Investigation of cannabidiol-induced cytotoxicity in human hepatic cells

Abstract

Cannabidiol (CBD) is one of the primary cannabinoids present in extracts of the plant Cannabis sativa L. A CBD-based drug, Epidiolex, has been approved by the U.S. FDA for the treatment of seizures in childhood-onset epileptic disorders. Although CBD-associated liver toxicity has been reported in clinical studies, the underlying mechanisms remain unclear. In this study, we demonstrated that CBD causes cytotoxicity in primary human hepatocytes and hepatic HepG2 cells. A 24-h CBD treatment induced cell cycle disturbances, cellular apoptosis, and endoplasmic reticulum (ER) stress in HepG2 cells. A potent ER stress inhibitor, 4-phenylbutyrate, markedly attenuated CBD-induced apoptosis and cell death. Additionally, we investigated the role of cytochrome P450 (CYP)-mediated metabolism in CBD-induced cytotoxicity using HepG2 cell lines engineered to express 14 individual CYPs. We identified CYP2C9, 2C19, 2D6, 2C18, and 3A5 as participants in CBD metabolism. Notably, cells overexpressing CYP2C9, 2C19, and 2C18 produced 7-hydroxy-CBD, while cells overexpressing CYP2C9, 2C19, 2D6, and 2C18 generated 7-carboxy-CBD. Furthermore, CBD-induced cytotoxicity was significantly attenuated in the cells expressing CYP2D6. Taken together, these data suggest that cell cycle disturbances, apoptosis, and ER stress are associated with CBD-induced cytotoxicity, and CYP2D6-mediated metabolism plays a critical role in decreasing the cytotoxicity of CBD.

Keywords: Apoptosis; CBD; CYPs; ER stress; Liver toxicity; Topoisomerases.

Fig. 1.. CBD causes cellular damage in primary human hepatocytes and HepG2 cells.

Primary human hepatocytes pooled from 10 donors (A) and HepG2 cells (B – D) were treated with DMSO (0 μM) or CBD at the concentrations indicated for 24 h. (A & B) Cell viability was measured using an ATP assay. Benchmark dose analysis was conducted for the concentration-responses data with a critical effect size of 0.5 (i.e., BMC₅₀). (C) Cellular growth was measured using a CellTiter-Blue cell viability assay and cell number counting. The values of BMC₅₀ were calculated. (D) The cytotoxicity of CBD was measured using an LDH assay. The data represent the mean ± SD from three independent experiments. *p < 0.05 compared with that of the DMSO control.

Fig. 2.. CBD induces cell cycle disturbance in HepG2 cells.

(A) Cell cycle distribution was analyzed by flow cytometry. Representative histograms show DNA content analyses for HepG2 cells treated with DMSO (0 μM) or CBD for 24 h. (B) Bar graph shows the percentage of cells in each cell cycle phase. The data represent the mean ± SD from three independent experiments. *p < 0.05 compared to the DMSO control. (C & D) Total cellular proteins were extracted at 24 h after treatment with 10 – 50 μM CBD or DMSO (0 μM). Levels of CDK2, CDK4, CDK6, cyclin D1, and cyclin D3 were determined by Western blotting. GAPDH was used as an internal control. Representative Western blots are shown in (C) and quantification from three independent experiments are shown in (D). *p < 0.05 compared to the DMSO control.

Fig. 3.. CBD activates caspase 3/7, increases expression of apoptosis-related proteins, and induces induction of γH2A.x.

(A) Cellular caspase 3/7 activity in HepG2 cells treated with CBD or DMSO (0 μM) for 24 h is expressed as fold change compared to DMSO-treated cells from three independent experiments. *p < 0.05 compared to the DMSO control. (B – D) Total cellular proteins were extracted at 24 h after treatment at 10 – 50 μM CBD or DMSO (0 μM). Levels of cleaved-caspase 3, 8, 9, cleaved-PARP, Bcl-2, and γH2A.x were determined by Western blotting. GAPDH was used as an internal control. Representative Western blots are shown in (B & D upper panel) and quantification from three independent experiments are shown in (C & D lower panel). *p < 0.05 compared to the DMSO control. (E) After HepG2 cells were treated with DMSO (0 μM) or CBD for 24 h, the expression of γH2A.x was determined using a MultiFlow assay in three independent experiments. MMS (50 μM) was used as a positive control (black bar). *p < 0.05 compared with the DMSO control.

Fig. 4.. The effect of CBD on DNA damage and the activities of topoisomerases.

