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 ...

<ol>
	<li>Gilbert-Barness, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Teratogenic+causes+of+malformations.">Teratogenic causes of malformations.</a> Ann Clin Lab Sci. 40 (2), 99-114 (2010).</li><li>Brent, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cause+and+prevention+of+human+birth+defects:+What+have+we+learned+in+the+past+50+years.">The cause and prevention of human birth defects: What have we learned in the past 50 years.</a> Con Anom. 41 (1), 3-21 (2001).</li><li>Lin, S., Zhao, Y., Nel, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish:+an+in+vivo+model+for+nano+EHS+studies.">Zebrafish: an in vivo model for nano EHS studies.</a> Small. 9 (9-10), 1608-1618 (2013).</li><li>Pamanji, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxicity+effects+of+profenofos+on+embryonic+and+larval+development+of+zebrafish+(Danio+rerio).">Toxicity effects of profenofos on embryonic and larval development of zebrafish (Danio rerio).</a> Environ Toxicol Pharmacol. 39 (2), 887-897 (2015).</li><li>Simmons, A. E., Karimi, I., Talwar, M., Simmons, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+nitrite+on+development+of+embryos+and+early+larval+stages+of+the+zebrafish+(Danio+rerio).">Effects of nitrite on development of embryos and early larval stages of the zebrafish (Danio rerio).</a> Zebrafish. 9 (4), 200-206 (2012).</li><li>Mantecca, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxicity+Evaluation+of+a+New+Zn-Doped+CuO+Nanocomposite+With+Highly+Effective+Antibacterial+Properties.">Toxicity Evaluation of a New Zn-Doped CuO Nanocomposite With Highly Effective Antibacterial Properties.</a> Toxicol Sci. , (2015).</li><li>Jensen, F. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitric+oxide+formation+from+nitrite+in+zebrafish.">Nitric oxide formation from nitrite in zebrafish.</a> J Exp Biol. 210, 3387-3394 (2007).</li><li>Scholz, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+embryo+model+in+environmental+risk+assessment--applications+beyond+acute+toxicity+testing).">The zebrafish embryo model in environmental risk assessment--applications beyond acute toxicity testing).</a> Environ Sci Pollut Res Int. 15 (5), 394-404 (2008).</li><li>Howe, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+reference+genome+sequence+and+its+relationship+to+the+human+genome.">The zebrafish reference genome sequence and its relationship to the human genome.</a> Nature. 496 (7446), 498-503 (2013).</li><li>CDC. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+abortions+possibly+related+to+ingestion+of+nitrate-contaminated+well+water--LaGrange+County,+Indiana,+1991-1994.">Spontaneous abortions possibly related to ingestion of nitrate-contaminated well water--LaGrange County, Indiana, 1991-1994.</a> MMWR Morb Mortal Wkly Rep. 45 (26), 569-572 (1996).</li><li>Brender, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prenatal+nitrate+intake+from+drinking+water+and+selected+birth+defects+in+offspring+of+participants+in+the+national+birth+defects+prevention+study.">Prenatal nitrate intake from drinking water and selected birth defects in offspring of participants in the national birth defects prevention study.</a> Environ Health Perspect. 121 (9), 1083-1089 (2013).</li><li>Huber, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal+dietary+intake+of+nitrates,+nitrites+and+nitrosamines+and+selected+birth+defects+in+offspring:+a+case-control+study.">Maternal dietary intake of nitrates, nitrites and nitrosamines and selected birth defects in offspring: a case-control study.</a> Nutr J. 12, 34 (2013).</li><li>Phillips, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Naturally+occurring+nitrate+and+nitrite+in+foods+in+relation+to+infant+methaemoglobinaemia.">Naturally occurring nitrate and nitrite in foods in relation to infant methaemoglobinaemia.</a> Food Cosmet Toxicol. 9 (2), 219-228 (1971).</li><li>Moncada, S., Palmer, R. M., Higgs, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitric+oxide:+physiology,+pathophysiology,+and+pharmacology.">Nitric oxide: physiology, pathophysiology, and pharmacology.</a> Pharmacol Rev. 43 (2), 109-142 (1991).</li><li>Gladwin, M. T., Crawford, J. H., Patel, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biochemistry+of+nitric+oxide,+nitrite,+and+hemoglobin:+role+in+blood+flow+regulation.">The biochemistry of nitric oxide, nitrite, and hemoglobin: role in blood flow regulation.</a> Free Radic Biol Med. 36 (6), 707-717 (2004).</li><li>Gupta, S. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recurrent+acute+respiratory+tract+infections+in+areas+with+high+nitrate+concentrations+in+drinking+water.">Recurrent acute respiratory tract infections in areas with high nitrate concentrations in drinking water.</a> Environ Health Perspect. 108 (4), 363-366 (2000).</li><li>Kross, B. C., Ayebo, A. D., Fuortes, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methemoglobinemia:+nitrate+toxicity+in+rural+America.">Methemoglobinemia: nitrate toxicity in rural America.