Incorporation of the food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) into hair of mice

The incorporation of the food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) into hair of newborn mice was investigated in order to contribute to the validation of PhIP in hair as a suitable biomarker for human dietary exposure. Black mice (C57BL/6J; 7-9 days old) were given graded doses of [³H]-PhIP subcutaneously during the start of the hair growth period. The distribution of [³H]-PhIP and incorporation into hair were investigated by tape-section autoradiography. Almost all the radioactivity in hair represented PhIP as shown by high performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS). A dose-response proportionality of incorporation into hair was found when incorporation was determined by liquid scintillation counting. Autoradiography showed that PhIP was rapidly cleared from the skin, but remained for at least 28 days in the part of the hair shafts which was formed during the exposure period. The present results obtained using the mouse as a model, further support the suggestion that PhIP in hair may be a suitable biomarker for human exposure to dietary PhIP.     

Chemopreventive effects from prebiotic inulin towards microbial 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine bioactivation

Aims:  Using a Simulator of the Human Intestinal Microbial Ecosystem (SHIME), we investigated the chemopreventive potential of prebiotic chicory inulin towards the in vitro bioactivation of 2-amino-1-methyl-6-phenylimidazo[4,5- b ]pyridine (PhIP) by human intestinal microbiota.
Methods and Results:  HPLC data revealed that inulin significantly decreased the formation of the genotoxic PhIP-M1 metabolite, with the highest inhibitory activity in the colon ascendens (87% decrease). Interestingly, this chemopreventive effect correlated with alterations of bacterial community composition and metabolism in the different colon compartments. Conventional culture-based techniques and PCR-DGGE analysis on the SHIME colon suspension revealed significant bifidogenic effects during inulin treatment, whereas the overall microbial community kept relatively unchanged. Additionally, the production of short-chain fatty acids increased with 12%, 3% and 7%, while ammonia concentrations decreased with 3%, 4% and 3% in the ascending, transverse and descending colon compartments, respectively.
Conclusions:  These results indicate that the prebiotic effects from inulin may also purport protective effects towards microbial PhIP bioactivation.
Significance and Impact of the Study:  As the colonic microbiota may contribute significantly to the carcinogenic potential of PhIP, the search for dietary constituents that decrease the formation of this harmful metabolite, may help in preventing its risk towards human health.     

Phytoalexin resveratrol attenuates the mutagenicity of the heterocyclic amines 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline

Resveratrol is a phytoalexin, that belongs to a family of naturally occurring stilbenes. It has been reported that resveratrol can inhibit chemical carcinogenesis in experimental animals and although the mechanisms involved are unknown, an anti-mutagen mechanism has been proposed. We have explored this hypothesis using mutagenicity assays based on bacterial (Salmonella typhimurium) and eukaryotic cells (Chinese hamster V79 cells). We found resveratrol to be potent in both systems, blocking the mutagenicity of the food-derived heterocyclic amines (HA) 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) at micromolar concentrations. Furthermore, in cells capable of activating 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine to cytotoxic derivatives, resveratrol was able to attenuate cytotoxicity. Paradoxically, in cells lacking the ability to activate PhIP, resveratrol itself was toxic and co-incubation with PhIP reduced this toxicity. Our data confirm the potent anti-mutagenic activity of resveratrol and support its potential as a chemopreventative.     

Analysis of DNA adducts by accelerator mass spectrometry in human breast tissue after administration of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and benzo[a]pyrene

Epidemiological evidence has suggested an association between meat consumption and the risk of breast cancer. 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a heterocyclic amine found in cooked meat, has been implicated in the aetiology of breast cancer and has been shown to induce tumour formation in rodent mammary glands. In addition, polycyclic aromatic hydrocarbons, such as benzo[a]pyrene (B[a]P) which has also been shown to induce tumour formation at a number of sites in rodents including the breast, are produced during the cooking of meat through the pyrolysis of fats. The aim of this study was to examine the bioavailability of these compounds to human breast tissue and their ability to bind to DNA to form DNA adducts. Patients undergoing breast surgery at York District Hospital were orally administered prior to surgery a capsule containing 20microg of 14C PhIP (182kBq, specific activity 2.05GBq/mmol) or 5microg of 14C B[a]P (36kBq, specific activity 1.81GBq/mmol). At surgery, normal and tumour breast tissue was resected and tissue concentrations of carcinogen measured by liquid scintillation counting and DNA adduct levels by accelerator mass spectrometry (AMS) were subsequently determined. It was found that both 14C PhIP and 14C B[a]P were able to reach the target organ where they had the ability to form DNA adducts. The level of adducts ranged from 26.22-477.35 and 6.61-208. 38 adducts/10(12) nucleotides following administration of 14C PhIP and 14C B[a]P, respectively, with no significant difference observed between levels in normal or tumour tissue. In addition, the data obtained in this study were comparable to adduct levels previously found in colon samples following administration of the same compounds to individuals undergoing colorectal surgery. This is the first report that these two carcinogens bind to human breast DNA after administration of a defined low dose.     

