Mutagenesis Advance Access originally published online on April 7, 2005
Mutagenesis 2005 20(3):165-171; doi:10.1093/mutage/gei023
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DNA damage induced by a quinoxaline 1,4-di-N-oxide derivative (hypoxic selective agent) in Caco-2 cells evaluated by the comet assay
Centro de Investigación en Farmacobiología Aplicada (CIFA), University of Navarra, C/Irunlarrea 1, 31008 Pamplona, Spain, 1Department of Biochemistry and Molecular Biology, University of Barcelona, Faculty of Chemistry, C/Martí Franqués 1, 08028 Barcelona, Spain and 2Department of Toxicology and Applied Hygiene, University Bordeaux 2, V. Ségalen, 146 Rue Léo Saignat, 33076 Bordeaux CEDEX, France
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Abstract |
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The DNA damage induced by 7-chloro-3-[[(N,N-dimethylamino)propyl]amino]-2-quinoxalinecarbonitrile 1,4-di-N-oxide hydrochloride (Q-85 HCl) in Caco-2 cells under hypoxic and well-oxygenated conditions has been studied by using the comet assay. This compound has shown a good in vitro profile of high selective toxicity in hypoxia, but its mechanism of action is unknown. The DNA damage has been evaluated by performing the comet assay after a 2-h treatment with Q-85 HCl (0.1, 0.2, 0.4 µM in hypoxia; 20, 40 µM in well-oxygenated conditions). The number of cells in apoptosis has also been assessed by flow cytometry analysis of Annexin V-FITC staining. The capability of the cells to repair the DNA damage and the proliferation rate was evaluated at different times after the treatment (24–168 h). Under hypoxic conditions, a clear dose-dependent increase in the number of nuclei with a comet was observed (comet score: 132 ± 13, 343 ± 30 and 399 ± 1; control comet score: 42 ± 14). Under well-oxygenated conditions, the number of nuclei with comet increased significantly with respect to the control (comet score: 273 ± 14 and 312 ± 9; control comet score: 27 ± 4). Cells in apoptosis were not detected by the comet assay nor by flow cytometry. The recovery from DNA damage was time- and concentration-dependent in hypoxia (cells treated with the highest concentration still showed DNA damage after 72 h) and rather time-dependent in well-oxygenated conditions (DNA was completely repaired after 24 h). In conclusion, Q-85 HCl acts by DNA damage and not only the reduced intermediate is genotoxic but also some other derivatives and Q-85 HCl itself may be acting.
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Introduction |
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The presence of hypoxic cells in solid tumours is one of the causes of cell resistance to anticancer treatments. The poor tumour vascular structure, the inefficient blood supply along with a high interstitial pressure generate a variable proportion of viable hypoxic cells (1
,2). They constitute 10–20% of the total viable tumour cell population and are already present in tumours with 1 mm of diameter (1,3). It has been demonstrated that hypoxic tumour cells are adapted to low pO2 and are able to survive under such conditions for a long time (3–5).
Hypoxic conditions confer resistance to standard radiotherapy because the generation of toxic radical oxygen species on DNA is not favoured. A resistance to chemotherapy is also postulated because of the relative low drug availability and the low proliferation rate of the hypoxic cells (3,5–10). Moreover, hypoxia induces the expression of gene products involved in the metastatic cascade and angiogenesis (5,7,8,10), and contributes to the selection of tumour cells with a diminished apoptotic potential (11,12). Therefore, hypoxia is an indicator of a more aggressive disease, independent of known prognostic parameters such as clinical stage (10,13–19).
Bioreductive drugs have been designed to take advantage of the particular metabolic characteristics of hypoxic cells. They are activated to a cytotoxic form in the hypoxic microenvironment and when oxygen is present, the active drug is back-oxidized to the non-toxic parental compound (1,3,67–8,10,20,21). The use of this type of drugs, in combination with traditional therapies, improves the outcome of treatment by increasing cytotoxicity to the hypoxic regions (6–8,10,21–23). This is an important advantage because these pro-drugs, only activated in hypoxic regions, offer the possibility of systemic treatment for solid tumours, including metastases (1). Moreover, the cytotoxic species are capable of diffusion to kill tumour cells surrounding the hypoxic core (2,10,21).
Several kinds of compounds are activated under hypoxic conditions: aromatic and aliphatic N-oxides, quinones, nitroaromatics and organo-metallic compounds. Tirapazamine (TPZ), a benzotriazine-di-N-oxide is the main drug among the bioreductive agents (1,8,21,24,25) and has already demonstrated significant activity in Phase II and III clinical trials in combination with radiotherapy and cisplatin based-chemotherapy (23). A great number of experiments have demonstrated that DNA is an important cellular target for TPZ that, under hypoxic conditions, generates base damage, single strand breaks (SSBs) and double strand breaks (DSBs) (26–31).
