Preston H. Grantham

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.

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.

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.

Enzymic N-Acetylation of 2,4-Toluenediamine by Liver Cytosols from Various Species

1. 2,4-Toluenediamine was incubated with liver cytosol from various species, or cytosol from various tissues of the hamster or rabbit, in the presence of [1-14C]acetyl-CoA. N-Acetylation occurred selectively at the p-amino group of 2,4-toluenediamine and to a much lesser extent on the o-amino group. 2. In hamsters and rabbits the highest N-acetyltransferase activity was present in the liver cytosol, followed by kidney intestinal mucosa and lung cytosols. 3. Hamster liver cytosol had the greatest activity followed by liver cytosols from guinea-pig, rabbit, mouse and rat. With human liver cytosol only a trace of an N-acetyl derivative of 2,4-toluenediamine was found while dog liver cytosol showed no activity. 4. N-Acetyltransferase activity was maximal at pH 7-5 in mouse, pH 6-0 in rat and man, and pH 7-0 in rabbit liver cytosols. 5. There was a slight difference in the levels of N-acetyltransferases in males and females; the female mouse had more enzyme activity than the male, but the male rat had more enzyme activity than the female.

Amino-terminal sequence analysis of six cytochrome P-450 isozymes purified by monoclonal antibody directed immunopurification.

Six hepatic microsomal cytochromes P-450 were isolated from 3-methylcholanthrene induced animals by immunopurification using two monoclonal antibodies. Two forms of cytochromes P-450 (MW 56K and 57K) were from Sprague-Dawley rats, two from C57BL/6 mice (56K and 57K), one form from DBA/2 mice (56K) and one form from guinea pigs (53K). NH2-terminal sequences of the first ten amino acids of these cytochromes P-450 were determined by automated Edman degradation. The 56K polypeptides from rats, C57BL/6 mice, and DBA/2 mice were shown to have identical NH2-terminal sequences. The 57K polypeptides from rats and C57BL/6 mice are homologous to each other but exhibit no homology to 56K polypeptides. The 53K polypeptide from guinea pigs has a unique NH2-terminal sequence with no apparent homology to the other five cytochromes P-450.

Metabolism of the carcinogen N-2-Fluorenylacetamide in germ-free and conventional rats

Abstract Because an appreciable difference was observed in the metabolism of N -hydroxy- N -2-fluorenylacetamide (N-OH-FAA) in germ-free and conventional control rats, the metabolism of the parent compound, N -2-fluorenylacetamide (FAA), was examined in this system. Germ-free and conventional rats injected i.p. with a single dose of FAA excreted similar levels of hydroxylated metabolites in urine, chiefly as conjugates with glucuronic acid. By quantitative rank, decreasing amounts of the 7- 5- 3- and N -hydroxy derivatives of FAA were present in both groups of rats. On the other hand, there were significant differences between the germ-free and control rats in metabolites in the cecum and in the feces. Substantial amounts of conjugated metabolites, glucosiduronic acids and sulfuric acid esters were found in the germ-free group. In the controls, the major portion of the metabolites occurred as free, unconjugated materials. Intracecal injection of FAA led to virtually complete absorption and metabolism similar to what was found after i.p. injection. The bacterial flora in conventional rats affects the metabolism of FAA by converting conjugates to free compounds in the lower portion of the intestinal tract ; these in turn are reabsorbed in part and undergo additional metabolism.

Alteration of the metabolism of the carcinogen N-2-fluorenylacetamide by acetanilide

Abstract In order to elucidate the mechanisms underlying the reduction of the toxicity and carcinogenicity of N -2-fluorenylacetamide by the simultaneous administration of acetanilide, the fate of the carcinogen under these conditions was investigated. In vitro experiments showed that nitrosobenzene, but not chloramphenicol, inhibited the binding of 2-nitrosofluorene to serum albumin. Acute experiments in male rats in which 250 and 500 μmoles of acetanilide were injected 2 hours prior to 10.4 μmoles of 14 C-labeled N -2-fluorenylacetamide yielded no significant difference in the urinary and tissue metabolites of the carcinogen. However, 4 groups of rats pretreated for 6 weeks with 0.02% N -2-fluorenylacetamide, or with 0.02% carcinogen plus 0.8% acetanilide, or with 0.8% acetanilide, or controls, then injected with a dose of tagged carcinogen, yielded appreciable differences in these parameters. Radio-activity in liver and plasma, and isotope bound to liver proteins as well as in the subcellular fractions of liver, showed the lowest binding in rats pretreated with unlabeled carcinogen. Rats fed acetanilide alone or acetanilide plus carcinogen had similar somewhat depressed binding, 65–70% of that of untreated controls. Radioactivity was also firmly bound to deoxyribonucleic acid in the same relative pattern in the 4 groups. As compared to controls, animals pretreated with N -2-fluorenylacet-amide excreted more radioactivity from the dose of labeled agent. The glucosiduronic fraction was higher, and in particular the active carcinogenic intermediate N -hydroxy- N -2-fluorenylacetamide, as glucuronide, amounted to 4.3% of dose compared to 0.21% in controls. Prefeeding of acetanilide, or acetanilide plus carcinogen gave a pattern of urinary carcinogen metabolites similar to that in controls. In particular the important N -hydroxy derivative increased only slightly in the 6-week feeding period. Thus, the reduction in toxicity and carcinogenicity of N -2-fluorenyl-acetamide by the simultaneous administration of acetanilide can be traced to the lower levels of active N -hydroxy metabolite and possibly also to decreased amounts of active carcinogen bound to cellular and molecular receptors.

The metabolism of N-2-fluorenylacetamide in the cat: Evidence for glucuronic acid conjugates

Abstract The urine and feces of adult female cats contained 20% and 20–30%, respectively, of the isotope from a single dose of 14 C-labeled N -2-fluorenylacetamide (FAA) in a 2- to 5-day period. The urinary metabolites consisted of 5% unconjugated compounds, 60–80% sulfuric acid conjugates, and about 15% glucuronic acid conjugates. There was unambiguous evidence for the latter class of metabolites. The free compounds were composed chiefly of FAA and its 7-hydroxyIated derivatives; the sulfuric acid fraction contained mainly the 7-hydroxy derivative and the glucuronic acid fraction the 5-, 7-, and N-hydroxy derivatives. Small percentages of a dose were found in the tissues examined, some in a protein-bound form.