Molecular and functional characterization of drug-metabolizing enzymes and transporter expression in the novel spontaneously immortalized human hepatocyte line HC-04

Abstract

In toxicological research, immortalized human hepatocytes provide a useful alternative to primary hepatocytes because interindividual variability in the expression of drug-metabolizing enzymes and drug transporters can largely be eliminated. However, it is essential that the cell line retain the original phenotype. The purpose of this study was to characterize a novel spontaneously immortalized human hepatocyte cell line, HC-04, with respect to the transcript and functional protein expression profile for the major drug-metabolizing enzymes and transmembrane transporters. HC-04 cells retained hepatocyte-specific function including albumin production and ornithine transcarbamoylase and glucose-6-phosphatase activity. Most of the major CYP forms were expressed at basal levels and responsive to inducing agents. In particular, CYP3A4 was expressed abundantly, and HC-04 cells were able to metabolize the CYP3A4 probe, midazolam, at a rate similar to primary human hepatocytes. Furthermore, the major human sulfotransferase and UDP-glucuronosyltransferase forms, as well as members of the ABC and SLC transporter superfamilies, nuclear receptors, and hepatic transcription factors were also expressed. HC-04 cells readily responded to standard hepatotoxicants that are dependent on CYP-mediated bioactivation, while another, tumor-derived cell line remained refractory to the drug challenge. Collectively, HC-04 cells provide a reliable, stable, and reproducible model for biomechanistic studies in drug toxicology.

Introduction

Cultured hepatocytes have been widely used to assess drug metabolism and toxicity. Due to species-specific metabolism, the use of human hepatocytes is preferred over rodent hepatocytes. Although primary cultures of human hepatocytes are available, their use has been limited due to rapid deterioration of the hepatocyte-specific functions (including drug metabolism) after several days in culture (LeCluyse, 2001). One approach to improve these shortcomings has been the introduction of collagen gel sandwich culture systems (Kono et al., 1995) and other techniques aimed at modifying the culture conditions with extracellular matrix components. However, this does not eliminate the fact that there are other challenges associated with primary human hepatocytes, such as limited availability and batch-to-batch differences in viability. In addition, and importantly, interindividual variation in the expression of drug-metabolizing enzymes and transporters (due to polymorphisms) makes it difficult to compare data from different preparations (Madan et al., 2003).

As a viable alternative, a number of human hepatocyte-derived cell lines are available. However, these cell lines suffer from the fact that many of them are of tumorigenic origin and that they often do not conserve the normal hepatocellular metabolizing capacities as a result of dedifferentiation. Hence, this greatly limits their application in pharmacology and toxicology (Wilkening et al., 2003, Clayton et al., 2005).

To overcome these drawbacks, immortalized human hepatocytes have been increasingly used (Mills et al., 2004). In most cases, hepatocytes were transfected with simian virus 40 large tumor antigen (Pfeifer et al., 1993). An alternative approach was the use of co-culture systems of human hepatocytes with rat liver epithelial cells (Roberts et al., 1994). Immortalized human hepatocytes can be easily maintained and cultured, and the problem of batch-to-batch variation in drug-metabolizing enzyme expression is largely eliminated. Moreover, immortalized human hepatocytes allow for high-throughput analysis and screening. However, not all immortalized hepatocytes express the full profile of liver-selective transporters and drug-metabolizing enzymes.

Recently, a spontaneously immortalized new cell line (HC-04) was developed from human hepatocytes (Sattabongkot et al., 2006). This cell line was generated without the use of SV40 or co-culture. Morphologically, HC-04 cells resemble liver parenchymal cells. They proliferate with a doubling time of ∼24 h. Cytogenetic analysis revealed that they exhibited a hyperdiploid karyotype with chromosomal abnormalities. Importantly, they expressed and secreted into the cell culture medium the liver-specific proteins α-fetoprotein, albumin, and transferrin. Ethoxyresorufin-O-dealkylase (EROD) and methoxyresorufin-O-dealkylase (MROD) activity (markers for CYP1A activity) were also detected in these cells. These enzymes were inducible by dibenzo[a,h]anthracene. The HC-04 cells were generated primarily for the study of the development of human malaria parasites, and the cells have not been characterized with respect to drug metabolism enzyme expression or hepatic transporters.

Because there is a great need for immortalized human hepatocytes that ideally express the full spectrum of drug-metabolizing enzymes and transporters, HC-04 cells need to be further evaluated as a potential alternative to other cell lines. Therefore, the purpose of this study was to further characterize HC-04 cells both at the transcript and protein level and to explore their response to a number of standard hepatotoxicants in order to evaluate their potential use in pharmacology and toxicology. We found that HC-04 cells not only exhibit transcript expression of a large panel of hepatocellular transcription factors, transporters, and drug-metabolizing enzymes, but also functionally expressed the major human CYP forms. Furthermore, these hepatocellular CYP forms were upregulated by standard inducers. More importantly, the cells responded to metabolic bioactivation-dependent hepatotoxic drugs in a similar way as primary human hepatocytes, while conventional tumor-derived cell lines are often refractory to such drugs.