Hemin potentiates nitric oxide-mediated nitrosation of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) to 2-nitrosoamino-3-methylimidazo[4,5-f]quinoline

Heme has been reported to be an important contributor to endogenous N-nitrosation within the colon and to the enhanced incidence of colon cancer observed with increased intake of red meat. This study uses the heterocyclic amine 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) as a target to evaluate hemin potentiation of nitric oxide (NO)-mediated nitrosation. Formation of 14C-2-nitrosoamino-3-methylimidazo[4,5-f]quinoline (N-NO-IQ) was monitored by HPLC following incubation of 10 microM IQ with the NO donor spermine NONOate (1.2 microM NO/min) at pH 7.4 in the presence or absence of hemin. N-NO-IQ formation due to autoxidation of NO was at the limit of detection (0.1 microM) and increased 22-fold in the presence of 10 microM hemin and an in situ system for generating H2O2 (glucose oxidase/glucose). A linear increase in N-NO-IQ formation was observed from 1 to 10 microM hemin. Significant nitrosamine formation occurred at fluxes of NO and H2O2 as low as 0.024 and 0.25 microM/min, respectively. Potentiation by hemin was not affected by a 400-fold excess flux of H2O2 over NO or a 4.8-fold excess flux of NO over H2O2. Reactive nitrogen species produced by hemin potentiation had a 46-fold greater affinity for IQ than those produced by autoxidation. Azide inhibited autoxidation, suggesting involvement of the nitrosonium ion, NO+. Hemin potentiation was inhibited by NADH, but not azide, suggesting oxidative nitrosylation with NO2* or a NO2*-like species. IQ and 2,3-diaminonaphthylene were much better targets for nitrosation than the secondary amine morpholine. Apc(min) mice with dextran sulfate sodium-induced colitis demonstrated increased levels of urinary nitrite and nitrate consistent with increased expression of iNOS and NO synthesis. As reported previously, identical conditions increased fecal N-nitroso compounds. Thus, hemin potentiation of NO-mediated nitrosation of heterocyclic amines provides a testable mechanism by which red meat consumption can generate N-nitroso compounds and initiate colon cancer under inflammatory conditions, such as colitis.     

Metabolism of 2-amino-3-methylimidazo[4,5-f]quinoline in the male rat

The metabolism of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) was studied in the male rat using the radiochemical labels 14C and 3H at positions 2 and 5 of the molecule, respectively. Adult male Fischer 344 rats were administered [2-14C]IQ or [5-3H]IQ by oral gavage at dose levels of 20 or 40 mg/kg body weight. Rats were also given [2-14C]IQ in the diet at a dose level of 300 ppm for 2 days and after administration of unlabelled IQ (300 ppm) in the diet for approximately 6.5 wk for an additional 2 days. In the initial 48 hr following oral administration of 20 or 40 mg [2-14C]IQ/kg body weight, about 40-50% radioactivity was recovered in the urine, and about 30-38% radioactivity was recovered in the faeces. In the initial 72 hr following consumption of [2-14C]IQ (300 ppm) in the diet about 26% radioactivity was recovered in the urine and about 61% radioactivity was recovered in the faeces. Following cannulation of the bile ducts, rats administered a single dose of [2-14C]IQ (40 mg/kg body weight) by oral gavage excreted about 15% of the administered dose in the bile over a period of 2 days. Urine from rats given [2-14C]IQ contained three main polar metabolites that included a glucuronide, a sulphate ester and IQ sulphamate, and a number of less polar metabolites that included IQ, 2-acetylamino-3-methylimidazo[4,5-f]quinoline, 2-aminoimidazo[4,5-f]quinoline and 2-amino-3,6-dihydro-3-methyl-7H-imidazo[4,5-f]quinoline-7-one (7-OH-IQ). Administration of [2-14C]IQ by oral gavage or in the diet gave the same metabolites, but in different amounts. In the faeces of rats given [2-14C] by oral gavage, IQ-sulphamate was the major metabolite in the polar fraction. Non-polar metabolites similar to those found in the urine were also present, but in different amounts. A major, non-polar faecal metabolite, 7-OH-IQ was probably formed as a result of the activity of the intestinal bacterial flora. In rats given a single gavage dose of [2-14C]IQ, excretion of metabolites was higher in the urine and lower in the faeces compared with that in animals fed [2-14C]IQ in the diet. One polar metabolite present in the urine, IQ-sulphamate (39%), was found at considerably higher levels in rats dosed orally with IQ compared with those fed IQ (less than 6%). Thus, IQ is extensively metabolized to give a number of polar and non-polar metabolites, the amounts of which depend, in part, on the mode of dosing.     

