Antioxidant Activity of Ruthenium Cyclopentadienyl Complexes Bearing Succinimidato and Phthalimidato Ligands

In these studies, we investigated the antioxidant activity of three ruthenium cyclopentadienyl complexes bearing different imidato ligands: (η5-cyclopentadienyl)Ru(CO)2-N-methoxysuccinimidato (1), (η5-cyclopentadienyl)Ru(CO)2-N-ethoxysuccinimidato (2), and (η5-cyclopentadienyl)Ru(CO)2-N-phthalimidato (3). We studied the effects of ruthenium complexes 1–3 at a low concentration of 50 µM on the viability and the cell cycle of peripheral blood mononuclear cells (PBMCs) and HL-60 leukemic cells exposed to oxidative stress induced by hydrogen peroxide (H2O2). Moreover, we examined the influence of these complexes on DNA oxidative damage, the level of reactive oxygen species (ROS), and superoxide dismutase (SOD) activity. We have observed that ruthenium complexes 1–3 increase the viability of both normal and cancer cells decreased by H2O2 and also alter the HL-60 cell cycle arrested by H2O2 in the sub-G1 phase. In addition, we have shown that ruthenium complexes reduce the levels of ROS and oxidative DNA damage in both cell types. They also restore SOD activity reduced by H2O2. Our results indicate that ruthenium complexes 1–3 bearing succinimidato and phthalimidato ligands have antioxidant activity without cytotoxic effect at low concentrations. For this reason, the ruthenium complexes studied by us should be considered interesting molecules with clinical potential that require further detailed research.


Introduction
In recent years, many different ruthenium complexes exhibiting various chemical and biological properties have been synthesized [1]. Complexes with anticancer potential are of particular interest [2][3][4]. Ruthenium complexes exhibit many features that are desirable among cancer drug candidates. They are less cytotoxic to normal cells compared to cancer cells, in which they can accumulate due to uptake by transferrin receptors. Many of them are active against cells that are metastatic and resistant to conventional chemotherapy. We recently demonstrated that ruthenium complex (η 5 -cyclopentadienyl)Ru(CO) 2 (η 1 -Nmaleimidato) has anticancer potential [4,5]. This complex was about ten times more cytotoxic to leukemic HL-60 cells compared to normal peripheral blood mononuclear cells (PBMCs). Moreover, complex (η 5 -cyclopentadienyl)Ru(CO) 2 (η 1 -N-maleimidato) induced DNA damage and apoptosis in HL-60 cells.
Peripheral blood mononuclear cells (PBMCs) were isolated from the buffy c obtained from healthy donors from Central Blood Bank, Lodz. Each donor agree donate blood. The first step of isolation of PBMCs was a mix of fresh blood from b coats with PBS at ratio 1:1. In the next step, the mixture was centrifuged in a de gradient of Lymphosep (Cytogen, Zgierz, Poland) at 2200 RPM for 20 min with the est values of acceleration and deceleration. Then the cells were washed three time centrifugation with 1% PBS. After isolation cells were suspended in RPMI 1640 med The study protocol was approved by the Committee for Research on Human Subjec the University of Lodz (17/KBBN-UŁ/III/2019).

Cell Viability
The cell viability resazurin assay was performed similarly to the method descr by O'Brien et al. [23]. Resazurin salt powder was dissolved in sterile PBS buffer. PB and HL-60 cells were seeded on 6-well plates at a density of 0.5 × 10 6 cells/mL. Cells pre-incubated with ruthenium complexes 1-3, succinimide, and RuCl3 at a concentr of 50 µM for 24 h at 37 °C in 5% CO2. Next, cells were washed with warm PBS. Then, were seeded on a 96-well plate in the count of 50,000 for PBMCs and 15,000 for H cells. Next, cells were incubated with H2O2 at concentrations of 0.2, 0.4, and 0.6 mM h at 37 °C in 5% CO2. Next, 10 μL of resazurin salt was added to each well, and the p again were incubated at 37 °C in 5% CO2 for 2 h. After that, fluorescence was meas with HT microplate reader Synergy HT (Bio-Tek Instruments, Winooski, VT, USA) u λex = 530/25 nm and an λem = 590/35 nm. The effects of ruthenium complexes, RuCl3 succinimide on cell viability were quantified as the percentage of control fluorescenc

