Expression of genes involved in xenobiotic metabolism and transport in end-stage liver disease: up-regulation of ABCC4 and CYP1B1

doi:10.1016/S1734-1140(12)70888-5

Pharmacological Reports

Volume 64, Issue 4, July–August 2012, Pages 927-939

https://doi.org/10.1016/S1734-1140(12)70888-5 Get rights and content

Abstract

Background

Expression of drug-metabolizing enzymes and drug transporters in liver is mainly regulated by a system of nuclear receptors. The aim of the current study was to investigate the expression of nuclear receptors, as well as these enzymes and transporters, in liver samples from patients suffering from end-stage liver disease of various etiologies (HCV infection, alcohol liver disease, and primary sclerosis cholangitis).

Methods

Gene expression was measured using quantitative real-time PCR with surgical specimens from livers of patients with end-stage liver disease, and non-tumoral liver tissue that served as control.

Results

Our study confirmed that the expression of most phase I enzymes is suppressed in end-stage liver disease, and is correlated with a decrease in NR1I2 and NR1I3, the main regulators of xenobiotic metabolism. While mRNA levels of phase II enzymes were generally unchanged, some ABC transporters were up-regulated. The most spectacular increases in expression were observed with ABCC4 (MRP4) – at the mRNA level, and CYP1B1 – at both the mRNA and protein levels. We also demonstrated that IL-6 can induce CYP1B1 expression independently of CYP1A1, in a human hepatocellular liver carcinoma cell line.

Conclusions

As CYP1B1 is an enzyme which converts various substrates into carcinogenous metabolites, its overexpression in liver may be one of the factors increasing the risk of hepatic cancers inpatients with liver disease. CYP1A1 and CYP1B1 are often referred to as model AHR target genes, but CYP1A1 was down-regulated in diseased liver samples. This points to the existence of differences in regulation of these two genes.

Introduction

Expression of drug-metabolizing enzymes (DME) and drug transporters (DT) in the liver is mainly regulated by a system of nuclear receptors activated by ligands: xenobiotics or physiological metabolites. Among these, the most important are: CAR (Constitutive Androstane Receptor, encoded by the NR1I3 gene) and PXR (Pregnane X Receptor, encoded by NR1I2) [22]. PXR activates transcription of genes containing XREM (Xenobiotic Responsive Enhancer Module) promoter sequences, e.g., CYP3A4, CYP2C9, SULT2A1, ABCB1, while CAR acts through PBREM (Phenobarbital Responsive Enhancer Module) sequences, e.g., CYP2B6, CYP3A4, CYP2C9, CYP2C19, UGT1A1, ABCC2. Since many genes contain both regulatory elements, their expression is determined by a cross-talk between CAR and PXR [36]. Additionally, Aryl hydrocarbon Receptor (AhR, encoded by the AHR gene), is a cytosolic transcription factor and activates many phase I (i.e., CYP1A1, CYP1A2, CYP1B1), and phase II (UGT1A1, UGT1A6, GSTA1) detoxification enzymes, as well as ABCtransporters. Expression of phase II enzymes is also regulated through the Nrf2 transcription factor (encoded by the NFE2L2 gene), which is an important element of the oxidative stress response [14]. Enzymes and transporters involved in xenobiotic metabolism are additionally regulated in the liver by nuclear receptors controlling endogenous metabolism (e.g., Liver X Receptor – LXR, encoded by NR1H3, and Farnesoid X Receptor – FXR, encoded by NR1H4), forming a complex network of regulators [22].

Hepatic fibrosis is an outcome of many liver diseases, and results from chronic damage to the liver in conjunction with the accumulation of extracellular matrix proteins. The main causes of liver fibrosis include chronic hepatitis C infection (HCV), alcohol abuse, and NonAlcoholic SteatoHepatitis (NASH), as well as chronic cholestatic disorders (i.e., Primary Sclerosis Cholangitis – PSC and Primary Biliary Cirrhosis – PBC) [2]. It is initiated by a cascade of events resulting in hepatocyte damage, recruitment of inflammatory cells to the injured liver, and activation of collagen-producing cells. The accumulation of extracellular matrix proteins distorts the hepatic architecture by forming a fibrous scar, and the subsequent development of nodules of regenerating hepatocytes defines cirrhosis, which result in hepatic insufficiency and portal hypertension, respectively [12].

