Urinary N2-(2'-deoxyguanosin-8-yl)PhIP as a biomarker for PhIP exposure
Min Fang1,
Robert J. Edwards1,
Michael Bartlet-Jones3,
Graham W. Taylor2,
Stephen Murray2,4 and
Alan R. Boobis1
1 Section of Experimental Medicine and Toxicology and 2 Section on Proteomics, Division of Medicine, Imperial College London, Hammersmith Campus, Du Cane Road, London W12 ONN, UK and 3 Cancer Research UK, Lincoln's Inn Fields, London WC2A 3PX, UK
4 To whom correspondence should be addressed Email: s.murray{at}imperial.ac.uk
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Abstract
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The food-derived, heterocyclic aromatic amine 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is genotoxic and is carcinogenic in experimental animals. Studies on the role of PhIP in human diet-related cancer would be aided considerably by the availability of a readily applicable biomarker of the internal dose of the ultimate genotoxic species. PhIP has been shown to adduct primarily at C-8 of deoxyguanosine in DNA and so the DNA repair product N2-(2'-deoxyguanosin-8-yl)PhIP is a potential biomarker of DNA adduction and repair after exposure to PhIP. An assay for N2-(2'-deoxyguanosin-8-yl)PhIP in urine has been developed based on liquid chromatography mass spectrometry, using a deuterated analogue of the nucleoside as an internal standard and an antibody-mediated extraction procedure. Polyclonal antibodies were raised against the PhIP-nucleotide, PhIP-nucleoside and PhIP-guanine base adducts conjugated to keyhole limpet haemocyanin. Following their evaluation, the immobilized PhIP nucleotide antibody was used for the extraction of N2-(2'-deoxyguanosin-8-yl)PhIP from urine. The limit of detection of the assay was 125 pg and the limit of quantification 200 pg for a 50 ml human urine sample. Following oral administration of PhIP (20 mg/kg body wt/day) to rats for 6 days, N2-(2'-deoxyguanosin-8-yl) PhIP was readily detected in the urine, reaching steady state over 3 days. This is the first direct demonstration of the urinary elimination of this adduct following exposure to parent amine. The half-life of the adduct with DNA was estimated to be
20 h. The total amount of PhIP recovered in the urine as adduct was <0.5 x 103% of the dose administered. Levels of the PhIP adduct in urine collections from human subjects ingesting the amine (4.9 µg) in cooked meat were below the limits of detection, indicating that humans are exposed to a bioactive dose of <3 x 104 of that associated with a non-carcinogenic level in rats.
Abbreviations: ELISA, enzyme-linked immunosorbent assay; HAA, heterocyclic aromatic amine; MRM, multiple reaction monitoring; OVA, ovalbumin; PBS, phosphate-buffered saline; PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine
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Introduction
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Epidemiological studies have established an apparent association between the consumption of red meat, particularly when well cooked, and certain types of cancer, including colon (1), breast (2) and prostate (3). There has been considerable speculation as to the reasons for such an association. Approximately two decades ago it was discovered that during the cooking of protein-based foods such as meat and fish natural constituents condense to form heterocyclic aromatic amines (HAAs), a group of structurally related compounds (46). It was subsequently established that most HAAs are extremely potent bacterial mutagens and that they are also genotoxic in mammalian cell lines (7,8) and carcinogenic in experimental animals, producing tumours in a variety of tissues (911). These discoveries raised the key question of what role, if any, HAAs play in the aetiology of human cancer.
Normal cooking procedures, especially those involving contact with a hot metal surface, have been shown to produce low levels of several HAAs, at concentrations of parts per billion (p.p.b.). One of the most abundant HAAs found in cooked meat is 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) (6) and this compound has been shown to be a potent mammalian carcinogen, targeting colon, breast and prostate in the rat (12,13) and mouse (14,15). While the concentration of PhIP in various cooked foods can be measured and an estimate of ingested dose made (16), of much greater relevance is the extent to which the compound is metabolized, as genotoxicity requires initial N-hydroxylation (17,18). However, inter-individual differences in metabolism in the human population mean that it is difficult to make a meaningful correlation between dietary exposure and carcinogenic risk (19). In fact, in epidemiological studies it has not been possible to demonstrate any association between measures of HAA intake and risk of colon cancer (20). Recently, the urinary excretion of a number of metabolites derived from the proximate genotoxic product, N-hydroxy-PhIP, have been assessed as possible biomarkers of exposure to the bioactive agent. These include N2-hydroxy-PhIP-N2-glucuronide and N2-hydroxy-PhIP-N3-glucuronide, as well as 4'-hydroxy-PhIP-sulphate (21,22). Whilst providing valuable information on exposure to parent compound and bioactive metabolite, measurement of these PhIP metabolites reflects events very distal to carcinogenicity.
Following metabolism, PhIP binds covalently to DNA, forming critical adducts resulting in mutation. Hence, an additional variable determining susceptibility to the carcinogenic effects of PhIP is the rate at which the adducts are repaired. Thus, if it were possible to measure the major DNA adduct of PhIP, this would enable a more effective evaluation of carcinogenic risk posed by this compound. Previous work in vitro has shown that the principal site of adduction of PhIP to DNA is at the C8 position of guanine (23,24) and in vivo it is probable that this adducted nucleotide will be excised by DNA repair mechanisms. While some degradation of the excised adduct might occur, it is probable that a significant percentage will be excreted as the dephosphorylated form N2-(2'-deoxyguanosin-8-yl)PhIP (dG-C8-PhIP) in urine, as it has been shown in the rat that almost all of an injected dose of dG-C8-PhIP is excreted intact and that the majority of PhIP adducts from exogenously modified DNA are excreted as dG-C8-PhIP (25). The fate of endogenously adducted DNA has not been studied previously. Nevertheless, it is apparent that dG-C8-PhIP could provide a measure of genotoxic damage caused by PhIP in the diet and serve as a biomarker of the biologically active dose and of the activity of nucleotide excision repair of PhIP-adducted DNA.
