Zebrafish as a Model to Assess the Teratogenic Potential of Nitrite

Summary

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.

Abstract

High nitrate levels in the environment may 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 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.

Introduction

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.

Underst....

Protocol

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

1. Maintain zebrafish at 28.5 °C, pH 7, conductivity between 500-1,500 µS, and a light/dark cycle of 14 hr light and 10 hr dark²⁴. Use wild-type strains such as Tü, AB or Tü/AB hybrid. Different strains may respond differently to chemical treatment²⁵.
2. 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 ....

Results

Exposure to 300 mM ethanol for 22 hr had no effect on survival (data not shown), consistent with previous reports^5,23,26. 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.......

Discussion

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

Materials

List of materials used in this article

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