Comparison of the phenylenediamine isomers bioactivation by the green alga Chlamydomonas reinhardtii.

The correlation between the chemical structure of arylamines meta-, orto-, para-phenylenediamine (m-PDA, o-PDA, p-PDA), and their mutagenic activity is known. It is accepted that these promutagenic compounds are metabolized to ultimate mutagens in mammals and higher plants. In our previous work, we used the alga Chlamydomonas reinhardtii as the activating organism and the bacteria Salmonella typhimurium and yeast Saccharomyces cerevisiae as the genetic indicators for m-PDA activation. In the present work, we used the same activation system for o-PDA and p-PDA activation. Different responses of the yeast and algal wild-type strain and of the repair-deficient strains to the toxic and mutagenic effects of o-PDA and p -PDA were observed. p-PDA had the most toxic effect on both intact yeast and algal cells and in the algal cell/microbe coincubation assays. Concerning repair-deficient algal strains, the recombination-deficient strain was the most sensitive to both compounds tested, indicating that the recombination process played an important role in the DNA repair of arylamines. The rank order of the PDA isomers mutagenicity (including m-PDA) was o-PDA > m-PDA > p-PDA for revertants in intact yeast and forward mutants in algae; m-PDA > o-PDA > p-PDA in the algal cell/S. typhimurium long-term coincubation assay, the algal cell/S. cerevisiae coincubation assay, and the intact S. cerevisiae assay for gene convertants as well.

Unicellular green alga Chlamydomonas reinhardtii as an activation system for 2-aminofluorene.

Despite the promutagenic/procarcinogenic potential, polycyclic aromatic amines are widely spread in the environment. Biotransformation of the polycyclic aromatic amine 2-aminofluorene (2-AF) was proved in mammals and higher plants. The algal cell/microbe coincubation assay is an additional system that complemented those proved in mammals and higher plants, useful for detection and conversion of environmental promutagens, mainly in aquatic environments. The unicellular green algae may be a good activating system in coincubation assays in that the algal cells exist as a natural system. To increase the effectiveness of this metabolizing system, different modifications of the standard experimental procedure were conducted. Algae can accumulate and metabolize promutagenic pollutants, some of which may differ from those activated by the animal microsome metabolizing system (S9 mix) and by the plant cell/microbe coincubation assay. 2-AF was activated in the algal cell/ microbe coincubation assay in which wild-type Chlamydomonas reinhardtii cells were used as an activating system and the bacteria Salmonella typhimurium TA98, YG1024, and yeast Saccharomyces cerevisiae D7 as the genetic indicator organisms. It was converted to the mutagenic product(s) for the strain YG1024, but the strain TA98 did not exhibit any increase in the mutant yield of His+ revertants. Consequently, metabolites from 2-AF are substrates for O-acetyltransferase. A direct comparison of algal 2-AF activation with mammalian activation system (S9 mix) proved the higher activity of mammalian microsome system (S9 mix). After the combination of both activation systems, a slight synergetic effect was found. Although the genetic endpoints induced by 2-AF using both modifications of the algal cell/S. cerevisiae coincubation assay and those obtained in intact yeast cells were similar at the equitoxic concentrations, 2-AF activation by the algal supernatant slightly increased the genetic endpoints studied.

Metabolic activation of meta-phenylenediamine by the alga Chlamydomonas reinhardtii.

Promutagens/procarcinogens arylamines are widely distributed in the environment. While it is accepted that these compounds can be metabolized to ultimate mutagens in mammals and higher plants, in aquatic plants they have not yet been explored. Intact wild-type and repair-deficient strains of Chlamydomonas reinhardtii and Saccharomyces cerevisiae D7 strain were assayed for their ability to activate meta-phenylenediamine (m-PDA) to an ultimate mutagen. The different responses of the algal wild-type strain and repair-deficient strains to the toxic and mutagenic effects of m-PDA were observed. Recombination repair played an important role in repair of damage induced to C. reinhardtii DNA by this arylamine. The examined isomer of phenylenediamine induced mutations in both algal and yeast cells. m-PDA was activated in the algal cell/microbe coincubation assay in which algal cells were used as an activating system and bacteria Salmonella typhimurium and yeast Saccharomyces cerevisiae as the genetic indicator organisms. This new assay is, in addition to the animal microsome metabolizing system and the plant cell/microbe coincubation assay, suitable for the detection of environmental promutagens and their conversion to mutagens mainly in aquatic environments.

