Evaluation of UBE3A antibodies in mice and human cerebral organoids.

UBE3A is an E3 ubiquitin ligase encoded by the neurally imprinted UBE3A gene. The abundance and subcellular distribution of UBE3A has been the topic of many previous studies as its dosage and localization has been linked to neurodevelopmental disorders including Autism, Dup15q syndrome, and Angelman syndrome. While commercially available antibodies have been widely employed to determine UBE3A localization, an extensive analysis and comparison of the performance of different UBE3A antibodies has not been conducted. Here we evaluated the specificities of seven commercial UBE3A antibodies in two of the major experimental models used in UBE3A research, mouse and human pluripotent stem cell-derived neural cells and tissues. We tested these antibodies in their two most common assays, immunofluorescence and western blot. In addition, we also assessed the ability of these antibodies to capture dynamic spatiotemporal changes of UBE3A by utilizing human cerebral organoid models. Our results reveal that among the seven antibodies tested, three antibodies demonstrated substantial nonspecific immunoreactivity. While four antibodies show specific localization patterns in both mouse brain sections and human cerebral organoids, these antibodies varied significantly in background signals and staining patterns in undifferentiated human pluripotent stem cells.

Activation of Lrrk2 and α-Synuclein in substantia nigra, striatum, and cerebellum after chronic exposure to arsenite.

Arsenic is a neurotoxin and environmental exposure to it correlates with an incidence of neurodegenerative diseases. Considering that arsenic has the potential to inhibit autophagic flux, it was hypothesized that arsenite (NaAsO2) may interplay with LRRK2 and α-Synuclein, affecting their phosphorylation in brain regions prone to neurodegeneration. After 15 weeks of chronic exposure to arsenite, a reduction in grip strength of C57BL/6 male mice was observed. Thirty minutes exposure to arsenite increased phosphorylation of Lrrk2 and α-Synuclein in organotypic brain slice cultures from the cerebellum and striatum, respectively. Chronic exposure of mice to a wide-range of concentrations of arsenite led to a significant induction of Lrrk2 phosphorylation in substantia nigra and cerebellum and α-Synuclein phosphorylation in substantia nigra and striatum. Strong correlations between phosphorylated forms of Lrrk2 and α-Synuclein in substantia nigra, Lrrk2 levels between substantia nigra and striatum, and between Lrrk2 in striatum and α-Synuclein in substantia nigra observed in control animals were completely disrupted by arsenic exposure at 50, 500, and 5000 ppb. A transcriptome analysis identified specific genes and canonical pathways that distinguish striatum, substantia nigra, and cerebellum from each other in control animals and compare individual brain regions to arsenite exposed animals. Chronic arsenite exposure altered transcripts of glutathione redox reactions and serotonin receptor signaling in striatum, axonal guidance signaling, NF-κB and androgen signaling in substantia nigra and mitochondrial dysfunction, oxidative phosphorylation, apoptosis and sirtuin signaling in the cerebellum. These data suggest that arsenite affects processes associated with neurodegenerative diseases in brain region specific manner.

Human Cerebral Organoids Reveal Early Spatiotemporal Dynamics and Pharmacological Responses of UBE3A.

Angelman syndrome is a complex neurodevelopmental disorder characterized by delayed development, intellectual disability, speech impairment, and ataxia. It results from the loss of UBE3A protein, an E3 ubiquitin ligase, in neurons of the brain. Despite the dynamic spatiotemporal expression of UBE3A observed in rodents and the potential clinical importance of when and where it is expressed, its expression pattern in humans remains unknown. This reflects a common challenge of studying human neurodevelopment: prenatal periods are hard to access experimentally. In this work, human cerebral organoids reveal a change from weak to strong UBE3A in neuronal nuclei within 3 weeks of culture. Angelman syndrome human induced pluripotent stem cell-derived organoids also exhibit early silencing of paternal UBE3A, with topoisomerase inhibitors partially rescuing UBE3A levels and calcium transient phenotypes. This work establishes human cerebral organoids as an important model for studying UBE3A and motivates their broader use in understanding complex neurodevelopmental disorders.

