Mutagenesis vol. 19 no. 3 pp. 207-213,
May 2004
© 2004 UK Environmental Mutagen Society/Oxford University Press
In vivo kinetics of micronuclei induction by bifunctional alkylating antineoplastics
Departamento de Genética, Instituto Nacional de Investigaciones Nucleares, Apartado Postal 18-1027, México, DF Mexico
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Abstract |
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The aim of the present study was to determine in vivo the kinetics of micronucleated polychromatic erythrocyte (MN-PCE) induction in mice, as an approach for studying the mechanism of micronuclei induction by mitomycin C, cis-diamine dichloroplatinum, busulfan and bis-chloroethylnitrosourea, bifuctional alkylating antineoplastic agents having different patterns of crosslink induction. The kinetics of MN-PCE induction was established by scoring the frequency of MN-PCE in 2000 PCE in peripheral blood, for periods of 8 or 10 h after acute treatment and up to 80 h, with different doses of the agent. The kinetics of MN-PCE induction and particularly the times of maximal induction by different bifunctional alkylating agents were compared with the kinetics previously obtained for ethylnitrosourea, methylnitrosourea and 6-mercaptopurine, agents that cause MN-PCE mainly in the first, second and third divisions after exposure, respectively. The results obtained in the present study allow us to conclude that: (i) bifunctional alkylating agents have very different efficiencies of genotoxic and cytotoxic action; (ii) all assayed bifunctional alkylating agents induced micronuclei during the first cell division, owing to the mistaken repair of primary lesions, e.g. excision; (iii) busulfan and bis-chloroethylnitrosourea showed an additional late mechanism of micronuclei induction, which is expressed at the third division and seems to be related to the mismatch repair process.
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Introduction |
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The study of the kinetics of micronucleated polychromatic erythrocyte (MN-PCE) induction by mutagens has led us to establish that this depends on different events which occur from the time of administration of the agent to the moment MN-PCE appear in the blood stream (Morales-Ramírez et al., 1997
; Morales-Ramírez and Vallarino-Kelly, 1998). However, some of these events seem to be determinant for the kinetics: (i) the pharmacokinetics of the agents, including the need for metabolic activation; (ii) the inhibitory effect of the agent on cell proliferation; (iii) the mechanism of micronucleous production (Morales-Ramírez and Vallarino-Kelly, 1999).
The effect of the pharmacokinetics was established by comparing the kinetics of MN-PCE induction by chemical agents with that of MN-PCE produced by 
-rays, using doses of the agent that do not cause cytotoxicity. The role of cytotoxicity was determined by the rate of PCE induction with respect to erythrocytes, which incidentally also allows one to determine the effect of the agent on cell proliferation (Morales-Ramírez and Vallarino-Kelly, 1999; Vallarino-Kelly and Morales-Ramírez, 2001).
With regard to the mechanism of micronuclei induction, the alkylating agents have different affinities for the nucleophilic sites on DNA (Beranek, 1990); this is why it is impossible to establish which lesion is involved in the production of micronuclei. Besides, the transformation mechanism of DNA alkylation in DNA breaks is mediated by a different enzymatic process related to DNA repair (Armstrong and Galloway, 1997; Allan et al., 1998).
Previous studies indicate that most mutagens cause maximal induction of MN-PCE in peripheral blood at 
30 h (Morales-Ramírez et al., 1997; Morales-Ramírez and Vallarino-Kelly, 1998; Vallarino-Kelly and Morales-Ramírez, 2001). However, evidence has been obtained showing that methylnitrosourea (MNU) and 6-mercaptopurine (6-MOP) require 8 and 19 h more, respectively (Morales-Ramírez et al., 1997; Morales-Ramírez and Vallarino-Kelly, 1999). These times are nearly one and two times the cell cycle duration in murine bone marrow cells (Rodríguez-Reyes and Morales-Ramírez, 2003); the delay does not seem to be explained by the pharmacokinetics or the cytotoxicity. In fact, the need for an additional cell division for MN-PCE induction agrees with results obtained by analyzing chromosome aberrations, which indicate that MNU caused persistent alkylation that was misprocessed by the cell as mismatches, which in turn produced DNA breaks that were expressed as chromosome breaks in the second division (Armstrong and Galloway, 1997). 6-Thioguanine (2-amino-6-mercaptopurine) was incorporated into DNA in the first cell division, which caused mismatches that could not be solved by the cell during the second division and generated the subsequent production of chromosome breaks in the third division (Armstrong and Galloway, 1997).
