Metabolism Of AAF Research Articles

N-Acetyl-2-Aminofluorene (AAF) Processing in Adult Rat Hepatocytes in Primary Culture Occurs by High-Affinity Low-Velocity and Low-Affinity High-Velocity AAF Metabolite-Forming Systems

N-acetyl-2-aminofluorene (AAF) is a procarcinogen used widely in physiological investigations of chemical hepatocarcinogenesis. Its metabolic pathways have been described extensively, yet little is known about its biochemical processing, growth cycle expression, and pharmacological properties inside living hepatocytes-the principal cellular targets of this hepatocarcinogen. In this report, primary monolayer adult rat hepatocyte cultures and high specific-activity [ring G-3 H]-N-acetyl-2-aminofluorene were used to extend previous observations of metabolic activation of AAF by highly differentiated, proliferation-competent hepatocytes in long-term cultures. AAF metabolism proceeded by zero-order kinetics. Hepatocytes processed significant amounts of procarcinogen (≈12 μg AAF/106 cells/day). Five ring-hydroxylated and one deacetylated species of AAF were secreted into the culture media. Extracellular metabolite levels varied during the growth cycle (days 0-13), but their rank quantitative order was time invariant: 5-OH-AAF > 7-OH-AAF > 3-OH-AAF > N-OH-AAF > aminofluorene (AF) > 1-OH-AAF. Lineweaver-Burk analyses revealed two principal classes of metabolism: System I (high-affinity and low-velocity), Km[APPARENT] = 1.64 × 10-7 M and VMAX[APPARENT] = 0.1 nmol/106 cells/day and System II (low-affinity and high-velocity), Km[APPARENT] = 3.25 × 10-5 M and VMAX[APPARENT] = 1000 nmol/106 cells/day. A third system of metabolism of AAF to AF, with Km[APPARENT] and VMAX[APPARENT] constants of 9.6 × 10-5 M and 4.7 nmol/106 cells/day, was also observed. Evidence provided in this report and its companion paper suggests selective roles and intracellular locations for System I- and System II-mediated AAF metabolite formation during hepatocarcinogenesis, although some of the molecules and mechanisms responsible for multi-system processing remain to be fully defined.

Metabolism of acetylaminofluorene in primary cultures of human hepatocytes: dose-response over a four-log range.

The metabolism of acetylaminofluorene (AAF) in human hepatocyte cultures from different donors was investigated for a four-log concentration range (500, 50, 5.0 and 0.5 microM) or at 3, 8 and 24 h at 500 microM. The metabolite profile was dependent on the concentration to which the cells were exposed. The hepatocyte cultures varied in the degree to which they metabolized AAF predominantly because of different levels of deacetylation. Ring-hydroxylation was the predominant pathway for AAF metabolism at low concentrations (5.0 and 0.5 microM) but saturated in three of four human cases at high concentrations of AAF; N-hydroxylation did not appear to become saturated. Human hepatocytes catalyzed the covalent binding of AAF metabolites to their DNA. A linear increase in DNA binding was observed when increasing concentrations of AAF were added to hepatocyte cultures; however, the increase in AAF metabolites binding to DNA was not proportional to the dose. While the concentration of AAF in the media was increased over a four-log range, both the production of N-hydroxy AAF and binding of metabolites to hepatocellular DNA increased over approximately a three-log range. These results with cultured human hepatocytes indicate that the pathways of AAF metabolism are qualitatively similar to those identified in experiments with rat hepatocytes as well as experiments conducted in vivo with human subjects. These studies confirm that the cultured human hepatocyte is a useful model for the investigation of human xenobiotic metabolism and indicate that the concentration of the xenobiotic used in the experiments is an important determinant of the metabolitic profile produced.

Mutagenicity-enhancing effect of quercetin on the active metabolites of 2-acetylaminofluorene with mammalian metabolic activation systems

The effects of quercetin on the mutagenicity of 2-acetylaminofluorene (AAF) and its 3 active metabolites, N-hydroxy-AAF ( N-OH-AAF), aminofluorene (AF) and N-acetoxy-AAF ( N-OAc-AAF) were investigated. The mutagenicity assays were carried out with Salmonella typhimurium TA98, and S9, microsomes and cytosol were used as metabolic activation systems. In the presence of S9, quercetin enhanced the mutagenicity of AAF, N-OH-AAF, AF and N-OAc-AAF by 6.9-, 4.3-, 3.6- and 3.9-fold, respectively. Quercetin enhanced the mutagenicity of these substrates with microsomes, whereas it depressed the mutagenicity of these substrates with cytosol. From these results, it seemed probable that quercetin promotes the N-hydroxylation and deacetylation in the microsomes, whereas it inhibits the deacetylation in the cytosol. It was shown that in the metabolism of AAF and its metabolites, quercetin modulates the balance between the mutagenicity activation and inactivation processes, which is catalysed by the enzymes in the microsomes and cytosol, and causes enhancement of the mutagenicity of AAF.

