Induction by cyproterone acetate of DNA synthesis and mitosis in primary cultures of adult rat hepatocytes in serum free medium

Abstract

The aim of this study was to elucidate whether serum-free conditions could be found in primary hepatocyte cultures under which the growth inducing properties of xenobiotics and hormones could be tested. Cyproterone acetate (CPA), a steroid with anti-androgenic and progestogenic activity, was chosen as a model compound because of its known strong mitogenic properties in rat liver in vivo. EGF served as a positive control. Induction of DNA synthesis was studied by [3H]-thymidine labeling and autoradiography. Mitoses were counted in hematoxylin stained specimens. The main steps which led to an efficient stimulation of DNA synthesis by CPA were (i) reduction of hormone concentrations to levels approaching (approx. 10x) physiological concentrations better than the previously used pharmacological ones (up to 2500x); (ii) supplementation with glucocorticoid (most effective at 10-100 nM dexamethasone); (iii) selection of the interval for cumulative labeling with thymidine at 44-68 h; (iv) lowering of cell density at seeding to 50,000 cells/cm2 (subconfluency); (v) treatment with concentrations of 10-100 microM CPA. With these conditions CPA labelling was 13-20% (increase 4- to 9-fold). Mitotic incidence was 0.56% (CPA) versus 0.08% in controls. From a dose response study (0.1-100 microM) a no-effect-level for induction of DNA synthesis was found in the range of 0.1-1 microM. None of the high concentrations of CPA did cause cytotoxicity as estimated by morphological observations or release of lactate dehydrogenase into the medium. This work demonstrates that CPA under appropriate, defined culture conditions induces DNA synthesis and mitosis.(ABSTRACT TRUNCATED AT 250 WORDS)