IDENTIFICATION OF METABOLITES IN URINE AND FECES FROM RATS DOSED WITH THE HETEROCYCLIC AMINE, 2-AMINO-3-METHYL-9H-PYRIDO[2,3-b]INDOLE (MeA C)

2-Amino-3-methyl-9H-pyrido[2,3-b]indole (MeA C) is a proximate mutagenic and carcinogenic heterocyclic amine formed during ordinary cooking. In model systems, MeA C can be formed by pyrolyses of either tryptophan or proteins of animal or vegetable origin. In the present study, the in vivo metabolism of MeA C in rats was investigated. Rats were dosed with tritium-labeled MeA C, and urine and feces were collected over 3 days. The metabolites of MeA C were identified by high performance liquid chromatography-mass spectrometry and quantified by liquid scintillation counting. Conjugated metabolites were characterized by enzymatic hydrolyzes with -glucuronidase or arylsulfatase. The data showed that the metabolic pattern of MeA C was similar in all rats. About 65% of the dose was excreted in urine and feces, and the major amount of MeA C-metabolites was excreted during the first 24 h. Thirty-four percent of the dose was found in the rat urine samples collected to 24 h. In addition to unmetabolized MeA C and two phase I metabolites, 6-OH-MeA C and 7-OH-MeA C, the following conjugated metabolites were identified: MeA C-N-glucuronide, A C-3-CH2O-glucuronide, 3-carboxy-A C and 3-carboxy-A C-glucuronide, and sulfate and glucuronide conjugates of 6-OH-MeA C and 7-OH-MeA C. Also, a large amount of a rather unstable compound proposed to be of MeA C-N1-glucuronide was found. About 21% of the dose was excreted in feces during the first 24 h, and MeA C and 7-OH-MeA C were the only compounds identified in feces. Any activated metabolites of MeA C were not detected in rat urine or feces. 2-Amino-9H-pyrido[2,3-b]indole (A C) and its methyl homolog 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeA C) are two foodborne mutagenic and carcinogenic heterocyclic amines (Wakabayashi et al., 1992). A C and MeA C are often referred to as -carbolines and are classified as unpolar heterocyclic aromatic amines. -Carbolines are formed as pyrolysis products of either tryptophan or proteins of animal or vegetable origin (e.g., albumin, casein, or soybean globulin) (Yoshida et al., 1978; Sugimura, 1997). -Carbolines are found in cooked food such as fried meat, chicken, fish, mushroom, and bouillon concentrates (Matsumoto et al., 1981; Gross and Gruter, 1992; Layton et al., 1995; Knize et al., 1997; Skog et al., 1998; Solyakov et al., 1999) and cigarette smoke condensates (Yoshida and Matsumoto, 1980; Matsumoto et al., 1981; Manabe et al., 1990; Wakabayashi et al., 1993). Finally, MeA C is also found in wine (Richling et al., 1997). -Carbolines are mutagenic in bacterial test systems (Matsumoto et al., 1977; Nagao et al., 1983). Dietary administration of -carbolines to rodents has shown that they are moderately potent carcinogens (Ohgaki et al., 1984; Tamano et al., 1994; Snyderwine et al., 1998). Like other heterocyclic amines, the metabolism of -carbolines usually follows two different pathways, detoxification and activation. The first step in the metabolism of heterocyclic amines is a phase I hydroxylation catalyzed by cytochrome P450 enzymes. Detoxified compounds are often ring-hydroxylated, followed by phase II conjugation. Activated compounds are hydroxylated in their characteristic exocyclic amino group, usually followed by O-esterification catalyzed by acetyltransferase or sulfotransferase (Eisenbrand and Tang, 1993). Enzymes from the CYP1A family are especially involved in the phase I metabolism of -carbolines to their corresponding N-OH-derivates (Niwa et al., 1982; Sugimura, 1985). Recently, we have shown that human CYP1A2 and rat CYP1A1 activate about 27% and 56%, respectively, of A C, whereas these enzymes only activate a few percent of MeA C (Frederiksen and Frandsen, 2003). Activated heterocyclic amines are able to form adducts with macromolecules such as protein and DNA. It was shown by P-postlabeling analysis that MeA C and A C both form one major DNA adduct (N-deoxyguanin-8-yl-compounds) in primary hepatocytes from rats (Pfau et al., 1996, 1997). The in vitro metabolism of A C in human and rodent hepatic microsomes and the metabolism of MeA C in hepatic microsomes from PCB-induced rat have been studied and some of the major metabolites characterized (Raza et al., 1996; Frandsen et al., 1998). We have previously studied and compared