SHORT COMMUNICATION: Uptake of the food-derived heterocyclic amine carcinogen 2-amino-1-methyl-6-phenylimidazo[4, 5-b]pyridine and its N-hydroxy metabolite into rat pancreatic acini and hepatocytes in vitro

Since DNA adducts of 2-amino-1-methyl-6-phenylimidazo[4, 5-b]pyridine(PhIP) are formed at relatively high levels in the rat pancreasbut not liver, we examined the uptake of PhIP and its N-hydroxymetabolite (N-OH-PhIP) into pancreatic acini and hepatocytesto determine if differential tissue uptake was a factor modulatingthe formation of PhIP-DNA adducts. In addition, since the precursorsof PhIP formation are two amino acids and since various aminoacid transporters have been identified in the pancreas, thepossible involvement of these transporters in the uptake ofPhIP and N-OH-PhIP was investigated. The uptake of both heterocycliccompounds into both tissue preparations was rapid, with maximaluptake occurring within 1–2 min. However, PhIP uptakeinto pancreatic acini was significantly (2-way ANOVA, P <0.05) greater than uptake of N-OH-PhIP into pancreatic aciniand the uptake of both PhIP and N-OH-PhIP into hepatocytes.Although uptake wasrapid, efflux of both compounds from bothtissue preparations was also rapid. However, the efflux rateconstant (1.86 ± 0.6/min, mean ± SEM) for PhIPwas significantly lower (Student's t-test, P < 0.05) thanthat for N-OH-PhIP (4.14 ± 0.04/min) from pancreaticacini. This,combined with the increased uptake of PhIP intopancreatic acini, suggests that there is substantial but reversiblebinding of PhIP in the pancreas. The uptake of both PhIP andN-OH-PhIP into pancreatic acini and hepatocytes was not affectedby the presence of various amino acids in the incubation buffer,indicating that amino acid transporters are not involved inuptake of these compounds. Furthermore, uptake of both compoundsdid not appear to be dependent on metabolic energy supply. Theabove data, together with the high octanol: buffer partitioncoefficients (logP = 1.322 and 1.301 for PhIP and N-OH-PhIPrespectively) suggest that both uptake and efflux of PhIP andN-OH-PhIP are consistent with a process of passive diffusion.The tissue binding characteristics for PhIP in the pancreasmay create conditions whereby pancreatic cytochrome P450 1A1can catalyse the formation of N-OH-PhIP While N-OH-PhIP is notthe ultimate reactive DNA binding species, it has been shownto directly bind to and form DNA adducts. Therefore, it is possiblethat the apparent selective accumulation of PhIP may contributeto the high level of PhIP-DNA adducts formed in the rat pancreas.     

In vitro formation and degradation of 2-amino-1-methyl-6-phenyliinidazo[4,5-b]pyridine(PhIP) protein adducts

Adduct formation between the food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b](PhIP)and rat serum albumin (RSA) was studied in vitro using hepaticmicrosomes isolated from polychiorinated biphenyl induced rats.With 1-methyl-2-nitro-6-phenylimidazo[4,5-b]pyridine (2-nitro-PhIP)as starting material, four main products were formed. Pretreatmentof RSA with ß-mercaptoethanol markedly increased theyield of one of them. In this adduct, the C-2 of PhIP was linkedto cysteine of RSA at position 34 in a C-S linkage. With N²-acetoxy-PhIPas starting material, unstable conjugates were formed with RSAas well as with glutathione (GSH) and cysteine. The suggestedstructures of the GSH-S-n² and cysteine conjug ates, GSH-S-N²-PhIPand cysteine-S-N²-PhIP respectively, are based on mass spectraand UV spectra. The degradation of the conjugates of GSH andcysteine as well as of the protein adduct were monitored. Theyall resulted in the same degradation product, identified as2-amino-5 hydroxy-1-methyl-6-phenylimidazo[4,5b]pyridine (5-hydroxy-PhIP).     

