QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Frederiksen, H. Articles by Frandsen, H. Search for Related Content PubMed PubMed Citation Articles by Frederiksen, H. Articles by Frandsen, H.

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

Institute of Food Safety and Nutrition, Danish Veterinary and Food Administration, Søborg, Denmark

(Received November 3, 2003; accepted March 12, 2004)

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
2-Amino-3-methyl-9H-pyrido[2,3-b]indole (MeAC) is a proximate mutagenic and carcinogenic heterocyclic amine formed during ordinary cooking. In model systems, MeAC can be formed by pyrolyses of either tryptophan or proteins of animal or vegetable origin. In the present study, the in vivo metabolism of MeAC in rats was investigated. Rats were dosed with tritium-labeled MeAC, and urine and feces were collected over 3 days. The metabolites of MeAC were identified by high performance liquid chromatography-mass spectrometry and quantified by liquid scintillation counting. Conjugated metabolites were characterized by enzymatic hydrolyzes with ß-glucuronidase or arylsulfatase. The data showed that the metabolic pattern of MeAC was similar in all rats. About 65% of the dose was excreted in urine and feces, and the major amount of MeAC-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 MeAC and two phase I metabolites, 6-OH-MeAC and 7-OH-MeAC, the following conjugated metabolites were identified: MeAC-N²-glucuronide, AC-3-CH₂O-glucuronide, 3-carboxy-AC and 3-carboxy-AC-glucuronide, and sulfate and glucuronide conjugates of 6-OH-MeAC and 7-OH-MeAC. Also, a large amount of a rather unstable compound proposed to be of MeAC-N1-glucuronide was found. About 21% of the dose was excreted in feces during the first 24 h, and MeAC and 7-OH-MeAC were the only compounds identified in feces. Any activated metabolites of MeAC were not detected in rat urine or feces.

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 N²-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 AC, whereas these enzymes only activate a few percent of MeAC (Frederiksen and Frandsen, 2003). Activated heterocyclic amines are able to form adducts with macromolecules such as protein and DNA. It was shown by ³²P-postlabeling analysis that MeAC and AC both form one major DNA adduct (N²-deoxyguanin-8-yl-compounds) in primary hepatocytes from rats (Pfau et al., 1996, 1997). The in vitro metabolism of AC in human and rodent hepatic microsomes and the metabolism of MeAC 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 AC and MeAC in hepatic microsomes from PCB-induced rat, uninduced rat, and human. AC was metabolized to two major detoxified metabolites, and MeAC was metabolized to three major detoxified metabolites. Amounts of both -carbolines were activated to their corresponding N²-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 PCB-induced 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 MeAC in rats. Metabolites excreted in urine and feces have been separated, quantified, and identified.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Chemicals. MeAC was obtained from Toronto Research Chemicals (Toronto, Ontario, Canada). Tritiation of MeAC ([³H]MeAC) was described previously (Frandsen et al., 1998). Reference compounds 3-CH₂OH-AC, 6-OH-MeAC, and 7-OH-MeAC were prepared in microsomal incubations, and N-OH-MeAC was prepared by chemical synthesis as previously described (Frederiksen and Frandsen, 2002). ß-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 [³H]MeAC (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 µl 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 µl, 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 µl of 50% dimethyl formamide and diluted with 300 µl 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 µl 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 µl 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 µl of 50% dimethyl formamide and diluted with 150 µl 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 5-µm, 150 x 3 mm column from Agilent Technologies. Injection volume was 12.5 µl, 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 MeAC 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 MeAC metabolites, individual metabolite peaks from the HPLC chromatograms were collected and treated with ß-glucuronidase or arylsulfatase. Two hundred fifty microliters of purified urine was injected in aliquots of 12.5 µl 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 µl of 50% dimethyl formamide, and diluted with 150 µl of water. Five microliters of each fraction were analyzed by liquid scintillation counting, and 5 µl were added to 45 µl of 50 mM acetate buffer, pH 5.5, and analyzed by HPLC-mass spectrometry. Hydrolysis was performed by addition of 195 µl of 50 mM acetate buffer, pH 5.5, and 5 µl of ß-glucuronidase or arylsulfatase to 50 µl 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 scintillation counting.

