Cytochrome P450 enzyme levels in HepG2 cells and cryopreserved primary human hepatocytes and their induction in HepG2 cells

Abstract

Early in vitro toxicity screening might improve the success rate of new chemical entities in pharmaceutical development. In previous studies, the advantage of cytotoxicity screening with the HepG2 cell line was shown. Cytotoxicity could be identified for 70% of the compounds in these assays as compared with known toxicity in either in vitro assays in primary hepatocytes, in in vivo assays in rats, or in (pre-)clinical development in humans. The low Phase I and II enzyme levels in HepG2 cells might have been responsible for the fact that 30% of the compounds scored negative. Therefore, we performed two follow-up studies in which Cytochrome P450 (CYP) enzymes and Phase II metabolism were examined.

In the present study, the transcript levels of CYP1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4 were measured with quantitative PCR. Results showed that transcripts of all CYPs were present in HepG2 cells, however, mRNA levels of most CYPs were dramatically lower than in primary human hepatocytes. These results were confirmed with luminometric assays which were used to measure the enzyme activities of CYP1A1, 1A2, 2C9, and 3A4.

Regulation of CYP1A1, 1A2, 2B6, 2C8, 2D6, 2E1, and 3A4 by the aryl hydrocarbon receptor, pregnane X receptor and constitutive androstane receptor was studied in HepG2 cells at the mRNA and/or enzyme level. Regulation of CYP1A1, 1A2, 2B6, and 3A4 mRNA levels was similar to the regulation in primary human hepatocytes. In contrast, CYP2C8 mRNA levels are inducible in primary human hepatocytes, but not in HepG2 cells, after treatment with PXR/CAR activators. Consistent with other studies, CYP2D6 and 2E1 transcript levels were not changed after treatment with AhR, PXR, and CAR activators. Moreover, CYP1A1 and 1A2 enzyme levels could be induced by AhR agonists and CYP3A4 by PXR agonists.

As a consequence of the low levels of CYPs in HepG2 cells, cytotoxicity of several compounds might have been missed or underestimated as compared with cytotoxicity in primary human hepatocytes. Inducing HepG2 cells with particular receptor stimulators might lead to higher toxicity for several of the tested compounds. Compared to primary human hepatocytes, HepG2 cells are a relatively easy-to-handle tool to study the up-regulation of CYP1A1, 1A2, 2B6, and 3A4.

Introduction

Approximately, 40% of the new drug candidates fail in the developmental phase due to toxicological side effects (Kola and Landis, 2004). Screening on toxicity and deselection in an early phase of development of drugs may improve the success rate of new chemical entities (Caldwell et al., 2001). However, this strategy implies a large number of compounds for which only a small amount of material is available. Therefore medium or high throughput screening methods are necessary.

In two previous studies (Schoonen et al., 2005a, Schoonen et al., 2005b) the advantage of early toxicity medium throughput screening was shown with seven different fluorometric assays on four different cell lines. In these studies cytotoxicity could be shown for 70% of the compounds with known toxicity in either in vitro assays in primary hepatocytes, in in vivo assays in rats, or in (pre-)clinical development in humans. Toxicity of the remaining 30% of compounds could not be established. The human hepatoma cell line HepG2 was chosen for further toxicity screening as detoxication and activation processes of compounds are studied most optimally in liver cells. However, a discrepancy exists between Cytochrome P450 (CYP) and Phase II metabolism of primary hepatocytes and HepG2 cells. Low CYP and Phase II enzyme levels in HepG2 cells might have been responsible for the fact that 30% of the compounds were falsely classified as non-toxic (Hewitt and Hewitt, 2004, Rodriguez-Antona et al., 2002, Wilkening et al., 2003). However, a recent study with Cellulomics techniques on HepG2 cells counteracts the hypothesis that the metabolic competence of HepG2 cells is significantly limiting the production of reactive metabolites (O’Brien et al., 2006). With this novel method, cytotoxicity in HepG2 cells could be established for more than 90% of 243 drugs with varying degrees of toxicity including drugs that produce their toxicities by a reactive metabolite. Nevertheless we performed two follow-up studies in which CYP enzymes and Phase II metabolism were examined. In the present study the focus is on CYPs.

The role of Phase I enzymes is to introduce a new functional group (e.g., hydroxylation) or modify an existing functional group (e.g., O-dealkylation) so as to facilitate Phase II conjugation reactions. Whilst Phase I reactions generally result in a more polar metabolite, it is the conjugation reactions (e.g., glucuronidation, sulphonation) that result in marked increases in water solubility and facilitate excretion.

This transformation in two steps facilitates excretion and detoxicates a wide variety of xenobiotics. For example the Phase II enzyme glutathione S-transferase P1-1 prevents 4-nitroquinoline 1-oxide (4-NQO) DNA-adduct formation by the formation of 4-NQO glutathione conjugates (Morrow et al., 1998, Townsend et al., 2002). However, many xenobiotics are also toxicated by CYPs and Phase II enzymes (Rooney et al., 2004). A well known example is the carcinogenicity of benzo[a]pyrene in which the activities of CYP1A1 and epoxide hydrolase lead to the formation of the carcinogenic metabolite benzo[a]pyrene-7,8-diol-9,10-epoxide (Bao et al., 2002, Huang et al., 2005).

The CYP and Phase II enzymes are mainly regulated by ligand-activated nuclear transcription factors, such as the aryl hydrocarbon receptor (AhR; for the CYP1A family), the constitutive androstane receptor (CAR; for the CYP2B family) and the pregnane X receptor (PXR; for the CYP3A family) (Bock and Kohle, 2004, Maglich et al., 2002, Mankowski and Ekins, 2003, Xie et al., 2003).

In the present study we characterized CYP metabolism in HepG2 cells. Transcript levels and enzyme activities in the HepG2 cell line were compared with levels and activities in cryopreserved primary human hepatocytes. Quantitative PCR was used to measure mRNA levels of CYP1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4. Enzyme activities of CYP1A1, 1A2, 2C9, and 3A4 were measured using luminometric P450-Glo substrates (Cali et al., 2006). Furthermore, the inducibility of CYPs in HepG2 cells was studied after exposure of HepG2 cells to agonists of the xenobiotic receptors AhR, CAR and PXR. Indigo, indirubin, β-naphthoflavone (BNF), 3-methylcholanthrene (3MC), and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were used as AhR agonists (Adachi et al., 2001, Runge et al., 2000, Yueh et al., 2005). Tularik T0901317, CITCO, rifampicin (RIF), and phenobarbital (PB) were used as PXR and/or CAR agonists. T0901317 is in principle a potent LXR ligand that is also capable of activating human PXR (Shenoy et al., 2004). CITCO and RIF are potent CAR and PXR activators, respectively (Maglich et al., 2003). PB is an activator of the PXR and although it does not appear to bind directly to CAR, it is also an activator of CAR (Chen et al., 2004).

A parallel study deals with Phase II metabolism and compares the mRNA levels and enzyme activities of 10 Phase II enzymes in HepG2 cells with those of cryopreserved primary human hepatocytes (Westerink and Schoonen, accepted for publication). Moreover, induction of Phase II enzymes by AhR, PXR and CAR activators was studied.