Cell Culture
HL-60 (human promyelocytic leukemia) cell line was obtained from the American Type Culture Collection (ATCC) and cultured in Iscove's Modified Dulbecco's Medium (IMDM) with 15% fetal bovine serum, streptomycin/penicillin solution (100 µg/mL and 100 U/mL). HL-60 cells were cultured in flasks at 37 • C in 5% CO 2 and sub-cultured every 2-3 days to maintain exponential growth.
Peripheral blood mononuclear cells (PBMCs) were isolated from the buffy coats obtained from healthy donors from Central Blood Bank, Lodz. Each donor agreed to donate blood. The first step of isolation of PBMCs was a mix of fresh blood from buffy coats with PBS at ratio 1:1. In the next step, the mixture was centrifuged in a density gradient of Lymphosep (Cytogen, Zgierz, Poland) at 2200 RPM for 20 min with the lowest values of acceleration and deceleration. Then the cells were washed three times by centrifugation with 1% PBS. After isolation cells were suspended in RPMI 1640 medium. The study protocol was approved by the Committee for Research on Human Subjects of the University of Lodz (17/KBBN-UŁ/III/2019).

Cell Viability
The cell viability resazurin assay was performed similarly to the method described by O'Brien et al. [23]. Resazurin salt powder was dissolved in sterile PBS buffer. PBMCs and HL-60 cells were seeded on 6-well plates at a density of 0.5 × 10 6 cells/mL. Cells were pre-incubated with ruthenium complexes 1-3, succinimide, and RuCl 3 at a concentration of 50 µM for 24 h at 37 • C in 5% CO 2 . Next, cells were washed with warm PBS. Then, cells were seeded on a 96-well plate in the count of 50,000 for PBMCs and 15,000 for HL-60 cells. Next, cells were incubated with H 2 O 2 at concentrations of 0.2, 0.4, and 0.6 mM for 4 h at 37 • C in 5% CO 2 . Next, 10 µL of resazurin salt was added to each well, and the plates again were incubated at 37 • C in 5% CO 2 for 2 h. After that, fluorescence was measured with HT microplate reader Synergy HT (Bio-Tek Instruments, Winooski, VT, USA) using λ ex = 530/25 nm and an λ em = 590/35 nm. The effects of ruthenium complexes, RuCl 3 , and succinimide on cell viability were quantified as the percentage of control fluorescence.

Cell Cycle
HL-60 cells were seeded on 6-well plates at a density of 0.5 × 10 6 cells/mL. The cells incubated with 100 ng/mL nocodazol (NOC) for 24 h at 37 • C were the positive control. The cells were incubated with ruthenium complexes 1-3, succinimide, and RuCl 3 at a concentration of 50 µM for 24 h at 37 • C in 5% CO 2 . Next, cells were washed with warm PBS. Then, cells were again seeded on 6-well plates. Next, cells were incubated with H 2 O 2 at concentrations of 0.1 and 0.2 mM for 24 h at 37 • C in 5% CO 2 . Then, cells were collected and washed twice with PBS. After that, cells were resuspended in PBS and put on ice for 15 min. Then, one volume of −20 • C absolute ethanol was added and the samples were stored at 4 • C. Before the analysis, samples were resuspended in 300 µL of staining solution containing 40 µg/mL PI (propidium iodide) 200 µg/mL RNase A. Samples were incubated for 30 min at 37 • C in the dark until analysis. DNA content was analyzed using an LSRII flow cytometer (Becton Dickinson, San Jose, CA, USA).

