The DNA Comet Assay for Evaluating Damage to Leukocyte DNA after Photodynamic Therapy

The aim of this research was (by using the DNA comet method) to study the level of DNA damage in leukocytes of the whole blood in tumor-bearing animals after photodynamic therapy (PDT) with the local administration of a photosensitizer.

Materials and Methods. The experiments were performed on 30 non-linear male albino rats. As a model of neoplasia, the rat renal carcinoma strain was used. The animals were divided into three groups: intact (n=10), without exposure (n=10) and with PDT exposure (n=10); each group was then divided into two subgroups according to the initial tumor volume: A — less than 0.3 cm³ and B — more than 0.5 cm³.

In the PDT group, 0.3% Photosens (SRC “NIOPIK”, Russia) was injected into the tumor. Then, 6–12 h after the injection, the tumor area was irradiated with a LED laser beam (λ=660±10 nm, P=100 mW/cm²). The PDT sessions were conducted on the 15^th and 19^th day after the transplantation.

The antitumor effect was evaluated by the absolute tumor growth rate. The DNA damage was assessed by the DNA comet assay adapted for this study. The %TDNA — the relative DNA content of the comet tail — was used for quantification.

Results. A direct correlation between the DNA damage and the absolute tumor growth was found (Spearman rank correlation coefficient r_s=0.85; p=0.006). Using the DNA comet method we observed an increased DNA damage in leukocytes of tumor-bearing animals exposed to PDT in the subgroup with initial tumor volumes <0.3 cm³; whereas no such changes were found in the subgroup with tumors >0.5 cm³. When PDT was preceded by a Photosens injection, the tumors regressed in 50% of rats regardless of the initial tumor volume. In rats resistant to PDT, the tumor growth was stimulated in rats with the initial tumors <0.3 cm³.

Conclusion. The level of DNA damage in blood leukocytes, determined with the alkaline version of the DNA comet method after author’s modification, can be used to indirectly evaluate the growth rate of a malignant neoplasm and predict the tumor response to photodynamic therapy combined with a locally-administered photosensitizer.

