Skip to main content

Cell-free DNA in the urine of rats exposed to ionizing radiation

Radiation and Environmental Biophysics volume 54, pages 297–304 (2015)Cite this article

Abstract

Investigation of cell-free DNA (cf-DNA) in body fluids, as a potential biomarker for assessing the effect of ionizing radiation on the organism, is of considerable interest. We investigated changes in the contents of cell-free mitochondrial DNA (cf-mtDNA) and cell-free nuclear DNA (cf-nDNA) in the urine of X-ray-exposed rats. Assays of cf-mtDNA and cf-nDNA were performed by a real-time PCR in rat urine collected before and after irradiation of animals with doses of 3 and 5 Gy. We also determined the presence of mutations in urine cf-mtDNA, as recognized by Surveyor nuclease. A sharp increase in cf-mtDNA and cf-nDNA in the urine of irradiated rats was observed within 24 h after exposure, followed by a decrease to normal levels. In all cases, the contents of cf-mtDNA fragment copies (estimated by gene tRNA) were significantly higher than those of cf-nDNA estimated by gene GAPDH. A certain portion of mutant cf-mtDNA fragments was detected in the urine of exposed rats, whereas they were absent in the urine of the same animals before irradiation. These preliminary data also suggest that the increased levels of urine cf-mtDNA and cf-nDNA may be a potential biomarker for noninvasive assessment of how the organism responds to ionizing radiation influence.

This is a preview of subscription content, access via your institution.

Access options

Buy single article

Instant access to the full article PDF.

43,34 €

Price includes VAT (Finland)
Tax calculation will be finalised during checkout.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

References

  • Abdullaev SA, Antipova VN, Gaziev AI (2009) Extracellular mutant mitochondrial DNA content is sharply elevated in the blood plasma of irradiated mice. Mol Biol (Mosk) 43(6):1063–1069

    Article  Google Scholar 

  • Bannwarth S, Procaccio V, Paquis-Flucklinger V (2006) Rapid identification of unknown heteroplasmic mutations across the entire human mitochondrial genome with mismatch-specific surveyor nuclease. Nat Protoc 1(4):2037–2047

    Article  Google Scholar 

  • Bannwarth S, Procaccio V, Lebre AS et al (2013) Prevalence of rare mitochondrial DNA mutations in mitochondrial disorders. J Med Genet 50:704–714

    Article  Google Scholar 

  • Bettegowda C, Sausen M, Leary RJ et al (2014) Detection of circulating tumor DNA in early- and late-stage human malignancies. Sci Transl Med 6(224):224ra24. doi:10.1126/s3007094

    Article  Google Scholar 

  • Botezatu I, Serdyuk O, Potapova G et al (2000) Genetic analysis of DNA excreted in urine: a new approach for detecting specific genomic DNA sequences from cells dying in an organism. Clin Chem 46:1078–1084

    Google Scholar 

  • Bouatra S, Aziat F, Mandal R et al (2013) The human urine metabolome. PLoS One 8(9):e73076. doi:10.1371/journal.pone.0073076

    ADS  Article  Google Scholar 

  • Bryzgunova OE, Skvortsova TE, Kolesnikova EV, Starikov AV, Rykova EY, Vlassov VV, Laktionov PP (2006) Isolation and comparative study of cell-free nucleic acids from human urine. Ann N Y Acad Sci 1075:334–340

    ADS  Article  Google Scholar 

  • Cheng C, Omura-Minamisawa M, Kang Y, Hara T, Koike I, Inoue T (2009) Quantification of circulating cell-free DNA in the plasma of cancer patients during radiation therapy. Cancer Sci 100:303–309

    Article  Google Scholar 

  • Cleaver JE (1992) Replication of nuclear and mitochondrial DNA in X-ray-damaged cells: evidence for a nuclear-specific mechanism that down-regulates replication. Radiat Res 131:338–344

    Article  Google Scholar 

  • Evdokimovsky EV, Ushakova TE, Kudriavtcev AA, Gubina NE, Gaziev AI (2011) Alteration of mtDNA copy number, mitochondrial gene expression and extracellular DNA content in mice after irradiation. Radiat Environ Biophys 50:181–188

    Article  Google Scholar 

  • Holdenrieder S, Stieber P (2009) Clinical use of circulating nucleosomes. Crit Rev Clin Lab Sci 46:1–24

    Article  Google Scholar 

  • Liao GJ, Gronowski AM, Zhao Z (2014) Non-invasive prenatal testing using cell-free fetal DNA in maternal circulation. Clin Chim Acta 428:44–50

    Article  Google Scholar 

  • Liu H, Ma Y, Fang F et al (2013) Wild-type mitochondrial DNA copy number in urinary cells as a useful marker for diagnosing severity of the mitochondrial diseases. PLoS One 8(6):e67146. doi:10.1371/journal.pone.0067146

    ADS  Article  Google Scholar 

  • Malik AN, Shahni R, Rodriguez-de-Ledesma A, Laftah A, Cunningham P (2011) 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 412:1–7

    Article  Google Scholar 

  • Melkonyan HS, Feaver WJ, Meyer E, Scheinker V, Shekhtman EM, Xin Z, Umansky SR (2008) Transrenal nucleic acids: from proof of principle to clinical tests; problems and solutions. Ann N Y Acad Sci 1137:73–81

    ADS  Article  Google Scholar 

  • Mittra I, Nair NK, Mishra PK (2012) Nucleic acids in circulation: are they harmful to the host? J Biosci 37:301–312

