Medical Radiology and radiation safety Medical Radiology and radiation safety Медицинская радиология и радиационная безопасность 1024-6177 2618-9615 30323 10.12737/1024-6177-2019-64-5-5-8 Радиационная биология Radiation biology Радиационная биология Increase of Cell-Free Nuclear and Mitochondrial DNA Content in the Urine of Rats after X-ray Irradiation or Bleomycin Administration Увеличение содержания внеклеточной ядерной и митохондриальной ДНК в моче крыс после рентгеновского облучения или введения блеомицина Минкабирова Г. М. Minkabirova G. M. Абдуллаев С. А. Abdullaev S. A.

кандидат биологических наук;

candidate of sciences in biology;

 Институт теоретической и экспериментальной биофизики РАН Пущино Россия Institute of Theoretical and Experimental Biophysics RAS Pushchino Russian Federation Институт теоретической и экспериментальной биофизики РАН Пущино Россия Institute of Theoretical and Experimental Biophysics RAS Pushchino Russian Federation 64 5 5 8 https://naukaru.ru/en/nauka/article/30323/view

Цель: Исследование содержания внеклеточной митохондриальной ДНК (вк-мтДНК) и внеклеточной ядерной ДНК (вк-яДНК) в моче крыс, подвергнутых воздействию ионизирующего излучения или цитостатического препарата блеомицина. Материал и методы: В экспериментах использовались самцы крыс линии Wistar 3-месячного возраста с массой тела 250 г. Облучение крыс проводили на рентгеновской установке в дозах 3, 5 и 8 Гр. Блеомицин вводили внутрибрюшинно в концентрациях 3, 7, и 10 мг/кг. Анализы содержания вк-мтДНК и вк-яДНК проводили методом ПЦР в режиме реального времени. Результаты: Результаты анализов показали увеличение уровня количества фрагментов вк-яДНК и вк-мтДНК в моче крыс после их облучения. Показано, что содержание вк-яДНК и вк-мтДНК в моче имеет линейную зависимость от дозы рентгеновского излучения. Так, максимальное количество копий мтДНК и яДНК регистрировалось на 12–24-е часы после воздействия. Количество продуктов ПЦР-амплификации вк-мтДНК в 2–3 раза выше по сравнению c вк-яДНК. Данные анализов содержания вк-яДНК и вк-мтДНК в моче у крыс после введения блеомицина также показали повышенные их уровни по сравнению с контрольными животными. Показано, что содержание вк-яДНК и вк-мтДНК имеет линейную зависимость от дозы химиотерапевтического препарата. Выводы: Таким образом, показана возможность преодоления у животных вк-мтДНК и вк-яДНК трансренального (почечного) барьера и перехода их в мочу после рентгеновского облучения, а также после введения блеомицина. Обнаружена дозовая зависимость выявленных эффектов. Повышенное содержание внеклеточной ДНК в моче можно рассматривать как потенциальный биомаркер для оценки уровня генотоксического груза при радиационном поражении организма, а также при воздействии других генотоксических агентов.

Purpose: To study the content of cell-free mitochondrial DNA (cf-mtDNA) and cell-free nuclear DNA (cf-nDNA) in urine of rats exposed to ionizing radiation, and after injection of a cytostatic drug bleomycin. Material and methods: Wistar male rats aged 3 months were used in the experiments. Rats were irradiated at a doses of 3, 5, and 8 Gy. Bleomycin was administered intraperitoneally in concentrations of 3, 7, and 10 mg/kg. The DNA content was measured by real-time PCR. Results: The results showed an increase in the level of the number of cf-nDNA and cf-mtDNA fragments in urine of irradiated rats. It was shown that the content of cf-nDNA and cf-mtDNA has a linear dependence on the X-ray dose. Thus, the maximum number of mtDNA and nDNA copies was recorded for 12–24th hours after irradiation. The number of PCR amplification products of cf-mtDNA is 2–3 times higher than those of cf-nDNA. Data analysis of the content of cf-nDNA and cf-mtDNA in rat urine after introduction of bleomycin also showed elevated levels compared with control animals. It was shown that the content of cf-nDNA and cf-mtDNA has a linear dependence on the dose of the chemotherapeutic drug. Conclusion: Thus, it has been shown that it is possible to overcome the transrenal (renal) barrier in animals with cf-mtDNA and cf-nDNA and pass them into the urine after X-ray irradiation, as well as after the administration of bleomycin. The dose dependence of the identified effects was found. The increased content of cell-free DNA in the urine can be considered as a potential biomarker for assessing the level of genotoxic load during radiation damage to the body, as well as when exposed to other genotoxic agents.

