Mutation Research, 28 (1975) 4 6 5 - 4 6 8 © Elsevier Scientific Publishing Company,
Amsterdam--Printed
in T h e N e t h e r l a n d s
465
Mutagenic activity of trimethylphosphate in Drosophila at low doses One of the characteristics of environmental mutagens is that they are normally present in very low doses. This is a problem for those wishing to assess their likely genetic hazards because testing in the laboratory has to be done at relatively high doses. However, it has been assumed that at low doses the mutagens pose their lowest threat and therefore compounds found to be of low mutagenic activity in the laboratory would not appear to be important environmental hazards TMP is known to be mutagenic in a variety of systems under various conditions, although normally of low activity 2. Evidence has now been obtained which suggests that, at least in Drosophila, TMP m a y be a more effective mutagen at low doses than at higher ones. This evidence comes partly from single generation experiments, in which larvae were allowed to develop in media containing TMP and the emerging adults tested for the induction of mutations. It also comes from population experiments in which initially identical populations of Drosophila were kept for periods of up to two years on media containing various doses of TMP. The materials and methods from these experiments are essentially those described in detail in our earlier papers1, ~. The results from the first group of experiments are presented in Table I. The salient feature of this table is the relatively high frequency of lethals at the lower doses (o.oo5 M and below) compared to those at the higher doses (o.oi M to o.o25 M). The mutation frequencies at the five lowest doses are all significantly higher than the figure predicted from a linear regression calculated for four higher doses and the mutation induction by o.oo4 M is significantly higher than o.oi M or o.o15 M. The overall lethal frequency at these five lower doses is 5.4% which is identical to that at the higher doses. Of course a concentration of o.ooi M TMP is still rather high and a number of TABLE
I
2 n d CHROMOSOME RECESSIVE LETHALS POUND IN SPERM OF DROSOPHILA WHICH HAD DEVELOPED AS LARVAE ON MEDIA CONTAINING VARYING CONCENTRATIONS OF TRIMETHYLPHOSPHATE
Concentration of T M P in feed
Number of tests
Number of lethals
Control~
11°5
4
389 747 336 4°2 595 521 523 848 186
22 22 19 31 4° 19 24 57 14
% Lethals
Linear regression of lethal frequencies based on control and values from highest four concentrations ( % )
0-4 i
0.6
o.oi
5.7 3.o 5-6 7.7 6-7 3.6 4.6 6.7 7.5
1.2 0.6 1.3 1.3 i.o 0.8 0.9 0.9 1.9
0.33 0.66 0.99 1.32 1.65 3.30 4.95 6.60 8-25
(M)
o.ooi 0.002 0.003 0.004 0.005 o.oi o-o15 0.02 0.025
± ± i i -E ± Jz ± ±
a Pooled results from three separate experiments.
Abbreviations : MMS, methyl methanesulphonate; phosphate.
TEM, triethylene melamine;
TMP, trimethyl-
466
SHORT COMMUNICATIONS
mutagens such as TEM or MMS are active in much lower concentrations than this in Drosophila. It would be expected from the figures in Table I that TMP would also be mutagenic at lower concentrations. The important point, however, is that it has already been shown that at doses down to o.oo2 M, TMP has a significant temporary sterilizing effect, whereas at o.ooi M no such sterilizing effect is observed 1. Furthermore it seems that this sterilizing effect is caused by killing initially meiotic stages and then premeiotic stages. One of the possible reasons for the increased mutagenic response at these low doses is that these cell stages which are most susceptible to mutation induction are also those most susceptible to killing. In order to test this hypothesis more directly, a further experiment was carried out. Adult male flies which had developed as larvae on media containing o.oo3, o.oo4 or o.oo 5 M TMP were collected and mated to Cy B I L virgins in the normal way. The time of regaining fertility was noted and they were then divided into two categories according to whether fertility was regained on days 3, 4, 5, 6 or 12, 13 and 14. There were flies which regained fertility on intermediate days, but these were not tested. The results from this experiment are shown in Table II. At each dose the total frequency of lethal mutations appearing from those males which recovered fertility late is lower than from those which regained