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     

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.     

Antimutagenic Effects of Autoxidized Linoleic and Oleic Acids on UV-Induced Mutagenesis in Escherichia coli

The antimutagenic effects of autoxidized linoleic and oleic acids on mutagenesis by UV irradiation were investigated in Escherichia coli B/r WP2 and WP2s uvrA. When added to an agar medium, these autoxidized acids greatly reduced the number of Trp⁺ revertants without significant effects on survival in WP2, but no such effect was observed with WP2s uvrA. The presence of autoxidized linoleic acid decreased the survival of WP2s uvrA greatly and CM571 recA somewhat. It thus appears that the autoxidized unsaturated fatty acid has antimutagenic effects on the wild type strain and lethal effects on the genetic repair-deficient strains.     

Non-enzymic activation of polycyclic aromatic hydrocarbons as mutagens.

Samples of 22 polycyclic aromatic hydrocarbons and related derivatives were subjected to ⁶⁰Co gamma radiation in air, and the irradiated samples were tested for mutagenicity with the Salmonella typhimurium strains TA 98, TA 1535, TA 1537, and TA 1538. Testing was conducted with the bacterial strains alone, thus not fortified with liver-microsomal enzymes or other metabolizing systems. Marked mutagen responses were obtained for several irradiated samples with the TA 98, TA 1537, and TA 1538 strains but not with the TA 1535 strain. Irradiated samples of benzo[a]anthracene, benzanthrone, benozo[g,h,i]perylene, benzo[a]pyrene, chrysene, fluorene, 9-methylanthracene, 1-methylphenanthrene, 2-methylphenanthrene, and pyrene gave positive mutagenic tests and dose-responses, whereas unirradiated control samples of these were inactive. Acenaphthene, phenanthrene, and phenanthrenequinone exhibited toxicity which interfered with interpretation of mutagenicity testing. Samples of 2-methylanthracene and tetracene were mutagenic with or without irradiation. Alizarin, anthracene, anthraquinone, anthrone, dobenzo[a,h]anthracene, picene, and triphenylene negative results. Samples of benzo[a]pyrene adsorbed on silica gel irradiated in air by ⁶⁰Co gamma radiation or by 254 nm ultraviolet light and samples adsorbed on filter paper irradiated by visible light yielded preparations mutagenic towards the TA 98, TA 1537, and TA 1538 strains. These results suggest that parent polycyclic aromatic hydrocarbons not themselves mutagenic towards S. typhimurium may be oxidized in air by radiation-induced processes to products whose mutagenicity resembles that of liver-microsomal metabolites of the parent polycyclic aromatic hydrocarbon.     

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.     

Mutagenicity of styrene and styrene oxide

Incubation of S. typhimurium strain TA 1535 with styrene increased the number of his⁺ revertants/plate in presence of a fortified S9 rat-liver fraction. Styrene was also highly cytotoxic for Salmonella cells. Styrene oxide, the presumed first metabolite, had a mutagenic effect towards strains TA 1535 and TA 100 both with and without metabolic activation. Styrene is probably mutagenic because it is metabolized to styrene oxide.     

Mutagenicity testing of benomyl, methyl-2-benzimidazole carbamate, streptozotocin and N-methyl-N'-nitro-N-nitrosoguanidine in Salmonella typhimurium in vitro and in rodent host-mediated assays.

The fungicide benomyl and its commercial preparations Fundazol 50WP and Benlate 50WP and the benomyl metabolite methyl-2-benzimidazole carbamate and its commercial preparation MBC 50WP were tested for mutagenicity in in vitro spot tests, in microsomal plate assay, in liquid-culture treatments, or in rodent host-mediated assay. The base-pair substitution Salmonella typhimurium mutant hisG46 and the hisG46-bearing uvrB excision-repair-deficient mutants TA100, TA1530, TA1535 or TA1950 were used as test organisms. Complete genotypic information of these mutants is given in Ames et al. [2]. Captain 50WP, streptozotocin (SZN), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 2-aminopurine and N-acetylaminofluorene were used as positive control compounds. In nonoverlay spot tests Benlate 50WP was not mutagenic over a dose range of 50-5000 microgram/spot in hisG46 and TA1535. In overlay spot tests 50 or 100 microgram/spot Benomyl, MBC, Fundazol 50WP, Benlate 50WP and MBC 50WP were tested in hisG46, TA1530 or TA1950. Only a non-commercial MBC sample at 100 microgram/spot showed weak mutagenic activity in hisG46. In microsomal activation plate assay MBC, benomyl, Fundazol 50WP and Benlate 50WP were tested in TA100 over a dose range of 50-2000 microgram/plate. None of the compounds showed mutagenicity. In a 20-h liquid-culture treatment 10, 100, 1000 and 10 000 microgram/ml Fundazol 50WP were not mutagenic in TA 30. In 1-h liquid-culture treatments benomyl, Benlate 50WP or Fundazol 50WP failed to induce mutations in hisG46, TA100 or TA1950 over a dose range of 0.25-1000 microgram/ml. Appropriate positive controls were mutagenic in each experiment. The consistently negative results in this study with commercial MBC and benomyl preparations are contrary to positive results reported earlier with similar methods and similar commercial preparations. Possible reasons to explain the different results are presented. The alkylating agents SZN and MNNG induced fewer mutations in TA1530 and TA1950 uvrB excision-repair-deficient strains than in the hisG46 excision-proficient strain, indicating that with these mutagens excision-repair is also a mutation-prone process. In rodent host-mediated assays with Fundazol 50WP in mice 3 consecutive subcutaneous hourly doses of 500 mg/kg in hisG46 and TA1950 and in rats or mice an oral dose of 4000 mg/kg in TA1950 were not mutagenic. The positive control SZN was mutagenic.     