Effect of Ruthenium Complexes 1-3 on DNA Oxidative Damage
We pre-incubated cells with complexes 1-3, succinimide, and RuCl 3 at a concentration of 50 µM for 24 h in 37 • C in 5% CO 2 , then cells were washed with PBS and incubated with H 2 O 2 at 0.025 or 0.05 mM for 15 min on ice. Cell viability after incubation with H 2 O 2 was in the range of 95-100% (data not shown). After incubation with H 2 O 2 cells were centrifuged, suspended in LMP agarose, and spread onto a microscope slide. The slides were processed as described below in the Section 2.6.

Comet Assay
The comet assay was performed under alkaline conditions according to the procedure of Tokarz et al. [24]. A freshly prepared cell suspension in 0.75% LMP agarose dissolved in PBS was layered onto microscope slides (Superior, Germany), which were pre-coated with 0.5% NMP agarose. Then, the cells were lysed for 1 h at 4 • C in a buffer containing 2.5 M NaCl, 0.1 M EDTA, 10 mM Tris, 1% Triton X-100, pH = 10. After cell lysis, the slides were placed in an electrophoresis unit. DNA was allowed to unwind for 20 min in the solution containing 300 mM NaOH and 1 mM EDTA, pH > 13.
Electrophoretic separation was performed in the solution containing 30 mM NaOH and 1 mM EDTA, pH > 13 at an ambient temperature of 4 • C (the temperature of the running buffer did not exceed 12 • C) for 20 min at an electric field strength of 0.73 V/cm (28 mA). Then, the slides were washed in water, drained, stained with 2 µg/mL DAPI, and covered with coverslips. To prevent additional DNA damage, the procedure described above was conducted under limited light or in the dark.

Comet Analysis
The comets were observed at 200× magnification in an Eclipse fluorescence microscope (Nikon, Tokyo, Japan) attached to a COHU 4910 video camera (Cohu, Inc., San Diego, CA, USA) equipped with a UV-1 A filter block and connected to a personal computer-based image analysis system Lucia-Comet v. 6.0 (Laboratory Imaging, Praha, Czech Republic). Fifty images (comets) were randomly selected from each sample and the mean value of DNA in the comet tail was taken as an index of DNA damage (expressed in percent).

Measurement of Reactive Oxygen Species
To measure reactive oxygen species (ROS), a 2 ,7 -dichlorofluorescein diacetate (H 2 DCFDA) probe was used. H 2 DCFDA is a cell membrane-permeable non-fluorescent probe. 2 ,7dichlorofluorescein diacetate is de-esterified intracellularly and turns into highly fluorescent 2 ,7 -dichlorofluorescein upon oxidation. PBMCs and HL-60 cells were seeded at a density of 2.5 × 10 6 cells/mL and 0.5 × 10 6 cells/mL, respectively, and were incubated with ruthenium complexes 1-3, succinimide, and RuCl 3 at a concentration of 50 µM for 24 h at 37 • C in 37 • C in 5% CO 2 . Next, the cells were washed twice with HBSS containing Ca 2+ and Mg 2+ and stained with 20 µM H 2 DCFDA (Sigma-Aldrich, St. Louis, MO, USA) for 30 min at 37 • C in darkness. Then, the cells were washed twice with HBSS and incubated with 1 mM and 5 mM H 2 O 2 at 37 • C in darkness. The intensity of fluorescence was measured after 30 min with λ ex = 495 nm and λ em = 530 nm using a microplate reader Synergy HT (Bio-Tek Instruments, Winooski, VT, USA). The data were analyzed according to the following formula: (T x − T 0 /T 0 ) × 100, where T x is the DCF fluorescence measured at the indicated time and T 0 is the DCF fluorescence measured at the beginning of the analysis.

