Mutagenesis vol. 19 no. 3 pp.

207±213, 2004

DOI: 10.1093/mutage/geh018

In vivo kinetics of micronuclei induction by bifunctional alkylating antineoplastics1

 Pedro Morales-Ramõrez1, Teresita Vallarino-Kelly,  Virginia L.Cruz-Vallejo, Rosario Lopez-Iturbe and Horacio Alvaro-Delgadillo
 Departamento de Genetica, Instituto Nacional de Investigaciones Nucleares,  Apartado Postal 18-1027, Mexico, DF Mexico

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 6mercaptopurine, agents that cause MN-PCE mainly in the ®rst, 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 ef®ciencies of genotoxic and cytotoxic action; (ii) all assayed bifunctional alkylating agents induced micronuclei during the ®rst 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.

Introduction 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-Ramõrez et al.,  1997; Morales-Ramõrez 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-Ramõrez 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 g-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-Ramõrez and Vallarino-Kelly, 1999; Vallarino-Kelly  and Morales-Ramõrez, 2001). With regard to the mechanism of micronuclei induction, the alkylating agents have different af®nities 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  Ramõrez et al., 1997; Morales-Ramõrez and Vallarino-Kelly,  1998; Vallarino-Kelly and Morales-Ramõrez, 2001). However, evidence has been obtained showing that methylnitrosourea (MNU) and 6-mercaptopurine (6-MOP) require 8 and 19 h  more, respectively (Morales-Ramõrez et al., 1997; Morales Ramõrez and Vallarino-Kelly, 1999). These times are nearly one and two times the cell cycle duration in murine bone   marrow cells (Rodrõguez-Reyes and Morales-Ramõrez, 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 ®rst 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 satis®ed, only a singlestrand 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), indicat207

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28 ±1.7 5. bis-chlorodiethylnitrosourea. resultant slope of the curve of PCE versus time (h).5 37.44 ±1. Table I. The aim of the present study was to determine the in vivo kinetics of MN-PCE induction in mice.13 0. times of maximal induction of MN-PCE.09 1.2 47.5 54.9 ND 5 3 4 4 4 4 5 5 5 5 5 5 T1max and T2max.0 7.2 Cytotoxicity M (PCE/h) ±0. cis-diamine dichloroplatin. P. Kinetics of MN-PCE induction caused by different doses of MMC.38 2.36 0.7 37. cis-Pt.0 31. 2012 Fig.5 32.5 Area beneath curve (ABC) PCE-MN time 654 1419 267 554 1394 70 179 619 574 900 1896 3091 Ef®ciency Rmax dose 24.5 35 70 33.23 n MMC cis-Pt Bus BCNU 1. Bus.17 1.3 3. Downloaded from http://mutage. no discerned.12 2. number of animals.9 12.7 51.7 32.2 44.6 134.26 ±0.0 32.82 ±1. Materials and methods Protocol The kinetics of MN-PCE induction was established by scoring the frequency of MN-PCE in 2000 PCE in peripheral blood. cis-Pt. Bus and BCNU.50 16.61 23. 1977).63 ±1.oxfordjournals.4 76.6 33. This process seems to be unexplainably slow.8 28.7 3. Points represent the average and standard error of the frequencies obtained in at least three mice.92 ABC dose 436 473 287 298 422 2.Morales-Ramõrez et al.42 ±0. MMC.8 45. evidence has been reported that the maximum frequency of CL caused by BCNU is obtained 6 h after treatment (Khon.7 20 30.53 1.1 70.24 0.9 25.0 38. m.3 30 60 80 100 17. 1.1 34.0 ND 32. Genotoxicity and cytotoxicity induced by different dose of bifunctional alkylating antineoplastic agents Agent Dose (mmol/kg) T1max (h) T2max (h) Maximal response (Rmax) PCE-MN 2000 PCE 37.0 33.21 0.6 21. ND. n.3 48 47 47.93 1. as an approach to analyzing the mechanism of MN induction by bifuctional 208 alkylating antineoplastic agents having different patterns of CL induction.5 by guest on February 5.9 76. mitomycin C.44 ±1.02 1. ing that CL formation is a two-step process.86 3. busulfan. for periods of 8 or 10 h after acute .60 1.0 0. BCNU.

