Mutagenesis, Vol. 17, No. 4, 317-320,
July 2002
© 2002 UK Environmental Mutagen Society/Oxford University Press
Cell studies of the DNA bis-intercalator 
- [µ-C4(cpdppz)2-(phen)4Ru2]4+: toxic effects and properties as a light emitting DNA probe in V79 Chinese hamster cells
Department of Physical Chemistry, Chalmers University of Technology, S-412 96 Gothenburg, Sweden and 1 Genetic and Cellular Toxicology, Wallenberg Laboratory, Stockholm University, S-106 91 Stockholm, Sweden
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Abstract |
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Coordination complexes of type [Ru(L)3]2+, where L is a nitrogen-containing aromatic bidentate ligand, can often be photolytically reduced, making them useful in studies of DNA- or protein-mediated electron transfer and in artifical photosynthesis model systems. Upon binding to DNA some Ru(L) complexes have been found to display strongly increased fluorescence compared with when free in solution, making those compounds interesting to test as DNA probes. Thus, they are becoming widely used in the chemistry community. Here, asynchronous cultures of V79 Chinese hamster cells were exposed to the DNA bis-intercalator
- [µ-C4(cpdppz)2-(phen)4Ru2]4+ at 10-10–10-4 M. The extraordinarily strong binding of the compound to DNA was the reason for testing its possible interference with DNA metabolism in intact mammalian cells. Exposure for 1 h to 10-10–10-4 M did not significantly decrease DNA synthesis. Cells exposed to 10-5 M for 27 h showed no staining of the nucleus, while DNA was stained in cells electroporated in the presence of the compound. However, the Ru dimer was probably taken up by pinocytosis, because numerous minute precipitates could be observed in the cytoplasm. Treatment for 24 h at concentrations of 10-10–10-5 M did not inhibit growth, as indicated by cell density and mitotic activity. Neither did it affect chromosomal arrangements during mitosis. However, at 10-4 M the density of cultures was reduced by ~45% and apoptotic cells were frequent, as opposed to mitoses. We also investigated the properties of the Ru dimer as a fluorescent DNA stain. The compound appears attractive as a red DNA stain when broad excitation in the visible range is desirable and extremely low background staining is essential. The low toxicity of the compound is a favourable trait in this context. |
Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionConclusionReferences |
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Chiral substitution inert ruthenium complexes with planar aromatic ligands have received much attention due to their interesting DNA binding (Friedman et al., 1990
; Hiort et al., 1993; Lincoln et al., 1996; Norden et al., 1996; Erkkila et al., 1999; Önfelt et al., 1999, 2001) and photophysical properties (Olson et al., 1997; Schoonover and Strouse, 1998; Önfelt et al., 2000). Their ability to be photolytically reduced has been used in studies of DNA- (Murphy et al., 1993; Lincoln et al., 1997) and protein-mediated (Di Bilio et al., 1998) electron transfer as well as in model artificial photosynthesis systems (Magnuson et al., 1999). Complexes with dipyridophenazine derivatives as ligands frequently show a large increase in fluorescence when bound to DNA compared with when in aqueous solution, a property that makes them interesting as DNA stains. A recent study showed that a newly developed ruthenium complex could be used as an optical probe in cell viability measurements (Jimenez-Hernandez et al., 2000). As these families of compounds are becoming more and more widespread in the chemistry community it is of importance to determine their toxicity.
The ruthenium complex - [µ-C4(cpdppz)2-(phen)4Ru2]4+ (Figure 1A) is a novel DNA binding agent. [C4(cpdppz)2 = N,N' bis-(cpdppz)-1,4-diaminobutane; cpdppz = 12-cyano-12,13-dihydro-11H-cyclopenta[b]dipyrido[3,2-h:2'3'-j]phenazine-12-carbonyl; dppz = dipyrido[3,2-a:2',3'-c]phenazine; phen = 1,10-phenanthroline.] The complex has a bis-intercalating binding mode where the dppz ligands are sandwiched between the DNA bases with the ruthenium atoms situated in one of the grooves. The affinity for DNA is very strong (108 at 200 mM NaCl) and the dissociation rate extremely slow (Önfelt et al., 1999, 2001). The Ru dimer absorbs light in the blue area of the spectrum and emits red light when it is bound to DNA (Figure 1B). In contrast, when the compound is free in water solution emission is almost totally quenched (Önfelt et al., 2000). The strong affinity for DNA together with the attractive spectroscopic properties led us to investigate the compound with regard to toxicity and properties as a fluorescent DNA probe. Earlier studies have shown that this compound, despite its relatively high charge (4+), can penetrate liposomes (Ardhammar et al., 1999). If the compound has the ability to penetrate cell membranes and reach the DNA inside the nucleus it would most probably inhibit DNA synthesis and repair. Here we have employed V79 Chinese hamster cells, widely used in mutagenicity studies, to investigate incorporation of thymidine, growth and mitotic and anaphase/metaphase indices after exposure to a range of concentrations. To estimate the degree of uptake microscopy was performed on living cells incubated with the Ru dimer for several hours or electroporated (Tsong, 1991) in the presence of the same concentration of the compound.
