Romano G et al. (2011), Nitric oxide mediates the stress response induc...

ECB-ART-42217

PLoS One 2011 Jan 01;610:e25980. doi: 10.1371/journal.pone.0025980.

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Nitric oxide mediates the stress response induced by diatom aldehydes in the sea urchin Paracentrotus lividus.

Romano G , Costantini M , Buttino I , Ianora A , Palumbo A .

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Diatoms are ubiquitous and abundant primary producers that have been traditionally considered as a beneficial food source for grazers and for the transfer of carbon through marine food webs. However, many diatom species produce polyunsaturated aldehydes that disrupt development in the offspring of grazers that feed on these unicellular algae. Here we provide evidence that production of the physiological messenger nitric oxide increases after treatment with the polyunsaturated aldehyde decadienal in embryos of the sea urchin Paracentrotus lividus. At high decadienal concentrations, nitric oxide mediates initial apoptotic events leading to loss of mitochondrial functionality through the generation of peroxynitrite. At low decadienal concentrations, nitric oxide contributes to the activation of hsp70 gene expression thereby protecting embryos against the toxic effects of this aldehyde. When nitric oxide levels were lowered by inhibiting nitric oxide synthase activity, the expression of hsp70 in swimming blastula decreased and the proportion of abnormal plutei increased. However, in later pluteus stages nitric oxide was no longer able to exert this protective function: hsp70 and nitric oxide synthase expression decreased with a consequent increase in the expression of caspase-8. Our findings that nitric oxide production increases rapidly in response to a toxic exogenous stimulus opens new perspectives on the possible role of this gas as an important messenger to environmental stress in sea urchins and for understanding the cellular mechanisms underlying toxicity during diatom blooms.

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Genes referenced: LOC100887844 LOC100893907 LOC105439191 LOC115929188

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	Figure 1. Detection of endogenous NO levels in sea urchin fertilized eggs.NO was revealed by the NO–specific indicator DAF-FM-DA. (A) Control eggs, (B,C and D) eggs treated for 10 min with 1, 2.5 and 5 µg/ml DD, respectively, and then fertilized. The images were acquired 20 min post-fertilization. (E) Relative fluorescence after 20 min post-fertilization for the same samples reported in the upper panel. White bars indicate relative fluorescence inside the eggs. Gray bars indicate fluorescence in the perivitelline space and fertilization membrane. Values are reported as mean ± S.D. , p<0.05; p<0.001 with respect to the control. Statistical significance of 5 µg/ml: * p<0.001 compared with 2.5 µg/ml. Control: N = 5; DD 1 µg/ml: N = 4; DD 2.5 µg/ml: N = 4; DD 5 µg/ml: N = 6.
	Figure 2. Mitocondrial functionality of developing sea urchin embryos.(A,C and E) Active mitochondria revealed by the mitochondrial-specific fluorescent dye Mitotracker after 50 min post-fertilization. (B,D and F) Corresponding bright field images. (A) Control embryos. (C) Embryos incubated with 5 µg/ml DD; (E) embryos incubated with 5 µg/ml DD in the presence of the peroxynitrite scavenger MnTBAP (10 µM).
	Figure 3. Effect of DD on the appearance of blebbing.Sea urchin embryos were treated with DD at different concentrations in the absence or presence of 800 µM c-PTIO as described in materials and method section. Left panel: embryos were monitored for blebbing appearance at 30, 60, 100, 130, 160 and 270 min after fertilization. Control (circle); DD 5 µg/ml (diamond), 2.5 µg/ml (square), 1 µg/ml (triangle), 0.5 µg/ml (star). Dashed lines with empty shapes indicate data obtained in the presence of c-PTIO. Right panel: Control and embryos treated with DD 1 µg/ml in the absence or presence of 800 µM c-PTIO observed at 270 min after fertilization. Values are reported as mean ± S.D.
	Figure 4. NO involvement in DD-induced teratogenesis.Embryo development was monitored after 48 hours post fertilization (hpf). (A) Control eggs and eggs treated with TRIM at 20, 50 and 100 µM. (B, C) Eggs treated with DD at 0.1 µg/ml and 0.25 µg/ml, respectively in the absence and presence of TRIM at different concentrations. *** p<0.001 compared to the corresponding DD concentration. Blue: normal plutei. Pink: abnormal plutei.
	Figure 5. Gene expression levels using Real Time qPCR.(A) hsp70, NOS and caspase-8 gene expression levels were followed by Real Time qPCR. Samples incubated with DD were collected at 5, 9, 24 and 48 hpf. (B) hsp70 in samples incubated with DD in the absence and presence of TRIM. 5T, 9T, 24T: samples treated with TRIM and collected at 5, 9 and 24 hpf, respectively. 5T+D, 9T+D, 24T+D: samples treated with TRIM and DD and collected at different times. (C) hsp70 in samples incubated with increasing concentrations of sper/NO. Spermine was used as a control. 5INE, 9INE, 24INE: samples treated with spermine at different times; 5NO5, 9NO5, 24NO5: samples treated with sper/NO 5 µM; 5NO10, 9NO10, 24NO10: samples treated with sper/NO 10 µM; 5NO20, 9NO20, 24NO20: samples treated with sper/NO 20 µM; 5NO40, 9NO40, 24NO40: samples treated with sper/NO 40 µM. Data in histograms are expressed as a fold difference from control and are reported as mean ± S.D. Fold differences greater than ±2 (see dotted horizontal guidelines at values of 2 and −2 on the histograms) were considered significant.

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