Complex human disorders such as ASDs involve a mixture of genetic and environmental risk and protective factors. This study was designed to reduce the complexity in a controlled experimental system by examining the effects of perinatal exposure to a single environmental toxin (BDE-47) in a genetically susceptible mouse model (Mecp2308/+). From this multidimensional investigation, several novel discoveries were made that contribute to understanding the complexities of gene by environment interactions relevant to human neurodevelopment. First, perinatal BDE-47 exposure produced sexually dimorphic effects on preweaning weights, brain DNA hypomethylation and behavioral interaction effects on social novelty preference and learning. Second, brain DNA hypomethylation in females corresponded with reduced sociability following perinatal BDE-47 exposure. Third, exposure to BDE-47 coincided with a reversal of deficits in the preference for social novelty test, which coincided with increased Dnmt3a levels. In contrast, impairments in learning in the MWM were exacerbated in BDE-47-exposed female mice with an Mecp2308/+ genotype. Lastly, the analysis of post-mortem human brain samples suggested that the relative low-dose BDE-47 exposures in our study are in the range of human exposures and are consistent with adverse effects on fertility and neurodevelopmental outcomes in genetically and epigenetically susceptible humans.
is an X-linked gene subject to XCI, sex differences were expected in the behavioral analyses, as males are hemizygous and females are heterozygous and mosaic for mutant and wild-type alleles (30
). A stronger genetic contribution may explain why males showed significant genotype-only effects in tests of anxiety, sociability, activity and social barrier interactions (Fig. ) that were predicted from prior behavioral studies of Mecp2308/y
mice on a 129/SvEv background (26
). Differences in genetic background, age of testing (2 versus 5 months), prior testing experiences and MWM-reduced pool size and spatial specificity of the probe trial are likely explanations for why the deficit in learning and memory previously described in Mecp2308/y
male mice (28
) was not observed in our study. Female Mecp2308/+
mice have not been previously analyzed for behavior because of the confounder of variable XCI (27
), even though RTT primarily affects females. Interestingly, similar defects in activity and social barrier interactions were demonstrated in Mecp2308/+
females that were not correlated with XCI in our study, suggesting that female mice of this model can be useful in therapy trials of RTT. The phenotype associated with the absence of Mecp2
), including abnormal clasping reflex, impaired motor ability, weight loss, respiratory abnormalities and early mortality, was not seen in our Mecp2308/+
mice at up to 130 days of age when testing was completed.
With the exception of USVs, which were lower in BDE-47-exposed pups of both sexes, BDE-47 and BDE-47 by Mecp2
interaction effects were exclusively found in females. C57BL/6J female mice are known to show increased latency relative to males in the MWM test (35
) and reduced sociability compared with males in the three-chamber test (36
). In our study, female mice appeared to be more susceptible to the added ‘hit’ of an environmental exposure in these specific behavioral tests. A study in rats showed that the half-life of BDE-47 was around one-third greater in females than in males (37
), providing a potential explanation for the greater impact of BDE-47 on females in our study. Similar to our study, gestational exposure to the pollutant bisphenol A resulted in social behavioral alterations specifically in female wild-type mice that corresponded to increased Dnmt3a
levels in exposed animals (38
). Consistent with the hypothesis that females may be more epigenetically susceptible to environmental exposures is the observation of significantly lower levels of global DNA methylation in human female peripheral blood compared with male (39
). Furthermore, a recent quantitative analysis of LINE-1 methylation levels in mouse tissues has shown that male mice show higher tissue DNA methylation than female mice in two different mouse strains (40
). Combined, these results suggest that because females of both species have lower levels of global DNA methylation than males, females may be more susceptible to environmental exposures that impact the saturation of DNA methylation.
