipoprotein (LDL) is a key part of the initiation and progression of atherosclerosis. Oxidized LDL contains a wide ranging set of toxic species, yet the molecular events that allow macrophages to withstand loading with these toxic species are not completely characterized. The transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a master regulator of the cellular stress response. However, the specific parts of the Nrf2-dependent stress response are diverse, with both tissue- and treatment-dependent components. The goal of these experiments was to develop and use a quantitative proteomic approach to characterize the Nrf2-dependent response in macrophages to oxidized LDL. Cultured mouse macrophages, the J774 macrophage-like cell line, were treated with a combination of oxidized LDL, the Nrf2-stabilizing reagent tert- butylhydroquinone (tBHQ), and/or Nrf2 siRNA. Protein expression was determined using a quantitative proteomics assay based on selected reaction monitoring. The assay was multiplexed to monitor a set of 28 antioxidant and stress response proteins, 6 housekeeping proteins, and 1 non-endogenous standard protein. The results have two components. The first component is the validation of the multiplexed, quantitative proteomics assay. The assay is shown to be fundamentally quantitative, precise, and accurate. The second component is the characterization of the Nrf2-mediated stress response. Treatment with tBHQ and/or Nrf2 siRNA gave statistically significant changes in the expression of a subset of 11 proteins. Treatment with oxidized LDL gave statistically significant increases in the expression of 7 of those 11 proteins plus one additional protein. All of the oxLDL-mediated increases were attenuated by Nrf2 siRNA. These results reveal a specific, multifaceted response of the foam cells to the incoming toxic oxidized LDL.
Inorganic arsenic (iAs) is an environmental toxicant and human carcinogen. The enzymatic methylation of iAs that is catalyzed by arsenic (+3 oxidation state)-methyltransferase (AS3MT) generates reactive methylated intermediates that contribute to the toxic and carcinogenic effects of iAs. We have shown that clonal human urothelial cells (UROtsa/F35) that express rat AS3MT and methylate iAs are more susceptible to acute toxicity of arsenite (iAsIII) than parental UROtsa cells that do not express AS3MT and do not methylate iAs. The current work examines transcriptional changes associated with AS3MT expression and identifies specific categories of genes expressed in UROtsa and UROtsa/F35 cells in response to a 24-h exposure to 1 or 50 μM iAsIII. Here, the expression of 21,073 genes was assessed using Agilent Human 1A(V2) arrays. Venn analysis showed marked concentration-dependent differences between gene expression patterns in UROtsa and UROTsa/F35 cells exposed to iAsIII. Among 134 genes altered by exposure to subtoxic 1 μM iAsIII, only 14 were shared by both cell lines. Exposure to cytotoxic 50 μM iAsIII uniquely altered 1389 genes in UROtsa/F35 and 649 genes in UROtsa cells; 5033 altered genes were associated with the chemical alone. In UROtsa, but not UROtsa/F35 cells exposure to 1 μM iAsIII altered expression of genes associated with cell adhesion. In contrast, expression of genes involved in cell cycle regulation was significantly altered in UROtsa/F35 cells at this exposure level. At 50 μM iAsIII, pathways regulating cell cycle, cell death, transcription, and metabolism were affected in both cell lines. However, only Urotsa/F35 cells showed numerous G-protein and kinase pathway alterations as well as alterations in pathways involved in cell growth and differentiation. These data link the AS3MT-catalyzed methylation of iAs to specific genomic responses in human cells exposed to iAsIII. Further analysis of these responses will help to characterize the role of AS3MT-catalyzed methylation in modulation of iAsIII toxicity.
