Mutagenesis, Vol. 15, No. 3, 261-269,
May 2000
© 2000 UK Environmental Mutagen Society/Oxford University Press
Cytokinesis-block micronucleus assay in WIL2-NS cells: a sensitive system to detect chromosomal damage induced by reactive oxygen species and activated human neutrophils
1 CSIRO Health Sciences and Nutrition, PO Box 10041, Adelaide BC, South Australia 5000, Australia and 2 Division of Applied Food Research, The National Institute of Health and Nutrition, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8636, Japan
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Abstract |
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We have developed a method that can detect the DNA-damaging and cytotoxic effects of physiological levels of reactive oxygen species (ROS) and activated human neutrophils. This was achieved using WIL2-NS cells, a human B lymphoblastoid cell line, as target cells and the cytokinesis-block micronucleus (CBMN) assay. With this method, we observed a 4- and a 30-fold increase in the frequency of micronucleated binucleated cells (MNed BNC) when cells were exposed to 10 and 30 µM hydrogen peroxide, for 1 h, respectively. A dose-dependent increase in the frequency of MNed BNC was also detected when cells were exposed to hypoxanthine (HX)/xanthine oxidase (XO), a superoxide generating system: a 50-fold increase in the frequency of MNed BNC was observed at the highest XO dose (12.5 mU/ml). In this CBMN assay, nucleoplasmic bridges (NPB) in BNC and necrotic cells were also readily detected, especially at the higher exposure doses of hydrogen peroxide or HX/XO. When WIL2-NS cells were exposed to neutrophils stimulated with phorbol 12-myristate acetate (PMA) for 1 h, the frequencies of MNed BNC in WIL2-NS cells increased in a dose-dependent manner (30-fold increase at 100 nM PMA) and with an increasing neutrophil:WIL2-NS co-culture ratio. The frequencies of MNed BNC were closely related to the production of ROS, especially hydrogen peroxide, by the neutrophils. Differentiated HL60 cells (DMSO-treated HL60) also produced ROS in response to PMA. In this case, we used a `Transwell' system to expose WIL2-NS cells to DMSO-treated HL60 cells, because direct contact with DMSO-treated HL60 cells impaired cell division in WIL2-NS target cells. Exposure to PMA-stimulated DMSO-treated HL60 cells resulted in a PMA dose-dependent increase in the frequency of MNed BNC in WIL2-NS cells. MNed BNC frequencies were positively correlated with NPB (r = 0.61–0.93) and necrosis (r = 0.55–0.86) and negatively correlated with nuclear division index (r = –0.72 to –0.91) in all of the above experiments. These results suggest that the CBMN assay using WIL2-NS cells is a sensitive assay system to examine ROS-induced chromosomal damage and necrosis by activated human neutrophils.
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Introduction |
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Reactive oxygen species (ROS), such as hydrogen peroxide and superoxide, induce oxidative damage in DNA and lipids, events that are implicated in the process of aging and cancer (Ames et al., 1993
). ROS are formed not only by treatment with radiation and chemicals, but also by inflammation, a common physiological response in our bodies to infection, antigen challenge and physical tissue damage (Winrow et al., 1993). In the inflammatory state, neutrophils are activated and produce ROS that damage DNA (Frenkel et al., 1986), lipoprotein (Abdalla et al., 1992) and cause cytotoxicity (Weiss, 1980; Schacter et al., 1988; Schraufstatter et al., 1988). DNA damage by inflammation has been suggested as an important causal event in cancer (Rosin et al., 1994). Therefore, studies on neutrophil activation and DNA damage performed at the cellular level would be appropriate to obtain some insight into the mechanism and extent of DNA damage caused by activated neutrophils. Development of practical methods for this purpose are needed to examine modifying factors such as environmental chemicals (Tuo et al., 1999) and allergens (Herrstrom et al., 1998). Recent preliminary results suggest that allergic reactions may cause chromosome damage in human B lymphocytes (Herrstrom et al., 1998).
