Base-displaced intercalation of the 2-amino-3-methylimidazo[4,5-f]quinolone N2-dG adduct in the NarI DNA recognition sequence

2-Amino-3-methylimidazo[4,5-f]quinolone (IQ), a heterocyclic amine found in cooked meats, undergoes bioactivation to a nitrenium ion, which alkylates guanines at both the C8-dG and N²-dG positions. The conformation of a site-specific N²-dG-IQ adduct in an oligodeoxynucleotide duplex containing the iterated CG repeat restriction site of the NarI endonuclease has been determined. The IQ moiety intercalates, with the IQ H4a and CH₃ protons facing the minor groove, and the IQ H7a, H8a and H9a protons facing the major groove. The adducted dG maintains the anti-conformation about the glycosyl bond. The complementary dC is extruded into the major groove. The duplex maintains its thermal stability, which is attributed to stacking between the IQ moiety and the 5′- and 3′-neighboring base pairs. This conformation is compared to that of the C8-dG-IQ adduct in the same sequence, which also formed a ‘base-displaced intercalated’ conformation. However, the C8-dG-IQ adopted the syn conformation placing the Watson−Crick edge of the modified dG into the major groove. In addition, the C8-dG-IQ adduct was oriented with the IQ CH₃ group and H4a and H5a facing the major groove. These differences may lead to differential processing during DNA repair and replication.     

Characterisation of metabolites of the food mutagens 2-amino-3-methylimidazo[4,5-f]quinoline and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline formed after incubation with isolated rat liver cells

The metabolism of 14C-labelled 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) was studied in suspensions of hepatocytes isolated from PCB-pretreated rats. The metabolites found after incubation of IQ/MeIQ (0.1 mM) with PCB-pretreated hepatocytes for 3 h were separated into three principal groups: ethyl acetate-extractable metabolites (2-4%), water soluble metabolites (94-98%) and covalently bound metabolites (0.4-0.5%). The water soluble metabolites were separated by HPLC. The metabolites were evaluated by beta-glucuronidase lability, sulphate incorporation and compared with glucuronides formed by microsomes. Mass spectroscopy and proton NMR were also run. The major metabolites formed were a N2-sulphamate, an O-sulphate in position 5 for IQ and 5 for MeIQ and an O-glucuronide in the same position. The MeIQ N2-sulphamate was much less abundant than the IQ N2-sulphamate. When compared with hepatocytes from uninduced rats, it was found that primarily the formation of ring-hydroxylated conjugates increased after PCB-pretreatment. The major ethyl acetate-extractable metabolites were the N2-acetyl derivatives and an unidentified metabolite. A small peak representing the 5-hydroxy-IQ or 5-hydroxy-MeIQ could also be seen in the HPLC chromatogram of the ethyl acetate extractable metabolites. All major water soluble products described in hepatocytes were also found in urine and bile of uninduced rats exposed to IQ/MeIQ in vivo.     

Fate of the food mutagen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) in Sprague-Dawley rats. I. Mutagens in the urine

2-Amino-3-methylimidazo[4,5-f]quinoline (IQ) is a potent bacterial mutagen formed during cooking of beef. IQ was administered intravenously to Sprague-Dawley rats at concentrations ranging from 7.5-50 mg/kg body weight. Urine was collected and analyzed for mutagenicity. Urinary mutagens were found which required activation by S9 mix, and reverted Ames test strains TA98 and TA100, but not TA1535 or TA1537. The amount of urinary mutagen(s) were related to IQ dose administered and were excreted within 48 h. Additional mutagenic activity was not released after incubation with beta-glucuronidase or aryl sulfatase. Analysis of urinary mutagens by HPLC indicates that the majority of mutagenic activity is due to unchanged IQ, but a small peak of mutagenic activity may correspond to N-acetyl or 3-N-demethylated metabolite. Since only 1% of the administered mutagenic activity is recovered in the urine, IQ may be readily detoxified in vivo.     

