Tests for recombinagens in somatic cells of Drosophila

Mutation Research, 284 (1992) 159-175

159

© 1992 Elsevier Science Publishers B.V. All rights reserved 0027-5107/92/$05.00

MUT 00382

Tests for recombinagens in somatic cells of Drosophila Ekkehart W. Vogel Department of Radiation Genetics and Chemical Mutagenesis, Leiden, Netherlands

(Accepted 30 March 1992)

Keywords: Recombination;Bioassay; Drosophila somatic cells

A widely accepted view in the field of genetic toxicology is that no single test procedure can satisfactorily predict mutagenicity or carcinogenieity to mammals of the test agent, and consequently a whole battery of genetic bioassays has been designed. Regarding the use of Drosophila, it was early recognized that it would be not practicable to assay for all the various genetic changes that can be detected in this system. In the period prior to 1980, this led to the recommendation of the multiple-locus sex-linked recessive lethal test (SLRLT) to serve as a screen for potential mammalian genotoxins (Abrahamson and Lewis, 1971; Sobels, 1974; Vogel and Sobels, 1976; Wfirgler et al., 1977). However, the low performance of the S L R L T as a predictor of mammalian genotoxicity in the Collaborative Study on Short-Term Tests (de Serres and Ashby, 1981), its low sensitivity (0.33-0.79) and low accuracy (0.50-0.73), when genotoxins other than direct-acting agents and simple procarcinogens (single-step activation) were included in the evaluation, led to a reappraisal of the potential use of Drosophila assays for mutagen testing (Vogel, 1987). It was against this background that attempts were made to develop genetic assays measuring genetic alterations in somatic cells of Drosophila

Correspondence: Dr. E.W. Vogel, Department of Radiation Genetics and Chemical Mutagenesis, Wassenaarseweg 72, 2300 RA Leiden, Netherlands.

(reviewed by WiJrgler and Vogel, 1986). One of the major reasons for this change in test strategy was the attractive idea that in terms of cost the performance of a somatic mutation assay would be just a fraction (5-10%) of that needed for an SLRLT, meaning that testing of a large number of chemicals would no longer be an unrealistic approach for Drosophila. It also meant that each individual somatic mutation assay could thoroughly be assessed against a wide array of genotoxic and non-genotoxic chemicals, because one of the lessons from the first collaborative study (de Serres and Ashby, 1981) and the two IPCS Collaborative Programs (Ashby et al., 1985, 1988) was that a solid database is required before a reliable judgement of an assay's advantages and limitations can be made. Therefore, in this review the main emphasis will be on those two assays which in the opinion of this author fulfil this requirement: the wing-spot system and the eye mosaic test with w / w +.

Assay systems Several short-term mutation assays have been developed for the detection of genetic alterations in somatic tissue of Drosophila. (In principle, these methods could also be used to monitor effects in germinal tissue.) They are all based on the generation of flies with a genotype such that a mutational event in a somatic cell leads to a genotypic change that is phenotypically manifested in all the progenitor cells of the mutant

160

mother cell, leading to a readily detectable clone. A further common feature of these assays is that Drosophila larvae are exposed to the chemical under test, whereby the target cells during or after treatment pass through several cell divisions. This is different from the SLRLT where primarily post-meiotic germ cells have been the stage examined in most routine tests. The four different systems (Table 1) available for use are: (1) The white-ivory (w i) test developed and recommended by Green et al. (1986). (2) The unstable white-zeste eye mosaic system (Rasmuson and Green, 1974; Rasmuson et al., 1984). (3) The wing mosaic system (Graf et al., 1984; WiJrgler et al., 1985). (4) The white/white + assay (Vogel and Zijlstra, 1987a,b).

The white-ivory system The white-ivory system depends upon the somatic reversion of the X-chromosomal, recessive eye color mutation, white-ivory (w i) to wild type (w+). Reversions are scored as clones of red (w +) facets in the white-ivory eyes of eclosing adults (Table 1). To increase the target site, a tandem quadruplication (Dp{1 : 1 : 1 : 1}wi) containing four w i mutations was synthesized by Green et al. (1986). Thus, in homozygous females eight w i

mutations are potentially reversible. Molecular analysis suggested that in most cases the induced reversions were associated with the loss of a 2.9-kb DNA sequence duplicated in the w ~ mutation. Based on calibration experiments with ethyl methanesulfonate (EMS), N-ethyl-N-nitrosourea (ENU), 1,2:3,4-diepoxybutane (DEB), triethylene melamine (TEM), mitomycin C and cisplatin (DDP), inclusion of this system as a test for environmental mutagens was recommended (Green et al., 1986). EMS, ENU, methyl methanesulfonate (MMS) and DEB were also the reference mutagens used in a study by Howe et al. (1990). Three out of four agents tested in another study (Xamena et al., 1991) were also alkylating agents, namely EMS, propylene oxide and cyclophosphamide (CP). In similar experiments, four anti-cancer drugs, bleomycin (BM), adriamycin (AM), mitoxantrone (MTX) and cyclophosphamide (CP), were found to significantly increase the white-ivory inversion frequency whereas cytosine arabinoside, methotrexate and vincristine did not give positive results (Clements et al., 1990).

The unstable zeste-white interaction system (UZ) This test system is also built on phenotypically visible changes in eye pigmentation, for which the activity of the w locus is responsible (Table 1).

TABLE 1 ASSAY SYSTEMS M E A S U R I N G G E N E T I C D A M A G E IN S O M A T I C CELLS O F D R O S O P H I L A Target tissue

Genotype tested

Genetic m a r k e r s / Location

Principle of method

Wing trichomes

Male or female

Multiple wing hairs,

mwh + / + fir; loss of (trans)heterozygosity expression of recessive marker genes mwh, fir

mwh / 3 - 0 . 0 Flare, flr/3-39.0 Eye pigment cells

Female ~

white, w / l - l . 5

Eye pigment cells

Male or female

Eye pigment cells

Male

Quadruplication of white-ivory, w i [Dp(1 : 1 : 1 : 1 ) ] / 1 - 1 . 5 white, w / l - 1 . 5 zeste, z / l - 1 . 0

a

a Use of the other sex also possible, but see Discussion.

w / w +; loss of heterozygosity expression of recessive marker w Loss of 2.9-kb duplicated D N A reversion of w i to w + Interaction between zeste and white; Insertion or loss of IS element in white locus; changes from zeste to wild-type phenotype or vice versa; use of unstable (UZ) chromosome; loss of the white locus can also be measured

161

The UZ system designed by Rasmuson et al. (1978, 1980) and recommended as a screening system for mutagens (Rasmuson et al., 1984), contains an unstable white locus in the X-chromosome, the instability presumably depending on an insertion (IS) of a piece of DNA in the locus (Rasmuson, 1985). This IS affects the regulatory part of the white + gene and interferes with the z-w interaction system. Depending on the acquisition or loss of a small IS element, shifts from zest° to red or vice versa occur and form the basis of this system as a test for mutagenicity (Rasmuson et al., 1984). Deletions and transpositions of the white locus, resulting in the occurrence of white clones, are also monitored by this assay. The extension of the aberrant sector depends on the number of cell divisions after the initial event, and visible spots or clones may comprise from 2 to more than 100 ommatidia. In the standard procedure of mutagen testing, only spots larger than 4 ommatidia are scored as mutations, since 1 and 2 ommatidium spots often occur spontaneously (Rasmuson, 1985).

