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Geh013 121.127Mutagenesis vol. 19 no. 2 pp. 121±127, 2004 Detection of mitotic recombination and sex chromosome loss induced by adriamycin, chlorambucil, demecolcine, paclitaxel and vinblastine in somatic cells of Drosophila melanogaster in vivo1 Rosario RodrõÂguez-Arnaiz1, AmeÂrica CastanÄeda Adriamycin (AD), an anthracycline antibiotic, is a topo- SortibraÂn and Guadalupe Ordaz TeÂllez isomerase-interactive agent that may induce mutations through Laboratorio de GeneÂtica, Facultad de Ciencias, UNAM, CoyoacaÂn, 04510 simple DNA intercalation or via generation of oxygen free MeÂxico, DF MeÂxico radicals (Anderson and Berger, 1994). The compound is The conventional w/w+ eye assay in Drosophila has been strongly mutagenic against frameshift-sensitive strain TA98 in used for the last 10 years for genotoxic evaluation of a the Salmonella microsome test (Brookes, 1990) and positive in broad number of chemicals with different mechanisms of the conventional w/w+ bioassay in Drosophila (Vogel and action. Although chemicals that induce genotoxic effects Nivard, 1993). Chlorambucil (CAB), a nitrogen mustard by mechanisms other than covalent binding to DNA are derivative used in the treatment of cancer, is a broad spectrum dif®cult to detect. The aim of this study was the parallel mutagen. CAB can both crosslink DNA in the absence of detection of both mitotic recombination and X chromo- metabolism (McLean et al., 1980) and monoalkylate DNA some loss induced by ®ve chemical compounds used world- following metabolic removal of one of its chloroethyl groups wide as antineoplastic drugs using the w/w+ somatic assay (Adams et al., 1996). CAB produces micronuclei in both spleen in Drosophila melanogaster. The compounds tested were and bone marrow cells of rats, while structural chromosome the intercalating agent adriamycin (AD), the alkylating aberrations were shown to be an end-point less sensitive to compound chlorambucil (CAB) and the spindle poisons damage (Moore et al., 1995). CAB induces 32P-labeled DNA demecolcine (DEM), paclitaxel (taxol, TX) and vinblastine adducts in calf thymus DNA used as a target for the direct (VBL). We used a cross between heterozygous females detection of adducts (Yourtee et al., 1992) and also induces with a rod-X and a ring-X chromosome mated with ywf inter-chromosomal mitotic recombination in the w/w+ assay in males. All four genotypes in the next generation are het- Drosophila (Vogel and Nivard, 1993). Spindle poisons may erozygous or hemizygous for the w+ reporter gene and inhibit the polymerization of tubulin into microtubules and were inspected for the occurrence of white clones in their block the cell cycle during mitosis in the transition from compound eyes. We found differences in the induction of prometaphase to metaphase and thus might be expected to mitotic recombination when compared with chromosome induce numerical chromosome aberrations (aneuploidy).
loss. Genotoxic pro®les obtained for the antineoplastic Demecolcine (DEM) exerts a selectively destructive action drugs studied indicate direct and indirect effects. While on rodent (basal cell) carcinomas, solar keratoses, Bowen's AD seems to be clastogenic due to its induction of X disease and keratoacanthomas, while sparing surrounding chromosome loss in XrX females; DEM, CAB and TX pro- normal tissues (Belisario, 1965). The vinca alkaloids have duced both structural chromosome aberrations through been used in cancer therapy for more than 30 years (Zhou and clastogenic activities and mitotic recombination through Rahmani, 1992). Among them, vinblastine (VBL) is used in the DNA interactions; the cytotoxic VBL induced rX loss only treatment of breast and lung cancer (Maral et al., 1984). VBL in XrY and intra-chromosomal recombination (XY) was classed as a weakly active genotoxin in the w/w+ assay and males, probably due to sister strand recombination, gener- positive in the mwh/¯r somatic test in Drosophila (Tiburi et al., ating a w+w+ duplication and a w± deletion, forward muta- 2002). Paclitaxel (taxol) is perhaps one of the most successful tions or small deletions at the white locus.
