Cytotoxic, clastogenic and genotoxic effects of cis- tetraammine(oxalato)ruthenium(III) dithionate on human peripheral blood lymphocytes

There is a concern about stablishing the clinical risk of drugs used for cancer treatment. In this study, the cytotoxic, clastogenic and genotoxic properties of cis-tetraammine(oxalato)ruthenium(III) dithionite cis-[Ru(C2O4)(NH3)4]2(S2O6), were evaluated in vitro in human lymphocytes. The mitotic index (MI), chromosomal aberrations (CA) and DNA damage by comet assay were also analyzed. The MTT test revealed that the ruthenium compound showed a slight cytotoxic effect at the highest concentration tested. The IC50 value for the compound after 24 hours of exposure was 185.4 μM. The MI values of human peripheral blood lymphocytes treated with 0.015, 0.15, 1.5 and 150 μM of cis-[Ru(C2O4)(NH3)4]2(S2O6) were 6.1, 3.9, 3.2 and 0.2%, respectively. The lowest concentration, 0.015 μM, did not show any cytotoxic activity. The CA values for the 0.015, 0.15 and 1.5 μM concentrations presented low frequency (1.5, 1.6 and 2.3%, respectively), and did not express clastogenic activity when compared to the negative control, although it was observed clastogenic activity in the highest concentration tested (150 μM). The results obtained by the comet assay suggest that this compound does not present genotoxic activity at lower concentrations. The results show that cis-[Ru(C2O4)(NH3)4]2(S2O6) has no cytotoxic, clastogenic or genotoxic in vitro effects at concentrations less than or equal to 0.015 μM. This information proves as promising in the treatment of cancer and is crucial for future trials.


Introduction
One of the major challenges in anti-cancer therapy is to overcome the high toxicity of the drugs applied to healthy cells and the multi-drug resistance of cells to chemotherapeutics (Matejczyk, Świderski, Świsłocka, Rosochacki, & Lewandowski, 2018). Doxorubicin (DOX) is an anthracycline drug widely used as a first chemotherapeutic drug treatment of a variety of solid tumors (breast, lung and ovary) (Adjei & Rowinsky, 2003) and hematological diseases, such as leukemia and Hodgkin's disease (Pilco-Ferreto & Calaf, 2016). However, the use of doxorubicin has been limited due to the significant toxicity and debility that causes unwanted side effects, including cardiac, renal, pulmonary, hematological and testicular toxicity (Matejczyk et al., 2018;Pilco-Ferreto & Calaf, 2016;Turner, Biganzoli, & Di Leo, 2015;Mitry & Edwards, 2016).
These unwanted side effects can occur during or some years after treatment, especially because DOX cannot discriminate healthy cells from malignant ones in a rapidly and non-selectively way, accumulating in healthy tissues (Mitry & Edwards, 2016).
Therefore, it has become essential to search for more selective, efficient and safer anti-cancer agents, which eliminate malignant cells and also maintain the integrity or minimally affect the normal cells. In particular, the ruthenium complexes have emerged against many types of cancers with great potential as antitumor agents with the aim of overcoming the unwanted effects (Bergamo & Sava, 2011;Sánchez-Suárez et al., 2008;Kamatchi, Chitrapriya, Kim, Fronczek, & Natarajan, 2013).
The antitumor activity pathway of ruthenium complexes works by the kinetic ligand exchange, favored by the octahedral molecular form; more specifically, such activity is related to the capacity to mimic iron in binding to biological molecules and to the reduction of the oxidative state of ruthenium III (RuIII) to ruthenium II (RuII), which is favored in hypoxic environments with low pH and high levels of glutathione (Allardyce & Dyson, 2001;Dougan, Habtemariam, McHale, Parsons, & Sadler, 2008;Clarke, 2003). The physiologic state in which cancer cells are found, gives to ruthenium the ability to become activated and to effectively bind to DNA (Keene, Smith, & Collins, 2009). Otherwise, the mechanism of action of doxorubicin includes an intercalation in DNA, the inhibition of topoisomerase II activity and induction of DNA breaks in cancer cells, resulting in DNA damage and cell death (Matejczyk et al., 2018).
