Heterocyclic aromatic amine formation in grilled bacon, beef and fish and in grill scrapings

The heterocyclic aromatic amines (HAAs) 2-amino-l-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-3, 8-di-methylimidazo[4,5-f)quinoxaline(MelQx), 2-amino-3, 4, 8-tri-methylimidazo[4, 5-f)quinoxaline(4, 8-DiMeIQx) and 2-amino     

Effect of cooking temperature on the formation of heterocyclic amines in fried meat products and pan residues

Frequent consumers of meat have an increased risk of colorectalcancer and possibly also of breast, stomach, pancreas and urinarybladder cancer. Bacon, ‘Falusausage’, ground beef,meatballs, pork belly, pork chops and sliced beef account formore than one-third of the intake of fried meat of the populationof Stockholm of age 50–75. These dishes were fried atfour temperatures (150, 175, 200 and 225 °C) representingnormal household cooking practices in Stockholm. Heterocyclicamines in these dishes were analysed using solid-phase extractionand HPLC. The heterocyclic amines 2-amino-3-methylimidazo[4,5-f]quinoline(IQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx),2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (DiMeIQx) and2-amino-1-methyl-6-phenylimidazo[4,5-b]-pyridine (PhIP) wererecovered. The formation of IQ was favoured by moderate cookingtemperatures; IQ was detected in one meat sample cooked at 150°Cand in some pan residues. The yield of MeIQx, DiMeIQx and PhIPincreased with the temperature. For several of the meat dishes,the content of heterocyclic amines in the pan residue was aslarge or larger than for corresponding piece of meat. The highestlevels of MeIQx were 23.7 ng/g in the meat and 233 ng/g in thepan residue. Corresponding data for DiMeIQx were 2.7 and 4.1ng/g and for PhIP 12.7 and 82.4 ng/g. The study leaves littledoubt that mutagenic heterocyclic amines are ingested by thepopulation of Stockholm, and added to previous epidemiologicalstudies from the same area, the combined data are consistentwith human carcinogenicity of heterocyclic amines. However,analytical epidemiological studies are needed before any statementon causality can be made.     

Synergistic enhancement of hepatic foci development by combined treatment of rats with 10 heterocyclic amines at low doses

Potential synergism between 10 carcinogenic heterocyclic amines[3-amino-l,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), 3-amino-1-methyl-5H-pyrido[4,3-b]indole(Trp-P-2), 2-amino-6 methyldipyrido[l,2-a:3',2'-d]imidazole(Glu-P-1), 2-ammo-dipyrido[l,2-a:3',2'-d]imidazoIe (Glu-P-2),2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethyl-imidazo[4,5-f]quinoline(MeIQ), 2-amino-3,8-dimethyl-imidazo[4,5-f]quinoxaline (MeIQx),2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeA     

The heterocyclic amines IQ and MeIQx show no promotive effect in a short-term in vivo liver carcinogenesis assay

The food-derived heterocyclic amines 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline(MeIQx) are carcinogenic in rodents as well as in non-humanprimates (IQ). Using a shortterm liver carcinogenesis modelthe compounds were found to be only weak initiators. It is alsopossible that these amines are involved in other phases of thecarcinogenesis process. In an attempt to study the promotiveeffects of IQ and MeIQx, these heterocyclic amines were repeatedlygiven by gavage, over a period of 10 days, to Wistar rats previouslyinitiated with diethylnitrosamine (DEN) and subjected to a partialhepatectomy. The rats were killed 1 month after DEN administrationand their livers examined for glutathione-S-transferase positive(GST-P) foci. The positive control, 2-acetylaminofluorene, significantlyincreased the development of GST-P foci in the DEN-treated ratscompared to the negative control, saline. However, neither theIQ- nor the MeIQx-treated groups differed from the controlsand thus these amines do not promote the growth of DEN-initiatedGST-P liver foci in this model.     

