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The mutagenic activity of diethyl sulphate in Drosophila melanogaster I. The dose-mutagenic response to larval and adult feeding
173
MUTATION RESEARCH
T H E MUTAGENIC A C T I V I T Y OF D I E T H Y L S U L P H A T E IN D R O S O P H I L A M E L A N O G A S T E R I. T H E DOSE-MUTAGENIC RESPONSE TO LARVAL AND ADULT F E E D I N G M. P E L E C A N O S * AND T. A L D E R S O N
Department of Genetics, Cambridge University, Cambridge (Great Britain) (Received March 6th, 1964)
SUMMARY
The dose-mutagenic response to diethyl sulphate by both a larval and an adult feeding method, shows initially a slight but significant increase in mutagenic effect until a concentration is reached at which there is a sudden and marked increase to a maximal level. The critical (or threshold) concentration to diethyl sulphate is approximately the same for the larval and adult feeding treatments (o.375 and o.4%, respectively). The simplest explanation for the threshold effect is that free diethyl sulphate is lost by reaction with food constituents until a concentration is reached where no further loss occurs. Although it is technically difficult to test this hypothesis for larval feeding the omission of the major food constituent (yeast) from the adult feeding method shows that this interpretation is likely to be the correct one. The frequency of recessive visible sex-linked mutation induced on mature spermatozoa b y adult feeding of diethyl sulphate is found to be approx. 25 times lower than the frequency of sex-linked recessive lethal mutations.
INTRODUCTION
The alkylating agents are one of the most important groups of chemical mutagens to have received attention, since they exhibit a triad of biological effects, m a n y of them being carcinogenic, carcinostatic, and mutagenic. Also, the alkylating agents have been classified as "radiomimetic" compounds because of the striking similarities between radiation-induced mutagenesis and mutagenesis induced by alkylation. The mutagenic activity of alkylating agents naturally focussed attention on their reaction with the cellular genetic material. The first alkylating agents studied were bifunctional (that is they contain two or more reactive centres per molecule); they were thought to be capable of causing cross-linkage of compounds in the cell. In particular, cross-linkage within or between chromosomes was postulated as a mechanism underlying their mutagenic activity. Although genetic experiments have shed little light on the mechanism(s) by which alkylating agents affect the chromosome, it is now clear that they need not act b y cross-linkage, since m a n y monofunctional alkylating agents produce mutations in a variety of test organisms. * P r e s e n t address: D e p a r t m e n t of Biology, U n i v e r s i t y of Thessaloniki, Salonika, Greece.
Mutation Research i (1964) 173-181
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M. P E L E C A N O S A N D T. A L D E R S O N
The most extensively studied alkylating agents, with respect to their mutagenic effects, are the monofunctional ethylating agents, such as diethyl sulphate, ethyl methanesulphonate and ethyl ethanesulphonate4. Following tests on a series of alkylating agents, only the ethylating agents, diethyl sulphate and ethyl methanesulphonate, were found to be mutagenic on extracellular bacteriophage T2 (ref. 5); it was concluded that ethylation is unique among the alkylation reactions in its ability to produce mutation in phage T2 after reaction iJz vitro. However, this uniqueness is not preserved in bacteria and "higher organisms", though ethylation appears to be a more efficient reaction than other alkylation reactions. Previous work has shown that diethyl sulphate is mutagenic in Drosophila both by a larval feeding method 8 and by an adult feeding methodT,, ; these reports, however, established little more than that diethyl sulphate possessed mutagenic activity. The present paper concerns the dose-mutagenic response to larval and adult feeding of diethyl sulphate, and the induction of visible sex-linked recessive mutations after treatment of mature spermatozoa. METHODS
The larval feeding method Newly emerged Oregon-K flies were fed for 3 days on a generous supply of yeast, and allowed to lay for 3 h onto a roughened agar surface. The collected eggs were then sterilised using SANG'S method 1° and spread evenly over a sterile 3°'u agar surface. After incubation at 25 ° for 20 h, the newly hatched larvae were transferred under aseptic conditions onto a treatment medium consisting of IO~)o glucose, IO% heatkilled Distillers' Co. Ltd. (D.C.L.) yeast, and 3/o oj agar; diethyl sulphate was added when the temperature of the autoclaved medium had fallen to 60 °. The medium was dispensed as 25-ml portions into bottles, and the larvae were transferred to give a density of IOO larvae per 25 ml of medium.
The adult feeding method Adult feeding of Drosophila was carried out using a modification of Li;ER'S method6. Several 1-pint bottles, the bottoms of which were covered with a double thickness of filter paper, had 2 drops of a 5% suspension of heat-killed D.C.L. yeast placed in the centre of the filter paper; this quantity of yeast sufficed for the duration of the treatment. The filter paper and yeast were then saturated with a solution containing: 2 parts of distilled water, I part of absolute alcohol, 5°/'0 glucose, diethyl sulphate (supplied by British Drug Houses and containing 1.177 to 1.18 g/ml at 20°). Since diethyl sulphate is not miscible with water, alcohol was added to the treatment solution to disperse the globules otherwise formed. The same diethyl sulphate treatment solution was used throughout an experiment despite loss by hydrolysis; the pH drop over the 48-h treatment period is 2.90 to 1.7o at a concentration of o.5°/{, diethyl sulphate. Fifty newly hatched Oregon-K males were placed in the treatment bottle for 48 h, during which time the treated yeast and glucose was their only source of food. The filter paper and yeast were kept saturated during the period of treatment by adding 3 drops of the treatment solution four times daily.
