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.     

Possible mechanisms for PhIP-DNA adduct formation in the mammary gland of female Sprague-Dawley rats

2-Amino-l-methyl-6-phenylimidazo[4, 5-b]pyridine (PhIP), themost abundant heterocyclic amine in fried beef, is a mammarygland carcinogen in rats. Using the ³²PP-postlabeling method,PhIP-DNA adduct levels were measured in mammary epithelial cellsisolated from female Sprague-Dawely rats given 10 daily dosesof PhIP (75 mg/ kg, p.o.) according to a protocol previouslyshown to induce mammary gland cancer. At 24 h, 48 h, 1 weekand 5 weeks after the last dose of PhIP, PhIP-DNA adduct levels[relative adduct labeling (RAL) x 10⁷, mean ± SD] were10.2 ± 0.7, 7.9 ± 2.7, 2.2 ± 0.6 and 0.9± 0.03 respectively. When isolated rat mammary epithelialcells (from untreated rats) were incubated in vitro with N-hydroxy-PhIP(45 µM, 1 h, 37°C), PhIP-DNA adducts were detectedin cell DNA (RAL =     

Synthesis, purification and mutagenicity of 2-hydroxyamino-3-methylimidazolo[4,5-f] quinoline

Synthesis of 2-hydroxyamino-3-methylimidazolo [4,5-f]quinoline(N-hydroxy-IQ), a reactive metabolite of 2-amino-3-methylimidazolo[4,5-f]quinoline(IQ), was achieved by a modification of an earlier method. N-Hydroxy-IQwas purified by a two-step procedure involving C₁₈ Sep-Packand semi-preparative h.p.l.c. Additional h.p.l.c. methods weredeveloped to monitor the synthesis of N-hydroxy-IQ, and to measureIQ and other IQ derivatives on the same h.p.l.c. profile. Thestructure of N-hydroxy-IQ was confirmed by mass spectral analysisfollowing derivatization to azoxy-IQ, phenyl-azoxy-IQ and acetoxy-acetamido-IQ,and by chemical reactivity studies. Mutagenicity studies withthe nitroreductase-deficient strain of Salmonella TA98 showedthat N-hydroxy-IQ is directly mutagenic, having a specific activityof 2 x 10⁴ revertants/nmol. The data confirm that N-hydroxy-IQis a mutagenic metabolite of IQ and further implicate the hydroxylaminein the carcinogenkity of IQ.     

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.     

Mutagenicity and in vitro covalent DNA binding of 2-hydroxyamino-3-methylimidazolo [4,5-f]quinoline

The 2-hydroxyamino-3-methylimidazolo[4,5-f]quinoline (N hydroxy-IQ),a metaholite of the food mutagen-carcinogen IQ, was mutagenicto Salmonella TA98 (nitroreductase deficient). When either rathepatic cytosol, NADPH (1 mM) or ascorbate (0.5 mM) wasaddedto the mutagenicity assay, mutagenicity increased up to 15-,10- and 50-fold respectively. In light of the effects of ascorbateand NADPH, it appears likely that hepatic cytosol may containfactors that protect N-hydroxy-IQ from oxidative decomposition.In contrast, hepatic monooxygenase metabolism of N-hydroxy-IQdecreased mutagenicity. When pentachiorophenol, an inhibitorof O-acetyltransferase and sulfotransferase, was added to themutagenicity assay, a dose-dependent inhibition of N-hydroxyIQ mutagenicity was observed. 2,6-Dichloro-4-nitrophenol, amore specific inhibitor of sulfotransferase than O-acetyltransferase,did not inhibit the mutagenicity of N-hydroxy IQ at concentrationswhich appear to selectively inhibit only bacterial sulfotransferase.The data suggest that bacterial O-acetyltransferase rather thansulfotransferase mutagenically activates N-hydroxy-IQ. N-hydroxy-IQcovalently bound to calf thymus DNA in vitro under non-enzymaticconditionsat pH 7.4. Rat hepatic cytosolic O-acetyltransferase and sulfotransferaseenhanced the covalent binding of N-hydroxy IQ to DNA 30- and5-fold respectively. The data suggest that the mutagenicityof N-hydroxy-IQ is due to the reactivity of N-hydroxy-IQ withDNA and the ability of N-hydroxy-IQ to be further activatedby bacterial O-acetyltransferase.     