As only a minute proportion (p.p.b.) of ingested PhIP would be expected to be excreted by this route, very sensitive analytical techniques would be required to detect and measure such a nucleoside. 32P-Postlabelling has been used widely to detect DNA adducts (2629) and can have a limit of detection as low as 1 modification in 109 nt for aromatic adducts. However the technique does not provide structural information and can suffer from interference leading to false-positive results. Accelerator mass spectrometry (3035) provides even greater sensitivity (with a detection limit of <1 adduct in 1011 nt) but this requires that a radiolabelled compound be administered and again no structural information is obtained, as isotope ratios only are measured, and false-positive results can be generated. Liquid chromatography mass spectrometry with ion fragmentation by collision-induced dissociation (LC/MS/MS) is the method that currently has the greatest potential for the analysis of DNA adducts. This is because of its great specificity, which allows for unequivocal structural identification, together with accurate quantification that can be obtained through the use of stable isotope-labelled internal standards. Until recently, LC/MS/MS lacked sensitivity in comparison with the methods described previously, mainly due to limitations in chromatography and sample transfer into the mass spectrometer. However reports have been published recently describing the use of capillary LC together with a microelectrospray interface (3638) that give the method a limit of detection for HAA nucleoside adducts similar to that of 32P-postlabelling.
The first aim of the studies described here was to develop an assay for dG-C8-PhIP in urine using LC/MS/MS. The assay would then be used to examine samples from rats and human subjects that had ingested PhIP to determine if the nucleoside could be detected and measured. LC/MS has been used previously to detect dG-C8-PhIP, generated by enzymatic hydrolysis of PhIP-adducted DNA, but, as purified DNA was used, relatively simple extraction procedures were sufficient (39,40). In this case, because of the complex nature of urine and the high sensitivity that would be required of such an assay, a very efficient and specific extraction procedure for dG-C8-PhIP was required. Antibody-mediated extraction can fulfil these requirements, as antibodies generated against a specific antigen often have a high affinity towards that molecule and are also highly specific in their binding characteristics, and so antibodies were generated against three different haptens containing PhIP covalently linked at the C8 of guanine. The antibody showing the highest capacity for binding dG-C8-PhIP was then used as the basis of an extraction procedure to isolate the nucleoside from urine.
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Materials and methods
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Materials
2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-1-trideuteromethyl-6-phenylimidazo[4,5-b]pyridine ([2H3]PhIP) and 1-methyl-2-nitro-6-phenylimidazo[4,5-b]pyridine (2-nitro-PhIP) were purchased from Toronto Research Chemicals (Toronto, Canada). N2-(2'-Deoxyguanosin-8-yl)PhIP (dG-C8-PhIP), N2-(2'-deoxyguanosin-8-yl)-[2H3]PhIP (dG-C8-[2H3]PhIP) and PhIP-adducted calf thymus DNA were synthesized as has been described elsewhere (39). The purities of dG-C8-PhIP and dG-C8-[2H3]PhIP were >98%, as judged by HPLC analysis, and both products showed no sign of degradation when stored in solution in methanol/water (3:7, v/v) at 80°C for 1 year. All other chemicals were obtained from Sigma-Aldrich (Poole, UK) unless stated otherwise. Acetonitrile (Rathburn Chemicals, Walkerburn, UK) was of HPLC grade and water was generated by a Milli-Q water purification system (Millipore, Watford, UK) and had a resistivity of 18.2 M
cm.
HPLC analysis of synthetic products
The purity and identity of synthetic intermediates and products were established using a Hewlett-Packard 1100 HPLC system consisting of a HP1100 pump, autosampler and photodiode array UV detector. Samples were chromatographed on a HiChrom (Theale, UK) RPB base deactivated column (25 cm x 4.6 mm i.d.) preceded by an RPB guard cartridge (1 cm x 4.6 mm i.d.) at a solvent flow rate of 0.8 ml/min using binary gradient elution [solvent A, 50 mM aqueous ammonium acetate (pH 6); solvent B, acetonitrile]. Two linear gradient programmes (method 1, 80% A:20% B to 50% A:50% B over 10 min followed by 50% A:50% B to 100%B over 10 min; method 2, 100% A to 90% A:10% B over 15 min) were used.
