Protective effects of solvent extracts from Taiwanese Agrocybe cylindracea strain B against DNA damage induced by environmental mutagens

Antioxidant activity of a water extract from Agrocybe cylindracea strain B (ACB) against iron‐mediated lipid peroxidation has been demonstrated. In addition, the protective effect of water extracts from ACB (WAC) on hydroxyl radical‐mediated DNA strand breaks was better than that of the shiitake mushroom (Lentinula edodes). Therefore, we decided to investigate whether different solvent extracts from ACB (ACES) protect DNA against oxidative stress induced by environmental mutagens, such as cooking oil fumes (COF) and benzo[a]pyrene (BaP). Oxidative DNA damage and intercellular DNA migration (tail length) were quantified by determining the decrease of extracellular supercoiled (SC) plasmid DNA and by using the ‘comet assay’ in the human adenocarcinoma CL‐3 cell line, respectively. IC₅₀ values of water, boiled water, methanol and acetone extracts from ACB were 108.97, 87.21, 970.52 and 1005.87 µg mL^?1, respectively, for the decrease in cupric/COF‐mediated SC plasmid DNA damage. The boiled water extract has the best protective effect. The ethyl acetate and ether extracts did not inhibit plasmid DNA damage. By using the comet assay, IC₅₀ values of the ether, methanol and acetone extracts from ACB were 672.95, 64.34, and 397.77 µg mL^?1, respectively, for the decrease in COF‐mediated DNA migration in CL‐3 cells. The methanol extract had the best protective effect. However, water, boiled water and ethyl acetate extracts from ACB showed no protective effect on COF‐mediated DNA migration. These results indicate that the protective capacity of ACES on DNA damage induced by environmental mutagens is different in pUC18 plasmid DNA and CL‐3 cell DNA. Copyright © 2006 Society of Chemical Industry     

Single‐cell gel electrophoresis: a tool for investigation of DNA protection or damage mediated by dietary antioxidants

One method of assessing DNA damage is the comet assay, which was developed in 1988. The comet assay enables the detection of DNA strand breaks in individual cells. This test has also been used to study the in vitro and in vivo genotoxic or genoprotective effects of certain agents such as dietary antioxidants. This paper aims to consolidate the antioxidant and pro‐oxidant effects of a series of dietary agents which have been evaluated by comet assay. Copyright © 2007 Society of Chemical Industry     

A new method to efficiently induce a site-specific double-strand break in the fission yeast Schizosaccharomyces pombe

Double‐strand DNA breaks are a serious threat to cellular viability and yeast systems have proved invaluable in helping to understand how these potentially toxic lesions are sensed and repaired. An important method to study the processing of DNA breaks in the budding yeast Saccharomyces cerevisiae is to introduce a unique double‐strand break into the genome by regulating the expression of the site‐specific HO endonuclease with a galactose inducible promoter. Variations of the HO site‐specific DSB assay have been adapted to many organisms, but the methodology has seen only limited use in the fission yeast Schizosaccharomyces pombe because of the lack of a promoter capable of inducing endonuclease expression on a relatively short time scale (~1 h). We have overcome this limitation by developing a new assay in which expression of the homing endonuclease I‐PpoI is tightly regulated with a tetracycline‐inducible promoter. We show that induction of the I‐PpoI endonuclease produces rapid cutting of a defined cleavage site (> 80% after 1 h), efficient cell cycle arrest and significant accumulation of the checkpoint protein Crb2 at break‐adjacent regions in a manner that is analogous to published findings with DSBs produced by an acute exposure to ionizing irradiation. This assay provides an important new tool for the fission yeast community and, because many aspects of mammalian chromatin organization have been well‐conserved in Sz. pombe but not in S. cerevisiae, also offers an attractive system to decipher the role of chromatin structure in modulating the repair of double‐stranded DNA breaks. Copyright © 2012 John Wiley & Sons, Ltd.     

