NONGENOTOXIC CARCINOGENS SHIFT CULTURED RAT HEPATOCYTES INTO G1 CELL-CYCLE PHASE - INFLUENCE OF TISSUE OXYGEN-TENSION ON CELLS WITH DIFFERENT PLOIDY

The transition of quiescent freshly isolated rat hepatocytes (GO phase ) into a prereplicative stage of the cell cycle (G1 phase) has been vi sualized by a decrease in fluorescence of quinacrine dihydrochloride ( QDH)-stained nuclei. This transition might be used as an early detecta ble and sensitive marker for the identification of chemicals with a mi togenic potential. This was tested with three presumedly nongenotoxic carcinogens in the rodent, namely cyproterone acetate (CPA), thioaceta mide (TA) and phenobarbital (PB). Freshly isolated hepatocytes were cu ltured under 13% or 4% O-2, representing the tissue oxygen tension in the periportal (13% O-2) and the pericentral area (4% O-2) in the live r lobules. Fluorescence intensities of QDH-stained hepatocyte nuclei o f different ploidy levels (2N, 4N or 8N) were quantified by image anal ysis. The epidermal growth factor induced GO-GI shift was affected by the oxygen tension and was reversible as shown after exposure to retin oic acid (RA). At subtoxic concentrations, all three nongenotoxic carc inogens induced a shift of quiescent hepatocytes of all ploidy levels into the G1 cell cycle phase within a 6-h period. CPA was the most eff ective compound, followed by PB and TA. The strongest induction of the G0-G1 shift was observed in most cases in 2N nuclei. The oxygen tensi ons applied and the individual compounds tested, differentially affect ed the response of the individual ploidy classes. The results correspo nd well with the site-specific mitogenic response within liver lobules as observed after exposure in vivo. The shift from GO to G1 cell cycl e phase as detected by changes in QDH staining is therefore a reliable , early detectable change for the identification of chemicals with a p otential mitogenic activity.