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.     

Tissue distribution of DNA adducts in CDF1 mice fed 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ)

Male and female CDF1 mice were administered a single oral dose of 3 mumol of the food mutagens 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) or 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) and killed 24 h later. DNA was isolated from the livers, lungs, kidneys, colon and forestomach and analysed by 32P-postlabelling for the presence of IQ and MeIQ adducts. Several adduct-enrichment procedures were investigated, including ATP-deficient labelling conditions, butanol extraction and nuclease P1 digestion, and only the ATP-deficient procedure was found to produce the same adduct pattern on polyethyleneimine--cellulose TLC as the standard procedure. Up to nine adduct spots were detected in liver DNA from IQ-treated mice, two of which were not detected in other tissues. The levels of binding in both male and female mice were in the order liver greater than kidney greater than colon greater than forestomach greater than lung. Analysis of DNA from MeIQ-treated mice revealed the presence of up to seven adducts, one of which was detected in liver but not in other tissues. The relative order of DNA binding was kidney greater than liver greater than or equal to colon greater than forestomach greater than lung. As dietary feeding of IQ induces liver, lung and forestomach tumours, and MeIQ induces liver and forestomach tumours in this mouse strain, these binding levels do not correlate with the susceptibility of the organs to carcinogenesis induced by these compounds; the results may indicate the importance of additional factors in determining organ specificity of carcinogenicity.     

Effects of sucrose and cornstarch on 2-amino-3-methylimidazo[4,5-f]quinoline (IQ)-induced colon and liver carcinogenesis in F344 rats

The purpose of the present study was to compare the effect of sucrose and cornstarch on colon and liver carcinogenesis induced by 0.02% of the food-borne carcinogen 2-amino-3-methylimidazo [4,5-f]quinoline (IQ) in the feed. Male F344 rats were allocated to four groups. Two groups were fed diets high in either cornstarch (68%) or sucrose (34% sucrose/34% cornstarch) and were initiated with IQ. The remaining two groups received the same two diets but did not receive any IQ. In both liver and colon, administration of IQ resulted in a higher level of DNA adducts. In animals not dosed with IQ, sucrose increased the adduct level in both organs but to a lower level than IQ. However, simultaneous administration of IQ and sucrose did not further increase the adduct level. Both IQ and sucrose increased the expression of the DNA-repair enzyme ERCC1 in the liver. In the colon, the number of large and medium aberrant crypt foci (ACF) of the group fed IQ and cornstarch was significantly higher than that in the other groups. There was no statistically significant difference in any tumour incidence in IQ dosed-animals fed either cornstarch or sucrose. In conclusion, no difference in effect on liver carcinogenesis was seen between sucrose and cornstarch-based diets, however, the number of tumours per animal tended to be slightly higher in the rats fed cornstarch (P = 0.08). Cornstarch enhanced ACF development induced by IQ when compared to sucrose whereas due to a low intestinal tumour incidence no correlation to diet and tumour incidence could be established.     

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.     

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.     

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.     

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.      

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.     

Mutagenic activation of 2-amino-3-methylimidazo[4,5-f]quinoline by complementary DNA-expressed human liver P-450

Eight forms of human liver microsomal P-450 were individually expressed in human hepatoma Hep G2 cells with a vaccinia virus cDNA expression system. Using the Ames test, each expressed P-450 was examined for its ability to activate to mutagenic products the compounds, 2-amino-3-methylimidazo[4,5-f]quinoline, 2-amino-3,4-dimethylimidazo[4,5-f]quinoline, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, and 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline, respectively. Three forms of human P-450 significantly activated 2-amino-3-methylimidazo[4,5-f]quinoline when the latter was at high substrate concentrations, but only a single form, P-450IA2, showed very high activation of all promutagens at lower substrate concentrations. Human IA2 had extraordinarily high affinity towards four promutagens tested and is likely the predominant P-450 enzyme responsible for their mutagenic activation in human liver.     

Inhibition of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ)-DNA binding in rats given chlorophyllin: dose-response and time-course studies in the liver and colon

Chlorophyllin (CHL), a water-soluble salt of chlorophyll, hasbeen shown to inhibit 2-amino-3-methylimidazo[4,5-f]quinoline(IO)-DNA binding in vitro by a mechanism that involves molecularcomplex formation with the carcinogen. Based on this mechanism,rats weighing     

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.     

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.     