(A) Supercoiled pHOT-1 plasmid DNA was incubated with topoisomerase I enzyme in the presence of DMSO or CBD at the concentrations of 500 and 1,250 μM at 37 °C for 30 min. (B) k-DNA was incubation with topoisomerase II enzyme in the presence of DMSO or CBD at concentrations of 250, 500, and 1,250 μM at 37 °C for 30 min. CBD-HQ at 500 μM was used as a positive topoisomerase II inhibitor. DNA samples were separated by electrophoresis on 1% agarose gel, stained with ethidium bromide. (C & D) HepG2 cells were exposed to DMSO (0 μM), 10 μM CBD, 20 μM CBD, or 50 μM MMS (positive control) for 24 h. The alkaline comet assay was conducted to detect the DNA damaging potential of CBD. Representative images are shown in (C) from one experiment for the alkaline comet assay. Quantification of % DNA in tails from three independent alkaline comet assay experiments is shown in (D). The data represent the mean ± SD. *p < 0.05 compared with that of the DMSO control. (E) HepG2 cells were treated with DMSO (0 μM), 10 μM CBD, 20 μM CBD, 30 μM CBD, or 75 nM mitomycin C (positive control) for 24 h. After a 24-h recover period, micronucleus formation was measured using an In Vitro MicroFlow kit. The red line indicates relative survival (right y-axis), and bars indicate the percentage of micronuclei (left y-axis). The data represent the mean ± SD from three independent experiments. *p < 0.05 compared with that of the DMSO control. (F) HepG2 cells were pretreated with DMSO (0 μM) or 10 μM Z-VAD-FMK (pan caspase inhibitor) for 2 h prior to treatment with CBD or DMSO (0 μM) for 24 h. The expression of γH2A.x was assessed using Western blotting. The intensity of each protein band was quantified using AlphaView software and normalized to its corresponding GAPDH band. *p < 0.05 versus treatment with CBD alone.

Fig. 5.. CBD induces ER stress in HepG2 cells.

(A) HepG2 or (B) HepG2-Fluc-Gluc cells were treated with various concentrations of CBD or DMSO (0 μM) for 2 or 5 h. (A) Cell viability was measured using cell number counting in HepG2 cells. (B) The relative activity of Gluc were measured using luciferase assays. The data represent the mean ± SD from three independent experiments. *p < 0.05 compared with the DMSO control. (C – D) Total cellular proteins were extracted at 2 and 5 h after treatment with 10 – 50 μM CBD or DMSO (0 μM). Levels of CHOP and XBP-1s were determined by Western blotting. GAPDH was used as an internal control. Representative Western blots are shown in (C) and quantification from three independent experiments are shown in (D & E). *p < 0.05 compared with the DMSO control. (E) After pretreatment with 1 mM 4-PBA for 2 h, HepG2 cells were treated with 10 – 50 μM CBD or DMSO (0 μM) for an additional 24 h. The effect of 4-PBA in reducing cell death and apoptosis caused by CBD was determined by LDH (upper panel) and caspase 3/7 (lower panel) assays. The data represent the mean ± SD from three independent experiments. *p < 0.05 versus treatment with CBD alone.

Fig. 6.. Metabolism of CBD in individual CYP-expressing HepG2 cells.

(A) LC-MS chromatograms for CBD in the extracts from HepG2-empty vector control (EV) and HepG2-CYP2C9 expressing cells treated with 5 μM CBD for 24 h. (B) 14 CYP-expressing HepG2 cell lines and empty vector control HepG2 cells were exposed to 5 μM CBD for 24 h. The total amount of CBD (B) and its metabolites 7-hydroxy-CBD (C) and 7-carboxy-CBD (D) in cell lysate and media were quantified using LC-MS. The results shown are the values of the compounds detected in cell lysate and media and are expressed as ng/million cells. Data represent mean ± S.D. from three independent experiments. *p < 0.05 compared with that of the EV control.

Fig. 7.. Effect of metabolism mediated by CYP2C9, 2C19, 2D6, 2C18, 3A5, and 3A4 on CBD-induced cytotoxicity.

Empty vector (EV)-transduced or CYP2C9, 2C19, 2D6, 2C18, 3A5, and 3A4-expressing HepG2 cells were treated with 10 – 30 μM CBD or DMSO (0 μM) for 24 h. Cytotoxicity was measured using a CellTiter-Blue cell viability assay. The results shown are mean ± S.D. from three independent experiments. *p < 0.05 compared with that of the EV control.