Metabolism of the food mutagen 2-amino-3-methylimidazo[4,5-f]quinoline in nonhuman primates undergoing carcinogen bioassay.

2-Amino-3-methylimidazo[4.5-f]quinoline (IQ) is a potent bacterial mutagen and rodent carcinogen which also produces hepatocellular carcinoma in monkeys. The metabolism and disposition of this procarcinogen were investigated in monkeys undergoing carcinogen bioassay and in monkeys given an acute dose of IQ. Analysis of urine, feces, and bile revealed that IQ was extensively metabolized. A number of metabolites in urine were purified by high-performance liquid chromatography and characterized by 1H NMR and mass spectroscopy. Metabolites resulted from cytochrome P450-mediated ring oxidation at the C-5 position or N-demethylation. These metabolites could be further transformed by conjugation to sulfate or beta-glucuronic acid. Glucuronidation and sulfamate formation at the exocyclic amine group were other major routes of metabolism. Enteric bacteria also contributed to IQ biotransformation by forming the 7-oxo derivatives of IQ and N-demethyl-IQ. The metastable N2-glucuronide conjugate of the carcinogenic metabolite, 2-(hydroxyamino)-3-methylimidazo[4,5-f]quinoline, was found in urine. This indicates that metabolic activation through cytochrome P450-mediated N-oxidation occurs in vivo and that glucuronidation is a means of transport of the carcinogenic metabolite to extrahepatic tissues.     

Prostaglandin-H synthase mediated metabolism and mutagenic activation of 2-amino-3-methylimidazo [4,5-f] quinoline (IQ)

 Prostaglandin-H synthase (PHS), a mammalian peroxidase of interest for the extrahepatic formation of reactive intermediates of carcinogens, catalyzes in vitro the metabolic activation of the mutagen and carcinogen 2-amino-3-methylimidazo-[4,5-f ]quinoline (IQ). Incubation of ¹⁴C-labeled IQ with ram seminal vesicle microsomes (RSVM), a rich source of PHS, resulted in protein binding and generated products mutagenic in S. typhimurium YG1024. The mutagenic activity produced in IQ/PHS incubations was stable and extractable with ethyl acetate. Upon fractionation of such extracts by HPLC and subsequent analysis, two metabolites were identified as 2,2′-azo-bis-3-methylimidazo[4,5-f ]quinoline (azo-IQ) and 3-methyl-2-nitro-imidazo[4,5-f ]quinoline (nitro-IQ) confirmed by comparison of HPLC retention times, UV/VIS-, ¹H-NMR-spectroscopy, and mass spectrometry of synthesized standards. Azo-IQ was obtained by chemical oxidation of IQ with metasodium periodate. It was the major metabolite in PHS incubations, but has not been detected in monooxygenase incubations. Azo-IQ, without metabolic activation, was much less mutagenic in S. typhimurium YG1024 (308 rev/nmol) than nitro-IQ and 3-methyl-2-nitroso-imidazo[4,5-f ]quinoline (nitroso-IQ), two other S9-independent mutagens which have been synthesized by chemical oxidation of IQ with sodium nitrite. Nitro-IQ was formed only in trace amounts but due to its potent mutagenicity in S. typhimurium YG1024 (2×10⁶ rev/nmol) it accounted for most of the mutagenic activity of the incubations. These data show that PHS-mediated in vitro metabolism of IQ results in its metabolic activation; thus PHS may contribute to the genotoxicity of IQ in extrahepatic tissues. Received: 25 July 1994/Accepted: 30 August 1994     

