The genetic activity of N6-hydroxyadenine and 2-amino-N6-hydroxyadenine in Escherichia coli, Salmonella typhimurium and Saccharomyces cerevisiae

The genetic activity of 2-amino-N6-hydroxyadenine or 2-amino-N-hydroxylaminopurine (AHA) and N6-hydroxyadenine or 6-N-hydroxylaminopurine (HAP) was studied in S. typhimurium, E. coli and Saccharomyces cerevisiae strains. AHA was a more potent mutagen for bacteria and a less potent mutagen for yeast than HAP. The mutagenic activity of analogs was not influenced by excision, mutagenic or double-strand DNA repair mutations. On the other hand, the uvrBdel mutation has a drastic effect on the mutagenicity and toxicity of both analogs in the Salmonella strains studied. HAP was a very potent mutagen in yeast with a low capability of inducing mitotic recombination contrary to common mutagens, possessed unique intergenic specificity and was able to induce mutations in diploids at rather high frequency.     

Genetic activity in Saccharomyces cerevisiae and thin-layer chromatographic comparisons of medical grades of pyrvinium pamoate and monopyrvinium salts

The pamoate, chloride, and iodide salts of pyrvinium, a cyanine dye with anthelmintic properties, were studied in a diploid mitotic recombination and gene conversion assay system (strain D5 of Saccharomyces cerevisiae) and a haploid yeast reversion assay (strain XV185-14C). With the use of a thin-layer chromatographic (TLC) detection technique, samples of pyrvinium pamoate from several sources were found to contain different numbers and quantities of impurities. All samples of pyrvinium pamoate and the monopyrvinium salts were recombinogenic in strain D5 and mutagenic in strain XV185-14C; the degree of genetic activity varied among the tested medical grades of pyrvinium pamoate. Monopotassium pamoate was found to be genetically inactive in both strains. Light-catalyzed degradation did not enhance the genetic activity of pyrvinium in either of the yeast strains; the degraded samples were not mutagenic.     

Genetic effects induced in Sacharomyces cerevisiae by cyclophosphamide in vitro without liver enzyme preparations

Cyclophosphamide induced forward mutation in Saccharomyces cerevisiae strain S288C and mitotic recombination in strains D3 and D5 but not in strain D4. The yeast cells were treated with the compound in phsphate buffer without recourse to metabolic activation protocols. Elevation of the treatment temperature increased the genetic activity of cyclophosphamide. Respiration-deficient isolates of strains S288C and D3 were more sensitive than the respiratory competent parent strains were for inducing forward mutation and mitotic recombination, respectively. Cyclophosphamide was incubated in phosphate buffer alone for increasing time intervals; strain D3 cells were added to aliquots for each time interval and incubated for an additional 30 min. The frequency of induced recombination increased as the time of compound incubation increased, showing that spontaneous degradation of cyclophosphamide to genetically active breakdown products was responsible for the genetic damage induced in the yeast cells.     

Genetic and biochemical consequences of thymidylate stress

We have examined the genetic and biochemical consequences of thymidylate stress in haploid and diploid strains of the simple eukaryote Saccharomyces cerevisiae (Bakers' yeast). Previously we reported that inhibition of dTMP biosynthesis causes "thymineless death" and is highly recombinagenic, but apparently not mutagenic, at the nuclear level; however, it is mutagenic for mitochondria. Concurrent provision of dTMP abolishes these effects. Conversely, excess dTMP is highly mutagenic for nuclear genes. It is likely that DNA strand breaks are responsible for the recombinagenic effects of thymidylate deprivation; such breaks could be produced by reiterative uracil incorporation and excision in DNA repair patches. In our experiments, thymidylate stress was produced both by starving dTMP auxotrophs for the required nucleotide and also by blocking de novo synthesis of thymidylate by various antimetabolites. We found that the antifolate methotrexate is a potent inducer of mitotic recombination (both gene conversion and mitotic crossing-over). This suggests that the gene amplification associated with methotrexate resistance in mammalian cells could arise, in part, by unequal sister-chromatid exchange induced by thymidylate stress. In addition, several sulfa drugs, which impede de novo folate biosynthesis, also have considerable recombinagenic activity.     

