Linalool induces relaxation of the mantle of golden apple snail (Pomacea canaliculata).

The objective of this study was to evaluate the possible relaxing effect of essential oils (EOs) (Aloysia triphylla and Lippia alba) and phytochemicals (citral and linalool) in the gastropod Pomacea canaliculata. Animals were exposed to compounds at the concentrations range of 25-750 µL L-1. Magnesium chloride (MgCl2, 10-50 g L-1) and control group (ethanol 6.75 mL L-1, highest concentration used for treatment dilution) were also tested. The EOs, citral and MgCl2 had no relaxing effect at the concentrations range tested, and citral caused aversive behavior (closure of the operculum) from 90 µL L-1. Exposure to linalool at 25, 50, 100, 200 and 400 µL L-1 relaxed 28, 76, 88, 96 and 100% of the animals, respectively. The concentrations of 25, 50 and 400 µL L-1 differed statistically from each other, while 100 and 200 µL L-1 were equal to 50 and 400 µL L-1. All animals recovered up to 40 min, except at of 400 µL L-1. Linalool is effective for relaxing P. canaliculata and can be useful in management techniques that require relaxation. However, further studies are needed to certify whether linalool is appropriate for maintaining animal welfare in invasive procedures that require total insensitivity.

GABAa receptor subunits expression in silver catfish (Rhamdia quelen) brain and its modulation by Nectandra grandiflora Nees essential oil and isolated compounds.

Studies using silver catfish (Rhamdia quelen) as experimental models are often applied to screen essential oils (EO) with GABAergic-mediated effects. However, the expression of GABAa receptors in the silver catfish brain remains unknown. Thus, we assessed whether silver catfish express GABAa receptor subunits associated with sedation/anesthetic process and/or neurological diseases. Additionally, we evaluated the brain expression of GABAa receptor subunits in fish sedated with Nectandra grandiflora EO and its isolated compounds, the fish anesthetic (+)-dehydrofukinone (DHF), and dehydrofukinone epoxide (DFX), eremophil-11-en-10-ol (ERM) and selin-11-en-4-α-ol (SEL), which have GABAa-mediated anxiolytic-like effects in mice. The expression of the subunits gabra1, gabra2, gabra3, gabrb1, gabrd and gabrg2 in the silver catfish brain were assessed after a 24h-sedation bath by real time PCR. Since qPCR data rarely describes mechanisms of action, which are usually found through interactions with receptors, we also performed an antagonist-driven experiment using flumazenil (FMZ). Real-time PCR detected the mRNA expression of all targeted genes in R. quelen brain. The expression of gabra1 was decreased in fish sedated with ERM; EO increased gabra2, gabra3, gabrb1 and gabrg2 expression; SEL increased gabrb1, gabrd and gabrg2 expression. EO and compounds DFX, SEL and ERM induced sustained sedation in fish and FMZ-bath prompted the recovery from ERM- and DFX-induced sedation. Our results suggest that the EO, SEL, ERM and DFX sedative effects involve interaction with the GABAergic system. Our findings support the use of the silver catfish as robust and reliable experimental model to evaluate the efficacy of drugs with putative GABAergic-mediated effects.

Diphenyl diselenide dietary supplementation protects against methylmercury-chloride-induced immunotoxicity in the head kidney and spleen of grass carp (Ctenopharyngodon idella) via regulation of purinergic signaling and the NLRP3 inflammasome.

This study aimed to evaluate whether dietary supplementation with diphenyl diselenide (Ph2Se2) would prevent the impaired immune and inflammatory responses elicited by methylmercury chloride (CH3HgCl) via protective effects on purinergic signaling in fish immune organs. Tissue and lymphocytic nucleoside triphosphate diphosphohydrolase (NTPDase) activity for adenosine triphosphate (ATP) and adenosine diphosphate (ADP) was downregulated in the head kidney and spleen of grass carp (Ctenopharyngodon idella) exposed to CH3HgCl. Concomitantly, adenosine deaminase (ADA) activity was upregulated. Further, nucleotide-binding oligomerization domain-like receptor (NLRP3) inflammasome gene expression was upregulated in the spleen and head kidney of CH3HgCl-exposed grass carp. Dietary supplementation with Ph2Se2 ameliorated these CH3HgCl-mediated alterations on purinergic enzymes, and their activities returned to baseline levels (except NTPDase activity for ADP). Based on these results, purinergic signaling in immune organs and lymphocytes can be considered a pathway linked to pro-inflammatory effects during exposure to environmental CH3HgCl concentrations, which may contribute to mortality of the affected fish. Since dietary supplementation with 3 mg Ph2Se2/kg in the feed prevented the CH3HgCl-induced alterations, it can be considered a potential suitable treatment to prevent impaired immune and inflammatory responses caused by Hg.

