Diethylstilbestrol and 11 derivatives: a mutagenicity study with Salmonella typhimurium.

Diethylstilbestrol was tested for mutagenicity with his- S. typhimurium strains under 10 different matabolic situations (no exogenous metabolizing system; S9 mix from liver homogenate of rats induced with Aroclor 1254, with or without inhibition of epoxide hydratase; liver and/or kidney S9 mix from control or hamsters treated with Aroclor 1254; horse-radish peroxidase + H2O2). Under none of these conditions did diethylstilbestrol give any indication of a mutagenic effect. Furthermore, 11 metabolites and other derivatives of diethylstilbestrol, 2 of them potent inducers of sister-chromatid exchange in cultured fibroblasts, were not mutagenic with any of the 4 tester strains (S. typhimurium TA100, TA98, TA1537, TA1535) in the presence or absence of S9 mix from liver homogenate of rats induced with Aroclor 1254. Thus, one of the few known human carcinogens is very resistant to detection by the mammalian enzyme-mediated Salmonella typhimurium mutagenicity test (Ames test). This is especially remarkable since the metabolizing systems used included: (1) some of very high metabolic activity (S9 mix from liver homogenate of rats and hamsters induced with Aroclor 1254); (2) metabolizing systems from organs susceptible to the carcinogenic activity of diethylstilbestrol (hamster kidney); as well as (3) a mixture of (1) and (2) in case both activities are required for the carcinogenic effect in the whole animal.     

Absence of mutagenic activity of ticlopidine in the Ames Salmonella/microsome test

Ticlopidine hydrochloride, 5-(o-chlorobenzyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridine hydrochloride, a platelet aggregation inhibitor, was tested for mutagenic activity in the Ames Salmonella/mammalian microsome test. Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538 were employed. Two of these strains (TA1535 and TA100) are sensitive to base-pair substitution mutagens, and the remaining 3 are sensitive to frame-shift mutagens. There was no evidence that ticlopidine hydrochloride had any mutagenic activity either in the presence or absence of a liver microsomal supplement.     

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.     

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.     

Determining the mutagenic activity of a tar, its vapors and aerosols

The Ames test was performed on Salmonella typhimurium, strain TA98, TA100, TA1535, TA1537, TA1538, to evaluate the mutagenic potential of a tar as well as its vapors and aerosols emitted at 250, 350 and 550 degrees C. Two chemical procedures were used: extractions of aromatics for DMSO; elimination of acids, alcohols and phenols. Weak mutagenic activity was demonstrated at each temperature. Then, using only Salmonella typhimurium strains TA98 and TA100, a study was made on the effects of the mutagenic compounds, benzo[a]pyrene, 2-aminoanthracene, nitrofluorene, methyl methanesulfonate and on the vapors and aerosols emitted at 350 degrees C by road-coating tar. For promutagenic compounds, an enhancing effect was observed before an inhibition effect. For direct mutagenic compounds, only the inhibition effect appeared. The mutagenic and/or carcinogenic activity was usually tested on a pure isolated chemical compound.     

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.     

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.     

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.     

Evaluation of the mutagenic potential of mycotoxins using Salmonella typhimurium and Saccharomyces cerevisiae.

The mutagenic effects of fiteen mycotoxins on Salmonella typhimurium strains TA1535, TA1537 and TA1538 and Saccharomyces cerevisiae strain D-3 were tested. Only aflatoxin B1 and sterigmatocystin were mutagenic. Both were active against S. typhimurium strain TA1538 and S. cerevisiae strain D-3; however, both required activation by the hepatic S-9 enzyme preparation. A positive correlation between the other mycotoxins reported to be carcinogenic and the two in vitro test systems employed was not demonstrated in our hands.     

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.     

Genotoxic activity of m-nitrobenzaldehyde

m-Nitrobenzaldehyde (MNB) was evaluated for mutagenic activity using the Ames microbial mutagenicity test and for its ability to induce DNA single-strand breaks in rat hepatocytes as measured by alkaline elution. MNB was tested in S. typhimurium strains TA1535, TA1537, TA1538, TA98, and TA100, both with and without pretreatment with liver microsomes (S9) isolated from rats pretreated with Aroclor 1254. MNB produced 2-fold or greater increases in revertants in TA1538, both with and without S9, and in TA100 with S9 only. A 2-fold increase in revertants was seen in TA98, but only at the highest dose tested which did not produce inhibition of background growth. MNB caused a greater than 3-fold increase in elution slope, with DNA alkaline elution assay, but only at highly cytotoxic doses and, therefore, is not considered genotoxic in this system. It is concluded that MNB possesses weak genotoxic activity.     

