Nondisjunction induced by ethanol in Drosophila melanogaster females

Mutation iiesearch, 268 (1992) 95-104 © 1992 Elsevier Science Publishers B.V. All rights reserved 0027-5107/92/$05.00

95

MUT 05104

Nondisjunction induced by ethanol in Drosophila melanogaster females M 6 n i c a R e y , Aria M. P a l e r m o a n d E n z o R . M u f i o z Departamento de Radiobiologfa, Comisidn Nacional de EnerglaAtdmica, 1429BuenosAires (Argentina) (Received 17 September 1991) (Revision received 6 January 1992) (Accepted 8 January 1992)

Keywords: Ethanol; Drosophila melanogaster; Nondisjunction

Summary The effect of ethanol on chromosomal segregation was investigated in Drosophila melanogaster females homozygous for a structurally normal X chromosome marked with the recessive mutation yellow (y/y). For chronic treatments the females were kept from eclosion in food supplemented with 10% or 15% (v/v) ethanol, mated 24 or 48 h later to wild-type males and brooded in freshly prepared ethanol food. For the acute treatments 24- or 48-h-old females were exposed for 60 rain to a 75% (v/v) ethanol solution by means of soaked tissue paper placed at the bottom of regular culture vials and brooded daily after mating. The results obtained show that: (1) both treatments significantly increased the frequency of X-chromosome nondisjunction; (2) after acute treatment this effect declined in later broods; (3) the yield of malformed flies in the progeny of acutely treated females was significantly higher than control values and also declined in later broods; (4) ovary analysis showed that chronic ethanol treatments caused a cessation of egg production. The induction pattern of nondisjunction and malformed flies is interpreted as giving support to the assumption that these effects may result from a direct action of ethanol. Ethanol toxicity was assessed by exposing females of different ages to a 50% or a 75% (v/v) solution for 60 min and counting the surviving flies 24 h later. The surviving fraction decreased steeply from 1-day-old (100%) to 5-day-old females (1.8%). it is suggested that toxicity may have been due to the action of a metabolite of ethanol, probably acetaldehyde.

Literature on the possible effect of ethanol on the disjunction of meiotic chromosomes is scanty. In Zea mays several anomalies suggestive of abnormal chromosomal behavior have been observed after exposing tassels at the meiotic stage to filter papers soaked with ethanol (Maguire, 1976).

Correspondence: Lic. M6nica Rey, Departamenlo de Radiobiologia, Comisi6n Nacional de Energla At6mica, Av. del Libertador 8250, 1429 Buenos Aires ~Argentina).

In mice the early claim of Kaufman (1983) that ethyl alcohol given orally to females after mating induced nondisjunction was later confirmed by the observation of a consistent increase of zygotes with hyperdiploid cells at the first cleavage mitosis (Kaufman and Bain, 1984; O'Neill and Kaufman, 1987). However, following essentially the same procedure Washington et al. (1985) failed to detect differences between experimental and control groups on the basis of trisomic zygotes, but found a suggestive and significant increase in the incidence of late death. Yet, positive as well as negative results on dominant lethality in rodents

96

after ethanol treatment have been repor:ed over the years (for a review see Obe and Anderson, 1987; Washington et al., 1985). Keeping in mind that at the time of birth all autosomal monosomies and trisomies are lethal in the mouse (Searle, 1981; cited by Washington et al., 1985) the picture that emerges from these conflicting results is not clear. The evidence in male mice and Chinese hamsters is also controversial (AIvarez, 1985; Hunt, 1987; Daniel and Roane, 1987). In Drosophila melanogaster no effect on the rate of nondisjunction could be detected in females fed with ethanol concentrations up to 15% (v/v) for 4 days (Traut, 1980) or in males fed for 24 h on a 10% concentration (Mittler, 1977). Studies have also been performed in mitotic cells exposed to ethanol. Disturbances of the normal distribution of chromosomes were observed in Allium cepa and Aspergillus nidulans (Obe and Anderson, 1987; K~ifer et al., 1986; CrebeUi et al., 1989) but not in cultured CHED (Chinese hamster embryonic diploid) cells (Dulout and Fumus, 1988). The widespread us~ of alcoholic beverages, the impact that aneuploidy has on human suffering and the above-mentioned results call for a deepening of our knowledge on the effect of ethanol on chromosome segregation and for the enlargement of the thus far limited data base available by using different test systems. With this purpose we have undertaken the analysis of X-chromosome nondisjunction in Drosophila melanogaster female germinal cells after acute and chronic treatments with ethanol. The mentioned limited data on ethanol are not an exception. When assessing the genetic hazard posed by environmental contaminants, monitoring for the possible induction of aneuploidy has been largely neglected. While it is true that once the clastogenic effect of an agent has been established its involvement in aneuploidy induction via chromosome loss can rightly be expected, direct testing to detect chemicals that affect the normal disjunction of chromosomes is mandatory, since the other source of aneuploidy is nondisjunction leading to chromosome gain. Drosophila is an ideal high eukaryote for the study of meiotic nondisjunction because part of the resulting exceptions involving the sex chromo-

