Frequent consumption of meat has been associated with an increased risk of colorectal cancer. Such a risk may be due to naturally occurring compounds in the meat, substances added to the meat, or agents formed during cooking. Concerning the latter alternative, mutagenic heterocyclic amines are multi-site animal carcinogens, but their relevance to human cancer has yet to be determined. In the present study, we made a population-based inventory of cooked meat dishes consumed in the county of Stockholm, ranked dishes according to cooking method and frequency of consumption and, in addition, determined levels of mutagenic activity in six commonly consumed fried meat dishes. Meat was consumed, on average, 493 times per year, giving 1.4 daily servings. Frying was the most common way to cook meat. When ranking meat dishes according to intake frequency, the top eight dishes were as follows: sausage, steak casserole, meatballs, pork chops, pork belly, bacon, ground beef patties, and finally, mince-meat sauce. The frying sessions were performed under controlled conditions at four different temperatures, and we documented the degree of surface browning and measured mutagenic activity in six frequently eaten dishes (sausage, meatballs, pork chops, pork belly, ground beef patties, and minute beef). We found extracts from all six dishes to be mutagenic, and a mean daily dose of exposure was calculated, giving 862 revertants. This investigation leaves no doubt that a major portion of the total meat consumption is fried before ingestion and that fried meat dishes frequently consumed by an elderly population in Stockholm contain mutagenic substances. Furthermore, the study provides usable information for future epidemiological research in which it is necessary to disentangle the effect of meat per se from the effect of potentially carcinogenic heterocyclic amines.

The cytochrome P-4501A1 and P-4501A2 enzymes are involved in the metabolic activation of a number of environmental precarcinogens including the food-derived heterocyclic amines. These compounds also induce CYP1A activity in rats, a feature they have in common with several of the xenobiotics (most notably benzo[a]pyrene) which are metabolically activated by the cytochrome P-4501A isozymes. Using an in vitro DNA binding assay and an in vivo functional (transactivating) assay, we have found that the cytochrome P-4501A1 induction response produced by the heterocyclic amines is mediated by the intracellular dioxin receptor. In the absence of ligand, the receptor is inactive and does not bind DNA. In this report we demonstrate a correlation between activation of DNA binding activity of the receptor by the heterocyclic amines and their ability to induce transcription from a minimal dioxin-response element-driven promoter construct. However, relatively high doses of these compounds are required to produce these effects. Therefore, despite a relatively high dietary intake of heterocyclic amines as compared to other cytochrome P-4501A1-inducing substances, the heterocyclic amines most probably only play a minor role in the induction of this enzyme activity. In contrast to the heterocyclic amines, indolo derivatives found in cruciferous vegetables exhibit high affinity for the dioxin receptor and represent potent activators of the DNA binding activity of the receptor. The indolo derivatives are therefore likely to have a larger impact than the heterocyclic amines on human CYP1A1 activity.

Humans frequently inhale as well as ingest cooked-food mutagens, among which the heterocyclic amines are the quantitatively most important. An extensive systemic distribution of these mutagens implies that most tissues in the body are exposed. Tissues containing cytochrome P450 (CYP) may be particularly susceptible to DNA damage. Accordingly, animal experiments have shown that oral exposure to heterocyclic amines leads to tumor formation at multiple sites. CYP1A2, which has only been demonstrated in the liver, seems to be the isozyme most efficient in metabolically activating the heterocyclic amines. In extrahepatic tissues, however, other CYP forms are likely to be important. Using Salmonella mutagenicity as an endpoint, we have studied the metabolic activation of 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) by isolated lung microsomes from rats and mice. Our studies show that CYP2A3, an isozyme that has hitherto not been investigated with regard to its capacity to activate heterocyclic amines, catalyses a major part of the IQ activating reactions in the uninduced lung. The formation of mutagens during cooking of meat is highly temperature dependent and meat extracts heated at 200 degrees C show a strong mutagenic activity in the Ames Salmonella assay. These extracts caused mutations at the HPRT locus in normal human fibroblasts as well as a pronounced decrease in survival of the cells. Furthermore, the heated meat extracts caused a decreased proliferative activity in primary cultures of normal mouse colonic epithelial cells as measured by autoradiography.

