[Show abstract][Hide abstract] ABSTRACT: Background:
Exposure to inorganic and organic arsenic compounds is a major public health problem that affects hundreds of millions of people worldwide. Exposure to arsenic is associated with cancer and non-cancer effects in nearly every organ in the body, and evidence is mounting for health effects at lower levels of arsenic exposure than previously thought. Building from a tremendous knowledge base with more than 1,000 scientific papers published annually with "arsenic" in the title, the question becomes, what questions would best drive future research directions?
The objective is to discuss emerging issues in arsenic research and identify data gaps across disciplines.
The National Institutes of Health's National Institute of Environmental Health Sciences Superfund Research Program convened a workshop to identify emerging issues and research needs to address the multi-faceted challenges related to arsenic and environmental health. This review summarizes information captured during the workshop.
More information about aggregate exposure to arsenic is needed, including the amount and forms of arsenic found in foods. New strategies for mitigating arsenic exposures and related health effects range from engineered filtering systems to phytogenetics and nutritional interventions. Furthermore, integration of 'omics data with mechanistic and epidemiological data is a key step towards the goal of linking biomarkers of exposure and susceptibility to disease mechanisms and outcomes.
Promising research strategies and technologies for arsenic exposure and adverse health effect mitigation are being pursued, and future research is moving toward deeper collaborations and integration of information across disciplines to address data gaps.
Environmental Health Perspectives 11/2015; DOI:10.1289/ehp.1510209 · 7.98 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Camptothecin (CPT), a topoisomerase I poison, is an important drug for the treatment of solid tumors in the clinic. Nitric oxide (·NO), a physiological signaling molecule, is involved in many cellular functions, including cell proliferation, survival and death. We have previously shown that ·NO plays a significant role in the detoxification of etoposide (VP-16), a topoisomerase II poison in vitro and in human melanoma cells. ·NO/·NO-derived species are reported to modulate activity of several important cellular proteins. As topoisomerases contain a number of free sulfhydryl groups which may be targets of ·NO/·NO-derived species, we have investigated the roles of ·NO/·NO-derived species in the stability and activity of topo I. Here we show that ·NO/·NO-derived species induces a significant down-regulation of topoisomerase I protein via the ubiquitin/26S proteasome pathway in human colon (HT-29) and breast (MCF-7) cancer cell lines. Importantly, ·NO treatment induced a significant resistance to CPT only in MCF-7 cells. This resistance to CPT did not result from loss of topoisomerase I activity as there were no differences in topoisomerase I-induced DNA cleavage in vitro or in tumor cells, but resulted from the stabilization/induction of bcl2 protein. This up-regulation of bcl2 protein in MCF-7 cells was wtp53 dependent as pifithrine-α, a small molecule inhibitor of wtp53 function, completely reversed CPT resistance, suggesting that wtp53 and bcl2 proteins played important roles in CPT resistance. Because tumors in vivo are heterogeneous and contaminated by infiltrating macrophages, ·NO-induced down-regulation of topoisomerase I protein combined with bcl2 protein stabilization could render certain tumors highly resistant to CPT and drugs derived from it in the clinic.
PLoS ONE 11/2015; 10(11):e0141897. DOI:10.1371/journal.pone.0141897 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Macrophage-solubilized indium-containing particles (ICPs) were previously shown in vitro to be cytotoxic. In this study, we compared macrophage solubilization and cytotoxicity of indium phosphide (InP) and indium tin-oxide (ITO) with similar particle diameters (~1.5 μm) and then determined if relative differences in these in vitro parameters correlated with pulmonary toxicity in vivo. RAW 264.7 macrophages were treated with InP or ITO particles and cytotoxicity was assayed at 24 hr. Ionic indium was measured in 24 hr culture supernatants. Macrophage cytotoxicity and particle solubilization in vitro were much greater for InP compared to ITO. To correlate changes in vivo, B6C3F1 mice were treated with InP or ITO by oropharyngeal aspiration. On days 14 and 28, bronchoalveolar lavage (BAL) and pleural lavage (PL) fluids were collected and assayed for total leukocytes. Cell differentials, LDH activity and protein levels were also measured in BAL. All lavage parameters were greatly increased in mice treated with InP compared to ITO. These data suggest that macrophage solubilization and cytotoxicity of some ICPs in vitro are capable of predicting pulmonary toxicity in vivo. In addition, these differences in toxicity were observed despite the two particulate compounds containing similar amounts of indium suggesting that solubilization, not total indium content, better reflects the toxic potential of some ICPs. Soluble InCl3 was shown to be more cytotoxic than InP to macrophages and lung epithelial cells in vitro further suggesting that ionic indium is the primary cytotoxic component of InP.
