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. Author manuscript; available in PMC: 2021 Mar 22.
Published in final edited form as: Chem Biol Interact. 2020 Dec 9;334:109353. doi: 10.1016/j.cbi.2020.109353

A mechanism of perhexiline’s cytotoxicity in hepatic cells involves endoplasmic reticulum stress and p38 signaling pathway

Zhen Ren a,**, Si Chen a, Sery Pak b, Lei Guo a,*
PMCID: PMC7982969  NIHMSID: NIHMS1674815  PMID: 33309543

Abstract

Perhexiline is a coronary vasodilator for angina treatment that was first developed in the 1960s. Perhexiline enjoyed worldwide success before reports of severe side effects, such as hepatotoxicity and neurotoxicity, caused its withdrawal from most of the markets. The underlying mechanism of the cytotoxicity of perhexiline, however, is not yet well understood. Here we demonstrated that perhexiline induced cellular damage in primary human hepatocytes, HepaRG cells and HepG2 cells. Analysis of gene and protein expression levels of endoplasmic reticulum (ER) stress markers showed that perhexiline caused ER stress in primary human hepatocytes and HepG2 cells. The splicing of XBP1 mRNA, a hallmark of ER stress, was observed upon perhexiline treatment. Using Gluc-Fluc-HepG2 cell line, we demonstrated that protein secretion was impaired upon perhexiline treatment, suggesting functional deficits in ER. Inhibition of ER stress using ER inhibitor 4-PBA or salubrinal attenuated the cytotoxicity of perhexiline. Directly knocking down ATF4 using siRNA also partially rescued HepG2 cells upon perhexiline exposure. In addition, inhibition of ER stress using either inhibitors or siRNA transfection attenuated perhexiline-induced increase in caspase 3/7 activity, indicating that ER stress contributed to perhexiline-induced apoptosis. Moreover, perhexiline treatment resulted in activation of p38 and JNK signaling pathways, two branches of MAPK cascade. Pre-treating HepG2 cells with p38 inhibitor SB239063 attenuated perhexiline-induced apoptosis and cell death. The inhibitor also prevented the activation of CHOP and ATF4. Overall, our study demonstrated that ER stress is one important mechanism underlying the hepatotoxicity of perhexiline, and p38 signaling pathway contributes to this process. Our finding shed light on the role of both ER stress and p38 signaling pathway in drug-induced liver injury.

Keywords: Cytotoxicity, ER stress, p38

1. Introduction

Perhexiline is a coronary vasodilator for angina treatment that was first developed in the 1960s [1]. Perhexiline enjoyed worldwide success before reports of severe side effects, such as hepatotoxicity and neurotoxicity, caused its withdrawal from most of the markets [2,3]. Due to its effectiveness, perhexiline is still used currently in a few countries, including Australia and New Zealand [4]. Despite all the usage, studies on the mechanisms of perhexiline-induced liver injury is limited. The endoplasmic reticulum (ER) is a critical organelle in the cell. It plays pivotal roles in the homeostasis of cellular calcium, protein modification and secretion, and many other cellular activities. In recent years, more and more research efforts have focused on the role of ER in cell death caused by diseases or drugs [5–8]. Particularly, multiple research groups including ours, have published various studies regarding the contribution of ER stress in drug-induced liver toxicity [9–15]. Exogenous stimuli could disturb the function of ER and cause the accumulation of unfolded proteins. This could result in the activation of unfolded protein response (UPR), an endogenous cellular process aims to restore the homeostasis in ER. The UPR signaling cascade is composed of three branches and starts from the upstream factors PKR-liken endoplasmic reticulum kinase (PERK), activating transcription factor 6 (ATF6) and inositol-requiring enzyme 1α (IRE1α). Each of these factors triggers the activation of the downstream factors including transcriptional factors, eventually results in gene expression changes. Activation of PERK leads to the phosphorylation of eukaryotic initiation factor 2α (eIF2α), which reduces overall influx of nascent polypeptides into the ER under stress whereas increases selective factors, such as activating transcription factor 4 (ATF4), which allows the cell to adapt to ER stress. Activation of ATF6 triggers the cleavage of the factor and the translocation of the cleaved form into the nucleus to function as transcriptional factor. For the IRE1α branch, phosphorylation of IRE1α causes the splicing of X-box binding protein 1 (XBP1) mRNA. Spliced XBP1 enters the nucleus and then regulates downstream gene expression. For mild ER stress, UPR strives to re-establish the balance in cell and promotes cell survival. However, excessive ER stress can eventually lead to cell death [7,11].

The mitogen-activated protein kinase (MAPK) cascade is considered one of the most important signaling pathways in cells. The MAPK cascade plays a pivotal role in various cellular functions, including cell proliferation, response to stress, survival, and cell death [16]. Previous study from our laboratory and others have shown that MAPK pathways also contribute to drug-induced liver toxicity [13,14,17–19]. The MAPK cascade is mainly composed of three pathways: the p38 pathway, the C-Jun N-terminal kinase (JNK) pathway, and the extracellular signal-regulated kinase 1 and 2 (ERK1/2) pathway. JNK and p38 pathways are generally pro-apoptosis, whereas activation of ERK1/2 pathways promotes cell survival [20,21].

In the current study, we used multiple hepatic cell models including HepG2 cells, HepaRG cells and primary human hepatocytes to study the molecular mechanisms of perhexiline-induced hepatotoxicity. We found that perhexiline caused ER stress in hepatic cells. Inhibition of ER stress attenuated the toxic effects of perhexiline. In addition, we demonstrated that P38 signaling pathway of the MAPK cascade also contributed to perhexiline-induced liver injury.

2. Material and methods

2.1. Chemicals and reagents

Williams’ Medium E, 4-Phenylbutyric acid (4-PBA), perhexiline maleate salt, salubrinal, dimethysulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO). Antibiotic-antimycotic were purchased from Life Technologies (Carlsbad, CA). Fetal bovine serum (FBS) was obtained from Atlanta Biologicals (Lawrenceville, GA). PureCol Bovine Collagen Solution was purchased from Advanced BioMatrix (San Diego, CA). SB239063 (p38 inhibitor) was from LC laboratories (Woburn, MA).

