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1.  Epigenetic Mediated Transcriptional Activation of WNT5A Participates in Arsenical-Associated Malignant Transformation 
Arsenic is a human carcinogen with exposure associated with cancer of the lung, skin, and bladder. Many potential mechanisms have been implicated as playing a role in the process of arsenical-induced malignancy including the perturbation of signaling pathways and aberrant epigenetic regulation. We initiated studies to examine the role of a member of the non-canonical WNT signaling pathway, WNT5A, in UROtsa cells and arsenite [URO-ASSC] and monomethylarsonous acid [URO-MSC] malignantly transformed variants. We present data herein that suggests that WNT5A is transcriptionally activated during arsenical-induced malignant transformation. This WNT5A transcriptional activation is correlated with the enrichment of permissive histone modifications and the reduction of repressive modifications in the WNT5A promoter region. The epigenetic activation of WNT5A expression and acetylation of its promoter remain after the removal of the arsenical, consistent with the maintenance of an anchorage independent growth phenotype in these cells. Additionally, treatment with epigenetic modifying drugs supports a functional role for these epigenetic marks in controlling gene expression. Reduction of WNT5A using lentiviral shRNA greatly attenuated the ability of these cells to grow in an anchorage independent fashion. Extension of our model into human bladder cancer cell lines indicate that each of the cell lines examined also express WNT5A. Taken together, these data suggest that the epigenetic remodeling of the WNT5A promoter is correlated with its transcriptional activation and this upregulation likely participates in arsenical-induced malignant transformation.
PMCID: PMC4438681  PMID: 19061910
WNT5A; Arsenic; Epigenetic; UROtsa
2.  Building a co-created citizen science program with gardeners neighboring a superfund site: The Gardenroots case study 
A research project that is only expert-driven may ignore the role of local knowledge in research, give low priority to the development of a comprehensive communication strategy to engage the community, and may not deliver the results of the study to the community in an effective way.
To demonstrate how a research program can respond to a community research need, establish a community-academic partnership, and build a co-created citizen science program.
A place-based, community-driven project was designed where academics and community members maintained a reciprocal dialogue, and together, we: 1) defined the question for study, 2) gathered information, 3) developed hypotheses, 3) designed data collection methodologies, 4) collected environmental samples (soil, irrigation water, and vegetables), 5) interpreted data, 6) disseminated results and translated results into action, and 7) discussed results and asked new questions.
The co-created environmental research project produced new data and addressed an additional exposure route (consumption of vegetables grown in soils with elevated arsenic levels). Public participation in scientific research improved environmental health assessment, information transfer, and risk communication efforts. Furthermore, incorporating the community in the scientific process produced both individual learning outcomes and community-level outcomes.
This approach illustrates the benefits of a community-academic co-created citizen-science program in addressing the complex problems that arise in communities neighboring a contaminated site. Such a project can increase the community's involvement in risk communication and decision-making, which ultimately has the potential to help mitigate exposure and thereby reduce associated risk.
PMCID: PMC4420190  PMID: 25954473
Arsenic; capacity building; citizen science; community-academic partnership; contaminated sites; gardening; public participation in scientific research; risk communication; environmental exposure assessment
3.  Estimated Exposure to Arsenic in Breastfed and Formula-Fed Infants in a United States Cohort 
Environmental Health Perspectives  2015;123(5):500-506.
Background: Previous studies indicate that concentrations of arsenic in breast milk are relatively low even in areas with high drinking-water arsenic. However, it is uncertain whether breastfeeding leads to reduced infant exposure to arsenic in regions with lower arsenic concentrations.
Objective: We estimated the relative contributions of breast milk and formula to arsenic exposure during early infancy in a U.S. population.
Methods: We measured arsenic in home tap water (n = 874), urine from 6-week-old infants (n = 72), and breast milk from mothers (n = 9) enrolled in the New Hampshire Birth Cohort Study (NHBCS) using inductively coupled plasma mass spectrometry. Using data from a 3-day food diary, we compared urinary arsenic across infant feeding types and developed predictive exposure models to estimate daily arsenic intake from breast milk and formula.
