Elizabeth K. Weisburger

Chemistry, carcinogenicity, and metabolism of 2-fluorenamine and related compounds.

Publisher Summary This chapter describes the chemistry, carcinogenicity, and metabolism of 2-flurorenamine and related compounds. It discusses the synthesis and chemistry of fluorenamine compounds and their derivatives, which gives evidence of the success achieved in furnishing materials for the investigation of the relationship of structure to carcinogenicity and of the metabolic fate of this class of compounds. Of the isomeric fluorenylacetamides, only N -2-fluorenylacetamide and some of its derivatives can be classed as active carcinogens. Carcinogenesis by N -2-fluorenylacetamide is influenced by some specific dietary factors. A high protein diet delays carcinogenesis and reduces the incidence of tumors at a number of sites. In some cases, a caloric restriction may decrease the carcinogenic effect. In addition, the fluorenamine carcinogens affect a number of body constituents, often reflecting a quickened metabolic turnover of these components. Thus, the half-life of riboflavin in the rat is decreased 6-fold. Numerous studies on the carcinogenic effect of 2-fluorenamine and related compounds under varying conditions of species, strain, diet, and hormonal environment have defined some of the limits within which the carcinogenic effect can be demonstrated.

Bioassay program for carcinogenic hazards of cancer chemotherapeutic agents.

Various compounds and combinations thereof, of promise in the treatment of neoplastic diseases, were tested for possible carcinogenicity in Swiss mice and Sprague‐Dawley rats. Some of the compounds were fairly active carcinogens, while others showed little or no evidence of carcinogenicity. The carcinogenicity of the drug combinations was generally less than that of the individual components.

The carcinogenic effect of 4,4′-methylene-bis-(2-chloroaniline) in mice and rats

Abstract As part of a series of bioassays of industrial and environmental chemicals for chronic toxicity or carcinogenicity, 4,4′-methylene-bis-(2-chloroaniline) was tested in male and female randombred albino mice and male Charles River CD rats. Preliminary studies established that the maximally tolerated dose of the chemical in the diet was 1000 mg/kg in rats and 2000 mg/kg in mice. The compound, at these dose levels and half the level, was fed to male and female mice, and male rats for 18 months, followed by control diet for another 6 months. In female mice but not in males, a statistically significant incidence of hepatoma at both dose levels was observed. In addition, a higher incidence of tumors was observed in treated animals than in controls as follows: hepatomas and lung adenomatosis in rats, hemangiosarcomas and hemangiomas in mice. Thus, 4,4′-methylene-bis-(2-chloroaniline) is definitely carcinogenic in female mice.

Metabolism of the carcinogen N-hydroxy-N-2-fluorenylacetamide in germ-free rats.

Abstract The question whether the carcinogen N-hydroxy-N-2-fluorenylacetamide (N-OH-FAA) was affected by the microbial flora in the intestinal tract was investigated by comparing the metabolism of this compound in germ-free and conventional Fischer strain rats, employing isotopic techniques. Although the amounts of total urinary glucosiduronic acids and sulfuric acid esters were similar in axenic and control rats after an i.p. dose of N-OH-FAA, there were important differences among the individual glucosiduronic acids. Germ-free rats excreted considerably larger amounts of the glucuronide of N-OH-FAA and appreciably less of the conjugates from the ring-hydroxylated metabolites. Furthermore, the cecal and fecal metabolites in the conventional rats were mostly free, unconjugated materials, wherease in axenic rats the major fraction was conjugated with sulfuric and glucuronic acid. Injection of N-OH-FAA into the cecum, or intraluminal administration of glucosiduronic acids of N-OH-FAA or of phenolphthalein showed that these materials could be absorbed readily from the gastrointestinal tract. In the cecum of germ-free rats, β-glucuronidase activity was low and had an optimum at a pH characteristic of mammalian enzyme, but in conventional rats it was higher and with a pH optimum more like that of the bacterial enzyme. The data indicate that N-OH-FAA is metabolized differently in germ-free rats: (1) because they lack the bacterial flora of the intestinal tract in conventional rats, which can hydrolyze glucosiduronic acids excreted into the gut by bile; and (2) because N-OH-FAA is a substrate for a bacterial N-dehydroxylase, also absent in axenic rats. In part, the enterohepatic cycle undergone by N-OH-FAA may be related to the liberation in the gut of readily absorbed metabolites, which are then further modified and excreted in urine or in bile.

