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Male C3H/He and C57BL/6 mice were given diets containing sodium phenobarbitone (PB) to allow a daily intake of 85 mg/kg. Control and treated animals were killed at 5, 30, 40, 60, and 80 wk. Other mice were killed in extremis or at the end of the respective experiments: 91 wk for C3H/He and 100 wk for the C57BL/6 animals. A basophilic nodule was found in 1/5 control C3H/He mice at 30 wk; these nodules increased in number with time so that nodules of this type were found in approximately 70% of animals by 91 wk. Nodules were not found in control C57BL/6 mice until 80 wk, when they were found in 4% of mice. PB treatment markedly increased the number of hepatic nodules in both strains of mice. The additional nodule burden was due to the development of a second nodule type formed of large cells with a predominantly eosinophilic cytoplasm. C3H/He animals given PB for 60 wk and then returned to a control diet bore fewer nodules at 91 wk than treated mice killed at 60 or 91 wk. The cumulative incidence of carcinoma in control C3H/He and C57BL/6 mice was 28 and 4%, respectively. The incidence of carcinoma was not increased by PB treatment in either strain. It is concluded that both strains of mice behave in a qualitively similar way to PB administration, although they show considerable quantitative differences in terms of the time and number of nodules that develop. Furthermore, the increased nodule numbers associated with PB treatment were not accompanied by an increase in the number of carcinomas.

Male C3H/He mice were given 0 (control) or 85 mg/kg/day phenobarbitone (PB) in the diet. At 40, 60 and 93 weeks, groups of mice were killed and the ultrastructure of spontaneous and PB-induced liver nodules was examined. Treated mice showed typical centrilobular hypertrophy and eosinophilic nodules which may be considered as an end stage lesion. The nodule cells were similar in appearance to those in areas of centrilobular hypertrophy except for the presence of convoluted membranes which are considered to be indicative of proliferation. The incidence of carcinoma was not increased by PB treatment. The carcinomas from control and treated animals differed in their ultrastructure in that increased levels of smooth endoplasmic reticulum (SER) were seen in the carcinomas of the PB animals. The presence of SER proliferation in the carcinomas of PB animals suggests that carcinoma may respond to the enzyme-inducing effects of PB.

The surface membrane glycoprotein patterns of spontaneous hepatic nodules, phenobarbitone induced nodules and hepatocellular carcinoma were studied in the C3H mouse using lectin histochemistry. The lectin binding patterns of hepatocellular carcinoma cells were markedly different to those of non-tumour cells and similar to the pattern in chemically induced hepatocellular carcinomas. This supports the hypothesis that changes in surface glycoprotein are a consistent feature associated with malignancy. Similar changes in the distribution of lectin binding sites were also seen in the phenobarbitone induced eosinophilic nodules and in a proportion of spontaneous basophilic nodules. Two populations of early basophilic nodules were identified on the basis of their lectin binding patterns, and this may indicate a link between one nodular type and carcinoma.

Alterations in the liver of rats 6 h after a dose of phenobarbitone have been studied by subcellular fractionation, conventional electron microscopy and morphometric analysis. The area immediately surrounding the central vein was the only area to undergo any alterations. There was a morphometrically measurable but not observable cellular hypertrophy of 71% whilst the hepatocyte complement of rough endoplasmic reticulum (RER) and smooth endoplasmic reticulum (SER) was increased by 72% and 93% respectively. The increases in RER and SER were not apparent by observation and it is assumed that they have been diluted by the cell hypertrophy to 1% and 22% which must be below the threshold for detection by subjective observation. Following subcellular fractionation and measurement of microsomal protein, there was no significant difference in the level of microsomes isolated from control or treated rats. Therefore, the morphometrically measured increase in RER and SER would appear to be restricted to a relatively small population of hepatocytes adjacent to the central vein. Such an increase would represent only a small percentage of total microsomes in a homogenate and would almost certainly be masked by variation in animals and techniques. Disruption of RER was also observed in hepatocytes that would proliferate their SER should phenobarbitone treatment have been continued. Therefore this RER disruption would seem in no way to interfere with the process of membrane and enzyme synthesis.