Responses of PhIP (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine) in MCF-7 cells are culture condition dependent

To compare the effects of the food toxin 2-amino-1-methyl-6-phenyl-imidazo[4,5-b]pyridine (PhIP) and estradiol in hormone-responsive MCF-7 cells, the cells were exposed to different concentrations of either PhIP or estradiol. The effect of various culture conditions (e.g. phenol red, FBS, vehicle (DMSO/EtOH) and seeding density) on responses was studied. Cells were continuously grown with steroid-containing or -deprived medium, or switched from steroid-containing to -deprived medium for the experiments to minimize the effect of background estrogenicity. Effects of PhIP and estradiol on cell viability and proliferation were determined by ATP analysis and Ki-67 immunocytochemistry. Expression of estrogen receptor alpha, cell stress markers (p53 and ERK) and estrogen responsive proteins (c-myc and ERK) were immunoblotted. All concentrations of estradiol induced cell proliferation, viability and changes in protein expression, typical for estrogenic responses. PhIP, however, increased viability only at low concentrations and depending on culture conditions. No changes in protein expressions by PhIP were noted, not even when switching cells from steroid-containing to -deprived medium which down-regulated the expression of proteins at basal level. Vehicle affected significantly viability, especially after exposure to PhIP, but not protein expression while medium changes affected both. In conclusion, the effects of PhIP and estradiol in MCF-7 cells are dependent on culture conditions. The detected PhIP-induced changes are weaker compared to those induced by estradiol.     

Inhibition of DNA adduct formation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and 2-amino-3-methylimidazo[4,5-f]quinoline by dietary indole-3-carbinol in female rats.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) are two important heterocyclic amines formed in proteinaceous foods during the cooking process. Both PhIP and IQ are carcinogenic in several strains of rats. PhIP induces mammary tumors in female F344 rats, while IQ induces principally mammary and liver tumors in female Sprague-Dawley rats. Both PhIP and IQ are activated enzymatically, first by N-hydroxylation, catalyzed by CYP1A1 and CYP1A2, and subsequently by esterification (O-acetylation or sulfation), to yield DNA adducts. Such DNA adduct formation, and persistence of adducts, is related to initiation of carcinogenesis, while inhibition of this process leads to prevention of carcinogenesis. Indole-3-carbinol (I3C), a constituent of cruciferous vegetables, has chemopreventive properties in various systems; it probably acts by induction of detoxification enzymes. We have examined the effect of dietary I3C on DNA adduct formation by PhIP in female F344 rats and on that by IQ in female Sprague-Dawley rats. In experiment 1, F344 rats were maintained on AIN-76A diet containing 0.1% (w/w) I3C and then given p.o. doses (10 or 50 mg/kg) of PhIP. These doses are known to induce CYP1A1 and CYP1A2. Groups of animals (4/time point) were euthanized 1, 2, 6, and 16 days later, and their blood (for isolation of white blood cells), mammary glands, liver, stomach, small intestine, cecum, colon, heart, lungs, kidneys, and spleen were removed for DNA isolation and quantitation of PhIP-DNA adducts by 32P-postlabeling. PhIP-DNA adduct formation was inhibited (40-100%) by I3C in virtually all organs, including the mammary gland (the target organ), at both doses of PhIP, and at almost all time points. In a second experiment, Sprague-Dawley rats were fed either control AIN-76A diet or this diet containing 0.02% I3C or 0.1% I3C for a total of 42 days. IQ was added to the diets (0.01%, w/w) from day 15 to day 42, after which all rats received diet free of IQ and I3C. Groups of animals (4/time point) were killed on days 43 and 57. In addition to the organs removed in experiment 1, the pancreas, uterus, and ovaries were also removed, and IQ-DNA adducts were quantitated by 32P-postlabeling. Both dietary concentrations of I3C inhibited IQ-DNA adduct formation in most organs (except in lungs, kidneys, and pancreas) on both days 43 and 57; in liver, stomach, mammary gland, and spleen, inhibition was evident only on day 43. Inhibitions ranged from 22.6 to 86.6% with the 0.02% I3C diet and from 32.2 to 89.6% with the 0.1% I3C diet. I3C diets did not affect rate of adduct removal in either experiment. It is concluded that dietary I3C inhibits PhIP- and IQ-DNA adduct formation in both target and nontarget organs of female rats, even with high doses of PhIP when CYP1A1 and CYP1A2, the enzymes responsible for the initial activation (N-hydroxylation) of PhIP, are expected to be induced.     