	Results

Top Abstract Materials and Methods Results Discussion References

 
Recovery of Radioactivity in Urine and Feces. Three rats were dosed with [³H]MeAC, and urine and feces were collected over 3 days. The total recovery of radioactivity excreted in urine and feces was 64, 61, and 70% of the dose in rat 1, rat 2, and rat 3, respectively. Table 1 shows the percentages of the dose recovered in urine and feces to 24, 48, and 72 h. In urine, a small amount of the radioactivity was bound to urine precipitate. The major amount of radioactivity (54.6%) was found in 0- to 24-h samples; about 33.9% of the dose was excreted in urine, and about 20.7% of the dose was excreted in feces during the first 24 h. A minor amount, about 5.7% of radioactivity, was found in feces in the 24- to 48-h collection. Only a few percent of the radioactivity were found in urine collected after 48 and 72 h and in feces collected after 72 h.

Separation of MeAC Metabolites. Figure 1 shows a radioactivity profile (Fig. 1A) of 1-min fractions collected from the HPLC separation (Fig. 1B) of metabolites in urine from rat 1, collected to 0 to 24 h. MeAC (peak 1) and 11 radioactive metabolites (peaks 2-12) associated with MeAC were detected in urine. In the 24-h urine samples, about 3.5% of the radioactive dose was identified as MeAC (Table 2). The major metabolite (peak 2) accounted for about 13.1% of the radioactive dose. Metabolite 3, 4, 6, 9, 10, and 12 were excreted in lower amounts, 1.1 to 4.8% each, whereas metabolites 5, 7, 8, and 11 were excreted in very low amounts, <1% each of the total radioactive dose.

View larger version (29K): [in this window] [in a new window]   FIG. 1. Comparison of chromatographic profiles of radioactivity in 1-min fractions (A) and UV chromatogram (B) of urine from rat 1, 0 to 24 h.

View this table: [in this window] [in a new window]   TABLE 2 Percentage of dose excreted as MeAC-metabolites in urine and feces Results are expressed as mean ± S.D., n = 3.

In addition to unmetabolized MeAC, metabolites 2, 3, 4, 10, and 12 were detected in urine collected after 48 h, and only metabolite 4 was found in urine collected after 72 h. Both MeAC and the five metabolites were excreted in very low amounts. Each of them accounted for <1% of the total radioactive dose.

A large amount of unmetabolized MeAC was excreted in feces collected after 24 and 48 h, about 17.1% and 5.3%, respectively. Only metabolite 10 (3.5%) was detected in the fecal sample collected after 24 h. The excretion pattern of MeAC metabolites in the three rats was rather similar (Table 2).

Identification of Excreted MeAC Metabolites. All metabolites, except metabolite 5, were eluted before the parent compound, MeAC (Fig. 1B). The mass spectra of metabolites 7 and 10 showed molecular ions [M + H]⁺ at m/z 214, indicating that they are hydroxylated metabolites. An earlier identification of the phase I metabolites from MeAC by electrospray mass spectrometry and ¹H NMR has shown three major detoxified metabolites, characterized as 3-CH₂OH-AC, 6-OH-MeAC, and 7-OH-MeAC (Frandsen et al., 1998). UV spectra of metabolites 7 and 10 were identical with the UV spectra of 6-OH-MeAC and 7-OH-MeAC, respectively (data not shown). Also, HPLC retention times (Rt) at 15.3 and 17.2 min of metabolite 7 and 10, respectively (Table 3), are identical with the retention times for 6-OH-MeAC and 7-OH-MeAC.

