Nitric oxide voltammetric measurements in the rat brain after gamma irradiation

The effects of a lethal gamma irradiation were investigated on cerebral NO-ergic system by using a voltammetric method in freely moving rats. It is reported that the cortical NO concentration increases right from the end of the radiation exposure (15 Gy) and reaches a maximal magnitude (+120%) 24 h later. A dose-effect relationship from 2 to 15 Gy for gamma-ray exposure has also been observed. The effects, obtained with either an NO synthase inhibitor nonselective for the different NO synthase isoforms or an NO synthase inhibitor selective for the constitutive isoform, suggest that the radiation-induced increase in NO is likely to be dependent on the inducible NO synthase isoform. Moreover, experiments performed under ex vivo conditions showed that the cortical mRNA level for Ca(++)-independent NO synthase, the brain NOS activity, and urinary nitrites/nitrates increased significantly 24 h after gamma-ray exposure. These results demonstrate that a supralethal whole-body irradiation alters the NO-ergic pathways. The increase in NO obtained under such conditions might constitute a good index of central nervous system radiosensitivity during the acute phase of the radiation syndrome.     

Stress reduces hemolymph ecdysteroid levels in the crab: mediation by the eyestalks

In decapod crustaceans, molt hormone (ecdysone) production by Y-organs is suppressed by an eyestalk neurosecretory product, molt-inhibiting hormone (MIH). Environmental stressors are known to delay or prevent molting in crabs. The present study assesses the function of the MIH-Y-organ neuroendocrine system in the crab Cancer antennarius under conditions of daily handling stress. After three days, stressed crabs showed significant suppression of hemolymph ecdysteroid levels, which continued to fall to 20% of controls by day 14. Ecdysteroid titers of stressed crabs returned to prestress levels seven days after stress termination. Ecdysteroid levels in de-eyestalked (DES) crabs rose 160% within 48 hr post-DES. Stressing DES crabs over 16 subsequent days did not significantly alter ecdysteroid levels compared with unstressed DES controls. Handling stress thus depresses hemolymph ecdysteroid levels in the crab, a response that is mediated by eyestalks and appears to result from stress-induced MIH release.     

Clearance of Vibrio campbellii injected into the hemolymph of Callinectes sapidus, the Atlantic blue crab: the effects of prior exposure to bacteria and environmental hypoxia

The Atlantic blue crab, Callinectes sapidus (Rathbun), lives in a bacteria-rich environment that experiences daily fluctuations in water quality. In the present study, we tested the hypothesis that crustaceans with prior or ongoing exposure to bacteria in their hemolymph have an increased susceptibility to subsequent infections, and that acute exposure to low dissolved oxygen (hypoxia) and elevated carbon dioxide levels (hypercapnia) may further confound the ability of blue crabs to counter a subsequent infection. Adult male blue crabs held in well-aerated (normoxic; P O2=20.7 kPA; CO(2)<0.06 kPa; pH 7.8-8.0) or hypercapnic hypoxic (HH; P O2=4 kPa; CO(2)=1.8 kPa; pH 6.9-7.2) seawater received an injection (pre-challenge dose) of 1 x 10(5)Vibrio campbellii g(-1) crab. Control animals were injected with an equivalent dose of HEPES-buffered saline (1 microl g(-1) crab). At 2h or 24h after the pre-challenge injection, both Vibrio and saline-pre-challenged animals were injected with a dose of live V. campbellii (1 x 10(5)g(-1) crab). This second injection will be referred to as a second injection or challenge injection. Degradation in or physical removal of intact bacteria from hemolymph was quantified using real-time PCR; bacteriostasis was quantified as the percentage of intact bacteria that could not be recovered by selective plating. We demonstrated that bacteriostasis occurs in the hemolymph of blue crabs. Furthermore, blue crabs that received a challenge injection 2h after a pre-challenge dose of V. campbellii cleared culturable bacteria from their hemolymph more rapidly when compared to animals that received a pre-challenge dose of saline. This enhanced clearance of culturable bacteria was associated with an increase in antibacterial activity in the cell-free hemolymph. However, the enhanced clearance of culturable bacteria disappeared when the time interval between the pre-challenge and challenge dose was extended to 24h and when crabs were held in HH seawater throughout the experiment. Neither the time interval between the pre-challenge and the challenge dose nor exposure to HH altered the pattern of intact bacterial clearance in blue crabs. These results demonstrate that prior exposure to bacteria does not increase the susceptibility of C. sapidus to a second, sublethal dose of V. campbellii. In fact, a recent exposure to V. campbellii enhances the ability of blue crabs to render bacteria non-culturable and the immune mechanisms/effectors responsible for this are short lived and appear to be sensitive to low dissolved oxygen and high carbon dioxide concentrations in the environment.     