Evaluation of genotoxity and mutagenicity of DL-p-chlorophenylalanine, its methyl ester and some N-acyl derivatives

DL-p-chlorophenylalanine (PCPA) and its derivatives were evaluated for genotoxic effects using Escherichia coli and Bacillus subtilis strains lacking various DNA-repair mechanisms in spottest and in suspension test. The mutagenic activity of studied compounds was determined by the Ames test. Reverse mutation test was performed with Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 without S9 mix. 0.02 M nitrosomethylurea (NMU) standard mutagen was used as a positive control. The results showed that the parent nonessential amino acid PCPA had no detectable genotoxic and mutagenic activities in bacteria. The methyl ester of this amino acid and its N-phenylacetyl derivative possessed weak genotoxicity. Meanwhile N-sec-butyloxycarbonyl, N-benzyloxycarbonyl, N-(p-nitrophenylacetyl) and N-(p-nitrophenoxyacetyl) derivatives of DL-p-chlorophenylalanine exhibited appreciable genotoxicity. Among the seven tested compounds only N-benzyloxycarbonyl and N-(p-nitrophenoxyacetyl) derivatives of DL-p-chlorophenylalanine have been found to be mutagenic. Only parent PCPA possessed antimutagenic properties in respect of nitrosomethylurea. The structural modification, which strongly affects genotoxicity and mutagenicity perhaps may be due to steric hydrance of the substituents, causing interference with enzyme and DNA interactions.     

Mutagenicity of anthraquinone and hydroxylated anthraquinones in the Ames/Salmonella microsome system.

The mutagenicity of anthracene, anthraquinone, and four structurally similar compounds of each was evaluated in the Ames/Salmonella microsome assay. Anthraquinone was shown to be mutagenic for strains TA1537, TA1538, and TA98 in the absence of rat liver homogenate. The four anthraquinone derivatives tested were mutagenic for TA1537 exclusively. None of the anthracenes exhibited mutagenic activity.     

The capacity of some nitro- and amino-heterocyclic sulfur compounds to induce base-pair substitutions

The capacity of 27 heterocyclic sulfur compounds to induce base-pair substitutions was investigated with Klebsiella pneumoniae ur- pro- and Salmonella typhimurium TA100 as test organisms. Among the compounds tested, all sulfur compounds with nitro groups and some thiazoles with an amino group were mutagenic. Among the nitrothiazoles, the most potent mutagen was niridazole, followed by 2-acetamido-5-nitrothiazole, 2-bromo-5-nitrothiazole, N-(5-nitrothiazol-2-yl)benzamide, and 2-amino-5-nitrothiazole. Of the nitrothiophenes, 2-nitrothiophene was more mutagenic than 3-nitrothiophene and 2,4-dinitrothiophene. 4-Nitroisothiazole was also mutagenic. Of the aminothiazoles, 2-amino-5-bromothiazole and 2-amino-5-chlorothiazole were mutagenic to both test organisms. With 2-amino-5-(p-nitrophenylsulfonyl)thiazole, a mutagenic action was only found with Salmonella typhimurium TA100, whereas 2-aminothiazole and 2-amino-4-methylthiazole were only mutagenic with Klebsiella pneumoniae. With the other 13 compounds, no mutagenic activity was observed. Of the coccidiostatics, 2-acetamido-5-nitrothiazole was also mutagenic on Escherichia coli K12 and Saccharomyces cerevisiae D4 but non-mutagenic on Salmonella typhimurium TA1530, TA1535, TA1537 and TA98, while 2-amino-5-nitrothiazole was mutagenic on Escherichia coli K12, Salmonella typhimurium TA1530, TA1535 and TA98, and non-mutagenic on strain TA1537 and on Saccharomyces cerevisiae D4.     

Bacterial mutagenicity of some methyl 2-cyanoacrylates and methyl 2-cyano-3-phenylacrylates

The mutagenic potential of three alkyl 2-cyanoacrylate adhesives, three commercial alkyl 2-cyanoacrylate adhesives and three methyl 2-cyano-3-phenylacrylates, was assessed using the Salmonella/microsome mutagenicity assay. Compounds were tested with and without Aroclor 1254-induced rat-liver homogenate (S9 mix). The methyl 2-cyanoacrylate adhesives were mutagenic in the standard plate test with S. typhimurium strain TA100 with and without S9 activation. Methyl 2-cyano-3-(2-bromophenyl)acrylate revealed a direct mutagenic action to S. typhimurium strain TA1535. The compounds most toxic towards the bacterium S. typhimurium, were the methyl 2-cyanoacrylate adhesives (greater than 500 micrograms/plate). All alkyl 2-cyanoacrylate adhesives were tested in a modified spot test for volatile compounds with tester strain TA100. Mutagenic and toxic effects were observed with the three methyl 2-cyanoacrylate adhesives. It can be concluded from the results that the bacterial toxicity and mutagenicity of methyl 2-cyanoacrylate adhesives may be due to the methyl 2-cyanoacrylate monomer.     