arsenite; AS3MT; human urothelial cells; transcriptional profiles
The stress response is one mechanism that the bladder urothelium could potentially employ to protect itself from cellular damage after exposure to arsenic and, in so doing, influence the shape of the dose-response curve at low concentrations of exposure to this environmental pollutant. In the present study, we used the cultured human urothelial cell line UROtsa, a model of human urothelium, to determine the expression of heat shock proteins hsp 27, hsp 60, hsc 70, and hsp 70 after acute and extended exposure of the cells to lethal and sublethal levels of sodium arsenite (NaAsO2). Acute exposure was modeled by exposing confluent cultures of UROtsa cells to 100 micro M NaAsO2 for 4 hr followed by a 48-hr recovery period. Extended exposure was modeled by exposing confluent UROtsa cells to 1, 4, and 8 micro M NaAsO2 for 16 days, with the highest concentration producing cell death by 4 days of exposure. The expression of hsp 27, hsp 60, hsc 70, and hsp 70 mRNA and protein was determined by reverse-transcription polymerase chain reaction and Western analysis. Cell viability was determined by the MTT [(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. The results demonstrated that the expression of hsp 27, hsp 60, and hsc 70 mRNA and protein were not consistently increased by either acute or extended exposure to NaAsO2. In contrast, hsp 70 expression was induced by NaAsO2 after both acute and extended exposure. The degree and duration of the induction of the hsp 70 protein in the extended time course of exposure to NaAsO2 correlated directly with UROtsa cell cytotoxicity. The substantial level of basal expression of hsp 27, hsp 60, and hsc 70 shown previously in human bladder urothelium, coupled with the inducible expression of hsp 70, could provide the human urothelium with a mechanism to withstand and recover from a low level of arsenite exposure.
Chronic exposure to drinking water contaminated with inorganic arsenic (iAs) is associated with an increased risk of urinary bladder (UB) cancers in humans. The exact role of specific iAs metabolite(s) in As-mediated carcinogenesis remains largely unknown. Experimental evidence suggests that trivalent arsenicals, namely arsenite (iAsIII) and two of its metabolites, monomethylarsonous acid (MMAIII) and dimethylarsinous acid (DMAIII), are possible proximate UB carcinogens. Here, we used a transcriptomics approach to examine perturbed molecular pathways in a human urothelial cell line (UROtsa) after short-term exposure to iAsIII, MMAIII and DMAIII. Molecular pathways containing genes that encode proteins implicated in UB cancer development were perturbed by both MMAIII and DMAIII. These pathways included those of the extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase (ERK 1/2 MAPK) and nuclear factor kappa beta (NF-κB). Together, these results may inform the current understanding of effects in the UB induced by acute As exposure and the relationship of these effects with As-mediated carcinogenesis.
Arsenite; gene expression; microarray; urinary bladder cancer; extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase; nuclear factor kappa beta; inflammation; monomethylarsonous acid; dimethylarsinous acid; UROtsa; MMAIII; DMAIII
Exposure to arsenic is associated with an increased risk of lung disease. Novel strategies are needed to reduce the adverse health effects associated with arsenic exposure in the lung. Nrf2, a transcription factor that mediates an adaptive cellular defense response, is effective in detoxifying environmental insults and prevents a broad spectrum of diseases induced by environmental exposure to harmful substances. In this report, we tested whether Nrf2 activation protects mice from arsenic-induced toxicity. We used an in vivo arsenic inhalation model that is highly relevant to low environmental human exposure to arsenic-containing dusts. Two-week exposure to arsenic-containing dust resulted in pathological alterations, oxidative DNA damage, and mild apoptotic cell death in the lung; all of which were blocked by sulforaphane (SF) in an Nrf2-dependent manner. Mechanistically, SF-mediated activation of Nrf2 alleviated inflammatory responses by modulating cytokine production. This study provides strong evidence that dietary intervention targeting Nrf2 activation is a feasible approach to reduce adverse health effects associated with arsenic exposure.
