Proc. Natl. Acad. Sci. USA
Vol. 74, No. 8, pp. 3523-3527, August 1977 Genetics
Mutation frequencies in female mice and the estimation of genetic hazards of radiation in women (germ-cell stage sensitivity/low-dose-rate irradiation)
W. L. RUSSELL Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830
Contributed by William L. Russell, June 9,1977
The female germ cell stage of primary imporABSTRACT tance in radiation genetic hazards is the immature, arrested oocyte. In the mouse, this stage has a near zero or zero sensitivity to mutation induction by radiation. However, the application of these mouse results to women has been questioned on the ground that the mouse arrested oocytes are highly sensitive to killing by radiation, while the human cells are not; and, furthermore, that the mature and maturing oocytes in the mouse, which are resistant to killing, are sensitive to mutation induction. The present results have a 2-fold bearing on this problem. First, a more detailed analysis of oocyte-stage sensitivity to killing and mutation induction shows that there is no consistent correlation, either negative or positive, between the two. This indicates that the sensitivity to cell killing of the mouse immature oocyte may not be sufficient reason to prevent its use in predicting the mutational response of the human immature oocyte. Second, if the much more cautious assumption is made that the human arrested oocyte might be as mutationally sensitive as the most sensitive of all oocyte stages in the mouse, namely the maturing and mature ones, then the present data on the duration of these stages permit more accurate estimates than were heretofore possible on the mutational response of these stages to chronic irradiation.
Because no transmitted genetic effects of radiation have been clearly detected in man, estimates of human genetic hazards from radiation are necessarily based on experimental animals, primarily the mouse. No special difficulties have been seen in extrapolating data from the male mouse. In applying the female mouse data to the human, however, there is a potential problem. The present paper deals with one aspect of this problem. The female germ cell stage of primary importance in radiation genetic hazards is the immature, arrested oocyte. In most of the conceptions in women who have had a prior exposure to radiation, most of the dose will have been accumulated in this oocyte stage. In 1965, the surprising discovery was reported (1) that, although the mature oocyte in the mouse is highly sensitive to mutation induction, the immature arrested oocyte appears to be completely resistant. This was shown first for acute neutron irradiation, but the finding was later extended to acute x-irradiation and chronic y irradiation. The results from eight separate experiments, summarized in a review paper (2), gave a total of 259,683 offspring from conceptions that occurred more than 7 weeks after irradiation of the mother. These conceptions involved oocytes that had received their radiation exposure in the immature arrested stage or, shortly after, in the beginning stages of follicle development. Only three mutations were detected by using our standard test for mutation induction at seven specific loci (3). This is slightly, but not, of course, significantly below the control (spontaneous) mutation frequency at the same seven loci, the latest totals for The costs of publication of this article were defrayed in part by the payment of page charges from funds made available to support the research which is the subject of the article. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
which are shown in Table 1. Thus, the mouse immature arrested oocyte shows no evidence of mutation induction, even after doses as high as 60 rads of neutrons and 400 roentgens (R) of y rays. (1 rad = 10-2 j/kg; 1 R = 2.6 X IO-4 C/kg). Since this discovery, committees charged with the responsibility for estimating genetic hazards of radiation have tended to regard the induced mutation frequency in women as negligible compared to that in men. However, as this author (2) and others have pointed out, and as the committees have recognized, the immature oocytes of the mouse, though resistant to mutation induction, are highly sensitive to killing by radiation, whereas human immature oocytes are resistant to killing. There are also species differences in the cytological appearance of the arrested oocytes. Thus, the question arises as to whether it is valid to assume that the human arrested oocyte will, like that of the mouse, be resistant to mutation induction. Our approach to this problem has been to investigate the mutational response of other oocyte stages in the mouse, including the maturing and mature ones, which are resistant to killing by radiation. These stages are highly mutationally sensitive to acute, high-dose-rate irradiation, but have proved to be insensitive to low-dose-rate irradiation. Furthermore, at low doses of high-dose-rate irradiation the mutation frequency drops well below that expected on a linear relationship with dose. Therefore, we have concluded that mouse oocyte stages covering a wide range of sensitivity to killing will, under the usual conditions of human exposure, show a low or zero mutation frequency (2). The latest, 1972, reports of the United Nations Scientific Committee on the Effects of Atomic Radiation (UNSCEAR) (5) and the U.S. National Academy of Sciences Committee on the Biological Effects of Ionizing Radiations (BEIR) (6) adopted a similar view. The mutational response of the mouse arrested oocyte was taken as zero or near zero, and the mutational sensitivity of the mouse maturing and mature oocytes to chronic irradiation was computed to be only about 1/20 of that to acute irradiation. Since the publication of these reports, Lyon and Phillips (7) have collected a most valuable set of data which support and extend the conclusions reached from the earlier results. In contrast, the interpretation of the earlier work has been challenged by Wolff (8) and Abrahamson and Wolff (9). In two papers, containing essentially the same material, they attempt to explain the mouse specific-locus mutation data on the basis of an old mathematical model for two-break chromosome aberration induction. In order to make the data fit this model, they have assumed a duration of oocyte stage sensitivity which, as the present paper will show, is inconsistent with the facts, and they have concluded from this that the mutational sensitivity Abbreviations: R, roentgen; UNSCEAR, United Nations Scientific Committee on the Effects of Atomic Radiation; BEIR, [Committee onl Biological Effects of Ionizing Radiations.
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Genetics: Russell
Proc. Natl. Acad. Sci. USA 74 (1977)
Table 1. Spontaneous mutation frequency at seven specific loci in female mice Number of offspring scored
Number of mutations
Ref.
166,826 37,813
3 or 8* 0
Russell (2)t Batchelor et al. (4)
204,639
3 or 8
demonstrated for comparisons restricted
to conceptions
oc-
curring within 2 weeks after accumulation of the dose (10). In order to utilize data collected from conceptions occurring more than 3 weeks after irradiation, in this example, it is necessary to compute the portion of the dose received in the effective period. This was done, but not published, in a rather crude way in computations used to arrive at the figure of 1/20 which, as was mentioned earlier, was accepted by the UNSCEAR and BEIR committees as the ratio of effects of chronic
* Three independent mutational events, one of which was a cluster of 6. t Also includes previously unpublished data.
