Mutagenesis, Vol. 14, No. 6, 605-612,
November 1999
© 1999 UK Environmental Mutagen Society/Oxford University Press
Necrosis, apoptosis, cytostasis and DNA damage in human lymphocytes measured simultaneously within the cytokinesis-block micronucleus assay: description of the method and results for hydrogen peroxide
CSIRO Human Nutrition, PO Box 10041, Adelaide BC, SA, Australia 5000
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
A method is described for the inclusion of apoptotic and necrotic cells in the cell counts obtained in the cytokinesis-block micronucleus (CBMN) assay, which is conventionally used solely for the assessment of chromosome breakage, chromosome loss and frequency of dividing cells. The morphological criteria for the recognition and discrimination between necrotic, apoptotic and viable cells are described. Using this comprehensive method we have evaluated the cytotoxic and genotoxic effects of hydrogen peroxide (0–100 µM) in lymphocytes exposed in RPMI 1640 medium. The results obtained indicated significant (P < 0.05) correlations between hydrogen peroxide concentration and the frequency of micronucleated cells (r = 0.39), necrotic cells (r = 0.73), apoptotic cells (r = –0.26) and binucleated cells (r = –0.55). Almost similar results were obtained using the cytosine arabinoside modification of the CBMN assay, which enables excision-repaired sites to be converted to micronuclei. Some of the above end-points were significantly (P < 0.05) correlated with each other (necrosis and apoptosis, R = –0.39; necrosis and micronucleated cell frequency, R = 0.46; necrosis and binucleated cells, R = –0.78; apoptosis and binucleated cells, R = 0.32). It was therefore necessary to use multiple regression analysis to identify the main event induced by hydrogen peroxide, which was necrosis (ß = 0.57, P = 0.0001) and not micronucleus formation (ß = 0.15, P = 0.1332). Using an ELISA assay we showed that hydrogen peroxide did not induce 8-hydroxydeoxyguanosine. Our data show that the proposed comprehensive test system may provide a better procedure for classifying potential toxic chemicals and enable discrimination between agents that primarily induce cytotoxic effects as opposed to genotoxic effects. The integration of apoptosis and necrosis into the micronucleus assay may also be of practical use in radiosensitivity studies.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
The cytokinesis-block micronucleus (CBMN) assay has become one of the most commonly used methods for assessing chromosome breakage and loss in human lymphocytes both in vitro and ex vivo (Fenech and Morley, 1985
; Fenech, 1997; Fenech et al., 1999). The method has the unique advantage that it is a multi-end-point assay for measuring the induction of acentric chromosome fragments and chromosome loss which can be distinguished by kinetochore or centromere detection using molecular methods (Kirsch-Volders et al., 1997; Schuler et al., 1997). The use of cytochalasin B not only makes the technique more precise by restricting scoring to cells that have completed one nuclear division but it also enables the frequency of dividing cells to be quantified rapidly (Fenech, 1997). The use of cytosine arabinoside during the first 16 h of culture converts excision repair sites to micronuclei (MN) within one cell cycle so that excision-repairable adducts can also be measured (Fenech and Neville, 1992). Recently it has been noted that one may also quantify apoptotic cells in this system using morphological criteria (Kirsch-Volders et al., 1997; Crott and Fenech, 1999).
During the past 10 years it has become increasingly evident that DNA or chromosome damage is only one of several critical events that happen following exposure to xenobiotic agents (Evan and Littlewood, 1998; Green and Reed, 1998). Furthermore, the probability of a cell surviving exposure to a DNA-damaging agent (endogenous or exogenous) is dependent on the propensity of that cell to undergo programmed cell death or apoptosis. A cell may also undergo necrosis rather than apoptosis depending on the intracellular oxidant/antioxidant status, the level of ATP in the cell and the extent of induced membrane damage (Hampton and Orrenius, 1997; Green and Reed, 1998; Lelli et al., 1998; Samali et al., 1999). Figure 1 describes the various pathways and events that may be expected to occur in cultured lymphocytes exposed to a toxic agent. Cytogenetic genotoxicity assays that require hypotonic treatment for the preparation of interphase cells (for the whole blood MN assay) or metaphase plates for chromosome analysis are not usable for cytotoxicity assays because hypotonic treatment may destroy necrotic cells and apoptotic cells, making them unavailable for assay. Inclusion of these parameters is important for the accurate description of the mechanism of action and measurement of cellular sensitivity to a chemical or radiation.
|
In contrast, the isolated lymphocyte culture assay does not require hypotonic treatment of cells for slide preparation, thus making it possible to preserve the morphology of both necrotic and apoptotic cells. The use of cytochalasin B should make it easier to score apoptotic cells because it is expected to inhibit the disintegration of apoptotic cells into smaller apoptotic bodies. The latter process requires microfilament assembly, which is readily inhibited by cytochalasin B (Carter, 1967
; Atencia et al., 1997). We have attempted to show the value of this fully integrated system by measuring in detail the full complement of cellular events that are visible in cytokinesis-blocked cultured lymphocytes exposed to increasing doses of hydrogen peroxide, an endogenous oxidant. The results from this study show that inclusion of necrosis and apoptosis end-points are crucial to identifying the main event caused by a particular xenobiotic agent. We also provide a detailed description of scoring methods and criteria. |
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Recruitment
The study was advertised to staff and students at CSIRO Human Nutrition. Six healthy, non-smoking males aged between 23 and 30 years were recruited after giving informed consent. The Human Ethics Committee of CSIRO Human Nutrition approved the study.
