RADIATION & RISK
Bulletin of the National Radiation and Epidemiological Registry
since 1992
About the Journal : 2018 : vol. 27, No. 1 : Application of MTT-assay to estimate long-term effects of acute γ-radiation on Daphnia magna

Application of MTT-assay to estimate long-term effects of acute γ-radiation on Daphnia magna

«Radiation and Risk», 2018, vol. 27, No. 1, pp.86-93

DOI: 10.21870/0131-3878-2018-27-1-86-93

Authors

Savina N.B.1 – Master's Degree Student. Obninsk Institute for Nuclear Power Engineering of the NRNU “MEPhI”, Obninsk
Uskalova D.V.1 – Post-Graduate Student. Obninsk Institute for Nuclear Power Engineering of the NRNU “MEPhI”, Obninsk
Sarapultseva E.I.1, 2 – D. Sc., Biol., Prof. OINPE NRNU MEPhI, Lead. Researcher. Obninsk Institute for Nuclear Power Engineering of the NRNU “MEPhI”. A. Tsyb MRRC, Obninsk. Contacts: 4 Korolyov str., Obninsk, Kaluga region, Russia, 249036. Tel. +7(910) 912-20-94; e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it. .

Abstract

We investigated applicability of MTT-assay for study of cytotoxic effects of low-level γ-radiation on multicellular crustacean D. magna. The species represent a very useful model for quick analysis of long-term cytotoxic effect of different agents including antitumor pharmaceuticals. The life span of D. magna is relatively short and seldom exceeds 10-11 weeks; they are also characterized by quite short duration of embryonic and postembryonic development, 3-4 days, and gestation of 5-8 days. At present, fast-growing application of radiopharmaceuticals (RFP) for diagnosis and treatment requires research into cytotoxicity and radiotoxicity of RFPs and products of their biotransformation in a body. Currently for preclinical study of radiopharmaceuticals mice and rats are used in biopharmacology. However, simple and express tests providing new insights into the mechanisms underlying the efficiency and toxicity of RFP are required. Over recent years we have studied long-term effects of low-dose radiation using D. magna as model for analysis of long-term effects of low-level γ-radiation and MTT-assay for assessment of the effects on metabolic activity in the species. In the article we present results of research on effects of low-level γ-radiation (10, 100 and 1000 mGy) on D. magna Straus and its non-irradiated offspring of the first generation. To assess cells metabolism in irradiated D. magna MTT-assay was used. The assay is based on the property of NAD(P)H-dependent oxidoreductase enzymes, contained in metabolically active cells, to reduce yellow tetrazolium salt (MTT) to the insoluble deep purple formazan. After dissolution of formazan crystals absorbance of the solution was measured. In our study we found that MTT-assay can be used for assessment of radiotoxic effects from exposure of D. magna to low-level γ-radiation; the toxic effect was passed to non-exposed offspring of the first generation; the toxic effect is low after exposure to 10 mGy. Our data also demonstrate that D. magna is a sensitive model that can be used for express analysis of long-term effects of low-level radiation.

Key words
МТТ-assay, Daphnia magna, gamma-radiation, acute radiation, radiation-induced biological effect, long-term radiobiological effects, cytotoxicity, low doses, viability, ecotoxicology.

References

1. Barata C., Campos B., Rivetti C., LeBlanc G.A., Eytcheson S., Street S., Tobor-Kaplon M., de Vries Buitenweg S., Choi S.-H., Choi J., Sarapultseva E.I., Coutellec M.-A., Coke M., Pandard P., Chaumo A., Quéau H., Delorme N., Geffard O., Martínez-Jerónimo F., Watanabe H., Tatarazako N., Lopes I., Pestana J., Soares A.M.V.M., De Schamphelaere K., Pereira C.M. Validation of a two-generational reproduction test in Daphnia magna: an interlaboratory exercise. Sci. Total Environ., 2017, vol. 579, pp.1073-1083.

2. Dallas L.J., Keith-Roach M., Lyons B.P., Jha A.N. Assessing the impact of ionising radiation on aquatic invertebrates: a critical review. Radiat. Res., 2012, vol. 177, pp. 693-716.

3. Fuller N., Lerebours A., Smith J.T., Ford A.T. The biological effects of ionising radiation on Crustaceans: a review. Aquat. Toxicol., 2015, vol. 167, pp. 55-67.