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.     

Microbiological mutagenicity studies of pesticides in vitro.

14 pesticides were tested as pure compounds for the induction of point mutations in four strains of Salmonella typhimurium--TA1535, TA1536, TA1537 and TA1538--in the presence and in the absence of rat-liver microsomal fractions and for the induction of resistance to low concentrations of streptomycin in the filamentous bacterium, Streptomyces coelicolor. The technique used was essentially the so-called "spot test". The pesticides investigated were: aminotriazole, Benomyl, Captafol, Captan, Dichlorvos, Dalapon-Na, Dinobuton, Dodine, Ioxynil, Mecoprop, Neburon, Picloram, Triallate and Trichlorphon. In Salmonella, Captan and Triallate were mutagenic on the TA1535 strain; Dichlorvos and Trichlorphon were negative in the spot test but mutagenic after incubation in liquid cultures of strain TA1535. By using the S. coelicolor forward-mutation test, aminotriazole, Dichlorvos, Picloram, Trichlorphon and Triallate were mutagenic with the "spot test" technique; Captan showed a weak mutagenic activity with a "plate-incorporated" technique.     

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.     

Fluroxene mutagenicity.

The commercially available volatile anesthetic fluroxene (2,2,2-trifluoroethyl vinyl ether) which contains the stabilizer N-phenyl-1-napthylamine, was tested for mutagenicity using four strains of S. typhimurium, TA1535, TA1537, TA98 and TA100, and one strain of E. coli, WP2. In addition, purified fluroxene; N-phenyl-1-napthylamine; trifluoroethanol, a major metabolite of fluoroxene; and urine from rats anesthetized with fluroxene were tested. Several procedures were utilized including exposure of bacteria to vapor in desiccators and in liquid suspension. Results indicate that fluroxene, but not its stabilizer, was mutagenic to strains TA1535, TA100 and WP2 only in liquid suspension and only in the presence of a rat-liver enzyme system. Trifluoroethanol and urine from fluroxene-treated rat were not mutagenic to any strain of bacteria. These findings indicate that fluroxene is a promutagen which requires preincubation before it is recognized. Further experiments were performed with enzymes prepared from mouse, hamster and human liver. Fluroxene was mutagenic only in the presence of enzymes prepared from Aroclor 1254 pretreated rodents. Since fluroxene was not mutagenic in the presence of enzymes prepared from three human livers, the significance of these findings to man are unclear.     

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.     

Mutagenicity in Salmonella typhimurium tester strains of XAD-2-ether extract, recovered from Katsura River water in Kyoto City, and its fractions

The mutagenic activity of XAD-2-ether extracts recovered from Katsura River water at monthly intervals during September to December 1980 was tested on S. typhimurium TA1538, TA1535, TA98 and TA100. The extracts showed strong mutagenic activity towards TA1538 nd TA98, especially in the presence of S9 mix. They were more active to TA1538 than to TA98. Some of each of the XAD-2-ether extracts were pooled and separated into neutral, basic and acidic fractions, and their mutagenic activities were tested on TA1538 and TA98 to determine their contribution to the total mutagenic activity of the parent extract. The neutral fraction was responsible for most of the total mutagenic activity of the parent extract. Although the basic fraction was only 5.4% by weight of the parent extract, it was much more mutagenic than any other fraction. Its contribution to the total mutagenic activity was higher than the acidic fraction which was 30.5% by weight of the parent extract.     

Mutagenicity in Salmonella typhimurium mutants of the benzene-soluble organic matter derived from air-borne particulate matter and its five fractions.

Air-borne particulate matter was collected on a filter, then extracted with benzene. The benzene-soluble material was separated into 5 fractions, namely acidic, basic, alipathic, polyaromatic and oxygenated fractions. The mutagenic activities of these fractions were examined with a set of Salmonella typhimurium mutants. The 6 mutants were from the TA1535 series, deep rough strains without excision repair, namely TA100 and TA98 (having a resistance-transfer factor) and the standard strain TA1535, TA1536, TA1537 and TA1538. Linear dose-response curves were obtained for the benzene-soluble organic matter, and its acidic, polyaromatic and oxygenated fractions with strain TA98 and a 9000 X g liver supernatant from both phenobarbital(PB)- and dibenz(a,h)anthracene(DBA)-treated rats. Among the 5 fractions tested, 3 fractions, namely the acidic, polyaromatic and oxygenated, played an important role in the mutagenicity of the benzene-soluble organic matter derived from air-borne particulate matter. The 9000 X g rat-liver supernatant was not required to make the acidic fraction mutagenic.     

