Inhibition of 2-amino-3-methylimidazo[4,5-f]quinoline-DNA adducts by indole-3-carbinol: dose-response studies in the rat colon

Indole-3-carbinol (I3C) inhibits the formation of colonic aberrant crypt foci and DNA adducts in rats given heterocyclic amine colon carcinogens, such as 2-amino-3-methylimidazo[4,5-f]quinoline (IQ). Mechanism studies indicate that I3C induces cytochromes P4501A1 and 1A2 (CYP1A1 and CYP1A2), isozymes that respectively metabolize IQ via ring hydroxylation or activate the carcinogen by N-hydroxylation. The present study examined the dose-response for induction of CYP1A1 versus CYP1A2 by I3C, and compared the profiles of induction with the dose- response for inhibition of IQ-DNA adducts in the colon of the F344 rat. Dietary equivalent doses of I3C in the range 100-1000 p.p.m. increased in a dose-related manner both ethoxyresorufin O-deethylase (EROD) and methoxyresorufin O-demethylase (MROD) activities in the liver and colonic mucosa, and Western blots showed a corresponding induction of CYP1A1 and CYP1A2 proteins. However, dietary equivalent doses of I3C in the range 10-25 p.p.m. (i) reduced hepatic EROD and MROD activities and CYP1A protein levels compared with controls, (ii) increased the ratio of CYP1A2 versus CYP1A1, and (iii) activated IQ to a more potent mutagen when liver microsomes from rats given I3C were used for metabolic activation in the Salmonella assay. Rats given a single oral dose of I3C shortly before administering IQ (5 mg/kg body wt, p.o.) exhibited dose-related inhibition of colonic IQ-DNA adducts in the range 25-100 p.p.m. I3C, reaching 95% inhibition at doses > or = 100 p.p.m. I3C, but IQ-DNA adducts were elevated slightly at the lowest I3C dose as compared with the controls. The possible significance of the low versus high dose effects of I3C are discussed in the context of human dietary exposures to I3C and the reported chemopreventive mechanisms of I3C in vivo.      

Accumulation of DNA adducts of 2-amino-3-methylimidazo[4, 5-f] quinoline (IQ) in tissues and white blood cells of the Fischer-344 rat after multiple oral dosing

The genotoxic effect of an environmental chemical may be estimatedfrom the concentration of its DNA adducts in peripheral whiteblood cells (WBCs). The food mutagen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) is carcinogenic in the Fischer-344 rat, affectingprincipally the liver, small intestine and large intestine.In the present study we have determined whether DNA adductsof IQ are present in circulating WBCs of rats after single ormultiple oral doses of IQ and how these adducts are relatedto those in internal organs. Male Fischer-344 rats receivedIQ as an oral dose (5or 50 mg/kg, starting on day 0) by dailygavage (1, 8 or 15 days of treatment). Using ³²P-postlabelingassays, IQ-DNA adducts were isolated and quantitated in organsand WBCs on days 1, 8 and 15. Adduct patterns in WBCs were qualitativelysimilar to those in the organs and adduct formation was highestin the liver, followed by the lungs, kidneys, stomach, largeintestine, WBC and small intestine. Accumulation of adductsoccurred in all organs and in WBCs in a dose- and time-dependentmanner. For all organs, IQ-DNA adduct formation was stronglycorrelated with those in WBCs. It is concluded that IQ-DNA adductsin WBCs are qualitatively and quantitatively directly relatedto those in internal organs, independent of the target organspecificity of the carcinogenic effect of IQ.     

Protection of conjugated linoleic acids against 2-amino-3-methylimidazo[4,5-f]quinoline-induced colon carcinogenesis in the F344 rat: a study of inhibitory mechanisms

