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.     

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.     

Effect of dietary fibre on the mutagenicity and distribution of 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ)

Female BALB/c mice were fed either a fibre-free diet or one supplemented with 30% wheat-bran for 5 weeks. The ability of these mice to convert MeIQ to a bacteria mutagen in vivo was determined using intrasanguinous host-mediated bacterial mutation assays. Less mutagenic activity was detected in the livers of mice fed the bran-supplemented diet compared with those fed the fibre-free diet. Subsequent experiments demonstrated that the effect of brain was not due to modifications in hepatic metabolism, but to changes in uptake of MeIQ from the gastrointestinal tract.     

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.     

Formation of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline in a model system by heating creatinine, glycine and glucose

A mixture of creatinine, glucose and glycine was heated in diethylene glycol containing 14% water for 2 h at 128 degrees C, and the mutagens formed were purified by XAD-2 column chromatography, acid-base partition, Sephadex LH-20 column chromatography, 'blue cotton' treatment and HPLC. Two mutagenic substances were isolated by HPLC. The major mutagen was identified by its UV absorption and mass and NMR spectra as 2-amino-3,8- dimethylimidazo [4,5-f]quinoxaline, which was originally isolated from fried beef. This finding supported the idea that creatinine, amino acids and sugars present in meat are precursors in the formation of the mutagenic imidazoquinoxaline derivative.     

Specific induction of hepatic cytochrome p4501a-2 in C57BL/6 and DBA/2 mice treated with 2-amino-3-methylimidazo[4,5-f]quinoline (IQ)

The food mutagen/carcinogen amino-3-methylimidazo[4,5-f]quinoline (IQ) is activated by cytochrome p4501a-2 via N-hydrox-ylation; various P450s may contribute to detoxification via ring hydroxylation. Alterations in P450 levels by IQ treatment might therefore influence its toxicity. To examine the role of Ah locus genotype on the biochemical effects of IQ, C57BL/6 (Ah^bAh^b; p450Ia-½ inducible) and DBA/2 (Ah_dAh_d, noninducible) mice of both sexes were given IQ at varying doses, with different vehicles and routes of administration. Livers taken after 24 hours were assessed for total cytochrome p450 and activities of ethoxyresorufin-O-deethylase (EROD, a p4501a-l activity, inducible in Ah^b mice), meth-oxyresorufin-O-demethylase (MROD, a p4501a-2 activity), and benzyloxyresorufin-O-dealkylase (BzROD, an activity of p4502b). There was little effect on total cytochrome p450, but all three enzyme activities were often induced, a maximum of 2.5-fold, in both sexes and in DBA/2 as well as C57BL/6 mice. However, Western immunoblot analysis with monoclonal antibodies demonstrated an increase only in p4501a-2 protein. p4501a-l remained undetectable. A monoclonal antibody to p4502-b recognized one protein band in liver mi-crosomes from males and two bands in female mice of both strains. Amounts of these proteins were not altered by IQ treatment. Thus, IQ specifically, if moderately, induces its activating enzyme, p4501a-2, in a process that was not clearly related to Ah responsiveness.     

Phytoalexin resveratrol attenuates the mutagenicity of the heterocyclic amines 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline

Resveratrol is a phytoalexin, that belongs to a family of naturally occurring stilbenes. It has been reported that resveratrol can inhibit chemical carcinogenesis in experimental animals and although the mechanisms involved are unknown, an anti-mutagen mechanism has been proposed. We have explored this hypothesis using mutagenicity assays based on bacterial (Salmonella typhimurium) and eukaryotic cells (Chinese hamster V79 cells). We found resveratrol to be potent in both systems, blocking the mutagenicity of the food-derived heterocyclic amines (HA) 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) at micromolar concentrations. Furthermore, in cells capable of activating 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine to cytotoxic derivatives, resveratrol was able to attenuate cytotoxicity. Paradoxically, in cells lacking the ability to activate PhIP, resveratrol itself was toxic and co-incubation with PhIP reduced this toxicity. Our data confirm the potent anti-mutagenic activity of resveratrol and support its potential as a chemopreventative.     

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.     

Formation of mutagens, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx), in heated fish meats

Fish meats were heated under conditions close to those used for cooking and processing. The mutagenic activity of the heated fish meats was estimated toward Salmonella typhimurium TA98 with metabolic activation after extraction with boiling water and adsorption to blue cotton. The numbers of His+ revertant colonies/5 g of the meat heat-dried without charring at 220 degrees C for 15 min were about 3000 for bonito, about 1000 for tunny, less than 500 for mackerel, salmon, swordfish, sardine, horse mackerel and cod, and 0 for cuttlefish. The mutagens in the heat-dried bonito meat were purified by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). They were identified as 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx) by comparison with the authentic specimen with respect to Rf values in TLC, retention times in HPLC, ultraviolet absorption spectra and mass spectra. The contents of MeIQx and 4,8-DiMeIQx in the bonito meat were estimated to be 5.2 and 5.4 ng/g, respectively. The major mutagens produced in the bonito, tunny and mackerel meats heated without charring at 100 degrees C for 48 h and at 220 degrees C for 15 min were found to be MeIQx and 4,8-DiMeIQx. It is interesting to note that the bonito and sardine meats grilled with charring for 15 min contained MeIQx and 4,8-DiMeIQx but higher mutagenicity was observed in the fraction that may contain 2-amino-3-methylimidazo[4,5-f] quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) and/or 2-aminodipyrido[1,2-a: 3',2'-d]imidazole (Glu-P-2).     

