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.     

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.     

Lack of a dose-response relationship for carcinogenicity in the rat liver with low doses of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline or N-nitrosodiethylamine.

For a long period, it has been generally considered that carcinogens, particularly genotoxic ones, have no threshold in exerting their potential for cancer induction. However, the non-threshold theory can be challenged with regard to assessment of cancer risk to humans. Here we show that a food-derived, genotoxic hepatocarcinogen, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, forms DNA adducts at low doses, but does not induce glutathione S-transferase placental form (GST-P)-positive foci (considered to be preneoplastic lesions) or 8-hydroxy-2'-deoxyguanosine in rat liver. Moreover a N-nitroso compound, N-nitrosodiethylamine, at low doses was also found not to induce GST-P-positive foci in rat liver. These results imply that there is a no-observed effect level for hepatocarcinogenesis by these genotoxic carcinogens.     

Lack of a Dose-response Relationship for Carcinogenicity in the Rat Liver with Low Doses of 2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline or N-Nitrosodiethylamine

For a long period, it has been generally considered that carcinogens, particularly genotoxic ones, have no threshold in exerting their potential for cancer induction. However, the non-threshold theory can be challenged with regard to assessment of cancer risk to humans. Here we show that a food-derived, genotoxic hepatocarcinogen, 2-amino-3,8-dimethylimidazo[4,5- f ]quinoxaline, forms DNA adducts at low doses, but does not induce glutathione S-transferase placental form (GST-P)-positive foci (considered to be preneoplastic lesions) or 8-hydroxy-2'-deoxyguanosine in rat liver. Moreover a N -nitroso compound, N -nitrosodiethylamine, at low doses was also found not to induce GST-P-positive foci in rat liver. These results imply that there is a no-observed effect level for hepatocarcinogenesis by these genotoxic carcinogens.     

Macromolecular adduct formation and metabolism of heterocyclic amines in humans and rodents at low doses.

2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) are heterocyclic amines formed during the cooking of meat and fish. Both are genotoxic in a number of test systems and are carcinogenic in rats and mice. Human exposure to these compounds via dietary sources has been estimated to be under 1 microg/kg body wt. per day, although most laboratory animal studies have been conducted at doses in excess of 10 mg/kg body wt. per day. We are using accelerator mass spectrometry (AMS), a tool for measuring isotopes with attomole sensitivity, to study the dosimetry of protein and DNA adduct formation by low doses of MeIQx and PhIP in rodents and comparing the adduct levels to those formed in humans. The results of these studies show: 1, protein and DNA adduct levels in rodents are dose-dependent; 2, adduct levels in human tissues and blood are generally greater than in rodents administered equivalent doses; and 3, metabolite profiles differ substantially between humans and rodents for both MeIQx and PhIP, with more N-hydroxylation (bioactivation) and less ring oxidation (detoxification) in humans. These data suggest that rodent models do not accurately represent the human response to heterocyclic amine exposure.     

Existence of a no effect level for MeIQx hepatocarcinogenicity on a background of thioacetamide-induced liver damage in rats

As exposure to heterocyclic amines might increase the risk of liver cancer, we investigated the carcinogenic potential of MeIQx under conditions of liver damage caused by TAA. Male, 6-week-old F344 rats (n = 280) were divided into 14 groups; groups 1-7 received TAA (0.03% in drinking water) and groups 8-14 received water for the first 12 weeks. Thereafter, the animals received MeIQx at doses from 0, 0.001, 0.01, 0.1, 1, 10 to 100 p.p.m. (groups 1-7 and 8-14, respectively) in pellet basal diet for 16 weeks. All survivors were killed at week 28 for assessment of numbers and areas of GST-P positive foci, considered to be pre-neoplastic lesions of the liver. Values were increased significantly in all the groups receiving TAA-->MeIQx compared to MeIQx alone (P < 0.01). Numbers of GST-P positive foci were significantly increased in groups 7 and 14 (treated with 100 p.p.m. MeIQx) as compared to 0 p.p.m.-MeIQx (groups 1 and 8) (P < 0.01), along with areas in group 14 compared to group 8 (P < 0.01). However, with the maximum likelihood method, the data for numbers of GST-P positive foci (groups 1-7 and groups 8-14) fitted the hockey stick regression model, representing no differences from groups 1-5 and from groups 8-13, despite a linear dose-dependent increase of MeIQx-DNA adducts from 0.1 to 100 p.p.m. We conclude that there is a no effect level for MeIQx hepatocarcinogenicity, even on a background of TAA-induced liver damage.     