The wing mosaic system Use of two genetic markers resulting in an altered phenotype of the trichomes (hairs) on the wing blade cells (among other tissues) constitutes the genetic principle of the wing mosaic system (Graf et al., 1984; Wiirgler et al., 1985). Both loci, i.e., multiple wing hairs (mwh) and flare (fir), are located on the left arm of the third chromosome

(Table 1). Multiple wing hairs is a homozygous viable recessive mutation producing multiple trichomes per cell instead of the normal unique trichome. The second marker, flare, is a zygotic recessive lethal but homozygous cells in the imaginal disc survive and lead to wing blade ceils with short, thickened, and misshapen trichomes. Because flare cannot be kept in homozygous strains, the flr alleles have to be balanced over inverted chromosomes such as TM1 or TM3. The genotypes under test in this system are either male or female larvae that are transheterozygous (mwh + / + f i r ) for the two markers. Expression of the recessive markers may be due to somatic recombination in the chromosome region between the centromere and fir, leading to m w h / / f l r twin spots (Fig. 1A), whereas single spots for mwh and for flr may arise from mutation or deletion of the respective wild-type alleles. However, somatic recombination between the markers mwh and flr also yields single spots of the mwh type (Fig. 1B). Since the introduction of the wing system about 8 years ago (Graf et al., 1984), this assay has been thoroughly evaluated against some 250 chemical agents. In addition, replacement of the original mwh-flr cross by the mwh-flr 3 version and the recent design of a new tester strain with high bioactivation properties led to a considerable refinement and improvement of the test (Fr61ich, 1989; Fr61ich and Wiirgler, 1990a,b). According to Graf and Wiirgler (1986), the following types of spots are evaluated sepa-

A I ~

Mrr¢~18

2 ~ m

~___

°

I ~ °

Idrl'~am

0

t OU.l.S m

°

0

0

°

-x;

o

°

a,

0

°

m.

g,

°

~

@

°

°

0

Fig. 1. Schemes illustrating clone formation in the wing-spot test. Recombination proximal to the fir marker results in twin spots (A), while recombination between mwh and fir produces mwh single spots only (Graf et al., 1984).

162

1 CELL

2

W

CELLS

~

q W*

I I

1 2

W ~

~N

~'

RED

014

I w+

1

ol3 RE

, ,,~

n

2 or4

WHITE

Fig. 2. Interchromosomal recombination between the two X-chromosomes is the most frequent event measured in w / w + heterozygotes after exposure to genotoxic agents or in controls.

rately: (i) small single spots (of mwh or fir phenotype) consisting of only 1 or 2 cells, (ii) large single spots (> 2 cells), and (iii) twin spots. The white/white + eye mosaic system The white/white + system (Vogel and Zijlstra, 1987a,b) monitors mosaic light spots in the eyes of adult females, resulting from the loss of heterozygosity and the expression of white in female genotypes heterozygous for this marker (Fig. 2). This method can be regarded as an improvement of the previously used white-coral / white bioassay (Mollet and Wiirgler, 1974; Mollet and Weileman, 1976), the improvement being the better detectability of mosaic sectors of a size between 2 and 8 ommatidia. Quantitative measurements revealed that in the original white-coral~white method the small size classes, mostly representing more than 90% of all clones induced, cannot be detected (Vogel and Zijlstra, 1987a). Although the induction of white spots in the w / w + test is mainly due to recombination, this assay in contrast to the m w h / f l r system does not detect 'recombinant spots' as twin spots. This is not to be considered a disadvantage of the w / w + assay, because in earlier experiments with the w / w c° assay not only the frequency of w C ° / / w twin spots but also that of single light spots was strongly reduced in inversion heterozygotes (Vogel and Zijlstra, 1987a). Depending on the type of genotoxin used, the reduction in single spot frequency varied between 23% (ENU) and 91% (cisplatin). Thus the ratio of twin spots to single spots does

not provide quantitative information on the contribution of recombination relative to other mutational events. The origin of mosaic clones

Before starting the discussion on the genetic principles involved in the formation of mosaic spots, considerations of some basic developmental aspects of the biology of Drosophila are essential. In both the wing and the eye mosaic assays, the target tissues under test are the imaginal discs of developing larvae. Total duration of the three larval instar periods at 25°C is 4 days (1 + 1 + 2 days), and during this period the undifferentiated cells of imaginal discs proliferate until they enter into differentiation at the pupal stage, where the distinct organs of the imago (eye, wing) are formed. The estimate for the average duration of a mitotic cycle during exponential growth of imaginal discs is 11.9 h for the eye disc and between 8.0 and 10.6 h for the wing (Bryant, 1970; Postlethwait, 1978). Cell proliferation of pre-ommatidia cells (eye) increases the number of target cells from about 20 at the end of the first instar, to 100-150 cells in second instar larvae, while reaching the final number of 780800 pre-ommatidia ceils at the end of the third instar (Becker, 1957, 1966). Cell proliferation is not strictly clonal, i.e., the average number of mitotic divisions is 3 in first instar, 2-3 in second instar and 3 during the last larval stage (Becker, 1957, 1966). Thus, primordial cells of the adult compound eye divide continuously throughout the larval period, and genetic alterations in these cells can be recognized as mutant clones in the imago when appropriate genetic markers are used. Similarly, growth of the imaginal wing discs results in an increase of cells from about 10 at the beginning of the first instar to approximately 30,000 at the beginning of pupation. Clones induced at the beginning of larval life will be large in size, whereas those produced later will successively be smaller. Hence, classification of mosaic spots into size classes and subsequent analysis of clone size distribution can provide information on the time point of the manifestation of the mutational event. (For further details see Wiirgler and Vogel, 1986, and references therein.)

163 An important characteristic of somatic mutation and recombination tests, which distinguishes them from most other short-term tests for mutagenicity, is that during and after mutagen treatment exposed cells still have to pass through a whole series of cell divisions under in vivo conditions. In this respect the somatic assays also differ from the S L R L T where (mostly) mutagenized post-division male germ cell stages are transferred to unexposed female germ cells. As a consequence, toxic effects on somatic ceils often limit the selection of high doses, meaning that the use of unphysiologically high concentrations is largely avoided in these in vivo systems.

combination in Xe but not in Xh causes cell lethality. For dissection of recombinagenic events at the level of individual genes, i.e., at the rosy and white loci, DNA sequencing and denaturing gradient gel electrophoresis have been applied in germinal but not yet in somatic tissue (Clark et al., 1988; Curtis et al., 1989; Green et al., 1986). However, the recent isolation of a large number of alkylation-induced intra-locus deletions at different locations within the white and r'ermilion loci (Pastink et al., 1988, 1989, 1991) opens new experimental possibilities for studying recombination at the gene level also in somatic cells of Drosophila.

Experimental approaches for analysis Analytical approaches for classification of somatic mosaic spots according to their genetic origin include both molecular and genetic techniques. The use of inverted chromosomes reduces but does not fully prevent reciprocal inter-chromosomal recombination in inversion heterozygotes. This technique enables one to measure the relative contribution of inter-chromosomal recombination to overall clone induction (Becker, 1976; Vogel and Zijlstra, 1987a). The combined use of the white alleles white-apricot (w a) and white-coral (w c°) on the X-chromosome, together with suppressor of forked (su-f ), makes it possible to distinguish events resulting from recombination in either euchromatic (Xe) or heterochromatic (Xh) regions (Haendle, 1979). Mitotic recombination in euchromatic regions, if not leading to reciprocal products, may often be lethal. This is not expected for heterochromatin consisting of highly repetitive sequences (Hilliker et al., 1980). Thus both reciprocal and unequal recombination in heterochromatic regions may be efficient mechanisms by which recessive markers become expressed and the study of this aspect is therefore of considerable theoretical interest. In fact, in a recent study with daunomycin (DM), ethyl methanesulfonate (EMS), N-ethyl-N-nitrosourea (ENU), monocrotaline (MC) and methyl methanesulfonate (MMS), the increase in clone frequencies was steeper for twin spots representing events in Xh, as compared with the category occurring in Xe (Vogel and Szakmary, 1990). This suggests that with increasing dose unequal re-

Consideration of genetic mechanisms The white-ivory system The molecular status of both somatic and germinal reversions of w i to w ÷ induced by chemical mutagens was analyzed by blot hybridizations of SalI-HindlII digests of DNA from revertants (Green et al., 1986). The mutagen-induced reversions all contained both w ÷ and w i DNA, suggesting that the reversions from w i to w ÷ were associated with the loss of the 2.9-kb DNA duplicated in the w i mutation. However, a genetic study on germinal mutation showed that not all the reversions may be due to precise excision of the 2.9-kb D N A duplicated region. Two of six phenotypic revertants (one was a partial revertant) carried insertions of moderately repetitive D N A from outside the locus, in addition to suffering deletions of some white locus DNA (Karess and Rubin, 1982). The fact that also ENU was active in this system, a strong point mutagen of low clastogenic efficiency, suggests that recombinagenic events might be involved in the reversion process. This interpretation seems consistent with the about 10-fold increase in spontaneous reversion frequency in Dp(1 : 1 : 1 : 1)w i males and females (0.9 and 1.5%, respectively), compared to genotypes carrying just one copy of the allele (0.09% in males, 0.18% in females) (Green et al., 1986). Apart from the suggested excision of a 2.9-kb piece of DNA as the ultimate change, many questions remain open regarding the type of genetic