Results: Urinary arsenic concentrations were generally low (median, 0.17 μg/L; maximum, 2.9 μg/L) but 7.5 times higher for infants fed exclusively with formula than for infants fed exclusively with breast milk (β = 2.02; 95% CI: 1.21, 2.83; p < 0.0001, adjusted for specific gravity). Similarly, the median estimated daily arsenic intake by NHBCS infants was 5.5 times higher for formula-fed infants (0.22 μg/kg/day) than for breastfed infants (0.04 μg/kg/day). Given median arsenic concentrations measured in NHBCS tap water and previously published for formula powder, formula powder was estimated to account for ~ 70% of median exposure among formula-fed NHBCS infants.
Conclusions: Our findings suggest that breastfed infants have lower arsenic exposure than formula-fed infants, and that both formula powder and drinking water can be sources of exposure for U.S. infants.
Citation: Carignan CC, Cottingham KL, Jackson BP, Farzan SF, Gandolfi AJ, Punshon T, Folt CL, Karagas MR. 2015. Estimated exposure to arsenic in breastfed and formula-fed infants in a United States cohort. Environ Health Perspect 123:500–506;
PMCID: PMC4421773  PMID: 25707031
4.  Differential Binding of Monomethylarsonous Acid Compared to Arsenite and Arsenic Trioxide with Zinc Finger Peptides and Proteins 
Chemical Research in Toxicology  2014;27(4):690-698.
Arsenic is an environmental toxin that enhances the carcinogenic effect of DNA-damaging agents, such as ultraviolet radiation and benzo[a]pyrene. Interaction with zinc finger proteins has been shown to be an important molecular mechanism for arsenic toxicity and cocarcinogenesis. Arsenicals such as arsenite, arsenic trioxide (ATO), and monomethylarsonous acid (MMA(III)) have been reported to interact with cysteine residues of zinc finger domains, but little is known about potential differences in their selectivity of interaction. Herein we analyzed the interaction of arsenite, MMA(III), and ATO with C2H2, C3H1, and C4 configurations of zinc fingers using UV–vis, cobalt, fluorescence, and mass spectrometry. We observed that arsenite and ATO both selectively bound to C3H1 and C4 zinc fingers, while MMA(III) interacted with all three configurations of zinc finger peptides. Structurally and functionally, arsenite and ATO caused conformational changes and zinc loss on C3H1 and C4 zinc finger peptide and protein, respectively, whereas MMA(III) changed conformation and displaced zinc on all three types of zinc fingers. The differential selectivity was also demonstrated in zinc finger proteins isolated from cells treated with these arsenicals. Our results show that trivalent inorganic arsenic compounds, arsenite and ATO, have the same selectivity and behavior when interacting with zinc finger proteins, while methylation removes the selectivity. These findings provide insights on the molecular mechanisms underlying the differential effects of inorganic versus methylated arsenicals, as well as the role of in vivo arsenic methylation in arsenic toxicity and carcinogenesis.
PMCID: PMC3998772  PMID: 24611629
5.  Expression Of Selected Pathway-Marker Genes In Human Urothelial Cells Exposed Chronically To A Non-Cytotoxic Concentration Of Monomethylarsonous Acid 
Toxicology reports  2014;1:421-434.
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 previous microarray analysis revealed only minor changes in gene expression at one and two months of chronic exposure to MMA(III), contrasting with substantial changes observed at three months of exposure. To address the lack of information between two and three months of exposure (the critical period of transformation), the expression of select pathway marker genes was measured by PCR array analysis on a weekly basis. Cell proliferation rate, anchorage-independent growth, and tumorigenicity in SCID mice were also assessed to determine the early, persistent phenotypic changes and their association with the changes in expression of these selected marker genes. A very similar pattern of alterations in these genes was observed when compared to the microarray results, and suggested that early perturbations in cell signaling cascades, immunological pathways, cytokine expression, and MAPK pathway are particularly important in driving malignant transformation. These results showed a strong association between the acquired phenotypic changes that occurred as early as one to two months of chronic MMA(III) exposure, and the observed gene expression pattern that is indicative of the earliest stages in carcinogenesis.