</a> Am Fam Physician. 46 (1), 183-188 (1992).</li><li>Greer, F. R., Shannon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infant+methemoglobinemia:+the+role+of+dietary+nitrate+in+food+and+water.">Infant methemoglobinemia: the role of dietary nitrate in food and water.</a> Pediatrics. 116 (3), 784-786 (2005).</li><li>Sanchez-Echaniz, J., Benito-Fernandez, J., Mintegui-Raso, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methemoglobinemia+and+consumption+of+vegetables+in+infants.">Methemoglobinemia and consumption of vegetables in infants.</a> Pediatrics. 107 (5), 1024-1028 (2001).</li><li>Virtanen, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitrate+and+nitrite+intake+and+the+risk+for+type+1+diabetes+in+Finnish+children.+Childhood+Diabetes+in+Finland+Study+Group.">Nitrate and nitrite intake and the risk for type 1 diabetes in Finnish children. Childhood Diabetes in Finland Study Group.</a> Diabet Med. 11 (7), 656-662 (1994).</li><li>Reimers, M. J., Flockton, A. R., Tanguay, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ethanol-+and+acetaldehyde-mediated+developmental+toxicity+in+zebrafish.">Ethanol- and acetaldehyde-mediated developmental toxicity in zebrafish.</a> Neurotoxicol Teratol. 26 (6), 769-781 (2004).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The zebrafish book: A guide for the laboratory use of zebrafish (Danio rerio). , (2007).</li><li>Loucks, E., Carvan, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strain-dependent+effects+of+developmental+ethanol+exposure+in+zebrafish.">Strain-dependent effects of developmental ethanol exposure in zebrafish.</a> Neurotoxicol Teratol. 26 (6), 745-755 (2004).</li><li>Bilotta, J., Barnett, J. A., Hancock, L., Saszik, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ethanol+exposure+alters+zebrafish+development:+a+novel+model+of+fetal+alcohol+syndrome.">Ethanol exposure alters zebrafish development: a novel model of fetal alcohol syndrome.</a> Neurotoxicol Teratol. 26 (2), 737-743 (2004).</li><li>Li, J., Jia, W., Zhao, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excessive+nitrite+affects+zebrafish+valvulogenesis+through+yielding+too+much+NO+signaling.">Excessive nitrite affects zebrafish valvulogenesis through yielding too much NO signaling.</a> PLoS One. 9 (3), e92728 (2014).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods for chemical analysis of water and wastes. , (1983).</li><li>Loucks, E., Ahlgren, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+teratogenic+changes+in+a+zebrafish+model+of+fetal+alcohol+exposure.">Assessing teratogenic changes in a zebrafish model of fetal alcohol exposure.</a> J Vis Exp. (61), (2012).</li><li>Addiscott, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fertilizers+and+nitrate+leaching.">Fertilizers and nitrate leaching.</a> Agricultural Chemicals and the Environment, Issues in Environmental Science and Technology. , 1-26 (1996).</li><li>Su, Y. F., Lu, L. H., Hsu, T. H., Chang, S. L., Lin, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Successful+treatment+of+methemoglobinemia+in+an+elderly+couple+with+severe+cyanosis:+two+case+reports.">Successful treatment of methemoglobinemia in an elderly couple with severe cyanosis: two case reports.</a> Journal of Medical Case Reports. 6 (290), (2012).</li><li>Harvey, M., Cave, G., Chanwai, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fatal+methaemoglobinaemia+induced+by+self-poisoning+with+sodium+nitrite.">Fatal methaemoglobinaemia induced by self-poisoning with sodium nitrite.</a> Emergency Medicine Australasia. 22, 463-465 (2010).</li><li>Nishiguchi, M., Nushida, H., Okudaira, N., Nishio, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+autopsy+case+of+fatal+methemoglobinemia+due+to+ingestion+of+sodium.">An autopsy case of fatal methemoglobinemia due to ingestion of sodium.</a> Forensic Research. 6, (2015).</li><li>Avdesh, A., Chen, M., Martin-Iverson, M. T., Mondal, A., Ong, D., Rainey-Smith, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regular+Care+and+Maintenance+of+a+Zebrafish+(Danio+rerio)+Laboratory:+An+Introduction.">Regular Care and Maintenance of a Zebrafish (Danio rerio) Laboratory: An Introduction.</a> J. Vis. Exp. (69), (2012).</li><li>Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., Schilling, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+embryonic+development+of+the+zebrafish.">Stages of embryonic development of the zebrafish.</a> Dev Dyn. 203 (3), 253-310 (1995).</li><li>White, R. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transparent+adult+zebrafish+as+a+tool+for+in+vivo+transplantation+analysis.">Transparent adult zebrafish as a tool for in vivo transplantation analysis.</a> Cell Stem Cell. 2 (2), 183-189 (2008).</li><li>Tsang, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish:+A+tool+for+chemical+screens.">Zebrafish: A tool for chemical screens.</a> Birth Defects Res C Embryo Today. 90 (3), 185-192 (2010).</li></ol>

