Activation of the food mutagens IQ and MeIQ by hepatic S9 fractions derived from various species

The ability of hepatic S9 mixes derived from different rodent species (rat, mouse, Syrian and Chinese hamster) to activate the mutagens 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) was investigated using Salmonella typhimurium strain TA98. In general, the mutagenicity of IQ and MeIQ was greatest in the presence of S9 fractions from Swiss albino mice and least from fractions derived from Chinese hamsters. However, treatment of rats or hamsters with Aroclor 1254 had little or no effect on the activation of IQ or MeIQ to mutagens.     

Comparative genotoxic effects of IQ and MeIQ in Salmonella typhimurium and cultured mammalian cells

The food mutagens 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) were studied for their genotoxic potential using hepatocytes isolated from untreated and Aroclor 1254 (PCB) pretreated rats as an activation system. Monolayers of hepatocytes co-incubated with Salmonella typhimurium TA98 activated IQ and MeIQ to bacterial mutagens, with MeIQ being about twice as potent as IQ. The mutagenic activities of IQ and MeIQ were increased by using hepatocytes from PCB-pretreated rats. IQ and MeIQ also caused primary DNA damage in the hepatocytes as determined by increases in the rate of alkaline elution of DNA, as well as increases in DNA-repair synthesis. Furthermore, exposure of V79 cells co-cultured with PCB-pretreated hepatocytes to IQ and MeIQ showed evidence of increased sister-chromatid exchanges and a low and variable increase in the number of 6-thioguanine-resistant mutants. The genotoxic potency of IQ and MeIQ in mammalian cells was low or virtually absent compared to their extreme potency in bacteria. This could be due to a lower capacity of mammalian cells to further metabolize the so-called directly acting bacterial mutagens, formed by a cytochrome P-450 dependent N-hydroxylation, to their ultimate reactive forms.     

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.     

Effects of plant-derived flavonoids and polyphenolic acids on the activity of mutagens from cooked food

The ability of 3 plant flavonoids (morin, myricetin and quercetin) and 4 polyphenolic acids (caffeic acid, chlorogenic acid, ellagic acid and ferulic acid) to inhibit the genotoxic effects of a number of cooked-food mutagens (IQ, MeIQ, MeIQx, Trp-P-1 and Trp-P-2), was investigated in a bacterial mutation assay using Salmonella typhimurium TA98 as indicator and hepatic S9 mixes from either SWR mice or Syrian hamster as metabolic activating systems. Although the polyphenolic acids failed to have an effect, the flavonoids generally inhibited IQ, MeIQ, MeIQx and Trp-P-1 induced mutagenesis in a dose-dependent manner, irrespective of the source of S9. This was not the case with Trp-P-2 where the flavonoids were only observed to inhibit when SWR mouse S9 but not Syrian hamster S9 was used. Of the 3 compounds, myricetin and quercetin were superior to morin in their inhibitory capacity.     

Inhibition of mutagenicity of food-derived heterocyclic amines by sulforaphane--a constituent of broccoli

Sulforaphane, a constituent of broccoli was investigated for its antimutagenic potential against different classes of cooked food mutagens (heterocyclic amines). These include imidazoazaarenes such as 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP); pyridoindole derivatives such as 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2); and, dipyridoimidazole derivative such as 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1). Tests were carried out by Ames Salmonella/reversion assay using Salmonella typhimurium TA98 (frame shift mutation sensitive) and TA100 (base pair mutation sensitive) bacterial strains in the presence of Aroclor 1254-induced rat liver S9. Results of these in vitro antimutagenicity studies strongly suggest that sulforaphane is a potent inhibitor of the mutagenicity induced by imidazoazaarenes such as IQ, MeIQ and MeIQx (approximately 60% inhibition) and moderately active against pyridoindole derivatives such as Trp-P-1 and Trp-P-2 (32-48% inhibition), but ineffective against dipyridoimidazole derivative (Glu-P-1) in TA 100.     

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.     

