Adalto Bianchini

Sodium turnover rate determines sensitivity to acute copper and silver exposure in freshwater animals

The mechanisms of acute copper and silver toxicity in freshwater organisms appear similar. Both result in inhibition of branchial sodium (and chloride) uptake initiating a cascade of effects leading to mortality. The inhibition of the branchial Na/K-ATPase in the basolateral membrane is generally accepted as the key component responsible for the reduced sodium uptake. We propose that branchial carbonic anhydrase and the apical sodium channel may also be important targets for both copper and silver exposure. Several attempts have been made to predict metal sensitivity. A prominent example is the geochemical-biotic ligand model. The geochemical-biotic ligand modeling approach has been successful in explaining variations in tolerance to metal exposure for specific groups of animals exposed at different water chemistries. This approach, however, cannot explain the large observed variation in tolerance to these metals amongst different groups of freshwater animals (i.e. Daphnia vs. fish). Based on the detailed knowledge of physiological responses to acute metal exposure, the present review offers an explanation for the observed variation in tolerance. Smaller animals are more sensitive than large animals because they exhibit higher sodium turnover rates. The same relative inhibition of sodium uptake results in faster depletion of internal sodium in animals with higher sodium turnover. We present a way to improve predictions of acute metal sensitivity, noting that sodium turnover rate is the key predictor for variation in acute copper and silver toxicity amongst groups of freshwater animals. We suggest that the presented sodium turnover model is used in conjunction with the Biotic Ligand Model for risk management decisions.

Salinity effects on osmoregulation and growth of the euryhaline flounder Paralichthys orbignyanus

The flounder, Paralichthys orbignyanus, is found in coastal and estuarine waters of the Western South Atlantic Ocean. It is being considered for aquaculture due to its high market price and wide tolerance to environmental factors such as salinity, pH, and nitrogenous compounds. The objective of this study was to characterize the ionic and osmotic regulation of P. orbignyanus over the range of its tolerated ambient salinities (0‐40x) and to evaluate the survival and growth in freshwater (0x) and seawater (30x) over 90 days. After 15 days of exposure to different salinities (0x ,1 0x, 20x ,3 0xand 40x), plasma osmolality and ionic (Na + ,C l ,K + and Ca 2+ ) concentrations slightly increased with salinity. The isosmotic point was estimated as 328.6 mOsm kg 1 H2O and corresponded to 10.9xsalinity. After 90 days, survival was similar in freshwater and seawater, but osmo- and ionoregulation was significantly affected in freshwater and flounders reared in this medium showed a lower growth rate than those reared in seawater. Based on the results from this study, P. orbignyanus can be characterized as a marine/estuarine euryhaline teleost capable of hyper/ hypo iono- and osmoregulation over the fluctuating salinity regime faced by this species in the environment. Furthermore, results suggest that the lower growth rate exhibited by P. orbignyanus in freshwater could be due, at least partially, to a higher energy expenditure associated to a higher branchial Na + ,K + -ATPase activity in this environment. D 2002 Elsevier Science B.V. All rights reserved.

Physiological and antioxidant enzyme responses to acute and chronic exposure of Laeonereis acuta (Polychaeta, Nereididae) to copper

Chronic (14 days) and acute (48 h) copper effects on the antioxidant defense system and some physiological variables of Laeonereis acuta (Polychaeta, Nereididae) were evaluated. In both assays, two nominal copper concentrations (chronic: C1=31.25 and C2=62.50 μg/l; acute: A1=250 and A2=500 μg/l) and one control group (Cc and Ac=0 μg/l) were tested. End points analyzed were antioxidant enzyme activity (catalase, CAT; superoxide dismutase, SOD; and glutathione S-transferase, GST), oxygen consumption, metahemoglobin concentration, and lipid peroxidation (LPO). In the chronic assay, CAT activity was significantly higher in worms exposed to both concentrations of copper tested (C1=3.36±0.07 U CAT/mg protein; C2=4.06 0.32 U CAT/mg protein) than in control worms (Cc=2.16±0.39 U CAT/mg protein). SOD activity was also increased in the two copper-exposed groups (C1=16.85±4.22 U SOD/mg protein; C2=38.19±4.31 U SOD/mg protein) than in control group (Cc=3.54±0.46 U SOD/mg protein). However, GST activity was increased only in worms exposed to the higher copper concentration (C2=0.022±9.10−4 U GST/mg protein) when compared to the other groups tested (Cc=0.012±3.10−3 U GST/mg protein; C1=0.016±9.10−4 U GST/mg protein). None of the physiological variables analyzed (oxygen consumption, metahemoglobin concentration, and lipid peroxidation) was affected by chronic copper exposure. In the acute assay, only GST activity was induced in worms exposed to copper. This induction was observed only in the A1 group (0.027±2.10−3 U GST/mg protein) when compared to Ac (0.017±2.10−3 U GST/mg protein) or A2 (0.016±7.10−4 U GST/mg protein) groups. On the other hand, lipid peroxidation was higher in A2 (481.9±49.2 nmol CHP/g ww) than in control worms (Ac=337.9±25.0 nmol CHP/g ww). Oxygen consumption was higher in worms acutely exposed to the lower copper concentration tested (A1=0.27±0.04 mg O2/g ww/h) than in the higher concentration (A2=0.14±0.01 mg O2/g ww/h). Changes in the swimming behavior of copper-exposed animals in both assays and edemas in the body wall of worms acutely exposed to copper were also observed. Results suggest that copper exposure favors reactive oxygen species generation and that enzymatic defense system is induced under chronic exposure, preventing oxygen consumption changes and lipid peroxidation and metahemoglobin formation. However, in acutely exposed worms, in spite of a transient peak of GST activity, no induction of antioxidant enzymes occurs, leading to morphological and physiological changes.

