Periodic Reporting for period 1 - EpiSTOX (EPIgenetic Signatures as biomarkers of ecoTOXicological effects)
Reporting period: 2019-02-01 to 2021-01-31
The evaluation of toxic effects in human health and in the environment has been focused on persistent and widely distributed chemical pollutants. Since early 1990s, plenty of studies have investigated the effect of environmental chemicals that interfere with hormonal signalling mechanisms, known as Endocrine Disrupting Chemicals (EDCs). EDCs are widely found in the environment and its exposure, especially during early development, disrupts endocrine function and causes deleterious effects in many species, including humans. While both animals and humans are constantly exposed to hundreds of EDCs, the chemicals have been suggested to contribute to dysfunctions and diseases such as obesity, diabetes, reproductive disorders, and cancers, in some cases affecting the health of future generations. Current studies focus on discerning the mode of action of EDCs at the molecular level by characterizing the transcriptomic responses (changes in mRNA levels) indicative of endocrine disruption. Evidence suggests that the long-lasting effects might be mediated by epigenetic mechanisms, proposing EDCs as epigenomic disruptors. The main objectives of EPISTOX were: Objective 1) To identify common epigenetic marks regulated by EDC exposures during early development; Objective 2) To integrate transcriptomic and epigenetic platforms to identify relevant biological pathways characteristic of EDCs exposures. The objectives of the proposed study are in line with seeking solutions to some of the challenges facing European societies. The European Commission under HORIZON 2020 has identified food security, water research and health as some of the challenges that need to be urgently address. The European Chemicals Agency (ECHA) identifies the study of EDCs as a hot scientific topic. Environmental epigenetics in ecotoxicological studies, such as EPISTOX, is an emerging field that provides very valuable information to fill the knowledge gaps required to develop cost-effective high-throughput methodologies to screen for epigenetic signatures after EDCs exposures. In the long-term, it is expected that compelling new information will be key for better assessing risks and developing new environmental policies, that in turn will positively impact and protect environmental and human health.
First, we exposed zebrafish embryos to different EDCs during early development. The selected compounds were: bisphenol A (BPA), tributyltin (TBT) and perfluorooctanesulfonate (PFOS). Then, we studied the effects of this compounds at the morphological levels and found that the three toxicants exerted distinct effects at the morphological level with BPA causing appearance of yolk sac malabsorption syndrome, TBT a general developmental delay (diapause-arrest effect) and PFOS muscle-skeletal alterations (Martinez et al., 2019; https://doi.org/10.1016/j.aquatox.2019.105232). Later to characterize epigenetic markers regulated by EDC (Objective 1), we studied epigenetic mechanisms, particularly the involvement of miRNAs and DNA methylation, using two different approaches. Using a target approach, we found significant changes in response to PFAS for muscle-, liver- and pancreas specific miRNAs (Tu et al., 2019; https://pubs.acs.org/doi/abs/10.1021/acs.est.9b03820). Moreover, we studied the effects of BPA on the epigenome in genes related to the retinoic acid pathway and to estrogen synthesis and found that effects of BPA were gene-specific. Our results also suggested that these effects were cell specific and highlighted the need for further studies at the tissue and cellular level (unpublished results). Finally, and with the aim to fulfil Objective 2 we performed an untargeted approach to study whole-genome alterations of DNA methylation after exposure to TBT. First, we observed a clear dose-response relationship between DNA methylation levels and TBT concentrations. Our results also revealed that differentially methylated regions were predominantly located in regulatory regions, suggesting that these may be responsible for driving alterations at the transcriptomic level. When comparing the effects of TBT on the transcriptome (previously published by Martinez et al., 2020, https://doi.org/10.1016/j.jhazmat.2020.122881) and the methylome, we observed a group of 32 genes showing a significant correlation between mRNA levels and DNA methylation. This subset of genes was mainly related to the brain and eye development, metabolic functions, organelle function and ion transport. Therefore, our results suggest that at least regulation of transcription of genes involved in those functions is mediated by DNA methylation. In conclusion, in the EPISTOX project has allowed us to discover correlations between DNA methylation levels and mRNA levels in key genes when animals exposed to EDCs. Moreover, we have demonstrated that effects of BPA might be cell specific and also a clear dose-response effect of the methylome when exposed to TBT. Therefore, we highlight the importance to include dose-response experimental designs to better elucidate EDCs modes of action in the epigenome and the need to introduce analysis that encompass cell specific responses in environmental epigenomics and ecotoxicology.
The European Commission has prioritized food security, water research and health as challenges that need to be urgently address. Due to their nature, EDCs are ubiquitous in the environment, especially in water bodies which also relate to other priorities outlined, such as “Healthy oceans, seas, coastal and inland waters”. For these reasons, the need for regulatory frameworks for EDCs has been included in the recent European Green Deal. Nowadays, one of the main challenges is to link molecular modes of action of EDCs to demonstrated long-lasting adverse effects of EDCs on human health and in wildlife. Although transgenerational tests have been included in risk assessment test guidelines, there are still many gaps on the understanding of the mode of action of EDCs through the epigenome, which makes it difficult for regulatory agencies to set guidelines for this matter. Results from our Project demonstrate the potential usefulness of targeted epigenetic biomarkers to develop cost-effective high-throughput methodologies to screen for epigenetic signatures after EDCs exposures, hereby predicting long-term and/or transgenerational effects. Therefore, projects such as the EPISTOX have the potential to positively impacting and protecting wildlife and human health through appropriate risk assessment, management and new environmental policies and regulation.