Transfluthrin- and prallethrin-based insecticides elicit specific enzymatic antioxidant responses in different tissue of zebrafish.

Pyrethroids prallethrin (P-BI) and transfluthrin (T-BI) are among the most commonly used molecules with insecticide action. These molecules comprise different formulations of insecticides largely used in household, agricultural, and animal production fields. However, the increased use of these molecules has led to concerns regarding their safety in animals and humans. Oxidative stress (OS) is believed to be easily established by xenobiotic contacts, such as pyrethroids. We aimed to evaluate and understand the impact of two household insecticides and two doses applied to different tissues of the antioxidant system of zebrafish (Danio rerio). We observed that the effect on the antioxidant system differed between tissues. The muscle was the most affected tissue in the body, the antioxidant enzymes were activated, and a mechanism of non-enzymatic antioxidants was activated; however, it could still cause cellular damage. The observed effect on muscle may be related to the progression of neurodegenerative conditions. In addition, in the brain, these compounds can inactivate the first line of enzymatic antioxidant defense, which is compensated for by the second line, avoiding cellular damage. Ultimately, the gill tissue did not appear to suffer lipid damage, but heme group formation was largely affected by the compounds.

Household based-pyrethroids on adult zebrafish (Danio rerio) exert behavioral and cholinergic changes in different brain regions.

Pyrethroid-based insecticides are largely used for mosquito control. These compounds have household and agricultural applications with different formulations. Two important compounds used as household insecticides are prallethrin and transfluthrin, both from the pyrethroid chemical group. With the mode of action centered on sodium channels, pyrethroids keep the ionic sodium channels open for a long time causing the death of the insect by nervous hyperexcitability. Given the increased use of household insecticides by humans and the incidence of disease outbreaks with unknown etiology such as autism spectrum disease, schizophrenia, and Parkinson's disease we investigate some physiological inputs of these compounds on zebrafish. In this study, we evaluated the social interaction, shoaling formation, and anxiety-like behavior of zebrafish exposed chronically to transfluthrin- and prallthrin-based insecticides (T-BI and P-BI). In addition, we quantified the activity of the enzyme acetylcholinesterase (AChE) in different brain regions. We observed that both compounds caused anxiolytic behavior and reduced shoaling formation and social interaction. Their behavioral biomarkers indicated a harmful ecological effect on the specie as well as a possible impact of these compounds on autism spectrum disorder (ASD) and schizophrenia (SZP). In addition, the AChE activity would change its activity in different brain regions modulating the anxiety-like behavior and social behavior in zebrafish. We conclude that P-BI and T-BI make us alert about the relationship of these compounds with nervous diseases related to cholinergic signaling.

Pyriproxyfen Exposure Impairs Cognitive Parameters and Alters Cortisol Levels in Zebrafish.

Pyriproxyfen is one of the most used larvicides and insecticides; it acts as an analog of juvenile insect hormone (a growth regulator). It is highly toxic during all stages of mosquito development, suppresses metamorphosis, and interferes in insect reproduction and proliferation. Pyriproxyfen and its main metabolite have been shown to affect brain development in rodents. This compound is employed mainly to eliminate outbreaks of the genus Aedes, even in potable water. Despite the increasing number of toxicological studies about larvicides and insecticides-with an indication of continuous use-there have been few studies about the effects of pyriproxyfen in non-target species such as fish. This study evaluated the effects of pyriproxyfen on behavioral, cognitive, and endocrine parameters in zebrafish. We exposed adult zebrafish to different pyriproxyfen (Pestanal®) concentrations (0.125, 0.675, and 1.75 mg/l) for 96 h. We analyzed behavioral parameters, memory, cortisol levels, and gene expression of glucocorticoid receptor (gr) and corticotrophin-releasing factor (crf) after pyriproxyfen exposure. This exposure did not alter locomotion (distance or mean speed), anxiety-like behavior (latency to enter to the top zone of the tank or time in the top zone of the tank), and social or aggressive behavior. However, there was impaired inhibitory avoidance memory at all tested pyriproxyfen concentrations. Cortisol levels were reduced in exposed groups when compared to control or vehicle. However, gr and crf gene expression in pyriproxyfen-treated animals were unaltered when compared to control or vehicle groups. Taken together, these findings indicate that pyriproxyfen may induce cognitive impairment and altered cortisol levels in zebrafish, a non-target species.

Novel object recognition and object location tasks in zebrafish: Influence of habituation and NMDA receptor antagonism.