Transgenerational Bisphenol A Causes Deficits in Social Recognition and Alters Postsynaptic Density Genes in Mice.

Bisphenol A (BPA) is a ubiquitous endocrine-disrupting chemical. Developmental exposure produces changes in behavior and gene expression in the brain. Here, we examined social recognition behaviors in mice from the third familial generation (F3) after exposure to gestational BPA. Second-generation mice were bred in one of four mating combinations to reveal whether characteristics in F3 were acquired via maternal or paternal exposures. After repeated habituation to the same mouse, offspring of dams from the BPA lineage failed to display increased investigation of a novel mouse. Genes involved in excitatory postsynaptic densities (PSDs) were examined in F3 brains using quantitative PCR. Differential expression of genes important for function and stability of PSDs were assessed at three developmental ages. Several related PSD genes-SH3 and multiple ankyrin repeat domains 1 (Shank1), Homer scaffolding protein 1c (Homer1c), DLG associated protein 1 (Gkap), and discs large MAGUK scaffold protein 4 (PSD95)-were differentially expressed in control- vs BPA-lineage brains. Using a second strain of F3 inbred mice exposed to BPA, we noted the same differences in Shank1 and PSD95 expression in C57BL/6J mice. In sum, transgenerational BPA exposure disrupted social interactions in mice and dysregulated normal expression of PSD genes during neural development. The fact that the same genetic effects were found in two different mouse strains and in several brain regions increased potential for translation. The genetic and functional relationship between PSD and abnormal neurobehavioral disorders is well established, and our data suggest that BPA may contribute in a transgenerational manner to neurodevelopmental diseases.

Bisphenol F has different effects on preadipocytes differentiation and weight gain in adult mice as compared with Bisphenol A and S.

Bisphenol S (2,2-bisulfone, BPS) and Bisphenol F (2,2-bis [4-hydroxyphenol]methane, BPF) are analogs of Bisphenol A (2,2-bis[4-hydroxyphenyl]propane, BPA), a widely used endocrine disrupting compound present in polycarbonate plastics, thermal receipts and epoxy resins that line food cans. Here we examined effects of BPA, BPS, and BPF in low concentrations on differentiation in murine 3T3-L1 preadipocytes. We also fed adult male mice chow with one of three doses of BPF (0, 0.5, 5, 50 mg/kg chow, or approximately 0.044, 0.44 and 4.4 mg/kg body weight per day) for 12 weeks, collected body weights, food intake, and tested for glucose tolerance. The doses of BPF used produced mean concentrations of 0, 6.2, 43.6, and 561 ng/mL in plasma. In 3T3-L1 cells BPS had the greatest effects, along with BPA, both increased expression of several genes required for preadipocyte differentiation over 12 days in culture. In contrast, BPF decreased expression of several genes late in differentiation. This dichotomy was also reflected in lipid accumulation as BPA and BPS treated cells had elevated lipid concentrations compared to controls or cells treated with BPF. Male mice fed either the highest or lowest concentrations of BPF gained less weight than controls with no effects on glucose levels or glucose tolerance. Plasma levels of BPF reflected doses in food with no overlap between doses. In summary, our results suggest that BPS has a strong potential to be obesogenic while effects of BPF are subtler and potentially in the opposite direction.

Transgenerational Effects of Bisphenol A on Gene Expression and DNA Methylation of Imprinted Genes in Brain.

