Effect of endotoxin on doxorubicin transport across blood–brain barrier and P-glycoprotein function in mice

Abstract

The aim of this study was to investigate whether Klebsiella pneumoniae endotoxin modifies transport of doxorubicin, a P-glycoprotein substrate, across the blood–brain barrier and P-glycoprotein function in mice. Doxorubicin (30 mg/kg) was administered into the tail vein or fluorescein isothiocyanate-labeled dextran (FD-4) was infused (20 μg/min) into the right jugular vein of mice intravenously injected with endotoxin (10 mg/kg) 6 or 24 h earlier. Blood and brain samples were collected 4 h after injection of doxorubicin or 1 h after infusion of FD-4. We examined using Western blotting the influence of endotoxin on the expression of P-glycoprotein in brains obtained 6, 12 and 24 h after injection. Endotoxin did not change the plasma and brain concentrations and brain-to-plasma concentration ratio (K_p value) of FD-4. No histopathological changes in brain capillaries were observed. These results suggest that endotoxin does not cause damage to brain capillaries. Plasma and brain concentrations of doxorubicin in mice treated 6 h earlier with endotoxin were significantly higher than those in control and mice treated 24 h earlier. However, endotoxin did not significantly change the K_p value of doxorubicin. The protein level of P-glycoprotein was significantly, but slightly down-regulated 6 h after endotoxin treatment. However, the levels remained almost unchanged after 12 and 24 h. The present results suggest that Klebsiella pneumoniae endotoxin has no effect on the brain capillary integrity and doxorubicin transport across the blood–brain barrier in mice. It is likely that P-glycoprotein function might be sufficient to transport doxorubicin in spite of decreased levels of P-glycoprotein in the brain.

Introduction

It is well known that numerous cancer cells overexpress multidrug resistance (MDR) gene-encoded P-glycoprotein, resulting in anticancer drug resistance Juliano and Ling, 1976, Ueda et al., 1987. P-glycoprotein, a member of the ATP-binding cassette superfamily of transport proteins, acts as an active efflux pump for hydrophobic and cationic anticancer drugs, such as Vinca alkaloids and anthracycline antibiotics, and plays an important role in their antitumor activity Endicott and Ling, 1989, Tsuruo, 1988, Tsuruo et al., 1982.

P-Glycoprotein is expressed largely, not only in anticancer drug resistant cells, but also in various normal tissues such as liver, kidneys, intestine and brain Cordon-Cardo et al., 1990, Thiebaut et al., 1987. In these tissues, this protein has a protective function of excluding endogenous and exogenous substances from the body. In brain, P-glycoprotein is expressed in the luminal membrane of the brain capillary endothelium Cordon-Cardo et al., 1989, Schinkel et al., 1994 and in the apical membrane of the choroid plexus epithelium (Rao, 1999). In particular, P-glycoprotein in the blood–brain barrier has an important role in limiting the distribution of various substances into the brain to protect from neurotoxicity Ohnishi et al., 1995, Tsuji, 1998, Watchko et al., 1998. It is likely that the existence of P-glycoprotein in the blood–brain barrier also leads to chemotherapy failure in brain cancers by limiting the effectiveness of many anticancer drugs since MDR 1 is expressed in human brain tumors (Nabors et al., 1991). Ohnishi et al. (1995) have reported that the brain distribution of doxorubicin, a substrate of P-glycoprotein, is mainly restricted by P-glycoprotein on the blood–brain barrier under normal physiological conditions. It is thought that the integrity of brain capillary cells and the function of P-glycoprotein in the blood–brain barrier may be modified by histopathological and physiological changes occurring in various disease states, including bacterial infectious disease.

Endotoxin, an active component in the outer membrane of Gram-negative bacteria, is known for its various biological and immunological activities. Endotoxin has been found to induce various histopathological and physiological changes in the body, such as damage to the central nervous system, liver, kidney, heart, gastrointestinal tract and lungs (Hewett and Roth, 1993). We have reported that K. pneumoniae endotoxin reduces hepatic drug-metabolizing enzyme activity and impairs biliary and renal excretion of various drugs by changing the ability of the biliary and tubular secretory systems Haghgoo et al., 1995, Hasegawa et al., 1994, Kitaichi et al., 1999, Nadai et al., 1993a, Nadai et al., 1993b, Nadai et al., 1993c, Nadai et al., 1996, Nadai et al., 1998. In addition, we recently reported that K. pneumoniae endotoxin impaired the P-glycoprotein-mediated transport of P-glycoprotein substrates, rhodamine 123 and sparfloxacin in rats Ando et al., 2001, Nadai et al., 2001.

There are many reports regarding the effects of endotoxin and endotoxin-induced inflammatory cytokines and mediators on brain permeability Boje, 1996, De Vries et al., 1996, Mayhan, 1998, Shukla et al., 1995. However, almost all the studies were carried out in vitro with brain endothelial cells and the in vivo studies used topical administration of endotoxin in animals. There is little in vivo information available regarding the effect of systemic administration of endotoxin on blood–brain barrier integrity and P-glycoprotein function.

The purpose of the present study was to investigate whether K. pneumoniae endotoxin changes the in vivo blood–brain barrier transport of doxorubicin and P-glycoprotein function. We measured the plasma and brain concentrations and brain-to-plasma concentration ratio of doxorubicin or of fluorescein isothiocyanate-labeled dextran and histopathological changes in brain capillaries. Furthermore, the effect of endotoxin on the constitutive level of P-glycoprotein in the brain was examined using Western blot analysis.