Metabolic activation of the food mutagens 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) to DNA binding species in human mammary epithelial cells.

When incubated in suspension with the heterocyclic aromatic amine food mutagens 2-amino-3-methylimidazo [4,5-f]-quinoline (IQ) and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), human mammary epithelial cell aggregates were found, by 32P-postlabelling analysis, to yield DNA that contained adducts. Analysis by HPLC of the 32P-labelled digests of mammary cell DNA indicated that in each case a major adduct peak corresponded to that produced in DNA in vitro by activated derivatives of the two compounds. The patterns of adducts obtained when DNA digests were separated by TLC on polyethyleneimine-cellulose plates were found to resemble those previously shown to be present in DNA of tissues of mice fed IQ or MeIQ. These results demonstrate the ability of human mammary epithelial cells to activate carcinogenic heterocyclic compounds known to be present in the human diet to DNA binding derivatives.     

Sex differences in the formation and persistence of DNA adducts of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) in CDF1 mice

The potent food mutagen 2-amino-3-methylimidazo[4,5-f]-quinoline (IQ) is carcinogenic in the CDF1 mouse, affecting the liver, lungs and forestomach. Females are approximately twice as sensitive as males to liver tumor induction. Using 32P-postlabeling assays, the dose-response of IQ-DNA adduct formation was determined in various organs of male and female CDF1 mice after single p.o. doses of IQ. To determine the possible correlation between IQ-DNA adduct formation, persistence and tumorigenicity in target organs, young adult male and female CDF1 mice were treated with a single p.o. dose (50 mg/kg) of IQ, and IQ-DNA adducts were isolated and quantitated in liver, lungs, forestomach, small intestine and large intestine over a 24 day period. In the range of 5-50 mg IQ/kg, IQ-DNA adduct formation was related to dose in all organs examined (liver, lungs, stomach, small intestine, large intestine). Total adduct formation (expressed as relative adduct labeling, RAL) 24 h after administration was highest in the liver (6.4-6.9 x 10(-7)) with lower levels, in decreasing order, in the large intestine, small intestine (non-target organs), forestomach and lungs (target organs). In all cases the major adduct, comprising 68-79% of the total, co-chromatographed with standard N-(deoxyguanosin-8-yl)-IQ. Up to three additional IQ-specific adducts could be detected. Except in the liver, adduct levels 24 h after administration of IQ were 2- to 3-fold higher in females than in males. There was no preferential retention of any one of the four adducts 1-24 days after administration of IQ. Beyond day 4 after administration, total adducts in the liver of females were approximately 2-fold higher than those in males, and the rate of removal from female lung was approximately 2-fold faster than that from male lung during the 1-24 day time period. Both these findings correspond to the known sex differences in IQ-induced tumor incidences in these organs. The higher adduct levels in non-target organs (intestines) as compared to target organs (lungs and stomach), combined with the absence of differential persistence of any individual adduct indicates that, in addition to adduct formation and persistence, other factors contribute to the target organ specificity of IQ in CDF1 mice.     

Beta-catenin mutations in liver tumors induced by 2-amino-3,4-dimethylimidazo[4,5-f]quinoline in CDF1 mice

Heterocyclic amines are potent mutagens and carcinogens formed in cooked protein rich foods. In this study, we screened liver tumors induced by 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) in CDF1 mice for beta-catenin and APC mutations and other genetic alterations shown to occur in human hepatocellular carcinomas (HCC), including mutations in the p53 and H-ras genes, c-myc amplification and E-cadherin promoter methylation. SSCP followed by direct DNA sequencing revealed mutations in exon 2 of the beta-catenin gene in 2 of 16 liver tumors (12.5%). Promoter methylation of the E-cadherin gene was detected in one liver tumor induced by MeIQ. There were no mutations in the mutation cluster region of the APC gene, in exons 5-8 of the p53 gene, or in codons 12, 13 and 61 of the H-ras gene, nor c-myc amplification in any of liver tumors induced by MeIQ. These data indicate that except for the occasional disruption of the Wnt pathway through beta-catenin mutations, the genetic pathways involved in the development of HCC differ significantly between human liver cancer and tumors induced in mice by MeIQ, but do not rule out the possibility that heterocyclic amines constitute a carcinogenic risk factor in humans.     