Metabolic activation pathway for the formation of DNA adducts of the carcinogen 2-amino-l-methyl-6-phenyUmidazo[4,5-b]pyridine (PhIP) in rat extrahepatic tissues

The food-borne mutagen 2-amino-l-methyl-6-phenylimidazo[ 4,5-b]pyridine(PhIP) induces tumors in colon of male rats and has been implicatedin the etiology of human cancers, particularly colorectal cancer.This study was conducted to examine: (1) the biliary and/orcirculatory transport of N-hydroxy- PhIP and its N-glucuronides,N-sulfonyloxy-PhIP and N-acetoxy-PhIP; (2) their role as proximateand ultimate carcinogenic metabolites of PhIP; (3) the potentialrole of glutathione in modulating PhIP-DNA adduct formation.PhIP-DNA adducts, measured by the ³²P-postlabeling method, werehighest in the pancreas (361 adducts/10⁸ nucleotides or 100%),followed by colon (56%), lung (28%), heart (27%) and liver (2%),at 24 h after a single oral dose of PhIP (220 µmol/kg)to male rats. In each tissue examined, we observed two majoradducts, each of which accounted for 35–45% of the total,and one minor adduct, which represented about 10–20% ofthe total. One of the major adducts was identified as N-(deoxyguanosin-8-yl)-2-amino-l-methyl-6-phenylimidazo[4,5-b]pyridine by chromatographic comparisonswith an authentic standard. The major urinary metabolites ofPhIP in these rats were 4'-hydroxy-PhIP and its glucuronideand sulfate conjugates, followed by N-hydroxy-PhIP N3-glucuronide,N-hydroxy-PhIP N-glucuronide and unchanged PhIP. In bile duct-ligatedrats, the urinary excretion of the N-OH-PhIP N3-glucuronidewas increased two-fold, but there was no effect on PhIP-DNAadduct formation in the colon, heart, lung, pancreas or liver.2,6-Dichloro-4-nitrophenol, which strongly inhibits arylsulfo-transferase-mediatedDNA binding in vivo, had no effect on PhIP-DNA adduct levelsin liver or in extrahepatic tissues. Pretreatment of rats withbuthionine sulfoximine, which results in hepatic glutathionedepletion, caused a five-fold increase in adduct formation inthe liver. Intravenous administration (10 µmol/kg) ofN-hydroxy-PhIP and N-acetoxy-PhIP each led to high levels ofPhIP-DNA adducts in each of the extrahepatic tissues examined.Adduct levels ranged from two- to six-fold higher (for N-hydroxy-PhIP)and four- to 28-fold higher (for N-acetoxy-PhIP) as comparedto that after an i.v. dose of the parent compound, indicatingthat these two bioactivated derivatives of PhIP are sufficientlystable to be transported through the circulation to extrahepatictissues. Analyses of whole blood obtained at 2—8 h afteroral administration of [³H]PhIP failed to detect N-hydroxy-PhIP(<0.1% of the radioactivity), however, a decomposition productof N-acetoxy-PhIP was found to account for about 80% of thetotal radioactivity in the blood. These results suggest thattransport of N-acetoxy-PhIP, and perhaps N-hydroxy-PhIP, viathe bloodstream and not biliary transport and deconjugationof N-hydroxy-PhIP N-glucuronides is primarily responsible forPhIP-DNA adduct formation in rat colon and other extrahepatictissues.     

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.     

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     

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     

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

A metabolite of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline(MeIQx) was incorrectly characterized in this paper. The metabolitewas thought to be an acetyl conjugate of the 5-hydroxyl-atedderivative of MeIQx. This assignment is incorrect. The correctassignment is a sulfate conjugate of 5-hydroxy-MeIQx, 2-amino-3,8-dimethylirnidazo[4,5-f]quinoxaUn-5-yl-sulfate.This conclusion is based upon repurified sample analyzed by¹H NMR and ¹³C NMR, enzyme hydrolysis assays, IR spectro-scopyand FAB/MS (accurate mass measurement).     

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.     