Measurement of SOD Activity
PBMCs and HL-60 cells were seeded at density 3 × 10 6 cells/mL and 1 × 10 6 cells/mL, respectively, in a 75 cm 2 cell culture flask. The cells were incubated with ruthenium complexes 1-3, succinimide, and RuCl 3 at a concentration of 50 µM for 24 h at 37 • C in 5% CO 2 . Next, HL-60 cells were washed with PBS and incubated with 0.1 mM H 2 O 2 for 15 min on ice. In the case of PBMCs, we used 0.25 mM H 2 O 2 for 15 min at 37 • C. Next, cells were sonicated under the ice in 0.5 mL of PBS for 30 s using the 4710 Series Ultrasonic Homogenizer (Cole-Parmer Instrument Co., Chicago, IL, USA) to obtain cell lysates. SOD activity was measured using the SOD Assay Kit-WST (Dojindo, Kumamoto, Japan) by following the manufacturer's instructions. Cells lysates served as the sample solution. The mixture was placed into each well with 200 µL of WST working solution. Twenty microliters of enzyme working solution were added into each well and mixed thoroughly. The plate was incubated at 37 • C for 20 min. The absorbance was read at 450 nm using a microplate reader Synergy HT (Bio-Tek Instruments, Winooski, VT, USA).

Statistical Analysis
Data of cell viability, ROS measurement, cell cycle, and SOD activity are presented as the mean values ± standard deviation (SD) of at least three replicates. Values in the comet test are expressed as mean values + standard error of the mean (SEM) of three experiments; data from three experiments were collected and statistical parameters were calculated. Statistical analysis was performed using the Mann-Whitney test (samples with distributions departing from normality) and the Student t-test (normal distribution of the sample). Differences were considered statistically significant when the p-value was <0.05.

Cell Viability
All experiments were performed with ruthenium complexes 1-3 at a concentration of 50 µM. We chose this concentration based on our previous studies in which we showed an increase in cell viability for both PBMCs and HL-60 after 24 h incubation with ruthenium complexes at 50 µM [5].
After pre-incubation, the cells were incubated with H 2 O 2 for 4 h and the viability was determined with a resazurin reduction assay. We observed a significant increase (p < 0.001) in the viability of normal and cancer cells pre-incubated with all ruthenium complexes compared to the cells that were not pre-incubated ( Figure 2). The cells that were pre-incubated with RuCl 3 under the same conditions showed a further decrease (p < 0.001) in viability following incubation with H 2 O 2 , except for the PBMCs which were incubated with H 2 O 2 at 0.4 and 0.6 mM. We also observed a decrease (p < 0.001) in the viability of the HL-60 cells that were pre-incubated with succinimide. In the case of the normal cells, we detected the opposite effect of pre-incubation with the ligand-an increase in the viability of PBMCs compared to the cells that were only incubated with H 2 O 2 at 0.4 and 0.6 mM (p < 0.001). The viability for individual samples was calculated relative to negative control (untreated cells) ± SD. Cell viability in the control was taken as 100%, n = 6, * p < 0.05, *** p < 0.001 vs. negative control, ## p < 0.01, ### p < 0.001 vs. H2O2.

Cell Cycle
After incubation of HL-60 cells with 0.1 mM H2O2, we observed an increase in the number of cells in the sub-G1 (p < 0.001), S (p < 0.001) and G2/M (p < 0.01) phases of the cell cycle and a decrease in the G0/G1 phase (p < 0.001) compared to the control cells

Cell Cycle
After incubation of HL-60 cells with 0.1 mM H 2 O 2 , we observed an increase in the number of cells in the sub-G1 (p < 0.001), S (p < 0.001) and G2/M (p < 0.01) phases of the cell cycle and a decrease in the G0/G1 phase (p < 0.001) compared to the control cells (Table 1) We showed a decrease in the number of cells in the sub-G1 phase after pre-incubation with ruthenium complexes 1-3 compared to the cells incubated only with H 2 O 2 , both at 0.1 and 0.2 mM (p < 0.001). A similar decrease in the number of cells was observed in the S phase (p < 0.001). In addition, we demonstrated cell arrest in the G2/M phase by pre-incubation of HL-60 cells with complexes 1-3 and incubation with 0.2 mM H 2 O 2 .
HL-60 cells that were pre-incubated with RuCl 3 or succinimide and then incubated with H 2 O 2 showed similar changes in the course of the cell cycle as the cells pre-incubated with ruthenium complexes 1-3 (Table 1).

