The dystonia gene THAP1 controls DNA double-strand break repair choice.

The Shieldin complex shields double-strand DNA breaks (DSBs) from nucleolytic resection. Curiously, the penultimate Shieldin component, SHLD1, is one of the least abundant mammalian proteins. Here, we report that the transcription factors THAP1, YY1, and HCF1 bind directly to the SHLD1 promoter, where they cooperatively maintain the low basal expression of SHLD1, thereby ensuring a proper balance between end protection and resection during DSB repair. The loss of THAP1-dependent SHLD1 expression confers cross-resistance to poly (ADP-ribose) polymerase (PARP) inhibitor and cisplatin in BRCA1-deficient cells and shorter progression-free survival in ovarian cancer patients. Moreover, the embryonic lethality and PARPi sensitivity of BRCA1-deficient mice is rescued by ablation of SHLD1. Our study uncovers a transcriptional network that directly controls DSB repair choice and suggests a potential link between DNA damage and pathogenic THAP1 mutations, found in patients with the neurodevelopmental movement disorder adult-onset torsion dystonia type 6.

The benzylisoquinoline alkaloids, berberine and coptisine, act against camptothecin-resistant topoisomerase I mutants.

DNA replication inhibitors are utilized extensively in studies of molecular biology and as chemotherapy agents in clinical settings. The inhibition of DNA replication often triggers double-stranded DNA breaks (DSBs) at stalled DNA replication sites, resulting in cytotoxicity. In East Asia, some traditional medicines are administered as anticancer drugs, although the mechanisms underlying their pharmacological effects are not entirely understood. In this study, we screened Japanese herbal medicines and identified two benzylisoquinoline alkaloids (BIAs), berberine and coptisine. These alkaloids mildly induced DSBs, and this effect was dependent on the function of topoisomerase I (Topo I) and MUS81-EME1 structure-specific endonuclease. Biochemical analysis revealed that the action of BIAs involves inhibiting the catalytic activity of Topo I rather than inducing the accumulation of the Topo I-DNA complex, which is different from the action of camptothecin (CPT). Furthermore, the results showed that BIAs can act as inhibitors of Topo I, even against CPT-resistant mutants, and that the action of these BIAs was independent of CPT. These results suggest that using a combination of BIAs and CPT might increase their efficiency in eliminating cancer cells.

Vitamin C and Cell Death.

Significance: Persistent oxidative stress is a common feature of cancer cells, giving a specific weapon to selectively eliminate them. Ascorbate in pharmacological concentration can contribute to the suspended formation of hydroxyl radical via the Fenton reaction; thus, it can be an important element of the oxidative stress therapy against cancer cells. Recent Advances: The main components of ascorbate-induced cell death are DNA double-strand breaks via the production of hydroxyl radical and ATP depletion due to the activation of poly (ADP-ribose) polymerase 1. Presumably, DNA damage can be the primary contributor to the anticancer activity of pharmacological ascorbate, as opposed to the rupture of bioenergetics. The caspase independency of high-dose ascorbate-induced cell death proposed the possible involvement of several types of cell death, such as ferroptosis, necroptosis, and autophagy. Critical Issues: Ascorbate can target at least two key molecular features of cancer cells as a part of the anticancer therapy: the intrinsic or acquired resistance to cell death and the dysregulated metabolism of cancer cells. It seems probable that different concentrations of ascorbate alter the nature of induced cell death. Autophagy and necroptosis may play a role at intermediate concentrations, but caspase-independent apoptosis may dominate at higher concentrations. However, ascorbate behaves as an effective inhibitor of ferroptosis that may have crucial importance in its possible clinical application. Future Directions: The elucidation of the details and the links between high-dose ascorbate-induced cancer selective cell death mechanisms may give us a tool to form and apply synergistic cancer therapies. Antioxid. Redox Signal. 34, 831-844.

A DNA Repair Inhibitor Isolated from an Ecuadorian Fungal Endophyte Exhibits Synthetic Lethality in PTEN-Deficient Glioblastoma.

