The mutagenicity of halogenated alkanols and their phosphoric acid esters for Salmonella typhimurium

9 halogenated alkanols, 9 corresponding tris(haloalkyl)phosphates, and 2 bis-(2,3-dibromopropyl)phosphate salts were evaluated for mutagenicity against Salmonella typhimurium TA98, TA100, TA1535, TA1537 and TA1538, with and without rat liver in vitro metabolic activation system (S9 mix). Most of the test samples showed mutagenic activity in the strains TA100 and TA1535, but not in the strains TA98, TA1537 and TA1538. In general, the mutagenic activities of the phosphates obtained with S9 mix were greater than the activities obtained without S9 mix. Among the phosphates, several structure—activity relationships were found; i.e., (i) the bromoalkyl derivatives were more mutagenic than the corresponding chloroalkyl derivatives, (ii) the β-haloethyl derivatives were more mutagenic than the γ-halopropyl derivatives, (iii) the phosphates having adjacent β and γ halogen atoms in the alkyl moiety, e.g., tris-(2,3-dibromopropyl)phosphate, were particularly potent mutagens, (iv) the branched carbon chain reduced the mutagenic activities in spite of the presence of β-halogen atoms, e.g., tris(1-bromomethyl-2-bromoethyl)phosphate. However, such relations did not necessarily apply to the halogenated alkanols. It is concluded that the metabolic activation pathway via haloalkanols to mutagens must not be in common with all of tris-BP-like phosphates.     

Assessment of the Mutagenicity of Sediments from Yangtze River Estuary Using Salmonella Typhimurium/Microsome Assay

Sediments in estuaries are of important environmental concern because they may act as pollution sinks and sources to the overlying water body. These sediments can be accumulated by benthic organisms. This study assessed the mutagenic potential of sediment extracts from the Yangtze River estuary by using the Ames fluctuation assay with the Salmonella typhimurium his (−) strain TA98 (frameshift mutagen indicator) and TA100 (baseshift mutagen indicator). Most of the sediment samples were mutagenic to the strain TA98, regardless of the presence or absence of exogenous metabolic activation (S9 induction by β-naphthoflavone/phenobarbital). However, none of the samples were mutagenic to the strain TA100. Thus, the mutagenicity pattern was mainly frameshift mutation, and the responsible toxicants were both direct (without S9 mix) and indirect (with S9 mix) mutagens. The mutagenicity of the sediment extracts increased when S9 was added. Chemical analysis showed a poor correlation between the content of priority polycyclic aromatic hydrocarbons and the detected mutagenicity in each sample. The concept of effect-directed analysis was used to analyze possible compounds responsible for the detected mutagenic effects. With regard to the mutagenicity of sediment fractions, non-polar compounds as well as weakly and moderately polar compounds played a main role. Further investigations should be conducted to identify the responsible components.     

Mutagenicity of the phenacetin metabolites: N-hydroxy-p-phenetidine and nitrosophenetol in S. typhimurium TA100 and derivatives deficient in nitroreductase or O-acetylase: probes for testing intrabacterial metabolic activation

Two mutagenic metabolites of phenacetin, p-nitrosophenetol and N-hydroxy-p-phenetidine, were tested in S. typhimurium strains TA100, its nitroreductase-deficient derivative TA100NR, and O-acetylase-deficient strains TA100 Tn5-1-8-DNP1011 and -DNP1012 in the presence or absence of an exogenous metabolic activation system. The results indicate that bacterial nitroreductase(s) and O-acetylase(s), shown to be involved in the conversion of certain nitroarenes, are not required for the intrabacterial activation of the two phenacetin metabolites to bacterial mutagens. In view of the low reactivity of nitrosoarenes towards nucleophiles at neutrality, the mechanism by which they exert such a high mutagenic effect in S. typhimurium strains remains to be clarified, but is discussed.     

The mutagenicity of halogenated alkanols and their phosphoric acid esters for Salmonella typhimurium.