Effects of ethanol extracts from stalked sea squirt (Styela clava) on antioxidant potential,oxidative DNA damage and DNA repair

The aim of this study was to assess the total radical trapping antioxidant potential and antigenotoxic effects by comet assay of ethanol extracts of stalked sea squirt, Styela clava, (tunic, substrate, and whole). All extracts of stalked sea squirt effectively scavenged ABTS^{· +} in a dose dependent manner. Pretreatment with each extract of stalked sea squirt produced significant reductions in oxidative DNA damage at concentrations of 1–50 μg/mL, with whole extract of stalked sea squirt showing higher inhibition (16.1 μg/mL) of H₂O₂ induced DNA damage than substrate or tunic extracts based on ED₅₀ values. The addition of 50 μg/mL of stalked sea squirt extracts to human leukocytes after oxidative stimulus (200 μM H₂O₂) for 5 min positively influences the kinetics of DNA repair during 24 hr of incubation. These results indicate that the ethanol extracts of tunic, substrate, and whole stalked sea squirt have significant antioxidant activities that protect against oxidative DNA damage and improve DNA repair capacity.     

SYNERGISM OF CRANBERRY PHENOLICS WITH ELLAGIC ACID AND ROSMARINIC ACID FOR ANTIMUTAGENIC AND DNA PROTECTION FUNCTIONS

Several studies have shown the antimutagenic DNA protective functions of some naturally occurring phenolic phytochemicals. Emerging research also indicates that synergistic functionality of these phytochemicals in whole foods benefits the management of many diseases. Here we have investigated the potential antimutagenic properties of cranberry phenolics, ellagic acid (EA), rosmarinic acid (RA) and their synergistic interactions on enhancing antimutagenic properties in Salmonella typhimurium tester system against mutagens sodium azide and N‐methyl‐N′‐nitro‐N‐nitrosoguanidine. Ability of these phytochemical treatments to protect oxidative damage to DNA was also investigated using the supercoiled DNA strand scission assay. Results showed that EA was most effective in inhibiting the mutations in S. typhimurium system, whereas RA and EA were equally effective in protecting the DNA from oxidative damage. Results also showed that the antimutagenic functionality of cranberry powder (CP) made from juice extracts was significantly enhanced when 30% (w/w) of phenolics in CP was substituted with RA and EA possibly because of synergistic redox modulation that can influence mutagen function. It is also suggested that the synergistic mixture of cranberry phenolics with RA could also be protecting the cell from mutations by modulating DNA repair systems.     

Genome‐wide map of Apn1 binding sites under oxidative stress in Saccharomyces cerevisiae

The DNA is cells is continuously exposed to reactive oxygen species resulting in toxic and mutagenic DNA damage. Although the repair of oxidative DNA damage occurs primarily through the base excision repair (BER) pathway, the nucleotide excision repair (NER) pathway processes some of the same lesions. In addition, damage tolerance mechanisms, such as recombination and translesion synthesis, enable cells to tolerate oxidative DNA damage, especially when BER and NER capacities are exceeded. Thus, disruption of BER alone or disruption of BER and NER in Saccharomyces cerevisiae leads to increased mutations as well as large‐scale genomic rearrangements. Previous studies demonstrated that a particular region of chromosome II is susceptible to chronic oxidative stress‐induced chromosomal rearrangements, suggesting the existence of DNA damage and/or DNA repair hotspots. Here we investigated the relationship between oxidative damage and genomic instability utilizing chromatin immunoprecipitation combined with DNA microarray technology to profile DNA repair sites along yeast chromosomes under different oxidative stress conditions. We targeted the major yeast AP endonuclease Apn1 as a representative BER protein. Our results indicate that Apn1 target sequences are enriched for cytosine and guanine nucleotides. We predict that BER protects these sites in the genome because guanines and cytosines are thought to be especially susceptible to oxidative attack, thereby preventing large‐scale genome destabilization from chronic accumulation of DNA damage. Information from our studies should provide insight into how regional deployment of oxidative DNA damage management systems along chromosomes protects against large‐scale rearrangements. Copyright © 2017 John Wiley & Sons, Ltd.     

