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Discrimination between the effects of X-ray irradiation of the mouse oocyte and uterus on the induction of dominant lethals and congenital anomalies
Mutation Research, 149 (1985) 231-238
231
Elsevier MTR 04007
Discrimination between the effects of X-ray irradiation of the mouse oocyte and uterus on the induction of dominant lethals and congenital anomalies II. Localised irradiation experiments John D. West *, K. Margaret Kirk, Yvonne Goyder and Mary F. Lyon MRC Radiobiology Unit, Harwell, Didcot, Oxon OXl l ORD (Great Britain)
(Received18 May 1984) (Revision received24 October 1984) (Accepted 31 October 1984)
Summary In order to evaluate whether irradiation of the postimplantation maternal environment contributed to the induction of postimplantation mortality or congenital anomalies, mouse ovaries were surgically exteriorised and selectively irradiated or shielded in a specially constructed apparatus. The results show that exposure of the mouse abdomen and uterus to 3.70 G y X-rays, 15-21 days prior to conception, has no significant effect on the incidence of either postimplantation mortality or congenital anomalies. Exposure of the ovaries to 3.27 Gy X-rays during the same period, however, increased the frequency of both postimplantation mortality and congenital anomalies.
The possibility of screening for congenital anomalies, such as malformed, dwarf or retarded fetuses offers an additional in vivo test for mutagenicity that can be applied in conjunction with the well-established dominant lethal test. A combined screen of this sort was suggested by Knudson et al. (1977). Nomura (1979, 1982) showed that exposure of germ cells of male or female mice to X-rays produced significant numbers of dominant lethals and congenital anomalies among their offspring. Kirk and Lyon (1982) demonstrated that the yields of dominant lethals and congenital anomalies from X-irradiated female mice were both dose- and time-dependent. However, their study did not determine whether X-irradiation of the * Present address: Department of Obstetrics and Gynaecology, Centre for ReproductiveBiology,37 Chalmers Street, Edinburgh EH3 9EW (Scotland).
uterus, or other aspects of the maternal environment, contributed to the observed incidence of dominant lethals and congenital anomalies. Embryo-transfer experiments, in which embryos were transferred from irradiated to untreated females and vice versa, showed that there was no significant effect of irradiation of the m a t e r n a l e n v i r o n m e n t on postimplantation mortality or fetal weight. However the result for congenital anomalies was equivocal (West et al., 1985). In this paper we described a novel technique which permits the selective irradiation of either the ovary or the uterus. This provides an experimentally more efficient means of testing for an effect of irradiating the maternal environment. We have used this method to investigate whether irradiation of the uterus contributes to the induction of postimplantation mortality and congenital anomalies.
0027-5107/85/$03.30 © 1985 ElsevierSciencePublishers B.V. (BiomedicalDivision)
232 Materials
and methods
Irradiation Virgin female ( C 3 H / H e H ? X 1 0 1 / H $ )El mice ( h e n c e f o r t h a b b r e v i a t e d to 3H1) were a n a e s t h e t i s e d with A v e r t i n a n d e x p o s e d to p a r t b o d y X - r a y t r e a t m e n t w h e n a p p r o x i m a t e l y 9 weeks old. T h e mice were d i v i d e d into 6 groups a c c o r d ing to the X - r a y t r e a t m e n t given ( T a b l e 1). M i c e in 4 of these g r o u p s u n d e r w e n t surgery to p e r m i t selective i r r a d i a t i o n or shielding of the ovaries using a specially c o n s t r u c t e d p e r s p e x b r i d g e a p p a r a t u s (Figs. 1 a n d 2). Incisions were m a d e in the d o r s a l skin a n d a b d o m i n a l wall. T h e ovaries a n d fat p a d s were p u l l e d t h r o u g h the incisions a n d the oviducts were p a s s e d t h r o u g h a n a r r o w slit in the p e r s p e x b r i d g e until the ovaries a n d fat p a d s rested in c o u n t e r s u n k holes in the bridge, as shown in Fig. 3. W h e n the ovaries were held in p o s i t i o n the uterus was s u s p e n d e d f r o m the bridge. A p p r o p r i a t e l e a d shielding was p l a c e d o n t o p of the a s s e m b l e d b r i d g e a p p a r a t u s to expose either the ovaries o r the rest of the b o d y p o s t e r i o r to the ovaries (including the uterus), to X - r a y s f r o m a source a b o v e the a p p a r a t u s , as shown in Figs. 4 a n d 5. T o m i n i m i s e the risk of i r r a d i a t i n g the top of the uterus with the ovary, the m o u s e ' s tail was p u l l e d gently, i m m e d i a t e l y b e f o r e i r r a d i a t i o n , in o r d e r to h o l d the uterus taut a n d a w a y f r o m the i r r a d i a t e d field. T h e fat p a d s a n d p r o b a b l y the
Fig. 1. The two parts of the perspex bridge apparatus used for selective irradiation or shielding of the ovaries. The top surfaces measure 76 nun long x 80 mm wide (larger bridge) and 70 mm x 86 mm (smaller bridge). oviducts were i r r a d i a t e d with the ovaries. F o l l o w ing i r r a d i a t i o n the ovaries a n d r e p r o d u c t i v e tract were r e t u r n e d to the a b d o m i n a l cavity a n d the incisions in the skin were closed with m e t a l w o u n d clips. All surgical i n s t r u m e n t s were flame sterilised. N e g a t i v e c o n t r o l s (groups A1 a n d A2) u n d e r w e n t surgery b u t were n o t i r r a d i a t e d . Mice in the positive c o n t r o l g r o u p s (B a n d D) were anaesthetised a n d i r r a d i a t e d f r o m above, without surgery, in groups of three to five. L e a d shielding was a r r a n g e d to cover the h e a d a n d t h o r a x a n d leave the a b d o m e n exposed.
TABLE 1 EXPERIMENTAL GROUPS Group
Anaesthetic
Surgery
X-Irradiation and shielding
X-Ray dose rate (Gy/min)
Absorbed dose X-rays (Gy)
A1
+
+
0
0
A2
+
+
0
0
B
+
-
0.79
3.27
C
+
+
0.69 a
3.27
D
+
-
0.79
3.70
E
+
+
Sham-irradiated in perspex bridge assembly. Shielded as shown in Fig. 4. Sham irradiated in perspex bridge assembly. Shielded as shown in Fig. 5. Irradiated in groups of 3-5. Head and thorax shielded. Ovaries irradiated in perspex bridge assembly. Shielded as shown in Fig. 4. Irradiated in groups of 3-5. Head and thorax shielded. Irradiated in perspex bridge assembly. Ovaries shielded as shown in Fig. 5.