9 halogenated alkanols, 9 corresponding tris (haloalkyl)phosphates, and 2 bis-(2,3-dibromopropyl)phosphate salts were evaluated for mutagenicity against Salmonella typhimurium TA98, TA100, TA1535, TA1537 and TA1538, with and without rat liver in vitro metabolic activation system (S9 mix). Most of the test samples showed mutagenic activity in the strains TA100 and TA1535, but not in the strains TA98, TA1537 and TA1538. In general, the mutagenic activities of the phosphates obtained with S9 mix were greater than the activities obtained without S9 mix. Among the phosphates, several structure--activity relationships were found; i.e., (i) the bromoalkyl derivatives were more mutagenic than the corresponding chloroalkyl derivatives, (ii) the beta-haloethyl derivatives were more mutagenic than the gamma-halopropyl derivatives, (iii) the phosphates having adjacent beta and gamma halogen atoms in the alkyl moiety, e.g., tris-(2,3-dibromopropyl)phosphate, were particularly potent mutagens, (iv) the branched carbon chain reduced the mutagenic activities in spite of the presence of beta-halogen atoms, e.g., tris(1-bromomethyl-2-bromoethyl)phosphate. However, such relations did not necessarily apply to the halogenated alkanols. It is concluded that the metabolic activation pathway via haloalkanols to mutagens must not be in common with all tris-BP-like phosphates.     

Mutagenic activities of ten imidazole derivatives in <Emphasis Type="Italic">Salmonella typhimurium</Emphasis>

Ten imidazole derivatives were tested for mutagenicity in Salmonella typhimurium strains TA98 and TA100 both in the absence and presence of metabolic activation by the microsomal fraction S9 mix. In a general manner, derivatives tested exhibited a greater mutagenic activity in the TA100 strain comparing to the responses in TA 98. In the standard plate incorporation assay, 8 of these substances (80%) were found to be mutagenic for at least one of the two strains in the presence or absence of metabolic activation. Two compounds showed positive results in TA98 and 6 compounds were also mutagenic in TA100 without S9. In the presence of S9 mix, all of the 10 substances were non-mutagenic in TA98, whereas 4 compounds were positive in TA100. The results suggested the mutagenic potentials of the imidazole derivatives particularly inducing the reversion of base-pair substitutions. According to the structure-activity relationships phenyl groups in position 2 with different substituents can confer the mutagenic activity of the tested compounds. Methyl groups in different positions of these phenyl substituents can cause different types of mutations. This mutagenic effect is observed more clearly when the phenyl group is inhibited with a nitro group.     

Mutagenicity of methylated fluorenes and benzofluorenes

Methylated fluorenes were assayed for mutagenic activity towards Salmonella typhimurium TA98 and TA100. None of these methylfluorenes were mutagenic in the absence of metabolic activation. In the presence of 900 × g supernatant from Aroclor-induced rats, 9-methylfluorene and 1,9-dimethylfluorene were active towards TA98 and TA100. The structural requirement for mutagenic activity within this series was the presence of a single methyl substituent at the 9-position. Enhanced mutagenic activity was also observed for benzofluorenes similarly methylated at their benzylic positions.     

Use of recombinant DNA techniques in cloning DNA repair genes and in the study of metagenesis in mammalian cells

18 polycyclic aromatic hydrocarbons (PAHs) and 7 quinones were tested for mutagenicity using Salmonella typhimurium TA97, TA98 and TA100 with or without metabolic activation. In the presence of metabolic activation, TA97 was more susceptible to mutation than either TA98 or TA100 by many of PAHs tested. PAHs such as 1-methylphenanthrene, fluoranthene, pyrene, benzo[a]pyrene, benzo[e]pyrene and perylene had high mutagenic effects on TA97 in the presence of metabolic activation. 1,6- and 1,8-pyrenequinones were also highly mutagenic on TA97 in the presence or absence of metabolic activation. It appears that pyrene is mutagenic through its metabolic conversion to pyrenequinones.     

Mutagenicity of aminophenyl and nitrophenyl ethers, sulfides, and disulfides.

The mutagenic activity of several aromatic amines and aromatic nitro compounds related to 4,4'-methylenedianiline towards Salmonella typhymurium tester strains TA100 and TA98 was evaluated. The heteroatomic analogs of 4,4'-methylenedianiline which include aminophenyl and nitrophenyl ethers, sulfides and disulfides were assayed in the presence of rat-liver homogenate. The relative mutagenic response of these analogs indicated the following order of activity, --S-- greater than --O-- greater than --CH2--CH2-- greater than or equal to --S--S--. In both tester strains 4-aminophenylsulfone was inactive with and without microsomal activation. The p-nitrophenyl ethers, sulfides and disulfides were relatively strong mutagens without microsomal activation towards TA100. While 4-nitrophenyldisulfide was found to possess significantly different mutagenic activity than 4-nitrothiophenol in TA98, 4-AMINOPHENYl disulfide has similar mutagenic properties to 4-aminothiophenol in both tester straains TA100 and TA98.     