of the maturing and mature mouse oocytes to chronic irradiation is much greater than had been supposed. The present paper, in addition to providing new data on spontaneous mutations, analyzes, in more detail than heretofore, the results of low-level irradiation of maturing oocytes, including the new data of Lyon and Phillips. It also discusses the interpretation of Abrahamson and Wolff, and presents some hitherto unpublished information on the distribution of mutations with time after irradiation that has an important bearing on the discussion. RESULTS AND DISCUSSION One of the difficulties in measuring the effect of low-dose-rate irradiation on mutation frequency in maturing oocytes is that the length of radiation exposure time necessary to accumulate a sizable dose may approach the duration of the oocyte stage under measurement. For example, suppose we want to determine the mutation frequency from chronic irradiation of maturing oocytes that are not more than 6 weeks from ovulation. Giving a dose of 258 R took 3 weeks. Therefore, any conceptions occurring more than 3 weeks after the end of irradiation would involve oocytes that received part of their dose earlier than 6 weeks before ovulation. When we discovered that the mutation frequency drops apparently to zero for conceptions occurring more than 6 or 7 weeks after irradiation (1), it was immediately apparent, and was pointed out at that same time, that the lowness of a total mutation frequency in offspring collected from a 6-week mating period after the end of irradiation could have resulted partly from a low-dose-rate effect on maturing oocytes and partly from a portion of the dose having been given to the oocytes in immature stage, more than 6 weeks before conception. However, as was shown long before that, at the beginning of our dose-rate studies in females, a very low mutation frequency from chronic irradiation could be
With additional data from Lyon and Phillips (7) now available, and with the mutation frequencies challenged by Abrahamson and Wolff (9), it now seems desirable to make more precise estimates. In order to do this, it was necessary to determine as sharply as possible the time interval after irradiation at which mutation frequency dropped. It was also necessary to find out if this was a sudden drop or a gradual one spread over several weeks. Pertinent data from our experiments are collected in Table 2. The radiation in these experiments was delivered in a few minutes or over about 8 hr. No mutations were obtained in conceptions occurring more than 6 weeks after irradiation, and the frequencies in the fifth and sixth weeks showed no decline compared with earlier weeks. The cut-off period appears to be very sharp, and according to Oakberg's latest timing of oocyte development (ref. 11, and personal communication) may be coincident with the oocyte stage in which formation of the zona pellucida begins, a stage that takes approximately 6 weeks to reach ovulation. From Table 2 and other data it also appears that offspring conceived in the first week after irradiation have a lower mutation frequency than those conceived during weeks 2 to 6 after irradiation. This will -be discussed in another
to acute irradiation in maturing oocytes.
paper.
With this information, it is now possible to compute an "effective dose" for offspring conceived within 6 weeks after the end of a period of chronic irradiation. By "effective dose" is meant the portion of the total dose that will have been received 6 weeks or less before ovulation. As an example of the computation, we can pick one that happened to work out in simple fashion, namely, the 400-R experiment, in which the dose was given at approximately 0.009 R/min over a period of 35 days. Offspring from conceptions that occurred in the first week after the end of irradiation came from oocytes that received all of their dose in less than 6 weeks before ovulation. For animals conceived in the second week the average dose received by the oocytes in the 6 weeks before ovulation turns out to be 90% of the total 400 R, or 360 R. For the third week it is 70%, or 280 R, and so on. In the sixth week it is only 40 R. By weighting these doses by the number of ani-
Table 2. Distribution of offspring and mutations at seven specific loci in conceptions occurring during successive weeks after irradiation of female mice with various doses and dose rates 400 R, 0.8 R/min 50 R, 90 R/min 200 R, 90 R/min No. of No. of No. of No. of No. of No. of mutations mutations mutations offspring offspring Week offspring 1 2 3 4 5 6 7 8 9 10 11 on
28,547 3,604 454 1,138 8,395 3,327 328 17 5 1
13 2 1 0 12 5 0 0 0 0
84,614 8,016 2,904 9,587 45,179 16,304 13,868 9,092
11,096 7,433 50,570
6 0 0 1 3 3 0 0 0 0 0
49,039 7,928
14 3
737 844
1 4 6
7,342 4,990 170 2 4
2 0 0 0
Proc. Natl. Acad. Sci. USA 74 (1977)
Genetics: Russell
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Table 3. Number of mutations at seven specific loci l4W offspritg conceived within 6 weeks after the end of exposure in various low-level radiation experiments on female mice
Weighted Ref.
Dose given, R
From Table 1 Lyon et al. (7) (group D) Russell (10) Lyon et al. (7) (group C) Russell (2, 12)1 Russell (2, 12) Carter (13)
0 215t 258 215t 400 400 615t
Dose rate 0 20 X 10.6 Rt 0.009 R/min 20 X 10.6 R§ 0.009 R/min 0.009 R/min 0.05 R/min
mean effective dose,* R
Number of offspring
Number of mutations
0 172 207 212 283 284 615
204,639 14,671 7,692 21,204 13,742 14,402 10,177
3 or 8 1 1 0 2 1 1
Mutation frequency per locus X 106 2.1 or 5.6 9.7 18.6 0.0 20.8 9.9 14.0
* Dose delivered to oocytes in the 6 weeks prior to ovulation (see text for detailed explanation). t Converted from rads. I Delivered over 4 weeks. § Delivered over 5 days. I Animals irradiated were old previously bred females.
mals in each week, the weighted mean dose for the whole population can be determined. In this case it is 283 R, as shown in Table 3. Table 3 shows the results of similar calculations made for two other experiments of ours and for the two sets of data from Lyon and Phillips. The Carter experiment required no adjustment of dose because all matings were made in less than 2 weeks after the 12-day exposure period. The Lyon and Phillips data were not from low-dose-rate irradiation, but from 20 fractions of 10 rad each spaced far enough apart to give a result that is apparently no more damaging than a low-dose-rate exposure. Two values are given for the control. There were three independent mutational events, one of which was a cluster of 6, representing six mutant germ cells from a single mutational event occurring early in development. This complicates the calculation of the spontaneous rate, and, as I have explained elsewhere (2), gives two extreme values. One can assume that, in spite of our happening to find one, clusters are extremely rare. On this basis there would be little error in taking the mutation frequency as 3 in 204,639. On the other hand, one can argue that our only estimate of the frequency of clusters is what was observed, namely one out of three mutational events. Then we must assume the mutation frequency to be 8 in 204,639. In this case, confidence limits and weighting should not be based on 204,639 independent observations: % of this, or 76,740, would be a better estimate. The frequency of independent mutational events would then be taken as 3 in 76,740 (the same as 8 in 204,639). The mutation frequencies shown in Table 3 are plotted in Fig. 1. The results for the similar doses of 207 and 212 R have been averaged in one point on the figure, but were treated as separate values in the regression analysis. The 283-R and 284-R data have not been combined because the 283-R experiment was the only one that used old animals. Other data to be presented elsewhere show consistently higher induced mutation rates for older females. Four weighted least squares regression lines were determined. The solid lines are based on all the data, the broken lines on all the data with the exception that old females are excluded. Each set has two fits, one for each of the two control values. The results may now be compared with the predictions of Abrahamson and Wolff (9). They assumed that the acute irradiation data will fit a simple quadratic equation: Y=C+ aD+
#D2
in which Y equals the expected yield of mutations, C equals the
