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VA Medical Center, St. Louis, Missouri (V.M.L., T.V.Z.); Division of Geriatric Medicine (V.M.L., T.V.Z.) and Department of Biochemistry and Molecular Biology (T.V.Z.), St. Louis University School of Medicine, St. Louis, Missouri; and Department of Medicine, Washington University, St. Louis, Missouri (F.F.H.)
(Received October 11, 2007; accepted March 18, 2008)
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Abstract |
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). More than 20 similarly formed HCAs have been identified. In the human diet, consumption of 400 g of cooked lean meat could result in exposure to several micrograms of mutagenic HCAs. These amines are detected in urine and their excretion, as unchanged amine, is increased by a cytochrome P450 (P450) inhibitor (Lynch et al., 1992; Boobis et al., 1994), indicating their absorption from cooked foods and P450 metabolism.
IQ is a potent carcinogen inducing tumors in multiple tissues and in different species (National Toxicology Program Report on Carcinogens: Background Document for Heterocyclic Amines: MeIQ, MeIQx, IQ, and PhIP, 2005, http://ntp.niehs.nih.gov/ntp/roc/eleventh/profiles/s092vhca.pdf). In mouse, oral administration of IQ caused forestomach, liver, and lung tumors in both sexes. Although IQ also caused tumors in several organs in both sexes of rats, mammary and clitoral gland tumors were only detected in females and colon and skin tumors only in males. IQ administered orally to monkeys or mice caused liver tumors. Using a colitis-induced carcinogenicity model with ApcMin/+ mice (Cooper et al., 2001), we have preliminary data indicating that induction of colitis by treatment with 4% dextran sulfate sodium (DSS) in drinking water followed by oral doses of 40 mg/kg IQ resulted in a 100% incidence of invasive colorectal tumors, whereas mice treated with only DSS had no tumors (Clapper et al., 2006). The strong association of intake of high-temperature cooked meat and colorectal cancer risk (Giovannucci et al., 1994), the presence of HCAs in cooked meat (Felton and Knize, 1990), and the initiation of colon cancer by HCAs (Kristiansen et al., 1997) suggest that HCAs present in high-temperature cooked meat may be responsible for the increased risk of colon cancer associated with this dietary component.
Activation of IQ is thought to involve N-oxidation by P450, followed by O-acetylation with subsequent formation of a reactive intermediate, nitrenium ion, that binds DNA (Turesky et al., 1991). These DNA adducts are thought to cause mutations that initiate carcinogenesis. After IQ administration, DNA adducts are found in a variety of rodent and nonhuman primate tissues (Nerurkar et al., 1995; Turesky et al., 1996). Whereas N-oxidation of IQ has been demonstrated with rat microsomes (Turesky et al., 1998), excretion of N-hydroxy-IQ has not been detected in rat. There is a considerable amount of evidence indicating that P450s play an important role in HCA genotoxicity.
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; Luks et al., 1989). The other major route of metabolism is direct conjugation of the exocyclic amine to form N2-glucuronide or sulfamate (Turesky et al., 1986; Inamasu et al., 1989; Luks et al., 1989). The 5-O-glucuronide and 5-sulfate were detected in urine and bile but not in feces (Inamasu et al., 1989; Luks et al., 1989). In contrast, sulfamate excretion was observed in urine, bile, and feces (Turesky et al., 1986; Inamasu et al., 1989; Luks et al., 1989). This finding is consistent with the conjugates being hydrolyzed by bacterial enzymes in the intestine and reabsorbed from the intestinal tract. Similar pathways for activation and inactivation have been reported in different species and for other aminoimidazole HCAs (Snyderwine et al., 1992; Langouët et al., 2001). Rat P450 oxidation appears to favor the 5-position, whereas in humans N-oxidation may be favored (Turesky et al., 1998). Metabolism by the monkey is different from that in rat in that 2-aminoimidazo[4,5-f]quinoline (demethyl-IQ) is formed as a major product (Snyderwine et al., 1992). Human hepatocytes metabolize 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, a structurally similar HCA, to an N-demethyl derivative, whereas rat hepatocytes do not (Langouët et al., 2001). Although N-acetylation plays a major role in metabolism of aromatic amines (Lakshmi et al., 1995a), the N-acetyltransferase pathway plays only a minor role in IQ rat metabolism (Inamasu et al., 1989).