Effects of benzyladenine on prokaryotic and eukaryotic cells.

Comparative studies of the effect of benzyladenine (BA) on the yeast Saccharomyces cerevisiae, the bacterium Salmonella typhimurium, the shallot Allium ascalonicum and Chinese hamster fibroblast cells were performed. The tested substance had no mutagenic activity on yeast, bacteria and cultured fibroblast cells. Changes in mitotic activity and cell division abnormalities were observed after BA treatment in shallot root-tip cells.     

DNA-damaging and mutagenic effects of 1,2-dimethylhydrazine on Bacillus subtilis repair-deficient mutants.

Mutagenic, DNA-damaging, and in vivo alteration of DNA have been demonstrated for 1,2-dimethylhydrazine (DMH), a potent inducer of adenocarcinomas of the large intestine and colon of rats. These activities are pH-dependent, with 6.5 giving optimum response. There was no requirement for metabolic activation with rat-liver S9 mix when the appropriate Bacillus subtilis mutant strains were used. The Rec- strains recA8 and mc-1 were greater than 300-fold more sensitive to the DNA-damaging activity of DMH than was their isogenic wild-type parent. The DNA isolated from DMH-treated mc-1 had altered spectroscopic characteristics, and gave a greatly reduced transformation efficiency. Treatment of B. subtilis strain TKJ6321 with DMH at pH 6.5 induced His+, Met+ mutations in substantial numbers at low concentrations of this chemical. The use of B. subtilis mutants in these studies has therefore made it possible to demonstrate mutagenic and DNA-damaging activity in bacteria for this potent carcinogenic chemical.     

Relationships between structure, toxicity and genetic effects in Salmonella typhimurium and Saccharomyces cerevisiae for substituted aniline mustards

A series of 4-substituted aniline mustards of widely varying reactivities have been evaluated for their mutagenic effects in Salmonella typhimurium strains of varying uvrB gene and plasmid status, and for their ability to cause mitotic crossing-over in Saccharomyces cerevisiae. The 4-methyl aniline mustard N,N-bis(2-chloroethyl)-4-methylaniline and its corresponding half-mustard N-(2-chloroethyl)-4-methylaniline showed widely different effects in the various bacterial strains, with the half-mustard being much less toxic than the full mustard in the uvrB- strain TA100. However, in the uvrB+ strain TA1978+, possessing an intact excision repair system, both compounds were equally toxic and the full mustard was the more mutagenic. Both compounds were equally effective in promoting mitotic crossing-over in yeast. For a series of 4-substituted full mustards, the toxicity in S. typhimurium strain TA100 correlated with substituent electronic parameters in the same way as does mammalian cell toxicity, supporting the view that the primary mode of toxicity is via DNA cross-linking, even for unreactive analogues. However, there were no obvious correlations between substituent physiochemical properties and mutagenic potential in bacteria, suggesting that mutagenic events are subject to a variety of influences other than the reactivity of the mustard group. In contrast, the most chemically reactive compounds were the most toxic and most recombinogenic in yeast.     

Mutagenicity and DNA-damaging activity for several pesticides tested with Bacillus subtilis mutants

The active pure compounds of 4 pesticides were tested for DNA-damaging and mutagenic activity in Bacillus subtilis and Salmonella typhimurium tester strains. Included were zinc ethylenebisdithiocarbamate (dithane), 1,2-dihydropyridazine-3,6-dione (maleic hydrazide), O,O-dimethylphosphorodithioate (malathion), and 1,2-dibromoethane (fumazone). These agents gave either weak or negative mutagenic responses with the Salmonella/microsome tests for mutagenicity, but were all positive when the tester was B. subtilis strain TKJ6321. Of the 4 chemicals, only fumazone required metabolic activation with rat-liver S9 mix. Upon activation, it produced a volatile mutagenic product. Dithane, maleic hydrazide, and malathion were all mutagenic and did not require metabolic activation. Among these agents, dithane was strongly mutagenic while fumazone, maleic hydrazide and malathion were moderately mutagenic. Only dithane gave significant DNA-damaging activity when applied to a battery of repair-deficient B. subtilis mutants. For the chemicals reported, it is concluded that B. subtilis is superior to S. typhimurium in the detection of mutagenic activity. We strongly recommend its use for prescreening procedures in combination with the S. typhimurium testers.     

