Effect of cruciferous vegetable consumption on heterocyclic aromatic amine metabolism in man

Stephen Murray1, Brian G. Lake3, Stuart Gray1,2, Anne J. Edwards3, Christine Springall3, Elizabeth A. Bowey3, Gary Williamson4, Alan R. Boobis1 and Nigel J. Gooderham2,5

1 Department of Clinical Pharmacology and
2 Department of Molecular Toxicology, Imperial College School of Medicine, Sir Alexander Fleming Building, London SW7 2AZ,
3 TNOBIBRA International, Carshalton, Surrey, SMS 4DS and
4 Institute of Food Research, Norwich, UK


    Abstract
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 Abstract
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 PhIP adducted to lymphocyte...
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The consumption of cooked meat appears to predispose individuals to colonic cancer and heterocyclic aromatic amines (HA), formed during the cooking of meat, have been suggested as aetiological agents. Consumption of cruciferous vegetables is thought to protect against cancer. To study the effect of cruciferous vegetables on heterocyclic aromatic amine metabolism in man, a three-period, dietary intervention study has been carried out with 20 non-smoking Caucasian male subjects consuming cooked meat meals containing known amounts of these carcinogens. A high cruciferous vegetable diet (250 g each of Brussels sprouts and broccoli per day) was maintained during period 2 but such vegetables were excluded from periods 1 and 3. At the end of each period, subjects consumed a cooked meat meal and urinary excretion of the HA 2-amino-3,8-dimethylimidazo(4,5-f)quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP) was measured. Following a 12 day period of cruciferous vegetable consumption (period 2), induction of hepatic CYP1A2 activity was apparent from changes in the kinetics of caffeine metabolism. Excretion of MeIQx and PhIP in urine at the end of this period of the study was reduced by 23 and 21%, respectively, compared with period 1. This reduction in excretion is probably due to an increase in amine metabolism that might be expected given the observed increase in CYP1A2 activity, since this enzyme has been shown to be primarily responsible for the oxidative activation of MeIQx and PhIP in man. In period 2, urinary mutagenicity was increased relative to period 1 by 52 and 64% in the absence and presence, respectively, of a human liver microsomal activation system, yet no evidence was found of PhIP adduction to lymphocyte DNA, a potential biomarker of the activation process. After another 12 days without cruciferous vegetables (period 3 of the study), the kinetics of caffeine metabolism had returned to original values but excretion of MeIQx and PhIP was still reduced by 17 and 30%, respectively, and urinary mutagenicity (with metabolic activation) was still elevated compared with period 1. This prolonged response of amine metabolism to the cruciferous vegetable diet, shown especially with PhIP, suggests that enzyme systems other than CYP1A2 are involved and affected by a cruciferous vegetable diet.

Abbreviations: DMSO, dimethyl sulphoxide; HA, heterocyclic aromatic amines; GC—MS, gas chromatography—mass spectrometry; MeIQx, 2-amino-3,8-dimethylimidazo(4,5-f)quinoxaline; PhIP, 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Dietary intervention study
 PhIP adducted to lymphocyte...
 Results
 Discussion
 References
 
A substantial proportion of human cancer is now believed to be associated with diet (1). Of the various dietary components that might be responsible for this association, epidemiological evidence indicates that consumption of cooked meat and cooked meat products predisposes individuals to neoplastic disease, especially of the colon (2,3). The cooking and processing of meat-containing food has been shown to generate a series of heterocyclic aromatic amines (HA) some of which are known to be present at concentrations of nanograms per gram of food (4,5). The majority of these compounds are mutagenic in bacterial mutagenicity assays and several have been found to be carcinogenic when administered to laboratory animals (610). Previous work has shown that in man two of these compounds, 2-amino-3,8-dimethylimidazo(4,5-f)quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP), are absorbed after ingestion of a cooked meat meal (11) and converted to genotoxic metabolites by the liver P450 enzyme CYP1A2 (1214).

The possible role of HA as aetiological agents in the initiation of human colonic cancer suggests it would be advantageous to reduce exposure to these compounds and particularly to their reactive metabolites. While cooking procedures that generate significant concentrations of amines in food can be modified or avoided (15,16), an alternative approach is to reduce the production in vivo of genotoxic metabolites of HA. This may be achievable by the consumption of dietary components that either reduce absorption of amines or alter metabolism, so that lower levels of genotoxic metabolites are generated. Epidemiological evidence has suggested that consumption of cruciferous vegetables is associated with a lower incidence of diet-related cancer (17). These vegetables are known inducers of xenobiotic metabolizing enzymes in animals and man (1820) and it is possible that a diet high in such foodstuffs might alter human metabolism of HA ingested in food.

A three-period dietary intervention study has therefore been undertaken with a panel of 20 non-smoking male subjects to examine the effect of cruciferous vegetables on the biological fate of HA in man. Subjects were asked to consume a standard cooked beef meal which had been analysed for MeIQx and PhIP content and urinary excretion of the two amines was measured. The effect of dietary intervention on CYP1A2 activity in vivo was determined from changes in the kinetics of caffeine metabolism (21) and excretion of genotoxic material was assessed from urinary mutagenicity. It has recently been shown that metabolic activation of HA, such as occurs in the N-hydroxylation of PhIP, generates products that react with nuclear and mitochondrial DNA (2224) and an assay based on gas chromatography–mass spectrometry (GC–MS) has been developed in this laboratory for the measurement of the major PhIP–DNA adduct, N2-(2 2-deoxyguanosin-8-yl)PhIP (25). To determine if PhIP adduction to lymphocyte DNA could be used as a biomarker for HA activation in vivo, a modified version of this assay has been used to check for the formation of these adducts in the three periods of the study.


