Phenotypic diversity as an early property of putative preneoplastic hepatocyte populations in liver carcinogenesis

This study was undertaken to answer the following question. Is the phenotypic diversity that is characteristic of hepatocellular carcinomas acquired early during carcinogenesis, or is it more likely to be a property added late in the process? This question was posed using a new model for the sequential analysis of hepatocarcinogenesis. This model utilizes a single initiating dose of a carcinogen, such as diethylnitrosamine, followed by the selective stimulation of the rare, initiated hepatocyte to proliferate under conditions in which the proliferation of the majority of uninitiated hepatocytes is inhibited. Under these conditions, discrete early foci of altered hepatocytes and hyperplastic foci and nodules are quite well synchronized for about 10 to 12 cell cycles, after which the synchrony is progressively lost. As phenotypic expressions, cell proliferation, judged by radioautography after the administration of [3H]thymidine and the activities of four enzyme markers, two positive ones, gamma-glutamyltranspeptidase and DT-diaphorase, and two negative ones, glucose-6-phosphatase and adenosine triphosphatase, all judged histochemically, were used. At the earliest time of observation, 7 days, and at subsequent time points thereafter, all histologically recognizable foci and nodules showed variable degrees of staining for each enzyme activity. Prior to selection, gamma-glutamyltranspeptidase activity was much more consistent than was that of the others; however, during and after the selection, the four markers showed almost the same consistency among developing lesions. During the period of selection, between 80 and 90% of hepatocytes in the proliferating nodules were labeled with [3H]thymidine, while only an occasional labeled hepatocyte was seen in the foci prior to selection and in the nodules following selection. In the postselection period, the majority of nodules acquired the histochemical and architectural properties of normal liver, while a minority persisted as typical hyperplastic nodules. This study suggests that phenotypes of carcinogen-altered hepatocytes are variable, but whether the histochemical diversity among the lesions is merely due to environmental variation or is a reflection of a more basic genotypic variability remains a fundamental question.