DNA Oxidative Damage
We induced DNA oxidative damage in PBMCs and HL-60 cells using H 2 O 2 at two concentrations of 0.025 and 0.05 mM. Then we investigated the influence of pre-incubation with ruthenium complexes 1-3 on oxidative DNA damage (Figure 3). In the PBMCs we observed a statistically significant decrease in oxidative DNA damage for complex 1 (p < 0.001) and complex 2 (p < 0.05), but only in the case of incubation with H 2 O 2 at 0.05 mM (Figure 3). After incubation of the PBMCs with H 2 O 2 at a lower concentration of 0.025 mM, we did not observe any of the complexes influencing the level of DNA oxidative damage.  In the experiment with HL-60 cells, we detected a significant decrease in oxidative DNA damage induced by H 2 O 2 at 0.025 and 0.05 mM for all ruthenium complexes (p < 0.001) (Figure 3). We showed a similar effect-reduction of DNA damage in the cells pre-incubated with succinimide and then incubated with H 2 O 2 at both 0.025 and 0.05 mM concentrations. In the HL-60 cells that were pre-incubated with RuCl 3 , we did not indicate any change in the oxidative DNA damage level (p > 0.05).

Reactive Oxygen Species Level
We used an H 2 DCF-DA probe to determine the effect of ruthenium complexes 1-3 on reactive oxygen species (ROS) induced by H 2 O 2 at concentrations of 1 and 5 mM (Figure 4). In the case of PBMCs, we observed a decrease (p < 0.001) in the level of ROS for complexes 1 and 2 and 1 mM H 2 O 2 (Figure 4). In contrast, complex 3 increased the level of H 2 O 2 induced ROS (p < 0.001). A similar effect to the one found in complex 3 was observed in the PBMCs pre-incubated with succinimide and then incubated with 5 mM H 2 O 2 (p < 0.001). Complex 2 has the greatest potential to scavenge ROS in normal cells; it also reduced endogenous ROS levels (p < 0.001).
In HL-60 cells, we clearly showed a significant reduction (p < 0.001) in ROS levels by all ruthenium complexes (Figure 4). We also observed a similar effect in the HL-60 cells pre-incubated with succinimide and then incubated with H 2 O 2 at 1 mM (p < 0.001) and 5 mM (p < 0.01). All ruthenium complexes and succinimide significantly reduced endogenous ROS levels in contrast to RuCl 3 .

SOD Activity
In this experiment, we investigated the influence of ruthenium complexes on SOD activity under oxidative stress conditions ( Figure 5). First, of all, we observed a decrease in SOD activity (p < 0.001) after the incubation of PBMCs ( Figure 4) and HL-60 cells ( Figure 5) with H 2 O 2 , which corresponds with the results of other research [25,26]. It should be emphasized that in the case of the incubation of HL-60 cells with 0.1 mM H 2 O 2 , we observed a decrease in SOD activity to 50%, whereas in the case of PBMCs the SOD activity decreased only to a level of about 93% after the use of 0.25 mM H 2 O 2 . Further increasing the concentration of H 2 O 2 to 0.6 mM did not cause any further inhibition of SOD activity in these cells (data not shown).
Our studies clearly showed that all ruthenium complexes increased SOD activity (p < 0.001) in HL-60 cells treated with H 2 O 2 ( Figure 5). We observed a similar effect after the pre-incubation of these cells with succinimide (p < 0.001). In the case of HL-60 cells pre-incubated with 50 µM RuCl 3 , we did not detect any changes in SOD activity compared to the cells that were not pre-incubated with RuCl 3 . In the case of PBMCs, we observed an increase in SOD activity (p < 0.001) after pre-incubation with complexes 1 and 2. Complex 3 as well as RuCl 3 and succinimide did not increase SOD activity (p > 0.05) ( Figure 5). Molecules 2022, 27, x FOR PEER REVIEW 11 of 16 Figure 4. Changes in reactive oxygen species (ROS) level in PBMCs and HL-60 cells pre-incubated with ruthenium complexes 1-3 at 50 μM for 24 h at 37 °C and then incubated with 1 mM or 5 mM H2O2 at 37 °C. Each value represents the mean ± SD, n = 6; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. negative control; ## p < 0.01, ### p < 0.001 vs. H2O2.