Inhibitory effect of dibenzoylmethane on mutagenicity of food-derived heterocyclic amine mutagens

Dibenzoylmethane (DBM), a structural analogue of curcumin (a bioactive phytochemical present in a widely used spice turmeric) was screened for its inhibitory effect against seven cooked food mutagens (heterocyclic amines): 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1), in both TA98 and TA100 strains of Salmonella typhimurium using Ames Salmonella/reversion assay in the presence of Aroclor1254-induced rat liver S9 homogenate. DBM has been reported to antagonize the mutagenicity of several chemical carcinogens in vitro and has recently been shown to be even more effective than curcumin in suppressing the 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumors in rats. But there are no reports regarding its antimutagenic properties against cooked food mutagens. Results of the present investigations clearly indicate that dibenzoylmethane is a very potent antimutagenic agent, that could effectively inhibit mutagenicity induced by all the tested cooked food mutagens in both the frame shift (TA98) as well as the base pair mutation sensitive (TA100) strains of S. typhimurium. These highly potent inhibitory effects of dibenzoylmethane against heterocyclic amines observed in our preliminary investigations strongly warrant further studies of its efficacy as a cancer chemopreventive agent.     

Dietary fat modifies the in vivo mutagenicity of some food-borne carcinogens

Female BALB/c mice were fed a low fat diet (1% safflower oil, by weight) or one supplemented with 25% (by weight) of beef fat or olive oil. The abilities of these diets to modify the in vitro and in vivo hepatic conversion of the dietary carcinogens aflatoxin B1, 2-amino-3, 4-dimethylimidazo[4,5-f]quinoline (MeIQ) and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) to bacterial mutagens was evaluated. Dietary olive oil appeared to increase the metabolism of both MeIQ and Trp-P-2 to bacterial mutagens in vivo using the intrasanguineous host-mediated assay. Feeding mice either of the high-fat diets increased hepatic conversion of these two compounds to bacterial mutagens in vitro. Dietary fat had no effect on the metabolism of aflatoxin B1. Subsequent experiments suggested that the in vivo effects of dietary olive oil on MeIQ and Trp-P-2 mutagenesis were due to the induction of hepatic enzyme activities rather than to increased rates of uptake of the carcinogen from the gut-lumen.     

Mutant yields and mutational spectra of the heterocyclic amines MeIQ and PhIP at the S1 locus of human-hamster AL cells with activation by chick embryo liver (CELC) co-cultures

Cooking meat and fish at high temperature creates heterocyclic amines (HA) including 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Several HA are mutagens in the Ames' S9/Salmonella assay. While PhIP is a substantial Ames' test mutagen, it is 1000-fold less active than the extraordinarily potent MeIQ. In contrast, MeIQ is significantly less mutagenic than PhIP in several mammalian cell assays, especially in repair-deficient Chinese hamster ovary (CHO) cells. HA are suspect human carcinogens on the basis of (i) epidemiological evidence, (ii) induction of tumors in rodents and monkeys, (iii) DNA adduct formation and (iv) mutagenic capacity. In this study, MeIQ and PhIP were significant mutagens at the S1 locus of co-cultivated human/hamster hybrid AL cells following metabolic activation by beta-napthoflavone (betaNF)-induced chick embryonic liver cultures (CELC). MeIQ was more mutagenic than PhIP in the CELC+AL cell assay. The mutant response curves increase with dose and then plateau (PhIP), or decrease (MeIQ). The inflections in these response curves coincide with dose-dependent decreases in cytochrome CYP1A1 activity. Molecular analysis of S1- mutants indicates that a substantial fraction, >65%, of the mutations induced by PhIP are deletions of 4.2 to 133 (Mbp); half are larger than 21 Mbp. Mutations induced by MeIQ were smaller, most (56%) being less than 5.7 Mbp. When appropriate metabolic activation is combined with a target locus, which can detect both small and large chromosomal mutations, both MeIQ and PhIP are significant mutagens and clastogens in repair proficient mammalian cells.     

Purified prostaglandin synthase activates aromatic amines to derivatives that are mutagenic to Salmonella typhimurium.

