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In recent years, obesity has become a global problem in children and adolescents, in parallel with the rapid increase in the use of information and communication technology. Recognizing the embryonic causes of obesity may help prevent adverse adult health outcomes. In our study, we hypothesized that radiofrequency-electromagnetic field (RF-EMF) exposure during embryogenesis would affect the molecular mechanisms related to adipogenesis and insulin resistance in zebrafish. To achieve this, we set up a system that emits RF-EMF in the 900 MHz band and subjected zebrafish embryos to its RF-EMF. We created two groups in which we exposed 30 min (EMF-30) and 60 min (EMF-60) per day, and a control group that was not exposed to RF-EMF. We ended the exposure at 96 hpf and analyzed the expression of lepa, ins, and pparg that are involved in the regulation of glucose and lipid metabolism. In addition, we analyzed oxidative stress parameters, embryonic development, and locomotor activity. We found decreased mRNA transcript abundance of lepa, ins, pparg, and activities of superoxide dismutase and acetylcholine esterase, along with increased lipid peroxidation (LPO), nitric oxide (NO), and glutathione S-transferase (GST). Locomotor activity increased in the EMF-30 group and decreased in the EMF-60 group. Our results showed that exposure to RF-EMF during the embryonic period disrupted the molecular pathways related to insulin resistance and adipogenesis in zebrafish. However, due to limited available resources, we were not able to appropriately quantify the actual RF exposure strength of the samples. Hence the results reported here should only be seen as preliminary, and further studies employing high quality exposure apparatus and dosimetry should be carried out in future.
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This study aimed to detect the biocompatibility of bioactivated polycaprolactone/silk fibroin-based nanofibers in vivo using zebrafish embryos. Anti-Bisphenol A (BPA) antibody or lactase enzyme was immobilized on electrospun nanofibers, for making the nanofiber bioactive. Lactase immobilized nanofiber was developed to hydrolyze lactose and produce milk with reduced lactose. Anti-BPA antibody immobilized nanofiber was developed to remove bisphenol A from liquids. To test the biocompatibility of the bioactive nanofibers, the zebrafish embryos were divided into 4 groups; control, raw nanofiber, lactase immobilized nanofiber, and anti-BPAantibody immobilized nanofiber groups. In nanofiber-based exposure groups; nanofibers were incubated separately in the embryonic development medium. Subsequently, the embryos were kept in these development mediums for 72 h post-fertilization (72 hpf) and their developmental analyzes were performed. At the end of 72 hpf, zebrafish embryos were homogenized. Lipid peroxidation and nitrite oxide levels, and superoxide dismutase and glutathione-S-transferase activities were determined to monitor the disturbance of oxidant-antioxidant balance in zebrafish embryos. Exposure to bioactive nanofibers slightly disrupted the oxidant-antioxidant balance, but this change did not affect the mortality and hatching times of the embryos. In conclusion, zebrafish embryos have been effectively used in biocompatibility testing for bioactive nanofibers suggesting that these materials are biocompatible.
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Compuestos de Bencidrilo , Embrión no Mamífero , Fibroínas , Lactasa , Nanofibras , Fenoles , Poliésteres , Pez Cebra , Animales , Pez Cebra/embriología , Compuestos de Bencidrilo/toxicidad , Fenoles/toxicidad , Fenoles/química , Nanofibras/química , Nanofibras/toxicidad , Embrión no Mamífero/efectos de los fármacos , Lactasa/metabolismo , Fibroínas/química , Fibroínas/toxicidad , Poliésteres/química , Poliésteres/toxicidad , Enzimas Inmovilizadas/química , Enzimas Inmovilizadas/metabolismo , Desarrollo Embrionario/efectos de los fármacos , Anticuerpos , Glutatión Transferasa/metabolismo , Superóxido Dismutasa/metabolismoRESUMEN
Benzoic acid, the most basic aromatic carboxylic acid, is produced industrially and used in cosmetic, hygiene, and pharmaceutical items as a flavoring ingredient and/or preservative. The significance of sodium benzoate, a metabolite of cinnamon, used as a food preservative and FDA-approved medication to treat urea cycle abnormalities in humans, has been shown to raise the levels of neurotrophic factors. Valproic acid (VPA), a commonly used anti-epileptic and mood-stabilizing medication, causes behavioral and intellectual problems and is a commonly used agent to induce animal model for autism. Aim of this study is to determine the effects of benzoic acid synthesized from Cinnamomum Cassia by green chemistry method on gene expressions related to autism development in case of VPA toxicity. Zebrafish embryos were exposed to low and high doses of benzoic acid for 72 h post-fertilization. Locomotor activities were determined. Acetylcholinesterase (AchE), lipid peroxidation, nitric oxide (NO), sialic acid (SA), glutathione (GSH)-S-transferase, catalase (CAT), and superoxide dismutase (SOD) activities were determined spectrophotometrically. eif4b, adsl, and shank3a expressions were determined by RT-PCR as autism-related genes. Although high-dose benzoic acid inhibited locomotor activity, benzoic acid at both doses ameliorated VPA-induced disruption in oxidant-antioxidant balance and inflammation in zebrafish embryos and was effective in improving the impaired expression of autism-related genes.