drugs used in the treatment of a variety of cancers, having been shown to be non-genotoxic in the Drosophila wing somatic mutation and recombination test (SMART) (Cunha et al., 2001). The spindle poison taxol exerts its action through stabilization of microtubules by promoting assembly, rather than disassembly, leading to the formation of non-functional Antineoplastic drugs are responsible for the survival of cancer microtubule bundles (Kumar, 1981; Ringel and Horwitz, patients around the world, however, like many other cancer therapeutics they may themselves cause mutation and second- Somatic cells are an indispensable target for the study of loss ary malignancies. Cancer induction is therefore a toxic of heterozygosity (LOH) produced by several mechanisms, consequence predicted by short-term tests of genotoxicty and including large deletions, mitotic recombination and chromo- should be weighed against the potential therapeutic bene®ts of some loss (Lasko et al., 1991). LOH by mitotic recombination, several antitumor drugs (Anderson and Berger, 1994). It is an important cancer-prone mechanism, can be detected in therefore essential that effective anticancer drugs should be Drosophila by several systems, such as the multiple wing/¯are tested not only for their cytotoxic potential but also for their wing spot system (Graf et al., 1994) and the white/white+ (w/ ability to disturb genomic integrity, in order to render a deeper w+) eye mosaic test (Vogel and Zijilstra, 1987). The basic understanding of the potential risks related to their clinical use principle of the w/w+ assay is the detection of phenotypically (Tiburi et al., 2002).
visible light spots in the red eyes of adult females, resulting 1To whom correspondence should be addressed. Tel: +52 5 622 4906; Fax: +52 5 622 4828; Email: firstname.lastname@example.org Mutagenesis vol. 19 no. 2 ã UK Environmental Mutagen Society 2004; all rights reserved.
R.RodrõÂguez-Arnaiz et al.
Table I. Number of eyes, distribution of spots (frequency of spots), average clone size, clones per 104 cells and statistical diagnoses induced in the eyes of ¯ies after acute (6 h) treatment with ®ve antineoplastic drugsGenotypea No. of spots scored (frequency of spots/100 eyes) diagnosesb (m = 2) Clone size (ommatidia affected) Chlorambucil (CAB) Demecolcine (DEM) Genotoxicicity of antineoplastic drugs Table I. Continued No. of spots scored (frequency of spots/100 eyes) diagnosesb (m = 2) Clone size (ommatidia affected) Vinblastine (VBL) aF, female; M, male.
bStatistical diagnosis according to Frei and WuÈrgler (1988): +, positive; ±, negative; ++, P < 0.01; +++, P < 0.001; m, multiplication factor for the assesment of negative results; c2 test for proportions.
from LOH and expression of the reporter gene white in female Visible light spots in the red compound eyes of the four regular phenotypes genotypes heterozygous for this marker (Vogel and Nivard, are produced by several mechanisms. (i) LOH in XX females by homologous 1993). The use of multiple inverted X chromosomes, which in inter-chromosomal recombination between the two rod-X chromosomes. (ii) In all three genotypes carrying the w+ allele several genetic end-points can be XX cells suppress recombination between the two X chromo- produced: unequal sister strand recombination generating a w+w+ duplication somes, identi®ed homologous (inter-chromosomal) mitotic and a w± deletion (intra-chromosomal recombination), forward mutations and recombination as the predominant cause generating loss of the small deletions at the white locus. Thus LOH predominantly monitors w+ reporter gene (Vogel and Szakmary, 1990). Recently, a homologous recombination in XX females while it represents rX loss due to novel method for the parallel monitoring of homologous breakage events in XrX females (Vogel and Nivard, 1999).