The food mutagen 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline: a conformational analysis of its major DNA adduct and comparison with the 2-amino-3-methylimidazo[4,5-f]quinoline adduct

The heterocyclic amine 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) is one of a group of heterocyclic amine carcinogens that exists in cooked meat and fish. It causes mutations in bacterial and mammalian assays and induces tumors in mammals. MeIQx is converted within cells to a reactive derivative which forms a major covalent adduct at carbon-8 of guanine in DNA. This adduct may alter the DNA conformation at critical stages of the replicative process, and cause mutations which initiate the carcinogenic process. Atomic resolution structures of the MeIQx-damaged DNA are not yet available experimentally. We have carried out an extensive molecular mechanics/energy minimization search to locate feasible structures for the major MeIQx adduct in DNA, using the sequence d(5'-C1-G2-C3-G4[IQ]-C5-G6-C7-3').d(5'-G8-C9-G10-C11-G12-C13-G14-3') with MeIQx modification at G4. We have created 1152 starting conformations which uniformly sampled each of the three flexible torsion angles that govern the MeIQx-DNA orientation at 15 degrees intervals, and minimized their energy. A mixture of conformations was generated, which were separated into families according to the position of the ring system of the carcinogenic amine: major groove, minor groove, and base-displaced-intercalated. While a generally similar mixture had been generated previously for the related carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) [Wu, X., et al. (1999) Chem. Res. Toxicol. 12, 895-905], differences were found which could be rationalized in terms of the additional methyl group in the MeIQx.     

Conformational analysis of the major DNA adduct derived from the food mutagen 2-amino-3-methylimidazo[4,5-f]quinoline.

The heterocyclic amine 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) is one of a number of carcinogens found in barbecued meat and fish. It is mutagenic in bacterial and mammalian assays and induces tumors in mammals. IQ is biochemically activated to a derivative which reacts with DNA to form a major covalent adduct at carbon 8 of guanine. This adduct may deform the DNA and consequently cause a mutation, which may be responsible for initiating IQ's carcinogenicity. Atomic resolution structures of the IQ-damaged DNA are not yet available experimentally. We have carried out an extensive molecular mechanics energy minimization search to locate feasible structures for the major IQ-DNA adduct in the representative sequence d(5'-G1-G2-C3-G4-C5-C6-A7-3'). d(5'-T8-G9-G10-C11-G12-C13-C14-3') with IQ modification at G4; this contains the GGCGCC mutational hotspot sequence known as NarI. The molecular mechanics program AMBER 5.0 with the force field of Cornell et al. [(1995) J. Am. Chem. Soc. 117, 5179-5197] was employed, including explicit Na(+) counterions and an implicit treatment for solvation. However, key parameters, the partial charges, bond lengths, bond angles, and dihedral parameters of the modified residue, are not available in the AMBER database. We carefully parametrized the force field, created 800 starting conformations which uniformly sampled at 18 degrees intervals each of the three flexible torsion angles that govern the IQ-DNA orientation, and minimized their energy. A conformational mix of structural types, including major groove, minor groove, and base-displaced intercalated carcinogen positions, was generated. This mixture may be related to the diversity of mutational outcomes induced by IQ.     

DNA damage of mammalian cells by the beef extract mutagen 2-amino-3-methylimidazo[4,5-f]quinoline

The beef-extract mutagen, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), was shown, by alkaline elution procedures, to induce DNA damage in radiation-induced mouse leukaemia cells. The effect, which was dose related, occurred in incubations containing S-9 mix derived from polychlorinated biphenyl-induced rat liver but not in the absence of this metabolic activation system. An increased alkaline elution of DNA was also observed following IQ addition to cultures of hepatocytes from 3,3',4,4'-tetrachloroazobenzene-induced rat liver, and the DNA damage was again dose related. IQ has thus been shown to be genotoxic to mammalian cells in the presence of an effective activation system.     

