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. 2013 Mar 1;54(2):203-9.
doi: 10.1093/jrr/rrs086. Epub 2012 Oct 11.

Radioprotective effect on HepG2 cells of low concentrations of cobalt chloride: induction of hypoxia-inducible factor-1 alpha and clearance of reactive oxygen species

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Radioprotective effect on HepG2 cells of low concentrations of cobalt chloride: induction of hypoxia-inducible factor-1 alpha and clearance of reactive oxygen species

Wensen Jin et al. J Radiat Res. .

Abstract

It has been found that low doses of certain toxicants might generate a protective response to cellular damage. Previous data have shown that elevated doses of cobalt (Co) induce injury to cells and organisms or result in radiological combined toxicity. Whether low doses of Co generate a protective effect or not, however, remains controversial. In this study, we investigated the effect and mechanism of action of low dose cobalt chloride (CoCl2, 100 μM) on the viability of irradiated cells. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay was used to observe the radio-sensitivity of HepG2 cells under different pretreatments. The alteration of intracellular DNA damage was further measured using micronucleus (MN) assay. Levels of hypoxia inducible factor-1α (HIF-1α) expression and its target gene, EPO, were monitored by western blot and reverse transcription polymerase chain reaction (RT-PCR), respectively, and intracellular reactive oxygen species (ROS) content was determined by 2',7'-dichlorofluorescein diacetate (DCFH-DA) probe staining. Our results show that low dose CoCl2does not influence HepG2 cell viability, but induces the expression of HIF-1α, followed by increased radio-resistance. Additionally, cells treated with HIF-1α siRNA retained a partial refractory response to irradiation concomitant with a marked reduction in intracellular ROS. The change of MN further indicated that the reduction of DNA damage was confirmed with the alteration of ROS. Our results demonstrate that low dose CoCl2may protect cells against irradiative harm by two mechanisms, namely HIF-1α expression and ROS clearance.

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Figures

Fig. 1.
Fig. 1.
Selection of the experimental concentration of CoCl2. MTT results showed that there was no significance in the decrease of cell viability until the concentration of CoCl2was at 100 μM (*P< 0.05, **P< 0.01, as compared with control).
Fig. 2.
Fig. 2.
Changes of HIF-1α and its target gene expression. (A) Induction of HIF-1α analyzed by western blot experiments. Similar results were obtained from three independent experiments. (B) Compared with the normoxic cell, HIF-1α expression in the irradiated and unirradiated cells was conspicuously induced by CoCl2pretreatment (##P< 0.01). After HIF-1α-siRNA transfection, a statistical difference was observed between cells treated with and without HIF-1α-siRNA under the same conditions (**P< 0.01). (C) Expression of EPO analyzed by RT-PCR experiments. Similar results were obtained from three independent experiments. (D) Compared with the normoxic cell, the expression of EPO in the irradiated cell or the unirradiated cell was increased by CoCl2pretreatment (##P< 0.01). Further results showed that the level of EPO in the cell with CoCl2was decreased again by HIF-1α-siRNA transfection, and a significant difference was observed between the transfected and untransfected cells (**P< 0.01).
Fig. 3.
Fig. 3.
Changes in intracellular ROS levels. One hundred micrometers of CoCl2pretreatment for 24 h significantly induced intracellular ROS levels induced by irradiation. Statistical analysis showed that, compared with the normoxic cells exposed to the same radiation dose, there was a significant difference in cells pretreated with CoCl2pretreatment (*P< 0.05, **P< 0.01).
Fig. 4.
Fig. 4.
Viability of irradiated cells determined by MTT assay. After 100 μM CoCl2pretreatment for 24 h, HepG2 cells were irradiated in different doses. Cell viability with or without HIF-1α-siRNA was significantly increased at the same radiation dose compared with normoxic cells (*P< 0.05, **P< 0.01). Subsequent analysis showed that the viability of HIF-1α-siRNA transfected cells irradiated with 3 and 5 Gy was reduced relative to untransfected cells (#P< 0.01).
Fig. 5.
Fig. 5.
MN ratios of irradiated cells. Cell irradiation in the absence of CoCl2resulted in the formation of elevated levels of MN relative to irradiated cells treated with 100 μM CoCl2for 24 h (*P< 0.05, **P< 0.01). Compared with untransfected cells, intracellular MN increased in cells treated with HIF-1α-siRNA and exposed to 3 Gy and 5 Gy irradiation (#P< 0.01).

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