Selenomethionine protects hematopoietic stem/progenitor cells against cobalt nanoparticles by stimulating antioxidant actions and DNA repair functions

Abstract

Hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) can differentiate into all blood lineages to maintain hematopoiesis, wound healing, and immune functions. Recently, cobalt-chromium alloy casting implants have been used extensively in total hip replacements; however, cobalt nanoparticles (CoNPs) released from the alloy were toxic to HSCs and HPCs. We aimed to investigate the mechanism underlying the toxic effect of CoNPs on HSCs/HPCs and to determine the protective effect of selenomethionine (SeMet) against CoNPs in vitro and in vivo. Human and rat CD34+ HSCs/HPCs were isolated from cord blood and bone marrow, respectively. CoNPs decreased the viability of CD34+ HSCs/HPCs and increased apoptosis. SeMet attenuated the toxicity of CoNPs by enhancing the antioxidant ability of cells. The protective effect of SeMet was not completely abolished after adding H2O2 to abrogate the improvement of the antioxidant capacity by SeMet. SeMet and CoNPs stimulated ATM/ATR DNA damage response signals and inhibited cell proliferation. Unlike CoNPs, SeMet did not damage the DNA, and cell proliferation recovered after removing SeMet. SeMet inhibited the CoNP-induced upregulation of hypoxia inducible factor (HIF)-1α, thereby disrupting the inhibitory effect of HIF-1α on breast cancer type 1 susceptibility protein (BRCA1). Moreover, SeMet promoted BRCA1-mediated ubiquitination of cyclin B by upregulating UBE2K. Thus, SeMet enhanced cell cycle arrest and DNA repair post-CoNP exposure. Overall, SeMet protected CD34+ HSCs/HPCs against CoNPs by stimulating antioxidant activity and DNA repair.

Keywords: DNA repair function; antioxidant action; cobalt nanoparticles; hematopoietic stem/progenitor cells; selenium.

Figure 1

Se attenuated toxic effect of CoNPs on human and rat CD34⁺ HSC/HPCs. (A) Human and rat CD34⁺ HSC/HPCs were isolated from cord blood and bone marrow, respectively, through flow cytometry. (B) CD34⁺ HSC/HPCs were exposed to CoNPs at concentrations from 0 to 400 μM for 24 h, followed by MTT assay. (C) Different dosages of Na₂SeO₃, SeMet and SeCys were added to CD34⁺ HSC/HPCs 15 h before treatment with 200 μM CoNPs for additional 24 h. MTT assay was conducted to assess the cell viability. (D) CD34⁺ HSC/HPCs were treated with 10 μM SeMet for 15 h and then subjected to 200 μM CoNPs for 24 h. Flow cytometry was conducted to evaluate apoptosis rate. *p < 0.05, **p < 0.01, and ***p < 0.001 vs. control cells that did no subjected to any treatments; ^##p < 0.01 vs. cells treated with CoNPs alone.

Figure 2

The protective effect of SeMet against CoNPs is partially associated to the improvement of anti-oxidant capacity. CD34⁺ HSC/HPCs were treated with 10 μM SeMet for 15 h and then subjected to 200 μM CoNPs for 24 h. Afterwards, cells underwent measurements of intracellular ROS level (A), T-AOC (B), GSH level (C) and GPx activity (D). *p < 0.05, and ***p < 0.001 vs. control cells that did no subjected to any treatments; ^#p < 0.05, ^##p < 0.01 and ^###p < 0.001 vs. cells treated with CoNPs alone. CD34⁺ HSC/HPCs were treated with 10 μM SeMet alone or in combination with 1 μM H₂O₂ for 15 h. Cells were then treated with 200 μM CoNPs for 24 h, followed by measurements of cell viability (E) and apoptosis rate (F). ***p < 0.001 vs. cells treated with CoNPs alone. ^#p < 0.05 vs. cells treated with SeMet and CoNPs.

Figure 3

Both CoNPs and SeMet led to the activation of γH2AX, but SeMet did not cause DNA damage. CD34⁺ HSC/HPCs were treated with 10 μM SeMet and 200 μM CoNPs, alone or in combination. (A) The phosphorylation level of H2AX (γH2AX) was determined by IF analysis. DNA damage degree was evaluated by 8-OHdG level (B) and comet assay (C). ***p < 0.001 vs. control cells that did no subjected to any treatments; ^#p < 0.05, ^##p < 0.01 and ^###p < 0.001 vs. cells treated with CoNPs alone.

