Single Cell Analysis of Human Colonoids Exposed to Uranium-Bearing Dust.

BACKGROUND: Uranium exposure remains an important environmental legacy and physiological health concern, with hundreds of abandoned uranium mines located in the Southwestern United States largely impacting underserved indigenous communities. The negative effects of heavy metals on barrier permeability and inhibition of intestinal epithelial healing have been described; however, transcriptomic changes within the intestinal epithelial cells and impacts on lineage differentiation are largely unknown. OBJECTIVES: Herein, we sought to determine the molecular and cellular changes that occur in the colon in response to uranium bearing dust (UBD) exposure. METHODS: Human colonoids from three biologically distinct donors were acutely exposed to UBD then digested for single cell RNA sequencing to define the molecular changes that occur to specific identities of colonic epithelial cells. Validation in colonoids was assessed using morphological and imaging techniques. RESULTS: Human colonoids acutely exposed to UBD exhibited disrupted proliferation and hyperplastic differentiation of the secretory lineage cell, enteroendocrine cells (EEC). Single-cell RNA sequencing also showed more EEC subtypes present in UBD-exposed colonoids. DISCUSSION: These findings highlight the significance of crypt-based proliferative cells and secretory cell differentiation using human colonoids to model major colonic responses to uranium-bearing particulate dust exposure. https://doi.org/10.1289/EHP13855.

Arsenic exposure is associated with alterations to multiple red blood cell parameters among adults in rural Bangladesh.

Chronic arsenic exposures are associated with multiple hematologic disturbances, including anemia. The goal of this study was to evaluate associations between arsenic exposures and hematological parameters among men and women who are chronically exposed to elevated levels of arsenic from drinking water. Hematologic analyses were performed on blood collected from 755 participants (45% male and 54% female) in the Health Effects of Arsenic Longitudinal Study (HEALS) cohort, Bangladesh. Herein, we used linear regression models to estimate associations between red blood cell (RBC) parameters (i.e., RBC counts, hematocrit (HCT), hemoglobin (Hgb), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC)) and measurements of arsenic exposure (urinary arsenic and urinary arsenic metabolites). Arsenic exposures showed trending associations with decreased RBC counts in both men and women, a positive association with MCV in males, and an inverse association with MCHC among males, but not among non-smoking females. Among men, those who smoked had stronger associations between arsenic exposures and MCHC than non-smoking males. Collectively, our results show that arsenic exposures affect multiple RBC parameters and highlight potentially important sex differences in arsenic-induced hematotoxicity.

Uranium-bearing dust induces differentiation and expansion of enteroendocrine cells in human colonoids.

Chronic exposure to environmental toxins and heavy metals has been associated with intestinal inflammation, increased susceptibility to pathogen-induced diseases, and higher incidences of colorectal cancer, all of which have been steadily increasing in prevalence for the past 40 years. The negative effects of heavy metals on barrier permeability and inhibition of intestinal epithelial healing have been described; however, transcriptomic changes within the intestinal epithelial cells and impacts on lineage differentiation are largely unknown. Uranium exposure remains an important environmental legacy and physiological health concern, with hundreds of abandoned uranium mines located in the Southwestern United States largely impacting underserved indigenous communities. Herein, using human colonoids, we defined the molecular and cellular changes that occur in response to uranium bearing dust (UBD) exposure. We used single cell RNA sequencing to define the molecular changes that occur to specific identities of colonic epithelial cells. We demonstrate that this environmental toxicant disrupts proliferation and induces hyperplastic differentiation of secretory lineage cells, particularly enteroendocrine cells (EEC). EECs respond to UBD exposure with increased differentiation into de novo EEC sub-types not found in control colonoids. This UBD-induced EEC differentiation does not occur via canonical transcription factors NEUROG3 or NEUROD1. These findings highlight the significance of crypts-based proliferative cells and secretory cell differentiation as major colonic responses to heavy metal-induced injury.

Exposure to arsenic and level of Vitamin D influence the number of Th17 cells and production of IL-17A in human peripheral blood mononuclear cells in adults.

