Developmental Arsenic Exposure Research Articles

Integrated miRNA-mRNA analysis reveals the dysregulation of lipid metabolism in mouse liver induced by developmental arsenic exposure.

Developmental arsenic exposure leads to increased susceptibility to liver diseases including nonalcoholic fatty liver diseases, but the mechanism is incompletely understood. In this study, C57BL/6J mice were used to establish a lifetime arsenic exposure model covering developmental stage. We found that arsenic-exposed offspring in later life showed hepatic lipid deposition and increased triglyceride content. Despite no significant hepatic pathological changes in the offspring at weaning, 86 miRNAs and 136 mRNAs were differentially expressed according to miRNA array and mRNA sequencing. The differentially expressed genes (DEGs) were crossed with the target genes predicted by differentially expressed miRNAs (DEMs), and 47 differentially expressed target genes (DETGs) were obtained. Functional annotation suggested that lipid metabolism related pathways were significantly enriched. The pivotal regulator in the four major pathways to maintain liver lipid homeostasis were further determined, with significant alterations found in FABP5, SREBP1, ACOX1 and EHHADH. Of note, miRNA-mRNA integration analysis revealed that miR-7118-5p, miR-7050-5p, miR-27a/b-3p, and miR-103-3p acted as key regulators of fatty acid metabolism genes. Taken together, miRNA-mRNA integration analysis indicates that the lipid metabolism pathway in the liver of weaned mice was dysregulated by developmental arsenic exposure, which may contribute to the development of NAFLD in later life.

Gestational arsenic exposure induces anxiety-like behaviors in F1 female mice by dysregulation of neurological and immunological markers.

Arsenic is a harmful heavy metal and a well-known developmental neurotoxicant. Previously, we have reported that gestational arsenic exposure resulted in impaired social behaviors in F1 and F2 male mice. However, little is known about the developmental arsenic exposure on anxiety-like behavior. This study aimed to detect the effect of gestational arsenic exposure on anxiety-like behavior and related gene expressions in 74-week-old F1 female mice. Pregnant C3H/HeN mice (F0) were given drinking water containing 85 ppm sodium arsenite (NaAsO2) from gestational day 8 to 18. The control mice were given tap water only. At 74-week-old, open field test was performed, then anxiety and apoptosis-related factors were determined by real_time RT_PCR and immunohistochemical analyses. The arsenite-exposed F1 female mice showed decreased center entry and center time in open field test. In addition, the number of grooming and fecal pallet was significantly increased in the arsenite-exposed F1 female mice compared to the control. Downregulation of brain-derived neurotrophic factor (BDNF), serotonin receptor (5HT1A) and upregulation of nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB), interleukin 1 β (IL-1β), cyclooxygenase 2 (COX2), caspase-3, Bcl2-associated X protein (Bax) were detected in the prefrontal cortex in the arsenite-exposed F1 female mice. Microglial marker ionized calcium-binding adapter molecule 1 (Iba1)-positive cells were increased in the arsenite-exposed F1 female mice. Moreover, a significantly increased plasma corticosterone level was observed in the arsenic-exposed F1 female mice. This study suggested that gestational arsenic exposure induced anxiety-like behavior accompanied with dysregulation of neurological and immunological markers, neuroinflammatory responses, neuronal apoptosis, and decreased neurogenesis in the prefrontal cortex of F1 female mice.

Nrf2 activation contributes to hepatic tumor-augmenting effects of developmental arsenic exposure.

