VK was funded by grants from the IUP Department of Biology and School of Graduate Studies and Research (Graduate Student Professional Development). CQD and TWS were supported by the IUP School of Graduate Studies and Research (Faculty Publication Costs/Incidental Research Expenses). We also thank members of the Diep laboratory for maintaining the zebrafish facility.

The procedures described in this protocol were approved by the Institutional Animal Care and Use Committee at the Indiana University of Pennsylvania.
1. Harvest Embryos
<ol>
	<li>Maintain zebrafish at 28.5 &#176;C, pH 7, conductivity between 500-1,500 &#181;S, and a light/dark cycle of 14 hr light and 10 hr dark24. Use wild-type strains such as T&#252;, AB or T&#252;/AB hybrid. Different strains may respond differently to chemical treatment25.</li>
	<li>Set up the fish for mating the night before harvesting eggs by adding fish system water into a mating tank. Add a male and female fish into the tank and separate the two fish with a divider. Each fish pair will produce a range of 50-300 eggs. To ensure that enough eggs will be produced, set up 30 pairs of fish. Typically, about 50% of fish pairs at the prime mating age (6-9 months old) will produce eggs, resulting up to 200 eggs per pair and a maximum of up to 3,000 eggs for this experiment.</li>
	<li>The next morning after the light turns on, remove the divider to initiate mating. Check the mating tanks for eggs every 15 min.</li>
	<li>Once the fish lay eggs, harvest all embryos using a tea strainer and combine them into one large container with E3 buffer (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4). At 1.5 hpf, remove and discard unfertilized eggs with a plastic transfer pipet under a dissecting microscope.&#160;Unfertilized eggs are opaque while fertilized eggs are transparent34.</li>
	<li>Transfer 50 embryos into a 100 x 15 mm glass Petri dish containing 50 ml E3 buffer for each treatment condition. A total of 33 dishes are needed for 11 treatment conditions and 3 replicates.</li>
</ol>
2. Treating Embryos
<ol>
	<li>Carry out treatments at 2 hr post-fertilization (hpf)35. Incubate embryos/larvae at 28.5 &#176;C and examine the larvae at 120 hpf. Perform at least three replicates of each treatment condition for statistical analysis.</li>
	<li>For 3 Petri dishes (each containing 50 embryos) at 2 hpf, remove the E3 buffer with a transfer pipet and add 50 ml of 300 mM ethanol diluted in E3 buffer. Cover the Petri dishes with Parafilm to minimize evaporation of ethanol.
	<ol>
		<li>Continue exposing the embryos to ethanol for 22 hr. Then remove the ethanol with a transfer pipet. Add 50 ml of E3 buffer and swirl the dish several times to wash out the ethanol. Remove this E3 buffer with a transfer pipet and repeat the washing step 2 more times.</li>
	</ol>
	</li>
	<li>For 3 Petri dishes (each containing 50 embryos) at 2 hpf, remove the E3 buffer with a transfer pipet and add 50 ml of new E3 buffer.</li>
