Mutagenesis, Vol. 15, No. 3, 215-221,
May 2000
© 2000 UK Environmental Mutagen Society/Oxford University Press
Evaluation and characterization of micronuclei induced by the antitumour agent ASE [3ß-hydroxy-13
-amino-13,17-seco-5-androstan-17-oic-13,17-lactam-p-bis(2-chloroethyl)amino phenylacetate] in human lymphocyte cultures
Division of Genetics, Cell and Developmental Biology, Department of Biology, University of Patras, 26500 Patras, Greece
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Abstract |
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3ß - Hydroxy - 13
- amino - 13, 17 - seco - 5 - androstan - 17 -oic-13,17-lactam-p-bis(2-chloroethyl)amino phenylacetate (ASE) is a homo-aza-steroidal ester of p-bis(2-chloroethyl) amino phenyl acetic acid and has been shown to display antineoplastic, mutagenic and genotoxic activity. In the present study an effort has been made to evaluate the ability of ASE to induce micronuclei (MN) in human lymphocytes treated in vitro using the cytokinesis-block assay. Lympocytes were treated with different concentrations of ASE (0.1, 0.25, 0.5, 1, 2.5, 5, 10 and 20 µg/ml) at two different cell culture times, 21 and 41 h after culture initiation. ASE treatment lasted until cell harvest, for 51 and 31 h, respectively. Two types of cultures were used, whole blood and isolated lymphocyte cultures. The content of induced MN was identified by FISH analysis, using an -satellite DNA probe, in binucleate cells. Our results suggest that ASE is capable of increasing MN frequencies in human lymphocytes under both culture conditions. This increase is related to the concentration in a linear dose-dependent manner and is also dependent on the duration of treatment. FISH analysis has shown that the induced MN resulted mainly from breakage events. Additionally, a weak aneugenic effect was found at the higher concentrations in whole blood cultures as well as in isolated lymphocyte cultures. Cytotoxic effects of ASE were observed under both cell culture conditions with a linear dose-dependent relationship according to CBPI evaluation and were more pronounced in isolated lymphocyte cultures. |
Introduction |
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3ß-Hydroxy-13
-amino-13,17-seco-5-androstan-17-oic-13,17-lactam-p-bis(2-chloroethyl)amino phenylacetate (ASE) is a homo-aza-steroidal ester of p-bis(2-chloroethyl)amino phenyl acetic acid whose chemical structure is shown in Figure 1
. It consists of a nitrogen mustard (alkylating molecule) bound to a modified steroid, a lactam, characterized by the group –CO-NH, via a phenylacetic bond and has been synthesized in pure form (Catsoulacos and Boutis, 1973; Wampler and Catsoulacos, 1977). Modified steroids, such as lactams, have been used as biological vehicles for transportation of alkylating molecules to target tumors in a rather specific manner (Catsoulacos et al., 1976). Compounds such as esters, in which the alkylating agent is linked to the steroid by an easily cleaved bond, produce satisfactory activity (Wall et al., 1969). ASE belongs to the above category and has been shown to display good antineoplastic activity. It has given a 50% increased lifespan over controls in the treatment of L1210 leukemia in mice and a 150% increased lifespan in the treatment of P388 leukemia (Wampler and Catsoulacos, 1977). Moreover, ASE has given good activity against B16 melanoma in C57b1 mice and T8 Guerrin tumors in rats (Catsoulacos and Wampler, 1982). In addition, ASE alone or in combination with a different modified steroid, the homo-aza-steroidal ester of o-bis(2-chloroethyl)aminobenzoic acid, proved to exert enhanced antitumor activity in a synergistic manner (Papageorgiou et al., 1999). It is noteworthy that although ASE exerts about the same antineoplastic activity as chlorambucil, it is less toxic with a more prolonged action (Stevenson and Patel, 1973; Catsoulacos et al., 1978).
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Additionally, ASE has shown mutagenic properties. It was found to be positive in strains TA1535 and TA100 as well as in strain TA102 with and without metabolic activation in the Salmonella/microsome system (Athanasiou and Arzimanoglou, 1986
). The compound has also been shown to induce sister chromatid exchange in human lymphocytes (Mourelatos et al., 1987) as well as in CHO cells (Athanasiou et al., 1983). ASE was also determined to decrease protein synthesis rate in ovaries of Drosophila melanogaster (Stephanou et al., 1991). Finally, ASE was identified as displaying genotoxic, cytostatic and antineoplastic properties (Nikolaropoulos et al., 1997).