Neutrophil-induced DNA damage has already been studied in experiments performed at the cellular level, using a DNA microfiltration assay in which DNA in target cells is radioactively labeled before oxidative challenge (Leanderson et al., 1994; Hahn et al., 1997). However, radioactive labeling may itself induce chromosomal damage and therefore confound the results obtained (Fenech and Morley, 1985). An alternative approach is to use the cytokinesis-block micronucleus (CBMN) assay, a reliable and established method to detect chromosome damage (Fenech, 1993). The CBMN assay, unlike the 8-hydroxydeoxyguanosine (8-OHdG) assay (Kasai, 1997), does not indicate the precise molecular nature of oxidative damage to DNA, however, it is very sensitive to the DNA-damaging effects of oxidants. For example, the CBMN assay has been reported to be about five times more sensitive in detecting DNA damage than the 8-OHdG assay in experiments with X-rays, a well-established method for generating oxidative stress (Kobus et al., 1993). In addition, the CBMN method is a multiple end-point assay because it can be effectively used to measure mitotic delay, apoptosis, necrosis and chromosomal damage simultaneously (Fenech, 1997; Kirsch-Volders et al., 1997; Fenech et al., 1999). Furthermore, assays for modified bases in DNA are unable to assess the overall effects of oxidative stress on mitosis and chromosome structure, but this is achievable with the CBMN assay.
The CBMN assay is based on the blocking of cytokinesis by cytochalasin B (cyt-B) so that chromosome damage events are scored only in dividing cells, which, unlike non-dividing cells, are cells that can express micronuclei produced as a result of chromosome breakage or damage to the mitotic apparatus (Fenech, 1993, 1997). The use of cyt-B is important as it eliminates the confounding effect of altered cell division kinetics on micronucleus (MN) expression (Fenech, 1997). In some specific neoplastic cell lines (e.g. EL4 and BW5147 cells) cyt-B may increase the baseline rate of DNA damage (Kolber et al., 1990), but this is not the case for normal human lymphocytes and commonly used mammalian cell lines (Fenech, 1997; Kolber et al., 1990; Prosser et al., 1988; Philpott and Buehring, 1999). The CBMN assay has so far been successfully applied to normal human lymphocytes, mouse spleen lymphocytes, mouse fibroblasts and Chinese hamster fibroblasts (Fenech, 1997). The human lymphocyte CBMN assay is a well-established system, however, a constant supply of lymphocytes for the assay is not always practical. In addition, different intakes of antioxidants by volunteers may result in modified responses of the lymphocytes to ROS (Umegaki et al., 1994; Duthie et al., 1996). Peripheral blood lymphocytes are suitable for treatment in G0 phase, which is a stage less sensitive to DNA damage than the S phase of the cell cycle (Duell et al., 1995), and cell lines are more appropriate for testing during S phase as it is relatively easy to obtain cells during the exponential phase of growth when a large proportion of cells are undergoing DNA synthesis. The use of animal cell lines is problematical in terms of species differences in response to DNA damage (Singh and Gupta, 1985). Accordingly, it seemed necessary to develop the CBMN assay using a human cell line which is sensitive to ROS.
In this study, we used WIL2-NS cells for the CBMN assay to detect chromosomal damage induced by hydrogen peroxide, superoxide, human neutrophils and differentiated HL60 cells. The latter is an accepted model for the study of human neutrophil activation (Takeuchi et al., 1994). WIL2-NS cells are a human B lymphoblastoid cell line isolated from the spleen of a Caucasian male (American Type Culture Collection, 1992) which has been reported to have a high mutational response to X-irradiation (Amundson et al., 1993). This cell line is ideal for the CBMN assay because of its high nuclear division index and the excellent morphology of the cells which facilitates MN scoring.
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Chemicals
Horseradish peroxidase, phorbol 12-myristate acetate (PMA), ferricytochrome c, superoxide dismutase (SOD), glutamine, hypoxanthine (HX), xanthine oxidase (XO), cyt-B and dimethylsulfoxide (DMSO) were purchased from Sigma (St Louis, MO). Ficoll-Paque and Dextran T-500 were obtained from Pharmacia Biotech (Uppsala, Sweden). RPMI 1640 medium, Hanks balanced salt solution (HBSS), fetal bovine serum (FBS) and antibiotics solution (5000 U/ml penicillin and 5 mg/ml streptomycin) were obtained from Trace Biosciences (Australia). Diff-Quik was purchased from Lab Aids (Australia) and hydrogen peroxide from Ajax (Australia). The WIL2-NS (ATCC no. CRL8155) and HL60 (ATCC no. CCL240) cell lines were obtained from the laboratories of Prof. A.A.Morley and Dr B.Sanderson (Flinders Medical Centre, Bedford Park, Australia).
Cell culture and cytokinesis-block micronucleus assay
WIL2-NS cells were cultured and maintained in RPMI 1640 medium containing 5% FBS, 2% antibiotics solution and 2 mM glutamine at 37°C in a humidified atmosphere with 5% CO2 (CO2 incubator). One day before the assay, the cells were seeded at a density of 0.3x106 cells/ml. On the assay day, the WIL2-NS cells were washed once with HBSS by centrifugation at 180 g for 5 min, resuspended in HBSS and used for respective exposure studies.
