EFFECTS OF OXYGEN FREE RADICAL SCAVENGERS ON THE HYPOXIA-REOXYGENATION INDUCED PROLIFERATION OF CULTURED HUMAN FIBROBLAST MALME-3 CELL LINE

Choi, Jae Won; Lee, Chang Won; Kim, Dong Koo; Kim, Yong Oock

Abstract

The aim of this study was to investigate the effect of hypoxia-reoxygenation on the proliferation of fibroblast, and to elucidate the role of oxygen free radicals in this process. Malme-3 fibroblast, derived from human skin fibroblast, was used for this study. The hypoxia or reoxygenation condition was made by exposing cultured cells to the environment of 95% N2, 5% CO2 or 95% room air, 5% CO2, respectively. Cell proliferation was estimated by the cell numbed, and DNA synthesis was measured by the [3H]-thymidine uptake. Release of oxygen free radicals was measured by the means of Ohkawa's method of lipid peroxidation. The effect of oxygen free radicals was confirmed by using dimethylthiourea(DMTU) and alpha-tocopherol, two known oxygen free radical scavengers. The results are as follows: 1. The dissolved oxygen of the culture medium was 8.97+/-1.23 ppm in the normal condition. When the culture dish was exposed to the hypoxic condition for 3 or 6 hours, the dissolved oxygen of the culture medium decreased markedly to the level of 3.10+/-0.46 ppm or 2.37+/-0.47 ppm, respectively 2. The number of cultured cells increased in a hypoxia duration-dependent manner up to 6 hours when the cells were cultured for 24 hours after hypoxia. The same pattern was observed in the cells cultured for 48 hours after hypoxia. Lipid peroxidation in the culture increased after the exposure to hypoxia-reoxygenation. DMTU or alpha-tocopherol blocked the increase in lipid peroxidation induced by the exposure to hypoxia-reoxygenation. 3. [3H]-thymidine uptake of the cultured cells increased after the exposure to hypoxia-reoxygenation. 4. DMTU or alpha-tocopherol blocked the proliferation of fibroblasts induced by the exposure to hypoxia-reoxygenation. The increase in lactate dehydrogenase (LDH) activity was also noted after the exposure to hypoxia-reoxygenation, and this increase was blocked by DMTU or alpha-tocopherol. These results indicate that the hypoxia-reoxygenation induces the proliferation of fibroblasts, and that oxygen free radicals play an important role in this process. Moreover, oxygen free radical scavengers may be of potential therapeutic value in preventing fibrosis.