Environmental Mutagenesis 6: 153-188 (1984)

Somatic Mutation and Recombination Test in Dmsophila melanogaster

U. Graf, F.E. Wurgler, A.J. Katz, H. Frei, H. Juon, C.B. Hall, and P.G. Kale
Institute of Toxicology, Swiss Federal Institute of Technology and University of Zurich, Schwerzenbach, Switzerland (U.G., F.E. W., A.J.K., H.F., H.J., P.G.K.); Department of Genetics, University of the Witwatersrand, Johannesburg, South Africa (U.G., C.B.H.); Department of Biological Sciences, Illinois State University, Normal, Illinois (A.J.K.): and Safety and Environmental Protection Division, Brookhaven National Laboratory, Upton, New York (P.G.K.)

A novel test system for the detection of mutagenic and recombinogenic activity of chemicals is described in detail. Drosophila melanogaster larvae trans-heterozygous for the mutations multiple wing hairs (mwh) and flare (flr) are exposed to the test compounds for various periods of time ranging from 96 hr to 1 hr. Induced mutations are detected as single mosaic spots on the wing blade of surviving adults that show either the multiple wing hairs or flare phenotype. Induced recombination leads to mwh and flr twin spots and also to a certain extent, to mwh single spots. Recording of the frequency and the size of the different spots allows for a quantitative determination of the mutagenic and recornbinogenic effects. This and earlier studies with a small set of well-known mutagens indicate that the test detects monofunctional and polyfunctional alkylating agents (ethyl methanesulfonate, diepoxybutane, mitomycin C, Trenimon), mutagens forming large adducts (aflatoxin B,), DNA breaking agents (bleomycin). intercalating agents (5-aminoacridine, ICR-170), spindle poisons (vinblastine). and antinietabolites (niethotrexate). In addition, the test detccts mutagens unstable in aqueous solution (0propiolactone), gaseous mutagens ( I , 2-dibromoethane). as well as proniutagens needing various pathways of metabolic activation (aflatoxin B I, diethylnitrosamine, dimethylnitrosamine, mitomycin C, and procarbazine). The rapidity and ease of performance as well as the low costs of the test necessitate a high priority for validation of this promising Drosophila short-term test.
Key words: genetic toxicology, mutagenicity test, somatic cells, mitotic recombination, Drosophila

Abbreviations: 5-Aa. 5-arninoacridine; AFB I , aflatoxin B ,; DBE, I .2-dibronioethane; DEB, diepoxybutane; DEN. diethylnitrosarnine: DMN. dimethylnitrosamine; DMSO, dirnethylsulfoxide: EMS, ethyl niethanesulfonate; MMC, mitomycin C; MMS. methyl methanesulfonate. Received August 9, 1983; revised and accepted October 3, 1983. Address reprint requests to Dr. U. Graf, Institute of Toxicology. Swiss Federal Institute of Technology and University of Zurich, Schorenstrasse 16. CH-8603 Schwerzenbach. Switzerland.

0 1984 Alan R. Liss, Inc.

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INTRODUCTION

For screening programs concerned with the detection of genotoxic activity of chemical compounds and complex mixtures, the test for sex-linked recessive lethal mutations in Drosophilu is routinely used. Based on this test, it was discovered that Drosophilu has a wide spectrum of metabolic activities capable of activating promutagens and procarcinogens. In some cases, Drosophilu is superior to microbial assays, as for example for the detection of mutagenic pyrrolizidine alkaloids [Candrian et al, 19841, while in other cases, in particular with the polycyclic hydrocarbons and aromatic amines [Vogel et al, 19831, the genotoxic effects are difficult to detect with the standard sex-linked recessive lethal assay. Mollet and Wiirgler [1974] suggested to use somatic cells of Drosophilu for mutagenicity testing. In individuals heterozygous for so-called visible marker mutations, certain mutagenic events can lead to the loss of the dominant wild-type allele opposite to the marker mutation, with subsequent expression of the recessive marker allele in a clone of ensuing mutant cells. Loss of the dominant allele can occur by a variety of mechanisms: homologous mitotic exchange or mutation in the dominant allele leads to clones of euploid cells, while nonhomologous mitotic exchange or deletion generates clones of aneuploid cells. Mollet and Wiirgler [ 19741 showed that alkylating agents induce clones in the eye imaginal discs of treated larvae. In the subsequent years, the test was not further developed, because at that time it appeared that somatic events which could not be studied further by genetic analysis were of minor interest compared with the sex-linked recessive lethal assay in which genetically transmissible damage is detected. In the meantime, a number of developments took place which reactivated our interest in somatic mutation assays in Drosophilu:

1. The correlation between mutagenic and carcinogenic activity of chemicals has generated much interest in the genotoxic activity of chemicals in somatic cells. In Drosophilu about 85% of the carcinogens tested are mutagenic in germ cells [Vogel et al, 19801. 2. Not only mutagenic but also recombinogenic activity of chemicals is now discussed in connection with their carcinogenicity [Cairns, 1981 ; Radman and Kinsella, 19801. 3. Drosophilu larvae possess metabolic activities that allow them to activate a large variety of xenobiotics. It has been shown by Vogel and co-workers that a number of polycyclic hydrocarbons and aromatic amines are mutagenic if larvae are treated [Vogel et al, 1980. 19831. In addition, they showed that some compounds are capable of inducing twin spots in the eye [Vogel et al, 19801. 4. A test system using wing tissue came to our attention that is advantageous because permanent preparations of the wings can be made. Therefore, verification and reconsideration are always possible on the basis of the original material. A second advantage compared with the eye system is the possibility to store the treated flies in 70% ethanol for mounting of the wings at a later time. A third advantage of the wing system is that in every exposed imaginal disc, literally thousands of target cells are potentially at risk.
In this paper, we report details of the techniques used and our experience with an initial set of compounds from different chemical classes.

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MATERIALS AND METHODS Chemicals The chemicals were obtained from the following sources: aflatoxin BI (Senn Chemicals, Dielsdorf, CH), 5-aminoacridine (Sigma, St. Louis), bleomycin (Calbiochem, Luzern, CH), 1,2-dibromoethane (Fluka, Buchs, CH), diepoxybutane (Schuchardt, Miinchen, FRG), diethylnitrosamine (Fluka, Buchs, CH), dimethylnitrosamine (Sigma, St. Louis), dimethylsulfoxide (Merck, Darmstadt, FRG), ethyl methanesulfonate (Sigma, St. Louis), ICR-170 (Polysciences, Warrington, PA), mitomycin C (Sigma, St. Louis), procarbazine (Natulan, Roche, Basel, CH), 0-propiolactone (Fluka, Buchs, CH), Trenimon (gift from Bayer, Leverkusen, FRG), vinblastine (Lilly, Giessen, FRG). With two exceptions, all the chemical mutagens could be dissolved in water. AFBl was used with 2 % DMSO and 5-Aa was dissolved in 10% ethanol. Drosophila Stocks

Two stocks are used. Virgin females are from the stock y; mwh jv and males from the stock y; Dp(1;3)scJ4, flr/TMl, MC ri sbd2. Detailed information on the genetic markers is found in Lindsley and Grell [ 19681. The mutation flr is described by Garcia-Bellido and Dapena [ 19741.
Treatments Larvae trans-heterozygous for multiple wing hairs (mwh) and flare (flr) are obtained from crosses between the two strains mentioned above. The trans-heterozygotes constitute half of the larval progeny, the other half consist of mwh/TMl heterozygotes. The TM1 chromosome is necessary to balance flr in the second parental strain, because flr is homozygous lethal; however, wing cells possessing the flr phenotype in somatic mosaics are viable [Garcia-Bellido and Dapena. 19741. The two types of larvae obtained are not distinguishable and thus are treated together. After metamorphosis, on the other hand, the flies carrying mwh + / + flr can be distinguished by their wild-type body color from the mwhiTM1 sibs which are yellow (y). In contrast to TMl, the third chromosome carrying flr also contains Dp(1;3)scJ4, an insertional duplication that includes y +. The X chromosomes from the two parental strains are all mutant y. Hence, the carriers of the flr chromosome are recognized among the progeny by the dominant expression of yf in an all-yellow sex-chromosoma1 background. Eggs are collected from optimally fertile parents during 8 hr. Three days later, when the larvae are 72 f 4 hr old, they are washed from the bottles according to the procedure of Nothiger [1970]. For rinsing we use a 20% NaCl solution instead of a sucrose solution. The larvae are then placed into plastic tubes (2.5-cm diameter and 8-cm height), which have the lower end covered with a fine nylon gauze. At the opposite end, the tube is closed with a foam rubber stopper. The tube is then placed into a 100-ml beaker containing approximately 0.3 gm of powdered cellulose (Merck, Darmstadt, FRG) and 1.5 ml of the mutagen solution. The larvae start immediately to feed through the gauze on the wet powdered cellulose. About 50 to 100 larvae are used per tube. Usually, the concentrations of the mutagens have to be adjusted according to the duration of the different treatment periods. To get comparable measures of exposure, we express the exposure as mM * hr if larvae were fed for short

