Role of tumor necrosis factor-α in down-regulation of hepatic cytochrome P450 and P-glycoprotein by endotoxin

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Abstract

We investigated the role of tumor necrosis factor-α (TNF-α) in the down-regulation of hepatic P-glycoprotein and cytochrome P450 (CYP) by endotoxin, using TNF-α gene-deficient (TNF-α−/−) mice. In the case of P-glycoprotein, endotoxin (10 mg/kg) significantly decreased the expression of hepatic P-glycoprotein in wild-type mice 6 h, but not 24 h, after intraperitoneal injection, with no significant differences in the constitutional expression of P-glycoprotein between wild-type mice and TNF-α−/− mice. However, endotoxin had no effect on the expression of P-glycoprotein in TNF-α−/− mice either 6 or 24 h after injection. When doxorubicin was administered intravenously to TNF-α−/− mice treated 6 h earlier with and without endotoxin, no significant differences in the plasma concentrations of doxorubicin 3 h after injection were observed between endotoxin-treated and untreated TNF-α−/− mice. These results suggest that TNF-α plays a pivotal role in the down-regulation of P-glycoprotein by endotoxin. In the case of CYP, the constitutive expression of hepatic CYP3A2 and CYP2C11 had a tendency to decline in TNF-α−/− mice compared with that in wild-type mice. Endotoxin significantly decreased the expression of hepatic CYP3A2 and CYP2C11 in wild-type mice 24 h after injection, and that decreased expression was significantly greater in TNF-α−/− mice than wild-type mice. When antipyrine was administered intravenously to wild-type mice and TNF-α−/− mice treated 24 h earlier with endotoxin, the plasma concentrations of antipyrine in TNF-α−/− mice 3 h after injection were significantly higher than those in wild-type mice. These findings suggest that TNF-α plays a key role in endotoxin-induced down-regulation of hepatic P-glycoprotein, as well as plays a protective role in the regulation of hepatic CYP3A2 and CYP2C11 against endotoxin-induced acute inflammatory response. In TNF-α−/− mice, other cytokines appear to function as compensation for the lack of endogenous TNF-α.

Introduction

It is well known that bacterial infections impair hepatic drug metabolism in humans, and that endotoxin (lipopolysaccharide), a major component of the cell wall of Gram-negative bacteria, plays a key role in this phenomenon. Endotoxin is known to secrete various inflammatory mediators such as nitric oxide (NO), tumor necrosis factor-α (TNF-α), interleukin-1, interleukin-2, and interleukin-6. These inflammatory mediators have been shown to regulate the content and activities of hepatic cytochrome P450 (CYP) in humans and animals (Chen et al., 1992, Manuel, 1996, Morgan, 1997, Shedlofsky et al., 1994). Among them, NO is significantly released after exposure to endotoxin, subsequent to the expression of inducible NO synthase (Bredt and Snyder, 1994, Khatsenko et al., 1993, Moncada et al., 1991). We and other investigators have demonstrated that NO is one of the important inflammatory mediators regulating the contents and activities of CYP (Kitaichi et al., 1999, Kitaichi et al., 2004, Gergel et al., 1997, Khatsenko and Kikkawa, 1997, Khatsenko et al., 1993, Minamiyama et al., 1997, Morgan, 1997, Sewer and Morgan, 1997, Takemura et al., 1999, Ueyama et al., 2004, Wink et al., 1993). On the other hand, it has also been reported that the endotoxin-induced down-regulation of CYP3A2, CYP2C11, and CYP2E1 is NO-independent (Sewer and Morgan, 1997, Sewer and Morgan, 1998). The mechanism responsible for the NO-mediated down-regulation of CYP isoforms is still unclear.

In addition to NO, TNF-α is also thought to be of major importance in the down-regulation of CYP isoforms in endotoxemia. It has been reported that TNF-α decreases the contents of the CYP isoforms CYP3A2 and CYP2C11 in rats (Monshouwer et al., 1996, Sewer and Morgan, 1997). In contrast, Warren et al. (1999), in animal experiments using TNF-α receptor (p55/p75)-deficient mice, reported that TNF-α does not play a key role in the endotoxin-induced down-regulation of CYP isoforms, including CYP1A and CYP3A.

