Márcio Alberto Geihs

Effects of melatonin in connection with the antioxidant defense system in the gills of the estuarine crab Neohelice granulata

Numerous studies have shown that melatonin exerts some influence on the antioxidant defense system (ADS) in vertebrates, but for crustaceans no such effect has been demonstrated till now. However, earlier reports did show a similar profile of daily variations in the ADS of the gills and the melatonin content of the eyestalk in the crab Neohelice granulata and, thus, the aim of this study was to take a closer look at the effects of melatonin in the gill ADS of N. granulata. Gill ADS is to a minor extent modulated by reactive oxygen species (ROS), because only the nonproteic sulfhydryl (NP-SH) content increases (p<0.05) in the presence of hydrogen peroxide (H(2)O(2)). No significant differences (p>0.05) were observed in the melatonin content of the hemolymph between intact and eyestalkless crabs. Gills from intact and eyestalkless crabs injected with physiological saline showed a daily variation in the total peroxyl radical scavenging capacity (TPRSC) (p<0.05) with two peaks, one at the photophase and another at the scotophase. However, in the gills of eyestalkless crabs injected with melatonin (2 x 10(-12)mol crab(-1)), the daily variation in TPRSC values was abolished (p>0.05). This molecule did not change the NP-SH content (p>0.05) in vitro, but decreased (p<0.05) the oxygen consumption in gills when incubated for 120 min. In the in vivo experiments melatonin also decreased (p<0.05) the oxygen consumption in eyestalkless crabs after 390 min. The results suggest that melatonin does not act directly on the ADS of the gills of N. granulata, but decreases the aerobic metabolism possibly involved in variations of tissue ADS.

Effect of melatonin in the antioxidant defense system in the locomotor muscles of the estuarine crab Neohelice granulata (Decapoda, Brachyura)

In vertebrates, many studies verified different effects of melatonin in the antioxidant defense system (ADS). In crustaceans, few studies have been conducted to verify this possibility. We verified the melatonin effects in the crab Neohelice granulata using low (0.002 and 0.02 pmol/crab) and high (2.0 and 20.0 pmol/crab) melatonin dosages in short-term (0.5h) and long-term (9.5h) experiments. We analyzed the antioxidant capacity against peroxyl radicals (ACAP), reactive oxygen species (ROS) concentration, levels of by products of lipid peroxidation (LPO), oxygen consumption (VO(2)), the activity of glutamate cysteine ligase (gamma-GCL) and catalase (CAT) and glutathione content (GSH). Finally, the effects of exogenous melatonin were verified in terms of melatonin and N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK) content in the muscles of N. granulata. In short-term experiment and low dosages, melatonin increased the VO(2), gamma-GCL activity and GSH content (p<0.05) and decreased melatonin content (p<0.05) without effects in ROS, ACAP and LPO (p>0.05). Possibly, melatonin is acting in the ADS increasing its efficiency and/or acting in mitochondrial activity and/or through signaling muscles to increase its consumption. AFMK was only detected in the eyestalk and cerebroid ganglia. In high dosages melatonin effects decreased, possibly by the desensitization of their receptors. In long-term experiment, melatonin decreased ACAP (p<0.05), and CAT activity (p<0.05) in low dosages. In high dosages melatonin reduced VO(2) (p<0.05) and increased ACAP (p<0.05), possibly stimulating others components of the ADS. In conclusion, melatonin in the locomotor muscles of N. granulata affects the antioxidant/pro-oxidant balance in a time and dosage dependent manner.

The effects of UV radiation on the visual system of the crab Neohelice granulata: A protective role of melatonin