Synthesis of 5'-phospho-dG-C8-PhIP conjugates
5'-Phospho-dG-C8-PhIP (I, Figure 1) was prepared from 2-nitro-PhIP using the method described by Lin et al. for the synthesis of the isomeric nucleotide 3'-phospho-dG-C8-PhIP (23). N-Hydroxy-PhIP, obtained by reduction of 2-nitro-PhIP with hydrazine in the presence of palladium on charcoal, was reacted with 5'-phospho-dG in the presence of acetic anhydride. The reaction product was purified on a C18 Sep-Pak cartridge (Waters, Watford, UK) and the overall yield of 5'-phospho-dG-C8-PhIP was
20%, with a purity of >90%, as judged by HPLC analysis [method 1, retention time (tR) 9.4 min]. Electrospray mass spectrometry of the nucleotide gave a negative ion mass spectrum with the base peak at m/z 568 (the deprotonated molecular ion). The 1H-NMR spectrum [dimethyl sulphoxide-[2H6] as solvent:
8.41 (H-5 PhIP), 8.01 (H-7 PhIP), 7.77 (H-2' and H-6' PhIP), 7.49 (H-3' and H-5' PhIP), 7.38 (H-4' PhIP), 3.60 (CH3 PhIP), 6.55 (H-1 guanine), 6.40 (NH2 guanine), 4.90 (OH ribose), 4.48 (H-3 ribose), 3.80 (H-4 ribose), 3.67 and 3.50 (2H-5 ribose)] showed the absence of the characteristic H-8 proton of guanine (normally seen at
7.85), indicating that attachment of PhIP was through the C-8 position of dG.

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Fig. 1. Synthesis of (a) KLH-5'-phospho-dG-C8-PhIP conjugate: 5'-phospho-dG-C8-PhIP ethylenediaminephosphoramidate (II) was synthesized from 5'-phospho-dG-C8-PhIP (I) and then conjugated to KLH to give the 5'-phospho-coupled PhIP-adducted nucleotide immunogen (III). (b) KLH-dG-C8-PhIP conjugate: dG-C8-PhIP-3'-O-hemisuccinate (V) was synthesized from 5'-O-(4,4'-dimethoxytrityl)-2'-deoxyguanosine-3'-O-hemisuccinate (IV) and then conjugated with KLH to give the 3'-hydroxy-coupled PhIP-adducted nucleoside immunogen (VI). (c) KLH-guanine-C8-PhIP conjugate: bromoacetylguanine-C8-PhIP (VIII) was synthesized from bromoacetylguanine (VII) and then conjugated to KLH to give the 2-amino-coupled PhIP-adducted guanine immunogen (IX).
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5'-Phospho-dG-C8-PhIP ethylenediaminephosphoramidate (II, Figure 1) was then prepared from 5'-phospho-dG-C8-PhIP using a modification of the method described by al-Deen et al. for the preparation of 5'-phospho-dG ethylenediaminephosphoramidate (41). A solution of 5'-phospho-dG-C8-PhIP (1 mg) and 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (1 mg) in imidazole buffer (1 M, pH 6, 1 ml) was left for 2 h at room temperature. Mass spectrometric analysis of the reaction mixture showed a protonated molecular ion of m/z 620, corresponding to the presence of 5'-phospho-dG-C8-PhIP phosphorimidazolide. Aqueous ethylenediamine solution (1 M, 1 ml) with pH adjusted to 7.4 was added and the reaction mixture left to stand a further 6 h at room temperature. The reaction mixture was passed through a C18 Sep-Pak cartridge, which was then washed with sodium phosphate buffer (0.1 M, pH 7.2, 5 ml) and acetonitrile/water (5:95, v/v, 5 ml). 5'-Phospho-dG-C8-PhIP ethylenediaminephosphoramidate was eluted with acetonitrile/water (70:30, v/v, 2 ml) and identified by mass spectrometry (MH+ m/z 612).
5'-Phospho-dG-C8-PhIP ethylenediaminephosphoramidate was coupled to keyhole limpet haemocyanin (KLH; Perbio Science UK, Tattenhall, UK) using glutaraldehyde (42). The reaction product was dialysed against phosphate-buffered saline (PBS; 10 mM sodium phosphate, 2.7 mM KCl, 137 mM NaCl, pH 7.4) at 4°C for 4 days with several changes of liquid to remove uncoupled material. The formation of a condensation product with KLH [KLH-5'-phospho-dG-C8-PhIP (III, Figure 1)] was confirmed by spectrometric analysis, which showed a characteristic absorption maximum at 360 nm. A condensation product with ovalbumin (OVA) instead of KLH (OVA-5'-phospho-dG-C8-PhIP) was prepared using the same procedure.
Synthesis of dG-C8-PhIP conjugates
5'-O-(4,4'-Dimethoxytrityl)-2'-deoxyguanosine-3'-O-hemisuccinate (IV, Figure 1) was prepared by reacting 5'-O-(4,4'-dimethoxytrityl)-2'-deoxyguanosine with succinic anhydride in anhydrous pyridine containing 4-dimethylaminopyridine (43). The positive ion electrospray mass spectrum of 5'-O-(4,4'-dimethoxytrityl)-2'-deoxyguanosine-3'-O-hemisuccinate showed a protonated molecular ion of m/z 671 and base peak of m/z 303 corresponding to the dimethoxytrityl group. The yield of product was
80% and purity as determined by HPLC (method 1, tR 13.0 min) was >95%.
A solution of N-acetoxy-PhIP prepared from 2-nitro-PhIP (1 mg) was added to 5'-O-(4,4'-dimethoxytrityl)-2'-deoxyguanosine-3'-O-hemisuccinate (5 mg) dissolved in 2,2,2-trifluoroethanol (3 ml). The reaction mixture was heated at 37°C for 3 h and then left at 4°C overnight. The dimethoxytrityl protecting group, being acid-labile, was removed by the addition of dichloroacetic acid in dichloromethane (3:97, v/v, 3 ml) to form dG-C8-PhIP-3'-O-hemisuccinate (V, Figure 1). Solvents were removed by evaporation under reduced pressure at room temperature and the residue redissolved in dimethyl sulphoxide/water (50:50, v/v, 1 ml). The solution was passed through a C18 Sep-Pak cartridge, which was then washed with water (5 ml), 5% acetonitrile in water (2.5 ml) and 10% acetonitrile in water (2.5 ml). dG-C8-PhIP-3'-O-hemisuccinate was eluted with 70% acetonitrile in water (2 ml) and stored at 20°C. The product was characterized by mass spectrometry (the positive ion electrospray mass spectrum showed a protonated molecular ion of m/z 590) and had a purity >90% as judged by HPLC (method 1, tR 10.1 min).