Collection of blood samples
Between 50 and 80 ml of blood was collected by venipuncture into vacutainer tubes containing lithium heparin as anticoagulant. Blood was taken between 8.30 and 9.30 a.m. after an overnight fast, before having breakfast, to minimize possible confounding effects of dietary factors.
CBMN assay
The CBMN assay was carried out using the protocol of Fenech (1993) with minor adaptations and modifications as described below. The blood sample was diluted with an equal volume of 0.85% isotonic saline and mixed. To isolate the lymphocytes, 12 ml of Ficoll Paque (Pharmacia Biotech, Uppsala, Sweden) was added to a 50 ml Falcon tube and 36 ml of diluted blood was gently overlaid. The tubes were centrifuged at 400 g for 25 min at room temperature. Following the first wash, the tubes were centrifuged at 280 g for 10 min at room temperature. The cell pellet in each Falcon tube was resuspended in 1 ml of RPMI 1640 medium (Trace Biosciences, Nobel Park, Victoria, Australia) without fetal bovine serum (FBS). Cells were counted using a Coulter Counter. Lymphocytes were cultured in round bottom, 10 ml capacity tissue culture grade tubes at a concentration of 1x106 cells/ml in RPMI 1640 medium without FBS. All cultures were prepared in duplicate. The volume of all cultures was 750 µl. Lymphocytes were challenged with four different concentrations of H2O2, i.e. 0, 25, 50 and 100 µM. Hydrogen peroxide dilutions were freshly prepared in Hanks balanced salt solution (Trace Biosciences). The cultures were pre-incubated for 30 min at 37°C and then the appropriate concentration of H2O2 was added to each tube and these were incubated for another 30 min at 37°C. The cell suspensions were centrifuged at 180 g for 2 min, the supernatant was aspirated to a minimal level and cells were resuspended by gentle agitation in the remaining medium. The cells were washed in 3 ml of RPMI 1640 without FBS. Cultures were centrifuged at 180 g for 5 min and the supernatant was aspirated to a minimal level, then the cells were resuspended in 750 µl RPMI 1640 with 10% (v/v) FBS (Trace Biosciences). Lymphocytes were stimulated to divide with 45 µg/ml phytohaemagglutinin (PHA) (Murex Biotech, Dartford, UK) and incubated at 37°C in a humidified atmosphere containing 5% CO2. Forty-four hours post-PHA stimulation, cytochalasin B was added to cultures to a final concentration of 4.5 µg/ml. Cells were harvested at 72 or 96 h after PHA stimulation, by transferring directly to a glass slide using a cytocentrifuge (Shandon Southern Products Ltd, Astmoor, UK). Aliquots of 120 µl of resuspended cells in culture medium per cytocentrifuge cup were spun at 600 r.p.m. for 5 min. The slides were air dried for 10 min, fixed in absolute methanol for 10 min and stained using Diff-Quik stain (Lab Aids, Narrabeen, NSW, Australia). To minimize any confounding effects, one batch of each reagent was used in all assays. For each individual, treatments were done in duplicate and separate cultures were set up for each treatment and harvest time, resulting in a total of 16 cultures/individual and a total of 96 cultures for all the subjects together.
Cytosine arabinoside (ARA) modification of the CBMN assay (CBMN-ARA assay)
Lymphocyte isolation, culture, CBMN assay and hydrogen peroxide challenge were all performed as described in the previous section. ARA cultures were prepared using protocol A described by Fenech and Neville (1992), with the adaptations and modifications described below. All cultures were 750 µl volume and cells were at a concentration of 1x106 cells/ml. ARA (1.0 µg/ml) (Sigma, St Louis, MO) was added to each culture just before PHA stimulation. All incubations were in a humidified atmosphere with 5% CO2 at 37°C. Cells were washed twice with 3 ml of RPMI 1640 without FBS 16 h after PHA stimulation and addition of ARA. Tubes were centrifuged at 180 g for 5 min before washing medium was aspirated to minimize loss of cells. Cells were then set up in 750 µl RPMI 1640 with 10% FBS supplemented with 10 U/ml of human interleukin-2 (Boehringer, Mannheim Australia Pty. Ltd, Rose Park, S. Australia) and 10.0 µg/ml 2'-deoxycytidine hydrochloride (DC) (Sigma). At 44 h post-PHA stimulation, cytochalasin B was added to a final concentration of 4.5 µg/ml. Cells were harvested at 72 or 96 h post-PHA stimulation and fixed/stained as described above. For each individual, treatments were done in duplicate and separate cultures were set up for each treatment and harvest time, resulting in a total of 16 cultures/individual and a total of 96 cultures for all the subjects together.