This study aims to establish a protocol for evaluating the object recognition memory and object location tasks in zebrafish. We evaluated novel the object recognition memory and analyzed the exploration time of the objects during training and testing. Zebrafish explored more the new object in comparison to the familiar object (61% of exploration time during test session). We also tested the object location task and measured the exploration time of each object in the familiar and novel object location. There was a preference to explore the object in the novel location (63% of exploration time during test session). The effect of the non-competitive NMDA receptor antagonist MK-801 was investigated on the object recognition and object location memory. Control (water only) and treated animals (5 µM MK-801) presented a significant preference in exploring the familiar object in comparison to the new object (66 and 68% of exploration time, respectively, during test session); however, 10 µM MK-801-treated animals did not show differences in the exploration time of the objects. In the object location task, the animals treated with the 5 or 10 µM MK-801 did not show a preference for the familiar or novel location whereas the control group had a higher preference in exploring the object in the familiar location (64% of exploration time during test session). Considering the different responses of the control group between original task and in the regimen treatment, we evaluated the impact of habituation on cortisol levels of animals in three different protocols: (1) habituated at the experiment apparatus for 3 days (C1 condition), (2) habituated at the experiment apparatus for 3 days plus treatment tank exposure at fourth day (C2 condition), (3) habituated at the treatment tank and experiment apparatus for 3 days and exposed to treatment tank again at fourth day (C3 condition). The results showed higher levels of cortisol in animals submitted to C2 and C3 conditions compared to animals submitted to C1. When introduced to an acute stressor during C1 condition, we observed an increase in the cortisol levels and an absence of preference for the objects in comparison to control group, which had a preference for novel object and novel location. Fluoxetine treatment induced a decrease in cortisol levels and an absence of preference for the objects in C2 and C3 conditions in comparison to control group, which had a preference for familiar object. However, fluoxetine treatment induced a preference to the novel location in C2 and C3 conditions in comparison to control group, which had a preference for familiar location. These results indicate that treatment tank exposure induced a different performance in object recognition and object location memory due to stress responses. Therefore, these tasks are prone to evaluate memory in physiological and pathological conditions, but its use is limited due to sensitivity to stress caused by manipulation.

Sex and the housing: Effects on behavior, cortisol levels and weight in zebrafish.

Studies with zebrafish use acclimatizing periods of at least one week immediately before the experiments. During this time, animals can be housed in sexually segregated conditions (only females or males in the tank) or in mixed-sex conditions (both sexes in the tank). The influence of sex and housing conditions regarding the presence of one or two sexes is largely unknown in zebrafish. Our aim was to evaluate the influence that sex and housing regarding the sex of animals had in the open tank task, in the inhibitory avoidance memory test, in cortisol levels and weight in zebrafish. Four groups of animals were used: 1) segregated housed females (only females were kept in the tank); 2) segregated housed males (only males were kept in the tank); 3) mixed-sex housed females (only females were analyzed from a tank containing 50% ratio of each sex); 4) mixed-sex housed males (only males were analyzed from a tank containing 50% ratio of each sex). Males showed higher total distance travelled and mean speed when compared to females. In the inhibitory avoidance memory, sexually segregated animals had higher latencies than their mixed-sex counterparts in the 1day test and sexually segregated females presented a memory that persisted longer and was able to be reinstated. Whole-body cortisol levels were higher in mixed-sex animals while weight was lower in these fish. To the best of our knowledge, this is the first time that effects of sex and housing regarding sex were investigated in behavior and physiology of zebrafish.

Glyphosate and Roundup® alter morphology and behavior in zebrafish.

Glyphosate has become the most widely used herbicide in the world, due to the wide scale adoption of transgenic glyphosate resistant crops after its introduction in 1996. Glyphosate may be used alone, but it is commonly applied as an active ingredient of the herbicide Roundup®. This pesticide contains several adjuvants, which may promote an unknown toxicity. The indiscriminate application poses numerous problems, both for the health of the applicators and consumers, and for the environment, contaminating the soil, water and leading to the death of plants and animals. Zebrafish (Danio rerio) is quickly gaining popularity in behavioral research, because of physiological similarity to mammals, sensitivity to pharmacological factors, robust performance, low cost, short spawning intervals, external fertilization, transparency of embryos through larval stages, and rapid development. The aim of this study was evaluate the effects of glyphosate and Roundup® on behavioral and morphological parameters in zebrafish larvae and adults. Zebrafish larvae at 3days post-fertilization and adults were exposed to glyphosate (0.01, 0.065, and 0.5mg/L) or Roundup® (0.01, 0.065, and 0.5mg/L) for 96h. Immediately after the exposure, we performed the analysis of locomotor activity, aversive behavior, and morphology for the larvae and exploratory behavior, aggression and inhibitory avoidance memory for adult zebrafish. In zebrafish larvae, there were significant differences in the locomotor activity and aversive behavior after glyphosate or Roundup® exposure when compared to the control group. Our findings demonstrated that exposure to glyphosate at the concentration of 0.5mg/L, Roundup® at 0.065 or 0.5mg/L reduced the distance traveled, the mean speed and the line crossings in adult zebrafish. A decreased ocular distance was observed for larvae exposed at 0.5mg/L of glyphosate. We verified that at 0.5mg/L of Roundup®-treated adult zebrafish demonstrated a significant impairment in memory. Both glyphosate and Roundup® reduced aggressive behavior. Our data suggest that there are small differences between the effects induced by glyphosate and Roundup®, altering morphological and behavioral parameters in zebrafish, suggesting common mechanisms of toxicity and cellular response.

