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Molecular dosimetry of chemical mutagens determination of molecular dose to the germ line
311
Mutation Research, 38 ( 1 9 7 6 ) 3 1 1 - - 3 1 6 © Elsevier Scientific Publishing C o m p a n y , A m s t e r d a m - - P r i n t e d in T h e N e t h e r l a n d s
M O L E C U L A R DOSIMETRY OF CHEMICAL MUTAGENS * D E T E R M I N A T I O N OF M O L E C U L A R DOSE TO THE GERM LINE
WILLIAM R. L E E
Department of Zoology and Physiology, Louisiana State University, Baton Rouge, Louisiana
70803 (U.S.A.) (Received December 8th, 1975} (Accepted May 13th, 1976}
To be able to extrapolate with confidence from the results of mutagenic tests with laboratory species to man, it is necessary to demonstrate our ability to extrapolate among laboratory test systems. In the three tier approach to mutagenic tests proposed by B.A. Bridges [ 5]; the first t w o tiers are essentially qualitative. That is, one asks only if a particular test system has a positive or negative response to the agents being tested; therefore, only the qualitative response of the test is extrapolated among laboratory test systems. Tier three, as proposed by Bridges, is far more demanding in that it asks for a quantitative estimation of the mutagenic risk-benefit t y p e analysis in which the benefit to mankind is weighed against the genetic risk of using the particular material in the environment. The most extensively studied mutagenic agent at the tier three level is that of ionizing radiation which is beneficial b o t h for medical purposes and energy production and yet has been known to be mutagenic for many years. In the study of ionizing radiation an ever increasing refinement of the quantitative estimation of risk is made and weighed against the potential benefit. This symposium will be concerned with meeting the dosimetry demand of chemical mutagens for this quantitative estimation of risk. In extrapolating from experimental organisms to man at the tier three level it is necessary to ask not only: "Will the agent be mutagenic in m a n ? " b u t also: "Can the mutational response in experimental species be related in a quantitative way to response in man?". Most experimental protocols confound the important question of g e n e t i c response with the physiological response; for example, will a material be metabolized in the same way in our experimental material as in man? In general, metabolism tends to vary among species more than does the genetic mechanism. In fact throughout the course of evolution the genetic mechanism has remained one of the most conserved elements of biology. Therefore, among diverse species, we would expect * S y m p o s i u m presented at the Sixth Annual Meeting of the Environmental Mutagen Society, May 9--12, 1975, Miami Beach, Florida.
312
the reaction of the genetic material to have greater similarity than the metabolism of chemical agents. Consequently, it is important to distinguish between differences in response in the metabolism of test organisms and man and differences in response of the genetic material to chemical mutagens. Therefore, we need to distinguish between comparative physiology and comparative mutagenesis. In order to separate comparative physiology from comparative mutagenesis it is necessary to distinguish between exposure and dose. In radiation biology dose defined in terms of " r a d s " is the absorption of energy at the place of interest. Actually what is referred to as dose in the chemical m u t a t i o n literature (for example, concentration of a chemical in the feeding media, a m o u n t and concentration injected, or length of time exposed to a gas) is really exposure of the whole organism [11]. The importance of the distinction between exposure and dose becomes apparent when we consider all the possible physiological effects and chemical interactions which could occur after exposure, for example: assimilation, circulation, transport across numerous cell membranes, the metabolic activity of the intact animal and enzymatic degradation or activation of the compound, as well as the physiological activity of microflora in the digestive tract. Saturation of any required enzyme system may alter relationship of exposure to dose. As a result of a recently completed experiment [2] we must add an additional variable, behavior. Feeding is one of the most c o m m o n methods of exposing insects to mutagens. We questioned the effect of concentration of the mutagen ethyl methanesulfonate (EMS) on the a m o u n t of solution imbibed. To answer this we prepared an EMS--I% sucrose feeding solution spiked with 14Clabeled sucrose. The concentration of the 14C label was the same in all solutions but the concentration of EMS was adjusted. In summary, our test strain of Drosophila melanogaster showed a t e n d e n c y to avoid EMS with concentrations above 1.25 mM, and flies consumed 10 times as much liquid with a concentration of 2.5 mM as with a concentration of 25 mM. This feeding reaction explained an apparent saturation type curve that we had obtained (unpublished) when the concentration of