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

Benorylate and its two major hydroyssis 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.     

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.     

Absence of mutagenic activity of acidity regulators in the Ames Salmonella/microsome test

The activities of various concentrations of 4 acidity regulators (anhydrous citric acid, phosphoric acid, malic acid and lactic acid) used in food industries in Iraq was assayed using the Salmonella/microsome mutagenicity assay. None of the samples was mutagenic in the absence or in the presence of S9 to any of the tester strains of Salmonella typhimurium.     

Absence of mutagenic activity of fodder proteins in the Ames Salmonella/microsome test

The mutagenicities of fodder proteins (Pekilo, L-lysine and Orsan) were tested towards Salmonella typhimurium in the plate-incorporation assay in the presence or absence of metabolic activation with a rat-liver S9 preparation. Filtrates and 2-, 5- and 10-fold-concentrated filtrates of saline- or ethanol-soluble fodder proteins were tested. No mutagenic activity was observed.     

Peroxyacetyl nitrate: measurement of its mutagenic activity using the Salmonella/mammalian microsome reversion assay

Exposures of Salmonella typhimurium strain TA100 with and without S9 metabolic activation to low ppm levels of pure peroxyacetyl nitrate (PAN) in the gas phase were conducted. Measurements of the gas-phase PAN exposure concentration and the concentration of its decomposition products in surrogate test media led to a measured mutagenic activity of 34 +/- 5 revertants/mumole. The data indicate that PAN is a relatively weak direct-acting mutagen with TA100.     

Adaptation of the Salmonella/mammalian microsome test to the determination of the mutagenic properties of mineral oils

Two techniques allowing the determination of the mutagenicity of lipophilic compounds such as mineral oils with the Ames test have been developed by using benzo[a]pyrene (BP) dissolved in white oil as a synthetic reference oil. The first technique involves prior extraction of polynuclear aromatic hydrocarbons (PAH) with dimethyl sulfoxide. In the second method, which proved simpler and of more general use, the compounds to be tested are directly dispersed in aqueous medium with Tween 80. The use of these techniques made possible the study of mutagenicity of various kinds of mineral oil. Mutagenic activity was found in used crankcase oils, and also in petroleum distillates but much less in solvent-refined oils. A good correlation was observed between mutagenic activity and PAH content but not BP content of oils.Because of their peculiar response to the test, petroleum distillates were studied in more detail. When added in low amounts to pure PAH compounds such as BP, they enhanced its mutagenic activity (enhancement). When added in higher amounts, on the contrary, these oils completely inhibited BP mutagenic activity (inhibition effect). Both effects correlated well with the PAH content and the mutagenic activity of the petroleum distillates tested. These results explain the abnormal dose—response curves obtained with these petroleum distillates and the negative results regarding their mutagenic activity reported in earlier studies. A likely explanation is discussed for the enhancement and inhibition effects.     

Evaluation of the mutagenic potential of endosulfan using the Salmonella/mammalian microsome assay

The mutagenic potential of endosulfan, a polychlorinated insecticide, was assessed using the highly sensitive Salmonella tester strains TA97(a), TA98, TA100 and TA102. It exhibited a toxic effect at dose levels of 50 micrograms/plate and higher. Plate incorporation studies did not show mutagenic response with any of the tester strains used. A modification of the assay using a preincubation procedure showed mutagenic activity with and without metabolic activation with TA97(a) only. Increased toxicity was observed after addition of phenobarbital-induced S9 mix.     

pH-Sensitive mutagenic activity of ozone-treated 1,2-dimethylhydrazine in the Salmonella/microsome assay

Treatment with ozone inactivates the mutagenicity of many carcinogens in aqueous solution. The colon carcinogen, 1,2-dimethylhydrazine (DMH) has been reported an exception; ozone treatment convenrts dimethylhydrazine from a non-mutagen into a mutagen. In the Salmonella/microsone assay, the mutagenicity of ozone-treated dimethylhydrazine was dependent on pH. The ozonation product was a strong mutagen in acidic but was not mutagenic in basic solution. The mutagenicity of the acidic ozonation product was inactivated by raising the pH of the solution. Unlike untreated dimethylhydrazine, its ozonation product in basic solution was not converted to a mutagen in this ozone-low pH system.     

