Nuclear and Mitochondrial DNA Methylation Patterns Induced by Valproic Acid in Human Hepatocytes

Abstract

Valproic acid (VPA) is one of the most widely prescribed antiepileptic drugs in the world. Despite its pharmacological importance, it may cause liver toxicity and steatosis through mitochondrial dysfunction. The aim of this study is to further investigate VPA-induced mechanisms of steatosis by analyzing changes in patterns of methylation in nuclear DNA (nDNA) and mitochondrial DNA (mtDNA). Therefore, primary human hepatocytes (PHHs) were exposed to an incubation concentration of VPA that was shown to cause steatosis without inducing overt cytotoxicity. VPA was administered daily for 5 days, and this was followed by a 3 day washout (WO). Methylated DNA regions (DMRs) were identified by using the methylated DNA immunoprecipitation–sequencing (MeDIP-seq) method. The nDNA DMRs after VPA treatment could indeed be classified into oxidative stress- and steatosis-related pathways. In particular, networks of the steatosis-related gene EP300 provided novel insight into the mechanisms of toxicity induced by VPA treatment. Furthermore, we suggest that VPA induces a crosstalk between nDNA hypermethylation and mtDNA hypomethylation that plays a role in oxidative stress and steatosis development. Although most VPA-induced methylation patterns appeared reversible upon terminating VPA treatment, 31 nDNA DMRs (including 5 zinc finger protein genes) remained persistent after the WO period. Overall, we have shown that MeDIP-seq analysis is highly informative in disclosing novel mechanisms of VPA-induced toxicity in PHHs. Our results thus provide a prototype for the novel generation of interesting methylation biomarkers for repeated dose liver toxicity in vitro.

Mitochondrial DNA: Epigenetics and environment

“…In a separate study using primary human hepatocytes, hypomethylation of seven mitochondrial genes was observed upon VPA treatment and within the same study, two nuclear genes crucial for DNA methylation, DNMT and MAT (methionine adenosyltransferase), were found to be hypermethylated, resulting in reduced levels of the DNMT enzymes and SAM. This may impact mtDNA methylation downstream, which is suggestive of crosstalk between the nucleus and mitochondria after VPA exposure (Wolters et al, 2017). The downstream molecular events after VPA exposure were also explored in human hepatocytes, where upregulation of DNMT caused transient hypermethylation in mtDNA and downregulation of mitochondrial proteins resulting in impaired mitochondrial function.…”

Data on novel DNA methylation changes induced by valproic acid in human hepatocytes

“…The data presented in this DIB demonstrate induced steatosis pathways by all DMRs during VPA-treatment, covering interesting drug-induced steatosis genes (persistent DMRs upon terminating VPA treatment and the EP300 network). This was illustrated in our associated article (Wolters et al, 2017) [1] . MeDIP-seq raw data are available on ArrayExpress (accession number: E-MTAB-4437).…”

“…1 B-C) and the gene names, gene symbols, and fold changes (FCs) of those neighbors were shown in Table 3 . A more detailed description of those findings can be found in Wolters et al [1] .

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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.…”