Zebrafish as a Translational Model: An Experimental Alternative to Study the Mechanisms Involved in Anosmia and Possible Neurodegenerative Aspects of COVID-19?

The Coronavirus disease-2019 (COVID-19) presents a variability of clinical symptoms, ranging from asymptomatic to severe respiratory and systemic conditions. In a cohort of patients, the Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV-2), beyond the classical respiratory manifestations, induces anosmia. Evidence has suggested SARS-CoV-2-induced anosmia can be the result of neurodegeneration of the olfactory pathway. Neurologic symptoms associated with COVID-19 have been reported; however, the precise mechanism and possible long-lasting effects remain poorly investigated. Preclinical models are valuable tools for describing and testing new possible treatments for neurologic disorders. In this way, the zebrafish (Danio rerio) organism model represents an attractive tool in the field of neuroscience, showing economic and logistic advantages besides genetic and physiologic similarities with mammalian, including the brain structure and functions. Besides, its external embryonic development, high availability of eggs, and fast development allows easy genetic manipulation and fast replications. In the present review, we suggest that the zebrafish model can be advantageous to investigate the neurologic features of COVID-19.

Cannabidiol did not induce teratogenicity or neurotoxicity in exposed zebrafish embryos.

Cannabidiol (CBD) is a non-psychotomimetic compound of the Cannabis sativa that has been used for the treatment of severe epilepsy as well as other diseases of nervous system. However, toxicity studies of CBD have great relevance to guarantee the patients safety. In this context, morphological analyses of zebrafish can contribute to evaluate the teratogenic potential, as well as evaluation of acetylcholinesterase activity and motor activity of zebrafish are valuable tools to verify the neurotoxicity potential. In the present work, we use this methodology to test the toxicity of CBD to zebrafish embryos. No malformation was observed in morphological analysis of embryos exposed to all tested concentrations of CBD. Although, twenty per cent of embryos exposed to maximal dose of CBD (300 µg/L) hatched after 96hpf, while embryos in control solution had already hatched in this period. Embryos exposed to CBD did not show differences in acetylcholinesterase activity, but embryos exposed to CBD 20-300 µg/L were 1.4 up to 1.7-fold more active when compared to the control. Despite that, at 48 hpf, motor activity returned to control values. Our results suggest that the effects observed after CBD exposure are intimately related to CB1 receptor that is present in zebrafish since early stages of development. The present work showed early light effects induced by CBD exposure in concentrations that did not alter biochemical activity.

Biochemical approaches to assess oxidative stress induced by exposure to natural and synthetic dyes in early life stages in zebrafish.

Zebrafish early life stages were found to be sensitive to several synthetic dyes widely used in industries. However, as environmental concentrations of such contaminants are often at sublethal levels, more sensitive methods are required to determine early-warning adverse consequences. The aim of this study was to utilize a multibiomarker approach to examine underlying oxidative stress mechanisms triggered by sublethal concentrations of synthetic azo dye Basic Red 51 (BR51), the natural dye erythrostominone (ERY), and its light-degraded product using zebrafish embryos. Biochemical biomarkers included parameters of detoxification and markers of antioxidant system, as well as oxidative damage. Results showed pro-oxidant mechanisms attributed to BR51 and ERY as evidenced by increased glutathione S-transferase (GST) activity, a phase II detoxification enzyme related to reactive oxygen species detoxification. BR51 also elevated total glutathione (GSH+GSSG) levels and catalase activity. However, both dyes induced oxidative damage as evidenced by elevated lipid peroxidation content. In contrast, when the natural dye was photodegraded, no marked effects were observed for all biomarkers assessed. Data indicate that such dyes are pro-oxidants at sublethal concentrations, predominantly involving GSH and/or related enzymes pathway.

Hepatotoxicity assessment of the azo dyes disperse orange 1 (DO1), disperse red 1 (DR1) and disperse red 13 (DR13) in HEPG2 cells.

During the dyeing process in baths approximately 10 to 15% of the dyes used are lost and reach industrial effluents, thus polluting the environment. Studies showed that some classes of dyes, mainly azo dyes and their by-products, exert adverse effects on humans and local biota, since the wastewater treatment systems and water treatment plants were found to be ineffective in removing the color and reducing toxicity of some dyes. In the present study, the toxicity of the azo dyes disperse orange 1 (DO1), disperse red 1 (DR1), and disperse red 13 (DR13) was evaluated in HepG2 cells grown in monolayers or in three dimensional (3D) culture. Hepatotoxicity of the dyes was measured using 3-(4,5-dimethylthiazol-2yl)2,5-diphenyltetrazolium (MTT) and cell counting kit 8 (CCK-8) assays after 24, 48, and 72 h of incubation of cells with 3 different concentrations of the azo dyes. The dye DO1 only reduced the mitochondrial activity in HepG2 cells grown in a monolayer after 72 h incubation, while the dye DR1 showed this deleterious effect in both monolayer and 3D culture. In contrast, dye DR13 decreased the mitochondrial activity after 24, 48, and 72 h of exposure in both monolayer and 3D culture. With respect to dehydrogenase activity, only the dye DR13 diminished the activity of this enzyme after 72 h of exposure in both monolayer and 3D culture. Our results clearly demonstrated that exposure to the studied dyes induced cytotoxicity in HepG2 cells.