Detection of mutagenicity of diphenyl ether herbicides in Salmonella typhimurium YG1026 and YG1021

Three kinds of diphenyl ether herbicides, 4-nitrophenyl 2,4,6-trichlorophenyl ether (CNP, chlornitrofen), 2,4-dichlorophenyl 3-methoxy-4-nitrophenyl ether (chlomethoxynil) and 2,4-dichlorophenyl 3-methoxycarbonyl-4-nitrophenyl ether (bifenox), were tested for mutagenicity in Salmonella typhimurium YG1026 and YG1021, which have high nitroreductase activity, and also in S. typhimurium TA100 and TA98. CNP and chlomethoxynil showed mutagenicity in S. typhimurium YG1026, without S9 mix, inducing 50 and 304 revertants per μg. These mutagenicities were suppressed by the addition of S9 mix. CNP and chlomethoxynil were also mutagenic to YG1021 with and without S9 mix, and their mutagenicities were lower than those to YG1026. On the other hand, bifenox was mutagenic to YG1026 only with S9 mix, inducing 3.0 revertants per μg. These three herbicides showed no mutagenicity in S. typhimurium TA100 and TA98 either with or without S9 mix.     

Mutagenicity of alcoholic beverages

The mutagenicities of evaporated residues of alcoholic beverages were tested by the Ames method with the modification of pre-incubation, by using Salmonella typhimurium TA100 and TA98. 12 of 13 brands of whisky were mutagenic to TA100 without S9 mix. Addition of S9 mix decreased or abolished these mutagenicities. 5 brands of brandy and 1 apple brandy were tested, and all showed a similar type of mutagenicity to that of whisky. A fraction of brand-K whisky, containing a major mutagen(s), eluted from XAD-2 column with water, gave 3800 revertants of TA100 per plate at a dose equivalent to 10 ml of whisky.     

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.     

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.     

Mutagenicity of products formed by ozonation of naphthoresorcinol in aqueous solutions

The mutagenicity of products formed by ozonation of naphthoresorcinol in aqueous solution was assayed with Salmonella typhimurium strains TA97, TA98, TA100, TA102 and TA104 in the presence and absence of S9 mix from phenobarbital- and 5,6-benzoflavone-induced rat liver. Ozonated naphthoresorcinol was mutagenic in TA97, TA98, TA100 and TA104 without S9 mix. By the addition of S9 mix, the mutagenic activity of ozonated naphthoresorcinol was markedly suppressed in TA98 and TA100, but became positive in TA102. High-performance liquid chromatography (HPLC) after derivatization to 2,4-dinitrophenylhydrazones demonstrated the formation of glyoxal as an ozonation product of naphthoresorcinol. Ion chromatographic technique also demonstrated the formation of o-phthalic acid, muconic acid, maleic acid, mesoxalic acid, glyoxylic acid and oxalic acid as ozonation products. The mutagenicity assays of these identified products with five Salmonella showed that glyoxal and glyoxylic acid were directly mutagenic; the former in TA100, TA102 and TA104, the latter in TA97, TA100 and TA104. In the presence of S9 mix, glyoxylic acid gave a positive response of mutagenicity for TA102. The experimental evidence supported that glyoxal and glyoxylic acid may contribute to the mutagenicity of ozonated naphthoresorcinol.     

Mutagenicity of five food additives in Ames/Salmonella/microsome test

The mutagenic activity of five food additives (K₂S₂O₅: potassium metabisulphite, KMB; K₂SO₄: potassium sulphate, KS; Na₂SO₃: sodium sulphite, SS; KNO₃: potassium nitrate, KN; NaNO₃: sodium nitrate, SN) were investigated using histidin auxotrophs TA98 and TA100 strains ofSalmonella typhimurium in the presence or absence of S9 mix. The test substance were investigated for their mutagenic effects at non toxic concentrations of 0.83, 1.66, 3.33 and 5.00 mg/plate with and without S9 mix. All the test substances were not mutagenic on TA98 and TA100 strains ofSalmonella typhimurium in the presence or absence of S9 mix except KS and SN. KS and SN showed a weak mutagenic effect on TA100 strain in the absence of S9 mix.     

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.     

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.     

Identification of methylglyoxal as a major mutagen in wood and bamboo pyroligneous acids

To identify the major mutagen in pyroligneous acid (PA), 10 wood and 10 bamboo pyroligneous acids were examined using the Ames test in Salmonella typhimurium strains TA100 and TA98. Subsequently, the mutagenic dicarbonyl compounds (DCs), glyoxal, methylglyoxal (MG), and diacetyl in PA were quantified using high-performance liquid chromatography, and the mutagenic contribution ratios for each DC were calculated relative to the mutagenicity of PA. Eighteen samples were positive for mutagens and showed the strongest mutagenicity in TA100 in the absence of S9 mix. MG had the highest mutagenic contribution ratio, and its presence was strongly correlated with the specific mutagenicity of PA. These data indicate that MG is the major mutagen in PA.     

