The mutagenic activity of 4-chloromethylbiphenyl (4CMB) and Benzyl Chloride (BC) in the bacterial/microsome assay

The mutagenic activity of 4CMB was investigated in agar layer cultures of Salmonella typhimurium TA1535, TA1537, TA1538, TA98 and TA100, and Escherichia coli WP2 and WP2 uvrA. The mutagenic activity of BC was investigated in the Salmonella strains only. Assays were performed both in the absence and in the presence of S9 microsomal fraction obtained from a liver homogenate from rats pretreated with Aroclor 1254.     

Fluctuation test data on 4-chloromethylbiphenyl (4CMB), 4-hydroxymethylbiphenyl (4HMB) and Benzyl Chloride (BC) using Salmonella typhimurium TA98 and TA100

Bacterial fluctuation tests (Green et al., 1976) were performed both with and without metabolic activation using the ‘Ames’ Salmonella typhimurium strains TA98 and TA100 (Ames et al., 1975) to assay the mutagenic potential of 4CMB, 4HMB and BC.4CMB and 4HMB were tested on the same occasion. However, 4CMB was only compared to BC in one assay. The results also show an independent test of BC.     

UKEMS trial: Bacterial mutation tests of 4-chloromethylbiphenyl, 4-hydroxymethylbiphenyl,and benzyl chloride,using E. coli WP2uvrA(pKM101) and S. typhimurium TA98 and TA100

4CMB, 4HMB and BC were assayed in plate tests, using E. coli WP2uvrA(pKM101), and S. typhimurium TA98 and TA100, in the presence or absence of microsomal activation. 4CMB was also assayed in fluctuation tests using E. coli WP2uvrA(pKM101). 4HMB was uniformly negative, and 4CMB was mutagenic to all 3 strains. BC was negative in TA98 and positive in TA100 and WP2uvrA(pKM101). The presence or absence of S9 made no substantial difference to the mutagenicity of 4CMB or BC.     

Inverse correlation between combined mutagenicity in Salmonella typhimurium and strength of coordinate bond in mixtures of cobalt(II) chloride and 4-substituted pyridines

Cobalt(II) chloride (CoCl₂), non-mutagenic by itself, has been tested for mutagenic activity in the presence of 4-substituted pyridines in the test strains of Salmonella typhimurium. CoCl₂ was found to be mutagenic in strains TA1537 and TA2637, when combined pyridine, with methyl isonicotinate, 4-methylpyridine, 4-ethylpyridine, 4-chloropyridine or 4-bromopyridine. Mixtures of CoCl₂ and isonicotinic acid, 4-cyanopyridine, 4-aminopyridine, or 4-dimethylaminopyridine exhibited no mutagenicity. Judging from the spectral observations, such combined mutagenicity may be due to the formation of moderate to weak complexes between these compounds and the Co(II) cation.     

UKEMS trial compounds: In vitro bacterial mutagenicity

4CMB, 4HMB and BC were examined in the Ames test using Salmonella typhimurium TA1535, TA1537, TA1538, TA98 and TA100. 4CMB was mutagenic for all of the indicator strains, 4HMB was inactive and BC was weakly mutagenic for TA100 only.     

Mutagenicity of glyceryl trinitrate (nitroglycerin) in Salmonella typhimurium

The recent finding that the clinical nitrovasodilator, glyceryl trinitrate (GTN), is mutagenic in Salmonella typhimurium strain TA1535 has been examined in closer detail, with emphasis on its mechanism of action. GTN increased the number of His⁺ revertants to a maximum of 4 times over background at a GTN dose of 5 μmol/plate. Hamster liver S9 depressed the toxicity of high GTN doses and increased the maximum number of revertants to 5 times over background at 10 μmol/plate. GTN did not cause significant reversion in any of the six other S. typhimurium strains tested (TA1975, TA102, TA1538, TA100, TA100NR, YG1026), although signs of toxicity were observed. Therefore, the mutagenicity of GTN was manifest only in the repair-deficient (uvrB and lacking in pKM101) strain which is responsive to single base changes. Oligonucleotide probe hybridization of TA1535 revertants showed that virtually all of the GTN-induced mutants contained C → T transitions in either the first or second base of the hisG46 (CCC) target codon, with a preference for the latter. A similar mutational spectrum was seen previously with a complex of spermine and nitric oxide (NO) which releases nitric oxide. This suggests that NO, which can be derived from GTN via metabolic reduction, may be responsible for GTN's mutagenic action. The known NO scavenger oxymyoglobin did not substantially alter the dose response of GTN, indicating that extracellular NO was not mediating reversion. The data are consistent with the hypothesis that intracellular nitric oxide is responsible for the observed mutations.     