fertility early. From each male which regained fertility up to ten gametes were tested and the percent lethal sperm from those males which had at least one lethal is recorded. This is a measure of the incidence and size of clusters of lethals and is indicative of the stage at which the mutations were induced. Those mutations which are induced in premeiotic stages are expected to give rise to clusters, those induced in postmeiotic stages are not. The differences between the two categories of flies are not consistent, but they do indicate a fairly high and increasing size of clusters in the early fertility flies, suggesting that an important proportion of their mutations are induced in premeiotic stages. The large cluster sizes indicated among the late fertility flies at o.oo5 M may indicate a relatively high rate of mutation induction amongst primary spermatogonia which survive the toxic effects of the TMP. Partly in order to check the long-term consequences of these findings, two populations of lethal flies were established on o.oo3 M and o.oi M TMP. These doses were chosen in order that populations of early fertile flies and late fertile flies could be compared. The expectation was that in the population exposed to o.oo3 M mainly early fertile flies would be tested and the resulting lethal frequency in this population would be as high or nearly so as that in the population exposed to o.oi M in which only late fertile flies would be exposed. However, when these populations were tested 18 months after their establishment, it was found that both populations were entirely composed of early fertile flies. In other words, the temporary sterilizing phenomenon at o.oi M TMP had completely disappeared, presumably as the result of selection favouring flies which regained fertility earlier. A sub-population from this was established in which the dose of TMP was slowly and progressively increased from o.oi M and this was tested for both sterilizing effects and the accumulation of recessive lethals. The dosage of TMP to larvae in this population has now reached o.o18 M but no temporary sterility has reappeared. The frequency of lethals in this population has rapidly increased. The mutation frequencies in these populations, and for comparison a population kept on a much higher dose s, are shown in Table III.
467
SHORT COMMUNICATIONS TABLE II
2nd CHROMOSOME RECESSIVE LETHALS FOUND IN SPERM OF DROSOPHILA WHICH HAD DEVELOPED AS LARVAE ON MEDIA CONTAINING VARYING CONCENTRATIONS OF TRIMETHYLPHOSPHATE Regained fertility days 3, 4, 5, 6
Number of lethals/number of tests Number of males with lethals/number of males tested Average cluster size Number of lethals/number of tests Number of males with lethals/number of males tested Average cluster size Number of lethals/number of tests Number of males with lethals/number of males tested Average cluster size
Regained fertility days i 2 , i 3 , 14
T M P concentration 0.003 l~I 44/847 = 5.4% 3/187 = 1.6%a 12/9o = 13.3% 3/3 ° = lO°/0
3"7
I
T~/[P concentration o.oo4 iVI 33/656 = 5.oO~ 4/I66 = 2.4(}.0 9/7 ° = 12.9% 4/29 ~ I3.8(}'o
3-7
I
T M P concentration o.oo5 M 19/111 = 17.1/o o/ 7/14 ° = 5.o% b 5/3 ° = I6.70~, 5 / 2 2 ~ 22.7(}~
3.8
1.4
a Significant5/0 o., level. b Significant i O,jolevel. TABLE III FREQUENCIES OF 2nd CHROMOSOME RECESSIVE LETHALS IN POPULATION'SEXPOSED TO VARYING CONCENTRATIONS OF TMP FOR 18 MONTHS OR 24 MONTHS Population treatment
N u m b e r o/ tests
N u m b e r of lethals
% Lethals
o.oo 3 M for 18 months o.oi M for 18 months o.oi M for 18 months increased to o.o12 M over 2 months o.oi M for 18 months increased to O.Ol5 M over 5 months o.oi M for 18 months increased to o.o18 M over 6 months 0.04 M for 24 months
241 223 iio lO8 119 126
82 155 74 89 99 lO5
34.0 69.5 67.3 82.4 83.2 83.3
~= 3.0 ~ 3.0 ± 3.6 ± 3.7 ± 3.3 ± 3.3
