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First published online October 21, 2005
Journal of Experimental Biology 208, 4109-4122 (2005)
Published by The Company of Biologists 2005
doi: 10.1242/jeb.01867

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Mitochondrial depolarization following hydrogen sulfide exposure in erythrocytes from a sulfide-tolerant marine invertebrate

David Julian^1,2,*, Kelly L. April¹, Shiven Patel¹, Jenny R. Stein¹ and Stephanie E. Wohlgemuth¹

¹ Department of Zoology, University of Florida, Gainesville, FL 32611-8525, USA
² Mount Desert Island Biological Laboratory, Bar Harbor, ME 24533, USA

View larger version (108K): [in a new window]   Fig. 1. Light micrographs and fluorescence micrographs of G. dibranchiata erythrocytes labeled with JC-1 (A-F) and TMRM (G-L) and exposed to control conditions (A,D,G,J), 1.9 mmol l-1 sulfide (B,E,H,K), or 0.10 mmol l-1 CCCP (C,F,I,L). Erythrocytes labeled with JC-1 were imaged in real color using a triple-bandpass filter set, as described in the text. Erythrocytes labeled with TMRM were counterstained with the nuclear dye Hoechst 33342, after which each dye was imaged in monochrome and pseudocolored for this figure. Scale bars, 10 µm.

View larger version (22K): [in a new window]   Fig. 2. Emission spectra of TMRM (A) and JC-1 (B) in sulfide-free incubation buffer (black, lower line) or in incubation buffer with 1.9 mmol l-1 sulfide (red, upper line). Note that the green fluorescence of the monomeric form of JC-1 did not contribute significantly to JC-1 emission spectra in this preparation.

View larger version (22K): [in a new window]   Fig. 3. Fluorescence of JC-1 (A,B) and TMRM (C,D) in G. dibranchiata erythrocytes exposed to sulfide (A,C; 0.11-1.9 mmol l-1) or CCCP (B,D; 0.10 mmol l-1) for 1 h. JC-1 fluorescence ratio and TMRM fluorescence emission at each sulfide concentration are normalized to that of control erythrocytes (erythrocytes incubated in sulfide-free buffer for 1 h). Values are means ± S.D. (N=8 for JC-1, N=4 for TMRM). CCCP (0.10 mmol l-1) was added as a positive control for loss of m. The regression line represents a sigmoidal dose-response curve (see equation in text) with correlation coefficient, significance and EC50 listed in each panel. Asterisks represent a significant effect of sulfide or CCCP compared to the control value (no toxin) by RM ANOVA (*P<0.05, **P<0.01, ***P<0.005).

View larger version (18K): [in a new window]   Fig. 4. Time course of TMRM fluorescence from G. dibranchiata erythrocytes exposed to control conditions (open symbols) or to 1.9 mmol l-1 sulfide (closed symbols) for 1 h. Fluorescence emission (595 nm) at each time point is normalized to the initial value (t=0) for each treatment. Values are means ± S.D. (N=5). Asterisks represent a significant effect over time compared to the initial value by RM ANOVA (*P<0.05).

View larger version (22K): [in a new window]   Fig. 5. TMRM fluorescence in G. dibranchiata erythrocytes exposed to sulfide (0.3, 0.5, 0.8 and 1.2 mmol l-1) for 1 h followed by recovery in sulfide-free buffer for 2 or 5 h. Fluorescence emission (595 nm) at each time point is normalized to the emission of control erythrocytes (sulfide-free buffer) at the same time point. Values are means ± S.D. (N=3). Statistical analyses were performed by two-factor, RM ANOVA followed by Fisher's LSD post-hoc test (note that this is less conservative than the Tukey HSD test). Asterisks adjacent to data points indicate a significant difference from the control value (no sulfide) at the same time point (*P<0.05, **P<0.01, ***P<0.005); daggers indicate a significant main effect of sulfide at that concentration (P<0.05, P<0.01, P<0.005). Note that there was no significant effect of time (P=0.83) and no significant interaction between sulfide and time (P=0.88).

View larger version (24K): [in a new window]   Fig. 6. Fluorescence of TMRM in G. dibranchiata erythrocytes exposed to the mitochondrial electron transport chain inhibitors rotenone (10 and 100 µmol l-1, N=10), antimycin (1, 10 and 100 µmol l-1, N=10, 10 and 4, respectively), azide (1 and 10 mmol l-1, N=10), and cyanide (0.1, 1 and 10 mmol l-1, N=4). CCCP (0.10 mmol l-1, N=10) was added as a positive control for loss of m. Fluorescence emission (595 nm) for each inhibitor is normalized to control conditions (buffer only). Values are means ± S.D. Asterisks represent a significant effect of toxin or CCCP compared to the control value (no toxin) by RM ANOVA (*P<0.05, ***P<0.005).

View larger version (14K): [in a new window]   Fig. 7. O2 consumption of G. dibranchiata erythrocytes under control conditions or exposed to the mitochondrial COX inhibitors sodium azide (1 mmol l-1) and sodium cyanide (1 mmol l-1). Values are means ± S.D. (N=5 for CN, N=3 for azide). O2 consumption is presented relative to the average at control conditions (i.e. consumption rate immediately prior to addition of inhibitor). Asterisks represent a significant decrease in consumption rate to the control value (no toxin) by one-tailed, paired t-test (**P<0.01, ***P<0.005).

View larger version (20K): [in a new window]   Fig. 8. TMRM fluorescence in G. dibranchiata coelomocytes exposed for 1 h to various sulfide concentrations (up to 0.73 mmol l-1) either alone (circles) or with CCCP (0.10 mmol l-1; squares) or PTP inhibitors (0.5 µmmol l-1 CsA and 5 µmol l-1 TFP; triangles). Fluorescence emission (595 nm) is normalized to control conditions (1 h exposure to buffer alone). Values are means ± S.D. (N=5). Asterisks represent a significant effect of sulfide, CCCP or CsA/TFP compared to the control value by two-factor, RM ANOVA (*P<0.05, **P<0.01, ***P<0.005). Refer to text for further results of statistical analyses.

View larger version (13K): [in a new window]   Fig. 9. Enhanced production of reactive oxygen species in G. dibranchiata erythrocytes exposed to sulfide (up to 1.9 mmol l-1) for 1 h. (A) Formation of fluorescent 2',7'-dichlorofluorescein (DCF) from nonfluorescent 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) in erythrocytes (closed circles) or incubation buffer alone (open circles). DCF fluorescence is normalized to DCF formation in erythrocytes under control conditions (no sulfide). (B) Oxidation of MitoSOXTM Red in erythrocytes. Fluorescence is normalized to control conditions (no sulfide). Values are means ± S.D. (N=4). Asterisks represent a significant effect of sulfide compared to the control value (no sulfide) by RM ANOVA (***P<0.005).

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