Metabolism of the food mutagen 2-amino-3-methylimidazo[4,5-f]quinoline in nonhuman primates undergoing carcinogen bioassay.

2-Amino-3-methylimidazo[4.5-f]quinoline (IQ) is a potent bacterial mutagen and rodent carcinogen which also produces hepatocellular carcinoma in monkeys. The metabolism and disposition of this procarcinogen were investigated in monkeys undergoing carcinogen bioassay and in monkeys given an acute dose of IQ. Analysis of urine, feces, and bile revealed that IQ was extensively metabolized. A number of metabolites in urine were purified by high-performance liquid chromatography and characterized by 1H NMR and mass spectroscopy. Metabolites resulted from cytochrome P450-mediated ring oxidation at the C-5 position or N-demethylation. These metabolites could be further transformed by conjugation to sulfate or beta-glucuronic acid. Glucuronidation and sulfamate formation at the exocyclic amine group were other major routes of metabolism. Enteric bacteria also contributed to IQ biotransformation by forming the 7-oxo derivatives of IQ and N-demethyl-IQ. The metastable N2-glucuronide conjugate of the carcinogenic metabolite, 2-(hydroxyamino)-3-methylimidazo[4,5-f]quinoline, was found in urine. This indicates that metabolic activation through cytochrome P450-mediated N-oxidation occurs in vivo and that glucuronidation is a means of transport of the carcinogenic metabolite to extrahepatic tissues.

A carcinogenicity study of instant coffee in Swiss mice.

Commercially available regular instant coffee was given in the diet to barrier-maintained, specified pathogen-free Swiss mice for 2 yr. Groups of 150 males and 150 females were fed diets containing 10, 25 or 50 g instant coffee powder/kg. The animals had already been exposed to coffee in utero. Coffee increased the energy expenditure of the animals as shown by increased daily calorific intake and depressed growth. The overall tumour incidence was inversely correlated to the coffee intake, and no unusual tumour or site of origin was found. The most frequent neoplasms were lymphosarcomas, bronchiolo-alveolar adenomas and adenocarcinomas, as well as hepatocellular adenomas. The incidence of total neoplasms (benign and malignant) decreased from 70.6 and 56.8% in control males and females, respectively, to 34.8 and 36.2%, respectively, in the high-dose group. This decrease, which was essentially due to a reduction in the number of lymphosarcomas and hepatocellular adenomas, was associated with a slower growth rate. The number of leiomyomas in the uterus was slightly increased due to coffee intake as shown by the analysis of positive trend (P less than or equal to 0.05). However, the incidence of this benign tumour was very low; 2.72% of mice affected in the high-dose group, 1.37% in the low-dose group and 0% in the control and medium-dose groups. From this study it is concluded that instant coffee did not increase the incidence of malignant neoplasms in mice when fed at dietary levels of up to 5% for 2 yr.

Purification of the food-borne carcinogens 2-amino-3-methylimidazo [4,5-f]quinoline and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline in heated meat products by immunoaffinity chromatography.

A rapid and simple scheme has been developed for the isolation and purification of two of the major mutagenic heterocyclic amines formed in heated beef products by affinity chromatography using monoclonal antibodies which recognize 2-amino-3-methylimidazo[4,5-f]quinoline (IQ). Two cell lines producing IgG antibodies were established following fusion of Sp2 or P3x.63 myeloma cells with spleen cells of immunized BALB/cby mice. The antigen was bovine gamma globulin haptenized with 2-(3-carboxypropylthio)-3-methylimidazo-[4,5-f]quinoline. The antibodies were immobilized on CNBr-activated Sepharose 4B. IQ and MeIQx formed in heated beef products were partially purified by XAD-2 chromatography and then applied to the affinity columns. Purification by affinity chromatography was adequate for subsequent quantitative analysis by HPLC with UV detection. With this purification scheme as little as 1 g of beef extract or 15 g of fried beef could be assayed for IQ and MeIQx at the part per billion level. Both antibodies had similar affinity constants for IQ (9.3 X 10(6) and 6.7 X 10(6) M-1) and for MeIQx (7.1 X 10(5) and 2.7 X 10(5) M-1) and both were suitable for immunoaffinity purification of IQ from complex mixtures. MAb2 could be used as well to selectively remove MeIQx from meat products after partial purification by XAD-2. MAb1, despite having a 3-fold higher affinity than MAb2 for MeIQx, could not be used for affinity chromatography for this mutagen.

