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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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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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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
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
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
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
Parkinson's disease (PD) is one of the most common forms of neurodegenerative diseases and research on potential therapeutic agents for PD continues. Rotenone is a neurotoxin that can pass the blood-brain barrier and is used to generate PD models in experimental animals. Boron is a microelement necessary for neural activity in the brain. Antioxidant, non-cytotoxic, anti-genotoxic, anti-carcinogenic effects of boric acid, the salt compound of boron has been reported before. Boronic acids have been approved for treatment by FDA and are included in drug discovery studies and pyridine boronic acids are a subclass of heterocyclic boronic acids used in drug design and discovery as substituted pyridines based on crystal engineering principles. The aim of our study was to determine the effect of 3-pyridinylboronic acid in rotenone-exposed zebrafish embryos, focusing on oxidant-antioxidant parameters and gene expression levels of nuclear factor erythroid 2-related factor 2 (Nrf2) target genes gclm, gclc, hmox1a, nqo1, and PD related genes, brain-derived neurotrophic factor, dj1, and tnfα. Zebrafish embryos were exposed to Rotenone (10 µg/l); Low Dose 3-Pyridinylboronic acid (100 µM); High Dose 3-Pyridinylboronic acid (200 µM); Rotenone + Low Dose-3-Pyridinylboronic acid (10 µg/l + 100 µM); Rotenone + High Dose-3-Pyridinylboronic acid (10 µg/l + 200 µM) in well plates for 96 h post-fertilization (hpf). Our study showed for the first time that 3-pyridinylboronic acid, as a novel sub-class of the heterocyclic boronic acid compound, improved locomotor activities, ameliorated oxidant-antioxidant status by decreasing LPO and NO levels, and normalized the expressions of bdnf, dj1, tnf⺠and Nrf2 target genes hmox1a and nqo1 in rotenone exposed zebrafish embryos. On the other hand, it caused the deterioration of the oxidant-antioxidant balance in the control group through increased lipid peroxidation, nitric oxide levels, and decreased antioxidant enzymes. We believe that these results should be interpreted in the context of the dose-toxicity and benefit-harm relationship of the effects of 3-pyridinylboronic.
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Fármacos Neuroprotectores , Enfermedad de Parkinson , Animales , Antioxidantes/metabolismo , Antioxidantes/farmacología , Boro/metabolismo , Boro/farmacología , Ácidos Borónicos/metabolismo , Ácidos Borónicos/farmacología , Fármacos Neuroprotectores/farmacología , Factor 2 Relacionado con NF-E2/metabolismo , Oxidantes , Estrés Oxidativo , Enfermedad de Parkinson/metabolismo , Piridinas/farmacología , Rotenona/toxicidad , Pez Cebra/metabolismoRESUMEN
Rotenone is used to generate Parkinson's disease (PD)-like symptoms in experimental animals. Octanoic acid (C8), is the principal fatty acid of medium-chain triglycerides in ketogenic diets. Beneficial effects of ketogenic diets were shown in PD. We applied proteomic methods to reveal the effects of octanoic acid in rotenone toxicity in zebrafish to gain information on the use of ketogenic diets in PD. Zebrafish were exposed to 5 µg/ml rotenone and octanoic acid (20 and 60 mg/ml) for 30 days. LC-MS/MS analysis was performed. Raw files were analyzed by Proteome Discoverer 2.4 software, peptide lists were searched against Danio rerio proteins. STRING database was used for protein annotations or interactions. 2317 unique proteins were quantified, 302 proteins were differentially expressed. Proteins involved in cell organization, biogenesis, transport, response to stimulus were most frequently expressed. Our study is first to report that the alterations in the expressions of proteins related to energy and redox system, stress response, and cytoskeleton proteins caused by rotenone exposure were normalized by octanoic acid treatment in zebrafish.