Published by Oxford University Press on behalf of the Society of Toxicology 2014. This work is written by US Government employees and is in the public domain in the US.
[Show abstract][Hide abstract] ABSTRACT: Background:
It has been proposed that cadmium (Cd) is an environmental "metalloestrogen" and that its action is mediated via the estrogen receptor (ER). Cd mimics the effects of estrogen in the rat uterus, and blood Cd concentrations positively correlate with ER levels in uteri of women with fibroids.
In the present study we explored whether Cd could stimulate proliferation of estrogen-responsive human uterine leiomyoma (ht-UtLM) cells and uterine smooth muscle cells (ht-UtSMCs) through classical interactions with ERα and ERβ, or by nongenomic mechanisms.
We used estrogen response element (ERE) reporters, phosphorylated receptor tyrosine kinase arrays, Western blot analysis, estrogen binding, and cell proliferation assays to evaluate the effects of Cd on ht-UtLM cells and ht-UtSMCs.
Cd stimulated growth of both cell types at lower concentrations and inhibited growth at higher concentrations (≥ 50 μM). Cd did not significantly bind to ERα or ERβ, nor did it show transactivation in both cell types transiently transfected with ERE reporter genes. However, in both cells types, Cd (0.1 μM and 10 μM) activated p44/42 MAPK (ERK1/2), and a MAPK inhibitor (PD98059) abrogated Cd-induced cell proliferation. Cd in ht-UtLM cells, but not in ht-UtSMCs, activated the growth factor receptors EGFR, HGFR, and VEGF-R1 upstream of MAPK. Additional studies in ht-UtLM cells showed that AG1478, an EGFR inhibitor, abolished Cd-induced phosphorylation of EGFR and MAPK.
Our results show that low concentrations of Cd stimulated cell proliferation in estrogen-responsive uterine cells by nongenomic activation of MAPK, but not through classical ER-mediated pathways.
Environmental Health Perspectives 10/2014; 123(4). DOI:10.1289/ehp.1408234 · 7.98 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Inorganic arsenic is a human carcinogen that likely targets the prostate. Chronic arsenic exposure malignantly transforms the RWPE-1 human prostate epithelial line to CAsE-PE cells, and a derivative normal prostate stem cell (SC) line, WPE-stem to arsenic-cancer SCs (As-CSCs). The KRAS oncogene is highly overexpressed in CAsE-PE cells and activation precedes transformation, inferring mechanistic significance. As-CSCs also highly overexpress KRAS. Thus, we hypothesize KRAS activation is key in causing and maintaining an arsenic-induced malignant phenotype, and hence, KRAS knockdown (KD) may reverse this malignant phenotype. RNA interference using shRNAmirs to obtain KRAS KD was used in CAsE-PE and As-CSC cells. Cells analyzed 2 weeks post transduction showed KRAS protein decreased to 5% of control after KD, confirming stable KD. KRAS KD decreased phosphorylated ERK, indicating inhibition of RAS/ERK signaling, a proliferation/survival pathway activated with arsenic transformation. Secreted metalloproteinase (MMP) activity was increased by arsenic-induced malignant transformation, but KRAS KD from 4 weeks on decreased secreted MMP-9 activity by 50% in As-CSCs. Colony formation, a characteristic of cancer cells, was decreased in both KRAS KD transformants. KRAS KD also decreased the invasive capacity of both cell types. KRAS KD decreased proliferation in As-CSCs, consistent with loss of rapid tumor growth. Genes predicted to impact cell proliferation (e.g. Cyclin D1, p16, and p21) changed accordingly in both KD cell types. Thus, KRAS silencing impacts aspects of arsenic-induced malignant phenotype, inducing loss of many typical cancer characteristics particularly in As-CSCs.
