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Effect of ethanol pretreatment on genetic damage induced by X-rays in Drosophila melanogaster sperm
Mutation Research, 232 (1990) 3-10
3
Hsevier MUT 02102
Effect of ethanol pretreatment on genetic damage induced by X-rays in Drosophilamelanogaster sperm Enzo R. Muhoz Departaraento de Radiobiologia, Comisibn Nacional de Energ[a Atbmica, 1429 - Buenos Aires (Argentina)
(Accepted25 February1990) Keywords: Drosophila melanogaster; Ethanolpretreatment; Geneticdamage,X-rays
Summary Drosophila melanogaster males were treated with 96% ethanol for 45 rain by means of soaked tissue paper placed at the bottom of regular culture vials before being exposed to 2 krad of X-rays. The use of ethanol was dictated by its high efficiency to scavenge hydroxyl radicals that play a substantial role in the indirect effect of ionizing radiation. The data obtained show that the frequency of sex-linked recessive lethals, reciprocal translocations and chromosome losses induced in postmeiotic cells were not reduced by ethanol pretreatment. Rather, in the combined treatments a significant increase in II-III translocations was observed in sperm. This effect declined in late and mid spermatids. Treatment with ethanol alone did not modify the frequencies of the genetic endpoints tested. It is tentatively suggested that: (i) ethanol or ethanol radicals impair the restitution of broken chromosome ends, thereby increasing the chances for rearrangement formation in the egg, or (ii) ethanol given prior to irradiation acts as a weak dose-modifying factor. If so, a slight increase in the effective dose could have resulted in a detectably higher frequency of translocations whose induction, unlike the other genetic damages investigated that increase linearly with dose, follows the slope of a 2-hit kinetic curve.
It has been well established that the genetic effects of ionizing radiation are the consequence of both direct and indirect actions and that the latter are exerted through free radicals originated as ultimate products of water radiolysis (Michaels and Hunt, 1978; Sehulte-Frolinde, 1985; T~oule, 1987; Goggle, 1983). Moreover by using scavengers such as dimethyl sulfoxide, thiourea, ethanol, tbutanol, mannitol, etc. it has been shown that the hydroxyl radical (HO') plays the major role in the
Correspondence: Dr. E.R. Muhoz,Departamentode Radiobiologla, Comisi6n Naeional de Energia Attmica, Av. del Libertador 8250, 1429 - BuenosAires (Argentina).
indirect effect (Johanssen and Howard-Flanders, 1965; Roots and Okada, 1972; Sasaki and Matsubara, 1977; Repine et al., 1981). The actual involvement of a particular free radical in a specific biological effect is difficult to assess, however in vitro studies of irradiated DNA strongly implicate HO" in the induction of about 80% of the single-strand breaks (Repine et al., 1981) and although the complexity of experimental models in which living cells are studied makes estimation less secure, it is clear that the induction of single-strand breaks, chromosome aberrations and mutations by this radical is significant (Roots and Okada, 1972; Sasaki and Matsubara, 1977; Oya et al., 1986; Imlay and Linn, 1988; Meneghini, 1988).
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In view of these facts and the high efficiency of ethanol to scavenge HO" the question arose as to whether, given prior to irradiation, it would diminish the genetic damage recovered after exposure of Drosophila melanogaster to X-rays. The present study was undertaken to determine the effect of combined treatments in postmeiotic male germ cells. To this end sex-linked recessive lethals, reciprocal translocations and chromosome loss tests were carried out. The results obtained show that under the experimental conditions used ethanol pretreatment afforded no protection against damage induced by X-rays. Quite unexpectedly, an increase in the frequency of reciprocal translocations was observed. Materials and methods Treatment procedures One hundred females or males were held for 45 rain in regular culture vials containing tissue paper soaked with 0.5 ml of 96% ethanol; a new bottle was opened for each experiment. In a few experiments the treatment was reduced to 35 min and also some experiments were run with a 50% ( v / v ) ethanol solution prepared with distilled water. Within 4 - 5 rain of treatment males were submitted to 2 or 3 krad of X-rays (200 r a d / m i n ) , delivered from a target distance of 37 cm by a Philips machine, operating at 200 kV, 10 mA, filtered with 1 m m A1. In all cases, untreated males were X-rayed simultaneously. Before mating, the males were allowed to recover for 24 h in vials with regular culture medium that were changed 6 times during the first 3 h. When ethanol was given as posttreatment a similar procedure was followed with the males exposed to ethanol within 4 - 5 min of irradiation. Ethanol-treated females were aerated by several transfers to empty vials and mated to just irradiated males 1 h later, a lapse of time required for the females' recovery. In all cases 7-day-old males and 4-5-day-old females were used and the experiments were carried out at 25 ° C. Genetic tests A description of the symbols can be found in Lindsley and Grell (1969).
(a) Sex-linked recessive lethal tests. To allow the detection of clusters of lethals treated wild-type Samarkand males were individually mated in numbered vials to 4 In (1) scSlLscSR + S, ScSlsc s w a B (Basc) virgin females and 2 successive 48-h broods were obtained. To sample fully mature motile sperm the males were individually mated to 1 female in empty vials and discarded after 1 observed mating. In both cases the inseminated females were subcultured 3 times at 3-day intervals. Eighteen F~ females per treated male were individually mated to 2 or 3 Basc males and their progeny inspected for the occurrence of sex-linked lethals. The criteria described by Wiirgler et al. (1977) were applied to score lethals carried by the tested chromosomes, with the difference that all suspected lethals were retested and those cultures containing only 1 wild-type male were retested in duplicate. Cultures were considered as containing a lethal only if no wild-type males were present. (b) Translocation experiments. Samarkand treated males were mass-mated to cn bw; e virgin females in a proportion of 1 : 4 and 2 or 3 successive broods of 24 h each were obtained. Fully mature motile sperm was sampled as above. In both cases the inseminated females were subcultured 3 times at 3-day intervals. In the experiments with ethanol-treated females, they were pair-mated for 24 h and the first 24-h egg-laying period was divided into two 4-h subcultures followed by a 16-h subculture (Osgood and Zimmering, 1972). After discarding the males, 4 additional subcultures were obtained. I I - I I I translocation tests were run in the conventional manner and all suspected translocations were retested. (c) Chromosome loss tests. Treated y / B s Y y + males were mated to y w f virgin females in a proportion of 1 male to 1.5 females. In series I and II only one 24-h brood was obtained and in series III 5 successive 24-h broods were obtained. The inseminated females were subcultured 3 times at 3-day intervals. The F1 was scored for entire X or Y chromosome loss ( y w f males) and for partial loss of the Y chromosome markers ( y w f B s and w f males), mosaics and gynandromorphs. The expected normal progeny consists of y females and w f B s males. The total progenies were used
to estimate the percentages of complete and partial losses. The probability values that figure in the tables are based on Kastenbaum and Bowman (1970) or chi square.
