DNA Damage Expression Research Articles

Thiotert Induces Myelodysplastic Syndromes Cells Apoptosis by Activating Oxidative Stress

To explore whether thiotert treatment can inhibit proliferation and induce apoptosis in myelodysplastic syndromes (MDS) cells. CCK-8 assay was used for determining the cytotoxicity of thiotert to MDS cell line SKM-1 and the reversal effect of GSH, NAC, and Z-VAD-FMK on thiotert-induced inhibition of cell viability. EdU assay was deployed to detect the cell proliferation ability. Intracellular reactive oxygen species (ROS) was measured by flow cytometry after DCFH-DA staining. The expression of DNA damage- and apoptosis-related proteins was detected by Western blot. Thiotert treatment significantly suppressed the cell viability and proliferation ability in SKM-1 cells. A large amount of ROS generation and markedly elevated C-PARP, C-Caspase 3, and γ-H2AX were observed after thiotert administration, while BCL-2 was significantly decreased. In addition, GSH, NAC, and Z-VAD-FMK were able to mitigate the cytotoxicity of thiotert on SKM-1 cells. Thiotert can promote MDS cell apoptosis by mediating ROS production and pro-apoptotic proteins expression.

The Impact of High Adiposity on Endometrial Progesterone Response and Metallothionein Regulation.

Obesity is a disease with deleterious effects on the female reproductive tract, including the endometrium. We sought to understand the effects of excess adipose on the benign endometrium. A physiologic in vitro coculture system was developed, consisting of multicellular human endometrial organoids, adipose spheroids, and menstrual cycle hormones. Native human endometrial tissue samples women with and without obesity were also analyzed. Academic institution. Benign endometrial tissues from premenopausal women were obtained following written consent. Gene expression, protein expression, chromatin binding, and expression of DNA damage and oxidative damage markers were measured. Under high-adiposity conditions, endometrial organoids downregulated endometrial secretory phase genes, suggestive of an altered progesterone response. Progesterone specifically upregulated the metallothionein (MT) gene family in the epithelial cells of endometrial organoids, while high adiposity significantly downregulated the MT genes. Silencing MT genes in endometrial epithelial cells resulted in increased DNA damage, illustrating the protective role of MTs. Native endometrium from women with obesity displayed increased MT expression and oxidative damage in the stroma and not in the epithelium, indicating the cell-specific impact of obesity on MT genes. Taken together, the in vitro and in vivo systems used here revealed that high adiposity or obesity can alter MT expression by decreasing progesterone response in the epithelial cells and increasing oxidative stress in the stroma.

Maslinic Acid Induces DNA Damage and Impairs DNA Repair in Human Cervical Cancer HeLa Cells.

Maslinic acid, a natural plant-derived triterpenoid compound, exhibits pharmacological activities, including anti-cancer. In the present study, we investigated the cytotoxic effects of maslinic acid on human cervical cancer HeLa cells in vitro and further investigated the molecular mechanism of maslinic acid-induced DNA damage and repair. Cell viability was measured by flow cytometry. DNA condensation (apoptotic cell death), DNA damage, and DNA fragmentation (DNA ladder) were assayed by DAPI staining, comet assay, and agarose gel electrophoresis, respectively. The expression of DNA damage and repair proteins was assayed by western blotting. Maslinic acid decreased total cell viability and induced DNA condensation, damage, and fragmentation in HeLa cells. Furthermore, maslinic acid elevated the levels of p-ATMSer1981, p-ATRSer428, p53, p-p53Ser151, p-H2A.XSer139, BRCA1 and PARP at 30-40 μM. However, it decreased the levels of DNA-PK and MGMT. Maslinic acid reduced the number of viable HeLa cells by inducing DNA damage and altering the expression of proteins involved in DNA damage and repair.

Abstract 713: BBI-608 modulates DNA repair pathways, induces apoptosis and impacts response to 5-Fluorouracil and ionizing radiation in pancreatic cancer cells