Developmental arsenic exposure increases cancer risk in later life with the mechanism elusive. Oxidative stress is a dominant determinant in arsenic toxicity. However, the role of Nrf2, a key regulator in antioxidative response, in tumor-augmenting effects by developmental arsenic exposure is unclear. In the present study, wild-type C57BL/6J and Nrf2-konckout (Nrf2-KO) were developmentally exposed to inorganic arsenic via drinking water. For hepatic tumorigenesis analysis, mice were intraperitoneally injected with diethylnitrosamine (DEN) at two weeks of age. Developmental arsenic exposure aggravated tumor multiplicity and burden, and expression of PCNA and AFP in hepatic tumors induced by DEN. Nrf2 activation as indicated by over-expression of Nrf2 and its downstream genes, including Gss, Gsr, p62, Gclc and Gclm, was found in liver tumors, as well as in the livers in developmentally arsenic-exposed pups at weaning. Notably, Nrf2 deficiency attenuated tumor-augmenting effects and over-expression of Nrf2 downstream genes due to developmental arsenic exposure. Furthermore, the levels of urinary DEN metabolite (acetaldehyde) and hepatic DNA damage markers (O6-ethyl-2-deoxyguanosine adducts and γ-histone H2AX) after DEN treatment were elevated by Nrf2 agonist, 2-Cyano-3,12-dioxooleana-1,9-dien-28-imidazolide. Collectively, our data suggest that augmentation of DEN-induced hepatic tumorigenesis by developmental arsenic exposure is dependent on Nrf2 activation, which may be related to the role of Nrf2 in DEN metabolic activation. Our findings reveal, at least in part, the mechanism underlying increased susceptibility to developing cancer due to developmental arsenic exposure.

Developmental arsenic exposure induces dysbiosis of gut microbiota and disruption of plasma metabolites in mice

Arsenic is a notorious environmental pollutant. Of note, developmental arsenic exposure has been found to increase the risk of developing a variety of ailments later in life, but the underlying mechanism is not well understood. Many elements of host health have been connected to the gut microbiota. It is still unclear whether and how developmental arsenic exposure affects the gut microbiota. In the present study, we found that developmental arsenic exposure changed intestinal morphology and increased intestinal permeability and inflammation in mouse pups at weaning. These alterations were accompanied by a significant change in gut microbiota, as evidenced by considerably reduced gut microbial richness and diversity. In developmentally arsenic-exposed pups, the relative abundance of Muribaculaceae was significantly decreased, while the relative abundance of Akkermansia and Bacteroides was significantly enhanced at the genus level. Metabolome and pathway enrichment analyses indicated that amino acid and purine metabolism was promoted, while glycerophospholipid metabolism was inhibited. Interestingly, the relative abundance of Muribaculaceae and Akkermansia showed a strong correlation with most plasma metabolites significantly altered by developmental arsenic exposure. These data indicate that gut microbiota dysbiosis may be a critical link between developmental arsenic exposure and metabolic disorders and shed light on the mechanisms underlying increased susceptibility to diseases due to developmental arsenic exposure.

Developmental arsenic exposure impairs cognition, directly targets DNMT3A, and reduces DNA methylation.

Developmental arsenic exposure has been associated with cognitive deficits in epidemiological studies, but the underlying mechanisms remain poorly understood. Here, we establish a mouse model of developmental arsenic exposure exhibiting deficits of recognition and spatial memory in the offspring. These deficits are associated with genome-wide DNA hypomethylation and abnormal expression of cognition-related genes in the hippocampus. Arsenic atoms directly bind to the cysteine-rich ADD domain of DNA methyltransferase 3A (DNMT3A), triggering ubiquitin- and proteasome-mediated degradation of DNMT3A in different cellular contexts. DNMT3A degradation leads to genome-wide DNA hypomethylation in mouse embryonic fibroblasts but not in non-embryonic cell lines. Treatment with metformin, a first-line antidiabetic agent reported to increase DNA methylation, ameliorates the behavioral deficits and normalizes the aberrant expression of cognition-related genes and DNA methylation in the hippocampus of arsenic-exposed offspring. Our study establishes a DNA hypomethylation effect of developmental arsenic exposure and proposes a potential treatment against cognitive deficits in the offspring of pregnant women in arsenic-contaminated areas.