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Ácido Benzoico , Cinnamomum aromaticum , Embrión no Mamífero , Tecnología Química Verde , Ácido Valproico , Pez Cebra , Animales , Pez Cebra/embriología , Ácido Valproico/toxicidad , Embrión no Mamífero/efectos de los fármacos , Embrión no Mamífero/metabolismo , Cinnamomum aromaticum/química , Ácido Benzoico/toxicidad , Ácido Benzoico/química , Síndromes de Neurotoxicidad/metabolismo , Síndromes de Neurotoxicidad/etiología , Estrés Oxidativo/efectos de los fármacos , Relación Dosis-Respuesta a DrogaRESUMEN
Bio-sourced insect repellents are becoming more popular due to their safer applications. Known for its strong fly-repellent property, Cis, trans-para-menthane-3,8-diol (PMD) is the main component of the lemon eucalyptus essential oil and is synthesized from citronellal. In April 2005, US Centers for Disease Control approved two fly repellents that do not contain N,N-diethyl-meta-toluamide (DEET), including PMD. Due to the intentional and pervasive human exposure caused by DEET as insect repellent, concerns have been raised about its toxicological profile and potential harm to people. We hypothesized PMD would have a different toxicological profile than DEET. We synthesized PMD from Eucalyptus citriodora using green chemistry methods and analyzed its structures by 1H-NMR,13C-NMR, and GC/MS spectral methods. We used MTS assay to determine the percentage inhibition of PMD and DEET on keratinocyte (human epidermal keratinocyte [HaCaT]) cells. The xCelligence system was used and followed at real time. Effects of PMD and DEET on zebrafish embryo development were monitored and levels of lipid peroxidation, glutathione-S-transferase (GST), superoxide dismutase (SOD), and acetylcholinesterase (AchE) were evaluated at 72 h post-fertilization using spectrophotometric methods. Our results showed that while DEET inhibited human keratinocyte cell growth, while imporved cell viability and proliferation was exposed in PMD exposed group. In zebrafish embryos, PMD was less toxic in terms of development, oxidant-antioxidant status, and AChE activities than DEET. Based on these results we suggest an efficient method using green chemistry for the synthesis of PMD, which is found to be less toxic in zebrafish embryos and human keratinocyte cells.
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Given that global prevalence of Parkinson's disease (PD) is expected to rise over the next few decades, understanding the mechanisms and causes of PD is critical. With emphasis on gut-brain axis, we sought to assess the impact of gentisic acid (GA), a diphenolic compound generated from benzoic acid, in rotenone (Rot) induced PD model in zebrafish. For thirty days, adult zebrafish were exposed to GA and rotenone. Tox-Track program was used to analyze locomotor behaviors in the control, GA, Rot, and Rot + GA groups. LC-MS/MS was performed in brain and intestinal tissues. Proteome Discoverer 2.4 was used to analyze raw files, peptide lists were searched against Danio rerio proteins. Protein interactions or annotations were obtained from STRING database. Tyrosine hydroxylase (Th) staining was performed immunohistochemically in the brain. PD-related gene expressions were determined by RT-PCR. Lipid peroxidation, nitric oxide, superoxide dismutase, glutathione S-transferase, and acetylcholinesterase were measured spectrophotometrically. Improved locomotor behaviors were observed by GA treatment in Rot group as evidenced by increased average speed, exploration rate, and total distance. 5214 proteins were identified in intestinal tissues, 4114 proteins were identified in brain by LC-MS/MS. Rotenone exposure altered protein expressions related to oxidative phosphorylation in brain and intestines. Protein expressions involved in ferroptis and actin cytoskeleton changed in brain and intestines. Altered protein expressions were improved by GA. GA ameliorated Th-immunoreactivity in brain, improved park2, park7, pink1, and lrrk2 expressions. Our results show that GA may be a candidate agent to be evaluated for its potential protective effect for PD.