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Chemicals
MeIQx, 2-amino-3,4,8-trimethylimidazo(4,5-f)quinoxaline (DiMeIQx) and PhIP were obtained from Toronto Research Chemicals (North York, Ontario, Canada). Syntheses of [13C,15N2]MeIQx, [2H5]PhIP, N2-(2,2-deoxyguanosin-8-yl)–PhIP and N2-(2,2-deoxyguanosin-8-yl)–[2H3]PhIP have been described previously (2527). Diazomethane was generated from Diazald and stored at –20°C. 3,5-Bistrifluoromethylbenzyl bromide, diisopropylethylamine, heptafluorobutyric acid anhydride, dodecane and calf thymus DNA were supplied by Sigma–Aldrich (Poole, UK). Acetonitrile, dichloromethane, ethyl acetate and methanol were all of Analar grade. Ethyl acetate was redistilled before use and, when required dry, stored over calcium hydride. Cofactors (NADPH, glucose-6-phosphate and glucose-6-phosphate dehydrogenase), caffeine and 2-nitrofluorene were purchased from Sigma-Aldrich. XAD-2 resin was obtained from Merck (Poole, UK). [3-Methyl-13C]caffeine from C/D/N Isotopes (Quebec, Canada) was purchased through Univar (Basingstoke, UK).


    Dietary intervention study
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Cruciferous vegetables
Fresh Brussels sprouts and broccoli were purchased as single batches. Sprouts were peeled and broccoli trimmed to remove most of the stalks. A 6 day menu plan was used which was repeated once. The subjects consumed 250 g of either Brussels sprouts or broccoli as a soup at breakfast and 250 g of the other vegetable at dinner (Table IGo). Fresh Brussels sprouts to be used in the soup recipes were cooked in the minimum amount of water necessary to be able to puree them and after pureeing were frozen at –20°C. For the stir-fry and bubble and squeak recipes, fresh Brussels sprouts were shredded, microwaved on full power for 3 min and then incorporated into the recipes. For the tortilla recipe the Brussels sprouts were halved, microwaved on full power for 3 min and then incorporated into the recipe. The fresh broccoli used for the soup recipes was microwaved on full power and then frozen at –20°C. After thawing, cooking was completed by simmering in boiling water and after pureeing was used for the soup recipes. Fresh broccoli for the lamb stew, pasta bake and cheese sauce recipes was cooked in the minimum amount of water necessary to be able to puree the vegetable. The puree was stored at –20°C prior to being incorporated into the broccoli-containing evening meals. After cooking all meals, individual portions were stored at –20°C, prior to being thawed, reheated and consumed by the subjects. In all instances all the liquid produced from cooking the vegetables was incorporated into the soup and other meals.


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Table I. Menu plan for cruciferous vegetable meals
 
Cooking of meat
Approximately 30 kg lean Aberdeen Angus rump beef steak was coarsely minced and moulded into patties (9 cm diameter, 1 cm thick) which were fried (without added fat or oil) for 6 min each side on a hot metal griddle at ~300°C until well browned. The patties, together with any juices released during the cooking process, were broken down in a food processor and finally mixed thoroughly in bulk. The bulk was randomly sampled (n = 8) and the HA content of the samples determined by GC–MS (11,27).

Study protocol
A group of 20 subjects were recruited to the three-period dietary intervention study that was based at a single centre and planned with the advice of a clinical nutritionist. To eliminate metabolic variables due to ethnic origin, sex and contraceptive steroids, the subjects were male, non-smoking Caucasians that were chosen from the TNOBIBRA Clinical Unit volunteer panel. Potential recruits were excluded if they had any history of drug abuse, chronic alcohol consumption (>28 units/week, 1 unit = 0.5 pint of beer or 1 glass of wine or 1 measure of spirits) or had taken prescribed drugs in a 1 month period prior to commencement of the study. All subjects underwent a medical examination including serum chemistry measurements before the start of the study. The mean age of the subjects was 33 years with an age range of 22–46 years. Mean body weight was 82.0 kg (range 68.6–103.2 kg) and all subjects were within 15% of the desirable weight range for their height.

Each of the three periods of the study was of 14 days duration. In periods 1 and 3, the subjects consumed their normal diet for 12 days with certain restrictions designed to avoid exposure to known modifiers of enzyme activity. Excluded dietary components were cruciferous vegetables and alliums such as Brussels sprouts, broccoli, calabrese, cabbage, cauliflower, swede, turnip, mustard, cress, onions, leeks, parsley, kale, radish, horseradish, garlic and spinach. On days 11–14 of all three periods, additional dietary restrictions were imposed which included avoidance of cooked and smoked meat and fish products, toasted items and drinks containing caffeine, chocolate, coffee, alcohol and grapefruit juice. The subjects were recommended to consume vegetarian meals based on eggs or cheese. The drugs paracetamol, aspirin and ibuprofen were also prohibited during these periods and to monitor compliance, subjects were asked to keep a written record of food intake during days 10, 11 and 12 of each period of the study. In period 2, subjects were fed 250 g of either Brussels sprouts or broccoli at both breakfast and dinner for 12 days. All meals were consumed under supervision in the TNOBIBRA Clinical Unit and subjects were provided with packed lunches for consumption outside the unit.

Following an overnight fast, subjects reported to the TNOBIBRA Clinical Unit on day 13 of each period of the study. After providing a urine sample, each subject was given a meal of 275 g well-cooked lean minced steak, which was consumed with vegetable stock and water. A 10 h urine collection was then commenced. Two hours later a 5 ml saliva sample was collected just prior to the administration of a 2 mg/kg body weight dose of caffeine, dissolved in a cup of decaffeinated coffee. Saliva samples for caffeine analysis were then collected 1, 2, 4 and 6 h after consumption of the caffeine containing drink. The subjects were provided with a vegetarian lunch and afternoon snack and were released from the unit after completing the 10 h urine collection. All subjects were supplied with containers for a 10–24 h urine collection and a 9 and 12 h saliva collection. On day 14, subjects returned to the TNOBIBRA Clinical Unit with their urine and saliva collections and provided a 22 h saliva sample together with, 24 h after the cooked meat meal, a 60 ml blood sample. The subjects were then provided with containers for a 24–48 h urine collection that were brought to the unit the following day. Figure 1Go summarizes period 1 of the study. Periods 2 and 3 immediately and sequentially followed period 1. Periods 1, 2 and 3 had identical formats except that a high cruciferous vegetable diet was consumed during period 2.



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Fig. 1. The period 1 study format. Periods 2 and 3 immediately and sequentially followed period 1. Periods 1, 2 and 3 had identical formats except that a high cruciferous vegetable diet was consumed during period 2.

 
Sample handling and storage
Urine samples were weighed, the weights recorded and samples stored at –80°C until required. Saliva samples were stored at –80°C. Blood samples were processed immediately for the isolation of lymphocytes which were then stored at –80°C.