somes, namely XXY females and XO males, are viable and easily identifiable. Despite these advantages, few data are also available on the induction of nondisjunction by chemical compounds in Drosophila and validation of this test system to detect agents with such an effect is evidently needed. This was another reason for starting the research reported here. The results obtained show that ethanol treatments lead to a significant increase of exceptions that are unambiguously nondisjunction products. Materials and methods

Several schemes have been developed to detect primary nondisjunction in Drosophila melanogaster and some of them permit the gathering of additional information (Zimmering et al., 1986). However, for this investigation we have preferred to employ a classic, simple and already successfully tested scheme (Traut, 1970; Traut and Schr/~der0 1978) that consists in mating females homozygous for a structurally normal X chromosome with the recessive yellow ( y / y ) t o wild-type ( + / Y ) males (for description of the mutants see Lindsley and Greli, 1968). Normal disjunction of the maternal Xs gives rise in the FI to phenotypically y+ females and y males, while nondisjunction leads to exceptional females (XXY) that are y and XO males that are y+. Both exceptions were scored in all experiments. However, since XO males can arise not only by nondisjunction but also from other causes such as breakage or lagging of the X chromosome, we have only used the bona fide products of nondisjunction, i.e., exceptional females, to calculate the frequencies reported below. The frequencies of X nondisjunction were calculated by the expression % X nondisjunction =

4(exc. ~) x 100 Total +exc.

The exceptional females are multiplied by 4 and the exceptions are added in the denominator to obtain a more accurate estimation of the actual frequency of nondisjunction induced by taking into account the other nondisjunctional products: XXX, XO, and YO (Grell et al., 1966;

97 Traut, 1970). Although when comparing relative values multiplication by 4 may not be really needed, it becomes important when assessing the ability of an agent to induce nondisjunction and for comparing data obtaine.d using other testing schemes that allow the recovery of different surviving classes. The females used as mothers were obtained from a stock with a Y chromosome marked with Bar of Stone (B s) and the normal allele of y (Bs.Y.y +) that permits the detection of females carrying an extra Y chromosome and thus eliminates secondary nondisjunction as a source of error. For acute treatments 0-5-h-old virgin females were kept in standard culture medium for 24 or 48 h before being held for 60 rain in regular culture vials (50 flies/vial) containing tissue paper (10 × 6 cm) soaked with 0.5 ml of 75% (v/v) ethanol (CAS No. 64-17-5) prepared with distilled water. Treated females were aerated by transfers to empty vials every 10 min and mated to 7-day-old wild-type Samarkand males in a proportion of 8 females to 16 males 1-3 h later, a lapse of time required for the females' recovery. The males were left with the females throughout the experiments and 6 subcultures were thus obtained: 2 of 12 h and 4 of 24 h. For the chronic treatments 0-5-h-old females were collected in culture vials containing standard medium with 10% or 15% (v/v) ethanol added shortly before hardening. When 24 or 48 h old the females were mated in a proportion of 4 to 8 Samarkand males. After a first 24-h subculture, females and males were transferred to vials containing newly prepared ethanol medium to obtain 2 additional 72-h and one 24-h subcultures. Since in the acute and chronic treatments females and males were transferred together to new vials, subculture and brood will he used as interchangeable terms throughout the paper. To investigate the possible retention of eggs as a consequence of the ethanol present in the culture medium, females were dissected in saline solution. Egg counts were made at the end of each subculture in ~n aliquot of 25 females (50 ovaries) from the 10% chronic experiments. The dissection was performed under a stereomicro-