Mutation Research i (i964) 173-181
MUTAGENIC
ACTIVITY
OF
DIETHYL
SULPHATE
I
175
RESULTS
The larval feeding method Using the larval feeding method, several concentrations of diethyl sulphate were tested in a series of experiments with the object of constructing a dose-response curve for the mutagenic activity of diethyl sulphate. After treatment of newly hatched larvae for the duration of l a r v a l life, emerging virgin males and females were examined by the Muller- 5 (Basc) method for the freTABLE
I
T H E D O S E - M U T A G E N I C R E S P O N S E TO D I E T H Y L S U L P H A T E W H E N MALE LARVAE ARE T R E A T E D FOR T H E I R E N T I R E LARVAL L I F E (FIRST BROOD ONLY)
Concentration of diethyl sulphate
(%) o.3 o.35 o-375 o. 4 o. 5 0. 7
o, .o of survivors 85.2 81.2 76-7 7°.0 53" 1
32.2
No. of No. of % of chromosomes sex-linked sex-linked tested recessive lethals recessive lethals I7o4 307 319 360 I OOO 149
31 9 33 35 I I6 16
TABLE
1.82 2.93 10"34 9"72 I 1,6 lO.74
0o of treated
males which yielded lethals I8. 7 20.0 4 7 .0 4 °'2 50.4 55.2
II
THE D O S E - M U T A G E N I C R E S P O N S E TO D I E T H Y L S U L P H A T E OF FEMALE LARVAE T R E A T E D FOR T H E E N T I R E LARVAL L I F E (FIRST BROOD ONLY)
Concentration of diethyl sulphate o,
o' o of survivors
o.3 o.375 0.5 o.7
87.5 77.2 50.5 31.4
(/o)
No. of No. of O/o of ° o of treated chromosomes sex-linked sex-linked females which tested recessive lethals resessive lethals yielded lelhals 6o 7 4o5 856 205
1o 6o 135 25
1.64 14.83 15.77 12.3o
I5.5 46. 2 5o.o 54.3
quency of sex-linked recessive lethal mutations. A single brood for both sexes was examined by individually mating I treated male to 2 virgin females for 3 days, and, in the case of females, I treated female to 2 males for 3 days. The results listed for males (Table I) and for females (Table II) over a concentration range o-o.7% diethyl sulphate are plotted graphically in Figs. I and 2. The dosemutagenic response curves for both sexes are seen not to be linear, but to show initially a slight increase in mutation with increasing dosage until a concentration is reached (o.35-o.375%) where a sudden and pronounced increase in the rate of mutation occurs. Further increase in concentration above this threshold level produces a slight increase in the mutation rate until at a concentration of o.5% the effect appears to be maximal; a slight fall in mutation rate at o.7% is probably due to the increased toxicity of the treatment at this concentration.
The adult feeding method In view of the results obtained for the dose-mutagenic response to larval feeding of diethyl sulphate, a study was carried out of the response to adult feeding of diethyl
Mutation Research i ( i 9 6 4 ) 173 181
176
M. PELECANOS AND T. ALDERSON
12
,,"
" - - - _
10
8
,q, < -r 6 uJ
•
4'
/
2" °
o
0 0
o.
.-"
o'.1
0;2
03
CONCENTRATION
o14 OF
oi~
DIETHYL
olo
o)
SULPHATE (°/o)
Fig. I. The d o s e - n l u t a g e n i e response to d i e t h y l s u l p h a t e , w h e n m a l e l a r v a e are t r e a t e d for t h e i r e n t i r e l a r v a l life.
16'
14
/
12'
10'
8" < -r 1-
26. 4'
o-O
o
oh
0;2
0;3
CONCENTRATION
0'.4
o'.s
OF DIETH"~L
o'.s
SULPHATE
o:7 (%)
Fig. 2. T h e d o s e - m u t a g e n i c r e s p o n s e to d i e t h y l s u l p h a t e , (0.5%) w h e n f e m a l e l a r v a e are t r e a t e d for t h e e n t i r e l a r v a l life.
Mutation
Research
• (1964) 173 i8x
MUTAGENIC ACTIVITY OF DIETHYL TABLE
SULPHATE
I
I77
III
THE DOSE-MUTAGENIC RESPONSE TO ADULT FEEDING OF DIETHYL SULPHATE WHEN DEAD YEAST IS PRESENT DURING TREATMENT
Concentration
o~
No. o/
of
(°/o)
survivors
chromosomes tested
Control
99 97 95 82 85 81 67 68
405 lO37 511 329 4oi lO4O 282 305
..o
0.3 °-375 0. 4 0.45 0. 5 0.55 0.575
Sex-linked recessive lethals
% of
% of treated males which yielded lethals
0.25 4 .60 4.7 ° 19.8o 19.oo 27.1o 26.6
0.24 35.5 ° 39.6o 83.30 84.40 88.oo 83.90 88.9o
sex-linked recessive lethals
t 48 24 65 76 282 75 85
27.90
I....
without yeast with yeast
32
28
24
20
<
16
"1" hi J
/
12
i
¢'
G¸
, o,
02
CONCENTRATION
03 OF
,
0.375 PIFTHYL
, 0.5
,
I
i
0.575
SULPNATE
(%)
F i g . 3. The d o s e - m u t a g e n i c response to a d u l t feeding of d i e t h y l sulphate, w h e n a d u l t males are t r e a t e d for 4 8 h in the presence and in t h e absence of yeast.
sulphate. A first brood dose-mutagenic response to adult feeding of males with diethyl sulphate was found to follow closely that obtained for larval feeding, initially there is a slight but significant increase in mutagenic effect with increasing dosage, until a concentration is reached (about 0.4% ) at which there is a sudden and marked increase which soon attains a maximal level (Table III, Fig. 3). The threshold concentration to diethyl sulphate for both the larval and adult feeding methods is strikingly similar (0.375 and 0.4%, respectively), and both dose-response curves follow essentially the same pattern.