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Materials and methods |
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Chemicals
- [µ-C4(cpdppz)2-(phen)4Ru2]Cl4 was prepared by condensation of [Ru(phen)2(1,10-phenanthroline-5,6-dione)]2+ with the appropriate bis-(5,6-diaminoindan) in acetonitrile solution (Lincoln et al., 1996; Önfelt et al., 1999). The crude product was isolated as the hexafluorophosphate salt and purified by chromatography on Al2O3, with acetonitrile as eluent. Finally, the hexafluorophosphate salt was converted to the water-soluble chloride salt by precipitation with tetra-n-butylammonium chloride in acetone.
The DNA dye YOYO (a homodimeric derivative of oxazole yellow) was purchased from Molecular Probes. The correct concentrations of Ru dimer and YOYO solutions used were obtained by diluting stock solutions where the concentration was determined by measuring the absorbance. The extinction coefficients used were 
440 nm = 40 000/M.cm for the Ru dimer and 457 nm = 96 100/M.cm for YOYO. Aphidicolin and propidium iodide (PI) were purchased from Sigma and prepared as stock solutions in DMSO and distilled water, respectively.
Cell culture
V79 Chinese hamster cells were grown in Eagle's minimum essential medium supplemented with 10% fetal calf serum, 90 U/ml penicillin, 90 µg/ml streptomycin, 1.8 mM L-glutamine and 45 µg/ml kanamycin (37°C, 3% CO2). Experiments were performed with asynchronous cultures set up 24 h before use.
Incorporation of radioactive thymidine
Cells in 24-well plates (0.75x105 cells/well) were incubated in fresh medium containing 1 µCi [3H]thymidine (sp. act. 25 Ci/mmol). The compound dissolved in either 0.9% NaCl (Ru dimer) or DMSO (aphidicolin) was added to give the final concentrations indicated in Figure 2. Parallel cultures with no added Ru dimer or aphidicolin were used as controls. After 1 h incubation at 37°C the medium was removed and 1 ml of ice-cold 5% trichloroacetic acid (TCA) was added. The cultures were left on ice for 30 min and then rinsed once with 1 ml of TCA before the cells were dissolved in 5% sodium lauryl sulfate. Scintillation liquid (4 ml of OptiPhase Hisafe 3) was added to each sample, before counting in a Wallace 1409 liquid scintillation counter. Parallel cultures were trypsinized and counted to allow correction for any loss of cells during treatment.
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, exposure time 1 h) and relative number of cells versus concentration of Ru dimer (
, exposure time 1 h; 
, exposure time 24 h). The known DNA antimetabolite aphidicolin was studied as a reference (
, relative number of cells; •, radioactivity, exposure time 1 h). (B) Relative number of cells in interphase (Observations of live cells
Cells were seeded on coverslips (1x105 cells) and incubated in complete medium containing 10 µM Ru dimer for 27.5 h and then mounted in a POC chamber (Bachofer GmbH) for acquisition of confocal images using a Zeiss LSM 510 microscope.
Electroporation
Washed cells (1.6x107) were suspended in 0.8 ml of Hank's medium containing 10 µM Ru dimer and held on ice for 10 min before electroporation at 1.01 kV and 25 µF (Gene Pulser; Bio-Rad). After 10 min on ice the cells were seeded on a coverslip and incubated in complete medium overnight before washing, fixation in methanol:glacial acetic acid (3:1) and image acquisition.
Microtubule immunostaining and chromosome staining with Ru dimer after formaldehyde fixation
Cells were fixed in 4% paraformaldehyde (Sigma) in phosphate-buffered saline (PBS), pH 7.3, for 15 min, permeabilized with 0.1% Triton X-100 in 0.1% BSA in PBS for 10 min and incubated at 4°C overnight with mouse anti-
-tubulin antibody (1:1000; Amersham Pharmacia), followed by the second antibody, rabbit-anti-mouse IgG (1:300; Zymed) for 30 min. DNA was stained with 10 µM Ru dimer in PBS for 10 min, before mounting (SlowFade Light Antifade Kit; Molecular Probes). Intervening rinses were with PBS.
Carnoy fixation and chromosome staining with Ru dimer
The medium was removed and the cells were fixed by addition of methanol and glacial acetic acid (3:1), rinsed with buffer (2x3 ml) and stained for 10–20 min in a solution of Ru dimer (10 µM) or for 10 min in a solution of PI (1 µM). Coverslips were rinsed twice with 3 ml of distilled water and left to dry before mounting (SlowFade Light Antifade Kit).