The N-hydroxysuccinimide ester of dG-C8-PhIP-3'-O-hemisuccinate was then prepared by reaction of dG-C8-PhIP-3'-O-hemisuccinate with dicyclohexylcarbodiimide and N-hydroxysuccinimide in dioxan (44). A precipitate of urea produced during the reaction was removed by centrifugation and excess carbodiimide eliminated from the reaction mixture by hexane extraction until infrared spectrometric analysis showed no carbodiimide remained in the sample (no characteristic absorption at 2120 cm1). The N-hydroxysuccinimide ester (8 mg) was dissolved in dioxan (0.4 ml), which was then added to sodium phosphate buffer (0.1 M, pH 7.2, 4.6 ml) containing KLH (10 mg) with continuous stirring. The reaction mixture was left at room temperature with gentle stirring overnight, and then dialysed against 20% (v/v) dioxan in PBS at 4°C for 2 days followed by PBS only for 2 days, both with several changes of liquid. The formation of a condensation product with KLH [KLH-dG-C8-PhIP (VI, Figure 1)] was confirmed by spectrometric analysis, which showed a characteristic absorption maximum at 360 nm. A condensation product with OVA instead of KLH (OVA-dG-C8-PhIP) was prepared using the same procedure.
Synthesis of guanine-C8-PhIP conjugates
Guanine (110 mg) was dissolved in water (10 ml), which was made alkaline (pH 10) with sodium hydroxide solution. Bromoacetyl bromide (100 µl) was then added dropwise with stirring over 1 min. HPLC analysis (method 2) showed the almost quantitative conversion of guanine (tR 7.3 min) to bromoacetylguanine (VII, Figure 1, tR 5.1 min), which was purified by preparative HPLC.
A solution of N-acetoxy-PhIP prepared from 2-nitro-PhIP (1 mg) was added with stirring to bromoacetylguanine (5 mg) dissolved in sodium phosphate buffer (0.1 M, pH 7.2, 3 ml). The reaction mixture was left at 37°C for 3 h, after which HPLC analysis (method 1) indicated that a single product (tR 8.8 min) had been formed. The reaction product was isolated by passage through a C18 Sep-Pak cartridge, which was then washed with water (10 ml) and 10% acetonitrile in water (10 ml). Bromoacetylguanine-C8-PhIP (VIII, Figure 1) was eluted with 70% acetonitrile in water (4 ml).
KLH (5 mg) was reduced with sodium borohydride (0.1 M) in sodium carbonate/bicarbonate buffer (0.1 M, pH 8.5, 2 ml) (45). Bromoacetylguanine-C8-PhIP (5.72 mg) in sodium carbonate/bicarbonate buffer (0.1 M, pH 8.5, 2 ml) containing 20 mM EDTA was then added to the KLH solution. The reaction was left at room temperature in the dark with stirring for 4 days and then dialysed against PBS at 4°C for 4 days with several changes of buffer. The formation of a condensation product with KLH [KLH-guanine-C8-PhIP (IX, Figure 1)] was confirmed by spectrometric analysis, which showed a characteristic absorption maximum at 360 nm. A condensation product with OVA instead of KLH (OVA-guanine-C8-PhIP) was prepared using the same procedure.
Antibody production and characterization
Seven male New Zealand White rabbits were immunized with repeated injections of KLH-5'-phospho-dG-C8-PhIP (III, two animals), KLH-dG-C8-PhIP (VI, three animals) and KLH-guanine-C8-PhIP (IX, two animals) and antisera prepared using a previously described protocol (46), except that here all booster injections were administered subcutaneously. This work was carried out by Harlan Sera-Lab (Loughborough, UK). Pre-immune sera and antisera were collected at 0, 5, 10 and 15 weeks and stored frozen at 20°C. The antisera collected at 15 weeks were characterized by enzyme-linked immunosorbent assay (ELISA) using 96-well NUNC Maxisorp microtitre plates (Fisher Scientific, Loughborough, UK) coated with OVA, PhIP conjugates of OVA, DNA or PhIP-adducted DNA in PBS (1 µg/ml, 100 µl/well). Thereafter, the procedure was as described previously (47) using 1,3-phenylenediamine dihydrochloride as substrate, except that the colour development was stopped after 10 min by the addition of sulphuric acid (1 M, 100 µl/well). The absorbance was then measured at 490 nm using an automatic colorimetric plate reader (Titertek Multiscan ELISA-plate reader, Labsystem, Basingstoke, UK).
