Oxidative Stress-induced DNA Damage Research Articles

Current status of auditory aging and anti‐aging research

The development of presbycusis, or age-related hearing loss, is determined by a combination of genetic and environmental factors. The auditory periphery exhibits a progressive bilateral, symmetrical reduction of auditory sensitivity to sound from high to low frequencies. The central auditory nervous system shows symptoms of decline in age-related cognitive abilities, including difficulties in speech discrimination and reduced central auditory processing, ultimately resulting in auditory perceptual abnormalities. The pathophysiological mechanisms of presbycusis include excitotoxicity, oxidative stress, inflammation, aging and oxidative stress-induced DNA damage that results in apoptosis in the auditory pathway. However, the originating signals that trigger these mechanisms remain unclear. For instance, it is still unknown whether insulin is involved in auditory aging. Auditory aging has preclinical lesions, which manifest as asymptomatic loss of periphery auditory nerves and changes in the plasticity of the central auditory nervous system. Currently, the diagnosis of preclinical, reversible lesions depends on the detection of auditory impairment by functional imaging, and the identification of physiological and molecular biological markers. However, despite recent improvements in the application of these markers, they remain under-utilized in clinical practice. The application of antisenescent approaches to the prevention of auditory aging has produced inconsistent results. Future research will focus on the identification of markers for the diagnosis of preclinical auditory aging and the development of effective interventions.

Protective role of the PG1036-PG1037-PG1038 operon in oxidative stress in Porphyromonas gingivalis W83.

As an anaerobe, Porphyromonas gingivalis is significantly affected by the harsh inflammatory environment of the periodontal pocket during initial colonization and active periodontal disease. We reported previously that the repair of oxidative stress-induced DNA damage involving 8-oxo-7,8-dihydroguanine (8-oxoG) may occur by an undescribed mechanism in P. gingivalis. DNA affinity fractionation identified PG1037, a conserved hypothetical protein, among other proteins, that were bound to the 8-oxoG lesion. PG1037 is part of the uvrA-PG1037-pcrA operon in P. gingivalis which is known to be upregulated under H2O2 induced stress. A PCR-based linear transformation method was used to inactivate the uvrA and pcrA genes by allelic exchange mutagenesis. Several attempts to inactivate PG1037 were unsuccessful. Similar to the wild-type when plated on Brucella blood agar, the uvrA and pcrA-defective mutants were black-pigmented and beta-hemolytic. These isogenic mutants also had reduced gingipain activities and were more sensitive to H2O2 and UV irradiation compared to the parent strain. Additionally, glycosylase assays revealed that 8-oxoG repair activities were similar in both wild-type and mutant P. gingivalis strains. Several proteins, some of which are known to have oxidoreducatse activity, were shown to interact with PG1037. The purified recombinant PG1037 protein could protect DNA from H2O2-induced damage. Collectively, these findings suggest that the uvrA-PG1037-pcrA operon may play an important role in hydrogen peroxide stress-induced resistance in P. gingivalis.

Oxidative Stress-Induced DNA Damage and Repair in Human Peripheral Blood Mononuclear Cells: Protective Role of Hemoglobin

BackgroundDNA repair is a cellular defence mechanism responding to DNA damage caused in large part by oxidative stress. There is a controversy with regard to the effect of red blood cells on DNA damage and cellular response.AimTo investigate the effect of red blood cells on H2O2-induced DNA damage and repair in human peripheral blood mononuclear cells.MethodsDNA breaks were induced in peripheral blood mononuclear cells by H2O2 in the absence or presence of red blood cells, red blood cells hemolysate or hemoglobin. DNA repair was measured by 3H-thymidine uptake, % double-stranded DNA was measured by fluorometric assay of DNA unwinding. DNA damage was measured by the comet assay and by the detection of histone H2AX phosphorylation.ResultsRed blood cells and red blood cells hemolysate reduced DNA repair in a dose-dependent manner. Red blood cells hemolysate reduced % double-stranded DNA, DNA damage and phosphorylation of histone H2AX. Hemoglobin had the same effect as red blood cells hemolysate on % double-stranded DNA.ConclusionRed blood cells, via red blood cells hemolysate and hemoglobin, reduced the effect of oxidative stress on peripheral blood mononuclear cell DNA damage and phosphorylation of histone H2AX. Consequently, recruitment of DNA repair proteins diminished with reduction of DNA repair. This suggests that anemia predisposes to increased oxidative stress induced DNA damage, while a higher hemoglobin level provides protection against oxidative-stress-induced DNA damage.