Detection of 2-amino-1-methyl-6-phenylimidazo [4,5-b]-pyridine (PhIP)-DNA adducts in human pancreatic tissues

Recent epidemiological investigations have observed an association between the consumption of grilled or barbecued meat and an increased risk of pancreatic cancer, suggesting that dietary exposure to heterocyclic aromatic amines (HCA) may contribute to the development of this disease. 2-Amino-1-methyl-6-phenylimidazo [4,5-b]-pyridine (PhIP) is the most abundant HCA found in well-done and grilled meats. To determine whether HCA-induced DNA damage is present in the human pancreas, immunohistochemistry and computer-assisted image analysis were used to measure PhIP-DNA adducts in 54 normal pancreatic tissues (N) from persons without pancreatic cancer and in 38 normal adjacent pancreatic tissues (A) and in 39 cancer tissues (T) from 68 patients with pancreatic adenocarcinoma. PhIP-DNA adducts were detected in 53 N, 34 A and 39 T samples. Mean values (±SD) of the absorbency for PhIP staining were 0.22±0.04, 0.24±0.04, and 0.24±0.03 for N, A, and T samples, respectively (p=0.004). Using the median absorbency (0.21) of the samples from normal controls as the cut-off, 71% of A and 77% of T tissues, compared with 48% of N tissues, were distributed in the higher range (p=0.009). The odds ratio of pancreatic cancer was 3.4 (95% confidence interval 1.5-7.5, p=0.002) for individuals with a higher level of PhIP-DNA adducts. This is the first report of the detection of PhIP-DNA adducts in human pancreatic tissue samples obtained from patients with unknown exposure to HCA. Although limited by the small sample size, these preliminary results suggest that PhIP exposure may contribute to human pancreatic cancer development.     

Mlh1-dependent suppression of specific mutations induced in vivo by the food-borne carcinogen 2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine (PhIP)

Disruption of the DNA mismatch repair (MMR) pathway results in elevated mutation rates, inappropriate survival of cells bearing DNA damage, and increased cancer risk. Relatively little is known about the potential impact of environmentally relevant carcinogens on cancer risk in individuals with MMR-deficiency. We determined the effect of MMR status (Mlh1+/+ versus Mlh1-/-) on mutagenesis induced by the cooked-meat mutagen, 2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine (PhIP) within cII and supFG1 transgene reporters. Despite being a lymphomagen in mice, PhIP was not mutagenic in thymus. In colon, PhIP exposure induced 3-fold more mutations in Mlh1-deficient mice compared to their Mlh1+/+ littermates. Similar induction was seen in Mlh1-/- small intestine. Analysis of mutational spectra revealed that G/C to T/A transversions, the "signature PhIP mutation", were induced to similar levels regardless of Mlh1 status. In contrast, Mlh1-/- mice exhibited hypermutability to frameshifts, G/C to A/T transitions, and G/C to C/G transversions. Thus, both the level and types of mutation induced by PhIP are influenced by the activity of the MMR system. MMR may suppress PhIP-induced mutation through recognition and processing of specific mispairs (PhIP-G/T, PhIP-G/G, and PhIP-G/loop mispairs). In contrast, the PhIP-G/A mispair is unlikely to be a MMR substrate. In addition, the similar induction of both transversions and transitions in Mlh1-/- mice suggests that mutagenic bypass of PhIP-G is similarly efficient with dATP, dTTP, and dGTP, in contrast to previously published conclusions. Our data suggests that MMR-deficiency would increase the likelihood of PhIP-induced carcinogenic mutations. Further evaluation of the risk that consumption of heterocyclic amines may impart to MMR-deficient individuals therefore is warranted.     