Cancer treatments with alkylating agents result in an increased frequency of chromosomal aberrations and they can elevate the chance of the patients develop a second neoplasia (Sánchez-Suárez et al., 2008). To detect clastogenic agents in peripheral human blood lymphocytes, chromosomal aberrations must be analyzed in detail (Albertini et al., 2000).
This study has the aim of analyzing the in vitro cytotoxic, clastogenic and genotoxic effects of the cis-[Ru(C2O4)(NH3)4]2(S2O6) complex on normal human blood lymphocytes.
The protocol (N. 043/2007) for these experiments was approved by the Research Ethics Committee at the Universidade Federal de Goiás, and prior to joining the study, all blood donors have signed an informed consent.

Cytogenetic Studies
Metaphase preparations were made to analyze chromosomal aberrations (CA). In brief, 5 mL of venous blood were collected from each donor in a heparinized vial (5.000 IU mL -1 ; Liquemine, Roche, São Paulo-SP, Brazil), and the cells were incubated (37 °C, 5% CO2) for 24 hours before cis-[Ru(C2O4)(NH3)4]2(S2O6) exposure. Then, the cells were exposed to cis-[Ru(C2O4)(NH3)4]2(S2O6) (0.015, 0.15, 1.5 and 150 µM) and to positive control doxorubicin (0.002 µM) and then incubated for 24 hours. Untreated cells were used as negative control. Colchicine was added at a final concentration of 0.0016% 1h prior to harvesting. Cells were harvested by centrifugation (800 rpm), treated with 0.075 M KCl at 37 °C for 20 min., centrifuged (800 rpm) again, and fixed in 1:3 (v v -1 ) acetic acid-methanol. Finally, the slides were air-dried, and stained with 3% Giemsa solution (pH 6.8) for 5 min. They were then analyzed using a light microscope, structural (chromosome/chromatid gaps and breaks) and numerical CA were examined in both the ruthenium complex-treated cultures and negative/positive controls. For assessment of chromosomal aberrations, one hundred cells were analyzed per treatment. The frequencies of chromosomal aberrations in 100 metaphases per culture and MI were determined. The MI was calculated for each treatment as the number of metaphases per 5,000 lymphocytes.
After exposure, 300 µL of the cell suspension were centrifuged for 5 min . (500 rpm). The resulting pellet was homogenized with 80 µL of a low melting-point agarose (0.5%), spread onto microscope slides pre-coated with normal melting-point agarose (1.5%), and covered with a coverslip. After 5 min. at 4 °C, the coverslip was removed, and the slides were immersed in cold lysis solution (2.4 M NaCl, 100 mM ethylenediamine tetraacetic acid (EDTA), 10 mM Tris, 10% DMSO, and 1% Tri ton-X, pH 10) for 24 hours. After lysis, the slides were placed in an electrophoresis chamber and covered with electrophoresis buffer (300 mM NaOH per 1 mM EDTA, pH>13) for further 20 min . to allow DNA unwinding. The electrophoresis proceeded for 20 min. at 25 V (0.7 V cm -1 ) and approximately 300 mA. Afterwards, the slides were submerged for 15 min. in a neutralization buffer (0.4 M Tris-HCl, pH 7.5), dried at room temperature, and fixed in 100% ethanol for 5 min. All steps were conducted in the dark to prevent additional DNA damage. Slide staining was performed immediately before analysis using ethidium bromide (20µg mL -1 ). Slides were prepared in duplicate, and 100 cells were screened per sample (50 cells from each slide) in a fluorescent microscope (Zeiss, Germany) equipped with an excitation filter of 515-560 nm and a barrier filter of 590 nm using a × 40 objective. The nucleus were classified according to the migration of the fragments using the software CometScore 15 as: class 0 (no damage); class 1 (little damage with a short tail length smaller than the diameter of the nucleus); class 2 (medium damage with a tail length once or twice the diameter of the nucleus); class 3 (significant damage with a tail length between two and a half to three times the diameter of the nucleus); class 4 (significant damage with a tail longer than three times the diameter of t he nucleus) (Burlinson et al., 2007).

