2 research outputs found
Nuclear and Mitochondrial DNA Methylation Patterns Induced by Valproic Acid in Human Hepatocytes
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 <i>EP300</i> 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 <i>in vitro</i>
Integrative “‑Omics” Analysis in Primary Human Hepatocytes Unravels Persistent Mechanisms of Cyclosporine A‑Induced Cholestasis
Cyclosporine
A (CsA) is an undecapeptide with strong immunosuppressant activities
and is used a lot after organ transplantation. Furthermore, it may
induce cholestasis in the liver. In general, the drug-induced cholestasis
(DIC) pathway includes genes involved in the uptake, synthesis, conjugation,
and secretion of bile acids. However, whether CsA-induced changes
in the cholestasis pathway <i>in vitro</i> are persistent
for repeated dose toxicity has not yet been investigated. To explore
this, primary human hepatocytes (PHH) were exposed to a subcytotoxic
dose of 30 ÎĽM CsA daily for 3 and 5 days. To investigate the
persistence of induced changes upon terminating CsA exposure after
5 days, a subset of PHH was subjected to a washout period (WO-period)
of 3 days. Multiple -omics analyses, comprising whole genome analysis
of DNA methylation, gene expression, and microRNA expression, were
performed. The CsA-treatment resulted after 3 and 5 days, respectively,
in 476 and 20 differentially methylated genes (DMGs), 1353 and 1481
differentially expressed genes (DEGs), and in 22 and 29 differentially
expressed microRNAs (DE-miRs). Cholestasis-related pathways appeared
induced during CsA-treatment. Interestingly, 828 persistent DEGs and
6 persistent DE-miRs but no persistent DMGs were found after the WO-period.
These persistent DEGs and DE-miRs showed concordance for 22 genes.
Furthermore, 29 persistent DEGs changed into the same direction as
observed in livers from cholestasis patients. None of those 29 DEGs
which among others relate to oxidative stress and lipid metabolism
are yet present in the DIC pathway or cholestasis adverse outcome
pathway (AOP) thus presenting novel findings. In summary, we have
demonstrated for the first time a persistent impact of repeated dose
administration of CsA on genes and microRNAs related to DIC in the
gold standard human liver <i>in vitro</i> model with PHH