Activation of the food-derived mutagen 2-amino-3-methylimidazo[4, 5-f]quinoline by human-liver microsomes

The ability of human-liver microsomes to metabolically activate the food-derived heterocyclic amine, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), and the model mutagen, 2-aminofluorene (AF), has been investigated using Salmonella typhimurium TA98. In 6 subjects tested the number of revertants produced by 0.1 micrograms IQ per mg microsomal protein varied from 11, 830 +/- 320 to 42, 830 +/- 290 (mean +/- SD). With the same livers and a dose of 10 micrograms AF per plate the number of revertants varied from 15,770 +/- 1600 to 29,380 +/- 810 per mg microsomal protein. Metyrapone and alpha-naphthoflavone caused differential inhibition of the mutagenesis of both IQ and AF indicating the involvement of different forms of cytochrome P450 in the metabolic activation of these amines in human-liver microsomes. In presence of human-liver microsomes IQ produced no detectable increase in mutations at the hypoxanthine phosphoribosyl transferase locus in lymphocytes and caused no increase in micronuclei formation at realistic exposure levels.     

Formation of 2-nitro-3-methylimidazo[4,5-f]quinoline, a directly mutagenic product, by near-ultraviolet irradiation of a mixture of 2-amino-3-methylimidazo[4,5-f]quinoline and N-nitrosodimethylamine

Direct-acting mutagens to Salmonella typhimurium TA98 were found to be formed from heterocyclic amines on exposure to near-ultraviolet light in the presence of N-nitrosodialkylamines. We have isolated the mutagenic photoproduct formed from 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and N-nitrosodimethylamine, and the product was identified as 3-methyl-2-nitromidazo[4,5-f]quinoline (IQ(NO₂)). The yield of IQ(NO₂) from IQ was estimated to be 17%. Similar light-dependent activation of IQ was noted with 4 different nitrosodialkylamines other than nitrosodimethylamine. Furthermore, MeIQ and MeIQx were also activated with nitrosamine and light. These reactions represent an example of interaction between 2 different classes of mutagens.     

Antimutagenic activity of selenium-enriched green tea toward the heterocyclic amine 2-amino-3-methylimidazo[4,5-f]quinoline

Both selenium and green tea have been reported to exhibit antigenotoxic and cancer chemopreventive properties. We compared the antimutagenic activities of regular green tea and selenium-enriched green tea obtained from Hubei Province, China, toward the heterocyclic amine, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) in the Salmonella assay. Selenium-enriched green tea obtained by foliar application of selenite exhibited concentration-dependent inhibition of IQ-induced mutagenesis in the presence of rat liver S9 and was significantly more effective than regular green tea tested under the same conditions. Analytical studies revealed no major differences in the polyphenol or caffeine content between regular green tea and selenium-enriched green tea, but the latter tea contained approximately 60-fold higher concentrations of selenium compared with regular green tea. The only soluble form of selenium was identified as selenite. The antimutagenic effects of certain individual tea constituents, such as epicatechin gallate and catechin, were enhanced by the addition of selenite to the Salmonella assay. Sodium selenite, sodium selenate, seleno-dl-cysteine, seleno-l-methionine, and l-Se-methylselenocysteine were not antimutagenic toward IQ when tested alone, but augmented significantly the inhibitory potency of green tea. The results suggested an enhancing (“coantimutagenic”) effect of selenium in combination with green tea in vitro, but in vivo studies are needed to assess whether there is a synergistic effect of tea and selenium to protect against heterocyclic amine-induced mutagenesis and carcinogenesis.     

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

A rat hepatocellular carcinoma, IQ7, induced by 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) gave two transformants of NIH 3T3 cells on DNA mediated gene transfer. One of these transformants was examined further and secondary and tertiary transformants were obtained. The secondary transformant was tumorigenic in nude mice. The activated oncogene in this primary transformant was identified as rat c-raf by Southern blot analysis.     

Comparative effects in rats of intact wheat bran and two wheat bran fractions on the disposition of the mutagen 2-amino-3-methylimidazo[4,5-f]quinoline

Wheat bran protects against mutations and cancer, but contains different plant cell types that are likely to have different protective effects. We previously described the production and chemical characterisation of an aleurone-rich fraction (ARF) and a pericarp-rich fraction (PRF) from wheat grain. We compared these with whole bran (WB), fed to rats as 10% of a high fat AIN-76 diet. All bran-supplemented diets increased faecal bulk, in the order PRF>WB>ARF. PRF increased the activity of NAD(P)H:quinone acceptor oxidoreductase only in the forestomach, whereas ARF and WB enhanced levels of glutathione S-transferase in the duodenum. ARF but not PRF was digested and fermented, and also encouraged bacterial growth. Rats were gavaged with the radioactive mutagen (14)C-labelled IQ (2-amino-3-methylimidazo[4,5-f]quinoline), and effects of the brans on plasma radioactivity measured. Compared with the control diet, all bran-supplemented diets reduced the concentration of radioactivity in plasma, in the order ARF>PRF>WB. All brans increased faecal elimination of radioactivity, but only ARF and PRF enhanced urinary radioactivity. These data suggest that wheat bran may reduce mutation and cancers through direct adsorption and enhanced elimination of a dietary mutagen and/or its metabolites, and that wheat bran enriched in pericarp or aleurone cell walls may exert protective effects through different mechanisms.     

