Impact of cell cycle delay on micronucleus frequency in TK6 cells

Abstract

Previous studies with TK6 cells have shown that extending the recovery period after pulse treatment allows for greater micronucleus expression for some compounds. This study explores the role of cell cycle delay in micronucleus expression after pulse treatment with three model genotoxins [mitomycin C, etoposide (ETOP), vinblastine]. Cells were treated for 4 hr and allowed to recover for 36 hr with samples removed at various time points during the recovery period and analyzed for cell cycle distribution, apoptosis and micronucleus frequency. Our results show that mitomycin C causes cell cycle delay for 20 hr after pulse treatment and cell cycle perturbation is no longer evident after 36 hr of recovery. The micronucleus frequency of cells sampled at 36 hr is doubled when compared with cells sampled at 20 hr after mitomycin C removal. When cells were treated with indirect acting genotoxins (ETOP, vinblastine), cell cycle perturbation was not observed at the 20 hr time point. Micronucleus frequency after treatment with either ETOP or vinblastine did not differ between the 20 hr and the 36 hr time point. All three compounds induced similar levels of apoptosis ranging from 4.5 to 5.6% with maximum induction occurring at the 36-hr time point. We conclude that TK6 cells exhibit extended cell cycle arrest after exposure to MMC and can go on to express micronuclei, after overcoming cell cycle arrest.

High-content imaging analyses of γH2AX-foci and micronuclei in TK6 cells elucidated genotoxicity of chemicals and their clastogenic/aneugenic mode of action

“…Cell cycle was analyzed in TK6 cells in a couple of previous reports. MMS and MMC induced arrest in the G2 or M phase (G2/M) [15] and VBS induced arrest in the M phase [41], and our results are consistent with these previous reports. In our results, ETP induced G2/M phase arrest in TK6 cells.…”

“…In our results, ETP induced G2/M phase arrest in TK6 cells. In other reports, TK6 cells were arrested in the S phase immediately after treatment with ETP while in a recovery period, and G2 phase cells increased [41]. Moreover, ETP was reported to induce G2/M phase arrest in 3T3-L1 mouse cells [42].…”

The in vitro micronucleus assay using imaging flow cytometry and deep learning

“…In the Cyt-B version of the assay, the critical events that must be scored are MONO cells, BN cells with and without MN, and POLY cells 9 . In the non-Cyt-B version of the MN assay, the critical events that must be scored are MONO cells with and without MN 9 , but it has also been demonstrated that scoring BN cells with MN can yield valuable information 7 , 8 . Example images selected from IFC data show that cytoplasm and DNA content (e.g.…”

“…4, 5). With microscope scoring, statistically 8 . Additionally, Doherty et al quantified MNed BN cells in the non-Cyt-B version of the assay using TK6 cells after showing that not scoring these cells could lead false negative results 7 .…”

“…Scoring of MN is typically performed in once-divided binucleated (BN) cells by blocking division with Cytochalasin-B (Cyt-B). As the post-cellular division fate of MN is generally not well understood, the use of Cyt-B ensures that MN can be associated to a single cell division, circumventing confounding factors such altered division kinetics or cell cycle delays [5][6][7][8] . The Cyt-B approach also allows the scoring of mononucleated (MONO) and polynucleated (POLY) cells permitting the evaluation of cytotoxicity 9 .…”