Antibodies to the food mutagens, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline: useful for immunoassay and immunoaffinity chromatography of biological samples

Monoclonal mouse IgG₁ and IgG₃ antibodies were developed tothe food mutagens, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine(PhIP) and 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline(4,8-DiMeIQx) in order to make specific and sensitive detectionand purification systems suitable for biological samples. Theantibodies were developed with the strategy that cross-reactionwith analogues modified in the N²-position was desirable. Competitiveenzyme-linked immunosorbent assays (ELISA) with 50% inhibitionby 0.4–6 pmol food mutagen were developed. The epitopesrecognized by the antibodies have been characterized by ELISAusing 52 synthetic analogues and metabolites of PhIP, 4,8-DiMeIQx,and other food mutagens. One of the anti-PhIP antibodies onlyrecognizes PhIP and those PhIP-analogues which have minor modificationsin the N²-amino group, whereas the other, 7B7-1, is less stringentand also recognizes several other modified metabolites, includingbulky adducts at the N²-amino group e.g. the major guanine anddeoxyguanosine adducts isolated from PhIP-modified DNA. Theantibodies to DiMeIQx also recognize the food mutagens 2-amino-3,4-dimethylimidazo[4,5-f]quinoxaline(4-MeIQx), 2-amino-3, 8-dimethylimidazo[4, 5-f]quinoxaline (8-MeIQx),and the corresponding quinolines (4-MeIQ and 8-MeIQ). Two ofthese antibodies only bind analogues with minor modificationsin the free amino group, whereas analogues with major modificationsin this position, including a deoxyguanosine adduct, react withthe third antibody. Urine samples and faecal extracts from ³H-PhIPor 2-¹⁴C-DiMeIQx dosed rats were analysed by these ELISA assays,and high correlations between radioactivity and response inthe ELISA assays were observed. Urine samples and faecal extractsfrom ³H-PhIP-dosed rats were purified on an affinity columncontaining the less stringent anti-PhIP antibody, 7B7-1. Theaffinity column was found by high performance liquid chromatography(HPLC) analysis to concentrate exclusively labelled material.This affinity column also bound PhIP-related materials fromdilute samples of acid hydrolysed PhIP-DNA with high efficiency.Only     

Mutational specificity of 2-nitro-3,4-dimethylimidazo[4,5-f]quinoline in the lacI gene of Escherichia coli

2-Nitro-3,4-dimethylimidazo[4,5-f]quinoline (NO₂-MeIQ) is thenitro derivative of 2-amino-3,4-dimethylimi-dazo[4,5-f]quinoline(MeIQ), one of the food pyrolysis product mutagens and carcinogens.The mutational specificity of NO₂-MeIQ was determined usingthe lacI system in an Escherichia coli strain, EE125, harbouringthe plasmid pKM101. NO₂-MeIQ acts as a direct-acting mutagentowards this strain, and induces a broad range of mutationalalterations. At higher doses; G:C     

Major routes of metabolism of the food-borne carcinogen 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline in the rat

The metabolic fate of 2-amino-3,8-dimethylimidazo[4,5-f] quinoxaline(MeIQx), a carcinogen formed in cooked meat and fish, has beeninvestigated in male Sprague-Dawley rats. Five metabolites wererecovered from bile of animals given an intragastric dose of{2-¹⁴C]MeIQx. These accounted for nearly all of the radioactivityin bile. The chemical structures of these metabolites were elucidatedby proton NMR, UV and mass spectroscopy. Three structures maybe assigned unambiguously: two sulfamates, N-(3,8.dimethylimidazo[4,5f]quinoxalin-2-yl)sulfamic acid and N-(8-hydroxymethyl-3-methylimidazo[4,5f]quinoxalin-2-yl)sulfamic acid, and N-(8-one glucuronide, N²(ß-1-glucosiduronyl)-2-amino-3,8-dimelhyliinidazo[4,5f]quinoxaline In addition, an acetyl and a glucosiduronylconjugate of 5-hydroxy-MeIQx were observed. The spectral evidencedid not allow an unambiguous assignment of the site of conjugation.The two glucuronides were excreted in urine and the sulfamateof MeIQx was found in feces as well as urine. All five metaboliteswere found to be non-mutagenic to Salmonella typhimurium TA98with or without metabolic activation. The glucuronide conjugateswere found also to be non-mutagenic when ß- glucuronidasewas incorporated with S-9 mixture in the mutation assay, andthus all appear to be detoxification products. The previouslyreported metabolite, 2-amino-8-hydroxymethyl-3-methylimidazo[4,5f]quinoxalinewhich is mutagenic to Salmonella typhimurium TA98 with metabolicactivation, was identified as a minor component in both urineand feces.     

