Diphenyl Diselenide Attenuates Mitochondrial Damage During Initial Hypoxia and Enhances Resistance to Recurrent Hypoxia.

Hypoxia plays a significant role in the development of various cerebral diseases, many of which are associated with the potential risk of recurrence due to mitochondrial damage. Conventional drug treatments are not always effective for hypoxia-related brain diseases, necessitating the exploration of alternative compounds. In this study, we investigated the potential of diphenyl diselenide [(PhSe)2] to ameliorate locomotor impairments and mitigate brain mitochondrial dysfunction in zebrafish subjected to hypoxia. Additionally, we explored whether these improvements could confer resistance to recurrent hypoxia. Through a screening process, an appropriate dose of (PhSe)2 was determined, and animals exposed to hypoxia received a single intraperitoneal injection of 100 mg/kg of the compound or vehicle. After 1 h from the injection, evaluations were conducted on locomotor deficits, (PhSe)2 content, mitochondrial electron transport system, and mitochondrial viability in the brain. The animals were subsequently exposed to recurrent hypoxia to assess the latency time to hypoxia symptoms. The findings revealed that (PhSe)2 effectively crossed the blood-brain barrier, attenuated locomotor deficits induced by hypoxia, and improved brain mitochondrial respiration by modulating complex III. Furthermore, it enhanced mitochondrial viability in the telencephalon, contributing to greater resistance to recurrent hypoxia. These results demonstrate the beneficial effects of (PhSe)2 on both hypoxia and recurrent hypoxia, with cerebral mitochondria being a critical target of its action. Considering the involvement of brain hypoxia in numerous pathologies, (PhSe)2 should be further tested to determine its effectiveness as a potential treatment for hypoxia-related brain diseases.

Forebrain glutamate uptake and behavioral parameters are altered in adult zebrafish after the induction of Status Epilepticus by kainic acid.

The development of new antiepileptic drugs is a high-risk/high-cost research field, which is made even riskier if the behavioral epileptic seizure profile is the unique approach on which the development is based. In order to increase the effectiveness of the screening conducted in the zebrafish model of status epilepticus (SE), the evaluation of neurochemical markers of SE would be of great relevance. Epilepsy is associated with changes in the glutamatergic system, and glutamate uptake is one of the critical parameters of this process. Therefore, we evaluated the levels of glutamate uptake in the zebrafish brain and analyzed its correlation with the progression of behavioral changes in zebrafish at different times after the administration of kainic acid (5 mg/kg). The results showed that the zebrafish suffered with lethargy while swimming for up to 72 h after SE, had reduced levels of GFAP cells 12 h after SE, reduced levels of S100B up to 72 h after SE, and reduced levels of glutamate uptake in the forebrain between 3 h and 12 h after SE. The forebrain region of adult zebrafish after SE present similar changes to the neurochemical limbic alterations that are seen in rodent models of SE. This study demonstrated that there is a time window in which to use the KA zebrafish model of SE to explore some of the known neurochemical alterations that have been observed in rodent models of epilepsy and epileptic human patients.

Induction of reactive oxygen species by diphenyl diselenide is preceded by changes in cell morphology and permeability in Saccharomyces cerevisiae.

Organoselenium compounds, such as diphenyl diselenide (PhSe)2 and phenylselenium zinc chloride (PhSeZnCl), show protective activities related to their thiol peroxidase activity. However, depending on experimental conditions, organoselenium compounds can cause toxicity by oxidising thiol groups of proteins and induce the production of reactive oxygen species (ROS). Here, we analysed the toxicity of (PhSe)2 and PhSeZnCl in yeast Saccharomyces cerevisiae. Cell growth of S. cerevisiae after 1, 2, 3, 4, 6, and 16 h of treatment with 2, 4, 6, and 10 µM of (PhSe)2 was evaluated. For comparative purpose, PhSeZnCl was analysed only at 16 h of incubation at equivalent concentrations of selenium (i.e. 4, 8, 12, and 20 µM). ROS production (DCFH-DA), size, granularity, and cell membrane permeability (propidium iodide) were determined by flow cytometry. (PhSe)2 inhibited cell growth at 2 h (10 µM) of incubation, followed by increase in cell size. The increase of cell membrane permeability and granularity (10 µM) was observed after 3 h of incubation, however, ROS production occurs only at 16 h of incubation (10 µM) with (PhSe)2, indicating that ROS overproduction is a more likely consequence of (PhSe)2 toxicity and not its determinant. All tested parameters showed that only concentration of 20 µM induced toxicity in samples incubated with PhSeZnCl. In summary, the results suggest that (PhSe)2 toxicity in S. cerevisiae is time and concentration dependent, presenting more toxicity when compared with PhSeZnCl.

