1 Division of Biological Engineering; Massachusetts Institute of Technology, Cambridge, 77 Massachusetts Avenue, MA 02139, USA and
2 Nutritional Epidemiology Branch, National Cancer Institute, NIH, Rockville, MD 20892, USA
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Abstract |
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Abbreviations: CYP1A2, cytochrome P4501A2; GC/MS, gas chromatography/mass spectrometry; HAA, heterocyclic aromatic amine; HPLC, highperformance liquid chromatography; MeIQx, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline; NAT2, N-acetyltransferase; NCI, negative ion chemical ionization; N-OH-PhIP, 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine; N-OH-PhIP-N2-glucuronide, N2-(ß-1-glucosiduronyl)-2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine; 2-OH-PhIP, 2-hydroxy-1-methyl-6-phenylimidazo[4,5-b]pyridine;; PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine;; SIM, selective ion monitoring
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Introduction |
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As a means of determining the potential health risk associated with PhIP, several dietary studies have been conducted on the metabolism and disposition of this compound in humans. Initial results showed that PhIP is readily absorbed and eliminated when ingested as part of a meat diet (5). Interindividual differences were also evident upon examination of the excretion levels of unchanged PhIP (5) and PhIP plus the amine-conjugated metabolite(s) in urine after ingestion of a controlled meat meal (6). Insight into the role of cytochrome P4501A2 (CYP1A2) in the metabolism of HAAs was later obtained in an in vivo study using the CYP1A2 inhibitor furafylline (7). The amount of PhIP recovered in urine after individuals were treated with furafylline increased 4.1-fold in comparison with untreated subjects; CYP1A2-mediated metabolism accounted for ~70% of the ingested dose (7). More recently, results based on the urinary profile data of [2-14C]PhIP metabolites confirmed that this HAA is subject to extensive N-hydroxylation in vivo (8,9). The N2- and N3-glucuronide conjugates of N-OH-PhIP (Figure 1) comprised a major portion of the urinary metabolite fraction among patients receiving an oral dose of radiolabeled PhIP (8). Furthermore, additional data obtained in the studies established that DNA and protein adduct formation occurred in the colon and blood, respectively, of subjects receiving dietary equivalents of [14C]PhIP (10).
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In assessing the N-oxidation metabolism of the related HAA 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) in humans, we recently determined the excretion level of the N2-glucuronide conjugate of N-OH-MeIQx in the urine of healthy subjects following consumption of a controlled meat diet (17). We have now extended our investigation to include the measurement of urinary N-OH-PhIP-N2-glucuronide [N2-(ß-1-glucosi-duronyl)-2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b] pyridine] within the same study group. N-OH-PhIP-N2-glucuronide is the principal human urinary metabolite of PhIP (8), and results indicate that it may serve as an index of the bioactivation pathway of metabolism. In the present study, we evaluate the extent and variability of the urinary excretion of N-OH-PhIP-N2-glucuronide, and determine its relation to individual metabolic phenotypes for CYP1A2 and NAT2. Measurement of the individual levels of N-OH-PhIP-N2-glucuronide in urine will increase our understanding of the in vivo capacity of humans to N-oxidize PhIP, as well as allow an assessment of the role of metabolic polymorphisms in mediating this pathway of metabolism.