SOD Activity
In this experiment, we investigated the influence of ruthenium complexes on SOD activity under oxidative stress conditions ( Figure 5). First, of all, we observed a decrease in SOD activity (p < 0.001) after the incubation of PBMCs ( Figure 4) and HL-60 cells ( Figure 5) with H2O2, which corresponds with the results of other research [25,26]. It should be emphasized that in the case of the incubation of HL-60 cells with 0.1 mM H2O2, we observed a decrease in SOD activity to 50%, whereas in the case of PBMCs the SOD activity decreased only to a level of about 93% after the use of 0.25 mM H2O2. Further pre-incubation of these cells with succinimide (p < 0.001). In the case of HL-60 cells pre-incubated with 50 µM RuCl3, we did not detect any changes in SOD activity compared to the cells that were not pre-incubated with RuCl3. In the case of PBMCs, we observed an increase in SOD activity (p < 0.001) after pre-incubation with complexes 1 and 2. Complex 3 as well as RuCl3 and succinimide did not increase SOD activity (p > 0.05) ( Figure 5).

Discussion
Here, we investigated the antioxidant activity of three ruthenium complexes with succinimidato and phthalimidato ligands against normal PBMCs and HL-60 leukemic cells. For this purpose, we used 24 h pre-incubation of cells with the complexes and then induced oxidative stress by incubating cells with H2O2. A correlation and gene ontology pathway analysis identified a rigid association with genes intertwined in cell cycle progression and proliferation after cell incubation with H2O2 [23]. The ten most substantially correlating genes were validated using qPCR, showing complete congruency with the microarray analysis findings. Western blotting confirmed the correlation of cell cycle-related proteins negatively correlating with H2O2 IC25. It was also shown that the top genes related to ROS production or antioxidant defense were only in modest correlation [27]. The intracellular concentration of H2O2 is regulated tightly, enabling its use as a cellular signaling molecule while minimizing its potential to cause cellular damage [28]. A high concentration of H2O2 induces necrosis and a low concentration induces apoptosis. In HL-60 cells H2O2-induced apoptosis is regulated by decreased BCL-2 [29]. Moreover, it was shown that H2O2 evoked apoptotic events through the increase of Ca 2+ in HL-60 cells, inducing caspase -9 and -3 Hydrogen peroxide, by producing hydroxyl radicals (OH • ) through the interaction with metal ions near DNA, induces DNA damage such as modified bases, apurinic/apyrimidinic (AP) sites, and single-strand breaks (SSBs). The addition of OH • at position C8 within the guanine ring generates the oxidative product, 8-oxo-7,8-dihydro-2 -deoxyguanosine (8-oxodG). Similarly, the addition of OH • at position C8 of deoxyadenosine generates the oxidative product 8-oxo-7,8-dihydro-2 -deoxyadenosine (8-oxodA). These radicals are capable of further reduction or oxidation forming 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) or 8-oxo-7,8-dihydroguanine (8-oxoG), in deoxyguanosine or 4,6-diamino-5formamidopyrimidine (FapyA) or 7,8-dihydro-8-oxoadenine (8-oxoA) in deoxyadenosine. Another prevalent oxidative product is thymine glycol, produced by the insertion of OH • at position C5 of the thymine rings. Similarly, another oxidation product of cytosine is cytosine glycol, which upon deamination leads to the formation of uracil glycol [31].