mitotic recombination (inter-chromosomal), intra-chromo- Test protocol and data analysis somal recombination and structural chromosome aberrations All crosses were set up as mass cultures, with 50 pairs of ¯ies/bottle. Chemicals were administered by acute treatment. Eggs from crosses were collected for 6 h has been developed (Vogel and Nivard, 1999). The assay is in culture bottles containing a solid agar base (5% w/v agar in water) covered based on the generation of four regular distinct genotypes, with a 5 mm layer of live baker's yeast supplemented with sucrose. Three days which can be recognized by their phenotypes. This study later, larvae were collected by washing them out with an aqueous solution of focused on the ability of several antineoplastic drugs to induce 20% sucrose and seeded in bottles (500 larvae/bottle) containing 50 ml of mitotic recombination and X sex chromosome loss in the in vivo standard medium and 2 ml of the solvent mixture with the test solution. Larvae were fed on the above medium for 6 h, then transferred to fresh medium until white/white+ eye somatic assay in Drosophila melanogaster.
the end of development. Newly hatched females were scored 1±5 days later.
Adult females were heterozygous for white and were inspected for the occurrence of white in their compound eyes. More details about the test Materials and methods procedure are given in Vogel and Nivard (1993). Spots separated from each other by at least four non-mutated ommatidia were counted as independent Chemical compounds and concentrations tested clones. Proliferation of pre-ommatidia cells in the imaginal discs of developing AD (CAS no. 23214-92-8), CAB (CAS no. 305-03-3), DEM (CAS no. 477-30- larvae increases the number of target cells from 20 at the end of ®rst instar, to 5), taxol (TX) (CAS no. 33069-62-4) and vinblastine sulfate (VBL) (CAS no.
100±150 cells in second instar, reaching a ®nal number of 780±800 pre- 143-67-9) were purchased from Sigma (St Louis, MO). The chemicals were ommatidia cells at the end of third instar (Becker, 1976). Thus, primordial cells dissolved in a mixture of 1% Tween-80 and 3% ethanol immediately prior to of the adult compound eye divide continuously throughout the larval period.
use. The ®nal concentration of this solvent mix was 4%. The higher exposure Mosaic white clones induced in early larvae will be large but less frequent, dose was determined as the LD whereas those produced later will be successively smaller and more frequent, as 50 and two additional lower doses were tested.
the number of potential target cells increases with larval age. The size of the white clone and their distribution among size classes therefore provides Wild-type (Hague-79) females were mated with rX, R(1)2; y (yellow) f information on the time of induction of a LOH event (Vogel and Nivard, 1999).
(forked)/y+ Y males. Virgin F1 females heterozygous for a ring-X and a rod-X For each experiment a concurrent control was run, in which larvae were chromosome were mated with ywf males (for a description of the genetic treated with the solvent mixture alone. For evaluation of the genotoxic effects markers see Lindsley and Zimm, 1992), generating in the F2 four regular recorded, the frequency of mosaic eyes of the treated series was compared to its classes: XX, y+w+f+/ywf female (wild-type phenotype); XY, y+w+f+/Y male concurrent negative control series. These statistical comparisons were done (wild-type); XrX, yw+f/ywf female (yellow, forked); XrY, yw+f/Y male (yellow, using the c2 test for proportions. Statistical analyses were done exclusively for forked). All four regular genotypes are heterozygous or hemizygous for the w+ the total number of spots recovered. To test the alternative hypothesis (HA) the reporter gene and thus express the red wild-type color in their eyes. Loss of the parameter m = 2 (multiplication factor) was used due to the relatively high w+ reporter gene in w/w+ (XX or XrX) or in w+ (XY or XrY) pre-ommatidia spontaneous incidence of total spots (Frei and WuÈrgler, 1988).
cells during larval development leads to white clones which become visible in the adult ¯y (Vogel and Nivard, 1999). Besides these four regular F2 classes, ®ve additional genotypes are expected from double crossovers in ring/rod heterozygotes and from rX loss. The frequency of these exceptional ¯ies in the present study in unexposed cultures was 0.016 (60 ¯ies among a total progeny Detection of mitotic recombination and X chromosome loss of 3690 ¯ies). According to Vogel and Nivard (1999) these exceptional induced by the ®ve antineoplastic drugs was assayed in two progeny have to be excluded from the scoring procedure.
