Heterocyclic Amines (HCA)

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Heterocyclic amines (HCAs) contain at least one heterocyclic ring (with atoms of at least 2 different elements) and one amine group, attached to the heterocyclic ring. Major groups of HCAs are Carbolines (indoles), Pyrrolidines, Pyrroles (in Hb and vitamin B12), Pyridines (vitamin B3 and B6) and Pyrimidines (vitamin B1), including Purines (adenine, guanine).

Many HCA are harmless, or even beneficial (eg vitamins), whereas others, created by cooking, may cause cancer or influence brain functioning.


Mutagenic HCA in cigarette smoke

Besides carcinogens such as polycyclic aromatic hydrocarbons (eg Benzo(alpha)Pyrene), acrylamide and nitrosamines (eg N-Nitrosodimethylamine), the following HCA are a few of the HCA found (besides in diesel exhaust [1]) in cigarette smoke:

But exactly the same compounds are also found in cooked foods, as Maillard reaction products:

Carcinogenic HCA in cooked foods

HCAs form when amino acids (particularly serine, tryptophan and glutamic acid[18]) are heated, particularly in the presence of creatinine (in meat and fish). Additional amino acids (particularly threonine) may increase total mutagenicity many times.[19] HCA concentration is associated with meat doneness [20] and cooking temperature [21][22]. The darker the surface colour of the meat, the higher the HCA concentrations [23]. Grilling yields over 10-fold more HCAs than cooking. [24] Various enzymes in the human liver (and tongue [25], pancreas[26], mammary gland (antimicrobial)[27] and prostate[28]) that are meant to detoxify specific HCAs actually activate others [29][30]:

  • HCAs are (phase 1) oxidized to hydroxylamino derivatives by cytochrome P450s (as humans age, the activity of P450s decreases[31]), and (phase 2) further converted to ester forms (with acetic acid, sulfuric acid, proline) by acetyltransferase and sulfotransferase. Eventually, they produce DNA adducts through the formation of N-C bonds at guanine bases [32], which actually exist in human tissues, and may be involved in human cancer development [33]
  • Instead, hydroxylamines (from phase 1, except for PhIP- and Trp-P-2 hydroxylates) may also get O-acetylated (phase 2) by arylamine N-acetyltransferase (NAT2).[34]
  • Alternatively, UDP-glucuronosyltransferases (UGTs) are also designed to detoxify HCAs, but may also activate them by glucuronidation of HCAs (eg AalphaC > AalphaC-N(2)-Gl)[35]
  • and in extrahepatic tissues ("other than the liver"), prostaglandin H synthase catalyzes one-electron oxidation, rendering free-radical metabolites.[36]

Adding PhIP to the diet at a concentration of 100 ppm (parts per million, or ng/g) for 2 years induced carcinomas in 47% of female rats.[37] There is a linear relation between DNA adducts in the liver and doses of MeIQx fed to rats [38]. These 10 HCA have been shown to be carcinogenic in rats and/or mice when administered in the diet (for 1 to 2 years) at concentrations of 100-800 ng / gram (ppm) : IQ, MeIQ, MeIQx, PhIP, Trp-P-!, Trp-P-2, Glu-P-1, Glu-P-2, AalphaC and MeAalphaC.[39] Daily HCA intake in humans extends beyond 2 years, and " the rat model that is used for carcinogenesis bioassays, significantly underestimates the potential hepatic genotoxicity of HCAs in humans ".[40] These amounts were found in cooked foods:

  • IQ; up to 87 ng/g [41]
  • MeIQ; up to 0.03 ng/g [42]
  • MeIQx; up to 45 ng/g [43]
  • PhIP; up to 258 ng/g [44]
  • Trp-P-1; up to 13 ng/g [45]
  • Trp-P-2; up t0 13 ng/g [46]
  • AalphaC; up to 650.8 ng/g [47]
  • MeAalphaC; up to 63.5 ng/g [48]
  • Glu-P-1; up to 3.2 ng/g [49]
  • Glu-P-2; up to 1068 ng/g [50]

Human tissues have been shown to be vulnerable to these HCAs [51][52][53][54]. Even very low doses of HCAs may cause cancer [55], because the carcinogenic effects of HCAs are additive / synergistic, particularly at low doses [56][57][58][59][60]. Epidemiological studies show associations between HCA intake and breast cancer, colon cancer[61], pancreatic cancer[62] and prostate cancer [63]. Diet and nutrition may be responsible for 60% of the total cancer incidence for women and greater than 40% for men.[64]

