Abstract
To clarify sensitivities of juvenile exposure to radiation on uterine carcinogenesis, female Donryu rats, a high yield strain of uterine corpus cancer, were exposed to 0.2 and 1.0 Gy of gamma radiation at postnatal day 14. Sequential changes in their reproductive organs and hematology, and the effects on uterine tumor development were compared to those in adult rats exposed to the same doses. Half number of the rats in each group was treated with N-ethyl-N’-nitro-N-nitrosoguanidine (ENNG) after the radiation to accelerate the development of uterine cancer. Severe apoptosis and depletion of oocytes in the primordial/primary follicles were immediately induced after juvenile exposure at 1.0 Gy only. The ovaries in rats exposed to 1.0 Gy at juvenile showed severe atrophy characterized by the loss of all types of follicles and a lack of corpora lutea by 2 months of age, and all rats elicited an early onset of persistent estrus corresponding to the atrophy. At the termination of 9 months of age, juvenile 1.0 Gy exposure with ENNG treatment increased the incidence of endometrial adenocarcinoma and the multiplicities of combined endometrial adenocarcinomas and their precancerous lesions. Enhancement of uterine cancer development was not apparent at the same exposure without ENNG. In comet assays, neither 0.2 nor 1.0 Gy juvenile exposure induced direct DNA damage to uteri though the damage was found in the ovary at 1.0 Gy. The present results indicated that juvenile exposure to gamma radiation indirectly enhanced uterine cancer development in rats through direct damage to oocytes resulting in serious atrophy of the ovary accompanying early onset of persistent estrus. The damage to ovary was more sensitive at juvenile than adults. The result in comet assay suggested that direct DNA damage to the uterus by radiation was excluded.
Key words: gamma ray, juvenile, oocyte depletion, persistent estrus, rat, uterine cancer
Introduction
Children have been considered to be more sensitive population to cancer development of external and internal exposure to radiation compared with adults. Adverse effects of in utero radiation exposure as well as cancer risk have been paid attention for workers at nuclear plants or victims of atomic bomb1–3). Preston and co-workers4) reported dose-related increases in solid cancer incidence rates among survivors of the atomic bombing of Hiroshima and Nagasaki who were internally or externally exposed in utero or young than 6 years at the time of bombing. Recently not only decreased pregnancy rates and early menopause in female cancer survivors5–7) but also increased opportunities given new techniques for diagnostic imaging such as computerized tomography (CT) for children and fetuses8) have raised concerns to be exposed to radiation externally. There has been no clear evidence for the effect of internal radiation via food on cancer development except several reports on leukemia or thyroid tumor exposed at childhood9). CODEX revised the guideline levels for radionuclides in food contaminated following a nuclear or radiological emergency, where the levels addressed two separate categories from infant foods, and from those for adults. The revised guidance levels for infant foods were lower in 7 of 20 radionuclides than those in adult foods10).
Preston and co-workers1) recently reported an increased risk of uterine corpus cancer from exposure to radiation during childhood. However, no experiment has yet been done to investigate the effects of radiation exposure during childhood on uterine corpus cancer using animal models and to study mechanisms of these effects. The perinatal or growing period is highly sensitive to exposure to xenobiotics as well as radiation, and exposure during critical periods sometimes induces irreversible effects, including cancer, in both humans and experimental animals11–13). Perinatal treatment with diethylstilbestrol (DES) or estrogenic compounds induces the development of vaginal adenocarcinomas in humans14,15) and uterine adenocarcinomas in rodents16,17). Although the relationship between atomic bomb explosions and the development of uterine adenocarcinoma in offspring had not been reported, endometrial carcinoma in the uterine corpus is among the most common malignant tumors of the female reproductive system in menopausal women18). Uterine cancers in the corpus are classified into 2 types with different pathogenesis19). Endometrioid adenocarcinoma is the predominant type, and excess estrogenic stimulation is accepted as a principal risk factor for this type of cancer, which occurs in the late and/or post-menopausal period. This estrogen-dependent cancer accounts for 80–85% of cases of endometrioid type. In contrast, the non-estrogen-dependent type of endometrial carcinoma, which accounts for the remaining 10–15% of cases, occurs in older postmenopausal women as serous or clear cell type cancers19).