A high antioxidant level in edible plants is associated with genotoxic properties

Aqueous extracts from 20 Malaysian edible plants were screened for total phenolic content (TPC), antioxidant activity and genotoxic effects on freshly isolated human lymphocytes. Ferric reducing antioxidant power (FRAP) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assays were performed to determine the antioxidant activity of the extracts. Single-cell gel electrophoresis (SCGE) or comet assay was carried out to determine the level of DNA damage in human lymphocytes. Of the 20 plant aqueous extracts tested, two exerted more than 50% DNA strand breaks (severe damage), nine exerted 25–50% strand breaks (moderate damage) and nine exerted <25% strand breaks (mild damage). Strong positive correlations between the extent of DNA damage and FRAP level (r = 0.816), DNA damage and TPC (r = 0.830) and DNA damage and DPPH radical scavenging activities (r = 0.859) were observed. It is evident from this study that plants rich in antioxidants have greater genotoxic effect.     

枸杞蜜的抗氧化活性及其对DNA氧化损伤的保护作用

以青海枸杞蜜为研究对象,在测定其总酚、总黄酮含量和抗氧化活性基础上,采用彗星电泳技术,研究了枸杞蜜对羟基自由基诱导的小鼠淋巴细胞DNA氧化损伤的保护作用。结果表明,枸杞蜜不仅能够有效地清除自由基,而且具有较强的抗氧化活性,枸杞蜜的抗氧化活性与其总酚含量有关(p<0.05),Fe²⁺络合力与其总黄酮含量相关(p<0.01)。彗星电泳结果表明,枸杞蜜能够显著降低羟基自由基诱导的小鼠淋巴细胞DNA的氧化损伤,与模型组相比,加入枸杞蜜的保护组彗星尾部DNA比例、尾矩(TM)和Olive尾矩(OTM)显著降低(p<0.05),且与枸杞蜜浓度存在明显的剂量-效应关系,线性回归方程决定系数依次为:R²=0.9775,R²=0.9341,R²=0.9425。本文的研究结果将为枸杞蜜功能性食品的开发利用提供依据。     

Genoprotective Effects of Essential Oil Compounds Against Oxidative and Methylated DNA Damage in Human Colon Cancer Cells

Essential oils (EO) are widely used in foods as flavoring and preservative agents. Many of the biological activities of EO have been attributed to major essential oil compounds (EOC) but their direct interaction with colonic epithelial cells and their genotoxic and genoprotective effects are not well established. In this study, the cytotoxicity and genotoxicity of EOC including nerolidol, thymol, geraniol, methylisoeugenol, eugenol, linalool, and a commercial blend (Agolin) were determined. Furthermore, the genoprotective effects of EOC against oxidative and methylating damage were assessed using the comet assay in HT‐29 colorectal adenocarcinoma cells. The majority of EOC were cytotoxic to HT‐29 cells at or above 250 ppm after 24 hr exposure. At noncytotoxic doses, none of the EOC was genotoxic in the comet assay. Genoprotection against oxidative DNA damage was observed for nerolidol (at 62.5 ppm), thymol (at 12.5 ppm), geraniol, and methylisoeugenol (both at 125 ppm), as well as linalool and Agolin (both at 250 ppm). Thymol was the most protective compound against oxidative DNA damage and geraniol (at 125 ppm) also protected cells against methylating DNA damage. This study highlights the potential of EOC such as thymol to protect the colonic epithelium against oxidative DNA damage and geraniol against methylating DNA damage. Further in vivo studies are needed to confirm these findings for safety and efficacy to exploit their potential pharmaceutical or nutraceutical uses for colonic health.     

