Deciphering DNA methyltransferase inhibitor mediated transgenerational effects on Daphnia: high-throughput analyses and Adverse Outcome Pathway assembly

Summary

Epigenetic marks can in many cases reflect the life-time exposure history of an organism to environmental stressors. While the rapid development of the OMICS techniques allows measurements of genome-wide epigenetic changes, the high costs for such analyses still limit our ability to fully understand the epigenetic effects across doses/concentrations, exposure durations and multiple generations in a population. Targeted high-throughput (HT) epigenetic bioassays are needed to allow the inclusion of more life stages and exposure conditions to yield comparative concentration-response data on a temporal scale, and to support universal mechanistic models such as Adverse Outcome Pathways (AOPs). The present study has therefore integrated and refined several HT bioassays to understand the relationships between chemical-mediated DNA methyltransferase (DNMT) inhibition, promoter and gene body methylation, gene expression and reproduction, and to develop a novel epigenetic AOP for DNMT inhibitor-mediated transgenerational effects in aquatic organisms. Adult female Daphnia magna were exposed to 0-80 µM of 5-azacytidine for 7 days and recovered in clean media for another 7 days. F1-F3 offspring were sub-cultured in clean media to investigate potential transgenerational effects. A suite of HT bioassays were employed, such as the total DNMT activity assay, methylation-sensitive high-resolution melt analysis (MS-HRM), and qPCR. The test genes are well-known biomarker genes or key regulators involved in major biological pathways. Cumulative fecundity was recorderd for each generation. After 7-day exposure, the total DNMT activity in F0 decreased in a concentration-dependent manner, with 80 µM 5-azacytidine caused significant reduction in DNMT acitvity. The majority of the test genes showed reduced promoter methylation and increased transcription in F0 after 7-day exposure, whereas increased promoter methylation and down-regulation in F0 after recovery. In F3, most of the genes displayed increased promoter methylation. The cumulative fecundity decreased in a concentration-dependent manner in F0 after exposure and recovery, and in the succesive generations. On the basis of the results, a conceptual AOP on DNMT inhibition leading to oocyte apoptosis associated population decline was proposed. The present study has developed a novel analytical pipeline for targeted HT analyses of epigenetic effects, and has unravelled the relationships between DNMT inhibition, DNA promoter methylation, gene body methylation, gene expression and transgenerational reproduction in D. magna exposed to 5-azacytidine. The world’s first epigenetic AOP was proposed for chemical-mediated transgenerational population effects in aquatic organisms.