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Altered hepatic foci (AHF) were analyzed by quantitative stereology on frozen serial sections stained sequentially for gamma-glutamyltranspeptidase (GGT), canalicular adenosine triphosphate (ATPase), glucose-6-phosphatase (G6Pase), and the placental isoenzyme of glutathione S-transferase (GST). Livers for these analyses were obtained from both male and female rats of different ages which had been subjected to initiation with a nonnecrogenic dose of diethylnitrosamine following a 70% partial hepatectomy with subsequent phenobarbital (PB) feeding. Different combinations of these four marker alterations (from single marker to four-marker combinations) were used to analyze the data, and the results were compared for their ability to detect AHF. In rats on the above protocol, GST was the single most effective marker, exhibiting a high sensitivity for scoring both number and volume of foci. There was a high degree of overlap with GGT. The combination of the four different markers, GST/GGT/ATPase/G6Pase, scored 80% more foci in number and 60% more in volume than the routinely used GGT/ATPase/G6Pase method. When all four markers were used to score AHF, PB promotion was equally effective in both sexes at weaning and at 6 months of age, but at 1 year of age males showed a dramatic reduction in the effectiveness of PB as a promoting agent, both for number and volume percentage of liver occupied by AHF. On the other hand, initiation was more effective in the male at weaning and at 6 months of age, although by the 12-month point no distinction between the sexes could be made. When only GGT was used as a marker, promotion by PB appeared to be markedly less effective in males than in females at all ages. In the absence of PB administration, both the number and volume fraction of AHF in the livers of both males and female increased with age. Likewise, both the number of AHF per liver and their volume fractions increased with age in both sexes when uninitiated animals were fed PB, although only after a 6-month lag in females. These experiments demonstrate that the stages of initiation and promotion in hepatocarcinogenesis in the rat as monitored by the number and volume percentage occupied of AHF are altered by both the age and the sex of the animal. The combination of GGT and GST identified all AHF scored by the GST/GGT/ATPase/G6Pase set of markers and thus may be the most efficient combination of markers of AHF resulting from promotion by PB.

The mathematical science of quantitative stereology has established relationships for the quantitation of elements in three-dimensional space from observations on two-dimensional planes. This report describes the utilization and importance of such mathematical relationships for the quantitative analysis of focal hepatic lesions in terms relative to the volume of the liver. Three examples are utilized to demonstrate the utility of such calculations in the three-dimensional quantitation of hepatic focal lesions. The first is that of a computer-simulated experiment based on defined hypothetical situations. The simulations demonstrate the applicability of the computations described in this report to the evaluation of two-dimensional data from typical animal experiments. The other two examples are taken from actual experiments and involve the transplantation of hepatic cell populations into the liver suitably prepared hosts and the quantitation of altered foci produced by initiation with diethylnitrosamine-partial hepatectomy followed by promotion with phenobarbital. The quantitation of altered foci by means of a two-dimensional analysis (simple enumeration of focal intersections/area of tissue section) is proportional to the quantitation of foci per volume of liver provided that the mean diameter of the foci for each treatment is sufficiently uniform, as exemplified in the text by the transplantation experiment. When such mean diameters are unequal as in the diethylnitrosamine-phenobarbital experiment described herein, quantitation from three-dimensional analysis gives significantly different results as compared with enumeration of focal intersections on two-dimensional areas. These studies clearly demonstrate that the frequency and size of foci intersections viewed on two-dimensional tissue sections do not necessarily reflect the number of size of foci in the three-dimensional tissue. Only by quantitating the number and size of the foci in relation to the three-dimensional volume of the tissue can one determine the validity of the proportionality of data from two-dimensional measurements to the total number of foci per volume of tissue. Such a conclusion has important implications for quantitative studies on hepatocarcinogenesis as well as for the enumeration of premalignant lesions which occur during the natural history of carcinogenesis in any solid tissue.

The effect of dietary exposure to synthetic estrogens on hepatocarcinogenesis was evaluated. Diethylnitrosamine-initiated and 0.85% NaCl solution-treated noninitiated female Sprague-Dawley rats were transferred to semisynthetic diets containing mestranol (0, 0.1, or 0.5 ppm), ethinyl estradiol (0.5 ppm), estradiol (0.6 ppm), or mestranol plus beta-methasone (0.5 and 0.2 ppm, respectively). gamma-Glutamyl transferase (GGT)-positive transections and hematoxylin and eosin-detectable nodules and carcinomas were scored at 9 and 12 months. Quantitative stereological calculations were performed to determine GGT lesion number and size. At 9 months, in diethylnitrosamine-initiated rats, ethinyl estradiol and mestranol caused 3.5- and 4.4-fold increases, respectively, in the number of GGT lesions per liver and an increased incidence of hepatocellular carcinomas while estradiol had no enhancing effect. Addition of beta-methasone to the mestranol-containing diet caused a significant decrease in GGT lesion number but not carcinoma incidence compared to mestranol alone. At 12 months, in diethylnitrosamine-initiated rats, mestranol caused a dose-dependent increase in GGT lesion number. The hepatocellular carcinoma incidence was significantly increased at the high mestranol dose. Small increases in the numbers of larger GGT lesions were also observed in noninitiated animals treated with mestranol and ethinyl estradiol and are most probably due to promotion of spontaneously initiated hepatocytes. These results indicated that the synthetic estrogens cause dramatic increases in the number of presumptive preneoplastic GGT lesions. Carcinoma incidence is also enhanced. Thus, these results confirm and extend our previous studies which together with the results of others have shown that synthetic estrogens can act as promoters of hepatocarcinogenesis.

In order to test the potential of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) as a promoter of hepatocarcinogenesis, rats which had received a single 10-mg/kg dose of diethylnitrosamine (DEN) following partial hepatectomy were given TCDD (0.14 and 1.4 micrograms/kg s.c. once every 2 weeks) for 7 months. Animals which received (a) only a single initiating dose of DEN after partial hepatectomy and no further treatment of (b) TCDD alone with no initiating dose of DEN exhibited relatively few enzyme-altered foci and no hepatocellular carcinomas. However, animals initiated with DEN and then given TCDD had a marked increase in enzyme-altered foci. At the higher dose of TCDD, hepatocellular carcinomas were present in five of seven rats. By means of three different enzyme markers used to evaluate the phenotypes of the enzyme-altered foci, a distinct phenotype heterogeneity of the foci was noted with a shift towards phenotypes exhibiting a greater deviation from normal liver when TCDD was given following DEN-partial hepatectomy. Quantitation of the numbers of enzyme-altered foci was performed by relating measurements made from two-dimensional tissue sections to the numbers of foci per unit volume of liver using relationships established in the field of stereology. The total volume of the liver occupied by the enzyme-altered foci, but not their number, increased with the dose of TCDD administered following DEN-partial hepatectomy. These studies demonstrate that TCDD is a potent promoting agent for hepatocarcinogenesis.