Salmonella/microsome mutagenicity tests of heat-processed milk samples

Samples of whole milk were heat treated by commercial heat-sterilization, by commercial heat-pasteurization or by a laboratory heat-pasteurization (65 degrees C for 30 min). Each sample was tested for mutagenicity in Salmonella typhimurium strains TA98 and TA100 with and without S-9 mix. Dichloromethane extracts of milk heated at 100, 135 and 150 degrees C for 5 hr were also tested for mutagenicity using the same assay. None of these samples exhibited mutagenicity in the Ames assay used.     

Biological effects of short-term feeding to rats of repeatedly used deep-frying fats in relation to fat mutagen content

The effects of short-term feeding of mutagen containing, heated deep-frying oils on urinary and faecal mutagenicity, plasma clinical biochemical parameters, peroxidative effects and cell proliferative indices in the gastro-intestinal tract were determined in rats. Repeatedly used frying oils [a saturated fatty acid-rich coconut oil (CO) and a polyunsaturated fatty acid (PUFA)-rich (greater than 60% PUFA) vegetable frying oil (PO)] were administered to groups of seven rats at a level of 10% (by weight) in the diet for 4 wk; control groups were fed equal amounts of the unheated oils. Both heated oils showed direct-acting mutagenicity to Salmonella tester strain TA97; heated PO was also mutagenic to strain TA100. Both heated CO and heated PO contained detectable amounts of thiobarbituric acid-reactive substances (TBA-RS). In heated PO, hydroperoxides of linoleic acid were also present. In groups fed heated oils the mutagenicity of urine and faeces to strain TA97 was not found to be increased in comparison with the control groups. Faecal mutagenicity to strain TA100 was also unaffected by consumption of heated oils. Urinary excretion of TA100 mutagens was significantly increased in rats fed heated PO. Plasma alkaline phosphatase activity was clearly raised in rats fed heated PO, in comparison with rats fed unheated oils or heated CO. In addition, other clinical biochemical plasma parameters showed a tendency to be increased in rats fed heated PO, indicating hepatic and renal cellular toxicity. Urinary and faecal excretion of thiobarbituric acid-reactive substances (TBA-RS) were also slightly, but not significantly, increased in rats fed heated PO. Feeding heated CO to rats did not result in increased plasma enzyme activities and excretion of TBA-RS, nor in increased cell proliferation in gastro-intestinal tissues. Cell proliferation of the oesophageal tissues were slightly, but significantly, increased in rats fed heated PO, in comparison with the group fed unheated PO. Tissues of the glandular stomach and colon/rectum did not show significantly enhanced cell proliferation in the group fed heated PO. The results obtained in this study indicated that consumption of heated oils containing TA100 mutagens and oxidation products of linoleic acid produced indications of cellular damage to liver and kidneys, and increased urine mutagenicity, as well as enhanced cell proliferation in the oesophagus.     

Ames mutagenicity tests of overheated brewed coffee

Five kinds of coffee samples were prepared from a commercial drip-grind coffee in order to examine the mutagenicity of brewed coffee using the Ames test. The samples prepared were a thick coffee syrup, coffee solid residues, dichloromethane and ethanol extracts of solid residues, a dichloromethane extract of a distillate from normally heated brewed coffee and dichloromethane extracts of distillates from overheated (150–300°C) brewed coffee. The samples were tested for mutagenicity towards Salmonella typhimurium strains TA98 and TA100 both with and without metabolic activation (S-9 mix). Only the extracts of the distillates obtained from coffee heated to 150° or 300°C exhibited mutagenicity towards strain TA98 with S-9 mix.     

Mutagenicity tests of cashewnut shell liquid, rice-bran oil and other vegetable oils using the Salmonella typhimurium/microsome system

In view of the shortage of edible oils in India, nutritional and toxicological evaluations have been carried out on some unconventional oils to determine whether they might be safe for human consumption. As part of these evaluations, eight unconventional oils were tested by the Ames mutagenicity assay, using Salmonella typhimurium strains TA98 and TA100 with and without metabolic activation with S-9 mix prepared from the livers of rats pretreated with sodium phenobarbitone or Aroclor 1254. Of the oils tested, metsa oil (Hibiscus sabdariffa) and cashewnut shell liquid were mutagenic with and without metabolic activation with S-9 of either source. No mutagenic activity (with or without S-9 of either source) was observed with any of the other oils tested (rice-bran oil, Cleome viscosa oil, mango-kernel oil, mahua oil, kapok oil and neem oil).     

