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St. Louis Veterans Affairs Medical Center, St. Louis, Missouri (H.J.A.,V.M.L., J.W., T.V.Z.); Division of Geriatric Medicine (H.J.A.,V.M.L., T.V.Z.) and Department of Biochemistry and Molecular Biology (H.J.A., T.V.Z.), St. Louis University School of Medicine, St. Louis, Missouri; and Department of Medicine, Washington University School of Medicine, St. Louis, Missouri (F.F.H.)
(Received October 26, 2006; accepted January 23, 2007)
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Abstract |
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). The reasons for this age dependence are complex, but altered metabolism of carcinogens with age can play a role. One such carcinogen is 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) (see Fig. 1), a heterocyclic amine (HCA) found in well done red meat. IQ is one of 17 new entries in the 2005 Eleventh Report on Carcinogens (NTP, 2005). IQ is a potent carcinogen in rodents (Takayama et al., 1984; Tanaka et al., 1985; Sugimura, 2000) and nonhuman primates (Adamson et al., 1990). In rodents, IQ induces tumors in liver, small and large intestine, pancreas, lung, and mammary gland. In humans, metabolites of IQ have been found in urine (Lynch et al., 1992), and HCAs have been implicated in the initiation of colon cancer (Kristiansen et al., 1997). This suggests that the strong association between well done red meat and colorectal cancer risk (Giovannani et al., 1994) may be mediated by HCAs such as IQ.
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; Luks et al., 1989). The other major route of metabolism is direct conjugation of the exocyclic amine by N2-glucuronidation or sulfamate formation (Turesky et al., 1986; Inamasu et al., 1989; Luks et al., 1989). Sulfamate excretion was observed in urine, bile, and feces (Turesky et al., 1986; Inamasu et al., 1989; Luks et al., 1989). In contrast, the 5-O-glucuronide and 5-sulfate were not detected in feces (Inamasu et al., 1989; Luks et al., 1989). This is consistent with the conjugates being hydrolyzed by bacterial enzymes in the intestine and reabsorbed from the intestinal tract. Despite nearly complete detoxification of IQ by the rat, a signification amount of oxidation of IQ seems to occur, forming DNA adducts and contributing to the presence of tumors (Takayama et al., 1984; Tanaka et al., 1985; Turesky and Markovic, 1995; Sugimura, 2000).
Although the metabolism of IQ has been well studied in young animals, there have been no studies of the metabolism of IQ in old animals. In general, the effect of age on metabolism depends on the compound being studied. For example, one dioxin compound (TCDD) shows greater concentrations in the tissues of old mice with less than 1% excreted (Pegram et al., 1995), whereas benzopyrene forms fewer adducts in old mice (Boerrigter et al., 1995).
The purpose of these studies was to determine whether the metabolism of IQ changed with age and with cytochrome P450 induction. Radiolabeled IQ was administered to young and old rats. The amount of radioactivity excreted in the urine and the urinary metabolite profile was determined. Because the cytochromes P450 play a major role in IQ metabolism, the effect of ß-naphthoflavone (BNF), an inducer of P450 metabolism, on IQ metabolism was also determined. Finally, as a possible indication of IQ adduct formation, the amount of IQ retained in specific tissues was determined as a function of age.
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Materials and Methods |
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Animals and Dosing. Young and old male F344 rats were purchased from Harlan (Indianapolis, IN). Young rats were 3 months old and old rats were retired breeders 22 to 24 months old. Food and deionized water were provided ad libitum. Animals were administered 20 mg/kg 14C-labeled IQ (20 µCi; 1.25 ml per 250 g injected i.p.; 20% dimethyl sulfoxide/80% 0.1 N HCl) and placed in metabolic cages, and a 24-h urine was collected. To induce hepatic phase I and II enzymes, these same animals were treated with 40 mg/kg BNF (2.5 ml per 250 g injected i.p.; 13% dimethyl sulfoxide/87% corn oil) for 3 consecutive days. Rats were then administered 20 mg/kg 14C-labeled IQ i.p., a 24-h urine was collected, and rats were euthanized by cervical dislocation.