Among the various quinoxaline 1,4-di-N-oxides that have shown a selective toxicity under hypoxic conditions, one of the most promising is 7-chloro-3-[[(N,N-dimethylamino)propyl]amino]-2-quinoxalinecarbonitrile 1,4-di-N-oxide hydrochloride (Q-85 HCl) (32). Two parameters describe the in vitro activity of a bioreductive agent: the potency under hypoxic conditions and the selectivity. The potency is the concentration of drug required that gives 1% of cell survival in hypoxia, and the selectivity, measured by the determination of the hypoxic cytotoxicity ratio, is the ratio of equitoxic concentrations of the drug under aerobic/anoxic conditions. High hypoxia potency and high selectivity index are required in this type of compounds. Q85-HCl proved to be more potent and selective than TPZ in different human tumour cell lines, with Caco-2 cells presenting the best profile (33).
Despite the promising in vitro data very little is known about the mechanism of action of Q-85 HCl. The quinoxaline di-N-oxides were first recognized as antibiotics whose activity was enhanced under hypoxic conditions (34). Suter et al. (35) suggested that the lethal effect in bacteria of quinoxaline-di-N-oxide was owing to a reduction by-product, probably a free radical. Subsequent studies have demonstrated that quinoxaline-di-N-oxide is mutagenic in bacteria and yeast (36,37) and carcinogenic in rats (38). Recently, Ganley et al. (39) have provided direct evidence that quinoxaline di-N-oxide is able to cleave DNA under hypoxic conditions in the presence of the xanthine/xanthine oxidase one-electron reducing system.
Following the evidence that Q-85 HCl is the best bioreductive agent among the new quinoxaline di-N-oxides (33), the aim of the present work was to study its DNA-damaging effects in Caco-2 cells under different conditions, since this was the putative mechanism of action. For this purpose, hypoxic and well-oxygenated cells were treated with several concentrations of Q-85 HCl for 2 h. The capability of Q-85 HCl to produce SSBs, as well as the capability of treated-cells to repair DNA, were evaluated by performing the comet assay at 0, 24, 48 and 72 h after the treatment, in parallel to the measurement of cell proliferation rate.
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Materials and methods |
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Chemicals
The structure of the compound Q-85 HCl is shown in Figure 1. It was synthesized at the R&D Unit of the University of Navarra following the method described by Monge et al. (32
).
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The identity of the synthesized compound was tested by elemental analysis (CHN). IR spectra was recorded on a Perkin-Elmer 681 instrument (Perkin-Elmer, Boston, MA). 1H-NMR and 13C-NMR were performed on a Bruker AC200 E instrument (Bruker Española, San Fernando de Henares, Madrid, Spain) at 200 and 50 MHz, respectively. Mass spectra were obtained on a Hewlett Packard model 5988A (Agilent Technologies, Palo Alto, CA) by direct insertion probe. Ionization was performed by electron impact at 70 eV. Purity was tested by high-performance liquid chromatography (Waters 600E) under the conditions described by Zamalloa et al. (40
).
Cell culture
Caco-2 cells (colorectal adenocarcinoma, ATCC/HTB-37), provided by the American Type Culture Collection (Manassas, VA), were grown in Dulbecco's Modified Eagle's Medium (DMEM, Gibco, Prat de Llobregat, Barcelona, Spain) supplemented with 10% foetal bovine serum (FBS, Gibco, Prat de Llobregat, Barcelona, Spain) and 1% antibiotic (10 000 U/ml penicillin and 10 000 µg/ml streptomycin, Gibco, Prat de Llobregat, Barcelona, Spain). Cells were maintained as monolayer cultures at 37°C in a humidified atmosphere with 5% CO2.
Cell treatment
Monolayer cells in exponential growth were trypsinized (Trypsin, Gibco, Prat de Llobregat, Barcelona, Spain) and suspension cultures were prepared in 50 ml glass flasks: 2.2 x 107 cells/ml in 30 ml of DMEM containing 10% FBS and 1% antibiotic. The glass flasks were topped with rubber caps perforated with two 21 G needles (MicrolanceTM3, Becton Dickinson, Fraga, Huesca, Spain) to provide gas inlet and outlet in order to generate hypoxia and well-oxygenated conditions. They were placed on a shaking device introduced in a water bath at 37°C, and were gassed with humidified air (oxygenated experiment) or with nitrogen (hypoxic experiment) during all the experiment.