Effect of microcystin on ion regulation and antioxidant system in gills of the estuarine crab Chasmagnathus granulatus (Decapoda,Grapsidae)

The objective of this work was to evaluate mechanisms of microcystin toxicity on crustacean species. Adult male crabs of Chasmagnathus granulatus (13.97+/-0.35 g) acclimated to low salinity (2 per thousand ) were injected with saline (control) or Microcystis aeruginosa aqueous extract (39.2 microg/l) at 24 h intervals for 48 h. After the exposure period, the anterior and posterior gills were dissected, measuring Na(+),K(+)-ATPase and glutathione-S-transferase (GST) activity. Total oxyradical scavenging capacity (TOSC) and lipid peroxides (LPO) content were also determined. Na(+),K(+)-ATPase activity in anterior gills was significantly lower in crabs injected with toxin than in control crabs, while no significant difference in the enzyme activity was detected in posterior gills. Both sodium and chloride concentration in the hemolymph were not affected by toxin exposure. Significant changes in GST activity were detected in posterior gills, with higher values being observed in the toxin-injected crabs. Crabs exposed to microcystin also showed a significant increase in the TOSC value against peroxyl radicals, for both anterior and posterior gills. Lipid peroxides level did not change in both gill types after exposure to the toxin. The increased levels of TOSC suggest the occurrence of a crab response against oxidative stress induced by toxin injection, which prevents lipid peroxidation.     

Physiological responses to emersion in the intertidal green crab,Carcinus maenas (L.)

The green shore crab, Carcinus maenas, undergoes on average 6?h periods of emersion during each low-tide cycle during the summer months. Under those conditions, the crab is cut off from its normal water environment and is exposed to potential stress from a suite of environmental and physiological changes: dehydration, compromised gas exchange and resultant internal hypoxia and hypercapnia, thermal stress, and ammonia toxicity. This study examined the comprehensive responses of the green crab in water and to a 6?h emersion period laboratory simulation of a tidal cycle followed by a 1?h re-immersion period, measuring indicators of dehydration, hemolymph osmolality, oxygen uptake, hemolymph acid–base status, heart and ventilatory rate, and hemolymph ammonia and ammonia excretion. Green crabs showed physiological responses of varying magnitude to 6?h of emersion. Individuals were found in the field exclusively under rocks and large clumps of seaweed where temperatures were approximately half those of exposed surfaces and relative humidity was about twice as high as ambient air. During emersion, crabs lost less than 5% of their wet weight, and hemolymph osmolality did not increase significantly. Oxygen uptake continued in air at about 50% of the control, aquatic values; and the gills continued to be ventilated by the scaphognathite, albeit at lower rates. Hemolymph lactate concentrations increased, indicating a shift to a greater reliance on anaerobic metabolism to support energetic needs. A slight acidosis developed in the hemolymph after 1?h of emersion, but it did not increase thereafter. Ammonia concentrations in the hemolymph were unchanged, as ammonia was volatilized by the gills and excreted into the air as NH₃ gas. These results show that the green crab copes with emersion by seeking refuge in microhabitats that mitigate the changes in the physical parameters of intertidal emersion. Physiologically, desiccation is avoided, cardio-respiratory processes are maintained at reduced levels, and hemolymph acid–base balance is minimally affected. Ammonia toxicity appears to be avoided by a shift to excreting NH₃ gas directly or indirectly to air.     

