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.     

Mutagenic and lethal effects of alpha-benzene hexachloride, dibutyl phthalate and trichloroethylene in Saccharomyces cerevisiae.

Saccharomyces cerevisiae strain XV185-14C for reversion studies was used to investigate the genetic activity of alpha-benzene hexachloride dibutyl phthalate and trichloroethylene. The results indicate that none of the three compounds was genetically active when yeast cells were treated in phosphate buffer (pH 7.0) in the absence of metabolic conversion. However, in the presence of the 9000 g supernatant of mice liver homogenate, NADP, glucose-6-phosphate, phosphate buffer (PH 7.4), MgCl2, KCl, the components which were used for the metabolic conversion, trichloroethylene porved to be a powerful mutagen. It increases the frequency of homoserine, histidine and lysine revertants over those of the control levels. Trichloroethylene appears to induce frameshift as well as base substitution mutations.     

Protective effect of two sunscreens against lethal and genotoxic effects of UVB in V79 Chinese hamster cells and Saccharomyces cerevisiae strains XV185-14C and D5.

Effects of p-aminobenzoic acid (PABA) and of 4-[(2-oxo-3-bornylidene)methyl]-phenyl trimethylammonium methylsulfate (OMM), two components used in sunscreen formulations, on the mutagenicity of UVB irradiation are compared in three genetic assay systems. A haploid strain of Saccharomyces cerevisiae XV185-14C was used to measure reverse mutations at three loci. The diploid strain D5 of Saccharomyces cerevisiae was used to screen for reciprocal mitotic recombination. The induction of forward mutations was measured in Chinese hamster V79 cells. Our results indicate that UVB irradiation induced HGPRT- mutants in V79 cells, reverse mutations in Saccharomyces cerevisiae strain XV185-14C, and mitotic crossing over and other genetic alterations in Saccharomyces cerevisiae strain D5. V79 Chinese hamster lung cells were the most sensitive to UVB irradiation, followed by Saccharomyces cerevisiae haploid strain XV185-14C and the diploid strain D5. PABA and OMM were both capable of protecting all three types of cells from UVB irradiation regarding both lethality and induction of various types of genetic alterations. At higher concentrations (above 10(-5) M), OMM was more effective in its photoprotective effect toward UVB irradiation than PABA.     

Simultaneous measurement of the frequencies of intrachromosomal recombination and chromosome gain using the yeast DEL assay

The yeast DEL assay measures the frequency of intrachromosomal recombination between two partially-deleted his3 alleles on chromosome XV. The his3Delta alleles share approximately 400bp of overlapping homology, and are separated by an intervening LEU2 sequence. Homologous recombination between the his3Delta alleles results in deletion of the intervening LEU2 sequence (DEL), and reversion to histidine prototrophy. In this study we have attempted to further extend the use of the yeast DEL assay to measure the frequency of chromosome XV gain events. Reversion to His(+)Leu(+) in the haploid yeast DEL tester strain RSY6 occurs upon non-disjunction of chromosome XV sister chromatids, coupled with a subsequent DEL event. Here we have tested the ability of the yeast DEL assay to accurately predict the aneugenic potential of the diversely-acting, known or suspected aneugens actinomycin D, benomyl, chloral hydrate, ethyl methanesulfonate (EMS), methyl methanesulfonate (MMS), and methotrexate. Actinomycin D and benomyl strongly induced aneuploidy. EMS and methotrexate modestly induced aneuploidy, while chloral hydrate and MMS failed to illicit any significant induction. In addition, by FACS-analysis of DNA content it was shown that the majority of both spontaneous- and chemically-induced His(+)Leu(+) revertants were heterodiploid. Thus, our results indicate endoreduplication of almost entire chromosome sets as a major mechanism of aneuploidy induction in haploid Saccharomyces cerevisiae.     

Mutagenicity of antiamebic and anthelmintic drugs in the Salmonella typhimurium microsomal test system

4 amebicides (chloroquine diphosphate, diiodohydroxyquin, iodochlorohydroxyquin and dehydroemetine) and 6 anthelmintics (bephenium hydroxynaphthoate, 4-hexylresorcinol, mebendazole, niclosamide, pyrantel pamoate and pyrvinium pamoate) were tested for mutagenicity in the Salmonella typhimurium microsomal test system. Frameshift mutations were induced by dehydroemetine and niclosamide following activation by microsomal enzymes, while pyrvinium pamoate induced both frameshift and base-pair substitution mutations with or without metabolic activation. The urine of mice treated with dehydroemetine or pyrvinium pamoate showed no mutagenic activity. However, urine obtained from mice treated with niclosamide was mutagenic in strains TA98 and TA1538. The fluctuation assay showed chloroquine diphosphate to be mutagenic in TA1537, a strain which detects frameshift mutations.     

