181
Mutation Research, 51 ( 1 9 7 8 ) 1 8 1 - - 1 8 8 9 E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press
DETERMINATION OF R A D I A T I O N EQUIVALENCE OF E T H Y L M E T H A N E S U L P H O N A T E (EMS) F O R INDUCTION OF GENE CONVERSION IN DIPLOID YEAST
M.S.S. M U R T H Y a n d N. S A N K A R A N A R A Y A N A N
Division of Radiological Protection, Bhabha Atomic Research Centre, Bombay-400 085 (India) (Received 5 J u l y 1 9 7 7 ) (Revision received 14 F e b r u a r y 1 9 7 8 ) (Accepted 7 March 1978)
Summary A unit Rad-Equivalent Chemical (REC) has been suggested for purposes of quantitating the mutagenic hazards of chemicals. The usefulness of this approach is demonstrated by the establishment of a constant relationship between the forward mutation frequency and haploid genome size in various organisms for both radiation and chemical EMS. However, it is necessary to determine the radiation equivalence of chemicals in as many organisms and for as many end-points as possible. For end-points we are limited to forward mutations. Another relevant genetic end-point of interest in this regard is gene conversion which can also monitor any kind of DNA damage in a suitable diploid system. Hence, we have determined the REC value for EMS in diploid yeast with gene conversion as the end-point. This agrees well with the REC values estimated in a number of organisms with forward mutation as the end-point. This finding further underlines the generality of the REC concept.
Introduction To quantitate the mutagenic hazards of chemicals, it has been proposed to express the exposure to chemical mutagens in terms of ionizing-radiation dose required to produce an equivalent effect [3]. For this purpose a unit called the Rad-Equivalent Chemical (REC) has been suggested [3,6]. REC can be defined as the dose of chemical (or concentration multiplied by time) that produces the same amount of genetic damage as 1 rad of ionizing radiation under chronic Work carried out under IAEA Contract No. 1961/RB.
Abbreviations: EMS, ethyl methanesulphonate; REC, Rad-Equivalent Chemical.
182
irradiation conditions. For radiation it has been shown that a predictable relationship exists between the forward mutation frequency and the haploid genome size of almost all organisms including man [1]. Recently, Heddle and Athanasiou [5] have shown that a similar relationship between forward mutation frequency and the haploid genome size exists for the mutagenic chemical ethyl methanesulphonate (EMS). Notwithstanding the problems involved, such as chemical penetration, specificity, stage dependence etc. in a theoretical consideration of such a relationship, if it can be established for other chemicals also, then the REC value for a given chemical determined for one organism can be extrapolated to any other organism including man. Quantitation and extrapolation of mutagenic hazards of chemicals can then be placed on a more scientific basis. (Since somatic effects such as induction of malignancies are also of concern in environmental health protection, it is necessary to extend the REC concept to cancer as the end-point. Here additional problems such as organ-specificity, etc. have to be taken into account.) To generalize the validity of the REC concept, however, it is necessary to establish the equivalence n o t only in various organisms, b u t also for various genetic end-points. For end-points we are limited to forward mutations, since back mutations are of little value because of their specificity. Another relevant genetic end-point in this regard is gene conversion, which is k n o w n to be induced during the process of repair of DNA damage caused by chemical and physical agents [12] and results in the expression of recessive genes. It can be easily detected by plating treated cells on selective media [12]. Zimmermann [12] in 1971 reviewed the induction of gene conversion by a b o u t 25 mutagenic chemicals and found a correlation between the mutagenic and convertogenic abilities of these chemicals. Further, there was no mutagen specificity in the induction of gene conversion. Since then many workers have used this endpoint to detect the genotoxicity of a variety of chemical substances (for example see refs. in ref. 13). Recently we have reviewed the literature on nearly 150 chemicals, comprising drugs, pesticides, herbicides, industrial chemicals, food additives etc., which have been tested for their ability to induce gene conversion in diploid yeast and have confirmed Zimmermann's findings {unpublished data). In this paper we report the determination of the REC value for EMS to induce gene conversion in diploid yeast, and compare this with the REC values reported in the literature for various organisms with forward mutation as the end-point. Methods and materials Yeast strain