Metabolites 9 and 12 (Rt 17.2 and 19.9 min), were identified as MeAC-6-O-sulfate and MeAC-7-O-sulfate, respectively (Table 3). Mass spectral analysis showed molecular ions [M + H]⁺ at m/z 294, both with daughter ions [M + H]⁺ at m/z 214, indicating the loss of SO₃^- groups. Enzymatic hydrolysis of these metabolites with arylsulfatase resulted in the liberation of 6-OH-MeAC and 7-OH-MeAC, respectively.

Metabolites 4, 8, and 11 (Rt 18.2, 13.1, and 14.4 min) were identified as glucuronic acid conjugates of 3-CH₂OH-AC, 6-OH-MeAC, and 7-OH-MeAC, respectively (Table 3). Mass spectral analysis showed primary molecular ions [M + H]⁺ at m/z 390, all with daughter ions [M + H]⁺ at m/z 214, formed by collision-induced decomposition in the interphase. The loss of a 176-Da fragment indicates cleavage of a hydrogenated glucuronic acid. Enzymatic hydrolysis of these metabolites with ß-glucuronidase showed that they were quite susceptible to hydrolysis with ß-glucuronidase. The resulting hydrolysis products were chromatographically, UV spectrally, and mass spectrally identical with 3-CH₂OH-AC, 6-OH-MeAC, and 7-OH-MeAC, respectively.

Metabolites 2 and 3 eluted at about 24.2 and 14.8 min, respectively (Table 3). Mass spectral analysis of both metabolites showed molecular ions [M + H]⁺ at m/z 374, both with daughter ions [M + H]⁺ at m/z 198. The loss of a 176-Da fragment indicates the cleavage of hydrogenated glucuronic acid. The tailing of peak 2 (Fig. 1B) indicates that metabolite 2 was very unstable and decomposed in the HPLC system with formation of MeAC. Reanalysis of metabolite 2 after fraction collection confirmed the instability of this metabolite, since MeAC was the only compound detected in the collected fraction. Also, storage of the HPLC sample for 8 h at room temperature before analysis resulted in disappearance of metabolite 2 and an increase in MeAC. Enzymatic hydrolysis of metabolite 3 showed that it was susceptible to hydrolysis with ß-glucuronidase. The resulting hydrolyzed compound was chromatographically, UV spectrally, and mass spectrally identical with the parent compound, MeAC, based on the above data. We therefore propose that metabolite 2 is MeAC-N1-glucuronide and metabolite 3 is MeAC-N²-glucuronide.

Metabolite 5 (Rt 28.6 min) is proposed to be 3-carboxy-AC (Table 3). Mass spectral analysis showed a molecular ion [M + H]⁺ at m/z 228. The 30-Da increase in molecular weight is in accordance with oxidation of the methyl group to a carboxyl group. A similar oxidation of a methyl substituent to a carboxylic acid resulting in a 30-Da increase in the molecular weight has previously been reported for 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) (Turesky et al., 2001). Metabolite 6 (Rt 20.6 min) is proposed to be a glucuronic acid conjugate of 3-carboxy-AC. Mass spectral analysis showed a molecular ion [M + H]⁺ at m/z 404 with a daughter ion [M + H]⁺ at m/z 228. The loss of 176 Da indicates cleavage of a hydrogenated glucuronic acid conjugate. Enzymatic hydrolysis of this metabolite with ß-glucuronidase resulted in a compound with mass, UV spectra, and retention time identical to those of the proposed 3-carboxy-AC. Further analysis is needed to fully characterize this metabolite.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
The phase I metabolism of MeAC has previously been characterized (Frandsen et al., 1998; King et al., 2000; Frederiksen and Frandsen, 2002, 2003); however, the phase II metabolism and the excretion of metabolites in vivo have not been reported before. In the present study, we have characterized the metabolites of MeAC excreted in urine and feces from three rats dosed with MeAC. The excretion pattern and the metabolic profile were similar in the three rats.

