Embryonic exposure to decitabine induces multiple neural tube defects in developing zebrafish.

Neural tube defects are severe congenital disorders of the central nervous system that originate during embryonic development when the neural tube fails to close completely. It affects one to two infants per 1000 births. The aetiology is multifactorial with contributions from both genetic and environmental factors. Dysregulated epigenetic mechanisms, in particular the abnormal genome-wide methylation during embryogenesis, have been linked to developmental abnormalities including neural tube defects. The current study investigated the influence of decitabine (DCT), a DNA methylation inhibitor, on embryonic development in zebrafish, with a focus on neural tube formation. The developing zebrafish embryos were exposed to graded concentrations of decitabine (from 13.69 µM to 1 mM) before the onset of neurulation. The developmental process was monitored at regular time intervals post fertilization. At 120 h post fertilization, the developing embryos were inspected individually to determine the incidence and severity of neural tube defects. Using alizarin red staining, the cranial and caudal neural tube morphology was examined in formaldehyde fixed larvae. Anomalies in neural tube and somite development, as well as a delay in hatching, were discovered at an early stage of development. As development continued, neural tube defects became increasingly evident, and there was a concentration-dependent rise in the prevalence and severity of various neural tube defects. 90% of growing embryos in the group exposed to decitabine 1 mM had multiple neural tube malformations, and 10% had isolated neural tube defects. With several abnormalities, the caudal region of the neural tube was seriously compromised. The histopathological studies supported the malformations in neural tube. Our study revealed the harmful impact of decitabine on the development of the neural tube in growing zebrafish. Moreover, these findings support the hypothesis that the hypomethylation during embryonic development causes neural tube defects.

Embryonic exposure to acetyl-L-carnitine protects against valproic acid-induced cardiac malformation in zebrafish model.

Worldwide, estimated counts of about 7.9 million children are born with serious birth defects. In addition to genetic factors, prenatal exposure to drugs and environmental toxicants represents a major contributing factor to congenital malformations. In earlier investigation, we explored cardiac malformation caused by valproic acid (VPA) during early developing stages of zebrafish. Since heart depends on mitochondrial fatty acid oxidative metabolism for energy demands in which carnitine shuttle has a major role, the present study aimed to investigate the effect of acetyl-L-carnitine (AC) against VPA-induced cardiac malformation in developing zebrafish. Initially, AC was subjected to toxicological evaluation, and two micromolar concentrations (25 µM and 50 µM) were selected for evaluation. A sub-lethal concentration of VPA (50 µM) was selected to induce cardiac malformation. The embryos were grouped and the drug exposures were made at 2.5 h post-fertilization (hpf). The cardiac development and functioning was monitored. A progressive decline in cardiac functioning was noted in group exposed to VPA 50 µM. At 96 hpf and 120 hpf, the morphology of heart was severely affected with the chambers which became elongated and string-like accompanied by histological changes. Acridine orange staining showed accumulation of apoptotic cells. Group exposed to VPA 50 µM with AC 50 µM showed a significant reduction in pericardial sac edema with morphological, functional and histological recovery in developing heart. Moreover, reduced number of apoptotic cells was noted. The improvement with AC might be due to restoration of carnitine homeostasis for cardiac energy metabolism in developing heart.

Heart malformation is an early response to valproic acid in developing zebrafish.

Valproic acid (VPA) is a branched short-chain fatty acid primarily used in epilepsy, but is also used in bipolar disorder, migraine, and psychotic disorders. Despite its wide range of use, it is a teratogen resulting in various congenital abnormalities. Although a large number of scientific studies evidenced the teratogenic effects, there are limited data on embryonic exposure to VPA at specific or different stages of early embryogenesis. Based on this, the present study was planned to investigate the embryonic exposure to VPA at specific and different hours post fertilization (hpf) in zebrafish embryonic model. In first set of experiments, embryos from spawning groups of adult zebrafish were exposed to different molar concentrations of VPA at 2.5 hpf, and in the second set of experiments, embryos were exposed to VPA 100 µM at 24 hpf, 36 hpf, 48 hpf, 72 hpf, and 96 hpf. The parameters examined were hatching rate, mortality, morphology, body length, pericardial sac size, heartrate, anatomical changes in heart, skeletal and notochord till 120 hpf. It was observed that the embryos exposed to VPA at 2.5 hpf suffered from cardiac abnormalities including heart malformation, bradycardia, circulatory failure, and pericardial sac enlargement which was more apparent in embryos exposed to 100 µM VPA. In the second set of experiments, embryos exposed to VPA 100 µM at 24 hpf and 36 hpf suffered from heart malformations, but there was no incidence of cardiac malformation in embryos exposed to VPA at 48 hpf, 72 hpf, and 96 hpf. From the results, it was evident that exposure to VPA at early developmental stage of embryogenesis produced congenital cardiac abnormalities. Since VPA is a selective HDAC inhibitor, histone acetylation with aberrant gene expression during cardiogenesis might be the underlying cause of cardiac malformation.