The diploid yeast strain BZ34 used in this investigation carries two noncomplementing heteroallelic mutants in the argino-succinase locus and hence is auxotrophic to arginine [4]. However, exposure to ionizing radiation [7] and mutagenic chemicals [9] induces arginine prototrophs in this strain by the mechanism of gene conversion. These can be scored by plating treated cells on medium from which arginine is omitted. Media
The cell line was maintained by routinely subculturing in yeast extract,
183
peptone and dextrose medium, at 30~ for 48 h in shaker incubators. Cell viability was determined by plating the cells on synthetic complete medium, and the convertants were detected by plating them on synthetic medium with only a trace a m o u n t (0.4 mg/1) of arginine. This medium will be referred to as arginine-omission medium. Details of the culture and media have been given elsewhere [ 7,8]. Chemical t r e a t m e n t The EMS used in this investigation was kindly provided by Dr. R. Fahrig of W. Germany. Stationary-phase cells were centrifuged and resuspended in 0.1 M potassium phosphate buffer (pH 7) to a concentration of 1 X 107 cells/ml. EMS was dissolved in phosphate buffer to the required concentrations. In each run, treatment was carried out in a given concentration but the duration of treatm e n t was varied from 30 min to 6 h. In a typical treatment protocol, 1 ml of cell suspension was added to 10 ml of EMS solution in a 100-ml flask and kept on a shaker water-bath at 30~ in the dark. At the end of the specified duration, 5 ml of the mixture was filtered on a Millipore filtration system, then resuspended in 5 ml sterile distilled water. Suitable dilutions were made to obtain 200--500 viable cells per ml, and 1 ml of each was plated on 4 arginineomission-medium plates for scoring arginine-independent convertants and on 4 complete medium plates for scoring viability. The plates were stored at 30~ in the dark for 3--5 days, after which the colonies were counted. Gene-conversion frequency was expressed as the ratio of the colony counts on the omission plates to those on the complete-medium plates multiplied by the dilution factor. A control flask was maintained to determine the background frequency. The same protocol was repeated for various concentrations of EMS from 0.0125 to 1% (v/v). Gamma-irradiation A 6~ cell with a dose rate of 2.6 krad/min was used for gammairradiation. Yeast cell suspension (5 • 106/ml) in buffer was exposed to various doses up to 10 krad. After irradiation, suitable dilutions were plated on omission as well as complete medium. Gene-conversion frequency was determined as mentioned in the previous paragraph.
Results Fig. 1 shows the dose--effect relationship for gamma-radiation. The geneconversion frequency increased linearly with dose. At all doses, the radiationinduced lethality was small. The slope of the line is 26.0 • 10 -2 conversion/106 survivors per rad. Earlier investigations had shown t h a t dose--effect linearity exists even at dose rates as low as a few rad/min [10]. While the events leading to biochemical lesions by ionizing radiations are extremely fast, this is not true for chemicals. They are dependent on various parameters such as penetration of the cell wall and the nuclear membrane by the chemical, reaction with the target molecule, etc. Consequently the dose-effect relationship for chemicals is more complicated than for radiation. Further, penetration itself m a y depend upon other parameters such as pH, tem-
184
o
3000
i
9
2000
z
o
--
~
2400
O >
t/3
(.Do 1000
1600
nUJ D_
0. ~D
u)
< I,,-. r > Z 0 U
Z
~
,
J
0
J
i
I
,
,
~
,
0.2
EMS
CONCENTRATION
Fig. 5. Variation of normalized
I
,
,
,
0.4
,
I
;r
0.6 (%
v/v
slope with concentration
1.0
) of EMS.
