The immobilization of GOX in slides for comet sssay provides a useful tool for investigation of the efficiency of the cellular DNA-integrity protecting system of the target cells.
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1
Institute of Theoretical&Experimental Biophysics RAS, Russia
Various modifications of Comet Assay are widely used for efficiency analysis of the cellular DNA-integrity protecting system (Azqueta et al., 2014). The nucleoids of eukaryotic cells immobilized in agarose gel and lyzed were used as a substrate for DNA modifying enzymes. Following gel-electrophoresis and analysis of the obtained DNA-comets finalizes the assay. Results of such experiment are well presented in the literature.
In the present work we have focused on application of the Comet Assay for analysis of intracellular DNA-protecting system ex vivo. We used glucose oxidase (GOX) for reactive oxygen species (ROS) generation. It is known that GOX produce hydrogen peroxide during its enzymatic cycle. Hydrogen peroxide on its turn induces DNA damage in living cells. Cells' treatment with GOX and its substrate was usually performed in a tube (Kaczara et al., 2010). In present work we immobilized GOX as well as mammalian cells within a single slide in low melting agarose. Such experimental design provided a ROS-generating system for induction of DNA damage in cells immobilized in agarose slides. Immobilization of the enzyme permits to setup the precise period of enzyme reaction product treatment without of necessity of subsequent time-consuming immobilization of treated cells into agarose, which researchers need to perform in case of treatment cells in a tube. It also permits precise time determination of the peroxide treatment of the cells even at short time periods. In our experiments splenocytes and bone marrow cells of mice and human whole blood leukocytes were used. Cells were immobilized in the middle (the second of the three) low-melting agarose layer. Enzyme solution was also mixed with low melting agarose solution and the mixture was used for the upper (the third) layer formation. Hydrogen peroxide production was induced by adding glucose solution on the top agarose layer of the slide. After that the slides were incubated for 1 or 5 min at 20-22 oC. Then the slides were placed in the lysis solution (2.5 M NaCl, 100 mM EDTA, 10 mMTris, pH 10, 1%Triton X-100) and incubated for 24 hours at 5-7 0C in the dark. Electrophoresis was carried out in a refrigerator. The electrophoretic solution (0.3 M NaOH and 1 mM EDTA, pH>13) and the chamber were preliminarily cooled to 4-6 oC. The electrophoresis was performed for 20 min at 2 V/cm. Then the slides were rinsed in distilled water and DNA of nucleoids was stained with ethidium bromide (2 µg/ml) in the PBS in the dark for 1 hour. The slides were rinsed with distilled water for 5-10 minutes to remove unbound dye before the microscopy. Digital images of DNA-comets were obtained with a Lyumam-I-3 microscope ("LOMO", Russia) and digital camera Nikon CoolPix 995 (Japan). For each slide 50 images of comets were acquired. Image analysis was performed using the software package developed at the Institute of Cell Biophysics of RAS (Chemeris et al., 2004).
Variation of DNA damage was evaluated by measuring changes of DNA amount of tails of the DNA-comets (%TDNA) within digital images of the DNA-comets. Reliability of the differences between the control and experimental data was estimated using Student’s t-test.
At first we optimized concentration of the ROS –generating system components (GOX and glucose). For this purpose we analyzed the influence of different concentration of GOX and glucose on the level of hydrogen peroxide induced DNA damage. We observed the non linear dependence between the increase of the concentration of glucose (Fig.1) or GOX (data not shown) and DNA damage. Prolongation of the incubation time of the slides with glucose also resulted in the increase of the DNA damage (Fig. 2).
In the second part of the work we studied the response of the DNA-integrity defense system of human whole blood leukocytes to the hydrogen peroxide using newly established GOX – glucose ROS-generating approach. We measured level of DNA damage immediately after the 5 minute treatment period and after the incubation of treated cells in PBS without glucose for 30 minutes. The results are present in the Table 1.
In conclusion we would like to summarize that in present work we have shown successful application of agarose-gel immobilized GOX – glucose ROS-generating system for inducing DNA damage and studying DNA-integrity defense system in mammalian cells. We suppose that this approach will be useful for measurement of the intracellular antioxidant systems efficiency and for many other applications for DNA damage studies.
References
Azqueta A, Slyskova J, Langie SA, O'Neill Gaivão I, Collins A. (2014) Comet assay to measure DNA repair: approach and applications. Front Genet.;5:288. doi: 10.3389/fgene.2014.00288. Review.
Kaczara P., Sarna T, and Burke J M.(2010) Dynamics of H2O2 Availability to ARPE-19 Cultures in Models ofOxidative Stress Free Radic Biol Med. 48(8): 1064. doi:10.1016/j.freeradbiomed.2010.01.022
Chemeris NK, Gapeyev AB, Sirota NP, Gudkova OY, Kornienko NV, Tankanag AV, Konovalov IV, Buzoverya ME, Suvorov VG, Logunov VA.(2004). DNA damage in frog erythrocytes after in vitro exposure to a high peak-power pulsed electromagnetic field. Mutat Res. 558(1-2):27.
Keywords:
Comet Assay,
Glucose,
GOx,
H2O2-induced DNA damage,
Leukocytes,
Bone Marrow Cells
Conference:
ICAW 2015 - 11th International Comet Assay Workshop, Antwerpen, Belgium, 1 Sep - 4 Sep, 2015.
Presentation Type:
Poster Presentation
Topic:
New applications and technical improvements
Citation:
Sirota
NP,
Kuznetsova
E,
Mitroshina
I,
Glukhov
S and
Sirota
T
(2015). The immobilization of GOX in slides for comet sssay provides a useful tool for investigation of the efficiency of the cellular DNA-integrity protecting system of the target cells..
Front. Genet.
Conference Abstract:
ICAW 2015 - 11th International Comet Assay Workshop.
doi: 10.3389/conf.fgene.2015.01.00033
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Received:
15 May 2015;
Published Online:
23 Jun 2015.
*
Correspondence:
Dr. Nikolay P Sirota, Institute of Theoretical&Experimental Biophysics RAS, Pushchino, Moscow Region, 142290, Russia, sirota@iteb.ru