0.79
3.70
a See text of Materials and Methods section for explanation of the low dose rate.
233
Fig. 2. The assembled perspex bridge apparatus. The alternative lead shields are shown (inverted) beside the bridge apparatus. The pegs on the larger lead shield locate in the semi-circular holes at the comers of the bridge assembly. The mouse lies on the perspex platform with its head beneath the smaller bridge (to the right of the picture).
Fig. 3. Diagram showing a mouse in position beneath the bridge apparatus. The oviducts are passed through a narrow slit at the edge of the larger perspex bridge (Fig. 1) until the ovaries and fat pads rest in the countersunk holes. The smaller perspex bridge is then put in position, next to the countersunk holes, to support the lead shield.
Fig. 4. Perspex bridge assembly and lead shield used to selectively irradiate ovaries (group C) with an X-ray source above the apparatus. The ovaries are irradiated through two 4-mm diameter holes, 17 mm apart, in the centre of the shield.
Fig. 5. Perspex bridge assembly and lead shields used to selectively shield ovaries while irradiating the uterus (group E) with X-rays from above.
Irradiation was designed to provide an absorbed dose of 3.60 G y (360 rad) of X-rays (250 kV, half-value thickness 1.1 mm Cu), to the appropriate region, in all the irradiated groups. However, while the experiment was in progress it was realised that selective irradiation of the ovary alone (group C) resulted in a lower than expected absorbed dose because of the small field size. Revised dosimetry showed that this group (C) received 3.27 Gy X-rays while other irradiated groups (D and E) each received 3.70 Gy X-rays. It was therefore necessary to include an extra 3.27 G y positive control group (B) as a separate control for group C (Table 1). The mice were mated to untreated 3H1 males 15-21 days after exposure to X-rays or sham irradiation.
Analysis of uterine contents Most females were killed 18 days after the detection of the vaginal plug so that the litters were 18.5 days post coitum (p.c.). A few females were killed a day earlier. Corpora lutea were counted and the uterine contents were examined and classified as described by Kirk and Lyon (1982) except that only dwarf or visibly abnormal fetuses and control littermates were fixed in Bouin's fluid for later dissection. A fetus was considered to be dwarf if it weighed less than 75% of the mean of the others in the litter. The index of % induced preimplantation loss
234 was calculated as [1-(
Implants, treated / _ _ _ _ I m pcontrol l a n t s ' . ] ] x.x. .t m . Corpora lutea, treated Corpora lutea, control ] ]
The index of % i n d u c e d p o s t i m p l a n t a t i o n domin a n t lethality was calculated as 1-
( Livefetuses, treated Live fetuses, control Total implants, treated / T ~ s ~ l )]
×100 Statistical tests were performed using a Hewlett Packard 97 p r o g r a m m a b l e calculator, p r o g r a m m e d b y M r r D.G. P a p w o r t h to c o m p u t e X 2, Fisher's exact test a n d b i n o m i a l s t a n d a r d errors. Results
I n giving the frequencies of e m b r y o n i c mortality a n d fetal a b n o r m a l i t i e s (Table 2) the two negative control groups (sham ovary irradiation a n d
s h a m uterus irradiation), shown as groups A1 a n d A2 i n T a b l e 1, have b e e n a m a l g a m a t e d as group A. There was n o significant difference b e t w e e n these two groups i n either the frequency of postimp l a n t a t i o n m o r t a l i t y or the frequency of a b n o r m a l fetuses. The frequency of p o s t i m p l a n t a t i o n mortality a m o n g the 22 females in group A1 was 2 3 / 2 2 0 (10.5%) c o m p a r e d with 1 8 / 1 8 6 (9.7%) for the 19 females in group A2 (X 2 = 0.0088). The frequency of a b n o r m a l fetuses was 3 / 1 9 7 (1.5%) for group A1 a n d 2 / 1 6 8 (1.2%) for group A2 (Fisher's Exact test gives P = 1.00).
Postimplantation mortality Significant n u m b e r s of p o s t i m p l a n t a t i o n domin a n t lethals were i n d u c e d when either the ovaries alone or the ovaries plus the uterus were irradiated (groups B, C a n d D in T a b l e 2) b u t negligible n u m b e r s were p r o d u c e d when the ovaries were selectively shielded from irradiation (group E). This is reflected b y the statistical tests o n the
TABLE 2 INDUCTION OF CONGENITAL ANOMALIES AND DOMINANT LETHALS BY X-RAY IRRADIATION Group: Anaesthetic: Surgery: Irradiated organs: Absorbed dose of X-rays (Gy):
C + + ovaries
0
B + ovaries plus uterus 3.27
Number of ¢ ¢ Corpora lutea
41 473
85 1187
Implantations Total Small moles Large moles Dead fetuses Abnormal fetuses (dwarf and other) a Normal fetuses
406 29 7 5 5(3 + 2) 360
Corpora lutea/females Implants/females % Induced preimplantation loss b % Postimplantation mortality % Induced postimplantation dominant lethality b % Abnormal fetuses c
A + + none
11.5 9.9 10.1 + 1.5 _ 1.4+0.6
a See Table 5 for classification of abnormal fetuses. b See Materials and Methods section for calculation. Abnormal fetuses c % Abnormal fetuses (Abnormal + normal fetuses)
×
100.