EMS in the feeding solution was plotted against the genetic effects. There is no justification in assuming linearity between exposure and dose to the germ line for chemical mutagens; therefore, the distinction between dose to the germ line and exposure of the organism is essential in developing dosim e t r y for comparative mutagenesis. Experimental protocols may be developed to give a linear relation between exposure and dose for a range of exposures, but this relation must be experimentally verified for each mutagen and protocol for exposure and cannot be assumed. When studying chemical mutagens dose must be determined within defined germ cell stages, for extreme differences in germ cell sensitivity are well established. For example, EMS induces a high frequency of mutations in late spermatid and mature spermatozoa, while inducing a low mutation frequency in spermatogonia [9]. In contrast, chloroethyl methanesulfonate induces a higher m u t a t i o n frequency in spermatogonia than in postmeiotic stages [8,16]. Loveless [15] has compiled a table of a number of alkylating agents that shows their varied effects on different germ cell stages. It has been found that the
313 range of response among different germ cell stages is equal to or possibly exceeds the limits of sensitivity of the genetic test [7]. In our first efforts at molecular dosimetry [13,20] we found differences in the amount of alkylation among different germ cell stages in Drosophila. A following paper [18] will discuss these differences with respect to the different germ cell stages in the mouse and the effect of repair following alkylation [17]. Because of cell stage specificity in both mutational response and products of alkylation we must identify the germ cell stage in all tests in order to make valid comparisons among test systems. There are several methods for dosimetry in the area of interest, the germ line. The earliest method determined the dose of a chemical mutagen by comparing its genetic effect with the effect of X-rays, the most studied mutagen of all. The difficulty of using a mutagen like X-rays as a measure of dose for other mutagens was recognized by Auerbach [3] in 1949 when she pointed out that this method of dosimetry depends on the similarity between X-ray induced lethals and chemically induced lethals. Unfortunately, most mutations induced by chemicals are different in some characteristics from those induced by radiation; for example, early experiments with mustard gas [4] showed there was a higher proportion of sex-linked recessive lethals to translocations than in the case of X-rays, and there was observed a high frequency of mosaics which are only rarely observed following treatment with X-rays. Because of differences in the mutational spectra of X-rays and chemical mutagens, the ratio between the mutational classes will change considerably. Table I shows a comparison of the frequency of mutations induced by X-ray with those induced by EMS. Both mutagens were tested using the same stocks and laboratory techniques. The Xray data were obtained from irradiating (250 KVP, 1-mm aluminum filter) 5day old males and allowing them to mate for one day following irradiation. Results of the same genetic test applied to progeny from males fed on 0.025 M EMS for 24 h, aged 24 h, and then allowed to mate for 24 h are also shown in Table I. Quantitative comparison of X-rays with chemical mutagens has been proposed as rem-equivalent-chemical (REC) [6] or rad-equivalent "radequiv" [5]. In computing the ltEC values in the appendix [6] of the Committee 17
TABLE I
Accumulated Controls 1820 tad X-ray 3640 tad X-ray 0 . 0 2 5 M EMS
F1 (% loss o f X or Y )
F2 (% l e t h a l s )
F3 (% l e t h a l s )
0.4 -+ 0.1 (5928) 2.4 + 0.3 (3047) 4.4 + 0.5 (1740) 1.2 -+ 0 . 2 (8227)
0.13 + 0.02 (21108) 5.1 -+ 0 . 4 (3189) 1 1 . 5 -+ 0.9 (1158) 43 -+ 1 ( 5 6 ) a (2390)
0 . 2 3 -+ 0 . 0 2 (13163) 0.9 -+ 0.2 (2833) 0.7 -+ 0.3 (926) 12 -+ 2 (491)
a C o r r e c t e d for t h e s a t u r a t i o n e f f e c t b y a s s u m i n g a Poisson d i s t r i b u t i o n o f m u t a t i o n s a m o n g t r e a t e d s p e r m cells,
314
Report there were questionable assumptions made in converting milligrams of hycanthone injected per fly to equivalent " h u m a n therapeutic doses (HTD)". Our work with radiolabeled mutagens suggests a number of problems in making this t y p e of calculation; these include non-linearity of exposure to dose as described above, rapid and variable loss of the labeled mutagen from the fly [19], and retention of the mutagenic agent in the fly past the time of exposure (unpublished), as well as basic differences in physiology. These problems of chemical dosimetry are largely avoided if, instead of expressing dose in terms of concentration, dose is expressed in terms of reaction products, as will be discussed later. However, even without a proper description of dose we can compare among the three genetic tests in Table I the tad equivalent (or for the X-ray treatment the rem equivalent) of the standard feeding procedure of 0.025 M EMS for 24 h [14] because all three genetic