Abstract

Epigenetic modifications have been hypothesized as a mode of action in programmed responses, i.e. responses upon toxic exposure early in life that are only expressed later in life, e.g. as increased sensitivity to develop chronic disease. Factors implicated in such toxic exposures included consumer goods such as maternal food substances and additives, food contaminants, cosmetic ingredients, and drugs. All such maternal factors may reach the embryo/fetus/lactating neonate, and thus affect the developing organism. There are presently no tests that address epigenetic effects, and therefore we here explored two models that have been suggested as suitable in the scientific literature for that purpose.

The first model was the zebrafish embryo. In this model, we tested a set of control compounds, including 5-azacytidine (5AC, a known DNA methylation inhibitor); 3 metals, nickel, arsenic, and cadmium; 3 estrogenic compounds ethynylestradiol (EE2), diethylstilbestrol (DES), bisphenol-A (BPA); and PFOA, a non-estrogenic endocrine active compound. After exposure to these compounds, starting within 2h after fertilization, epigenetic effects were evaluated after 72 hours of exposure in whole embryo DNA extracts. This was done through analysis of 1) global DNA methylation after DNA digestion and single nucleotide HPLC chromatography, and 2) methylation at three specific loci with shown (vasa, vtgI) or suggested (cyp19a2) DNA methylation plasticity, through pyrosequencing of defined DNA fragments. In this analyis, there were compound specific responses in a locus specific way, with vasa as the most responsive target, showing responses with 7 out of 8 tested compounds. Global methylation was only affected by 5AC, and therefore not an informative parameters. Arsenite did not affect any of the methylation parameters, whereas the other two metals induced methylation effects at concentrations that also produced developmental (morphological) effects. 5AC and the endocrine active compounds, on the other hand, induced methylation responses at subtoxic concentrations. Such epigenetic effects at subtoxic concentrations are of particular interest because these could be hypothesized to predict adverse health effects later in life. Our results confirm that compound induced epigenetic effects can be analyzed in the zebrafish embryo model.

The second model was the primary human umbilical cord derived mesenchymal stem cells, which can be differentiated into adipocytes. Epigenetic changes play an important role in cell differentiation. Therefore, this model could perhaps serve to causally relate epigenetic changes in differentiation-related genes to morphological effects on cell differentiation. We tested a set of 5 compounds that have known effects on adipocyte differentiation. Although we observed patterns suggesting compound specific effects on adipocyte differentiation, there was considerable variation of responses between and within the explored cell lines. Overall, we conclude that this model is not sufficiently robust to serve a phenotype controlled evaluation of epigenetic effects.