	<li>For 9 Petri dishes (each containing 50 embryos) at 2 hpf, remove the E3 buffer with a transfer pipet and add 50 ml of 1,000 mg/L sodium nitrite dissolved in E3 buffer. Confirm the concentration of the stock nitrite solution beforehand using a modification of the diazotization (USEPA Method 354.1) spectrophotometric method28.
	<ol>
		<li>Continue exposing 3 dishes for 46 hr, 3 dishes for 70 hr and 3 dishes for 94 hr. Replace the nitrite solution with freshly made nitrite solution daily.</li>
		<li>After each exposure time, remove the nitrite with a transfer pipet. Add 50 ml of E3 buffer and swirl the dish several times to wash out the nitrite. Remove this E3 buffer with a transfer pipet and repeat the washing step 2 more times.</li>
	</ol>
	</li>
	<li>For 3 Petri dishes (each containing 50 embryos) at 2 hpf, remove the E3 buffer with a transfer pipet and add 50 ml of 200 mg/L sodium nitrite. Repeat this with 400, 600, 800, and 1,000 mg/L of sodium nitrite.
	<ol>
		<li>Continue exposing the embryos to nitrite for 70 hr. Replace the nitrite solution with freshly made nitrite solution daily.</li>
		<li>After the exposure time, remove the nitrite with a transfer pipet. Add 50 ml of E3 buffer and swirl the dish several times to wash out the nitrite. Remove this E3 buffer with a transfer pipet and repeat the washing step 2 more times.</li>
	</ol>
	</li>
	<li>For 3 Petri dishes (each containing 50 embryos) at 2 hpf, remove the E3 buffer with a transfer pipet and add 50 ml of 1,000 mg/L sodium nitrate dissolved in E3 buffer. Confirm the concentration of the stock nitrate solution beforehand using a modification of the cadmium reduction (USEPA Method 353.3) spectrophotometric method28.
	<ol>
		<li>Continue exposing the embryos to nitrate for 70 hr. Replace the nitrate solution with freshly made nitrate solution daily.</li>
		<li>After the exposure time, remove the nitrate with a transfer pipet. Add 50 ml of E3 buffer and swirl the dish several times to wash out the nitrate. Remove this E3 buffer with a transfer pipet and repeat the washing step 2 more times.</li>
	</ol>
	</li>
	<li>During each day of the exposure, count the number of dead embryo/larvae using a stereomicroscope. Signs of death include lack of heartbeat and blood circulation, or lack of motility after 1 min of observation. Remove dead embryos/larvae with a transfer pipet to reduce contamination of the E3 buffer.</li>
	<li>When experiments end at 120 hpf, euthanize the larvae.
	<ol>
		<li>Remove the E3 buffer with a transfer pipet. Then add 50 ml of 0.2% MS-222 (buffered to pH 7) and wait for 10 min.</li>
		<li>Remove the MS-222 with a transfer pipet. Add 50 ml of E3 buffer and swirl to wash out the MS-222.</li>
		<li>Remove the E3 buffer with a transfer pipet and add 20 ml of 4% paraformaldehyde (PFA) to fix the larvae. Swirl the dish several times. Use a transfer pipet to transfer the larvae into a glass vial along with enough PFA to fill the vial. Store the vials in the refrigerator overnight (O/N).</li>
	</ol>
	</li>
	<li>Take pictures of fixed larvae using a stereoscope with a digital camera. Use 30X magnification, ISO 200, and 200 msec&#160;exposure time. Orient the larvae so that the anterior to the left and the dorsal is to the top of the field.</li>
</ol>