Validity of a self-administered food frequency questionnaire in the assessment of heterocyclic amine intake using 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) levels in hair

Several case-control studies have reported possible associations between heterocyclic amine (HCA) intake and the risk of cancer. The validity of questionnaires used to assess HCA intake has hardly been examined, however; in particular, no biomarker able to serve as an independent measure of habitual HCA intake has been established. In this study, we examined the validity of HCA intake estimated from a food frequency questionnaire (FFQ) using 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) level in hair as a reference method. Study subjects were 20 volunteers (7 men and 13 women) aged 25-57 years residing in Tokyo or neighboring cities in Japan. The subjects completed the FFQ, and gave 3-5g of hair twice at an interval of 1-3 months for use in establishing validity. Results showed that intakes of PhIP, MeIQ, Trp-P-1, and total HCA by the FFQ were significantly correlated with PhIP levels in hair when adjustment was made for melanin content (r=0.47, r=0.50, r=0.55, and r=0.51, respectively). The present study indicates that HCA intake estimated from this FFQ provides a reasonable ranking of individuals to allow the analysis of associations between HCA intake and risk of cancer in large-scale epidemiological studies.     

Validity of a self-administered food frequency questionnaire in the assessment of heterocyclic amine intake using 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) levels in hair

Several case-control studies have reported possible associations between heterocyclic amine (HCA) intake and the risk of cancer. The validity of questionnaires used to assess HCA intake has hardly been examined, however; in particular, no biomarker able to serve as an independent measure of habitual HCA intake has been established. In this study, we examined the validity of HCA intake estimated from a food frequency questionnaire (FFQ) using 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) level in hair as a reference method. Study subjects were 20 volunteers (7 men and 13 women) aged 25–57 years residing in Tokyo or neighboring cities in Japan. The subjects completed the FFQ, and gave 3–5 g of hair twice at an interval of 1–3 months for use in establishing validity. Results showed that intakes of PhIP, MeIQ, Trp-P-1, and total HCA by the FFQ were significantly correlated with PhIP levels in hair when adjustment was made for melanin content (r = 0.47, r = 0.50, r = 0.55, and r = 0.51, respectively). The present study indicates that HCA intake estimated from this FFQ provides a reasonable ranking of individuals to allow the analysis of associations between HCA intake and risk of cancer in large-scale epidemiological studies.     

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) level in human hair as biomarkers for dietary grilled/stir-fried meat and fish intake

Several case-control studies have reported possible associations between heterocyclic amine (HCA) intake and the risk of cancer. However, the validity of a questionnaire to assess HCA intake has hardly been examined. In particular, no biomarker which could serve as an independent measure of habitual HCA intake has been established. Therefore, the validity of a questionnaire to assess HCA intake by means of a biomarker remains to be investigated. In this study, we examined the availability of hair HCAs as a biochemical indicator of dietary intake of HCAs. Study subjects were 20 volunteers (7 men and 13 women) aged 25-57 years, either residents of Tokyo or the neighboring cities in Japan. We collected individual weighed dietary records (DR) over 28 consecutive days. Approximately 3-5 g of hair was collected twice from all subjects before and after DR at intervals of 1-3 months. The mean (S.D.) 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) level of hair was 1376.0 pg/g hair (928.9) and 16.6 ng/g melanin (12.3). A steady increase in the mean PhIP level in hair from the lowest to the highest tertile of the grilled/stir-fried meat intake was observed (P = 0.009), but not in the grilled/stir-fried fish intake (P = 0.461). The PhIP level in hair was highly correlated with the grilled/stir-fried meat intake (r = 0.68) but not with the grilled/stir-fried fish intake (r = 0.28). These observations were made of hair with and without melanin adjustment. The present study indicates that the PhIP level in hair can be used as a biological indicator of dietary intake of HCAs.     

