Large scale toxicoepigenetics on histones: a mass spectrometry-based screening assay applied to developmental toxicity

Toxicoepigenetics is an emerging field that studies the toxicological impact of compounds on protein expression through heritable, non-genetic mechanisms, such as histone post-translational modifications (hPTMs). Due to substantial progress in the large-scale study of hPTMs, integration into the field of toxicology is a promising addition that offers the opportunity to gain novel insights into toxicological phenomena. Moreover, there is a growing demand for high-throughput human-based in vitro assays for toxicity testing and especially for developmental toxicity. Consequently, we developed a mass spectrometry based proof-of-concept to develop an assay to screen the histone code and hence detecting multiple hPTMs changes simultaneously in human embryonic stem cells. To prove the applicability and performance, we first validated the untargeted workflow with valproic acid (VPA), a histone deacetylase inhibitor. These results demonstrate that our workflow is capable of mapping the hPTM-dynamics, with a general increase in acetylations as an internal control. To illustrate the scalability, the workflow was applied on a proof-of-concept dose-response library of a total of ten compounds with either i) a known effect on the hPTMs (BIX-01294, 3-Deazaneplanocin A, Trichostatin A, and VPA), ii) a presumed developmental toxicity, including compounds of abuse (Caffeine, Ethanol, Nicotine, Methotrexate, and All-trans retinoic acid), or iii) no proven embryotoxicity (Penicillin G). In conclusion, we show that toxicoepigenetic screening on histones is feasible and yields very rich data that holds great potential, not only for applications in the pharmaceutical industry, but also for environmental toxicity and food safety.