ATF2 knockdown reinforces oxidative stress‐induced apoptosis in TE7 cancer cells

Cancer cells showing low apoptotic effects following oxidative stress-induced DNA damage are mainly affected by growth arrest. Thus, recent studies focus on improving anti-cancer therapies by increasing apoptosis sensitivity. We aimed at identifying a universal molecule as potential target to enhance oxidative stress-based anti-cancer therapy through a switch from cell cycle arrest to apoptosis. A cDNA microarray was performed with hydrogen peroxide-treated oesophageal squamous epithelial cancer cells TE7. This cell line showed checkpoint activation via p21WAF1, but low apoptotic response following DNA damage. The potential target molecule was chosen depended on the following demands: it should regulate DNA damage response, cell cycle and apoptosis. As the transcription factor ATF2 is implicated in all these processes, we focused on this protein. We investigated checkpoint activation via ATF2. Indeed, ATF2 knockdown revealed ATF2-triggered p21WAF1 protein expression, suggesting p21WAF1 transactivation through ATF2. Using chromatin immunoprecipitation (ChIP), we identified a hitherto unknown ATF2-binding sequence in the p21WAF1 promoter. p-ATF2 was found to interact with p-c-Jun, creating the AP-1 complex. Moreover, ATF2 knockdown led to c-Jun downregulation. This suggests ATF2-driven induction of c-Jun expression, thereby enhancing ATF2 transcriptional activity via c-Jun-ATF2 heterodimerization. Notably, downregulation of ATF2 caused a switch from cell cycle arrest to reinforced apoptosis, presumably via p21WAF1 downregulation, confirming the importance of ATF2 in the establishment of cell cycle arrest. 1-Chloro-2,4-dinitrobenzene also led to ATF2-dependent G2/M arrest, suggesting that this is a general feature induced by oxidative stress. As ATF2 knockdown also increased apoptosis, we propose ATF2 as a target for combined oxidative stress-based anti-cancer therapies.

APE2 is required for ATR-Chk1 checkpoint activation in response to oxidative stress

The base excision repair pathway is largely responsible for the repair of oxidative stress-induced DNA damage. However, it remains unclear how the DNA damage checkpoint is activated by oxidative stress at the molecular level. Here, we provide evidence showing that hydrogen peroxide (H2O2) triggers checkpoint kinase 1 (Chk1) phosphorylation in an ATR [ataxia-telangiectasia mutated (ATM) and Rad3-related]-dependent but ATM-independent manner in Xenopus egg extracts. A base excision repair protein, Apurinic/apyrimidinic (AP) endonuclease 2 (APE2, APN2, or APEX2), is required for the generation of replication protein A (RPA)-bound single-stranded DNA, the recruitment of a checkpoint protein complex [ATR, ATR-interacting protein (ATRIP), and Rad9] to damage sites, and H2O2-induced Chk1 phosphorylation. A conserved proliferating cell nuclear antigen interaction protein box of APE2 is important for the recruitment of APE2 to H2O2-damaged chromatin. APE2 3'-phosphodiesterase and 3'-5' exonuclease activity is essential for single-stranded DNA generation in the 3'-5' direction from single-stranded breaks, referred to as single-stranded break end resection. In addition, APE2 associates with Chk1, and a serine residue (S86) in the Chk1-binding motif of APE2 is essential for Chk1 phosphorylation, indicating a Claspin-like but distinct role for APE2 in ATR-Chk1 signaling. Our data indicate that APE2 plays a vital and previously unexpected role in ATR-Chk1 checkpoint signaling in response to oxidative stress. Thus, our findings shed light on a distinct mechanism of how an ATR-Chk1-dependent DNA damage checkpoint is mediated by APE2 in the oxidative stress response.