4. Sarapultseva E.I., Riabchenko N.I., Igolkina Iu.V., Ivannik B.P. Methyltetrazolium bromide (MTT) was applied for bioassay of environmental radioactivity in vivo. Radiatsionnaya biologiya. Radioekologiya – Radiation Biology. Radioecoogy, 2013, vol. 53, no. 6, pp. 634-638. (In Russian).

5. Hayon T., Dvilansky A., Shpilberg O., Nathan I. Appraisal of the MTT-based assay as a useful tool for predicting drug chemosensitivity in leukemia. Leukemia and Lymphoma, 2003, vol. 44, no. 11, pp. 1957-1962.

6. Melekhova O.P. Svobodnoradical’nye prosessy v razvitii genomnoy epiregulyacii [Free radical processes in the development of genomic epiregulation]. Moscow, Nauka Publ., 2010. 324 p. 7. Moskalev A.A., Aliper A.M., Smit-McBride Z., Buzdin A., Zhavoronkov A. Genetics and epigenetics of aging and longevity. Cell Cycle, 2014, vol. 13, no. 7, pp. 1063-1077.

8. Loed L.A., Wallace D.C., Martin G.M. The mitochondrial theory of aging and its relationship to reactive oxygen species damage and somatic mtDNA mutations. Proc. Natl. Acad. Sci. USA, 2005, vol. 102, no. 52, pp. 18769-18770.

9. Gaziev A.I. Pathways for maintenance of mitochondrial DNA integrity and mitochondrial functions in cells exposed to ionising radiation. Radiatsionnaya biologiya. Radioekologiya – Radiation Biology. Radioecology, 2013, vol. 53, no. 2, pp. 117-136. (In Russian).

10. Sarapultseva E.I., Melekhova O.P., Kossova G.V., Igolkina J.V., Alexeeva N.V. Free radical reactions in Daphnia after low doses of γ-irradiation in vivo. Radiatsionnaya biologiya. Radioekologiya – Radiation Biology. Radioecology, 2014, vol. 54, no. 3, pp. 305-308. (In Russian).

11. Bernas T., Dobrucki J. Mitochondrial and nonmitochondrial reduction of MTT: interaction of MTT with TMRE, JC-1, and NAO mitochondrial fluorescent probes. Cytometry, 2002, vol. 47, pp. 236-242.

12. Buch K., Peters T., Nawroth Th., Sänger M., Schmidberger H., Langguth P. Determination of cell survival after irradiation via clonogenic assay versus multiple MTT assay – a comparative study. Radiat. Oncol., 2012, vol.7, no. 1. doi: 10.1186/1748-717X-7-1.

13. Guideline for the Testing of Chemicals No. 211. Daphnia magna Reproduction Test. Organization for Economic Cooperation and Development. Paris, 2012. 202 p.

14. Cancer Cell Culture. Methods and Protocols. Ed.: I.A. Cree. Second ed. Springer. New York, Dordrecht, Heidelberg, London, Human Press, 2011, pp. 237-244.

15. Tryapitsina G.A. Reaktsiya biotsenozov vodnykh ekosistem na khronicheskoe radiatsionnoe vozdeystvie [Reaction biocenoses of aquatic ecosystems to chronic radiation exposure]. Avtoref. diss. doct. biol. nauk. Moscow, MSU, 2011. 46 p.

16. Sarapultseva E.I., Gorski A.I., Malina J.J. Radiation risk of mortality and reduced lifespan of daphnids gamma-irradiated at low doses. Radiatsiya i risk – Radiation and Risk, 2011, vol. 20, no. 1, pp. 32-38. (In Russian).

17. Sarapultseva E.I., Bychkovskay I.B., Fedortseva R.F. Inherited dose independent effects of the low-dose irradiation. The model experiments on Daphnia Magna. Meditsinskaya radiologiya i radiatsionnaya bezopas-nost' – Medical Radiology and Radiation Safety, 2009, vol. 54, no. 6, pp. 11-15. (In Russian).

18. Sarapultseva E.I., Dubrova Y.E. The long-term effects of acute exposure to ionising radiation on survival and fertility in Daphnia magna. Environ. Res., 2016, vol. 150, pp. 138-143.

Full-text article (in Russian)