Disruption of the tumor suppressor PTEN, either at the protein or genomic level, plays an important role in human cancer development. The high frequency of PTEN deficiency reported across several cancer subtypes positions therapeutic approaches that exploit PTEN loss-of-function with the ability to significantly impact the treatment strategies of a large patient population. Here, we report that an endophytic fungus isolated from a medicinal plant produces an inhibitor of DNA double-strand-break repair. Furthermore, the novel alkaloid product, which we have named irrepairzepine (1), demonstrated synthetic lethal targeting in PTEN-deficient glioblastoma cells. Our results uncover a new therapeutic lead for PTEN-deficient cancers and an important molecular tool toward enhancing the efficacy of current cancer treatments.

Genotoxic activity of 1,2,3-triazolyl modified furocoumarins and 2,3-dihydrofurocoumarins.

The furocoumarin backbone is a promising platform for chemical modifications aimed at creating new pharmaceutical agents. However, the high level of biological activity of furocoumarins is associated with a number of negative effects. For example, some of the naturally occurring ones and their derivatives can show genotoxic and mutagenic properties as a result of their forming crosslinks with DNA molecules. Therefore, a particularly important area for the chemical modification of natural furocoumarins is to reduce the negative aspects of their bioactivity. By studying a group of 21 compounds-1,2,3-triazolyl modified derivatives of furocoumarin and peucedanin-using the SOS chromotest, the Ames test, and DNA-comet assays, we revealed modifications that can neutralize the structure's genotoxic properties. Theoretical aspects of the interaction of the compound library were studied using molecular modeling and this identified the leading role of the polyaromatic molecular core that takes part in stacking-interactions with the pi-systems of the nitrogenous bases of DNA.

Protective Effects of Aqueous Extracts of Flos lonicerae Japonicae against Hydroquinone-Induced Toxicity in Hepatic L02 Cells.

Hydroquinone (HQ) is widely used in food stuffs and is an occupational and environmental pollutant. Although the hepatotoxicity of HQ has been demonstrated both in vitro and in vivo, the prevention of HQ-induced hepatotoxicity has yet to be elucidated. In this study, we focused on the intervention effect of aqueous extracts of Flos lonicerae Japonicae (FLJ) on HQ-induced cytotoxicity. We demonstrated that HQ reduced cell viability in a concentration-dependent manner by administering 160 µmol/L HQ for 12 h as the positive control of cytotoxicity. The aqueous FLJ extracts significantly increased cell viability and decreased LDH release, ALT, and AST in a concentration-dependent manner compared with the corresponding HQ-treated groups in hepatic L02 cells. This result indicated that aqueous FLJ extracts could protect the cytotoxicity induced by HQ. HQ increased intracellular MDA and LPO and decreased the activities of GSH, GSH-Px, and SOD in hepatic L02 cells. In addition, aqueous FLJ extracts significantly suppressed HQ-stimulated oxidative damage. Moreover, HQ promoted DNA double-strand breaks (DSBs) and the level of 8-hydroxy-2'-deoxyguanosine and apoptosis. However, aqueous FLJ extracts reversed HQ-induced DNA damage and apoptosis in a concentration-dependent manner. Overall, our results demonstrated that the toxicity of HQ was mediated by intracellular oxidative stress, which activated DNA damage and apoptosis. The findings also proved that aqueous FLJ extracts exerted protective effects against HQ-induced cytotoxicity in hepatic L02 cells.

Sinomenine hydrochloride sensitizes cervical cancer cells to ionizing radiation by impairing DNA damage response.

