Metabolism and biomarkers of heterocyclic aromatic amines in humans

Abstract

Heterocyclic aromatic amines (HAAs) form during the high-temperature cooking of meats, poultry, and fish. Some HAAs also arise during the combustion of tobacco. HAAs are multisite carcinogens in rodents, inducing cancer of the liver, gastrointestinal tract, pancreas, mammary, and prostate glands. HAAs undergo metabolic activation by N-hydroxylation of the exocyclic amine groups to produce the proposed reactive intermediate, the heteroaryl nitrenium ion, which is the critical metabolite implicated in DNA damage and genotoxicity. Humans efficiently convert HAAs to these reactive intermediates, resulting in HAA protein and DNA adduct formation. Some epidemiologic studies have reported an association between frequent consumption of well-done cooked meats and elevated cancer risk of the colorectum, pancreas, and prostate. However, other studies have reported no associations between cooked meat and these cancer sites. A significant limitation in epidemiology studies assessing the role of HAAs and cooked meat in cancer risk is their reliance on food frequency questionnaires (FFQ) to gauge HAA exposure. FFQs are problematic because of limitations in self-reported dietary history accuracy, and estimating HAA intake formed in cooked meats at the parts-per-billion level is challenging. There is a critical need to establish long-lived biomarkers of HAAs for implementation in molecular epidemiology studies designed to assess the role of HAAs in health risk. This review article highlights the mechanisms of HAA formation, mutagenesis and carcinogenesis, the metabolism of several prominent HAAs, and the impact of critical xenobiotic-metabolizing enzymes on biological effects. The analytical approaches that have successfully biomonitored HAAs and their biomarkers for molecular epidemiology studies are presented.

Keywords: Biomarkers; Cancer; Cooked meat; Heterocyclic aromatic amines; Metabolism; Mutagens.

Fig. 1

Chemical structures of prevalent HAAs

Fig. 2

Major pathways AαC, IQ, MeIQx, and PhIP metabolism in experimental laboratory animals and humans

Fig. 3

Levels of CYP1A2 expression in rat and human liver microsomes correlates with CYP1A2 expression, MeIQx, and PhIP N-oxidation rates. The checkered lines depicted in the regression curves show the upper levels of CYP1A2 expression and MeIQx and PhIP N-oxidation rates in rat liver microsomes. Adapted with permission from [107]

Fig. 4

Kinetic parameters of MeIQx, PhIP N-oxidation, and methoxyresoruin oxidative demethylation by rat and recombinant human CYP1A2

Fig. 5

Chemical structures of HONH-PhIP Gluc conjugates

Fig. 6

Characterization of isomeric HONH-PhIP Gluc conjugates by multistage scanning (MSⁿ) with an Orbital trap by electrospray ionization in the negative ion mode. a PhIP-HON²-Gluc, MS² at m/z 415.1259, b MS³ at 415.1259 > 239.0938 >, c PhIP-HN²-O-Gluc; MS² at m/z 415.1259, d MS³ at 415.1259 > 223.0989 >, and e MS³ at m/z 415.1259 > 191.0197 >). Adapted with permission from [160]

Fig. 7

Reaction pathways of a HONH-Trp-P-2 and b N-acetoxy-PhIP intermediates with GSH and GSTs

Fig. 8

Structures of HAA-DNA adducts

Fig. 9

Proposed mechanism of dG-C8-Ar adduct formation, induction of abasic sites, DNA strand breakage, and 8-oxo-dG formation. Ar represents an aromatic amine or HAA

Fig. 10

Reconstructed mass chromatograms at the MS² scan stage of a human prostate sample targeting dG-C8-PhIP. One subject is shown with dG-C8-PhIP below the detection limit, and a second patient is positive for dG-C8-PhIP. [¹³C₁₀]-dG-C8-PhIP was employed as the internal standard at a level of 3 adducts per 10⁸ nucleotides. The MS³ scan stage product in spectra confirmed the identities of dG-C8-PhIP and its internal standard. Proposed MS³ fragmentation pathways are displayed; isotopically labeled ¹³C atoms of the internal standard are marked in red. Adapted with permission from [214]

Fig. 11

Co-oxidation of HbO₂ by N-hydroxylated aromatic amines and HAAs in human RBCs and met-Hb formation. The mass spectrum of the AαC sulfinamide formed at the β-Cys⁹³ of Hb was acquired by ion trap mass spectrometry employing electrospray ionization in the positive ion mode. The AαC-modified Hb was digested with Glu-C, and the peptide sequence encompassing the AαC sulfinamide adduct formed at Hb β-Cys⁹³ is reported. Adapted with permission from [226]

