Major routes of metabolism of the food-borne carcinogen 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline in the rat

The metabolic fate of 2-amino-3,8-dimethylimidazo[4,5-f] quinoxaline(MeIQx), a carcinogen formed in cooked meat and fish, has beeninvestigated in male Sprague-Dawley rats. Five metabolites wererecovered from bile of animals given an intragastric dose of{2-¹⁴C]MeIQx. These accounted for nearly all of the radioactivityin bile. The chemical structures of these metabolites were elucidatedby proton NMR, UV and mass spectroscopy. Three structures maybe assigned unambiguously: two sulfamates, N-(3,8.dimethylimidazo[4,5f]quinoxalin-2-yl)sulfamic acid and N-(8-hydroxymethyl-3-methylimidazo[4,5f]quinoxalin-2-yl)sulfamic acid, and N-(8-one glucuronide, N²(ß-1-glucosiduronyl)-2-amino-3,8-dimelhyliinidazo[4,5f]quinoxaline In addition, an acetyl and a glucosiduronylconjugate of 5-hydroxy-MeIQx were observed. The spectral evidencedid not allow an unambiguous assignment of the site of conjugation.The two glucuronides were excreted in urine and the sulfamateof MeIQx was found in feces as well as urine. All five metaboliteswere found to be non-mutagenic to Salmonella typhimurium TA98with or without metabolic activation. The glucuronide conjugateswere found also to be non-mutagenic when ß- glucuronidasewas incorporated with S-9 mixture in the mutation assay, andthus all appear to be detoxification products. The previouslyreported metabolite, 2-amino-8-hydroxymethyl-3-methylimidazo[4,5f]quinoxalinewhich is mutagenic to Salmonella typhimurium TA98 with metabolicactivation, was identified as a minor component in both urineand feces.     

Mutagenic metabolites in urine and feces of rats fed with 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, a carcinogenic mutagen present in cooked meat

To study the in vivo fate of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), a carcinogenic mutagen present in cooked meat, rats were fed MeIQx in the diet and their urine and feces were analyzed for the metabolites. The isolation procedure included specific adsorption of MeIQx derivatives to blue cotton and subsequent fractionations by thin layer chromatography on silica gel and by high pressure liquid chromatography. Attention was focused on mutagenically active metabolites. Three metabolites were isolated from the urine, and their structures were elucidated on the basis of 1H nuclear magnetic resonance, ultraviolet, and mass spectra. The first metabolite characterized was 2-amino-8-hydroxymethyl-3-methylimidazo[4,5-f]quinoxaline (Compound I), the second was 2-acetylamino-3,8-dimethylimidazo[4,5-f]quinoxaline (Compound II), and the third was 2-amino-8-methylimidazo[4,5-f]quinoxaline (Compound III). Compound I was isolated also from the feces. Compounds I-III were mutagenic to Salmonella typhimurium TA98 with metabolic activation. The mutagenic potency of Compounds I and II was as high as that of MeIQx, and that of Compound III was much lower than that of MeIQx.     

Biomonitoring of heterocyclic aromatic amine metabolites in human urine.

Human exposure to heterocyclic aromatic amines such as MeIQx (2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline) may be monitored by measuring the levels of the heterocyclic aromatic amine in urine. In order to investigate the contribution of N-oxidation to the metabolism of MeIQx in vivo, we developed a biomonitoring procedure for the analysis and quantification of the N2-glucuronide conjugate of 2-hydroxyamino-3,8-dimethylimidazo[4,5-f]quinoxaline in human urine. Subjects (n = 66) in the dietary study ingested a uniform diet of cooked meat containing known amounts of MeIQx, and urine was collected after consumption of the test meal. A method based on solid-phase extraction and immunoaffinity separation was used to isolate N2-(beta-1-glucosiduronyl)-2-hydroxyamino-3,8-dimethylimidazo++ +[4,5-f]quinoxaline and its stable isotope-labeled internal standard from urine. The isolated conjugate was converted to the deaminated product 2-hydroxy-3,8-dimethylimidazo[4,5-f]quinoxaline by treatment with acetic acid under moderate heating. 2-Hydroxy-3,8-dimethylimidazo[4,5-f]quinoxaline and the [2H3]methyl analog were derivatized to form the corresponding 3,5-bis(trifluoromethyl)benzyl ether derivatives and quantified by capillary gas chromatography-negative ion chemical ionization mass spectrometry employing selected ion monitoring procedures. The amounts of N2-(beta-1-glucosiduronyl)-2-hydroxyamino-3,8-dimethylimidazo++ +[4,5-f]quinoxaline recovered in urine collected 0-12 h after the test meal accounted for 2.2-17.1% of the ingested dose, with a median value of 9.5%. The variability in the proportion of the dose excreted among the subjects may be reflective of several factors, including interindividual variation in the enzymic activity of CYP1A2 and/or conjugation reactions of the N-hydroxylamine metabolite with N-glucuronosyltransferase(s).     

