Alcohol Induced Teratogenesis: How Different Concentrations of Ethanol Affect Zebrafish Embryonic Development.

Nick Breugom

Introduction

This study aimed to examine the effects of alcohol-induced teratogenesis on the embryonic development of Zebrafish (Danio rerio). Under normal conditions, zebrafish embryos progress through fertilization, cleavage, blastula, gastrula, segmentation, pharyngula, and hatching within approximately 48 hours (Kimmel et al. 1995). Gastrulation is a particularly critical stage, as cell movements such as epiboly establish the body axis and guide cellular differentiation. Disruptions during this phase can lead to significant developmental abnormalities.

Alcohol-induced teratogenesis, a process known to impair gene expression and disrupt developmental pathways, has been associated with defects in prechordal plate formation (Blader and Str?hle 1998). This study simulated the effects of ethanol exposure on zebrafish embryos at the 30% epiboly stage to assess how it impacts their development.

Materials and Methods

Zebrafish (Danio rerio) embryos were incubated at 28°C until they reached the 30% epiboly stage. Embryos were maintained in zebrafish culture medium (ZCM; 0.06 g/L Instant Ocean salt) prior to the application of treatments.

For the control group, embryos remained in ZCM within the incubator to continue normal development. To simulate the effects of alcohol exposure, experimental groups of embryos were transferred into solutions of ethanol at concentrations of 1%, 2%, or 3%. Embryos were exposed to the ethanol solutions for 3 hours while remaining in the incubator. Following exposure, they were transferred back into 50 mL of ZCM to resume development.

Embryos were retrieved from the incubator at 24 and 48 hours post-treatment for observation. Assessments focused on developmental deformities such as cyclopia, truncated trunks and tails, and mortality rates.

Results and Discussion

At 24 hours post-treatment, ethanol-exposed zebrafish embryos displayed an increased incidence of morphological abnormalities compared to the control group, which showed normal development. These abnormalities included truncated tails and cyclopia, with a clear dose-dependent relationship observed. Embryos exposed to higher ethanol concentrations (2% and 3%) exhibited more pronounced deformities than those exposed to 1%.

By 48 hours, the effects became increasingly severe, with higher concentrations of ethanol correlating with both a greater number of affected embryos and more significant abnormalities. Mortality rates were also elevated in embryos exposed to 2% and 3% ethanol, suggesting the compounding impact of both concentration and exposure duration. In contrast, control embryos exhibited typical development, highlighting the role of ethanol in inducing these deformities.

Statistical analysis using ANOVA revealed significant differences between the control group and embryos exposed to 3% ethanol at 48 hours (p = 0.003). Pairwise comparisons further demonstrated significant differences among ethanol concentrations, with 1% and 3% (p ≤ 0.001) and 2% and 3% (p = 0.003) ethanol treatments showing clear separation in their effects. These findings underscore a dose-responsive trend where higher ethanol concentrations resulted in more severe morphological disruptions.

The observed abnormalities, such as cyclopia and axial truncations, suggest disruptions to specific developmental processes during embryogenesis. The timing of exposure appears critical, as the 30% epiboly stage marks a period of active cell movement and axis establishment. The increased severity of deformities at 48 hours indicates that the effects of ethanol may continue to compound as development progresses, potentially interfering with later stages of differentiation.

Interestingly, not all embryos displayed abnormalities, even within the same ethanol treatment groups. This variability may be influenced by genetic factors, such as differences in metabolic rates or gene expression patterns, which could mitigate or exacerbate the effects of ethanol. Additionally, microenvironmental conditions may have played a role in the diffusion of ethanol into individual embryos, potentially shielding some from the full extent of exposure.

These results highlight the nuanced interplay of ethanol concentration, developmental timing, and intrinsic variability among embryos in determining the extent of morphological disruptions. Further exploration of these factors could provide deeper insights into the mechanisms underlying ethanol-induced teratogenesis.

Conclusion

Ethanol exposure during zebrafish embryogenesis revealed a distinct dose-dependent relationship, with increasing ethanol concentrations leading to a higher incidence of developmental abnormalities and mortality among embryos. Morphological defects such as truncated tails, shortened trunks, and cyclopia were observed with greater frequency and severity as ethanol concentration increased, particularly at the 2% and 3% levels. The findings illustrate the susceptibility of developing embryos to environmental factors during critical windows of development and underscore the significant teratogenic potential of ethanol. These results emphasize the role of precise developmental timing, as embryos exposed at the 30% epiboly stage exhibited heightened sensitivity to ethanol’s disruptive effects.

This dose-dependent pattern supports the broader principles of developmental biology, where environmental insults during key stages of growth can profoundly affect normal development. The observed abnormalities highlight the intricate interplay between gene regulation, cell differentiation, and external influences. Such findings provide insight into the mechanisms by which ethanol interferes with normal embryonic pathways, ultimately resulting in phenotypic deformities. These observations underscore the importance of minimizing alcohol exposure during pregnancy, particularly in light of the stage-specific vulnerabilities demonstrated in this study.

The variability in individual embryo responses to ethanol exposure also raises questions about genetic and environmental factors that may influence sensitivity. Genetic diversity among embryos, differences in metabolic capacity, or variations in the microenvironment of individual embryos may contribute to resilience or susceptibility to ethanol-induced damage. Such factors could help explain why certain embryos exhibited more pronounced abnormalities while others appeared less affected despite identical treatment conditions.

Further research should prioritize elucidating the molecular and genetic pathways disrupted by ethanol during embryogenesis. Investigating how ethanol affects critical regulatory processes such as gene expression, protein synthesis, and cell signalling could provide valuable insights into its teratogenic mechanisms. Additionally, exploring potential genetic or environmental modifiers that influence embryonic resilience could offer avenues for targeted interventions. These findings could ultimately pave the way for strategies to mitigate the harmful effects of ethanol on embryonic development and improve our understanding of how teratogens interact with developmental biology.

By identifying specific pathways and critical periods of vulnerability, future studies may inform public health recommendations and therapeutic approaches, reducing the risks associated with alcohol exposure during early development. This research not only builds upon existing knowledge of teratogenicity but also contributes to a deeper understanding of the complex interplay between genetics, environment, and embryonic growth.

References

Bilotta J, Barnett JA, Hancock L, Saszik S. 2004. Ethanol exposure alters zebrafish development: a novel model of fetal alcohol syndrome. Neurotoxicology and teratology. 26(6):737–43. doi:https://doi.org/10.1016/j.ntt.2004.06.011. https://www.ncbi.nlm.nih.gov/pubmed/15451038.

Blader P, Str?hle U. 1998. Ethanol Impairs Migration of the Prechordal Plate in the Zebrafish Embryo. Developmental Biology. 201(2):185–201. doi:https://doi.org/10.1006/dbio.1998.8995.

Kimmel CB, Ballard WW, Kimmel SR, Ullmann B, Schilling TF. 1995. Stages of embryonic development of the zebrafish. Developmental Dynamics. 203(3):253–310.

Terzin, Tomislav. 2024. AUBIO 338 Developmental Biology Lab Manual. Camrose (AB). University of Alberta.