Donryu rats have a high rate of uterine endometrial adenocarcinoma. Uterine cancer in this rat strain has many similarities to the endometrioid type of adenocarcinoma in humans for the following reasons13,20–23): 1) There is a prolonged increase in estrogen levels relative to progesterone levels. This increase is associated with persistent estrus, as determined by vaginal cytology, and enhanced tumor development.; 2) There is multistep cancer progression from atypical hyperplasia to adenocarcinoma.; and 3) The cancer of the rats has morphological similarities to human cases. In addition, estrogenic compounds and chemicals that induce persistent estrus have been reported to increase tumor development24). Due to these biological and morphological similarities to human endometrioid-type endometrial adenocarcinomas, we used this rat strain in the present study.
This study was conducted to determine the effects of juvenile exposure to radiation on uterine carcinogenesis in Donryu rats, and the tumor development was compared to that in rats exposed to radiation at adulthood. In addition, to investigate direct DNA damage from gamma ray exposure in the ovary and uterus, comet assays on these organs obtained from rats exposed to radiation as juveniles or adults were examined, as whole-body radiation increases the probability of direct DNA damage in all organs, including the uterus. Comet assays are accepted as a useful tool to detect direct DNA damage to any organ by xenobiotics and radiation in in vivo studies25).
Materials and Methods
Animals
Pregnant female Crj/Donryu rats at gestation day 14 were purchased from Charles River (Atsugi, Japan), and their female offspring were used for the present study. The animals were maintained in an SPF barrier animal room under controlled temperature, moisture, lighting, and air exchange. Animals were fed CRF-1 (Oriental Yeast Japan, Kanagawa) and chloride water ad libitum. Low concentration chloride water was supplied to prevent infection of radiation-exposed animals, which have weakened immune systems. Animal treatment and experimental procedures were in strict accordance with the Guidelines for Handling of Laboratory Animals for Biomedical Research compiled by the Safety and Ethical Handling Regulations Committee for Laboratory Animal Experiments of the National Institute of Radiological Sciences and the Animal Committee of the National Institute of Health Science, which were former institutions of the first author.
Animal Treatment
The experimental protocol for the study of the effects of juvenile or adult radiation exposure on the female reproductive tract is shown in Fig. 1. At 13 days of age, female pups were allocated into 10 groups, avoiding the placement of littermates in the same group. For the group receiving juvenile radiation exposure without N-ethyl-N’-nitro-N-nitrosoguanidine (ENNG, Nakarai Tesque, Tokyo Japan), female rats were given a single exposure to gamma radiation at 0, 0.2, or 1.0 Gy at age 14 days, in the morning using Gammacell® 40 Exactor (Best Theratronics, Ottawa, Canada). The high dose of Gamma radiation was chosen based on 1.0 to 1.5 Gy was reported as threshold for an effect on fertility in the exposure at childhood26). Intrauterine treatment with ENNG has been reported to accelerate uterine carcinogenesis27–29). For the group receiving juvenile exposure plus ENNG, irradiation was performed in the same manner and at the same ages as in the juvenile exposure group, but these were given intrauterine treatment with 20 mg/kg body weight ENNG dissolved in polyethylene glycol (Wako Pure Chemical, Osaka) at 10 weeks of age via the vagina using a stainless steel catheter. The age of 14 days was selected for irradiation because 14 days after the birth is a time when uterine glands are starting to develop; the epithelia of these glands are accepted as the site of development of endometrial adenocarcinoma. For the group exposed as adults without ENNG treatment, female rats at 10 weeks of age were exposed to 0.2 or 1.0 Gy of gamma radiation. For the group exposed as adults and treated with ENNG, the same exposure protocol was used at the same ages, but these were then treated with ENNG in the same dose and manner as for the group receiving radiation as juveniles and ENNG. ENNG treatment is known to have no modulating effect on estrous cycling from our previous studies28,29).
Fig. 1.
The protocol of present study to conduct for detecting effects of juvenile or adult exposure to radiation on female reproductive tracts or uterine carcinogenesis. h, hours; d, day(s); w, weeks; m, months, ENNG, N-ethyl-N’-nitro-N-nitrosoguanidine; ▲, intra-uterine treatment with ENNG at 10 weeks of age; △, the timing of interim kill;, the timing of irradiation (14 days of age for juvenile group and 10 weeks of age for adult group). Gray or black in color is 0.2 or 1.0 Gy, respectively.
Short-term Effects of Gamma Radiation on the Reproductive Tract
To investigate the short-term effects of radiation on the female reproductive system, 4 to 5 rats per group were killed at 6 hours (juvenile groups only), 1 day, or 1 or 9 weeks after irradiation (Fig. 1). After withdrawal of blood under deep anesthesia for differential leucocyte counts, all animals were necropsied and fixed for histopathological examination.
