(HESLOT and FERRARY, 1958; EHRENBERG, 1960 a). The high mutagenic efficiency of EMS was not unexpected since previous experiments had shown that ...
HERE 56-22
ON THE MUTAGENIC ACTION OF ALKANESULFONIC ESTERS IN BARLEY By L . EHRENBERG, U . LUNDQVIST, S . OSTERMAN and B . SPAR RM A N INSTITUTE OF BIOCHEMISTRY, UNIVERSITY OF STOCKHOLM, AND INSTITUTE OF GENETICS, UNIVERSITY OF LUND, SWEDEN
(Received September 7th, 1966)
-
INTRODUCTION
I
T was early found that ethyl methanesulfonate (EMS) is a much more efficient mutagen in higher plants than, e.g., ionizing radiations (HESLOT and FERRARY, 1958; EHRENBERG, 1960 a ) . The high mutagenic efficiency of EMS was not unexpected since previous experiments had shown that monomers of ethylene oxide and ethyleneimine were able to provoke much higher mutation rates than the strongly cytotoxic difunctional and trifunclional derivatives, respectively, of these compounds (EHRENBERG and GUSTAFSSON, 1957; EHRENBERG, 1960 a ) , and since the mutagenically most efficient difunctional alkylating agent so far found was the Myleran [ bis (niethanesulfonyloxy)butane] (EHRENBERG, 1960 a) which may be described formally as a double EMS molecule. The present paper will describe a few experiments from the years 1956-64, performed in order to characterize the mutagenic and other biological effects of the treatment of barley seeds with EMS and some homologues and isomers of the alkyl alkanesulfonate series. The experiments were designed and performed within the frame of a mutation breeding programme in barley in cooperation with A. GUSTAFSSON, and certain results with respect to viable mutations have been published elsewhere (EHRENBERG ef al., 1961, 1966). Since pairs of homologues and isomers of the investigated compounds sometimes reveal striking differences in the pattern of biological effects, a preliminary effort to understand these differences was made by the determination, for certain compounds, of the hydrolysis rates and the rates of reaction with the hydroxyl (OH-) and thiosulfate (S208*-)ions. The biological action of the alkanesulfonic esters has chiefly to be ascribed to alkylation of nucleophilic oxygen, nitrogen, and sulfur, the reactivity of which mostly increases in the order given (cf. ROSS, 1962). Hydroxyl and thiosulfate were therefore chosen to represent a relatively
278
EHRENBERG, LUNDQVIST, OSTERMAN AND SPARRMAN
slow alkylation of oxygen and a relatively fast alkylation of sulfur. (Details and extensions of this work will be presented in another context.)
Material and methods Seeds of the two-rowed barley varieties Bonus and Fonia were used in earlier and later experiments, respectively. In general these two closely related varieties react in the same way to the chemicals, and therefore no effort will be done to analyse the varietal influence on the sensitivity to the treatments. The compounds listed in Table 1, which also gives the abbreviated names used in the following, were tested. In the beginning of the experimental period all compounds were synthesized according to methods Later, several of suggested and developed by Dr C. A. WACHTMEISTER. the compounds have become commercially available. The monoesters, most of which are previously known, were prepared from the appropriate alkyl bromide or alkyl iodide with silver methanesulfonate or silver ethanesulfonate in acetonitrile (method A; EMMONS 1953); from the alcohol with methanesulfonyl chloride in and FERRIS, and MOSHER, 1954 a, b ) , from the pyridine (method B or C; WILLIAMS alcohol with methanesulfonic anhydride alone (method D; WACHTMEISTER et al., 1966) or in the presence of 2,6-dimethylpyridine (method et al., 1966). The diethyl propane-l,3-disulfonate E ; WACHTMEISTER which has been described in a n impure state (LICHTENBERGER and TRITSCH, 1961) has now been prepared (OSTERMAN and WACHTMEISTER, 1966) from 1,3-dibromopropane via propane-1,3-dithiol which was oxidized to the disulfonic acid and converted to the diethyl ester through method A. Certain experiments with these compounds have been published by other authors (MOUTSCHEN, 1964; RAO and NATARAJAN,1965; MARIANI and SPARRMAN, 1965). The standard procedure consisted in the treatment of initially dry seeds for 24 h at 20' C with solutions of the compounds, the volume to weight ratio of solution to seeds being 10 ml/g. Aeration and homogeneity of treatment (discussed by ZACHARIAS and EHRENBERG, 1962) were secured by shaking the flasks containing the seeds during the whole period of treatment. The rate of the shaking machine used was predetermined as the one permitting a normal germination of submersed seeds. In experimental series comprising shortlived esters, such as iPMS or sBMS, shorter treatment times were applied, mostly 3-5 h. In certain instances presoaked seeds were treated.