Quantitation of 2-amino-3-methylimidazo[4,5-f]quinoline and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline DNA adducts in specific sequences using alkali or uvrABC excinuclease

2-Amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MelQx) are carcinogens found in cooked meats that form DNA adducts upon metabolic activation. Purified DNA from Chinese hamster ovary (CHO) cells was reacted in vitro with the active metabolites N-acetoxy-lQ or N-acetoxy-MelQx, and the adduct levels in the 5′ dihydrofolate reductase (DHFR) gene and downstream region were quantitated by Southern hybridization. Adducted and restricted DNA was treated with Escherichia coli uvrABC excinuclease or alkali (0.1 N NaOH, 37°C, 60 min) to incise DNA at IQ and MeIQx adduct sites. The DNA was then denatured with formamide, electrophoresed on a neutral agarose gel, transferred to a support membrane, and hybridized with sequence-specific DNA probes. Both uvrABC and alkali reduced the intensity of Southern hybridization in proportion to the number of IQ or MeIQx adducts in DNA, indicating that these adducts are substrates for uvrABC and that they form alkali-labile lesions in DNA. IQ and MeIQx adduct levels were the same in the 5′ DHFR gene and in the downstream region. Southern hybridization analysis of pBR322 containing known levels of IQ or MeIQx adducts showed that the efficiency of cutting IQ or MeIQx adducts by uvrABC excinuclease and alkali was approximately 30% and 15%, respectively. ³²P-postlabeling studies examining adduct level in bulk DNA further showed that the adduct profiles were identical in pBR322, CHO DNA, and cultured CHO cells exposed to the reactive metabolites of IQ or MeIQx. The results indicate that IQ and MeIOx adducts can be quantitated in specific genomic sequences and that this method should be directly applicable to studies of gene-specific repair of these adducts in cultured cells.     

Fluorometric detection of 2-amino-3-methylimidazo[4,5-f]quinoline, 2-amino-3,4-dimethylimidazo[4,5-f]quinoline and their N-acetylated metabolites excreted by the rat

A sensitive fluorometric method has been developed for the determinationof carcinogenic amino imidazoazaarenes (AIA compounds) whichare formed during the cooking of food. They are separated byt.l.c. followed by spraying with dimethylsulfoxide and visualizationby long-wave u.v. light. Quantification is done by fluorescencescanning. This method was applied to the determination of themetabolites in urine and feces from rats given 5 mg/kg bodywt i.p. of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ). Metabolitesextractable by organic solvents were identified as the parentcompound and their N-acetylated derivatives (AcIQ, AcMeIQ).Between 0.1 and 1% of the administered dose was recovered asthese metabolites in urine during 24 h. The amounts of MeIQand AcMeIQ found in feces were 1 and 3% respectively. AcIQ wasnot detected in feces while IQ, MeIQ and their N-acetylatedderivatives were found in bile. The metabolites in urine wereidentified by m.s. and in feces by t.l.c. and their fluorescentproperties. When IQ or MeIQ were incubated with rat liver cytosolin the presence of acetyl-CoA, N-acetylated derivatives wereformed. The reverse reaction, deacetylation, also took placein the liver cytosol, with K_m values of 67 and 32 µM forAcIQ and AcMeIQ respectively.     

Organ-dependent susceptibility of p53 knockout mice to 2-amino-3-methylimidazo[4,5-f]quinoline (IQ)

p53 knockout mice are now being frequently used to identify the carcinogenic potential of chemicals, thus it is important to precisely assess the susceptibility of the animals to various test chemicals. In the present study the susceptibility of p53 nullizygous((-/-)), heterozygous((+/-)), and wild-type((+/+)) mice to 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) was investigated. Mice of all three genotypes were first fed a diet containing 100 or 300 p.p.m. IQ for 15 weeks in a short-term experiment. p53((+/-)) and ((+/+)) mice were then treated with IQ for 40 weeks and maintained without further treatment for an additional 12 weeks in the long-term experiment. In the forestomach, the incidence of squamous cell hyperplasia was significantly higher in p53((-/-)) than in ((+/-)) and ((+/+)) mice at 15 weeks and higher in ((+/-)) mice than ((+/+)) mice with long-term IQ treatment, indicating an elevated susceptibility of p53 knockout mice. In contrast, in the liver, various hepatocellular lesions developed mainly in female mice with long-term IQ exposure but no significant differences were evident between p53 knockout and wild-type mice, indicating a lack of elevated susceptibility in the knockout animals. Furthermore, polymerase chain reaction-single strand conformation polymorphism and sequencing analysis revealed relatively high (13/30) and low (1/15) incidences of p53 mutations (exons 5-8) in squamous cell hyperplasia and hepatocellular tumors, respectively. These results clearly indicate that the susceptibility of p53 knockout mice is organ-dependent, coinciding to some extent with the likelihood of p53 gene alteration in the induced tumors.     