This means that an exposure to 1.7 X 10 -s M concentration of EMS for 1 h produces the same number of gene convertants as 1 tad of 6~ gamma-radiation. Discussion The REC value obtained with gene conversion as the end-point in this system for EMS is 1.7 X 10 -s M-h. It is of interest to compare this with the REC values obtained for EMS in other systems with forward mutation as the end-point. Not much literature is available on this subject. In what is available, the time parameter is not explicitly taken into account. Arbitrary treatment periods are taken, and hence the REC values are expressed only in terms of concentration of the chemical instead of in units of concentration multiplied by time. However, it is to be emphasized that, unless the reciprocity between concentration and treatment time is established, different REC values will be obtained for different treatment periods. With these limitations in view the following comparisons can be made. Heddle and Athanasiou [5], in their review of the forward m u t a t i o n frequency induced by EMS in various organisms, arrived at an average REC value of 4 X 10 -6 M (250 rad/mM) based on the whole curves of m u t a t i o n rate versus genome size. It we take the specific case of, say S. pombe, for a treatment period of 2 h, the REC value is f o u n d to be 5.1 X 10 -6 M, which is close to the average value. The report of the Environmental Mutagen Society of America [3] has also estimated the REC values for EMS from data available in the literature for rodent cells in culture. The REC value for the induction of forward m u t a t i o n at the TK locus for a treatment period of 2 h was estimated to be in the range of 0.62 to 0.83 mg/kg. Expressed in molar concentrations this will be 5 X 10 -6 to 6.7 X 10 -6 M. Recently Ban, Suzuki and Horikawa [2] have reported the induction of mutation to thymidine a u x o t r o p h y in Chinese hamster hai cells when treated with X-rays, EMS, MNNG, 4NQO and AF-2. Taking the ratio of the EMS concentration (for 2-h treatment) and the X-ray dose to produce the same frequency of mutation, the REC value can be estimated to be 2 X 10 -s M. Considering that none of these original investigations was conducted with the objective of obtaining the REC value for EMS, the agreement among the
187 TABLE 1 C O M P A R I S O N O F T H E R E C V A L U E S F O R EMS O B T A I N E D W I T H V A R I O U S O R G A N I S M S W I T H FORWARD MUTATION AND GENE CONVERSION AS THE END-POINTS Biological system
Treatment t i m e (h) a
End-point
A v e r a g e o f a n u m b e r o f species
--
S. p o m b e
2
R o d e n t cells
2
C h i n e s e h a m s t e r hai cells
2
D i p l o i d y e a s t S a c c h a r o m y c e s cerevisiae BZ34
2
Forward mutation Forward mutation Forward mutation Forward mutation Gene conversion
R E C value (M)
Ref.
2.5 X 10 - 6 b
5
5.1 • 1 0 - 6
5
5 - - 6 . 7 X 1 0 -6
3
2
X 1 0 -5
8.5 • 10-6
2 Present investigation
o a T r e a t m e n t t e m p e r a t u r e s : m a m m a l i a n cells, 3 7 ~ y e a s t , S. p o m b e , 2 5 C; S. cerevisiae, 3 0 ~ b C a l c u l a t e d f o r A r a b i d o p s i s w h i c h falls o n t h e g e o m e t r i c m e a n o f t h e r a n g e o f o r g a n i s m s s t u d i e d .
