Evaluation of the in vitro cytogenotoxic profile of the silver(I) metal complex with furosemide (Ag-FSE)

The aim of the present study was to evaluate the cytotoxic potential of a new complex of silver(I) with furosemide (Ag-FSE) over a panel of tumor and non-tumor human cells, as well as its genotoxicological safety. According to the results obtained, cell line derived from colorectal adenocarcinoma (Caco-2) was the most sensitive to the Ag-FSE complex, and among the non-tumor cells was the non-transforming fibroblasts known as GM07492A, which stimulated the characterization of cytotoxic properties against these cells. In addition to interfering with the cellular metabolic profile (evidenced by the resazurin method), Ag-FSE alters the integrity of cell membranes (results obtained by the Trypan blue assay) regardless of the cell line evaluated. However, interference with clonogenic capacity is dependent on the cell being tested; the cytotoxic activity evaluated by the clonogenic survival assay was evidenced against Caco-2 cells, but not against GM07492A cells. Furthermore, Ag-FSE did not induce an increase in the extent of DNA damage detectable by the comet assay, nor in the frequency of micronuclei compared to the negative control, demonstrating the absence of genotoxicity under the experimental conditions used. The data of the present study provide information on various biological aspects of the Ag-FSE complex and help in the generation of new drug candidates with potential to impact the health area, targeting cancer treatment.


Introduction
Metal ions associated with suitable ligands originate metal complexes that can have different biological and/or pharmacological properties (Yan et al., 2017), due to the numerous characteristics of coordination geometry, oxidation state, redox, kinetic and thermodynamic state of an inorganic molecule (Thompson & Orvig, 2006;Cohen, 2007).
Scientific interest in this line of research intensified due to the accidental discovery of cisplatin around 1960; one of the first metallic drugs, completely inorganic, with chemotherapy activity used until today in the therapy of testicular, ovarian, cervical, bladder and lung cancer (Cirri et al., 2019;Ghosh, 2019), which despite the benefits, it has several undesirable effects and therefore serves as an inspiration for the search for new compounds for the treatment of cancer (Cohen, 2007;Cirri et al., 2019;Ghosh, 2019).
As a result, it promoted the development of several metal-based drugs, essential and non-essential, with the potential to be used in imaging diagnosis, in addition to having different pharmacological activities (Barry & Sadler, 2013;Mjos & Orvig, 2014;Mahapatra et al., 2019).
The antibacterial potential is of particular interest and has been widely explored due to bacterial multi-resistance acquired by the excessive use of antibiotics (Mosconi et al., 2014).
According to Lustri et al. (2017), the complex of silver with furosemide (Ag-FSE) has shown a significant in vitro antibacterial activity against Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), and yeast Candida albicans (ATCC 90028), and the absence of mutagenic activity by Ames assay.
Silver complexes have the ability to interfere with bacterial metabolism, break the cell wall and bind to its DNA, preventing bacterial replication and the possibility of resistance development (Rocha et al., 2011). Currently, with new technologies, it has been possible to adopt other forms of this metal, such as refined silver salts, colloidal silver and nanoparticles, further expanding its application, making it present in food packaging coatings, fabrics, dressings, ointments, purifiers. air and water (Möhler et al., 2018). Studies also show that silver has antiparasitic, antifungal and antitumor activity, which compared to cisplatin has less toxicity and more cytotoxicity depending on the ligand (Medici et al., 2015;Ciol et al., 2018;Liang et al., 2018;Rocha et al., 2019;Esteban-Parra et al., 2019;Kizrak et al., 2019;Favarin et al., 2019). Therefore, in the perspective of the discovery of new drugs, in this study we explore the in vitro antitumor activity of Ag-FSE, as well as its genotoxic potential by the Comet and micronucleus assays.

Methodology
This is an experimental research, carried out in a laboratory, with a quantitative approach (Pereira et al., 2018) that aimed to evaluate the cytotoxic potential of a new complex of silver(I) with furosemide (Ag-FSE), through assays that assess cell viability (resazurin redox assay, trypan blue exclusion test and clonogenic survival assay) in a panel of tumor and nontumor human cells and its genotoxicological profile by the comet and micronucleus assays.   In 96-well microplates, the cells (1.5 x10 4 cells/well) were exposed to different concentrations of Ag-FSE, silver nitrate (AgNO3, metallic salt) and furosemide (FSE, ligand) that ranged from 2.2 to 285 µmol•L -1 . In addition, methyl methanesulfonate (MMS, 150 µmol•L -1 ) was used as positive control (cytotoxic drug) and dimethyl sulfoxide (DMSO 0.1%
After 24 h treatment, 0.01% w/v of resazurin solution (50 μL) was added to each well and the plates were incubated for 2h. The reading was performed in a microplate Synergy H1 (BioTek®) reader using excitation and emission filters at wavelengths of 560 and 590 nm, respectively. The assay was performed in three individual experiments in triplicate.