Protective versus promotional effects of white tea and caffeine on PhIP-induced tumorigenesis and beta-catenin expression in the rat

A 1 year carcinogenicity bioassay was conducted in rats treatedwith three short cycles of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine(PhIP)/high-fat (HF) diet, followed by 2% white tea (wt/vol),0.05% epigallocatechin-3-gallate (EGCG) or 0.065% caffeine assole source of fluid intake. Thirty-two percent of the PhIP/HFcontrols survived to 1 year, compared with 50, 48.7 and 18.2%in groups given white tea, EGCG and caffeine, respectively.After 1 year, PhIP/HF controls had tumors in the colon, skin,small intestine, Zymbal’s gland, salivary gland and pancreas.For all sites combined, excluding the colon, tumor incidencedata were as follows: PhIP/HF 69.5%, PhIP/HF + EGCG 48.7%, PhIP/HF+ white tea 46.9% and PhIP/HF + caffeine 13.3%. Unexpectedly,a higher incidence of colon tumors was detected in rats post-treatedwith white tea (69%) and caffeine (73%) compared with the 42%incidence in PhIP/HF controls. In the colon tumors, β-cateninmutations were detected at a higher frequency after caffeineposttreatment, and there was a shift toward more tumors harboringsubstitutions of Gly34 with correspondingly high protein andmessenger RNA expression seen for both β-catenin and c-Myc.c-Myc expression exhibited concordance with tumor promotion,and there was a concomitant increase in cell proliferation versusapoptosis in colonic crypts. A prior report described suppressionof PhIP-induced colonic aberrant crypts by the same test agents,but did not incorporate a HF diet. These findings are discussedin the context of epidemiological data which do not supportan adverse effect of tea and coffee on colon tumor outcome—indeed,some such studies suggest a protective role for caffeinatedbeverages. Abbreviations: ACF, aberrant crypt foci; BrdU, 5-bromo-2'-deoxyuridine; Ctnnb1, β-catenin gene (rat); EGCG, epigallocatechin-3-gallate; HF, high fat; mRNA, messenger RNA; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine; Tcf, T-cell factor Received January 16, 2008; revised February 4, 2008; accepted February 8, 2008.     

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.     

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.     

32P-Postlabeling analysis of IQ, MelQx and PhIP adducts formed in vitro in DNA and polynucleotides and found in vivo in hepatic DNA from IQ-, MelQx- and PhIP-treated monkeys

The ³²P-postlabeling method was used to examine the adductsin DNA, polynucleotides, and mononucleotides reacted in vitrowith the N-hydroxy and N-acetoxy derivatives of 2-amino-3-methylimidazo[4,5-f]quinoline(IQ), 2-amino-3, 8-dimethylimidazo[4,5-f]quinoxaline (MelQx)or 2-amino-1-methy1-6-phenylimidazo[4,5-b]pyridine (PhIP). Adductprofiles were compared to those found in vivo in liver of cynomolgusmonkeys fed IQ, MeIQx or PhIP. The N-acetoxy derivatives ofIQ, MelQx and PhIP (generated in situ from the correspondingN-hydroxylamine in the presence of acetic anhydride) each formedthree principal adducts in DNA. Adduct 1 of IQ, MeIQx and PhlPwas chromatographically identical to the ³²P-labeled bis(phosphate)derivative of N-(deoxyguanosin-8-yI)-IQ, N-(deoxyguanosin-8-yI)-MeIQx,and N-(deoxyguanosin-8-yl)-PhIP respectively, and this adductcomprised     

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     

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.     

