Viera Vlčková

Selenium: a double-edged sword for defense and offence in cancer

Selenium (Se) is an essential dietary component for animals including humans and is regarded as a protective agent against cancer. Although the mode of anticancer action of Se is not fully understood yet, several mechanisms, such as antioxidant protection by selenoenzymes, specific inhibition of tumor cell growth by Se metabolites, modulation of cell cycle and apoptosis, and effect on DNA repair have all been proposed. Despite the unsupported results of the last SELECT trial, the cancer-preventing activity of Se was demonstrated in majority of the epidemiological studies. Moreover, recent studies suggest that Se has a potential to be used not only in cancer prevention but also in cancer treatment where in combination with other anticancer drugs or radiation, it can increase efficacy of cancer therapy. In combating cancer cells, Se acts as pro-oxidant rather than antioxidant, inducing apoptosis through the generation of oxidative stress. Thus, the inorganic Se compound, sodium selenite (SeL), due to its prooxidant character, represents a promising alternative for cancer therapy. However, this Se compound is highly toxic compared to organic Se forms. Thus, the unregulated intake of dietary or pharmacological Se supplements mainly in the form of SeL has a potential to expose the body tissues to the toxic levels of Se with subsequent negative consequences on DNA integrity. Hence, due to a broad interest to exploit the positive effects of Se on human health and cancer therapy, studies investigating the negative effects such as toxicity and DNA damage induction resulting from high Se intake are also highly required. Here, we review a role of Se in cancer prevention and cancer therapy, as well as mechanisms underlying Se-induced toxicity and DNA injury. Since Saccharomyces cerevisiae has proven a powerful tool for addressing some important questions regarding Se biology, a part of this review is devoted to this model system.

Antimutagenic potential of homoisoflavonoids from Muscari racemosum

The potential antimutagenic effect of the plant extract of Muscari racemosum bulbs, rich on 3-benzylidene-4-chromanones, was evaluated on three genetic model organisms. The mixture of three homoisoflavonoids was applied together with diagnostic mutagens in the Ames assay on four bacterial strains Salmonella typhimurium TA97, TA98, TA100, TA102, in the toxicity and mutagenicity/antimutagenicity assay on the yeast strain Saccharomyces cerevisiae D7, and in the simultaneous phytotoxicity and clastogenicity/anticlastogenicity assay on Vicia sativa (L.). The extract exerted antimutagenic and anticlastogenic effects due to the presence of homoisoflavonoids, which may be included in the group of natural antimutagens. This genotoxicological study suggests that homoisoflavonoids from M. racemosum (L.) owing to antimutagenic and anticlastogenic properties are of great pharmacological importance, and might be beneficial for prevention of cancer.

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.

Rad52 has a role in the repair of sodium selenite-induced DNA damage in Saccharomyces cerevisiae

Selenium (Se) is a chemo-preventive agent that has been shown to have a protective role against cancer. The inorganic form of Se, sodium selenite (Na2SeO3), has frequently been included in various chemo-prevention studies, and this commercially available form of Se is used as dietary supplement by the public. Because high doses of this Se compound can be toxic, the underlying molecular mechanisms of sodium selenite toxicity need to be elucidated. Recently, we have reported that sodium selenite is acting as an oxidizing agent in the budding yeast Saccharomyces cerevisiae, producing oxidative damage to DNA. This pro-oxidative activity of sodium selenite likely accounted for the observed DNA double-strand breaks (DSB) and yeast cell death. In this study we determine the genetic factors that are responsible for repair of sodium selenite-induced DSB. We report that the Rad52 protein is indispensable for repairing sodium selenite-induced DSB, suggesting a fundamental role of homologous recombination (HR) in this repair process. These results provide the first evidence that HR may have a fundamental role in the repair of sodium selenite-induced toxic DNA lesions.

The Escherichia coli recA gene increases resistance of the yeast Saccharomyces cerevisiae to ionizing and ultraviolet radiation

SummaryThe Escherichia coli recA protein coding region was ligated into an extrachromosomally replicating yeast expression vector downstream of the yeast alcohol dehydrogenase promoter region to produce plasmid pADHrecA. Transformation of the wild-type yeast strains YNN-27 and 7799-4B, as well as the recombination-deficient rad52-t C5-6 mutant, with this shuttle plasmid resulted in the expression of the bacterial 38 kDa RecA protein in exponential phase cells. The wild-type YNN27 and 7799-4B transformants expressing the bacterial recA gene showed increased resistance to the toxic effects of both ionizing and ultraviolet radiation. RecA moderately stimulated the UV-induced mutagenic response of 7799-4B cells. Transformation of the rad52-t mutant with plasmid pADHrecA did not result in the complementation of sensitivity to ionizing radiation. Thus, the RecA protein endows the yeast cells with additional activities, which were shown to be error-prone and dependent on the RAD52 gene.

