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.     

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 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 and DNA-damaging activity caused by decomposed products of potassium sorbate reacting with ascorbic acid in the presence of Fe salt.

Although potassium sorbate (PS), ascorbic acid and ferric or ferrous salts (Fe-salts) are used widely in combination as food additives, the strong reactivity of PS and oxidative potency of ascorbic acid in the presence of Fe-salts might form toxic compounds in food during its deposit and distribution. In the present paper, the reaction mixture of PS, ascorbic acid and Fe-salts was evaluated for mutagenicity and DNA-damaging activity by means of the Ames test and rec-assay. Effective lethality was observed in the rec-assay. No mutagenicity was induced in either Salmonella typhimurium strains TA98 (with or without S-9 mix) or TA100 (with S-9 mix). In contrast, a dose-dependent mutagenic effect was obtained when applied to strain TA100 without S-9 mix. The mutagenic activity became stronger increasing with the reaction period. Furthermore, the reaction products obtained in a nitrogen atmosphere did not show any mutagenic and DNA-damaging activity. PS, ascorbic acid and Fe-salts were inactive when they were used separately. Omission of one component from the mixture of PS, ascorbic acid and Fe-salt turned the reaction system inactive. These results demonstrate that ascorbic acid and Fe-salt oxidized PS and the oxidative products caused mutagenicity and DNA-damaging activity.     

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.     

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.     

Mutagenicity studies of selected antihistamines, their metabolites and products of nitrosation

Methapyrilene, four structurally related antihistamines, three metabolites of methapyrilene and two products of the reaction of methapyrilene with nitrite were all tested for mutagenicity to Salmonella typhimurium. The two products of the methapyrilene-nitrite reaction have also been identified as metabolites of methapyrilene. None was mutagenic alone, either with or without rat liver S-9 activation. After reaction with sodium nitrite in acetic acid solution (nitrosation), the products of five of the ten compounds were mutagenic. These compounds were methaphenilene, 2-thiophenemethanol, 2-thiophenecarboxylic acid, N-(2-pyridyl)-N'N'-dimethylethylenediamine and N-(2-thenylmethyl)-2-aminopyridine.     

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.     

Detection of IQ-type mutagens in canned roasted eel

Basic extracts of canned roasted eel exhibited the highest mutagenicity among seven kinds of canned products assayed with Salmonella typhimurium TA98 in the presence of S-9 mix. The major mutagenic compounds of canned roasted eel extracts were purified and analysed by HPLC. The mutagenic fractions corresponding to the peaks of standard 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline(MeIQx) and 2-amino-3,7,8-trimethylimidazo[4,5-f]quinoxaline(7,8-DiMeIQx ) were further confirmed by the comparison of UV spectra, tester strain specificity and nitrite treatment. The estimated contents of MeIQx and 7,8-DiMeIQx were 1.1 ng and 5.3 ng per gram of canned roasted eel, respectively. Cooking temperature and time seemed to be the major factors affecting mutagen formation in fried eel. The type and amount of mutagenic compounds detected in canned roasted eel are likely to be correlated with the relative levels of specific free amino acids in raw eel.     

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.     

Antimutagenicity of some flowers grown in Thailand

The mutagenicity of dichloromethane, methanol and water extracts of Antigonon leptopus Hook. & Arn., Curcuma sessilis Gage, Hibiscus rosa-sinensis Linn., Ixora coccinea Linn., Millingtonia hortensis Linn., Nelumbo nucifera Gaertn., Plumeria obtusa Linn., Punica granatum Linn., Rhinacanthus nasutus ((Linn.) Kurz.) and Syzygium malaccense ((Linn.) Merr.& Perry) before and after nitrite treatment was firstly investigated in the Ames test. Their antimutagenicity against the product of the reaction mixture of 1-aminopyrene nitrite model in the absence of metabolic activation on Salmonella typhimurium TA 98 and TA 100 was evaluated. The results showed that none of the samples was mutagenic. Most nitrite-treated samples but dichloromethane extracts of Hibiscus rosa-sinensis, Plumeria obtusa, Syzygium malaccense, methanol extract of Syzygium malaccense and water extract of Hibiscus rosa-sinensis were mutagenic. The nitrite treated methanol extract of Nelumbo nucifera exhibited the highest mutagenicity on both strains. All dichloromethane extracts of flowers decreased the mutagenicity induced by the product of 1-aminopyrene nitrite model on both tester strains. Methanol extract of Curcuma sessilis and Punica granatum (15 mg/plate) showed the highest antimutagenic activity in TA 98 and TA 100, respectively. The protective effects of these flower extracts might be due to the presence of antimutagenic components that were supposed to be flavonoids.     

