Background: Hair is a promising tissue to assess exposure to 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a carcinogen formed in cooked meats. However, an understanding of how dietary exposure to PhIP, cytochrome P450 1A2 activity—a key enzyme involved in PhIP metabolism, and hair pigmentation affect the level of PhIP accrued in hair is required to determine the reliability of the PhIP hair level as a biomarker of exposure to this carcinogen. Methods: We examined the impact of PhIP exposure, cytochrome P450 1A2 activity, and hair pigmentation on the levels of PhIP accumulated in the hair of volunteers on a 4-week semicontrolled diet of cooked meat containing known quantities of PhIP. Results: The amount of PhIP in hair increased, on average, 15-fold in light- and dark-haired individuals during consumption of cooked meat. PhIP levels in hair were correlated to PhIP intake (ρ = 0.53; P < 0.001), and the relationship was strengthened when PhIP levels were normalized for the melanin content of hair (ρ = 0.71; P < 0.001). However, PhIP accrual in hair was not correlated to cytochrome P450 1A2 activity, as assessed by the caffeine test, or to the levels of unmetabolized PhIP in urine or to the metabolic ratio of the major urinary metabolite N2-(β-1-glucosiduronyl-2-(hydroxyamino)-1-methyl-6-phenylimidazo[4,5-b]pyridine to unmetabolized PhIP. Conclusions: The use of the PhIP hair biomarker should take hair pigmentation into account for accurate exposure assessment of PhIP. Impact: PhIP hair levels can serve as a biomarker in epidemiologic studies investigating the association of heterocyclic aromatic amine (HAA), cooked meat, and cancer risk. Cancer Epidemiol Biomarkers Prev; 22(3); 356–64. ©2013 AACR.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a heterocyclic aromatic amine (HAA) that is formed during the cooking of meat, poultry, and fish. PhIP is a rodent carcinogen and is thought to contribute to several diet-related cancers in humans. PhIP is present in the hair of human omnivores but not in the hair of vegetarians. We have now identified PhIP in the fur of 14 out of 16 healthy dogs consuming different brands of commercial pet food. The levels of PhIP in canine fur varied by over 85-fold and were comparable to the levels of PhIP present in human hair. However, high density fur containing PhIP covers a very high proportion of the body surface area of dogs, whereas high density terminal hair primarily covers the scalp and pubis body surface area of humans. These findings signify that the exposure and bioavailability of PhIP are high in canines. A potential role for PhIP in the etiology of canine cancer should be considered.

Abstract 2-Amino-9H-pyrido[2-3-b]indole (AαC) is a heterocyclic aromatic amine (HAA) and considered to be a possible human carcinogen by the International Agency for Research on Cancer (IARC). Like other HAAs, AαC is metabolized in the liver and can undergo bioactivation to produce highly electrophilic derivatives that can form DNA adducts by covalent binding to DNA. Among the HAAs, AαC is of particular interest because it is present at high levels in tobacco smoke and ubiquitous in the environment. AαC was shown to form high levels of DNA adducts in primary cultured human hepatocytes. The adduct levels were higher than those formed by other HAAs such as 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) or 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and comparable to those derived from 4-aminobiphenyl (4-ABP), a known human carcinogen. An understanding of the metabolism of AαC and its potential to form DNA adducts would provide a better assessment of the human health risk posed by AαC. Indeed, the metabolism pathways of MeIQx, PhIP and AαC are different. These differences in metabolic processing of HAAs can lead to variable levels of DNA adducts formed in humans and impact the interindividual susceptibility towards these procarcinogens. Knowledge about the metabolism of AαC in humans is unknown. We have analyzed the role of human cytochromes P450, especially CYP1A2, in the metabolism of AαC and DNA adduct formation. The ring-oxidized 3-HO-AαC and 6-HO-AαC sulfate and glucuronide conjugates, AαC-N2-Gl, N2-acetyl-AαC and the glucuronide conjugates of 2-hydroxyamino-9H-pyrido[2,-3-b]indole (HONH-AαC) and the deoxyguanosine DNA adduct, dG-C8-AαC, were identified and quantified by liquid chromatography coupled with multistage mass spectrometry in human hepatocytes in primary culture. The levels of DNA adducts formed after a 24hr-treatment with AαC were (2 folds) lower when cells were pre-treated with furafylline, a specific CYP1A2 inhibitor; however, the levels of most of the metabolites formed were not significanly affected by furafylline. Our data suggests that CYPs other than CYP1A2 may be involved in AαC metabolism, while CYP1A2 is the main enzyme involved in the production of reactive metabolites leading to the formation of DNA adducts. The identification of CYPs and Phase II enzymes involved in AαC metabolism in human hepatocytes are under study to fully understand the health risk of AαC to humans. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5739. doi:1538-7445.AM2012-5739

