Biol Trace Elem Res (2009) 128:258–268 DOI 10.1007/s12011-008-8272-y
Cytotoxic and Genotoxic Effects of cis-Tetraammine (oxalato)Ruthenium(III) Dithionate on the Root Meristem Cells of Allium cepa Flávia de Castro Pereira & Cesar Augusto Sam Tiago Vilanova-Costa & Aliny Pereira de Lima & Alessandra de Santana Braga Barbosa Ribeiro & Hugo Delleon da Silva & Luiz Alfredo Pavanin & Elisângela de Paula Silveira-Lacerda
Received: 28 August 2008 / Accepted: 29 October 2008 / Published online: 20 November 2008 # Humana Press Inc. 2008
Abstract Ruthenium complexes have attracted much attention as possible building blocks for new transition-metal-based antitumor agents. The present study examines the mitotoxic and clastogenic effects induced in the root tips of Allium cepa by cis-tetraammine(oxalato) ruthenium(III) dithionate {cis-[Ru(C2O2)(NH3)4]2(S2O6)} at different exposure durations and concentrations. Correlation tests were performed to determine the effects of the time of exposure and concentration of ruthenium complex on mitotic index (MI) and mitotic aberration index. A comparison of MI results of cis-[Ru(C2O2)(NH3)4]2(S2O6) to those of lead nitrate reveals that the ruthenium complex demonstrates an average mitotic inhibition F. C. Pereira : C. A. S. T. Vilanova-Costa (*) : A. P. Lima : A. S. B. B. Ribeiro : H. D. Silva : E. P. Silveira-Lacerda Laboratório de Genética Molecular e Citogenética, Instituto de Ciências Biológicas–ICB I–Sala 200, Universidade Federal de Goiás, Campus Samambaia (Campus II), Cx. Postal: 2425-0, Goiânia, GO 74690-970, Brazil e-mail:
[email protected] F. C. Pereira e-mail:
[email protected] A. P. Lima e-mail:
[email protected] A. S. B. B. Ribeiro e-mail:
[email protected] H. D. Silva e-mail:
[email protected] E. P. Silveira-Lacerda e-mail:
[email protected] L. A. Pavanin Instituto de Química, Universidade Federal de Uberlândia–UFU, Uberlândia, Minas Gerais, Brazil e-mail:
[email protected]
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eightfold higher than lead, with the frequency of cellular abnormalities almost fourfold lower and mitotic aberration threefold lower. A. cepa root cells exposed to a range of ruthenium complex concentrations did not display significant clastogenic effects. Cistetraammine(oxalato)ruthenium(III) dithionate therefore exhibits a remarkable capacity to inhibit mitosis, perhaps by inhibiting DNA synthesis or blocking the cell cycle in the G2 phase. Further investigation of the mechanisms of action of this ruthenium complex will be important to define its clinical potential and to contribute to a novel and rational approach to developing a new metal-based drug with antitumor properties complementary to those exhibited by the drugs already in clinical use. Keywords Cis-tetraammine(oxalato)ruthenium(III) dithionate . Cytotoxicity . Genotoxicity . Allium cepa . Ruthenium compounds
Introduction Several metallic compounds have been recognized as potent antitumor agents and rigorously tested in vivo and in vitro [1]. Examples of established antitumor metallodrugs that are routinely used in the clinic are cisplatin [cis-diamminedichloroplatinum(II)] and its analogues carboplatin and oxaliplatin [2]. Nevertheless, the clinical utility of these drugs has been proven limited due to the relatively narrow range of tumors affected and the development of acquired drug resistance. Resistance to treatment can occur in certain disease types (ovarian and small cell lung cancers) or present as intrinsic drug resistance in less responsive cases (non-small cell lung and colon cancers) [3, 4]. Furthermore, cisplatin is administered intravenously due to its limited solubility in water and severe side effects [5]. Limitations of platinum-based complexes have prompted a search for more effective and less toxic metal-based antitumor agents. Some of these efforts have been directed towards the design of non-platinum, transition-metal-based compounds. Within this category, ruthenium complexes have attracted much interest as alternatives to cisplatin in cancer chemotherapy [2]. During the last two decades, a number of ruthenium(III) complexes have been synthesized and tested as anticancer drugs [6, 7]. In comparison to the platinum(II) antitumor complexes currently used in the clinic, ruthenium compounds offer potentially reduced toxicity, a novel mechanism of action, the prospect of non-cross-resistance, and a different spectrum of activity [8–11]. The reduced toxicity is in part due to the ability of ruthenium complexes to mimic the binding of iron to molecules of biological significance, exploiting the mechanisms that the body has evolved for non-toxic transport of iron. This reduced toxicity, together with non-cross-resistance in cisplatin-resistant cancer cells, is particularly attractive attributes of these complexes [12]. In addition, some chemical properties make these compounds well suited as alternatives to platinum-based antitumor drugs, such as the rate of ligand exchange, the range of accessible oxidation states, and the ability of ruthenium to mimic iron in binding to certain biological molecules. Hence, complexes based on ruthenium have been proposed to have potential antitumor and antimetastatic activities and generally show lower systemic toxicity than platinum compounds [13]. Plant bioassays are more sensitive and simpler than most methods used to detect the genotoxic effects of environmental pollutants. These assays have been validated in international collaborative studies under the United Nations Environmental Program, the World Health Organization, and the United States Environmental Protection Agency and demonstrated to be efficient approaches for monitoring the genotoxicity of environmental
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pollutants [14]. Since Levan introduced the first Allium test in 1938, various chemicals have been tested for cytogenetic activities using this method [15]. The test uses meristematic plant cells to investigate universal mechanisms and permits extrapolation to animal cells, thus providing valuable information regarding possible genotoxic effects of these chemicals in mammals and especially in humans [16–18]. This study was carried out with the goal of evaluating the cytotoxic effects induced by cis-tetraammine(oxalato)ruthenium(III) dithionate {cis-[Ru(C2O2)(NH3)4]2(S2O6)} and the relevance of its biological activity. Mitotoxic and clastogenic effects induced in the root tips of Allium cepa by the chemical were assessed for different exposure periods and concentrations. The results of this study will contribute to a better understanding of the mechanisms of action of cis-tetraammine(oxalato)ruthenium(III) dithionate upon the cellular context.