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The authors have nothing to disclose.

The method described here demonstrates the utility of zebrafish in assessing the teratogenic potential of nitrite and nitrate. Compared to other vertebrates, zebrafish have advantages that include high fecundity, external fertilization, optical transparency, and rapid development. Available mutants that lack pigmentation (such as the casper zebrafish36) also help to enhance visibility of internal organs. It is also easy to generate transgenic zebrafish with reporter genes to facilitate analysis in live fish<su...

Teratogenesis is a process that disrupts the normal development of an embryo or fetus by causing permanent structural and functional abnormalities, growth retardation, or miscarriage in severe cases1. It can be caused by certain natural agents (teratogens), which interfere with embryonic development in multiple ways2. During human fetal development, common teratogens such as radiation, infectious agents, toxic metals, and organic chemicals have been reported to cause defects in epicanthic folds (the skin fold in the upper eye lid) and clinodactyly (curved finger or toe) through morphogenetic errors1.
Understanding the molecular mechanism of teratogenesis is the first step towards developing treatment and prevention. Several vertebrate models such as the African clawed frog (Xenopus laevis) and zebrafish (Danio rerio) have been used to determine the molecular pathways affected by teratogens. Previous studies have used zebrafish as a model for epidemiology, toxicology and teratogenesis3-7. Scholz et al. considered zebrafish as a &#34;gold standard&#34; for environmental toxicity assessment. This is due, in part, to the transparency of the zebrafish embryo, which allows researchers to visualize the developmental defect as it occurs8. Approximately 70% of human genes have orthologues in zebrafish, making zebrafish a desirable vertebrate model for studying human defects9.
Some epidemiological studies have suggested that nitrate and nitrite, commonly present in farm foods and water, are associated with birth defects or spontaneous abortions10,11, while other studies do not support this association12. Nitrate (NO3-) and nitrite (NO2-) are naturally present in soil and water. They are a source of nitrogen for plants and are a part of the nitrogen cycle13. Foods such as green beans, carrots, squash, spinach, and beets from farms that use fertilizers high in nitrate have significantly augmented levels of nitrate and nitrite7. Milk from cows fed with high nitrate foods and fish in high nitrate water (mainly from soil runoff30) can lead to humans consuming large amounts of nitrate and nitrite14. Nitrate and nitrite are also commonly used in food preservation, which dramatically increases the amount ingested by humans12.
Optimal levels of nitrate and nitrite play fundamental roles in physiological processes like vascular homeostasis and function, neurotransmission and immunological host defense mechanisms13-15. However, exposure to high levels of nitrate and nitrite may lead to adverse effects, especially in infants and children16. Ingested nitrate is further converted to nitrite in the oral cavity by microflora and in the gastrointestinal tract by intestinal microflora17.
Nitrate puts infants at a high risk for blue baby syndrome by oxidation of hemoglobin to methemoglobin, impairing hemoglobin from its oxygen carrying ability18. This results in the blue color of skin that extends to peripheral tissues in more severe cases. Inhibited oxygenation of tissues results in other symptoms, most severely leading to coma and death19,20. Similar symptoms are observed in babies and adults at higher concentrations of nitrate21. Elevated levels of methemoglobin in adults due to nitrite poisoning results in cyanosis, headache, breathing disorders31, and death if not treated due to complications related to vital tissue hypoxia32,33.
Nitrate ingested at higher levels can also result in various health complications. Childhood diabetes, recurrent diarrhea, and recurrent respiratory tract infections in children have been linked with high nitrate intake11,17,22. Chronic exposure to a high level of nitrate is associated with urination and spleen hemorrhaging. Acute high dose exposure to nitrates can lead to a wide spectrum of medical conditions like abdominal pain, muscle weakness, blood in stools and urine, fainting, and death11. Prenatal exposure to nitrate at high levels has been associated with neural tube and musculoskeletal defect11.
A recent report showed that treating zebrafish embryos with nitrite led to yolk sac edema, craniofacial and axial malformations, and swim bladder noninflation5. In this study, we demonstrate a method for treating zebrafish embryos with nitrate and nitrite to determine their teratogenic potential. Embryos were exposed to nitrite at different concentrations and different lengths of time. Ethanol was used as a positive control, since it is an established teratogen23. Our method showed that both high concentrations and long exposure times to nitrite were detrimental to survival and resulted in various phenotypes, ranging from mild (edema) to severe (gross developmental defects). Therefore, the zebrafish is a useful model for directly exploring the potential teratogenic effects of nitrate and nitrite on embryos to complement epidemiological studies.

<table><tbody><tr><td>DREL/2010 instrument</td><td>Hach</td><td>26700-03</td><td></td></tr><tr><td>Ethanol</td><td>Sigma-Aldrich</td><td>E7023</td><td></td></tr><tr><td>KIMAX glass Petri Dish</td><td>VWR</td><td>89001-244</td><td></td></tr><tr><td>MS-222</td><td>Sigma-Aldrich</td><td>E10521</td><td></td></tr><tr><td>NitraVer 5 Nitrate Reagent</td><td>Hach</td><td>14034-46</td><td></td></tr><tr><td>NitriVer 3 Nitrite Reagent</td><td>Hach</td><td>14065-99</td><td></td></tr><tr><td>Parafilm</td><td>Fisher Scientific</td><td>3-374-10</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>158127</td><td></td></tr><tr><td>S6E stereomicroscope</td><td>Leica</td><td>10446294</td><td></td></tr><tr><td>Sodium nitrate</td><td>Fisher Scientific</td><td>S343</td><td></td></tr><tr><td>Sodium nitrite</td><td>Fisher Scientific</td><td>S347</td><td></td></tr><tr><td>Transfer pipets</td><td>Laboratory Products Sales</td><td>L320072</td><td></td></tr><tr><td>Glass vials</td><td>Fisher Scientific</td><td>03-338B</td><td></td></tr></tbody></table>

