THE SPECIFICITIES OF THE COMUTAGENESIS PHENOMENON FORMATION IN THE IRRADIATED CELLS OF CANCER PATIENTS

Domina E.A.¹, Pylypchuk A.P.²

• ¹R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology, NAS of Ukraine, Kyiv, Ukraine
• ²Medical diagnostic center «Diagnostics», Kyiv, Ukraine

Aim: to determine the specificities of formation of radiation-induced chromosome aberrations in human peripheral blood lymphocytes under conditions of comutagenesis (in vitro studies). Object and methods: the cytogenetic study was carried out on peripheral blood T-lymphocytes of 24 primary gynecologic cancer patients (study group) and 67 conditionally healthy subjects (comparison group). Isolation and cultivation of peripheral blood lymphocytes and metaphase analysis of radiation-induced chromosome aberrations was performed according to the standard protocol. Comutagens were introduced into the cell culture immediately after X-irradiation in therapeutic concentrations: verapamil — 1.0 μg/ml blood, ascorbic acid — 20.0 μg/ml blood, as well as in concentrations exceeding their values. Polymerase chain reaction (PCR) method was used to study microsatellite DNA instability of blood lymphocytes; Immunocytochemical method was used to study the process of apoptosis. Statistical methods were used. Results: ascorbic acid and verapamil in the therapeutic concentration did not affect the initial level of aberrations of chromosomes in non-irradiated lymphocytes of oncological patients. With the increase of verapamil concentration up to 4.0 μg/ml the frequency of chromosomal rearrangements in blood lymphocytes of cancer patients increased by 20.0%. By means of immunocytochemical analysis, we established that under irradiation with low doses (0.3 Gy) and additional action of verapamil (1.0 and 4.0 μg/ml blood) on lymphocytes of patients the highest number of caspase-3-positive nuclei in these cells is observed under the action of comutagen at 1.0 μg/ml. Under irradiation of lymphocyte culture at low doses (0.3 Gy) and under the additional action of ascorbic acid (20.0 and 80.0 μg/ml) the comutagenic effect — increase of aberration frequency by 1.7–1.4 times in comparison with radiation effect (19.0 ± 0.5 and 15.0 ± 0.7 vs 11.0 ± 0.4) was found. For the first time was detected DNA microsatellite instability due to comutagenic action of ascorbic acid on blood lymphocytes of irradiated in low dose patients. This fact could indicate the disturbance of misfolded nucleotides repair system in cells of circulating blood pools of patients. Conclusions: in irradiation (0.3 Gy) of blood lymphocytes of oncological patients, ascorbic acid exhibits comutagenic properties in a therapeutic concentration (20 μg/ml), and verapamil — at an increased concentration (4.0 μg/ml of blood). The increase in ascorbic acid (20.0; 40.0; 80.0 μg/ml) and verapamil (1.0; 1.5; 2.0 and 4.0 μg/ml) had stimulating effect on mitotic activity of irradiated lymphocytes of cancer patients. It was established for the first time that in the mechanisms of formation of comutagenic effects of ascorbic acid in the irradiated blood lymphocytes an important role is played by the disturbances of the system of reparation of wrongly paired nucleotides, causing changes in the DNA structural organization (microsatellite instability). The phenomenon of comutagenesis that develops in the irradiated lymphocytes of cancer patients is based on the processes of chromosomal and microsatellite instability, apoptosis and stimulation of cell proliferation.