control rate, a and # are coefficients, and D is dose. For females, they fitted this equation to the control point and to our 50-R, 200-R, and 400-R single acute dose points. The. a derived from this was used to predict what the mutation frequency should be at low-dose rates. Actually three separate fits were made based on three different control rates. Two of these, like ours, 100-
80-
*~~~~~~/ ' 0
/0/
a
20ig
0
100
200
300 400 Dose, R
500
660
700
FIG. 1. Weighted least squares regression lines of data in Table 3, and theoretical fits by Abrahamson and Wolff (8). See text for details. O. Only data point for irradiated old breeding females. Remaining points are for females. irradiated as young virgins. Intercepts, C, and slopes, a, are as follows:
Regression line Fit of data a, all data b, all data c, excluding old females d, excluding old females Abrahamson-Wolff theoretical fit Cluster = 0 Cluster = 1
C X 107
aX
107
21.2 55.4 21.3 54.3
0.296 0.178 0.221 0.113
8.39 16.7
1.43 1.26
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Proc. Natl. Acad. Sci. USA 74 (1977)
Genetics: Russell
were based on treating the cluster as one or six mutations. The third one was based on excluding the cluster altogether, on the argument that it must have occurred in oogonia and the induced mutations can occur only in oocytes. This is a departure from the usual consideration of a spontaneous rate as a pergeneration rate. It will also undoubtedly lead to bias, particularly where the sibships are smaller in the irradiated population and, therefore, less likely to detect clusters. However, this is the fit that Abrahamson and Wolff prefer. All three a values were then used to compute the number of mutations expected in the various experiments on females. The authors focus on the fits derived from treating the cluster as 0 and 1, apparently rejecting the remaining fit, where the cluster is taken equal to six mutations, as giving a poor match with the data. They recognize that the other two fail to give good agreement with all three of the low-dose-rate experiments considered by them, namely, Carter's and our 258-R and 400-R data. Then, in an extensive discussion, they try to explain away the discrepancy. Their first step is in keeping with our procedure. The data cited are for all offspring conceived within 7 weeks after the end of irradiation, and Abrahamson and Wolff realize that the effective dose to the maturing oocyte stages will average less than the total given. Errors arise when they try to compute the effective dose on the basis of number of estrous cycles and an oocyte maturation scheme that seems to have no basis in reality. Thus, they compute that the effective dose in the Carter experiment would be only one-half or less of the dose given. The irradiation time in this experiment was 12 days, and all matings occurred within the 2 weeks of the end of irradiation. In view of Table 2, and the well-known fact that maturation and loss of oocytes occur at the same rate in breeding and nonbreeding females (14), it is hard to see how any of the oocytes in Carter's experiment could have received less than the total dose during the maturing stages that are sensitive to acute irradiation. It should be noted that the two mutations in the 283-R experiment in Table 3 were from conceptions occurring in the fifth week after the end of irradiation, a period long after the time when Abrahamson and Wolff would have had the effective dose down to zero. Although Abrahamson and Wolff include the new Lyon and Phillips (7) data for a single 200-R dose of acute irradiation, they omit any consideration of the fractionated exposures. If they had computed their expected value (assuming cluster = 0) for this set of data, it would have been 9.5 mutations expected where only one was observed. With a correct adjustment for effective dose they would still have predicted 7.8 mutations for the one observed. Abrahamson and Wolff, on the one hand, admit that their corrections for effective dose are crude, but, on the other hand, conclude "it is reasonable to assume" that the wide discrepancies between their theoretical expectations and the actual data are "caused by some of the radiation occurring during less mutable stages." Because the data shown in Fig. 1 have already been adjusted for this factor by our criteria, it is of interest to see how much discrepancy is still left between the corrected observed values and the Abrahamson-Wolff prediction. The two values of a for the fits preferred by Abrahamson and Wolff are shown in Fig. 1. The third fit would be much closer to the observed data, but gives a very poor fit to some other data points, and, for that reason, was apparently not acceptable to Abrahamson and Wolff. The slope of the upper Abrahamson-Wolff line exceeds the slopes of the actual data by multiples of 4.8, 6.5, 8.0, and 12.7; the slope of the lower line (cluster = 1) exceeds them by 4.3, 5.7, 7.1, and 11.2. Both Abrahamson-Wolff slopes are highly statistically significantly above all the slopes of the actual data. The
lowest t value in these comparisons is 7.84, while the critical value of t for 5% probabality is 2.57. Thus the Abrahamson-Wolff derivation, from acute irradiation, of the mutation frequency theoretically expected, on their hypothesis, for low-level irradiation exceeds the observed value by from 4- to 13-fold. It should be clear that their approach is not a reliable one for estimating hazards. * The observed data are still meager in terms of number of mutations obtained, and the confidence limits are, therefore, very wide for each point. Nevertheless the fitted lines presumably provide a better estimate of the mutation frequency from low-level irradiation of mature and maturing oocytes than has been available before. APPLICATIONS OF THE DATA TO HUMAN HAZARD EVALUATION For mature and maturing oocytes, the UNSCEAR and BEIR committees, in their 1972 reports (5, 6), accepted 1/20 as the ratio of mutation rate from chronic irradiation to that from acute irradiation. The rates presented in this paper (slopes a, b, c, and d in Fig. 1) for low-level irradiation of mature and maturing oocytes, when compared to the mutation rate for our 400-R acute irradiation data (ref. 15, and unpublished), yield ratios of 1/18, 1/29, 1/24, and 1/46, respectively. The true ratios would be expected to be even somewhat lower, because the 400-R acute irradiation data contain a higher proportion of offspring from oocytes that received all of their radiation in the week before ovulation, when the mutational sensitivity is lower than for radiation received from 2 to 6 weeks before ovulation. In any case, with the more precise estimation of the effective doses than was available to the committees, and with the addition of the new data of Lyon and Phillips (7), it turns out that the committees' statements did not underestimate the risks from low-level irradiation of mature and maturing oocytes. It is of interest to compare the specific-locus mutation rates obtained for mature and maturing oocytes with the mutation rate in the male mouse for chronic irradiation of the germ-cell stage primarily at risk in human hazards, namely, the spermatogonia. The mutation rate, for the same seven loci, in mouse spermatogonia irradiated at dose rates of 0.009 R/min and below, was calculated in the review paper by Searle (16) to be 6.59 X 10-8 per locus, per R. The rates for low-level irradiation of mature and maturing oocytes (slopes a, b, c, and d in Fig. 1) are, respectively, only 0.44, 0.27, 0.33, and 0.17 times as effective. Furthermore, it should be pointed out that only in the first of these four possible estimates (slope a) is the induced mutation rate in oocytes significantly above the control rate. Thus, the ratio of effectiveness to the spermatogonial mutation rate could be zero. Similarly, the ratio of effectiveness to acute irradiation given in the previous paragraph could also be zero.
With regard to the validity of extrapolating from mouse immature arrested oocytes to human immature arrested oocytes, the results given in Table 2 have an indirect bearing on this problem. In these data, and in others to be presented elsewhere, the mutation frequency is lower for the most mature oocytes, those that produced the offspring for week 1 in Table * Note Added in Proof: Abrahamson and Wolff have already used their calculations in two reports on radiation hazards: (a) Report of the Nuclear Energy Policy Study Group (1977) Nuclear Power Issues
and Choices (Ballinger Publishing Co., Cambridge, MA); and (b) National Academy of Sciences Committee on Safe Drinking Water (1977) Drinking Water and Health (National Academy of Sciences,
Washington, DC).
