Integrative omics data analyses of repeated dose toxicity of valproic acid in vitro reveal new mechanisms of steatosis induction

Abstract

Valproic acid (VPA) is a very potent anti-cancer and neuro-protective drug probably by its HDAC inhibiting properties, which may cause steatosis in the liver. The present study investigates the effect of repetitive VPA treatment of primary human hepatocytes (PHH) on whole genome gene expression-, DNA methylation-, and miRNA changes, using microarrays and integrated data analyses. PHH were exposed to a non-cytotoxic dose of VPA for 5days daily which induced lipid accumulation. Part of the PHH was left untreated for 3days for studying the persistence of 'omics' changes. VPA treatment appeared to inhibit the expression of the transcription factors HNF1A and ONECUT1. HNF1A interacted with 41 differentially expressed genes of which 12 were also differentially methylated. None of the genes present in this network were regulated by a DE-miR. The subnetwork of ONECUT1 consisted of 44 differentially expressed genes of which 15 were differentially methylated, and 3 were regulated by a DE-miR. A number of genes in the networks are involved in fatty acid metabolism, and may contribute to the development of steatosis by increasing oxidative stress thereby causing mitochondrial dysfunction, and by shifting metabolism of VPA towards β-oxidation due to reduced glucuronidation. Part of the changes remained persistent after washing out of VPA, like PMAIP1 which is associated with cellular stress in liver of patients with NASH. The MMP2 gene showed the highest number of interactions with other persistently expressed genes, among which LCN2 which is a key modulator of lipid homeostasis. Furthermore, VPA modulated the expression and DNA methylation level of nuclear receptors and their target genes involved in the adverse outcome pathway of steatosis, thereby expanding our current knowledge of the pathway. In particular, VPA modulated PPARγ, and PPARα, AHR and CD36 on both the gene expression and the DNA methylation level, thereby inhibiting β-oxidation and increasing uptake of fatty acid into the hepatocytes, respectively. Overall, our integrative data analyses identified novel genes modulated by VPA, which provide more insight into the mechanisms of repeated dose toxicity of VPA, leading to steatosis.

Butyrate prevents valproate‐induced liver injury: In vitro and in vivo evidence

“…11 Indeed, VPA-induced liver injury appears to be dose-and time-dependent, even if it also occurs at an early stage and at low therapeutic doses. 17 Preclinical and clinical evidence suggest that early supplementation with l-carnitine could improve survival in severe VPA-induced toxicity, even if this treatment does not appear to be determined. 13,14 Valproyl-CoA, its main non-metabolizable metabolite, 15 can inhibit hepatic carnitine palmitoyl-transferase (CPT) 1A, 16 a pivotal enzyme in mitochondrial fatty acid β-oxidation that may be crucial in the drug-induced hepatotoxic mechanisms and the weight gain frequently observed in patients on VPA therapy.…”

Epigenetic Effects Mediated by Antiepileptic Drugs and their Potential Application

“…The liver damage caused by VPA can be explained by epigenetic effects. In a previous study, VPA exposure to human primitive liver cells caused a decrease in the methylation levels of the PPARγ, PPARα, CD36 and AHR genes, thereby inhibiting β-oxidation and increasing fatty acid entry into hepatocytes, possibly explaining the molecular mechanism of VPA-induced hepatic steatosis [ 117 ]. VPA-induced cataracts are a rare side effect.…”

The application of omics-based human liver platforms for investigating the mechanism of drug-induced hepatotoxicity in vitro

“…Facilitated by transcriptomic and epigenomic approaches, this in vitro culture system revealed interactions between alterations in DNA methylation and mRNA expression changes during the development of AFB1-induced hepatocellular carcinoma (Rieswijk et al 2016) and revealed the persistent changes in gene expression and microRNA expression in response to CsA-associated cholestasis (Wolters et al 2016). Wolters et al (2017Wolters et al ( , 2018 and Van Breda (van Breda et al 2018) also used the sandwich-cultured PHH and multiple omics approaches (transcriptomics, epigenomic and proteomics) to examine the roles of epigenetic factors in liver steatosis development induced by repeated exposures (3-5 days) to valproic acid (VPA), a drug to treat epilepsy and bipolar disorders. Through the integrative cross-omics analyses, they discovered several treatment-initiated reactions (e.g.…”

“…Through the integrative cross-omics analyses, they discovered several treatment-initiated reactions (e.g. the cross-talk between nuclear DNA and mitochondrial DNA hypermethylation (Wolters et al 2017), the persistent epigenetic and transcriptomic alterations in the mitochondrial genome coupled with the measurable mitochondrial dysfunction (Wolters et al 2018) and the inhibition of nuclear receptors at DNA methylation and mRNA levels (van Breda et al 2018), deepening the understanding of the VPA-induced hepatosteatosis.…”