Inhibition of plasmid reporter gene expression in cho cells by DNA adducts of 2-amino-3-methylimidazo[4,5-f]quinoline and 2-amino-1-methyl-6-phenylimidazo[4,5–b]pyridine

2-Amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-1-methyl-6-phenylimidazo[4,5-b)]pyridine (PhIP) are two members of a family of carcinogenic heterocyclic amines (HAs) found in cooked meats that form DNA adducts after activation to N-acetoxy derivatives. The ability of IQ- and PhlP-DNA adducts to inhibit gene expression was investigated using a human growth hormone (hGH) reporter gene in a pUC12-based mammalian expression vector under the control of either the herpes simplex virus-1 thymidine kinase promoter or the human immunodeficiency virus-1 long terminal repeat. The plasmids were treated in vitro with 0, 5, 10, or 40 μM N-hydroxy-IQ or N-hydroxy-PhIP in the presence of a 10-fold molar excess of acetic anhydride to generate the N-acetoxy derivatives in situ. The adduct levels in the plasmids were quantitated by the ³²P-postlabeling method. The adducted (and control) plasmids were each transfected into repair-deficient or -proficient Chinese hamster ovary cells, and expression of hGH was measured by immunoassay of growth hormone secreted into the cell medium. The results showed that IQ- and PhlP-DNA adducts inhibited gene expression in both plasmids and that the degree of inhibition of hGH production was proportional to the levels of IQ- and PhlP-DNA adducts. The degree of inhibition, however, was independent of the promoter, despite the differences in the strengths of the two promoters to drive hGH production. Repair capacity influenced the extent of inhibition of gene expression by HA adducts since, in general, fewer adducts were needed to inhibit reporter gene expression in repair-deficient cells than in repair-proficient cells. In both cell lines, DNA adducts of PhIP appeared to be more potent in inhibiting hGH expression than adducts of IQ. Whether alteration of gene expression by HA adducts plays a role in the carcinogenicity of these compounds deserves further study. © 1994 Wiley-Liss, Inc.     

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).     

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     

Binding of 2-amino-3-methylimidazo[4,5-f]quinoline to hemoglobin and albumin in vivo in the rat. Identification of an adduct suitable for dosimetry

Blood protein binding by the food-borne carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline(IQ) was investigated using male Sprague-Dawley rats. Amongthe many blood proteins modified in rats dosed intragastricallywith [³H(G)]IQ, hemoglobin and albumin were modified in a dosedependent fashion. Albumin bound 3 – 5 times more IQ thanhemoglobin at doses from 2 to 150 µmol. IQ-modified serumalbumin was enzymatically digested using Pronase and analyzedby h.p.l.c. Many peptide fragments containing radioactivitywere detected but the low level of protein modification (0.01– 0.04% of dose) prevented spectroscopic analyses of theseadducts. An in vitro system containing hepatic microsomes metabolizedIQ to a reactive species which could bind to serum albumin.One major adduct was formed at the cysteine³⁴ residue usingthis activation system and was identical to an adduct isolatedfrom in vivo-modified albumin. Chemical and spectroscopic analysesof the Pronase fragment proved the adduct was a tripeptide containingN²-cysteinesulfinyl-IQ. A chemically identical adduct was formedin vitro when N-hydroxy-IQ was incubated with serum albumin.As much as 10% of the IQ bound to serum albumin in vivo waspresent as this sulfur-linked adduct based on h.p.l.c. analysisof the Pronase digest fragments and on the acid-labile activitywhich could be recovered as IQ.     

Reaction of N-hydroxylamine and N-acetoxy derivatives of 2-amino-3-methylimidazolo[4,5-f]quinoline with DNA. Synthesis and identification of N-(deoxyguanosin-8-yl)-IQ