DNA adduct formation of the food carcinogen 2-amino-3-methylimidazo[4, 5-f]quinoline (IQ) in liver, kidney and colo-rectum of rats

DNA adducts of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ)have been measured in the liver, kidney, and colorectum of maleFischer-344 rats given a single oral dose of IQ (20 mg/kg).The pattern and distribution of DNA adducts examined by ³²P-postlabelingwas similar in all tissues. N-(Deoxyguanosin-8-yl)-2-amino-3-methylimidazo-[4,5-f]quinoline(dG-C8-IQ) was the principal adduct identified and it accountedfor     

Occurrence of mutagenic/carcinogenic heterocyclic amines in meat and fish products, including pan residues, prepared under domestic conditions

Some typical Swedish meat and fish products, e.g. bacon, beefburgers,meatballs, Baltic herring, salmon, smoked fish, black puddingand sausages, and their corresponding pan residues, were analysedby HPLC for their content of mutagenic/carcinogenic heterocyclicamines (HAs). The products were cooked using recommended domesticcooking conditions concerning temperature, time and frying equipmentThe amount of HAs was low in most products, though the amountwas higher in the pan residues, especially in the pan residuefrom the frying of Falun sausage, which contained 18.5 ng HAs/gcooked product Mostly MelQx (2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline)and 4,8-DiMeIQx (2-amino-3,4,8-trimethylimidazo[4,5-f]-quinoxaline)were found, being 0.03–2.8 ng MelQx/g and n.d.-3.4 ng4,8-DiMeIQx/g cooked product in the food products and 0.05–73ng MelQx/g and n.d.-2.8 ng 4,8-DiMelQx/g cooked product in thepan residues. High levels of IQ (2-amino-3-methylimidazo[4,5-f]quinoline),10.5 ng/g, were only found in well-done bacon and a correlationwas seen between fat content and IQ formation. Low levels ofMelQ (2-amino-3,4-dimethylimidazo[4,5-f]quinoline) and PhIP(2-amino-l-methyl-6-phenylimidazo[4,5-b]pyridine) were foundin the foods.     

Structural determination of a new mutagenic heterocydic amine, 2-amino-1,7,9-trimethylimidazo[4,5-g]quinoxaline (7,9-DiMeIgQx), present in beef extract

We previously found two new mutagens, compounds I and II, inbacteriological-grade beef extract by monitoring the mutagenicityto a new Salmonella strain, YG1024; compound I was identifiedas 2-amino-4-hydroxymethyl-3,8-dimethyli-midazo[4,5-f]quinoxaline(4-CH₂OH-8-MeIQX) In the present study, we isolated compoundII from the beef extract, which accounted for 2% of the totalmutagenicity of materials adsorbed on blue cotton. Further,we found that a large quantity of compound II was produced byheating a mixture of creatine, threonine and glucose (1:1:0.5)at 200^°C for 5 h, the level being 860-fold of that in thebeef extract. The structure of this compound was determinedto be 2-amino-1,7,9-trimethylimidazo[4,5-g]quinoxaline (7,9-DiMeIgQx)by X-ray crystallography. The amount of 7,9-DiMeIgQx in bacteriological-gradebeef extract was estimated to be 53 ng/g. This compound induced13 800 and 670 revertants of S.typhimurium YG1024 and TA98 respectively,per µg in the presence of S9 mix.     