Genetics: Russell
2, than for the less mature oocytes that pkoduced thbfig listed for weeks 2 to 6. The fully mature oocytes are less siiive to killing than the less mature ones. Thus, here there is a positive correlation between killing and mutational sensitivity. This is in contrast to the negative correlation found for immature arrested oocytes, which give no evidence of mutation induction but which are highly sensitive to killing. It appears, from the lack of consistent correlation, that mutation induction and killing are independent events. In this connection, the new data of Cox and Lyon (17) on x-ray induction of dominant lethal mutations in mature and immature oocytes of guinea pigs and golden hamsters are of great importance. In both species, they found lower mutation yields from immature than from mature oocytes, despite the fact that the immature oocytes of the guinea pig are less sensitive to killing than the mature ones, while the reverse is true for the golden hamster, which, therefore, resembles the mouse. Thus again, as the authors state, there is "no general pattern ... of correlation, either positive or negative, in the sensitivity of oocytes to killing and to dominant lethal induction." This is in contrast to the statement by Abrahamson (18) that "I suspect that cell killing, chromosome rearrangements and gene mutations are rather closely linked." The fact that cell killing of oocytes is apparently not linked to the other two effects is not surprising to us. Death of the cells occurs rapidly, before cell division, and is, therefore, not related to the kind of death that results from aneuploidy after cell division. Cox and Lyon question whether dominant lethals and gene mutations behave similarly, but their doubts are based on the fact that Searle and Beechey (19) had shown an increase in dominant-lethal frequency over the first 3 weeks after irradiation of female mice. Cox and Lyon believed that this disagreed with the gene mutation results, because they were aware only of the decline in mutation frequency with time after irradiation. However, there is no disagreement: the decline in gene mutation frequency occurs only after 6 weeks, and, with the new data reported in Table 2, it is now clear that, as with dominant lethals, there is, in fact, an increase in gene mutation frequency after the first week. In summary, there would now seem to be less reason than before for rejecting an application of the mouse immature arrested oocyte data to the human immature arrested oocyte. In the immature oocytes, the specific-locus mutation frequency and X-chromosome loss frequency (20) in the mouse and the dominant lethal frequency in guinea pig and golden hamster are all low compared to the frequencies in mature oocytes. The fact that this is so, despite the differences in sensitivity to cell killing, and in chromosomal morphology, in the immature oocytes of these species, indicates that the sensitivity to cell killing of the mouse immature oocyte may not be sufficient reason to prevent its use in predicting the mutational response of the human immature oocyte. If, on the side of caution, one continues to consider the possibility that the human immature arrested oocyte might be as mutationally sensitive as the most sensitive of all oocyte stages in the mouse, namely the maturing and mature oocytes, then the present paper provides estimates for this extrapolation. With low-level irradiation, the estimates of mutational frequency in these stages average from 0.17 to 0.44 times that in spermatogonia, but in three of the four estimates the frequencies are not significantly above control values. Thus, even in the event that the response of the human immature arrested oocyte is unlike that of the mouse arrested oocyte, but similar to that of the most
Proc. Natl. Acad. Sci. USA 74 (1977)
3527
monsltive oocyte stages in the mouse, it seems likely that the genetic hazard of radiation in the female will still be less than in the male. For the statistical analyses I gratefully acknowledge the help of D. G. Gosslee, Computer Sciences Division, Union Carbide Corp., Nuclear Division. Research was sponsored by the Energy Research and Development Administration under contract with the Union Carbide Corp. 1. Russell, W. L. (1965) "Effect of the interval between irradiation and conception on mutation frequency in female mice," Proc. NatI. Acad. Sci. USA 54, 1552-1557. 2. Russell, W. L. (1972) "The genetic effects of radiation," in Peaceful Uses of Atomic Energy (United Nations, New York), Vol. 13, pp. 487-500. 3. Russell, W. L. (1951) "X-ray-induced mutations in mice," Cold Spring Harbor Symp. Quant. Biol. 16, 327-36. 4. Batchelor, A. L., Phillips, R. J. S. & Searle, A. G. (1969) "The ineffectiveness of chronic irradiation with neutrons and gamma rays in inducing mutations in female mice," Br. J. Radiol. 42, 448-451. 5. United Nations Scientific Committee on the Effects of Atomic Radiation (1972) Ionizing Radiation: Levels and Effects (United Nations, New York), Vol. 2. 6. National Academy of Sciences-National Research Council Advisory Committee on the Biological Effects of Ionizing Radiations (1972) The Effects on Populations of Exposure to Low Levels of Ionizing Radiation (National Academy of Sciences, Washington, DC). 7. Lyon, M. F. & Phillips, R. J. S. (1975) "Specific locus mutation rates after repeated small radiation doses to mouse oocytes," Mutat. Res. 30, 375-382. 8. Wolff, S. (1975) "Estimation of the effects of chemical mutagens: Lessons from radiation genetics," Mutat. Res. 33, 95-102. 9. Abrahamson, S. & Wolff, S. (1976) "Re-analysis of radiationinduced specific locus mutations in the mouse," Nature 264, 715-719. 10. Russell, W. L., Russell, L. B. & Cupp, M. B. (1959) "Dependence of mutation frequency on radiation dose rate in female mice," Proc. Natl. Acad. Sci. USA 45,18-23. 11. Oakberg, E. F. & Palatinus, D. T. (1976) "The stage of the mouse oocyte at which the change in sensitivity to radiation-induced mutations occurs," Genetics 83, s56 (abstr.). 12. Russell, W. L. (1963) in Repair from Genetic Radiation Damage, ed. Sobels, F. (Pergamon Press, Oxford), pp. 205-217; 231235. 13. Carter, T. C. (1958) "Radiation-induced gene mutation in adult female and foetal male mice," Br. J. Radiol. 31, 407-411. 14. Oakberg, E. F. (1966) in Radiation and Ageing, eds. Lindop, P. J. & Sacher, G. A. (Taylor and Francis, London), pp. 293-306. 15. Russell, W. L. (1965) "The nature of the dose-rate effect of radiation on mutation in mice," Jpn. J. Genet. Suppl. 40, 128140. 16. Searle, A. G. (1974) "Mutation induction in mice," Adv. Radiat. Biol. 4, 131-207. 17. Cox, B. D. & Lyon, M. F. (1975) "X-ray induced dominant lethal mutations in mature and immature oocytes of guinea pigs and golden hamsters," Mutat. Res. 28, 421-436. 18. Abrahamson, S. (1976) in "Discussion" in Biological and Envi-
ronmental Effects of Low-Level Radiation (International Atomic Energy Agency, Vienna), Vol. 1, p. 18. 19. Searle, A. G. & Beechey, C. V. (1974) "Cytogenetic effect of xrays and fission neutrons in female mice," Mutat. Res. 24, 171-186. 20. Russell, W. L., Hunsicker, P. R., Kelly, E. M., Vaughan, C. M. & Guinn, G. M. (1973) "X-chromosome loss in the offspring of
irradiated female mice," Oak Ridge National Laboratory, Biology Division, Annual Progress Report, ORNL-4915 (Oak Ridge National Laboratory, Oak Ridge, TN), p. 98.