Induction of preneoplastic lesions by a low dose of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) in the livers of rats treated with carbon tetrachloride.

The multifactorial nature of carcinogenesis in man has impelled us to study the effects of various chemicals and conditions in combination. In the present investigation, we examined the effects of low doses of 2-amino-3,8-dimethyl-imidazo[4,5-f]quinoxaline (MeIQx) in combination with carbon tetrachloride (CCl4) on the formation of glutathione S-transferase placental form (GST-P)-positive foci in rat liver. Administration of diet containing MeIQx at 0.4, 4 or 40 p.p.m., representing one-thousandth, one-hundredth and one-tenth of the dose proved to induce hepatocellular carcinomas (400 p.p.m.), for 8 or 12 weeks did not induce GST-P-positive foci. However, 40 p.p.m. of MeIQx when co-administered with CCl4 (0.7 ml/kg, s.c. twice a week) induced preneoplastic lesions: 7- and 3-fold increases in the numbers and areas of GST-P positive foci in week 8, and 8- and 6-fold increases respectively in week 12, over those with CCl4 alone. Furthermore, a marked increase in the number of hyperplastic nodules was observed in this group of rats in week 12. No significant increases of GST-P-positive foci were observed at doses of 0.4 or 4 p.p.m. MeIQx in combination with CCl4. Thus, it is predicted that chronic exposure to 40 p.p.m. of MeIQx eventually results in induction of hepatocellular carcinomas in injured rat liver.     

N-hydroxy-MeIQx is the major microsomal oxidation product of the dietary carcinogen MeIQx with human liver.

2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), one of the most abundant of the heterocyclic aromatic amines formed during the cooking of meat, is genotoxic and carcinogenic in rodents. MeIQx requires metabolic activation by P450 before it can exert these effects. Whilst there is indirect evidence that the mutagenic product is N-hydroxy-MeIQx (N-OHMeIQx), we have now identified this unequivocally following incubation of the amine with human hepatic microsomal fraction. A mixture of unlabelled MeIQx, [13C,15N2]MeIQx and [14C]MeIQx was used as substrate and the products analysed by HPLC-thermospray mass spectrometry. Characteristic doublet ions, 3 mass units apart, were found at m/z 214/217 ([M+H]+) from the parent compound, MeIQx and at 230/233 ([M+H]+) from N-OHMeIQx. The presence of a doublet ion at m/z 214/217 with the doublet at 230/233 [M+H+] provided additional evidence that this was N-OHMeIQx, as facile loss of 'O' is characteristic of N-hydroxylamines. Further evidence for the identity of the major metabolite, which accounted for approximately 90% of all microsomal metabolism, was obtained by comparing the mutagenicity of the HPLC eluate using Salmonella typhimurium YG1024, which is particularly sensitive to N-hydroxylamines, and TA98/1,8-DNP6 which is resistant to most N-hydroxylamines. Ninety-five per cent of direct-acting mutagenicity present in the reaction mixture was associated with a single peak, which co-eluted with N-OHMeIQx, as indicated by mass spectrometry. In the presence of a metabolic activation system, only one additional mutagenic peak, corresponding to unchanged MeIQx, could be detected. MeIQx (5 microM) was N-hydroxylated at a rate of 77 +/- 11 pmol/mg/min (mean +/- SEM, n = 4) by human liver microsomes. The specific inhibitor of human CYP1A2, furafylline (5 microM) inhibited the N-hydroxylation of MeIQx by > 90%. These data show that N-OHMeIQx is both the major oxidation product and the major genotoxic product of MeIQx generated by microsomal fractions of human liver and that the reaction is catalysed almost exclusively by CYP1A2.     