Verapamil as a co-mutagen in the Salmonella/mammalian microsome mutagenicity test

Verapamil is a calcium-channel blocking agent, commonly used for chronic treatment of heart conditions. We have previously demonstrated that verapamil acts as a co-mutagen in a bacterial mutagenicity test for some experimental anilinoacridine antitumour drugs. Within the anilinoacridines series there are several compounds which are apparently non-mutagenic (or very weak mutagens) in the absence of verapamil, but strong mutagens in its presence. We have now tested a wider range of materials for verapamil enhancement of mutagenicity, to include some of those to which persons on verapamil therapy might be exposed through life-style or occupation. Some verapamil enhancement of mutagenicity was seen with most mutagenic compounds including anticancer drugs, antiparasitic agents, one biological stain and one hair dye. A number of tricyclic antidepressants and biological stains were tested and found to be non-mutagenic. If these results extrapolate to mammalian cells, long-term verapamil therapy could potentially increase the effects of certain environmental mutagens.     

Comparative mutagenicity of halogenated pyridines in the Salmonella typhimurium/mammalian microsome test

The Salmonella/microsome assay with strains TA97, TA98, TA100 and TA102 was used to examine the potential mutagenicity and structure-activity of 16 mono- and di-halogenated pyridines. The chemical reactivity of the halopyridines suggests that nucleophilic displacement of halogens can occur with halogens at positions 2, 4 and 6 being displaced in addition-elimination reactions. 2-Chloropyridine gave a positive result with rat-liver metabolic activation, and 2-fluoropyridine gave equivocal results under these conditions. Mutagenic responses were also obtained with 2-chloromethyl pyridine and 3-chloromethyl pyridine, in both the presence and absence of rat-liver S9. These results suggest that the halogenated pyridines, especially with halogens at the 2-position, and singly on a methyl substituent, have mutagenic activity in the Salmonella assay.     

Mutagenicity studies on coffee. The influence of different factors on the mutagenic activity in the Salmonella/mammalian microsome assay

Recently, mutagenic activity on several strains of Salmonella typhimurium has been found in many heat-processed foodstuffs. The previously reported direct-acting mutagenic activity of coffee in Salmonella typhimurium TA100 (Ames assay) was confirmed in our study. In addition to TA100, a mutagenic effect of coffee was also found by using the newly developed strain TA102. The mutagenic activity was abolished by the addition of rat-liver homogenate. 10% S9 mix completely eliminated the mutagenic activity of 30 mg of coffee per plate. The addition of reduced glutathione to active S9 further decreased the mutagenic activity and also reduced the mutagenicity together with inactivated S9. The compound or compounds responsible for this inactivation are heat-labile and seem to be located in the cytosol fraction of the S9. Part of the mutagenicity of coffee was also lost spontaneously upon incubation at temperatures between 0 degrees and 50 degrees C. The loss of activity was dependent on temperature, being more pronounced at 50 degrees C compared to 0 degrees C (at 50 degrees C approximately 50% of the mutagenic activity was lost after 6 h). As anaerobic conditions prevented this loss of mutagenicity almost totally, oxidative processes are probably responsible for the inactivation. The stability of the mutagen was not influenced by incubation at low pH values (pH 1-3), with or without the addition of pepsinogen. The mutagenic properties of methylglyoxal, which to some extent could be responsible for the mutagenic activity of coffee, were compared with those of coffee. Methylglyoxal was strongly mutagenic towards Salmonella typhimurium TA100 and TA102. Its mutagenic activity was partially inactivated by the addition of 10% S9. Glyoxalase I and II together with reduced glutathione abolished the mutagenic activity of methylglyoxal but reduced the mutagenicity of coffee by only 80%. Since these enzymes occur in mammalian cells, the mutagenic compound(s) of coffee could also be degraded in vivo. This conclusion is supported by the fact that a long-term carcinogenicity study with rats was negative. These results clearly demonstrate that the effects observed in vitro do not necessarily also occur in vivo, but that in vitro experiments may contribute to the understanding of fundamental mechanisms of chemical carcinogenesis.     