On the other hand, inflammatory cytokines, including TNF-α, interleukin-1, interleukin-2, and interleukin-6, might play an important role in endotoxin-induced changes in certain drug transporter-mediated hepatobiliary excretion systems (Hirsch-Ernst et al., 1998, Simpson et al., 1997). We previously reported that Klebsiella pneumoniae endotoxin significantly reduces hepatobiliary excretion of the β-lactam antibiotic, cefoperazone, which is a substrate for multidrug resistance-associated protein 2 (Mrp2) (Haghgoo et al., 1995, Nadai et al., 1998), suggesting that some inflammatory mediators released by endotoxin contribute to the impairment of the hepatobiliary excretion of drugs by reducing the expression and/or function of Mrp2 in the canalicular membrane of hepatocytes. Endotoxin is known to induce cholestasis and hyperbilirubinemia by down-regulating Mrp2, an efflux pump for bile acids and bilirubin, due to the secretion of some cytokines, including TNF-α and interleukin-1 (Green et al., 1996, Nakamura et al., 1999, Trauner et al., 1997). In contrast, there is evidence that TNF-α induces the up-regulation of transporter genes or MRP1 protein in human colon carcinoma cells and mdr1 in rat hepatoma cells (Chapekar et al., 1991, Stein et al., 1997). However, the precise role of TNF-α in the down-regulation or up-regulation of hepatic Mrp2 by endotoxins remains unclear.

Like Mrp2, the ATP-binding cassette transport protein, P-glycoprotein, is expressed in many eliminating organs such as the liver and kidney (Cordon-Cardo et al., 1989, Thiebaut et al., 1987), and acts as the efflux transport protein for endogenous and exogenous toxic substances (Schinkel et al., 1996, Thiebaut et al., 1987). Thus, these two drug transport proteins, P-glycoprotein and Mrp2, might exert a protective function of excluding various lipophilic substrates from the liver. There is evidence that TNF-α reduces the expression of P-glycoprotein (Sukhai et al., 2000). In our previous studies, it was suggested that endotoxin dramatically decreases the P-glycoprotein-mediated hepatobiliary excretion of rhodamine 123 by reducing the expression of mdr1a due to increased plasma TNF-α levels (Ando et al., 2001). From the above findings, the expression of both hepatic P-glycoprotein and Mrp2 appears to be regulated by inflammatory cytokines, including TNF-α. Interestingly, there is evidence that the numerous substrates of P-glycoprotein, CYP3A, and Mrp2 largely overlap, and that these proteins are located at hepatocytes and have similar functions of removing various drugs from the body (Mayer et al., 1995, Oude Elferink et al., 1995, Wacher et al., 1995). On the basis of these observations, it is possible that endotoxin might simultaneously down-regulate hepatic P-glycoprotein, CYP3A, and Mrp2. However, to our knowledge, there is no information confirming whether endotoxin simultaneously regulates the expression of CYP3A, P-glycoprotein, and Mrp2. Taken together, it is, at present, difficult to clarify the role of endogenous TNF-α in the regulation of hepatic CYP isoforms and drug transporters.

In the present study, we focused on the effect of TNF-α on the expression of the drug transporter P-glycoprotein and the major CYP isoforms, CYP3A2 and CYP2C11. It is considered that mice with targeted deletions of the TNF-α gene (TNF-α−/− mice) are useful as an animal model for evaluating the role of endogenous TNF-α in the down-regulation of CYP isoforms by endotoxin. The aim of the present study was to clarify the role of TNF-α in the endotoxin-induced down-regulation of P-glycoprotein and these CYP isoforms, using mice with a targeted disruption of the gene encoding TNF-α (Taniguchi et al., 1997), which can block the effects of TNF-α.

Section snippets

Chemicals

Endotoxin was obtained from K. pneumoniae LEN-1 (O3:K1), which was identical to that used in previous studies (Ando et al., 2001, Kitaichi et al., 1999, Nadai et al., 1998, Zhao et al., 2002). Doxorubicin hydrochloride and daunorubicin hydrochloride were purchased from Sigma (St. Louis, MO, USA). Doxorubicin hydrochloride, in the form of a commercial preparation for injection, was purchased from Kyowa Hakko Kogyo (Adriamycin; Tokyo, Japan). Antipyrine, testosterone, 6β-hydroxytestosterone,

Effect of endotoxin on histopathological findings in wild-type and TNF-α−/− mice

Light micrographs obtained by the histopathological examinations revealed that there was no difference in the light microscopy of liver tissues between wild-type mice and TNF-α−/− mice, indicating that the livers of both possess almost the same morphological characteristics. Endotoxin induced only a mild infiltration with no evidence of a massive necrotic or apoptotic area in either the wild-type or TNF-α−/− mice.

Effect of endotoxin on expression of hepatic P-glycoprotein in wild-type and TNF-α−/− mice

The time-dependent effects of endotoxin on the expression of P-glycoprotein in

Discussion

Endotoxin is known to increase the levels of cytokines, including TNF-α, and the elevation of these cytokines might play an important role in endotoxin-induced changes in certain transporter-mediated biliary excretion systems (Hirsch-Ernst et al., 1998, Simpson et al., 1997). We previously reported that the expression of P-glycoprotein mRNA (mdr1a mRNA) in the liver of rats treated 6 h earlier with endotoxin declined, and returned to control levels after 24 h, and that pentoxifylline, which

Acknowledgements

This work was supported by a Grant-in-Aid for Scientific Research (15590484) and a Grant-in-Aid for Scientific Frontier Research Project of Meijo University from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

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