There is limited evidence on the effects of environmental exposure to arsenic (As) on the immune system in adults. In a population-based study, we have found that urinary As (UAs), and its metabolites [inorganic As (InAs), monomethylated arsenicals (MMA+3/+5), and dimethylated arsenicals (DMA+3/+5)] modulate or influence the number of T-helper 17 (Th17) cells and IL-17A cytokine production. In non-smoking women, we observed that UAs and DMA+3/+5 were associated with changes in Th17 cell numbers in a nonlinear fashion. In smoking males, we found that UAs was associated with a significant decrease of Th17 cell numbers. Similar association was observed among non-smoking males. Likewise, UAs, DMA+3/+5 and MMA+3/+5 were associated with diminished production of IL-17A among non-smoking males. When stratified by Vitamin D levels defined as sufficient (≥20 ng/ml) and insufficient (<20 ng/ml), we found a substancial decrease in Th17 cell numbers among those with insufficient levels. Individuals with sufficient VitD levels demonstrated significant inhibition of IL-17A production in non-smoking males. Collectively, we find that exposure to As via drinking water is associated with alterations in Th17 numbers and IL-17A production, and that these associations may be modified by Vitamin D status. Our findings have significance for health outcomes associated with As exposure.

Uptake and Toxicity of Respirable Carbon-Rich Uranium-Bearing Particles: Insights into the Role of Particulates in Uranium Toxicity.

Particulate matter (PM) presents an environmental health risk for communities residing close to uranium (U) mine sites. However, the role of the particulate form of U on its cellular toxicity is still poorly understood. Here, we investigated the cellular uptake and toxicity of C-rich U-bearing particles as a model organic particulate containing uranyl citrate over a range of environmentally relevant concentrations of U (0-445 µM). The cytotoxicity of C-rich U-bearing particles in human epithelial cells (A549) was U-dose-dependent. No cytotoxic effects were detected with soluble U doses. Carbon-rich U-bearing particles with a wide size distribution (<10 µm) presented 2.7 times higher U uptake into cells than the particles with a narrow size distribution (<1 µm) at 100 µM U concentration. TEM-EDS analysis identified the intracellular translocation of clusters of C-rich U-bearing particles. The accumulation of C-rich U-bearing particles induced DNA damage and cytotoxicity as indicated by the increased phosphorylation of the histone H2AX and cell death, respectively. These findings reveal the toxicity of the particulate form of U under environmentally relevant heterogeneous size distributions. Our study opens new avenues for future investigations on the health impacts resulting from environmental exposures to the particulate form of U near mine sites.

Arsenite and monomethylarsonous acid disrupt erythropoiesis through combined effects on differentiation and survival pathways in early erythroid progenitors.

Strong epidemiological evidence demonstrates an association between chronic arsenic exposure and anemia. We recently found that As+3 impairs erythropoiesis by disrupting the function of GATA-1; however the downstream pathways impacted by the loss of GATA-1 function have not been evaluated. Additionally, our previous findings indicate that the predominant arsenical in the bone marrow of mice exposed to As+3 in their drinking water for 30 days was MMA+3, but the impacts of this arsenical on erythorpoisis also remain largely unknown. The goal of this study was to address these critical knowledge gaps by evaluating the comparative effects of arsenite (As+3) and the As+3 metabolite, monomethyarsonous acid (MMA+3) on two critical regulatory pathways that control the differentiation and survival of early erythroid progenitor cells. We found that 500 nM As+3 and 100 and 500 nM MMA+3 suppress erythropoiesis by impairing the differentiation of early stage erythroid progenitors. The suppression of early erythroid progenitor cell development was attributed to combined effects on differentiation and survival pathways mediated by disruption of GATA-1 and STAT5. Our results show that As+3 primarily disrupted GATA-1 function; whereas, MMA+3 suppressed both GATA-1 and STAT5 activity. Collectively, these findings provide novel mechanistic insights into arsenic-induced dyserythropoiesis and suggest that MMA+3 may be more toxic than As+3 to early developing erythroid cells.

Modulation of PARP activity by Monomethylarsonous (MMA+3) acid and uranium in mouse thymus.