Influence of amino acids on the formation of mutagenic/carcinogenic heterocydic amines in a model system

Mixtures of creatinine, glucose and various single amino acidswere heated at 180°C for 10 min in an aqueous model system.The heated mixtures all showed mutagenic activity, ranging from80 to 2400 TA98 revertant colonies/µmol creatinine withmetabolic activation. Testing of HPLC fractions for mutagenicactivity showed each mixture to contain several mutagenic components,some of which corresponded to known heterocyclic amines andothers to unknown compounds. The presence of 2-amino-3-methyl-imidazo[4,5-f]quinoxaline,2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline and 2-amino-3,7,8-trimethylimidazo[4,5-f]quinoxalinein most of the samples was established using HPLC with photodiodearray detection and liquid chromatography/mass spectrometrywith electrospray interface and single ion monitoring. In addition,2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine,3-amino-1,4-di-methyl-5H-pyrido[4,3-b]indole and 3-amino-1-methyl-5H-pyrido[4,3-b]indoleand the co-mutagenic compounds 9H-pyrido[3,4-bindole and 1-methyl-9H-pyrido[3,4-b]indolewere detected in some samples.     

An efficient and convenient method for the purification of mutagenic heterocyclic amines in heated meat products

A simple and efficient method for the purification of mutagenicheterocyclic amines from heated meat products has been developed.In only two steps, namely extraction of raw material on Kieselgurfollowed by medium pressure liquid chromatography on SephasorbHP, very clean fractions with high recovery rates of mutageniccompounds were obtained, thus allowing isolation and quantitationby high performance liquid chromatography (HPLC) with UV detection.The method was validated on both food grade and bacterial beefextracts as well as fried beef. In 1–5 gsamples of beefextracts, levels up to 70 p.p.b. (ng/g) of 2-amino-3-methyl-imidazo[4,5-f]quinoline(IQ), 8–90 p.p.b. of 2-amino-3, 8-di-methylimidazo[4,5-f]quinoxaline (MeIQx) and up to 8 p.p.b. of 2-amino-3, 4,8-trimethylimidazo[4, 5-f]quinoxaline (4, 8-DiMeIQx) were determined.In fried beef, 1p.p.b. of 2-amino-1-methyl-6phenylimidazo[4,5-b]pyridine(PhIP) and 1 p.p.b. of MeIQx were measured. Thequantitative results of beef samples were in agreement withresults from determinations using immunoaffinity chromatography/HPLCor liquid chromatography coupled with mass spectrometry. MeIQxcould be quantified in fried beef down to 1ng/g of fresh beefmaterial. According to assays performed with reference standardsof tryptophan and glutamic acid pyrolysis products, the methodcould also be extented to quantitate other heterocyclic amines.     

SHORT COMMUNICATION: Metabolic activation and DNA binding of food mutagens and other environmental carcinogens in human mammary epithelial cells

Cultures of human mammary epithelial cells were treated withone of seven heterocyclic amine food mutagens [2-amino-3-methylimidazo[4,5-f)quinoline (IQ), 2-amino-3, 4-dimethylimidazo[4, 5-f)quinoline(MelQ), 2-amino-3, 8-di-methylimidazo[4, 5-f]quinoxaline (MeIQx),2-amino-3, 4, 8-trimethylimidazo[4, 5-f]quinoxaline (4, 8-DiMelQx)2-amino-3, 7, 8-trimethylimidazo[4, 5-f]quinoline (7, 8-DiMelQx),2-amino-3, 4, 7, 8-tetramethylimidazo[4, 5-f]quinoxaline (4,7, 8-TriMelQx) or 2-amino-1-methy1–6-phenylimidazo[4,5-b] pyridine (PhlP)], four nitropyrenes (1-nitropyrene (1-NP),1, 3-dinitropyrene (1, 3-DNP), 1, 6-dinitropyrene (1, 6-DNP)or 1, 8-dinitropyrene (1, 8-DNP) or the Polycyclic aromatichydrocarbon dibenzo[a, l]pyrene (DB[a, l]P). DNA isolated fromthe cultures was analysed by ³²P-post-labelling and in eachcase the presence of carcinogen-DNA adducts was detected. Thepatterns and numbers of adducts obtained when human mammarycell DNA digests were separated on polyethyleneimine-celluloseTLC were found to closely resemble those previously demonstratedto be present in the DNA of tissues from rodents and other primatestreated with the same agents. Up to six DNA adducts were detectedin human breast cells treated with IQ and MelQ. Fewer adducts(1–3) were detected following treatment with MelQx orits methylated derivatives, whilst PhIP gave rise to at leastfour distinct adduct spots. Five adduct spots were detectedin breast cells treated with DB[a, l]P or with 1-NP, but feweradduct spots were formed by 1, 3-, 1, 6- and 1, 8-DNP. Thesedata demonstrate the ability of human breast epithelial cellsto activate to DNA binding species a range of carcinogenic compoundsknown to be present in the human diet or to which humans areknown to be exposed environmentally.     