Determination of 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (PhIP) and its metabolite 2-hydroxyamino-PhIP by liquid chromatography/electrospray ionization-ion trap mass spectrometry.

2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is the most abundant heterocyclic aromatic amine found in cooked meat. It is metabolically activated by the human cytochrome P450 enzymes to form the carcinogenic metabolite N2-OH-PhIP. PhIP has been found to induce tumors in rats and is a suspected human carcinogen. In the present work, we have developed and validated a liquid chromatography-electrospray ionization/ion trap mass spectrometry (LC-ESI/ITMS) method for the determination of PhIP and N2-OH-PhIP. PhIP was incubated with microsomes prepared from the human liver; the PhIP and N2-OH-PhIP formed were isolated from the biomatrices by solid-phase extraction using C18 cartridges, with recoveries greater than 86%. Subsequently, the products were separated on a microbore reversed-phase C18 liquid chromatograph coupled to an ESI-ITMS. The ESI interface and the ITMS were tuned for various parameters, and data acquisition was performed in selective ion monitoring mode. The detection limit of PhIP and N2-OH-PhIP was 1 and 10 pg, respectively. The method is highly sensitive and selective, has simple sample preparation protocols, and should be applicable to the study of the metabolic activation of PhIP in various human tissues.     

Analysis of DNA adducts by accelerator mass spectrometry in human breast tissue after administration of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and benzo[a]pyrene

Epidemiological evidence has suggested an association between meat consumption and the risk of breast cancer. 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a heterocyclic amine found in cooked meat, has been implicated in the aetiology of breast cancer and has been shown to induce tumour formation in rodent mammary glands. In addition, polycyclic aromatic hydrocarbons, such as benzo[a]pyrene (B[a]P) which has also been shown to induce tumour formation at a number of sites in rodents including the breast, are produced during the cooking of meat through the pyrolysis of fats. The aim of this study was to examine the bioavailability of these compounds to human breast tissue and their ability to bind to DNA to form DNA adducts. Patients undergoing breast surgery at York District Hospital were orally administered prior to surgery a capsule containing 20 μg of ¹⁴C PhIP (182 kBq, specific activity 2.05 GBq/mmol) or 5 μg of ¹⁴C B[a]P (36 kBq, specific activity 1.81 GBq/mmol). At surgery, normal and tumour breast tissue was resected and tissue concentrations of carcinogen measured by liquid scintillation counting and DNA adduct levels by accelerator mass spectrometry (AMS) were subsequently determined. It was found that both ¹⁴C PhIP and ¹⁴C B[a]P were able to reach the target organ where they had the ability to form DNA adducts. The level of adducts ranged from 26.22–477.35 and 6.61–208.38 adducts/10¹² nucleotides following administration of ¹⁴C PhIP and ¹⁴C B[a]P, respectively, with no significant difference observed between levels in normal or tumour tissue. In addition, the data obtained in this study were comparable to adduct levels previously found in colon samples following administration of the same compounds to individuals undergoing colorectal surgery. This is the first report that these two carcinogens bind to human breast DNA after administration of a defined low dose.     

Adenomatous polyposis coli truncation mutations in 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced intestinal tumours of multiple intestinal neoplasia mice

The heterocyclic amine 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) induces intestinal tumours in C57BL/6J-multiple intestinal neoplasia (Min)/+ mice. The main mechanism for PhIP-induced tumour induction in Min/+ mice is loss of the wild-type adenomatous polyposis coli (Apc) allele, i.e. loss of heterozygosity (LOH). In this study, single injections of either 10, 17.5 or 25 mg/kg PhIP on days 3-6 after birth all increased the mean number of small intestinal tumours two to three-fold, from 37.7 in controls to 124.8 in the PhIP-treated Min/+ mice. In total, we analysed 292 small intestinal tumours and 253 of these had LOH. The frequency of LOH in the Apc gene was 88, 93, 83 and 84% in tumours of 0, 10, 17.5 and 25 mg/kg PhIP-treated mice, respectively. Therefore, these lower doses of PhIP did not reduce the frequency of LOH, as found in our previous study with a single injection of 50 mg/kg PhIP (Mutat. Res. 1-2 (2002) 157). In the second part of this study, we wanted to characterise Apc truncation mutations from tumour samples apparently retaining the Apc wild-type allele from this and two previous experiments with PhIP-exposed Min/+ mice. In the first half of exon 15 in Apc, we verified 25 mutations from 804 tumour samples of PhIP-treated mice. Of these were 60% G-->T transversions, and 16% G deletions, indicating that these are the predominant types of PhIP-induced truncation mutations in the Apc gene in Min/+ mice. Most of the mutations were located between codon 989 and 1156 corresponding to the first part of the beta-catenin binding region. We also identified two Apc truncation mutations from 606 spontaneously formed intestinal tumours from untreated Min/+ mice, one C-->T transition and one T insertion, which were different from those induced by PhIP.     