Genotoxicity of the boldine aporphine alkaloid in prokaryotic and eukaryotic organisms

The aporphine alkaloid boldine, present in Peumus boldus (boldo-do-Chile) widely used all over the world, was tested for the presence of genotoxic, mutagenic and recombinogenic activities in microorganisms. This alkaloid did not show genotoxic activity with or without metabolic activation in the SOS chromotest and Ames tester strains TA100, TA98 and TA102. It was not able to induce point and frameshift mutations in haploid Saccharomyces cerevisiae cells. However, mitotic recombinational events such as crossing-over and gene conversion were weakly induced in diploid yeast cells by this alkaloid. Also, boldine was able to induce weakly cytoplasmic 'petite' mutation in haploid yeast cells.     

Mutagenic and recombinogenic consequences of DNA-repair inhibition during treatment with 1,3-bis(2-chloroethyl)-1-nitrosourea in Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae has been used as a model system to explore whether the clinical combination of the antitumour agent BCNU (1,3-bis(2-chloroethyl)-1-nitrosourea) with DNA-repair inhibitors would affect the drug's mutagenic or recombinogenic potential. Preliminary experiments suggested that mitotic crossing-over and other mutagenic events are controlled in a separate fashion. BCNU was more toxic in yeast derivatives with specific defects in any of the three recognised major DNA repair pathways than in the DNA-repair-proficient parent strain. However, in a diploid homozygous for rad18, BCNU showed enhanced mutagenic and recombinogenic potential. Both of these effects were reduced in a comparable rad3 strain, and mitotic crossing-over but not other types of mutagenic event eliminated in the rad52 derivative. Experiments were performed in the presence of three DNA-repair inhibitors which are currently in clinical use and which might be available for combination chemotherapy. Hydroxyurea and amsacrine themselves caused mitotic crossing-over and other events, and did not reduce mutagenic or recombinogenic potential of the BCNU. Hydroxyurea actually decreased toxicity of the BCNU. Caffeine, however, showed some effect in enhancing toxicity and decreasing both mutagenic and recombinogenic potential of the drug. Development of more specific repair inhibitors related to amsacrine or to caffeine, using these repair-deficient strains as model systems, might lead to an enhanced clinical potential of this bisalkylating drug and related compounds.     

A role for RAD54B in homologous recombination in human cells.

In human somatic cells, homologous recombination is a rare event. To facilitate the targeted modification of the genome for research and gene therapy applications, efforts should be directed toward understanding the molecular mechanisms of homologous recombination in human cells. Although human genes homologous to members of the RAD52 epistasis group in yeast have been identified, no genes have been demonstrated to play a role in homologous recombination in human cells. Here, we report that RAD54B plays a critical role in targeted integration in human cells. Inactivation of RAD54B in a colon cancer cell line resulted in severe reduction of targeted integration frequency. Sensitivity to DNA-damaging agents and sister-chromatid exchange were not affected in RAD54B-deficient cells. Parts of these phenotypes were similar to those of Saccharomyces cerevisiae tid1/rdh54 mutants, suggesting that RAD54B may be a human homolog of TID1/RDH54. In yeast, TID1/RDH54 acts in the recombinational repair pathway via roles partially overlapping those of RAD54. Our findings provide the first genetic evidence that the mitotic recombination pathway is functionally conserved from yeast to humans.     

Inducibility of error-prone DNA repair in yeast?

Whereas some experimental evidence suggests that mutagenesis in yeast after treatment with DNA-damaging agents involves inducible functions, a general-acting error-prone repair activity analogous to the SOS system of Escherichia coli has not yet been demonstrated. The current literature on the problem of inducibility of mutagenic repair in yeast is reviewed with emphasis on the differences in the experimental procedures applied.     