Antigenotoxic potential of glucomannan on four model test systems

Antimutagenic, anticlastogenic, and bioprotective effect of polysaccharide glucomannan (GM) isolated fromCandida utilis was evaluated in four model test systems. The antimutagenic effect of GM against 9-aminoacridine (9-AA)- and sodium azide (NaN3)-induced mutagenicity was revealed in theSalmonella typhimurium strains TA97 and TA100, respectively. GM showed anticlastogenic effect against N-nitroso-N′-methylurea (NMU) induced chromosome aberrations in theVicia sativa assay. The bioprotective effect of GM co-treated with methyl-methane-sulphonate (MMS) was also established inChlamydomonas reinhardtii repair deficient strainsuvs10 anduvs14. The statistically significant antimutagenic potential of GM was not proved against 4-nitro-quinoline-1-oxide (4-NQO)-induced mutagenicity inSaccharomyces cerevisiae D7 assay. It may be due to bioprotectivity of α-mannan and β-glucan, which are integral part ofS. cerevisiae cell walls. Due to the good water solubility, low molecular weight (30 kDa), antimutagenic/anticlastogenic, and bioprotective activity against chemical compounds differing in mode of action, GM appears to be a promising natural protective (antimutagenic) agent.

The E. coli recA gene can restore the defect in mutagenesis of the pso4-1 mutant of S. cerevisiae.

The E. coli recA gene was introduced into the pso4-1 mutant of S. cerevisiae and transformants were treated with 8-MOP+UVA and 254-nm UV light. The results showed that the recA gene increased the resistance to the toxic effect of 8-MOP+UVA and restored the frequency of reversion of the pso4-1 mutants after both treatments. The presence of the recA gene stimulated expression of the small subunit of the ribonucleotide reductase (Rnr2) in the pso4-1 mutants. Thus the E. coli recA gene is functional in yeast. Moreover, it was shown that the pso4-1 mutant is epistatic to pso1-1 and rad6-1, which belong to a mutagenic repair pathway. We propose here that the PSO4 gene has some role in the control of mutagenic repair in yeast.

Genotoxicity and antigenotoxicity evaluation of non-photoactivated hypericin.

The potential genotoxicity and antigenotoxicity of non‐photoactivated hypericin was investigated in five experimental models. Hypericin was non‐mutagenic in the Ames assay, with and without metabolic activation. It did not exert a protective effect against mutagenicity induced by 9‐aminoacridine. In a yeast (Saccharomyces cerevisiae) assay, hypericin did not increase the frequency of mitotic crossovers or total aberrants at the ade2 locus, the number of convertants at the trp5 locus, or the number of revertants at the ilv1 locus. In combined application with 4‐nitroquinoline‐1‐oxide, it significantly enhanced the number of revertants at the ilv1 locus at the highest concentration used. Hypericin was not mutagenic in the alga Chlamydomonas reinhardtii. However, in combined application with methyl methane sulfonate, toxicity and mutagenicity were slightly reduced. In a chromosome aberration assay using three mammalian cell lines, hypericin did not alter the frequency of structural chromosome aberrations, and in the DPPH radical scavenging assay, it did not exert any antioxidant effects. Copyright

Increased DNA double strand breakage is responsible for sensitivity of the pso3-1 mutant of Saccharomyces cerevisiae to hydrogen peroxide.

Escherichia coli endonuclease III (endo III) is the key repair enzyme essential for removal of oxidized pyrimidines and abasic sites. Although two homologues of endo III, Ntgl and Ntg2, were found in Saccharomyces cerevisiae, they do not significantly contribute to repair of oxidative DNA damage in vivo. This suggests that an additional activity(ies) or a regulatory pathway(s) involved in cellular response to oxidative DNA damage may exist in yeast. The pso3-1 mutant of S. cerevisiae was previously shown to be specifically sensitive to toxic effects of hydrogen peroxide (H2O2) and paraquat. Here, we show that increased DNA double strand breakage is very likely the basis of sensitivity of the pso3-1 mutant cells to H2O2. Our results, thus, indicate an involvement of the Pso3 protein in protection of yeast cells from oxidative stress presumably through its ability to prevent DNA double strand breakage. Furthermore, complementation of the repair defects of the pso3-1 mutant cells by E. coli endo III has been examined. It has been found that expression of the nth gene in the pso3-1 mutant cells recovers survival, decreases mutability and protects yeast genomic DNA from breakage following H2O2 treatment. This might suggest some degree of functional similarity between Pso3 and Nth.

Effect of bacterial recA expression on DNA repair in the rad51 and rad52 mutants of Saccharomyces cerevisiae

Molecular and functional homology between yeast proteins pRad51 and pRad52 and Escherichia coli pRecA involved in recombinational DNA repair led us to investigate possible effects of recA gene expression on DNA repair in rad51 and rad52 mutants of Saccharomyces cerevisiae. The mutant cells were subjected to one of the following treatments: preincubation with 8-methoxypsoralen and subsequent irradiation with 360-nm ultraviolet (UVA) (8-MOP + UVA), irradiation with 254-nm UV light or treatment with methyl methane sulfonate (MMS). While recA expression did not repair lethal DNA lesions in mutant rad51, it was able to partially restore resistance to 8-MOP + UVA and MMS in rad52. Expression of recA could not complement the sensitivity of rad51rad52 double mutants, indicating that pRad51 may be essential for the repair-stimulating activity of pRecA in the rad52 mutant. Spontaneous mutagenesis was increased, and 8-MOP-photoinduced mutagenesis was decreased by the presence of pRecA in rad52, whereas pRecA decreased UV-induced mutagenesis in rad51. Thus, pRecA may function in yeast DNA repair either as a member of a protein complex or as an individual protein that binds to mutagen-damaged DNA.