Lack of Carcinogenicity of 2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) in Cynomolgus Monkeys

The carcinogenic potential of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) was evaluated in cynomolgus monkeys. The animals received MeIQx, beginning at the age of one year, at doses of 10 or 20 mg/kg body weight by gavage five times a week for 84 months and were autopsied 8 months thereafter. Although sporadic development of aberrant crypt foci in the colon and glutathione S-transferase π-positive foci in the liver as well as hyper plastic changes of the lymphatic tissue in the lung and gastro-intestinal tract were observed in several monkeys, this was not treatment-related. No neoplastic or preneoplastic lesions were found in other organs. Serum chemistry data and organ weights were also within the normal ranges. From these data, it is concluded that MeIQx is not carcinogenic in the cynomolgus monkey under the conditions examined. This lack of carcinogenicity is probably related to the poor activation of MeIQx due to the lack of constitutive expression of CYP1A2 as well as an inability of other cytochrome P450s to catalyze N- hydroxylation of MeIQx in the cynomolgus monkey.     

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.     

Hepatocellular Carcinoma Induction in LEC Rats by a Low Dose of 2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline

A food-borne heterocyclic amine, 2-amino-3,8-dimethylimidazo[4,5- f ]qninoxaline (MeIQx), induces hepatocellular carcinomas (HCCs) in F344 male rats at an incidence of 95%, when fed in the diet at 400 ppm for 61 weeks. In this study, the effect of a low dose of MeIQx was examined in Long-Evans with cinnamon-like coat color (LEC) rats, which have a mutation in Atp7b and suffer from hereditary hepatitis and HCCs, with high levels of copper accumulation in the liver. Rats of the LEC and Long-Evans with agouti coat color (LEA) sibling lines were given a diet containing 40 ppm MeIQx from the age of 23 weeks to 63 weeks, for a total administration period of 40 weeks. In LEC rats, HCCs were observed in 8/8 animals administered MeIQx, and 2/8 rats receiving a normal diet. The number of HCCs per rat (mean±SD) was 2.8±2.0 and 0.3±0.5, respectively. In the LEA rats, however, no tumors were induced by administration of MeIQx. These results indicate that damaged liver associated with compensatory cell proliferation is much more susceptible to chemical hepatocarcinogens, including MeIQx, than the normal liver.     

Mechanism of Oxidative DNA Damage Induced by a Heterocyclic Amine, 2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline

Adduct formation has been considered to be a major causal factor of DNA damage by carcinogenic heterocyclic amines. By means of experiments with ³²P-labeled DNA fragments and an electrochemical detector coupled to a high-pressure liquid chromatograph, we investigated whether the N -hydroxy metabolite of 2-amino-3,8-dimethylimidazo[4,5- f ]quinoxaline (MeIQx) can cause oxidative DNA damage or not. This metabolite [MeIQx(NHOH)] was found to cause Cu(II)-mediated DNA damage, including 8-oxo-7,8-dihydro-2'-deoxyguanosine formation. When an endogenous reductant, β-nicotinamide adenine dinucleotide (NADH), was added, the DNA damage was greatly enhanced. Catalase and bathocuproine, a Cu(I)-specific chelator, inhibited the DNA damage, suggesting the involvement of H₂O₂ and Cu(I). MeIQx(NHOH) frequently induced DNA cleavage at thymine and cytosine residues in the presence of NADH and Cu(II). A UV-visible spectroscopic study showed that little decomposition of MeIQx(NHOH) occurred in the absence of Cu(II), whilst rapid spectral change was observed in the presence of Cu(II), suggesting that Cu(II) catalyzes the autoxidation. The addition of NADH reduced the oxidized product back to MeIQx(NHOH). These results suggest that a copper-peroxo intermediate, derived from the reaction of Cu(I) with H₂O₂, participates in Cu(II)-dependent DNA damage by MeIQx(NHOH), and NADH enhances the DNA damage via a redox cycle. We conclude that in addition to DNA adduct formation, oxidative DNA damage plays an important role in the carcinogenic process 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.     

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.     

Carcinogenicity in rats of a mutagenic compound, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline

Carcinogenicity of 2-amino-3,8-dimethylimidazo[4,5-f]-quinoxaline(MeIQx), which is a potent mutagen first isolated from friedbeef and widely present in various cooked foods, was testedin both sexes of F344 rats. Rats were continuously given a dietcontaining 0.04% MeIQx or basal diet and the experiment wasfinished on day 429. In experimental animals, the incidenceof liver, Zymbal gland, clitoral gland and skin tumors was significantlyhigher than in control animals. The incidence of liver tumorswas 100% in males and 53% in females; most liver tumors of maleswere hepatocellular carcinomas and all liver tumors of femaleswere neoplastic nodules. The incidence of Zymbal gland tumorswas 75% in males and 53% in females. Clitoral gland tumors wereinduced in 63% and skin tumors were observed in 35% of malesand 5% of females. Most of these three types of tumors werediagnosed as squamous cell carcinoma. In the control rats, liver,Zymbal gland, clitoral gland and skin tumors were not observedin either sex.     