Although metabolism can influence tumorigenicity, only balance studies of IQ excretion (Sjödin and Jagerstad, 1984) and its organ distribution (Bergman, 1985; Alldrick and Rowland, 1988) have been assessed in mouse. Because the mouse is an attractive model for testing tumorigenicity and its use in this area is likely to continue and even increase, we have evaluated pathways for mouse IQ metabolism. Results indicate that mouse IQ metabolism is similar to that for monkey and human but different from that for rat. Thus, parameters affecting IQ tumorigenicity in rat and mouse may be different.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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-naphthoflavone, furafylline, SKF-525A, ketoconazole, sulfaphenazole, Escherichia coli β-glucuronidase (type VII-A), and abalone sulfatase (type VIII) were purchased from Sigma-Aldrich (St. Louis, MO). Ultima-Flo AP was purchased from PerkinElmer Life and Analytical Sciences (Shelton, CT). Animals and Dosing. Six-week-old C57BL/6 female mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Mice were fed AIN-76A from Bio-Serv (Frenchtown, NJ) for 8 weeks before use. Female F344 rats at 13 weeks of age were purchased from Harlan (Indianapolis, IN) and fed the AIN-76A diet for 8 weeks before use. Experiments were performed according to the National Institutes of Health standards for care and use of experimental animals and the St. Louis VA Medical Center Animal Care and Use Committee. Animals were group-housed, maintained on a 12-h light/dark cycle, and had access to food and water ad libitum. Mice were administered 40 mg/kg [14C]IQ (20 µCi; 0.2 ml/20 g oral dose; 20% DMSO-80% 0.05 N HCl) and placed in metabolic cages, and a 24-h urine sample was collected. Rats were administered 40 mg/kg [14C]IQ (20 µCi; 2.5 ml/250 g oral dose; 20% DMSO-80% 0.05 N HCl) and placed in metabolic cages, and a 24-h urine sample was collected. The recoveries of radioactivity in the 24-h urine sample from rats and mice were 42 ± 2 and 55 ± 3% of the total radioactivity administered, respectively (p < 0.05). The 24- to 48-h urinary excretions of radioactivity from rats and mice were 4.2 ± 0.3 and 3.4 ± 0.5%, respectively. To assess a different route for administering IQ, F344 rats were also administered 20 mg/kg [14C]IQ (20 µCi; 1.25 ml/250 g injected i.p.; 20% DMSO-80% 0.1 N HCl) and placed in metabolic cages, and a 24-h urine sample was collected. The recovery of radioactivity in the 24-h urine sample was 55 ± 7% of the total radioactivity administered.
HPLC Analysis of Metabolites. Urine was analyzed for IQ metabolites by HPLC. Metabolites were assessed using a Beckman HPLC with System Gold software and a C18 ultrasphere column (5 µm, 4.6 x 150 mm) attached to a guard column. Analysis and purification of metabolites required the use of the following solvent systems at a flow rate of 1 ml/min: solvent system 1, the mobile phase contained 20 mM ammonium acetate (pH 5.0) in 2% acetonitrile for 0 to 5 min; 2 to 5% acetonitrile for 5 to 23 min; 5 to 10% acetonitrile for 24 to 29 min; 10 to 40% acetonitrile for 35 to 40 min; and 40 to 2% acetonitrile for 40 to 45 min; solvent system 2, the mobile phase contained 20 mM ammonium formate (pH 3.1) in 0% acetonitrile for 0 to 2 min; 0 to 4% acetonitrile for 2 to 22 min; 4 to 40% acetonitrile for 22 for 27 min; and 40 for 0% acetonitrile for 27 to 32 min; solvent system 3, the mobile phase was the same as solvent system 1, except the aqueous phase contained 20 mM ammonium formate (pH 3.1); solvent system 4, the mobile phase contained 20 mM ammonium acetate (pH 5.0) in 5% methanol for 0 to 3 min; 5 to 10% for 3 to 13 min; 10 to 90% for 18 to 23 min; and 90 to 5% for 25 to 30 min; solvent system 5, the mobile phase contained 20 mM ammonium acetate (pH 5.0) in 10% methanol for 0 to 15 min; 10 to 20% for 15 to 20 min; 20 to 90% for 20 to 25 min; and 90 to 10% for 30 to 35 min; solvent system 6, the mobile phase contained 20 mM ammonium acetate (pH 5.0) in 10% methanol for 0 to 2 min; 10 to 20% for 2 to 7 min; 20 to 30% for 7 to 17 min; 30 to 90% for 20 to 25 min; and 90 to 10% for 25 to 30 min; solvent system 7, the mobile phase contained 20 mM ammonium acetate (pH 5.0) in 6% acetonitrile with 6 to 12% acetonitrile for 0 to 6 min; 12 to 35% acetonitrile for 14 to 19 min; and 35 to 6% acetonitrile for 20 to 25 min; and solvent system 8, the mobile phase contained 20 mM ammonium formate (pH 3.1) in 6% acetonitrile with 6 to 7% acetonitrile for 0 to 10 min; 7 to 35% acetonitrile for 10 to 15 min; and 35 to 6% acetonitrile for 15 to 20 min. Radioactivity in HPLC eluents was continuously measured using a FLO-ONE (PerkinElmer Life and Analytical Sciences) radioactive flow detector. Data are expressed as a percentage of total radioactivity or nanomoles recovered after HPLC.