Vol. 74, No. 8, pp. 3523-3527, August 1977 Genetics
Mutation frequencies in female mice and the estimation of genetic hazards of radiation in women (germ-cell stage sensitivity/low-dose-rate irradiation)
W. L. RUSSELL Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830
Contributed by William L. Russell, June 9,1977
The female germ cell stage of primary imporABSTRACT tance in radiation genetic hazards is the immature, arrested oocyte. In the mouse, this stage has a near zero or zero sensitivity to mutation induction by radiation. However, the application of these mouse results to women has been questioned on the ground that the mouse arrested oocytes are highly sensitive to killing by radiation, while the human cells are not; and, furthermore, that the mature and maturing oocytes in the mouse, which are resistant to killing, are sensitive to mutation induction. The present results have a 2-fold bearing on this problem. First, a more detailed analysis of oocyte-stage sensitivity to killing and mutation induction shows that there is no consistent correlation, either negative or positive, between the two. This indicates that the sensitivity to cell killing of the mouse immature oocyte may not be sufficient reason to prevent its use in predicting the mutational response of the human immature oocyte. Second, if the much more cautious assumption is made that the human arrested oocyte might be as mutationally sensitive as the most sensitive of all oocyte stages in the mouse, namely the maturing and mature ones, then the present data on the duration of these stages permit more accurate estimates than were heretofore possible on the mutational response of these stages to chronic irradiation.
Because no transmitted genetic effects of radiation have been clearly detected in man, estimates of human genetic hazards from radiation are necessarily based on experimental animals, primarily the mouse. No special difficulties have been seen in extrapolating data from the male mouse. In applying the female mouse data to the human, however, there is a potential problem. The present paper deals with one aspect of this problem. The female germ cell stage of primary importance in radiation genetic hazards is the immature, arrested oocyte. In most of the conceptions in women who have had a prior exposure to radiation, most of the dose will have been accumulated in this oocyte stage. In 1965, the surprising discovery was reported (1) that, although the mature oocyte in the mouse is highly sensitive to mutation induction, the immature arrested oocyte appears to be completely resistant. This was shown first for acute neutron irradiation, but the finding was later extended to acute x-irradiation and chronic y irradiation. The results from eight separate experiments, summarized in a review paper (2), gave a total of 259,683 offspring from conceptions that occurred more than 7 weeks after irradiation of the mother. These conceptions involved oocytes that had received their radiation exposure in the immature arrested stage or, shortly after, in the beginning stages of follicle development. Only three mutations were detected by using our standard test for mutation induction at seven specific loci (3). This is slightly, but not, of course, significantly below the control (spontaneous) mutation frequency at the same seven loci, the latest totals for The costs of publication of this article were defrayed in part by the payment of page charges from funds made available to support the research which is the subject of the article. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
which are shown in Table 1. Thus, the mouse immature arrested oocyte shows no evidence of mutation induction, even after doses as high as 60 rads of neutrons and 400 roentgens (R) of y rays. (1 rad = 10-2 j/kg; 1 R = 2.6 X IO-4 C/kg). Since this discovery, committees charged with the responsibility for estimating genetic hazards of radiation have tended to regard the induced mutation frequency in women as negligible compared to that in men. However, as this author (2) and others have pointed out, and as the committees have recognized, the immature oocytes of the mouse, though resistant to mutation induction, are highly sensitive to killing by radiation, whereas human immature oocytes are resistant to killing. There are also species differences in the cytological appearance of the arrested oocytes. Thus, the question arises as to whether it is valid to assume that the human arrested oocyte will, like that of the mouse, be resistant to mutation induction. Our approach to this problem has been to investigate the mutational response of other oocyte stages in the mouse, including the maturing and mature ones, which are resistant to killing by radiation. These stages are highly mutationally sensitive to acute, high-dose-rate irradiation, but have proved to be insensitive to low-dose-rate irradiation. Furthermore, at low doses of high-dose-rate irradiation the mutation frequency drops well below that expected on a linear relationship with dose. Therefore, we have concluded that mouse oocyte stages covering a wide range of sensitivity to killing will, under the usual conditions of human exposure, show a low or zero mutation frequency (2). The latest, 1972, reports of the United Nations Scientific Committee on the Effects of Atomic Radiation (UNSCEAR) (5) and the U.S. National Academy of Sciences Committee on the Biological Effects of Ionizing Radiations (BEIR) (6) adopted a similar view. The mutational response of the mouse arrested oocyte was taken as zero or near zero, and the mutational sensitivity of the mouse maturing and mature oocytes to chronic irradiation was computed to be only about 1/20 of that to acute irradiation. Since the publication of these reports, Lyon and Phillips (7) have collected a most valuable set of data which support and extend the conclusions reached from the earlier results. In contrast, the interpretation of the earlier work has been challenged by Wolff (8) and Abrahamson and Wolff (9). In two papers, containing essentially the same material, they attempt to explain the mouse specific-locus mutation data on the basis of an old mathematical model for two-break chromosome aberration induction. In order to make the data fit this model, they have assumed a duration of oocyte stage sensitivity which, as the present paper will show, is inconsistent with the facts, and they have concluded from this that the mutational sensitivity Abbreviations: R, roentgen; UNSCEAR, United Nations Scientific Committee on the Effects of Atomic Radiation; BEIR, [Committee onl Biological Effects of Ionizing Radiations.
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Proc. Natl. Acad. Sci. USA 74 (1977)
Table 1. Spontaneous mutation frequency at seven specific loci in female mice Number of offspring scored
Number of mutations
Ref.
166,826 37,813
3 or 8* 0
Russell (2)t Batchelor et al. (4)
204,639
3 or 8
demonstrated for comparisons restricted
to conceptions
oc-
curring within 2 weeks after accumulation of the dose (10). In order to utilize data collected from conceptions occurring more than 3 weeks after irradiation, in this example, it is necessary to compute the portion of the dose received in the effective period. This was done, but not published, in a rather crude way in computations used to arrive at the figure of 1/20 which, as was mentioned earlier, was accepted by the UNSCEAR and BEIR committees as the ratio of effects of chronic
* Three independent mutational events, one of which was a cluster of 6. t Also includes previously unpublished data.
