Busulfan induces steatosis in HepaRG cells but not in primary human hepatocytes: Possible explanations and implication for the prediction of drug‐induced liver injury

Abstract

BackgroundThe antineoplastic drug busulfan can induce different hepatic lesions including cholestasis and sinusoidal obstruction syndrome. However, hepatic steatosis has never been reported in patients.ObjectivesThis study aimed to determine whether busulfan could induce steatosis in primary human hepatocytes (PHH) and differentiated HepaRG cells.MethodsNeutral lipids were determined in PHH and HepaRG cells. Mechanistic investigations were performed in HepaRG cells by measuring metabolic fluxes linked to lipid homeostasis, reduced glutathione (GSH) levels, and expression of genes involved in lipid metabolism and endoplasmic reticulum (ER) stress. Analysis of two previous transcriptomic datasets was carried out.ResultsBusulfan induced lipid accumulation in HepaRG cells but not in six different batches of PHH. In HepaRG cells, busulfan impaired VLDL secretion, increased fatty acid uptake, and induced ER stress. Transcriptomic data analysis and decreased GSH levels suggested that busulfan‐induced steatosis might be linked to the high expression of glutathione S‐transferase (GST) isoenzyme A1, which is responsible for the formation of the hepatotoxic sulfonium cation conjugate. In keeping with this, the GST inhibitor ethacrynic acid and the chemical chaperone tauroursodeoxycholic acid alleviated busulfan‐induced lipid accumulation in HepaRG cells supporting the role of the sulfonium cation conjugate and ER stress in steatosis.ConclusionWhile the HepaRG cell line is an invaluable tool for pharmacotoxicological studies, it might not be always an appropriate model to predict and mechanistically investigate drug‐induced liver injury. Hence, we recommend carrying out toxicological investigations in both HepaRG cells and PHH to avoid drawing wrong conclusions on the potential hepatotoxicity of drugs and other xenobiotics.

The dithiocarbamate pesticides maneb and mancozeb disturb the metabolism of lipids and xenobiotics in an in vitro model of metabolic dysfunction-associated steatotic liver disease

“…Altogether, our results indicate that maneb and mancozeb can have broad hepatocellular effects with dire consequences on metabolism, oxidative stress and cell death. Of note, a maximum of 30% decrease in ATP content in HepaRG cells did not impede the study of lipid accumulation and related mechanisms (Allard et al 2021; Allard et al 2024). One key finding in this study was the exacerbation of steatosis in +FA-HepaRG cells by maneb, mancozeb and MnCl 2 , whereas these molecules did not induce neutral lipid accumulation in -FA-HepaRG cells.…”

“…Furthermore, our study disclosed that steatosis aggravation was associated with increased fatty acid uptake and reduced VLDL secretion whereas mitochondrial FAO and DNL were unchanged. Interestingly, impaired VLDL secretion was associated with a reduction in the expression of APOB and APOC3 (two major apolipoproteins in VLDL) and of MTTP (also referred to as MTP ), a key enzyme for the assembly and secretion of this lipoprotein (Allard et al 2024; Heeren and Scheja 2021). However, the decrease in VLDL secretion and expression of APOB , APOC3 and MTTP were not associated with ER stress, contrary to what occurred with different pharmaceuticals inducing steatosis in HepaRG cells such as 5-fluorouracil, indomethacin, rifampicin, troglitazone (Allard et al 2021) and busulfan (Allard et al 2024).…”

“…Interestingly, impaired VLDL secretion was associated with a reduction in the expression of APOB and APOC3 (two major apolipoproteins in VLDL) and of MTTP (also referred to as MTP ), a key enzyme for the assembly and secretion of this lipoprotein (Allard et al 2024; Heeren and Scheja 2021). However, the decrease in VLDL secretion and expression of APOB , APOC3 and MTTP were not associated with ER stress, contrary to what occurred with different pharmaceuticals inducing steatosis in HepaRG cells such as 5-fluorouracil, indomethacin, rifampicin, troglitazone (Allard et al 2021) and busulfan (Allard et al 2024). Thus, further investigations will be needed to determine the exact mechanism(s) whereby maneb, mancozeb and MnCl 2 reduce APOB , APOC3 and MTTP expression and VLDL secretion in hepatocytes.…”

Chemotherapy-Induced Liver Injury: Unveiling Emerging Mechanisms and Exploring Mitigation Strategies

“…In addition, investigation into Pirfenidone's protective potential reveals a promising strategy for mitigating busulfan-induced hepatotoxicity, specifically by targeting hepatic sinusoidal endothelial cells (HSECs) and inhibiting collagen formation and hepatic stellate cell activation. These findings highlight the multifaceted nature of busulfaninduced liver injury and the significance of prospective therapeutic interventions, providing new avenues for enhancing patient outcomes in the context of transplantation conditioning (54)(55)(56).…”