164

alterations leading to spot formation. In contrast to the wing mosaic test and the w / w + system, the white-it~ory test did not pick up the antimetabolites cytosine arabinoside and methotrexate, the spindle poisons vincristine and vinblastine, and the recombinagen strychnine (Clements et al., 1990; WiJrgler and K~igi, 1991). However, for a more profound evaluation of this system's advantages and limitations, a larger range of genotoxic agents should be examined. The unstable zeste-white (UZ) interaction system In the U Z interaction system developed by B. Rasmuson (Rasmuson et al., 1978, 1980, 1984), shifts from zeste to red seem to be associated with the acquisition of a small IS element. Mutations to a pigmentless phenotype are also possible, and were interpreted as deletions of the white locus, because they always are irreversible and show non-complementation with whitespotted (Rasmuson, 1985). The exact functioning of the U Z system is still obscure. One explanation given for this instability is that the inserted element consists partly of foldback sequences that form loops; the element can either change orientation or leave its position, so that the white locus achieves different levels of activity. This would mean that DNA breakage and repair are necessary factors for the shifts to occur (Rasmuson, 1984). However, in a study with 12 mutagens and clastogens of widely differing mode of action, no association was found between the ability of an agent to act as a clastogen and the recovery of aberrant (red spots) sectors in the U Z strain (Vogel, 1989). In contrast, reversions from zeste to red phenotypes were most abundant with ENU, suggesting a link between its high efficiency for gene mutation induction and the occurrence of shifts in pigmentation. This led to the hypothesis that the process causing the genetic instability in the U Z strain is under strict genetic control, and that strong point mutagens such as ENU through efficient gene mutation induction can interfere with it (Vogel, 1989). The eye assay with w / w + and the wing mosaic system with mwh and fir Theoretically, both versions of the somatic mutation and recombination test should detect a

broad spectrum of genetic damage, ranging from point mutations and deletions at the genetic marker loci, reciprocal and non-reciprocal recombination within and between chromosomes, to other types of structural chromosome aberrations, and non-disjunction. However, the extent to which these various types of genetic alterations contribute to the overall yield of aberrant spots has been an issue of extensive debate and will be discussed in the following. Gene mutations and deletions A view expressed from the beginning of studies on somatic cell mutagenesis in Drosophila has been that these bioassays, among other genetic endpoints, monitor mutations and deletions (Becker, 1976). Thus the induction of a non-complementing new mutation at the site of one of the three wild-type alleles (w +, mwh ÷, fi r÷) will result in a detectable single spot. However, an often ignored fact is that one is just dealing with one or two genetic loci in those assays. At least for the w / w + system it is rather unlikely that gene mutations and deletions represent a significant fraction of the spots induced. In the w / w ÷ assay the frequency of spontaneous light spots varied between 2.0 and 7.5 x 10 - 4 in the standard cross (no inversion on one of the two X-chromosomes), and was approximately 10 s in inversion heterozygotes (Vogel and Zijlstra, 1987a), suggesting that in non-exposed individuals the majority of spots may be due to reciprocal recombination. (It should be noted here that the background spot frequencies show considerable variation, depending on the genetic constitution of the strain selected (Vogel et al., 1991).) In germ ceils of Drosophila, recessive lethal mutations occur with a frequency of about 2-5 x 10-6 per locus per generation. This calculation is based on the estimate that the number of loci which can mutate to a recessive lethal mutation is 700 for the X-chromosome (Abrahamson et al., 1980). Induction experiment with EMS and ENU revealed a similar per locus frequency for recessive lethals as compared to visible mutations at the t,ermilion locus (Pastink et al., 1989, 1991). Thus on the assumption that the spontaneous and chemically induced mutability of X-linked loci are similar in somatic ceils, for the example

165 TABLE 2 COMPARISON ON A PER LOCUS BASIS OF THE FREQUENCIES OF GENETIC DAMAGE INDUCED IN THE SMART AND THE SLRLT a Mutagen

MMS (5.0 raM) EMS (10.0 mM) ENU (1.0 raM) Spontaneous rate

SLRLT (F×I0 -6)

SMART w/w ~ (FxI0 4)

Ratio1

10

164

0.00061

7

18

0.0039

105

67

0.016

5b

3.7

SLRLT/w/w+

0.014

Mutagen exposure of female genotypes. From Vogel and Zijlstra, 1987b. b From Vogel and Zijlstra, 1987b (Tables 1 and 2). (strain) given here, a 40-375-fold increased mutation frequency at the white locus would just double the frequency of spots in the w / w + system. Even with a strong point mutagen like ENU such activities are difficult to reach at non-toxic dose levels (Table 2). This reasoning is supported by the low ratios obtained when comparing on a per locus basis the activities measured in the SLRLT and in the w / w + assay (Table 2). Moreover, in experiments with genotoxic agents of different mode of action, mosaic spot frequencies were reduced up to 95% in inversion heterozygotes (w +~In-w) in comparison with w+/w genotypes. The 'inhibitory effect' of the inversion was stronger for cross-linking agents than for those having monofunctional properties (Vogel and Szakmary, 1990). Another fraction of spots could be due to unequal sister-strand recombination, an event which should not be affected by the presence of an inversion. In conclusion, apart from strong point mutagens like ENU it is questionable whether mutations and deletions at the white locus significantly contribute to the activities measured by the w / w + system. In the wing spot test with the mwh-flr 3 cross, 0.2-0.4 spots per wing were obtained for small single mwh spots and 0.03-0.10 for large single mwh spots (Frei et al., personal communication; Fr61ich and Wiirgler, 1990a,b; van Schaik and Graf, 1991; Zordan et al., 1991), which is equiva-

lent to a frequency of about 2 x 10 -5 events per cell and per cell division, when applying the equation suggested by Szabad et al. (1983). Mitotic recombination and gene concersion The various recombinagenic events which may lead to homozygosity of recessive markers are mitotic crossing-over, i.e., the reciprocal exchange of genetic information between homologous chromosomes at the four-strand stage, unequal sisterstrand recombination between the chromatids carrying the wild-type allele, and gene conversion, i.e., the nonreciprocal transfer of information from one DNA duplex to another. In meiosis, gene conversion is frequently associated with crossing-over (Curtis et al., 1989; Hurst et al., 1972; Szostak et al., 1983), and both events are envisioned in many recombination models as alternative outcomes of a single underlying process (Curtis et al., 1989). Most models of recombination mechanisms involve formation of a Holliday junction (Meselson and Radding, 1975; Szostak et al., 1983). This structure can be resolved as either a crossover or non-crossover, depending on the choice of strands which are cleaved and religated. Although the various types of recombination have not yet been analyzed at the DNA sequence level, there is indirect evidence from genetic experiments for their occurrence also in somatic cells of Drosophila. From a quantitative point of view, reciprocal mitotic recombination between the two X-chromosomes seems the major event measured by the white/white + system (Vogel and Zijlstra, 1987a; Vogel and Szakmary, 1990). As already mentioned, in a study with seven carcinogens, an up to 95% reduction in mosaic spot frequencies was seen in inversion heterozygotes (w+/ln-w), as opposed to marked spot induction in w +/w not carrying an inversion (Vogel and Szakmary, 1990). Similarly, in the wing spot test reductions in spot frequencies between 35% and 99% were found in inversion heterozygotes (Fig. 3). There is also good experimental evidence for the occurrence of unequal recombination in somatic cells of Drosophila. In females, a dose-dependent increase of mosaic twin spots in Xh but not in Xe was interpreted to indicate an increase with dose of unequal recombination, leading to

166 WING MOSAIC T E S T cJ~.6

lOO

80

I.-

,eI

oo

I,,,,,

40

o n

20

o AZS

AMP

STZ

EPT

MXT

CHEMICAL

Fig. 3. Percentage of spot reduction ( = % recombination) in

mwh/TM1 (AZS, AMP, STZ) or in mwh/TM3 (EPT, MXT) inversion heterozygotes in comparison to mwh/fir genotypes. AZS, azaserine; AMP, aminopterin; STZ, streptozotocin; EPT, ellipticine; MXT, mitoxantrone. (Unpublished data from H. Frei, U. Graf and F.E. Wiirgler; personal communication).

