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.     

Synthesis and mutagenicity of A-ring reduced analogues of 7,12-dimethylbenz[a]anthracene

The synthesis and mutagenicity of two derivatives of 7,12-dimethylbenz[a]anthracene (DMBA; 1), i.e., 1,2-H2DMBA (4) and 1,2,3,4-H4DMBA (5), are reported. These analogues (4 and 5) represent dihydro and tetrahydro A-ring reduced forms of DMBA, a region in the parent hydrocarbon (1) proposed to be involved in metabolism to the ultimate carcinogen. The synthesis for 4 without isolation of intermediates from the tosylhydrazone of 1,2,3,4-tetrahydrobenz[a]anthracene-4,7,12-trione (10) by successive reaction with 8 molar equiv of CH3Li, HI, and NaBH4 represents a novel approach to this hydrocarbon now available in sufficient quantity for biological studies. Interestingly, both of these reduced analogues 4 and 5 exhibited mutagenic activity in the Ames assay in the presence or absence of microsomal activation for strains TA98 and TA100. In these strains, DMBA was active only in the presence of S-9 fraction. In the plasmid-deficient strain TA1537, only tetrahydro analogue 5 exhibited mutagenic activity both in the absence and presence of S-9 fraction.     

Mutagenicity evaluation of phthalic acid esters and metabolites in Salmonella typhimurium cultures

The mutagenic potential of dimethyl phthalate (DMP), diethyl phthalate (DEP), dibutyl phthalate (DBP), and di-2-ethylhexyl phthalate (DEPH), as well as metabolites of DEHP--i.e., mono-2-ethylhexyl phthalate (MEHP), 2-ethylhexanol (2-EH), and phthalic acid (PA)--were tested in Salmonella typhimurium cultures using the Ames test procedure. The compounds were tested on strains TA98, TA100, TA1535, TA1537, TA1538, and TA2637 for base-pair substitution or frameshift-type mutations. Spot tests yielded negative responses for all compounds with the strains tested. Each compound was tested for a dose-effect relationship in the TA98, TA100, TA1535, and TA1538 systems. DEP and DBP exhibited a mildly positive response in both TA100 and TA1535 cultures, and DMP showed a similar response in TA1535. Normalization of the data for cytotoxicity of DMP suggests TA100 has a mildly positive effect. The higher doses of these compounds exhibited some cytotoxic effects. The mutagenic effects were apparently abolished by the addition of S9 fraction in TA100 and TA1535 cultures, while no effect, other than cytotoxicity, was observed in the TA98 and TA1538 systems. DEHP, MEHP, 2-EH, and PA exhibited no mutagenicity in any of the strains of Salmonella typhimurium tested, with or without S9 metabolic activation. MEHP and 2-EH, however, exhibited a moderate cytotoxic effect in most cultures.     

Synthesis,characterization and mutagenicity of new cis-[Pt(2-substituted-benzimidazole)2Cl2] complexes

In this study, four new platinum(II) complexes with the structures cis-[Pt(Ligand)2Cl2] (ligand = 2-(p-methoxy-/or-p-chlorobenzyl or p-methoxyphenyl)benzimidazol (1, 2, 4 respectively) and 5(6)-methyl-2-phenoxymethylbenzimidazole (3) were synthesized and characterized by their elemental analysis, and IR and 1H NMR spectra. The potentials of the Pt(II) complexes for short-term bacterial mutagenicity were tested in reverse-mutation assays using Salmonella typhimurium frame-shift strain T 98 and S. typhimurium TA 100 and TA 102 strains, which carry mutations particularly sensitive to reversion by DNA base-pair substitution. The tests were performed in the absence of S9 rat liver fraction. Among the complexes tested 1 had no mutagenic activity. Complex 4 was found to be weakly mutagenic in TA 98 only. The Pt(II) complexes 2 and 3 were found to be mutagenic in TA 98, TA 100 and TA 102.     

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.     

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.     

