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.     

Mutagen formation in the reaction of maillard browning products, 2-acetylpyrrole and its analogues, with nitrite

Three 2-substituted pyrroles (2-acetylpyrrole, pyrrole-2-carboxaldehyde and pyrrole-2-carboxylic acid), which are products of the Maillard browning reaction, were reacted with nitrite in buffer solution (pH 3) at 50°C for 24 hr. The reaction mixtures were extracted with methylene chloride and the extracts were tested for mutagenicity using Salmonella typhimurium strains TA97, TA98, TA100, TA102 and TA104, with and without S-9 metabolic activation. The methylene chloride extract of the 2-acetylpyrrole-nitrite reaction mixture showed strong mutagenicity to all the tester strains, both in the presence and absence of S-9 mix. The reaction product of pyrrole-2-carboxaldehyde with nitrite only gave a weak mutagenic response with strain TA100, while the pyrrole-2-carboxylic acid-nitrite reaction product did not produce a mutagenic response in any of the tester strains. Two mutagenically active fractions, separated by thin-layer chromatography, were found in the reaction of 2-acetylpyrrole with nitrite. The formation of mutagenic products in the latter reaction was found to vary with reaction pH, time and temperature, with nitrite level and with 2-acetylpyrrole concentration.     

Mutagenicity studies on N-nitrosated products of the Maillard browning reaction: N-nitroso-fructose-amino acids

The N-nitroso derivatives of D-fructose-L-glycine, D-fructose-L-alanine, D-fructose-L-phenylalanine, D-fructose-L-serine, Dfructose-L-aspartic acid and D-fructose-L-tryptophan (a mixture of alpha-N-nitroso-D-fructose-L-tryptophan and 'indolyl-nitrosamine'-D-fructose-L-tryptophan) were tested for mutagenicity in five auxotrophic strains of Salmonella typhimurium with and without metabolic activation (S-9 mix). The alanine, phenylalanine and aspartic acid compounds were not mutagenic. The glycine and serine compounds showed a very low but reproducible increase in the numbers of his+ revertants in strain TA1535 without S-9 mix. The mixture containing both nitrosated D-fructose-L-tryptophan compounds was mutagenic in all five strains, with or without metabolic activation. The alpha-N-nitroso-D-fructose-L-tryptophan component of the mixture, which is nitrosated at the amino group, was isolated and tested without S-9 mix. It was mutagenic in three strains. Unnitrosated D-fructose-L-amino acids, D-fructose, and the individual L-amino acids were non-mutagenic when tested under those conditions for which a positive response had been obtained with the corresponding nitrosated compounds. These results indicate the potential value of developing analytical methods to identify alpha-N-nitroso-D-fructose-L-tryptophan in food or food extracts that are to be screened for mutagenic components.     

Sources of mutagenicity in cooked Finnish foods

The mutagenic activities of the alkaline fractions derived from various heat-processed, ready-made meat, fish and poultry foods were studied using the Salmonella mutagenicity assay with strain TA98 and S-9 mix to provide information about the mutagenicity of everyday Finnish foods. The majority of the food samples tested were mutagenic. The mutagenic activities of various commercial ready-made products. Mutagenicity varied remarkably between different samples. The cooking temperature clearly affected the mutagenicities of fried or reheated food samples, mutagenicity increasing with increasing temperature. The results indicate that the main sources of cooked-food mutagens in everyday Finnish foods are grilled and broiled products and foods fried at restaurants and at home. By comparison, commercial ready-made fried foods are only a minor source of mutagenicity. Variations between equivalent food samples indicated that heat processing has a marked effect on the mutagenic activity of the product, which might therefore be reduced by modifying the cooking process.     

Mutagenicity of some monoaromatic hydroxamic acids

The mutagenicity of some monoaromatic hydroxamic acids was tested in the presence and absence of rat liver S-9 with Salmonella typhimurium tester strains TA98 and TA100. Of the five N-(chlorophenyl)-substituted hydroxamic acids and seven N-arylformohydroxamic acids tested, 2 of the first and 4 of the latter series were mutagenic to both strains upon metabolic activation. None of the four N-acetyl-type hydroxamic acids was mutagenic to either strain, even upon activation. Because some of the N-acetyl-derived hydroxamic acids were inactive, whereas the same aromatic nucleus possessing a formyl group displayed significant activity, a consideration of the nature of the aryl group in hydroxamic acid mutagenicity is important.     

The mutagenicity of butadiene towards salmonella typhimurium

Gaseous butadiene (BUT) was mutagenic towards S. typhimurium strain TA 1530 when the incubation mixture was supplemented with a NADPH-fortified rat liver microsomal preparation; mutagenicity increased with the dose.A significant mutagenic effect was similarly observed when the petri dishes, containing the bacteria but no metabolic activation system, were incubated in the presence of butadiene, in a desiccator in which plates containing the S-9 rat liver fraction had been placed. This indirect mutagenic effect was attributed to the formation, by the S-9 mix, of volatile intermediate(s) that migrated and induced mutations in neighbouring bacteria.     