scope and the stages of development of the maturing oocytes classified according to King (1970). Ethanol toxicity was evaluated by exposing females of different ages to 50% and 75% (v/v) solutions for 60 mill and the surviving flies were counted 24 h later. The teratogenic effect of ethanol was assessed in part of the progeny of 48-h-old females acutely exposed to a 75% (v/v) ethanol solution. No effort was made to detect minute changes and only gross abnormalities were recorded. In all cases parallel control series were ran in standard conditions. The temperature was maintained at 25 + I°C. The results were analyzed for statistical significance using the normal test described by Margolin et al. (1983). The significance of the difference between the means of stage 14 oocytes was estimated with Student's t test and that of malformations recorded in the progeny of treated and control flies by using Kastenbaum and Bowman (1966) tables. Results

Toxicity Females of 6 different ages were tested with a 50% ethanol solution and the results, shown in Table 1, indicate that toxicity is directly related to the female's age. With a 75% solution the surviving fractions followed the same trend though in this case the testing was limited to females 24 and 48 h old. This ethanol concentration, which allows the recovery of around 50% of the exposed females, was used in our acute tests. After the 60-min treatment with either concentration the TABLE ! FREOUENCIES OF SURVIVING FEMALES AFTER ACUTE (60-min)ETHANOLTREATMENT Female's Ethanol 50% (v/v)

Ethanol 75% (v/v)

age

Surviving flies/ treated flies

%

Surviving flies/ treated flies

0-5 h 24 h 48 h 72 h 4 days 5 days

300/300 144/150 148/302 14/150 32/655 16/899

100 3504/5 306 96 49.01 2744/5 816 9.33 4.88 1.78

%

66.04 47.18

98

flies were asleep, recovering within the following 3h. At the beginning of the chronic treatment with a 10% ethanol concentration the exposed females ate and mated normally. In the first brood of females kept in ethanol-supplemented food for 24 h before mating, we recorded a production of 7.3 flies/treated female vs. 7.4 flies/female in the control. However, in succeeding broods an increasingly higher number of exposed females died and the few surviving became sterile around 7-9 days after the beginning of the treatment. Females kept in ethanol food for 48 h before mating produced 6.8 flies/treated female in the first brood vs. 29.3 flies/female in the control. Chronic exposure to ethanol 15% resulted in a still more pronounced progeny reduction (2.7 flies/female in the first brood) that extended to all broods. it was observed that in food supplemented with 10% or 15% ethanol the flies became increasingly uncoordinated and remained motionless at the bottom of the culture vials.

Teratogenicity Abnormal flies were detected in the progeny of treated females in all series and were routinely scored in a subset of females acutely exposed to a 75% solution. The ntalformations observed, mainly fused and detbrmed tergites and leg and wing defects ranging from slight deformities to complete absence, have been grouped in Table 2.

Since some flies exhibited more than one defect, we chose to calculate the frequency of malformations observed in each subculture and not the frequency of malformed flies, which may be slightly lower. Even if the total value was somehow underestimated on account of minor changes that may have been overlooked, it can be seen that the frequencies of malformations decreased constantly from brood I to V and were significantly higher than the control values up to the fourth brood.

Ovary analysis It

has clearly been

established

that

in

Drosophila melanogaster the ovariole number and the rate at which oocytes complete their development vary greatly in relation to genotype, age, culture conditions, etc. (King, 1970). Therefore the results of the dissections carried out to determine whether egg retention took place in the females kept in ethanol-supplemented food from eclosion onward should be interpreted only from this comparative point of view. In Table 3 it can be seen that in females mated 24 or 48 h after eclosion the number of stage 14 oocytes decreased steadily throughout the four broods tested, clearly indicating lack of egg retention. A continuous decline in the number of oocytes in other advanced stages of development was also observed and it should be pointed out that no other cells were seen between these maturing cells and the early germinal cells in the germar-

TABLE 2 FREQUENCIES OF MALFORMATIONS E T H A N O L 75% (v/v) F O R 60 rain

Brood ! II II!

