Kinetics of excretion of 2-acetylaminofluorene in normal and xenobiotic-treated rats and in rats with hepatocyte nodules.

This study was designed to explore further the hypothesis that the special resistance phenotype seen in hepatocyte nodules during liver carcinogenesis could have a physiologic correlate in the manner with which a carcinogenic xenobiotic is handled. Hepatocyte nodules were induced in male rats by continuous or intermittent exposure to dietary 2-acetylaminofluorene over a 25-week period. Two or 5 weeks after the exposure, the animals were given a single dose of 9-14C-2-acetylaminofluorene. The amounts and rates of excretion of unconjugated compound and derivatives and of the glucuronic acid metabolites in the bile and urine and the amounts in the blood and liver were measured over a period of 180 minutes. For comparison, animals fed the basal diet alone, animals injected with phenobarbital or 3-methylcholanthrene, animals receiving a single dose of cobalt heme and animals fed the 2-acetylaminofluorene for only 2 weeks were studied. These groups were used as controls for different patterns of drug metabolism, especially relating to the cytochromes P-450. The nodule-bearing animals showed a pattern of handling of the carcinogen that is quite different than that of the animals of any other group. They excreted in the bile plus urine from 20 to 30% less. However, relatively much more was in the urine. The free and glucuronide-conjugated metabolic products of the carcinogen were assessed by high performance liquid chromatography. The nodule-bearing animals and the animals treated with 3-methylcholanthrene excreted much more glucuronic acid esters. The pattern of distribution of labeled 2-acetylaminofluorene is different in the nodule-bearing rats than in other animals in which variations in phase I and phase II drug-metabolizing enzymes were induced by treatment with cobalt heme, phenobarbital, 3-methylcholanthrene or short-term exposure to dietary 2-acetylaminofluorene.

The use of rat urine extracted on XAD-2 resin and assayed in a microsuspension-modified Ames test as an in vivo indicator of genotoxic exposure.

The measurement of urinary mutagenicity is a non-invasive monitoring tool which often reflects an animal's recent exposure to genotoxic agents. Although studies in man are indispensable for monitoring industrial and/or environmental exposure to genotoxins, a sensitive laboratory animal model is necessary for mechanistic studies on the role of specific chemical exposure in altering urinary mutagenicity. The objective of this study was to enhance the sensitivity of the methodology used for detecting urinary mutagenicity in rats by using XAD-2 resin to extract and concentrate the urine and a microsuspension-modified Ames test to quantify mutagenicity. The polycyclic aromatic hydrocarbon benzo[a]pyrene (BP) and the aromatic amine 2-acetylaminofluorene (AAF) were used as test compounds. Under the conditions of our study, AAF administered to rats by gavage at doses of 1 mg/kg or higher induced a dose-dependent increase in urine mutagenicity. The greatest mutagenic response was seen when S9 was present during the microsuspension-modified Ames test and beta-glucuronidase (BG) was not included. Similarly, BP administered to rats by gavage at doses of 10 mg/kg or higher induced a dose-dependent increase in urinary mutagenicity. The relative importance of BG and S9 were quite different with BP than with AAF. With BP, mutagenicity was greatest when both S9 and BG were present during the microsuspension-modified Ames test, and least with S9 and without BG. In both AAF- and BP-treated animals, extraction of the urine on XAD-2 resin markedly enhanced the mutagenic response compared to neat urine, but partitioning of the XAD-2 eluate into methylene chloride always diminished the mutagenicity of the urine extract. The results demonstrate the sensitivity and reproducibility of rodent urinary mutagenicity assays when XAD-2 resin is used to extract and concentrate the urine and a microsuspension-modified Ames test is used to quantify mutagenicity. This sensitive method should facilitate mechanistic studies on the roles of specific environmental agents in affecting urinary mutagenicity and, in addition, may be used during acute, subchronic and chronic rodent bioassays as a non-invasive in vivo indicator of genotoxic exposure.