Genotoxic properties of the newly synthesized antineoplastic agents amidox, didox and trimidox.

Toxic and genotoxic effects of three polyhydroxy-substituted benzohydroxamates (amidox, didox and trimidox), having antineoplastic activities by the mechanism of the ribonucleotid reductase activity inhibition, were evaluated by reverse mutation assay on Salmonella typhimurium strains TA97, TA98, TA100, TA102. While amidox did not exhert any toxic effect, didox and trimidox were toxic. The toxicity of the test chemicals was dependent on the structure of their molecule and the repair capacity of the test strains. Trimidox exhibited the highest toxicity, and it was proved as a direct-acting frameshift mutagen. Its mutagenic effect was increased after a metabolic activation. Amidox and didox can be classified as frameshift promutagens.

Bioactivation of promutagens by the unicellular green alga Chlamydomonas reinhardtii.

The aromatic amine 2-aminofluorene (2-AF) was activated by the intact Chlamydomonas reinhardtii cells to a mutagen that exhibited toxic and mutagenic effects comparable to those of the direct-acting mutagen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). There were different responses of the wildtype and repair-deficient strains to the toxic and mutagenic effect of 2-AF. The recombination repair plays a major role in repair of damages induced in the C. reinhardtii DNA by the aromatic amine promutagen 2-AF and the direct-acting mutagen MNNG. The 2-AF activation has also been analyzed by algal cells/microbe coincubation assay. This new assay is used in addition to animal microsome-metabolizing system (S9 fraction) and plant cell/microbe coincubation assay. This additional system is suitable for detection of environmental promutagens and their conversion to mutagens, mainly in aquatic environments.

A Chlamydomonas reinhardtii UV-sensitive mutant uvs15 is impaired in a gene involved in several repair pathways.

In this report, three DNA repair-deficient mutants of Chlamydomonas reinhardtii (uvs13, uvs14, uvs15) were characterized by using genetic, mutational and biochemical analyses. The mutant strain uvs15 belongs to the most sensitive repair-deficient mutants following exposure to all agents used. It is deficient in the nuclear excision-repair pathway, whereas uvs13 and uvs14 are not blocked in removal of pyrimidine dimers. Mutation study also revealed differences among strains. The mutant uvs15 does not mutate after UV and X-ray irradiation, and there is very low mutation rate after MNNG. These findings might indicate the involvement of UVS15 gene product in regulation of several repair pathways. Contrary to this, uvs14 showed higher mutation frequency, both spontaneous and induced after UV and MNNG treatments. Tetrad dissection proved that the uvs13 and uvs14 genes are located on the right arm of the linkage group I in the vicinity of the previously mapped uvs10 gene. Both mutants belong to the same repair pathway, which is different from that of uvs10 and uvs15.

The comparative genotoxicological study of new local anesthetics, 3-(2-alkoxyphenylcarbamoyloxy)quinuclidium chlorides, on Salmonella typhimurium, Saccharromyces cerevisiae, Vicia faba, Hordeum vulgare and Drosophila melanogaster.

Potential gentoxicity of five new local anesthetics, derivatives of phenylcarbamic acid differing in the length of the alkyl chain of the alkoxy substituent, was studied on five test systems. There was a direct relationship with increased toxic effect in bacteria and yeast as a function of the elongation of the alkyl chain of the alkoxy substituents of the phenylcarbamic acid esters. On the other hand, no structure-toxicity relationship was found after application of 3-(2-alkoxyphenylcarbamoyloxy)-quinuclidium chlorides on plants and Drosophila. All anesthetics were nonmutagenic to Salmonella typhimurium strains TA97, TA98, TA100, and TA102 in the absence and in the presence of S9 mix. Pentyloxy and heptyloxy derivatives increased rates of genetic changes in Saccharomyces cerevisiae, mainly revertants at the isoleucine locus. Pentyloxy and hexyloxy derivatives increased the frequency of chromosome aberrations in Vicia faba root-tip meristems. No chlorophyll mutations were detected after treatment of Hordeum vulgare with pentyloxy, hexyloxy and heptyloxy derivatives. No sex-linked recessive lethals were scored in Drosphila melanogaster males. The rates of aneuploids induced in their germ cells were significantly increased after treatment with butoxy and octyloxy derivatives. However, the local toxic and genotoxic effects of test anesthetics on the microorganisms of the anesthetized tissues may be of some importance. In particular, the genotoxic effect exhibited in fungi by the heptyloxy derivative, a potent local anesthetic, was remarkable.