Scoring method and criteria for MN, cytostasis, necrosis and apoptosis
All slides were coded prior to scoring. Scoring was carried out by a single individual (S.B.) using a Olympus BH-2 light microscope at 1000x magnification under oil immersion. Criteria for scoring MN were as described by Fenech (1993, 1996). ARA-induced MN cells were calculated by subtracting the difference between micronucleated (MNed) cell frequency in ARA-treated cultures and corresponding cultures without ARA treatment.
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 damaged cytoplasmic membrane with a fairly intact nucleus as well as cells exhibiting loss of cytoplasm and damaged/irregular nuclear membrane with a partially intact nuclear structure were classified as necrotic. Figure 2 illustrates the various cell types scored according to the scheme described in Figure 1
. Five hundred cells were counted and scored as either mononucleate, binucleate, trinucleate, tetranucleate, apoptotic or necrotic and ratios for these types of cells were calculated. The number of MN in 1000 binucleated (BN) cells were scored for each treatment and the frequency of MN and MNed cells and distribution of MN in BN cells were calculated. The cell classification and MN counts were obtained by scoring an equal number of cells from cytocentrifuge preparations from duplicate cultures. For some cultures exposed to the higher doses of H2O2 it was not possible to determine the MN and MNed cell frequency as there were too few BN cells due to extensive necrosis.
|
8-Hydroxy-2'-deoxyguanosine (8-OHdG) enzyme-linked immunosorbent assay (ELISA)
Lymphocyte isolation and culture and hydrogen peroxide challenges were all performed as described above. Separate cultures were set up for the ELISA assay. At 24, 48 and 72 h after PHA addition, culture medium samples were taken to assess the extent of excretion of 8-OHdG by the cultured cells. Cytochalasin B was not added to any of these cultures. At the relevant time point, the culture tubes were centrifuged at 300 g for 10 min. Two 250 µl supernatant samples were removed into separate cryovials, frozen in liquid nitrogen and stored at –80°C until required. Culture medium samples were assayed for 8-OHdG using the 8-OHdG Check ELISA from the Japan Institute for the Control of Aging (Fukuroi City, Japan). The ELISA was conducted using the method described by Sri Kantha et al. (1996). It was conducted on polystyrene 96-well flat bottom plates (ImmunoPlate Maxisorb; Nalge Nunc International, Naperville, IL) using the 8-OHdG ELISA kit. A monoclonal antibody (N45.1) specific for 8-OHdG was used in this ELISA with horseradish peroxidase-conjugated anti-mouse polyclonal antibody with substrate o-phenylenediamine. The absorbance was measured at 492 nm using a computerized plate reader (SpectraMax, Sunnyvale CA). The 8-OHdG standards ranged between 0.64 and 2000 ng/ml. The concentration of 8-OHdG in the culture medium samples was determined from a standard curve plotted on a log scale.
Statistical methods
The significance of dose–response effects was evaluated using the non-parametric Kruksall–Wallis ANOVA test and comparisons for results at different doses of H2O2 were done using Dunn's multiple comparison test. To determine whether results for cells harvested at 72 and 96 h were significantly different, we pooled the data for all H2O2 treatments and compared them using Wilcoxon's test. The correlation matrix values were obtained using the Spearman rank order correlation test. Multiple regression analysis was used to identify which of the variables measured was most strongly related to H2O2 dose. These tests were performed using Prism 2.0 (GraphPad Inc., San Diego, CA) and CSS Statistica (Statsoft Inc., Tulsa, OK) software.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
The results for the CBMN assay at 72 and 96 h harvest (Table I
) were not significantly different. We therefore combined the results from these two time points and found that H2O2 induces a significant increment in MNed cell frequency (P = 0.0048) and necrosis (P < 0.0001) and a reduction in the BN cell ratio (P < 0.0001) and apoptosis (P = 0.051) (Figure 3).
|
|
The results obtained with CBMN-ARA cultures for cells at 72 and 96 h harvest (Table II
) were not significantly different and showed similar trends to those for the CBMN assay. Combined data from these time points indicated that H2O2 causes a significant increment in ARA-induced MNed cell frequency (P < 0.001) and necrosis (P < 0.0001) and a decrease in BN cell ratio (P = 0.001) and apoptosis (P = 0.1657) (Figure 4). The ratios for ARA-induced MNed cells (in the CBMN-ARA cultures) to baseline MNed cells (in the CBMN cultures) in H2O2-treated cells and untreated cells were 2.2 and 2.0, respectively (the corresponding values for MN were 2.9 and 2.4, respectively).
|
|
In both the CBMN and CBMN-ARA cultures the frequency of necrotic cells was 40–200 times greater than that of apoptotic cells depending on the H2O2 dose.