Effect of dexamethasone on cytochrome P-450 mediated metabolism of 2-acetylaminofluorene in cultured rat hepatocytes

The metabolism of 2-acetylaminofluorene (AAF) to its six oxidative metabolites has been used to investigate the effect of dexamethasone on cytochrome P-450 activity in cultured rat hepatocytes. In control hepatocytes the metabolism of AAF to its 1-, 5-, 7-, 9- and N-hydroxylated metabolites rapidly declined in culture over the first 24 hr while 3-hydroxylation remained relatively constant. These activities either remained unchanged or increased slightly during the next 48 hr in culture. The addition of dexamethasone (100 nM) to the culture medium had little effect in arresting the initial decline but by 72 hr the 7-, 5- and 3-hydroxylations increased to values 2.5, 16 and 21 times the respective 24-hr values. The inductive effect of dexamethasone on the 3- and 5-hydroxylations of AAF was maximal at 100 nM whereas the 7-hydroxylation increased linearly as a function of the dexamethasone concentration up to l μM. Cortisol and corticosterone and the non-glucocorticoids fluoxymesterone and methyl-testosterone induced a pattern of AAF metabolism resembling that in dexamethasone-treated cultures, suggesting that a range of steroids not restricted to glucocorticoids may induce multiple cytochrome P-450 isozymes via related mechanisms. Pregnenolone 16α-carbonitrile induced only the 7-hydroxylation of AAF probably reflecting induction of cytochrome P-450p. While dexamethasone was a strong inducer of the 3- and 5-hydroxylations of AAF in hepatocyte culture, assay of these activities in freshly isolated cells after in vivo treatment with dexamethasone showed a strong induction of 7-hydroxylation but only small effects on 3- and 5-hydroxylations. Indeed the profile of AAF metabolism induced in culture by dexamethasone resembles more closely the profile induced by 3-methylcholanthrene in vivo. These data suggest that factors yet to be identified strongly influence the steroid-induced pattern of cytochrome P-450 gene expression.

Effects of harman and norharman on the metabolism and genotoxicity of 2-acetylaminofluorene in cultured rat hepatocytes.

Monolayers of rat hepatocytes metabolize 0.25 mM 2-acetylaminofluorene (AAF) to various ether-extractable, water-soluble as well as covalently bound products. The major ether-extractable metabolite formed is 2-aminofluorene (AF), followed by 7-OH-AAF and 9-OH-AAF. Pretreatment of rats with the inducer Aroclor 1254 (PCB) increased the metabolism of AAF and caused an increased DNA repair synthesis in hepatocytes exposed to AAF or AF. With N-OH-AAF, a decreased genotoxic response in PCB-treated cells compared to control cells was seen. The addition of harman and norharman decreased the metabolism of AAF to ether-extractable metabolites, water-soluble metabolites and metabolites covalently bound to macromolecules. In contrast, the DNA-repair synthesis caused by the same concentrations of AAF was increased by harman. One explanation for this apparent discrepancy could be that the aromatic amines changed the metabolism of harman and norharman in such a way that these compounds were converted into genotoxic metabolites.

Absence of genotoxic effects of nonasbestos mineral fibers.

The biological activity of natural and synthetic mineral fibers has been examined. Natural attapulgite [(Mg, Al)2Si4O10(OH).4H2O], synthetic xonotlite [Ca3Si3O8(OH)2] and natural sepiolite [Mg2Si3O8.2H2O] were selected. Genotoxic effects were investigated by means of a well established cellular model based upon the measurement of unscheduled DNA synthesis (UDS) in rat hepatocytes in primary culture. The intrinsic capacity of the fibers (1 and 10 micrograms/ml) to induce UDS was first tested. None of the fiber types showed detectable UDS-eliciting activity. Also, the possible modulation of the cellular response to genotoxic agents by the materials was examined by exposing the cells to mixtures of 2-acetylaminofluorene (AAF) (0.05 and 0.25 micrograms/ml) and fibers (1 and 10 micrograms/ml). In these experiments, the UDS response was significantly diminished in the presence of xonotlite. This phenomenon may reflect changes in the uptake and/or metabolism of AAF or may result from an inhibition of DNA repair processes, the latter suggesting a possible cocarcinogenic potential for this synthetic silicate. These results point to the immediate necessity of studying more extensively the biological effects of fibrous materials that can be used as substitutes for asbestos.

