Elsevier

Biochemical Pharmacology

Volume 83, Issue 2, 15 January 2012, Pages 269-278
Biochemical Pharmacology

Ivermectin induces P-glycoprotein expression and function through mRNA stabilization in murine hepatocyte cell line

https://doi.org/10.1016/j.bcp.2011.10.010Get rights and content

Abstract

Ivermectin is widely used in human and veterinary medicine for the control of helminth infections. Ivermectin is known to interact with P-glycoprotein (P-gp/MDR1), being a good substrate and a potent inhibitor, however, the influence of ivermectin on the expression of the transporter has not been investigated. Expression of P-glycoprotein was investigated in cultured mouse hepatocytes acutely exposed to ivermectin. The two P-glycoprotein murine isoforms, Mdr1a and Mdr1b, mRNA levels were assessed by real-time RT-PCR. Ivermectin induced a clear time- and concentration-dependent up-regulation of Mdr1a and Mdr1b mRNA levels (as early as a 12-h exposure and up to 2.5-fold at 10 μM). Moreover, ivermectin-treated cells displayed enhanced cellular efflux of the P-glycoprotein substrate calcein that was inhibited by the P-glycoprotein blocker valspodar, providing evidence that the ivermectin-induced P-glycoprotein was functional. The mechanisms underlying these effects were investigated. Ivermectin-mediated Mdr1 mRNA induction was independent of the two nuclear receptors CAR and PXR, which are known to be involved in drug transporters regulation. Moreover, by using reporter cell lines that detects specific ligand-activated transcription factors, we showed that ivermectin did not displayed CAR, PXR or AhR ligand activities. However, studies with actinomycin D revealed that the half-life of Mdr1a and Mdr1b mRNA were significantly prolonged by two-fold in ivermectin-treated cells suggesting a post-transcriptional mode of ivermectin regulation. This study demonstrates for the first time that ivermectin induces P-glycoprotein overexpression through post-transcriptional mRNA stabilization, thus offering insight into the mechanism of reduced therapeutic efficacy and development of ivermectin-resistant parasites.

Introduction

Macrocyclic lactones (MLs), such as ivermectin, are a large family of broad spectrum antiparasitic drugs intensively used worldwide in livestock to treat diseases caused by gastrointestinal nematodes and external parasites. In humans, ivermectin is used through mass drug administration programs for the treatment of onchocerciasis, a tropical parasitic disease caused by the filarial nematode Onchocerca volvulus. MLs resistance has become a worsening problem in veterinary medicine and recent reports suggest that human ivermectin-resistant parasites are emerging, thus threatening the sustainable efficacy of this drug [1].

P-glycoprotein (P-gp, MDR1/ABCB1) is a plasma membrane protein belonging to the ATP-binding cassette (ABC) transporters family. This transporter is an ATP-dependent efflux pump that reduces the intracellular concentration of a broad range of hydrophobic compounds and is involved in the multidrug resistance (MDR) [2]. The activity of efflux ABC transporters can be subject to regulation by inhibition or induction and considerable evidence has accumulated to indicate that MDR gene expression can be rapidly and transiently induced following acute exposure to some xenobiotics [3], [4]. The regulation of ABC transporters occurs by transcriptional or post-transcriptional events. At transcriptional level, the constitutive androstane receptor (CAR, NR1I3) and the pregnane X receptor (PXR, SXR, NR1I2), two nuclear receptors primarily expressed in the liver, are now both widely recognized to play the most important role in the regulation of MDR1 [5], [6], [7]. Other specific ligand-activated transcription factors have been implicated in regulation of drug transporters expression such as aryl-hydrocarbon receptor (AhR) [8]. Apart from transcriptional regulation, overexpression of P-gp has also been shown to be controlled partially through post-transcriptional mechanisms, such as mRNA stability alteration [9], [10].

P-gp has been clearly identified as the main factor that controls the concentration of ivermectin by affecting its absorption, distribution, and elimination in the host [11], [12], but also its neurotoxicity by limiting its penetration in the brain [13]. Despite the key role of P-gp in metabolism, toxicity, efficacy and drug–drug interactions of many other xenobiotics [14], the question of whether ivermectin can affect the expression levels of P-gp has not been addressed. Several arguments from the literature imply that ivermectin may modulate P-gp expression. (i) Many P-gp function modulators are often found to influence the expression of the transporter in cells [15], and ivermectin is reported as good substrate and potent inhibitor of P-gp [16], [17]. (ii) Ivermectin was previously shown to induce the expression and activity of cytochrome P450 isoenzymes, including CYP1A, 2B and 3A subfamilies in vivo [18], [19], suggesting that ivermectin could interact with cellular transcription factors such as AhR, CAR and PXR which are involved in chemical induction of these cytochromes [20]. Since several studies have shown that the biotransformation systems (phase I/II) and efflux proteins are regulated by the same network of transcription factors, one can speculate that ivermectin may also regulate ABC efflux transporters expression. (iii) P-gp homologs expression levels were shown to be increased in ivermectin-resistant nematodes [21], [22], [23].

In this context, this study aimed at investigating P-gp regulation in response to acute ivermectin treatment. Hepatocytes were chosen as a suitable in vitro model system for studying the potential of xenobiotics to modulate P-gp gene expression [24] and elucidating the role of specific transcription factors in the regulation of gene involved in the metabolism of xenobiotics [25], [26]. The objectives were to evaluate the ability of ivermectin to modulate P-gp gene expression, to determine the functional significance of this modulation and to further establish molecular mechanisms underlying this effect. Our finding demonstrate for the first time that acute exposure to ivermectin led to overexpression of functional P-gp in mouse liver cells through increased stability of mRNA in the cell.

Section snippets

Materials

1,4-Bis[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP), pregnenolone 16α-carbonitrile (PCN), dimethylsulfoxide (DMSO), T0901317, geneticin (G418), Triton X-100, calcein-AM, actinomycin D (Act-D) and ivermectin were purchased from Sigma–Aldrich Chimie (St Quentin Fallavier, France). Luciferin was from Promega (Charbonnières-les-bains, France). Valspodar (VP) was a generous gift from Novartis (Basel, Switzerland). 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD, dioxin) was purchased from Promochem

Effects of ivermectin on cell viability

The aim of these studies was to determine subtoxic concentrations of ivermectin which could be used in the subsequent studies on hepatocytes JWZ cells. The cytotoxicity of ivermectin towards JWZ cells was examined using the MTS assay and LDH release. The dose-dependent viability of JWZ cells treated with ivermectin for 24, 48 or 72 h is presented in Fig. 1. These results showed that ivermectin had cytotoxic effects in a concentration- and time-dependent manner. A 24-h treatment with ivermectin

Discussion

Despite the well documented interactions of ivermectin, a commonly used anthelmintic, with P-gp, there is no data on a possible modulation of the expression of this transporter by ivermectin. We have used murine cultured hepatocytes to study the effects of short-term exposure to ivermectin on P-gp expression and function.

In this study, ivermectin was found to up-regulate the two mouse P-gp isoforms Mdr1a and Mdr1b gene expression in both a time- and dose-dependent manner, demonstrating the

Acknowledgments

We gratefully acknowledge Abdel Boulahtouf and Virginie Bellet for reporter cell lines studies. We also thank Arnaud Polizzi (Integrative Toxicology and Metabolism, INRA UMR1331) for helpful advices in qPCR studies.

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