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For purification of mouse urine, metabolites were first separated using solvent system 1, individual fractions were collected, and samples were concentrated and applied to the next solvent system for further purification. N-Glucuronide was further purified with solvent systems 2 and 4, 5-O-glucuronide with systems 2 and 4, sulfamate with systems 2 and 5, 5-sulfate with systems 3 and 5, and demethyl-IQ and IQ with systems 3 and 6.
Qualitative Identification of Urinary and Liver Slice Metabolites. Metabolites were identified by their HPLC elution times relative to those of authentic products identified previously by us and by susceptibility to specific treatments (Armbrecht et al., 2007). Urine metabolites were purified before their susceptibility was assessed. Slice media was partially purified by extraction with ethyl acetate and metabolites in the aqueous fraction collected from a C18 Sep-Pak column before susceptibility was assessed. The 5-O-glucuronide was susceptible to E. coli β-glucuronidase (type VII-A) with 10 units of enzyme, 50 mM sodium acetate buffer at pH 6.8, and 0.3 µg of substrate/0.1 ml at 37°C for 4 h. 5-Sulfate was susceptible to abalone sulfatase (type VIII) with 200 units, 50 mM sodium acetate buffer at pH 5.0, and 0.3 µg of substrate/0.1 ml at 37°C for 72 h. N2-Glucuronide and sulfamate were hydrolyzed with 1 N HCl at 60°C for 2 h.
To determine whether N-acetyl-IQ was present in mouse urine samples, we synthesized this compound (see below) and then determined its elution profile with different HPLC solvent systems. Although it coeluted with IQ in our standard solvent system 1, N-acetyl-IQ was separated from IQ in system 3. Under this condition, a peak, which coelutes with N-acetyl-IQ (approximately 1% of recovered radioactivity), was observed in mouse urine. Urine samples were treated with carboxyesterase, which converts N-acetyl-IQ to IQ. When urine was incubated with esterase from porcine liver (25 units) in sodium phosphate buffer (pH 8.0) for 7 h at 37°C, no loss of the peak, which coelutes with N-acetyl-IQ, or increase in IQ was detected. Under these conditions, synthetic N-acetyl-IQ (0.025 mM) and N-acetylbenzidine (0.03 mM) were completely hydrolyzed to their corresponding free amine. Furthermore, whereas adding synthetic N-acetyl-IQ to urine resulted in complete hydrolysis of synthetic standard to IQ, residual radioactivity (approximately 1%) previously present remained unhydrolyzed. These results suggest that N-acetyl-IQ is not present in mouse urine. Similar studies with rat urine were unsuccessful. Subsequent studies determined that N-acetyl-IQ was not synthesized from IQ by mouse liver cytosol fractions containing N-acetyl-transferase activity for benzidine (see below).