Human lymphocytes cultured in vitro have been widely used as an appropriate system to identify the genetic activity of various chemical compounds. They are a more or less homogeneous cell population, being all in the G0 phase, and they start DNA synthesis 20–24 h after phytohemagglutinin (PHA) stimulation. The duration of their cycle is well known (Watt and Stephen, 1986). By treating lymphocyte cultures at different cell cycle phases with a given chemical compound, information on the mechanism of action on lymphocyte DNA can be obtained.
On the other hand, the whole blood culture methodology is quite different from isolated human lymphocyte culture with respect to metabolic activation or detoxification of the compound concerned. Erythrocytes present in whole blood cultures may play an important role in metabolic activation (Norppa et al., 1983; Elhajouji et al., 1994). Moreover, plasma, also present in whole blood cultures, can act as an antioxidant (Ames and Gold, 1991). Isolated human lymphocyte cultures contain no erythrocytes or plasma.
In recent years the cytokinesis-block micronucleus assay (Fenech and Morley, 1985; Fenech, 1993) applied to human lymphocyte cultures has been widely evaluated and used for the identification of genetic damage in genetic toxicology studies (Evans, 1997; Kirsch-Volders, 1997). In combination with other methodologies, such as application of antikinetochore antibody and immunofluorescence techniques (CREST) and fluorescence in situ hybridization (FISH) with centromere probes, this method has been proved valuable to follow up clastogenic as well as aneugenic phenomena (Degrassi and Tanzarella, 1988; Eastmond and Tucker, 1989; Vlachodimitropoulos et al., 1997; Stephanou et al., 1997; Vlastos and Stephanou, 1998).
In the present study an effort has been made to evaluate the ability of ASE to induce micronuclei (MN) in human lymphocytes treated in vitro. Lymphocyte cultures were treated with different concentrations of ASE. Treatment of cells with ASE started at two different cell culture times, 21 and 41 h after PHA stimulation, reflecting different phases of the lymphocyte cycle, namely G1 and G2 (Watt and Stephen, 1986; Van Hummelen and Kirsch-Volders, 1992; Vian et al., 1995; Kirsch-Volders et al., 1996), and the total exposure times were 51 and 31 h, respectively. ASE was studied in whole blood cultures as well as in isolated lymphocyte cultures. The content of induced MN was identified by FISH using an -satellite pancentromeric probe. Our aim was to better understand and evaluate ASE genotoxic activity in human lymphocytes.
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Materials and methods |
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Chemical compound
The test compound, ASE, was kindly provided by the late Prof. P.Catsoulacos. The procedure for synthesis of ASE has been published previously (Catsoulacos and Boutis, 1973
) and briefly is as follows. To a solution of 7 g of 13ß-hydroxy-13-amino-13,17-seco-5-androstan-17-oic-13,17-lactam in 250 ml of anhydrous benzene was added 9 g of p-bis(2-chloroethyl)amino phenylacetic chloride. The mixture was heated under reflux for 24 h. The solvent was evaporated under reduced pressure. The residue was chromatographed on a column of silica gel prepared with chloroform. Elution with chloroform gave the steroidal ester (ASE) in pure form. Yield 65%; m.p. 131–132°C (acetone/n-hexane); IR, vmax 3170, 3050, 1725, 1675, 1650 per cm; NMR, 
6.52 (-CH2CO-), 6.3 [N(CH2CH2)2], 5.25 (C3-H), in addition to common peaks.
