Effects of ceruloplasmin and the haptoglobin-hemoglobin complex on the oxygen-dependent reduction in growth of human diploid fibroblasts in serum-free, albumin containing medium.

Abstract

ABSTRACT. We examined that growth-promoting activity of two different human albumin (HSA) preparations for human diploid fibroblasts in serumfree RITC 80-7 medium. The activity of one preparation (sample A) was affected markedly by environmental oxygen, whereas the other (sample B) was little affected. Sample B contained ceruloplasmin (Cp) and haptoglobin (Hp) as impurities. To detect the generation of superoxide anion in the media the amount of reduction of cytochrome c that is inhibited by superoxide dismutase (SOD) was determined. In an aerobic environment it was relatively large in comparison with reduction inhibited in a hypoxic environment. Reduction in the sample A with HSA-supplemented medium was relatively large in comparison with that in sample B with HSA-supplemented medium. The reduction of cytochrome c also was inhibited by Cp (25 mg/1) and catalase (4000 units/ml). Moreover, SOD, Cp, catalase and Hp.Hb (but not Hp) partially prevented oxygen-dependent reduction in growth in an aerobic environment when added to sample A HSA-supplemented medium. These results suggest that Cp and Hp.Hb act as an antioxidants in culture.We developed a serum-free culture medium RITC 80-7, for human diploid fibroblasts and succeeded in the long term culture of these cells using this medium supplemented with human fibronectin (10 mg/l, FN) and bovine serum albumin (5 g/l, BSA, Cohn's fraction V) in a hypoxic (7 %) environment (11,27). Similar results were obtained with human serum albumin (HSA) instead of BSA. The growth promoting activities of both BSA and HSA were affected markedly by the oxygen tension in the culture environment. In an aerobic environment, a reduction in cell growth and a decrease in the in vitro lifespan were apparent in comparison to results in a hypoxic environment. The degree of reduction differed for each BSA commercial preparation (28). In contrast, cells cultured with 10 % fetal bovine serum (FBS)-supplemented