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(acute) periods. At the end of feeding, the plastic tube with the larvae is removed and the larvae are flushed with tap water of room temperature to one spot at the edge of the gauze. The tube is then inverted on a culture vial containing 5 ml of dry Instant Medium (Carolina Biological Supply Company, Burlington, NC), and the larvae are flushed with about 5 ml of tap water into the vial. The cultures are kept at 25C until the adult flies hatch. In some cases, treatments of longer (chronic) duration are chosen. Small culture vials are prepared containing 5 ml of Instant Medium and 5 ml of mutagen solution. In these experiments, 72-hr larvae are placed directly into the culture vials. The treated individuals remain in the vials until the emergence of the surviving adult flies. In another type of treatment the larvae are exposed during the whole iarval period. Adult flies are allowed to lay eggs in vials with Instant Medium. When all larvae are hatched, the chemical under test is added to the vials. For details see Results section. The possibility to test volatile mutagens was examined by using 72-hr larvae, which were exposed to DBE. Twenty milliliters of saturated DBE vapor were injected into a I , 150-ml glass bottle containing the larvae [Kale and Baum, 19821. After I hr, the larvae were transferred into vials with Instant Medium. A summary of the various possible exposure schedules is given in Figure 1. Continuous larval feeding may start at an age of 24, 48, or 72 hr roughly coinciding with the beginning of each of the three larval instars. We estimate that in these cases the feeding period is 96, 72 and 48 hr, respectively. These latter figures are only approximations, because repellent chemicals. toxic effects leading to retarded development, or premature pupation may be responsible for variations in feeding time. With continuous feeding procedures in which the chemical is added to Instant Medium, the actual exposure concentration may be lower than the initial concentration used. This was demonstrated for nialondialdehyde by Szabad et a1 [1983]. In the various exposure schedules used, no attempts were made to determine the toxicity of the treatment in a quantitative way. Usually about 100 larvae were

Development: Egg
0

I
24

Larval stages 1-111 I I 48 72

H H

Pupa (Metamorphosis)

Adult fly

120 hours

Mutagen exposure:

2h 4h

6h

48h 72h

Scoring of wings for mutant mwh andlor fk spots

Fig. I . Exposure schedules for feeding of trans-heterozygous mwh + / + flr larvae with chemical mutagens. Acute exposures of 2 to 6 hr can bc performed with 48-hr or 72-hr larvae. Duration of chronic treatments may vary from 48 hr to 96 hr. The wings of thc flies reaching adulthood are scored for single spots (either mwh or flr) and twin spots (adjacent mwh and flr clones).

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treated in each series; the number of surviving flies per vial gives an indication of the toxicity of the treatment. In general, the experiments were done at exposure levels below the LDSO. In those cases where exposure-effect relationships were established, the treatment conditions reached highly toxic levels.
Preparation of Wings

To make permanent preparations of wings they are embedded in Faures solution (gum arabic 30 gm, glycerol 20 ml, chloral hydrate 50 gm, H 2 0 50 ml). Faures solution was obtained from the Kantonsapotheke, University Hospital, Zurich, CH. Fresh flies are brought into a drop of Faures solution. Flies from storage in 70% ethanol are first washed in water and then transferred into Faures solution. The wings are first separated from the body and are then, with forceps, lined up on a clean slide, and allowed to spread out. If becoming too sticky, Faures solution can be diluted with distilled water. The wings arranged on the slide are kept in a dust-free atmosphere (eg, in a Petri dish) for at least 24 hr to become firmly glued to the slide. A droplet of Faures solution is put on a cover slip, which is then, with the hanging drop, lowered on top of the wings. The cover slip is charged for at least 24 hr with several metal cubes (about 40 gm each) while the preparation is drying and hardening. A permanent preparation is obtained by sealing the cover slip with nail polish. In cases, in which the wings have to be analyzed as quickly as possible, one may mount them on the same day the flies hatch. Hereby, it is important to use a very small drop of well-liquified Faures solution to glue the wings onto the slide. After a few hours of drying, a cover slip, again with a very small drop of Faures solution, is placed onto the wings. Provided the preparation is handled carefully, the slide can be analyzed after a few hours of drying under metal cubes. Other authors use Gurrs water mounting medium [Bryant, 1970; Haynie and Bryant, 1977) or Euparal [Baker et al, 19781. In the latter case, the flies are preserved in three parts 70% ethanol and one part glycerol. For mounting, the wings are washed free of preservative, placed on a slide into n-propanol, and embedded in Euparal.
Microscopic Analysis of Wings

The wings (both the dorsal and ventral surface) are analyzed under a compound microscope at 400x magnification. Every cell on the wing blade forms a single cell processus, ie, a hair or so-called trichome [Dobzhansky, 19291. The genetic markers (mwh and flr) alter, if expressed, the final differentiation of the trichomes. Spotinitiating events change the genetic information in one of the continuously dividing primordial wing cells in the larva or the early pupa. The cell lineage derived from such a genotypically altered cell, forms a clone which is recognized as a mutant spot in the adult, when the appropriate markers become phenotypically expressed. During the microscopic analysis of the wings, the position of the spots is noted according to the sector of the wing (see Fig. 2). Only the distal wing compartment [Garcia-Bellido et al, 19761 is scored for clones. The size of a spot is recorded, ie. the number of affected cells, as well as its type, ie, whether it is a mwh or flr spot, or a twin spot. Large clones mostly show an elongated shape and usually extend parallel to the longitudinal axis of the wing [Garcia-Bellido and Merriam, 1971a; Haynie and Bryant, 19771. Marked clones on the wing blade appear in general as contiguous, noninterrupted.spots. However, sometimes the spots are split into two or more cell

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Fig. 2 . Normal half mesothorax showing the regions A-E of the wing surface scored for spots (after Garcia-Bellido and Merriam [ 1971al).

groups of different size arranged along the axis of the main growth direction [GarciaBellido and Merriam, 1971al. The different parts of such split spots can be separated by one or several rows of normal cells. Garcia-Bellido and Merriam [ 197la] interpret these clones as due to partial separation of the cells of a clone during development owing to internal tissue pressure or independent cell movements. Split clones have not been observed by all authors [see eg, Baker et al, 19781. We have occasionally seen split clones. To arrive at an uniform record of split clones and neighboring individual clones, we decided to count two spots as separate spots as soon as they are separated by three or more wild-type cell rows.
Classification of Spots

The classification of small spots consisting of one or two affected cells poses some problems. According to our experience, continously more of the small spots are detected with increasing practice. Because small spots make up the large majority of spots in untreated controls, we had to differentiate true mutant spots from developmental disturbances in trichome formation or trichome pattern formation. mwh. We classified all those cases as mwh in which a wing cell contained three or more hairs instead of one hair per cell as in wild-type (see Fig. 3). We are aware of the fact that on the wings of mwh homozygotes, cells with only two hairs instead of three or more can be found. On the other hand, cells with two hairs seem to appear frequently in sector D of the mwh +/+ flr wings and, at least there, seem to be formed independently of the mwh mutation. To be on the safe side, we decided to exclude from the counts of mwh single spots all single cells with only two hairs. In contrast to this, if a cell with two hairs appeared within a rnwh spot or at its margin, it was included in the determination of the size of the clone. flr. Single flr cells have not been observed. According to Szabad et a1 [I9831 the flr allele used in the present experiments is not, or not fully expressed in clones smaller than four cells per clone. In larger spots, flr exhibits a quite variable

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expression, ranging from pointed, shortened and thickened hairs to amorphic, sometimes balloon-like extrusions of melanotic chitinous material (see Fig. 3). Theoretically, the size of a spot reflects the number of cell divisions that occurred after the induction of the genetic change in the cell lineage. Provided all cells in a clone divide at the same rate, the spot size would directly reflect the number of divisions (n) with the size ranging from 2 ' to 2" cells. Although this strict relation is usually not retained, grouping of the clones according to the inferred minimal number of cell divisions nevertheless provides useful information. We classified the spots according to the following size classes: 1, 2, 3-4, 5-8, 9-16, 17-32 cells etc, and numbered the classes in increasing order beginning with 1. To avoid having one or a few large spots among many small ones biasing too much the calculation of the mean spot size, we decided to calculate the mean size class rather than the mean number of affected cells as a measure for the average spot size. We observed that in untreated controls the very small inwh spots clearly predominate. To improve the power of the statistical analysis, we classified the single spots into two groups: small single spots with one or two affected cells were distinguished from large single spots with three or more affected cells. This differentiation made it possible to test the frequencies of large single spots in treated series against low control values. As twin spots, too, are rare in controls, no particular difficulties were encountered in statistical testing with this type of spots. The induction of small single spots, however, had to be tested against a high, and often variable background of spontaneous events.
Statistical Analysis

The central statistical analysis judges whether the fraction of wings showing at least one spot of a particular type (small single, large single, or twin spot) is increased in the treated series as compared with a matched control series. We employed the

Fig. 3. Trichornes on the wing blade. a) Normal. b) deviate trichornes not counted as rnwh or flr, c) configurations indicative of mwh. d) typical manifestations of flr.

