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Molecular dosimetry of chemical mutagens
317
Mutation Research, 3 8 ( 1 9 7 6 ) 3 1 7 - - 3 2 6 © Elsevier Scientific Publishing Company,
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 * M E A S U R E M E N T OF M O L E C U L A R DOSE AND DNA R E P A I R IN MAMMALIAN GERM CELLS **
G A R Y A. S E G A
Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tenn. 37830 (U.S.A.) (Received December 8th, 1975) (Revision received April 13th, 1976) (Accepted May 13th, 1976)
Introduction At the most basic level, measurement of molecular dose is aimed at determining the number of chemical alterations (lesions) induced in the GST by an administered concentration of a chemical agent. Once this molecular dose has been established for genetic test systems it becomes possible to relate mutagenic response to the number of chemical alterations in the GST and to make valid comparisons between the mutagenic responses of different species to the chemical agent. Since DNA is generally assumed to be the GST, it will be used in this discussion as the STM referred to by Lee [17] and Aaron [1] in this Symposium, and alkylated DNA will be taken as the SRP. However, it should be kept in mind that other cellular targets m a y also have roles as GST's [39, 40]. An indirect approach to the study of t h e chemical dose received by the DNA in the germ cells of mice may be possible by making use of the recently demonstrated DNA repair which- occurs in certain meiotic and post-meiotic germ-cell stages of male mice following treatment with chemical mutagens [37]. Studies in our laboratory are currently being done to determine the level of DNA repair found in the germ cells as a function of administered concentrations of chemical mutagens. Some care must be exercised in such studies, however, be* S y m p o s i u m presented at the Sixth A n n u a l Meeting of the Environmental Mutagen Society, May 9--12, 1975, Miami Beach, Florida. ** Research jointly sponsored by the National Center for Toxicological Research and t h e U.S. Energy Research and Development Administration under contract with the Union Carbide Corporation. By acceptance of this article, t h e publisher or recipient acknowledges the right of t h e U.S. Government to retain a nonexclusive, royalty-free license in and t o any copyright covering the article. Abbreviations: GST, genetically significant target; STM, selected target molecule: SRP, selected reaction product: EMS, ethyl methanesulfonate: MMS, m e t h y l m e t h a n e s u l f o n a t e ; PMS, propyl methanesulfonate; IMS, isopropyl methanesulfonate.
318 cause not all germ-cell stages that have been studied undergo DNA repair [37], even though it is known that chemical mutagens can reach the DNA in such cells [39--41]. Ultimately the particular site or sites in the GST that are the direct cause of the mutagenic events need to be determined. For example, not all DNA bases are equally susceptible to alkylation, and different sites within the same DNA base show differential sensitivity to chemical attack depending on such factors as nucleophilicity [28], the involvement of Watson--Crick hydrogen bonding [15,19], the position of the site in the DNA double helix (wide or narrow groove) [14], etc. Right now, in vivo molecular dosimetry studies being carried out with male mice are still at the level of simply trying to determine the total number of chemical alterations occurring in the GST for a given administered concentration of a chemical agent and relating this total number of chemical alterations to the genetic effects observed. Molecular dosimetry in the germ cells of male mice In vivo alkylation of sperm heads In vivo molecular dosimetry studies with mice have been carried out in our laboratory [38--41] using [2-3H] ethyl methanesulfonate ([3H]EMS). In this study a single intraperitoneal injection of 200 mg/kg of [3H] EMS was given to (C3Hf × 101) F1 males 10--12 weeks old at the time of treatment. At each of 11 time points from 4 h to 16 days after the [3HI EMS injections, four mice were killed and the sperm from caput and caudal epididymides and vasa deferentia were recovered using procedures already described [41]. Sperm ethylations were determined from the specific activity of the [3H] EMS and from [2-3H]ethyl activity measured in samples of purified sperm heads by liquid scintillation counting. The number of ethylations per sperm reached a peak in the caput epididymis ~ 2 days post-injection, in the caudal epididymis at ~ 6 days, and in the vas at 9--10 days [39,40]. Most of the [3H] EMS reacts by 4 h after injection [8] so that the ethylations measured in the sperm of the epididymides and vas through 16 days post-treatment represent ethylations that occur within hours after treatment. The ethylation pattern for the vas sperm, over the 16-day period studied, closely parallels the pattern of dominant lethals induced in postmeiotic germ cells of treated males given similar doses of EMS [3,9,11]. When the dominantlethal frequency is low (1--3 days and 13--16 days after treatment) the sperm ethylations are relatively low, and when the dominant-lethal frequency is high (7--10 days after treatment) the sperm ethylations are also high. Thus there is a strong positive correlation between dominant-lethal frequency and the number of ethylations per sperm cell. It is n o t e w o r t h y that by 16 days after treatment with the [3H] EMS almost no ethylation can be detected in the sperm cells recovered from the vas. This is in spite of the fact that all germ-cell stages in the testes are apparently ethylated [8]. One possible explanation for the decrease in sperm ethylation in the vas by 16 days post-treatment is that histones which may be ethylated in the earlier germ-cell stages are removed and replaced by protamine as the germ cells
319 mature [22]. Another possibility is that ethylated DNA contained in the earlier germ-cell stages at the time of treatment may undergo excision repair before these germ cell stages have matured into spermatozoa in the vas. More will be said below a b o u t the repair of alkylated germ-cell DNA.