 внеклеточная ДНК в моче рентгеновское облучение блеомицин крысы cell-free DNA in urine X-ray irradiation bleomycin rats

Bettegowda C, Sausen M, Leary RJ, et al. Detection of circulating tumor DNA in early- and late-stage human malignancies. Sci Transl Med. 2014; 6(224). 224ra24. Bettegowda C, Sausen M, Leary RJ, et al. Detection of circulating tumor DNA in early- and late-stage human malignancies. Sci Transl Med. 2014; 6(224). 224ra24. Zhang L, Zhang M, Yang S, et al. A new biodosimetric method: branched DNA-based quantitative detection of B1 DNA in mouse plasma. Br J Radiol. 2010;83:694-701. Zhang L, Zhang M, Yang S, et al. A new biodosimetric method: branched DNA-based quantitative detection of B1 DNA in mouse plasma. Br J Radiol. 2010;83:694-701. Zhang M, Zhang B, Guo Y, et al. Alteration of circulating mitochondrial DNA concentration after irradiation. Adv Exp Med Biol. 2013;765:371-7. Zhang M, Zhang B, Guo Y, et al. Alteration of circulating mitochondrial DNA concentration after irradiation. Adv Exp Med Biol. 2013;765:371-7. Strelkova IYu, Abdullaev SA, Snigireva GP, et al. Share of extracellular mutated mitochondrial DNA increases in plasma of lung cancer patients following radiotherapy. Biomed Khim. 2010;56:517-25. (in Russian). Strelkova IYu, Abdullaev SA, Snigireva GP, et al. Share of extracellular mutated mitochondrial DNA increases in plasma of lung cancer patients following radiotherapy. Biomed Khim. 2010;56:517-25. (in Russian). Sun W, Sun Y, Zhu M, et al. The role of plasma cell-free DNA detection in predicting preoperative chemoradiotherapy response in rectal cancer patients. Oncol Rep. 2014;31:1466-72. Sun W, Sun Y, Zhu M, et al. The role of plasma cell-free DNA detection in predicting preoperative chemoradiotherapy response in rectal cancer patients. Oncol Rep. 2014;31:1466-72. Abdullaev SA, Anishchenko ES, Gaziev AI. Mutant copies of mitochondrial DNA in tissues and plasma of X-rays exposed mice. Radiats Biol Radioecol. 2010;50(3):318-28. (in Russian). Abdullaev SA, Anishchenko ES, Gaziev AI. Mutant copies of mitochondrial DNA in tissues and plasma of X-rays exposed mice. Radiats Biol Radioecol. 2010;50(3):318-28. (in Russian). Abdullaev SA, Antipova VN, Gaziev AI. Extracellular mutant mitochondrial DNA content is sharply elevated in the blood plasma of irradiated mice. Mol Biol (Mosk). 2009;43(6):1063-9. (in Russian). Abdullaev SA, Antipova VN, Gaziev AI. Extracellular mutant mitochondrial DNA content is sharply elevated in the blood plasma of irradiated mice. Mol Biol (Mosk). 2009;43(6):1063-9. (in Russian). Della Latta V, Cecchettini A, Del Ry S, et al. Bleomycin in the setting of lung fibrosis induction: From biological mechanisms to counteractions. Pharmacol Res 2015;97:122-30. Della Latta V, Cecchettini A, Del Ry S, et al. Bleomycin in the setting of lung fibrosis induction: From biological mechanisms to counteractions. Pharmacol Res 2015;97:122-30. Oberoi HS, Nukolova NV, Kabanov AV, Bronich TK. Nanocarriers for delivery of platinum anticancer drugs. Adv Drug Deliv Rev. 2013;65:1667-85. Oberoi HS, Nukolova NV, Kabanov AV, Bronich TK. Nanocarriers for delivery of platinum anticancer