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Apoptosis can be a confusing factor in in vitro clastogenic assays
S Meintières1, A Biola, M Pallardy
1Laboratoire de Toxicologie Génétique, Institut Pasteur de Lille, 1 Rue du Pr Calmette, BP 245, 59019 Lille Cedex, France.
Mutagenesis
|April 26, 2001
Summary
Apoptosis, programmed cell death, can cause DNA fragmentation, leading to false positive results in mutagenicity testing. A new cell line helps accurately assess chemical genotoxicity by distinguishing apoptosis from true mutagenic effects.
Area of Science:
- Toxicology
- Molecular Biology
- Genetics
Background:
- Chromosomal aberrations and micronucleus assays are crucial for evaluating chemical mutagenic potential.
- Apoptosis, characterized by DNA fragmentation, can interfere with these assays, potentially yielding false positive results.
- Accurate assessment of genotoxicity is vital for chemical safety and risk evaluation.
Purpose of the Study:
- To develop and validate a cell line model for demonstrating and quantifying the interference of apoptosis in mutagenicity testing.
- To differentiate between apoptosis-induced DNA fragmentation and true genotoxic events in chemical safety assessments.
- To improve the accuracy of in vitro mutagenicity assays, specifically the micronucleus and chromosomal aberration tests.
Main Methods:
- Utilized a murine cytotoxic T cell line (CTLL-2) stably transfected with the bcl2 gene (CTLL-2 Bcl2) to inhibit apoptosis.
- Compared mutagenicity assay results (in vitro micronucleus and metaphase analysis) between parental CTLL-2 cells and apoptosis-resistant CTLL-2 Bcl2 cells.
- Treated cells with non-genotoxic apoptosis inducers (dexamethasone, gliotoxin), a clastogen inducing apoptosis (etoposide), and an aneugen (nocodazole).
Main Results:
- Non-genotoxic apoptosis inducers caused false positive results in parental CTLL-2 cells, but not in CTLL-2 Bcl2 cells, confirming apoptosis interference.
- Etoposide treatment showed significantly higher percentages of aberrant cells and micronuclei in CTLL-2 cells compared to CTLL-2 Bcl2 cells, indicating apoptosis overestimates genotoxicity.
- Nocodazole results in CTLL-2 Bcl2 cells demonstrated the model's ability to detect pure genotoxic potential, allowing for better estimation of genotoxic thresholds.
Conclusions:
- Apoptosis can significantly interfere with in vitro mutagenicity assays, leading to overestimation of a chemical's genotoxic potential.
- The developed CTLL-2 Bcl2 cell line model effectively distinguishes apoptosis-related artifacts from true genotoxic effects.
- This model enhances the reliability of mutagenicity testing, enabling more accurate risk assessment of chemical compounds.