Ascorbic Acid Protects Bone Marrow from Oxidative Stress and Transient Elevation of Corticosterone Caused by X-ray Exposure in Akr1a-Knockout Mice

Abstract

Bone marrow cells are the most sensitive to exposure to X-rays in the body and are selectively damaged even by doses that are generally considered permissive in other organs. Ascorbic acid (Asc) is a potent antioxidant that is reported to alleviate damages caused by X-ray exposure. However, rodents can synthesize Asc, which creates difficulties in rigorously assessing its effects in such laboratory animals. To address this issue, we employed mice with defects in their ability to synthesize Asc due to a genetic ablation of aldehyde reductase (Akr1a-KO). In this study, concentrations of white blood cells (WBCs) were decreased 3 days after exposure to X-rays at 2 Gy and then gradually recovered. At approximately one month, the recovery rate of WBCs was delayed in the Akr1a-KO mouse group, which was reversed via supplementation with Asc. Following exposure to X-rays, Asc levels decreased in plasma, bone marrow cells, and the liver during an early period, and then started to increase. X-ray exposure stimulated the pituitary gland to release adrenocorticotropic hormone (ACTH), which stimulated corticosterone secretion. Asc released from the liver, which was also stimulated by ACTH, appeared to be recruited to the bone marrow. Since corticosterone in high doses is injurious, these collective results imply that Asc protects bone marrow via its antioxidant capacity against ROS produced via exposure to X-rays and the cytotoxic action of transiently elevated corticosterone.

Keywords: adrenal; adrenocorticotropic hormone; reactive oxygen species.

Figure 1

Akr1a-KO delayed the recovery of WBCs following exposure to X-rays. Blood samples were analyzed using a hematology analyzer following exposure to X-rays at 2 Gy in WT and Akr1a-KO mice with or without Asc. In WT ADX mice, the measurement was begun 2 weeks after adrenalectomy. (A) Total WBC counts were measured following exposure. (B) Lymphocyte counts were measured after exposure. Data are expressed as the mean ± SE for 5–7 animals per group. * p < 0.05 vs. 0 Gy and # p < 0.05 vs. WT mice. WBC, white blood cells; WT ADX, mouse with adrenalectomy.

Figure 2

Exposure to X-rays decreasing the Asc levels in plasma, BMCs and liver. Asc levels in plasma (A), BMCs (B), liver (C), and adrenal glands (D) were measured using Naph-DiPy in WT and Akr1a-KO mice with or without Asc following exposure to X-rays at 2 Gy. The fluorescence intensity was measured using a microplate reader (Ex: 310 nm, Em: 410 nm). Data are expressed as the mean ± SE for 3–5 animals per group. * p < 0.05 vs. 0 Gy and # p < 0.05 vs. WT mice and Akr1a-KO mice supplemented with Asc. n.s., not significant.

Figure 3

Increased SVCT1 and SVCT2 mRNA levels in the liver following exposure to X-rays. The expression of SVCT1 (A) and SVCT2 (B) mRNA in the liver was measured via quantitative RT-PCR following exposure to X-rays in WT and Akr1a-KO mice. Data are expressed as the mean ± SE for 5 animals per group. * p < 0.05 vs. 0 Gy and # p < 0.05 vs. WT mice.

Figure 4

Akr1a-KO enhanced the radiation-induced increase in plasma corticosterone levels following exposure to X-rays. ACTH levels (A) and corticosterone levels (B) in plasma were measured using an ELISA kit in WT and Akr1a-KO mice with or without Asc following X-ray exposure at 2 Gy. Data are expressed as the mean ± SE for 5 animals per group. * p < 0.05 vs. 0 Gy and # p < 0.05 vs. WT mice and Akr1a-KO mice supplemented with Asc.

Figure 5

Exposure to X-rays, which did not affect the levels of the primary steroidogenic proteins but transiently decreased corticosterone concentrations. (A) The proteins associated with steroid hormone synthesis evaluated via Western blotting in representative blots of StAR, P450_scc, AKR1A, and Actin. (B) Corticosterone levels in adrenal glands measured using an ELISA kit in WT and Akr1a-KO mice with or without Asc following X-ray exposure at 2 Gy. Data are expressed as the mean ± SE for 3–5 animals per group. * p < 0.05 vs. 0 Gy and # p < 0.05 vs. WT mice and Akr1a-KO mice supplemented with Asc.

Figure 6

Radiation-induced LDH release from cultured BMCs reduced via Asc treatment but increased via dexamethasone treatment. BMCs were isolated and cultured overnight. The BMCs were treated either with 50 μmol/L Asc or 200 nmol/L dexamethasone (DEX) 2 h before exposure to X-rays at 5 Gy. LDH release from cultured BMCs was measured one day following exposure. Data are expressed as the mean ± SE for 3 samples per group. * p < 0.05.

Figure 7

Schematic diagram outlining the results of this study.