In vivo alkylation o f sperm DNA After the number of ethylations per sperm in the vas was determined as a function of time after injection with the [3H] EMS, the DNA was extracted from the sperm heads recovered from 4 h through 10 days after treatment [39, 40]. The extraction procedures that were used assured the removal of at least 99% of the protamine and other proteins present and resulted in no preferential recovery of either (G + C)-rich or (A + T)-rich regions of the mouse genome [41]. When the ethylations per nucleotide were determined for these vas sperm, it was found that the number of DNA ethylations stayed at a relatively constant value of between 2 to 3 • 10 -s ethylations per nucleotide for all of the time points studied. Although it is possible that some ethylated DNA bases may have been lost in the recovery of the vas sperm DNA, especially 3-ethyladenine and 7-ethylguanine [15], the decrease in the amount of ethyl-[2-3H] associated with the sperm DNA compared with that present in the whole sperm cells is so large that it does not seem likely that selective loss of ethylated DNA in the isolation procedures could account for the difference. Since it appears that the increased number of ethylations found in the vas sperm at 9--10 days post-injection are n o t due to increased ethylation of DNA, it is possible that ethylation of another nuclear component, such as protamine, may account for the increase. Possible alkylation o f sperm protamine Mouse protamine is an attractive candidate as a target for ethylation because of the nucleophilic sites it contains. It possesses a high proportion of the basic amino acids arginine and lysine and also contains aspartate and glutamate [13]. The free NH2 groups of the basic amino acids and the free carboxy groups of the acidic amino acids are potential sites for ethylation [30]. In addition, mammalian sperm protamine obtained from the bull has been found to contain cysteine [6], and were have evidence that mouse protamine also contains cysteine [36]; the sulfhydryl group of this amino acid is another potential site for alkylation [43]. Thus, it is reasonable to consider ethylation of protamine as a possible cause of at least a portion of the dominant lethals produced by EMS in the mouse. In fact t h e germ-cell stages most sensitive to the action of EMS are those that have just replaced the usual chromosomal histones with protamine at the time of treatment [13,22,38]. As dominant lethals are generally attributed to chromosomal aberrations, it is possible that ethylated protamine could lead to dominant lethals through a weakening of the chromatin structure, perhaps by ethylating the sulfhydryl groups of cysteine and blocking normal disulfide bond formation in the condensing sperm nucleus. From the results discussed above it is apparent that the GST for the induction of dominant lethals in the germ cells of male mice b y EMS has not yet been clearly established. Although the germ-ceU D N A is the most likely GST, it m a y n o t be the only one. Until the relationship between the extent of prot-
320 amine ethylation and dominant-lethal frequency is established, it will not be possible to say whether or not mouse protamine is a GST. Therefore, the possible role of mouse protamine in the induction of dominant lethals by various alkylating agents is being investigated in our laboratory. DNA repair in the germ cells of male mice Another important area of investigation in molecular mechanisms of mutagenesis in mammals is repair of damaged germ-cell DNA. Repair studies may also prove useful as an indirect measure of the molecular dose received by the DNA, for as the molecular dose is raised the number of DNA lesions and the amount of repair should also increase. Of course at very high doses the repair system may be inhibited, and a reduction in repair could occur. In general, though, repair of damaged germ-cell DNA after exposure to a suspected chemical mutagen would immediately indicate that the chemical agent or a metabolite was able to penetrate the nucleus of the germ cells and produce "repairable" lesions in the DNA. It is possible to study DNA repair in meiotic and post-meiotic germ cells of male mice by making use of the well-studied sequence of events that occur during spermatogenesis and spermiogenesis [21,23,24]. In developing male germ cells the last DNA synthesis takes place during a 14-h period [21] in preleptotene spermatocytes. After DNA synthesis the spermatocytes continue to develop through spermatogenesis and spermiogenesis for a b o u t 28--30 days [23,24] before the late spermatids (young spermatozoa) finally leave the testes and enter the caput epididymides. Two to three days later the maturing sperm reach the caudal epididymides, and in two or three days more they enter the vasa deferentia. If, at any meiotic or postmeiotic germ-cell stage, the DNA is damaged and repair occurs, the repair can be detected by an unscheduled incorporation of [3H] thymidine ([3H] dT) which is measured after the developing cells have left the testes and entered the remainder of the reproductive tract. The basic design of the experiment to establish if a chemical agent is capable of inducing a DNA repair response in mouse germ cells is straightforward. Two groups of (C3Hf × 101)F1 male mice, 10--14 weeks old at the start of the experiment, are both given testicular injections of [3H] dT (usually between 70 and 100 pCi per animal) to maximize DNA labeling in the testes. Each animal from one group is also given an intraperitoneal injection of the chemical agent under study, usually dissolved in a carrier solution such as Hanks' balanced salts, while each animal from the second group receives an equivalent injection of the carrier solution alone. Then at various times after treatment, four animals from each group are killed and the [3H] dT activity in the sperm recovered from the reproductive tracts is determined by liquid scintillation counting. These procedures have already been used to study the ability of meiotic and post-meiotic germ-cell stages of male mice to "repair" DNA lesions induced by EMS [37]. (The use of the word "repair" does not necessarily imply that the original state of the DNA molecule is restored, b u t merely that some process is acting which attempts to reverse damage to DNA.) When a 250 mg/kg dose of EMS was used, an unscheduled presence of [3H] dT was seen in sperm found in
321 the vas from 14 to 27 days after treatment. Sperm from control animals showed no such uptake of labeled thymidine during the same period. The time interval when the vas sperm showed the unscheduled presence of [3H] dT established that germ-cell stages from early to middle meiotic prophase through early to middle spermatid were able to repair EMS-induced lesions in DNA. Isolation and analysis of purified DNA from sperm cells recovered from the vas verified that the 3H label was, indeed, being incorporated into the germ-cell DNA of the treated animals [37]. Another finding was that the most advanced germ-cell stages {late spermatids to mature spermatozoa), including those which give rise to EMS-induced dominant-lethal mutations [3,9,11], do not appear capable of repairing DNA lesions, although there is no question that the DNA in these germ cells is being ethylated [38--41]. While one might like to postulate that EMS-induced dominant lethals may result, at least in part, from failure of DNA repair to occur, the time correlation may be simply coincidental. Other chemical agents may show entirely different relationships between dominant-lethal patterns and repair of lesions in germ-cell DNA. For example, IMS induces dominant-lethal mutations in early spermatid stages [10] that exhibit DNA repair, although the frequency of dominant lethals is lower than in more mature germ-cell stages not showing DNA repair. A possibly more general finding is the observation that after EMS- or X-ray treatment (see below) no DNA repair is seen in the male germ cells in which protamine has replaced the usual chromosomal histones [37,38]. The protamine begins to appear in mid-spermatid stages and is present through all the later germ-cell stages [22]. It may be that once protamine replaces the chromosomal histones and the chromatin becomes tightly packed [18], any damaged DNA is no longer accessible to the repair system. An alternative explanation is that the repair system itself is no longer present and functioning in the later germ-cell stages. Most of the cytoplasm of the developing spermatids is eventuaUy lost [22], and the repair machinery of the germ cells might also be lost at this time. Studies of DNA repair after exposure of male mice to 600 R of X-rays have shown that repair occurs in the same meiotic and postmeiotic germ-cell stages that show repair after EMS treatments although the level of unscheduled incorporation of [3H]dT after X-ray treatment is much less (Fig. 1) [38]. This is in spite of the fact that a 250 mg/kg dose of EMS and 600-R of X-rays both produce 50% or more dominant-lethal mutations in the sensitive germ-cell stages [ 11,12,32,35]. The 600-R X-ray dose is not inhibiting the repair process, because we find an elevated level of repair when 1200 R is used. Painter [27] has estimated that for every single-strand break induced in DNA by X-rays, between two and three bases are inserted into the repaired site, while Roberts [29] estimates that for every alkylation-induced lesion in DNA, approximately 100 bases are inserted in the repaired site. It is, therefore, possible that the much lower level of DNA repair measured in male germ cells using 600 R of Xrays compared with 250 mg/kg of EMS is a manifestation of the number of bases inserted into the repaired lesions. The occurrence of DNA repair in some germ-cell stages of male mice but not in others gives the mammalian geneticist a method for determining whether
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VAS SPERM :~ r.z:
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I EMS 25Omg/kg /1 /
Z
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_o
40-
F- 20,q
,~ /
X RAY . I . 600 R
0 ~--ql--~--ct~ . . . . . 3 9 15 2f DAYS AFTER TREATMENT
27
Fig, 1. T h e r e s p o n s e p a t t e r n o f m e i o t i c a n d p o s t m e i o t i e germ-cell stages of m a l e m i c e t o t h e i n d u c t i o n of u n s c h e d u l e d D N A s y n t h e s i s a f t e r t r e a t m e n t w i t h X - r a y s (o) or EMS (o). 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 v a r i o u s germ-ceU stages is a s s a y e d b y m e a s u r i n g t h e u n s c h e d u l e d p r e s e n c e of [ 3 H I d T in t h e g e r m cells a f t e r t h e y h a v e m a t u r e d t o s p e r m a t o z o a in t h e vasa d e f e r e n t i a . C o n t r o l values for all t i m e p o i n t s cons i d e r e d are in the base llne. T h e e r r o r bars r e p r e s e n t +-1 s t a n d a r d d e v i a t i o n .