2.2. Cell culture and drug treatment

The human hepatoma cell line HepG2 was cultured in Williams’ Medium E supplemented with 10% FBS and 1 × antibiotic-antimycotic, as described previously [14]. The passage number did not exceed 10 for all the experiments. Cells were either seeded at a density of 2.5 × 105 cells/ml in volumes of 100 μl in the wells of 96-well tissue culture plates for toxicity assays, or in volumes of 5 ml in 60 mm plates for biochemical assays. Unless otherwise specified, cells were maintained in growth medium for approximately 24 h before exposed to various concentrations of perhexiline and/or inhibitors, as indicated in the text. DMSO treated cells were served as control (the final concentration did not exceed 0.1%).

Primary human hepatocytes were purchased from In Vitro ADMET Laboratories (Columbia, MD) and were pooled from 10 donors. Plates used for primary human hepatocytes were pre-coated overnight with PureCol® (Advanced BioMatrix, Carlsbad, CA) following manufacturer’s protocol. Primary human hepatocytes were seeded at a density of 4 × 105 cells/ml in volumes of 100 μl in the wells of 96-well cell culture plates for toxicity assays, or in volumes of 5 ml in 60 mm petri dishes for isolation of RNA or protein extraction, according to supplier’s protocol. Cells were maintained in Universal Primary Cell Plating Medium (UPCM™) supplied by the manufacturer.

The human hepatoma cell line HepaRG was purchased from Bio-predic International (Saint Grégoire, France) and cultured following the supplier’s instruction. Briefly, cells were counted and plated at a density of 0.7 × 106 cells in a 100 mm cell culture dish and maintained in 10 ml Williams’ Medium E supplemented with 1% GlutaMax (ThermoFisher Scientific, Waltham, MA), 100 μg/ml primocin (Invivogen, San Diego, CA), and a growth supplement (Lonza, Walkersville, MD) at 37 °C for 14 days. Cell differentiation was induced by adding differentiation supplement (Lonza) to the culture medium for an additional 14 days. Afterwards differentiated cells were seeded at a density of 2.5 × 105 cells/ml in volumes of 100 μl in the wells of 96-well cell culture plates for 72 h before drug treatment as described above.

2.3. Lactate dehydrogenase assay

Lactate dehydrogenase (LDH) release was measured and calculated as described previously [22].

2.4. Measurement of caspase 3/7 activity

The enzymatic activities of caspase 3/7 was measured using Caspase-Glo® 3/7 Assay System (Promega Corporation) following the supplier’s instruction. The intensity of luminescent signals was detected using a Cytation 5 Microplate Reader and the results in drug treated cells were normalized to those in DMSO vehicle controls.

2.5. Isolation of RNA and quantitative real-time PCR assay

Total RNA was isolated using the RNeasy Mini Kit purchased from Qiagen (Germantown, MD) upon the completion of drug treatment. The yield of the extracted RNA was assessed by measurement of absorption at 260 nm using a NanoDrop 2000 spectrometer (Thermo Scientific, Waltham, MA). cDNAs were generated through reverse transcription of 2 μg of total RNA using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystem, Foster City, CA) following the manufacturer’s instruction. Quantitative real-time PCR assay for ATF6, ERN1, EIF2AK3, ATF4, and DDIT3 were conducted as described previously and GAPDH was used as an internal control.

2.6. X-box binding protein 1 (XBP1) mRNA splicing assay

The cDNAs were prepared from total RNAs (1 μg) using high capacity cDNA reverse transcription kits (Life Technologies) according to the manufacturer’s protocol. The primer information and amplification conditions are detailed in our previous publication [9]. GAPDH was used as internal control. The unspliced and spliced XBP1 were analyzed by electrophoresis using 4% (w/v) agarose gels, stained with 0.5 μg/ml ethidium bromide solution, and visualized under UV light.

2.7. Luciferase activity

Upon the completion of drug treatment, 5 μL of medium from each well of the 96-well plate was saved for Gaussia luciferase assay, to measure the level of secreted Gaussia luciferase. The cells in the plate were lysed and proceed for Firefly Luciferase assay as internal control. Gaussia luciferase (Glue) activity and firefly luciferase (Flue) was measured by NanoFuel Glow Assay kit and Firefly Luciferase kit purchased from NanoLight™ Technology (Pinetop, AZ) following manufacturer’s instruction. The Gaussia luciferase activity in each cell was first normalized to the Firefly luciferase activity level to compensate the difference in cell number, then expressed as a percentage of the DMSO control.

2.8. siRNA transfection

HepG2 cells were reverse transfected with Silencer® ATF4 siRNA, or Silencer® Negative Control #1 siRNA (Life Technologies) by Lipofect-amine® RNAiMAX Transfection Reagent (Life Technologies), as described before [13]. Briefly, HepG2 cells were seeded to 10 cm dishes at a density of 3 × 106 cells/dish in 10 ml transfection solution (8 ml antibiotic-free growth medium, 2 ml of Opti-MEM, 20 μl of Lipofect-amine RNAiMAX, and 200 pmol of desired siRNA). Cells were changed to normal growth medium 24 h after transfection and plated into 96-well plates for toxicity assays or 6 cm dishes for biochemical assays, as described above. Drug treatments started approximately 48 h after transfection, and the silencing efficiency of the siRNA was determined by Western blotting.

2.9. Western blot analysis

Upon completion of drug treatments, cells were harvested using RIPA buffer supplemented with Halt Protease Inhibitor Cocktail (ThermoFisher Scientific). The concentrations of the samples were determined by Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules, CA). Standard Western blot analysis was performed using antibodies against CHOP, phosphor(p)-eIF2α (Ser 51), eIF2α, p-p38, p38, p-JNK, JNK, p-ERK1/2 (Thr202/Tyr204), ERK1/2 (Cell Signaling Technology, Danvers, Massachusetts), ATF-4 and GAPDH (as internal control, Santa Cruz Biotechnology, Santa Cruz, CA). The bands were detected by FluorChem E and M Imager (ProteinSimple, San Jose, CA) and were quantified using ImageJ software (NIH, Bethesda, MD).

2.10. Statistical analysis

Data are presented as the mean ± standard deviation (SD) of at least three independent experiments. Analyses were performed using GraphPad Prism 5 (GraphPad Software, San Diego, CA). Statistical significance was determined by one-way analysis of variance (ANOVA) followed by the Dunnett’s tests for pairwise-comparisons or two-way ANOVA followed by the Bonferroni post-test. The difference was considered statistically significant when p was less than 0.05.

