Introduction

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One of the metabolic activation pathways of carcinogenic arylamines is conversion to N-hydroxy and N-acyloxy derivatives (Crebelli and Iorio, 1985; Heflich and Neft, 1994). These arylamines are also acylated to N-arylacetamide and N-arylformamide, which are themselves toxic. Several reports have indicated that arylacetamides and arylformamides are further activated by hydroxylation to the corresponding hydroxamic acids, and N-hydroxy-2-acetylaminofluorene is further activated by sulfate conjugation in mammalian species (Miller and Miller, 1983). In contrast, these N-arylacylamides are deacylated to the corresponding arylamines, and this process also contributes to the toxicity of N-arylacylamides (Glinsukon et al., 1980; Aune et al., 1985; Land et al., 1989). 2-Acetylaminofluorene (AAF¹) and 2-formylaminofluorene (FAF), typical carcinogenic aromatic acylamines, are also reported to be deacylated in animal bodies (Heymann, 1982; Sertkaya and Gorrod, 1988; Ueda et al., 1996).

When polycyclic arylamines were administered to rabbits, these acetylamino and formylamino derivatives were isolated from the urine and feces (Tatsumi et al., 1989). In mammalian liver, arylamines undergo N-acetylation and N-formylation. Acetylation with acetyl-CoA is catalyzed by arylamine acetyltransferase in liver cytosol (Weber and Hein, 1985). Recently, we demonstrated that the formylation of arylamines is catalyzed by cytosolic formamidase in the presence of N-formyl-L-kynurenine, and some arylformamides were concomitantly deacylated by liver preparations (Tatsumi et al., 1989). However, the enzymes responsible for these deacylations have not been extensively examined. In the current study, we examined the deacylation in rat liver in detail and showed that formamidase plays an important role in the deacylation of N-arylacylamides.

	Materials and Methods

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Chemicals. AF, AAF, 4-aminobiphenyl, 1-aminopyrene, 1-aminonaphthalene, 2-aminonaphthalene were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). L-Kynurenine was obtained from Sigma-Aldrich (St. Louis, MO). N-Formyl-L-kynurenine was prepared from L-kynurenine by the method of Dalgliesh (1952). FAF and other N-arylformamides were prepared from arylamines by formylation according to the method of Tatsumi et al. (1989).

Liver Preparations. Male Wistar (Slc:Wistar/ST) rats (190-230 g), male ddY mice (25-32 g), male Hartley guinea pigs (210-250 g), male golden hamsters (74-77 g), and male Japanese albino rabbits (3.1-3.4 kg) were used. The animals were exsanguinated, and the livers were immediately perfused with 1.15% KCl, then homogenized in 4 volumes of the KCl solution. Microsomes and cytosol were obtained from the homogenate by successive centrifugation at 9,000g for 20 min and 105,000g for 60 min. The microsomal fraction was washed by resuspension in the KCl solution and resedimentation for 60 min at 105,000g for 60 min.

Purification of Formamidase from Rat Liver. Formamidase was purified from rat liver cytosol by the method of Shinohara and Ishiguro (1970). Briefly, formamidase was purified from rat liver cytosol by ammonium sulfate fractionation, heat treatment (55°C for 15 min), and DEAE-cellulose and hydroxyapatite column chromatographies. The specific activity was 0.06 unit/mg protein. Formamidase activity was measured in terms of the increase in absorbance at 366 nm due to hydrolysis of N-formyl-L-kynurenine according to the method of Arndt et al. (1973).

Assay of Deacylase Activity. The incubation mixture consisted of 0.1 µmol of FAF or AAF and a liver preparation equivalent to 0.25 g of liver in a final volume of 2 ml of 0.1 M potassium sodium-phosphate buffer (pH 7.4). The incubation was performed at 37°C for 30 min in air. After incubation, 30 µg of phenacetin was added to the mixture as an internal standard, and then the mixture was extracted with 5 ml of ethyl acetate. The extract was evaporated to dryness in vacuo and the residue was subjected to high-performance liquid chromatography (HPLC). AF formed was determined from its peak area. The time course of the cytosolic reductase activity toward FAF and AAF was linear at least for the initial 30 min. HPLC showed that the extract contained a metabolite with the retention time corresponding to that of authentic AF. This metabolite was purified by HPLC. AF formed by liver preparations was identified as a metabolite of FAF or AAF by comparing the mass and UV spectra with those of an authentic sample as shown in previous paper (Ueda et al., 2001).