Materials and Methods Cis-[Ru(C2O2)(NH3)4]2(S2O6) Synthesis The cis-[Ru(C2O2)(NH3)4]2(S2O6) complex was prepared as follows: Pentaamminechlororuthenium(III) dichloride (1.0 g, 0.0034 mol) was refluxed in deaerated concentrated ammonium hydroxide (25 ml) under a blanket of argon until the solution turned dark pink. A stoichiometric amount of sodium dithionate (0.70 g, 0.0034 mol) was then added to the hot solution. Upon stirring in an ice bath, pentaaminehydroxoruthenium(III) dithionate precipitated from this solution. Addition of 100% ethanol completely precipitated the salt (1.2 g), which was filtered and air-dried. The off-white salt was then dissolved in a saturated solution of oxalic acid dihydrate (12 ml), and the mixture was refluxed under argon for about 5 min until a yellow solid precipitated and the mother liquor turned brown. The mixture was cooled and the yellow cis-tetraammine(oxalato)ruthenium(III) dithionate was collected by filtration, washed with ethanol, and air-dried. The compound was characterized by electronic spectra at room temperature with an HP 8453 spectrophotometer with diodes arrangement interfaced with a compatible PC HP Vectra XM using quartz cells. The electronic spectra of the synthesized tetraammineoxalatoruthenium(III) dithionate complex contained bands at 260 and 350 nm. Carbon and hydrogen microanalyses were performed by the staff of the Central Analítica of the Chemistry Institute of Universidade de São Paulo. A. cepa (Onion) Test Preparation Onion meristems (A. cepa) were used in this study, as they are considered to be one of the best biological models for the study of environmental pollutants [19]. Onions are easy to store and handle, and the root tip cells constitute a convenient system for macroscopic measurements (e.g., growth, EC50 values) as well as for assessment of microscopic parameters (e.g., c-mitosis, stickiness, chromosome breaks). Since these cells possess important plant activation enzymes, the Allium test has a broad range of applications. Furthermore, results from this test have shown good agreement with results from other test systems, both eukaryotic and prokaryotic [19, 20]. Inhibition of onion bulb root growth was measured according to the methods defined by Fiskesjö [20]. Briefly, onion bulbs, similar in size, shape and color, with an average weight of 90 g, were placed in filtered water, which was changed every 24 h and kept at a constant
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temperature of 25°C, with a continuous airflow, for 120 h. Each day, the bulbs were weighed and the roots were counted and measured. Once the roots had reached an average length of 4–5 cm, they were placed in an aqueous solution of cis-tetraammine(oxalato) ruthenium(III) dithionate at concentrations of 0.01 mg mL−1 (38 μM), 0.05 mg mL−1 (190 μM), or 0.1 mg mL−1 (380 μM) for 24 or 48 h. The solution was changed every 24 h. Prior to ruthenium complex exposure, a quarter of the roots of every bulb was collected for negative controls. All tests were repeated. Each iteration also included a second negative control in which the roots were only exposed to distilled water, as well as a positive control, in which lead(II) nitrate [50 μM Pb(NO3)2] was used in place of the ruthenium complex. The bulb weights, root numbers, and root lengths of these treatment groups were recorded daily. Preparation of Root Tip Cells and Mitotic Index Determination All experiments were carried out when the roots reached 4–5 cm in length. Ten roots were cut for each exposure period and concentration. Root tissues were placed in Carnoy’s solution [ethanol (99%) and glacial acetic acid (3:1)] for 30 min, washed with distilled water three times, and submitted to hydrolysis with an aqueous solution of 1 N hydrochloric acid (HCl) at 60°C. Roots were then dried and their apical regions (approximately 0.5 cm of root tip) were macerated and dyed with acetic-hydrochloric orcein. For the study of the mitotic index (MI) and chromosomal aberrations, an average of 5,000 root tip cells from each bulb were used in analysis. The MI was calculated for each treatment as the number of dividing cells per 100 cells. Cytological abnormalities were scored in the mitotic cells and the results were tabulated. Most abnormalities were presented with micrographs in the results section. Statistical Analysis Mean and SD values were calculated from the results of four bulbs for each experimental group. Correlation tests were performed to determine the effects of the period of exposure (time) and concentration of ruthenium complex on MI and mitotic aberration index. Oneway analysis of variance and Dunnet’s t test were used to determine the significance of observed differences (F=19.04, p
