Administration of ON 01210.Na after exposure to ionizing radiation protects bone marrow cells by attenuating DNA damage response

Abstract

Background: Ionizing radiation-induced hematopoietic injury could occur either due to accidental exposure or due to diagnostic and therapeutic interventions. Currently there is no approved drug to mitigate radiation toxicity in hematopoietic cells. This study investigates the potential of ON 01210.Na, a chlorobenzylsulfone derivative, in ameliorating radiation-induced hematopoietic toxicity when administered after exposure to radiation. We also investigate the molecular mechanisms underlying this activity.

Methods: Male C3H/HeN mice (n = 5 mice per group; 6-8 weeks old) were exposed to a sub-lethal dose (5 Gy) of γ radiation using a ¹³⁷Cs source at a dose rate of 0.77 Gy/min. Two doses of ON 01210.Na (500 mg/kg body weight) were administered subcutaneously at 24 h and 36 h after radiation exposure. Mitigation of hematopoietic toxicity by ON 01210.Na was investigated by peripheral white blood cell (WBC) and platelet counts at 3, 7, 21, and 28 d after radiation exposure. Granulocyte macrophage colony forming unit (GM-CFU) assay was done using isolated bone marrow cells, and terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) was performed on bone marrow sections at 7 d post-exposure. The DNA damage response pathway involving ataxia telangiectasia mutated (ATM) and p53 was investigated by Western blot in bone marrow cells at 7 d post-exposure.

Results: Compared to the vehicle, ON 01210.Na treated mice showed accelerated recovery of peripheral WBC and platelet counts. Post-irradiation treatment of mice with ON 01210.Na also resulted in higher GM-CFU counts. The mitigation effects were accompanied by attenuation of ATM-p53-dependent DNA damage response in the bone marrow cells of ON 01210.Na treated mice. Both phospho-ATM and phospho-p53 were significantly lower in the bone marrow cells of ON 01210.Na treated than in vehicle treated mice. Furthermore, the Bcl2:Bax ratio was higher in the drug treated mice than the vehicle treated groups.

Conclusions: ON 01210.Na treatment significantly mitigated the hematopoietic toxicity induced by a sub-lethal radiation dose. Mechanistically, attenuation of ATM-p53 mediated DNA damage response by ON 01210.Na is contributing to the mitigation of radiation-induced hematopoietic toxicity.

Figure 1

Post-exposure peripheral blood cell count with or without ON 01210.Na treatment. A) Absolute peripheral WBC counts. B) Absolute neutrophil count (ANC). C) Absolute monocyte counts (AMC). D) Platelet count expressed as percent of normal count. R+V: 5 Gy radiation+vehicle; R: 5 Gy radiation; R+D: 5 Gy radiation+drug (ON 01210.Na). *p < 0.05 compared to radiation+vehicle.

Figure 2

Quantification of bone marrow cellularity in H&E stained sections at 7 d post-radiation. A) Photomicrograph showing H&E stained sections of bone marrow at 40× magnification. B) Relative quantification of total bone marrow cellularity. *p < 0.004 compared to radiation+vehicle (R+V). C) Megakaryocyte counts in H&E stained bone marrow sections. *p < 0.02 compared to radiation+vehicle. Control: no radiation, ON 01210.Na or vehicle; R: 5 Gy radiation; R+V: 5 Gy radiation+vehicle; R+D: 5 Gy radiation+drug (ON 01210.Na).

Figure 3

Granulocyte-macrophage colony forming units (GM-CFU) assay of bone marrow cells at 7 d post-radiation. *p < 0.005 compared to radiation+vehicle (R+V). Control: no radiation, ON 01210.Na or vehicle; R: 5 Gy radiation; R+V: 5 Gy radiation+vehicle; R+D: 5 Gy radiation+drug (ON 01210.Na).

Figure 4

TUNEL staining of bone marrow and spleen sections at 7 d post-radiation. A) Photomicrograph showing TUNEL staining of bone marrow sections at 20× magnification. B) Quantification of TUNEL positive cells in bone marrow sections. *p < 0.01 compared to radiation+vehicle (R+V). C) Photomicrograph showing TUNEL staining of spleen sections at 20× magnification. D) Quantification of TUNEL positive cells in spleen sections. *p < 0.05 compared to radiation+vehicle. Control: no radiation, ON 01210.Na or vehicle; R: 5 Gy radiation; R+V: 5 Gy radiation+vehicle; R+D: 5 Gy radiation+drug (ON 01210.Na).

Figure 5

Western blot analysis of DNA damage response in bone marrow cells at 7 d post-radiation. A) Scanned images showing Western blot of p53, Bcl2, and Bax. B) Densitometric quantification of p53, Bcl2, and Bax. *p < 0.002 for p53, < 0.001 for Bax, and < 0.007 for Bcl2 compared to respective radiation+vehicle groups C) Bcl2/Bax ratio. *p < 0.005 compared to radiation+vehicle. D) Western blot images of phospho-ATM and phospho-p53. E) Densitometric quantification of phospho-ATM and phospho-p53. *p < 0.0007 for p-ATM and < 0.0002 for p-p53 compared to radiation+vehicle treated mice. R+V: 5 Gy radiation+vehicle; R: 5 Gy radiation; R+D: 5 Gy radiation+drug (ON 01210.Na).