Expression Of DNA Repair Enzymes Research Articles

Gentiopicroside and swertiamarin induce non-selective oxidative stress-mediated cytotoxic effects in human peripheral blood mononuclear cells

Gentiopicroside (Gp) and swertiamarin (Sm), secoiridoid glycosides commonly found in plants of the Gentianaceae family, differ in one functional group. They exhibit promising cytotoxic effects in cancer cell lines and overall protective outcomes, marking them as promising molecules for developing novel pharmaceuticals. To investigate potential variations in cellular sensitivity to compounds of similar molecular structures, we analyzed the mode of Gp and Sm induced cell death in human peripheral blood mononuclear cells (PBMCs) after 48 h of treatment. The lowest tested concentration that significantly reduces cell viability, 50 μM, was applied. Oxidative stress parameters were estimated by measuring the levels of prooxidative/antioxidative balance, lipid peroxidation products, and 8-oxo-7,8-dihydro-2-deoxyguanosine, while gene expression of DNA repair enzymes was evaluated by employing quantitative real-time PCR. Cellular morphology was analyzed by fluorescent microscopy, and immunoblot analysis of apoptosis and necroptosis-related proteins was used to assess the type of cell death induced by the treatments. The discriminatory impact of Gp/Sm treatments on apoptosis and necroptosis-induced cell death was evaluated by monitoring the cell survival in co-treatment with specific cell death inhibitors. Obtained results show greater cytotoxicity of Gp than Sm suggesting that variations in the molecular structures of the tested compounds can substantially affect their biological effects. Gp/Sm co-treatment with apoptosis and necroptosis inhibitors revealed a distinct, albeit non-specific mechanism of PBMCs cell death. Although the therapeutic may not directly cause a specific type of cell death, its extent can be pivotal in assessing the safety of therapeutic application and developing phytopharmaceuticals with improved features. Since phytopharmaceuticals affect all exposed cells, identification of cytotoxic mechanisms on PBMCs after Gp and Sm treatment is important for addressing the formulation and dosage of potential phytopharmaceuticals.

WT1 regulates expression of DNA repair gene Neil3 during nephrogenesis.

Mammalian nephrons arise from a population of nephron progenitor cells (NPCs) expressing the master transcription factor Wilms tumor-1 (WT1), which is crucial for NPC proliferation, migration, and differentiation. In humans, biallelic loss of WT1 precludes nephrogenesis and leads to the formation of Wilms tumor precursor lesions. We hypothesize that WT1 normally primes the NPC for nephrogenesis by inducing expression of NPC-specific DNA repair genes that protect the genome. We analyzed transcript levels for a panel of DNA repair genes in embryonic day 17.5 (E17.5) versus adult mouse kidneys and noted seven genes that were increased >20-fold. We then isolated Cited1+ NPCs from E17.5 kidneys and found that only one gene, nei-like DNA glycosylase 3 (Neil3), was enriched. RNAscope in situ hybridization of E17.5 mouse kidneys showed increased Neil3 expression in the nephrogenic zone versus mature nephron structures. To determine whether Neil3 expression is WT1 dependent, we knocked down Wt1 in Cited1+ NPCs (60% knockdown efficiency) and noted a 58% reduction in Neil3 transcript levels. We showed that WT1 interacts with the Neil3 promoter and that activity of a Neil3 promoter-reporter vector was increased twofold in WT1+ versus WT1- cells. We propose that Neil3 is a WT1-dependent DNA repair gene expressed at high levels in Cited1+ NPCs, where it repairs mutational injury to the genome during nephrogenesis. NEIL3 is likely just one of many such lineage-specific repair mechanisms that respond to genomic injury during kidney development.NEW & NOTEWORTHY We studied the molecular events leading to Wilms tumors as a model for the repair of genomic injury. Specifically, we showed that WT1 activates DNA repair gene Neil3 in nephron progenitor cells. However, our observations offer a much broader principle, demonstrating that the embryonic kidney invests in lineage-specific expression of DNA repair enzymes. Thus, it is conceivable that failure of these mechanisms could lead to a variety of "sporadic" congenital renal malformations and human disease.

