Toxicity evaluation of nanocrystalline silver-impregnated coated dressing on the life cycle of worm Caenorhabditis elegans.

In recent times, however, due to the emergence of bacterial strains with resistance to conventional antibiotics, silver has again gained attention as an alternative for developing new efficient bactericides, including the use of silver nanoparticles (AgNPs). However, the improper disposal of these items after use may cause toxicological effects on organisms in the environment. To evaluate the potential environmental hazard of nanosilver-coated dressings, the nematode Caenorhabditis elegans was chosen as a test organism. The assays were conducted in 24-well plates that contain four different sizes of coated dressing to obtain different concentrations. L1 and L4 C. elegans larval stages were exposed to these nanosilver concentrations. Dressing cutouts were arranged between two layers of agar for 3 days and Escherichia coli (OP 50 strain) was added as food source for the worms. After the exposure period, growth, reproduction, fertility, silver concentration in the medium and the concentration of reactive oxygen species (ROS) in the worms were evaluated. Scanning and transmission electron microscopy analyses were performed on the coated dressings, as well as analyses of zeta potential, ionic release and antibacterial power in two bacterial strains (Pseudomonas aeruginosa and Staphylococcus aureus). It was verified the antibacterial power of the coated dressing, in both bacteria strains tested. Characterization of the coated dressing indicated heterogeneous nanoparticles, as well as distinct zeta potentials for the medium in water and saline medium (0.9% NaCl). L1 larval worms exposed to nanosilver-coated dressing showed a high ROS concentration and reductions in growth, fertility and reproduction. Worms exposed to the coated dressing during the L4 stage showed almost no response. Overall, the obtained results indicate the potential environmental hazard of nanosilver-coated dressings.

Interference of single walled carbon nanotubes (SWCNT) in the measurement of lipid peroxidation in aquatic organisms through TBARS assay.

Nanomaterials (NM) exhibit unique properties due their size and relative area, but the mechanisms and effects in the living organisms are yet to be unfold in their totality. Potential toxicity mechanisms concerning NM as carbon nanotubes include oxidative stress generation. Several fluorimetric and colorimetric methods have been systematically used to measure NM toxicity, and controversial results have been reported. One of the problems can be related to the interference effects induced by NM, leading to artifacts that can lead to misleading conclusions. In present study, it was performed in vitro assays with two aquatic species: the zebrafish Danio rerio and the polychaete Laeonereis acuta to evaluate the potential interference capacity of single-wall carbon nanotubes (SWCNT) in a fluorometric method (TBARS assay) to measure lipid peroxidation. Obtained results indicated that gills and brain of zebrafish presented a lowered fluorescence only at extremely high concentrations (50 and 500mg/L). Determinations in anterior, middle, and posterior body regions of L. acuta showed a quite different pattern: high fluorescence at low SWCNT concentrations (0.5mg/L) and lowering at the highest (500mg/L). To eliminate matrix effect of biological samples, tests employing the standard for TBARS assay, 1,3,3-tetramethoxipropane, were run and the results showed again higher fluorescence values at low concentrations (0.5-5mg SWCNT/L), a technique artifact that could lead to misleading conclusions since higher fluorescence values implicate higher TBARS concentration, implying oxidative stress. Using the colorimetric FOX assay with cumene hydroperoxide as standard presented remarkable better results since no artifacts were observed in the same SWCNT concentration range that employed with the TBARS technique.

Age protects from harmful effects produced by chronic intermittent hypoxia.