Role of poly(ADP-ribosyl)ation in a ‘two-hit’ model of hypoxia and oxidative stress in human A549 epithelial cells in vitro

A preceding hypoxic insult can sensitize the cells or the organism to a subsequent, second insult. The aim of the present study was to investigate the molecular mechanism of this phenomenon (often termed ‘two-hit’ injury paradigm), in an in vitro model of hypoxia/oxidative stress injury in A549 epithelial cells, with special emphasis on the role of the nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP-1) in the process. Pre-exposure of the cells to 24 h hypoxia significantly reduced intracellular glutathione (GSH) levels, reduced mitochondrial activity and adenosine triphosphate (ATP) levels. However pre-exposure to hypoxia failed to induce any change in PARP-1 expression and activation, DNA single-strand breaks or plasma membrane integrity. Pre-exposure to hypoxia markedly increased the sensitivity of the cells to subsequent oxidative stress-induced DNA damage. Hydrogen peroxide (H2O2) induced a concentration-dependent increase in DNA breakage, PARP activation, depletion of intracellular ATP, inhibition of mitochondrial activity and two distinct parameters that quantify the breakdown of plasma membrane integrity (propidium iodide uptake or lactate dehydrogenase release). PARP-1 activation played a significant role in the H2O2-induced cell death response because PARP activation, depletion of intracellular ATP, inhibition of mitochondrial activity, and the breakdown of plasma membrane integrity were attenuated in cells with permanently silenced PARP-1. Based on measurement of the endogenous antioxidant GSH, we hypothesized that the mechanism of hypoxia-mediated enhancement of H2O2 involves depletion of the GSH during the hypoxic period, which renders the cells more sensitive to a subsequent DNA single-strand break elicited by H2O2. DNA strand breakage then activates PARP-1, leading to the inhibition of mitochondrial function, depletion of ATP and cell necrosis. PARP-1 deficiency protects against the cytotoxicity, to a lesser degree, by protecting against GSH depletion during the hypoxic period, and, to a larger degree, by maintaining mitochondrial function and preserving intracellular ATP levels during the subsequent oxidative stress period.

Werner's syndrome protein limits the error‐prone 8‐oxo‐G lesion bypass activity of human DNA polymerase kappa by promoting the error‐free bypass

Oxidative stress induced DNA damage plays a key role in cancer and aging. 7,8‐dihydro‐8oxo‐2′‐deoxyguanosine (8‐oxo‐dG), produced during DNA oxidation is highly mutagenic and known to block replication. Previous studies show that the human DNA polymerase kappa (hpol κ) bypasses 8‐oxo‐dG by preferentially inserting dATP opposite the lesion. Recent studies indicated that hpol κ coordinates with Werner's syndrome protein (WRN) during the 8‐oxo‐dG bypass. Our data show that WRN stimulates the 8‐oxo‐dG bypass activity of hpol κ by enhancing the correct base insertion opposite the lesion and diminishing the efficiency of dATP mispair insertion. Kinetic analysis reveals that in the presence of WRN, hpol κ inserts the dCTP opposite 8‐oxo‐dG ~ 1.7‐fold more efficiently than dCTP opposite dG. Currently we are investigating the fidelity and catalytic efficiency of hpol κ bypass of 8‐oxo‐dG in the presence of WRN using pre‐steady state kinetic analysis and mass spectrometric detection of polymerization products. WRN may function in coordination with hpol κ to limit the error‐prone bypass of 8‐oxo‐dG, which could suppress the mutagenicity associated with hpol κ and prevent genomic instability. This work is supported in part by USPHS R00 GM084460 (R.L.E).

Oxidative Stress Induced DNA Damage and Repair in Heart Failure Patients Supported by Continuous Flow Ventricular Assist Devices

Purpose The blood contacting surfaces of a left ventricular assist device (LVAD) is made of biologically inert compounds such as titanium, polytetrafluoroethylene, and acrylics. These inert materials as well as non-physiological high shear stress can affect different cellular processes in recipients. To evaluate the effects of LVAD among congestive heart failure (CHF) patients on oxidative stress and DNA damage repair, the present study have been conducted. Methods and Materials We recruited 5 CHF patients implanted with continuous flow LVADs as bridge to transplant and 5 non-smoking healthy control. Reactive oxygen species (ROS) generation in blood leukocytes and the levels of antioxidant enzyme superoxide dismutase (SOD) in erythrocytes were measured by flow cytometry/immunofluorescence microscopy and spectrophotometry respectively. ELISA was used to measure oxidized low density lipoproteins ( ox LDL) in plasma. γ-H2AX foci count as DNA double strand break marker and different key proteins involved in DNA repair via non-homologous-end-joining (NHEJ) and homologous recombination (HR) were done by immunocytochemistry in lymphocytes. Results Our study clearly indicated that the levels of ROS and ox LDL were significantly higher in CHF patients after LVAD implantation compared to baseline and controls. Moreover, SOD levels were decreased with increasing post-operative days. All the changes indicated oxidative stress among LVAD recipients. Increasing numbers of γ-H2AX foci in lymphocytes were positively correlated with the ROS (r=0.9429) and oxLDL (r=0.9753) and negatively with SOD (r=–0.9924) levels (p Conclusions Our result, for the first time showed that, LVAD not only induce oxidative stress and DNA damage in lymphocytes but also modulates the DNA repair pathways among CHF patients.