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multiple-decision procedure of Selby and Olson [ 19811, which is based on testing the following two hypotheses: 1) the mutation frequency (induced spontaneous) of treated flies is not higher than the mutation frequency in the appropriate pooled control, and 2) the induced mutation frequency of the treated flies is no less than m times as high as the observed spontaneous mutation frequency in the control (we use m = 1, or m=4; see below). Each hypothesis was tested at the 5% significance level. The following four decisions are possible 1) accept both hypotheses; these cannot be true simultaneously, so no conclusions can be drawn-inconclusive result; 2) accept the first hypothesis and reject the second hypothesis-negative result; 3) reject the first hypothesis and accept the second hypothesis-positive result; 4) reject both hypotheses-weak effect. Because small single spots and total single spots have high spontaneous frequencies, m is fixed at a value of one (testing for the exclusion of a doubling of the frequency to define a negative result). For the rare spontaneous large single and twin spots, m=4 is used in accordance with the practice followed with the mouse spot test [Russell et al, 19811. In a second analysis, the spot density distributions (number of spots per wing) of treated and control series are studied with the chi-square test (if necessary, data from different columns are pooled so that no expected value is below 1 and so that only up to 20% of the expected values are between 1 and 5 [Cochran, 1954; Roscoe and Byars, 19711). For comparison, the statistically equivalent or even preferable Kolmogorov-Smirnov test is calculated [Sachs, 1974; Lindgren, 19761. The chisquare test is used two-sided, whereas the Kolmogorov-Smirnov test is applied onesided. For both tests, the significance level of 5% is chosen. The statistical analyses are performed with the computer program SMART (Somatic Mutation and Recombination Test [Wurgler, unpublished]). For an initial visualization of the data, the spot size distributions for single and twin spots (see Fig. 7, 8), as well as the spot density distributions are plotted [Juon, unpublished].

RESULTS Water Controls Control wings were obtained from flies that were grown on standard medium or on Instant Medium or from flies fed for 6 hr on a water-cellulose suspension. To contrast these controls with solvent controls, we termed this series water controls. Wings from water controls were analyzed in several laboratories. The mean number of spots per wing and the mean size class are given in Table I. In addition, the percentage of wings showing one or more spots is given. Most of the laboratories analyzed individual slides with 40 to 48 wings. In Johannesburg, only 20 wings per slide were used. By adding up sets of two sequential slides, the data from Johannesburg could be made directly comparable to other control sets. The unweighted mean frequencies of spots (averaged over slides) and standard deviations are presented for small single, large single, and twin spots. Clone size of single spots. The spot size distributions for single spots in different water control sets are found in Figure 4a. The graph is constructed in the same way as the corresponding figures in Baker et al 119781. The clones are grouped according to size into integral size classes with upper cell number limits corresponding

Somatic Mutagenicity Test in Drosophila

161

to 2; size class (n + 1) is thus related to the minimal number of cell divisions required to produce a spot of a particular size from a single precursor cell. If the probability of an event generating a marked clone is the same for all cells throughout development, clones of one cell should be twice as frequent as those of two cells, etc. Thus a plot of spot size versus the logarithm of clone frequency should be a straight line with a slope equal to log 0.5 = -0.30 [Baker et al, 19781. Figure 4b shows that in contrast to the expectation, the clone size distribution is bipartite. This may be explained by assuming that two types of clones exist: 1) aneuploid clones that are characterized by a small size, and 2) euploid clones that are of normal (expected) size based on the number of cell divisions taking place between clone induction and differentiation. Variation of spot frequencies in different control sets. As can be seen from the clone size distributions (Figure 4a), the small spots (one or two cells affected) predominate in all data sets. They show comparatively large frequency variations. This is obviously the class of spots where difkrent experience in scoring of slides, somewhat different identification criteria for mwh spots, as well as observer-independent interlaboratory variations of spontaneous spot frequencies are most important. For the small single spots in the water controls, we plotted (Fig. 5) the mean number of small single spots per wing versus their mean spot size (described as the mean size class, see Materials and Methods for the calculations). Accounting for the variation, we accept a statistically significant increase of the frequency of spots only, if the frequency observed with the treated group is above the variation in the control, ie, above the control mean plus 2 standard deviations (SD). The numerical values for the SDs are included in Table I.
Solvent Controls

In this study, we had to include two different solvent controls. Because AFBl was dissolved in DMSO, a solvent control was run with 72-hr larvae fed 2 % DMSO during 6 hr. 5-Aa was dissolved in ethanol. Therefore, a solvent control with 72-hr larvae fed 10%ethanol for 6 hr is included. The data of both control sets are shown in Table I. Neither 2% DMSO nor 10% ethanol lead to an increased frequency of somatic spots on the wings (Table I).
Mutagen-Treated Series

A compilation of the experimental results and the outcome of the statistical analysis is given in Table 11. Individual standard mutagens used to evaluate the test system, lead to the following results:
Chemicals inducing single and twin spots. Atlafoxin B7. This mycotoxin was used because it represents a strong mutagen needing bioactivation via cytochrome P-450-dependent epoxidation. The compound was used in three experiments with 72-hr larvae to evaluatethe influence of the duration of the feeding period. At a concentration of 0.16 mM, a 3-hr feeding period was quite sufficient to induce single as well as twin spots. A 4-hr exposure gave a maximum of spots, whereas with 6-hr feeding no further increase in spot frequency could be obtained. The exposure-effect curves are shown in Figure 9 and the linear regression coefficients calculated for the initial portion of the curves are found in Table 111. The ratio of the induction of large single spots to twin spots is 6.7. The

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statistical analyses presented in Table I1 as well as the nonsignificant regression for small single spots (Table 111, Figure 9) indicate that this spot type is not induced by AFBl under the experimental conditions studied. 7,2-Dibromoefhane. This volatile compound allowed us to evaluate the gas exposure procedures. In adult males, it induces sex-linked recessive lethal mutations at concentrations ranging from 0.2 to 2 parts per million [Kale and Baum. 19791. The exposure-effect relationship has not been studied with the wing spot test, but an exposure of 72-hr larvae to 20 ml of saturated vapor of DBE in 1150 ml air for 1 hr
TABLE I. Spontaneous Spots on Wings of mwh + I
Small singles Spots Per wing Mean size class
.~

+ flr Flies
Large singles Mean % size with class spots
-.

Twins
%

Wings Water controls 40 48 48 48 48 48 48 48 48 48 46 48 48 36 48 48 total SD

% with spots

Spots Per wing

spots Mean size Per w i n g- class

with spots -

Schwerfenbach 0.32 I .08 0.17 1.13 1.13 0.3 1 0.15 1.43 0.12 1.17 0.04 1S O 0.06 1.67 0.2 I 1.60 0.19 1.33 0.37 1.28 0.24 1.36 I .28 0.37 0.33 1.25 0.08 2.00 0.23 I .oo 0.29 I .07

30.0 14.6 25 .O 9.3 12.5 4.2 6.2 18.8 16.7 31.3 19.6 25.0 27.1 8.3 21.8 25 .O

0.050 0.02 1 O.OO0 0.02 1 0.021 0.000 0.02 1 0.OOO 0.000 0.021 0.022 0.083
0.03-1

4.50 6.00
-

4 .OO 3.00 4.00 .-

0.028 0.02 I 0.02 1 0.022 0.02 1

3.00 4.00 5.25 4.00 7.00 3.00 5.00 4.40 1.24

5.0 2. I 0.0 2.1 2.1 0.0 2.1 0.0 0.0 2.1 2.2 6.3 2.1 2.8 2.1 2.1

0.025 O.OO0 0.021 0.OOO 0.000 0.063 0.000 0.021 0.000 0.000 0.022 0.02 I 0.042 0.056 0.000 0.02 1
0.018

5.00 7.00 -

5.33 6.00 -

746

0.22 0.12

I .33 0.26

18.5 8.6

2.1 1.7

0.021

5.00 5.00 6.00 6.00 6.00 5.70 0.68

2.5 0.0 2. I 0.0 0.0 6.2 0.0 2.1 0.0 0.0 2.2 2. I 4.2 5.6 0.0 2. I 1.8 2.1

Water controls 40 40 38 40 36 40 total 234 SD Water controls 48 48 48 32 48 total 224 SD

Johannesburg 0.15 1.17 0.50 I .20 0.45 1.47 0.30 1.17 0.47 1.35 0.32 I .54 0.36 1.32 0.13 0. I6 Normal, IL 0.37 1.33 0.56 1.15 0.50 1.08 0.47 1.20 0.46 1.14 1.18 0.47 0.07 0.09

15.0 40.0 38.9 30.0 30.6 20.0 29. I 10.0

0.025 0. loo 0.053 0.025 0. I67 0.050 0.070 0.055

3.00 3.oo 3.00 3.00 3.67 4.00 3.28 0.44

2.5 10.0 5.3 2.5 16.7 5.0 7.0 5.5

0.050 0 .Ooo 0.000 0.050 0.028 0.000 0.021 0.025

0.042 O.OO0 O.OO0 O.OO0 O.Oo0 0.008 0.019

5.50
-

4.50 7.00
-

4.27 1.27

5.0 0.0 0.0 5.0 2.8 0.0 2.1 2.5

4.2 0.0 0.0 0.0 0.0 0.8 1.9

32.5 41.7 39.6 40.6 35.4 38 .O 3.9

0.021 0.02 I 0.021 0.031 0.021 0.023

0.004

4.00 3.00 3.00 3.00 6.00 3.80 1.30

2.1 2. I 2.1 3.1 2. I 2.3 0.4

5.00 5.00

Continued

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163

gave statistically significant positive results for all types of spots. Diepoxybutane. This is a direct-acting mutagcn that was shown by Shukla and Auerbach [ 19801 to induce many small deletions in germ cells. Our data indicate that in addition to a high frequency of single spots, DEB also induces large numbers of twin spots. Diethylnitrosamine. DEN was chosen to reprcsent the mutagenic nitrosamines that require metabolic activation. Seventy-two-hour larvae were treated either in acute exposures or chronically during the whole third larval instar. DEN showed genotoxic

TABLE I. (continued)
Spots Per wing Small singles % Mean size with spots class
1.11

Wings Water controls 38 40 40 40 40 40 40 40 40 40 total SD

Spots Per wing ~.