32P-Post-labelling analysis of DNA adducts formed by food-derived heterocyclic amines: evidence for incomplete hydrolysis and a procedure for adduct pattern simplification

Food-derived aminoimidazoazarenes have been shown to be mutagenicand carcinogenic and to form covalent DNA adducts. ³²P-Post-labellinganalysis of DNA modified with these heterocyclic amines (HA),including 2-amino-3-methyl-imidazo[4,5-f]quinoline (IQ), 2-amino-1-methyl-6-phenylimid-azo[4,5-b]pyridine(PhIP), 2-amino-3,4-dimethylimidazo [4,5-fquinoline (MeIQ),2-amino-3,4,8-trimethylimidazo [4,5-f1 quinoxaline (4,8-DiMeIQx),2-amino-3,7,8-trimethylimidazo[4,5-f]quinoxaline (7,8-DiMeIQx)and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) hasresulted in considerable interlaboratory variation in the characteristicpatterns of DNA adduct spots, with up to six being detectedfor each compound. Similar complex patterns were observed whenazido-derivatives of HA were photoreacted with calf thymus DNA.When deoxyguanosine 3'-monophosphate was modified with the azidoderivatives and analysed using the ³²P-post-labelling procedure,one major spot was observed for IQ, 4,8-DilMeIQx, 7,8-DiMeIQxor PhIP and two major spots for MeIQ or MeIQx. In each case,these adducts were chromatographically indistinguishable fromthe major adducts formed with DNA. No major adduct spots wereobserved when 3'-phosphate derivatives of deoxyadenosine, deoxycytidineor thymidine were reacted with the azido-derivatives of HA.In an attempt to identify the additional spots, azido derivativesof PhIP or IQ were reacted with the synthetic homopolymer poly(dG)·poly(dC),the alternating copolymer poly(dC-dG) or a synthetic oligonucleotide(TTT-GTTTTTTCTTTCCCT): in each case a reduced number of adductspots were detected. The introduction of an additional nucleaseP1 hydrolysis step following the labelling reaction furtherreduced the number of adduct spots to only one or two majorspots. Reversed-phase HPLC analysis showed that the number ofpeaks of radioactivity was also reduced to one or two, presumablycorresponding to the [³²P]-5'-monophosphate deoxyguanosine adducts.We suggest that many of the additional spots commonly observedin conventional ³²P-post-labelling analysis of HA-modified DNAare adducted oligonucleotides that are partly resistant to hydrolysisby micrococcal nuclease and spleen phosphodiesterase but aresusceptible to hydrolysis by nuclease P1.     

Enzymatic phase II activation of the N-hydroxylamines of IQ, MelQx and PhIP by various organs of monkeys and rats

