Using CTLL‐2 and CTLL‐2 bcl2 cells to avoid interference by apoptosis in the in vitro micronucleus test

Abstract

In vitro assays for chromosome aberrations (i.e., in vitro micronucleus and in vitro metaphase analysis tests) frequently produce false-positive or exaggerated-positive results. Our previous work suggested that apoptosis interferes with these tests, producing misleading results. These previous studies were conducted by performing the in vitro micronucleus test in CTLL-2 cells and a CTLL-2 cell derivative stably transfected with the apoptosis inhibitor gene bcl2. In the present study, these previous observations were extended by examining micronucleus induction with a larger number of compounds in both CTLL-2 and CTLL-2 bcl2 cells and measuring apoptosis with annexin V-FITC. Both cell lines were treated with different classes of compounds that were anticipated to be exclusively apoptosis inducers, or compounds known to be clastogens or aneugens, some of which were anticipated to be both genotoxic and apoptotic. We were able to confirm that compounds that are only apoptogenic induced micronuclei in CTLL-2 but not CTLL-2 bcl2 cells, indicating that the positive responses are due to apoptosis in CTLL-2 cells. Some genotoxins (clastogens and aneugens) did not produce apoptosis by the annexin V assay and gave similar responses in CTLL-2 and CTLL-2 bcl2 cells. Finally, higher responses were induced in CTLL-2 cells compared to CTLL-2 bcl2 cells that were treated with aneugens or clastogens that were also apoptosis inducers, suggesting that the greater response in CTLL-2 cells is a consequence of both genotoxicity and apoptosis. Finally, it was demonstrated that just eliminating CTLL-2 cells having three or more micronuclei from scoring was not adequate for correctly evaluating agents that only produce apoptosis. The results indicate that coupling the in vitro micronucleus test in both CTLL-2 and CTLL-2 bcl2 cells with the measurement of apoptosis is able to distinguish the genotoxic effects of a test compound from its apoptotic potential and is able to avoid interference from apoptosis in the in vitro micronucleus test. These observations may provide the basis for a useful genotoxicity assay.

Interlaboratory evaluation of a flow cytometric, high content in vitro micronucleus assay

“…Clearly the number and classes of compounds analyzed needs to be expanded. Even so, these results are very encouraging, as the non-genotoxic cytotoxicants DEX and STS tend to be extreme challenges to mammalian cell genotoxicity assays [16][17]. Furthermore, we chose 24 hrs of continuous treatment as a particularly challenging experimental design to study, as previous work by lab L4 had demonstrated that assay specificity is reduced in this case [23].…”

“…Reports by Mentieres et al [16][17] and others suggest that the apoptotic program, perhaps especially nuclear fragmentation, can lead to the presence of MN-like events that may enhance the rate of false positive results. STS data presented here may represent a case in point, as elevated MN frequencies were observed by microscopy (L3).…”

Absence of genotoxic effects after exposure of mammalian cells to the recombinant human bone morphogenetic protein 2 (BMP‐2) prepared from E. coli

“…The micronuclei were identified according to the criteria of Countryman and Heddle [23]: the size of the micronucleus is less than one-third of that of the main nucleus, the micronucleus lies within the cytoplasm of the cells, is surrounded by a distinct nuclear membrane and shows no overlapping or linkage to the main nucleus. No evidence for apoptosis, which may interfere with the micronucleus assay [24], was observed during the testing.…”

Relationships between p53 status, apoptosis and induction of micronuclei in different human and mouse cell lines in vitro: Implications for improving existing assays

“…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, leading to possible 'false positive' results [16,40] which provides an explanation for the false-positive results observed for curcumin but not for the reference mutagen MMS.…”

“…Rat liver S-9 (aroclor 1253 induced) Treatment times (h) o-Anthranilic acid [14,15] 137.14 118-92-3 + 3 Curcumin [16] 368.38 458-37-7 − 3 2,4-Dichlorophenol [14,17,18] 163.00 120-83-2 + 3 Ethyl acrylate [14,19] 100.12 140-88-5 − 24 or 30 Eugenol [14,20,21] 164.20 97-53-0 + 3 p-Nitrophenol [17] 139.11 100-02-7 + 3 Phthalic anhydride [17,18] 148.12 85-44-9 − 3 Propyl gallate [17] 212.20 121-79-9 − 3 and 24 or 30 1,3-Dihydroxybenzene (resorcinol) [17] 110.11 108-46-3 − 3 genotoxic potential which is not verified). In a review [1] of a large database of over 700 chemicals, the cumulative incidence of ' [4,5]) would prohibit such in vivo follow-up.…”