RF07 | PSUN384 Hepatocyte Deletion of IGF2 Prevents DNA Damage and Tumor Formation in Hepatocellular Carcinoma

Abstract Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide with a 18% 5-year survival rate. Mice lacking the splicing factor SRSF3 in hepatocytes (SKO mice) overexpress IGF2 and are predisposed to developing HCC with age. Loss of Igf2 in the SKO background prevented hepatic fibrosis and inflammation, and completely prevented tumor formation. This was associated with decreased proliferation, apoptosis and DNA damage, and restored DNA repair enzyme expression. Mechanistically, IGF2 treatment of HepG2 cells in vitro caused DNA damage and decreased DNA repair enzyme expression, and tumors from the SKO mice show mutational signatures consistent with double strand break and defective mismatch repair. Looking at human data, HCC patients with high IGF2 mRNA expression had worse survival compared to patients with normal IGF2 expression. The patients also showed a switch in IGF2 promoter usage that correlated with increased expression. HCC patients having high SRSF3 mRNA expression also showed poor survival as did patients with alterations in known SRSF3-dependent splicing events. The level of SRSF3 protein was decreased 6-fold in human HCC tissues compared with normal liver tissues. The results indicate that IGF2 overexpression in conjunction with reduced SRSF3 splicing activity could be a major cause of DNA damage and driver of liver cancer. Presentation: Saturday, June 11, 2022 1:18 p.m. - 1:23 p.m., Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m.

Hepatocyte Deletion of IGF2 Prevents DNA Damage and Tumor Formation in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide. Serine‐arginine rich splicing factor 3 (SRSF3) plays a critical role in hepatocyte function and its loss in mice promotes chronic liver damage and leads to HCC. Hepatocyte‐specific SRSF3 knockout mice (SKO mice) also overexpress insulin‐like growth factor 2 (IGF2). In the present study, double deletion of Igf2 and Srsf3 (DKO mice) prevents hepatic fibrosis and inflammation, and completely prevents tumor formation, and is associated with decreased proliferation, apoptosis and DNA damage, and restored DNA repair enzyme expression. This is confirmed in vitro, where IGF2 treatment of HepG2 hepatoma cells decreases DNA repair enzyme expression and causes DNA damage. Tumors from the SKO mice also show mutational signatures consistent with homologous recombination and mismatch repair defects. Analysis of frozen human samples shows that SRSF3 protein is decreased sixfold in HCC compared to normal liver tissue but SRSF3 mRNA is increased. Looking at public TCGA data, HCC patients having high SRSF3 mRNA expression show poor survival, as do patients with alterations in known SRSF3‐dependent splicing events. The results indicate that IGF2 overexpression in conjunction with reduced SRSF3 splicing activity could be a major cause of DNA damage and driver of liver cancer.

Reduced expression of OXPHOS and DNA damage genes is linked to protection from microvascular complications in long-term type 1 diabetes: the PROLONG study

Type 1 diabetes is a chronic autoimmune disease requiring insulin treatment for survival. Prolonged duration of type 1 diabetes is associated with increased risk of microvascular complications. Although chronic hyperglycemia and diabetes duration have been considered as the major risk factors for vascular complications, this is not universally seen among all patients. Persons with long-term type 1 diabetes who have remained largely free from vascular complications constitute an ideal group for investigation of natural defense mechanisms against prolonged exposure of diabetes. Transcriptomic signatures obtained from RNA sequencing of the peripheral blood cells were analyzed in non-progressors with more than 30 years of diabetes duration and compared to the patients who progressed to microvascular complications within a shorter duration of diabetes. Analyses revealed that non-progressors demonstrated a reduction in expression of the oxidative phosphorylation (OXPHOS) genes, which were positively correlated with the expression of DNA repair enzymes, namely genes involved in base excision repair (BER) machinery. Reduced expression of OXPHOS and BER genes was linked to decrease in expression of inflammation-related genes, higher glucose disposal rate and reduced measures of hepatic fatty liver. Results from the present study indicate that at transcriptomic level reduction in OXPHOS, DNA repair and inflammation-related genes is linked to better insulin sensitivity and protection against microvascular complications in persons with long-term type 1 diabetes.