estimated values is good. These can be compared with the REC value with gene conversion as the end-point obtained in this investigation. For this purpose, the slope of the linear portion of the concentration--effect curve for a treatment period of 2 h can be considered (Fig. 4). This slope should be equal to twice the normalized slope at the plateau region (Fig. 5). Hence for a 2-h treatment, the REC value will become (1.7 • 1 0 - s ) / 2 = 8.5 • 10-6-M. This is compared in Table 1 with the other values estimated above for forward mutation in different systems. Here again, the agreement between the REC value obtained with gene conversion as the end-point on the one hand and forward mutation on the other, is remarkably good. This finding underlines the generality of the REC concept. References 1 A b r a h a m s o n , S., M . A . B e n d e r , A . D . C o n g e r a n d S. W o l f f , U n i f o r m i t y o f r a d i a t i o n - i n d u c e d m u t a t i o n rates among different species, Nature (London), 245 (1973) 460--462. 2 B a n , S., F . S u z u k i a n d M. H o r i k a w a , S t u d i e s o n s o m a t i c cell m u t a t i o n s , 1. R a d i a t i o n a n d c h e m i c a l i n d u c t i o n o f n u t r i t i o n a l l y d e f i c i e n t a n d s u f f i c i e n t m u t a n t s in C h i n e s e h a m s t e r hai cells i n v i t r o , J a p a n . J. G e n e t . , 51 ( 1 9 7 6 ) 2 3 7 - - 2 5 1 . 3 Environmental Mutagenic Hazards, Report of Committee 17, Science, 187 (1975) 503--514. 4 F o g e l , S., a n d R . K . M o r t i m e r , I n f o r m a t i o n a l t r a n s f e r in m e i o t i c g e n e - c o n v e r s i o n , P r o c . N a t l . A c a d . Sci. (U.S.A.), 62 (1969) 96--103. 5 H e d d l e , J . A . , a n d A . A t h a n a s i o u , M u t a t i o n r a t e , g e n o m e size a n d t h e i r r e l a t i o n t o t h e rec c o n c e p t , Nature (London), 258 (1975) 359--361. 6 M u x t h y , M.S.S., R e c o m m e n d a t i o n s o f C o m m i t t e e 17 o n e n v i r o n m e n t a l m u t a g e n e s i s : I m p l i c a t i o n s t o radiation safety, Health Phys., 32 (1977) 567--569. 7 M u r t h y , M.S.S., B.S. R a o , N . M . S . R e d d y 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 e v e r s i o n s t u d i e s , M u t a t i o n R c s . , 2 7 ( 1 9 7 5 ) 2 1 9 - - 2 2 3 . 8 M u r t h y , M.S.S., B.S. R a o a n d V . V . D e o r u k h a k a r , D e p e n d e n c e o f t h e e x p r e s s i o n o f t h e r a d i a t i o n induced gene conversion to arginine independence in diploid yeast on the amino acid concentration: E f f e c t o n allelic m a p p i n g , M u t a t i o n R e s . , 3 5 ( 1 9 7 6 ) 2 0 7 - - 2 1 2 . 9 M u x t h y , M . S . S . , a n d N . S a n k a r a n a r a y a n a n , Use o f d i p l o i d y e a s t as a t e s t s y s t e m f o r s c r e e n i n g c h e m i cals c a p a b l e o f i n d u c i n g g e n e c o n v e r s i o n , S y m p o s i u m o n m u t a g e n i c i t y , c a r c i n o g e n i c i t y a n d t e r a t o g e n i c i t y o f c h e m i c a l s , D e c . 1 9 7 5, M.S. U n i v e r s i t y , B a r o d a , I n d i a . 1 0 M a d h v a n a t h , U., P. S u b r a h m a n y a m , N. S a n k a r a n a r a y a n a n a n d M.S.S. M u r t h y , R B E o f C a l i f o r n i u m -
188 252 neutrons at low dose rates, International Symposium on Californium-252 Utihzation, IAEA, Brussels, 2 2 - - 2 4 A p r i l 1 9 7 6 . 11 O s t e r m a n - G o l k a r , S., L. E h r e n b e r g a n d C.A. W a c h t m e i s t e r , R e a c t i o n k i n e t i c s a n d b i o l o g i c a l a c t i o n in b a r l e y o f m o n o f u n c t i o n a l m e t h a n e s u l f o n i c esters, R a d i a t . B o t a n y , 1 0 ( 1 9 7 0 ) 3 0 3 - - 3 2 7 . 1 2 Z i m m e r m a n n , F . K . , I n d u c t i o n of m i t o t i c g e n e c o n v e r s i o n b y m u t a g e n s , M u t a t i o n R e s . , 11 ( 1 9 7 1 ) 327--337. 1 3 Z i m m e r m a n n , F . K . , P r o c e d u r e s u s e d in t h e i n d u c t i o n o f m i t o t i c r e c o m b i n a t i o n a n d m u t a t i o n in y e a s t Saccharomyces cerevisiae, M u t a t i o n R e s . , 31 ( 1 9 7 5 ) 7 1 - - 8 6 .