tests are sampled from the same pool of treated spermatozoa. The REC for this feeding procedure is 730 for loss of sex chromatin and 19,000 for sex-linked recessive lethals observed in the F2. For gonads mosaic for sex-linked recessive lethals (lethals detected in the F3) the computation of REC becomes ridiculous because the denominator, X-ray induced mosaics, is close to zero. Therefore, we obtain substantially different values for REC from the same pool of treated spermatozoa depending on which genetic test we use. Because of differences in the mutational spectra induced by EMS and by X-rays, the change in REC values was not surprising. It is not reasonable to compute a single ratio between one mutational spectrum and another if the spectra are quite different. Nevertheless, Auerbach in 1949 pointed o u t that using a class of mutations as a comparative measure of dose is "preferable to simple determination of the concentration in the outside media, and is also superior to dosage estimates based on toxicity for the exposed flies" [3]. As she points out, toxicity as a measure for mutagenicity may be very misleading because the mechanism for toxicity may be quite different from that for mutagenicity [3]. The relationship between toxicity and mutagenicity becomes especially complex in resistant strains. In essence what is needed is a physical measure of dose. It is only the germ cells in multi-cellular organisms that must be considered in the case of heritable mutations; therefore, only the a m o u n t of the mutagen that penetrates to the germ line is of genetic significance. The concentration of the active form of a mutagen in the immediate neighborhood of the genome has been defined as the Genetically Significant Concentration (GSC) by Committee 17 Report [6]. The concentration of a chemical mutagen in the gonad can be compared to the dose rate of ionizing radiation to the gonad; that is, the dose is the integral over time of the concentration. In a typical X-ray experiment the length of time is the time the organism is exposed to a constant intensity of radiation; however, for chemical mutagens, concentration varies with time. The curve of the GSC is time dependent and may be very complex and quite different for different mutagens. Also, due to retention of the chemical within the body, a GSC may persist b e y o n d the time when treatment stops (Fig. 1 ). To simplify dosimetry when there are variations in concentration with time following exposure to a chemical mutagen, I defined the dose for chemical mutagens as the a m o u n t of a chemical that reacts in the germ line with a selected
315
GENETICALLY SIGNIFICANT CONCENTRATION
(
EXPOSURE
) TIME
)
Fig. 1. D o ~ as t h e i n ~ g r M o f the ~ m e d e p e n d e n t variable Genetically Significant C o n e e n ~ a t i o n ( G S C ) . T h e r e l a t i o n o f the GSC to t i m e i n t h m f i g u r e m h y p o t h e ~ c M .
target molecule (STM) [11]. Ideally one should select as the STM the genetically significant target (GST) [18] which is generally assumed to be DNA even though the genetically significant moiety of the DNA may not be known. By selecting target molecules that react with the chemical mutagen to produce stable products one can measure the accumulated products and thereby obtain a measure of dose. The selected reaction products (SRP) [12] chosen to measure the reaction of chemical agents with the STM should be not only stable and of sufficient quantity to permit accurate measurement within germ cell stages b u t also should be sufficiently uniform among species to permit dosimetry comparisons among species. The SRP may not be the actual products that cause mutational lesions. The actual p r o d u c t that causes the alteration of DNA may not be known, and if it is known, it m a y be chemically unstable and/or present in concentrations that are t o o low for detection with present techniques. Nevertheless, if the SRP can be correlated with the mutation frequency, the SRP can be an effective measure of dose. However, such a correlation between SRP and mutation frequency cannot be taken as evidence that the STM is actually the GST, for, as will be discussed by Aaron [ 1 ], there should be a constant relationship between alkylation of the GST and other nucleophilic sites within the subcellular genetic compartment. Although the experimentalist's choice of the SRP is somewhat arbitrary because of incomplete information, previous work suggests that the SRP could be alkylated DNA [11,13,20,21,22] or alkylated bases [10] which can be measured using radio tracer techniques. The actual correlation of mutation frequency with the SRP must be determined experimentally among several test species. If the SRP used as a measure of dose is sufficiently representative of nuclear events among species, it can then be hoped that a basis for comparative mutagenesis among species can be developed which is independent of comparative physiology. The next paper [1] by Aaron will discuss the chemical consideration in selecting appropriate target molecules and reaction products for molecular dosimetry.