2-Amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline(MelQx) and 2-amino-1-methy1-6-phenylimidazo[4,5-b]pyridine(PhIP) are mutagenic and carcinogenic heterocyclic amines producedduring the ordinary cooking of meat. These compounds undergometabolic activation via both cytochrome P450-mediated N-oxidationand phase II esterification in order to exert their genotoxicity.In the current study, we examined the in vitro phase II activationof N-hydroxy-IQ, N-hydroxy-PhIP and N-hydroxy-MelQx by cytosolicacetyltransferase, sulfotransferase, aminoacyl-tRNA synthetaseand phosphatase from a number of tissues including liver, kidney,colon and heart. These tissues were chosen for study becauseeach is either a target organ for carcinogenicity or has displayedhigh levels of DNA adducts in in vivo studies with the heterocyclicamines. Cytosol from various tissues of both monkeys and ratswas incubated with and without the respective cofactors, andcarcinogen binding to calf thymus DNA was measured by ³²P-postlabelinganalysis. Our results show that all four phase II enzymes mayparticipate in the activation of the N-hydroxylamines. However,the degree of activation depends on the substrate, tissue andanimal species. For example, in both monkeys and rats, the highestacetyl CoA-enhanced binding was observed with N-hydroxy-IQ andthe lowest acetyl CoA-enhanced binding was observed with N-hydroxy-MelQx.In contrast, no significant adenosine 3'-phosphate 5'-phosphosulfate-dependentactivation of N-hydroxy-IQ was observed with monkey cytosolfrom liver, kidney, heart or colon but the sulfotransferase-mediatedactivation of N-hydroxy-PhIP was at least 10 times higher inall four tissues of monkeys than in rats. Prolylation appearsimportant in the activation of all three N-hydroxylamines byrat liver and heart cytosol, whereas in monkeys, prolylationappears important in kidney cytosol. The differences observedin the phase II activation of heterocyclic amines may have implicationsfor DNA adduct formation, toxicity and carcinogenicity.     

Presence of N2-(deoxyguanosin-8-yl)-2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (dG-C8-MeIQx) in human tissues

One of the mutagenic and carcinogenic heterocyclic amines (HCAs),2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MelQx), is presentin cooked foods and we are chronically exposed to this compoundin our daily life. To study the role of HCAs in human carcinogenesis,we analyzed MelQx-DNA adducts in 38 DNA samples obtained fromsurgical and autopsy specimens by the ³²P-postlabeling methodunder adduct-intensification conditions with the modificationof additional digestion with nuclease P1 and phosphodiesteraseI after ³²P-labeling at 5' -hydroxyl termini. This modified³²P-postlabeling method can detect N²-(deoxyguanosin-8-yl)-2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline 5'-monophos-phate (5'-pdG-C8-MeIQx) at levelsdown to 1/10¹⁰ nucleo-tides. The DNA samples from colon andrectum surgical specimens and a kidney taken at autopsy werefound to contain an adduct spot corresponding to that of standard5'-pdG-C8-MeIQx on TLC at levels of 14, 18 and 1.8 per 10¹⁰nucleotides, respectively. Each adduct spot was extracted fromTLC and identified to be 5'-pdG-C8-MeIQx by HPLC. Thus, MelQx-DNAadducts actually exist in human tissues and this adduct formationmay be involved in human cancer development.     

Contribution of CYP1A1 and CYP1A2 to the activation of heterocyclic amines in monkeys and human

The activation of heterocyclic amines to mutagenic productsby hepatic microsomal fractions from cynomolgus monkey, marmosetmonkey and man was compared with the respective levels of cytochromeP450 enzymes CYP1A1 and CYP1A2. The rate of activation of 2-amino-3,8-dimethylidazo[4,5-fquinoxaline (MeIQx), 2-amino-3-methylidazo[4,5-fquino-line (IQ)and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) tomutagens by hepatic microsomal fraction from cynomolgus monkeywas very low. This was associated with a lack of constitutiveexpression of CYP1A1 and CYP1A2. In contrast, human hepaticmicrosomal fraction readily activates these heterocyclic aminesand this is associated with constitutive expression of CYP1A2.Treatment of cynomolgus monkey with 2,3,7,8-tetrachlorodibenzo-p-dioxin(TCDD) causes a very modest induction of CYP1A2, and a smallincrease in the activation of MeIQx and IQ. However, there wasmarked induction of CYP1A1 which was accompanied by > 10-foldincreases in PhIP activation and 7-ethoxyresorufin O-deethylase(EROD), 7-methoxyresorufin O-demethyiase (MROD) and aryl hydrocarbonhydroxylase activities. Following treatment of cynomolgus monkeywith 3-methylcholanthrene, induction of CYP1A1, but not CYP1A2,was evident. In untreated marmoset monkey the activations ofMeIQx and PhIP, as well as pbenacetin O-deethylase, EROD, MRODand aryl hydrocarbon hydroxylase activities, are similar tothose in man, although the activations of IQ and coumarin 7-hydroxylaseactivity are lower than in man. The presence of constitutiveCYP1A2, and the absence of CYP1A1, in the liver of this speciescorrespond to the situation in man. Treatment of marmoset monkeywith TCDD results in increased CYP1A2 levels (4-fold), accompaniedby proportional increases in the activation of MeIQx and IQand phenacetui O-deethylase, EROD and MROD activities. The activationof PhIP is increased disproportionately, by 8-fold, most likelydue to the activity of CYP1A1 which is also induced by TCDDin this species. Overall, the hepatic metabolism of heterocyclicamines by CYP1A enzymes in the untreated marmoset monkey resemblesthat in human more closely than that in the cynomolgus monkey.Therefore, marmoset monkey may be a more suitable model thanthe cynomolgus monkey for carcinogenicity studies involvingMeIQx and PhIP, but not IQ     

