Mutagenic action of a series of epoxides.

The mutagenicity of a series of 13 epoxide compounds was studied using a bacterial plate assay system. The histidine-dependent tester strains TA98 (for frameshift mutagens) and TA100 (for base-pair substitution mutagens) of Salmonella typhimurium were used. Mutagenicity was evaluated both with and without the additon of rat liver microsomal extract. Dieldrin, diglycidyl ether of bis phenol A and 3 of its homologues were not mutagenic. Allyl glycidyl ether, n-butyl glycidyl ether, vinly cyclohexene diepoxide, glycidol, glycidal-dehyde, diglycidyl ether, diepoxybutane and diglycidyl ether of substituted glycerine were mutagenic in the TA100 strain, causing reversion of the bacteria to histidine independence. Dose-reponse curves of the mutagenicity of the latter 4 compounds were obtained. On a molar basis, glycidaldehyde was about 20-50 times more potent in producing mutation that were the other 3 epoxides in the dose-response test. In general, the mutagenicity of the epoxides was not enhanced or diminished by the addition of microsomal extract.     

Mutagenicity of aromatic glycidyl ethers with Salmonella

6 aromatic glycidyl ethers containing naphthyl, biphenyl or benzylphenyl substituents were synthesized. These epoxides together with the commercially available compounds 2-biphenylyl glycidyl ether were examined for dose-mutagenicity relationships using the plate incorporation Ames test with Salmonella typhimurium strains TA100 and TA1535. Structure-mutagenicity relationships were further examined for these compounds and 3 phenyl glycidyl ethers by concurrent testing at a single dose with strain TA100. Meaningful correlations could not be established for the mutagenicity of these epoxides to their molecular volumes, partition values, nor to their reactivities with the model nucleophile, 4-(4-nitrobenzyl) pyridine. However, it was noted that increased conjugated aromatic unsaturation with its resulting planarity led to increased mutagenicity and that this effect decreased when it was further removed from the epoxide moiety.     

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.     

Mutagenicities of glycidyl ethers for Salmonella typhimurium: relationship between mutagenic potencies and chemical reactivity

The lethal and mutagenic effects of ethyl, benzyl, 1-naphthylmethyl, 2-naphthylmethyl, 1-naphthylethyl, 2-naphthylethyl and 9-anthrylmethyl glycidyl ethers on Salmonella typhimurium (TA100, TA1535, TA98 and TA1538) were investigated. LD30-value became smaller with an increase in compound hydrophobicity. The mutagenicities of these compounds in TA100 increased in the order: 1-naphthylethyl glycidyl ether less than 2-naphthylethyl glycidyl ether less than benzyl glycidyl ether less than 2-naphthylmethyl glycidyl ether less than 1-naphthylmethyl glycidyl ether less than 9-anthrylmethyl glycidyl ether. 1-Naphthylmethyl and 2-naphthylmethyl glycidyl ethers were mutagenic toward TA1535. In TA98, 1-naphthylmethyl and 9-anthrylmethyl glycidyl ethers showed mutagenic activity and 9-anthrylmethyl glycidyl ether was more mutagenic than 1-naphthylmethyl glycidyl ether. 9-Anthrylmethyl glycidyl ether was also active in TA1538. In the reaction of glycidyl ethers with deoxyguanosine and related compounds, glycidyl ethers attacked at only N-7 of guanine. The alkylation rates of glycidyl ethers toward guanine residues in DNA were determined and the exciplex-formation ability of 7-substituted guanines was studied. The reactivity of glycidyl ethers with guanine residues in DNA has not provided a sufficient explanation for the variation in mutagenic potencies of glycidyl ethers.     