316
Acknowledgement This work is supported in part by PHS Research Grant No. ES 00320-08 from the National Institute of Environmental Health Sciences and the Energy Research and Development Administration Contract No. AT-(40-1)-3728. References 1 A a r o n , C.S., Molecular d o s i m e t r y of chemical m u t a g e n s . Selection of a p p r o p r i a t e t a r g e t m o l e c u l e s f o r d e t e r m i n i n g m o l e c u l a r dose t o t h e g e r m llne, M u t a t i o n Res., 38 ( 1 9 7 6 ) 3 0 3 - - 3 1 0 . 2 A a r o n , C.S. a n d W.R. Lee, R e j e c t i o n of e t h y l m e t h a n e s u l f o n a t e feeding s o l u t i o n by Drosophila melanogaster a d u l t males, s u b m i t t e d f o r p u b l i c a t i o n t o D r o s o p h i l a I n f o r m a t i o n Service. 3 A u e r b a c h , C., Chemical m u t a g e n e s i s , Biological Reviews, 24 ( 1 9 4 9 ) 3 5 5 - - 3 9 7 . 4 A u e r b a c h , C., J.M. R o b s o n a n d J.G. Cart, The c h e m i c a l p r o d u c t i o n of m u t a t i o n s , Science, 1 0 5 ( 1 9 4 7 ) 243--247. 5 Bridges, B.A., The three-tier a p p r o a c h to m u t a g e n i e i t y screening a n d the c o n c e p t o f r a d i a t i o n - e q u i v a l e n t dose, M u t a t i o n Res., 26 ( 1 0 7 4 ) 3 3 5 - - 3 4 0 . 6 D r a k e , J.W., C h a i r m a n , C o m m i t t e e 17, E n v i r o n m e n t a l m u t a g e n i c h a z a r d s , Science, 1 8 7 ( 1 9 7 5 ) 5 0 3 - 514. 7 Ehling, U.H., R.B. C u m m i n g a n d H.V. Mailing, I n d u c t i o n of d o m i n a n t lethal m u t a t i o n s b y a l k y l a t i n g agents in male mice, M u t a t i o n Res, 5 ( 1 9 6 8 ) 4 1 7 - - 4 2 8 . 8 F a h m y , O.G. a n d M.J. F a h m y , M u t a g e n i e i t y o f 2 - c h l o r o e t h y l m e t h a n e s u l f o n a t e in Drosophila melanogaster, N a t u r e , L o n d . , 1 7 7 ( 1 9 5 6 ) 9 9 6 - - 9 9 7 . 9 J e n k i n s , J.B., Mutagenesis at a c o m p l e x locus in D r o s o p h i l a with t h e m o n o f u n e t i o n a l a l k y l a t i n g a g e n t , e t h y l m e t h a n e s u l f o n a t e , Genetics, 57 ( 1 9 6 7 ) 7 8 3 - - 7 9 3 . 10 L a w l e y , P.D., Effects o f s o m e chemical m u t a g e n s a n d c a r c i n o g e n s o n nucleic acids, Prog. Nucleic A c i d Res. Molee. Biol., 5 ( 1 9 6 6 ) 8 9 - - 1 3 1 . 11 Lee~ W.R., C o m p a r i s o n of the m u t a g e n i c effects of c h e m i c a l s a n d ionizing r a d i a t i o n using Drosophila melangaster test systems, in O.F. N y g a a r d , H.I. Adler a n d W.K. Sinclair (eds.), R a d i a t i o n R e s e a r c h , B i o m e d i c a l , Chemical a n d Physical Perspectives, P r o c e e d i n g s of the 5th I n t e r n a t i o n a l Congress of R a d i a t i o n R e s e a r c h , A c a d e m i c Press, New Y o r k , 1 9 7 5 , pp. 976---983. 1 2 Lee~ W.R., Chemical Mutagenesis, in M. A s h b u r n e r a n d E. Novitski (eds.), The Genetics a n d Biology of D r o s o p h i l a , in press. 13 Lee, W.R. a n d G.A. Sega, Dosage d e t e r m i n a t i o n of c h e m i c a l m u t a g e n s in the g e r m line o f Drosophila melanogaster, EMS N e w s l e t t e r o f the E n v i r o n m e n t a l M u t a g e n Society, 3 ( 1 9 7 0 ) 37. 14 Lewis, E.B. a n d F. Bacher, M e t h o d of feeding e t h y l m e t h a n e s u l f o n a t e (EMS) t o D r o s o p h i l a males, D r o s o p h i l a I n f o r m a t i o n Service, 4 3 ( 1 9 6 8 ) 193. 15 Loveless, A., Genetic a n d allied effects of a l k y l a t i n g agents, P e n n s y l v a n i a State University Press, 1 9 6 6 , p. 2 7 0 (see Fig. 1 2 , p. 51). 16 M a t t h e w , C., The n a t u r e of d e l a y e d m u t a t i o n a f t e r t r e a t m e n t w i t h c h l o r o e t h y l m e t h a n e s u l f o n a t e a n d o t h e r a l k y l a t i n g agents, M u t a t i o n Res., 1 ( 1 9 6 4 ) 1 6 3 - - 1 7 2 . 17 Sega, G.A., U n s c h e d u l e d D N A s y n t h e s i s in t h e g e r m cells of male mice e x p o s e d in vivo t o the chemical m u t a g e n e t h y l m e t h ~ n e s u l f o n a t e , Proc. Natl. Aead. Sci., 71 ( 1 9 7 4 ) 4955---4959. 