Exposure to ionizing radiation causes long-term increase in serum estradiol and activation of PI3K-Akt signaling pathway in mouse mammary gland

Abstract

Purpose: Exposure to ionizing radiation is an established risk factor for breast cancer. Radiation exposure during infancy, childhood, and adolescence confers the highest risk. Although radiation is a proven mammary carcinogen, it remains unclear where it acts in the complex multistage process of breast cancer development. In this study, we investigated the long-term pathophysiologic effects of ionizing radiation at a dose (2 Gy) relevant to fractionated radiotherapy.

Methods and materials: Adolescent (6-8 weeks old; n = 10) female C57BL/6J mice were exposed to 2 Gy total body γ-radiation, the mammary glands were surgically removed, and serum and urine samples were collected 2 and 12 months after exposure. Molecular pathways involving estrogen receptor-α (ERα) and phosphatidylinositol-3-OH kinase (PI3K)-Akt signaling were investigated by immunohistochemistry and Western blot.

Results: Serum estrogen and urinary levels of the oncogenic estrogen metabolite (16αOHE1) were significantly increased in irradiated animals. Immunostaining for the cellular proliferative marker Ki-67 and cyclin-D1 showed increased nuclear accumulation in sections of mammary glands from irradiated vs. control mice. Marked increase in p85α, a regulatory sub-unit of the PI3K was associated with increase in Akt, phospho-Akt, phospho-BAD, phospho-mTOR, and c-Myc in irradiated samples. Persistent increase in nuclear ERα in mammary tissues 2 and 12 months after radiation exposure was also observed.

Conclusions: Taken together, our data not only support epidemiologic observations associating radiation and breast cancer but also, specify molecular events that could be involved in radiation-induced breast cancer.

Fig. 1

(A) Serum estradiol level in 2-month postirradiation samples. (B) Serum estradiol level in 12-month postirradiation samples. (C, D) Urinary estrogen metabolites 2OHE1 and 16αOHE1 in 12-month postirradiation samples. (E) Ratio of 2OHE1/16αOHE1 in 12-month postirradiation samples. *p < 0.05.

Fig. 2

(A) Mammary gland sections showing immunohistochemical staining for ERα in 2-month and 12-month postirradiation samples. (B) Quantitation of ERα-positive nuclei in 2-month and 12-month postirradiation samples. (C, D) Western blot analysis for ERα in 2-month and 12-month postirradiation samples. *p < 0.05.

Fig. 3

(A) Ki-67 immunostaining in 2-month and 12-month postexposure samples. (B) Quantitation of Ki-67–positive nuclei in 2-month and 12-month postirradiation samples. *p < 0.05.

Fig. 4

(A) Cyclin-D1 immunostaining in 2-month and 12-month postexposure samples. (B) Quantitation of Cyclin-D1–positive nuclei in 2-month and 12-month postirradiation samples. *p < 0.05.

Fig. 5

(A) Western blot analysis of PI3K–Akt pathway in 2-month and 12-month postexposure samples. (B, C) Quantification of Western blots in 2-month and 12-month postexposure samples. *p < 0.05.

Fig. 6

Summary of radiation-induced long-term alterations, local and systemic, in relation to mammary glands.