The N-hydroxylamine of the carcinogen 2-amino-3-methylimidazolo[4,5-f]quinoline(IQ) covalently bound to calf thymus DNA at pH 7, and the bindingwas 11% higher under acidic conditions (pH 5). The extent ofN-hydroxy-IQ binding to single-stranded polynucleotides at neutralpH was in the following order: polyguanylic acid and polyadenylicacid >polycytidylic acid = polyuridylic acid. The bindingof the carcinogen to DNA, polyguariyllc acid and polyadenylicacid at neutral pH was enhanced 6-, 4- and 2-fold respectivelyby the presumed in situ formation of N-acetoxy-IQ from N hydroxy-IQand acetic anhydride. N-(Deoxyguanosin-8-yl) IQ was synthesizedby reaction at pH 7 of N-acetoxy-IQ (formed in situ with N-hydroxy-IQand acetic anhydnde) with deoxyguanosine and the structure characterizedby NMR, mass spectral and UV absorption spectral analyses. Reversephase HPLC of enzymatically hydrolyzed DNA which had been reactedwith N-hydroxy-IQ in vitro showed a major adduct which was chromatographicallyidentical to synthetic N-(deoxyguanosin-8-yl)-IQ. In addition,N-acetoxy-IQ, generated chemically by acetic anhydride or enzymatlcallywith mammalian acetyltransferase, formed one major adduct withDNA which was chromatographically identical to the syntheticN-(deoxyguanosin-8-yl)-IQ. The results indicate that N-hydroxy-IQand N-acetoxy-IQ react with DNA forming primarily the N-(deoxyguanosin-8-yl)-IQadduct.     

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.     

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     

DNA adduct levels of 2-amino-l-methyl-6-phenylimidazo-[4,5-b]pyridine (PhIP) in tissues of cynomolgus monkeys after single or multiple dosing

DNA adducts of 2-amino-l-methyl-6-phenylimidazo[4,5-b]-pyridine(PhIP), a heterocyciic amine derived from cooked meat, weremeasured by the ³²P-postlabeling method in tissues of cynomolgusmonkeys given PhIP. Monkeys received either a single dose ofPhIP (20 mg/kg orally) or nine daily doses of PhIP (20 mg/kgorally, days 1–5 and 8–l1) and tissue samples wereobtained 24 h after the last dose. Over 28 different tissueswere examined for PhIP—DNA adducts. Adducts were detectedin all tissues examined except the fat and bone marrow. Aftera single dose, adduct levels (mean value/10⁷ nucleotides, n= 2 monkeys) were highest in the liver (2.1), followed by thelung (1.7), gall bladder (1.7) and pancreas (0.9). Low adductlevels were detected in the brain and aorta (0.06 and 0.02 respectively).Following multiple doses of PhIP, adduct levels (mean value/10⁷nucleotides ± SE, n = 3 monkeys) were highest in theheart (5.7 ± 2.0) followed by the liver (3.8 ±0.8), submandibular gland (2.7 ± 1.8) and pancreas (2.2± 0.5). Comparison of the adduct levels after a singledose with those found after multiple doses indicates that accumulationof PhlP—DNA adducts occurred in certain tissues. Adductlevels in liver, pancreas, kidney, small intestine and colonincreased about 1.5- to 2.4-fold. PhlP—DNA adduct levelsin submandibular gland and brain increased 4- to 5-fold. Adductlevels in heart increased 10-fold and levels in the aorta increased31-fold. Adducts in white blood cell DNA increased with dailydosing for 9 days. No apparent changes in adduct levels wereseen in the lung, stomach, bladder, muscle and spleen. The widedistribution of PhlP-DNA adducts and their presence in whiteblood cells suggests that there is transport of reactive metabolitesfrom the liver to extrahepatic tissues. The relatively highadduct levels in the gall bladder in comparison with the liversuggests biliary excretion and possible reabsorption of reactivemetabolites. The presence of DNA adducts in tissues implicatesPhIP as a potential carcinogen in non-human primates. The possibilitythat PhlP-DNA adducts in tissues such as the heart and aortamay have toxicological consequences is discussed.     

Heterocyclic aromatic amine-DNA-adducts in bacteria and mammalian cells detected by 32P-postlabeling analysis