 1998). The dried smear was stained using the May±Grunwald±Giemsa technique (Schmid. With respect to the curves induced by cis-Pt. MMC was the most ef®cient BAA with a range between 24. as with MMC. from zero time up to the time of lowest frequency.86 mmol cis-Pt produced slopes of +1.9 and 25.6 and +1. the kinetics of the frequency of PCE (Figure 2) varies substantially. Whereas the kinetics of MN-PCE induction is very regular and reproducible (Figure 1). some interesting ®ndings can be noted from zero time to the time of maximal cytotoxicity. For example. as with the two higher doses of Bus. 1999).17 and from ±1. which is the cell cycle duration reported earlier for murine bone marrow cells   in vivo (Rodrõguez-Reyes and Morales-Ramõrez. The ef®ciency was also estimated as the maximal induction of MN-PCE. second and third divisions after exposure. they ®rst showed an increase. but in other instances. 1975) and mounted in resin.44. Low doses seem to induce additional small increases in MN-PCE after the times of the main curve. The scoring of micronuclei was done according to the following criteria: (i) that the MN were round.42.Micronuclei induction by antineoplastics treatment with different doses of the agent and up to 80 h. but the highest dose caused a reduction. Doses of 0. as was previously mentioned. However. 2003). ENU induced MN in the ®rst division. The least ef®cient agent was Bus.44 and doses of 30 and 60 mmol Bus resulted in slopes of +1. The response obtained on BCNU administration strongly suggests the presence of two successive curves of MN-PCE induction that increase proportionally to dose. The ®gure shows that all BAA induced MN-PCE in the period 209 Downloaded from http://mutage.93 and 1. 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-Ramõrez and Vallarino-Kelly. Interestingly enough. The two higher doses of Bus dramatically decreased the frequency of PCE to 20%. and fed with Purina chow for small rodents and water ad libitum. 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. Results The kinetic curves of MN-PCE induction by different doses of MMC. in at least three mice per group. re¯ected in the slopes. respectively. The relative clastogenic ef®ciency was calculated by dividing the total or the maximal ef®ciency by the dose. in which the maximum frequency was determined in the adjusted curves using the Microcal Origin V-6 PC program.24. Slide Four samples were obtained from each mouse at each time. followed by BCNU with 1. Table I shows the maximal induction of genotoxicity and cytotoxicity caused by different doses of BAA. MNU mainly in the second and 6-MOP did so in partly the second but mostly in the third. we observed that cytotoxicity did not affect the kinetics of MN-PCE induction. but a higher increase at nearly 50 h. Bus and BCNU are shown in Figure 1. With regard to the cytotoxicity index obtained from the slope of the curve of PCE frequency versus time. with a range between 0.6±23. The 80 mmol/kg dose produced a curve with a shoulder at 30 three-month-old BALB/c male mice weighing 30 g were used in the study. The curves for the two lower doses of BCNU indicated a marginal reduction in PCE. The ef®ciencies obtained from the maximal response were very reproducible. a clear reduction in PCE percentage was observed. such as that of BCNU.6 PCE-MN/mmol/kg. agents that cause MN-PCE mainly in the ®rst. between 46 and 48 h (T2). MN-PCE analysis The frequency of MN-PCE was determined in 2000 erythrocytes per mouse. The genotoxic ef®ciency measured as the area beneath the curve. under controlled conditions of temperature and dark-light periods. or the maximal response with respect to dose. The high dose produced a 50% reduction in PCE. in groups of at least three mice.. 400 erythrocytes were scored at different times before and after the treatment. the curves could clearly be adjusted to a straight line (r = 0. Morales-Ramõrez and Vallarino-Kelly.53 and +1. MNU (Morales-Ramõrez and Vallarino-Kelly. 1999) and 6-MOP  (Vallarino-Kelly and Morales-Ramõrez. (iii) that they were stained deep purple. BCNU and higher doses of Bus also caused a late response. the lower doses caused a substantial increase in PCE. Figure 2 shows the kinetics curves for cytotoxicity measured in terms of the difference in percentage with respect to the initial PCE frequency. The earlier curve rises to a maximum at 30 h and the later one rises to the maximum at nearly 50 h. The curves for MMC are similar and suggest a dose-dependent response. 1998. Forty-eight hours after treatment. low doses of cis-Pt or Bus caused an increase in PCE. respectively. The lower doses (30 and 60 mmol/kg) showed an increment proportional to dose and a maximum induction at 30 h. is very different for the BAA by guest on February 5. which at the last time point scored was about twice the initial frequency.oxfordjournals. but a signi®cant recovery appeared at the last time point scored. the production of MN by high doses of Bus was by a late response mechanism. MNU and 6-MOP   (Morales-Ramõrez et al.28 to ±1. which was more pronounced 40 h after treatment.13 and 0. In Figure 3. except for those found with Bus. the pro®les of the kinetics of MN-PCE obtained with the BAA are compared with those previously obtained in the same system by exposure to ENU. whose low doses caused half the ef®ciency observed with high doses. The two low doses of Bus caused a rise in PCE after 50 h. 1997. Kinetics of MN-PCE induction The kinetics of MN-PCE induction was established by the curves of MN-PCE frequency with respect to time. as shown in Figure 2. 2012 . MMC is clearly cytotoxic even at a lower dose. but there remained a small increase at 30 h. then a smear was prepared. 2001).38 PCE-MN/ mmol/kg. Clastogenic ef®ciency The total clastogenic ef®ciency was determined as the area beneath the curve of MN-PCE frequency with respect to time. Animals Two. (ii) that they had a diameter of ~1/20±1/5 of the erythrocyte. However. All agents induced a maximum response at nearly 32 h (T1). The kinetic curves obtained for treatment with Bus reveal a peculiar behavior. which is re¯ected in positive slopes. Cytotoxicity Cytotoxicity was estimated as the slope of the curve of PCE rate with respect to time. the tendency of the curves is quite variable: in some cases.98). The animals were maintained and bred in our laboratory. which were ±1. The 100 mmol/kg dose showed a maximum increase at 50 h and then suddenly fell. The difference in the times of maximal frequency was nearly 9 h. however. With the highest dose both curves merge and their presence is only suggested by a wider and deformed pro®le.92±2.12 and cis-Pt with an ef®ciency of 16. The MMC curve for both doses did not cause an immediate reduction in PCE. a strong dispersion was found. The high doses of cis-Pt and Bus brought about an increase in cytotoxicity. although the time of maximal response is clearly delayed as the dose rises. cis-Pt. The curves of MN-PCE induction caused by cis-Pt are also similar and dose-dependent. but rather an increase. and to almost 0% at the last sampling time. this was also exempli®ed by the response obtained on treatment with MMC. then a dramatic decrease in the frequency of PCE.