Antibody immobilization
The IgG fraction of serum samples was prepared by affinity chromatography using a Protein Aagarose column and then coupled to cyanogen bromide activated Sepharose 4B resin at a ratio of 5 mg IgG/ml of swollen resin using the protocols provided by the manufacturer (Amersham Biosciences, Little Chalfont, UK). The resin was suspended in PBS containing 4 mM sodium azide (5 ml) for storage at 4°C. The binding capacity of antibody-coupled Sepharose 4B resin was measured by taking aliquots of blank human urine (1 ml) in Eppendorf tubes (1.5 ml) to which had been added dG-C8-PhIP [1 ng in 10 µl, 5 ng in 5 µl or 10 ng in 10 µl methanol/water (3:7, v/v)]. After washing with PBS to remove traces of sodium azide, a suspension of antibody-coupled Sepharose 4B resin (200 µl/sample) was added and urine samples mixed by inversion for 3 h. Resin was isolated by centrifugation and washed with PBS (2x 1 ml) followed by deionized water (2x 1 ml). dG-C8-PhIP was eluted from resin samples with methanol/water/formic acid (10:90:2, v/v/v, 3x 0.4 ml). dG-C8-[2H3]PhIP [5 ng in 50 µl methanol/water (3:7, v/v)] was added to each eluate and the samples were thoroughly mixed. Solvent was evaporated to dryness under vacuum at room temperature and residues redissolved in acetonitrile/water (20:80, v/v, 20 µl) for LC/MS/MS analysis.
Rat urine samples
Dosing of rats with PhIP was carried out and urine collections made at BIBRA International (Carshalton, Surrey, UK). Eight male SpragueDawley rats of body weight 120150 g were purchased from Harlan UK (Bicester, UK). The rats were housed individually in Jencons Metabowls so that separate urine collections could be made. A 24 h pre-study urine collection was made for each animal. Five rats then received PhIP suspended in Gum Tragacanth at a dose of 20 mg/kg body wt administered by gavage, while the three other animals received the vehicle only, and urine was collected for 24 h. The procedure of PhIP or vehicle dosing and urine collection was repeated for a total of 6 days. At the end of each 24 h urine collection, cages were washed with small volumes of water, which were then combined with the relevant sample. Faeces were removed by centrifugation and the 56 urine samples collected (510 ml in volume) were stored at 80°C.
Human urine samples
Urine samples were available as part of a dietary intervention study that had been carried out to assess the effect of cruciferous vegetable consumption on the metabolism of HAAs in man (48).
Extraction and quantification of urinary dG-C8-PhIP
Rat urine samples were thawed and centrifuged to remove particulate matter. If necessary, pH was adjusted to 7 with Tris buffer (1 M, pH 8) and samples of <10 ml made up to that volume with deionized water. dG-C8-[2H3]PhIP [5 ng in 50 µl methanol/water (3:7, v/v)] was added to each sample and the samples were thoroughly mixed. After washing with PBS to remove traces of sodium azide, a suspension of anti-KLH-5'-phospho-dG-C8-PhIP IgG-coupled Sepharose 4B resin (200 µl/sample) was added and the samples mixed by inversion for 3 h. Resin was isolated by centrifugation and washed with PBS (2x 5 ml) followed by deionized water (2x 5 ml). Bound adducts were eluted from resin samples with methanol/water/formic acid (10:90:2, v/v/v, 3x 1 ml). Eluates were lyophilized and residues redissolved in acetonitrile/water (20:80, v/v, 20 µl) for LC/MS/MS analysis. Human urine samples were processed in a similar way to rat urine samples except that a larger volume of urine (50 ml), more dG-C8-[2H3]PhIP [10 ng in 100 µl methanol/water (3:7, v/v)] and more anti-KLH-5'-phospho-dG-C8-PhIP IgG-coupled Sepharose 4B resin (400 µl/sample) were used.
Extracts of urine samples (20 µl) were chromatographed on a Hichrom (Theale, UK) RPB base deactivated column (10 cm x 2.1 mm i.d.) preceded by an RPB guard cartridge (1 cm x 2.1 mm i.d.) and coupled to a Waters 616 HPLC pump fitted with a 20 µl injection loop. Elution was isocratic, using acetonitrilewater (2:3, v/v) containing 0.1% formic acid as solvent, and at a flow-rate of 100 µl/min the retention times of dG-C8-PhIP and dG-C8-[2H3]PhIP were 4.2 min. The column was connected to a VG Quattro II mass spectrometer (Micromass, Manchester, UK) by a fused-silica capillary and the mass spectrometer was operated in the positive ion electrospray, multiple reaction monitoring (MRM) mode with a source temperature of 70°C and a cone voltage of 80 V. The instrument was tuned to monitor ion transitions of m/z 490.2
m/z 374.2 for dG-C8-PhIP and m/z 493.2
m/z 377.2 for dG-C8-[2H3]PhIP and data acquisition and handling were performed with MassLynx software.
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Results
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The nucleotide 5'-phospho-dG-C8-PhIP possesses three functional groups (5'-phospho, 3'-hydroxy and 2-amino) that can potentially be used to form covalent links with amino and sulphydryl groups in protein (Figure 2). In order to allow conjugation reactions with KLH and OVA to be directed to each of these sites, analogues of 5'-phospho-dG-C8-PhIP, dG-C8-PhIP and guanine C8-PhIP were prepared with suitable modifications to the 5'-phospho, 3'-hydroxy and 2-amino groups, respectively. 5'-Phospho-dG-C8-PhIP ethylenediaminephosphoramidate (II, Figure 1) was coupled to KLH and OVA with glutaraldehyde, a bis-aldehyde that forms imine linkages with the amino groups of both the phosphoramidate and the protein. The ratios of 5'-phospho-dG-C8-PhIP bound to KLH (3.2 molecules/100 kDa, 216 mol/mol) and OVA (4.9 molecules/100 kDa, 2.2 mol/mol) were estimated by UV spectroscopy, using the reported extinction coefficient of 33 300 M1 cm1 (39) for the absorption maximum at 360 nm due to the PhIP chromophore in dG-C8-PhIP (23,49) and molecular weights of 6.75 mDa and 45 kDa for KLH and OVA, respectively. This chromophore was common to all of the antigenprotein conjugates and was therefore used to estimate the conjugation ratios of each of the synthetic products. dG-C8-PhIP-3'-O-hemisuccinate (V, Figure 1) was conjugated to KLH (dG-C8-PhIP/KLH, 9.1 molecules/100 kDa, 613 mol/mol) and OVA (dG-C8-PhIP/OVA, 6.0 molecules/100 kDa, 2.7 mol/mol) via an N-hydroxysuccinimide ester. Bromoacetylguanine-C8-PhIP (VIII, Figure 1) was covalently linked to KLH and OVA by nucleophilic displacement of bromine by sulphydryl groups to give stable thioether bonds. The ratios of hapten to protein in the conjugates KLH-guanine-C8-PhIP (IX) and OVA-guanine-C8-PhIP were 2.4 molecules/100 kDa (161 mol/mol) and 0.9 molecules/100 kDa (0.4 mol/mol), respectively.