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Eje Cerebro-Intestino , Encéfalo , Modelos Animales de Enfermedad , Fármacos Neuroprotectores , Rotenona , Pez Cebra , Animales , Fármacos Neuroprotectores/farmacología , Rotenona/toxicidad , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Eje Cerebro-Intestino/efectos de los fármacos , Eje Cerebro-Intestino/fisiología , Neurotoxinas/toxicidad , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/tratamiento farmacológico , Locomoción/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacosRESUMEN
Exposure of zebrafish embryos to glucose is a suitable model for the fetal hyperglycemia seen in gestational diabetes. Diethylhexyl phthalate (DEHP), which is considered an endocrine-disrupting chemical, is one of the most common phthalate derivatives used in stretching plastic and is encountered in every area where plastic is used in daily life. In the present study, the effects of DEHP on pathways related to insulin resistance and obesity were examined in zebrafish embryos exposed to glucose as a fetal hyperglycemia model. Zebrafish embryos were exposed to DEHP, glucose, and glucose + DEHP for 72 h post-fertilization (hpf), and developmental parameters and locomotor activities were monitored. At 72 hpf ins, lepa, pparγ, atf4a, and il-6 expressions were determined by RT-PCR. Glucose, lipid peroxidation (LPO), nitric oxide (NO) levels, glutathione S-transferase (GST), superoxide dismutase (SOD), and acetylcholine esterase (AChE) activities were measured spectrophotometrically. Compared with the control group, glucose, LPO, GST activity, il6, and atf4a expressions increased in all exposure groups, while body length, locomotor, and SOD activities decreased. While AChE activity decreased in the DEHP and glucose groups, it increased in the glucose + DEHP group. Although glucose exposure increased pparγ and lepa expressions, DEHP significantly decreased the expressions of pparγ and lepa both in the DEHP and glucose + DEHP groups. Our findings showed that DEHP amplified oxidant and inflammatory responses in this fetal hyperglycemia model, predisposing insulin resistance in zebrafish embryos.
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Dietilhexil Ftalato , Hiperglucemia , Resistencia a la Insulina , Animales , Dietilhexil Ftalato/toxicidad , Pez Cebra/metabolismo , Oxidantes , PPAR gamma , Glucosa/metabolismo , Hiperglucemia/inducido químicamente , Superóxido DismutasaRESUMEN
Prenatal exposure to environmental factors may play an important role in the aetiopathogenesis of autism spectrum disorder (ASD). We aim to investigate the potential effects of low-dose x-rays from dental diagnostic x-rays on neurodevelopment and molecular mechanisms associated with ASD in developing zebrafish embryos. Zebrafish embryos were divided into four groups and exposed using a dental x-ray unit: control, 0.08, 0.15 and 0.30 seconds, which are exemplary exposure settings for periapical imaging. These exposure times were measured as 7.17, 23.17 and 63.83 mSv using optical stimulated luminescence dosimeters. At the end of 72 hours post-fertilization, locomotor activity, oxidant-antioxidant status, and acetylcholine esterase (AChE) activity were analyzed. Expression of genes related to apoptosis (bax, bcl2a, p53), neurogenesis (α1-tubulin, syn2a, neurog1, elavl3) and ASD (eif4eb, adsl2a, shank3) was determined by RT-PCR. Even at reduced doses, developmental toxicity was observed in three groups as evidenced by pericardial edema, yolk sac edema and scoliosis. Deleterious effects of dental x-rays on neurogenesis through impaired locomotor activity, oxidative stress, apoptosis and alterations in genes associated with neurogenesis and ASD progression were more pronounced in the 0.30s exposure group. Based on these results we suggest that the associations between ASD and low-dose ionizing radiation need a closer look.