Abstract Background: BBI-608 has been shown to modulate multiple oncogenic cellular pathways which are often constitutively active in pancreatic ductal adenocarcinoma (PDAC). Among these are key mediators of DNA repair and cellular survival pathways. We hypothesized that BBI-608 would sensitize PDAC cells to chemoradiotherapy. Methods: The combined effects of BBI-608 and chemoradiotherapy (5-FU + IR) were evaluated in human (MIA PaCa-2) and murine (PANC-02) PDAC cell lines using a Br-dU cell proliferation assay. To elucidate the mechanism of action, effects on the expression of DNA damage and repair (pATM, γ-H2AX, p53; pATR, and Rad51) molecules were examined by Western blot. Apoptosis was assessed via Annexin V staining and flow cytometric analysis to evaluate mechanism of cell death following treatment of MIA PaCa-2 and PANC-02 cell lines. The in vivo activity of BBI-608, 5-FU + IR, alone and in combination, were evaluated in C57BL/6 mice bearing subcutaneous PANC-02 tumors. Results: The combination of BBI-608 and chemoradiotherapy significantly decreased (p<0.001) cell proliferation and induced Annexin V positive cells in both MIA PaCa-2 and PANC-02 cell lines. BBI-608 and chemoradiotherapy also decreased the activation of pATR, MDM2, and Rad51 and increased pATM, γ-H2AX and p53. Administration of BBI-608 potentiated the effects of chemoradiotherapy in vivo in mice bearing subcutaneous PANC-02 tumors as measured by tumor volume. Conclusion: These data identify a role for BBI-608 as a modulator of DNA damage and apoptosis. These effects enhanced the efficacy of chemoradiotherapy in cell lines and in vivo models of PDAC and provide rationale for continued investigation in the pre-clinical and clinical setting. Note: This abstract was not presented at the meeting. Citation Format: Batoul Farran, Ganji P. Nagaraju, Sneha G. Shipra, Shipra R. Bethi, Gregory B. Lesinski, Bassel El-Rayes. BBI-608 modulates DNA repair pathways, induces apoptosis and impacts response to 5-Fluorouracil and ionizing radiation in pancreatic cancer cells [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 713.

Abstract 205: BBI-608 enhances the activity of chemoradiotherapy in colorectal cancer pre-clinical models

Abstract Background: Chemoradiotherapy is a standard treatment modality for early stage rectal cancer (CRC). The pathologic complete response rate after chemoradiation in CRC remains low at 15%. Given the potential for BBI608 to elicit effects on multiple oncogenic cellular pathways, we hypothesized that BBI608 can sensitize colorectal cell lines to the effects of chemoradiotherapy. The aim of this study was to evaluate if BBI-608 enhances the response of chemoradiotherapy against CRC. Methods: The combined effects of BBI-608 and chemoradiotherapy [5-FU + ionizing radiation (IR)] were evaluated in CRC cell lines (HCT 116 and HT-29) using Br-dU cell proliferation and clonogenic assays. Effects on the expression of DNA damage and repair (pATM, pATR, Rad51 and γ-H2AX) molecules and angiogenesis (pSTAT-3 and VEGF) pathways were examined by Western blot. Egg CAM assay was also performed to evaluate effects of the drugs on angiogenesis (quantification was performed by AngioQuant software). The in vivo efficacy of BBI-608 alone or combined with 5-FU + IR, was evaluated in nude mice bearing subcutaneous human HCT116 xenograft tumors Results: The combination of BBI-608 and chemoradiotherapy significantly decreased cell proliferation (p<0.001), colony formation (p<0.001) and inhibited angiogenesis (p<0.001) in CRC cell lines as compared to chemoradiotherapy. BBI-608 decreased angiogenesis as determined by both matrigel plug and egg CAM assays. Immunoblot analysis indicated BBI-608 and chemoradiotherapy also decreased expression of pSTAT-3, MDM2, Rad51 and VEGF while increasing pATM, γ-H2AX and p53, in comparison to chemoradiotherapy alone. In animal models, combined therapy with BBI-608 and 5-FU + IR led to significant inhibition of longitudinal tumor growth as compared to chemoradiotherapy alone (p<0.001). No overt signs of systemic toxicity were evident and animals showed no loss of body weight in any of the treatment groups, indicating the combination was well-tolerated. Conclusion: These observations provide preclinical proof-of-principle data that BBI-608 can enhance the anti-tumor effects of chemoradiotherapy. BBI608 plus chemoradiotherapy is a rational approach for potential future development in early stage rectal cancer. Citation Format: Ganji Purnachandra Nagaraju, Rajitha Balney, Shipra R. Bethi, Sneha Govardhanagiri, Gregory B. Lesinski B. Lesinski, Bassel F. El-Rayes. BBI-608 enhances the activity of chemoradiotherapy in colorectal cancer pre-clinical models [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 205.

Over expression of DNA damage and cell cycle dependent proteins are associated with poor survival in patients with adrenocortical carcinoma

Adrenocortical carcinoma is a rare and aggressive malignancy with poor survival. With limited treatment options and high risk of relapse, identifying improved targets and therapies for adrenocortical carcinoma is important. We hypothesized that analysis of the database of The Cancer Genome Atlas could identify important novel biomarkers for improved therapeutic targeting of adrenocortical carcinoma. We utilized the University of Alabama interactive web resource to identify novel biomarkers observed in 79 adrenocortical carcinoma patients. Identified biomarkers were then examined for prognostic correlations using the cBioPortal and analyzed for statistical significance using STATA 13.0. The Cancer Genome Atlas data mining in the University of Alabama interactive web resource for pathways associated with poor survival of patients with adrenocortical carcinoma revealed significant upregulation of genes involved in DNA damage and regulation of cell-cycle pathways, such as AURKA, AURKB, CDK1, CDK4, CDK6, PLK1, CHEK1, CHEK2, CDC7, BUB3, and MCM3 (P < .001-.05). On outcome correlation, greater expression levels of all the genes except CDK4 were associated with worse survival compared with medium or low levels of gene expression (P < .001 all) irrespective of age orsex. Consistent with our University of Alabama interactive web resource findings, data mining in the cBioPortal also revealed upregulation of genes regulating DNA-damage and cell cycle-related genes in 82% of patients (z score = 1.5). Large data mining from the The Cancer Genome Atlas and cBioPortal databases identified overexpression of genes involved in DNA damage and those regulating pathways of the cell cycle, which correlated with poorer overall survival in adrenocortical carcinoma patients.