After an exposure treatment, WIL2-NS cells were washed with HBSS and RPMI 1640, then resuspended in RPMI 1640 medium containing 10% FBS, 2% antibiotics solution, 2 mM glutamine, and 4.5 µg/ml cyt-B at a cell density of ~0.5x106 cells/ml. After a predetermined culture time, the cells were harvested. Slides were prepared using a cytocentrifuge (Shandon Southern Products. Cheshire, UK). Before cytocentrifugation, DMSO was added at a final concentration of 5% to minimize clumping of the cells and thus optimize recognition of cytoplasmic boundaries. The slides were air dried for 10 min and then fixed and stained using Diff-Quik.
Micronuclei in BN cells (Figure 1A) were scored using established criteria (Fenech, 1993, 1996). Inclusion of apoptosis and necrosis scoring in the CBMN assay was performed as described (Fenech et al., 1999). Briefly, the following guidelines for scoring necrotic and apoptotic cells were used: (i) cells showing chromatin condensation with intact cytoplasmic and nuclear boundaries as well as cells exhibiting nuclear fragmentation into smaller nuclear bodies within an intact cytoplasm/cytoplasmic membrane were classified as apoptotic; (ii) cells exhibiting a pale cytoplasm with numerous vacuoles and/or damaged cytoplasmic membrane and/or loss of cytoplasm with a fairly intact nucleus were classified as necrotic. Cells in the late stages of necrosis (i.e. exhibiting only a disintegrating nuclear structure with no cytoplasm) were not scored as they were difficult to distinguish from other cellular debris on the slide.
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A total of 400 cells [mononucleated, binucleated (BNC), other multinucleated, apoptotic and necrotic cells] were scored to determine cell ratios and 1000 BNC were scored for micronuclei and nucleoplasmic bridges (NPB) (Figure 1
). NPB were scored because they represent dicentric chromosome bridges, thus providing a measure of chromosome rearrangement (Fenech, 1997). Chromosomal damage rates are expressed as the number of micronucleated binucleate cells (MNed BNC) per 1000 BNC and NPB per 1000 BNC. The nuclear division index (NDI) was calculated by the formula suggested by Eastmond and Tucker (1989) using only viable cells to determine the ratios (i.e. excluding necrotic and apoptotic cells). Necrosis is expressed as a percentage of total cell numbers. In untreated WIL2-NS cells, 3–4% were not viable as assessed by trypan blue exclusion; these cells may have been spontaneous necrotic cells. All assays were performed in triplicate, i.e. for each dose tested, three cell suspensions were treated separately with ROS or activated neutrophils and each of these cell suspensions provided a separate culture for MN assay. The results shown, unless otherwise indicated, represent the means ± SE for these triplicate experiments.
Exposure of cells to hydrogen peroxide and superoxide
WIL2-NS cell suspension (0.95 ml, 0.5x106 cells/ml) in HBSS was pre-incubated for 10 min at 37°C in a CO2 incubator, then exposed to 50 µl of hydrogen peroxide solution or superoxide generating system (HX/XO) for 1 h. Superoxide generation by HX/XO was linear with concentration of XO when the concentration of HX was 25 µM. Therefore, exposure of WIL2-NS cells to increasing doses of superoxide was performed using different concentrations of XO in the presence of 25 µM HX. After exposure, 2 ml of HBSS were added to the cell suspension. The latter was centrifuged at 180 g for 5 min and the cells washed twice with HBSS and RPMI 1640, respectively, then cultured in the presence of cyt-B.
In our preliminary experiments, we used HBSS containing HEPES buffer when WIL2-NS cells were exposed to ROS. Exposure to HEPES markedly impaired the formation of BNC during the cytokinesis-block (CB) culture and many necrotic cells were detected. Even when cells were extensively washed with HBSS without HEPES after exposure to HEPES, we observed high frequencies of MNed BNC (~20 MNed BNC/1000 BNC) in untreated samples. For this reason, we did not use HEPES in this study.