lethality when the event took place in euchromatic regions (Vogel and Szakmary, 1990). In males which are hemizygous for an X-chromosome, unequal mitotic recombination between the two sister strands will lead to a twin spot, provided the daughter cell that receives the deficient chromosome remains viable. After exposure of white-coral (w c°) male larvae to MMS, EMS, diethylnitrosamine (DEN), N-ethyl-N'-nitro-Nnitrosoguanidine (ENNG), ENU or DDP, low rates (5-20% of the rates in females) of w C ° / / w twin spots were indicative of unequal sister-strand recombination in the X-chromosome. From this it can be inferred that in inversion heterozygotes 5 - 2 0 % of the activity is due to unequal sisterstrand recombination. This is in line with the observation that after exposure to MMS, ENU or DDP total spot frequencies were similar in males and in females heterozygous for the inversions In(1)sc sH~ sc ~R and ln(1)dl-49 (Vogel and Zijlstra, 1987a). Unequal mitotic recombination had been predicted by Auerbach (1945) to occur in males, and this event was later demonstrated after exposure to X-rays and triethylenemelamine (Becker, 1975). It is not yet clear whether and to what extent gene conversion contributes to overall clone in-

duction. In his work, Becker (1975, 1976) suggested that the ratio of twin spots to single spots in the white-coral cross might give an indication of the relative proportion between recombination and mutation. This seems not to be the case because it was later shown that not only the frequency of twin spots but also that of white spots was strongly reduced in inversion heterozygotes (wC°/ln-w) compared to wC°/w females. Depending on the type of genotoxic agents examined (monofunctional vs. cross-linking), the reduction rates varied from 23% to 91% (Vogel and Zijlstra, 1987a). The nature of events causing white single spots is not yet clear but gene conversion as a mechanism seems one realistic possibility. Although it is realized that 'strong' clastogens, i.e., cross-linking agents, are powerful inducers of both recombination and structural chromosome aberrations, from a mechanistic point of view it seems not justified to equate the two categories. There is first the phenomenon that in Drosophila, irrespective of their clastogenic efficiency, genotoxic agents of widely different mode of action are recombinagenic (Wiirgler and Vogel, 1986). Of particular relevance in this discussion are agents like ENU and DEN which, because of their preference for O-alkylation, are among the weakest clastogens known in Drosophila. Weak or negative responses were found in assays measuring translocations or chromosome loss in germ ceils of Drosophila, in spite of high mutation rates induced under similar conditions (Vogel and Natarajan, 1979a,b; Vogel, 1986). Recently, sequence analysis of ENU-induced vermilion and rosy mutations demonstrated the dominance of single base-pair substitutions (Gray et al., 1991; Pastink et al., 1989). In spite of its low relative clastogenic efficiency, in both the wing and the eye mosaic test ENU is an efficient recombinagen. Even with this type of genotoxin, a 60% reduction in clone frequencies was found in inversion heterozygotes (Vogel and Szakmary, 1990). Models for the function of post-replication repair include recombinational exchange whereby discontinuities in nascent strands are eliminated (Kaufman, 1989). Regions of single-stranded template containing damage may be substrates for

167

the activity of enzymes involved in homologous recombination. In this case the region of template containing the blocking lesion for DNA replication is annealed to its original (pre-replication) partner in the duplex. In this model, structures are created which are composed of either both parental strands or both daughter strands. Resolution of these intermediates will generate either a recombination patch or a sister-chromatid exchange (Holliday, 1964; Ishii and Bender, 1980; Kaufman, 1989). While this recombination pathway of post-replication repair appears to be capable of error-free elimination of discontinuities interfering with DNA replication, the parental strand still may contain the (persistent) lesion. This might be a mechanism by which new recombinagenic events could repeatedly be generated in successive cell divisions. This would explain why in spite of acute exposure of first instar larvae to ENU - an unstable chemical which rapidly decomposes hydrolytically within minutes - the majority of induced clones were small (size classes 2 and 3 + 4), meaning that they represented events which became manifest only in third instar larvae (Vogel and Zijlstra, 1987a). Such a 'delayed' occurrence of aberrant sectors was less apparent with MMS, which is also an unstable, direct-acting alkylating agent (Fig. 4).

CON

~

k/l'vlS

~

ENU

15 u3 W >W O O rr w n co I.0 I! cO

12 9

6

3 0 1+2

.3+4 SIZE

CLASSES

5-8

>" 8

(OMMATIDIA)

Fig. 4. Spot size distribution after exposure of w / w + first instar larvae to either methyl methanesulfonate (MMS) or N-ethyl-N-nitrosourea (ENU). The majority of mosaic spots induced by ENU are small, indicating 'delayed' occurrence in relation to those produced by MMS (data from Vogel and Zijlstra, 1987a).

Chromosome aberrations Most mutagens produce deletions, translocations, inversions and other types of structural chromosome aberrations. It is still not clear whether these different types of structural chromosome aberrations represent a significant fraction in clone formation. There is some indirect evidence that this should be the case because generally fewer males than females survived treatments with a series of strong clastogens (Vogel, 1989). In addition, low yields of large sectors in male genotypes were invariably related to a high incidence of DNA damage leading to chromosome breaks, suggesting that certain chromosome aberrations (presumably deletions) are more frequently lethal in hemizygous condition. For further discussion of chromosome aberrations see Wfirgler and Vogel (1986). Non-disjunction and chromosome loss Theoretically, mis-segregation of autosomes or X-chromosomes as a consequence of non-disjunction by spindle poisons may produce aneuploid daughter cells. In both the wing and the w / w + assay, the cytostatics vinblastine, vincristine and podophyllotoxin were shown to be active (Wfirgler and Vogel, 1986; Vogel and Nivard, 1992). However, the low rates of spots formed by these agents in the eye were generally accompanied by toxic effects, i.e., deformed eyes with irregular ommatidia structure and other malformations were frequently seen. Furthermore, there is experimental evidence supporting the view that chemically induced non-disjunction and chromosome loss, compared to recombination, is not a major source of clone formation. For instance, in w / R X females heterozygous for a ring-X-chromosome, the frequency of light spots was only approximately one tenth of that of the control (w+/w) group after exposure to MMS or DDP (Vogel and Zijlstra, 1987a). This suggested that the majority of mutational events resulting in chromosome loss did not lead to viable clones. This is concordant with the concept that either XO genotypes act as a cell lethal, or more likely such clones succumb to cell competition (Morata and Ripoll, 1975), when generated in XX genotypes after dosage compensation and sex determination took place at or shortly after blastoderm

168

stage (Sfinchez and N6thiger, 1983). Similarly, Szabad and Wfirgler (1987a,b) also came to the conclusion that malsegregation, i.e., single or double non-disjunction of homologous chromosomes or loss of an entire chromosome, plays little, if any, role in the formation of mosaic spots. Studies with X-rays, TEM, EMS and colchicine support this conclusion (Becker, 1975; Szabad, 1986). In those cases where formation of aneuploid daughter cells was shown, as after exposure to X-rays or vincristine (Szabad and Wfirgler, 1987b), the observed activities were a factor 10-100 lower than those measured in assays for recombination. For instance, small alkylating agents such as MMS and EMS produced 7.1 x 10 -s (EMS) and 1.7 x 10 2 (MMS) mutant clones per 104 cells at non-toxic dose levels, and 60-80% of this activity could be suppressed by an inversion (Vogel and Zijlstra, 1987a). The highest yields reported for X-ray or vincristine induced aneuploidy reached 7 . 4 × 10 4 and 6.1 X 10 -4, respectively, and this included some cases (chromosome loss) that could have resulted from chromosome breaks (Szabad and Wfirgler, 1987b). S t a t u s

of

v a l i d a t i o n

Technical conduct Both the eye ( w / w +) and the wing mosaic test are technically simple and need, besides a microscope and a 25°C incubator, no expensive equipment or laboratory facilities. The technical details with respect to the use of suitable markers, the exposure methods, and the registration and interpretation of data have been worked out in detail. Descriptions and recommendations of test protocols have been published (Graf et al., 1984; Vogel and Zijlstra, 1987a; Wfirgler and Vogel, 1986; Wiirgler, 1989). The purpose of this section is to briefly summarize the current status of validation, and to highlight critical steps in the protocol on the basis of experience made during the evaluation program. Selection of chemicals and test results When selections of test chemicals for calibration studies were made it was considered essential to compare compounds differing in their physico-chemical properties, such as stability in

