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