José M. Monserrat

Oxidative stress generation by microcystins in aquatic animals: Why and how

Microcystins (MICs) are potent toxins produced worldwide by cyanobacteria during bloom events. Phosphatases inhibition is a well recognized effect of this kind of toxins as well as oxidative stress. However, it is not fully understood why and how MICs exposure can lead to an excessive formation of reactive oxygen species (ROS) that culminate in oxidative damage. Some evidences suggest a close connection between cellular hyperphosphorylation state and oxidative stress generation induced by MICs exposure. It is shown, based on literature data, that MICs incorporation per se can be the first event that triggers glutathione depletion and the consequent increase in ROS concentration. Also, literature data suggest that hyperphosphorylated cellular environment induced by MICs exposure can modulate antioxidant enzymes, contributing to the generation of oxidative damage. This review summarizes information on MICs toxicity in aquatic animals, focusing on mechanistic aspects, and rise questions that in our opinion needs to be further investigated.

Acetylcholinesterase activity and antioxidant capacity of zebrafish brain is altered by heavy metal exposure

Pollution is a world problem with immeasurable consequences. Heavy metal compounds are frequently found as components of anthropogenic pollution. Here we evaluated the effects of the treatment with cadmium acetate, lead acetate, mercury chloride, and zinc chloride in acetylcholinesterase activity and gene expression pattern, as well as the effects of these treatments in antioxidant competence in the brain of an aquatic and well-established organism for toxicological analysis, zebrafish (Danio rerio, Cyprinidae). Mercury chloride and lead acetate promoted a significant decrease in acetylcholinesterase activity whereas they did not alter the gene expression pattern. In addition, the antioxidant competence was decreased after exposure to mercury chloride. The data presented here allowed us to hypothesize a signal transmission impairment, through alterations in cholinergic transmission, and also in the antioxidant competence of zebrafish brain tissue as some of the several effects elicited by these pollutants.

Arsenic toxicity in mammals and aquatic animals: a comparative biochemical approach.

Arsenic (As) is a widespread pollutant in the world and its toxicity is related to its chemical form, with inorganic forms being considered more toxic than the organic form, and huge differences in effects and processes of metabolism. This paper reviews the potential biochemical mechanisms of uptake of arsenic by aquaporins, capacity for metabolism and cellular efflux of As. It is known that As can affect signaling pathways since it can activate proteins such as ERK2, p38 and JNK, as shown in mammals. A comparison between phosphorylation sites of these proteins is presented in order to determine whether the same effect triggered by As in mammals might be observed in aquatic animals. The toxicity resulting from As exposure is considered to be linked to an imbalance between pro-oxidant and antioxidant homeostasis that results in oxidative stress. So, present review analyzes examples of oxidative stress generation by arsenic. Biotransformation of As is a process where firstly the arsenate is converted into arsenite and then transformed into mono-, di-, and trimethylated products. In the methylation process, the role of the omega isoform of glutathione-S-transferase (GST) is discussed. In addition, a phylogenetic tree was constructed for aquaporin proteins of different species, including aquatic animals, taking into account their importance in trivalent arsenic uptake.

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.

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.

The anesthetic efficacy of eugenol and the essential oils of Lippia alba and Aloysia triphylla in post-larvae and sub-adults of Litopenaeus vannamei (Crustacea, Penaeidae)

The aim of this study was to evaluate the anesthesia induction and recovery times of sub-adult and post-larvae white shrimp (Litopenaeus vannamei) that were treated with eugenol and the essential oils (EOs) from Lippia alba and Aloysia triphylla. Oxidative stress parameters in the hemolymph of this species were also analyzed. The concentrations of eugenol, A. triphylla EO and L. alba EO recommended for anesthesia were 200, 300 and 750 μL L(-1) for sub-adults and 175, 300 and 500 μL L(-1) for post-larvae, respectively. The concentrations studied during the transport of sub-adults were between 20 and 50 μL L(-1) eugenol, 20-30 μL L(-1)A. triphylla EO and 50 μL L(-1)L. alba EO. For post-larvae, the optimal concentrations for transport were 20 μL L(-1) eugenol and between 20 and 50 μL L(-1)A. triphylla EO. The white shrimp sub-adults that were exposed to A. triphylla EO (20 μL L(-1)) showed increases in their total antioxidant capacities (150%), catalase (70%) and glutathione-S-transferase (615%) activity after 6 h. L. alba EO (50 μL L(-1)) and eugenol (20 μL L(-1)) also increased GST activity (1292 and 1315%) after 6 h, and eugenol (20 μL L(-1)) decreased the total antioxidant capacity (100%). Moreover, concentrations above 30 μL L(-1) for the EOs of A. triphylla and L. alba and 20 μL L(-1) eugenol were effective at inducing anesthesia and improving the antioxidant system against reactive oxygen species (ROS) after 6 h.

Effects of arsenic (As) exposure on the antioxidant status of gills of the zebrafish Danio rerio (Cyprinidae)

In fishes, arsenic (As) is absorbed via the gills and is capable of causing disturbance to the antioxidant system. The objective of present study was to evaluate antioxidant responses after As exposure in gills of zebrafish (Danio rerio, Cyprinidae). Fish were exposed for 48 h to three concentration of As, including the highest As concentration allowed by current Brazilian legislation (10 microg As/L). A control group was exposed to tap water (pH 8.0; 26 degrees C; 7.20 mg O(2)/L). As exposure resulted in (1) an increase (p<0.05) of glutathione (GSH) levels after exposure to 10 and 100 microg As/L, (2) an increase of the glutamate cysteine ligase (GCL) activity in the same concentrations (p<0.05), (3) no significant differences in terms of glutathione reductase, glutathione-S-transferase and catalase activities; (4) a significantly lower (p<0.05) oxygen consumption after exposure to 100 microg As/L; (4) no differences in terms of oxygen reactive species generation and lipid peroxidation content (p>0,05). In the gills, only inorganic As was detected. Overall, it can be concluded that As affected the antioxidant responses increasing GCL activity and GSH levels, even at concentration considered safe by Brazilian legislation.

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.

Kinetic and toxicological characteristics of acetylcholinesterase from the gills of oysters (Crassostrea rhizophorae) and other aquatic species

The aim of this work was to characterize the cholinesterases from gills of Crassostrea rhizophorae in order to use them as biomarkers. Gills were homogenized and then centrifuged (9,000 x g, 4 degrees C, 30 min). S9 and Triton X-100 S9 treated (TX S9) fractions were employed as enzyme source. Km(ap) and Vmax were estimated, using acetylthiocholine iodide as substrate. Inhibition assays were performed with iso-OMPA and eserine. The Km(ap) for S9 and TX S9 fractions were 0.05 and 0.06 mM, whereas the Vmax were 1.92 and 5.84 nmol/min/mg protein. respectively. No inhibition was detected when the samples were incubated with iso-OMPA, suggesting the presence of acetylcholinesterases (AChE) in oyster gill homogenates. Sensitivity to eserine inhibition of AChE in the gills of oysters is intermediate when compared with other aquatic species.

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.