PMCID: PMC4144464  PMID: 25177542
6.  In utero arsenic exposure and infant infection in a United States cohort: A prospective study 
Environmental research  2013;126:10.1016/j.envres.2013.05.001.
Arsenic (As), a ubiquitous environmental toxicant, has recently been linked to disrupted immune function and enhanced infection susceptibility in highly exposed populations. Drinking water As levels above the EPA maximum contaminant level occur in our US study area and are a particular health concern for pregnant women and infants. As part of the New Hampshire Birth Cohort Study, we investigated whether in utero exposure to As affects risk of infant infections. We prospectively obtained information on four-month-old infants (n=214) using a parental telephone survey on infant’s infections and symptoms, including respiratory infections, diarrhea and specific illnesses, as well as the duration and severity of infections. Using logistic regression and Poisson models, we evaluated the association between maternal urinary As during pregnancy and infection risks adjusted for potentially confounding factors. Maternal urinary As concentrations were related to total number of infections requiring a physician visit (Relative Risk (RR) per one-fold increase in As in urine =1.5; 95% confidence interval (CI)=1.0, 2.1) or prescription medication (RR=1.6; 95% CI =1.1, 2.4), as well as lower respiratory infections treated with prescription medication (RR=3.3; 95% CI =1.2, 9.0). Associations were observed with respiratory symptoms (RR=4.0; 95% CI =1.0, 15.8), upper respiratory infections (RR=1.6; 95% CI =1.0, 2.5), and colds treated with prescription medication (RR=2.3; 95% CI =1.0, 5.2). Our results provide initial evidence that in utero As exposure may be related to infant infection and infection severity and provide insight into the early life impacts of fetal As exposure.
PMCID: PMC3808159  PMID: 23769261
Arsenic; infant respiratory infection; prenatal exposure; pregnancy; US cohort
7.  Differential Susceptibility of Human Peripheral Blood T Cells to Suppression by Environmental Levels of Sodium Arsenite and Monomethylarsonous Acid 
PLoS ONE  2014;9(10):e109192.
Human exposure to arsenic in drinking water is known to contribute to many different health outcomes such as cancer, diabetes, and cardiopulmonary disease. Several epidemiological studies suggest that T cell function is also altered by drinking water arsenic exposure. However, it is unclear how individual responses differ to various levels of exposure to arsenic. Our laboratory has recently identified differential responses of human peripheral blood mononuclear cell (HPMBC) T cells as measured by polyclonal T cell activation by mitogens during sodium arsenite exposure. T cells from certain healthy individuals exposed to various concentrations (1–100 nM) of arsenite in vitro showed a dose-dependent suppression at these extremely low concentrations (∼0.1–10 ppb) of arsenite, whereas other individuals were not suppressed at low concentrations. In a series of more than 30 normal donors, two individuals were found to be sensitive to low concentration (10 nM equivalent ∼1 ppb drinking water exposure) to sodium arsenite-induced inhibition of T cell proliferation produced by phytohemagglutinin (PHA) and anti-CD3/anti-CD28. In an arsenite-susceptible individual, arsenite suppressed the activation of Th1 (Tbet) cells, and decreased the percentage of cells in the double positive Th17 (RORγt) and Treg (FoxP3) population. While the majority of normal blood donors tested were not susceptible to inhibition of proliferation at the 1–100 nM concentrations of As+3, it was found that all donors were sensitive to suppression by 100 nM monomethylarsonous acid (MMA+3), a key metabolite of arsenite. Thus, our studies demonstrate for the first time that low ppb-equivalent concentrations of As+3 are immunosuppressive to HPBMC T cells in some individuals, but that most donor HPBMC are sensitive to suppression by MMA+3 at environmentally relevant exposure levels.