The effects of 4'-alkyl substituents on the mutagenic activity of 4-amino- and 4-nitrostilbenes in Salmonella typhimurium

Six derivatives of trans-4-aminostilbene bearing different alkyl groups in the 4'-position and six of the corresponding nitro compounds were synthesized and tested for their mutagenic potency in Salmonella typhimurium strains TA98 and TA100. Regarding the test series in presence of S9-mix, maximum activity was observed for those trans-4-aminostilbenes and trans-4-nitrostilbenes bearing small alkyl substituents like methyl and ethyl. More bulky substituents reduced the mutagenic potential in the order iso-propylethyl>iso-propyl>sec-butyl>tert-butyl). These trends have been compared with quantitative structure activity relationship (QSAR) model predictions, leading to the conclusion that steric demand is an important factor for mutagenicity of substituted aminostilbenes and nitrostilbenes. The unexpected result for the tert-butyl nitrostilbene tested with metabolic activation may be attributed to a different metabolic pathway.     

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.     

Mutagenic activity of carcinogenic and noncarcinogenic nitrofurans and of urine of rats fed these compounds

The nitrofurans, 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2), N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT), nitrofurantoin, 5-nitro-2-furoic acid, 5-nitro-2-furamidoxime, 5-nitrofurfurylidene diacetate and the urine of rats fed these compounds, were assayed for mutagenic activity in Salmonella typhimurium strains TA100 and TA100FR1. All the nitrofurans were mutagenic in the order: AF-2 and FANFT > nitrofurantoin > 5-nitro-2-furamidoxime > 5-nitrofurfurylidene diacetate > 5-nitro-2-furoic acid. Strain TA100 was more sensitive than TA100FR1 to the mutagenic influence of these nitrofurans. Only the urine of rats fed AF-2, FANFT and nitrofurantoin had mutagenic activity. Again, TA100 was more sensitive than TA100FR1. The mutagenicity of the urine was not increased by treatment with β-glucuronidase. AF-2, 2-amino-4-(5-nitro-2-furyl)thiazole (deformylated product of FANFT) and nitrofurantoin were excreted in the urine of rats fed these compounds; whereas the other nitrofurans were not excreted.     

The hair-dye reagent 2-(2',4'-diaminophenoxy)ethanol is mutagenic to Salmonella typhimurium

A new hair-dye ingredient, 2-(2',4'-diaminophenoxy)ethanol (2,4-DAPE), was described as being devoid of any genotoxic activity on the basis of a multi-laboratory study. Since 2,4-DAPE is a close analogue of 2,4-diaminoanisole (2,4-DAA), which is mutagenic and carcinogenic, we tested this claim by assaying 2,4-DAPE for bacterial mutagenicity. Two samples of 2,4-DAPE X 2HCl were synthesized by reduction of the corresponding dinitrophenoxyethanol and identity and purity were established by elemental analysis, NMR spectrometry, mass-spectrometry, UV-spectrophotometry, TLC and HPLC. Fresh aqueous solutions of 2,4-DAPE X 2HCl were assayed in several separate plate tests using S. typhimurium TA1538, TA97, TA98 and TA100, and E. coli WP2uvrA (pKM101), 3 plates per dose and 0%, 4%, 10% and 30% Aroclor 1254-induced rat-liver S9 in S9 mixes. We obtained negative results in TA100 and E. coli. Reproducible, statistically significant dose-related increases in revertants (up to 14 times the background) were obtained in frame-shift mutants of S. typhimurium in the dose range 10-80 micrograms per plate. Mutagenicity was S9-dependent, significant increases in revertants being obtained only with 50 microliter per plate or more of S9. 2,4-DAPE induced significant mutagenic effects at doses of less than 1 micrograms per ml in TA1538 and TA98 in fluctuation tests using 2% S9 in the S9 mix. In plate tests, 2,4-DAPE was less mutagenic (by a factor of about 8) than 2,4-DAA, which gave the highest mutant yields with 20 microliter S9 per plate (4% S9 in the S9 mix). 2,4-DAPE obtained commercially was about 8 times more mutagenic than our sample of 2,4-DAPE. After purification, the commercial product, now chromatographically identical with our own sample, gave plate-test results close to those obtained for our samples of 2,4-DAPE. A review of the published reports (in which 2,4-DAPE was claimed to be inactive in a variety of short-term tests) revealed: (a) the use of protocols for bacterial mutagenicity testing which, in the light of our own results, were probably too limited in scope, especially in the choice of conditions for metabolic activation; (b) insufficient information on the identification and purity of the samples of 2,4-DAPE tested in the published collaborative study.     