the in vitro metabolism of A C and MeA C in hepatic microsomes from PCB-induced rat, uninduced rat, and human. A C was metabolized to two major detoxified metabolites, and MeA C was metabolized to three major This study has been carried out with financial support from the commission of the European communities, specific RTD program “Quality of life and management of living resources” QLK1-CT99-01197, Heterocyclic amines in cooked foods—Role in human health. It does not necessarily reflect its views and in no way anticipates the commission’s future policy in this area. 1 Abbreviations: A C, 2-amino-9H-pyrido[2,3-b]indole; HPLC, high performance liquid chromatography; MeA C, 2-amino-3-methyl-9H-pyrido[2,3-b]indole; MeIQx, 2-amino-4,8-dimethylimidazo[4,5-f]quinoxaline; PCB, Aroclor 1254 (polychlorinated biphenyl); Rt, retention time. Address correspondence to: Henrik Frandsen, Institute of Food Safety and Nutrition, Danish Veterinary and Food Administration, Mørkhøj Bygade 19, DK 2860 Søborg, Denmark. E-mail: [email protected] 0090-9556/04/3206-661–665$20.00 DRUG METABOLISM AND DISPOSITION Vol. 32, No. 6 Copyright © 2004 by The American Society for Pharmacology and Experimental Therapeutics 1320/1154722 DMD 32:661–665, 2004 Printed in U.S.A. 661 at A PE T Jornals on A uust 4, 2017 dm d.aspurnals.org D ow nladed from detoxified metabolites. Amounts of both -carbolines were activated to their corresponding N-OH-derivates. The distribution between the detoxified and activated metabolites in the different types of hepatic microsomes showed the same pattern for both -carbolines. In PCBinduced microsomes about 90% of the metabolites were detoxified, and in the uninduced rat microsomes there was a 50/50 distribution between detoxification and activation, whereas the major part of the metabolites in the human microsomes (about 60%) were activated and reacted to form dimers and protein adducts (Frederiksen and Frandsen, 2002). However, although the in vitro metabolism of -carbolines has been studied, there have only been a very few in vivo studies of -carbolines focused on DNA-adduct formation (Yamashita et al., 1986; Pfau et al., 1997; Snyderwine et al., 1998). Compared with other heterocyclic amines, little is known about the in vivo metabolism of -carbolines. In the present study, we have investigated the metabolism of MeA C in rats. Metabolites excreted in urine and feces have been separated, quantified, and identified. Materials and Methods Chemicals. MeA C was obtained from Toronto Research Chemicals (Toronto, Ontario, Canada). Tritiation of MeA C ([H]MeA C) was described previously (Frandsen et al., 1998). Reference compounds 3-CH2OH-A C, 6-OH-MeA C, and 7-OH-MeA C were prepared in microsomal incubations, and N-OH-MeA C was prepared by chemical synthesis as previously described (Frederiksen and Frandsen, 2002). -Glucuronidase (Escherichia coli) and arylsulfatase type VI (Aerobacter aerogenes) were obtained from Roche Diagnostics (Basel, Switzerland) and Sigma-Aldrich (St. Louis, MO), respectively. Isolute 101 columns were obtained from International Sorbent Technology (Glamorgan, UK) and Strata SDB-L columns were obtained from Phenomenex (SubWare, Hillerød, Denmark). High performance liquid chromatography (HPLC)-grade acetonitrile and formic acid (distilled before use) were obtained from Romil (Cambridge, UK). Soluene-350 and Hionic Fluor were obtained from PerkinElmer Life and Analytical Sciences (Boston, MA). All other chemicals were of analytical grade. Animals. Three adult male Wistar rats (age 7–8 weeks, weight 200 g) were delivered from Taconic M&B (Lille Skensved, Denmark). After a 5-day acclimatization period, the animals were placed in metabolism cages. The animals received one single dose of 0.2 ml of [H]MeA C (8.6 mg/ml dissolved in 50% dimethyl formamide) by oral gavage. Urine and feces were collected on dry ice to 24, 48, and 72 h and stored at 80°C until analyses. Purification of Urinary Metabolites. Collected urine samples were thawed and mixed, and 2-ml aliquots of each were centrifuged. The amount of tritium-labeled compounds in the urine was quantified by liquid scintillation analyses of 50 l of supernatant. Two hundred microliters of water and 1 ml of Soluene-350 were added to the urinary precipitate, mixed, and incubated for 30 min at room temperature. Six hundred microliters of the precipitate-Soluene solution was analyzed by liquid scintillation