3. Results

3.1. Perhexiline causes cellular damage and ER stress in primary human hepatocytes

We first examined the cytotoxicity of perhexiline in primary human hepatocytes. Cells were exposed to 5, 10, 15, 20, and 25 μM perhexiline for 4 h before LDH release was assessed. As shown in Fig. 1A, 20 and 25 μM perhexiline increased the LDH release in primary human hepatocytes to 39.6% and 47.3%, a significant elevation from those in DMSO treated controls. The increase was in a concentration-dependent manner.

Fig. 1.

Fig. 1.

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Perhexiline causes cellular damage and ER stress in primary human hepatocytes. (A) HepG2 cells, HepaRG cells, and primary human hepatocytes were exposed to 5, 10, 15, 20 and 25 μM perhexiline for 4 h. The cytotoxicity of perhexiline was measured using LDH release. (B and C) Primary human hepatocytes were treated with 20 μM perhexiline. Relative mRNA levels of ER markers were assessed after 1 h (B), and protein levels were analyzed using Western blotting at 2 h (C). The results shown are mean ± S.D. from 3 independent experiments. *p < 0.05 compared to DMSO control.

In addition to primary human hepatocytes, we exposed HepaRG cells and HepG2 cells to perhexiline under the same conditions. In both cell lines, perhexiline induced similar cytotoxicity as we observed in primary human hepatocytes. In HepG2 cells, LDH release climbed to 55% at 25 μM perhexiline treatment, suggesting severer toxicity.

Perhexiline is known to cause mitochondrial damage [23]. Our own investigation also demonstrated that perhexiline induced mitochondrial dysfunction in hepatic cells [24]. Mitochondrial dysfunction and ER stress are two closely related mechanisms in drug-induced liver injury due to the physical and functional ties between the two cellular organelles [25–29]. Therefore, in the current study we explored whether ER stress could be an underlying mechanism for perhexiline induced hepatotoxicity.

We analyzed the mRNA levels of UPR hallmark proteins ATF6, IRE1α (gene ERN1), PERK (gene EIF2AK3), ATF4, and CHOP (transcriptional factor C/EBP homologous protein, gene DDIT3) in primary human hepatocytes after exposure to 20 μM perhexiline for 1 h. As demonstrated in Fig. 1B, all the mRNA tested showed 3–5 folds increase compared to those in DMSO treated controls. Western blotting confirmed this observation: 20 μM perhexiline induced an increase in expression of CHOP and ATF4 after 2 h, suggesting perhexiline caused ER stress in primary human hepatocytes Fig. 1C.

3.2. Perhexiline induces ER stress in HepG2 cells

When exposed to perhexiline, HepG2 cells generated similar responses as we observed in primary hepatocytes. As demonstrated in Fig. 2A, when exposed to 1 h perhexiline treatment, HepG2 cells displayed an increase in mRNA levels for ATF6, ERN1, EIF2AK3, ATF4 and DDIT3. Similar trends were observed at the protein levels. Western blotting analysis demonstrated that the protein levels of CHOP, ATF4, and p-eIF2α were elevated at 2 h and 4 h timepoints when exposed to perhexiline (Fig. 2B and C). The increase was concentration-dependent; quantification of Western blots at 2 h suggested that the increase reached 3–4 folds for the three ER stress markers. The total level of eIF2α, however, was unaltered. Furthermore, we observed the splicing of XBP1 mRNA, a hallmark of the occurrence of ER stress, upon exposure to perhexiline in HepG2 cells (Fig. 2D).

Fig. 2.

Fig. 2.

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Perhexiline induces ER stress in HepG2 cells. (A–C) HepG2 cells were exposed to 5–25 μM perhexiline. Relative mRNA levels of gene ATF6, ERN1, EIF2AK3, ATF4 and DDIT3 were measured after 1 h and expressed as fold-changes (A). Protein levels of ER stress markers CHOP, ATF4, P-eIF2α, and eIF2α after 2 h and 4 h exposure to perhexiline were measured using Western blotting. Representative Western blots were shown in (B), and quantification of band densities at 2 h was shown in (C). (D) Representative DNA gel showing semi-quantitative RT-PCR analysis of spliced XBP1 mRNA. GAPDH was used as an internal control. (E) Gluc-Fluc-HepG2 cells were treated with 5–20 μM perhexiline for 0.5, 1, and 2 h, and relative Gaussia Luciferase level was expressed as percentage of control. The results shown are mean ± S.D. from 3 to 4 independent experiments. *p < 0.05 compared to DMSO control.

One major function of ER is protein secretion; thus, measurement of protein secretion could be an indicator for the function of ER. Previously we have established a stable Gluc-Fluc-HepG2 cell line that uses reporter assays to measure the level of protein secretion. Our previous publication detailed the protocol in establishing and using the cell line [30]. The Gluc-Fluc-HepG2 cells permanently express a naturally secreted protein Gaussia luciferase (Gluc) and firefly luciferase (Fluc). The Gluc reporter is measured from the medium while the Fluc reporter is measured after cells are lysed and used as an internal control to normalize the difference in cell numbers in each sample. The level of Gluc is first normalized to the Fluc level in that sample, then expressed as a percentage of the results obtained from DMSO treated control. The inhibition of Gaussia luciferase activity represents decreased protein secretion and thus indicates ER stress [9,13,14,30]. To test whether the changes we observed in UPR related proteins indicated functional deficits in ER, we exposed Gluc-Fluc-HepG2 cells to 5–20 μM perhexiline for 0.5, 1, and 2 h, and examined the amount of secreted Gaussia luciferase (Fig. 2E). The level of secreted proteins showed significant drop as early as 0.5 h, suggesting the negative effect on ER was rapid. For both 1 h and 2 h treatment, small but significant decrease in ER function was observed at concentration as low as 5 μM perhexiline. Moreover, exposure to 10 μM perhexiline for 2 h resulted in almost a 50% decrease in the function of the ER. The detrimental effect on ER function was both time- and concentration-dependent. Together with the changes of UPR markers at both genetic and protein levels, these observations suggested that perhexiline caused ER stress in HepG2 cells.