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Materials and methods |
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Reference compounds
The 2-nitro derivatives of PhIP and [2H3]methyl-PhIP were synthesized according to the method of Grivas (18), with the modifications described by Turesky et al. (19), except that 2-nitro-PhIP was purified by high-performance liquid chromatography (HPLC) using a C-18 EPS platinum column (250x4.6 mm, Alltech, Deerfield, IL). Elution of 2-nitro-PhIP was performed isocratically with 55% methanol in 45% water containing 0.2% acetic acid. The N-OH derivatives of PhIP and [2H3]methyl-PhIP were prepared by reduction of the corresponding 2-nitro compounds as reported by Turesky et al. (19). N-OH-PhIP-N2-glucuronide and its [2H3] analog were biosynthesized from the corresponding PhIP and 2-hydroxyamino-PhIP derivatives using human liver S-9 homogenates (20). The incubation mixture consisted of 4 ml of 100 mM KH2PO4 (pH 7.4), 5 mM MgCl2, 5 mM glucose-6-phosphate, 6 mM uridine 5'-diphosphoglucuronic acid, 5 U glucose-6-phosphate dehydrogenase, 0.3 mM Na2NAD, 0.4 mM NADPH, 1 ml of pooled human liver S-9 homogenate (containing 20 mg protein) and 150 µg of substrate dissolved in 150 µl DMSO/ethanol (4:1). After incubation at 37°C for 3 h the homogenate was placed on ice and the proteins were precipitated with 10 volume of cold methanol:acetone (1:1, by volume). The organic layer was decanted and concentrated by rotary evaporation to a volume of 10 ml. After centrifugation, the organic layer was then transferred to a glass vial and dried under vacuum. The dried residue was suspended in 5 ml of a 50 mM ammonium acetate solution (pH 6.8) containing 2% methanol and applied to a C-18 solid-phase extraction cartridge. The cartridge was washed with 10 ml of 50 mM ammonium acetate (pH 6.8), followed by 10 ml of H2O and the metabolites eluted with 10 ml of 40% methanol in water. The methanolic solution was dried under vacuum to a volume of 1 ml, filtered and the metabolites chromatographed and characterized by HPLC using a HP 1100 liquid chromatograph (Agilent Technologies, Palo Alto, CA) equipped with diode array detection. Metabolites were purified and collected using a 5 µm particle size C18 DB column (250x4.6 mm, Supelco, Bellefonte, PA) with a mobile phase consisting of 50 mM ammonium acetate (pH 5.5) containing 5% methanol, followed by a linear gradient to 100% methanol over 30 min at a flow rate of 1 ml/min. N-OH-PhIP-N2-glucuronide eluted at a retention time of 15.4 min; the on-line HPLC-UV absorption spectrum was consistent with that reported previously (21). Metabolite fractions were collected and evaporated to dryness under vacuum centrifugation. Additional purification of the collected N-OH-PhIP-N2-glucuronide and the [2H3] analog was conducted using HPLC with a linear gradient from 0 to 100% methanol in 5 mM ammonium acetate (pH 5.0). N-OH-PhIP-N2-glucuronide and its deuterated isotopomer were further characterized by LC/MS using a Finnigan 7000 TSQ instrument operated in positive ion mode. Full scan mass spectra of the biosynthesized products exhibited protonated molecular ions at m/z 417 and 420, respectively, for the d0 and d3-labeled compounds. The extinction coefficient of purified N-OH-PhIP-N2-glucuronide at 316 nm in methanol was estimated to be 17 200 M-1cm-1 by using the reported value of 18 133 M/cm for PhIP at 316 nm (22) and a correction value derived from the ratio of the molar extinction coefficients of the related compounds N-OH-MeIQx-N2-glucuronide and MeIQx (23).
2-Hydroxy-1-methyl-6-phenylimidazo[4,5-b]pyridine (2-OH-PhIP) was prepared synthetically by treating 2-nitro-PhIP (0.5 mg) with 0.4 ml of 1.0 N NaOH and heating for 2 h at 37°C. After neutralization of the reaction mixture with 0.4 ml of 1 N HCl, the product was purified by solid-phase extraction (17). HPLC-UV analysis of the extract using the procedure described above exhibited a major component whose on-line spectrum had a -max at 309 nm. The hydrolysis product was characterized by NCI-gas chromatography/mass spectroscopy (GC/MS) after derivatization with 3,5-bis(trifluoromethyl)benzyl bromide (17). The full scan mass spectrum under NCI conditions revealed only one significant ion at m/z 224 (100%) corresponding to the loss of the CH2-bis(trifluoromethyl)benzyl moiety from the molecular anion (M-227). On a smaller scale, the reference compound [2H3]methyl-2-OH-PhIP was prepared from the corresponding 2-nitro-PhIP analog using the above conditions. This compound was further purified by HPLC using the chromatographic separation described above.