Arsenic exposure is well established to impair the function of zinc finger proteins, including PARP-1. Previous studies from our lab show that early developing T cells in the thymus are very sensitive to arsenite (As+3)-induced genotoxicity mediated through PARP-1 inhibition. Additionally, it has been shown that uranium (in the form of uranyl acetate, UA) also suppresses PARP-1 activity in HEK cells. However, very little is known about whether the As+3 metabolite, monomethylarsonous acid (MMA+3), also inhibits PARP-1 activity and if this is modified by combined exposures with other metals, such as uranium. In the present study, we found that MMA+3 significantly suppressed PARP-1 function, whereas UA at high concentrations significantly increased PARP-1 activity. To evaluate whether the effects on PARP-1 activity were mediated through oxidative stress, we measured the induction of hemoxygenase-1 (Hmox-1) expression by qPCR. MMA+3, but not UA, significantly induced oxidative stress; however, the inhibition of PARP-1 produced by MMA+3 was not reversed by the addition of the antioxidant, Tempol. Further evaluation revealed minimal interactive effects of MMA+3 and UA on PARP-1 function. Collectively, our results show that contrary to As+3, the suppressive effects of MMA+3 on PARP-1 were not substantially driven by oxidative stress. in mouse thymus cells. Results for this study provide important insights into the effects of MMA+3 and uranium exposures on PARP-1 function, which is essential for future studies focused on understanding the effects of complex environmentally relevant metal mixtures.

Inhibition of red blood cell development by arsenic-induced disruption of GATA-1.

Anemia is a hematological disorder that adversely affects the health of millions of people worldwide. Although many variables influence the development and exacerbation of anemia, one major contributing factor is the impairment of erythropoiesis. Normal erythropoiesis is highly regulated by the zinc finger transcription factor GATA-1. Disruption of the zinc finger motifs in GATA-1, such as produced by germline mutations, compromises the function of this critical transcription factor and causes dyserythropoietic anemia. Herein, we utilize a combination of in vitro and in vivo studies to provide evidence that arsenic, a widespread environmental toxicant, inhibits erythropoiesis likely through replacing zinc within the zinc fingers of the critical transcription factor GATA-1. We found that arsenic interacts with the N- and C-terminal zinc finger motifs of GATA-1, causing zinc loss and inhibition of DNA and protein binding activities, leading to dyserythropoiesis and an imbalance of hematopoietic differentiation. For the first time, we show that exposures to a prevalent environmental contaminant compromises the function of a key regulatory factor in erythropoiesis, producing effects functionally similar to inherited GATA-1 mutations. These findings highlight a novel molecular mechanism by which arsenic exposure may cause anemia and provide critical insights into potential prevention and intervention for arsenic-related anemias.

Exposures to uranium and arsenic alter intraepithelial and innate immune cells in the small intestine of male and female mice.

Human exposures to environmental metals, including uranium (U) and arsenic (As) are a global public health concern. Chronic exposures to U and As are linked to many adverse health effects including, immune suppression and autoimmunity. The gastrointestinal (GI) tract is home to many immune cells vital in the maintenance of systemic immune health. However, very little is known about the immunotoxicity of U and As at this site. The present study examined the burden of U and As exposure in the GI tract as well as the resultant immunotoxicity to intraepithelial lymphocytes (IELs) and innate immune cells of the small intestine following chronic drinking water exposures of male and female mice to U (in the form of uranyl acetate, UA) and As (in the form of sodium arsenite, As3+). Exposure to U or As3+ resulted in high levels of U or As in the GI tract of male and female mice, respectively. A reduction of small intestinal CD4+ IELs (TCRαß+, CD8αα+) was found following As3+ exposure, whereas U produced widespread suppression of CD4- IEL subsets (TCRαß+ and TCRγδ+). Evaluation of innate immune cell subsets in the small intestinal lamina propria revealed a decrease in mature macrophages, along with a corresponding increase in immature/proinflammatory macrophages following As3+ exposures. These data show that exposures to two prevalent environmental contaminants, U and As produce significant immunotoxicity in the GI tract. Collectively, these findings provide a critical framework for understanding the underlying immune health issues reported in human populations chronically exposed to environmental metals.

Arsenic exposure associated T cell proliferation, smoking, and vitamin D in Bangladeshi men and women.