The formation of DNA adducts by the fried meat mutagen and carcinogen2-amino-3-methylimidazo[4,5-f]quinoline (IQ) was studied bymeans of ³²P-postlabeling of DNA digests and four-directionalt.l.c. Three major and five minor adducts were detected in assaysof DNA digests obtained from Salmonella typhimurium TA98 cellsafter treatment with IQ and rat liver postmitochondrial supernatant(S9). A qualitatively identical adduct pattern was obtainedwith nitro-IQ (3-methyl-2-nitrounidazo[4,5-f]quinoline), a newanalogue of IQ with a nitro instead of the amino group. Thesetwo compounds, therefore, form the same ultimate metabolite.The same adduct pattern was also found after TA98/1,8-DNP⁶ (acetyltransferase-deficient)cells were treated with nitro-IQ; this is probably due to aresidual acetyltransferase activity in this strain. Upon treatmentof TA98 cells with 1 mM IQ for 3 h one adduct was detected in4.7 x 10⁵ total bases; a considerably higher adduct frequency,one in 4.2 x 10³, was induced by nitro-IQ (70 µM, 30 min).The IQ isomer 2-amino-1-methyliniidazo[4,5-f]quinoline (isoIQ)and its nitro-analogue nitro-isoIQ (1-methyl-2-nitroimidazo[4,5-f]-quinoline)also produced identical adducts. Their common adduct patternwas very similar to the IQ adduct pattern but was located ina position different from that of the IQ adduct pattern. DNAfrom Syrian hamster embryo (SHE) cells treated with IQ and S9exhibited adducts apparently identical with those of SalmonellaDNA.     

Excretion of food-derived heterocyclic amine carcinogens into breast milk of lactating rats and formation of DNA adducts in the newborn

The distribution, DNA adduction and excretion into breast milkof 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) were examined in lactating female F344 ratswith 5 day old pups. Six hours after a single dose (10 mg/kg,p.o.) of radiolabeled IQ, MelQx or PhIP to lactating dams, radioactivityin the dams was highest in the liver and kidney followed, indescending order, by the mammary gland, omental fat and brain.By 24 h after carcinogen administration, all tissues of thedams showed significantly reduced levels of radioactivity exceptfor omental fat which changed only marginally from 6 to 24 h.³²P-Postlabeling analysis showed that the level of DNA adductsin mammary gland 6 h after dosing was 2.2, 0.7 and 0.2 adducts/10⁷nucleotides for PhIP, IQ and MelQx respectively. In contrast,in hepatic DNA, the levels of IQ-DNA adducts (5.5 adducts/10⁷nucleotides) were 11-fold higher than those of PhIP or MelQx.The stomach contents, liver, kidney and urine of pups nursedby dams given radiolabeled IQ, MelQx or PhIP were radioactive,indicating that these carcinogens (and/or metabolites) wereexcreted into breast milk and absorbed by the pups. After a6 h suckling period, the amount of PhlP-derived radioactivityin the stomach contents of the pups was     

ACCELERATED PAPER: Mutational spectra of the dietary carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) at the Chinese hamster hprt locus

The mutagenic ‘fingerprint’ of the cooked food carcinogen2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) was determinedin a Chinese hamster cell line genetically engineered to expresshuman CYP1A2 (XEMh1A2-MZ). The parental Chinese hamster V79and XEMh1A2-MZ cells were exposed to PhIP at various concentrationsfor 24 h. There was a dose-dependent increase in frequency ofmutations at the hypoxanthine-guanine phosphoribosyl-transferase(hprt)locus only in the metabolically competent XEMh1A2-MZ cells.The mutant frequency ranged from 25 to 90x10⁶ with final concentrationsof 2.5 to 100 µM PhIP compared to 8x10⁶ in the solventcontrols and the V79MZ cells. The molecular nature of PhlP-inducedmutations in XEMh1AZ-MZ cells was determined by examining DNAsequence modifications at the hprt locus in forty five 6-thioguanineresistant (6-TG¹) mutant clones. Single base substitutions,predominantly GC     

DNA adduct formation in B6C3F1 mice and Fischer-344 rats exposed to 1, 2, 3-trichloropropane