Metabolite profile in milk of lactating rats after treatment with a carcinogen, N-2-fluorenylacetamide.

Metabolites were determined in milk and urine of lactating rats 4 to 5 hr after each of 3 to 6 ip injections of N-2-fluorenylacetamide (2-FAA) at 0.2 mmol/kg of body weight. Milk contained 2-FAA as the major free compound, variable amounts of N-2-fluorenamine (2-FA) and phenols (7- greater than 5- greater than 3-hydroxy-2-FAA) chiefly as glucuronides, and very small amounts of the glucuronide of N-hydroxy-2-FAA. Urine contained large amounts of the phenols and N-hydroxy-2-FAA as free and conjugated compounds, but in contrast to milk, only small amounts of 2-FAA and no 2-FA. Pretreatment of rats with beta-napthoflavone, an inducer of microsomal C- and N-hydroxylations of 2-FAA, increased the amounts of 3- and 5-hydroxy-2-FAA in milk and of 3-hydroxy-2-FAA in urine. However, the total amounts of the compounds excreted in 1 ml of milk or urine, i.e. 0.05 to 0.13% or 0.6% of the dose of 2-FAA, respectively, were similar in the uninduced and induced groups. Protein hydrolysates of milk of 2-FAA-treated rats and of milk interacted with 2-nitrosofluorene (2-NOF) in vitro both contained 2-FA and 9-oxo-2-FA. This suggested formation of 2-NOF in vivo possibly by peroxidative metabolism of N-OH-2-FAA. Since 2-NOF has been reported to form adducts with unsaturated lipids, the effect of treatment of lactating rats with 2-FAA on the fatty acid composition of milk lipids was examined.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection of urinary metabolites of 2-acetylaminofluorene by cultured hamster embryo cells and high performance liquid chromatography.

Urine was collected from Sprague-Dawley rats injected intraperitoneally with a suspension of 2-acetylaminofluorene (AAF) or with an inert suspension vehicle. Morphological transformation was observed in clonal cultures of secondary passage hamster embryo cells (HEC) after the addition of AAF-treated rat urine or concentrated urine extracts prepared by incubating the urine with beta-glucuronidase. No morphological alteration was observed in cultures treated with urine (or urine extracts) from rats injected with the suspension vehicle alone. The presence of N-hydroxy- and ring-hydroxylated metabolites of AAF in the AAF-treated concentrated urine extract was confirmed by high-performance liquid chromatography (HPLC).

The effects of a marginally lipotrope-deficient diet on the hepatic levels of S-adenosylmethionine and on the urinary metabolites of 2-acetylaminofluorene in rats.

Hepatic levels of S-adenosylmethionine (AdoMet), of glutathione, and of the microsomal enzymes p-nitroanisole demethylase and benzo(a)pyrene hydroxylase were measured in male and female rats fed a diet marginally deficient in choline and methionine and void of folic acid (lipotrope deficient) or an adequate diet for 0 to 14 weeks with and without added 2-acetylaminofluorene (AAF). The urinary metabolites of AAF were determined throughout the experimental period. After 2 to 4 weeks of dietary administration, the hepatic AdoMet levels were 43% lower in male rats fed the lipotrope-deficient diet than in male rats fed the lipotrope-adequate diet; no differences were found in hepatic AdoMet of females fed the lipotrope-deficient or lipotrope-adequate diets for 2 to 14 weeks. Administration of AAF to lipotrope-deficient female rats for 2 weeks led to a transient decrease in hepatic levels of AdoMet. The administration of AAF for 2 to 14 weeks did not significantly affect hepatic AdoMet in female rats fed the lipotrope-adequate diet or in male rats fed either diet. Female rats fed the lipotrope-deficient diet and treated with AAF excreted decreased proportions of N-hydroxy-2-acetylaminofluorene and increased proportions of 5-hydroxy-2-acetylaminofluorene in their urine. However, the urine of lipotrope-deficient male rats treated with AAF contained increased proportions of N-hydroxy-2-acetylaminofluorene and decreased levels of 5-hydroxy-2-acetylaminofluorene. The urinary excretion of 7-hydroxy-2-acetylaminofluorene by male and female lipotrope-deficient rats treated with AAF was generally similar to that in lipotrope-adequate rats. The lipotrope-deficient diet did not appear to alter the hepatic levels of glutathione, p-nitroanisole demethylase, or benzo(a)pyrene hydroxylase activity was lower in the livers of lipotrope-deficient male rats treated with AAF for 8 to 14 weeks than in the livers of lipotrope-deficient rats not receiving the carcinogen. The altered metabolism of AAF correlated well with the previously reported effects of a marginal lipotrope deficiency on AAF carcinogenesis.

