Multiple cytochrome P-450 subfamilies are co-induced with P-glycoprotein by both phenothiazine and 2-acetylaminofluorene in rats

doi:10.1016/S0304-3835(98)00374-7

Cancer Letters

Volume 138, Issues 1–2, 26 April 1999, Pages 73-79

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Abstract

We studied the effects of two P-glycoprotein (P-gp) inducers, 2-acetylaminofluorene (2-AAF) and phenothiazine (PTZ), administered intraperitoneally, on the activities and content of hepatic cytochrome P-450 (CYP) subfamilies in hepatic microsomes of Sprague–Dawley rats. After 4-day administration of 2-AAF or PTZ, the P-gp content was increased. The total CYP content after PTZ treatment was significantly increased compared with that of controls. The CYP1A, CYP2B and CYP3A2 contents were induced, while the CYP2C6, CYP2C11 and CYP2E1 contents remained unaffected. A marked increase in CYP1A1 was found after administration of each compound. Ethoxyresorufin O-deethylase, pentoxyresorufin O-deethylase, and testosterone 6β hydroxylation activities showed a significant increase after both 2-AAF and PTZ treatments. In particular, ethoxyresorufin O-deethylase exhibited more than ten times greater activity than that of the controls after the treatments. These results suggest that P-gp inducers affect several CYP subfamilies in addition to CYP3A, which is reported to be up-regulated coordinately with P-gp by a CYP3A inducer.

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.

Section snippets

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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Multiple cytochrome P-450 subfamilies are co-induced with P-glycoprotein by both phenothiazine and 2-acetylaminofluorene in rats

Abstract

Introduction

Section snippets

Chemicals

Results

Discussion

J. Hepatol.

J. Biol. Chem.

J. Biol. Chem.

Cancer Lett.

Regul. Toxicol. Pharm.

The clinical relevance of multidrug resistance

Cancer Invest.

Function and regulation of the human multidrug resistance gene

Adv. Cancer Res.

Absence of the mdrla P-Glycoprotein in mice affects tissue distribution and pharmacokinetics of dexamethasone, digoxin, and cyclosporin A

J. Clin. Invest.

Overlapping substrate specificities and tissue distribution of cytochrome P4503A and P-glycoprotein: implications for drug delivery and activity in cancer chemotherapy

Mol. Carcinogen.

Modulators and substrates of P-glycoprotein and cytochrome P4503A coordinately up-regulate these proteins in human colon carcinoma cells

Mol. Pharmacol.