Kinetics of CHO A_L mutant expression after treatment with γ radiation, EMS, and asbestos

Abstract

The flow cytometry mutation assay (FCMA) uses hybrid CHO A L cells to measure mutations of the cd59 gene located on human chromosome 11 by the absence of fluorochrome-conjugated antibody binding to the CD59 surface antigen. Mutant expression peaks between 6 and 12 days, then decreases to a stable plateau, instead of a constant mutant fraction obtained by clonogenic assays. To evaluate this variable mutant expression time, cells were treated with radiation, EMS or asbestos and cell proliferation and survival were measured at times leading up to peak mutant expression. Potential doubling time (T pot ) values increased by at least 75% for each agent by 3 h after treatment but returned to control levels after only 3 days. Survival returned to 90% of control within a week, close to the peak expression day for all three agents. The survival of CD59 2 cells sorted on the peak day of expression was roughly half that of CD59 1 cells. Cloned EMS-treated CD59 2 cells had a doubling time of 16.7 vs. 14.1 h for CD59 1 cells. Triple mutants (CD59 2 /CD44 2 /CD90 2 ) were preferentially lost from the population over time, while the proportion of CD59 2 /CD90 2 mutants increased. In conclusion, the peak day of mutant expression occurs only when cells recover from the toxic effects of the mutagen. A fraction of cells originally quantified as mutants are lost over time due to lethal deletions and slower growth. ' 2009 International Society for Advancement of Cytometry Key terms mutation; CD59; CHO; flow cytometry; EMS; radiation; asbestos PEOPLE may be exposed to a wide range of potentially genotoxic substances in the environment. Furthermore, regulatory agencies require many commercial compounds to be tested for mutagenicity prior to production (1). As it was discovered that many mutagens are also carcinogens, there have been efforts to develop more sensitive methods to detect mutations caused by various agents, including mammalian cell assays such as the mouse lymphoma assay and Chinese hamster ovaryhuman hybrid (CHO A L ) assay (2-4). We and others have developed a flow cytometry mutation assay (FCMA) using the hybrid CHO A L cell line that measures mutant yield in less than 2 weeks (5-7). Mutations in the cd59 gene located on human chromosome 11 are measured by the absence of fluorochrome-conjugated antibody binding to the CD59 antigen on the cell surface, instead of relying on clonogenic survival.We have previously characterized the sensitivity of the FCMA to detect mutations induced by a wide range of genotoxic agents, including low responders, while discriminating between true mutagens and false positives (8). Because of the significant differences in mutant detection between clonogenic assays and the FCMA, it is important to determine if mutant fractions generated by the two methods are similar. Significantly larger mutant fractions for the same agents have been measured using the FCMA when compared with clonogenic assays such as the mouse lymphoma assay (MLA) or the HPRT, which is expected since only a small reg...

EMS mutant spectra generated by multi-parameter flow cytometry

“…This discrepancy may have several explanations; first, radiation causes large deletions which are of no consequence when they occur along chromosome 11, which for the most part is not essential for survival, while large deletions of pigA on the X chromosome would often be lethal. Second, we have reported that CD59 − cells that are likely GPI mutants increase from a small fraction to the majority of total CD59 − cells measured with the FCMA over several weeks [3]. This increase could be caused by the importance of GPI linkage in the cell membrane for proper cell death signaling [24,25] allowing GPI mutants that would normally die to propagate.…”

“…We have previously characterized the flow cytometry mutation assay (FCMA) as a sensitive and rapid method of measuring the genotoxicity of known and unknown chemical and physical agents [1–3]. The FCMA uses the CHO A L cell line that has an incorporated human chromosome 11 as well as the entire hamster genome.…”

Hypoxia/Reoxygenation-Induced Mutations in Mammalian Cells Detected by the Flow Cytometry Mutation Assay and Characterized by Mutant Spectrum

“…We have reported previously that the survival of cells sorted from the mutant regions on the peak day of mutant expression after treatment with EMS or γ radiation was lower than that of cells sorted from the CD59 + peak of the same population (23). The surviving fraction of cells from the CD59 − region (0.69 ± 0.07) was significantly less than those from the treated positive peak (0.83 ± 0.05) ( P < 0.02).…”

“…We showed previously that the return to control survival of treated cells is an indicator of the peak day of mutant expression after treatment with genotoxic agents (23). Cells were sorted from hypoxia-treated and control populations from days 2–12 after treatment and grown into colonies for survival analysis.…”

“…It is important to note that cells treated with hypoxia responded similarly in that the peak day of expression was day 12. We reported that clonogenic survival of treated cells returns to control levels at roughly the same time as the peak day of mutant expression (23). Hypoxia is no different given that the survival of the treated cells was ~90% of control on day 12.…”