water, solubility, lipophilic vs. hydrophilic character, chemical reactivity, and the Swain-Scott s value (1953). For volatile chemicals special inhalation procedures were developed. The second criterion applied was to select representatives from various classes of genotoxins including those which served as standards in other calibration studies. The following criteria were used. Representatives from different groups of metabolism-dependent and direct-acting agents; Antimetabolites, DNA synthesis inhibitors; - Heavy metals; Spindle poisons; - Tumor promoters; - Complex mixtures (only wing spot test); Non-genotoxic carcinogens; - Non-carcinogens; - The 'pair-testing' strategy of the I P C S / C S S T T collaborative programs; - Chemicals from the NTP list (Tennant et al., 1987); Human carcinogens; Salmonella test negatives. To date some 350 compounds have been tested in either the wing (162 chemicals) or the eye (97 chemicals) mosaic test, and for 91 chemicals information for both assays is available (Graf et al., 1984, 1989; van Schaik and Graf, 1991; Surjan et al., 1985; Vogel and Zijlstra, 1987a,b; Vogel and Szakmary, 1989; Vogel and Nivard, 1992; Wi~rgler, 1989; Wfirgler and Vogel, 1986; Wfirgler, personal communication). These compounds belong to 27 different chemical classes. Among them are at least 90 chemicals that need metabolic activation. The two methods do not seem to differ in their test performance, because with the 91 chemicals tested in both assays, differences in response were obtained only for five substances (Table 3). Apart from the fact that they were all weak mutagens, strain differences in bioactivation (see below) a n d / o r effects related to toxicity rather than intrinsic differences in test performance seem to account for the few discrepancies. Thus, the principal conclusion is that both the wing spot test and the w / w + system detect a broad spectrum of genetic alterations, including various classes of antimetabolites, DNA synthesis inhibitors, and spindle poisons. These somatic assays are therefore suitable to screen for genotoxi-

-

-

-

-

169 TABLE 3 THE FIVE CASESOUT OF 91 CHEMICALSFOR WHICH THE RESULTS OF THE WING SPOT TEST AND THE w / w + SYSTEM WERE AT VARIANCE Chemical

Wing spot w/w + test test

Possiblecause of diverse response

Amitrole

+~

Toxicity, also weak response in wing Strain differences

-

Benz[a]anthra- +'~ cene Di(2-ethylhexyl)- +w _ Exposure dose phthalate (200 mM) (20 mM) p-Dimethyl+w Strain differences aminoazobenzene O,L-Ethionine -+ "~ Toxicity-related response

+W,weakly positive (mutation rate significantly enhanced compared to both concomitant and pooled controls).

city in general. Problems were encountered with heavy metals (highly toxic but mostly inactive), non-genotoxic carcinogens, and certain nitrated and aminated polycyclic hydrocarbons carrying e i t h e r - N O 2 o r - N H 2 groups.

Genetic ~,ariability: different genotypes Bioactiuation Genotype-dependent variability in response to xenobiotics has long been known to occur in Drosophila (Casida, 1969; D~ivring, 1969; Wilkinson and Brattsten, 1972). Analysis of basic characteristics of the enzyme systems involved in oxidative metabolism of xenobiotics in Drosophila revealed considerable differences in some enzyme activities between wild-type (WT) and insecticide-resistant (IR) strains (Zijlstra et al., 1984). Aminopyrine (AP) demethylation and pnitroanisole (pNA) demethylation were substantially higher in all IR strains, while no correlation was found between their increased insecticide resistance and BP hydroxylating capacity or cytochrome P-450 content of microsomes. Furthermore, studies on the enzyme distribution pattern in larvae and adult flies have established that the spectral and enzyme features of the larval cytochromes differ considerably from those present in adult flies (Zijlstra et al., 1984; Zijlstra, 1987).

Studies on the consequences for mutation induction of genetic variation revealed marked genotype-dependent responses to nitrosamines, triazenes and to 7,12-dimethylbenz[a]anthracene (DMBA) (Forbes, 1981; H~illstr6m et al., 1982; H~illstr6m, 1984; Vogel, 1980; Zijlstra and Vogel, 1984; Zijlstra et al., 1984). Of particular interest has been the up to 15-fold variation in X-linked recessive lethal frequency after treatment with DMBA, because for many years a major problem in Drosophila was the weak mutagenic effectiveness of aromatic amines and polycyclic hydrocarbons in assays measuring genotoxic damage in the germ line. It was against this background that attempts were made to develop new tester strains from naturally occurring genetic variants or from strains experimentally selected for enhanced metabolic capacity, in order to improve the detectability of procarcinogens in the somatic mutation and recombination test. Using this approach for the wing-spot test, Fr61ich and Wiirgler (1990b) showed an increased detection capability of a 'high bioactivation cross' (HB) for the three PAHs benzo[a]pyrene, benz[a]anthracene and 7,12-dimethylbenz[a]anthracene, for ethyl carbamate (Fr61ich and Wfirgler, 1990a), diethylnitrosamine (Fr61ich and Wiirgler, 1989), and aflatoxin B~

BENZO[s]PYRENE ' (~

STANDARD

--~-

HB-CROSS

2.00

z 1,60 I,U D.. 1.20 I--

o a.

0.80

t~ ,,J

0.40

I'-

o t-

o.o5 0

0.25

0.50

1

2

4

EXPOSURE DOSE (raM)

Fig. 5. Improved detectability in the wing spot test of the genotoxicity of benzo[a]pyrene (BP) in the HB (high bioactivation) cross compared to a standard cross. Pooled results from 48-h or 72-h exposures (from Fr61ich and Wfirgler, 1990b).

170

(Fr61ich, 1989). Fig. 5 shows as an example the higher response to benzo[a]pyrene of the HB cross. The HB strain was constructed by substituting chromosomes 1 and 2 of the conventional strain by the corresponding chromosomes from an Oregon R(R) strain with increased cytochrome P-450-dependent metabolism linked to a gene on chromosome 2 (Hfillstr6m, 1984). Recently, an increased cytochrome P-450 content was detected in the larval microsomes of the HB cross and the Oregon R(R) strain in comparison with the standard cross, resulting in modified capacities with respect to the epoxidation of aldrin (Fr61ich and Wfirgler, 1992). In a similar study with the w / w + system, ENU-induced white mutations were introduced into three wild-type (Berlin-K, Oregon-K, and 91-C) and three insecticide-resistant strains (Hikone-R, 91-R and Haag-79), and this new set of tester strains was tested against 2-naphthylamine (2-NA), 9,10-dimethylanthracene (DA) and benzo[a]pyrene (BP). An up to 20-fold variation in induced spot frequencies between different genotypes was found (Vogel et al., 1991). BP (Fig. 6), DA and 2-NA were readily detectable in both Hikone-R (IR) and Oregon-K (WT), less so in 91-C (WT) and Haag-79 (IR), whereas the performance of strain Berlin-K (WT) was rather poor. The special problem with strain 91-R was the high frequency of mosaic light spots in non-ex-

Benzo[a]pyrene 8mall

Large

~

Tot=l

5O

40

.

.

.

.

.

.

.

.

.

.

.

.

o HR

OK

E~

91C

H79

91R

STRAINS

Fig. 6. Illustration of genotype-dependent variation in response to benzo[a]pyrene in the w / w + assay. Strains: HR, Hikone-R (insecticide-resistant, IR); OK, Oregon-K (wild type, WT); BK, Berlin-K (WT); 91C (WT); H79, Haag-79 (IR); 91R (IR). (From Vogel et al., 1991.)

posed flies. Interestingly, in both the wing and the eye mosaic system the performance of the Oregon-type strains was excellent (Figs. 5 and 6). Thus, development of uniform Oregon tester strains carrying both the markers white and mwh / f l r 3 seems desirable.