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Traditionally, morphological, conventional, and toxicological approaches have been used to demonstrate neurotoxicity; however, there has been a growing interest in animal behavioral methods for assessing neurotoxicity, both at the scientific and regulatory levels. Zebrafish (Danio rerio) is a small tropical freshwater fish currently recognized as a suitable model organism for investigating developmental neurotoxicity. There are many animal-tracking software programms used for behavioral analysis in biomedical research. Some of these software programms require a fee, which may exceed the laboratory budget and require detailed technical equipment. As a solution, freely available programs can be used. However, animal tracking may not be possible due to the glare from the aquatic environment of fish, and the small size of zebrafish embryos makes animal tracking difficult. In our laboratory, we developed a semi-automatic system to overcome these difficulties by using three different software available for free. This chapter explains the system for zebrafish embryos and adult zebrafish.
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Investigación Biomédica , Síndromes de Neurotoxicidad , Perciformes , Animales , Pez Cebra , Agua Dulce , LaboratoriosRESUMEN
Craniofacial abnormalities are one of the most frequent birth malformations in humans, affecting around one in every thousand live births. The zebrafish (Danio rerio), a model organism that has seen increased usage in toxicological research in recent years, is ideal for assessing the effects of various chemicals on bone and cartilage structures. Chondrogenesis developed in zebrafish embryos by embryonic day 2, and supporting cartilage components are apparent at hatching (72 h post-fertilization). Individual cartilage may be observed using Alcian Blue staining as early as 2 days post-fertilization (dpf). The preferential binding of Alcian Blue causes the staining of zebrafish cartilage to acidic glycoproteins in an acidic solution (pH 2.2). In 72-120 hpf embryos, the cranial skeleton is easily visible after cartilage staining using Alcian Blue. Various cranial lengths and structures can be determined by measuring specific distances and angles to optimize the quantitative analysis of cranial malformations in zebrafish after exposure to various toxic agents. This chapter explains the Alcian Blue staining procedure to identify craniofacial cartilaginous structures in zebrafish embryos.
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Perciformes , Pez Cebra , Humanos , Animales , Azul Alcián , Cartílago , Cráneo , Coloración y EtiquetadoRESUMEN
A commonly employed technique in molecular biology to evaluate the temporal and spatial expression of a certain gene is in situ hybridization. This method is an effective strategy to construct synexpression groups, co-expressed genes acting in shared biological processes, and to find new members of genes engaged in the same signaling pathways to discover similar spatial and temporal expression patterns in zebrafish embryos. The major disadvantage of this method is that RNA probes can penetrate within 2 days of post-fertilization embryos, and therefore, in later developmental stages, the probe can only reach the surface tissues. Further application of the method in histological sections will be required for a complete and accurate gene expression investigation. However, this method is highly effective at late embryogenesis and early larval stages for observing gene expression in endodermal derivatives and sensory organs. RNA probes for in situ hybridization can be prepared through in vitro transcription from plasmids carrying specific promoter elements and mRNA-specific cDNA, or an alternative polymerase chain reaction (PCR) method can be used through PCR amplification. This chapter describes the procedures for detecting gene expression in zebrafish embryos using whole-mount RNA in situ hybridization.
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Perciformes , ARN , Animales , Sondas ARN , Pez Cebra/genética , ARN Mensajero/genética , Hibridación in SituRESUMEN
The fast zebrafish embryonic development offers an opportunity for the study of genes crucial for developmental processes. Several genes associated with human diseases have orthologs in zebrafish. Enhanced comprehension of a gene's function can be achieved by knowing when and where it is expressed. This knowledge also makes it possible to alter a gene-by-gene knockdown in a time- and place-specific manner. Moreover, gene expression analyses contribute greatly to teratogenicity studies in zebrafish embryos. Therefore, the importance of examining the differences between the expressions of these genes has increased day by day. The incorporation of reverse transcription (RT) as the initial step prior to thermal cycling in quantitative real-time polymerase chain reaction (RT-qPCR) has made a significant contribution to RNA research. RT-qPCR is the gold standard and an effective method for gene expression analysis. Quick readout, high sensitivity, reproducibility, as well as high potential throughput, along with reliable quantification, are just a few advantages of RT-qPCR. However, there are drawbacks to its application, such as RNA's inherent variability, impurities during RNA extraction, and variations in reverse transcription and PCR efficiencies. Implementing a precise normalization technique is crucial to account for these inaccuracies. In this chapter, the protocol for gene expression analysis by RT-qPCR for zebrafish embryos is explained.