Grape pomace flour alleviates Pseudomonas aeruginosa-induced hepatic oxidative stress in grass carp by improving antioxidant defense.

Pseudomonas aeruginosa is a Gram-negative opportunistic bacterial pathogen in aquaculture systems being associated to extensive liver damage caused by oxidative stress in both marine and freshwater fish. Dietary supplementation with natural antioxidants is considered a rational strategy to prevent hepatic diseases involved with oxidative stress. Bio-residues resulting from the wine industry, such as grape pomace, are potential sources of bioactive phenolic compounds that can be applied as supplement for animal production. Thus, the aim of this study was to evaluate whether dietary supplementation with grape pomace flour (GPF) was able to prevent or reduce the hepatic oxidative damage of grass carp, Ctenopharyngodon idella, experimentally infected by P. aeruginosa. Hepatic reactive oxygen species (ROS), metabolites of nitric oxide (NOx), thiobarbituric acid reactive substances, and protein carbonylation levels were higher in fish experimentally infected by P. aeruginosa compared to the control group. Hepatic superoxide dismutase and catalase activities and antioxidant capacity against peroxyl radical levels were also higher in fish experimentally infected by P. aeruginosa compared to the control group. Dietary supplementation with 300 mg/kg GPF prevented all alterations elicited by P. aeruginosa, with the exception of protein carbonylation levels. The dietary supplementation with 150 mg/kg GPF was not able to avoid alteration of the analyzed variables, being results similar to those infected (positive control). Based on these results, dietary supplementation with 300 mg/kg GPF prevented P. aeruginosa-induced liver damage in grass carp, and this protective effect occurred through prevention on excessive ROS and NOx production, as well as via prevention of lipid damage. Moreover, 300 mg/kg GPF exerted its hepatoprotective effects by improving enzymatic and non-enzymatic antioxidant defense system. In summary, this supplementation can be an interesting approach to prevent P. aeruginosa-induced liver damage.

A caffeine-supplemented diet modulates oxidative stress markers and prevents oxidative damage in the livers of Nile tilapia (Oreochromis niloticus) exposed to hypoxia.

Recent evidence has revealed the involvement of oxidative stress and oxidative damage with health impairment and mortality in fish exposed to hypoxia. Thus, natural compounds with antioxidant and free-radical-scavenging properties, such as caffeine, might help to prevent or reduce hepatic damage elicited by hypoxia. Thus, the aim of this study was to evaluate whether dietary supplementation with caffeine could prevent or reduce oxidative damage in the livers of Nile tilapia (Oreochromis niloticus) exposed to hypoxia. Hepatic reactive oxygen species, lipid peroxidation levels, and xanthine oxidase (XO) activity were higher in fish exposed to hypoxia compared with normoxia. Hepatic catalase, glutathione peroxidase, and glutathione S-transferase activities, as well as the antioxidant capacity against peroxyl radical levels, were lower in fish exposed to hypoxia compared with normoxia. No significant difference between groups was observed regarding hepatic superoxide dismutase activity. Dietary supplementation with 8% caffeine prevented all alterations elicited by hypoxia. Based on this evidence, the use of dietary supplementation with 8% caffeine can be an interesting approach to preventing hepatic lipid damage and impairment of the antioxidant defense system elicited by hypoxia, and this effect can be mediated by protective effects on XO activity.

Disturbance of energetic homeostasis and oxidative damage provoked by trichlorfon as relevant toxicological mechanisms using silver catfish as experimental model.

Recent evidences have suggested the involvement of phosphoryl transfer, catalyzed by creatine kinase (CK), adenylate kinase (AK) and pyruvate kinase (PK), to metabolic alterations and impairment of bioenergetics homeostasis linked to adenosine triphosphate (ATP) production, and utilization during exposure to pesticides. It is recognized that sublethal concentrations of trichlorfon alter hepatic and branchial metabolism, but the pathways involved in this process remains unknown. Thus, the aim of this study was to evaluate whether phosphoryl transfer network can be a pathway involved in the hepatic and branchial metabolic alterations during exposure to sublethal concentrations of trichlorfon using silver catfish Rhamdia quelen as experimental model. Hepatic and branchial CK (cytosolic and mitochondrial isoforms) and PK activities were inhibited after 48 h of exposure to 11 and 22 mg/L trichlorfon compared to control group, while AK activity did not differ between groups. In addition, sodium-potassium pump (Na+, K+-ATPase) activity was lower after 48 h of exposure to 22 mg/L trichlorfon compared to control group. Thiobarbituric acid reactive substances (TBARS) were higher in liver samples after 24 h of exposure to 22 mg/L trichlorfon compared to control group, as well as after 48 h of exposure to 11 and 22 mg/L trichlorfon in liver and gills. Finally, hepatic and branchial non-protein thiol (NPSH) levels were lower after 48 h of exposure to 11 and 22 mg/L trichlorfon. All evaluated parameters did not recover after 48 h in clean water. Based on these evidence, the impairment of phosphoryl transfer network can be considered a pathway involved in the hepatic and branchial metabolic alterations during exposure to sublethal concentrations of trichlorfon. Moreover, alterations on CK and PK activities provoke an impairment on Na+, K+-ATPase activity, which can be mediated by lipid oxidative damage and reduction of NPSH content.