Liver Slice IQ Incubation. Mouse liver slices were prepared with a Stadie-Riggs microtome as described previously (Lakshmi et al., 1995b) and incubated with Dulbecco's modified Eagle's media with high glucose containing 3.7 µg/ml NaHCO3 and 6 µg/ml Hepes. Approximately 150 to 200 mg of liver were placed in a 20-ml scintillation vial with 1 ml of media, gassed with 5% O2/95% CO2 for 1 min, and incubated at 37°C for 60 min. Media contained 0.06 mM IQ and where indicated 0.5 mM ellipticine or SKF-525A. To stop incubations, 1 ml of cold methanol was added, and samples were frozen. Thawed samples were sonicated three times for 15 s, samples were spun to remove protein and cell debris, and the supernatant was analyzed by HPLC using solvent system 1. Activity is expressed as a percentage of radioactivity recovered by HPLC.
Microsomal IQ Incubation. To identify specific pathways for IQ metabolism, liver microsomes were prepared using a procedure described previously (Zenser et al., 1978). Mice were sacrificed by cervical dislocation and open thoracotomy. Samples were stored in small aliquots at –70°C for the assays; unused material was discarded after one freeze-thaw cycle. Microsomes (1 mg/ml) were incubated in sodium phosphate buffer (pH 7.4), containing 0.1 mM diethylenetriaminepentaacetic acid, 1 mM NADPH, 5 mM MgCl2, and 0.06 mM [14C]IQ for 30 min at 37°C. Microsomes were preincubated with furafylline for 10 min before [14C]IQ addition. The reaction was linear with respect to protein concentration and time and was stopped by addition of an equal volume of methanol with 1 mM vitamin C. After 30 min on ice, the mixture was centrifuged to remove the precipitated protein. Supernatants were analyzed by HPLC, using solvent system 7. Activity is expressed as nanomoles or percentage of radioactivity recovered by HPLC as product. 7-Ethoxyresorufin O-deethylation (EROD) was determined by measuring the fluorescence of resorufin with 0.002 mM ethoxyresorufin (Pohl and Fouts, 1980).
Synthesis of N-Acetyl-2-amino-3-methylimidazo[4,5-f]quinoline and N-Acetyl-2-aminoimidazo[4,5-f]quinoline. To prepare synthetic N-acetyl-IQ, 26 µgof[14C]IQ was added to 1 mM acetic anhydride in 0.1 ml of pyridine for 15 min at 60°C. The reaction was stopped by the addition of distilled water (0.2 ml). This mixture was later diluted with 10 ml of water, applied to a C18 Sep-Pak column and washed with 10 ml of water, and the N-acetylated product was eluted with 3 ml of methanol. The product was purified by solvent systems 8, 7, and 6 and then identified by electrospray ionization (ESI) mass spectrometry (MS) in the positive-ion mode. The product exhibited [M + H]+, [M + Na]+, and [M + K]+ ions at m/z 241, 263, and 279, respectively. The [M + H]+ at m/z 241 gave rise to prominent ions at m/z 199, 184, and 157, representing IQ and consecutive losses of CH3 and HCN. This fragmentation pattern is consistent with the product being N-acetyl-2-amino-3-methylimidazo[4,5-f]quinoline.
N-Acetyl-2-aminoimidazo[4,5-f]quinoline was synthesized from demethyl-IQ as described above for N-acetyl-IQ, except for being only partially purified by immediate methylene chloride extraction because of its lability in methanol and aqueous solutions. The N-acetylated product was identified by ESI MS in the positive-ion mode. The product exhibited an [M + H]+ ion at m/z 227, which gave rise to prominent ions at m/z 185, 158, and 143 representing demethyl-IQ and consecutive losses of HCN and NH2-CN. N-Acetyl-2-aminoimidazo[4,5-f]quinoline was not stable in aqueous solution and hydrolyzed to demethyl-IQ.
We also attempted to prepare synthetic N-acetyl-IQ from mouse liver supernatant. The supernatant was saved from the microsomal preparation described above and used to assess N-acetyltransferase activity. Samples were stored in small aliquots at –70°C for the assays; unused material was discarded after one freeze-thaw cycle. Supernatant (1 mg/ml) was incubated (total volume 0.1 ml) in sodium phosphate buffer (pH 7.4), containing 1 mM acetyl CoA, 1 mM dithiothreitol, and 0.06 mM [14C]IQ or 3H-N-acetylbenzidine for 60 min at 37°C. The reaction was stopped by addition of 0.1 ml of methanol. Supernatants were analyzed by HPLC, using solvent system 8. Under these conditions, 70% of N-acetylbenzidine is converted to N,N'-diacetylbenzidine. However, no formation of N-acetyl-IQ could be detected.