Bisphenol A (BPA) is a ubiquitous man-made endocrine disrupting compound (EDC). Developmental exposure to BPA changes behavioral and reproductive phenotypes, and these effects can last for generations. We exposed embryos to BPA, producing two lineages: controls and BPA exposed. In the third filial generation (F3), brain tissues containing the preoptic area, the bed nucleus of the stria terminalis, and the anterior hypothalamus were collected. RNA sequencing (RNA-seq) and subsequent data analyses revealed 50 differentially regulated genes in the brains of F3 juveniles from BPA vs control lineages. BPA exposure can lead to loss of imprinting, and one of the two imprinted genes in our data set, maternally expressed gene 3 (Meg3), has been associated with EDCs and neurobehavioral phenotypes. We used quantitative polymerase chain reaction to examine the two imprinted genes in our data set, Meg3 and microRNA-containing gene Mirg (residing in the same loci). Confirming the RNA-seq, Meg3 messenger RNA was higher in F3 brains from the BPA lineage than in control brains. This was true in brains from mice produced with two different BPA paradigms. Next, we used pyrosequencing to probe differentially methylated regions of Meg3. We found transgenerational effects of BPA on imprinted genes in brain. Given these results, and data on Meg3 methylation in humans, we suggest this gene may be a biomarker indicative of early life environmental perturbation.

Metabolomic profiles of arsenic (+3 oxidation state) methyltransferase knockout mice: effect of sex and arsenic exposure.

Arsenic (+3 oxidation state) methyltransferase (As3mt) is the key enzyme in the pathway for methylation of inorganic arsenic (iAs). Altered As3mt expression and AS3MT polymorphism have been linked to changes in iAs metabolism and in susceptibility to iAs toxicity in laboratory models and in humans. As3mt-knockout mice have been used to study the association between iAs metabolism and adverse effects of iAs exposure. However, little is known about systemic changes in metabolism of these mice and how these changes lead to their increased susceptibility to iAs toxicity. Here, we compared plasma and urinary metabolomes of male and female wild-type (WT) and As3mt-KO (KO) C57BL/6 mice and examined metabolomic shifts associated with iAs exposure in drinking water. Surprisingly, exposure to 1 ppm As elicited only small changes in the metabolite profiles of either WT or KO mice. In contrast, comparisons of KO mice with WT mice revealed significant differences in plasma and urinary metabolites associated with lipid (phosphatidylcholines, cytidine, acyl-carnitine), amino acid (hippuric acid, acetylglycine, urea), and carbohydrate (L-sorbose, galactonic acid, gluconic acid) metabolism. Notably, most of these differences were sex specific. Sex-specific differences were also found between WT and KO mice in plasma triglyceride and lipoprotein cholesterol levels. Some of the differentially changed metabolites (phosphatidylcholines, carnosine, and sarcosine) are substrates or products of reactions catalyzed by other methyltransferases. These results suggest that As3mt KO alters major metabolic pathways in a sex-specific manner, independent of iAs treatment, and that As3mt may be involved in other cellular processes beyond iAs methylation.

The Association of Arsenic Exposure and Metabolism With Type 1 and Type 2 Diabetes in Youth: The SEARCH Case-Control Study.

OBJECTIVE: Little is known about arsenic and diabetes in youth. We examined the association of arsenic with type 1 and type 2 diabetes in the SEARCH for Diabetes in Youth Case-Control (SEARCH-CC) study. Because one-carbon metabolism can influence arsenic metabolism, we also evaluated the potential interaction of folate and vitamin B12 with arsenic metabolism on the odds of diabetes. RESEARCH DESIGN AND METHODS: Six hundred eighty-eight participants <22 years of age (429 with type 1 diabetes, 85 with type 2 diabetes, and 174 control participants) were evaluated. Arsenic species (inorganic arsenic [iAs], monomethylated arsenic [MMA], dimethylated arsenic [DMA]), and one-carbon metabolism biomarkers (folate and vitamin B12) were measured in plasma. We used the sum of iAs, MMA, and DMA (∑As) and the individual species as biomarkers of arsenic concentrations and the relative proportions of the species over their sum (iAs%, MMA%, DMA%) as biomarkers of arsenic metabolism. RESULTS: Median ∑As, iAs%, MMA%, and DMA% were 83.1 ng/L, 63.4%, 10.3%, and 25.2%, respectively. ∑As was not associated with either type of diabetes. The fully adjusted odds ratios (95% CI), rescaled to compare a difference in levels corresponding to the interquartile range of iAs%, MMA%, and DMA%, were 0.68 (0.50-0.91), 1.33 (1.02-1.74), and 1.28 (1.01-1.63), respectively, for type 1 diabetes and 0.82 (0.48-1.39), 1.09 (0.65-1.82), and 1.17 (0.77-1.77), respectively, for type 2 diabetes. In interaction analysis, the odds ratio of type 1 diabetes by MMA% was 1.80 (1.25-2.58) and 0.98 (0.70-1.38) for participants with plasma folate levels above and below the median (P for interaction = 0.02), respectively. CONCLUSIONS: Low iAs% versus high MMA% and DMA% was associated with a higher odds of type 1 diabetes, with a potential interaction by folate levels. These data support further research on the role of arsenic metabolism in type 1 diabetes, including the interplay with one-carbon metabolism biomarkers.

