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.

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.

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.

Exposure to arsenic, a common environmental toxin found in drinking water, leads to a host of neurological pathologies. We have previously demonstrated that developmental exposure to a low level of arsenic (50ppb) alters epigenetic processes that underlie deficits in adult hippocampal neurogenesis leading to aberrant behavior. It is unclear if arsenic impacts the programming and regulation of embryonic neurogenesis during development when exposure occurs. The master negative regulator of neural-lineage, REST/NRSF, controls the precise timing of fate specification and differentiation of neural stem cells (NSCs). Early in development (embryonic day 14), we observed increased expression of Rest, its co-repressor, CoREST, and the inhibitory RNA binding/splicing protein, Ptbp1, and altered expression of mRNA spliced isoforms of Pbx1 that are directly regulated by these factors in the male brain in response to prenatal 50ppb arsenic exposure. These increases were concurrent with decreased expression of microRNA-9 (miR-9), miR-9*, and miR-124, all of which are REST/NRSF targets and inversely regulate Rest expression to allow for maturation of NSCs. Exposure to arsenic decreased the formation of neuroblasts in vitro from NSCs derived from male pup brains. The female response to arsenic was limited to increased expression of CoREST and Ptbp2, an RNA binding protein that allows for appropriate splicing of genes involved in the progression of neurogenesis. These changes were accompanied by increased neuroblast formation in vitro from NSCs derived from female pups. Unexposed male mice express transcriptomic factors to induce differentiation earlier in development compared to unexposed females. Thus, arsenic exposure likely delays differentiation of NSCs in males while potentially inducing precocious differentiation in females early in development. These effects are mitigated by embryonic day 18 of development. Arsenic-induced dysregulation of the regulatory loop formed by REST/NRSF, its target microRNAs, miR-9 and miR-124, and RNA splicing proteins, PTBP1 and 2, leads to aberrant programming of NSC function that is perhaps perpetuated into adulthood inducing deficits in differentiation we have previously observed.