Gill Na(+),K(+)-ATPase and osmoregulation in the estuarine crab, Chasmagnathus granulata Dana, 1851 (Decapoda, Grapsidae).

Some kinetic properties of gill Na(+),K(+)-ATPase of the estuarine crab, Chasmagnathus granulata, and its involvement in osmotic adaptation were analyzed. Results suggest the presence of different Na(+),K(+)-ATPase isoforms in anterior and posterior gills. They have different affinities for Na(+), but similar affinity values for K(+), Mg(2+), ATP and similar enzymatic profiles as a function of temperature of the incubation medium. Ouabain concentrations which inhibit 50% of enzyme activity were also similar in the two types of gills. Enzyme activity and affinity for Na(+) are higher in posterior gills than in anterior ones. Furthermore, affinities of Na(+),K(+)-ATPase of posterior gills for Na(+) and K(+) were similar to or higher than those of gills or other structures involved in the osmoregulation in several euryaline decapod crustaceans. Acclimation to low salinity was related to a significant increase in the maximum Na(+), K(+)-ATPase activity, mainly in posterior gills. On the other hand, crab acclimation to high salinity induced a significant decrease in maximum enzyme activity, both in anterior and posterior gills. These results are in accordance to the osmoregulatory performance showed by C. granulata in diluted media, and point out the major role of posterior gills in the osmoregulation of this species.

Toxic effects of microcystins in the hepatopancreas of the estuarine crab Chasmagnathus granulatus (Decapoda, Grapsidae)

Microcystins are toxins produced by cyanobacteria, being toxic to aquatic fauna. It was evaluated alternative mechanisms of microcystins toxicity, including oxidative stress and histopathology in the hepatopancreas of the estuarine crab Chasmagnathus granulatus (Decapoda, Grapsidae). Microcystins was administered to crabs (MIC group) over 1 week, whereas the control (CTR group) received the saline from cyanobacteria culture medium. At day 7, catalase activity was higher in the MIC than in the CTR group, although a decrease of activity was verified in both groups with respect to time 0. Glutathione-S-transferase activity augmented in MIC with respect to CTR, suggesting a higher conjugation rate of the toxins with glutathione. No differences were detected in the superoxide dismutase activity. Lipid peroxidation remained stable in both groups. Histopathological analyses showed that the number of B cells decreased significantly in the CTR as a possible effect of starvation, while no significant change was observed in the MIC group. The hepatopancreas from the MIC group exhibited some necrotic tubules and melanin-like deposits. Overall, results showed that some enzymes of the antioxidant defense system were activated after microcystins exposure, this response being able to maintain lipid peroxidation levels, but insufficient to completely prevent histological damage.

Reactive oxygen species generation and expression of DNA repair-related genes after copper exposure in zebrafish (Danio rerio) ZFL cells

Copper is an essential metal to aquatic animals, but it can be toxic when in elevated concentrations in water. The objective of the present study was to analyze copper effects in zebrafish hepatocytes (ZFL cell-line). The number of viable cells and copper accumulation were determined in hepatocytes exposed in vitro to different copper concentrations (5-30mgCu/L). Intracellular reactive oxygen species (ROS) formation, total antioxidant capacity against peroxyl radicals, and expression of genes related do DNA repair system were also measured in hepatocytes exposed to 5 and 20mgCu/L. After 24h of exposure, hepatocytes showed an exponential kinetics of copper accumulation. Copper exposure (24 and 48h) significantly reduced hepatocyte number in all concentrations tested, except at the lowest one (5mgCu/L). Exposure to 20mgCu/L for 6, 12 and 24h significantly increased intracellular ROS formation. However, no significant change in total antioxidant capacity was observed. After 12 and 24h of exposure to 20mgCu/L, a significant decrease in expression of p53 and CDKI genes was observed. Conversely, expression of Gadd45alpha, CyclinG1 and Bax genes was significantly induced after 24h of exposure to 20mgCu/L. In hepatocytes exposed to 5mgCu/L, any significant alteration in expression of these genes was observed. In a broad view, most of genes encoding for DNA repair proteins were inhibited after copper exposure, especially in hepatocytes exposed to 20mgCu/L. Taken all together, results obtained suggest that the increased intracellular ROS formation induced by copper exposure would be responsible for the alteration in gene expression pattern observed.