Different concentrations of Q-85 HCl in 100% dimethylsulfoxide (DMSO, Panreac, Montcada i Reixac, Barcelona, Spain) were prepared just before dosing. After 30 min of gassing, 200 µl of each solution were added to reach the following final concentrations: 20 and 40 µM in oxygenated conditions, and 0.1, 0.2 and 0.4 µM in hypoxia. Two controls were included under both conditions: one of them with solely the solvent and the other one without any treatment. After 2 h of treatment, cells were centrifuged at 175 g and resuspended in 1 ml of DMEM supplemented with 10% FBS and 1% antibiotic mixture. The number of viable cells was determined by the Trypan blue exclusion method, according to the procedure described by Pappenheimer (41). For each drug concentration tested, the percentage of viable cells with respect to the control, was calculated.
Proliferation assay
After the 2-h treatment a cell suspension was prepared to seed 1.8 x 105 TB-negative cells per well in 6-well plates, in a final volume of 3 ml of medium (DMEM supplemented with 10% FBS and 1% antibiotic). Four wells for each concentration of Q-85 and the controls were seeded. Plates were maintained at 37°C in 5% CO2. After 24, 48, 72 and 168 h of incubation, wells were washed with phosphate-buffered saline (Gibco, Prat de Llobregat, Barcelona, Spain) and the cells were trypsinized and counted in a haemocytometer (Neubauer Improved, Paul Marienfeld, Lauda-Koenigshofen, Germany), using the Trypan blue exclusion method. Each cell suspension was counted twice.
Comet assay
Just after the 2-h treatment with Q-85 HCl and also 24, 48 and 72 h later, the comet assay was carried out. The technique described by Singh et al. (42) and Tice et al. (43) with some modifications was followed. One hundred and sixty microlitres of 0.5% agarose-LMP containing 4.5 x 104 cells were distributed quickly on each ring of the commercially available Comet slide (Trevigen, Gaithersburg, MD). A cover slip was added and the agarose was allowed to set for 9 min on ice. Then the cover slip was removed and the cells were lysed immediately by immersion of the slide in a cold solution (pH 10.5) of 2.5 M NaCl, 100 mM Na2EDTA, 10 mM Trizma–HCl, 1% n-lauryl sarcosine and 1% Triton X-100 for 2 h. The slides were placed on a horizontal gel electrophoresis platform and covered with an alkaline solution made up of 300 mM NaOH and 1 mM Na2EDTA (pH >13). The slides were left in the solution for 20 min to allow unwinding of the DNA and expression of alkali-labile sites. The power supply was set at 0.7 V/cm (300 mA). The DNA was electrophoresed for 15 min and the slides were rinsed gently three times with 400 mM Trizma (pH 7.5) to neutralize the excess alkali. Each slide was stained with 30 µl of DAPI (Sigma-Aldrich, St Louis, MO), covered with a cover slip and coded before microscopic analysis.
DAPI-stained nuclei were evaluated with a Nikon Eclipse TE 300 fluorescence microscope (Nikon, Tokyo, Japan). A total of 50 comets on each ring of the comet slide were visually scored and classified as belonging to one of five classes according to the tail intensity. Each comet class was given a value between 0 and 4: (0) = no damage and (4) = maximum damage. The total score was calculated by the following equation: (percentage of cells in class 0 x 0) + (percentage of cells in class 1 x 1) + (percentage of cells in class 2 x 2) + (percentage of cells in class 3 x 3) + (percentage of cells in class 4 x 4). Consequently, the total score was in the range from 0 to 400. Two comet slides were used for each condition, which makes a total of 200 comets.
To check the performance of the comet assay, a positive control was included in all the experiments: Caco-2 cells were treated with 300 µM methyl methanesulfonate during 2 h, without nitrogen or air gassing.
Assessment of apoptosis
Just after the 2-h treatment with Q-85 HCl and also 2, 4, 6, 8, 12 and 24 h later, detection of apoptosis was performed using Annexin V-FITC kit binding assay. The basis of this assay is that during apoptosis, phosphatidyl serine is translocated from the inner side to the outer side of the plasma membrane, where it can be detected by Annexin V conjugates. Annexin V-positive/propidium iodine (PI)-negative cells are considered apoptotic; Annexin V-negative/PI-negative cells are considered alive; Annexin V-positive/PI-positive cells and Annexin V-negative/PI-negative cells are considered dead. Briefly, cells were resuspended in binding buffer (10 mM HEPES/NaOH, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2). Annexin V-FITC (Bender System Kit) was added according to the product insert and incubated for 30 min at room temperature in the dark. One minute before fluorescence-activated cell sorter (FACS) analysis, PI was added at a concentration of 20 µg/ml. Cells were analysed by an FACS. Experiments were performed in triplicate.