The total recovery of the radiolabeled MeAC in urine and feces was about 65% following oral exposure. This is a low amount compared with other in vivo studies of heterocyclic amines; e.g., the total recovery of urinary metabolites in rats was about 90% after dosing with 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and MeIQx and about 70 to 80% after dosing with imidazo[4,5-f]quinoxaline; however, the metabolism of heterocyclic amines is highly species- and compound-dependent (King et al., 2002). The largest amount (55%) of radiolabeled MeAC was excreted during the first 24 h, 34% in urine and 21% in feces. HPLC analyses revealed the presence of 11 metabolites in addition to the parent compound, MeAC. The metabolites were identified by UV spectroscopy and mass spectrometry, and conjugated metabolites were further characterized by hydrolysis with arylsulfatase or ß-glucuronidase. In urine, unmetabolized MeAC was excreted in low amounts (about 3.5%). Two metabolites were readily identified as 6-OH-MeAC and 7-OH-MeAC (metabolites 7 and 10, respectively) by comparison with standards. Four metabolites (8, 9, 11, and 12) were characterized as sulfate and glucuronic acid conjugates of 6-OH-MeAC and 7-OH-MeAC, since these compounds were liberated following treatment with arylsulfatase or ß-glucuronidase. Metabolite 4 was characterized as a glucuronic acid conjugate of 3-CH₂OH-AC by hydrolysis with ß-glucuronidase followed by comparison with a standard. Among these metabolites, the major metabolites found in urine were AC-3-CH₂O-glucuronide and MeAC-7-O-sulfate, accounting for about 2.7% and 4.8% of the radioactive dose. Mass spectral analysis of metabolites 2 and 3 showed that both were glucuronic acid conjugates of MeAC; metabolite 3 was stable, whereas metabolite 2 spontaneously lost the glucuronic acid moiety.

Several studies on the in vivo metabolism of heterocyclic amines showed that the exocyclic amine groups are conjugated to stable N-glucuronides (King et al., 2002; Yanazoe and Nagata, 2002); therefore, it is proposed that metabolite 3, which was a stable N-glucuronide conjugate, is MeAC-N²-glucuronide. This leaves two possibilities for the position of the glucuronic acid moiety of metabolite 2: N1- or N9-position. Direct phase II conjugation of pyridine nitrogen with glucuronic acid has previously been reported. The major urinary metabolite of cotinine in smokers was identified as cotinine glucuronide (Caldwell et al., 1992), and the major metabolite in baboon urine after treatment with thiadiazinone was the quaternary N-glucuronide (McKillop et al., 1990). In both cases, analyses confirmed the site of glucuronic acid conjugation as the nitrogen in the pyridine ring, which results in a positive charge on the pyridine nitrogen. These compounds appear to be unstable, resulting in broad peaks in the chromatographic systems. They are easily decomposed under physical conditions, such as mass spectrometry (McKillop et al., 1990). Another study confirms that humans and rats are able to form and excrete unstable N-glucuronide conjugates of tripelennamine (Chaudhuri et al., 1976). Based on the reported instability of pyridine-nitrogen-glucuronic acid conjugates, we propose that the similar unstable metabolite 2 is MeAC-N¹-glucuronide. The proposed MeAC-N¹-glucuronide metabolite accounts for the largest amount of excreted radioactivity (13% of the radioactive dose), but because of the labile behavior of this metabolite, the excreted amount could be even higher.

