Development of HepG2-derived cells expressing cytochrome P450s for assessing metabolism-associated drug-induced liver toxicity

Highlights

• We developed a battery of HepG2-derived cell lines that express 14 individual CYPs.

• We confirmed the functionality of the CYPs at the mRNA, protein and enzymatic activity levels.

• We examined the metabolism-related toxicity of three drugs in our cell system.

Abstract

The generation of reactive metabolites from therapeutic agents is one of the major mechanisms of drug-induced liver injury (DILI). In order to evaluate metabolism-related toxicity and improve drug efficacy and safety, we generated a battery of HepG2-derived cell lines that express 14 cytochrome P450s (CYPs) (1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 3A4, 3A5 and 3A7) individually using a lentiviral expression system. The expression/production of a specific CYP in each cell line was confirmed by an increased abundance of the CYP at both mRNA and protein levels. Moreover, the enzymatic activities of representative CYPs in the corresponding cell lines were also measured. Using our CYP-expressed HepG2 cells, the toxicity of three drugs that could induce DILI (amiodarone, chlorpromazine and primaquine) was assessed, and all of them showed altered (increased or decreased) toxicity compared to the toxicity in drug-treated wild-type HepG2 cells. CYP-mediated drug toxicity examined in our cell system is consistent with previous reports, demonstrating the potential of these cells for assessing metabolism-related drug toxicity. This cell system provides a practical in vitro approach for drug metabolism screening and for early detection of drug toxicity. It is also a surrogate enzyme source for the enzymatic characterization of a particular CYP that contributes to drug-induced liver toxicity.

Introduction

Drug-induced liver injury (DILI) is a leading cause of drug failures in clinical trials and the major reason of drug withdrawals from the market [1]. Identification of drugs that cause liver injury at the early stage of drug development poses a challenge to both the pharmaceutical industry and the U.S. Food and Drug Administration (FDA). Preclinical testing is one of the important approaches to early detect drug toxicity, and there is a constant need for the development of improved tools to facilitate toxicity assessment and risk identification.

The causes of DILI are multifactorial, including toxic effects caused by reactive metabolites, reactive oxygen species, inflammatory reactions, mitochondrial dysfunction, and imbalances between cellular damage and protective responses [1], [2], [3], [4], [5]. Metabolism-related toxicity is generally mediated by the generation of reactive metabolites from non-toxic parenteral compounds, particularly via cytochrome P450 (CYP) enzyme pathways [6]. The inter-individual variability in the expression of drug metabolizing genes predisposes certain individuals to increased susceptibility to DILI [7], [8]. Therefore, it is important to examine the roles of drug metabolizing enzymes and identify specific metabolizing enzymes that contribute to drug-induced liver toxicity.

Numerous in vitro models, such as recombinant enzymes, liver microsomes, liver cytosolic fractions, hepatic cells, liver slices and isolated perfused livers, have been used to examine drug-related hepatotoxicity [9]. Traditionally, cell-based assays have been performed using human primary hepatocytes, either freshly isolated or cryopreserved, to evaluate drug metabolism and drug–drug interactions [10], [11]. Indeed, the application of primary human hepatocytes in drug metabolism and toxicity studies is considered as a “gold standard”, because, under appropriate conditions, these cells retain functional activity of the major drug-metabolizing enzymes [12]. However, phenotypic instability, short life span, batch-to-batch variation and limited availability of primary human hepatocytes constrain their broad use.

Human hepatoma cell lines, such as HepG2, Hep3B, and Huh7, have been widely used in toxicity screening and mechanistic studies, owing to their high stability, unlimited life-span and ready availability. However, lower or no expression of the majority of drug-metabolizing genes is the most critical drawback associated with using these cell lines for drug metabolism and toxicity studies [13], [14]. As a strategy to overcome this limitation, genetically modified hepatic cell lines expressing human drug metabolizing genes have been developed and used for assessing drug metabolism and toxicity. For example, using adenoviral or lentiviral infection systems, cells that transiently or stably express individual CYPs, such as CYP1A1, CYP2C8, CYP2C9 or CYP3A4, have been generated [15], [16], [17], [18]. These cells responded appropriately to known toxic chemicals, demonstrating their values for toxicity testing and mechanistic studies. However, not all of them are publicly available.

In this study, we aimed to develop a comprehensive set of cell lines that express the major human CYPs individually, to provide surrogate hepatic cell lines for the study of metabolism-mediated drug hepatotoxicity and the identification of specific CYP isoforms responsible for the metabolism of a drug. Toward this goal, using the lentiviral expression system, HepG2-derived cell lines expressing 14 individual CYPs (1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 3A4, 3A5 and 3A7) were generated, and the functionality of these CYPs was confirmed at the mRNA, protein, and enzymatic activity levels. In addition, three drugs that could cause metabolism-mediated DILI were examined to evaluate the utility of these cells in drug metabolism and toxicity screening.