In Life Parameters and Uterine Carcinogenesis
All animals were checked for symptoms of abnormalities and death at least once per day, and their body weights were measured every 2 weeks. After the start of puberty (the date of vaginal opening), all animals were checked for estrous cycling by vaginal cytology throughout the study. If a rat showed a regular cycle (i.e., sequential changes from proestrus, estrus, metestrus and diestrus in one cycle usually 4 or 5 days) or irregular cycle (i.e., no sequential change in each cycle), the rat was diagnosed as having normal cycling or abnormal cycling, respectively. In particular, continuous order of estrus and/or proestrus for 3 days or longer in abnormal cycling, it was categorized as persistent estrus.
Pathology
The animals killed for examination of short-term effects were necropsied, and the weights of ovaries (excluding 14 to 21-day old rats), uteri (excluding 14 to 21-day old rats), spleen, thymus, and liver were determined. These organs and other representative organs were fixed in 10% buffered neutral formalin solution, dissected, and processed for histopathological examination. Paraffin-embedded sections of ovaries and uteri from the juvenile groups at 14 to 21 days of age were examined by TUNEL assay to check for increases in the numbers of apoptotic cells in these organs. Ovarian follicles was classified into small including primordial and primary ones, medium and large follicles including preantral and antral ones in accordance with criteria by Petersen’s criteria30).
All survivors were euthanized at 9 months of age to examine the effects of exposure on uterine cancer development. The same organs were examined as described above for short-term effects. All uteri were dissected into four parts: upper, middle, lower, and cervical. Each part was dissected into 3 or 4 blocks and examined microscopically. Proliferating lesions in the uterus were classified into atypical glandular hyperplasia and adenocarcinoma27,31). The incidences and multiplicities of uterine proliferating lesions (adenocarcinomas and endometrial hyperplasias) were counted.
Comet Assay
To examine direct DNA damage to the ovary or uterus by gamma irradiation, comet assays were conducted using the ovary, uterus, and liver from rats exposed to 0.2, 1.0, or 4.0 Gy of gamma radiation at 14-day old. Bilateral ovaries, small pieces of left lobe in the liver, and mucosal surfaces of the uterus obtained from curettage using spatula were minced and processed in the manner previously reported. The experimental procedure basically conformed to the JaCVAM protocol for international validation study25). Cell suspensions from each organs were mixed with 0.5% low melting-point agarose (NuSieve GTG Agarose, Lonza Rockland Inc., Basel, Switzerland) and deposited on MAS-coated glass slides (Matsunami Glass Ind. Ltd., Osaka, Japan). After electrophoresis, we stained the cells with SYBR gold (Life Technologies, Carlsbad, CA) and analyzed them using a fluorescence microscope connected to a comet image analyzer (comet assay IV, Perceptive Instruments Ltd, Suffolk, UK). For each sample, 100 cells (50 cells/slide) were analyzed to calculate the percentage of DNA in the tail (% Tail DNA). Heavily damaged cells, commonly referred to as ‘‘hedgehogs’’ were separately scored by counting 100 cells (50 cells/slide), and the frequency of hedgehogs was calculated. In the present study, we sued following three parameters: tail length (the length of tail excluding nucleus), tail intensity, tail moment (tail distance multiplies the ratio of sum of tail intensity to the sum of cell intensity).
Statistical Analysis
Following Bartlett’s test, variance in data for body weights, organ weights, and hematological parameters, multiplicities (the number of lesion per organ) of uterine proliferating lesions including atypical hyperplasia and adenocarcinoma, parameters of comet assay were compared with the control group by one way analysis of variance or the Kruskal-Wallis test. Statistical comparisons were conducted among the treatment groups with or without ENNG treatment and corresponding control groups. The incidence of histologic finding was compared using Fisher’s exact probability test. In these tests, the level of significance was set at 0.05 or 0.01.
Results
Body Weights and Clinical Signs
Neither gamma irradiation nor ENNG treatment affected growth curves, except that there were lower body weights among the 1.0 Gy juvenile exposure group at 21 days of age. No exposure-related clinical signs were noted throughout the study.