An in vitro study of the metabolism of MeAC in rat hepatic microsomal incubations has shown that the major phase I metabolite was 3-CH₂OH-AC (Frederiksen and Frandsen, 2002). It would therefore be expected that this metabolic pathway would also dominate in the in vivo phase I metabolism of MeAC. However, only a small amount of conjugated 3-CH₂OH-AC (metabolite 4) was found in the urine of rats dosed with MeAC. Isoenzymes of alcohol dehydrogenase oxidize larger aliphatic and aromatic alcohols to aldehydes, which are further oxidized to carboxylic acids by aldehyde dehydrogenase, which is a common metabolic pathway in the liver in both humans and rats (Parkinson, 1996). Metabolite 5 is proposed to be 3-carboxy-AC, formed by oxidation of the alcohol moiety of 3-CH₂OH-AC to the carboxylic acid. Such a structure is in accordance with the mass spectral data. A similar oxidation of a methyl substituent on a heterocyclic amine to a carboxylic acid has previously been reported for MeIQx (Turesky et al., 2001). Mass spectral data indicate that metabolite 6 is a glucuronic acid conjugate, which by enzymatic hydrolysis was degraded to 3-carboxy-AC. Further analysis is needed to fully characterize this metabolite. Figure 2 shows a proposed metabolic pathway for MeAC.

View larger version (13K): [in this window] [in a new window]   FIG. 2. Proposed in vivo metabolic pathway of MeAC in the rat.

All the identified metabolites were detoxification products, which means that at least 65% of the MeAC dose was detoxified or unmetabolized. Previously, we have found a 50/50 distribution between detoxification and activation in an in vitro study in which rat microsomes were incubated with MeAC (Frederiksen and Frandsen, 2002). Hydroxylation of the exocyclic amino group, followed by glucuronic acid conjugation, is a common route for detoxification of phase I activated heterocyclic amines (King et al., 2002; Yanazoe and Nagata, 2002), but in this in vivo study, we did not detect any activated metabolites excreted in urine or feces. About 2% of the dose was bound to urine precipitate, and it is possible that activated N²-OH-MeAC could be bound to proteins or other macromolecules in this precipitate. The total recovery of the radiolabeled MeAC in urine and feces in this study was only about 65%; therefore, further studies of the covalent binding of MeAC to macromolecules in rat organs are apparently needed.

	Acknowledgments

 
We thank Helle E. Gluver for technical assistance.

	Footnotes

 
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: AC, 2-amino-9H-pyrido[2,3-b]indole; HPLC, high performance liquid chromatography; MeAC, 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: hf{at}fdir.dk

	References

Top Abstract Materials and Methods Results Discussion References

 


Caldwell WS, Greene JM, Byrd GD, Chang KM, Uhrig MS, deBethizy JD, Crooks PA, Bhatti BS, and Riggs RM (1992) Characterization of the glucuronide conjugate of cotinine: a previously unidentified major metabolite of nicotine in smokers' urine. Chem Res Toxicol 5: 280-285.[CrossRef][Medline]

Chaudhuri NK, Servando OA, Manniello MJ, Luders RC, Chao DK, and Bartlett MF (1976) Metabolism of tripelennamine in man. Drug Metab Dispos 4: 372-378.[Abstract]

Eisenbrand G and Tang W (1993) Food-borne heterocyclic amines. Chemistry, formation, occurrence and biological activities. A literature review. Toxicology 84: 1-82.[CrossRef][Medline]

Frandsen H, Damkjaer S, Grivas S, Andersson R, Binderup ML, and Larsen JC (1998) Microsomal metabolism and activation of the environmental carcinogen 2-amino-3-methyl-9H-pyrido[2,3-b]indole. Mutagenesis 13: 181-185.[Abstract/Free Full Text]

Frederiksen H and Frandsen H (2002) In vitro metabolism of two heterocyclic amines, 2-amino-9H-pyrido[2,3-b]indole (AC) and 2-amino-3-methyl-9H-pyridol2,3-b]indole (MeAC) in human and rat hepatic microsomes. Pharmacol Toxicol 90: 127-134.[CrossRef][Medline]

Frederiksen H and Frandsen H (2003) Impact of five cytochrome P450 enzymes on the metabolism of two heterocyclic aromatic amines, 2-amino-9H-pyrido[2,3-b]indole (AC) and 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeAC). Pharmacol Toxicol 92: 246-248.[CrossRef][Medline]

Gross GA and Gruter A (1992) Quantitation of mutagenic/carcinogenic heterocyclic aromatic amines in food products. J Chromatogr 592: 271-278.[CrossRef][Medline]

King RS, Kadlubar FF, and Turesky R (2002) In vivo metabolism, in Food Borne Carcinogens: Heterocyclic Amines (Nagao M and Sugimura T eds) pp 90-111, John Wiley & Sons Ltd., West Sussex, UK.