Simple methods for quantifying mutagenic heterocyclic aromatic amines in food products

Two solid-phase extraction methods were developed for the determinationof mutagenic heterocyclic aromatic amines in heated meat products.The copper phthalocyanine (CPC) tandem extraction was performedon coupled cartridges of diatomaceous earth and CPC-derivatizedSephasorb HP, followed by further clean-up on Sephasorb HP.Parts per billion levels of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline(MeIQx) and its homologs as well as 2-amino-3-methylimidazo[4,5-f]quinoline(IQ), 2-amino-3,8-dimethyl-imidazo[4,5-f]quinoline (MeIQ), 2-amino-1-methyl-6-phenyl-imidazo[4,5-b]pyridine(PhIP), amino--carboline (AC), 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole(Trp-P-1), 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2),harman (H) and norharman (NH) can then be simultaneously quantifiedby HPLC with UV detection. The propylsulfonyl silica gel (PRS)tandem extraction is a one-step clean-up method on coupled cartridgesof diatomaceous earth and PRS, suitable for the determinationof MeIQx, IQ and their homologs, as well as the glutamic acidpyrolysates 2-amlno-6-methyldipyrido[1,2-a:3',2']imidazole (Glu-P-1)and 2-aminodipyrido[1,2-a:3',2'-d]imidazole (Glu-P-2). 4,7,8-TriMeIQxor 7,8-DiMeIQx were used as internal standards. Four grams ofsample or less are required for analysis. The recovery of theamineswas between 46 and 83% and the detection limit was in the lowp.p.b. range with coefficients of variation ranging between5 and 18%. The major mutagenic contaminant found in meat extractswas MeIQx (from <1 to 44 p.p.b.), followed by 4,8-DiMeIQx(1.3–5 p.p.b.) whereas the major contaminant in friedmeat was PhIP (23–48 p.p.b.), followed by MeIQx (5.1–8.3p.p.b.), AC (3.2–8.9 p.p.b.) and 4,8-DiMeIQx (1.3–2p.p.b.).The co-mutagens NH and H were found in fried meat atlevels of 8.7–19 p.p.b. and 3–4.8 p.p.b. respectively.     

Identification of N-(deoxyguanosin-8-yl)-2-amino-3,4-dimethylimidazo[4,5-f]quinoline (dG-C8-MeIQ) as a major adduct formed by MeIQ with nucleotides in vitro and with DNA in vivo

The N-hydroxylamine of a carcinogenic heterocyclic amine, 2-amino-3,4-dimethylimidazo[4,5-f]quinoline(MeIQ), was reacted with four 2'-deoxynucleoside 3'-monophosphatesafter O-acetylation. ³²P-Postlabeing analysis demonstrated thatthe adduct was formed with only the guanine nucleotide, andthe structure of the compound in the obtained adduct spot wasdetermined to be N-(deoxyguanosin-8-yl)-MeIQ 3',5'-diphosphate(3',5'-pdGp-C8-MeIQ). DNA samples from livers of mice fed MelQwere also ³²P labeled under standard conditions and additionallytreated with nuclease P1 and phosphodiesterase I. A single adductspot was obtained and the structure of the adduct was identifiedas 5'-pdG-C8-MeIQ. Thus, MelQ binds at the C-8 position of guaninein vitro and in vivo, like other heterocyclic amines.     