Trypan blue exclusion test
The protocol was performed in 6-well plates. Caco-2 and GM 07492A cells (1 × 10 5 cells/well) were exposed to different concentrations of Ag-FSE, silver nitrate (AgNO3, metallic salt) and furosemide (FSE, ligand) that ranged from 1.4 a 22.8 µmol•L -1 . Experimental controls constituted of negative (without any treatment), vehicle (DMSO 0.1% (v/v)) and positive (MMS, 50 µmol•L -1 ) controls were also included in the treatment. After 24 h treatment, 10 µL of the cell suspension was homogenized with trypan blue dye (0.2%), and then quantified on TC20 Automated Cell Counter (Bio-RAD). The experiments were performed in triplicate.

Clonogenic survival assay
The colony-forming assay in Caco-2 and GM 07492A cells was conducted under the same experimental conditions as the trypan blue assay. After 24 h treatment, 200 cells were seeded per well in 6-well plates. The experiments were carried out for 7 days. At the end of the growth period, the culture medium was removed and cells were washed with phosphate-buffered saline (PBS, pH 7.4), fixed in methanol/acetic acid/distilled water (1:1:8) for 30 min, and stained with 3% Giemsa for 30 min.
After staining, the colonies were washed with distilled water, and then counted. The experiments were performed in triplicate.

Cytotoxicity (IC50)
The inhibitory concentration to 50% (IC50), which represents the required concentration of the test sample capable of inhibiting 50% of cell growth, was calculated by plotting the viability or survival of the cells in relation to the respective concentrations of the test substances by the GraphPad Prism 9.0 program (GraphPad Software, San Diego, CA, USA). This parameter was calculated for all cytotoxicity assays.
After 24 h of treatment, the mixture of 20 µL of cell suspension of each treatment group and 120 µL of 0.5% low melting point agarose (dissolved in PBS) was added to the microscope slides coated with 1.5% normal-melting agarose.
The microscope slides were immersed in a lysis buffer with 1% Triton X for 18 h and then placed in an electrophoretic tank and dipped into a cool electrophoresis solution (NaOH 300 mmol•L -1 and EDTA 1 mmol•L -1 , pH 13) for 20 min. The settings of electrophoresis were 25 V and 350 mA for 20 min. The slides were then submerged in a neutralization buffer (0.4 mol•L -1 Tris-HCl, pH 7.5) for 15 min, dried at room temperature, and fixed in 100% ethanol for 5 min.

5
The percentage of DNA in the tail was established visually. A total of 300 cells (in three independent assays) were examined per treatment and the total score was calculated by multiplying the number of damaged cells by the value of the respective comet class (0, 1, 2 or 3) and then taking the sum for each treatment.
After the incubation period, 6.25 µmol•L -1 of pre-prepared cytochalasin B (Sigma-Aldrich) solution in culture medium were added for blocking cytokinesis, and the plates were placed in the CO2 incubator for 1.5 normal cell cycle lengths.
At harvest time, the cells were rinsed with PBS, trypsinized, and the pellet was hypotonized in 1% sodium citrate at 37°C and homogenized. After a new centrifuge, the pellet was resuspended in methanol:acetic acid (3:1) and again homogenized. Fixed cells were then transferred to slides, stained with 3% Giemsa, and analyzed under a light microscope.
Interphase cells were harvested and stained to analyze the presence of MN. The criteria established by Fenech (2007) were employed for the analysis of MN and binucleated cells. A total of 3000 binucleated cells were scored per treatment, corresponding to 1000 cells/treatment/repetition.
In addition, 500 viable cells per repetition between mononucleated, binucleated and multinucleated (three or more nuclei) were counted to calculate the Cytokinesis-Block Proliferation Index (CBPI), which allows the assessment of cell where, CBPI⊤ represents the CBPI of the cells that received different concentrations of the Ag-FSE treatment and CBPIင represents the CBPI of the negative control cells.

Statistical analysis
The data were analyzed statistically by the Graph Pad Prism 9.0 software (Graph-Pad Software Inc., San Diego, CA, USA) using analysis of variance one way ANOVA to determine the statistical significance, followed by the Tukey test considering p < 0.05. The tests were performed in triplicate and the experimental criterion was the significance of the response to treatment in relation to the negative control.