Here, we observed a statistically significant decrease in oxidative DNA damage in PBMCs for ruthenium complex 1 (p < 0.001) and ruthenium complex 2 (p < 0.05) (Figure 3). In the case of HL-60 cells, we noticed a significant decrease in oxidative DNA damage for all ruthenium complexes (p < 0.001) ( Figure 3). Previously, we detected a protective effect on H 2 O 2 -induced oxidative DNA damage by tricarbonyldichlororuthenium (II) dimer (CORM-2) and the CO-depleted molecule (iCORM-2) [32]. This may indicate that not only the released CO but also the iCORM-2, to which new ligands attach, have antioxidant properties. Here, we also observed that ruthenium complexes reduced the level of ROS induced by H 2 O 2 in both types of cells ( Figure 4). We used the fluorogenic 2 ,7dichlorodihydrofluorescein-diacetate (H 2 DCFDA) probe to detect ROS. The acetate groups on H 2 DCF allow for diffusion across the plasma membrane, after which both groups are cleaved by intracellular esterases to form H 2 DCF. H 2 DCF is reactive toward many types of oxidants, including nitrogen dioxide ( • NO 2 ), the carbonate radical anion (CO 3 •-), the hydroxyl radical (OH • ), Fe 2+ , Cu + , thiyl radicals (e.g., the glutathione radical; GS • ), and peroxidases (e.g., cytochrome c peroxidase) [33,34]. Importantly, none of the studied ruthenium complexes induced oxidative stress (Figure 4).
Superoxide dismutases (SODs) including MnSOD, Cu/Zn-SOD, and extracellular SOD along with catalase are composed as the first line of defense against ROS. Some studies also showed that oxidative stress downregulated MnSOD in several oxidative stress models induced by H 2 O 2 and other oxidants. The oxidative stress-induced downregulation of MnSOD activity causes superoxide radical accumulation and superoxide can be converted to OH • in the presence of transition metals (e.g., Fenton reaction) [26]. Research carried out by Gottfredsen et al. [31] showed that hsEC-SOD is inhibited and fragmented by H 2 O 2 . The enzyme was inhibited by H 2 O 2 (37 • C, 1 h) in a dose-dependent manner, with an IC 50 value of 0.8 mM and complete inhibition at~2 mM. The mechanism of inhibition was similar to that of bovine Cu,Zn-SOD, including the oxidation of proline (Pro 112) and histidine (His 98, His 163) residues proximal to the active site Cu [35,36].
We detected that all ruthenium complexes increased SOD activity (p < 0.001) in HL-60 cells treated with H 2 O 2 ( Figure 5). We observed a similar effect after the pre-incubation of these cells with succinimide (p < 0.001). In the case of PBMCs, we observed an increase in SOD activity (p < 0.001), decreased by H 2 O 2 , after pre-incubation with complexes 1 and 2 ( Figure 5). We showed that ruthenium complexes 1 and 2 reversed the H 2 O 2 -induced downregulation of SOD to normal levels in PBMCs. We assume that ruthenium complexes are responsible for the restoration of SOD activity in the case of PBMCs and its increase in HL-60 cells due to their ROS scavenging capacity. The numerous studies mentioned in the Introduction have shown that various ruthenium complexes synthesized in recent years have the ability to scavenge ROS and RNS [6][7][8][9][10][11]16].
Our results show that ruthenium complexes 1-3 bearing succinimidato and phthalimidato ligands at a low concentration of 50 µM protect cells against oxidative stress and do not exhibit cytotoxic activity. Here, we have experimentally confirmed our previous theoretical calculation results on the electronic structure of ruthenium complexes bearing different imidato ligands. We showed that in the case of maleimide, the HOMO-LUMO (highest occupied molecular orbital and lowest unoccupied molecular orbital) energy gap was lower by 1.36 eV when compared with succinimide [5]. Therefore, we suppose that the antioxidant properties of ruthenium complexes, which we studied here, may be due to the high HOMO-LUMO energy gap. Our current comparative studies of ruthenium complexes bearing succimidato ligands with succinimide, and RuCl 3 indicate that the succinimide ligand, rather than the ruthenium metal center, is responsible for the antioxidant activity of complexes 1-3. We plan to undertake further electrochemical investigations of succinimide and complexes 1-3 to shed some light on this problem. Taking into account the fact that oxidative stress is involved in many human diseases, including cancer, antioxidant treatment involving new molecules that exhibit antioxidant properties in biological systems could be of potential interest from a clinical point of view.