The ELISA assays for 8-OHdG in the culture medium showed no significant difference for measurements at 24, 48 and 72 h. The mean values for these measurements were therefore combined and showed that H2O2 does not increase the level of 8-OHdG in the medium (Figure 5).
|
To obtain an understanding of the interrelationship between the parameters measured we estimated the Spearman rank order correlation factors using data from both the CBMN and CBMN-ARA cultures (Table III
). It was evident that H2O2 was significantly correlated with all measured parameters excluding 8-OHdG and that there was good agreement for correlation factors estimated using results from the CBMN and CBMN-ARA cultures. Necrosis was the event most strongly correlated with H2O2 dose (r = 0.73, 0.65) as well as the BN ratio (r = –0.78, –0.74) in both CBMN and CBMN-ARA cultures, respectively. 8-OHdG was negatively correlated (r = –0.50) with ARA-induced MNed cell frequency.
|
Because MNed cell frequency, ARA-induced MNed cell frequency, apoptosis, necrosis and BN ratio were significantly related to H2O2 dose and to each other it was necessary to perform multiple regression analysis to identify which of these was the main event caused by H2O2. Two independent multiple regression analyses, based on the results from the CBMN and CBMN-ARA cultures (Table IV
), showed that only necrosis is significantly related to H2O2 dose (ß = 0.56, 0.57, respectively, P < 0.0004).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
The extent of observed DNA damage induced by a chemical or physical agent may depend on whether the agent induces or inhibits necrosis and/or apoptosis. Induction of necrosis could result in the intracellular release of degradative enzymes from subcellular particles such as lysosomes, which may cause partial digestion of DNA during the early stages of necrosis. Inhibition of apoptosis may allow cells that have experienced a significant level of DNA damage to proceed through the cell cycle and survive as mutated/MNed cells. Until very recently genotoxicity assays have tended to ignore the role of necrosis and apoptosis in determining the outcome of cytogenetic and point mutation assays. In those assays, such as the single cell gel electrophoresis assay, when DNA damage is assessed shortly after exposure to test chemicals there is some concern that positive results for strand breakage may be a side-effect of necrosis (Henderson et al., 1998
). In cytogenetic assays requiring cell division in culture for the expression of the end-point measured (e.g. MN or chromosome aberrations) there is concern that not all damaged cells are observed because some have undergone apoptosis or necrosis instead of completing nuclear division (Fenech et al., 1997). It is therefore essential to integrate necrosis and apoptosis into current DNA damage assays. As we have shown, it is relatively easy to integrate these additional biomarkers within the CBMN assay, because apoptotic cells and necrotic cells can be readily counted alongside the MNed BN cells, as well as obtaining a proliferation index, which is essential for determining whether a chemical has cytostatic effects. Scoring necrotic and apoptotic cells is enabled by the lack of hypotonic treatment that is possible when using isolated lymphocyte cultures. In addition, this approach takes into account all cells, both viable and non-viable, which enables a precise ratio of all cells to be obtained. For example, in the original CBMN assay, non-viable cells are ignored when estimating the BN cell ratio or the nuclear division index, which leads to an inaccurate picture of what is actually happening within the culture system used. This discrepancy is now corrected by including all cells, regardless of their viability status, in the ratio estimates.
In terms of event frequency it was clear that necrosis was more prevalent than apoptosis by a factor of at least 40-fold, increasing to 200-fold at higher H2O2 doses. This suggests that while apoptosis has an important role in the elimination of cells with DNA damage, the great majority of damaged cells appear to be eliminated by necrosis in lymphocyte cultures. The baseline rate of necrosis (~23%) observed in our cultures appears to be quite high, however, it may be that a significant proportion of observed necrotic cells are non-dividing B cells rather than T cells. Up to 30% of isolated lymphocytes may be B cells (Natarajan and Obe, 1982). It is also important to note that in the proposed protocol for scoring apoptosis both mononuclear and BN apoptotic cells are scored. However, it may be useful to consider scoring mononuclear and BN apoptotic cells separately because the former may be more representative of the normal process of DNA damage-induced apoptosis occurring prior to metaphase, while in the latter case apoptosis may have also been induced by an extended cytokinesis-block period. Even if the majority of apoptotic cells scored were BN this would be expected to have a negligible effect on MN frequency in BN cells as the great majority (>98%) do not undergo apoptosis during the assay period (data derived from Figures 3 and 4).