TABLE 1 FREQUENCIES OF SEX-LINKED RECESSIVE LETHALS INDUCED BY 2 krad OF X-RAYS IN POSTMEIOTIC MALE CELLS PRETREATED WITH 96% ETHANOL FOR 45 rain Expt.
R ~
Although few flies died during the exposure to ethanol, the survival fraction, 24 h after the treatments, indicates that concentrated solutions are highly toxic for Drosophila melanogaster. Treatment with 50% ethanol ( v / v ) for 45 rain resulted in the killing of approximately 50% of the exposed females and males. In one of the first experiments (not shown in the tables) fully mature sperm were sampled from males treated with this concentration before being X-rayed with 2 krad and I I - I I I translocations were scored. N o effect of ethanol was detected in the combined treatment: 3.93% translocations in 1094 chromosomes tested as compared to 3.66% in 1340 chromosomes tested from males that were only irradiated. With 96% ethanol, the concentration used throughout the experiments reported here, the flies became uncoordinated within the first 15 rain of treatment and most of them were motionless after 35 min. When the treatment was extended to 45 min all the flies fell asleep and only slight movements of tarsus and proboscis could be seen. 4 0 50% females and males survived the 35-min treatment and only 10-15% males and 20% females exposed only for 45-min to ethanol were alive 24 h later. The results obtained in the sex-linked recessive lethal tests are shown in Table 1. It can be seen that pretreatment with ethanol did not increase the frequency of lethals induced by 2 krad of X-rays. In the progeny derived from a male only irradiated (Expt. I) a cluster of 7 lethals was detected and all (lethal and non-lethal) cultures from this male were excluded from final calculations, because it could have resulted from a spontaneous mutation in a premeiotic cell. The frequency of lethals detected in males exposed only to ethanol agreed with control values. Tables 2 and 3 summarize the results of tests carried out to study the effect of ethanol pretreatment on the induction of I I - I I I translocations by
Treatment
Lethals/Chromosomes tested (%) Brood A
B
I
R E+ R
67/1380 (4.85) 86/1340 (6.42)
51/1 407 (3.62) 52/1323 (3.93)
II
R E+R
63/1185 (5.32) 75/1375 (5.45)
34/599 (5.68) 32/603 (5.31)
III
R E+ R E C
76/1348 (5.64) 60/1272 (4.72) 4/3 963 (0.10) 4/3409 (0.12)
55/1428 (3.85) 54/1002 (5.39) 3/3 368 (0.09) 4/3313 (0.12)
One observed mating I
R E+ R
40/816 (4.90) 58/1113 (5.21)
II
R E+R
26/625 (4.16) 65/1417 (4.59)
Total
R E+ R
66/1441 (4.58) 123/2 530 (4.86)
Broods A and B: 48 h each; R: 2 krad; E: ethanol; C: control.
2 and 3 krad of X-rays in different postmeiotic male cells. The results obtained in 3 successive 24-h broods consisting mainly of mature sperm, later and mid spermatids are shown in Table 2. It can be seen that the pretreatment consistently increased the frequency of translocations induced by radiation in cells sampled in brood A; this effect decreased in brood B and disappeared in brood C. Exposure to ethanol alone induced no translocations in about 15 000 chromosomes tested. The results obtained with fully mature motile sperm (one observed mating) are shown in Table 3. In the first series the males were exposed to ethanol for 35 min prior to irradiation. The frequency of translocations recovered was significantly higher than that yielded by males only irradiated. An increase in translocation frequency was also observed when the exposure to ethanol was extended to 45 min, although in this case the difference did not reach significance. Since similar frequencies were actually obtained in the 2 series
TABLE 2 F R E Q U E N C I E S OF II-III TRANSLOCATIONS I N D U C E D BY 2 krad OF X-RAYS IN POSTMEIOTIC MALE CELLS PRETREATED WITH 96% ETHANOL FOR 45 min Expt.
Treatment
Translocations/Chromosomes tested (%) Brood A
B
C
I
R1 E+R 1
58/1597 81/1571
(3.63) * (5.16)
8/734 27/659
II
R1 E+R 1
81/1827 118/1909
(4.43) * (6.18)
III
R1 E+ R 1
47/1252 76/1251
IV
R1 E+ R 1
V VI VII
(1.09) * * * (4.09)
4/77 6/74
(5.19) (8.11)
24/1419 (1.69) * * 55/1574 (3.49)
10/199 20/330
(5.02) (6.06)
(3.75) * (6.08)
33/1099 (3.00) 35/950 (3.68)
31/409 24/194
(7.58) (12.37)
60/1507 67/1218
(3.98) (5.50)
33/1416 (2.33) 39/1056 (3.69)
R1 E+ R t
34/1390 59/1369
(2.45) * * (4.31)
R2 E+R 2
115/1725 146/1617
(6.67) * (9.03)
R2 E+ R 2 E
117/1544 (7.58) * 118/1173 (10.06) - / 5 940
108/1 335 (8.09) 112/1333 (8.40)
45/605 (7.44) 60/775
(7.72)
- / 5 557
-/4039
Broods A, B and C: 24 h each; RI: 2 krad; R 2 : 3 krad; E: ethanol. *P~0.05; **P<0.01; ***P<0.001.
the results were combined. As can be seen at the bottom of Table 3, the enhancement of translocations recovered in the combined treatment over irradiation alone is significant at the 0.01 level. The rate of sterile F 1 males is homogeneous in all the experiments. TABLE 3 F R E Q U E N C I E S OF I I - I I I T R A N S L O C A T I O N S IND U C E D BY 2 krad OF X-RAYS IN F U L L Y M A T U R E MOTILE SPERM (ONE OBSERVED MATING) PRET R E A T E D WITH 96% ETHANOL F O R 35 A N D 45 rain
To investigate whether ethanol posttreatment had any effect on the recovery of I I - I I I translocations, 3 experimental series were run in which the
TABLE 4 F R E Q U E N C I E S O F I I - I I I T R A N S L O C A T I O N S IND U C E D BY 2 krad OF X-RAYS IN POSTMEIOTIC MALE CELLS POSTTREATED WITH 96% ETHANOL F O R 45 min Expt. Treatment
Translocations/Chromosomes tested (%) Brood
Exposure time 35 rain 45 rain Total
Treatment
Translocations/ Chromosomes tested (%)
Steriles No. %
A
B
I
R R+E
70/1837 (3.81) 61/2 024 (3.01)
34/1615 (2.10) 46/1500 (3.07)
R E+R
23/960 38/885
(2.40) * (4.29)
88 69
8.40 7.23
II
R E+R
19/755 24/568
(2.52) (4.22)
68 40
8.26 6.70
R R+E
91/1950 (4.67) 66/1711 (3.86)
42/2002 (2.10) 27/1470 (1.84)
III
R E+ R
42/1715 (2.45) * * 62/1 453 (4.27)
156 109
8.34 7.08
R R+ E
67/1722 (3.89) 83/1 942 (4.27)
16/970 (1.65) 40/1899 (2.11)
Total
R R+E
228/5509 (4.14) 210/5677 (3.70)
92/4587 (2.01) 113/4864 (2.32)
R: 2 krad; E: ethanol. * P < 0.05; * * P < 0.01.
