Apoptosis can be a confusing factor in in vitro clastogenic assays.
Among the tests used to determine the mutagenic potential of chemicals, the chromosomal aberrations and micronucleus assays play an important role. These tests score either chromosomal structural aberrations at metaphase or micronuclei at interphase. One of the hallmarks of apoptosis is DNA fragmentation into 50-300 kpb leading to oligonucleosomal fragmentation that can interfere with the results of clastogenic assays. In this case, apoptosis may be a confusing factor in the evaluation of the mutagenic potential of molecules and lead to false positive results. For these reasons we have developed a cell line able to demonstrate the interference of apoptosis in two mutagenicity tests: the in vitro micronucleus test and metaphase analysis in vitro. We used a murine cytotoxic T cell line, CTLL-2 Bcl2, in which a stably transfected bcl2 gene is known to protect these cells from apoptosis induced by various stimuli. A comparison between results obtained in parental CTLL-2 cells and in CTLL-2 Bcl2 cells treated with non-genotoxic apoptosis inducers, such as dexamethasone or gliotoxin, leads us to conclude that apoptosis could give false positive results due to DNA fragmentation. Moreover, with etoposide, a clastogen that also induces apoptosis, we observed that the percentages of aberrant cells and numbers of micronuclei were significantly increased in CTLL-2 cells compared with CTLL-2 Bcl2 cells. This observation suggests that apoptosis leads to an overestimation of the genotoxic potential of chemicals. Finally, with nocodazole, an aneugen, we confirm that this model can also detect agents that have only genotoxic potential and thus allows a better estimation of the genotoxic threshold in studies with aneugens, thus avoiding overestimation of the mutagenic risk of such a compound.
Meintières
S
S
Laboratoire de Toxicologie Génétique, Institut Pasteur de Lille, 1 Rue du Pr Calmette, BP 245, 59019 Lille Cedex, France.
Biola
A
A
Pallardy
M
M
Marzin
D
D
D000818:Animals; D017304:Annexin A5 / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000633:toxicity; D018931:Antineoplastic Agents, Hormonal / Q000633:toxicity; D017209:Apoptosis; D002460:Cell Line; D053938:DNA Fragmentation; D003907:Dexamethasone / Q000633:toxicity; D004305:Dose-Response Relationship, Drug; D005047:Etoposide / Q000633:toxicity; D005912:Gliotoxin / Q000633:toxicity; D015151:Immunoblotting; D007166:Immunosuppressive Agents / Q000633:toxicity; D008677:Metaphase; D051379:Mice; D009152:Mutagenicity Tests / Q000379:methods*; D009153:Mutagens; D015739:Nocodazole / Q000633:toxicity; D019384:Nucleic Acid Synthesis Inhibitors / Q000633:toxicity; D014162:Transfection
D017304:Annexin A5; D000970:Antineoplastic Agents; D018931:Antineoplastic Agents, Hormonal; D007166:Immunosuppressive Agents; D009153:Mutagens; D019384:Nucleic Acid Synthesis Inhibitors; D005912:Gliotoxin; D005047:Etoposide; D003907:Dexamethasone; D015739:Nocodazole