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Perciformes , Pez Cebra , Femenino , Humanos , Animales , Reacción en Cadena en Tiempo Real de la Polimerasa , Reproducibilidad de los Resultados , Pez Cebra/genética , ARN , Expresión GénicaRESUMEN
Chemical exposure in humans begins from the zygote stage and continues throughout the development of the embryo and the fetus. Zebrafish are one of the most powerful model organisms used in many research areas, including genetics, environmental toxicology, development, DNA damage and repair, cancer, and other diseases. Among the advantages that facilitate the use of zebrafish as a model for studies are features such as high homology with the human genome, small size, and high reproductive potential in short periods. The use of zebrafish embryos in research has increased rapidly due to their advantageous properties, including extrauterine development and the transparent feature of the embryos. However, there are thousands of genes that can be encountered in research, and in this case, the workforce is too much. This workload has been alleviated with the developed technologies. Microarray is one of these technologies. An important parameter in this assay is the RIN value. The RIN value ranges from 1 to 10, indicating mRNA degradation, and therefore helps to decide whether to continue the study. In this chapter, microarray analysis, which is one of the main techniques used in the determination of gene expression in zebrafish embryos, is described.
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Perciformes , Pez Cebra , Humanos , Animales , Pez Cebra/genética , Análisis por Micromatrices , Bioensayo , Daño del ADN , Expresión GénicaRESUMEN
Salicylic acid topical is used to treat variety of skin conditions. However, salicylic acid in these products is generated through industrial synthesis and has been shown to negatively impact fetal development and cause congenital abnormalities. We hypothesized that teratogenic effects reported in salicylic acid can be prevented by naturally synthesizing salicylic acid from wintergreen oil using green chemistry method. For this purpose, we investigated the effects of natural salicylic acid (NSA) synthesized from wintergreen oil using green chemistry and synthetic salicylic acid (SSA) on keratinocyte cell (HaCaT) proliferation and zebrafish embryo development. NSA structures were analyzed by 1H NMR, 13C NMR, and GC/MS methods. Percentage inhibition against HaCaT cell was determined by MTS assay. xCelligence system was used for cellular activities. Zebrafish embryos were exposed to NSA and SSA for 72 h post-fertilization. Lipid peroxidation, nitric oxide, sialic acid, glutathione-S-transferase, catalase, and superoxide dismutase were evaluated using biochemical methods. Expressions of nqO1, gfap, bdnf, vtg, egr, cyp1a, and igf2 were determined by RT-PCR as developmental indicators. MTS and RT-cell analysis showed increased cell viability by NSA, whereas SSA decreased cell viability. NSA beneficially affected zebrafish embryo development while SSA exerted deleterious effects through oxidant-antioxidant status, inflammation, and development. Results of our study showed for the first time that synthesis of salicylic acid from wintergreen oil by green chemistry overcomes its cytotoxicity in keratinocyte cells and teratogenicity in zebrafish embryos. This finding is important for drug research on safe topical applications during pregnancy, when preventing exposure to drug and chemical-derived teratogens is vital.
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Aceites Volátiles , Extractos Vegetales , Ácido Salicílico , Pez Cebra , Animales , Ácido Salicílico/toxicidad , Ácido Salicílico/metabolismo , Embrión no Mamífero , Queratinocitos , SalicilatosRESUMEN
Panoramic x-ray units are widely used in dental radiodiagnostics. Patients are exposed to relatively low radiation doses with panoramic imaging, but considering lifetime frequency of exposure, even a small risk can have serious health consequences. Our aim was to assess the effects of panoramic x-rays at two different exposure times on developing zebrafish embryos, focusing on oxidative stress, inflammation, apoptotic pathways, and development. Zebrafish embryos were divided into three groups: control, standard panoramic (SPE, 5.5 s exposure time) and pedodontic panoramic x-ray group (PPE, 4.8 s exposure time). Optically stimulated luminescence dosimeters were used to measure absorbed doses. Mean radiation doses for SPE and PPE were 7.83 mSv and 5.83 mSv respectively. At the end of 96 h post-fertilization, lipid peroxidation (LPO), nitric oxide (NO), reduced glutathione (GSH), glutathione S-transferase and superoxide dismutase were measured in the embryos. Expressions of genes related with inflammation (tnfα, il6, ill15, il21), immunoregulation (ifng) and apoptosis (p53, bax, casp2, casp3, casp8) were determined by RT-PCR. Even at reduced doses at high-speed mode, developmental toxicity was observed in both groups as evidenced by decreased pigmentation, yolk sac oedema, and spinal curvature. While deterioration of oxidant-antioxidant balance, suppression of immune response, induction of inflammation and apoptosis were observed through increased LPO, NO, decreased GSH, ifng, and increased expressions of genes related with inflammation and apoptosis, these effects were more pronounced in the SPE group. These results demonstrate the influence of exposure time and indicate the need for further consideration of optimal panoramic modes from a radiation-induced damage perspective.