Minocycline reverses developmental arsenic exposure-induced microglia activation and functional alteration in BALB/c mice

Arsenic activates microglia and exerts bystander effects on neuron. The present study is focused to test whether minocycline, a second generation antibiotic, can reverse the effect of developmental arsenic exposure on microglial activation and function. Pregnant Balb/c dams were gavaged with sodium arsenite (0.38 mg/kg bd wt) from gestational day 5 (GD5) till post natal day 21 (PND21) and then one group of pups continued till PND59 with arsenic gavage. Minocycline (33 mg/kg bd wt) was administered intraperitoneally two weeks till sacrifice, every alternate day. Mice were sacrificed on PND22 and PND60 and used for various assays. Primary microglial were isolated (ex vivo microglia) from experimental animals and used to measure reactive oxygen species (ROS), nitric oxide (NO), cytokine production and phagocytosis. The whole brain lysate was used for western blot analysis of microglial marker CD68 and synaptic marker, post synaptic density protein 95 (PSD95). For real-time PCR analysis of triggering receptor expressed on myeloid cells 2 (TREM2) and PSD95, RNA isolated from whole brain was used. The study reveals that minocycline administration reversed arsenic-induced increased expression of CD68, ROS, NO, cytokine production, phagocytosis and TREM2 expression. Arsenic-induced reduced expression of PSD95 protein was reversed by minocycline, although the mRNA of PSD95 was unaltered among different groups. Finally, we have checked the learning and memory response of the experimental animals using Y-maze test to correlate the arsenic-induced altered level of synaptic protein. Taken together, the present study finds minocycline to reduce arsenic-induced microglial activation and function which in turn reverses the arsenic-induced impaired learning and memory response.

Genome-wide alteration of histone methylation profiles associated with cognitive changes in response to developmental arsenic exposure in mice

Inorganic arsenic is a xenobiotic entering the body primarily through contaminated drinking water and food. There are defined mechanisms that describe arsenic’s association with increased cancer incidence, however mechanisms explaining arsenic exposure and neurodevelopmental or aging disorders are poorly defined. In recent years, arsenic effects on epigenome have become a particular focus. We hypothesize that human relevant arsenic exposure during particular developmental windows, or long-term exposure later in life induce pathophysiological neural changes through epigenomic alterations, in particular histone methylation profile, manifesting as cognitive decline. C57BL/6 wild-type mice were continually exposed to sodium arsenite (100 µg/L) in drinking water prior to mating through weaning of the experimental progeny. A second cohort of aged APP/PS mice were chronically exposed to the same level of arsenic. Cognitive testing, histological examination of brains and genome-wide methylation levels of H3K4me3 and H3K27me3 examined after ChIP-seq were used to determine the effects of arsenic exposure. Developmental arsenic exposure caused significantly diminished cognition in wild-type mice. The analysis of ChIP-seq data and experiments with mouse embryonic stem cells demonstrated that epigenetic changes induced by arsenic exposure translated into gene expression alterations associated with neuronal development and neurological disease. Increased hippocampal amyloid plaques levels of APP/PS mice and cognitive decline provided evidence that arsenic exposure aggravated an existing Alzheimer’s disease-like phenotype. We show developmental arsenic exposure significantly impacts histone modifications in brain which remain present into adulthood and provide a potential mechanism by which developmental arsenic exposure influences cognitive functions. We also show that human relevant, chronic arsenic exposure has deleterious effects on adult APP/PS mice and exacerbates existing Alzheimer’s disease-like symptoms. The results demonstrate how developmental arsenic exposure impacts the brain epigenome, leading to altered gene expression later in life.

Integrated Mirna-Mrna Analysis Reveals the Dysregulation of Lipid Metabolism in Mouse Liver Induced by Developmental Arsenic Exposure

Integrated Mirna-Mrna Analysis Reveals the Dysregulation of Lipid Metabolism in Mouse Liver Induced by Developmental Arsenic Exposure

Early Low-Level Arsenic Exposure Impacts Post-Synaptic Hippocampal Function in Juvenile Mice.