There are limited data examining the consequences of environmental exposure to arsenic on the immune system in adults, particularly among smokers. Smoking has been shown to exacerbate or contribute to impaired immune function in men chronically exposed to arsenic. In contrast, vitamin D (VitD) is known to have a positive influence on innate and adaptive immune responses. The effect of circulating VitD on arsenic-associated immune dysfunction is not known. Here we examine the relationship of arsenic exposure and T cell proliferation (TCP), a measure of immune responsiveness, and circulating VitD among adult men and women in Bangladesh. Arsenic exposure was assessed using total urinary arsenic as well as urinary arsenic metabolites all adjusted for urinary creatinine. TCP was measured ex vivo in cryopreserved peripheral blood mononuclear cells from 614 adult participants enrolled in the Bangladesh Health Effects of Arsenic Longitudinal Study; serum VitD was also evaluated. The influence of cigarette smoking on arsenic-induced TCP modulation was assessed only in males as there was an inadequate number of female smokers. These studies show that arsenic suppressed TCP in males. The association was significantly strong in male smokers and to a lesser extent in male non-smokers. Interestingly, we found a strong protective effect of high/sufficient serum VitD levels on TCP among non-smoking males. Furthermore, among male smokers with low serum VitD (⊔20 ng/ml), we found a strong suppression of TCP by arsenic. On the other hand, high VitD (>20 ng/ml) was found to attenuate effects of arsenic on TCP among male-smokers. Overall, we found a strong protective effect of VitD, when serum levels were >20 ng/ml, on arsenic-induced inhibition of TCP in men, irrespective of smoking status. To our knowledge this is the first large study of immune function in healthy adult males and females with a history of chronic arsenic exposure.

An increase in circulating B cells and B cell activation markers in peripheral blood is associated with cigarette smoking in a male cohort in Bangladesh.

In a cohort of approximately 200 Bangladeshi men, equally divided into smokers and non-smokers and equally divided by exposure to high and low levels of drinking water arsenic, we examined ex vivo a series of immune markers and immune function tests in peripheral blood mononuclear cells (PBMC). These immune parameters included PBMC cell surface markers (CSM) for B, T, monocytes, and NK cells, activated T and B cell markers, cytokine production in vitro, and analysis of CD4 subsets (Th1, Th2, Treg, and Th17 cells). We found that the effects of cigarette smoke were quite different than those associated with arsenic or polycyclic aromatic hydrocarbon (PAH)-DNA adducts. Cigarette smoking was associated with a significant increase in the number of PAH-DNA adducts as well as an increase in urinary levels of 1-hydropxypyrene (1-OHP). After correcting for arsenic exposure and PAH-DNA adducts, we found that cigarette smoking was associated with an increase in the percentage of CD19+ B cells, as well as the percentage of activated B cells (CD19+, HLA-DRbright cells) found in PBMC. These findings demonstrate activation of the immune system during chronic exposure to cigarette smoke, which is a known risk factor for autoimmune diseases.

Changes in human peripheral blood mononuclear cell (HPBMC) populations and T-cell subsets associated with arsenic and polycyclic aromatic hydrocarbon exposures in a Bangladesh cohort.

Exposures to environmental arsenic (As) and polycyclic aromatic hydrocarbons (PAH) have been shown to independently cause dysregulation of immune function. Little data exists on the associations between combined exposures to As and PAH with immunotoxicity in humans. In this work we examined associations between As and PAH exposures with lymphoid cell populations in human peripheral blood mononuclear cells (PBMC), as well as alterations in differentiation and activation of B and T cells. Two hundred men, participating in the Health Effects of Arsenic Longitudinal Study (HEALS) in Bangladesh, were selected for the present study based on their exposure to As from drinking water and their cigarette smoking status. Blood and urine samples were collected from study participants. We utilized multiparameter flow cytometry in PBMC to identify immune cells (B, T, monocytes, NK) as well as the T-helper (Th) cell subsets (Th1, Th2, Th17, and Tregs) following ex vivo activation. We did not find evidence of interactions between As and PAH exposures. However, individual exposures (As or PAH) were associated with changes to immune cell populations, including Th cell subsets. Arsenic exposure was associated with an increase in the percentage of Th cells, and dose dependent changes in monocytes, NKT cells and a monocyte subset. Within the Th cell subset we found that Arsenic exposure was also associated with a significant increase in the percentage of circulating proinflammatory Th17 cells. PAH exposure was associated with changes in T cells, monocytes and T memory (Tmem) cells and with changes in Th, Th1, Th2 and Th17 subsets all of which were non-monotonic (dose dependent). Alterations of immune cell populations caused by environmental exposures to As and PAH may result in adverse health outcomes, such as changes in systemic inflammation, immune suppression, or autoimmunity.