T h e r e l a t i v e l y h i g h levels of m u t a t i o n s in t h e p o p u l a t i o n s o n l o w e r d o s e s a r e i m m e d i a t e l y a p p a r e n t . A l t h o u g h t h e d i f f e r e n t l e n g t h s of t r e a t m e n t s a n d sizes of t h e p o p u l a t i o n s r e n d e r m o r e p r e c i s e c o m p a r i s o n s i m p o s s i b l e a t t h e p r e s e n t t i m e , it is c l e a r t h a t d o s e s of 0 . 0 I M u p t o 0.018 M a r e r e s u l t i n g in a t l e a s t as m a n y m u t a t i o n s as d o s e s of 0.04 M , w h e n t h e t r e a t m e n t is o v e r e x t e n d e d p e r i o d s of t i m e . This paper has shown that TMP may be a relatively greater genetic hazard at l o w d o s e s t h a n it is a t h i g h e r o n e s , p r o b a b l y b e c a u s e t h e killing of s e n s i t i v e cells is m u c h less a t l o w e r doses. I t h a s also s h o w n t h a t s e l e c t i o n p r e s s u r e m a y a l l o w t h e d e v e l o p m e n t of r e s i s t a n c e t o t h i s killing w h i l e still a l l o w i n g m u t a t i o n i n d u c t i o n a n d t h e r e b y m a r k e d l y i n c r e a s e t h e p o t e n t i a l g e n e t i c t h r e a t . F u r t h e r w o r k is r e q u i r e d t o i n v e s t i g a t e h o w c o m m o n t h i s s o r t of p h e n o m e n o n m a y b e a m o n g o t h e r e n v i r o n m e n t a l m u t a g e n s . S o m e of t h i s w o r k is a l r e a d y u n d e r w a y in o u r l a b o r a t o r y . I w i s h t o t h a n k Ms. SUZANNE WRIGHT for t e c h n i c a l a s s i s t a n c e a n d Mr. PETER HANNA for m a n y f r u i t f u l d i s c u s s i o n s .
468 Department of Genetics, Monash University, Clayton, Victoria 3168 (Australia) Present address: Department of Genetics, Adelaide University, Adelaide, S.A. 5ooz (Australia)
SHORT COMMUNICATIONS
K. F. DYER
I DYER, K. F., AND P. J. HANNA, Mutagenic and antifertility activity of t r i m e t h y l p h o s p h a t e hi Drosophila melanogaster, Mutation Res., 16 (1972) 327-331. 2 HANNA, P. J., AND K. F. DYER, Mutagenicity of o r g a n o p h o s p h o r u s c o m p o u n d s in bacteria and Drosophila, Mutation Res., 28 (1975) 405-42o.
Received February I8th, 1975
Amsterdam--Printed
in T h e N e t h e r l a n d s
465
Mutagenic activity of trimethylphosphate in Drosophila at low doses One of the characteristics of environmental mutagens is that they are normally present in very low doses. This is a problem for those wishing to assess their likely genetic hazards because testing in the laboratory has to be done at relatively high doses. However, it has been assumed that at low doses the mutagens pose their lowest threat and therefore compounds found to be of low mutagenic activity in the laboratory would not appear to be important environmental hazards TMP is known to be mutagenic in a variety of systems under various conditions, although normally of low activity 2. Evidence has now been obtained which suggests that, at least in Drosophila, TMP m a y be a more effective mutagen at low doses than at higher ones. This evidence comes partly from single generation experiments, in which larvae were allowed to develop in media containing TMP and the emerging adults tested for the induction of mutations. It also comes from population experiments in which initially identical populations of Drosophila were kept for periods of up to two years on media containing various doses of TMP. The materials and methods from these experiments are essentially those described in detail in our earlier papers1, ~. The results from the first group of experiments are presented in Table I. The salient feature of this table is the relatively high frequency of lethals at the lower doses (o.oo5 M and below) compared to those at the higher doses (o.oi M to o.o25 M). The mutation frequencies at the five lowest doses are all significantly higher than the figure predicted from a linear regression calculated for four higher doses and the mutation induction by o.oo4 M is significantly higher than o.oi M or o.o15 M. The overall lethal frequency at these five lower doses is 5.4% which is identical to that at the higher doses. Of course a concentration of o.ooi M TMP is still rather high and a number of TABLE
I
2 n d CHROMOSOME RECESSIVE LETHALS POUND IN SPERM OF DROSOPHILA WHICH HAD DEVELOPED AS LARVAE ON MEDIA CONTAINING VARYING CONCENTRATIONS OF TRIMETHYLPHOSPHATE
Concentration of T M P in feed
Number of tests
Number of lethals
Control~
11°5
4
389 747 336 4°2 595 521 523 848 186
22 22 19 31 4° 19 24 57 14
% Lethals
Linear regression of lethal frequencies based on control and values from highest four concentrations ( % )
0-4 i
0.6
o.oi
5.7 3.o 5-6 7.7 6-7 3.6 4.6 6.7 7.5
1.2 0.6 1.3 1.3 i.o 0.8 0.9 0.9 1.9
0.33 0.66 0.99 1.32 1.65 3.30 4.95 6.60 8-25
(M)
o.ooi 0.002 0.003 0.004 0.005 o.oi o-o15 0.02 0.025
± ± i i -E ± Jz ± ±
a Pooled results from three separate experiments.