IV V

Tergites C

4

E C E C E C E C E

36 9 15 4 19 5 7 2 5

DETECTED

IN T H E

Genitalia

Wings

Legs

4

6

12

2

8

16

5

4 I I

I I

3

I

2

C, control E, ethanol; Broods: I-II, 12 h each; Ill-IV, 24 h each. * P < 0.05; ** P <0.01.

PROGENY

Thorax

3

O F 48-h-old F E M A L E S

Total malf./ total flies

EXPOSED

%

4/10 ! 43

0.039

58/4 394 9/22 706 44/5 910 4/21266 28/7 311 7/I 1938 12/7981 2/4814 8/5 367

1.32 0.040 0.745 0.019 0.383 0.059 0.150 0,042 0.149

TO

99 ium. Thus, the frequency of fully atrophic ovaries with no stage 14 oocytes increased in succeeding broods reaching 42% in brood IV. This picture contrasted with that seen in the ovaries of control females where representatives of all post-oogonial stages could be found. Virgin females were dissected at similar time intervals. The number of stage 14 oocytes in the ovaries of females kept in ethanol for up to 120 h (equivalent to those dissected at the end of brood II) was much lower than in the control. This indicates that in ethanol-exposed flies egg maturation was retarded. The number of mature eggs increased in the following samples reaching the control value when the females were 236 h old (end of brood IV) but the gap between mature eggs and germarial cells was similar to that described for mated females.

of those kept in that medium for 48 h before mating are significantly higher than control values. The analysis further shows that the frequencies of nondisjunction in broods III and IV in both series are also higher than controls, though evaluation of the significance of the differences would have required samples 2 - 6 times larger. The results obtained after chronic treatment with 15% ethanol are alsc, shown in Table 4. It can be seen that in three out of the four broods tested the frequencies of nondisjunction were higher than in the controls. Although the sample sizes are not large enough to perform the statistical test mentioned above, the magnitude of the increment suggests that a significant difference with the controls would have been obtained had the numbers been increased. As shown in Table 5 a highly significant increase in the frequency of nondisjunction was detected in the first three broods of 24- and 48-h-old females after acute treatments. In the later series the increase, though less pronounceg, persisted up to the fifth brood and disappeared in the last brood. It can further be seen that in this series the frequency of exceptional females in the second 12-h brood was significantly higher than in the first. The y+ males, representing the other viable exceptional class (XO) resulting from nondisjunction of the X chromosomes outnumber the XXY

Nondisjunction As mentioned above, chronic treatment in 10% ethanol-supplemented food profoundly affected the fly production, so obtaining progenies large enough to be evaluated by the test described by Margolin et al. (1983) was a real problem, particularly in the last broods. Analysis of the data shown in Table 4 indicates that the frequencies of nondJsjunction in the first .'ood of females kept in ethanol food for 24 h before mating and in the first and second broods

TABLE 3 NUMBER OF STAGE 14 OOCYTES IN FEMALES KEPT IN ETHANOL FOOD (10% v/v) FROM HATCHING Dissection time after eclosion 24 h End of brood i (24h) End of brood I! (72h) End of brood I!I (72 h) End of brood IV (24 h)

Virgin females a C 3.8 + 0.5

E

Female's age at mating 24h

48h

C

E

C

E

0.04 + 0.03 *

12 +0.3

6

+0.1 *

0.7+0.1

1.1+0.3

1.5+0.2

!.4+0.3

12 +0.2

7.6 +0.3"

3.3+0.4

1.3+0.2 *

1.9+0.2

1.1+0.2 *

11.4+0.4

8.5 -I-0.3 *

2.7+0.3

1.1 +0.2 *

2.4_+0.2

0.8_+0.2*

10.1-I-0.7

9.2 _+0.3

2.7+0.2

0.5 +0.1 *

3.2+0.2

0.7+0.2 *

C, control females kept in regular food; E, exposed females. a Virgin females were dissected at similar time intervals to those mated when 24 h old. Each value represents the mean + SE of 50 ovaries. * P < 0.05.

72

72

24

I!I

1V

3

3

6

l

3

4

3

8

8

10

XO 0.123 *** (20/16 252) 0.067 (16/23831) 0.295 (12/4064) 0.132 (4/3023)

ND% (4XXY/Total Prog.)

2

3

8

5

XXY

48h old

1

1

9

5

XO 0.155 *** (20/12 913) 0.153 *** (32/20952) 0.197 (12/6074) 0.318 (8/2512)

ND% (4XXY/Total Prog.)