Metabolic processing of 2-acetylaminofluorene by microsomes and six highly purified cytochrome P-450 forms from rabbit liver.

Kinetic analysis of oxidative metabolism of 2-acetylaminofluorene (AAF) was studied in control and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced microsomes and with six highly purified cytochrome P-450 isoenzymes from rabbit liver. Kinetic parameters were defined for 7-, 5-, 3-, 1- and N-hydroxylations of AAF. 7-Hydroxylation was best defined by a two enzyme system, displaying a high affinity and relatively low capacity and a low affinity high capacity components in both control and TCDD induced microsomes. All the purified cytochrome P-450 isoenzymes were capable of catalyzing the 7-hydroxylation of AAF and, with the exception of form 4, this was the only oxidation on the AAF molecule catalyzed by these forms. It is probable that forms 1, 4 and 6 accounted for a substantial part (greater than or equal to 25%) of total metabolic capacity corresponding to the high affinity component of 7-hydroxylation, whereas forms 3b and 3c accounted for less than 5% of the metabolic capacity displayed by the low affinity component in control microsomes. However, forms 4 and 6 could account for greater than 90% of the metabolic capacity of the high affinity component of 7-hydroxylation in TCDD microsomes, whereas the form(s) responsible for the metabolic capacity of the low affinity component were not identified. Each of the 1-, 3-, 5- and N-hydroxylations were best defined by a single enzyme system in both control and TCDD microsomes (3- and 5-hydroxylations could not be defined in TCDD microsomes). Close agreements were found between the apparent Km for N-hydroxylation in control, TCDD induced microsomes and with form 4. alpha-Naphthoflavone inhibited AAF N-hydroxylation to a similar extent in control and TCDD microsomes and in form 4. These date indicate that: a subpopulation of cytochrome P-450 isoenzymes, which includes all the purified P-450 forms tested in the present study, is solely involved in detoxification (i.e., 7-hydroxylation) of AAF, and as such probably behave as a functional unit in vivo; modulation of cytochrome P-450 content by inducers such as TCDD results in emergence of relatively few cytochrome P-450 isoenzymes that can account for most of the oxidative metabolism of AAF; and a single cytochrome P-450 isoenzyme (i.e., form 4) is responsible for catalyzing N-hydroxylation of AAF, the first and the obligatory step in the metabolic activation of this carcinogen.

Kinetic evidence for the involvement of multiple forms of human liver cytochrome P-450 in the metabolism of acetylaminofluorene.

The kinetics of 2-acetylaminofluorene (AAF) metabolism have been studied in liver microsomes from four human subjects over a substrate range of 0.02-200 microM. The N-hydroxylation of AAF was best described by a single enzyme system with a mean Km of 1.63 microM and a mean Vmax of 61 pmol mg-1 protein min-1. Biphasic kinetics for the 7-hydroxylation of AAF in all four subjects were observed and a two enzyme system best described the data. The mean Km and Vmax for the high affinity, low-capacity enzyme were 0.69 microM and 30 pmol mg-1 protein min-1, respectively, while for the low affinity, high capacity enzyme the mean Km was 75 microM and the mean Vmax was 286 pmol mg-1 protein min-1. In one of the four subjects studied the 5-hydroxylation of AAF was similarly resolved into a two enzyme system. The 1-hydroxylation of AAF in human microsomes was a major reaction and was best described by a single enzyme system. The 3- and 5-hydroxylations of AAF were minor metabolic pathways. Non-classical Michaelis-Menten kinetics were observed for the 9-hydroxylation of AAF and at high substrate concentrations this was the major metabolite formed. These data demonstrate the involvement of at least two forms of human cytochrome P450 in the metabolism of AAF.

Effect of 1,8-pyrenequinone, a mutagenicity enhancing agent, on metabolisms of 2-acetylaminofluorene in liver microsomal fraction.