Comparison of the mutagenic activity of 5-hydroxymethyldeoxyuridine with 5-substituted 2'-deoxyuridine analogs in the Ames Salmonella/microsome test

4 antiviral drugs 5-hydroxymethyldeoxyuridine (HMUdR), 5-trifluorothymidine (F3TdR), 5-methoxymethyldeoxyuridine (MMUdR) and 5-ethyldeoxyuridine (EtUdR) have been evaluated for mutagenic activity in the Ames Salmonella/microsome test. The antimetabolites F3TdR and HMUdR were mutagenic in a dose-dependent manner in strain TA100. F3TdR also was mutagenic in strain TA1535. Rat-liver post-mitochondrial supernatant (S9) was not required for mutagenicity.     

Mutagenic activity of nine N,N-disubstituted hydrazines in the Salmonella/mammalian microsome assay.

The mutagenic activity of N,N-dimethyl-, N,N-diethyl-, N,N-dibutyl-, N,N-diisobutyl-, N,N-di(p-tolyl)-, N-ethyl-N-phenyl-, N,N-dibenzyl-, N,N-diphenyl- and N,N-diisopropylhydrazine was examined in the Salmonella/mammalian microsome assay using the strains TA1535, TA1537, TA97, TA98, TA100, TA102 and TA1530. All nine hydrazines were mutagenic in at least one tester strain, although of borderline significance for some of the compounds. The mutagenic potencies of the hydrazines varied 2-3 orders of magnitude, from very weak to moderate mutagenic activity. In general, the addition of S9 resulted in a lowering of the mutagenic activity and a lowering of the toxic properties of the hydrazines. The test results were relatively difficult to evaluate due to toxic effects of many of the test compounds on the test bacteria which may have resulted in an underestimation of the mutagenic potencies of some of the compounds. The pattern of mutagenic activity of the hydrazines in the different tester strains indicates that more than one mechanism of action may be involved in the mutagenicity.     

Mutagenicity of N-nitrosodiethanolamine in the Salmonella/microsome test

Mutagenicity of a commercially available N-nitrosodiethanolamine (NDELA) and purified NDELA was examined, using Salmonella typhimurium TA100 as a tester strain. Purified NDELA was positive in the presence of liver activation system from either rats or hamsters, but the mutagenicity was completely lost when dimethyl sulfoxide (DMSO) was used as a solvent. In contrast, the commercial NDELA which was chemically of 93.8% purity showed positive mutagenicity without metabolic activation, and the liver activation system and DMSO had no effect on the direct mutagenic activity. These results indicate that an apparent discrepancy among previous findings of several investigators with the mutagenic response of NDELA might be due to an impurity in NDELA samples and the solvent, DMSO.     

Mutagenicity of N,N'-dimethylurea and methylamine hydrochloride in the Ames Salmonella/microsome test: absence of mutagenic response.

Methyl isocyanate (MIC) in aqueous solution forms methylamine (MA) and N,N'-dimethylurea (DMU). MA in buffered system further converts into its salt form, methylamine hydrochloride (MAH). Therefore, MAH and DMU were evaluated for their mutagenic activity in the in vitro Ames Salmonella/microsome mutagenicity test. The liquid preincubation protocol was followed, using tester strains TA98, TA100 and TA104 of Salmonella typhimurium, in the presence of 0, 5, 15 and 30% Aroclor 1254-induced rat liver S9 mixture. DMU and MAH did not induce a mutagenic response in any of the tester strains, both in the presence and in the absence of S9 mixture. The results therefore confirm that MIC in its native form or as its unknown metabolites is responsible for the mutagenic activity reported earlier by us in the his tester strains TA100 and TA104 of Salmonella typhimurium (Mutation Res., 204 (1988) 123-129) and not due to its hydrolysis products, MA or DMU.     