Mutation Research, 51 ( 1 9 7 8 ) 1 8 1 - - 1 8 8 9 E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press
DETERMINATION OF R A D I A T I O N EQUIVALENCE OF E T H Y L M E T H A N E S U L P H O N A T E (EMS) F O R INDUCTION OF GENE CONVERSION IN DIPLOID YEAST
M.S.S. M U R T H Y a n d N. S A N K A R A N A R A Y A N A N
Division of Radiological Protection, Bhabha Atomic Research Centre, Bombay-400 085 (India) (Received 5 J u l y 1 9 7 7 ) (Revision received 14 F e b r u a r y 1 9 7 8 ) (Accepted 7 March 1978)
Summary A unit Rad-Equivalent Chemical (REC) has been suggested for purposes of quantitating the mutagenic hazards of chemicals. The usefulness of this approach is demonstrated by the establishment of a constant relationship between the forward mutation frequency and haploid genome size in various organisms for both radiation and chemical EMS. However, it is necessary to determine the radiation equivalence of chemicals in as many organisms and for as many end-points as possible. For end-points we are limited to forward mutations. Another relevant genetic end-point of interest in this regard is gene conversion which can also monitor any kind of DNA damage in a suitable diploid system. Hence, we have determined the REC value for EMS in diploid yeast with gene conversion as the end-point. This agrees well with the REC values estimated in a number of organisms with forward mutation as the end-point. This finding further underlines the generality of the REC concept.
Introduction To quantitate the mutagenic hazards of chemicals, it has been proposed to express the exposure to chemical mutagens in terms of ionizing-radiation dose required to produce an equivalent effect [3]. For this purpose a unit called the Rad-Equivalent Chemical (REC) has been suggested [3,6]. REC can be defined as the dose of chemical (or concentration multiplied by time) that produces the same amount of genetic damage as 1 rad of ionizing radiation under chronic Work carried out under IAEA Contract No. 1961/RB.
Abbreviations: EMS, ethyl methanesulphonate; REC, Rad-Equivalent Chemical.
182
irradiation conditions. For radiation it has been shown that a predictable relationship exists between the forward mutation frequency and the haploid genome size of almost all organisms including man [1]. Recently, Heddle and Athanasiou [5] have shown that a similar relationship between forward mutation frequency and the haploid genome size exists for the mutagenic chemical ethyl methanesulphonate (EMS). Notwithstanding the problems involved, such as chemical penetration, specificity, stage dependence etc. in a theoretical consideration of such a relationship, if it can be established for other chemicals also, then the REC value for a given chemical determined for one organism can be extrapolated to any other organism including man. Quantitation and extrapolation of mutagenic hazards of chemicals can then be placed on a more scientific basis. (Since somatic effects such as induction of malignancies are also of concern in environmental health protection, it is necessary to extend the REC concept to cancer as the end-point. Here additional problems such as organ-specificity, etc. have to be taken into account.) To generalize the validity of the REC concept, however, it is necessary to establish the equivalence n o t only in various organisms, b u t also for various genetic end-points. For end-points we are limited to forward mutations, since back mutations are of little value because of their specificity. Another relevant genetic end-point in this regard is gene conversion, which is k n o w n to be induced during the process of repair of DNA damage caused by chemical and physical agents [12] and results in the expression of recessive genes. It can be easily detected by plating treated cells on selective media [12]. Zimmermann [12] in 1971 reviewed the induction of gene conversion by a b o u t 25 mutagenic chemicals and found a correlation between the mutagenic and convertogenic abilities of these chemicals. Further, there was no mutagen specificity in the induction of gene conversion. Since then many workers have used this endpoint to detect the genotoxicity of a variety of chemical substances (for example see refs. in ref. 13). Recently we have reviewed the literature on nearly 150 chemicals, comprising drugs, pesticides, herbicides, industrial chemicals, food additives etc., which have been tested for their ability to induce gene conversion in diploid yeast and have confirmed Zimmermann's findings {unpublished data). In this paper we report the determination of the REC value for EMS to induce gene conversion in diploid yeast, and compare this with the REC values reported in the literature for various organisms with forward mutation as the end-point. Methods and materials Yeast strain