Neonatal Metabolomic Profiles Related to Prenatal Arsenic Exposure.

Prenatal inorganic arsenic (iAs) exposure is associated with health effects evident at birth and later in life. An understanding of the relationship between prenatal iAs exposure and alterations in the neonatal metabolome could reveal critical molecular modifications, potentially underpinning disease etiologies. In this study, nuclear magnetic resonance (NMR) spectroscopy-based metabolomic analysis was used to identify metabolites in neonate cord serum associated with prenatal iAs exposure in participants from the Biomarkers of Exposure to ARsenic (BEAR) pregnancy cohort, in Gómez Palacio, Mexico. Through multivariable linear regression, ten cord serum metabolites were identified as significantly associated with total urinary iAs and/or iAs metabolites, measured as %iAs, %monomethylated arsenicals (MMAs), and %dimethylated arsenicals (DMAs). A total of 17 metabolites were identified as significantly associated with total iAs and/or iAs metabolites in cord serum. These metabolites are indicative of changes in important biochemical pathways such as vitamin metabolism, the citric acid (TCA) cycle, and amino acid metabolism. These data highlight that maternal biotransformation of iAs and neonatal levels of iAs and its metabolites are associated with differences in neonate cord metabolomic profiles. The results demonstrate the potential utility of metabolites as biomarkers/indicators of in utero environmental exposure.

Oxidation state specific analysis of arsenic species in tissues of wild-type and arsenic (+3 oxidation state) methyltransferase-knockout mice.

Arsenic methyltransferase (As3mt) catalyzes the conversion of inorganic arsenic (iAs) to its methylated metabolites, including toxic methylarsonite (MAsIII) and dimethylarsinite (DMAsIII). Knockout (KO) of As3mt was shown to reduce the capacity to methylate iAs in mice. However, no data are available on the oxidation states of As species in tissues of these mice. Here, we compare the oxidation states of As species in tissues of male C57BL/6 As3mt-KO and wild-type (WT) mice exposed to arsenite (iAsIII) in drinking water. WT mice were exposed to 50mg/L As and As3mt-KO mice that cannot tolerate 50mg/L As were exposed to 0, 15, 20, 25 or 30mg/L As. iAsIII accounted for 53% to 74% of total As in liver, pancreas, adipose, lung, heart, and kidney of As3mt-KO mice; tri- and pentavalent methylated arsenicals did not exceed 10% of total As. Tissues of WT mice retained iAs and methylated arsenicals: iAsIII, MAsIII and DMAsIII represented 55%-68% of the total As in the liver, pancreas, and brain. High levels of methylated species, particularly MAsIII, were found in the intestine of WT, but not As3mt-KO mice, suggesting that intestinal bacteria are not a major source of methylated As. Blood of WT mice contained significantly higher levels of As than blood of As3mt-KO mice. This study is the first to determine oxidation states of As species in tissues of As3mt-KO mice. Results will help to design studies using WT and As3mt-KO mice to examine the role of iAs methylation in adverse effects of iAs exposure.

Association Between Variants in Arsenic (+3 Oxidation State) Methyltranserase (AS3MT) and Urinary Metabolites of Inorganic Arsenic: Role of Exposure Level.

