Jake Beckman
Laura Twichell
Effect of Inorganic Arsenic (V) on the Development of Zebrafish Embryos
Introduction
Arsenic, a metal pollutant found naturally in groundwater and unnaturally in mine waste sites and agricultural runoff, has been considered toxic to humans for several millennia (Shalat et al., 1996). Arsenic has similarly toxic effects on a range of organisms, with effects on grown organisms ranging from cancers and nerve damage in humans (Shalat et al., 1996) to metabolic changes and learning deficiencies in rats (Rodríguez et al., 2002). Many studies have also examined arsenic’s effect on embryological development to determine the signaling pathways that the toxin impacts. Studies have suggested that arsenic is teratogenic, and it has been shown to cross the mammalian placenta, affecting developing embryos whose mothers undergo exposure (Wlodarczyk et al., 1996).
Arsenic exists in both trivalent and pentavalent ionic forms, and is often administered as either sodium arsenate (AsV) or arsenic trioxide (AsIII), with sometimes opposing results. Only the trivalent ion of arsenic has direct biological ramifications: AsV must convert to AsIII to be active, so the greater influence of pentavalent arsenic in some cases is surprising. Ultimately, response to arsenic is species dependent and cannot be generalized based on broader phylogenies.
Neural tube defects have been identified as a common defect among mammals, possibly reflecting the fact that arsenic accumulates in the neuroepithelium of developing fetuses (Shalat et al., 1996). Inorganic arsenic, of which sodium arsenate and arsenic trioxide are two kinds, may cause neural tube defects by repressing cell replication through microtubule organization or by inhibiting cell shape changes necessary to neural tube formation (Shalat et al., 1996). Wlodarczyk and coworkers (1996) suggest that arsenic damaging of DNA is responsible for inhibition of cell propagation, thus delaying and preventing a normal neural tube closure.
This experiment examines the effect of inorganic pentavalent arsenic, administered in the form of sodium arsenate, on developing zebrafish (Danio rerio) embryos. The effects of arsenic on fish have environmental ramifications as arsenic is present in some water systems throughout the world and accumulates in aquatic animals (Tchounwou et al., 2001) sometimes to concentrations that would be toxic to humans (Chapman 1926, as cited by Dabrowski 1975). Zebrafish make good test subjects as embryos can be easily harvested year round and at selected times of day. Their advantage over other fish species lies in their relatively rapid development, and their optically clear embryos. Arsenic’s impact on fish embryos may vary greatly from its impact on embryos from other classes because fish undergo a different process of neurulation from many other vertebrates. Their neural tube does not close over to form a tube but rather hollows from a solid core of cells (J. Cebra-Thomas, personal communication, Spring 2004). Thus, neural tube defects may manifest themselves differently.
Zebrafish embryos will be exposed to various concentrations of arsenic at five and eight hours to observe the effects of this teratogen on neural tube development. At five hours, most embryos are at approximately 50% epiboly. At this stage, the embryonic shield is just forming, so cells have not yet differentiated. At eight hours, embryos are between 60 and 100% epiboly and the axes begin to form as cells converge to create the basic form of the embryo curled around the yolk sac. This stage was chosen because neurulation succeeds it closely.
Materials
Zebrafish embryos at 5h, 8h post fertilization
Zebrafish embryo medium
Sodium arsenate (AsHNa2O4 * 7H2O) obtained from Sigma-Aldrich Co., St. Louis, MO.
Distilled water
Petri dishes
Incubator
Procedure
Embryo Harvesting
1) Induce adult zebrafish to mate at the beginning of their light cycle by covering the bottom of the aquarium with a layer of marbles the night before. This will both excite the fish and protect the embryos from predation. Leave overnight.
2) Harvest the embryos between one and three hours after the fish have awakened (at the appearance of light) by using a suction vacuum to clean the bottom, removing embryos from under the marbles into a filter.
3) Wash embryos from filter into a dish of aquarium water. Sort the embryos from extraneous debris by using a wide-mouth pipet to remove them into a Petri dish of ZE solution.
4) Stage the embryos and allow to develop to desired stage.
Solution Preparation
1) Dilute arsenic to desired concentrations (preliminary assay should be performed to determine effective range) using extreme caution at high concentrations, as arsenic is highly toxic. The earliest dilutions may be made with distilled water; later solutions should be diluted in ZE solution.
2) Put approximately 10 mL of solutions into labeled Petri dishes. Make a control solution with 10 mL ZE solution.
2) Carefully clean or dispose of pipets and other exposed equipment.
Embryo Treatment
1) Divide embryos between the prepared solutions. Cover the dishes to prevent excessive evaporation.
2) Place in incubator and allow to develop.
Embryo Observation
1) Examine embryos under the dissecting microscope approximately 24 hours after fertilization.
2) Stage embryos and note any deformities observed.
3) Photograph selected embryos to document results.
References
Dabrowski, K. R. (1975). The effect of arsenic on embrional development of rainbow trout (Salmo gairdneri, Rich.). Water Research, 10: 793-796.
Peterková, R. and L. Puzanová. (1974). Effect of trivalent and pentavalent arsenic on early developmental stages of the chick embryo. Folia Morphologica, 24: 5-13.
Rodríguez, V. M., L. Carrizales, M. S. Mendoza, O. R. Fajardo, M. Giordano. (2002). Effects of sodium arsenate exposure on development and behavior in the rat. Neurotoxicology and Teratology, 24: 743-750.
Shalat, S. L., D. B. Walker, R. H. Finnell. (1996). Role of arsenic as a reproductive toxin with particular attention to neural tube defects. Journal of Toxicology and Environmental Health, 48: 253-272.
Stump, D. G., J. F. Holson, T. L. Fleeman, M. D. Nemec, C. H. Farr. (1999). Comparative effects of single intraperitoneal or oral doses of sodium arsenate or arsenic trioxide during in utero development. Teratology 60: 283-291.
Tchounwou, P. B., B. A. Wilson, A. B. Ishaque. (2001). Embryotoxicity of arsenic trioxide and atrazine to Japanese medaka (Oryzias latipes), and cytotoxicity to human liver carcinoma cells (HepG2). Environmental Toxicology and Risk Assessment: Science, Policy, and Standardisation – Implications for Environmental Decisions: Tenth Volume, ASTM STP 1403. B. M. Greenberg, R. N. Hull, M. H. Roberts, Jr., and R. W. Gensemer, Eds., American Society for Testing and Materials, West Conshohocken, PA. 83-95.
Wlodarczyk, B. A., G. D. Bennett, J. A. Calvin, R. H. Finnell. (1996). Arsenic-induced neural tube defects in mice: alterations in cell cycle gene expression. Reproductive Toxicology, 10: 447-454.
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