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.     

An Anthelmintic Drug,Pyrvinium Pamoate,Thwarts Fibrosis and Ameliorates Myocardial Contractile Dysfunction in a Mouse Model of Myocardial Infarction

Metabolic adaptation to limited supplies of oxygen and nutrients plays a pivotal role in health and disease. Heart attack results from insufficient delivery of oxygen and nutrients to the heart, where cardiomyocytes die and cardiac fibroblasts proliferate – the latter causing scar formation, which impedes regeneration and impairs contractility of the heart. We postulated that cardiac fibroblasts survive metabolic stress by adapting their intracellular metabolism to low oxygen and nutrients, and impeding this metabolic adaptation would thwart their survival and facilitate the repair of scarred heart. Herein, we show that an anthelmintic drug, Pyrvinium pamoate, which has been previously shown to compromise cancer cell survival under glucose starvation condition, also disables cardiac fibroblast survival specifically under glucose deficient condition. Furthermore, Pyrvinium pamoate reduces scar formation and improves cardiac contractility in a mouse model of myocardial infarction. As Pyrvinium pamoate is an FDA-approved drug, our results suggest a therapeutic use of this or other related drugs to repair scarred heart and possibly other organs.     

Relationship between the induction of mitotic gene conversion and the formation of thymine glycols in yeast S. cerevisiae treated with hydrogen peroxide

The effects of hydrogen peroxide on yeast Saccharomyces cerevisiae were assessed by measuring gene conversion at the trp 5 locus and the amount of thymine glycols in DNA using a monoclonal antibody specific to this base modification.Our results show that: (a) hydrogen peroxide-induced mitotic gene conversion in yeast strain D7M1 was dose-dependent in the low dose range where no toxicity was observed; (b) in the low dose range, the frequency of gene conversion depended on the temperature of the treatment, with more conversion at 25°C than at 15°C; (c) thymine glycols were induced in DNA in a dose-dependent manner following exposure of cells to up to 400 mM hydrogen peroxide; (d) there was little difference in the amount of thymine glycols formed in DNA when treatment occurred at either 25°C or 15°C.     

Ultraviolet-Induced Reversion of cyc1 Alleles in Radiation Sensitive Strains of Yeast. II. rev2 Mutant Strains

The range of specificity of the rev2-1 mutation, an allele that reduces the frequency of ochre revertants induced by UV in Saccharomyces cerevisiae (LEMONTT 1971a), has been investigated by examining its influence on the reversion of eleven well-defined and contrasting cyc1 mutations. We have shown, in support of a suggestion of LEMONTT (1971a), that the REV2 gene product is concerned only with the reversion of ochre alleles; it plays virtually no role in the reversion of amber, missense or frameshift mutations. We have also shown that its effect is specific and confined to only some highly revertible ochre alleles. The REV2 gene product appears to enhance reversion at these sites by facilitating the conversion of two otherwise nonmutagenic photo-products into a single premutational lesion. UV-induced killing of rev2-1 strains was found to be significantly greater on fermentable rather than on nonfermentable media.     