Exposure of cells to PMA-stimulated neutrophils
Blood was obtained from healthy volunteers using lithium heparin-coated tubes. Neutrophils were isolated using the Dextran T-500 method (Rollet-Labelle et al., 1998). Briefly, blood was mixed with 0.5 vol of 2% Dextran T-500 in saline and allowed to stand at room temperature for 20 min to sediment erythrocytes. The leukocyte-rich plasma was overlayed on Ficoll-Paque (0.5 vol of the plasma fraction) and centrifuged at 400 g for 20 min. The resulting cell pellet at the bottom of the tube was treated with 0.2% NaCl for 40 s to destroy residual erythrocytes and then an equal amount of 1.6% NaCl was added to restore normal osmolarity. The resulting suspension of neutrophils was washed twice with HBSS and used for the assay. Cell viability, as checked by the trypan blue method, was >99%.
Suspensions of WIL2-NS cells (0.96 ml, 1x106 cells/ml) and neutrophils (0.96 ml, 2x106 cells/ml) in HBSS were mixed in a 5 ml culture tube and preincubated for 10 min at 37°C in a CO2 incubator. To stimulate neutrophils, 80 µl of PMA were added and the cells incubated for another 1 h. After incubation, the cell suspension was underlayed with 1 ml of Ficoll-Paque and centrifuged at 400 g for 20 min. In this procedure, neutrophils precipitated to the bottom of the tube and WIL2-NS cells stayed at the surface of the Ficoll-Paque. The WIL2-NS cells were carefully transferred to another tube, washed twice with HBSS followed by one wash with RPMI 1640 medium and then cultured using the CB method.
Exposure of DMSO-treated HL60 cells
DMSO-treated HL60 cells were prepared according to the method of Collins et al. (1978). Briefly, HL60 cells were cultured in RPMI 1640 medium containing 1.3% DMSO, 10% FBS, 2% antibiotics and 2 mM glutamine at 37°C in a humidified atmosphere with 5% CO2. After 7 days culture, the cells were harvested, washed twice with HBSS and used for an experiment. After culture in DMSO-supplemented medium, HL60 cells differentiate to a neutrophil-like morphology and are henceforth referred to as DMSO-treated HL-60 cells. Exposure of WIL2-NS cells to DMSO-treated HL60 cells was performed using a Transwell (12 mm insert diameter and 3 µm pore size; Corning Cortstar, Cambridge, MA). DMSO-treated HL60 cells (2x106 cells/ml, 1.42 ml) were placed in the bottom well and WIL2-NS cells (1x106 cells/ml, 0.5 ml) in the top well and they were preincubated for 10 min at 37°C. DMSO-treated HL60 cells were stimulated by adding PMA (80 µl) to the bottom well of the Transwell and incubated together with the WIL2-NS cells (in the top well) for 1 h at 37°C in a CO2 incubator. After the incubation, WIL2-NS cells were collected from the top well and transferred to a culture tube, washed twice with HBSS and RPMI 1640 medium, then cultured using the CB method.
Analysis of hydrogen peroxide and superoxide production by neutrophils and DMSO-treated HL60 cells
Production of hydrogen peroxide was evaluated using the phenol red method (Pick and Keisari, 1980). Briefly, a cell suspension of neutrophils or DMSO-treated HL60 cells (1x106 cells/ml, 0.96 ml) containing horseradish peroxidase (50 µg/ml) and phenol red (100 µg/ml) was stimulated with 40 µl of PMA for 30 min at 37°C. After incubation, the cell suspension was centrifuged at 10 000 g for 10 s, then 950 µl of the supernatant was immediately transferred to a tube containing 66 µl of 1 N NaOH to adjust the pH to 12.5. The absorbance of the mixture was measured at 610 nm. Production of superoxide was determined by the cytochrome c method (Takeuchi et al., 1994). Briefly, a cell suspension of neutrophils or DMSO-treated HL60 cells (1x106 cells/ml, 0.96 ml) containing cytochrome c (50 µM) in the presence or absence of SOD (20 µg/ml) was stimulated with 40 µl of PMA for 10 min at 37°C. After incubation, the cell suspension was immediately centrifuged at 10 000 g for 10 s and the absorbance in the supernatant was measured at 550 nm. Under these conditions, SOD completely prevented the increase in absorbance due to PMA stimulation.
Statistical analysis
The data are presented as means ± SE. Statistical analysis of the data was carried out using ANOVA followed by a post hoc test of Fisher's protected least significant difference. In the study of hydrogen peroxide and HX/XO exposure, Student's t-test was also applied to determine the significance of increases in MNed BNC in treated cultures relative to control cultures. Correlation factors were calculated using Spearman's method. A P value <0.05 was considered to be significant. The statistical analyses were performed using a computer program (StatView 4.5, Abacus Concepts, CA, USA and Prism 2.1 GraphPad Inc., San Diego, USA).