Inhibitory effect of dietary 4-ipomeanol on DNA adduct formation by the food mutagen 2-amino-3-methylimidazo [4,5-f]quinoline (IQ)in male CDF1 mice

The food mutagen 2-amino-3-methlimidazo[4,5-f]quinoline (IQ)is carcinogenic in the CDF₁ mouse liver, lungs and stomach.IQ Is activated to its ultimate carcinogenic form by N-hydroxylation,catalyzed principally by hepatic microsomal cytochrome P450IA2,and further esterification, resulting in the formation of N-(deoxyguanosin-8-yl)-IQand other adducts. The furanoterpenold 4-ipomeanol (IPO) isa naturally occurring pneumotoxin which exerts its specifictoxicity in Clara cells of the lung after activation by microsomalcytochrome P450. Because IPO is activated in the liver by acytochrome P450IA2 enzyme, we evaluated IPO as a possible chemopreventiveagent by assessing its ability to inhibit IQ-DNA adduct formationin the CDF₁ mouse. Mice were put on an AIN-76A diet with orwithout 0.075% IPO from day 0 to 54. IQ (0.01%) was added tothe diets from day 22 to 41 and animals were killed (four animals/timepoint) on days 42, 44, 46, 48, 50 and 54. Blood (for white bloodcell isolation), liver, lungs, stomach, small intestine, cecun,colon, kidneys, spleen and heart were collected for analysisof IQ-DNA adducts by .³²P-post-labeling. During the 12 day periodafter cessation of IQ exposure (days 42–54) IQ-DNA adductformation was significantly inhibited in the liver (33.6–46.4%),lungs (29.9–58.6%), stomach (33.2–51.5%) and whiteblood cells (24.5–63.7%), but not in the other organs.Except in the colon, adduct removal from organs during days42–54 was relatively slow (36.0–81.9% of day 42levels remaining on day 54, 9.4–16.7% in the colon), butthe presence of IPO in the diet did not influence the rate ofadduct removal. Measurement of hepatic microsomal ethoxyresorufindeethylase, an activity specific for cytochrome P450IA isozymes,showed that the enzyme could be inhibited (14.1–68.1%)by IPO (0.05–10.0 mM) in vitro. It is concluded that IPOinhibits IQ-DNA adduct formation in target organs of the CDF₁mouse and that IPO may act by inhibiting N-hydroxylation ofIQ. It is therefore possible that IPO may be a candidate chemopreventiveagent against IQ-induced carcinogenesis.     

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.     

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.     

Glucuronidation of N-hydroxy heterocyclic amines by human and rat liver microsomes

The food-borne carcinogenic and mutagenic heterocyclic aromaticamines undergo bioactivation to the corresponding N-hydroxy(OH)-arylamines and the subsequent N-glucuronidation of thesemetabolites is regarded as an important detoxification reaction.In this study, the rates of glucuronidation for the N-OH derivativesof 2-amino-3-methylimidazo[4,5-f]-quinoline (IQ), 2-amino-l-methyl-6-phenylimidazo[4,5-b]-pyridine (PhIP), 2-amino-6-methyi-dipyrido[l,2-a:3',2'-d]imidazole(Glu-P-1) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline(MelQx) by liver microsomal glucuronosyltransferase were comparedto that of the proximate human urinary bladder carcinogen, N-OH-aminobiphenyl(N-OH-ABP) and the proximate rat colon carcinogen N-OH-3,2'-dimethyl-4-aminobiphenyl(N-OH-DMABP). Human liver microsomes catalyzed the uridine 5'-diphosphoglucuronicacid (UDPGA)-dependent glucuronidation of N-OH-IQ, N-OH-PhIP,N-OH-Glu-P-1 and N-OH-MeIQx at rates of 59%, 42%, 35% and 27%,respectively, of that measured for N-OH-ABP (11.5 nmol/min/mg).Rat liver microsomes also catalyzed the UDPGA-dependent glucuronidationof N-OH-PhIP, N-OH-Glu-P-1 and N-OH-IQ at rates of 30%, 20%and 10%, respectively of that measured for N-OH-DMABP (11.2nmol/min/mg); activity towards N-OH-MelQx was not detected.Two glucuronide(s) of N-OH-PhIP, designated I and II, were separatedby HPLC. Conjugate II was found to be chromatographically andspectrally identical with a previously reported major biliarymetabolite of PhlP in the rat, while conjugate I was identicalwith a major urinary metabolite of PhIP in the dog. Hepaticmicrosomes from rat, dog and human were found to catalyze theformation of both conjugates. The rat preferentially formedconjugate II (I to II ratio of 1:15), while the dog and humanformed higher relative amounts of conjugate I (I to II ratioof 2.5:1.0 and 1.3:1.0 respectively). Fast atom bombardmentmass spectrometry of conjugates I and II gave the correspondingmolecular ions and showed nearly identical primary spectra.However, collision-induced spectra were distinct and were consistentwith the identity of conjugates I and II as structural isomers.Moreover, the UV spectrum of conjugate I exhibited a     

