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Int J Mol Sci
2024 Aug 08;2516:. doi: 10.3390/ijms25168661.
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Effect of Synthetic Vitreous Fiber Exposure on TMEM16A Channels in a Xenopus laevis Oocyte Model.
Zangari M
,
Zabucchi G
,
Conti M
,
Lorenzon P
,
Borelli V
,
Constanti A
,
Dellisanti F
,
Leone S
,
Vaccari L
,
Bernareggi A
.
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Many years ago, asbestos fibers were banned and replaced by synthetic vitreous fibers because of their carcinogenicity. However, the toxicity of the latter fibers is still under debate, especially when it concerns the early fiber interactions with biological cell membranes. Here, we aimed to investigate the effects of a synthetic vitreous fiber named FAV173 on the Xenopus laevis oocyte membrane, the cell model we have already used to characterize the effect of crocidolite asbestos fiber exposure. Using an electrophysiological approach, we found that, similarly to crocidolite asbestos, FAV173 was able to stimulate a chloride outward current evoked by step membrane depolarizations, that was blocked by the potent and specific TMEM16A channel antagonist Ani9. Exposure to FAV173 fibers also altered the oocyte cell membrane microvilli morphology similarly to crocidolite fibers, most likely as a consequence of the TMEM16A protein interaction with actin. However, FAV173 only partially mimicked the crocidolite fibers effects, even at higher fiber suspension concentrations. As expected, the crocidolite fibers' effect was more similar to that induced by the co-treatment with (Fe3+ + H2O2), since the iron content of asbestos fibers is known to trigger reactive oxygen species (ROS) production. Taken together, our findings suggest that FAV173 may be less harmful that crocidolite but not ineffective in altering cell membrane properties.
decreto n. 2100/SPS dd. 2/11/20 Regione Friuli Venezia Giulia (Contributi alle Aziende Sanitarie per la realizzazione di progetti di ricerca sulle malattie correlabili all'amianto, decreto n. 2100/SPS dd. 2/11/20), Italian League for the Fight Against Cancer (Lega Italiana Lotta contro
Figure 1. (a) Example of SEM images of Croc and FAV173 fibers. (b) Vitelline membrane of non-treated (Ctrl), Croc-treated, and ground FAV173-treated oocytes. Note, in (b), a Croc fiber partially inserted into the pore of the vitelline membrane. Croc = 15 μg/mL, FAV173 = 200 μg/mL.
Figure 2. Dose–response effect of FAV173 fiber exposure (from 15 to 400 μg/mL) on resting potential (RP) in (a) and membrane resistance (Rm) in (b). In (a,b), the green bars show the effect of 15 μg/mL of Croc for comparison. The effect of FAV173 and Croc is expressed as % of decrease with respect to untreated cells (Ctrl) from the same donor. * p < 0.05, *** p < 0.001 fibers vs. Ctrl (unpaired t-test). In (c), the time course effect of Croc and FAV treatments on RP (n ≥ 3 donors). §§§ p < 0.001 FAV173 vs. Croc, ## p < 0.01 Croc (15–20) vs. Croc (5–10), (unpaired t-test). In (d), the effect on RP was irreversible only at higher fiber concentrations in Croc-treated cells (120 min, Croc: 45 μg/mL; FAV173: 600 μg/mL). *** p < 0.001 Croc vs. Ctrl (one-way ANOVA with Tukey’s post hoc).
Figure 3. In (a), examples of current traces recorded by stepping the membrane potential from −80 to +40 mV (10 mV steps, 3 s, Vh = −40 mV) in non-treated (Ctrl, black), Croc-treated (green), and FAV173-treated (red) cells. (b) I–V relationships from Ctrl, Croc-treated, and FAV173-treated cells (same batch). Note the outward rectification in treated cells. (c) Percentage increase in the evoked current amplitude measured at −80 mV and +40 mV and normalized to their respective Ctrl (of the same donor). *** p < 0.001, Fiber-treatments vs. Ctrl; §§ p < 0.001, Croc vs. FAV173 (unpaired t-test). Croc: 15 μg/mL; FAV173: 200 μg/mL.
Figure 4. (a, left) Examples of recording traces from Ctrl cells in normal bathing solution, in presence of [Ca2+]e = 11 mM (Ca11) and Ca11 + Ani9 (1 μM). (a, right) I–V relationships in Ctrl (normal bathing solution), Ca11, and Ca11 + Ani9 (1 μM) conditions. Note that, in Ca11, the currents at negative potentials increased with respect to those recorded in normal bathing solution, and the evident blocking effect of Ani9. (b, left) Evoked currents from FAV173-treated (200 μg/mL) cells in presence of Ca11 and Ca11 + Ani9 (1 μM). (b, right) The I–V relationships revealed a significant increase in the evoked currents at positive and negative potentials in presence of Ca11 and a partial blocking effect induced by Ani9. Ctrl: n = 5; FAV173: n = 7, same donor. * p < 0.05, *** p < 0.001, Fibers vs. Ctrl (unpaired t-test); § p < 0.05, §§ p < 0.01, §§§ p < 0.001, Ca11 vs. Ca11 + Ani9 (paired t-test).
Figure 5. Example of SEM images of microvilli in (a) Ctrloocyte, and oocytes in Ca11 bath condition in the absence and in the presence of FAV173 (200 μg/mL). In the inset of Ctrl, the vitelline membrane (VM) above the microvilli (MV) is shown. The FAV173 effect on the diameter (b) and density (c) of the microvilli (c), *** p < 0.001 vs. Ca11 (unpaired t-test). In (d) microvilli of Croc and (Fe3+ + H2O2)-treated oocytes. Comparison of microvilli diameter (e) and density (f) values measured under the 3 test conditions (* p < 0.05, *** p < 0.001 vs. Ctrl, § p < 0.05, §§§ p < 0.001 (Fe3+ + H2O2) vs. Croc, One-Way ANOVA with Tukey’s post hoc). Cells were treated for 60 min. (values are in the text). Scale bar 1 μm.
Figure 6. (a) Comparison of RP values in oocytes in the presence of CyTD alone (n = 5), or with FAV173 (n = 13) and Ani9 (n = 13), Croc (n = 12) and Ani9 (n = 15), (Fe3+ + H2O2) (n = 12), and Ani9 (n = 14). * p < 0.05, ** p < 0.01, *** p < 0.001 vs. CyTD, §§§ p < 0.001 CyTD + Croc vs. CyTD + Croc + Ani9 (ANOVA with Tukey’s post hoc). (b) Examples of SEM images of the plasmalemma from CyTD, CyTD + Croc, and (Fe3+ + H2O2) + CyTD-treated oocytes. In both cases, damage on the plasmalemma are visible (red asterisks). In (c), the presence of Ani9 fully recovered the membrane damage in the Croc-treated cells, while it is still visible in (Fe3+ + H2O2)-treated cells (white asterisk). Scale bar: 1 μm.