the mammalian repair system in vivo is error-free. For example, Russell's specific-locus test [31] has been used to look for point mutations in the mouse after EMS treatment. Six presumed specific-locus mutations were observed in 10,941 offspring derived from cells that were in post-spermatogonial stages at the time of treatment [33]. However, most of the data and all of the mutants came from the first 2 weeks post-injection [34], when the sperm in the vas show no unscheduled presence of [3H] dT after EMS treatment. It would be very informative to compare the specific-locus m u t a t i o n frequency for progeny conceived from 16 to 24 days after EMS treatment (when the sperm in the vas do show an unscheduled presence of [3H] dT) with the m u t a t i o n frequency found for progeny conceived in the first 2 weeks after EMS treatment. Such a comparison might shed some light on whether the mammalian repair system is actually correcting premutational DNA lesions. Since establishment of the meiotic and post-meiotic germ-ceU stages that undergo DNA repair when exposed to EMS, three chemical homologs -- MMS, PMS and IMS -- have also been studied for their ability to induce DNA repair in mouse germ cells [42]. In particular, sperm from the caudal epididymides were studied 16 days after treatment. (Caudal sperm are used because there are about twice as many per animal compared with the a m o u n t of sperm in the caput epididymis or in the vas.) This means that at the time of treatment these caudal sperm were early spermatids [23,24,38]. For each of these chemicals a dose-response curve has been obtained which measures the relative, unscheduled uptake of [3HI dT into early spermatids as a function of administered concentration of the chemical mutagen. Also, the relative a m o u n t of repair occurring in early spermatids over a 3-day period following administration of each chemical has been studied by injecting [3H]dT at various times after chemical treatment. The duration of repair could be
323 studied in this way since we have found that after injection of the [3H]dT its removal from the thymidine pool of the testes is quite rapid, with most of the free [3H] dT disappearing from the testes within a b o u t 1 h after injection [37]. This result is in agreement with the work of others using mammalian systems to measure the kinetics of incorporation of [3H] dT [4,5]. When [3HI dT was administered simultaneously with each of the four chemical mutagens there was an unscheduled uptake of the label into the DNA of early spermatids. This means that all four of these mutagens must reach the DNA of early spermatids and begin producing repairable lesions within 1 h after treatment. However, as discussed below, repair can extend over several days. The dose-response effects of all four chemicals in the induction of germcell DNA repair appear to be linear over the dose ranges studied (MMS, 5--75 mg/kg; EMS, 10--300 mg/kg; PMS, 50--700 mg/kg; IMS, 10--170 mg/kg.) The greatest DNA repair response (unscheduled [3H]dT uptake) was seen when MMS was used, followed by EMS, IMS, and finally PMS. MMS, EMS, and PMS have the same relative order of effectiveness in the induction of dominantlethal mutations in sperm and late spermatids [3,9,10,11]. However, IMS falls between MMS and EMS in this case [10]. Comparing MMS and EMS, for example, it was found that at equimolar administered concentrations of the two chemicals, MMS was a b o u t 4 times as effective as EMS in inducing DNA repair in early spermatids and also a b o u t 4 times as effective as EMS in producing dominant-lethal mutations in sperm and late spermatids [9]. A similar relationship has been found between these two chemicals in the induction of DNA repair in cultured mammalian cells [29], although in that case the difference was tenfold. Roberts et al. [29] have found that in their cultured mammalian cells "repair synthesis" is directly related to the overall level of DNA alkylation in the cells. If the same result holds true for the mouse germ cells treated in vivo, then measurement of DNA repair in these germ cells may become a practical way of determining chemical dose in the genetic material once relationships have been established between the number (and types) of chemical lesions in DNA and the a m o u n t of repair induced. On the basis of the lowest injected chemical dose which can cause a measurable effect, DNA repair in mouse germ cells appears to be a considerably
TABLE I C O M P A R I S O N O F T H E L O W E S T C H E M I C A L D O S E S O F MMS, EMS, PMS, A N D IMS N E E D E D T O DETECT DOMINANT-LETHAL MUTATIONS OR TRANSLOCATIONS WITH DOSES THAT STILL INDUCE DNA REPAIR Chemical
M i n i m u m dose (mg/kg) for d o m i n a n t lethal * or translocation t detection
D N A r e p a i r in g e r m ceils still r e a d i l y m e a s u r e d at a d o s e (mg/kg) of:
MMS EMS
~50 ~150 ~50 ~400 ~50
5 10
PMS IMS
* [ref.9] * [ref. 12] t" [ r e f . 1 2 ] * [ref. 10] ( [ref.lO]
50 10