excision. homologous or non-homologous recombination and even mismatch repair. Under these circumstances the BAA must be particularly ef®cient in producing double-strand breaks.Morales-Ramõrez et by guest on February 5. MNU induces MN in the second  division post-treatment (Morales-Ramõrez and VallarinoKelly. 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. 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. 2003). nevertheless. However. but a necrotic process could also be involved (Fuertesa et al. 200 and 250 times higher. The latter. Based on our earlier studies with g-rays and ENU. and high doses of Bus induce the major increase in this division as well. although it is also a monofunctional alkylating agent. the ef®ciencies for Bus. MMC and cis-Pt seem to induce MN mainly in the ®rst division. Discussion BAA that cause CL are widely used as antineoplastics (Erlichman. BAA have the possibility not only to produce lesions in different sites on DNA. Curves of the PCE percentage with respect to the initial PCE frequency at different times after exposure to various doses of crosslinkers. but also to cause different types of adducts and different kinds of CL. respectively. 2. they can generate CL between DNA and other molecules. The results obtained in the present study indicate that BAA have variable ef®ciencies in MN-PCE induction. BCNU additionally induces a substantial increase in MN in the third division. This period corresponds to the early induction of MN by Bus and BCNU. which in turn can result in chromosome breaks and MN. Setting the ENU ef®ciency to unity (21 MN-PCE/214 mmol/kg). These agents have a second peak that corresponds to the third division. The data represent the average of the frequencies obtained in the same animals scored for MN-PCE frequency. as did ENU. 1988). 2012 Fig. corresponding to the ®rst division. 20.6. 1998). BCNU. in DNA. MNU is 20 times more ef®cient than ENU. seems to be the factor responsible for late induction of MN (Armstrong and Galloway. 1999) and probably through failed mismatch repair (Armstrong and Galloway. if not exclusively.. both intrastrand and interstrand. 1997). Downloaded from http://mutage. 210 they all proved to be more ef®cient than the monofunctional alkylating agent ENU in the same experimental model  (Morales-Ramõrez and Vallarino-Kelly. as mentioned before. such as proteins. The results obtained in the present study indicate that all BAA are capable of inducing MN in the ®rst division. Furthermore. cis-Pt and MMC were 3. P.oxfordjournals. Evidence has been obtained suggesting that their cytotoxicity is due to apoptosis induced by failed repair of DNA lesions. we have found that this effect is . 1997).