Statistical Analysis
Statistical analyses of the results were performed using One-way analysis of variance followed by Tukey's post hoc test for multiple comparisons with a control. All statistical analyses were undertaken using the statistical software GraphPad 5.0 for windows (San Diego California USA, www.graphpad.com). A probability of 0.05 or less was deemed as statistically significant. The following notation is used throughout the manuscript: *, p < 0.05 and **, p < 0.01 relative to the control.

Mitotic Index
The results from the analysis of mitotic index (MI), (which estimates the frequency of cellular division), revealed a concentration-dependent decrease of MI in the treated peripheral human blood lymphocytes (PBL). The cells treated with 0.015, 0.15, 1.5 and 150 µM of cis-[Ru(C2O4)(NH3)4]2(S2O6) compound presented a mitotic index of 6.1, 3.9, 3.2 and 0.2%, respectively (Figure 1). The mitotic index of PBL exposed to the positive control drug (doxorubicin) was 2.9% and the negative control was 5.6%.
A concentration of 0.015 µM of cis-[Ru(C2O4)(NH3)4]2(S2O6) did not show cytotoxic effect, did not inhibit the mitotic index of the cells, and resulted in increased proliferation, when compared to the negative control (p > 0.01). It was observed a decrease in MI for 0.15 and 1.5 µM concentrations, and a highly significant reduction in the MI in the 150 µM concentration, of 0.2 % (**p < 0.01). The positive control, doxorubicin (0.002 µM), presented a MI similar to that found to 1.5 µM of the ruthenium complex, which represents a concentration approximately 750 x higher than that used for doxorubicin ( Figure 1).  Table 2 contains the results of the chromosomal aberrations assay in the PBL that were exposed to different concentrations of cis-[Ru(C2O4)(NH3)4]2(S2O6), negative control (cells with no exposition) and positive control (cells exposed to doxorubicin). The chromosomal aberrations of cells treated with ruthenium complex at concentrations of 0.015, 0.15 and 1.5 µM were similar to values found in the negative control (2.0%), with frequencies of 1.5, 1.6 and 2.3%, respectively. These values compared to the negative control were not statistically significant (p > 0.05).

Chromosomal Aberration
The ruthenium complex concentration of 150 µM presented approximately one-third of chromosomal aberrations observed for doxorubicin (p < 0.01). For each treatment 100 cells were analyzed; Ctg=chromatid gaps, Ctb = chromatid breaks, Chg = chromosome gaps, Chb = chromosome breaks, DXR = doxorubicin. ** Indicates significant difference in comparison to negative control (p < 0.01). Data are mean ± S.D. *** Frequency estimates a total of 195 metaphases, different from the other concentrations, which were calculated on a total of 600 metaphases per treatment. Figure 2 shows chromosomal aberrations observed on metaphase of peripheral human blood lymphocytes exposed to 150 µM of cis-[Ru(C2O4)(NH3)4]2(S2O6) (Figure 2C), compared to negative control ( Figure 2A) and positive control ( Figure 2B).
The concentration of 150 µM induced DNA damages and breaks (red arrows 2 and 3, demonstrating significant clastogenic activity when compared to the negative control (p < 0.01), predominantly through chromatidic failure, although a greater amount of chromosomal aberrations was observed for positive control (red arrow 1).
All the chromosomal aberrations analyzed for doxorubicin were higher than that found for the ruthenium complex tested, with exception to chromatid breaks and chromosome breaks, for which the highest concentration of the complex (150 µM) presented similar results of CA related to doxorubicin (0.002 µM). The total number of CA verified for doxorubicin were approximately the triple of the number for the highest concentration of ruthenium complex and 49 times higher than found for the concentration of 1.5 µM of cis-[Ru(C2O4)(NH3)4]2(S2O6).

Genotoxicity assay
Comet assay was performed to analyze the genotoxicity potential in peripheral human blood lymphocytes treated with different concentrations of cis-[Ru(C2O4)(NH3)4]2(S2O6). The concentration of 0.015 µM did not present DNA damage and was similar to that observed in the negative control (p > 0.01). The DNA damages indexes were concentration dependent, as the highest concentrations of cis-[Ru(C2O4)(NH3)4]2(S2O6) caused more DNA damages when compared to the negative control (Figure 3).
