Techniques in Measuring DNA Synthesis and Mitosis Induced by Tumor Promoters in Hepatocyte Primary Cultures

Summary

Repeated regenerative liver growth and adaptive liver growth stimuli (of hormonal or xenobiotic nature) applied in vivo have been closely connected with liver tumor promotion. Therefore, from a practical point in toxicology it is of interest to elucidate whether or not a given chemical is a liver growth stimulator. Identification of a test compound as liver growth stimulator justifies the suspicion that on long term administration tumor development may be a corollary event. Such a conclusion may be often confirmed by detection of the induction of drug metabolizing enzymes. Short term tests in vivo for the induction of liver growth and drug metabolizing enzymes have been described for different classes of compounds. Short term tests in vitro are currently under development. They would provide the possibility to check for hormonal and xenobiotic actions on liver cell replication not only in animal cells but more importantly on human material as well.

During the last 5 years it has become obvious that onset of DNA synthesis in primary hepatocyte cultures is inversely related to cell density. More importantly progression of these cells through G2 into mitosis seems to take place only in cultures of sufficiently low cell density in cultures. Nevertheless it appears that control of this step in the hepatocyte cell cycle is not yet fully understood. Two main techniques have been used to monitor DNA synthetic activity which are discussed: biochemical measurement of incorporated tritium-labelled nucleotide precursors into DNA by liquid scintillation counting and autoradiograpy. The first technique has been widely used because results are on hand rapidly. Their drawbacks, however, were noticed soon, as there were unspecifically incorporated label due to nucleotide metabolism, inadequate relation of data to cell protein or per culture dish instead on purified hot acid soluble DNA leading to unreliable results, and finally lacking information on the number of non-parenchymal cells incorporating label and on cells undergoing mitosis.

Autoradiographic techniques, although more laborious for the collection of data, had to be used in order to look for hepatocytes in mitotis. And this finally has helped to identify cell density as one of the regulators of hepatocyte replication in culture. Induction of hepatocyte DNA replication by hormones and tumor promoters under various conditions are discussed as examples and the use of both techniques will be demonstrated.