Glucosinolates in vegetables
Samples of the fresh vegetables were frozen at –80°C and transported to the Institute of Food Research (Norwich, UK) for analysis of glucosinolate content as previously described (28).

Thiocyanates in blood plasma
Levels of thiocyanates in plasma were determined by the method of Pettigrew and Fell (29).

Caffeine in saliva
Thawed 1 ml saliva samples were spiked with 0.1 µg/ml [13C]caffeine (internal standard) and treated with 1 ml 5% (w/v) zinc sulfate and 1 ml saturated barium hydroxide. After centrifugation, 25 µl 96% (w/v) formic acid was added to 1 ml of the supernatant and a 25 µl aliquot injected onto a Platinum EPS C18 100 Å 3 µm column (100x4.6 mm) and eluted with 50% (v/v) methanol at a flow rate of 1 ml/min. A Hewlett-Packard HP1100 series liquid chromatograph was used which was coupled, using a splitter, to a Micromass Quattro LC Triple Quadrupole Mass Spectrometer (LC-MS-MS) using an Electrospray Z-spray® source. Levels of caffeine and [13C]caffeine were selectively detected by multiple reaction monitoring.

Heterocyclic amines in cooked meat
MeIQx, DiMeIQx and PhIP were measured by an assay based on GC–MS as has been described in detail elsewhere (11,27). Samples of cooked meat (2 g) to which had been added the internal standards [13C,15N2]MeIQx (10 ng) and [2H5]PhIP (50 ng) were homogenized in 10 ml 0.25 M hydrochloric acid. A portion of the acidic extract (5 ml) was then washed with dichloromethane (3x5 ml), made alkaline with 1 M sodium carbonate (3 ml) and extracted with ethyl acetate (2x5 ml). The organic extract was evaporated to dryness and the residue treated with a 5% solution of 3,5-bistrifluoromethylbenzyl bromide in acetonitrile (80 µl) and diisopropylethylamine (20 µl) overnight at room temperature, which converted the amines into their di-(3,5-bistrifluoromethylbenzyl) derivatives. The derivatives were chromatographed on a 15 m DB5 J&W fused silica capillary column and analysed by selected ion monitoring MS (11,27). The mass spectrometer was operated in the electron capture negative ion mode, with an electron energy of 100 eV and ammonia as reagent gas, and all three compounds could be measured in a single chromatographic run.

MeIQx and PhIP in urine
MeIQx and PhIP were measured by a GC–MS assay which has been reported in detail elsewhere (11). Urine (5 ml), to which had been added the internal standards 2.5 ng [13C,15N2]MeIQx and 12.5 ng [2H5]PhIP, was made alkaline with 2.5 ml 1 M sodium carbonate solution and extracted twice with 10 ml ethyl acetate. The organic extract was in turn extracted with 1 ml 0.1 M hydrochloric acid. After washing with ethyl acetate, the acid was evaporated to dryness and the residue derivatized and analysed by GC–MS as for cooked meat extracts.

Urinary mutagenicity
Glass columns were packed with XAD-2 resin (4 g resin per 100 g urine) and washed once with 150 ml methanol and twice with 150 ml distilled water. Thawed 600 g aliquots of the 0–10 h urine samples were then passed through the XAD-2 resin columns and the columns rinsed with 600 ml distilled water. Urinary mutagens were extracted from the column with 20 ml acetone and the eluent centrifugally evaporated to dryness at 65°C. Dried extracts were redissolved in 0.5 ml dimethyl sulphoxide (DMSO) and stored at –80°C prior to analysis. In vitro urinary mutagenicity assays with Salmonella typhimurium strain YG1024 were performed both with and without metabolic activation according to the method of Maron and Ames (30). For the with metabolic activation studies, duplicate 10, 20 and 25 µl aliquots of the DMSO urine extracts were pre-incubated for 60 min at 37°C in tubes containing 0.5 ml of an activating system [containing 0.5 mg of pooled (n = 5) human liver microsomal protein, 1 µmol NADPH, 1 µmol NADP+, 2.5 µmol DL-isocitric acid, 4 µmol MgCl2 and 3 U isocitrate dehydrogenase in 0.1 M phosphate buffer pH 7.4] which was sterilized by passage through a 0.45 µm syringe filter, 0.1 ml of an overnight bacterial culture (>=2x109 cells/ml) and 0.1 M phosphate buffer in a final volume of 1 ml. The final DMSO concentration of all tubes including control (no urine extract) tubes was 2.5% (v/v) and, as a measure of hepatic CYP1A2, the 7-ethoxyresorufin O-deethylase activity of the pooled human liver microsomal preparation was 65 pmol/min/mg protein. After pre-incubation, a 2 ml volume of molten top agar containing 50 µM histidine and 50 µM biotin was added to the incubation tube and the mixture overlaid onto a 20 ml Vogel-Bonner agar plate. The plates were incubated for 72 h at 37°C and the number of revertant colonies scored with an automatic colony counter. The direct acting mutagenicity of duplicate 10, 20 and 25 µl aliquots of DMSO urine extracts was determined as described above, but in the absence of a human liver microsome metabolic activating system. To serve as positive controls for the mutagenicity studies, plates were also treated with either 0.5 and 2 µg/plate of PhIP (with metabolic activation) or 2.5 and 5 µg/plate 2-nitrofluorene (without metabolic activation). The mutagenicity data were used to calculate revertants per 0.5 ml DMSO urine extract and then to calculate total revertants per 0–10 h urine sample.


    PhIP adducted to lymphocyte DNA
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 Dietary intervention study
 PhIP adducted to lymphocyte...
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Preparation of lymphocytes
Fresh blood was mixed with heparinized phosphate buffered saline and the mixture layered over Ficoll-Paque lymphocyte separation media (Amersham Pharmacia Biotech, Little Chalfont, UK). The discontinuous gradient was centrifuged according to the manufacturer's instructions. Enriched lymphocyte preparations were harvested from the Ficoll–plasma interface.

Purification of lymphocyte DNA
DNA was isolated from lymphocytes using a Stratagene DNA Extraction kit (Stratagene, La Jolla, CA) and quantified by UV absorbance using a GeneQuant spectrophotometer (Amersham Pharmacia Biotech).