Developmental changes in hemolymph ecdysteroid level and prothoracicotropic hormone activity during the fifth larval instar of the Eri silkworm, Samia cynthia ricini

Developmental changes in hemolymph ecdysteroid level, ecdysteroid synthesis by prothoracic glands （PGs） in vitro, prothoracicotropic hormone （PTTH） activity in brain extracts, and PTTH activity in the hemolymph were measured during the fifth larval instar of the Eri silkworm, Samia cynthia ricini. The changing patterns of hemolymph ecdysteroid level and ecdysteroid synthesis by laGs in vitro are similar to each other, with maximums on day 9. However, on this day, hemolymph ecdysteroid level was substantially higher than ecdysteroid synthesis by PGs in vitro suggesting a high PTTH activity in the hemolymph on day 9. Moreover, the changing pattern of PTTH activity in brain extracts is also similar to that of PTTH activity in the hemolymph, both peaking on day 9. However, on this day, activity in brain extracts was much smaller than PTTH activity in the hemolymph implying that most PTTH synthesized by the brain is secreted to the hemolymph and the brain stores a very little amount of PTTH. This study provides unique insights onto the hormonal regulation of ecdysteroid synthesis in the Eri silkworm and is useful for our future studies on signal transduction of insect neurolaelatides.     

Nitric Oxide Production in Striatum and Pallidum of Cirrhotic Rats

Ammonium and manganese are neurotoxic agents related to brain metabolic disturbances observed after prolonged liver damage. The aim of this study was to assess the production of nitric oxide (NO) in the brain of cirrhotic rats exposed to manganese. We induced cirrhosis by bile duct ligation for 4 weeks in rats. From brain, striatum and globus pallidus were dissected out, and NO synthase activity and the content of nitrites plus nitrates (NOx) were determined. In pallidum we found a diminished constitutive NO synthase activity from cirrhotic rats, independently of manganese exposure. This result was confirmed by low levels of NOx in the same brain area (P<0.05, two-way ANOVA). This finding was not related to protein expression of NO synthase since no differences were observed in immunoblot signals between cirrhotic and sham-operated animals. Results from present study suggest that the production of NO is reduced in basal ganglia during cirrhosis.     

A differential proteomic approach to assess the effects of chemotherapeutics and production management strategy on giant tiger shrimp Penaeus monodon

The intensification of shrimp farming has been related to the increasing use of chemotherapeutics and potentially suboptimal rearing conditions. For the purpose of assessing the stress level of cultured giant tiger shrimp Penaeus monodon, a proteomic analysis (2D-DIGE) was performed on hemolymph. On the one hand, shrimp were exposed for 7 days to the antibiotics enrofloxacin or furazolidone via feed (4 g kg^?1) under laboratory conditions. On the other hand, shrimp were submitted to enrofloxacin directly in field conditions in Vietnam, for which two different culture systems were distinguished (intensive and improved extensive). No significant different protein abundance pattern was induced by antibiotics under laboratory conditions, while only one protein spot displayed a 1.53-fold reduction in intensity after exposure to enrofloxacin in improved extensive ponds. When we compared the proteome of shrimp bred either in intensive or in improved extensive system, we observed 9 protein spots displaying significant difference in abundance. Among them, 3 spots of hemocyanin were under-expressed in shrimp from improved extensive ponds. At the opposite 2 spots corresponding to Sarcoplasmic Calcium-binding Protein (SCP) were less abundant in hemolymph of shrimp from intensive ponds. These results demonstrate that the very subtle effects of tested antibiotics on patterns of hemolymph protein expression are overwhelmed by the effects of conditions encountered in different production management systems, such as different oxygen and nitric concentrations.     

Osmotic and ionic regulation in shore crabsCarcinus maenas inhabiting a tidal estuary

Shore crabsCarcinus maenas were exposed to salinities fluctuating according to the natural tidal rhythm. To this end they were maintained in net cages positioned in the estuarine waters of the river Elbe. The cages were lifted every hour, and between 8–12 specimens were analyzed for hemolymph concentrations of Na, K, Ca, Mg, and osmolality. The results obtained were compared with the respective data measured in external brackish water. In addition, the specific activity of Na−K-ATPase in a posterior gill was determined. Hemolymph Na and Mg as well as branchial Na−K-ATPase were also determined in crabs collected in the North Sea and the Baltic. The results show that inC. maenas living in salinities fluctuating with the tides by approx. 15‰ S, Na, K and Ca were hyperregulated, and Mg was effectively hyporegulated. The concentrations of all hemolymph ions and the activity of the Na−K-ATPase were kept constant over the whole tidal cycle. In Baltic crabs, Na was effectively hyperregulated and gill Na−K-ATPase was significantly elevated by a factor of ca 2 when compared with North Sea crabs. It is suggested that long-term hyperregulation of Na in constant salinities results from an increased number of Na−K-ATPase molecules which may change by synthesis or degradation following salinity stress. Constant hemolymph levels of hyperregulated Na in crabs inhabiting fluctuating brackish water are accomplished by activation of existing Na−K-ATPase by low Na and inhibition by higher ambient concentrations. This work is part of the first author's doctoral thesis submitted to the Department of Biology at the University of Hamburg.     

