Flow-cytometric analysis of in vitro micronucleus formation: comparative studies with WIL2-NS human lymphoblastoid and L5178Y mouse lymphoma cell lines.
The development of a flow cytometry-based micronucleus (FCMMN) assay for measuring the micronucleus (MN) frequency in cells following exposure to test chemicals has potential for improving reproducibility and reducing turn-around time when compared with the traditional microscopy-based micronucleus method. A major drawback of the FCMMN assay is that a false-positive interpretation could result from the presence of large numbers of apoptotic or necrotic bodies in the measured sample. Although several studies have reported ways in which the FCMMN assay could be improved using different staining techniques or electronic gating strategies, to date none of these protocols are suitable for use as a screening assay. To reduce the interference from apoptosis, performing the FCMMN assay with an apoptosis-resistant cell line may be an alternative approach. This study reports the use of p53-mutated cell lines to minimise the interference found in the FCMMN assay. Two commonly used cell lines (WIL2-NS and L5178Y) were investigated by comparison of (1) cytotoxicity and micronucleus induction in the FCMMN assay following treatment with model genotoxicants and (2) apoptotic responses after exposure to inducers of apoptosis. Both cell lines were responsive to all genotoxicants, producing concentration-dependent results with respect to genotoxicity. WIL2-NS cells were found to be more tolerant to apoptosis induction than L5178Y cells. This characteristic could be beneficial to minimise the interference from apoptotic nuclei in the FCMMN genotoxicity-screening assay.
Laingam
S
S
Australian Water Quality Centre, Salisbury, Australia. somprasong.laingam@sawater.com.au
Froscio
S M
SM
Humpage
A R
AR
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D053148:Caspase 3 / Q000378:metabolism; D045744:Cell Line, Tumor; D003603:Cytotoxins / Q000633:toxicity; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D051379:Mice; D048629:Micronuclei, Chromosome-Defective / Q000139:chemically induced*; D015162:Micronucleus Tests / Q000379:methods*; D009153:Mutagens / Q000633:toxicity; D013395:Sucrose / Q000633:toxicity
D003603:Cytotoxins; D009153:Mutagens; D013395:Sucrose; D053148:Caspase 3