Chemical structure and mutagenic activity of aminoimidazoquinolines and aminonaphthimidazoles related to 2-amino-3-methylimidazo[4,5-f]quinoline

We have synthesized 11 heterocyclic aromatic amines with chemical structures related to that of 2-amino-3-methylimidazo [4,5-f] quinoline (IQ), a potent mutagen occurring in broiled sardines, fried beef and beef extract. The mutagenic activity of these IQ analogs was studied and compared with that of IQ using the Ames test with strain TA98 of Salmonella typhimurium in presence of a metabolic activation system (S9 mix) derived from rat liver. The mutagenic activities of the IQ analogs vary over a million-fold; structure-activity comparisons indicate major contributions of the methyl substitution in the imidazole ring and of the quinoline-N, and significant contributions of methylation of the exocyclic amino group and of the geometry of the entire ring system.     

Inhibition of DNA adduct formation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and 2-amino-3-methylimidazo[4,5-f]quinoline by dietary indole-3-carbinol in female rats.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) are two important heterocyclic amines formed in proteinaceous foods during the cooking process. Both PhIP and IQ are carcinogenic in several strains of rats. PhIP induces mammary tumors in female F344 rats, while IQ induces principally mammary and liver tumors in female Sprague-Dawley rats. Both PhIP and IQ are activated enzymatically, first by N-hydroxylation, catalyzed by CYP1A1 and CYP1A2, and subsequently by esterification (O-acetylation or sulfation), to yield DNA adducts. Such DNA adduct formation, and persistence of adducts, is related to initiation of carcinogenesis, while inhibition of this process leads to prevention of carcinogenesis. Indole-3-carbinol (I3C), a constituent of cruciferous vegetables, has chemopreventive properties in various systems; it probably acts by induction of detoxification enzymes. We have examined the effect of dietary I3C on DNA adduct formation by PhIP in female F344 rats and on that by IQ in female Sprague-Dawley rats. In experiment 1, F344 rats were maintained on AIN-76A diet containing 0.1% (w/w) I3C and then given p.o. doses (10 or 50 mg/kg) of PhIP. These doses are known to induce CYP1A1 and CYP1A2. Groups of animals (4/time point) were euthanized 1, 2, 6, and 16 days later, and their blood (for isolation of white blood cells), mammary glands, liver, stomach, small intestine, cecum, colon, heart, lungs, kidneys, and spleen were removed for DNA isolation and quantitation of PhIP-DNA adducts by 32P-postlabeling. PhIP-DNA adduct formation was inhibited (40-100%) by I3C in virtually all organs, including the mammary gland (the target organ), at both doses of PhIP, and at almost all time points. In a second experiment, Sprague-Dawley rats were fed either control AIN-76A diet or this diet containing 0.02% I3C or 0.1% I3C for a total of 42 days. IQ was added to the diets (0.01%, w/w) from day 15 to day 42, after which all rats received diet free of IQ and I3C. Groups of animals (4/time point) were killed on days 43 and 57. In addition to the organs removed in experiment 1, the pancreas, uterus, and ovaries were also removed, and IQ-DNA adducts were quantitated by 32P-postlabeling. Both dietary concentrations of I3C inhibited IQ-DNA adduct formation in most organs (except in lungs, kidneys, and pancreas) on both days 43 and 57; in liver, stomach, mammary gland, and spleen, inhibition was evident only on day 43. Inhibitions ranged from 22.6 to 86.6% with the 0.02% I3C diet and from 32.2 to 89.6% with the 0.1% I3C diet. I3C diets did not affect rate of adduct removal in either experiment. It is concluded that dietary I3C inhibits PhIP- and IQ-DNA adduct formation in both target and nontarget organs of female rats, even with high doses of PhIP when CYP1A1 and CYP1A2, the enzymes responsible for the initial activation (N-hydroxylation) of PhIP, are expected to be induced.     

