Andrea Sevcovicova

Assessment of Antioxidative, Chelating, and DNA-Protective Effects of Selected Essential Oil Components (Eugenol, Carvacrol, Thymol, Borneol, Eucalyptol) of Plants and Intact Rosmarinus officinalis Oil

Selected components of plant essential oils and intact Rosmarinus officinalis oil (RO) were investigated for their antioxidant, iron-chelating, and DNA-protective effects. Antioxidant activities were assessed using four different techniques. DNA-protective effects on human hepatoma HepG2 cells and plasmid DNA were evaluated with the help of the comet assay and the DNA topology test, respectively. It was observed that whereas eugenol, carvacrol, and thymol showed high antioxidative effectiveness in all assays used, RO manifested only antiradical effect and borneol and eucalyptol did not express antioxidant activity at all. DNA-protective ability against hydrogen peroxide (H2O2)-induced DNA lesions was manifested by two antioxidants (carvacrol and thymol) and two compounds that do not show antioxidant effects (RO and borneol). Borneol was able to preserve not only DNA of HepG2 cells but also plasmid DNA against Fe(2+)-induced damage. This paper evaluates the results in the light of experiences of other scientists.

Borneol administration protects primary rat hepatocytes against exogenous oxidative DNA damage

Experimental evidences suggest that most essential oils possess a wide range of biological and pharmacological activities that may protect tissues against oxidative damage. In this study, we investigated DNA-protective effect of borneol, a component of many essential oils, against oxidative DNA damage induced in primary cultures of rat hepatocytes. Borneol was added to drinking water of Sprague-Dawley rats and DNA resistance against oxidative agents was compared in hepatocytes originated from control and borneol-treated rats. Oxidative stress induced by visible light-excited methylene blue (MB/VL) or 2,3-dimethoxy-1,4-naphthoquionone (DMNQ) resulted in increased levels of DNA lesions measured by the modified single cell gel electrophoresis. Borneol (17 or 34 mg/kg body weight) added to drinking water of rats for 7 days reduced the level of oxidative DNA lesions induced in their hepatocytes by MB/VL or DMNQ. To explain the increased resistance of DNA towards oxidative stress, we measured the base-excision repair (BER) capacity in liver cell extracts of control and borneol-supplemented rats on DNA substrate of HepG2 cells containing oxidative damage. Our results showed that administration of borneol in drinking water had no effect on incision activity of hepatocytes isolated from supplemented rats. The spectrophotometric assessment of enzymatic antioxidants superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities and the flow cytometric assessment of total intracellular glutathione (iGSH) in primary hepatocytes of borneol-supplemented rats showed no changes in SOD and GPx activities but higher iGSH content particularly in hepatocytes of higher borneol dose (34 mg/kg) supplemented rats in comparison to control animals. Despite the fact that borneol had no effect either on BER of oxidative DNA damage or on the levels of antioxidant enzymes and manifested no reducing power and radicals scavenging activity, it increased significantly the level of non-enzymatic antioxidant iGSH which could reduce the oxidative DNA lesions induced by MB/VL or DMNQ.

Chlamydomonas reinhardtii: a convenient model system for the study of DNA repair in photoautotrophic eukaryotes

The green alga Chlamydomonas reinhardtii is a convenient model organism for the study of basic biological processes, including DNA repair investigations. This review is focused on the studies of DNA repair pathways in C. reinhardtii. Emphasis is given to the connection of DNA repair with other cellular functions, namely the regulation of the cell cycle. Comparison with the results of repair investigations that are already available revealed the presence of all basic repair pathways in C. reinhardtii as well as special features characteristic of this alga. Among others, the involvement of UVSE1 gene in recombinational repair and uniparental inheritance of chloroplast genome, the specific role of TRXH1 gene in strand break repair, the requirement of PHR1 gene for full activity of PHR2 gene, or encoding of two excision repair proteins by the single REX1 gene. Contrary to yeast, mammals and higher plants, C. reinhardtii does not appear to contain the ortholog of RAD6 gene, which plays an important role in DNA translesion synthesis and mutagenesis. Completed genome sequences will be a basis for molecular analyses allowing to explain the differences that have been observed in DNA repair of this alga in comparison with other model organisms.