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 been investigated in male Sprague-Dawley rats. Five metabolites were recovered from bile of animals given an intragastric dose of [2-14C]MeIQx. These accounted for nearly all of the radioactivity in bile. The chemical structures of these metabolites were elucidated by proton NMR, UV and mass spectroscopy. Three structures may be assigned unambiguously: two sulfamates, N-(3,8-dimethylimidazo [4,5-f]quinoxalin-2-yl)sulfamic acid and N-(8-hydroxy-methyl-3-methylimidazo[4,5-f]quinoxalin-2-yl) sulfamic acid, and one glucuronide, N2-(beta-1-glucosiduronyl)-2-amino-3,8-dimethylimidazo[4,5-f ]quinoxaline. In addition, an acetyl and a glucosiduronyl conjugate of 5-hydroxy-MeIQx were observed. The spectral evidence did not allow an unambiguous assignment of the site of conjugation. The two glucuronides were excreted in urine and the sulfamate of MeIQx was found in feces as well as urine. All five metabolites were found to be non-mutagenic to Salmonella typhimurium TA98 with or without metabolic activation. The glucuronide conjugates were found also to be non-mutagenic when beta-glucuronidase was incorporated with S-9 mixture in the mutation assay, and thus all appear to be detoxification products. The previously reported metabolite, 2-amino-8-hydroxymethyl-3-methylimidazo[4,5-f]quinoxaline which is mutagenic to Salmonella typhimurium TA98 with metabolic activation, was identified as a minor component in both urine and feces.

Metabolism of the food-borne mutagen/carcinogen 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline in the rat: assessment of biliary metabolites for genotoxicity.

The absorption and kinetics of excretion of [14C]2-amino-3,8-dimethylimidazo[4,5-f]-quinoxaline (MeIQx) was studied in male Sprague-Dawley rats. Within 72 hr of an oral dose of [14C]MeIQx (20 mg/kg) 33-56% of the radioactivity was excreted in the urine and 37-75% of the radioactivity in the faeces, which accounted for greater than 99% of the dose. Only low levels of radioactivity remained in the body. Radioactivity, when expressed per gram of tissue, was highest in the liver and kidney with smaller amounts detected in the lung and both the small and large intestines. Between 25 and 50% of a dose of MeIQx was recovered in the bile within 24 hr. Biliary metabolites were excreted over a long period of time with one radioactive fraction rapidly excreted at 2-3 hr and a second fraction excreted at 10-12 hr. The metabolites present in bile were assessed for genotoxicity using Salmonella typhimurium TA98 with or without hepatic S-9 activation and were found to be present as detoxified products. The residual mutagenic activity present in bile was attributed primarily to unmetabolized MeIQx.

Comparison of the mutagenic activity of various brands of food grade beef extracts.

The mutagenic activity of 9 brands of commercial meat extracts were compared using Ames tester strain TA 98 with hepatic S-9 activation prior to concentration by solvent extraction. In a preincubation test system where 20 mg of intact meat extract preparations were tested per plate, about half of the brands were non-mutagenic whereas the other half increased the spontaneous revertant number by a factor of 2.5-3 (MF). The mutagenic principals could be extracted into methylene chloride under alkaline conditions and the mutagenic activity of these basic organic extracts ranged in potency from a mutation factor of 7-40 (280-1600 revertants) per 2 g equivalents of intact meat extracts in the standard Ames test. A quantitative correlation was obtained between the two trials (intact and extracted samples). The commercial meat extract with the greatest activity contained approximately 70 ng/g of total amino-imidazoquinoline and amino-imidazoquinoxaline compounds while the weakest brand of meat extract contained less than 10 ng/g of these heterocyclic aromatic amines. The human intake of these heterocyclic amines from meat extract in bouillon soups was estimated at maximally 100 ng/day and is approximately 10(3) times less than the estimated total daily intake of all heterocyclic amines which are known to be formed in protein-rich heated foods.

[Food toxicology]. / Zur Problematik der Lebensmittel-Toxikologie.

The complex problems of food toxicology and especially of mutagenesis and carcinogenesis require continuing efforts for a better understanding of the mechanisms, risk evaluation and prevention. Essential progress was the recognition of mutation. In vitro tests now provide reproducible results within a short time. Risk evaluation remains a difficult problem, since is has not been possible yet to establish thresholds values for genotoxic substances. However, threshold levels for carcinogen promoters gain increasing importance as demonstrated for 3 representative classes of substances: mycotoxines , nitrosamines and pesticides.

An evaluation of instant and regular coffee in the Ames mutagenicity test.

High concentrations of "home brew" and instant coffe induced revertants 2--3-fold the spontaneous level with the Ames Salmonella tester strain TA 100 but not with the strains TA 98, TA 1535, TA 1537 and TA 1538. This borderline effect, which may also have been due to non-mutagenic interactions (false positives) occurred only at bacterial levels of coffees and was completely abolished in the presence of the microsomal "metabolic activation system". Negative results were obtained in host-mediated assays when mice received up to 6 g instant coffee/kg body weight. An extrapolation in respect of possible carcinogenic risks is dubious.

Individual susceptibility of mice to a mutagen in both germ and somatic cells.

Male Swiss mice received simultaneously, by gavage, 500 mg methylurea/kg body weight and 25 mg sodium nitrite/kg body weight, and in a further experiment 330 mg of methylurea/kg and 17 mg sodium nitrite/kg, for 5 consecutive days. This treatment resulted in a dose-dependent mutagenic effect in both the dominant-lethal and the micro-nucleus tests. Furthermore, in both mammalian tests, some animals were more susceptible to the mutagenic compounds than were others. However, the liver microsomal metabolizing system did not seem to be responsible for this variation of mutagenic induction.