[Show abstract][Hide abstract] ABSTRACT: In mice, inorganic arsenic in the drinking water in the parts per million range via the dam during in utero life or with whole-life exposure is a multi-site carcinogen in the offspring. However, human arsenic exposure is typically in the parts per billion (ppb) range. Thus, we studied "whole-life" inorganic arsenic carcinogenesis in mice at levels more relevant to humans. Breeder male and female CD1 mice were exposed to 0, 50, 500 or 5,000 ppb arsenic (as sodium arsenite) in the drinking water for 3 weeks prior to breeding, during pregnancy and lactation, and after weaning (at week 3) groups of male and female offspring (initial n = 40) were exposed for up to 2 years. Tumors were assessed in these offspring. Arsenic exposure had no effect on pregnant dam weights or water consumption, litter size, offspring birthweight or weight at weaning compared to control. In male offspring mice, arsenic exposure increased (p < 0.05) bronchiolo-alveolar tumor (adenoma or carcinoma) incidence at 50-ppb group (51 %) and 500-ppb group (54 %), but not at 5,000-ppb group (28 %) compared to control (22 %). These arsenic-induced bronchiolo-alveolar tumors included increased (p < 0.05) carcinoma at 50-ppb group (27 %) compared to controls (8 %). An increase (p < 0.05) in lung adenoma (25 %) in the 50-ppb group compared to control (11 %) occurred in female offspring. Thus, in CD1 mice whole-life arsenic exposure induced lung tumors at human-relevant doses (i.e., 50 and 500 ppb).
Archive für Toxikologie 07/2014; 88(8). DOI:10.1007/s00204-014-1305-8 · 5.98 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Inorganic arsenic (iAs), a human carcinogen, potentially targets the prostate. iAs malignantly transforms the RWPE-1 human
prostate epithelial line to CAsE-PE cells, and a derivative normal stem cell (SC) line, WPE-stem, to As-Cancer SC (As-CSC)
line. MicroRNAs (miRNA) are noncoding but exert negative control on expression by degradation or translational repression
of target mRNAs. Aberrant miRNA expression is important in carcinogenesis. A miRNA array of CAsE-PE and As-CSC revealed common
altered expression in both for pathways concerning oncogenesis, miRNA biogenesis, cell signaling, proliferation, and tumor
metastasis and invasion. The KRAS oncogene is overexpressed in CAsE-PE cells but not by mutation or promoter hypomethylation, and is intensely overexpressed
in As-CSC cells. In both transformants, decreased miRNAs targeting KRAS and RAS superfamily members occurred. Reduced miR-134, miR-373, miR-155, miR-138, miR-205, miR-181d, miR-181c, and let-7 in CAsE-PE
cells correlated with increased target RAS oncogenes, RAN, RAB27A, RAB22A mRNAs, and KRAS protein. Reduced miR-143, miR-34c-5p, and miR-205 in As-CSC correlated with increased target RAN mRNA, and KRAS, NRAS, and RRAS proteins. The RAS/ERK and PI3K/PTEN/AKT pathways control cell survival, differentiation, and
proliferation, and when dysregulated promote a cancer phenotype. iAs transformation increased expression of activated ERK
kinase in both transformants and altered components of the PI3K/PTEN/AKT pathway including decreased PTEN and increases in
BCL2, BCL-XL, and VEGF in the absence of AKT activation. Thus, dysregulated miRNA expression may be linked to RAS activation in both transformants.