3.3. Inhibition of ER stress attenuates the cytotoxicity of perhexiline

To confirm further the role of ER stress in perhexiline induced hepatotoxicity, we pre-treated HepG2 cells with 1 mM 4-Phenylbutyrate (4-PBA), an established chemical chaperone, for 1 h before exposing the cells to 25 μM perhexiline. As shown in Fig. 3A, 4-PBA partially rescued cells from the toxic effects of perhexiline. LDH release was reduced from 40% to 20% in the 4-PBA treated group, compared to perhexiline-only group. Western blotting analysis showed that the increase in protein levels of CHOP and ATF4 were also attenuated by 4-PBA, confirmed the inhibition of ER stress (Fig. 3B).

Fig. 3.

Fig. 3.

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Inhibition of ER stress attenuates perhexiline-indueed cytotoxicity. (A and B) HepG2 cells were pre-treated with 1 mM 4-PBA for 1 h before exposed to 25 μM perhexiline. The cytotoxicity of perhexiline was measured using LDH release (A). The protein levels of CHOP and ATF4 was assessed by Western blotting (B). (C) HepG2 cells were pre-treated with 20 μM salubrinal for 1 h before exposed to 20 and 25 μM perhexiline. (D and E) HepG2 cells were transfected with Ctrl siRNA or ATF4 siRNA for 24 h before exposed to 20 and 25 μM perhexiline. Cytotoxicity was assessed using LDH release (D), and the knockdown efficiency was assessed using Western blotting (E). The cytotoxicity of perhexiline was measured using LDH release. The results shown are mean ± S.D. from 3 independent experiments. *p < 0.05 compared to DM SO control.

To exclude the possibility that the observed effect of 4-PBA was due to off-target function of 4-PBA, we applied another ER stress inhibitor salubrinal. Salubrinal selectively inhibits the dephosphorylation of eIF2α and has been shown to have protective effect against ER stress [14,31,32]. HepG2 cells were pre-treated with 20 μM salubrinal for 1 h before exposed to 20 or 25 μM perhexiline. As shown in (Fig. 3C), similar to 4-PBA, salubrinal attenuated the increase of LDH release induced by perhexiline treatment. These results suggested that blocking ER stress could protect HepG2 cells from the cytotoxicity of perhexiline.

We also used siRNA to directly knockdown components of UPR pathways, to test the role of ER stress in the cytotoxicity of perhexiline. HepG2 cells were transfected with ATF4 siRNA or control siRNA for 24 h before exposed to 20 or 25 μM perhexiline. As shown in Fig. 3D, compared to control siRNA transfected cells, ATF4 siRNA transfected cells showed significantly lower increase in LDH release. The knockdown efficiency was confirmed by Western blotting, as demonstrated in Fig. 3E.

Previous study in our laboratory demonstrated that perhexiline treatment caused apoptosis in hepatic cells [24]. Exposure to perhexiline treatment rapidly increased caspase 3/7 activity to 20 folds of control (Fig. 4A), suggesting the activation of apoptotic pathways. To explore whether perhexiline-induced ER stress is involved in this process, we tested caspase 3/7 activity upon perhexiline treatment when the ER stress is inhibited, either by treatment of chemical inhibitors or siRNA knockdown. As depicted in Fig. 4B, pre-treating HepG2 cells with 4-PBA attenuated the increase in caspase 3/7. Similar effects were observed in salubrinal pre-treated cells (Fig. 4C). Transiently knockdown of ATF4 resulted in more robust effect on caspase 3/7 activity (Fig. 4D). In control siRNA transfected HepG2 cells, caspase 3/7 activity upon a 2 h-treatment with 20 and 25 μM perhexiline was 17.3-fold and 22.8-fold greater than the DMSO treated control, whereas in the ATF4 siRNA-transfected HepG2 cells the increases were only 4.4 folds and 8.7 folds. These results suggested that ER stress facilitates the onset of apoptosis in HepG2 cells upon perhexiline treatment.

Fig. 4.

Fig. 4.

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Inhibition of ER stress attenuates perhexiline-induced apoptosis. (A) HepG2 cells were exposed to 5–25 μM perhexiline for 2 h and caspase 3/7 activity was measured. (B) HepG2 cells were pre-treated with 1 mM 4-PBA for 1 h before exposed to 25 μM perhexiline, and caspase 3/7 activity was measured. (C) HepG2 cells were pre-treated with 20 μ salubrinal for 1 h before exposed to 20 and 25 μM perhexiline, and caspase 3/7 activity was measured. (D) HepG2 cells were transfected with Ctrl siRNA or ATF4 siRNA 24 h before exposing to 20 or 25 μM perhexiline for 2 h, and caspase 3/7 activity was measured. The results shown are mean ± S.D. from 3 independent experiments. *p < 0.05 compared to DMSO control.

3.4. Perhexiline treatment activated MAPK signaling pathways

MAPK signaling cascade plays critical roles in various cellular functions. Studies from our laboratory and others have demonstrated that MAPK pathways are also important contributors to drug-induced cytotoxicity [13,14,18,22,33,34]. Therefore, in the current study we tested whether MAPK cascade could be involved in perhexiline-induced hepatotoxicity. HepG2 cells were treated with 5–25 μM perhexiline for 1 or 2 h, and then whole cell extracts were harvested for Western blotting analysis. As demonstrated in Fig. 5, the p-p38 and p-JNK showed a concentration-dependent increase upon perhexiline treatment, suggesting the activation of the p38 and p-JNK pathways. On the other hand, p-ERK1/2 and the overall levels of p38, JNK, and ERK1/2 showed little change under the same conditions.

Fig. 5.

Fig. 5.

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Perhexiline activates MAPK signaling cascade. HepG2 cells were treated with 5–25 μM perhexiline for 1 or 2 h, and the whole cell extracts were analyzed using Western blotting. (A) Representative Western blots showing activation of MAPK signaling pathways. (B) Quantification of bands density for 2 h perhexiline treatment. The results shown are mean ± S.D. from 3 independent experiments. *p < 0.05 compared to DMSO control.