Whole blood cultures
Whole blood (0.5 ml) derived from two healthy adult men was added to 6.5 ml Ham's F10 medium (Gibco), 1.5 ml fetal calf serum (Gibco), 0.2 ml PHA (Gibco). Separate cultures were established from each donor corresponding to the different experimental points. Cytochalasin B (6 µg/ml; Sigma) was added to the culture medium 44 h after culture initiation. Whole blood cultures were also set up for Giemsa and FISH analysis. For Giemsa analysis the appropriate ASE solutions, diluted in DMSO (Sigma) to give final concentrations of 0.1, 0.25, 0.5, 1, 2.5, 5, 10 and 20 µg/ml, were added at two different time intervals, 21 and 41 h after initiation of the culture, for total exposure periods of 51 and 31 h, respectively. The final concentration of DMSO in the cultures did not exceed 0.2%. Mitomycin C (Sigma) at a final concentration of 0.1 µg/ml was used as a positive control. For FISH analysis ASE was added to the cultures 31 h before cell harvesting to give final concentrations of 1, 5 and 10 µg/ml. The cultures were incubated at 37°C for 72 h in a 5% CO2 atmosphere with 95% humidity.
Isolated lymphocyte cultures
Purified lymphocytes were used in FISH analysis and obtained as follows. Heparinized blood was diluted 1:1 with phosphate-buffered saline (PBS) and was then carefully packed with about the same volume of lymphocyte separation medium (Histopaque 1077; Sigma). Cells were centrifuged for 30 min at 400 g. The buffy layer was carefully taken, transferred to a new centrifuge tube and washed three times with PBS. After the third wash, the cell pellet was obtained by centrifugation for 10 min at 250 g and resuspended in 1 ml fresh medium. Lymphocyte cultures were initiated at a concentration of 0.8–1x106 cells/ml in complete medium. The growth medium was the same as for the whole blood cultures. ASE solution was added to the culture medium to give final concentrations of 1, 5 and 10 µg/ml 31 h before cell harvesting. Cytochalasin B (6 µg/ml; Sigma) was added to the culture medium 44 h after culture initiation.
Micronucleus analysis
Giemsa analysis.
Cells were harvested 72 h after culture initiation. Cells were collected by centrifugation at 400 g for 10 min. A mild hypotonic treatment with a solution of Ham's F10:ddH2O (1:3) supplemented with 2% serum was given for 3 min at room temperature and was followed by a 10 min fixation with a fresh solution of methanol:acetic acid (5:1) (Merck). Cell drops were layered onto clean slides from a very low height. Cells were stained with 7% Giemsa (Ferak). For the estimation of MN frequency at least 1000 binucleate cells were scored for each donor and for each treatment. Giemsa analysis was performed on slides from whole blood cultures in both male donors.
FISH analysis.
Cells were harvested 72 h after PHA stimulation. They were treated with a hypotonic solution of Ham's F10:ddH2O (1:1) for 2 min at 37°C. Fixation of the cells was achieved instantly at least three times by treating them with a solution of methanol:acetic acid (3:1) at room temperature. Cell drops were layered onto clean slides from a very low height. FISH was performed using an -satellite probe for all human centomeres (P 5095:DG 5; Oncor). Slides were pretreated in pepsin (Sigma) solution in 0.1 M HCl, pH 3, for 5 min and then washed in distilled sterile water and PBS, for 2 min each time, and then submerged in 1% formaldehyde (Merck) in PBS at 4°C for 5 min and washed with PBS and distilled sterile water, for 2 min each time, followed by dehydration with an increasing series of ethanol (Merck). Nuclear DNA denaturation was achieved with 70% formamide (Merck) in 2x SSC at 70°C for 2 min. The denatured DNA was then chilled in 70% ethanol and dehydrated in an increasing series of ethanol. Probe denaturation was performed at 70°C for 5 min and the denatured probe chilled on ice. Slides were then incubated with the probe (15 µl for each slide) to hybridize overnight at 37°C in a humidified atmosphere. At the end of the hybridization time slides were washed twice for 10 min with 50% formamide in 2x SSC, followed by two washes in 2x SSC for 4 min each and one 5 min wash in 4x SSC, 0.05% Tween-20 (Sigma) buffer. Slides were then incubated with 5% skimmed milk as blocking reagent at 37°C for 15 min. After a short wash in 4x SSC, Tween-20 buffer, an anti-digoxigenin antibody (D-8156; Sigma) dialyzed against 0.5% skimmed milk (1:250) was laid onto the slides and allowed to bind to the probe by incubation at 37°C for 30 min. Three washes with 4x SSC, Tween-20 buffer followed and the slides were then incubated with anti-mouse antibody (Boehringer Mennheim) dialyzed against 0.5% skimmed milk (1:20) at 37°C for 30 min followed by a subsequent incubation with FITC-conjugated anti-sheep antibody (Sigma), also dialyzed against 0.5% skimmed milk (1:20) at 37°C for 30 min. Every incubation was followed by extensive washes in 4x SSC, Tween-20 buffer. Counterstaining was performed with a mixture of DAPI and propidium iodide (Sigma) in 4x SSC, Tween-20 buffer at room temperature for 5 min. The counterstained slides were washed in tap water, air dried and mounted in Vectashield Mounting Medium (Vector Laboratories). Slides were kept in the dark at 4°C and analyzed in a Zeiss Axioskop fluorescence microscope. The passband filters used were of 546, 490 and 360 nm for green, blue and ultraviolet light, respectively. At least 50 MN were analysed for the presence of a centromere-positive signal for each experimental point. To identify a MN as containing a centromere-positive signal, this should be of the same intensity as those of the main nuclei. FISH analysis was performed on slides from whole blood cultures as well as from isolated lymphocyte cultures from one of the male donors.