acute and independent experiments. The data from each R.RodrõÂguez-Arnaiz et al.
experiment were compared and analyzed before being pooled DEM induced LOH by homologous inter-chromosomal for statistical testing by Fisher's exact test (P < 0.05) (Statistica recombination in XX females, indicating ef®cient induction of Program version 6.0). Table I shows the results with the ®ve DNA damage. It also induced intra-chromosomal recombina- compounds in this novel somatic w/w+ assay in tion, as can be seen from the signi®cant spot induction in XY males. In addition, DEM induced rX loss due to chromosome The effects induced by all ®ve antineoplastic drugs were breakage events in both XrX females and XrY males. The mainly related to increases in the frequencies of small spots (1± genetic pro®le of DEM appeared to be more recombinagenic 2 ommatidia affected). Large spots (>2 ommatidia affected) than clastogenic (Figure 3).
were by far less frequent; the ratio between small and large TX induced structural chromosome aberrations more ef®- spots varied between 0.94 and 19.0. Size classes are related to ciently in both XrY males and XrX females through its the number of cell divisions that had occurred between the time clastogenic activity. It also induced inter-chromosomal re- of clone induction in the imaginal larval cells and the combination as well as mitotic intra-chromosomal recombina- beginning of eye differentiation. In acute treatments the tion. For both end-points males seemed to be more affected number of cell divisions before pupation was between 2 and than females (Figure 4).
3. Average clone size as a measure of genotoxicty was between VBL induced frequent irregularities of the compound eye, 2.15 and 4.91 in all genotypes and concentrations tested reduced survival of the four types of larvae and several malformations at the highest LD50 concentration assayed. It The total number of spots per 100 eyes induced by AD were appeared to be clearly positive for intra-chromosomal not signi®cantly above solvent control levels for XX females recombination as well as for the induction of rX loss in and XY and XrY males. In contrast, for XrX females the results males, while at all concentrations tested it was negative for the obtained were positive at all concentrations tested (Figure 1).
XX and XrX female genotypes (Figure 5).
Spot frequencies obtained after exposure to CAB are similar Since large spots are rare events, and in order to make the for XX and XrX female genotypes and XrY males, thus differences between the activity patterns of the antineoplastic homologous mitotic recombination and late rX loss, but clearly drugs more visible, several size classes were grouped together not intra-chromosomal events, dominate the activity of this into three larger groups: group 1 (all spots >8 ommatidia), alkylating agent. Thus mutations at the white locus do not group 2 (spots between 3 and 8 ommatidia) and group 3 (1±2 measurably contribute to the observed activities; otherwise ommatidia affected). An average of all dose groups was spot frequencies in XY males should have been higher obtained before they were pooled, then corrected for spontan- Fig. 1. Frequencies of total white spots induced by AD.
Fig. 3. Frequencies of total white spots induced by DEM.
Fig. 2. Frequencies of total white spots induced by CAB.
Fig. 4. Frequencies of total white spots induced by TX.
Genotoxicicity of antineoplastic drugs eous events in parallel controls. The results of these analyses showed a similar general response for both female and male genotypes (Figure 6). The genetic end-point induced by AD The conventional w/w+ assay seems to be a good predictor of was only signi®cant for rX chromosome loss in females. DEM, the genotoxicity of compounds with different mechanisms of CAB and TX were very ef®cient in the induction of both rX interaction with DNA, while positive spindle poisons with an loss due to breakage events and homologous inter-chromo- aneuploidogenic effect have typically shown weak effects somal mitotic recombination producing LOH in females. In (Vogel and Nivard, 1993). This behavior was found with the addition, VBL was shown to be active only in male genotypes, micronucleus antagonist chloral hydrate (Zordan et al., 1994), producing a positive response for structural chromosome VBL, vincristine and vinorelbine, which induce signi®cant aberrations (XrY) and intra-chromosomal recombination, increases only in the frequency of single (1±2 cells) spots; large mutations and small deletions at the white locus (XY).