sBAIS
iBhlS tBhlS XeolIS AhIS ClEMS blOE3IS
blgleran
sec-butyl methanesulfonate isobutyl methanesulfonate tert-butyl methanesulfonate neopentyl methanesulfonate ally1 methanesulfonate 2-chloroethyl methanesulfonate 2-methoxyethgl methanesulfonate 2. Di- and trifunctional: 1,4-bis(methanesulfonyloxS)butane
DEPD 111
BMS
n-butyl methanesulfonate
diethyl 1,3-propanedisulfonate l,l,l-tris(methanesu1fonyloxymethy1)propane
hlES EMS EES PBIS iPlIS
b lh l S
Abbreviated name
1. Monofunctional: methyl methanesulfonate methyl ethanesulfonate ethyl methanesulfonate ethyl ethanesulfonate n-propyl methanesulfonate isopropyl methanesulfonate
Chemical name
C,H,0S0,CH,CH,CH,S0,0CgH5 /CH, - 0 - SOZCH, CH3 - CH, - C - CH, - 0 - S0,CH.q \CH, - 0 - SO,CH,
CH,S0,0CH,CH2CH,CH,0SOzCH3
A
- CH, - CH = CH, - CH,CH,Cl - CH,CH,OCH,
Commercial A
B B
B
C
B
C
B, A
13 E
A A, D A
A, D
Method (cf. text)
- CH, - C(CH&
- CH,CH’CH3 \,C H,
- CH,CH,CH,CH,
- CH/CH3 \CH,
- CH, - CH, - CH,CH, - CHSCH, - CH,CH,CH,
R=
Formula R’S0,OR
(“Busulphan” from Burrough Wellcome & Co) cf. text WACHTMEISTER et al., 1963
PATTISON and ~ I I L L I N G T O N , 1956 WACHTMEISTER, to be published ROBERTSOS and LAUCHTON, 1957 EnrhioNs and FERRIS, 1953 Ross and DAVIS,1957 PATTISON and hfILLINGTOS, 1936
WILLIAMS and MOSHER, 1954
EnrMons and FERRIS,1953 CARIUS,1870 EMMONS and FERRIS,1953 KURBATOW, 1874 WILLIAMSand n l O S H E R , 1954 WACHTHEISTER, PRING, OSTERMANand EHREXBERG, 1966 WILLIAMS and NOSHER, 1954
Reference
Synthesis
TABLE 1. Formulae, abbreviated names and synthesis o f the compounds studied.
280
EHRENBERG. LUNDOVIST. OSTERMAN AND SPARRMAN
In order to avoid errors of the concentrations of the solutions, dilution series were never used, but all solutions were prepared directly by mixing a (freshly made) stock solution with water in different proportions. After treatment the seeds were rinsed for around one minute in running tap water, and were then sown in the field, mostly within a few hours after the end of the treatment. - As indicated in experiments where the treated seeds were stored for some time before sowing, further reactions of absorbed substance may produce appreciable aftereffects, especially with slowly reacting substances (cf. FROESE-GERTZEN et al., 1964; GICHNER and EHRENBERG, 1966). According to WALLES (1966) the washing out of inreacted EMS is so slow (50 per cent/h) that an effective liberation of the seeds from unreacted alkylating substance can hardly be done without profound physiological changes during the washing period. Since experiments with Arabidopsis and barley (WALLESand A H N S T R ~ M1965; , WALLES, 1966) show that presoaked seeds take up the substance more effectively than dry seeds which exhibit a “lag period” in this respect, it is clear that experiments aiming at quantitative comparisons of alkylating agents with respect to their effects in seeds, should be planned as follows: 1-2 h presoaking, followed by 2-5 h treatment, and, then, washing for at least 8 h, all treatments calculated for 18-22’ C (cf. WALLES, 1966). Smaller seeds are advantageous in this respect because of shorter diffusion ranges. Nothing is known about the diffusion of compounds from seeds to the soil, although this process is presumably enhanced by rain. The variations obtained between “simultaneous” replications and between identical experiments run in different years may therefore depend on many factors, such as (a) a slight variation of the time between end of treatment and sowing; (b) different conditions such as temperature and soil moisture for the leakage out from the seeds of unreacted substance; (c) different conditions for later consequences of alkylation of the types discussed by GICHNERand EHRENBERG (1.c.). In the field material the following effects were determined: germination, survival at maturity, and sterility (for methods, see NYBOMet al., 1953; GUSTAFSSON, 1940). The frequencies of chlorophyll mutations were determined in the following generation, by analysis of the spike progenies in the greenhouse (cf. NYBOM, 1954). Mostly samples of 200 seeds were sown giving around 160 plants in the control and fully surviving materials. The mutation rates were determined from analyses of 200-600 spike progenies (in critical cases
MUTAGENIC ACTION O F ALKANESULFONIC ESTERS
28 1
the numbers are given in the Figure legends) except in cases of low survival. The sterilities were determined from 20 plants corresponding to about 100 spikes. The hydrolysis rates were determined by titration of the acid formed in 0.05 M solutions thermostatted at temperatures 20-37' C. The reactions with OH- were studied at the same temperatures, using about 0.4 M NaOH and 0.05 M alkyl alkanesulfonate. The OH- consumed was determined by titration of the remaining OH-. The reactions with S,O,'were followed by iodometric titration. Details of this work, extended to other compounds, will be given in another context.