Early Changes after Juvenile Or Adult Exposure to Radiation (Tables 1–3, Figures 2 and 3)
Table 1. Sequential histopathological changes after single exposure to gamma rays.
| Organs/Findings | Juvenile exposure | Adult exposure | ||||
|---|---|---|---|---|---|---|
| Groups | Cont | 0.2 Gy | 1.0 Gy | Cont | 0.2 Gy | 1.0 Gy |
| Time after the exposure (a) | 6 h 1d 1w 9w | 6 h 1d 1w 9w | 6 h 1d 1w 9w | 1d 1w 9w | 1d 1w 9w | 1d 1w 9w |
| No. of rats examined | 5 5 5 4 | 5 5 5 4 | 5 5 5 4 | 4 4 4 | 4 4 4 | 4 4 4 |
| Ovary | ||||||
| Apoptosis, Oocytes | 0 0 0 0 | 0 0 0 0 | 5 0 0 0 | 0 0 0 | 0 0 0 | 0 0 0 |
| Apoptosis, granulosa
cells/ Increased apoptosis, granulosa cells |
||||||
| 0 0 0 0 | 0 0 0 0 | 5 0 0 0 | 0 0 0 | 0 0 0 | 0 0 0 | |
| Depletion, oocytes of small follicles | 0 0 0 0 | 0 0 0 0 | 0 5 5 4 | 0 0 0 | 0 0 0 | 0 0 0 |
| Atrophy, marked | 0 0 0 0 | 0 0 0 0 | 0 0 0 4 | 0 0 0 | 0 0 0 | 0 0 0 |
| Uterus | ||||||
| Apoptosis, luminal epithelium and stroma/Increased apoptosis, luminal epithelium and stroma | ||||||
| 0 0 0 0 | 0 0 0 0 | 5 0 0 0 | 0 0 0 | 0 0 0 | 0 0 0 | |
| Vagina | ||||||
| Apoptosis, mucosal epithelium, stroma | 0 0 0 0 | 0 0 0 0 | 5 0 0 0 | 0 0 0 | 0 0 0 | 0 0 0 |
| Thymus | ||||||
| Increased apoptosis,
lymphocytes |
0 0 0 0 | 0 0 0 0 | 5 0 0 0 | 0 0 0 | 0 0 0 | 0 0 0 |
| Intestine | ||||||
| Increased apoptosis, mucosal epithelium | 0 0 0 0 | 0 0 0 0 | 4 0 0 0 | 0 0 0 | 0 0 0 | 0 0 0 |
| Spleen | ||||||
| Increased apoptosis,
lymphocytes, follicle |
0 0 0 0 | 0 0 0 0 | 4 0 0 0 | 0 0 0 | 0 0 0 | 0 0 0 |
(a), 6 h, 6 hours; 1d, 1 days; 1w, 1 weeks; 9w, 9 weeks after the exposure
Table 2. Sequential changes in organ weights after exposure.
Table 3. Sequential changes in leukocytes after exposure.
| 24hrs after exposure | 1 wks after exposure | 9 wks after exposure | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| WBC | NEUT | LYMPH | MONO | WBC | NEUT | LYMPH | MONO | WBC | NEUT | LYMPH | MONO | ||
| Group | X10^2/uL | % | % | % | X10^2/uL | % | % | % | X10^2/uL | % | % | % | |
| Juvenile | Age | PND 15 | PND 21 | 11wks | |||||||||
| Control | Mean | NE | 25.8 | 31.6 | 60.7 | 6.7 | 38.8 | 28.3 | 58.7 | 10.0 | |||
| SD | 12.3 | 2.1 | 2.7 | 1.0 | 16.0 | 7.2 | 10.5 | 4.4 | |||||
| ENNG | Mean | NE | NE | 39.4 | 22.8 | 70.2 | 4.8 | ||||||
| SD | 9.4 | 3.1 | 2.0 | 1.9 | |||||||||
| 0.2 Gy at juvenile | Mean | NE | 20.3 | 25.5 | 71.6 | 1.6 | 51.9 | 27.5 | 66.0 | 5.1 | |||
| SD | 10.8 | 7.3 | 7.8 | 1.6 | 16.2 | 0.8 | 1.1 | 1.3 | |||||
| 1.0 Gy at juvenile | Mean | NE | 13.5* | 34.1 | 60.6 | 4.1 | 37.1 | 22.6 | 69.3 | 6.7 | |||
| SD | 9.1 | 14.1 | 11.5 | 7.4 | 8.9 | 5.4 | 2.7 | 2.7 | |||||
| 0.2 Gy at juvenile+ENNG | Mean | NE | NE | 36.1 | 27.9 | 66.6 | 3.4 | ||||||
| SD | 10.6 | 9.9 | 6.9 | 2.7 | |||||||||
| 1.0 Gy at juvenile+ENNG | Mean | NE | NE | 42.1 | 28.5 | 64.5 | 5.2 | ||||||
| SD | 8.0 | 7.9 | 6.7 | 1.7 | |||||||||
| Adult | Age | 10 wks | 11 wks | 19 wks | |||||||||
| Control (a) | Mean | 56.2 | 26.0 | 66.9 | 5.0 | 38.8 | 28.3 | 58.7 | 10.0 | 36.7 | 19.1 | 73.7 | 4.6 |
| SD | 10.6 | 3.9 | 4.2 | 1.8 | 16.0 | 7.2 | 10.5 | 4.4 | 6.8 | 1.7 | 2.4 | 2.3 | |
| ENNG (a) | Mean | NE | 39.4 | 22.8 | 70.2 | 4.8 | NE | ||||||
| SD | 9.4 | 3.1 | 2.0 | 1.9 | |||||||||
| 0.2 Gy at adult | Mean | 30.7* | 20.0 | 73.5 | 4.2 | 29.1 | 31.1 | 62.6 | 4.5 | 41.4** | 21.8* | 67.7* | 7.1 |