Effect of glutathione depletion and inhibition of glucuronidation and sulfation on 2-amino-l-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) metabolism, PhIP-DNA adduct formation and unscheduled DNA synthesis in primary rat hepatocytes

The potent rat colon carcinogen 2-amino-l-methyl-6-phenylimidazo[4,5-d]pyridine (PhIP), unlike other food-borne heterocyclicamines, does not induce tumors in rat liver. This correlateswith an extremely low level of PhIP-DNA adducts formed in thistissue, and together these observations suggest that PhIP isefficiently detoxified in the liver. In order to identify possibledetoxification mechanisms, we assessed the effect of inhibitionof glucuronidation, glutathione (GSH) conjugation and sulfationon PhIP metabolism and PhlP-induced DNA damage in rat hepatocytes.Hepatocytes isolated from rats pretreated with Aroclor 1254metabolized PhIP to the same products found in vivo. N-Hydroxy-PhIPN3-glucuronide and N-hydroxy-PhIP N²-glucuronide were majorand minor metabolites respectively. ³²P-Postlabeling analysisof DNA from the PhIP-treated hepatocytes indicated the presenceof two major adducts, one of which was identified as N-(deoxyguanosin-8-yl)-PhIP,and one minor adduct. There was no unscheduled DNA synthesis(UDS) in these cells. However, pretreatment of the hepatocyteswith 1-bromoheptane and buthionine sulfoximine, which depletesGSH and prevents its resynthesis, resulted in a 15-fold increasein the formation of PhIP-DNA adducts, as well as in a high levelof UDS. GSH depletion had no effect on the formation of detectablePhIP metabolites. Hepatocyte pretreatment with D-galactosamine,which inhibits glucuronidation, increased the formation of DNAadducts two-fold and UDS was increased similarly. D-Galactosaminedecreased the formation of the two N-glucuronides of N-hydroxy-PhIPby 50–60%, but had no effect on other metabolites. Pentachlorophenol,which strongly inhibits sulfotransferases, decreased adductformation slightly, but had essentially no effect on UDS oron the formation of PhIP metabolites. These results indicatethat metabolic conjugation pathways involvingGSH and glucuronidationmay play an important role in protecting rat liver against PhIPcarcinogenesis.     

N-acetyltransferase-dependent activation of 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine: formation of 2-amino-1-methyl-6-(5-hydroxy)phenylimidazo [4,5-b]pyridine, a possible biomarker for the reactive dose of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine

2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a mutagenic and carcinogenic heterocyclic amine formed during ordinary cooking. PhIP is metabolically activated to the ultimate mutagenic metabolite by CYP P450-mediated N-hydroxylation followed by phase II esterification. Incubation of N-hydroxy-PhIP (N-OH-PhIP) with cytosol, acetyl coenzyme A (AcCoA) and 2'-deoxyguanosine for 24 h resulted in the formation of three different adducts:N(2)-(deoxyguanosin-8-yl)-PhIP, N(2)-(guanosin-8-yl)-PhIP and PhIP-xanthine. One additional product, 5-hydroxy-PhIP (5-OH-PhIP), was also identified in the incubation mixtures. 5-hydroxy-PhIP is formed as a degradation product of conjugates formed from N-acetoxy-PhIP and protein, glutathione or buffer constituents. A similar spectrum of products was obtained using 3'-phosphoadenosine-5'-phosphosulfate (PAPS) instead of acetyl CoA. Addition of glutathione (3 mM) to the incubation mixture resulted in a 50% reduction in both adducts and 5-hydroxy-PhIP formation in liver cytosol. The main product detected was PhIP, suggesting glutathione-dependent reduction of the N-acetoxy-PhIP. Addition of glutathione to incubation mixtures from the other cytosolic preparations had less dramatic effects. In addition, increasing the amount of N-OH-PhIP in the incubation mixture resulted in proportional increased amounts of total adducts and 5-OH-PhIP. Incubation of rat and human S9 with PhIP resulted in the formation of only traces of 5-OH-PhIP. Fortification with AcCoA clearly increased the formation of 5-OH-PhIP. Addition of the CYP 450 1A2 inhibitor, furafylline, completely inhibited the formation of 5-OH-PhIP in incubations with human S9. These results indicate that both PhIP adducts and 5-OH-PhIP are formed by similar routes of activation of N-OH-PhIP. 5-OH-PhIP may therefore serve as a biomarker for the formation of the ultimate mutagenic metabolite of PhIP. A rat dosed orally with PhIP excreted 1% of the dose as 5-OH-PhIP in the urine at 24 h and 0.05 and 0.01% at 48 and 72 h, respectively. This shows that 5-OH-PhIP is also formed in vivo and indicates the possible use of 5-OH-PhIP as a urinary biomarker.     