PMCID: PMC4182801  PMID: 25271956
8.  Exposure Assessment of Organochlorine Pesticides, Arsenic, and Lead in Children From the Major Agricultural Areas in Sonora, Mexico 
There is a lack of information of exposure to organochlorine pesticides (OCP) and some metals, such as lead (Pb) and arsenic (As), both of which were used as arsenicals pesticides, in children living in the major agricultural areas of Mexico. The objective of this study was to assess the exposure of children to different OCP, As, and Pb in the Yaqui and Mayo valleys of Sonora to generate population baseline levels of these toxins. A cross-sectional study was undertaken in 165 children (age 6–12 years old) from 10 communities from both valleys during 2009. Blood samples were analyzed for OCP and Pb and first morning void urine for inorganic As (InAs). All of the blood samples had detectable levels of dichlorodiphenyltrichloroethylene (p,p′-DDE) ranging from 0.25 to 10.3 μg/L. However lindane, dichlorodiphenyltrichloroethane (p,p′-DDT), aldrin, and endosulfan were detected in far less of the population (36.4, 23.6, 9.1, and 3 %, respectively). Methoxychlor and endrin were not found in any sample. The average value of Pb in this population was 3.2 μg Pb/dL (range 0.17–9.0) with 8.5 % of the samples having levels <5.0 μg Pb/dL. Urinary As levels ranged from 5.4 to 199 μg As/L with an average value of 31.0 μg As/L. Levels > 50 μg/L were observed in 12.7 % of the samples. Our results show that is important to start a risk-reduction program to decrease exposure to these toxins in Mexican communities. In addition, the results can be used to establish the baseline levels of exposure to these toxins in this agricultural region and may be used as a reference point for regulatory agencies.
PMCID: PMC4023507  PMID: 23254566
9.  Arsenite Selectively Inhibits Mouse Bone Marrow Lymphoid Progenitor Cell Development In Vivo and In Vitro and Suppresses Humoral Immunity In Vivo 
PLoS ONE  2014;9(4):e93920.
It is known that exposure to As+3 via drinking water causes a disruption of the immune system and significantly compromises the immune response to infection. The purpose of these studies was to assess the effects of As+3 on bone marrow progenitor cell colony formation and the humoral immune response to a T-dependent antigen response (TDAR) in vivo. In a 30 day drinking water study, mice were exposed to 19, 75, or 300 ppb As+3. There was a decrease in bone marrow cell recovery, but not spleen cell recovery at 300 ppb As+3. In the bone marrow, As+3 altered neither the expression of CD34+ and CD38+ cells, markers of early hematopoietic stem cells, nor CD45−/CD105+, markers of mesenchymal stem cells. Spleen cell surface marker CD45 expression on B cells (CD19+), T cells (CD3+), T helper cells (CD4+) and cytotoxic T cells (CD8+), natural killer (NK+), and macrophages (Mac 1+) were not altered by the 30 day in vivo As+3 exposure. Functional assays of CFU-B colony formation showed significant selective suppression (p<0.05) by 300 ppb As+3 exposure, whereas CFU-GM formation was not altered. The TDAR of the spleen cells was significantly suppressed at 75 and 300 ppb As+3. In vitro studies of the bone marrow revealed a selective suppression of CFU-B by 50 nM As+3 in the absence of apparent cytotoxicity. Monomethylarsonous acid (MMA+3) demonstrated a dose-dependent and selective suppression of CFU-B beginning at 5 nM (p<0.05). MMA+3 suppressed CFU-GM formation at 500 nM, a concentration that proved to be nonspecifically cytotoxic. As+5 did not suppress CFU-B and/or CFU-GM in vitro at concentrations up to 500 nM. Collectively, these results demonstrate that As+3 and likely its metabolite (MMA+3) target lymphoid progenitor cells in mouse bone marrow and mature B and T cell activity in the spleen.
PMCID: PMC3979857  PMID: 24714590
10.  Hedgehog Signaling Regulates Bladder Cancer Growth And Tumorigenicity 
Cancer research  2012;72(17):10.1158/0008-5472.CAN-11-4123.
