DNA susceptibility of Saccharomyces cerevisiae to Zeocin depends on the growth phase

The aim of this study was to evaluate the level of Zeocin-induced double-strand breaks (DSBs) in Saccharomyces cerevisiae cells in a different growth phase, using constant-field gel electrophoresis (CFGE). Saccharomyces cerevisiae diploid strain D7ts1 with enhanced cellular permeability was used. The effects of growth phase and treatment time were evaluated based on Zeocin-induced DSBs, measured by CFGE. Survival assay was also applied. No protoplast isolation was necessary for the detection of DSBs in strain D7ts1. Differences in the response of cells depending on the growth phase were obtained. Cells in exponential growth phase had increased DSB levels only after Zeocin treatment with concentrations equal or higher than 200 μgml−1. Increasing treatment time did not result in higher DSB levels. Oppositely, treatment of cells at the beginning of stationary phase with Zeocin concentrations resulted in more than 1.5-fold increase in DSB levels in comparison with those in untreated cells. Increased DSB levels were measured for all the treatment times. A dose-dependent decrease in cell survival was observed after Zeocin treatment with concentrations in the range of lethality LD20-LD50. A strong negative correlation was calculated between the levels of DSBs and cell survival. New information is provided concerning DNA susceptibility depending on the growth phase. DNA susceptibility is higher in cells at the beginning of stationary phase than those in exponential phase. Data presented here illustrate that the optimized by us CFGE protocol is sensitive and could be used successfully for DSB measurement in Saccharomyces cerevisiae strains with enhanced cellular permeability

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DNA susceptibility of Saccharomyces cerevisiae to Zeocin depends on the growth phase.

The aim of this study was to evaluate the level of Zeocin-induced double-strand breaks (DSBs) in Saccharomyces cerevisiae cells in a different growth phase, using constant-field gel electrophoresis (CFGE). Saccharomyces cerevisiae diploid strain D7ts1 with enhanced cellular permeability was used. The effects of growth phase and treatment time were evaluated based on Zeocin-induced DSBs, measured by CFGE. Survival assay was also applied. No protoplast isolation was necessary for the detection of DSBs in strain D7ts1. Differences in the response of cells depending on the growth phase were obtained. Cells in exponential growth phase had increased DSB levels only after Zeocin treatment with concentrations equal or higher than 200 µgml-1. Increasing treatment time did not result in higher DSB levels. Oppositely, treatment of cells at the beginning of stationary phase with Zeocin concentrations resulted in more than 1.5-fold increase in DSB levels in comparison with those in untreated cells. Increased DSB levels were measured for all the treatment times. A dose-dependent decrease in cell survival was observed after Zeocin treatment with concentrations in the range of lethality LD20-LD50. A strong negative correlation was calculated between the levels of DSBs and cell survival. New information is provided concerning DNA susceptibility depending on the growth phase. DNA susceptibility is higher in cells at the beginning of stationary phase than those in exponential phase. Data presented here illustrate that the optimized by us CFGE protocol is sensitive and could be used successfully for DSB measurement in Saccharomyces cerevisiae strains with enhanced cellular permeability.

Antioxidant, antimutagenic, and anticarcinogenic effects of Papaver rhoeas L. extract on Saccharomyces cerevisiae.

The aim of this work was to analyze the antioxidant and antimutagenic/anticarcinogenic capacity of Papaver rhoeas L. water extract against standard mutagen/carcinogen methyl methanesulfonate (MMS) and radiomimetic zeocin (Zeo) on a test system Saccharomyces cerevisiae. The following assays were used: 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity, quantitative determination of superoxide anion (antireactive oxygen species [antiROS test]), DNA topology assay, D7ts1 test--for antimutagenic--and Ty1 transposition test--for anticarcinogenic effects. Strong pro-oxidative capacity of Zeo was shown to correlate with its well-expressed mutagenic and carcinogenic properties. The mutagenic and carcinogenic effects of MMS were also confirmed. Our data concerning the antioxidant activity of P. rhoeas L. extract revealed that concentration corresponding to IC(50) in the DPPH assay possessed the highest antioxidant activity in the antiROS biological assay. It was also observed that a concentration with 50% scavenging activity expressed the most pronounced antimutagenic properties decreasing Zeo-induced gene conversion twofold, reverse mutation fivefold, and total aberrations fourfold. The same concentration possessed well-expressed anticarcinogenic properties measured as reduction of MMS-induced Ty1 transposition rate fivefold and fourfold when Zeo was used as an inductor. Based on the well-expressed antioxidant, antimutagenic, and anticarcinogenic properties obtained in this work, the P. rhoeas L. extract could be recommended for further investigations and possible use as a food additive.