Inhibition of cell death in human mammary epithelial cells by the cooked meat-derived carcinogen 2-amino-1-methyl-6-phenylimidazo[4, 5-b]pyridine

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a mammary gland carcinogen present in the human diet in cooked meat. To examine if PhIP and its reactive metabolite N-hydroxy-PhIP inhibit apoptosis in human mammary epithelial MCF-10A cells, confluent cultures deprived of serum and growth factors were incubated for 24 h with either compound. The percentages of dead cells (mean +/- SEM, n = 3) as measured by trypan blue exclusion were 5.7 +/- 0.6, 3.4 +/- 0.3, 2.7 +/- 0.3, and 0.2 +/- 0.003%, in control, 1 microM N-hydroxy-PhIP-, 5 microM N-hydroxy-PhIP-, and 100 microM PhIP-treated dishes, respectively. The expression of Bcl-2 and Bcl-x(L) as quantitated by Western blotting was 1.2- to 1.9-fold higher in the treated groups. PhIP-DNA adducts induced by N-hydroxy-PhIP in MCF-10A cells measured by the (32)P-postlabeling assay were low (<1 x 10(7), relative adduct labeling). No adducts were detected after incubation with PhIP. Western blot analysis indicated that PhIP increased ERK2 phosphorylation concomitant with Bcl-2. The results suggest that the inhibition of cell death in mammary epithelial cells by PhIP occurs independently of PhIP-DNA adducts and may involve enhanced signaling through the MAP kinase pathways.     

Detection and quantitation of N-(deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine adducts in DNA using online column-switching liquid chromatography tandem mass spectrometry

The heterocyclic aromatic amine, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is formed by the grilled cooking of certain foods such as meats, poultry and fish. PhIP has been shown to induce tumours in the colon, prostate and mammary glands of rats and is regarded as a potential human dietary carcinogen. PhIP is metabolically activated via cytochrome P450 mediated oxidation to an N-hydroxylamino-PhIP intermediate that is subsequently converted to an ester by N-acetyltransferases or sulfotransferases and undergoes heterolytic cleavage to produce a PhIP-nitrenium ion, which reacts with DNA to form the N-(deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP-C8-dG) adduct. Thus far, the detection and quantification of PhIP-DNA adducts has relied to a large extent on ³²P-postlabelling methodologies. In order to expand the array of available techniques for the detection and improved quantification of PhIP-C8-dG adducts in DNA we have developed an online column-switching liquid chromatography (LC)–electrospray ionization (ESI)-tandem mass spectrometry (MS/MS) selected reaction monitoring (SRM) method incorporating an isotopically [¹³C₁₀]-labelled PhIP-C8-dG internal standard for the analysis of DNA enzymatically hydrolysed to 2′-deoxynucleosides. A dose-dependent increase was observed for PhIP-C8-dG adducts when salmon testis DNA was reacted with N-acetoxy-PhIP. Analysis of DNA samples isolated from colon tissue of mice treated by oral gavage daily for 5 days with 50 mg/kg body weight of PhIP resulted in the detection of an average level of 14.8 ± 3.7 PhIP-C8-dG adducts per 10⁶ 2′-deoxynucleosides. The method required 50 μg of hydrolysed animal DNA on column and the limit of detection for PhIP-C8-dG was 2.5 fmol (1.5 PhIP-C8-dG adducts per 10⁸ 2′-deoxynucleosides). In summary, the LC–ESI-MS/MS SRM method provides for the rapid automation of the sample clean up and a reduction in matrix components that would otherwise interfere with the mass spectrometric analysis, with sufficient sensitivity and precision to analyse DNA adducts in animals exposed to PhIP.     