Mutation Research i (1964) 173-181
178
M. P E L E C A N O S
AND
T. A L D E R S O N
The simplest explanation to account for the threshold concentration to diethyl sulphate feeding, is that free diethyl sulphate is lost by reaction with food constituents until a concentration is reached where the free compound is present. It is technically difficult to test this hypothesis for larval feeding, since reduction of food constituents in the treatment medium has a marked effect on the length of larval life and on the toxicity of the treatment medium. Consequently, an attempt was made to test the hypothesis by the adult feeding method. The dose-mutagenic response to adult feeding of diethyl sulphate was compared for 2 series of experiments. In one experiment, newly emerged adult males were fed for 48 h with 2 drops of a 5% heat-killed yeast suspension placed in the centre of the filter paper to which the glucose-diethyl sulphate treatment solution was added ; this experiment is the one which has already been described as showing a threshold TABLE
IV
THt~ DOSE-MUTAGIgNIC RESPONSE TO ADULT F E E D I N G OF DIETHYL SULPHATE
WHEN DEAD YEAST IS NOT PRESENT DURING TREATMENT
Concentration o, (Jo)
Oo of survivors
No. of chromosomes tested
,qex-linked recessive lethals
o~, oj sex-linked recessive lethals
~o of lreated males which yielded lethals
o. I 0.2 0. 3 o.5
94 92 90 6t
012 {)I 5 457 138
35 80 11 I 37
5.72 13.00
4t.9 ~3.2 84.7 81 .(-)
24.70 26.80
concentration at o.4/o o/ diethyl sulphate. A second experiment was also carried out where the yeast was omitted, but otherwise the conditions of treatment were identical. The results of this experiment (Table IV, Fig. 3) clearly show that the dose-mutagenic response now has an immediate linear response to increasing concentration; there is no sign of a threshold effect. Both dose-response curves have mutational responses which coincide at a concentration of 0.5/o o/ diethyl sulphate, and they show parallel mutational responses over the sensitive concentration ranges (Fig. 3). The threshold effect for adult feeding of diethyl sulphate, and probably also for larval feeding, would thus appear to be due to loss of free diethyl sulphate by reaction with the major food constituent (yeast) until, at the critical threshold concentration, free diethyl sulphate is present. It is remarkable that the dose-mutagenic response curves for larval and adult feeding of diethyl sulphate should show such a similar behaviour, particularly with regard to the threshold level of response. There are several notable differences between the two methods of administration which are expected to influence the loss of diethyl sulphate by reaction with food constituents. By larval feeding method, diethyl sulphate is added at 6o ° to the yeast glucose treatment medium, and its reactivity with yeast is expected to be greater than by adult feeding where reaction with yeast is at 25 °. Also in the adult feeding method, the diethyl sulphate is placed not only on the small area of yeast, but on the surrounding filter paper, and unless the flies concentrate solely on the yeast for nourishment, the dose-response curve would not be expected to be so similar to that of larval feeding. The type of dose-response curve shown by the feeding of diethyl sulphate might be expected for any mutagenic chemicals which reacts extensively with food eonstit-
Mutation Research i (1964) 173 181
M U T A G E N I C A C T I V I T Y OF D I E T H Y L
SULPHATE
I
179
uents. However, formaldehyde is the only other mutagenic chemical whose dosemutagenic response curve (by larval feeding) has been studied in detail1. Although formaldehyde reacts extensively with the food constituents in the larval treatment medium a, its dose-mutagenic response is linear until it reaches a maximal level; there is no indication of a threshold effect. Formaldehyde-induced mutagenesis by larval feeding is, however, unique, in that its mutagenic activity is completely mediated by reaction with a food constituent, adenylic acid 2.
Induction of sex-linked visible mutations after treatment of spermatozoa with diethyl sulphate Adult feeding of diethyl sulphate is of particular interest since it is the most effective mutagenic treatment yet recorded on Drosophila. The results (for the first brood sample) represent the effect on germ cell predominantly treated as mature spermatozoa, and the lethals must consequently have arisen independently of one another. As reported by RAPOPORT~, a n uncommonly high proportion of semi-lethals is induced on mature spermatozoa after adult feeding of diethyl sulphate; however, these have not been recorded in the data presented here. In view of the high frequency of sex-linked recessive lethals induced by diethyl sulphate, an investigation was undertaken of the frequency of sex-linked visible mutations induced in spermatozoa after diethyl sulphate treatment. Two independent experiments were carried out where newly hatched Oregon-K males were treated b y adult feeding with 0.5% diethyl sulphate for 48 h. Sex-linked visible mutations were detected by mating each treated male to two attached-X females for 3 days, giving a first brood sample where the progeny arose from germ cells which were treated as spermatozoa. Visible sex-linked mutations were scored in the F1 from this mating, and all F1 males which deviated from the wild type phenotype were provisionally classified as m u t a n t ; these mutants were then confirmed by remating to attached-X females. First experiment. In this experiment an attached-X stock was used where both sex chromosomes carried the recessive gene for yellow body colour, and the second and third chromosomes were homozygous for the recessive genes, brown eyes and scarlet eyes, respectively; the brown and scarlet genes interact to produce a whiteeyed phenotype. 641 males arising from 51 F1 cultures were examined for mutant phenotypes; the sex ratio of males to females from these F1 cultures was 1:2. 44 F1 males from the total of 641 males were provisionally classified as m u t a n t on the basis of their phenotypic deviation from wild type; 52.3~o of these males were mosaic for the character affected. Since a mosaic fly consists of m u t a n t and non-mutant tissue, the gonads too m a y be mosaic for both m u t a n t and non-mutant germ cells, and such a gonosomic mosaic expectedly produces both m u t a n t and non-mutant progeny. However, if the m u t a n t is not present in germinal tissue it is lost, or alternatively, the size of the m u t a n t area m a y be too small for the m u t a n t to be recovered in the progeny sample. Of the 44 suspected mutants, 50% failed to produce progeny due to the sterility or death of the fly. Consequently, only 22 mutants could be tested, and the majority of these failed to produce m u t a n t progeny. Only 5 mutants seemed likely to be sexlinked visible mutations; they all arose in different males. (i) A miniature Ft male yielded all miniature males. (2) An F1 male with yellow thorax and microchaete bred
Mutation Research i (1964) 173-I81