Large singles Mean % size with class spots

Spots Per ~. wing O.Oo0 0.050 0.025 0.025 0.025 0.075 0.050 0.025 0.000 0.000 0.028 0.025

Twins Mean size class

-

% with spots

398

Zurich 0.74 0.47 0.35 0.25 0.55 0.17 0.17 0.05 0.35 0.30 0.34 0.20

1.16 1.07 1.00 1.23 1. O O 1.14 1. O O 1.14 1.08 1.09 0.08

52.6 40.0 30.0 20.0 47.5 12.5 15.0 5.0 30.0 25.0 27.8 15.4

0.026 0.025 0.150 0.000 0.000 0.000 0.000 O.Oo0 0.025 0.000 0.023 0.046

3. O O 7.00 4.17 5.00

-

4.79 1.68

2.6 2.5 12.5 0.0 0.0 0.0 0.0 0.0 2.5 0.0 2.0 3.9

6.00 7.00 7.00 5.00 4.33 5.00 6.00 5.76 I .03

0.0 5.0 2.5 2.5 2.5 7.5 2.5 2.5 0.0 0.0 2.5 2.4

Water controls 48 48 40 total SD

Normal, IL (mwh/TMI progeny) 0.17 16.7 0.OO0 1.12 14.6 0.020 0.17 1.12 22.5 O.OO0 0.22 1.00

0.00 3.00 0.00 3.00 1.73

136

0.18 0.03

I .08 0.07

17.6 4.1

0.010 0.012

0.0 2.1 0.0 0.7 1.2

6.2 1.7 1.9 3.3 2.5

O.OO0 0.OO0 O.OO0 O.OO0

0.00 0.00 0.00 -

0.0 0.0 0.0 0.0

-

2%'DMSO controls 48 0.33 60 0.22 54 0.33 total 162 0.29 SD 0.06

10% Ethanol controls

Schwerzenbach 1.44 27.1 1.15 20.0 33.3 1.00

1.19 0.22

26.8 6.7

0.063 0.017 0.019 0.033 0.033

3.67 3.00 3.00 3.22 0.39 4.50 9.00 7.33 6.94 2.27

O.Oo0 0.017 0.037 0.018 0.019

5.00 5.50 5.25 0.35

0.0 1.7 3.7

I .8 1.9

total SD

32 46 40 40 158

0.22 0.43 0.43 0.30 0.35 0.10

Johannesburg 19.8 1.29 1.35 26.1 1.35 40.0 1.25 22.5 1.31 27.1 0.05 9.0

O.Oo0 0.043 0.025 0.075 0.036 0.032

0.0 2.2 2.5 7.5 3.0 3.2 --

0.03 1 O.OO0 0.025 0.050 0.026 0.021

5.00 8.00 5.00 6.00 1.73-

3.1 0.0 2.5 5.0 2.6 2. I

Water controls from different laboratories and solvent controls. For the mean numbers of spots per wing the standard deviations are given. The data from Zurich are unpublished results of P. Schweizer.

1 1
6 7

5
6

size class

100

:
-32 -64

2
3
4

5
9-16

size class
spot size

A
2
3-4
5-8 -64

3-4 -32

5-8

9-16

spot size

Fig. 4. Spot size distribution of single spots in different water control series. a) Individual data sets from different laboratories: J = Johannesburg, N = Normal, S = Schwerzenbach, Z = Zurich. b) t = theoretical expectation for the case that every dividing cell has the same probability of generating a clone recognizable on the wing blade (after Baker et a1 [ 19781); e = experimental data (*) obtained by pooling the data shown in panel a; two linear regressions are fitted by eye.

Somatic Mutagenicity Test in Drosophila

165

SMFlLL
0 0
0

SINGLE SPOTS
Schwer zenbach Johannesburg

Normal
Zurich

. .
0

0 0

.O

--

Fig. 5 . Frequency variation of small single spots (one or two cells) observed in different water control series. For different control samples of 40 to 48 wings the mean spot frequency per wing (abscissa) is plotted against the mean size class (ordinate). Means are shown as surrounded symbols.

activity in both types of treatments. The presence of twin spots indicates that DEN has recombinogenic activity. The statistical tests in Table I1 show that, based on the Selby-Olson analysis, all exposures resulted in large single spots. By contrast, the acute exposures (2 to 6 hr) gave inconclusive or negative results for small single spots. These are only found when the larvae are treated during the whole third larval instar, ie, when the chemical is still present near the time of cessation of cell division in the early pupa. Twin spots are always found in statistically significant frequencies with the exception of the 2-hr exposure. The Selby-Olson diagnosis inconclusive indicates that this exposure is too low. Dimethylnitrosamine. After exposure to this promutagen, not only wings of mwh +/ flr progeny, but also wings of inversion-heterozygous mwh/TM 1 progeny were analyzed. Single spots, small and large ones, were obtained in similar and statistically significant frequencies with both types of wings. Twin spots, by contrast,

TABLE 11. Spot Data Obtained After Mutagen Exposure of mwh

Wings with spots

+ I + flr Trans-heterozygousLarvae of Drosophila rnelanogaster

Density-distribution SPIW C
%

Chemical N 746
S

s c
K

conc

mM 19.03 1-2 1.26 >2 29.63 1-2 3.10

1.90

Treatment 1.55

Type

Conclusion

Mean class size

Controls Sla
s s
t

18.10 2.01 1.74

0.23 range 0.02-0.46 0.22 range 0.04-0.37 0.02 0.02

Slb
s

162 26.54

4.56 5.69 1.48 1.28

s >2
t

Jla
S

234
s

31.20 1-2 27.35

3.40 5.33 1.65 1.33

s >2
t

6.84 2.14

3.38 5.40 29.75 27.20 1.65 1.33

0.32 range 0.23-0.40 0.29 range 0.22-0.33 0.03 0.02 0.48 range 0.17-0.64 0.36 range 0.15-0.50 0.02 0.02

Jlb
S

158 s 1-2
s
t

>2

3.16 2.53

3.38 5.40

0.39 range 0.22-0.48 0.35 range 0.22-0.43 0.04 0.03

N/a
s 1-2

224
s

38.80 38.00 2.30 0.80 18.38 17.65 0.74 POS INC 3.00 1.08 3.80 5.00 1.15 1.18

1.29

s >2
136
t s

Nlay

s 1-2 s >2
t

0.50 range 0.40-0.58 0.47 range 0.37-0.56 0.023 0.008 0.19 range 0.17-0.22 0.18 range 0.17-0.22 0.01 -

Aflatoxin B1 0.16 Slb 38

s s 1-2 s >2

3 hr

Pos
POS
PoS

4 hr
s 1-2 s >2
t

46

6 hr

22
s s 1-2 s >2
t

63.16 36.84 47.37 15.79 65.22 23.91 52.17 13.04 59.09 27.27 50.00 18.18 NEG POS POS POS NEG POS POS 2.45 I .46
5.10

3.98 1.57 5.10 5.14 4.31 1.46 5.11 5.10 3.25 1.29 4.06 6.20

1.16 0.37 0.79 0.18 1.28 0.28 1 .00 0.22 1.09 0.32 0.77 0.23

5-Aminoacridine 40.2 f

6 hr
Jlb
69
t

s s 1-2 s >2
s s 1-2 s >2
t

+ + +-

5' b

20. I

6 hr

54

+ + + +
59.42 52.17 24.64 4.35 44.44 37.04 18.52 7.41 POS POS POS NEG WEAK NEG POS INC 5.33 2.35 1.31 5 .oo 5.75 1.12 0.81 0.35 0.04 0.63 0.53 0.19 0.07

2 .
E
Continued

2 5

TABLE 11. Spot Data Obtained After Mutagen Exposure of mwh

Wings with spots Density-distribution SP/W

+ I + flr Trans-heterozygous Larvae of Drosophila mehogaster (Continued)

