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Reading Report

Reading Report. Reporter：Yong Jiang Guiding Teacher：Yong-liang Zhang Professor.

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Reading Report

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  1. Reading Report Reporter：Yong Jiang Guiding Teacher：Yong-liang Zhang Professor

  2. Oxidative stress,DNA damageandantioxidant enzyme geneexpre-ssion in the Pacificwhiteshrimp,Litopenaeus vannamei whenex-posed to acutepHstress Comparative Biochemistry and Physiology, Part C（2009）

  3. Content • Background • Introduction • Materials and methods • Results • Discussions

  4. Background Fluctuations in pH, caused by acid rain, acidic effluent and red tides,can affect the growth and survival ofcultured shrimp (Boyd,1990;WangandWang,1995;Wang et al., 2002; Zhanget al., 2007).The physiologicaland biochemical responses of shrimp have been studiedextensively toelucidate both responses and adaptations to pH change (Wang et al.2002); there is, however, little information about the effects of pHchanges on the induction of oxidative stress in the Pacific white shrimp(Zhou et al., 2008) or its defense responses to such changes.

  5. In recent years, there has been considerable interest in theinduction of acute injuries to shrimp as a resultof exposure toenvironmental stress. However, the exact molecular mechanismof pHstress-induced injury remains largely unknown, along with how totreat such injury in vivo. In view of this situation，the author takes out a study to assess theeffects of acidic (pH 5.6) and alkaline (pH 9.3) exposure (compared topH 7.4; control) on ROS production and DNA damage in shrimp, and toinvestigate any changes occurring in the gene expression ofantioxidant enzymes (cMnSOD, CAT, GPx and TRx)

  6. in the hepatopancreasof the shrimp, L. vannamei.The author also investigated the intracellularCa2+ con-centration ([Ca2+]i), the percentage of dead anddama-gedhaemocytes in shrimp when exposed to pHstress.

  7. Introduction An organism is subjected to chemical (i.e. environ-mental pHchange), physical or biological stress, experiencesabnormal oxidativereactions in their aerobic metabolic pathways, resulting in oxidativestress (Ranby and Rabek, 1978). For example, reactive oxygen species(ROS) are produced as a result of awide range of environmental stimuli(e.g. physical parameters and xenobiotics) (Morel and Barouki, 1999).ROS are continuously generatedby an aerobic metabolism and can damage important biomolecules,such as DNA, proteins, and lipids (Halliwell and Gutteridge, 1999).

  8. ROS are commonly used as messenger molecules in normal cellfunctions. However, at increased concen-trations they can disruptnormal physiological pathways and cause cell death; such a switch islargely mediated through Ca2+ signaling (Ermak and Davies, 2002).To counteract ROS-induced DNA damage, cells have evolveddefense mechanisms that act at different levels to prevent or repairsuch damage (Hegde et al., 2008). Antioxidant enzyme systems are awell-developed regulatory mechanism protecting against oxidativestress.

  9. Materials and methods 1、Animal collection and maintenance Shrimp (L. vannamei), 6.86±0.39 cm long and weighing 3±0.53 g,were collected from a local shrimp farm in Panyu (Guangdong, China)and reared in 20 m3 cycling-filtered fiberglass tanks in which thesalinity (10‰) and temperature (20–22 °C) were maintained at thesame levels as in standard shrimp culture ponds. Prior to experimentaluse, animals were acclimated to the laboratory conditions for 1 weekand fed twice daily with commercial shrimp feed until 24 h before theexperimental treatments began.

  10. 2、pH experiment In order to assess the effects of pH on the shrimp and their oxidative stress profiles,18 plastic aquaria (50×35×30 cm, water volume 52 L)containing 10‰ saltwater (pH 7.4) were prepared, and approximately40 shrimpwere placed in each aquarium. The pHwas then raised to 9.3in six aquaria by the gradual addition of 1 mol/L each of Na2CO3 andNaHCO3, and reduced to 5.6 in another set of six aquaria by adding 1mol/L HCl.

  11. 3、Haemolymph samples The haemolymph of 24 shrimp from each experimental group (pH5.6, 7.4, and 9.3) was collected 0, 3, 6, 12 and 24 h after the start of theexperiment.Haemolymph was drawn directly from the heart of theanimals using sterile syringes with an anticoagulant (ModifiedAlsever's) (Söderhäll and Smith, 1983). Haemolymph from eachshrimp was transferred into an individual eppendorf tube held on ice.Samples of haemolymph were pooled (×4) from six individuals.Pooled haemolymph samples were filtered through an 80 mm meshtoeliminate aggregates and large pieces of debris.

  12. 4、Assays of respiratory burst, intracellular [Ca2+]i and levels of deadand damaged haemocytes 4.1 Respiratory burst by phagocytes To monitor the level of respiratory burst, the author used the cellpermeantprobe 2',7'-dichlorofluorescein diacetate (DCFH-DA)method, as described by Bass et al. (1983) and Lambert et al.(2003). A volume of 2 mL DCFH-DA was added to 200mL haemocytesamples from each of the three pooled samples. After incubation withDCFH-DA for 30 min in the dark, the mixture was diluted with 200mLofmodified Alserver's solution (MAS) to obtain a final concen-trationof 1×106 cells/mL; it was then analyzed by flow cytometry

  13. 4.2Measurement of intracellular [Ca2+]i Intracellular calciumconcentrationwasmonitored using the calciumsensitive dye Fluo-3.Cell suspensions from each of thethree sets of pooled samples were stained with Fluo-3 (finalconcen-trationof 10 mM) and incubated for 30 min in a 37 °C wa-ter bath in thedark. The cells were then centrifuged for 5 min at 700×g, washed andresus-pended in phosphate buffer solution (PBS), and transferred to flowcytometry tubes (1–2×106 cells/mL). The control cells were incubatedunder identical conditions, except that Fluo-3 was absent (Bailey andMacardle, 2006; Chang et al., 2009).

  14. 4.3 Percentage of dead and damaged haemocytes Cell viability was monitored using 7-AAD (7-amino-actinomycin D,Contents: 2.0 mL in PBS and FBS wash buffer, stored at 4 °C) (Oyamaet al.,1996;Mehta and Shaha, 2006).First, cells were washed twice with cold PBS andthen the cells were resuspended in 1× binding buffer (10 mM Hepes/NaOH (pH 7.4), 140 mM NaCl, 2.5 mM CaCl2, stored at 4 °C) at aconcentration of 1–2×106 cells/mL. One hundredmicroliters (1–2×105cells) of the solutionwas transferred to a 5 mL culture tube and 5 mL of7-AAD was added.The solution was gently vortexed and incubated for15 min at room temperature (25 °C) in the dark. Then 400 mL of 1×binding buffer was added to each tube and analyzed by flowcytometrywithin 1 h (Dieye et al., 2000; Colonna-Romano et al., 2004).

  15. From the abovementioned experimental sample, 10,000 cells were analyzed for the FL1 or FL3fluore-scent signals (Hegaret etal.,2003).The percentages of cells and mean fluorescence valueswerecalculated(Chang et al., 2009).

  16. Fig. 1. Changes in respiratory burst activity (A), [Ca2+]i (B) and percentage of dead anddamaged haemocytes (C) in the Pacific white shrimp at different ambient pH levels.Each bar represents the mean±SD (standard deviation), n=4. Significant differences(Pb0.05) between control (pH 7.4) and stressed specimens are indicated with asterisks.

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