Bifunctional alkylating agents (BAA) in general produce crosslinks (CL) in DNA, which can be interstrand, as are those induced by mitomycin C (MMC) (Millard et al., 1991; Tomasz, 1995) and by bis-chloroethylnitrosourea (BCNU) (Bedford and Eisenbrand, 1984), or they can be intrastrand, as are those induced by cis-diamine dichloroplatin (cis-Pt) (Jones et al., 1991), or DNA–protein, as are those that are preferentially induced by acetaldehyde (Merk and Speit, 1998) or busulfan (Bus) (Pacheco et al., 1989, 1990; Hincks et al., 1990). These agents, however, also cause single-strand adducts. This is because CLs require two nucleophilic sites appropriately separated so as to allow the BAA molecule to react with both. If this condition is not satisfied, only a single-strand adduct can be formed. In fact, it has been reported that elimination by DNA methyltransferase of O6-chloroethylguanine methylguanine adducts induced by BCNU reduces the probability of CL formation (Mitra and Kaina, 1993), indicating that CL formation is a two-step process. This process seems to be unexplainably slow; evidence has been reported that the maximum frequency of CL caused by BCNU is obtained 6 h after treatment (Khon, 1977).
The aim of the present study was to determine the in vivo kinetics of MN-PCE induction in mice, as an approach to analyzing the mechanism of MN induction by bifuctional alkylating antineoplastic agents having different patterns of CL induction.
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Materials and methods |
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Protocol
The kinetics of MN-PCE induction was established by scoring the frequency of MN-PCE in 2000 PCE in peripheral blood, for periods of 8 or 10 h after acute treatment with different doses of the agent and up to 80 h, in groups of at least three mice.
Rationale
The kinetics of MN-PCE induction and particularly the times of maximal induction of different BAA agents were compared with the kinetics previously obtained for ethylnitrosourea (ENU) (Morales-Ramírez and Vallarino-Kelly, 1998), MNU (Morales-Ramírez and Vallarino-Kelly, 1999) and 6-MOP (Vallarino-Kelly and Morales-Ramírez, 2001), agents that cause MN-PCE mainly in the first, second and third divisions after exposure, respectively.
Animals
Two- to three-month-old BALB/c male mice weighing 30 g were used in the study. The animals were maintained and bred in our laboratory, under controlled conditions of temperature and dark-light periods, and fed with Purina chow for small rodents and water ad libitum.
Slide
Four samples were obtained from each mouse at each time. A small segment of the tail end was cut and a drop of blood was placed on a slide containing a drop of fetal calf serum, then a smear was prepared. The dried smear was stained using the May–Grunwald–Giemsa technique (Schmid, 1975) and mounted in resin.
MN-PCE analysis
The frequency of MN-PCE was determined in 2000 erythrocytes per mouse, in at least three mice per group. The scoring of micronuclei was done according to the following criteria: (i) that the MN were round; (ii) that they had a diameter of 1/20–1/5 of the erythrocyte; (iii) that they were stained deep purple.
Kinetics of MN-PCE induction
The kinetics of MN-PCE induction was established by the curves of MN-PCE frequency with respect to time, in which the maximum frequency was determined in the adjusted curves using the Microcal Origin V-6 PC program.
Clastogenic efficiency
The total clastogenic efficiency was determined as the area beneath the curve of MN-PCE frequency with respect to time. The efficiency was also estimated as the maximal induction of MN-PCE. The relative clastogenic efficiency was calculated by dividing the total or the maximal efficiency by the dose.
Cytotoxicity
Cytotoxicity was estimated as the slope of the curve of PCE rate with respect to time, from zero time up to the time of lowest frequency. 400 erythrocytes were scored at different times before and after the treatment.