The first and main target-structure of ultraviolet (UV) radiation in animals is the body surface, including the skin and eyes. Here, we investigated cell damage in the visual system of the crab Neohelice granulata acclimated to constant light and exposed to UVA or UVB at 12:00 h for 30 min. The reactive oxygen species (ROS) production, antioxidant capacity against peroxyl radicals (ACAP), lipid peroxidation (LPO) damage, catalase (CAT) activity, and the melatonin immunohistochemical reactivity in the eyestalks were evaluated. The animals that received melatonin and were exposed to UVA and UVB radiation showed a decreased ROS concentration (p<0.05).The ACAP test showed a decrease (p<0.05) in their values when the animals received 2 pmol/crab of melatonin (physiological dose) before the exposure to UVA radiation. The animals exposed to UVB radiation after receiving the same dose of melatonin showed an increase (p<0.05) in the ACAP test compared with the animals exposed to UVB radiation after receiving only crab physiological saline. The CAT activity increased (p<0.05) in the animals that received melatonin and were exposed to UVA and UVB radiation. Animals exposed to UVA and UVB displayed an increase (p<0.05) in the LPO levels, whereas animals treated with melatonin showed lower (p<0.05) LPO levels when irradiated. The results indicate that the specific oxidative parameters altered by UV radiation can be modulated by a physiological dose of melatonin. Moreover, the melatonin regularly produced by virtually all eyestalk cells suggests that it may function to modulate the noxious effects of radiation, at least in the crab N. granulata.

Antioxidant activity stimulated by ultraviolet radiation in the nervous system of a crustacean.

Ultraviolet (UV) radiation can produce biological damage, principally oxidative stress, by increasing the production of reactive oxygen species (ROS). This study evaluated biochemical impairments related to the oxidative stress induced by UVA, UVB and UVA+UVB (solar simulator-SIM) in environmental doses, during five consecutive days of exposure, in the brain and eyestalk of the crab Ucides cordatus. We evaluated these regions by sampling on the 1st, 3rd and 5th days of UV exposure for lipid peroxidation (LPO), antioxidant capacity against the peroxyl radical (ACAP), and the activities of catalase (CAT), glutathione peroxidase (GPX) and glutathione-S-transferase (GST). Immunohistochemical and immunoblotting assays were performed for anti-activated-caspase 3 in the brains. After the first day of exposure, LPO increased in the eyestalks and brains of the UV-exposed animals; ACAP, and CAT, GPX and GST activities also increased in the brains. On the third day, the LPO values in the eyestalk remained high in the UV-exposed groups, while ACAP decreased in the brain and eyestalk and CAT activity remained high in all irradiated groups in both regions. On the fifth day, LPO decreased in the eyestalk and brain of the UV-exposed groups. These results may have been a consequence of the antioxidant defense system (ADS) activity, since CAT activity was high in both regions, ACAP was high in the eyestalks of the SIM group, and GPX activity remained high in the eyestalks of the UVA and UVB groups. Immunohistochemical assays and immunoblotting showed that there was apoptosis in the brains of the UV-exposed crabs. In conclusion, environmental doses of UV can cause oxidative damage to the CNS cells, including apoptosis.

Melatonin as a Signaling Molecule for Metabolism Regulation in Response to Hypoxia in the Crab Neohelice granulata

Melatonin has been identified in a variety of crustacean species, but its function is not as well understood as in vertebrates. The present study investigates whether melatonin has an effect on crustacean hyperglycemic hormone (CHH) gene expression, oxygen consumption (VO2) and circulating glucose and lactate levels, in response to different dissolved-oxygen concentrations, in the crab Neohelice granulata, as well as whether these possible effects are eyestalk- or receptor-dependent. Melatonin decreased CHH expression in crabs exposed for 45 min to 6 (2, 200 or 20,000 pmol·crab−1) or 2 mgO2·L−1 (200 pmol·crab−1). Since luzindole (200 nmol·crab−1) did not significantly (p > 0.05) alter the melatonin effect, its action does not seem to be mediated by vertebrate-typical MT1 and MT2 receptors. Melatonin (200 pmol·crab−1) increased the levels of glucose and lactate in crabs exposed to 6 mgO2·L−1, and luzindole (200 nmol·crab−1) decreased this effect, indicating that melatonin receptors are involved in hyperglycemia and lactemia. Melatonin showed no effect on VO2. Interestingly, in vitro incubation of eyestalk ganglia for 45 min at 0.7 mgO2·L−1 significantly (p < 0.05) increased melatonin production in this organ. In addition, injections of melatonin significantly increased the levels of circulating melatonin in crabs exposed for 45 min to 6 (200 or 20,000 pmol·crab−1), 2 (200 and 20,000 pmol·crab−1) and 0.7 (200 or 20,000 pmol·crab−1) mgO2·L−1. Therefore, melatonin seems to have an effect on the metabolism of N. granulata. This molecule inhibited the gene expression of CHH and caused an eyestalk- and receptor-dependent hyperglycemia, which suggests that melatonin may have a signaling role in metabolic regulation in this crab.