We have used hydrogen peroxide as the test chemical to evaluate the improved CBMN assay because we had previously observed a high level of necrosis relative to MN induction (Crott and Fenech, 1999). These studies had also suggested a strong positive correlation between necrosis and MN induction and a negative correlation between necrosis and apoptosis. The results from the present study have confirmed that MNed cell frequency, necrosis, apoptosis and the BN cell ratio are significantly interrelated to varying extents with each other and with hydrogen peroxide concentration. However, multiple regression analysis clearly shows that necrosis is the main event induced by hydrogen peroxide when supplied extracellularly. This implies that hydrogen peroxide is primarily a necrosis-inducing agent and only secondarily a genotoxin.
Furthermore, this study also shows that 8-OHdG was not induced at doses of hydrogen peroxide that are clearly cytotoxic. This observation is in agreement with those of Takeuchi et al. (1994), who showed that 8-OHdG was not induced in HL-60 cells exposed to up to 500 µM hydrogen peroxide in culture medium when measured using the HPLC method for 8-OHdG in isolated DNA. If 8-OHdG was an important adduct induced by hydrogen peroxide one would have expected a significant positive correlation between the ARA-induced MNed cell frequency and 8-OHdG concentration in culture medium, but this was not the case; in fact the correlation factor was negative. The latter is difficult to explain unless 8-OHdG in culture medium originated from necrotic cell nuclei and indirectly acts as a biomarker of reduced capacity to perform excision repair. A reduced ability to perform excision repair, as has been demonstrated with aging in the CBMN-ARA assay, could minimize ARA-induced MNed cell frequency (Fenech and Neville, 1992).
These results do not exclude the possibility that H2O2 generated intracellularly may be genotoxic but to do so it would have to penetrate the nuclear membrane and participate in the Fenton reaction in close proximity to DNA because the hydroxyl radical is highly reactive (Wiseman and Halliwell, 1996). The results we obtain do not agree with data from the single cell gel electrophoresis assay which classify exogenous H2O2 as a genotoxic rather than a cytotoxic agent (Singh et al., 1988; Collins et al., 1993; Henderson et al., 1998) and highlight the possibility that cells classified as exhibiting DNA damage in this assay may in some instances be early necrotic or early apoptotic cells.
The data obtained also suggest a negative correlation between necrosis and apoptosis. Recent experiments with Jurkat T lymphocytes showed that 50 µM H2O2 increases caspase activity and induces apoptosis, but at higher concentrations caspase activity is inhibited and the cells instead die by necrosis (Hampton and Orrenius, 1997). This mechanism may explain why apoptosis was inhibited and necrosis increased with increasing H2O2 concentration in our experiments. Other studies have also shown a shift from apoptosis to necrosis as H2O2 concentration is increased above 100 µM in cultures of human lung fibroblasts (Teramoto et al., 1999). H2O2 may operate by primarily damaging mitochondrial membranes, which results in the release of cytochrome c. Release of the latter into the cytoplasm is thought to commit the cell to die by either a rapid apoptotic mechanism involving Apaf-1-mediated caspase activation or a slower necrotic process due to defective electron transport and decreased production of ATP (Green and Reed, 1998). In evaluating the data obtained in our study it is important to note that the levels of H2O2 used (25–100 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 ~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 MNed 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.
The results from this study and the proposed methodology have implications with regard to classification of chemicals, cellular sensitivity testing and baseline genotoxicity data in human populations. In evaluating genotoxicity it is important to consider not only DNA damage but also necrosis and apoptosis and cytostatic effects, all of which are likely to be interrelated, as shown by the results from our study. The use of a multiple regression model can help to determine whether the agent studied is primarily a genotoxin or whether the apparent genotoxic effects are simply a side-effect of necrotic changes. We suggest that simultaneous measurement of these end-points in the one test system may be required to obtain comprehensive and reliable results. The proposed CBMN test system is likely to provide more detailed information than shorter term assays because all the morphology of viable and non-viable cells is maintained and sufficient time is allowed for the relatively slow process of necrosis and apoptosis to occur.
The same test system is of relevance in evaluating radiation sensitivity of cells. This is becoming increasingly important, both in the prediction of cancer susceptibility and in identifying cancer patients with normal tissue sensitivity to ionizing radiation prior to radiotherapy (O'Driscoll et al., 1998; Scott et al., 1999). Recent studies with the MN assay suggest that in vitro tests on fibroblasts in tissue close to the radiotherapy site is predictive of normal tissue reaction (Nachtrab et al., 1998), however, results with lymphocytes appear to be less predictive (Rached et al., 1998). In addition, there is sometimes a discordance between results using colony assays and MN assays when measuring radiosensitivity of cell lines (Johansen et al., 1998; O'Driscoll et al., 1998). The discordance may well be due to differences between tissues and cell lines in the extent of induced necrosis and apoptosis so that damaged cells would, to varying degrees, die by necrosis or apoptosis rather than survive to express DNA damage as MN; the converse may also apply. However, the proposed integrated approach described in this paper would take account of all these events and may therefore provide a better prediction of radiosensitivity.
