We have studied the co-mutagenic effect of verapamil and ascorbic acid on formation of radiation-induced chromosomal aberrations in T-lymphocytes of peripheral blood (PBL) of healthy donors. Test system of PBL treated in vitro with subsequent metaphase analysis of chromosomal aberrations has been used. Cells were exposed to γ-ray radiation in G0- and G2- periods of cell cycle and treated with verapamil (1.5; 2 and 4.0 μg/ml of blood) and ascorbic acid (20; 40 and 80 μg/ml of blood). The post-irradiation treatment of PBL with ascorbic acid in concentrations of 40 and 80 μg/ml, which exceeded therapeutic concentration value 2 and 4 times, increased overall chromosome aberrations frequency in 1.4 times compared to low dose (0.3 Gy) radiation effect. Verapamil at a concentration of 4.0μg/ml also increased the damaging effect of low dose of radiation in 1.5 times. Modification of radiation-induced cytogenetic effects in human PBL treated with co-mutagens was dependent on the concentration of the drugs, the absorbed dose of radiation, as well as cell radiosensitivity. It could be concluded that high concentrations of comutagens potentiate the damaging effect of low doses of ionizing radiation.
There is some uncertainty in the impact assessment of physical and chemical factors on human cells. Uncertainty becomes even more pronounced in case of successive action of agents . This concerns primarily the co-mutagens, since their impact on the human genome is one of the important components of primary cancer prevention . The co-mutagenesis is a phenomenon that contributes to the burdening of human genetic load, and can be interpreted as the effect potentiation .
In 1962, Taylor JH et al. found that the inhibitor of DNA synthesis fluorodeoxyuridine increased the yield of chromosomal aberrations, induced by irradiation in counterfoils beans . In 1978, Lychnik N.V. introduced the term psevdomutagen referred to chemical agents that “increase the yield of mutations not by inducing new local damage in the chromosomes, but by inhibiting the repair of spontaneous (or those caused by another mutagen) hidden damage, which in its absence would be repaired” . According to the modern concepts, the process of psevdomutagenesis is treated as co- mutagenesis .
Co-mutagens are compounds that, while not having own mutagenic properties can significantly modify (enhance) effects of known mutagens of chemical nature. Drugs with co-mutagenic properties remain insufficiently investigated, since lack of own mutagenic activity making them difficult to detect at genotoxic screening [6,7].
It was shown that calcium antagonist — verapamil (Vp), enhances the effect of bleomycin. Ascorbic acid (AA) may increase the damage caused by hydrogen peroxide or bleomycin. Caffeine expands toxicity of methotrexate and others drugs. The uncontrolled presence of comutagens in the human environment may increase the negative effects of industrial, medical and other mutagens.
Possible co-mutagenic effects of some common drugs, for instance AA, are of high scientific and practical interest . In series of studies, ambiguous effect of AA on human cells has been revealed [9-12]. As opposed to the animals, AA is not produced in human organism, and its food deficiency contributes to cancer development of gastric, esophagus, oral cavity and cervix origin . There is a contradictory view that vitamins, including AA, are impractical to use as preventive treatment to lower carcinogenic risk . Data on antitumor effect of AA at breast cancer , gastric cancer , prostate cancer  and other tumor localizations have been obtained.
Due to existing environmental situation in post-Chernobyl period, probabilistic development of carcinogenic effect of low dose ionizing irradiation (IR) and oncogenic threat caused by increased level of chromosome changes in cell population, study of AA effect on formation of radiation-induced instability of human somatic cells genome is relevant.
Researchers fairly state that interpretation of data on radioprotective and co-mutagenic effects of some drugs received on experimental animals, including vitamins-antioxidants, is “wrong to extrapolate to human” . Data on nature of AA impact is not always confirmed even in methodically close studies .
Ionizing radiation as a global environmental factor, even in small (above background) doses, can cause instability of the human genome, the development of somatic disease, reproductive problems, and increased carcinogenic risk.
However, the influence of co-mutagens on the pattern of radiationinduced effects remains unclear. Nevertheless, there has been a suggestion that the combined effect of co-mutagens and radiation can lead to “the emergence of nonlinear synergistic effects” .