With a smaller preparation, 2-OH-PhIP was synthesized by heating 12 µg of N-OH-PhIP-N2-glucuronide in 0.1 ml of 0.1 N HCl at 92°C for 75 min. After neutralization of the reaction mixture with 3% ammonium hydroxide solution, the sample was dried under vacuum centrifugation. The residue was dissolved in water and the products analyzed by HPLC using the conditions described above. The major component was identified as 2-OH-PhIP in comparison with the synthetic standard prepared by base treatment of 2-nitro-PhIP (described above). Small amounts of N-OH-PhIP were also detected by HPLC analysis with UV detection at 316 nm; this product accounted for ~25% of the total.
Study design
A fully detailed account of the study design, enrollment criteria and the individuals participating in the study has been published previously (24,25). In brief, the 66 subjects (33 males and 33 females) were recruited from the Rockville, Maryland area and ranged in age from 27 to 62 years. The enrollment guidelines included being in good health, being a non-smoker for at least 6 months, not consuming any atypical diet, having weight not <90% or >130% of the 1983 Metropolitan Life Insurance desirable weights and taking no medication other than an occasional analgesic.
Meat preparation and dietary protocol
The meat preparation and the controlled dietary protocol were described earlier (24,25). In summary, the subjects consumed a diet containing differing portions of ground beef based on their body weight (3.1 4.0 g meat/kg body weight). The well-done cooked meat contained 32.8 ng/g of PhIP. Timed urine collections were made at 012 and 1224 h after consumption of the meal. These collections obtained in a previous study dating from 1993 were stored frozen at 80°C until thawed and samples were aliquoted for analysis.
CYP1A2 and NAT2 phenotype
The individuals were phenotyped for CYP1A2 and NAT2 status by measurement of urinary caffeine metabolites as described earlier (25,26).
Isolation of urinary N-OH-PhIP-N2-glucuronide
N-OH-PhIP-N2-glucuronide was isolated from urine using a Certify II extraction procedure (17). The solid-phase cartridges were washed and conditioned before use with 40 ml of methanol, then with 25 ml of water, followed by 25 ml of 50 mM ammonium acetate buffer (pH 7.1). Stock solutions of the internal standard [2H3]methyl-N-OH-PhIP-N2-glucuronide were prepared in methanol and quantified with a Hewlett-Packard 8452A UV/vis spectrometer using an extinction coefficient of 17 200 M-1cm-1 for the standard at 316 nm. Urine samples (typically 4 ml) were spiked with 12 ng of [2H3]methyl-N-OH-PhIP-N2-glucuronide and applied to the preconditioned Certify II cartridges by gravity flow. The cartridge was washed in succession with 15 ml of 50 mM ammonium acetate buffer (pH 7.1), 10 ml of distilled water and 1 ml of methanol. The conjugate fraction was eluted with 14 ml of methanol:1 N acetic acid (80:20, by volume). The eluant was neutralized by the addition of 0.10.2 ml of 29.9% ammonium hydroxide, and then taken to dryness under reduced pressure. The dried sample was reconstituted using 4 ml of distilled water, transferred to 50 ml glass-stoppered glass tubes, and acidified by the addition of 1 ml of 0.5 N HCl. The acidified solution was heated in a water bath at 92 ± 2°C for 75 min. After cooling, the hydrolyzed sample was neutralized by addition of 2 N NaOH and transferred to a C-18 Sep-pak (500 mg) cartridge for extraction and clean up. The cartridges were preconditioned before use with methanol (10 ml) and water (15 ml). Following application of the sample, the cartridge was washed with 5 ml of distilled water, then with 4 ml of 30% methanol in water and the metabolite fraction was eluted with 4 ml of 80% methanol in water. The eluant was taken to dryness under vacuum centrifugation and the residue extracted with ethyl acetate using the procedure described previously (17). 2-OH-PhIP and its deuterated analog were derivatized with 3,5-bis(trifluoromethyl)benzyl bromide and analyzed by NCI-GC/MS (17).
