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Mutation Research, 37 (1976) 193--200 © Elsevier/North-Holland Biomedical Press

EFFECT OF DIMETHYL SULFOXIDE (DMSO) ON RADIATION-INDUCED HETEROALLELIC REVERSION IN DIPLOID YEAST

D.R. SINGH, J.M. MAHAJAN and D. KRISHNAN

Division of Radiological Protection, Bhabha Atomic Research Centre, Trombay, Bombay-400 085 (India)

{Received November 18th, 1975) {Revision received May 13th, 1976) (Accepted June 8th, 1976)

Summary

Dimethyl sulfoxide has cryoprotective and radioprotective properties. It is also an efficient scavenger of radicals produced by radiolysis of water. Gamma- induced reversions of diploid yeast in the presence of this chemical during irra- diation have been studied. The dose-modifying factor was in the same range as for survival. When the yeast was irradiated in the frozen state the observed protection by DMSO disappeared. The results are discussed in terms of direct and indirect actions of radiations and the radical-scavenging ability of this chemical.

Introduction

Dimethyl sulfoxide (DMSO) is a well-known chemical protector against radiation damage. The protective effect of the chemical is known for bacteria [3,5] as well as for mammalian cells grown in vitro [1,4,15] and for organisms [2,6,8]. Work reported in the literature shows the modifying effect mainly with respect to survival of the cells. Not much work has been done on the protective ability of DMSO on mutations or DNA damage. In this note the protection of radiation-induced reversion frequency of S a c c h a r o m y c e s cerevi- siae, BZ34 by DMSO is reported.

Freezing below --30°C eliminates part or all indirect radical effect within a cell [ 13,14]. The experiment has been repeated on cells in the frozen condition to see whether DMSO can protect the direct damage due to radiation.

Materials and methods

The genotype of the yeast and the materials used in this laboratory are mentioned elsewhere [ 10]. The genotype of the yeast strain BZ34 is

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a + arg4-4 + thr l + trp5-48 + ura3his5-2 lys l -1 ade2-1 a pe t1 + arg4-17 + leu1-12 trp5-48 me t1 + his5-2 lys l -1 ade2-1

It has a non-complementary mutant allele in the argino succinase locus and requires arginine in the medium for growth. Exposure to radiation can induce reversion to arginine independence by a process of intragenic recombination. These recombinants can be scored by plating the irradiated cells in a medium lacking arginine.

Media (1) YEPD liquid growth medium: 1% Oxoid yeast extract, 2% Difco

peptone, 2% dextrose. (2) YEPD solid growth medium: same as above but with 2% agar. (3) Arginineless solid medium: Difco yeast nitrogen base without amino acids, 6.4 g; adenine, uracil 20 mg; threonine 300 mg; dextrose 20 g; agar 20 g; amino acids (except arginine) histidine, leucine, lysine, methionine, t ryptophan 20 mg (all per litre).

The cells were grown in 20 ml YEPD liquid growth medium in a shaker at 30°C for two days. These cells were tested for spontaneous reversions and the one with minimal reversions (usually 5--10 revertants per million cells) was taken for experiments. The proportion of budding cells was usually only about 5%.

The toxicity of the chemical, DMSO, on the viability of the cells was tested for 4 h in water at room temperature in the concentration range 5 to 30% (v/v). No appreciable killing was seen up to 10% DMSO, and at 30% the survival was 50%. Therefore in most of the experiments 10% DMSO was used. In all the experiments on reversions, reversions were estimated for 106 surviving cells, as measured by colony formation on YEPD plates.

Colony growth was evident after 3 days incubation at 30°C for YEPD plates, and 5 days for arg- plates.

G a m m a irradiation The stationary phase cells, at the concentration of 1.0 × 106 per ml in

distilled water, were irradiated at various doses with a 6°Co gamma cell having a dose rate of 4.5 kR per min. After appropriate dilutions, 1 ml of the culture was plated on Arg- and YEPD plates. The YEPD medium was used to assay the surviving cells and arg- medium to detect the number of radiation-induced revertants.

Freez ing e x p e r i m e n t s The samples were slowly cooled by keeping them overnight at --10°C with

thick thermocole insulation. Just before irradiation the samples were dipped in liquid nitrogen with thermocole insulation and irradiated. The samples, which remained frozen during the short irradiation period, were quickly dipped into water kept at about 40 °C. Appropriate dilutions were plated on Arg- and YEPD plates.

The killing effect of the cooling process used on cells was negligible in both the controls (i.e. with or without DMSO) for the yeast strain used. If sudden cooling with liquid nitrogen took place, cells in water without DMSO were preferentially killed. However, when slow overnight cooling was done, as in

195

our experimental procedure, the cells survived. The slow cooling procedure also improved the survival of cells in 10% DMSO.