Mutagenic activity of the methyl and phenyl derivatives of the food mutagen 2-amino-3-methylimidazo[4,5-f]quinoxaline (IQx) in the Ames test

The mutagenic activity of 15 different mono-, di-, tri-, and tetramethyl derivatives of the food mutagen IQx (2-amino-3-methylimidazo[4,5-f]quinoxaline), one diphenyl derivative of IQx and two phenyl derivatives of 5-MeIQx (2-amino-3,5-dimethylimidazo[4,5-f]quinoxaline) were studied in the Ames test with Salmonella typhimurium TA98 and enzymatic activation (S9). The number and positioning of the methyl groups strongly affected the mutagenic activity. The phenylated compounds showed weak mutagenic potency. It seems that both resonance stabilization of the nitrenium ion and steric effects are important in determining mutagenic potency.     

Protective effects of epigallocatechin gallate on colon preneoplastic lesions induced by 2-amino-3-methylimidazo[4,5-f ] quinoline in mice

Epigallocatechin gallate (EGCG), a key active ingredient in green tea, has multiple anticarcinogenic effects. The aim of the present study was to investigate if EGCG could prevent the formation of colon aberrant crypt foci (ACF) induced by 2-amino-3-methylimidazo[4,5-f ]quinoline (IQ) and to explore possible mechanisms for resultant effects. Sixty male BALB/cA nude, immunodeficient mice were divided into six groups including a normal unexposed control, mice induced with IQ alone, three groups treated with varying doses of EGCG post-IQ induction, and a EGCG-treated control population. Six weeks later, the mice were killed, and tissues subjected to hematoxylin-eosin (H&E) and 0.2% methylene blue staining to observe histopathological alterations of colon mucus and the formation of ACF, respectively. Protein expression of NF-E2-related factor 2 (Nrf2) was assessed via immunohistochemistry (IHC) and Western analysis, and mRNA levels of Nrf2 and uridine 5'-diphosphate-glucuronosyltransferase (UGT)1A10 were determined in colon tissues. Our results demonstrate that, compared with IQ-induced controls, the degree of atypical hyperplasia decreased and the number of total ACF and total AC also decreased significantly (P < 0.05 and P < 0.01, respectively) in mice belonging to all EGCG dosing groups. At the same time, the protein levels of Nrf2 detected by IHC and Western blotting increased (both P < 0.01 compared with IQ group), and the mRNA levels of Nrf2 and UGT1A10 increased (both P < 0.01 compared with IQ group). In conclusion, EGCG had preventive effects on preneoplastic lesions induced by IQ. Our observations suggest that this effect may be the result of activation of the Nrf2-UGT1A10 signaling pathway.     

Effects of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) on somatic mutation in a soybean test system.

The mutagenic activity of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) was assayed in heterozygous soybean plants (Y11y11), based on the appearance of mutational spots (yellow, dark green and twin) on the leaves. When soybean seeds were treated with IQ at concentrations of 0.01-0.1 microgram/ml, the frequency of mutational spots per leaf increased significantly in proportion to the concentration of IQ. At higher concentrations IQ was toxic. The mutagenicity of IQ was enhanced by pretreatment with the hepatic S9 fraction from Aroclor-induced rats. The numbers of yellow and dark green spots per leaf increased markedly by the treatment with IQ and S9-activated IQ, but the number of twin spots did not increase.     

Nuclear damage of colon epithelial cells by the food carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) is modulated by dietary lipids

Intestinal damage in C57BL/6J female mice was quantified by measuring the frequency of nuclear aberrations in colonic crypts. The animals were maintained on the following diets: standard (5% lipids, 5% cellulose); low- and high-cellulose (0-20% cellulose); high lipids (20% maize oil or 20% olive oil). All groups of animals were treated by gavage either with saline or 250 mg/kg of the dietary carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ). After 24 h their colons were removed and stained and the nuclear aberrations scored under the microscope. The administration of IQ markedly increased the number of colon aberrations in all of the treated animals. Variations in dietary fiber did not modify the colon-damaging activity of this compound. Maize oil slightly increased the colon-damaging activity, whereas significant protection was observed in the animals on a high-lipid olive-oil diet. These results show that composition of the diet may vary the genotoxic effect of this dietary carcinogen.     

