Mutagenesis, Vol. 15, No. 5, 375-378,
September 2000
© 2000 UK Environmental Mutagen Society/Oxford University Press
Induction of chromosomal aberrations by the rhodium(III) complex cis-[Rh(biq)2Cl2]Cl in cultured human lymphocytes
Department of Biological Sciences and 1 Department of Chemistry, Faculty of Sciences, Yarmouk University, Irbid and 2 Department of Paediatrics and Medical Laboratory Sciences, Jordan University of Science and Technology, Irbid, Jordan
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Abstract |
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The genotoxicity of the rhodium(III) complex cis-[Rh(biq)2Cl2]Cl (complex R) in cultured human lymphocytes was studied using the chromosome aberrations (CAs) assay. Lymphocyte cultures were initiated from two adult healthy non-smoking male volunteers and were exposed to the complex for a duration of 3 or 20 h prior to cell collection. The reduction in mitotic indices (MI) and the induction of CAs represented the toxic and clastogenic effects of the different treatments, respectively. Complex R proved to be an intermediate toxic clastogen with a MI50 of 1.0 µg/ml and a minimum positive dose (MPD) of 0.1 µg/ml. Like bleomycin, complex R exerted its clastogenic effects without the need for metabolic activation and induced CAs of all types in lymphocytes treated in the G2 and late S phases and, therefore, can be considered a radiomimetic. In addition, it induced more total CAs of chromatid-type than of chromosome-type. The reduction in the frequencies of CAs following the 20 h treatment as compared with those induced following the 3 h treatments indicated that human lymphocytes in the presence of complex R can partially tolerate the lesions involved in CA production. Based on the biological effects of complex R and the similarities between its functional group and that of bleomycin, possible mechanisms for complex R genotoxicity are discussed.
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Introduction |
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Interest in studying the biological properties of transition metal complexes arose after the discovery of the antitumor activity of cisplatin, cis-[Pt(NH3)2Cl2] (Rosenberg et al., 1969
). In the same year, a large number of rhodium(III) complexes with nitrogen-donor ligands such as pyridine, ethylenediamine, 2,2'-bipyridine and 1,10-phenanthroline were reported to have antibacterial activity (Bromfield et al., 1969). Recently we have investigated the mutagenic activity of a series of rhodium(III) complexes with 2,2'-biquinoline (biq) and 2-(2'-pyridyl)quinoline towards S.typhimurium strains TA100 and TA98. Among this series, we found that the rhodium(III) complex cis-[Rh(biq)2Cl2]Cl (complex R) exhibited the highest mutagenic and least toxic activity without the need for complex metabolic activation (Sadiq and Zaghal, 1997). These results encouraged us to further investigate the genotoxicity of complex R in human cells. Genotoxic and clastogenic compounds have been used as chemotherapeutic agents to prevent carcinogenicity by killing the potentially dividing carcinogenic cells (Radman et al., 1982; Preston et al., 1987). Due to the correlation between the chromosome aberrations (CAs) assay and both the clastogenicity and mutagenicity of the tested chemicals in humans (Holstein et al., 1979; Radman et al., 1982; Preston et al., 1987), we have investigated the clastogenicity of complex R by testing its ability to induce CAs in cultured human lymphocytes. |
Materials and methods |
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Lymphocyte cultures and chemical treatment
Blood cultures were prepared using two healthy, non-smoking, same age, adult, male volunteers. These cultures were initiated by the addition of 1 ml of fresh, heparinized whole blood to 9 ml of RPMI 1640 medium (Sigma) supplemented with 20% heat-inactivated fetal bovine serum, penicillin, streptomycin (Gibco) and L-glutamine (Serva). Lymphocytes were stimulated by phytohemagglutinin (Gibco) and were incubated in humidified incubators at 37°C for a total of 72 h.
