Charged-Iron-Particles Found in Galactic Cosmic Rays are Potent Inducers of Epithelial Ovarian Tumors

Abstract

Astronauts traveling in deep space are exposed to high-charge and energy (HZE) particles from galactic cosmic rays. We have previously determined that irradiation of adult female mice with iron HZE particles induces DNA double-strand breaks, oxidative damage and apoptosis in ovarian follicles, causing premature ovarian failure. These effects occur at lower doses than with conventional photon irradiation. Ovarian failure with resultant loss of negative feedback and elevated levels of gonadotropin hormones is thought to play a role in the pathophysiology of ovarian cancer. Therefore, we hypothesized that charged-iron-particle irradiation induces ovarian tumorigenesis in mice. In this study, three-month-old female mice were exposed to 0 cGy (sham) or 50 cGy iron ions and aged to 18 months. The 50 cGy irradiated mice had increased weight gain with age and lack of estrous cycling, consistent with ovarian failure. A total of 47% and 7% of mice irradiated with 50 cGy had unilateral and bilateral ovarian tumors, respectively, whereas 14% of mice in the 0 cGy group had unilateral tumors. The tumors contained multiple tubular structures, which were lined with cells positive for the epithelial marker cytokeratin, and had few proliferating cells. In some tumors, packets of cells between the tubular structures were immunopositive for the granulosa cell marker FOXL2. Based on these findings, tumors were diagnosed as tubular adenomas or mixed tubular adenoma/granulosa cell tumors. In conclusion, charged-iron-particle-radiation induces ovarian tumors in mice, raising concerns about ovarian tumors as late sequelae of deep space travel in female astronauts.

FIG. 1.

Charged-iron-particle irradiation leads to increased weight gain with aging. Graph shows the means ± SEM body weights. Body weight was monitored for up to 15 months after 50 cGy or sham (0 cGy) irradiation at 12 weeks of age. There was a trend towards higher weight gain in the irradiated group beginning 40 weeks postirradiation (P = 0.06, effect of radiation on body weight, GEE).

FIG. 2.

Mice irradiated with charged-iron particles develop ovarian tumors. Treatment groups were the same as for Fig. 1, and ovaries were harvested at necropsy 15 months postirradiation. Ovarian sections were H&E stained. Panel A: Ovarian section from 0 cGy control group has antral follicle. Panel B: Tubular structure within nontumor-bearing ovary from 50 cGy iron-irradiated mouse. Panels C and D: Representative ovarian tumor histology from the 50 cGy iron-irradiated group showing tubular structures within the ovarian parenchyma and complete absence of follicles. Tumors were histologically classified as tubular adenomas.

FIG. 3.

Charged-iron-particle-radiation-induced ovarian tumors are positive for the epithelial cell marker cytokeratin. Representative images of cytokeratin immunostaining. Cyst (panel A) and ovarian tumors (panels b and C) tumors from irradiated mice show positive (brown) cytokeratin immunostaining in cells lining tubular structures. Ovary shown in panel A is the same ovary as is shown in Fig. 2B and ovary in panel C is same ovary as in Fig. 2D. Panel D: Section from ovary collected 15 months after sham irradiation has primary follicle and is cytokeratin positive only in ovarian surface epithelial cells (arrow). Panel E: Ovary collected at 8 weeks after 50 cGy charged-iron-particle irradiation shows absence of follicles and cytokeratin immunostaining in ovarian surface epithelial cells (arrow). Panel F: Lack of immunostaining in technical negative control with primary antibody replaced by nonimmune IgG. Scale bars = 50 μm.

FIG. 4.

Few proliferating cells in ovarian tumors induced by charged-iron-particle irradiation. Representative images of Ki67 immunostaining. Panels A and B: Ovaries from irradiated mice show few Ki67-immunopositive (brown) cells. Panel C: Ovarian sections from control ovary with immunopositive granulosa cells. Panel D: Lack of immunostaining in technical negative control with primary antibody replaced by nonimmune IgG. Scale bars = 50 μm. Black arrows indicate representative immunopositive cells.

FIG. 5.

ALDH immunostaining in ovaries of control and charged-iron-particle-irradiated mice. Representative images of ALDH1 immunostaining. Panels A and B: Ovaries from 0 cGy control mice aged 15 months and 8 weeks, respectively, with ALDH1-positive (brown stain) in the ovarian surface epithelium (black arrow in panel A), hilar epithelium and stroma, but minimal staining in corpus luteum (green asterisk in panel A) or granulosa cells of follicles (arrow in panel B). Panels C and D: Ovaries from 50 cGy irradiated mice show increased immunostaining at the ovarian surface epithelium and epithelial tubular structure (green asterisk in panel D, compare with Fig. 3A), hilum region (arrow in panel C) and stroma compared to control ovaries. Panels E and F: Ovarian tumors from 50 cGy irradiated mice show immunostaining in the cells lining the tubules, which were positive for cytokeratin (black arrows), and tumor cells that were negative for cytokeratin (green asterisk in panel E, compare with Fig. 3C). Scale bars = 50 μm.

FIG. 6.

Charged-iron-particle-radiation-induced ovarian tumors have granulosa cell components. Representative images of FOXL2 immunostaining. Panel A: Ovary from 0 cGy control mouse with FOXL2-positive (brown) granulosa cells. Panel B: Ovary from 50 cGy irradiated mouse has FOXL2-positive cells in cell packets between tubular structures, but not in tubule epithelial cells. Panel C: Ovary from another 50 cGy irradiated mouse with similar staining pattern as in panel B. Panel D: Lack of FOXL2 immunostaining in technical negative control with primary antibody replaced by nonimmune IgG. Arrows indicate representative immunopositive cells. Scale bars = 50 μm.