Extraction of unadducted PhIP from DNA
[2H5]PhIP in methanol (5 ng/20 µl) was added to aqueous solutions of lymphocyte DNA (<=600 µg) which were then taken to dryness under vacuum at room temperature. A 200 µl aliquot of 0.5 M sodium hydroxide solution was added to the DNA samples, which were then extracted twice with 600 µl ethyl acetate. The organic extracts were evaporated to dryness under nitrogen and the residues derivatized for analysis by GC–MS.

Extraction of adducted PhIP from DNA
N2-(2,2-Deoxyguanosin-8-yl)–[2H3]PhIP (internal standard) in methanol– water (10 ng/25 µl of 1/1 v/v) was added to aqueous solutions of lymphocyte DNA (<=600 µg) in 1 ml ReactivialsTM (Perbio Science, Chester, UK). The samples were then taken to dryness under vacuum at room temperature. A 200 µl aliquot of 0.5 M sodium hydroxide solution was added to each vial and, after purging with nitrogen, the vials were securely capped and heated at 100°C for 24 h. After cooling, the hydrolysed samples were extracted twice with 600 µl ethyl acetate. The organic extracts were then evaporated to dryness under nitrogen and the residues derivatized for analysis by GC–MS.

Derivatization
A 200 µl aliquot of 1:9 (v/v) heptafluorobutyric acid anhydride/dry ethyl acetate was added to standards and sample extracts and the derivatization reactions left to stand in capped glass vials overnight at room temperature. The next day, reagents were removed by evaporation under nitrogen and diazomethane in ether (500 µl) was added to each vial. The methylation reaction was allowed to proceed for 1 h at room temperature, after which samples were evaporated to dryness under nitrogen and the residues reconstituted in dodecane (40 µl for standards and 20 µl for sample extracts). Aliquots (2 µl) were then injected into the gas chromatograph mass spectrometer.

Gas chromatography—mass spectrometry
A ThermoQuest Trio 1000 gas chromatograph quadrupole mass spectrometer (ThermoQuest, San Jose, CA) fitted with a CTC-A200S autosampler was used. The gas chromatograph was equipped with a 15 mx0.25 mm ID DB5 J&W fused-silica capillary column, with helium as carrier gas at a head pressure of 70 kPa, and a Grob-type capillary injector operated in the splitless mode at a temperature of 270°C. The gas chromatograph oven temperature was held at 190°C for 1 min and then raised to 310°C at 20°C/min. Under these conditions, the retention times of the heptafluorobutyryl/methyl derivatives of PhIP, [2H3]PhIP and [2H5]PhIP, were 5.1 min. The mass spectrometer was operated in the electron-capture negative ion mode with an electron energy of 70 eV and with ammonia as the reagent gas. The instrument was set to monitor negative ions of m/z 434, m/z 437 and m/z 439, and data acquisition and processing were performed with MassLynx software.

Assay validation
The alkaline hydrolysis procedure for the cleavage of adducted PhIP from DNA has been described previously by us (25) while the use of a heptafluorobutyryl/methyl derivative for the quantitative analysis of PhIP by selected ion monitoring mass spectrometry has been reported by other workers (31). For the measurement of PhIP not covalently bound to DNA, a six-point standard curve was prepared using standards containing 5 ng [2H5]PhIP and 0–2 ng PhIP and, plotting the intensity ratio I434/I439 against the amount of PhIP, this was found to be linear (y = 0.251x + 0.003, r2 = 1.000). The slope and intercept of an extracted standard curve, constructed from standards extracted from aqueous solutions of calf thymus DNA, were essentially the same as for the unextracted standard curve and so the latter was used for the quantitative analysis of samples. The limit of detection of PhIP was set at 20 pg, which gave a peak area ratio more than twice the blank value and was clearly distinguishable from the latter. For the analysis of PhIP adducted to DNA, six standards containing 10 ng N2-(2,2-deoxyguanosin-8-yl)–[2H3]PhIP and 0–5 ng N2-(2,2-deoxyguanosin-8-yl)–PhIP were prepared. These were hydrolysed, derivatized and analysed by GC–MS and, plotting the intensity ratio I434/I437 against amount of N2-[2,2-deoxyguanosin-8-yl]–PhIP, the standard curve obtained was linear (y = 0.101x + 0.008, r2 = 1.000). The slope and intercept of an extracted standard curve, constructed from standards hydrolysed in aqueous solutions of calf thymus DNA, were essentially the same as for the unextracted standard curve and so the latter was used for the quantitative analysis of samples. The limit of detection of N2-(2,2-deoxyguanosin-8-yl)–PhIP was set at 50 pg, which gave a peak area ratio almost twice the blank value and was clearly distinguishable from the latter.

Statistical analysis
Data were examined by analysis of variance (ANOVA or repeated measures ANOVA) and by non-parametric methods as appropriate.


    Results
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The three periods of the dietary intervention study were completed successfully by the 20 subjects. Side effects were negligible with five subjects reporting incidences of mild flatulence and two subjects experiencing bloatedness and minor stomach cramps during period 2 of the study. One subject experienced a headache for a few hours in each period following caffeine withdrawal. A small but significant drop in the mean bodyweight of the subjects in periods 2 and 3, compared with period 1, was detected (Table IIGo).


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Table II. Subject body weight and plasma thiocyanate data
 
Samples of fresh Brussels sprouts and broccoli used in the dietary intervention study were analysed for glucosinolate content (Table IIIGo). These showed values that were typical for both vegetables and within the normal ranges for shop-purchased Brassicas. In agreement with previous studies the broccoli contained high levels of the methylsulphinylbutyl glucosinolate, with none in the Brussels sprouts; whereas the Brussels sprouts contained high levels of the propenyl glucosinolate with none in the broccoli. On absorption from the diet, glucosinolates are metabolized to thiocyanates (32) and so the concentration of thiocyanate in plasma of the 20 subjects was analysed as a measure of glucosinolate intake (33). Compared with period 1, plasma thiocyanate levels were increased around 2.5-fold at the end of period 2 (Table IIGo), but had returned to lower levels at the end of period 3. The mean plasma thiocyanate concentration at the end of period 2 was significantly higher (P < 0.001) than at the end of period 1, indicating that substantial absorption of glucosinolates had occurred during period 2 of the study.