Grilled ground beef contains a number of heterocyclic aminecarcinogens, such as 2-amino-3-methylimidazo[4,5-/Iquinoline(IQ), as well as anticarcinogenic conjugated linoleic acids(CLA). In the present study, CLA was administered to male F344rats by gavage on alternating days in weeks 1–4, whileIQ was given by gavage every other day in weeks 3 and 4 (100mg/kg body wt). Rats were killed 6 h after the final carcinogendose in order to quantify IQ-DNA adducts or after week 16 inorder to score colonic aberrant crypt foci (ACF). In the ACFstudy, CLA had no effect on the size of the foci, but inhibitedsignificantly (P <0.05) the number of ACF/colon, from 4.3± 2.4 in controls to 1.1 ± 1.3 in CLA-treatedrats (mean ± SD,n = 10). Rats given CLA also had significantlylower IQ—DNA adducts in the colon as determined by 32P-postlabelinganalysis; relative adduct labeling levels (RAL x 10⁷) for themajor adduct were 9.13 ± 2.6 in controls versus 5.42± 1.8 in CLA-treated animals (P < 0.05). Mechanismstudies indicated that CLA and other fatty acids interact withcertain heterocyclic amines in a manner consistent with substrate—ligandbinding. However, no such interaction occurred with IQ, andCLA failed to inhibit significantly the mutagenicity ofN-hydroxy-IQin the Salmonella assay, Liver microsomes from CLA-treated ratsexhibited lower activities for dealkylation of 7-ethoxyresorufinand methoxyresorufin and activated IQ to DNA binding speciesless effectively than microsomes from control animals. Directaddition of CLA to the in vitro incubation inhibited IQ-DNAbinding and was associated with increased recovery of unmetabolizedparent compound. In the Salmonella assay, CLA inhibited themuta-genic activity of IQ in the presence of S9 or ram seminalvesicle microsomes. Collectively, these results support a mechanisminvolving inhibition of carcinogen activation by CLA, as opposedto direct interaction with the procarcin-ogen, scavenging ofelectrophiles or selective induction of phase I detoxificationpathways.     

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.     

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

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

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     

Cytochrome P4501A2 constitutively expressed from transduced DNA mediates metabolic activation and DNA-adduct formation of aromatic amine carcinogens in NIH 3T3 cells

We transduced mouse cytochrome P4501A2 DNA into NIH 3T3 cells by retrovirus-mediated gene transfer. The capacity of the transduced cytochrome P4501A2 for metabolic activation and DNA-carcinogen adduct formation of aromatic amine carcinogens was investigated. Clones of NIH 3T3 cells that constitutively express cytochrome P4501A2 and controls were exposed to a prototype food-derived carcinogenic heterocyclic amine, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), and an aromatic amine, 2-acetylaminofluorene (AAF), and their genomic DNAs were analyzed for adducts by 32P-postlabeling assays. Kinetic analysis of DNA-carcinogen adducts indicated that adduct formation was dependent on the level of the enzyme, the dose of carcinogen, and the duration of exposure. Addition of 7,8-benzoflavone, an inhibitor of P4501A2, blocked both the enzyme activity and DNA-adduct formation, indicating the specific role of P4501A2 in metabolic activation and adduct formation. Three specific IQ-DNA adducts were detected in cells expressing P4501A2. Fingerprints of the in situ DNA adducts were similar to those of the in vivo adducts in rodent hepatic DNA after the administration of IQ. A single AAF-DNA adduct was observed in cells exposed to AAF, but other minor adducts were also detected in vivo. These results show that cells expressing constitutive levels of single cytochrome P450s provide an excellent in situ model system for analyzing the catalytic specificity, metabolic activation, and genotoxicity of putative toxic, mutagenic, and carcinogenic substances.     

Selective induction of rat hepatic CYP1 and CYP4 proteins and of peroxisomal proliferation by green tea

Rats were exposed to freshly prepared aqueous extracts of greentea (2.5%w/v) as the sole source of drinking water for 4 weeks.Hepatic cytochrome P450 activity was determined using chemicalprobes, showing selectivity for particular isoforms, and byiminunoblot analysis employing polyclonal antibodies. Exposureto green tea gave rise to increases in the O-demethylation ofmethoxyresorufin and, to a lesser extent, in the dealkylationsof ethoxyresorufin and pentoxy resorulin. An Increase was alsoseen In lauric acid hydroxylation but, In contrast, the N-demethylationof erythromycln was Inhibited. p-Nitrophenol oxidase activitywas unaffected by the same treatment. Iminunoblot analysis revealedIncreases In the apoprotein levels of CYP1A2 and CYP4A1 followingtreatment with green tea. A significant Increase was also notedin the CN^–-insensitive palmitoyl CoA oxidation and thiswas paralleled by an increase In the levels of the peroxisomaltrifunctional protein determined immunologically. Hepatic S9and microsomal preparations from tea-treated animals were moreeffective than controls in activating 2-amlno-3-methylimidazol[4,5-f]quinolineand 2-aminoanthracene to mutagens In the Ames test. When N-nitrosopyrrolidineserved as the promutagen, tea did not Influence its mutagenicltywhen isolated microsomes comprised the activation system buta significant inhibition was observed when hepatic S9 was used.The above findings are discussed within the context of the establishedanti-carcinogenic and anti-mutagenic properties of green tea.     

