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First published online November 28, 2008
Journal of Experimental Biology 211, 3816-3825 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.021303
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Hypotaurine and sulfhydryl-containing antioxidants reduce H2S toxicity in erythrocytes from a marine invertebrate

J. A. Ortega, J. M. Ortega and D. Julian*

University of Florida, P.O. Box 118525, Department of Zoology, Gainesville, FL 32611, USA


Figure 1
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  		Fig. 1. Comparison of total dissolved sulfide concentration following 2 h exposure to 0–3.2% H2S in cell-free incubation buffer alone (A–F, blue symbols, N=15, with the same data set shown in each plot) or in cell-free incubation buffer with added hypotaurine (HT) or antioxidants (A–F, red symbols). (A) 50 mmol l–1 HT (hypotaurine), (N=3–9). (B) 3 mmol l–1 GEE (glutathione ethyl ester) (N=6). (C) 1.0 mmol l–1 NAC (N-acetylcysteine) (N=6). (D) 0.10 mmol l–1 ASC (L-ascorbic acid) (N=6). (E) 3.0 mmol l–1 Tempol (4-hydroxyl-2,2,6,6-tetramethylpiperidine-1-oxyl) (N=6). (F) 0.10 mmol l–1 Trolox (6-hydoxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) (N=6). Data are means ± s.e.m. (G) Comparison of total dissolved sulfide concentration following 2 h exposure to 0–3.2% H2S in cell-free incubation buffer alone (0 HT) and with 0.50, 5.0 and 50 mmol l–1 HT. For each H2S percentage, the sulfide concentration is expressed relative to the concentration in the absence of HT. Data are means ± 1 standard deviation. For all plots, asterisks represent significant differences from the dissolved sulfide concentration in incubation buffer alone at the same H2S percentage.

 

Figure 2
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  		Fig. 2. Viability of G. dibranchiata erythrocytes exposed to H2S in vitro. (A–E) Epifluorescence micrographs of cells exposed to control conditions (A) or H2S at 0.10% (B), 0.32% (C), 1.0% (D) and 3.2% (E) for 2 h, followed by labeling with calcein (green) and propidium iodide (PI; red). Scale bar=100 µm. (F) Quantitative analysis of calcein and PI labeling following H2S exposure. `Calcein' (green circles) represents cells with cytoplasmic calcein labeling; `NPI' (red triangles) represents cells with nuclear PI labeling; `Total PI' (red circles) represents cells with nuclear or cytoplasmic PI labeling. Asterisks indicate a significant difference from control conditions by one-way analysis of variance (ANOVA) with Dunnett's post-hoc test, N=3.

 

Figure 3
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  		Fig. 3. Effects of hypotaurine and thiotaurine on the viability of G. dibranchiata erythrocytes exposed to H2S in vitro. (A–D) Epifluorescence micrographs of cells in incubation buffer that were exposed for 2 h to air (A) and 0.32% H2S (B), and cells in incubation buffer with hypotaurine (50 mmol l–1) or thiotaurine (5.0 mmol l–1) that were exposed for 2 h to air (C) or 0.32% H2S (D), followed by labeling with calcein (green) and propidium iodide (PI; red). (E–F) Quantitative analysis of calcein labeling (E) and total PI labeling (F) in cells incubated with various concentrations of hypotaurine (0–50 mmol l–1) or thiotaurine (0–5 mmol l–1) and exposed for 2 h to 0–3.2% H2S. N=5. Results of statistical analysis presented in Table 1.

 

Figure 4
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  		Fig. 4. Effects of antioxidants on the viability of G. dibranchiata erythrocytes exposed to H2S in vitro. (A–D) Epifluorescence micrographs of cells in incubation buffer that were exposed for 2 h to air (A) and 0.32% H2S (B), and cells in incubation buffer with GEE (glutathione ethyl ester) (3 mmol l–1), NAC (N-acetylcysteine) (1.0 mmol l–1), ASC (L-ascorbic acid) (0.10 mmol l–1), Tempol (4-hydroxyl-2,2,6,6-tetramethylpiperidine-1-oxyl) (3.0 mmol l–1) or Trolox (6-hydoxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) (0.10 mmol l–1) that were then exposed for 2 h to air (C) or 0.32% H2S (D), followed by labeling with calcein (green) and propidium iodide (PI; red). (E–F) Quantitative analysis of calcein labeling (E) and total PI labeling (F) in cells incubated with various concentrations of antioxidant (0–3.0 mmol l–1 GEE, 0–1 mmol l–1 NAC, 0–0.1 mmol l–1 ASC, 0–3.0 mmol l–1 Tempol or 0–0.1 mmol l–1 Trolox) and exposed for 2 h to 0–3.2% H2S. N=5. Results of statistical analysis presented in Table 1.

 

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