Susan K. Healy

Mutagens from the cooking of food: I. Improved extraction and characterization of mutagenic fractions from cooked ground beef☆

Ground beef was fried at 200 degrees C (392 degrees F) to a well-done, non-charred state, and the extracted organic base fraction was found to be highly mutagenic in Salmonella strain TA1538 (6300 revertants/100 g equivalent, gE, fresh weight). The neutral and acidic extracts showed no mutagenic activity in any of the 5 standard strains of Salmonella. A new procedure based upon extraction and protein precipitation with acetone is described, which is simpler and more efficient than previously described methods. The organic base fraction was mutagenic only in Salmonella strains TA1537, TA1538, and TA98, all sensitive to frameshift mutations. Strains sensitive to base-substitution mutations showed no activity. Metabolic activation was an absolute requirement for mutagenesis; however cell toxicity was decreased by the presence of S9 activation mixture. After normal cooking, more than 20 times as much mutagenic material remained in the meat as was recovered in the pan grease and vapors. The results confirm that mutagens are formed under conventional frying conditions, and show that mutagen can be isolated by an improved extraction method.

Effects of temperature, patty thickness and fat content on the production of mutagens in fried ground beef

The high-pressure liquid chromatography (HPLC) profiles of mutagenic components were compared for extracts of ground beef patties fried at 200, 250 and 300 degrees C for 6 min/side. The HPLC profiles of the mutagenic samples were similar, although total mutagenic activity in Salmonella typhimurium TA1538 was roughly four times as high after the 300 degrees C than after the 200 degrees C frying. Six mutagenic peaks were analysed quantitatively at different temperatures and meat thicknesses. Two major components, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline and 2-aminotrimethylimidazo[4,5-f]quinoxaline, and a minor component, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), were all present at the three different temperatures. Thus, in general, cooking temperature seems to have a major effect on the quantities of mutagens produced but not on their HPLC profiles. The thickness of the meat patty did not affect the total yield of mutagens except at longer cooking times (8-10 min/side) and, in general, neither did it affect the HPLC profiles of the mutagenic components. Total mutagenic activity increased with increasing cooking times. Increasing the fat content lowered the total mutagenicity, with 150,000 revertants/kg of fresh beef at 30% fat compared with 230,000 revertants/kg at 15%, but had little effect on the mutagenicity due to IQ.

An XAD-2 resin method for efficient extraction of mutagens from fried ground beef☆

4 methods of extraction of mutagens from fried ground beef were compared for total mutagen recovery and chromatographic profile of isolated substances. A method which employs Amberlite XAD-2 resin to isolate mutagenic activity from an initial aqueous acid extract of fried beef was found to yield approx. 4 times more activity than other aqueous or organic solvent extraction procedures. Chromatographic profiles of mutagenic extracts isolated by the 4 methods suggest that the XAD-2 resin method does not recover different mutagens, but is primarily a more efficient isolation procedure. The resin method is rapid, inexpensive, simple, and requires approx. half the time of the other methods.

Base-change analysis of revertants of the hisD3052 allele in Salmonella typhimurium

This report is an investigation of the specific sequence changes in the DNA of Salmonella hisD3052 revertants induced by a set of specific frameshift mutagens found in our diet. They include B[a]P, aflatoxin B1, and the cooked-food mutagens, IQ, MeIQ, and PhIP. The Salmonella DNA was cleaved with restriction enzymes Sau3A, EcoR1, and Alu1 to give a 620-bp fragment containing the hisD3052 site. The size-fractionated fragments were ligated to the bacteriophage vector M13mp8. After transformation into E. coli, the recombinants were screened with a nick-translated hisD+ gene probe, and the isolated single-stranded DNA was sequenced. All IQ (13), MeIQ (3), PhIP (5), and aflatoxin B1 (3) induced revertants isolated had a 2-base (-CG- dinucleotide) deletion situated 10 bases upstream from the original hisD3052 -C- deletion. In contrast, 9 of 24 revertants induced by B[a]P had extensive deletions varying from 8 to 26 nucleotides in length and located at various sites along a 45-base-pair sequence beginning at nucleotide 2085 of the his operon. The other 15 B[a]P-induced revertants had a -CG- deletion at the same location as the revertants induced by the other food mutagens. 7 spontaneous revertants were also analyzed; they showed 3 -CG- deletions, 1 insertion and 3 distinct deletions (varying from 2 to 11 bases in size). In total, 13 distinct base changes are described which lead to reversion of the hisD3052 mutation.