Prenatal toxicity and lack of carcinogenicity of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) following transplacental exposure.

Accumulating evidence from human and experimental animal studies indicates that consumption of heterocyclic amines (HA), derived from cooked meat and fish, may be associated with an increased incidence of cancer. Experiments were initiated to assess the role of one of these compounds, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), as a potential transplacental carcinogen, as well as to evaluate whether in utero exposure to IQ results in the induction of fetal cytochrome P4501A1 (Cyp1a1), P4501B1 (Cyp1b1), and/or glutathione S-transferase (GST). Inducible, or responsive, backcrossed fetuses resulting from a cross between congenic C57BL/6 (Ah(d)Ah(d)) nonresponsive female mice and C57BL/6 (Ah(b)Ah(b)) responsive male mice were transplacentally exposed to olive oil or 6.25, 12.5, or 25 mg/kg of IQ on day 17 of gestation. No macroscopically or microscopically visible liver, lung, or colon tumors were found in the transplacentally treated offspring by one year after birth. Ethoxyresorufin O-deethylase (EROD) and 1-chloro-2,4-dinitrobenzene assays were performed to evaluate whether transplacental exposure to IQ results in the induction of fetal Cyp1a1 and GST, respectively, in lung and liver tissues. Results showed levels of EROD and GST activity in tissues of IQ-treated mice to be very close, if not identical, to those of mice treated with olive oil. Similarly, ribonuclease protection assay data showed that the levels of Cyp1a1 and Cyp1b1 RNA in tissues of IQ-treated mice were not significantly different from those of oil-treated controls. Previous studies have shown that the developing organism expresses very low levels of Cyp1a2. Thus, in utero exposure to IQ does not lead to induction of Cyp1a1, Cyp1a2, or Cyp1b1 in the fetal compartment, thereby maintaining the low levels of these activating enzymes in the developing organism. Taken together, these data imply that, at least under the conditions employed for these experiments, IQ may not play an important role in transplacentally induced tumorigenesis.     

2-Nitrosoamino-3-methylimidazo[4,5-f]quinoline stability and reactivity

N-Nitrosamines and nitrosamides can initiate cancer. These studies evaluated the stability and reactivity of 2-nitrosoamino-3-methylimidazo[4,5-f]quinoline (N-NO-IQ) to assess its possible role in the initiation of colon cancer by 2-amino-3-methylimidazo[4,5-f]quinoline (IQ). (14)C-N-NO-IQ was incubated with different solvents and pHs in the presence and in the absence of nucleophiles and analyzed by HPLC. The products identified by electrospray ionization mass spectrometry include 2-chloro-3-methylimidazo[4,5-f]quinoline (2-Cl-IQ), 2,2'-azo-3,3'-dimethylimidazo[4,5-f]quinoline (AZO-IQ), 2-azido-IQ (2-N(3)-IQ), 3-methylimidazo[4,5-f]quinoline (deamino-IQ), and IQ. A variety of organic solvents were tested with 0.1 N HCl. 2-Cl-IQ and IQ were formed following acidification of all solvents. AZO-IQ was only formed in methanol. Deamino-IQ was the major product formed in all of the alcohols tested, except for methanol. Under acidic conditions that completely convert N-NO-IQ in 5 min (acetonitrile with 0.1 N HCl), 62% of N-NO-IQ remains after 30 min if dimethyl sulfoxide is substituted for acetonitrile. N-NO-IQ was stable in the physiologic pH range of 5.5-9.0 and did not react with nucleophiles over a 4 h period at pH 7.4 and 37 degrees C. At acidic pH (pH < or =2.0) for 30 min and 37 degrees C, N-NO-IQ becomes labile forming electrophile(s), which combine with biologically relevant nucleophiles. The reaction of N-NO-IQ at pH 2.0 followed first-order kinetics (t(1/2) = 10 +/- 2 min) and was significantly increased in 10 mM NaN(3) (t(1/2) = 2 +/- 0.1 min). 2-N(3)-IQ was the major product observed in the latter incubation. N-NO-IQ binding to DNA at pH 2.0 is 100-fold more than that at pH 7.4. At pH 2.0, greater than 90% of the binding was inhibited by 10 mM NaN(3). Thus, N-NO-IQ forms a reactive electrophile(s) at acidic pH, which binds DNA. N-NO-IQ reaction products may depend on the pH and the hydrophobic milieu of cells or tissues.     