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Table III. Glucosinolate analysis of fresh vegetables
 
Compared with period 1, at the end of period 2 caffeine half-life was significantly reduced by 11% (P < 0.05, Table IVGo), caffeine clearance was significantly increased by 7% (P < 0.001) and the area under the salivary concentration/time curve (AUC0–{infty}) was significantly reduced by 15% (P < 0.001). By the end of period 3, all of these parameters had returned to period 1 levels.


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Table IV. Effect of cruciferous vegetable consumption on caffeine kinetics in saliva
 
Analysis of the cooked meat used in the study showed the presence of the heterocyclic amines MeIQx, DiMeIQx and PhIP. In order to obtain an estimate of amines in the standard cooked meat meal that was as accurate as possible, the cooked meat was bulked, reduced to crumbs and thoroughly mixed in a large food blender and eight samples were taken from different parts of the mixture. These samples were then analysed and mean values of the amine concentrations calculated (Table VGo). Concentrations of MeIQx, DiMeIQx and PhIP were very similar to those reported previously by us (11,26,27). The coefficients of variation were low and suggested that the amine content of the meat was evenly distributed throughout the bulk and would therefore be consistent from meal to meal. Hence in a meal of 275 g cooked meat each of the subjects ingested 924 ng MeIQx, 382 ng DiMeIQx and 4.90 µg PhIP.


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Table V. Heterocyclic amine content of the bulked fried beef
 
Urine samples collected during the three periods of the dietary intervention study were analysed for MeIQx, DiMeIQx and PhIP and measurable levels of MeIQx and PhIP were found in the 0–10 h urine collections of all 20 subjects. In agreement with our previous studies, DiMeIQx could not be detected in any of these samples because of the lower intake of DiMeIQx relative to MeIQx and PhIP and the poorer limit of detection of this amine in urine (11,14). No MeIQx or PhIP could be detected in any of the pre-meal urine samples or in any of the 10–24 and 24–48 h urine collections. MeIQx and PhIP excretion in the 0–10 h urine collections of period 1 of the study (Figure 2Go) was consistent with our previous human clinical studies (11,14), with 2.61 ± 0.23% (mean ± SEM) of ingested MeIQx and 0.69 ± 0.07% (mean ± SEM) of ingested PhIP being excreted unchanged (Figure 3Go). At the end of period 2 of the study, excretion was significantly lower (P < 0.05) than for period 1 for MeIQx (2.04 ± 0.20%). After period 3 of the study, excretion of MeIQx (2.20 ± 0.19%) was higher than after period 2 and lower than after period 1 but not significantly different from either value. In comparison to period 1, excretion of PhIP was significantly lower after period 2 (0.56 ± 0.06%, P < 0.05) and after period 3 (0.50 ± 0.04%, P < 0.005) (Figure 3Go).



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Fig. 2. Excretion of MeIQx and PhIP in the 0–10 h urine collections of 20 subjects in period 1 of the study.

 


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Fig. 3. Excretion of MeIQx and PhIP in the 0–10 h urine collections of 20 subjects in the three periods of the study (mean ± SEM). *Significantly different from period 1 (P < 0.05). **Significantly different from period 1 (P < 0.005).

 
Urinary mutagenicity (0–10 h samples) after the three test meals of the study was measured both in the absence and presence of a human liver microsomal metabolic activation system. Mutagenicity was detected in the urine of all subjects in period 1 of the study (Figure 4Go). Urinary mutagenicity in the absence of metabolic activation after period 2 (3206 ± 470 revertant colonies, mean ± SEM) was significantly higher than that found after period 1 (2116 ± 338) (Figure 5Go). After period 3, mutagenicity (2605 ± 375) had fallen below that found after period 2 but the difference was not significant. In the presence of a metabolic activation system, mutagenicity of the urine samples was increased after periods 2 and 3 (4032 ± 803 and 4535 ± 630, respectively) compared with period 1 (2454 ± 375) (Figure 5Go). The mutagenicity found in individual urine samples failed to consistently correlate with the levels of either MeIQx (correlation coefficients ranged from –0.14 to 0.67) or PhIP (correlation coefficients ranged from –0.05 to 0.68) in these samples.



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Fig. 4. Mutagenicity in the 0–10 h urine collections of 20 subjects in period 1 of the study. (Note: subject 7 sample in the presence of metabolic activation was lost.)

 


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Fig. 5. Mutagenicity in the 0–10 h urine collections of 20 subjects in the three periods of the study (mean with SEM). *Significantly different from period 1 (P < 0.05).

 
To obtain a more integrated and direct measure of HA activation in vivo, DNA isolated from lymphocytes was analysed for the presence of PhIP adducts using a stable isotope dilution assay based on capillary column gas chromatography–negative ion mass spectrometry. N2-(2,2-Deoxyguanosin-8-yl)–PhIP adducts of DNA were hydrolysed by alkaline hydrolysis (25) and the PhIP released was converted to a heptafluorobutyryl/methyl derivative (31) for analysis. Yields of DNA from the lymphocytes collected in each period of the study ranged from 300 to 600 µg/60 ml blood sample. Consequently, two or three individual DNA preparations from a particular period of the study were combined to give a total of 1000–1200 µg. These combined samples were then divided in half so that ~500 µg DNA could be assayed before and after alkaline hydrolysis. This was done to ensure that any PhIP detected was covalently adducted to DNA and not merely associated with the sample. Upon analysis, however, no PhIP was detected in any of the DNA samples either before or after hydrolysis. The limit of detection of adducted PhIP was 50 pg N2(2,2-deoxyguanosin-8-yl)–PhIP/500 µg DNA, corresponding to one adduct in 107 nucleotides, and so any PhIP adduction in these samples was below this level.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Dietary intervention study
 PhIP adducted to lymphocyte...
 Results
 Discussion
 References
 
The increased plasma thiocyanate levels seen after period 2 of the study indicated that glucosinolates present in the cruciferous vegetable diet had been absorbed and metabolized. Consumption of the cruciferous vegetable diet had a significant effect on caffeine metabolism in this period of the study. Changes in the kinetics of caffeine in saliva indicated CYP1A2 induction, with a reduction in half-life and AUC0–{infty} and an increase in clearance being observed. These results are consistent with previous studies in which induction of CYP1A2 and other drug metabolizing enzymes in humans by cruciferous vegetables has been reported (17,18,32,3436). The changes in caffeine kinetics were completely reversed after withdrawal of cruciferous vegetables from the diet (period 3), which also resulted in much reduced plasma thiocyanate concentrations.