N,N-diethylphenylacetamide, an insect repellent: absence of mutagenic response in the in vitro Ames test and in vivo mouse micronucleus test

N,N-diethylphenylacetamide (DEPA), a promising new insect repellent, was tested for mutagenicity in the in vitro Ames Salmonella/microsome mutagenicity test and the in vivo mouse micronucleus test. For the Ames test, DEPA was assayed both in the presence and absence of Aroclor 1254-induced rat-liver S-9 mix (5 and 20% S-9 fraction), using five tester strains of Salmonella typhimurium--TA97a, TA98, TA100, TA102 and TA104. For the micronucleus test, mice were exposed to DEPA through ip injection for 2 and 5 days in separate experiments, and bone marrow and peripheral blood were sampled 6 and 48 hr after the final injection, respectively. DEPA did not induce a mutagenic response in the Ames test, and mouse bone marrow and peripheral blood micronucleus tests. DEPA was not considered cytotoxic, as a depression of the percentage PCE was not observed at any dose in the range of 1 to 100 mg/kg body weight with either treatment protocol of the micronucleus test.     

Effect on cooking time on mutagen formation in smoke, crust and pan residue from pan-broiled pork

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 degrees C for various times between 2 and 10 min. Broiled meat was reheated up to 5 times at 200 degrees C, each time to a centre temperature of 70 degrees C. Reheating was also performed in a microwave oven for 2 min and in an electric oven at 200 degrees C for 10 min. In addition, broiled patties were kept warm at 60 degrees 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 degrees 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.     

Effect of pyrolysis temperature on the mutagenicity of tobacco smoke condensate.

Tobacco smoke aerosols with fewer mutagens in the particulate fraction may present reduced risk to the smoker. The objective of this study was to test the hypothesis that the temperature at which tobacco is pyrolyzed or combusted can affect the mutagenicity of the particulate fraction of the smoke aerosol. Tobacco smoke aerosol was generated under precisely controlled temperature conditions from 250 to 550 degrees C by heating compressed tobacco tablets in air. The tobacco aerosols generated had a cigarette smoke-like appearance and aroma. The tobacco smoke aerosol was passed through a Cambridge filter pad to collect the particulate fraction, termed the smoke condensate. Although condensates of tobacco smoke and whole cigarette mainstream smoke share many of the same chemical components, there are physical and chemical differences between the two complex mixtures. The condensates from smoke aerosols prepared at different temperatures were assayed in the Ames Salmonella microsome test with metabolic activation by rat liver S9 using tester strains TA98 and TA100. Tobacco smoke condensates were not detectably mutagenic in strain TA98 when the tobacco smoke aerosol was generated at temperatures below 400 degrees C. Above 400 degrees C, condensates were mutagenic in strain TA98. Similarly, condensates prepared from tobacco smoke aerosols generated at temperatures below 475 degrees C were not detectably mutagenic in strain TA100. In contrast, tobacco tablets heated to temperatures of 475 degrees C or greater generated smoke aerosol that was detectably mutagenic as measured in TA100. Therefore, heating and pyrolyzing tobacco at temperatures below those found in tobacco burning cigarettes reduces the mutagenicity of the smoke condensate. Highly mutagenic heterocyclic amines derived from the pyrolysis of tobacco leaf protein may be important contributors to the high temperature production of tobacco smoke Ames Salmonella mutagens. The relevance of these findings regarding cancer risk in humans is difficult to assess because of the lack of a direct correlation between mutagenicity in the Ames Salmonella test and carcinogenicity.     