of the maturing and mature mouse oocytes to chronic irradiation is much greater than had been supposed. The present paper, in addition to providing new data on spontaneous mutations, analyzes, in more detail than heretofore, the results of low-level irradiation of maturing oocytes, including the new data of Lyon and Phillips. It also discusses the interpretation of Abrahamson and Wolff, and presents some hitherto unpublished information on the distribution of mutations with time after irradiation that has an important bearing on the discussion. RESULTS AND DISCUSSION One of the difficulties in measuring the effect of low-dose-rate irradiation on mutation frequency in maturing oocytes is that the length of radiation exposure time necessary to accumulate a sizable dose may approach the duration of the oocyte stage under measurement. For example, suppose we want to determine the mutation frequency from chronic irradiation of maturing oocytes that are not more than 6 weeks from ovulation. Giving a dose of 258 R took 3 weeks. Therefore, any conceptions occurring more than 3 weeks after the end of irradiation would involve oocytes that received part of their dose earlier than 6 weeks before ovulation. When we discovered that the mutation frequency drops apparently to zero for conceptions occurring more than 6 or 7 weeks after irradiation (1), it was immediately apparent, and was pointed out at that same time, that the lowness of a total mutation frequency in offspring collected from a 6-week mating period after the end of irradiation could have resulted partly from a low-dose-rate effect on maturing oocytes and partly from a portion of the dose having been given to the oocytes in immature stage, more than 6 weeks before conception. However, as was shown long before that, at the beginning of our dose-rate studies in females, a very low mutation frequency from chronic irradiation could be
With additional data from Lyon and Phillips (7) now available, and with the mutation frequencies challenged by Abrahamson and Wolff (9), it now seems desirable to make more precise estimates. In order to do this, it was necessary to determine as sharply as possible the time interval after irradiation at which mutation frequency dropped. It was also necessary to find out if this was a sudden drop or a gradual one spread over several weeks. Pertinent data from our experiments are collected in Table 2. The radiation in these experiments was delivered in a few minutes or over about 8 hr. No mutations were obtained in conceptions occurring more than 6 weeks after irradiation, and the frequencies in the fifth and sixth weeks showed no decline compared with earlier weeks. The cut-off period appears to be very sharp, and according to Oakberg's latest timing of oocyte development (ref. 11, and personal communication) may be coincident with the oocyte stage in which formation of the zona pellucida begins, a stage that takes approximately 6 weeks to reach ovulation. From Table 2 and other data it also appears that offspring conceived in the first week after irradiation have a lower mutation frequency than those conceived during weeks 2 to 6 after irradiation. This will -be discussed in another
to acute irradiation in maturing oocytes.
paper.
With this information, it is now possible to compute an "effective dose" for offspring conceived within 6 weeks after the end of a period of chronic irradiation. By "effective dose" is meant the portion of the total dose that will have been received 6 weeks or less before ovulation. As an example of the computation, we can pick one that happened to work out in simple fashion, namely, the 400-R experiment, in which the dose was given at approximately 0.009 R/min over a period of 35 days. Offspring from conceptions that occurred in the first week after the end of irradiation came from oocytes that received all of their dose in less than 6 weeks before ovulation. For animals conceived in the second week the average dose received by the oocytes in the 6 weeks before ovulation turns out to be 90% of the total 400 R, or 360 R. For the third week it is 70%, or 280 R, and so on. In the sixth week it is only 40 R. By weighting these doses by the number of ani-
Table 2. Distribution of offspring and mutations at seven specific loci in conceptions occurring during successive weeks after irradiation of female mice with various doses and dose rates 400 R, 0.8 R/min 50 R, 90 R/min 200 R, 90 R/min No. of No. of No. of No. of No. of No. of mutations mutations mutations offspring offspring Week offspring 1 2 3 4 5 6 7 8 9 10 11 on
28,547 3,604 454 1,138 8,395 3,327 328 17 5 1
13 2 1 0 12 5 0 0 0 0
84,614 8,016 2,904 9,587 45,179 16,304 13,868 9,092
11,096 7,433 50,570
6 0 0 1 3 3 0 0 0 0 0
49,039 7,928
14 3
737 844
1 4 6
7,342 4,990 170 2 4
2 0 0 0
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Table 3. Number of mutations at seven specific loci l4W offspritg conceived within 6 weeks after the end of exposure in various low-level radiation experiments on female mice
Weighted Ref.
Dose given, R
From Table 1 Lyon et al. (7) (group D) Russell (10) Lyon et al. (7) (group C) Russell (2, 12)1 Russell (2, 12) Carter (13)
0 215t 258 215t 400 400 615t
Dose rate 0 20 X 10.6 Rt 0.009 R/min 20 X 10.6 R§ 0.009 R/min 0.009 R/min 0.05 R/min
mean effective dose,* R
Number of offspring
Number of mutations
0 172 207 212 283 284 615
204,639 14,671 7,692 21,204 13,742 14,402 10,177
3 or 8 1 1 0 2 1 1
Mutation frequency per locus X 106 2.1 or 5.6 9.7 18.6 0.0 20.8 9.9 14.0
* Dose delivered to oocytes in the 6 weeks prior to ovulation (see text for detailed explanation). t Converted from rads. I Delivered over 4 weeks. § Delivered over 5 days. I Animals irradiated were old previously bred females.
mals in each week, the weighted mean dose for the whole population can be determined. In this case it is 283 R, as shown in Table 3. Table 3 shows the results of similar calculations made for two other experiments of ours and for the two sets of data from Lyon and Phillips. The Carter experiment required no adjustment of dose because all matings were made in less than 2 weeks after the 12-day exposure period. The Lyon and Phillips data were not from low-dose-rate irradiation, but from 20 fractions of 10 rad each spaced far enough apart to give a result that is apparently no more damaging than a low-dose-rate exposure. Two values are given for the control. There were three independent mutational events, one of which was a cluster of 6, representing six mutant germ cells from a single mutational event occurring early in development. This complicates the calculation of the spontaneous rate, and, as I have explained elsewhere (2), gives two extreme values. One can assume that, in spite of our happening to find one, clusters are extremely rare. On this basis there would be little error in taking the mutation frequency as 3 in 204,639. On the other hand, one can argue that our only estimate of the frequency of clusters is what was observed, namely one out of three mutational events. Then we must assume the mutation frequency to be 8 in 204,639. In this case, confidence limits and weighting should not be based on 204,639 independent observations: % of this, or 76,740, would be a better estimate. The frequency of independent mutational events would then be taken as 3 in 76,740 (the same as 8 in 204,639). The mutation frequencies shown in Table 3 are plotted in Fig. 1. The results for the similar doses of 207 and 212 R have been averaged in one point on the figure, but were treated as separate values in the regression analysis. The 283-R and 284-R data have not been combined because the 283-R experiment was the only one that used old animals. Other data to be presented elsewhere show consistently higher induced mutation rates for older females. Four weighted least squares regression lines were determined. The solid lines are based on all the data, the broken lines on all the data with the exception that old females are excluded. Each set has two fits, one for each of the two control values. The results may now be compared with the predictions of Abrahamson and Wolff (9). They assumed that the acute irradiation data will fit a simple quadratic equation: Y=C+ aD+
#D2
in which Y equals the expected yield of mutations, C equals the