1

2

3

1

XXY

1

5

3

1

XO

E 15% (v/v) 9 48 h old

0.121 (4/3 315) 0.125 (12/9596) 0.099 (8/8096) 0.348 (4/1149)

ND% (4XXY/Total Prog.)

For calculation of nondisjunction frequencies see Materials and methods. In the treated series the females were kept in ethanol-supplemented food from hatching. *** p < 10-5.

l

9

8

5

I!

9

0.015 (8/54 714) 0.041 (32/77390) 0.099 (36/36326) 0.033 (4/12186)

24

!

2

XXY

ND% (4XXY/ToIal Prog.)

XXY

XO

5 days old

h

N

E 10% (v/v) ¢ 24h old

Control ~

Brood

I'

FREQUENCIES OF NONDISJUNCTION (ND) DETECTED IN THE PROGENY OF FEMALES CHRONICALLY EXPOSED TO ETHANOL

TABLE 4

101 TABLE 5 FREQUENCIES OF NONDISJUNCTION (ND) DETECTED IN THE PROGENY OF FEMALES EXPOSED TO ETHANOL 75% (v/v) FOR 60 rain Brood N

Control ~ h

Treated 9

5 days old

24 h old

48 h old

XXY

XO

ND % (4XXY/Total Prog.)

XXY

XO

ND % (4XXY/Total Prog.)

XXY

XO

ND % (4XXY/Total Prog.)

0.087 (8/9 238) 0.029 (4/13 816) 0.025 (4/15918) 0.095 (12/12651) 0.046 (4/8 781)

57

69

34

41

49

55

32

39

17

21

22

35

5

8

7

29

8

19

9

31

5

11

1.572 *** (228/14 504) 1.392 *** ( 196/14 083) 0.109 ** (68/62491) 0.047 (20/42464) 0.068 (32/47 057) 0.070 (20/28 384)

1

14

0.753 *** (136/18050) 1.250 *** (128/10 236) 0.218 *** (88/40373) 0.109 (28/25581) 0.128 * (36/28028) 0.019 (4/20 848)

I

12

2

3

II

12

1

4

III

24

!

1

IV

24

3

3

V

24

1

4

VI

24

For calculation of nondisjunction frequencies see Materials and methods. * P<0.02; * * P < 0 . 0 1 ; * * * P < I 0 -s.

females found in all series. This was expected since XO males can arise not only by nondisjunc(ion but also by other causes leading to chromosome loss. All suspected XO males were tested for fertility and the very few that died during the testing were included in the sterile class (Tokunaga, 1970). It was found that 100% (36/36) of the y ÷ males recovered in the controls were sterile as compared with 96% (427/445) in the treated series.

Discussion In Drosophila melanogaster females the frequency of spontaneous X-chromosome nondisjunction is low and may be affected by the female's age. In the progeny of newly eclosed females brooded daily for 16 days under normal conditions Tokunaga (1970) detected a tendency towards an increase of nondisjunction in later broods as the females became older. However, under similar conditions Traut and Schr6der (1978) and Traut (1980) found that the frequency of exceptions was not modified or diminished with maternal age. In the control run for the chronic treatments we observed an increase of nondisjunction in the

second and third broods, when the females were 6 and 9 days old respectively. It is difficult to ascertain whether this was due to the age of the females, because although the sample analyzed was very large, the increment was no longer seen in the following and last brood, nor in the last broods of the control run for the acute treatment with 5-day-old females. In the progeny of females chronically exposed to ethanol a significant increase of nondisjunction was already seen in the first brood. Since at the end of this brood the oldest tested females were only 3 days old, it is clear that their age playe,? no role in the results obtained. A different age effect, not related to the age of the females but to that of mature oocytes, has been reported by Traut (1980) who observed that, when virgin females were prevented from laying eggs for 4 days, there was an increase in the frequency of XXY exceptions recovered after mating. This effect, not found in females aged under conditions favoring oviposition, was restricted to the first 2-day brood, obtained by this author, implying that only mature or maturing oocytes are susceptible to aging. This age effect seems relevant to the data reported here seeing that in some Drosophila melanogaster strains