Early Thymoquinone Injections and Expression of DNA Repair Enzymes in Hamster Buccal Pouch-Induced Dysplasia

Because carcinogenesis is a multi-step process, Sporn (1976)suggested that the best time to control cancer is in its “preneoplastic” phase, before it has a chance to develop into “invasive” cancer. Therefore, the aim of the present work was to evaluate the effect of TQ on the dysplasia induced in that model, through IHC expression of both enzymes.6 weeks of DMBA painting resulted in irreversible genetic changes that did not resolve by the studied repair enzymes. So it can be proposed that TQ had resulted in regression of the carcinogenesis process through the following mechanisms. First: TQ as a potent antioxidant that antagonizes the adverse effect resulting from elevated ROS levels due to carcinogenesis i.e. reduction of the intracellular ROS allowed the repair enzymes to fulfill their main target.Second: TQ through inactivation of the TNF-Nf-κB pathway, and in turn induced p53 activation, i.e. stimulated apoptosis of malignant cells. Third: TQ has the ability to arrest cells at different phases of the cell cycle leading to growth inhibition, so allowing repair of mutated cells

Tolerance induction in memory CD4 T cells is partial and reversible.

Memory T cells respond rapidly in part because they are less reliant on a heightened levels of costimulatory molecules. This enables rapid control of secondary infecting pathogens but presents challenges to efforts to control or silence memory CD4 T cells, for example in antigen-specific tolerance strategies for autoimmunity. We have examined the transcriptional and functional consequences of reactivating memory CD4 T cells in the absence of an adjuvant. We find that memory CD4 T cells generated by infection or immunisation survive secondary activation with antigen delivered without adjuvant, regardless of their location in secondary lymphoid organs or peripheral tissues. These cells were, however, functionally altered following a tertiary immunisation with antigen and adjuvant, proliferating poorly but maintaining their ability to produce inflammatory cytokines. Transcriptional and cell cycle analysis of these memory CD4 T cells suggests they are unable to commit fully to cell division potentially because of low expression of DNA repair enzymes. In contrast, these memory CD4 T cells could proliferate following tertiary reactivation by viral re-infection. These data indicate that antigen-specific tolerogenic strategies must examine multiple parameters of Tcell function, and provide insight into the molecular mechanisms that may lead to deletional tolerance of memory CD4 T cells.

Molecular evaluation of the chemotherapeutic effect of thymoquinone loaded on gold nanoparticles through expression of DNA repair enzymes in induced oral squamous cell carcinoma

Different types of DNA damages occur during the induced oral carcinogenesis, which can be eliminated through several DNA repair pathways. XRCC1 and ERCC1 are the main repair enzymes involved in repair of oral squamous cell carcinoma. A combination of thymoquinone with gold nanoparticles as a novel chemotherapeutic modality is the aim of the present work against chemically induced SCC in the classic model of HBP/DMBA carcinogenesis. One hundred male Syrian golden hamsters were divided into: Group A: Ten animals (negative control), group B: Ten animals (positive control) painted with DMBA only 3times / week/ 12weeks. The rest of animals were painted with DMBA (3times / week/ 12weeks) then painted and injected intraperitoneal with TQ only, 0.01mg/kg TQ-GNps, 0.001mg/kg TQ-GNps or GNps only for 6- and 12- weeks, intervals. By end of the experiment, both pouches from all groups were surgically excised, fresh samples from each pouch were processed for RT-PCR technique. The rest of the pouches were fixed and processed for H&E evaluation. Loading of thymoquinone on gold nanoparticles was promising chemotherapeutic combination, through regression of well-differentiated SCC (positive control) to dysplasia and enhanced expression of the studied DNA repair enzymes compared to either thymoquinone or gold nanoparticles groups.

Abstract GMM-037: CARBONIC ANHYDRASE 9 AND TUMOR HYPOXIA ARE ASSOCIATED WITH RECURRENCE AND RESISTANCE IN BRCA1-EXPRESSING HGSOC