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 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 effect of furocoumarin monoadducts and cross-links on bacteriophage lambda

The mutagenicities of 17 closely related oxiranes were determined in 4 tester strains (Salmonella typhimurium TA98, TA100, TA1535, TA1537). The test compounds comprised all possible oxides of benzene and its partially hydrogenated congeners. In TA100 and TA1535, 12 of the tested oxiranes were weak to moderate mutagens. 4 of these were also active in TA98. No mutagenicity was observed with the remaining 5 compounds in any of the 4 strains.The presence of a double bond in formal conjugation with the epoxide ring increased the mutagenicity relative to that of the saturated oxirane. Interestingly, additional epoxide rings within the same molecule did not markedly increase the mutagenic activity, and for the oxiranes that are not activated by a double bond, the relationship between mutagenic activity and the number of epoxide rings in the molecule was even inverse.The influence of bromo and hydroxyl substitution on oxirane mutagenicity is discussed. Most notably, a compound having a 4-hydroxyl group in syn position to a 1,2-epoxide ring fused to the cyclohexane ring, a structure which has been suggested to increase the electrophilic reactivity of dihydrodiol epoxides through hydrogen bonding, was almost inactive.     

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.     

Synthesis and enantioselective mutagenicity of azidoalanine in Salmonella typhimurium

Azide mutagenicity involves the requisite formation of the putative novel aminoacid metabolite, beta-azidoalanine. The role of this metabolite, however, is unclear. In order to confirm the identity of this metabolite and provide additional information on possible stereochemical requirements for mutagenicity, authentic racemic and L-azidoalanine were synthesized by an unambiguous route and tested for mutagenicity in Salmonella typhimurium TA100, TA1535, hisG46 and Escherichia coli WP2-. A marked antipodal potency ratio was observed in strains TA100 and TA1535 when racemic and L-azidoalanine were compared. The mutagenic activity resided primarily in the L-isomer. The molar potency of L-azidoalanine in TA100 and TA1535 was nearly identical to that of azide. The lack of mutagenic response for racemic or L-azidoalanine in hisG46 and E. coli WP2- was like that reported for azide and is consistent with similar modes of action for these agents.     

A comparative study of mutagenic and SOS-inducing activity of biphenyls, phenanthrenequinones and fluorenones

A total of 23 chemicals--biphenyls, phenanthrenequinones and fluorenones--were tested for mutagenicity towards Salmonella typhimurium strains TA1538, TA1535 and TA98. SOS-inducing activity of the same chemicals was studied in terms of the SOS-inducing potency in Escherichia coli PQ37, using an automated instrument controlled by a dedicated computer program for the SOS Chromotest. Of the 23 chemicals studied 14 induced His+ revertants in S. typhimurium TA1538 hisD305 (-1 frameshift); none induced His+ reversions in TA1535 (base-pair substitution). The mutagenicity of the chemicals in S. typhimurium TA98 (pKM 101) was lower than in TA1538. There was a close correlation between mutagenicity and SOS-inducing activity of fluorenones and phenanthrenequinones. None of the biphenyls tested induced SOS response and this property does not depend upon the mutagenic activity of the chemicals. SOS Chromotest is particularly valid in detecting chemicals which give rise to base-pair substitutions through SOS induction. If positive results are obtained, the Salmonella assay may be omitted. However, this test cannot replace the Ames test especially for the primary screening of mutagenicity of chemicals with unknown structure.     

The mutagenicities of safrole, estragole, eugenol, trans-anethole, and some of their known or possible metabolites for Salmonella typhimurium mutants.

Safrole, estragole, anethole, and eugenol and some of their known or possible metabolites were tested for mutagenic activity for S. typhimurium TA1535, TA100, and TA98. Highly purified 1'-hydroxyestragole and 1'-hydroxysafrole were mutagenic (approximately 15 and 10 revertants/micromole, respectively) for strain TA100 in the absence of fortified liver microsomes; trans-anethole and estragole appeared to have very weak activity. 3'-Hydroxyanethole was too toxic for an adequate test. Supplementation with NADPH-fortified rat-liver microsomes and cytosol converted 3'-hydroxyanethole to a mutagen(s) and increased the mutagenic activities for strain TA100 of 1'-hydroxyestragole, 1'-hydroxysafrole, estragole, and anethole. No mutagenicity was detected for safrole or eugenol with or without added NADPH-fortified liver preparations. The electrophilic 2',3'-oxides of safrole, 1'-hydroxysafrole, 1'-acetoxysafrole, 1'-oxosafrole, estragole, 1'-hydroxyestragole, and eugenol showed dose-dependent mutagenic activities for strain TA1535 in the absence of fortified liver microsomes. These mutagenic activities ranged from about 330 revertants/micromole for 1'-oxosafrole-2',3'-oxide to about 7000 revertants/micromole for safrole-2',3'-oxide. The arylalkenes, their hydroxylated derivatives, or their epoxides did not show mutagenic activity for strain TA98, except for 1'-oxosafrole-2',3'-oxide, which had weak activity. Since the arylalkenes are hydroxylated and/or epoxidized by hepatic microsomes, hydroxy and epoxide derivatives appear to be proximate and ultimate mutagenic metabolites, respectively, of the arylalkenes.     