Obstructive sleep apnoea (OSA) affects an estimated 3­7% of the adult population, the frequency doubling at ages >60­65 years. As it evolves, OSA becomes frequently associated with cardiovascular, metabolic and neuropsychiatric pathologies defining OSA syndrome (OSAS). Exposing experimental animals to chronic intermittent hypoxia (CIH) can be used as a model of the recurrent hypoxic and O2 desaturation patterns observed in OSA patients. CIH is an important OSA event triggering associated pathologies; CIH induces carotid body (CB)-driven exaggerated sympathetic tone and overproduction of reactive oxygen species, related to the pathogenic mechanisms of associated pathologies observed in OSAS. Aiming to discover why OSAS is clinically less conspicuous in aged patients, the present study compares CIH effects in young (3­4 months) and aged (22­24 months) rats. To define potential distinctive patterns of these pathogenic mechanisms, mean arterial blood pressure as the final CIH outcome was measured. In young rats, CIH augmented CB sensory responses to hypoxia, decreased hypoxic ventilation and augmented sympathetic activity (plasma catecholamine levels and renal artery content and synthesis rate). An increased brainstem integration of CB sensory input as a trigger of sympathetic activity is suggested. CIH also caused an oxidative status decreasing aconitase/fumarase ratio and superoxide dismutase activity. In aged animals, CIH minimally affected CB responses, ventilation and sympathetic-related parameters leaving redox status unaltered. In young animals, CIH caused hypertension and in aged animals, whose baseline blood pressure was augmented, CIH did not augment it further. Plausible mechanisms of the differences and potential significance of these findings for the diagnosis and therapy of OSAS are discussed.

Effects of lipoic acid on growth and biochemical responses of common carp fed with carbohydrate diets.

Lipoic acid (LA) is an antioxidant that also favors glucose uptake in mammals. Until now, there are no studies evaluating the potential effect of this molecule on glycemic control in fish. It was evaluated LA effects on glucose uptake in common carp Cyprinus carpio fed with carbohydrate diets from two carbohydrate sources: glucose (GLU) and starch (STA), and supplemented or not with LA, being the diets: +GLU/-LA (GLU); +GLU/+LA (GLU + LA); +STA/-LA (STA); and +STA/+LA (STA + LA). Carp juveniles (6.5 ± 0.1 g) were fed with each diet ad libitum 4 times a day, during 68 days. Muscle glycogen concentration was higher (p < 0.05) in GLU and GLU + LA than in STA and STA + LA groups. On fish fed with starch, muscle cholesterol and triglyceride concentrations were higher (p < 0.05) in fish fed diets supplemented with LA. Muscle protein levels were higher in fish fed with LA, independent of the diet carbohydrate source. Lipid peroxidation was significantly reduced (p < 0.05) in fish muscle on fish fed the STA + LA diets when compared with the STA diet. Our findings indicate that LA modulates lipid, proteins and carbohydrate metabolism together with the well-known antioxidant effect. Also, LA showed to enhance starch utilization taking into account muscle cholesterol and triglyceride levels.

Antioxidant, phase II and III responses induced by lipoic acid in the fish Jenynsia multidentata (Anablapidae) and its influence on endolsulfan accumulation and toxicity.

Antioxidants like lipoic acid (LA) are known to trigger augmented antioxidant and phase II and III responses. This study aimed to evaluate the effect of LA in P-glycoprotein (Pgp) expression, glutathione-S-transferase (GST) activity, total antioxidant competence, levels of lipid peroxides (TBARS) and accumulation of the organochlorine insecticide endosulfan (Endo: α-, ß-isomers and sulfate metabolite) in different organs of the fish Jenynsia multidentata. One hundred and twenty females (1.55±0.07 g) were fed during 8 days with (n=60) or without (n=60) a LA enriched ration (6000 mg/kg). Four experimental groups were defined: -LA/-Endo; +LA/-Endo; -LA/+Endo; and +LA/+Endo. Endo groups were exposed during 24 h to 1.4 µg of insecticide/L. Results showed that only LA induced a significant increment in liver Pgp expression. GST activity was augmented in liver after exposure to LA or Endo. TBARS levels were lowered in liver and gills after LA pre-treatment. Total antioxidant capacity was lowered in liver of Endo exposed fish, a result that was reversed by LA pre-treatment. It is concluded that LA induced the expected effects in terms of Pgp expression, GST activity and reduced TBARS levels although favored α-Endo accumulation in brain. However, the Endo metabolism to the more persistent endosulfan sulfate was not facilitated by LA pre-treatment.