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Fig. 2. Functional groups ( ) in the nucleotide 5'-phospho-dG-C8-PhIP (R = H2PO3) that can potentially be linked to amino and sulphydryl groups in protein (KLH). In practice, three conjugates were synthesized by linking (a) amino groups in protein with the phosphate group of the nucleotide 5'-phospho-dG-C8-PhIP (R = H2PO3). (b) Amino groups in protein with the 3'-hydroxyl group of the nucleoside dG-C8-PhIP (R = H). (c) Sulphydryl groups in protein with the 2-amino group of guanine-C8-PhIP.
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The three conjugates [KLH-5'-phospho-dG-C8-PhIP (III), KLH-dG-C8-PhIP (VI) and KLH-guanine-C8-PhIP (IX)] were used to immunize rabbits. The binding of the antisera (designated anti-KLH-5'-phospho-dG-C8-PhIP, anti-KLH-dG-C8-PhIP and anti-KLH-guanine-C8-PhIP, respectively) for each antigen was determined by examining binding to a conjugate of that antigen with OVA, namely a protein other than the one used as a carrier for immunization. The data presented here summarize the results obtained with antisera produced after 15 weeks immunization. The protein conjugates proved to be efficient antigens, eliciting immune responses in all seven rabbits. Each antibody showed the strongest binding to its own immunogen with poor binding to the other two PhIP adducts, this being most marked with the anti-KLH-dG-C8-PhIP antibody (Figure 3). All three antisera were found to bind to PhIP-adducted DNA, although the avidity of the anti-KLH-guanine-C8-PhIP was less than that of the other two antibodies (Figure 4).

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Fig. 3. Direct ELISA of the three polyclonal antibodies (a) anti-KLH-5'-phospho-dG-C8-PhIP, (b) anti-KLH-dG-C8-PhIP and (c) anti-KLH-guanine-C8-PhIP using the homologous antigen and the heterologous antigens coupled to OVA [i.e. 5'-phospho-dG-C8-PhIP (empty circle), dG-C8-PhIP (filled triangle) and guanine-C8-PhIP (filled diamond) conjugates of OVA] to coat the plates. OVA (empty square) was included as a negative control. Pre-immune sera from the rabbits gave binding curves for the three conjugates of OVA that were similar to those obtained for the equivalent antiserum for OVA (data not shown). The data points shown are means of duplicate determinations that were within 5% of each other and are for antisera obtained from single animals immunized with each of the conjugates. Results similar to those shown in (b) and (c) were obtained using antisera from other animals immunized with the same antigen. Of the two rabbits immunized with KLH-5'-phospho-dG-C8-PhIP, one showed weaker binding than the other and the binding curves shown (a) are for the stronger binding antiserum of the two.
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Fig. 4. Direct ELISA of the three polyclonal antibodies (a) anti-KLH-5'-phospho-dG-C8-PhIP, (b) anti-KLH-dG-C8-PhIP and (c) anti-KLH-guanine-C8-PhIP using native DNA (filled square) and PhIP-adducted DNA (filled triangle) to coat the wells of microtitre plates. Pre-immune sera from the rabbits gave binding curves for PhIP-adducted DNA that were similar to those obtained for the equivalent antiserum binding to native DNA (data not shown). The data points shown are means of duplicate determinations that were within 5% of each other and are for antisera obtained from single animals immunized with each of the three conjugates. Results similar to those shown in (a), (b) and (c) were obtained using antisera from other animals immunized with the same antigen.
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We have developed previously an assay for dG-C8-PhIP, following its cleavage from adducted DNA, using selected ion monitoring LC/MS and a deuterated analogue, dG-C8-[2H3]PhIP, as internal standard (39). In order to obtain improved selectivity for the current method, the mass spectrometer was operated in the MRM mode. The positive ion electrospray mass spectrum of dG-C8-PhIP shows a protonated molecular ion at m/z 490 which, when subjected to collision-induced dissociation, undergoes fragmentation to yield an aglycone ion of m/z 374 (40). dG-C8-[2H3]PhIP generates analogous ions of m/z 493 and m/z 377, respectively. The mass spectrometer was therefore set to monitor ion transitions of m/z 490.2
m/z 374.2 for dG-C8-PhIP and of m/z 493.2
m/z 377.2 for dG-C8-[2H3]PhIP. Eight-point unextracted standard curves were prepared using standards containing first dG-C8-PhIP (010 ng) and dG-C8-[2H3]PhIP (10 ng) and secondly dG-C8-PhIP (05 ng) and dG-C8-[2H3]PhIP (5 ng). Plots of the peak area intensity ratio I490.2
374.2/I493.2
377.2 against the amount of dG-C8-PhIP for the two standard curves were linear (r2 = 0.999 and 0.995, respectively). The limit of detection (LOD) of dG-C8-PhIP, defined as a signal-to-noise ratio for the adducted nucleoside of 4:1, was 50 pg.