Effect of Zinc Oxide nanoparticles on cellular oxidative stress and antioxidant defense mechanisms in mouse liver

Zinc oxide nanoparticles (ZnO2), a common ingredient of cosmetics has a huge variety of applications. Previous studies reported oxidative stress mediated toxicity of ZnO2 nanoparticles on various mammalian cell lines. Although zinc (Zn) is an essential mineral at higher concentrations this metal is toxic. The present study focused on size determination by monitoring changes in activities of antioxidant defense mechanism in response to oxidative stress induced by ZnO2 nanoparticles using mouse liver tissue homogenates. The study also investigated effects of oxidative stress induced DNA damage by determining formation of 8-OHdG in mouse liver homogenate. A cytotoxicity assay was also carried out in L929 cells to determine cell viability. The results of the study indicated that 50μg/ml of ZnO2 nanoparticles induced 50% cell death. Alterations in antioxidant parameters and 8-OHdG were also noted. Data showed that there was a concentration-dependent fall in cell viability, decrease antioxidant enzyme levels and increase formation of DNA adduct (8-OHdG) when mouse liver tissue homogenate were exposed to ZnO2 nanoparticles.

Genetic polymorphism in hOGG1 is associated with triple-negative breast cancer risk in Chinese Han women

8-hydroxy-2′-deoxyguanine (8-OHdG), a typical product of oxidative stress-induced DNA damage, can cause a G–T transversion during DNA replication if it is not removed. Human 8-oxoguanine glycosylase 1 (hOGG1), a key DNA repair gene, recognizes and excises 8-OHdG from damaged DNA accurately; however, a c.977C>G (Ser326Cys) polymorphism in hOGG1 can inhibit the gene's ability to remove 8-OHdG. The aim of present study was to investigate the association between the c.977C>G polymorphism in hOGG1 and the risk of breast cancer in Chinese Han women. We used high-resolution melting and sequencing to analyze the genotypes of 630 patients with sporadic breast cancer patients and 777 healthy controls. We also performed risk-stratified subgroup analyses to determine the association between the c.977C>G polymorphism and other characteristics of breast cancer subgroups. Breast cancer patients and healthy controls did not have significantly different of c.977C/G genotypes (odds ratio [OR] = 1.10, 95% confidence interval [CI] = 0.82–1.49, p = 0.57) and c.977G/G genotypes (OR = 1.34, 95% CI = 0.97–1.84, p = 0.09). However, the c.977G/G genotype was especially prevalent in breast cancer patients who were younger than 55 years (OR = 1.58, 95% CI = 1.05–2.39, p = 0.04), were premenopausal status (OR = 1.87, 95% CI = 1.14–3.06, p = 0.02), had triple-negative disease (OR = 2.14, 95% CI = 1.06–4.29, p = 0.04), or p53-positive disease (OR = 1.56, 95% CI = 1.14–2.12, p = 0.005). These findings suggest that the c.977C>G polymorphism in hOGG1 is associated with an increased risk of breast cancer in Chinese Han women who are younger than 55 years, premenopausal, triple-negative, or p53-positive subgroups.

Early transformative changes in normal ovarian surface epithelium induced by oxidative stress require Akt upregulation, DNA damage and epithelial–stromal interaction