control rate, a and # are coefficients, and D is dose. For females, they fitted this equation to the control point and to our 50-R, 200-R, and 400-R single acute dose points. The. a derived from this was used to predict what the mutation frequency should be at low-dose rates. Actually three separate fits were made based on three different control rates. Two of these, like ours, 100-
80-
*~~~~~~/ ' 0
/0/
a
20ig
0
100
200
300 400 Dose, R
500
660
700
FIG. 1. Weighted least squares regression lines of data in Table 3, and theoretical fits by Abrahamson and Wolff (8). See text for details. O. Only data point for irradiated old breeding females. Remaining points are for females. irradiated as young virgins. Intercepts, C, and slopes, a, are as follows:
Regression line Fit of data a, all data b, all data c, excluding old females d, excluding old females Abrahamson-Wolff theoretical fit Cluster = 0 Cluster = 1
C X 107
aX
107
21.2 55.4 21.3 54.3
0.296 0.178 0.221 0.113
8.39 16.7
1.43 1.26
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Genetics: Russell
were based on treating the cluster as one or six mutations. The third one was based on excluding the cluster altogether, on the argument that it must have occurred in oogonia and the induced mutations can occur only in oocytes. This is a departure from the usual consideration of a spontaneous rate as a pergeneration rate. It will also undoubtedly lead to bias, particularly where the sibships are smaller in the irradiated population and, therefore, less likely to detect clusters. However, this is the fit that Abrahamson and Wolff prefer. All three a values were then used to compute the number of mutations expected in the various experiments on females. The authors focus on the fits derived from treating the cluster as 0 and 1, apparently rejecting the remaining fit, where the cluster is taken equal to six mutations, as giving a poor match with the data. They recognize that the other two fail to give good agreement with all three of the low-dose-rate experiments considered by them, namely, Carter's and our 258-R and 400-R data. Then, in an extensive discussion, they try to explain away the discrepancy. Their first step is in keeping with our procedure. The data cited are for all offspring conceived within 7 weeks after the end of irradiation, and Abrahamson and Wolff realize that the effective dose to the maturing oocyte stages will average less than the total given. Errors arise when they try to compute the effective dose on the basis of number of estrous cycles and an oocyte maturation scheme that seems to have no basis in reality. Thus, they compute that the effective dose in the Carter experiment would be only one-half or less of the dose given. The irradiation time in this experiment was 12 days, and all matings occurred within the 2 weeks of the end of irradiation. In view of Table 2, and the well-known fact that maturation and loss of oocytes occur at the same rate in breeding and nonbreeding females (14), it is hard to see how any of the oocytes in Carter's experiment could have received less than the total dose during the maturing stages that are sensitive to acute irradiation. It should be noted that the two mutations in the 283-R experiment in Table 3 were from conceptions occurring in the fifth week after the end of irradiation, a period long after the time when Abrahamson and Wolff would have had the effective dose down to zero. Although Abrahamson and Wolff include the new Lyon and Phillips (7) data for a single 200-R dose of acute irradiation, they omit any consideration of the fractionated exposures. If they had computed their expected value (assuming cluster = 0) for this set of data, it would have been 9.5 mutations expected where only one was observed. With a correct adjustment for effective dose they would still have predicted 7.8 mutations for the one observed. Abrahamson and Wolff, on the one hand, admit that their corrections for effective dose are crude, but, on the other hand, conclude "it is reasonable to assume" that the wide discrepancies between their theoretical expectations and the actual data are "caused by some of the radiation occurring during less mutable stages." Because the data shown in Fig. 1 have already been adjusted for this factor by our criteria, it is of interest to see how much discrepancy is still left between the corrected observed values and the Abrahamson-Wolff prediction. The two values of a for the fits preferred by Abrahamson and Wolff are shown in Fig. 1. The third fit would be much closer to the observed data, but gives a very poor fit to some other data points, and, for that reason, was apparently not acceptable to Abrahamson and Wolff. The slope of the upper Abrahamson-Wolff line exceeds the slopes of the actual data by multiples of 4.8, 6.5, 8.0, and 12.7; the slope of the lower line (cluster = 1) exceeds them by 4.3, 5.7, 7.1, and 11.2. Both Abrahamson-Wolff slopes are highly statistically significantly above all the slopes of the actual data. The
lowest t value in these comparisons is 7.84, while the critical value of t for 5% probabality is 2.57. Thus the Abrahamson-Wolff derivation, from acute irradiation, of the mutation frequency theoretically expected, on their hypothesis, for low-level irradiation exceeds the observed value by from 4- to 13-fold. It should be clear that their approach is not a reliable one for estimating hazards. * The observed data are still meager in terms of number of mutations obtained, and the confidence limits are, therefore, very wide for each point. Nevertheless the fitted lines presumably provide a better estimate of the mutation frequency from low-level irradiation of mature and maturing oocytes than has been available before. APPLICATIONS OF THE DATA TO HUMAN HAZARD EVALUATION For mature and maturing oocytes, the UNSCEAR and BEIR committees, in their 1972 reports (5, 6), accepted 1/20 as the ratio of mutation rate from chronic irradiation to that from acute irradiation. The rates presented in this paper (slopes a, b, c, and d in Fig. 1) for low-level irradiation of mature and maturing oocytes, when compared to the mutation rate for our 400-R acute irradiation data (ref. 15, and unpublished), yield ratios of 1/18, 1/29, 1/24, and 1/46, respectively. The true ratios would be expected to be even somewhat lower, because the 400-R acute irradiation data contain a higher proportion of offspring from oocytes that received all of their radiation in the week before ovulation, when the mutational sensitivity is lower than for radiation received from 2 to 6 weeks before ovulation. In any case, with the more precise estimation of the effective doses than was available to the committees, and with the addition of the new data of Lyon and Phillips (7), it turns out that the committees' statements did not underestimate the risks from low-level irradiation of mature and maturing oocytes. It is of interest to compare the specific-locus mutation rates obtained for mature and maturing oocytes with the mutation rate in the male mouse for chronic irradiation of the germ-cell stage primarily at risk in human hazards, namely, the spermatogonia. The mutation rate, for the same seven loci, in mouse spermatogonia irradiated at dose rates of 0.009 R/min and below, was calculated in the review paper by Searle (16) to be 6.59 X 10-8 per locus, per R. The rates for low-level irradiation of mature and maturing oocytes (slopes a, b, c, and d in Fig. 1) are, respectively, only 0.44, 0.27, 0.33, and 0.17 times as effective. Furthermore, it should be pointed out that only in the first of these four possible estimates (slope a) is the induced mutation rate in oocytes significantly above the control rate. Thus, the ratio of effectiveness to the spermatogonial mutation rate could be zero. Similarly, the ratio of effectiveness to acute irradiation given in the previous paragraph could also be zero.
With regard to the validity of extrapolating from mouse immature arrested oocytes to human immature arrested oocytes, the results given in Table 2 have an indirect bearing on this problem. In these data, and in others to be presented elsewhere, the mutation frequency is lower for the most mature oocytes, those that produced the offspring for week 1 in Table * Note Added in Proof: Abrahamson and Wolff have already used their calculations in two reports on radiation hazards: (a) Report of the Nuclear Energy Policy Study Group (1977) Nuclear Power Issues
and Choices (Ballinger Publishing Co., Cambridge, MA); and (b) National Academy of Sciences Committee on Safe Drinking Water (1977) Drinking Water and Health (National Academy of Sciences,
Washington, DC).