zebrafish as a model to assess the teratogenic potential of nitrite

High nitrate levels in the environment may&#160;result in congenital defects or miscarriages in humans. Presumably, this is due to the conversion of nitrate to nitrite by gut and salivary bacteria. However, in other mammalian studies, high nitrite levels do not cause birth defects, although they can lead to poor reproductive outcomes. Thus, the teratogenic potential of nitrite is not clear. It would be useful to have a vertebrate model system to easily assess teratogenic effects of nitrite or any other chemical of interest. Here, we demonstrate the utility of zebrafish (Danio rerio) to screen compounds for toxicity and embryonic defects. Zebrafish embryos are fertilized externally and have rapid development, making them a good model for teratogenic studies. We show that increasing the time of exposure to nitrite negatively affects survival. Increasing the concentration of nitrite also adversely affects survival, whereas nitrate does not. For embryos that survive nitrite exposure, various defects can occur, including pericardial and yolk sac&#160;edema, swim bladder noninflation, and craniofacial malformation. Our results indicate that the zebrafish is a convenient system for studying the teratogenic potential of nitrite. This approach can easily be adapted to test other chemicals for their effects on early vertebrate development.

Exposure to 300 mM ethanol for 22 hr had no effect on survival (data not shown), consistent with previous reports5,23,26. This is expected, as ethanol is a known teratogen and served as a positive control. Observed phenotypes included pericardial edema, swim bladder noninflation (Figure 1), craniofacial defects, and developmental delay (data not shown).
Treatment with nitrite resulted in mild to sever...

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Zebrafish as a Model to Assess the Teratogenic Potential of Nitrite

Exposure to teratogens may cause birth defects. Zebrafish are useful for determining the teratogenic potential of chemicals. We demonstrate the utility of zebrafish by exposing embryos to various levels of nitrite and also at different times of exposure. We show that nitrite can be toxic and cause severe developmental defects.

High nitrate levels in the environment may&#160;result in congenital defects or miscarriages in humans. Presumably, this is due to the conversion of ...

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9.4K Views.  Indiana University of Pennsylvania. Exposure to teratogens may cause birth defects. Zebrafish are useful for determining the teratogenic potential of chemicals. We demonstrate the utility of zebrafish by exposing embryos to various levels of nitrite and also at different times of exposure. We show that nitrite can be toxic and cause severe developmental defects.

Video: Zebrafish as a Model to Assess the Teratogenic Potential of Nitrite

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, including mental retardation due to disordered and damaged brain development. Fetal alcohol spectrum disorder (FASD) is a term used to cover a continuum of birth defects that occur due to maternal alcohol consumption, and occurs in approximately 4% of children born in the United States. With 50% of child-bearing age women reporting consumption of alcohol, and half of all pregnancies being unplanned, unintentional exposure is a continuing issue2. In order to best understand the damage produced by ethanol, plus produce a model with which to test potential interventions, we developed a model of developmental ethanol exposure using the zebrafish embryo. Zebrafish are ideal for this kind of teratogen study3-8. Each pair lays hundreds of eggs, which can then be collected without harming the adult fish. The zebrafish embryo is transparent and can be readily imaged with any number of stains. Analysis of these embryos after exposure to ethanol at different doses and times of duration and application shows that the gross developmental defects produced by ethanol are consistent with the human birth defect. Described here are the basic techniques used to study and manipulate the zebrafish FAS model.

In order to understand the molecular mechanisms of the ethanol-induced developmental damage, we have developed a zebrafish model of ethanol exposure and are exploring the physical, cellular, and genetic alterations that occur after ethanol exposure1. We then seek to find potential interventions and rapidly test them in this animal model.

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, ...

Medicine

Studying Neurobehavioral Effects of Environmental Pollutants on Zebrafish Larvae

Recent years more and more environmental pollutants have been proved neurotoxic, especially at the early development stages of organisms. Zebrafish larvae are a preeminent model for the neurobehavioral study of environmental pollutants. Here, a detailed experimental protocol is provided for the evaluation of the neurotoxicity of environmental pollutants using zebrafish larvae, including the collection of the embryos, the exposure process, neurobehavioral indicators, the test process, and data analysis. Also, the culture environment, exposure process, and experimental conditions are discussed to ensure the success of the assay. The protocol has been used in the development of psychopathic drugs, research on environmental neurotoxic pollutants, and can be optimized to make corresponding studies or be helpful for mechanistic studies. The protocol demonstrates a clear operation process for studying neurobehavioral effects on zebrafish larvae and can reveal the effects of various neurotoxic substances or pollutants.