The diploid yeast strain BZ34 used in this investigation carries two noncomplementing heteroallelic mutants in the argino-succinase locus and hence is auxotrophic to arginine [4]. However, exposure to ionizing radiation [7] and mutagenic chemicals [9] induces arginine prototrophs in this strain by the mechanism of gene conversion. These can be scored by plating treated cells on medium from which arginine is omitted. Media
The cell line was maintained by routinely subculturing in yeast extract,
183
peptone and dextrose medium, at 30~ for 48 h in shaker incubators. Cell viability was determined by plating the cells on synthetic complete medium, and the convertants were detected by plating them on synthetic medium with only a trace a m o u n t (0.4 mg/1) of arginine. This medium will be referred to as arginine-omission medium. Details of the culture and media have been given elsewhere [ 7,8]. Chemical t r e a t m e n t The EMS used in this investigation was kindly provided by Dr. R. Fahrig of W. Germany. Stationary-phase cells were centrifuged and resuspended in 0.1 M potassium phosphate buffer (pH 7) to a concentration of 1 X 107 cells/ml. EMS was dissolved in phosphate buffer to the required concentrations. In each run, treatment was carried out in a given concentration but the duration of treatm e n t was varied from 30 min to 6 h. In a typical treatment protocol, 1 ml of cell suspension was added to 10 ml of EMS solution in a 100-ml flask and kept on a shaker water-bath at 30~ in the dark. At the end of the specified duration, 5 ml of the mixture was filtered on a Millipore filtration system, then resuspended in 5 ml sterile distilled water. Suitable dilutions were made to obtain 200--500 viable cells per ml, and 1 ml of each was plated on 4 arginineomission-medium plates for scoring arginine-independent convertants and on 4 complete medium plates for scoring viability. The plates were stored at 30~ in the dark for 3--5 days, after which the colonies were counted. Gene-conversion frequency was expressed as the ratio of the colony counts on the omission plates to those on the complete-medium plates multiplied by the dilution factor. A control flask was maintained to determine the background frequency. The same protocol was repeated for various concentrations of EMS from 0.0125 to 1% (v/v). Gamma-irradiation A 6~ cell with a dose rate of 2.6 krad/min was used for gammairradiation. Yeast cell suspension (5 • 106/ml) in buffer was exposed to various doses up to 10 krad. After irradiation, suitable dilutions were plated on omission as well as complete medium. Gene-conversion frequency was determined as mentioned in the previous paragraph.
Results Fig. 1 shows the dose--effect relationship for gamma-radiation. The geneconversion frequency increased linearly with dose. At all doses, the radiationinduced lethality was small. The slope of the line is 26.0 • 10 -2 conversion/106 survivors per rad. Earlier investigations had shown t h a t dose--effect linearity exists even at dose rates as low as a few rad/min [10]. While the events leading to biochemical lesions by ionizing radiations are extremely fast, this is not true for chemicals. They are dependent on various parameters such as penetration of the cell wall and the nuclear membrane by the chemical, reaction with the target molecule, etc. Consequently the dose-effect relationship for chemicals is more complicated than for radiation. Further, penetration itself m a y depend upon other parameters such as pH, tem-
184
o
3000
i
9
2000
z
o
--
~
2400
O >
t/3
(.Do 1000
1600
nUJ D_
0. ~D
u)
< I,,-. r > Z 0 U
Z
~
,
J
0
J
i
I
,
,
~
,
0.2
EMS
CONCENTRATION
Fig. 5. Variation of normalized
I
,
,
,
0.4
,
I
;r
0.6 (%
v/v
slope with concentration
1.0
) of EMS.
