Alteration in oxidative stress responses by the pretreatment with cobalt

Cytotoxicity by reactive oxygen species (ROS) are mainly resulted from the reaction with biological molecules. Recently, it was reported that mitochondrial DNA (mtDNA) damage was induced by CoCl₂ through ROS. However, there are only a few reports indicating cytotoxicity of CoCl₂ at the same concentration. Here, we evaluated cell growth, DNA damage and expression of hypoxia inducible factor-1α relevant to the treatment with CoCl₂ in order to determine the responses of 293 cells in detail. For the estimation of DNA damage, we used long PCR method for mtDNA and nuclear-encoded β-globin gene (nDNA). When cells were incubated with 0.4 mM H₂O₂ for 1 hr, mtDNA damage occurred but nDNA remained unchanged. The cell growth was suppressed completely 2 days after the treatment and cell number decreased within 4 days. In the case of CoCl₂ treatment at 100 μM for 24 hr, cell number was the same as that without any treatment throughout the experiment, suggesting that CoCl₂ did not cause cytotoxicity. Furthermore, western blots revealed the activation of hypoxia inducible factor-1α under the conditions. When cells was exposed to 100 μM CoCl₂ for 24 hr prior to 0.4 mM H₂O₂ treatment for 1 hr, we observed a transient growth arrest and the cell number increased after 4days of the treatment of H₂O₂. These data indicate that the pretreatment of CoCl₂ cause the suppression of cytotoxicity by H₂O₂ rather than the induction. [Jpn J Physiol 55 Suppl:S77 (2005)]