Genotoxicity of diphenyl diselenide in bacteria and yeast

Diphenyl diselenide (DPDS) is an electrophilic reagent used in the synthesis of a variety of pharmacologically active organic selenium compounds. This may increase the risk of human exposure to the chemical at the workplace. We have determined its mutagenic potential in the Salmonella/microsome assay and used the yeast Saccharomyces cerevisiae to assay for putative genotoxicity, recombinogenicity and to determine whether DNA damage produced by DPDS is repairable. Only in exponentially growing cultures was DPDS able to induce frameshift mutations in S. typhimurium and haploid yeast and to increase crossing over and gene conversion frequencies in diploid strains of S. cerevisiae. Thus, DPDS presents a behavior similar to that of an intercalating agent. Mutants defective in excision-resynthesis repair (rad3, rad1), in error-prone repair (rad6) and in recombinational repair (rad52) showed higher than WT-sensitivity to DPDS. It appears that this compound is capable of inducing single and/or double strand breaks in DNA. An epistatic interaction was shown between rad3-e5 and rad52-1 mutant alleles, indicating that excision-resynthesis and strand-break repair may possess common steps in the repair of DNA damage induced by DPDS. DPDS was able to enhance the mutagenesis induced by oxidative mutagens in bacteria. N-acetylcysteine, a glutathione biosynthesis precursor, prevented mutagenesis induced by DPDS in yeast. We have shown that DPDS is a weak mutagen which probably generates DNA strand breaks through both its intercalating action and pro-oxidant effect.     

Removal of Frameshift Intermediates by Mismatch Repair Proteins in Saccharomyces cerevisiae

Frameshift mutations occur when the coding region of a gene is altered by addition or deletion of a number of base pairs that is not a multiple of three. The occurrence of a deletion versus an insertion type of frameshift depends on the nature of the transient intermediate structure formed during DNA synthesis. Extrahelical bases on the template strand give rise to deletions, whereas extrahelical bases on the strand being synthesized produce insertions. We previously used reversion of a +1 frameshift mutation to analyze the role of the mismatch repair (MMR) machinery in correcting -1 frameshift intermediates within a defined region of the yeast LYS2 gene. In this study, we have used reversion of a -1 frameshift mutation within the same region of LYS2 to analyze the role of the MMR machinery in the correction of frameshift intermediates that give rise to insertion events. We found that insertion and deletion events occur at similar rates but that the reversion spectra are very different in both the wild-type and MMR-defective backgrounds. In addition, analysis of the +1 spectra revealed novel roles for Msh3p and Msh6p in removing specific types of frameshift intermediates.     

Genotoxicity of selenite in diploid yeast

Selenite, a chemical of industrial importance and also an antimutagenic/anticarcinogenic agent, was tested for mutagenic and recombinogenic effects in 2 diploid yeast strains, Saccharomyces cerevisiae BZ 34 and D7. Selenite induced gene conversion and toxicity in BZ 34 and a variety of genetic events, viz. back-mutation, gene conversion, mitotic crossing-over, aberrant colony formation and also toxicity in the D7 strain. In both strains, the genetic effects of selenite showed a peak and a decline during 5 h of treatment while its toxicity increased marginally during 1-5 h. In the BZ 34 strain, the presence of glutathione (GSH) during selenite treatment greatly enhanced the convertogenic and toxic effects of selenite.     

Genetic effects of 5-azacytidine in Saccharomyces cerevisiae

The base analog 5-azacytidine induced a variety of genetic and epigenetic effects in different organisms. It was tested in two diploid strains of the yeast Saccharomyces cerevisiae to study the induction of point mutation, mitotic reciprocal crossing-over, mitotic gene conversion (strain D7) and mitotic aneuploidy (strain D61.M). It was used on cells growing in its presence for 4-5 generations. There was a strong induction of both types of mitotic recombination and point mutation. However, there was no induction of mitotic chromosomal malsegregation under the same conditions.     

Decreased chemical mutagenesis in cdc8, a DNA replication mutant of Saccharomyces cerevisiae.

In the DNA replication mutant of yeast cdc8 the frequency of chemically induced reversion of lys2-1 and hom2-1 was found to be reduced. Mutation induced by ethyl methanesulfonate (EMS) were greatly diminished in the strain homozygous for the cdc8-1 gene.     

The RBE of neutrons for induced mitotic gene conversion in "error-prone repair" defective yeast