1. Kaplan M.A., Kapinus V.N., Popuchiev V.V., Romanko Yu.S., Yaroslavtseva-Isaeva E.V., Spichenkova I.S., Shubina A.M., Borgul O.V., Goranskaya E.V. Photodynamic therapy: results and prospects. Radiatsiya i risk (Byulleten’ NRER) 2013; 22(3): 115–123.
2. Gamayunov S.V., Grebenkina Е.V., Ermilina А.А., Karov V.А., König K., Korchagina К.S., Skrebtsova R.R., Terekhov V.M., Terentiev I.G., Turchin I.V., Shakhova N.М. Fluorescent monitoring of photodynamic therapy for skin cancer in clinical practice. Sovremennye tehnologii v medicine 2015; 7(2): 75–83, https://doi.org/10.17691/stm2015.7.2.10.
3. Fargnoli M.C., Peris K. Photodynamic therapy for basal cell carcinoma. Future Oncol 2015; 11(22): 2991–2996, https://doi.org/10.2217/fon.15.208.
4. Duvanskiy V.A., Knyazev M.V., Pravednikov P.V. Modern aspects of photodynamic therapy of esophageal. Eksperimental’naya i klinicheskaya gastroenterologiya 2011; 10: 111–116.
5. Yi E., Yang C.K., Leem C., Park Y., Chang J.E., Cho S., Jheon S. Clinical outcome of photodynamic therapy in esophageal squamous cell carcinoma. J Photochem Photobiol B 2014; 141: 20–25, https://doi.org/10.1016/j.jphotobiol.2014.09.001.
6. Yaitsky N.A., Gerasin V.A., Orlov S.V., Butenko A.B., Molodtsova V.P., Derevyanko A.V., Stelmakh L.V., Urtenova M.A., Gerasin A.V. Photodynamic therapy in treatment of lung cancer. Vestnik khirurgii im. I.I. Grekova 2010; 169(5): 31–34.
7. Simone C.B. 2nd, Cengel K.A. Photodynamic therapy for lung cancer and malignant pleural mesothelioma. Semin Oncol 2014; 41(6): 820–830, https://doi.org/10.1053/j.seminoncol.2014.09.017.
8. Chen K.C., Hsieh Y.S., Tseng Y.F., Shieh M.J., Chen J.S., Lai H.S., Lee J.M. Pleural photodynamic therapy and surgery in lung cancer and thymoma patients with pleural spread. PLoS One 2015; 10(7): e0133230, https://doi.org/10.1371/journal.pone.0133230.
9. Kubasova I.Yu., Vakulovskaya E.G., Ermakova K.V., Smirnova Z.S. Fluorescent detection and photodynamic therapy in treatment of brain tumors. Rossiyskiy bioterapevticheskiy zhurnal 2006; 5(4): 54–63.
10. Uzdensky A.B., Berezhnaya E., Kovaleva V., Neginskaya M., Rudkovskii M., Sharifulina S. Photodynamic therapy: a review of applications in neurooncology and neuropathology. J Biomed Opt 2015; 20(6): 61108, https://doi.org/10.1117/1.jbo.20.6.061108.
11. Trushina O.I., Chissov V.I., Sokolov V.V., Novikova Ye.G., Filonenko Ye.V., Frank G.A., Volchenko N.N. The antiviral and antitumor efficiency of photodynamic therapy in precancer and early cancer of the cervix uteri. Rossiyskiy onkologicheskiy zhurnal 2009; 4: 15–18.
12. Hillemanns P., Garcia F., Petry K.U., Dvorak V., Sadovsky O., Iversen O.E., Einstein M.H. A randomized study of hexaminolevulinate photodynamic therapy in patients with cervical intraepithelial neoplasia 1/2. Am J Obstet Gynecol 2015; 212(4): 465.e1–465.e7, https://doi.org/10.1016/j.ajog.2014.10.1107.
13. Krikunova L.I., Mkrtchyan L.S., Kaplan M.A., Rykova E.V., Kapinus V.N. Possibilities of photodynamic therapy for vulvar cancer. Radiatsiya i risk (Byulleten’ NRER) 2015; 24(2): 107–115.
14. Patel J., Rizk N., Kahaleh M. Role of photodynamic therapy and intraductal radiofrequency ablation in cholangiocarcinoma. Best Pract Res Clin Gastroenterol 2015; 29(2): 309–318, https://doi.org/10.1016/j.bpg.2015.02.008.
15. Stranadko E.P., Titova V.A., Riabov M.V., Petrovsky V.Y. Photodynamic therapy as a component of combined and complex treatment of head and neck cancer. Al’manakh klinicheskoy meditsiny 2006;
16. Ulupov M.J. A method for interstitial photodynamic treatment of head and neck malignancies. Rossiyskaya otorinolaringologiya 2010; 1(44): 137–140.
17. Polkin V.V., Kaplan M.A., Medvedev V.S., Semin D.Yu., Kapinus V.N., Spichenkova I.S., Isayev P.A., Derbugov D.N., Shubina A.M. Place of photodynamic therapy in organ-sparing treatment programs for squamous cell carcinoma of the oral mucosa. Opukholi golovy i shei 2012; 1: 23–28.