Nrf2; Keap1; Arsenic; Antioxidant response
Previous studies have shown that ethanol exposure causes apoptosis in cranial neural crest cells (NCCs), an ethanol-sensitive cell population implicated in Fetal Alcohol Spectrum Disorders (FASD). Additionally, induction of endogenous antioxidants through activation of nuclear factor-erythroid 2-related factor 2 (Nrf2) has been shown to prevent oxidative stress and apoptosis in ethanol-exposed mouse embryos. The objective of this study was to test whether tert-butylhydroquinone (tBHQ), an Nrf2 inducer, can protect NCCs against ethanol-induced apoptosis. Ethanol exposure was shown to cause a moderate increase in the protein expression of Nrf2 and its downstream antioxidants in the NCCs. Treatment of NCCs with tBHQ alone significantly increased the protein expression of Nrf2 and its downstream antioxidants and also significantly increased the activities of the antioxidant enzymes. In NCCs exposed to ethanol, the tBHQ-mediated antioxidant response prevented oxidative stress and apoptosis. These results clearly demonstrate that activation of Nrf2 signaling confers protection against ethanol-induced apoptosis in NCCs.
Neural crest cells; Nrf2; ethanol; antioxidant; apoptosis
Arsenic is a known human bladder carcinogen; however, the mechanisms underlying arsenical-induced bladder carcinogenesis are not understood. Previous research has demonstrated that exposure of a nontumorigenic human urothelial cell line, UROtsa, to 50nM monomethylarsonous acid (MMAIII) for 52 weeks resulted in malignant transformation. To focus research on the early mechanistic events leading to MMAIII-induced malignancy, the goal of this research was to resolve the critical period in which continuous MMAIII exposure (50nM) induces the irreversible malignant transformation of UROtsa cells. An increased growth rate of UROtsa cells results after 12 weeks of MMAIII exposure. Anchorage-independent growth occurred after 12 weeks with a continued increase in colony formation when 12-week exposed cells were cultured for an additional 12 or 24 weeks without MMAIII exposure. UROtsa cells as early as 12 weeks MMAIII exposure were tumorigenic in severe combined immunodeficiency mice with tumorigenicity increasing when 12-week exposed cells were cultured for an additional 12 or 24 weeks in the absence of MMAIII exposure. To assess potential underlying mechanisms associated with the early changes that occur during MMAIII-induced malignancy, DNA methylation was assessed in known target gene promoter regions. Although DNA methylation remains relatively unchanged after 12 weeks of exposure, aberrant DNA methylation begins to emerge after an additional 12 weeks in culture and continues to increase through 24 weeks in culture without MMAIII exposure, coincident with the progression of a tumorigenic phenotype. Overall, these data demonstrate that 50nM MMAIII is capable of causing irreversible malignant transformation in UROtsa cells after 12 weeks of exposure. Having resolved an earlier timeline in which MMAIII-induced malignant transformation occurs in UROtsa cells will allow for mechanistic studies focused on the critical biological changes taking place within these cells prior to 12 weeks of exposure, providing further evidence about potential mechanisms of MMAIII-induced carcinogenesis.
arsenic; monomethylarsonous acid; bladder cancer; UROtsa; epigenetic
Exposure of human bladder urothelial cells (UROtsa) to 50 nM of the arsenic metabolite, monomethylarsonous acid (MMAIII), for 12 weeks results in irreversible malignant transformation. The ability of continuous, low-level MMAIII exposure to cause an increase in genotoxic potential by inhibiting repair processes necessary to maintain genomic stability is unknown. Following genomic insult within cellular systems poly(ADP-ribose) polymerase-1 (PARP-1), a zinc finger protein, is rapidly activated and recruited to sites of DNA strand breaks. When UROtsa cells are continuously exposed to 50 nM MMAIII, PARP-1 activity does not increase despite the increase in MMAIII-induced DNA single-strand breaks through 12 weeks of exposure. When UROtsa cells are removed from continuous MMAIII exposure (2 weeks), PARP-1 activity increases coinciding with a subsequent decrease in DNA damage levels. Paradoxically, PARP-1 mRNA expression and protein levels are elevated in the presence of continuous MMAIII indicating a possible mechanism to compensate for the inhibition of PARP-1 activity in the presence of MMAIII. The zinc finger domains of PARP-1 contain vicinal sulfhydryl groups which may act as a potential site for MMAIII to bind, displace zinc ion, and render PARP-1 inactive. Mass spectrometry analysis demonstrates the ability of MMAIII to bind a synthetic peptide representing the zinc-finger domain of PARP-1, and displace zinc from the peptide in a dose-dependent manner. In the presence of continuous MMAIII exposure, continuous 4-week zinc supplementation restored PARP-1 activity levels and reduced the genotoxicity associated with MMAIII. Zinc supplementation did not produce an overall increase in PARP-1 protein levels, decrease the levels of MMAIII-induced reactive oxygen species, or alter Cu-Zn superoxide dismutase levels. Overall, these results present two potential interdependent mechanisms in which MMAIII may increase the susceptibility of UROtsa cells to genotoxic insult and/or malignant transformation: elevated levels of MMAIII-induced DNA damage through the production of reactive oxygen species, and the direct MMAIII-induced inhibition of PARP-1.
