High throughput adaptive stress response pathway activation to support read across of valproic acid analogue-induced liver steatosis

Hepatic steatosis is a common liver disease that can lead to hepatotoxicity. Many drugs, including the anticonvulsant valproic acid (VPA), can induce hepatic steatosis as a side effect. Besides human clinical data, VPA and some of its structural analogues have shown to induce steatosis in rodents. Transcriptomics data derived from yeast cells suggest that VPA activates several stress response pathways, which are related to hepatotoxicity. This study aims to investigate stress response pathway activation by VPA and its structural analogues by using HepG2 BAC-GFP reporter cell lines in a high throughput screening platform. Activation of different stress responses were visualized by GFP tagged to target proteins SRXN1 (Nrf2-dependent oxidative stress response), P21 (p53-dependent DNA damage), BiP (unfolded protein response) and A20 (NFkB-dependent inflammatory signaling). Cells were treated with VPA and 18 structural analogous to generate dynamic concentration and time response datasets on pathway activation using high content confocal imaging. SRXN1 activation could discriminate between stimulation of VPA and structural in vivo steatosis positive analogues and in vivo negative analogues. P21 and A20 expression was also changed upon VPA exposure, where BiP did not show a response. Additionally, we observed no cytotoxic effects in our dose range. However, cells treated with valproic analogues that cause steatosis and co-stimulated with TNFa showed an increase in apoptosis, further supporting activation of common signaling pathways by the various toxic valproic analogues that drive onset of cytotoxicity. In conclusion, our current data support the application of a high content imaging stress response reporter platform to provide biological weight-of-evidence support for read across-based risk assessment strategies by providing mechanistic mode-of-action information.