OXIDATIVE STRESS-INDUCED BY HUMIC-ACID SOLVENT-EXTRACTION FRACTION INCULTURED RABBIT ARTICULAR CHONDROCYTES

Kashin-Beck disease (KBD), an endemic, chronic osteoarthritic disorder with necrosis of chondrocytes, commonly occurs in China. The humic su bstance present in the drinking water of endemic areas has been propos ed as one of the causative factors. In this study an in vitro cell cul ture system was used to investigate the damaging effects of humic acid (HA), a constituent of humic substance, on cultured rabbit articular chondrocytes. The commercial Aldrich humic acid (AHA) was fractionated With a series of organic solvents including n-hexane, benzene, ethyl acetate, and methanol. Among the several fractions of AHA, the ethyl a cetate fraction (AHA-[EA]) displayed the most potent inhibitory effect on the survival of chondrocytes in clonogenic assays. Cellular injury induced by AHA-[EA] was evaluated by measuring cell viability with me thylthiazol tetrazolium (MTT) and by determining the release of intrac ellular lactate dehydrogenase (LDH). Incubation of chondrocytes with A HA-[EA] (100-500 mu g/ml) for 12 h produced a concentration-dependent decrease in cell viability and increase in LDH release. in addition, A HA-[EA] triggered lipid peroxidation manifested by elevated malondiald ehyde (MDA) formation. In chemiluminescence assay, AHA-[EA] at the con centrations of 150-600 mu g/ml caused 6- to 15-fold increases of lumin ol-amplified chemiluminescence responses, which are considered to refl ect the production of hydrogen peroxide (H2O2). Moreover, pretreating the cells with 500-750 U/ml of catalase significantly prevented the lo ss of cell viability, while superoxide dismutase (SOD) enhanced the ad verse effect of 300 mu g/ml AHA-[EA]. Data suggest that the injury to chondrocytes induced by AHA[EA] may be first through O-2(.-) productio n, which is then converted into H2O2, thus initiating lipid peroxidati on and leading to chondronecrosis observed in KBD.