324 more sensitive biological end point than measurement of dominant lethals or translocations. Table I shows that the measurement of DNA repair is at least 5--10 times more sensitive than the dominant-lethal tests or translocation studies using these four chemical mutagens. However, it should be stressed that the germ-cell stages showing peak sensitivity to the induction of dominant lethals with these four chemicals are stages where the DNA repair assay would be insensitive. The results from the study of the duration of DNA repair in early spermatids of the mouse after treatment with MMS, EMS, PMS, and IMS indicate that as long as 3 days after exposure to these chemicals the early spermatid stages are still undergoing measurable DNA repair. This extended repair is n o t due to continuing alkylation of the DNA during this time period, because these four chemical homologs are quite reactive in aqueous solutions [26] and even more reactive in biological systems [8], probably disappearing within several hours, at most, after injection. For MMS and EMS there is a rapid decrease in the level of DNA repair in the early spermatids in the first half-day following treatment. This is followed by a much slower, exponential decrease in the level of repair o u t to 3 days posttreatment. The curves suggest that the a m o u n t of repair occurring between 1/2 and 3 days post-treatment is simply proportional to the number of repairable lesions still present in the DNA. The half-life for the DNA repair occurring between 1/2 and 3 days posttreatment with MMS and EMS is between 1 and 3 days. Craddock [7] and Margison et al. [20] have both found the removal of 7-methylguanine from rat liver DNA to be exponential, with a half-life of 1 [ref. 7] to 3 [ref. 20] days. It is possible that the decrease in repair with time could be explained by removal of 7-alkylguanine from the germ-cell DNA, especially since the N-7 position of guanine should be the most frequently alkylated site in the DNA [2,15]. However, other alkylation products in the DNA, such as 3-alkyladenine, O6-alkylguanine, various alkylpyrimidines, and phosphotriesters may also be targets for DNA repair [16,25]. Another aspect of the study of chemical dosimetry and DNA repair in mouse germ cells is the question of the proportion of the germ-cell population that is damaged by a particular chemical agent and the proportion of cells that can undergo repair. Is it 100% of the cell population or something less than this? This question cannot be answered by liquid scintillation counting, so autoradiographs were also made of sperm samples taken from each mouse. The results indicated that while most of the sperm from each animal had the expected number of grains, a portion showed either considerably fewer or more grains than would be expected from a Poisson distribution. Earlier work with EMS [37] had shown that the unscheduled presence of [3H]dT in the caudal sperm had reached a constant level by 16 days posttreatment which was maintained through 24 days post-treatment. Thus on the average, all treated sperm cells in the caudal epididymides 16 days after treatment had incorporated [3HI dT. The sperm present in the caudal epididymides at that time (16 days) did n o t represent a mixture of germ-cell stages, some of which showed DNA repair and some of which did not. Therefore, the deviations from a Poisson distribution seen in the frequencies of grains over the chemically-treated sperm are not likely due to differences in sensitivities of the
325
germ-cell stages represented in the caudal epidymides at 16 days post-treatment. It will be important in future work studying the molecular mechanisms of mutagenesis in mammalian germ cells to determine what biochemical events are occurring in these small fractions of the germ-cell populations. Are the numbers of chemically induced lesions in some cells fewer (or much more) than the average, and is the repair of chemically induced lesions in the DNA of these germ cells much less efficient (or much more efficient) than the average? References 1 A a r o n , C.S., M o l e c u l a r d o s i m e t r y o f c h e m i c a l m u t a g e n s . S e l e c t i o n o f 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 d o s e t o t h e g e r m line, M u t a t i o n Res., 3 8 ( 1 9 7 6 ) 3 0 3 - - 3 1 0 . 2 B r o o k e s , P. a n d P.D. L a w l e y , T h e r e a c t i o n o f m o n o - a n d d i f u n c t i o n a l a l k y l a t i n g a g e n t s w i t h n u c l e i c a c i d s , B i o c h e m . J., 8 0 ( 1 9 6 1 ) 4 9 6 - - 5 0 3 . 3 C a t t a n a c h , B.M., C.E. P o l l a r d a n d J . H . I s a a c s o n , E t h y l m e t h a n e s u l f o n a t e - i n d u c e d c h r o m o s o m e b r e a k a g e in t h e m o u s e , M u t a t i o n Res., 6 ( 1 9 6 8 ) 2 9 7 - - 3 0 7 . 4 C h a n g , L . O . a n d W.B. L o o n e y , A b i o c h e m i c a l a n d a u t o r a d i o g r a p h i c s t u d y o f t h e in vivo u t i l i z a t i o n o f t r i t i a t e d t h y m i d i n e i n r e g e n e r a t i n g r a t liver, C a n c e r Res., 2 5 ( 1 9 6 5 ) 1 8 1 7 - - 1 8 2 2 . 5 Cleaver, J . E . , T h y m i d i n e m e t a b o l i s m a n d cell k i n e t i c s , in A. N e u b e r g c r a n d E.L. T a t u m (eds.), F r o n tiers in B i o l o g y , Vol. 6 , J o h n Wiley a n d S o n s , N e w Y o r k , 1 9 6 7 , p p . 5 7 - - 5 9 . 6 Coelingh, J.P., T.H. Rozijn and C.H. Monfoort, Isolation and partial characterization of a basic protein from bovine sperm heads, Biochim. Biophys. Acta, 188 (1969) 353--356. 7 C r a d d o c k , V.M., T h e P a t t e r n o f m e t h y l a t e d p u r i n e s f o r m e d i n D N A o f i n t a c t a n d r e g e n e r a t i n g liver of rats treated with the carcinogen dimethyinitrosamine, Biochim. Biophys. Acta, 312 (1973) 202-210. 