Bernareggi A , Conte G , Constanti A , Borelli V , Vita F , Zabucchi G .

	Figure 1. Time-dependency effect of crocidolite (Croc) on the electrical membrane properties of Xenopus oocytes. (A) I-V curve relationships recorded in 4 untreated (Ctrl, black square) and 4 Croc-treated (Croc: 15 μM/ml for 7, 20, 30 and 54 minutes respectively) oocytes. Vh = −40 mV, voltage steps: −100 mV to +40 mV, 10 mV intervals. (B) Averages of the RP and Rm values obtained in Ctrl condition and in cells incubated with Croc at different incubation intervals (5–30 min and more than 120 min). Note at 5–30 min interval there is a significant depolarization of the RP (Ctrl: n = 29; Croc: n = 37) and a decrease of Rm (Ctrl: n = 29; Croc: n = 37). Oocytes incubated for more than 120 min showed a partial but not significant recovery of the RP (n = 9), while the Rm was similar to Ctrl cells (n = 9). Mean ± SEM. *P < 0.05, ***P < 0.001, One-Way ANOVA (with Tukey’s post hoc).
	Figure 2. H2O2 mimics the effect induced by crocidolite. (A) Comparison of H2O2 released by oocytes before and after incubation with Croc (mean ± SD, **P < 0.01, t-test, values are in the text). (B) left, I-V relationships of Croc-treated cells (15 μg/ml, 5–30 min) without (Croc: n = 5) or in the presence of 250 U/ml CAT (Croc + CAT: n = 7). Vh = −40 mV, voltage steps: −100 mV to + 40 mV, 10 mV intervals. right, The RP and Rm values of the same oocytes. *P < 0.05, t-test, oocytes from same donor. (C) left, I-V relationships of Ctrl (n = 20), CAT- (250 U/ml, 5–30 min, n = 8), H2O2− (1 mM, 5–30 min, n = 12), and H2O2 + CAT-treated cells (1 mM H2O2, 250 U/ml CAT, 5–30 min, n = 7). Vh = −40 mV, voltage steps: −100 mV to + 40 mV, 10 mV intervals. right, Comparison of the RP and Rm values of same oocytes. Mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, One-Way Anova test (with Tukey’s post hoc). Oocytes from same donors.
	Figure 3. Time-dependency effect of H2O2 on the oocyte electrical membrane properties. (A) Example of currents induced by the voltage step protocol in an untreated oocyte (Ctrl) and in two oocytes treated with Croc (15 μg/ml) or H2O2 (1 mM). Vh = −40 mV, voltage steps: −100 mV to + 40 mV, 10 mV intervals. (B) I-V relationships of Ctrl cells (n = 5), H2O2 - treated cells (n = 5, time interval 5–30 min) and in Croc (n = 4, time interval 5–30 min). Oocytes were from the same donor. (C) Time course of H2O2 effect recorded after 5, 17, 23 and 125 min, respectively. (D) Effect of H2O2 on RP and Rm after 5–30 min of incubation (n = 5) and after more than 120 min (n = 3; Ctrl: n = 5). *P < 0.05, One-Way Anova test (Tukey’s post hoc). Oocytes from same donor.
	Figure 4. Effect of Fe2+ or Fe3+ ions on oocyte membrane properties. (A) left, I-V relationships of Ctrl cells and cells treated with Fe2+ (1 mM), Vh = −40 mV, voltage steps: −100 mV to +40 mV, 10 mV intervals. Vh = −40 mV, voltage steps: −100 mV to +40 mV, 10 mV intervals. right, The treatment did not change the RP and Rm (n = 6, ns, t-test). Both experiments were performed at pH 5. Oocytes from the same donor. (B) left, Example of recording traces of a Ctrl oocyte and an oocyte after incubation with Fe3+ (400 μM). right, I-V relationships of Ctrl cells (n = 8) and cells after a treatment with Fe3+ for 5–30 min (n = 13) or more than 120 min (n = 3). below, Comparison of RP and Rm values of the same cells (Ctrl: n = 13; Fe3+ more that 120 min: n = 3). Mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001, One-Way Anova test (Tukey’s post hoc). Oocytes from same donor.
	Figure 5. Combined effect of Fe3+ and H2O2 on oocyte membrane properties. (A) left, I-V relationships of Ctrl cell (n = 5) and a cell treated with Fe3+ (400 μM, n = 4, 5–30 min), H2O2 (1 mM, n = 5, 5–30 min) or Fe3+ + H2O2 (400 μM and 50 μM respectively, n = 3, 5–30 min). right, Comparison of I-V relationships obtained in same cells, after the treatment with Fe3+ (400 μM) and H2O2 (50 μM) up to 23 min. Vh = −40 mV, voltage steps: −100 mV to + 40 mV, 10 mV intervals. (B) The combined treatment significantly depolarized the RP (Ctrl: n = 5; Fe3+ + H2O2: n = 3) as well the Rm (Ctrl: n = 5; Fe3+ + H2O2: n = 3), both values were partially recovered after a prolonged treatment (n = 3). Mean ± SEM. *P < 0.05 One-Way Anova test (Tukey’s post hoc). Oocytes from same donor.