Improved gap repair cloning in yeast: treatment of the gapped vector with Taq DNA polymerase avoids vector self‐ligation

Gap repair is a fast and efficient method for assembling recombinant DNA molecules in Saccharomyces cerevisiae. This method produces a circular DNA molecule by homologous recombination between two or more linear DNA fragments, one of which is typically a vector carrying replicative sequences and a selective marker. This technique avoids laborious and costly in vitro purification and ligation of DNA. The DNA repair machinery can also close and ligate the linear vector by mechanisms other than homologous recombination, resulting in an empty vector. The frequency of these unwanted events can be lowered by removing the 5′‐phosphate groups using phosphatase, which is the standard method used for in vitro ligation. However, phosphatase treatment is less effective for gap repair cloning than for in vitro ligation, presumably due to the ability of the S. cerevisiae DNA repair machinery to efficiently repair the missing phosphate group to allow religation. We have developed a more efficient method to prevent vector religation, based on treatment of the vector fragment with Taq DNA polymerase and dATP. This procedure prevents vector recircularization almost completely, facilitating the screening for true recombinant clones. Copyright © 2012 John Wiley & Sons, Ltd.     

Transformation-associated recombination between diverged and homologous DNA repeats is induced by strand breaks

Rearrangements within plasmid DNA are commonly observed during transformation of eukaryotic cells. One possible cause of rearrangements may be recombination between repeated sequences induced by some lesions in the plasmid. We have examined the mechanisms of transformation-associated recombination in the yeast Saccharomyces cerevisiae using a plasmid system which allowed the effects of physical state and/or extent of homology on recombination to be studied. The plasmids contain homologous or diverged (19%) repeats of the URA3 genes (from S. cerevisiae or S. carlsbergensis) separated by the genetically detectable ADE2 colour marker. Recombination during transformation for covalently closed circular plasmids was over 100-fold more frequent than during mitotic growth. The frequency of recombination is partly dependent on the method of transformation in that procedures involving lithium acetate or spheroplasting yield higher frequencies than electroporation. When present in the repeats, unique single-strand breaks that are ligatable, as well as double-strand breaks, lead to high levels of recombination between diverged and identical repeats. The transformation-associated recombination between repeat DNAs is under the influence of the RAD52 and RAD1 genes.     

Tryptophan biosynthesis is important for resistance to replicative stress in Saccharomyces cerevisiae

Acute tryptophan depletion is used to induce low levels of serotonin in the brain. This method has been widely used in psychiatric studies to evaluate the effect of low levels of serotonin, and is generally considered a safe and reversible procedure. Here we use the budding yeast Saccharomyces cerevisiae to study the effects of tryptophan depletion on growth rate upon exposure to DNA‐damaging agents. Surprisingly, we found that budding yeast undergoing tryptophan depletion were more sensitive to DNA‐damaging agents such as methyl methanesulphonate (MMS) and hydroxyurea (HU). We found that this defect was independent of several DNA repair pathways, such as homologous recombination, base excision repair and translesion synthesis, and that this damage sensitivity was not due to impaired S‐phase signalling. Upon further analysis, we found that the DNA‐damage sensitivity of tryptophan depletion was likely due to impaired protein synthesis. These studies describe an important source of variance in budding yeast when using tryptophan as an auxotrophic marker, particularly on studies focusing on DNA repair, and suggest that further testing of the effect of tryptophan depletion on DNA repair in mammalian cells is warranted. Copyright © 2016 John Wiley & Sons, Ltd.     