Mechanisms by which resistant starches and non-starch polysaccharide sources affect the metabolism and disposition of the food carcinogen, 2-amino-3-methylimidazo[4,5-f]quinoline

Although both non-starch polysaccharides (NSP) and resistant starches (RS) are included in current definitions of dietary fibre, our previous work has suggested fundamental differences in the way in which these two classes of material affect the disposition and absorption of a dietary carcinogen. The present studies explore whether different effects on carcinogen metabolism could play a role in the contrasting patterns seen previously. Groups of female Wistar rats were pre-fed for 4 weeks one of five types of defined diet (AIN-76). The control diet contained 35% maize starch and no dietary fibre. The RS-containing diets had all the maize starch substituted with either Hi-maize or potato starch. In the NSP-containing diets, 10% of the maize starch was substituted with dietary fibre in the form of either lignified plant cell walls (wheat straw) or soluble dietary fibre (apple pectin). Pre-fed rats were gavaged with the food carcinogen, [2-14C] 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), and plasma and urinary metabolites characterized using HPLC at various time intervals after administration. After 4 h gavage, plasma from rats on both RS-containing diets contained significantly higher levels of intact IQ and lower levels of the major metabolites, IQ-5-O-glucuronide and IQ-5-sulfate, as compared with plasma from the negative control group at this time. In contrast, plasma from animals on the NSP-containing wheat straw diet (and to a lesser extent the apple pectin diet) showed significantly lower levels of intact IQ, and significantly higher levels of the two major metabolites, as compared with those from the control rats. These different metabolite profiles were also reflected in different urinary excretion profiles. Urine from rats pre-fed RS-containing diets revealed significantly slower metabolite excretion as compared with urine from rats that had been given the NSP-containing diets. Western blotting methodologies also profiled differences between the effects of these two types of dietary fibre in the expression of xenobiotic metabolizing enzymes. We conclude that changes in activity and expression of xenobiotic metabolising enzymes could play a role in the contrasting effects of these two types of dietary fibre on carcinogen uptake and disposition.     

Modulation of the mutagenic effects of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) in bacteria with rat-liver 9000 x g supernatant or monolayers of rat hepatocytes as an activation system

An in vitro protocol was designed to separate the process of metabolic activation from the mutational events. Cultured rat hepatocytes were first incubated with the food mutagens 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) or 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ). After the incubation period the medium was removed and further incubated with Salmonella typhimurium TA98. A high direct mutagenic activity of the culture medium was then measured. The half-lives of the mutagenic metabolites formed from IQ and MeIQ were in the order of 45 min. The presence of the cytochrome P450 inhibitors alpha-naphthoflavone and metyrapone during the pre-incubation period reduced the accumulation of mutagenic metabolites. No effects of ascorbate on the mutagenic effects of IQ and MeIQ were seen. (+)-Catechin, another antioxidant and free-radical scavenger, markedly enhanced the number of IQ/MeIQ-induced revertants when added to the hepatocytes. In contrast, (+)-catechin clearly decreased the number of revertants when 9000 X g supernatant from rat liver (S9) was used as an activation system. No marked effect of pentachlorophenol, an inhibitor of hepatocyte sulfation and bacterial O-acetylation, was seen using hepatocytes as an activation system, while the mutagenic activity of both IQ and MeIQ was reduced by 90% in the S9/Salmonella system. The addition of an inhibitor of glucuronidation, galactosamine, or the nucleophile glutathione caused no or only minor decreases in the genotoxic effects of the IQ compounds. With both S9 and hepatocytes as activation systems the relative mutagenic effects observed in the S. typhimurium strains TA98 and TA98 NR were in the same order of magnitude, while a large decrease was seen with TA98/1,8-DNP6. The results show that this in vitro test protocol may be useful as a tool to study mechanisms involved in the formation of mutagenic metabolites.     