Genetically determined rariability in spontaneous spot frequencies The most striking and unexpected result of the comparative study with six tester strains was the large variation in spontaneous occurrence of mosaic light spots (Vogel et al., 1991). The spontaneous frequencies varied from 3.5% (Hikone-R), 6.3% (Oregon-K), 4.3% (Berlin-K), 9.1% (91-C), 20.5% (Haag-79) to 49.1% (91-R), corresponding to a 14-fold difference in spot frequencies between the two extremes. This drastic variation in spot frequencies in non-exposed larvae was due to small spots (2-4 ommatidia in size), i.e., events which became manifest in third instar larvae (Becker, 1976). Mosaic spots > 4 ommatidia in size occurred with frequencies of 0.4% (HikoneR) to 2.9% (Haag-79). A further examination of strain 91-R, the genotype with the highest yield of light spots in non-treated condition, also revealed high frequencies of small light spots in the homozygous (w+/w +) strain. The cause of this genetic instability is not yet known. One possibility to explain this phenomenon includes a mechanism of transposon-mediated unequal crossingover between mobile elements, belonging to the same family and displaced by ~< 60 kb. Tartof (1988) hypothesized that slightly displaced transposons appear to be a significant cause of unequal crossing over, with an average genomic frequency of about 3 × 10 3 per meiosis. It is not known whether similar processes are responsible for the variability observed in somatic cells. However, the consequence for genotoxicity testing is that strains Haag-79 and 91-R are not suited for this specific purpose. Repair-defl'cient genotypes A question posed by several investigators has been whether somatic cell mutagenesis could be potentiated by repair deficiencies. Using the zeste-white interaction system, Fujikawa and Kondo (1986) introduced either a post-replication

171 repair-deficient m u t a t i o n (mei-41DS, mus(1) 104 TM, or mus(1)101TM) or an excision repair deficiency (mei-9a). After treatment with EMS, MMS, M N N G or E N N G , w ÷(TE) was somatically more mutable in the mei-9 strain and less mutable in the rnei-41 and mus-104 strains than in the repair-proficient situation. However, the 'hypermutability' effects seen in mei-9 relative to mei-9 ÷, i.e., 1.2 for EMS, 1.2 for MMS, 1.8 for M N N G , and 1.6 for E N N G , except with E N N G are generally small compared to similar studies in germ cells (Vogel et al., 1985). In a study on eight genotoxins belonging to different chemical classes, G r a f et ai. (1990) concluded that introducing the mei-9 L1 mutation into the wing system did not improve the detection capacity of the assay. The pertinacious problems were (i) the about 4 - 5 times higher frequencies of spontaneous spots per wing in the mei-9 excision repair-defective progeny compared to the wild type, (ii) the greater variability of both the spontaneous and induced frequencies in mei-9 wings. (iii) the strongly reduced reproduction capacity of the repair mutant, and (iv) the smaller size of the single spots recovered in control and treated series in mei-9 wings. Due to these shortcomings, the scoring of the mei-9 wings was generally more laborious and tedious, and the relative increases seen in induction experiments were smaller compared to

MMS .~

¢3 Z

WILD-TYPE

0.1

1

20

:

r~ I11 a.

16

a uJ

12

:

0.5 40 U) IO 13. ¢0 a t/J

0

a _Z

,

,

:

:

100

: ::~:::

:

:

:::::::

o

8 _z co

I0 a.

o9

4 0 0.1

1

10

EXPOSURE

100

DOSE

(mM)

Fig. 7. Dose-response relationships in the wing mosaic test for total spots after treatment of 3-day-old larvae with varying concentrations of MMS for 2 h. Comparison of mei-9 genotype to repair proficiency(data from Graf et al., 1990). the wild type, as shown in Fig. 7 for MMS. In the w / w + system the presence of the excision repair mutation mus-201 had no (ENU) or only weak (MMS) effects on the activity of these agents. With adriamycin and daunomycin even a lowering of the clone frequencies compared to the repair-proficient genotype was found (Fig. 8A,B) (Vogel, unpublished results). There may be one

DAUNOMYCIN t

MUS-201

--tr-

1 , , I

10

:::::::

ADRIAMYCIN WILD-TYPE

MEI-9

--tr-

,

0.02 3O

,

WlI~-'I'Y~

--~'-

MUS-201

0.1 :

:

:

:

:

;

:

I

:..,

:

"

:&

~- -." ~1"[ "''~"

I

30 ~0

20

20

10

O 0.8

0 0.02

1

EXPOSURE

DOSE

(mM)

....

i

0.1 EXPOSURE

DOSE

(mM)

Fig. 8. Percentages of mosaic spots induced by adriamycin (A) or daunomycin (B) in either repair-proficient (wild-type) or excision repair-deficient (mus-201)genotypes, w/w-- assay, data from Vogel (unpublished).

172

exception where the use of exr strains might be of some advantage. In a recent calibration study on 181 test chemicals (Vogel and Nivard, 1992) certain chemicals of high nucleophilic selectivity, i.e., acrylamide, iodomethane and chloroethyl isocyanate, due to efficient repair of N-alkylation damage were weak somatic cell mutagens or were even inactive in repair-competent strains. This class of genotoxins might show some potentiating effects in exr genotypes. In general, however, the use of repair-defective strains seems of little value and is therefore not recommended for routine screening purposes.

Conclusions All four bioassays considered in this overview measure genetic alterations in somatic cells of Drosophila melanogaster. However, only the wing spot test (with the genetic markers mwh and fir) and the w / w + system have been evaluated against various categories of genotoxic and nongenotoxic chemicals, together some 350 compounds. As there are no indications for intrinsic differences in test performance between the two tests, the assays seem to be interchangeable. By and large, these assays are capable of detecting a broad spectrum of genetic alterations, with mitotic recombination of different types representing by far the major events. Since distinct types and classes of genotoxic agents are monitored by these assays, somatic mutation tests in Drosophila are suitable to screen for genotoxicity in general. The limited database available for both the zeste-white interaction system and the white-ivory system, the problems encountered with clastogenic chemicals in the former system, and the small number of procarcinogens (e.g., aromatic amines and polycyclic hydrocarbons) examined, do not enable one to consider their possible value for mutagenicity testing. Both systems certainly need more systematic calibration studies before any relevant judgement regarding their advantages and limitations as tools for mutagenicity testing can be made.

Acknowledgement This work was supported by a research grant from the Dutch Cancer Society.

References Abrahamson, S. and Lewis, E.B. (1971) The detection of mutations in Drosophila melanogaster, in: A. Hollaender (Ed.), Chemical Mutagens, Principles and Methods for their Detection. Plenum, New York, pp. 461-487. Abrahamson, S., Wiirgler, F.E., De Jongh, C. and Meyer, H.U. (1980) How many loci on the X-chromosome of Drosophila melanogaster can mutate to recessive lethals, Environ. Mutagen., 2, 447-453. Ashby, J., de Serres, F.J., Draper, M., Ishidate, M., Margolin, B.H., Matter, B.E. and Shelby, M.D. (1985) Evaluation of •short-term tests for carcinogens. Report of the IPCS Collaborative Study on In Vitro Assays, Prog. Mutation Res., 5, 1-752. Ashby, J., de Serres, F.J., Shelby, M.D., Margolin, B.H., lshidate, M. and Becking, G.C. (Eds.) (1988) Evaluation of Short Term Tests for Carcinogens, Vol. 2, Cambridge University Press, WHO, pp. 2.277-2.328 Auerbach, C. (1945) The problem of chromosome rearrangements in somatic cells of Drosophila melanogaster, Proc. R. Soc. Edinburgh, B62, 120-127. Becker, H.J. (1957) Llber R6ntgenmosaikflecken und Defektmutationen am Auge von Drosophila und die Entwicklungsphysiologie des Auges, Z. Indukt. AbstammungsVererbungslehre, 88, 333-373. Becker, H.J. (1966) Genetic and variegation mosaics in the eye of Drosophila, Curr. Topics Dev. Biol., 1, 155-171. Becker, H.J. (1975) X-Ray- and TEM-induced mitotic recombination in Drosophila melanogaster, Mol. Gen. Genet., 138, 11-24. Becker, H.J. (1976) Mitotic recombination, in: M. Ashburner and E. Novitski (Eds.), The Genetics and Biology of Drosophila, Vol. lc, Academic Press, New York, pp. 1019-1087. Bryant, P.J. (1970) Cell lineage relationships in the imaginal wing disc of Drosophila melanogaster, Dev. Biol., 22, 389411. Casida, J.E. (1969) Insect microsomes and insecticide chemical oxidations, in: J.R. Gillette, A.H. Conney, G.J. Cosmides, R.W. Estabrook, J.R. Fouts and G.J. Mannerig (Eds.), Microsomes and Drug Oxidations. Academic Press, New York, pp. 517-531. Clark, S.H., Hilliker, A.J. and Chovnick, A. (1988) Recombination can initiate and terminate at a large number of sites within the rosy locus of Drosophila melanogaster. Genetics, 118, 261-268. Clements, J., Howe, D., Lowry, A. and Phillips, M. (1990) The effects of a range of anti-cancer drugs in the white-ivory somatic mutation test in Drosophila, Mutation Res., 228, 171-176. Curtis, D., Clark, S.H., Chovnick, A. and Bender, W. (1989) Molecular analysis of recombination events in Drosophila, Genetics, 122, 653-661. D~ivring, L. (1969) The reaction of different Drosophila populations to treatment with pesticides, Hereditas, 62, 303313. de Serres, F.J. and Ashby, J. (1981) Evaluation of short-term