102

adults are somehow reluctant to lay eggs in highly concentrated ethanol solutions. The question arose as to whether the significant increase in nondisjunction observed in the first broods of the chronic series was actually due to ethanol or to the aging of retained mature oocytes. Keeping in mind that after eclosion at least 21 h are required for the oocytes to reach full maturity and to be ready for fertilization (King, 1970) it seems evident that the eggs sampled in our first 24-h broods were by no means aged. Moreover, the number of stage 14 oocytes present in the ovaries of females dissected during the treatment clearly shows a lack of retention in females kept in ethanol medium since hatching and mated 24 or 48 h later. This is in agreement with the similar fly production in the first brood of females held in ethanol food for 24 h before mating and the control. The steady progeny decline observed in succeeding broods from females chronically exposed to ethanol was due to a continuous decline of both stage 14 oocytes and maturing ooeytes in their ovaries. As a consequence, egg laying by these females came to a virtual stop in the fourth brood. This picture is consistent with the view that ethanol, probably by affecting mitotically dividing early germinal cells, impaired their further development causing a cessation of egg production. Oviposition stopped once the females used up the supply of eggs that at the time of treatment had already passed a critical stage. Inhibition of cell proliferation by ethanol has been observed in several experimental models (Pennington et al., 1984; Pierdomenico and Alpi, 1991; Cook et al., 1990) and may be due not only to a genotoxie effect but also to a generalized disruption of the cells' organization. It should be mentioned that the chronic series with 15% ethanol were discontinued because in several runs the first broods were very poor, egg retention being suspected. However, the dissection of ovaries later showed that eggs were not actually retained but that their production was severely altered. This effect on the germinal cell line plus the diminishect egg hatchability (Bijlsma-Meeles, 1979)and the reduced larva-toadult survival (Heinstra et ai., 1987) in concentrated ethanol food led to the low fly production in the cronic series.

Acute ethanol treatments also induced a highly significant increase of X-chromosome nondisjunction in the first three broods of 24- and 48-h females pointing to mature and maturing oocytes as the most affected cells. Whether this resulted from a higher intrinsic sensitivity as compared to that of younger cells or to the longer time available to the latter for recovery or detoxification before meiosis cannot be stated at the moment. In the series of 48-h-old females the increase in nondisjunction extended up to broods IV and V. This could be due to their higher supply of more mature oocytes, also to an inability of these cells to reverse, once established, the deleterious effects of ethanol. The view that ethanol acts only during meiosis seems less tenable, because to explain its effect beyond the first brood one would need to assume that an active concentration was still present several days after acute treatment. In Drosophila melanogaster adults exogenous ethanol disappears rapidly attaining the level detected in unexposed flies in a few hours (Middleton and Kacser, 1983). It is converted into acetaldehyde mainly by alcohol dehydrogenase (ADH), an en~ m e with a dual function that by further oxidizing this metabolite to acetate prevents its accumulation (Heinstra et al., 1983; Anderson and Barnett, 1991). The induction pattern of teratogenic effects is also puzzling. Our results show that the longer the time elapsed between acute treatment of the females and egg sampling, the lower the incidence of malformed flies, perhaps reflecting the action of a detoxifying mechanism. However, the frequency of malformed flies in the fifth brood sampled 4 days after the treatment was still 3.5 times higher than that of the controls. This would imply that either the teratogenic effect resulted from damage inflicted before fertilization or that ethanol persisted in the eggs, despite their ADH activity (Bijlsma-Meeles, 1979), for several days in a concentration high enough to damage the embryo. Our results showing that ethanol strongly affects the development of segments, genitalia, wings and legs are otherwise in line with previous reports in Drosophila (Ranganathan et al., 1987a). If the distribution pattern of exceptional females and malformed flies in the progeny of acutely treated females is, at least in part, the outcome of a detoxifying process, it could suggest