Biomonitoring of antioxidant and oxidative stress responses in Perinereis gualpensis (Polychaeta: Nereididae) in Chilean estuarine regions under different anthropogenic pressure.

This study aimed to analyze oxidative stress parameters, including levels of the antioxidant glutathione (GSH), activity of glutamate-cysteine ligase (GCL) and glutathione-S-transferase (GST), total antioxidant capacity and protein oxidation, in the polychaete Perinereis gualpensis (Nereididae) collected from the Biobío, Itata, Valdivia and Lingue estuaries in Chile, which present different degrees of anthropogenic pressure. Sampling sites were characterized considering a geographic information system and the physicochemical characteristics of water and sediment. Significant differences (p<0.05) were observed between the sampling sites for most of the responses (GSH, GCL, GST and antioxidant capacity), mainly related to human activities such as agriculture, industry, among others. Multivariate correlation analysis indicates a certain relationship of antioxidant responses with human activities, salinity, and worm weight, this last employed to standardize GST and antioxidant capacity. These results clearly indicate biomarker responses in P. gualpensis in Biobío and Valdivia estuaries, the more affected by human activities.

Neurotoxicity in zebrafish exposed to carbon nanotubes: Effects on neurotransmitters levels and antioxidant system.

Given the increasing use of carbon nanotubes (CNT) in several industries and technological applications, it is essential to perform in vivo toxicological studies with these nanomaterials to evaluate their potential ecotoxicity. Dopamine (DA) and serotonin (5HT) are key neurotransmitters for brain functions and behavioral responses. Determination of DA and 5HT were performed in brain samples from zebrafish Danio rerio exposed i.p. to single-walled CNT (SWCNT), besides analyzing acetylcholinesterase (AChE) and ectonucleotidases activity, lipid peroxidation and total antioxidant capacity. Results showed that treatment with SWCNT increased between 3 and 6-fold the concentration of DA and 5HT (p < 0.05). Similarly, a significant reduction (p < 0.05) in AChE activity was observed in the brains of SWCNT exposed zebrafish when compared to the control groups. Cholinergic, serotonergic, and dopaminergic systems, through AChE activity and serotonin and dopamine levels, respectively were affected by SWCNT in the zebrafish brain. Alterations in these neurotransmitters can potentially affect several physiological and behavioral that they control.

Metabolic rate and reactive oxygen species production in different genotypes of GH-transgenic zebrafish.

Growth hormone overexpression increases growth and consequently increases the metabolic rate in fishes. Therefore, the objective of this study was to evaluate the effects of growth hormone overexpression in zebrafish Danio rerio in terms of growth, oxygen consumption, reactive oxygen species production, lipid hydroperoxide content, antioxidant enzyme activity and glutamate-cysteine ligase catalytic subunit gene expression. The employed models were wild type and transgenic (hemizygous and homozygous) zebrafish expressing the Odonthestes argentinensis growth hormone gene directed by the Cyprinus carpio beta-actin promoter. Higher growth parameters were observed in the hemizygous group. The homozygous group possessed higher oxygen consumption and reactive oxygen species production. Growth hormone transgenesis causes a decrease in glutamate-cysteine ligase catalytic subunit expression, an enzyme responsible for glutathione synthesis. Although the lipid hydroperoxide content was similar between groups, we demonstrate that growth hormone overexpression has the potential to generate oxidative stress in fishes.

Microcystin-induced oxidative stress in Laeonereis acuta (Polychaeta, Nereididae).

Oxidative stress induced by microcystins was evaluated in an estuarine worm, Laeonereis acuta (Nereididae). Ten organisms were exposed to lyophilized cells of a toxic Microcystisaeruginosa strain RST9501 ( approximately 2 microg/mL microcystins, MC); 10 were exposed to lyophilized cells of a nontoxic Aphanotece sp. strain RSMan92 and 10 were maintained without cyanobacterial cells. Exposure time was 48 h. The enzymatic antioxidant defenses, as well as the oxidative damage, were analyzed. Toxic and nontoxic cyanobacteria lowered catalase activity with no changes in glutathione reductase and glutathione-S-transferase activities. This may have led to toxin intracellular accumulation, which should favor oxidative stress generation, observed by the high lipid peroxide and DNA-protein crosslink levels in the group exposed to MC.