The emission of polycyclic aromatic hydrocarbons (PAH) was quantitated in the smoke from the grilling of meat using hardwood charcoal as fuel in a table grill set. Smoke samples were collected with an ice-cooled condenser and subsequently cleaned up employing both open column- and high performance-liquid chromatography techniques. Using a PAH standard mixture, 23 PAH were identified in the smoke using gas chromatography-mass spectrometry. Emission levels of PAH ranged from several tens of μg kg−1 meat for some 3- and 4-ringed PAH, to sub μg kg−1 meat levels for the 5- and 6-ringed PAH (sum of PAH approximately 0.1 mg kg−1 meat), originating from a 10 min grilling of 0.33 kg minced lean pork. The major source of PAH emitted to the local air environment from charcoal grilling is from the combustion of the charcoal itself. The amount of PAH emitted from charcoal grilling in Sweden, time period June to August, is estimated to approximately 2 kg which is a minor source to the total emissions of PAH to the environment. However, relatively large peak exposures of PAH can be expected in a more local environment, e.g. during household grilling.

The food-derived heterocyclic amines 2-amino-3-methyl-imidazo[4,5-f]quinoline (IQ) and 2-amino-3,8-dimethyl-imidazo[4,5-f]quinoxaline (MeIQx) are carcinogenic in rodents as well as in non-human primates (IQ). Using a short-term liver carcinogenesis model the compounds were found to be only weak initiators. It is also possible that these amines are involved in other phases of the carcinogenesis process. In an attempt to study the promotive effects of IQ and MeIQx, these heterocyclic amines were repeatedly given by gavage, over a period of 10 days, to Wistar rats previously initiated with diethylnitrosamine (DEN) and subjected to a partial hepatectomy. The rats were killed 1 month after DEN administration and their livers examined for glutathione-S-transferase positive (GST-P) foci. The positive control, 2-acetylaminofluorene, significantly increased the development of GST-P foci in the DEN-treated rats compared to the negative control, saline. However, neither the IQ- nor the MeIQx-treated groups differed from the controls and thus these amines do not promote the growth of DEN-initiated GST-P liver foci in this model.

The metabolic activation of the promutagens 2-amino-3,8- dimethylimidazo[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) by rat and mouse lung microsomes was studied using Salmonella mutagenicity (strain TA98). Lungs from uninduced animals were found to activate all three compounds. A 4-6 fold higher mutagenic activity was obtained with IQ compared to MeIQx and the mutagenic response of PhIP was 1-2 orders of magnitude lower than that of IQ. In order to characterize the forms of P450 in the lung responsible for the metabolic activation of these food mutagens Western blots were performed with microsomes and partially purified P450 fractions from the lung. Western blots revealed the presence of cytochrome P450 2A, 2B and 4A forms in untreated rats. In the lung CYP 1A1 was only detectable after BNF treatment of rats. The CYP 4A isozymes, which have not previously been described in the rat lung, were further identified after PCR amplification from lung mRNA as 4A2 and 4A8. Antibody inhibition studies showed that CYP 2A3 catalyzed a major part (70%) of the metabolic activation of IQ by uninduced rat lung microsomes. The metabolic activation of MeIQx was not influenced by this antibody. An antibody against CYP 2B isozymes also partially inhibited the activation of IQ by uninduced rat lung microsomes. However, since induction of CYP 2B isozymes in the liver by phenobarbital treatment did not increase the metabolic activation of the heterocyclic amines over controls it is unlikely that the rat lung CYP 2B1 is participating in the activation of heterocyclic amines. The inhibition of the IQ-dependent mutagenicity by the CYP 2B antibody is probably due to cross-reaction with CYP 2A3. Alfa-naphthoflavone (ANF), considered to be a specific inhibitor of CYP 1A isozymes at 10 microM, partly inhibited the activation of IQ (30-40%) and MeIQx (60-80%) by uninduced rat and mouse lung microsomes. Upon pretreatment of rats with BNF, lung microsomes activated MeIQx at a rate that was 2-10-fold higher than control lung microsomes, whereas the increase in EROD activity was approximately 100-fold in the same lung preparations. These results suggest that CYP 1A1 may not be the enzyme responsible for the activation of MeIQx in the control rat despite the inhibition with ANF. It is likely that ANF can inhibit other P450 enzymes in the lung, including CYP 2A3.(ABSTRACT TRUNCATED AT 400 WORDS)