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     

The role of aberrant crypt foci induced by the two heterocyclic amines 2-amino-3-methyl-imidazo[4,5-f]quinoline (IQ) and 2-amino-1-methyl-6-phenyl-imidazo[4,5-b]pyridine (PhIP) in the development of colon cancer in mice

Aberrant crypt foci (ACF) have recently been identified as early putative preneoplastic lesions which appear in the colons of experimental animals treated with colon carcinogens. In a recent study the two heterocyclic amines, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-1-methyl-6-phenyl-imidazo[4,5-b]pyridine (PhIP) were shown to be able to induce ACF in the colon of mice after, respectively, 4 and 10 weeks of exposure. In spite of the induction of ACF in colon of mice, IQ and PhIP have not been found to have colon as target organ in carcinogenicity studies. Therefore, one may question that ACF induced by IQ and PhIP in mice represent early stages of colon cancer. In order to investigate the possible role of PhIP- and IQ-induced aberrant crypt foci in the development of colon cancer in mice, colons from mice participating in other IQ- and PhIP-studies of much longer duration were analyzed for ACF. The results of these studies showed that the number of ACF increased statistically significantly over time, and that the small ACF were predominant (95–100%) at all time-points. In conclusion, this finding suggests that the detection of a high number of ACF with low crypt multiplicity (1–3 AC/Focus) in mice colon after IQ- or PhIP-treatment is not indicative for the end-point colon cancer, and thus supports the hypothesis that only the presence of a high number of ACF with high crypt multiplicity is predictive for tumor outcome.     

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.     

Similar patterns of DNA adduct formation of 2-amino-3-methylimidazo [4,5-f]quinoline in the Fischer 344 rat, CDF1 mouse, cynomolgus monkey and Salmonella typhimurium

2-Amino-3-methylimidazo [4,5-f]quinoline (IQ) is a known liver carcinogen in the Fischer 344 rat, the CDF1 mouse and in the cynomolgus monkey. Using 32P-postlabeling assays, we compared IQ-DNA adduct formation in the liver of IQ-treated Fischer 344 rats, CDF1 mice and cynomolgus monkeys with that in Salmonella typhimurium (strain TA98) incubated with IQ (in the presence of a liver S9 activating system) or N-hydroxy-IQ. Up to five adducts could be detected, the pattern of which was identical in all cases. The major adduct co-chromatographed with standard N-(deoxyguanosin-8-yl)-IQ in all cases and comprised 54.7-82.8% of the total. The four minor adducts were not identified. It is concluded that N-(deoxyguanosin-8-yl)-IQ is the major IQ-DNA adduct under all experimental conditions and that the pattern of N-hydroxy-IQ-DNA adducts is identical to that found in the liver of animals exposed to IQ, and to that found after reacting IQ with DNA in the presence of a liver S9 activating system. Thus, N-hydroxylation of IQ is a critical step in the formation of IQ-DNA adducts.     

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.     

Effect of carcinogen dose fractionation, diet and source of F344 rat on the induction of colonic aberrant crypts by 2-amino-3-methylimidazo[4,5-f]quinoline

Carcinogen dose fractionation, diet and source of laboratory animal were examined as variables in the induction of colonic aberrant crypt foci (ACF) by the heterocyclic amine 2-amino-3-methylimidazo [4, 5-f]quinoline (IQ). In the first experiment, male F344 rats from the National Cancer Institute (NCI rats) were fed AIN-93G diet and, starting in the third week, IQ was given by gavage on alternating days, the total carcinogen dose of 105 mg being fractionated proportionally over 2, 4, 8 or 14 weeks. Only the high dose (2 week) treatment with IQ was effective for the induction of ACF at 16 weeks, producing on average 3.8 ACF/colon versus 0.5 ACF/colon in all other groups (P < 0.05). The 2 week IQ dosing protocol was used in a second experiment in which male F344 rats from Simonsen Laboratories (SN) or NCI were fed AIN-93G, AIN-76A or chow diet. On average, SN rats on chow diet had twice the number of aberrant crypts compared with NCI rats given the same diet and three to four times as many aberrant crypts as NCI rats fed AIN diets. Hepatic cytochrome P4501A1 (CYP1A1) levels were essentially unaffected by diet, but methoxyresorufin O-demethylase activities and CYP1A2 protein levels were increased 2- to 3-fold in animals fed chow versus AIN diets. During the 2 week period of carcinogen administration, IQ markedly induced CYP1A proteins and negated the differences among groups related to diet. No consistent diet-related changes were detected in the activities of aryl sulfotransferase or N-acetyltransferase, but UDP-glucuronosyltransferase activities were elevated 2- to 3-fold in rats given chow versus AIN diets. In summary, high dose treatment with IQ was required for the induction of ACF, rats on the chow diet had more aberrant crypts than those given AIN diets and male F344 rats purchased from different vendors and fed chow diet differed with respect to their sensitivity to induction of ACF.     