| SD | 6.6 | 4.9 | 5.8 | 1.9 | 14.3 | 7.5 | 6.3 | 2.1 | 3.6 | 1.9 | 5.0 | 5.3 | |
| 1.0 Gy at adult | Mean | 12.2* | 31.2 | 61.1 | 4.6 | 22.0 | 41.8* | 49.6 | 7.2 | 26.7* | 24.4* | 65.0* | 7.1 |
| SD | 5.0 | 5.4 | 6.3 | 1.3 | 13.8 | 8.2 | 3.7 | 3.7 | 7.6 | 1.4 | 4.4 | 3.0 | |
| 0.2 Gy at adult+ENNG | Mean | NE | 37.6 | 40.1 | 51.5 | 6.3 | NE | ||||||
| SD | 25.6 | 15.0 | 12.3 | 2.9 | |||||||||
| 1.0 Gy at adult+ENNG | Mean | NE | 25.2* | 43.4 | 47.9 | 6.3 | NE | ||||||
| SD | 1.7 | 6.9 | 7.2 | 1.9 | |||||||||
Significantly different from relevant controls at 5% (*) and 1% (**).
(a), The values of 1 wks after the exposure at adult (11 wks of age) are the same as those of 9 wks after the exposure at pup.
WBC, white blood cells; NEUT, neutrophils; LYMPH, lymphocytes; MONO, monocytes; PND, postnatal days; SD, Standard deviation
Fig. 2.
Early changes in the ovary after radiation exposure on 14-day of age. A and B were 6 hours after radiation. C was 24 hours after radiation. D was the ovary from control rats at the same age. A, The oocytes in the primordial and primary follicles became condensed, indicating apoptosis (arrowheads). B, Apoptosis in the granulosa cells in preantral follicles (*). C, Depletion of oocytes in the primordial or primary cells (arrowheads). D, Normal structure. S, Small follicle. PreA, Preantral follicle. H-E stain. Magnification A-D, x20
Fig. 3.
Early changes in the uterus (A and B) and thymus (C and D) after radiation exposure on 14-day of age (A and C) and control (B and D) rats. A, 6 hours after radiation. Apoptotic bodies were detected (arrowheads). B, Control rats at the same age as A. C, Increased apoptotic bodies of lymphocytes in the thymus. D, Normal structure in rats at the same age as C. A, B; TUNEL stain. C, D; H-E stain. Magnification A-D, x20
Histopathological changes in female reproductive organs, thymus, intestine, and spleen are shown in Table 1. Morphological changes were immediately detected in the various organs, but these changes were only found in rats exposed as juveniles to 1.0 Gy of gamma radiation (Figs 2 and 3). The characteristic finding observed in these organs was apoptosis, which was detected in oocytes and the granulosa cells of the ovary, positive by TUNEL assay (Fig. 2), the luminal epithelium of the uterus (Fig. 3), the mucosal epithelium of the vagina, lymphocytes in the thymus (Fig. 3) and spleen, and epithelial cells of the intestine at 6 hours after exposure. Apoptosis of oocytes was limited in primordial and primary follicles and the intensity was severe. It was microscopically characterized as irregularly shaped and condensed oocytes. These apoptotic figures were not observed in these organs/tissues over 24 hours after exposure, and these organs except the ovary morphologically recovered normal structure. In the primordial and primary follicles no oocytes remained in the primordial and primary follicles after 24 hours. The apoptosis in granulosa cells were not found over 24 hours and 7 days after the radiation. These ovaries were morphologically similar to those of intact control except oocyte depletion in small follicles. At 9 weeks after exposure, severely atrophic ovaries expressing oocyte depletion, no normal follicles, no CL or a few CL were observed in the rats exposed as juveniles to 1.0 Gy. No exposure-related abnormalities were detected in other organs/tissues in both juvenile and adult exposure rats.