Copper effects on key metabolic enzymes and mitochondrial membrane potential in gills of the estuarine crab Neohelice granulata at different salinities

The estuarine crab Neohelice granulata was exposed (96h) to a sublethal copper concentration under two different physiological conditions (hyperosmoregulating crabs: 2ppt salinity, 1mg Cu/L; isosmotic crabs: 30ppt salinity, 5mg Cu/L). After exposure, gills (anterior and posterior) were dissected and activities of enzymes involved in glycolysis (hexokinase, phosphofructokinase, pyruvate kinase, lactate dehydrogenase), Krebs cycle (citrate synthase), and mitochondrial electron transport chain (cytochrome c oxidase) were analyzed. Membrane potential of mitochondria isolated from anterior and posterior gill cells was also evaluated. In anterior gills of crabs acclimated to 2ppt salinity, copper exposure inhibited hexokinase, phosphofructokinase, pyruvate kinase, and citrate synthase activity, increased lactate dehydrogenase activity, and reduced the mitochondrial membrane potential. In posterior gills, copper inhibited hexokinase and pyruvate kinase activity, and increased citrate synthase activity. In anterior gills of crabs acclimated to 30ppt salinity, copper exposure inhibited phosphofructokinase and citrate synthase activity, and increased hexokinase activity. In posterior gills, copper inhibited phosphofructokinase and pyruvate kinase activity, and increased hexokinase and lactate dehydrogenase activity. Copper did not affect cytochrome c oxidase activity in either anterior or posterior gills of crabs acclimated to 2 and 30ppt salinity. These findings indicate that exposure to a sublethal copper concentration affects the activity of enzymes involved in glycolysis and Krebs cycle, especially in anterior (respiratory) gills of hyperosmoregulating crabs. Changes observed indicate a switch from aerobic to anaerobic metabolism, characterizing a situation of functional hypoxia. In this case, reduced mitochondrial membrane potential would suggest a decrease in ATP production. Although gills of isosmotic crabs were also affected by copper exposure, changes observed suggest no impact in the overall tissue ATP production. Also, findings suggest that copper exposure would stimulate the pentose phosphate pathway to support the antioxidant system requirements. Although N. granulata is very tolerant to copper, acute exposure to this metal can disrupt the energy balance by affecting biochemical systems involved in carbohydrate metabolism.     

Hyper-osmoregulatory capacity of the Chinese mitten crab (Eriocheir sinensis) exposed to cadmium; acclimation during chronic exposure

The aim of this study was to evaluate the effects of waterborne cadmium on hyper-osmoregulatory capacity of the Chinese mitten crab Eriocheir sinensis acclimated to freshwater. For this purpose, crabs were submitted to acute (0.5 mg Cd L(-1) for 1, 2 or 3 days), chronic (10 or 50 microg Cd L(-1) for 30 days) or chronic, immediately followed by acute, exposure. While no effect was observed after 1 or 2 days, hemolymph osmolality, Na(+) and Cl(-) concentrations were significantly reduced after 3 days of acute exposure. Under this latter condition, the respiratory anterior gill ultrastructure, Na(+)/K(+)-ATPase and cytochrome c oxidase activities were significantly impaired. In contrast, the osmoregulatory posterior gill was unaffected for all treatments. As a consequence, we suggest that the observed hyper-osmoregulatory capacity impairment is the result of increased dissipative flow of ions and/or water through anterior gills. In contrast to acute exposure, chronic exposure did not induce any observable effect. However, crabs submitted to a known deleterious acute condition (0.5 mg Cd L(-1) for 3 days) directly after chronic exposure to 50 microg Cd L(-1) for 30 days showed normal hyper-osmoregulatory capacity with no change in gill Na(+)/K(+)-ATPase activity, and only little disturbance of anterior gill ultrastructure. These results demonstrate that a chronic cadmium exposure can induce acclimation mechanisms related to osmoregulation in this euryhaline decapod crustacean.     