The addition of 1, 8-pyrenequinone into the assay system containing rat liver homogenates (S9) caused an approximately 10-fold increase in the mutagenicity of 2-acetylaminofluorene (AAF) in the current Salmonella reversion assay system. Since no chemical reaction between 1, 8-pyrenequinone and AAF was observed, the in vitro effects of 1, 8-pyrenequinone on the metabolisms of AAF with S-9 mix were studied. The enhancement of mutagenicity by 1, 8-pyrenequinone was not dependent on the dose of NADPH under the present assay condition. The mutagenicity of AAF was increased approximately 4-fold by the addition of 1, 8-pyrenequinone into microsomes, whereas it remained at the spontaneous level in the presence of cytosol. However, by reconstituting microsomes with cytosol, the mutagenicity enhancing activity was recovered to the original level. Since 1, 8-pyrenequinone inhibited the AAF hydroxylase activity, chemical analysis of the incubation mixture of AAF was tried. This indicated that a higher amount of unmetabolized AAF remained and higher amounts of 2-aminofluorene and N-hydroxy-2-acetylaminofluorene were accumulated in the presence of 1, 8-pyrenequinone compared with those in the absence of 1, 8-pyrenequinone. From these results, it seems probable that 1, 8-pyrenequinone inhibits C-hydroxylation (the detoxifying pathway) and promotes N-hydroxylation (the activating pathway) as well as deacetylation in the AAF metabolism.

The metabolism of N-acetyl-2-aminofluorene to a mutagen in L5178Y/TK +/− mouse lymphoma cells

Direct treatment of L5178Y mouse lymphoma TK +/− cells with N-acetyl-2-aminofluorene (AAF) from two commercial sources produced small, but reproducible increases in mutant frequency over background in the absence of exogenous microsomal enzymes. Unlike most direct-acting mutagens which typically produce regular, dose-dependent increases in mutant frequency; AAF treatment caused very slight dose-related increases or a saturation phenomenon which could be overcome by increased exposure time. Direct mutagenicity following prolonged (24 h) exposure was confirmed when a third highly purified (99.9%) AAF sample was tested. Microsomal enzyme analyses of disrupted L5178Y cell preparations revealed negligible benzo[ a]pyrene hydroxylase but measurable AAF- N-hydroxylase activity. These data demonstrate that L5178Y mouse lymphoma cells are capable of limited metabolism of AAF to an active mutagen.

Metabolism of 2-acetylaminofluorene in primary rat hepatocyte cultures.

Primary cultures of adult rat parenchymal hepatocytes were developed as an in vitro model to investigate the biochemical fate of 2-acetylaminofluorene (AAF), a potent hepatocarcinogen. More than 5 x 10(8) viable cells were routinely isolated by collagenase perfusion in rat liver; the cells were cultured 2-5 d on collagen-coated dishes in serum-free culture medium containing hormones and other factors to retard the decline of cytochrome P-450. All of 137 ng or 13.7 microgram AAF was metabolized in 21-24 h by 2 x 10(6) cultured hepatocytes in 4.0 ml defined medium. At the higher dose, water-soluble metabolites appeared at 70% of the rate of metabolism at the lower dose, which was 17 ng/h for the initial 4 h. As the parent compound was consumed, bound AAF residues were recovered with exhaustively extracted, trichloro-acetic acid-precipitated hepatocellular macromolecules, accounting for a maximum of 5% of the 137-ng dose. Addition of hormones to the culture medium stimulated the rate of appearance of water-soluble metabolites, AAF, correlating with the enhanced cytochrome P-450 levels of hormone-treated cells. Metabolism of AAF was diminished 50% during 3 h of incubation with 10(-4) M SKF 525A and 100% with 10(-3) M SKF 525A. At a dose of 40 microgram AAF per 2 X 10(6) cells, only 31% of the carcinogen was recovered from the culture medium as water-soluble products after 24 h; the cells were sown to be capable of metabolizing a subsequent 40-microgram dose at an undiminished rate, suggesting that saturation of metabolizing enzymes rather than toxicity occurred. These results support the validity of primary hepatocyte cultures as a model system for quantitative investigations of the biochemical fate of AAF in mammalian cells, and provide preliminary characterization of the cells' processes of detoxification and metabolic activation of a chemical carcinogen.