spots were not produced (Tiburi et al., 2002). Chemicals which are able to induce genotoxic effects by mechanisms other than covalent binding to DNA are of special interest. Thus, genotoxicity tests able to detect drugs that cause genetic damage by interaction with other cellular targets, such as enzymes and microtubules, are particularly interesting because they play a signi®cant role in DNA replication or in segregation of chromosomes during cell division. The w/w+ assay for parallel monitoring of both mitotic recombination and clastogenicity in somatic cells in vivo in Drosophila seems to separate the two processes from each other (Vogel and Nivard, In this paper we report genotoxic damage induced by several chemotherapeutic agents which act on different targets, as well as topoisomerase (AD), spindle poisons (DEM, TX and VBL) and DNA (CAB). The events that cause visible light spots in the four regular F1 phenotypes are: (i) LOH in XX females by Fig. 5. Frequencies of total white spots induced by VBL.
homologous inter-chromosomal recombination between the Fig. 6. Distribution of white spots induced per chemical in XX, XrX, XY and XrY cells (several size classes are grouped together).
R.RodrõÂguez-Arnaiz et al.
two rod-X chromosomes; (ii) unequal sister strand recombina- CAB produced structural chromosome aberrations more tion generating a w+w+ duplication and a w± deletion (intra- consistently through chromosome breakage events than mitotic chromosomal recombination), forward mutations or small recombination through its DNA crosslinking activity (Moore deletions at the white locus in all three genotypes carrying et al., 1995; Yourtee et al., 1992). Thus CAB is more the w+ allele on their rod-X chromosome; (iii) structural clastogenic than recombinogenic. The recombinogenic activity chromosome aberrations and sister-chromatid exchanges in responsible for induced somatic cell recombination in cells carrying a ring-shaped chromosome, resulting in its loss.
Drosophila observed for CAB is in agreement with the results Whole ring-X chromosome loss as a result of clastogenicity is previously reported in the conventional w/w+ assay (Vogel and cell lethal when generated at or shortly after the blastoderm Nivard, 1993).
stage, however, if rX loss is induced in preommatidia cells of The data obtained with DEM seem to indicate that this third instar larvae, viable clones visible as small white patches antitubulin compound induces mitotic intra- and inter-chromo- in the red compound eye are generated (Vogel and Nivard, somal recombination equally, as well as rX loss. The type of 1999). In somatic cells, ring chromosome loss may result either response for both genetic end-points was very similar between from chromosome breakage or from sister-chromatid ex- male and female genotypes (Figure 6). DEM has been shown to changes (Ashburner, 1989). In XrX cells mitotic recombination be active without requiring any cytochrome P450 isoform between rX and the rod-X in heterochromatin, loss of the (Dvorak et al., 2000).
dicentric element and death of the rX/0 cell (Becker, 1976) While TX was found to be non-genotoxic in the wing might produce a light spot. Another cause of rX loss could be somatic assay in Drosophila in the standard cross as well as in non-disjunction, resulting in aneuploidy. With the antineoplas- the higher bioactivation cross (Cunha et al., 2001), in the bone tic drugs analyzed a relatively low clone induction, generally marrow micronucleus test TX give a strong positive response.
no more than a 2- to 4-fold increase in the number of mosaic Some of the micronuclei induced by this antineoplastic drug spots, was found in this study. In addition, with the three are either large or of aberrant morphology (Tinwell and Ashby, spindle poisons tested clone induction was always associated 1994) and may be associated with metaphase spindle disturb- with cytotoxicity: reduced survival and frequent irregularities ances and consequently with an aneugenic action of this of the structure of the compound eye. This effect was also inhibitor of tubulin disassembly. In our study TX was found to observed with ®ve spindle poisons in the conventional w/w+ be a potent inducer of both mitotic recombination as well as X assay (Vogel and Nivard, 1993).
chromosome loss (Figure 4), with a very similar dose±response The direct and indirect genotoxic potential of antineoplastic relationship for male and female genotypes. It is well known drugs, evaluated by means of LOH of the reporter white gene, that biotransformation mediated by cytochrome 450 enzymes could be predominantly due to whole X chromosome loss (AD, leads to the generation of active metabolites, in particular, TX CAB, TX and VBL) as well as through mitotic inter- has been shown to undergo two pathways of metabolism; the chromosomal recombination and intra-chromosomal recombi- major one leads to 6-hydroxylation catalyzed by CYP2C8 nation (CAB, DEM, TX and VBL).