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.

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	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:191px;">DREL/2010 instrument</td>
			<td style="width:185px;">Hach</td>
			<td style="width:104px;">26700-03</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:191px;">Ethanol</td>
			<td style="width:185px;">Sigma-Aldrich</td>
			<td style="width:104px;">E7023</td>
			<td></td>
		</tr>
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			<td height="20" style="height:20px;width:191px;">KIMAX glass Petri Dish</td>
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			<td style="width:104px;">89001-244</td>
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			<td height="20" style="height:20px;width:191px;">MS-222</td>
			<td style="width:185px;">Sigma-Aldrich</td>
			<td style="width:104px;">E10521</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:191px;">NitraVer 5 Nitrate Reagent</td>
			<td style="width:185px;">Hach</td>
			<td style="width:104px;">14034-46</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:191px;">NitriVer 3 Nitrite Reagent</td>
			<td style="width:185px;">Hach</td>
			<td style="width:104px;">14065-99</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:191px;">Parafilm</td>
			<td style="width:185px;">Fisher Scientific</td>
			<td style="width:104px;">3-374-10</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:191px;">Paraformaldehyde</td>
			<td style="width:185px;">Sigma-Aldrich</td>
			<td style="width:104px;">158127</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:191px;">S6E stereomicroscope</td>
			<td style="width:185px;">Leica</td>
			<td style="width:104px;">10446294</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:191px;">Sodium nitrate</td>
			<td style="width:185px;">Fisher Scientific</td>
			<td style="width:104px;">S343</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:191px;">Sodium nitrite</td>
			<td style="width:185px;">Fisher Scientific</td>
			<td style="width:104px;">S347</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:191px;">Transfer pipets</td>
			<td style="width:185px;">Laboratory Products Sales</td>
			<td style="width:104px;">L320072</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:191px;">Glass vials</td>
			<td style="width:185px;">Fisher Scientific</td>
			<td style="width:104px;">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...

Watch this Scientific Journal Video about Zebrafish as a Model to Assess the Teratogenic Potential of Nitrite at JoVE.com

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 ...

