John H. Weisburger

Isolation and long-term cell culture of epithelial-like cells from rat liver.

Abstract Epithelial-like cells from livers of 10-day-old rats were separated from fibroblast-like cells on the basis of differential attachment to a culture surface. After further elimination of persisting fibroblast-like cells from the cultures of epithelial-like cells, lines of the latter cell type were initiated and these maintained their morphology for up to nine months of continuous culture and displayed no tumorigenicity upon injection into syngeneic hosts.

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.

Antioxidants and carcinogenesis: butylated hydroxytoluene, but not diphenyl-p-phenylenediamine, inhibits cancer induction by N-2-fluorenylacetamide and by N-hydroxy-N-2-fluorenylacetamide in rats.

Abstract The effect of butylated hydroxytoluene (BHT) and diphenyl-p-phenylenediamine (DPPD) on cancer induction by N-2-fluorenylacetamide (FAA) and its N-hydroxy derivative (N-OH-FAA) has been studied in two strains of rat. Groups of Charles River rats were fed carcinogen and antioxidant in a molar ratio of 1:30 (223 ppm FAA or 239 ppm N-OH-FAA (1 mM) and 7850 ppm DPPD or 6600 ppm BHT) in Wayne Chow for 24 wk (males) or 32 wk (females). Male and female rats then continued on control diet for another 12 wk. With FAA alone 70% of male rats had hepatoma and 20% of females had mammary adenocarcinoma; with N-OH-FAA, 60% of males had hepatoma and 70% of females had mammary adenocarcinoma. The simultaneous administration of DPPD failed to alter cancer induction by these agents in male or female rats. However, BHT reduced the incidence of hepatoma in males to 20% when the carcinogen was FAA. With N-OH-FAA, BHT lowered the incidence of liver tumours to 15% and of mammary cancer in females to 35%. Similar data on inhibition by BHT were obtained using different levels of carcinogens in rats of the Fischer strain. Equimolar levels of sulphate failed to increase liver-tumour incidence in animals given N-OH-FAA and BHT. Also, BHT failed to depress the excretion of free urinary inorganic sulphate. Liver- and oesophageal-tumour induction with 51 ppm diethylnitrosamine in drinking water for 24 wk was not affected by BHT or DPPD, nor was tumour induction by propane sultone.

Effect of the antioxidant butylated hydroxytoluene (BHT) on the metabolism of the carcinogens N-2-fluorenylacetamide and N-hydroxy-N-2-fluorenylacetamide

Abstract The effect of the antioxidant butylated hydroxytoluene (BHT) on the metabolism of N-2-fluorenylacetamide (FAA) and N-hydroxy-N-2-fluorenylacetamide (N-OH-FAA) was studied in male and female rats. The administration of 6600 ppm BHT in the diet for 4 wk increased the liver-to-body weight ratio and led to the excretion in the urine of a larger percentage of a single ip dose of either carcinogen. This higher level of excretion was accounted for chiefly by glucuronic acid conjugates. Sulphuric acid esters were decreased. Also lower were the levels of radioactivity in blood, in liver and bound to liver DNA 48 hr after injection of the labelled carcinogen. It is concluded that BHT increases the detoxification metabolites of FAA and N-OH-FAA, and thus lowers the amount of a given dose available for activation reactions.

Enzymic reduction of carcinogenic aromatic nitro compounds by rat and mouse liver fractions

Abstract The enzymatic reduction of 2-nitroaphthalene and similar aromatic nitro compounds by rat liver extracts was investigated. The incubation of 2-nitronaphthalene with the post-mitochondrial fraction of rat and mouse liver led to the slow formation of 2-naphthylamine and to the disappearance of 2-nitronaphthalene; the reaction also occurred with the cytoplasmic and microsomal fractions of rat liver and was generally accelerated by NADPH and FMN. No 2-naphthylhydroxylamine could be observed at any time during the reduction, even using a gas-liquid chromatographic system capable of detecting 5 μg of 2-naphthylhydroxylamine added to the incubation mixture. The similar incubation of 4-nitrobiphenyl and 1-nitronaphthalene also led to the slow formation of the corresponding arylamine, with no evidence of hydroxylamine accumulation. 2-Naphthylamine was also produced both chemically and enzymatically by incubating 2-naphthylhydroxylamine with rat liver post-mitochondrial supernatant. In contrast to 2-nitronaphthalene, 4-nitroquinoline- N -oxide was rapidly reduced by rat liver post-mitochondrial fraction to yield high levels of the 4-hydroxylamine derivative, as well as small but significant quantities of the corresponding amine.

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.

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.

Prevention by arginine glutamate of the carcinogenicity of acetamide in rats

Abstract In confirmation of an earlier report, it was found that administration of 2.5% acetamide in the diet of rats for 12 months led to malignant liver tumors after 12–15 months of observation in approximately 50% of the animals at risk. Additional animals exhibited hyperplastic nodules and similar precancerous lesions. The combined feeding of an equimolar amount of arginine glutamate (5.6%) with 2.5% acetamide, led to virtually complete inhibition of the carcinogenic process. Untreated controls or rats fed 5.6% arginine glutamate showed no lesions in the liver. The findings are consistent with, but not necessarily related to, the hypothesis that acetamide is carcinogenic to the liver because of chronic intracellular liberation of ammonia. However, an effort to demonstrate the possible carcinogenic effect of chronic treatment with ammonium ions was equivocal inasmuch as the feeding of 4.8% ammonium citrate alone (equimolar with 2.5% acetamide) did not lead to lesions in the liver.