Conformational differences of the C8-deoxyguanosine adduct of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) within the NarI recognition sequence

2-amino-3-methylimidazo[4,5-f]quinoline (IQ) is a highly mutagenic heterocyclic amine found in cooked meats. The major DNA adduct of IQ is at the C8-position of dGuo. We have previously reported the incorporation of the C8-IQ adduct into oligonucleotides, namely, the G1-position of codon 12 of the N-ras oncogene sequence (G1G2T) and the G3-position of the NarI recognition sequence (G1G2CG3CC) (Elmquist et al. (2004) J. Am. Chem. Soc. 126, 11189-11201). Ultraviolet spectroscopy and circular dichroism studies indicated that the conformation of the adduct in the two oligonucleotides was different, and they were assigned as groove-bound and base-displaced intercalated, respectively. The conformation of the latter was subsequently confirmed through NMR and restrained molecular dynamics studies (Wang et al. (2006) J. Am. Chem. Soc. 128, 10085-10095). We report here the incorporation of the C8-IQ adduct into the G1- and G2-positions of the NarI sequence. A complete analysis of the UV, CD, and NMR chemical shift data for the IQ protons are consistent with the IQ adduct adopting a minor groove-bound conformation at the G1- and G2-positions of the NarI sequence. To further correlate the spectroscopic data with the adduct conformation, the C8-aminofluorene (AF) adduct of dGuo was also incorporated into the NarI sequence; previous NMR studies demonstrated that the AF-modified oligonucleotides were in a sequence-dependent conformational exchange between major groove-bound and base-displaced intercalated conformations. The spectroscopic data for the IQ- and AF-modified oligonucleotides are compared. The sequence-dependent conformational preferences are likely to play a key role in the repair and mutagenicity of C8-arylamine adducts.     

Nitrosation and nitration of 2-amino-3-methylimidazo[4,5-f]quinoline by reactive nitrogen oxygen species

Both cooked red meat intake and chronic inflammation/infection are thought to play a role in the etiology of colon cancer. The heterocyclic amine 2-amino-3-methylimidazo[4,5-f ]quinoline (IQ) is formed during cooking of red meat and may be involved in initiation of colon cancer. Reactive nitrogen oxygen species (RNOS), components of the inflammatory response, contribute to the deleterious effects attributed to inflammation on normal tissues. This study assessed the possible chemical transformation of IQ by RNOS. RNOS were generated by various conditions to react with (14)C-IQ, and samples were evaluated by HPLC. Myeloperoxidase (MPO)-catalyzed reaction was dependent upon both H(2)O(2) and NO(2)(-). This reaction produced an azo-IQ dimer and IQ dimer along with two nitrated IQ products identified by ESI/MS. 2-Nitro-IQ was not detected. Product formation was inhibited by 2 mM cyanide. Reduction in nitrated products observed with 100 mM chloride was not altered with 0.5 mM taurine. Nitrated products were also produced by other conditions, ONOO(-) and NO(2)(-) + HOCl, which generate nitrogen dioxide radical. In contrast, conditions which generate N(2)O(3), such as diethylamine NONOate, produced only small amounts of nitrated products with the major product identified by MS and NMR as N-nitroso-IQ. MPO activation of IQ to bind DNA was dependent upon both H(2)O(2) and NO(2)(-). RNOS generated by ONOO(-) and DEA NONOate also activated IQ DNA binding. The nitrated IQ products were not activated by MPO to bind DNA. In contrast, N-nitroso-IQ was activated to bind DNA by MPO +/- NO(2)(-). HOCl activated N-nitroso-IQ, but not IQ. RAW cells produced N-nitroso-IQ and increased amounts of NO(2)(-)/NO(3)(-), when incubated with 0.1 mM IQ and stimulated with lipopolysaccharide and interferon gamma. Results demonstrate chemical transformation and activation of IQ by RNOS and activation of its N-nitroso product by biological oxidants, events which may contribute to initiation of colon cancer.     