Pharmacology & ToxicologyVolume 71, Issue 6 p. 457-460 Tissue Distribution of the Food Mutagen MeIQx in Control and BNF-Treated Mice Eva Brittebo, Eva Brittebo Department of Pharmacology, University of Lund, Sölvegatan 10, S-223 62 Lund, SwedenSearch for more papers by this authorCatarina Eriksson, Catarina Eriksson Department of Pharmacology and Toxicology, SLU, Box 573, S-751 23 Uppsala, SwedenSearch for more papers by this authorEva Övervik, Eva Övervik Department of Medical Nutrition, Karolinska Institute, Novum F 60, Huddinge University Hospital, S-141 86 Huddinge, SwedenSearch for more papers by this authorJan-Åke Gustafsson, Jan-Åke Gustafsson Department of Medical Nutrition, Karolinska Institute, Novum F 60, Huddinge University Hospital, S-141 86 Huddinge, SwedenSearch for more papers by this authorIngvar Brandt, Ingvar Brandt Department of Pharmacology and Toxicology, SLU, Box 573, S-751 23 Uppsala, SwedenSearch for more papers by this author Eva Brittebo, Eva Brittebo Department of Pharmacology, University of Lund, Sölvegatan 10, S-223 62 Lund, SwedenSearch for more papers by this authorCatarina Eriksson, Catarina Eriksson Department of Pharmacology and Toxicology, SLU, Box 573, S-751 23 Uppsala, SwedenSearch for more papers by this authorEva Övervik, Eva Övervik Department of Medical Nutrition, Karolinska Institute, Novum F 60, Huddinge University Hospital, S-141 86 Huddinge, SwedenSearch for more papers by this authorJan-Åke Gustafsson, Jan-Åke Gustafsson Department of Medical Nutrition, Karolinska Institute, Novum F 60, Huddinge University Hospital, S-141 86 Huddinge, SwedenSearch for more papers by this authorIngvar Brandt, Ingvar Brandt Department of Pharmacology and Toxicology, SLU, Box 573, S-751 23 Uppsala, SwedenSearch for more papers by this author First published: December 1992 https://doi.org/10.1111/j.1600-0773.1992.tb00578.xCitations: 1 We thank Dr. Spiros Grivas for the synthesis of 14C-MeIQx and Margareta Mattson for skilful technical assistance. Financial support was given by the Swedish Council for Forestry and Agricultural Research, the Swedish Cancer Society and the Faculty of Medicine, University of Lund. Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Citing Literature Volume71, Issue6December 1992Pages 457-460 RelatedInformation

Extracts from lean pork heated at 200-degrees-C have a strong mutagenic activity in the Ames Salmonella assay (strain TA98 + S9). The formation of mutagenicity is highly temperature dependent, thus an extract heated at 100-degrees-C is not mutagenic in this system. This paper shows that the 200-degrees-C extract also causes mutations at the hprt locus in normal human fibroblasts, as demonstrated by a dose-dependent increase of 6-thioguanine resistant mutants. The mutation frequencies were increased 6 and 13 times, respectively, for extract concentrations corresponding to 100 and 200 mg meat/ml medium. Heterocyclic amines, previously shown to be present in the 200-degrees-C extract are conceivably responsible for at least part of the observed mutagenicity. The extracts prepared at 100-degrees-C had no significant effect on the mutant frequency in human fibroblasts. A pronounced, dose-dependent, decrease in cell survival was observed with both the 100 and the 200-degrees-C extracts. The nature of the cytotoxic components is not clear and might be different in the two extracts.

Lactic acid bacteria have been reported to have antimutagenic properties in vitro. In order to investigate whether Lactobacillus acidophilus supplements have antimutagenic effects in humans, 11 healthy subjects on a standardised diet consumed fried beef patties twice daily for 3 d. The diets were supplemented with ordinary Lactococcus fermented milk (phase 1) and thereafter with L. acidophilus fermented milk (phase 2), whereby the excretion of mutagenic activity was determined in urine and faeces. In both faeces and urine high levels of mutagenicity were detected during phase 1. There was an increase in lactobacilli in the intestinal microflora in seven of 11 subjects by the L. acidophilus supplement (phase 2), and the mutagenic activity in urine was 72 per cent lower on day 2 (P < 0.01) and 55 per cent lower on day 3 (P < 0.05) compared to days 2 and 3 in phase 1. The total faecal and urinary mutagen excretion on day 3 during phase 2 was 47 per cent lower compared to day 3, phase 1 (P<002). Thus, L. acidophilus given together with fried meat lowered mutagen excretion in humans.