Incorporation of carbon atoms from glucose into the food mutagens MeIQx and 4,8-DiMeIQx using 14C-labelled glucose in a model system

Mixtures of creatinine, glucose and threonine with the additionof a small amount, 250 µCi, of [U-¹⁴C]glucose, [1-¹⁴C]glucoseor [6-¹⁴C]glucose were heated at 180°C for 30 min in anaqueous model system. The mixtures were purified and analysedusing HPLC, scintillation and Ames tests. 2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline(MeIQx) and 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline(4,8-DiMeIQx) were detected as the main radioactive mutagens.The amount of MeIQx and 4,8-DiMeIQx produced from threoninewas estimated at 18 and 60 nmol/mmol glucose respectively. Radioactivecarbon atoms originating from glucose were also shown to beincorporated into 2-amino-3-methylimidazo[4,5-f]quinoxaline(IQx). The specific activity was calculated to be 0.6, 0.3 and0.1–0.3 mCi/mmol for MeIQx, 4,8-DiMeIQx and IQx respectivelyfor all three labelled forms of glucose. By the incorporationof carbon atoms originating from glucose into the imidazoquinoxalinemutagens it was clearly demonstrated that glucose is a precursorin the formation of these food mutagens.     

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.     

Isolation and identification of a new mutagen, 2-amino-4-hydroxy-methyl-3, 8-dimethylimidazo[4,5-f] quinoxaline (4-CH2OH-8-MeIQx), from beef extract

By monitoring the mutagenicity to a new Salmonella tester strain,YG1024, which has a much higher level of 0- acetyltransferaseactivity than S.typhimurium TA98, we found two new mutageniccompounds in bacteriological-grade beef extract. One of them(compound I), which had a similar UV spectrum to that of 2-amlno-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx), was isolated and shown toaccount for {small tilde}2% of the total mutagenicity of thematerials adsorbed to blue cotton, and its concentration wasestimated to be 6.0 ng/g beef extract. This amount of compoundin beef extract was insufficient to allow measurements of variousspectra, but its level was increased {small tilde}9-fold byheating beef extract with creatine and threonine at 200°Cfor 5 h. From UV and mass spectra of the compound obtained frombeef extract heated with creatine plus threonine, it was deducedto be a hydroxymethyl derivative of anminodimethylimidazoquinoxaline.Compound I was isolated from the urine of rats given 4,8-DiMeIQxand identified as 2-amino-4-hydroxymethyl-3,8-dimethylimidazo[4,5-f]quinoxaline (4-CH₂OH-8-MeIQx) by ¹H-NMR analysis. 4-CH²OH-8-MeIQxinduced 326 000 revertants of YG1024 and 99 000 revertants ofTA98 per µg in the presence of S9 mix.     

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.