521 202 8 3 20(16 + 4) 288 14.0 6.1 48.9 + 1.9 40.9 + 2.2 34.2 + 2.6 6.5+1.4
3.27
D + ovaries plus uterus 3.70
E + + uterus (and abdomen) 3.70
82 1056
85 1237
46 609
550 240 5 1 12(5 + 7) 292 12.9 6.7 39.3 + 2.1 44.7 ___2.1 38.5 + 2.6 3.9+1.1
537 254 15 0 11(5 + 6) 257 14.6 6.3 49.4 + 1.9 50.1 + 2.2 44.5 + 2.6 4.1+1.2
465 42 7 8 4{3 + 1) 404 13.2 10.1 11.0 ___2.6 12.3 + 1.5 2.4 + 2.3 1.0+0.5
235 TABLE 3 DIFFERENCES IN F R E Q U E N C I E S O F P O S T I M P L A N T A T I O N M O R T A L I T Y A N D A B N O R M A L FETUSES B E T W E E N GROUPS Groups compared
Postimplantation mortality
Abnormal fetuses
Frequency
Significance
Frequency
Significance
0varies+uterus irradiated, Sham irradiated
3.27 G y (B) (A)
213/521 41/406
X 2 =
107.2 ***
20/308 5/365
P = 0.00070 ***
0varies+uterus irradiated, Sham irradiated
3.70 Gy (D) (A)
269/537 41/406
X2 =
165.8 ***
11/268 5/365
P = 0.039 *
0varies+uterus irradiated, Ovaries + uterus irradiated,
3.70 Gy (D) 3.27 Gy (B)
269/537 213/521
X 2 =
8.68 **
11/268 20/308
P = 0.267
Ovaries irradiated, Sham irradiated
3.27 Gy (C) (A)
246/550 41/406
X 2 = 131.7 ***
12/304 5/365
P = 0.047 *
Ovaries +uterus irradiated, Ovaries irradiated,
3.27 Gy (B) 3.27 G y (C)
213/521 246/550
X2 =
20/308 12/304
P = 0.203
0varies+uterus irradiated, Uterus irradiated,
3.70 G y (D) 3.70 G y (E)
269/537 57/465
X 2 = 160.8 ***
11/268 4/408
P = 0.013 *
Uterus irradiated, Sham irradiated
3.70 G y (E) (A)
57/465 41/406
Ovaries irradiated, Uterus irradiated,
3.27 G y (C) 3.70 G y (E)
246/550 57/465
X2 =
1.46
0.81
X 2 = 125.3 ***
4/408 5/365 12/304 4/408
P = 0.742
P = 0.010 *
P values for abnormal fetuses were calculated using a two-sided Fisher's exact test. Asterisks are used for easy comparison with the X 2 results for postimplantation mortality as follows: * P < 0.05; ** P < 0.005; ***P < 0.001.
postimplantation mortality figures shown in Table 3. As expected, the frequency of postimplantation mortality was very significantly higher after irradiation of ovaries plus uterus (groups B and D) than after sham irradiation (A). Comparison of the two positive control groups shows that the higher absorbed dose of 3.70 Gy (group D) produced significantly more postimplantation mortality than the 3.27 Gy dose (group B). Irradiation of the ovaries alone (group C) also produced a very significant increase in postimplantation mortality over the sham-irradiated level (A) and this was not significantly lower than for the relevant positive Control group (B). Thus, irradiation of the ovaries alone can account for all of the postimplantation mortality produced by irradiation of the entire abdomen. In order to rule out the possibility that the anterior part of the uterus was irradiated, along with the ovaries, and contributed to the postimplantation mortality in group C, anterior and post-
erior implants were analysed separately. In uterine horns containing an odd number of implants the middle implant was allocated to the posterior group. Otherwise, implants were allocated equally to anterior and posterior groups (Table 4). Although the difference in frequency of postimplantation mortality between the two regions approaches significance using a 1-sided test (X 2= 2.46; P = 0.058) this is not relevant since the frequency in the anterior group is actually lower than that in the posterior group. There is, therefore, no evidence for higher postimplantation mortality in the anterior part of the uterus. Irradiation of the uterus alone (group E) produced very significantly lower postimplantation mortality levels than either irradiation of the whole abdomen with the same absorbed dose (group D) or irradiation of the ovaries alone with a lower absorbed dose (group C) (Table 3). This low level was not significantly different from the frequency of postimplantation mortality found among the sham irradiated, negative controls (group A).
236 TABLE 4 COMPARISON OF THE INCIDENCE OF POSTIMPLANTATION MORTALITY IN ANTERIOR AND POSTERIOR IMPLANTATION SITES FOLLOWING X-IRRADIATION OF THE OVARIES (GROUP C IN TABLE 1) Anterior implants Total implantations Small moles Large moles Dead fetuses Abnormal fetuses Normal fetuses Postimplantation mortality Abnormal fetuses
"
Posterior implants
236 93 3 0 7 133
Significance
314 147 2 1 5 159
96/236 (40.7%) 7/140 (5.0%)
150/314 (47.8%) 5/164 (3.0%)
X2 = 2.46 (not significant)a P = 0.396 (not significant)b
a See text for discussion. b p was calculated using a two-sided Fisher's exact test. These results show that i r r a d i a t i o n of the uterus or a b d o m e n , 15-21 days prior to mating, makes n o significant c o n t r i b u t i o n to the incidence of p o s t i m p l a n t a t i o n mortality.