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.     

Comparison of initiation potential of 2-amino-3-methylimidazo[4,5-f]quinoline and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline in an in vivo carcinogen bioassay system

A new approach to low-dose assessment of carcinogenic potential was applied to food contaminant pyrolysis products. Single intragastric doses of the carcinogenic pyrolysates, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) or 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline MeIQx), were given 12 h after two-thirds partial hepatectomy (PH) to F344 male rats. Two weeks thereafter the animals were placed on a basal diet containing 0.05% phenobarbital (PB) for 6 weeks combined with an i.p. administration of D-galactosamine (300 mg/kg) to facilitate growth of initiated cells. Both IQ and MeIQx clearly caused initiation of hepatocarcinogenesis as revealed by induction of preneoplastic placental-form glutathione-S-transferase-positive (GST-P+) hepatocyte foci composed of more than three cells (approximately 30 microns in diameter). A similar protocol without performance of PH before pyrolysate administration gave a positive result only for the IQ-treated group indicating that cell proliferation is essential during the low-dose, one-shot initiation step. IQ was found to be two to three times more potent in inducing GST-P+ foci using both protocols. The current approach could find application in practical carcinogenicity screening of chemicals, for which only small amounts are available.     

Purification of the food-borne carcinogens 2-amino-3-methylimidazo [4,5-f]quinoline and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline in heated meat products by immunoaffinity chromatography

A rapid and simple scheme has been developed for the isolation and purification of two of the major mutagenic heterocyclic amines formed in heated beef products by affinity chromatography using monoclonal antibodies which recognize 2-amino-3-methylimidazo[4,5-f]quinoline (IQ). Two cell lines producing IgG antibodies were established following fusion of Sp2 or P3x.63 myeloma cells with spleen cells of immunized BALB/cby mice. The antigen was bovine gamma globulin haptenized with 2-(3-carboxypropylthio)-3-methylimidazo-[4,5-f]quinoline. The antibodies were immobilized on CNBr-activated Sepharose 4B. IQ and MeIQx formed in heated beef products were partially purified by XAD-2 chromatography and then applied to the affinity columns. Purification by affinity chromatography was adequate for subsequent quantitative analysis by HPLC with UV detection. With this purification scheme as little as 1 g of beef extract or 15 g of fried beef could be assayed for IQ and MeIQx at the part per billion level. Both antibodies had similar affinity constants for IQ (9.3 X 10(6) and 6.7 X 10(6) M-1) and for MeIQx (7.1 X 10(5) and 2.7 X 10(5) M-1) and both were suitable for immunoaffinity purification of IQ from complex mixtures. MAb2 could be used as well to selectively remove MeIQx from meat products after partial purification by XAD-2. MAb1, despite having a 3-fold higher affinity than MAb2 for MeIQx, could not be used for affinity chromatography for this mutagen.     

Formation and persistence of DNA adducts of 2-amino-3-methylimidazo[4,5-f] quinoline in male Fischer-344 rats

The mutagenic heterocyclic amine 2-amino-3-methylimidazo[4,5-f] quinoline (IQ) is carcinogenic in the Fischer-344 rat, affecting the liver and small and large intestines, as well as several other organs. In male animals the incidences of tumors in the liver, small intestine, and large intestine were reported to be 67.5, 30.0, and 62.5%, respectively. Using 32P-postlabeling assays, the formation and persistence of IQ-DNA adducts in the liver and small and large intestines were studied in male Fischer-344 rats. Young, adult animals were either given a single p.o. dose (5, 25, or 50 mg/kg) of IQ and were killed 24 h later or were given a single p.o. or i.p. dose (50 mg/kg) of IQ and were killed at different time points, from 6 h to 31 days after p.o. treatment and from 6 h to 6 days after i.p. treatment, to follow adduct persistence. Up to five specific adducts could be isolated, and adduct formation was dose related in all three organs. Adduct 1, previously shown to be N-(hydroxyguanosin-8-yl)-IQ, was the major adduct in all cases, comprising up to 78% of the total. After p.o. administration (6-24 h) adduct levels in the liver were 3- to 4-fold higher than after i.p. administration, while levels in the intestines during this time period were independent of the route of administration. At 24 h after p.o. administration total adduct levels in the liver were 13.5-41.4 and 9.2-18.4 times higher than those in the small intestine and large intestine, respectively. Maximum adduct levels were observed between 6 and 24 h after administration, and from 1 to 6 days later, rates of removal from the liver were 7-fold and 2-fold slower, respectively, than from the small and large intestine. Rates of adduct removal from the intestines after i.p. administration were similar to those after p.o. administration. Beyond day 15 adduct levels in all organs constituted less than 12% of those on day 1, and low levels of adducts persisted for up to 31 days. In all cases there was no preferential loss or persistence of any of the adducts. It is concluded that total adduct levels and persistence in target organs may, in part, be related to susceptibility to IQ-induced carcinogenesis in the Fischer-344 rat.     

