Enhancement of the clonability of adult parenchymal hepatocytes with the liver tumor promoter phenobarbital

An in vivo clonogenic assay system was utilized to investigatethe effect of the tumor promoter, phenobarbital (PB), on adultparenchymal hepatocyte proliferation. Enzymatically dispersedhepatocytes from female Fischer 344 rats were injected intothe interscapular and mammary fat pads of isogeneic recipientanimals where they proliferate to form hepatocyte colonies within3 weeks. The number of hepatocytes required to form a colonyin 50% of the transplantation sites (LND₅₀) was 23 700 cellsand 520 cells when normal adult liver cells were injected intonon-hepatectomized and 2/3 hepatectom-ized normal recipientanimals, respectively. Thus, a partial hepatectomy increasedthe hepatocyte clonogenicity by a factor of 40. A 2-week pre-treatmentof both the donor and recipient animals with PB (0.1% in thedrinking water) significantly increased the clonogenicity ofthe liver cells when transplanted into non-hepatectomized (15-fold)and 2/3 hepatectomized (2-fold) animals. However, PB treatmentof the recipient animals was not required for the majority ofthis mitogenk effect since the clonability of PB-treated donorcells was increased (92% of the maximum stimulation observed)even when they were transplanted into untreated control animals.Furthermore, the PB-induced effect on hepatocyte clonabilitywas reversible since the removal of PB from the donor animals2 weeks prior to their use reduced the clonability of the hepatocytes(LND₅₀=20 500 cells) to that observed for cells which were neverexposed to PB. These results are consistent with the postulatethat rather than PB being directly mitogenic, it primarily increasesthe clonability of adult parenchymal hepatocytes by inducinga reversible cellular alteration which enhances their responsivenessto endogenous growth stimuli.