The effect of dose and cytochrome P450 induction on the metabolism and disposition of the food-borne carcinogen 2-amino-3,8-dimethylimidazo[4,5-f] quinoxaline (MeIQx) in the rat

The effect of dose and cytochrome P450 induction on the metabolism and disposition of the food-borne carcinogen 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) was investigated in the male Sprague-Dawley rat. Animals were given MeIQx by gavage at doses of 0.01, 0.2 or 20 mg/kg body wt. The phase II conjugates, MeIQx-N2 sulfamate and MeIQx-N2 glucuronide were the predominant metabolites found in urine of non-induced animals at the highest dose treatment. Animals induced with polychlorinated-biphenyl (PCB) produced greater amounts of metabolites hydroxylated at the 5 position of MeIQx which were excreted as glucuronide or sulfate conjugates. At the lowest dose studied, the urinary excretion profile was nearly identical for both animal groups and cytochrome P450 induction had little influence on metabolism. In contrast to high dose exposure, where sulfamate formation was a major route of detoxification, N2 glucuronide formation was the most important metabolic pathway for elimination of MeIQx at low doses. Liver microsomes transformed MeIQx to the genotoxic metabolite 2-hydroxyamino-3,8-dimethylimidazo[4,5-f]quinoxaline (HNOH-MeIQx) and N-hydroxylase activity was 20-fold greater in microsomes obtained from PCB-treated animals than in untreated control animals. The increase in N-hydroxylase activity was discerned in vivo through formation of the metastable N-glucuronide conjugate of HNOH-MeIQx (MeIQx-[HO-N]-Gl). This metabolite accounted for approximately 3% of the dose in bile of PCB-treated rats. In contrast, in the non-induced rat, MeIQx-[HO-N]-Gl was preferentially excreted in urine and accounted for approximately 0.2-1% of the total dose. These results demonstrate that the metabolism of MeIQx in the rat is influenced by both dose and cytochrome P450 induction. The absence of intestinal tumors in the non-induced rat may be partially attributed to the low levels of formation and poor biliary excretion of the N-glucuronide conjugate of the genotoxic metabolite HNOH-MeIQx.     

Tissue distribution of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) in two strains of male rats.

Albino and hooded male rats were administered 14C-labeled 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) by gavage. The tissue distribution was investigated by means of whole-body autoradiography and liquid scintillation counting. MeIQx was rapidly absorbed from the alimentary tract and distributed to several tissues. The major predilection sites were the liver and kidneys. The amount of radioactivity decreased dramatically within a few days. However, unextractable radioactive material was still detectable in these organs 6 days after the administration.     

Metabolism of the food carcinogen 2-amino-3, 8-dimethylimidazo[4, 5-f]quinoxaline in isolated rat liver cells

2-Amino-3, 8-dimethylimidazo[4, 5-f]quinoxaline (MelQx) wastransformed to at least 10 metabolites in suspensions of hepatocytesisolated from Aroclor 1254 treated rats. Combining biochemicaldata such as effects on MeIQx metabolism of metabolic modulatorsand incorporation of radioisotopic sulfur with UV, mass and¹H-NMR spectroscopy, we elucidated the structures of six metabolites.About 40% of the MeIQx was transformed to 2-amino-3, 8-dimethylimidazo[4,5-f]quinoxalin-4(or5)-yl sulfate. Other oxygenated metaboliteswere 2-amino-8-hydroxymethy1–3-methylimidazo[4, 5-f)quinoxalin-4(or5)-yl sulfate and 2-amino-4(or5)-ß-D-glucuronopyranosyloxy-3,8-dimethylimidazo[4, 5-f]quinoxaline. Evidence was obtainedthat a glutathione conjugate was formed. This metabolite, andthe other oxygenated metabolites were probably formed in P-450catalyzed reactions. Two metabolites, 2-ß-D-glucurono-pyranosylamino-3,8-dimethylimidazo[4, 5-f)quinoxaline and the N(3, 8-dimethylimidazo[4,5-f]quinoxaline-2-yl)sulfamate, were direct conjugates of MeIQx.     