Mutagenicity of 2-amino-3-methylimidazo[4,5-f]quinoline in human lymphoblastoid cells

2-Amino-3-methylimidazo[4,5-f]quinoline (IQ), a heterocyclic aromatic amine that has been identified in cooked meats and cigarette condensates, is mutagenic in human lymphoblastoid TK6 cells at the thymidine kinase and hypoxanthine-guanine phosphoribosyl transferase (hprt) loci. Treatment of the cells with IQ following activation with either an exogenous metabolizing mixture (S9) or following photoactivation of the azido-derivative of IQ (N3-IQ) showed that the photolytic-derivative of N3-IQ was more active. This observation is consistent with other reports that indicate that the weak mutagenicity of IQ in mammalian cells is caused by the lack of enzymes required for the ultimate activation of the compound within the cells. Two DNA adducts were found by 32P-post-labelling in the cells treated with the photoactivated N3-IQ. The major adduct was identified as N- (deoxyguanosin-8-yl)-2-amino-3-methylimidazo[4,5-f]quinoline (dG-C8-IQ) and the minor adduct as 5-(deoxyguanosin-N2-yl)-2-amino-3- methylimidazo[4,5-f]quinoline (dG-N2-IQ). The ratio of the dG-C8IQ to the dG-N2-IQ adducts was approximately 3:1 and did not significantly change in cultures treated with different concentrations of the mutagen. Approximately 50% of the adducts were removed 9 h after treatment with IQ and <10% of these adducts remained after 24 h. There was no significant preferential repair of either adduct under the experimental conditions used. The identification of 15 mutations induced at the hprt locus (of the 44 mutants analysed) showed IQ to be efficient at inducing single base deletions in a run of guanines. Six single guanine deletions were observed in the run of six guanines in exon III and one deletion of a single guanine was observed in a non- repetitive sequence in exon VI. Other mutations observed were two GC-- >TA transversions, two GC-->CG transversions, one AT-->TA transversion and one GC-->AT transition. In addition, two multiple mutations were found. The majority of the identified mutations (12/15) occurred at GC base pairs and suggests either the dG-C8-IQ or the dG-N2-IQ adduct to be the pre-mutagenic lesion.      

Effect of enzyme inducers on the metabolism of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) in the rat

The effect of enzyme inducers 3-methylcholanthrene (3-MC) and Aroclor 1254 (A-1254) on the metabolic fate of the dietary mutagen and carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) in male F344 rats was studied in relation to single dose corn oil and untreated controls. The latter two groups were similar as regards metabolism of IQ. However, the ratio of total metabolites excreted in urine compared with those in feces was higher in A-1254 pretreated rats. In fact, this distinct excretory pattern stemmed from a lower level of IQ-N-sulfamate, and a considerably higher level of 5-OH-IQ sulfate ester, a major metabolite in urine of A-1254-injected rats. Interestingly, 5-OH-IQ glucuronide urinary levels were unaffected by the treatment. Thus, the direct 5-hydroxylation of IQ appears to be considerably increased by 3-MC and more so by A-1254, and under those conditions the resulting 5-OH-IQ is preferentially converted to the sulfate ester, in turn readily excreted in urine.     

Detection of 2-amino-3-methylimidazo[4,5-f]quinoline in cigarette smoke condensate

2-Amino-3-methylimidazo[4,5-f]quinoline (IQ), which is mutagenic and carcinogenic, was found to be present in cigarette smoke condensate by liquid chromatography with an electrochemical detector and a photodiode-array detector. The amount of IQ was estimated to be 0.26 ng per cigarette.     

Formation of a nitro derivative of 2-amino-3,4-dimethylimidazo[4,5-f]quinoline by photo-irradiation

A direct-acting mutagen to Salmonella typhimurium TA98 was found to be formed by exposing 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) in acetone to sunlight for 60 min. The direct-acting mutagen in the irradiated sample was purified by HPLC and identified as 3,4-dimethyl-2-nitroimidazo-[4,5-f]quinoline (NO2-MeIQ). The yield of NO2-MeIQ from MeIQ was estimated to be 0.3%.     