	Figure 6. Comparison of currents activated by a voltage-ramp protocol. Example of currents activated by a ramp voltage protocol (from −120 mV to +40 mV, 1 s, Vh = −40 V) in a Ctrl cell (A) and in oocytes treated with crocidolite (15 μg/ml, 5–30 min, n = 4, B) or Fe3+ + H2O2 (400 μM and 50 μM, 5–30 min, n = 5, C). The arrows indicate the mean intersection points with the Ctrl I-V (Croc: −20.64 ± 2.5 mV; Fe3+ + H2O2: −14.45 ± 4.76 mV).
	Figure 7. Ultrastructural appearance of the plasma membrane of Xenopus oocytes under SEM, taken in those regions that following processing, revealed fractures of the vitelline membrane and showed underneath the surface of the oocyte plasma membrane. (A) Shows the plasma membrane appearance of an untreated oocyte and (B) following the exposure to asbestos fibers (arrow). The surface appearance was completely changed when the oocyte was treated with cytochalasin D (CyTD, C). In (D) a CyTD-treated oocyte exposed to [Croc exposure (60 min); CyTD alone (60 min); CyTD + Croc co-incubation (60 min)].
	Figure 8. Reversible effect of crocidolite on the oocyte cell membrane was prevented by the presence of cytochalsin D (CyTD). (A) I-V curve relationships of oocytes treated with CyTD (CyTD: 5 μM, n = 8), Croc (Croc: 15 μg/ml, n = 41) or CyTD + Croc (CyTD + Croc, n = 36). Vh = −40 mV, voltage steps: −80 mV to + 40 mV, 10 mV intervals. (B) Comparison of RP and (C) Rm values of the same oocytes and those left in Croc after a 30 min of co-treatment with Croc and CyTD (Croc recovery from CyTD). (D) Co-treatment for more than 120 min killed 100% of the cells (CyTD 0%, Croc 10%, Croc + CyTD 32%). Mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, One-Way Anova test (with Tukey post hoc). Oocytes from same donors.
	Figure 9. Effect of Fe3+ or H2O2 on the oocyte cell membrane treated in the presence of cytochalsin D. (A) I-V curve relationships of oocytes treated with cytochalsin D (CyTD: 5 μM, n = 5), Fe3+ (400 μM, n = 5) or Fe3+ + CyTD (5–30 min, n = 8; more than 120 min, n = 4). Vh = −40 mV, voltage steps: −100 mV to + 40 mV, 10 mV intervals. (B) Comparison of RP and Rm values of the same oocytes. Mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001, One-Way Anova test (Tukey post hoc). Oocytes from same donor. (C) I-V curve relationships of oocytes treated with cytochalsin D (CyTD: 5 μM, n = 5), H2O2 (1 μM, n = 5), H2O2 (1 mM) + CyTD (5 μM, 5–30 min, n = 5). Vh = −40 mV, voltage steps: −100 mV to +40 mV, 10 mV intervals. (D) Comparison of RP and Rm values of the same oocytes. Note that long incubation with H2O2 + CyTD killed 100% of the cells. Mean ± SEM *P < 0.05, One-Way Anova test (Tukey post hoc). Oocytes from same donor.
	Figure 10. Crodidolite-mediated effect may involve Ca2+-activated Cl− channels. Effect of Mn2+ (5 mM) on a non-treated (A) and in two crocidolite-treated oocytes (oocytes form the same donor, B,C). Vh = −70 mV (voltage steps: −100 mV to +60 mV, 20 mV intervals). In (B) Mn2+ produces a substantial reduction of the Croc-induced outward currents (on average the currents at +20 mV decreased from 324.63 ± 96.63 nA to 189.22 ± 27.68 nA, mean ± SD, n = 4, *P < 0.05) whereas in (C), it has a minimal effect, suggesting that the effects of Croc may involve multiple conductance mechanisms in different cells.
	Figure 11. Schematic summary of the sequence of common mechanisms which either crocidolite or H2O2 + Fe3+ may share for inducing electrophysiological changes and cell damage in Xenopus oocytes: (1) Exposure to crocidolite fibers allows more H2O2 to become available, which in turn induces Fe3+ to be released from the fibers themselves (2). Free Fe3+ also derives from ferritin in the presence of H2O2 (3). Fe3+ can be reduced to Fe2+ by H2O2. Fe2+ in turn, triggers ROS production by reacting with H2O2 (4). The same reaction can be triggered by the exogenous addition of either H2O2, which can react with ferritin-released Fe3+, or Fe3+ that reacts with the small amount of H2O2 produced by resting Xenopus oocytes (5). As expected, iron chelators or catalase (CAT) prevent the changes induced by either crocidolite or H2O2 + Fe3+ (6). The main character for inducing membrane lesion is most likely OH°, as superoxide dismutase (SOD), which allows O2− dismutation, failed to inhibit these changes. The final membrane effect/lesion may be twofold:(7) a modification of the function of an endogenous chloride channel (possibly a calcium-activated chloride channel CACC) and (8) the formation of membrane “pores”, revealed morphologically when the cortical actin repair mechanism is inactivated by CyTD.

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