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Results |
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Optimal culture time for the cytokinesis-block assay
Untreated WIL2-NS cells divide quickly with ~65% becoming BNC after 28–42 h culture in the presence of 4.5 µg/ml cyt-B. The parameters (mean ± SE) of the CBMN assay in seven untreated WIL2-NS cultures performed on different days were 2.38 ± 0.08 for NDI, 61.0 ± 3.2 for BNC (%), 7.4 ± 0.5 for MNed BNC/1000 BNC. In the same cultures the frequency of mononucleated cells was 9.7 ± 1.6%, indicating that ~90% of the cells had divided during the 42 h cyt-B culture treatment and that the use of cyt-B did not impair nuclear division.
However, preliminary experiments indicated that NDI and BNC (%) were markedly decreased when the cells were exposed to hydrogen peroxide before CB culture. Therefore, in the next experiment relationships between the parameters of the CBMN assay and culture time were examined in WIL2-NS cells exposed to 20 µM hydrogen peroxide for 1 h. As shown in Figure 2, NDI and BNC (%) increased with increasing culture time. At 42 h after culture, BNC (%) increased to ~50%. The frequency of MNed BNC also increased significantly with culture time, achieving a maximum at 42 h. In an earlier experiment with untreated control cultures of WIL-NS cells we observed an increase in NDI from 2.0 to 2.9, a decline in BNC (%) from 75.5 to 35.0 and an increase in MNed BNC frequency from 4.5 to 10.5 when harvest time was increased from 24 to 44 h after cyt-B addition. As chromosomal damage is evaluated by the frequencies of MNed BNC, culture conditions that maximize the number of BNC in the ROS-treated cultures are preferable for efficient scoring of slides. In view of the above results a culture time of 42 h in the presence of cyt-B was considered optimal for the CBMN assay in this system.
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Effect of hydrogen peroxide and superoxide
WIL2-NS cells were exposed to various concentrations of hydrogen peroxide (0, 3, 10, 20 and 30 µM) for 1 h and the influence of the exposure on parameters of the CBMN assay was determined (Table I
). The values of NDI and BNC (%) decreased and those of MNed BNC and necrosis increased in a dose-dependent manner. Appearance of NPB in BNC (as shown in Figure 1B) also increased at the higher doses of hydrogen peroxide. Apoptotic cells were rarely observed even at the highest dose of hydrogen peroxide. A significant increase in MNed BNC was detected at 10 µM hydrogen peroxide. With exposure to 30 µM hydrogen peroxide, ~20% of the MNed BNC had more than two MN within a cell. There was a linear relationship between log(MNed BNC) and hydrogen peroxide (Figure 3).
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WIL2-NS cells were exposed to superoxide generated by the HX/XO system. An exposure to various concentrations of XO (0, 0.3, 1, 3 and 12.5 mU/ml) in the presence of HX (25 µM) for 1 h decreased NDI and BNC (%) and increased MNed BNC, NPB in BNC and necrosis in an XO dose-dependent manner (Table II
). Production of superoxide by HX (25 µM)/XO (3 mU/ml) in a cell-free system was ~0.6 µmol/min. As in the hydrogen peroxide experiment, apoptotic cells were not induced by HX/XO exposure. A significant increase in MNed BNC frequency was detected at 3 mU/ml XO, when the data were analyzed by ANOVA followed by a post hoc test. When statistical analysis of the data was performed using the t-test, a significant 3-fold increase over controls in MNed BNC was observed at 1 mU/ml XO. There was a linear relationship between log(MNed BNC) and XO dose up to 3 mU/ml (Figure 4).
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Effect of exposure to PMA-activated human neutrophils
When neutrophils were stimulated with various concentrations of PMA (0, 0.3, 1, 3, 10, 30 and 100 nM), they produced hydrogen peroxide and superoxide (Figure 5
). The production of hydrogen peroxide and superoxide was significantly increased and reached a plateau at the PMA dose of 10 nM. WIL2-NS cells were exposed for 1 h to neutrophils activated by various concentration of PMA and the parameters of the CBMN assay were determined (Table III). NDI and BNC (%) were significantly decreased at PMA doses of 3 nM and higher. MNed BNC, necrosis and NPB increased in a PMA dose-dependent manner up to 10 nM, then reached a plateau. The frequencies of MNed BNC in WIL2-NS cells correlated positively with increased production of hydrogen peroxide and superoxide in neutrophils (Figure 6). Interestingly, the relationship between hydrogen peroxide production and MNed BNC was linear (correlation coefficient = 0.99). At the higher PMA dose, ~50% of the cells were detected as undivided necrotic cells. To examine the PMA effect per se on WIL2-NS cells, a CBMN assay was performed in a separate experiment. Exposure to PMA up to 100 nM did not increase MNed BNC [7.3 ± 1.3 (n = 3) in control and 6.3 ± 0.9 (n = 3) in PMA-treated cultures] and did not affect necrotic cell frequency [1.5 ± 0.1 (n = 3) in control and 1.8 ± 0.1 (n = 3) in PMA-treated cultures]. In addition, WIL2-NS cells did not produce hydrogen peroxide and superoxide in response to PMA (data not shown).
