Elsevier

Journal of Hepatology

Volume 35, Issue 6, December 2001, Pages 765-773
Journal of Hepatology

Decreased expression of an ATP-binding cassette transporter, MRP2, in human livers with hepatitis C virus infection

https://doi.org/10.1016/S0168-8278(01)00216-1Get rights and content

Abstract

Background/Aims: To understand hepatic injury during the process of hepatitis viral infection, determination of liver-specific functions at molecular levels is critical. Because the transport of endogenous/exogenous toxic substances is an intrinsically important hepatic function, we examined whether expression of the ATP-binding cassette (ABC) transporter gene was affected in patients with hepatitis viral infection.

Methods: To determine which ABC transporter was expressed differently in patients with hepatic viral infection, we assayed the expression of MDR1, MDR3, MRP1, MRP2, and MRP3 in non-cancerous regions in the liver of 42 patients with hepatic tumors using both quantitative RT-PCR and immunological staining analysis, and compared the hepatic expression levels between patients with hepatitis viral infection and non-infected controls.

Results: Of the five ABC transporter genes studied, the mRNAs of MRP2 and MRP3 were highly expressed in the human liver. There was a significant reduction in MRP2 expression to 29% in the virus-infected liver. Treatment of hepatic cells with inflammatory cytokines resulted in decreased mRNA levels of MRP2 and decreased MRP2 promoter activity.

Conclusions: The down-regulation of MRP2 might induce a failure in the transport of various genotoxic substances in the liver with hepatitis virus infection.

Introduction

Several types of transporters on the liver plasma membrane have recently been identified as being associated with the disposition and detoxication of xenobiotics. On sinusoidal membranes, many types of drugs and toxins are transported into hepatocytes by secondary active transport systems, and then subjected to metabolic conversion and biliary excretion [1]. Of all the transporters on the plasma membrane, the ATP binding cassette (ABC) superfamily transporters play perhaps the most important roles in the disposition and detoxication of xenobiotics in the liver. Several kinds of primary active transport systems for xenobiotics and endogenous substrates, all of them driven by ATP hydrolysis, have been identified on the human canalicular membrane [2], [3], [4], [5].

Two ABC superfamily transporters, P-glycoprotein (P-gp) encoded by multidrug resistance (MDR) MDR1 and multidrug resistance protein1 (MRP1), are well-known to confer multidrug resistance to cancer cells through enhanced drug efflux [2], [6]. In addition to MDR1 and MRP1, two MDR subfamily members, MDR3 and BSEP, which show structural similarity to MDR1, and five MRP subfamily members, MRP26, which show structural similarity to MRP1, have been further identified [3], [4], [5], [7], [8], [9], [10]. Of all the transporters located on the plasma membrane, ABC superfamily transporters play important roles in disposition and detoxication of xenobiotics in the liver. Three of the ABC family genes, P-gp/MDR1, BSEP and MRP2, appear to mediate the ATP-dependent transport of a number of conjugates, including leukotriene C4 and the glucuronide complex of bilirubin and thyroxine in the liver canalicular membranes [4], [10], [11], [12], [13]. MDR3 is a phospholipid flippase in the liver canalicular membranes [3]. MRP2 and MRP3 are highly expressed in the normal liver [5], [7], [14], [15] and compensate each other functionally [16]. The presence or absence of ABC transporters is expected to play a critical role in detoxication or self-defense mechanisms in livers.

The mechanisms of tissue injury by acute and chronic hepatitis virus infection are not well understood. Several studies have suggested that chronic liver injury by hepatitis virus infection is due to immunological reaction [17], [18]. Because the many types of chronic liver injury caused by HCV-infection cannot be explained by cytotoxic T lymphocytes-mediated liver injury alone [17], another mechanism is thought to exist. It has been shown that the HCV viral capsid protein and core protein have a number of regulatory functions and can activate or suppress the expression of viral and cellular genes [19], [20], [21].

One might expect that dysfunction of the liver, including side-effects by various drugs, is partly due to a failure in the expression of various ABC transporters in liver cells. Determination of the expression levels of ABC transporters should thus contribute to clarification of the liver's self-defense mechanisms against endogenous and exogenous toxic substances on a molecular basis.

In this study, we examined whether hepatitis C virus infection modulates the expression of five liver-related ABC transporters, MDR1, MDR3, MRP1, MRP2 and MRP3, in non-cancerous regions in the liver of 42 patients with hepatic tumor. Among these five ABC transporter genes, the canalicular multispecific organic anion transporter, MRP2, was rather specifically reduced by hepatitis C virus infection. We demonstrated that pro-inflammatory cytokine such as IL-1β, TNF-α and IL-6 might affect the reduced expression of human MRP2 mRNA and promoter activity in a hepatocarcinoma cell line, HepG2. This reduced expression of ABC transporters is therefore discussed in association with inflammatory cytokines.

Section snippets

Patients

Between July 1996 and February 1999, 42 Japanese male and female patients with hepatic tumors underwent partial hepatectomy in the Kyushu University Hospital. Table 1 shows the clinicopathogenic characteristics of these 42 patients, who were classified by virus infection and were histologically determined to have both hepatic inflammation and fibrosis. A new European classification system was used developed by Anthony et al. [22], in which S indicates the staging of hepatic fibrosis (S0: non;

Results

We examined whether the expression of ABC transporters was altered by hepatitis C virus infection in patients with liver tumors. To determine the mRNA levels of MDR1, MDR3, MRP1, MRP2, and MRP3 in human hepatic non-cancerous tissue, we examined surgically removed hepatic samples from 42 patients using quantitative RT-PCR with specific primers and probes. The data were standardized against GAPDH mRNA levels of non-cancerous regions. Fig. 1 shows the messenger RNA levels of ABC transporters in

Discussion

The mechanisms underlying tissue injury by acute and chronic hepatitis virus infection remain unclear because of a lack of suitable animal models and of a conventional tissue culture system [30]. In our present study, we investigated the expressions of five ABC transporters to find any association of ABC transporters with liver injury. The expression of these five ABC transporter genes showed a surprisingly wide range, suggesting a great difference in individual responses to endogenous and

Acknowledgements

We thank Morimasa Wada, Takanori Nakamura, Sei Haga, Takayuki Hamatsu and Masahiro Okamoto for helpful discussion. Supported by a Grant-in-Aid for Scientific Research on the Priority Area of ABC Proteins, by CREST (Core Research for Evolutional Science and Technology) of the Japan Science and Technology Corporation (JST), and by the Second-Term Comprehensive Ten-Year Strategy for Cancer Control from the Ministry of Health and Welfare of Japan.

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