Definition of the mechanisms that underlie the decrease in expression of the cytochrome P450s in primary hepatocytes

A physiological understanding of how biological systems respond to xenobiotics and endobiotics is fundamental to the prediction of the consequences of exposure to dietary, environmental, and pharmaceutical products. Currently, the investigation of mammalian responses to these products is reliant on whole animal studies, partly due to the lack of reliable, stable and cost effective in vitro alternatives that faithfully reflect their disposition in an integrated physiological system. This is particularly true for metabolism studies where isolation and/or culture of liver cells leads to de-differentiation and loss of multiple enzyme functions. The ideal in vitro model for understanding the physiological consequences of exposure of the liver to drugs and chemicals is a metabolically-competent hepatocyte. To be a physiologically adequate model, hepatocytes should contain a normal complement of the cytochrome P450 proteins (CYP450s) essential for the chemical modification and clearance of the majority of foreign compounds, as well as many endogenously-formed compounds. Unfortunately, expression of most of the relevant CYP450s in freshly isolated hepatocytes is lost within days of their transfer into laboratory culture conditions. The loss of the CYP450s from these cells is associated with a reduction in the activity of a number of regulatory proteins. However, the identity of all the transcription factors contributing to the maintenance of the CYP450s in primary cells and the extent to which these determine the basal level of CYP450s in hepatocytes is not known. Therefore, the main question that I wish to address in this proposal is what are the molecular mechanisms that determine the decrease in expression of the CYP450s in primary hepatocytes in culture? In parallel with this question, I will attempt to test the hypothesis that the manipulation of positive and/or negative regulators of CYP450 expression in primary hepatocytes can yield a more physiologically relevant Phase I phenotype. A twin-track approach is proposed in this application that aims to help determine the events that occur during the loss of the CYP450s from hepatocytes in culture. This will consist of a measurement of the principal candidate transcriptional regulators in a primary hepatocyte model, alongside an unbiased global screen of nuclear transcription factor changes that accompany the classical loss of the Phase 1 metabolic phenotype in these cells. The technology that will be employed in this study benefits from the great advances in gene transfection and RNA interference in primary cells that have occurred only recently. This will allow the manipulation of the regulatory factors that are implicated in the control of the CYP450s, and represents a fresh approach to tackle this important question.

The main objective of this study is to define the regulatory factors responsible for maintenance of Phase 1 metabolic competence in primary hepatocytes. The four stages of the proposal designed to address this objective are as follows: 1. Measurement of changes in primary mouse hepatocyte transcription factors before and after isolation and culture, using a twin-track approach, i.e. a. a targeted transcription factor approach, in which the nuclear levels of liver-enriched transcription factors (LETFs) believed to be implicated in Phase I drug metabolism (hepatocyte nuclear factors plus others) as well as the levels of a number of nuclear receptors which may play a role in P450 expression (PXR, RXR, CAR etc) will be measured in primary mouse hepatocytes during their isolation and up to 96 hours after their isolation during culture, and related to the expression of a small panel of relevant P450s, b. an unbiased transcription factor approach: the global nuclear protein profile of the hepatocytes will be determined using the differential proteomic technology of two-dimensional difference gel electrophoresis (2D DIGE) and this will be related to the levels of the panel of P450s. These experiments will inform our selection of regulator(s) for the following expression and knock-down studies. 2. Metabolic phenotype rescue by transduction of primary mouse hepatocytes transfected with the transcription factors identified in section 1, and the other factors previously implicated in the expression of the P450s. 3. Regulation of P450 expression by RNAi of regulatory transcription factors under culture conditions of the hepatocytes selected from Section 1 that would normally allow maintenance of the P450s. 4. Significance of the regulatory proteins in human hepatocytes. As an initial step in this direction, genes coding for the regulatory proteins will be introduced into several novel regulatable human hepatoma cell lines.