1999). 1998.. However. 1996.. 1999). 2003). According to what was mentioned above and considering that MMC and cis-Pt did not cause late PCE-MN. 1996. 2000.. agents that have been shown to be capable of producing DNA breaks as late as the third division (Armstrong and Galloway. Allan et al. 1991. Vilpo et al... not due to cell toxicity. as their incorporation into DNA in the ®rst division supposedly causes breaks due to mismatch repair in the second DNA duplication. since a small dose range determines the appearance of the late mechanism of MN induction. The curves were selected as the low dose that causes a clear response.. Furthermore. the processing of lesions induced by cis-Pt seems to be different from that of the lesions induced by BCNU. but not O6-alkylguanine-DNA methyltransferase (Gustafson et al. MMC speci®cally and ef®ciently induces CpG interstrand CL (Millard et al. it also induces single-strand breaks with kinetics which suggest that these breaks were induced by two mechanisms. The Bus response is more by guest on February 5. which would imply that this repair system is related to the genotoxic consequences of the lesions induced by cis-Pt (Fink et al. This last alternative is supported by evidence that maximum CL induction by BCNU is achieved 6 h after treatment (Khon. Both the Bus and BCNU effects clearly differ from this behavior.. 2001. This response is homologous to that obtained in the present study. 1997.. Schwartz et al. 1977). with only a slight presence of the early mechanism. Pluth et al. in order to diminish the effect of toxicity. 1997. Curves are compared with   those previously obtained by treatment with 6-mercaptopurine (Morales-Ramõrez et al... 2003). 1985b. but rather caused a gradual delay in MN-PCE induction. an early one around 15 h after exposure and another late one at ~36 h (Bedford and Eisenbrand. 2012 Fig. Besides. 1993) and that CL formation is a slow process which occurs in two steps. 1987).oxfordjournals. Allan et al.. Moynahan et al. cis-Pt causes 85% intrastrand CL (Jones et al. 1985a.. 1998. Vernole et al. Bodell et al. 2001. Kokkinakis et al. 1989. ENU and MNU (Morales-Ramõrez and Vallarino-Kelly. BCNU preferably induces interstrand CL. 3. 1995). 1997). Donoho et al. which in turn causes breaks in the third division (Armstrong and Galloway. Pro®les of the curves of MN-PCE frequency versus time induced by different bifunctional alkylating antineoplastic agents. These data indicate that a fault in elimination of O6chloroethylguanine by O6-alkylguanine-DNA methyltransferase raises the probability of inducing CL (Mitra and Kaina.. Ochs et al. 1997). Maze et al..Micronuclei induction by antineoplastics Downloaded from http://mutage.. 1995). 1999) and by excision (Engelward et al... as is excision repair (Engelward et al. 1997). 211 . Aida and Bodell.. 1998a. because it represents the transition dose effect. Numbers near the mutagen names represent the dose in mmol/kg.. 1989. Besides. because cytotoxicity usually causes a gradual delay in MN-PCE production and only early induction.. There is evidence that BCNUinduced lesions are repaired by O6-alkylguanine-DNA methyltransferase (Khon. 1998).. 1977. the cytotoxic doses of MMC and cis-Pt did not produce two peaks. 1984). Tomasz. 1999). 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.. the processing of lesions by excision or homologous recombination does not seem to be related to the late induction of MN. Contradictory results have been published with respect to the effect of the loss of mismatch repair on cell resistance to cis-Pt. because cell mutants resistant to BCNU were not resistant to cis-Pt (Bodell et al. 1991) and excision of nucleotides seems to be involved in the repair of such lesions (Sibghat-Ullah et al. This is so because these agents require three divisions to cause MN. there is evidence that homologous recombination is involved in the repair of these lesions (Essers et al. Glassner et al. 1996. it is not possible to compare the action of Bus and BCNU with that of 6-MOP and 6-thiogunanine..  Morales-Ramõrez and Vallarino-Kelly. except for the 80 mmol/kg dose of busulfan.