Radioimmunoassays for monitoring exposure to potential carcinogens.

As the numbers and production of environmental pollutants and hazardous industrial compounds increase, the necessity for monitoring the environment and workplace to avoid or to limit human exposure to toxic substances becomes more urgent. In support of the occupational safety program at the National Center for Toxicological Research, very sensitive and specific radioimmunoassays for measuring two known animal carcinogens, 2-acetylaminofluorene and diethylstilbestrol and their metabolites were developed. Radioimmunoassay (RIA) techniques offer unique advantages over ixisting methodologies in the field of toxicology; these include exquisite specificity and sensitivity, simplicity in sample handling and rapid analysis. These RIAs are currently being used for monitoring possible human exposure to carcinogenic compounds.

Effect of pair-feeding of carcinogen on the incidence of bladder tumors in hamsters. Role of indole, age, and sex.

The role of indole in 2-acetylaminofluorene (AAF) bladder tumorigenesis was critically evaluated in hamsters. To avoid the influence of a nutritional factor on the tumor incidence, the animals were pair-fed for 8, 10, and 12 months. Addition of indole to the AAF diet resulted in a higher incidence of bladder tumors in both males and females (P less than 0.05, P less than 0.05). Indole definitely increased the bladder tumor incidence at 8 months (P less than 0.05). Also significantly different was the tumor incidence between males and females irrespective of the diet administered (P less than 0.05). Indole did not alter the urinary output of N-hydroxy-2-acetylaminofluorene (N-OH-AAF) in males.

Mechanisms of the inhibitory action of p-hydroxyacetanilide on carcinogenesis by N-2-fluorenylacetamide or N-hydroxy-N-2-fluorenylacetamide.

The metabolism of N-2-fluorenylacetamide (FAA) and N-hydroxy-2-fluorenylacetamide (N-OH-FAA) was studied in groups of rats that had been prefed the protective agent p-hydroxyacetanilide (p-OH-AA) alone or in combination with each of the carcinogens for 4 weeks. Compared with controls, pretreatment increased the percentage of metabolites in the urine, chiefly as glucuronic acid conjugates, whereas the fecal excretion of FAA metabolites was lowered. The levels of total and tissue-bound material in the liver and blood plasma were also lower after prefeeding. Liver aryl hydrocarbon hydroxylase and liver deacetylase were not affected by p-OH-AA pretreatment. However, liver glucuronyl transferase was increased by either prefeeding with p-OH-AA and/or the carcinogen. The protective effect of p-OH-AA against liver tumor induction with FAA or N-OH-FAA may in part result from a combination of the decreased binding of carcinogen to hepatic cellular macromolecules and the increased excretion as the glucuronide conjugates.

Metabolism of N-2-fluorenylacetamide in X/Gf mice: lack of correlation between biochemical interaction and carcinogenicity.

A comparative study of the metabolism of the carcinogen N-2-fluorenylacetamide by mice of the X/Gf strain (resistant to the tumorigenic action) and the NiH Swiss strain (susceptible to the tumorigenic action) showed slight but not outstanding differences in metabolic patterns. The activated metabolite N-hydroxy-N-2-fluorenylacetamide was excreted by both strains, and the levels of carcinogens or metabolites bound to liver macromolecular constituents were comparable in the X/Gf and NiH Swiss mice.