Selenium effects on the carcinogenicity and metabolism of 2-acetylaminofluorene

Addition of 4 ppm Se to the drinking water of male albino rats fed diets containing 0.03% 2-acetylaminofluorene (AAF) provided protection against hepatic damage and also resulted in at least 50% reduction in liver tumor incidence. An in vitro assay system utilizing microsomes from Se supplemented or non-supplemented 3-methylcholanthrene (MC) induced rats was used to determine the effect of oral Se intake on the metabolism of AAF. Oral Se administration led to an increase in ring hydroxylation and a decrease in N-hydroxylation. Addition of Se to the microsomal assay system increased 3-OH AAF formation and decreased N-OH AAF formation, thus shifting the balance of metabolism toward detoxification pathways.

Effects of selenium on chemical carcinogenesis

Chemical carcinogenesis can be characterized by a sequence of events leading to the development of tumors. Selenium (Se) inhibition of colon, liver, and lung carcinogens is demonstrated. Using the male Sprague Dawley rat model Se inhibited the colon tumor incidence in 1,2-dimethylhydrazine (DMH) treated rats and reduced the total number of colon tumors in methylazoxymethanol (MAM) treated rats. Selenium inhibited 2-acetylaminofluorene (AAF) and 3'-methyl-4-dimethylaminoazobenzene (3'-MeDAB) hepatocarcinogenesis. The hepatic tumor incidence induced by 3'-MeDAB was reduced by both inorganic Se (Na2SeO3) and by organic Se (Se-yeast) supplements.In vitro systems have been studied in an effort to decipher the inhibitory properties of Se on the multistage origin of tumors induced by chemical carcinogens. Current studies suggest that the protective effect of Se against AAF hepatocarcinogenesis may be correlated with a change in AAF metabolism. The mutagenicity of AAF and AAF metabolites inSalmonella typhimurium TA1538 is decreased by Se. Additionally, Se reduced N-t-OH-AAF induction of sister chromatid exchange (SCE) frequencies in whole blood cultures, and also reduced aryl hydrocarbon hydroxylase activity using benzo(a) pyrene as substrate.The comparative effects of antioxidants on DMH induction of colon tumors are presented in detail. Supplements of 4 ppm Se to the drinking water, 1.2% ascorbic acid (V c ) to the diet or 0.5% butylated hydroxytoluene (BHT) to the diet of DMH-treated rats reduced the colon tumor incidence of DMH controls from 64 to 31% (Se), 38% (V c ), and 43% (BHT). The colon tumor incidence in DMH-treated rats receiving a combination of Se+V c increased to 83%, while the combination of Se+BHT decreased the colon tumor incidence to 55%. The growth and survival of rats provided long-term supplements of 4 ppm Se in the drinking water are compared with untreated controls.

Inhibitory effects of selenium on the mutagenicity of 2-acetylaminofluorene (AAF) and AAF derivatives

Selenium (Se) decreased the mutagenicity of 2-acetylaminofluorene (AAF), N-hydroxy-2-acetylaminofluorene (N-OH-AAF) and N-hydroxyaminofluorene (N-OH-AF) in the Salmonella typhimurium TA 1538 bacterial tester system. Metabolism of AAF and N-OH-AFF to the active mutagen, N-OH-AF, was accomplished by rat liver extracts. Graded decreases in mutagenicity with increasing Se concentrations were observed for each of the three mutagens. Se decreased the mutagenicity of AAF, N-OH-AAF and N-OH-AF to 65, 68 and 61% of their respective controls with mutagen alone. The effective molar ratios of Se to mutagen yielding these decreases were approximately 10:1 (Se:AAF), 10:1 (Se:N-OH-AAF) and 300:1 (Se:N-OH-AF). The largest Se effect observed was accomplished by a molar ratio of 100:1 (Se:N-OH-AAF) yielding 28% of the mutagenicity elicited by N-OH-AAF alone.

An oxime analogue of atropine—some pharmacological observations

Addition of 4 ppm Se to the drinking water of male albino rats fed diets containing 0.03% 2-acetylaminofluorene (AAF) provided protection against hepatic damage and also resulted in at least 50% reduction in liver tumor incidence. An in vitro assay system utilizing microsomes from Se supplemented or non-supplemented 3-methylcholanthrene (MC) induced rats was used to determine the effect of oral Se intake on the metabolism of AAF. Oral Se administration led to an increase in ring hydroxylation and a decrease in N-hydroxylation. Addition of Se to the microsomal assay system increased 3-OH AAF formation and decreased N-OH AAF formation, thus shifting the balance of metabolism toward detoxification pathways.