Broods A and B: 24 h each; R: 2 krad; E: ethanol.
TABLE 5 F R E Q U E N C I E S O F I I - I I I T R A N S L O C A T I O N S REC O V E R E D A F T E R M A T I N G F O R 24 h X - R A Y E D MALES (2 krad) TO E T H A N O L - T R E A T E D F E M A L E S (96% F O R 45 min) Sub-
Expt.
culture
T r a n s l o c a t i o n s / C h r o m o s o m e s tested (%) ~R× ~
8 R× ~E
1
I II
14/445 (3.15) 6 9 / 1 5 8 2 (4.36)
12/366 (3.28) 5 4 / 1 3 6 9 (3.94)
2
I II
7 5 / 2 2 0 2 (3.41) 12/317 (3.79)
104/2827 (3.68) 10/430 (2.33)
3
I II
3 9 / 1 1 6 2 (3.36) 5 3 / 1 195 (4.44)
3 8 / 1 1 9 3 (3.19) 69/1 808 (3.82)
4
I II
8 8 / 2 2 9 6 (3.83) 34/952 (3.57)
80/2071 (3.86) 4 0 / 1 1 4 3 (3.50)
5
I II
17/612 39/891
(2.78) (4.38)
33/636 (5.19) 49/1133(4.32)
6
I II
22/640 22/674
(3.44) (3.26)
38/790 37/873
(4.81) (4.24)
7
I
14/347
(4.03)
19/486
(3.91)
Sub. 1 : 4 h; Sub. 2: 4 h; Sub. 3 : 1 6 h; Sub. 4 : 2 4 h; Sub. 5 : 2 4 h; Sub. 6 and 7 : 4 8 h each; R: 2 krad; E: ethanol. * P < 0.05.
males were exposed to ethanol immediately after irradiation. It is shown in Table 4 that the frequency of translocations induced by 2 krad in
cells sampled in two 24-h successive broods was not modified by the posttreatment. Two replicate experiments were carried out to determine if ethanol affects the female germinal cells' capacity to handle chromosome breaks induced in sperm by radiation. It is known that when mature females are treated, physiological differences may exist among the eggs to be layed during the first 24-h oviposition period (Sankaranarayanan and Sobels, 1976). Thus, to sample eggs that at the time of treatment were homogeneous, females exposed to ethanol were mated to irradiated males within 1 h and the egg-laying periods were divided as indicated in Materials and methods. From the data in Table 5 it is evident that ethanol given to the females did not modify the frequency of radiation-induced translocations in sperm. The higher frequency of translocations observed in subculture 5 of one experiment can hardly be taken as a true increase since it coincides with an atypically low frequency scored in the experiment with untreated females. The frequency of translocations was homogeneous throughout the cell stages sampled. The results of experiments performed to test the effect of ethanol pretreatment on the frequency of radiation-induced chromosome loss are presented in Table 6. It is clear that no differences were detected between the frequencies of complete
TABLE 6 F R E Q U E N C I E S OF C O M P L E T E LOSS (CL), P A R T I A L LOSS (PL), A N D T O T A L LOSS (TL) I N D U C E D BY 2 krad O F X-RAYS IN POSTMEIOTIC M A L E G E R M CELLS P R E T R E A T E D W I T H 96% E T H A N O L F O R 45 min Expt.
Treatment
Normal progeny
CL
d
~/8
No.
%
Loss of Y+ or B s
Mosaics and Gyn.
PL
TL
No.
%
No.
No.
%
No.
%
I
R E+R
2592 2390
2362 2350
1.1 1
22 25
0.44 0.52
7 8
0.14 0.17
1 3
8 11
0.16 0.23
30 36
0.60 0.75
II
R E+R
2826 3229
2837 2823
1 1.1
34 31
0.59 0.51
12 14
0.21 0.23
1 -
13 14
0.23 0.23
47 45
0.82 0.74
I+II
R E+R
5418 5619
5199 5193
1 1.1
56 56
0.52 0.52
19 22
0.18 0.20
2 3
21 25
0.20 0.23
77 81
0.72 0.74
III
R E+R E O
3804 3584 6 382 4414
3126 2977 5 327 3699
1.2 1.2 1.2 1.2
54 60 6 6
0.83 0.90 0.05 0.07
7 5 1 1
0.11 0.07 0.008 0.01
3 3 2
10 8 1 3
0.15 0.12 0.008 0.04
64 68 7 10
0.98 1.02 0.06 0.12
R: 2 krad; E: ethanol.
and partial chromosome losses induced by 2 krad and those of the combined treatments. In series I and II the sampling of treated cells was limited to one 24-h brood; the frequencies of complete and partial losses observed are within the range of values previously found in our laboratory for 2 krad using the same stock and cell sampling (Mazar Barnett and Muhoz, 1989). In series III, although 5 successive 24-h broods were obtained from the treated males, the results have been pooled due to the small proportion of losses. The frequencies of complete chromosome losses with and without pretreatment may be higher than those observed in series I and II, a difference that could be attributed to the sampling of younger cells with a higher radiosensitivity in series III.
Discussion
The lack of genetic effect of ethanol reported here in postmeiotic male germ cells of Drosophila melanogaster is in line with previous findings in this organism and differs from evidence obtained with other experimental models showing that ethanol is genotoxic (for a review see Obe and Anderson, 1987). Whether this effect of ethanol is exerted directly or via its first metabolite, acetaldehyde, is a matter being actively investigated. It has been established that the indirect effect of ionizing radiation contributes substantially to the total genetic damage recovered and that this effect is mainly due to the hydroxyl radical (HO') originated from the radiolysis of water (Johansen and Howard-Flanders, 1965; Roots and Okada, 1972; Sasaki and Matsubara, 1977; Repine et al., 1981). Therefore, since ethanol is an efficient scavenger of H O ' , a reduction of radiation-induced genetic damage could be fairly expected in the combined treatments. However, our data consistently show that ethanol administered to the males prior to irradiation did not reduce the yield of recessive lethals, reciprocal translocations or chromosome losses induced by X-rays in postmeiotic cells. These negative results cannot be taken as meaning that the indirect effect of ionizing radiation and more
specifically the HO" plays no role in the induction of genetic damage in Drosophila. Perhaps factors such as cell stage, X-ray dose and dose rate, intracellular ethanol concentration, etc. could have been responsible for the lack of sparing action observed. Quite surprisingly, ethanol-pretreated males yielded a higher frequency of translocations than those exposed only to 2 or 3 krad of X-rays. This effect was consistent in fully mature motile sperm and in the first 24-h brood but less consistent thereafter, indicating either that sperm are the cells more affected by ethanol pretreatment or that its action on younger cells is reversed during the longer time available between treatment of these cells and fertilization. Although chromosome breaks induced in Drosophila sperm do not rejoin until after fertilization (Muller, 1954), extensive evidence has been presented showing that the yield of genetic damage recovered can be modified by postirradiation treatment of these cells with gases and chemicals. In view of these results it was proposed that pre-repair processes may actually take place in sperm. Posttreatments, by interfering with restitution of chromosome breaks or repair of potential breaks, favor the formation of translocations later on in the egg (for a thorough discussion and bibliography, see Sankaranarayanan and Sobels, 1976). At low concentrations ethanol is well tolerated by adult Drosophila and is rapidly metabolized (Van Herrewege and David, 1974; DeltombeLietaert et al., 1979) but at the acute doses used in the experiments reported here the males, irradiated immediately after the treatment, showed symptoms of being heavily intoxicated, recovering slowly after the irradiation. This implies that ethanol was present at high concentrations in the flies, not only during but also after the irradiation and the question then arose whether the observed increase of translocations was due to the pretreatment or to a postirradiation effect of ethanol upon the chromosome breaks induced by radiation. The results with ethanol-posttreated males show that the frequency of translocations was not modified, leading to the conclusion that ethanol has to be present during the irradiation to exert an enhancing effect.