Immunochemical detection of rodent hepatic and urinary metabolites of cooking-induced food mutagens

Monoclonal antibodies, previously selected to bind either 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) or 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) were evaluated to determine their binding properties with several known metabolites of these compounds purified from rat hepatocyte cultures. Both 2-N- and 5-substituted MeIQx metabolites were recognized by antibodies AIA-2 and AIA-11, while antibodies AIA-1 and AIA-12 bound N-substituted metabolites only. Anti-PhIP antibodies bound N-hydroxy-PhIP, N-sulfinamide-glutathione-PhIP and N-hydroxy-glucuronide-PhIP. Immunoaffinity columns incorporating these antibodies were used to concentrate and purify both the unmetabolized parent compounds and several relatively non-polar metabolites from the urine of rats exposed either to MeIQx or PhIP. Several of these metabolites corresponded with available standards of previously identified polar conjugate metabolites, e.g. N-sulfamate-MeIQx and N(OH)-gluc-PhIP, while others are as yet unidentified. Immunoaffinity chromatography is demonstrated as a promising means of selectively concentrating metabolites of PhIP and MeIQx from urine.     

Intra- and interindividual variability in systemic exposure in humans to 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline and 2-amino-1-methyl- 6-phenylimidazo[4,5-b]pyridine, carcinogens present in cooked beef.

During the cooking of beef, the genotoxic heterocyclic aromatic amines 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (DiMeIQx), and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) are formed. Little is known about the fate of these compounds in humans or the factors affecting it. We have developed assays based on capillary column gas chromatography-negative ion mass spectrometry capable of the simultaneous measurement of MeIQx, DiMeIQx, and PhIP in cooked meat and in human urine using stable isotope labeled analogues. Ten normal, healthy male volunteers were invited to consume a standard cooked meat meal (400-450 g lean beef, cooked as patties on a griddle hotplate) on four separate occasions over a period of 14 months. Following consumption of the test meals, urine was collected from 0 to 8 h, during which time all free amines were excreted and analyzed for MeIQx, DiMeIQx, and PhIP. Subjects ingested 240 +/- 9 (SEM) g cooked meat, which contained 2.2 +/- 0.2 ng MeIQx/g meat, 0.7 +/- 0.1 ng DiMeIQx/g meat, and 16.4 +/- 2.1 ng PhIP/g meat. The variability in relative systemic bioavailability was assessed from the percentage of ingested amine excreted unchanged in the urine. Subjects excreted 2.1 +/- 1.1% of MeIQx and 1.1 +/- 0.5% of PhIP ingested as unchanged amine in the urine. Levels of DiMeIQx in urine, if present, were below the sensitivity of our assay (20 pg/ml) and could not be detected in any of the samples analyzed. Irrespective of dose, urinary excretion of unchanged MeIQx or PhIP (expressed as a percentage of the ingested dose) remained constant for each individual subject. The intraindividual coefficients of variation for MeIQx (28.4%) and PhIP (23.7%) were low and the pooled interday (intrasubject) coefficients of variation for both compounds were only 19 and 3.4%, respectively. In contrast, inter-subject (intraday) variation was greater, with pooled coefficients of variation of 145% for MeIQx and 71% for PhIP. Based on these studies, it should be possible to use the percentage excretion of MeIQx and PhIP to assess the relative bioavailability of these compounds in humans.     

The presence of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline in smoked dry bonito (katsuobushi)

Smoked dry bonito (katsuobushi), an everyday food item for most Japanese people, was found to contain 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), the content of which was estimated at about 2 ng/g. This content is similar to the known MeIQx content of cooked beef. The katsuobushi also contained another mutagenic component, the total activity of which was 1/6-1/3 that of the MeIQx. This component was similar to 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx) with respect to its behavior in high-pressure liquid chromatography and its ultraviolet absorption spectrum.     

N-oxidative metabolism of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) in humans: excretion of the N2-glucuronide conjugate of 2-hydroxyamino-MeIQx in urine.