Results and discussion

The survival curve of yeast BZ34 exposed to 6°Co gamma rays is shown in Fig. 1. This was obtained by normalizing control plates to 100%. The survival experiment was repeated with 10% DMSO (v/v). The curve shows that 10% DMSO gave a DMF of 1.66 at 10% survival for survival of yeast which is in general agreement with published data [1,3]. It may appear from Fig. 1 that the presence of 10% DMSO produces a change in extrapolation number from 2 to 3. Detailed experiments were not undertaken to see whether this is true. Earlier works [1,3,15] have shown the effect of DMSO to be dose mod- ifying. Fig. 1 is used to establish that the survival of this strain of yeast is protected by 10% DMSO, and for the same reason DMF is estimated at the 10% survival level, rather than from the slopes.

The reversion frequency with 6°Co gamma rays with and without 10% DMSO is given in Fig. 2. It is seen that 10% DMSO also protected the yeast against radiation-induced reversions. A DMF of 1.8 was obtained. The gamma doses were kept low to prevent appreciable cell killing. Because of its high reaction rate with hydroxyl radicals (5.8 X 109 M -1 sec -1) [11], DMSO could protect indirect radiation damage by radical scavenging.

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G A M M A D O S E ( k r o d s )

Fig. 1. E f f e c t o f 10% D M S O on survival o f yeas t . (o) 10% DMSO; (e ) cont ro l .

196

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Freezing of the samples to liquid nitrogen temperature at which the free- radical mobilities are negligible, eliminates most of the indirect radiation damage. The extent of protection by DMSO for irradiations done at room temperature and in the frozen state is given in Fig. 3. In liquid nitrogen control samples, only doses up to 6 krad could be given because the samples started melting at higher doses. The reversion frequency when DMSO was

300

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F i g . 3. E f f e c t o f 1 0 % D M S O o n r e v e r s i o n f r e q u e n c y in y e a s t a t r o o m t e m p e r a t u r e a n d at l i q u i d n i t r o g e n t e m p e r a t u r e . ( o ) C o n t r o l ( r o o m t e m p e r a t u r e ) : (A) 1 0 % D M S O ( r o o m t e m p e r a t u r e ) ; (n ) 1 0 % D M S O (l iq . N 2 ) ; (o ) c o n t r o l ( l iq . N 2 ) .

197

present during irradiation at both these temperatures was the same. This in- dicates that DMSO can only protect against indirect radiation damage.

Wood [18] and Wood and Taylor [19] have studied the effect of phase- state changes, various freezing temperatures, dehydrating chemicals and cellular water content on the radiosensitivity of yeast. They found that when large cells such as yeast or mammalian cells are quickly frozen to temperatures down to --72°C cellular water probably freezes inside the cells before dehydration takes place. In such case, the radiosensitivity of the frozen cells is of the same order as at 0°C (i.e. in the liquid state). However, slow freezing occurs down to --33°C and probably the dehydration is complete during the freezing. They find that such cells (i.e. slowly cooled to - -33°C)are protected from radiation effects. In our experiments the overnight slow cooling process to --10°C probably dehydrated the cell, resulting in the protect ion observed when irradia- tion was subsequently done at liquid nitrogen temperature.

The direct and the indirect components of radiation damage are estimated using the formulae

slope of revers ion f r equency graph for f rozen samples pe rcen tage d i rec t ac t ion . . . . . . . . . . . . . . . . . . × 100 slope of revers ion f r e que nc y graph for r o o m t e m p e r a t u r e cont ro l s

percentage indirect action = 100 -- (percentage direct action) The values from Fig. 3 are 44 and 56% for direct and indirect action, respectively. These values are in general agreement with those reported in the literature [ 14,17 ].

Johansen and Howard-Flanders [7] observed a correlation between protec- tion of bacteria by various alcohols and their OH rate constants. Roots and Okada [12] found similar results with strand breaks in DNA. Sanner and Pihl [13] observed that enzyme inactivation follows the same pattern. A correlation was observed between hydroxyl scavenging and protection.

Experiments with DNA in vitro [11] suggest that DMSO reacts with primary radical species, especially OH. Making the assumption that the cellular target molecules and DMSO compete for hydroxyl radicals, one can estimate the reac- tion rate of cellular target molecules by using the relations

(DNA) + OH kDNA (reversions) (protector) + O H k_~ (scavenging of OH)

when the concentration of protector is such as to offer half the maximal possible protection by the scavenger

kDNA • (DNA) • (OH) = kp. (protector) • (OH)

Chapman et al. [4] , on the basis of survival of mammalian cells, arrived at a DMSO concentration of 0.12 M for half maximal protection.