103

that ethanol itself and not a metabolite is responsible for these effects. In Drosophila the direct involvement of ethanol in teratogenicity has been proposed by Ranganathan et al. (1987a,b). In adult flies the ADH content increases from eclosion and reaches a maximum around the fifth day of life, remaining unchanged thereafter up to the tenth day (Dunn et al., 1969; Maroni and Stamey, 1983). The fact that a significant increase of exceptions was already seen in the progeny of young females, with lower ADH activity than that of older flies, adds some support to the suggestion that nondisjunetion may result from a direct action of ethanol. This would be in line with the findings in AspergiUus nidulans where chromosome malsegregation does not seem to be dependent on the conversion of ethanol into acetaldehyde since these two compounds have quite different patterns of activity tCrebelli et al., 1989). Whether the increase in nondisjunction reported here resulted from primary damage inflicted in DNA or to other cellular constituents that in turn disrupted chromosomal segregation is not yet known. In this connection it is worth mentioning that thus far we have failed to detect a mutagenic or clastogenic effect after acute ethanol treatment in Drosophila (Mufioz, 1990). A different picture emerges from the toxicity testing. The data presented here plus those obtained earlier with ethanol 96% (Mufioz, 1990) show that, as expected, toxicity is directly related to the time of exposure and the concentrations used. But the age of the flies also plays a decisive role, since while 100% of 1-day-old females survived the acute treatment with 50% ethanol, mortality increased steeply as the females became older. This increase coincides with that of the ADH content mentioned above, suggesting that at the concentration used death may have been mainly due not to a direct action of ethanol but to that of a metabolite, probably the highly toxic acetaldehyde.

Acknowledgements We are indebted to Dr. Beatriz Mazar Barnett for reading the manuscript and correcting the English and to Ms. Vilma B. de Fernfindez, Ms.

Mabel N. Quevedo and Mr. Pedro A. Giordano for their excellent technical assistance.

References Alvarez, M.R. (1985) Aneuploidy in male germ cells induced by alcohol ingestion, Gamete Res., 15, 165-170. Anderson, S.M., and S.E. Barnett (1991) The involvement of alcohol dehydrogenase and aldehyde dehydrogenase in alcohol/aldehyde metabolism in Drosophila melanogaster, Genetica, 83, 99-106. Bijlsma-Mceles, E. (1979) Viability in Drosophila melanogaster in relation to age and ADH activity of eggs transferred to ethanol food, Heredity, 42, 79-89. Crebelli, R., G. Conti, L. Conti and A. Carere (1989) A comparative study on ethanol and acetaldehyde as inducers of chromosome malsegregation in Aspergillus nidulans, Mutation Res., 215, 187-195. Cook, R.T., J. Keiner, A. Yen and J. Fishbaugh (1990) Ethanol induced growth inhibition and growth adaptation in vitro. Cell cycle delay in late G1, Alcohol Alcohol., 25, 33-43. Daniel, A., and D. Roane (1987) Aneuploidy is not induced by ethanol during spermatogenesis in the Chinese hamster, Cytogcnct. Cell Genet., 44, 43-48. Dulout, F.N., and C.C. Furnus (1988) Acetaldehyde-induced aneuploidy in cultured Chinese hamster cells, Mutagenesis, 3, 207-211. Dunn, G.R., T.G. Wilson and K.B. Jacobsen (1969) Age-dependent changes in alcohol dehydrogenase in Drosophila, J. Exp. Zool., 171, 185-190. Geer, B.W., M.L. Langevin and S.W. McKechnie (1985) Dietary ethanol and lipid synthesis in Drosophila mdanogaste, Biochem. Genet., 23, 607-622. Grell, R.F., E.R. Mufioz and W.F. Kirschbaum (1966) Radiation-induced nondisjunction and loss of chromosomes in Drosophila melanogaster females. I. The effect of chromosome size, Mulation Res., 3, 494-502. Heinstra, P.W.H., W, Schoonen, W. Aben, A.J, De Winter, D.J, Van Der Horst, W.J.A. Van Marrewijk, A.M.Th. Beenakkers, W. Schadoo and G.E.W. Th6rig (1983) A dual function of alcohol dehydrogenase in Drosophila, Genetica, 60, 129-137. Heinstra, P.W.H., W. Scharloo and G.E.W. Th6rig (1987) Physiological significance of the alcohol dehydrogenase polymorphism in larvae of Drosophila, Genetics, 117, 7584. Hunt, P.A. (1987) Ethanol-induced aneuploidy in male germ cells of the mouse, Cytogenet. Cell Genet., 44, 7-10. Kiifer, E., B.R. Scott and A. Kappas (1986) Systems and results of test for chemical induction of mitotic malsegregation and aneuploidy in Aspergillus nidulans, Mutation Res., 167, 9-34. Kastenbamn, M.A., and K.O. Bowman (1966) The minimun Number of Successes in a Binomial Sample, ORNL-3909, Oak Ridge Natl. Lab., Oak Ridge, TN. Kaufman, M.H. (1983) Ethanol-induced cbrGmosomal abnormalities at conception, Nature (London), 302, 258-260.

