Basic Study
Copyright ©The Author(s) 2017. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Nov 14, 2017; 23(42): 7519-7530
Published online Nov 14, 2017. doi: 10.3748/wjg.v23.i42.7519
Pregnane X receptor and constitutive androstane receptor modulate differently CYP3A-mediated metabolism in early- and late-stage cholestasis
Daniela Gabbia, Arianna Dalla Pozza, Laura Albertoni, Roberta Lazzari, Giorgia Zigiotto, Maria Carrara, Vincenzo Baldo, Tatjana Baldovin, Annarosa Floreani, Sara De Martin
Daniela Gabbia, Arianna Dalla Pozza, Giorgia Zigiotto, Maria Carrara, Sara De Martin, Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Padova 35131, Italy
Laura Albertoni, Department of Medicine, General Pathology and Cytopathology Unit, University of Padova, Padova 35131, Italy
Roberta Lazzari, Vincenzo Baldo, Tatjana Baldovin, Department of Cardiac, Thoracic, and Vascular Sciences, Hygiene and Public Health Unit, University of Padova, Padova 35131, Italy
Annarosa Floreani, Department of Surgery, Oncology and Gastroenterology, University of Padova, Padova 35131, Italy
Author contributions: Gabbia D, Dalla Pozza A, Albertoni L, Lazzari R and Zigiotto G performed the research; Baldovin T, Carrara M, Baldo V and De Martin S contributed new reagents/analytic tools; Gabbia D, Alberton L, Baldovin T, Carrara M, Floreani A and De Martin S analyzed the data; De Martin S designed and coordinated the research; Gabbia D, Floreani A and De Martin S wrote the paper.
Supported by the University of Padova, No. CPDA138721/13.
Institutional review board statement: The study was reviewed and approved by the Institutional Review board of the “Organismo Preposto al Benessere Animale (OPBA)” of the University of Padova.
Institutional animal care and use committee statement: All procedures involving animals were reviewed and approved by the Ethics Committee of University of Padova, and by the Italian Ministry for the care and use of laboratory animals [Prot. no. 24, 2015].
Conflict-of-interest statement: The authors have no conflict of interest to report.
Data sharing statement: Statistical analyses and dataset available from the corresponding author at sara.demartin@unipd.it.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Correspondence to: Sara De Martin, PharmD, PhD, Assistant Professor, Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Largo Meneghetti 2, Padova 35131, Italy. sara.demartin@unipd.it
Telephone: +39-49-8275776 Fax: +39-49-8275093
Received: August 4, 2017
Peer-review started: August 7, 2017
First decision: August 30, 2017
Revised: September 18, 2017
Accepted: October 17, 2017
Article in press: October 17, 2017
Published online: November 14, 2017
Processing time: 99 Days and 21.4 Hours
ARTICLE HIGHLIGHTS
Research background

Primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC) are the two main classes of cholestatic disease, and chronic cholestasis and liver inflammation are their main pathophysiological components[2]. To escape the deleterious effects of high concentrations of bile acids (BAs), a peculiar feature of cholestatic disease, the liver triggers an adaptive response to cholestasis, activating a complex network of nuclear receptors (NRs) that tightly regulate the BA transporters to maintain proper BA homeostasis. Among liver NRs, both the constitutive androstane receptor (CAR) and the pregnane X receptor (PXR), which control the expression of CYP3A drug-metabolizing enzymes, play a major role in these adaptive responses. PXR and CAR act as xenobiotic sensors, as one of their main functions is to regulate the expression of enzymes and transporters involved in xenobiotic elimination. These NRs are also involved in controlling hepatic processes closely related to the progression of cholestatic diseases, such as BA homeostasis, lipid metabolism, fibrosis and inflammation[5]. Changes in CAR and PXR, and CYP3A expression have already been identified in the course of cholestatic liver disease, but different effects have been documented depending on the etiology and severity of the cholestasis. The aim of this study was to analyze expression and enzymatic activity of CYP3A1 and CYP3A2, as well as the nuclear expression of CAR and PXR in a validated animal model of cholestasis rigorously stratified on the basis of the degree of liver dysfunction.