This means that an exposure to 1.7 X 10 -s M concentration of EMS for 1 h produces the same number of gene convertants as 1 tad of 6~ gamma-radiation. Discussion The REC value obtained with gene conversion as the end-point in this system for EMS is 1.7 X 10 -s M-h. It is of interest to compare this with the REC values obtained for EMS in other systems with forward mutation as the end-point. Not much literature is available on this subject. In what is available, the time parameter is not explicitly taken into account. Arbitrary treatment periods are taken, and hence the REC values are expressed only in terms of concentration of the chemical instead of in units of concentration multiplied by time. However, it is to be emphasized that, unless the reciprocity between concentration and treatment time is established, different REC values will be obtained for different treatment periods. With these limitations in view the following comparisons can be made. Heddle and Athanasiou [5], in their review of the forward m u t a t i o n frequency induced by EMS in various organisms, arrived at an average REC value of 4 X 10 -6 M (250 rad/mM) based on the whole curves of m u t a t i o n rate versus genome size. It we take the specific case of, say S. pombe, for a treatment period of 2 h, the REC value is f o u n d to be 5.1 X 10 -6 M, which is close to the average value. The report of the Environmental Mutagen Society of America [3] has also estimated the REC values for EMS from data available in the literature for rodent cells in culture. The REC value for the induction of forward m u t a t i o n at the TK locus for a treatment period of 2 h was estimated to be in the range of 0.62 to 0.83 mg/kg. Expressed in molar concentrations this will be 5 X 10 -6 to 6.7 X 10 -6 M. Recently Ban, Suzuki and Horikawa [2] have reported the induction of mutation to thymidine a u x o t r o p h y in Chinese hamster hai cells when treated with X-rays, EMS, MNNG, 4NQO and AF-2. Taking the ratio of the EMS concentration (for 2-h treatment) and the X-ray dose to produce the same frequency of mutation, the REC value can be estimated to be 2 X 10 -s M. Considering that none of these original investigations was conducted with the objective of obtaining the REC value for EMS, the agreement among the
187 TABLE 1 C O M P A R I S O N O F T H E R E C V A L U E S F O R EMS O B T A I N E D W I T H V A R I O U S O R G A N I S M S W I T H FORWARD MUTATION AND GENE CONVERSION AS THE END-POINTS Biological system
Treatment t i m e (h) a
End-point
A v e r a g e o f a n u m b e r o f species
--
S. p o m b e
2
R o d e n t cells
2
C h i n e s e h a m s t e r hai cells
2
D i p l o i d y e a s t S a c c h a r o m y c e s cerevisiae BZ34
2
Forward mutation Forward mutation Forward mutation Forward mutation Gene conversion
R E C value (M)
Ref.
2.5 X 10 - 6 b
5
5.1 • 1 0 - 6
5
5 - - 6 . 7 X 1 0 -6
3
2
X 1 0 -5
8.5 • 10-6
2 Present investigation
o a T r e a t m e n t t e m p e r a t u r e s : m a m m a l i a n cells, 3 7 ~ y e a s t , S. p o m b e , 2 5 C; S. cerevisiae, 3 0 ~ b C a l c u l a t e d f o r A r a b i d o p s i s w h i c h falls o n t h e g e o m e t r i c m e a n o f t h e r a n g e o f o r g a n i s m s s t u d i e d .