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Enfermedad de Parkinson , Rotenona , Animales , Caprilatos , Cromatografía Liquida , Proteínas del Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Oxidación-Reducción , Enfermedad de Parkinson/metabolismo , Proteómica , Rotenona/toxicidad , Espectrometría de Masas en Tándem , Pez Cebra/metabolismoRESUMEN
Parkinson's disease (PD) is one of the most common neurodegenerative diseases due to the loss of dopaminergic neurons in the midbrain in the substantia nigra. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a neurotoxic agent causing disruptions in mitochondria of dopaminergic neurons leading to impaired oxidant-antioxidant balance. Both zebrafish and zebrafish embryos are sensitive to MPTP. In zebrafish embryos, MPTP decreases the dopaminergic cells in the diencephalon by damaging dopaminergic neurons. Morphine is an opioid pain killer and a strong analgesic that is used to treat chronic pain. Until today morphine has been shown to regulate the survival or death of neurons and both protective and destructive effects of morphine have been reported in the central nervous system. This study aimed to evaluate the effects of morphine in MPTP-exposed zebrafish embryos. Developmental parameters were monitored and documented daily during embryonic development. Locomotor activity of zebrafish embryos at 96 h postfertilization (hpf) was determined. Acetylcholinesterase (AChE) activity and oxidant-antioxidant parameters were analyzed by biochemical methods. RT-PCR was used to evaluate bdnf, dj1, lrrk and pink1 expressions. Morphine treatment improved mortality and hatching rates, locomotor activity, AChE, and antioxidant enzyme activities as well as the expressions of bdnf, dj1, lrrk and pink1 in a dose-dependent manner that were altered by MPTP. Increased lipid peroxidation supports the role of morphine to induce autophagy to prevent PD-related pathologies. Our study provided important data on the possible molecular mechanism of the therapeutic effects of morphine in PD.
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Intoxicación por MPTP , Fármacos Neuroprotectores , Síndromes de Neurotoxicidad , Animales , 1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina/metabolismo , 1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina/farmacología , 1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina/uso terapéutico , Acetilcolinesterasa/metabolismo , Analgésicos Opioides/metabolismo , Analgésicos Opioides/uso terapéutico , Antioxidantes/metabolismo , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Modelos Animales de Enfermedad , Morfina/farmacología , Intoxicación por MPTP/tratamiento farmacológico , Intoxicación por MPTP/prevención & control , Intoxicación por MPTP/metabolismo , Fármacos Neuroprotectores/farmacología , Oxidantes/metabolismo , Proteínas Quinasas/metabolismo , 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.
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1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a neurotoxin that damages dopaminergic neurons. Zebrafish has been shown to be a suitable model organism to investigate the molecular pathways in the pathogenesis of Parkinson's disease and also for potential therapeutic agent research. Boron has been shown to play an important role in the neural activity of the brain. Boronic acids are used in combinatorial approaches in drug design and discovery. The effect of 3-pyridinylboronic acid which is an important sub-class of heterocyclic boronic acids has not been evaluated in case of MPTP exposure in zebrafish embryos. Accordingly, this study was designed to investigate the effects of 3-pyridinylboronic acid on MPTP exposed zebrafish embryos focusing on the molecular pathways related to neurodegeneration and apoptosis by RT-PCR. Zebrafish embryos were exposed to MPTP (800 µM); MPTP + Low Dose 3-Pyridinylboronic acid (50 µM) (MPTP + LB) and MPTP + High Dose 3-Pyridinylboronic acid (100 µM) (MPTP + HB) in well plates for 72 hours post fertilization. Results of our study showed that MPTP induced a P53 dependent and Bax mediated apoptosis in zebrafish embryos and 3-pyridinylboronic acid restored the locomotor activity and gene expressions related to mitochondrial dysfunction and oxidative stress due to the deleterious effects of MPTP, in a dose-dependent manner.