Extract from Armoracia rusticana and Its Flavonoid Components Protect Human Lymphocytes against Oxidative Damage Induced by Hydrogen Peroxide

DNA damage prevention is an important mechanism involved in cancer prevention by dietary compounds. Armoracia rusticana is cultivated mainly for its roots that are used in the human diet as a pungent spice. The roots represent rich sources of biologically active phytocompounds, which are beneficial for humans. In this study we investigated the modulation of H2O2 genotoxicity using the A. rusticana root aqueous extract (AE) and two flavonoids (kaempferol or quercetin). Human lymphocytes pre-treated with AE, kaempferol and quercetin were challenged with H2O2 and the DNA damage was assessed by the comet assay. At first we assessed a non-genotoxic concentration of AE and flavonoids, respectively. In lymphocytes challenged with H2O2 we proved that the 0.0025 mg·mL−1 concentration of AE protected human DNA. It significantly reduced H2O2-induced oxidative damage (from 78% to 35.75%). Similarly, a non-genotoxic concentration of kaempferol (5 μg·mL−1) significantly diminished oxidative DNA damage (from 83.3% to 19.4%), and the same concentration of quercetin also reduced the genotoxic effect of H2O2 (from 83.3% to 16.2%). We conclude that AE, kaempferol and quercetin probably act as antimutagens. The molecular mechanisms underlying their antimutagenic activity might be explained by their antioxidant properties.

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

Analysis of some barley chlorophyll mutants and their response to temperature stress

Six barley chlorophyll (Chl) mutants, viridis, flavoviridis, chlorina, xanhta, lutea, and albina, differed in the contents of Chl (a+b) and carotenoids (Cars). In accordance with their Chl-deficient phenotype, the Chl a and b and Car contents of mutants decreased from viridis to albina, only xantha had the same or even higher concentration of Cars as the wild type plant. The albina mutant completely lacked and xantha had a significantly reduced photosynthetic activity. We found quantitative differences in protein contents between wild type and mutant plants, with the lowest concentration per fresh mass in the albina mutant. Chl fluorescence analysis revealed that heat-treated barley leaves of both the wild type and Chl mutants had a lower photosystem 2 efficiency than the untreated ones. With 35S-methionine labelling and SDS-PAGE we found that six to nine de novo synthetized proteins appeared after heat shock (2 h, 42 °C) in the wild type and Chl mutants. In albina the expression of heat shock proteins (HSPs) was reduced to 50 % of that in the wild type. Hence mainly albina mutants, with a completely destroyed proteosynthetic apparatus of the chloroplasts, are able to synthesize a small set of HSPs. The albina mutant is a very useful tool for the study of different gene expression of chloroplast and nuclear DNA.

DNA-protective activities of hyperforin and aristoforin

The aim of this study was to explain the molecular mechanisms of action of hyperforin, a phluoroglucinol derivative found in Hypericum perforatum L. and its more stable derivative aristoforin. DNA-topology assay revealed partial DNA-protective activities of hyperforin and aristoforin against Fe(2+)-induced DNA breaks. In order to assess molecular mechanisms underlying DNA-protective activity, the potential antioxidant activity of hyperforin and aristoforin was investigated using DPPH and OH scavenging assays, reducing power assay and Fe(2+)-chelating assay. We also studied interaction of hyperforin and aristoforin with DNA using established protocols for fluorescence titration. The ability of the studied compounds to relax topoisomerase I with electrophoretic techniques was investigated. The reduction in the fluorescence of hyperforin indicated an interaction between hyperforin and DNA with a binding constant of 0.2×10(8)M(-1). We suggest that a mechanism of hyperforin/aristoforin DNA-protective abilities is based on free radicals (mainly OH) scavenging activity.