The benefits and drawbacks of nicotine exposure in the cortex and hippocampus of old rats.

Nicotine is the main alkaloid of tobacco and possesses well-established stimulant effects. Previous reports show that nicotine at low doses improves memory functions, while high doses impair memory. This study aims to analyze the effects of nicotine (NIC) on inhibitory avoidance task and on DNA damage, reactive oxygen species (ROS) concentration, total antioxidant capacity, and lipid peroxidation in cortex and hippocampus of old rats. Male Wistar rats of 24-26 months old (620-700g) were exposed i.p. to two doses (0.3 and 1mg/kg) of NIC daily during 9 days. The treatment NIC 0.3 enhanced long-term memory (p<0.05), whereas NIC 1 improved both short and long-term memories (p<0.05). DNA damage was observed only in hippocampus (p<0.05) after NIC 1 exposure. A similar result was obtained for ROS: higher levels were detected at NIC 1 treatment in hippocampus (p<0.05). No alterations in the total antioxidant capacity were verified after NIC exposure (0.3 and 1mg/kg) in both tissues (p>0.05). Finally, evidence of oxidative damage was observed in terms of lipid peroxides levels, being higher at NIC 1 in hippocampus (p<0.05). Overall the results indicate that deleterious effects paralleled the improved short and long-term memories at the highest NIC dose, since augmented DNA damage, ROS concentration and lipid peroxides levels were registered.

Effects of microcystins over short- and long-term memory and oxidative stress generation in hippocampus of rats.

Microcystins produced by cyanobacteria are potent inhibitors of some protein phosphatases, but recent evidence also indicates its potential to generate oxidative stress. In the present study, the effects of microcystin raw extracts (Mic; 0.01 and 20microg/L) and purified okadaic acid (OA; 0.01 and 10microg/L) on short- and long-term memory alteration and antioxidant and oxidative damage were investigated in hippocampus of rats. The results showed an amnesic effect with 0.01 and 20microg/L Mic on retrieval and only with 0.01microg/L Mic on spatial learning. Parallel to these effects oxidative damage was observed as evidenced by augmented levels of lipid peroxides and DNA damage and the absence of antioxidant responses in terms of total oxyradical scavenging capacity. Phase II reactions catalyzed by glutathione-S-transferase were not modified after microcystins exposure. Overall this study showed physiological events (retrieval and spatial learning) that can be related to the classical toxic effects of microcystins (i.e., phosphatase inhibition). In addition, evidence of alternative toxicity mechanisms via oxidative stress generation was also obtained. The fact that organic anion transporter polypeptides (OATP) involved in microcystins uptake are expressed not only in liver but also in brain points to the environmental relevance of the observed effects.

Estradiol valerate and tibolone: effects on memory.

This study investigated the effects of estradiol valerate (EV) and tibolone (TB) treatments on some memory parameters of ovariectomized young (2 months), adult (8 months) and old (20 months) female rats. A Sham-operated group was used as control and the animals were treated daily, by oral gavage, with saline (Sham and placebo NR group), EV (0.3 mg/kg) or TB (0.5 or 1 mg/kg, TB1 and TB2, respectively). In step-down inhibitory avoidance task, the latency of old TB2-treated females in the short-term test was significantly inferior (p<0.05), compared to TB2 adults. In the elevated plus maze, adult NR females spent significantly less time (p<0.05) in the open arms as compared with EV and TB2-treated animals. Additionally, adult TB2-treated females spent significantly less time in the closed arms compared to Sham, NR and TB1 groups. Finally, in the water maze retention test, young TB1-treated animals performed worse when compared to Sham, EV and TB2 females. In the old animals, EV treatment hampered subject performance as compared to all other treatments. Taken together, these results indicate that ovarian hormones differently affect female memory in an age-dependent manner.

Importance of cholinesterase kinetic parameters in environmental monitoring using estuarine fish.