Assessment of arsenic and polycyclic aromatic hydrocarbon (PAH) exposures on immune function among males in Bangladesh.

Arsenic and polycyclic aromatic hydrocarbons (PAH) are environmental pollutants to which people around the world are exposed through water, food and air. In mouse and in vitro studies of human cells, both of these chemicals have been shown to modulate the immune system. In some experimental studies, a synergistic disruption of immune function was observed by a combined exposure to arsenic and PAH. However, a joint effect of arsenic and PAH on immune function has not been studied in humans. We have conducted an epidemiological investigation to examine effects of chronic arsenic and PAH exposures on immune function. We assessed T-cell proliferation (TCP) and cytokine production of anti-CD3/anti-CD28 stimulated lymphocytes in human peripheral blood mononuclear cells (HPBMC) among 197 healthy men enrolled to the Health Effects of Arsenic Longitudinal (HEALS) cohort in Bangladesh. By design, approximately half were active smokers and the rest were never smokers. Our analyses demonstrated that IL-1b, IL-2, IL-4 and IL-6 were significantly stimulated as a function of urinary arsenic levels in models adjusted for age, body mass index (BMI), smoking status and PAH-DNA adducts. After correcting for false detection rate (FDR), only IL-1b remained statistically significant. We found a U-shaped dose response relationship between urinary arsenic and IL-1b. On the other hand, PAH-DNA adducts were associated with an inhibition of TCP and appeared as an inverted U-shape curve. Dose response curves were non-monotonic for PAH-DNA adduct exposures and suggested that cytokine secretion of IFNg, IL-1b, IL-2, IL-10 and IL17A followed a complex pattern. In the majority of donors, there was a trend towards a decrease in cytokine associated with PAH-DNA adducts. We did not observe any interaction between urinary arsenic and PAH-DNA adducts on immune parameters. Our results indicate that long-term exposures to arsenic and PAH have independent, non-monotonic associations with TCP and cytokine production.

Minimal uranium immunotoxicity following a 60-day drinking water exposure to uranyl acetate in male and female C57BL/6J mice.

Historical uranium (U) mining in the Southwestern United States resulted in significant environmental contamination throughout this region and presents a significant risk of chronic metal exposure and toxicity for communities living in close proximity to mine waste sites. Uranium exposure is associated with numerous deleterious health effects including immune dysfunction; however, its effects on the immune system have yet to be fully characterized. We recently published that drinking water exposure to U, in the form of uranyl acetate (UA), results in low overall tissue retention of U (<0.01%), with very little accumulation in immune organs (blood, bone marrow, spleen, and thymus) of male and female mice. In the present study we characterized the immunotoxicity of U, in the form of UA, following a 60-day drinking water exposure to 5 and 50 ppm in male and female C57BL/6J mice. The following immunotoxicity endpoints were evaluated: hematology, immune tissue weights and total cell recoveries, immunophenotying of the spleen and thymus, and immune cell function (lymphocyte mitogenesis and T-dependent antibody response). Uranium exposure had subtle impacts on the immune endpoints evaluated, likely due to low U accumulation at these sites. The only significant alterations were a slight decrease in the percentages of splenic natural killer T-cells and macrophages in exposed male mice. Despite minimal immunological effects, this study highlights the importance of investigating toxicological endpoints in both sexes and developing accurate animal models that model epidemiological exposures in the future.

Minimal uranium accumulation in lymphoid tissues following an oral 60-day uranyl acetate exposure in male and female C57BL/6J mice.