[Show abstract][Hide abstract] ABSTRACT: Inorganic arsenic (iAs) and its toxic methylated metabolite, methylarsonous acid (MMA(III)), both have carcinogenic potential. Prior study shows iAs-induced malignant transformation in both arsenic methylation-proficient (liver) and methylation-deficient (prostate) cells, but only methylation-proficient cells show oxidative DNA damage (ODD) during this transformation. To further define whether arsenic methylation is necessary for transformation or ODD induction, here we chronically exposed these same liver or prostate cell lines to MMA(III) (0.25-1.0 μM) and tested for acquired malignant phenotype. Various metrics of oncogenic transformation were periodically assessed along with ODD during chronic MMA(III) exposure. Methylation-deficient and methylation-proficient cells both acquired a cancer phenotype with MMA(III) exposure at about 20 weeks, based on increased matrix metalloproteinase secretion, colony formation, and invasion. In contrast, prior work showed iAs-induced transformation took longer in biomethylation-deficient cells (~30 weeks) than in biomethylation-proficient cells (~18 weeks). In the present study, MMA(III) caused similar peak ODD levels at similar concentrations and at similar exposure times (18-22 weeks) in both cell types. At the approximate peak of ODD production, both cell types showed similar alterations in arsenic and oxidative stress adaptation factors (i.e., ABCC1, ABCC2, GST-π, SOD-1). Thus, MMA(III) causes oncogenic transformation associated with ODD in methylation-deficient cells, indicating that further methylation is not required to induce ODD. Together, these results show that MMA(III) and iAs cause an acquired malignant phenotype in methylation-deficient cells, yet iAs does not induce ODD. This indicates iAs likely has both genotoxic and non-genotoxic mechanisms dictated by the target cell's ability to methylate arsenic.
Archives of Toxicology 10/2013; 88(2). DOI:10.1007/s00204-013-1141-2 · 5.98 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Accumulating data suggest arsenic may be an endocrine disruptor and tentatively linked to breast cancer by some studies. Therefore, we tested the effects of chronic inorganic arsenic exposure on the normal estrogen receptor (ER)-negative breast epithelial cell line, MCF-10A. Cells were chronically exposed to a low-level arsenite (500 nM) for up to 24 weeks. Markers of cancer cell phenotype and the expression of critical genes relevant to breast cancer or stem cells (SCs) were examined. After 24 weeks, chronic arsenic-exposed breast epithelial (CABE) cells showed increases in secreted MMP activity, colony formation, invasion, and proliferation rate, indicating an acquired cancer cell phenotype. These CABE cells presented with basal-like breast cancer characteristics, including ER-α, HER-2, and progesterone receptor negativity, and overexpression of K5 and p63. Putative CD44(+)/CD24(-/low) breast SCs were increased to 80 % over control in CABE cells. CABE cells also formed multilayer cell mounds, indicative of loss of contact inhibition. These mounds showed high levels of K5 and p63, indicating the potential presence of cancer stem cells (CSCs). Epithelial-to-mesenchymal transition occurred during arsenic exposure. Overexpression of aromatase, a key rate-limiting enzyme in estrogen synthesis, occurred with arsenic starting early on in exposure. Levels of 17β-estradiol increased in CABE cells and their conditioned medium. The aromatase inhibitor letrozole abolished arsenic-induced increases in 17β-estradiol production and reversed cancer cell phenotype. Thus, chronic arsenic exposure drives human breast epithelia into a cancer cell phenotype with an apparent overabundance of putative CSCs. Arsenic appears to transform breast epithelia through overexpression of aromatase, thereby activating oncogenic processes independent of ER.