Previous studies have shown that absorption of HA from a cooked beef meal is essentially complete and that urinary excretion of unchanged amine is indicative of the extent to which they are metabolized (11,14). Cruciferous vegetables contain glucosinolates, some of which are bifunctional inducers in that they induce both phase 1 and phase 2 xenobiotic metabolizing enzymes such as CYP1A2 (34), glucuronyl transferase (19) and glutathione S-transferase (20), while others such as sulphoraphane selectively induce phase 2 enzymes such as quinone reductase and glutathione S-transferases (38). In the present study, urinary excretion of MeIQx and PhIP after period 2 was significantly decreased relative to period 1, suggesting that consumption of cruciferous vegetables had increased the metabolism of food-derived HA. This effect, while more pronounced with MeIQx, was more persistent in the case of PhIP. The transient increase in MeIQx metabolism was mirrored by the increase found in the CYP1A2 mediated metabolism of caffeine. This is entirely consistent with the human oxidative metabolism of MeIQx, which is known to be predominantly CYP1A2 mediated (12,14) to generate the genotoxic N-hydroxy derivative. The observed prolonged alteration of PhIP metabolism in response to the cruciferous vegetable diet is longer lived than the induction of CYP1A2 (as measured by caffeine) and suggests the involvement of other enzyme systems. The half life of proteins is dependent upon the rate of their degradation and will vary between different enzymes. Thus withdrawal of the inducing influence of cruciferous vegetables will affect different enzymes to differing extents. The results of the current study therefore suggest that cruciferous vegetable consumption exerts effects on amine metabolism that are both CYP1A2 dependent and independent. This is also in line with the known metabolism of PhIP, the metabolic clearance of which is less reliant on CYP1A2 activity (14) and includes glucuronidation, conjugation with glutathione and oxidation by CYP1A1 and CYP1B1 (39,40). Induction of the enzymes involved in competing metabolic routes such as the formation of non-genotoxic oxidation products, direct conjugation and conjugation of the N-hydroxy metabolite, is likely to promote detoxification. In support of this, the recent reports by Turesky et al. (41) and Malfatti et al. (40) describing the urinary metabolites of MeIQx and PhIP, respectively, show that the N-hydroxy derivatives are extensively conjugated, with glucuronides being the major metabolites.

After consumption of the standard cooked meat meal, subjects' urine was found to be mutagenic with a promutagenic component. The XAD-2 extraction procedure we used to clean up urine samples is not designed to enrich for a specific class of mutagen, but subjects were told to avoid known sources of dietary mutagen. However, without detailed chemical analysis of the urine samples, it is difficult to estimate the contribution, if any, that the heterocyclic amines make to urinary mutagenicity in the post test-meal period. MeIQx is a substantially more potent bacterial promutagen than PhIP and this fact along with the levels of amine being excreted would suggest that of the two, MeIQx is the more likely to contribute to urinary mutagenicity. However, under identical test conditions and despite a large variation between subjects, urinary mutagenicity was elevated after period 2. Furthermore, while period 3 CYP1A2 activity (caffeine metabolism) returned to period 1 levels, urinary mutagenicity like urinary amine levels did not. If the heterocyclic amines do contribute to the urine mutagenicity, this activity is unlikely to be due to their N-hydroxy metabolites (direct acting genotoxins) per se but could be a conjugate that decomposes to give a direct acting mutagen in the Ames test. In support of this concept, Alexander et al. (42) have reported that N-hydroxy PhIP N3-glucuronide can be de-conjugated by bacteria liberating the genotoxic N-hydroxy derivative. Such a mechanism is compatible with cruciferous vegetable-mediated induction of conjugation reactions.

An attempt to obtain direct evidence of HA genotoxicity was made by measuring covalent adduction of PhIP to DNA. Although not a recognized target tissue for PhIP, it has been reported that treatment of both laboratory animals and humans with heterocyclic amines can lead to lymphocyte–DNA adducts (43). We used a recently developed GC–MS assay which specifically measures N2-(2,2-deoxyguanosin-8-yl)–PhIP adducts (25). With a limit of detection of one adduct in 107 nucleotides, this assay is one of the most sensitive specific assays currently available for population studies. However, the data suggest that measurement of N2-(2,2-deoxyguanosin-8-yl)–PhIP in human lymphocyte DNA is unlikely to be a realistic biomarker for amine activation in man since all the samples analysed for all three periods of the study were below the limits of detection for the assay.

Our results indicate that consumption of cruciferous vegetables increases the metabolism of food-derived HA, thereby lowering the levels of amines excreted unchanged in the urine. This effect, more pronounced with MeIQx, was more persistent in the case of PhIP. Since metabolic clearance of PhIP is thought to be less reliant on CYP1A2 activity (14), the prolonged response of PhIP to the cruciferous vegetable diet implies that enzyme systems other than CYP1A2 are involved in the clearance and that they may also be affected by a cruciferous vegetable diet. There is clearly a requirement to develop robust assays for the comprehensive measurement of HA metabolites in body fluids as biomarkers of exposure and activation. In line with this, the recent reports by Turesky et al. (39) and Malfatti et al. (40) are significant advances and should facilitate routine analysis of HA metabolites in humans.