Results and Discussion
In order to investigate new molecules in the search for potential therapeutic agents, the synthesis, spectroscopic characterization, in addition to the antimicrobial and mutagenic activities of the Ag-FSE complex were evaluated and the results described by Lustri et al. (2017).
Due to the promising antimicrobial potential found, and the absence of mutagenic effect by the Ames test (Lustri et al., 2017), the present study focuses on evaluating cytotoxicity over a panel of tumoral and non tumoral human cells, and mutagenicity by the micronucleus and comet assay, in order to complement the data already obtained from this complex and provide reliable data to support future clinical research.
The effectiveness of silver complexes against bacteria and cancer cells depends on several factors: lipophilicity, redox propensity, solubility and stability in water, and rate of release of silver ions. These factors are strictly controlled by the characteristics of the ligands and their requirements on steric and electronic properties (Medici et al., 2019).
FSE is a sulfonylamide that belongs to the loop diuretics group. These compounds produce an increase in sodium excretion and urinary flow of about 30 times when compared to normal excretion transporters, blocking Na+, K+ and Cl in the ascending limb of the loop of Henle. They are drugs used clinically mainly in the treatment of kidney diseases, liver cirrhosis, hypertension and edema (Barroso et al., 1996;Malode et al., 2011).
The FSE ligand was not cytotoxic under the conditions of this study, demonstrating that the cytotoxicity profile of the complex is related to the metallic salt, AgNO3, which can be considered a source of free Ag(I) ions.
Tables 1 and 2 presents the inhibitory concentration to 50% (IC50) values observed through the evaluation of the cytotoxic activity of Ag-FSE and AgNO3 against the tumor and non-tumor cells obtained by resazurin redox assay, trypan blue exclusion and clonogenic survival assay.
The results showed varying degrees of sensitivity of the cell lines after treatment with the test substances, being that Caco-2 was the tumor cell most sensitive to the inhibitory activity of the Ag-FSE complex. Among the non-tumor cell lines, the GM 07492A cells were the most sensitive to this complex, which encouraged us to further studies with these two cells to investigate the mechanisms that induce cytotoxicity.
The clonogenic survival assay was performed under the same experimental conditions as the trypan blue assay. Ag-FSE and AgNO3 induced similar effects; both did not interfere in the capacity of GM 07492A cells to divide and proliferate, and thus form colonies, at all concentrations evaluated, while in Caco-2 cells, IC50 values were 5.4 ± 0.5 µmol•L -1 for Ag-FSE and 5.1 ± 1.2 µmol•L -1 for AgNO3 (Table 2).
The results of these assays suggest that Ag-FSE and AgNO3 interfere with the cellular metabolic profile (evidenced by the resazurin method) and alter the membrane integrity (results obtained by the trypan blue assay) regardless of the cell lineage evaluated. However, interference in clonogenic capacity is dependent on the cell being tested.
Furthermore, this study also showed the greater sensitivity of Caco-2 cells (tumor cell line) compared to the nontumor cell line GM 07492A to different treatments, which corroborates other studies in the literature that show a special sensitization of Caco-2 cells by silver nanoparticles and silver ions (Iqbal et al., 2013;Oberemma et al., 2016;Zande et al., 2016;Vila et al., 2017). Ciol et al. (2018) described the synthesis, characterization and biological activities of the complex of Ag(I) with cycloserine (AgCIC). Among the results obtained, the authors showed that the complex was able to inhibit the growth of all tumor cells tested, being particularly more active in vitro against breast cancer cells and leukemia cells than doxorubicin, in addition to its remarkable selectivity in relation to cancer cells when compared to the non-tumor cell line, suggesting less toxicity than commercial chemotherapeutics. Manzano et al. (2020) synthesized and characterized three Ag(I) complexes with fluoroanthranilic acid (fa) isomers, which were named Ag4fa, Ag5fa and Ag6fa, in addition to analyzing their antibacterial, antiproliferative and mutagenic activities. Cytotoxic activity assays revealed that the Ag4fa complex was more selective against Caco-2 cells when compared to non-tumor cell lines (GM 07492A, MRC-5 and HUVEC). Aquaroni et al. (2020) carried out a study demonstrating the antibacterial and antiproliferative activity of the complex of Ag with 4-aminobenzoic acid (Ag-pABA). The complex exhibited antiproliferative activity in a panel of eight types of tumor cell lines and no mutagenic activity. By analysis of the selectivity index, the concentration of Ag-pABA needed to inhibit the growth of leukemic cells (K562) was 6X lower than that needed to inhibit non-tumor human keratinocytes. As can be seen, several studies show promising results considering the search for new bioactive Ag compounds for the treatment of cancer. Table 2. Inhibitory concentration to 50% (IC50, µmol•L -1 ) values of the Ag-FSE complex and metallic salt (AgNO3), in the Caco-2 and GM 07492A cells obtained by the trypan blue exclusion and clonogenic survival assays IC50 (µmol•L -1 ± SD)