Biochemical and physiological adaptations in the estuarine crab Neohelice granulata during salinity acclimation.

Neohelice granulata (Chasmagnathus granulatus) is an intertidal crab species living in salt marshes from estuaries and lagoons along the Atlantic coast of South America. It is a key species in these environments because it is responsible for energy transfer from producers to consumers. In order to deal with the extremely marked environmental salinity changes occurring in salt marshes, N. granulata shows important and interesting structural, biochemical, and physiological adaptations at the gills level. These adaptations characterize this crab as a euryhaline species, tolerating environmental salinities ranging from very diluted media to concentrated seawater. These characteristics had led to its use as an animal model to study estuarine adaptations in crustaceans. Therefore, the present review focuses on the influence of environmental salinity on N. granulata responses at the ecological, organismic and molecular levels. Aspects covered include salinity tolerance, osmo- and ionoregulatory patterns, morphological and structural adaptations at the gills, and mechanisms of ion transport and their regulation at the gills level during environmental salinity acclimation. Finally, this review compiles information on the effects of some environmental pollutants on iono- and osmoregulatory adaptations showed by N. granulata.

Effects of copper and zinc on growth, feeding and oxygen consumption of Farfantepenaeus paulensis postlarvae (Decapoda: Penaeidae)

The effect of chronic exposure (35 days) to sub-lethal concentrations of copper (17-212 ppb) and zinc (41-525 ppb) on growth of Farfantepenaeus paulensis postlarvae 17 days old (PL(17)) was analysed. The effects of acute exposure of PL(17) to the same metal on food ingestion and oxygen consumption were also evaluated. Studies were performed using copper and zinc singly, and in a mixture of equipotent concentrations (1:2.5). Chronic exposure to copper (85 and 212 ppb) and zinc (106, 212 and 525 ppb) reduced PL(17) growth. Acute exposure to copper (212 ppb) and zinc (525 ppb) reduced the number of Artemia sp. predated during 30 min and the positive feeding response induced by L-isoleucine. Despite of the lower positive feeding response when PL(17) were exposed to zinc, a significant difference from control condition was not seen. Oxygen consumption was reduced by all copper and zinc concentrations tested. The mean reduction was approximately 32%. The copper zinc-mixture did not modify food consumption and feeding response, or the oxygen consumption of the PL(17). The inhibition of food and oxygen consumption induced by copper and zinc could explain, at least in part, the long-term reduction of growth observed in chronically exposed PL(17). Our results also suggest that the inhibition of food consumption induced by copper is possibly due to an effect on chemosensory mechanisms. Finally, an antagonism between copper and zinc was observed, when were employed to analyse feeding behaviour and aerobic metabolism after acute exposure.

Antioxidant responses in different body regions of the polychaeta Laeonereis acuta (Nereididae) exposed to copper

Antioxidant enzymes, total antioxidant capacity (TOSC) and concentration of reactive oxygen species (ROS) were measured in anterior (A), middle (M) and posterior (P) body regions of Laeonereis acuta after copper (Cu; 62.5 microg/l) exposure. A catalase (CAT) activity gradient observed in control group (lowest in A, highest in P) was not observed in Cu exposed group. Glutathione-S-transferase (GST) activity in A region of Cu group was higher than in A region of the control group. DNA damage (comet assay) was augmented in the A region of Cu group. Since copper accumulation was similar in the different body regions, sensitivity to copper in A regions seems to be related to lowest CAT activity. In sum, copper exposure lowered TOSC, a result that at least in part can be related to lowering of antioxidant enzymes like CAT. DNA damage was induced in the anterior region, where a lower CAT activity was observed.

Aluminum as an endocrine disruptor in female Nile tilapia (Oreochromis niloticus)

The effects of aluminum on plasma ion, lipid, protein and steroid hormone concentration were evaluated in Oreochromis niloticus broodstock females. Lipid and protein concentrations from the gonads and liver were also measured. Experiments were performed at neutral and acidic water pH. Four groups of fish were tested for 96h: 1) control conditions at neutral water pH; 2) control conditions at acidic water pH (CTR-Ac); 3) aluminum at neutral water pH (Al-N); and 4) aluminum at acidic water pH (Al-Ac). Aluminum and acidic water pH exposure caused no ionoregulatory disturbances. Total lipid concentration increased in the mature gonads and decreased in the liver, suggesting an acceleration of lipid mobilization to the ovaries in animals exposed to aluminum. However, a decreased protein concentration in ovaries was also observed. Exposure of control fish to acidic water pH caused an increased concentration of plasma 17alpha-hydroxyprogesterone. However, females exposed to aluminum at acidic water pH showed a decreased of plasma 17alpha-hydroxyprogesterone and cortisol. No differences in plasma 17beta-estradiol were observed. The physiological mechanisms underlying the disturbances observed are discussed focusing on reproduction. We suggest that aluminum can be considered an endocrine disrupting compound in mature O. niloticus females.