Antioxidant and HSP70B responses in Chlamydomonas reinhardtii genotypes with different resistance to oxidative stress.

Today, the information from model species that differ in their resistance to oxidative stress and the determination of suitable plant markers for screening stress-resistant genotypes are essential for better understanding of plant stress responses and for selection. Here we aimed to assess the differences in antioxidant and HSP70B responses to paraquat treatment between genotypes susceptible and resistant to oxidative stress. Four genotypes of Chlamydomonas reinhardtii were chosen as a model of plant cells: two susceptible genotypes: wild type and paraquat-sensitive; and two paraquat-resistant genotypes: with high and moderate resistance. Varying responses to paraquat treatment were found depending on the genotype and paraquat concentrations. High paraquat concentrations (>50µM) were shown to be very stressful for all C. reinhardtii genotypes, leading to inhibition of enzyme activity. Only the paraquat-sensitive genotype responded to low-level paraquat treatment with a marked enhancement of SOD, CAT, GST activities. The lack of statistically significant response measured as SOD, CAT, GST activities in WT and resistant genotypes could be considered as an indication of absence of strong oxidative stress. This could relate to higher levels of endogenous SOD and CAT activities characteristic of moderately and highly paraquat-resistant genotypes. The response to lower paraquat concentrations evaluated as HSP70B accumulation was proportional to the level of genotype susceptibility to PQ. New evidence is provided that low-level oxidative stress impacts the antioxidant and HSP70B responses differently depending on the genotype resistance. In light of the still unresolved challenge for identification of reliable characters for screening of genotype resistance/susceptibility to oxidative stress, our study demonstrates that HSP70B accumulation could be used as an early marker for induced oxidative stress in the studied genotypes. The obtained results that the most pronounced differences in the antioxidant and HSP70B response were found between the two susceptible genotypes provoke us to convey future experiments with other susceptible genotypes.

Heat shock protein HSP70B as a marker for genotype resistance to environmental stress in Chlorella species from contrasting habitats.

The cellular response of three Chlorella species that differ in their temperature preferences and tolerance - Chlorella vulgaris - Antarctic, C. vulgaris strain 8/1 - thermophilic, and Chlorella kesslery - mesophilic - is analysed. Interspecies variability is found for the sensitivity of the species, with respect to the constitutive and induced levels of HSP70B. Based on their sensitivity to heat, the species were ranked as follows: C. vulgaris>C. vulgaris 8/1>C. kesslery. A higher constitutive level and the well-expressed overproduction of HSP70B estimated for C. vulgaris are most likely among the factors that allow it to survive in the extreme Antarctic environment. The overexpression of HSP70B could be characterised as a short-lived stress response as the most pronounced enhancement is measured at 2h after the stress. We also show that HSP70B may be used as a marker of thermal stress in these organisms. Our results contribute to the hypothesis of the conserved functional properties of HSP70B as a mechanism of thermotolerance in plants.

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.

Adaptive response: some underlying mechanisms and open questions: [review]

Organisms are affected by different DNA damaging agents naturally present in the environment or released as a result of human activity. Many defense mechanisms have evolved in organisms to minimize genotoxic damage. One of them is induced radioresistance or adaptive response. The adaptive response could be considered as a nonspecific phenomenon in which exposure to minimal stress could result in increased resistance to higher levels of the same or to other types of stress some hours later. A better understanding of the molecular mechanism underlying the adaptive response may lead to an improvement of cancer treatment, risk assessment and risk management strategies, radiation protection, e.g. of astronauts during long-term space flights. In this mini-review we discuss some open questions and the probable underlying mechanisms involved in adaptive response: the transcription of many genes and the activation of numerous signaling pathways that trigger cell defenses - DNA repair systems, induction of proteins synthesis, enhanced detoxification of free radicals and antioxidant production.

Genetic variability in Daphnia magna and ecotoxicological evaluation.

The fresh water crustacean Daphnia magna is widely used as a test organism in aquatic toxicology to assess the adverse effects of individual substances or complex mixtures, e.g. industrial wastewaters. Cultures are held in several European testing laboratories and testing is typically carried out according to internationally standardised protocols. However, despite accounting for many potential confounding factors these guidelines do not currently take into account any specification related to the use of a specific clone. Cultures from seven laboratories were used to assess genetic variability by random-amplified polymorphic DNA polymerase chain reaction. Results pointed out the existence of two main clone clusters Responses in the acute Daphnia immobilisation test showed no direct correlation with genetic clusters resulting from random genetic markers (random-amplified polymorphic DNA) analysis. Considering that genetic differences are the most probable cause for the ecotoxicological test data, further analysis concerning gene expression and genetic stability should be performed.