Ames mutagenicity tests on purified 3-nitropropionic acid

3- Nitropropionic acid is a toxic compound produced by several moulds involved in food fermentation or spoilage. An impure commercial sample of this compound was previously reported as being mutagenic to Salmonella typhimurium strains TA1535 and TA100. In the present study, a sample from the same lot of 3- nitropropionic acid was mutagenic in strain TA100 without metabolic activation, but this activity was diminished after recrystallization. This sample was not mutagenic in strain TA98, before or after recrystallization. A new, purer commercial sample was non-mutagenic in strains TA98, TA100 and TA1538, with or without metabolic activation. Therefore the mutagenicity reported to be due to 3- nitropropionic acid was considered to be due to the impurity(ies).     

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.     

Effect of micronutrients,antioxidants and related compounds on the mutagenicity of 3,2′-dimethyl-4-aminobiphenyl,a colon and breast carcinogen

The possible antimutagenic effects of butylated hydroxytoluene (BHT), disulfiram, indole-3-carbinol, indole-3-acetonitrile, sodium selenite and α-tocopherol on 3,2′-dimethyl-4-aminobiphenyl-induced mutagenicity were studied using the Ames Salmonella/mammalian microsome assay system with strains TA98 and TA100. All seven compounds were nonmutagenic in both bacterial tester strains. The addition of 50–250 μg of sodium selenite, 5–50 mg of α-tocopherol of 50–250 μg of BHT per plate inhibited DMAB-induced mutagenicity in TA98 and/or TA100. Ethoxyquin, disulfiram and indole-3-carbinol increased DMAB-induced mutagenicity in TA100, whereas these compounds had little or no effect in TA98. Indole-3-acetonitrile had very little effect in either strain.     

Synthesis and mutagenicity of 5,11-dimethylchrysene and some methyl-oxidized derivatives of 5-methylchrysene.

A series of compounds structurally related to the carcinogen and mutagen 5-methylchrysene (1) was synthesized and tested for mutagenicity toward S. typhimurium TA 100. The compounds prepared were 5,11-dimethylchrysene (2), 5-(hydroxymethyl)chrysene (3), 5-(acetoxymethyl)chrysene (4), 5-carbomethoxychrysene (5), 5-(hydroxymethyl)-1,2,3,4-tetrahydrochrysene (6), 5-carbomethoxy-1,2,3,4-tetrahydrochrysene (7), and 5H-chryseno[4,5-bcd]pyran-5-one (31). When tested in the presence of rat liver homogenate, 1 and 2 were active while 3--7 were less mutagenic than 1; 31 was highly mutagenic. The mutagenicity of 1 and 2 contrasts with the low activity of 5,12-dimethylchrysene, which supports the generalization that the structural requirements favoring activity are a bay-region methyl group and a free peri position, both adjacent to an unsubstituted angular ring. The low activity of 3--7 indicates that methyl oxidation is not an important activation process for 1. This agrees with previous studies in which the major proximate mutagen and carcinogen of 1 was identified as 1,2-dihydro-1,2-dihydroxy-5-methylchrysene.     

Mutagenicity and cytotoxicity of N-methyl-2-pyrrolidinone and 4-(methylamino)butanoic acid in the Salmonella/microsome assay

The industrial solvent N-methyl-2-pyrrolidinone (NMP) and its hydrolysis product, 4-(methylamino)butanoic acid (N-MeGABA), were examined for mutagenicity and cytotoxicity in the Ames Salmonella/microsome assay. In order to detect a broad range of possible mutagenic endpoints, the following strains were used in the assay: base-pair substitution strains TA100, TA102 and TA104; frameshift strains TA97 and TA98; and repair proficient strains TA2638, UTH8413 and UTH8414. In the standard plate incorporation assay, six log-linear doses of each compound were tested; doses ranged from 0.01 to 1000 mumol/plate for NMP, and 0.01 to 316 mumol/plate for N-MeGABA. Neither compound was detectably mutagenic when tested in the presence and absence of metabolic activation by Aroclor-induced rat liver S9. NMP did show significant responses with strains TA102 and TA104 that were less than two-fold over background, but no clear dose-response relationships were evident. A preincubation modification of the assay was also performed, using strains TA98 and TA104. Mutagenic activity was not observed for NMP, while N-MeGABA showed significant responses with TA104 but dose-related mutagenicity was not established. Preincubation testing revealed both NMP and N-MeGABA to be cytotoxic to the test population of Salmonella at the highest treatment doses.     