Prostaglandin H synthase (PHS) is widely distributed in mammalian tissues and has the ability to oxidize a variety of mutagens and carcinogens. It may therefore play a key role in the metabolic activation of xenobiotics. The present study documents that highly purified PHS can be used in conjunction with 5-phenyl-4-pentenyl-1-hydroperoxide (PPHP), a relatively stable and non-mutagenic hydroperoxide substrate, for the metabolic activation of aromatic amines to mutagenic derivatives that can be detected in short-term Salmonella typhimurium mutagenesis assays. The PHS-based activation system alone was not mutagenic for these tester strains, nor were the test compounds significantly toxic for the bacteria over the concentration range tested. When used in conjunction with Salmonella strains TA98 and TA100 in a modified Ames assay, this system should prove useful for screening of a wide range of compounds for metabolic activation by this mammalian peroxidase. The potential broad utility of this purified PHS-dependent metabolic activation system was investigated by evaluating the activation of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), which are representative of a group of mutagenic and carcinogenic heterocyclic arylamines to which humans are exposed via their diet. Both IQ and MeIQ were activated by PHS to potent mutagens and confirm the utility of the PPHP/PHS system for the activation of premutagens. Whereas the extent of activation of aromatic amines by S9-based systems is significantly greater than for the PHS activation system described herein, PHS may play a significant role in target tissues in which it is present at significantly greater levels than P450 isoenzymes. Moreover, it is likely that the substrate specificity of PHS differs sufficiently from that of P450 isoenzymes so that PHS may activate some compounds that are not efficiently activated by mixed-function oxidase based systems.     

Developmental changes in hepatic activation of dietary mutagens by mice

Metabolic activation of the food mutagens 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) and aflatoxin B1 by female BALB/c mice of different ages (2-24 weeks) was investigated in vivo and in vitro using Salmonella typhimurium TA98 as the indicator organism. The in vivo activation of the three mutagens was investigated in 4- and 24-week-old mice using an intrasanguineous host-mediated assay. All three compounds showed reduced levels of activation with the older hosts. Hepatic S9 fractions from female mice of varying ages between 2 and 24 weeks were used in the in vitro mutagenicity assay. To achieve optimal activation to bacterial mutagens, 5% S9 was required for aflatoxin B1 and Trp-P-2 and 10% S9 for MeIQ; age of donor generally had little effect on the profile of these protein activation curves. Under these optimal conditions MeIQ and Trp-P-2 both exhibited, as before, age-dependent decreases in activation over a wide range of mutagen concentrations, however the in vitro activation of aflatoxin showed no consistent change with age. Spectrophotometric measurements of S9 cytochrome P-450 content showed a decrease in concentration with increasing age, but this was not sufficient to account for changes observed in hepatic mutagen activation. However, changes in the activities of certain cytochrome P-450 isoenzymes and cytosolic GSH-transferases, which in turn result in changes in the activation and detoxification capacity of the liver, would appear to explain age-dependent changes in the activity of mutagens in vivo.     

Mutagenic activation of 2-amino-3-methylimidazo[4,5-f]-quinoline (IQ) and 2-amino-3,4-dimethylimidazo[4,5-f]-quinoline (MeIQ) by subcellular fractions and cells isolated from small intestine,kidney and liver of the rat

The mutagenic activity of the pyrolysis products 2-amino-3-methylimidazo[4,5-f]-quinoline and 2-amino-3,4-dimethylimidazo[4,5-f]-quinoline in Salmonella typhimurium TA98 using rat intestinal and renal subcellular fractions as activation systems was approximately 1 and 5 revertants per nmol, respectively. This was 1,000 times less than the activity with a subcellular fraction from rat liver. The mutagenic activity of both compounds was considerably increased using intestinal, renal and hepatic preparations isolated from PCB (Aroclor 1254)-pretreated rats, compared to preparations from control animals. In addition, both compounds displayed a moderate direct-acting mutagenic activity at concentrations above 10^-5 M. Isolated cells from small intestine, kidney and liver incubated in nucleopore chambers were able to convert both compounds into products which mutated bacteria outside the chambers. The concentrations of chemicals required to yield responses of a similar magnitude were approximately 3 orders of magnitude higher in the intestinal and renal systems compared to the hepatic system. The formation of metabolites mutagenic for Salmonella typhimurium by hepatic subcellular and cellular systems was shown to be superior to the respective intestinal and renal systems.Abbreviations AHH arylhydrocarbon hydroxylase - IQ 2-amino-3-methylimidazo[4,5-f]-quinoline - MC 3-methylcholanthrene - MeIQ 2-amino-3,4-dimethylimidazo[4,5-f]-quinoline - PCB polychlorinated biphenyls (Aroclor 1254) - S9 the 9,000 g supernatant tissue fraction - TCDD 2,3,7,8-tetrachlorodibenzo-p-dioxin     

Binding activity of natto (a fermented food) and Bacillus natto isolates to mutagenic-carcinogenic heterocyclic amines.