Ovarian cancer is the deadliest gynecological malignancy due to detection of cancer at a late stage when the disease has metastasized. One likely progenitor cell type of ovarian cancer is the ovarian surface epithelium (OSE), which proliferates rapidly in the presence of inflammatory cytokines and oxidative stress following ovulation. To determine whether oxidative stress induces DNA damage leading to spontaneous transformative changes in normal OSE, an immortalized mouse OSE cell line (MOSE cells) or normal mouse ovarian organoids were treated with hydrogen peroxide (H2O2) and loss of contact inhibition was assessed by soft agar assay. In response to H2O2, OSE cells grown in 3D exhibited growth in soft agar but MOSE cells grown on 2D plastic did not, indicating a critical role for epithelial-stromal interactions in neoplastic initiation. Loss of contact inhibition in response to H2O2 correlated with an increase in proliferation, DNA damage and upregulation of the oncogene Akt1. Use of a reactive oxygen species scavenger or Akt inhibitor blocked H2O2-induced proliferation and growth in soft agar. Although parental MOSE cells did not undergo transformation by H2O2, MOSE cells stably overexpressing constitutively active myristoylated Akt or knockdown of phosphatase and tensin homolog (PTEN) exhibited loss of contact inhibition and increased proliferation. This study indicates that normal OSE undergo transformative changes induced by oxidative stress and that this process requires Akt upregulation and activation. A 3D model that retains tissue architecture is critical for studying this process and may lead to development of new intervention strategies directed at early stages of ovarian cancer.

Intrauterine exposure to flavonoids modifies antioxidant status at adulthood and decreases oxidative stress-induced DNA damage

Maternal intake of flavonoids, known for their antioxidant properties, may affect the offspring's susceptibility to developing chronic diseases at adult age, especially those related to oxidative stress, via developmental programming. Therefore, we supplemented female mice with the flavonoids genistein and quercetin during gestation, to study their effect on the antioxidant capacity of lung and liver of adult offspring. Maternal intake of quercetin increased the expression of Nrf2 and Sod2 in fetal liver at gestational day 14.5. At adult age, in utero exposure to both flavonoids resulted in the increased expression of several enzymatic antioxidant genes, which was more pronounced in the liver than in the adult lung. Moreover, prenatal genistein exposure induced the nonenzymatic antioxidant capacity in the adult lung, partly by increasing glutathione levels. Prenatal exposure to both flavonoids resulted in significantly lower levels of oxidative stress-induced DNA damage in liver only. Our observations lead to the hypothesis that a preemptive trigger of the antioxidant defense system in utero had a persistent effect on antioxidant capacity and as a result decreased oxidative stress-induced DNA damage in the liver.

Epigenetic Regulation of Survivin by Bmi1 Is Cell Type Specific During Corticogenesis and in Gliomas

Polycomb group proteins are essential regulators of stem cell function during embryonic development and in adult tissue homeostasis. Bmi1, a key component of the Polycomb Repressive Complex 1, is highly expressed in undifferentiated neural stem cells (NSC) as well as in several human cancers including high-grade gliomas--highly aggressive brain tumors. Using a conditional gene activation approach in mice, we show that overexpression of Bmi1 induces repressive epigenetic regulation of the promoter of Survivin, a well-characterized antiapoptotic protein. This phenomenon is cell type-specific and it leads to apoptotic death of progenitor cells exclusively upon commitment toward a neuronal fate. Moreover, we show that this is triggered by increased oxidative stress-induced DNA damage. In contrast, undifferentiated NSC as well as glioma-initiating cells display an open chromatin configuration at the Survivin promoter and do not undergo apoptotic death. These findings raise the possibility that normal and neoplastic stem cells depend on the same mechanism for surviving the hyperproliferative state induced by increased Bmi1 expression.

Abstract 18300: Myocardial Ischemia Induces Telomerase Activity in the Mouse Heart