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.     

DNA adducts formed by 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline in rat liver: dose-response on chronic administration

The effect of administration of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) at various doses on DNA adduct formation in male rats was examined by 32P-postlabeling analysis. Administration of MeIQx in the diet at 0.4 ppm, 4 ppm, 40 ppm and 400 ppm for one week resulted in the formations of 0.04, 0.28, 3.34 and 39.0 adducts per 10(7) nucleotides in rat liver cells. Continuous administration of 400 ppm of MeIQx in the diet for 61 weeks to rats induced hepatocellular carcinomas in all rats. The carcinogenicity of MeIQx at doses of 40 ppm or less is not known yet, but the above results show a linear relationship between the level of MeIQx administered and the adduct level. In rats treated with low doses of 0.4, 4 and 40 ppm of MeIQx, adduct levels increased linearly with time of treatment, the levels in week 12 being two to three times those in week 1. In contrast, on treatment with 400 ppm of MeIQx, the adduct level in the liver increased until week 4, when it was 110 adducts per 10(7) nucleotides, and then remained constant for the next 8 weeks. Induction of the multidrug-resistance gene was suggested to be involved in development of this plateau level.     

Heterozygous p53-deficient mice are not susceptible to 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) carcinogenicity.

2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) is a very potent mutagen which induces tumors in the liver, lung and hematopoietic system of CDF1 mice and the liver, Zymbal gland and skin in F344 rats. The recent development of transgenic knockout mice allows their introduction for sensitive screening of environmental carcinogens due to the rapid development of tumors. P53 gene deficient mice (p53-/-) were found to spontaneously develop malignant lymphoma and hemangiosarcoma, whereas heterozygotes (p53+/-) mice display a high incidence of tumors of the urinary bladder when treated with N-butyl-N-(4-hydroxybutyl)nitrosamine. In the present study, to determine whether p53 gene knockout mice can be utilized in a short-term assay model for the screening of heterocyclic amines (HCAs), the effects of MeIQx, as a representative compound, at low doses were examined. Male and female p53+/- mice and wild type littermates (p53+/+) were continuously given diets containing 0, 0.1, 1, 10 and 100 ppm MeIQx for 1 year. No significant difference in tumor induction was observed other than an increase in liver adenomas in males receiving 10 ppm MeIQx treatment. The results indicate that p53+/- mice have no practical advantages for use in short-term carcinogenicity tests of HCAs.     

DNA Adducts Formed by 2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline in Rat Liver: Dose-Response on Chronic Administration

The effect of administration of 2-amino-3,8-dimethylimidazo[4,5- f ]quinoxaline (MeIQx) at various doses on DNA adduct formation in male rats was examined by ³²P-postlabeling analysis. Administration of MeIQx in the diet at 0.4 ppm, 4 ppm, 40 ppm and 400 ppm for one week resulted in the formations of 0.04, 0.28, 3.34 and 39.0 adducts per 10⁷ nucleotides in rat liver cells. Continuous administration of 400 ppm of MeIQx in the diet for 61 weeks to rats induced hepatocellular carcinomas in all rats. The carcinogenicity of MeIQx at doses of 40 ppm or less is not known yet, but the above results show a linear relationship between the level of MeIQx administered and the adduct level. In rats treated with low doses of 0.4, 4 and 40 ppm of MeIQx, adduct levels increased linearly with time of treatment, the levels in week 12 being two to three times those in week 1. In contrast, on treatment with 400 ppm of MeIQx, the adduct level in the liver increased until week 4, when it was 110 adducts per 10⁷ nucleotides, and then remained constant for the next 8 weeks. Induction of the multidrug-resistance gene was suggested to be involved in development of this plateau level.     