Comparative direct-acting mutagenicity of 1- and 2-nitropyrene: Evidence for 2-nitropyrene mutagenesis by both guanine and adenine adducts

The mutations and DNA adducts produced by the environmental pollutant 2-nitropyrene were examined in Salmonella typhimurium tester strains. 2-Nitropyrene was a stronger mutagen than its extensively studied structural isomer 1-nitropyrene in strains TA96, TA97, TA98, TA100, TA102, TA104 and TA1538. Both 1- and 2-nitropyrene were essentially inactive in TA1535. The mutagenicity of 1- and 2-nitropyrene in TA98 was much higher than in TA98NR and the activity of these compounds in TA100 was much higher than in TA100NR. While 1-nitropyrene exhibited similar mutagenicity in strains TA98 and TA98/1,8-DNP₆, the mutagenicity of 2-nitropyrene in TA98/1,8-DNP₆ was much lower than in TA98. Analysis of DNA from TA96 and TA104 incubated with 2-nitropyrene indicated the presence of two adducts, N-(deoxyguanosin-8-yl)-2-aminopyrene and N-deoxyadenosin-8-yl)-2-aminopyrene. The results suggest that 2-nitropyrene is metabolized by bacterial nitroreductase(s) to N-hydroxy-2-aminopyrene, and possibly by activation to a highly mutagenic O-acetoxy ester. DNA adduct formation with deoxyguanosine and deoxyadenosine correlates with the mutagenicity of 2-nitropyrene in tester strains possessing both G:C and A:T mutational targets.     

Detoxication of aliphatic epoxides by diol formation and glutathione conjugation

The Ames procedure with Salmonella typhimurium strain TA100 was used to follow the detoxication by rat liver fractions of two series of aliphatic epoxides. The epoxides employed were 3-chloro-, 3,3-dichloro- and 3,3,3-trichloropropylene oxides and also p-methoxyphenyl-, phenyl- and p-nitrophenylglycidyl ethers. In our procedure with preincubation of the epoxides with rat liver fractions prior to the Ames tests, there was more detoxication of both systems by glutathione conjugation (non-enzymatic and transferase promoted) than by the hydrolase pathways. Non-enzymatic reaction with glutathione was more pronounced for the chloro series than for the glycidyl ethers. An HPLC system was developed which was capable of quantitative measurements of the phenylglycidyl ethers together with their diol and glutathione conjugate products. A comparison of the HPLC and Ames test results indicates that the glutathione transferase reported to be present in Salmonella could be playing a role in detoxication by the Ames test. Diols were measured more readily by HPLC than by use of the Ames test in the microsomal fraction and were detected in the cytosol with the glycidyl ethers while they were not by the Ames procedure. However, all three epoxides were converted to a greater extent to their glutathione conjugates than to their diols. Thus, while literature references question the availability of the glutathione detoxication system for epoxides produced by membrane-bound enzymes, such detoxication would be of primary importance where direct-acting environmental epoxides come into contact with the cytosolic enzymes prior to possible reaction with bionucleophiles.     