8 C u m m i n g , R . B . a n d M . F . W a l t o n , F a t e a n d m e t a b o l i s m o f s o m e m u t a g e n i c a l k y l a t i n g a g e n t s in t h e m o u s e . I. E t h y l m e t h a n e s u l f o n a t e a n d m e t h y l m e t h a n e s u l f o n a t e a t s u b l e t h a l d o s e in h y b r i d m a l e s , Mutation Res., 10(1970) 365---377. 9 E h l l n g , U . H . , R . B . C u m m i n g a n d HH.V. Mailing, I n d u c t i o n o f d o m i n a n t l e t h a l m u t a t i o n s b y a l k y l a t i n g a g e n t s in m a l e m i c e , M u t a t i o n Res., 5 ( 1 9 6 8 ) 4 1 7 - - 4 2 8 . 1 0 E h l i n g , U . H . , D . G . D o h e r t y a n d H . V . Mailing, D i f f e r e n t i a l s p e r m a t o g e n i c r e s p o n s e o f m i c e t o t h e induction of dominant-lethal mutations by n-propyl methanesulfonate and isopropyl methanesulfonate, Mutation Res., 15 (1972) 175--184. 11 G e n e r o s o , W.M. a n d W.L. R u s s e l l , S t r a i n a n d s e x v a r i a t i o n in t h e s e n s i t i v i t y o f m i c e t o d o m i n a n t lethal induction with ethyl methanesulfonate, Mutation Res., 8 (1969) 589--598. 1 2 G e n e r o s o , W.M., W.L. R u s s e l l , S.W. H u f f , S.K. S t o u t a n d D . G . Gosslee, E f f e c t s o f d o s e o n t h e ind u c t i o n o f d o m i n a n t - l e t h a l m u t a t i o n s a n d h e r i t a b l e t r a n s i o c a t i o n s w i t h e t h y l m e t h a n e s u l f o n a t e in male mice, Genetics, 77 (1974) 741--752. 13 Lain, D.M.K. and W.R. Bruce, The biosynthesis of protamine during spermatogenesis of the mouse. E x t r a c t i o n , p a r t i a l c h a r a c t e r i z a t i o n a n d site o f s y n t h e s i s , J. Cell P h y s i o l . , 7 8 ( 1 9 7 1 ) 1 3 - - 2 4 . 14 Lawley, P.D., Some chemical aspects of dose-response relationships in alkylation mutagenesis, Mutation Res., 23 (1974) 283--295. 1 5 L a w l e y , P . D . a n d P. B r o o k e s , F u r t h e r s t u d i e s o n t h e a l k y l a t i o n o f n u c l e i c a c i d s a n d t h e i r c o n s t i t u e n t n u c l e o t i d e s , B i o c h e m . J., 8 9 ( 1 9 6 3 ) 1 2 7 - - 1 3 8 . 16 L a w l e y , P.D., a n d D.J. 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A . , u n p u b l i s h e d d a t a . 3 9 S e g a , G . A . a n d R . B . C u m m i n g , E t h y l a t i o n p a t t e r n in m o u s e s p e r m as a f u n c t i o n o f d e v e l o p m e n t a l s t a g e a n d t i m e a f t e r e x p o s u r e t o 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., 2 6 ( 1 9 7 5 ) 4 4 8 - - - 4 4 9 . 4 0 Sega, G . A . , R . B . C u m m i n g a n d M . F . W a l t o n , E t h y l a t i o n p a t t e r n in m o u s e s p e r m as a f u n c t i o n of dev e l o p m e n t a l s t a g e a n d t i m e a f t e r e x p o s u r e t o e t h y l m e t h a n e s u l f o n a t e , Biol. Div. A n n u . P r o g r . R e p . June 30, 1974, ORNL-4993, pp. 126--127. 4 1 Sega, G . A . , R . B . C u m m i n g a n d M . F . 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Mutation Research, 3 8 ( 1 9 7 6 ) 3 1 7 - - 3 2 6 © Elsevier Scientific Publishing Company,
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 * M E A S U R E M E N T OF M O L E C U L A R DOSE AND DNA R E P A I R IN MAMMALIAN GERM CELLS **
G A R Y A. S E G A
Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tenn. 37830 (U.S.A.) (Received December 8th, 1975) (Revision received April 13th, 1976) (Accepted May 13th, 1976)
Introduction At the most basic level, measurement of molecular dose is aimed at determining the number of chemical alterations (lesions) induced in the GST by an administered concentration of a chemical agent. Once this molecular dose has been established for genetic test systems it becomes possible to relate mutagenic response to the number of chemical alterations in the GST and to make valid comparisons between the mutagenic responses of different species to the chemical agent. Since DNA is generally assumed to be the GST, it will be used in this discussion as the STM referred to by Lee [17] and Aaron [1] in this Symposium, and alkylated DNA will be taken as the SRP. However, it should be kept in mind that other cellular targets m a y also have roles as GST's [39, 40]. An indirect approach to the study of t h e chemical dose received by the DNA in the germ cells of mice may be possible by making use of the recently demonstrated DNA repair which- occurs in certain meiotic and post-meiotic germ-cell stages of male mice following treatment with chemical mutagens [37]. Studies in our laboratory are currently being done to determine the level of DNA repair found in the germ cells as a function of administered concentrations of chemical mutagens. Some care must be exercised in such studies, however, be* S y m p o s i u m presented at the Sixth A n n u a l Meeting of the Environmental Mutagen Society, May 9--12, 1975, Miami Beach, Florida. ** Research jointly sponsored by the National Center for Toxicological Research and t h e U.S. Energy Research and Development Administration under contract with the Union Carbide Corporation. By acceptance of this article, t h e publisher or recipient acknowledges the right of t h e U.S. Government to retain a nonexclusive, royalty-free license in and t o any copyright covering the article. Abbreviations: GST, genetically significant target; STM, selected target molecule: SRP, selected reaction product: EMS, ethyl methanesulfonate: MMS, m e t h y l m e t h a n e s u l f o n a t e ; PMS, propyl methanesulfonate; IMS, isopropyl methanesulfonate.