__

d
K -

Chemical conc mM

Treatment
6 0

N
Type

Conclusion

Mean class size

z a
c

20.1
s s 1-2 s >2 t
s

4 hr

+
-

43.33 38.33 8.33 0

WEAK NEG INC NEG

I .84 1.25 5.20 0

0.62 0.44 0.08 0

Bleomycin 0.01 % S/a 48

96 hr

+ +
1.48 1.31 0.17 0.02

+ +
-

s 1-2 s >2
t

68.75 66.67 14.58 2.08

POS POS POS INC

I .77 1.22 6.13 5.00

1 hr

N/a
s

38
s 1-2 s >2
t

+
-

1,2-Dibromoethane g 20 ml in I150 ml 52.63 39.47 26.32 13.16 NEG NEG POS POS 2.13 1.08 4.42 5.17

+ +
-

1.08 0.68 0.32 0.16

Diepoxybutane 12.9 S/a

s

f
s t

2 hr 1-2 s >2 14
s s 1-2

32

+
6.40 I .50 7.33 6.20 0.78 0.12 0.66 0.16

53.12 12.50 50.00 12.50

POS NEG POS POS

+ +
-

4 hr s >2
t

42.86 7.14 42.86 50.00

POS NEG POS POS

6 hr 8
s

5.60 2 .OO 6.00 6.38 6.80 1. O O 8.25 1-2 s >2 t 62.50 12.50 50.00 0

0.71 0.07 0.64 0.57 0.62 0.12 0.50

POS INC POS INC

Diethylnitrosarnine 20 f Sla 48
S

2 hr
s 1-2 s >2
t

4 hr
S

48
s 1-2 s >2
t

6 hr
S

45
s 1-2 s >2

+
-

+
-

35.42 27.08 10.42 2.08 27.08 22.92 8.33 12.50 57.78 22.22

44.44

+
-

6 33
S

48 hr

s 1-2 s >2
t

8.89 84.85 72.73 45.45 30.30

POS INC POS INC INC NEG POS POS POS NEG POS POS POS POS POS POS

2.05 1.33 4.20 5.00 2.13 1.42 4.25 5.00 3.53 1.33 4.73 5.50 1.97 1.22 4.81 5.27

0.42 0.31 0.10 0.02 0.33 0.25 0.08 0.12 0.76 0.27 0.49 0.09 2.30 1.82 0.48 0.33

+ + +
5.58 3.38 2.21 0.67 3.00 2.05 0.95
-

Dimethylnitrosarnine 50 f Nla
S

6 hr 24

s 1-2
s
t

>2

POS POS POS

100.00 87.50 91.97 33.33

POS
POS POS POS

2.46 1.41 4.08 5.19 2.13 1.22 4.11 -

+ + + +

6 hr
s 1-2 s >2 t

mwh/TMI progeny 16 s 95.00 95 .oo 60.00 POS POS POS

+ +

+ +

Ethyl methanesulfonate 46. I f J/a

s s 1-2 s >2
t

2 hr

40

+ + +
POS
2.70 1.49 3.95 5.45 2.96 1.41 4.41 5.44
I .92 0.98 0.95 0.28 3.07 1.48 1.59 0.35

+ +
+
POS POS POS POS

4 hr

46

s s 1-2 s >2
t

85.00 60.00 57.50 17.50 95.65 71.74 76.09 26.09

+ +

+ + +

+
Continued

z 2
E
C

TABLE 1 1 . Spot Data Obtained After Mutagen Exposure of mwh

Wings with spots N Type

+ I + tlr Trans-heterozygous Larvae of Drosophila melanogaster (Continued)

Density-distribution
SPIW

Chemical conc

mM

Treatment

Conclusion

Mean class size

6 hr
S

38

s 1-2 s >2
1

+ +

+ + +
-

96 hr
POS POS

71

s s 1-2 s >2 t
S

71.05 55.26 39.47 15.79 95.77 88.73 63.38 29.58

POS POS POS POS POS POS

2.91 1.52 4.58 4.58 2.14 1.27 4.05 5.38

1.39 0.76 0.63 0.18 3.94 2.70 1.24 0.34

+ + + +
+

+ + + + + + +

ICR-170 20.86
Nla
s 1-2 s >2 t

f
40

6 hr

75.00 57.50 45.00 5.00

POS WEAK POS INC

2.21 1.23 3.86 4.50

I .40 0.87 0.52 0.05

Mitomycin C 0.625 Jla

S

f
s 1-2 s >2
t

6 hr

80

0.625
Sla
S

30

+ + + + +
s 1-2 s >2 t

+ + + + +
67.50 30.00 56.25 28.75 100.00 36.67 93.33 56.67
POS NEG POS POS POS POS POS POS

4.75 1.22 6.19 7.00 5.61 1.63 6.12 6.55

1.38 0.40 0.98 0.35 4.73 0.53 4.20 0.97

Procarbazine
S/a

45

48 hr 23

s s 1-2 s >2
t

95.65 91.30 69.57 39.13

POS POS POS POS

2.06 1.32 4.08 5.13
6.22 4.57 1.65 0.65

+ +

300 95

6 hr

s s 1-2
POS POS
POS POS
1.66
0.70

s >2
t

70.53 51.58 34.74 17.89 2.28 1.33 4.22 5.20

I .28 0.86 0.42 0.2 I

+ + + + +

+ + + + + + + +
+ +

8-Propiolactone Sla
50

200

f
s s 1-2 s >2
t

6 hr 72.00 50.00 48.00 32.00

POS POS POS POS

3.40 1.14 5.04 5.83

0.96 0.36

100 48

+
1.13 0.62 0.50 0.27

+ + +

s 1-2 s >2
t

68.75 50.00 39.58 16.67

POS POS POS POS

2.93 I .23 5.04 6.23

Trenirnon Sla
48

0.3

f
s s 1-2 s >2
t

2 hr 41.67 35.42 12.50 4.17

2.43 1.23 6.83 6.00

0.58 0.46 0.12 0.04

4 hr 48

s s 1-2 s >2
t

37.50 31.25 12.50 0

POS (POS) POS INC POS (POS) POS

NEG

3.31 1.33 6.00 0

0.54 0.31 0.23 0

6 hr 180

s s 1-2 s >2
t

57.78 36.11 36. I1 8.89

POS POS POS POS

4.03 1.43 6.02 6.60

1.10 0.48 0.62 0.11

Continued
w

TABLE 11. Spot Data Obtained After Mutagen Exposure of mwh

Wings with spots N Sla 78 Type
%

+ I + tlr Trans-heterozygous Larvae of Drosophila mefanoguster (Continued)

Conclusion size SPlW Mean class Density-distribution

Chemical conc

mM

Treatment

Vinblastine 0.1 s s 1-2 s >2 t

s

6 hr

+ +
0.77 0.63 0. I4 0.01

+
+
-

44.87 41.03 10.26 1.28

POS POS POS NEG

1.73 I .22 4.00 7.00

0.005

48 hr

36

s 1-2
s >2 .t

+ +
2.34 I .36 4.50 5.00

69.44 50.00 27.78 2.78

POS POS POS INC

0.89 0.61 0.28 0.03

+ + +
-

All short-time treatments were done with 72 h old larvae. Data are not corrected for spontaneous spots. Codes: Controls: Sla, Schwerzenbach, water; S/b, 2 % DMSO; Jla, Johannesburg, water; Jlb, 10% ethanol; Nla, Normal IL, water; N/ay, water (mwhl TM I flies). Treatments: f, feeding; g, gas exposure; 2 hr, exposure period 2 h. Wings with spots: Type-s, total of single spots; s 1-2, small single spots; s >2, large single spots; t, twin spots. Statistics: Conclusion, SELBY-OLSON analysis; m, induced frequency no less than m times control; for total of single spots and small single spots m = 1 is chosen and for large single spots and twin spots m = 4 (see text for details). NEG, negativc; INC, inconclusive; POS, positive; (POS) SELBY-OLSON diagnosis not accepted because mean is within control range; WEAK, weak positive; Density distribution-SP/W, mean number of spots per wing; C, CHIsquare at 5 % level of significance (2-sided); K, KOLMOGOROV-SMIRNOV-test at 5 % level of significance (I-sided); significant; (+), statistical diagnosis not accepted because mean is within control range; -, not significant; (blank), calculation not possible (sample too small).

