Development of in vitro toxicity tests with cultures of freshly isolated rat hepatocytes.

Freshly isolated and cultured hepatocytes were analyzed by two-parameter flow cytometry. The combined analysis of DNA and cellular protein content allowed the contribution of ploidy classes and of subpopulations within a ploidy class to be defined. Analysis of hepatocytes during exposure to dimethylsulfoxide (DMSO), phenobarbital (PB), low oxygen tension (4% O2) or fetal calf serum (FCS), provided insight into the dynamic response of individual ploidy classes as a function of culture time. By analogy with the age-dependent ploidy shifts in vivo, hepatocyte-cultures shift towards adult animals during exposure to DMSO and towards young animals when cultured at low pO2 (4% O2). FCS and phenobarbital disturb this constitutive ploidy balance. FCS increased the 2 N cell population, where stem cells probably respond to the proliferative stimuli provided by growth factors in the serum. Phenobarbital affects the liver-specific 4 N hepatocytes, which agrees with effects seen in liver after exposure in vivo. It is suggested that drug-induced pathological alterations in ploidy in hepatocyte cultures could serve as indicators of compounds, such as liver tumor promoters, which interfere with cell differentiation in liver. The heterotypic cell-cell interaction of freshly isolated hepatocytes with isolated, in vitro cultured, rat liver epithelial cells in co-cultures proved to be a valuable concept in toxicity testing: aldrin epoxidase, an enzyme system involved in xenobiotic metabolism, was stabilized for more than two weeks. After exposure to the three chemicals, 2-acetylaminofluoren, procarbazine and cyproterone-acetate, a preferential toxicity for each compound and cell population was established. Thus heterotypic cell cultures can considerably increase the amount of information available from in vitro studies. The final concept, combining monitoring of cellular DNA (ploidy) and protein content in hepatocyte cultures during and after exposure to a given test compound at tissue oxygen tension with the heterotypic cell-cell interaction, would create a more in vivo-like culture system. This would enhance the predictability of hepatocyte cultures and contribute to a more widespread use of the test system and as a result help to reduce the number of whole-animal tests.