Nitric oxide production by hemocytes of the ascidian Styela plicata

Ascidian hemolymph contains various types of blood cells (hemocytes), which are believed to be involved in defense mechanisms. We have studied nitric-oxide (NO) synthase activity in hemocytes of the ascidian Styela plicata after exposure to lipopolysaccharide (LPS). To investigate which cell types are involved in NO production, we first identified, by electron microscopy, the types of hemocytes previously described, mainly by light microscopy, by others. Five types of blood cells could be recognized in the hemolymph: granulocytes, hemoblasts, lymphocyte-like cells, morula cells, and pigment cells. The lymphocyte-like cells produced the most NO. In agreement with studies of other invertebrates, nitrite generation did not change after LPS stimulation in assays in vitro, under either different concentrations of LPS or different time periods. Therefore, we performed an in vivo assay by injecting a known quantity of Escherichia coli into the tunic of the ascidians in order to investigate possible differences in NO levels. No increase of NO occurred accompanying the inflammatory reaction suggesting that another molecule in the pathway was involved. We found that nuclear factor κB (NFκB) was activated. Since NFκB is involved in the production of many substances related to immune responses, additional molecules might also be generated in response to E. coli infection. These observations may improve our understanding of the reaction of animals to eutrophic conditions.     

Influence of Echinostoma paraensei (Lie and Basch, 1967) infection on the calcium content in Biomphalaria glabrata (Say, 1818)

The calcium content in the hemolymph and shell of Biomphalaria glabrata (Say, 1818) was determined after exposure to different parasite burdens (5 and 50 miracidia) of Echinostoma paraensei (Lie and Basch, 1967). The snails were dissected 1, 2, 3, and 4 weeks after infection to collect the hemolymph and shell. An increase in calcemia was observed in snails infected with both miracidial doses. A significant decrease in the calcium ions in the shell was observed, coinciding with the calcemia peak in the hemolymph. This indicates greater mobilization of calcium between the shell and hemolymph to regulate the calcium content in the body when the snail is exposed to stress conditions, as has also been observed in some other infected snail species. The results obtained indicate that in this model, the calcium metabolism depends on the miracidial dose used.     

Cytoplasmic superoxide dismutase activity is a sensitive indicator of the antioxidant status of the rat liver and brain

Several parameters of the cytoplasmic enzymatic antioxidant system of the liver and brain of the rat have been investigated under conditions of immobilization stress and of an antioxidant preparation in the diet of animals. These included superoxide dismutase (SOD) and glutathione reductase (GR) activities and nonspecific NADPH oxidation. Only changes in the activity of SOD both in the liver and brain were revealed. In the liver of animals that receive no preparation, a decrease in the activity of SOD after 30-min immobilization and its restoration after a 360-min immobilization were observed. In the brain, the activity of SOD decreased only in preconditioned animals after 30 and 360 min of exposure to stress. In addition, the activity of SOD in the brain of preconditioned animals, both stressed and unstressed, was lower than in the corresponding groups of control animals. It is probable that, under the conditions of immobilization stress, the level of reactive oxygen species (ROS) and as a consequence the activity of SOD decrease. The intake of an antioxidant preparation under these conditions seems to be not correct.     

The effects of exposure to critical thermal maxima on the physiological,metabolic, and immunological responses in adult white shrimp Litopenaeus vannamei (Boone)

Exposure to thermal stress was shown to have a significant effect on the osmotic pressure of the hemolymph, glucose levels, total count of hemocyte (TCH), and proPO activity in adult white shrimp Litopenaeus vannamei. Exposure of the shrimp to CTMax significantly increased the osmotic pressure of the hemolymph relative to the control group. In organisms reaching CTMax, temperature elicited a secondary stress response that included an increase in hemolymph glucose of 31?mg?mL^?1. Metabolites in hemolymph such as cholesterol, acylglycerides, and total protein were not significantly affected by exposure to CTMax. CTMax exposure affected several immunological parameters causing decreases in TCH and proPO activity. We suggested that biomarkers such as osmolality, glucose levels, TCH, and proPO activity could be used as sensitive predictors of exposure to CTMax in white shrimp.     