Interactions between photolyase and dark repair processes in Chlamydomonas reinhardtii.

The participation of DNA photolyase in dark repair processes has been reported in some heterotrophic organisms. To assess the role of photolyase in dark repair in photoautotrophs, double mutants of Chlamydomonas reinhardtii deficient in dark repair and photoreactivation were constructed and assayed for UV sensitivity in different posttreatment light conditions (with or without subsequent photoreactivation). We found that a functional PHR1 gene enhanced dark survival in the excision deficient (uvs9, uvs12) and in the recombination deficient (uvs10) genetic backgrounds but failed to do so in the strain deficient in a repair pathway other than excision and recombination (uvs13). Therefore we can conclude that photolyase may stimulate dark repair processes in C. reinhardtii also via pathway(s) other than nucleotide excision repair. The fact that some of the double mutants deficient in dark repair and photoreactivation survived better in the light than in the dark supports the idea that additional photorepair might be active and may enhance survival in a specific genetic background.

Genotoxicity evaluation of Rastim 30 DKV, the plant growth regulator, on five test systems.

The possible mutagenic activity of Rastim 30 DKV, a new plant growth regulator, was studied on five model test systems. It did not increase the frequency of His+ revertants in Salmonella typhimurium strains TA97, TA98, TA100, TA1535, TA1538 in the absence and the presence of S9 mix. It slightly increased rates of genetic changes in Saccharomyces cerevisiae, mainly convertants at the tryptophan locus. No clastogenic effect was observed after Vicia faba root-tip meristem treatment, and at the lowest concentration used its mitotic activity was significantly increased. No chlorophyll mutants after the treatment of two cultivars of barley were observed. Though no sex-linked recessive lethals were scored in Drosophila melanogaster males, the rates of aneuploids induced in their germ cells were significantly increased.

Mutagenic activity of phosmet, the active component of the organophosphorus insecticide Decemtione EK 20 in Salmonella and Saccharomyces assays.

Phosmet, the active component of the organophosphorus insecticide Decemtione EK 20, was shown to be mutagenic in the standard Ames Salmonella/mammalian microsome assay in the absence and presence of metabolic activation. It appears to be a direct mutagen inducing base substitution mutations (TA100) as well as a weak frameshift mutagen (TA97). This compound was genotoxic in the Saccharomyces cerevisiae D7 strain. It significantly increased reverse mutation, mitotic crossing-over and slightly, but not significantly, increased gene conversion at the highest concentration used.

New DNA repair-deficient mutants of Chlamydomonas reinhardtii.

Two new UV-sensitive mutants of Chlamydomonas reinhardtii, uvs12 and uvs13, were characterized. Genetic analysis proved that they were non-allelic. They complemented the other repair-deficient mutants uvs8, uvs9, uvs10 and uvs11. While uvs12 may have an impaired excision-repair pathway, uvs13 is, by its UV sensitivity under non-photoreactivating conditions, very similar to uvsE1 and uvs10, but differs in the effect of caffeine on its survival. After UV, survival of some repair-deficient mutants was, under photoreactivating conditions, much lower than that of phr1, while survival of other repair-deficient mutants did not differ from that of a wild-type strain. A lower UV survival of some dark-repair-defective mutants of Chlamydomonas reinhardtii, under photoreactivating conditions, can perhaps be used as an additional criterion for mutants defective in the excision-repair pathway.

Effects of supercypermethrin, a synthetic developmental pyrethroid, on four biological test systems.

The genotoxic potential of the insecticide supercypermethrin, a second-generation pyrethroid, was studied on four different test systems. It was non-mutagenic to Salmonella typhimurium strains TA1535, TA100, TA1538, TA98 and TA97 in the presence and absence of S9 mixture. It induced gene conversion at the tryptophan locus and induced point mutations at the isoleucine locus in Saccharomyces cerevisiae cells. A slight increase in the frequency of aberrant anaphases and telophases in root tips of Hordeum vulgare and Vicia faba was observed, but no genotoxic effects were detected in Drosophila melanogaster.

General characteristics, molecular and genetic analysis of two new UV-sensitive mutants of Chlamydomonas reinhardtii.