For the scoring of MN standard criteria were used (Fenech, 1993) for both analyses. To determine possible cytotoxic effects, 2000 cells were counted for the calculation of cytokinesis-block proliferation index (CBPI) (Surrallés et al., 1995), which is given by the equation CBPI = M1 + 2M2 + 3(M3 + M4)/n, where M1, M2, M3 and M4 correspond to the numbers of cells with one, two, three and four nuclei and n is the total number of cells.
Statistical analysis
Statistical analysis of MN data comparing each treatment with the control was achieved with the G-test (Sokal and Rohlf, 1981) for independence on 2x2 tables. This test is based on the general assumptions of 
2 analysis, but offers theoretical and computational advantages. In order to test whether there is a dose–response relationship between ASE concentration and the end-points studied, linear regression analysis was achieved and correlation coefficient (r) and probability (P) were calculated. P values <0.05 indicate a linear dose–response relationship (Snedecor and Cochran, 1972).
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Results |
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Data on MN induction in whole blood cultures by ASE, determined by Giemsa analysis, are presented in Table I
. After 51 h exposure, a statistically significant increase in MN was observed in relation to the control, even at the lowest concentration tested, 0.1 µg/ml. At this concentration the frequencies of micronucleated binucleate cells (BNMN) were 36 and 30.77
(0.0001 
P < 0.001 and P < 0.01 for the first and second donor, respectively) and of MN were 37 and 33.85 (P < 0.01 for both donors). An ASE concentration increase was followed by an increase in BNMN and MN frequencies. At the highest concentration tested, 20 µg/ml, it was not possible to determine BNMN and MN frequencies because the CBPI was too low (1.07 and 1.08 for the first and second donor, respectively) and the yield of binucleate cells was too poor to estimate MN frequency. Regression analysis has shown a linear dose–response increase for BNMN (treg = 7.33, r = 0.948, P = 0.000 for the first donor; treg = 13.07, r = 0.983, P = 0.000 for the second) and MN frequencies (treg = 9.72, r = 0.969, P = 0.000 for the first donor; treg = 14.61, r = 0.986, P = 0.000 for the second). Taking into consideration the CBPI, a clear reduction was obvious in both donors and a linear dose–response decrease was observed (treg = –16.67, r = –0.988, P = 0.000 for the first donor; treg = –9.21, r = –0.961, P = 0.000 for the second). Additionally, mitomycin C (0.1 µg/ml), used as a positive control, induced statistically significant MN frequencies, in comparison with the control, in both donors (P < 0.01).