Organophosphate pesticide trichlorfon induced neurotoxic effects in freshwater silver catfish Rhamdia quelen via disruption of blood-brain barrier: Implications on oxidative status, cell viability and brain neurotransmitters.

The aim of this study was to evaluate whether rupture on blood-brain barrier (BBB) can be a pathway for trichlorfon-induced neurotoxic effects, and to investigate its implications on oxidative status, cell viability and brain neurotransmitters in silver catfish (Rhamdia quelen). The BBB permeability was increased in fish exposed for 24 h to 22 mg/L of trichlorfon compared to the control group, as well as in those exposed to 11 and 22 mg/L of trichlorfon for 48 h. Compared to the control group, brain reactive oxygen species and lipid peroxide levels were higher when exposed to 22 mg/L of trichlorfon and 11 and 22 mg/L of trichlorfon after 24 h and 48 h, respectively, while the antioxidant capacity against peroxyl radical levels was lower. Exposure to 22 mg/L of trichlorfon for 24 h reduced brain cell viability compared to the control group, together with 11 and 22 mg/L of trichlorfon for 48 h. Also, brain AChE, Na+ and K+-ATPase activities were reduced in those fish exposed to trichlorfon compared to the control group. Thus, the rupture of BBB can be considered an important pathway involved in trichlorfon-induced neurotoxic effects, which contributes to brain oxidative damage and important changes on brain neurotransmitters.

Caffeine modulates brain purinergic signaling in Nile tilapia (Oreochromis niloticus) under hypoxia conditions: improvement of immune and inflammatory responses.

Purinergic signaling is linked to neurodegenerative and proinflammatory damage during pathological conditions such as hypoxia, but involvement of this pathway in brain damage in fish exposed to environmental hypoxia remains unknown, and we propose dietary supplementation with caffeine in order to improve the immune response. Therefore, the aim of the study was to evaluate whether the enzymatic purinergic signaling pathway is associated with inflammatory brain damage in Nile tilapia (Oreochromis niloticus) exposed to environmental hypoxia and whether dietary supplementation with caffeine (5% and 8%) can prevent these changes in purinergic signaling. Animals were randomly divided into six groups (A-F, n = 6 per group, in triplicate), as follows: groups A-C were submitted to normoxia, while groups D-F were submitted to hypoxia. Groups A and D received the basal diet, while groups B and D and groups C and F received a diet containing 5% and 8% caffeine, respectively, and fed with their respective diets for 21 days. After 21 days, aeration was disconnected (groups D-F) and the dissolved oxygen levels were maintained as follows: group A (6.55 ± 0.23 mg/L), group B (6.51 ± 0.24 mg/L), group C (6.58 ± 0.22 mg/L), group D (1.23 ± 0.11 mg/L), group E (1.20 ± 0.15 mg/L), and group F (1.18 ± 0.13 mg/L). Cerebral triphosphate diphosphohydrolase (NTPDase) using adenosine triphosphate (ATP) as a substrate and 5'-nucleotidase activities decreased in fish exposed to 72 h of hypoxia compared with the normoxia group, while adenosine deaminase (ADA) activity and levels of nitric oxide (NOx) metabolites were higher. Dietary supplementation with 5% and 8% caffeine prevented all alterations elicited by hypoxia, with the exception of ADA activity in the case of 5% caffeine. Based on this evidence, our findings reveal that nucleotide/nucleoside hydrolysis is modified in the brains of fish exposed to 72 h of hypoxia, contributing to inflammatory damage, which apparently is mediated by excessive ATP content in the extracellular medium and by excessive NOx production. Also, the use of a diet containing 5% and 8% caffeine prevented these alterations (except 5% of dietary caffeine on ADA activity) and can be considered an interesting approach to preventing the impairment of immune and inflammatory responses elicited by hypoxia, principally the inclusion of 8% caffeine.

Purinergic signalling displays a pro-inflammatory profile in spleen and splenic lymphocytes of Rhamdia quelen fed with a diet contaminated by fungal mycotoxin: Involvement on disease pathogenesis.

