We have shown previously that approximately 1 in 10,000 primary hepatocytes isolated from untreated rats undergo clonal growth in soft
agar in vitro in response to the synergistic action of
nafenopin, a peroxisome proliferator (PP) and
epidermal growth factor (
EGF), a naturally occurring liver growth regulator. Here, we demonstrate that prior treatment of the animals with the genotoxic hepatocarcinogen
diethylnitrosamine (DEN) caused a dose-dependent increase in soft
agar colony numbers formed in vitro. These data suggest that the colony assay may offer a method of detecting in vitro hepatocytes transformed in vivo by DEN. It is known that rats treated with DEN develop
enzyme altered foci prior to the development of tumours. The majority of these foci express high levels of
gamma-glutamyl transpeptidase (GGT). However, foci promoted by PPs do not show this increased
enzyme activity. In the present study, the colonies we have generated in vitro mimicked this pattern since the majority (approximately 80%) of the spontaneous colonies expressed GGT whereas colonies promoted by the synergistic action of
nafenopin and
EGF were mainly (75%) GGT negative. The proportion of colonies positive for GGT were similar using either hepatocytes isolated from control or from DEN-initiated rats. Further studies are required to assess if the hepatocytes selected for clonal expansion by this
EGF/
nafenopin regime reflect the presumed pre-neoplastic cells induced by
genotoxin in vivo and associated with an increased propensity to
cancer.