A short overview of chlorophyll biosynthesis in algae

Chlorophylls are the most abundant classes of natural pigments and their biosynthesis is therefore a major metabolic activity in the ecosphere. Two pathways exist for chlorophyll biosynthesis, one taking place in darkness and the other requiring continuous light as a precondition. The key process for Chl synthesis is the reduction of protochlorophyllide (Pchlide). This enzymatic reaction is catalysed by two different enzymes — DPOR (dark-operative Pchlide oxidoreductase) or the structurally distinct LPOR (light-dependent Pchlide oxidoreductase). DPOR which consists of three subunits encoded by three plastid genes in eukaryotes was subject of our study. A short overview of our present knowledge of chlorophyll biosynthesis in Chlamydomonas reinhardtii in comparison with other plants is presented.

Extract of Lillium candidum L. Can Modulate the Genotoxicity of the Antibiotic Zeocin

Lilium candidum L. extract (LE) is well known in folk medicine for the treatment of burns, ulcers, inflammations and for healing wounds. This work aims to clarify whether the genotoxic potential of the radiomimetic antibiotic zeocin (Zeo) could be modulated by LE. Our results indicate that LE exerts no cytotoxic, DNA-damaging and clastogenic activity in in Chlamydomonas reinhardtii, Pisum sativum L. and Hordeum vulgare L. test systems over a broad concentration range. Weak but statistically significant clastogenic effects due to the induction of micronuclei and chromosome aberrations have been observed in H. vulgare L. after treatment with 200 and 300 μg/mL LE. To discriminate protective from adverse action of LE different experimental designs have been used. Our results demonstrate that the treatment with mixtures of LE and Zeo causes an increase in the level of DNA damage, micronuclei and “metaphases with chromatid aberrations” (MwA). Clear evidence has been also obtained indicating that pretreatment with LE given 4 h before the treatment with Zeo accelerates the rejoining kinetics of Zeo-induced DNA damage in P. sativum L. and C. reinhardtii, and can decrease clastogenic effect of Zeo measured as frequencies of micronuclei and MwA in H. vulgare L. Here, we show for the first time that LE can modulate the genotoxic effects of zeocin. The molecular mode of action strongly depends on the experimental design and varies from synergistic to protective effect (adaptive response–AR). Our results also revealed that LE-induced AR to zeocin involves up-regulation of DSB rejoining in C. reinhardtii and P. sativum L. cells.

CDKA and CDKB kinases from Chlamydomonas reinhardtii are able to complement cdc28 temperature-sensitive mutants of Saccharomyces cerevisiae

Summary.Cyclin-dependent kinases (CDK) play a key role in coordinating cell division in all eukaryotes. We investigated the capability of cyclin-dependent kinases CDKA and CDKB from the green alga Chlamydomonas reinhardtii to complement a Saccharomyces cerevisiae cdc28 temperature-sensitive mutant. The full-length coding regions of algal CDKA and CDKB cDNA were amplified by RT-PCR and cloned into the yeast expression vector pYES-DEST52, yielding pYD52-CDKA and pYD52-CDKB. The S. cerevisiae cdc28-1N strain transformed with these constructs exhibited growth at 36 °C in inducing (galactose) medium, but not in repressing (glucose) medium. Microscopic observation showed that the complemented cells had the irregular cylindrical shape typical for G2 phase-arrested cells when grown on glucose at 36 °C, but appeared as normal budded cells when grown on galactose at 36 °C. Sequence analysis and complementation tests proved that both CDKA and CDKB are functional CDC28/cdc2 homologs in C. reinhardtii. The complementation of the mitotic phenotype of the S. cerevisiae cdc28-1N mutant suggests a mitotic role for both of the kinases.