NCI-GC/MS analysis
GC/MS was performed with a Hewlett-Packard 5989A GC-MS system. For negative ion chemical ionization (NCI), with methane as moderating gas, the source pressure was 1.7 torr (indicated), the electron energy was 200 eV, the source temperature was 220°C and the emission current was 350 µA. Chromatographic separations were performed in splitless mode using a DB-35ms capillary column [15 mx0.25 mm i.d. with 0.25 µm stationary phase film thickness (J & W Scientific, Folsom, CA)]. The temperature program was as follows: 140°C for 0.5 min, then 20°C/min to 280°C at which point the temperature was held constant for 4 min. For analyses of the samples, the mass spectrometer was operated in the selective ion monitoring (SIM) mode using the negative ions at m/z 224 for 2-OH-PhIP and m/z 227 for the [2H3]methyl analog. Quantification of the analyte in the sample was performed using the peak areas as determined by the integration routine in the data system.
Control urine samples obtained from subjects who consumed no meat 24 h prior to collection were assayed under the same conditions described above to determine any background present in the analysis.
Pooled urine samples from the study group were analyzed without acid hydrolysis to determine the presence (if any) of preformed 2-OH-PhIP in urine. For these analyses, urine (4 ml) was spiked with 15 ng of [2H3]methyl-2-OH-PhIP. Following purification by C-18 solid-phase extraction, ethyl acetate extraction and derivatization using 3,5-bis(trifluoromethyl)benzyl bromide the samples were assayed by NCI-GC/MS. Pooled urine samples were also analyzed after enzymatic hydrolysis to determine the possible presence of conjugated 2-OH-PhIP. In this procedure, urine (3 ml) was buffered with sodium acetate to 0.1 M and the pH adjusted to 5.0 with acetic acid. Following the addition of 8 ng of [2H3]methyl-2-OH-PhIP, the samples were incubated at 37°C for 6 h with either arylsulfatase at a concentration of 100 U/ml or ß-glucuronidase at a concentration of 500 U/ml. Following C-18 Sep-pak purification, ethyl acetate extraction and derivatization (see above) the samples were analyzed by NCI-GC/MS.
Recovery studies
Experiments were conducted with a pooled sample to evaluate the effect of various heating conditions, acid normality and length of time on the recovery of 2-OH-PhIP from urine. The percent recovery of 2-OH-PhIP derived from N-OH-PhIP-N2-glucuronide in urine was determined by comparing the gas chromatographic peak areas of the extracted analyte to the peak area of a deuterated standard of 2-OH-PhIP.
Precision of the assay
Assay precision was determined by duplicate analyses of aliquots from representative individual urine samples (n = 12) during the course of the study.
A calibration plot for 2-OH-PhIP was generated by adding increasing amounts of N-OH-PhIP-N2-glucuronide (030 ng) to a constant amount (12 ng) of the [2H3]methyl analog. After acid hydrolysis, the standard compounds were extracted, derivatized and analyzed by NCI-GC/MS in SIM mode. A plot of the peak-area ratios of analyte:internal standard of 2-OH-PhIP versus the concentration ratios of the amounts in nanograms of analyte:internal standard was constructed.
Specificity of the assay
In order to examine the specificity of the procedure in the conversion of N-OH-PhIP-N2-glucuronide to form 2-OH-PhIP with acid treatment, we treated PhIP and the related metabolite N-OH-PhIP-N3-glucuronide with 0.1 N HCl for 75 min at 90°C. The reaction mixtures were neutralized with a 3% ammonium hydroxide solution and evaporated to dryness under vacuum. After drying, the samples were reconstituted in methanol:water (50:50) and analyzed by HPLC and UV/visible detection using the above described procedure.