Variants in AS3MT, the gene encoding arsenic (+3 oxidation state) methyltranserase, have been shown to influence patterns of inorganic arsenic (iAs) metabolism. Several studies have suggested that capacity to metabolize iAs may vary depending on levels of iAs exposure. However, it is not known whether the influence of variants in AS3MT on iAs metabolism also vary by level of exposure. We investigated, in a population of Mexican adults exposed to drinking water As, whether associations between 7 candidate variants in AS3MT and urinary iAs metabolites were consistent with prior studies, and whether these associations varied depending on the level of exposure. Overall, associations between urinary iAs metabolites and AS3MT variants were consistent with the literature. Referent genotypes, defined as the genotype previously associated with a higher percentage of urinary dimethylated As (DMAs%), were associated with significant increases in the DMAs% and ratio of DMAs to monomethylated As (MAs), and significant reductions in MAs% and iAs%. For 3 variants, associations between genotypes and iAs metabolism were significantly stronger among subjects exposed to water As >50 versus ≤50 ppb (water As X genotype interaction P < .05). In contrast, for 1 variant (rs17881215), associations were significantly stronger at exposures ≤50 ppb. Results suggest that iAs exposure may influence the extent to which several AS3MT variants affect iAs metabolism. The variants most strongly associated with iAs metabolism-and perhaps with susceptibility to iAs-associated disease-may vary in settings with exposure level.

Biological and behavioral factors modify urinary arsenic metabolic profiles in a U.S. population.

BACKGROUND: Because some adverse health effects associated with chronic arsenic exposure may be mediated by methylated arsenicals, interindividual variation in capacity to convert inorganic arsenic into mono- and di-methylated metabolites may be an important determinant of risk associated with exposure to this metalloid. Hence, identifying biological and behavioral factors that modify an individual's capacity to methylate inorganic arsenic could provide insights into critical dose-response relations underlying adverse health effects. METHODS: A total of 904 older adults (≥45 years old) in Churchill County, Nevada, who chronically used home tap water supplies containing up to 1850 µg of arsenic per liter provided urine and toenail samples for determination of total and speciated arsenic levels. Effects of biological factors (gender, age, body mass index) and behavioral factors (smoking, recent fish or shellfish consumption) on patterns of arsenicals in urine were evaluated with bivariate analyses and multivariate regression models. RESULTS: Relative contributions of inorganic, mono-, and di-methylated arsenic to total speciated arsenic in urine were unchanged over the range of concentrations of arsenic in home tap water supplies used by study participants. Gender predicted both absolute and relative amounts of arsenicals in urine. Age predicted levels of inorganic arsenic in urine and body mass index predicted relative levels of mono- and di-methylated arsenic in urine. Smoking predicted both absolute and relative levels of arsenicals in urine. Multivariate regression models were developed for both absolute and relative levels of arsenicals in urine. Concentration of arsenic in home tap water and estimated water consumption were strongly predictive of levels of arsenicals in urine as were smoking, body mass index, and gender. Relative contributions of arsenicals to urinary arsenic were not consistently predicted by concentrations of arsenic in drinking water supplies but were more consistently predicted by gender, body mass index, age, and smoking. CONCLUSIONS: These findings suggest that analyses of dose-response relations in arsenic-exposed populations should account for biological and behavioral factors that modify levels of inorganic and methylated arsenicals in urine. Evidence of significant effects of these factors on arsenic metabolism may also support mode of action studies in appropriate experimental models.

Expression of the Longest RGS4 Splice Variant in the Prefrontal Cortex Is Associated with Single Nucleotide Polymorphisms in Schizophrenia Patients.