Mitotic gene conversion was induced in the diploid yeast strain D7.rad6 which lacks "error-prone repair" and thus does not mutate. Neutrons (14.5 MeV), 60Co gamma rays, and 150 kVp X rays delivered under oxic or anoxic conditions were compared for their ability to induce gene conversion. Doses were chosen to minimize cell killing. A lack of induced mutation in this strain at the ilv1-92 allele was confirmed. Gene conversion of the trp5-27/trp5-12 alleles was induced with a linear dose response, and the yield of convertants per gray was significantly enhanced over yields reported previously for a wild-type stain. The relative biological effectiveness (RBE) of neutrons relative to low-LET radiations was found to be about 2.2 for either oxic or anoxic radiation in contrast to wild-type where the oxic RBE was 1.7 and the anoxic RBE 2.7. Absence of the rad6 function was therefore associated with an altered RBE for the conversional end point. The oxygen enhancement ratio (OER) for gene conversion was found to be about 1.7 for all radiations in contrast to the wild type where the OER for neutrons was 1.7, but for low-LET radiations it was 2.7. As repair of ionizing damage in the rad6 strain did not lead to mutation, owing to the loss of "error-prone repair," the changes in yield, RBE, and OER were consistent with the hypothesis that some of the lesions processed by wild type to generate mutations could, in the rad6 strain, lead instead to gene conversion.     

Frameshift mutagenesis in Escherichia coli by reversible DNA intercalators: sequence specificity

The mutagenic potency of the simple reversible intercalators isopropyl-OPC (iPr-OPC) and 9-aminoacridine (9-AA) is assessed in E. coli using reversion assays based on plasmids derived from pBR322 carrying various frameshift mutations within the tetracycline resistance gene in repetitive sequences: +/- 2 frameshift mutations within alternating GC sequences; +/- 1 frameshift mutation at runs of guanines. The results obtained show that iPr-OPC and 9-AA have a sequence specificity for mutagenesis: they revert +1 and -1 frameshift mutations within runs of monotonous G:C base pairs. The precise determination of the size of a small restriction fragment which contains the mutation allowed us to demonstrate that reversion occurred by -1 deletions for the +1 frameshift mutations and by +1 additions for the -1 frameshift mutations. The possible relations of this specific reversion with the base sequence specificity of the mutagenesis are briefly discussed.     

Multiple correcting COL17A1 mutations in patients with revertant mosaicism of epidermolysis bullosa

Revertant mosaicism by somatic reversion of inherited mutations has been described for a number of genetic diseases. Several mechanisms can underlie this reversion process, such as gene conversion, crossing-over, true back mutation, and second-site mutation. Here, we report the occurrence of multiple corrections in two unrelated probands with revertant mosaicism of non-Herlitz junctional epidermolysis bullosa, an autosomal recessive genodermatosis due to mutations in the COL17A1 gene. Immunofluorescence microscopy and laser dissection microscopy, followed by DNA and RNA analysis, were performed on skin biopsy specimens. In patient 1, a true back mutation, 3781T-->C, was identified in the specimen from the arm, and a second-site mutation, 4463-1G-->A, which compensated for the frameshift caused by the inherited 4424-5insC mutation, was identified in the 3' splice site of exon 55 in a specimen from the middle finger. Patient 2 showed--besides two distinct gene conversion events in specimens from the arm and hand sites, both of which corrected the 1706delA mutation--a second-site mutation (3782G-->C) in an ankle specimen, which prevented the premature ending of the protein by the 3781C-->T nonsense mutation (R1226X). Thus, both inherited mutations, paternal as well as maternal, reverted at least once by different reversion events in distinct cell clusters in the described patients. The occurrence of multiple correcting mutations within the same patient indicates that in vivo reversion is less unusual than was generally thought. Furthermore, in the male patient, mosaic patterns of type XVII collagen-positive keratinocytes were present in clinically unaffected and affected skin. This latter observation makes it likely that reversion may be overlooked and may happen more often than expected.     

The paromomycin resistance mutation (parr-454) in the 15 S rRNA gene of the yeast Saccharomyces cerevisiae is involved in ribosomal frameshifting

Summary The leaky expression of the yeast mitochondrial geneoxi1, containing a frameshift mutation (+1), is caused by natural frameshift suppression, as shown previously (Fox and Weiss-Brummer 1980). A drastic decrease in the natural level of frameshifting is found in the presence of thepar ^r-454 mutation, localized at the 3′ end of the 15 S rRNA gene. This mutation causes resistance to the antibiotic paronomycin in the yeast strains D273-10B and KL14-4A (Li et al. 1982; Tabak et al. 1982). The results of this study imply that in the yeast strain 777-3A this mutation alone is sufficient for restriction of the level of natural frameshifting but is insufficient to confer resistance to paromomycin. A second mutation, arising spontaneously with a frequency of 10⁻⁴ leads, in combination with thepar ^r-454 mutation, to full paromomycin resistance in strain 777-3A.