The use of plant­based compounds derived from traditional medicine to improve human diseases has been gaining momentum, due to their high bioavailability and moderate adverse effects. Sinomenine is one such biomonomer alkali compound derived from Sinomenium acutum and is known for its anti­inflammatory and antitumor effects. However, the molecular mechanism(s) of its antitumor properties are not fully characterized. In the present study, we evaluated the radiosensitizing effects of the water­soluble sinomenine, sinomenine hydrochloride (SH) in human cervical cancer cell line (HeLa). SH sensitized HeLa cells to ionizing radiation (IR) by promoting accumulation of IR­induced DNA double­strand breaks (DSBs) and also by interfering with DNA damage checkpoint activation. We then investigated the molecular mechanisms underlying the SH­mediated cellular sensitization to IR and found that SH inhibited the expression of DNA damage response (DDR) factors Ku80 and Rad51 at the transcription level. Finally, the radiosensitizing activity of SH was confirmed in a cervical cancer mouse xenograft model. The combinatorial treatment of SH and IR significantly slowed the tumor growth rate compared with IR alone. Collectively, our study not only provides molecular insights into the novel role of SH in cellular response to IR, but also suggests a therapeutic potential of SH as a radiosensitizer in cervical cancer therapy.

Effects of electromagnetic field, cisplatin and morphine on cytotoxicity and expression levels of DNA repair genes.

Morphine (Mor) is widely used as an analgesic drug in cancers and in combination with chemotherapy is known to have DNA damaging effects on non-targeted cell. This study surveyed the effect of Mor in combination with 50-Hz electromagnetic field (EMF) and co-treatment of cisplatin in combination with Mor and EMF on the expression of genes involved in DNA repair pathways. MCF-7 and SH-SY5Y cells were treated with 5.0 µM Mor and then exposed to 50-Hz 0.50 mT EMF in the intermittent pattern of 15 min field-on/15 min field-off. Gene expression, cisplatin and bleomycin cytotoxicity were measured using real-time PCR and MTT assay. Mor treated cells showed significant down-regulation of the examined genes, while in "Mor + EMF" treatments the genes were not significantly changed. IC50 of cisplatin was significantly elevated in both cell lines when co-treated with "Mor + EMF" compared with Mor treated cells. Non-homologous end joining (NHEJ) related genes were significantly decreased in co-treatment of cisplatin and "Mor + EMF" which led to bleomycin higher cytotoxicity in SH-SY5Y not in MCF-7. Our data is promising for providing a cell line-specific sensitization by combination of cisplatin and "Mor + EMF" treatment with local administration of double strand breaking agents.

Resveratrol-Inspired Benzo[b]selenophenes Act as Anti-Oxidants in Yeast.

Resveratrol is a natural (poly)phenol primarily found in plants protecting them against pathogens, as well as harmful effects of physical and chemical agents. In higher eukaryotic cells and organisms, this compound displays a remarkable range of biological activities, such as anti-oxidant, anti-inflammatory, anti-cancer, anti-aging, cardio- and neuro-protective properties. Here, biological activities of synthetic selenium-containing derivatives of resveratrol-benzo[b]selenophenes-have been studied in lower eukaryotes Saccharomyces cerevisiae. Their toxicity, as well as DNA damaging and reactive oxygen species (ROS) inducing potencies, manifested through their ability to act as redox active anti-microbial agents, have been examined. We show that some benzo[b]selenophenes can kill yeast cells and that the killing effects are not mediated by DNA damage types that can be detected as DNA double-strand breaks. These benzo[b]selenophenes could potentially be used as anti-fungal agents, although their concentrations relevant to application in humans need to be further evaluated. In addition, most of the studied benzo[b]selenophenes display redox-modulating/anti-oxidant activity (comparable or even higher than that of resveratrol or Trolox) causing a decrease in the intracellular ROS levels in yeast cells. Therefore, after careful re-evaluation in other biological systems these observations might be transferred to humans, where resveratrol-inspired benzo[b]selenophenes could be used as supra-anti-oxidant supplements.

Analysis of antioxidant, cytotoxic and mutagenic potential of Agave sisalana Perrine extracts using Vero cells, human lymphocytes and mice polychromatic erythrocytes.