A detailed experimental protocol is presented in this paper for the evaluation of neurobehavioral toxicity of environmental pollutants using a zebrafish larvae model, including the exposure process and tests for neurobehavioral indicators.

Recent years more and more environmental pollutants have been proved neurotoxic, especially at the early development stages of organisms. Zebrafish larvae ...

Environment

Zebrafish Larvae as a Model to Evaluate Potential Radiosensitizers or Protectors

Zebrafish are extensively used in several kinds of research because they are one of the easily maintained vertebrate models and exhibit several features of a unique and convenient model system. As highly proliferative cells are more susceptible to radiation-induced DNA damage, zebrafish embryos are a front-line in vivo model in radiation research. In addition, this model projects the effect of radiation and different drugs within a short time, along with major biological events and associated responses. Several cancer studies have used zebrafish, and this protocol is based on the use of radiation modifiers in the context of radiotherapy and cancer. This method can be readily used to validate the effects of different drugs on irradiated and control (non-irradiated) embryos, thus identifying drugs as radio sensitizing or protective drugs. Although this methodology is used in most drug screening experiments, the details of the experiment and the toxicity assessment with the background of X-ray radiation exposure are limited or only briefly addressed, making it difficult to perform. This protocol addresses this issue and discusses the procedure and toxicity evaluation with a detailed illustration. The procedure describes a simple approach for using zebrafish embryos for radiation studies and radiation-based drug screening with much reliability and reproducibility.

The zebrafish has recently been exploited as a model to validate potential radiation modifiers. The present protocol describes the detailed steps to use zebrafish embryos for radiation-based screening experiments and some observational approaches to evaluate the effect of different treatments and radiation.

Zebrafish are extensively used in several kinds of research because they are one of the easily maintained vertebrate models and exhibit several features ...

Cancer Research

Rapid Evaluation of Toxicity of Chemical Compounds Using Zebrafish Embryos

The zebrafish is a widely used vertebrate model organism for the disease and phenotype-based drug discovery. The zebrafish generates many offspring, has transparent embryos and rapid external development. Zebrafish embryos can, therefore, also be used for the rapid evaluation of toxicity of the drugs that are precious and available in small quantities. In the present article, a method for the efficient screening of the toxicity of chemical compounds using 1-5-day post fertilization embryos is described. The embryos are monitored by stereomicroscope to investigate the phenotypic defects caused by the exposure to different concentrations of compounds. Half-maximal lethal concentrations (LC50) of the compounds are also determined. The present study required 3-6 mg of an inhibitor compound, and the whole experiment takes about 8-10 h to be completed by an individual in a laboratory having basic facilities. The current protocol is suitable for testing any compound to identify intolerable toxic or off-target effects of the compound in the early phase of drug discovery and to detect subtle toxic effects that may be missed in the cell culture or other animal models. The method reduces procedural delays and costs of drug development.

Zebrafish embryos are used for evaluating the toxicity of chemical compounds. They develop externally and are sensitive to chemicals, allowing detection of subtle phenotypic changes. The experiment only requires a small amount of compound, which is directly added to the plate containing embryos, making the testing system efficient and cost-effective.

The zebrafish is a widely used vertebrate model organism for the disease and phenotype-based drug discovery. The zebrafish generates many offspring, has ...

A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology due to their considerable degree of genetic conservation with humans. Chemical genetics entails testing the effect that small molecules have on a biological process and is becoming a popular translational research method to identify therapeutic compounds. Zebrafish are specifically appealing to use for chemical genetics because of their ability to produce large clutches of transparent embryos, which are externally fertilized. Furthermore, zebrafish embryos can be easily drug treated by the simple addition of a compound to the embryo media. Using whole-mount in situ hybridization (WISH), mRNA expression can be clearly visualized within zebrafish embryos. Together, using chemical genetics and WISH, the zebrafish becomes a potent whole organism context in which to determine the cellular and physiological effects of small molecules. Innovative advances have been made in technologies that utilize machine-based screening procedures, however for many labs such options are not accessible or remain cost-prohibitive. The protocol described here explains how to execute a manual high-throughput chemical genetic screen that requires basic resources and can be accomplished by a single individual or small team in an efficient period of time. Thus, this protocol provides a feasible strategy that can be implemented by research groups to perform chemical genetics in zebrafish, which can be useful for gaining fundamental insights into developmental processes, disease mechanisms, and to identify novel compounds and signaling pathways that have medically relevant applications.