King RS, Teitel CH, and Kadlubar FF (2000) In vitro bioactivation of N-hydroxy-2-amino--carboline. Carcinogenesis 21: 1347-1354.[Abstract/Free Full Text]

Knize MG, Salmon CP, Hopmans EC, and Felton JS (1997) Analysis of foods for heterocyclic aromatic amine carcinogens by solid-phase extraction and high-performance liquid chromatography. J Chromatogr A 763: 179-185.[CrossRef][Medline]

Layton DW, Bogen KT, Knize MG, Hatch FT, Johnson VM, and Felton JS (1995) Cancer risk of heterocyclic amines in cooked foods: an analysis and implications for research. Carcinogenesis 16: 39-52.[Abstract/Free Full Text]

Manabe S, Wada O, and Kanai Y (1990) Simultaneous determination of amino--carbolines and amino--carbolines in cigarette smoke condensate by high-performance liquid chromatography. J Chromatogr 529: 125-133.[Medline]

Matsumoto T, Yoshida D, Mizusaki S, and Okamoto H (1977) Mutagenic activity of amino acid pyrolyzates in Salmonella typhimurium TA 98. Mutat Res 48: 279-286.[Medline]

Matsumoto T, Yoshida D, and Tomita H (1981) Determination of mutagens, amino--carbolines in grilled foods and cigarette smoke condensate. Cancer Lett 12: 105-110.[CrossRef][Medline]

McKillop D, Pickup KJ, Swaisland AJ, and Holmes BF (1990) The metabolic disposition of an orally active pyridyl thiadiazinone cardiotonic agent (MPTD) in rat and baboon. Xenobiotica 20: 401-415.[Medline]

Nagao M, Fujita Y, Wakabayashi K, and Sugimura T (1983) Ultimate forms of mutagenic and carcinogenic heterocyclic amines produced by pyrolysis. Biochem Biophys Res Commun 114: 626-631.[CrossRef][Medline]

Niwa T, Yamazoe Y, and Kato R (1982) Metabolic activation of 2-amino-9H-pyrido[2,3-b]indole by rat-liver microsomes. Mutat Res 95: 159-170.[CrossRef][Medline]

Ohgaki H, Matsukura N, Morino K, Kawachi T, Sugimura T, and Takayama S (1984) Carcinogenicity in mice of mutagenic compounds from glutamic acid and soybean globulin pyrolysates. Carcinogenesis 5: 815-819.[Abstract/Free Full Text]

Parkinson A (1996) Biotransformation of xenobiotics, in Casarett and Doull's Toxicology: The Basic Science of Poisons, 5th ed (Klaassen CD ed) pp 113-189, The McGraw-Hill Companies Inc., New York.

Pfau W, Brockstedt U, Schulze C, Neurath G, and Marquardt H (1996) Characterization of the major DNA adduct formed by the food mutagen 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeAC) in primary rat hepatocytes. Carcinogenesis 17: 2727-2732.[Abstract/Free Full Text]

Pfau W, Schulze C, Shirai T, Hasegawa R, and Brockstedt U (1997) Identification of the major hepatic DNA adduct formed by the food mutagen 2-amino-9H-pyrido[2,3-b]indole (AC). Chem Res Toxicol 10: 1192-1197.[CrossRef][Medline]