180
M. P E L E C A N O S A N D T. A L D E R S O N
true. (3) A mosaic F1 male for yellow body colour (on its left side), produced true breeding male progeny all of which had dumpy wings. (4) An F1 male, mosaic for brown eye/normal eye (left/right) produced male progeny in a I : I : I : I segregation for light red eye:white eye:pink eye:orange eye. (5) An F1 male, mosaic for brown eye/normal eye (left/right) produced male progeny in a I : I : I : I segregation for light red eye : white eye : pink eye : apricot eye. Both these latter two mutant cultures (4 and 5) showed the expected i : i : i : i backcross segregations for females, i.e. wild type eye:white eye:brown eye:scarlet eye, since the attached-X stock used in this experiment was homozygous for brown eye and scarlet eye on the second and third chromosome, respectively. Of these 5 mutants only 2 (I and 2) were simple true breeding sex-linked mutants; 5 and 6 are probably mutants which dilute eye colour expression, possibly alleles of white eye which show a dilution effect on the other eye colour genes. The rate of induced sex-linked visible mutations would thus appear to be of the order of 0.78% (5/641). In addition 3 dominant visible mutations were recorded, A Minute, Hairless, and Curly Wing. Second Experiment. In this experiment an attached-X stock was used where both sex chromosomes carried the recessive mutant yellow body; no other markers were present. 687 males arising from 83 F1 cultures were examined for mutant phenotypes; as in the first experiment, the sex ratio was approximately 1:2 = males:females. 49 F1 males showed phenotypes which were suspected of being mutant. 49% of the F~ mutant progeny showed mosaicism for the character affected. Only 33 of the 49 suspected mutants produced progeny, and only 6 of these showed the mutant in their progeny. The 6 mutants arose in different males. (I) An F1 male with orange eyes produced all orange-eyed males. (2) A miniature F1 male produced all miniature males. (3) An F1 male, mosaic for rough eye/wild type eye (right/left) produced male progeny all of which lacked ocellar and post verticle macrochaetae; probably ascute allele. (4) An F1 male, mosaic for a short left wing produced male progeny all of which were dwarfed; I out of 8 also had reduced wings. (5) An F~ male with rough eyes produced male progeny all of which had barred eyes. (6) An F~ male with rough eyes and forked anterior head chaete produced male progeny all of which had rough eyes and forked microchaete. The rate of induced sex-linked visible mutations would thus appear to be of the order of 0.87% (6/687). In addition 2 dominant visible mutations were recorded, Curly Wing and Hairless. In both experiments, the percentage of sex-linked visible mutations was similar, 0.78 and o.87°/~ ; the ratio of sex-linked visible to sex-linked recessive lethals is thus approximately I to 25. Both treatments show similar percentages of mosaicism, 52.3 and 49~'o; however, the left/right distribution of mutant/non-mutant tissue is 17/6 and 7/17, respectively, for the first and second experiment. Two dominant mutations, Curly Wing and Hairless, and the sex-linked recessive mutant, minature, were common to both experiments. It was not possible to carry out allelism tests on the visible mutations recovered after diethyl sulphate treatment, since a general food crisis, consequent on changing to another agar source, resulted in the loss of these mutants. The tests for the induction of visible sex-linked mutations on mature sperm after diethyl sulphate treatment, were not as satisfactory as had been hoped. This was
Mutation Research i (i964} 173-181
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181
primarily due to the high frequency of "mutant" flies which proved to be sterile, or showed mosaicism for the "mutation" but failed to produce mutant progeny on testing. Further, several mosaic "mutants" produced progeny showing a mutant phenotype different from the "mutation" for which they were originally tested. Undoubtedly, the estimated frequency of sex-linked visible mutations from the data presented is an underestimation of the actual frequency produced after diethyl sulphate treatment. The criterion used for scoring mutants in these tests, was that an F 1 "mutant" is recoverable in its progeny from a back-cross to attached-X females, and behaves as a sex-linked mutation on further testing. If "mutants" have been scored simply as F~ progeny from the treated males which showed a mutant phenotype, frequencies of 6.8 and 7.2% visible sex-linked mutations would have been recorded for the first and second experiments, respectively. Also many possible mosaic mutants for one slightly shorter wing were observed, but these were not recorded or tested. The experiments reported on the frequency of sex-linked visible mutations after diethyl sulphate treatment of mature sperm can only be regarded as pilot experiments, and a more detailed analysis is obviously required: This should involve the backcrossing of all F~ male progeny to attached-X females in order to detect hidden mosaicism. ACKNOWLEDGEMENTS
Dr. ALDERSON wishes to make acknowledgements to the British Empire Cancer Campaign, and Dr. PELECANOS to the British Council and the Greek State Scholarship Foundation, for financial support of this work. Thanks are due to Professor J. M. THODAY for supplying the laboratory facilities, and Mrs. V. C. FYFE for drawing the figures. REFERENCES 1 ALDERSON, T. Conditional antimutagenesis i n Drosophila melanogaster. Nature, 182 (~958) 508 51o. 2 ALDERSON, T. Mechanism of formaldehyde-induced mutagenesis. The uniqueness of adenylic acid in the mediation of the mutagenic activity of formaldehyde. Nature, 187 (196o) 485 489 . 3 BARKER, J. S. F. The mutagenicity of formaldehyde medium to Drosophila melanogaster larvae. Austr. J. Biol. Sci., 11 (1959) 4 1 8 - 4 2 4 . FREESE, E. Molecular mechanism of mutation. Chapter V of 31olecular Genetics, A c a d e m i c Press, New York, 1963. LOVELESS, A. The influence of radiomimetic substances on D N A synthesis and function in E. coil~phage s y s t e m s . I I I Mutation of T2 bacteriophage as a consequency of alkylation in vitro: The uniqueness of ethylation. Proc. Roy. Soc. Londo*z, Ser. B, 15o (t959) 497 5 °8. 6 LOERS, H . Untersuehung tiber die Mutagenit/~t des Tri~thylenmelamin (TEM) an Drosophila melanogaster. Arch. Geschwulstforsch., 6 (1953) 7 7 - 7 9 . 7 PELECANOS, M. AND T. ALDERSON. The mutagenic response to adult feeding of diethyl sulphate. Drosophila Inform. Service, 37 (1963) 1 I G - I I 7. 8 RAPOPORT, I. A. O n the mutagenic action of dimethyl and diethyl sulphate (in Russian). Dokl. Vses. Akad. Sel'skokhoz. Nauk., 12 (1947) 12-2.5. 9 RAPOPORT, I. A. Chemical mutations in sex-chromosomes with a frequency above fifty per c e n t and increased proportions of semi-lethals (in Russian). Dokl. Biol. Sci. Sect., I 4 t (1961) 1 4 7 0 1479. lo SANG, J. H . The quantitative nutritional requirements of Drosophila melanogaster..]. Exptl. Biol., 33 (19.56 ) 4 5 - 7 2 .