Statistical analysis
The statistical analysis was performed by using the software SPSS 11.0. Data are presented by descriptive analysis (mean ± SD for three independent experiments). The comparisons between total comet scores of the different groups were performed by the non-parametric Mann–Whitney U-test. The P 
0.05 probability was accepted as the level of significance.
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Results |
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Effect of Q-85 HCl on cell viability and cell proliferation
Q-85 HCl is not cytotoxic after 2 h incubation at the concentrations tested under hypoxic and well-oxygenated cell conditions. The percentage of survival with respect to the untreated cells was >90% in both conditions (data non shown). Cells treated with the solvent (DMSO) showed a cell survival of 100% in both conditions.
Just after the treatment, 1.8 x 105 cells per well were seeded in 6-well plates and incubated at 37°C for 24, 48, 72 and 168 h. The numbers of viable (TB-negative) cells counted after the different incubation times are presented in Figure 2. Under hypoxic conditions, the proliferation rate of cells treated with 0.1 µM Q-85 HCl was similar to that of the control cells during all the periods studied. Meanwhile, 24 h after the treatment, cells which had been treated with 0.2 µM Q-85 HCl showed a significant decrease in the number of TB-negative cells with respect to the control (mean number = 0.6 x 105 versus 1.9 x 105 ); only some resistant cells could proliferate in order to increase significantly the number of cells after 168 h (mean number = 12.1 x 105) (Figure 2). On the contrary, very few cells could survive treatment with 0.4 µM Q-85 HCl (mean number = 1.3 x 104) and they were completely unable to proliferate, the number of TB-negative cells remaining constant during all the periods studied (mean number between 1.3 x 104 and 1.9 x 104).
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Under well-oxygenated conditions, the two concentrations tested (20 and 40 µM) decreased the number of TB-negative cells significantly with respect to the control cells. At 24 h, the viability percentages were 65.6 ± 6.3 and 45 ± 9.3, respectively (Figure 2).
Genotoxic effect of Q-85 HCl
The evaluation of Caco-2 DNA damage was performed at time 0, 24, 48 and 72 h after the 2-h treatment with different concentrations of Q-85 HCl, under hypoxic and well-oxygenated conditions. The results are presented in Figures 3 and 4.
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There was no difference between the total comet score of the cells treated with the solvent (DMSO) and the untreated cells under both conditions (Figure 3). The hypoxic atmosphere per se induces a slight DNA damage in the cells: at time 0, after 2.5 h of gasification, the hypoxic control cells showed a total comet score slightly higher than the well-oxygenated control cells (42 ± 14 versus 27 ± 4, P < 0.05) (Figure 3), although the proportion of comets in class 0, 1 and 2 remained very similar (Figure 4).
In hypoxic conditions, at time 0 the DNA was damaged at all the doses tested in a dose-dependent manner (Figures 4 and 3A).
After 24 h, there was no significant difference at P < 0.05 between the total comet score of the control and the total comet scores of the cells treated with 0.1 and 0.2 µM Q-85 HCl (Figure 3A). Cells that survived to the highest concentration (0.4 µM) showed a decrease in the total comet score at 24, 48 and 72 h, but they remained significantly higher than the controls (P < 0.05) (Figure 3A).
In well-oxygenated conditions, the DNA was also damaged after the treatment, although the difference between the two concentrations was not significant (Figures 4 and 3B). After 24 h, there was no significant difference between the total comet score of the control and the total comet score of the cells treated with 20 and 40 µM (Figure 3B).
Assessment of apoptosis
The detection of apoptotic cells was performed at different times after the 2-h treatment with all the concentrations of Q-85 HCl, which had been assayed under hypoxic and well-oxygenated conditions. Cells in apoptosis were not detected at any time or condition. The results obtained just after the treatment and 12 h later with cells treated with 0.4 µM of Q-85 HCl in hypoxia and 40 µM of Q-85 HCl in well-oxygenated conditions, are presented in Figure 5.
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In hypoxic conditions, the percentage of dead cells just after the treatment with 0.4 µM of Q-85 HCl was similar to the control (Figure 5). Results were similar 2, 4, 6 and 8 h later (data not shown), and 12 h later there was an increase in the percentage of dead cells in the treated cells with respect to the control (Figure 5).