Ratliver cells in culture: Effect of storage, long-term culture, and transformation on some enzyme levels

SummaryAryl hydrocarbon hydroxylase (AHH) and tyrosine aminotransferase (TAT) activities were determined in rat liver cell lines after frozen storage, long-term culture, and transformation in vitro. Levels of AHH activity after 17 months in frozen storage were comparable to levels prior to freezing. During long-term culture the AHH levels of the cell lines tended to decrease. Transformed lines had variable levels of AHH activity. Cell lines retained measurable TAT activity following long-term culture and frozen storage. TAT activity of transformed cells was comparable to that of normal lines. Prolonged frozen storage did not induce transformation up to one year.

Participation of liver fractions and of intestinal bacteria in the metabolism of N-hydroxy-N-2-fluorenylacetamide in the rat

Abstract The level of the active carcinogenic intermediate, N -hydroxy- N -2-fluorenylacetamide (N-OH-FAA) in rats is controlled in part by enzymic reduction to N -2-fluorenylacetamide (FAA). These reactions are performed not only by enzymes in liver but also by systems from the microbial flora in the gut. N-OH-FAA was secreted via the bile into the intestinal tract as a glucuronic acid conjugate. Cecum contents contained β-glucuronidase of a bacterial type with maximal activity at pH 6. Lower glucuronidase activity was present in the cecum of germ-free rats with an optimum at pH s, typical of mammalian β-glucuronidase. Rat cecal contents readily hydrolyzed glucosiduronic acids and reduced N-OH-FAA to FAA. The N -dehydroxylation was faster after preincubation of an extract of cecal contents with N-OH-FAA. Strain K 32 of Escherichia coli contained the enzyme required for this reduction, The microflora in the gut, in particular coliform organisms, was inhibited by N-OH-FAA. N -dehydroxylase activity in liver was chiefly in the soluble fraction, although a small amount was also associated with the microsome fraction. On chronic feeding of 160 ppm of N-OH-FAA for 8 weeks, there was a progressive shift in microbial flora in the gut with a reduction in coliform type organisms and an increase in yeast cells. As a result there was a lowering in cecal β-glucuronidase and in N -dehydroxylase. Soluble liver N -dehydroxylase increased slightly at the 4-week point, then decreased. During the same period, urinary excretion of the glucuronide of N-OH-FAA increased from 1.8 per cent of the dose to a peak of 19.4 per cent after 4 weeks. In part, the alterations in intestinal microflora with consequent shifts in enzyme content may account for the increased excretion of this metabolite of N-OH-FAA. Other factors play a role, since alteration of the microbial flora in the cecum by administration of Neomycin failed to yield a proportional increase in the excretion of the glucuronide of N-OH-FAA. A new analytical method to determine the substrate N-OH-FAA was devised. Oxidation of solutions of N-OH-FAA by potassium permanganate was followed by the colorimetric assay of the intermediate product with trisodium pentacyanamino ferrate at 560 nm. Concentrations of 5–100 μg/ml of N-OH-FAA could thus be determined readily.

Phenobarbital-mediated increase in ring- and N-hydroxylation of the carcinogen N-2-fluorenylacetamide, and decrease in amounts bound to liver deoxyribonucleic acid

