Comparative developmental and phenotypic properties of altered hepatocyte foci and hepatic tumors in rats.

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)

Effects of separate and combined treatments with gamma radiation and diethylnitrosamine in neonatal rats on the induction of altered hepatocyte foci and hepatic tumors.

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.

Effects of rat strain, diet composition, and phenobarbital on hepatic gamma-glutamyl transpeptidase histochemistry and on the induction of altered hepatocyte foci and hepatic tumors by diethylnitrosamine.

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.

Characterization of histochemically detectable altered hepatocyte foci and their relationship to hepatic tumorigenesis in rats treated once with diethylnitrosamine or benzo(a)pyrene within one day after birth.

A new experimental system was used to examine the stages of chemically induced hepatic neoplasia in the rat. The treatment protocol involved the i.p. injection of a single dose of carcinogen [diethylnitrosamine or benzo(a)pyrene] into male and female rats within 1 day after birth, followed by dietary exposure to promoter (0.05% dietary phenobarbital) beginning at weaning. Rats were killed at intervals, and their livers were examined for tumors and for histochemically detectable foci of altered hepatocytes using six histochemical markers. Through serial frozen-sectioning techniques and computer-assisted image analysis, foci containing between one and six markers were identified, and their average diameters were calculated. The same complement of histochemical tests was applied to the primary hepatic tumors observed in this study. The principal findings were the following. (a) Both the diethylnitrosamine and benzo(a)pyrene treatments were tumorigenic and produced foci with similar phenotypic properties (numbers and identities of histochemical markers). (b) Foci relative growth rates and growth capacities (ranges of possible growth rates) were directly related to foci phenotypic complexity levels (numbers of markers per focus). (c) Individual foci were phenotypically stable; i.e., they neither gained nor lost markers. (d) A substantial fraction of the tumors observed in this study had fewer markers than the most complex foci. On the basis of these observations, we suggest that foci emerge as the result of a specific set of cellular changes solely inducible by carcinogenic stimuli, but the foci do not evolve through progressively more deviated forms into tumors. Instead, we postulate that tumorigenesis involves a separate transformation event that may occur in a susceptible fraction of foci subsequent to their induction or, alternatively, may occur at the time of exposure to carcinogen, in parallel with the carcinogen-mediated events leading to focus formation.

Relationship of histochemically detectable altered hepatocyte foci to hepatic tumorigenesis.

A new experimental system was used to examine the stages of chemically induced hepatic neoplasia in the rat. The treatment protocol involved the intraperitoneal injection of a single non-necrogenic dose of carcinogen [N-nitrosodiethylamine (NDEA) or benzo[a]pyrene (BP)] into male and female rats within one day after birth, followed by dietary exposure to promoter (0.05% phenobarbital) from weaning. Rats were killed at intervals, and their livers were examined for tumours and for histochemically detectable foci of altered hepatocytes. Six histochemical markers were used. Through serial frozen-sectioning techniques and computer-assisted image analysis, foci containing between one and six markers were identified, and their average sizes were calculated. The same complement of histochemical tests was applied to the primary hepatic tumours observed in this study. The data showed that (1) the new treatment protocol was highly efficient in foci and tumour production; (2) growth rates and incidence levels of foci were directly related to hepatocarcinogenic effectiveness (NDEA greater than BP), whereas both carcinogens had similar effects on foci phenotypic properties; (3) after their formation, foci at a given level of phenotypic complexity did not become progressively more complex; (4) incidence levels of foci were sex-dependent (females greater than males), but growth rates of foci were the same for both sexes; (5) growth rates and growth capacities (ranges of possible growth rates) of foci were directly related to phenotypic complexity levels of foci; (6) frequencies and phenotypic complexities of foci were inversely related; the reverse was true for tumours, although 10% of the tumours were relatively simple (three markers or fewer); (7) marker frequency distribution patterns were completely different in foci and in tumours. From these observations, we suggest that the foci are not direct tumour progenitors but, instead, are manifestations of a mosaic of subtumorigenic effects of the carcinogenic stimulus on cellular functions associated with the control of cell division and phenotypic character. The observed foci and tumour characteristics suggest that these carcinogen-induced cellular changes define elements of the mechanism whereby a specific neoplastic transformation site is rendered more accessible to carcinogenic attack.