+,

Somatic Mutagenicity Test in Drosophila

TABLE III. Regression Analysis of Exposure Effect Relationships
Compound 5-Aminoacridine Spot tYPe
S

173

Reg. coeff 0.00301 0.00178 0.00135

SD

Significance

Ratio f SD NC NC NC 6.8 f 1.9 NC 6.7 f 1.7 7.8 k 2.6 3.4 f 1.1 4.8 f 1.6 NC NC NC 2.8 f 0.16 1.1 f 0.11 1.7 f 0.07

s 1-2 s >2
t

O.oooO87
1.558 0.018 1.531 0.229 0.0140 0.00607 0.00851 0.00179 0.428 0.105 0.318 0.0383 54.08 20.57 33.38

Aflatoxin B1

s 1-2 s >2
t

Ethyl methane sulfonate

s 1-2 s >2
t
S

0.000428 0.000491 0.000214 0.000195 0.200 0.070 0.054 0.058 0.000908 0.000376 0.000908 0.000595 0.129 0.0857 0.0828 0.0343 I .27 2.04 0.87

* * *
NS

*
NS

* * *

*
* * *
NS

Trenimon

s 1-2 s >2 t

* * * *

NS

Methotrexate

s 1-2 s >2

The dose-response curves were recorded using different exposure periods (0 to 6 hr) for one mutagen concentration (see Table 11). Only in the case of methotrexate [Wurgler et al, 1983b] different concentrations for a constant chronic exposure of 48 hr were used. Level of significance: * = significant at the 5% level, NS = not significant. Ratio: regression coefficient single spotdregression coefficient twin spots. NC = not calculated because at least one regression coefficient was not significantly different from zero.

are found only on the wings of the double heterozygotes, because the TMl chromosome lacks flr. Ethyl mefhanesulfonate. This classical monofunctional alkylating agent was used to study the influence of increased feeding periods. In a first set of tests, EMS was fed to 72-hr larvae for 2, 4 and 6 hr, respectively. For chronic treatment with EMS, eggs were collected during 24 hr, and 24 hr after the removal of the adult flies, EMS was pipetted onto the surface of the culture medium containing the hatched young larvae. This treatment induced about the same frequency of spots as a 4 hr treatment of 72 hr larvae (Table 11). With all exposures studied, EMS induced small single, large single, and twin spots. The small and the large single spots predominate over the twin spots (Fig. 9). Based on the regression coefficients (Table 111) calculated for the initial portions of the exposure-effect curves, the induction ratio (+SD) of large to small single spots is 1.4 0.17. Mifornycin C. This well-known promutagen was tested independently in two laboratories. Both laboratories found that MMC induces single spots as well as twin spots. Unexpectedly, the same exposure (0.625 mM fed for 6-hr to 72-hr larvae) gave about twice the number of spots in Schwerzenbach as compared with Johannesburg. Because the samples of the chemical were of different age, it cannot be decided whether the difference is due to the chemical or to experimental conditions. This

174

Grafet a1

1 Cell

Mitosis

2 Cells

Ensuing clones

e
mvuh

W h mwh

'1

+
+-mwh
flr

mwh

flr

Point mutation
Fig. 6. Genetic schemes illustrating various ways of spot formation in the somatic mutation and recombination test with the wing cell markers multiple wing hairs (mwh) and flare (flr). Twin spots are obtained by recombination proximal to the flr marker (b), while more distal recombination produces mwh single spots only (d). Deficiencies ( c ) . point mutations (e) and nondisjunction events (0 give rise

Somatic Mutagenicity Test in Drosophila

175

1 Cell

Mitosis

2 Cells
mwh

Ensuing clones

+
I

mwrh

fir

Recombination

flr

+
fir

+
I

Recombination
f

f Ir

mwh

+
I

mwh

+
1

/
\
mwh
I

mWh

+
I

+
I

f Ir

f Ir

t +
I

+ Nondisjunction

flr
U

to mwh single spots, or in analogous ways to flr single spots (not illustrated). Only the relevant left arms of chromosome 3 are shown in the panels.

176

Grafet al
BLEOMYCI N
trmtnent8 86
h

ETHYL METHFINESULFONRTE
conc.8 96.1 mft ffl treated2 38 wings treotmentz 6 h ncon!roli 234 uings

slnals spots

spot

slze

tuln s w t s

I
tuln spots

DIETHYLNITROSRMINE
conc.8 20 nM treated8 45 uinas treotmenl8 6 h Ucontrol: 746 uinas

DIETHYLNITROSRMINE
conc.I 6 nM treated, 33 uings

treotaentr 48 h u c o n t r o l : 746 uings

4I
30
\

single spots

0 ':

$0
0

.$
uo
I

I-Y

6-1 4-16

-a2

-c(

-120

-266 -612 -ION >(OH

spot size
I

spot slze

tuln soots

.$/

twin spots

5 5
2 :
1

1 4

6-8

9-16

-32

-8y

-126

-266 -6lZ - l W >ION

spot size

Fig. 7. Wing spots induced by treatment with bleomcyin, EMS, and DEN. For details of the duration of feeding (treatment), exposure concentration. and number of wings analyzed, see the individual panels. Open columns: controls: hatched columns; treated series. Note that the ordinates giving the frequencies of spots per wing have different scales.

observation points to the need for a coordinated study of interlaboratory variations. Procarbazine. We studied the effects of procarbazine as a representative of a promutagen of the hydrazine type. It proved active when fed chronically at low concentration as well as in an acute treatment at a six times higher concentration. 0-propiolactone. This compound with its known instability in aqueous solution was included in our study to test the efficiency of acute treatment. When injected into adult males, but not when fed, /3-propiolactone was a potent inducer of sex-linked recessive lethals [Kortselius, 19791. In our study, feeding of 72-hr larvae with 100 or 200 mM for 6-hr gave a clear-cut positive result. Trenimon. As an example for the genotoxic efficiency of polyfunctional alkyl-

Somatic Mutagenicity Test in Drosophilu

177

ating agents, Trenimon was studied. This compound induces increasing frequencies of both small and large single spots with increasing exposure (Fig. 9). Due to fluctuations at higher exposure levels, the ratio of small to large single spots cannot be estimated. Twin spots are induced at the highest exposure level (6-hr feeding) in a statistically significant frequency (Table II), but more data are needed to obtain a significant exposure-effect relationship (Figure 9, Table 111). Chemicals inducing only single spots.
5-Arninoacridine. This frameshift mutagen induced significant frequencies of single spots. After an acute exposure of 72-hr larvae, the induction of large single spots, but not of twins, was observed. In the 6-hr exposure the frequency of twin

PROCRRBRZINE
conc. i US mtl treated: 23 uinps
lrealmmtr 48 h O c o n t r o l : 746 uinps

B-PROPIOLRCTONE
conc.8 LOO mtl treated: 48 UinQs
treatmenti 6 h Ocontrol: 7% uings

sinole spots

soot size I

I
tuin scots

spot

size

VINBLRSTINE
conc.8 0.1 mM treated: 78 winos
treatmenti 6 h Ocontroli 746 uings

5-RMINORCRIDINE
conc.8 1 1 0 . 2 . M treated, 69 winos treatmentr 6 h Ocontroli 158winos

tuln spots
\

q
H

: 33
I 2 2-4
6-8
9-16

-32

-84

-128

-26(1 -612 -IW r l W

spot size

Fig. 8. Wing spots induced by treatment with procarbazine. P-propiolactone, vinblastine. and 5-Aa. The duration of feeding (treatment), exposure concentration, and number of wings analyzed are given with the individual panels. Open bars: controls: hatched bars: treated series. Note that the ordinates giving the frequencies of spots per wing have different scales.

178

Grafet al

5-FIMINOFICRIDINE

QFLFITOXIN

B1

30

0.25

0.50

0.75

exposure

(mM.h)

1 .oo

ETHYL METHQNESULFONQTE
0 l n1

TREN I MON

60

exposure ( m M . h )

120

180

240

0.00

0.40

0.80

exposure

(mK.h)

1.20

1.60

e
@-------*

........

small singles large singles a twins

Fig. 9. Exposure-effect curves for mutagen-induced wing spots. For all four chemicals, the values on the abscissa are given as mM-hr and on the ordinate as mean number of spots per wing. Note that for EMS the scale of the ordinate is different from that of the other panels.

Somatic Mutagenicity Test in Drosophila

179

spots was higher than in the control, but it did not reach statistical significance. The exposure-effect relationships (Fig. 9) indicate that small and large single spots increase linearly with exposure. The regression lines are parallel with a common regression coefficient of 0.00156. This indicates that the curves for small and large single spots differ only in the control frequency, which is higher with the small spots. The values for the twin spots are of the same order as in the control without statistical significance (Table 11). In addition, the regression analysis gives no statistically significant regression (Table 111). Under the experimental conditions tested so far, 5 Aa does induce single spots but no twin spots. Bleomycin. This compound is a very efficient inducer of DNA breaks [Vig and Lewis, 19781. Unexpectedly, we were not able to find exposure conditions that led to a statistically significant increase of the twin spots. Almost all treatments resulted in increased frequencies of single spots (data not shown). We observed a significant induction of large single spots when we collected eggs in vials with Instant Medium for 30 hr and added the bleomycin at the end of the egg collection period (Table 11). ICR-770. Structurally, this chemical consists of a flat ring system, the intercalating part of the molecule, and an alkylating part which can fix the molecule to DNA. Feeding ICR-170 for 6 hr to larvae, resulted in increased frequencies of single spots. The clearest positive result was obtained for large single spots. Vinblastine. Vinblastine represents the class of mutagenically active spindle poisons. A 6-hr treatment of 72-hr larvae induced large single spots but no twin spots. Essentially, the same is true for a chronic treatment of 72-hr larvae.
DISCUSSION Genetic Basis of the Effects Detected