The genotoxicity of enantiomeric aliphatic epoxides

The (R)- and (S)-optical isomers of 9 epoxides, benzyloxymethyloxirane, epichlorohydrin, glycidol, glycidyl 3-nitrobenzenesulfonate, glycidyl 4-nitrobenzoate, glycidyl tosylate, styrene oxide, glycidyl 1-naphthyl ether and glycidyl 4-nitrophenyl ether, have been compared for their in vivo and in vitro genotoxicity. The in vitro short-term test employed was the Ames mutagenicity assay with Salmonella strain TA100. The in vivo tests were chromosomal aberrations (CA) as well as sister-chromatid exchange (SCE) in bone-marrow cells of mice following intraperitoneal administration of these epoxides. Differences in mutagenicity between isomers were established with TA100 for all the compounds. While 13 of the isomers were genotoxic compared to a negative control by CA measurements, only in the case of glycidyl 4-nitrobenzoate could a significant difference be found between isomers by this test. However, with SCE evaluations, differences were detected between the (R)- and (S)-isomers for all the pairs of compounds with the exception of those for benzyloxymethyloxirane and glycidyl 4-nitrophenyl ether. At least in part, differences in the patterns of genotoxicity among compounds can be related to their differences in reaction pathways.     

Absence of mutagenic activity of benorylate, paracetamol and aspirin in the Salmonella/mammalian microsome test

Benorylate and its two major hydrolysis products, paracetamol and aspirin were examined for mutagenicity in the Salmonella/mammalian microsome screening test. The compounds were tested in 6 strains of Salmonella typhimurium (TA1535, TA1537, TA1538, TA100, TA97 and TA98) in the presence and absence of a rat-liver microsome activation system. Benorylate did not show evidence of mutagenic activity in the 6 strains tested with or without metabolic activation at concentrations ranging from 0.006 to 3 mg per plate. Paracetamol and aspirin likewise did not show any evidence of mutagenic activity at concentrations ranging from 0.1 to 50 mg per plate for the former and 0.01 to 50 mg per plate for the latter.     

Further mutagenicity studies on pesticides in bacterial reversion assay systems

A total of 228 pesticides (88 insecticides, 60 fungicides, 62 herbicides, 12 plant-growth regulators, 3 metabolites and 3 other compounds) was tested for mutagenicity in bacterial reversion-assay systems with 5 strains (TA100, TA98, TA1535, TA1537 and TA1538) of Salmonella typhimurium and a strain (WP2 hcr) of Escherichia coli. 50 pesticides (25 insecticides, 20 fungicides, 3 herbicides, 1 plant-growth regulator and 1 other compound) were found to be mutagenic. 5 of them required metabolic activation (S9 mix) for their activities. Among various chemical groups, organic phosphates, halogenated alkanes and dithiocarbamates showed higher ratios of mutagens. Although 22 of the pesticides tested have been reported to be carcinogenic, 7 of them, i.e., captain, DBCP, EDB, EDC, ETU, HEH and nitrofen, were detected as mutagens in the present assay. Most of the other 15 non-mutagenic carcinogens were organochlorine pesticides such as alpha-BHC, chlorobenzilate, p,p'-DDT, dieldrin and quintozene.     

Structure-activity considerations and in vitro approaches to assess the genotoxicity of 19 methane-, benzene- and toluenesulfonic acid esters