Genotoxicity of stannous chloride in yeast and bacteria

Stannous chloride was found genotoxic in microbial test systems of the yeast Saccharomyces cerevisiae, in one strain of Salmonella typhimurium and in the Mutoxitest of Escherichia coli. Five isogenic haploid yeast strains differing only in a particular repair-deficiency had the following ranking in Sn2+ -sensitivity: rad52delta>rad6delta>rad2delta>rad4delta>RAD, indicating a higher relevance of recombinogenic repair mechanisms than nucleotide excision in repair of Sn2+ -induced DNA damage. Sn2+ -treated cells formed aggregates that lead to gross overestimation of toxicity when not undone before diluting and plating. Reliable inactivation assays at exposure doses of 25-75 mM SnCl2 were achieved by de-clumping with either EDTA- or phosphate buffer. Sn2+ -induced reversion of the yeast his1-798, his1-208 and lys1-1 mutant alleles, in diploid and haploid cells, respectively, and putative frameshift mutagenesis (reversion of the hom3-10 allele) was observed. In diploid yeast, SnCl2 induced intra-genic mitotic recombination while inter-genic (reciprocal) recombination was very weak and not significant. Yeast cells of exponentially growing cultures were killed to about the same extend at 0.1% of SnCl2 than respective cells in stationary phase, suggesting a major involvement of physiological parameters of post-diauxic shift oxidative stress resistance in enhanced Sn2+ -tolerance. Superoxide dismutases, but not catalase, protected against SnCl2-induced reactive oxygen species as sod1delta had a three-fold higher sensitivity than the WT while the sod2delta mutant was only slightly more sensitive but conferred significant sensitivity increase in a sod1delta sod2delta double mutant. In the Salmonella reversion assay, SnCl2 did not induce mutations in strains TA97, TA98 or TA100, while a positive response was seen in strain TA102. SnCl2 induced a two-fold increase in mutation in the Mutoxitest strain IC203 (uvrA oxyR), but was less mutagenic in strain IC188 (uvrA). We propose that the mutagenicity of SnCl2 in yeast and bacteria occurs via error-prone repair of DNA damage that is produced by reactive oxygen species.     

Testing of chemicals for genetic activity with Saccharomyces cerevisiae: a report of the U.S. Environmental Protection Agency Gene-Tox Program

The yeast Saccharomyces cerevisiae is a unicellular fungus that can be cultured as a stable haploid or a stable diploid . Diploid cultures can be induced to undergo meiosis in a synchronous fashion under well-defined conditions. Consequently, yeasts can be used to study genetic effects both in mitotic and in meiotic cells. Haploid strains have been used to study the induction of point mutations. In addition to point mutation induction, diploid strains have been used for studying mitotic recombination, which is the expression of the cellular repair activities induced by inflicted damage. Chromosomal malsegregation in mitotic and meiotic cells can also be studied in appropriately marked strains. Yeast has a considerable potential for endogenous activation, provided the tests are performed with appropriate cells. Exogenous activation has been achieved with S9 rodent liver in test tubes as well as in the host-mediated assay, where cells are injected into rodents. Yeast cells can be recovered from various organs and tested for induced genetic effects. The most commonly used genetic end point has been mitotic recombination either as mitotic crossing-over or mitotic gene conversion. A number of different strains are used by different authors. This also applies to haploid strains used for monitoring induction of point mutations. Mitotic chromosome malsegregation has been studied mainly with strain D6 and meiotic malsegregation with strain DIS13 . Data were available on tests with 492 chemicals, of which 249 were positive, as reported in 173 articles or reports. The genetic test/carcinogenicity accuracy was 0.74, based on the carcinogen listing established in the Gene-Tox Program. The yeast tests supplement the bacterial tests for detecting agents that act via radical formation, antibacterial drugs, and other chemicals interfering with chromosome segregation and recombination processes.     