Processing Urine and Purification of Metabolites. Urine was analyzed for IQ metabolites. The samples were treated with an equal volume of methanol and spun at 1500g for 20 min to precipitate protein and debris. After evaporation of the methanol, the urine was applied to a Sep-Pak column, previously prepared with methanol/water, washed with 5 ml of water, and eluted with 10 ml of 100% methanol. The latter was concentrated by evaporating the methanol and reconstituted in 100 mM ammonium acetate buffer (pH 7.4). The resuspended material was extracted twice with 2 volumes of ethyl acetate and then with equal volumes of n-butanol three times. There was no significant radioactivity in the ethyl acetate extract. The combined n-butanol extracts were back-extracted with an equal volume of pH 7.4 buffer, and the organic layer was evaporated under N2 gas. The total recovery of radioactivity in the aqueous (37%) and n-butanol (48%) fractions was 85%. Urine from each rat was analyzed using solvent system 1 (see below). For identification of metabolites, urine from a young rat was purified using solvent systems 1, 2, and 3, respectively. After each purification step, metabolite peaks were concentrated and applied to the next solvent system for further purification.
HPLC Analysis of Products. Products were assessed using a Beckman Coulter (Fullerton, CA) HPLC apparatus with System Gold software and a 5-µm, 4.6 x 150 mm C-18 Ultrasphere column attached to a guard column. For solvent system 1, the mobile phase contained 20 mM ammonium acetate (pH 5.0) in 2% acetonitrile, 0 to 5 min; 2 to 5% acetonitrile, 5 to 23 min; 5 to 40% acetonitrile, 24 to 29 min; 40 to 2% acetonitrile, 35 to 40 min; flow rate 1 ml/min. For solvent system 2, the mobile phase contained 20 mM ammonium acetate (pH 6.8) in 4% acetonitrile, 0 to 5 min; 4 to 5% acetonitrile, 5 to 23 min; 5 to 40% acetonitrile, 24 to 29 min; 40 to 4% acetonitrile, 35 to 40 min; flow rate 1 ml/min. For solvent system 3, the mobile phase contained 20 mM ammonium acetate (pH 5.0) in 10% methanol, 0 to 15 min; 10 to 90% methanol, 15 to 20 min; 90 to 10% methanol, 25 to 30 min; flow rate 1 ml/min. Radioactivity in HPLC eluents was measured using a Flo-One (PerkinElmer LAS, Inc.) radioactive flow detector. Data are expressed as a percentage of total radioactivity or picomoles recovered by HPLC.
Mass Spectral Analysis. Electrospray ionization (ESI) mass spectrometry (MS) analyses were performed on a Finnigan TSQ-7000 triple stage quadrupole mass spectrometer (Thermo Electron Corp., Waltham, MA) equipped with a Finnigan ESI source and controlled by Finnigan ICIS software operated on a DEC alpha 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 collision-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.
Qualitative Identification of Urinary Metabolites. To assist in identifying IQ metabolites in individual urine samples, their elution time and susceptibility to specific treatments were determined (Turesky et al., 1986; Inamasu et al., 1989; Luks et al., 1989). The 5-O-glucuronide is susceptible to E. coli ß-glucuronidase (type VII-A; Sigma-Aldrich) with 100 units of enzyme at pH 6.8 and 0.3 µg of substrate per 0.1 ml at 37°C for 24 h. 5-Sulfate is susceptible to Abalone sulfatase (type VIII; Sigma-Aldrich) with 10 units at pH 5.0 and 0.3 µg of substrate per 0.1 ml at 37°C for 24 h. N2-Glucuronide and sulfamate were hydrolyzed with 1 N HCl at 60°C for 4 h.