The Regulator of G protein signaling 4 (RGS4) gene is a candidate susceptibility gene for schizophrenia (SCZ). Previous studies showed that the mRNA level of the longest splice variant RGS4-1 was decreased in the dorsolateral prefrontal cortex (DLPFC) of SCZ patients compared with healthy controls. In this pilot study, we examined the possible mechanisms of RGS4-1 mRNA reduction in SCZ. We genotyped SNP1 (rs10917670), rs2661347, SNP4 (rs951436), SNP7 (rs951439), SNP18 (rs2661319), and rs10799897 (SNP9897) and tested the methylation status of CpG islands of the RGS4 gene in the postmortem DLPFC samples obtained from subjects with SCZ and bipolar disorder as well as healthy controls. RGS4-1 mRNA level was associated with five SNPs (SNP1, rs2661347, SNP4, SNP7, and SNP18) and their haplotypes but not with SNP9897. In addition, this study revealed that RGS4-1 mRNA was low in subjects with specific genotypes of SNP1, rs2661347, SNP4, SNP7, and SNP18. Lower RGS4-1 mRNA expression in the DLPFC of SCZ is associated with SNPs in the 5' regulatory region of the RGS4 gene but not with the methylation status of its CpG islands.

Analysis of maternal polymorphisms in arsenic (+3 oxidation state)-methyltransferase AS3MT and fetal sex in relation to arsenic metabolism and infant birth outcomes: Implications for risk analysis.

Arsenic (+3 oxidation state) methyltransferase (AS3MT) is the key enzyme in the metabolism of inorganic arsenic (iAs). Polymorphisms of AS3MT influence adverse health effects in adults, but little is known about their role in iAs metabolism in pregnant women and infants. The relationships between seven single nucleotide polymorphisms (SNPs) in AS3MT and urinary concentrations of iAs and its methylated metabolites were assessed in mother-infant pairs of the Biomarkers of Exposure to ARsenic (BEAR) cohort. Maternal alleles for five of the seven SNPs (rs7085104, rs3740400, rs3740393, rs3740390, and rs1046778) were associated with urinary concentrations of iAs metabolites, and alleles for one SNP (rs3740393) were associated with birth outcomes/measures. These associations were strongly dependent upon the male sex of the fetus but independent of fetal genotype for AS3MT. These data highlight a potential sex-dependence of the relationships among maternal genotype, iAs metabolism and infant health outcomes.

Chronic Exposure to Arsenic and Markers of Cardiometabolic Risk: A Cross-Sectional Study in Chihuahua, Mexico.

BACKGROUND: Exposure to arsenic (As) concentrations in drinking water > 150 µg/L has been associated with risk of diabetes and cardiovascular disease, but little is known about the effects of lower exposures. OBJECTIVE: This study aimed to examine whether moderate As exposure, or indicators of individual As metabolism at these levels of exposure, are associated with cardiometabolic risk. METHODS: We analyzed cross-sectional associations between arsenic exposure and multiple markers of cardiometabolic risk using drinking-water As measurements and urinary As species data obtained from 1,160 adults in Chihuahua, Mexico, who were recruited in 2008-2013. Fasting blood glucose and lipid levels, the results of an oral glucose tolerance test, and blood pressure were used to characterize cardiometabolic risk. Multivariable logistic, multinomial, and linear regression were used to assess associations between cardiometabolic outcomes and water As or the sum of inorganic and methylated As species in urine. RESULTS: After multivariable adjustment, concentrations in the second quartile of water As (25.5 to < 47.9 µg/L) and concentrations of total speciated urinary As (< 55.8 µg/L) below the median were significantly associated with elevated triglycerides, high total cholesterol, and diabetes. However, moderate water and urinary As levels were also positively associated with HDL cholesterol. Associations between arsenic exposure and both dysglycemia and triglyceridemia were higher among individuals with higher proportions of dimethylarsenic in urine. CONCLUSIONS: Moderate exposure to As may increase cardiometabolic risk, particularly in individuals with high proportions of urinary dimethylarsenic. In this cohort, As exposure was associated with several markers of increased cardiometabolic risk (diabetes, triglyceridemia, and cholesterolemia), but exposure was also associated with higher rather than lower HDL cholesterol. CITATION: Mendez MA, González-Horta C, Sánchez-Ramírez B, Ballinas-Casarrubias L, Hernández Cerón R, Viniegra Morales D, Baeza Terrazas FA, Ishida MC, Gutiérrez-Torres DS, Saunders RJ, Drobná Z, Fry RC, Buse JB, Loomis D, García-Vargas GG, Del Razo LM, Stýblo M. 2016. Chronic exposure to arsenic and markers of cardiometabolic risk: a cross-sectional study in Chihuahua, Mexico. Environ Health Perspect 124:104-111; http://dx.doi.org/10.1289/ehp.1408742.