In phenobarbitone-treated starved male rats 1 g/kg 3-amino-1,2,4-triazole produced moderate liver necorsis and increased the serum glutamic-pyruvic transaminase activity. If half an hour after the administration of aminotriazole animals were exposed for 4 h to 2.0 mg/l CS2, the necrotic damage in the liver was larger and the serum glutamic-pyruvic transaminase activity higher than in rats not exposed to CS2. Carbon-disulphide in phenobarbitone-treated starved male rats caused only a very slight increase in the serum transminase activity in spite of the widespread hydropic degeneration in the liver. These experiments indicated that increase in serum transaminase activity is the consequence of necrosis and not hydropic degeneration; aminotriazole is hepatotoxic in rats when microsomal enzymes are induced and the hepatotoxicity of aminotriazole and carbon disulphide is potentiated by the administration of the other compound.

Male inbred Fischer rats were fed phenobarbitone-Na at a level of 500 parts/10(6) in the diet for 1 week, followed by 1000 parts 10(6) for 103 weeks at which time the survivors were killed. Thirty-three treated rats survived to 80 weeks. Before 80 weeks, no animals showed hyperplastic lesions. Of the 33 rats surviving 80 weeks and more, 11 had foci of nodular hyperplasia. These foci were usually small, but one animal killed at 102 weeks had a lesion of 0.75 cm diameter, which compressed the surrounding liver. In one case was evidence of local invasion or metastasis found. All the livers had evidence of parenchymal cell damage. No evidence of nodular hyperplasia was found in the controls. It is concluded that there is no evidence to suggest that phenobarbitone-Na induced neoplasm in the liver of male Fischer rats.

Male C3H/He mice were given 0 (control) or 85 mg/kg/day phenobarbitone (PB). Groups of five control and five treated animals were killed at 5, 10, 25, 40 and 55 weeks; additional animals were allowed to live out their natural life span or killed at the termination of the experiment at 100 weeks. Small nodules of basophilic cells were seen in control animals at 55 weeks. These increased both in size and number so that at term nodules were found in 17/35 animals examined. Animals given PB showed typical centrilobular hypertrophy. Basophilic nodules were also seen in the treated animals and these were similar in form and in number to those seen in control animals. The total number of liver nodules seen in animals given PB was, however, greater than the number seen in control animals. The increased nodule burden was a result of the development of a second nodule type that was first seen in animals killed at 40 weeks. These nodules were formed of large eosinophilic cells that had a similar appearance to the hypertrophied cells of the centrilobular region. In contrast the incidence of carcinoma in PB-treated animals was not increased over that of the control group.

Alterations in the liver of rats subjected to 24 days of continuous administration of phenobarbitone have been supplied bu subcellular fractionation, conventional electron microscopy and morphometric analysis. The increase in wet weight of the liver was found to result from a combination of cellular hypertrophy, hyperplasia and an enlarged hepatic blood space. In the centrilobular zone all the hepatocytes underwent a substantial proliferation of total ER, became enlarged and had an increased blood supply. However, in the periportal zone phenobarbitone caused changes in only 45% of the hepatocytes, the remainder being apparently resistent or tardy. An overall dramatic increase in hepatic RER was both measured and observed but the response involved hepatocytes in which the RER had proliferated as well as those which were depleted of RER or had stacks and cisternae that were severely shortened and dispersed. These alterations are discussed in relation to changes in RER after administration of agents causing hepatonecrosis. Possible reasons for the inability of other workers to detect a phenobarbitone-induced increase in RER are also put forward. After subcellular fractionation and corection for centrifugation losses into the 9500 g pellet, using the microsomal marker cytochrome P-450, phenobarbitone-induced increase in total ER was substantially less than that found by morphometric analysis. This indicates that during the preparation of microsomes a substantial proportion of intracellular membranes, having different metabolic and synthetic properties to those finally isolated, are discarded and emphasizes the need to exercise care when using microsomal preparations.