Abbreviations : MMS, methyl methanesulphonate; phosphate.
TEM, triethylene melamine;
TMP, trimethyl-
466
SHORT COMMUNICATIONS
mutagens such as TEM or MMS are active in much lower concentrations than this in Drosophila. It would be expected from the figures in Table I that TMP would also be mutagenic at lower concentrations. The important point, however, is that it has already been shown that at doses down to o.oo2 M, TMP has a significant temporary sterilizing effect, whereas at o.ooi M no such sterilizing effect is observed 1. Furthermore it seems that this sterilizing effect is caused by killing initially meiotic stages and then premeiotic stages. One of the possible reasons for the increased mutagenic response at these low doses is that these cell stages which are most susceptible to mutation induction are also those most susceptible to killing. In order to test this hypothesis more directly, a further experiment was carried out. Adult male flies which had developed as larvae on media containing o.oo3, o.oo4 or o.oo 5 M TMP were collected and mated to Cy B I L virgins in the normal way. The time of regaining fertility was noted and they were then divided into two categories according to whether fertility was regained on days 3, 4, 5, 6 or 12, 13 and 14. There were flies which regained fertility on intermediate days, but these were not tested. The results from this experiment are shown in Table II. At each dose the total frequency of lethal mutations appearing from those males which recovered fertility late is lower than from those which regained fertility early. From each male which regained fertility up to ten gametes were tested and the percent lethal sperm from those males which had at least one lethal is recorded. This is a measure of the incidence and size of clusters of lethals and is indicative of the stage at which the mutations were induced. Those mutations which are induced in premeiotic stages are expected to give rise to clusters, those induced in postmeiotic stages are not. The differences between the two categories of flies are not consistent, but they do indicate a fairly high and increasing size of clusters in the early fertility flies, suggesting that an important proportion of their mutations are induced in premeiotic stages. The large cluster sizes indicated among the late fertility flies at o.oo5 M may indicate a relatively high rate of mutation induction amongst primary spermatogonia which survive the toxic effects of the TMP. Partly in order to check the long-term consequences of these findings, two populations of lethal flies were established on o.oo3 M and o.oi M TMP. These doses were chosen in order that populations of early fertile flies and late fertile flies could be compared. The expectation was that in the population exposed to o.oo3 M mainly early fertile flies would be tested and the resulting lethal frequency in this population would be as high or nearly so as that in the population exposed to o.oi M in which only late fertile flies would be exposed. However, when these populations were tested 18 months after their establishment, it was found that both populations were entirely composed of early fertile flies. In other words, the temporary sterilizing phenomenon at o.oi M TMP had completely disappeared, presumably as the result of selection favouring flies which regained fertility earlier. A sub-population from this was established in which the dose of TMP was slowly and progressively increased from o.oi M and this was tested for both sterilizing effects and the accumulation of recessive lethals. The dosage of TMP to larvae in this population has now reached o.o18 M but no temporary sterility has reappeared. The frequency of lethals in this population has rapidly increased. The mutation frequencies in these populations, and for comparison a population kept on a much higher dose s, are shown in Table III.