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.     

Mutagenicity of vinyl chloride,chloroethyleneoxide, chloroacetaldehyde and chloroethanol

Exposure of S. typhimurium strains TA 1530, TA 1535 and G-46 to vinyl chloride increased the number of his⁺ rev./plate 16, 12 or 5 times over the spontaneous mutation rate. The mutagenic response for TA 1530 strain was enhanced 7, 4 or 5-fold when fortified S-9 liver fractions from humans, rats or mice were added. In TA 1530 strain, chloroacetic acid showed only toxic effects, while chloroacetaldehyde, chloroethanol and chloroethyleneoxide caused a mutagenic response. The latter compound was shown to be a strong alkylating agent.     

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.     

N(4)-amino-and N(4)-hydroxycytosines as base analogue mutagens

N(4)-Aminocytosine [N(4)NH₂C] and N(4)-amino-2′-deoxycytidine [N(4)NH₂dC] are highly mutagenic for Escherichia coli and phage φ 80 but not for T₄. There is some evidence that they are incorporated into the φ 80 DNA but $\text{[}{}^{14}\text{C}\text{]-N(4)}\text{NH}{}_2\text{C}$ could not be detected in the bacterial DNA.N(4)-Hydroxy-5,6-dihydro-6-hydroxylaminodeoxycytidine (di-NHOH-dC) is mutagenic for φ 80 and E. coli, but N(4)-hydroxydeoxycytidine [N(4)OH-dC] only has a strong inactivating effect.     

Metabolic and mutagenicity studies on DDT and 15 derivatives. Detection of 1,1-bis(p-chlorophenyl)-2,2-dichloroethane and 1,1-bis(p-chlorophenyl)-2,2,2-trichloroethyl acetate (kelthane acetate) as mutagens in Salmonella typhimurium and of 1,1-bis(p-chlorophenyl) ethylene oxide, a likely metabolite, as an alkylating agent.

Using a novel in vitro technique, whereby microsomal enzymes were embedded in an agar layer to prolong their viability, 1,1-bis(p-chlorophenyl) ethylene(DDNU), a mammalian metabolite of 1,1-bis(p-chlorophenyl)-2,2,2-trichloroethane (DDT), was converted by microsomal mono-oxygenases of mouse liver into 1,1-bis(p-chlorophenyl)-1,2-ethanediol (DDNU-diol). The putative epoxide intermediate, 1,1-bis(p-chlorophenyl)ethylene oxide (DDNU-oxide), a new compound, was synthesized; it showed weak alkylating activity with 4-(4-nitrobenzyl)pyridine but was not mutagenic in Salmonella typhimurium strains TA100 and TA98. DDT and 13 of its metabolites or putative synthetic derivatives, including 1,1-bis(p-chlorophenyl)-2,2-dichloroethylene (DDE), 1 1,1-bis(p-chlorophenyl)-2-chloroethylene (DDMU), 1,1-bis(p-chlorophenyl)-2-chloroethane (DDMS)-DDNU, 2,2-bis(p-chlorophenyl)ethanol (DDOH), bis(p-chlorophenyl)acetic acid (DDA) and 1,1-bis(p-chlorophenyl)-2,2,2-trichloroethanol (Kethane), caused no mutagenic effects in S. typhimurium strains TA100 or TA98, either in the presence or absence of a mouse-liver microsomal fraction. 1,1-Bis(p-chlorophenyl)-2,2,2-trichloroethyl acetate (Kelthane acetate) was a direct-acting mutagen in strain TA100, whereas 1,1-bis(p-chlorophenyl)-2,2-dichloroethane (DDD) was mutagenic in TA98, only in the presence of a mouse-liver microsomal system. The results are discussed in relation to possible pathways whereby DDT is activated to mutagenic and/or carcinogenic metabolites.     

Mutagenicity of benzo[b]phenanthro[2,3-d]thiophene (BPT) and its metabolites in TA100 and base-specific tester strains (TA7001-TA7006) of Salmonella typhimurium: evidence of multiple pathways for the bioactivation of BPT