(1997) Mismatch repair provokes chromosome aberrations in hamster cells treated with methylating agents or 6-thioguanine.. Khon.F.N. 44. Vogel. Somat.C.O.J.J.. and Hopkins..3-bis(2-chloroethyl)-1nitrosourea. 36. but a de®ciency in mismatch repair confers cell resistance to treatment with Bus. Cancer Genet. (1998) Pharmacokinetic parameters determined from the clastogenic activity of ethylnitrosourea (ENU) and dimethylnitrosamine (DMN) in mice in vivo. and Williams. nitrogen mustard and cis-diamminedichloroplatinum(II) in human glialderived cell lines. Wilczynski. Maze. suggesting that this repair mechanism plays a role in the genotoxic effect of Bus (Fink et al. 104.. and Kaina. Different hypotheses could be proposed: saturation of the process involved in early expression. Proc. 58. 412.M.. 315±322. 292±307.M.. Kurahara. In another study.S.B. e.I. 1998a). Cell Mol. Res. 119±126 Bodell. Res..H. Fink. Jones. (1999) DNA repair methyltransferase (Mgmt) knockout mice are sensitive to the lethal effects of chemotherapeutic alkylating agents..D. Fink. and Gibson.. Cancer Res. 7559±7563. Nucleic Acid Res.. Dahl. The Basic Science of Oncology. Adlakha. (2003) Deletion of Brca2 exon 27 causes hypersesitivity to DNA crosslinks. (1998b) Enrichment for DNA mismatch repair-de®cient cells during treatment with cisplatin. and Speit. whereas no sensitivity is caused to MMC (Lindor et al. Miguel Angel Garcõa and   Felipe Beltran for their excellent technical assistance and Rosa Marõa Noriega for English editing.J.H.D.R. Cannistra....A.A.L. 17.S.J.T. Kirchner. Huszard. and Howell.. Aida.M. New York. Bedford. J. Biol. Trotter.J.J. 31±38.M. 3±7.J. Chen. Cancer Res. and Bohr.W. Perfecto Aguilar. Earlier publications indicate that Bus mainly causes DNA± protein or intrastrand CL (Hincks et al. (1982) Repair of DNA interstrand crosslinks after busulphan.A.B.P.. Acknowledgements  We wish to thank Angel Reyes.D. Parker.W.. Weidner.H.J. 266. Armstrong. Allan. Mutagen.R.. Prog.P.M. and Fox...J. 9±17. 1997). Natl Acad. Nucleic Acids Res. 62.P.. Alonsoa.A.T. Pharmacol.N. Baergen.. (1977) Interstrand crosslinking of DNA by 1.J. de Wit. Cancer Res. and Hill. (1988) The pharmacology of anticancer drugs. (1996) Repair de®cient 3 methyladenine DNA glycosylase homozygous mutant mouse cells have increased sensitivity to alkylationinduced chromosome damage and cell killing.P.. 5.M. NY. Bus does not produce adducts susceptible to repair by O6-alkylguanine-DNA methyltransferase (Westerhof et al. 1989. excision.W. Cunningham.S. Curr. Swagemakers.. Biol. Hoeijmakers.g. Aida. Med.B..G. et al. (1998) Search for chromosome instability in lymphocytes with germ-line mutations in DNA mismatch repair genes. 48±51.B. 231.A.. Nebel.M. Hincks. Downloaded from http://mutage. and Kanaar.. Weeda.. 