  • In a Polish study total daily exposure to IQ, MeIQ, MeIQx, DiMeIQx and PhIP was estimated at 200 to 7700 ng per person.[65]
  • In a Malaysian study the average daily intake level of HCAs was 554 ng per person (lower than those reported from other regions).[66]
  • in a Swiss study the average daily exposure to HCAs was estimated at 5 ng/kg body mass. (about 400 ng per person)[67]
  • In a Spanish study the average total daily intake of mutagenic HCAs through meat dishes was estimated at 286 ng per person. [68]
  • Another Spanish study estimated total daily intake of mutagenic HCAs at 606 ng per person.[69]
  • In a German study the assessed average daily dietary intake of total HCA was 103 ng per person.[70] (69 ng from meat alone[71])
  • In a Japanese study average daily HCA intake levels were estimated at 1.08 ng/kg bodyweight. (about 58 ng per person)[72]
  • In a Chinese study estimated daily exposure to HCAs was 50 ng per person, but 50% higher among 20-39 yr olds.[73]

Substitutions on the structural rings of HCAs may block detoxication reactions [74]. The capacity of detoxification mechanisms may account for several hundred-fold difference in mutagenicity of HCAs.[75]

  • Antioxidants (present in the food when cooked) inhibit HCA formation [76], particularly phenols (as in olive oil, tea) [77] vitamin E [78] and vitamin C [79].
  • Dietary fat increases activity of the enzymes that activate and/or detoxify HCAs [80]. Fatty acids (and fiber[81]) may also bind and inactivate HCAs[82]), but unsaturated fatty acids in cooking oil may also prevent the degradation of HCAs.[83] Cooking in fresh (more so than old) virgin olive oil inhibits HCA formation by between 30 and 50%.[84], though (similar to the addition of iron[85]) may double the yield of MeIQx.
  • Adding sugar may either enhance or inhibit mutagenicity.[86]
  • Particularly juice from sweet cherries, and (to a lesser extend) from kiwi, plum and blueberry inhibits genotoxicity of IQ, more so than the juices from watermelon, blackberry, strawberry, black currant and Red delicious apple. OJ was inactive. Vegetables incl. broccoli moderately inhibited IQ genotoxicity[87], even though sulforaphane (in broccoli) inhibits mutagenicity of some HCAs (incl IQ, but not Glu-P-1, Glu-P-2).[88] OJ, however, contains naringenin (also in grapefruits and tomatoes), which inhibits P450 enzymes (that activate/detoxify HCAs).[89][90]
  • Besides containing various other toxic or mutagenic compounds[91], specific phenols in turmeric (a ginger; Indian saffron; Curcuma longa); curcumin (C), demethoxycurcumin (dmC; both > 80%) and bisdemethoxycurcumin (bdmC; 40% - 80%) strongly inhibit mutagenicity of all HCAs.[92]
  • Lemon grass inhibits mutagenicity of HCAs.[93]
  • Bacteria from a healthy intestinal microflora may (irreversibly) bind HCAs (50% of PhIP, almost 100% Trp-P-2) [94]

HCA inhibitors

Retinol, retinal, riboflavin, riboflavin 5'-phosphate, FAD, vitamins K1, K3, K4, 1,4-naphthoquinone, and coenzyme Q10 caused a concentration-dependent decrease in the mutagenicity of all six mutagens (IQ, MeIQ, MeIQx, PhIP, Glu-P-1 and Trp-P-2).[95]

Mutagenic HCA in cooked foods

The IQ-type HCAs are derived from amino acids and from creatinine in raw meat and fish, or sugars [96] additionally. IQ-type HCAs are readily formed from dideoxyosones[97]. Non-IQ-type HCAs are obtained by heating tryptophan (indoles / gamma-carbolines) or glutamic acid (imidazoles). The actual number of mutagenic HCAs is multiple-fold higher, as for each HCA listed, there may be a serie of mutagenic isomeres. (eg 12 TMIP isomeres[98])