Abstract Tumor hypoxia contributes to poor outcome in multiple cancer types, in part by influencing the expression of DNA repair enzymes. The objective of this study was to examine the possible relationship between Carbonic Anhydrase 9 (CA-IX, an indicator of tumor hypoxia), BRCA1 expression (a potential indicator of competent DNA repair), and patient outcome in epithelial ovarian cancer. BRCA1 and CA-IX were evaluated by immunohistochemistry in a tissue microarray of 167 epithelial ovarian cancer (EOC) patient samples, and their respective expression correlated to other biological parameters in all patients. The correlations between BRCA1 and CA-IX expression, and clinical outcome were determined in the High Grade Serous Ovarian Carcinoma (HGSOC) samples (N = 101) on the array using Spearman's correlation and log-rank (Mantel-Cox) tests. Elevated expression of BRCA1 strongly correlated with shorter intervals for disease recurrence and mortality (median time to recurrence = 1.05 yrs vs 1.94 yrs, P <0.001; median time to mortality: 2.01 yrs vs 4.72 yrs, P <0.001). There was a significant positive correlation between CA-IX and BRCA1 expression (Spearman's r = 0.486, P < 0.001). CA-IX expression was significantly associated with recurrence-free survival (restricted to first 1500 days, P <0.01). When patients were stratified by BRCA1 expression, co-expression of CA-IX and BRCA1 correlated with the shortest recurrence-free interval (P <0.05). In complementary in vitro studies, exposure of multiple BRCA1-wild-type cell lines to hypoxia (1% O2, mimicking general tumor hypoxia) had only a minimal effect on BRCA1 expression. Additionally, in MTT viability assays, application of the hypoxia-activated pro-drug TH-302 preferentially killed cells in 1% O2 compared to standard conditions (21% O2). In contrast, the PARP inhibitor olaparib was 2-5 times less effective in hypoxic conditions than in standard growth conditions, suggesting that tumor hypoxia promotes a more resistant phenotype. CONCLUSION: The co-expression of CA-IX and BRCA1 may be a useful prognostic indicator for rapid recurrence in high grade serous ovarian cancer. Dissecting the mechanisms regulating therapeutic resistance in hypoxic ovarian cancer cells could lead to more effective treatment options for HGSOC patients. Citation Format: Adam J. Krieg, Shawn Campbell, Paulette Mhawech-Fauceglia, Jeong Youn Lim, Tanja Pejovic. CARBONIC ANHYDRASE 9 AND TUMOR HYPOXIA ARE ASSOCIATED WITH RECURRENCE AND RESISTANCE IN BRCA1-EXPRESSING HGSOC [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr GMM-037.

Abstract 5244: BET inhibition decreases the expression of DNA repair enzymes in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC), the most common type of pancreatic cancer (PC), is now the third leading cause of cancer related deaths in the US. This is a highly aggressive disease in that 80% of patients present with locally advanced or metastatic disease and their only treatment option is systemic chemotherapy. Ultimately patient tumors develop resistance to therapy and resulting in a median survival of ~6 months. Therefore, there is an imperative need to identify therapies that provide a more durable response for this patient population. The family of bromodomain and extraterminal domain (BET) proteins has recently become a target of interest for the treatment of PDAC. The BET proteins (BRD2, BRD3, BRD4, and BRDT) function to regulate transcription by recruiting positive transcriptional activators to the promoters of genes. Our lab has shown that inhibition of BET bromodomain function using the BET inhibitor JQ1 suppresses PDAC tumor growth in vivo and inhibits cell viability in vitro. Importantly, PDAC cells and tumors exposed to JQ1 show evidence of DNA damage. We hypothesized that because of the role BET proteins are known to play in regulating gene expression, that the observed JQ1-induced DNA damage may result from JQ1 inhibiting the expression of DNA repair genes whose expression are dependent on BET protein transcriptional complexes. qRT-PCR and immunoblot analysis demonstrated that treatment of the pancreatic cancer cell line BxPC3 with JQ1 inhibited the expression of two double strand break repair proteins, Ku80 and RAD51, both of which have been shown to be overexpressed in cancer including PDAC. Although it has been established that JQ1 co-occupies 99% of the genomic loci that BRD4 is known to bind to, specific gene products whose expression is dependent on BRD4 in PDAC have not been thoroughly investigated. The goal of the current study was to determine if the expression of DNA repair genes Ku80 and RAD51 were specific gene targets of BRD4 transcriptional complexes. We exposed BxPC3 cells to JQ1 for 48 hours and assessed changes in the association of BRD4 with the promoter loci of Ku80 and RAD51 using chromatin immunoprecipitation (ChIP) assays. qRT-PCR of the ChIP demonstrated that treatment with JQ1 significantly (p<0.05) decreased the association of BRD4 with the promoters of both genes. To further confirm that these gene products were dependent on BRD4 for their expression, we down-regulated BRD4 using shRNA and evaluated the effect on the expression of Ku80 and RAD51. Immunoblot revealed that by reducing the expression of BRD4 by 53% in BxPC3 cells, the expression of Ku80 and RAD51 were also decreased by 35% and 93% respectively. We conclude that the expression of Ku80 and RAD51 are dependent on BRD4 transcriptional complexes and are gene targets that contribute to the anti-tumor efficacy of JQ1 in PDAC. Citation Format: Aubrey Lynn Miller, Samuel C. Fehling, Patrick L. Garcia, Karina J. Yoon. BET inhibition decreases the expression of DNA repair enzymes in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5244.

