Development of an in vitro high content imaging assay for quantitative assessment of CAR-dependent mouse, rat, and human primary hepatocyte proliferation

Highlights

• Multiplexed analysis of cytotoxicity and CAR-dependent DNA synthesis in hepatocytes

• Development of an optimized medium formulation for initiating cell cycle progression

• Automated high content image analysis of concentration-response profiles

Abstract

Rodent liver tumors promoted by constitutive androstane receptor (CAR) activation are known to be mediated by key events that include CAR-dependent gene expression and hepatocellular proliferation. Here, an in vitro high content imaging based assay was developed for quantitative assessment of nascent DNA synthesis in primary hepatocyte cultures from mouse, rat, and human species. Detection of DNA synthesis was performed using direct DNA labeling with the nucleoside analog 5-ethynyl-2′-deoxyuridine (EdU). The assay was multiplexed to enable direct quantitation of DNA synthesis, cytotoxicity, and cell count endpoints. An optimized defined medium cocktail was developed to sensitize hepatocytes to cell cycle progression. The baseline EdU response to defined medium was greatest for mouse, followed by rat, and then human. Hepatocytes from all three species demonstrated CAR activation in response to the CAR agonists TCPOBOP, CITCO, and phenobarbital based on increased gene expression for Cyp2b isoforms. When evaluated for a proliferation phenotype, TCPOBOP and CITCO exhibited significant dose-dependent increases in frequency of EdU labeling in mouse and rat hepatocytes that was not observed in hepatocytes from three human donors. The observed species differences are consistent with CAR activators inducing a proliferative response in rodents, a key event in the liver tumor mode of action that is not observed in humans.

Introduction

The use of hepatocyte cultures from rodent and non-rodent species has utility in exploring modes of action (MOA) for liver tumor responses seen in chronic bioassays (Hirose et al., 2009, James and Roberts, 1996, Parzefall et al., 1991). As one example, activation of the constitutive androstane receptor (CAR) by xenobiotics such as phenobarbital (PB) triggers a series of key events in rats and mice that ultimately leads to hepatocellular adenomas and carcinomas (Elcombe et al., 2014, Jones et al., 2009). The key events contributing to this MOA in mice and rats include activation of CAR-dependent gene expression, increased cell proliferation, development of altered foci, and ultimately tumor formation (Elcombe et al., 2014). In addition, CAR activation is associated with characteristic gene expression changes (Oshida et al., 2015) and induction of hepatic enzymes (e.g. Cyp2b isoforms > Cyp3a isoforms) that can serve as markers of a CAR-mediated effect (Elcombe et al., 2014, Yang and Wang, 2014). Mice lacking CAR exhibit loss of PB-induced Cyp2b expression and hepatocyte proliferation (Ross et al., 2010, Wei et al., 2000), and knockout of the CAR receptor in mice also prevents liver tumor formation (Yamamoto et al., 2004).

Human hepatocytes and those of other species (e.g. hamsters) also contain species-specific forms of the CAR nuclear receptor and are capable of displaying induction of Cyp2b enzymes and associated increases in liver weight, but humans and hamsters do not experience the cell proliferative response and do not get tumors after long-term treatment (Diwan et al., 1986, Whysner et al., 1996). Testing the response of xenobiotics in hepatocyte culture systems from various species is a valuable tool for exploring potential MOAs for a xenobiotic (e.g. via activation of CAR or alternative nuclear receptors such as aryl hydrocarbon receptor (AhR) and peroxisome proliferator activated receptor alpha (PPARα)), and for demonstrating species differences in response to that same xenobiotic.

Previous rodent hepatocyte culture systems have employed the measurement of new DNA synthesis, as a precursor to a cell proliferation key event, to evaluate effects of rat liver tumor promoters on cell cycle progression (Goll et al., 1999, Hirose et al., 2009, James and Roberts, 1996, Parzefall et al., 1991, Plant et al., 1998, Sawada et al., 1987), however sample throughput, simultaneous detection of cytotoxicity, and automated methods for determining quantitative responses in vitro has been limited. Prior hepatocyte studies classically have used direct DNA labeling agents such as bromodeoxyuridine (BrdU) for detecting an increase in DNA synthesis during S-phase. The immunochemical detection procedure for this method is often confounded by non-specific staining of cellular debris in hepatocyte cultures, and the typical manual counting procedure is both labor intensive, and requires observational determination of positively stained cells from non-specific signals. In this study, a thymidine analog (EdU) that is amenable to direct fluorescent labeling and reduction of non-specific background is explored as a means to quantify induction of DNA synthesis in isolated hepatocytes via high content imaging.

To enhance the sensitivity of these in vitro culture models, additives like recombinant growth factors or hormone supplements have been explored as ways to sensitize primary hepatocytes to initiate DNA replication in rat (Hodges et al., 2000, McGowan et al., 1981, Yusof and Edwards, 1990), mouse (Bowen et al., 2014), and human cells (Block et al., 1996, Runge et al., 1999). Factors that may contribute to variability in isolated hepatocytes for key events in the CAR MOA include differential cell enrichment methods, the relatively short time that hepatocytes remain viable in culture, differences in baseline donor gene expression, and the contribution of non-parenchymal cells to proliferative signaling processes that are absent in a monoculture environment.

The primary objective of this study was to establish an in vitro assay for high-content imaging-based quantitative assessment of DNA synthesis in multiple species of primary hepatocytes exposed to prototypical CAR activators. Assay parameter optimization and establishment of a defined medium was initially conducted in mouse hepatocytes, then extended into rat and human species. The endpoints for analysis were CAR-dependent gene expression (as markers of CAR activation), DNA synthesis (as an early indicator of proliferation), and multiplexed cytotoxicity markers for identifying appropriate bioactivity ranges. The cross-species assay platform provides an in vitro approach for directly comparing rodent and human responses to CAR-dependent non-genotoxic rodent liver carcinogens and demonstrates utility for both screening MOAs of new compounds, as well as assessing the human relevance of rodent liver effects.