The IgG fraction was isolated from pre-immune and immune sera, from rabbits immunized with each of the three PhIP conjugates. The IgG preparations were then coupled to Sepharose 4B resin and the binding capacities of the antibody-coupled resins determined by measuring the recovery of dG-C8-PhIP added to control human urine at concentrations of 1, 5 and 10 ng/ml (Table I). The internal standard dG-C8-[2H3]PhIP was added after the extraction so that comparison with equivalent unextracted standards would enable absolute recovery to be calculated. At levels of up to 5 ng dG-C8-PhIP, resins coupled to control antibodies gave <2% recovery of dG-C8-PhIP. Resins coupled to antibodies from immunized rabbits showed much higher recoveries of up to 60%, with the anti-KLH-guanine-C8-PhIP resin giving a slightly lower value. When the concentration of dG-C8-PhIP was increased to 10 ng/ml, recoveries using control antibodies increased to
6%, whilst the anti-PhIP adduct antibody resins showed evidence of saturation with recoveries falling to
25%. As anti-KLH-5'-phospho-dG-C8-PhIP gave the highest recovery at non-saturating concentrations of dG-C8-PhIP, this antibody was used for further development of the assay.
Extracted standard curves were generated from blank human urine to which known amounts of dG-C8-PhIP had been added. These curves did not differ from the unextracted standard curves covering the same concentration ranges. The LOD of dG-C8-PhIP in urine, defined as a signal-to-noise ratio for the adducted nucleoside of 4:1, was 125 pg and the limit of quantification (defined as the lowest point on the extracted standard curve where multiple analysis gave a coefficient of variation of
20%) was 200 pg. The accuracy and precision of the assay at 200 pg were 94.5 ± 17.5% (189 ± 35 pg, mean ± SD, n = 4).
Rats were treated by gavage with PhIP, 20 mg/kg, for 6 days and 24 h urine samples were collected prior to and during the dosing interval. The PhIP dosed animals had an initial body weight of 125 ± 4 g (mean ± SD, n = 5), which increased to 153.8 ± 5.8 g (mean ± SD) by the end of the study. Urine samples were analysed for the presence of dG-C8-PhIP using the analytical procedure described above. No nucleoside could be detected in the urine of the control animals at any time or in the pre-study collections from the PhIP dosed animals. In contrast, measurable levels of dG-C8-PhIP were found in all of the urine collections from animals that received PhIP. The specific peak at m/z 490.2
374.2 corresponding to dG-C8-PhIP can be seen clearly in the chromatogram of a urine sample from a rat that had received PhIP but was absent in a control animal (Figure 5). In contrast, the peak at m/z 493.2
377.2, corresponding to the trideuterated internal standard is clearly evident in both treated and control samples. Whilst there was some inter-animal variability, one rat in particular excreting appreciably more dG-C8-PhIP than the other four, the pattern of excretion was quite consistent. It increased linearly over the first 3 days of treatment and then plateaued for the remainder of the study (Figure 6).

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Fig. 6. Excretion of dG-C8-PhIP in urine of rats receiving an oral dose of PhIP (20 mg/kg body wt) daily for 6 days (filled circle; mean ± SD, n = 5) and in control rats (empty circle; n = 3). Urine samples were collected over 24 h intervals.
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Urine samples from subjects who participated in a dietary intervention study and consumed meat meals containing a known amount of PhIP were analysed. Subjects consumed cooked meat containing 4.9 µg of PhIP on three occasions and urine collections were made 010, 1024 and 2448 h after the test meal (48). Four of the 20 participants were chosen at random and urine samples (12 for each subject) analysed as described above. No dG-C8-PhIP could be detected in any of the 48 samples, indicating that excretion was <5 ng/24 h, assuming a urine volume of 2 l/24 h.
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Discussion
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Previous work published by Vanderlaan et al. (5052) and Dragsted et al. (53) has described the production of monoclonal antibodies against PhIP. These were used in an ELISA for the detection of HAAs in meat and for the immunoaffinity purification of PhIP metabolites from urine. More recently, Shirai and Takahashi have raised a polyclonal antibody against PhIP-adducted DNA (54,55) and used this for immunohistochemical staining of tissue sections from rats that had been dosed with PhIP. In the present work, we have produced polyclonal antibodies against the major PhIP adduct with guanine. The strategy adopted was to synthesize haptens comprising PhIP coupled through the C8 of guanine such that the adduct was presented in different orientations and included different moieties of the nucleotide. Hence, one hapten incorporated 2'-deoxyguanosine 5'-monophosphate coupled to carrier protein via the 5'-hydroxy group of deoxyribose [i.e. KLH-5'-phospho-dG-C8-PhIP (III)]. A second hapten included deoxyguanosine coupled to KLH via the 3'-hydroxy group of deoxyribose [i.e. KLH-dG-C8-PhIP (VI)] whilst the third involved coupling guanine via the 2-amino group [i.e. KLH-guanine-C8-PhIP (IX)]. It was anticipated that these immunogens would generate subtly different immunological responses resulting in antibodies recognizing different parts of the PhIPguanine adduct.