Fig. 12

a Mechanism of acid-labile PhIP sulfinamide adduct formation at SA-Cys³⁴. b Kinetics of formation and removal of this acid-labile PhIP-SA adduct in plasma of subjects on a cooked meat diet containing known quantities of PhIP. Box and whiskers plot showing the distribution of PhIP-SA measured over the entire study, separately for Group 3 (N = 13) and Group 4 (N = 7) ingested PhIP during the meat-feeding phase (days 22–49). Adapted with permission from [252]

Fig. 13

N-Acetoxy-PhIP modified human SA. Reconstructed mass chromatograms of a AW*[PhIP] AVAR ([M + 2H]²⁺ m/z 448.2385 > m/z 225.1135, m/z 671.3624; mass tolerance 2 ppm), b MS² product ion spectrum of AW*[PhIP] AVAR ([M + 2H]²⁺, c Product ion spectrum of W*PhIP adduct following digestion with Pronase E, and d Product ion ion spectrum of the H*PhIP adduct following digestion with Pronase E and proposed structures. The base peak at m/z 332.2 is attributed to the loss of formic acid. Adapted with permission from [255]

Fig. 14

Measurement of PhIP in the hair of omnivores and vegetarians. The full scan product ion spectrum acquired by triple quadrupole mass spectrometry confirmed the identity of PhIP. Adapted with permission from [263]

Fig. 15

Correlation of post-feeding PhIP scalp hair levels normalized for melanin in healthy volunteers with variable dietary PhIP intake 5 days a week over 4 weeks. The 0 value represents PhIP hair levels before commencing the feeding study. Adapted with permission from [268]

Fig. 16

HAA and HONH-HAA cytotoxicity and DNA adduct formation in LNCaP cells. a and b LNCaP cells were treated with DMSO (0.1%), HAAs, and HONH-HAAs (0.1 μM – 10 μM), for 24 h. Cell viability was evaluated by the MTS assay. c and d HAA and HONH-HAA DNA adduct formation in LNCaP cells after 24 h were measured by UPLC-ESI/MS³. Data are representative of at least three different experiment and are expressed as mean ± SD. ND: not detected. Ctrl: control

Fig. 17

dG-C8-PhIP DNA adduct formation as a function of HONH-PhIP (10 nM – 1000 nM) dose in LNCaP cells treated for 8 h. dG-C8-PhIP was measured by UPLC-ESI/MS³. Data are representative of at least three different experiments and are expressed as mean ± SD. LOQ: limit of quantification

Fig. 18

LNCaP cells (1 million cells) were incubated with test compounds HNOH-PhIP (10, 100 or 100 nM), DHT (1 nM), or t-BuOOH (500 μM) for 24 h [302]. Cells were lysed with 80% methanol/20% water containing 0.2% formic acid and extracts, concentrated by vacuum centrifugation, and analyzed by LC/MS with the Orbitrap Lumos at 120 K resolution [302]. Extracted ion chromatograms (5 ppm tolerance) for selected metabolite precursor ions and box and whisker plots depicting altered profiles for a glutamine, b glutamic acid, c GSH, d SAM, and e the product ion spectrum and the match to NIST 2019 mass spectrometry small molecule library entry supports the the identity of SAM

Fig. 19

PCA plots and molecular networks of LNCaP cells following treatment with HONH-PhIP (10, 100 or 1000 nM), DHT (1 nM), or t-BuOOH (500 μM). Data were processed using MetaboAnalyst 3.0 [309], R3.5 for statistics, and mummichog for molecular ID and pathways analysis [308]. a Statistical clustering result using ropls R package for the HRMS metabolite intensities showing the separation of the treatment groups. b mummichog molecular network of detected metabolite masses contributing toward the enrichment of multiple metabolomic pathways. The network of nodes and lines are molecules from metabolic networks detected using high-resolution accurate m/z values and mummichog with a database of possible m/z values and ion types for metabolites in humans. Round, colored nodes each represent a metabolite ion detected in the experiment. Pink and blue nodes depict molecules that increased or decreased (p < 0.01), respectively, in the HONH-PhIP-treated cells compared to the DMSO control. Lines indicate connections between metabolites within the metabolic network. Red labeled metabolites are profiled by LC/MS² in Fig. 18