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

BACKGROUND: 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 i...

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Autores principales: Mangia, Carlo, Vismarra, Alice, Genchi, Marco, Epis, Sara, Bandi, Claudio, Grandi, Giulio, Bell-Sakyi, Lesley, Otranto, Domenico, Passeri, Benedetta, Kramer, Laura
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6069974/
https://www.ncbi.nlm.nih.gov/pubmed/30064465
http://dx.doi.org/10.1186/s13071-018-3020-4
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author Mangia, Carlo
Vismarra, Alice
Genchi, Marco
Epis, Sara
Bandi, Claudio
Grandi, Giulio
Bell-Sakyi, Lesley
Otranto, Domenico
Passeri, Benedetta
Kramer, Laura
author_facet Mangia, Carlo
Vismarra, Alice
Genchi, Marco
Epis, Sara
Bandi, Claudio
Grandi, Giulio
Bell-Sakyi, Lesley
Otranto, Domenico
Passeri, Benedetta
Kramer, Laura
author_sort Mangia, Carlo
collection PubMed
description BACKGROUND: 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. 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. RESULTS: Cell 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). CONCLUSIONS: The 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.
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spelling pubmed-60699742018-08-06 Exposure to amitraz, fipronil and permethrin affects cell viability and ABC transporter gene expression in an Ixodes ricinus cell line Mangia, Carlo Vismarra, Alice Genchi, Marco Epis, Sara Bandi, Claudio Grandi, Giulio Bell-Sakyi, Lesley Otranto, Domenico Passeri, Benedetta Kramer, Laura Parasit Vectors Research BACKGROUND: 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. 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. RESULTS: Cell 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). CONCLUSIONS: The 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. BioMed Central 2018-07-31 /pmc/articles/PMC6069974/ /pubmed/30064465 http://dx.doi.org/10.1186/s13071-018-3020-4 Text en © The Author(s). 2018 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Research
Mangia, Carlo
Vismarra, Alice
Genchi, Marco
Epis, Sara
Bandi, Claudio
Grandi, Giulio
Bell-Sakyi, Lesley
Otranto, Domenico
Passeri, Benedetta
Kramer, Laura
Exposure to amitraz, fipronil and permethrin affects cell viability and ABC transporter gene expression in an Ixodes ricinus cell line
title Exposure to amitraz, fipronil and permethrin affects cell viability and ABC transporter gene expression in an Ixodes ricinus cell line
title_full Exposure to amitraz, fipronil and permethrin affects cell viability and ABC transporter gene expression in an Ixodes ricinus cell line
title_fullStr Exposure to amitraz, fipronil and permethrin affects cell viability and ABC transporter gene expression in an Ixodes ricinus cell line
title_full_unstemmed Exposure to amitraz, fipronil and permethrin affects cell viability and ABC transporter gene expression in an Ixodes ricinus cell line
title_short Exposure to amitraz, fipronil and permethrin affects cell viability and ABC transporter gene expression in an Ixodes ricinus cell line
title_sort exposure to amitraz, fipronil and permethrin affects cell viability and abc transporter gene expression in an ixodes ricinus cell line
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6069974/
https://www.ncbi.nlm.nih.gov/pubmed/30064465
http://dx.doi.org/10.1186/s13071-018-3020-4
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