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Embrión no Mamífero , Pez Cebra , Animales , Humanos , Pez Cebra/genética , Pez Cebra/metabolismo , Rayos X , Embrión no Mamífero/metabolismo , Estrés Oxidativo/genética , Apoptosis/genética , Inflamación/inducido químicamente , Inflamación/metabolismoRESUMEN
Porphyromonas gingivalis (P. gingivalis), a key pathogen in periodontal diseases, is also associated with hyperglycemia-associated systemic diseases, including diabetes mellitus (DM). Gingipains are the most important endotoxins of P. gingivalis, and in vivo studies using gingipains are scarce. Zebrafish (Danio rerio) is a vertebrate with high physiological and genetic homology with humans that has multiple co-orthologs for human genes, including inflammation-related proteins. The aim of our study was to determine the effects of gingipain in a hyperglycemia-induced zebrafish model by evaluating inflammation, oxidant-antioxidant status, and the cholinergic system. Adult zebrafish were grouped into the control group (C), hyperglycemia-induced group subjected to 15 days of overfeeding (OF), gingipain-injected group (GP), and gingipain-injected hyperglycemic group (OF + GP). At the end of 15 days, an oral glucose tolerance test (OGTT) was performed, and fasting blood glucose (FBG) levels were measured. Lipid peroxidation (LPO), nitric oxide (NO), glutathione (GSH), glutathione S-transferase, catalase, acetylcholinesterase (AChE), alkaline phosphatase (ALP), and sialic acid (SA) levels were determined spectrophotometrically in the hepatopancreas. The expression levels of tnf-âº, il-1ß, ins, crp, and the acute phase protein YKL-40 analogs chia.5 and chia.6 were evaluated by RTâPCR. After two weeks of overfeeding, significantly increased weight gain, FBG, and OGTT confirmed that the zebrafish were hyperglycemic. Increased oxidative stress, inflammation, and AChE and ALP activities were observed in both the overfeeding and GP groups. Amplification of inflammation and oxidative stress was evident in the OF + GP group through increased expression of crp, il-1ß, chia.5, and chia.6 and increased LPO and NO levels. Our results support the role of gingipains in the increased inflammatory response in hyperglycemia-associated diseases.
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Parkinson's disease (PD) is the second most common neurodegenerative disease caused by the degeneration of dopaminergic neurons and the accumulation of Lewy bodies. Pain is one of the most common non-motor symptoms in PD, but the molecular mechanism of pain in PD is not fully understood, which prevents early diagnosis of PD. We aimed to determine the changes in opioidergic pathways when external pain is inflicted by inducing pain intraperitoneally in zebrafish, for which we generated a rotenone-induced PD model. After behavioural analyses in control(C), acetic acid (AA), rotenone (ROT), and rotenone+ acetic acid (ROT+AA) groups, catecholamine levels in brain tissue were determined by LC-MS/MS, expression of opioid peptides and their receptors by RT-PCR, expression of tyrosine hydroxylase by immunohistochemical method, and analyses of oxidant-antioxidant parameters by spectrophotometric methods. In the ROT group, distance travelled, average speed, and brain dopamine levels decreased, while LPO (lipid peroxidation) and NO (nitric oxide) increased as indicators of oxidative damage, and the SOD activity decreased. The mRNA expression of lrrk, pink1, and park7 genes associated with PD increased, while the mRNA expression of park2 decreased. This indicates that rotenone exposure is a suitable means to induce PD in zebrafish. The fact that body curvature was higher in the AA group than in the ROT and ROT+AA groups, as well as the decreased expression of penka, pdyn, and ion channels associated with the perception of peripheral pain in the ROT+AA group, suggest that mechanisms associated with pain are impaired in the rotenone-induced PD model in zebrafish.