Screening for the mutagenicity of nitro-group containing hypoxic cell radiosensitizers using Salmonella typhimurium strains TA 100 and TA 98

A series of sixteen 2-, 4- and 5-nitroimidazoles, four nitrobenzenes, five nitrofurans, and a nitropyrrole, most of which have been studied previously as hypoxic cell specific radiosensitizers, have been screened for their mutagenicity using the Salmonella typhimurium strains TA 100 and TA 98 developed by Ames and co-workers. Most of these compounds were mutagenic and had a one to two order of magnitude greater mutagenicity towards TA 100 (base-pair substitution sensitive) than TA 98 (frame-shift sensitive). The spectrum of mutagenic efficiencies for the drugs which was observed could be correlated to some extent with the electron affinity of these compounds. Exceptions to this correlation may indicate drugs of interest for further studies both as mutagens and hypoxic cell radiosensitizers.     

Mutagenic activity of 6-aminoquinoxalines in Salmonella typhimurium

Mutagenicity of 6-aminoquinoxaline derivatives was tested with Salmonella typhimurium strains Ta98 and TA100 in the presence and absence of S9 mix from the viewpoint that the 6-aminoquinoxaline skeleton is a common unit of mutagenic imidazoquinoxalines. We tested nine compounds: 5-methyl-6-methylaminoquinoxaline (1), 3,5-dimethyl-6-methylaminoquinoxaline (2), 2,5-dimethyl-6-metnylaminoquinoxaline (3), 6-methylamino-2,3,5-trimethylquinoxaline (4), 2,3-diethyl-5-methyl-6-methylaminoquinoxaline (5), 5-methyl-6-methylamino 3-phenylquinoxaline (6), 6-amino-2,3,5-trimethylquinoxaline (7), 6-dimethylamino-2,3-5-trimethylaminoquinoxaline (8), 6-amino-2,3-dimethylquinoxaline (9). These compounds showed the mutagenic activity for both TA98 and TA100 in the presence of S9 mix, where they were more sensitive for TA100 strain. Methyl groups at the 2, 3 and/or 5 positions increased the potency of mutagenicity (1 < 2 < 3 ⪡ 4, 9 < 7). However, ethyl groups at the 2 and 3 positions lowered the mutagenicity of the methyl substitute but elevated it of the parental compound (1 < 5 < 4). A methyl group at the N⁶ position decreased the mutagenicity (7 > 4 > 8).     

Mutagenicity of glycerol chlorohydrines and of their esters with higher fatty acids present in protein hydrolysates

3-Chloro-1,2-propanediol and 1,3-dichloro-2-propanol caused base substitutions in Salmonella typhimurium TA1535 both with and without metabolic activation. Metabolic activation seemed to act mainly by decreasing the toxicity of these compounds. A difference in the growth of the wild-type and repair-deficient strains of Escherichia coli was observed only for 1,3-dichloro-2-propanol with S9 mix. Esters of both chlorohydrines with fatty acids had smaller mutagenic effects than unesterified compounds.     

The mutagenicity analysis of imidapril hydrochloride and its degradant,diketopiperazine derivative,nitrosation mixtures by in vitro Ames test with two strains of Salmonella typhimurium

AimThe evaluation of mutagenic properties of imidapril hydrochloride (IMD) and its degradation impurity, diketopiperazine derivative (DKP), nitrosation mixtures was conducted in order to analyze the carcinogenic risk of IMD long-term treatment in patients. In this study an in vitro Ames test with Salmonella enterica serovar Typhimurium TA 98 and TA 100 strains was used.BackgroundIMD and DKP contain nitrogen atoms, which makes them theoretically vulnerable to in vivo nitrosation with the production of N-nitroso compounds (NOC). NOC, in turn, are known animal mutagens indicating that their endogenous production from nitrosable drugs constitutes a carcinogenic hazard.Materials and methodsPure IMD sample was exposed to forced degradation conditions of increased temperature and dry air in order to achieve a DKP sample. Both samples were then treated with a nitrosating agent and the obtained nitrosation mixtures were subjected to mutagenicity analysis by the Ames test with S. typhimurium TA 98 and TA 100 strains in the presence and absence of metabolic activation system (S9 mix) using a commercial Ames MPF 98/100 microplate format mutagenicity assay kit.ResultsNone of the six concentrations of the investigated nitrosation mixtures exhibited any mutagenic potential in both S. typhimurium strains. The addition of S9 mix did not alter the non-mutagenic properties of the studied compounds.ConclusionsThe nitrite treatment of both studied compounds has no impact on their mutagenic properties under the conditions of the present studies. Hence, IMD and DKP nitrosation mixtures are classified as non-mutagens in this test.     

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.     