The aims of this study were a) to examine in the comparative aspect the influence of the drugs Vp and AA on the frequency and spectrum of radiation-induced chromosomal aberrations in the culture of peripheral blood lymphocytes of healthy individuals; b) to evaluate the effect of different drugs concentrations on cytogenetic damage depending on dose of radiation and stage of mitotic cycle.
In this work, we used the test system of human PBL and metaphase analysis of chromosomal aberrations. Human PBL were analyzed for level and spectrum of chromosomal aberrations, that gives objective information about genome integrity in human somatic cells and are acknowledged to be one of the most sensitive to radiation. This methodology is recommended WHO, IAEA and UNSCEAR for biological indication of the radiation injury of human organism [16- 18]. Peripheral blood from healthy donors (47 samples) has been cultivated by semi-micro method with some modifications during 52 h. This study was guided by regulations of Helsinki Declaration of the World Medical Association (2008), which provides informed consent of donors for participation in study. Study algorithm is shown graphically in Figure 1.
PBL culture was exposed to radiation in G0- and G2- phases of cell cycle (on 0 and 46 hr of cells incubation correspondingly) on γ-installation “Rokus”(a source of γ-rays 60Co). The irradiation dose rate was 1.0 Gy/min, studied doses range 0.3 – 2.0 Gy. As modifiers of radiation effect, AA and Vp have been used. They were added to PBL culture immediately after exposure to radiation in the range of concentrations: AA – 20.0-80.0 μg/ml and Vp – 1.5-4.0 mg/ml of blood, respectively.
Cells were cultured according to the standard procedures with modifications, followed by slides preparation and metaphase analysis . PBL were incubated in RPMI 1640 medium(“Biowest”, France), containing 0,1 μg/ml PHA (M form, Gibco-Invitrogen, USA), 0.5 ml fetal calf serum (“PPA”, Austria), 10 ml gentamicin (“Health”, Ukraine) at 37°C for 52 h. This procedure designed for cells analysis in the first post-radiation mitosis. Last 3 h cells were incubated with colcemid (“Biowest”, France) at a concentration of 0.5 μg/ml of culture medium, in order to collect cells on metaphase stage. After incubation cells were treated in hypotonic 0.075 M solution of KC? for 10 minutes at 37°C. The cell suspension was fixed by cold mixture of ethanol and glacial acetic acid in a ratio of 3:1 (3 times). Slides were stained with 5% Giemsa solution (Gibco, USA). All slides were coded and scored blindly at 1000 × magnification under oil immersion. Metaphase chromosomes analysis was conducted according to standard procedure . All types of chromosomal aberrations were analyzed. As chromosomal type aberrations were taken into account the acentric fragments (paired and point), centric rings and dicentric chromosomes. The method allows registering a certain part of stable aberrations - reciprocal translocations (approximately 20% of their total amount). As the chromatid type aberrations were analyzed the acentric fragments and metabolic aberrations.
On each observation, an average 300 metaphases have been analyzed. The differences between group average values were estimated. Error bars represent variations between donors. Statistical analysis of obtained results was performed using of descriptive methods and Student’s t-criteria .
It is shown that the AA in the investigated concentration range (20.0-80.0 μg/ml) does not affect the magnitude of spontaneous level of chromosomal aberrations in the lymphocytes from healthy donors, which corresponds to the average values (2.0 ± 0.86 on 100 metaphases) . The results are consistent with the data  that showed the absence of genotoxic effect of antioxidant vitamins on human lymphocytes.
The analysis of the frequency of chromosome damage induced by irradiation of PBL in the G0-period of cell cycle (dose range 0.3-2.0 Gy) and post-radiation AA exposure in therapeutic concentrations (20.0 μg/ml of blood), showed that radio modifying effect of this drug was ambiguous (Figure 2).
Combined treatment with X-ray radiation in the low dose (0.3 Gy) and AA in the therapeutic concentration (20.0 μg/ml of blood), caused decrease in the total frequency of chromosomal aberrations by 1.5 times as compared to the effect of irradiation. This is consistent with the conclusions of , according to which the AA in therapeutic doses exhibits radioprotective properties by utilizing free radicals and improving antioxidant status of cells. Irradiation of PBL in relatively high doses (2.0 Gy) followed by treatment with AA in the same concentration led to “potentiation of radiation effect” increase in the total frequency of chromosomal aberrations in 1.4 times , thus indicating the co-mutagenic properties of the drug.