Arsenic; Monomethylarsonous acid; Bladder cancer; UROtsa; Genotoxicity; Poly(ADP-ribose) polymerase-1
The association between chronic human exposure to arsenicals and bladder cancer development is well recognized; however, the underlying molecular mechanisms have not been fully determined. We propose that inflammatory responses can play a pathogenic role in arsenic-related bladder carcinogenesis. In previous studies, it was demonstrated that chronic exposure to 50 nM monomethylarsenous acid [MMA(III)] leads to malignant transformation of an immortalized model of urothelial cells (UROtsa), with only 3 mo of exposure necessary to trigger the transformation-related changes. In the three-month window of exposure, the cells over-expressed pro-inflammatory cytokines (IL-1β, IL-6 and IL-8), consistent with the sustained activation of NFKβ and AP1/c-jun, ERK2, and STAT3. IL-8 was over-expressed within hours after exposure to MMA(III), and sustained over-expression was observed during chronic exposure. In this study, we profiled IL-8 expression in UROtsa cells exposed to 50 nM MMA(III) for 1 to 5 mo. IL-8 expression was increased mainly in cells after 3 mo MMA(III) exposure, and its production was also found increased in tumors derived from these cells after heterotransplantation in SCID mice. UROtsa cells do express both receptors, CXCR1 and CXCR2, suggesting that autocrine cell activation could be important in cell transformation. Supporting this observation and consistent with IL-8 over-expression, CXCR1 internalization was significantly increased after three months of exposure to MMA(III). The expression of MMP-9, cyclin D1, bcl-2, and VGEF was significantly increased in cells exposed to MMA(III) for 3 mo, but these mitogen-activated kinases were significantly decreased after IL-8 gene silencing, together with a decrease in cell proliferation rate and in anchorage-independent colony formation. These results suggest a relevant role of IL-8 in MMA(III)-induced UROtsa cell transformation.
bladder cancer; arsenic; monomethylarsonous acid; IL-8; CXCL8; CXCR1; inflammation
Inorganic arsenic is an environmental carcinogen that may act through multiple mechanisms including formation of methylated derivatives in vivo. Sodium arsenite (up to 5.0 μM) renders arsenic methylation–competent TRL1215 rat liver epithelial cells tumorigenic in nude mice at 18 weeks of exposure and arsenic methylation-deficient RWPE-1 human prostate epithelial cells tumorigenic at 30 weeks of exposure. We assessed the role of arsenic biomethylation in oxidative DNA damage (ODD) using a recently developed immuno-spin trapping method.
Immuno-spin trapping was used to measure ODD after chronic exposure of cultured TRL1215 vs RWPE-1 cells, or of methylation-competent UROtsa/F35 vs methylation-deficient UROtsa human urothelial cells, to sodium arsenite. Secreted matrix metalloproteinase (MMP)-2 and -9 activity, as analyzed by zymography, cellular invasiveness by using a transwell assay, and colony formation by using soft agar assay were compared in cells exposed to arsenite with and without selenite, an arsenic biomethylation inhibitor, to assess the role of ODD in the transition to an in vitro cancer phenotype.