The aim of the present study was to determine the kinetic parameters and cholinesterase (ChE) activity in two teleost fish, the white mouth croaker Micropogonias furnieri (Scianidae) and the Madamango sea catfish Cathorops spixii (Ariidae), to verify their suitability as sentinel species of aquatic pollution by anticholinergic compounds. Individuals of each species were captured in one reference and one polluted site in the Southern Brazilian coast. Brain tissue was used as enzyme source. Inhibition kinetic parameters indicated that ChE from C. spixii collected at the reference site showed more affinity (Ka) for eserine that from those collected at the polluted site. The opposite was observed for the carbamylation constants (Kc). Thus, similar inhibition constants (Ki) were observed. M. furnieri brain showed an extremely low sensitivity to in vitro inhibition by eserine, indicating that it is not a suitable biomarker to be employed in environmental monitoring of anticholinergic compounds. Results from the present study also point to the need for considering kinetic analysis when cholinesterase activity is employed as a biomarker.

Uptake, metabolism and sub-lethal effects of BDE-47 in two estuarine invertebrates with different trophic positions.

Two microcosm types -sediment-biota and biota-biota- were constructed to simulate different pathways of BDE-47 uptake, metabolism and oxidative stress effects in two key estuarine invertebrates (polychaete Laeonereis acuta and crab Cyrtograpsus angulatus). In the sediment-biota experiment, both species were exposed to spiked sediments; an environmentally reported and a high concentration of BDE-47 for 2 weeks. In the biota-biota experiment, crabs were fed with polychaetes pre-exposed to BDE-47 in the sediment-biota experiment. The sediment-biota experiment first revealed that polychaetes significantly accumulated BDE-47 (biota-sediment accumulation factor >2; p < 0.05) to a much greater extent than the crab organs (muscle, hepatopancreas, gills) at both sediment concentrations. For oxidative stress responses, polychaete and crab tissues exposed to spiked sediment showed a significant increase (p < 0.05) of only glutathione S-transferase (GST) activity with respect to controls in both BDE-47 concentrations. No lipid peroxidation (TBARS) or total antioxidant capacity (ACAP) changes were evident in the species or organs exposed to either BDE-47 sediment concentration. The biota-biota experiment showed that feeding crabs with pre-exposed polychaetes caused BDE-47 accumulation in organs as well as significant amounts of BDE-47 eliminated through feces (p < 0.05). Unlike the sediment-biota exposure, crabs fed with pre-exposed BDE-47 polychaetes showed the most conspicuous oxidative stress responses. Significant changes in GST and ACAP in both hepatopancreas and gills, in addition to enhanced TBARS levels in the hepatopancreas with respect to controls (p < 0.05), revealed that BDE-47 assimilated by invertebrates represents a potential source of toxicity to their predators. No methoxylated metabolites (MeO-PBDEs) were detected during BDE-47 metabolism in the invertebrates in either of the two different exposure types. In contrast, hydroxylated metabolites (OH-PBDEs) were detected in polychaetes and crab organs/feces in both experiments. Our results demonstrate that PBDE hydroxylation is one of the main biotransformation routes of BDE-47 in estuarine animals, which could be associated with the oxidative stress responses found.

Oxidative status of the myocardium in response to different intensities of physical training.

The intensity of exercise determines the metabolic pathway and the energetic substrate that is spent. Our study sought to identify the effects of different intensities of swimming on myocardial oxidative status and the blood lipid profile. Eighty Wistar rats (male and female) submitted to different intensities of a swimming regimen (low, LS; moderate, MS; or high, HS) for 16 weeks. Samples of blood and myocardium from the left ventricle were collected to determine lipid profiles and oxidative status. Reactive oxygen species (ROS) and antioxidant capacity against peroxyl radicals (ACAP), lipid profiles and lipid peroxidation was analyzed. ROS levels and ACAP were higher in male rats than in female rats overall (p<0.05). However, ACAP in the myocardium was significantly elevated in LS female rats compared to the MS and HS female rats, which had a significantly lower ACAP compared to all other groups. LS and MS training in both sexes and HS training (in females) led to significant decreases in the heart's lipid peroxidation. Amelioration of the lipid profile and reduction in oxidative damage contributed to a physiological state that benefits cardiovascular function in exercised animals. The results show that low and moderate intensity exercise promotes beneficial adaptations.

