Assessment of Developmental Neurotoxicity Using Semi-automatic Behavior Analysis System for Zebrafish.

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.

Panoramic dental X-ray exposure leads to oxidative stress, inflammation and apoptosis-mediated developmental defects in zebrafish embryos.

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.

The effect of acetic acid-induced pain in Parkinson's disease model in zebrafish.

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.

Quantitative phosphoproteomics to resolve the cellular responses to octanoic acid in rotenone exposed zebrafish.

Ketosis is a potentially beneficial metabolic state for health especially in neurological conditions including Parkinson's disease (PD). Medium-chain-triglycerides (MCT) have specific metabolic properties and they are described as ketogenic even without restriction of carbohydrate. Octanoic acid (C8) is the main MCT showing this effect. Rotenone is a neurotoxin that is used to induce experimental PD model. Rotenone inhibits mitochondrial respiratory complex 1 (MRC1) and causes reactive oxygen species formation. Mass spectrometry (MS)-based phosphoproteomic methods enable discovering specific signaling events in special molecular pathways through identification and quantification of phosphoproteins. Signaling networks involved in rotenone-mediated biological processes and beneficial effects of MCTs on neurodegenerative diseases are not well understood. We aimed to gain comprehensive molecular perspective on the global phosphoproteome differences in rotenone-exposed zebrafish treated with octanoic acid. Raw files obtained from MS analysis were processed and searched against the Danio rerio protein database using SEQUEST-HT algorithm to identify and quantify phosphopeptides with 2,569 unique phosphoproteins and 4,161 unique phosphopeptides corresponding to 2005 proteins. Microtubule-associated protein (MAP) family members were significantly lower in rotenone group. Phosphoproteins involved in ion binding (calcium, magnesium, zinc ion), oxygen binding, microtubule binding, ATP- and GTP-binding were among differentially expressed 347 proteins in rotenone group and they were reversed after octanoic acid treatments. Phosphoproteins and phosphorylation sites were identified for future exploration of signaling pathways involved in rotenone toxicity. We believe our findings might help in the formulation of effective therapeutic strategies for the treatment of PD using ketogenic formulations involving MCTs. PRACTICAL APPLICATIONS: Ketosis is a potentially beneficial metabolic state for health especially in neurological conditions including Parkinson's disease (PD). Medium-chain-triglycerides (MCT) (C6-C12) have specific metabolic properties making them described as ketogenic even without restriction of carbohydrate. Octanoic acid (caprylic acid, C8) is the main MCT showing this effect. Our findings might help in the formulation of effective therapeutic strategies for the treatment of Parkinson's disease using ketogenic formulations involving Medium-chain-triglycerides.

Caprylic acid ameliorates rotenone induced inflammation and oxidative stress in the gut-brain axis in Zebrafish.

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 tnf⍺, 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 tnf⍺, 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.

Oxidative stress and apoptosis in electromagnetic waves exposed Zebrafish embryos and protective effects of conductive nonwoven fabric.

The amount of technological products including television, radio transmitters, and mobile phone that have entered our daily life has increased in recent years. But these devices may cause adverse effects on human health. Electromagnetic shielding fabrics may limit and inhibit electromagnetic waves. Aim of our study was to evaluate electromagnetic wave blocking performance of nonwoven textile surfaces on zebrafish embryos that were exposed to electromagnetic waves at specific frequencies. Oxidant-antioxidant system parameters were evaluated spectrophotometrically. The expressions of tp53 and casp3a were evaluated by RT-PCR. Results showed that electromagnetic shielding fabrics produced as conductive nonwoven textile surfaces improved oxidant-antioxidant status and tp53 expression that were impaired in electromagnetic waves exposed zebrafish embryos. Also, electromagnetic shielding fabrics decreased casp3a expression responsible for the execution phase of apoptosis that increased in electromagnetic waves exposed zebrafish embryos.

Methylparaben induces malformations and alterations on apoptosis, oxidant-antioxidant status, ccnd1 and myca expressions in zebrafish embryos.

Methylparabens (MP) are widely used as preservatives in cosmetics, pharmacy, and food industry. Although acute toxicity studies in animals indicated that parabens are not significantly toxic, the effects of chronic exposure under sublethal doses are still unknown and the number of related studies is limited. Our aim was to evaluate the effects of MP on the development of zebrafish embryos focusing on development, locomotor activity, oxidant-antioxidant status, apoptosis, and ccnd1 and myca expressions. The expressions of ccnd1 and myca were determined by RT-PCR. Lipid peroxidation (LPO), nitric oxide (NO), and glutathione-S-transferase (GST) activities were determined spectrophotometrically. Apoptosis was determined using acridine orange staining. Locomotor activity was measured using touch-evoked movement test. MP exposure increased malformations, LPO, apoptosis, ccnd1 and myca expressions, and decreased GST activities and NO levels compared with the control group. Our findings will lead to further understanding of the mechanism of MP toxicity, and merit further research.

Evaluation of the interaction between proliferation, oxidant-antioxidant status, Wnt pathway, and apoptosis in zebrafish embryos exposed to silver nanoparticles used in textile industry.

Antimicrobial textile products are developing rapidly as an important part of functional textiles. Silver nanoparticles (AgNPs) are nanotechnology products with antimicrobial properties. However, exposure to nanoparticles in daily life is an important issue for public health, still being updated. Aim was to evaluate the effects of AgNPs on the development of zebrafish embryos focusing on Wnt pathway, proliferation, oxidant-antioxidant status, and apoptosis. The expressions of ccnd1 and gsk3ß were determined by RT-PCR, whereas ß-catenin and proliferative cell antigen (PCNA) expressions were determined immunohistochemically. Lipid peroxidation, superoxide dismutase, and glutathione-S-transferase activities were determined spectrophotometrically. Apoptosis was determined using acridine orange staining. Oxidant status, apoptosis, immunohistochemical PCNA, and ß catenin staining increased, whereas ccnd1 and antioxidant enzyme activities decreased in AgNPs-exposed embryos in a dose-dependent manner. Our results indicate the interaction of possible mechanisms that may be responsible for the toxic effects of AgNPs in zebrafish embryos.