(Cresteil et al., 1994) and a minor human metabolite results Both chromosome breakage events and sister chromatid from hydroxylation of the lateral chain by CYP3A (Royer et al., recombination remain as major rX loss generating mechan- 1996). The constitutive overexpression of CYP6 subfamily isms. If it occurs in the ring a dicentric ring is formed which genes in resistant Drosophila strains (Waters and Nix, 1988; Brun et al., 1996; Ffrench-Constant et al., 1999), which is will be eliminated leading to single white clones, presuming similar to the CYP3 subfamily in humans (Aoyama et al., they are viable (Becker, 1975). The distribution of small spots 1989), could be responsible for the LOH observed.
among the size classes revealed strict correlations of spot size Spindle poisons such as VBL are non-DNA targeting and larval age: the later the treatment the smaller the size of the mutagens and do not damage, intercalate in or form adducts white clone induced by the compound. Small spots must with DNA directly, as evidenced by their non-mutagenicity in represent independent events because there are no more than bacterial and most mammalian gene mutation assays (Dickins two or three mitotic divisions left for the production of visible et al., 1985; Galloway and Ivett, 1986; Mortelmans et al., white patches in the context of red eyes. In order to avoid an 1986). VBL induced somatic recombination in the wing underestimation of the total induced spot frequency, particu- somatic assay while the vinca analogs vincristine and larly at higher dose levels (data not shown, but calculated), the vinorelbine were shown to induce mutagenic and recombina- frequency of spots per eye, rather than the frequency of mosaic genic events almost equally (Tiburi et al., 2002). In this study eyes, was determined (Vogel and Nivard, 1999).
VBL did not produce inter-chromosomal recombination or X Mitotic aneuploidy may contribute to tumorigenesis by chromosome loss in females, although it was active for inter- facilitating loss of a chromosome involving tumor suppressor chromosomal recombination and whole X chromosome loss in genes that harbor oncogenes (Pihan and Doxsey, 1999; males. In the white-ivory test in Drosophila VBL induces non- Duesberg et al., 1999). Chemicals that can interact with the disjunction (Clements et al., 1990).
spindle apparatus or interfere with spindle function, preventing Our data indicate that the genotoxicity of spindle poisons is normal segregation of chromosomes or chromatids (Bourner due to their clastogenic effects, an activity that is expected for et al., 1998), are proven carcinogens (Oshimura and Barret compounds that are potent inhibitors of mitotic cellular 1986; Dellarco et al., 1985).
division. Microtubules play an important role in cell prolifer- Direct DNA intercalation induced by AD (Anderson and ation and inhibition of microtubule dynamics appears to be the Berger, 1994) could be the mechanism underlying the mechanistic basis underlying the antitumor effects of most genotoxic effect observed in XrX females. LOH due to antimitotic compounds. Coupled with their chemical ef®cacy homologous mitotic recombination was not observed, in in cancer chemotherapy, spindle poisons seem to disturb the contrast to the results obtained by Vogel and Nivard (1993) integrity of the genome, mainly inducing loss of whole in the conventional w/w+ assay.
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ORIGINAL RESEARCH A structured judgement method toenhance mortality case note review:development and evaluation Allen Hutchinson,1 Joanne E Coster,1 Katy L Cooper,1 Michael Pearson,2Aileen McIntosh,1 Peter A Bath3 ▸ Additional material is has resulted in a major public debate.1 2 published online only. To view Background Case note review remains a prime Concerns about hospital deaths in well-