Statistical methods
We used linear regression analysis to determine the association between the excretion level of N-OH-PhIP-N2-glucuronide in urine and the ingested dose of PhIP. Spearman rank-correlation tests were conducted to determine associations between the data sets. The median urinary N-OH-PhIP-N2-glucuronide level (normalized for the amount of meat ingested) in males versus females was compared using the Student's t-test. To assess whether the excreted amount of N-OH-PhIP-N2-glucuronide in urine (controlled for the amount of meat consumed) was related to intersubject variation in the activity levels of CYP1A2 and NAT2, we performed linear regression analysis of total N-OH-PhIP-N2-glucuronide versus the levels of these enzymes. A standard transformation (log10) was used to increase the accuracy of the statistical methods and to decrease the sensitivity of the analysis to individual points. Significance was accepted at P < 0.05 and all P values quoted were two-sided.
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Results |
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Standard measurements and analysis of urine
When synthetic N-OH-PhIP-N2-glucuronide and 2-nitro-PhIP were hydrolyzed with acid and base, respectively, the major product was identified as 2-OH-PhIP. Mass spectral and UV/visible analyses of the product were consistent with a structure in which the 2-amino moiety of PhIP has been replaced by a hydroxyl group. The structural identification of 2-OH-PhIP is in agreement with the earlier characterization of this compound derived as a by-product in the synthesis of 2-nitro-PhIP (27).
Tests for the specificity of the assay were conducted using PhIP and N-OH-PhIP-N3-glucuronide. PhIP was stable to acid treatment and demonstrated no conversion to 2-OH-PhIP. As expected, the metabolite N-OH-PhIP-N3-glucuronide was not stable to acid and was not detected after treatment with 0.1 N HCl for 75 min at 90°C. Analysis of the reaction mixture by HPLC with UV/visible detection showed no detectable evidence of 2-OH-PhIP formation.
Analysis of control urine samples obtained from subjects not consuming meat 24 h prior to collection established no chromatographic presence of N-OH-PhIP-N2-glucuronide. In addition, pooled urine collections obtained in the dietary study demonstrated no evidence of preformed 2-OH-PhIP in the samples; this compound, if present in urine, was below the analysis detection limit (<20 pg/ml). Analysis of pooled urine samples after enzymatic hydrolysis using either arylsulfatase or ß-glucuronidase treatment further showed no detectable presence of 2-OH-PhIP.
In order to optimize the hydrolysis conditions, various reaction times and acid concentrations were investigated (data not shown). It was found that the best yield of 2-OH-PhIP in urine was obtained when the reaction was carried out for 75 min at 92 ± 2°C using a final concentration of 0.1 N HCl. Overall, the recovery of 2-OH-PhIP derived from N-OH-PhIP-N2-glucuronide in the urine assay was estimated to range between 32 and 44% of the total, based on GC/MS comparison of a derivatized standard of [2H3]2-OH-PhIP. The precision of the assay was determined by duplicate analysis of aliquots of representative urine samples (n = 12) during the course of the study. The relative standard deviation between the replicates ranged between 5.5 and 22.2%; the mean value was 9.4 ± 5.1% (± SD).