Mutagenicity assessment of Salacia chinensis by bacterial reverse mutation assay using histidine dependent Salmonella typhimurium tester strains

Background and objectiveGenotoxicity analysis is one of the most important non-clinical environmental safety investigations required for pharmaceutical and agrochemical product registration. Any medicinal product must undergo a risk evaluation to determine its mutagenicity and carcinogenicity.Materials and methodsThe Ames test is a commonly used in vitro test for determining a test chemical's mutagenic activity. Histidine-dependent Salmonella typhimurium strains with a defective gene that causes the bacteria to synthesis the necessary amino acid histidine for life were tested for mutagenic potential. In order to reveal pro-mutagens and mutagens, the mutagenic potential of both plate integration and pre-incubation techniques was examined in the presence and absence of metabolizing system. Salacia chinensis has been widely used in ayurveda to treat various ailments. However, the information of mutagenicity of Salacia chinensis is scarce as per available literature.ResultsThe mutagenicity of a Salacia chinensis root extract was investigated utilizing the Ames assay with plate incorporation and pre-incubation protocols using the appropriate Salmonella typhimurium tester strains: TA98, TA100, TA1537, TA1535, and TA102 in the presence and absence of S9. The concentrations used were 0.3123, 0.625, 1.25, 2.5 and 5 mg/plate. The extract of Salacia chinensis root did not show any mutagenic effect in any of the Salmonella typhimurium strains at the concentrations tested in the absence or presence of metabolic activation.ConclusionThe root of Salacia chinensis was hence confirmed to be non-mutagenic and at least according to the results of this genotoxicity evaluation can be regarded as being safe for human use.     

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.     

Evaluation of the mutagenic properties of the coccolithophorePleurochrysis carterae (Haptophyceae) as a potential human food supplement

The mutagenicity of the algaPleurochrysis carterae for use as human food was tested by the Ames method with the modification of pre-incubation, by usingSalmonella typhimurium TA98, TA100, TA1535, TA1537 andEscherichia coli WP2uvrA. The freeze-dried powder ofP. carterae was not mutagenic to any strain either with or without S9 mix. In view of the absence of adverse effects ofP. carterae in this mutagenicity study, it is suggested thatP. carterae is safe for human consumption as a human food supplement.Author for correspondence     

Non-mutagenicity of toluene,o-, m- and p-xylene,o-methylbenzylalcohol and o-methylbenzylsulfate in the Ames assay

Toluene, o-, m- and p-xylene, o-methylbenzylalcohol and o-methylbenzylsulfate were assayed for mutagenicity in the Ames assay. These compounds were unable to revert Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100, either with or without metabolic activation by S9 mix derived from livers of rats either untreated or induced with Aroclor 1254.     

Mutagenicity and genotoxicity of dicapthon insecticide

Mutagenic and genotoxic effects of dicapthon were investigated by using the bacterial reverse mutation assay in Salmonella typhimurium TA97, TA98, TA100 and TA102 strains with or without metabolic activation system (S9 mix), and chromosome aberrations (CAs), sister chromatid exchanges (SCEs), and micronucleus (MN) tests in human peripheral blood lymphocytes in vitro. Dicapthon was dissolved in dimethyl sulfoxide for all test systems. 0.1, 1, 10 and 100 μg/plate doses of dicapthon were found to be weakly mutagenic on S. typhimurium TA 98 without S9 mix. The human peripheral lymphocytes were treated with four experimental concentrations of dicapthon (25, 50, 100, and 200 μg/mL) for 24 and 48 h. Dicapthon increased the frequency of SCE only at the 100 μg/mL concentration for the 24 and 48 h applications. Dicapthon also induced abnormal cell frequency, CA/cell ratio and frequency of MN dose dependently for 24 and 48 h. Dicapthon showed a statistically significant cytotoxic effect by decreasing the mitotic index in all concentrations and a cytostatic effect by decreasing nuclear division index in 100 and 200 μg/mL concentrations for both treatment periods when compared with both untreated and solvent controls. These values decreased also in a dose dependent manner.     

Mutagenicity of 2-methylacrolein, 2-ethylacrolein and 2-propylacrolein in Salmonella typhimurium TA100. A comparative study.

The C2-alkylated acrolein derivatives 2-methylacrolein, 2-ethylacrolein and 2-propylacrolein are mutagenic in Salmonella typhimurium TA100. They are direct mutagens, their mutagenic potency being inversely proportional to the size of the alkylating substituent in the C2 position. In the presence of S9 mix, the mutagenicity of all these substances is considerably reduced; the reduction in mutagenicity is inversely proportional to the direct mutagenic potential of the substance. As shown for 2-methylacrolein, the reduction in mutagenicity is dependent on the concentration of S9 in the S9 mix and is not significantly influenced by heat inactivation of the S9 mix or by addition of TCPO, an inhibitor of epoxide hydrolase, to the testing system. There are no indications of enzymatic activation by the metabolizing microsomal system.