DNA Repair in Primary Cultures of Rat Hepatocytes

This report describes a precise quantitative analysis of DNA repair in cultured rat hepatocytes following exposure to ultraviolet light, 2-acetylaminofluorene, and two of its more active derivatives, N-hydroxy-2-acetylaminofluorene and N-acetoxy-2-acetylaminofluorene. Hepatocytes were isolated from young adult male Wistar rats with the collagenase perfusion technique and maintained in short-term monolayer culture on collagen-coated plates in a serumfree modified Waymouth's medium. The nuclear [³H]thymidine ([³H]dThd)-labeling index of control cultures was less than 0.1%, but significant cytoplasmic labeling was evident in autoradiographs. Of the total acid-precipitable radioactivity present in control cultures following exposure to [³H]dThd, 54% of the³H was found in protein, demonstrating the ability of these nonreplicating hepatocytes to catabolize [³H]dThd and reutilize the labeled metabolites. Ultraviolet light irradiation of the cultured hepatocytes resulted in a dose-dependent increase of [³H]dIThd incorporation into DNA. That this represented nuclear DNA repair synthesis was demonstrated by detecting nonsemiconservative DNA synthesis (repair replication) with Nal isopycnic centrifugation and autoradiography. Hydroxyurea (10 and 100 mM) had only a small inhibitory effect, while both 1-β-o-arabinofuranosylcytosine (25 and 100 μm) and ethidium bromide (at 25 μm) dramatically inhibited the ultraviolet light-induced increase in [³H]dThd incorporation. Repair synthesis also occurred in response to treatment of the hepatocytes with 2-acetylaminofluorene, demonstrating their ability to metabolize this prohepatocarcinogen to a form capable of damaging DNA. N-Hydroxy-2-acetylaminofluorene and N-acetoxy-2-acetylaminofluorene were more effective in inducing a repair response. These results represent additional characterization of the primary hepatocyte culture system and demonstrate its potential for studies on mechanisms of carcinogenesis and as a potential screening system for environmental chemicals suspected of being capable of damaging DNA and causing cancer.