A calibration plot constructed from the NCI-GC/MS analysis of 2-OH-PhIP versus its [2H3]methyl-labeled isotopomer, derived from the corresponding N-OH-PhIP-N2-glucuronide conjugates, was linear over the range of 030 ng/sample (r2 = 0.99; slope = 0.97, with an intercept close to zero). Based on peak areas of the fragment ions at m/z 224 and 227, unlabeled 2-OH-PhIP was present to the extent of 0.6%. The chromatographic properties and sensitivity of detection were good for the 3,5-bis(trifluoromethyl)benzyl derivative of 2-OH-PhIP in SIM mode. The on-column limit of detection of the standard compound was 5 pg with signal-to-noise ratio of 3:1. The 3,5-bis(trifluoromethyl)benzyl derivative 2-OH-PhIP was stable over many months, and low coefficients of variation (between 1 and 2%) were observed for replicate injections of the standard compounds and urine samples using NCI-GC/MS SIM analysis. Before each analysis of a urine sample, a blank sample (hexane) was injected to show that no interference was present at the retention time of the analyte. Figure 2 illustrates a typical SIM analysis of 2-OH-PhIP derived from N-OH-PhIP-N2-glucuronide in urine. The amount of N-OH-PhIP-N2-glucuronide in the sample was 3.1 ng/ml. The inset panel in Figure 2
depicts a control urine sample analyzed under the same conditions.
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Table I presents the values for the mean, median and range of the amount of N-OH-PhIP-N2-glucuronide (measured as 2-OH-PhIP) in 012 h and 1224 h urine collections. The quantity of N-OH-PhIP-N2-glucuronide recovered in the 012 h urine, for all subjects, ranged from 0.7 to 6.3 µg, with an average value of 3.0 ± 1.4 µg (± SD). The percentages of the ingested dose eliminated in the 012 and 1224 post-meal period are also given in Table I
. N-OH-PhIP-N2-glucuronide in 012 h urine for all subjects ranged from 5.4 to 39.6% of the ingested dose of PhIP with a median value of 18.8%; the average value was 20.2 ± 8.0% (± SD). The amount of N-OH-PhIP-N2-glucuronide in the 1224 h urine was determined for a subset of 18 subjects. The level was found to range from 2.2 to 10.5% of the ingested dose with an average value of 4.4 ± 2.5% (± SD).
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Figure 4 illustrates the relationship between the excretion levels of N-OH-PhIP-N2-glucuronide and combined PhIP (unmetabolized PhIP plus amine-conjugated PhIP), values determined in a previous study (6). A significant association was found between the urinary levels of these two metabolic parameters for all subjects (r = 0.43; P = <0.001). Spearman's rank-correlation test gave a value of rs = 0.37; P = 0.004.
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CYP1A2 and NAT2 phenotypes versus excretion level of N-OH-PhIP-N2-glucuronide
The individuals in the study were phenotyped for CYP1A2 and NAT2 activity by measurement of the ratio of urinary caffeine metabolites in the interval prior to the ingestion of high-temperature cooked beef (24). The enzyme values were reported previously (6,25). In order to ascertain whether the urinary level of N-OH-PhIP-N2-glucuronide was related to the interindividual variation of CYP1A2 and NAT2 phenotypes, we examined the effect of the activities of these enzymes in a regression that controlled for the amount of meat eaten. Log10-transformed values were used in these assessments to reduce the sensitivity of the analysis to single points. Figure 5 shows the effect of CYP1A2 activity on the individual urinary excretion level of N-OH-PhIP-N2-glucuronide (normalized to a constant amount of meat eaten). In the combined group of males and females, CYP1A2 activity exhibited a low association with N-OH-PhIP-N2-glucuronide excretion (r = 0.25; P = 0.05, slope = 0.20). When one outlier is excluded from this group, a stronger correlation was found (r = 0.30, P = 0.015, slope = 0.23). A plot of the linear regression line for the male group of subjects exhibited a steeper slope (B = 0.33), whereas for the females the slope was much less steep (B = 0.04). The linear regression analysis of NAT2 activity versus the normalized value of urinary N-OH-PhIP-N2-glucuronide among the combined group of males and females showed no relation (r = 0.16; P = 0.22).