Arsenic is a well-established carcinogen known to increase mortality, but its effects on the central nervous system are less well understood. Epidemiological studies suggest that early life exposure is associated with learning deficits and behavioral changes. Studies in arsenic-exposed rodents have begun to shed light on potential mechanistic underpinnings, including changes in synaptic transmission and plasticity. However, previous studies relied on extended exposure into adulthood, and little is known about the effect of arsenic exposure in early development. Here, we studied the effects of early developmental arsenic exposure in juvenile mice on synaptic transmission and plasticity in the hippocampus. C57BL/6J females were exposed to arsenic (0, 50 ppb, 36 ppm) via drinking water two weeks prior to mating, with continued exposure throughout gestation and parturition. Electrophysiological recordings were then performed on juvenile offspring prior to weaning. In this paradigm, the offspring are exposed to arsenic indirectly, via the mother. We found that high (36 ppm) and relatively low (50 ppb) arsenic exposure both decreased basal synaptic transmission. A compensatory increase in pre-synaptic vesicular release was only observed in the high-exposure group. These results suggest that indirect, ecologically relevant arsenic exposure in early development impacts hippocampal synaptic transmission and plasticity that could underlie learning deficits reported in epidemiological studies.

Effects of maternal exposure to arsenic on social behavior and related gene expression in F2 male mice

BackgroundArsenic is a developmental neurotoxicant. It means that its neurotoxic effect could occur in offspring by maternal arsenic exposure. Our previous study showed that developmental arsenic exposure impaired social behavior and serotonergic system in C3H adult male mice. These effects might affect the next generation with no direct exposure to arsenic. This study aimed to detect the social behavior and related gene expression changes in F2 male mice born to gestationally arsenite-exposed F1 mice.MethodsPregnant C3H/HeN mice (F0) were given free access to tap water (control mice) or tap water containing 85 ppm sodium arsenite from days 8 to 18 of gestation. Arsenite was not given to F1 or F2 mice. The F2 mice were generated by mating among control F1 males and females, and arsenite-F1 males and females at the age of 10 weeks. At 41 weeks and 74 weeks of age respectively, F2 males were used for the assessment of social behavior by a three-chamber social behavior apparatus. Histological features of the prefrontal cortex were studied by ordinary light microscope. Social behavior-related gene expressions were determined in the prefrontal cortex by real time RT-PCR method.ResultsThe arsenite-F2 male mice showed significantly poor sociability and social novelty preference in both 41-week-old group and 74-week-old group. There was no significant histological difference between the control mice and the arsenite-F2 mice. Regarding gene expression, serotonin receptor 5B (5-HT 5B) mRNA expression was significantly decreased (p < 0.05) in the arsenite-F2 male mice compared to the control F2 male mice in both groups. Brain-derived neurotrophic factor (BDNF) and dopamine receptor D1a (Drd1a) gene expressions were significantly decreased (p < 0.05) only in the arsenite-F2 male mice of the 74-week-old group. Heme oxygenase-1 (HO-1) gene expression was significantly increased (p < 0.001) in the arsenite-F2 male mice of both groups, but plasma 8-hydroxy-2′-deoxyguanosine (8-OHdG) and cyclooxygenase-2 (COX-2) gene expression were not significantly different. Interleukin-1β (IL-1β) mRNA expression was significantly increased only in 41-week-old arsenite-F2 mice.ConclusionsThese findings suggest that maternal arsenic exposure affects social behavior in F2 male mice via serotonergic system in the prefrontal cortex. In this study, COX-2 were not increased although oxidative stress marker (HO-1) was increased significantly in arsnite-F2 male mice.

Developmental arsenic exposure is associated with sex differences in the epigenetic regulation of stress genes in the adult mouse frontal cortex