Isolation and studies of the mutagenic activity in the Ames test of flavonoids naturally occurring in medical herbs

Quercetin, rhamnetin, isorhamnetin, apigenin and luteolin were isolated from medicinal herbs: Erigeron canadensis L., Anthyllis vulneraria L. and Pyrola chloranta L. The mutagenicity of these naturally occurring flavonoids was tested by the Ames method with S. typhimurium strains TA1535, TA1538, TA97, TA98, TA100 and TA102 in the presence and absence of metabolic activation. Of the above flavonoids only quercetin and rhamnetin revealed mutagenic activity in the Ames test. Quercetin induced point mutations in strains TA97, TA98, TA100 and TA102 of S. typhimurium. The presence of S9 rat liver microsome fraction markedly enhanced the mutagenic activity of quercetin in these strains. Rhamnetin appeared to be a much weaker mutagen in the Ames test. The compound induced mutations in strains TA97, TA98 and TA100 of S. typhimurium but only in the presence of metabolic activation.Comparison of the structure of the studied flavonoids with their mutagenic activity indicates that the mutagenicity of flavonoids is dependent on the presence of hydroxyl groups in the 3′ and 4′ positions of the B ring, and that the presence of a free hydroxy or methoxy group in the 7 position of the A ring also probably contributes to the appearance of mutagenic activity of flavonoids in the Ames test. It also appeared that the presence of methoxy groups, particularly in the B ring of the flavonoid molecule, markedly decreases the mutagenic activity of the compound.     

Δ1-Tetrahydrocannabinol and 1α,2α-epoxyhexahydrocannabinol: Mutagenicity investigation in the ames test

1,2-Epoxyhexahydrocannabinol is a metabolite of Δ¹-tetrahydrocannabinol. Because many epoxides are mutagens, we investigated 1,2-epoxyhexahydrocannabinol as well as Δ¹-tetrahydrocannabinol for mutagenicity with Salmonella typhimurium TA1535, TA1537, TA98 and TA100 in the presence and in the absence of S9 mix from liver homogenate of rats treated with Aroclor 1254. Additionally, an epoxide hydratase inhibitor was used in some experiments. Whereas several other epoxides and further positive controls, not requiring activation or activated under the same conditions, respectively, showed strong mutagenicity, no indications of a mutagenic hazard by 1,2-epoxyhexahydrocannabinol or by Δ¹-tetrahydrocannabinol were found.     

Mutagenic activity of furfural in Salmonella typhimurium TA100

The mutagenic activity of furfural was tested in Salmonella typhimurium strains TA98 and TA100. Furfural produced mutations in the TA100 strain, but not in the TA98 strain. A rat-liver microsomal fraction did not increase the mutagenic activity of furfural in either strain. Mutagenic activity of furfural in the TA100 strain was not increased by benzo[a]pyrene in the presence of metabolic activation.     

Evaluation of the mutagenicity of 'pan masala', a chewing substitute widely used in India

Mutagenicity of polar and non-polar extracts of a popular brand of 'pan masala' was examined using the Salmonella/mammalian microsome test (Ames assay) and 2 tester strains of Salmonella typhimurium, TA98 and TA100. These extracts were also subjected to pretreatment with sodium nitrite at acidic pH, to simulate conditions for endogenous nitrosation. The aqueous, aqueous:ethanolic and chloroform extracts as well as their nitrosated mixtures were non-mutagenic in the Ames assay, in the presence and absence of metabolic activation. Only the ethanolic extract elicited a weak mutagenic response in strain TA98 without metabolic activation demonstrating the presence of direct-acting frameshift mutagens in 'pan masala'.     