Strong anti-mutagenic activity of the novel lipophilic antioxidant 1-O-hexyl-2,3,5-trimethylhydroquinone against heterocyclic amine-induced mutagenesis in the Ames assay and its effect on metabolic activation of 2-Amino-6-methyldipyrido[1,2-a:3', 2'-d]imidazole (Glu-P-1)

Antimutagenic effects of a novel lipophilic antioxidant, 1-O-hexyl-2,3,5-trimethylhydroquinone(HTHQ), and other known antioxidants against heterocyclic amine-or other mutagen-induced mutagenesis were examined in the Amesassay using Salmonella strain TA 98 to access the chemopreventiveeffects of antioxidants on heterocyclic amineinduced carcinogenesis.Further the mechanisms of inhibition by HTHQ were accessed.HTHQ was shown to potently inhibit mutagenesis induced by allof 8 different heterocyclic amines at rates between 100% and63% in the presence of S9 mix. When the protection of HTHQ against2-amino-6-methyldipyrido[1,2-a: 3',2'-d]imidazole (Glu-P-1)-inducedmutagenesis was compared with known antioxidants t-butylhydroquinone,propyl gallate, BHA, BHT and     

Metabolism of the food carcinogen 2-amino-3, 8-dimethylimidazo[4, 5-f]quinoxaline in isolated rat liver cells

2-Amino-3, 8-dimethylimidazo[4, 5-f]quinoxaline (MelQx) wastransformed to at least 10 metabolites in suspensions of hepatocytesisolated from Aroclor 1254 treated rats. Combining biochemicaldata such as effects on MeIQx metabolism of metabolic modulatorsand incorporation of radioisotopic sulfur with UV, mass and¹H-NMR spectroscopy, we elucidated the structures of six metabolites.About 40% of the MeIQx was transformed to 2-amino-3, 8-dimethylimidazo[4,5-f]quinoxalin-4(or5)-yl sulfate. Other oxygenated metaboliteswere 2-amino-8-hydroxymethy1–3-methylimidazo[4, 5-f)quinoxalin-4(or5)-yl sulfate and 2-amino-4(or5)-ß-D-glucuronopyranosyloxy-3,8-dimethylimidazo[4, 5-f]quinoxaline. Evidence was obtainedthat a glutathione conjugate was formed. This metabolite, andthe other oxygenated metabolites were probably formed in P-450catalyzed reactions. Two metabolites, 2-ß-D-glucurono-pyranosylamino-3,8-dimethylimidazo[4, 5-f)quinoxaline and the N(3, 8-dimethylimidazo[4,5-f]quinoxaline-2-yl)sulfamate, were direct conjugates of MeIQx.     

Identification of N2-(deoxyguanosin-8-yl)-2-amino-3,8-dimethylimidazo[4,5-f)quinoxaline 3',5'-diphosphate, a major DNA adduct, detected by nuclease P1 modification of the 32P-postlabeling method, in the liver of rats fed MeIQx

The carcinogenic heterocyclic amine 2-amino-3,8-dimethyl-imidazo[4,5-f]quinoxaline(MeIQx) is widely distributed in cooked foods. The nucleaseP1 method increased the sensitivity of the standard ³²P-postlabelinganalysis about 1000-fold for detection of MeIQx-DNA adducts.The recovery of MeIQx-DNA adducts by the nuclease P1 methodwas determined to be about 50% using liver DNA of a rat treatedwith [¹⁴C]MeIQx intragastrically. By the nuclease P1 methodfive adducts were detected in the liver DNA of rats fed MeIQxand two of them, including the most abundant one, were identifiedas MeIQx-deoxyguanosine adducts by comparison with the adductsformed in in vitro reactions of N-acetoxy-2-amino-3,8-dimethylimidazo[4,5-f)quinoxalinewith the four 2'-deoxyribonucleotides. The most abundant adductin vivo was identified as N²-(deoxyguanosin-8-yl)-MeIQx 3',5'-diphosphate(3',5'-pdGp-C8-MeIQx). MeIQx-DNA adduct levels in human tissuescould be determined by the nuclease P1 modification of the ³²P-postlabelingmethod in combination with HPLC, and thus provide informationon the roles of MeIQx in human carcinogenesis.     