18 Sega, G.A., Molecular d o s i m e t r y of c h e m i c a l m u t a g e n s . M e a s u r e m e n t of m o l e c u l a r dose a n d D N A repair in m a m m a l i a n g e r m cells, M u t a t i o n Res., 38 ( 1 9 7 6 ) 3 1 7 - - 3 2 6 . 19 Sega, G.A. a n d W.R. Lee, A v a c u u m i n j e c t i o n t e c h n i q u e f o r o b t a i n i n g u n i f o r m dosages in Drosophila melanogaster, D r o s o p h i l a I n f o r m a t i o n Service, 4 5 ( 1 9 7 0 ) 179. 2 0 Sega, G.A. a n d W.R. Lee, A q u a n t i t a t i v e s t u d y o f the effects o f t h e a l k y l a t i n g a g e n t e t h y l m e t h a n e s u l f o n a t e (EMS) o n the germ line of Drosophila melaaogaster, Genetics, 6 4 ( 1 9 7 0 ) $58. 21 Sega, G.A., R.B. C u m m i n g a n d M.F. Walton, D o s i m e t r y studies o n t h e e t h y l a t i o n of m o u s e s p e r m D N A a f t e r in vivo e x p o s u r e t o [3H] e t h y l m e t h a n e s u l f o n a t e , M u t a t i o n Res, 24 ( 1 9 7 4 ) 3 1 7 - - 3 3 3 . 22 Sega, G.A., P.A. Gee a n d W.R. Lee, Dosirnetry of the c h e m i c a l m u t a g e n e t h y l m e t h a n e s u l f o n t e in s p e r m a t o z o a n D N A f r o m Drosophila melanogaster, M u t a t i o n Res., 16 ( 1 9 7 2 ) 2 0 3 - - 2 1 3 .
Mutation Research, 38 ( 1 9 7 6 ) 3 1 1 - - 3 1 6 © Elsevier Scientific Publishing C o m p a n y , A m s t e r d a m - - P r i n t e d in T h e N e t h e r l a n d s
M O L E C U L A R DOSIMETRY OF CHEMICAL MUTAGENS * D E T E R M I N A T I O N OF M O L E C U L A R DOSE TO THE GERM LINE
WILLIAM R. L E E
Department of Zoology and Physiology, Louisiana State University, Baton Rouge, Louisiana
70803 (U.S.A.) (Received December 8th, 1975} (Accepted May 13th, 1976}
To be able to extrapolate with confidence from the results of mutagenic tests with laboratory species to man, it is necessary to demonstrate our ability to extrapolate among laboratory test systems. In the three tier approach to mutagenic tests proposed by B.A. Bridges [ 5]; the first t w o tiers are essentially qualitative. That is, one asks only if a particular test system has a positive or negative response to the agents being tested; therefore, only the qualitative response of the test is extrapolated among laboratory test systems. Tier three, as proposed by Bridges, is far more demanding in that it asks for a quantitative estimation of the mutagenic risk-benefit t y p e analysis in which the benefit to mankind is weighed against the genetic risk of using the particular material in the environment. The most extensively studied mutagenic agent at the tier three level is that of ionizing radiation which is beneficial b o t h for medical purposes and energy production and yet has been known to be mutagenic for many years. In the study of ionizing radiation an ever increasing refinement of the quantitative estimation of risk is made and weighed against the potential benefit. This symposium will be concerned with meeting the dosimetry demand of chemical mutagens for this quantitative estimation of risk. In extrapolating from experimental organisms to man at the tier three level it is necessary to ask not only: "Will the agent be mutagenic in m a n ? " b u t also: "Can the mutational response in experimental species be related in a quantitative way to response in man?". Most experimental protocols confound the important question of g e n e t i c response with the physiological response; for example, will a material be metabolized in the same way in our experimental material as in man? In general, metabolism tends to vary among species more than does the genetic mechanism. In fact throughout the course of evolution the genetic mechanism has remained one of the most conserved elements of biology. Therefore, among diverse species, we would expect * S y m p o s i u m presented at the Sixth Annual Meeting of the Environmental Mutagen Society, May 9--12, 1975, Miami Beach, Florida.