Mutagenic factors in cooked foods.

The charred surface of fish and beef showed strong mutagenic activity in Salmonella typhimurium test strains when activated by S-9 mix of rat liver. The pyrolysis products of proteins and amino acids were also highly mutagenic. Among the pyrolysis products of amino acids, those of tryptophan, serine, and glutamic acid were most active. The new gamma-carboline derivatives, 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole and 3-amino-1-methyl-5H-pyrido[4,3-b]indole, were purified from the pyrolysis products of tryptophan. These new compounds were stronger mutagens than aflatoxin B1 towards S. typhimurium TA98, a frameshift type mutant, and they also transformed cryopreserved Syrian hamster embryo cells in vitro. Tryptophan pyrolysate also contained the beta-carboline derivatives, norharman and harman, which are not mutagenic alone, but act as comutagens. A mixture of norharman or harman and nonmutagenic aniline or o-toluidine was strongly mutagenic. The mutagenicities of charred products of other foods, such as seaweed and garlic, are reviewed in this article. Flavonoids, such as kaempferol and quercetin, and glycosides of these flavonoles were mutagenic. The mutagenicity of cooked vegetables depends partly on these flavonoid derivatives. The already-known existence of benzol[a]pyrene and nitroso compounds in cooked food is also reviewed.     

Mutagenicity of the C-nitroso analog of fenitrothion

The chemicals fenitrothion, nitroso fenitrothion, amino fenitrothion and 3-methyl-4-nitrophenol were tested for mutagenicity to Salmonella typhimurium strains TA98 and TA100, both in the presence and absence of rat liver S-9 mix. The strong mutagenicity of nitroso fenitrothion to both strains either in the presence or absence of S-9 mix contrasted with the observation that fenitrothion displayed no mutagenicity in these tester strains. The results suggest that the normal nitroreductases present in TA98 and TA100 cannot metabolize fenitrothion to a mutagenic metabolite. This inability of the tester strains to effect partial nitroreduction results in the failure of this screening system to predict the potential genotoxicity of this pesticide.     

Mutagenicity of deep-frying fat, and evaluation of urine mutagenicity after consumption of fried potatoes

Mutagen formation during deep-frying was evaluated using standard frying conditions. Portions of pre-fried, sliced potatoes were fried in a commercial brand of hydrogenated vegetable frying fat, which was used repeatedly and for a prolonged period of time. Concentrations of polar oxidation and degradation products, and of dimeric and polymeric triglycerides, were found to increase in the frying fat as well as in fried potatoes with prolonged use of the fat. Thiobarbituric acid-reactive substances were detectable neither in the frying fat nor in the fried potatoes. Polar fractions of repeatedly used frying fat significantly increased the number of revertants in Salmonella typhimurium strain TA97 without S-9 mix. In the presence of S-9 mix mutagenic activity was reduced. As a consequence of ongoing formation of polar degradation and oxidation products, the mutagenicity of the fat increased after repeated use. Polar fractions of lipids extracted from commercially obtained pre-fried potatoes, as well as from fried potatoes, marginally increased the number of revertants in strain TA97 without S-9 mix. The mutagenicity of the lipid fractions of fried potatoes was not related to the heating time of the fat. Methanol extracts of fat-free residues of fried potatoes significantly increased numbers of revertants in strain TA97 after metabolic activation, which indicated that a different class of mutagens had been isolated. The mutagenicity of methanol extracts was not increased after either prolonged or repeated use of the fat. Urine samples of six healthy, non-smoking volunteers, collected during the 24 hr following consumption of portions of potatoes fried in repeatedly used fat, showed no increase in mutagenicity compared with control samples. Since the exact identity of mutagens formed during deep-frying, as well as their metabolic fate in man, is unclear at present, evaluation of possible adverse biological effects associated with consumption of fried foods will require strictly controlled metabolic studies.     

Mutagenicity tests of lipid oxidation products in Salmonella typhimurium: Monohydroperoxides and secondary oxidation products of methyl linoleate and methyl linolenate