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Results |
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The kinetic curves of MN-PCE induction by different doses of MMC, cis-Pt, Bus and BCNU are shown in Figure 1. The curves for MMC are similar and suggest a dose-dependent response. The curves of MN-PCE induction caused by cis-Pt are also similar and dose-dependent, although the time of maximal response is clearly delayed as the dose rises. Low doses seem to induce additional small increases in MN-PCE after the times of the main curve.
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The kinetic curves obtained for treatment with Bus reveal a peculiar behavior. The lower doses (30 and 60 µmol/kg) showed an increment proportional to dose and a maximum induction at 30 h. The 80 µmol/kg dose produced a curve with a shoulder at 30 h, but a higher increase at nearly 50 h. The 100 µmol/kg dose showed a maximum increase at 50 h and then suddenly fell, but there remained a small increase at 30 h.
The response obtained on BCNU administration strongly suggests the presence of two successive curves of MN-PCE induction that increase proportionally to dose. The earlier curve rises to a maximum at 30 h and the later one rises to the maximum at nearly 50 h. With the highest dose both curves merge and their presence is only suggested by a wider and deformed profile.
Figure 2 shows the kinetics curves for cytotoxicity measured in terms of the difference in percentage with respect to the initial PCE frequency. The MMC curve for both doses did not cause an immediate reduction in PCE, but rather an increase. Forty-eight hours after treatment, a clear reduction in PCE percentage was observed. With respect to the curves induced by cis-Pt, the lower doses caused a substantial increase in PCE, but the highest dose caused a reduction, which was more pronounced 40 h after treatment. The two low doses of Bus caused a rise in PCE after 50 h, which at the last time point scored was about twice the initial frequency, however, a strong dispersion was found. The two higher doses of Bus dramatically decreased the frequency of PCE to 20%, and to almost 0% at the last sampling time. The curves for the two lower doses of BCNU indicated a marginal reduction in PCE. The high dose produced a 50% reduction in PCE, but a significant recovery appeared at the last time point scored.
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Table 1 shows the maximal induction of genotoxicity and cytotoxicity caused by different doses of BAA. All agents induced a maximum response at nearly 32 h (T1); BCNU and higher doses of Bus also caused a late response, between 46 and 48 h (T2). The genotoxic efficiency measured as the area beneath the curve, or the maximal response with respect to dose, is very different for the BAA agents. The least efficient agent was Bus, with a range between 0.13 and 0.38 PCE-MN/µmol/kg, followed by BCNU with 1.92–2.12 and cis-Pt with an efficiency of 16.6–23.24. MMC was the most efficient BAA with a range between 24.9 and 25.6 PCE-MN/µmol/kg.
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The efficiencies obtained from the maximal response were very reproducible, except for those found with Bus, whose low doses caused half the efficiency observed with high doses. However, as was previously mentioned, the production of MN by high doses of Bus was by a late response mechanism.
With regard to the cytotoxicity index obtained from the slope of the curve of PCE frequency versus time, some interesting findings can be noted from zero time to the time of maximal cytotoxicity. For example, low doses of cis-Pt or Bus caused an increase in PCE, which is reflected in positive slopes. Doses of 0.93 and 1.86 µmol cis-Pt produced slopes of +1.6 and +1.44 and doses of 30 and 60 µmol Bus resulted in slopes of +1.53 and +1.44. The high doses of cis-Pt and Bus brought about an increase in cytotoxicity, reflected in the slopes, which were –1.17 and from –1.28 to –1.42, respectively. MMC is clearly cytotoxic even at a lower dose, such as that of BCNU. However, the tendency of the curves is quite variable: in some cases, as with the two higher doses of Bus, the curves could clearly be adjusted to a straight line (r = 0.98), but in other instances, as with MMC, they first showed an increase, then a dramatic decrease in the frequency of PCE, as shown in Figure 2.
Interestingly enough, we observed that cytotoxicity did not affect the kinetics of MN-PCE induction; this was also exemplified by the response obtained on treatment with MMC. Whereas the kinetics of MN-PCE induction is very regular and reproducible (Figure 1), the kinetics of the frequency of PCE (Figure 2) varies substantially.