Exposure of methylation-competent TRL1215 cells to up to 1.0 μM sodium arsenite was followed by a substantial increase in ODD at 5–18 weeks (eg, at 16 weeks with 1.0 μM arsenite, 1138% of control, 95% confidence interval [CI] = 797% to 1481%), whereas exposure of methylation-deficient RWPE-1 cells to up to 5.0 μM arsenite did not increase ODD for a 30-week period. Inhibition of arsenic biomethylation with sodium selenite abolished arsenic-induced ODD and invasiveness, colony formation, and MMP-2 and -9 hypersecretion in TRL1215 cells. Arsenic induced ODD in methylation-competent UROtsa/F35 cells (eg, at 16 weeks, with 1.0 μM arsenite 225% of control, 95% CI = 188% to 262%) but not in arsenic methylation-deficient UROtsa cells, and ODD levels corresponded to the levels of increased invasiveness, colony formation, and hypersecretion of active MMP-2 and -9 seen after transformation to an in vitro cancer phenotype.
Arsenic biomethylation appears to be obligatory for arsenic-induced ODD and appears linked in some cells with the accelerated transition to an in vitro cancer phenotype.
Type II endometrial cancer, which mainly presents as serous and clear cell types, has proved to be the most malignant and recurrent carcinoma among various female genital malignancies. The transcription factor, Nrf2, was first described as having chemopreventive activity. Activation of the Nrf2-mediated cellular defense response protects cells against the toxic and carcinogenic effects of environmental insults by upregulating an array of genes that detoxify reactive oxygen species (ROS) and restore cellular redox homeostasis. However, the cancer-promoting role of Nrf2 has recently been revealed. Nrf2 is constitutively upregulated in several types of human cancer tissues and cancer cell lines. Furthermore, inhibition of Nrf2 expression sensitizes cancer cells to chemotherapeutic drugs. In this study, the constitutive level of Nrf2 was compared in different types of human endometrial tumors. It was found that Nrf2 was highly expressed in endometrial serous carcinoma (ESC), whereas complex hyperplasia (CH) and endometrial endometrioid carcinoma (EEC) had no or marginal expression of Nrf2. Likewise, the ESC derived SPEC-2 cell line had a higher level of Nrf2 expression and was more resistant to the toxic effects of cisplatin and paclitaxel than that of the Ishikawa cell line, which was generated from EEC. Silencing of Nrf2 rendered SPEC-2 cells more susceptible to chemotherapeutic drugs while it had a limited effect on Ishikawa cells. Inhibition of Nrf2 expression by overexpressing Keap1 sensitized SPEC-2 cells or SPEC-2-derived xenografts to chemotherapeutic treatments using both cell culture and SCID mouse models. Collectively, we provide a molecular basis for the use of Nrf2 inhibitors to increase the efficacy of chemotherapeutic drugs and to combat chemoresistance, the biggest obstacle in chemotherapy.
Nrf2; chemoresistance; and endometrial cancer
Peroxiredoxin 6 (Prdx6) is a unique antioxidant enzyme that can reduce phospholipid and other hydroperoxides. A549 cells, a human lung-derived cell line, express both Prdx6 and Nrf2, a transcription factor that binds to antioxidant response elements (AREs) and promotes expression of antioxidant genes. Treatment of A549 cells with 500μM H2O2 increased Prdx6 mRNA levels 2.5 fold while treatment with 400μM H2O2 or 200μM tert-butylhydroquinone (tBHQ) triggered a corresponding 2.5 fold increase in reporter gene activity in A549 cells transfected with the pSEAP2-Basic vector (BD, Bioscience), containing 1524 nucleotides of the human Prdx6 promoter region. Deletion of a consensus ARE sequence present between positions 357 and 349 before the start of transcription led to a striking decrease in both basal and H2O2 or tBHQ-induced activation in A549 cells and H2O2-induced activation in primary rat alveolar type II cells. Co-transfection with Nrf2 stimulated the Prdx6 promoter in an ARE-dependent manner, while it was negatively regulated by Nrf3. siRNA targeting Nrf2 down-regulated reporter gene expression whereas siRNA targeting the Nrf2 repressor, Keap1, up-regulated it. Binding of Nrf2 to the ARE sequence in chromatin was confirmed by PCR following chromatin immunoprecipitation. These data demonstrate that the ARE within the Prdx6 promoter is a key regulator of basal transcription of the Prdx6 gene and of its inducibility under conditions of oxidative stress.
