Protection by antioxidants of copper-induced decline of proliferation and SOD activity

The effect of Cu plate on the cellular function was investigated by two different methods: an extraction method (Method I) and a direct contact method (Method II). In Method I, the supernatant of the culture medium, which had been pre-incubated with Cu plate, was added to mouse macrophage-like Raw 264.7 cells. This supernatant dose-dependently inhibited the proliferation and nitric oxide (NO) production by lipopolysaccharide-stimulated Raw 264.7 cells. In Method II, human promyelocytic leukemic HL-60 cells in suspension were incubated with culture medium which contained Cu plate. The direct contact with Cu plate rapidly suppressed the proliferation and MnSOD and Cu/ZnSOD activities. The suppressed proliferation and SOD activity reverted to or exceeded the control level by sodium ascorbate, whereas N-acetyl-L-cysteine (NAC) only reactivated the proliferation, but not the SOD activity. ESR spectroscopy showed that contact with Cu plate slightly diminished the hydroxyl radical (generated by Fenton reaction), without affecting the intensity of NO (produced from NOC-7) and DPPH radical. The present study suggests that two representative antioxidants, such as sodium ascorbate and NAC, protect the cells from Cu-induced cytotoxicity via different mechanisms.