    Notes
 
5 To whom correspondence should be addressed Email: n.gooderham{at}ic.ac.uk Back


    Acknowledgments
 
These studies were carried out with the financial support of the Food Standards Agency. The analysis of dietary glucosinolates was supported by the BBSRC.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Dietary intervention study
 PhIP adducted to lymphocyte...
 Results
 Discussion
 References
 

  1. Doll,R. and Peto,R. (1981) The Causes of Cancer. Oxford Medical Publications, Oxford University Press, Oxford, pp. 1226–1235.
  2. Doll,R. (1992) The lessons of life. Cancer Res. (suppl.), 52, 2024s–2029s.[Abstract]
  3. Gerhardsson de Verdier,M., Hagman,U., Peters,R.K., Steineck,G. and Overvik,E. (1991) Meat, cooking methods and colorectal cancer. A case referent study in Stockholm. Int. J. Cancer, 49, 520–525.[Medline]
  4. Felton,J.S., Knize,M.G., Shen,N.H., Andresen,B.D., Bjeldanes,L.F. and Hatch,F.T. (1986) Identification of the mutagens in cooked beef. Environ. Health Perspect., 67, 17–24.[Medline]
  5. Felton,J.S. and Knize,M.G. (1990) Heterocyclic-amine mutagens/carcinogens in foods. In Cooper,C.S. and Grover,P.L. (eds) Handbook of Experimental Pharmacology, Vol. 94. Springer-Verlag, Berlin, pp. 471–502.
  6. Ohgaki,H., Hasegawa,H., Suenaga,M., Sato,S., Takayama,S. and Sugimura,T. (1987) Carcinogenicity in mice of a mutagenic compound, 2-amino-3,8-dimethylimidazo(4,5-f)quinoxaline (MeIQx) from cooked foods. Carcinogenesis, 8, 665–668.[Abstract]
  7. Kato,T., Ohgaki,H., Hasegawa,H., Sato,S., Takayama,S. and Sugimura,T. (1988) Carcinogenicity in rats of a mutagenic compound, 2-amino-3,8-dimethylimidazo(4,5-f)quinoxaline. Carcinogenesis, 9, 71–73.[Abstract]
  8. Esumi,H., Ohgaki,H., Kohzen,E., Takayama,S. and Sugimura,T. (1989) Induction of lymphoma in CDF1 mice by the food mutagen 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine. Jpn. J. Cancer Res., 80, 1176–1178.[Medline]
  9. Ochiai,M., Ogawa,K., Wakabayashi,K., Sugimura,T., Nagase,S., Esumi,H. and Nagao,M. (1991) Induction of intestinal adenocarcinomas by 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine in Nagase analbuminemic rats. Jpn. J. Cancer Res., 82, 363–366.[Medline]
  10. Ito,N., Hasegawa,R., Sano,M., Tamano,S., Esumi,H., Takayama,S. and Sugimura,T. (1991) A new colon and mammary carcinogen in cooked food, 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP). Carcinogenesis, 12, 1503–1506.[Abstract]
  11. Lynch,A.M., Knize,M.G., Boobis,A.R., Gooderham,N.J., Davies,D.S. and Murray,S. (1992) Intra- and interindividual variability in systemic exposure in humans to 2-amino-3,8-dimethylimidazo(4,5-f)quinoxaline and 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine, carcinogens present in cooked beef. Cancer Res., 52, 6216–6223.[Abstract]
  12. Rich,K., Murray,B.P., Lewis,I., Rendell,N., Davies,D.S., Gooderham,N.J. and Boobis,A.R. (1992) N-Hydroxy-MeIQx is the major microsomal oxidation product of the dietary carcinogen MeIQx with human liver. Carcinogenesis, 13, 2221–2226.[Abstract]
  13. Zhao,K., Murray,S., Davies,D.S., Boobis,A.R. and Gooderham,N.J. (1994) Metabolism of the food derived mutagen and carcinogen 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP) by human liver microsomes. Carcinogenesis, 15, 1285–1288.[Abstract]
  14. Boobis,A.R., Lynch,A.M., Murray,S., de la Torre,R., Solans,A., Farre,M., Segura,J., Gooderham,N.J. and Davies,D.S. (1994) CYP1A2-catalyzed conversion of dietary heterocyclic amines to their proximate carcinogens is their major route of metabolism in humans. Cancer Res., 54, 89–94.[Abstract]
  15. Skog,K., Steineck,G., Augustsson,K. and Jagerstad,M. (1995) Effect of cooking temperature on the formation of heterocyclic amines in fried meat products and pan residues. Carcinogenesis, 16, 861–867.[Abstract]
  16. Skog,K.I., Johansson,M.A. and Jagerstad,M.I. (1998) Carcinogenic heterocyclic amines in model systems and cooked foods: a review on formation, occurrence and intake. Food Chem. Toxicol., 36, 879–896.[Medline]
  17. Verhoeven,D.T., Verhagen,H., Goldbohm,R.A., van den Brandt,P.A. and van Poppel,G. (1997) A review of mechanisms underlying anticarcinogenicity by brassica vegetables. Chem. Biol. Interact., 103, 79–129.[Medline]
  18. Pantuck,E.J., Pantuck,C.B., Garland,W.A., Min,B.H., Wattenberg,L.W., Anderson,K.E., Kappas,A. and Conney,A.H. (1979) Stimulatory effect of brussels sprouts and cabbage on human drug metabolism. Clin. Pharm. Ther., 25, 88–95.[Medline]
  19. Pantuck,E.J., Pantuck,C.B., Anderson,K.E., Wattenberg,L.W., Conney,A.H. and Kappas,A. (1984) Effect of brussels sprouts and cabbage on drug conjugation. Clin. Pharm. Ther., 35, 161–169.[Medline]
  20. Bogaards,J.J.P., Verhagen,H., Willems,M.I., van Poppel,G. and van Bladeren,P.J. (1994) Consumption of brussels sprouts results in elevated {alpha}-class glutathione S-transferase levels in human blood plasma. Carcinogenesis, 15, 1073–1075.[Abstract]
  21. Fuhr,U., Rost,K.L., Engelhardt,R., Sachs,M., Liermann,D., Belloc,C., Beaune,P., Janezic,S., Grant,D., Meyer,U.A. and Staib,A.H. (1996) Evaluation of caffeine as a test drug for CYP1A2, NAT2 and CYP2E1 phenotyping in man by in vivo versus in vitro correlations. Pharmacogenetics, 6, 159–176.[Medline]
  22. Frandsen,H., Grivas,S., Andersson,R., Dragsted,L. and Larsen,J.C. (1992) Reaction of the N2-acetoxy derivative of 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP) with 2'-deoxyguanosine and DNA. Synthesis and identification of N2-(2'-deoxyguanosin-8-yl)–PhIP. Carcinogenesis, 13, 629–635.[Abstract]