Introduction: The incidence of cardiovascular disease rises with age. Ischemia and oxidative stress-induced DNA damage and telomere erosion may accelerate cardiac senescence. The enzyme telomerase has been shown to protect cardiovascular cells from mitochondrial oxidative stress independent of telomere elongation. We examined whether myocardial ischemia impacts on telomere biology and cellular aging in the infarcted mouse heart. Methods and results: C57Bl/6 mice (10 weeks old, n=6-8 per group) were subjected to anterior myocardial ischemia induced by proximal ligation of the left anterior descendant coronary artery or sham operation. Cardiac MRI was used to verify infarct localization and measure LV function, showing that ejection fraction was reduced in MI mice (47±5% vs. 68±3% in sham, p<0.01). Sham and MI mice were compared 3 days, 7 days, or 4 weeks after LAD ligation and myocardial infarct zone (IZ) and remote zone (RZ) were analyzed. Telomerase activity as measured by TRAP assays was elevated in the infarct zone 3 days after LAD ligation (187±9% vs. sham mice, p<0.01), but not in the remote zone (113±35%). Seven days after myocardial infarction, telomerase was up-regulated both, in the infarct zone (177±31%) and the remote zone (154±26%). This effect was even more pronounced 4 weeks after LAD ligation (IZ: 271±14%; RZ 223±28%). In concert with telomerase activation, telomere repeat binding factor (TRF) 2 protein expression was up-regulated in the infarct zone after three days (337±25%) and in both areas after 7 days (IZ: 431±48%; RZ: 380±31%) and less strongly after 4 weeks (IZ: 170±23%; RZ: 141±27%). In parallel to telomerase activity and TRF2 expression, the apoptosis and senescence markers p53 (189±11%), Chk2 (167±32%), bax/bcl2-ratio (278±27%) and p16 (518±42%) were up-regulated in the myocardium of MI mice after 4 weeks (all shown values p<0.05 vs. myocardial tissue of sham-operated mice). Conclusion: Myocardial telomerase and telomere-associated factors are up-regulated in a time- and region-specific manner in the infarcted mouse heart in concert with apoptosis and cell-cycle regulators. This regulatory process may be of relevance for myocardial survival, inflammation, and remodelling after ischemia.

Mapping histological levels of 8-hydroxy-2′-deoxyguanosine in female reproductive organs

Oxidative stress is associated with many disease states including gynecologic disease. This process can damage lipids, proteins and DNA. The present study highlights the role of oxidative stress induced DNA damage as measured by 8-hydroxy-2-deoxyguanosine in development of benign gynecological conditions (BGC). Our aim was to map the oxidative DNA damage on female reproductive organs and highlight the high amount found in a variety of benign gynecologic disorders. Seventeen biopsy specimens from female pelvic organs were divided in two groups: healthy organs tissue and BGC tissue. Healthy organs biopsy tissue included the cervix, tubes, uterus, peritoneum, and topic endometrium in secretory phase. Benign gynecological biopsy tissue included hydrosalpinges, leiomyoma, adenomyosis and tubal cysts. Immunohistochemical staining showed significantly higher levels of DNA damage between BGC and healthy organs [19.36% (6.20; 32.51) vs. 4.61% (0.63; 8.53); P<0.0344]. Our results highlight the involvement of oxidative stress DNA damage in female benign pelvic disease. Hydrosalpinges, leiomyoma, and adenomyosis exhibit the highest amounts of oxidative DNA damage in the pelvic cavity.

Preferential recognition of catechol-estrogen modified DNA by circulating autoantibodies in cancer patients

Catecholestrogens [4-hydroxyestradiol (4-OHE(2))] have been implicated in human carcinogenesis, although the mechanism remains unestablished. In this study pUC 18 plasmid DNA was modified with 4-OHE(2) and nitric oxide (NO). The modification induced in native DNA exhibited hyperchromicity, single strand breaks, damage to restriction sites, modification of bases, decrease in Tm and change in ellipticity. Modified DNA was found to be highly immunogenic in experimental animal, eliciting high titer antibodies. Circulating cancer autoantibodies showed preferable recognition of 4-OHE(2)-NO-DNA over native form (p < 0.001) and the oxidative epitopes on the DNA isolates from cancer patients were immunochemically detected by using experimentally induced anti-4-OHE(2)-NO-DNA antibodies as a probe. Preferential recognition of 4-OHE(2)-NO-DNA by cancer autoantibodies coupled with enhanced binding of induced antibodies to DNA isolated from cancer patients is an indicative of oxidative stress induced DNA damage in cancer. Possible involvement of unique epitopes on modified DNA in cancer autoantibody induction has been suggested.

In vitro antioxidative and antigenotoxic capacity of wheat aleurone extracted with solvents

This study investigated the antioxidant and antigenotoxic ability of different wheat aleurone (WA) extracts. Oxygen radical absorbance capacity (ORAC), which was the highest in the aleurone ethanol extract (AEE) and the lowest in the acetone extract (AAE), was highly correlated with total polyphenol content (r=0.897, p<0.01). DPPH radical scavenging activity was highest in AAE followed by AEE and the aleurone methanol extract (AME). The ABTS·+ quenching properties of the WA extracts were similar to their DPPH radical scavenging activity. Furthermore, WA extracts had a protective effect against oxidative stress-induced DNA damage in both human leukocytes and HT-29 human colon carcinoma cells. This inhibitory activity was attributed to their unique phytochemical content, with a strong correlation between DPPH radical scavenging activity and inhibition of DNA damage (r=0.779, p<0.01 at 10 ∝g/mL AAE). Therefore, WA might be a promising component of future nutraceuticals or value-added products.

