In MEFs adduct formation increased with time at 20 μM but at 50 μM after 48 h resulted in lower adduct levels (compare Fig. 2F). As indicated above, it may be possible that the increased cytotoxicity at this condition may have impacted metabolic activation of the compound and/or DNA adduct formation. Highest DNA binding in MEFs was observed at 50 μM after 24 h with 2810 ± 1048

adducts per 108 nucleotides which was 468-fold higher than the adduct levels observed under the same experimental conditions in ES cells (6 ± 3 adducts per 108 nucleotides). AAI-induced Histone Acetyltransferase inhibitor DNA damage in MEFs was associated with a strong induction of the DNA damage response proteins p53 and p21 ( Fig. 4B). Interestingly, AAI exposure also led to a

strong p53 induction in ES cells and also subsequently its downstream target p21 but at considerably lower DNA adduct levels than in MEFs. In ES cells neither Nqo1 nor Cyp1a1 mRNA expression was significantly altered after AAI treatment (Figs. 5E and 6E). In contrast, we found a significant induction of Nqo1 and Cyp1a1 in MEFs (Figs. 5F and 6F) but the levels of transcriptional alterations in MEFs are very small, and thus do not explain GDC-0980 the differences of AAI–DNA adduct formation observed in the two cell types. Further, as the basal Cyp1a1 and Nqo1 mRNA expression levels in untreated ES cells and MEFs were only marginally different, if at all (see legends to Fig. 5 and Fig. 6), this also did not provide

an explanation for the huge differences in AAI–DNA adduct formation between cell types. Therefore we investigated whether the observed alterations in AAI-induced DNA damage are linked to epigenetic changes. Tumours are characterized by a global reduction in DNA methylation (hypomethylation) and/or a locus-specific increase in DNA methylation (hypermethylation) (Esteller, 2008). DNA methylation can regulate gene expression and it has been shown in cancer cells that DNA hypermethylation of CpG islands near tumour suppressor genes switches off the expression of these genes (Tommasi et al., 2014). Further, it has been suggested that epigenetic mechanisms may function as an interface between environmental factors and the genome and that aberrant epigenetic changes associated with environmental TCL exposures might deregulate not only key cellular processes such as DNA damage response and DNA repair but also carcinogen metabolism (Herceg and Vaissiere, 2011). Several environmental pollutants have been shown to affect DNA methylation in mammalian cells in vitro. Tabish et al. (2012) demonstrated for example that benzene, hydroquinone, styrene, carbon tetrachloride and trichloroethylene induced global DNA hypomethylation in human TK6 cells. However, little is known about equivalent mechanisms in embryonic stem cells or MEFs.