High promutagen activating capacity of yeast microsomes containing human cytochrome P-450 1A and human NADPH-cytochrome P-450 reductase

Yeast Saccharomyces cerevisiae strains have been constructedthat co-express cDNAs coding for the human cytochrome P-450enzymes CYP1A1 or CYP1A2 in combination with human NADPH-cytochromeP-450 reductase (oxidoreductase). Microsomal fractions preparedfrom the strains were able to efficiently activate various drugsto Salmonella mutagens. These experiments demonstrated thata functional interaction occurred between the respective humanenzymes in the yeast microsomes. For every drug tested, themicrosomes containing CYP enzymes and oxidoreductase were 2-to 4-fold better in activation than the corresponding microsomesthat contained CYP alone. Interestingly, co-expression of CYP1A2with oxidoreductase resulted in a decrease of 7-ethoxyresorufin-O-deethylaseactivity, a problem which is related to this specific substrate.Using the microsomes, it was demonstrated that aflatoxin B₁,was activated to a mutagen not only by CYP1A2 but also by CYP1A1.In contrast, benzo[a]pyrene was exclusively activated by CYP1A1whereas CYP1A2 was inactive. The drug 3-amino-1-methyl-5H-pyrido[4,3-b]indole(Trp-P-2) was activated by CYP1A2 and to a lesser extent byCYP1A1. A strong substrate specificity was observed with thetwo structurally related heterocyclic arylamines 2-amino-3,4-dimethylimidazo[4,5-f]quinoline(MeIQ) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQ_x).MeIQ_x was activated efficiently by both CYP enzymes, whereasMeIQ was only activated by CYP1A2 and not by CYP1A1. The factthat microsomes from vector transformed control strains wereunable to activate any of the drugs studied underlines the suitabilityof these microsomes for metabolic studies. Moreover, the presenceof suitable marker genes in the yeast strains will enable usto study mitotic recombination and gene conversion events inducedby drugs that require metabolic activation.     

Mutagenic activation of IQ, PhIP, and MeIQx by hepatic microsomes from rat, monkey and man: low mutagenic activation of MeIQx in cynomolgus monkeys in vitro reflects low DNA adduct levels in vivo

Cooked meat, poultry and fish contain a number of mutagenicand carcinogenic heterocyclic amines, including 2-amino-3-methylimidazo[4,5-f]quinoline(IQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx)and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP).In the present study we examined the capacity of hepatic microsomesfrom Fischer 344 rats, cynomolgus monkeys and humans to metabolicallyactivate IQ, MeIQx and PhIP in vitro using the Ames Salmonellamutagenicity assay. The mutagenic activation of IQ was similaramong the three species; however, there were significant differencesamong the species in the activation of PhIP and MeIQx. Livermicrosomes from humans showed the greatest capacity to activatePhIP and MeIQx, followed by rats, and then monkeys. The largestdifferences between the species were observed when MeIQx wasused as the mutagen. MeIQx–DNA adducts formed in vivowere then compared among rats and monkeys given MeIQx by gavage(20 mg/kg/day, 10 doses). ³²P-Postlabeling analysis, carriedout under intensification conditions, was used to examine MeIQx–DNAadducts in the liver, kidney, heart, colon and white blood cells.MeIQx–DNA adducts were highest in all tissues examinedfrom male rats, followed by female rats, and much lower in monkeys.In the liver, the total MeIQx–DNA adduct levels of monkeyswere {small tilde}19 and {small tilde}10 times lower than inmale and female rats respectively. In extrahepatic tissues,the differences in MeIQx–DNA adduct levels between monkeysand rats were even greater. The results suggest that the lowlevel of MeIQx–DNA adducts found in vivo in cynomolgusmonkeys reflects a low capacity to activate MeIQx via the hepaticcytochrome P450 monooxygenase system.     