w h e n the ovaries were selectively shielded (group E) or n o i r r a d i a t i o n was given (group A). A classification of the a b n o r m a l fetuses is s h o w n in T a b l e 5. T h e tests in table 3 show that the frequency of a b n o r m a l fetuses was significantly higher after i r r a d i a t i o n of the entire a b d o m e n (groups B a n d D) t h a n after sham i r r a d i a t i o n (group A). The yield of a b n o r m a l fetuses after irradiation with 3.70 G y (group D) was somewhat lower t h a n after 3.27 G y (group B). A l t h o u g h this difference was n o t significant, it was reflected b y the difference in
Congenital anomalies The incidence of congenital anomalies ( a b n o r mal fetuses) paralleled the incidence of postimp l a n t a t i o n m o r t a l i t y discussed above (Table 2). Higher frequencies of a b n o r m a l fetuses occurred when either the ovaries alone or the entire abdom e n were irradiated (groups B, C a n d D) t h a n TABLE 5 CLASSIFICATION OF ABNORMAL FETUSES Treatment group: Irradiated organs:
A none
Absorbed dose of X-rays (Gy):
0
B
C
D
E
ovaries plus uterus 3.27
ovaries 3.27
ovariesplus uterus 3.70
uterus (and abdomen) 3.70
Abnormality Dwarf Dwarf with open eyelids Dwarf with cleft palate Dwarf with liver and spleen abnormalities Dwarf with umbilical hernia Dwarf with microphthalmia, exencephaly and kidney abnormality
3 0 0 0 0
16 0
5 2
5 1
3 0
1 1
0 0
2 0
0 0
0
0
1
0
0
0
0
1
0
Umbilical hernia Open eyelids Exencephaly Hare-lip, cleft palate and polydactyly Polydactyly Isolated dextrocardia and ear abnormality
0 1 0 1 0 0
2 0 0 0 0 0
0 3 2 0 0 0
0 0 0 0 1 0
0 0 0 0 0 1
237
significance levels attained by the first two comparisons shown in Table 3. The incidence of abnormal fetuses after irradiation of ovaries alone (group C) was significantly higher than for the sham-irradiated negative control group (A). This yield, however, was not significantly lower than when the whole abdomen was irradiated with the same absorbed dose of X-rays (group B). Separate analysis of anterior and posterior implants (Table 4), as described in the previous section, revealed no significant difference between the frequency of abnormal fetuses between the two regions. Thus, irradiation of the ovaries alone can account for all of the abnormal fetuses irroduced by irradiation of the whole abdomen. Irradiation of the uterus alone (group E) produced significantly fewer abnormal fetuses than either irradiation of the whole abdomen with the same absorbed dose of 3.70 Gy (group D) or irradiation of the ovaries alone with the lower absorbed dose of 3.27 Gy (group C). This small yield was not significantly different from the yield of abnormal fetuses found among the sham-irradiated negative controls (group A). Although the levels of statistical significance are less impressive, the results for the abnormal fetuses parallel those for postimplantation mortality. The results show that irradiation of the uterus, 15-21 days prior to mating, makes no significant contribution to the incidence of abnormal fetuses. Discussion
The results of the localised irradiation experiments, reported here, confirm the results for the induction of postimplantation mortality and resolve the ambiguous results for the production of congenital anomalies that were obtained in the embryo-transfer experiments, reported in our companion paper (West et al., 1985). Thus, it appears that exposure of the uterus or abdomen of the female mouse to X-rays, 15-21 days prior to conception, makes no significant contribution to the incidence of either postimplantation mortality or congenital anomalies. Kirk and Lyon (1982) reported a dose-related yield of congenital anomalies following exposure of female mice to X-rays before mating. We can
now be confident that, for the group that were exposed 15-21 days before mating, any effect of X-rays that is mediated via the maternal reproductive environment and potentiates the production of congenital anomalies is trivial compared with the mutagenic effect on female germ cells. In principle, therefore, congenital anomalies, identified as dwarf or malformed fetuses, provide a valid in vivo assay for mutagenesis. The routine combination of a screen for congenital anomalies with the dominant lethal test for mutagenicity could provide useful additional information on genetic risks. In man, congenital malformations are relatively common in comparison with genetic diseases due to well defined gene or chromosome mutations. Consequently, the incidence of congenital malformations is sometimes used as an indicator for possible mutagenic effects in man (Blatt et al., 1980; Miller, 1983; Mulvihill, 1982; Rustin et al., 1982; Schull et al., 1981). However, the genetic basis for susceptibility to malformation is complex and not well understood. Although it is possible to predict the effect of a rise in mutation rate on the incidence of diseases with a clear genetic basis, including those due to autosomal or X-linked dominant genes or to chromosome anomalies, present genetic theory does not clearly predict the effect on incidence of diseases of irregular inheritance. Thus, it is valuable to have data on the induction of congenital malformation in the offspring in mice, for comparison with results obtained in man. This is the more important as a high proportion of the incidence of genetic disease in man is accounted for by conditions of irregular inheritance. Congenital malformations may therefore serve as a model for predicting the effect of a rise in mutation rate on the incidence of such conditions in general. Although our results show that the increased incidence of malformations among the offspring of irradiated female mice is indeed due to a mutagenic effect, there is still the question whether the underlying genetic basis of the anomalies is similar to that of malformations in man. Malformations, both in mouse and in man, may be due to chromosome aberrations or to genic effects. In man, the majority are not due to detectable chromosome changes (Carter, 1969; Hook, 1983). The genetic
238 basis of the a n o m a l i e s f o u n d in o u r w o r k with the mouse is n o t known. K i r k a n d L y o n (1984) a r g u e d that they p r o b a b l y result f r o m several different types of genetic change. However, this p o i n t n e e d s further work. This is p a r t i c u l a r l y so in view of the t e n d e n c y for the frequency of m a l f o r m a t i o n s to vary with the incidence of d o m i n a n t lethals, which have a m a i n l y c h r o m o s o m a l basis. O n the o t h e r hand, the o b s e r v a t i o n of N o m u r a (1979, 1982), that u r e t h a n e induces significant n u m b e r s of congenital a n o m a l i e s b u t n o t d o m i n a n t lethals, suggests some d i s s o c i a t i o n b e t w e e n these two tests.
Acknowk~lgements W e are grateful to Mr. S. A l l e n a n d his colleagues for c o n s t r u c t i n g the p e r s p e x b r i d g e a n d lead shield assembly. W e also t h a n k Mr. M. C o r p a n d Mr. P. A d a m s for d o s i m e t r y a n d i r r a d i a t i n g the mice, Mr. D . G . P a p w o r t h for help with statistical tests, Mr. G. W i l k i n s for p h o t o g r a p h y a n d Mr. G. F i s h e r a n d Ms. G. Collier for p r e p a r i n g Fig. 3. K . M . K . was s u p p o r t e d b y a g r a n t from the N a t i o n a l R a d i o l o g i c a l P r o t e c t i o n Board.