The carcinogenic heterocyclic amines, which are formed upon cooking of protein-rich food, are activated in the body mainly by cytochrome P450 IA1 and IA2. Several of these co-called food mutagens have, by enzymatic and immunoblotting techniques, been shown to be weak inducers of cytochrome P450 IA in the rat. To elucidate whether this induction occurs via the dioxin receptor, the capacity of the heterocyclic amines to activate in vitro the dioxin receptor to a DNA-binding form was determined. The activation of the receptor in Hepa cell cytosol was analyzed with a gel-retardation assay using a [32P]3'-end-labeled synthetic oligonucleotide, corresponding to nucleotides -1026 to -999 of the rat cytochrome P450 IA1 gene (XRE1), as probe. Five out of 14 heterocyclic amines had the ability to induce specific DNA-binding activity in the Hepa cell cytosol in a dose-dependent manner. The concentrations eliciting 50% of the maximal effect (EC50) were found to be between 30 and 135 microM. Thus, in comparison to high-affinity ligands such as benzo[a]pyrene (B[a]P; EC50 11.2 nM), and 2,3,7,8-tetrachloro-1,6-dibenzo-p-dioxin (TCDD; EC50 1.9 nM), the activating capacity of the heterocyclic amines is low. Binding to the dioxin receptor has been shown to be dependent on the three-dimensional size of a molecule in relation to a 6.8 x 13.7 A rectangle. The relatively low EC50 values found for the heterocyclic amines might be explained by the smaller size of these molecules when compared to that of B[a]P and TCDD.

Most heterocyclic amines formed during the cooking of meat and fish have been shown to form adducts in the livers of rats. Recently, however, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), administered in the diet to Fischer 344 (F344) rats for 4 weeks, was shown to produce the highest levels of adducts in the heart. In the present study 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,8-dimethylimidazo [4,5-f]quinoxaline (MeIQx), 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and 2-amino-6-methyldipyrido[1,2-a:1',2'-d]imidazole (Glu-P-1) were given to F344 rats at carcinogenic dose levels (IQ 0.03%, MeIQx 0.04%, Trp-P-1 0.015%, Glu-P-1 0.05%) in the diet for 4 weeks. DNA adducts in the liver and heart were analyzed by 32P-postlabeling. DNA adducts were demonstrated to appear in the hearts of all animals exposed to heterocyclic amines at the following levels: IQ, 1.8 adducts/10(7) nucleotides, MeIQx, 3.8/10(7) ntd, Trp-P-1, 20/10(7) ntd and Glu-P-1, 7.2/10(7) ntd. Values for the heart were 10-20% of the respective liver adduct levels. Heart adducts increased linearly throughout the observed period when MeIQx was administered for up to 40 weeks. When MeIQx feeding was discontinued after 20 weeks and the animals subsequently given the basal diet, the adduct level at 20 weeks did not change during the following 20 weeks. A possible role for heart DNA alterations caused by food-borne heterocyclic amines in the development of age-related myopathies and cardiovascular disease is not inconceivable.

Mutagenic and carcinogenic heterocyclic aromatic amines are formed in protein-rich food cooked under household conditions. The compounds have been shown to be activated mainly by cytochromes P-450 IA1 and IA2. In the conventional rat, these P-450 enzymes were increased in the intestinal mucosa following oral administration of fried meat at a level comparable to human consumption. No such increase was observed in germfree rats. Furthermore, the metabolic disposition of the mutagenic material differed between the germfree and the conventional animals. A different urinary pattern of mutagenic metabolites and a substantially lower fecal mutagen excretion was observed in the germfree rats. The results implies a role of the intestinal microflora in the metabolism of fried meat mutagens.

Journal Article Cooked-food mutagens: current knowledge of formation and biological significance Get access Eva Övervik, Eva Övervik 1 Department of Medical Nutrition, Karolinska Institute, NOVUM, F60 Huddinge University HospitalS-141 86 Huddinge, Sweden 1To whom correspondence should be addressed Search for other works by this author on: Oxford Academic PubMed Google Scholar Jan-Åke Gustafsson Jan-Åke Gustafsson Department of Medical Nutrition, Karolinska Institute, NOVUM, F60 Huddinge University HospitalS-141 86 Huddinge, Sweden Search for other works by this author on: Oxford Academic PubMed Google Scholar Mutagenesis, Volume 5, Issue 5, September 1990, Pages 437–446, https://doi.org/10.1093/mutage/5.5.437 Published: 01 September 1990 Article history Received: 12 January 1990 Accepted: 03 May 1990 Published: 01 September 1990