Although the DNA damage indexes were considered statistically significant when compared to the negative control at concentrations of 0.15, 1.5 and 150 µM, just the two highest concentrations presented significant percentage of DNA damage. Otherwise, doxorubicin, despite widely used in the treatment of many types of cancer, when used in this study in a very low concentration, presented significant DNA damage in human blood lymphocytes.
The use of current antineoplastic drugs requires the knowledge about their cytotoxicity, clastogenicity and genotoxicity, in order to obtain treatments with greater selectivity and less side effects.
Ruthenium complexes have demonstrated selective cytotoxic activity to tumor cells with low toxicity to normal cells. Ribeiro et al. (2009) has demonstrated that cis-(dichloro)tetraammineruthenium(III) complex did not cause DNA damage in human lymphocytes. Furthermore, this ruthenium(III) complex also presented immune stimulatory activity, and a high concentration of this complex had induced damage to the normal peripheral blood mononuclear cells (Silveira-Lacerda et al., 2010a). Studies with cis-tetraammine(oxalato)ruthenium(III) dithionate complex tested on the root meristem cells of Allium cepa showed no significant cytotoxic and genotoxic activity . The cis-[Ru(C2O4)(NH3)4]2(S2O6) compound has presented in vitro cytotoxicity activity in sarcoma 180 cells (Pereira et al., 2015) and in chronic myeloid leukemia K562 cells (Pereira et al., 2014).
Peripheral human blood lymphocyte cultures are an excellent method for testing the capacity of chemical agents to cause DNA damage (Lima et al., 2007).
The ruthenium (III) complex has been studied because it has favorable antitumor properties, so there is a need to test it for cytotoxic and genotoxic effects in human normal cells, using the test established by ANVISA (National Health Surveillance Agency) and FDA (Food and Drug Administration) for the release of new drugs.
Because of the promising results from previous cis-tetraammine(oxalato)ruthenium(III) studies Pereira et al., 2014), the need has risen for research into the possible cytotoxic, clastogenic and genotoxic effects of this complex in peripheral human blood lymphocytes in vitro.
In this study, using the MTT assay, we found that cis-[Ru(C2O4)(NH3)4]2(S2O6) reduced the viability of peripheral human blood lymphocyte cells only at the highest concentration tested (150 µM) relative to untreated cells (p < 0.05); on the other concentrations tested (0.015, 0.15 and 1.5 µM) there were no significant reduction in cellular viability (p > 0.05).
Otherwise, Pereira et al. (2014) and Silveira-Lacerda et al. (2010a;2010b) demonstrated that the cis-[Ru(C2O4)(NH3)4]2(S2O6) and cis-[RuCl2(NH3)4]Cl complexes, respectively, showed expressive selectivity for tumor cells such as Jurkat, A549, S-180 and K562 with IC50 of 18.6, 19.1, 24.5 and 29.2 µM, respectively. In contrast to the verified cytotoxicity in tumor cells, this study using human primary normal cells demonstrated that cytotoxic, clastogenic and genotoxic effects were observed at a much higher concentration than that required to cause cytotoxicity in tumor cells. On the other hand, the positive control doxorubicin has caused cytotoxic effects in these normal cells in a very low concentration (0.002 µM). The cytotoxic properties of other ruthenium complexes have been investigated comparing tumor and normal cells; and these complexes are selective for tumor cells when compared to normal cells (Djinovic, Todorovic, Zizak, Sabo, & Juranic, 2009).
Based on the IC50 value of earlier studies with tumor cells, we suggest that the cytotoxicity of cis-[Ru(C2O4)(NH3)4]2(S2O6) for human cancer cells is much stronger than its cytotoxicity for normal peripheral human blood lymphocyte cells (IC50 184.5 µM). This value was ten times higher than the IC50 for K562 tumor cell found by Pereira et al (2014), which was 18.28 μM, revealing a great selectivity associated to this ruthenium complex, with values of SI ≥ 2.0 considered significant. The same study noticed that K562 cells suffered apoptosis and presented genotoxic effects after treatment with this complex, with a significant increase in DNA damage index in all of the concentrations tested in tumour cells, in a dose-dependent manner (10 -150 µM).