Nine hydroperoxy and hydroperoxy-epidioxy oxidation products derived from either autoxidation (AO) or photosensitized oxidation (PO) of methyl linoleate (MLo) or methyl linolenate (MLn) were tested for mutagenic activity by the Salmonella typhimurium his⁺ reversion assay using strains TA100, TA98, TA102, TA97 and TA1537. All nine oxidation products, monohydroperoxides from AO-MLn (I) or from PO-MLn (II), dihydroperoxides from PO-MLo (III), AO-MLn (IV) or PO-MLn (V), hydroperoxy epidioxides from PO-MLo (VI), AO-MLn (VII) or PO-MLn (VIII) and hydroperoxy bis-epidioxides from PO-MLn (IX), were weakly mutagenic in strains TA97 and/or TA100. The hydroperoxy epidioxides (VI–IX) exhibited significantly greater activity in strain TA97 than did the monohydroperoxides (I, II) or the dihydroperoxides (III–V). In strain TA100, all of the oxidation products tested exhibited similar activity. No major differences between products derived from autoxidized and photooxidized MLn (I v. II, IV v. V, VII v. VIII) were obtained. Rat-liver S-9 reduced the toxicity of all oxidation products to the tester strains. The greatest mutant yields were usually obtained in the presence of S-9, but mutagenic potency was sometimes greater without S-9. The structural feature common to all of the mutagenic oxidation products was the presence of a hydroperoxy group, suggesting that this characteristic is responsible for the observed mutagenicity, either directly or through a common degradative pathway to reactive products of lower molecular weight.     

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).     

Mutagenic potential of allyl and allylic compounds: Structure-activity relationship as determined by alkylating and direct in vitro mutagenic properties

A series of compounds, each containing an allylic moiety, has been tested using Salmonella typhimurium TA 100 in a modified Ames mutagenicity assay system. In the absence of activating enzymes (S-9) mix, those allylic compounds possessing chemically good leaving groups show direct mutagenic activity. Their activity decreases in the following order: allyl methanesulfonate > -iodide > -bromide > -chloride. This is in good agreement with the alkylating properties measured in the nitrobenzyl-pyridine (NBP) test. For all allyl and allylic compounds found to be directly mutagenic, a decrease in, and sometimes total loss, of mutagenicity is registered after addition of S-9 supernatants. Compared to the direct mutagenic activity of allyl chloride, epichlorohydrin shows a much higher mutagenicity, whereas propyl chloride has proven to be nonmutagenic. The direct mutagenic effect of this type of compound is theoretically explained by sn-1, Sn-2 and SN-2' mechanisms.     

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.     

Negative Ames tests of epoxide fatty methyl esters derived from hemolysis of linoleic acid hydroperoxides

Five isomeric epoxyhydroxyene and epoxyoxoene fatty esters derived from hemolytic decomposition of linoleic acid hydroperoxide were tested for mutagenicity by the "Ames' top-agar incorporation method using S-9 mix derived from livers of male rats pretreated with Aroclor 1254. The epoxide fatty esters tested--methyl trans-12,13-epoxy-erythro-11-hydroxy-cis(trans)-9-octadecenoate and methyl trans-12,13-epoxy-threo-11-hydroxy-cis(trans)-9-octadecenoate (each composed of approximately 80% cis-9-ene and 20% trans-9-ene), methyl trans-12,13-epoxy-9-oxo-(trans-10-octadecenoate, methyl trans-12,13-epoxy-9-hydroxy-trans-10-octadecenoate and methyl cis-12,13-epoxy-9-oxo-trans-10-octadecenoate--had structural characteristics similar to certain potent mutagens. However, these esters were not mutagenic in Salmonella typhimurium strains TA100, TA98 or TA1537 at concentrations up to 2000 micrograms/test plate. Under the same test conditions, the methyl ester of hydroperoxy linoleic acid, from which these epoxides were derived, was weakly mutagenic in strain TA100 and possibly also in strain TA98.     

Mutagenicity evaluation of riot control agent o-chlorobenzylidene malononitrile (CS) in the Ames Salmonella/microsome test.

o-Chlorobenzylidene malononitrile (CS), a riot control agent, was evaluated for its possible mutagenic activity in the Ames Salmonella/mammalian microsome mutagenicity test. Five histidine-deficient (His-) mutant tester strains of Salmonella typhimurium--TA97a, TA98, TA100, TA102 and TA104--were used. The liquid preincubation procedure was used with metabolic activation (presence of S9 mixture) and without metabolic activation (absence of S9 mixture). For the experiments with metabolic activation, three different concentrations of S9 fraction (supernatant of Aroclor 1254-induced rat liver homogenate at 9000 g)--5%, 15% and 30% in S9 mixture--were used. Along with mutagenic activity, CS was also evaluated for cytotoxic activity in all the five tester strains of Salmonella typhimurium, both in the presence and absence of S9 mixture. The mutagenic and cytotoxic activities of CS were assessed by counting the His+ revertant colonies and by counting the microcolonies (His-, auxotrophs in the background lawn), respectively, and the respective mean values were compared with the relative negative (solvent) control. A dose range of 12.5-800 micrograms plate-1 for CS did not induce a mutagenic response either in the presence or absence of S9 mix. No change in the negative mutagenic response of CS has been observed even in the presence of an elevated level of S9 fraction in the S9 mix. A dose of 200 micrograms plate-1 for CS was found to be cytotoxic by decreasing the surviving cells as well as His+ revertant colonies; however, the effect was reduced in the presence of an elevated level of S9 fraction in the S9 mix.     