23..M..M. Vermeij.C.. Cancer Res.. Merk.A. Environ.L. 15. Bedford.C.S. (eds). Norris. Cui.. (1991) Sequence preferences of DNA interstrand crosslinking agents: quantitation of interstrand crosslink locations in DNA duplex fragments containing multiple crosslinkable sites.T. 427. Int. Cook.L.J.. Essers. (iii) the increased resistance to Bus of cells de®cient in mismatch repair (Fink et al. In Tannock.S. 1990).T...3-bis(2-chloroethyl)1-nitrosourea and other 1-(2-haloethyl)-nitrosoureas. Dix. Sci. 1703±1710.. Glassner. Pegg.. Genes Chromosomes Cancer. Aebi.M.G.T.S. Environ. Res.P.N.M.J. USA.. Glassner. Snead.3-bis(2-chloroethyl)1-nitrosourea. Dreslin. This study was supported by the Consejo Nacional de  Ciencia y Tecnologõa (National Council of Science and Tecnology).J.S. (ii) all assayed BAA induced MN during the ®rst cell division.J.M.. Pergamon Press.S.. Health Perspect.D. 1984) with the late time of MN induction observed in the present study.T.E. Mutat. Cancer Res.F. (1999) Relationship between the kinetics of micronuclei induction and the mechanism of chromosome break formation by methylnitrosourea in mice in vivo. References Aida. Res. and Vallarino-Kelly.. formation of a new dosedependent lesion and even formation of a secondary lesion..M.  Morales-Ramõrez.. 47. Tomasz. (1996) Association of busulfan area under the curve with veno-occlusive disease following BMT. Ribeiro. Bone Marrow Transplant.J.. 167±178..G. 1998a).J. 44.P. 14.. Mutat.. 77. Carney.R.P.S. and Schold. Cytogenet. Dix et al. Lindor.N..J. Brenneman. (1990) Distribution of methyl and ethyl adducts following alkylation with monofunctional alkylating agents. (iii) Bus and BCNU reveal an additional late mechanism of MN induction. Haas. 4..B.R.C. 45. (1985b) Investigation of resistance to DNA cross-linking agents in 9L cell lines with different sensitivities to chloroethylnitrosoureas.D.V.M..A.C. Millard. The results obtained in the present study allow us to conclude the following: (i) BAA have very different ef®ciencies of genotoxic and cytotoxic action. 19. EMBO J. 1998. Castillab. 32.S.S... 109±142. 19. Allan.D.C.oxfordjournals. Gustafson. 50. 3460± 3464. this agent has been used extensively in bone marrow ablation prior to transplant (Schuler et al. van Steeg. 1982). et al.M.. 11±30.B. (1996) Increasing DNA repair methyltransferase levels via bone marrow stem cell transduction rescues mice from the toxic effects of 1....W.J. Mutagenesis.. Jalal. Zhen. 2012 212 . Cancer Res. (ii) the homology of the late breaks induced by BCNU (Bedford and Eisenbrand.A.S. Berger. (2000) Homologous and nonhomologous recombination affect DNA damage repair in mice. by guest on February 5.S.G.P.J. (1997) Expression of human O6-methyl guanine methyl transferase (MGMT) in post replication repair (PRR) de®cient CHO-UV-1 cells: compensation for hypersensitivity to methylating and ethylating agents but not to mitomycin C.. and Galloway. (1999) Eradicaction of human medulloblastoma tumor xenografts with a combination of O6-benzyl-2¢-deoxyguanosine and 1. (1984) DNA damage and repair in the bone marrow of rats treated with four chloroethylnitrosoureas.P.. but not with ethylating agents.P. Kelley.J. Mutat. Moschel. chromosomal instability and reduced life span in mice.Morales-Ramõrez et al. and Samson.B. and Hasty. Clin.D. 1450±1454. 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 6MOP (Armstrong and Galloway.P.. Cancer. Pacheco et al.E.D. Cancer Chemother. pp. Engelward. Gerosa. Erlichman. A possible mode of resistance. 514±518.M. 257±266. 1990. Johnson. 206±210. 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