Baicalein (5,6,7-trihydroxyflavone) reduces oxidative stress-induced DNA damage by upregulating the DNA repair system

Oxidative stress caused by reactive oxygen species (ROS) induces DNA base modifications and DNA strand breaks. In this study, the protective effect of baicalein against H(2)O(2)-induced DNA damage was investigated in V79-4 Chinese hamster fibroblast cells. H(2)O(2) treatment increased the levels of intracellular ROS and DNA double-strand breaks (DSBs) and decreased the level of Ku70 protein and the phosphorylation (activation) of DNA-dependent protein kinase catalytic subunit (DNA-PKcs), which are involved in the repair of DSBs by nonhomologous end joining. Baicalein effectively scavenged intracellular ROS induced by H(2)O(2), reduced DSBs, and rescued Ku70 protein level and phosphorylation of DNA-PKcs. In cellular response to DNA base damage, 8-oxoguanine DNA glycosylase 1 (OGG1) plays a vital role in the removal of 8-oxoguanine (8-OxoG), which is formed mainly by oxidative stress. Baicalein significantly decreased the levels of 8-OxoG induced by H(2)O(2), and this correlated with increases in OGG1 promoter activity and OGG1 mRNA and protein expression. The phosphorylated form of Akt kinase, which is a regulator of OGG1, was sharply decreased by H(2)O(2), but was prevented by baicalein. A specific Akt inhibitor abolished the cytoprotective effects of baicalein, suggesting that OGG1 induction by baicalein involves the Akt pathway. In conclusion, baicalein exerted protective effects against DNA damage induced by oxidative stress by activating DNA repair systems and scavenging ROS.

Genotoxicity assessment in iron deficiency anemia patients using sister chromatid exchanges and chromosomal aberrations assays

Previous studies have shown that iron deficiency anemia is associated with oxidative stress produced by a decrease in antioxidant enzyme activities and/or a high level of oxidants. Because oxidative stress induces DNA damage, we investigated genotoxicity in lymphocytes from patients with iron deficiency anemia (IDA) using chromosomal aberrations (CAs) and sister chromatid exchanges (SCEs) assays. Eighteen IDA subjects and a similar number of age-matched healthy controls were included in the study. The results demonstrated that IDA was associated with a slight increase in the frequency of spontaneous CAs and a decrease in the frequency of SCEs (P<0.05). In addition, the level of SCEs was positively correlated with both the ferritin concentration (r=0.485, P<0.05) and hemoglobin content (r=0.514, P<0.05) in subjects. Moreover, vitamin E treatment reduced the frequency of SCEs in IDA patients and control subjects by the same percentage (∼30%) without affecting the magnitude of the difference in the levels of SCEs between the two groups. In conclusion, our results demonstrated that IDA has a differential effect on the frequency of spontaneous CAs and SCEs.

Antiproliferation and Induction of Apoptosis in Ca9-22 Oral Cancer Cells by Ethanolic Extract of Gracilaria tenuistipitata

The water extract of Gracilaria tenuistipitata have been found to be protective against oxidative stress-induced cellular DNA damage, but the biological function of the ethanolic extracts of G. tenuistipitata (EEGT) is still unknown. In this study, the effect of EEGT on oral squamous cell cancer (OSCC) Ca9-22 cell line was examined in terms of the cell proliferation and oxidative stress responses. The cell viability of EEGT-treated OSCC cells was significantly reduced in a dose-response manner (p < 0.0001). The annexin V intensity and pan-caspase activity of EEGT-treated OSCC cells were significantly increased in a dose-response manner (p < 0.05 to 0.0001). EEGT significantly increased the reactive oxygen species (ROS) level (p < 0.0001) and decreased the glutathione (GSH) level (p < 0.01) in a dose-response manner. The mitochondrial membrane potential (MMP) of EEGT-treated OSCC cells was significantly decreased in a dose-response manner (p < 0.005). In conclusion, we have demonstrated that EEGT induced the growth inhibition and apoptosis of OSCC cells, which was accompanied by ROS increase, GSH depletion, caspase activation, and mitochondrial depolarization. Therefore, EEGT may have potent antitumor effect against oral cancer cells.