Mefenamic Acid Attenuates Chronic Alcohol Induced Cognitive Impairment in Zebrafish: Possible Role of Cholinergic Pathway.

Based on the scientific evidence supporting the neuroinflammatory response contributes the cognitive impairment associated with chronic alcoholism and the neuroprotective actions of mefenamic acid with reversal of memory loss and brain inflammation in mice, this study was designed to evaluate the effect of mefenamic acid against chronic alcohol induced cognitive impairment in zebrafish model. Zebrafish were grouped and subjected to normal behavioral analysis in light-dark chamber for 10 days. The preference to dark compartment was noted in zebrafish. Zebrafish were grouped and exposed to escalating doses of alcohol for 28 days with and without mefenamic acid exposure (100 and 200 µg/L) and subjected to a fear conditioning passive avoidance task from day 13 of 28. The cognitive evaluation was performed for 10 days and the brain tissue was isolated to estimate acetylcholinesterase activity. In cognitive evaluation study, the normal zebrafish retained the memory of the learned task and avoided the dark. The alcohol exposed zebrafish showed impairment in retaining the memory of learned task. Mefenamic acid exposed zebrafish showed a significant protection against cognitive impairment caused by alcohol and retained the memory of learned task with a significant decrease in AChE activity in brain homogenate compared to alcohol exposed zebrafish. The results of this study suggest that the memory enhancing activity of mefenamic acid might be due to activation of cholinergic transmission that has protected neuroinflammatory and neurodegenerative conditions caused by alcohol.

Cognition Enhancing Activity of Sulforaphane Against Scopolamine Induced Cognitive Impairment in Zebra Fish (Danio rerio).

Several epidemiological studies have shown that consumption of large quantities of vegetables especially cruciferous vegetables (Broccoli and Brussels sprouts) can protect against chronic diseases. Sulforaphane, an isothiocynate found in cruciferous vegetables has been demonstrated to have neuroprotective effects in several experimental paradigms. This study was undertaken to examine the effect of sulforaphane on cognitive impairment in zebra fish model using a novel method of fear conditioning. Initially, the normal behaviour of zebra fishes was studied in light-dark tank for 10 min daily for 10 days. Fishes were then divided into seven groups of twelve in each. Group I served as normal, group II served as fear conditioned control, group III and group IV were sulforaphane (25 µM/L) and piracetam (200 mg/L) treated respectively. Group V served as scopolamine (400 µM/L) induced memory impairment fishes. Group VI and VII were sulforaphane (25 µM/L) and piracetam (200 mg/L) treated scopolamine induced memory impairment groups respectively. In normal behavioural analysis, fishes preferred to stay in dark compartment. The average number of entries into the dark and time spent in dark were significantly more. Fishes in group II to VII were individually subjected to fear conditioning passive avoidance task and evaluated for learned task memory. It was observed that the average number of entries into dark and time spent in dark were significantly decreased. After exposure to respective treatment fishes in group III to VII were subjected to cognitive evaluation. There was no significant difference in cognition of group III and IV fishes exposed to sulforaphane and piracetam alone respectively. Fishes exposed to scopolamine showed a significant cognitive impairment. Sulforaphane exposure prior to scopolamine significantly retained the memory of learned task. These findings suggest that sulforaphane might be a promising therapeutic agent for cognitive enhancement in Alzheimer's disease.