2-Amino-9H-pyrido[2,3-b]indole (AαC) is a carcinogenic heterocyclic aromatic amine (HAA) that arises in tobacco smoke. UDP-glucuronosyltransferases (UGTs) are important enzymes that detoxicate many procarcinogens, including HAAs. UGTs compete with P450 enzymes, which bioactivate HAAs by N-hydroxylation of the exocyclic amine group; the resultant N-hydroxy-HAA metabolites form covalent adducts with DNA. We have characterized the UGT-catalyzed metabolic products of AαC and the genotoxic metabolite 2-hydroxyamino-9H-pyrido[2,3-b]indole (HONH-AαC) formed with human liver microsomes, recombinant human UGT isoforms, and human hepatocytes. The structures of the metabolites were elucidated by 1H NMR and mass spectrometry. AαC and HONH-AαC underwent glucuronidation by UGTs to form, respectively, N2-(β-d-glucosidurony1)-2-amino-9H-pyrido[2,3-b]indole (AαC-N2-Gl) and N2-(β-d-glucosidurony1)-2-hydroxyamino-9H-pyrido[2,3-b]indole (AαC-HON2-Gl). HONH-AαC also underwent glucuronidation to form a novel O-linked glucuronide conjugate, O-(β-d-glucosidurony1)-2-hydroxyamino-9H-pyrido[2,3-b]indole (AαC-HN2-O-Gl). AαC-HN2-O-Gl is a biologically reactive metabolite and binds to calf thymus DNA (pH 5.0 or 7.0) to form the N-(deoxyguanosin-8-yl)-AαC adduct at 20–50-fold higher levels than the adduct levels formed with HONH-AαC. Major UGT isoforms were examined for their capacity to metabolize AαC and HONH-AαC. UGT1A4 was the most catalytically efficient enzyme (Vmax/Km) at forming AαC-N2-Gl (0.67 μl·min−1·mg of protein−1), and UGT1A9 was most catalytically efficient at forming AαC-HN-O-Gl (77.1 μl·min−1·mg of protein−1), whereas UGT1A1 was most efficient at forming AαC-HON2-Gl (5.0 μl·min−1·mg of protein−1). Human hepatocytes produced AαC-N2-Gl and AαC-HN2-O-Gl in abundant quantities, but AαC-HON2-Gl was a minor product. Thus, UGTs, usually important enzymes in the detoxication of many procarcinogens, serve as a mechanism of bioactivation of HONH-AαC.

Background: 2-Amino-9H-pyrido[2,3-b]indole (AαC) is a carcinogen formed in tobacco smoke, but little is known about its metabolism in humans. Result: UDP-Glucuronosyltransferases catalyze the binding of N-oxidized-AαC to DNA. Conclusion: Glucuronidation, normally a detoxication pathway, contributes to the genotoxicity of AαC. Significance: The exposure to and UGTbioactivation of AαC provides a biochemical mechanism for the elevated risk of liver and digestive tract cancers in smokers.

Some epidemiological investigations have revealed that frequent consumption of well-done cooked meats and tobacco smoking are risk factors for breast cancer in women. 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a heterocyclic aromatic amine that is formed in well-done cooked meat, and 4-aminobiphenyl (4-ABP) is an aromatic amine that arises in tobacco smoke and occurs as a contaminant in the atmosphere. Both compounds are rodent mammary carcinogens, and putative DNA adducts of PhIP and 4-ABP have been frequently detected, by immunohistochemistry (IHC) or (32)P-post-labeling methods, in mammary tissue of USA women. Because of these findings, PhIP and 4-ABP have been implicated as causal agents of human breast cancer. However, the biomarker data are controversial: both IHC and (32)P-post-labeling are non-selective screening methods and fail to provide confirmatory spectral data. Consequently, the identities of the lesions are equivocal. We employed a specific and sensitive liquid chromatography/mass spectrometry (MS) method, to screen tumor-adjacent normal mammary tissue for DNA adducts of PhIP and 4-ABP. Only 1 of 70 biopsy samples obtained from Minneapolis, Minnesota breast cancer patients contained a PhIP-DNA adduct. The level was three adducts per 10(9) nucleotides, a level that is 100-fold lower than the mean level of PhIP adducts reported by IHC or (32)P-post-labeling methods. The occurrence of 4-ABP-DNA adducts was nil in those same breast tissues. Our findings, derived from a specific mass spectrometry method, signify that PhIP and 4-ABP are not major DNA-damaging agents in mammary tissue of USA women and raise questions about the roles of these chemicals in breast cancer.