The overall goal of this procedure is to treat zebrafish embryos with nitrite and determine its teratogenic effect. This method can help answer some of the key questions in the field of developmental toxicology such as assessing the teratogenic effects of chemicals on embryonic development. The main advantage of this procedure is that it uses zebrafish embryo which develop externally and can be observed under a microscope.For the experiments demonstrated here maintain wild-type zebrafish strains, such as the Tuebingen strain, at 28.5 degrees Celsius and a light-dark cycle of 14 hours and 10 hours, respectively. The night before harvesting eggs, add fish water and dividers into mating tanks and place male and female fish on separate sides of the divider. To ensure that enough eggs will be produced set up 30 pairs of fish.The next morning, after the lights turn on, remove the dividers to initiate mating. Check the mating tanks for eggs every 15 minutes. Once the fish lay eggs, use a tea strainer to harvest the embryos and combine them all into a large container with E3 buffer.Incubate the eggs at 28.5 degrees Celsius. At 1.5 hours post fertilization, or HPF, under a dissecting microscope use a plastic transfer pipette to discard unfertilized eggs that will appear opaque. Transfer 50 embryos each into 100 by 15 millimeter glass petri dishes containing 50 milliliters of E3 buffer in triplicate for each treatment condition.For example for 11 treatment conditions, prepare 33 dishes of 50 embryos each. To three petri dishes of embryos at 2 HPF remove the E3 buffer and add 50 milliliters of 300 millimolar ethanol diluted in E3 buffer. Cover the dishes with parafilm to minimize evaporation of ethanol.Incubate the embryos in the ethanol buffer for 22 hours at 28.5 degrees Celsius, then remove the solution and use E3 buffer to wash out the ethanol, swirling the dish several times. Repeat the wash two more times. To 3 petri dishes of embryos at 2 HPF remove the E3 and replace with 50 milliliters of fresh E3.For nine petri dishes at 2 HPF remove the E3 and add 50 milliliters of 1, 000 milligrams per milliliter of sodium nitrite dissolved in E3 and incubate at 28.5 degrees Celsius, replacing the sodium nitrite solution daily.After 46 hours, remove three of the nine sodium nitrite plates, remove the solution, and use E3 buffer to wash the plates three times. 24 hours later remove three different sodium nitrite plates from the incubater, remove the solution, and use fresh E3 buffer to wash the embryos three times. 24 hours later, perform similar wash steps for the last three plates.Next, for another set of three petri dishes, replace the E3 buffer with 200 milligrams per milliliter of sodium nitrite, then set up groups of three dishes of 50 embryos each with 400, 600, 800, and 1, 000 milligrams per milliliter of sodium nitrite. Incubate the embryos for 70 hours, replacing the sodium nitrite solution daily. After the incubation, remove the solution from the plates, and use E3 buffer to wash the plates three times.For another three petri dishes of embryos, replace the E3 buffer with 50 milliliters of 1, 000 milligrams per milliliter of sodium nitrate in E3 buffer. After exposing the embryos for 70 hours, use E3 to wash the embryos as previously demonstrated. During each day of exposure, under a stereo microscope count the number of dead embryos, looking for a lack of heartbeat and blood circulation, or a lack of mobility after one minute of observation.Remove the dead embryos. At the end of the experiments, to euthanize the larvae, use a transfer pipette to remove the E3 buffer, then add 50 milliliters of 0.2%MS-222 and incubate for 10 minutes. After one E3 wash to remove the MS-222, leaving some volume to properly pipette the embryo, add the larvae to 4%PFA, swirl the dish several times, and use a transfer pipette to place the larvae into a glass vial with enough PFA to fill the vial.Then fix the larvae in the refrigerator overnight. After fixation, use a stereoscope and a digital camera at 30X magnification to take pictures of the larvae. Orient the larvae so that the anterior is to the left and the dorsal surface is at the top of the field.Exposure to 300 millimolar ethanol for 22 hours had no effect on survival, consistent with previous results. As shown here, observed phenotypes included pericardial edema and swim bladder non-inflation. Cranial facial defects, and developmental delay were also observed and are not shown here.Treatment with sodium nitrite resulted in mild to severe effects on survival, depending on the concentration, with higher concentrations of sodium nitrite resulting in lower survival rates. This figure illustrates that at higher concentrations, such as 1, 000 milligrams per milliliter, longer incubation times also have a more severe impact on survival. Finally, as shown here, unlike nitrate treated fish, nitrite treated larvae have developmental defects.Once mastered, this technique can be done in 3 hours and it's important to remember to remove the embryos daily to minimize the contamination. Following this procedure, other methods, like in situ hybridization, can be performed to assess gene expression. This technique is communion for researchers to explore teratogenic potentials of chemicals on development in zebrafish.

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9.3K visualizações.  Indiana University of Pennsylvania. The overall goal of this procedure is to treat zebrafish embryos with nitrite and determine its teratogenic effect. This method can help answer some of the key questions in the field of developmental toxicology such as assessing the teratogenic effects of chemicals on embryonic development. The main advantage of this procedure is that it uses zebrafish embryo which develop externally and can be observed under a microscope.For the experiments demonstrated here maintain wild-type zebrafi...