EP300-HDAC1-SWI/SNF functional unit defines transcription of some DNA repair enzymes during differentiation of human macrophages

Differentiation of human macrophages predisposes these cells to numerous tasks, i.e. killing invading pathogens, and this entails the need for enhanced intracellular defences against stress, including conditions that may increase DNA damage. Our study shows that expression of DNA repair enzymes, such as PARP1, BRCA1 and XRCC1, are activated during macrophage development by the SWI/SNF chromatin remodelling complex, which serves as a histone acetylation sensor. It recognises and displaces epigenetically marked nucleosomes, thereby enabling transcription. Acetylation is controlled both in monocytes and macrophages by the co-operation of EP300 and HDAC1 activities. Differentiation modulates the activities of individual components of EP300-HDAC1-SWI/SNF functional unit and entails recruitment of PBAF to gene promoters. In monocytes, histone-deacetylated promoters of repressed PARP1, BRCA1 and XRCC1 respond only to HDAC inhibition, with an opening of the chromatin structure by BRM, whereas in macrophages both EP300 and HDAC1 contribute to the fine-tuning of nucleosomal acetylation, with HDAC1 remaining active and the balance of EP300 and HDAC1 activities controlling nucleosome eviction by BRG1-containing SWI/SNF. Since EP300-HDAC1-SWI/SNF operates at the level of gene promoters characterized simultaneously by the presence of E2F binding site(s) and CpG island(s), this allows cells to adjust PARP1, BRCA1 and XRCC1 transcription to the differentiation mode and to restart cell cycle progression. Thus, mutual interdependence between acetylase and deacetylase activities defines the acetylation-dependent code for regulation of histone density and gene transcription by SWI/SNF, notably on gene promoters of DNA repair enzymes.

Effects of Watermelon Powder and l-arginine Supplementation on Azoxymethane-Induced Colon Carcinogenesis in Rats

Diets high in fruits and vegetables may help prevent colorectal cancer (CRC). Watermelon consumption may reduce CRC risk due to its concentration of l-citrulline and its role in endothelial nitric oxide (NO) production. Research suggests that increased NO levels have tumoricidal effects. The purpose of this study was to determine the effects of watermelon powder supplementation on aberrant crypt foci (ACF) formation, precancerous lesions, and expression of genes associated with colon carcinogenesis. Thirty-two male Sprague-Dawley rats were assigned into three groups: control, 0.36% l-arginine, or 0.5% watermelon powder and injected with azoxymethane (15 mg/kg body weight). Both l-arginine and watermelon powder groups exhibited lower total numbers of ACF and high multiplicity ACF (P < 0.01). The watermelon powder group exhibited higher NO levels and lower 8-hydroxyguanosine DNA damage (P < 0.05). Watermelon powder and l-arginine downregulated 8-oxoguanine DNA glycosylase gene expression and upregulated O6-methylguanine DNA methyltransferase gene expression (P < 0.05). Cyclooxgenase-2 gene expression was lower for rats fed with watermelon powder (P < 0.05). These results suggest that watermelon powder or l-arginine supplementation may reduce the risk of colon cancer by suppressing ACF formation through lowering oxidative DNA damage and inflammation, modulating DNA repair enzyme expression, and/or enhancing NO production.

A role for β,β-xanthophylls in Arabidopsis UV-B photoprotection.