In order to evaluate the effect of cis-[Ru(C2O4)(NH3)4]2(S2O6) on the mitotic index in human lymphocytes, we used four concentrations of the complex (0.015 -150 µM), as well as a positive control, doxorubicin, at 0.002 µM. The MI at a 0.015 µM concentration of ruthenium complex did not reveal cytotoxicity for human lymphocytes (Figure 1). Similarly, Pereira et al. (2009) evaluated the cytotoxic and genotoxic activities in Allium cepa roots treated with cis-[Ru(C2O4)(NH3)4]2(S2O6), showing that a higher concentration of the complex was necessary for a significant reduction in MI.
Previous studies using the cis-[RuCl2(NH3)4]Cl ruthenium complex showed that this compound inhibited cellular division (MI=0%) ) and presented cytotoxicity, reduction of tumor growth and increased survival time of animals on murine sarcoma S180 using in vitro and in vivo assays. Additionally, the cytotoxic activity of the cis-[RuCl2(NH3)4]Cl ruthenium complex in peripheral blood mononuclear cells was lesser than that in tumor cells, presenting a high IC50 of 8157 µg ml -1 (De Paula Silveira-Lacerda et al., 2010a). This expresses the higher selectivity of this ruthenium complex towards tumor cells in relation to normal cells. These results corroborate with the findings of the present study, once the increased concentrations of the cis-[Ru(C2O4)(NH3)4]2(S2O6) complex were necessary to induce cytotoxicity in peripheral human blood lymphocytes.
The low toxicity related to ruthenium compounds can be explained by the ability of ruthenium to mimic iron in respect of binding to several biomolecules, such as transferrin and albumin. Since cancer cells divide rapidly, the demand for iron is greater, so they increase the number of transferrin receptor on their surface. Consequently, ruthenium-loaded transferrin is sequestrated in higher quantity than in normal cells. Additionally, regarding to Ru(III) complexes, it is believed that their anticancer activity occurs with initial reduction to Ru(II) at the cancer tissue, due to the altered and more acidic physicochemical environment shown in tumour cells (Allardyce & Dyson, 2001).
The CA analysis of metaphases after treatments showed that 0.015, 0.15 and 1.5 µM of cis-[Ru(C2O4)(NH3)4]2(S2O6) did not induce CA, but instead presented similar values to those of the negative control. The positive control doxorubicin presented a higher frequency of chromosomal abnormalities (Table 2), predominantly through chromatin failures and breaks.
The cis-[RuCl2(NH3)4]Cl complex did not show any metaphase at the highest concentration of 380 µM, probably because the cell cycle was blocked at the interphase, and thus interfered with the repair mechanism and blocked mitosis ).
In the present study, the CA assay did not reveal cytotoxic or clastogenic effects in human lymphocytes exposed to cis-[Ru(C2O4)(NH3)4]2(S2O6). The CA frequency in the negative control of human lymphocytes was 2.0%, which is within the range for healthy individuals (around 2%) (Julian Preston, San Sebastian, & McFee, 1987). The 0.015, 0.15 and 1.5 µM concentrations of the complex did not demonstrate clastogenic effects when compared to the negative control either. However, there was an increase in the clastogenic activity of cis-[Ru(C2O4)(NH3)4]2(S2O6) in human lymphocytes when the 150 µM was used, predominantly manifested as chromatic breaks and failures.
The present study also used the comet assay to evaluate DNA damage. This test is a sensitive method for detecting simple breaks, double breaks and crosslinking of DNA (Tice et al., 2000). In this study, the lowest concentration (0.015 µM) of cis-[Ru(C2O4)(NH3)4]2(S2O6) complex did not demonstrate statistically significant difference of DNA damage when compared to the negative control (p > 0.01). On the other hand, DOX has caused approximately four times more damage to DNA in a concentration seven times lower than that of the minor concentration of ruthenium complex (0.015 µM).