This observed potentiation of radiation-induced cytogenetic effect is due to formation of the radiation markers - dicentrics (10/100 metaphases, compared with radiation - 5/100 metaphases) (Figure 3). The formation of metabolic aberrations - dicentrics - requires local double chromosome breaks. Increased yield of this type aberration due to additional treatment with AA can suggest accumulation of primary radiation damage under the influence of this drug. The resulting effects could identify the problem of clastogenic action of AA on the human genome .
Special attention is given to the modification of cytogenetic effects in PBL treated with low doses of IR and AA in concentrations exceeding the therapeutic value. An additional post-radiation effect of AA in concentrations of 40.0 and 80.0 μg/ml of blood increased the overall frequency of chromosome aberrations in 1,2-1.4 times when compared to the effect of irradiation with 0.3 Gy, respectively (Figure 4). It may also indicate the co-mutagenic activity of AA in a range of concentrations exceeding by 2 and 4 times the therapeutic value.
Since irradiation in low doses along with chromosome breaks can also induce premutational changes in PBL, the additional potentiating effect of co-mutagens in the high concentrations range may contribute to the structural rearrangements of chromosomes, also due to the inhibition of repair enzymes .
We made an estimation of the universal indicator of the functional activity of T lymphocytes - their ability to proliferate after treatment with mitogenic factors. Analysis of the proliferative activity of PBL under the combined action of irradiation (0.3-2.0 Gy) and AA (20.0 μg/ml of blood) in G0 - period of the cell cycle showed on Figure 5. The most pronounced inhibition of mitotic activity was observed in cells irradiated at a dose of 2.0 Gy, but exposure to AA in therapeutic concentrations, on the contrary, increased mitotic activity in 2 times, even exceeding the value of intact control (Figure 5). This effect may be caused by removal of the radiation-induced block (delay of mitosis) under the influence of the drug, which reduces the time of primary repair damage. This is confirmed by the increase in the overall frequency of chromosome aberrations under these experimental conditions (Figure 2).
The analysis of co-mutagenic activity of the drug and its relationship with the cell radiosensitivity has specific scientific interest. In this regard, we have carried out a study in the most radiosensitive G2-period of the cell cycle of cultured lymphocytes . It is shown that PBL irradiation at 0.3 Gy dose and AA treatment in therapeutic concentrations, as well as during the experiments in G0- period, resulted in radioprotective effect with reduced in 1.4 times overall frequency of chromosome aberrations as compared to the effect of irradiation. However, the comutagenic effect in the G2-period appears earlier starting from the dose of 1.0 Gy (12.0 ± 1.1 and 18.0 ± 1.4, respectively) in comparison with the G0-period. The potentiation the co-mutagenic effect in 1.5 times suggests increase of cells radiosensitivity in this period of mitotic cycle (Figures 1 and 6).
Thus, we have studied the regularities of formation of radiationinduced structural rearrangements in PBL chromosomes of healthy individuals under an additional modifying effect of AA. This vitamin, depending on the used concentration for treatment of irradiated cells, can have both radioprotective and co-mutagenic properties. Comutagenic effect of AA was most pronounced after the irradiation of human somatic cells in a relatively high dose, resulted in increased frequency of the radiation markers and depended on the period of the cell cycle. High concentrations of AA potentiated the damaging effect of low doses of IR.
Co-mutagenic effect of AA preferably caused by its impact on the duration of radiation-induced block of mitosis, and thus on the proliferative potential of cells.
The drug Vp in the range of indicated concentrations had no effect on the spontaneous level of chromosomal aberrations in the lymphocytes of healthy donors. This is consistent with the studies in which the long-term therapy with calcium antagonists does not lead to an increase in the frequency of chromosomal changes [29,30]. PBL irradiation in the range of 0.3-2.0 Gy and additional treatment with Vp in different concentrations led to an increase in overall frequency of chromosomal aberrations compared to radiation alone. The observed increase in the level of chromosome aberrations correlated with the Vp concentration (Figure 7).