The spleen is an important secondary lymphatic organ that plays a key role in the immune and inflammatory responses of teleost fish. The purinergic signalling has been associated to these types of responses under pathological conditions by the regulation of extracellular adenosine triphosphate (ATP) and its metabolite adenosine (Ado), where both exert potent pro-inflammatory and anti-inflammatory profiles, respectively. The exact pathway involved on the immunotoxic effects of aflatoxin B1 (AFB1) in fish fed with diets containing this mycotoxin remains poorly understood. Thus, the aim of this study was to evaluate whether purinergic signalling exerts anti or pro-inflammatory effects in spleen and splenic lymphocytes of Rhamdia quelen fed with a diet contaminated by AFB1. Ectonucleoside triphosphate diphosphohydrolase (NTPDase) activity (ATP as substrate) decreased in spleen and splenic lymphocytes of fish fed with an AFB1-contaminated diet on day 21 post-feeding compared to fish fed with a basal diet; while adenosine deaminase (ADA) activity increased. No differences were observed between groups or over time regarding NTPDase (adenosine diphosphate as substrate) and 5'-nucleotidase activities. In summary, the purinergic signalling can be a pathway involved in the impairment of the immune and inflammatory responses in fish fed with an AFB1-contaminated diet, contributing to the immunotoxic effects of AFB1 in spleens of fish.

Serum and hepatic oxidative damage induced by a diet contaminated with fungal mycotoxin in freshwater silver catfish Rhamdia quelen: Involvement on disease pathogenesis.

It has been recognized that oxidative stress is implicated in the initiation and progression of diseases due to the excessive formation of free radicals and impairment of the antioxidant defense system, contributing to the mortality of affected animals. The occurrence of a disequilibrium between the antioxidant/oxidant status in serum and liver of freshwater fish fed with aflatoxin B1 (AFB1) remains poorly understood and limited to only a few oxidant variables. Thus, the aim of this study was to evaluate whether an AFB1-contaminated diet causes disturbance on the antioxidant and oxidant status in silver catfish (Rhamdia quelen) of freshwater. Serum and hepatic reactive oxygen species (ROS), metabolites of nitric oxide (NOx), and lipid hydroperoxide increased on days 14 and 21 post-feeding in animals that received AFB1 contaminated diet compared to the control group (basal diet), while protein carbonylation levels increased on day 21 post-feeding. On the other hand, serum and hepatic antioxidant capacity against peroxyl radical and vitamin C levels, as well as glutathione peroxidase and catalase activities were lower on days 14 and 21 post-feeding in animals that received AFB1 contaminated diet compared to the control group. No difference was observed between groups regarding the superoxide dismutase activity and glutathione levels. Based on these evidences, an AFB1-contaminated diet causes a disturbance on serum and hepatic antioxidant/oxidant system due to lipid and protein damage elicited by excessive ROS and NOx production. Also, the antioxidant defense system was unable to avoid or minimize ROS and NOx deleterious effects, and consequently, the oxidative damage. In summary, this disturbance can contribute to understand the pathophysiology and mortality of fish after the consumption of AFB1-contaminated diets.

Aflatoxin B1-contaminated diet disrupts the blood-brain barrier and affects fish behavior: Involvement of neurotransmitters in brain synaptosomes.

It is known that the cytotoxic effects of aflatoxin B1 (AFB1) in endothelial cells of the blood-brain barrier (BBB) are associated with behavioral dysfunction. However, the effects of a diet contaminated with AFB1 on the behavior of silver catfish remain unknown. Thus, the aim of this study was to evaluate whether an AFB1-contaminated diet (1177 ppb kg feed-1) impaired silver catfish behavior, as well as whether disruption of the BBB and alteration of neurotransmitters in brain synaptosomes are involved. Fish fed a diet contaminated with AFB1 presented a behavioral impairment linked with hyperlocomotion on days 14 and 21 compared with the control group (basal diet). Neurotransmitter levels were also affected on days 14 and 21. The permeability of the BBB to Evans blue dye increased in the intoxicated animals compared with the control group, which suggests that the BBB was disrupted. Moreover, acetylcholinesterase (AChE) activity in brain synaptosomes was increased in fish fed a diet contaminated with AFB1, while activity of the sodium-potassium pump (Na+, K+-ATPase) was decreased. Based on this evidence, the present study shows that silver catfish fed a diet containing AFB1 exhibit behavioral impairments related to hyperlocomotion. This diet caused a disruption of the BBB and brain lesions, which may contribute to the behavioral changes. Also, the alterations in the activities of AChE and Na+, K+-ATPase in brain synaptosomes may directly contribute to this behavior, since they may promote synapse dysfunction. In addition, the hyperlocomotion may be considered an important macroscopic marker indicating possible AFB1 intoxication.