Effects of oxygen concentrations on human fibroblasts treated with Fe3+-NTA

Free radicals derived from the reaction of iron and oxygen are thought to be one of the causes of tissue injury. In order to identify whether oxygen concentrations are an important factor in iron-mediated damage to cells, cytotoxic effects of Fe3+-NTA on human fibroblasts (KMST-6 line) were studied under the conditions of 1% and 20% oxygen concentrations in an incubator. A comparison of the effects of Fe3+-NTA on cells cultured in 1% and 20% oxygen environments showed that the following features were more prominent under the usual culture concentrations of 20% oxygen: i) cytotoxicity, ii) increase in intracellular reactive oxygen species, iii) increase in H2O2 production in the cells, and iv) formation of 8-hydroxydeoxyguanosine. To elucidate the roles of endogenous antioxidants, the levels of manganese superoxide dismutase (MnSOD) and catalase were measured by Western blotting. The increase in MnSOD in the presence of Fe3+-NTA was greater under the condition of 20% O2 than under the condition of 1% O2. The expression of catalase was significantly up-regulated at 20% O2. However, when the cells were treated with Fe3+-NTA, the expression of catalase was markedly down-regulated under the condition of 20% O2. Hydroxyl radical scavengers such as vitamin E, dimethyl-sulfoxide (DMSO) and mannitol reduced endogenous ROS generation and alleviated the cytotoxic effects of iron. On the other hand, superoxide dismutase (SOD), vitamin C and catalase did not show any protective effects against Fe3+-NTA. These findings suggest that enhanced cytotoxic effects of Fe3+-NTA at 20% O2 are due to endogenously produced hydroxyl radicals.