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Discussion |
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A comparison may be made between the results presented here and a prior investigation conducted by Malfatti et al. (8) in elderly patients administered radiolabeled PhIP in capsule form. The latter study revealed that the N2-glucuronide conjugate of N-OH-PhIP accounted for 4760% of the recovered counts in 024 h urine or ~2454% of the administered dose. Our data show a lower and more variable fraction of N-OH-PhIP-N2-glucuronide recovered in the 024 h urine to that reported above. Differences between the two studies may be due to the fact that in the current work PhIP was ingested as a part of a meat meal and not in capsule form. Variation may also arise because of the possible effects of concomitant disease and drug therapies in the patients involved in the previous study. Variability in elimination pathways within the two groups may also influence the proportion of the dose excreted in urine. A more recent investigation conducted by Kulp et al. (29) in normal female subjects consuming a cooked chicken meal found highly variable levels of N-OH-PhIP-N2-glucuronide as well as other phase II metabolites of PhIP in urine. It was suggested that the variation in the metabolic ratios might be due to individual digestion, metabolism or other components present in the diet that can influence the absorption and amounts of metabolic products formed from PhIP.
The N-oxidation of PhIP is mediated primarily by hepatic CYP1A2, although other cytochrome P450 enzymes may also be involved (30,31). Differences in the metabolism of PhIP in relation to CYP1A2 phenotypes were reported by Malfatti et al. (8). It was noted that subjects with the highest CYP1A2 activity had the highest level of N-OH-PhIP-N2-glucuronide in the urine and plasma. In our study, we found a low correlation between the activity levels of CYP1A2 and the urinary excretion level of N-OH-PhIP-N2-glucuronide among the combined male and female subjects. A more positive relationship was observed among the male subjects in comparison with the female subjects. Wide variability in CYP1A2 activity has been described in humans, and in general CYP1A2 expression levels are higher in males than in females (15). The lack of a strong correlation between CYP1A2 and N-OH-PhIP-N2-glucuronide excretion in the subjects may involve several factors, such as variability in N-glucuronidation reactions with N-OH-PhIP, competing reactions of the N-hydroxylamine metabolite or differences in elimination pathways. In addition, prior work by King et al. (32) found evidence for the enzymatic reduction of N-hydroxyarylamines to the parent amines in human microsomes. The participation of this pathway would limit the bioavailability of the N-hydroxylamine metabolite of PhIP to undergo glucuronidation reactions. In humans, at least four UDP-glucuronosyltransferases are capable of catalyzing the biotransformation of N-OH-PhIP (28). Hormonal factors may contribute to the variability in the metabolic clearance of N-OH-PhIP-N2-glucuronide within the males and females. In cases where gender differences have been noted, the capacity of glucuronidation was found to be greater in males than in females (33). In the present work, the median value of N-OH-PhIP-N2-glucuronide in the 012 h urine, normalized to a constant amount of meat ingested, was somewhat higher in the males when compared with the females; however, the difference between the groups was not statistically significant (P = 0.10).
Preceding this investigation, our laboratory examined the urinary excretion level of N-OH-MeIQx-N2-glucuronide among the same individuals (17). We found that the amount of this conjugate in urine averaged 9.4% of the ingested dose, and furthermore that the level in urine was not correlated with CYP1A2 activity within the subjects. In contrast, in the present investigation, a low correlation was observed between CYP1A2 activity and the excretion level of N-OH-PhIP-N2-glucuronide. The results may be explained in part as reflecting the two different metabolic pathways of oxidation of MeIQx and PhIP mediated by CYP1A2. That is, for the latter HAA, the major urinary product is N-OH-PhIP-N2-glucuronide, whereas for MeIQx the predominant urinary metabolite is 2-amino-3-methylimidazo[4,5-f]quinoxaline-8-carboxylic acid, a newly identified metabolite of MeIQx, formed via CYP1A2 oxidation of the 8-methyl group (34). Active participation of this pathway would be a competing reaction and would therefore limit the amount of MeIQx available for N-oxidation.
We did not observe an inverse association between the amount of N-OH-PhIP-N2-glucuronide in urine and the activity level of NAT2 within the subjects. This result is comparable with previous findings (6,17,25) and suggests that the effect of NAT2 metabolism, if any, in relation to in vivo HAA clearance is too small to measure by these methods.