2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), a major heterocyclic aromatic amine (HAA) formed in cooked meats, is metabolically transformed to mutagenic/carcinogenic intermediates. Cytochrome P4501A2 (CYP1A2)-mediated N-hydroxylation followed by phase II O-esterification by N-acetyltransferase (NAT2) are generally regarded as activation processes in which MeIQx and other HAAs are converted to genotoxic species. In this study, we determined the relationship between the activities of these two enzymes and the urinary excretion level of the N2-glucuronide conjugate of 2-hydroxyamino-MeIQx--N2-(beta-1-glucosiduronyl)-2-hydroxyam ino-3,8-dimethylimidazo[4,5-f]quinoxaline (N-OH-MeIQx-N2-glucuronide)--among healthy subjects fed a uniform diet containing high-temperature cooked meat. The individuals (n = 66) in the study ate meat containing known amounts of MeIQx, and urine was collected from 0 to 12 h after the meal. After addition of the deuterium-labeled internal standard to urine, N-OH-MeIQx-N2-glucuronide was isolated using solid-phase extraction and immunoaffinity separation. The isolated conjugate was converted to the deaminated product 2-hydroxy-3,8-dimethylimidazo[4,5-f]quinoxaline (2-OH-MeIQx) by heating with acetic acid. 2-OH-MeIQx and its deuterated analogue were derivatized to form the corresponding 3,5-bis(trifluoromethyl)benzyl ether derivatives and analyzed by capillary gas chromatography-negative ion chemical ionization mass spectrometry using selected ion monitoring procedures. The subjects in the study excreted an average of 9.4 +/- 3.0% (+/-SD) of an ingested dose of MeIQx as N-OH-MeIQx-N2-glucuronide in urine; the range varied from 2.2 to 17.1%. A significant correlation was found between the level of N-OH-MeIQx-N2-glucuronide in urine and the amount of MeIQx ingested (r(s) = 0.44; P = 0.0002). The excretion level of N-OH-MeIQx-N2-glucuronide in urine was not associated with the enzyme activities of NAT2 or CYP1A2. This is expected with the latter enzyme because the metabolism of MeIQx is first order and very rapid at the amounts ingested. The amount of N-OH-MeIQx-N2-glucuronide in urine was not correlated with the age or sex of the individuals. Our results indicate that biotransformation of MeIQx via CYP1A2 oxidation to form the N-hydroxylamine followed by N2-glucuronidation is a general pathway of MeIQx metabolism in humans; the variability in the excreted levels of N-OH-MeIQx-N2-glucuronide is probably due to interindividual differences in UDP-glucuronosyltransferase activity and/or excretion pathways.     

Presence of carcinogenic heterocyclic amines in urine of healthy volunteers eating normal diet, but not of inpatients receiving parenteral alimentation

For estimation of human exposures to carcinogenic heterocyclic amines, the amounts of four compounds, 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-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), in human urine were measured. Twenty-four hour urine specimens were collected from ten healthy volunteers eating normal diet (five males and five females) and three inpatients (two males and a female) receiving parenteral alimentation, and the levels of the four heterocyclic amines were measured by HPLC after partial purification by treatment with blue cotton and ion exchange column chromatography. Trp-P-1, Trp-P-2, PhIP and MeIQx were detected in the 24 h urine samples of all healthy volunteers at levels of 0.04-1.43 ng, 0.03-0.68 ng, 0.12-1.97 ng and 11-47 ng respectively. As 1.8-4.9% of an oral dose of MeIQx is reported to be excreted unchanged in the urine, the daily exposure of humans to MeIQx was estimated to be 0.2-2.6 micrograms/person. The four heterocyclic amines were not detected in the urine of parenterally fed inpatients. These results indicate that humans are continually exposed to carcinogenic heterocyclic amines in food, and these compounds may not be formed endogenously.     