Effect of mangiferin, a naturally occurring glucoxylxanthone, on fear memory in rats.

Mangiferin (1,3,6,7-tetrahydroxy-2-[3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl] -xanthen-9-one, CAS 4773-96-0), a naturally occurring glucosylxanthone, is widely distributed in higher plants and a constituent of folk medicine. In the present study the effect of systemic administration of mangiferin on behavioural outcomes of neurological function in normal rats was investigated. A single intraperitoneal injection of mangiferin (10, 50 and 100 mg/kg body weight) immediately post-training produced an impairment of long-term memory for aversive training and a reduced freezing in a dose independent manner, when given immediately post-training. The administration of mangiferin 6 h post-training did not affect fear memory. The results indicate that mangiferin might induce deficits of emotionally motivated memory.

A Valepotriate Fraction of Valeriana glechomifolia Shows Sedative and Anxiolytic Properties and Impairs Recognition But Not Aversive Memory in Mice.

Plants of the genus Valeriana (Valerianaceae) are used in traditional medicine as a mild sedative, antispasmodic and tranquilizer in many countries. This study was undertaken to explore the neurobehavioral effects of systemic administration of a valepotriate extract fraction of known quantitative composition of Valeriana glechomifolia (endemic of southern Brazil) in mice. Adult animals were treated with a single intraperitoneal injection of valepotriate fraction (VF) in the concentrations of 1, 3 or 10 mg kg(-1), or with vehicle in the pre-training period before each behavioral test. During the exploration of an open field, mice treated with 10 mg kg(-1) of VF showed reduced locomotion and exploratory behavior. Although overall habituation sessions for locomotion and exploratory behavior among vehicle control and doses of VF were not affected, comparison between open-field and habituation sessions within each treatment showed that VF administration at 1 and 10 mg kg(-1) impaired habituation. In the elevated plus-maze test, mice treated with VF (10 mg kg(-1)) showed a significant increase in the percentage of time spent in the open arms without significant effects in the number of total arm entries. VF at 3 mg kg(-1) produced an impairment of novel-object recognition memory. In contrast, VF did not affect fear-related memory assessed in an inhibitory avoidance task. The results indicate that VF can have sedative effects and affect behavioral parameters related to recognition memory.

Possible enhancement of long-term fear conditioning performance by cisplatin administration in rats / Possível facilitação do desempenho em condicionamento aversivo por administração de cisplatina em ratos

Neurocognitive deficits associated with chemotherapy represent an increasing concern, and the development of animal models to investigate chemotherapy-induced alterations in memory is warranted. Aims: to examine the effects of systemic injection of cisplatin on formation of fear-motivated memory in rats. Methods: male Wistar rats were given an intraperitoneal (i.p.) injection of saline or cisplatin followed by inhibitory avoidance (IA) training. Memory retention was tested 1 and 7 days after training. Control experiments using an open field were carried out to confirm the specificity of the cisplatin-induced alteration in IA performance. Results: cisplatin induced a unexpected enhancement of IA performance measured 7 days after drug injection and training. Control experiments suggested that the effect could not be attributed to sensorimotor alterations or toxic effects. Discussion: the findings are discussed in the light of previous preclinical evidence that cancer chemotherapy can, under some conditions, lead to memory enhancement

É crescente a preocupação com disfunções cognitivas associadas ao uso de quimioterapia para tratamento de câncer. É necessário o desenvolvimento de modelos experimentais que permitam avaliar alterações na memória induzidas por antineoplásicos. Objetivos: avaliar os efeitos da administração sistêmica de cisplatina sobre a formação de memória motivada por medo em ratos. Métodos: ratos Wistar machos receberam uma injeção intraperitoneal (i.p.) de solução salina (controles) ou cisplatina antes de uma sessão de treino em esquiva inibitória (EI). A retenção da memória de EI foi avaliada em testes realizados 1 e 7 dias depois do treino. Experimentos controle em um campo aberto foram usados para confirmar a especificidade das alterações induzidas por cisplatina no desempenho em EI. Resultados: a administração de cisplatina levou a um inesperado aumento do desempenho de EI medido 7 dias após o treino. Os experimentos controle indicam que esse efeito não deve estar relacionado à toxicidade ou alterações em funções sensoriais e motoras. Discussão: os resultados são discutidos em relação a estudos prévios que indicam que, em algumas condições, quimioterápicos antineoplásicos podem levar a uma facilitação da memória

Mangiferin, a naturally occurring glucoxilxanthone improves long-term object recognition memory in rats.

