DNA repair in primary cultures of rat hepatocytes
Yager, J.D.; Miller, J.A.
Cancer Research 38(12): 4385-4394
1978
ISSN/ISBN: 0008-5472 PMID: 719626 Document Number: 128527
A precise quantitative analysis of DNA repair in cultured rat hepatocytes following exposure to UV light, 2-acetylaminofluorene and 2 of its more active derivatives, N-hydroxy-2-acetylaminofluorene and N-acetoxy-2-acetylaminofluorene is described. 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 serum-free 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 the 3H was found in protein, demonstrating the ability of these nonreplicating hepatocytes to catabolize [3H]dThd and reutilize the labeled metabolites. UV light irradiation of the cultured hepatocytes resulted in a dose-dependent increase of [3H]dThd 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-.beta.-D-arabinofuranosylcytosine (25 and 100 .mu.M) and ethidium bromide (at 25 .mu.M) 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. The primary hepatocyte culture system is further characterized. 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 is demonstrated.