312
the reaction of the genetic material to have greater similarity than the metabolism of chemical agents. Consequently, it is important to distinguish between differences in response in the metabolism of test organisms and man and differences in response of the genetic material to chemical mutagens. Therefore, we need to distinguish between comparative physiology and comparative mutagenesis. In order to separate comparative physiology from comparative mutagenesis it is necessary to distinguish between exposure and dose. In radiation biology dose defined in terms of " r a d s " is the absorption of energy at the place of interest. Actually what is referred to as dose in the chemical m u t a t i o n literature (for example, concentration of a chemical in the feeding media, a m o u n t and concentration injected, or length of time exposed to a gas) is really exposure of the whole organism [11]. The importance of the distinction between exposure and dose becomes apparent when we consider all the possible physiological effects and chemical interactions which could occur after exposure, for example: assimilation, circulation, transport across numerous cell membranes, the metabolic activity of the intact animal and enzymatic degradation or activation of the compound, as well as the physiological activity of microflora in the digestive tract. Saturation of any required enzyme system may alter relationship of exposure to dose. As a result of a recently completed experiment [2] we must add an additional variable, behavior. Feeding is one of the most c o m m o n methods of exposing insects to mutagens. We questioned the effect of concentration of the mutagen ethyl methanesulfonate (EMS) on the a m o u n t of solution imbibed. To answer this we prepared an EMS--I% sucrose feeding solution spiked with 14Clabeled sucrose. The concentration of the 14C label was the same in all solutions but the concentration of EMS was adjusted. In summary, our test strain of Drosophila melanogaster showed a t e n d e n c y to avoid EMS with concentrations above 1.25 mM, and flies consumed 10 times as much liquid with a concentration of 2.5 mM as with a concentration of 25 mM. This feeding reaction explained an apparent saturation type curve that we had obtained (unpublished) when the concentration of EMS in the feeding solution was plotted against the genetic effects. There is no justification in assuming linearity between exposure and dose to the germ line for chemical mutagens; therefore, the distinction between dose to the germ line and exposure of the organism is essential in developing dosim e t r y for comparative mutagenesis. Experimental protocols may be developed to give a linear relation between exposure and dose for a range of exposures, but this relation must be experimentally verified for each mutagen and protocol for exposure and cannot be assumed. When studying chemical mutagens dose must be determined within defined germ cell stages, for extreme differences in germ cell sensitivity are well established. For example, EMS induces a high frequency of mutations in late spermatid and mature spermatozoa, while inducing a low mutation frequency in spermatogonia [9]. In contrast, chloroethyl methanesulfonate induces a higher m u t a t i o n frequency in spermatogonia than in postmeiotic stages [8,16]. Loveless [15] has compiled a table of a number of alkylating agents that shows their varied effects on different germ cell stages. It has been found that the
313 range of response among different germ cell stages is equal to or possibly exceeds the limits of sensitivity of the genetic test [7]. In our first efforts at molecular dosimetry [13,20] we found differences in the amount of alkylation among different germ cell stages in Drosophila. A following paper [18] will discuss these differences with respect to the different germ cell stages in the mouse and the effect of repair following alkylation [17]. Because of cell stage specificity in both mutational response and products of alkylation we must identify the germ cell stage in all tests in order to make valid comparisons among test systems. There are several methods for dosimetry in the area of interest, the germ line. The earliest method determined the dose of a chemical mutagen by comparing its genetic effect with the effect of X-rays, the most studied mutagen of all. The difficulty of using a mutagen like X-rays as a measure of dose for other mutagens was recognized by Auerbach [3] in 1949 when she pointed out that this method of dosimetry