3.5. Activation of p38 pathway contributes to perhexiline-induced hepatotoxicity

To explore further the potential role of p38 pathway in perhexiline induced cell damage, we pre-treated HepG2 cells with 10 μM p38 kinase specific inhibitor SB239063 for 1 h before exposing the cells to 25 μM perhexiline. In the presence of SB239063, the perhexiline-induced elevation of LDH was attenuated, as depicted in Fig. 6A. The inhibitory effect on p-p38 was assessed using Wester blotting (Fig. 6B). No significant change in total p38 level was observed. Interestingly, the application of SB239063 also inhibited the increase in CHOP and ATF4 (Fig. 6B), suggesting the activation of p38 pathway is essential for the perhexiline induced UPR. In addition, SB239063 showed decreased caspase 3/7 activation upon perhexiline treatment, in comparison to the control group, indicating p38 pathway contributed to perhexiline-induced apoptosis (Fig. 6C). Therefore, these results showed that the activation of p38 pathway is involved in both perhexiline induced ER stress and apoptosis, thus contributed to the drug’s hepatotoxicity.

Fig. 6.

Fig. 6.

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Inhibition of p38 signaling pathway attenuates the cytotoxicity of perhexiline. HepG2 cells were pre-treated with 10 μM SB239063 before exposed to 25 μM perhexiline. (A) Cytotoxicity of perhexiline was assessed using LDH release. (B) The effectiveness of SB239063 was assessed using Western blotting. (C) Caspase 3/7 activity was assessed after 2 h perhexiline treatment. The results shown are mean ± S.D. from 3 independent experiments. *p < 0.05 compared to DMSO control.

4. Discussion

Perhexiline is a highly effective treatment for angina and has been used as last line of medicine in patients with severe conditions. The reported cases of hepatotoxicity and neurotoxicity have greatly limited the use of the drug [1]. The underlying mechanism, however, is not clear yet. In our current study, we identified ER stress as one of the mechanisms for the cytotoxicity of perhexiline, and the activation of p38 pathway contributed to this process.

Clinically, in the countries where perhexiline is used, plasma concentrations of the drug are monitored to be within 600 μg/L (2.16 μM) [1,35]. Neurological or hepatic negative effects have been reported when perhexiline plasma concentration is between 720 and 2680 μg/L (2.59 and 9.65 μM) [1]. In this study we used perhexiline concentration 5–25 μM. The lower concentrations we used are within the range that encompasses clinical concerns. Although the high concentrations exceeded the clinical plasma concentrations, for risk identification especially for identifying the idiosyncratic hepatotoxic potential of a drug, using the concentrations up to 100-fold of the Cmax has been considered for in vitro studies. In the current study, we used concentrations of 5–25 μM for perhexiline, and cytotoxic effects were observed starting at 10 μM, so we believe that the data presented here are meaningful.

Our major findings of the current study are twofold. First, we identified ER stress as one of the mechanisms underlying the toxicity of perhexiline. ER stress has been recognized as an important contributing factor for multiple drugs and diseases, and thus is receiving increasing research interest in recent years [5,6,10,13,14,36–38]. In the case of perhexiline, we found that ER stress occurs rapidly. The mRNA levels of all the ER stress markers tested were elevated at 1 h perhexiline exposure (Fig. 2A), and the protein levels were increased in a concentration-dependent manner at 2 h (Fig. 2B and C). Splicing of XBP1 mRNA, a hallmark of ER stress, was also observed upon perhexiline treatment (Fig. 2D). More importantly, using the Gluc-Fluc-HepG2 cells, we demonstrated that the functional deficits in ER could be observed as early as 0.5 h (Fig. 2E). In addition, we found that the occurrence of ER stress contributed to perhexiline-induced apoptosis. Inhibition of ER stress attenuated the increase in caspase 3/7 upon perhexiline treatment (Fig. 4B–D). All these observations suggested that ER stress happened rapidly and may be one of the earliest mechanisms activated by perhexiline exposure.

The toxicity of perhexiline is mostly caused by the parent drug; clinically severe side effects was only observed in patients with extra accumulation of the parent drug in their plasma due to poor metabolism. Therefore, although we are aware that HepG2 cells have limited metabolism capacity [39], we do not consider it a limitation for the current mechanistic study. In addition, we observed activation of ER stress and UPR in primary human hepatocytes, with elevated gene and protein expression levels of ER stress hallmarks (Fig. 1B and C). These results are consistent with the observation in HepG2 cells.

Another major finding of our study is the role of p38 in perhexiline-induced toxicity. Among the three branches of MAPK cascade, the activation of p38 and JNK pathways is generally pro-apoptosis, whereas the activation of ERK1/2 pathway usually promotes cell survival. This is consistent with our observation in the current study. In the presence of p38 inhibitor SB239063, we found that the cell damage induced by perhexiline was partially rescued (Fig. 6A). Moreover, blocking p38 function attenuated the perhexiline-induced increase in caspase 3/7 activity (Fig. 6C), suggesting that p38 contributes to perhexiline-induced apoptosis. Reduced activation of CHOP and ATF4 upon application of SB239063 (Fig. 6B) indicated p38 is critical for ER stress. Previous studies have shown that p38 could phosphorylate CHOP and thus activates it [40]. Although we observed activation of the JNK pathway upon perhexiline treatment for 1 and 2 h (Fig. 5), pre-treating HepG2 cells with JNK inhibitor SP600125 did not attenuate perhexiline-induced increase in either LDH release or caspase 3/7 activity level (Supplemental Fig. 1), suggesting a limited rescue effect against the cytotoxicity of perhexiline. It has been reported that p38 was involved in regulation of early apoptosis, whereas JNK was involved in the late apoptosis of the cells [41], this could be one explanation for why we did not observe a substantial contribution of JNK signaling in our study. JNK may play a role during late apoptosis and cell death, whereas the current study looks at the early stage, thus little effect of JNK was detected in this time frame.

The ER is both physically and functionally related to mitochondria [28,29]. Studies in our laboratory and by other research groups have suggested that ER stress and mitochondrial dysfunction simultaneously contribute to the toxicity of multiple drugs [13,14,25,27,42–45]. Our own study and studies from other research groups showed that perhexiline caused mitochondrial damage and induced apoptosis [23,24], providing additional evidence for the close relation in the functions of mitochondria and ER, especially in drug-induced toxicity. Activation of CHOP and ATF4 has been shown to regulate genes encoding Bcl2-family proteins, such as Bcl2, Mcl1, and Bcl2-associated X protein (Bax) [46–48], which could facilitate induction of apoptosis, as we observed in the current study. Lim et al. reported that mitochondrial dysfunction could trigger ER stress responses, including the increase in expression level of CHOP and p-eIF2α [49]. Interestingly, they suggested that p38 is a critical element in this cross-talk between ER and mitochondria. The application of p38 inhibitor or dominant-negative p38 could block the mitochondrial dysfunction-induced elevation of 78-kDa glucose-regulated protein (Grp78), an UPR regulator. Alleviation of ER stress using 4-PBA, on the other hand, reduced p38 activation. In addition, p38 was shown to be involved in the regulation of both mitochondrial death pathway and ER stress in the early apoptosis [41]. Moreover, p38 activation has been demonstrated to participate in apoptosis triggered by multiple signaling pathways [50,51]. All these reports are consistent with our observation, and further suggest an important role of p38 in orchestrating mitochondrial dysfunction, ER stress, and apoptosis.