1, 2, 3-Trichloropropane (TCP) is a multispecies, multisitecarcinogen which has been found to be an environmental contaminantIn this study, we have characterized and measured DNA adductsformed in vivo following exposure to TCP. [¹⁴C]TCP was administeredto male B6C3F1 mice and Fischer-344 rats by gavage at dosesused in the NTP carcinogenesis bioassay. Both target and nontargetorgans were examined for the formation of DNA adducts. Adductswere hydrolyzed from DNA by neutral thermal or mild acid hydrolysis,isolated by HPLC, and detected and quanti-tated by measurementof radioactivity. The HPLC elution profile of radioactivitysuggested that one major DNA adduct was formed. To characterizethis adduct, larger yields were induced in rats by intraperitonealadministration of TCP (300 mg/kg). The DNA adduct was isolatedby HPLC based on coelution with the radiolabeled adduct, andcompared to previously identified adducts. The isolated adductcoeluted with S-[1-(hydroxymethyl)-2-(N⁷-guanyl)-ethyljglutathione,an adduct derived from the structurally related carcinogen 1,2-dibromo-3-chloropropane (DBCP). Analysis by electrospray massspectrometry suggested that the TCP-induced adduct and the DBCP-derivedadduct were identical. The ¹⁴C-labeled DNA adduct was distributedwidely among the organs examined. Adduct levels varied dependingon species, organ, and dose. In rat organs, adduct concentrationsfor the low dose ranged from 0.8 to 6.6 µmol per mol guanineand from 7.1 to 47.6 µmol per mol guanine for the highdose. In the mouse, adduct yields ranged from 0.32 to 28.1 µmolper mol guanine for the low dose and from 12.2 to 208.1 µmolper mol guanine for the high dose. The relationship betweenDNA adduct formation and organ-specific tumorigenesis was unclear.Although relatively high concentrations of DNA adducts weredetected in target organs, several nontarget sites also containedhigh adduct levels. Our data suggest that factors in additionto adduct formation may be important in TCP-induced carcinogenesis.     

Persistence, gestation stage-dependent formation and interrelationship of benzo[a]pyrene-induced DNA adducts in mothers, placentae and fetuses of Erythrocebus patas monkeys

Since DNA adducts have been detected in the placentae of pregnantwomen who smoke cigarettes, the importance of these adductsas biomarkers of fetal exposure and risk has been evaluatedusing a non-human primate as a model. Pregnant Erythrocebuspatas monkeys on days 50, 100 or 150 of gestation (term = 160± 5 days) were treated once with 5–50 mg/kg benzo[a]pyrene(B[a]P), p.o. Fetuses were removed by Cesarean section 1–50days after treatment and analyzed for DNA adducts by the nucleaseP₁ version of the ³²P-postlabeling method. B[a]P induced highlevels of DNA adducts in all fetal organs, placentae and maternallivers in all three trimesters of gestation. DNA adduct levelswere higher in mid-gestation compared to early and late gestation.The major adduct detected was 10ß-(deoxyguanosin)-N²-yl-7ß,8     

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     

Formation and persistence of benzo[a]pyrene-DNA adducts in mouse epidermis in vivo: importance of adduct conformation

The formation and repair of benzo[a]pyrene diol epoxide-N²-deoxyguanosineadducts (BPDE-N²-dG) in DNA isolated from the skin of mice treatedtopically with benzo[a]pyrene (BP) was studied by ³²P-postlabelingand by low-temperature fluorescence spectroscopy under low resolutionand under high resolution fluorescence line narrowing (FLN)conditions. In agreement with earlier studies, total BP-DNAbinding reached a maximum at 24 h after treatment (dose: 1 µmol/mouse),then declined rapidly until 4 days after treatment and muchmore slowly thereafter. An HPLC method was developed which resolvedthe ³²P-postlabeled (–)-trans- from (–)-cis-anti-BPDE-N²-dG,and (+)-trans- from (+)-cis-anti-BPDE-N² High performance liquidchromatography analysis of the major TLC adduct spot (containing>80% of the total adducts) obtained by postlabeling BP-modifiedmouse skin DNA showed that it consisted of a major componentthat coeluted with (–)-cis-/(+)-trans-anti-BPDE-N²-dGand a minor component that coeluted with (–)-trans-/(+)-cis-anti-BPDE-N²-dGand that the minor component was repaired at a slower rate thanthe major component. Low-temperature fluorescence spectroscopyof the intact DNA identified the major adduct as (+)-trans-anti-BPDE-N²-dGand the minor adduct fraction consisted mainly of (+)-cis-anti-BPDE-N²In agreement with the ³²P-postlabeling results it was observedby fluorescence spectroscopy that the (+)-cis-adducts were repairedmore slowly than most other adducts. Moreover, the (+)-trans-adductsexhibited a broad distribution of base-stacked, partially base-stackedand helix-external conformations. Mouse skin DNA samples obtainedat early timepoints (2–8 h) after treatment with BP containedsubstantially more of the ‘external’ adducts, whilesamples at later timepoints (24–48 h) contained relativelymore adducts in the base-stacked conformation, indicating alsothat the latter adducts are repaired less readily than the former.The possible biological significance of these novel observationsof conformation-dependent rates of DNA adduct repair and theirpossible dependence on DNA sequence, are discussed.