Metabolism of 2-amino-3-methylimidazo[4,5-f]quinoline IQ and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) in suspensions of isolated rat-liver cells

Suspensions of rat-liver cells metabolized ¹⁴C-labelled 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) to metabolites that were ethyl acetate extractable, water soluble or covalently bound to macromolecules. The major ethyl acetate extractable metabolite(s) had a retention time on the high-performance liquid chromatograph (HPLC), an ultraviolet spectrum and an R_f value on the thin-layer chromatograph that corresponded to those of the N-acetylated derivative. Relatively more of this metabolite was formed from MeIQ than from IQ. In contrast, IQ was more easily converted to water-soluble metabolites than was MeIQ. The amount of covalently bound metabolite(s) found with MeIQ as test substance was larger than that found with IQ. No major increase in the ethyl acetate-extractable metabolites was obtained after incubation of the aqueous phase with β-glucuronidase or aryl sulphatase in comparison with untreated controls. HPLC analysis showed that after acid hydrolysis of the water-soluble metabolites, about 55 and 20% of the hydrolysed metabolites had retention times that were the same as those of IQ and MeIQ, respectively. The ratio between covalently bound (activated) metabolites and water-soluble (detoxified) metabolites was larger for MeIQ than for IQ. From these data, one would expect MeIQ to be more potent than IQ as a liver carcinogen in male rats.     

DNA adduct levels and intestinal lesions in congenic rapid and slow acetylator syrian hamsters admi food mutagens 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) or 2-amino-3-methylimidazo[4,5-f]quinoline (IQ)

Epidemiological studies indicate that rapid acetylators with a high intake of well-done red meat have an increased risk of colorectal cancer. Arylamine N-acetyltransferase enzymes (E.C. 2.3.1.5) activate carcinogenic heterocyclic amines found in the crust of fried meat via O-acetylation of their N-hydroxylamines to reactive intermediates that bind covalently to DNA and produce mutations. Syrian hamsters as well as humans express two N-acetyltransferase isozymes (NAT1 and NAT2) which differ in substrate specificity and genetic control. Nucleic acid substitutions in the NAT2 gene segregate individuals into rapid, intermediate and slow acetylator phenotypes. In the present paper, we examined the role of the polymorphic NAT2 acetylator genotype in carcinogenesis induced by the food mutagens 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) or 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) by comparing Syrian hamster lines congenic at the NAT2 locus. No differences were found between rapid and slow acetylator congenic hamsters in levels of intestinal PhIP-DNA adducts. In contrast to previous studies in rats, no carcinogen-related induction of the preneoplastic lesions aberrant crypt foci or tumors was found in the intestines of rapid and slow acetylator congenic Syrian hamsters administered PhIP or IQ.     