Brazilian Northeast is the world's largest producer of Agave sisalana Perrine for the supply of the sisal fiber. About 95% of plant biomass, which comprise the mucilage and sisal juice, is considered a waste residual is discarded in the soil. However, the sisal juice is rich in steroidal saponins, which exhibits different pharmacological properties. Despite this, natural products are not necessarily safe. Based on this, this study analyzed the antioxidant, cytotoxic and mutagenic potential of three extracts derived from acid hydrolysis (AHAS), dried precipitate (DPAS) and hexanic of A. sisalana (HAS). These analyses were performed by in vitro and in vivo methods, using Vero cells, human lymphocytes and mice. Results showed that AHAS 50 and 100 can be considered a useful antineoplastic candidate due to their antioxidant and cytotoxic activity, with no genotoxic/clastogenic potential in Vero cells and mice. Although the comet assay in human lymphocytes has showed that the AHAS 25, AHAS 50 and AHAS 100 can lead to DNA breaks, these extracts did not promote DNA damages in mice bone marrow. Considering the different mutagenic responses obtained with the different methods employed, this study suggest that the metabolizing pathways can produce by-products harmful to health. For this reason, it is mandatory to analyze the mutagenic potential by both in vitro and in vivo techniques, using cells derived from different species and origins.

4ß-Hydroxywithanolide E selectively induces oxidative DNA damage for selective killing of oral cancer cells.

Reactive oxygen species (ROS) induction had been previously reported in 4ß-hydroxywithanolide (4ßHWE)-induced selective killing of oral cancer cells, but the mechanism involving ROS and the DNA damage effect remain unclear. This study explores the role of ROS and oxidative DNA damage of 4ßHWE in the selective killing of oral cancer cells. Changes in cell viability, morphology, ROS, DNA double strand break (DSB) signaling (γH2AX foci in immunofluorescence and DSB signaling in western blotting), and oxidative DNA damage (8-oxo-2'deoxyguanosine [8-oxodG]) were detected in 4ßHWE-treated oral cancer (Ca9-22) and/or normal (HGF-1) cells. 4ßHWE decreased cell viability, changed cell morphology and induced ROS generation in oral cancer cells rather than oral normal cells, which were recovered by a free radical scavenger N-acetylcysteine (NAC). For immunofluorescence, 4ßHWE also accumulated more of the DSB marker, γH2AX foci, in oral cancer cells than in oral normal cells. For western blotting, DSB signaling proteins such as γH2AX and MRN complex (MRE11, RAD50, and NBS1) were overexpressed in 4ßHWE-treated oral cancer cells in different concentrations and treatment time. In the formamidopyrimidine-DNA glycolyase (Fpg)-based comet assay and 8-oxodG-based flow cytometry, the 8-oxodG expressions were higher in 4ßHWE-treated oral cancer cells than in oral normal cells. All the 4ßHWE-induced DSB and oxidative DNA damage to oral cancer cells were recovered by NAC pretreatment. Taken together, the 4ßHWE selectively induced DSB and oxidative DNA damage for the ROS-mediated selective killing of oral cancer cells.

BRCA2 protects mammalian cells from heat shock.

PURPOSE: Heat shock induces DNA double-strand breaks (DSBs) in mammalian cells. Mammalian cells are capable of repairing DSBs by utilising the homologous recombination (HR) pathway. Breast cancer susceptibility gene 2 (BRCA2) is known to regulate the HR pathway. Here, we investigate the role of BRCA2 in repairing DNA damage induced by heat shock. MATERIALS AND METHODS: Chinese hamster lung fibroblast cell lines and human tongue squamous cell carcinoma SAS cells were used. RAD51 foci formation assay was used as an HR indicator. Heat sensitivity was analysed with colony forming assays. Phosphorylated histone H2AX (γH2AX) intensity, which correlates with the number of DSBs, was analysed with flow cytometry. RESULTS: RAD51 foci appeared with heat shock, and the number of cells with RAD51 foci was maximal at about 4 h after heat shock. Heat-induced RAD51 foci co-localised with γH2AX foci. BRCA2-deficient cells were sensitive to heat when compared to their parental wild-type cells. Heat-induced γH2AX was higher in BRCA2-deficient cells compared to parental cells. In SAS cells, cells transfected with BRCA2-siRNA were more sensitive to heat than cells transfected with negative control siRNA. Apoptotic bodies increased in number more rapidly in BRCA2-siRNA transfected cells than in cells transfected with negative control siRNA when cells were observed at 48 h after a heat treatment. In addition, cells deficient in BRCA2 were incapable of activating heat-induced G2/M arrest. CONCLUSION: BRCA2 has a protecting role against heat-induced cell death. BRCA2 might be a potential molecular target for hyperthermic cancer therapy.