Previous study by Jones and colleagues measured the concentration-time profile for perhexiline in patients and reported that the peak plasma concentration occurred around 6 h post-dose [52], therefore we believe the data we presented here could shed some light on the understanding of perhexiline toxicity during the acute phase. It is important, however, to explore the cytotoxicity of perhexiline in a longer time frame. The ongoing project in our laboratory tests the hepatotoxicity of perhexiline in hepatic cells for up to 24 h. We are investigating whether ER stress plays a role at 24 h and longer time, and if other mechanisms also contribute to the adverse effects of perhexiline. In addition, perhexiline is known to be a substrate of CYP2D6, and its cytotoxic effects were mostly observed in poor metabolizers. However, whether the hepatotoxicity of perhexiline is solely due to the parental drug but not its oxidative metabolites has not yet been clarified. In our laboratory we have established a battery of CYP-expressing HepG2 cells which have been shown a useful tool in studies of metabolism-related drug toxicity [53–55]. Currently we are using these cells and other hepatic cells to explore the role of CYP2D6 and other CYPs in the toxicity of perhexiline. The outcome of the current efforts will be published in our upcoming paper soon.

Overall, our study demonstrated that ER stress is one of the early mechanisms underlying the cytotoxicity of perhexiline. Activation of p38 contributed to perhexiline-induced ER stress and apoptosis. These findings shed light on the importance of ER stress and MAPK signaling pathway in drug-induced liver injury.

Supplementary Material

Fig. S1. Inhibition of JNK signaling pathway had little effect on perhexiline-induced toxicity. HepG2 cells were pre-treated with 10 μM SP600125 before exposed to 25 μM perhexiline. (A) Cytotoxicity of perhexiline was assessed using LDH release. (B) Caspase 3/7 activity was assessed after 2 h perhexiline treatment. The results shown are mean ± S.D. from 3 independent experiments. *p < 0.05 compared to DMSO control

Acknowledgments

We thank Drs. Ji-Eun Seo and Xiaoqing Guo for culturing HepaRG cells. SP was supported by NCTR Summer Science Research Program.

Footnotes

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This article is not an official guidance or policy statement of the U.S. FDA. No official support or endorsement by the U.S. FDA is intended or should be inferred.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A.: Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.cbi.2020.109353.