Figure 4

Cell damage was evaluated by cell proliferation assay and electron microscope observation. (A) CD34⁺ HSC/HPCs were treated with 10 μM SeMet for 15 h or 200 μM CoNPs for 24 h. Alternatively, CD34⁺ HSC/HPCs were treated with 10 μM SeMet for 15 h before additional treatment with 200 μM CoNPs for 24 h. Edu staining was performed immediately after these treatments were finished. In addition, after these treatments were finished, cells were culture in fresh medium for 24 h before Edu staining. (B) CD34⁺ HSC/HPCs were treated with 10 μM SeMet and 200 μM CoNPs, alone or in combination, followed by electron microscope observation. *p < 0.05, **p < 0.01, and ***p < 0.001 vs. control cells that did no subjected to any treatments.

Figure 5

The regulatory effects of CoNPs and SeMet on DNA damage response signal. CD34+ HSC/HPCs were treated with 10 μM SeMet and 200 μM CoNPs, alone or in combination, followed by western blot (A) and Co-IP assay (B). In Co-IP assay, HIF-1α, BRCA1 and cyclin B proteins were immunoprecipitated and the enrichment of ubiquitin in these proteins was further determined by western blot. *p < 0.05, **p < 0.01, and ***p < 0.001 vs. control cells that did no subjected to any treatments. #p < 0.05, ##p < 0.01 and ###p < 0.001 vs. cells treated with CoNPs alone.

Figure 6

Suppression of DNA damage response signal attenuated the protection of SeMet against CoNPs. An inhibitor of ATM/ATR (CGK733) was added to CD34⁺ HSC/HPCs treated with SeMet and CoNPs. Afterwards, cells underwent cell viability (A) and apoptosis (B) assays. BRCA1 and UBE2K were silenced in CD34⁺ HSC/HPCs, followed by cell viability (C), apoptosis (D), cell cycle (E) and western blot assays (F). ***p < 0.001 vs. control cells that did no subjected to any treatments. ^##p < 0.01 and ^###p < 0.001 vs. cells treated with CoNPs alone. ^&p < 0.05, ^&&p < 0.01 and ^&&&p < 0.001 vs. cells treated with SeMet and CoNPs in combination.

Figure 7

HIF-1α is implicated in the toxic effect of CoNPs in CD34⁺ HSC/HPCs. This study used an activator of HIF-1α (DMOG) to imitate the activation of HIF-1α by CoNPs. Furthermore, HIF-1α was knocked down to determine whether HIF-1α partially mediates the toxic effect of CoNPs. After these treatments, cells underwent cell viability (A) and apoptosis (B) and western blot assays (C). *p < 0.05, **p < 0.01 and ***p < 0.001 vs. control cells that did no subjected to any treatments. ^##p < 0.01 and ^###p < 0.001 vs. cells treated with CoNPs alone.

Figure 8

SeMet attenuated toxic effect of CoNPs on CD34⁺ HSC/HPCs in rat. Male SD rats were 6-8 weeks old and weighed approximate 800 g at the start of the experiments. CoNPs particles were suspended in a vehicle of 1:1 rat serum: phosphate buffered saline (PBS; Oxoid) by sonication and administered to rats. 50 μL CoNPs (1000 μg/kg BW)-containing vehicle was injected in the right hip joint. The rats were exposed to three injections of the particles at three week intervals. Sham treated rat received 15 ml of vehicle alone. In addition, rats received an oral dose of SeMet (2 mg SeMet/kg BW/day). All rats were sacrificed three weeks after final injection of CoNPs. Bone marrow was collected for analysis of CD34⁺ HSC/HPCs number by flow cytometry (A), for the measurements of biochemical parameters, including, T-AOC (B), GSH level (C), GPx activity (D) and 8-OHdG level (E), and for comet assay (F). (G) The mechanism diagram shows the mechanism by which SeMet attenuates toxic effect of CoNPs on CD34⁺ HSC/HPCs. *p < 0.05, **p < 0.01 and ***p < 0.001 vs. control cells that did no subjected to any treatments. ^##p < 0.01 and ^###p < 0.001 vs. cells treated with CoNPs alone.