Mutagens from the cooking of food. III. Survey by Ames/Salmonella test of mutagen formation in secondary sources of cooked dietary protein

A survey of mutagen formation during the cooking of a variety of protein-rich foods that are minor sources of protein intake in the American diet is reported (see Bjeldanes, Morris, Felton et al. (1982) for survey of major protein foods). Milk, cheese, tofu and organ meats showed negligible mutagen formation except following high-temperature cooking for long periods of time. Even under the most extreme conditions, tofu, cheese and milk exhibited fewer than 500 Ames/Salmonella typhimurium revertants/100 g equivalents (wet weight of uncooked food), and organ meats only double that amount. Beans showed low mutagen formation after boiling and boiling followed by frying (with and without oil). Only boiling of beans followed by baking for 1 hr gave appreciable mutagenicity (3650 revertants/100g equivalents). Seafood samples gave a variety of results: red snapper, salmon, trout, halibut and rock cod all gave more than 1000 revertants/100 g wet weight equivalents when pan-fried or griddle-fried for about 6 min/side. Baked or poached rock and deep-fried shrimp showed no significant mutagen formation. Broiled lamb chops showed mutagen formation similar to that in red meats tested in the preceding paper: 16,000 revertants/100 g equivalents. These findings show that as measured by bioassay in S. typhimurium, most of the foods that are minor sources of protein in the American diet are also minor sources of cooking-induced mutagens.

The metabolic disposition of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine in the induced mouse

The toxicokinetics of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a member of the aminoimidazoazaarene family of mutagens, was studied in C57BL/6 male mice after ip and gavage exposure to a single dose of 100 mg/kg body weight. Twenty-four hr after administration of the dose, 39% of the [14C]PhIP had been found in the urine of the animals exposed ip and 12% had been found in the faeces. By comparison, at 24 hr, gavage-exposed animals had excreted 31% of the administered radioactivity in the urine and 30% in the faeces. Significantly higher levels of labelled compounds could be detected in the blood of animals dosed ip than in that of those dosed by gavage at 6 and 12 hr after dosing. Tissue concentrations of labelled compound were highest in the liver and gastro-intestinal tract of the ip group and the large intestine of the gavage group. Considerable radioactivity was also detected in the contents of the large and small intestine of the ip group suggesting biliary excretion of PhIP. Peak tissue concentrations were found 6-12 hr after administration of the dose by both routes. High-performance liquid chromatography of urine demonstrated the presence of 11 metabolites while only two major metabolites were found in the faeces, one of which was not present in the urine. None of the metabolites appeared to be formed by simple N-acetylations or N-demethylations of PhIP. No differences were seen in the metabolite profiles between the ip and the gavage groups, but significant differences were seen in the kinetics of PhIP excretion between animals dosed by these two routes. These data indicate that the toxicokinetics of PhIP metabolism differ depending on the route of administration and should be considered when performing animal studies to assess the significance of human dietary PhIP consumption.

Activation of mutagens in cooked ground beef by human-liver microsomes

The total organic base fraction purified from fried ground beef is metabolized by human-liver microsomes to form mutagens detectable by the Ames/Salmonella bacterial assay. The mutagens produced have an absolute requirement for metabolic activation; without it, no increase in the number of revertants over background is seen. Microsomes from human liver activate the mutagens significantly more than microsomes from uninduced mouse or rat liver; the microsomes from one individual were nearly as active as those of Aroclor-induced mice and rats. alpha-Naphthoflavone (ANF) inhibits activation of these mutagenic bases, implying that the metabolism is mediated by the inducible form(s) of cytochrome P-448. Thus, the human liver has the potential to metabolize the cooked beef mutagen(s) to active intermediates, posing a possible mutagenic risk. However, unlike the animal metabolizing system, which needs to be artificially induced, the human system appears to be naturally induced through diet or environmental exposure.

Identification of the mutagenic quinoxaline isomers from fried ground beef.

Two mutagens isolated from fried-beef patties were compared to a series of synthetic structural isomers of 2-aminodimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-aminotrimethylimidao[4,5-f]quinoxaline (DiMeIQx). Comparison by NMR spectrometry and HPLC coelution showed that one beef mutagen (molecular weight of 213) was identical to the 8-MeIQx isomer not the 7-Me isomer. Another quinoxaline beef mutagen, having 3 methyl groups (molecular weight of 227), had an NMR spectrum different from the 5,8- or 7,8-DiMeIQx isomers, but not clearly distinguishable from the 4,8- or 4,7-DiMeIQx isomers. The HPLC separation of the DiMeIQx isomers and subsequent addition of the beef mutagen showed the beef-derived compound to coelute with the 4,8-DiMeIQx and not with the 4,7-DiMeIQx. The number and position of methyl groups was responsible for a 7-fold range of mutagenic response in the Ames/Salmonella assay. In conclusion, the major quinoxaline mutagens isolated from fried beef were identified as 8-MeIQx and 4,8-DiMeIQx isomers.