Chronic Treatment with Paraquat Induces Brain Injury, Changes in Antioxidant Defenses System, and Modulates Behavioral Functions in Zebrafish.

Paraquat (PQ) administration consists in a chemical model that mimics phenotypes observed in Parkinson's disease (PD), due to its ability to induce changes in dopaminergic system and oxidative stress. The aim of this study was to evaluate the actions of PQ in behavioral functions of adult zebrafish and its influence on oxidative stress biomarkers in brain samples. PQ (20 mg/kg) was administered intraperitoneally with six injections for 16 days (one injection every 3 days). PQ-treated group showed a significant decrease in the time spent in the bottom section and a shorter latency to enter the top area in the novel tank test. Moreover, PQ-exposed fish showed a significant decrease in the number and duration of risk assessment episodes in the light-dark test, as well as an increase in the agonistic behavior in the mirror-induced aggression (MIA) test. PQ induced brain damage by decreasing mitochondrial viability. Concerning the antioxidant defense system, PQ increased catalase (CAT) and glutathione peroxidase (GPx) activities, as well as the non-protein sulfhydryl content (NPSH), but did not change ROS formation and decreased lipid peroxidation. We demonstrate, for the first time, that PQ induces an increase in aggressive behavior, alters non-motor patterns associated to defensive behaviors, and changes redox parameters in zebrafish brain. Overall, our findings may serve as useful tools to investigate the interaction between behavioral and neurochemical impairments triggered by PQ administration in zebrafish.

Brain zinc chelation by diethyldithiocarbamate increased the behavioral and mitochondrial damages in zebrafish subjected to hypoxia.

The increase in brain levels of chelatable zinc (Zn) in dysfunctions involving oxygen deprivation has stimulated the treatment with Zn chelators, such as diethyldithiocarbamate (DEDTC). However, DEDTC is a redox-active compound and it should be better evaluated during hypoxia. We use the hypoxia model in zebrafish to evaluate DEDTC effects. The exploratory behavior, chelatable Zn content, activities of mitochondrial dehydrogenases, reactive species levels (nitric oxide, superoxide anion, hydroxyl radical scavenger capacity) and cellular antioxidants (sulfhydryl, superoxide dismutase) of zebrafish brain were assessed after recovery, with or without 0.2 mM DEDTC. The increased brain levels of chelatable Zn induced by hypoxia were mitigated by DEDTC. However, the novel tank task indicated that DEDTC did further enhance the exploratory deficit caused by hypoxia. Furthermore, these behavioral impairments caused by DEDTC were more associated with a negative action on mitochondrial activity and brain oxidative balance. Thus, due to apparent pro-oxidant action of DEDTC, our data do not support its use for neuroprotection in neuropathologies involving oxygen deprivation.

Virtual screening of acetylcholinesterase inhibitors using the Lipinski's rule of five and ZINC databank.

Alzheimer's disease (AD) is a progressive and neurodegenerative pathology that can affect people over 65 years of age. It causes several complications, such as behavioral changes, language deficits, depression, and memory impairments. One of the methods used to treat AD is the increase of acetylcholine (ACh) in the brain by using acetylcholinesterase inhibitors (AChEIs). In this study, we used the ZINC databank and the Lipinski's rule of five to perform a virtual screening and a molecular docking (using Auto Dock Vina 1.1.1) aiming to select possible compounds that have quaternary ammonium atom able to inhibit acetylcholinesterase (AChE) activity. The molecules were obtained by screening and further in vitro assays were performed to analyze the most potent inhibitors through the IC50 value and also to describe the interaction models between inhibitors and enzyme by molecular docking. The results showed that compound D inhibited AChE activity from different vertebrate sources and butyrylcholinesterase (BChE) from Equus ferus (EfBChE), with IC50 ranging from 1.69 ± 0.46 to 5.64 ± 2.47 µM. Compound D interacted with the peripheral anionic subsite in both enzymes, blocking substrate entrance to the active site. In contrast, compound C had higher specificity as inhibitor of EfBChE. In conclusion, the screening was effective in finding inhibitors of AChE and BuChE from different organisms.