Why would females have lower global DNA methylation and be thereby more influenced by BDE-47 exposure? While differences in sex hormones cannot be ruled out in our experiments, the most likely explanation is the genomic influence of X chromosome number. Females have an inactive X chromosome that is globally hypomethylated compared with the active X chromosome (41
). In addition, a recent study of blood DNA samples with XO, XY, XX, XXY and XXXXY chromosome content showed that LINE-1 methylation levels at both X-linked and autosomal loci correlated with genome size, with the lowest methylation levels observed in Klinefelter's males carrying extra inactive X chromosomes (42
). The effect on autosomal methylation levels may be due to a larger genomic ‘sink’ size that requires additional methyl donors to maintain high global methylation levels (43
). In future studies, it would be informative to investigate global DNA methylation in individuals with copy number variation gains that are observed at a higher frequency in individuals with autism (44
In the emerging area of environmental epigenetics, the vast majority of environmental toxins contribute to global DNA hypomethylation (12
). Exposure-linked hypermethylation has been limited to a few specific gene promoters, which are a small fraction of the genome (reviewed in 12
). The supply of methyl donors to DNA is dependent on the one-carbon cycle, which is biochemically linked to the glutathione (GSH) synthesis pathway, the major redox buffer in virtually all brain cells. Therefore, an enhanced GSH response to conjugate unwanted toxic chemicals in the body is predicted to reduce the supply of methyl donors, leading to global DNA hypomethylation (45
). The suppression of GSH levels also promotes oxidative stress. Interestingly, deficiencies in global DNA methylation, GSH and oxidative stress pathways have been observed in autism blood samples (5
). The human brain has particularly high levels of DNA methylation compared with other tissues genome-wide, and neuron-specific methylation domains have been observed to be enriched for genes implicated in neurodevelopment and ASDs (47
). Together, these results suggest that environmental exposures may have a widespread epigenomic impact on the developing brain.
Female mice exposed to BDE-47 during the perinatal period showed reduced sociability independent of Mecp2
genotype, coincident with reduced global methylation in the brain. There appeared to be a compensatory mechanism in female mice that were both Mecp2
deficient and BDE-47 exposed for social novelty learning that may be in part explained by upregulated Dnmt3a
levels seen specifically in the interaction group. Consistent with our findings, both Dnmt1
were upregulated in response to diet-induced methyl deficiency in the liver (48
). In addition, Dnmt3a
is increased in response to neuronal activity (49
) and targets the non-proximal promoter regions of neuronal genes in postnatal neural precursor cells (50
), suggesting a specific and targeted role in the brain rather than a global one. Whereas Dnmt1 protein is predominantly nuclear and levels are relatively constant in the brain, Dnmt3a is found in both cytosolic and nuclear fractions and increases with postnatal development (51
). The social novelty test was dependent on short-term recognition of a mouse previously encountered 10 min prior to the test, whereas the MWM was conducted over several days and therefore required long-term memory storage of the platform position. Therefore, the reversing interaction effect observed in the social novelty test may be a reflection of a compensatory mechanism when there are ‘two hits’ to the methylation system in the brain (global hypomethylation and Mecp2
mutation) that can be observed within a specific behavioral test. These results suggest that increased Dnmt3a
levels in females with two hits of global hypomethylation and Mecp2
mutation may be a compensatory mechanism of the more dynamically expressed DNA methyltransferase gene utilized in the brain. Future studies that examine the earlier developmental dynamics of brain DNA methylation and Dnmt3a
levels in females versus males in this system would be needed to determine whether these epigenetic changes are independent or related events.
Brain levels of BDE-47 were at a level comparable with those measured in human post-mortem brain samples from individuals of the general population born in the last half of the 1900s. Human brain levels for individuals born in the USA within the past decade are expected to be higher, based on recent blood level detection in children (52
). To our knowledge, this is the first study to quantify BDE-47 levels in the lipid-rich human brain, where these organic pollutants bioaccumulate. The dose-dependent reduction in reproductive success observed in our study is consistent with a study showing a correlation between levels of some PBDE congeners and reduced fecundity (53
). Since the reproductive effect of perinatal BDE-47 exposure was not observed previously for wild-type C57Bl/6J mice (54
), our results suggest that genetically susceptible mice and humans could be most impacted by PBDE exposures. Combined genetic and environmental studies are important to future investigations of both mice and humans to further understand the impact these persistent flame retardants have on fertility and brain development.