In the current study, a procedure was developed for measurement of the excretion level of N-OH-PhIP-N2-glucuronide in human urine. Acidic metabolites are isolated by solid-phase extraction (Certify II cartridges) and the metabolite N-OH-PhIP-N2-glucuronide is converted to 2-OH-PhIP prior to analysis by GC-MS. A labeled internal standard was used to compensate for any loss of the analyte or conversion to other products during the work-up procedure. To our knowledge, none of the other metabolites of PhIP thus far identified in human urine (8) can be converted to 2-OH-PhIP. 2-Nitro-PhIP upon acid treatment will form the product 2-OH-PhIP; however, 2-nitro-PhIP has not been identified in human urine. Data obtained in our laboratory show further that PhIP does not undergo deamination with acid treatment (0.1 N HCl at 90°C for 75 min) to form 2-OH-PhIP. Experiments testing whether the related metabolite N-OH-PhIP-N3-glucuronide forms 2-OH-PhIP upon acid treatment also were negative. In addition, we did not detect any preformed 2-OH-PhIP in urine. Nonetheless, there may exist unknown or unidentified PhIP metabolites in urine that undergo a similar hydrolysis reaction to form 2-OH-PhIP; thus, the amount of N-OH-PhIP-N2-glucuronide measured as 2-OH-PhIP may be overestimated using the present protocol. Such metabolites would in all probability also be oxidatively activated on the N2 position and represent potential carcinogens. Direct quantitative analysis of PhIP metabolites such as N-OH-PhIP-N2-glucuronide and N-OH-PhIP-N3-glucuronide in urine is at the present time a lengthy method requiring a series of separation techniques (29). Our earlier study with MeIQx (17) employed a protocol for the detection of 2-OH-MeIQx via hydrolysis of N-OH-MeIQx-N2-glucuronide in urine. That method employing monoclonal antibody separation for the isolation of urinary N-OH-MeIQx-N2-glucuronide before acid treatment to 2-OH-MeIQx was in agreement with the more direct assay of the metabolite. Our present protocol for the analysis of N-OH-PhIP-N2-glucuronide (clean-up step with solid-phase extraction) ensures a purified acidic extract before the hydrolysis procedure.
Monitoring HAAs and their metabolites in urine gives an overall picture in assessing the bioactivation and detoxification pathways of the particular HAA (35,36). The relatively large sample size of the present investigation allowed for a detailed examination of human variation in metabolism of the HAA. With respect to PhIP and MeIQx, a comparison of the data of the excretion levels of the corresponding N-OH-N2-glucuronide conjugates yielded interesting results. For example, in the case of MeIQx the amount of N-OH-N2-glucuronide in the 012 h urine accounts for an average of 9.4% of the dose with little excreted at the later time points. For PhIP, an average of 20.2% of the ingested dose is found in the 012 h period, and an additional 4.4% in the 1224 h time point, indicating a relatively slower clearance. Moreover, the urinary level of N-OH-PhIP-N2-glucuronide varied considerably among the individuals and exhibited a positive correlation to CYP1A2 activity, whereas for MeIQx, although the range of the excretion level was as extensive, no correlation was observed with CYP1A2 activity. The results and those of others suggest that PhIP may be the more significant HAA in terms of carcinogenic risk to humans because of its abundance in cooked meats relative to the other HAAs, and the fact that an appreciable fraction is transformed via N-oxidation. In general, N-OH-PhIP-N2-glucuronide may serve as an indicator of the balance between an individual's capacity to bioactivate and to detoxify PhIP. Concerning the epidemiological implications and cancer risk assessment in relation to PhIP, further studies in determining comparative exposure in humans ingesting unrestricted diets is warranted (3739). In addition, analysis of relevant biomarkers of exposure such as protein or DNA adducts (40) may allow a better evaluation of metabolic activation and individual exposure to this HAA.
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Notes |
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Acknowledgments |
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