318 cause not all germ-cell stages that have been studied undergo DNA repair [37], even though it is known that chemical mutagens can reach the DNA in such cells [39--41]. Ultimately the particular site or sites in the GST that are the direct cause of the mutagenic events need to be determined. For example, not all DNA bases are equally susceptible to alkylation, and different sites within the same DNA base show differential sensitivity to chemical attack depending on such factors as nucleophilicity [28], the involvement of Watson--Crick hydrogen bonding [15,19], the position of the site in the DNA double helix (wide or narrow groove) [14], etc. Right now, in vivo molecular dosimetry studies being carried out with male mice are still at the level of simply trying to determine the total number of chemical alterations occurring in the GST for a given administered concentration of a chemical agent and relating this total number of chemical alterations to the genetic effects observed. Molecular dosimetry in the germ cells of male mice In vivo alkylation of sperm heads In vivo molecular dosimetry studies with mice have been carried out in our laboratory [38--41] using [2-3H] ethyl methanesulfonate ([3H]EMS). In this study a single intraperitoneal injection of 200 mg/kg of [3H] EMS was given to (C3Hf × 101) F1 males 10--12 weeks old at the time of treatment. At each of 11 time points from 4 h to 16 days after the [3HI EMS injections, four mice were killed and the sperm from caput and caudal epididymides and vasa deferentia were recovered using procedures already described [41]. Sperm ethylations were determined from the specific activity of the [3H] EMS and from [2-3H]ethyl activity measured in samples of purified sperm heads by liquid scintillation counting. The number of ethylations per sperm reached a peak in the caput epididymis ~ 2 days post-injection, in the caudal epididymis at ~ 6 days, and in the vas at 9--10 days [39,40]. Most of the [3H] EMS reacts by 4 h after injection [8] so that the ethylations measured in the sperm of the epididymides and vas through 16 days post-treatment represent ethylations that occur within hours after treatment. The ethylation pattern for the vas sperm, over the 16-day period studied, closely parallels the pattern of dominant lethals induced in postmeiotic germ cells of treated males given similar doses of EMS [3,9,11]. When the dominantlethal frequency is low (1--3 days and 13--16 days after treatment) the sperm ethylations are relatively low, and when the dominant-lethal frequency is high (7--10 days after treatment) the sperm ethylations are also high. Thus there is a strong positive correlation between dominant-lethal frequency and the number of ethylations per sperm cell. It is n o t e w o r t h y that by 16 days after treatment with the [3H] EMS almost no ethylation can be detected in the sperm cells recovered from the vas. This is in spite of the fact that all germ-cell stages in the testes are apparently ethylated [8]. One possible explanation for the decrease in sperm ethylation in the vas by 16 days post-treatment is that histones which may be ethylated in the earlier germ-cell stages are removed and replaced by protamine as the germ cells
319 mature [22]. Another possibility is that ethylated DNA contained in the earlier germ-cell stages at the time of treatment may undergo excision repair before these germ cell stages have matured into spermatozoa in the vas. More will be said below a b o u t the repair of alkylated germ-cell DNA.
In vivo alkylation o f sperm DNA After the number of ethylations per sperm in the vas was determined as a function of time after injection with the [3H] EMS, the DNA was extracted from the sperm heads recovered from 4 h through 10 days after treatment [39, 40]. The extraction procedures that were used assured the removal of at least 99% of the protamine and other proteins present and resulted in no preferential recovery of either (G + C)-rich or (A + T)-rich regions of the mouse genome [41]. When the ethylations per nucleotide were determined for these vas sperm, it was found that the number of DNA ethylations stayed at a relatively constant value of between 2 to 3 • 10 -s ethylations per nucleotide for all of the time points studied. Although it is possible that some ethylated DNA bases may have been lost in the recovery of the vas sperm DNA, especially 3-ethyladenine and 7-ethylguanine [15], the decrease in the amount of ethyl-[2-3H] associated with the sperm DNA compared with that present in the whole sperm cells is so large that it does not seem likely that selective loss of ethylated DNA in the isolation procedures could account for the difference. Since it appears that the increased number of ethylations found in the vas sperm at 9--10 days post-injection are n o t due to increased ethylation of DNA, it is possible that ethylation of another nuclear component, such as protamine, may account for the increase. Possible alkylation o f sperm protamine Mouse protamine is an attractive candidate as a target for ethylation because of the nucleophilic sites it contains. It possesses a high proportion of the basic amino acids arginine and lysine and also contains aspartate and glutamate [13]. The free NH2 groups of the basic amino acids and the free carboxy groups of the acidic amino acids are potential sites for ethylation [30]. In addition, mammalian sperm protamine obtained from the bull has been found to contain cysteine [6], and were have evidence that mouse protamine also contains cysteine [36]; the sulfhydryl group of this amino acid is another potential site for alkylation [43]. Thus, it is reasonable to consider ethylation of protamine as a possible cause of at least a portion of the dominant lethals produced by EMS in the mouse. In fact t h e germ-cell stages most sensitive to the action of EMS are those that have just replaced the usual chromosomal histones with protamine at the time of treatment [13,22,38]. As dominant lethals are generally attributed to chromosomal aberrations, it is possible that ethylated protamine could lead to dominant lethals through a weakening of the chromatin structure, perhaps by ethylating the sulfhydryl groups of cysteine and blocking normal disulfide bond formation in the condensing sperm nucleus. From the results discussed above it is apparent that the GST for the induction of dominant lethals in the germ cells of male mice b y EMS has not yet been clearly established. Although the germ-ceU D N A is the most likely GST, it m a y n o t be the only one. Until the relationship between the extent of prot-