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     

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.     

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     

Protection by chlorophyllin against the covalent binding of 2-amino-3-methylimidazo[4,5-f]qiiinoline (IQ) to rat liver DNA

Chlorophyllin (CHL), a sodium/copper salt of chlorophyll usedin the treatment of geriatric patients, exhibits potent anti-mutagenicactivity in a range of assays in vitro and in vivo. The protectiveeffects of CHL were studied in Sprague-Dawley rats using inhibitionof carcinogen-DNA binding as an end-point. Animals were administeredCHL (150 mg/kg body wt) and [2-¹⁴C]2-amino-3-methylimidazo[4,5-f]quinoline(IQ, 50 mg/kg body wt) by single oral gavage. Covalent IQ-DNAbinding in liver was determined 8, 24 and 48h after dosing;CHL inhibited binding at these times by 58, 56 and 46% respectively,compared with rats given IQ alone. The total liver burden ofIQ-derived radioactivity was reduced in CHL-treated rats, aswas the total amount of radiolabel eliminated in the urine andbile. However, elimination via the feces was increased in ratsgiven CHL, both in terms of total radiolabel eliminated andamount of unmetabolized IQ in dichloromethane extracts of feces.Finally, pretreatment with CHL in the drinking water, or injectionof CHL into isolated loops of intestine in situ, reduced theabsorption of IQ from the gut. Collectively, these findingsindicate that, when administered simultaneously with the carcinogen,CHL attenuates IQ-DNA binding in rat liver by interacting withIQ in the gut and reducing carcinogen uptake, distribution andmetabolism. The results suggest that further studies shouldbe conducted with respect to the protective mechanisms and possibleanti-carcinogenic properties of CHL.     

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.     

Effect of ellagic acid and 3-O-decylellagic acid on the formation of benzo[a]pyrene-derived DNA adducts in vivo and on the tumorigenicity of 3-methylcholanthrene in mice

The effect of ellagic acid and its more lipophilic derivative,3-O-decylellagic acid, on the amount of DNA-bound adducts inthe epidermis or lung of CD-I mice treated with [³H]benzo-[a]pyrene([³H]B[a]P) was evaluated using several different treatmentprotocols. The i.v. administration of 50µmol/kg of ellagicacid or 3-O-decyIellagk acid either together with or 5 min beforea 0.2 µmol/kg i.v. dose of [³H]B[a]P did not inhibit theformation of pulmonary DNA-bound adducts. Feeding mice a dietthat contained 1% ellagic acid for 10 days or the i.p. administrationof 120 µmol/kg of ellagic acid 30 min before the i.v.administration of 0.2 µmol/kg of [³H)B(a)P did not inhibitthe formation of DNA-bound adducts in the lung. The applicationof 2500 nmol of ellagic acid or 3-O-decylellagic acid to mouseskin 5 min before the application of 2, 10 or 50 nmol of [³H]B[a]Phad little or no effect on the covalent binding of [³H]B[a]Pmetabolites to epidermal DNA. Feeding mice a diet containing1% ellagic acid for 10 days did not inhibit the formation ofepidermal DNA-bound adducts after a topical dose of 2 nmol of[³H]B[a]P. Similarly, the topical application of 2500 nmol ofellagic acid at 2 h, 1 h and 5 min before and at 10 min afterthe application of 2 nmol of [³H]B[a]P did not inhibit the formationof DNA-bound adducts, but the same dosing regimen of 3-O-decylellagicacid (total dose of 10 000 nmol) resulted in a modest inhibitionin the formation of DNA-bound adducts. The topical applicationof 1500 nmol of ellagic acid 1 h before the application of 1500nmol of 3-methylcholanthrene (3-MC) to CD-I or BALB/c mice twiceweekly did not inhibit the development of skin tumors. Our resultsindicate that ellagic acid and 3-O-decylellagic acid are noteffective in inhibiting [³H]B[a]P DNA adduct formation in mouseskin and lung and that ellagic acid does not inhibit 3-MC-inducedskin tumori-genesis in BALB/c or CD-I mice.