References Blatt, J., J.J. Mulvihill, J.L. Ziegler, R.C. Young and D.G. Poplack (1980) Pregnancy outcome following cancer chemotherapy, Am. J. Med., 64, 828-832. Carter, C.O. (1969) Genetics of common disorders, Br. Med. Bull., 25, 52-57. Hook, E.B. (1983) ICPEMC Working paper 5/3, Perspectives in mutation epidemiology, 3. Contribution of chromosome abnormalities to human morbidity and mortality and some
comments upon surveillance of chromosome mutation rates, Mutation Res., 114, 389-423. Kirk, M., and M.F. Lyon (1982) Induction of congenital anomalies in offspring of female mice exposed to varying doses of X-rays, Mutation Res., 106, 73-83. Kirk, M., and M.F. Lyon (1984) Induction of congenital malformations in the offspring of male mice treated with X-rays at pre-meiotic and post-meiotic stages, Mutation Res., 125, 75-85. Knudsen, I., E.V. Hansen, O.A. Meyer and E. Poulson (1977) A proposed method for the simultaneous detection of germ cell mutations leading to fetal death (dominant lethality) and of malformations (male teratogenicity) in mammals, Mutation Res., 48, 267-270. Miller, J.R. (1983) ICPEMC Working paper 5/4, Perspectives in mutation epidemiology, 4. General principles and considerations, Mutation Res., 114, 425-447. Mulvihill, J.J. (1982) Towards documenting human germinal mutagens: epidemiologic aspects of ecogenetics in human mutagenesis, T. Sugimura, S. Kondo and H. Takebe (Eds.), In: Environmental Mutagens and Carcinogens, Liss, New York, pp. 625-637. Nomura, T. (1979) Changed urethan and radiation response of the mouse germ cell to tumour induction, in: L. Severi, A.G. Knudson and J.F. Franmeri, (Eds.), Tumours of Early Life in Man and Animals, Perugia Univ. Press, Italy, pp. 873-891. Nomura, T. (1982) Parental exposure to X-rays and chemicals induces heritable tumours and anomalies in mice, Nature (London), 296, 575-577. Rustin, G.J.S., M. Booth, J. Dent, S. Salt, F. Rustin and K.D. Bagshawe (1984) Pregnancy after cytotoxic chemotherapy for gestational trophoblastic tumours, Br. Med. J., 288, 103-106. Schull, W.J., M. Otake and J.V. Neel (1981) Genetic effects of the atomic bombs: a reappraisal, Science, 213, 1220-1227. West, J.D., K.M. Kirk, Y. Goyder and M.F. Lyon (1985) Discrimination between the effects of X-ray irradiation of the mouse oocyte and uterus on the induction of dominant lethals and congenital anomalies, I. Embryo-transfer experiments, Mutation Res., 149, 221-230.
231
Elsevier MTR 04007
Discrimination between the effects of X-ray irradiation of the mouse oocyte and uterus on the induction of dominant lethals and congenital anomalies II. Localised irradiation experiments John D. West *, K. Margaret Kirk, Yvonne Goyder and Mary F. Lyon MRC Radiobiology Unit, Harwell, Didcot, Oxon OXl l ORD (Great Britain)
(Received18 May 1984) (Revision received24 October 1984) (Accepted 31 October 1984)
Summary In order to evaluate whether irradiation of the postimplantation maternal environment contributed to the induction of postimplantation mortality or congenital anomalies, mouse ovaries were surgically exteriorised and selectively irradiated or shielded in a specially constructed apparatus. The results show that exposure of the mouse abdomen and uterus to 3.70 G y X-rays, 15-21 days prior to conception, has no significant effect on the incidence of either postimplantation mortality or congenital anomalies. Exposure of the ovaries to 3.27 Gy X-rays during the same period, however, increased the frequency of both postimplantation mortality and congenital anomalies.
The possibility of screening for congenital anomalies, such as malformed, dwarf or retarded fetuses offers an additional in vivo test for mutagenicity that can be applied in conjunction with the well-established dominant lethal test. A combined screen of this sort was suggested by Knudson et al. (1977). Nomura (1979, 1982) showed that exposure of germ cells of male or female mice to X-rays produced significant numbers of dominant lethals and congenital anomalies among their offspring. Kirk and Lyon (1982) demonstrated that the yields of dominant lethals and congenital anomalies from X-irradiated female mice were both dose- and time-dependent. However, their study did not determine whether X-irradiation of the * Present address: Department of Obstetrics and Gynaecology, Centre for ReproductiveBiology,37 Chalmers Street, Edinburgh EH3 9EW (Scotland).
uterus, or other aspects of the maternal environment, contributed to the observed incidence of dominant lethals and congenital anomalies. Embryo-transfer experiments, in which embryos were transferred from irradiated to untreated females and vice versa, showed that there was no significant effect of irradiation of the m a t e r n a l e n v i r o n m e n t on postimplantation mortality or fetal weight. However the result for congenital anomalies was equivocal (West et al., 1985). In this paper we described a novel technique which permits the selective irradiation of either the ovary or the uterus. This provides an experimentally more efficient means of testing for an effect of irradiating the maternal environment. We have used this method to investigate whether irradiation of the uterus contributes to the induction of postimplantation mortality and congenital anomalies.
0027-5107/85/$03.30 © 1985 ElsevierSciencePublishers B.V. (BiomedicalDivision)
232 Materials
and methods
Irradiation Virgin female ( C 3 H / H e H ? X 1 0 1 / H $ )El mice ( h e n c e f o r t h a b b r e v i a t e d to 3H1) were a n a e s t h e t i s e d with A v e r t i n a n d e x p o s e d to p a r t b o d y X - r a y t r e a t m e n t w h e n a p p r o x i m a t e l y 9 weeks old. T h e mice were d i v i d e d into 6 groups a c c o r d ing to the X - r a y t r e a t m e n t given ( T a b l e 1). M i c e in 4 of these g r o u p s u n d e r w e n t surgery to p e r m i t selective i r r a d i a t i o n or shielding of the ovaries using a specially c o n s t r u c t e d p e r s p e x b r i d g e a p p a r a t u s (Figs. 1 a n d 2). Incisions were m a d e in the d o r s a l skin a n d a b d o m i n a l wall. T h e ovaries a n d fat p a d s were p u l l e d t h r o u g h the incisions a n d the oviducts were p a s s e d t h r o u g h a n a r r o w slit in the p e r s p e x b r i d g e until the ovaries a n d fat p a d s rested in c o u n t e r s u n k holes in the bridge, as shown in Fig. 3. W h e n the ovaries were held in p o s i t i o n the uterus was s u s p e n d e d f r o m the bridge. A p p r o p r i a t e l e a d shielding was p l a c e d o n t o p of the a s s e m b l e d b r i d g e a p p a r a t u s to expose either the ovaries o r the rest of the b o d y p o s t e r i o r to the ovaries (including the uterus), to X - r a y s f r o m a source a b o v e the a p p a r a t u s , as shown in Figs. 4 a n d 5. T o m i n i m i s e the risk of i r r a d i a t i n g the top of the uterus with the ovary, the m o u s e ' s tail was p u l l e d gently, i m m e d i a t e l y b e f o r e i r r a d i a t i o n , in o r d e r to h o l d the uterus taut a n d a w a y f r o m the i r r a d i a t e d field. T h e fat p a d s a n d p r o b a b l y the
Fig. 1. The two parts of the perspex bridge apparatus used for selective irradiation or shielding of the ovaries. The top surfaces measure 76 nun long x 80 mm wide (larger bridge) and 70 mm x 86 mm (smaller bridge). oviducts were i r r a d i a t e d with the ovaries. F o l l o w ing i r r a d i a t i o n the ovaries a n d r e p r o d u c t i v e tract were r e t u r n e d to the a b d o m i n a l cavity a n d the incisions in the skin were closed with m e t a l w o u n d clips. All surgical i n s t r u m e n t s were flame sterilised. N e g a t i v e c o n t r o l s (groups A1 a n d A2) u n d e r w e n t surgery b u t were n o t i r r a d i a t e d . Mice in the positive c o n t r o l g r o u p s (B a n d D) were anaesthetised a n d i r r a d i a t e d f r o m above, without surgery, in groups of three to five. L e a d shielding was a r r a n g e d to cover the h e a d a n d t h o r a x a n d leave the a b d o m e n exposed.