320 amine ethylation and dominant-lethal frequency is established, it will not be possible to say whether or not mouse protamine is a GST. Therefore, the possible role of mouse protamine in the induction of dominant lethals by various alkylating agents is being investigated in our laboratory. DNA repair in the germ cells of male mice Another important area of investigation in molecular mechanisms of mutagenesis in mammals is repair of damaged germ-cell DNA. Repair studies may also prove useful as an indirect measure of the molecular dose received by the DNA, for as the molecular dose is raised the number of DNA lesions and the amount of repair should also increase. Of course at very high doses the repair system may be inhibited, and a reduction in repair could occur. In general, though, repair of damaged germ-cell DNA after exposure to a suspected chemical mutagen would immediately indicate that the chemical agent or a metabolite was able to penetrate the nucleus of the germ cells and produce "repairable" lesions in the DNA. It is possible to study DNA repair in meiotic and post-meiotic germ cells of male mice by making use of the well-studied sequence of events that occur during spermatogenesis and spermiogenesis [21,23,24]. In developing male germ cells the last DNA synthesis takes place during a 14-h period [21] in preleptotene spermatocytes. After DNA synthesis the spermatocytes continue to develop through spermatogenesis and spermiogenesis for a b o u t 28--30 days [23,24] before the late spermatids (young spermatozoa) finally leave the testes and enter the caput epididymides. Two to three days later the maturing sperm reach the caudal epididymides, and in two or three days more they enter the vasa deferentia. If, at any meiotic or postmeiotic germ-cell stage, the DNA is damaged and repair occurs, the repair can be detected by an unscheduled incorporation of [3H] thymidine ([3H] dT) which is measured after the developing cells have left the testes and entered the remainder of the reproductive tract. The basic design of the experiment to establish if a chemical agent is capable of inducing a DNA repair response in mouse germ cells is straightforward. Two groups of (C3Hf × 101)F1 male mice, 10--14 weeks old at the start of the experiment, are both given testicular injections of [3H] dT (usually between 70 and 100 pCi per animal) to maximize DNA labeling in the testes. Each animal from one group is also given an intraperitoneal injection of the chemical agent under study, usually dissolved in a carrier solution such as Hanks' balanced salts, while each animal from the second group receives an equivalent injection of the carrier solution alone. Then at various times after treatment, four animals from each group are killed and the [3H] dT activity in the sperm recovered from the reproductive tracts is determined by liquid scintillation counting. These procedures have already been used to study the ability of meiotic and post-meiotic germ-cell stages of male mice to "repair" DNA lesions induced by EMS [37]. (The use of the word "repair" does not necessarily imply that the original state of the DNA molecule is restored, b u t merely that some process is acting which attempts to reverse damage to DNA.) When a 250 mg/kg dose of EMS was used, an unscheduled presence of [3H] dT was seen in sperm found in
321 the vas from 14 to 27 days after treatment. Sperm from control animals showed no such uptake of labeled thymidine during the same period. The time interval when the vas sperm showed the unscheduled presence of [3H] dT established that germ-cell stages from early to middle meiotic prophase through early to middle spermatid were able to repair EMS-induced lesions in DNA. Isolation and analysis of purified DNA from sperm cells recovered from the vas verified that the 3H label was, indeed, being incorporated into the germ-cell DNA of the treated animals [37]. Another finding was that the most advanced germ-cell stages {late spermatids to mature spermatozoa), including those which give rise to EMS-induced dominant-lethal mutations [3,9,11], do not appear capable of repairing DNA lesions, although there is no question that the DNA in these germ cells is being ethylated [38--41]. While one might like to postulate that EMS-induced dominant lethals may result, at least in part, from failure of DNA repair to occur, the time correlation may be simply coincidental. Other chemical agents may show entirely different relationships between dominant-lethal patterns and repair of lesions in germ-cell DNA. For example, IMS induces dominant-lethal mutations in early spermatid stages [10] that exhibit DNA repair, although the frequency of dominant lethals is lower than in more mature germ-cell stages not showing DNA repair. A possibly more general finding is the observation that after EMS- or X-ray treatment (see below) no DNA repair is seen in the male germ cells in which protamine has replaced the usual chromosomal histones [37,38]. The protamine begins to appear in mid-spermatid stages and is present through all the later germ-cell stages [22]. It may be that once protamine replaces the chromosomal histones and the chromatin becomes tightly packed [18], any damaged DNA is no longer accessible to the repair system. An alternative explanation is that the repair system itself is no longer present and functioning in the later germ-cell stages. Most of the cytoplasm of the developing spermatids is eventuaUy lost [22], and the repair machinery of the germ cells might also be lost at this time. Studies of DNA repair after exposure of male mice to 600 R of X-rays have shown that repair occurs in the same meiotic and postmeiotic germ-cell stages that show repair after EMS treatments although the level of unscheduled incorporation of [3H]dT after X-ray treatment is much less (Fig. 1) [38]. This is in spite of the fact that a 250 mg/kg dose of EMS and 600-R of X-rays both produce 50% or more dominant-lethal mutations in the sensitive germ-cell stages [ 11,12,32,35]. The 600-R X-ray dose is not inhibiting the repair process, because we find an elevated level of repair when 1200 R is used. Painter [27] has estimated that for every single-strand break induced in DNA by X-rays, between two and three bases are inserted into the repaired site, while Roberts [29] estimates that for every alkylation-induced lesion in DNA, approximately 100 bases are inserted in the repaired site. It is, therefore, possible that the much lower level of DNA repair measured in male germ cells using 600 R of Xrays compared with 250 mg/kg of EMS is a manifestation of the number of bases inserted into the repaired lesions. The occurrence of DNA repair in some germ-cell stages of male mice but not in others gives the mammalian geneticist a method for determining whether
322
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F- 20,q
,~ /
X RAY . I . 600 R
0 ~--ql--~--ct~ . . . . . 3 9 15 2f DAYS AFTER TREATMENT
27
Fig, 1. T h e r e s p o n s e p a t t e r n o f m e i o t i c a n d p o s t m e i o t i e germ-cell stages of m a l e m i c e t o t h e i n d u c t i o n of u n s c h e d u l e d D N A s y n t h e s i s a f t e r t r e a t m e n t w i t h X - r a y s (o) or EMS (o). 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 v a r i o u s germ-ceU stages is a s s a y e d b y m e a s u r i n g t h e u n s c h e d u l e d p r e s e n c e of [ 3 H I d T in t h e g e r m cells a f t e r t h e y h a v e m a t u r e d t o s p e r m a t o z o a in t h e vasa d e f e r e n t i a . C o n t r o l values for all t i m e p o i n t s cons i d e r e d are in the base llne. T h e e r r o r bars r e p r e s e n t +-1 s t a n d a r d d e v i a t i o n .
