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. 2010 Jan 15;48(2):348-56.
doi: 10.1016/j.freeradbiomed.2009.11.005. Epub 2009 Dec 2.

Total body irradiation causes residual bone marrow injury by induction of persistent oxidative stress in murine hematopoietic stem cells

Yong Wang  1 , Lingbo Liu, Senthil K Pazhanisamy, Hongliang Li, Aimin Meng, Daohong Zhou
Affiliations

Total body irradiation causes residual bone marrow injury by induction of persistent oxidative stress in murine hematopoietic stem cells

Yong Wang et al. Free Radic Biol Med. .

Abstract

Ionizing radiation (IR) and/or chemotherapy causes not only acute tissue damage but also late effects including long-term (or residual) bone marrow (BM) injury. The induction of residual BM injury is primarily attributable to the induction of hematopoietic stem cell (HSC) senescence. However, the molecular mechanisms by which IR and/or chemotherapy induces HSC senescence have not been clearly defined, nor has an effective treatment been developed to ameliorate the injury. Thus, we investigated these mechanisms in this study. The results from this study show that exposure of mice to a sublethal dose of total body irradiation (TBI) induced a persistent increase in reactive oxygen species (ROS) production in HSCs only. The induction of chronic oxidative stress in HSCs was associated with sustained increases in oxidative DNA damage, DNA double-strand breaks (DSBs), inhibition of HSC clonogenic function, and induction of HSC senescence but not apoptosis. Treatment of the irradiated mice with N-acetylcysteine after TBI significantly attenuated IR-induced inhibition of HSC clonogenic function and reduction of HSC long-term engraftment after transplantation. The induction of chronic oxidative stress in HSCs by TBI is probably attributable to the up-regulation of NADPH oxidase 4 (NOX4), because irradiated HSCs expressed an increased level of NOX4, and inhibition of NOX activity with diphenylene iodonium but not apocynin significantly reduced TBI-induced increases in ROS production, oxidative DNA damage, and DNA DSBs in HSCs and dramatically improved HSC clonogenic function. These findings provide the foremost direct evidence demonstrating that TBI selectively induces chronic oxidative stress in HSCs at least in part via up-regulation of NOX4, which leads to the induction of HSC senescence and residual BM injury.