TABLE 1 EXPERIMENTAL GROUPS Group
Anaesthetic
Surgery
X-Irradiation and shielding
X-Ray dose rate (Gy/min)
Absorbed dose X-rays (Gy)
A1
+
+
0
0
A2
+
+
0
0
B
+
-
0.79
3.27
C
+
+
0.69 a
3.27
D
+
-
0.79
3.70
E
+
+
Sham-irradiated in perspex bridge assembly. Shielded as shown in Fig. 4. Sham irradiated in perspex bridge assembly. Shielded as shown in Fig. 5. Irradiated in groups of 3-5. Head and thorax shielded. Ovaries irradiated in perspex bridge assembly. Shielded as shown in Fig. 4. Irradiated in groups of 3-5. Head and thorax shielded. Irradiated in perspex bridge assembly. Ovaries shielded as shown in Fig. 5.
0.79
3.70
a See text of Materials and Methods section for explanation of the low dose rate.
233
Fig. 2. The assembled perspex bridge apparatus. The alternative lead shields are shown (inverted) beside the bridge apparatus. The pegs on the larger lead shield locate in the semi-circular holes at the comers of the bridge assembly. The mouse lies on the perspex platform with its head beneath the smaller bridge (to the right of the picture).
Fig. 3. Diagram showing a mouse in position beneath the bridge apparatus. The oviducts are passed through a narrow slit at the edge of the larger perspex bridge (Fig. 1) until the ovaries and fat pads rest in the countersunk holes. The smaller perspex bridge is then put in position, next to the countersunk holes, to support the lead shield.
Fig. 4. Perspex bridge assembly and lead shield used to selectively irradiate ovaries (group C) with an X-ray source above the apparatus. The ovaries are irradiated through two 4-mm diameter holes, 17 mm apart, in the centre of the shield.
Fig. 5. Perspex bridge assembly and lead shields used to selectively shield ovaries while irradiating the uterus (group E) with X-rays from above.
Irradiation was designed to provide an absorbed dose of 3.60 G y (360 rad) of X-rays (250 kV, half-value thickness 1.1 mm Cu), to the appropriate region, in all the irradiated groups. However, while the experiment was in progress it was realised that selective irradiation of the ovary alone (group C) resulted in a lower than expected absorbed dose because of the small field size. Revised dosimetry showed that this group (C) received 3.27 Gy X-rays while other irradiated groups (D and E) each received 3.70 Gy X-rays. It was therefore necessary to include an extra 3.27 G y positive control group (B) as a separate control for group C (Table 1). The mice were mated to untreated 3H1 males 15-21 days after exposure to X-rays or sham irradiation.
Analysis of uterine contents Most females were killed 18 days after the detection of the vaginal plug so that the litters were 18.5 days post coitum (p.c.). A few females were killed a day earlier. Corpora lutea were counted and the uterine contents were examined and classified as described by Kirk and Lyon (1982) except that only dwarf or visibly abnormal fetuses and control littermates were fixed in Bouin's fluid for later dissection. A fetus was considered to be dwarf if it weighed less than 75% of the mean of the others in the litter. The index of % induced preimplantation loss
234 was calculated as [1-(
Implants, treated / _ _ _ _ I m pcontrol l a n t s ' . ] ] x.x. .t m . Corpora lutea, treated Corpora lutea, control ] ]
The index of % i n d u c e d p o s t i m p l a n t a t i o n domin a n t lethality was calculated as 1-
( Livefetuses, treated Live fetuses, control Total implants, treated / T ~ s ~ l )]
×100 Statistical tests were performed using a Hewlett Packard 97 p r o g r a m m a b l e calculator, p r o g r a m m e d b y M r r D.G. P a p w o r t h to c o m p u t e X 2, Fisher's exact test a n d b i n o m i a l s t a n d a r d errors. Results
I n giving the frequencies of e m b r y o n i c mortality a n d fetal a b n o r m a l i t i e s (Table 2) the two negative control groups (sham ovary irradiation a n d
s h a m uterus irradiation), shown as groups A1 a n d A2 i n T a b l e 1, have b e e n a m a l g a m a t e d as group A. There was n o significant difference b e t w e e n these two groups i n either the frequency of postimp l a n t a t i o n m o r t a l i t y or the frequency of a b n o r m a l fetuses. The frequency of p o s t i m p l a n t a t i o n mortality a m o n g the 22 females in group A1 was 2 3 / 2 2 0 (10.5%) c o m p a r e d with 1 8 / 1 8 6 (9.7%) for the 19 females in group A2 (X 2 = 0.0088). The frequency of a b n o r m a l fetuses was 3 / 1 9 7 (1.5%) for group A1 a n d 2 / 1 6 8 (1.2%) for group A2 (Fisher's Exact test gives P = 1.00).
Postimplantation mortality Significant n u m b e r s of p o s t i m p l a n t a t i o n domin a n t lethals were i n d u c e d when either the ovaries alone or the ovaries plus the uterus were irradiated (groups B, C a n d D in T a b l e 2) b u t negligible n u m b e r s were p r o d u c e d when the ovaries were selectively shielded from irradiation (group E). This is reflected b y the statistical tests o n the
TABLE 2 INDUCTION OF CONGENITAL ANOMALIES AND DOMINANT LETHALS BY X-RAY IRRADIATION Group: Anaesthetic: Surgery: Irradiated organs: Absorbed dose of X-rays (Gy):
C + + ovaries
0
B + ovaries plus uterus 3.27
Number of ¢ ¢ Corpora lutea
41 473
85 1187
Implantations Total Small moles Large moles Dead fetuses Abnormal fetuses (dwarf and other) a Normal fetuses
406 29 7 5 5(3 + 2) 360
Corpora lutea/females Implants/females % Induced preimplantation loss b % Postimplantation mortality % Induced postimplantation dominant lethality b % Abnormal fetuses c
A + + none
11.5 9.9 10.1 + 1.5 _ 1.4+0.6
a See Table 5 for classification of abnormal fetuses. b See Materials and Methods section for calculation. Abnormal fetuses c % Abnormal fetuses (Abnormal + normal fetuses)
×
100.