The zebrafish is an excellent experimental organism to study vertebrate developmental processes and model human disease. Here, we describe a protocol on how to perform a manual high-throughput chemical screen in zebrafish embryos with a whole-mount in situ hybridization (WISH) read-out.

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology ...

Biology

Acute Kidney Injury Model Induced by Cisplatin in Adult Zebrafish

Cisplatin is commonly used as chemotherapy. Although it has positive effects in cancer-treated individuals, cisplatin can easily accumulate in the kidney due to its low molecular weight. Such accumulation causes the death of tubular cells and can induce the development of Acute Kidney Injury (AKI), which is characterized by a quick decrease in kidney function, tissue damage, and immune cells infiltration. If administered in specific doses cisplatin can be a useful tool as an AKI inducer in animal models. The zebrafish has appeared as an interesting model to study renal function, kidney regeneration, and injury, as renal structures conserve functional similarities with mammals. Adult zebrafish injected with cisplatin shows decreased survival, kidney cell death, and increased inflammation markers after 24 h post-injection (hpi). In this model, immune cells infiltration and cell death can be assessed by flow cytometry and TUNEL assay. This protocol describes the procedures to induce AKI in adult zebrafish by intraperitoneal cisplatin injection and subsequently demonstrates how to collect the renal tissue for flow cytometry processing and cell death TUNEL assay. These techniques will be useful to understand the effects of cisplatin as a nephrotoxic agent and will contribute to the expansion of AKI models in adult zebrafish. This model can also be used to study kidney regeneration, in the search for compounds that treat or prevent kidney damage and to study inflammation in AKI. Moreover, the methods used in this protocol will improve the characterization of tissue damage and inflammation, which are therapeutic targets in kidney-associated comorbidities.

This protocol describes the procedures to induce Acute Kidney Injury (AKI) in adult zebrafish using cisplatin as a nephrotoxic agent. We detailed the steps to evaluate the reproducibility of the technique and two techniques to analyze inflammation and cell death in the renal tissue, flow cytometry and TUNEL, respectively.

Cisplatin is commonly used as chemotherapy. Although it has positive effects in cancer-treated individuals, cisplatin can easily accumulate in the kidney ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

Behavioral Test Battery to Assess Neurotoxic Effects in Adult Zebrafish

Neurotoxicity Assessment in Adult Danio rerio using a Battery of Behavioral Tests in a Single Tank

The presence of neuropathological effects proved to be, for many years, the main endpoint for assessing the neurotoxicity of a chemical substance. However, in the last 50 years, the effects of chemicals on the behavior of model species have been actively investigated. Progressively, behavioral endpoints were incorporated into neurotoxicological screening protocols, and these functional outcomes are now routinely used to identify and determine the potential neurotoxicity of chemicals. Behavioral assays in adult zebrafish provide a standardized and reliable means to study a wide range of behaviors, including anxiety, social interaction, learning, memory, and addiction. Behavioral assays in adult zebrafish typically involve placing the fish in an experimental arena and recording and analyzing their behavior using video tracking software. Fish can be exposed to various stimuli, and their behavior can be quantified using a variety of metrics. The novel tank test is one of the most accepted and widely used tests to study anxiety-like behavior in fish. The shoaling and social preference tests are useful in studying the social behavior of zebrafish. This assay is particularly interesting since the behavior of the entire shoal is studied. These assays have proven to be highly reproducible and sensitive to pharmacological and genetic manipulations, making them valuable tools for studying the neural circuits and molecular mechanisms underlying behavior. Additionally, these assays can be used in drug screening to identify compounds that may be potential modulators of behavior.
We will show in this work how to apply behavioral tools in fish neurotoxicology, analyzing the effect of methamphetamine, a recreational drug, and glyphosate, an environmental pollutant. The results demonstrate the significant contribution of behavioral assays in adult zebrafish to the understanding of the neurotoxicological effects of environmental pollutants and drugs, in addition to providing insights into the molecular mechanisms that may alter neuronal function.