The role of HEDGEHOG (HH) signaling in bladder cancer remains controversial. The gene encoding the HH receptor and negative regulator PATCHED1 (PTCH1) resides on a region of chromosome 9q, one copy of which is frequently lost in bladder cancer. Inconsistent with PTCH1 functioning as a classic tumor suppressor gene, loss-of-function mutations in the remaining copy of PTCH1 are not commonly found. Here, we provide direct evidence for a critical role of HH signaling in bladder carcinogenesis. We show that transformed human urothelial cells and many urothelial carcinoma (UC) cell lines exhibit constitutive HH signaling, which is required for their growth and tumorigenic properties. Surprisingly, rather than originating from loss of PTCH1, the constitutive HH activity observed in UC cell lines was HH ligand-dependent. Consistent with this finding, increased levels of HH and the HH target gene product GLI1 were found in resected human primary bladder tumors. Furthermore, based on the difference in intrinsic HH dependence of UC cell lines, a gene expression signature was identified that correlated with bladder cancer progression. Our findings therefore indicate that therapeutic targeting of the HH signaling pathway may be beneficial in the clinical management of bladder cancer.
PMCID: PMC3809830  PMID: 22815529
Hedgehog signaling; bladder cancer; urothelial carcinoma; HEDGEHOG; GLI
11.  A Population-based Case–Control Study of Urinary Arsenic Species and Squamous Cell Carcinoma in New Hampshire, USA 
Environmental Health Perspectives  2013;121(10):1154-1160.
Background: Chronic high arsenic exposure is associated with squamous cell carcinoma (SCC) of the skin, and inorganic arsenic (iAs) metabolites may play an important role in this association. However, little is known about the carcinogenicity of arsenic at levels commonly observed in the United States.
Objective: We estimated associations between total urinary arsenic and arsenic species and SCC in a U.S. population.
Methods: We conducted a population-based case–control SCC study (470 cases, 447 controls) in a U.S. region with moderate arsenic exposure through private well water and diet. We measured urinary iAs, monomethylarsonic acid (MMA), and dimethylarsinic acid (DMA), and summed these arsenic species (ΣAs). Because seafood contains arsenolipids and arsenosugars that metabolize into DMA through alternate pathways, participants who reported seafood consumption within 2 days before urine collection were excluded from the analyses.
Results: In adjusted logistic regression analyses (323 cases, 319 controls), the SCC odds ratio (OR) was 1.37 for each ln-transformed microgram per liter increase in ln-transformed ΣAs concentration [ln(ΣAs)] (95% CI: 1.04, 1.80). Urinary ln(MMA) and ln(DMA) also were positively associated with SCC (OR = 1.34; 95% CI: 1.04, 1.71 and OR = 1.34; 95% CI: 1.03, 1.74, respectively). A similar trend was observed for ln(iAs) (OR = 1.20; 95% CI: 0.97, 1.49). Percent iAs, MMA, and DMA were not associated with SCC.
Conclusions: These results suggest that arsenic exposure at levels common in the United States relates to SCC and that arsenic metabolism ability does not modify the association.
Citation: Gilbert-Diamond D, Li Z, Perry AE, Spencer SK, Gandolfi AJ, Karagas MR. 2013. A population-based case–control study of urinary arsenic species and squamous cell carcinoma in New Hampshire, USA. Environ Health Perspect 121:1154–1160;
PMCID: PMC3801199  PMID: 23872349
Toxicology  2011;291(1-3):102-112.
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.
PMCID: PMC3245769  PMID: 22108045
Arsenic; Monomethylarsonous Acid; Bladder Cancer; UROtsa; Gene Expression
13.  Arsenic Exposure and Calpain-10 Polymorphisms Impair the Function of Pancreatic Beta-Cells in Humans: A Pilot Study of Risk Factors for T2DM 
PLoS ONE  2013;8(1):e51642.
The incidence of type 2 diabetes mellitus (T2DM) is increasing worldwide and diverse environmental and genetic risk factors are well recognized. Single nucleotide polymorphisms (SNPs) in the calpain-10 gene (CAPN-10), which encodes a protein involved in the secretion and action of insulin, and chronic exposure to inorganic arsenic (iAs) through drinking water have been independently associated with an increase in the risk for T2DM. In the present work we evaluated if CAPN-10 SNPs and iAs exposure jointly contribute to the outcome of T2DM. Insulin secretion (beta-cell function) and insulin sensitivity were evaluated indirectly through validated indexes (HOMA2) in subjects with and without T2DM who have been exposed to a gradient of iAs in their drinking water in northern Mexico. The results were analyzed taking into account the presence of the risk factor SNPs SNP-43 and -44 in CAPN-10. Subjects with T2DM had significantly lower beta-cell function and insulin sensitivity. An inverse association was found between beta-cell function and iAs exposure, the association being more pronounced in subjects with T2DM. Subjects without T2DM who were carriers of the at-risk genotype SNP-43 or -44, also had significantly lower beta-cell function. The association of SNP-43 with beta-cell function was dependent on iAs exposure, age, gender and BMI, whereas the association with SNP-44 was independent of all of these factors. Chronic exposure to iAs seems to be a risk factor for T2DM in humans through the reduction of beta-cell function, with an enhanced effect seen in the presence of the at-risk genotype of SNP-43 in CAPN-10. Carriers of CAPN-10 SNP-44 have also shown reduced beta-cell function.
