Effect of lipopolysaccharide on P-glycoprotein-mediated intestinal and biliary excretion of rhodamine123 in rats

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Abstract

The effects of lipopolysaccharide (LPS) on the ileal and biliary excretion of rhodamine123 were investigated in rats at different times after intraperitoneal (i.p.) injection (1 mg/kg and 5 mg/kg of body weight). P-gp protein decreased 8 h after injection of LPS and returned to the control level 24 h after i.p. injection of LPS in the ileum. There was a marked decrease in the expression level of mdr1a mRNA in the ileum and liver 8 h after i.p. injection of LPS when compared with the control condition. Also, the ileal and biliary clearance of rhodamine123 significantly decreased 8 h after i.p. injection of LPS, but returned to the control levels 24 h after i.p. injection of LPS. These results suggest that LPS-induced decreases in P-gp-mediated ileal and biliary excretion of rhodamine123 were probably due to impaired P-gp-mediated transport ability. The levels of iNOS and IL-1β mRNA in the ileum and liver increased 2 and 8 h after i.p. injection of LPS, respectively, and returned to the control levels 24 h after injection of LPS. These findings suggest that LPS markedly decreases P-gp-mediated ileal and biliary excretion of rhodamine123, probably by partly decreasing the expression of P-gp protein levels, likely due to increased lipid peroxidation levels through iNOS mRNA and inflammatory mediators such as IL-1β.

Introduction

Endotoxin, which is an active component of the cell wall of Gram-negative bacteria, is well known to damage numerous organs, including the intestine and liver, which have crucial functions for the absorption and elimination of endogenous and exogenous substances. These are converted to more hydrophilic compounds by cytochrome P-450 and/or conjugating enzyme, and then excreted into the bile and urine. A series of studies suggested that endotoxin reduces biliary excretion of various organic anionic drugs as a result of changes in the biliary secretory systems (Hasegawa et al., 1985, Haghgoo et al., 1995, Nadai et al., 1996). Also, it has been demonstrated that endotoxin reduces hepatic cytochrome P-450-dependent drug-metabolizing enzymes 24 h after intraperitoneal (i.p.) injection in rats, due to the overproduction of nitric oxide in plasma (Kitaichi et al., 1999); however, less is known about the effect of endotoxin treatment on intestinal absorption and the hepatobiliary transport system.

It is well known that hepatobiliary excretion of organic anionic drugs is mediated by a bile canalicular multispecific organic anion transporter (cMOAT), which belongs to the ATP-binding cassette transporter superfamily (Ito et al., 1997). Another important transporter, P-glycoprotein (P-gp), acts as an ATP-dependent efflux pump for various drugs, such as vinca alkaloid and anthracycline anticancer drugs, calcium blocker, immunosuppressive agents, and macrolide antibiotics (Tsuruo et al., 1982, Twentyman et al., 1987, Wang et al., 2000), in normal tissues such as the brain, liver, intestine and kidney (Fojo et al., 1987, Thiebaut et al., 1987, Kamimoto et al., 1989, Hsing et al., 1992, Schinkel et al., 1994). Interestingly, there is evidence that the substrate specificities of P-gp, cytochrome P-450 3A4 (CYP3A4), and cMOAT overlap, and these proteins are located in hepatocytes and have similar functions for removing various drugs from the body (Mayer et al., 1995, Oude et al., 1995, Wacher et al., 1995, Kiso et al., 2000). On the basis of these observations, endotoxin may possibly modify the pharmacokinetics of drugs mediated by P-gp; however, there is little information regarding the effect of endotoxin on P-gp-mediated absorption and the biliary transport system and the expression of P-gp transport genes.

The aim of the present study was to clarify whether lipopolysaccharide (LPS) modifies P-gp-mediated intestinal absorption and the biliary transport systems in rats. Rhodamine123 is a substrate of P-gp and thus, it was chosen as the model drug in this study. This compound is primarily excreted into the intestinal lumen and bile in unchanged form (Kunihara et al., 1998). In order to evaluate the contribution of P-gp-mediated transport to the luminal and hepatobiliary excretion of rhodamine123, we used reverse transcriptase PCR (RT-PCR) to measure the expression of mRNA of the multidrug resistance proteins 1a and 1b (Mdr1a and Mdr1b), which are members of the P-gp subfamily, isolated from the ileum and liver at different times after i.p. injection of LPS.

Section snippets

Materials

LPS was isolated from Escherichia coli o111:B4, which was identical to that used in this study. Rhodamine123 was purchased from Sigma Co. Ltd. (St. Louis, MO). All other reagents are commercially available and were of analytical grade or better.

Animal and experimental protocol

Eight nine-week-old male Wistar rats weighing 250–280 g (Japan SLC, Hamamatsu, Japan) were used for all experiments. The rats were housed under controlled environmental conditions (temperature, 23 °C ± 1 °C, humidity, 55% ± 5%) with a commercial food diet and

Effects of LPS on the level of mdr1a mRNA expression in ileal tissue

We examined the level of mdr1a mRNA expression at different times after i.p. injection of LPS. The significant decrease in mdr1a mRNA expression level from the control value in ileal tissue 8 h after injection of LPS was observed in a dose-dependent manner, but the levels under both 1 mg/kg and 5 mg/kg LPS injection groups returned to the control 24 h after i.p. injection of LPS (Fig. 1). No marked changes were observed at 4 h after both doses of LPS when compared with the control condition (Fig. 1

Discussion

The present study focused on the effect of i.p. injection of LPS on P-gp mediated ileal and biliary excretion of the P-gp substrate, rhodamine123, which has been used as a marker for evaluating the role of P-gp in anticancer drug-resistant cells and various normal tissues, such as the small intestine, liver, kidney and brain (Chin et al., 1989, Neyfakh et al., 1989, Lee et al., 1994, Wang et al., 1995, de Lange et al., 1998, Kunihara et al., 1998). According to our studies, it is considered

Acknowledgments

This work was supported in part by a Grant-in-Aid for Scientific Research (18590156) from the Ministry of Education, Science, Sport, and Culture of Japan.

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