High levels of uranium (U) exist in soil, water, and air in the Southwestern United States due, in part, to waste generated from more than 160,000 abandoned hard rock mines located in this region. As a result, many people living in this region are chronically exposed to U at levels that have been linked to detrimental health outcomes. In an effort to establish a relevant in vivo mouse model for future U immunotoxicity studies, we evaluated the tissue distribution of U in immune organs; blood, bone marrow, spleen, and thymus, as well as femur bones, kidneys, and liver, following a 60-d drinking water exposure to uranyl acetate (UA) in male and female C57BL/6J mice. Following the 60-d exposure, there was low overall tissue retention of U (<0.01%) at both the 5 and the 50 ppm (mg/L) oral concentrations. In both male and female mice, there was limited U accumulation in immune organs. U only accumulated at low concentrations in the blood and bone marrow of male mice (0.6 and 16.8 ng/g, respectively). Consistent with previous reports, the predominant sites of U accumulation were the femur bones (350.1 and 399.0 ng/g, respectively) and kidneys (134.0 and 361.3 ng/g, respectively) of male and female mice. Findings from this study provide critical insights into the distribution and retention of U in lymphoid tissues following chronic drinking water exposure to U. This information will serve as a foundation for immunotoxicological assessments of U, alone and in combination with other metals.

Isolation, Cryopreservation, and Immunophenotyping of Human Peripheral Blood Mononuclear Cells.

This unit describes procedures for the isolation, cryopreservation, and thawing of human peripheral blood mononuclear cells (HPBMC) and analysis of cell surface markers (CSM) for immunophenotyping using polychromatic flow cytometry. This methodology can be used to ensure that cell integrity and phenotype stability are not altered through cryopreservation and extended storage. For this analysis, HPBMC were isolated from 7 healthy individuals, and 11-color flow cytometry was performed on freshly isolated samples as well as samples cryopreserved for short- and long-term periods. There is no significant difference in the percentage of cells expressing the CSM CD3, CD4, CD8, CD45RO, CD16, CD19, or CD56 between freshly isolated and cryopreserved HPBMC. Hence, cryopreservation of HPBMC does not influence the phenotype of distinct cellular subsets in isolated mononuclear cells. This protocol for HPBMC isolation, cryopreservation, and thawing of HPBMC is intended for long-term studies of large cohorts requiring sample shipment and subsequent batch analysis. © 2017 by John Wiley & Sons, Inc.

Intracellular Cytokine Detection by Flow Cytometry in Surface Marker-Defined Human Peripheral Blood Mononuclear T Cells.

In a recent unit in this series, protocols for the isolation, cryopreservation, thawing, and immunophenotyping of HPBMC isolated from peripheral whole blood using cell surface marker (CSM) staining and multi-color flow cytometry analysis were presented. The current procedure describes the detection and quantification of CSM and intracellular markers (ICM), including transcription factors and cytokines, following activation and differentiation of CD4+ T-cells using multi-color flow cytometry. Results indicated that repeatable and robust detection of ICM could be obtained in surface marker-defined T cells that identify functional subsets of cells. There were no observed differences between fresh and cryopreserved HPBMC in eight phenotypes analyzed (T-CD3, Th-CD4, Tmem-CD45RO, activated T-CD3/CD25, Treg- Foxp3/CD25, Th1-IFNγ, Th2- IL-4, Th17-IL-17A). There was an observed difference in activated T- CD3/CD69 in the short term (30-90 days) cryopreserved samples as compared to the freshly isolated samples, which may have resulted from the variance in controls or small sample size. © 2017 by John Wiley & Sons, Inc.

Genotoxicity induced by monomethylarsonous acid (MMA+3) in mouse thymic developing T cells.