The assay consists of exposing to a mutagen populations of cells that are destined to multiply in relatively fixed configurations so that an induced mutation in one of the exposed cells will give rise to a detectable clone. To ensure the clone is identifiable on the surface of the adult fly, one chooses genetic markers that are expressed autonomously in wing cells. In our experiments, the exposed cells of the larval wing imaginal discs are trans-heterozygous for two recessive markers located on the left arm of chromosome 3 . Multiple wing hairs (rnwh) is at position 0.0 and flare (flr) at 39.0, while the centromere is located at 47.7 [Lindsley and Grell, 1968; GarciaBellido and Dapena, 19741. In all the experimental series analyzed, the occurrence of the various types of spots was as follows: most frequent were single spots expressing the mwh phenotype, less frequent twin spots with both a mwh and a flr sub-clone, and quite rare single spots with the flr phenotype. There exist several mechanisms that lead to genetically marked clones (see Fig. 6). An important possibility is a mitotic recombination event between two non-sister chromatids. Twin spots are expected if recombination occurs between flr and the centromere and if an X-type segregation follows so that each daughter cell receives one recombined and one nonrecombined chromosome [see Becker, 19761. A recombination event between mwh and flr may result in a mwh single spot. If both types of recombination events (one between flr and the centromere and a second between mwh and flr) take place within the same cell, a flr single spot may result. Selection against cells in one of the homozygous marked subclones in a growing

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twin spot with extinction of one and persistence of the other of the two subclones results in a single spot. The same may result from lethal damage to one of the cell lineages in an original twin spot. A mwh single spot can also appear if in a small twin spot flr is not phenotypically expressed [Szabad et al, 19831. The appearance of a single spot can, theoretically, have a number of other genetic causes as well: A gene mutation or gene conversion in the wild-type allele. as well as the deletion (small or large) of a chromosomal segment involving the wildtype allele may result in a single spot. Phenocopies might mimic genetically determined spots. Nondisjunctional or other loss of the chromosome carrying the wildtype allele represents another mechanism that may lead to single spots. The observation of twin spots after X-ray treatment [Haynie and Bryant, 19771 as well as after chemical treatment (AFB1, MMC, etc) clearly indicates that mitotic recombination between flr and the centromere occurs in wing disc cells. From this, we infer that at least some single spots are relics of twin spots. For X-ray treatments, there exists genetic as well as cytological evidence for lethally damaged cells that lead to the loss of a subclone [Merriam and Garcia-Bellido, 1972; Haynie and Bryant, 19771. For chemicals, such studies are not available, but the finding of Becker [1975] that unequal mitotic recombination in euchromatin can be induced by X-rays and by triethylene melamine is most suggestive. It was shown by Merriam and Garcia-Bellido [1972] that X-ray induced rnwh spots are very much reduced in frequency in larvae that are inversion-heterozygous for the third chromosome. Therefore, it is expected that those mutagens that lead to spots predominantly by recombination events should strongly depend on the absence of inversion-heterozygosity . On the other hand, mutagens acting through mechanisms independent of recombination are expected to lead to roughly the same rnwh frequency in structurally normal larvae as in inversion-heterozygotes. Data are accumulating which show that certain chemicals induce single spots on wings of inversionheterozygous mwh/TM 1 flies: DMN (Table n), fluorodeoxyuridine [Katz, unpublished], methotrexate [Wurgler, unpublished], and senkirkine [Frei, unpublished]. Overall, the data suggest that gene mutations and/or small deletions are induced in cells of the larval wing discs. Since it is known that particular genes can respond quite differently to the same mutagen exposure (eg, in E cofi, see Aaron et a1 [1980]), other genes in addition to mwh should be studied. Gene conversion remains a theoretical possibility without proof or disproof. Gene conversion has been shown to occur in the germ line of Drosophilu. Observations at different loci (rosy and maroon-like [Chovnik et al, 19771, rudimentary [Carlson, 19711, garnet [Hexter, 19631, shibire [Kahn and Sick, 19821) indicate that nonreciprocal intragenic recombination is a general phenomenon in Drosophifu. Kahn and Sick [1982] tried to promote gene conversion in germ cells by EMS. The effect was not sufficiently large or reproducible to prove an enhanced gene conversion. How the situation in somatic cells contrasts that one in germ cells remains to be determined. That mitotic gene conversion is a realistic possibility is substantiated by the fact that meiotic as well as mitotic gene conversion is known in fungi, eg, yeast [Zimmerman et al, 19751. Nondisjunction seqms to be detected by the wing test, since vinblastine, a spindle poison interfering with tubulin assembly [Wilson et al, 1975; Snyder and Mclntosh, 19761, does induce single spots, but no twin spots. Phenocopies, if they occur, would falsify the frequency of spots. Some pheno-

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copies have been observed after irradiation of wing disc cells with lo00 R. Phenocopies of the mwh marker were never found, but y phenocopies apparently form a low percentage of modifications that change the color of the bristles [Garcia-Bellido and Merriam, 1971bl. These modifications are restricted to a limited period of time toward the end of development. In the cross used for the present study, some rare mwh phenocopies could be expected from position effects connected with the Dp(1;3)scJ4insertion present on one of the third chromosomes. The frequency of phenocopies has not yet been determined after treatment with chemical mutagens. The examination of 48 wings from a Berlin wild stock did not give any indication for the existence of spontancous phenocopies.
Mutagen Treatments

In this paper, we present mostly data on chemicals that were fed to the larvae. As an example of gas exposure, we report on the clear-cut positive outcome of an assay with DBE. Acute exposures. The initial idea was to treat young larvae that would develop large, easily recognizable wing spots. On the other hand, older larvae have the advantage that larger numbers of cells in the wing discs are exposed simultaneously. We compromised for these two contradictory aspects by using 72-hr larvae. All chemicals used for acute feeding could be fed as solutions mixed to powdered cellulose without addition of yeast. In this way, possible influences of live yeast on the mutagen exposure of the larvae can be avoided. This is particularly important because Callan and Philpot [1977] have shown that under certain growth conditions Succhuromyces cerevisiue is capable of activating promutagens such as benzo(a)pyrene. Some chemicals tested with acute feeding gave, in terms of a developmental analysis of spot formation, easily interpretable results with respect to the size of the induced spots. After an acute exposure of 72-hr larvae, the wing disc cells are passing through about six to seven cell divisions until final differentiation occurs at metamorphosis. Those of the spot-generating events that take place at the time of exposure mostly lead to large spots. In the case of DEB for example, both the large single spots as well as the twin spots have, as expected, a mean size class of 4.4 and 5.2, respectively. Depending on the chemical studied, induced small single spots may also be found in addition to large spots. While the small spots are absent with AFBl (Fig. 9, Table 111), DEN (Fig. 7) and DEB (Table 11), they are present for example with EMS (Fig. 7) and 0-propiolactone (Fig. 8). These small spots affecting only one or two cells may have been induced by mutagen molecules remaining in the body after cessation of the exposure or may represent slow growing, eg, aneuploid clones [Baker et al, 19781. How important the persistance of compounds with long biological half life in larval and pupal tissues may be is at present unknown. The bimodal spot size distributions seen with compounds such as EMS (Fig. 7) and 6-propiolactone (Fig. 8) seem to be more in favor of an immediate induction of two types of clones, ie, clones with normal proliferation capacity lead to twin and large single spots, while clones with reduced proliferation capacity lead to small single spots. ICR-170 is highly mutagenic in adult Drosophilu males. If administered by abdominal injection, it induces sex-linked recessive lethals in all stages of spermatogenesis and 2;3-translocations as well as chromosome loss in mature sperm (for a

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review see Vogel et a1 [1981]). In feeding experiments with ring-X males, ICR-170 did not induce sex-chromosome loss, whereas abdominal injection of the same type of males yielded a statistically significant frequency of ring-X loss [Graf, unpublished]. The observation that acute feeding of trans-heterozygous larvae with ICR-170 leads to a statistically significant increase in the frequency of wing spots, demonstrates the ease with which genotoxic chemicals are identified in the wing spot test without laborious injection experiments. Chronic exposure. Theoretically, different spectra of spot size are expected after chronic feeding as compared with acute exposures. For single spots, the two types of size spectra are exemplified by the DEN data presented graphically in Figure 7. A continuous treatment during 48 hr results in a distribution in which the small spots predominate and in which the larger spots are represented with continuously decreasing frequencies. Such a picture is expected: in the larvae feeding on the DENcontaining medium, the imaginal discs are growing and thus increasingly more target cells become exposed, which increases the number of spots initiated in a disc. Yet, the number of cell divisions following a spot initiating event becomes less as development proceeds. This consequently leads to gradually smaller spots. For twin spots, the continuous and acute feeding procedures lead primarily to quantitative differences, the continuous treatment yielding higher spot frequencies. The spot size distributions are very similar, most of the twin spots comprising from 9 to 32 cells (see Fig. 7 for DEN, Fig. 8 for procarbazine, and Table I1 for EMS). Certain compounds such as bleomycin induced only small single spots upon acute feeding. After a most massive exposure during the entire larval period of development, however, a statistically significant frequency of large single spots was induced in addition to the small single spots. The induction of twin spots could not be assessed, although the treatment occurred under conditions that may lead to twin spots in the eye [Vogel, personal communication]. Bleomycin was also reported to induce somatic recombination spots in dorsal abdominal histoblasts as well as crossing-over in male germ cells [Demopoulos et al. 19801. It is not clear at present how this apparent difference with respect to twin spot induction has to be interpreted. The studies performed so far with a large variety of mutagens indicate that acute exposures of different duration are possible and may allow the detection of genotoxic activity. On the other hand, our experience with weak mutagens and nonmutagens is not sufficient at present for recommending a detailed test system. Detection of direct-acting mutagens. Strong direct-acting mutagens such as methylating and ethylating monofunctional alkylating agents, eg, methyl nitrosourea [Wurgler et al, 1983b], ethyl nitrosourea wurgler et al, 1983a1, and EMS, or polyfunctional ablating agents, eg, Trenimon and DEB, are readily detected in the wing spot test. In these cases, short acute treatments of just a few hours duration are sufficient to detect the genotoxic activity. Detection of promutagens. Biochemical studies [Baars, 1980; Hallstrom and Grafstrom, 1981; Hallstrom et al, 1981; Holzer, 1981; Forster, 19821 as well as genetic tests with a large variety of promutagens and procarcinogens [Vogel, 1977, 19821 have shown that Drosophila possesses a potent xenobiotics metabolism. Corresponding enzymatic activities have been demonstrated in homogenates of adult flies as well as larvae. Genetic studies indicated an active metabolism in germ cells of adults and larvae. The situation in the somatic cells of the wing imaginal discs was largely unknown when we started our experiments. For a first check of the metabolic