Treatments
Trypan blue Clonogenic survival Caco-2 GM 07492A Caco-2 GM 07492A AgNO3 7.3 ± 0.5 27.2 ± 3.0 5.1 ± 1.2 >58.9 Ag-FSE 4.5 ± 1.1 9.3 ± 1.1 5.4 ± 0.5 >22.8 IC50 values were determined from three independent experiments using concentration (µmol•L -1 ) in triplicate (mean ± standard deviation, SD). The ligand (furosemide) did not show cytotoxic activity, under the conditions used in this study (IC50 >30.2 µmol•l -1 ) Regarding genotoxicity, the comet assay detects DNA damage before the repair mechanism. The basic principle of this test is the migration of DNA in an agarose matrix under electrophoretic conditions. When observed under a microscope, Research, Society andDevelopment, v. 11, n. 13, e21111334986, 2022 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v11i13.34986 8 the cells have the appearance of a comet, with a head (the nuclear region) and a tail containing the DNA fragments that migrate towards the positive pole (Lu et al., 2017;Singh et al., 1988;Tice et al., 2000). The MN test, which assesses the induction of chromosomal mutations, is currently one of the best-established cytogenetic tests in the field of toxicological genetics, with international validation and applicable to any nucleated cell population (Fenech, 2007;Kirsch-Volders et al., 2014). Table 3 details the data obtained by the comet assay, where it is observed that Ag-FSE, at concentrations tested for a period of 24 h in HepG2 cells, does not present genotoxic potential, as it did not induce a statistically significant difference in DNA damage when compared to the negative control, under the conditions used in this study.
In the different treatments, most of the cells analyzed are in classes 0 and 1 and the calculated score values ranged from 25.7 to 34.2 for the groups treated with Ag-FSE and 19.7 ± 3.5 for the negative control, while in the positive control this value was 162 ± 8.6.
Ag-FSE also did not induce a statistically significant increase in the frequency of MNs compared to the negative control, regardless of treatment time (3 or 24 hours) and cell used (CHO or HepG2), showing that Ag-FSE or possible metabolites are not mutagenic (Table 4).
About cytostasis, another parameter evaluated in this study, there was a statistically significant reduction in CBPI values only at the highest concentration evaluated, which made the counting of MNs unfeasible. In CHO, treatment with 11.4 µmol•L -1 of FSE-Ag caused 63.2% inhibition of cell proliferation (CBPI: 1.32 ± 0.10) and in HepG2 86.6% inhibition (1.14 ± 0.06) ( Table 4). These data corroborate the toxicity profile of Ag-FSE and helped in choosing the doses for the experiments. Values are presented as mean ± SD, based on three independent experiments. A total of 300 cells were examined per treatment group and the total score was calculated by multiplying the number of cells scored by the respective comet class value (0, 1, 2 or 3) and then calculating the sum of each treatment. NC: negative control (complete culture medium), SC: solvent control (DMSO, 0.0025%), PC: positive control (hydrogen peroxide, 50 µmol•L -1 ). * p < 0.05 statistically different from the negative control. Furthermore, previously evaluated by our group, Ag-FSE was also not able to induce gene mutations in the Ames test (Lustri et al., 2017).
The systematic discovery and development of Ag complexes have produced a large number of promising antibacterial, antifungal and anticancer compounds (Liang et al., 2018). Despite the versatility and promising activities of metal complexes, studies investigating the safety profile of these complexes are essential, as characteristics such as mutagenicity, carcinogenicity and toxicity may limit their use in therapy, since, for example, mutagenicity can lead to the development of secondary tumors (Pracharova et al., 2018).
Given this context, the absence of genotoxicity of Ag-FSE is highly relevant, as it is extremely important to have a balance between the therapeutic and toxicological effects of a pharmacological compound. Overall, the literature shows that genotoxicity data for Ag+ are limited. Ag+ is proven to be cytotoxic but did not induce genotoxicity in several different assays (Eliopoulos & Mourelatos, 1998;Demir et al., 2011). Ag iodide, for example, was not mutagenic in the Ames test and in the sister chromatid exchange assay (Eliopoulo & Mourelatos, 1998). AgNO3 did not induce mutations, while Ag nanoparticles were positive in the Drosophila wing cell somatic mutation assay (Demir et al., 2011). Thus, in vitro toxicity studies with Ag exposed to cell lines or by other methodologies are useful to confirm its actual mutagenic effects or carcinogenic alterations. Research, Society andDevelopment, v. 11, n. 13, e21111334986, 2022 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v11i13.34986

Conclusion
In conclusion, this study allowed us to explore various biological aspects of the Ag-FSE complex. The data from the present study, in addition to contributing to the search for new metal complexes with promising biological activities, provide an understanding of their interaction with genetic material. We demonstrate that the cytotoxicity of Ag-FSE is not related to genomic instability with direct damage or its metabolites to DNA, as Ag-FSE was not genotoxic when evaluated by the comet and MN assays. Further studies are needed to confirm this hypothesis, and to clarify their mechanisms of action, besides many more biological and physicalchemical studies to develop it into new antitumor drug or with other pharmacological application.