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Considering the results at 31 h exposure in Table I
it can be seen that the effect of ASE on human lymphocytes is less intensive than that at 51 h exposure. The first statistically significant increase in MN induction appeared at a higher concentration, 0.5 µg/ml, at which the BNMN and MN frequencies were 30 (P < 0.01) and 33 (P < 0.01) for the first donor and 24 (P < 0.01) and 25 (P < 0.01) for the second, respectively, compared with the control. Treatment with ASE at 0.1 and 0.25 µg/ml produced a slight but not statistically significant effect in both donors. At the highest concentration tested, 20 µg/ml, the corresponding frequencies were 70 (0.0001 P < 0.001) and 79 (0.0001 P < 0.001) for the first donor and 67 (0.0001 P < 0.001) and 73 (0.0001 P < 0.001) for the second. As can be seen from Table I, regression analysis has also shown that there is a linear dose–response enhancement of BNMN frequency (treg = 6.31, r = 0.922, P = 0.000 for the first donor; treg = 6.18, r = 0.919, P = 0.000 for the second) as well as of MN frequency (treg = 6.13, r = 0.918, P = 0.000 for the first donor; treg = 5.87, r = 0.912, P = 0.001 for the second). With regard to CBPI, a linear dose–response decrease was also shown for both donors (treg = –8.36, r = –0.953, P = 0.000 for the first donor; treg = –10.72, r = –0.971, P = 0.000 for the second) after this exposure period. At the highest concentration tested CBPI was 1.42 and 1.39 for the first and second donor, respectively. Mitomycin C also induced statistically higher MN frequencies in comparison with the control after 31 h exposure (P < 0.05 and P < 0.01 for the first and second donor, respectively).
Taking into account the fact that the induced MN frequencies are homogeneous between the two donors, the results presented in Table II were pooled. In Table II the distribution of MN in binucleate cells is presented. For both exposure periods, 51 and 31 h, the majority of BNMN contain only one MN, very few binucleates contain two MN and even less contain three or more MN.
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FISH experiments were carried out in order to identify the content of MN after 31 h treatment. Isolated human lymphocyte cultures were also used in parallel with whole blood cultures in order to see if the effect of ASE is affected by the presence of erythrocytes and plasma in the culture. Three ASE concentrations were used, 1, 5 and 10 µg/ml, at which a statistically significant MN induction was determined from Giemsa experiments for both time intervals studied. The results of these experiments are presented in Table III
. As can be seen, the results from Table I on MN frequencies and CBPI are confirmed. An MN frequency increase and a CBPI decrease were also observed.
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Analysis of MN content has shown that at all concentrations tested the frequency of centromere-negative MN (C– MN) (14.82, 32.40 and 36.15
at 1, 5 and 10 µg/ml) in whole blood cultures is statistically higher than the respective control frequency (6.26, 0.0001 P < 0.001). In isolated lymphocyte cultures the same observation also seems to be true. At the same concentrations a statistically significant enhancement of the corresponding MN frequencies (16.02, P < 0.05; 30.82 and 35.18, 0.0001 P < 0.001) has been shown compared with the control frequency (9.15). Thus it can be said that the effect of ASE on human lymphocytes, cultured by two different methods, is mainly via breakage events. In addition, statistical analysis has shown that at 5 and 10 µg/ml in whole blood cultures the centromere-positive MN frequencies (C+ MN) (11.25 and 9.20) were also significantly higher than the corresponding control (2.96, 0.0001 P < 0.001). The same observation also holds true in isolated lymphocyte cultures. At the same concentrations the corresponding frequencies are 8.20 and 9.64, being statistically higher in comparison with the control (2.58, 0.0001 P < 0.001). This is evidence that, at higher concentrations, ASE can also act as an aneugenic factor through chromosome loss. |
Discussion |
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Several in vitro studies have shown that ASE is able to interact with DNA in both bacteria and mammalian cells. In our study an effort has been made to determine the in vitro effect of ASE on the formation of MN in human lymphocyte cultures. The mechanism, chromosome breakage or chromosome loss, by which the induced MN are formed was also investigated by FISH analysis using an
-satellite probe. Our results show that ASE is able to induce an enhanced MN frequency in human lymphocytes treated in vitro. The comparison of the induced MN frequency in both donors (Table I) between the two different exposure periods has shown that this induction is higher when cells are treated at 21 h after PHA stimulation for a further 51 h until cell harvest than at 41 h for a further 31 h. Although the lymphocyte cultures were not perfectly synchronous, the first exposure period began before DNA replication, in G1 phase, while the second was in G2 phase (Watt and Stephen, 1986; Van Hummelen et al., 1992; Vian et al., 1995; Kirsch-Volders et al., 1996). Hence, when cells were treated for 51 h the total observed MN originated from chromosome damage induced not only in G2 but also in the G1 and S phases of the same cell cycle. The cytotoxicity of the compound seems also to be influenced by the duration of ASE treatment, taking into account the lower CBPI values observed for the 51 h exposure period.