3lutation Research I (1964) I73 181
MUTATION RESEARCH
T H E MUTAGENIC A C T I V I T Y OF D I E T H Y L S U L P H A T E IN D R O S O P H I L A M E L A N O G A S T E R I. T H E DOSE-MUTAGENIC RESPONSE TO LARVAL AND ADULT F E E D I N G M. P E L E C A N O S * AND T. A L D E R S O N
Department of Genetics, Cambridge University, Cambridge (Great Britain) (Received March 6th, 1964)
SUMMARY
The dose-mutagenic response to diethyl sulphate by both a larval and an adult feeding method, shows initially a slight but significant increase in mutagenic effect until a concentration is reached at which there is a sudden and marked increase to a maximal level. The critical (or threshold) concentration to diethyl sulphate is approximately the same for the larval and adult feeding treatments (o.375 and o.4%, respectively). The simplest explanation for the threshold effect is that free diethyl sulphate is lost by reaction with food constituents until a concentration is reached where no further loss occurs. Although it is technically difficult to test this hypothesis for larval feeding the omission of the major food constituent (yeast) from the adult feeding method shows that this interpretation is likely to be the correct one. The frequency of recessive visible sex-linked mutation induced on mature spermatozoa b y adult feeding of diethyl sulphate is found to be approx. 25 times lower than the frequency of sex-linked recessive lethal mutations.
INTRODUCTION
The alkylating agents are one of the most important groups of chemical mutagens to have received attention, since they exhibit a triad of biological effects, m a n y of them being carcinogenic, carcinostatic, and mutagenic. Also, the alkylating agents have been classified as "radiomimetic" compounds because of the striking similarities between radiation-induced mutagenesis and mutagenesis induced by alkylation. The mutagenic activity of alkylating agents naturally focussed attention on their reaction with the cellular genetic material. The first alkylating agents studied were bifunctional (that is they contain two or more reactive centres per molecule); they were thought to be capable of causing cross-linkage of compounds in the cell. In particular, cross-linkage within or between chromosomes was postulated as a mechanism underlying their mutagenic activity. Although genetic experiments have shed little light on the mechanism(s) by which alkylating agents affect the chromosome, it is now clear that they need not act b y cross-linkage, since m a n y monofunctional alkylating agents produce mutations in a variety of test organisms. * P r e s e n t address: D e p a r t m e n t of Biology, U n i v e r s i t y of Thessaloniki, Salonika, Greece.
Mutation Research i (1964) 173-181
174
M. P E L E C A N O S A N D T. A L D E R S O N
The most extensively studied alkylating agents, with respect to their mutagenic effects, are the monofunctional ethylating agents, such as diethyl sulphate, ethyl methanesulphonate and ethyl ethanesulphonate4. Following tests on a series of alkylating agents, only the ethylating agents, diethyl sulphate and ethyl methanesulphonate, were found to be mutagenic on extracellular bacteriophage T2 (ref. 5); it was concluded that ethylation is unique among the alkylation reactions in its ability to produce mutation in phage T2 after reaction iJz vitro. However, this uniqueness is not preserved in bacteria and "higher organisms", though ethylation appears to be a more efficient reaction than other alkylation reactions. Previous work has shown that diethyl sulphate is mutagenic in Drosophila both by a larval feeding method 8 and by an adult feeding methodT,, ; these reports, however, established little more than that diethyl sulphate possessed mutagenic activity. The present paper concerns the dose-mutagenic response to larval and adult feeding of diethyl sulphate, and the induction of visible sex-linked recessive mutations after treatment of mature spermatozoa. METHODS
The larval feeding method Newly emerged Oregon-K flies were fed for 3 days on a generous supply of yeast, and allowed to lay for 3 h onto a roughened agar surface. The collected eggs were then sterilised using SANG'S method 1° and spread evenly over a sterile 3°'u agar surface. After incubation at 25 ° for 20 h, the newly hatched larvae were transferred under aseptic conditions onto a treatment medium consisting of IO~)o glucose, IO% heatkilled Distillers' Co. Ltd. (D.C.L.) yeast, and 3/o oj agar; diethyl sulphate was added when the temperature of the autoclaved medium had fallen to 60 °. The medium was dispensed as 25-ml portions into bottles, and the larvae were transferred to give a density of IOO larvae per 25 ml of medium.
The adult feeding method Adult feeding of Drosophila was carried out using a modification of Li;ER'S method6. Several 1-pint bottles, the bottoms of which were covered with a double thickness of filter paper, had 2 drops of a 5% suspension of heat-killed D.C.L. yeast placed in the centre of the filter paper; this quantity of yeast sufficed for the duration of the treatment. The filter paper and yeast were then saturated with a solution containing: 2 parts of distilled water, I part of absolute alcohol, 5°/'0 glucose, diethyl sulphate (supplied by British Drug Houses and containing 1.177 to 1.18 g/ml at 20°). Since diethyl sulphate is not miscible with water, alcohol was added to the treatment solution to disperse the globules otherwise formed. The same diethyl sulphate treatment solution was used throughout an experiment despite loss by hydrolysis; the pH drop over the 48-h treatment period is 2.90 to 1.7o at a concentration of o.5°/{, diethyl sulphate. Fifty newly hatched Oregon-K males were placed in the treatment bottle for 48 h, during which time the treated yeast and glucose was their only source of food. The filter paper and yeast were kept saturated during the period of treatment by adding 3 drops of the treatment solution four times daily.