Archives of Toxicology 09/2013; 88(2). DOI:10.1007/s00204-013-1131-4 · 5.98 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Genome-wide disruption of the epigenetic code is a hallmark of malignancy that encompasses many distinct, highly interactive modifications. Delineating the aberrant epigenome produced during toxicant-mediated malignant transformation will help identify the underlying epigenetic drivers of environmental toxicant-induced carcinogenesis. Gene promoter DNA methylation and gene expression profiling of arsenite-transformed prostate epithelial cells showed a negative correlation between gene expression changes and DNA methylation changes; however, less than 10% of the genes with increased promoter methylation were downregulated. Studies described herein confirm that a majority of the DNA hypermethylation events occur at H3K27me3 marked genes that were already transcriptionally repressed. In contrast to aberrant DNA methylation targeting H3K27me3 pre-marked silent genes, we found that actively expressed C2H2 zinc finger genes (ZNFs) marked with H3K9me3 on their 3' ends, were the favored targets of DNA methylation linked gene silencing. DNA methylation coupled, H3K9me3 mediated gene silencing of ZNF genes was widespread, occurring at individual ZNF genes on multiple chromosomes and across ZNF gene family clusters. At ZNF gene promoters, H3K9me3 and DNA hypermethylation replaced H3K4me3, resulting in a widespread downregulation of ZNF gene expression, which accounted for 8% of all the downregulated genes in the arsenical-transformed cells. In summary, these studies associate toxicant exposure with widespread silencing of ZNF genes by DNA hypermethylation-linked H3K9me3 spreading, further implicating epigenetic dysfunction as a driver of toxicant associated carcinogenesis.
Epigenetics: official journal of the DNA Methylation Society 08/2013; 8(10). DOI:10.4161/epi.25926 · 4.78 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Indium-containing particles (ICPs) are used extensively in the microelectronics industry. Pulmonary toxicity is observed after inhalation exposure to ICPs; however, the mechanism(s) of pathogenesis is unclear. ICPs are insoluble at physiological pH and are initially engulfed by alveolar macrophages (and likely airway epithelial cells). We hypothesized that uptake of ICPs by macrophages followed by phagolysosomal acidification results in the solubilization of ICPs into cytotoxic indium ions. To address this, we characterized the in vitro cytotoxicity of indium phosphide (InP) or indium tin-oxide (ITO) particles with macrophages (RAW cells) and lung-derived epithelial (LA-4) cells at 24 hours using metabolic (MTT) and membrane integrity (LDH) assays. InP and ITO were readily phagocytosed by RAW and LA-4 cells; however, the particles were much more cytotoxic to RAW cells and cytotoxicity was dose-dependent. Treatment of RAW cells with cytochalasin D blocked particle phagocytosis and reduced cytotoxicity. Treatment of RAW cells with bafilomycin A1, a specific inhibitor of phagolysosomal acidification, also reduced cytotoxicity but did not block particle uptake. Based on direct indium measurements, the concentration of ionic indium was increased in culture medium from RAW but not LA-4 cells following 24 hour treatment with particles. Ionic indium derived from RAW cells was significantly reduced by treatment with cytochalasin D. These data implicate macrophage uptake and solubilization of InP and ITO via phagolysosomal acidification as requisite for particle-induced cytotoxicity and the release of indium ions. This may apply to other ICPs and strongly supports the notion that ICPs require solubilization in order to be toxic.
[Show abstract][Hide abstract] ABSTRACT: Cadmium is a known human lung carcinogen. Here, we attempt to develop an in vitro model of cadmium-induced human lung carcinogenesis by chronically exposing the peripheral lung epithelia cell line, HPL-1D, to a low level of cadmium. Cells were chronically exposed to 5 μM cadmium, a noncytotoxic level, and monitored for acquired cancer characteristics. By 20 weeks of continuous cadmium exposure, these chronic cadmium treated lung (CCT-LC) cells showed marked increases in secreted MMP-2 activity (3.5-fold), invasion (3.4-fold), and colony formation in soft agar (2-fold). CCT-LC cells were hyperproliferative, grew well in serum-free media, and overexpressed cyclin D1. The CCT-LC cells also showed decreased expression of the tumor suppressor genes p16 and SLC38A3 at the protein levels. Also consistent with an acquired cancer cell phenotype, CCT-LC cells showed increased expression of the oncoproteins K-RAS and N-RAS as well as the epithelial-to-mesenchymal transition marker protein Vimentin. Metallothionein (MT) expression is increased by cadmium, and is typically overexpressed in human lung cancers. The major MT isoforms, MT-1A and MT-2A were elevated in CCT-LC cells. Oxidant adaptive response genes HO-1 and HIF-1A were also activated in CCT-LC cells. Expression of the metal transport genes ZNT-1, ZNT-5, and ZIP-8 increased in CCT-LC cells culminating in reduced cadmium accumulation, suggesting adaptation to the metal. Overall, these data suggest that exposure of human lung epithelial cells to cadmium causes acquisition of cancer cell characteristics. Furthermore, transformation occurs despite the cell's ability to adapt to chronic cadmium exposure.