Damage caused during hypoxia and reoxygenation in the locomotor muscle of the crab Neohelice granulata (Decapoda: Varunidae).

The aim of this work was to determine whether different durations of severe hypoxia (0.5 mg O2 L(-1)) followed by reoxygenation cause damage to the locomotor muscle of the crab Neohelice granulata. We evaluated reactive oxygen species (ROS), lipid peroxidation (LPO), mitochondrial membrane potential, and aerobic fiber area of the locomotor muscle after different periods of hypoxia (1, 4, or 10h) followed by 30 or 120 min of reoxygenation. Additionally, changes in cell volume, mitochondrial dysfunction, and infiltration of hemocytes were evaluated after hypoxia and a subsequent 2, 24, or 48 h of reoxygenation. After hypoxia, neither ROS nor LPO increased. However, mitochondrial membrane potential and aerobic fiber area decreased in a time-dependent manner. After reoxygenation, the ROS and LPO levels increased and mitochondrial membrane potential decreased, but these quickly recovered in crabs exposed to 4h of hypoxia. On the other hand, alterations of mitochondria resulted in morphological changes in aerobic fibers, which required more time to recover during reoxygenation after 10h of hypoxia. The locomotor muscles of the crab N. granulata suffer damage after hypoxia and reoxygenation. The intensity of this damage is dependent on the duration of hypoxia. In all experimental situations analyzed, the locomotor muscle of this crab was capable of recovery.

Effects of hypoxia and reoxygenation on the antioxidant defense system of the locomotor muscle of the crab Neohelice granulata (Decapoda, Varunidae).

Crustaceans often occur in areas with variations in oxygen and experience situations known as hypoxia and reoxygenation. Consequences of such situations are increased levels of reactive oxygen species. To avoid oxidative damage intertidal crabs appear to possess an efficient antioxidant defense system (ADS). However, to date, studies have not addressed the strategies that are adopted by the crabs when exposed to hypoxia/reoxygenation cycles. Towards this end we evaluated the ADS and the role of melatonin as an antioxidant in the locomotor muscle of the crab Neohelice granulata under conditions of severe hypoxia and reoxygenation. Total antioxidant capacity against peroxyl radicals and the enzymes superoxide dismutase, catalase, glutathione peroxidase (GPx), and glutathione-S-transferase as well as the key enzyme of glutathione synthesis, glutamate cysteine ligase (GCL), were evaluated. Furthermore, GSH, GSH/GSSG index as well as hemolymph and cellular melatonin levels were evaluated. During hypoxia, increased GPx and GCL activity and decreased GSH and mitochondrial melatonin levels were observed, but during reoxygenation catalase activity increased and cytosolic melatonin levels decreased. It appears that the ADS in the locomotor muscle of N. granulata exert a modulating effect when being confronted with hypoxia and reoxygenation to avoid oxidative stress. During hypoxia, the ADS appear to target GPX activity as well as GSH and mitochondrial melatonin. During reoxygenation, however, evidence suggests that catalase and cytosolic melatonin are involved in the recovery of the locomotor muscle from oxidative damage and the suppression of further damage.

Induction of oxidative stress by chlorothalonil in the estuarine polychaete Laeonereis acuta