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In the next experiment, WIL2-NS cells were exposed to different concentrations of neutrophils to examine the effect of the ratio of neutrophils to WIL2-NS cells because a higher ratio was expected to increase exposure to ROS. The neutrophils were stimulated with 3 nM PMA for 1 h. When the ratio of neutrophils to WIL2-NS cells was between 0.5 and 2.0, the parameters of the CBMN assay changed in a cell ratio-dependent manner (Table IV
). However, when the cell ratio was 4.0, NDI and BNC (%) increased and MNed BNC and NPB decreased. These changes were probably related to the appearance of large numbers of necrotic cells under these exposure conditions.
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Effect of exposure to PMA-activated DMSO-treated HL60 cells
Unlike the experiments with normal neutrophils, separation of WIL2-NS cells and DMSO-treated HL60 cells using a Ficoll-Paque gradient was unsuccessful because both types of cells remained at the surface of the Ficoll-Paque layer after centrifugation. In addition, intimate contact with DMSO-treated HL60 cells during the CBMN assay inhibited nuclear division of WIL2-NS cells. Therefore, in this case separation of DMSO-treated HL60 cells was required before the CB culture of WIL2-NS cells. For these reasons, exposure of DMSO-treated HL60 cells to WIL2-NS cells was performed using a Transwell system. DMSO-treated HL60 cells produced hydrogen peroxide and superoxide in response to PMA in a dose-dependent manner (Figure 7
). Exposure of PMA-activated DMSO-treated HL60 cells to WIL2-NS cells decreased NDI and increased BNC (%), MNed BNC, necrotic cells and NPB in a PMA dose-dependent manner (Table V). When diffusion of hydrogen peroxide from the bottom to the top of the Transwell was checked in cell-free conditions, the concentration in the top well was 50, 86 and 100% of the equilibrium level at 15, 30 and 60 min, respectively.
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Correlations of cystostatic effect, necrosis and NPB with MNed BNC
Using the results from Tables I–V
we estimated the correlation factors of the various measures of cytotoxicity with MNed BNC (Table VI). These results show that MNed BNC in WIL2-NS cells is strongly negatively correlated with NDI and frequency of BN cells and strongly positively correlated with necrosis and NPB following exposure to hydrogen peroxide, superoxide and activated normal human neutrophils. The results for exposure of WIL2-NS cells to DMSO-treated HL60 cells were somewhat different, exhibiting a positive correlation with frequency of BN cells and weaker positive correlations with necrosis and NPB. The correlation factors obtained for data on exposure to activated human neutrophils appeared to best match those obtained for hydrogen peroxide.
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Discussion |
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It has been suggested that chronic inflammation may be related to cancer at sites of infection and tissue damage (Rosin et al., 1994
). In the inflammation process neutrophils are activated and produce ROS, which induce oxidative damage in DNA, proteins and lipids of bystander normal cells that are not necessarily the target of the immune response (Weiss, 1980; Frenkel et al., 1986; Schacter et al., 1988; Schraufstatter et al., 1988; Abdalla et al., 1992). In evaluating the data obtained in our study it is important to note that the levels of H2O2 used (3–30 µM) are physiologically relevant with regard to inflammatory processes as it has been shown that 25–50 µM is bacteriostatic and the concentration of H2O2 at abscess sites in subcutaneous infections is approximately 100 µM (Hyslop et al., 1995). For comparison, plasma H2O2 levels in normotensive and hypertensive subjects were estimated to be 2.5 and 3.2 µM, respectively (Lacy et al., 1998). It is also important to note that similar levels of micronucleated cells are induced when lymphocytes are exposed to 500 µM H2O2 in plasma or 100 µM H2O2 in culture medium (Fenech et al., 1997; Crott and Fenech, 1999), suggesting the presence of a much higher capacity of plasma to neutralize H2O2. Experiments using a human cell system would be appropriate for clearly understanding the mechanism of neutrophil-mediated oxidative damage to DNA and for investigating factors that modify the harmful effects of cells involved in inflammation. The CBMN assay is a genotoxicity assay method that simultaneously provides many complementary parameters, namely NDI, necrosis, apoptosis and a variety of chromosomal damage end-points (e.g. chromosome loss, chromosome breakage and nucleoplasmic bridges, a measure of chromosomal rearrangement) (Fenech, 1997; Kirsch-Volders et al., 1997; Fenech et al., 1999). As shown in the results of this study, WIL2-NS cells are well suited for the study of the DNA damaging effects of ROS using the CBMN assay because: (i) the baseline MN frequency in the presence of cyt-B is low and within the expected range for normal human B lymphocytes, confirming their appropriateness as a surrogate for normal B cells (Herrstrom et al., 1998; Wuttke et al., 1993); (ii) the cells are very sensitive to expression of MN when exposed to physiological levels of ROS.