RESULTS The reaction rate constants are summarized in Table 2 and the biological data in Tables 3 and 4. The biological effects are also exemplified in a few diagrams (Figs. 1-1 1). Comparing experiments in different years with one and the same compound, the effects are astonishingly reproducible in view of the variability of the conditions as indicated in the preceding section. This fact which is in contrast to the great variation in corresponding experiments with ethyleneimine (EHRENBERG et al., unpublished; cf. DUMANOVIC et al., 1966) increases the usefulness of alkanesulfonic esters in plant breeding work. With few exceptions the survival and sterility and also the mutation rates are typical multihit functions of the concentrations (cf. Figs. 1-1 1), and the biological effects should therefore preferently be described in terms of the whole curves, determined under precise conditions. In the present material a rough characterization of the biological reaction pattern is obtained by giving, in Table 3, the (interpolated) concentrations producing 50 per cent survival (CL,,) , 50 per cent sterility (CS,,) and by giving the highest mutation rate obtained (mutagenic efficiency, cf. EHRENBERG, 1960 a, b) . The mutagenic efficiency relative to other effects may also be expressed by the mutation rate, e.g., a t CL,, or CS,,, although in the present material values rather close to the maximum mutation rate are obtained. The multihit shape of the mutation curve is expressed by giving the (interpolated) mutation rate at 1/5 of the concentration giving the maximum mutation rate. In case of a linear concentration dependence 1/5 of the maximum mutation rate should then be obtained. Table 3 gives also the sterilities at CL,,.
282
EHRENBERG, LUNDQVIST, OSTERMAN AND SPARRMAN
TABLE 2 . Rate constants for reactions of certain alkyl alkanesulfonates with water (h-I), O H - ( l . mole-' h-') and S,O,"-(l- mole-' h-'),
-
-
Substance
MMS
I R:iyI HZO OH-
~
s,o,2EMS
HZO OHSz0,2-
iPMS
H,O OH-
BkIS
HZO OH-
szo,2-
k (25" C)
k (20°C)
0.009~0.001 -0.9 34.6f 1.1 0.0072~0.0005 0.12f0.03 1.3fO.l
i
I
H2O
OHs20,2-
DEPD
H2O OHs20,2-
k (37" C)
0.017f 0.002 1.7 51.2f2.2
0.072 f0.004
0.0143f 0.0006 0.23f0.02 1.9f 0.1
0.060& 0.003 0.93f0.14 8.030.4
0.0102f 0.0005 0.13f0.02 1.5f 0.2
0.035&0.003 0.4950.09 5.050.3
125h20
0.38 0.6430.05 0.00533 0.0003 0.059f0.008 1 . 2 3 0.1
0.00043 -
MOE hIS
I
--
(57f2). 10-5 0.015f0.002 0.250&0.013
( 2 6 f i ) . 10-5 0.010~0.001 0.151*0.007
-
0.07 0.8 9
0.002 0.015 (290f20). 0.042% 0.004 0.9fO.1
-
0.39; 2nd step-0.1 3; 2nd step- 1 40
With reference to biological as well as chemical data we may point out certain characteristics of the individual compounds: MMS and MES. These methylating agents are appreciably more toxic than the higher homologues of the series, and the killing of seeds already at low concentrations (CL,, 0.5-1 mmole/l) is probably the factor limiting the maximum mutation rates. (Cf. Fig. 1). Comparing the rate constants for the reactions at 20' C of MMS and EMS with H,O, OH- and S,O,'- (cf. Table 2), we obtain the relationship log k M M S z l . 5 9 106 kEMs+ 1.37;
MUTAGENIC ACTION O F ALKANESULFONIC ESTERS
283
(cf. SWAINand SCOTT,1953; HUDSONand HARPER, 1958) which means that the faster the reactions are, i.e. the more nucleophilic is the other reactant, the greater beconies kMMS relative to kEMs. The above expression examplifies the fact that whereas MMS reacts by a pure S N ~ mechanism, the reaction type of E M S is partly s N 1 with the consequence that E M S is less dependent on the nucleophilic character of the attacked atom (cf. ROSS, 1962). According to the given relationship and Table 2 the ratio kMMs/kEMs for negative oxygen (as in OH-) is about 7.5 and for negative sulfur (as in S,O,'-) about 27. Although a quantitative comparison of biological data is not strictly allowed, because possible aftereffects were not controlled (cf. p. 280), and although differences in reaction mechanisms may occur (BROOKES and LAWLEY, 1963; ALEXANDER, P., cf. ROSS,I.c., p. 81), such a comparison may be used as a preliminary indication of reaction types involved. Extracting from available data (from 1961-62, when parallel series were sown) the concentrations of MMS and E M S giving 50 per cent survival (CL5J, 10 per cent mutation (CM,,), and 10 per cent sterility (CS,,), we obtain:
1961
hIhIS I 200 13.5 14
115 178 75 80 38 16 u 1008
N
5 50 0.4 ~ 4 0 0.4 -10 -1 1.5-3 N1 2 5 5.4 4 130 9 >200 13 9 5 12
low ster. no ster. 5.5 >200 18" low ster.
-
100 180 58-72 72 33 22 85
29 47 20 d20 30 19 22
145 180 1208 >100 38 >22
0