To evaluate a possible role of the intestinal microflora in the metabolism of the highly mutagenic compounds formed in fried meat, conventional and germfree male AGUS rats were fed a semi-synthetic diet containing fried meat. Changes in mutagen excretion in urine and faeces over time were studied using the Ames Salmonella assay. The faecal and urinary extracts were separated by means of high-performance liquid chromatography (HPLC), and the mutagenicity of the collected fractions was determined. Cytochrome P-450 IA (IA1 and/or IA2) were detected by the use of antibodies with the Western blot technique, and the corresponding enzyme activities were measured in microsomes from the small intestine and the liver. A quantitative as well as qualitative difference in excretion of mutagens between germfree and conventional rats was observed. The total excreted of mutagenicity was significantly higher for the conventional than for the germfree rats, as a result of a higher faecal excretion of mutagens in the conventional animals. The HPLC separations of urinary and faecal extracts showed a different mutagenic metabolite pattern between the germfree and conventional rats. An increased activity of the cytochrome P-450-dependent enzyme ethoxyresorufin-O-deethylase was observed in the small intestine of conventional rats on the fried meat diet, whereas no effect of this diet was observed in the germfree rats. Similar results were obtained in immunoblotting experiments using a P-450 IA antiserum. The present study indicates that the excretion pattern and thus also the metabolism of compounds present in fried meat are affected by the germfree status.

The effect of cooking time on mutagenic activity in crust, pan residue and smoke from pan-broiled pork patties was studied in the Ames Salmonella mutagenicity test system. The effect on mutagenicity of reheating the cooked patties and of keeping them warm was also studied. The meat was broiled at 200°C for various times between 2 and 10 min. Broiled meat was reheated up to 5 times at 200°C, each time to a centre temperature of 70°C. Reheating was also performed in a microwave oven for 2 min and in an electric oven at 200°C for 10 min. In addition, broiled patties were kept warm at 60°C in an incubator for up to 9 hr. The mutagenic activity increased rapidly in all fractions except the volatile phase over the first 6 min of cooking, after which time only a slight increase was seen. At cooking times below 4 min no mutagenic activity was detected in the smoke. Reheating or keeping the meat warm for up to 9 hr had very little effect on the mutagenic activity of the meat. Reversed-phase high-performance liquid chromatography mutagenicity profiles of the aerosol, crust and pan-residue extracts showed no major qualitative differences in samples cooked at different times. It is concluded that during pan broiling at 200°C the major part of the mutagenic activity is formed during the first 6 min of cooking. Reheating the meat or keeping it warm does not significantly affect the mutagenic activity. No major additional mutagens are formed during continued heating for up to 25 min.

Cooking fumes constitute a major part of the mutagenic activity detected in indoor air samples. The chemical nature of the smoke mutagens has not been determined, but existing data indicate the presence of carcinogenic heterocyclic amines, previously identified in crust and pan residue from fried meat. Reports on the chemical nature and biological activity of these compounds are reviewed.

The promutagenic 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) found in cooked food are converted to their active forms mainly by cytochrome P450 forms IA1 and IA2. By induction of these isoenzymes the food mutagens could thus influence their own rate of activation. Male and female Wistar rats were given MeIQx, PhIP, beta-naphthoflavone (BNF) or saline i.p. at 50 mg/kg body wt on three consecutive days. On the fourth day the rats were killed and lungs, kidneys, liver and intestines taken. The microsomal fraction from each organ was prepared as well as 9000 g supernatant from the liver. The induction of cytochrome P450IA was measured at the protein level by enzymatic assays (ethoxyresorufin-O-deethylation, Ames' mutagenicity test) and immunoassays (Western blot) and at the pretranslational level by RNA hybridization (Northern blot). The binding affinities of MeIQx, PhIP and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) for the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-receptor were studied by evaluation of the competition with 3H-labelled TCDD for specific binding. Ethoxyresorufin-O-deethylase (EROD) activity was significantly increased in the liver (males 2.1-fold, females 3.3-fold), kidneys (males 2.1-fold, females 1.8-fold) and lungs (males 4.3-fold, females 3-fold) of the MeIQx-treated rats. Furthermore, the levels of cytochrome P450IA proteins were increased in these animals. It was not possible, however, to detect the corresponding mRNA. In the case of the PhIP-treated animals a significantly increased EROD activity (2.7-fold) and an increased cytochrome P450IA protein level were seen only in the male lungs. Only a very weak TCDD-receptor affinity was observed for PhIP, whereas MeIQx or IQ did not appear to compete significantly with [3H]TCDD for binding to the TCDD-receptor. It is concluded that MeIQx is a weak inducer of cytochrome P450IA in several organs of the rat, while PhIP induced these isoenzymes only in the male lungs. More work is needed to clarify the mechanism(s) whereby this induction occurs.