467
SHORT COMMUNICATIONS TABLE II
2nd CHROMOSOME RECESSIVE LETHALS FOUND IN SPERM OF DROSOPHILA WHICH HAD DEVELOPED AS LARVAE ON MEDIA CONTAINING VARYING CONCENTRATIONS OF TRIMETHYLPHOSPHATE Regained fertility days 3, 4, 5, 6
Number of lethals/number of tests Number of males with lethals/number of males tested Average cluster size Number of lethals/number of tests Number of males with lethals/number of males tested Average cluster size Number of lethals/number of tests Number of males with lethals/number of males tested Average cluster size
Regained fertility days i 2 , i 3 , 14
T M P concentration 0.003 l~I 44/847 = 5.4% 3/187 = 1.6%a 12/9o = 13.3% 3/3 ° = lO°/0
3"7
I
T~/[P concentration o.oo4 iVI 33/656 = 5.oO~ 4/I66 = 2.4(}.0 9/7 ° = 12.9% 4/29 ~ I3.8(}'o
3-7
I
T M P concentration o.oo5 M 19/111 = 17.1/o o/ 7/14 ° = 5.o% b 5/3 ° = I6.70~, 5 / 2 2 ~ 22.7(}~
3.8
1.4
a Significant5/0 o., level. b Significant i O,jolevel. TABLE III FREQUENCIES OF 2nd CHROMOSOME RECESSIVE LETHALS IN POPULATION'SEXPOSED TO VARYING CONCENTRATIONS OF TMP FOR 18 MONTHS OR 24 MONTHS Population treatment
N u m b e r o/ tests
N u m b e r of lethals
% Lethals
o.oo 3 M for 18 months o.oi M for 18 months o.oi M for 18 months increased to o.o12 M over 2 months o.oi M for 18 months increased to O.Ol5 M over 5 months o.oi M for 18 months increased to o.o18 M over 6 months 0.04 M for 24 months
241 223 iio lO8 119 126
82 155 74 89 99 lO5
34.0 69.5 67.3 82.4 83.2 83.3
~= 3.0 ~ 3.0 ± 3.6 ± 3.7 ± 3.3 ± 3.3
T h e r e l a t i v e l y h i g h levels of m u t a t i o n s in t h e p o p u l a t i o n s o n l o w e r d o s e s a r e i m m e d i a t e l y a p p a r e n t . A l t h o u g h t h e d i f f e r e n t l e n g t h s of t r e a t m e n t s a n d sizes of t h e p o p u l a t i o n s r e n d e r m o r e p r e c i s e c o m p a r i s o n s i m p o s s i b l e a t t h e p r e s e n t t i m e , it is c l e a r t h a t d o s e s of 0 . 0 I M u p t o 0.018 M a r e r e s u l t i n g in a t l e a s t as m a n y m u t a t i o n s as d o s e s of 0.04 M , w h e n t h e t r e a t m e n t is o v e r e x t e n d e d p e r i o d s of t i m e . This paper has shown that TMP may be a relatively greater genetic hazard at l o w d o s e s t h a n it is a t h i g h e r o n e s , p r o b a b l y b e c a u s e t h e killing of s e n s i t i v e cells is m u c h less a t l o w e r doses. I t h a s also s h o w n t h a t s e l e c t i o n p r e s s u r e m a y a l l o w t h e d e v e l o p m e n t of r e s i s t a n c e t o t h i s killing w h i l e still a l l o w i n g m u t a t i o n i n d u c t i o n a n d t h e r e b y m a r k e d l y i n c r e a s e t h e p o t e n t i a l g e n e t i c t h r e a t . F u r t h e r w o r k is r e q u i r e d t o i n v e s t i g a t e h o w c o m m o n t h i s s o r t of p h e n o m e n o n m a y b e a m o n g o t h e r e n v i r o n m e n t a l m u t a g e n s . S o m e of t h i s w o r k is a l r e a d y u n d e r w a y in o u r l a b o r a t o r y . I w i s h t o t h a n k Ms. SUZANNE WRIGHT for t e c h n i c a l a s s i s t a n c e a n d Mr. PETER HANNA for m a n y f r u i t f u l d i s c u s s i o n s .
468 Department of Genetics, Monash University, Clayton, Victoria 3168 (Australia) Present address: Department of Genetics, Adelaide University, Adelaide, S.A. 5ooz (Australia)
SHORT COMMUNICATIONS
K. F. DYER
I DYER, K. F., AND P. J. HANNA, Mutagenic and antifertility activity of t r i m e t h y l p h o s p h a t e hi Drosophila melanogaster, Mutation Res., 16 (1972) 327-331. 2 HANNA, P. J., AND K. F. DYER, Mutagenicity of o r g a n o p h o s p h o r u s c o m p o u n d s in bacteria and Drosophila, Mutation Res., 28 (1975) 405-42o.
Received February I8th, 1975