depends on the similarity between X-ray induced lethals and chemically induced lethals. Unfortunately, most mutations induced by chemicals are different in some characteristics from those induced by radiation; for example, early experiments with mustard gas [4] showed there was a higher proportion of sex-linked recessive lethals to translocations than in the case of X-rays, and there was observed a high frequency of mosaics which are only rarely observed following treatment with X-rays. Because of differences in the mutational spectra of X-rays and chemical mutagens, the ratio between the mutational classes will change considerably. Table I shows a comparison of the frequency of mutations induced by X-ray with those induced by EMS. Both mutagens were tested using the same stocks and laboratory techniques. The Xray data were obtained from irradiating (250 KVP, 1-mm aluminum filter) 5day old males and allowing them to mate for one day following irradiation. Results of the same genetic test applied to progeny from males fed on 0.025 M EMS for 24 h, aged 24 h, and then allowed to mate for 24 h are also shown in Table I. Quantitative comparison of X-rays with chemical mutagens has been proposed as rem-equivalent-chemical (REC) [6] or rad-equivalent "radequiv" [5]. In computing the ltEC values in the appendix [6] of the Committee 17
TABLE I
Accumulated Controls 1820 tad X-ray 3640 tad X-ray 0 . 0 2 5 M EMS
F1 (% loss o f X or Y )
F2 (% l e t h a l s )
F3 (% l e t h a l s )
0.4 -+ 0.1 (5928) 2.4 + 0.3 (3047) 4.4 + 0.5 (1740) 1.2 -+ 0 . 2 (8227)
0.13 + 0.02 (21108) 5.1 -+ 0 . 4 (3189) 1 1 . 5 -+ 0.9 (1158) 43 -+ 1 ( 5 6 ) a (2390)
0 . 2 3 -+ 0 . 0 2 (13163) 0.9 -+ 0.2 (2833) 0.7 -+ 0.3 (926) 12 -+ 2 (491)
a C o r r e c t e d for t h e s a t u r a t i o n e f f e c t b y a s s u m i n g a Poisson d i s t r i b u t i o n o f m u t a t i o n s a m o n g t r e a t e d s p e r m cells,
314
Report there were questionable assumptions made in converting milligrams of hycanthone injected per fly to equivalent " h u m a n therapeutic doses (HTD)". Our work with radiolabeled mutagens suggests a number of problems in making this t y p e of calculation; these include non-linearity of exposure to dose as described above, rapid and variable loss of the labeled mutagen from the fly [19], and retention of the mutagenic agent in the fly past the time of exposure (unpublished), as well as basic differences in physiology. These problems of chemical dosimetry are largely avoided if, instead of expressing dose in terms of concentration, dose is expressed in terms of reaction products, as will be discussed later. However, even without a proper description of dose we can compare among the three genetic tests in Table I the tad equivalent (or for the X-ray treatment the rem equivalent) of the standard feeding procedure of 0.025 M EMS for 24 h [14] because all three genetic tests are sampled from the same pool of treated spermatozoa. The REC for this feeding procedure is 730 for loss of sex chromatin and 19,000 for sex-linked recessive lethals observed in the F2. For gonads mosaic for sex-linked recessive lethals (lethals detected in the F3) the computation of REC becomes ridiculous because the denominator, X-ray induced mosaics, is close to zero. Therefore, we obtain substantially different values for REC from the same pool of treated spermatozoa depending on which genetic test we use. Because of differences in the mutational spectra induced by EMS and by X-rays, the change in REC values was not surprising. It is not reasonable to compute a single ratio between one mutational spectrum and another if the spectra are quite different. Nevertheless, Auerbach in 1949 pointed o u t that using a class of mutations as a comparative measure of dose is "preferable to simple determination of the concentration in the outside media, and is also superior to dosage estimates based on toxicity for the exposed flies" [3]. As she points out, toxicity as a measure for mutagenicity may be very misleading because the mechanism for toxicity may be quite different from that for mutagenicity [3]. The relationship between toxicity and mutagenicity becomes especially complex in resistant strains. In essence what is needed is a physical measure of dose. It is only the germ cells in multi-cellular organisms that must be considered in the case of heritable mutations; therefore, only the a m o u n t of the mutagen that penetrates to the germ line is of genetic significance. The concentration of the active form of a mutagen in the immediate