The effect of various concentrations of DMSO ,on reversions in yeast is given in Fig. 4. Even though protection is still increasing at 30% (4 M) DMSO, the half maximal concentration will be around 1.2 M or higher. This large difference in concentration of the chemical at half maximal protect ion could be due to one or more of various factors such as differences in the two cell

198

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F i g . 4 . E f f e c t o f v a r i o u s c o n c e n t r a t i o n s o f D M S O o n g a m m a i n d u c e d r e v e r s i o n s in y e a s t a t r o o m t e m p e r a -

t u r e . (o ) C o n t r o l ; (n ) 5% D M S O ; (A) 1 0 % D M S O ; (~) 2 0 % D M S O ; (~') 3 0 % D M S O .

systems, the difference in the two biological end-points, the possible difference in permeability of the chemical etc. Further, it is also possible that the product kDNA X (DNA) in the above equation is much higher in yeast than in mam- malian cells. If this is so, then it will suggest that in yeast (at least in this strain) the number of radiation-sensitive reversion sites (on DNA) are quite high or the hydroxyl reaction Late constants of the sensitive sites are high, as compared with similar sites for killing in mammalian cells.

Sanner and Pihl [13] used ethanol or glycerol on Escherichia coil at 0°C and at liquid nitrogen temperatures. They found that ethanol gave appreciable protect ion at 0°C whereas protect ion in the frozen state was negligible. With glycerol, the relative protection at --196°C was even greater than that at 0°C. They concluded that ethanol protects primarily by decreasing indirect action of radiation, whereas protection by glycerol may possibly be due to its ability to dehydrate cells and replace bound water. This property of glycerol also makes it a cryoprotective agent. DMSO also has equally good cryoprotective properties. Manney et al. [9] found that t reatment with 6M glycerol protects haploid Saccharomyces cerevisiae against both low and high LET radiations. The DMF was of the order of 2. Wood [18] also found protection by 6.9 M glycerol for yeast. The concentrations that are necessary for radiation protec- tion by both these is also similar, and much higher than other chemical protec- tors [5]. Thus Vos and Kaalen [15] found increasing radioprotection even at 4 M (30%) DMSO in survival of mammalian cells. In Fig. 4 we see protection no t ye t complete at 30% DMSO for reversion in yeast.

These similarities indicate a similar mechanism of action as suggested by Ashwood-Smith [1] and Webb [16]. However, the inability of DMSO to protect yeast in the frozen state (Fig. 3) indicates a radical-scavenging radioprotective mechanism in addition to its cryoprotective property. The radioprotective ability of DMSO is probably a radical-scavenging ability as suggested by its very high reaction rate with hydroxyl radicals.

199

References

1 A s h w o o d - S m i t h , M.J. , R a d i o p r o t e c t i v e a n d c r y o p r o t e c t i v e p r o p e r t i e s o f DMSO in ce l lu la r sy s t ems , A n n . N.Y. A c a d . Sci . , 141 ( 1 9 6 7 ) 4 5 - - 6 2 .

2 B a r n e t t , B.M., R a d i o p r o t e c t i v e e f f e c t o f d i m e t h y l su l fox ide in D r o s o p h i l a m e l a n o g a s t e r , R a d i a t i o n Res . , 51 ( 1 9 7 2 ) 1 3 4 - - 1 4 1 .

3 Bridges , B.A. , C h e m i c a l p r o t e c t i o n o f p s e u d o m o n a s species aga ins t i on iz ing r a d i a t i o n , R a d i a t i o n Res . , 17 ( 1 9 6 2 ) 8 0 1 - - 8 0 8 .

4 C h a p m a n , J .D . , A.P. Reuver s , J . B o r s a a n d C.L. G r e e n s t o c k , C h e m i c a l r a d i o p r o t e c t i o n a n d r ad io - s ens i t i za t ion o f m a m m a l i a n cells g r o w i n g in v i t ro , R a d i a t i o n Res . , 56 ( 1 9 7 3 ) 2 9 1 - - 3 0 6 .

5 D e w e y , D.L . , E f f e c t o f p r o t e c t o r s o n Serra t ia m a r c e s c e n s d u r i n g a n a e r o b i c X - i r r a d i a t i o n , In t . J . R a d i a t i o n Biol . , 7 ( 1 9 6 3 ) 1 5 1 - - 1 5 4 .

6 H a g e m a n n , R . F . , T.C. Evans a n d E .F . Ri ley , M o d i f i c a t i o n of r a d i a t i o n e f f e c t o n the eye b y t o p i c a l a p p l i c a t i o n o f d i m e t h y l su l fox ide , R a d i a t i o n Res. , 4 4 ( 1 9 7 0 ) 3 6 8 - - 3 7 8 .

7 Johansen , I. and P. H o w a r d - F l a n d e r s , M a c r o m o l e c u l a r r e p a i r a n d f ree r ad i ca l s caveng ing in the p r o t e c - t i on o f b a c t e r i a aga in s t X-rays , R a d i a t i o n Res . , 24 ( 1 9 6 5 ) 1 8 4 - - 2 0 0 .