Measurement of CYP1A1 Protein Levels. Rat liver, duodenum, and renal cortex were homogenized (150 mg tissue/ml) in ice-cold homogenizing buffer (25 mM NaCl, 1 mM Tris, pH 8.0). Protein levels of CYP1A1 were measured by Western blotting with chemiluminescence detection as described previously (Armbrecht et al., 1999). The polyclonal antiserum used was CYP1A1 (G-18): sc-9828 from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). This antiserum recognizes CYP1A1 from mouse, rat, and human. Homogenate proteins were separated out by SDS-polyacrylamide gel electrophoresis using 10% gels. Separated proteins were transferred to Hybond-P membranes (Amersham-GE Healthcare, Piscataway, NJ), and membranes were incubated for 1 h with 1:1000 of the primary antiserum followed by 30 min with 1:20,000 of the secondary antiserum, donkey anti-goat IgG-horseradish peroxidase (Santa Cruz). Antigen-antibody complexes were visualized using an ECL Western blotting kit and ECL Plus Hyperfilm (Amersham-GE Healthcare). Bands were quantitated by optical densitometry using an Amersham Biosystems ImageScanner. Based on actin rehybridization, sample loading was uniform. Therefore, absorbance was routinely normalized to total protein.
Statistical Analysis. The data from these experiments are reported as means ± S.E. of the number of animals indicated. Experiments with a 2 x 2 design (age versus BNF treatment) were first analyzed by two-way ANOVA. If needed, individual comparisons were performed using Student's two-tailed t test. A confidence level of 95% or greater was considered significant.
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Results |
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Analysis of IQ Metabolites in Rat Urine by HPLC. The HPLC profile of IQ urinary metabolites from untreated rats is illustrated in Figs. 3 and 4 (top). Peaks eluting at 15.0, 20.1, 22.8, and 28.5 min were tentatively identified as N-glucuronide, 5-O-glucuronide, sulfamate, and 5-sulfate, respectively, in both age groups (Figs. 3 and 4, top). The metabolite profile was changed after administration of BNF. Major peaks were only observed for 5-O-glucuronide and 5-sulfate (Figs. 3 and 4, bottom). Positive peak identity was determined either by ESI collision-activated dissociation tandem mass spectrometry or by their elution time and susceptibility to specific treatments.
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; Inamasu et al., 1989; Luks et al., 1989). Identification of IQ Metabolites by Susceptibility to Specific Treatments. To assist in identifying IQ metabolites in individual urine samples, the Sep Pak-purified fraction was subjected to specific enzyme or acid treatment and then analyzed by HPLC. The N-glucuronide was susceptible to treatment with 1 N HCl at 60°C, 5-O-glucuronide to ß-glucuronidase, sulfamate to 1 N HCl at 60°C, and 5-sulfate to sulfatase. Metabolites were only susceptible to the treatments indicated. Because of the small amounts of N-glucuronide and sulfamate present in urine from BNF-treated rats, these apparent metabolites were not further identified. These results are consistent with their ESI/MS-assigned structures as above.
Effect of Age and BNF on Urinary IQ Metabolites. The metabolites identified in Figs. 3 and 4 were quantitated by HPLC (Table 1) and analyzed statistically by two-way ANOVA (Table 2). BNF treatment had a highly significant effect in both age groups. It increased 5-O-glucuronide 200 to 286% and increased 5-sulfate by 48 to 56% in both age groups. This is consistent with the fact that BNF increases both phase 1 (P450s) and phase 2 (UDP-glucuronosyltransferases) enzymes. Likewise, it decreased N-glucuronide to 18 to 22% of control and sulfamate to 2 to 3% of control in both age groups. With age there was a significant increase in 5-O-glucuronide and significant decrease in 5-sulfate (Table 2). This indicated a significant metabolic shift in older animals from 5-sulfate to 5-O-glucuronide metabolism. There was no difference in the effect of BNF with age.
