Objective Intro Materials Procedure Results 1 Results 2 Results 3 Discussion Lit. Cited Prep Sheet
		Discussion No arsenic related mortalities or deformities were observed in any of the treatments performed. At 48 hrs after fertilization, there were no notable mortalities or deformations at any of the concentrations in any of the treatments, aside from a few isolated incidences of unviable embryos and malformation. Embryos from treatments 1 and 3 were euthanized at 48 hrs after fertilization. When treatment 2 was viewed at 96 hrs after fertilization, the mortality rate of embryos exposed to arsenic in the 5 h and 8 h groups showed no correlation to arsenic concentration. Neither the difference in the age of exposure nor concentration seemed to affect embryo development. It is possible that embryo mortality in treatment two at 96 hrs after fertilization could be attributed to the onset of starvation, as the embryos had nearly exhausted their endogenous supply of nutrients at this stage of development (discussion with Judy Cebra-Thomas, Ph.D., April 2004). One possible reason for the arsenic’s apparent lack of effect on zebrafish development is that neural patterning defects in vertebrates, such as those associated with arsenic exposure, are notoriously difficult to detect from morphological observation alone (Furutani-Seiki et al 1996). The neural tube forms by the process of apoptotic cavitation in fish, as compared to neural groove invagination in mammals, and as a result, makes gross morphological observation of neural tube defects much harder to recognize. Alternate methods for observing neural tube defects include in situ hybridization of neural associated proteins as well as staining for molecular apoptosis markers that are associated with neural development in zebrafish (Furutani-Seiki et al 1996). In further studies, we would employ these types of assays in addition to morphological observations. We examined the possibility that the chorion, a semi-permeable protective membrane that surrounds the developing embryo, might prevent arsenic from reaching the developing embryo and found there to be no observable difference in development between chorionated and dechorionated embryos. Studies suggest that effective embryonic exposure to certain chemicals requires the dechorionation of zebrafish embryos (Hammerschmidt et al 1999). Permeation assays of glycerol and DMSO have suggested that the chorion retards the free exchange of solutes into and out of the interchorionic space (Harvey et al 1983). Our results, however, show no difference in the effect of arsenic exposure on chorionated versus dechorionated embryos, given that all embryos in treatment 3 survived. Both embryos exposed to arsenic and control embryos showed an accelerated rate of growth when compared to their expected stage. There are several reasons for the discrepancy between the actual age of the embryos and the stage at which we categorized them. Incubation at a temperature slightly different from the optimal temperature of 28.5? C affects the rate of development (Kimmel et al 1995), as does individual variability even within a clonal strain of zebrafish (Streisinger et al., 1981 viewed in Kimmel et al 1995). Staging numbers (given as hpf) reflect the developmental stage of the embryo and do not necessarily relate to the actual age of the embryo. The range of arsenic exposure levels chosen was based on studies undertaken in other vertebrate systems. The concentration ranges used in previous studies to test the effect of arsenic exposure on developing embryos were 10-400 µM As(V) in mouse embryo cultures (Chaineau et al 1990), 0.6-667 µM As(V) in rainbow trout (Dabrowski 1975), and 1-5 µM As(III) in Japanese medaka (Tchounwou et al 2001). As(III) and As(V) have been shown to cause similar effects in development, except that As(III) is ten times stronger than As(V) (Chaineau et al 1990). There appears to be a species specific response to arsenic that varies not only across the vertebrate phylum, but within the teleost class as well. Rainbow trout embryos died at higher rates in lower concentrations of arsenic (Dabrowski 1975), while Japanese medaka exhibited higher mortality at higher concentrations of arsenic (Tchounwou 2001). Our results suggest that zebrafish might also exhibit a unique response to arsenic. In two separate treatments (8 h group in 1200 µM, viewed at 96 hr and 5 h group in 100 µM, viewed at 24 hr), cell blebbing in the head region was observed. We do not think this to be an arsenic-related incident. Rather, we propose that this blebbing was associated with cell death (Hagstrom et al 1999) and might reflect chemical or microbial contamination. It is important to note that due to the inaccuracy of our weighing devices (+/- 0.1 g) a large number of dilutions had to be made, with the possible effect of creating a disparity between expected arsenic concentration and actual concentration. Regardless, our results are puzzling especially in light of the fact that other studies show 80%-100% mortality at the 400-650 µM As(V) equivalents (Dabrowski 1975, Tchounwou 2001, Chaineau et al 1990). [Lab Protocols | Students | Cebra-Thomas | Course | Links ]