Reduction of ultraviolet light-induced DNA damage in human colon cancer cells treated with a lactoferrin-derived peptide

Treatment of Caco-2 cells with the peptide lactoferricin_4–14, results in reduction of the growth rate by prolongation of the S phase of the cell cycle. Lactoferricin_1–25 is formed in the gut by cleavage from lactoferrin and the bioactive amino acids are found within lactoferricin_4–14. Our hypothesis is that the reduction of the rate of S phase progression may result in increased DNA repair. To test this hypothesis, Caco-2 cells were subjected to UV light that caused DNA lesions and then the cells were grown in the absence or presence of 2.0 μM lactoferricin_4–14. Evaluation of DNA strand breaks using the comet assay showed that lactoferricin_4–14 treatment indeed resulted in a reduction of comets showing damaged DNA. In the search for a mechanism, we have investigated the levels of several proteins involved in cell cycle regulation, DNA replication, and apoptosis using Western blot. Lactoferricin_4–14 treatment resulted in an increased expression of flap endonuclease-1 pointing to increased DNA synthesis activity. Lactoferricin_4–14 treatment decreased the expression of the proapoptotic protein B-cell lymphoma 2-associated X protein (or Bax), indicating decreased cell death. As we have found previously, lactoferricin_4–14 treatment reduced the expression of cyclin E involved in the G₁/S transition. Immunofluorescence microscopy showed that a lower γ-H2AX expression in lactoferricin_4–14-treated cells, pointing to more efficient DNA repair. Thus, altogether our data show that lactoferricin_4–14 treatment has beneficial effects.     

Modulation of meiotic homologous recombination by DNA helicases

DNA helicases are ATP‐driven motor proteins which translocate along DNA capable of dismantling DNA‐DNA interactions and/or removing proteins bound to DNA. These biochemical capabilities make DNA helicases main regulators of crucial DNA metabolic processes, including DNA replication, DNA repair, and genetic recombination. This budding topic will focus on reviewing the function of DNA helicases important for homologous recombination during meiosis, and discuss recent advances in how these modulators of meiotic recombination are themselves regulated. The emphasis is placed on work in the two model yeasts, Saccharomyces cerevisiae and Schizosaccharomyces pombe, which has vastly expanded our understanding of meiotic homologous recombination, a process whose correct execution is instrumental for healthy gamete formation, and thus functioning sexual reproduction. Copyright © 2016 John Wiley & Sons, Ltd.     

Modulation of doxorubicin-induced genotoxicity by squalene in Balb/c mice

The present study aims to evaluate the protective effect of squalene against the genotoxicity of the chemotherapeutic agent doxorubicin (Dox) using two genotoxicity assays, the micronucleus assay and the comet assay. Different groups of mice were fed squalene at the doses of 1 and 4 mmol g(-1) body weight (100 or 400 μl as squalene oil) either at 4 h before or 1 h after Dox (20 mg kg(-1)) treatment. 24 h after the Dox treatment, bone marrow erythrocytes were evaluated for the incidence of micronuclei, and the induced DNA strand breaks were examined in heart tissue by the alkaline comet assay. As expected, Dox significantly induced micronuclei in polychromatic (immature) erythrocytes, as well as in total erythrocytes. The frequency of Dox-induced micronucleated erythrocytes was significantly reduced in the mice treated with squalene both before and after Dox administration. Squalene itself obviously did not induce any micronuclei in bone marrow erythrocytes. The comet assay also demonstrated a significant increase in DNA damage, especially DNA single strand breaks in the Dox-treated group of mice as compared to the control. The Dox-induced DNA damage was also effectively reduced by squalene when it was administered either before or after the Dox treatment. Squalene did not induce any significant DNA damage by itself. Compared to the pre-treatment of squalene, post treatment gave rise to more effective prevention against Dox-induced DNA damage. The data suggest that the complimentary use of squalene with Dox will be beneficial to reduce the adverse effect of Dox in cancer chemotherapy, such as the increased incidence of undesirable mutagenic side effects.     