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.     

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.     

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.     

Structure-affinity relationship study on N-(1,2,3,4-tetrahydronaphthalen-1-yl)-4-aryl-1-piperazinealkylamides, a new class of 5-hydroxytryptamine7 receptor agents

A series of N-(1,2,3,4-tetrahydronaphthalen-1-yl)-4-aryl-1-piperazinealkylamides was prepared and their affinity for serotonin (5-hydroxytryptamine, 5-HT) 5-HT7, 5-HT(1A), and 5-HT(2A) receptors was measured by in vitro binding assays. In relation to 5-HT7 receptor affinity, receptor binding studies indicated that (i) the optimal alkyl chain length was five methylenes, (ii) an unsubstituted 1,2,3,4-tetrahydronaphthalenyl nucleus was preferred, and (iii) the substitution pattern of the aryl ring linked to the piperazine ring played a crucial role. Several compound with high affinity for 5-HT7 receptors were identified. Among them, 4-(2-methoxyphenyl)-N-(1,2,3,4-tetrahydronaphthalen-1-yl)-1-piperazinehexanamide (28), 4-(2-acetylphenyl)-N-(1,2,3,4-tetrahydronaphthalen-1-yl)-1-piperazinehexanamide (34), 4-(2-methylthiophenyl)-N-(1,2,3,4-tetrahydronaphthalen-1-yl)-1-piperazinehexanamide (44), 4-(2-hydroxyphenyl)-N-(1,2,3,4-tetrahydronaphthalen-1-yl)-1-piperazinehexanamide (46), and 4-(2-methylphenyl)-N-(1,2,3,4-tetrahydronaphthalen-1-yl)-1-piperazinehexanamide (49) were assayed for the 5-HT7 receptor-mediated relaxation of substance P-induced guinea pig ileum contraction. Compounds 28, 44, and 49 behaved as full agonists and compound 34 as a partial agonist, whereas derivative 46 acted as an antagonist. Among the compounds presented here, it emerged that 44 was identified as a potent 5-HT7 receptor agonist (Ki = 0.22 nM, EC50 = 2.56 microM), endowed with selectivity over 5-HT(1A) and 5-HT(2A) receptors (200-fold and >1000-fold, respectively).     

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 and anti-mutagenicity of Acanthopanax divaricatus var. albeofructus

The beneficial effects of Acanthopanax divaricatus var. albeofructus (ADA) extracts have been assessed by mutagenic and anti-mutagenic activities by Ames test. Mutation of Salmonella typhimurium strains TA 98, TA 100, TA1535, TA1537, and Escherichia coli WP2 uvr A was assayed in duplicates by the procedure of Maron and Ames in the presence or absence of S9 mix. As a result, ADA extracts were not mutagenic for S. typhimurium strains TA 98, TA 100, TA1535, TA1537, and E. coli by the Ames assay. Anti-mutagenic activity was assayed by the Ames mutagenicity assay using histidine mutant of S. typhimurium strains TA 98 and TA 100, using the plate-incorporation method. 2-Aminoanthrancene (2-AA), 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide (AF-2), and sodium azide (NaN(3)) were used as the mutagens. ADA extracts showed a strong anti-mutagenic activity against 2-AA-induced mutagenesis which requires liver-metabolizing enzymes, and the same extract exhibited inhibitory effects on AF-2 and NaN(3)-induced mutagenesis in the absence of liver-metabolizing enzymes. The data indicate that ADA extracts contain anti-mutagenic activities against typical mutagens. The anti-mutagenic property of ADA provides additional health supplemental value to the other claimed therapeutic properties of the plant.     

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.     

Mutagenicity testing of agent orange components and related chemicals

Components of the herbicide Agent Orange--2,4-dichlorophenoxyacetic acid (2,4,-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) and their esters, and the contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)--and related chemicals were tested for mutagenicity using Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537. No mutagenic activity was observed for any of the chemicals tested.     

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.