Research motivation

Both in vitro and in vivo studies have shown a decrease in CYP3A activity in cholestatic liver disease, but a clear demonstration of the mechanism(s) responsible for it is still lacking. To our knowledge, no studies published to date simultaneously analyzed CYP3A enzyme expression and activity and the activation of NRs responsible for their transcriptional control in different stages of cholestatic disease. CYP3A enzymes are the most abundant CYPs in human beings, and the most important enzymes in terms of drug metabolism, because they have a key role in the first-pass and systemic metabolism of many drugs. On the basis of these considerations, the aim of this study was to analyze expression and enzymatic activity of CYP3A1 and CYP3A2, as well as the nuclear expression of CAR and PXR. For this purpose, we used a validated animal model of cholestasis based on the bile duct ligation technique in animals rigorously stratified by degree of liver injury.

Research objectives

In this study we investigated whether cholestasis affects the gene and protein expression, and the enzymatic activity of CYP3A1 and CYP3A2 enzymes, as well as the activation of CAR and PXR, the nuclear receptors controlling their transcription. Our results let us hypothesize that PXR and CAR work synergistically to maintain CYP3A induction in the early stages of cholestasis, and their detoxification function fails when the liver dysfunction becomes severe.

Research methods

The procedures involving the animals were managed in accordance with national and international laws and policies (Directive 2010/63/EU on the protection of animals used for scientific purposes). The study design was approved by the Ethics Committee of University of Padova, and by the Italian Ministry for the care and use of laboratory animals (Prot. no. 24, 2015). Gene and protein expressions of PXR, CAR, CYP3A1 and CYP3A2 were assessed by means of qRT-PCR and Western blot, respectively. Alterations in CYP3A activity were measured by calculating the kinetic parameters of marker reactions for CYP3A enzymes. Statistical analyses were performed with the GraphPad Prism 5.0 software (GraphPad Software Inc., San Diego, CA, United States). The experimental results were compared by one-way analysis of variance or Student’s t-test, as appropriate. In the case of statistically significant differences (α = 0.05), the analysis of variance was followed by the Newman-Keuls post-hoc test. A P value < 0.05 was considered statistically significant.

Research results

On the basis of the results obtained in this study, we could hypothesize that PXR and CAR work synergistically to maintain CYP3A induction in the early stages of cholestasis, and their detoxification function fails when the liver dysfunction becomes severe. The mechanism by which cholestasis affects “cross-talk” between PXR, CAR and other NRs in the liver remains to be described in detail.

Research conclusions

The findings of this study can have two clinical consequences. For start, cholestatic patients may have an altered drug metabolism: in the early stage due to the induction of CYP3A enzymes; and in the late stage due to the high deposition of fibrotic liver and consequent hepatocyte loss. This effect could be particularly relevant in humans because a single isoform, i.e., CYP3A4, is responsible for metabolizing more than 50% of the drugs used in medical practice. Secondly, since PXR activation is known to induce alternative hepatic export routes (MRP3 and MRP4) and detoxification enzymes (SULTs, UGTs and CYPs), the induction of these cellular pathways with PXR and/or CAR agonists could be exploited as a therapeutic strategy for the management of cholestatic diseases.

Research perspectives

On the basis of the results obtained in this study, we hypothesized that cholestatic patients may have an altered drug metabolism and suggested the induction of liver detoxification by means of PXR and/or CAR agonists as a therapeutic strategy for the management of cholestatic diseases. To test the first hypothesis, clinical studies analyzing the pharmacokinetic parameters of cholestatic patients stratified by degree of liver injury can be performed after the administration of selected drugs. Preclinical studies analyzing the effects of the administration of PXR/CAR agonists on the cholestatic liver are currently in progress in our laboratory.