Creatine or one of 15 amino acids were mixed with minced pork before broiling at 200 degrees C. Total mutagenic activity and reversed-phase HPLC-separated mutagenicity profiles were determined for the crust and pan residue of all samples and also in the aerosol fraction of the smoke formed during cooking of the creatine-fortified samples. Addition of 5% (w/w) creatine increased the total mutagenicity 4-fold without changing the mutagenicity profile of either crust, pan residue or aerosol. Amino acid addition (1% w/w) increased the total mutagenicity between 1.5 (lysine) and 43 times (threonine). In most cases the mutagenicity profiles of crust and pan residues were changed by amino acid addition. Dry-heated mixtures of amino acids and creatine were all mutagenic with a 250-fold range between the amino acids. The production of known food mutagens in these mixtures was analyzed by LC-MS of HPLC-fractionated mutagenic peaks. Serine, threonine, phenylalanine, alanine, leucine and tyrosine were all shown to give rise to one of the known food mutagens 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) or 2-amino-trimethylimidazopyridine (TMIP). Lyophilized and subsequently fried meat patties and a heated powder of lyophilized meat juice were both mutagenic, with mutagenicity profiles similar to the regular meat crust, showing that water is not a prerequisite for mutagen formation in meat. MeIQx, 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-di-MeIQx) and PhIP were shown, by LC-MS, to be present in the dry-heated meat juice. It is concluded that creatine and free amino acids are the main reactants of the mutagen-forming reactions that occur during frying of meat. Creatine is probably a necessary part of all of these reactions; what specific compounds are formed in each case therefore depends upon the levels in the meat of certain free amino acids and their interactions with other, as yet unknown, compounds in the meat.

The initiating activity of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and a mutagenic meat extract obtained from cooked meat was examined, using the resistant hepatocyte model (RH-model). Male Wistar rats were given a single i.p. injection of PhIP (50 or 75 mg/kg body weight) or MeIQx (50 mg/kg body weight) after a 2/3 partial hepatectomy (PH). The meat extract (corresponding to 1050 g meat/animal) was given by gastric feeding at three time points after PH. Two weeks after initiation the rats received a diet containing 0.02% 2-acetylaminofluorene for a period of 2 weeks. In the middle of this period a single dose of carbon tetrachloride was given. Rats were killed 6 weeks after the experimental start. The number of enzyme-altered (gamma-glutamyl-transferase positive) hepatic foci was significantly increased in the animals given MeIQx (P less than 0.05) and the highest dose of PhIP (P less than 0.01) whereas no effect of the meat extract was observed. The mutagenic meat extract was also studied with regard to promotive capacity in vivo, the meat extract was given in the diet of diethylnitrosamine initiated rats for a period of 6 weeks. No significant changes were detected after administration of the meat extract in the diet. It is concluded that both MeIQx and PhIP are weak initiators in the RH-model, in the same order of magnitude as the previously investigated, structurally related pyrolysis products. The meat extract was not active in the RH-model under the conditions used in this study.

Fried meat was included in the diet of Sprague-Dawley rats during one week. Ingested and excreted amounts of mutagenic activity were determined daily by the use of the Ames' Salmonella/mammalian microsome test on extracts of the diet, urine and feces. In addition, the effect of the fried meat on ethoxyresorufin-O-deethylase activity in the small intestine and in the liver was measured. The results were compared to those from a control group of rats receiving boiled instead of fried meat as their source of protein. The diet containing boiled meat was not mutagenic and none of the samples from the control group, neither urine nor feces, contained any mutagenicity. The activity of ethoxyresorufin-O-deethylase in the small intestine was increased by fried meat whereas this enzyme activity in the liver was unaffected. O-deethylation of ethoxyresorufin is catalyzed by cytochrome P-450 dependent enzyme(s) and induction of the enzyme activity might indicate an increased metabolicactivation of premutagens and precarcinogens in the intestine. It is not yet known whether the mutagens excreted by the animals receiving the fried meat diet represent unmetabolized dietary mutagens or are formed as a result of metabolic activation of compounds in fried food.