  • 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) in broiled sardines, cooked beef, fried fish[99], up to levels of 1.9 ng/g in "medium" barbequed sardines.[100] IQ is activated to N-hydroxy-2-amino-3-methyl-imidazo[4,5-f]quinoline (N-OH-IQ). Its mutagenicity is distinctly reduced by riboflavin 5'-phosphate (vitamin B2 analog). Vitamin A, K3 and K4 also inhibit IQ induced mutagenesis.[101]
  • 2-amino-3-methylimidazo[4,5-f]quinoxaline (IQx) in broiled sardines[102], grilled beef[103], may be metabolized in various (>15), more or less mutagenic derivatives.[104]
  • 2-amino-1-methylimidazo[4,5-b]quinoline (IQ[4,5-b]) [105] in grilled beef. Also present in urine of vegetarians; may be formed endogenously from creatinine and 2-aminobenzaldehyde.[106]
  • 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) in fried fish [107][108][109], cooked meat [110].
  • 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx or 8-MeIQx) in various cooked foods[111][112], up to 45.5 ng/g [113], up to 21.2 ng/g in commercial beef flavors[114], activated to IQx-8-COOH, HONH-MeIQx, 7-oxo-MeIQx and various other metabolites.[115]
  • 2-amino-3,4,7,8-tetramethylimidazo[4,5-f]quinoxaline (TriMeIQx); 80 ng/g in griddled bacon [116]
  • 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx) in fried fish [117], beef extract [118], poultry products[119].
  • 2-amino-3,7,8-trimethylimidazo[4,5-f]quinoxaline (7,8-DiMeIQx; in roasted eel) [120] (creatinine + glycine + glucose[121])
  • 2-amino-1,7,9-trimethylimidazo[4,5-g]quinoxaline (7,9-DiMeIgQx) 53 ng/g in beef extract. [122]
  • 2-amino-4-hydroxymethyl-3,8-dimethylimidazo[4,5-f]quinoxaline (4-CH2OH-8-MeIQx; in beef extract) [123]
  • 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in cooked meat and fish[124][125], 6 to 54 ng/g in roasted pork[126], 258 ng/g in griddled pork loin[127], 13.3 ng/g in barbequed salmon[128].
  • 2-amino-(1,6-dimethylfuro[3,2-e]imidazo[4,5-b])pyridine (IFP) [129] in fried chicken breast[130]
  • 2-amino-1-methyl-6-(4-hydroxyphenyl)imidazo[4,5-b]pyridine (4'-OH-PhIP); 21.0 ng/g in broiled beef [131] (creatine + tyrosine + glucose)
  • 2-amino-n,n-dimethylimidazopyridine (DMIP) [132] in fried chicken [133]
  • 2-amino-n,n,n-trimethylimidazopyridine (TMIP) [134] in fried chicken breast[135] and fried ground beef[136]. All 11 heated-muscle-meat TMIP isomeres tested also proved mutagenic.[137]
  • 2-Amino-5-phenylpyridine (2-APP or Phe-P-1)[138] (its ultimate acetoxy reactive species) [139]
  • 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) [140] in broiled beef[141], broiled fish (13.3 ng of Trp-P-1 / g. broiled sardines)[142] and griddled beef steak (0.35 ng/g)[143].
  • 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) [144] in cooked beef and fish[145](13.1 ng of Trp-P-2 / g. broiled sardines.)[146]; also inhibits L-Dopa synthesis (similar to other alpha- and gamma-carbolines, and PhIP) [147]
  • 2-amino-9H-pyrido[2,3-b]indole (A alpha C) in soybean globulin and cooked meat [148] 0.2 to 1.4 ng/g in griddled beef steak [149], 17.7 ng/g in well done barbequed sardines[150], 650.8 ng/g in grilled beef[151], activated to 2-hydroxyamino-9H-pyrido[2,3-b]indole (HONH-AalphaC) and subsequently penultimate ester species, which bind to DNA[152].
  • 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeA alpha C); soybean globulin, cooked meat, fried fish [153][154][155], up to 10.6 ng/g in "well done" barbequed sardines[156], 63.5 ng/g in grilled beef[157]. About 17% is activated to mutagenic N2-hydroxy-MeA alpha C [158].
  • 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1; a delta-carboline) in roasted mackerel and pork[159], 3.18 ng/g in barbequed salmon[160], and activated to N-hydroxylated Glu-P-1, and its mutagenicity is further stimulated by zinc-superoxide dismutase (CuZn-SOD), without changing its structure.[161]. Activated Glu-P-1 produces a large amount of superoxide.[162] HCAs are eventually metabolized into less mutagenic compounds, such as N-acetyl-Glu-P-1 [163]
  • 2-aminodipyrido[1,2-a:3',2'-d]imidazole (Glu-P-2; a delta-carboline) in cooked salmon [164] and Worcestershire sauce [165], 286 to 1068 ng/g in grilled fish patty[166]
  • methyl-2-methylamino-IH,6H-pyrrolo[3,4-f]benzimidazole-5,7-dione (Cre-P-1) [167]
  • 2,6-diamino-3,4-dimethyl-7-oxo-pyrano[4,3-g]benzimidazole from the natural meat components creatine, glutamic acid and glucose. [168]