The fermented food, whole meal Natto, viscous polymeric material from Natto, Natto bean, cooked soya bean, and 28 bacterial isolates from Natto were studied for their binding capacity to foodborne mutagenic-carcinogenic heterocyclic amines. The mutagenic heterocyclic amines used were Trp-P-1 (3-amino-1,4-dimethyl-5H-pyrido(4,3-b)indole); Trp-P-2 (3-amino-1-methyl-5H-pyrido(4,3-b)indole); Glu-P-1 (2-amino-6-methyldipyrido(1,2-a:3'2'-d)imidazole); PhIP (2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine); IQ (2-amino-3-methylimidazo(4,5-f)quinoline); MeIQ (2-amino-3,4-dimethylimidazo(4,5-f)quinoxaline); MeIQx (2-amino-3,8-dimethylimidazo(4,5-f)quinoxaline); and MeAalphaC (2-amino-3-methyl-9H-pyrido(2,3)indole). The lyophilized Natto and other fractions of Natto exhibited high binding activity towards Trp-P-1, Trp-P-2, PhIP, and MeAalphaC, while Glu-P-1, IQ, and MeIQ were not effectively bound. The binding capacity of bacterial isolates (Bacillus natto) were isolate-mutagen dependent. Heat treated lyophilized cells, cell wall, and cytoplasmic contents of the bacterial isolate with the highest binding capacity were analyzed for their ability to bind different heterocyclic amines. The results indicate the importance of the cell wall in binding to heterocyclic amines, whereas the cytoplasmic contents were less effective. Heat-treated cells were not much different from that of viable cells in their binding. The impact of different factors, such as pH, incubation time, metal ions, different concentrations of sodium chloride and alcohol, various enzymes, and acetylation of mutagens on binding of Trp-P-1 and IQ, were discussed. The significance of the present results is also discussed from the viewpoint that Natto, a fermented food, is able to scavenge dietary mutagenic heterocyclic amines through binding.     

Enhancement of the mutagenicity of IQ and MeIQ by nitrite in the Salmonella system.

The fried food mutagens IQ, MeIQ, Glu-P-1 and Trp-P-2 were treated with nitrite at pH 3.0 for 1 h at 37 degrees C. The resulting reaction mixtures were tested for mutagenicity towards Salmonella typhimurium TA97, TA98, TA100 and TA1535. Glu-P-1 and Trp-P-2 were readily converted to weak or non-mutagenic deaminated compounds, whereas IQ and MeIQ were converted to extremely strong mutagenic derivatives in both the presence and the absence of rat liver S9 mix. The mutagenicity of MeIQ in TA98 was enhanced by nitrite up to 3-fold, while that of nitrosated MeIQ was further enhanced by S9 mix up to 15-fold. The nitrosation products of MeIQ were resolved into 7 bands by TLC on silica gel plate. Bands I, III, V and VI were highly mutagenic to both TA98 and TA100. The experimental results suggest that the non-enzymatic formation of direct-acting mutagens from indirect-acting mutagens such as IQ or MeIQ might be physiologically important, especially with regard to the etiology of human gastrointestinal tract tumors.     

Studies on the mutagenicity of p-phenylenediamine in Salmonella typhimurium. Presence of PCB's in rat-liver microsomal fraction induced by Aroclor.

