Haemocyte apoptosis of the tiger shrimp Penaeus monodon exposed to cadmium.

This study investigated the effect of ambient Cadmium (Cd) on haemocyte apoptosis of the shrimp, Penaeus monodon. Cellular response was determined in Cd-exposed (0, 0.05, 0.5 and 5 mg L(-1)) shrimp. Results showed that 0.05 mg L(-1) Cd(2+) had no significant effect on the haemocyte parameters during the 48 h exposure. Cadmium at doses of 0.5 and 5 mg L(-1) depressed the total haemocyte count (THC), and increased reactive oxygen species (ROS) production and apoptosis ratio in haemocytes. Esterase activity increased in shrimp exposed to 0.5 mg L(-1) Cd(2+) for 6 h, and decreased to the initial level later. Depressed esterase activity could be observed in shrimp after 24 and 48 h exposure to 5 mg L(-1) Cd(2+). These results demonstrated that Cd(2+) modified esterase activity and induced ROS generation, which led to haemocyte apoptosis and THC reduction. Oxidative stress is one of the induction mechanisms for Cd-caused apoptosis of shrimp haemocytes.

Measurement of intracellular nitric oxide (NO) production in shrimp haemocytes by flow cytometry.

A flow cytometric method to measure the production of intracellular nitric oxide (NO) was adapted for use with shrimp haemocytes. We applied fluorescent probe 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM DA) for NO detection in haemocytes from the tiger shrimp Penaeus monodon, and used flow cytometry to quantify fluorescence intensity in individual haemocyte. The optimized protocol for intracellular NO analysis consists to incubate haemocytes with DAF-FM DA at 10 µM for 60 min to determine the mean fluorescence intensity. Result showed that NO was also produced in the untreated shrimp haemocytes. NO level in granular cells and semigranular cells were much higher than that in hyaline cells. Defined by different characteristic of NO content, three subsets of haemocytes were observed. Zymosan A at dose of 10 or 100 particles per haemocyte triggered higher DAF-FM fluorescence intensity in granular and semigranular cells, than PMA that had no significant impact on all three cell types. These results indicate that granular and semigranular cells are the primary cells for NO generation. Cytochalasin B significantly inhibited the NO level induced by zymosan A. NG-Monomethyl-L-arginine (L-NMMA) and diphenylene iodonium chloride (DPI) significantly suppressed the DAF-FM fluorescence in haemocytes, but apocynin could not modulate it, indicating that the DAF-FM fluorescence was closely related to the activity of NO-synthase pathway. The NO donor sodium nitroprusside (SNP) improved the DAF-FM fluorescence in haemocytes, while the NO scavenger C-PTIO (2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide) significantly decreased the fluorescence, demonstrating that the fluorescence intensity of DAF-FM is mainly dependent on the intracellular NO level.

In vitro toxicity of nitrite on haemocytes of the tiger shrimp, Penaeus monodon, using flow cytometric analysis.

This study investigated the in vitro effects of nitrite on reactive oxygen species (ROS) production, NO production, esterase activity and cell apoptosis of Penaeus monodon haemocytes. Haemocytes were in vitro exposed to different dose of nitrite (0, 0.1, 0.5, 1, 5 and 10 µM). Cellular responses of nitrite-treated haemocytes were determined by flow cytometry. The results revealed that haemocytes treated by nitrite in vitro showed conspicuous time- and dose-dependent decreases in ROS and NO production as well as esterase activity. Additionally, 0.1 and 0.5 µM nitrite did not affect the apoptotic cell ratio during the 3h experimental time, while significant increases in apoptotic cells were observed after haemocyte exposure to nitrite at 1 µM for 3h, and at 5 or 10 µM for 1h. These results indicated that nitrite suppresses cellular functions, including production of ROS and NO, and activity of esterase. Cell apoptosis of haemocytes would be induced by extracellular nitrite as doses exceed 1 µM.