In vitro screening of carcinogens using DNA of the His- mutant of Salmonella typhimurium

Carcinogens: 7,12-dimethylbenz(a)anthracene (DMBA), 2-acetylaminofluorene (AAF), 3-methylcholanthrene (MC), daunorubicin, thio-TEPA, cyclophosphamide, 1-(2-chloroethyl)2-cyclohexyl-1-nitrosourea (CCNU) and mitomycin C, which mutate Salmonella typhimurium tester strains in the Salmonella/microsome mutagenicity test, strongly stimulate the in vitro synthesis of DNA isolated from His- mutants (TA-1538, TA-1537, TA-1535), and induce in vitro His- DNA strand separation. Four other carcinogens, ethionine, actinomycin D, bleomycin and reserpine, which were not known as mutagens in the Ames test, also strongly stimulate His- DNA synthesis and His- DNA strand separation in local areas. Those substances which are neither carcinogenic nor mutagenic: d-lactose, fluorene, cholesterol, and saccharin, do not stimulate either His- DNA (or His+ DNA) synthesis or DNA in vitro strand separation. Steroid hormones, which are carcinogenic only for steroid target tissues, do not react with bacterials DNA.     

The bacterial mutagenicity of three naturally occurring indoles after reaction with nitrous acid

Three naturally occurring indoles were evaluated for potential nitrosatability using the Nitrosation Assay Procedure (NAP test) as recommended by the World Health Organisation. All three indoles i.e. tryptophan, tryptamine and 5-hydroxy-tryptamine were nitrosated to products which were directly mutagenic for S. typhimurium TA1537. In addition, the products of nitrosation of tryptamine and 5-hydroxytryptamine were also mutagenic for strains TA1538, TA98 and TA1535 without the need for metabolic activation. The sensitivities of the frameshift-detecting strains TA1537, TA1538 and TA98 were of particular interest, since nitroso compounds are characteristically base-substitution mutagens. The mutagenic effects of the products formed after nitrosation of each indole at pH 3.6, were eliminated in the presence of S9 mix. This was not the case when the nitrosation assay was carried out at pH 2.6. At this pH the mutagenicity of the nitrosated products varied in the presence of S9 mix and depended upon the nature of the indole undergoing nitrosation, and the bacterial test strain utilised for the mutagenicity assay. This indicated that more than one mutagenic product was responsible for the observed effects. As well as pH, a number of other factors influenced the formation of mutagenic nitroso products. Most notably, the concentrations of precursor compounds (sodium nitrite, and indole) present in the NAP test were of critical importance. As the sodium nitrite concentration was reduced from that recommended by the W.H.O. (40 mM), so the mutagenicity decreased. For all three compounds significant mutagenic effects were lost at sodium nitrite concentrations below 15 mM. In conclusion the data presented in this paper clearly demonstrates that individuals are chronically exposed to naturally occurring substances which readily nitrosate in excess nitrous acid and yield bacterial mutagens.     

Bioindication of mutagenic and carcinogenic pollutants in waters of the O?awa River

Samples of raw waters from the O?awa River, chlorinated raw water, raw water filtered through activated charcoal and treated and chlorinated water before and after ozonization were examined with the use of the Ames test for potential carcinogenic activity. Positive results were obtained for raw water with Salmonella typhimurium strains TA 98 and TA 1535 and for chlorinated raw water with strain TA 1537.     

Isolation and characterization of an isogenic set of Salmonella typhimurium strains analogous to the "Ames" tester strains

The standard Ames tester strains of Salmonella typhimurium contain a number of genetic differences at loci other than his. The fact that these strains contain independently isolated uvrB-bio-gal deletions and rfa mutations implies that these are likely to vary from strain to strain. Since the strains were isolated from different parental stocks of S. typhimurium LT-2, they differ in their ability to metabolize arabinose. Other, unknown differences may exist because the isolation of some of the strains involved ultraviolet and chemical mutagenesis. We have isolated a set of isogenic S. typhimurium strains that contain the relevant genetic markers of the standard Ames tester strains. These strains all contain the same uvrB-bio-gal deletion and the same rfa mutation; they differ only in the nature of their his mutations and in the presence or absence of the plasmid pKM101. We have assessed the responsiveness of these strains to a number of mutagens and conclude that their mutagenic specificities are the same as those of the corresponding Ames strains: TA98, TA100, TA1535, TA1537 and TA1538. Therefore, the specificity of the standard Ames strains with respect to these mutagens is a result solely of the differences in the nature of their his mutations and the effects of pKM101.     

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.