Mutational analysis of the liver, colon and kidney of Big Blue rats treated with 2-amino-3-methylimidazo[4,5-f]quinoline

The heterocyclic aromatic amine (HAA) 2-amino-3-methylimidazo[4, 5-f]quinoline (IQ) induces intestinal tumours and hepatocellular carcinomas in rats, but no tumourigenic effects have been identified in the kidney. The tissue-specific mutagenicity of IQ was studied at the lacI locus in the liver, colon and kidney of Big Blue transgenic rats. At the highest dosing regime of IQ (20 mg/kg for 5 consecutive days) the mean mutant frequencies were significantly increased above background (P < 0.05) and were highest in the liver (12.9 +/- 6.2 x 10(-5)), followed by colon (7.4 +/- 1.4 x 10(-5)) and kidney (5.9 +/- 0.8 x 10(-5)). The mutational spectra from the livers of IQ-treated rats was statistically significantly different to that from the livers of control rats (P < 0.01). The lacI mutation spectra of the liver, colon and kidney from IQ-treated rats were similar. These were characterized by an increase in GC transversions in the liver and colon and an increase in the proportion of 1 bp G:C deletions in the liver and kidney. A single G deletion in the sequence 5'-CGGGA-3', characteristic of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine exposure, was detected in the liver and colon. A 2 bp GC deletion was identified at an identical position in the liver, colon and kidney. The colon was the only organ to contain two larger deletions of 13 and 33 bp. A preference was observed for base substitution mutations at guanine in the sequence 5'-CGC/T-3' and for 1 bp deletions at the guanine doublet in the sequence 5'-CGGA-3', especially in the liver and colon. Using the lacI gene as marker in the Big Blue rat model, the mutations identified in the IQ spectra have similarities to those identified for other HAAs studied in the same experimental system, but not to mutations identified in IQ-induced tumours.     

Activated oncogenes in a rat hepatocellular carcinoma induced by 2-amino-3-methylimidazo[4,5-f]quinoline

The presence of activated oncogenes in several rat hepatocellular carcinomas induced by 2-amino-3-methylimidazo[4,5-f]-quinoline (IQ) was examined by NIH 3T3 cell transfection assay and Southern blot analysis. In one tumor, IQ4, rat H-ras-1 and another oncogene that did not belong to the ras family were found to be activated.     

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.     

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.     

Multipotential carcinogenicity of the fried food mutagen 2-amino-3-methylimidazo[4,5-f]quinoline in rats

The carcinogenicity of the mutagenic compound 2-amino-3-methylimidazo[4,5-f]-quinoline (IQ), identified in fried and broiled foods, was investigated in female Sprague-Dawley rats. Administration by gavage of IQ (0.4 mmol/kg body weight) on a weekly regimen for 31 weeks starting with 6-week-old animals induced a high yield of multiple mammary gland carcinomas in a limited bioassay lasting a total of 52 weeks. In a positive control group receiving 4-aminobiphenyl (4-AB), the expected carcinomas in the mammary gland developed. IQ, but not 4-AB, also yielded neoplasms in the ear duct and to a lesser degree, in the liver, pancreas, and urinary bladder. In addition, with IQ, a high incidence of preneoplastic lesions in the pancreas was observed. Vehicle and untreated controls did not have any lesions. These findings demonstrate that IQ is a powerful multipotent carcinogen in the rat.     

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.     

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.     

Inhibition of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ)-DNA binding by chlorophyllin: studies of enzyme inhibition and molecular complex formation.

Chlorophyllin (CHL), a copper/sodium salt of chlorophyll used in the treatment of geriatric patients, is an anti-mutagen that has been demonstrated to inhibit carcinogen--DNA binding in vivo. To study the mechanism of inhibition, the microsomal metabolism of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and the kinetics of IQ--DNA binding were investigated in the presence and absence of CHL. In time-course studies, CHL produced greater than 80% inhibition of IQ--DNA binding and blocked the metabolism of IQ, such that 80% of the initial dose of carcinogen was recovered unmetabolized from the incubations after 1 h. Kinetic constants were determined for the in vitro DNA binding reaction, with the reaction rate measured as 'pmol IQ bound/mg DNA/min/mg microsomal protein'. Without altering V(max), the Km of the IQ--DNA binding reaction was increased by CHL, and the replot of Km/V(max) versus CHL concentration yielded a straight line with an inhibitor constant of 58.3 microM CHL. Spectrophotometric studies provided evidence in vitro for the formation of a non-covalent complex between CHL and IQ. The CHL--IQ complex had a stoichiometric ratio of 2:1 (mole ratio method) and an apparent dissociation constant from the Benesi-Hilderbrand plot of 1.41 x 10(-4)M at pH 7.4. These results are discussed in the context of a CHL inhibitory mechanism involving enzyme inhibition and molecular complex formation.