Evaluation of ABCB gene expression in an Ixodes ricinus cell line exposed to acaricides

Abstract

Over-expression of ATP-binding cassette (ABC) transporter proteins has been implicated in resistance of ticks to acaricides. Tick cell lines are useful for investigating resistance mechanisms, and development of an in vitro model for the study of acaricide resistance would greatly facilitate screening for drug resistance in ticks. In the present study, cultures of the Ixodes ricinus-derived cell line IRE/CTVM19 were treated with the acaricides ivermectin amitraz, permethrin or fipronil to determine cytopathic effects of treatment and modulation of ABC transporter gene expression. In experiment 1 IRE/CTVM19 cells were treated with different concentrations of ivermectin (0, 11, 22 or 33 μM) and incubated for 10 days. Evaluation of viability and relative expression of ABCB1, ABCB6, ABCB8 and ABCB10 genes were carried out at day 10 post treatment. Cell viability ranged between 84% and 92% with no significant differences between untreated and treated cells. Analysis of qRT-PCR results showed that ABC pump expression was not significantly modulated by ivermectin treatment. Expression of the ABCB8 gene revealed a biphasic trend, based on the ivermectin concentration. ABCB6 and ABCB10 gene expression was not modulated by ivermectin treatment and ABCB1 expression was not detected. In experiment 2, cells were treated with different drug concentrations of ivermectin amitraz, permethrin or fipronil and incubated for 10 days. Cytology, trypan blue exclusion, MTT assay, and relative expression of ABC (ABCB1, ABCB6, ABCB8 and ABCB10) genes were determined at day 10 post treatment. Cell morphology was altered following treatment with all drugs, but only at high concentrations. Cell viability, as determined by trypan blue exclusion, was not significantly different from untreated controls (p>0.1) following treatment with amitraz and permethrin, but high concentrations of fipronil and ivermectin caused a significant decrease (63% for fipronil and 58% for ivermectin, p<0.01). Fipronil and permethrin induced significant (p<0.01), dose-dependent reduction on MTT assay at all drug concentrations. Quantitative RT-PCRs showed that the drugs significantly affected expression of ABC genes. Fipronil treatment led to down-regulation of ABCB1 (p<0.001) and up-regulation of ABCB6, ABCB8 and ABCB10 (p<0.01). Amitraz treatment resulted in down-regulation of ABCB1 (significant difference between 25 μM and 150 μM, p<0.001) and up-regulation of ABCB10 at lower concentrations (50 µM, p<0.05). Permethrin led to up-regulation of ABCB6, ABCB8 and ABCB10 only at 150 μM (p<0.01). Ivermectin treatment led to a down-regulation of all the genes under investigation, above all at higher concentrations. The up- and down-regulation of the expression of different ABC transporter genes detected in IRE/CTVM19 cells, following treatment with amitraz, fipronil, permethrin, and ivermectin supports the proposed application of tick cell lines as in vitro models for the study of resistance to these acaricides in ticks.