PMCID: PMC3551951  PMID: 23349674
14.  Adjusting for Urinary Creatinine Overestimates Arsenic Concentrations in Diabetics 
Cardiorenal Medicine  2011;2(1):26-32.
Arsenic (As) is linked to insulin resistance in animal studies, but the effect of low-level As exposure on the prevalence of diabetes in humans is uncertain. An optimal method to report inorganic As in humans has not been established. Measurements of As in spot urine are usually adjusted to creatinine (Cr). However, urinary Cr is an independent variable in diabetes. Our aims are to optimize reporting of urinary As in the setting of diabetes and insulin resistance.
Urinary inorganic As was measured in 24-hour or first-void spot urine from diabetic (n = 31) and non-diabetic (n = 12) subjects and normalized to Cr or specific gravity (SG). The relation of normalized urinary inorganic As to glycemia and surrogate measures of insulin resistance was investigated. Blood pressure, waist circumference, and glycated hemoglobin were also assessed. Homeostasis model assessment was used to determine insulin resistance.
A strong correlation was found between spot urinary As adjusted to Cr (R2 = 0.82) or SG (R2 = 0.61) to 24-hour urinary As (p < 0.001), while non-adjusted urinary As did not correlate well (R2 = 0.03, p = 0.46). Adjusting for Cr revealed significant differences in total 24-hour urinary As when comparing diabetic to normal subjects. In contrast, no differences were found when As was adjusted to SG using either 24-hour or spot urine. Moreover, adjusted urinary spot or 24-hour As measures did not correlate with measures of glycemia or insulin resistance. Conclusions: Urinary Cr is an independent variable in diabetes, therefore adjusting spot As for SG is preferred.
PMCID: PMC3318926  PMID: 22493600
Arsenic; Creatinine; Diabetes; Insulin resistance; Specific gravity
15.  Monomethylarsonous Acid Produces Irreversible Events Resulting in Malignant Transformation of a Human Bladder Cell Line Following 12 Weeks of Low-Level Exposure 
Toxicological Sciences  2010;116(1):44-57.
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.
PMCID: PMC2886861  PMID: 20375083
arsenic; monomethylarsonous acid; bladder cancer; UROtsa; epigenetic
16.  Arsenicals Produce Stable Progressive Changes in DNA Methylation Patterns that are Linked to Malignant Transformation of Immortalized Urothelial Cells 
Toxicology and applied pharmacology  2009;241(2):221-229.
Aberrant DNA methylation participates in carcinogenesis and is a molecular hallmark of a tumor cell. Tumor cells generally exhibit a redistribution of DNA methylation resulting in global hypomethylation with regional hypermethylation; however, the speed in which these changes emerge has not been fully elucidated and may depend on the temporal location of the cell in the path from normal, finite lifespan to malignant transformation. We used a model of arsenical-induced malignant transformation of immortalized human urothelial cells and DNA methylation microarrays to examine the extent and temporal nature of changes in DNA methylation that occur during the transition from immortal to malignantly transformed. Our data presented herein suggest that during arsenical-induced malignant transformation, aberrant DNA methylation occurs non-randomly, progresses gradually at hundreds of gene promoters, alters expression of the associated gene, and these changes are coincident with the acquisition of malignant properties, such as anchorage independent growth and tumor formation in immunocompromised mice. The DNA methylation changes appear stable, since malignantly transformed cells removed from the transforming arsenical exhibited no reversion in DNA methylation levels, associated gene expression, or malignant phenotype. These data suggest that arsenicals act as epimutagens and directly link their ability to induce malignant transformation to their actions on the epigenome.