Raza H, King RS, Squires RB, Guengerich FP, Miller DW, Freeman JP, Lang NP, and Kadlubar FF (1996) Metabolism of 2-amino--carboline. A food-borne heterocyclic amine mutagen and carcinogen by human and rodent liver microsomes and by human cytochrome P4501A2. Drug Metab Dispos 24: 395-400.[Abstract]

Richling E, Decker C, Haring D, Herderich M, and Schreier P (1997) Analysis of heterocyclic aromatic amines in wine by high-performance liquid chromatography-electrospray tandem mass spectrometry. J Chromatogr A 791: 71-77.[CrossRef][Medline]

Skog K, Solyakov A, Arvidsson P, and Jagerstad M (1998) Analysis of nonpolar heterocyclic amines in cooked foods and meat extracts using gas chromatography-mass spectrometry. J Chromatogr A 803: 227-233.[CrossRef][Medline]

Snyderwine EG, Sadrieh N, King RS, and Schut HA (1998) Formation of DNA adducts of the food-derived mutagen 2-amino-9H-pyrido-[2,3-b]indole (AC) and bioassay of mammary gland carcinogenicity in Sprague-Dawley rats. Food Chem Toxicol 36: 1033-1041.[CrossRef][Medline]

Solyakov A, Skog K, and Jagerstad M (1999) Heterocyclic amines in process flavours, process flavour ingredients, bouillon concentrates and a pan residue. Food Chem Toxicol 37: 1-11.[CrossRef][Medline]

Sugimura T (1985) Carcinogenicity of mutagenic heterocyclic amines formed during the cooking process. Mutat Res 150: 33-41.[CrossRef][Medline]

Sugimura T (1997) Overview of carcinogenic heterocyclic amines. Mutat Res 376: 211-219.[Medline]

Tamano S, Hasegawa R, Hagiwara A, Nagao M, Sugimura T, and Ito N (1994) Carcinogenicity of a mutagenic compound from food, 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeAC), in male F344 rats: Carcinogenesis 15: 2009-2015.[Abstract/Free Full Text]

Turesky RJ, Parisod V, Huynh-Ba T, Langouet S, and Guengerich FP (2001) Regioselective differences in C(8)- and N-oxidation of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline by human and rat liver microsomes and cytochromes P450 1A2. Chem Res Toxicol 14: 901-911.[CrossRef][Medline]

Wakabayashi K, Nagao M, Esumi H, and Sugimura T (1992) Food-derived mutagens and carcinogens. Cancer Res 52: 2092-2098.

Wakabayashi K, Ushiyama H, Takahashi M, Nukaya H, Kim SB, Hirose M, Ochiai M, Sugimura T, and Nagao M (1993) Exposure to heterocyclic amines. Environ Health Perspect 99: 129-134.[Medline]

Yamashita K, Takayama S, Nagao M, Sato S, and Sugimura T (1986) Aminomethyl--carboline-induced DNA modification in rat salivary glands and pancreas detected by phosphorus-31 postlabeling method. Proc Jpn Acad Ser B Phys Biol Sci 62: 45-48.

Yanazoe Y and Nagata K (2002) In vitro metabolism, in Food Borne Carcinogens: Heterocyclic Amines (Nagao M and Sugimura T eds) pp 74-89, John Wiley & Sons Ltd., West Sussex, UK.

Yoshida D and Matsumoto T (1980) Amino--carbolines as mutagenic agents in cigarette smoke condensate. Cancer Lett 10: 141-149.[CrossRef][Medline]

Yoshida D, Matsumoto T, Yoshimura R, and Matsuzaki T (1978) Mutagenicity of amino--carbolines in pyrolysis products of soybean globulin. Biochem Biophys Res Commun 83: 915-920.[CrossRef][Medline]

This Article Abstract Full Text (PDF) Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Frederiksen, H. Articles by Frandsen, H. Search for Related Content PubMed PubMed Citation Articles by Frederiksen, H. Articles by Frandsen, H.