Evaluation of zinc effect on cadmium action in lipid peroxidation and metallothionein levels in the brain.

Cadmium (Cd) is a known hepato- and nephrotoxic pollutant and zinc (Zn) metalloproteins are important targets of Cd. Hence, the administration of Zn may mitigate Cd toxic effects. However, the interaction of Cd and Zn has been little investigated in the brain. Previously, we reported a protective effect of Zn on mortality caused by Cd in rats. Here, we tested whether the protective effect of Zn could be related to changes in brain Zn-proteins, metallothionein (MT) and δ-aminolevulinate dehydratse (δ-ALA-D). Male adult rats were daily administered for 10 days with Zn (2 mg kg-1), Cd (0.25 and 1 mg kg-1) and 0.25 mg kg-1 of Cd plus Zn and 1 mg kg-1 of Cd plus Zn. The body weight loss, food intake deprivation, and mortality occurred in 1 mg kg-1 of Cd, but Zn co-administration did mitigate these effects. The brain Zn content was not modified by treatment with Cd, whereas cerebral Cd levels increased in animals exposed to Cd. The administration of 0.25 mg kg-1 of Cd (with or without Zn) induced lipid peroxidation and decreased MT concentration, but 2 mg kg-1 of Zn and 1 mg kg-1 of Cd did not change these parameters. Brain δ-ALA-D was not modified by Cd and/or Zn treatments. Since the co-administration of Zn did not attenuate the changes induced by Cd in the brain, our results suggest that the protective effect of Zn on impairments caused by Cd in animal status is weakly related to a cerebral interaction of these metals.

Topographical analysis of reactive zinc in the central nervous system of adult zebrafish (Danio rerio).

Reactive zinc (Zn) is crucial for neuronal signaling and is largely distributed within presynaptic vesicles of some axon terminals of distinct vertebrates. However, the distribution of reactive Zn throughout the central nervous system (CNS) is not fully explored. We performed a topographical study of CNS structures containing reactive Zn in the adult zebrafish (Danio rerio). Slices of CNS from zebrafish were stained by Neo-Timm and/or cresyl violet. The Zn specificity of Neo-Timm was evaluated with Zn chelants, N,N,N',N'-Tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), sodium diethyldithiocarbamate (DEDTC), Zn sulfide washing solution, and hydrochloric acid (HCl). Unfixed slices were also immersed in the fluorescent Zn probe (zinpyr-1). Yellow-to-brown-to-black granules were revealed by Neo-Timm in the zebrafish CNS. Telencephalon exhibited slightly stained regions, while rhombencephalic structures showed high levels of staining. Although stained granules were found on the cell bodies, rhombencephalic structures showed a neuropil staining profile. The TPEN produced a mild reduction in Neo-Timm staining, while HCl and mainly DEDTC abolished the staining, indicating a large Zn content. This result was also confirmed by the application of a Zn probe. The present topographical study revealed reactive Zn throughout the CNS in adult zebrafish that should be considered in future investigation of Zn in the brain on a larger scale.

Evaluation of spontaneous recovery of behavioral and brain injury profiles in zebrafish after hypoxia.