counting. Urine-supernatant, 900 l, was added to 4 ml of water and applied on an activated Isolute 101 column (washed before use with 2 ml of methanol followed by 2 ml of water). The column was washed with 1 ml of water and eluted with 1 ml of methanol. Fifty microliters of each fraction were analyzed by liquid scintillation counting. The eluate was evaporated to dryness at 30°C under a stream of nitrogen, and redissolved in 200 l of 50% dimethyl formamide and diluted with 300 l of water. Extraction and Purification of Fecal Metabolite. Collected feces samples were added to 2 volumes of water and homogenized by an Ultra-Turrax homogenizer (IKA, Staufen, Germany). The amount of tritium-labeled compounds in the feces was quantified by liquid scintillation analyses. Aliquots of 100 mg of homogenized feces were added to 1 ml of Soluene-350 and incubated for 1 h at 50°C, followed by addition of 500 l of 2-propanol and 2-h incubation at 50°C. Two hundred microliters of 30% hydrogen peroxide was added dropwise and, after 30 min, incubated at room temperature. Four milliliters of Hionic-Fluor was added and the sample was incubated for 3 days in the dark, followed by liquid scintillation counting. Aliquots of 100 mg of homogenized feces were added to 5 ml of 50% dimethyl formamide. After mixing and centrifugation, the supernatants were collected. The extractions of the precipitates were repeated twice, and 100 l of each supernatant were analyzed by liquid scintillation counting. Aliquots of 5 ml of the pooled feces extracts were added to 20 ml of water and applied in small portions on an activated Strata SDB-L column (washed before use with 5 ml of acetone, 5 ml of methanol, and 5 ml of water). The column was washed with 2 ml of water and eluted with 6 ml of methanol. One hundred microliters of each fraction was analyzed by liquid scintillation counting. The eluate was evaporated to dryness at 30°C under a stream of nitrogen, and redissolved in 100 l of 50% dimethyl formamide and diluted with 150 l of water. Analyses of Metabolites. Purified urine and feces samples were analyzed by HPLC, performed on an Agilent Technologies model 1100 liquid chromatograph equipped with a photodiode array detector (Agilent Technologies, Wallbronn, Germany). The metabolites were separated on a Zorbax SB-C3 5m, 150 3 mm column from Agilent Technologies. Injection volume was 12.5 l, flow rate 0.4 ml/min, and oven temperature 40°C. Solvents were A (0.001% formic acid) and B (acetonitrile). Solvent programming was: 0 to 2 min, 2% B; 8 min, 15% B; 30 min, 30% B; 34 min, 100% B; 37 min, 100% B. One-minute fractions were collected and analyzed by liquid scintillation counting. Positive ion electrospray mass spectra were obtained with an Agilent Technologies MSD 1100 mass spectrometer equipped with an electrospray interface. The following interphase settings were used: nebulizer pressure, 60 psi; drying gas (nitrogen), 10 l/min, 350°C; capillary voltage, 4000 V; fragmentor voltage, 70 V. Extracted ion chromatograms were used to identify peaks with masses corresponding to hydroxylated and conjugated MeA C metabolites. Liquid Scintillation Counting. Before analyses by liquid scintillation counting, all samples were added to 4 ml of Hionic-Fluor. Liquid scintillation counting was performed on a Tri Carb 3100TR with external standardization (PerkinElmer Life and Analytical Sciences). Enzymatic Hydrolysis. To investigate the presence of conjugated MeA C metabolites, individual metabolite peaks from the HPLC chromatograms were collected and treated with -glucuronidase or arylsulfatase. Two hundred fifty microliters of purified urine was injected in aliquots of 12.5 l on the HPLC column, and 1.5-min fractions were collected. The collected fractions were evaporated to dryness on a SpeedVac evaporator (Thermo Savant, Holbrook, NY), redissolved in 100 l of 50% dimethyl formamide, and diluted with 150 l of water. Five microliters of each fraction were analyzed by liquid scintillation counting, and 5 l were added to 45 l of 50 mM acetate buffer, pH 5.5, and analyzed by HPLC-mass spectrometry. Hydrolysis was performed by addition of 195 l of 50 mM acetate buffer, pH 5.5, and 5 l of -glucuronidase or arylsulfatase to 50 l of each of the fractions, followed by argonpurging and 2-h incubation at 37°C in a shaking water bath. The hydrolyzed compounds were analyzed by HPLC-mass spectrometry and by liquid scintil-