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Study of the roles of cytochrome P450 (CYPs) in the metabolism and cytotoxicity of perhexiline

  • Toxicokinetics and Metabolism
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Abstract

Perhexiline is a prophylactic antianginal agent developed in the 1970s. Although, therapeutically, it remained a success, the concerns of its severe adverse effects including hepatotoxicity caused the restricted use of the drug, and eventually its withdrawal from the market in multiple countries. In the clinical setting, cytochrome P450 (CYP) 2D6 is considered as a possible risk factor for the adverse effects of perhexiline. However, the role of CYP-mediated metabolism in the toxicity of perhexiline, particularly in the intact cells, remains unclear. Using our previously established HepG2 cell lines that individually express 14 CYPs (1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 3A4, 3A5, and 3A7) and human liver microsomes, we identified that CYP2D6 plays a major role in the hydroxylation of perhexiline. We also determined that CYP1A2, 2C19, and 3A4 contribute to the metabolism of perhexiline. The toxic effect of perhexiline was reduced significantly in CYP2D6-overexpressing HepG2 cells, in comparison to the control cells. In contrast, overexpression of CYP1A2, 2C19, and 3A4 did not show a significant protective effect against the toxicity of perhexiline. Pre-incubation with quinidine, a well-recognized CYP2D6 inhibitor, significantly attenuated the protective effect in CYP2D6-overexpressing HepG2 cells. Furthermore, perhexiline-induced mitochondrial damage, apoptosis, and ER stress were also attenuated in CYP2D6-overexpressing HepG2 cells. These findings suggest that CYP2D6-mediated metabolism protects the cells from perhexiline-induced cytotoxicity and support the clinical observation that CYP2D6 poor metabolizers may have higher risk for perhexiline-induced hepatotoxicity.

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Funding

This study was funded by the National Center for Toxicological Research/U.S. Food and Drug Administration. Drs. Feng Li and Xuan Qin were supported by the National Institute of Diabetes and Digestive and Kidney Diseases (R01-DK121970) and the Eunice Kennedy Shriver National Institute of Child Health and Human Development (R61HD099995) to Feng Li.

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Authors and Affiliations

  1. Division of Biochemical Toxicology, HFT-110, National Center for Toxicological Research (NCTR), U.S. Food and Drug Administration (FDA), 3900 NCTR Road, Jefferson, AR, 72079, USA

    Zhen Ren, Si Chen & Lei Guo

  2. Department of Pathology and Immunology, Center for Drug Discovery, Baylor College of Medicine, Houston, TX77030, USA

    Xuan Qin & Feng Li

Authors
  1. Zhen Ren
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  2. Si Chen
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  3. Xuan Qin
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  4. Feng Li
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  5. Lei Guo
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Correspondence to Lei Guo.

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204_2022_3369_MOESM1_ESM.pptx

Supplementary file 1. Figs. S1 CYP2D6-mediated metabolism reduces the mitochondrial damage, apoptosis and ER stress induced by perhexiline. (A) CYP2D6-overexpressing HepG2 cells and EV control cells were changed into glucose-containing media or galactose-containing media and exposed to various concentrations of perhexiline for 24 h, and cell viability was measured. *p < 0.05 for comparison of cell viability in glucose medium and galactose medium within the same type of cell for the same concentration of perhexiline. (B and C) CYP2D6-overexpressing HepG2 cells and EV-control cells were exposed to 5, 7.5, or 10 μM perhexiline for 24 h and mitochondrial membrane potential by JC-1 staining (B) and caspase 3/7 activity (C) were measured. *p < 0.05 compared to EV control. (D) CYP2D6-overexpressing HepG2 cells and EV control cells were exposed to 10 μM perhexiline or DMSO for 24 h and mRNA levels of ER stress markers were measured using quantitative real-time PCR. (E) The protein levels of CHOP in EV control and CYP2D6-overexpressing HepG2 cells upon perhexiline treatment for 24 h were assessed using Western blots. GAPDH protein levels were used as internal control. The results shown are mean ± S.D. from 3 biological replicates. *p < 0.05 for comparison between perhexiline treatment and DMSO treatment in EV control cells. #p < 0.05 for comparison between perhexiline treated CYP2D6-overexpressing HepG2 cells and EV control cells. Fig. S2. CYP3A4-mediated metabolism marginally attenuates perhexiline-induced mitochondrial damage (A). CYP3A4-overexpressing HepG2 cells were changed into glucose-containing media or galactose-containing media and exposed to various concentrations of perhexiline for 24 h, and cell viability was measured. *p < 0.05 for comparison of cell viability in glucose medium and galactose medium for the same concentration of perhexiline treatment. (B and C) CYP3A4-overexpressing HepG2 cells and EV-control cells were exposed to 5–10 μM perhexiline for 24 h and mitochondrial membrane potential by JC-1 staining (B) and caspase 3/7 activity (C) were measured. The results shown are mean ± S.D. from 3 biological replicates. *p < 0.05 compared to EV control. Fig. S3. Generation of M1 and M3 in CYP2D6- and CYP3A4-overexpressing HepG2 cells upon 24 h exposure to perhexiline. CYP2D6- and 3A4-overexpressing HepG2 cell and EV control cells were treated with 5 μM perhexiline for 24 h. The cell extracts were then subjected to mass spectrometry to determine the abundance of the two types of trans-OH-Phx, M1 and M3, in CYP2D6 and CYP3A4 cells. The relative abundance of M1 and M3, measured as peak areas, were shown. The results shown are mean ± S.D. from 4 independent experiments. Fig. S4. CYP2B6-overexpressing HepG2 cells do not show protective effect against the cytotoxicity of perhexiline. CYP2B6-overexpressing HepG2 cells were exposed to 2.5 – 10 μM perhexiline for 24 h and the cytotoxicity of perhexiline was measured using cellular ATP contents (A) and LDH release (B). The results shown are mean ± S.D. from 3 biological replicates (PPTX 233 KB)

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Ren, Z., Chen, S., Qin, X. et al. Study of the roles of cytochrome P450 (CYPs) in the metabolism and cytotoxicity of perhexiline. Arch Toxicol 96, 3219–3231 (2022). https://doi.org/10.1007/s00204-022-03369-0

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