Mutation Research i (i964) 173-181
MUTAGENIC
ACTIVITY
OF
DIETHYL
SULPHATE
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175
RESULTS
The larval feeding method Using the larval feeding method, several concentrations of diethyl sulphate were tested in a series of experiments with the object of constructing a dose-response curve for the mutagenic activity of diethyl sulphate. After treatment of newly hatched larvae for the duration of l a r v a l life, emerging virgin males and females were examined by the Muller- 5 (Basc) method for the freTABLE
I
T H E D O S E - M U T A G E N I C R E S P O N S E TO D I E T H Y L S U L P H A T E W H E N MALE LARVAE ARE T R E A T E D FOR T H E I R E N T I R E LARVAL L I F E (FIRST BROOD ONLY)
Concentration of diethyl sulphate
(%) o.3 o.35 o-375 o. 4 o. 5 0. 7
o, .o of survivors 85.2 81.2 76-7 7°.0 53" 1
32.2
No. of No. of % of chromosomes sex-linked sex-linked tested recessive lethals recessive lethals I7o4 307 319 360 I OOO 149
31 9 33 35 I I6 16
TABLE
1.82 2.93 10"34 9"72 I 1,6 lO.74
0o of treated
males which yielded lethals I8. 7 20.0 4 7 .0 4 °'2 50.4 55.2
II
THE D O S E - M U T A G E N I C R E S P O N S E TO D I E T H Y L S U L P H A T E OF FEMALE LARVAE T R E A T E D FOR T H E E N T I R E LARVAL L I F E (FIRST BROOD ONLY)
Concentration of diethyl sulphate o,
o' o of survivors
o.3 o.375 0.5 o.7
87.5 77.2 50.5 31.4
(/o)
No. of No. of O/o of ° o of treated chromosomes sex-linked sex-linked females which tested recessive lethals resessive lethals yielded lelhals 6o 7 4o5 856 205
1o 6o 135 25
1.64 14.83 15.77 12.3o
I5.5 46. 2 5o.o 54.3
quency of sex-linked recessive lethal mutations. A single brood for both sexes was examined by individually mating I treated male to 2 virgin females for 3 days, and, in the case of females, I treated female to 2 males for 3 days. The results listed for males (Table I) and for females (Table II) over a concentration range o-o.7% diethyl sulphate are plotted graphically in Figs. I and 2. The dosemutagenic response curves for both sexes are seen not to be linear, but to show initially a slight increase in mutation with increasing dosage until a concentration is reached (o.35-o.375%) where a sudden and pronounced increase in the rate of mutation occurs. Further increase in concentration above this threshold level produces a slight increase in the mutation rate until at a concentration of o.5% the effect appears to be maximal; a slight fall in mutation rate at o.7% is probably due to the increased toxicity of the treatment at this concentration.
The adult feeding method In view of the results obtained for the dose-mutagenic response to larval feeding of diethyl sulphate, a study was carried out of the response to adult feeding of diethyl
Mutation Research i ( i 9 6 4 ) 173 181
176
M. PELECANOS AND T. ALDERSON
12
,,"
" - - - _
10
8
,q, < -r 6 uJ
•
4'
/
2" °
o
0 0
o.
.-"
o'.1
0;2
03
CONCENTRATION
o14 OF
oi~
DIETHYL
olo
o)
SULPHATE (°/o)
Fig. I. The d o s e - n l u t a g e n i e response to d i e t h y l s u l p h a t e , w h e n m a l e l a r v a e are t r e a t e d for t h e i r e n t i r e l a r v a l life.
16'
14
/
12'
10'
8" < -r 1-
26. 4'
o-O
o
oh
0;2
0;3
CONCENTRATION
0'.4
o'.s
OF DIETH"~L
o'.s
SULPHATE
o:7 (%)
Fig. 2. T h e d o s e - m u t a g e n i c r e s p o n s e to d i e t h y l s u l p h a t e , (0.5%) w h e n f e m a l e l a r v a e are t r e a t e d for t h e e n t i r e l a r v a l life.
Mutation
Research
• (1964) 173 i8x
MUTAGENIC ACTIVITY OF DIETHYL TABLE
SULPHATE
I
I77
III
THE DOSE-MUTAGENIC RESPONSE TO ADULT FEEDING OF DIETHYL SULPHATE WHEN DEAD YEAST IS PRESENT DURING TREATMENT
Concentration
o~
No. o/
of
(°/o)
survivors
chromosomes tested
Control
99 97 95 82 85 81 67 68
405 lO37 511 329 4oi lO4O 282 305
..o
0.3 °-375 0. 4 0.45 0. 5 0.55 0.575
Sex-linked recessive lethals
% of
% of treated males which yielded lethals
0.25 4 .60 4.7 ° 19.8o 19.oo 27.1o 26.6
0.24 35.5 ° 39.6o 83.30 84.40 88.oo 83.90 88.9o
sex-linked recessive lethals
t 48 24 65 76 282 75 85
27.90
I....
without yeast with yeast
32
28
24
20
<
16
"1" hi J
/
12
i
¢'
G¸
, o,
02
CONCENTRATION
03 OF
,
0.375 PIFTHYL
, 0.5
,
I
i
0.575
SULPNATE
(%)
F i g . 3. The d o s e - m u t a g e n i c response to a d u l t feeding of d i e t h y l sulphate, w h e n a d u l t males are t r e a t e d for 4 8 h in the presence and in t h e absence of yeast.
sulphate. A first brood dose-mutagenic response to adult feeding of males with diethyl sulphate was found to follow closely that obtained for larval feeding, initially there is a slight but significant increase in mutagenic effect with increasing dosage, until a concentration is reached (about 0.4% ) at which there is a sudden and marked increase which soon attains a maximal level (Table III, Fig. 3). The threshold concentration to diethyl sulphate for both the larval and adult feeding methods is strikingly similar (0.375 and 0.4%, respectively), and both dose-response curves follow essentially the same pattern.