Exposure of females to ethanol did not alter the yield of translocations induced by X-rays in sperm, suggesting either that the maternal repair system was not affected by the treatment or that a deleterious effect was reversed by the metabolically active oocytes during the time elapsed between treatment and fertilization. In line with previous unpublished results obtained in our laboratory with the same female strain, the frequency of radiation-induced translocations remained unchanged throughout all the subcultures tested from the first 4-h oviposition period up to 5 days. This is worth noting in view of the findings of Wiirgler and Maier (1972) and Biirki and Wiirgler (1973) with 4-day-old females that yielded a higher rate of chromosome loss in the first 1-day brood than in the following broods, an increase that might result from a diminished repair capacity of aged oocytes. It should be kept in mind that the role played by repair is instrumental in the recovery of translocations, being less significant perhaps in chromosome loss. Thus, the data presented showing that the frequency of radiation-induced complete and partial losses were unaffected by the pretreatment give support to the assumption that the rejoining of breaks would be the step more affected by ethanol. The basis of the observations reported here remains unclear. However, one cannot rule out the possibility, although at this time it seems highly speculative, that ethanol or ethanol radicals, by temporarily altering the physiology of the treated cells or chromosomal proteins, impairs the restitution of broken chromosome ends thereby increasing the changes for rearrangement formation in the egg. Evidence that ethanol radicals formed during irradiation can cause modification and inactivation of proteins comes from studies with ribonuclease, bovine serum albumin and hemoglobin irradiated in vitro in the presence of ethanol (Schuessler et al., 1976; Schuessler and Freundl, 1983; Puchala and Schuessler, 1986). Another interpretation, related to the induction kinetics of the genetic endpoints scored, could be that ethanol given prior to irradiation acts as a weak dose modifier. From the early days of radiation genetics it was known that the frequency of sex-linked lethals increases linearly with X-ray
dose (Muller, 1954). Similarly, at 2 krad the induction of chromosome losses is also in the linear range, although at higher exposures the dose exponent is greater than 1 (Traut et al., 1970). On the other hand the induction of translocations follows 2-hit kinetics, the dose exponent being between 2 and 1.5 (Sobels and Broerse, 1970; Gonzhlez, 1972; Sankaranarayanan and Sobels, 1976). This means that owing to the slope of the curve, even slight increases in sensitivity (or effective dose) are likely to result in translocation frequencies high enough to reach significance. This suspicion was brought about by the fact that, albeit significant in all the experiments, the translocation increments observed were rather low (about 1.5 times). The way in which this presumptive dose-modifying effect of ethanol is exerted can only be surmised at this time.
Acknowledgements The author is indebted to Ms Vilma B. de Fernfindez, N o b e r E. Pereyra and B. del Carmen Paz for their excellent technical assistance and to Dr. Beatriz Mazar Barnett for her help in preparing the manuscript and correction of the English.
References Btirki, K., and F.E. Wiirgler (1973) Maternal effects on X-rayinduced sex chromosomeloss in mature sperm of Drosophila melanogaster: a storage effect in mature oocytes, Arch. Genet., 46, 53-61. Deltombe-Lietaert, M.C., J. Delcour, N. Lenelle-Monfort and A. Elens (1979) Ethanol metabolism in Drosophila rnelanogaster, Experientia, 35, 579-581. Goggle, J.E. (1983) Biological Effects of Radiation, Taylor and Francis Ltd., London. Gonzhlez, F.W. (1972) Dose-response kinetics of genetic effects induced by 250-kVp X-rays and 0.68 MeV neutrons in mature sperm of Drosophila melanogaster, Mutation Res., 15, 303-324. Imlay, J.A., and S. Lima (1988) DNA damage and oxygen radical toxicity, Science, 240, 1302-1309. Johansen, I., and P. Howard-Flanders (1965) Macromolecular repair and free radical scavenging in the protection of bacteria against X-ray, Radiat. Res., 24, 184-200. Kastenbaum, M.A., and K.O. Bowman (1966) The Minimum Number of Successes in a Binomial Sample, ORNL-3909, Oak Ridge Natl. Lab., Oak Ridge, TN. Lindsley, D.L., and E.H. Grell (1968) Genetic variations of Drosophila melanogaster, Carnegie Inst. Wash. Publ., 627.
