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.     

Use of the 32P-postlabeling method to detect DNA adducts of 2-amino-3-methylimidazolo[4, 5-f]quinoline (IQ) in monkeys fed IQ: identification of the N-(deoxyguanosin-8-yl)-IQ adduct

Eight DNA adducts of 2-amino-3-methylimidazolo[4, 5-f]-quinoline(IQ) were found by the standard ³²P-postlabeling method in liversfrom male Cynomolgus monkeys fed IQ (5 days/week, 3 weeks, 20mg/kg,nasal-gastric intubation). The IQ-DNA adduct fingerprints observedin monkeys were identical to those observed in rats that receivedIQ (0.03%) in the diet for 2 weeks. The C8-guanine-IQ adductwas identified by comigration with the synthetic 3‘, 5’-bisphosphatederivative of N(-deoxyguanosin-8-yl)-Q. DNA modified in vitrowith N-hydroxy-IQ showed seven adducts, including the C8-guanine-IQadduct, that were identical to those found in monkeys and rats.Thus it appears that N-hydroxy-IQ, the reactive metabolite ofIQ, was responsible for all adducts found in vivo, except one.In order to detect adducts in other organs that were presentat lower levels, the intensification (ATP-deficient) methodfor ³²P-postlabeling was used. Five of the adducts detectedunder standard conditions, including the C8-guanine-IQ adduct,were also detected under intensification conditions. The totallevel of DNA-IQ adducts was highest in the liver, followed bythe kidney, colon and stomach, and bladder. The adduct patternswere identical in all organs examined. The results indicatethat IQ is potentially genotoxic in primates and therefore alikely human carcinogen.     

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     

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     

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.     

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.     

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.     

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     

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.     

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     

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 heterocyclic amines and beef extract on chromosome aberrations and sister chromatid exchanges in cultured human lymphocytes

Two of the major bacterial mutagens formed in heated meat products,2-amino-3-methylimidazo[4, 5-f]quinoline and 2-amino-3, 8-dimethylimidazo[4,5-f quinoxaline or the basic fraction of beef extract induceda low frequency of sister chromatid exchanges in human lymphocytecultures in the presence of metabolic activation. Structuralchromosome aberrations were not induced at comparable high concentrationsin human lymphocytes with intact repair system, suggesting thatrepair or induction of point mutations are involved in the DNA-damagingeffect of heterocyclic amines rather than structural chromosomeaberrations. Accordingly it may be concluded that mammaliancells with both intact repair and enzyme systems are more relevantthan bacterial systems for evaluating the carcinogenic potentialof heterocyclic amines.     

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.     

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.     

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.     

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     

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.     

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.     

Mouse models for the study of colon carcinogenesis

The study of experimental colon carcinogenesis in rodents hasa long history, dating back almost 80 years. There are manyadvantages to studying the pathogenesis of carcinogen-inducedcolon cancer in mouse models, including rapid and reproducibletumor induction and the recapitulation of the adenoma–carcinomasequence that occurs in humans. The availability of recombinantinbred mouse panels and the existence of transgenic, knock-outand knock-in genetic models further increase the value of thesestudies. In this review, we discuss the general mechanisms oftumor initiation elicited by commonly used chemical carcinogensand how genetic background influences the extent of disease.We will also describe the general features of lesions formedin response to carcinogen treatment, including the underlyingmolecular aberrations and how these changes may relate to thepathogenesis of human colorectal cancer. Abbreviations: AA, arachidonic acid; ACF, aberrant crypt foci; AOM, azoxymethane; Apc, adenomatous polyposis coli; BCAC, β-catenin-accumulated crypt; COX-2, cyclooxygenease-2; cPLA₂, cytosolic phospholipase A₂; CRC, colorectal cancer; DFMO, difluoromethylornithine; DMAB, 3,2'-dimethyl-4-aminobiphenyl; DMH, 1,2-dimethylhydrazine; DSS, dextran sodium sulfate; HCA, heterocyclic amine; IBD, inflammatory bowel disease; i.p., intraperitoneal; IQ, 2-amino-33-methylimidazo[4,5-f]quinoline; MAM, methylazoxymethanol; miRNA, microRNA; MNNG, N-methyl-N'-nitro-N-nitrosoguanidine; MNU, methylnitrosourea; ODC, ornithine decarboxylase; PGDH, 15-hydroxyprostaglandin dehydrogenase; PGE₂, prostaglandin E2; PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine; s.c., subcutaneous; TGF, transforming growth factor; TNF, tumor necrosis factor Received August 13, 2008; revised October 31, 2008; accepted November 20, 2008.