Identification of novel gene targets and putative regulators of arsenic-associated DNA methylation in human urothelial cells and bladder cancer.

There is strong epidemiologic evidence linking chronic exposure to inorganic arsenic (iAs) to myriad adverse health effects, including cancer of the bladder. We set out to identify DNA methylation patterns associated with arsenic and its metabolites in exfoliated urothelial cells (EUCs) that originate primarily from the urinary bladder, one of the targets of arsenic-induced carcinogenesis. Genome-wide, gene-specific promoter DNA methylation levels were assessed in EUCs from 46 residents of Chihuahua, Mexico, and the relationship was examined between promoter methylation profiles and the intracellular concentrations of total arsenic and arsenic species. A set of 49 differentially methylated genes was identified with increased promoter methylation associated with EUC tAs, iAs, and/or monomethylated As (MMAs) enriched for their roles in metabolic disease and cancer. Notably, no genes had differential methylation associated with EUC dimethylated As (DMAs), suggesting that DMAs may influence DNA methylation-mediated urothelial cell responses to a lesser extent than iAs or MMAs. Further analysis showed that 22 of the 49 arsenic-associated genes (45%) are also differentially methylated in bladder cancer tissue identified using The Cancer Genome Atlas repository. Both the arsenic- and cancer-associated genes are enriched for the binding sites of common transcription factors known to play roles in carcinogenesis, demonstrating a novel potential mechanistic link between iAs exposure and bladder cancer.

A concurrent exposure to arsenic and fluoride from drinking water in Chihuahua, Mexico.

Inorganic arsenic (iAs) and fluoride (F-) are naturally occurring drinking water contaminants. However, co-exposure to these contaminants and its effects on human health are understudied. The goal of this study was examined exposures to iAs and F- in Chihuahua, Mexico, where exposure to iAs in drinking water has been associated with adverse health effects. All 1119 eligible Chihuahua residents (>18 years) provided a sample of drinking water and spot urine samples. iAs and F- concentrations in water samples ranged from 0.1 to 419.8 µg As/L and from 0.05 to 11.8 mg F-/L. Urinary arsenic (U-tAs) and urinary F- (U-F-) levels ranged from 0.5 to 467.9 ng As/mL and from 0.1 to 14.4 µg F-/mL. A strong positive correlation was found between iAs and F- concentrations in drinking water (rs = 0.741). Similarly, U-tAs levels correlated positively with U-F- concentrations (rs = 0.633). These results show that Chihuahua residents exposed to high iAs concentrations in drinking water are also exposed to high levels of F-, raising questions about possible contribution of F- exposure to the adverse effects that have so far been attributed only to iAs exposure. Thus, investigation of possible interactions between iAs and F- exposures and its related health risks deserves immediate attention.

Metabolomic characteristics of arsenic-associated diabetes in a prospective cohort in Chihuahua, Mexico.

