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To characterize the effects of combined treatments with gamma radiation and diethylnitrosamine (DEN) on the induction of histochemically detectable altered hepatocyte foci and hepatic tumors, we assessed the yields of these lesions in the livers of 150-day-old rats that had been treated neonatally with a single dose of gamma radiation (75 rad, whole body) and i.p.-injected DEN (0.15 mumol/g body wt), either separately or in combination. The combined treatments involved the administration of the two stimuli in both possible sequences, with the interval between treatments set at 1 h. The focus population was examined for two histochemical markers (elevated gamma-glutamyl transpeptidase [GGT(+)] and iron exclusion [FE(-)], giving rise to three detectable focus phenotypes, i.e. GGT(+) foci, FE(-) foci, and GGT(+), FE(-) foci. Frequencies of the three phenotypes were quantitated through the use of serial frozen sectioning techniques and computer-assisted image analysis. GGT(+) focus induction was synergistically enhanced by the combined treatment irrespective of the order in which the two stimuli were administered; the remaining two phenotypes did not show such enhancement. The magnitude of the GGT(+) focus response was significantly greater when the treatment sequence was gamma----DEN as opposed to DEN----gamma. Tumor yields in rats receiving combined gamma--DEN treatment were similar to those in rats receiving the DEN alone, irrespective of the gamma--DEN treatment sequence. These results suggest that phenotypically distinguishable lesions, including foci with different histochemical marker patterns and tumors, originate from specific types of damage at different genetic loci and are developmentally independent; and the expression of the GGT(+) marker per se in altered hepatocyte foci is not a reliable index of incipient hepatic neoplasia.

Previous investigations in this laboratory have provided evidence that histochemically detectable altered hepatocyte foci and hepatic tumors appearing in rats given a single neonatal treatment with a low dose of carcinogen followed by chronic dietary phenobarbital administration are developmentally independent. The present investigation further evaluates developmental relationships among these lesions. Altered hepatocyte foci were divided into two subclasses consisting of foci that were detectable by histochemical as well as by hematoxylin-eosin staining [designated hist(+)/morph(+) foci] and those foci that were detectable solely by histochemical staining [designated hist(+)/morph(-) foci]. The developmental and phenotypic properties of the hist(+)/morph(-) foci, hist(+)/morph(+) foci, and hepatic tumors were compared in rats initiated once neonatally with different doses of diethylnitrosamine and promoted with dietary phenobarbital from weaning. The morph(+) and morph(-) lesion subclasses were distinguishable on the basis of several developmental characteristics. Hist(+)/morph(+) foci were present at low frequency until at least 150 days after initiation. Although the development of hist(+)/morph(-) foci was essentially complete at that point, the rate of appearance of hist(+)/morph(+) increased significantly. The diethylnitrosamine dose response of the hist(+)/morph(+) foci followed the histochemical marker patterns of the tumor lesion class more closely than that of the hist(+)/morph(-) group. The rates of expression of the hist(+)/morph(+) foci increased with the increasing level of histochemical complexity, whereas the rates of expression of the hist(+)/morph(-) foci groups were inversely correlated to their complexity level. Although the average focus size or diameter in the hist(+)/morph(+) groups was greater than that of the hist(+)/morph(-) foci, the focus growth rates of morph(+) and morph(-) subsets matched for histochemical phenotype were comparable. The complexity level and individual marker distribution patterns of the hist(+)/morph(+) focus class were more similar to tumor patterns than to the distribution patterns of the hist(+)/morph(-) lesion class. The results suggest the following. (a) The development of lesion classes with successively greater deviation from normalcy does not occur via lineal progression from less to more deviated forms within a given lesion class. The three lesion classes appear to develop independently, with the developmental characteristics of each lesion class determined at the time of initiation.(ABSTRACT TRUNCATED AT 400 WORDS)

To extend our ongoing characterization of modulatory influences on hepatic tumorigenesis, we examined effects of rat strain (Sprague-Dawley versus Fischer), diet composition (semipurified diet versus standard nonpurified laboratory chow), and dietary phenobarbital on the production of gamma-glutamyl transpeptidase (GGT)-positive hepatocyte foci and hepatic tumors initiated by diethylnitrosamine. In addition to GGT-positive foci, we observed, under certain conditions, the appearance of extensive hepatic GGT staining not associated with focal lesions. This elevated nonfocal GGT was found in rats of both strains fed the nonpurified rather than the purified diet, but the level of staining was higher in Fischer than in Sprague-Dawley rats. Enhancement of this nonfocal staining by dietary phenobarbital appeared insignificant. By comparison, frequencies of GGT-positive foci were generally higher in rats fed the semipurified rather than the nonpurified diet, and the frequencies of GGT-positive foci were invariably higher in Sprague-Dawley than in Fischer rats. Moreover, dietary phenobarbital generally enhanced focus production. Assessments of focus and tumor yields among these experimental groups showed that differences in focus frequencies did not correspond closely to differences in subsequent tumor formation. These results document the need to consider the influences of diet and rat strain on experimental end points in designing protocols for hepatocarcinogenesis studies, especially those involving GGT histochemistry. The data also raise questions about the mechanistic relevance of GGT induction to hepatocarcinogenesis and support our prior evidence against the putative lineal relationship between foci and tumors.