Acute and chronic effects of cadmium on blood homeostasis of an estuarine crab, Chasmagnathus granulata, and the modifying effect of salinity.

Whole body oxygen consumption and some hemolymph parameters such as pH, partial pressure of gases, level of ions and lactate were measured in the estuarine crab Chasmagnathus granulata after both acute (96 h) and chronic (2 weeks) exposure to cadmium at concentrations ranging from 0.4 to 6.3 mg/l. In all instances, the crabs developed hemolymph acidosis, but no respiratory (increased PCO2) or lactate increases were evident. Hemolymph levels of sodium and calcium were always increased by cadmium exposure. The chronic toxicity of cadmium was enhanced at 12 per mil salinity, even causing a significantly higher mortality in comparison with the higher salinity (30 per mil ) used. A general metabolic arrest took place at 12 per mil salinity in the crabs chronically exposed to cadmium, as indicated by decreases of oxygen consumption and PCO2, an increase of PO2, along with no changes in lactate levels. These imbalances were associated with severe necrosis and telangiectasia in the respiratory gills, probably leading to respiratory impairment and finally histotoxic hypoxia and death of the animals.

Some kinetic and toxicological characteristics of thoracic ganglia cholinesterase of Chasmagnathus granulata (Decapoda, Grapsidae).

In this study, some kinetic and toxicological parameters of the thoracic ganglia cholinesterase from the estuarine crab Chasmagnathus granulata were determined. Effects of the type and concentration of substrate, pH (6.80-8.50), incubation temperature (5-35 degrees C) and eserine on the enzyme activity were studied. Enzymatic activity was higher at pH 7.40 and 8.00 and significantly reduced at lower temperatures (5-10 degrees C). Employing acetylthiocholine iodide (ATCh) as substrate, the K(m) and Vmax were estimated as 0.28 mM and 1.75 mumol.mg protein-1.min-1, respectively. A 14.11 and 24.51% decrease in enzyme activity were registered at 4.62 and 9.24 mM of ATCh, respectively. Using propionylthiocholine iodide as substrate, the K(m) and Vmax were estimated as 0.16 mM and 0.91 mumol.mg protein-1.min-1, respectively. The IC50 for eserine was estimated as 5.3 x 10(-4) mM. The Ki estimated for eserine (8.10 mM-1.min-1) indicates that the thoracic ganglia cholinesterase from C. granulata showed a low ability to generate an irreversible enzyme-inhibitor complex. The higher enzymatic activity registered with ATCh and the enzyme inhibition observed at high concentration of this substrate, suggest that thoracic ganglia cholinesterase from C. granulata is an acetylcholinesterase.

Inhibitory effects of cadmium on carbonic anhydrase activity and ionic regulation of the estuarine crab Chasmagnathus granulata (Decapoda, Grapsidae).

This work was aimed at evaluating the gill carbonic anhydrase (CA) activity of the estuarine crab Chasmagnathus granulata exposed in vivo to cadmium, at different salinities. The in vivo effect of the specific inhibitor acetazolamide (AZ) was also assayed. Besides, the inhibition of CA activity by different heavy metals (cadmium, copper, zinc) and AZ were evaluated under in vitro conditions. For the in vivo assays, adult males were acclimated to salinities of 2.5 or 30/1000. The corresponding 96-h LC50 of cadmium was 2.69 mg l-1 at 2.5/1000, and > 50 mg l-1 at 30/1000. Cadmium only caused a significant lower CA activity than control at 2.5/1000. EC50 for CA inhibition was estimated to be 1.59 mg l-1 at 2.5/1000. Statistical differences in Na+ hemolymphatic levels (P < 0.05) were only detected at 2.5/1000, between 0 and 1.25 mg l-1 of cadmium, but no statistical differences were observed for Cl- levels at any assayed salinity. As CA inhibition registered at 2.5/1000 was followed by only changes in Na+ concentration, it is likely that cadmium exposure could differentially affect ions permeability, among others factors. The concentrations that inhibited in vitro 50% of enzymatic activity (IC50) were 2.15 x 10(-5), 1.62 x 10(-5), 3.75 x 10(-6) and 4.4 x 10(-10) M for cadmium, copper, zinc and AZ, respectively. The comparison with IC50 values of other aquatic species, indicates a higher CA sensitivity for C. granulata to pollutants.

