The Imidazolium Ionic Liquids Toxicity Is Due to Their Effect on the Plasma Membrane

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Abstract

Ionic liquids (ILs) are organic salts with a low melting point, which is due to the fact that alkyl side chains chemically bonded to the ion hinder the crystallization of ILs. The low melting point of ILs has led to their widespread use as relatively harmless solvents. However, ILs have toxic properties, the mechanism of which is largely unknown, so identifying the cellular targets of ILs is of practical importance. In our work, we showed that imidazolium ILs are not able to penetrate model membranes without damaging them. We also found that inactivation of multidrug resistance (MDR) pumps in yeast cells does not increase their sensitivity to imidazolium ILs. The latter indicates that the target of the toxicity of imidazolium ILs is not in the cytoplasm. Thus, our data suggest that the disruption of the plasma membrane barrier function is the main cause of the toxicity of low concentrations of imidazolium ILs. We also showed that supplementation with imidazolium ILs restores the growth of cells with kinetically blocked glycolysis. Our data indicate that IL-induced minor disruption of the plasma membrane may, in some cases, be beneficial for the cells.

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S. S. Sokolov

Lomonosov Moscow State University

Email: severin@belozersky.msu.ru
Russian Federation, Moscow

E. A. Smirnova

Lomonosov Moscow State University

Email: severin@belozersky.msu.ru
Russian Federation, Moscow

T. I. Rokitskaya

Lomonosov Moscow State University

Email: severin@belozersky.msu.ru
Russian Federation, Moscow

F. F. Severin

Lomonosov Moscow State University

Author for correspondence.
Email: severin@belozersky.msu.ru
Russian Federation, Moscow

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Supplementary files

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2. Fig. 1. Structural formulae of the most toxic imidazolium ILs: C12Mim-Cl (a); C14Mim-Cl (b) and C16Mim-Cl (c)

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3. Fig. 2. Recordings of the kinetics of the current through the BLM after applying a voltage of 50 mV at the initial time point before (light grey curves) and after the addition of 10 μM (black curves) and 20 μM (dark grey curves) C12Mim-Cl. BLMs were formed from diphytanyl-FC (a) or diphytanoyl-FC (b)

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4. Fig. 3. Measurement of the ability of imidazolium ILs to transfer protons across the liposomal membrane in a model system of liposomes loaded with the fluorescent probe pyranine. a - Kinetics of pH change within liposomes in control (curve 1, control), in the presence of 0.1 μM CCF (curve 2), in the presence of 2 μM P66614 (curve 3) and 20 μM P66614 (curve 4). b - Kinetics of pH change within liposomes in control (curve 1), in the presence of 20 μM C12Mim-Cl (curve 2), 2 μM (curve 3) or 20 μM (curve 4) C14Mim-Cl

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5. Fig. 4. ILs increase Pdr5-GFP content. a - Comparison of Pdr5-GFP increase 1 h after addition of ILs and the standard Pdr5-GFP inducer clotrimazole by flow cytometry. The X-axis is plotted as the logarithmic scale of substance concentration in μM; the Y-axis is the logarithmic scale of Pdr5-GFP fluorescence in cells relative to cells without added substances (value 0). Each point is an independent biological repeat. b - Light and fluorescence microscopy of cells containing Pdr5-GFP 1 h after addition of C12Mim-Cl and the standard Pdr5-GFP inducer clotrimazole. Size cut-off is 5 μm

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6. Fig. 5. MDU proteins do not affect the growth rate of S. cerevisiae in the presence of different concentrations of ILs. Typical cell culture growth curves of S. cerevisiae wild-type W303 and delta strain pdr1pdr3 cells with impaired MLU activation. X-axis is time in hours; Y-axis is OD at 550 nm of cell culture

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7. Fig. 6. Comparison of S. cerevisiae cell survival after exposure to imidazolium ILs, C12Mim-Cl, C14Mim-Cl and C16Mim-Cl, by CFU number (blue curves) and the proportion of PI-negative cells (red curves). X-axis shows PI concentration in μM; Y-axis shows survival relative to control without addition of substances, in per cent

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8. Fig. 7. ILs are able to restore growth of delta tps1 cells in the presence of glucose. The tested concentrations of ILs that do not induce stimulation are shown in grey. Concentrations corresponding to the restoration of delta tps1 strain growth on YP-ethanol medium in the presence of glucose are shown in green colour

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