The effects of exposure on organ weights are shown in Table 2. The changes in organ weights were consistent with the morphological changes. The weights of thymus and spleen were still reduced 24 hours after juvenile and adult exposure at 1.0 Gy. The reductions in weights of these organs were found 1 week later in the juvenile group, but not in the adult exposure group. At 9 weeks after both juvenile and adult exposure, no differences were found in the weights of thymus, spleen, liver, and uteri.
Sequential changes in leukocytes and its composed cells are shown in Table 3. Adult exposure at either dose statistically significantly reduced leukocyte counts after 24 hours in a dose-dependent manner. Although leukocyte counts recovered in the 0.2 Gy group with and without ENNG treatment at 1 week after exposure, the reduction continued until 9 weeks in the 1.0 Gy group (Table 3). A lower ratio of lymphocytes to neutrophils indicated that the main cause of the decrease in leukocytes was loss of lymphocytes in the 1.0 Gy exposure group, while the cause was not clear in the 0.2 Gy exposure group. Similar effects on leukocytes were observed in the juvenile exposure rats 1 week after exposure, but there was no difference at 9 weeks after exposure (Table 3).
Change in Estrous Cycling (Figure 4)
Fig. 4.
The change in number of rats showing persistent estrus. Rats exposed to radiation on 14-day of age, both with and without ENNG clearly showed early onset of abnormal cycling, particularly persistent estrus. At 4 months of age, all rats showed persistent estrus instead of normal cycling in the juvenile exposure groups.
The incidence of PE is shown in Fig. 4. In the groups receiving juvenile irradiation at 1.0 Gy, both with or without ENNG treatment, persistent estrus appeared in several animals at 9 weeks of age, and all animals in both groups showed persistent estrus by 22 weeks of age. Estrous cycling in the adult exposure groups was comparable to that in the control group.
Uterine Carcinogenesis (Table 4 and Figure 5)
Table 4. Incidence and multiplicity of uterine neoplastic and pre-neoplastic lesions.
| Incidences | Multiplicity (a) | |||||
|---|---|---|---|---|---|---|
| Groups | No. of rats examined |
Atypical hyperplasias | Adenocarcinomas | |||
| Slight | Moderate | Marked | ||||
| Intact control | 24 | 0 | 0 | 0 | 0 | 0 |
| ENNG only | 24 | 0 | 9 | 0 | 1 | 0.46 ± 0.59 |
| Juvenile exposuree | ||||||
| 0.2 Gy at juvenile | 24 | 0 | 0 | 1 | 0 | 0.04 ± 0.20 |
| 1.0 Gy at juvenile | 24 | 0 | 2 | 1 | 0 | 0.17 ± 0.85 |
| 0.2 Gy at juvenile + ENNG | 24 | 5 | 6 | 0 | 1 | 0.71 ± 0.75 |
| 1.0 Gy at juvenile + ENNG | 24 | 5 | 8 | 1 | 7* | 1.36 ± 0.66 * |
| Adult exposuret | ||||||
| 0.2 Gy at adult | 24 | 0 | 0 | 1 | 0 | 0.04 ± 0.20 |
| 1.0 Gy at adults | 24 | 1 | 1 | 0 | 0 | 0.08 ± 0.28 |
| 0.2 Gy at adult + ENNG | 23 | 8 | 2 | 2 | 1 | 0.70 ± 0.70 |
| 1.0 Gy at adults + ENNG | 24 | 4 | 6 | 5 | 1 | 0.58 ± 0.78 |
(a), The average number of neoplastic and pre-neoplastic lesions per rat
(b), Number of rats examined
*, Significantly different from values of relevant control group (ENNG only) at 5%
Fig. 5.