The spectrum of damage was dominated by the aberrations of chromosomal type, the level of which was also dependent on the concentration of the co-mutagen Vp (Figure 3 and 8). Co-mutagenic effect of Vp was formed primarily by dicentric chromosomes (for example, at a dose of 0.3 Gy, 7.0 / 100 metaphases) and their level was 2 times higher than after irradiation.
The manifestation of Vp co-mutagenic properties in human cells exposed to low dose irradiation has particular interest (Figure 9.). In the concentration range of 1.5-2.0 mg/ml of blood, Vp did not significantly affect the level of radiation-induced chromosome aberrations. However, its effect in the concentration of 4.0 μg/ml of blood (4-time excess of therapeutic value) potentiated the damaging effect of radiation and enhanced the overall frequency of chromosomal aberrations in about 1.5 times (14.0 ± 1.12 / 100 metaphases) (Figure 9). The formation of co-mutagenic effect of Vp has also occurred mainly due to the chromosomal type aberrations (Figures 3 and 10). The γ-irradiation of cultured PBL in a dose of 1.0 Gy in the most radiosensitive G2-period of the cell cycle followed by treatment with Vp in a concentration of 4.0 μg/ml of blood increased 2.4 times genotoxic damage as compared to the effect of irradiation.
It has been shown that Vp at a concentration of 1.5 μg / ml of blood had no effect on the mitotic activity of the lymphocytes (Figure 11). However, after combined influence of radiation (0.3 Gy) and Vp we observed 30% inhibition of cells proliferative capacity in comparison with the effect of radiation and intact control. Elevation of γ-irradiation dose up to 2.0 Gy combined with Vp treatment did not change the level of PBL mitotic activity.
Earlier, a pilot study that used the analysis of chromosomal aberrations in bone marrow cells of mice was established the effect of calcium channel blockers - diltiazem and nifedipine on the mutagenic effects of dioxide and cyclophosphamide . These drugs exhibited co-mutagenic activity by potentiating the chromosomes damage by mutagens in 1.3 and 2.9 times depending on the dose. Moreover, co-mutagenic effects of the drugs were most pronounced after their repeated administration.
Vp and its analogues enhanced the cytotoxic effects of some antibiotics , including the frequency of microkernels in human blood lymphocytes treated with erythromycin . The researchers explain the mechanism of co-mutagenic effect of Vp in terms of “the theory of accumulation”, according to which the calcium antagonists inhibited the excretion of cytotoxins from the cells, thereby increasing their potential mutagenic effect [34,35].
In accordance with the hypothesis , an increase in the level of intracellular calcium is associated with DNA damage due to oxidative stress. However, until now the influence of Vp on the formation of genetic lesions in irradiated human cells, in particular immunocompetent T-lymphocytes, that are responsible for the antitumor defense of the organism, stays unexplained .
The lack of conclusive data related to characteristics and mechanisms of co-mutagenic effects manifestation makes it impossible to estimate their actual genetic risk to humans and develop ways to correct the possible negative consequences.
DNA double-strand breaks (DSB) are critical damages and incorrect repair or absence of repair is involved in the formation of chromosomal aberrations. Homologous recombination and DNA non homologous end-joining represent the two major DSB repair mechanisms in mammalian cells . Elevated level of chromosomal aberrations in irradiated cells induced by co-mutagens may be caused by increased formation of DSB and/or aberrant processing of DSB by inhibited or altered repair system.
We have studied qualitative and quantitative features of the formation of radiation-induced chromosomal aberrations in the most radiosensitive cells (T-lymphocytes of peripheral blood) of healthy donors exposured to the drugs (Vp, AA) with co-mutagenic properties.
The intensity of co-mutagenic effects depended on the concentration of the drugs, the absorbed dose of IR and the extent of cell radiosensitivity. High concentrations of co-mutagens potentiated the damaging effect of low (above background) doses of radiation.
The obtained results should be taken into consideration when prescribing drugs with co-mutagenic activity to the patients that were in contact with the sources of IR, including those living in the radiation-contaminated areas.
The authors declare that there is no conflict of interests regarding the publication of this manuscript.