Prdx6 promoter; Transcription factor; Nrf2; Keap1; lung cells; reporter gene assay
Traumatic brain injury (TBI) can induce intestinal inflammatory response and mucosal injury. Antioxidant transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) has been shown in our previous studies to prevent oxidative stress and inflammatory response in gut after TBI. The objective of this study was to test whether tert-butylhydroquinone (tBHQ), an Nrf2 inducer, can protect against TBI-induced intestinal inflammatory response and mucosal injury in mice. Adult male ICR mice were randomly divided into three groups: (1) sham + vehicle group, (2) TBI + vehicle group, and (3) TBI + tBHQ group (n = 12 per group). Closed head injury was adopted using Hall's weight-dropping method. Intestinal mucosa apoptosis and inflammatory-related factors, such as nuclear factor kappa B (NF-κB), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6) and intercellular adhesion molecule-1 (ICAM-1), were investigated at 24 h after TBI. As a result, we found that oral treatment with 1% tBHQ prior to TBI for one week markedly decreased NF-κB activation, inflammatory cytokines production, and ICAM-1 expression in the gut. Administration of tBHQ also significantly attenuated TBI-induced intestinal mucosal apoptosis. The results of the present study suggest that tBHQ administration could suppress the intestinal inflammation and reduce the mucosal damage following TBI.
Arsenic, a human carcinogen that is associated with an increased risk of bladder cancer, is commonly found in drinking water. An important mechanism by which arsenic is thought to be carcinogenic is through the induction of epigenetic changes that lead to aberrant gene expression. Previously, we reported that the SAS2 gene is required for optimal growth of yeast in the presence of arsenite (AsIII). Yeast Sas2p is orthologous to human MYST1, a histone 4 lysine 16 (H4K16) acetyltransferase. Here, we show that H4K16 acetylation is necessary for the resistance of yeast to AsIII through the modulation of chromatin. The role of MYST1 and H4K16 acetylation in arsenic toxicity and carcinogenesis was further explored in human bladder epithelial cells. The expression of MYST1 was knocked down in UROtsa cells, a model of bladder epithelium that has been used to study arsenic-induced carcinogenesis. Silencing of MYST1 reduced acetylation of H4K16, and induced sensitivity to AsIII and to its more toxic metabolite monomethylarsonous acid (MMAIII) at doses relevant to high environmental human exposures. In addition, both AsIII and MMAIII treatments decreased global H4K16 acetylation levels in a dose- and time-dependent manner. This indicates that acetylated H4K16 is required for resistance to arsenic and that a reduction in its levels as a consequence of arsenic exposure may contribute to toxicity in UROtsa cells. Based on these findings, we propose a novel role for the MYST1 gene in human sensitivity to arsenic.
Arsenic; H4K16; histone acetylation; epigenetics; bladder cancer; yeast
UROtsa is an authentic, immortalized human urothelial cell line that is used to study the effects of metals and other toxic substances, mostly in the context of bladder cancer carcinogenesis. Unusual properties on the molecular level of a provided UROtsa cell line stock prompted us to verify its identity.