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Intoxicación por MPTP , Pez Cebra , 1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina/metabolismo , 1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina/farmacología , Animales , Ácidos Borónicos/metabolismo , Ácidos Borónicos/uso terapéutico , Modelos Animales de Enfermedad , Intoxicación por MPTP/tratamiento farmacológico , Intoxicación por MPTP/metabolismo , Intoxicación por MPTP/patología , Ratones , Ratones Endogámicos C57BL , Piridinas , Pirrolidinas/metabolismo , Pirrolidinas/uso terapéutico , Pez Cebra/metabolismoRESUMEN
Obesogens affect lipid metabolism, and genetic or epigenetic factors may also contribute to the progression of obesity. Endocrine-disrupting chemicals (EDCs) are the most striking among obesogens. Bisphenol A (BPA) is an estrogenic EDC used in food containers, adhesives, dye powders, and dental fillers. We aimed to elucidate molecular mechanisms of BPA's obesogenic effects focusing on obesogenic pathways in the liver including fibroblast growth factor (FGF) and Dnmt3a which is its epigenetic regulator, oxidant-antioxidant status, and inflammatory cytokines. Zebrafish were divided into three groups as control, low-dose BPA (1 µm BPA), and high-dose BPA groups (10 µm BPA). At the end of 30 days, oral glucose tolerance test (OGTT) was performed, fasting blood glucose levels were measured, and hepatopancreas tissues were taken. Malondialdehyde (MDA) levels, superoxide dismutase (SOD), glutathione S-transferase (GST), and nitric oxide (NO) activities were examined in the hepatopancreas. Inflammatory cytokines, lepa, fgf21, and dnmt3a expressions were determined by RT-PCR. BPA exposure increased the body weights, il1ß, tnfα, il6, lepa, fgf21, and dnmt3a expressions, impaired glucose tolerance, and oxidant-antioxidant status in a dose-dependent manner. Hepatocyte degeneration, lipid vacuolization, and vasocongestion were observed in both BPA-exposed groups. Our study suggests impaired glucose tolerance, oxidant-antioxidant balance, increased inflammatory response, fgf21 expression, and dnmt3a expressions as the possible mechanisms for the BPA-induced obesity model in zebrafish.
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Antioxidantes/metabolismo , Compuestos de Bencidrilo/toxicidad , Citocinas/metabolismo , Factores de Crecimiento de Fibroblastos/metabolismo , Hígado/metabolismo , Obesidad/inducido químicamente , Fenoles/toxicidad , Pez Cebra/metabolismo , Animales , ADN Metiltransferasa 3A/metabolismo , Prueba de Tolerancia a la Glucosa , Metabolismo de los Lípidos , Estrés OxidativoRESUMEN
BACKGROUND: Dysfunction of the gastrointestinal tract (GIT) is one of the most common non-motor symptom of Parkinson's Disease (PD). Pathological processes causing PD were suggested to initiate in the enteric nervous system (ENS) and proceed to the central nervous system (CNS). There are studies showing that low-carbohydrate ketogenic diets can improve motor symptoms of PD. Caprylic acid (C8) is the principal fatty acid component of the medium-chain triglycerides in the ketogenic diets. In this study, we aimed to evaluate the effects of caprylic acid, in neurotoxin exposed zebrafish focusing on the relationship between intestinal and brain oxidative stress and inflammation. METHODS: Adult zebrafish were exposed to rotenone (5 µg/L) (R group) and caprylic acid (20 and 60 mg/mL) (L + HDCA and R + HDCA groups) for 30 days. At the end of 30 days locomotor activities were determined. Levels of lipid peroxidation (LPO), nitric oxide, glutathione and superoxide dismutase and glutathione S-transferase activities were determined by spectrophotometric methods and gene expressions of tnfâº, il1, il6, il21, ifnÉ£ and bdnf were evaluated by RT-PCR in the brain and intestinal tissues of zebrafish. RESULTS: Caprylic acid ameliorated LPO, NO, SOD and the expressions of tnfâº, il1, il6, il21, ifnÉ£ and bdnf in brain and intestines. Locomotor activities were only ameliorated in high dose R + HDCA group. CONCLUSIONS: Caprylic acid ameliorated the neurotoxin-induced oxidative stress and inflammation both in the brain and intestines and enhanced locomotor activity in zebrafish.
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Eje Cerebro-Intestino/fisiología , Caprilatos/farmacología , Animales , Encéfalo/metabolismo , Eje Cerebro-Intestino/efectos de los fármacos , Caprilatos/metabolismo , Modelos Animales de Enfermedad , Tracto Gastrointestinal/metabolismo , Tracto Gastrointestinal/microbiología , Glutatión/metabolismo , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Peroxidación de Lípido/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Enfermedad de Parkinson/metabolismo , Rotenona/efectos adversos , Superóxido Dismutasa/metabolismo , Pez Cebra , Proteínas de Pez CebraRESUMEN
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
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
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.
Asunto(s)
Investigación Biomédica , Síndromes de Neurotoxicidad , Perciformes , Animales , Pez Cebra , Agua Dulce , LaboratoriosRESUMEN
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.
Asunto(s)
Perciformes , ARN , Animales , Sondas ARN , Pez Cebra/genética , ARN Mensajero/genética , Hibridación in SituRESUMEN
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.