Previously, we reported sex-dependent changes to global histone modifications and impairment of executive function in adult mice exposed to moderate levels of arsenic during early development. The present study investigated the effects of developmental arsenic exposure (DAE) on gene specific histone 3 lysine 9 acetylation (H3K9ac) and tri-methylation (H3K9me3) in the frontal cortex of male and female adult C57BL/6J mice. Dams were exposed to 50ppb arsenic in drinking water prior to and throughout gestation, and until pup weaning; once weened, the pups were given tap water (<5ppb arsenic) and raised to postnatal day 70. Using chromatin immunoprecipitation and qPCR, we assessed changes in H3K9 regulatory markers and mRNA expression levels of the stress signaling genes, Nr3c1, Crh, Crhr1, Hsd11b1, and Fkbp5. In female DAE mice, H3K9ac association with the Crh promoter was increased and this corresponded with increased Crh mRNA expression. Crhr1 mRNA expression was increased in female DAE mice, while expression was reduced in male DAE mice. DAE resulted in chromatin and mRNA changes associated with decreased glucocorticoid signaling in male but not female frontal cortex: increased H3K9ac association with Fkbp5 and a corresponding increase in Fkbp5 mRNA expression. In addition, there was a decrease of Hsd11b1 mRNA expression in DAE males. These results suggest a sex-dependent response to arsenic during embryonic development leading to different patterns of gene regulation and expression.

Potential facet for prenatal arsenic exposure paradigm: linking endocrine disruption and epigenetics

Environmental exposure to toxicants/heavy metals during critical periods of development can influence changes in embryo and germline of the offspring; and later on affect the disease susceptibility in adults. Exposures to toxic metals or endocrine disruptors are particularly harmful during fetal development. Arsenic, a well-known toxic metalloid and reproductive toxicant is one the major concern because of its adverse and delayed health effects. Considering the complex and numerous adverse health effects of prenatal arsenic exposure, it is very difficult to identify the one single mechanism for arsenic-induced toxicity. This is further complicated due to biphasic response reported where arsenic has very different effects at low and high doses particularly during early life exposure scenario. In this review, we are focusing on prenatal arsenic exposure and its lifelong adverse effects, and their association with endocrine disruption and epigenetic changes. We provide evidence that developmental arsenic exposure alters the functional fetal epigenome in a tissue-specific manner by alterations in DNA methylation patterns, histone modifications and changes in micro RNA. Arsenic as an endocrine disruptor also affects the reproductive potential of the organism. These adverse effects of arsenic could manifest directly through classical hormone imprinting or through irreversible epigenetic modulation. Thus, understanding the association of epigenetic changes and endocrine disruption by prenatal arsenic exposure may help unravel the crucial mechanism for the development of disease later in life.

Effects of Developmental Arsenic Exposure on the Social Behavior and Related Gene Expression in C3H Adult Male Mice.

Arsenic is carcinogenic and teratogenic. In addition, it is also a developmental neurotoxicant. Little is known however about the effect of arsenic exposure during brain development on social behavior. This study aimed to detect the effect of developmental arsenic exposure on social behavior and related gene expression in C3H adult male mice. Pregnant C3H mice were exposed to sodium arsenite (NaAsO2, 85 ppm in the drinking water) from gestational day (GD) 8 to 18. The F1 generation male pups from different mothers were taken and social behavior tasks were examined. Social behavioral-related gene expression in the prefrontal cortex was determined by the real-time RT-PCR method. The mice with developmental arsenic exposure showed poor sociability and poor social novelty preference. Glutamate receptor expression (NMDA and AMPA receptor subunits) showed no significant difference, but gene expressions of serotonin receptor 5B (5-HT 5B) and brain-derived neurotrophic factor (BDNF) were significantly decreased (p < 0.05) in the arsenic-exposed group compared to control group. The heme oxygenase-1 (HO-1) and cyclooxygenase-2 (COX-2) gene expressions were not significantly different. Our findings indicate that developmental arsenic exposure might affect social behavior by modulating serotonin receptors and reducing BDNF. Some oxidative stress markers and inflammatory markers were not affected.

Sex-Dependent Effects of the Histone Deacetylase Inhibitor, Sodium Valproate, on Reversal Learning After Developmental Arsenic Exposure.

