Primary monolayer cultures of rat hepatocytes at confluence were exposed to an exogenously added source of superoxide, and its influence on collagen synthesis was examined. Superoxide was generated by the addition of dihydroxyfumarate to the culture medium. Exposure of hepatocytes to dihydroxyfumarate greatly stimulated the activity of prolyl hydroxylase and the synthesis of collagen. A significant increase in prolyl hydroxylase activity was observed with 5 pg per ml dihydroxyfumarate in 24 hr relative to that in the untreated cultures. Maximum stimulation of greater than %fold compared to the control value was elicited by 25 pg per ml dihydroxyfumarate. When scavengers of superoxide such as superoxide dismutase and Cu(Ly& were added in the medium, the increase in prolyl hydroxylase activity induced by dihydroxyfumarate was nearly abolished. Experiments with actinomycin D indicated that synthesis of new RNA was involved in the stimulation of prolyl hydroxylase activity. Analysis of collagen synthesis in cultures exposed to dihydroxyfumarate also showed a marked increase compared to that of the untreated cultures. The presence of superoxide dismutase in the medium significantly reduced the increase in collagen synthesis. Our results indicate that superoxide mediates the stimulation of collagen synthesis in hepatocytes. These findings may provide a possible explanation for excess collagen formation during induced liver fibrosis.
Normal liver contains very little collagen compared to the amount present in the total protein of the mammalian body (1). The rate of collagen synthesis and its accumulation however, greatly increases in chronic liver disease and in cirrhosis induced by chemical injury resulting in hepatic fibrosis (2-8). The induction of collagen synthesis is almost always preceded by inflammation (9-13). Although the precise mechanisms involved in the stimulation of collagen proliferation are not known, it is recognized that inflammation may play an important role in this process (14, 15).
Nonproliferating primary cultures of isolated rat hepatocytes prepared from either normal liver or regenerating liver after partial hepatotectomy have been shown