Production of frameshift mutations in Salmonella by phenanthridinium derivatives: enzymatic activation and photoaffinity labeling

The effect of metabolic activation on the mutagenic potential of some phenanthridinium compounds was examined in Salmonella typhimurium strains TA1538 and TA1978 . All of the compounds tested were mutagenic in TA1538, a DNA excision-repair-deficient strain, when metabolizing enzymes were included in the assay. Reversions were not detected when these compounds were examined under the same conditions in TA1978 , the isogenic strain of TA1538 proficient in DNA repair. The mutagenic activity of an azido analog of propidium iodide was also examined using photoactivation and enzymatic activation, and with both conditions, reversions were observed in TA1538 but not in TA1978 . Furthermore, the ranking of mutagenic activity of propidium azide relative to ethidium azide analogs was comparable for both types of activation. The evidence from several studies suggests that the structural requirements for mutagenic activity for this series of phenanthridinium compounds appear to be the same whether mutagenesis is induced via photoactivation or metabolic activation. The interaction with DNA resulting in covalent alteration of the DNA is implicated as the mutagenic mechanism whether the active species is generated by metabolic- or photo-activation.     

Mutagenic action of methyl 2-cyanoacrylate vapor

Alkyl 2-cyanoacrylate adhesives were tested for mutagenicity in Salmonella typhimurium strains TA98, TA100, TA1535 and TA1538. Both a normal spot test and a spot test specially designed to test volatile compounds were used. The adhesives were also tested in the plate incorporation assay. These investigations showed that methyl 2-cyanoacrylate adhesives are mutagenic in strain TA100. The spot test for volatile compounds showed that it is the vapors from the methyl 2-cyanoacrylate monomer that are responsible for the mutagenic effect. One can conclude that working with methyl 2-cyanoacrylate adhesives entails exposure to vapors with a mutagenic effect and may therefore pose a carcinogenic hazard. Because the adhesives are used in industry, their mutagenic effect has a special importance in work environment.     

Mutagenicity studies with N6, O2'-dibutyryl cyclic adenosine 3',5'-monophosphate (DBcAMP)

N6,O2'-Dibutyryl cyclic adenosine 3,5-monophosphate (DBcAMP) was studied for mutagenicity using the rec assay, the Ames method, and in vitro cytogenetics. DBcAMP had no mutagenic effect on B. subtilis in the rec assay, or on S. typhimurium (TA1535, TA1537, TA1538, TA98 and TA100) or E. coli (WP2 uvrA). In the cytogenetic study, a significant increase in chromosomal aberrations was observed at a concentration of 50,000 micrograms/ml, but it was considered that this effect could be attributed to the secondary effect of the high osmotic pressure in the culture medium. These results suggest that DBcAMP has no mutagenic potential.     

The mutagenicity and DNA-damaging activity of cyclic aliphatic sulfuric acid esters.

The cyclic aliphatic sulfuric acid esters 1,2-ethylene sulfate (ESF), 1,3-propylene sulfate (PSF) and 1,3-butylene sulfate (BSF) have been tested for their mutagenic and DNA-damaging activity. Mutagenicity of the compounds was established with his-auxotrophic indicator strains of Salmonella typhimurium using the in vitro plate test and the host-mediated assay technique with mice as host animals. The DNA-damaging activity was tested in a repair test with Proteus mirabilis mutants defective in DNA repair.In the repair test with a set of P. mirabilis strains (PG713 hcr^?rec^?: PG273 hcr⁺rec⁺) PSF and BSF showed a preferential growth inhibition of the repair-defective strain suggesting DNA-damaging activity of these chemicals. No such activity was found for ESF using the same concentrations of 5 and 15 μmol/plate.All cyclic sulfates revert the tester strain TA1535 of S. typhimurium in vitro indicating their ability to induce base substitutions. Compared with the reference compounds dimethyl sulfate (DMS), diethyl sulfate (DES), 1,3-propane sulfone (PPS) and 1,4-butane sulfone (BTS) the mutagenic activity in the plate test can be described as follows: PPS > PSF > BSF > BTS > ESF > DES > DMS.Dose-response studies in the host-mediated assay with tester strain TA1950 of S. typhimurium as genetic indicator system revealed a linear dosedependency of mutagenic activity. For PPS and PSF the lowest effective dose (LED) has been established as 10 μmol/kg. The LED for BSF and BTS was 50 μmol/kg, DMS and DES were mutagenic in doses of 2500 μmol/kg, while ESF was only weakly mutagenic with a LED of 5000 μmol/kg.The dose-response studies in the host-mediated assay and the results obtained in the in vitro spot test demonstrate similarities in the mutagenic action of the cyclic sulfates PSF and BSF and the respective sulfones, while the stronger alkylating compound ESF was a weak mutagen both in vitro and in vivo.     

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.     

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.