Down-regulation of 2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx)-induced CYP1A2 Expression Is Associated with Bovine Lactoferrin Inhibition of MeIQx-induced Liver and Colon Carcinogenesis in Rats

The inhibitory influence of bovine lactoferrin (bLF) on induction of preneoplastic hepatic glutathione S -transferase placental form-positive (GST-P⁺) cell foci and colon aberrant crypt foci (ACF) by diethylnitrosamine (DEN) and 2-amino-3,8-dimethylimidazo[4,5- f ]quinoxaline (MeIQx) was investigated in F344 rats. Rats were initially treated with DEN, then placed on basal diet containing MeIQx (200 ppm) alone, MeIQx plus 2% bLF, or MeIQx plus 0.2% bLF from week 2 to week 8, with partial hepatectomy performed at week 3. Concomitant administration of 2% or 0.2% bLF with MeIQx caused significant dose-dependent decreases in both number and unit area of GST-P⁺ cell foci (2% bLF, P <0.001; 0.2% bLF, P <0.01). Similar results were observed for MeIQx-induced colon ACF in the groups without DEN treatment (2% and 0.2% bLF, P <0.05). To investigate the underlying mechanisms, we analyzed the influence of bLF on levels of cytochrome P4501A2 (CYP1A2), a metabolically activating enzyme of MeIQx in the liver. The results demonstrated that combined administration of 2% bLF significantly reduced levels of MeIQx-induced CYP1A2 mRNA ( P <0.05) and protein ( P <0.05) to the normal levels, in association with reduced values for MeIQx-DNA adducts ( P <0.05), liver GST-P⁺ cell foci and colon ACF. These results suggest that bLF is a chemopreventive agent for DEN alone or DEN plus MeIQx-induced liver, and MeIQx-induced colon carcinogenesis in rats. One possible mechanism is a normalizing down-regulation of CYP1A2 expression by bLF, with consequent reduction of carcinogen activation and adduct formation.     

Lack of carcinogenicity of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) in cynomolgus monkeys.

The carcinogenic potential of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) was evaluated in cynomolgus monkeys. The animals received MeIQx, beginning at the age of one year, at doses of 10 or 20 mg/kg body weight by gavage five times a week for 84 months and were autopsied 8 months thereafter. Although sporadic development of aberrant crypt foci in the colon and glutathione S-transferase pi-positive foci in the liver as well as hyperplastic changes of the lymphatic tissue in the lung and gastro-intestinal tract were observed in several monkeys, this was not treatment-related. No neoplastic or preneoplastic lesions were found in other organs. Serum chemistry data and organ weights were also within the normal ranges. From these data, it is concluded that MeIQx is not carcinogenic in the cynomolgus monkey under the conditions examined. This lack of carcinogenicity is probably related to the poor activation of MeIQx due to the lack of constitutive expression of CYP1A2 as well as an inability of other cytochrome P450s to catalyze N-hydroxylation of MeIQx in the cynomolgus monkey.     

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.     

Formation of 2-Amino-3-methylimidazo[4,5-f]quinoline- and 2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline-sulfamates by cDNA-expressed Mammalian Phenol Sulfotransferases

In rat liver cytosol systems, 2-amino-3-methylimidazo[4,5- f ]quinoIine (IQ) and 2-amino-3,8-dimethyl-imidazo[4,5- f ]quinoxaline (MelQx) were converted into their sulfamates in the presence of 3'-phosphoadenosine 5'-phosphosulfate at rates of 51.2 and 50.7 pmol/min/mg cytosol in the male, and 23.7 and 22.5 pmol/min/mg cytosol in the female, respectively. IQ-sulfamate formation was low (0.24 pmol/min/mg cytosol) in human liver cytosols, and MeIQx-sulfamate was not detected (<0.1 pmol/ min/mg cytosol). These results suggest only a minor contribution of IQ- and MeIQx-sulfamate formation to the detoxification of both heterocyclic amines in humans. Using sulfotransferase cDNA-expression systems, a rat ST1A1 arylsulfotransferase has been shown to catalyze the formation of the sulfamates, suggesting a role of the ST1A type of sulfotransferase in the N-sulfation of heterocyclic amines.     

Carcinogenicity in mice of a mutagenic compound, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) from cooked foods

2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), whichis a mutagenic compound present in fried beef and beef extracts,was given orally to CDF₁ mice at a concentration of 0.06% inthe diet for 84 weeks. Liver tumors were induced in 43% of malesand 91% of females fed MeIQx. The incidences of liver tumorsin mice of both sexes were significantly higher in groups fedMeIQx than in control groups. The incidences of lung tumorsin females fed MeIQx and of lymphomas and leukemias in bothsexes fed MeIQx were also significantly higher than in the respectivecontrols.     

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.     

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.     