Chronic exposure to inorganic arsenic (iAs) has been linked to an increased risk of diabetes, yet the specific disease phenotype and underlying mechanisms are poorly understood. In the present study we set out to identify iAs exposure-associated metabolites with altered abundance in nondiabetic and diabetic individuals in an effort to understand the relationship between exposure, metabolomic response, and disease status. A nested study design was used to profile metabolomic shifts in urine and plasma collected from 90 diabetic and 86 nondiabetic individuals matched for varying iAs concentrations in drinking water, body mass index, age, and sex. Diabetes diagnosis was based on measures of fasting plasma glucose and 2-h blood glucose. Multivariable models were used to identify metabolites with altered abundance associated with iAs exposure among diabetic and nondiabetic individuals. A total of 132 metabolites were identified to shift in urine or plasma in response to iAs exposure characterized by the sum of iAs metabolites in urine (U-tAs). Although many metabolites were altered in both diabetic and nondiabetic 35 subjects, diabetic individuals displayed a unique response to iAs exposure with 59 altered metabolites including those that play a role in tricarboxylic acid cycle and amino acid metabolism. Taken together, these data highlight the broad impact of iAs exposure on the human metabolome, and demonstrate some specificity of the metabolomic response between diabetic and nondiabetic individuals. These data may provide novel insights into the mechanisms and phenotype of diabetes associated with iAs exposure.

Maternal arsenic exposure, arsenic methylation efficiency, and birth outcomes in the Biomarkers of Exposure to ARsenic (BEAR) pregnancy cohort in Mexico.

BACKGROUND: Exposure to inorganic arsenic (iAs) from drinking water is a global public health problem, yet much remains unknown about the extent of exposure in susceptible populations. OBJECTIVES: We aimed to establish the Biomarkers of Exposure to ARsenic (BEAR) prospective pregnancy cohort in Gómez Palacio, Mexico, to better understand the effects of iAs exposure on pregnant women and their children. METHODS: Two hundred pregnant women were recruited for this study. Concentrations of iAs in drinking water (DW-iAs) and maternal urinary concentrations of iAs and its monomethylated and dimethylated metabolites (MMAs and DMAs, respectively) were determined. Birth outcomes were analyzed for their relationship to DW-iAs and to the concentrations and proportions of maternal urinary arsenicals. RESULTS: DW-iAs for the study subjects ranged from < 0.5 to 236 µg As/L. More than half of the women (53%) had DW-iAs that exceeded the World Health Organization's recommended guideline of 10 µg As/L. DW-iAs was significantly associated with the sum of the urinary arsenicals (U-tAs). Maternal urinary concentrations of MMAs were negatively associated with newborn birth weight and gestational age. Maternal urinary concentrations of iAs were associated with lower mean gestational age and newborn length. CONCLUSIONS: Biomonitoring results demonstrate that pregnant women in Gómez Palacio are exposed to potentially harmful levels of DW-iAs. The data support a relationship between iAs metabolism in pregnant women and adverse birth outcomes. The results underscore the risks associated with iAs exposure in vulnerable populations.

Prenatal arsenic exposure and the epigenome: identifying sites of 5-methylcytosine alterations that predict functional changes in gene expression in newborn cord blood and subsequent birth outcomes.

Prenatal exposure to inorganic arsenic (iAs) is detrimental to the health of newborns and increases the risk of disease development later in life. Here we examined a subset of newborn cord blood leukocyte samples collected from subjects enrolled in the Biomarkers of Exposure to ARsenic (BEAR) pregnancy cohort in Gómez Palacio, Mexico, who were exposed to a range of drinking water arsenic concentrations (0.456-236 µg/l). Changes in iAs-associated DNA 5-methylcytosine methylation were assessed across 424,935 CpG sites representing 18,761 genes and compared with corresponding mRNA expression levels and birth outcomes. In the context of arsenic exposure, a total of 2919 genes were identified with iAs-associated differences in DNA methylation. Site-specific analyses identified DNA methylation changes that were most predictive of gene expression levels where CpG methylation within CpG islands positioned within the first exon, the 5' untranslated region and 200 bp upstream of the transcription start site yielded the most significant association with gene expression levels. A set of 16 genes was identified with correlated iAs-associated changes in DNA methylation and mRNA expression and all were highly enriched for binding sites of the early growth response (EGR) and CCCTC-binding factor (CTCF) transcription factors. Furthermore, DNA methylation levels of 7 of these genes were associated with differences in birth outcomes including gestational age and head circumference.These data highlight the complex interplay between DNA methylation, functional changes in gene expression and health outcomes and underscore the need for functional analyses coupled to epigenetic assessments.