Chlorpromazine toxicity is associated with disruption of cell membrane integrity and initiation of a pro-inflammatory response in the HepaRG hepatic cell line

Katie Morgan, Nicole Martucci, Ada Kozlowska, Wesam Gamal, Fillip Brzeszczynki, Philipp Treskes, Kay Samuel, Peter Hayes, Lenny Nelson, Pierre Bagnaninchi, Joanna Brzeszczynska, John Plevris

Research output: Contribution to journalArticle

Abstract

Chlorpromazine (CPZ) is a neuroleptic drug and prototype compound used to study intrahepatic cholestasis. The exact mechanisms of CPZ induced cholestasis remain unclear. Rat hepatocytes, or a sandwich culture of rat and human hepatocytes, have been the most commonly used models for studying CPZ toxicity in vitro. However, to better predict outcomes in pre-clinical trials where cholestasis may be an unwanted consequence, a human in vitro model, based on human HepaRG cells, capable of real-time, non-invasive and label free monitoring, alongside molecular investigations would be beneficial. To address this we used the human hepatic HepaRG cell line, and established concentrations of CPZ ranging from sub-toxic, 25 μM and 50 μM, to toxic 100 μM and compared them with untreated control. To assess the effect of this range of CPZ concentrations we employed electrical cell-substrate impedance sensing (ECIS) to measure viability and cell membrane interactions alongside traditional viability assays, immunocytostaining and qRT-PCR to assess genes of interest within adaptive and inflammatory pathways. Using these methods, we show a concentration dependant response to CPZ involving pro-inflammatory pathway, loss of tight junctions and membrane integrity, and an adaptive response mediated by Cytochrome P450 (CYP) enzyme activation and up-regulation of membrane phospholipid and xenobiotic transporters. In conclusion, structural changes within the membrane caused by sub-toxic and toxic concentrations of CPZ negatively impact the function of the cellular membrane. Damage to efflux transport proteins caused by CPZ induce cholestasis alongside downstream inflammation, which activates compensatory responses for cell survival.
Original languageEnglish
Pages (from-to)1408-1416
Number of pages9
JournalBiomedicine & Pharmacotherapy
Volume111
DOIs
Publication statusPublished - 19 Jan 2019
Externally publishedYes

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Chlorpromazine
Hepatocytes
Cell Membrane
Cell Line
Poisons
Cholestasis
Membranes
Cytochrome P-450 Enzyme System
Intrahepatic Cholestasis
Enzyme Activation
Tight Junctions
Xenobiotics
Electric Impedance
Cell Communication
Antipsychotic Agents
Phospholipids
Cell Survival
Carrier Proteins
Up-Regulation
Clinical Trials

Cite this

Morgan, Katie ; Martucci, Nicole ; Kozlowska, Ada ; Gamal, Wesam ; Brzeszczynki, Fillip ; Treskes, Philipp ; Samuel, Kay ; Hayes, Peter ; Nelson, Lenny ; Bagnaninchi, Pierre ; Brzeszczynska, Joanna ; Plevris, John. / Chlorpromazine toxicity is associated with disruption of cell membrane integrity and initiation of a pro-inflammatory response in the HepaRG hepatic cell line. In: Biomedicine & Pharmacotherapy. 2019 ; Vol. 111. pp. 1408-1416.
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abstract = "Chlorpromazine (CPZ) is a neuroleptic drug and prototype compound used to study intrahepatic cholestasis. The exact mechanisms of CPZ induced cholestasis remain unclear. Rat hepatocytes, or a sandwich culture of rat and human hepatocytes, have been the most commonly used models for studying CPZ toxicity in vitro. However, to better predict outcomes in pre-clinical trials where cholestasis may be an unwanted consequence, a human in vitro model, based on human HepaRG cells, capable of real-time, non-invasive and label free monitoring, alongside molecular investigations would be beneficial. To address this we used the human hepatic HepaRG cell line, and established concentrations of CPZ ranging from sub-toxic, 25 μM and 50 μM, to toxic 100 μM and compared them with untreated control. To assess the effect of this range of CPZ concentrations we employed electrical cell-substrate impedance sensing (ECIS) to measure viability and cell membrane interactions alongside traditional viability assays, immunocytostaining and qRT-PCR to assess genes of interest within adaptive and inflammatory pathways. Using these methods, we show a concentration dependant response to CPZ involving pro-inflammatory pathway, loss of tight junctions and membrane integrity, and an adaptive response mediated by Cytochrome P450 (CYP) enzyme activation and up-regulation of membrane phospholipid and xenobiotic transporters. In conclusion, structural changes within the membrane caused by sub-toxic and toxic concentrations of CPZ negatively impact the function of the cellular membrane. Damage to efflux transport proteins caused by CPZ induce cholestasis alongside downstream inflammation, which activates compensatory responses for cell survival.",
author = "Katie Morgan and Nicole Martucci and Ada Kozlowska and Wesam Gamal and Fillip Brzeszczynki and Philipp Treskes and Kay Samuel and Peter Hayes and Lenny Nelson and Pierre Bagnaninchi and Joanna Brzeszczynska and John Plevris",
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Morgan, K, Martucci, N, Kozlowska, A, Gamal, W, Brzeszczynki, F, Treskes, P, Samuel, K, Hayes, P, Nelson, L, Bagnaninchi, P, Brzeszczynska, J & Plevris, J 2019, 'Chlorpromazine toxicity is associated with disruption of cell membrane integrity and initiation of a pro-inflammatory response in the HepaRG hepatic cell line' Biomedicine & Pharmacotherapy, vol. 111, pp. 1408-1416. https://doi.org/10.1016/j.biopha.2019.01.020