Contribution of coffee aroma constituents to the mutagenicity of coffee

About 40 coffee aroma constituents belonging to the classes of dicarbonyls, sulphur-containing compounds, furfuryls, N-heterocyclics and others were systematically evaluated in three Ames tester strains. Only aliphatic dicarbonyl compounds showed notable direct mutagenic activity, which mainly affected 'base-pair substitution' in Ames tester strains TA100 and TA102. Very weak effects were also seen with some N-heterocyclics, mainly affecting frameshift tester strain TA98 upon metabolic activation. However, it was shown that these N-heterocyclics do not contribute substantially to the mutagenicity in coffee. The hydrogen peroxide and methylglyoxal contents of coffee were determined up to 26 hr after preparation. Their concentrations tended to decrease whereas mutagenic activity decreased significantly with time in tester strains TA100 and TA102. It is concluded that several highly labile coffee constituents contribute to the bacterial mutagenicity and also that the synergism between hydrogen peroxide and methylglyoxal is not the main factor. The absence of coffee mutagenicity/carcinogenicity in rodents with these highly reactive coffee aroma compounds can be explained in part by detoxification of microsomal enzyme systems.     

猫眼草水煎液体外致突变性的研究

目的检验猫眼草水煎液的致突变性;改进经典的A-mes试验体系使之适应于中药体外致突变性检验。方法通过经典的Ames试验检测猫眼草的体外致突变性;通过哺乳动物骨髓细胞染色体畸变试验检测猫眼草的致畸作用;改进的Ames试验通过增设含补充组氨酸(含量对应于猫眼草水煎液中组氨酸浓度)的阴性对照,排除样品中组氨酸成分对试验结果的影响。结果猫眼草水煎液在经典的Ames试验中为强阳性;对哺乳动物骨髓细胞染色体致畸作用为阴性;在改进的Ames试验中猫眼草水煎液的致突变性为阴性。结论经典的Ames试验不适合猫眼草水煎液致突变性检测,改进后的Ames实验体系适合。猫眼草水煎液在体外和体内均没有致突变性。     

Comparative mutagenicity studies of azo dyes and their reduction products in Salmonella typhimurium

The arabinose-resistant and Ames assay systems of Salmonella typhimurium were used to evaluate the mutagenic potential of azo dyes and their aromatic amine reduction products. Azo dyes, namely direct black 38, direct blue 15, and direct red 2, were mutagenic in the arabinose-resistant and Ames assays with both hamster and rat liver S9 activation. Both assays gave relatively higher mutagenic responses with hamster S9. Reduction products of these dyes, namely benzidine, o-dianisidine, and o-tolidine, were mutagenic in the Ames assay. Benzidine was weakly mutagenic and o-dianisidine and o-tolidine were nonmutagenic in the arabinose-resistant assay. These results indicate that both arabinose-resistant tester SV50 and Ames tester TA98 were sensitive in detecting mutagenicity of azo dyes. The use of the standard plate protocol with Ames tester TA98 is more efficient than the modified azo dye protocol in detecting mutagenicity of aromatic amine reduction products. Additional modifications in either the standard plate or modified azo dye protocols may improve detection of mutagenicity of these compounds in the arabinose-resistant assay system.     

Limitations of urinary mutagen assays for monitoring occupational exposure to antineoplastic drugs

The sensitivity of the Salmonella reversion test of Ames as a screen for accidental absorption of 17 antineoplastic agents by drug handlers was evaluated. Dilutions of each drug were added to agar inoculated with each of two Salmonella typhimurium strains (TA98 and TA100); control plates contained no test drug. Colonies were counted after incubation at 36 degrees C for 48 hours. The drugs were tested in the presence of a liver preparation to provide metabolic activation of mutagenicity. Urine samples collected from patients after doses of three mutagenic drugs were extracted and tested with the Ames test. For 11 of the 17 drug solutions, no mutagenic activity was seen, but many of these 11 were toxic to the organisms. The most highly mutagenic drugs were doxorubicin and cisplatin, with mechlorethamine, carmustine, dacarbazine, and cyclophosphamide exhibiting less mutagenic activity. Urine from patients treated with doxorubicin or cyclophosphamide showed mutagenicity, but the results suggested that the quantity of these drugs that would have to be absorbed to produce a definite reaction in urine is unlikely to be achieved by drug handlers who use standard precautions. Because of its lack of sensitivity and the potential effects of environmental and dietary factors on the results, this bacterial mutagenicity test should not be used routinely for detection of accidental absorption of antineoplastic drugs.     