Plastidial isoprenoids, such as carotenoids and tocopherols, are important anti-oxidant metabolites synthesized in plastids from precursors generated by the methylerythritol 4-phosphate (MEP) pathway. In this study, we found that irradiation of Arabidopsis thaliana plants with UV-B caused a strong increase in the accumulation of the photoprotective xanthophyll zeaxanthin but also resulted in slightly higher levels of γ-tocopherol. Plants deficient in the MEP enzymes 1-deoxy-D-xylulose 5-phosphate synthase and 1-hydroxy-2-methyl-2-butenyl 4-diphosphate synthase showed a general reduction in both carotenoids and tocopherols and this was associated with increased DNA damage and decreased photosynthesis after exposure to UV-B. Genetic blockage of tocopherol biosynthesis did not affect DNA damage accumulation. In contrast, lut2 mutants that accumulate β,β-xanthophylls showed decreased DNA damage when irradiated with UV-B. Analysis of aba2 mutants showed that UV-B protection was not mediated by ABA (a hormone derived from β,β-xanthophylls). Plants accumulating β,β-xanthophylls also showed decreased oxidative damage and increased expression of DNA-repair enzymes, suggesting that this may be a mechanism for these plants to decrease DNA damage. In addition, in vitro experiments also provided evidence that β,β-xanthophylls can directly protect against DNA damage by absorbing radiation. Together, our results suggest that xanthophyll-cycle carotenoids that protect against excess illumination may also contribute to protection against UV-B.

Microsatellite Instability Occurs Rarely in Patients with Cholangiocarcinoma: A Retrospective Study from a German Tertiary Care Hospital.

Immune-modulating therapy is a promising therapy for patients with cholangiocarcinoma (CCA). Microsatellite instability (MSI) might be a favorable predictor for treatment response, but comprehensive data on the prevalence of MSI in CCA are missing. The aim of the current study was to determine the prevalence of MSI in a German tertiary care hospital. Formalin-fixed paraffin-embedded tissue samples, obtained in the study period from 2007 to 2015 from patients with CCA undergoing surgical resection with curative intention at Johann Wolfgang Goethe University hospital, were examined. All samples were investigated immunohistochemically for the presence of MSI (expression of MLH1, PMS2, MSH2, and MSH6) as well as by pentaplex polymerase chain reaction for five quasimonomorphic mononucleotide repeats (BAT-25, BAT-26, NR-21, NR-22, and NR-24). In total, 102 patients were included, presenting intrahepatic (n = 35, 34.3%), perihilar (n = 42, 41.2%), and distal CCA (n = 25, 24.5%). In the immunohistochemical analysis, no loss of expression of DNA repair enzymes was observed. In the PCR-based analysis, one out of 102 patients was found to be MSI-high and one out of 102 was found to be MSI-low. Thus, MSI seems to appear rarely in CCA in Germany. This should be considered when planning immune-modulating therapy trials for patients with CCA.

Oxidatively-induced DNA damage and base excision repair in euthymic patients with bipolar disorder

Oxidatively-induced DNA damage has previously been associated with bipolar disorder. More recently, impairments in DNA repair mechanisms have also been reported. We aimed to investigate oxidatively-induced DNA lesions and expression of DNA glycosylases involved in base excision repair in euthymic patients with bipolar disorder compared to healthy individuals. DNA base lesions including both base and nucleoside modifications were measured using gas chromatography-tandem mass spectrometry and liquid chromatography-tandem mass spectrometry with isotope-dilution in DNA samples isolated from leukocytes of euthymic patients with bipolar disorder (n = 32) and healthy individuals (n = 51). The expression of DNA repair enzymes OGG1 and NEIL1 were measured using quantitative real-time polymerase chain reaction. The levels of malondialdehyde were measured using high performance liquid chromatography. Seven DNA base lesions in DNA of leukocytes of patients and healthy individuals were identified and quantified. Three of them had significantly elevated levels in bipolar patients when compared to healthy individuals. No elevation of lipid peroxidation marker malondialdehyde was observed. The level of OGG1 expression was significantly reduced in bipolar patients compared to healthy individuals, whereas the two groups exhibited similar levels of NEIL1 expression. Our results suggest that oxidatively-induced DNA damage occurs and base excision repair capacity may be decreased in bipolar patients when compared to healthy individuals. Measurement of oxidatively-induced DNA base lesions and the expression of DNA repair enzymes may be of great importance for large scale basic research and clinical studies of bipolar disorder.