8 L a p p e n b u s c h , W.L. , O n the m e c h a n i s m of r a d i o p r o t e c t i v e a c t i o n o f d i m e t h y l su l fox ide , R a d i a t i o n . Res . , 4 6 ( 1 9 7 1 ) 2 7 9 - - 2 8 9 .

9 M a n n e y , T .R . , T. B rus t ad a n d C.A. Tob ia s , E f fec t s o f g lyce ro l a n d o f a n o x i a o n the r ad io sens i t i v i t y o f h a p l o i d y e a s t s to dense ly ion iz ing pa r t i c l e s , R a d i a t i o n . Res . , 1 8 ( 1 9 6 3 ) 3 7 4 - - 3 8 8 .

10 M u r t h y , M.S.S. , B.S. R a o , N.M.S. R e d d y , P. S u b r a h m a n y a m a n d U. M a d h v a n a t h , N o n - e q u i v a l e n c e o f Y E P D a n d s y n t h e t i c c o m p l e t e m e d i a in y e a s t r eve r s ion s tud ies , M u t a t i o n Res . , 27 ( 1 9 7 5 ) 2 1 9 - - 2 2 3 .

11 Rcuver s , A.P. , C.L. G r e e n s t o c k , J. B o r s a a n d J .D. C h a p m a n , S tud i e s o n the m e c h a n i s m of c h e m i c a l r a d i o p r o t e c t i o n b y d i m e t h y l su l fox ide , In t . J . R a d i a t i o n Biol . , 24 ( 1 9 7 3 ) 5 3 3 - - 5 3 6 .

12 R o o t s , R. a n d S. O k a d a , P r o t e c t i o n o f D N A m o l e c u l e s o f c u l t u r e d m a m m a l i a n cel ls f r o m r a d i a t i o n i n d u c e d single s t r a n d sciss ions b y va r i ous a l c o h o l s a n d SH c o m p o u n d s , In t . J . R a d i a t i o n Biol . , 21 ( 1 9 7 2 ) 3 2 9 - - 3 4 2 .

13 S a n n e r , T. a n d A. Pihl , S ign i f i cance a n d m e c h a n i s m of i n d i r e c t e f f e c t in bac t e r i a l cells. The re la t ive p r o t e c t i v e e f f ec t of a d d e d c o m p o u n d s in E. coli. i r r a d i a t e d in l iqu id a n d in f r o z e n suspens ion , Rad ia - t ion . Res . , 3 7 ( 1 9 6 9 ) 2 1 6 - - 2 2 7 .

14 Sha lek , B.J . , C.E. S m i t h a n d J. H u n t e r , T e m p e r a t u r e d e p e n d e n c e a n d e f f e c t o f g l y c e r o l o n the r ad ia - t i on sens i t iv i ty o f b a c t e r i o p h a g e E. co l i c o m p l e x e s r e l a t e d to d i r e c t a n d i n d i r e c t r a d i a t i o n e f fec t , R a d i a t i o n Rcs . , 31 ( 1 9 6 7 ) 4 6 7 - - 4 8 5 .

15 Vos , O. a n d M.C.A.C. Kaa l en , P r o t e c t i o n o f t issue c u l t u r e cells aga ins t i on i z ing r a d i a t i o n . II. The ac t i v i t y o f h y p o x i a , d i m e t h y l su l fox ide , d i m e t h y l su l fone , g l y c e r o l a n d c y s t e a m i n e a t r o o m t e m p e r a t u r e a n d a t - - 1 9 6 ° C , In t . J. R a d i a t i o n Biol . , 5 ( 1 9 6 2 ) 6 0 9 - - 6 2 1 .

16 Webb , R.B. G l y c e r o l a n d w a t e r e f f ec t s o n X-ray sens i t iv i ty in S t a p h y l o c o c c u s aureus , R a d i a t i o n Res . , 18 ( 1 9 6 3 ) 6 0 7 - - 6 1 9 .

17 W o o d , T .H. , Cel lu la r r a d i o b i o l o g y , A n n . Rev. Nuc . Sci. , 8 ( 1 9 5 8 ) 3 4 3 - - 3 8 6 . 18 W o o d , T .H. , I n h i b i t i o n o f cell d ivis ion, R a d i a t i o n Res. Supp l . , 1 ( 1 9 5 9 ) 3 3 2 - - 3 4 6 . 19 W o o d , T .H. a n d A.L. T a y l o r , X- ray i n a c t i v a t i o n o f y e a s t a t f r eez ing t e m p e r a t u r e s , R a d i a t i o n . Res . , 7

( 1 9 5 7 ) 99- - -106 .