Mutagenicity of the products obtained from heated milk systems

Methylene chloride extracts of the browning reaction products prepared from model systems consisting of major milk components (casein and/or lactose, and non-fat dried milk) were tested for mutagenicity in the Ames Salmonella/microsome assay. Samples obtained by heating aqueous solutions of these components under either neutral or basic (pH 10) conditions exhibited no significant mutagenic activity when tested with or without S-9 mix. The addition of common food additives, such as sodium nitrite, butylated hydroxyanisole and butylated hydroxytoluene, to the aqueous solutions did not enhance the mutagenic activity of the browning samples. On the other hand, the tar samples prepared by heating the same milk components in the dry state exhibited strong mutagenicity, primarily to Salmonella typhimurium strain TA98 and only with S-9 mix. A casein/lactose mixture and non-fat dried milk were also heated with baking soda in the dry state. The presence of the baking soda enhanced the mutagencity of the browning products; the tar from the non-fat dried milk heated with baking soda was the most potently mutagenic of all the samples towards strain TA98 and also produced a positive response in strain TA100 in the presence of S-9 mix.     

Possible mutagenic constituents in nitrite-treated soy sauce

Soy sauce was heated with 100, 500, 1000 or 2000 ppm sodium nitrite for 30 min at 80°C and pH 3. The reaction mixtures were extracted with dichloromethane followed by ethyl acetate. After removal of the solvents, the extracts were subjected to analysis (gas chromatograph-thermal energy analyser and gas chromatograph-mass spectrometer) and Ames mutagenicity tests. N-Nitrosodimethylamine and N-nitrosodiethylamine were found in the dichloromethane extract of the soy sauce treated with 2000 ppm nitrite at levels of 10 and 120 μg/ml, respectively. N-Nitrosoproline was identified in the ethyl acetate extract of the same sample at a level of 0.5 μg/ml. Both extracts exhibited dose-related mutagenicity in Salmonella typhimurium strain TA100 with S-9 mix. The dichloromethane extract showed much higher mutagenicity than did the ethyl acetate extract. The samples obtained from soy sauce treated with 100, 500 and 1000 ppm nitrite were not mutagenic, but N-nitrosodiethylamine was detected by thermal energy analysis in the soy sauce treated with 1000 ppm nitrite. The addition of 10,000 ppml-ascorbic acid, along with 2000 ppm nitrite, to soy sauce prevented the formation of mutagenic materials or detectable nitrosamines.     

Ames mutagenicity tests of products from a heated potato-starch system

A charred sample was prepared from potato starch heated with ammonium carbonate at 600°C in a flask under a nitrogen stream. The water produced was collected and extracted with methylene chloride. The basic fraction obtained from the extract exhibited strong mutagenicity in Ames assays using Salmonella typhimurium strains TA98 or TA100 with metabolic activation (rat-liver S-9 mix). The basic fraction was further fractionated by silica gel column chromatography and subsequently by Scphadex column chromatography. Some of the resulting fractions exhibited strong mutagenic activities in S. typhimurium strain TA98 with S-9 mix.     

Biological and chemical studies on overheated brewed coffee

Vapour formed from overheated decaffeinated coffee was condensed and tested for mutagenicity using the Ames assay in Salmonella typhimurium strains TA98 and TA100. Vapour produced at 73 and 100 degrees C exhibited no mutagenicity. The basic fraction of vapour produced at 350 degrees C showed weak mutagenicity towards strains TA98 with metabolic activation. The chemical analysis of this fraction identified pyridines and pyrazines as the major constituents. None of the compounds identified in this fraction has been reported as mutagenic when tested in the Ames assay.     

Lysinoalanine: Absence of mutagenic response in the salmonella/mammalian-microsome mutagenicity assay

Lysinoalanine (N^?-(dl-2-amino-2-carboxyethyl)-l-lysine) was tested for mutagenicity in the Ames Salmonella/mammalian-microsome mutagenicity assay. No mutagenic response was detected at doses up to 5 mg/plate when samples were pre-incubated without S-9 mix, nor when they were pre-incubuted with S-9 mix prepared from Aroclor 1254-induced rat liver or kidney. The results indicate that the stereoisomers of lysinoalanine likely to be present in the greatest proportions in processed foods are not mutagenic in the Ames assay.     

