Jyotshna Kanungo

The era of using zebrafish (Danio rerio) as a model to investigate human disease began in the 1960s with the pioneering work by a molecular biologist, George Streisinger, who established its role as a genetic vertebrate model

Arsenic induces apoptosis in both cancerous and non-cancerous cells. The mechanism of arsenic-induced apoptosis is complex. We previously demonstrated that the antioxidant acetyl L-carnitine prevented sodium arsenite-induced apoptosis in zebrafish embryos. To gain more insight into the mechanism of arsenic-induced apoptosis, we explored the effect of another antioxidant, N-acetylcysteine (NAC). Co-treatment of sodium arsenite with 1 or 2 mM NAC had no effect on zebrafish development. There was a significant but partial reduction in apoptosis in the embryos co-treated with sodium arsenite and 1 mM NAC, while embryos treated with 1 mM NAC alone showed the loss of normal apoptosis that was observed in the control embryos. Complete abolition of apoptosis occurred in embryos co-treated with sodium arsenite and 2 mM NAC; however, 2 mM NAC alone resulted in 100% mortality, indicating antioxidant toxicity at high doses. NAC (1 mM) did not prevent sodium arsenite-induced increase in motor neurons, suggesting that arsenic-induced apoptosis and supernumerary motor neuron development are mediated via distinct pathways. To determine whether NAC prevented arsenic-induced apoptosis via reactive oxygen species (ROS) signaling, we assessed ROS levels and oxidative modification of proteins (carbonylation) using an OxyBlot assay. Neither sodium arsenite nor NAC altered protein oxidation, ROS levels, or p53, a pro-apoptotic protein, transcript levels. Additionally, dicoumarol, an inducer of p53 protein degradation, did not inhibit sodium arsenite-induced apoptosis. These results indicate that protein oxidation and p53 signaling are not involved in arsenic-induced apoptosis and that NAC prevents arsenic toxicity in zebrafish embryos through a hitherto unknown mechanism.

In 1996, a special zebrafish (Danio rerio) issue of the journal Development, with a 481-page volume containing 37 papers from four different laboratories, published genetic and phenotypic details of hundreds of different mutants, obtained through mutagenesis screening

Verapamil (a P‐glycoprotein inhibitor) and inorganic arsenic cotreatment has been shown to be toxic in chick cardiomyocytes. Previously, we have shown that sodium arsenite at 200 mg/L did not cause developmental toxicity or cardiotoxicity in zebrafish embryos. Here, we investigated the effect of verapamil and sodium arsenite cotreatment on the zebrafish embryos. Embryos at 5 h post‐fertilization (hpf) were exposed to sodium arsenite (100–400 mg/L; 0.77–3.08 mM) in the presence or absence of 20 μM verapamil for 67 h. At 72 hpf, all the embryos treated with sodium arsenite or verapamil alone were alive, while only ~23% and ~17% survived in the groups cotreated with 20 μM verapamil and 100 mg/L or 200 mg/L arsenite, respectively. However, 10 μM of verapamil and 200 mg/L sodium arsenite cotreatment resulted in 100% embryo survival. Inductively coupled plasma mass spectrometry analysis showed that in the verapamil and sodium arsenite cotreated group, the internal arsenic concentration was significantly higher than in the group treated with only sodium arsenite, suggesting that verapamil inhibited arsenic efflux. Surprisingly, verapamil, a calcium channel blocker, reduced sodium arsenite–induced apoptosis but caused developmental toxicity and cardiotoxicity in the sodium arsenite cotreated embryos, without affecting arsenite‐induced supernumerary motor neuron development. Furthermore, acetyl L‐carnitine (ALCAR) completely abolished both developmental toxicity and cardiotoxicity induced by sodium arsenite and verapamil cotreatment. We show for the first time that ALCAR prevents toxicities induced by arsenic and verapamil cotreatment in zebrafish embryos, a vertebrate model for investigating chemical toxicity.

The exact mechanisms of the development of autism, a multifactorial neurological disorder, are not clear. The pathophysiology of autism is complex, and investigations at the cellular and molecular levels are ongoing to provide clarity. Mutations in specific genes have been identified as risk factors for autism. The role of heavy metals in the pathogenesis of autism is subject to many studies and remains debatable. Although no exact neuronal phenotypes have been identified linked to autistic symptoms, overproduction and reduction of specific neurons have been implicated. A growing literature on generating genetic and non-genetic models of autism aims to help with understanding mechanistic studies that can explain the complexity of the disorder. Both genetic and non-genetic methods of zebrafish have been used to model autism. For several human autism risk genes, validated zebrafish mutant models have been generated. There is growing evidence indicating a potential link between autism and inorganic arsenic exposure. We have previously shown that inorganic arsenic induces supernumerary spinal motor neurons via Sonic hedgehog (Shh) signaling pathway, and Cdk5 knockdown causes an overproduction of cranial and spinal motor neurons in zebrafish. Here, in this review, we provide a perspective on what these findings of neurogenic phenotypes mean in terms of dysregulated pathways of motor neuron development and their applicability to understanding cellular and molecular underpinnings of autism.