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Effect of Age on IQ Metabolites in Tissue. After urine collection, the BNF-treated rats were euthanized and the tissues were assessed for the distribution of radioactivity (Fig. 5). Levels of radioactivity were similar in the intestine and liver of the two age groups, but the amount of radioactivity in the kidneys of the old rats was dramatically higher than that of the young. Because of the large amount of radioactivity in the old kidneys, the radioactivity present was further characterized (Fig. 6). The renal cortical homogenates from three old rats were mixed with an equal volume of methanol, forming a precipitate. The supernatant of this was precipitated with 5% TCA. The total precipitates (methanol and TCA) contained 31 ± 0.7% of the radioactivity and the soluble fractions contained 66 ± 7%. The precipitates were pooled and incubated with proteinase K for 3 h at 60°C. After this treatment, 33% of this material was now soluble. The soluble fractions remaining after the TCA precipitation (66 ± 7% of the total) were pooled and applied to a C18 solid-phase extraction column (1 g, PrepSep) for further purification (Fig. 6). Of this material, 61% was eluted from the column and 39% was retained. HPLC analysis of the elutant indicated that it contained 60% 5-O-glucuronide and 24% 5-sulfate. Treatment with ß-glucuronidase and sulfatase confirmed the identification of these metabolites. This distribution of metabolites in renal tissue is quite similar to the percentages seen in urine from old, BNF-treated rats (Table 1).
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Effect of BNF and Age on Expression of CYP1A1. The effect of BNF on CYP1A1 expression was determined in the liver and kidney (Fig. 7). CYP1A1 protein levels were undetectable in the absence of BNF in both tissues and age groups. BNF markedly increased CYP1A1 protein levels in the liver and the kidney. There was no significant difference with age in either tissue.
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Discussion |
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). However, of relevance to these studies, BNF increases CYP1A1 activity to a greater degree than 1A2 activity in rodents (Turesky et al., 1998). Preliminary studies with liver slices from young rats have demonstrated almost complete blockage of 5-O-glucuronide and 5-sulfate formation by ellipticine, a CYP1A1 inhibitor (data not shown). This finding supports the metabolism of IQ by CYP1A1 in the rat. In the old rat there is a significant increase in 5-O-glucuronide and a decrease in 5-sulfate compared with young rats (Table 1). BNF markedly increases 5-O-glucuronide and decreases sulfamate in both age groups, but there is no significant change in the effect of BNF with age (Table 2). This result suggests that there is no change in the P450 metabolic pathway with age either basally or after BNF stimulation. This conclusion is supported by the fact that the induction of CYP1A1 protein by BNF does not change with age in the liver or kidney (Fig. 7). The major age-related change in IQ metabolism is an increase in glucuronic acid conjugation and a decrease in sulfate conjugation.
A major finding of this study is that there was decreased excretion and increased renal binding of IQ metabolites with age. The amount of radiolabeled IQ retained by the old kidney is much higher than in the young kidney or any other tissue regardless of age (Fig. 5). When this radioactivity was further characterized (Fig. 6), it was found that 31% was precipitable and 26% was retained by the C18 column. Thus, 57% of the radioactivity probably represents metabolites bound to macromolecules in the kidney. Only 24% was recovered as soluble IQ-5-O-glucuronide and 10% as IQ-5-sulfate. These findings suggest that much more of the IQ metabolites are complexed with proteins and nucleic acids in the old kidney compared with the young kidney. However, even in the old kidneys, the amount of radioactivity bound is a very small percentage of the total dose administered. Binding itself would not explain the decreased radiolabeled IQ excretion by the old animals. It is of interest that 3-methylcholanthrene also decreased urinary excretion of a heterocyclic amine carcinogen in young mice (Buonarati et al., 1992). In this study, 3-methylcholanthrene increased binding in the liver but decreased binding in the kidney.