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.     

Formation of a mutagenic diazoquinone by interaction of phenol with nitrite

Reaction of phenol with nitrite under mildly acidic conditions produced p-nitrosophenol, p-diazoquinone and o-diazoquinone. p-Diazoquinone showed mutagenicity in Salmonella typhimurium strains TA98 and TA100 without metabolic activation; the number of his+ revertant colonies in strain TA98 was 85 with a dose of 20 nmol (after deduction of the background (control) number of about 20). Higher doses of the compound were bactericidal. While the reaction of phenol with an equivalent amount of nitrite at pH 3 produced p-nitrosophenol in high yield, reaction with excess nitrite produced a high yield of p-diazoquinone. p-Nitrosophenol was converted to p-diazoquinone by reaction with nitrite. Formation of o-diazoquinone also increased with increasing amounts of nitrite. The formation of these compounds was not greatly affected by the presence of dimethylamine. Nitrosation of dimethylamine with nitrite was stimulated by phenol because of the formation of p-nitrosophenol and its stimulatory effect. Thus, the reaction of phenol with nitrite can produce mutagenic p-diazoquinone, and can also stimulate nitrosation of secondary amines by production of p-nitrosophenol.     

Non-clastogenicity in mouse bone marrow of fructose/lysine and other sugar/amino acid browning products with in vitro genotoxicity

Heated sugar/amino acid reaction mixtures, known to contain products that are clastogenic and/or mutagenic to cells in vitro, were evaluated for clastogenic activity in mouse bone marrow using the erythrocyte micronucleus assay. Heated (i.e. browned) fructose/lysine reaction mixtures were also evaluated in the Salmonella his-reversion assay and the Chinese hamster ovary (CHO) cell chromosomal aberration assay to confirm and extend previous in vitro observations. Significant mutagenicity of fructose/lysine mixtures was observed in Salmonella typhimurium strains TA100, TA2637, TA98 and TA102, with greater activity in mixtures heated at pH 10 than at pH 7. S-9 decreased the activity in strains TA100, TA2637 and TA98, but increased the activity in strain TA102. Both pH 7 and pH 10 reaction mixtures of the fructose/lysine browning reaction were highly clastogenic in CHO cells. Heated mixtures of fructose and lysine, and of glucose or ribose with lysine, histidine, tryptophan or cysteine, did not increase the frequency of micronucleated erythrocytes in mice when administered by the oral route. This indicates the absence of chromosomal aberrations in erythrocyte precursor cells, and indicates that the genotoxic components of the browned mixtures are not absorbed and distributed to bone marrow cells in amounts sufficient to induce micronuclei when given orally. Because sugar/amino acid browning reactions occur commonly in heated foods, it is important to evaluate further the in vivo genotoxicity of browning products in cell populations other than bone marrow.     

In vitro mutagenicity of water contaminants in complex mixtures

Trihalomethanes, Carbon tetrachloride and trichloroethylene were tested in single, binary and multi-complex mixtures using standard tester strains TA1535, TA1537, TA98 and TA100 of Salmonella typhimurium with and without addition of an in vitro metabolizing fraction S-9. Chloroform (CHCl3) was found to be mutagenic in all strains without S-9 activation. However, when tested with Bromoform (15%), which was nonmutagenic singly, the combined effect of the mixture was nonmutagenic. CCl4 was a direct mutagen (without S-9) in all strains except TA 1535. When combined with 85% CHCl3, only strains TA1535 and TA1537 were mutagenic. When tested with mammalian activation (S-9), CCl4 was mutagenic in all strains. However, when tested with CHCl3 (CHCl3 and CCl4-85:15), the mutagenic capability was lost. With or without S-9 Activation multi-complex mixture of CHCl3, CCl4 and TCE (85:8:7) was mutagenic for a narrow range of doses in all strains.     