The mutagenicity of fresh solutions of p-phenylenediamine (PPD) and Aroclor 1254 was investigated. The histidine-requiring strains of Salmonella typhimurium were used in the absence and presence of uninduced and/or Aroclor-induced rat-liver homogenate. The presence of polychlorinated biphenyls (PCBs) was also examined by chromatographic methods in Aroclor-induced rat-liver homogenate. In the absence of metabolic activation, as well as in the presence of uninduced rat-liver homogenate, PPD was not mutagenic in the strains used. In the presence of Aroclor-induced S9 a twofold increase (or less) was observed in the number of revertant colonies over those of the controls in TA1538 and TA98. There was no increase in the number of revertant colonies over those of the controls when PPD was dissolved in NH4OH solution and the solution mixed with H2O2 before the addition of S9 mix. Aroclor 1254 was not mutagenic in TA1538 or TA98. However, the presence of PCBs in Aroclor-induced rat-liver homogenate (induced S9) was identified by gas-liquid chromatography (GLC), high-performance liquid chromatography (HPLC) and gas--liquid chromatography/mass spectrometry (GC/MS).     

Evaluation of genotoxicity of N-nitrosodibenzylamine in Chinese hamster V79 cells and in Salmonella

Health concerns have arisen due to the formation of N-nitrosodibenzylamine (NDBzA; CAS No. 5336-53-8) in pork processed in a new type of rubber netting. In view of the potent carcinogenicity of related nitrosamines (e.g. N-nitroso-n-dibutylamine and N-nitrosodiethylamine), NDBzA was evaluated for genotoxicity in vitro in both Chinese hamster V79 cells and in Salmonella. In V79 cells, concentrations up to 25 micrograms/ml were tested with and without activation by rat or hamster hepatocytes. Significant elevation of SCE frequency was seen only at 25 micrograms/ml in the presence of uninduced hamster hepatocytes. Mutation to 6-thioguanine resistance was observed at 25 micrograms/ml, in the absence of hepatocytes and in the presence of induced (Aroclor 1254) or uninduced hamster hepatocytes, but not with rat hepatocytes. With uninduced rat hepatocytes, a small but significant (p less than 0.05) increase in the mutation frequency was seen with 10 micrograms/ml NDBzA. In the Salmonella assay, using a pre-incubation protocol and concentrations up to 1000 micrograms/ml, NDBzA was negative in strain TA98, and in TA100 with rat S9, but was positive at the highest dose in TA100 with hamster S9, and more strongly with Aroclor 1254-induced hamster S9. When activated by uninduced rat or hamster hepatocytes, as opposed to S9, NDBzA was negative with all tester strains. Hamster hepatocytes activated more than rat in the V79 studies, and hamster S9 was more strongly activating in the Salmonella assay. These results indicate that NDBzA is weakly mutagenic to both Salmonella and V79 cells.     

Mutagenic and carcinogenic heterocyclic amines in Chinese cooked foods

Samples of 7 foods commonly eaten in the Northeast of China (i.e. fried and broiled fishes and broiled meat) were tested for mutagenicity on Salmonella typhimurium TA98 with S9 mix. The basic fractions of the samples were mutagenic, inducing 33-2930 revertants/g of cooked food. Fried walleye pollack (a kind of cod fish heated on a stainless steel pan) showed the highest mutagenicity, so attempts were made to isolate mutagens from the basic fraction of this food. The mutagens were purified by treatment with blue cotton and HPLC on a semi-preparative ODS column and analytical cation exchange and ODS columns. 5 mutagens were isolated and identified as 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). 1 g of fried fish was estimated to contain 0.16 ng of IQ, 0.03 ng of MeIQ, 6.44 ng of MeIQx, 0.10 ng of 4,8-DiMeIQx and 69.2 ng of PhIP. MeIQx and PhIP accounted for 24% and 4.7%, respectively, of the total mutagenicity. The other 3 heterocyclic amines were each responsible for only 0.3-1.2% of the total mutagenicity.     

Metabolic activation of food mutagens by liver and lung enzymes from rats, pigs and oxen

Mutagenic activation of the 3 cooked food mutagens 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) was compared in liver and lung enzyme preparations from oxen, pigs and rats. Liver preparations from oxen were the most efficient in activating the mutagens, while the rat enzymes were more active than those from pigs. The different cooking mutagens showed different mutagenic potential. MeIQ was the most potent mutagen, followed by IQ and MeIQx in descending order. In oxen, MeIQx was as potent as IQ. The activation with the lung enzymes was 2-3 orders of magnitude lower than with liver. Furthermore, species differences in mutagenic activation with lung enzymes were small compared with liver enzymes. In lung preparations the differences between IQ and MeIQ were small, but in all 3 animal species the mutagenicity of MeIQx was 1 order of magnitude lower than that of the other 2 mutagens.     