  23. Lin,D., Kaderlik,K.R., Turesky,R.J., Miller,D.W., Lay,J.O.Jr and Kadlubar,F.F. (1992) Identification of N-(deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenylimidazo-(4,5-b)pyridine as the major adduct formed by the food-borne carcinogen, 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine, with DNA. Chem. Res. Toxicol., 5, 691–697.[Medline]
  24. Snyderwine,E.G., Davis,C.D., Nouso,K., Roller,P.P. and Schut,H.A. (1993) 32P-postlabelling analysis of IQ, MeIQx and PhIP adducts formed in vitro in DNA and polynucleotides and found in vivo in hepatic DNA from IQ-, MeIQx- and PhIP-treated monkeys. Carcinogenesis, 14, 1389–1395.[Abstract]
  25. Crosbie,S.J., Murray,S., Boobis,A.R. and Gooderham,N.J. (2000) Mass spectrometric detection and measurement of N2-(2'-deoxyguanosin-8-yl)PhIP adducts in DNA. J. Chromatogr., B744, 55–64.
  26. Murray,S., Gooderham,N.J., Boobis,A.R. and Davies,D.S. (1988) Measurement of MeIQx and DiMeIQx in fried beef by capillary column gas chromatography electron capture negative ion chemical ionisation mass spectrometry. Carcinogenesis, 9, 321–325.[Abstract]
  27. Murray,S., Lynch,A.M., Knize,M.G. and Gooderham,N.J. (1993) Quantification of the carcinogens 2-amino-3,8-dimethyl- and 2-amino-3,4,8-trimethylimidazo(4,5-f)quinoxaline and 2-amino-1-methyl-6-phenyl-imidazo(4,5-b)pyridine in food using a combined assay based on gas chromatography–negative ion mass spectrometry. J. Chromatogr., 616, 211–219.[Medline]
  28. Falkner,K., Williamson,G. and Mithen,R. (1998) Selective increase of the anticarcinogenic 4-methylsulphinylbutyl glucosinolate in broccoli. Carcinogenesis, 19, 605–609.[Abstract]
  29. Pettigrew,A.R. and Fell,G.S. (1972) Simplified colorimetric determination of thiocyanate in biological fluids, and its application to the investigation of the toxic amblyopias. Clin. Chem., 18, 996–1000.[Medline]
  30. Maron,D.M. and Ames,B.M. (1983) Revised method for the Salmonella mutagenicity test. Mutat. Res., 113, 173–215.[Medline]
  31. Reistad,R., Rossland,O.J., Latva-Kala,K.J., Rasmussen,T., Vikse,R., Becher,G. and Alexander,J. (1997) Heterocyclic aromatic amines in human urine following a fried meat meal. Food Chem. Toxicol., 35, 945–955[Medline]
  32. McDanell,R., McLean,A.E., Hanley,A.B., Heaney,R.K. and Fenwick,G.R. (1988) Chemical and biological properties of indole glucosinolates (glucobrassicins): a review. Food Chem. Toxicol., 26, 59–70.[Medline]
  33. Nijhoff,W.A., Grubben,M.J., Nagengast,F.M., Jansen,J.B., Verhagen,H., van Poppel,G. and Peters,W.H. (1995) Effects of consumption of Brussels sprouts on intestinal and lymphocytic glutathione S-transferases in humans. Carcinogenesis, 16, 2125–2128.[Abstract]
  34. Kall,M.A., Vang,O. and Clausen,J. (1996) Effects of dietary broccoli on human in vivo drug metabolizing enzymes: evaluation of caffeine, oestrone and chlorzoxazone metabolism. Carcinogenesis, 17, 793–799.[Abstract]
  35. Vistisen,K., Poulsen,H.E. and Loft,S. (1992) Foreign compound metabolism capacity in man measured from metabolites of dietary caffeine. Carcinogenesis, 13, 1561–1568.[Abstract]
  36. Lampe,J.W., King,I.B., Li,S., Grate,M.T., Barale,K.V., Chen,C., Feng,Z. and Potter,J.D. (2000) Brassica vegetables increase and apiaceous decrease cytochrome P4501A2 activity in humans: changes in caffeine metabolite ratios in response to controlled vegetable diets. Carcinogenesis, 21, 1157–1162.[Abstract/Full Text]
  37. Jongen,W.M. (1996) Glucosinolates in Brassica: occurrence and significance as cancer-modulating agents. Proc. Nutr. Soc., 55, 433–446.[Medline]
  38. Zhang,Y., Talalay,P., Cho,C.-G. and Posner,G.H. (1992) A major inducer of anticarcinogenic protective enzymes from broccoli: isolation and elucidation of structure. Proc. Natl Acad. Sci. USA, 89, 2399–2403.[Abstract]
  39. Crofts,F.G., Sutter,T.R. and Strickland,P.T. (1998) Metabolism of 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine by human cytochrome P4501A1, P4501A2 and P4501B1. Carcinogenesis, 19, 1969–1973.[Abstract]
  40. Malfatti,M.A., Kulp,K.S., Knize,M.G., Davis,C., Massengill,J.P., Williams,S., Nowell,S., MacLeod,S., Dingley,K.H., Turtletaub,K.W., Lang,N.P. and Felton,J.S. (1999) The identification of (2-14C)2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine metabolites in humans. Carcinogenesis, 20, 705–713.[Abstract/Full Text]
  41. Turesky,R.J., Garner,R.C., Welti,D.H., Richoz,J., Leveson,S.H., Dingley,K.H., Turtletaub,K.W. and Fay,L.B. (1998) Metabolism of the food-borne mutagen 2-amino-3,8-dimethylimidazo(4,5-f)quinoxaline in humans. Chem. Res. Toxicol., 11, 217–225.[Medline]
  42. Alexander,J., Wallin,H., Rossland,O.J., Solberg,K.E., Holm,J.A., Becher,G., Andersson,R. and Grivas,S. (1991) Formation of a glutathione conjugate and a semistable transportable glucuronide conjugate of N2-oxidised species of 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP) in rat liver. Carcinogenesis, 12, 2239–2245.[Abstract]
  43. Dingley,K.H., Curtis,K.D., Nowell,S., Felton,J.S., Lang,N.P. and Turtletaub,K.W. (1999) DNA and protein adduct formation in the colon and blood of humans after exposure to a dietary-relevent dose of 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine. Cancer Epidemiol. Biomarkers Prev., 8, 507–512.[Abstract/Full Text]
Received March 22, 2001; revised May 29, 2001; accepted May 31, 2001.