In a previous report (Fenech et al., 1997) on the use of the MN assay in human biomonitoring, we suggested that a more comprehensive picture of the extent of cellular damage could be obtained if both pre-exisiting MN in cells as well as cell death events during culture were enumerated. In the latter case cells that experienced extensive damage to DNA or other cellular components in G0, whilst in the blood circulation, may not survive the first cell cycle in tissue culture to express MN but rather may undergo necrosis or apoptosis. Thus assessment of these alternative end-points may provide the complete picture needed.
In conclusion, we suggest that the cytokinesis-block micronucleus assay can be used for integrated measurements of chromosome breakage and loss, excision-repaired sites, necrosis, apoptosis and cytostatic effects. Furthermore, this system may provide a better way of estimating the relative importance of these events when cells are stressed by a variety of agents.
|
Acknowledgments |
|---|
We are very thankful to the volunteers who participated in the study and to Dr Peter Clifton and Sister Rosemary McArthur for collecting blood samples. Special acknowledgement is due to Clare Aitken who performed the initial experiments indicating the feasibility of measuring apoptotic cells in cytokinesis-blocked lymphocyte cultures.
|
Notes |
|---|
1 To whom correspondence should be addressed. Tel: +61 8 8303880; Fax: +61 8 83038899; Email: michael.fenech{at}hsn.csiro.au
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
-
Atencia,R., Garciasanz,M., Perezyarza,G., Asumendi,A., Hilario,E. and Arechaga,J. (1997) A structural analysis of cytoskeletal components during the execution phase of apoptosis. Protoplasma, 198, 163–169.
Carter,S.B. (1967) Effects of cytochalasins on mammalian cells. Nature, 213, 261–264.[Medline]
Collins,A.R., Duthie,S.J. and Dobson,V.L. (1993) Direct enzymic detection of endogenous oxidative base damage in human lymphocyte DNA. Carcinogenesis, 14, 1733–1735.
Crott,J.W. and Fenech,M. (1999) Effect of vitamin C supplementation on chromosome damage, apoptosis and necrosis ex vivo. Carcinogenesis, 20, 1035–1041.
Evan,G. and Littlewood,T. (1998) A matter of life and cell death. Science, 281, 1317–1322.
Fenech,M. (1993) The cytokinesis-block micronucleus technique: a detailed description of the method and its application to genotoxicity studies in human populations. Mutat. Res., 285, 35–44.[Web of Science][Medline]
Fenech,M. (1996) The cytokinesis-block micronucleus technique. In Pfeifer,G.P. (ed.), Technologies for Detection of DNA Damage and Mutations. Plenum Press, New York, NY, pp. 25–36.
Fenech,M. (1997) The advantages and disadvantages of the cytokinesis-block micronucleus method. Mutat. Res., 392, 11–18.[Web of Science][Medline]
Fenech,M. and Morley,A.A. (1985) Measurement of micronuclei in human lymphocytes. Mutat. Res., 148, 29–36.
Fenech,M. and Neville,S. (1992) Conversion of excision-repairable DNA lesions to micronuclei within one cell cycle in human lymphocytes. Environ. Mol. Mutagen., 19, 27–36.[Web of Science][Medline]
Fenech,M., Perepetskaya,G. and Mikhalevich,L. (1997) A more comprehensive application of the micronucleus technique for biomonitoring of genetic damage rates in human populations—experiences from the Chernobyl catastrophe. Environ. Mol. Mutagen., 30, 112–118.[Web of Science][Medline]
Fenech,M., Holland,N., Chang,W.P., Zeiger,E. and Bonassi,S. (1999) The Human Micronucleus Project—an international collaborative study of the micronucleus technique for measuring DNA damage in humans. Mutat. Res., in press.
Green,D.R. and Reed,J.C. (1998) Mitochondria and apoptosis. Science, 281, 1309–1312.
Hampton,M.B. and Orrenius,S. (1997) Dual regulation of caspase activity by hydrogen peroxide: implications for apoptosis. FEBS Lett., 414, 552–556.[Web of Science][Medline]
Henderson,L., Wolfreys,A., Fedyk,J., Bourner,C. and Windebank,S. (1998) The ability of the comet assay to discriminate between genotoxins and cytotoxins. Mutagenesis, 13, 89–94.