18. Mironov A.F. Photodynamic cancer therapy: a novel effective method for the malignant tumors diagnostics and treatment. Sorosovskiy obrazovatel’nyy zhurnal 1996; 8: 32–40.
19. Meerovich I.G., Meerovich G.A., Oborotova N.A., Baryshnikov A.Yu. Distribution of light along the depth of the tumor lesion and efficiency of utilization of therapeutic irradiation during the photodynamic therapy. Rossiyskiy bioterapevticheskiy zhurnal 2006; 5(3): 93–97.
20. Tsyb A.F., Kaplan M.A., Romanko Yu.S., Popuchiev V.V. Fotodinamicheskaya terapiya [Photodynamic therapy]. Moscow: Meditsinskoe informatsionnoe agentstvo; 2009; 212 p.
21. Lagoda T.S., Kaplan M.A., Krivosheev Ia.V., Zhavoronkov L.P., Bokova M.B. The optimization of a plan for the photodynamic therapy of sarcoma M1 using photosens. Voprosy onkologii 2000; 46(3): 327–331.
22. Yaroslavtseva-Isaeva Е.V., Kaplan M.A., Romanko Ju.S., Sokol N.I. Method of photodynamic therapy of experimental tumor (sarcoma-MI) with local administration of photosensitizer. Rossiyskiy bioterapevticheskiy zhurnal 2003; 2(4): 19–22.
23. Lagoda T.S., Kaplan M.A., Bondar’ A.M., Bokova M.B., Ivanov E.A., Brovin A.I., Spichenkova O.N. Use of photosense treatment to enhance photodynamic effect on solid sarcoma-1 in Wistar rats. Voprosy onkologii 2005; 51(1): 103–107.
24. Bonnet R., Berenbaum M. Porphyrins as photosensitizers. In: Photosensitising compounds: their chemistry, biology and clinical use. Bock G., Harnett S. (editors). John Wiley & Sons, Chichester; 1989; p. 40–49.
25. Moan J., Berg K., Kvam E., Western A., Malik Z., Rock A., Schneckenburger H. Intracellular localization of photosensitizers. In: Ciba Foundation Symposium 146. Wiley, Chichester; 1989; p. 95–111.
26. Moan J. On the diffusion length of singlet oxygen in cells and tissues. J Photochem Photobiol B Biol 1990; 6: 343–347, https://doi.org/10.1016/1011-1344(90)85104-5.
27. McNair F.I., Marples B., West C.M., Moore J.V. A comet assay of DNA damage and repair in K562 cells after photodynamic therapy using haematoporphyrin derivative, methylene blue and meso-tetrahydroxyphenylchlorin. Br J Cancer 1997; 75(12): 1721–1729, https://doi.org/10.1038/bjc.1997.295.
28. Haylett A.K., Ward T.H., Moore J.V. DNA damage and repair in Gorlin syndrome and normal fibroblasts after aminolevulinic acid photodynamic therapy: a comet assay study. Photochem Photobiol 2003; 78(4): 337–341, https://doi.org/10.1562/0031-8655(2003)0780337:ddarig2.0.co;2.
29. Mozaffarieh M., Schötzau A., Josifova T., Flammer J. The effect of ranibizumab versus photodynamic therapy on DNA damage in patients with exudative macular degeneration. Mol Vis 2009; 15: 1194–1199.
30. Eksperimental’naya otsenka protivoopukholevykh preparatov v SSSR i SShA [Experimental evaluation of antitumor drugs in the USSR and USA]. Pod red. Sof’inoy Z.P., Syrkina A.B., Goldina A., Klyayna A. [Sof’ina Z.P., Syrkin A.B., Goldin A., Klyayn A. (editors)]. Moscow: Meditsina; 1980; 295 p.
31. Bures J., Bureshova O., Huston J.P. Metodiki i osnovnye eksperimenty po izucheniyu mozga i povedeniya [Techniques and basic experiments on the study of the brain and behavior]. Moscow: Vysshaya shkola; 1991; 399 p.
32. Chernigina I.A., Shcherbatyuk T.G. A new version of comet assay. Sovremennye tehnologii v medicine 2016; 8(1): 20–27, https://doi.org/10.17691/stm2016.8.1.03.
33. Stepanov V.N. Metody i programmnye sredstva avtomatizatsii analiza izobrazheniy mediko-biologicheskikh mikroob»ektov. Avtoref. dis. … kand. tekhn. nauk [The techniques and software tools for automation of image analysis of biomedical micro-objects. PhD Thesis]. Moscow; 2005.
34. Sirota N.P., Kuznetsova E.A. The comet assay application in radiobiological investigations. Radiatsionnaya biologiya. Radioekologiya 2010; 50(3): 329–339.

Chernigina I.A., Plekhanova E.S., Scherbatyuk T.G. The DNA Comet Assay for Evaluating Damage to Leukocyte DNA after Photodynamic Therapy. Sovremennye tehnologii v medicine 2017; 9(4): 89, https://doi.org/10.17691/stm2017.9.4.11