References

  • [1].Ashrafian H, Horowitz JD, Frenneaux MP, Perhexiline, Cardiovasc. Drug Rev 25 (2007) 76–97. [DOI] [PubMed] [Google Scholar]
  • [2].Paliard P, Vitrey D, Fournier G, Belhadjali J, Patricot L, Berger F, Perhexiline maleate-induced hepatitis, Digestion 17 (1978) 419–427. [DOI] [PubMed] [Google Scholar]
  • [3].Poupon R, Rosensztajn L, Prudhomme de Saint-Maur P, Lageron A, Gombeau T, Darnis F, Perhexiline maleate-associated hepatic injury prevalence and characteristics, Digestion 20 (1980) 145–150. [DOI] [PubMed] [Google Scholar]
  • [4].Inglis S, Stewart S, Metabolic therapeutics in angina pectoris: history revisited with perhexiline, Eur. J. Cardiovasc. Nurs 5 (2006) 175–184. [DOI] [PubMed] [Google Scholar]
  • [5].Chen S, Melchior WB Jr., Guo L, Endoplasmic reticulum stress in drug- and environmental toxicant-induced liver toxicity, J. Environ. Sci. Health C Environ. Carcinog. Ecotoxicol. Rev 32 (2014) 83–104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [6].Iurlaro R, Munoz-Pinedo C, Cell death induced by endoplasmic reticulum stress, FEBS J 283 (2016) 2640–2652. [DOI] [PubMed] [Google Scholar]
  • [7].Sano R, Reed JC, ER stress-induced cell death mechanisms, Biochim. Biophys. Acta Mol. Cell Res 1833 (2013) 3460–3470. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [8].Tabas I, Ron D, Integrating the mechanisms of apoptosis induced by endoplasmic reticulum stress, Nat. Cell Biol 13 (2011) 184–190. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [9].Chen S, Xuan J, Couch L, Iyer A, Wu Y, Li QZ, Guo L, Sertraline induces endoplasmic reticulum stress in hepatic cells, Toxicology 322 (2014) 78–88. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [10].Chen S, Zhang Z, Wu Y, Shi Q, Yan H, Mei N, Tolleson WH, Guo L, Endoplasmic reticulum stress and store-operated calcium entry contribute to usnic acid-induced toxicity in hepatic cells. Toxicological sciences, an official journal of the Society of Toxicology 146 (2015) 116–126. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [11].Iorga A, Dara L, Kaplowitz N, Drug-induced liver injury: cascade of events leading to cell death, apoptosis or necrosis, Int. J. Mol. Sci 18 (2017). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [12].Li X, Wang Y, Wang H, Huang C, Huang Y, Li J, Endoplasmic reticulum stress is the crossroads of autophagy, inflammation, and apoptosis signaling pathways and participates in liver fibrosis, Inflamm. Res 64 (2015) 1–7. [DOI] [PubMed] [Google Scholar]
  • [13].Ren Z, Chen S, Qing T, Xuan J, Couch L, Yu D, Ning B, Shi L, Guo L, Endoplasmic reticulum stress and MAPK signaling pathway activation underlie leflunomide-induced toxicity in HepG2 Cells, Toxicology 392 (2017) 11–21. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [14].Ren Z, Chen S, Zhang J, Doshi U, Li AP, Guo L, Endoplasmic reticulum stress induction and ERK1/2 activation contribute to nefazodone-induced toxicity in hepatic cells, Toxicol. Sci. : an official journal of the Society of Toxicology (2016). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [15].Uzi D, Barda L, Scaiewicz V, Mills M, Mueller T, Gonzalez-Rodriguez A, Valverde AM, Iwawaki T, Nahmias Y, Xavier R, Chung RT, Tirosh B, Shibolet O, CHOP is a critical regulator of acetaminophen-induced hepatotoxicity, J. Hepatol 59 (2013) 495–503. [DOI] [PubMed] [Google Scholar]
  • [16].Plotnikov A, Zehorai E, Procaccia S, Seger R, The MAPK cascades: signaling components, nuclear roles and mechanisms of nuclear translocation, Biochim. Biophys. Acta 1813 (2011) 1619–1633. [DOI] [PubMed] [Google Scholar]
  • [17].Chen S, Xuan J, Wan L, Lin H, Couch L, Mei N, Dobrovolsky VN, Guo L, Sertraline, an antidepressant, induces apoptosis in hepatic cells through the mitogen-activated protein kinase pathway. Toxicological sciences, an official journal of the Society of Toxicology 137 (2014) 404–415. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [18].Darling NJ, Cook SJ, The role of MAPK signalling pathways in the response to endoplasmic reticulum stress, Biochim. Biophys. Acta 1843 (2014) 2150–2163. [DOI] [PubMed] [Google Scholar]
  • [19].Pal S, Sarkar A, Pal PB, Sil PC, Protective effect of arjunolic acid against atorvastatin induced hepatic and renal pathophysiology via MAPK, mitochondria and ER dependent pathways, Biochimie 112 (2015) 20–34. [DOI] [PubMed] [Google Scholar]
  • [20].Lawrence MC, Jivan A, Shao C, Duan L, Goad D, Zaganjor E, Osborne J, McGlynn K, Stippec S, Earnest S, Chen W, Cobb MH, The roles of MAPKs in disease, Cell Res 18 (2008) 436–442. [DOI] [PubMed] [Google Scholar]
  • [21].Pyakurel A, Savoia C, Hess D, Scorrano L, Extracellular regulated kinase phosphorylates mitofusin 1 to control mitochondrial morphology and apoptosis, Mol Cell 58 (2015) 244–254. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [22].Chen S, Ren Z, Yu D, Ning B, Guo L, DNA damage-induced apoptosis and mitogen-activated protein kinase pathway contribute to the toxicity of dronedarone in hepatic cells, Environ. Mol. Mutagen 59 (2018) 278–289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [23].Deschamps D, DeBeco V, Fisch C, Fromenty B, Guillouzo A, Pessayre D, Inhibition by perhexiline of oxidative phosphorylation and the beta-oxidation of fatty acids: possible role in pseudoalcoholic liver lesions, Hepatology 19 (1994) 948–961. [PubMed] [Google Scholar]
  • [24].Ren Z, Chen S, Seo JE, Guo X, Li D, Ning B, Guo L, Mitochondrial dysfunction and apoptosis underlie the hepatotoxicity of perhexiline, Toxicol. Vitro (2020) 104987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [25].Grimm S, The ER-mitochondria interface: the social network of cell death, Biochim. Biophys. Acta 1823 (2012) 327–334. [DOI] [PubMed] [Google Scholar]
  • [26].Karna KK, Choi BR, You JH, Shin YS, Soni KK, Cui WS, Lee SW, Kim CY, Kim HK, Park JK, Cross-talk between ER stress and mitochondrial pathway mediated adriamycin-induced testicular toxicity and DA-9401 modulate adriamycin-induced apoptosis in Sprague-Dawley rats, Canc. Cell Int 19 (2019) 85. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [27].Malhotra JD, Kaufman RJ, ER stress and its functional link to mitochondria: role in cell survival and death. Cold Spring Harb Perspect Biol 3, a004424, 2011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [28].Pizzo P, Pozzan T, Mitochondria–endoplasmic reticulum choreography: structure and signaling dynamics, Trends Cell Biol 17 (2007) 511–517. [DOI] [PubMed] [Google Scholar]
  • [29].Szabadkai G.r., Bianchi K, Várnai P.t., De Stefani D, Wieckowski MR, Cavagna D, Nagy AI, Balla T.s., Rizzuto R, Chaperone-mediated coupling of endoplasmic reticulum and mitochondrial Ca2+ channels, JCB (J. Cell Biol.) 175 (2006) 901–911. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [30].Ren Z, Chen S, Guo L, Methods for Establishing and Using a Stable Cell Line Expressing Both Gaussia Luciferase and Firefly Luciferase to Screen for Endoplasmic Reticulum Stress, in: Keohavong P, Singh KP, Gao W (Eds.), Molecular Toxicology Protocols, Springer US, New York, NY, 2020, pp. 531–555. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [31].Boyce M, Bryant KF, Jousse C, Long K, Harding HP, Scheuner D, Kaufman RJ, Ma D, Coen DM, Ron D, Yuan J, A selective inhibitor of eIF2alpha dephosphorylation protects cells from ER stress, Science 307 (2005) 935–939. [DOI] [PubMed] [Google Scholar]