Our previous study showed that sodium arsenite (200 mg/L) affected the nervous system and induced motor neuron development via the Sonic hedgehog pathway in zebrafish larvae. To gain more insight into the effects of arsenite on other signaling pathways, including apoptosis, we have performed quantitative polymerase chain reaction array‐based gene expression analyses. The 96‐well array plates contained primers for 84 genes representing 10 signaling pathways that regulate several biological functions, including apoptosis. We exposed eggs at 5 h postfertilization until the 72 h postfertilization larval stage to 200 mg/L sodium arsenite. In the Janus kinase/signal transducers and activators of transcription, nuclear factor κ‐light‐chain‐enhancer of activated B cells, and Wingless/Int‐1 signaling pathways, the expression of only one gene in each pathway was significantly altered. The expression of multiple genes was altered in the p53 and oxidative stress pathways. Sodium arsenite induced excessive apoptosis in the larvae. This compelled us to analyze specific genes in the p53 pathway, including cdkn1a, gadd45aa, and gadd45ba. Our data suggest that the p53 pathway is likely responsible for sodium arsenite‐induced apoptosis. In addition, sodium arsenite significantly reduced global DNA methylation in the zebrafish larvae, which may indicate that epigenetic factors could be dysregulated after arsenic exposure. Together, these data elucidate potential mechanisms of arsenic toxicity that could improve understanding of arsenic's effects on human health.

Ketamine is an N‐methyl‐D‐aspartate (NMDA) receptor antagonist. Used as an anesthetic, potential neurotoxic and cardiotoxic effects of ketamine in animal models have been reported. The underlying mechanisms of ketamine‐induced toxicity are not clear. The zebrafish is an ideal model for toxicity assays because of its predictive capability in chemical testing, which compares well with that of mammalian models. To gain insight into potential mechanisms of ketamine effects, we performed real‐time quantitative polymerase chain reaction‐based gene expression array analyses. Gene expression analysis was conducted for multiple genes (a total of 84) related to 10 major signaling pathways including the transforming growth factor β (TGFβ), Wingless and Int‐1 (Wnt), nuclear factor κ‐light‐chain‐enhancer of activated B cells (NF‐κB), Janus kinase/signal transducers and activators of transcription (JAK/STAT), p53, Notch, Hedgehog, peroxisome proliferator‐activated receptor (PPAR), oxidative stress, and hypoxia pathways. Our results show that ketamine altered the expression of specific genes related to hypoxia, p53, Wnt, Notch, TGFβ, PPAR, and oxidative stress pathways. Thus, we can further focus on these specific pathways to elucidate the mechanisms by which ketamine elicits a toxic response.

Recently, the United States Food and Drug Administration approved esketamine, the S-enantiomer of ketamine, as a fast-acting therapeutic drug for treatment-resistant depression. Although ketamine is known as an N-methyl-d-aspartate (NMDA) receptor antagonist, the underlying mechanisms of how it elicits an antidepressant effect, specifically at subanesthetic doses, are not clear and remain an advancing field of research interest. On the other hand, high-dose (more than the anesthetic dose) ketamine-induced neurotoxicity in animal models has been reported. There has been progress in understanding the potential pathways involved in ketamine-induced antidepressant effects, some of which include NMDA-receptor antagonism, modulation of voltage-gated calcium channels, and brain-derived neurotrophic factor (BDNF) signaling. Often these pathways have been shown to be linked. Voltage-gated L-type calcium channels have been shown to mediate the rapid-acting antidepressant effects of ketamine, especially involving induction of BDNF synthesis downstream, while BDNF deficiency decreases the expression of L-type calcium channels. This review focuses on the reported studies linking ketamine’s rapid-acting antidepressant actions to L-type calcium channels with an objective to present a perspective on the importance of the modulation of intracellular calcium in mediating the effects of subanesthetic (antidepressant) versus high-dose ketamine (anesthetic and potential neurotoxicant), the latter having the ability to reduce intracellular calcium by blocking the calcium-permeable NMDA receptors, which is implicated in potential neurotoxicity.