the mammalian repair system in vivo is error-free. For example, Russell's specific-locus test [31] has been used to look for point mutations in the mouse after EMS treatment. Six presumed specific-locus mutations were observed in 10,941 offspring derived from cells that were in post-spermatogonial stages at the time of treatment [33]. However, most of the data and all of the mutants came from the first 2 weeks post-injection [34], when the sperm in the vas show no unscheduled presence of [3H] dT after EMS treatment. It would be very informative to compare the specific-locus m u t a t i o n frequency for progeny conceived from 16 to 24 days after EMS treatment (when the sperm in the vas do show an unscheduled presence of [3H] dT) with the m u t a t i o n frequency found for progeny conceived in the first 2 weeks after EMS treatment. Such a comparison might shed some light on whether the mammalian repair system is actually correcting premutational DNA lesions. Since establishment of the meiotic and post-meiotic germ-ceU stages that undergo DNA repair when exposed to EMS, three chemical homologs -- MMS, PMS and IMS -- have also been studied for their ability to induce DNA repair in mouse germ cells [42]. In particular, sperm from the caudal epididymides were studied 16 days after treatment. (Caudal sperm are used because there are about twice as many per animal compared with the a m o u n t of sperm in the caput epididymis or in the vas.) This means that at the time of treatment these caudal sperm were early spermatids [23,24,38]. For each of these chemicals a dose-response curve has been obtained which measures the relative, unscheduled uptake of [3HI dT into early spermatids as a function of administered concentration of the chemical mutagen. Also, the relative a m o u n t of repair occurring in early spermatids over a 3-day period following administration of each chemical has been studied by injecting [3H]dT at various times after chemical treatment. The duration of repair could be
323 studied in this way since we have found that after injection of the [3H]dT its removal from the thymidine pool of the testes is quite rapid, with most of the free [3H] dT disappearing from the testes within a b o u t 1 h after injection [37]. This result is in agreement with the work of others using mammalian systems to measure the kinetics of incorporation of [3H] dT [4,5]. When [3HI dT was administered simultaneously with each of the four chemical mutagens there was an unscheduled uptake of the label into the DNA of early spermatids. This means that all four of these mutagens must reach the DNA of early spermatids and begin producing repairable lesions within 1 h after treatment. However, as discussed below, repair can extend over several days. The dose-response effects of all four chemicals in the induction of germcell DNA repair appear to be linear over the dose ranges studied (MMS, 5--75 mg/kg; EMS, 10--300 mg/kg; PMS, 50--700 mg/kg; IMS, 10--170 mg/kg.) The greatest DNA repair response (unscheduled [3H]dT uptake) was seen when MMS was used, followed by EMS, IMS, and finally PMS. MMS, EMS, and PMS have the same relative order of effectiveness in the induction of dominantlethal mutations in sperm and late spermatids [3,9,10,11]. However, IMS falls between MMS and EMS in this case [10]. Comparing MMS and EMS, for example, it was found that at equimolar administered concentrations of the two chemicals, MMS was a b o u t 4 times as effective as EMS in inducing DNA repair in early spermatids and also a b o u t 4 times as effective as EMS in producing dominant-lethal mutations in sperm and late spermatids [9]. A similar relationship has been found between these two chemicals in the induction of DNA repair in cultured mammalian cells [29], although in that case the difference was tenfold. Roberts et al. [29] have found that in their cultured mammalian cells "repair synthesis" is directly related to the overall level of DNA alkylation in the cells. If the same result holds true for the mouse germ cells treated in vivo, then measurement of DNA repair in these germ cells may become a practical way of determining chemical dose in the genetic material once relationships have been established between the number (and types) of chemical lesions in DNA and the a m o u n t of repair induced. On the basis of the lowest injected chemical dose which can cause a measurable effect, DNA repair in mouse germ cells appears to be a considerably
TABLE I C O M P A R I S O N O F T H E L O W E S T C H E M I C A L D O S E S O F MMS, EMS, PMS, A N D IMS N E E D E D T O DETECT DOMINANT-LETHAL MUTATIONS OR TRANSLOCATIONS WITH DOSES THAT STILL INDUCE DNA REPAIR Chemical
M i n i m u m dose (mg/kg) for d o m i n a n t lethal * or translocation t detection
D N A r e p a i r in g e r m ceils still r e a d i l y m e a s u r e d at a d o s e (mg/kg) of:
MMS EMS
~50 ~150 ~50 ~400 ~50
5 10
PMS IMS
* [ref.9] * [ref. 12] t" [ r e f . 1 2 ] * [ref. 10] ( [ref.lO]
50 10
324 more sensitive biological end point than measurement of dominant lethals or translocations. Table I shows that the measurement of DNA repair is at least 5--10 times more sensitive than the dominant-lethal tests or translocation studies using these four chemical mutagens. However, it should be stressed that the germ-cell stages showing peak sensitivity to the induction of dominant lethals with these four chemicals are stages where the DNA repair assay would be insensitive. The results from the study of the duration of DNA repair in early spermatids of the mouse after treatment with MMS, EMS, PMS, and IMS indicate that as long as 3 days after exposure to these chemicals the early spermatid stages are still undergoing measurable DNA repair. This extended repair is n o t due to continuing alkylation of the DNA during this time period, because these four chemical homologs are quite reactive in aqueous solutions [26] and even more reactive in biological systems [8], probably disappearing within several hours, at most, after injection. For MMS and EMS there is a rapid decrease in the level of DNA repair in the early spermatids in the first half-day following treatment. This is followed by a much slower, exponential decrease in the level of repair o u t to 3 days posttreatment. The curves suggest that the a m o u n t of repair occurring between 1/2 and 3 days post-treatment is simply proportional to the number of repairable lesions still present in the DNA. The half-life for the DNA repair occurring between 1/2 and 3 days posttreatment with MMS and EMS is between 1 and 3 days. Craddock [7] and Margison et al. [20] have both found the removal of 7-methylguanine from rat liver DNA to be exponential, with a half-life of 1 [ref. 7] to 3 [ref. 20] days. It is possible that the decrease in repair with time could be explained by removal of 7-alkylguanine from the germ-cell DNA, especially since the N-7 position of guanine should be the most frequently alkylated site in the DNA [2,15]. However, other alkylation products in the DNA, such as 3-alkyladenine, O6-alkylguanine, various alkylpyrimidines, and phosphotriesters may also be targets for DNA repair [16,25]. Another aspect of the study of chemical dosimetry and DNA repair in mouse germ cells is the question of the proportion of the germ-cell population that is damaged by a particular chemical agent and the proportion of cells that can undergo repair. Is it 100% of the cell population or something less than this? This question cannot be answered by liquid scintillation counting, so autoradiographs were also made of sperm samples taken from each mouse. The results indicated that while most of the sperm from each animal had the expected number of grains, a portion showed either considerably fewer or more grains than would be expected from a Poisson distribution. Earlier work with EMS [37] had shown that the unscheduled presence of [3H]dT in the caudal sperm had reached a constant level by 16 days posttreatment which was maintained through 24 days post-treatment. Thus on the average, all treated sperm cells in the caudal epididymides 16 days after treatment had incorporated [3HI dT. The sperm present in the caudal epididymides at that time (16 days) did n o t represent a mixture of germ-cell stages, some of which showed DNA repair and some of which did not. Therefore, the deviations from a Poisson distribution seen in the frequencies of grains over the chemically-treated sperm are not likely due to differences in sensitivities of the
325
germ-cell stages represented in the caudal epidymides at 16 days post-treatment. It will be important in future work studying the molecular mechanisms of mutagenesis in mammalian germ cells to determine what biochemical events are occurring in these small fractions of the germ-cell populations. Are the numbers of chemically induced lesions in some cells fewer (or much more) than the average, and is the repair of chemically induced lesions in the DNA of these germ cells much less efficient (or much more efficient) than the average? References 1 A a r o n , C.S., M o l e c u l a r d o s i m e t r y o f c h e m i c a l m u t a g e n s . S e l e c t i o n o f 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 d o s e t o t h e g e r m line, M u t a t i o n Res., 3 8 ( 1 9 7 6 ) 3 0 3 - - 3 1 0 . 2 B r o o k e s , P. a n d P.D. 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