Complex R was prepared as described earlier (Zaghal and Qaseer, 1991). Directly before addition to the lymphocyte cultures, complex R was freshly dissolved in distilled water at a concentration of 50 µg/ml and diluted to the proper concentrations in fresh medium. Untreated cultures were set up as negative controls, while cultures treated with 10 µg/ml bleomycin (CAS no. 9041-93-4; Sigma) were set up as positive controls. Before collection of the cells, cultures were treated with complex R or bleomycin for either 3 or 20 h and were then treated with colchicine (Sigma) at a final concentration of 10–4 M for the last hour of incubation. Collected cells were then subjected to hypotonic treatment followed by fixation in ice-cold 3:1 methanol:glacial acetic acid mixture. Metaphase spread slides were prepared and stained with 5% Giemsa in Sorensen buffer, pH 6.8.
Analysis of cell kinetics and CAs
All metaphase slides were independently coded and examined by a blind study, using a x100 oil immersion objective mounted on a Labrolux 11 bright field microscope (Zeiss, Germany). Scoring of CAs was done according to previously proposed criteria, where only chromatid and chromosome breaks from normally damaged cells with not more than 30 breaks were considered (Scott et al., 1983; Hsu, 1987; Preston et al., 1987; Carrano and Natarajan, 1988). Gaps (achromatid lesions without dislocations in the chromatid) and exchanges were also scored. In each treatment a total of 100 well-spread metaphases were studied in the CAs analyses and about 1000–2000 lymphocytes were examined in the cell kinetic studies.
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Statistical analysis |
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The Mantel–Haenszel
2 test was used for statistical evaluation of the association between CA production and the different concentrations of complex R. The evaluation was done by analyzing the data obtained from each donor separately, as well as the data obtained after controlling for the donor effect (Agresti, 1996). |
Results |
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In this study the cytotoxic and genotoxic effects of complex R were investigated in cultured human lymphocytes. The effects of complex R were compared with those of the positive control bleomycin, because both compounds share similarities in their functional groups. The cytotoxic activity of complex R is presented in Table I
as significant reductions in the mitotic indices (MI). The cytotoxic effects of all the treatments were not significantly different (P < 0.001) in the cultures initiated from both donors. All treatments with complex R and bleomycin were significantly (P < 0.001) toxic to lymphocyte cultures, but the toxicity of complex R was significantly less than that of bleomycin. The toxicity of complex R was dose dependent and was proportional to the duration of treatment at 0.1 µg/ml, but plateaued and was independent of concentration and duration of treatment at concentrations 
0.5 µg/ml. The frequencies of the different types of CAs induced by complex R are shown in Table II. The baseline frequencies of CAs in the untreated cultures of both donors were not significantly (P < 0.001) different. These frequencies varied between no and one chromosomal break, two and four chromatid breaks and no chromatid exchanges per 100 metaphases. All cultures treated with complex R had significant increases (P < 0.001) in total CA frequencies and in chromatid and chromosomal exchanges over the untreated controls. Both the complex R and bleomycin treatments induced more total CAs of chromatid-type than of chromosome-type. The frequencies of CAs, including only breaks and exchanges, induced by a concentration of 0.1 µg/ml complex R were directly proportional to the duration of treatment.
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In the presence of complex R, cultured human lymphocytes were able to partially tolerate the lesions involved in the production of CAs, leading to lower CA frequencies following the 20 h treatments compared with those induced following the 3 h treatments.
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Discussion |
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TopAbstractIntroductionMaterials and methodsStatistical analysisResultsDiscussionReferences |
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This work aimed to further evaluate the mutagenic effects of complex R reported earlier in S.typhimurium (Sadiq and Zaghal, 1997
). The results show that complex R is toxic and exerts its clastogenic effects in human lymphocytes without the need for metabolic transformation. The concentration of complex R which would reduce the MI by 50% (MI50), was found to be ~1.0 µg/ml, while the minimum positive dose which induced significant breaks (MPD) was 0.1 µg/ml. The 10-fold difference between these two parameters indicated that complex R is an intermediate toxic clastogen for cultured human lymphocytes. This was confirmed by the relative lower toxicity of complex R at a concentration of 20 µg/ml as compared with that obtained by treatment with 10 µg/ml bleomycin. Similar to bleomycin, complex R induced CAs of all types in lymphocytes treated in G2 and late S phase. This is in accordance with the reported clastogenicity of bleomycin (Povirk and Austin, 1991; Darroudi et al., 1992; Chu, 1994; McLeod et al., 1994; Sadiq et al., 2000) and indicates that complex R is a clastogen with radiomimetic effects similar to bleomycin and other structurally related compounds. The cytotoxicities of complex R and bleomycin can be explained by the effects they exert on the cellular control mechanisms (checkpoints) that temporarily arrest the cell cycle following damage to the DNA. Excessive DNA damage induced by high concentrations of complex R was, however, highly toxic and resulted in cell disintegration.
