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.

中文翻译：

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丙戊酸在人肝细胞中诱导的核和线粒体DNA甲基化模式

丙戊酸（VPA）是世界上处方最广泛的抗癫痫药之一。尽管具有重要的药理作用，但它可能通过线粒体功能障碍引起肝脏毒性和脂肪变性。这项研究的目的是通过分析核DNA（nDNA）和线粒体DNA（mtDNA）中甲基化模式的变化，进一步研究VPA诱导的脂肪变性机制。因此，将原代人肝细胞（PHH）暴露于一定浓度的VPA中，该浓度显示可引起脂肪变性而不会引起明显的细胞毒性。每天施用VPA，持续5天，然后进行3天的冲洗（WO）。通过使用甲基化DNA免疫沉淀测序（MeDIP-seq）方法鉴定甲基化DNA区域（DMR）。VPA治疗后的nDNA DMR确实可以分为氧化应激和脂肪变性相关的途径。特别是脂肪变性相关基因的网络EP300为VPA治疗诱导的毒性机理提供了新颖的见解。此外，我们建议VPA诱导nDNA高度甲基化和mtDNA甲基化之间的串扰，在氧化应激和脂肪变性的发展中发挥作用。尽管大多数VPA诱导的甲基化模式在终止VPA治疗后似乎可逆，但在WO期后仍保留了31种nDNA DMR（包括5个锌指蛋白基因）。总体而言，我们已经证明MeDIP-seq分析在揭示VPA诱导的PHHs毒性的新机制方面非常有用。因此我们的结果为新型有趣的甲基化生物标志物的体外重复剂量肝毒性提供了一个原型。