Several studies have demonstrated that exposure to arsenic in drinking water adversely affects brain development and cognitive function in adulthood. While the mechanism by which arsenic induces adverse neurological outcomes remains elusive, studies suggest a link between reduced levels of histone acetylation and impaired performance on a variety of behavioral tasks following arsenic exposure. Using our developmental arsenic exposure (DAE) paradigm, we have previously reported reduced histone acetylation and associated histone acetyltransferase enzyme expression in the frontal cortex of C57BL/6J adult male mice, with no changes observed in the female frontal cortex. In the present study, we sought to determine if DAE produced sex-dependent deficits in frontal cortical executive function using the Y-maze acquisition and reversal learning tasks, which are specific for assessing cognitive flexibility. Further, we tested whether the administration of valproic acid, a class I–IIa histone deacetylase inhibitor, was able to mitigate behavioral and biochemical changes resulting from DAE. As anticipated, DAE inhibited acquisition and reversal learning performance in adult male, but not female, mice. Valproate treatment for 2 weeks restored reversal performance in the male arsenic-exposed offspring, while not affecting female performance. Protein levels of HDACs 1, 2, and 5 were elevated following behavioral assessment but only in DAE male mice; restoration of appropriate HDAC levels occurred after valproate treatment and was concurrent with improved behavioral performance, particularly during reversal learning. Female frontal cortical levels of HDAC enzymes were not impacted by DAE or valproate treatment. Finally, mRNA expression levels of brain-derived neurotrophic factor, Bdnf, which has been implicated in the control of frontal cortical flexibility and is regulated by HDAC5, were elevated in DAE male mice and restored to normal levels following HDACi treatment. Levels of mRNA encoding glutamate receptor ionotropic NMDA type subunits, which have been linked to cognitive flexibility, were not related to the reversal learning deficit in the DAE mice and were not altered by HDACi treatments. These findings demonstrate that DAE alters frontal cortical HDAC levels and Bdnf expression in males, but not females, and that these molecular changes are associated with sex-dependent differences in cognitive flexibility in a reversal-learning task.

Height and blood chemistry in adults with a history of developmental arsenic poisoning from contaminated milk powder

BackgroundArsenic poisoning interferes with bone metabolism in laboratory animal studies, and human studies suggest lowered bone mass density at elevated exposures. As the long-term consequences of developmental arsenic toxicity are poorly known, we carried out a clinical pilot study of survivors of the mass arsenic poisoning of bottle-fed infants in Japan in 1955. ObjectivesThe purpose was to evaluate the association between developmental arsenic exposure and physical stature and routine blood chemistry reflecting major organ functions more than 50 years later. MethodsThe study sample consisted of 50 individuals recruited at two hospitals in Okayama Prefecture, Japan: 27 known poisoning victims (14 men and 13 women), and 23 non-exposed local controls of similar age (10 men, 13 women). We collected information from physical examinations that included routine blood counts and blood biochemistry. ResultsThe average height of the exposed group was 6.5cm below that of the unexposed group (p=0.02), while the latter was in accordance with national data for both sexes. In addition, the exposed participants had a higher mean (SD) serum concentration of alkaline phosphatase (ALP) of 233 (63) U/L than the unexposed participants (191 (44) U/L) (p=0.01). No other statistically significant difference was observed, and liver enzymes were within normal ranges. ConclusionsAdults who had suffered arsenic poisoning during infancy showed decreased height and elevated ALP that suggests abnormalities in bone metabolism possibly induced by arsenic incorporated in the bone matrix.

Neurological and neuropsychological functions in adults with a history of developmental arsenic poisoning from contaminated milk powder