PMCID: PMC2783766  PMID: 19716837
DNA Methylation; Epigenetic; Arsenic; Histone acetylation; Bladder
17.  Monomethylarsonous acid induces transformation of human bladder cells 
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.
PMCID: PMC2851136  PMID: 16806342
Arsenic methylation; Monomethylarsonous acid; Bladder cancer; Cell culture; UROtsa
18.  Identification of a stem cell candidate in the normal human prostate gland 
European journal of cell biology  2005;84(2-3):341-354.
Stem cells of the human prostate gland have not yet been identified utilizing a structural biomarker. We have discovered a new prostatic epithelial cell phenotype-expressing cytokeratin 6a (Ck6a+ cells). The Ck6a+ cells are present within a specialized niche in the basal cell compartment in fetal, juvenile and adult prostate tissue, and within the stem cell-enriched urogenital sinus. In adult normal prostate tissue, the average abundance of Ck6a+ cells was 4.9%. With proliferative stimuli in the prostate organ culture model, in which the epithelial–stromal interaction was maintained, a remarkable increase of Ck 6a expression was noticed to up to 64.9%. The difference in cytokeratin 6a expression between the normal adult prostate and the prostate organ culture model was statistically significant (p<0.0001). Within the prostate organ culture model the increase of cytokeratin 6a-expressing cells significantly correlated with increased proliferation index (r = 0.7616; p = 0.0467) The Ck6a+ cells were capable of differentiation as indicated by their expression of luminal cell markers such as ZO-1 and prostate specific antigen (PSA). Our data indicate that Ck6a+ cells represent a prostatic epithelial stem cell candidate possessing high potential for proliferation and differentiation. Since the development of benign prostatic hyperplasia and prostate carcinogenesis are disorders of proliferation and differentiation, the Ck6a+ cells may represent a major element in the development of these diseases.
PMCID: PMC2730953  PMID: 15819412
Prostate; Glandular epithelium; Stem cells; Cytokeratin 6
19.  Epigenetic remodeling during arsenical-induced malignant transformation 
Carcinogenesis  2008;29(8):1500-1508.
Humans are exposed to arsenicals through many routes with the most common being in drinking water. Exposure to arsenic has been associated with an increase in the incidence of cancer of the skin, lung and bladder. Although the relationship between exposure and carcinogenesis is well documented, the mechanisms by which arsenic participates in tumorigenesis are not fully elucidated. We evaluated the potential epigenetic component of arsenical action by assessing the histone acetylation state of 13 000 human gene promoters in a cell line model of arsenical-mediated malignant transformation. We show changes in histone H3 acetylation occur during arsenical-induced malignant transformation that are linked to the expression state of the associated gene. DNA hypermethylation was detected in hypoacetylated promoters in the select cases analyzed. These epigenetic changes occurred frequently in the same promoters whether the selection was performed with arsenite [As(III)] or with monomethylarsonous acid, suggesting that these promoters were targeted in a non-random fashion, and probably occur in regions important in arsenical-induced malignant transformation. Taken together, these data suggest that arsenicals may participate in tumorigenesis by altering the epigenetic terrain of select genes.
PMCID: PMC2516486  PMID: 18448484
20.  Nrf2 protects human bladder urothelial cells from arsenite and monomethylarsonous acid toxicity 
Toxicology and applied pharmacology  2007;225(2):206-213.