UROtsa cell line stocks from different sources were tested on several molecular levels and compared with other cell lines. MicroRNA and mRNA expression was determined by Real-Time PCR. Chromosome numbers were checked and PCR of different regions of the large T-antigen was performed. DNA methylation of RARB, PGR, RASSF1, CDH1, FHIT, ESR1, C1QTNF6, PTGS2, SOCS3, MGMT, and LINE1 was analyzed by pyrosequencing and compared with results from the cell lines RT4, T24, HeLa, BEAS-2B, and HepG2. Finally, short tandem repeat (STR) profiling was applied.
All tested UROtsa cell line stocks lacked large T-antigen. STR analysis unequivocally identified our main UROtsa stock as the bladder cancer cell line T24, which was different from two authentic UROtsa stocks that served as controls. Analysis of DNA methylation patterns and RNA expression confirmed their differences. Methylation pattern and mRNA expression of the contaminating T24 cell line showed moderate changes even after long-term culture of up to 56 weeks, whereas miRNAs and chromosome numbers varied markedly.
It is important to check the identity of cell lines, especially those that are not distributed by major cell banks. However, for some cell lines STR profiles are not available. Therefore, new cell lines should either be submitted to cell banks or at least their STR profile determined and published as part of their initial characterization. Our results should help to improve the identification of UROtsa and other cells on different molecular levels and provide information on the use of urothelial cells for long-term experiments.
Transcriptional regulation of the antioxidant response element (ARE) by Nrf2 is important for the cellular adaptive response to toxic insults. New data show that primary skin-derived fibroblasts from the long-lived Snell dwarf mutant mouse, previously shown to be resistant to many toxic stresses, have elevated levels of Nrf2 and of multiple Nrf2-sensitive ARE genes. Dwarf-derived fibroblasts exhibit many of the traits associated with enhanced activity of Nrf2/ARE, including higher levels of glutathione and resistance to plasma membrane lipid peroxidation. Treatment of control cells with arsenite, an inducer of Nrf2 activity, increases their resistance to paraquat, hydrogen peroxide, cadmium, and UV light, rendering these cells as stress resistant as untreated cells from dwarf mice. Furthermore, mRNA levels for some Nrf2-sensitive genes are elevated in at least some tissues of Snell dwarf mice, suggesting that the phenotypes observed in culture may be mirrored in vivo. Augmented activity of Nrf2 and ARE-responsive genes may coordinate many of the stress resistance traits seen in cells from these long-lived mutant mice.
Liver regeneration can be impaired by permanent oxidative stress and activation of nuclear factor erythroid 2–related factor 2 (Nrf2), known to regulate the cellular antioxidant response, and has been shown to improve the process of liver regeneration. A variety of factors regulate hepatic tissue regeneration, among them augmenter of liver regeneration (ALR), attained great attention as being survival factors for the liver with proproliferative and antiapoptotic properties. Here we determined the Nrf2/antioxidant response element (ARE) regulated expression of ALR and show ALR as a target gene of Nrf2 in vitro and in vivo. The ALR promoter comprises an ARE binding site and, therefore, ALR expression can be induced by ARE-activator tertiary butylhydroquinone (tBHQ) in hepatoma cells and primary human hepatocytes (PHH). Promoter activity and expression of ALR were enhanced after cotransfection of Nrf2 compared with control and dominant negative mutant of Nrf2. Performing partial hepatectomy in livers from Nrf2+/+ mice compared with Nrf2−/− knock-out (KO) mice, we found increased expression of ALR in addition to known antioxidant ARE-regulated genes. Furthermore, we observed increased ALR expression in hepatitis B virus (HBV) compared with hepatitis C virus (HCV) positive hepatoma cells and PHH. Recently, it was demonstrated that HBV infection activates Nrf2 and, now, we add results showing increased ALR expression in liver samples from patients infected with HBV. ALR is regulated by Nrf2, acts as a liver regeneration and antioxidative protein and, therefore, links oxidative stress to hepatic regeneration to ensure survival of damaged cells.