Early appearance of histochemically altered hepatocyte foci and liver tumors in female rats treated with carcinogens one day after birth.

A single i.p. injection of diethylnitrosamine (DEN) or benzo[a]pyrene (BAP) in 1-day-old female rats produced a high incidence of gamma-glutamyltranspeptidase-(GGT)-positive hepatocyte foci within 4 weeks after the rats were weaned onto a 0.05% phenobarbital diet; the injection of benzo[e]pyrene did not produce foci under these conditions. Liver tumors appeared in rats treated with DEN within 8 weeks after weaning and in BAP-treated rats within 16 weeks after weaning. The results suggest that the treatment protocol used in this study may increase the utility of the liver tumorigenesis model as a broadly applicable in vitro system for the rapid detection of tumorigenic potential in environmental contaminants.

Odontomas in Peromyscus leucopus.

A colony of Peromyscus leucopus was established 15 years ago from animals trapped in the deciduous forest at Argonne National Laboratory, Argonne, Illinois. A roentgenographic survey of the skeletons of 189 of these untreated animals dying during a 13-month period disclosed 48 odontogenic growths in 21 of the mice. These growths were diagnosed on histopathologic examination as complex odontomas, the incidence of which was higher in males than in females. In this relatively small sample, these benign tumors appeared to be associated with youth rather than old age.

Comparative effects of aminotriazole on normal and acatalasemic mice.

Some pharmacological and toxicological effects of dietary 3-amino-1,2,4-triazole (AT), a known catalase inhibitor, antithyroid agent, and carcinogen, have been examined, using acatalasemic (Csb) and normal catalase, "wild-type" (Csa) substrains of highly inbred C3H and C57BL mice. It was found that (a) the acatalasemic substrains are more resistant to weight loss and death on the AT diet than are their normal catalase counterparts; (b) Csb and Csa substrains of C57BL mice are more resistant to weight loss and death on the AT diet than are the Csb and Csa substrains of the C3H mouse; (c) the liver catalase, as well as the whole body catalase, of the two C57BL substrains is less inhibited by the AT diet than is that of the C3H substrains; (d) mice consuming the same quantity of either normal or AT-containing diet gain much more weight on the normal diet; (e) temporary consumption of the AT diet causes a considerable increase in thyroid weight, with an extremely slow, and only partial, return toward normal weight; and (f) the C3H/Csa mouse on an AT diet develops a scaly, necrotic tail very similar in appearance to the so-called rodent ringtail; this lesion is never observed in the acatalasemic mouse on the same diet.

Carcinogenic and antitumor effects of aminotriazole on acatalasemic and normal catalase mice.

Dietary 3-amino-1H-1,2,4-triazole (AT), although carcinogenic when administered alone, was an antitumor agent when combined with certain other carconogenic stimuli. The carcinogenic effect was prominent in the livers of C3H mice; thyroid tumors were less common because they required a longer period of development, and the life-span of the animal was shortened by the AT diet. The antitumor effects of AT included: delay in appearance of mammary tumors, striking reduction in gamma-radiation-induced lymphomas, and sharp reduction in neutron radiation-induced harderian gland and ovarian tumors. On an AT diet, the inbred C3H acatalasemic mouse substrain developed more liver tumors, starting earlier, than did the C3H normal catalase substrain. We suggest that our findings pointed to a possible relevance of catalase and H2O2 in carcinogenesis. The most probable mechanism for the increased incidence of liver tumors in AT-treated acatalasemic mice was the diminished rate of degradation of endogenous H2O2.