    Article  Google Scholar 

  • Shekhtman EM, Anne K, Melkonyan HS, Robbins DJ, Warsof SL, Umansky SR (2009) Optimization of transrenal DNA analysis: detection of fetal DNA in maternal urine. Clin Chem 55:723–729

    Article  Google Scholar 

  • Strelkova IY, Abdullaev SA, Snigireva GP, Bezlepkin VG, Gaziev AI (2010) Share of extracellular mutated mitochondrial DNA increases in plasma of lung cancer patients following radiotherapy. Biomed Khim 56(4):517–525

    Article  Google Scholar 

  • Su YH, Wang M, Brenner DE, Ng A, Melkonyan H, Umansky S, Syngal S, Block TM (2004) Human urine contains small, 150 to 250 nucleotide sized, soluble DNA derived from the circulation and may be useful in the detection of colorectal cancer. J Mol Diagn 6:101–107

    Article  Google Scholar 

  • Su YH, Song J, Wang Z, Wang XH, Wang M, Brenner DE, Block TM (2008) Removal of high-molecular-weight DNA by carboxylated magnetic beads enhances the detection of mutated K-ras DNA in urine. Ann N Y Acad Sci 1137:82–91

    ADS  Article  Google Scholar 

  • Sun W, Sun Y, Zhu M, Wang Z, Zhang H, Xin Y, Jiang G, Guo X, Zhang Z, Liu Y (2014) The role of plasma cell-free DNA detection in predicting preoperative chemoradiotherapy response in rectal cancer patients. Oncol Rep 31:1466–1472

    Google Scholar 

  • Tsui NB, Jiang P, Chow KC, Su X, Leung TY, Sun H, Chan KC, Chiu RW, Lo YM (2012) High resolution size analysis of fetal DNA in the urine of pregnant women by paired-end massively parallel sequencing. PLoS One 7(10):e48319. doi:10.1371/journal.pone.0048319

    ADS  Article  Google Scholar 

  • Umansky SR, Tomei LD (2006) Transrenal DNA testing: progress and perspectives. Expert Rev Mol Diagn 6:155–163

    Article  Google Scholar 

  • Vasiléva IN (2001) Low-molecular-weight DNA in blood plasma as an index of the influence of ionizing radiation. Ann N Y Acad Sci 945:221–228

    ADS  Article  Google Scholar 

  • Wallace DC (2005) A mitochondrial paradigm of metabolic and degenerative diseases, aging, and cancer: a dawn for evolutionary medicine. Annu Rev Genet 39:359–407

    Article  Google Scholar 

  • Xia P, Radpour R, Zachariah R, Fan AXC, Kohler C, Hahn S, Holzgreve W, Zhong XY (2009) Simultaneous quantitative assessment of circulating cell-free mitochondrial and nuclear DNA by multiplex real-time PCR. Genet Mol Biol 32(1):20–24

    Article  Google Scholar 

  • Yu SC, Lee SW, Jiang P, Leung TY, Chan KA, Chiu RW, Lo YD (2013) High-resolution profiling of fetal DNA clearance from maternal plasma by massively parallel sequencing. Clin Chem 59(8):1228–1237

    Article  Google Scholar 

  • Zhang J (2013) Autophagy and mitophagy in cellular damage control. Redox Biol 1(1):19–23

    Article  Google Scholar 

  • Zhang H, Maguire D, Swarts S et al (2009) Replication of murine mitochondrial DNA following irradiation. Adv Exp Med Biol 645:43–48

    Article  Google Scholar 

  • Zhang L, Zhang M, Yang S, Cao Y, Zhang SB, Yin L, Tian Y, Ma Y, Zhang A, Okunieff P, Zhang L (2010) A new biodosimetric method: branched DNA-based quantitative detection of B1 DNA in mouse plasma. Br J Radiol 83:694–701

    Article  Google Scholar 

  • Zhang M, Zhang B, Guo Y et al (2013) Alteration of circulating mitochondrial DNA concentration after irradiation. Adv Exp Med Biol 765:371–377

    Article  Google Scholar 

Download references

Acknowledgments

This work has been supported by the Russian Foundation for Basic Research (Grant 12-04-31070). We are grateful to Margaret Il’ina for her excellent help in the preparation of this manuscript.

Conflict of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

Author information

Affiliations

  1. Institute of Theoretical and Experimental Biophysics, Russian Academy of Science (RAS), Pushchino, Moscow Region, 142290, Russia

    Serazhutdin A. Abdullaev, Gulchachak M. Minkabirova, Vladimir G. Bezlepkin & Azhub I. Gaziev

  2. Pushchino State Institute of Natural Sciences, Pushchino, Moscow Region, 142290, Russia

    Gulchachak M. Minkabirova

Authors
  1. Serazhutdin A. Abdullaev
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Gulchachak M. Minkabirova
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Vladimir G. Bezlepkin
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Azhub I. Gaziev
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Serazhutdin A. Abdullaev.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Abdullaev, S.A., Minkabirova, G.M., Bezlepkin, V.G. et al. Cell-free DNA in the urine of rats exposed to ionizing radiation. Radiat Environ Biophys 54, 297–304 (2015). https://doi.org/10.1007/s00411-015-0599-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00411-015-0599-0

Keywords

  • Urine mitochondrial and nuclear DNA
  • Irradiated rats
  • Real-time PCR