Lymphocytes have been shown to be more sensitive to the DNA-damaging effects of ROS when compared with other cell types (Schraufstatter et al., 1988). However, comparison with published data suggests that WIL2-NS cells may be more sensitive than normal lymphocytes when exposed to hydrogen peroxide. A 10-fold increase in the frequency of MNed BNC over baseline was obtained at 20 µM hydrogen peroxide in this study (Table I) but an exposure to 128 µM hydrogen peroxide, under similar conditions, was required to produce a similar increment in MNed BNC when normal lymphocytes were exposed in the G0 phase (Fenech et al., 1997). This difference in sensitivity could be explained by the fact that WIL2-NS cells were exposed during the sensitive S and G2 phases of the cell cycle and that lymphocytes in the G0 phase of the cell cycle are not sensitive to DNA damage. In fact, in recent experiments, integrating measures of necrosis, apoptosis, cytostasis and MN formation, we have shown that the main event induced by hydrogen peroxide in G0 lymphocytes is necrosis and not MN formation (Fenech et al., 1999). Furthermore, unlike phytohemagglutinin-stimulated lymphocytes, which are mainly T cells, WIL2-NS cells are a B cell lymphoblastoid cell line. B cells have been shown to be more sensitive than T cells to X-irradiation (Wuttke et al., 1993). An additional explanation can be derived from the observation that we could not detect appreciable numbers of apoptotic cells when WIL2-NS cells were exposed to either hydrogen peroxide or superoxide or PMA-stimulated neutrophils or DMSO-treated HL60 cells. This fact may be related to mutation of the p53 gene in this cell line (Xia et al., 1995), which may allow cells that would normally be eliminated by apoptosis (prior to completing nuclear division) to proceed through the cell cycle and express their chromosome damage as MN in the accumulated BN cells. All of the above facts may explain the high response of WIL2-NS cells to DNA damage by ROS.
MN are formed during nuclear division. Therefore, factors which influence WIL2-NS nuclear division impair the ability to utilize the MN assay. Impaired expression of MN in WIL2-NS cells was observed in the neutrophil experiment shown in Table IV, i.e. when the ratio of neutrophils to WIL2-NS cells was 4:1, NDI and BNC (%) increased markedly but MNed BNC frequency decreased. This finding suggests that when cells are damaged due to direct contact with more than one activated neutrophil, they become necrotic instead of forming MNed BNC. These findings led us to speculate that extremely damaged cells do not survive to complete nuclear division during CB culture, but instead proceed to become necrotic cells. In any case, simultaneous evaluation of necrosis, cytostasis, apoptosis and the frequencies of MNed BNC is important for performing and interpreting the results of the CBMN assay, as we have suggested recently (Fenech et al., 1999).
Co-culture of WIL2-NS cells with other cells during CB culture may also interfere with the CBMN assay. In the DMSO-treated HL60 cell experiment, many WIL2-NS cells became necrotic if they were in direct contact with DMSO-treated HL60 cells during the CB culture. This finding seemed to be unrelated to ROS production, because necrosis was also observed with DMSO-treated HL60 cells that were not PMA-stimulated. For these reasons, intimate contact between DMSO-treated HL60 and WIL2-NS cells is to be avoided for successful co-culture. The exposure of PMA-activated DMSO-treated HL60 cells to WIL2-NS cells in the Transwell was satisfactory, although the DNA damage response was weak when compared with that of the neutrophil experiment (Tables III–V). Cell cycle kinetics in WIL2-NS cells appear to have been altered differently by exposure to DMSO-treated HL60 cells when compared with neutrophils, because with the latter both NDI and BNC (%) decreased with PMA dose increment but with DMSO-treated HL60 cells NDI decreased but BNC (%) increased. The weak MN response with DMSO-treated HL60 cells might be related to a slow diffusion of ROS between the two wells in the Transwell and with reduced production of ROS in the DMSO-treated HL60 cells in response to PMA. Nevertheless, the Transwell method is still an effective alternative way to study the DNA-damaging effect of cells that generate ROS on co-cultured target cells.