Chemopreventive Potential of Ethanolic Extracts of Luobuma Leaves (Apocynum venetum L.) in Androgen Insensitive Prostate Cancer.

Luobuma (Apocynum venetum L. (AVL)) is a popular beverage in Asia and has been reportedly to be associated with the bioactivities such as cardiotonic, diuretic, antioxidative, and antihypertensive. However, its biofunction as chemoprevention activity is seldom addressed. Herein, we aimed to characterize the anti-androgen-insensitive-prostate-cancer (anti-AIPC) bioactive compounds of Luobuma, and to investigate the associated molecular mechanisms. Activity-guided-fractionation (antioxidative activity and cell survivability) of Luobuma ethanolic extracts was performed to isolate and characterize the major bioactive compounds using Ultra Performance Liquid Chromatography (UPLC), Liquid Chromatography-Mass Spectrometry (LC-MS), and Nuclear Magnetic Resonance (NMR). Plant sterols (lupeol, stigamasterol and β-sitosterol) and polyphenolics (isorhamnetin, kaempferol, and quercetin) were identified. Lupeol, a triterpene found in the fraction (F8) eluted by 10% ethyl acetate/90% hexane and accounted for 19.3% (w/w) of F8, inhibited the proliferation of PC3 cells. Both lupeol and F8 induced G2/M arrest, inhibition of β-catenin signaling, regulation of apoptotic signal molecules (cytochrome c, Bcl-2, P53, and caspase 3 and 8), and suppression DNA repair enzyme expression (Uracil-DNA glycosylase (UNG)). To our knowledge, our study is the first report that lupeol inhibited the expression of UNG to elicit the cytotoxicity against androgen-insensitive-prostate-cancer cells. Collectively, Luobuma, which contains several antitumor bioactive compounds, holds the potential to be a dietary chemopreventive agent for prostate cancer.

Oxidative stress and DNA damage induced by spinosad exposure in Spodoptera frugiperda Sf9 cells

ABSTRACT Spinosad, a neurotoxic insecticide, is widely used for crop protection. In order to elucidate the effects of spinosad on oxidative stress and genotoxicity in Sf9 cells, the levels of lipid peroxidation, the activity of antioxidative enzymes, and DNA damage were measured. The results showed that spinosad caused a time-dependent increase in the formation of malondialdehyde and decrease in the activity of superoxide dismutase and catalase. Further studies confirmed that spinosad induced 8-oxoguanine accumulation in Sf9 cells, which is accompanied by increased expression of DNA repair enzymes (OGG1 and MTH1). The neutral comet assay revealed that spinosad induced significant time-related increases of DNA double-strand breaks in Sf9 cells. Our results indicate that spinosad effectively induced oxidative stress and DNA damage in Sf9 cells.

Effect of Watermelon Powder Supplementation on Azoxymethane‐Induced Colon Carcinogenesis in Rats

Colorectal cancer (CRC) is a public health concern and one of the most common cancers in men and women globally. Given the substantial morbidity and mortality associated with this disease, methods for prevention, in part through a diet high in fruits and vegetables, are under investigation. Specifically, watermelon may be a practical method for reducing CRC risk factors due to its high concentration of L‐citrulline, a precursor to L‐arginine, which plays a crucial role in endothelial nitric oxide (NO) production. Evidence suggests that increased levels of NO have tumoricidal effects; therefore, the purpose of this study was to determine the effects of watermelon powder supplementation on aberrant crypt foci (ACF) formation, precancerous lesions, and biomarkers that are associated with colon carcinogenesis in rats. Thirty‐two male Sprague‐Dawley rats, at 21 days old, were divided into three groups: control, 0.36% L‐arginine, or 0.5% watermelon powder. The 0.5% watermelon powder diet provided 0.36% of L‐arginine+L‐citrulline. Rats were injected subcutaneously with azoxymethane (dose of 15 mg/kg body weight), a carcinogenic agent, at the beginning of weeks 3 and 4. The watermelon group exhibited higher blood NO levels compared to the control group (P &lt; 0.05). Both the L‐arginine and watermelon powder groups exhibited lower total numbers of ACF and high multiplicity aberrant crypt foci (HMACF, &gt; 4 aberrant crypts/focus) (P &lt; 0.01). The watermelon powder group exhibited lower 8‐hydroxyguanosine DNA damage in the blood (P &lt; 0.05) compared to controls. Watermelon powder and L‐arginine downregulated 8‐oxoguanine DNA glycosylase (OGG1) gene expression (P &lt; 0.05) and upregulated O6‐methylguanine DNA methyltransferase (MGMT) gene expression (P &lt; 0.01). Cyclooxgenase‐2 (COX‐2) gene expression in the colon mucosa of the watermelon group was lower compared to the control group (P &lt; 0.05). These results suggest that watermelon powder or L‐arginine consumption may reduce the risk of colon cancer by suppressing ACF formation through lowering oxidative DNA damage and inflammation, modulating DNA repair enzyme expression, and enhancing NO production.Support or Funding InformationThe study was funded by The US National Watermelon Promotion Board.

