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High-throughput microscopy of adaptive stress response pathway activation by steatosis-inducing valproic acid analogues

: Vrijenhoek, N.G.; Gräpel, Rabea; Escher, Sylvia E.; Water, Bob van de

The Toxicologist 162 (2018), No.1, pp.522, Abstract PS 3159
ISSN: 0731-9193
Society of Toxicology (Annual Meeting) <57, 2018, San Antonio/Tex.>
Fraunhofer ITEM ()

Hepatic steatosis is a common liver disease that can lead to hepatotoxicity1. Many drugs, such as the anticonvulsant valproic acid (VPA), induce liver steatosis as a side effect2. In addition to human clinicaldata, rodent in vivo studies show induction of hepatic steatosis by VPA3, 4, 5 and some structural VPA analogues. Transcriptomics data derived from yeast cells suggests that VPA activates several stress response pathways6. This study aims to investigate activation of stress response pathways by VPA and structural analogues using HepG2 BAC reporter cell lines as a screening platform for microvesicular steatosis/hepatotoxicity and application in a biological read across. Activation of stress response pathways was visualized using HepG2 BAC GFP reporter cells for the target proteins SRXN1 (oxidative stress), BiP and Chop (unfolded protein response) and p21 (DNA damage response). Cells were treated with VPA and 18 structural analogues (5 in vivo positive; 6 in vivo negative and 8 unknown compounds) to generate dose - (8 concentrations) and time (up to 72h) - response curves. In addition, cells were treated with increasing concentrations of fatty acids (2:1 ratio oleate & palmitate) in lipid free medium to observe the influence on lipid accumulation (using HCS LipidTox dye) and pathway activation. High content confocal imaging was used to capture images of cells for analysis. VPA and in vivo positive analogues activate the oxidative stress pathway and the p53-mediated activation of the p21 upregulation in a dose and time dependent manner. Little activation of stress responses was observed for in vivo negative analogues. Unknown compounds were categorized according to their response. Controlled addition of fatty acids to lipid free medium allows the assessment of altered stress pathway activation and steatosis formation under different, physiologically relevant conditions. This high content imaging platform to monitor adaptive stress response activation has the potential to contribute to support risk assessment by providing quantitative mechanistic biological information that may support a read across approach. 1. Browning et al., 2004 2. Vinken M. 2015 3. Tong et al., 2005 4. RepDose (www.fraunhofer-repdose. de) 5. eToxSys ( 6. Golla U. et al., 2016.