Mode of action and human relevance analysis for nuclear receptor-mediated liver toxicity: A case study with phenobarbital as a model constitutive androstane receptor (CAR) activator

Abstract

The constitutive androstane receptor (CAR) and pregnane X receptor (PXR) are important nuclear receptors involved in the regulation of cellular responses from exposure to many xenobiotics and various physiological processes. Phenobarbital (PB) is a non-genotoxic indirect CAR activator, which induces cytochrome P450 (CYP) and other xenobiotic metabolizing enzymes and is known to produce liver foci/tumors in mice and rats. From literature data, a mode of action (MOA) for PB-induced rodent liver tumor formation was developed. A MOA for PXR activators was not established owing to a lack of suitable data. The key events in the PB-induced liver tumor MOA comprise activation of CAR followed by altered gene expression specific to CAR activation, increased cell proliferation, formation of altered hepatic foci and ultimately the development of liver tumors. Associative events in the MOA include altered epigenetic changes, induction of hepatic CYP2B enzymes, liver hypertrophy and decreased apoptosis; with inhibition of gap junctional intercellular communication being an associative event or modulating factor. The MOA was evaluated using the modified Bradford Hill criteria for causality and other possible MOAs were excluded. While PB produces liver tumors in rodents, important species differences were identified including a lack of cell proliferation in cultured human hepatocytes. The MOA for PB-induced rodent liver tumor formation was considered to be qualitatively not plausible for humans. This conclusion is supported by data from a number of epidemiological studies conducted in human populations chronically exposed to PB in which there is no clear evidence for increased liver tumor risk.

Figure 1

MoA for Phenobarbital-induced rodent liver tumour formation. Proposed key events, associative events and modulating factors for the mode of action (MOA) for PB-induced rodent liver tumor formation. The initial key event is CAR activation which results in altered gene expression, increased cell proliferation, clonal expansion? leading to altered foci and subsequently in the formation of liver tumors. Associative events which can serve as reliable biomarkers of key events include epigenetic changes, induction of CYP2B enzymes and liver hypertrophy and decreased apoptosis; whereas inhibition of gap junctional intercellular communication constitutes an associative event or modulating factor.

Figure 2

Human Relevance Framework Analysis for Phenobarbital-induced rodent liver tumour formation. Evaluation of the human relevance of the proposed mode of action (MOA) for PB-induced rodent liver tumor formation. A robust MOA has been developed for PB-induced liver tumor formation in rodents. By reference to the IPCS scheme (Boobis et al., 2006), the animal MOA is considered to be likely not qualitatively plausible for humans. While PB can result in CAR activation in both rodents and humans, the key events of increased cell proliferation, clonal expansion leading to altered foci and subsequently to liver tumors are considered not plausible in humans.