Isolation and identification of mutagens from a fried Norwegian meat product

Two samples of a typical Norwegian minced meat emulsion were fried at 215 degrees C. One had the regular composition while 4.2% creatine was added to the second sample prior to frying. Mutagens in both samples were purified using aqueous acid extraction, XAD-2 adsorption and a series of semipreparative and analytical high performance liquid chromatography (HPLC) purification steps monitored by the Ames/Salmonella mutagenicity test. Mutagenic activity in the creatine-fortified product was enhanced 15-fold. Mutagenicity profiles from reverse-phase and normal-phase HPLC were qualitatively similar for both samples indicating no major production of new mutagens due to the presence of additional creatine. A total of 8 distinct mutagenic peaks could be separated after three additional HPLC steps. These compounds fall into a class of compounds called amino-imidazoazaarenes (AIA). The majority of mutagenic activity is made up by the known cooking mutagens 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (DiMeIQx), 2-amino-3-methylimidazo[3,5-f]quinoline (IQ) and 2-amino-n,n,n-trimethylimidazopyridine (TMIP). Smaller contributions are from 2-amino-3-methylimidazo- [4,5-f]quinoxaline (IQx), 2-amino-n,n-dimethylimidazopyridine (DMIP), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and two oxygen-containing AIA. With respect to mass, MeIQx and PhIP were the dominating mutagens.     

Mutagenesis of mouse intestine in vivo using the Dlb-1 specific locus test: studies with 1,2-dimethylhydrazine, dimethylnitrosamine, and the dietary mutagen 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline

The ability of three model carcinogens, 1,2-dimethylhydrazine, dimethylnitrosamine, and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, to induce mutation in a novel in vivo assay in mouse intestine has been examined. The assay is based on mutations at the Dlb-1 locus which determines the tissue specific pattern of expressio of the binding site for the lectin Dolichos biflorus agglutinin. In C57BL/6J x SWR F1 mice Dlb-1 mutants are recognized as clones of epithelial cells not staining with a peroxidase conjugate of D. biflorus agglutinin. Chronic administration of 1,2-dimethylhydrazine (20 mg/kg/week s.c. for 10 weeks) induced Dlb-1 mutants, whereas administration of a single dose did not. Similarly, chronic dimethylnitrosamine treatment p.o. (0.001% in drinking water for 8 weeks) induced Dlb-1 mutants, but acute administration did not. In contrast, neither chronic nor acute treatment of the mice with 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline induced Dlb-1 mutations. The activities of 1,2-dimethylhydrazine, dimethylnitrosamine, and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline in the Dlb-1 assay more accurately reflect their carcinogenic potential than do many in vitro bioassays.     

A model system for studying covalent binding of food carcinogens MeIQx, MeIQ and IQ to DNA and protein

We have studied the covalent binding of carcinogenic aminoimidazoazaarene compounds to macromolecules in a microsomal model system. The 14C-labelled compounds were incubated with rat-liver microsomes, and binding to macromolecules was measured after their precipitation on glass filters, which were washed several times in organic solvents. The amount of radioactivity was determined by liquid scintillation counting. Covalent binding was dependent on the addition of NADPH, with an optimal concentration of about 1 mM. The binding appeared to follow saturation kinetics when carcinogen concentrations were lower than 200 microM, with Km values of less than 20 microM. At 50 microM, 2-amino-3,4-dimethylimidazo[4,5-f]-quinoline (MeIQ) and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) bound more effectively than 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx). When DNA was included in the incubations, binding to this macromolecule was ten-fold less per milligram than binding to proteins. In comparison with microsomes from untreated animals, those from rats treated with beta-naphthoflavone caused up to nine-fold more binding of MeIQx, six-fold more of IQ and three times as much of MeIQ. Induction by Aroclor 1254 caused up to 17-fold more binding, whereas induction by phenobarbital caused up to three-fold more binding. The effects of the inducers were greatest for MeIQx and IQ, while smaller effects were seen for MeIQ. The results are most consistent with cytochrome P450-dependent metabolic activation of the carcinogens to hydroxylamine metabolites, for which an isoenzyme(s) inducible by polyaromatic and polychlorinated hydrocarbons is most effective. To our knowledge, this is the first report that MeIQx is metabolized to reactive species capable of covalent binding to macromolecules.     

Metabolism of the food-derived carcinogen 2-amino-3,8-dimethylimidazo[4,5,-f)quinoxaline (MeIQx) in nonhuman primates

The metabolism and disposition of the food mutagen and rodentcarcinogen 2-amino-3, 8-dimethylimidazo[4, 5-f)quinoxaline wasinvestigated in cynomolgus monkeys. Monkeys were administereda single dose of radiolabeled [¹⁴C]MeIQx (2.2 or 50 µmol/kg).Peak blood levels of radioactivity were observed within 1–3h after dosing and declined rapidly thereafter. By 72 h afterdosing, approximately 50% and 70% of the 2.2 µmol/kg,and 50 µmol/kg dose, respectively, was excreted in theurine. Approximately 15–20% of either dose was recoveredin the feces. Eight metabolites and the parent compound weredetected in urine by HPLC. The parent compound accounted for     

Interspecies differences in metabolism of heterocyclic aromatic amines by rat and human P450 1A2.