Cerebral hypoxia-ischemia can lead to motor and sensory impairments which can be dependent on the extent of infarcted regions. Since a better understanding of the neurochemical mechanisms involved in this injury is needed, the use of zebrafish as a cerebral hypoxia model has become quite promising because it could improve the knowledge about hypoxia-ischemia. In the current study, we aimed to investigate the spontaneous recovery of brain and behavioral impairments induced by hypoxia in adult zebrafish. Brain injury levels were analyzed by spectrophotometric measurement of mitochondrial dehydrogenase activity by staining with 2,3,5-triphenyltetrazolium chloride, and behavioral profiles were assessed by the open tank test. The induction of hypoxia substantially decreased mitochondrial activity in the brain and impaired behavior. The spontaneous recovery of fish subjected to hypoxia was assessed after 1, 3, 6, 24, and 48h under normoxia. The quantification of brain injury levels showed a significant increase until 24h after hypoxia, but after 48h this effect was completely reversed. Regarding behavioral parameters, we verified that locomotor activity and vertical exploration were impaired by hypoxia and these effects were reversed after 3h under normoxia. Taken together, these results show that zebrafish exhibited transient cerebral and behavioral impairments when submitted to hypoxia, and 1h under normoxic conditions was insufficient to reverse both effects. Therefore, our data help to elucidate the time window of spontaneous recovery in zebrafish after hypoxia and also the behavioral phenotypes involved in this phenomenon.

Behavioral effects of taurine pretreatment in zebrafish acutely exposed to ethanol.

Taurine (TAU) is an amino sulfonic acid that plays protective roles against neurochemical impairments induced by ethanol (EtOH). Mounting evidence shows the applicability of zebrafish for evaluating locomotor parameters and anxiety-like behavioral phenotypes after EtOH exposure in a large scale manner. In this study, we assess the effects of TAU pretreatment on the behavior of zebrafish in the open tank after acute 1% EtOH (v/v) exposure (20 and 60 min of duration) and on brain alcohol contents. The exposure for 20 min exerted significant anxiolytic effects, which were prevented by 42, 150, and 400 mg/L TAU. Conversely, the 60-min condition induced depressant/sedative effects, in which the changes on vertical activity were associated to modifications on the exploratory profile. Although all TAU concentrations kept locomotor parameters at basal levels, 150 mg/L TAU, did not prevent the impairment on vertical activity of EtOH[60]. Despite the higher brain EtOH content detected in the 60-min exposure, 42, 150, and 400 mg/L TAU attenuated the increase of alcohol content in EtOH[60] group. In conclusion, our data suggest that both protocols of acute EtOH exposure induce significant changes in the spatio-temporal behavior of zebrafish and that TAU may exert a preventive role by antagonizing the effects induced by EtOH possibly due to its neuromodulatory role and also by decreasing brain EtOH levels. The hormetic dose-response of TAU on vertical exploration suggests a complex interaction between TAU and EtOH in the central nervous system.

Cd modifies hepatic Zn deposition and modulates δ-ALA-D activity and MT levels by distinct mechanisms.

Cadmium (Cd) is a pollutant that is harmful to human and animals. The liver is a target for Cd accumulation and it can disrupt Zn homeostasis. Here we examined the interaction of Zn and Cd to determine how these two metals could affect δ-aminolevulinate-dehydratase (δ-ALA-D) and metallothionein (MT), two potential molecular endpoints for Cd hepatotoxicity. Cd exposure (0.25 and 1 mg kg1 body weight, i.p., for 10 days) caused a marked increase in hepatic Zn deposition, which was not modified by treatment with Zn (2 mg kg1 , i.p.). Cd caused a dose-dependent increase in hepatic Cd content that was not modified by Zn. Zn and/or Cd treatment increased hepatic δ-ALA-D activity, although the increase caused by Cd was less marked. Reactivation index of δ-ALA-D by DTT was decreased by Zn and Cd exposure, which indicates that Zn protects enzyme from oxidation. Hepatic MT was increased only after exposure to 1 mg kg(-1) Cd and Zn reduced the stimulation of MT synthesis. The results presented here indicate that Cd can redistribute Zn from non-hepatic tissues to liver and the increase in hepatic Zn deposition can account for the increase in hepatic δ-ALA-D activity after Cd exposure. However, Zn blocked the increase in hepatic MT levels caused by Cd. Thus, the modulation of the two molecular endpoints of Cd toxicity used here was distinct, which indicates that the mechanism(s) involved in Zn and Cd distribution, δ-ALA-D and MT regulation are not coincident.

Sistema de monitoramento da estratégia de saúde da família: o desenvolvimento do aplicativo / Monitoring system of the family health strategy: the development of application

Estudo sobre a criação, em janeiro de 2008, do Sistema de Monitoramento e Avaliação da ESF (SMAESF) composto por um conjunto de 17 indicadores (Quadro I). O foco deste trabalho é descrever a criação do aplicativo para os cálculos destes indicadores.