1H nuclear magnetic resonance spectroscopy-based studies of the metabolism of food-borne carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline by human intestinal microbiota

2-Amino-3-methylimidazo[4,5-f]quinoline (IQ) is a mutagenic/carcinogenic compound formed from meat and fish during cooking. Following ingestion, IQ is metabolized mainly by liver xenobiotic-metabolizing enzymes, but intestinal bacteria may also contribute to its biotransformation. The aim of this study was to investigate the metabolism of IQ by the human intestinal microbiota. Following incubation of IQ (200 microM) under anoxic conditions with 100-fold dilutions of stools freshly collected from three healthy volunteers, we quantified residual IQ by high-pressure liquid chromatography (HPLC) analysis and characterized the production of IQ metabolites by in situ (1)H nuclear magnetic resonance ((1)H-NMR) spectroscopic analysis of crude incubation media. In addition, we looked for IQ-degrading bacteria by screening collection strains and by isolating new strains from the cecal contents of human-microbiota-associated rats gavaged with IQ on a regular basis. HPLC and (1)H-NMR analyses showed that the three human microbiota degraded IQ with different efficiencies (range, 50 to 91% after 72 h of incubation) and converted it into a unique derivative, namely, 7-hydroxy-IQ. We found 10 bacterial strains that were able to perform this reaction: Bacteroides thetaiotaomicron (n = 2), Clostridium clostridiiforme (n = 3), Clostridium perfringens (n = 1), and Escherichia coli (n = 4). On the whole, our results indicate that bacteria belonging to the predominant communities of the human intestine are able to produce 7-hydroxy-IQ from IQ. They also suggest interindividual differences in the ability to perform this reaction. Whether it is a metabolic activation is still a matter of debate, since 7-hydroxy-IQ has been shown to be a direct-acting mutagen in the Ames assay but not carcinogenic in laboratory rodents.     

Preferential induction of the rat hepatic P450 I proteins by the food carcinogen 2-amino-3-methyl-imidazo[4,5-f]quinoline

1. Administration of the food carcinogen, 2-amino-3-methyl-imidazo[4,5-f]quinoline (IQ) to rats gave rise to significant dose-dependent increases in the microsomal O-deethylations of ethoxycoumarin and ethoxyresorufin but had no effect on the O-dealkylation of pentoxyresorufin and the NADPH-dependent reduction of cytochrome c, and decreased the N-demethylation of dimethylnitrosamine. Microsomal cytochrome b5 and total cytochrome P-450 levels decreased following the administration of the carcinogen. 2. Hepatic microsomal preparations from IQ-treated animals were much more efficient than control in activating the premutagen 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole to mutagenic intermediates in the Ames test. 3. Immunoquantification of two of the major families of cytochrome P-450, namely P450 I and P450 II B, using ELISA techniques showed that treatment with IQ induced the apoprotein levels of the P450 I family but not of P450 II B. 4. Immunoblot analysis employing polyclonal antibodies against P450 I revealed that IQ induced both isoenzymes of this family, namely P450 I A1 and A2. 5. It is concluded that IQ is an inducer of the rat hepatic monooxygenases, selectively inducing the P450 I family as predicted by a computer-graphic analysis of its dimensions which showed that it is a large, essentially planar, molecule.     

Biochemical basis of genotoxicity of heterocyclic arylamine food mutagens: Human DNA polymerase eta selectively produces a two-base deletion in copying the N2-guanyl adduct of 2-amino-3-methylimidazo[4,5-f]quinoline but not the C8 adduct at the NarI G3 site

Heterocyclic arylamines are highly mutagenic and cause tumors in animal models. The mutagenicity is attributed to the C8- and N2-G adducts, the latter of which accumulates due to slower repair. The C8- and N 2-G adducts derived from 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) were placed at the G1 and G3 sites of the NarI sequence, in which the G3 site is an established hot spot for frameshift mutation with the model arylamine derivative 2-acetylaminofluorene but G1 is not. Human DNA polymerase (pol) eta extended primers beyond template G-IQ adducts better than did pol kappa and much better than pol iota or delta. In 1-base incorporation studies, pol eta inserted C and A, pol iota inserted T, and pol kappa inserted G. Steady-state kinetic parameters were measured for these dNTPs opposite the C8- and N 2-IQ adducts at both sites, being most favorable for pol eta. Mass spectrometry of pol eta extension products revealed a single major product in each of four cases; with the G1 and G3 C8-IQ adducts, incorporation was largely error-free. With the G3 N 2-IQ adduct, a -2 deletion occurred at the site of the adduct. With the G1 N 2-IQ adduct, the product was error-free at the site opposite the base and then stalled. Thus, the pol eta products yielded frame-shifts with the N 2 but not the C8 IQ adducts. We show a role for pol eta and the complexity of different chemical adducts of IQ, DNA position, and DNA polymerases.