This inflammatory process leads to a decrease in hepatic drug metabolism and transport, which has been previously demonstrated in in vitro models [23, 26]. It has also been shown that DME and DT expression might be decreased in liver disease caused by HCV infection [7, 18]. The aim of the current study was to investigate the expression of nuclear receptors, as well as DME and DT that were regulated by these factors, in liver samples collected from patients suffering from end-stage liver disease of various etiologies (HCV infection, alcohol liver disease – ALD, and PSC). Liver fibrosis is associated with major alterations in both quantity and composition of extracellular matrix, mainly provided by activated hepatic stellate cells (HSCs) [2]. As HSC activation, as well subsequent inflammatory processes leading to liver disease and fibrosis, are mediated by pro-inflammatory cytokines [2], we additionally tested in vitro the influence of selected cytokines involved in liver disease pathogenesis on the expression of ABCC4 and CYP1B1 genes in human hepatoma cell culture.

Section snippets

Patients

Surgical specimens were obtained from explanted livers from 36 patients with end-stage liver disease (mean age 53.3 ± 7.7 years), who underwent transplantation at the Division of Hepatobiliary Surgery and Liver Transplantation, Marie Curie Hospital, Szczecin, Poland. The studied group included patients with chronic HCV (n = 18), PSC (n = 10), and ALD (n = 8). Severity of the chronic liver disease was assessed using the Child-Pugh classification (grade A = 5–6; grade B = 7–9; grade C = 10–15). The score was

Gene expression in end-stage liver disease

The expression of genes encoding nuclear receptors involved in drug metabolism regulation – CAR (NR1I3) and AHR (AHR) – was significantly decreased (to about 50%) in all groups of patients, compared to the controls. In contrast, PXR (NR1I2) expression was not significantly lower when compared with controls. The mRNA levels of other receptors (LXR – NR1H3, FXR – NR1H4, RXR – RXRA) were decreased to a lesser extent (to 60–70%), but the difference was still significant in relation to control

Discussion

We have observed that the expression of nuclear receptors (NR) involved in the regulation of drug metabolism (NR1I2, NR1I3, AHR, NR1H3, NR1H4, RXRA) was decreased in end-stage liver disease compared to controls, regardless of disease pathology. Although there is no strong evidence for a direct relationship between NR expression and DME and DT activity, it was demonstrated by Wortham et al. [40] that NR1I3 (CAR) expression levels were associated with expression of coregulated

Acknowledgment

The study was supported by grant No. NN405165739 from the Ministry of Science and Higher Education, Poland.

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Expression of genes involved in xenobiotic metabolism and transport in end-stage liver disease: up-regulation of ABCC4 and CYP1B1

Abstract

Background

Methods

Results

Conclusions

Introduction

Section snippets

Patients

Gene expression in end-stage liver disease

Discussion

Acknowledgment

Biochim Biophys Acta

Bioorg Med Chem

Toxicol Appl Pharmacol

Biochem Pharmacol

Toxicol Lett

Drug Metab Pharmacokinet

Biochem Pharmacol

Biochem Pharmacol

J Hepatol

J Biol Chem

J Biol Chem

Toxicol Sci

Mutat Res

Pharmacol Rep

Expression of cytochromes P450, conjugating enzymes and nuclear receptors in human hepatoma HepaRG cells

Drug Metab Dispos

Liver fibrosis

J Clin Invest

Role of epigenetic mechanisms in differential regulation of the dioxin-inducible human CYP1A1 and CYP1B1 genes

Mol Pharmacol

Genetic polymorphism of metabolic enzymes P450 (CYP) as a susceptibility factor for drug response, toxicity, and cancer risk

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Coordinate regulation of metabolic enzymes and transporters by nuclear transcription factors in human liver disease

J Gastroenterol Hepatol

Multidrug resistance and cancer: the role of the human ABC transporter ABCG2

Curr Protein Pept Sci

Hepatic cytochrome P450 enzyme alterations in humans with progressive stages of nonalcoholic fatty liver disease

Drug Metab Dispos