Dose-dependence of promotion of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline-induced rat hepatocarcinogenesis by ethanol: evidence for a threshold

Although ethanol is thought to be a tumor-promoter, there are conflicting results concerning its effects on experimental hepatocarcinogenesis. Furthermore, the relationship between the amount of ethanol consumed and tumor promoting effects has hitherto not been investigated in detail. In the present study, 21-day-old F344/DuCrj rats were fed 200 p.p.m. 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) in their diet for 8 weeks and thereafter received ethanol at doses of 0, 0.1, 0.3, 1, 3, 10 and 20% in drinking water ad libitum for 16 weeks. The incidences of hepatocellular adenoma and total tumors increased dose-dependently with statistical significance at doses of 10% and 20%, compared to the initiated control value. Similarly, dose dependence was observed for the incidence of hepatocellular carcinoma, which was elevated significantly at the dose of 20%. No alteration in development of preneoplastic glutathione-S-transferase placental form positive foci or tumors was observed with 0.1-1%. Cell proliferation also increased dose-dependently and CYP2E1 protein induction was recognized in centrilobular regions without alteration in mRNA levels, but no effects were evident on formation of 8-hydroxy-2'-deoxyguanosine, an oxidative DNA damage marker, or lipid peroxidation in any of the initiated groups. The mRNA expression of cyclin D1 increased dose dependently. The results demonstrated that ethanol dose-dependently promotes hepatocarcinogenesis induced by MeIQx, but with no adverse influence at doses of 1% or less, comparable to sensible drinking levels in humans.     

Formation and Removal of DNA Adducts in the Liver of Rats Chronically Fed the Food-borne Carcinogen, 2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline

Effects of chronic administration of 2-amino-3,8-dimethylimidazo[4,5- f ]quinoxaline (MeIQx) at 0.4, 8 and 400 ppm in the diet on DNA adduct formation and removal in the rat liver were examined by the ³²P-postlabeling method. The 0.4 and 8 ppm doses for 40 weeks resulted in time-dependent increases in MeIQx-DNA adduct levels until 16 and 8 weeks, respectively, with constant values being maintained thereafter. In the case of a carcinogenic dose (400 ppm) of MeIQx, the adduct levels reached a maximum at week 12, and then gradually decreased. Alteration of metabolism of MeIQx during liver carcinogenesis might be related to this decrease in DNA adduct levels. When MeIQx administration was stopped at week 20, 60–90% of the MeIQx-DNA adducts formed with the three doses (0.4, 8 and 400 ppm) of MeIQx were removed in a biphasic manner after return to a basal diet, with initial rapid removal followed by a slow change. No difference in the pattern of MeIQx-DNA adducts was detected on thin layer chromatography at any dose at any time point. Thus, it is suggested that there may be at least two types of damaged DNA, susceptible and resistant to removal of MeIQx-DNA adducts, after chronic administration of MeIQx.     

Dose-dependent Induction of 8-Hydroxyguanine and Preneoplastic Foci in Rat Liver by a Food-derived Carcinogen, 2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline, at Low Dose Levels

Male F344 rats were administered 2-amino-3,8-dimethylimidazo[4,5- f ]quinoxaline (MeIQx) in the diet at doses of 200, 50, 12.5, 3.2, 0.8, 0.2 and 0.05 ppm for 6 weeks, and partially hepatectomized 1 week after the beginning of MeIQx administration. Quantitative values for glutathione S-transferase placental form (GST-P)-positiye foci in the liver were dose-dependently increased by the MeIQx treatment. 8-Hydroxyguanine (8-OHG) levels assessed after 1 week of dietary MeIQx administration were also dose-dependently increased, although the effect was no longer observed at the end of the treatment period. The correlation between numbers of GST-P-positive foci at week 6 and 8-OHG levels at week 1 was linear, values for both parameters being higher than the control levels even in the 0.8 ppm dose group. These findings indicate that, in addition to the previously reported MeIQx-DNA adduct formation, DNA modifications due to oxidative damage may play an important role in MeIQx liver carcinogenesis in rats.     