IQ (2-amino-3-methylimidazo[4,5-f]quinoline)-induced aberrant crypt foci and colorectal tumour development in rats fed two different carbohydrate diets.

In most aberrant crypt foci (ACF) and colorectal tumour studies, chemical carcinogens not normally found in food have been used as initiators. In the present study the food-related compound, IQ (2-amino-3-methylimidazo[4,5-f]quinoline), has been used. A diet high in refined carbohydrates has been associated with enhanced development of ACF and colorectal cancer in humans. The present study was designed as an integrated part of our earlier published ACF study and follows the animals until tumour development. The aim of the study was to investigate (1) the effect of a refined carbohydrate-rich diet on the development of IQ-induced ACF over time and (2) possible correlation between early and late ACF and/or colorectal tumour development. The study showed that a feeding regimen with continuous doses of 0.03% IQ in the diet for 14 weeks, followed by 32 weeks without IQ was able to induce tumours in the rat colon, liver, skin and Zymbal gland. The data demonstrate that a sucrose-rich diet enhance ACF development. A correlation between the outcome of early and late ACF was seen. However, as the tumour incidence of this study was very low it was not possible to obtain a meaningful correlation between ACF development and colorectal tumour incidence.     

Parp-1 deficiency does not enhance liver carcinogenesis induced by 2-amino-3-methylimidazo[4,5-f]quinoline in mice

The susceptibility of poly(ADP-ribose) polymerase-1 (Parp-1) knockout mice to 2-amino-3-methylimidazo[4,5-f]quinoline (IQ)-induced liver carcinogenesis was analyzed. Twelve-week-old male Parp-1(+/+), Parp-1(+/-) and Parp-1(-/-) mice of the C57BL/6 congenic strain were fed a diet containing IQ at a concentration of 300 ppm or a control diet for 60 weeks. Hepatocellular carcinomas were observed only in 1/19, 2/18 and 1/17 of the Parp-1(-/-), Parp-1(+/-) and Parp-1(+/+) mice, respectively. Parp-1 deficiency did not affect the susceptibility of mice to carcinogenicity of IQ, which produces bulky DNA adducts that are repaired mainly through the nucleotide excision repair pathway. This result is in sharp contrast to the increased susceptibility of Parp-1(-/-) mice to carcinogenesis induced by alkylating agents that produce DNA damage repaired mainly through base excision repair and DNA strand break repair pathways.     

Genotoxicity of the food mutagen 2-amino-3-methylimidazo-[4,5-f]quinoline (IQ) and analogs

The food mutagen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ)is an analogue of quinoline, a hepatocarcinogen. 2-Aminofluorene,benzldine and 3,2'-dimethyl-4-aminobiphenyl (DMAB) are potentinducers of unscheduled DNA repair in primary culture rat liverhepatocytes, as was IQ (151 grains/ nucleus at 1 x 10^–6M). Quinoline, on the other hand, is only weakly positive inthis assay (15 grains/nucleus at 1 x 10^–3 M). IQ, quinolineand DMAB were applied topically to shaved skin of Sencar micewith promotion by 12-O-tetradecanoylphorbol 13-acetate (TPA)for 20 weeks, when 14 of 20 mice in the quinoline group had25 tumors, but only one of 30 animals in the IQ group and fiveof 30 in the DMAB group were tumor-bearing. Analogs of IQ synthesizedby substitution at the 2- or 3-position with amino or methylgroups were assayed with the Ames Salmonella typhimurium testerstrains TA98 and TA100. Mutagenicity for TA98 is reduced inthe absence of the 3-methyl group and is completely abolishedwith removal of the 2-amino moiety. None of these analogs arestrong mutagens for TA100. Exocyclic N-oxidation is a likelyobligatory step in the activation of IQ to a mutagen.     

Tissue-specific mutational spectra of 2-amino-3,4-dimethylimidazo[4,5-f]quinoline in the liver and bone marrow of lacl transgenic mice

2-Ammo-3,4-dimethylimldazo[4,5-f]quinoline (MeIQ) is a food-borneheterocyclic amine, so clarification of its mutational spectrumis important for evaluation of its carcinogenic risk to humans.The mutational spectrum of MeIQ was investigated in the liverand bone marrow of transgenic mice carrying the lacI gene. ByPCR-single-strand conformation polymorphism analysis and sequencingof the lacI gene, 81 and 61 mutations were identified in 80and 59 mutants obtained from the liver and bone marrow respectivelyof three transgenic mice given food containing 300 p.p.m. MeIQ.In the liver, G     

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.