10 Mazar Barnett, B., and E.R. Mufaoz (1989) Effect of glyoxal pre-treatment on radiation induced genetic damage in Drosophila melanogaster, Mutation Res., 212, 173-179. Meneghini, R. (1988) Genotoxicity of active oxygen species in mammalian cells, Mutation Res., 195, 215-230. Michaels, H.B., and J.W. Hunt (1978) A model for radiation damage in cells by direct effect and by indirect effect: a radiation chemistry approach, Radiat. Res., 74, 23-34. Muller, H.J. (1954) The manner of production of mutations by radiation, in: A. Hollaender (Ed.), Radiation Biology, Vol. 1, Part 1, McGraw-Hill, New York. 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. Osgood, C., and S. Zimmering (1972) Measurement of radiation-induced dominant lethals in stage-14 oocytes of Drosophila, Mutation Res., 15, 355-357. Oya, Y., K. Yamamoto and A. Tonomura (1986) The biological activity of hydrogen peroxide. I. Induction of chromosome-type aberrations susceptible to inhibition by scavengers of hydroxyl radicals in human embryonic fibroblasts, Mutation Res., 172, 245-253. Puchala, M., and H. Schuessler (1986) Radiation-induced binding of methanol, ethanol and 1-butanol to haemoglobin, Int. J. Radiat. Biol., 50, 535-546. Repine, J.E., O.W. Pfenninger, D.W. Talmage, E.M. Berger and D.E. Pettijohn (1981) Dimethyl sulfoxide prevents DNA nicking mediated by ionizing radiation or iron/ hydrogen peroxide-generated hydroxyl radical, Proc. Natl. Acad. Sci. (U.S.A.), 78, 1001-1003. Roots, R., and S. Okada (1972) Protection of DNA molecules of cultured mammalian cells from radiation-induced single-strand scission by various alcohols and SH compounds, Int. J. Radiat. Biol., 21, 329-342. Sankaranarayanan, K., and F.H. Sobels (1976) Radiation genetics, in: M. Asbburner and E. Novitski (Eds.), The Genetic and Biology of Drosophila, Academic Press, New York. Sasaki, M.S., and S. Matsubara (1977) Free radical scavenging
in protection of human lymphocytes against chromosome aberration formation by gamma-ray irradiation, Int. J. Radiat. Biol., 32, 439-445. Schuessler, H., and K. Freundl (1983) Reactions of formate and ethanol radicals with bovine serum albumin studied by electrophoresis, Int. J. Radiat. Biol., 44, 17-29. Schuessler, H., L.K. Mee and S.J. Adelstein (1976) Reaction of ethanol radicals with ribonuclease, Int. J. Radiat. Biol., 30, 467-472. Schulte-Frolinde, D. (1985) Comparison of mechanisms for DNA strand break formation by direct and indirect effect of radiation, in: M.G. Simic, L. Grossman and A.C. Upton (Eds.), Mechanisms of DNA Damage and Repair, Implications for carcinogenesis and risk assessment, Plenum Press, New York. Sobels, F.H., and J.J. Broerse (1970) RBE values of 15-MeV neutrons for recessive lethals and translocations in mature spermatozoa and late spermatids of Drosophila, Mutation Res., 9, 395-406. T6oule, R. (1987) Review: Radiation-induced DNA damage and its repair, Int. J. Radiat. Biol., 51,573-589. Traut, H., W. Scheid and H. Wind (1970) Partial and total sex-chromosome loss induced by X-ray in mature spermatozoa of Drosophila melanogaster, Mutation Res., 9, 489499. Van Herrewege, J., and M.J. David (1974) Utilisation de ralcool &hylique darts le m&abolisme ~nerg~tique d'un insecte: influence sur ta dur6e de survie des adultes de Drosophila rnelanogaster, C.R. Acad. Sci. Paris, 279, 335338. Wiirgier, F.E., and P. Maier (1972) Genetic control of mutation induction in Drosophila melanogaster. I. Sex-chromosome loss in X-rayed mature sperm, Mutation Res., 15, 41-53. Wiirgler, F.E., F.H. Sobels and E. Vogel (1977) Drosophila as assay system for detecting genetic changes, in: B.J. Kilbey, M. Legator, W. Nichols and C. Ramel (Eds.), Handbook of Mutagenicity Test Procedures, Elsevier/North-Holland, Amsterdam, pp. 335-373.
3
Hsevier MUT 02102
Effect of ethanol pretreatment on genetic damage induced by X-rays in Drosophilamelanogaster sperm Enzo R. Muhoz Departaraento de Radiobiologia, Comisibn Nacional de Energ[a Atbmica, 1429 - Buenos Aires (Argentina)
(Accepted25 February1990) Keywords: Drosophila melanogaster; Ethanolpretreatment; Geneticdamage,X-rays
Summary Drosophila melanogaster males were treated with 96% ethanol for 45 rain by means of soaked tissue paper placed at the bottom of regular culture vials before being exposed to 2 krad of X-rays. The use of ethanol was dictated by its high efficiency to scavenge hydroxyl radicals that play a substantial role in the indirect effect of ionizing radiation. The data obtained show that the frequency of sex-linked recessive lethals, reciprocal translocations and chromosome losses induced in postmeiotic cells were not reduced by ethanol pretreatment. Rather, in the combined treatments a significant increase in II-III translocations was observed in sperm. This effect declined in late and mid spermatids. Treatment with ethanol alone did not modify the frequencies of the genetic endpoints tested. It is tentatively suggested that: (i) ethanol or ethanol radicals impair the restitution of broken chromosome ends, thereby increasing the chances for rearrangement formation in the egg, or (ii) ethanol given prior to irradiation acts as a weak dose-modifying factor. If so, a slight increase in the effective dose could have resulted in a detectably higher frequency of translocations whose induction, unlike the other genetic damages investigated that increase linearly with dose, follows the slope of a 2-hit kinetic curve.
It has been well established that the genetic effects of ionizing radiation are the consequence of both direct and indirect actions and that the latter are exerted through free radicals originated as ultimate products of water radiolysis (Michaels and Hunt, 1978; Sehulte-Frolinde, 1985; T~oule, 1987; Goggle, 1983). Moreover by using scavengers such as dimethyl sulfoxide, thiourea, ethanol, tbutanol, mannitol, etc. it has been shown that the hydroxyl radical (HO') plays the major role in the
Correspondence: Dr. E.R. Muhoz,Departamentode Radiobiologla, Comisi6n Naeional de Energia Attmica, Av. del Libertador 8250, 1429 - BuenosAires (Argentina).
indirect effect (Johanssen and Howard-Flanders, 1965; Roots and Okada, 1972; Sasaki and Matsubara, 1977; Repine et al., 1981). The actual involvement of a particular free radical in a specific biological effect is difficult to assess, however in vitro studies of irradiated DNA strongly implicate HO" in the induction of about 80% of the single-strand breaks (Repine et al., 1981) and although the complexity of experimental models in which living cells are studied makes estimation less secure, it is clear that the induction of single-strand breaks, chromosome aberrations and mutations by this radical is significant (Roots and Okada, 1972; Sasaki and Matsubara, 1977; Oya et al., 1986; Imlay and Linn, 1988; Meneghini, 1988).
0027-5107/90/$03.50 © 1990 ElsevierSciencePublishersB.V.(BiomedicalDivision)
In view of these facts and the high efficiency of ethanol to scavenge HO" the question arose as to whether, given prior to irradiation, it would diminish the genetic damage recovered after exposure of Drosophila melanogaster to X-rays. The present study was undertaken to determine the effect of combined treatments in postmeiotic male germ cells. To this end sex-linked recessive lethals, reciprocal translocations and chromosome loss tests were carried out. The results obtained show that under the experimental conditions used ethanol pretreatment afforded no protection against damage induced by X-rays. Quite unexpectedly, an increase in the frequency of reciprocal translocations was observed. Materials and methods Treatment procedures One hundred females or males were held for 45 rain in regular culture vials containing tissue paper soaked with 0.5 ml of 96% ethanol; a new bottle was opened for each experiment. In a few experiments the treatment was reduced to 35 min and also some experiments were run with a 50% ( v / v ) ethanol solution prepared with distilled water. Within 4 - 5 rain of treatment males were submitted to 2 or 3 krad of X-rays (200 r a d / m i n ) , delivered from a target distance of 37 cm by a Philips machine, operating at 200 kV, 10 mA, filtered with 1 m m A1. In all cases, untreated males were X-rayed simultaneously. Before mating, the males were allowed to recover for 24 h in vials with regular culture medium that were changed 6 times during the first 3 h. When ethanol was given as posttreatment a similar procedure was followed with the males exposed to ethanol within 4 - 5 min of irradiation. Ethanol-treated females were aerated by several transfers to empty vials and mated to just irradiated males 1 h later, a lapse of time required for the females' recovery. In all cases 7-day-old males and 4-5-day-old females were used and the experiments were carried out at 25 ° C. Genetic tests A description of the symbols can be found in Lindsley and Grell (1969).
