USE OF PRIMARY CULTURES OF ADULT-RAT HEPATOCYTES ON COLLAGEN GEL-NYLON MESH TO EVALUATE CARCINOGEN-INDUCED UNSCHEDULED DNA-SYNTHESIS

  • 1 January 1980
    • journal article
    • research article
    • Vol. 40  (9) , 3259-3267
Abstract
The procarcinogen 2-acetylaminofluorene and the carcinogens methyl methanesulfonate, 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- or to 28-h cultures along with [methyl-3H]thymidine (1 .mu.Ci/ml medium) in the presence of 10 mM hydroxyurea. After 12 h, 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 .mu.g DNA of the carcinogen-treated cultures as compared with that of controls. 2-Acetylaminofluorene and methyl methanesulfonate demonstrated a concentration-dependent stimulation of unscheduled DNA synthesis in the 6-h hepatocyte cultures. The response of the 28-h cultures to these 2 carcinogens was absent unless the hepatocytes were preincubated for 22 h 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 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. Specific hormones may be important in maintaining the capability for repair of DNA damage produced by carcinogenic and mutagenic chemicals in cultured hepatocytes.