Mutation Research i (1964) 173-181
178
M. P E L E C A N O S
AND
T. A L D E R S O N
The simplest explanation to account for the threshold concentration to diethyl sulphate feeding, is that free diethyl sulphate is lost by reaction with food constituents until a concentration is reached where the free compound is present. It is technically difficult to test this hypothesis for larval feeding, since reduction of food constituents in the treatment medium has a marked effect on the length of larval life and on the toxicity of the treatment medium. Consequently, an attempt was made to test the hypothesis by the adult feeding method. The dose-mutagenic response to adult feeding of diethyl sulphate was compared for 2 series of experiments. In one experiment, newly emerged adult males were fed for 48 h with 2 drops of a 5% heat-killed yeast suspension placed in the centre of the filter paper to which the glucose-diethyl sulphate treatment solution was added ; this experiment is the one which has already been described as showing a threshold TABLE
IV
THt~ DOSE-MUTAGIgNIC RESPONSE TO ADULT F E E D I N G OF DIETHYL SULPHATE
WHEN DEAD YEAST IS NOT PRESENT DURING TREATMENT
Concentration o, (Jo)
Oo of survivors
No. of chromosomes tested
,qex-linked recessive lethals
o~, oj sex-linked recessive lethals
~o of lreated males which yielded lethals
o. I 0.2 0. 3 o.5
94 92 90 6t
012 {)I 5 457 138
35 80 11 I 37
5.72 13.00
4t.9 ~3.2 84.7 81 .(-)
24.70 26.80
concentration at o.4/o o/ diethyl sulphate. A second experiment was also carried out where the yeast was omitted, but otherwise the conditions of treatment were identical. The results of this experiment (Table IV, Fig. 3) clearly show that the dose-mutagenic response now has an immediate linear response to increasing concentration; there is no sign of a threshold effect. Both dose-response curves have mutational responses which coincide at a concentration of 0.5/o o/ diethyl sulphate, and they show parallel mutational responses over the sensitive concentration ranges (Fig. 3). The threshold effect for adult feeding of diethyl sulphate, and probably also for larval feeding, would thus appear to be due to loss of free diethyl sulphate by reaction with the major food constituent (yeast) until, at the critical threshold concentration, free diethyl sulphate is present. It is remarkable that the dose-mutagenic response curves for larval and adult feeding of diethyl sulphate should show such a similar behaviour, particularly with regard to the threshold level of response. There are several notable differences between the two methods of administration which are expected to influence the loss of diethyl sulphate by reaction with food constituents. By larval feeding method, diethyl sulphate is added at 6o ° to the yeast glucose treatment medium, and its reactivity with yeast is expected to be greater than by adult feeding where reaction with yeast is at 25 °. Also in the adult feeding method, the diethyl sulphate is placed not only on the small area of yeast, but on the surrounding filter paper, and unless the flies concentrate solely on the yeast for nourishment, the dose-response curve would not be expected to be so similar to that of larval feeding. The type of dose-response curve shown by the feeding of diethyl sulphate might be expected for any mutagenic chemicals which reacts extensively with food eonstit-
Mutation Research i (1964) 173 181
M U T A G E N I C A C T I V I T Y OF D I E T H Y L
SULPHATE
I
179
uents. However, formaldehyde is the only other mutagenic chemical whose dosemutagenic response curve (by larval feeding) has been studied in detail1. Although formaldehyde reacts extensively with the food constituents in the larval treatment medium a, its dose-mutagenic response is linear until it reaches a maximal level; there is no indication of a threshold effect. Formaldehyde-induced mutagenesis by larval feeding is, however, unique, in that its mutagenic activity is completely mediated by reaction with a food constituent, adenylic acid 2.
Induction of sex-linked visible mutations after treatment of spermatozoa with diethyl sulphate Adult feeding of diethyl sulphate is of particular interest since it is the most effective mutagenic treatment yet recorded on Drosophila. The results (for the first brood sample) represent the effect on germ cell predominantly treated as mature spermatozoa, and the lethals must consequently have arisen independently of one another. As reported by RAPOPORT~, a n uncommonly high proportion of semi-lethals is induced on mature spermatozoa after adult feeding of diethyl sulphate; however, these have not been recorded in the data presented here. In view of the high frequency of sex-linked recessive lethals induced by diethyl sulphate, an investigation was undertaken of the frequency of sex-linked visible mutations induced in spermatozoa after diethyl sulphate treatment. Two independent experiments were carried out where newly hatched Oregon-K males were treated b y adult feeding with 0.5% diethyl sulphate for 48 h. Sex-linked visible mutations were detected by mating each treated male to two attached-X females for 3 days, giving a first brood sample where the progeny arose from germ cells which were treated as spermatozoa. Visible sex-linked mutations were scored in the F1 from this mating, and all F1 males which deviated from the wild type phenotype were provisionally classified as m u t a n t ; these mutants were then confirmed by remating to attached-X females. First experiment. In this experiment an attached-X stock was used where both sex chromosomes carried the recessive gene for yellow body colour, and the second and third chromosomes were homozygous for the recessive genes, brown eyes and scarlet eyes, respectively; the brown and scarlet genes interact to produce a whiteeyed phenotype. 641 males arising from 51 F1 cultures were examined for mutant phenotypes; the sex ratio of males to females from these F1 cultures was 1:2. 44 F1 males from the total of 641 males were provisionally classified as m u t a n t on the basis of their phenotypic deviation from wild type; 52.3~o of these males were mosaic for the character affected. Since a mosaic fly consists of m u t a n t and non-mutant tissue, the gonads too m a y be mosaic for both m u t a n t and non-mutant germ cells, and such a gonosomic mosaic expectedly produces both m u t a n t and non-mutant progeny. However, if the m u t a n t is not present in germinal tissue it is lost, or alternatively, the size of the m u t a n t area m a y be too small for the m u t a n t to be recovered in the progeny sample. Of the 44 suspected mutants, 50% failed to produce progeny due to the sterility or death of the fly. Consequently, only 22 mutants could be tested, and the majority of these failed to produce m u t a n t progeny. Only 5 mutants seemed likely to be sexlinked visible mutations; they all arose in different males. (i) A miniature Ft male yielded all miniature males. (2) An F1 male with yellow thorax and microchaete bred
Mutation Research i (1964) 173-I81
180
M. P E L E C A N O S A N D T. A L D E R S O N