Heterocyclic amine mixture carcinogenesis and its enhancement by caffeine in F344 rats.

In order to elucidate whether mixed exposure to environmental carcinogens and caffeine increases the risk of cancer induction, we investigated the relationship between preneoplastic lesion development in the liver and colon and drug metabolizing enzyme induction and DNA adduct formation, in rats treated with a mixture of heterocyclic amines (HCAs) and caffeine. In Experiment 1, male F344 rats were administered 3 different HCAs, the food carcinogens, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), alone or in combinations of 2 or 3 at 50 ppm in the diet for 16 weeks. The numbers of hepatic glutathione-S-transferase P form positive (GST-P+) foci and colonic aberrant crypt foci (ACF) were greater in the IQ + MeIQx group than expected from simple summation and increased levels of HCA-DNA adducts were noted. However, no summation was obtained when combined with PhIP, which rather caused inhibition. In Experiment 2, the effects of concurrent caffeine administration on the PhIP carcinogenicity were assessed. Caffeine at 1000 and 500 ppm in the drinking water for 2 weeks significantly increased levels of CYP1A2. Ten weeks concurrent administration of caffeine (1000 ppm) and PhIP (400 ppm) resulted in significant increase of colon ACFs and CYP1A2 expression. Thus, concurrent administration of IQ and MeIQx caused elevation of their carcinogenicity but other mixtures with PhIP did not enhance carcinogenicity. However, a non-carcinogen, caffeine, enhanced PhIP colon carcinogenesis, possibly due to induction of CYP1A2.     

Inhibition of 2-Amino-3, 8-dimethylimidazo[4,5-f]quinoxaline-mediated DNA-adduct Formation by Chlorophyllin in Drosophila

The effect of chlorophyllin on 2-ammo-3,8-dimethylimidazo[4,5- f ]quinoxaline (MeIQx)-mediated DNA-adduct formation in Drosophila was studied. Third-instar larvae of Drosophila were fed MeIQx at 1 mg/6.5 g-feed/bottle, with or without chlorophyllin (100–300 ing). After a 6 h feeding exposure to MeIQx, the larvae were divided into 2 groups. The first group was examined for covalent DNA adducts by ³²P-postlabeIing assay. The second group was assayed for DNA damage by allowing the larvae to develop to adults and measuring the male/female ratio (males, DNA repair-deficient; females, DNA repair-proficient). The ³²P-postlabeling results indicated a significant decrease in DNA adduct levels in larvae treated with MeIQx and 300 mg chlorophyllin (1.7±0.7 adducts/10⁷ nucleotides) as compared with MeIQx-treated larvae (6.5±2.1 adducts/10⁷ nucleotides). The results on male/female sex ratios also indicated a chlorophyllin-indnced decrease in DNA damage by exposure to MeIQx. The suppressive effect of chlorophyllin on the genotoxic actions of a polycyclic mutagen, MeIQx, may be a result of complex formation between chlorophyllin and the mutagen.     

Essential similarities between spontaneous and MeIQx-promoted aberrant crypt foci in the F344 rat colon

Aberrant crypt foci (ACFs) in the Fischer 344 (F344) rat colon, of control or 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx)-treated groups, were compared morphologically, immunohistochemically, and at the molecular biological level in order to elucidate their biological characteristics. Male 3-week-old rats were fed a diet supplemented with or without MeIQx at doses of 100 ppm or less for 16 weeks. The incidence of ACFs was the highest (90%) in animals given 100 ppm MeIQx but that in untreated rats was also surprisingly high (57%). Nine ACFs from nine MeIQx-treated rats and ten ACFs from ten untreated control rats were selected for detailed examination for their large size. There were no morphological differences in macroscopic and microscopic features between MeIQx-promoted and spontaneous ACFs. There were also no differences in immunohistochemical labeling for proliferating cell nuclear antigen (PCNA) and p53 protein between these ACFs although in both cases labeling was higher than in normal crypts. Dot blot hybridization revealed no c-K-ras mutations in codon 12 except in one ACF (11.1%) developing in a rat treated with 100 ppm MeIQx, in which a GGT-->GAT single base substitution was detected. Our results thus suggest that in terms of morphology, cell proliferation, P53 expression and c-K-ras mutation, most ACFs found in rats given 100 ppm MeIQx are essentially identical to their spontaneous counterparts.     