Abstract The effect of phenobarbital (PB) pretreatment of young male rats on the metabolism in vivo and in vitro of the carcinogen N -2-fluorenylacetamide (FAA) was studied. PB increased the urinary excretion of 14 C from labeled FAA, mainly as met-abolites conjugated with glucuronic acid. There was a small drop in excretion of sulfuric acid conjugates. In the glucosiduronic acid fraction, there were increases in the N - and 7-hydroxy derivatives of FAA and a decrease in the 5-hydroxy compound, while the 3-hydroxy metabolite was virtually unchanged. The concentration of metabolites of FAA in the liver was considerably lower, as was the amount of isotope bound to DNA. This important finding, correlated with a reduced carcinogenicity of FAA in rats given PB, is ascribed to increased conjugation with glucuronic acid and lesser formation of sulfate esters. The microsomal fraction in vitro from the livers of young rats yielded metabolites of FAA, in decreasing order, hydroxylated at 7-, 5-, N - and 3-. Carbon monoxide significantly inhibited formation of the 7-hydroxy metabolite but elevated somewhat that of the N -hydroxy compound. Pretreatment with PB increased cytochrome P-450 and hydroxylation at all positions 2- to 4-fold, the greatest effect being with N -hydroxy-FAA. With microsomes from PB-treated rat livers, carbon monoxide depressed hydroxylation at the 3-position, while N -hydroxylation was least affected. Because hydroxylation of FAA occurs at several well-defined ring positions and on the amido nitrogen, FAA is a good substrate to explore the mechanisms of hydroxylation reactions. The data obtained suggest that these metabolic reactions are performed by a family of related enzyme systems.

Part V. Pharmacodynamics of carcinogenic azo dyes, aromatic amines, and nitrosamines

In this review we touch upon factors affecting the carcinogenicity of three broad classes of chemicals: the carcinogenic azo dyes, the aromatic amines, and the alkyl nitrosamines. Present knowledge of their mode of action indicates that they share some points in common but differ in other respects.

Dehydroxylation and deacetylation of N-hydroxy-N-2-fluorenylacetamide by rat liver and brain homogenates

Abstract Two reactions, dehydroxylation and deacetylation of the proximate carcinogen N-hydroxy-N-2-fluorenylacetamide were studied with liver and brain homogenates, or with a soluble fraction from liver of young or adult male or female rats of two strains. Incubation of N-hydroxy-N-2-[9-14C]fluorenylacetamide with tissue homogenate in 0.2 M Tris buffer (pH 8.1) gave progressively decreasing levels of substrate. The products, N-2-fluorenylacetamide and 2-fluorenamine, were resolved and identified by solvent partition, paper chromatography, ultraviolet spectroscopy, and color tests. Liver homogenate from 2 rat strains was most active, the soluble liver fraction slightly less so, and brain homogenate least. Deacetylation was but dehyroxylation was not inhibited by 0.1 M fluoride. Both reactions proceeded equally well in nitrogen or in air. The dehydroxylase was inactive after 1 h at 37°. The decaylase was somewhat more stable. Linear increases in protein-binding of 14C from the substrate were found. The final level was lower in liver from adult female rats than from males, but was similar with weanling rats. Binding was inhibited somewhat by fluoride and more so by a nitrogen atmosphere. Some of the binding may have been due to the oxidation of amine or hydroxylamine in the presence of tissue. Isotope from synthetic N-2-[ 14 C] fluorenylhydroxylamine and 2-nitroso[9−14C]fluorene bound to serum proteins, liver homogenate or soluble liver fractions, hemoglobin, albumin, γ-globulin, or histones. 2-Nitrosofluorene reacted similarly in air and in nitrogen. It may be the active intermediate.

The mouse liver tumor as an endpoint in carcinogenesis tests.

Abstract Available carcinogenesis and mutagenesis information was reviewed for 85 chemicals that were demonstrated to increase significantly the incidence of liver tumors in mice. Sixty-one chemicals (73%) induced tumors in other tissues of mice and/or rats. For 45 of the 61 chemicals for which bacterial mutagenesis information was available, 65% were mutagenic. Of 24 chemicals inducing only mouse liver tumors, 5 of 14 tested (35%) were mutagenic. Of all 59 chemicals causing liver tumors in mice for which mutagenesis information is available, 58% were mutagenic for bacteria, primarily Salmonella typhimurium . Several classes of chemicals were represented among those inducing only mouse liver tumors or mouse liver tumors and other tumors. The findings provide evidence that the mouse liver tumor is an important endpoint in carcinogenesis tests. Limited information on the subject also suggests that certain classes of chemicals may not be mutagenic and induce only mouse liver tumors. More importantly, several nonmutagenic mouse hepatocarcinogens also induce other tumors in mice or rats. The interpretation and significance of these findings awaits further research into the mechanisms of carcinogenesis.