Mutagenesis 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: [email protected]
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)
Genotoxicicity of antineoplastic drugs
Table I. Continued
No. of spots scored (frequency of spots/100 eyes)
diagnosesb (m = 2)
Clone size (ommatidia affected)
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
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.
chromosomes. The responsiveness of the antineoplastic drugs
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St. PETER'S UNIVERSITY St. Peter's Institute of Higher Education and Research (Declared under section 3 of UGC Act 1956) Avadi, Chennai – 600 054. M.Sc. (CHEMISTRY) PROGRAMME (Full Time) (I to IV SEMESTERS) REGULATIONS AND SYLLABI REGULATIONS – 2014 (Effective from the Academic Year 2014-'15)
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-