The measurement of MeIQx adducts with mouse haemoglobin in vitro and in vivo: implications for human dosimetry

We have investigated covalent binding of radiolabelled [14C]2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) to mouse haemoglobin in vitro and in vivo. Furthermore, we report the development of a capillary column gas chromatography negative ion mass spectrometry (GC-MS) assay capable of detecting MeIQx liberated from haemoglobin after acid or base hydrolysis. Following microsomal activation, the amount of radiolabelled material associated with haemoglobin in vitro increased with incubation time to 0.67 +/- 0.15 nmol/mg haemoglobin at 2 h (initial concentration 0.47 mM [14C]MeIQx, mean +/- SD, n = 6). Hydrolysis of these samples with acid revealed that 47-60% of the radiolabelled material covalently bound to haemoglobin was acid labile. Of this, 7.2-9.8% was recovered as MeIQx as determined by GC-MS. This liberated fraction should reflect the amount of sulphinic acid amide present which is formed when N-hydroxy-MeIQx reacts with sulphydryl-containing amino acids present in haemoglobin. In vivo, no radiolabelled material bound to haemoglobin could be detected in animals treated with the lowest dose of MeIQx (0.2 mg/kg). At higher doses, there was a dose-dependent increase in the covalent binding of radiolabel to haemoglobin (2.0-200 mg/kg). However, the GC-MS assay for hydrolysable adducts of MeIQx yielded detectable quantities of MeIQx (32.2 +/- 17.5 fmol MeIQx/mg haemoglobin) only at the highest dose used. Application of the GC-MS assay to human haemoglobin samples showed that acid-labile adducts of MeIQx, if present, were below the limit of detection of the assay. These results show that levels of sulphinamide adducts of the dietary aromatic amine MeIQx, with haemoglobin, are very low and the implications for future human dosimetry of this carcinogen are discussed.     

Induction of DNA-adducts and increase of 8-hydroxy-2-deoxyguanosine, but no development of preneoplastic lesions in offspring liver with transplacental and trans-breast milk exposure to 2-amino-3,8-dimethylimidazo [4,5-f ]quinoxaline (MeIQx) in rats

Humans may be exposed to 2-amino-3,8-dimethylimidazo[4,5-f ]quinoxaline (MeIQx) at low doses during the period of gestation and lactation, and thereafter throughout life. The current study was designed to examine the possibility that early exposure may increase the risk of liver tumor development and related genetic changes. Male and female F344 rats were therefore administered MeIQx in diet (1, 10 and 100 ppm) for 4 weeks before mating and also during gestation and lactation. We also examined the carcinogenic risk of low-dose maternal and post-weaning exposure (MeIQx at doses of 1 and 10 ppm). Surviving male F1 rats were sacrificed under ether anesthesia at 19 weeks of age for analyses of glutathione S-transferase placental form-positive foci in the liver and aberrant crypt foci in the colon, as putative preneoplastic lesions. Transplacental and trans-breast milk exposure to MeIQx did not enhance development of the lesions, and levels of cell proliferation in the liver also did not differ from control values. However, excretion of MeIQx into breast milk and transfer to the fetus and offspring were observed with resultant hepatic MeIQx-DNA adducts and 8-hydroxy-2'-deoxyguanosine formation. Thus, our data suggest that maternal exposure to MeIQx during the period of pregnancy and lactation may not increase the risk of hepatocarcinogenesis in male offspring, despite causing genetic damage. If this result can be extrapolated to humans, exposure to MeIQx may not increase carcinogenic risk in offspring at usual human exposure levels.