Structural specificity of aromatic compounds with special reference to mutagenic activity in Salmonella typhimurium — a series of chloro- or fluoro-nitrobenzene derivatives

The mutagenicity of 21 chloro- or fluoronitrobenzene compounds and 9 chloro- or fluorobenzene compounds in Salmonella typhimurium (strains TA98, TA1538, TA1537, TA100 and TA1535) was examined. The tests were carried out under the conditions of absence and presence of liver microsomal activation.15 nitro-group compounds had mutagenic activity; above all, compounds of fluoronitrobenzene were mutagenic for both types of strain. On the other hand, chloronitrobenzene compounds were mutagenic for base-pair substitution strains only. Mutagenic activity was exhibited by all compounds having a chloro or fluoro substituent at the para and ortho position in the nitrobenzene nucleus. All compounds without a nitro substituent showed no mutagenic activity.     

Relationship between mutagenic potency in Salmonella typhimurium and chemical structure of amino- and nitro-substituted biphenyls

All positional isomers of mononitro- and monoaminobiphenyls and those of dinitro-, diamino- and aminonitrobiphenyls, which have one substituent on each benzene ring, were assayed for mutagenicity in Salmonella typhimurium by the Ames method. The results suggest that the structural requirements favoring mutagenic activity are the presence of substituents at the 4-position and their absence at the 2'-position. The introduction of an amino group to the 3'- or 4'-position of 4-nitrobiphenyl or a nitro group to 3'- or 4'-position of 4-aminobiphenyl enhanced the mutagenicity. Among the mutagenic compounds, 4-nitro analogues were mutagenic in strains TA98 and TA100 in the absence of a microsomal metabolic activation system. Strain TA98NR was not reverted by the direct-acting mutagens, whereas strain TA98/1,8-DNP6 was as revertible as strain TA98; these results suggest that the direct-acting mutagenicity involves the reduction of the nitro group by bacterial nitroreductase but does not involve specific esterification enzymes.     

Mutagenicity of flavones,chromones and acetophenones in Salmonella typhimurium: New structure—activity relationships

28 flavones and 11 structurally-related flavonoids, chromones, and acetophenones, were tested for mutagenicity in the Salmonella typhimurium his reversion assay. 7 flavones, all of which were hydroxy- or methoxy-substituted at position 8, were moderate to strong mutagens in strain TA100 in the presence of rat liver S9 mix. In each case, the response of strain TA98 was either not significant or was very much weaker than that observed in strain TA100. The activation by S9 is not mediated by the microsomal cytochrome P₄₅₀ system, since activation was not diminished when microsomes were removed by centrifugation at 100000 × g. The observed strain specificity and structural requirements for activity indicate a mutagenic mechanism different from that associated with previously reported mutagenic flavonols (3-hydroxy-flavones) which are most active in strain TA98. The most mutagenic flavone investigated, 5,7,8-trihydroxy-flavone (norwogonin), had a potency of 17 revertants/nmole.Simplification of the chemical structures to hydroxy-substituted chromone and acetophenone systems revealed similar strain specificity, hydroxylation requirements, and S9 dependence within these structural classes, suggesting a similar activation pathway and mutagenic mechanism. The greatest mutagenic potency was observed within the flavone series, but significant potency was retained by similarly hydroxylated chromones and acetophenones. No mutagenic activity was observed in the absence of the aryl ketone moiety.     

Fecal mutagens from subjects consuming a mixed-western diet

Because of potential significance of fecal mutagens (presumptive carcinogens) in the pathogenesis of colon cancer, feces from 99 healthy subjects from the New York metropolitan area were studied. The diet histories indicate that all participants were consuming a mixed-western diet which is high in total fat and low in fiber. Fecal samples that were incubated under anaerobic conditions at 37 degrees C for 96 h or frozen without incubation, were extracted with hexane: peroxide-free diethyl ether (1:1), partially purified on a silica Sep-pak cartridge and assayed for mutagenicity using the Salmonella typhimurium/mammalian microsome system. Aliquots of fecal samples incubated anaerobically showed a higher frequency of mutagenic activity (per cent samples showing activity) in strains TA98 and TA100 with and without microsomal (S9) activation. In addition, the mutagens requiring S9 activation, were more frequently inactivated when the fecal samples were frozen immediately after defecation and transported to the laboratory. Compared with hexane: ether, extraction of fecal samples with acetone increased the mutagenic activity mostly with TA98 with S9 activation. The HPLC fractionation of hexane: ether extract with methanol: water gradient using reverse phase C-18 column and UV detector at 254 nm indicated that the mutagenic activity (TA98 with S9 activation) is concentrated in several peaks. This is the first demonstration of HPLC profile of fecal samples that are active in TA98 with S9 activation. HPLC profile of fecal extracts and mutagenic activity of these extracts in strains TA98 and TA100 suggest the presence of several types of mutagens in the feces of healthy subjects consuming a high-fat, low-fiber mixed-western diet.     

delta 1-Tetrahydrocannabinol and 1 alpha, 2 alpha-epoxyhexahydrocannabinol: mutagenicity investigation in the Ames test.