Effects of extracts from the cyanobacterium Microcystis aeruginosa on ion regulation and gill Na+,K+-ATPase and K+-dependent phosphatase activities of the estuarine crab Chasmagnathus granulata (Decapoda, Grapsidae).

Recent discoveries indicate that microcystins affect enzymes, such as Na(+),K(+)-ATPase, involved in ion regulation of aquatic animals, through K(+)-dependent phosphatase inhibition. In vitro studies showed the inhibitory effect of Microcystis aeruginosa extracts on Na(+),K(+)-ATPase and K(+)-dependent phosphatase activities in gills of Chasmagnathus granulata (Decapoda, Grapsidae). Extracts of M. aeruginosa were prepared from lyophilized or cultures cells of the cyanobacterium. For lyophilized cells, IC(50) values were estimated as 0.46 microg/L (95% confidence interval [CI]=0.40-0.52 microg/L) and 1.31 microg/L (95% CI=1.14-1.51 microg/L) for Na(+),K(+)-ATPase and K(+)-dependent phosphatase, respectively. However, extracts prepared from cultured cells presented a much lower inhibitory potency against both enzymes. Gas chromatography revealed long-chain fatty acids in the lyophilized cell extracts, indicating that they are in part responsible for the enzyme inhibition. In vivo studies showed that the toxin inhibited Na(+),K(+)-ATPase activity in anterior gills, whereas an increased augmented activity of glutathione-S-transferase was observed in both kind of gills, indicating that the crab has increased its ability to conjugate the toxin. No significant differences in hemolymph sodium or chloride concentration were detected. This result is in agreement with the lack of effects of microcystin on Na(+),K(+)-ATPase activity of posterior (osmoregulating) gills.

Effects of Anabaena spiroides (Cyanobacteria) aqueous extracts on the acetylcholinesterase activity of aquatic species.

The effects of aqueous extracts from a cyanobacteria species, Anabaena spiroides, on fish (Odontesthes argentinensis), crab (Callinectes sapidus), and purified eel acetylcholinesterase (AChE) activity were studied. In vitro concentrations of A. spiroides aqueous extract that inhibited 50% of enzyme activity (IC50) were 23.0, 17.2, and 45.0 mg/L of lyophilized cyanobacteria for eel, fish, and crab AChE, respectively. Eel AChE inhibition follows pseudo-first-order kinetics, the same expected for organophosphorus pesticides. Inhibition of purified eel AChE using mixtures of bioxidized malathion and aqueous extract of A. spiroides showed a competitive feature (p < 0.05), suggesting that the toxin(s) could be structurally similar to an organophosphorus pesticide and that toxins present in the aqueous extract inhibit the active site of the enzyme. The inhibition recovery assays using 2-PAM (0.3 mM) showed that (1) bioxidized malathion inhibited 27.0 +/- 1.1% of crab and 36.5 +/- 0.1% of eel AChE activities; (2) with bioxidized malathion + 2-PAM the registered inhibition was 13.2 +/- 2.1% and 3.7 +/- 0.5% in crab and eel AChE, respectively; (3) the aqueous extract from A. spiroides inhibited 17.4 +/- 2.2% and 59.9 +/- 0.5% of crab and eel AChE activity, respectively; and (4) aqueous extract + 2-PAM inhibited 22.3 +/- 2.6 and 61.5 +/- 0.2% of crab and eel AChEs. The absence of enzyme activity recovery after 2-PAM exposure could imply that the enzyme aging process was extremely quick.