  • [32].Goswami P, Gupta S, Biswas J, Joshi N, Swarnkar S, Nath C, Singh S, Endoplasmic reticulum stress plays a key role in rotenone-induced apoptotic death of neurons, Mol. Neurobiol 53 (2016) 285–298. [DOI] [PubMed] [Google Scholar]
  • [33].Cassidy H, Radford R, Slyne J, O’Connell S, Slattery C, Ryan MP, McMorrow T, The role of MAPK in drug-induced kidney injury, J Signal Transduct 2012 (2012) 463617. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [34].Zhang C, Jia X, Bao J, Chen S, Wang K, Zhang Y, Li P, Wan JB, Su H, Wang Y, Mei Z, He C, Polyphyllin VII induces apoptosis in HepG2 cells through ROS-mediated mitochondrial dysfunction and MAPK pathways, BMC Compl. Alternative Med 16 (2016) 58. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [35].Killalea SM, Krum H, Systematic review of the efficacy and safety of perhexiline in the treatment of ischemic heart disease, Am. J. Cardiovasc. Drugs 1 (2001) 193–204. [DOI] [PubMed] [Google Scholar]
  • [36].Mohammad MK, Avila D, Zhang J, Barve S, Arteel G, McClain C, Joshi-Barve S, Acrolein cytotoxicity in hepatocytes involves endoplasmic reticulum stress, mitochondrial dysfunction and oxidative stress, Toxicol. Appl. Pharmacol 265 (2012) 73–82. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [37].Tsutsumi S, Gotoh T, Tomisato W, Mima S, Hoshino T, Hwang HJ, Takenaka H, Tsuchiya T, Mori M, Mizushima T, Endoplasmic reticulum stress response is involved in nonsteroidal anti-inflammatory drug-induced apoptosis, Cell Death Differ. 11 (2004) 1009–1016. [DOI] [PubMed] [Google Scholar]
  • [38].Xu C, Bailly-Maitre B, Reed JC, Endoplasmic reticulum stress: cell life and death decisions, J. Clin. Invest 115 (2005) 2656–2664. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [39].Ren Z, Chen S, Ning B, Guo L, Use of Liver-Derived Cell Lines for the Study of Drug-Induced Liver Injury, in: Chen M, Will Y (Eds.), Drug-Induced Liver Toxicity, Humana Press, New York, NY, 2018, pp. 151–177. [Google Scholar]
  • [40].Wang X, Ron D, Stress-induced phosphorylation and activation of the transcription factor CHOP (GADD153) by p38 MAP kinase, Science 272 (1996) 1347–1349. [DOI] [PubMed] [Google Scholar]
  • [41].Wei H, Li Z, Hu S, Chen X, Cong X, Apoptosis of mesenchymal stem cells induced by hydrogen peroxide concerns both endoplasmic reticulum stress and mitochondrial death pathway through regulation of caspases, p38 and JNK, J. Cell. Biochem 111 (2010) 967–978. [DOI] [PubMed] [Google Scholar]
  • [42].Gupta S, Cuffe L, Szegezdi E, Logue SE, Neary C, Healy S, Samali A, Mechanisms of ER stress-mediated mitochondrial membrane permeabilization, Int J Cell Biol 2010 (2010) 170215. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [43].Rainbolt TK, Saunders JM, Wiseman RL, Stress-responsive regulation of mitochondria through the ER unfolded protein response, Trends Endocrinol. Metabol 25 (2014) 528–537. [DOI] [PubMed] [Google Scholar]
  • [44].Silva AM, Barbosa IA, Seabra C, Beltrao N, Santos R, Vega-Naredo I, Oliveira PJ, Cunha-Oliveira T, Involvement of mitochondrial dysfunction in nefazodone-induced hepatotoxicity, Food Chem. Toxicol 94 (2016) 148–158. [DOI] [PubMed] [Google Scholar]
  • [45].Xuan J, Ren Z, Qing T, Couch L, Shi L, Tolleson WH, Guo L, Mitochondrial dysfunction induced by leflunomide and its active metabolite, Toxicology 396–397 (2018) 33–45. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [46].Hu H, Tian M, Ding C, Yu S, The C/EBP homologous protein (CHOP) transcription factor functions in endoplasmic reticulum stress-induced apoptosis and microbial infection. Frontiers in immunology 9, 2019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [47].Ramírez-Peinado S, Alcázar-Limones F, Lagares-Tena L, El Mjiyad N, Caro-Maldonado A, Tirado OM, Muñoz-Pinedo C, 2-Deoxyglucose induces noxa-dependent apoptosis in alveolar rhabdomyosarcoma, Cane. Res 71 (2011) 6796–6806. [DOI] [PubMed] [Google Scholar]
  • [48].Tsukano H, Gotoh T, Endo M, Miyata K, Tazume H, Kadomatsu T, Yano M, Iwawaki T, Kohno K, Araki K, Mizuta H, Oike Y, The endoplasmic reticulum stress-C/EBP homologous protein pathway-mediated apoptosis in macrophages contributes to the instability of atherosclerotic plaques, Arterioscler. Thromb. Vasc. Biol 30 (2010) 1925–1932. [DOI] [PubMed] [Google Scholar]
  • [49].Lim JH, Lee HJ, Ho Jung M, Song J, Coupling mitochondrial dysfunction to endoplasmic reticulum stress response: a molecular mechanism leading to hepatic insulin resistance, Cell. Signal 21 (2009) 169–177. [DOI] [PubMed] [Google Scholar]
  • [50].Mishra R, Karande AA, Endoplasmic reticulum stress-mediated activation of p38 MAPK, Caspase-2 and Caspase-8 leads to abrin-induced apoptosis, PloS One 9 (2014) e92586. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [51].Xia Z, Dickens M, Raingeaud J, Davis RJ, Greenberg ME, Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis, Science 270 (1995) 1326–1331. [DOI] [PubMed] [Google Scholar]
  • [52].Jones TE, Morris RG, Horowitz JD, Concentration-time profile for perhexiline and hydroxyperhexiline in patients at steady state, Br. J. Clin. Pharmacol 57 (2004) 263–269. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [53].Chen S, Wu Q, Li X, Li D, Fan M, Ren Z, Bryant M, Mei N, Ning B, Guo L, The role of hepatic cytochrome P450s in the cytotoxicity of sertraline, Arch. Toxicol 94 (2020) 2401–2411. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [54].Wu Q, Ning B, Xuan J, Ren Z, Guo L, Bryant MS, The role of CYP 3A4 and 1A1 in amiodarone-induced hepatocellular toxicity, Toxicol. Lett 253 (2016) 55–62. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [55].Xuan J, Chen S, Ning B, Tolleson WH, Guo L, Development of HepG2-derived cells expressing cytochrome P450s for assessing metabolism-associated drug-induced liver toxicity, Chem. Biol. Interact 255 (2016) 63–73. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Fig. S1. Inhibition of JNK signaling pathway had little effect on perhexiline-induced toxicity. HepG2 cells were pre-treated with 10 μM SP600125 before exposed to 25 μM perhexiline. (A) Cytotoxicity of perhexiline was assessed using LDH release. (B) Caspase 3/7 activity was assessed after 2 h perhexiline treatment. The results shown are mean ± S.D. from 3 independent experiments. *p < 0.05 compared to DMSO control
NIHMS1674815-supplement-Fig__S1__Inhibition_of_JNK_signaling_pathway_had_little_effect_on_perhexiline-induced_toxicity__HepG2_cells_were_pre-treated_with_10__M_SP600125_before_exposed_to_25__M_perhexiline___A__Cytotoxicity_of_perhexiline_was_.jpg (93.7KB, jpg)

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