In well-oxygenated conditions the percentage of dead cells after the 2-h treatment with 40 µM of Q-85 HCl was similar to the control. Results were similar 2, 4, 6 and 8 h later (data not shown), and 12 h after the treatment there was an increase in the percentage of dead cells in the treated cells with respect to the control.
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Discussion |
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The quinoxaline di-N-oxide derivative Q-85 HCl has shown a good in vitro profile in different cell lines (V79, Caco-2, HT-29, MCF-7 and Tk-10), the Caco-2 cells being the most sensitive in hypoxia to this compound (33
). For this reason, the Caco-2 cell line was chosen to study the genotoxic potential of Q-85 HCl, which has not been evaluated so far.
Owing to the high hypoxic selectivity of this compound, different concentrations were assayed under hypoxic and well-oxygenated conditions, based on previous data obtained in this cell line (33). The hypoxic concentrations were selected within a range that yielded high survival percentages, measured by the Trypan exclusion method after the 2-h treatment with the compound, but having an effect on the clonogenic efficiencies (<10%), evaluated after 14 days of incubation: 0.1–0.4 µM (33). As the selectivity ratio is 
155 in this cell line, the concentrations tested under well-oxygenated conditions were chosen within a range 155-fold higher: 15.5–62 µM.
In this study, it was confirmed that after the 2-h treatment with Q-85 HCl at the concentrations tested, the percentages of survival with respect to the controls were >90%, under both well-oxygenated and hypoxic conditions. Thus, these are good conditions to evaluate the potential genotoxicity of the compound. One may assume that, under our experimental conditions, there was no apoptosis and no significant toxicity. Moreover, the possibility that this compound could induce the apoptotic process has been discarded in a specific fluorometric assay to detect cells in apoptosis. Thus, the data of the comet assay reflect a direct genotoxicity.
The time course of DNA breaks repair has been studied previously by different groups: SSBs appear to be more rapidly repaired than that of DSBs [for a review see (44)]. Moreover, some cells repair more efficiently than some others (45). For all these reasons we have decided to allow more time for DNA repair. The capability of Caco-2 cells to repair the DNA damage induced by Q-85 HCl was then evaluated by the comet formation at different times after the treatment (24–72 h).
In the current study, the visual scoring method has been applied since it has been shown that there is a clear relationship between visual scoring and the percentage of DNA appearing in the tail, as measured by computer image analysis (45,46).
Under hypoxic conditions, a clear dose-dependent increase in the number of nuclei with a comet was observed, with a maximum effect at 0.4 µM (virtually all the comets being of category 4). The recovery from DNA damage is time- and concentration-dependent. For 0.1 µM, DNA damage was completely repaired after 24 h and the cell proliferation was very similar to the control. For 0.2 µM, the cells that survived showed a comet score similar to the control, whereas the very few cells that survived at 0.4 µM, showed always high comet scores, indicating much more DNA damage. It seems that the time dependency is less important than the concentration dependency for Caco-2 cells to repair DNA damage induced by Q-85 HCl. Indeed, SSBs are normally repaired in <1 h. In contrast to hypoxic conditions, the recovery from DNA damage induced by Q-85 HCl in well-oxygenated cells is rather time-dependent. After 24 h the DNA damage is completely repaired for both concentrations.
To evaluate the effectiveness of a bioreductive agent in vivo is difficult because of two obstacles: one, the inaccuracy of existing methods to evaluate the number of hypoxic cells in tumours, second, the poor predictiveness of reductive enzyme testing in biopsies. This justifies in vitro testing as designed in the present experiment. For the prediction of efficiency of a given drug in specific tumour, one may need to prepare cells from biopsies taken from this tumour and assay the DNA damage produced by the bioreductive drugs on these cells. This conclusion is in line with that of Siim et al. (27) who have worked on TPZ.
Nothing is known about the metabolism of Q-85 HCl, but from our data it can be said that Q-85 HCl or its active forms bear genotoxic properties under hypoxic conditions. The genotoxicity is also found in oxygenated conditions although the concentrations used are higher. It could be concluded that not only the reduced intermediate is genotoxic but that some other derivatives and Q-85 HCl itself may be acting.
In conclusion, Q-85 HCl is an efficient and selective bio-reductive quinoxaline-di-N-oxide compound in Caco-2 cells and proceeds by damaging DNA.
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Acknowledgments |
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This work has received financial support from the Government of Navarra, Aquitania and Catalunya within the framework of the program ITT of the Work Community of Pyrenees.
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Notes |
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* To whom correspondence should be addressed. Tel: +34 948 425653; Fax: +34 948 425652; Email: acerain{at}unav.es
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Received on January 17, 2005; revised on February 14, 2005; accepted on March 7, 2005.
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