Author Spotlight: Assessment of Environmental and Drug-Induced Behavioral Changes in Adult Zebrafish 

Here, we present a comprehensive behavioral test battery, including the novel tank, Shoaling, and social preference tests, to effectively determine the potential neurotoxic effects of chemicals (e.g., methamphetamine and glyphosate) on adult zebrafish using a single tank. This method is relevant to neurotoxicity and environmental research.

The presence of neuropathological effects proved to be, for many years, the main endpoint for assessing the neurotoxicity of a chemical substance. ...

Neuroscience

High-throughput Measurement of Gut Transit Time Using Larval Zebrafish

Zebrafish are used as alternative model organisms for drug safety testing. The gastrointestinal (GI) tract of zebrafish has genetic, neuronal, and pharmacological similarities to that of the mammals. GI intolerance during clinical testing of drug candidates is common and may pose a serious threat to human health. Testing for GI toxicity in preclinical mammalian models can be expensive in terms of time, test compound, and labor. The high-throughput method presented here may be used to predict GI safety issues. Compared to mammalian models, this method allows for more expedient assessment of test compound effects on GI transit while using low quantities of compound. In this method, larval zebrafish (7 days post fertilization) are fed food containing a fluorescent label. After feeding, each larval fish is placed into a well of a 96-conical-bottom-well plate and dosed with test compound (dissolved in water) or the vehicle. As gut transit occurs, fecal matter accumulates on the bottom of the wells, and the rate at which this happens is monitored by measuring fluorescence from the bottom of the well repeatedly over time using a plate spectrophotometer. The fluorescence from larvae in a given treatment group are averaged and these values are graphed along with standard error, for each measurement time, yielding a curve representing average transit of food over time. Effects on gut transit time are identified by comparing the area under the curve for each treatment group to that of the vehicle-treated group. This method detected changes in zebrafish GI transit time induced by drugs with known clinical GI effects; it can be employed to interrogate dozens of treatments for GI effects per day. As such, safer compounds can be quickly prioritized for mammalian testing, which expedites discovery and proffers 3Rs advancement.

The goal of this protocol is to measure the transit time of fluorescently labeled food through the gut of larval zebrafish in a high throughput fashion.

Zebrafish are used as alternative model organisms for drug safety testing. The gastrointestinal (GI) tract of zebrafish has genetic, neuronal, and ...

Startle Assay for Toxicity Evaluation in Zebrafish Embryo

Evaluating Toxicity of Chemicals using a Zebrafish Vibration Startle Response Screening System

We developed a simple screening system for the evaluation of neuromuscular and general toxicity in zebrafish embryos. The modular system consists of electrodynamic transducers above which tissue culture dishes with embryos can be placed. Multiple such loudspeaker-tissue culture dish pairs can be combined. Vibrational stimuli generated by the electrodynamic transducers induce a characteristic startle and escape response in the embryos. A belt-driven linear drive sequentially positions a camera above each loudspeaker to record the movement of the embryos. In this way, alterations to the startle response due to lethality or neuromuscular toxicity of chemical compounds can be visualized and quantified. We present an example of the workflow for chemical compound screening using this system, including the preparation of embryos and treatment solutions, operation of the recording system, and data analysis to calculate benchmark concentration values of compounds active in the assay. The modular assembly based on commercially available simple components makes this system both economical and flexibly adaptable to the needs of particular laboratory setups and screening purposes.

Author Spotlight: High-Throughput Toxicity Screening Using Zebrafish Embryo Startle Response Assay

We describe a screening system's workflow and data analysis for evaluating chemical compound toxicity based on the zebrafish embryo vibration startle response. The system records the movements of zebrafish embryos upon exposure to a vibration stimulus and allows for an integrated evaluation of general toxicity/lethality and neuromuscular toxicity.

We developed a simple screening system for the evaluation of neuromuscular and general toxicity in zebrafish embryos. The modular system consists of ...