In the CBMN assay, chromosomal damage is evaluated by the frequency of MNed BNC. Accordingly, the culture time that yields the highest BNC ratio allows for more rapid scoring as much less time is spent searching for suitable BNCs in which to score MN. In the case of WIL2-NS cells, 42 h culture in the presence of cyt-B is optimal for this purpose and it also corresponds to the optimal expression time for MN. The 42 h culture time also has practical import for performance of the assay as the culture can be commenced in the late afternoon and cells harvested 2 days later in the morning. It is not clear why MNed BNC increased with increasing culture time after exposure. However, it is reasonable to speculate that the more the cells are damaged, the more time is necessary for them to recover/repair and divide to form MNed BNC, which is suggested by the trend for higher BNC (%) and NDI with increased culture time (Figure 2). Similar findings were reported by Mitchell and Norman (1987) and Scott et al. (1998) in experiments with X-irradiated lymphocytes. Falck et al. (1997) also reported that spontaneous frequencies of MNed BNC in human lymphocytes increased marginally with culture time, although the increase in this case was mainly due to centromere-positive MN.
Previous studies have shown that PMA-stimulated neutrophils release superoxide and hydrogen peroxide and result in DNA damage to the neutrophil itself and neighboring cells (Weiss, 1980; Schacter et al., 1988; Schraufstatter et al., 1988; Takeuchi et al., 1994). It was also shown that DNA damage is prevented by catalase, but not SOD, indicating that hydrogen peroxide is responsible for the observed DNA damage (Schacter et al., 1988; Takeuchi et al., 1994). It has been known that hydrogen peroxide can easily penetrate membranes and the nucleus, where it has a high probability of being converted to hydroxyl radical in the presence of reduced transition metals (Halliwell and Gutteridge, 1985). In our experiment, production of superoxide in neutrophils in response to PMA was higher than that of hydrogen peroxide (Figure 5). Although chromosomal damage in WIL2-NS cells is in proportion to the amount of both hydrogen peroxide and superoxide generated in neutrophils, a linear relationship (r = 0.99) was observed in the case of hydrogen peroxide (Figure 6). These findings, together with the very similar correlation factors between MNed BNC and other cytotoxicity markers (Table VI), suggest that hydrogen peroxide is a major cause of chromosomal damage in WIL2-NS cells induced by exposure to neutrophils and DMSO-treated HL60 cells.
An additional important aspect of this study is the use of NPB as a biomarker of DNA damage induced by ROS. The strong positive correlation between NPB and MNed BNC strengthens the validity of the assumption that NPB represent dicentric chromosomes (whose centromeres are being pulled apart to opposite poles of the cell) because single- and double-strand DNA breaks (and their misrepair and/or misreplication) induced by ROS are expected to result in the production of both acentric fragments and dicentric chromosomes (IAEA, 1986). In addition, acentric chromosomes (which lead to MN formation) are expected to be a by-product of dicentric chromosome formation (IAEA, 1986), which explains why a large proportion (>50%) of binucleated cells with NPB also contain MN (data not shown). The use of the NPB biomarker increases the usefulness of the CBMN assay as a comprehensive test system of chromosome damage because NPB can only be effectively observed in binucleate cells. Chromosome painting studies are needed to confirm the hypothesis that NPB represent dicentric chromosomes.
In conclusion, the CBMN assay using WIL2-NS cells is an effective system to detect ROS-induced chromosomal damage. Using this experimental system, it would be relatively easy to examine the mechanism of activated neutrophil-induced chromosomal damage and modifying factors such as environmental chemicals, food components and allergens.
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We greatly appreciate the technical support provided by Philip Thomas. This study was financially supported by the Human Science Foundation of Japan.
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* To whom correspondence should be addressed. Tel: +618 8303 8880; Fax: +618 8303 8899; Email: michael.fenech{at}hsn.csiro.au
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