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Figures

Fig. 1
Fig. 1
Schematic illustration of the isolation of BM-MNCs, Lin− cells, HPCs (Lin− c-kit+ Sca1− or LKS− cells), and HSCs (Lin− c-kit+ Sca1+ or LKS+ cells).
Fig. 2
Fig. 2
A representative analysis of ROS production in HPCs and HSCs by flow cytometry.
Fig. 3
Fig. 3. TBI induces persistent oxidative stress selectively in HSCs
A. Intracellular ROS in BM-MNCs, HPCs and HSCs were measured at 1, 3, 7, 14, 28, and 56 days after TBI. The data are expressed as fold increases in DCF MFI compared to that of cells from control un-irradiated mice. B. Fold-increase in ROS production by HPCs and HSCs 56 days after TBI from control. Data are presented as mean ± SE (N = 3 independent assays). C. A representative analysis of ROS production in HSCs by flow cytometry using DCFDA, DHR and DHE. Data presented in the histograms are MFI of DCF, R123 and ethidium ± SD of triplicates. Numbers in parenthesis are percent of control. D. A representative analysis of ROS production in HSCs after incubation with NAC (200 μM) or MnTE-2-PyP (MnTE, 100 μM) at 37 °C for 1 h prior to ROS assay with DCFDA. Control, HSCs from un-iradiated mice; TBI, HSCs from mice 56 days after exposure to TBI. * Not assayed due to inability to obtain sufficient number of HPCs and HSCs for the assay.
Fig. 4
Fig. 4. TBI induces sustained DNA damage selectively in HSCs
A. Analysis of 8-OH-dG oxidative DNA damage in HSCs from control un-irradiated mice and mice 56 days after exposure to TBI. Representative photomicrographs of 8-OH-dG immunofluorescent staining (Red) and nucleic counterstaining with Hoechst-33342 (Blue) are shown. A similar result was observed in another experiment and in Fig. 8B. B & C. Analysis of DNA double-strand breaks in HSCs from control un-irradiated mice and mice 56 days after exposure to TBI by immunofluorescent microscopy using an antibody specific against γH2AX. Representative photomicrographs of γH2AX immunofluorescent staining (Red) and nucleic counterstaining with Hoechst-33342 (Blue) are shown in B. Numbers of γH2AX foci/cells are presented in C as a mean ± SE of three independent assays. * p<0.05 vs control.
Fig. 5
Fig. 5. TBI selectively inhibits HSC clonogenic function via induction of senescence
Mice were exposed to TBI or were not irradiated as control and BM-MNCs were isolated from irradiated and control mice as previously described. A. The clonogenic function of HPCs and HSCs in BM-MNCs from control and TBI mice was measured by CFC assay and day-35 CAFC assay, respectively. The number of total CFUs and day-35 CAFCs is expressed as a function of HPCs and HSCs according to the frequencies of HPCs and HSCs in BM-MNCs quantified by flow cytometry. Data are presented as mean ± SE (N = 5 for CFC assay and N = 3 for CAFC assay). B. Representative analysis of apoptosis in HPCs and HSCs from control and irradiated mice by Annexin V staining and flow cytometry. Numbers presented in the histograms are mean ± SD (triplicates) of percentage of Annexin V positive cells. C. A representative analysis of p16Ink4a expression in HPCs and HSCs from control mice and irradiated mice by immunostaining with anti-p16Ink4a antibodies and flow cytometry. Numbers presented in the histograms are mean ± SD (triplicates) of percentage of p16Ink4a positive cells.
Fig. 6
Fig. 6. NAC attenuates TBI-induced inhibition of HSC clonogenic function and enhances HSC long-term engraftment after TBI
A & B. Mice were exposed to TBI and then were treated with NAC (40 mM in drinking water) 6 hr after TBI (TBI + NAC) or untreated (TBI). BM-MNCs were isolated from control and irradiated mice 56 days after TBI. The frequencies of HSCs in BM-MNCs were quantified by flow cytometry (A). The clonogenic function of HSCs was measured by day-35 CAFC assay and is expressed as a function of HSCs according to the frequencies of HSCs (B). Data are presented as mean ± SE (n = 3). a, p<0.05 vs control. C-F. Hematopoietic engraftment was determined at 4 months in lethally irradiated recipients after transplantation of BM-MNCs from TBI mice treated with or without NAC as described above in a competitive repopulating assay. The percentage of total donor-derived peripheral blood leukocytes (C) and multilineage reconstitution of myeloid (GM-granulocyte- monocyte/macrophage), T and B cells (D-F) are presented as mean ± SE (11 mice/group).
Fig. 7
Fig. 7. TBI up-regulates NOX4 expression in HSCs
A. ROS production in HSCs from mice 56 days after TBI was analyzed after the cells were pre-incubated with vehicle, DPI (10 μM) or apocynin (10 μM) at 37 °C for 1 hr. Data are expressed as percent of control from the un-irradiated and vehicle-treated cells and presented as mean ± SE (N = 3 independent assays). B. Analysis of NOX1, 2, 3, and 4 and GAPDH mRNA expression in BM-MNCs, Lin- cells, HPCs, and HSCs from control mice by RT-PCR. C. A representative analysis of NOX1, 2, and 4 and GAPDH mRNA expression in HSCs from control un-irradiated mice and mice 56 days after exposure to TBI by RT-PCR. Similar results were observed in three independent experiments. D. Analysis of NOX3 and 4 expression in HSCs from control un-irradiated mice and mice 56 days after exposure to TBI by immunofluorescent microscopy. Representative photomicrographs of NOX3 and 4 immunofluorescent staining (Green) and nucleic counterstaining with Hoechst-33342 (Blue) are shown. Similar results were observed in two independent experiments. ** p<0.01 vs vehicle treated cells.
Fig. 8
Fig. 8. DPI inhibits TBI-induced chronic oxidative stress in HSCs and attenuates TBI-induced suppression of HSC clonogenic funtion
Mice were exposed to TBI and then 6 hr later were treated with DPI (1mg/kg) (TBI + DPI) or vehicle (TBI) by sc injection every other day for 30 days. HSCs were isolated from control un-irradiated and irradiated mice and analyzed for ROS production (A), oxidative DNA damage (B), and DNA DSBs (C) by flow cytometeric measurement of DCF oxidation, and 8-OH-dG and γH2AX immunofluorescent staining, respectively. The clonogenic function of HSCs was measured by day-35 CAFC assay (D). Data are presented as mean ± SE (N = 4). a, p<0.05 vs control; and b, p<0.05 vs TBI.
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