neighborhood of the genome has been defined as the Genetically Significant Concentration (GSC) by Committee 17 Report [6]. The concentration of a chemical mutagen in the gonad can be compared to the dose rate of ionizing radiation to the gonad; that is, the dose is the integral over time of the concentration. In a typical X-ray experiment the length of time is the time the organism is exposed to a constant intensity of radiation; however, for chemical mutagens, concentration varies with time. The curve of the GSC is time dependent and may be very complex and quite different for different mutagens. Also, due to retention of the chemical within the body, a GSC may persist b e y o n d the time when treatment stops (Fig. 1 ). To simplify dosimetry when there are variations in concentration with time following exposure to a chemical mutagen, I defined the dose for chemical mutagens as the a m o u n t of a chemical that reacts in the germ line with a selected
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GENETICALLY SIGNIFICANT CONCENTRATION
(
EXPOSURE
) TIME
)
Fig. 1. D o ~ as t h e i n ~ g r M o f the ~ m e d e p e n d e n t variable Genetically Significant C o n e e n ~ a t i o n ( G S C ) . T h e r e l a t i o n o f the GSC to t i m e i n t h m f i g u r e m h y p o t h e ~ c M .
target molecule (STM) [11]. Ideally one should select as the STM the genetically significant target (GST) [18] which is generally assumed to be DNA even though the genetically significant moiety of the DNA may not be known. By selecting target molecules that react with the chemical mutagen to produce stable products one can measure the accumulated products and thereby obtain a measure of dose. The selected reaction products (SRP) [12] chosen to measure the reaction of chemical agents with the STM should be not only stable and of sufficient quantity to permit accurate measurement within germ cell stages b u t also should be sufficiently uniform among species to permit dosimetry comparisons among species. The SRP may not be the actual products that cause mutational lesions. The actual p r o d u c t that causes the alteration of DNA may not be known, and if it is known, it m a y be chemically unstable and/or present in concentrations that are t o o low for detection with present techniques. Nevertheless, if the SRP can be correlated with the mutation frequency, the SRP can be an effective measure of dose. However, such a correlation between SRP and mutation frequency cannot be taken as evidence that the STM is actually the GST, for, as will be discussed by Aaron [ 1 ], there should be a constant relationship between alkylation of the GST and other nucleophilic sites within the subcellular genetic compartment. Although the experimentalist's choice of the SRP is somewhat arbitrary because of incomplete information, previous work suggests that the SRP could be alkylated DNA [11,13,20,21,22] or alkylated bases [10] which can be measured using radio tracer techniques. The actual correlation of mutation frequency with the SRP must be determined experimentally among several test species. If the SRP used as a measure of dose is sufficiently representative of nuclear events among species, it can then be hoped that a basis for comparative mutagenesis among species can be developed which is independent of comparative physiology. The next paper [1] by Aaron will discuss the chemical consideration in selecting appropriate target molecules and reaction products for molecular dosimetry.
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Acknowledgement This work is supported in part by PHS Research Grant No. ES 00320-08 from the National Institute of Environmental Health Sciences and the Energy Research and Development Administration Contract No. AT-(40-1)-3728. References 1 A a r o n , C.S., Molecular d o s i m e t r y of chemical m u t a g e n s . Selection of a p p r o p r i a t e t a r g e t m o l e c u l e s f o r d e t e r m i n i n g m o l e c u l a r dose t o t h e g e r m llne, M u t a t i o n Res., 38 ( 1 9 7 6 ) 3 0 3 - - 3 1 0 . 2 A a r o n , C.S. a n d W.R. Lee, R e j e c t i o n of e t h y l m e t h a n e s u l f o n a t e feeding s o l u t i o n by Drosophila melanogaster a d u l t males, s u b m i t t e d f o r p u b l i c a t i o n t o D r o s o p h i l a I n f o r m a t i o n Service. 3 A u e r b a c h , C., Chemical m u t a g e n e s i s , Biological Reviews, 24 ( 1 9 4 9 ) 3 5 5 - - 3 9 7 . 4 A u e r b a c h , C., J.M. R o b s o n a n d J.G. 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