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Enfermedades Neurodegenerativas , Fármacos Neuroprotectores , Enfermedad de Parkinson , Animales , Pez Cebra , Rotenona/toxicidad , Ácido Acético/farmacología , Cromatografía Liquida , Espectrometría de Masas en Tándem , Estrés Oxidativo , ARN Mensajero , Modelos Animales de Enfermedad , Fármacos Neuroprotectores/farmacologíaRESUMEN
Sodium-dependent glucose co-transporter-2 (SGLT2) inhibitor empagliflozin (EMP), is the new class of oral hypoglycemic agent approved as a treatment for Type 2 diabetes. SGLT2 inhibitors may induce ketogenesis through inhibiting the renal reabsorption of glucose. In recent years, positive effects of ketogenic diets on neurodegenerative diseases such as Parkinson's disease (PD) have been reported by improving autophagy. We aimed to evaluate the effects of EMP treatment as a SGLT2 inhibitor that can mimic the effects of ketogenic diet, in rotenone induced PD model in zebrafish focusing on ketogenesis, autophagy, and molecular pathways related with PD progression including oxidative stress and inflammation. Adult zebrafish were exposed to rotenone and EMP for 30 days. Y-Maze task and locomotor analysis were performed. Neurotransmitter levels were determined by liquid chromatography tandem- mass spectrometry (LC-MS/MS). Lipid peroxidation (LPO), nitric oxide (No), alkaline phosphatase, superoxide dismutase, glutathione, glutathione S-transferase (GST), sialic acid, acetylcholinesterase, and the expressions of autophagy, ketogenesis and PD-related genes were determined. Immunohistochemical staining was performed for the microglial marker L-plastin (Lcp1) and tyrosine hydroxylase (Th). EMP treatment improved DOPAC/DA ratio, Y-Maze task, locomotor activity, expressions of Th and Lcp-1, autophagy and inflammation related (mTor, atg5, tnfα, sirt1, il6, tnfα); PD-related (lrrk2, park2, park7, pink1), and ketone metabolism-related genes (slc16a1b, pparag, and pparab), and oxidant-damage in brain in the rotenone group as evidenced by decreased LPO, No, and improved antioxidant molecules. Our results showed benefical effects of EMP as a SGLT2 inhibitor in neurotoxin-induced PD model in zebrafish. We believe our study, will shed light on the mechanism of the effects of SGLT2 inhibitors, ketogenesis and autopahgy in PD.
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Disruption of the gut-brain axis in Parkinson's disease (PD) may lead to motor symptoms and PD pathogenesis. Recently, the neuroprotective potential of different PPARδ-agonists has been shown. We aimed to reveal the effects of erucic acid, peroxisome proliferator-activated receptors (PPARs)-ligand in rotenone-induced PD model in zebrafish, focusing on the gut-brain axis. Adult zebrafish were exposed to rotenone and erucic acid for 30 days. Liquid chromatography-mass spectrometry and tandem mass spectrometry (LC-MS/MS) analysis was performed. Raw files were analysed by Proteome Discoverer 2.4 software; peptide lists were searched against Danio rerio proteins. STRING database was used for protein annotations or interactions. Lipid peroxidation (LPO), nitric oxide (No), alkaline phosphatase, superoxide dismutase, glutathione S-transferase (GST), acetylcholinesterase and the expressions of PD-related genes were determined. Immunohistochemical tyrosine hydroxylase (TH) staining was performed. LC-MS/MS analyses allowed identification of over 2000 proteins in each sample. The 2502 and 2707 proteins overlapped for intestine and brain. The 196 and 243 significantly dysregulated proteins in the brain and intestines were found in rotenone groups. Erucic acid treatment corrected the changes in the expression of proteins associated with cytoskeletal organisation, transport and localisation and improved locomotor activity, expressions of TH, PD-related genes (lrrk2, park2, park7, pink1) and oxidant-damage in brain and intestines in the rotenone group as evidenced by decreased LPO, No and increased GST. Our results showed beneficial effects of erucic acid as a PPARδ-ligand in neurotoxin-induced PD model in zebrafish. We believe that our study will shed light on the mechanism of the effects of PPARδ agonists and ω9-fatty acids in the gut-brain axis of PD.