Chlorpromazine toxicity is associated with disruption of cell membrane integrity and initiation of a pro-inflammatory response in the HepaRG hepatic cell line. / Morgan, Katie; Martucci, Nicole; Kozlowska, Ada; Gamal, Wesam; Brzeszczynki, Fillip; Treskes, Philipp; Samuel, Kay; Hayes, Peter; Nelson, Lenny; Bagnaninchi, Pierre; Brzeszczynska, Joanna; Plevris, John.

In: Biomedicine & Pharmacotherapy, Vol. 111, 19.01.2019, p. 1408-1416.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Chlorpromazine toxicity is associated with disruption of cell membrane integrity and initiation of a pro-inflammatory response in the HepaRG hepatic cell line

AU - Morgan, Katie

AU - Martucci, Nicole

AU - Kozlowska, Ada

AU - Gamal, Wesam

AU - Brzeszczynki, Fillip

AU - Treskes, Philipp

AU - Samuel, Kay

AU - Hayes, Peter

AU - Nelson, Lenny

AU - Bagnaninchi, Pierre

AU - Brzeszczynska, Joanna

AU - Plevris, John

PY - 2019/1/19

Y1 - 2019/1/19

N2 - Chlorpromazine (CPZ) is a neuroleptic drug and prototype compound used to study intrahepatic cholestasis. The exact mechanisms of CPZ induced cholestasis remain unclear. Rat hepatocytes, or a sandwich culture of rat and human hepatocytes, have been the most commonly used models for studying CPZ toxicity in vitro. However, to better predict outcomes in pre-clinical trials where cholestasis may be an unwanted consequence, a human in vitro model, based on human HepaRG cells, capable of real-time, non-invasive and label free monitoring, alongside molecular investigations would be beneficial. To address this we used the human hepatic HepaRG cell line, and established concentrations of CPZ ranging from sub-toxic, 25 μM and 50 μM, to toxic 100 μM and compared them with untreated control. To assess the effect of this range of CPZ concentrations we employed electrical cell-substrate impedance sensing (ECIS) to measure viability and cell membrane interactions alongside traditional viability assays, immunocytostaining and qRT-PCR to assess genes of interest within adaptive and inflammatory pathways. Using these methods, we show a concentration dependant response to CPZ involving pro-inflammatory pathway, loss of tight junctions and membrane integrity, and an adaptive response mediated by Cytochrome P450 (CYP) enzyme activation and up-regulation of membrane phospholipid and xenobiotic transporters. In conclusion, structural changes within the membrane caused by sub-toxic and toxic concentrations of CPZ negatively impact the function of the cellular membrane. Damage to efflux transport proteins caused by CPZ induce cholestasis alongside downstream inflammation, which activates compensatory responses for cell survival.

AB - Chlorpromazine (CPZ) is a neuroleptic drug and prototype compound used to study intrahepatic cholestasis. The exact mechanisms of CPZ induced cholestasis remain unclear. Rat hepatocytes, or a sandwich culture of rat and human hepatocytes, have been the most commonly used models for studying CPZ toxicity in vitro. However, to better predict outcomes in pre-clinical trials where cholestasis may be an unwanted consequence, a human in vitro model, based on human HepaRG cells, capable of real-time, non-invasive and label free monitoring, alongside molecular investigations would be beneficial. To address this we used the human hepatic HepaRG cell line, and established concentrations of CPZ ranging from sub-toxic, 25 μM and 50 μM, to toxic 100 μM and compared them with untreated control. To assess the effect of this range of CPZ concentrations we employed electrical cell-substrate impedance sensing (ECIS) to measure viability and cell membrane interactions alongside traditional viability assays, immunocytostaining and qRT-PCR to assess genes of interest within adaptive and inflammatory pathways. Using these methods, we show a concentration dependant response to CPZ involving pro-inflammatory pathway, loss of tight junctions and membrane integrity, and an adaptive response mediated by Cytochrome P450 (CYP) enzyme activation and up-regulation of membrane phospholipid and xenobiotic transporters. In conclusion, structural changes within the membrane caused by sub-toxic and toxic concentrations of CPZ negatively impact the function of the cellular membrane. Damage to efflux transport proteins caused by CPZ induce cholestasis alongside downstream inflammation, which activates compensatory responses for cell survival.

U2 - 10.1016/j.biopha.2019.01.020

DO - 10.1016/j.biopha.2019.01.020

M3 - Article

VL - 111

SP - 1408

EP - 1416

JO - Biomedicine & Pharmacotherapy

JF - Biomedicine & Pharmacotherapy

SN - 0753-3322

ER -