521 202 8 3 20(16 + 4) 288 14.0 6.1 48.9 + 1.9 40.9 + 2.2 34.2 + 2.6 6.5+1.4
3.27
D + ovaries plus uterus 3.70
E + + uterus (and abdomen) 3.70
82 1056
85 1237
46 609
550 240 5 1 12(5 + 7) 292 12.9 6.7 39.3 + 2.1 44.7 ___2.1 38.5 + 2.6 3.9+1.1
537 254 15 0 11(5 + 6) 257 14.6 6.3 49.4 + 1.9 50.1 + 2.2 44.5 + 2.6 4.1+1.2
465 42 7 8 4{3 + 1) 404 13.2 10.1 11.0 ___2.6 12.3 + 1.5 2.4 + 2.3 1.0+0.5
235 TABLE 3 DIFFERENCES IN F R E Q U E N C I E S O F P O S T I M P L A N T A T I O N M O R T A L I T Y A N D A B N O R M A L FETUSES B E T W E E N GROUPS Groups compared
Postimplantation mortality
Abnormal fetuses
Frequency
Significance
Frequency
Significance
0varies+uterus irradiated, Sham irradiated
3.27 G y (B) (A)
213/521 41/406
X 2 =
107.2 ***
20/308 5/365
P = 0.00070 ***
0varies+uterus irradiated, Sham irradiated
3.70 Gy (D) (A)
269/537 41/406
X2 =
165.8 ***
11/268 5/365
P = 0.039 *
0varies+uterus irradiated, Ovaries + uterus irradiated,
3.70 Gy (D) 3.27 Gy (B)
269/537 213/521
X 2 =
8.68 **
11/268 20/308
P = 0.267
Ovaries irradiated, Sham irradiated
3.27 Gy (C) (A)
246/550 41/406
X 2 = 131.7 ***
12/304 5/365
P = 0.047 *
Ovaries +uterus irradiated, Ovaries irradiated,
3.27 Gy (B) 3.27 G y (C)
213/521 246/550
X2 =
20/308 12/304
P = 0.203
0varies+uterus irradiated, Uterus irradiated,
3.70 G y (D) 3.70 G y (E)
269/537 57/465
X 2 = 160.8 ***
11/268 4/408
P = 0.013 *
Uterus irradiated, Sham irradiated
3.70 G y (E) (A)
57/465 41/406
Ovaries irradiated, Uterus irradiated,
3.27 G y (C) 3.70 G y (E)
246/550 57/465
X2 =
1.46
0.81
X 2 = 125.3 ***
4/408 5/365 12/304 4/408
P = 0.742
P = 0.010 *
P values for abnormal fetuses were calculated using a two-sided Fisher's exact test. Asterisks are used for easy comparison with the X 2 results for postimplantation mortality as follows: * P < 0.05; ** P < 0.005; ***P < 0.001.
postimplantation mortality figures shown in Table 3. As expected, the frequency of postimplantation mortality was very significantly higher after irradiation of ovaries plus uterus (groups B and D) than after sham irradiation (A). Comparison of the two positive control groups shows that the higher absorbed dose of 3.70 Gy (group D) produced significantly more postimplantation mortality than the 3.27 Gy dose (group B). Irradiation of the ovaries alone (group C) also produced a very significant increase in postimplantation mortality over the sham-irradiated level (A) and this was not significantly lower than for the relevant positive Control group (B). Thus, irradiation of the ovaries alone can account for all of the postimplantation mortality produced by irradiation of the entire abdomen. In order to rule out the possibility that the anterior part of the uterus was irradiated, along with the ovaries, and contributed to the postimplantation mortality in group C, anterior and post-
erior implants were analysed separately. In uterine horns containing an odd number of implants the middle implant was allocated to the posterior group. Otherwise, implants were allocated equally to anterior and posterior groups (Table 4). Although the difference in frequency of postimplantation mortality between the two regions approaches significance using a 1-sided test (X 2= 2.46; P = 0.058) this is not relevant since the frequency in the anterior group is actually lower than that in the posterior group. There is, therefore, no evidence for higher postimplantation mortality in the anterior part of the uterus. Irradiation of the uterus alone (group E) produced very significantly lower postimplantation mortality levels than either irradiation of the whole abdomen with the same absorbed dose (group D) or irradiation of the ovaries alone with a lower absorbed dose (group C) (Table 3). This low level was not significantly different from the frequency of postimplantation mortality found among the sham irradiated, negative controls (group A).
236 TABLE 4 COMPARISON OF THE INCIDENCE OF POSTIMPLANTATION MORTALITY IN ANTERIOR AND POSTERIOR IMPLANTATION SITES FOLLOWING X-IRRADIATION OF THE OVARIES (GROUP C IN TABLE 1) Anterior implants Total implantations Small moles Large moles Dead fetuses Abnormal fetuses Normal fetuses Postimplantation mortality Abnormal fetuses
"
Posterior implants
236 93 3 0 7 133
Significance
314 147 2 1 5 159
96/236 (40.7%) 7/140 (5.0%)
150/314 (47.8%) 5/164 (3.0%)
X2 = 2.46 (not significant)a P = 0.396 (not significant)b
a See text for discussion. b p was calculated using a two-sided Fisher's exact test. These results show that i r r a d i a t i o n of the uterus or a b d o m e n , 15-21 days prior to mating, makes n o significant c o n t r i b u t i o n to the incidence of p o s t i m p l a n t a t i o n mortality.