Drinking water exposure to arsenic is known to cause immunotoxicity. Our previous studies demonstrated that monomethylarsonous acid (MMA+3) was the major arsenical species presented in mouse thymus cells after a 30 d drinking water exposure to arsenite (As+3). MMA+3 was also showed to be ten times more toxic than As+3 on the suppression of IL-7/STAT5 signaling in the double negative (DN) thymic T cells. In order to examine the genotoxicity induced by low to moderate doses of MMA+3, isolated mouse thymus cells were treated with 5, 50 and 500nMMMA+3 for 18h in vitro. MMA+3 suppressed the proliferation of thymus cells in a dose dependent manner. MMA+3 at 5nM induced DNA damage in DN not double positive (DP) cells. Differential sensitivity to double strand breaks and reactive oxygen species generation was noticed between DN and DP cells at 50nM, but the effects were not seen at the high dose (500nM). A stronger apoptotic effect induced by MMA+3 was noticed in DN cells than DP cells at low doses (5 and 50nM), which was negated by the strong apoptosis induction at the high dose (500nM). Analysis of intracellular MMA+3 concentrations in DN and DP cells, revealed that more MMA+3 accumulated in the DN cells after the in vitro treatment. Collectively, these results suggested that MMA+3 could directly induce strong genotoxicity in the early developing T cells in the thymus. The DN cells were much more sensitive to MMA+3 induced genotoxicity and apoptosis than DP cells, probably due to the higher intracellular levels of MMA+3.

Arsenic exposures alter clinical indicators of anemia in a male population of smokers and non-smokers in Bangladesh.

Drinking water arsenic (WAs) exposure has been linked to a number of detrimental health outcomes including anemia, primarily among pregnant women. Little is known about the effects of arsenic (As) on hematological disorders among men. We have examined the role of As exposure on hematological indicators of anemia in a group of men exposed to a wide range of As in their drinking water. We conducted a cross-sectional investigation among 119 healthy men in the Health Effects of As Longitudinal Study (HEALS) cohort, in rural Bangladesh. The participants are part of an ongoing study focused on evaluating the influence of As and smoking on immune function. Samples were collected at recruitment and analyzed for water As, urinary As (UAs) and UAs metabolites to assess As exposure. Blood samples were also collected at recruitment and assayed immediately for hematological parameters. We found that increased WAs levels were associated with decreased red blood cell counts [ß=-0.13, p<0.0001] as well as hematocrit packed cell volumes [ß=-0.68, p=0.008] following adjustment for age, smoking, body mass index and polycyclic aromatic hydrocarbon-DNA adducts. Other measures of As exposure (UAs and its metabolites) demonstrated similar associations. Slightly stronger effects were observed among smokers. We also observed an effect of As on hemoglobin among smokers in relation to UAs [ß=-0.54, p<0.05]. Our analysis revealed effects of As exposure on hematological indicators of anemia in a group of healthy male smokers and non-smokers.

Efflux Transporters Regulate Arsenite-Induced Genotoxicity in Double Negative and Double Positive T Cells.

Arsenite (As+3) exposure is known to cause immunotoxicity in human and animal models. Our previous studies demonstrated that As+3 at 50-500 nM concentrations induced both genotoxicity and nongenotoxicity in mouse thymus cells. Developing T cells at CD4-CD8- double negative (DN) stage, the first stage after early T cells are transported from bone marrow to thymus, were found to be more sensitive to As+3 toxicity than the T cells at CD4 + CD8 + double positive (DP) stage in vitro. Induction of Mdr1 (Abcb1) and Mrp1 (Abcc1), 2 multidrug resistance transporters and exporters of As+3, was associated with the reversal of As+3-induced double strand breaks and DNA damage. In order to confirm that the thymus cell populations have different sensitivity to As+3in vivo, male C57BL/6J mice were exposed to 0, 100, and 500 ppb As+3 in drinking water for 30 d. A significant decrease in DN cell percentage was observed with exposure to 500 ppb As+3. Low to moderate concentrations of As+3 were shown to induce higher genotoxicity in sorted DN than DP cells in vitro. Calcein AM uptake and Mdr1/Mrp1 mRNA quantification results revealed that DN cells not only had limited As+3 exporter activity, but also lacked the ability to activate these exporters with As+3 treatments, resulting in a higher accumulation of intracellular As+3. Knockdown study of As+3 exporters in the DN thymic cell line, D1 using siRNA, demonstrated that Mdr1 and Mrp1 regulate intracellular As+3 accumulation and genotoxicity. Taken together, the results indicate that transporter regulation is an important mechanism for differential genotoxicity induced by As+3 in thymocytes at different developmental stages.