Lack of modification of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) rat hepatocarcinogenesis by caffeine, a CYP1A2 inducer, points to complex counteracting influences

2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), one of the most abundant carcinogenic heterocyclic amines in cooked foods, is speculated to be a human liver carcinogen. To test the hypothesis that it is metabolically activated by CYP1A2, we here investigated the effects of caffeine as a CYP1A2 inducer on MeIQx induced rat hepatocarcinogenesis in a medium-term liver bioassay system. Unexpectedly, no modifying effects of caffeine on MeIQx-induced hepatocarcinogenesis were evident, although up-regulation of CYP1A2 and NAT2 were detected. Therefore, mRNAs extracted from GST-P positive foci and the surrounding liver tissue in each group were analyzed to explore mechanisms in detail. The results suggest that suppression of syndecan-2 (Sdc2) and induction of cell cycle arrest through a p21-dependent pathway might have counter-acted any promotion effects of up-regulation of CYP1A2.     

Enhancement by Cigarette Smoke Exposure of 2-Amino-3,8-dimethylimidazo[4,5- f]quinoxaline-induced Rat Hepatocarcinogenesis in Close Association with Elevation of Hepatic CYP1A2

The modifying effects of cigarette smoke (CS) exposure on a heterocyclic amine (HCA) 2-amino-3,8-dimethylimidazo[4,5- f ]quinoxaline (MeIQx)-induced carcinogenesis were investigated in male F344 rats. Groups 1 and 2 were fed MeIQx at a dose of 300 ppm, and simultaneously received CS and sham smoke (SS) for 16 weeks, respectively. Groups 3–5 were given the MeIQx diet for 4 weeks, and simultaneously exposed to CS for 4 weeks (group 3), exposed to CS for 12 weeks after the MeIQx treatment (group 4) or received SS for 16 weeks (group 5). Groups 6 and 7 were fed basal diet and respectively received CS and SS for 16 weeks. In terms of the mean number or area, the development of glutathione S-transferase placental form-positive (GST-P⁺) liver cell foci was significantly ( P <0.01) greater in group 1 than in group 2. The mean number of colonic aberrant crypt foci (ACFs) per animal was increased by continuous CS exposure regardless of MeIQx feeding, the differences between groups 4 and 5 ( P <0.05), and between groups 6 and 7 ( P <0.05) being significant. Immunoblot analysis confirmed that the hepatic CYP1A2 level in group 6 was remarkably increased as compared to that in group 7. In addition, liver S9 from rats in group 6 consistently increased the mutagenic activities of six HCAs including MeIQx as compared to those in group 7. Thus, our results clearly indicate that CS enhances hepatocarcinogenesis when given in the initiation phase via increasing intensity of metabolic activation for MeIQx and possibly colon carcinogenesis when given in the post-initiation phase in rats induced by MeIQx.     

Quantitation of 2-amino-3-methylimidazo[4,5-f]quinoline and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline DNA adducts in specific sequences using alkali or uvrABC excinuclease

2-Amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MelQx) are carcinogens found in cooked meats that form DNA adducts upon metabolic activation. Purified DNA from Chinese hamster ovary (CHO) cells was reacted in vitro with the active metabolites N-acetoxy-lQ or N-acetoxy-MelQx, and the adduct levels in the 5′ dihydrofolate reductase (DHFR) gene and downstream region were quantitated by Southern hybridization. Adducted and restricted DNA was treated with Escherichia coli uvrABC excinuclease or alkali (0.1 N NaOH, 37°C, 60 min) to incise DNA at IQ and MeIQx adduct sites. The DNA was then denatured with formamide, electrophoresed on a neutral agarose gel, transferred to a support membrane, and hybridized with sequence-specific DNA probes. Both uvrABC and alkali reduced the intensity of Southern hybridization in proportion to the number of IQ or MeIQx adducts in DNA, indicating that these adducts are substrates for uvrABC and that they form alkali-labile lesions in DNA. IQ and MeIQx adduct levels were the same in the 5′ DHFR gene and in the downstream region. Southern hybridization analysis of pBR322 containing known levels of IQ or MeIQx adducts showed that the efficiency of cutting IQ or MeIQx adducts by uvrABC excinuclease and alkali was approximately 30% and 15%, respectively. ³²P-postlabeling studies examining adduct level in bulk DNA further showed that the adduct profiles were identical in pBR322, CHO DNA, and cultured CHO cells exposed to the reactive metabolites of IQ or MeIQx. The results indicate that IQ and MeIOx adducts can be quantitated in specific genomic sequences and that this method should be directly applicable to studies of gene-specific repair of these adducts in cultured cells.     

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.     

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.     

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

A metabolite of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline(MeIQx) was incorrectly characterized in this paper. The metabolitewas thought to be an acetyl conjugate of the 5-hydroxyl-atedderivative of MeIQx. This assignment is incorrect. The correctassignment is a sulfate conjugate of 5-hydroxy-MeIQx, 2-amino-3,8-dimethylirnidazo[4,5-f]quinoxaUn-5-yl-sulfate.This conclusion is based upon repurified sample analyzed by¹H NMR and ¹³C NMR, enzyme hydrolysis assays, IR spectro-scopyand FAB/MS (accurate mass measurement).