The mechanism by which complex R exerts its action at the molecular level is not known. However, similarities in the functional groups of complex R and bleomycin suggest similar interactions between each of these compounds and cellular components such as DNA and proteins. Both complex R and bleomycin have an octahedral environment around their transition metal ion, bulky nitrogen ligands and other moderately labile leaving groups which undergo some changes in solution to facilitate attachment of their metal ion to DNA bases. For bleomycin to be active, a transition metal ion such as Fe(II), Mn(II), Cu(II) or Co(III) is required as a cofactor. These metal ions are bound to sterically hindered nitrogen-donor ligands in an octahedral geometry (McGall and Stubbe, 1988). Similarly, complex R includes Rh(III) as a transition metal ion which has an octahedral geometry with two bidentate bulky biq ligands and two chlorides in a cis arrangement (Zaghal and Qasser, 1997). The presence of two moderately labile cis leaving groups is essential for biological activity of the transition metal complex (Umapathy, 1989; Cotton et al., 1999). Hydrolysis of the two cis chlorides in complex R is expected to play a role in the interactions of this complex with DNA and facilitate its covalent binding to two sites. The binding of similar transition metal ions to DNA breaks the hydrogen bonds between the nitrogen bases on opposite DNA strands (Umapathy, 1989; Cotton et al., 1999) and induces a variety of intrastrand and interstrand DNA adducts and protein crosslinks (Huang et al., 1994; Zamble and Lippard, 1995). The ability of complex R to separate the two strands of the DNA molecule would produce single-stranded DNA lesions which could then be subjected to degradation by single-strand nucleases, possibly similar to the DNA scissions induced by the nuclease reported in HeLa cells (Slor and Lev, 1973). Cell tolerance to complex R could be due to an increase in the capacity of their DNA repair systems or a result of binding of the DNA adducts to high mobility group-domain proteins, which may enhance or reduce access of the repair complex to the site of damage (Zamble and Lippard, 1995). Other possible mechanisms for repair of the induced DNA damage could be similar to repair of that induced by bleomycin, including removal of blocked DNA termini by excision with a DNA polymerase-associated nuclease, followed by resynthesis of the DNA (Miller and Chinault, 1982). In addition, the possibility that complex R is not stable for long times under culture conditions cannot be excluded. This instability may result from reaction of complex R with serum proteins and/or from an increase in intracellular scavenging by GSH or metallothionines, as in the case for bleomycin and cisplatin (Green et al., 1984; Hamilton et al., 1985; Chatterjee et al., 1989).
Moreover, the possibility of a redox reaction occurring in solution cannot be excluded, since complex R induces a significant increase in exchanges, which are mainly produced by DNA breaks as a result of attacks by free radicals. Rhodium(III) is known to undergo reduction to rhodium(I) (Cotton et al., 1999). Complexes of rhodium(I) are also known to exhibit biological activity (Monti-Bragadin et al., 1987). Such a reaction, which releases electrons, may result in the formation of radicals that attack DNA and cause damage. Finally, further in vitro studies in the presence of metabolic activation would be useful to determine whether the clastogenic and cytotoxic effects of complex R can be increased or decreased, as would be expected from a potential antitumor drug.
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Acknowledgments |
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The authors thank the two volunteer donors, O.Khabour and M.Shboul. Special appreciation is due to O.Khabour for his technical assistance and to Dr H.Samawi for performing the statistical analysis. This study was partially supported by grant 12/97 to M.F.S. from the Deanship of Research and Graduate Studies at Yarmouk University, Jordan.
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Notes |
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3 To whom correspondence should be addressed. Tel: +962 2 7271100; Fax: +962 2 7274725; Email: may{at}yu.edu.jo
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Received on February 7, 2000; accepted on April 14, 2000.
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