Sistema de monitoramento da estratégia de saúde da família: o desenvolvimento do aplicativo

Estudo sobre a criação, em janeiro de 2008, do Sistema de Monitoramento e Avaliação da ESF (SMAESF) composto por um conjunto de 17 indicadores (Quadro I). O foco deste trabalho é descrever a criação do aplicativo para os cálculos destes indicadores.

Alteraçöes clínicas em bezerros desmamados submetidos à hiperinfecçäo experimental por Strongyloides papillosus (WEDL, 1856) (Nematoda: Rhabdiasoidea) / Clinical observations in weaned calves subjected to experimental hiperinfection with Strongyloides papillosus (WEDL, 1856) (Nematoda: Rhabdiasoidea)

Com o objetivo de avaliar as alteraçöes clínicas em bezerros submetidos à infecçäo experimental por Strongyloides papillosus foram utilizados 24 animais mestiços zebu x holandeses com 42 a 50 dias de idade, divididos aleatoriamente em seis grupos com quatro bezerros. Os animais dos grupos A, B, C, D e E receberam aproximadamente 350.000, 700.000, 1.500.000, 3.200.000 e 10.000.000 larvas infectantes por 100kg de peso corporal respectivamente, sendo que o grupo F näo recebeu inóculo. Todos os animais inoculados desenvolveram prurido cutâneo e hiperemia nas primeiras 24 a 48 horas após infecçäo. A morbidade ou mortalidade observada foi decorrente da debilidade orgânica geral ou emaciaçäo, geradas pelo autoconsumo das reservas energérticas do organismo. Os sintomas clínicos como hiperorexia, poliúria, diarréia, palidez das mucosas e perda de peso ocorreram entre 15 e 29 dias, tendo sido relacionados com a quantidade da dose infectante.

Avaliação preliminar da atividade anti-helmíntica da folha de bananeira (Musa sp) em bovinos

A ação anti-helmíntica da folha de bananeira (Musa sp) foi estudada em 10 bezerros, mestiços, de ambos os sexos, com idadeentre três e cinco meses, infectados naturalmente por Haemonchus sp, Cooperia sp, Trichostrongylus sp e Oesophagostomumsp. A atividade anti-helmíntica da bananeira foi avaliada através de OPG (técnica de Gordon e Whitlock, 1939-modificada) ecoprocultura (técnica de Roberts e O"sullivan, 1950). Ao final do tratamento observou-se redução significativa na média de OPG por coleta e na quantidade de L 3 de Haemonchus sp no grupo teste.

Prevalência de anticorpos contra Babesia bovis (Babés, 1888) e B. bigemina (Smith e Kilborne, 1893) (Apicomplexa: Babesiidae) em bovinos de quatro municípios do Estao do Rio de Janeiro / Prevalence of antibodies against Babesia bovis (Babés, 1888) and B. bigemina (Smith & Kilborne, 1893) (Apicomplexa: Babesiidae) en cattle from four municipality of Rio de Janeiro State

Realizou-se um levantamento sorológico para o diagnóstico de anticorpos da classe IgG contra B. bovis e B. bigemina utilizando-se o teste de imunofluorescência indireta (IFI). No total examinou-se 218 soros colhidos em quatro municípios de diferentes regiöes do Estado do Rio de Janeiro. A soroprevalência encontrada foi de 82,11 por cento para B. bovis e 94,03 por cento para B. bigemina. Oitenta e três soros foram examinados comparativamente nos testes de IFI e teste de conglutinaçäo rápida em placa (TCR). Os resultados, soropositivos, foram 87,95 por cento e 92,77 por cento na IFI e 85,54 por cento e 98,79 por cento no TCR, respectivamente, para B. bovis e B. bigemina. Estes dados indicam que os municípios estudados säo áreas de estabilidade enzoótica. A análise de variância indicou que o TCR tem sensibilidade idêntica a da IFI, näo diferindo significativamente (P(3) 0,05), tanto no diagnóstico de anticorpos contra B. bovis, como contra B. bigemina. Estes dados indicam que o TCR é uma técnica viável para estudos soroepidemiológicos.