true. (3) A mosaic F1 male for yellow body colour (on its left side), produced true breeding male progeny all of which had dumpy wings. (4) An F1 male, mosaic for brown eye/normal eye (left/right) produced male progeny in a I : I : I : I segregation for light red eye:white eye:pink eye:orange eye. (5) An F1 male, mosaic for brown eye/normal eye (left/right) produced male progeny in a I : I : I : I segregation for light red eye : white eye : pink eye : apricot eye. Both these latter two mutant cultures (4 and 5) showed the expected i : i : i : i backcross segregations for females, i.e. wild type eye:white eye:brown eye:scarlet eye, since the attached-X stock used in this experiment was homozygous for brown eye and scarlet eye on the second and third chromosome, respectively. Of these 5 mutants only 2 (I and 2) were simple true breeding sex-linked mutants; 5 and 6 are probably mutants which dilute eye colour expression, possibly alleles of white eye which show a dilution effect on the other eye colour genes. The rate of induced sex-linked visible mutations would thus appear to be of the order of 0.78% (5/641). In addition 3 dominant visible mutations were recorded, A Minute, Hairless, and Curly Wing. Second Experiment. In this experiment an attached-X stock was used where both sex chromosomes carried the recessive mutant yellow body; no other markers were present. 687 males arising from 83 F1 cultures were examined for mutant phenotypes; as in the first experiment, the sex ratio was approximately 1:2 = males:females. 49 F1 males showed phenotypes which were suspected of being mutant. 49% of the F~ mutant progeny showed mosaicism for the character affected. Only 33 of the 49 suspected mutants produced progeny, and only 6 of these showed the mutant in their progeny. The 6 mutants arose in different males. (I) An F1 male with orange eyes produced all orange-eyed males. (2) A miniature F1 male produced all miniature males. (3) An F1 male, mosaic for rough eye/wild type eye (right/left) produced male progeny all of which lacked ocellar and post verticle macrochaetae; probably ascute allele. (4) An F1 male, mosaic for a short left wing produced male progeny all of which were dwarfed; I out of 8 also had reduced wings. (5) An F~ male with rough eyes produced male progeny all of which had barred eyes. (6) An F~ male with rough eyes and forked anterior head chaete produced male progeny all of which had rough eyes and forked microchaete. The rate of induced sex-linked visible mutations would thus appear to be of the order of 0.87% (6/687). In addition 2 dominant visible mutations were recorded, Curly Wing and Hairless. In both experiments, the percentage of sex-linked visible mutations was similar, 0.78 and o.87°/~ ; the ratio of sex-linked visible to sex-linked recessive lethals is thus approximately I to 25. Both treatments show similar percentages of mosaicism, 52.3 and 49~'o; however, the left/right distribution of mutant/non-mutant tissue is 17/6 and 7/17, respectively, for the first and second experiment. Two dominant mutations, Curly Wing and Hairless, and the sex-linked recessive mutant, minature, were common to both experiments. It was not possible to carry out allelism tests on the visible mutations recovered after diethyl sulphate treatment, since a general food crisis, consequent on changing to another agar source, resulted in the loss of these mutants. The tests for the induction of visible sex-linked mutations on mature sperm after diethyl sulphate treatment, were not as satisfactory as had been hoped. This was
Mutation Research i (i964} 173-181
M U T A G E N I C A C T I V I T Y OF D I E T H Y L S U L P H A T E
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181
primarily due to the high frequency of "mutant" flies which proved to be sterile, or showed mosaicism for the "mutation" but failed to produce mutant progeny on testing. Further, several mosaic "mutants" produced progeny showing a mutant phenotype different from the "mutation" for which they were originally tested. Undoubtedly, the estimated frequency of sex-linked visible mutations from the data presented is an underestimation of the actual frequency produced after diethyl sulphate treatment. The criterion used for scoring mutants in these tests, was that an F 1 "mutant" is recoverable in its progeny from a back-cross to attached-X females, and behaves as a sex-linked mutation on further testing. If "mutants" have been scored simply as F~ progeny from the treated males which showed a mutant phenotype, frequencies of 6.8 and 7.2% visible sex-linked mutations would have been recorded for the first and second experiments, respectively. Also many possible mosaic mutants for one slightly shorter wing were observed, but these were not recorded or tested. The experiments reported on the frequency of sex-linked visible mutations after diethyl sulphate treatment of mature sperm can only be regarded as pilot experiments, and a more detailed analysis is obviously required: This should involve the backcrossing of all F~ male progeny to attached-X females in order to detect hidden mosaicism. ACKNOWLEDGEMENTS
Dr. ALDERSON wishes to make acknowledgements to the British Empire Cancer Campaign, and Dr. PELECANOS to the British Council and the Greek State Scholarship Foundation, for financial support of this work. Thanks are due to Professor J. M. THODAY for supplying the laboratory facilities, and Mrs. V. C. FYFE for drawing the figures. REFERENCES 1 ALDERSON, T. Conditional antimutagenesis i n Drosophila melanogaster. Nature, 182 (~958) 508 51o. 2 ALDERSON, T. Mechanism of formaldehyde-induced mutagenesis. The uniqueness of adenylic acid in the mediation of the mutagenic activity of formaldehyde. Nature, 187 (196o) 485 489 . 3 BARKER, J. S. F. The mutagenicity of formaldehyde medium to Drosophila melanogaster larvae. Austr. J. Biol. Sci., 11 (1959) 4 1 8 - 4 2 4 . FREESE, E. Molecular mechanism of mutation. Chapter V of 31olecular Genetics, A c a d e m i c Press, New York, 1963. LOVELESS, A. The influence of radiomimetic substances on D N A synthesis and function in E. coil~phage s y s t e m s . I I I Mutation of T2 bacteriophage as a consequency of alkylation in vitro: The uniqueness of ethylation. Proc. Roy. Soc. Londo*z, Ser. B, 15o (t959) 497 5 °8. 6 LOERS, H . Untersuehung tiber die Mutagenit/~t des Tri~thylenmelamin (TEM) an Drosophila melanogaster. Arch. Geschwulstforsch., 6 (1953) 7 7 - 7 9 . 7 PELECANOS, M. AND T. ALDERSON. The mutagenic response to adult feeding of diethyl sulphate. Drosophila Inform. Service, 37 (1963) 1 I G - I I 7. 8 RAPOPORT, I. A. O n the mutagenic action of dimethyl and diethyl sulphate (in Russian). Dokl. Vses. Akad. Sel'skokhoz. Nauk., 12 (1947) 12-2.5. 9 RAPOPORT, I. A. Chemical mutations in sex-chromosomes with a frequency above fifty per c e n t and increased proportions of semi-lethals (in Russian). Dokl. Biol. Sci. Sect., I 4 t (1961) 1 4 7 0 1479. lo SANG, J. H . The quantitative nutritional requirements of Drosophila melanogaster..]. Exptl. Biol., 33 (19.56 ) 4 5 - 7 2 .
3lutation Research I (1964) I73 181