Neuro-active HCAs; natural ones and in cooked foods

In the human body (and human milk [169]), various endogenous beta-carbolines (eg tetrahydro-beta-carboline (tryptoline), 6-methoxytetrahydro-beta-carboline, tetrahydro-harman, harman, harmalan [170][171]) act on the Benzodiazepine receptors as neurotransmitters (regulating behavioral habituation [172], appetite [173] and modulating acetylcholine release [174][175]), and are monoamine oxidase (MAO) inhibitors [176]. beta-Carbolines may be anti-mutagenics, anti-genotoxic [177] and often associated with (acetyl)cholinesterase inhibition [178][179]

Various foods, such as fish, meat and fruits naturally also contain beta-carbolines, and cooking may dramatically increase their level and form different ones, that may be moderately naturally present in other foods. When heating tryptophan, various beta-carbolines (indoles) are formed [180]), such as harman and norharman. [181] Studies show long-term retention of these specific neurotoxic beta-carbolines in brain neuromelanin [182]. Nitrosation (in the presence of nitrite) of beta-carbolines generally produces mutagenics [183], but also reaction with aniline may produce mutagenics [184][185]. Unlike 'normal' beta-carbolines (such as harman and norharman), tetrahydro-beta-carbolines are generally anti-oxidants. [186] Reaction of tryptophan with aldehydes (benzaldehyde, vanillin, syringaldehyde, salicylaldehyde, anisaldehyde; particularly in fermented foods[187]) may already result in beta-carbolines at 70°C [188], and with the addition of glucose, copper and iron ('fortified foods'), beta-carbolines may already form at 40°C. [189]

beta-Carbolines in cooked foods

Cooking may induce complex beta-carbolines. All simple beta-carbolines foods have been tested for, were actually present in cooked foods, and yet, many simple beta-carbolines have not been tested for occurence in cooked foods. The following (simple and complex) beta-carbolines have been identified.

  • Norharman(e) (9H-pyrido[3,4-b]indole = β-carboline) in cigarette smoke, cooked meat [190][191][192], cooked fish, toasted bread [193] and coffee [194] is a neurotoxin [195] has synergistic effects with Trp-P-2[196] and (N-hydroxylated) Glu-P-1[197] and may contribute to idiopathic Parkinson's disease [198]). Endogenous norharman formation is about 50-100 ng per kg bodyweight, whereas total daily dietary exposure is estimated at max 4000 ng / kg. [199] Griddled bacon contains up to 413 ng/g noirharman.[200]
  • Harman(e) (1-methyl-β-carboline) in cigarette smoke, cooked meat [201], particularly chicken[202], cooked fish, toasted bread [203][204] and coffee [205]. Harman is a a tremor-producing neurotoxin. Meat consumption is higher in men with essential tremor [206]. Harman also impairs learning [207][208] through the nicotinic cholinergic system [209] and alters behaviour [210]). Daily total dietary exposure is estimated at max 1000 ng/kg bodyweight (daily endogenous formation 20 ng/kg)[211] Griddled pork loin contains up to 991 ng/g harman.[212]
  • Harmine (7-methoxy-1-methyl-β-carboline); in smoked salmon and soft cheese [213]; genotoxic [214], induces dopamine release [215], inhibits the enzymes MAO-A (which may cause accumulation of mono-amines), DYRK1A, CLK1, CLK2 [216], phosphodiesterase [217] and acetylcholinesterase [218]) UV light exposure increases toxicity of harmine.[219]
  • Harmaline (7-methoxy-1-methyl-4,9-dihydro-3H-pyrido[3,4-b]indole) in beer, coffee and cheese [220]; psychoactive, induces dopamine [221] and nitric oxide release and inhibits phosphodiesterase [222], acetylcholinesterase [223] and MAO-A.
  • Harmalol (1-Methyl-4,9-dihydro-3H-pyrido[3,4-b]indol-7-ol) in beer, coffee and cheese [224]; induces melanogenesis [225] interferes with DNA synthesis [226] and inhibits the enzymes acetylcholinesterase and butyrylcholinesterase. [227]
  • 1-acetyl-β-carboline-3-carboxylic acid (in ketchup and heated tomato concentrate[228]; selectively decreases responding [229] and may interact with other beta-carbolines[230])
  • 3,4-dinitro-1-methyl-β-carboline-3-carboxylic acid in soy sauce and beer [231]
  • Tryptoline (1,2,3,4-tetrahydro-β-carboline) in sausages[232]; a MAO-A inhibitor [233])
  • 1-furyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid in soy sauce and bean paste [234]
  • 1-(5’-hydroxymethylfuryl)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid in bean paste [235]
  • 1-hydroxymethyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid in smoked foods. [236]
  • 1-pentahydroxypentyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid in fruit- and vegetable- (heat-involved) products (jams etc) [237]
  • Flazin (1-(5'- hydromethyl-2'-furyl)-β-carboline-3-carboxylic acid) in soy sauce; cytotoxic [238] and induces quinone reductase (QR) activity [239])
  • Perlolyrin (in soy sauce [240]; weakly cytotoxic [241] induces quinones reductase (QR) activity [242])
  • 1-(1,3,4,5-tetrahydroxypent-1-yl)-β-carboline
  • 1-(1,4,5-trihydroxypent-1-yl)-β-carboline
  • 1-(1,5-dihydroxypent-3-en-1-yl)-β-carboline; in various foods, but particularly ketchup, soy sauce, and fish sauce.[243].
  • 1-(1,4-dihydroxybutyl)-β-carboline
  • 1-(1,3,4-trihydroxybutyl)-β-carboline (tryptophan + xylose) [244]