Mangiferin (2-beta-D-glucopyranosyl-1,3,6,7-tetrahydroxyxanthone) is a xanthone widely distributed in higher plants showing antioxidative, antiviral, anticancer, antidiabetic, immunomodulatory, hepatoprotective, and analgesic effects. In the present study, we have investigated the effects of systemic administration of mangiferin on behavioral outcomes of neurological function in normal rats. A single intraperitoneal injection of mangiferin (10, 50, or 100mg/kg body weight) enhanced novel object recognition (NOR) memory when given immediately post-training. The administration of mangiferin 6h post-training did not affect NOR memory. There were no significant differences between groups in the total time exploring both objects, indicating that mangiferin did not affect locomotion or motivation. Mangiferin stimulated cell proliferation and induced a significant increase in the supernatant levels of nerve growth factor (NGF) and tumor necrosis factor (TNF)-alpha in vitro in human U138-MG glioblastoma cells. The results indicate that mangiferin enhances recognition memory through a mechanism that might involve an increase in neurotrophin and cytokine levels.

Systemic administration of doxorubicin impairs aversively motivated memory in rats.

There is growing clinical evidence of cognitive impairment in cancer patients treated with chemotherapy, especially in women treated with drug combinations for breast cancer. Clinical studies have a difficult task of defining which drugs individually are responsible for the cognitive changes and published papers evaluating single agents in experimental models are scanty. In the present study we have investigated the effect of single escalating doses of doxorubicin (DOX) on memory for inhibitory avoidance conditioning (IA) in rats. The doses used were comparable to those applied in the clinic. When given systemically before training, higher doses of DOX impaired IA memory retention measured 24h and 7days, but not 3h after training. DOX did not affect IA retention when given either before or after training in a multiple-trial IA training protocol. Control experiments showed that DOX produced a decrease in exploratory behavior assessed by the number of rearings performed during exploration of an open field. The results indicate that a single systemic administration of DOX might impair long-term aversive learning.

Alterations in oxidative markers in the cerebellum and peripheral organs in MPS I mice.

Mucopolysaccharidosis type I is a lysosomal storage disease with alterations in several organs. Little is known about the pathways that lead to the pathology. Evidences point oxidative stress on lysosomal storage diseases and mucopolysaccharidosis type I. The aim of the present study was to evaluate oxidative biomarkers on mucopolysaccharidosis type I mice model. We evaluated antioxidant enzymatic activity, protein damage and lipid peroxidation in the forebrain, cerebellum, heart, lung, diaphragm, liver, kidney and spleen. Superoxide dismutase activity was increased on cerebellum, lung, diaphragm, liver and kidney of mucopolysaccharidosis type I mice. Catalase activity was increased on cerebellum, spleen and lung. There was no alteration on glutathione peroxidase activity on any of the analyzed organs. Mucopolysaccharidosis type I mice showed increased carbonyl groups on cerebellum, heart and spleen. There was a decrease of thiobarbituric acid-reactive substances on the cerebellum of mucopolysaccharidosis type I mice. The results indicate a oxidative imbalance in this model. As lysosomes are very susceptible to oxidative damage, leading inclusive to cellular death, and lysosomal storage diseases present several alterations on this organelles, this finding can help to elucidate the cellular damage pathways on mucopolysaccharidosis type I.

Long-term memory for aversive training is impaired in Idua(-/-) mice, a genetic model of mucopolysaccharidosis type I.

Mucopolysaccharidosis type I (MPS I) is a lysosomal storage disease that leads to neurodegeneration and neurological deficits, among other pathological and clinical consequences. The aim of the present study was to evaluate neurobehavioral parameters in a genetic mouse model of mucopolysaccharidosis type I (MPS I). During exploration of an open field, adult MPS I (Idua(-/-)) mice showed normal locomotion and anxiety but reduced number of rearings. Idua(-/-) mice performed normally in a novel object recognition memory task and showed normal short-term retention of inhibitory avoidance training. By contrast, long-term retention of inhibitory avoidance was impaired in Idua(-/-) mice. The deficit in inhibitory avoidance memory could not be attributed to reduced footshock reactivity. The results indicate that Idua(-/-) mice present deficits in long-term memory for aversive training and reduced exploratory behavior.