Use of 2-color flow cytometry to assess radiation-induced genotoxic damage on CHO-KI cells

Authors

  • Luma Ramirez de Carvalho Institute of Energy an Nuclear Research
  • Letícia Bonfim Instituto de Pesquisas Energéticas e Nucleares
  • Daniel Perez Vieira Instituto de Pesquisas Energéticas e Nucleares

DOI:

https://doi.org/10.15392/bjrs.v7i2A.570

Keywords:

micronucleus, radiation, flow cytometry

Abstract

The micronucleus assay is an important technique used to evaluate genotoxic damage of chemical or physical agents (as ionizing radiations) on cells, based on quantification of cells bearing micronuclei, which are fragments derived from damage (breakage) of the DNA. Currently, this technique was updated to an automated approach that relies on plasma membrane dissolution to analyze fluorescent dye-labelled nuclei and micronuclei by flow cytometry. Cell suspensions were irradiated in PBS by a 60Co source in doses between 0 and 16 Gy, and incubated by 72h. Cell membranes were lysed in the presence of SYTOX Green and EMA dyes, so EMA-stained nuclei could be discriminated as from dead cells, and nuclei and micronuclei could be quantified. Amounts of micronuclei (percent of events) in the samples, were found to be proportional to radiation doses, and could be fitted to a linear-quadratic model (R² = 0.993). Only higher doses (8 and 16 Gy) and positive control could induce relevant increases in micronucleus amounts. The incorporation EMA showed an increase in irradiated cells. Mid- to high doses (4, 8 and 16 Gy) induced reduction of cell proliferation. Experiments showed the suitability of the technique to replace traditional microscopy analysis in evaluation of the effects of ionizing radiations on cells, with possibility to use in biological dosimetry.

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References

SPEIT, G.; ZELLER, J.; NEUSS, S. The in vivo or ex vivo origin of micronuclei measured in human biomonitoring studies. Mutagenesis, v. 26, n. 1, p. 107–110, 2011.

OECD – Organisation for Economic Co-operation and Development. Oecd guideline for the testing of chemicals draft proposal for a new guideline 487 : in vitro micronucleus test. 2012. Available at: < http://www.oecd.org/env/ehs/testing/TG487%20Oct%202012%20updated%2029oct.pdf>. Last accessed: 22 Nov 2017.

HEDDLE, J. A. et al. Reflections on the development of micronucleus assays. Mutagenesis, v. 26, n. 1, p. 3–10, 2011.

OCAMPO, I.Z.; PASSOS, P.Q.S.P.; de CARVALHO, L.R.; LIRA DA CRUZ, C.A.; ESTEVES-PEDRO, N.M.; da SILVA, F.M.; HIGA, O.Z.; DIAS, L.A.; OKAZAKI, K.; VIEIRA, D.P. In vitro cytotoxic and genotoxic evaluation of peptides used in nuclear medicine (DOTATATE and Ubiquicidin(29-41)) in CHO-K1 cells. Cytotechnology, v. 6, p. 2301-2310, 2016.

BRYCE, S. M; BEMIS, J.C.; AVLASEVICH, S.L.; DERTINGER, S.D. In vitro micronucleus assay scored by flow cytometry provides a comprehensive evaluation of cytogenetic damage and cytotoxicity. Mutation Research - Genetic Toxicology and Environmental Mutagenesis, v. 630, p. 78–91, 2007.

GARTY, G.; BIGELOW, A.W.; REPNI, M.; TURNER, H.C.; BIAN, D.; BALAJEE, A.S.; LYULKO, O.V.; TAVERAS, M.; YAO, Y.L.; BRENNER, D.J. An automated imaging system for radiation biodosimetry. Microscopy Research and Technique, v. 598, p. 587–598, 2015.

HALL, E. J. et al. Basic radiobiology. American journal of clinical oncology., v. 11, p. 220–252.

BRYCE, S.M.; SHI, J.; NICOLETTE, J.; DIEHL, M.; SONDERS, P.; AVLASEVICH, S.; RAJA, S.; BEMIS, J.C.; DERTINGER, S.D. High content flow cytometric micronucleus scoring method is applicable to attachment cell lines. Environ Mol Mutagen, v. 51, p.260-266, 2010.

SANTOS, G.S.; TSUTSUMI, S.; VIEIRA, D.P.; BARTOLINI, P.; OKAZAKI, K. Effect of Brazilian propolis (AF-08) on genotoxicity, cytotoxicity and clonogenic death of Chinese hamster ovary (CHO-K1) cells irradiated with (60)Co gamma-radiation. Mutat Res Genet Toxicol Environ Mutagen, v. 762, p.17-23, 2014.

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Published

2019-02-07

How to Cite

de Carvalho, L. R., Bonfim, L., & Vieira, D. P. (2019). Use of 2-color flow cytometry to assess radiation-induced genotoxic damage on CHO-KI cells. Brazilian Journal of Radiation Sciences, 7(2A (Suppl.). https://doi.org/10.15392/bjrs.v7i2A.570

Issue

Section

The Meeting on Nuclear Applications (ENAN)