Zerumbone Regulates DNA Repair Responding to Ionizing Radiation and Enhances Radiosensitivity of Human Prostatic Cancer Cells.

INTRODUCTION: Radiation therapy using ionizing radiation is widely used for the treatment of prostate cancer. The intrinsic radiation sensitivity of cancer cells could be enhanced by modulating multiple factors including the capacity to repair DNA damage, especially double-strand breaks (DSBs). We aimed to examine the effect of zerumbone on radiation sensitivity and its protective effects against ionizing radiation-induced DSB in human prostate cancer cells. MATERIALS AND METHODS: The human prostate cancer PC3 and DU145 cell lines were used. A colony formation assay was performed to analyze the radiation survival of cells. DNA histogram and generation of reactive oxygen species (ROS) were examined using flow cytometry. Western blotting was used to examine the expression of regulatory molecules related to DNA damage repair. RESULTS: Pretreatment with zerumbone enhanced the radiation effect on prostate cancer cells. Zerumbone delayed the abrogation of radiation-induced expression of γ-H2AX, an indicator of DNA DSB. Zerumbone pretreatment markedly reduced ionizing radiation-induced upregulated expression of phosphorylated ATM (ataxia telangiectasia-mutated), which was partially reversed by the ATM agonist methyl methanesulfonate. Ionizing radiation augmented and zerumbone pretreatment reduced the expression of Jak2 and Stat3, which are involved in DNA damage repair signaling. No significant effect on the generation of ROS and expression of ATR was noted after zerumbone treatment. CONCLUSION: Zerumbone sensitized DU145 and PC3 prostatic cancer cells to ionizing radiation by modulating radiation-induced ATM activation during repair of DNA DSBs.

N-Acetyl-L-cysteine protects thyroid cells against DNA damage induced by external and internal irradiation.

We evaluated the effect of the antioxidant N-acetyl-L-cysteine (NAC) on the levels of reactive oxygen species (ROS), DNA double strand breaks (DSB) and micronuclei (MN) induced by internal and external irradiation using a rat thyroid cell line PCCL3. In internal irradiation experiments, ROS and DSB levels increased immediately after 131I addition and then gradually declined, resulting in very high levels of MN at 24 and 48 h. NAC administration both pre- and also post-131I addition suppressed ROS, DSB and MN. In external irradiation experiments with a low dose (0.5 Gy), ROS and DSB increased shortly and could be prevented by NAC administration pre-, but not post-irradiation. In contrast, external irradiation with a high dose (5 Gy) increased ROS and DSB in a bimodal way: ROS and DSB levels increased immediately after irradiation, quickly returned to the basal levels and gradually rose again after >24 h. The second phase was in parallel with an increase in 4-hydroxy-2-nonenal. The number of MN induced by the second wave of ROS/DSB elevations was much higher than that by the first peak. In this situation, NAC administered pre- and post-irradiation comparably suppressed MN induced by a delayed ROS elevation. In conclusion, a prolonged ROS increase during internal irradiation and a delayed ROS increase after external irradiation with a high dose caused serious DNA damage, which were efficiently prevented by NAC. Thus, NAC administration even both after internal or external irradiation prevents ROS increase and eventual DNA damage.

The effect of DHEA on apoptosis and cohesin levels in oocytes in aged mice.