HPLC. HPLC was performed in a Hitachi L-6000 chromatograph (Tokyo, Japan) fitted with a column of 125 × 4 mm LiChrosorb RP-18. The chromatograph was operated at a flow rate of 0.6 ml/min acetonitrile-H₂O (4:6), using phenacetin as an internal standard, with the detector set at 254 nm. Retention times of authentic samples were as follows: FAF, 17.4 min; AAF, 19.6 min; AF, 24.4 min; 4-formylaminobiphenyl, 21.5 min; 4-aminobiphenyl, 28.9 min; 1-formylaminonaphthalene, 15.4 min; 1-aminonaphthalene, 20.6 min; 2-formylaminonaphthalene, 14.5 min; 2-aminonaphthalene, 18.7 min; 1-formylaminopyrene, 9.8 min; and 1-aminopyrene, 19.8 min.

	Results and Discussion

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Metabolism of FAF and AAF by Rat Liver Preparations. When FAF was incubated with liver microsomes or cytosol of rats without cofactors, one metabolite, having a retention time corresponding to that of AF, was detected in HPLC chromatograms of the extracts of these incubation mixtures (data not shown). Liver cytosol exhibited a significant deacylase activity toward FAF but little activity toward AAF. In contrast, liver microsomes exhibited a significant deacylase activity toward AAF but a low activity toward FAF. The microsomal and cytosolic deacylase activities toward FAF and AAF were inhibited by the addition of paraoxon (diethyl 4-nitrophenylphosphate) and diisopropyl fluorophosphate (DFP), inhibitors of carboxylesterase and formamidase (Ahmad and Forgash, 1976). The microsomal activities toward these arylacylamides were inhibited by bis(4-nitrophenyl)phosphate and phenylmethylsulfonyl fluoride, inhibitors of carboxylesterase (Heymann, 1980). The cytosolic activities toward FAF and AAF were inhibited by potassium cyanide (Table 1). These results suggest that the microsomal activities toward FAF and AAF were exhibited by carboxylesterase, and the cytosolic activities toward these arylacylamides, by formamidase.

DEAE-Cellulose Column Chromatography of Rat Liver Cytosol. Next, we examined the components in liver cytosol that mediate the deacylation of FAF and AAF. The liver cytosol of untreated rat was subjected to DEAE-cellulose column chromatography, and the fractions were assayed for deacylase activity toward FAF and AAF, and for formamidase activity. The fractions that exhibited deformylase activity toward FAF also showed formamidase activity, which was assayed in terms of the hydrolysis of N-formyl-L-kynurenine, as described under Materials and Methods. The active fractions also showed weak deacetylase activity toward AAF (data not shown). These facts suggest that formamidase in rat liver cytosol was involved in the deacylation of FAF and AAF.

Deformylation of N-Arylformamides by Formamidase. Formamidase purified from rat liver cytosol exhibited a significant deformylase activity toward FAF (13.8 nmol/min/mg protein) and also deacetylase activity toward AAF (1.3 nmol/min/mg protein). The activity was inhibited by paraoxon, DFP, and potassium cyanide. When 1-formylaminopyrene, 4-formylaminobiphenyl, 2-formylaminonaphthalene, and 1-formylaminonaphthalene was incubated with formamidase, deformylase activities toward these N-arylformamides were also observed at 4.2, 13.2, 29.2, and 16.0 nmol/min/mg protein, respectively.

Deacylation by Formamidase in Liver Cytosol of Various Animals. Deacylating activities of liver cytosol of various animals toward FAF and AAF were examined. A marked variation of the deformylating activity was observed among the animals examined. The highest deformylating activity toward FAF was observed in mice, followed by rats, hamsters, and guinea pigs. The lowest activity was observed in rabbits. Deacylating activity toward AAF also showed similar variation among animals. The deacetylase activities were much lower than the deformylase activities in these animals (Fig. 1).

View larger version (28K): [in this window] [in a new window]   Fig. 1.   Deacylation of FAF and AAF by liver cytosol of several mammalian species. Each bar represents the mean ± S.D. of four animals. FAF or AAF was incubated with liver cytosol of various animals. The amounts of AF formed were determined by HPLC as described under Materials and Methods.

Interconversion between AAF and FAF. Formamidase, a cytosolic enzyme, is known to participate in tryptophan metabolism, catalyzing the deformylation of N-formyl-L-kynurenine to kynurenine with simultaneous liberation of formic acid (Arndt et al., 1973). The importance of formamidase in the metabolism of xenobiotics, including carcinogenic arylamines and N-arylacylamides, has not been recognized, and the toxicological and pharmacological implications are of significant concern.

View larger version (8K): [in this window] [in a new window]   Fig. 2.   Postulated mechanism for the interconversion between 2-formylaminofluorene and 2-acetylaminofluorene in rats.