Mutagenicity of bovine lactoferrin in reverse mutation test

The mutagenicity of bovine lactoferrin, which is an iron-binding glycoprotein in milk, was evaluated by the Ames mutagenicity test. A total of 5 test strains including 3 base-pair substitution-type strains, Salmonella typhimurium TA100, TA1535 and Escherichia coli WP2uvrA, and 2 frameshift-type strains, TA98 and TA1537, were used in the test. The test was performed by both the direct method and the metabolic activation method with preincubation applied in each instance. The concentration range of the test solution was 0.16 to 5.00 mg/100 microliters (plate). Results of the test revealed that the number of revertant colonies at each concentration of the test solutions was less than 1.4 times that of the control group. In the test system used, bovine lactoferrin did not exhibit mutagenicity.     

Mutagenicity of human urine after the consumption of fried salted salmon

Mutagenicity in the urine of four non-smoking individuals who had eaten salted salmon cooked at home for both lunch and supper was monitored by means of Salmonella/microsome mutagenicity tests. Extracts from fresh and salted salmon had the same level of mutagenicity after being cooked for 10 min at 200 degrees C, but no activity was detected before cooking. Salmonella strains TA98 and TA1538 were equally sensitive to the mutagens and required metabolic activation. No mutagenicity was shown with TA100 and TA1535. Urine samples were tested using a concentrate prepared by means of an XAD-2 resin column. Mutagenicity was detected mainly in urine excreted during 4-5 hr after the ingestion of cooked salmon, but only weak mutagenicity, or none at all, was detected in the urine after the ingestion of vegetables. The levels of urinary mutagenicity due to salmon consumption were not affected when cabbage was eaten simultaneously. The excretion of mutagenic substances was completed within about 20 hr, and there were almost no mutagens in the urine 24 hr after the ingestion of cooked salmon.     

An evaluation of the sensitivity of the Ames assay to discern low-level mutagenic impurities

Low level impurities often reside in active pharmaceutical ingredients (API). Some of these impurities are potentially genotoxic since reactive intermediates are used in the synthetic route for the production of API. Routine mutagenicity testing is conducted in support of clinical trials with the intent to identify genotoxic hazards associated with API. Depending on the amount of impurity present in the API tested, the potency of the impurities and the relative sensitivity of the Ames assay, it is possible that mutagenicity associated with the presence of genotoxic impurities could also be detected while testing API. Therefore, we evaluated published data and generated new information to understand the sensitivity of the Ames assay. Based on a literature survey of approximately 450 mutagens, it was estimated that 85% of mutagens are detected at concentrations of 250 microg/plate or less. Based on this estimate, most mutagens should be detected in an Ames assay testing API concentrations up to 5000 microg/plate if present at a 5% or greater concentration. Data from experiments where several direct and indirect-acting mutagens were spiked into representative API further support the literature-based evaluation. Some limitations of this approach, including toxicity of API and competing metabolism are discussed.     

Fate and toxicity assessment of linear alkylbenzene sulfonates in drinking water using the ames test

The genotoxic activity of the methanolic water extracts of prechlorinated water from Barcelona (NE Spain) using the Ames test was studied. High performance liquid chromatography (HPLC) and mass spectrometry in the mass spectroscopy/fast atom bombardment mode (MS/FAB) was employed to tentatively identify organic compounds responsible of genotoxic activity. Methanolic extracts of prechlorinated water were highly mutagenic in the Ames test, mainly with the TA98 strain for concentration lesser than 1 L. On the other hand, the TA100 strain showed higher mutagenicity for tap water extracts and concentrations higher than 1 L. Also, a strong toxic effect was observed when methanolic extracts were analyzed by the Ames test. Toxicity showed a reduction of the genotoxic ratio by a characteristic negative slope for the concentration vs genotoxicity curve. Toxicity was usually observed using the TAlOO strain and at a higher concentration than mutagenicity does. Both mutagenicity and toxicity in the Ames test showed a characteristic pattern depending on their origin (tap or prechlorinated water). It was possible to separate mutagenic from toxic fractions by HPLC. These subfractions were analyzed by MS/FAB in order to identify the organic compounds responsible for these effects, but unsuccessful results were obtained for mutagenic subfractions. Alkylbenzenesulfonates (LA3) were the sole compounds identified in toxic subfractions. The correlation between toxicity of samples (TA100 strain) and the presence of LAS was proved by comparison of toxicity from a standard LAS and those observed from real samples. An EC ₅₀ of 9.8 mg/L for LAS has been established by the Ames test using the TAlOO strain. © 1993 John Wiley & Sans, Inc.