Effects on mitosis after 24 h exposure
Cells were fixed in methanol:glacial acetic acid (3:1) as above, stained with Giemsa (4%, 5 min) and permanently mounted in DPX. All slides were coded before the frequencies of interphases, mitoses and apoptotic cells were determined among 1000 cells/concentration. The anaphase/metaphase index was estimated among 100 cells/slide.
Comparison of Ru dimer, propidium iodide and YOYO in bleaching experiments
The software Time series of the Zeiss LSM 510 was applied to estimate bleaching of the fluorescence of cells stained with Ru dimer, PI and YOYO (cells were stained as described above). The Ar laser (
= 488 nm; 12.5% effect corresponding to 1.9 mW) was used for excitation and the emitted light was collected through a 505 nm cut-off filter. Decay curves displayed the average light intensity/pixel of a select area as a function of irradiation time.
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Results and discussion |
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TopAbstractIntroductionMaterials and methodsResults and discussionConclusionReferences |
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Figure 2
shows the results of the toxicity test. The incorporation of thymidine during exposure for 1 h was not significantly different from controls and there was no extensive loss of cells except at the highest concentration. This loss was relatively more prominent than the decrease in DNA synthesis. The Ru dimer precipitated at the higher concentrations (10-4 and 10-5 M), which made it necessary to rinse all the wells carefully, which in turn may have led to an increased loss of cells. More efficient trypsinization and thereby fewer doublets in the suspension is the most likely cause of slightly higher cell numbers with concentrations of 10-8–10-7 M. Taken together, the results suggest that the Ru dimer weakly induced rounding and detachment rather than inhibition of DNA synthesis. When exposure was extended to 24 h only the highest concentration applied appeared toxic; cultures were less dense at 10-4 M and the frequency of apoptosis was found to increase drastically. There was no sign of a block in metaphase at 10-10–10-5 M; the anaphase/metaphase ratios were found to be similar to that among controls. At 10-4 M the ratio could not be estimated due to a lack of mitoses. This concentration is about one order of magnitude higher than concentrations normally used in spectroscopic studies.
Figure 3A shows confocal images of live cells incubated with complete medium containing 10 µM Ru dimer for 27.5 h before study. No emission was found from the DNA inside the nucleus, indicating low membrane penetration. However, small areas of low emission could be found in the cytoplasm, showing that the compound was taken up by the cells, probably by pinocytosis. Here, the intensity of emission is a poor measure of the amount of compound, since the emission quantum yield of this chromophore is very sensitive to the environment. When the cells were electroporated in the presence of Ru dimer, emission was detected from the nucleus several hours later, showing that the drug was bound to DNA (Figure 3C). Importantly, after the thorough washing procedure before fixation most of the cells left were those that remained viable after electroporation and invasion of the drug.
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Figure 3D and E
shows examples of cells where the chromosomes were stained with Ru dimer after fixation. Figure 4 illustrates the results of the comparative study of the bleaching properties of Ru dimer, YOYO (a homodimeric derivative of oxazole yellow) and PI. Both YOYO and PI bind to DNA by intercalation (similarly to the Ru dimer, YOYO bis-intercalates) and are commonly used as stains for nucleic acids and are therefore interesting as a comparison. Stability under these conditions was found to be in the order PI > YOYO > Ru dimer. However, the experimental conditions used were very rough, e.g. the output energy of the laser was about 10 times higher than what is needed for the generation of good images. The lower stability was not a problem when the compound was used in ordinary confocal laser scanning microscopy (CLSM) studies.
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The strong affinity of the Ru dimer for DNA along with its spectroscopical properties make it suitable as a DNA marker. The fact that the wavelength interval of excitation is large, reaching from the green to the blue area of the spectrum, means that many different light sources can be used. The wide absorption spectrum and relatively large Stokes shift for emission also makes it possible to see Ru–DNA fluorescence simultaneously with fluorescence at other wavelengths. The same excitation source may be used but, for example, red/green fluorescence be separated (Jimenez-Hernandez et al., 2000
). The fact that emission is quenched in aqueous solutions makes the chromosome staining selective and the problem of stray light from other parts of the cell is minimal. Similarly to DAPI, background staining was clearly lower with the Ru dimer than with YOYO and PI. |
Conclusion |
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TopAbstractIntroductionMaterials and methodsResults and discussionConclusionReferences |
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The DNA bis-intercalator
- [µ-C4(cpdppz)2-(phen)4Ru2]4+ was found to have no immediate effect on DNA synthesis and survival of V79 Chinese hamster cells, probably due to low membrane penetration. Toxicity was observed only with long treatment at concentrations of Ru dimer >10-5 M. The compound is an excellent fluorescent marker for DNA, for example in studies of chromosome/spindle arrangements.
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Notes |
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2 To whom correspondence should be addressed. Tel: +46 31 7725120; Fax: +46 31 7723858; Email: onfelt{at}phc.chalmers.se
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References |
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Received on September 12, 2001; accepted on February 25, 2002.
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