The effect of UDP-glucuronosyltransferase 1A1 expression on the mutagenicity and metabolism of the cooked-food carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine in CHO cells

UDP-glucuronosyltransferase proteins (UGT) catalyze the glucuronidation of both endogenous and xenobiotic compounds. In previous studies, UGT1A1 has been implicated in the detoxification of certain food-borne carcinogenic-heterocyclic amines. To determine the importance of UDP-glucuronosyltransferase 1A1 (UGT1A1) in the biotransformation of the cooked-food carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), genetically modified CHO cells that are nucleotide excision repair-deficient, and express cytochrome P4501A2 (UV5P3 cell line) were transfected with a cDNA plasmid of human UGT1A1 to establish the UDP-glucuronosyltransferase 1A1 expressing 5P3hUGT1A1 cell line. Expression of the UGT1A1 gene was verified by screening neo gene expressing clonal isolates (G-418 resistant) for their sensitivity to cell killing from PhIP exposure. Five of 11 clones were chosen for further analysis due to their resistance to cell killing. Western blot analysis was used to confirm the presence of the UGT1A1 and CYP1A2 proteins. All five clones displayed a 52-kDa protein band, which corresponded to a UGT1A1 control protein. Only four of the clones had a protein band that corresponded to the CYP1A2 control protein. Correct fragment size of the cDNAs in the remaining four clones was confirmed by RT-PCR and quantification of the mRNA product was accomplished by real-time RT-PCR. Expression of UGT1A1 in the transfected cells was 10⁴–10⁵-fold higher relative to the UV5P3 parental cells. One clone (#14) had a 10-fold higher increase in expression at 1.47 × 10⁵ over the other three clones. This clone was also the most active in converting N-hydroxy-PhIP to N-hydroxy-PhIP glucuronide conjugates in microsomal metabolism assays. Based on the D₅₀ values, the cytotoxic effect of PhIP was decreased 350-fold in the 5P3hUGT1A1 cells compared to the UV5P3 control cells. In addition, no significant increase in mutation frequency was observed in the transfected cells. These results clearly indicate that UGT1A1 plays a critical role in PhIP biotransformation, providing protection against PhIP-mediated cytotoxicity and mutagenicity.     

One dose of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) or 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) induces tumours in Min/+ mice by truncation mutations or LOH in the Apc gene

The C57BL/6J-Min/+ (multiple intestinal neoplasia) mouse has a heterozygous nonsense Apc(Min) (adenomatous polyposis coli) mutation, and numerous adenomas spontaneously develop in the intestine. Neonatal exposure of Min/+ mice to the food carcinogens 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) or 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) (one injection of 50mg/kg) increased the number of small intestinal tumours about three- and two-fold, respectively. The number of colonic tumours was only increased in males. We examined whether the wild-type Apc allele was affected in intestinal tumours induced by either PhIP or IQ. In spontaneously formed and in IQ-induced small intestinal and colonic tumours from these mice, the main mechanism for tumour induction was loss of wild-type Apc allele, i.e. loss of heterozygosity (LOH). In contrast to the IQ-induced (84% LOH) and spontaneously (88% LOH) formed tumours, only 55% of the PhIP-induced small intestinal tumours from males showed LOH. Tumours that apparently had retained the wild-type Apc allele were further analysed for the presence of truncated Apc proteins by the in vitro synthesised protein (IVSP) assay. Truncated Apc proteins, indicating truncation mutations in exon 15 of the Apc gene, were detected in two of the 12 PhIP-induced tumours in segment 2 (codons 686-1217), and two of five IQ-induced tumours, one in segment 2 and the other in segment 3 (codons 1099-1693). Three of these four mutations, all in segment 2 of the Apc gene, were confirmed by sequencing. The PhIP-induced mutations were detected at codon 1125 (C deletion) and 1130 (G-T transversion), and the IQ-induced mutation was at codon 956 (C-T transition). Importantly, no truncated proteins were detected in tumours from unexposed mice with apparently retained wild-type Apc allele. These results show that one injection of either PhIP or IQ induces intestinal tumours in the Min/+ mice by inactivation of the wild-type Apc allele either by causing LOH or truncation mutations.