Genetics: Russell
2, than for the less mature oocytes that pkoduced thbfig listed for weeks 2 to 6. The fully mature oocytes are less siiive to killing than the less mature ones. Thus, here there is a positive correlation between killing and mutational sensitivity. This is in contrast to the negative correlation found for immature arrested oocytes, which give no evidence of mutation induction but which are highly sensitive to killing. It appears, from the lack of consistent correlation, that mutation induction and killing are independent events. In this connection, the new data of Cox and Lyon (17) on x-ray induction of dominant lethal mutations in mature and immature oocytes of guinea pigs and golden hamsters are of great importance. In both species, they found lower mutation yields from immature than from mature oocytes, despite the fact that the immature oocytes of the guinea pig are less sensitive to killing than the mature ones, while the reverse is true for the golden hamster, which, therefore, resembles the mouse. Thus again, as the authors state, there is "no general pattern ... of correlation, either positive or negative, in the sensitivity of oocytes to killing and to dominant lethal induction." This is in contrast to the statement by Abrahamson (18) that "I suspect that cell killing, chromosome rearrangements and gene mutations are rather closely linked." The fact that cell killing of oocytes is apparently not linked to the other two effects is not surprising to us. Death of the cells occurs rapidly, before cell division, and is, therefore, not related to the kind of death that results from aneuploidy after cell division. Cox and Lyon question whether dominant lethals and gene mutations behave similarly, but their doubts are based on the fact that Searle and Beechey (19) had shown an increase in dominant-lethal frequency over the first 3 weeks after irradiation of female mice. Cox and Lyon believed that this disagreed with the gene mutation results, because they were aware only of the decline in mutation frequency with time after irradiation. However, there is no disagreement: the decline in gene mutation frequency occurs only after 6 weeks, and, with the new data reported in Table 2, it is now clear that, as with dominant lethals, there is, in fact, an increase in gene mutation frequency after the first week. In summary, there would now seem to be less reason than before for rejecting an application of the mouse immature arrested oocyte data to the human immature arrested oocyte. In the immature oocytes, the specific-locus mutation frequency and X-chromosome loss frequency (20) in the mouse and the dominant lethal frequency in guinea pig and golden hamster are all low compared to the frequencies in mature oocytes. The fact that this is so, despite the differences in sensitivity to cell killing, and in chromosomal morphology, in the immature oocytes of these species, indicates that the sensitivity to cell killing of the mouse immature oocyte may not be sufficient reason to prevent its use in predicting the mutational response of the human immature oocyte. If, on the side of caution, one continues to consider the possibility that the human immature arrested oocyte might be as mutationally sensitive as the most sensitive of all oocyte stages in the mouse, namely the maturing and mature oocytes, then the present paper provides estimates for this extrapolation. With low-level irradiation, the estimates of mutational frequency in these stages average from 0.17 to 0.44 times that in spermatogonia, but in three of the four estimates the frequencies are not significantly above control values. Thus, even in the event that the response of the human immature arrested oocyte is unlike that of the mouse arrested oocyte, but similar to that of the most
Proc. Natl. Acad. Sci. USA 74 (1977)
3527
monsltive oocyte stages in the mouse, it seems likely that the genetic hazard of radiation in the female will still be less than in the male. For the statistical analyses I gratefully acknowledge the help of D. G. Gosslee, Computer Sciences Division, Union Carbide Corp., Nuclear Division. Research was sponsored by the Energy Research and Development Administration under contract with the Union Carbide Corp. 1. Russell, W. L. (1965) "Effect of the interval between irradiation and conception on mutation frequency in female mice," Proc. NatI. Acad. Sci. USA 54, 1552-1557. 2. Russell, W. L. (1972) "The genetic effects of radiation," in Peaceful Uses of Atomic Energy (United Nations, New York), Vol. 13, pp. 487-500. 3. Russell, W. L. (1951) "X-ray-induced mutations in mice," Cold Spring Harbor Symp. Quant. Biol. 16, 327-36. 4. Batchelor, A. L., Phillips, R. J. S. & Searle, A. G. (1969) "The ineffectiveness of chronic irradiation with neutrons and gamma rays in inducing mutations in female mice," Br. J. Radiol. 42, 448-451. 5. United Nations Scientific Committee on the Effects of Atomic Radiation (1972) Ionizing Radiation: Levels and Effects (United Nations, New York), Vol. 2. 6. National Academy of Sciences-National Research Council Advisory Committee on the Biological Effects of Ionizing Radiations (1972) The Effects on Populations of Exposure to Low Levels of Ionizing Radiation (National Academy of Sciences, Washington, DC). 7. Lyon, M. F. & Phillips, R. J. S. (1975) "Specific locus mutation rates after repeated small radiation doses to mouse oocytes," Mutat. Res. 30, 375-382. 8. Wolff, S. (1975) "Estimation of the effects of chemical mutagens: Lessons from radiation genetics," Mutat. Res. 33, 95-102. 9. Abrahamson, S. & Wolff, S. (1976) "Re-analysis of radiationinduced specific locus mutations in the mouse," Nature 264, 715-719. 10. Russell, W. L., Russell, L. B. & Cupp, M. B. (1959) "Dependence of mutation frequency on radiation dose rate in female mice," Proc. Natl. Acad. Sci. USA 45,18-23. 11. Oakberg, E. F. & Palatinus, D. T. (1976) "The stage of the mouse oocyte at which the change in sensitivity to radiation-induced mutations occurs," Genetics 83, s56 (abstr.). 12. Russell, W. L. (1963) in Repair from Genetic Radiation Damage, ed. Sobels, F. (Pergamon Press, Oxford), pp. 205-217; 231235. 13. Carter, T. C. (1958) "Radiation-induced gene mutation in adult female and foetal male mice," Br. J. Radiol. 31, 407-411. 14. Oakberg, E. F. (1966) in Radiation and Ageing, eds. Lindop, P. J. & Sacher, G. A. (Taylor and Francis, London), pp. 293-306. 15. Russell, W. L. (1965) "The nature of the dose-rate effect of radiation on mutation in mice," Jpn. J. Genet. Suppl. 40, 128140. 16. Searle, A. G. (1974) "Mutation induction in mice," Adv. Radiat. Biol. 4, 131-207. 17. Cox, B. D. & Lyon, M. F. (1975) "X-ray induced dominant lethal mutations in mature and immature oocytes of guinea pigs and golden hamsters," Mutat. Res. 28, 421-436. 18. Abrahamson, S. (1976) in "Discussion" in Biological and Envi-
ronmental Effects of Low-Level Radiation (International Atomic Energy Agency, Vienna), Vol. 1, p. 18. 19. Searle, A. G. & Beechey, C. V. (1974) "Cytogenetic effect of xrays and fission neutrons in female mice," Mutat. Res. 24, 171-186. 20. Russell, W. L., Hunsicker, P. R., Kelly, E. M., Vaughan, C. M. & Guinn, G. M. (1973) "X-chromosome loss in the offspring of
irradiated female mice," Oak Ridge National Laboratory, Biology Division, Annual Progress Report, ORNL-4915 (Oak Ridge National Laboratory, Oak Ridge, TN), p. 98.