(a) Sex-linked recessive lethal tests. To allow the detection of clusters of lethals treated wild-type Samarkand males were individually mated in numbered vials to 4 In (1) scSlLscSR + S, ScSlsc s w a B (Basc) virgin females and 2 successive 48-h broods were obtained. To sample fully mature motile sperm the males were individually mated to 1 female in empty vials and discarded after 1 observed mating. In both cases the inseminated females were subcultured 3 times at 3-day intervals. Eighteen F~ females per treated male were individually mated to 2 or 3 Basc males and their progeny inspected for the occurrence of sex-linked lethals. The criteria described by Wiirgler et al. (1977) were applied to score lethals carried by the tested chromosomes, with the difference that all suspected lethals were retested and those cultures containing only 1 wild-type male were retested in duplicate. Cultures were considered as containing a lethal only if no wild-type males were present. (b) Translocation experiments. Samarkand treated males were mass-mated to cn bw; e virgin females in a proportion of 1 : 4 and 2 or 3 successive broods of 24 h each were obtained. Fully mature motile sperm was sampled as above. In both cases the inseminated females were subcultured 3 times at 3-day intervals. In the experiments with ethanol-treated females, they were pair-mated for 24 h and the first 24-h egg-laying period was divided into two 4-h subcultures followed by a 16-h subculture (Osgood and Zimmering, 1972). After discarding the males, 4 additional subcultures were obtained. I I - I I I translocation tests were run in the conventional manner and all suspected translocations were retested. (c) Chromosome loss tests. Treated y / B s Y y + males were mated to y w f virgin females in a proportion of 1 male to 1.5 females. In series I and II only one 24-h brood was obtained and in series III 5 successive 24-h broods were obtained. The inseminated females were subcultured 3 times at 3-day intervals. The F1 was scored for entire X or Y chromosome loss ( y w f males) and for partial loss of the Y chromosome markers ( y w f B s and w f males), mosaics and gynandromorphs. The expected normal progeny consists of y females and w f B s males. The total progenies were used
to estimate the percentages of complete and partial losses. The probability values that figure in the tables are based on Kastenbaum and Bowman (1970) or chi square.
TABLE 1 FREQUENCIES OF SEX-LINKED RECESSIVE LETHALS INDUCED BY 2 krad OF X-RAYS IN POSTMEIOTIC MALE CELLS PRETREATED WITH 96% ETHANOL FOR 45 rain Expt.
R ~
Although few flies died during the exposure to ethanol, the survival fraction, 24 h after the treatments, indicates that concentrated solutions are highly toxic for Drosophila melanogaster. Treatment with 50% ethanol ( v / v ) for 45 rain resulted in the killing of approximately 50% of the exposed females and males. In one of the first experiments (not shown in the tables) fully mature sperm were sampled from males treated with this concentration before being X-rayed with 2 krad and I I - I I I translocations were scored. N o effect of ethanol was detected in the combined treatment: 3.93% translocations in 1094 chromosomes tested as compared to 3.66% in 1340 chromosomes tested from males that were only irradiated. With 96% ethanol, the concentration used throughout the experiments reported here, the flies became uncoordinated within the first 15 rain of treatment and most of them were motionless after 35 min. When the treatment was extended to 45 min all the flies fell asleep and only slight movements of tarsus and proboscis could be seen. 4 0 50% females and males survived the 35-min treatment and only 10-15% males and 20% females exposed only for 45-min to ethanol were alive 24 h later. The results obtained in the sex-linked recessive lethal tests are shown in Table 1. It can be seen that pretreatment with ethanol did not increase the frequency of lethals induced by 2 krad of X-rays. In the progeny derived from a male only irradiated (Expt. I) a cluster of 7 lethals was detected and all (lethal and non-lethal) cultures from this male were excluded from final calculations, because it could have resulted from a spontaneous mutation in a premeiotic cell. The frequency of lethals detected in males exposed only to ethanol agreed with control values. Tables 2 and 3 summarize the results of tests carried out to study the effect of ethanol pretreatment on the induction of I I - I I I translocations by
Treatment
Lethals/Chromosomes tested (%) Brood A
B
I
R E+ R
67/1380 (4.85) 86/1340 (6.42)
51/1 407 (3.62) 52/1323 (3.93)
II
R E+R
63/1185 (5.32) 75/1375 (5.45)
34/599 (5.68) 32/603 (5.31)
III
R E+ R E C
76/1348 (5.64) 60/1272 (4.72) 4/3 963 (0.10) 4/3409 (0.12)
55/1428 (3.85) 54/1002 (5.39) 3/3 368 (0.09) 4/3313 (0.12)
One observed mating I
R E+ R
40/816 (4.90) 58/1113 (5.21)
II
R E+R
26/625 (4.16) 65/1417 (4.59)
Total
R E+ R
66/1441 (4.58) 123/2 530 (4.86)
Broods A and B: 48 h each; R: 2 krad; E: ethanol; C: control.
2 and 3 krad of X-rays in different postmeiotic male cells. The results obtained in 3 successive 24-h broods consisting mainly of mature sperm, later and mid spermatids are shown in Table 2. It can be seen that the pretreatment consistently increased the frequency of translocations induced by radiation in cells sampled in brood A; this effect decreased in brood B and disappeared in brood C. Exposure to ethanol alone induced no translocations in about 15 000 chromosomes tested. The results obtained with fully mature motile sperm (one observed mating) are shown in Table 3. In the first series the males were exposed to ethanol for 35 min prior to irradiation. The frequency of translocations recovered was significantly higher than that yielded by males only irradiated. An increase in translocation frequency was also observed when the exposure to ethanol was extended to 45 min, although in this case the difference did not reach significance. Since similar frequencies were actually obtained in the 2 series
TABLE 2 F R E Q U E N C I E S OF II-III TRANSLOCATIONS I N D U C E D BY 2 krad OF X-RAYS IN POSTMEIOTIC MALE CELLS PRETREATED WITH 96% ETHANOL FOR 45 min Expt.