Elevated DNA Oxidation and DNA Repair Enzyme Expression in Brain White Matter in Major Depressive Disorder.

Background:Pathology of white matter in brains of patients with major depressive disorder (MDD) is well-documented, but the cellular and molecular basis of this pathology are poorly understood.Methods:Levels of DNA oxidation and gene expression of DNA damage repair enzymes were measured in Brodmann area 10 (BA10) and/or amygdala (uncinate fasciculus) white matter tissue from brains of MDD (n=10) and psychiatrically normal control donors (n=13). DNA oxidation was also measured in BA10 white matter of schizophrenia donors (n=10) and in prefrontal cortical white matter from control rats (n=8) and rats with repeated stress-induced anhedonia (n=8).Results:DNA oxidation in BA10 white matter was robustly elevated in MDD as compared to control donors, with a smaller elevation occurring in schizophrenia donors. DNA oxidation levels in psychiatrically affected donors that died by suicide did not significantly differ from DNA oxidation levels in psychiatrically affected donors dying by other causes (non-suicide). Gene expression levels of two base excision repair enzymes, PARP1 and OGG1, were robustly elevated in oligodendrocytes laser captured from BA10 and amygdala white matter of MDD donors, with smaller but significant elevations of these gene expressions in astrocytes. In rats, repeated stress-induced anhedonia, as measured by a reduction in sucrose preference, was associated with increased DNA oxidation in white, but not gray, matter.Conclusions:Cellular residents of brain white matter demonstrate markers of oxidative damage in MDD. Medications that interfere with oxidative damage or pathways activated by oxidative damage have potential to improve treatment for MDD.

Oxidative DNA damage is involved in cigarette smoke-induced lung injury in rats.

Reactive oxygen species (ROS) induced by exogenous toxicants are suggested to be involved in carcinogenesis by oxidative modification of DNA. 8-Hydroxyl-2-deoxyguanosine (8-OHdG) has been considered as a reliable biomarker for oxidative DNA damage both in vivo and in vitro studies. But the effect of smoking on oxidative damage has not yet been fully elucidated. Wistar rats were exposed to cigarette smoke at concentrations of 20 and 60% for 30min, twice/day for 45weeks. Then the histopathology of lung tissues, levels of ROS, 8-OHdG, and total antioxidant (T-AOC), expression of DNA repair enzymes, e.g. 8-oxyguaine DNA glycosylase (OGG1), and MutThomolog 1 (Oxidized Purine Nucleoside Triphosphatase, MTH1) were determined in urine, peripheral blood lymphocytes, and lung tissue. The results showed that long-term cigarette smoke exposure can cause obvious damages of lung tissue in rats. In addition, a significant and cigarette smoke concentration-dependent increase in ROS and 8-OHdG were observed compared with the non-exposed control rats. In contrast, the expression of OGG1 and MTH1, and T-AOC levels were obviously decreased after long-term exposure to cigarette smoke. These findings indicate that long-term exposure to cigarette smoker increases ROS levels, decreases total antioxidant capacity, and interferes DNA repair capacity that eventually induces oxidative DNA damage, which appears to play an important role in cigarette smoke-induced lung injury in rats, and determination of 8-OHdG levels might be a useful method for monitoring oxidative damage in cigarette smokers.