beta-Carbolines in fruits and plants

Some beta-carbolines may be natural in some raw foods, whereas the product of cooking in others (or in the same food). MTCA is naturally present in fruits; oranges, mandarins, bananas, pears etc. As fruit ripens and gets softer during storage, MTCA levels increase. [245] Besides fruits, THCA is naturally present in raw fish, and raw meat (and humans) and partly converted in MTCA due to cooking, whereas the levels of both THCA and MTCA (and mutagenic harman and norharman) are increased by cooking. [246][247] Tetrahydro-beta-carbolines generally cannot induce mutation. [248]

  • 1-methyltryptoline (1-methyl-1,2,3,4-tetrahydro-β-carboline = tetrahydro-harman) in sausages [249], tomato and kiwi; antioxidant [250]and MAO-A inhibitor [251])
  • THCA (1,2,3,4-Tetrahydro-beta-carboline-3-carboxylic acid) in fruits [252], raw (and cooked) fish and meat [253], toasted bread, beer, cider, wine vinegar, soy and tabasco sauce and blue cheese. [254]
  • MTCA (1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid) acting as antioxidant [255] in fruits [256][257], cooked fish [258], soy sauce [259], vinegar [260], wine, beer, yoghurt, tabasco, blue cheese [261] and fermented garlic. [262] MTCA is a xanthine oxidase (XO) inhibitor, thus inhibiting uric acid formation [263] MTCA is (co)mutagenic in the presence of nitrite, which is inhibited by components present in oranges.[264]
  • MTCdiC (1-methyl-1,2,3,4-tetrahydro-β-carboline-1,3-dicarboxylic acid) in aged garlic (not raw[265]); a superoxide scavenger [266])
  • 6-hydroxy-1-methyl-1,2,3,4-tetrahydro-β-carboline in alcoholic beverages [267], bananas, pineapple and tomato; acting as an antioxidant [268]
  • 1-(2-pyrrolidinethione)-3-yl)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid in fermented radish root. [269]

beta-Carbolines in the seeds of Peganum harmala Also known as Harmal or Syrian Rue.

  • Harmine (7-methoxy-1-methyl-β-carboline)
  • Harmaline (7-methoxy-1-methyl-4,9-dihydro-3H-pyrido[3,4-b]indole)
  • Harmalan (1-methyl-3,4-dihydro-beta-carboline)
  • Harmol [270]
  • Harmalol (1-methyl-4,9-dihydro-3H-pyrido[3,4-b]indol-7-ol)
  • Tetrahydroharmine ((7-methoxy-1-methyl-1,2,3,4-tetrahydro-β-carboline)
  • Harmine (methyl-7-methoxy-β-carboline-1-carboxylate)
  • Harmilinic acid (7-methoxy-3,4-dihydro-β-carboline-1-carboxylic acid)
  • Harmanamide (1-carbamoyl-7-methoxy-β-carboline)
  • Acetylnorharmine (1-acetyl-7-methoxy-β-carboline)

Author

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Author of this article is Thijs Klompmaker, born in 1966