During the summer of 1955, mass arsenic poisoning of bottle-fed infants occurred in the western part of Japan due to contaminated milk powder, and more than 100 died; some childhood victims were later found to suffer from neurological sequelae in adolescence. This unique incident enabled us to explore infancy as a critical period of arsenic exposure in regard to developmental neurotoxicity and its possible persistence through adulthood. The purpose of this work is to evaluate the association between developmental arsenic exposure and the neurological outcomes more than 50years later. We conducted a retrospective cohort study during the period from April 2012 to February 2013 in two hospitals in Okayama Prefecture, Japan. The study sample consisted of 50 individuals: 27 known poisoning victims from Okayama Prefecture, and 23 non-exposed local controls of similar age. In addition to neurological examination, we adapted a battery of neurophysiological and neuropsychological tests to identify the types of brain functions affected by early-life arsenic exposure. While limited abnormalities were found in the neurophysiological tests, neuropsychological deficits were observed. Except for Finger tapping, all test scores in the exposed group – Vocabulary and Block Design from Wechsler Adults Intelligent Scale III, Design memory subtest from Wide Range Assessment of Memory and Learning 2, and Grooved pegboard test – were substantially below those obtained by the unexposed. The exposed group showed average performance at least 1.2 standard deviations below the average for the controls. Exposed participants performed less well than controls, even after exclusion of subjects with recognized disabilities or those with a high level of education. Adults who had suffered arsenic poisoning during infancy revealed neuropsychological dysfunctions, even among those subjects not recognized as having disabilities. Developmental neurotoxicity due to arsenic likely results in permanent changes in brain functions.

ChIP-Seq analysis of the adult male mouse brain after developmental exposure to arsenic.

Exposure to the common environmental contaminant arsenic impacts the epigenetic landscape, including DNA methylation and histone modifications, of several cell types. Developmental arsenic exposure (DAE) increases acetylation and methylation of histone proteins and the protein expression of several chromatin-modifying enzymes in the dentate gyrus (DG) subregion of the adult male mouse brain [26]. To complement and support these data, ChIP-Seq analysis of DNA associated with trimethylation of histone 3 lysine 4 (H3K4me3) derived from the adult male DG after DAE was performed. DAE induced differential H3K4me3 enrichment on genes in pathways associated with cellular development and growth, cell death and survival, and neurological disorders, particularly as they relate to cancer, in the adult male brain. Comparison of H3K4me3 enrichment in controls revealed mechanisms that are potentially lacking in arsenic-exposed animals, including neurotransmission, neuronal growth and development, hormonal regulation, protein synthesis, and cellular homeostasis. New pathways impacted by arsenic include cytoskeleton organization, cell signaling, and potential disruption of immune function and warrant further investigation using this DAE paradigm in the mouse brain.

Developmental exposure to 50 parts-per-billion arsenic influences histone modifications and associated epigenetic machinery in a region- and sex-specific manner in the adult mouse brain

Epidemiological studies report that arsenic exposure via drinking water adversely impacts cognitive development in children and, in adults, can lead to greater psychiatric disease susceptibility, among other conditions. While it is known that arsenic toxicity has a profound effect on the epigenetic landscape, very few studies have investigated its effects on chromatin architecture in the brain. We have previously demonstrated that exposure to a low level of arsenic (50ppb) during all three trimesters of fetal/neonatal development induces deficits in adult hippocampal neurogenesis in the dentate gyrus (DG), depressive-like symptoms, and alterations in gene expression in the adult mouse brain. As epigenetic processes control these outcomes, here we assess the impact of our developmental arsenic exposure (DAE) paradigm on global histone posttranslational modifications and associated chromatin-modifying proteins in the dentate gyrus and frontal cortex (FC) of adult male and female mice. DAE influenced histone 3K4 trimethylation with increased levels in the male DG and FC and decreased levels in the female DG (no change in female FC). The histone methyltransferase MLL exhibited a similar sex- and region-specific expression profile as H3K4me3 levels, while histone demethylase KDM5B expression trended in the opposite direction. DAE increased histone 3K9 acetylation levels in the male DG along with histone acetyltransferase (HAT) expression of GCN5 and decreased H3K9ac levels in the male FC along with decreased HAT expression of GCN5 and PCAF. DAE decreased expression of histone deacetylase enzymes HDAC1 and HDAC2, which were concurrent with increased H3K9ac levels but only in the female DG. Levels of H3 and H3K9me3 were not influenced by DAE in either brain region of either sex. These findings suggest that exposure to a low, environmentally relevant level of arsenic during development leads to long-lasting changes in histone methylation and acetylation in the adult brain due to aberrant expression of epigenetic machinery based on region and sex.