Effects of cadmium on carbohydrate and protein metabolisms in the freshwater crab Sinopotamon yangtsekiense

The physiological impact of Cd(2+) on Sinopotamon yangtsekiense was evaluated through changes of selected parameters considered as key elements of carbohydrate and protein metabolisms. Crab were exposed to 0.725, 1.45, 2.9mg·L(-1) Cd(2+) for 7, 14 and 21 days. A time- and/or concentration- dependent decrease in muscle glycogen and increase in LDH activity suggested that glycolysis was accelerated during the treatments. Increased protease activity, lowering of FAA and the initially increased and subsequently decreased aminotransferase activities suggest an enhanced protein mobilization during early Cd(2+) exposure followed by a metabolic impairment during late exposure. Decreased hemolymph glucose level was observed in the crabs treated with 2.9mg·L(-1) Cd(2+) for 21d, suggesting an impaired gluconeogenesis. Ammonia level barely changed during the 14d Cd(2+) exposure most likely due to the increased urea and glutamine production; After 1.45 and 2.9mg·L(-1) Cd(2+) treatment for 21d, ammonia was observed increased followed by an exclusive increase in glutamine. Taken together, our results indicate that carbohydrate and protein are mobilized to a varying degree as a compensatory metabolism to response to the energy stress during acute Cd(2+) exposure. As the time lapsed, some symptoms on metabolism obstacle reflect the toxic effect of sublethal Cd(2+).     

Cu/Zn‐ and Mn‐superoxide dismutases are specifically up‐regulated in neurons after focal brain injury

In previous studies, we have demonstrated that damaged neurons within a boundary area around necrosis fall into delayed cell death due to the cytotoxic effect of microglial nitric oxide (NO), and are finally eliminated by activated microglia. In contrast, neurons in a narrow surrounding region nearby this boundary area remain alive even though they may encounter cytotoxic NO. To investigate the mechanism by which neurons tolerate this oxidative stress, we examined the in vitro and in vivo expression levels of superoxide dismutase (SOD) under pathological conditions. Results from our in situ hybridization and immunohistochemical studies showed up‐regulation of Cu/Zn‐SOD only in neurons outside the boundary area, whereas up‐regulation of Mn‐SOD was detected in both neurons and glial cells in the same region. In vitro experiments using rat PC12 pheochromocytoma and C6 glioma cell lines showed that induction of both Cu/Zn‐ and Mn‐SOD mRNA could only be detected in PC12 cells after treatment with NO donors, while a slight induction of Mn‐SOD mRNA alone could be seen in C6 glioma cells. The mechanism of resistance toward oxidative stress therefore appears to be quite different between neuronal and glial cells. It is assumed that these two types of SOD might play a critical role in protecting neurons from NO cytotoxicity in vivo, and the inability of SOD induction in damaged neurons seems to cause their selective elimination after focal brain injury. © 2000 John Wiley & Sons, Inc. J Neurobiol 45: 39–46, 2000     

Age-Dependent Effects of ELF-MF on Oxidative Stress in the Brain of Mongolian Gerbils

The aim of study was to investigate the effects of extremely low frequency magnetic field (ELF-MF; 50 Hz; 0.1, 0.25 and 0.5 mT) on oxidative stress in the brain of 3- (adult) and 10-month-old (middle-aged) gerbils. Nitric oxide (NO) level, superoxide (O₂ ^?) production, superoxide dismutase (SOD) activity, and index of lipid peroxidation (ILP) were measured in the forebrain cortex, striatum, hippocampus, and cerebellum immediately and 3 days after cessation of 7-day exposure. In all gerbils, ELF-MF significantly increased oxidative stress in all tested brain regions. This effect was correlated with the value of magnetic induction and was higher in middle-aged gerbils. Three days after cessation of exposure, the values of examined parameters were closer to control levels. In adult gerbils, the effect of ELF-MF of 0.1 mT on NO level, O₂ ^? production and SOD activity was almost fully disappeared, and ILP was at the control level regardless of the value of magnetic induction. In middle-aged gerbils, the effect of ELF-MF was still present but to a lesser degree than those observed immediately after cessation of exposure. These findings pointed out the ability of ELF-MF to induce age- and magnetic induction-dependent modification of oxidative stress in the brain.