DNA adduct formation of the aromatic amine, 4-aminobiphenyl (4-ABP), a known human carcinogen present in tobacco smoke, and the heterocyclic aromatic amines (HAAs), 2-amino-9H-pyrido[2,3-b]indole (AαC), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), and 2-amino-3,8-dimethylmidazo[4,5-f]quinoxaline (MeIQx), potential human carcinogens, which are also present in tobacco smoke or formed during the high-temperature cooking of meats, was investigated in freshly cultured human hepatocytes. The carcinogens (10 μM) were incubated with hepatocytes derived from eight different donors for time periods up to 24 h. The DNA adducts were quantified by liquid chromatography-electrospray ionization mass spectrometry with a linear quadrupole ion trap mass spectrometer. The principal DNA adducts formed for all of the carcinogens were N-(deoxyguanosin-8-yl) (dG-C8) adducts. The levels of adducts ranged from 3.4 to 140 adducts per 10(7) DNA bases. The highest level of adduct formation occurred with AαC, followed by 4-ABP, then by PhIP, MeIQx, and IQ. Human hepatocytes formed dG-C8-HAA-adducts at levels that were up to 100-fold greater than the amounts of adducts produced in rat hepatocytes. In contrast to HAA adducts, the levels of dG-C8-4-ABP adduct formation were similar in human and rat hepatocytes. These DNA binding data demonstrate that the rat, an animal model that is used for carcinogenesis bioassays, significantly underestimates the potential hepatic genotoxicity of HAAs in humans. The high level of DNA adducts formed by AαC, a carcinogen produced in tobacco smoke at levels that are up to 100-fold higher than the amounts of 4-ABP, is noteworthy. The possible causal role of AαC in tobacco-associated cancers warrants investigation.

A targeted liquid chromatography/tandem mass spectrometry-based metabolomics type approach, employing a triple stage quadrupole mass spectrometer in the product ion scan and selected reaction monitoring modes, was established to profile 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), and their principal metabolites in the urine of omnivores. A mixed-mode reverse phase cation exchange resin enrichment procedure was employed to isolate MeIQx and its oxidized metabolites, 2-amino-8-(hydroxymethyl)-3-methylimidazo[4,5-f]quinoxaline (8-CH(2)OH-IQx) and 2-amino-3-methylimidazo[4,5-f]quinoxaline-8-carboxylic acid (IQx-8-COOH), which are produced by cytochrome P450 1A2 (P450 1A2). The phase II conjugates N(2)-(beta-1-glucosiduronyl)-2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline and N(2)-(3,8-dimethylimidazo[4,5-f]quinoxalin-2-yl)-sulfamic acid were measured indirectly, following acid hydrolysis to form MeIQx. The enrichment procedure permitted the simultaneous analysis of PhIP, N(2)-(beta-1-glucosidurony1)-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, N3-(beta-1-glucosidurony1)-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, 2-amino-1-methyl-6-(4'-hydroxy)-phenylimidazo[4,5-b]pyridine (4'-HO-PhIP), and the isomeric N(2)- and N3-glucuronide conjugates of the carcinogenic metabolite, 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine (HONH-PhIP), which is formed by P450 1A2. The limit of quantification (LOQ) for MeIQx, PhIP, and 4'-HO-PhIP was approximately 5 pg/mL; the LOQ values for 8-CH(2)OH-IQx and IQx-8-COOH were, respectively, <15 and <25 pg/mL, and the LOQ values for the glucuronide conjugates of PhIP and HONH-PhIP were 50 pg/mL. The metabolism was extensive; less than 9% of the dose was eliminated in urine as unaltered MeIQx, and <1% was eliminated as unaltered PhIP. Phase II conjugates of the parent amines accounted for up to 12% of the dose of MeIQx and up to 2% of the dose of PhIP. 8-CH(2)OH-IQx and IQx-8-COOH accounted for up to 76% of the dose of MeIQx, and the isomeric glucuronide conjugates of HONH-PhIP accounted for up to 33% of the dose of PhIP that were eliminated in urine within 10 h of meat consumption. P450 1A2 significantly contributes to the metabolism of both HAAs but with marked differences in substrate specificity. P450 1A2 primarily catalyzes the detoxification of MeIQx by oxidation of the 8-methyl group, whereas it catalyzes the bioactivation of PhIP by oxidation of the exocyclic amine group.

The cooked meat carcinogens 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), and their principal metabolites produced by cytochrome P450 and/or uridine diphosphate glucuronosyl transferases were simultaneously measured at the parts per trillion level in urine of omnivores, by ultraperformance liquid chromatography (UPLC) with a Michrom Advance CaptiveSpray source and a triple stage quadrupole mass spectrometer. Quantitation was performed in the selected reaction monitoring mode. The UPLC method is much more rapid and sensitive than our earlier capillary HPLC method: the duty cycle of the UPLC method is 19 min compared to 57 min for capillary HPLC. The performance of the UPLC assay was evaluated with urine samples from three subjects over 4 different days. The intraday and interday precisions of the estimates of PhIP, MeIQx, and their metabolites, reported as the coefficients of variation, were ≤10%. The limit of quantification (LOQ) values for PhIP and MeIQx were about 5 pg/mL, whereas the LOQ values of their metabolites ranged from 10 to 40 pg/mL. Furthermore, the identities of the analytes were corroborated by acquisition of full scan product ion spectra, employing between 0.5 and 5 pg of analyte for assay.