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Fármacos Neuroprotectores , PPAR delta , Enfermedad de Parkinson , Animales , Enfermedad de Parkinson/metabolismo , Rotenona , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/uso terapéutico , Pez Cebra , Eje Cerebro-Intestino , Acetilcolinesterasa , Cromatografía Liquida , Ácidos Erucicos , Ligandos , Espectrometría de Masas en Tándem , Modelos Animales de Enfermedad , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina , Proteínas de Pez CebraRESUMEN
Zebrafish (Danio rerio) is becoming an increasingly important model in epilepsy research. Pentylenetetrazole (PTZ) is a convulsant agent that induces epileptic seizure-like state in zebrafish and zebrafish embryos and is most commonly used in antiepileptic drug discovery research to evaluate seizure mechanisms. Classical antiepileptic drugs, such as valproic acid (VPA) reduce PTZ-induced epileptiform activities. Opioid system has been suggested to play a role in epileptogenesis. The aim of our study is to determine the effects of morphine in PTZ-induced epilepsy model in zebrafish embryos by evaluating locomotor activity and parameters related to oxidant-antioxidant status, inflammation, and cholinergic system as well as markers of neuronal activity c-fos, bdnf, and opioid receptors. Zebrafish embryos at 72 hpf were exposed to PTZ (20 mM), VPA (1 mM), and Morphine (MOR) (100 µM). MOR and VPA pretreated groups were treated with either MOR (MOR + PTZ) or VPA (VPA + PTZ) for 20 min before PTZ expoure. Locomotor activity was quantified as total distance moved (mm), average speed (mm/sec) and exploration rate (%) and analyzed using ToxTrac tracking programme. Oxidant-antioxidant system parameters, acetylcholinesterase activity, and sialic acid leves were evaluated using spectrophotometric methods. The expression of c-fos, bdnf, oprm1, and oprd1 were evaluated by RT-PCR. MOR pretreatment ameliorated PTZ-induced locomotor pattern as evidenced by improved average speed, exploration rate and distance traveled. We report the restoration of inflammatory and oxidant-antioxidant system parameters, c-fos, bdnf, and opioid receptor oprm1 as the possible mechanisms involved in the ameliorative effect of MOR against PTZ-induced epileptogenic process in zebrafish embryos.
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Epilepsia , Morfina , Pentilenotetrazol , Animales , Acetilcolinesterasa , Anticonvulsivantes/uso terapéutico , Antioxidantes/metabolismo , Factor Neurotrófico Derivado del Encéfalo , Epilepsia/inducido químicamente , Epilepsia/tratamiento farmacológico , Inflamación , Morfina/uso terapéutico , Estrés Oxidativo , Pentilenotetrazol/toxicidad , Receptores Opioides/genética , Convulsiones/inducido químicamente , Convulsiones/tratamiento farmacológico , Ácido Valproico/uso terapéutico , Pez CebraRESUMEN
We aimed to evaluate how different types of toothpaste (TP) for children affected molecular mechanisms of odontogenesis in zebrafish embryos. Commercially available TPs were selected according to their detergent contents as the cocamidopropyl betaine (CAPB) containing TP (TP1) and sodium lauryl sulfate (SLS) containing TP (TP2). TP3 contained no detergent. Effects of SLS, and CAPB alone were also examined. TP and detergent concentrations affecting development were determined as 750 mg/L and 4 mg/L, respectively. Embryos were exposed to TP1, TP2, TP3, SLS, CAPB, and embryo medium (control) for 72 h post fertilization. Acetylcholinesterase (AChE) activity and oxidant-antioxidant parameters were analyzed spectrophotometrically. Expressions of tooth development genes were evaluated by reverse transcription PCR (RT-PCR). Intraocular distance, lower jaw, and ceratohyal cartilage length were displayed using Alcian Blue staining. axin2 and wnt10a expressions increased in SLS and TP2 groups. igf2a and eve1 expressions decreased in all groups except TP3. nrOb1 expression decreased in TP1, SLS, and CAPB groups. Oxidant-antioxidant balance was disturbed in all groups except TP3, evidenced by increased lipid peroxidation, nitric oxide. SLS, and CAPB groups were more affected in terms of AChE, glutathione-S-transferase, and superoxide dismutase; perturbations were observed in cartilage structures. Altered expression of tooth development gene axin2 correlated with wnt10a, and with changes in cartilage structures in SLS and TP2 groups. TP3 group presented no disruptions in oxidant-antioxidant balance. Our study shows the availability of externally developing zebrafish embryos in examining the effects of TP' contents on embryogenesis.