No effects of chlorophyllin on IQ (2-amino-3-methylimidazo[4,5-f]-quinoline)-genotoxicity and -DNA adduct formation in Drosophila

Previously we demonstrated that chlorophyllin suppressed the genotoxicities of many carcinogens. However, the genotoxicity of IQ (2-amino-3-methylimidazo[4,5-f]quinoline), a carcinogenic heterocyclic amine, was not suppressed in Drosophila. On the contrary, it has been reported that chrolophyllin suppressed the genotoxicity of IQ in rodents, rainbow trout and Salmonella. We demonstrated that the chlorophyllin-induced suppression of MeIQx (2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline)-genotoxicity was associated with a decrease in MeIQx-DNA adduct formation in Drosophila larval DNA. MeIQx represents another type of heterocyclic amine and is similar to IQ in structure. In this study we utilized (32)P-postlabeling to examine whether chlorophyllin reduced IQ-DNA adduct formation in Drosophila DNA in the same way as MeIQx. The results revealed that the formation of IQ-DNA adducts was unaffected by treatment with chlorophyllin. This was consistent with the absence of any inhibitory effect on genotoxicity as observed in the Drosophila repair test. These results suggest that IQ-behavior in Drosophila is not affected by chlorophyllin, indicating that the process of IQ-DNA adduct formation followed by expression of genotoxicity in Drosophila may be different from that in other organisms.     

Characterization of antimutagenic mechanism of 3-allyl-5-substituted 2-thiohydantoins against 2-amino-3-methylimidazo[4,5-f]quinoline.

3-Allyl-5-substituted 2-thiohydantoins (ATH-amino acids) derived from allyl isothiocyanate and amino acids can inhibit the mutagenicity of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) in the Salmonella assay. In this report, we studied possible mechanisms for the inhibition using rat liver S9 in assays for ethoxyresorufin O-deethylase (EROD), a marker activity for cytochrome P450 1A (CYP1A), which activates heterocyclic amines, and the Salmonella assays with the direct-acting mutagen 2-hydroxyamino-3-methylimidazo[4,5-f]quinoline (N-hydroxy-IQ). Quantitative analysis of ATH-amino acids and IQ during incubation with rat liver S9 fraction by HPLC showed that ATH-amino acids could act as S9-inhibitors, thereby inhibiting metabolic activation of IQ. Among the tested ATH-amino acids, ATH-Phe, ATH-Trp, ATH-Leu and ATH-Val showed a dose-dependent inhibition of EROD activity. ATH-Gly, ATH-Glu, and ATH-Asp behaved as blocking agents toward N-hydroxy-IQ, but exhibited no inhibition of EROD activity.     

Contrasting effects of non-starch polysaccharide and resistant starch-based diets on the disposition and excretion of the food carcinogen, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), in a rat model.

It has commonly been believed that increasing fibre in the diet should reduce the incidence of cancers, especially those of the colon and rectum. The earliest definitions of dietary fibre restricted the term to plant cell walls in which non-starch polysaccharides are key chemical components. However, new definitions encompass a wider range of materials, including starches resistant to digestion in the colon (resistant starches). Nevertheless, most definitions require that "dietary fibres" show physiological effects considered beneficial against cancer, including enhanced laxation and faecal bulking. On theoretical grounds, such properties might be expected to dilute the concentration of any carcinogen present and move it more rapidly through the colon, thereby reducing bioavailability. We have compared the properties of two dietary fibre preparations that are primarily non-starch polysaccharides with two resistant starch preparations for effects on carcinogen disposition in a rodent model. Although both preparations enhanced laxation and faecal bulking, only the non-starch polysaccharide preparation reduced carcinogen biovailability. Indeed, carcinogen biovailability was significantly enhanced by resistant starch. We suggest that there may be fundamental differences in the manner by which non-starch polysaccharides or resistant starches affect carcinogen disposition, and express concern that the events seen with the resistant starches [RS] are unlikely to be beneficial with respect to protection against cancer by exogenous carcinogens. Furthermore, the data reveal that the observation of enhanced laxation and faecal bulking does not necessarily imply a reduction in carcinogen bioavailability.     