estimated values is good. These can be compared with the REC value with gene conversion as the end-point obtained in this investigation. For this purpose, the slope of the linear portion of the concentration--effect curve for a treatment period of 2 h can be considered (Fig. 4). This slope should be equal to twice the normalized slope at the plateau region (Fig. 5). Hence for a 2-h treatment, the REC value will become (1.7 • 1 0 - s ) / 2 = 8.5 • 10-6-M. This is compared in Table 1 with the other values estimated above for forward mutation in different systems. Here again, the agreement between the REC value obtained with gene conversion as the end-point on the one hand and forward mutation on the other, is remarkably good. This finding underlines the generality of the REC concept. References 1 A b r a h a m s o n , S., M . A . B e n d e r , A . D . C o n g e r a n d S. W o l f f , U n i f o r m i t y o f r a d i a t i o n - i n d u c e d m u t a t i o n rates among different species, Nature (London), 245 (1973) 460--462. 2 B a n , S., F . S u z u k i a n d M. H o r i k a w a , S t u d i e s o n s o m a t i c cell m u t a t i o n s , 1. R a d i a t i o n a n d c h e m i c a l i n d u c t i o n o f n u t r i t i o n a l l y d e f i c i e n t a n d s u f f i c i e n t m u t a n t s in C h i n e s e h a m s t e r hai cells i n v i t r o , J a p a n . J. G e n e t . , 51 ( 1 9 7 6 ) 2 3 7 - - 2 5 1 . 3 Environmental Mutagenic Hazards, Report of Committee 17, Science, 187 (1975) 503--514. 4 F o g e l , S., a n d R . K . M o r t i m e r , I n f o r m a t i o n a l t r a n s f e r in m e i o t i c g e n e - c o n v e r s i o n , P r o c . N a t l . A c a d . Sci. (U.S.A.), 62 (1969) 96--103. 5 H e d d l e , J . A . , a n d A . A t h a n a s i o u , M u t a t i o n r a t e , g e n o m e size a n d t h e i r r e l a t i o n t o t h e rec c o n c e p t , Nature (London), 258 (1975) 359--361. 6 M u x t h y , M.S.S., R e c o m m e n d a t i o n s o f C o m m i t t e e 17 o n e n v i r o n m e n t a l m u t a g e n e s i s : I m p l i c a t i o n s t o radiation safety, Health Phys., 32 (1977) 567--569. 7 M u r t h y , M.S.S., B.S. R a o , N . M . S . R e d d y 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 e v e r s i o n s t u d i e s , M u t a t i o n R c s . , 2 7 ( 1 9 7 5 ) 2 1 9 - - 2 2 3 . 8 M u r t h y , M.S.S., B.S. R a o a n d V . V . D e o r u k h a k a r , D e p e n d e n c e o f t h e e x p r e s s i o n o f t h e r a d i a t i o n induced gene conversion to arginine independence in diploid yeast on the amino acid concentration: E f f e c t o n allelic m a p p i n g , M u t a t i o n R e s . , 3 5 ( 1 9 7 6 ) 2 0 7 - - 2 1 2 . 9 M u x t h y , M . S . S . , a n d N . S a n k a r a n a r a y a n a n , Use o f d i p l o i d y e a s t as a t e s t s y s t e m f o r s c r e e n i n g c h e m i cals c a p a b l e o f i n d u c i n g g e n e c o n v e r s i o n , S y m p o s i u m o n m u t a g e n i c i t y , c a r c i n o g e n i c i t y a n d t e r a t o g e n i c i t y o f c h e m i c a l s , D e c . 1 9 7 5, M.S. U n i v e r s i t y , B a r o d a , I n d i a . 1 0 M a d h v a n a t h , U., P. S u b r a h m a n y a m , N. S a n k a r a n a r a y a n a n a n d M.S.S. M u r t h y , R B E o f C a l i f o r n i u m -
188 252 neutrons at low dose rates, International Symposium on Californium-252 Utihzation, IAEA, Brussels, 2 2 - - 2 4 A p r i l 1 9 7 6 . 11 O s t e r m a n - G o l k a r , S., L. E h r e n b e r g a n d C.A. W a c h t m e i s t e r , R e a c t i o n k i n e t i c s a n d b i o l o g i c a l a c t i o n in b a r l e y o f m o n o f u n c t i o n a l m e t h a n e s u l f o n i c esters, R a d i a t . B o t a n y , 1 0 ( 1 9 7 0 ) 3 0 3 - - 3 2 7 . 1 2 Z i m m e r m a n n , F . K . , I n d u c t i o n of m i t o t i c g e n e c o n v e r s i o n b y m u t a g e n s , M u t a t i o n R e s . , 11 ( 1 9 7 1 ) 327--337. 1 3 Z i m m e r m a n n , F . K . , P r o c e d u r e s u s e d in t h e i n d u c t i o n o f m i t o t i c r e c o m b i n a t i o n a n d m u t a t i o n in y e a s t Saccharomyces cerevisiae, M u t a t i o n R e s . , 31 ( 1 9 7 5 ) 7 1 - - 8 6 .