Arsenic is widely spread in our living environment and imposes a big challenge on human health worldwide. Arsenic damages biological systems through multiple mechanisms including the generation of reactive oxygen species. The transcription factor Nrf2 regulates the cellular antioxidant response that protects cells from various insults. In this study, the protective role of Nrf2 in arsenic toxicity was investigated in a human bladder urothelial cell line, UROtsa. Using an UROtsa cell line stably infected with Nrf2-siRNA, we clearly demonstrate that compromised Nrf2 expression sensitized the cells to As(III)- and MMA(III)-induced toxicity. On the other hand, the activation of the Nrf2 pathway by tert-butylhydroquinone (tBHQ) and sulforaphane (SF), the known Nrf2-inducers, rendered UROtsa cells more resistant to As(III)- and MMA(III). Furthermore, the wild type mouse embryo fibroblast (WT-MEF) cells were protected from As(III)- and MMA(III)-induced toxicity following Nrf2 activation by tBHQ or SF whereas neither tBHQ nor SF conferred protection in the Nrf2−/−-MEF cells, demonstrating that tBHQ- or SF-mediated protection against As(III)- and MMA(III)-induced toxicity depends on Nrf2 activation. These results, obtained by both loss of function and gain of function analyses, clearly demonstrate the protective role of Nrf2 in arsenic-induced toxicity. The current work lays the groundwork for using Nrf2 activators for therapeutic and dietary interventions against adverse effects of arsenic.
PMCID: PMC2610476  PMID: 17765279
Nrf2; Keap1; arsenic; arsenite; MMA(III); UROtsa
21.  Developmental and Genetic Modulation of Arsenic Biotransformation: A Gene by Environment Interaction? 
Toxicology and applied pharmacology  2006;222(3):381-387.
The complexity of arsenic toxicology has confounded the identification of specific pathways of disease causation. One focal point of arsenic research is aimed at fully characterizing arsenic biotransformation in humans, a process that appears to be quite variable, producing a mixture of several arsenic species with greatly differing toxic potencies. In an effort to characterize genetic determinants of variability in arsenic biotransformation, a genetic association study of 135 subjects in western Sonora, Mexico was performed by testing 23 polymorphic sites in three arsenic biotransformation candidate genes. One gene, arsenic 3 methyltransferase (AS3MT), was strongly associated with the ratio of urinary dimethylarsinic acid to monomethylarsonic acid (D/M) in children (7-11 years) but not in adults (18-79 years). Subsequent analyses revealed that the high D/M values associated with variant AS3MT alleles were primarily due to lower levels of monomethylarsonic acid as percent of total urinary arsenic (%MMA5). In light of several reports of arsenic-induced disease being associated with relatively high %MMA5 levels, these findings raise the possibility that variant AS3MT individuals may suffer less risk from arsenic exposure than non-variant individuals. These analyses also provide evidence that in this population, regardless of AS3MT variant status, children tend to have lower %MMA5 values than adults, suggesting that the global developmental regulation of arsenic biotransformation may interact with genetic variants in metabolic genes to result in novel genetic effects such as those in this report.
PMCID: PMC2040165  PMID: 17306849
Arsenic; Genetic Association; Biotransformation; AS3MT; MMA; DMA
22.  Developmentally Restricted Genetic Determinants of Human Arsenic Metabolism: Association between Urinary Methylated Arsenic and CYT19 Polymorphisms in Children 
Environmental Health Perspectives  2005;113(6):775-781.
We report the results of a screen for genetic association with urinary arsenic metabolite levels in three arsenic metabolism candidate genes, PNP, GSTO, and CYT19, in 135 arsenic-exposed subjects from the Yaqui Valley in Sonora, Mexico, who were exposed to drinking water concentrations ranging from 5.5 to 43.3 ppb. We chose 23 polymorphic sites to test in the arsenic-exposed population. Initial phenotypes evaluated included the ratio of urinary inorganic arsenic(III) to inorganic arsenic(V) and the ratio of urinary dimethylarsenic(V) to monomethylarsenic(V) (D:M). In the initial association screening, three polymorphic sites in the CYT19 gene were significantly associated with D:M ratios in the total population. Subsequent analysis of this association revealed that the association signal for the entire population was actually caused by an extremely strong association in only the children (7–11 years of age) between CYT19 genotype and D:M levels. With children removed from the analysis, no significant genetic association was observed in adults (18–79 years). The existence of a strong, developmentally regulated genetic association between CYT19 and arsenic metabolism carries import for both arsenic pharmacogenetics and arsenic toxicology, as well as for public health and governmental regulatory officials.
PMCID: PMC1257606  PMID: 15929903
arsenic metabolism; CYT19; genetic association; GSTO; pharmacogenetics; PNP; polymorphism; SNP

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