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.     

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.     

Detection of the carcinogen 2-amino-1-methyl-6-phenyl-imidazo[4,5-b]pyridine (PhIP) in beer and wine

A carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine(PhIP), was measured in beer and wine by HPLC. PhIP was foundto be present in all brands of beer and wine analyzed. The concentrationsof PhIP in beer and wine were 14.1 ± 6.18 ng/l (mean± SD, n = 11) and 30.4 ± 16.4 ng/l (n = 10) respectively.     

2-hydroxyamino-1-methyl-6-phenylimidazo [4,5-b] pyridine induction of recombinational mutations in mammalian cell lines as detected by dna fingerprinting

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is the most abundant mutagenic heterocyclic amine in cooked foods. Two mouse tumor cell lines, BMT11 and FM3A, were exposed to its proximate form, 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-OH-PhIP). Fifty-six subclones of BMT11 and 39 subclones of FM3A were isolated and analyzed by DNA fingerprinting. Treatment with 10–20 μM N-OH-PhIP gave rise to extra bands or shifted bands, but treatment without N-OH-PhIP did not. This suggests that mutations resulting from recombination were induced. The mutation frequencies were 21–53% and 22–35% for BMT11 and FM3A, respectively. These findings suggest that PhIP induces recombinational mutations. © 1994 Wiley-Liss, Inc.     

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     

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.     

Comparative tumorigenicity of benzo[a]pyrene, 1-nitropyrene and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine administered by gavage to female CD rats

Agents that are ubiquitous in the environment and are knowninducers of mammary cancer in rodents can be regarded as potentialcauses of human cancer and need to be evaluated more completely.Therefore, the purpose of this study was to determine underidentical conditions the relative carcinogenic potency in themammary glands of rats of benzo[a]pyrene (B[a]P), 1-nitropyrene(1-NP) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyidne (PhIP).Thirty-day-old female CD rats were gavaged once weekly for 8weeks with B[a]P, 1-NP or PhIP. Each compound was given at 50µmol/rat/week in 0.5 ml trioctanoin for a total dose of400 µmoul/rat. Forty-one weeks after the last carcinogenadministration, rats were killed. In the 1-NP-treated rats,treatment elicited primarily benign tumors. In contrast, theB[a]P- and PhIP-treated rats developed both malignant and benigntumors. The incidence of adenocarcinomas in rats treated withB[a]P or PhIP was comparable and significantly higher than thatin animals receiving trioctanoin only. The incidence of benigntumors (fibroadenomas, desmoplastic adenomas and adenomas) observedin animals treated with B[a]P or 1-NP was comparable and significantlyhigher than that in animals given PhIP or trioctanoin. Thisis the first report describing the carcinogenic activity ofPhIP, given by gavage, in the mammary gland of CD rats and rankingthe carcinogenic potency observed under identical conditions,of three agents (B[a]P     

4-(2-amino-1-methylimidazo pyrid-6-yl)[4,5-b]phenyl sulfate--a major metabolite of the food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b] (PhIP) in the rat

Isolated rat hepatocytes (4 ? 10⁶ cells/ml metabolized the foodmutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)(100 µM) to at least eight different metabolites in a4 h duration. The major metaboilte formed was 4-(2-amino-1-methylimldazo[4,5-b]pyrid-6-yl)sulfate (4'-PhIP sulfate). Its rate of formation was increasedin hepatocytes from PCB pretreated animals in comparison tohepatocytes from untreated animals. One of the other metaboliteswas the unconjugated derivative of the sulfate (4'-OH-PhIP).This metabolite was also found after in-vitro incubations ofrat liver microsomes from PCB or ß-naphtho flavonepretreated animals. The evidence for the proposed structureof the major metabolite is based on [¹H]NMR and UV spectroscopy,incorporation of radiolabelled sulfate and arylsulfatase-labilhty.The formation of 4'-PhIP sulfate was inhibited by the P-450inhibitors -naphthoflavone and metyrapone and when incubatedin sulfate-free medium added the sulfotransferase Inhibitorpentachlorophenol. 4'-PhIP sulfate was also the major metaboliteof PhIP in the urine of exposed rat.     

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.