drugs. Adv Drug Deliv Rev. 2013;65:1667-85. Umansky SR, Tomei LD. Transrenal DNA testing: progress and perspectives. Expert Rev Mol. 2006;6:155-63. Umansky SR, Tomei LD. Transrenal DNA testing: progress and perspectives. Expert Rev Mol. 2006;6:155-63. Bouatra S, Aziat F, Mandal R, et al. The human urine metabolome. PLoS ONE. 2013;8(9) e73076. Bouatra S, Aziat F, Mandal R, et al. The human urine metabolome. PLoS ONE. 2013;8(9) e73076. Liu H, Ma Y, Fang F, et al. Wild-type mitochondrial DNA copy number in urinary cells as a useful marker for diagnosing severity of the mitochondrial diseases. PLoS ONE. 2013;8(6) e67146. Liu H, Ma Y, Fang F, et al. Wild-type mitochondrial DNA copy number in urinary cells as a useful marker for diagnosing severity of the mitochondrial diseases. PLoS ONE. 2013;8(6) e67146. Dasgupta S, Shao C, Keane TE, et al. Detection of mitochondrial deoxyribonucleic acid alterations in urine from urothelial cell carcinoma patients. Int J Cancer. 2012;131:158-64. Dasgupta S, Shao C, Keane TE, et al. Detection of mitochondrial deoxyribonucleic acid alterations in urine from urothelial cell carcinoma patients. Int J Cancer. 2012;131:158-64. Umansky SR. From transrenal DNA to stem cell differentiation: an unexpected twist. Clinical Chem. 2009;55:602-4. Umansky SR. From transrenal DNA to stem cell differentiation: an unexpected twist. Clinical Chem. 2009;55:602-4. Abdullaev SA, Minkabirova GM, Bezlepkin VG, et al. Cell-free DNA in the urine of rats exposed to ionizing radiation. Radiat Environ Biophys. 2015;54:297-304. Abdullaev SA, Minkabirova GM, Bezlepkin VG, et al. Cell-free DNA in the urine of rats exposed to ionizing radiation. Radiat Environ Biophys. 2015;54:297-304. Malik AN, Shahni R, Rodriguez-de-Ledesma A, et al. Mitochondrial DNA as a non-invasive biomarker: accurate quantification using real time quantitative PCR without co-amplification of pseudogenes and dilution bias. Biochem Biophys Res Commun. 2011;412:1-7. Malik AN, Shahni R, Rodriguez-de-Ledesma A, et al. Mitochondrial DNA as a non-invasive biomarker: accurate quantification using real time quantitative PCR without co-amplification of pseudogenes and dilution bias. Biochem Biophys Res Commun. 2011;412:1-7. Holdenrieder S, Stieber P. Clinical use of circulating nucleosomes. Crit Rev Clin Lab Sci. 2009;46:1-24. Holdenrieder S, Stieber P. Clinical use of circulating nucleosomes. Crit Rev Clin Lab Sci. 2009;46:1-24. Lichtenstein AV, Melkonyan HS, Tomei LD, Umansky SR. Circulating nucleic acids and apoptosis. Ann NY Acad Sci. 2001;945:239-49. Lichtenstein AV, Melkonyan HS, Tomei LD, Umansky SR. Circulating nucleic acids and apoptosis. Ann NY Acad Sci. 2001;945:239-49. Kim I, Lemasters JJ. Mitophagy selectively degrades individual damaged mitochondria after photoirradiation. Antioxid Redox Signal. 2011;14:1919-28. Kim I, Lemasters JJ. Mitophagy selectively degrades individual damaged mitochondria after photoirradiation. Antioxid Redox Signal. 2011;14:1919-28. Zhang J. Autophagy and mitophagy in cellular damage control. Redox Biology. 2013;1:19-23. Zhang J. Autophagy and mitophagy in cellular damage control. Redox Biology. 2013;1:19-23.