[Show abstract][Hide abstract] ABSTRACT: Background: Cancer stem cells (CSCs) drive tumor initiation, progression, and metastasis. The microenvironment is critical to the fate of CSCs. We have found that a normal stem cell (NSC) line from human prostate (WPE-stem) is recruited into CSC-like cells by nearby, but noncontiguous, arsenic-transformed isogenic malignant epithelial cells (MECs).
Objective: It is unknown whether this recruitment of NSCs into CSCs by noncontact co-culture is specific to arsenic-transformed MECs. Thus, we used co-culture to examine the effects of neighboring noncontiguous cadmium-transformed MECs (Cd-MECs) and N-methyl-N-nitrosourea–transformed MECs (MNU-MECs) on NSCs.
Results: After 2 weeks of noncontact Cd-MEC co-culture, NSCs showed elevated metalloproteinase-9 (MMP-9) and MMP-2 secretion, increased invasiveness, increased colony formation, decreased PTEN expression, and formation of aggressive, highly branched duct-like structures from single cells in Matrigel, all characteristics typical of cancer cells. These oncogenic characteristics did not occur in NSCs co-cultured with MNU-MECs. The NSCs co-cultured with Cd-MECs retained self-renewal capacity, as evidenced by multiple passages (> 3) of structures formed in Matrigel. Cd-MEC–co-cultured NSCs also showed molecular (increased VIM, SNAIL1, and TWIST1 expression; decreased E-CAD expression) and morphologic evidence of epithelial-to-mesenchymal transition typical for conversion to CSCs. Dysregulated expression of SC-renewal genes, including ABCG2, OCT-4, and WNT-3, also occurred in NSCs during oncogenic transformation induced by noncontact co-culture with Cd-MECs.
Conclusions: These data indicate that Cd-MECs can recruit nearby NSCs into a CSC-like phenotype, but MNU-MECs do not. Thus, the recruitment of NSCs into CSCs by nearby MECs is dependent on the carcinogen originally used to malignantly transform the MECs.
Environmental Health Perspectives 05/2013; 121(8). DOI:10.1289/ehp.1306714 · 7.98 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Today's developed world faces a major public health challenge in the rise in the obese population and the increased incidence in fatty liver disease. There is a strong association among diet induced obesity, fatty liver disease and development of nonalcoholic steatohepatitis but the environmental link to disease progression remains unclear. Here we demonstrate that in obesity, early steatohepatitic lesions induced by the water disinfection byproduct bromodichloromethane are mediated by increased oxidative stress and leptin which act in synchrony to potentiate disease progression. Low acute exposure to bromodichloromethane (BDCM), in diet-induced obesity produced oxidative stress as shown by increased lipid peroxidation, protein free radical and nitrotyrosine formation and elevated leptin levels. Exposed obese mice showed histopathological signs of early steatohepatitic injury and necrosis. Spontaneous knockout mice for leptin or systemic leptin receptor knockout mice had significantly decreased oxidative stress and TNF-α levels. Co-incubation of leptin and BDCM caused Kupffer cell activation as shown by increased MCP-1 release and NADPH oxidase membrane assembly, a phenomenon that was decreased in Kupffer cells isolated from leptin receptor knockout mice. In obese mice that were BDCM-exposed, livers showed a significant increase in Kupffer cell activation marker CD68 and, increased necrosis as assessed by levels of isocitrate dehydrogenase, events that were decreased in the absence of leptin or its receptor. In conclusion, our results show that exposure to the disinfection byproduct BDCM in diet-induced obesity augments steatohepatitic injury by potentiating the effects of leptin on oxidative stress, Kupffer cell activation and cell death in the liver.