Induction of oxidative stress biomarkers following whole-body irradiation in mice

Abstract

Dose assessment is an important issue for radiation emergency medicine to determine appropriate clinical treatment. Hematopoietic tissues are extremely vulnerable to radiation exposure. A decrease in blood cell count following radiation exposure is the first quantitative bio-indicator using hematological techniques. We further examined induction of oxidative stress biomarkers in residual lymphocytes to identify new biomarkers for dosimetry. In vivo whole-body radiation to mice exposed to 5 Gy significantly induces DNA double-strand breaks, which were visualized by γ-H2AX in mouse blood cells. Mouse blood smears and peripheral blood mononuclear cells (PBMC) isolated from irradiated mice were used for immunostaining for oxidative biomarkers, parkin or Nrf2. Parkin is the E3 ubiquitin ligase, which is normally localized in the cytoplasm, is relocated to abnormal mitochondria with low membrane potential (ΔΨm), where it promotes clearance via mitophagy. Nrf2 transcription factor controls the major cellular antioxidant responses. Both markers of oxidative stress were more sensitive and persistent over time than nuclear DNA damage. In conclusion, parkin and Nrf2 are potential biomarkers for use in radiation dosimetry. Identification of several biological markers which show different kinetics for radiation response is essential for radiation dosimetry that allows the assessment of radiation injury and efficacy of clinical treatment in emergency radiation incidents. Radiation-induced oxidative damage is useful not only for radiation dose assessment but also for evaluation of radiation risks on humans.

Fig 1. Radiation damage of hematopoietic tissues numbers of cells in WBC, lymphocytes, monocytes, granulocytes, platelets and red blood cells on day1 (open bar) and 7 (closed bar) after irradiation with indicated doses.

Fig 2. Detection of DNA damage, mitochondrial damage and Nrf2 activation in PBMCs.

Image of parkin (A), Nrf2 (B) and γ-H2AX (C) staining in non-irradiated control and irradiated cells at day 1 after irradiation. Magnified image was inserted. Fluorescence intensity of parkin is shown in graph at right.

Fig 3. FACS analyses for detecting parkin-staining cells and mitochondrial membrane potential.

(A) FACS result for parkin staining on 1 day after irradiation in PBMCs that were either non-irradiated (dotted line) or irradiated (solid line) at the indicated doses. Mean fluorescence intensity of parkin staining is shown in the graph. (B) FACS results for JC-1 staining in non-irradiated cells and 5 Gy-irradiated cells. Blood cells were stained with JC-1 on day 1 after exposure. Asterisks indicate significant differences in FL2/FL1 ratio in irradiated cells as compared with non-irradiated cells.

Fig 4. Immunostaining for parkin, Nrf2 and γ-H2AX in blood smear.

Image of parkin (A), Nrf2 (B) and γ-H2AX (C) staining in non-irradiated control and irradiated cells at day 1 after irradiation. Magnified image was inserted. Fluorescence intensity of parkin is shown at indicated day in graph at right.

Fig 5. Schematic diagram of sequential induction of γ-H2AX, parkin and Nrf2 following radiation in irradiated mouse blood cells.