Study of the mutagenic activity of 6 hepatotoxic pharmaceutical drugs in the Salmonella typhimurium microsome test, and the HGPRT and Na+/K+ ATPase system in cultured mammalian cells

Several pharmaceutical drugs show strong hepatotoxicity during therapeutic use. We have studied 6 of them: aminophenazone, clofibrate, nifuroxazide, oxamniquine, perhexiline maleate, tienilic acid. Their mutagenicity was assessed in the Ames test on 6 strains of Salmonella typhimurium, and in V79 Chinese hamster lung cells using a rat-hepatocyte-mediated metabolic activation system and the HGPRT and Na+/K+ ATPase assay. Nifuroxazide was positive in the Ames test in two Salmonella strains (TA100, and TA100 Fr1). In the hepatocyte-mediated mammalian V79 cell system, nifuroxazide, clofibrate and aminophenazone were negative; oxamniquine and tienilic acid were positive with and without metabolic activation in tests looking for ouabain and 6-thioguanine resistance. Perhexiline maleate was negative for the direct induction of 6-thioguanine resistance without metabolic activation, and positive after metabolisation mediated by primary rat's hepatocytes. These results suggest the need for some caution in the use of some pharmaceutical drugs because of hepatotoxicity and because 3 out of 6 drugs were shown to be slightly mutagenic in mammalian cells.     

Evaluation of mutagenic and antimutagenic activities of alpha-bisabolol in the Salmonella/microsome assay

alpha-Bisabolol (BISA) is a sesquiterpene alcohol found in the oils of chamomile (Matricaria chamomilla) and other plants. BISA has been widely used in dermatological and cosmetic formulations. This study was undertaken to investigate the mutagenicity and antimutagenicity of BISA in the Salmonella/microsome assay. Mutagenicity of BISA was evaluated with TA100, TA98, TA97a and TA1535 Salmonella typhimurium strains, without and with addition of S9 mixture. No increase in the number of his+ revertant colonies over the negative (solvent) control values was observed with any of the four tester strains. In the antimutagenicity assays, BISA was tested up to the highest nontoxic dose (i.e. 50 and 150 microg/plate, with and without S9 mix, respectively) against direct-acting (sodium azide, SA; 4-nitroquinoline-N-oxide, 4-NQNO; 2-nitrofluorene, 2-NF; and nitro-o-phenylenediamine, NPD) as well as indirect-acting (cyclophosphamide, CP; benzo[a]pyrene, B[a]P; aflatoxin B1, AFB1; 2-aminoanthracene, 2-AA; and 2-aminofluorene, 2-AF) mutagens. BISA did not alter mutagenic activity of SA and of NPD, and showed only a weak inhibitory effect on the mutagenicity induced by 4-NQNO and 2-NF. The mutagenic effects of AFB1, CP, B[a]P, 2-AA and 2-AF, on the other hand, were all markedly and dose-dependently reduced by BISA. It was also found that BISA inhibited pentoxyresorufin-o-depentylase (PROD, IC50 2.76 microM) and ethoxyresorufin-o-deethylase (EROD, 33.67 microM), which are markers for cytochromes CYP2B1 and 1A1 in rat liver microsomes. Since CYP2B1 converts AFB1 and CP into mutagenic metabolites, and CYP1A1 activates B[a]P, 2-AA and 2-AF, results suggest that BISA-induced antimutagenicity could be mediated by an inhibitory effect on the metabolic activation of these promutagens.