Mass Spectral Analysis. Metabolites were identified by ESI MS. Analyses were performed with a Finnigan TSQ-7000 triple-stage quadrupole mass spectrometer (Thermo Electron Corporation, San Jose, CA) equipped with a Finnigan ESI source and controlled by Finnigan ICIS software operated on a DEC workstation. Samples were loop injected onto the ESI source with a Harvard syringe pump, which was continuously infused with methanol at a flow rate of 5 µl/min. The skimmer was at ground potential, and the electrospray needle was at 4.5 kV. The heated capillary temperature was 250°C. To obtain collisionally activated dissociation tandem mass spectra, the collision energy was set at 22 eV, and argon (2.3 mTorr) was used as target gas. The product ion spectra were acquired in the profile mode at the scan rate of one scan per 3 s.
Statistical Analysis. Data are expressed as mean ± S.E., and significant differences were evaluated using a Student's unpaired t test with p < 0.05.
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; Inamasu et al., 1989; Luks et al., 1989; Armbrecht et al., 2007). These include N-glucuronide, 5-O-glucuronide, sulfamate, and 5-sulfate. Although these same metabolites were also present in mouse urine, there was an additional metabolite observed, demethyl-IQ (Fig. 2, bottom). Table 1 reports the relative abundance of excreted metabolites using HPLC data illustrated in Fig. 2. The relative abundance of metabolites in these species was different. For rat, the proportion of metabolites excreted was 5-O-glucuronide > sulfamate > 5-sulfate > N-glucuronide. The presence of demethyl-IQ in rat urine was not confirmed by mass spectrometry. For mouse, the proportion of metabolites excreted was 5-O-glucuronide > demethyl-IQ > sulfamate > N-glucuronide > 5-sulfate. With HPLC solvent system 3, the amount of unmetabolized IQ in mouse urine was 2.3 ± 0.4% of recovered radioactivity. N-Acetyl-2-amino-3-methylimidazo[4,5-f]quinoline was not detected. The urinary IQ metabolite profile in mouse and rat is different, and demethyl-IQ is a major new metabolite in mouse. To determine whether the route of IQ administration is responsible for the lack of demethyl-IQ formation in rat, rats were administered IQ by i.p. injection. In these experiments, the proportion of metabolites excreted was sulfamate (48 ± 1%) > 5-O-glucuronide (15 ± 0.5%) = 5-sulfate (15 ± 0.5%) > N-glucuronide (4.8 ± 0.4%). The amount of radioactivity that coeluted with demethyl-IQ was 1.9 ± 0.2% of the total radioactivity after HPLC and not further identified.
Identification of Urinary IQ Metabolites. Rat metabolites were identified by their HPLC elution profile relative to authentic products previously identified by us (Armbrecht et al., 2007) and their susceptibility to specific treatments. The N-glucuronide and sulfamate were susceptible to treatment with 1 N HCl at 60°C, whereas 5-O-glucuronide and 5-sulfate were susceptible to β-glucuronidase and sulfatase, respectively. Rat metabolites were only susceptible to the treatments indicated.
