Towards understanding the mechanisms of chlorpromazine-induced hepatic toxicity using a human HepaRG-based model

Background and Aims
We have previously shown, using impedance biosensor technology, that chlorpromazine (CPZ) induces early, dose-dependent disruption of tight junctions even at sub-toxic CPZ levels (25 μM)1. In this study we investigated the molecular mechanisms that may mediate the hepatic alterations induced by CPZ in the human HepaRG model and in particular those related to inflammation and oxidative stress.

Method
HepaRG cells were exposed to CPZ concentrations of 25, 50 and 100 μM for 24 h. Cell viability was assessed by ATP-depletion assay (CellTiter-Glo). Assessment of cytoskeleton intergrity was performed by TJ-associated/F-actin expression following CPZ challenge. A number of molecular markers were analysed using qPCR (N = 3). The fold change in expression of target genes relative to the internal control genes (TOP1, UBC and GAPDH) was calculated. QRT-PCR data were presented as the fold change in gene expression normalised to the average value of 3 common endogenous reference genes and relative to control (untreated cells).

Results
CPZ induced extensive cell death at 100 μM. At concentrations of 25 and 50 μM, cell viability by ATP-depletion assay was not significantly impaired. As previously demonstrated, cytoskeletal changes suggestive of tight junction disruption were seen. CPZ provoked a dose dependent inflammatory response [3-fold at 25 μM and 10-fold at 50 μM) for IL-6 as well as 2.7-fold at 25 μM and 8-fold at 50 μM for TNFα. Τhis response was associated with 0.5-fold down regulation of HNF4α transcript and cytochrome 3A4 at 50 μM. mRNA expression of bile canalicular transporters ABCB11 (bile salt exporter pump), ABCB4 (phospholipids transporter) and ABCB1 (drug efflux transporter) was decreased. 50 μM of CPZ induced 20-fold increase in the expression of ABCB11 likely to represent a hepatoportective response to accumulation of toxic bile acids (BA). GGT1 a key antioxidative compound was up regulated. Expression of Bax (pro apoptotic gene) was increased at 50 μM, while the Bcl2 anti apoptotic gene was upregulated in 25 and 50 μM CPZ treated cells. AlF1 and p53 gene expressions were unchanged. Nrf2 was up regulated by 2.3-fold in 50 μM CPZ treated cells.

Conclusion
Our results show that an inflammatory response is induced by CPZ at 25 and 50 μM, resulting in activation of anti oxidant and anti apoptotic defence pathways Nrf2 and Bcl2 with evidence of intracellular oxidative stress perhaps caused by accumulation of bile acids with reduction of cytochrome 3A4 activity and adaptive upregulation of GGT1. In conclusion we demonstrate that CPZ appears to cause disruption of hepatic cytoskeleton through an inflammatory response and induction of intracellular oxidative stress.

Reference
1. Kozlowska et al. Mechanistic insight into chlorpromazine-induced hepatic tight junction disruption using a human HepaRG-based LiverBioChip Impedance biosensor. Abstract UEGW 2015.