Synthesis of oligonucleotides containing the N2-deoxyguanosine adduct of the dietary carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline

2-amino-3-methylimidazo[4,5-f]quinoline (IQ) is a highly mutagenic heterocyclic amine formed in all cooked meats. IQ has been found to be a potent inducer of frameshift mutations in bacteria and carcinogenic in laboratory animals. Upon metabolic activation, IQ forms covalent adducts at the C8- and N2-positions of deoxyguanosine with a relative ratio of up to approximately 4:1. We have previously incorporated the major dGuo-C8-IQ adduct into oligonucleotides through the corresponding phosphoramidite reagent. We report here the sequence-specific synthesis of oligonucleotides containing the minor dGuo-N2-IQ adduct. Thermal melting analysis revealed that the dGuo-N2-IQ adduct significantly destabilizes duplex DNA.     

Effects of dietary antioxidants and 2-amino-3-methylimidazo[4,5-f]- quinoline (IQ) on preneoplastic lesions and on oxidative damage, hormonal status, and detoxification capacity in the rat.

The potential beneficial or adverse affect of prolonged dietary administration of moderate to high doses (1-100 mg/kg diet) of the antioxidants, lycopene, quercetin and resveratrol or a mixture of lycopene and quercetin was investigated in male F344 rats. Selected markers for toxicity and defense mechanisms were assayed in blood, liver and colon and the impact of the antioxidant administrations on putative preneoplastic changes in liver and colon was assessed. The dietary carcinogen, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) (200 mg/kg diet) served as a pro-oxidant, genotoxicity and general toxicity control. IQ increased the levels of protein and DNA oxidation products in plasma, the area of glutathione S-transferase-placental form positive (GST-P) foci in the liver as well as the number of colonic aberrant crypt foci (ACF). All antioxidants and the antioxidant combination significantly increased the level of lymphocytic DNA damage, to an extent comparable with the effect induced by IQ. In contrast to the control group where no GST-P foci were detected, GST-P foci were detected in animals exposed to quercetin, lycopene and the combination of the two. However, the increase in the volume of GST-P foci did not reach statistical significance. The present results indicate that moderate to high doses of common dietary antioxidants can damage lymphocyte DNA and induce low levels of preneoplastic liver lesions in experimental animals. Long-term exposure to moderate to high doses of antioxidants may thus via pro-oxidative mechanisms and non-oxidative mechanisms modulate carcinogenesis.     

Bioactivation of the food mutagen 2-amino-3-methyl-imidazo[4, 5-f]quinoline (IQ) by prostaglandin-H synthase and by monooxygenases: DNA adduct analysis.

2-Amino-3-methylimidazo[4,5-f]quinoline (IQ) is a known multisite carcinogen in rodents and a potent mutagen in acetyltransferase-proficient Salmonella typhimurium strains on activation by either monooxygenases (MFO) or by prostaglandin H synthase (PHS). The primary metabolites formed by MFO- or PHS-mediated IQ-oxidation are different ([Wolz]), but secondary metabolism could ultimately result in the same DNA-binding intermediates. For further investigations, the DNA adduct pattern was now studied by means of (32)P-postlabelling analysis in vitro on PHS-activation and compared to that formed on MFO-mediated activation of IQ in hepatocytes. The C8-dG-IQ-adduct N-(deoxyguanosin-8-yl)-IQ was the major adduct in all samples, that is, in DNA isolated from S. typhimurium YG1024 treated with PHS-oxidized IQ or its nitro-derivative, from ovine seminal vesicle cells, and from hepatocytes exposed to IQ or nitro-IQ. This speaks for the formation of a common DNA-reactive species, presumably an arylnitrenium ion, generated by different pathways in these cellular model systems. The similarity of critical biochemical DNA lesions suggests that PHS can contribute to the bioactivation of IQ in vivo: this is of particular interest in extrahepatic tissues since expression of cytochrome P450 isoenzymes known to be involved in the N-oxidation of IQ is largely confined to the liver.