The antibodies generated against each of the conjugates bound best to their target antigen, as shown by ELISA, although the anti-KLH-dG-C8-PhIP antibody had the greatest specificity. Despite the relative specificity of the antibodies for the respective adduct with OVA, they all recognized PhIP-adducted DNA, although those raised against conjugates that included a deoxyribose moiety had greater avidity than that against guanine-C8-PhIP. Consistent with this, all of the antibodies were effective in immunoaffinity purification of dG-C8-PhIP, those against conjugates including a deoxyribose moiety having the greater binding capacity. The objective of the present study was to develop an assay for dG-C8-PhIP, on the premise that this was likely to be the major PhIP-derived adduct present in urine. Specificity would be provided by the use of mass spectrometry. Hence, the immunopurification step was to improve sensitivity and, as a consequence, antibody binding capacity was more important than absolute specificity. For these reasons, the antibody directed against 5'-phospho-dG-C8-PhIP was selected for further assay development. Whilst the antibodies reported here have been evaluated for immunoaffinity extraction of dG-C8-PhIP, they have considerable additional potential, such as in structural identification of the intermediate products of adduct disposition and in immunolocalization of adducts. However, this would require further characterization of their properties.
The combination of immunoaffinity extraction and MRM LC/MS enabled the development of a satisfactory assay for dG-C8-PhIP in urine. Interference from other constituents in the urine was minimal. The limit of detection was in the 10 s of picogram range (varying with the volume of urine processed), whilst the response was linear to 10 ng. The assay was used to analyse urine samples from rats that had been dosed with PhIP by oral gavage, to establish proof of concept. Adduct could be readily detected in all urine samples from rats that had been exposed to PhIP, whilst no adduct could be detected in the urine of control animals or in the pre-treatment samples from the treated rats. These data demonstrate that the adduct arises as a consequence of treatment of animals with PhIP itself and, for the first time, establish directly the presence of dG-C8-PhIP in urine following exposure to PhIP. Like several other bulky aromatic genotoxins, such as benzo[a]pyrene (56) and aflatoxin B1 (57), the urinary deoxyguanosine adduct most probably arises following nucleotide excision repair of adducted tissue DNA.
Whilst there was some inter-animal variation, the pattern of excretion of dG-C8-PhIP in the urine was relatively consistent, increasing linearly over the first 3 days and then plateauing for the remainder of the study. Nucleoside excretion varied from 1.04 ng to 10.3 ng/24 h urine collection, representing 0.42 to 4.1 x 104% of the ingested dose of PhIP over 24 h. The absorption and elimination of PhIP following oral administration are rapid (58), and hence the kinetics of adduct excretion in urine will reflect the kinetics of DNA repair, rather than those of bioactivation. The study design adopted did not include a washout period, during which the decay curve for adduct excretion could be followed. However, from the time to achieve steady state excretion, it can be estimated that the half-life of the dG-C8-PhIP in the rat is
20 h. This, of course, is averaged over the entire body and does not preclude the possibility of marked differences in adduct persistence in some, potentially critical, sites. Nevertheless, the assay clearly has the potential to define the kinetics of global excision repair of dG-C8-PhIP in PhIP-treated animals, which would be of use in studying, for example, the possible influence of chemopreventative agents (59). From the half-life of the adduct, it is apparent that urinary dG-C8-PhIP levels observed any time after 3 days of exposure will reflect both the bioactive dose and DNA repair whilst those after cessation of exposure will reflect only DNA repair. Hence, it should be possible to devise a relatively simple protocol utilizing the current assay that would enable both parameters to be assessed.
We have investigated previously the effect of cruciferous vegetable consumption on the disposition of PhIP, present in a test meal of cooked meat, in healthy volunteers (48). As part of this study, urine samples were analysed for dG-C8-PhIP but no dG-C8-PhIP could be detected in any of the samples investigated. The metabolic activation of PhIP is more efficient in humans than in untreated rats (60) and so, if anything, humans might be expected to excrete more of the adduct of PhIP than rats for a given dose. With this is mind, and assuming excretion is linear with dose, it is possible to calculate the minimum expected level of dG-C8-PhIP in human urine. Rats excrete on average 2 x 104% of an oral dose of PhIP in the urine as dG-C8-PhIP in 24 h. Given that the dose of PhIP ingested by the human volunteers was 5 µg, this would result in excretion of 10 pg of the nucleoside in 24 h. Assuming a urine volume of 2 l in 24 h, then a 50 ml urine sample would contain <1 pg dG-C8-PhIP, well below the limit of detection of the assay of 125 pg for a 50 ml urine sample. However, this limit of detection can be used to estimate the minimum risk margin on exposure to PhIP. At a dose of 25 p.p.m. (equivalent to 1.25 mg/kg body wt/day) there was no significant increase in any tumour type in rats treated with PhIP in the diet (61). Hence, an intake of 5 µg/day of PhIP by humans represents a bioactive dose of <3 x 104 of that associated with a non-carcinogenic level in rats. However, it should be noted that, as PhIP is a genotoxic carcinogen, it is not yet known whether there is a safe threshold for human exposure to this compound.
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Acknowledgments
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The authors are grateful to the UK Food Standards Agency (Contract Number T01009) for financial support of these studies. M.B.J. is a visiting worker in the joint Imperial College/MRC Section on Proteomics, Hammersmith Campus.
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Received October 7, 2003;
revised January 9, 2004;
accepted January 10, 2004.