Sex-dependent effects of developmental arsenic exposure on methylation capacity and methylation regulation of the glucocorticoid receptor system in the embryonic mouse brain

Previously we have shown that prenatal moderate arsenic exposure (50 ppb) disrupts glucocorticoid receptor (GR) programming and that these changes continue into adolescence in males. However, it was not clear what the molecular mechanisms were promoting these GR programming changes or if these changes occurred in arsenic-exposed females. In the present studies, we assessed the effects of arsenic on protein and mRNA of the glucocorticoid receptor (GR) and 11β-hydroxysteroid dehydrogenase (Hsd) isozymes and compared the levels of methylation within the promoters of the Nr3c1 and Hsd11b1 genes in female fetal brain at embryonic days (E) 14 and 18. Prenatal arsenate exposure produced sex specific effects on the glucocorticoid system. Compared to males, females were resistant to arsenic induced changes in GR, 11β-Hsd-1 and 11β-Hsd-2 protein levels despite observed elevations in Nr3c1 and Hsd11b2 mRNA. This sex-specific effect was not due to differences in the methylation of the GR promoter as methylation of the Nr3c1 gene was either unchanged (region containing the egr-1 binding site) or similarly reduced (region containing the SP-1 transcription factor binding site) in both males and females exposed to arsenic. Arsenic did produce sex and age-specific changes in the methylation of Hsd11b1 gene, producing increased methylation in females at E14 and decreased methylation at E18.These changes were not attributed to changes in DNMT levels. Since arsenate metabolism could interfere with the generation of methyl donor groups, we assessed glutathione (GSH), S-adenosylmethionine (SAM) and As 3 methyltransferase (As3MT). Exposed males and females had similar levels of As3MT and SAM; however, females had higher levels of GSH/GSSH. It is possible that this greater anti-oxidative capacity within the females provides protection against low to moderate arsenate. Our data suggest that the GR signaling system in female offspring was not as affected by prenatal arsenic and predicts that female arsenic-exposed mice should have normal GR feedback regulation.

Adult Hippocampal Neurogenesis and mRNA Expression are Altered by Perinatal Arsenic Exposure in Mice and Restored by Brief Exposure to Enrichment

Arsenic is a common and pervasive environmental contaminant found in drinking water in varying concentrations depending on region. Exposure to arsenic induces behavioral and cognitive deficits in both human populations and in rodent models. The Environmental Protection Agency (EPA) standard for the allotment of arsenic in drinking water is in the parts-per-billion range, yet our lab has shown that 50 ppb arsenic exposure during development can have far-reaching consequences into adulthood, including deficits in learning and memory, which have been linked to altered adult neurogenesis. Given that the morphological impact of developmental arsenic exposure on the hippocampus is unknown, we sought to evaluate proliferation and differentiation of adult neural progenitor cells in the dentate gyrus after 50 ppb arsenic exposure throughout the perinatal period of development in mice (equivalent to all three trimesters in humans) using a BrdU pulse-chase assay. Proliferation of the neural progenitor population was decreased by 13% in arsenic-exposed mice, but was not significant. However, the number of differentiated cells was significantly decreased by 41% in arsenic-exposed mice compared to controls. Brief, daily exposure to environmental enrichment significantly increased proliferation and differentiation in both control and arsenic-exposed animals. Expression levels of 31% of neurogenesis-related genes including those involved in Alzheimer’s disease, apoptosis, axonogenesis, growth, Notch signaling, and transcription factors were altered after arsenic exposure and restored after enrichment. Using a concentration previously considered safe by the EPA, perinatal arsenic exposure altered hippocampal morphology and gene expression, but did not inhibit the cellular neurogenic response to enrichment. It is possible that behavioral deficits observed during adulthood in animals exposed to arsenic during development derive from the lack of differentiated neural progenitor cells necessary for hippocampal-dependent learning. This study is the first to determine the impact of arsenic exposure during development on adult hippocampal neurogenesis and related gene expression.