Mouse metabolites were further identified by their elution profile relative to rat metabolite standards (Armbrecht et al., 2007), susceptibility to specific treatments described above for rat, and ESI MS spectra in Fig. 3. In the negative-ion mode, the 12-min peak exhibited an [M – H]– at m/z 373 and gave prominent ions at m/z 239 and 197, representing N-acetyl-IQ and IQ. The 12-min metabolite is IQ-N2-glucuronide (Fig. 3A). The 17-min peak, in the negative-ion mode, yielded deprotonated [M – H]– molecular ion at m/z 389 and gave prominent ions at m/z 213 and 198, representing 5-OH-IQ and loss of CH3. This is consistent with the 17-min product being IQ-5-O-glucuronide (Fig. 3B). The 19.5-min peak in the negative-ion mode exhibited an [M – H]– ion at m/z 277 and gave rise to prominent ions at m/z 197 and 182, representing IQ and loss of CH3. The ion at m/z 80 corresponded to SO3. This fragmentation pattern is consistent with the 19.5-min peak being IQ-sulfamate (Fig. 3C). For the 26-min peak, the negative-ion mode displayed an [M – H]– ion at m/z 293 and gave prominent ions at m/z 213 and 198, representing 5-OH-IQ and loss of CH3. The ion at m/z 80 corresponds to SO3. In the positive-ion mode, the product yielded [M + Na]+ at m/z 317. The 26-min metabolite is IQ-5-sulfate (Fig. 3D). Urinary IQ was identified by its coelution with authentic commercial material and by its mass spectra (Fig. 3E). In the positive-ion mode, the [M + H]+ at m/z 199 gave prominent ions at m/z 184, 157, and 131 representing consecutive losses of CH3, HCN, and C2H2, respectively. The spectra of the commercial starting material were identical to that of urinary IQ. The ESI MS fragmentation patterns of all the metabolites matched their corresponding metabolite in rat. This is consistent with the mouse and rat metabolites being identical to one another and not isomers.
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The ESI MS spectra of the new metabolite eluting at 31.5 min is illustrated in Fig. 4. In the positive mode, this urinary metabolite exhibited [M + H]+ at m/z 185. The product ion spectra of m/z 185 along with its fragmentation pattern is consistent with it being 2-aminoimidazo[4,5-f]quinoline (demethyl-IQ).
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Evaluation of Demethyl-IQ Formation by Mouse Liver Microsomes. To examine P450 N-demethylase catalyzed metabolism of IQ in more detail, liver microsomal metabolism was examined. Consistent with liver slice data, Demethyl-IQ was a product of liver microsomal metabolism and its formation was inhibited by ellipticine (Fig. 6). Demethyl-IQ was the only metabolite of IQ observed. In similar studies with rat microsomes, Demethyl-IQ was not detected (not shown). The ESI MS spectrum of the microsomal product was identical to that illustrated in Fig. 4. To determine the P450(s) responsible for demethyl-IQ formation, inhibitors of different P450s previously used to assess N-demethylation were examined (Iribarne et al., 1996; Yamagata et al., 1998). In Table 3, ellipticine, a P450 1A inhibitor, was the only inhibitor to completely prevent demethyl-IQ formation at 0.02 mM. At this concentration, inhibition was also observed with -naphthoflavone (27% of total nanomoles; 1A1/1A2 inhibitor), SKF-525A (74%; nonspecific inhibitor), and ketoconazole (64%; 3A inhibitor). No inhibition was detected with 0.02 mM furafylline (1A2 inhibitor) or 0.1 mM sulfaphenazole (2C inhibitor). To assess the efficiency of ellipticine inhibition of N-demethylation, a range of concentrations was used and a Dixon plot determined the Ki to be 0.001 mM. EROD is thought to represent 1A1 activity and was used to further evaluate ellipticine inhibition. By using Kinetic plots EROD Ki values for ellipticine and furafylline were determined to be 0.0008 and 0.011 mM, respectively. These results are consistent with P450 1A1 mediating N-demethylation of IQ in mouse liver.
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Analysis of Mouse Urine for Demethyl-IQ Metabolites. To determine whether demethyl-IQ is further metabolized, purified demethyl-IQ (10 µg) was injected i.p. into a mouse, and a 24-h urine sample was collected. No metabolism of demethyl-IQ was detected (not shown). We have synthesized N-acetyl-demethyl-IQ and determined that it elutes at 41.5 min (Fig. 2). Thus, this N-acetylated product was not detected in urine from a mouse injected with either IQ or demethyl-IQ. Additional studies demonstrated that demethyl-IQ was not metabolized by liver microsomes or N-acetylated by liver cytosol (not shown). Thus, demethyl-IQ does not further contribute to mouse metabolites derived from IQ.
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Discussion |
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). Demethyl-IQ is a major metabolite in monkey (Snyderwine et al., 1992). Mice have a high capacity for metabolizing IQ as little IQ was found unmetabolized (1.2 ± 0.3%) in urine after administration of a high dose of IQ (40 mg/kg). Results are consistent with mouse IQ metabolism being significantly different from that in rat and with demethyl-IQ being a major new rodent metabolite unique to the mouse.