Female fertility declines with age as the number of ovarian follicles decreases and aneuploidy increases. Degradation of the cohesin complex might be responsible for age-related aneuploidy. Dehydroepiandrosterone (DHEA) can improve the ovarian reserve and reduce the rate of aneuploidy, but the relationship between DHEA and cohesin levels in oocytes is still unknown. The aim of the current study was to evaluate the effect of the supplement DHEA on ovarian function, including the number of follicles and cohesin levels in oocytes. C57BL/6J mice at 3 weeks, 6 weeks, 12 weeks, 6 months, and 10 months of age were used to obtain a systematic view into follicle apoptosis and cohesin levels in oocytes. Nine-month-old C57BL/6J mice were administered saline (n = 5), 17ß-estradiol (100 µg/kg per day, n = 5), or DHEA (5mg/Kg per day, n = 5). After 4 weeks, aged mice were weighed and sacrificed, and ovarian tissue samples were prepared. Anti-VASA staining and HE staining were used to count the number of follicles. Anti-γH2AX staining and TUNEL were used to measure follicle apoptosis and immunofluorescent staining was used to detect the levels of three oocyte cohesin subunits: REC8, SMC1ß, and SMC3. Administration of the supplements 17ß-estradiol and DHEA to aged mice increased the number of primordial and primary follicles and decreased the age-related apoptosis of follicles. Levels of the cohesin subunits REC8 and SMC1ß declined with age, but DHEA and 17ß-estradiol tended to delay that decline. The supplement DHEA increased the number of primordial and primary follicles in aged mice by inhibiting follicle apoptosis and tended to delay the decrease in cohesin levels in oocytes.

Anti-cancer agents in Saudi Arabian herbals revealed by automated high-content imaging.

Natural products have been used for medical applications since ancient times. Commonly, natural products are structurally complex chemical compounds that efficiently interact with their biological targets, making them useful drug candidates in cancer therapy. Here, we used cell-based phenotypic profiling and image-based high-content screening to study the mode of action and potential cellular targets of plants historically used in Saudi Arabia's traditional medicine. We compared the cytological profiles of fractions taken from Juniperus phoenicea (Arar), Anastatica hierochuntica (Kaff Maryam), and Citrullus colocynthis (Hanzal) with a set of reference compounds with established modes of action. Cluster analyses of the cytological profiles of the tested compounds suggested that these plants contain possible topoisomerase inhibitors that could be effective in cancer treatment. Using histone H2AX phosphorylation as a marker for DNA damage, we discovered that some of the compounds induced double-strand DNA breaks. Furthermore, chemical analysis of the active fraction isolated from Juniperus phoenicea revealed possible anti-cancer compounds. Our results demonstrate the usefulness of cell-based phenotypic screening of natural products to reveal their biological activities.

The preclinical discovery of vosaroxin for the treatment of acute myeloid leukemia.

INTRODUCTION: Acute myeloid leukemia (AML) represents a disease with a very poor outcome and remains an area of significant unmet need necessitating novel therapeutic strategies. Among novel therapeutic agents, vosaroxin is a first-in-class anticancer quinolone derivative that targets topoisomerase II and induces site-selective double-strand breaks in DNA, leading to tumor cell apoptosis. Areas covered: Herein, the authors provide a comprehensive review of the preclinical development of vosaroxin. This includes coverage of vosaroxin's mechanism of action in addition to its pharmacology and of the main studies reported over the past few years with vosaroxin when used to treat adult AML. Expert opinion: Given that vosaroxin is associated with fewer potential side effects, it may be of benefit to elderly patients with relapsed/refractory AML and to those with additional comorbidities who have previously received an anthracycline and cytarabine combination. Furthermore, vosaroxin also was seen to be active in multidrug-resistant preclinical models. However, further studies have to be performed to better evaluate its place in the armamentarium against AML.

Chemical Composition and Biological Activity of Allium cepa L. and Allium × cornutum (Clementi ex Visiani 1842) Methanolic Extracts.