Mutagenicity study of nine monoalkyl phthalates and a dialkyl phthalate using Salmonella typhimurium and Escherichia coli

Nine monoalkyl (C1-C8) phthalates and di-(2-ethylhexyl) phthalate (DEHP) were assayed for mutagenicity in two strains of Salmonella typhimurium (TA98 and TA100) and two strains of Escherichia coli WP2 try- (uvrA+ and uvrA-) with and without metabolic activation with S-9 mix. The procedure of Ames et al. (Mutation Res. 1975, 31, 347) was used, with minor modifications. None of the compounds tested showed any mutagenic activity, but all the monoalkyl phthalates showed some lethality towards the S. typhimurium strains, the most toxic being monoheptyl phthalate. A marginally lethal effect on the Salmonella strains was shown by DEHP, but only at the highest concentration tested (2000 micrograms/plate) and in the absence of S-9 mix.     

Characterization of water-soluble glucuronide and sulphate conjugates of aflatoxin B1. 2. Studies in primary cultures of rat hepatocytes

Aflatoxin B1 and some of its metabolites were released from water-soluble aflatoxin conjugates isolated from rat primary hepatocyte cultures and hydrolysed by enzymes (beta-glucuronidase and sulphatase), by acid or by a combination of both treatments. The presence of AFB1 in the hydrolysates was detected on TLC plates, or indicated indirectly by the Ames mutagenicity assay. The aflatoxin conjugates were not mutagenic to Salmonella typhimurium strain TA98 in the presence of rat-liver S-9 mix. However, following enzymatic hydrolysis, the chloroform extract of the hydrolysate was highly mutagenic to the bacteria, indicating the presence of mutagenic AFB1. The conjugates AFB1-glucuronide and AFB1-sulphate are therefore produced from AFB1 in primary cultures of rat hepatocytes.     

Mutagenicity of extracts from Ceylon cinnamon in the rec assay

The extraction of about 1.9 kg of Ceylon cinnamon (Cinnamomum zeylanicum Nees) with 10 litres each of petroleum ether, chloroform and ethanol in a Soxhlet apparatus produced extracts weighing 76, 28 and 270 g respectively for the three solvents. In the preliminary test the ethanol extract showed no mutagenic activity. However, both the petroleum ether and the chloroform extracts showed mutagenicity when tested in the rec assay using Bacillus subtilis strains H17 (rec+) and M45 (rec-). When these extracts were studied quantitatively by the liquid and spore rec-assay methods, the minimum inhibitory concentrations of the extracts against strain H17 were higher than those against strain M45. However, in the presence of the liver S-9 mix, the minimum inhibitory concentrations of the petroleum ether and chloroform extracts against both strains of B. subtilis were equal, indicating that the mutagenicity of the extracts had been inactivated.     

Screening of tabacco smoke constituents for mutagenicity using the Ames' test

To clarify the mutagenic activity of individual smoke components, 239 compounds, representative of the gaseous and semivolatile phases of tobacco smoke, were assayed for mutagenicity towards 4 histidine-requiring mutants of Salmonella typhimurium (TA 98, TA 100, TA 1535 and TA 1537). All compounds were tested qualitatively both with and without metabolic activation using a liver fraction (S-9) from Aroclor 1254 or methylcholanthrene induced rats. Without S-9, only 2,3-dimethylindole and 2,3,5-trimethylindole showed mutagenic activity that was not enhanced by hte metabolic activation system. 2,6-Diaminotoluene and coronene, which like the above compounds are not documented carcinogens were found to be mutagenic for strain TA 98 with S-9. Mutagenic activity was also observed for the previously known mutagens benz[a]pyrene, chrysene, benz[a]-anthracene, perylene and β-naphthylamine, on exposure to strains TA 98 and/or TA 100 with S-9.     

In vitro antimutagenicity of curcumin against environmental mutagens

The effects of curcumin, the yellow pigment of the spice, turmeric (Curcuma longa) on the mutagenicity of several environmental mutagens were investigated in the Salmonella/microsome test with or without Aroclor 1254-induced rat-liver homogenate (S-9 mix). With Salmonella typhimurium strain TA98 in the presence of S-9 mix, curcumin inhibited the mutagenicity of bidi and cigarette smoke condensates, tobacco and masheri extracts, benzo[a]pyrne and dimethyl benzo[a]anthracene in a dose-dependent manner. Curcumin did not influence the mutagenicity without S-9 mix of sodium azide, monoacetylhydrazine and streptozocin in strain TA100 nor of 4-nitrophenylenediamine in strain TA98. Our observations indicate that curcumin may alter the metabolic activation and detoxification of mutagens.