Regarding FISH experiments, the results on induced MN frequency confirmed the findings from Giemsa analysis. Furthermore, it was shown that the main effect of ASE on lymphocyte DNA is chromosome breakage, as far as at all tested concentrations the frequency of induced C– MN, which hence contained only acentric chromosome fragments, was always higher than the control. These results, indicating ASE chromosome breakage and cytotoxic potential, are in accord with previously published findings showing that ASE induces chromosome abnormalities and sister chromatid exchange in CHO cells (Athanasiou et al., 1983) as well sister chromatid exchange and cytotoxic effects in human lymphocytes (Mourelatos et al., 1987; Nikolaropoulos et al., 1997; Papageorgiou et al., 1999). ASE is an alkylating compound and its genetic activity is mediated through DNA alkylation, although the specific DNA lesions induced by this chemical are not yet known.
However, at the two higher concentrations, 5 and 10 µg/ml, it was found that ASE also induced high frequencies of C+ MN, i.e. MN that include whole chromosomes as a consequence of chromosome loss. This is not the first observation that compounds mainly characterized as clastogens also express aneugenic activity. Butadiene and its major metabolites, epoxybutene and diepoxybutane, which are known to express strong clastogenic activity in mice (Cunningham et al., 1986; Autio et al., 1994), were also found to be weak aneugens (Xiao et al., 1996; Stephanou et al., 1997, 1998; Vlachodimitropoulos et al., 1997). The exact mechanism by which clastogens can induce C+ MN is not yet known. Previously it has been proposed that some mutagens interact with the centromeric region and may cause aneugenic effects (Brinkley et al., 1985). As has been recently suggested (Abramson Zetterberg, 1997), the aneugenic activity of clastogens could well be due to asymmetrical chromosomal exchange yielding chromosomal bridges or interlocked chromatids and C+ MN may arise from aggregation of the chromosomes involved in such exchanges which lag behind during anaphase. In addition, it was recently reported (Kirsch-Volders et al., 1996) that when primary human cells are irradiated in vitro by X-rays in G1 and G2 phase an aneugenic effect was determined, which was attributed to both chromosome loss and non-disjunction. Higher frequencies of chromosome loss were observed in G1 phase and it is assumed that aneuploidy during G1 phase might be preferentially attained by interaction with DNA targets.
Fixation time can influence baseline frequency of MN in human lymphocyte cultures using the cytochalasin B method, especially of MN harbouring whole chromosomes, and also non-disjunction rate. Recently (Zijno et al., 1994), after treatment of cells with cytochalasin B at 6 µg/ml with a 72 h fixation time, a small but insignificant increase in aberrant binucleate cells was found. An earlier collection of cells was proposed, in order to obtain low rates of aberrant binucleates. Also, support was found (Sgura et al., 1997) that MN frequencies vary with culture time (56, 72, 80 and 92 h) and this was attributed to second division binucleate cells. Comparing the percentage of multinucleated (tri- and tetranucleated) cells in control cultures between 56 and 72 h, it was found that 20–30% of the cells were multinucleated at the second sampling time. It was suggested that at a fixation time of 72 h a considerable fraction of the cell population had already progressed to the second mitosis in the presence of 6 µg/ml cytochalasin B. In our experiments, cells were cultured for 72 h and treated with 6µg/ml cytochalasin B. As can be seen in Figure 2, the percentage of multinucleated cells in both the Giemsa and FISH experiments in the control cultures was clearly below 20% (lowest yield 7.75%). This is also true for all ASE concentrations tested. In most cultures the multinucleated cells were below 15 or 10%, excluding two experimental points at which this was slightly above 20% (20.78). In another experiment recently carried out by us (data not shown) on control cultures from the first donor, as well as in cultures treated with 5 µg/ml ASE, cytokinesis was blocked by the same concentration of cytochalasin B (6 µg/ml), fixed at 66 h and stained with Giemsa. Although at this earlier fixation time the multinucleated percentage was lower than that at 72 h, it was found that ASE enhanced MN frequency by about the same factor as in the cultures that were fixed at 72 h (3.67 and 3.83 times, respectively). From the above results it seems that in our experiments a small proportion of cells had passed to a second mitosis at the fixation time used, but this proportion does not seem to influence the effect of