It is not clear whether rats N-demethylate IQ. Although we reported demethyl-IQ in rat urine (Table 1), this finding was not confirmed by ESI MS. In a prior study in which rats were given an oral dose of IQ (40 mg/kg), the 24-h urine was thought to contain demethyl-IQ along with some other nonpolar metabolites, including IQ, N-acetyl-IQ, and 7-OH-IQ (Inamasu et al., 1989). This nonpolar urine fraction represented less than 1% of the dose administered. The metabolites were only identified by their coelution with synthetic standards. Another study by this group provided MS and nuclear magnetic resonance structural identification for the sulfamate, 5-O-glucuronide, 5-sulfate, and N-glucuronide conjugates of IQ, but no further mention of demethyl-IQ or the other nonpolar metabolites (Luks et al., 1989). Studies of rat IQ metabolism by other investigators have not demonstrated the formation of demethyl-IQ (Alexander et al., 1989). However, rat liver microsomes N-demethylate dacarbazine by 1A enzymes (Yamagata et al., 1998). Thus, structural proof for the presence of demethyl-IQ in rat urine has not been demonstrated and, if present, N-demethylation would only be a minor pathway compared with that in mouse.
Results suggest that the route of IQ administration does not affect the formation of demethyl-IQ in the rat. With either oral or i.p. administration of IQ, less than 2% of radioactivity recovered after HPLC coeluted with demethyl-IQ compared with 26 ± 1.6% in mouse. Because this could not be confirmed by ESI MS, it is not clear whether rats N-demethylate IQ. Although excretion of sulfamate and 5-O-glucuronide was similar after oral IQ administration in the rat, the excretion of sulfamate was 3-fold greater than that of 5-O-glucuronide after i.p. injection. This change in metabolite distribution is probably due to higher plasma concentrations of IQ after i.p. injection with higher concentrations of IQ within hepatocytes saturating P450 metabolism and allowing more sulfamate formation.
Studies with monkey identified three demethyl-IQ metabolites in urine (Snyderwine et al., 1992): demethyl-IQ, demethyl-IQ N-glucuronide, and demethyl-7-oxo-IQ. The latter was thought to be derived from intestinal bacterial metabolism of demethyl-IQ. Approximately, 16 to 22% of the dose given at 2.2 µmol/kg IQ in two monkeys was derived from N-demethylation. In contrast to monkey, we observed no further metabolism of demethyl-IQ. Human hepatocytes N-demethylate a HCA structurally similar to IQ (Langouët et al., 2001). Demethyl-IQ is mutagenic (Barnes et al., 1985). Thus, human, monkey, and mouse N-demethylate HCAs.
Although N-acetylation is a characteristic feature of aromatic amine metabolism, this pathway does not appear to be active in heterocyclic amine metabolism. We were unable to detect N-acetyl-IQ in mouse urine or as a product of mouse liver cytosol N-acetyltransferase activity. The lack of N-acetyltransferase (NAT1 and NAT2) N-acetylation of IQ and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline has been attributed to their 3-methyl group. Results with N-acetylation of ortho-toluidine compared with those for para-toluidine suggested that there is steric hindrance of the exocyclic amine group resulting from methyl group substitution ortho to the amine (Hein et al., 1993). However, because N-acetylation of demethyl-IQ was not demonstrated either in vitro or in vivo, steric hindrance by the 3-methyl group does not appear to be the reason for the lack of N-acetylation of these heterocyclic amines. In aqueous solutions, chemically synthesized N-acetyl-IQ was stable, whereas N-acetyl-demethyl-IQ was not stable. In addition, the N-glucuronides of aromatic amines are quite acid-labile, i.e., the N-glucuronide of N-acetylbenzidine has a half-life at pH 5.5 of approximately 8 min (Babu et al., 1995), whereas N-glucuronides of these heterocyclic amines are relatively acid-stable (see Materials and Methods). Results suggest that the chemistry involving the exocyclic amine of these heterocyclic compounds is different from that of aromatic amines and may also contribute to the lack or low rate of N-acetylation of heterocyclic amines. For example, the electron withdrawing effects of the nitrogen substituents of the imidazole ring may reduce the reactivity of the exocyclic amine or the amine may preferentially exist as an imine tautomer, making it less likely to be N-acetylated.