The catalytic efficiences of cytochrome P450 (P450)-mediated N-oxidation of the 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) by recombinant human P450 1A2 were 10-19-fold higher than rat P450 1A2, while methoxyresorufin O-demethylation activity was comparable for both P450s. Similar findings were observed with rat and human liver microsomal samples. Interspecies differences in P450 enzyme expression and catalytic activities may be significant and must be considered when assessing human health risk.     

Low-dose carcinogenicity of a heterocyclic amine, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline: relevance to risk assessment

Male, 21-day-old, F344 rats were administered 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) in the diet at various low doses and a high dose, 100 ppm for 16 weeks. Quantitative values for glutathione-S-transferase placental form (GST-P)-positive foci in their livers were similar among the 0, 0.001, 0.01, 0.1 and 1 ppm MeIQx group while 10 ppm MeIQx administration slightly and 100 ppm MeIQx significantly increased their numbers. These results indicate that MeIQx has a no-observed effect level for induction of preneoplastic lesions in rats. Transplacental and trans-breast milk exposure to low doses of MeIQx also did not exert carcinogenic potential in F344 rats and 20% of calorie restriction clearly inhibited development of GST-P-positive foci. The results are of direct significance to human risk assessment.     

Existence of no-observed effect levels for 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline on hepatic preneoplastic lesion development in BN rats

There is increasing evidence that dose-response curve of genotoxic carcinogen is nonlinear and a practical threshold dose exists. However, little is known about differences in the dose-response relationship of genotoxic carcinogen among different strain rats. Herein, we showed that low doses of genotoxic carcinogen 2-amino-3,8-dimethylimidazo[4,5-f] quinoxaline (MeIQx) had no effects on induction of liver glutathione S-transferase placental form (GST-P)-positive foci in both BN and F344 rats, and therefore demonstrated the existence of no-observed effect level for hepatocarcinogenicity of this genotoxic carcinogen irrespective of strains. These findings further support our notion that a practical threshold dose for MeIQx hepatocarcinogenicity exists in rats.     

Evidence of direct generation of oxygen free radicals from heterocyclic amines by NADPH/cytochrome P-450 reductase in vitro.

Highly purified cytochrome P-450 reductase (also called cytochrome c reductase; EC 1.6.2.4.) and NADPH were used to generate superoxide radical (O2.-) from 11 different heterocyclic amines (HCAs) as identified by electron spin resonance spectroscopy using the spin trapping method with 5,5-dimethyl-1-pyrroline N-oxide (DMPO). The signal intensity of DMPO-OOH(-O2-) (i.e., the DMPO spin adduct of O2.-) was strongest for 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ). The O2.- generation with HCAs decreased in the following order: 2-amino-3,8-dimethyl-imidazo[4,5-f]quinoxaline (MeIQx) = 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) > 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (diMeIQx) > or = other HCAs; O2.- generation was lowest with 2-amino-3-methyl-9H-pyrido[2,3-b]indole .CH3COOH (MeA alpha C). By using Lineweaver-Burk plots, Km values of cytochrome P-450 reductase for mitomycin C, IQ, and MeIQ were determined to be 1.60 x 10(-6) M, 1.97 x 10(-5) M, and 2.83 x 10(-6) M, respectively. The present findings have important implications for carcinogenesis because of the known effect of oxygen radicals on cell proliferation.     

Lack of initiation activity in rat liver of low doses of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline

It has been generally accepted that genotoxic carcinogens have no threshold in exerting their potential for cancer induction. However, the non-threshold theory can be challenged for cancer risk assessment in humans. Here we examined low dose carcinogenicity of a food-derived, genotoxic hepatocarcinogen, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), using an in vivo medium-term bioassay to detect initiating activity for rat hepatocarcinogenesis. With MeIQx initiation at various doses followed by administration of phenobarbital, a well known hepatopromoter, no induction of glutathione S-transferase placental form-positive foci, assessed as preneoplastic lesions, was noted at doses of 0.001-1 ppm. The results imply a no-observed effect level for hepatocarcinogenicity with this genotoxic agent.