Chlorothalonil is an active biocide applied in antifouling paints, and also used as fungicide in agricultural activities with the purpose to protect plants from foliar and seed diseases. Thus, the aim of this study was to evaluate the effects of chlorothalonil exposure on biochemical biomarkers of oxidative metabolism as well as on cholinesterases in the estuarine polychaete Laeonereis acuta. Animals were exposed for 24 and 96 h to the following nominal concentrations of chlorothalonil: 0.1, 10.0 and 100.0 μg/L. The antioxidant capacity against peroxyl radicals (ACAP) and the activity of the enzymes catalase (CAT), superoxide dismutase (SOD), glutathione S-transferase (GST), glutamate cysteine ligase (GCL), acetylcholinesterase (AChE) and propionylcholinesterase (PChE) were evaluated in whole-body tissue. In addition, the levels of reduced glutathione (GSH), lipid peroxidation (LPO), glycogen and lactate levels were also analyzed. A reduction in ACAP levels was observed in animals exposed to the higher chlorothalonil concentration, concomitantly with an induction of GST activity as well as diminution in GSH content in these animals. This disturbance in the redox state of animal tissues leads to an oxidative stress situation, resulting in an induction in LPO levels. It was also demonstrated that chlorothalonil exposure causes alteration in AChE activity, possibly related to damage to membrane lipids. These results demonstrated that chlorothalonil possesses harmful effects to estuarine animals and its use as antifouling biocide has to be carefully reconsidered in risk analysis studies.

Effects of the herbicide Roundup on the polychaeta Laeonereis acuta: Cholinesterases and oxidative stress.

Glyphosate based herbicides, including Roundup, are widely employed in agriculture and urban spaces. The objective of this study was to evaluate the toxicological effects of Roundup on the estuarine polychaeta Laeonereis acuta. Biomarkers of oxidative stress as well as acetylcholinesterase and propionilcholinesterase activities were analyzed. Firstly, the LC50 96h for L. acuta was established (8.19mg/L). After, the animals were exposed to two Roundup concentrations: 3.25mg/L (non-observed effect concentration - NOEC) and 5.35mg/L (LC10) for 24h and 96h. Oxygen consumption was determined and the animals were divided into three body regions (anterior, middle and posterior) for biochemical analysis. An inhibition of both cholinesterase isoforms were observed in animals exposed to both Roundup concentrations after 96h. A significant reactive oxygen species (ROS) reduction was observed in the posterior region of animals in both periods, while antioxidant capacity against peroxyl radicals (ACAP) was reduced in the posterior region of animals exposed for 24h. Considering the antioxidant defense system, both GSH levels and enzyme activities (catalase, superoxide dismutase, glutathione s-transferase, glutathione peroxidase and glutamate cysteine ligase) were not altered after exposure. Lipid peroxidation was reduced in all analyzed body regions in both Roundup concentrations after 24h. Animals exposed to the highest concentration presented a reduction in lipid peroxidation in the anterior region after 96h, while animals exposed to the lowest concentration presented a reduction in the middle region. Overall results indicate that Roundup exposure presents toxicity to L. acuta, causing a disruption in ROS and ACAP levels as well as affects the cholinergic system of this invertebrate species.

Air exposure behavior of the semiterrestrial crab Neohelice granulata allows tolerance to severe hypoxia but not prevent oxidative damage due to hypoxia-reoxygenation cycle.

The air exposure behavior of the semi-terrestrial crab Neohelice granulata during severe hypoxia was studied. This study also verified whether this behavior mitigates possible oxidative damage, namely lipoperoxidation, caused by hypoxia and reoxygenation cycles. The lethal time for 50% of the crabs subjected to severe hypoxia (0.5 mgO2 · L(-1)) with free access to air was compared to that of crabs subjected to severe hypoxia without access to air. Crabs were placed in aquaria divided into three zones: water (when the animal was fully submersed), land (when the animal was completely emerged) and intermediate (when the animal was in contact with both environments) zones. Then the crabs were held in this condition for 270 min, and the time spent in each zone was recorded. Lipid peroxidation (LPO) damage to the walking leg muscles was determined for the following four experimental conditions: a--normoxic water with free access to air; b--hypoxic water without access to air; c--hypoxic water followed by normoxic water without air access; and d--hypoxic water with free access to air. When exposed to hypoxic water, N. granulata spent significantly more time on land, 135.3 ± 17.7 min, whereas control animals (exposed to normoxic water) spent more time submerged, 187.4 ± 20.2 min. By this behavior, N. granulata was able to maintain a 100% survival rate when exposed to severe hypoxia. However, N. granulata must still return to water after periods of air exposure (~ 14 min), causing a sequence of hypoxia/reoxygenation events. Despite increasing the survival rate, hypoxia with air access does not decrease the lipid peroxidation damage caused by the hypoxia and reoxygenation cycle experienced by these crabs.