Treatment
Translocations/Chromosomes tested (%) Brood A
B
C
I
R1 E+R 1
58/1597 81/1571
(3.63) * (5.16)
8/734 27/659
II
R1 E+R 1
81/1827 118/1909
(4.43) * (6.18)
III
R1 E+ R 1
47/1252 76/1251
IV
R1 E+ R 1
V VI VII
(1.09) * * * (4.09)
4/77 6/74
(5.19) (8.11)
24/1419 (1.69) * * 55/1574 (3.49)
10/199 20/330
(5.02) (6.06)
(3.75) * (6.08)
33/1099 (3.00) 35/950 (3.68)
31/409 24/194
(7.58) (12.37)
60/1507 67/1218
(3.98) (5.50)
33/1416 (2.33) 39/1056 (3.69)
R1 E+ R t
34/1390 59/1369
(2.45) * * (4.31)
R2 E+R 2
115/1725 146/1617
(6.67) * (9.03)
R2 E+ R 2 E
117/1544 (7.58) * 118/1173 (10.06) - / 5 940
108/1 335 (8.09) 112/1333 (8.40)
45/605 (7.44) 60/775
(7.72)
- / 5 557
-/4039
Broods A, B and C: 24 h each; RI: 2 krad; R 2 : 3 krad; E: ethanol. *P~0.05; **P<0.01; ***P<0.001.
the results were combined. As can be seen at the bottom of Table 3, the enhancement of translocations recovered in the combined treatment over irradiation alone is significant at the 0.01 level. The rate of sterile F 1 males is homogeneous in all the experiments. TABLE 3 F R E Q U E N C I E S OF I I - I I I T R A N S L O C A T I O N S IND U C E D BY 2 krad OF X-RAYS IN F U L L Y M A T U R E MOTILE SPERM (ONE OBSERVED MATING) PRET R E A T E D WITH 96% ETHANOL F O R 35 A N D 45 rain
To investigate whether ethanol posttreatment had any effect on the recovery of I I - I I I translocations, 3 experimental series were run in which the
TABLE 4 F R E Q U E N C I E S O F I I - I I I T R A N S L O C A T I O N S IND U C E D BY 2 krad OF X-RAYS IN POSTMEIOTIC MALE CELLS POSTTREATED WITH 96% ETHANOL F O R 45 min Expt. Treatment
Translocations/Chromosomes tested (%) Brood
Exposure time 35 rain 45 rain Total
Treatment
Translocations/ Chromosomes tested (%)
Steriles No. %
A
B
I
R R+E
70/1837 (3.81) 61/2 024 (3.01)
34/1615 (2.10) 46/1500 (3.07)
R E+R
23/960 38/885
(2.40) * (4.29)
88 69
8.40 7.23
II
R E+R
19/755 24/568
(2.52) (4.22)
68 40
8.26 6.70
R R+E
91/1950 (4.67) 66/1711 (3.86)
42/2002 (2.10) 27/1470 (1.84)
III
R E+ R
42/1715 (2.45) * * 62/1 453 (4.27)
156 109
8.34 7.08
R R+ E
67/1722 (3.89) 83/1 942 (4.27)
16/970 (1.65) 40/1899 (2.11)
Total
R R+E
228/5509 (4.14) 210/5677 (3.70)
92/4587 (2.01) 113/4864 (2.32)
R: 2 krad; E: ethanol. * P < 0.05; * * P < 0.01.
Broods A and B: 24 h each; R: 2 krad; E: ethanol.
TABLE 5 F R E Q U E N C I E S O F I I - I I I T R A N S L O C A T I O N S REC O V E R E D A F T E R M A T I N G F O R 24 h X - R A Y E D MALES (2 krad) TO E T H A N O L - T R E A T E D F E M A L E S (96% F O R 45 min) Sub-
Expt.
culture
T r a n s l o c a t i o n s / C h r o m o s o m e s tested (%) ~R× ~
8 R× ~E
1
I II
14/445 (3.15) 6 9 / 1 5 8 2 (4.36)
12/366 (3.28) 5 4 / 1 3 6 9 (3.94)
2
I II
7 5 / 2 2 0 2 (3.41) 12/317 (3.79)
104/2827 (3.68) 10/430 (2.33)
3
I II
3 9 / 1 1 6 2 (3.36) 5 3 / 1 195 (4.44)
3 8 / 1 1 9 3 (3.19) 69/1 808 (3.82)
4
I II
8 8 / 2 2 9 6 (3.83) 34/952 (3.57)
80/2071 (3.86) 4 0 / 1 1 4 3 (3.50)
5
I II
17/612 39/891
(2.78) (4.38)
33/636 (5.19) 49/1133(4.32)
6
I II
22/640 22/674
(3.44) (3.26)
38/790 37/873
(4.81) (4.24)
7
I
14/347
(4.03)
19/486
(3.91)
Sub. 1 : 4 h; Sub. 2: 4 h; Sub. 3 : 1 6 h; Sub. 4 : 2 4 h; Sub. 5 : 2 4 h; Sub. 6 and 7 : 4 8 h each; R: 2 krad; E: ethanol. * P < 0.05.
males were exposed to ethanol immediately after irradiation. It is shown in Table 4 that the frequency of translocations induced by 2 krad in
cells sampled in two 24-h successive broods was not modified by the posttreatment. Two replicate experiments were carried out to determine if ethanol affects the female germinal cells' capacity to handle chromosome breaks induced in sperm by radiation. It is known that when mature females are treated, physiological differences may exist among the eggs to be layed during the first 24-h oviposition period (Sankaranarayanan and Sobels, 1976). Thus, to sample eggs that at the time of treatment were homogeneous, females exposed to ethanol were mated to irradiated males within 1 h and the egg-laying periods were divided as indicated in Materials and methods. From the data in Table 5 it is evident that ethanol given to the females did not modify the frequency of radiation-induced translocations in sperm. The higher frequency of translocations observed in subculture 5 of one experiment can hardly be taken as a true increase since it coincides with an atypically low frequency scored in the experiment with untreated females. The frequency of translocations was homogeneous throughout the cell stages sampled. The results of experiments performed to test the effect of ethanol pretreatment on the frequency of radiation-induced chromosome loss are presented in Table 6. It is clear that no differences were detected between the frequencies of complete
TABLE 6 F R E Q U E N C I E S OF C O M P L E T E LOSS (CL), P A R T I A L LOSS (PL), A N D T O T A L LOSS (TL) I N D U C E D BY 2 krad O F X-RAYS IN POSTMEIOTIC M A L E G E R M CELLS P R E T R E A T E D W I T H 96% E T H A N O L F O R 45 min Expt.
Treatment
Normal progeny
CL
d
~/8
No.
%
Loss of Y+ or B s
Mosaics and Gyn.
PL
TL
No.
%
No.
No.
%
No.