The ovary (A and B), and proliferative lesions in the uterus (C and D) at termination of 9-month of age. A, Severe atrophic ovary showing no follicles or CL. B, Age-matched atrophic ovary from the control group. An ovary with cystic atretic follicles (*) and a few CL. C, Moderate endometrial hyperplasia. C, Endometrial adenocarcinoma. C and D were obtained from the rats exposed to radiation as juveniles at 1.0 Gy. The morphological features in these proliferating lesions were comparable to those in control group. A-D, H-E stain. Magnification, A,B x4, C,D x10.
The incidences and multiplicities of proliferating lesions (adenocarcinomas and hyperplasias) in uteri are shown in Table 4. Both the incidence of adenocarcinoma and the multiplicity of proliferating lesions were significantly increased in the rats exposed to 1.0 Gy as juveniles with ENNG treatment compared to the controls. The incidences and multiplicities in other groups, including the 0.2 Gy juvenile and 1.0 Gy adult exposure groups with and without ENNG, were comparable to those in the corresponding control groups. The rats treated with ENNG including control group increased atypical hyperplasia or adenocarcinoma indicating that ENNG treatment was effective to enhance uterine carcinogenesis. The morphologic features of endometrial hyperplasia and adenocarcinoma were comparable between the exposure and control rats (Fig. 5).
At termination, microscopic examination of ovaries in the 1.0 Gy juvenile exposure rats with and without ENNG indicated that the atrophy of these ovaries continued to progress over time. No follicles or CL were detected in the ovaries, and they were composed entirely of stromal and fibrous components (Fig. 5). Focal hyperplasia of granulosa cells was observed in each rat exposed to 1.0 Gy as juveniles with and without ENNG as a negative-feedback effect of continuous stimulation on severe atrophy of the ovary. In the other exposure and the control groups, age-matched atrophic ovaries, which were consistent with cystic atretic follicles and the presence of few or no CL, were observed (Fig. 5).
Comet Assay at Juvenile Exposure (Figure 6)
Fig. 6.
The results of comet assays for the ovary, uterus, and liver in rats exposed to radiation at 14 days of age. The parameters of tail length, intensity, and moment were changed at 0.2 Gy exposure. At 1.0 Gy exposure, these parameters changed in the ovary and liver, but not in the uterus.
The results of comet assays for the ovary, uterus, and liver are summarized in Fig. 6. At 1.0 Gy exposure the tail intensity as well as tail moment were statistically significantly increased in the ovary and liver but not in the uterus. All three indicators showed no effects on the liver, ovary and uterus at 0.2 Gy exposure. At 4 Gy, all parameters tended to increase in all organs.
Discussion
The present results that juvenile exposure to gamma irradiation seriously affected oocytes in the primordial and primary follicles provided a clear evidence that irradiation during the juvenile period caused severe damage to the ovary, similar to exposure in the neonatal period, which is known as the sensitive period for radiation26). One day after the irradiation, all oocytes in small follicles were depleted. On the other hand, oocytes in larger follicles including preantral and antral follicles were considered to be recovered quickly from apoptosis of the granulosa cells observed 6 hours after irradiation, because any morphological changes weres not found in medium and large follicles one day after the exposure. The lack of morphological abnormalities in large follicles was related to normal estrous cycling occurrence in the 1.0 Gy juvenile exposure group for several weeks after puberty. Larger follicles functionally survived after the irradiation could develop Graafian follicles over several weeks. Thus, the present result indicates that small follicles and not large follicles are the main target of gamma radiation during the juvenile period. Several weeks after the period of normal follicle growth, the growing follicles were sold out from stock in the ovary, and the ovary became atrophic eliciting as abnormal estrous cyclicity, i.e. anovulation status. Although the ovaries exposed in the juvenile period to 1.0 Gy were morphologically different from age-matched atrophy which were a few atretic follicles and no CL in this strain rats, both the atrophic ovaries resulted in persistent estrus in vaginal cytology in the present study.
The present study revealed that ovaries are more sensitive to radiation exposure in the juvenile period than in adulthood. We did not examine the effects of radiation after 6 hours in the adult exposure groups; however, comparable attitudes of estrous cycling and ovarian morphology in the adult exposure and control rats suggested that there was little effect of either dose of radiation on the ovary immediately after exposure. The dose of 1.0 Gy was reported a threshold value of infertility for humans exposed in childhood26). Therefore, the sensitivity in rats might be similar to that in humans.
Another evidence of the present study was that juvenile exposure to 1.0 Gy of gamma radiation enhanced the development of uterine adenocarcinoma and its precancerous lesions in both their incidence and multiplicity. Lack of effects on uterine carcinogenesis in rats exposed to 0.2 Gy at juvenile and at both doses in adulthood indicated the presence of dose dependency of the effects and less sensitivity of the effects of exposure in adulthood.
