Multiple cytochrome P-450 subfamilies are co-induced with P-glycoprotein by both phenothiazine and 2-acetylaminofluorene in rats
Introduction
The P-glycoprotein (P-gp), originally identified in multidrug-resistant cell membranes, has been demonstrated to be a transmembrane pump for a number of xenobiotics, including several commonly used chemotherapeutic agents [1], [2]. The localization of P-gp at the bile canalicular surface of hepatocytes suggests that it has a physiological role in the excretion of xenobiotics into bile and plays a major role in the elimination of some drugs from the body [3], [4]. Since many substrates or modulators of P-gp also affect CYP3A, the P-gp and CYP3A gene families are considered to be regulated coordinately [5]. In addition, a corresponding increase in expressions of P-gp and CYP3A mRNAs by several CYP3A inducers has been shown in cultured cells [6].
Since the CYP family consists of multiple structurally and functionally distinct isoenzymes [7], an inducer of P-gp might affect some CYP isoenzymes in addition to CYP3A. Little information is available on the influence of inducers of P-gp on the expression of CYP subfamily members. For example, 2-acetylaminofluorene (2-AAF) and phenothiazine (PTZ) are known to induce P-gp and CYP1A2 mRNAs [8], but their influence on CYP3A expression has not been studied. If induction of P-gp and CYP3A are coordinated, the greatest induction by 2-AAF and PTZ might occur in CYP3A. We therefore, designed a study to examine how 2-AAF and PTZ affect CYP subfamily expression in the rat liver.
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Chemicals
2-AAF, PTZ, ethoxyresorufin, pentoxyresorufin, resorufin, aniline, p-aminophenol, testosterone, 16α-hydroxytestosterone, 6β-hydroxytestosterone, progesterone and deoxycorticosterone were purchased from Sigma Chemical Co. (St. Louis, MO). Anti-rat CYP1A, CYP2B, CYP2C6, CYP2C11, CYP2E1 and CYP3A2 antibodies were purchased from Daiichi Pure Chemicals (Tokyo, Japan). Anti-human MDR1 IgG was from Oncogene Science (Uniondale, NY). All other chemicals were obtained from Wako Chemicals Co. (Osaka,
Results
After the 4-day administration, the total CYP content in the rat liver microsomes was higher in the PTZ administration group than in the control group, whereas neither 2-AAF nor PTZ affected the body or liver weights (Table 1).
Fig. 1 shows the results of Western blot analysis of P-gp and CYP subfamilies. The relative intensity of the bands of P-gp and each CYP isoenzyme is shown in Fig. 2, Fig. 3, respectively. P-gp protein levels increased in the 2-AAF and PTZ groups. Although CYP2C6, CYP2C11
Discussion
We have described the effect of 2-AAF and PTZ, inducers of P-gp, on the activities and expressions of CYP isoenzymes in hepatic microsomes. The P-gp protein levels expressed in the livers of the 2-AAF and PTZ groups were significantly greater than the level in the control group. The total CYP content was significantly increased after the 4-day treatment of PTZ, compared to that in the control group. In order to demonstrate how the 2-AAF and PTZ treatments affect these isoenzymes individually,
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