%
I
R E+R
2592 2390
2362 2350
1.1 1
22 25
0.44 0.52
7 8
0.14 0.17
1 3
8 11
0.16 0.23
30 36
0.60 0.75
II
R E+R
2826 3229
2837 2823
1 1.1
34 31
0.59 0.51
12 14
0.21 0.23
1 -
13 14
0.23 0.23
47 45
0.82 0.74
I+II
R E+R
5418 5619
5199 5193
1 1.1
56 56
0.52 0.52
19 22
0.18 0.20
2 3
21 25
0.20 0.23
77 81
0.72 0.74
III
R E+R E O
3804 3584 6 382 4414
3126 2977 5 327 3699
1.2 1.2 1.2 1.2
54 60 6 6
0.83 0.90 0.05 0.07
7 5 1 1
0.11 0.07 0.008 0.01
3 3 2
10 8 1 3
0.15 0.12 0.008 0.04
64 68 7 10
0.98 1.02 0.06 0.12
R: 2 krad; E: ethanol.
and partial chromosome losses induced by 2 krad and those of the combined treatments. In series I and II the sampling of treated cells was limited to one 24-h brood; the frequencies of complete and partial losses observed are within the range of values previously found in our laboratory for 2 krad using the same stock and cell sampling (Mazar Barnett and Muhoz, 1989). In series III, although 5 successive 24-h broods were obtained from the treated males, the results have been pooled due to the small proportion of losses. The frequencies of complete chromosome losses with and without pretreatment may be higher than those observed in series I and II, a difference that could be attributed to the sampling of younger cells with a higher radiosensitivity in series III.
Discussion
The lack of genetic effect of ethanol reported here in postmeiotic male germ cells of Drosophila melanogaster is in line with previous findings in this organism and differs from evidence obtained with other experimental models showing that ethanol is genotoxic (for a review see Obe and Anderson, 1987). Whether this effect of ethanol is exerted directly or via its first metabolite, acetaldehyde, is a matter being actively investigated. It has been established that the indirect effect of ionizing radiation contributes substantially to the total genetic damage recovered and that this effect is mainly due to the hydroxyl radical (HO') originated from the radiolysis of water (Johansen and Howard-Flanders, 1965; Roots and Okada, 1972; Sasaki and Matsubara, 1977; Repine et al., 1981). Therefore, since ethanol is an efficient scavenger of H O ' , a reduction of radiation-induced genetic damage could be fairly expected in the combined treatments. However, our data consistently show that ethanol administered to the males prior to irradiation did not reduce the yield of recessive lethals, reciprocal translocations or chromosome losses induced by X-rays in postmeiotic cells. These negative results cannot be taken as meaning that the indirect effect of ionizing radiation and more
specifically the HO" plays no role in the induction of genetic damage in Drosophila. Perhaps factors such as cell stage, X-ray dose and dose rate, intracellular ethanol concentration, etc. could have been responsible for the lack of sparing action observed. Quite surprisingly, ethanol-pretreated males yielded a higher frequency of translocations than those exposed only to 2 or 3 krad of X-rays. This effect was consistent in fully mature motile sperm and in the first 24-h brood but less consistent thereafter, indicating either that sperm are the cells more affected by ethanol pretreatment or that its action on younger cells is reversed during the longer time available between treatment of these cells and fertilization. Although chromosome breaks induced in Drosophila sperm do not rejoin until after fertilization (Muller, 1954), extensive evidence has been presented showing that the yield of genetic damage recovered can be modified by postirradiation treatment of these cells with gases and chemicals. In view of these results it was proposed that pre-repair processes may actually take place in sperm. Posttreatments, by interfering with restitution of chromosome breaks or repair of potential breaks, favor the formation of translocations later on in the egg (for a thorough discussion and bibliography, see Sankaranarayanan and Sobels, 1976). At low concentrations ethanol is well tolerated by adult Drosophila and is rapidly metabolized (Van Herrewege and David, 1974; DeltombeLietaert et al., 1979) but at the acute doses used in the experiments reported here the males, irradiated immediately after the treatment, showed symptoms of being heavily intoxicated, recovering slowly after the irradiation. This implies that ethanol was present at high concentrations in the flies, not only during but also after the irradiation and the question then arose whether the observed increase of translocations was due to the pretreatment or to a postirradiation effect of ethanol upon the chromosome breaks induced by radiation. The results with ethanol-posttreated males show that the frequency of translocations was not modified, leading to the conclusion that ethanol has to be present during the irradiation to exert an enhancing effect.
Exposure of females to ethanol did not alter the yield of translocations induced by X-rays in sperm, suggesting either that the maternal repair system was not affected by the treatment or that a deleterious effect was reversed by the metabolically active oocytes during the time elapsed between treatment and fertilization. In line with previous unpublished results obtained in our laboratory with the same female strain, the frequency of radiation-induced translocations remained unchanged throughout all the subcultures tested from the first 4-h oviposition period up to 5 days. This is worth noting in view of the findings of Wiirgler and Maier (1972) and Biirki and Wiirgler (1973) with 4-day-old females that yielded a higher rate of chromosome loss in the first 1-day brood than in the following broods, an increase that might result from a diminished repair capacity of aged oocytes. It should be kept in mind that the role played by repair is instrumental in the recovery of translocations, being less significant perhaps in chromosome loss. Thus, the data presented showing that the frequency of radiation-induced complete and partial losses were unaffected by the pretreatment give support to the assumption that the rejoining of breaks would be the step more affected by ethanol. The basis of the observations reported here remains unclear. However, one cannot rule out the possibility, although at this time it seems highly speculative, that ethanol or ethanol radicals, by temporarily altering the physiology of the treated cells or chromosomal proteins, impairs the restitution of broken chromosome ends thereby increasing the changes for rearrangement formation in the egg. Evidence that ethanol radicals formed during irradiation can cause modification and inactivation of proteins comes from studies with ribonuclease, bovine serum albumin and hemoglobin irradiated in vitro in the presence of ethanol (Schuessler et al., 1976; Schuessler and Freundl, 1983; Puchala and Schuessler, 1986). Another interpretation, related to the induction kinetics of the genetic endpoints scored, could be that ethanol given prior to irradiation acts as a weak dose modifier. From the early days of radiation genetics it was known that the frequency of sex-linked lethals increases linearly with X-ray
dose (Muller, 1954). Similarly, at 2 krad the induction of chromosome losses is also in the linear range, although at higher exposures the dose exponent is greater than 1 (Traut et al., 1970). On the other hand the induction of translocations follows 2-hit kinetics, the dose exponent being between 2 and 1.5 (Sobels and Broerse, 1970; Gonzhlez, 1972; Sankaranarayanan and Sobels, 1976). This means that owing to the slope of the curve, even slight increases in sensitivity (or effective dose) are likely to result in translocation frequencies high enough to reach significance. This suspicion was brought about by the fact that, albeit significant in all the experiments, the translocation increments observed were rather low (about 1.5 times). The way in which this presumptive dose-modifying effect of ethanol is exerted can only be surmised at this time.
Acknowledgements The author is indebted to Ms Vilma B. de Fernfindez, N o b e r E. Pereyra and B. del Carmen Paz for their excellent technical assistance and to Dr. Beatriz Mazar Barnett for her help in preparing the manuscript and correction of the English.
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