A variety of inhibitors were used to determine the P450(s) responsible for N-demethylation of IQ. The 1A inhibitor ellipticine was effective in liver slices and also in microsomal incubations with a Ki for demethyl-IQ formation of 0.001 mM. At the concentration of 0.02 mM used for all inhibitors (Table 3), other compounds exhibited inhibition with -naphthoflavone > ketoconazole > SKF-525A. The former, like ellipticine, is an inhibitor of both 1A1 and 1A2 and would be expected to be an effective inhibitor of 1A1-catalyzed reactions. Ketoconazole has been reported to be an effective inhibitor of 1A2 in rat (Kobayashi et al., 2003) and may exhibit some 1A1-like activity in mouse. SKF-525A is a nonspecific inhibitor. The lack of effect of furafylline, a 1A2-selective inhibitor, further demonstrates the apparent selectivity of 1A1 for IQ N-demethylation. Although P450 3A does not mediate mouse liver IQ N-demethylation, previous studies have shown 3A to be expressed in this tissue and to catalyze N-demethylation of cocaine (Pellinen et al., 1994; Martignoni et al., 2006). EROD activity demonstrated preferential inhibition by ellipticine compared with furafylline similar to that observed for IQ N-demethylation. In addition, mouse liver benzo[a]pyrene hydroxylase, an activity attributed to 1A1, was inhibited by 0.005 mM -naphthoflavone but not by 0.02 mM furafylline (Tsyrlov et al., 1993). That study also demonstrated preferential inhibition of mouse 1A2 by furafylline compared with 1A1. Thus, mouse IQ N-demethylation appears to be catalyzed by hepatic P450 1A1.
Because of our interest in the role of colitis in colon cancer (Cooper et al., 2000; Clapper et al., 2006), we assessed IQ metabolism in female C57BL/6 mice administered DSS in their drinking water. After three cycles of DSS to induce colitis, an oral dose of 40 mg/kg IQ was administered, and analysis was performed as described in this report. Although urinary 5-O-glucuronide excretion decreased from 30.8 ± 2.5% in controls to 17.2 ± 2.1% of total radioactivity for DSS-treated mice, the distribution of other excreted metabolites was not different. These results are consistent with a similar study in which rats were administered DSS at 3% in drinking water for 7 days. Decreases in several hepatic P450 enzyme activities, including P450 1A1 and A2 phenacetin O-deethylation activity, were reported (Masubuchi and Horie, 2004). Inflammation decreases P450-mediated metabolism (for review, see Morgan, 1997), and this is a likely mechanism by which DSS decreased 5-O-glucuronide formation.
In conclusion, this study demonstrated for the first time that N-demethylation is an important pathway for IQ metabolism in mouse, possibly mediated by P450 1A1. However, the constitutive expression of 1A1 in mouse liver is low (Dey et al., 1999), and additional studies may be needed to determine the P450 responsible for demethyl-IQ formation. Demethyl-IQ is mutagenic (Barnes et al., 1985). N-Demethylation is a prominent pathway in mouse, monkey, (Snyderwine et al., 1992), and human (Langouët et al., 2001) and may play a role in IQ tumorigenicity in these species. Differences in metabolism between mouse and rat, especially demethyl-IQ formation, suggests that there is more similarity of mouse to human metabolism of HCAs than to rat metabolism and that the mouse is a useful model for testing tumorigenicity of these chemicals.
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Acknowledgments |
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Footnotes |
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
ABBREVIATIONS: IQ, 2-amino-3-methylimidazo[4,5-f]quinoline; HCA, heterocyclic amine; P450, cytochrome P450; DSS, dextran sulfate sodium; demethyl-IQ, 2-aminoimidazo[4,5-f]quinoline; SKF-525A, 2-diethylaminoethyl 2:2-diphenylvalerate hydrochloride; DMSO, dimethylsulfoxide; HPLC, high-performance liquid chromatography; N-acetyl-IQ, N-acetyl-2-amino-3-methylimidazo[4,5-f]quinoline; EROD, 7-ethoxyresorufin O-deethylation; ESI, electrospray ionization; MS, mass spectrometry.
Address correspondence to: Dr. Terry V. Zenser, VA Medical Center (11G-JB), St. Louis, MO 63125. E-mail: zensertv{at}slu.edu
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