Use of 5-azacytidine in a proof-of-concept study to evaluate the impact of pre-natal and post-natal exposures, as well as within generation persistent DNA methylation changes in Daphnia

Abstract

Short-term exposures at critical stages of development can lead to delayed adverse effects long after the initial stressor has been removed, a concept referred to as developmental origin of adult disease. This indicates that organisms’ phenotypes may epigenetically reflect their past exposure history as well as reflecting chemicals currently present in their environment. This concept has significant implications for environmental monitoring. However, there is as yet little or no implementation of epigenetics in environmental risk assessment. In a proof-of-principle study we exposed Daphnia magna to 5-azacytidine, a known DNA de-methylating agent. Exposures covered combinations of prenatal and postnatal exposures as well as different exposure durations and recovery stages. Growth, the transcription of genes and levels of metabolites involved in regulating DNA methylation, and methylation levels of several genes were measured. Our data shows that prenatal exposures caused significant changes in the methylome of target genes, indicating that prenatal stages of Daphnia are also susceptible to same level of change as post-natal stages of Daphnia. While the combination of pre- and postnatal exposures caused the most extreme reduction in DNA methylation compared to the control group. Furthermore, some of the changes in the methylation patterns were persistent even after the initial stressor was removed. Our results suggest that epigenetic biomarkers have the potential to be used as indicators of past chemical exposure history of organisms and provide strong support for implementing changes to the current regimes for chemical risk assessment to mimic realistic environmental scenarios.

The Importance of Validating the Demethylating Effect of 5-aza-2′-deoxycytidine in Model Species

“…In other arthropod model systems, DNA methylation is present. In Daphnia, for instance, 5-aza-dC had a clear demethylating effect on four out of the five tested genes, which differed among CpG sites (Athanasio et al 2018). However, few insect studies have shown a consistent effect of 5-aza-dC.…”

“…Also, the use of demethylating agents such as 5-aza-2 0deoxycytidine (5-aza-dC) has been popular in showing the impact of DNA methylation on the phenotype. The 5-aza-dC is incorporated into the DNA, where it inhibits DNA methylation (Momparler 1985), as has been shown in plants (Bossdorf et al 2010;Vergeer et al 2012), invertebrates ( Vandegehuchte et al 2010;Amarasinghe et al 2014;Athanasio et al 2018), and vertebrates (Christman 2002;Dasmahapatra et al 2017).…”

Prospects for incorporation of epigenetic biomarkers in human health and environmental risk assessment of chemicals

“…In spite of the large amount of research that remains to be done, epigenetic biomarkers are promising as tools in retrospective ERA as they can discriminate exposure and effects in real environmental scenarios (Athanasio et al ., ; Jeremias et al ., ). The recommendations outlined in Section V.2 will apply here, with the species selected for study being perhaps the most important distinguishing (from ERAC) consideration.…”

The anti-lipidemic drug simvastatin modulates Epigenetic Biomarkers in the Amphipod Gammarus locusta

“…Understanding the long-term effects of epigenetic changes can improve our knowledge about the adaptive regulation and/or disruptive responses, addressing the concerns about how these effects may modulate the populations´ evolution [73]. Therefore, the identification and validation of biomarkers of epigenetic modifications can be of great interest as early warning tools in risk assessment frameworks to mitigate the risks of environmental exposures [12,23,74,75].…”

“…Therefore, epigenetic signatures are interesting candidates to link environmental exposures to altered phenotypes, providing new insights into the heritability of multigenerational exposure history [12,[20][21][22]. Despite the fact that biomarkers of epigenetic modifications are still not integrated into hazard and risk assessment frameworks [12,23], a number of recent studies has suggested that risk assessment can benefit from the integration of chemical-induced epigenetic effects into toxicity testing strategies and hazard assessment [24][25][26][27].…”