1,2-Epoxyhexahydrocannabinol is a metabolite of delta 1-tetrahydrocannabinol. Because many epoxides are mutagens, we investigated 1,2-epoxyhexahydrocannabinol as well as delta 1-tetrahydrocannabinol for mutagenicity with Salmonella typhimurium TA1535, TA1537, TA98 and TA100 in the presence and in the absence of S9 mix from liver homogenate of rats treated with Aroclor 1254. Additionally, an epoxide hydratase inhibitor was used in some experiments. Whereas several other epoxides and further positive controls, not requiring activation or activated under the same conditions, respectively, showed strong mutagenicity, no indications of a mutagenic hazard by 1,2-epoxyhexahydrocannabinol or by delta 1-tetrahydrocannabinol were found.     

The bacterial mutagenicity of nitropolychlorinated dibenzo-p-dioxins

The bacterial mutagenicity of 2-nitrodibenzo-p-dioxin, a mixture of 2-nitro-7-chloro- and 2-nitro-8-chlorodibenzo-p-dioxin, 7-nitro-2,3-dichloro-, 8-nitro-2,3,7-trichloro-, 2-nitro-1,3,7,8-tetrachloro- and 3-nitro-1,2,4,7,8-pentachlorodibenzo-p-dioxin was determined using Salmonella typhimurium tester strains TA98 and TA100 with and without rat hepatic S9 for metabolic activation. All the nitro-PCDDs exhibited some direct-acting mutagenicity with both tester strains, however, the activity was significantly lowered in the presence of exogenous S9 and the compounds were more mutagenic to tester strain TA98. The mutagenicity of the nitro-PCDDs was also dependent on structure because there was a marked decrease in activity with increasing chlorine content. Because nitro-PCDDs have recently been identified as incomplete combustion products of municipal waste, this study confirms that this new class of compounds contains some bacterial mutagens.     

Mutagenicity to Salmonella typhimurium of some Aspergillus and Penicillium mycotoxins

17 mycotoxins produced by various Aspergillus and Penicillium species were screened for their mutagenic activity to Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537, both with and without metabolic activation. Austdiol, austocystins A and D, kojic acid and viridicatumtoxin were found to be mutagenic after metabolic activation, while austdiol was also mutagenic per se. Aflatoxin B₁, sterigmatocystin and versicolorin A, which were used as positive controls were also mutagenic. No mutagenic activity was evident in the case of citrinin, cyclopiazonic acid, fumitremorgen B, griseofulvin, luteoskyrin, O-methylsterigmatocystin, mycophenolic acid, ochratoxin A, patulin, penicillic acid, secalonic acid D and TR₂-toxin.A good relationship was found between the mutagenic activity, or lack of it, of most of the mycotoxins with existing data on carcinogenicity. Inadequate information on the carcinogenicity of austdiol, austocystins A and D, kojic acid and viridicatumtoxin precluded correlations with mutagenicity to S. typhimurium. The relationship between chemical structure and mutagenicity of the mycotoxins is discussed.     

Mutagenicity of aliphatic epoxides

The mutagenicity of 17 aliphatic epoxides was determined using the specially constructed mutants of Salmonella typhimurium developed by Ames. The activity of these epoxides together with those reported in the literature as mutagens in strains TA100 and TA1535 depended on the degree of substitution around the oxirane ring. Monosubstituted oxiranes were the most potent mutagens in both strains. 1,1-Disubstitution resulted in the complete loss or reduction of mutagenicity. trans-1,2-Disubstituted, and tetrasubstituted oxiranes all lacked mutagenicity, while the cis-1,2-disubstituted oxiranes tested were weakly mutagenic in strain TA100 only. For the monosubstituted compounds the presence of electron-withdrawing substituents increased mutagenicity.