Protective effects of hepatocellular canalicular conjugate export pump (Mrp2) on sodium arsenite-induced hepatic dysfunction in rats

Abstract

Arsenic is a double-edged sword to human health. The excretion of various organic anions into bile is mediated by an adenosine triphosphate-dependent conjugate export pump, which has been identified as the canalicular isoform of the multidrug resistance protein 2 (Mrp2). It has been proved that Mrp2 can transport arsenite in vitro, but its effects in vivo are not clear. The aim of this study was to investigate whether Mrp2 plays a role in exportation of arsenic in vivo and its protective effects on liver function. Mrp2 protein level in rat liver was determined by Western blot analysis. Total arsenic concentrations in whole blood and bile were measured using hydride generation atomic absorption spectrometry. Alanine aminotransferase (ALT) activity, aspartate aminotransferase activity (AST), glutathione peroxidase (GSH-PX) activity, malon dialdehyde (MDA) and total bilirubin were measured by biochemical assays. The morphological changes were observed by electron microscopy. Total arsenic levels in blood and bile of arsenite-treated rats were significantly higher than those of control rats (P<0.05) at all three different time points. The overexpression of Mrp2 was 36.61%, 32.36% and 12.73% at 2, 4 and 6 weeks, respectively (percentage of controls, P<0.05), which was significantly higher than controls. A positive correlation between Mrp2 expression level and total arsenic concentration in bile indicated that Mrp2 accelerated the transport of arsenic. Electron microscopy showed that microvilli of bile canaliculi became swollen and sparse. ALT and AST activities in serum were markedly raised at 6 weeks. MDA level in serum increased (P<0.05) and GSH-PX activity in serum decreased except for 2 weeks. Damage of liver function became worse following decreased expression of Mrp2. In conclusion, overexpression of Mrp2 may explain increased biliary excretion of arsenic and it may protect liver function.

Introduction

Arsenic is a double-edged sword to human health. Namely, chronic adverse effects of arsenic exposure have been reported in Latin America, South Asia, Taiwan, Japan and China, and people exposed to arsenic are prone to develop skin, bladder and lung cancer and occlusive vascular disease (blackfoot disease) (Chen et al., 1985; Tsai et al., 1998; Nordstrom, 2002; Yu et al., 2006). On the other hand, arsenic trioxide (As₂O₃) is a potent antitumor agent used to treat acute promyelocytic leukemia and, more recently, solid tumors (Liu et al., 2006). Much has been done to investigate the mechanisms, including growth inhibition, apoptosis, oxidative stress, chromosomal abnormality and alteration of growth factors.

As one of the most important organs in the body, the liver is specially designed to perform many essential functions, such as the metabolism and elimination of various endo- and xenobiotic compounds, many of which are excreted into bile after conversion into amphiphilic anionic conjugates with glutathione, glucuronate or sulfate. However, its distinctive characteristics and activities render it susceptible to damage from a variety of sources, and such damage can have enormous impacts on health. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are the two main serum parameters for liver function and released from the liver cells into the bloodstream, often in liver disease, resulting in abnormally high serum levels that may not return to normal for days or weeks.

Bilirubin is removed from the blood by the liver and excreted into the bile, and it gives the bile its pigmentation. Total bilirubin is a by-product of the breakdown of old red blood cells in the liver and a good indication of the liver function. It is elevated in liver disease, mononucleosis, hemolytic anemia and drug treatment. GSH-Px is an important enzyme to remove the free radicals and oxidative products.

At present, researches about the action modes of arsenic are concentrated on oxidative stress, chromosomal abnormalities, altered DNA repair, p53 suppression, DNA methylation patterns and cellular signaling (Kitchin, 2001; Miller et al., 2002) and less attention is paid to the transport of arsenic and its metabolites. In the work reported here, the transport mechanism was also investigated. The multidrug resistance-associated proteins (Mrps) belong to family C of the adenosine triphosphate (ATP)-binding cassette (ABC) superfamily and function as ATP-dependent export pumps for a variety of organic solutes (Borst et al.,1999; Konig et al., 1999). One member of this family, rodent multidrug resistance protein 2 (Mrp2) (Abcc2), is localized to the apical membrane of excretory organs, and in particular the canalicular membrane of hepatocytes, where it functions to export a diverse group of compounds, including sulfate, glutathione (GSH) and glucuronide conjugates (Keppler et al, 1997; Trauner et al.,1997; Paulusma et al.,1999). In this study, we examined several relevant issues such as: (a) the possible protective effects of Mrp2 on liver function and (b) the relationship between Mrp2 and transportation of arsenite into bile.