Here, we report a comparative study of the phytochemical profile and the biological activity of two onion extracts, namely Allium cepa L. and Allium × cornutum (Clementi ex Visiani 1842), members of the family Amaryllidaceae. The identification of flavonoids and anthocyanins, and their individual quantities, was determined by high-performance liquid chromatography (HPLC). The potency of both extracts to scavenge free radicals was determined by the DPPH (2,2'-diphenyl-1-picrylhydrazyl) radical-scavenging activity and oxygen radical absorbance capacity (ORAC) methods. The DNA protective role was further tested by the single-cell gel electrophoresis (COMET) assay and by Fenton's reagent causing double-strand breaks on the closed circular high copy pUC19 plasmid isolated from Escherichia coli. In the presence of both extracts, a significant decrease in DNA damage was observed, which indicates a protective role of Allium cepa and Allium × cornutum on DNA strand breaks. Additionally, cytotoxicity was tested on glioblastoma and breast cancer cell lines. The results showed that both extracts had antiproliferative effects, but the most prominent decrease in cellular growth was observed in glioblastoma cells.

Mechanism of synergistic DNA damage induced by the hydroquinone metabolite of brominated phenolic environmental pollutants and Cu(II): Formation of DNA-Cu complex and site-specific production of hydroxyl radicals.

2,6-Dibromohydroquinone (2,6-DBrHQ) has been identified as an reactive metabolite of many brominated phenolic environmental pollutants such as tetrabromobisphenol-A (TBBPA), bromoxynil and 2,4,6-tribromophenol, and was also found as one of disinfection byproducts in drinking water. In this study, we found that the combination of 2,6-DBrHQ and Cu(II) together could induce synergistic DNA damage as measured by double strand breakage in plasmid DNA and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) formation, while either of them alone has no effect. 2,6-DBrHQ/Cu(II)-induced DNA damage could be inhibited by the Cu(I)-specific chelating agent bathocuproine disulfonate and catalase, but not by superoxide dismutase, nor by the typical hydroxyl radical (•OH) scavengers such as DMSO and mannitol. Interestingly, we found that Cu(II)/Cu(I) could be combined with DNA to form DNA-Cu(II)/Cu(I) complex by complementary application of low temperature direct ESR, circular dichroism, cyclic voltammetry and oxygen consumption methods; and the highly reactive •OH were produced synergistically by DNA-bound-Cu(I) with H2O2 produced by the redox reactions between 2,6-DBrHQ and Cu(II), which then immediately attack DNA in a site-specific manner as demonstrated by both fluorescent method and by ESR spin-trapping studies. Further DNA sequencing investigations provided more direct evidence that 2,6-DBrHQ/Cu(II) caused preferential cleavage at guanine, thymine and cytosine residues. Based on these data, we proposed that the synergistic DNA damage induced by 2,6-DBrHQ/Cu(II) might be due to the synergistic and site-specific production of •OH near the binding site of copper and DNA. Our findings may have broad biological and environmental implications for future research on the carcinogenic polyhalogenated phenolic compounds.

In vitro screening of radioprotective properties in the novel glucosylated flavonoids.

Novel glucosyl flavonoids are developed by the addition of glucose to naturally occurring flavonoids. Flavonoids are known antioxidants that possess radioprotective properties. In order to investigate the radioprotective properties of novel glucosyl flavonoids, in vitro DNA double-strand breaks (DSBs) analysis was carried out. In the present study, Quercetin, Naringenin, and Hesperetin groups of flavonoids included in the natural and novel glucosyl 13 flavonoids were investigated. Flavonoids were mixed with Lambda DNA, and subsequently exposed to gamma­rays. Furthermore, DNA DSB yields were visualized by gel electrophoresis. Quercetin derivatives displayed reduced DNA DSB formation at 10 µM. At a high concentration, the majority of flavonoids displayed radioprotective properties as a reduction of DSB yields. Suppression of DSB formation was confirmed via the molecular combing assay for Quercetin, and three monoglucosyl flavonoids. Glucosylation showed positive effects for radioprotection and monoglucosyl-Rutin showed superior radioprotective properties when compared to monoglucosyl-Naringin and Hesperidin. In addition, Quercetin derivatives had greater total antioxidant capacities and DPPH radical scavenging ability than other flavonoid groups. Since Quercetin, Isoquercetin, and Rutin display poor water solubility, monoglucosyl-Rutin, maltooligosyl-Isoquercetin, and maltooligosyl-Rutin may be better radioprotective agents and easily bioavailable with increased water solubility.