Activation of carcinogenic N-nitrosopropylamines to mutagens by lung and pancreas S9 fractions from various animal species and man

The mutagenic potential of 7 carcinogenic N-nitrosopropylamines was examined by the Ames liquid incubation assay, using lung and pancreas 9000 × g supernatant (S9) fractions from rats, hamsters, mice, rabbits, monkeys and humans for metabolic activation. N-Nitroso(2-hydroxypropyl)(2-oxopropyl)amine (HPOP), N-nitrosobis(2-oxopropyl)amine (BOP) and N-nitrosomethyl(2-oxopropyl)amine (MOP) showed positive mutagenicity in strain TA100 in the presence of lung S9 from each of the uninduced animals and humans. Besides the 3 N-nitrosopropylamines, N-nitrosomethyl(2-hydroxypropyl)amine (MHP) was also positive in the presence of lung S9 from polychlorinated biphenyl (PCB)-induced rats, hamsters and mice. On the other hand, in the presence of pancreas S9 from uninduced or PCB-induced animals, only HPOP and BOP showed positive mutagenicity. In contrast, N-nitrosobis(2-hydroxypropyl)amine (BHP), N-nitrosobis (2-acetoxypropyl)amine (BAP) and N-nitroso-2,6-dimethylmorpholine (NDMM) showed negative mutagenicity in the presence of lung and pancreas S9 from either uninduced or PCB-induced animals and humans. HPOP was a direct-acting mutagen, and lung and pancreas S9 from 5 animal species and man did not affect the activity. BOP was mutagenic even in the presence of bovine serum albumin. The mutagenic activation of MHP by lung S9 from PCB-induced rats, hamsters and mice was completely inhibited by preincubation in an atmosphere of carbon monoxide or by addition of cytochrome c or metyrapone to the S9 mixture, whereas 7,8-benzoflavone totally lacked this effect. However, that of MOP was insensitive to these inhibitors. These results of mutagenicity assay indicate that only the methyl derivatives of N-nitrosopropylamines, MHP and MOP are activated by the lung from 5 animal species and man, whereas the pancreas from all the tested animals did not activate the 7 N-nitrosopropylamines to mutagens, and that the phenobarbital-inducible major cytochrome P-450 in the lung of rodents is involved in the mutagenic activation of MHP.     

Screening of molecular cell targets for carcinogenic heterocyclic aromatic amines by using CALUX® reporter gene assays

Heterocyclic aromatic amines (HCAs) are compounds formed when meat or fish are cooked at high temperatures for a long time or over an open fire. To determine which pathways of toxicity are activated by HCAs, nine out of the ten HCAs known to be carcinogenic in rodents (2-amino-9H-pyrido[2,3-b]indole (AαC), 2-aminodipyrido[1,2-a:3′,2-d]imidazole (Glu-P-2), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeAαC), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2)) were tested in the estrogen receptor α (ERα), androgen receptor (AR), glucocorticoid receptor (GR), peroxisome proliferator-activated receptor γ2 (PPARγ2), polycyclic aromatic hydrocarbons (PAH), Nrf2, and p53 CALUX® reporter gene assays. Trp-P-1 was the only HCA that led to a positive response in the ERα, PPARγ2, and Nrf2 CALUX® assays. In the PAH CALUX® assay, Trp-P-2, MeAαC, and AαC induced luciferase activity to a greater extent than MeIQ and PhIP. In the p53 CALUX® assay without a coupled metabolic activation, only Trp-P-1 and Trp-P-2 enhanced luciferase expression; when a metabolic activation step was coupled to the p53 CALUX® assay, Trp-P-1, Glu-P-2, MeIQ, MeIQx, and PhIP induced a positive response. No HCA was positive in the AR and GR CALUX® assays. Taken together, the results obtained show that the battery of CALUX® assays performed in the present study can successfully be used to screen for molecular cell targets of carcinogenic compounds such as HCAs.