Cytotoxicity, DNA adduct formation and DNA repair induced by 2-hydroxyamino-3-methylimidazo[4,5-f]quinoline and 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine in cultured human mammary epithelial cells

2-Amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine(PhIP) are heterocyclic amines (HAs) found in cooked meats.Both compounds are mammary gland carcinogens in rats. The initiationof carcinogenesis by the HAs is believed to be associated withtheir DNA adduct formation that occurs after metabolic activationof the parent amines via cytochrome P-450-mediated N-hydroxylationand esterification. To assess the capacity of the human mammaryepithelium to metabolically activate the HAs, we used the ³²P-postlabelingmethod to measure the levels of DNA adducts in a culture ofhuman mammary epithelial cells exposed to IQ, PhIP or theirN-hydroxylamine metabolites. Whereas 50 µM parent aminesdid not form detectable levels of DNA adducts in cultured humanmammary epithelial cells after 24 h incubations, concentrationsas low as 1 µM N-hydroxylamines produced detectable levelsof adducts after 2 h incubations. N-Hydroxy-PhIP formed higheradduct levels than N-hydroxy-IQ at all concentrations tested.For example, following a 2 h incubation at 50 µM, adductlevels (per 10⁷ nucleotides) were 674 and 16 for N-hydroxy-PhIPand N-hydroxy-IQ, respectively. At similar initial adduct levels(10–11/10⁷ nucleotides), 60–80% of IQ- and PhIP—DNAadducts were removed after 24 h, indicating that the mammaryepithelial cell culture showed efficient repair of HA adducts.Whereas neither IQ nor PhIP was cytotoxic, both N-hydroxy-IQand N-hydroxy-PhIP were cytotoxic as assessed by a dose-dependentinhibition of colony formation. After exposure to 0.1, 1, 10or 50 µM N-hydroxy-PhIP (or N-hydroxy-IQ), colony formationwas 103 (94), 84 (74), 37 (29) and 3 (2)% of the control values,respectively. Only N-hydroxy-PhIP (at 10 and 50 µM), however,was cytotoxic as assessed by the MTT cell survival assay (whichmeasures the capacity of mitochondria to metabolize a tetrazoliumsalt). The ability of the N-hydroxylamines to form DNA adductsand to be cytotoxic in a culture of human mammary epithelialcells may implicate these metabolites as proximate carcinogenicforms of IQ and PhIP in the human mammary gland. However, whetherthere are inter-individual differences in N-hydroxylamine metabolism,adduct formation and repair in human mammary epithelial cellsrequires further study. The results from this study support the usefulness of culturedhuman mammary epithelial cells for studies on the genotoxicityand metabolism of the N-hydroxylamines of HA food mutagens.     

Adduction of the heterocyclic amine food mutagens IQ and PhIP to mitochondrial and nuclear DNA in the liver of Fischer-344 rats

The heterocyclic amines 2-amino-3-methylimidazo[4,5-f]quinoline(IQ) and 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP)are carcinogens that form DNA adducts. In the present study,we used the ³²P-postlabeling method to measure the levels ofIQ and PhIP adducts in hepatic nuclear and mitochondrial DNAof Fischer-344 rats given a single dose (100 mg/kg, p.o.) or10 doses of either carcinogen. After a single dose of IQ, adductlevels were > 2-fold higher in hepatic nuclear than in mitochondrialDNA; however, after repeated IQ exposure, the levels of adductsin nuclear and mitochondrial DNA were not significantly different.In contrast, after a single dose of PhIP, there were no significantdifferences in adduct levels in nuclear and mitochondrial DNA;however, after multiple doses of PhIP, adduct levels were significantlyhigher in mitochondrial DNA than in nuclear DNA. The percentagesof individual IQ or PhIP adducts were different between nuclearDNA and mitochondrial DNA, particularly after 10 doses. WithIQ, the C8-guanine adduct accounted for 72% of the total IQadduct levels in nuclear DNA but only 40% of total adduct levelsin mitochondrial DNA. After 10 doses of PhIP, the C8-guanineadduct accounted for 48% and 15% of total adduct levels in nuclearDNA and mitochondrial DNA respectively. In addition, the percentageof an uncharacterized PhIP adduct was 14% In nuclear DNA but< 1% in mitochondrial DNA. The percentages of individualadducts were approximately the same 3, 24, 120 and 240 h aftera single dose of either compound, though total IQ and PhIP adductlevels appeared to decline over time In both organelles. Thesignificance of IQ and PhIP mitochondrial DNA adduction andthe influence of distinct heterocyclic amine adducts on cardnogenesismerit further investigation.