DNA damage, a biomarker of carcinogenesis: its measurement and modulation by diet and environment

Free radicals and other reactive oxygen or nitrogen species are constantly generated in vivo and can cause oxidative damage to DNA. This damage has been implicated to be important in many diseases, including cancer. The assessment of damage in various biological matrices, such as tissues, cells, and urine, is vital to understanding this role and subsequently devising intervention strategies. During the last 20 years, many analytical techniques have been developed to monitor oxidative DNA base damage. High-performance liquid chromatography-electrochemical detection and gas chromatography-mass spectrometry are the two pioneering contributions to the field. Currently, the arsenal of methods available include the promising high-performance liquid chromatography-tandem mass spectrometry technique, capillary electrophoresis, 32P-postlabeling, antibody-base immunoassays, and assays involving the use of DNA repair glycosylases such as the comet assay. The objective of this review is to discuss the biological significance of oxidative DNA damage, evaluate the effectiveness of several techniques for measurement of oxidative DNA damage in various biological samples and review current research on factors (dietary and non-dietary) that influence DNA oxidative damage using these techniques.     

Bolus ingestion of white and green tea increases the concentration of several flavan‐3‐ols in plasma,but does not affect markers of oxidative stress in healthy non‐smokers

White tea (WT) is rich in flavan‐3‐ols as green tea (GT) and might provide health protective effects due to the strong antioxidant properties of flavan‐3‐ols. Since intervention studies with WT are lacking, we evaluated the effects of WT consumption on antioxidant status, antioxidant capacity and biomarkers of oxidative stress compared to water and GT. After an overnight fast, 70 healthy non‐smokers were randomized to consume 600 mL of WT, GT or water (control). Plasma (epi‐)catechin and epi(gallo)catechingallate, antioxidant capacity (Folin assay, trolox equivalent antioxidant capacity test), 8‐iso‐prostaglandin F_2α, ascorbic acid and uric acid were determined before and several times within 8 h after consumption. DNA strand breaks were measured in vivo and ex vivo (H₂O₂ stimulation) in leukocytes. Plasma flavan‐3‐ols significantly increased after WT and GT ingestion. Trolox equivalent antioxidant capacity was lower after 5 h in controls versus WT (p=0.031) and GT (p=0.005). Folin‐Ciocalteu reducing capacity, ascorbic and uric acid as well as markers of oxidative stress (8‐iso‐prostaglandin‐F_2α, DNA strand breaks) were not affected by the beverages. A short‐term increase of catechins does not change plasma antioxidant capacity in healthy subjects. Conclusions with respect to health protective effects of WT and GT on the basis of these biomarkers can, thus, not be drawn.     

Mutagenic effect of freezing on nuclear DNA of Saccharomyces cerevisiae

Although fragmentation of DNA has been observed in cells undergoing freezing procedures, a mutagenic effect of sub‐zero temperature treatment has not been proved by induction and isolation of mutants in nuclear DNA (nDNA). In this communication we supply evidence for mutagenicity of freezing on nDNA of Saccharomyces cerevisiae cells. In the absence of cryoprotectors, cooling for 2 h at +4°C and freezing for 1 h at ?10°C and 16 h at ?20°C, with a cooling rate of 3°C/min, resulted in induction of frame‐shift and reverse mutations in microsatellite and coding regions of nDNA. The sub‐zero temperature exposure also has a strong recombinogenic effect, evidenced by induction of gene‐conversion and crossing‐over events. Freezing induces mutations and enhances recombination with a frequency equal to or higher than that of methylmethanesulphonate at comparable survival rates. The signals for the appearance of nDNA lesions induced by freezing are detected and transduced by the DNA damage pathway. Extracellular cryoprotectors did not prevent the mutagenic effect of freezing, while accumulation of trehalose inside cells reduced nDNA cryodamage. Freezing of cells is accompanied by generation of high ROS levels, and the oxidative stress raised during the freeze–thaw process is the most likely reason for the DNA damaging effect. Experiments with mitochondrial rho^– mutants or scavengers of ROS indicated that mutagenic and recombinogenic effects of sub‐zero temperatures can be decreased but not eliminated by reduction of ROS level. The complete protection against cryodamage in nDNA required simultaneous usage of intracellular cryoprotector and ROS scavenger during the freeze–thaw process. Copyright © 2012 John Wiley & Sons, Ltd.