173 tests for carcinogens. Report of the IPCS Collaborative Study on In Vitro Assays. Prog. Mutation Res., 1, 1-827. Forbes, C. (1981) Sex-linked lethal frequencies produced by 7,12-dimethylbenz[a]anthracene in five Drosophila melanogaster wild strains, Mutation Res., 90, 255-260. Frei, H., Graf, U. and Wiirgler, F.E., personal communication. Fr61ich, A. (1989) Genotoxizitit~itspriJfung mit Drosophila melanogaster: Neue Testst~imme mit erh6hter metabolischer Kapazit~it, Thesis, Swiss Federal Institute of Technology, Ziirich. Fr61ich, A. and Wiirgler, F.E. (1989) New tester strains with improved bioactivation capacity for the Drosophila wingspot test, Mutation Res., 216, 179-187. Fr61ich, A. and Wiirgler, F.E. (1990a) Genotoxicity of ethyl carbamate in the Drosophila wing spot test: dependence on genotype-controlled metabohc capacity, Mutation Res., 244, 201-208. Fr/51ich, A. and Wiirgler, F.E. (1990b) Drosophila wing-spot test: improved detectability of genotoxicity of polycyclic aromatic hydrocarbons, Mutation Res., 234, 71-80. Fr61ich, A. and Wiirgler, F.E. (1992) Increased aldrin epoxidase activity and P450 content in an improved set of tester strains for the Drosophila SMART assay with the wing, Mutation Res., in press. Fujikawa, K. and Kondo, S. (1986) DNA repair dependence of somatic mutagenesis of transposon-caused white alleles in Drosophila melanogaster after treatment with alkylating agents, Genetics, 112, 505-522. Graf, U., Wiirgler, F.E., Katz, A.J., Frei, H., Juon, H., Hall, C.B. and Kale, P.G. (1984) Somatic mutation and recombination test in Drosophila melanogaster, Environ. Mutagen., 6, 153-188. Graf, U. and Wiirgler, F.E. (1986) The present status of validation of the wing spot test in Drosophila, in: Genetic Toxicology of Environmental Chemicals, Part B: Genetic Effects and Applied Mutagenesis, Liss, New York, pp. 391-398. Graf, U., Juon, H., Katz, A.J., Frei, H.J. and Wiirgler, F.E. (1989) Thirty compounds tested in the Drosophila wing spot test, Mutation Res., 222, 359-373. Graf, U., Hall, C.B. and van Schaik, N. (1990) On the use of excision repair defective cells in the wing somatic mutation and recombination test in Drosophila melanogaster, Environ. Mol. Mutagen., 16, 225-237. Gray, M., Charpentier, A., Walsh, K., Wu, P. and Bender, W. (1991) Mapping point mutations in the Drosophila rosy locus using denaturing gradient gel blots, Genetics, 127, 139-149. Green, M.M., Todo, T., Ryo, H and Fujikawa, K. (1986) Genetic-molecular basis for a simple Drosophila melanogaster somatic system that detects environmental mutagens, Proc. Natl. Acad. Sci. USA, 83, 6667-6671. H~illstr6m, I. (1984) Cytochrome P-450 in Drosophila melanogaster. Activity, genetic variation and regulation. Ph.D. Thesis, Stockholm. H~illstr6m, I., Magnusson, J. and Ramel, C. (1982) Relation between the somatic toxicity of dimethylnitrosamine and a

genetically determined variation in the level and induction of cytochrome P-450 in Drosophila, Mutation Res., 92, 161-168. Haendle, J. (1979) X-Ray induced mitotic recombination in Drosophila melanogaster. IV. Distribution within eu- and heterochromatin, Mutation Res., 62, 467-475. Hilliker, A.J., Appels, R. and Schalet, A. (1980) The genetic analysis of D. melanogaster, Cell, 21, 607-619. Holliday, R. (1964) A mechanism of gene conversion in fungi. Genet. Res., 5, 282-304. Howe, D. and Clements, J. (1990) The white-iuory somatic mutation test in Drosophila. The effects of larval age, increasing the number of copies of w i and the response to some reference mutagens, Mutation Res., 228, 193-202. Hurst, D.D., Fogl, S. and Mortimer, R.K. (1972) Conversionassociated recombination in yeast. Proc. Natl. Acad. Sci. USA, 69, 101-105. Ishii, Y. and Bender, M.A (1980) Effects of inhibitors of DNA synthesis on spontaneous and ultraviolet light-induced sister-chromatid exchanges in Chinese hamster cells. Mutation Res., 79, 19-32. Karess, E.R. and Rubin, G.M. (1982) A small tandem duplication is responsible for the unstable white-iL,ory mutation in Drosophila, Cell, 30, 63-69. Kaufman, W.K. (1989) Pathways of human cell post-replication repair, Carcinogenesis, 10, 1-11. Meselson, M.S. and Radding, C.M. (1975) A general model for genetic recombination. Proc. Natl. Acad. Sci. USA, 72, 358-361. Mollet, P. and Weileman, W. (1976) Characteristics of a new mutagenicity test, induction of somatic recombination and mutation in Drosophila by different chemicals, Mutation Res., 38, 131-132. Mollet, P. and Wiirgler, F.E. (1974) Detection of somatic recombination and mutation in Drosophila. A method for testing genetic activity of chemical compounds. Mutation Res., 25, 421-424. Morata, G. and Ripoll, P. (1974) Minutes: Mutants of Drosophila autonomously affecting cell division rate, Dev. Biol., 42, 211-221. Pastink, A., Vreeken, C., Schalet, A.P. and Eeken, J.C.J. (1988) DNA sequence analysis of X-ray-induced deletions at the white locus of Drosophila melanogaster, Mutation Res., 207, 23-28. Pastink, A., Vreeken, C., Nivard, M.J.M., Searles, L. and Vogel, E.W. (1989) Sequence analysis of N-ethyi-Nnitrosourea-induced uermilion mutations in Drosophila melanogaster. Genetics, 123, 123-129. Pastink, A., Heemskerk, E., Nivard, M.J.M., van Vliet, C.J. and Vogel, E.W. (1991) Mutational specificity of ethylmethanesulfonate in excision repair-proficient and -deficient strains of Drosophila melanogaster, Mol. Gen. Genet., 229, 213-218. Postlethwait, J.H. (1978) Clonal analysis of Drosophila cuticular patterns, in: M. Ashburner and T.R.F. Wright (Eds.), The Genetics and Biology of Drosophila, Vol. 2c, Academic Press, New York, pp. 359-441. Rasmuson, A. (1984) Effects of DNA-repair-deficient mutants

