Exposure to amitraz, fipronil and permethrin affects cell viability and ABC transporter gene expression in an Ixodes ricinus cell line

Carlo Mangia,Claudio Bandi,Marco Genchi,Domenico Otranto,Giulio Grandi,Benedetta Passeri,Alice Vismarra,Laura Kramer,Sara Epis,Lesley Bell-Sakyi

doi:10.1186/s13071-018-3020-4

Abstract

BackgroundOver-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, as development of an in vitro model for the study of acaricide resistance would contribute to improving knowledge of the molecular basis behind drug processing and exclusion in ticks. In the present study, cultures of the Ixodes ricinus-derived cell line IRE/CTVM19 were treated with the acaricides amitraz, permethrin or fipronil to determine modulation of ABC transporter gene expression. Cells were treated with different drug concentrations (25, 50, 100, 150 μM) and incubated for ten days. Cell morphology, viability, metabolic activity and relative expression of ABC (B1, B6, B8 and B10) genes were determined at day 10 post-treatment.ResultsCell morphology determined by light microscopy was altered following treatment with all drugs, but only at high concentrations, while total cell numbers decreased with increasing drug dose. Cell viability 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 caused decrease (up to 37%, P < 0.01) in viability. At all drug concentrations, fipronil and permethrin induced dose-dependent reduction in cell metabolic activity measured by MTT assay (P < 0.01). Quantitative RT-PCR showed that the drugs significantly affected expression of ABC genes. In particular, fipronil treatment downregulated ABCB1 (P < 0.001) and upregulated ABCB6, ABCB8 and ABCB10 (P < 0.01); amitraz treatment down regulated ABCB1 (significant difference between 25 and 150 μM, P < 0.001) and upregulated ABCB8 and ABCB10 at lower concentrations (25 and 50 μM, P < 0.05); and permethrin upregulated ABCB6, ABCB8 and ABCB10 only at 150 μM (P < 0.01).ConclusionsThe adverse effects on cell viability and metabolic activity, and changes in expression of different ABC transporter genes, detected in IRE/CTVM19 cells following treatment with amitraz, permethrin and fipronil, support the proposed application of tick cell lines as in vitro models for the study of resistance to these acaricides in ticks.

Highlights

Over-expression of ATP-binding cassette (ABC) transporter proteins has been implicated in resistance of ticks to acaricides
In order to further elucidate the role of ATP-binding cassette transporter (ABCT) in the response of tick cells to a variety of acaricides, the present study evaluated cell viability and metabolic activity and ABC gene expression (ABCB1, ABCB6, ABCB8, ABCB10) in an I. ricinus cell line following treatment with amitraz, fipronil or permethrin
IRE/CTVM19 viability and metabolic activity All acaricides, when used at high concentrations (100– 150 μM), resulted in decreased cell adherence and density, and increase in cell size and vacuolation (Fig. 1), while there was no difference in morphology of live cells between the lowest drug concentration and the untreated and the dimethyl sulfoxide (DMSO)-treated control for all drugs

Summary

Introduction

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, as development of an in vitro model for the study of acaricide resistance would contribute to improving knowledge of the molecular basis behind drug processing and exclusion in ticks. Cultures of the Ixodes ricinus-derived cell line IRE/CTVM19 were treated with the acaricides amitraz, permethrin or fipronil to determine modulation of ABC transporter gene expression. Ticks are among the most important vectors of pathogens affecting livestock, companion animals and humans worldwide [1]. Tick cell lines have been recently investigated for their potential in studying acaricide resistance and, to elucidate the molecular mechanism(s) underlying the lack of efficacy of acaricides [9,10,11]

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