Recombinogenic phenotype of human activation-induced cytosine deaminase

Class switch recombination, gene conversion, and somatic hypermutation that diversify rearranged Ig genes to produce various classes of high affinity Abs are dependent on the enzyme activation-induced cytosine deaminase (AID). Evidence suggests that somatic hypermutation is due to error-prone DNA repair that is initiated by AID-mediated deamination of cytosine in DNA, whereas the mechanism by which AID controls recombination remains to be elucidated. In this study, using a yeast model system, we have observed AID-dependent recombination. Expression of human AID in wild-type yeast is mutagenic for G-C to A-T transitions, and as expected, this mutagenesis is increased upon inactivation of uracil-DNA glycosylase. AID expression also strongly induces intragenic mitotic recombination, but only in a strain possessing uracil-DNA glycosylase. Thus, the initial step of base excision repair is required for AID-dependent recombination and is a branch point for either hypermutagenesis or recombination.     

Genotoxic activity of pulp mill effluent in Salmonella and Saccharomyces cerevisiae assays

An XAD-2 resin concentrate of chlorination-stage pulp mill effluent was found to induce mutations in Salmonella typhimurium strains TA1535, TA100 and TA98 but not in strains TA1537 or TA1538. The presence of either S9 mix, S9 mix without cofactors, or heat-inactivated S9 mix, reduced the mutagenic effects. Dose-related increases in gene conversion, mitotic recombination and aberrant colony formation in Saccharomyces cerevisiae strain D7 also were found.     

Genetic activity of bleomycin: differential effects on mitotic recombination and mutations in yeast.

The genetic effects of the antitumor antibiotic, bleomycin were studied in different strains of Saccharomyces cerevisiae. It was observed that the drug induced a high frequency of mitotic recombination and gene conversion. In contrast, it produced only a few mutations from adenine independence to adenine dependence and histidine dependence (a missense mutant) to histidine independence. In the strains carrying ochre-suppressible auxotrophic markers, no prototrophs were induced by this drug. The results indicating the specific activity of bleomycin are discussed and, in this connection, the usefulness of yeast as a test organism in mutagenicity screening is emphasized.     

Conservation of genetic linkage in nonisogenic isolates of Candida albicans

A number of laboratories are now engaged in the genetic analysis of Candida albicans. This diploid yeast, the major fungal pathogen of humans, is imperfect. Parasexual techniques have been devised for complementation and recombination analysis in this organism. This paper attempts to address the question of the extent to which nonisogenic strains of C. albicans have conserved a common genetic map. This analysis is a prerequisite for the integration of work done in different laboratories and may also provide useful information on the taxonomy of the genus Candida. The paper also reports the analysis of an interspecific hybrid between C. albicans and Candida stellatoidea. The method employed in these studies was the analysis of the mitotic recombination relationships of a group of linked genes and their centromere. Strains carrying linked auxotrophic mutations were fused with isogenic and nonisogenic complementary strains to form tetraploids. The mitotic recombination analyses of these tetraploids suggest that in the isolates studied the genetic map is conserved. A comparison of tetraploid and diploid mitotic recombination analyses is also presented.     

Mitotic recombination and inactivation in Saccharomyces cerevisiae induced by UV-radiation (254 nm) and hyperthermia depend on UV fluence rate

In experiments with wild-type diploid yeast cells of Saccharomyces cerevisiae, the synergistic interaction of ultraviolet (UV) light (wavelength, 254 nm) and heat (45--60 degrees C) was studied both for mutagenic and inactivation effects. Simultaneous hyperthermia and UV light treatments increase the frequency of UV-induced mitotic intergenic recombination (crossing-over) and cell inactivation. The enhancing effect was a function of UV light fluence rate. It is concluded that the effect of hyperthermia on low fluence UV or high fluence UV irradiation results in comparable effects on survival and mitotic recombination suggesting similar modulation by hyperthermia of the effects induced by UV at different fluence rates. The interpretation of the data obtained was carried out within the widely accepted point of view considering the synergistic effects as a result of repair ability damage.