Two new UV-sensitive mutants of Chlamydomonas, UVS10 and UVS11, were isolated. Both behave as single nuclear mutations. UVS10 was mapped to linkage group I. UVS11 is a separate, unlinked mutation but has not yet been located to a specific linkage group. Both mutants are proficient in the excision of pyrimidine dimers from nuclear DNA. The survival of UV-irradiated UVS11 is increased when plated in the presence of 1.5 mM caffeine, similar to wild-type. Caffeine has no effect on the survival of UV-irradiated UVS10. UV-irradiated UVS11 frequently divides at least once before dying, in contrast to UVS10 or wild-type. UVS11 also exhibits a much increased frequency of mutation to streptomycin resistance after UV irradiation.

Evaluation of the mutagenic effect of the new fungicide trimorphamide.

The new Czechoslovak fungicide trimorphamide was tested for its mutagenic activity. To evaluate the potential mutagenic effects on Drosophila, trimorphamide at 0.5, 1.0, 5.0, 10.0% was administered into the cultivation medium, and the sex-linked recessive lethal mutation detection test and the chromosome nondisjunction test were used. After administration of trimorphamide to mice at 60, 150 and 300 mg . kg-1 b.w. perorally, and 30, 70 and 150 mg . kg-1 b.w. intraperitoneally in single and repeated (5X) doses, a cytogenetic analysis of chromosomal aberrations in bone-marrow cells was performed. The cytogenetic analysis of human peripheral lymphocytes for chromosomal aberrations in vitro was performed 24 h after trimorphamide had been applied into the culture in concentrations 19.1 X 10(-3), 19.1 X 10(-4) and 19.1 X 10(-5) M. Under our testing conditions the trimorphamide concentrations used did not show any mutagenic effect upon Drosophila, compared with the controls. Also, under the conditions of the cytogenetic analysis, no significant increase in the frequency of chromosomal abnormalities in mouse bone marrow or in human peripheral lymphocyte was observed compared with the group of controls.

The mutagenic effect of the new insecticide and acaricide pyridathion.

The potential mutagenic effect of the new organophosphate insecticide and acaricide, pyridathion, was tested on Escherichia coli, strains WP2, WP2 uvrA, and on Salmonella typhimurium, strains TA100 and TA98, both with and without metabolic activation. The compound was tested at 1, 10 and 100 micrograms/ml. The analysis of chromosomal aberrations in mouse bone marrow was performed after a single peroral application of pyridathion at doses of 0.6, 1.11, 6.0, 12.0 and 24.0 mg . kg-1 b.w., i.p. application at 6.0 mg . kg-1 b.w., and repeated application (5 times) p.o. at 1.0, 2.5 and 5.0 mg . kg-1 b.w. Analysis of rat bone marrow was performed after a 3-month peroral application of pyridathion at 0.07, 0.175 and 0.35 mg . kg-1 b.w. An analysis of chromosomal aberrations in human peripheral lymphocytes in vitro was performed 24 h after the application of pyridathion into the culture in concentrations of 9.2 X 10(-3), 9.2 X 10(-4) and 9.2 X 10(-5) moles. The insecticide did not exert any mutagenic effect on the bacteria. There was no significant increase in the frequency of chromosomal abnormalities in bone marrow of mice of rats, or in human peripheral lymphocytes in vitro, compared with controls.

Genetic variability of Chlamydomonas reinhardtii induced by six urea derivatives.

The lethal and mutagenic effect of six urea derivatives applied to the cells of Chlamydomonas reinhardtii Dang was investigated. The mutagenic effect of two N-alkylnitroso derivatives (N-methyl-N-nitroso- and N-ethyl-N-nitrosourea), two monoalkyl derivatives (N-methyl- and N-ethylurea) and two dialkyl derivatives (N,N'-dimethyl- and N,N'-diethylurea) was compared at equal molar concentrations and different cell survival or at equitoxic concentrations, i.e. at equal survival. The former type of appraisal shows the alkylnitroso derivatives to be strong mutagenic agents, exceeding in their effect several times both monoalkyl- and dialkyl derivatives. MNU is seen to be a stronger mutagen than ENU. Alkyl derivatives are generally weak mutagens, the strongest being DEU and the least potent DMU. On using the latter type of evaluation MNU is seen to be clearly the strongest mutagen while the other five compounds, including ENU, have, at equitoxic concentrations, approximately the same effect.