In Figure 3, the profiles of the kinetics of MN-PCE obtained with the BAA are compared with those previously obtained in the same system by exposure to ENU, MNU and 6-MOP (Morales-Ramírez et al., 1997; Morales-Ramírez and Vallarino-Kelly, 1998, 1999). ENU induced MN in the first division, MNU mainly in the second and 6-MOP did so in partly the second but mostly in the third. The difference in the times of maximal frequency was nearly 9 h, which is the cell cycle duration reported earlier for murine bone marrow cells in vivo (Rodríguez-Reyes and Morales-Ramírez, 2003). The figure shows that all BAA induced MN-PCE in the period corresponding to the first division, as did ENU. This period corresponds to the early induction of MN by Bus and BCNU. These agents have a second peak that corresponds to the third division.
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Discussion |
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BAA that cause CL are widely used as antineoplastics (Erlichman, 1988
). Evidence has been obtained suggesting that their cytotoxicity is due to apoptosis induced by failed repair of DNA lesions, but a necrotic process could also be involved (Fuertesa et al., 2003). BAA have the possibility not only to produce lesions in different sites on DNA, but also to cause different types of adducts and different kinds of CL, both intrastrand and interstrand, in DNA. Furthermore, they can generate CL between DNA and other molecules, such as proteins. The generation of CL is a special problem for the cell because they could cause close single-strand breaks or double-strand breaks during excision repair. Under these circumstances the BAA must be particularly efficient in producing double-strand breaks, which in turn can result in chromosome breaks and MN.
The results obtained in the present study indicate that BAA have variable efficiencies in MN-PCE induction, nevertheless, they all proved to be more efficient than the monofunctional alkylating agent ENU in the same experimental model (Morales-Ramírez and Vallarino-Kelly, 1998). Setting the ENU efficiency to unity (21 MN-PCE/214 µmol/kg), the efficiencies for Bus, BCNU, cis-Pt and MMC were 3.6, 20, 200 and 250 times higher, respectively. MNU is 20 times more efficient than ENU, although it is also a monofunctional alkylating agent. However, MNU induces MN in the second division post-treatment (Morales-Ramírez and Vallarino-Kelly, 1999) and probably through failed mismatch repair (Armstrong and Galloway, 1997).
The fact that the alkylating agents were not capable of directly inducing DNA breaks implies that breaks are the result of the processing of lesions by the different repair processes, excision, homologous or non-homologous recombination and even mismatch repair. The latter, as mentioned before, seems to be the factor responsible for late induction of MN (Armstrong and Galloway, 1997).
The results obtained in the present study indicate that all BAA are capable of inducing MN in the first division. MMC and cis-Pt seem to induce MN mainly in the first division, if not exclusively. BCNU additionally induces a substantial increase in MN in the third division, and high doses of Bus induce the major increase in this division as well. Based on our earlier studies with -rays and ENU, we have found that this effect is not due to cell toxicity, because cytotoxicity usually causes a gradual delay in MN-PCE production and only early induction. Both the Bus and BCNU effects clearly differ from this behavior. Besides, the cytotoxic doses of MMC and cis-Pt did not produce two peaks, but rather caused a gradual delay in MN-PCE induction.
However, it is not possible to compare the action of Bus and BCNU with that of 6-MOP and 6-thiogunanine, agents that have been shown to be capable of producing DNA breaks as late as the third division (Armstrong and Galloway, 1997; Morales-Ramírez and Vallarino-Kelly, 1997). This is so because these agents require three divisions to cause MN, as their incorporation into DNA in the first division supposedly causes breaks due to mismatch repair in the second DNA duplication, which in turn causes breaks in the third division (Armstrong and Galloway, 1997).
An approach to establishing the mechanism involved in late MN production is to compare the process related to repair of lesions caused by MMC and cis-Pt with that involved in the repair of lesions caused by BCNU and Bus.