Proposing a plausible mechanism on the enhancement of uterine carcinogenesis is important for extrapolation of the finding to humans. Basically estrogens play a crucial role of uterine carcinogenesis in both humans and rodents13,19). Regarding the increased uterine carcinogenesis found in the present study, the early occurrence of persistent estrus, found by monitoring of estrous cycling using vaginal smears, was characteristic of rats exposed to 1.0 Gy in the juvenile period. Persistent estrus has been accepted to reflect an increased ratio of 17β-estradiol (E2) to progesterone (P4) in the blood in rodents13,24,31). Our results for estrous cycling and atrophic ovaries in the 1.0 Gy juvenile exposure group suggest that early onset of persistent estrus due to atrophic ovaries is crucial to the increase in uterine cancer in this group. ENNG treatment did not disturb estrous cycling in the present study, consistent with previous reports29).
Radiation is well known to damage DNA in every organ and tissue. Therefore, we should consider the possibility that DNA damage in the uterus is a mechanism for the increase in uterine cancer in the juvenile exposure group. Our results from comet assays found no effects on the uterus at 1.0 Gy exposure. Although the comet assay (single-cell gel electrophoresis) is now a popular method for measuring low levels of damage in cellular DNA32,33), the comet assay is not a test protocol to detect all mutagenesis or repair from the DNA damage. However, the induction of apoptosis in the uterus observed immediately after irradiation was limited to the luminal epithelium and not seen in the glandular epithelium which is considered to be the cellular origin of uterine adenocarcinoma. Prolonging persistent estrus due to an increased ratio of E2 to P4 has been accepted one of key events in uterine carcinogenesis in both rodents13,24,31) and humans20,21). On the basis of integrating the present results described above, the major pathway for increased endometrial adenocarcinoma induced by juvenile exposure to 1.0 Gy of gamma radiation is the secondary effect of prolonging persistent estrus, reflecting an increased E2 to P4 ratio resulting from early depletion of oocytes in small follicles, which was the primary effect of irradiation. The direct effect of radiation on uterine carcinogenesis could be excluded. A proposed mechanism of the uterine tumor development in the present study is figured (Fig. 7)
Fig. 7.
Proposed mechanism of uterine adenocarcinoma development by exposure to radiation at juveniles.
Children have been considered to be more sensitive to solid cancer development of external and internal exposure to radiation compared with adults. There are a few reports that juvenile exposure to radiation increases uterine cancer and sold tumors1,4). The present results did not indicate direct effect of radiation on uterine cancer, however the results indicated that high sensitivity of ovary during juvenile period to radiation was a trigger to development of uterine cancer occurring at later in life. Although the present study was conducted under external exposure to gamma ray, and the affected dose, 1,0 Gy was extremely high, our results could give information that conservative regulation might be necessary for establishing tolerance levels of radioactive nuclides in foods for childhood9,10). The present results also suggest that women treated with radiation as a therapy for cancer in childhood have an increased risk for uterine cancer later in life. Long-term follow-up observation of these patients is expected.
The systemic effects of irradiation could be detected in many organs/tissues in the present study. In particular, the decreased leucocyte count due to the decrease in lymphocytes was a very sensitive parameter in both juvenile and adult exposure to radiation. Corresponding to the decrease in lymphocytes, the weights of lymphoid tissues, such as the thymus and spleen, were also reduced by the exposure. These results indicate that, although exposure in adulthood did not have an effect on cancer development, it did have systemic effects.
In conclusion, the present results suggest that atrophic ovaries resulting from early depletion of oocytes by juvenile exposure to gamma radiation indirectly promotes rat uterine cancer development through early occurrence of persistent estrus. The damage to ovary was more sensitive at juvenile than adults. The possibility that direct DNA damage in the uterus from radiation exposure is a major factor in cancer development could be excluded.
Acknowledgment
We do express our sincere thanks to great contribution of all members in our laboratories in National Institute of Radiological Sciences and National Institute of Health Sciences to their excellent assistances of animal care and histopathological preparation. We do appreciate to Dr. Naoki Koyama, Division of Genetics and Mutagenesis, National Institute of Health Sciences, for his scientific support of interpretation of micronuclear assay.
Footnotes
Conflict of interest statement: The authors have no conflicts of interest to disclose.
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