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Use of Primary Cultures of Adult Rat Hepatocytes on Collagen Gel-Nylon Mesh to Evaluate Carcinogen-induced Unscheduled DNA Synthesis¹

Departments of Anatomy [A. E. S.] and of Oncology [C. G. H., G. L. S., H. C. P.] and Pathology [H. C. P.], McArdle Laboratory for Cancer Research, The Medical School, University of Wisconsin, Madison, Wisconsin 53706

The procarcinogen, 2-acetylaminofluorene, the direct-acting carcinogen, methyl methanesulfonate, and two other hepatocarcinogens, thioacetamide and urethan, were tested for their ability to elicit unscheduled DNA synthesis in adult rat hepatocytes maintained in primary culture on collagen gel-nylon mesh. The carcinogens, dissolved in dimethyl sulfoxide, were added to 6-hr or to 28-hr cultures along with [methyl-³H]thymidine (1 µCi/ml medium) in the presence of 10 mM hydroxyurea. Twelve hr later, the hepatocytes were harvested from the cultures with collagenase, and their DNA was purified on CsCl isopyknic gradients. Unscheduled DNA synthesis was measured as the increase in [methyl-³H]thymidine radioactivity incorporated per µg DNA of the carcinogen-treated cultures as compared with that of control cultures. Both 2-acetylaminofluorene and methyl methanesulfonate demonstrated a concentration-dependent stimulation of unscheduled DNA synthesis in the 6-hr hepatocyte cultures. However, the response of the 28-hr cultures to these two carcinogens was absent unless the hepatocytes were preincubated for 22 hr in culture medium supplemented with 10^-5 M dexamethasone and 10^-6 M glucagon or in a more complete hormone-supplemented medium. Thioacetamide and urethan, on the other hand, failed to elicit a concentration-dependent unscheduled DNA synthesis under these conditions. The results obtained with this culture system are similar to those of other short-term tests for chemical carcinogenicity and support the potential use of the collagen gel-nylon mesh-hepatocyte primary culture as an in vitro screen for chemical carcinogens. Furthermore, this study suggests the importance of specific hormones in maintaining the capability for repair of DNA damage produced by carcinogenic and mutagenic chemicals in cultured hepatocytes.

¹ Presented in part at the 70th annual meeting of the American Association for Cancer Research, New Orleans, Louisiana, May, 1979 (28). This work was supported by Contract NO1-CP-85609 from the National Cancer Institute, NIH.

² To whom requests for reprints should be addressed.

Received 8/29/79. Accepted 6/ 4/80.