MMC specifically and efficiently induces CpG interstrand CL (Millard et al., 1991; Tomasz, 1995). Furthermore, there is evidence that homologous recombination is involved in the repair of these lesions (Essers et al., 2000; Moynahan et al., 2001; Pluth et al., 2001; Donoho et al., 2003), as is excision repair (Engelward et al., 1996; Allan et al., 1998; Ochs et al., 1999), but not O6-alkylguanine-DNA methyltransferase (Gustafson et al., 1997; Glassner et al., 1999). cis-Pt causes 85% intrastrand CL (Jones et al., 1991) and excision of nucleotides seems to be involved in the repair of such lesions (Sibghat-Ullah et al., 1989; Vilpo et al., 1995). Contradictory results have been published with respect to the effect of the loss of mismatch repair on cell resistance to cis-Pt, which would imply that this repair system is related to the genotoxic consequences of the lesions induced by cis-Pt (Fink et al., 1998a; Vernole et al., 2003). Besides, the processing of lesions induced by cis-Pt seems to be different from that of the lesions induced by BCNU, because cell mutants resistant to BCNU were not resistant to cis-Pt (Bodell et al., 1985b; Aida and Bodell, 1987).
According to what was mentioned above and considering that MMC and cis-Pt did not cause late PCE-MN, the processing of lesions by excision or homologous recombination does not seem to be related to the late induction of MN.
BCNU preferably induces interstrand CL; it also induces single-strand breaks with kinetics which suggest that these breaks were induced by two mechanisms, an early one around 15 h after exposure and another late one at 36 h (Bedford and Eisenbrand, 1984). This response is homologous to that obtained in the present study. There is evidence that BCNU-induced lesions are repaired by O6-alkylguanine-DNA methyltransferase (Khon, 1977; Bodell et al., 1985a; Schwartz et al., 1989; Maze et al., 1996; Kokkinakis et al., 1999) and by excision (Engelward et al., 1996; Allan et al., 1998). These data indicate that a fault in elimination of O6-chloroethylguanine by O6-alkylguanine-DNA methyltransferase raises the probability of inducing CL (Mitra and Kaina, 1993) and that CL formation is a slow process which occurs in two steps. This last alternative is supported by evidence that maximum CL induction by BCNU is achieved 6 h after treatment (Khon, 1977).
The Bus response is more complex, since a small dose range determines the appearance of the late mechanism of MN induction, with only a slight presence of the early mechanism. Different hypotheses could be proposed: saturation of the process involved in early expression, formation of a new dose-dependent lesion and even formation of a secondary lesion. However, although there is little experimental support, this agent has been used extensively in bone marrow ablation prior to transplant (Schuler et al., 1998; Dix et al., 1996).
Earlier publications indicate that Bus mainly causes DNA–protein or intrastrand CL (Hincks et al., 1990; Pacheco et al., 1989, 1990). In another study, evidence was obtained showing that Bus produces interstrand DNA CL (Bedford and Fox, 1982). Bus does not produce adducts susceptible to repair by O6-alkylguanine-DNA methyltransferase (Westerhof et al., 2001), but a deficiency in mismatch repair confers cell resistance to treatment with Bus, suggesting that this repair mechanism plays a role in the genotoxic effect of Bus (Fink et al., 1998a).
Three lines of evidence suggest that mismatch repair is involved in the mechanism of late induction of MN by Bus and BCNU: (i) the analogy of late MN induction by MNU and 6-MOP (Armstrong and Galloway, 1997); (ii) the homology of the late breaks induced by BCNU (Bedford and Eisenbrand, 1984) with the late time of MN induction observed in the present study; (iii) the increased resistance to Bus of cells deficient in mismatch repair (Fink et al., 1998a), whereas no sensitivity is caused to MMC (Lindor et al., 1998) and only slight sensitivity is produced to cis-Pt (Fink et al., 1998b).
The results obtained in the present study allow us to conclude the following: (i) BAA have very different efficiencies of genotoxic and cytotoxic action; (ii) all assayed BAA induced MN during the first cell division, owing to mistaken repair of primary lesions, e.g. excision; (iii) Bus and BCNU reveal an additional late mechanism of MN induction, which is expressed at the third division and seems to be related to the mismatch repair process.
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Acknowledgements |
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We wish to thank Angel Reyes, Perfecto Aguilar, Miguel Angel García and Felipe Beltrán for their excellent technical assistance and Rosa María Noriega for English editing. This study was supported by the Consejo Nacional de Ciencia y Tecnología (National Council of Science and Tecnology), Project 33167-N.
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Notes |
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1To whom correspondence should be addressed. Tel: +52 5329 7200; Fax: +52 5329 7310; Email pmr{at}nuclear.inin.mx
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References |
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