w h e n the ovaries were selectively shielded (group E) or n o i r r a d i a t i o n was given (group A). A classification of the a b n o r m a l fetuses is s h o w n in T a b l e 5. T h e tests in table 3 show that the frequency of a b n o r m a l fetuses was significantly higher after i r r a d i a t i o n of the entire a b d o m e n (groups B a n d D) t h a n after sham i r r a d i a t i o n (group A). The yield of a b n o r m a l fetuses after irradiation with 3.70 G y (group D) was somewhat lower t h a n after 3.27 G y (group B). A l t h o u g h this difference was n o t significant, it was reflected b y the difference in
Congenital anomalies The incidence of congenital anomalies ( a b n o r mal fetuses) paralleled the incidence of postimp l a n t a t i o n m o r t a l i t y discussed above (Table 2). Higher frequencies of a b n o r m a l fetuses occurred when either the ovaries alone or the entire abdom e n were irradiated (groups B, C a n d D) t h a n TABLE 5 CLASSIFICATION OF ABNORMAL FETUSES Treatment group: Irradiated organs:
A none
Absorbed dose of X-rays (Gy):
0
B
C
D
E
ovaries plus uterus 3.27
ovaries 3.27
ovariesplus uterus 3.70
uterus (and abdomen) 3.70
Abnormality Dwarf Dwarf with open eyelids Dwarf with cleft palate Dwarf with liver and spleen abnormalities Dwarf with umbilical hernia Dwarf with microphthalmia, exencephaly and kidney abnormality
3 0 0 0 0
16 0
5 2
5 1
3 0
1 1
0 0
2 0
0 0
0
0
1
0
0
0
0
1
0
Umbilical hernia Open eyelids Exencephaly Hare-lip, cleft palate and polydactyly Polydactyly Isolated dextrocardia and ear abnormality
0 1 0 1 0 0
2 0 0 0 0 0
0 3 2 0 0 0
0 0 0 0 1 0
0 0 0 0 0 1
237
significance levels attained by the first two comparisons shown in Table 3. The incidence of abnormal fetuses after irradiation of ovaries alone (group C) was significantly higher than for the sham-irradiated negative control group (A). This yield, however, was not significantly lower than when the whole abdomen was irradiated with the same absorbed dose of X-rays (group B). Separate analysis of anterior and posterior implants (Table 4), as described in the previous section, revealed no significant difference between the frequency of abnormal fetuses between the two regions. Thus, irradiation of the ovaries alone can account for all of the abnormal fetuses irroduced by irradiation of the whole abdomen. Irradiation of the uterus alone (group E) produced significantly fewer abnormal fetuses than either irradiation of the whole abdomen with the same absorbed dose of 3.70 Gy (group D) or irradiation of the ovaries alone with the lower absorbed dose of 3.27 Gy (group C). This small yield was not significantly different from the yield of abnormal fetuses found among the sham-irradiated negative controls (group A). Although the levels of statistical significance are less impressive, the results for the abnormal fetuses parallel those for postimplantation mortality. The results show that irradiation of the uterus, 15-21 days prior to mating, makes no significant contribution to the incidence of abnormal fetuses. Discussion
The results of the localised irradiation experiments, reported here, confirm the results for the induction of postimplantation mortality and resolve the ambiguous results for the production of congenital anomalies that were obtained in the embryo-transfer experiments, reported in our companion paper (West et al., 1985). Thus, it appears that exposure of the uterus or abdomen of the female mouse to X-rays, 15-21 days prior to conception, makes no significant contribution to the incidence of either postimplantation mortality or congenital anomalies. Kirk and Lyon (1982) reported a dose-related yield of congenital anomalies following exposure of female mice to X-rays before mating. We can
now be confident that, for the group that were exposed 15-21 days before mating, any effect of X-rays that is mediated via the maternal reproductive environment and potentiates the production of congenital anomalies is trivial compared with the mutagenic effect on female germ cells. In principle, therefore, congenital anomalies, identified as dwarf or malformed fetuses, provide a valid in vivo assay for mutagenesis. The routine combination of a screen for congenital anomalies with the dominant lethal test for mutagenicity could provide useful additional information on genetic risks. In man, congenital malformations are relatively common in comparison with genetic diseases due to well defined gene or chromosome mutations. Consequently, the incidence of congenital malformations is sometimes used as an indicator for possible mutagenic effects in man (Blatt et al., 1980; Miller, 1983; Mulvihill, 1982; Rustin et al., 1982; Schull et al., 1981). However, the genetic basis for susceptibility to malformation is complex and not well understood. Although it is possible to predict the effect of a rise in mutation rate on the incidence of diseases with a clear genetic basis, including those due to autosomal or X-linked dominant genes or to chromosome anomalies, present genetic theory does not clearly predict the effect on incidence of diseases of irregular inheritance. Thus, it is valuable to have data on the induction of congenital malformation in the offspring in mice, for comparison with results obtained in man. This is the more important as a high proportion of the incidence of genetic disease in man is accounted for by conditions of irregular inheritance. Congenital malformations may therefore serve as a model for predicting the effect of a rise in mutation rate on the incidence of such conditions in general. Although our results show that the increased incidence of malformations among the offspring of irradiated female mice is indeed due to a mutagenic effect, there is still the question whether the underlying genetic basis of the anomalies is similar to that of malformations in man. Malformations, both in mouse and in man, may be due to chromosome aberrations or to genic effects. In man, the majority are not due to detectable chromosome changes (Carter, 1969; Hook, 1983). The genetic
238 basis of the a n o m a l i e s f o u n d in o u r w o r k with the mouse is n o t known. K i r k a n d L y o n (1984) a r g u e d that they p r o b a b l y result f r o m several different types of genetic change. However, this p o i n t n e e d s further work. This is p a r t i c u l a r l y so in view of the t e n d e n c y for the frequency of m a l f o r m a t i o n s to vary with the incidence of d o m i n a n t lethals, which have a m a i n l y c h r o m o s o m a l basis. O n the o t h e r hand, the o b s e r v a t i o n of N o m u r a (1979, 1982), that u r e t h a n e induces significant n u m b e r s of congenital a n o m a l i e s b u t n o t d o m i n a n t lethals, suggests some d i s s o c i a t i o n b e t w e e n these two tests.
Acknowk~lgements W e are grateful to Mr. S. A l l e n a n d his colleagues for c o n s t r u c t i n g the p e r s p e x b r i d g e a n d lead shield assembly. W e also t h a n k Mr. M. C o r p a n d Mr. P. A d a m s for d o s i m e t r y a n d i r r a d i a t i n g the mice, Mr. D . G . P a p w o r t h for help with statistical tests, Mr. G. W i l k i n s for p h o t o g r a p h y a n d Mr. G. F i s h e r a n d Ms. G. Collier for p r e p a r i n g Fig. 3. K . M . K . was s u p p o r t e d b y a g r a n t from the N a t i o n a l R a d i o l o g i c a l P r o t e c t i o n Board.
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