Hyslop,P.A., Hinshaw,D.B., Scraufstatter,I.U., Cochrane,C.G., Kunz,S. and Vosbeck,K. (1995) Hydrogen peroxide as a potent bacteriostatic antibiotic: implications for host defense. Free Radic. Biol. Med., 19, 31–37.[Web of Science][Medline]
Johansen,J., Streffer,C., Fuhrmann,C., Bentzen,S.M., Stausbolgron,B., Overgaard,M. and Overgaard,J. (1998) Radiosensitivity of normal fibroblasts from breast cancer patients assessed by the micronucleus and colony assays. Int. J. Radiat. Biol., 73, 671–678.[Web of Science][Medline]
Kirsch-Volders,M., Elhajouji,A., Cundari,E. and Van Hummelen,P. (1997) The in vitro micronucleus test: a multi-end-point assay to detect simultaneously mitotic delay, apoptosis, chromosome breakage, chromosome loss and non-disjunction. Mutat. Res., 392, 19–30.[Web of Science][Medline]
Lacy,F., O'Connor,D.T., Schmid-Schonbein,G.W. (1998) Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertension. J. Hypertens., 16, 291–303.[Web of Science][Medline]
Lelli,J.L., Becks,L.L., Dabrowska,M.I. and Hinshaw,D.B. (1998) ATP converts necrosis to apoptosis in oxidant-injured cells. Free Radic. Biol. Med., 25, 694–702.[Web of Science][Medline]
Nachtrab,U., Oppitz,U., Flentje,M. and Stopper,H. (1998) Radiation-induced micronucleus formation in human skin fibroblasts of patients showing severe and normal tissue damage after radiotherapy. Int. J. Radiat. Biol., 73, 279–287.[Web of Science][Medline]
Natarajan,A.T. and Obe,G. (1982) Mutagenicity testing with cultured mammalian cells: cytogenetic assays. In Heddle,J.A. (ed.), Mutagenicity: New Horizons in Genetic Toxicology. Academic Press, New York, NY, pp. 171–213.
O'Driscoll,M.C., Scott,D., Orton,C.J., Kiltie,A.E., Davidson,S.E., Hunter,R.D. and West,C.M.L. (1998) Radiation-induced micronuclei in human fibroblasts in relation to clonogenic radiosensitivity. Br. J. Cancer, 78, 1559–1563.[Web of Science][Medline]
Rached,E., Schindler,R., Beer,K.T., Vetterli,D. and Greiner,R.H. (1998) No predictive value of the micronucleus assay for patients with severe acute reaction of normal tissue after radiotherapy. Eur. J. Cancer, 34, 378–383.
Samali,A., Nordgren,H., Zhivotovsky,B., Peterson,E. and Orrenius,S. (1999) A comparative study of apoptosis and necrosis in HepG2 cells: oxidant-induced caspase inactivation leads to necrosis. Biochem. Biophys. Res. Commun., 255, 6–11.[Web of Science][Medline]
Schuler,M., Rupa, D.S. and Eastmond,D.A. (1997) A critical evaluation of centromeric labelling to distinguish micronuclei induced by chromosome loss and breakage in vitro. Mutat. Res., 392, 81–95.[Web of Science][Medline]
Scott,D., Barber,J.B.P., Spreadborough,A.R., Burrill,W. and Roberts,S.A. (1999) Increased chromosomal radiosensitivity in breast cancer patients: a comparison of two assays. Int. J. Radiat. Biol., 75, 1–10.[Web of Science][Medline]
Singh,N.P., McCoy,M.T., Tice,R.R. and Schneider,E.L. (1988) A simple technique for quantitation of low levels of DNA damage in individual cells. Exp. Cell Res., 175, 184–191.[Web of Science][Medline]
Sri Kantha,S., Wada,S., Takeuchi,M., Watabe,S. and Ochi,H. (1996) A sensitive method to screen for hydroxyl radical scavenging activity in natural food extracts using competitive inhibition ELISA for 8-hydroxy-deoxyguanosine. Biotechnol. Tech., 10, 933–936.
Takeuchi,T., Nakajima,M. and Morimoto,K. (1994) Establishment of a human system that generates superoxide and induces 8-hydroxydeoxyguanosine, typical of oxidative damage, by a tumour promoter. Cancer Res., 54, 5837–5840.
Teramoto,S., Tomita,T., Matsue,H., Ohga,E., Matsuse,T. and Ouchi,Y. (1999) Hydrogen peroxide-induced apoptosis and necrosis in human lung fibroblasts: protective roles of glutathione. Jpn J. Pharmacol., 79, 33–40.[Medline]
Wiseman,H. and Halliwell,B. (1996) Damage to DNA by reactive oxygen and hydrogen species: role of inflammatory diseases and progression to cancer. Biochem. J., 313, 17–29.