ASE on human lymphocytes, although fixation time could have partially affected MN frequencies. On the other hand, the same authors (Sgura et al., 1997) did not find any statistically significant difference in the percentage of C+ MN with respect to culture time, although a slight increase was observed. On the contrary, non-disjunction rates were found to increase with time and they suggested that a low percentage of multinucleated cells should be maintained in culture in order to ensure a minimal contribution of dividing binucleate cells, especially when tested for non-disjunction. However, in human lymphocyte cultures treated with 3 µg/ml cytochalasin B it was demonstrated (Falck et al., 1997) that the baseline MN frequency increases with culture time due to an increase in C+ MN, with no influence of C– MN. Nevertheless, a protocol in which a later fixation time (70–72 h) is used is followed by many authors even for non-disjunction rates (Elhajouji et al., 1998; Chang et al., 1999; González et al., 1999; Migliore et al., 1999a,b; Thierens et al., 1999).
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We obtained the same results in both whole blood and isolated lymphocyte cultures. Whole blood cultures contain all the cell types found in normal blood and as it is known that this culture type has some metabolizing capacity of its own. Styrene oxide, a known mutagen, was found to induce sister chromatid exchange in whole blood cultures and this induction was dependent on the presence and number of erythrocytes in the culture (Norppa et al., 1983
). Differences in sensitivity of lymphocytes in whole blood cultures in comparison with isolated lymphocyte cultures have also been found for other chemicals, such as griseofulvin (Kolachana and Smith, 1994) and malathion (Titenko-Holland et al., 1997). Although whole blood cultures are suggested as a better experimental model for the detection of MN induced by chemicals in vitro (Migliore et al., 1989), the evaluation of MN formation in both culture systems would give information on the influence of metabolic activation on the genotoxic and cytotoxic activity of the compounds. From our results it seems that ASE exerts the same clastogenic and aneugenic genotoxic activity in both culture types. On the other hand, the cytotoxicity of the compound seems to be enhanced in isolated lymphocyte cultures, at least at higher concentrations. This could be explained by the assumption that in whole blood cultures ASE is converted to a metabolite by erythrocytes that exerts less cytotoxicity but has the same genotoxic effect as the original compound. A second explanation for the lower cytotoxicity could be that ASE is bound by a specific receptor on the erythrocyte membrane, thus decreasing the ASE concentration in culture, compared with isolated lymphocyte cultures, in which there are no erythrocytes. However, if this explanation was true a decreased genotoxicity of the compound would possibly be observed as well. Hence, the first explanation seems to be preferable. In conclusion our results can be summarized as follows.
- ASE is capable of inducing increased MN frequencies in human lymphocytes. This increase is related to the concentrations in a linear dose-dependent way and is also dependent on the duration of treatment. Enhanced MN frequencies are observed in both culture methods at the same level.
- ASE is cytotoxic in human lymphocytes as evaluated by a reduction in the CBPI. The cytotoxicity is also linearly dose dependent in both cell culture types. However, it seems that ASE cytotoxicity in isolated lymphocyte cultures is significantly greater than in whole blood cultures.
- The study of the mechanism by which MN are created by ASE has shown that it acts mainly via chromosome breakage. However, a weak chromosome loss cannot be excluded, at least at the higher concentrations, in both culture methods.
Taking into consideration the above results as well as the known antineoplastic activity of ASE, it could be said that this activity may be due to genotoxic damage induced by this agent although a straightforward relationship cannot be drawn.
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Notes |
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* To whom correspondence should be addressed. Tel: +30 61 997 168; Fax: +30 61 997 185; Email: geosteph{at}biology.upatras.gr
This paper is dedicated to the memory of P. Catsoulacos, late Professor of Pharmaceutical Sciences at the University of Patras
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References |
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Received on September 14, 1999; accepted on January 21, 2000.
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