Effect of common Brassica vegetables (Brussels sprouts and red cabbage) on the development of preneoplastic lesions induced by 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) in liver and colon of Fischer 344 rats

Aim of the present study was the investigation of effects of juices from commonly consumed Brassica vegetables (two cultivars of Brussels sprouts and two cultivars of red cabbage) on formation and development of preneoplastic lesions in colons (aberrant crypt foci, ACF) and livers (glutathione-S-transferase placental form, GST-P+) in male F344 rats. The foci were induced by 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), a widespread carcinogenic heterocyclic aromatic amine which is found in fried meats. Recently, we reported on pronounced protective effects in the two-organ foci model when the vegetable juices were given during the carcinogen treatment but several findings by other groups indicated that breakdown products of glucosinolates contained in Brassica vegetables cause tumour promotion in various organs of laboratory rodents. In the present study, the animals received the juices in the drinking water (5%) over a period of 20 days after treatment with IQ (100 mg/kg bw on 10 alternate days). To increase the foci yield (which facilitates the detection of modifying effects), the animals were fed with a modified (high fat, fibre free) AIN-76 diet. With exception of the sprout variety "Cyrus", all juices lowered the number of GST-P+ foci as well as the foci area in the liver, but none of these effects was statistically significant. In the colon, none of the juices had an impact on crypt multiplicity (number of crypts/focus), whereas the number of ACF was decreased; only with the sprout variety Maximus the protective effect was significant (reduction 49%). The present findings show that administration of vegetable juices to the animals after the carcinogen does not increase the number and size of IQ-induced preneoplastic lesions in liver and colon.     

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.     

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.     

Low dose genotoxicity of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) in gpt delta transgenic mice

Although humans are chronically exposed to most environmental chemicals at low doses, genotoxicity assays with rodents are usually performed at high doses with short treatment period. To investigate the dose-response of genotoxicity at lower doses, gpt delta transgenic mice were fed a diet containing 300, 30 or 3 parts per million (ppm) of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) for 12 weeks and the gpt mutations in the liver were analyzed. In addition, the mice were continuously fed a diet containing MeIQx at a dose of 300 ppm for 78 weeks to examine the effect of a long-term treatment. In the mice treated for 12 weeks, the gpt mutant frequencies (MFs) were 8.6-, 2.3- and 1.2-fold higher than the control level at the doses of 300, 30 and 3 ppm, respectively. G:C to T:A transversion was the most predominant type of mutations and the fold increases in the specific MF of G:C to T:A were 58.2, 4.4 and 1.7 above the control at the three doses, respectively. The increases in the whole gpt and specific MFs at 3 ppm were not statistically significant. In the mice treated with 300 ppm of MeIQx for 78 weeks, the gpt MF was about 20 times higher than that of the untreated mice fed a control diet for 78 weeks, which was about two times higher than that of the untreated mice at 12 weeks. These results suggest that no obvious genotoxic effects can be detectable at the dose of MeIQx at 3 ppm in the liver and a longer treatment substantially enhances the genotoxicity. Factors constituting the practical threshold dose are discussed.     

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.     

One dose of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) or 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) induces tumours in Min/+ mice by truncation mutations or LOH in the Apc gene

The C57BL/6J-Min/+ (multiple intestinal neoplasia) mouse has a heterozygous nonsense Apc(Min) (adenomatous polyposis coli) mutation, and numerous adenomas spontaneously develop in the intestine. Neonatal exposure of Min/+ mice to the food carcinogens 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) or 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) (one injection of 50mg/kg) increased the number of small intestinal tumours about three- and two-fold, respectively. The number of colonic tumours was only increased in males. We examined whether the wild-type Apc allele was affected in intestinal tumours induced by either PhIP or IQ. In spontaneously formed and in IQ-induced small intestinal and colonic tumours from these mice, the main mechanism for tumour induction was loss of wild-type Apc allele, i.e. loss of heterozygosity (LOH). In contrast to the IQ-induced (84% LOH) and spontaneously (88% LOH) formed tumours, only 55% of the PhIP-induced small intestinal tumours from males showed LOH. Tumours that apparently had retained the wild-type Apc allele were further analysed for the presence of truncated Apc proteins by the in vitro synthesised protein (IVSP) assay. Truncated Apc proteins, indicating truncation mutations in exon 15 of the Apc gene, were detected in two of the 12 PhIP-induced tumours in segment 2 (codons 686-1217), and two of five IQ-induced tumours, one in segment 2 and the other in segment 3 (codons 1099-1693). Three of these four mutations, all in segment 2 of the Apc gene, were confirmed by sequencing. The PhIP-induced mutations were detected at codon 1125 (C deletion) and 1130 (G-T transversion), and the IQ-induced mutation was at codon 956 (C-T transition). Importantly, no truncated proteins were detected in tumours from unexposed mice with apparently retained wild-type Apc allele. These results show that one injection of either PhIP or IQ induces intestinal tumours in the Min/+ mice by inactivation of the wild-type Apc allele either by causing LOH or truncation mutations.     

Anaerobic metabolism of 2-amino-3-methyl-3H-imidazo[4,5-f]quinoline (IQ) by human fecal flora

Incubation of the heterocyclic cooked food mutagen IQ with mixed human fecal microflora under anaerobic conditions yielded 2-amino-3,6-dihydro-3-methyl-7H-imidazo[4,5-f]quinoline-7-one (2) as the major detectable metabolite.