Discussion

Physical deformities were observed only among the zebrafish embryos cultured in 3% ethanol-ZEM solution and not in 1% and 2%, suggesting that the critical ethanol concentration may be anywhere from 2% to 3%, including 3% (Table 1). That range is overlaps significantly with Blader and Strahle’s critical ethanol concentration of 2.4% (1998). However, the frequency of the deformities obtained in the experiment was different from Blader and Strahle’s 74.1% (1998). 48 hours after the ethanol treatment, at 3% ethanol solution, approximately 20% displayed any physical deformities. Such discrepancy may be due to mistakes made when staging the embryos for the experiment. According to Blader and Strahle, only ethanol treatment during a narrow time window comprising late blastula and early gastrula stages (dome/30% epiboly) causes cyclopia (1998). Moreover, deformities of the trunk and the tail also represent dome/30% epiboly stage treatment effects. Thus, it can be suggested that the embryos used in the experiment were generally past their 30% epiboly stages. However, the low percentage of deformed embryos is statistically insignificant due to the small sample size used in the experiment; only 10 embryos were used for each ethanol concentration as opposed to Blader and Strahle’s 700 (1998).

Though in low numbers, cyclopia was observed in embryos treated with 3% ethanol, supporting the fact that ethanol does induce cyclopia in zebrafish embryos (Blader and Strähle, 1998). Cyclopia is induced in amphibians and chick embryos when the prechordal plate is removed, suggesting that the prechordal mesoderm is required to separate the monolithic eye field into two lateral domains (Blader and Strähle, 1998). Therefore, it can be suggested that the embryo with separate, but more closely spaced eyes had its prechordal plate mildly affected by ethanol. The role of the prechordal plate is further evidenced in the cylopic embryo; it was clearly observed that what looked like a single medial eye was actually two eye vesicles fused at the center of the head when viewed under the microscope (Figures 1D, 2D). However, it is not clear as to why embryos treated with ethanol displayed significant lack of pigmentation when observed 24 hours after the ethanol treatment but not after 48 hours. One possible explanation could be that ethanol slowed down the zebrafish’s development. In the presence of ethanol, eye development may proceed abnormally slowly, resulting in retarding of the proliferation of the pigment cells of the eyes. Another possible explanation is that ethanol might have caused apoptosis of the eye pigment cells at 24 h and neighboring cells compensated for the loss by differentiating into eye pigment cells by 48 h.

Ethanol did cause defective development in the posterior structures in zebrafish embryos. Although the mechanisms behind the observed deformities of the trunk and the tail are not known, it can be conjectured that the impairment of epiboly may be responsible. Blader and Strahle noted that epiboly was marginally impaired by the treatment, with the advance of the blastoderm margin lagging behind untreated controls by 10 to 15% toward the end of gastrulation (1998). Epiboly establishes the posterior area of the embryo including the trunk region and the tail bud and, therefore, it seems reasonable that impairing the last 10-15% of epiboly may cause defective trunk and tail (Gilbert, 2003). In addition to a defective trunk and tail, it was also observed that there lack of pigmentation in the truck regions in embryos exposed to 3% ethanol (Figures 2D, 2E, and 4D). One possible explanation for the pigmentation deficiency is that ethanol might have caused deformities in the spinal cord from which neural crest cells destined to become melanocytes arise (Gilbert, 2003). However, it is not conclusive as to whether lack of pigmentation can be attributed to ethanol exposure. Largely due to small sample size, further experimentation is needed to confirm ethanol’s role in pigmentation defect.

Another noticeable effect of ethanol was the mortality rate (Table 2). Though Blader and Strahle specifically noted that ethanol does not seem to cause death in embryos, the experiment suggests that increasing ethanol concentration increased the number of deaths (1998). Six out of 10 embryos incubated in the 3% ethanol solution died as opposed to 1 out of 10 of the controls dying seem significant. Moreover, it is difficult to simply attribute the linear relationship displayed in the data to chance. It was conjectured that abnormal apoptosis, an accompanying symptom caused by ethanol exposure, may have caused the deaths of embryos. It is thought that the enzymes that cells use to degrade and clear ethanol out of the system produce free radicals which chemically react with important components of the cell, such as proteins, DNA, and lipids (Sulik et al., 1988). Therefore, it does not seem farfetched to suggest that ethanol induced apoptosis may ultimately cause death by damaging the essential cellular structures of the embryos.

This experiment demonstrated that ethanol induces physical deformities, more specifically cyclopia and trunk and tail deformities, in zebrafish embryos. It is known that, in humans, severe fetal alcohol syndrome causes mild forms of holoprosencephalies, a group of disorders that include cyclopia (Sulik et al., 1988). Therefore, the findings of this experiment seem to bear at least some relevance to experiments investigating fetal alcohol syndrome in humans. This experiment, however, did little to investigate the presence of ethanol induced abnormal apoptosis and its role on physical defects and deaths. In another experiment, embryos may be exposed to varying concentrations of ethanol, stained for apoptosis using vital stains such as the Acridine Orange, and observe whether there are correlation between deformities and cell death.

 

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