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.     

Mutagenicity of thionitrites in the Ames test. The biological activity of thiyl free radicals

Thiols, such as glutathione and cysteine, are mutagenic in the Ames test, using Salmonella typhimurium strain TA 100 and rat kidney S-9 preparation [Glatt et al. Science 220, (1983)]. Formation of thiyl free radicals, RS., has been implicated in this effect. We have prepared thionitrite (nitrosylmercaptan) derivatives of glutathione and other thiols. These unstable derivatives decompose by homolysis, yielding RS. radicals. Glutathione thionitrite is mutagenic to strains TA 100 and TA 102, in the absence of activation by mammalian S-9 preparations. We suggest that this mutagenicity is evidence for the role of thiyl free radicals as biological reactive intermediates. Since alkyl nitrites readily convert thiols to thionitrites, our findings have implications for the toxicology of nitrosating drugs, such as amyl nitrite.     

The mutagenicities of seven coumarin derivatives and a furan derivative (nimbolide) isolated from three medicinal plants

Seven coumarin derivatives (imperatorin, heraclenin, xanthotoxin, marmesin, chelepin, oxypeucedanin, esculin) and a furan derivative (nimbolide) were screened on 6 Ames tester strains (TA92, TA94, TA97, TA98, TA100, TA102). The eight compounds are chemicals isolated from three Nigerian medicinal plants: Afraegle paniculata, Clausena anisata, and Azadirachta indica. Different preparations of the former are taken by Nigerians for gut disturbances, and a concoction of the latter called "Agbo" is taken as an antimalarial. Marmesin and imperatorin were mutagenic in all tester strains except TA94 and TA102. The mutagenicity potencies of marmesin and imperatorin were 20 and 0.2 respectively. Mutagenicity was highest in TA98 and TA100 in both compounds. Marmesin was optimally mutagenic at a dose of 1.04 micrograms, and imperatorin at 30.0 micrograms. Microsomal activation was not required for mutagenicity in both compounds.     

Enzyme-mediated mutagenicity in Salmonella typhimurium of contaminants of synthetic indigo products

The mutagenic potential of two natural and seven synthetic, commercial indigo dye products was investigated. The natural products showed no mutagenicity in Salmonella typhimurium stains TA98 and TA100. In the presence of rat-liver homogenate from Aroclor 1254 pretreated rats all of the synthetic products were mutagenic towards strain TA98 but not towards strain TA100. The mutagenic effect produced was highly dependent on the amount of rat-liver homogenate added. Because of its high mutagenic potential, one product was further investigated. In the presence of rat-liver homogenate this product was weakly mutagenic towards strain TA1537 and strongly mutagenic towards strain TA1538. No mutagenicity was observed in strain TA1535. Experiments with purified synthetic indigo and natural indigo revealed that the mutagenic activity of the synthetic commercial products can be ascribed to one or more contaminants.     

Chemical fate and mutagenic formation potentials of phenothiazine and related compounds during water chlorination

The reactions of hetero-tricyclic aromatic hydrocarbons (H-TCAHs) with hypochlorite in an aqueous solution were investigated under conditions that simulate wastewater disinfection. H-TCAH-hypochlorite reaction products were determined by gas chromatographic-mass spectrometric (GC-MS) analyses. For 20 μM, 10H-phenothiazine, 10H-phenoxazine, and phenoxathiin reacted rapidly with active chlorine in neutral pH (7.0), but no phenazine-hypochlorite reaction was observed over pH values of 5-9 for 1 hr. The 10H-phenothiazine-hypochlorite reaction began by oxidation with active chlorine to form its dioxides, followed by chloro-substitution in water. The extent of the reactions depended on the chlorine dose, solution pH and compound structures. Ames assays for the chlorination byproducts of 10H-phenothiazine and 10H-phenoxazine also showed to be weak mutagenicity in TA98 and TA100 strains without S9 mix, but no chlorination byproducts of phenoxathiin exhibited any mutagenicity in both tester strains with and without S9 mix.