title: Infrared spectroscopy detects changes in an amphibian cell line induced by fungicides: Comparison of single and mixture effects.
abstract: Amphibians are regarded as sensitive sentinels of environmental pollution due to their permeable skin and complex life cycle, which usually involves reproduction and development in the aquatic environment. Fungicides are widely applied agrochemicals and have been associated with developmental defects in amphibians; thus, it is important to determine chronic effects of environmentally-relevant concentrations of such contaminants in target cells. Infrared (IR) spectroscopy has been employed to signature the biological effects of environmental contaminants through extracting key features in IR spectra with chemometric methods. Herein, the Xenopus laevis (A6) cell line was exposed to low concentrations of carbendazim (a benzimidazole fungicide) or flusilazole (a triazole fungicide) either singly or as a binary mixture. Cells were then examined using attenuated total reflection Fourier-transform IR (ATR-FTIR) spectroscopy coupled with multivariate analysis. Results indicate significant changes in the IR spectra of cells induced by both agents at all concentrations following single exposures, primarily in regions associated with protein and phospholipids. Distinct differences were apparent in the IR spectra of cells exposed to carbendazim and those exposed to flusilazole, suggesting different mechanisms of action. Exposure to binary mixtures of carbendazim and flusilazole also induced significant spectral alterations, again in regions associated with phospholipids and proteins, but also in regions associated with DNA and carbohydrates. Overall these findings demonstrate that IR spectroscopy is a sensitive technique for examining the effects of environmentally-relevant levels of fungicides at the cellular level. The combination of IR spectroscopy with the A6 cell line could serve as a useful model to identify agents that might threaten amphibian health in a rapid and high throughput manner.
authors.affiliation: Lancaster Environment Centre, Lancaster University, Bailrigg, Lancaster LA1 4YQ, UK.
authors.forename: Rebecca J
authors.initials: RJ
authors.lastname: Strong
authors.affiliation: Lancaster Environment Centre, Lancaster University, Bailrigg, Lancaster LA1 4YQ, UK. Electronic address: c.halsall@lancaster.ac.uk.
authors.forename: Crispin J
authors.initials: CJ
authors.lastname: Halsall
authors.affiliation: Lancaster Environment Centre, Lancaster University, Bailrigg, Lancaster LA1 4YQ, UK.
authors.forename: Kevin C
authors.initials: KC
authors.lastname: Jones
authors.affiliation: Centre for Ecology and Hydrology, Lancaster University, Bailrigg, Lancaster LA1 4YQ, UK.
authors.forename: Richard F
authors.initials: RF
authors.lastname: Shore
authors.affiliation: Lancaster Environment Centre, Lancaster University, Bailrigg, Lancaster LA1 4YQ, UK; School of Pharmacy and Biomedical Sciences, University of Central Lancashire, Preston PR1 2HE, UK. Electronic address: f.martin@lancaster.ac.uk.
authors.forename: Francis L
authors.initials: FL
authors.lastname: Martin
mesh_terms: D000818:Animals; D001562:Benzimidazoles / Q000633:toxicity; D015415:Biomarkers / Q000378:metabolism; D002219:Carbamates / Q000633:toxicity; D018929:Cell Culture Techniques; D002460:Cell Line; D016000:Cluster Analysis; D004305:Dose-Response Relationship, Drug; D004784:Environmental Monitoring / Q000295:instrumentation / Q000379:methods*; D004847:Epithelial Cells / Q000187:drug effects* / Q000378:metabolism; D005659:Fungicides, Industrial / Q000633:toxicity*; D006801:Humans; D015999:Multivariate Analysis; D012821:Silanes / Q000633:toxicity; D017550:Spectroscopy, Fourier Transform Infrared; D014230:Triazoles / Q000633:toxicity; D014874:Water Pollutants, Chemical / Q000633:toxicity*; D029867:Xenopus Proteins / Q000378:metabolism; D014982:Xenopus laevis
chemical_list: D001562:Benzimidazoles; D015415:Biomarkers; D002219:Carbamates; D005659:Fungicides, Industrial; D012821:Silanes; D014230:Triazoles; D014874:Water Pollutants, Chemical; D029867:Xenopus Proteins; C061365:flusilazole; C006698:carbendazim
keywords: A6 cells; ATR-FTIR spectroscopy; Carbendazim; Flusilazole; Xenopus laevis