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The Chicken Egg Genotoxicity Assay (CEGA): Assessing Target Tissue Exposure and Metabolism in the Embryo-Fetal
Yax Thakkar1,2, T Kobets2, Anne Marie Api1
1Research Institute for Fragrance Materials, Inc., Mahwah, New Jersey, USA.
Abstract:
The Chicken Egg Genotoxicity Assay (CEGA) is an avian egg-based model that utilizes the livers of developing chicken embryo-fetuses to assess the ability of chemicals to produce direct DNA damage. The main goal of the study was to evaluate target tissue exposure and metabolism in the CEGA to assess its suitability as a biologically relevant new approach methodology (NAM) for detecting the genotoxic potential of chemicals. An imaging study using two-photon excitation microscopy after the administration of a fluorescent dye (acridine orange) verified that chemicals following administration into the air sac of the fertilized chicken egg reach the target organ, liver. A metabolism study using liquid chromatography with high resolution mass spectrometry (LC/MS), conducted after the administration of benzo(a)pyrene (B(a)P) according to the CEGA protocol, confirmed the formation of sufficient amounts of reactive metabolite(s) responsible for the genotoxic effects of a parent compound upon reaching the target tissue. Moreover, an RNA sequencing study revealed that B(a)P in embryo-fetal chicken livers significantly upregulated several genes responsible for the activity of the CYP1A1 enzyme, which is critical for the bioactivation of B(a)P. These findings, along with the previously reported DNA damage (i.e., DNA adducts and single-strand breaks) produced by B(a)P in CEGA, support sufficient target tissue exposure to B(a)P and the ability of avian fetal livers to bioactivate B(a)P to a reactive intermediate. Overall, the findings in the study support the conclusion that the CEGA can be considered a robust potential alternative to the animal testing strategy for assessing the genotoxic potential of chemicals.
Insights
The Chicken Egg Genotoxicity Assay (CEGA) effectively assesses chemical genotoxicity by confirming target organ exposure and metabolic activation in avian embryos. This new approach methodology (NAM) shows promise as a robust alternative to traditional animal testing for genotoxicity evaluation.
Area of Science:
- Toxicology
- Genetics
- Developmental Biology
Background:
- The Chicken Egg Genotoxicity Assay (CEGA) is an avian model for detecting chemical-induced DNA damage.
- Evaluating target tissue exposure and metabolism is crucial for validating CEGA as a New Approach Methodology (NAM).
Purpose of the Study:
- To assess target tissue exposure and metabolic activation within the CEGA model.
- To determine the CEGA's suitability as a biologically relevant NAM for genotoxicity testing.
Main Methods:
- Two-photon excitation microscopy to track fluorescent dye distribution to the liver.
- Liquid chromatography with high-resolution mass spectrometry (LC/MS) to analyze chemical metabolism.
- RNA sequencing to investigate gene expression changes related to chemical bioactivation.
Main Results:
- Chemicals administered via the air sac successfully reached the embryonic liver.
- Benzo(a)pyrene (B(a)P) was metabolized into reactive intermediates within the avian fetal liver.
- B(a)P exposure upregulated CYP1A1 enzyme activity, indicating bioactivation.
Conclusions:
- The CEGA model demonstrates sufficient target tissue exposure and metabolic capacity for genotoxicity assessment.
- The CEGA assay is a robust potential alternative to animal testing for evaluating chemical genotoxicity.