Sulfonic acid esters are considered as potentially alkylating agents that may exert genotoxic effects in bacterial and mammalian cell systems. One possible source of human exposure stems from drug synthesis when the salt-forming agents methanesulfonic acid, benzenesulfonic acid or p-toluenesulfonic acid are used together with alcoholic solvents such as methanol, ethanol and propanol. In this study computer-assisted structural considerations and in vitro approaches (Ames mutagenicity test using Salmonella typhimurium strains TA98 and TA100, and the micronucleus test using L5178Y mouse lymphoma cells) were used to assess the genotoxic properties of 19 sulfonic esters. While all esters may be principally active as genotoxicants based on the presence of the sulfonate moiety, the statistical correlative multiple computer automated structure evaluation (MCASE) system (MC4PC version 1.0) using the Ames mutagenicity A2I module (version 1.54), rank-ordered the activity of the benzenesulfonic acid esters in the Ames test negligible due to an inactivating modulator and a deactivating fragment, whereas the methane- and toluenesulfonic acid esters were predicted to be positive in this test. In the Ames test, with the exception of the p-toluenesulfonic acid ethyl and iso-butyl esters, all compounds came out positive in Salmonella strain TA100. Methanesulfonic iso-propyl, sec-butyl and benzenesulfonic acid iso-propyl ester also showed mutagenic potential in strain TA98. In general, differences between results seen in Ames tests performed with or without metabolic activation were rather small. In L5178Y mouse lymphoma cells, benzenesulfonic acid n- and iso-butyl ester and p-toluenesulfonic acid iso-butyl ester did not increase the number of cells containing micronuclei. The other esters were positive in this micronucleus test; however, methanesulfonic acid iso-butyl ester was found to be only weakly positive at excessively cytotoxic concentrations. These compounds were generally found to be more potent with regard to micronucleus induction when tested without metabolic activation (20 h treatment). In conclusion, the iso-propyl esters of the three sulfonic acids under study were found to be the strongest mutagens, either when tested in the Ames test or the micronucleus assay, whereas p-toluenesulfonic acid iso-butyl ester was the only compound shown to be devoid of a genotoxic potential in both tests.     

Isolation of a mutant of Salmonella typhimurium strain TA1535 with decreased levels of glutathione (GSH-). Primary characterization and chemical mutagenesis studies

A mutant of Salmonella typhimurium strain TA1535 with decreased glutathione (GSH) levels was isolated after treatment with UV and selection for N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG) resistance; this GSH- mutant also exhibited increased resistance to MNNG, the methyl analog of ENNG. Estimation of the cellular GSH content showed that the GSH- derivative contained about 20% of the GSH levels found in TA1535. In mutagenicity tests (hisG46 leads to His+), the GSH- strain required the presence of GSH or L-cysteine in the medium for an optimal phenotypic expression and/or growth of spontaneous and induced His+ revertants, and may, therefore, be allelic to cys mutants of Salmonella described earlier. The mutagenic activity of MNNG, ENNG and 1,2-dibromoethane (DBE), but not that of N-ethylnitrosourea (ENU), was strongly reduced in TA1535/GSH-; pretreatment of the strain with GSH restored the mutagenicity of the first 3 chemicals to levels normally found in TA1535. The results support the current view that MNNG, ENNG and DBE, but not ENU, can be activated via reaction with GSH to species of higher reactivity and mutagenicity. It is concluded that the present GSH- strain can be used to study more systematically the role of GSH in the bioactivation and -deactivation of xenobiotics to mutagenic factors.     

Mutagenicity of some commercially available nitro compounds for Salmonella typhimurium.

Benzoyl chloride and 53 commercially available aromatic heterocyclic and aliphatic nitro compounds were tested for mutagenicity in Salmonella typhimurium TA98 and TA100. 34 of 53 nitro compounds (64%) were mutagenic, 4 in TA100 only, 15 in TA98 only, and 15 in both strains. 13 of the heterocyclic derivatives of pyridine, indole, indazole, quinoline, and benzimidazole were mutagenic. 21 of 34 mutagenic nitro compounds were bactericidal. Nitromethane was the only aliphatic tested and was not mutagenic. Benzoyl chloride, a human carcinogen, was mutagenic for TA98.     

Mutagenicity of aryl propylene and butylene oxides with salmonella

10 aryl propylene oxides and 6 aryl butylene oxides were synthesized. Dose-mutagenicity relationships were studied for these compounds and for 1,2-epoxybutane, using both the preincubation and plate incorporation Ames tests with Salmonella typhimurium strains TA100 and TA1535. Structure-mutagenicity relationships were further examined by concurrent testing at single doses with the plate incorporation assay in strain TA100. In both series of compounds, mutagenicity showed very correlation to chemical reactivity, molar volume and partition values. However, all compounds were mutagenic in at least one system with the propylene oxides being more mutagenic than the corresponding butylene oxide derivatives. The naphthyl derivatives in each series were the most mutagenic.