Received on June 4, 1999; accepted on July 19, 1999.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  A. Kabil, E. Silva, and A. Kortenkamp Estrogens and genomic instability in human breast cancer cells--involvement of Src/Raf/Erk signaling in micronucleus formation by estrogenic chemicals Carcinogenesis, October 1, 2008; 29(10): 1862 - 1868. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. A. El-Zein, M. Fenech, M. S. Lopez, M. R. Spitz, and C. J. Etzel Cytokinesis-Blocked Micronucleus Cytome Assay Biomarkers Identify Lung Cancer Cases Amongst Smokers Cancer Epidemiol. Biomarkers Prev., May 1, 2008; 17(5): 1111 - 1119. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Ferk, M. Misik, C. Hoelzl, M. Uhl, M. Fuerhacker, B. Grillitsch, W. Parzefall, A. Nersesyan, K. Micieta, T. Grummt, et al. Benzalkonium chloride (BAC) and dimethyldioctadecyl-ammonium bromide (DDAB), two common quaternary ammonium compounds, cause genotoxic effects in mammalian and plant cells at environmentally relevant concentrations Mutagenesis, November 1, 2007; 22(6): 363 - 370. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. G. Cox and M. B. Hampton Bcl-2 over-expression promotes genomic instability by inhibiting apoptosis of cells exposed to hydrogen peroxide Carcinogenesis, October 1, 2007; 28(10): 2166 - 2171. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. J.S. Jenkins, F. R. D'Souza, S. H. Suzen, Z. S. Eltahir, S. A. James, J. M. Parry, P. A. Griffiths, and J. N. Baxter Deoxycholic acid at neutral and acid pH, is genotoxic to oesophageal cells through the induction of ROS: the potential role of anti-oxidants in Barrett's oesophagus Carcinogenesis, January 1, 2007; 28(1): 136 - 142. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Chen, M. Arjomandi, H. Qin, J. Balmes, I. Tager, and N. Holland Cytogenetic damage in buccal epithelia and peripheral lymphocytes of young healthy individuals exposed to ozone Mutagenesis, March 1, 2006; 21(2): 131 - 137. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Dhawan, M. A. Kayani, J. M. Parry, E. Parry, and D. Anderson Aneugenic and clastogenic effects of doxorubicin in human lymphocytes Mutagenesis, November 1, 2003; 18(6): 487 - 490. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Miltyk, C. N Craciunescu, L. Fischer, R. A Jeffcoat, M. A Koch, W. Lopaczynski, C. Mahoney, R. A Jeffcoat, J. Crowell, J. Paglieri, et al. Lack of significant genotoxicity of purified soy isoflavones (genistein, daidzein, and glycitein) in 20 patients with prostate cancer Am. J. Clinical Nutrition, April 1, 2003; 77(4): 875 - 882. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. Greenrod and M. Fenech The principal phenolic and alcoholic components of wine protect human lymphocytes against hydrogen peroxide- and ionizing radiation-induced DNA damage in vitro Mutagenesis, March 1, 2003; 18(2): 119 - 126. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Thomas, K. Umegaki, and M. Fenech Nucleoplasmic bridges are a sensitive measure of chromosome rearrangement in the cytokinesis-block micronucleus assay Mutagenesis, March 1, 2003; 18(2): 187 - 194. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. W. Crott, S. T. Mashiyama, B. N. Ames, and M. Fenech The Effect of Folic Acid Deficiency and MTHFR C677T Polymorphism on Chromosome Damage in Human Lymphocytes in Vitro Cancer Epidemiol. Biomarkers Prev., October 1, 2001; 10(10): 1089 - 1096. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Bishay, K. Ory, M.-F. Olivier, J. Lebeau, C. Levalois, and S. Chevillard DNA damage-related RNA expression to assess individual sensitivity to ionizing radiation Carcinogenesis, August 1, 2001; 22(8): 1179 - 1183. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Gualandi, L. Giselico, M. Carloni, F. Palitti, P. Mosesso, and A. M. Alfonsi Enhancement of genetic instability in human B cells by Epstein-Barr virus latent infection Mutagenesis, May 1, 2001; 16(3): 203 - 208. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Crott and M. Fenech Preliminary study of the genotoxic potential of homocysteine in human lymphocytes in vitro Mutagenesis, May 1, 2001; 16(3): 213 - 217. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Laffon, E. Pasaro, and J. Mendez Effects of styrene-7,8-oxide over p53, p21, bcl-2 and bax expression in human lymphocyte cultures Mutagenesis, March 1, 2001; 16(2): 127 - 132. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Vanhauwaert, P. Vanparys, and M. Kirsch-Volders The in vivo gut micronucleus test detects clastogens and aneugens given by gavage Mutagenesis, January 1, 2001; 16(1): 39 - 50. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Kirsch-Volders and M. Fenech Inclusion of micronuclei in non-divided mononuclear lymphocytes and necrosis/apoptosis may provide a more comprehensive cytokinesis block micronucleus assay for biomonitoring purposes Mutagenesis, January 1, 2001; 16(1): 51 - 58. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Fenech A mathematical model of the in vitro micronucleus assay predicts false negative results if micronuclei are not specifically scored in binucleated cells or in cells that have completed one nuclear division Mutagenesis, July 1, 2000; 15(4): 329 - 336. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Basso and A. Russo Detection and characterization of micronuclei in a murine liver epithelial cell line, by application of the in vitro cytokinesis block MN assay and PRINS Mutagenesis, July 1, 2000; 15(4): 349 - 356. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Umegaki and M. Fenech Cytokinesis-block micronucleus assay in WIL2-NS cells: a sensitive system to detect chromosomal damage induced by reactive oxygen species and activated human neutrophils Mutagenesis, May 1, 2000; 15(3): 261 - 269. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||