Michael A. Collins

The Histology and Development of Hepatic Nodules and Carcinoma in C3H/He and C57BL/6 Mice Following Chronic Phenobarbitone Administration

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.

Studies on the metabolism of dimethylnitrosamine in vitro by rat-liver preparations.: I. Comparison with mixed-function oxidase enzymes

1. The metabolism of dimethylnitrosamine (DMN) to formaldehyde by rat-liver preparations has been studied at substrate concn. of 0.5, 5 and 50 mM and compared with mixed-function oxidase enzyme activities. 2. The microsomal metabolism of low (0.5 and 5 mM) and high (50 mM) substrate concn. of DMN was differentially affected by acetone addition or KI treatment. 3. A series of heterocyclic compounds related to pyrazole were potent inhibitors of metabolism of 0.5 and 5 mM DMN at concn. which had little effect on mixed-function oxidase activities. In contrast, purine addition slightly stimulated the metabolism of low but not high concn. of DMN. 4. The results are consistent with the suggestion that multiple enzymic pathway(s) are involved in hepatic DMN metabolism and that some of these pathway(s) may be independent of cytochrome P-450.

Studies on the metabolism of dimethylnitrosamine in vitro by rat-liver preparations.: II. Inhibition by substrates and inhibitors of monoamine oxidase

1. The metabolism of dimethylnitrosamine (DMN) to formaldehyde by rat-hepatic postmitochondrial supernatant fractions has been compared with the activities of several cytochrome P-450-dependent mixed-function oxidase enzymes and the Ziegler mixed-function amine oxidase enzyme (EC 1.14.13.8). 2. A variety of monoamine oxidase (MAO, EC 1.4.3.4) inhibitors of diverse chemical structure inhibited the metabolism of DMN. In parallel studies a number of MAO substrates, but not their deaminated products, also inhibited DMN metabolism, whereas substrates of diamine oxidase were ineffective. 3. At concentrations which inhibited DMN metabolism several MAO substrates and inhibitors did not inhibit the N-oxidation of N, N-dimethylaniline and an inhibitor and an activator of the Ziegler enzyme had no corresponding effect on DMN metabolism. 4. The metabolism of DMN and a number of MAO enzyme activities were stable to storage under conditions where mixed-function oxidase enzymes were not. 5. These results are consistent with the suggestion that DMN may, at least in part, be metabolized by hepatic enzyme(s) not dependent on cytochrome P-450 and that a microsomal amine oxidase enzyme, unrelated to the Ziegler enzyme, may be involved in the hepatic degradation of this nitrosamine. The present data does, however, suggest a role for microsomal NADPH-cytochrome c reductase in hepatic DMN metabolism.

Sustained induction of hepatic xenobiotic metabolising enzyme activities by phenobarbitone in C3H/He mice: Relevance to nodule formation

Phenobarbitone (PB) was administered to male C3H/He mice at a dose of 85 mg/kg/day in a semisynthetic diet for up to 90 weeks. Throughout the treatment period a sustained induction of a number of parameters of hepatic Phase I and Phase II xenobiotic metabolism was observed. Histological examination revealed hypertrophy of the centrilobular cells of the liver lobule in PB treated mice and after 25 weeks small basophilic nodules were found in control and PB treated animals. In addition eosinophilic nodules, which were often large, developed in PB treated mice. Xenobiotic metabolising enzyme activities in large excised nodules after 70 or 90 weeks of PB treatment were either similar to or greater than those present in surrounding host tissue. Both phenobarbitone- and polycyclic hydrocarbon-type mixed function oxidase enzyme activities were induced in large nodules. In conclusion, PB produced a sustained induction of xenobiotic metabolising enzymes both in host tissue and in large eosinophilic nodules. The formation of these nodules in C3H/He mice was thus not associated with any failure of induction of hepatic xenobiotic metabolism.

Studies on the metabolism of dimethylnitrosamine in vitro by rat-liver preparations. III. Effect of cobaltous chloride treatment.

1. The effects of CoCl2 administration to rats on xenobiotic metabolism, dimethylnitrosamine (DMN) metabolism to formaldehyde and methanol, and monoamine oxidase (MAO) enzyme activities in hepatic subcellular fractions have been studied. 2. CoCl2 treatment markedly decreased hepatic mixed-function oxidase enzyme activities and microsomal cytochrome P-450 content. In contrast, the N-oxidation of N, N-dimethylaniline and the activity of microsomal NADPH-cytochrome c reductase was unaffected. 3. The metabolism of DMN to formaldehyde by postmitochondrial supernatant fractions was decreased at substrate concn. of 0 . 5, 5 and 50 mM by CoCl2 treatment but the metabolism of 5 and 50 mM DMN to methanol was affected less. 4. CoCl2 had little effect on MAO activities in whole homogenates, but microsomal MAO activities were markedly inhibited. 5. The inhibition of microsomal MAO indicates that CoCl2 is not a specific inhibitor of cytochrome P-450-dependent biotransformations and consequently the inhibition of DMN metabolism is not evidence of a wholly cytochrome P-450-dependent process.

The effect of treatment with some phase II substrates on hepatic xenobiotic metabolism and the urinary excretion of metabolites of the d-glucuronic acid pathway in the rat

Abstract Investigations have been conducted to study the relationship between the activities of certain enzymes of Phase I and Phase II hepatic xenobiotic metabolism and the urinary excretion of some metabolites of the d -glucuronic acid pathway. Male Sprague-Dawley rats were treated for 7 days with daily ip injections of either diphenylacetic acid, guaiacol, 4-hydroxybiphenyl, 2-naphthol, 1-naphthylacetic acid, paracetamol (acetaminophen), 2-phenylpropionic acid, n -propyl gallate, or phenylacetic acid. None of the compounds administered had any effect on a number of parameters of hepatic Phase I xenobiotic metabolism, namely mixed function oxidase enzyme activities, cytochrome P -450 and the microsomal content of protein. However, some of the compounds did stimulate hepatic microsomal UDP-glucuronyltransferase activity. While none of the compounds stimulated the urinary excretion of either d -glucaric acid, l -gulonic acid, or xylitol, all of the compounds except phenylacetic acid increased the urinary excretion of total (free and conjugated) d -glucuronic acid. The results indicate that when hepatic xenobiotic conjugative activity is increased without the stimulation of mixed function oxidases and cytochrome P -450, the urinary excretion of certain metabolites of the d -glucuronic acid pathway, other than d -glucuronic acid, remains unchanged.