104 Kaufman, M.H., and I.M. Bain (1984) Influence of ethanol on chromosome segregation during the first and second meiotic divisions in the mouse egg, J. Exp. Zooi., 230, 315-320. King, R.C. (1970) Ovarian Development in Drosophila melanogaster, Academic Press, New York. Lindsley, D.L., and E.H. Grail (1968) Genetic variations of Drosophila melanogaster, Carnegie Inst. Wash. Publ. 627. Maguire, M.P. (1976) The effect of ethanol on meiotic chromosome behavior in maize, Caryologia, 29, 41-52. Margolin, B.H., B.J. Collings and J.M. Mason (1983) Statistical analysis and sample-size determinations for mutagenicity experiments with binomial responses, Environ. Mutagen., 5, 705-716. Maroni, G., and S.C. Stamey (1983) Developmental profile and tissue distribution of alcohol dehydrogenase, Drosophila Inf. Sew., 59, 77-79. Middleton, R.J., and H. Kacser (1983) Enzyme variation, metabolic flux and fitness: alcohol dehydrogenase in Drosophila melanogaster, Genetics, 105, 633-650. Mittler, S. (1977) Feeding water insoluble compounds that are alcohol soluble to adult flies, Drosophila Inf. Sew., 52, 171. Mufioz, E.R. (1990) Effect of ethanol pretreatment on genetic damage induced by X-rays in Drosophila melanogaster sperm, Mutation Res., 232, 3-10. Obe, G., and D. Anderson (1987) Genetic effects of ethanol, International Commission for Protection against Environmental Mutagens and Carcinogens (ICPEMC), Working paper No. 15/1, Mutation Res,, 186, 177-200. O'Neill, G.T., and M.H. Kaufman (1987) Cytogenetic analysis of first cleavage fertilized mouse eggs following in vivo exposure to ethanol shortly before and at the time of conception, Development, 100, 441-448. Pennington, S.N., W.A. Taylor, D,H. Cowan and G.W. Kalmus

(1984) A single dose of ethanol suppresses rat embryo development in vivo, Alcohol Clin. Exp. Res., 8, 326-329. Pierdomenico, P., and A. Alpi (1991) Ethanol-induced injuries to carrot cells, Plant Physiol., 95, 748-752. Ranganathan, S., D.G. Davis and R.D. Hood (1987a) Developmental toxicity of ethanol in Drosophila melanogaster, Teratology, 36, 45-49. Ranganathan, S., D.G. Davis, J.D. Leeper and R.D. Hood (1987b) Effects of differential alcohol dehydrogenase activity on the developmental toxicity of ethanol in Drosophila melanogaster, Teratology, 36, 329-334. Searle, A.G. (1981) Chromosomal variants: numerical variants and structural rearrangements, in: M.C. Green (Ed.), Genetic Variants and Strains of the Laboratory Mouse, Fisher, New York, pp. 324-357. Tokunaga, C. (1970) The effects of low temperature and aging on nondisjunction in Drosophila, Genetics, 65, 76-94. Traut, H. (1970) Nondisjunction induced by X-rays in oocytes of Drosophila melanogaster, Mutation Res., 10, 125-132. Traut, H. (1980) X-chromosomal nondisjunction induced by aging oocytes of Drosophila melanogaster: the special susceptibility of mature eggs, Can. J. Genet. Cytol., 22, 433437. Traut, H., and FJ. Sch/~der (1978) The increase in the frequency of X-chromosomal aneuploidy in Drosophila melanogaster as a consequence of suppressed oviposition, Mutation Res., 49, 225-232. Washington, W.J., K.T. Cain, N.L.A. Cacheiro and W.M. Generoso (1985) Ethanol-induced late fetal death in mice exposed around the time of fertilization, Mutation Res., 147, 205-210.

Zimmering, S,, J,M, Mason and C. Osgood (1986) Current status of ane-,ploidy testing in Drosophila, Mutation Res,, 167, 71-87.