174 on somatic and germ line mutagenesis in the UZ system in Drosophila melanogaster, Mutation Res., 141, 29-33. Rasmuson, A. (1985) Comparative studies of the induction of somatic eye-colour mutations in an unstable strain of Drosophila melanogaster by MMS and X-rays at different developmental stages, Mutation Res.,148, 65-70. Rasmuson, B. and Green, M.M. (1974) Genetic instability in Drosophila melanogaster: A mutable tandem duplication, Mol. Gen. Genet., 133, 249-260. Rasmuson, B., Rasmuson, A. and Nygren, J. (1984) Eye pigmentation changes in Drosophila melanogaster as a sensitive test for mutagenicity, in: B. Kilbey, M. Legator, W. Nichols, and C. Ramel (Eds.), Handbook of Mutagenicity Test Procedures, Elsevier, Amsterdam, pp. 603613. Rasmuson, B., Svahlin, H., Rasmuson, A., Montell, I. and Olofsson, H. (1978) The use of a mutationally unstable X-chromosome in Drosophila melanogaster for mutagenicity testing, Mutation Res., 54, 33-38. Rasmuson, B., Montell, I., Rasmuson, A., Svahlin, H. and Westerberg, B.-M. (1980) Genetic instability in Drosophila melanogaster, Evidence for regulation, excision and transposition at the white locus, Mol. Gen. Genet., 117, 567570. Sfinchez, L. and N6thiger (1983) Sex determination and dosage compensation in Drosophila melanogaster: production of male clones in XX females, EMBO J., 2, 485-491. Sobels, F.H. (1974) The advantage of Drosophila for mutation studies. Mutation Res., 26, 277-284. Surjan, A., Kocsis, Z., Csik, M., Pinter, A., T6r6k, G., B6rzs6nyi, M. and Szabad, J. (1985) Analysis of the genotoxic activity of four N-nitroso compounds by the Drosophila mosaic test, Mutation Res., 144, 177-181. Swain, C.G. and Scott, C.B. (1953) Quantitative correlation of relative rates: Comparison of hydroxide ion with other nucleophilic reagents towards alkyl halides, esters, epoxides and acyl halides, J. Am. Chem. Soc., 75, 141-147. Szabad, J. (1986) A genetic assay for the detection of aneuploidy in Drosophila melanogaster, Mutation Res., 164, 305 -326. Szabad, J. and Wiirgler, F.E. (1987a) A genetic assay to detect chromosome gain a n d / o r loss in somatic cells of Drosophila melanogaster, Mutation Res., 180, 201-206. Szabad, J. and Wiirgler, F.E. (1987b) Malsegregation of chromosomes is not a major source of somatic mosaicism in Drosophila melanogaster, Mutation Res., 180, 207-214. Szabad, J., Soos, I., Polgar, G. and Hejja, G. (1983) Testing by mutagenicity of malondialdehyde and formaldehyde by the Drosophila mosaic and the sex-linked recessive lethal tests, Mutation Res., 113, 117-133. Szostak, J.W., Orr-Weaver, T.L. and Rothstein, R.J. (1983) The double-strand-break repair model for recombination. Cell, 33, 25-35. Tartof, K.D. (1988) Anecdotal, historical and critical commentaries on genetics, Genetics, 120, 1-6. Tennant, R.W., Margolin, B.H., Shelby, M.D., Zeiger, E., Haseman, J.K., Spalding, J., Caspary, W., Resnick, M., Stasiewicz, S., Anderson, B. and Minor, R. (1987) Predic-

tion of chemical carcinogenicity in rodents from in vitro genetic toxicity assays, Science, 236, 933-941. van Schaik, N. and Graf, U. (1991) Genotoxicity of five tricyclic antidepressants in the wing somatic mutation and recombination test of Drosophila melanogaster, Mutation Res., 260, 99-104. Vogel, E.W. (1980) Genetical relationship between resistance to insecticides and procarcinogens in two Drosophila populations, Arch. Toxicol., 43, 201-211. Vogel, E.W. (1986) O-alkylation in DNA does not correlate with the formation of chromosome breakage events in D. melanogaster, Mutation Res., 162, 201-213. Vogel, E.W. (1987) Discussion Forum. Evaluation of potential mammalian genotoxins using Drosophila: the need for a change in test strategy, Mutagenesis, 2, 161-171. Vogel, E.W. (1989) Somatic cell mutagenesis in Drosophila: recovery of genetic damage in relation to the types of DNA lesions induced in mutationally unstable and stable X-chromosomes, Mutation Res., 211, 153-170. Vogel, E.W. and Sobels, F.H. (1976) The function of Drosophila in genetic toxicology testing, in: A. Hollaender (Ed.), Chemical Mutagens, Principles and Methods for their Detection. Plenum, New York, pp. 31-142. Vogel, E.W. and Natarajan, A.T. (1979a) The relation between reaction kinetics and mutagenic action of monofunctional alkylating agents in higher eukaryotic systems. I. Recessive lethal mutations and translocations in Drosophila, Mutation Res., 62, 51-100. Vogel, E.W. and Natarajan, A.T. (1979b) The relation between reaction kinetics and mutagenic action of monofunctional alkylating agents in higher eukaryotic systems. II. Total and partial sex-chromosome loss in Drosophila. Mutation Res., 62, 101-123. Vogel, E.W. and Zijlstra, J.A. (1987a) Mechanistic and methodological aspects of chemically induced somatic mutation and recombination in Drosophila melanogaster, Mutation Res., 182, 243-264. Vogel, E.W. and Zijlstra, J.A. (1987'o) Somatic cell mutagenesis in Drosophila melanogaster in comparison with genetic damage in early germ-cell stages, Mutation Res., 180, 189-200. Vogel, E.W. and Szakmary, A. (1990) Basic principles and evaluation of results of assays measuring genotoxic damage in somatic cells of Drosophila, in: Mutation and the Environment, Part B, Wiley-Liss, pp. 149-158. Vogel, E.W. and Nivard, M.J.M. (1992) Performance of 181 chemicals in a Drosophila assay predominantly monitoring interchromosomal mitotic recombination, Mutagenesis, in press. Vogel, E.W., Dusenbery, D.L. and Smith, P.D. (1985) The relationship between reaction kinetics and mutagenic action of monofunctional alkylating agents in higher eukaryotic systems. VL The effect of the excision-defective rnei9 LI and mus(2)201TM mutants on alkylation-induced genetic damage in Drosophila, Mutation Res., 149, 193-207. Vogel, E.W., Nivard, M.J.M. and Zijlstra, J.A. (1991) Variation of spontaneous and induced mitotic recombination in different Drosophila populations: a pilot study on the

175 effects of polyaromatic hydrocarbons in six newly constructed tester strains, Mutation Res., 250, 291-298. Wilkinson, C.F. and Brattsten, L.B. (1972) Microsomal drug metabolizing enzymes in insects. Drug Metab.-'Rev., 1, 153-228. Wiirgler, F.E. (1989) Drosophila techniques for somatic genotoxicity assays. In: Laboratory Manual, Second Southeast Asian Workshop on Short-Term Assays for Detecting Environmental Mutagens, Carcinogens and Teratogens. Bangkok-Chiang Mai Press, Bangkok, pp. 33-47. Wiirgler, F.E. and K~igi, A. (1991~ Genotoxicity testing with the somatic white-iL'ory system in the eye of Drosophila melanogaster, Mutation Res., 263, 33-39. WiJrgler, F.E. and Vogel, E.W. (1986) In vivo mutagenicity testing using somatic cells of Drosophila melanogaster, in: F.J. de Serres (Ed.), Chemical Mutagens, Vol. 10, Plenum, New York, pp. 1-59. Wiirgler, F.E., Graf, U. and Frei, H. (1985) Somatic mutation and recombination in wings of Drosophila melanogaster, in: J. Ashby, F.J. de Serres, M. Draper, M. Ishidate Jr., B. Margolin, B. Matter and M.D. Shelby (Eds.), Progress in Mutation Research, Collaborative Study of Short-Term Tests for Carcinogens, Vol. 5, Elsevier/North-Holland, Amsterdam, pp. 325-340. Wiirgler, F.H., Sobels, F.H. and Vogel, E.W. (1977)

Drosophila as an assay system for detection of genetic changes, in: Kilbey et al. (Eds.), Handbook of Mutagenicity Test Procedures Elsevier Amsterdam, pp. 335-373. Xamena, N., Egido, A., Velfizquez, A., Creus, A. and Marcos, R. (1991) Additional data in support of the quadruplicated white-icor), reversion system to test for somatic genotoxicity in Drosophila melanogaster, Mutation Res., 252, 305312. Zijlstra, J.A. and Vogel, E.W. (1984) Mutagenicity of 7,12-dimethylbenz[a]anthracene and some other aromatic mutagens in Drosophila melanogaster, Mutation Res., 125,243261. Zijlstra, J.A., Vogel, E.W. and Breimer, D.D. (1984) Strain differences and inducibility of microsomal oxidative enzymes in Drosophila melanogaster flies, Chem.-Biol. Interact., 48, 317-338. Zijlstra, J.A. (1987) Pharmacological and mechanistic aspects of chemically induced mutagenesis in Drosophila melanogaster, Thesis, University of Leiden. Zordan, M., Graf, U., Singer, D., Beltrame, C., Dalla Valle, L., Osti, M., Costa, R. and Levis, A.G. (1991) The genotoxicity of nitrilotriacetic acid (NTA) in a somatic mutation and recombination test in Drosophila melanogaster, Mutation Res., 262, 253-261.