Benzo[b]phenanthro[2,3-d]thiophene (BPT), and a number of its metabolites, including BPT-3,4-diol, BPT sulfoxide, BPT sulfone, and 3-hydroxyBPT were assessed for their mutagenic activity in Salmonella typhimurium strain TA100, and S. typhimurium base-specific strains TA7001, TA7002, TA7003, TA7004, TA7005, and TA7006. Among the compounds tested in strain TA100, BPT, BPT sulfone, and 3-hydroxyBPT did not show any significant mutagenic response in the presence of S9. In contrast BPT sulfoxide and BPT-3,4-diol (a precursor to the bay-region diol epoxide of BPT) showed significant mutagenic activity in the presence of S9. Surprisingly, BPT sulfoxide was nearly 3.3-fold more mutagenic than BPT-3,4-diol in the presence of S9. BPT sulfoxide also displayed intrinsic mutagenic activity, which was nearly 1.5-fold less than that displayed by BPT-3,4-diol in the presence of S9. In base specific tester strains, BPT sulfoxide was the most active metabolite in strains TA7002, TA7004, and TA7005 with S9 activation. In these strains, BPT-3,4-diol was 2- to 7-fold less mutagenic than BPT sulfoxide in the presence of S9. Only in strain TA7006, BPT-3,4-diol was four-fold more mutagenic than BPT sulfoxide. The fact that BPT sulfoxide is significantly more mutagenic than BPT-3,4-diol in S. typhimurium strain TA100 suggests that the formation of sulfoxide may be the principal pathway for the metabolic activation of BPT to mutagenic products. Based on the results from Tester Strain TA7005, it indicate that BPT and its most mutagenic metabolite BPT sulfoxide induce predominantly CG --> AT transversion, which is observed as the most frequent base substitution mutation of p53 tumor-suppressor gene in human lung cancer.     

Mutagenicity of polycyclic aromatic compounds (PAC) identified in source emissions and ambient air

Several polycyclic aromatic compounds (PAC) including nitrated and oxygenated derivatives of polycyclic aromatic hydrocarbons (PAH) were tested for mutagenic activity in the Salmonella/microsome assay. Among the compounds tested the isomer mix of nitro-1-hydroxypyrenes showed the highest direct mutagenic response in both the Salmonella strain TA98 and TA100 (1251 revertants/micrograms and 463 revertants/micrograms, respectively). The direct-acting mutagenicity of the nitro-1-hydroxypyrene isomer mix was dependent upon reduction of the nitro function as evidenced by the decrease in activity observed with the nitroreductase-deficient and arylhydroxylamine esterifying-deficient tester strains. The oxygenated derivatives of PAH containing aldehyde or keto groups showed weak or no mutagenic responses. In most cases addition of S9 was essential for any mutagenic activity and the strain TA100 was more sensitive than the strain TA98. Within this group, 7H-dibenzo[c,g]fluoren-7-one showed the highest mutagenic effect; 7 and 22 revertants/micrograms using the strains TA98 and TA100, respectively.     

Mutagenesis by photoaffinity labeling using selected azidofluorenes

Several 2-azidofluorenes have been synthesized for use as photoaffinity labels inside bacteria. In the dark they were not mutagenic for any Salmonella typhimurium tested. When photolyzed inside the bacteria, all were mutagenic for strain TA1538 to varying degrees, and were considerably less mutagenic in the corresponding repair positive TA1978. None were mutagenic for strain TA1535 or TA1537, although most compounds were toxic for those strains when photolyzed.     

Relationships between structures and mutagenic potencies of 16 heterocyclic nitrogen mustards (ICR compounds) in Salmonella typhimurium

16 heterocyclic nitrogen mustards (ICR compounds), which were synthesized for use as possible antitumor agents by Creech and coworkers, were tested for mutagenicity in Salmonella typhimurium strains TA1535, TA1536, TA1537, TA1538, TA98 and TA100. The compounds were incorporated into the top agar at 5 doses: 0.5, 1, 2.5, 5 and 10 micrograms/plate. All of the compounds were negative in TA1535 except ICR 449, which was positive in all 6 strains. The other 15 compounds were positive in the remaining strains with the following exceptions: ICR 371 and 355 were negative in TA100; ICR 445 was negative in TA98 and TA100; and ICR 360 was negative in TA1537, TA1538, TA98 and TA100. Good qualitative agreement was observed between the mutagenic and antitumor activities of the 16 compounds, and between the mutagenic and carcinogenic activities of the 5 compounds that have been tested for carcinogenicity by Peck and coworkers. However, no significant correlation was found between mutagenic potency in Salmonella and antitumor potency in mice for the 16 compounds. Also, for the 5 compounds that have been tested for carcinogenicity, no significant correlation was found between their mutagenic potency in Salmonella and their carcinogenic potency in mice. In Salmonella, the secondary (2 degrees) amines generally were more mutagenic than their tertiary (3 degrees) amine homologs, although the opposite result has been reported in certain eukaryotes. Relationships between structures and potencies for the different nuclei of the 16 ICR compounds are discussed, as are similarities and differences in strain sensitivities. We conclude that the Salmonella his reversion test is not a good predictor of the antitumor and carcinogenic potencies of these ICR compounds.     

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.