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capacity of wing disc cells (and possibly of other larval cells capable of producing long-lived mutagenic metabolites), we included several promutagens in the sample of compounds to be studied during the initial phase of test development. Among the mutagens detected with the Drosophila wing system, the following promutagens activated via various metabolic pathways proved to be active: cyclophosphamide, seneciphylline Wiirgler et al, 1983al; DEN (Fig. 7); procarbazine (Fig. 8); AFB1, DMN, and MMC (Table 11). More promutagens need to be tested to arrive at a validation comparable to the sex-linked recessive lethal assay [Lee et al, 19831. However, the results with compounds tested so far indicate that the high versatility characterizing the sex-linked recessive lethal assay may also extend to tests with mitotically dividing somatic cells treated in larvae. Detection of unstable mutagens. /3-Propiolactone was selected as a model mutagen with a known short half life of about 3.5 hr in aqueous solution [Kortselius, 19791. The genotoxicity of this chemical was difficult to detect after feeding to adult flies. When injected into adult males, /3-propiolactone was a potent inducer of sexlinked recessive lethals [Kortselius, 19791. Using larvae, this unstable compound was detected in feeding experiments already with a few hours of exposure. Figure 8 shows the induction of twin spots and the bimodal distribution of single spots observed in a 6-hr exposure experiment. This clearly illustrates the ease of detecting the mutagenicity of an unstable compound in the wing assay by means of an acute feeding regime. In tests with adults, injection of an unstable mutagen is generally required when the chemical has a half life of only a few hours. Detection of mutagens with different modes of action. Alkylating agents, which form small adducts on the DNA as premutational lesions, are easily detectable in the wing spot test. Compounds forming large adducts on the DNA, for example AFB1, are active, also. Cross-linking agents such as MMC and Trenimon, exhibit genotoxic activity as well. The mutagenic activities of the low molecular aldehydes, formaldehyde, and malondialdehyde were demonstrated by Szabad et a1 [ 19831. 5-Aa, a frameshift mutagen, induced single spots (Fig. 8). Under the conditions used so far, no recombinogenic activity could be detected. This is unexpected since in yeast other frameshift mutagens, such as acridine orange, induce recombination [Simmon, 19791. The same situation is found with the frameshift mutagen ICR-170. In Drosophila larvae, it induced single spots but not twin spots, whereas in yeast it was found to induce homozygosity by genetic recombination and gene conversion Prusick and Andrews, 19741. It remains to be determined whether these differences seen between yeast and Drosophila result from differences in mutational mechanisms or whether in Drosophila the induction of recombinogenic events leading to twin spots needs higher concentrations of the frameshift mutagens than could be applied so far. The wing test system also detects mutagens that do not directly interact with DNA. Out of the class of spindle poisons, we tested vinblastine (Fig. 8) and observed an induction of single spots. The absence of twin spots seems to support the supposed mechanism of action via somatic nondisjunction. Methotrexate (amethopterin) was tested [Wiirgler et al, 1983bI as an example of an antimetabolite that in other test systems such as Saccharomyces cerevisiae or mammalian cells in culture proved to be mutagenic. The effect is due to disturbances arising in the DNA precursor nucleotide pools [Kunz, 19821. In Drosophila, methotrexate proved to be a potent genotoxic agent inducing wing spots at high frequencies.

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Exposure-effect relationships. For some chemicals data from three different exposure levels are available (Table 11). For the first (preliminary) analysis, we fitted linear regressions to the more or less linear low-exposure portions of the exposureeffect curves (see Table 111. Fig. 9). As more data become available the use of other regression models has to be evaluated. At present, the linear approximations allow for a preliminary classification of the chemicals with respect to the preference of induction of particular types of spots or the absence of certain types of spots. The analysis of exposure-effect curves (Fig. 9, Table 111) allows the substantiation of the following points:

1 . Absence of induction of certain types of spots: With 5-Aa, no significant regression for twin spots is found. This indicates that under the conditions tested so far, 5-Aa does not induce twin spots. In the same way, it appeared that small single spots are not induced by AFB1. With the twin spots induced by Trenimon, the situation is less clear. No significant regression is obtained, but the frequency at the highest exposure is statistically significantly different from the control. From this, we conclude that at an exposure of 1.6 mM.h, Trenimon does induce twin spots, but that more data are needed before a regression analysis can show a significant exposureeffect relationship. 2. Relative induction frequencies: The data presented in Table I1 indicate that particular chemicals might be characterized by different relative induction frequencies for the different types of spots. The small and large single spots induced by 5-Aa can be approximated by two parallel regression lines with a common regression coefficient. This shows that despite the different spontaneous frequencies, the two types of spots are induced with the same probability. On the other hand, the different regression coefficients obtained for different spot types with AFBl and EMS, allow to estimate the relative induction probabilities for different types of spots. For example, the ratio of induction of large single spots to twin spots is 6.7 for AFB1, but only 4.8 for EMS (Table 111). Although additional data are needed to substantiate the difference at the 5 % level of statistical significance, we assume that in the future such analyses will allow for a classification of different types of chemicals. 3. Shape of the curves at high exposure levels: There are two examples demonstrated in Figure 9 in which at the highest exposure level, substantially fewer spots than expected are observed: this concerns the large single spots after AFBl treatment and all three types of spots after EMS treatment. Several factors can contribute to the reduction of spots at higher exposure levels. Repellent effects of high concentrations of chemicals such as EMS are known for adult feeding [Lee, 1976; Aaron et al, 19771 and may lead to a reduced uptake of the mutagen in larval feeding experiments. This is particularly a problem with 72-hr larvae, which can survive and pupate without any additional feeding [Simpson, 19791. At toxic exposure levels, a preferential survival of individuals having fed less than average, yields surviving adults which, overall, will have less than the expected average spot frequency. The regeneration dynamics are another phenomenon which might interfere with the phenotypic manifestation of the induced marked cell clones [Haynie and Bryant, 19771. To establish the exact relationship between clones induced and clones observed at high exposures remains a task for the future. With promutagens, such as AFB 1, which need enzymatic biotransformation to form the ultimate mutagen, the enzymatic systems responsible for the metabolic transformation may become satu-

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rated at high exposure levels. Blijleven and Vogel [1977] showed that this can occur in exposed spermatids.
CONCLUSIONS

This paper gives a detailed description of the somatic mutation and recombination test with wing imaginal disc cells in Drosophilu melunoguster. The data presented document the high efficiency with which this bioassay detects the genotoxic activity of mutagens selected from various chemical classes. The spectrum of compounds whose genotoxicity can be detected with this assay ranges from the strong directacting alkylating agents to a number of promutagens activated via different pathways of enzymatic biotransformation. The versatility of the test procedure makes it possible to test stable as well as unstable compounds. It is also possible to test not only dissolved, but also gaseous chemicals. Usually an experimental series begins with a preliminary test involving a large array of concentrations. This allows to detect the toxic and nontoxic exposure ranges. A potent mutagen is already detected with a small number of wings analyzed for spots (10 to 20 wings). In the present exploratory phase of test development, additional wings are usually scored to obtain quantitative information suitable for a variety of statistical comparisons. In this way, basic information is obtained concerning the spectra of spots induced by different types of mutagens and promutagens, the influence of larval age and the effects of other treatment conditions. Experiments will continue with attention to reproducibility of results and studies of weak mutagens to explore the limits of detectability. The wing spot test as a short-term test assay is relatively inexpensive in comparison with the sex-linked recessive lethal test of Drosophilu. A potent mutagen is detected within ten days. The requirements in laboratory space and other facilities are minimal. Over all, the wing spot test is fast, reliable, and easy to perform.

ACKNOWLEDGMENTS

We thank Rita de Castro and Doris Singer for excellent technical help and Ruth Todd for the careful typing of the manuscript. We thank Pascal Schweizer, Institute of Radiobiology, University of Zurich, for the permission to use his unpublished control data for comparison. The studies in Schwerzenbach are supported by the Swiss National Science Foundation grant No. 3.670-0.80. U. Graf was supported by a Postdoctoral Research Fellowship of the University of the Witwatersrand, Johannesburg, SA.
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