One possible explanation for a sharp increase in IQ binding in the old rat kidney is an age-related decrease in antioxidant defenses. In the F344 rat kidney, there is an age-related decrease in glutathione and glutathione transferase activity (Liu and Dickinson, 2003) and in superoxide dismutase and catalase activity (Tian et al., 1998). This leads to an increase in reactive oxygen species in the old rat kidney that manifests itself in a number of ways. The formation of protein carbonyl groups (Greenberg et al., 2000) and advanced glycation end products (Teillet et al., 2000) increases with age in the kidney. Renal telomere shortening, due in part to free radical damage, and an increase in cell senescence markers have been observed in the old kidney (Tarry-Adkins et al., 2006). Of direct relevance to the present study is the fact that there is an increase in DNA adducts (8-hydroxy-2'-deoxyguanosine) in the F344 rat kidney with age (Hamilton et al., 2001). Thus, the environment of the old kidney may favor the binding of IQ metabolites. There are also physiological changes in the old rat kidney, such as decreased glomerular filtration rate, which may contribute to decreased excretion, increased metabolite exposure, and increased binding to macromolecules (Baylis and Corman, 1998). Conversely, this increased binding to macromolecules may, in turn, contribute to physiological changes with age.
It is of interest that there is no change in the induction of CYP1A1 by BNF with age (Fig. 7). In general, the effect of age on the induction of the hepatic P450 xenobiotic-metabolizing pathways is complex. In a series of experiments, Horbach et al. (1990a,b) administered multiple doses of various inducing agents i.p. to rats and then measured P450 induction at the level of mRNA and protein. At the mRNA level, a number of differences in induction with age were found. Induction of CYP1A2, CYP2B1, and CYP2B2 by isosafrole declined with age (Horbach et al., 1990a,b). Induction of CYP2B1 and CYP2B2 by pentobarbital also declined with age (Horbach et al., 1990a). However, at the protein level, there was no difference in the maximal levels of P450 protein induced as a function of age (Horbach et al., 1992). In another study, low doses of pentobarbitol showed no effect of age in terms of inducing a whole range of hepatic P450s (Agrawal and Shapiro, 2003). The capacity of TCDD to induce some forms of P4501A either remained unchanged or increased with age (Pegram et al., 1995). In the present studies, the fact that the BNF induction of CYP1A1 protein does not change with age would be consistent with previous aging studies.
These studies in the rat raise the possibility that the metabolic products of some carcinogens may accumulate preferentially in tissues of older animals. This may occur without major changes in the metabolism of those carcinogens with age. This is supported by a study of the effect of age on the distribution of a single oral dose of a dioxin compound (TCDD) (Pegram et al., 1995). In this study, the blood concentrations of TCDD did not change with age, but the TCDD concentrations in skin, kidney, and muscle in old mice were twice those of the young. On the other hand, after a single intraperitoneal dose of benzopyrene, there were significantly fewer adducts formed in organs of old mice compared with young mice (Boerrigter et al., 1995). Thus, the effect of age may be specific to a particular compound and tissue.
In the human diet, consumption of 400 g of cooked lean meat could result in exposure to several micrograms of mutagenic heterocyclic amines (Pais et al., 1999), which have been detected in urine (Lynch et al., 1992), indicating their absorption from cooked foods. The strong association of high-temperature cooked meat intake and colorectal cancer risk (Giovannucci et al., 1994), the presence of heterocyclic amines in cooked meat (Pais et al., 1999), and the initiation of colon cancer by heterocyclic amines (Kristiansen et al., 1997) suggest that heterocyclic amines present in high-temperature cooked meat may be responsible for the increased risk of colon cancer associated with this dietary component. The present studies should further our understanding of IQ-induced colon cancer.
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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; BNF, ß-naphthoflavone; HCA, heterocyclic amine; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; ESI, electrospray ionization; MS, mass spectrometry; ANOVA, analysis of variance; TCA, trichloroacetic acid.
Address correspondence to: Dr. Harvey J. Armbrecht, Geriatric Center (11G-JB), St. Louis VA Medical Center, St. Louis, MO 63125. E-mail: hjarmbrec{at}aol.com
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