Abstract 4086: RNAseq hiPSC-cardiomyocytes reveals altered expression of DNA damage and cell cycle genes in response to doxorubicin

Abstract Introduction: Doxorubicin is a highly effective chemotherapeutic agent, but its use is limited due to cumulative and dose-dependent effects that can lead to irreversible heart damage. Despite efforts in understanding the mechanisms, the precise genes and pathways implicated in doxorubicin-induced cardiotoxicity still remain elusive. We hypothesized that by exploring time- and dose-dependent changes in gene expression in human induced pluripotent stem cell derived-cardiomyocytes (hiPSC-CM), we can identify genes and signaling pathways related to doxorubicin-induced cardiotoxicity. Methods: hiPSC-CM were treated with 0, 50, 150, or 450 nM of doxorubicin for 2, 7, or 12 days then subjected to RNA sequencing for assessment of gene expression. Sequenced reads were aligned to the human reference genome (GRCh37) with STAR, and a read count matrix was then generated with the R package GenomicAlignments. Global variation and differential gene expression analyses were performed using DESeq2 software (P&amp;lt;0.05). For time course analysis, log2 fold-changes in gene expression for pairwise comparisons between any two time points or doses were calculated using DESeq2 and a Time x Dosage interaction model. For Ingenuity Pathway Analysis of time course genes, input genes were selected based on a cutoff &amp;gt; 2.0 and &amp;lt;-2.0 average log2 fold-change in expression. Results: Hierarchical clustering of expression profiles of 40,861 genes produced by DESeq2 revealed 192 genes downregulated following doxorubicin exposure related to mitotic roles of polo-like kinase, cell cycle control chromosomal replication, and DNA damage checkpoint regulation pathways. This included a set of nine genes with the highest variation in expression, MCM5, PRC1, NUSAP1, CENPF, CCNB1, MELK, AURKB, RACGAP1, and TROAP, with functions in microtubule stabilization and cell cycle regulation. Following a 2-day exposure to doxorubicin, 242 genes, including the top nine genes, were differentially expressed between untreated and treated with enrichment in ribosomal processes, DNA repair and damage, and oxidation-reductase activity. Time course analysis revealed 289 genes between 0 nM and 150 nM on days 2, 7 and 12 with enrichment in fatty acid oxidation, MIF-mediated glucocorticoid regulation, and relaxin signaling pathways. Transcription regulator, SMARCA4, was identified as the only upstream regulator with elevated expression following a later recovery time (day 7 to day 12) compared to shorter or earlier time point comparisons at 150 nM dose. Conclusion: Overlapping genes and pathways identified in these analyses provide additional support for a role of reactive oxygen species and mitochondrial dysfunction linked to DNA damage in doxorubicin-induced cardiotoxicity. This study also warrants further investigation of SMARCA4 since it is a direct regulator of genes with overlapping functions described for the top nine genes. Citation Format: Monica E. Reyes, Jianzhong Ma, Megan L. Grove, Joann L. Ater, Alanna C. Morrison, Michelle A. Hildebrandt. RNAseq hiPSC-cardiomyocytes reveals altered expression of DNA damage and cell cycle genes in response to doxorubicin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4086. doi:10.1158/1538-7445.AM2017-4086

Abstract P1-07-05: Integrated transcriptional analysis of the triple negative 'proliferation paradox': High proliferation, chemosensitivity, and poor prognosis

Abstract Background: In triple-negative breast cancers (TNBC), high proliferation is associated with greater chemosensitivity but, paradoxically, also associated with poor prognosis. We hypothesized that this subset of TNBC has distinct transcriptional features that contribute to poor prognosis. Approach: To evaluate transcriptional signatures associated with this 'proliferation paradox,' we identified 17 study cohorts of TNBC treated with neoadjuvant chemotherapy (NAC) that reported receptor status, pathologic response, and had expression data from biopsies obtained prior to NAC (n=446). In 6 studies, distant metastasis-free survival (DMFS) data was available for 235 patients with a median follow-up of 31.2 months. We calculated scores for 135 published gene expression signatures for each tumor and evaluated the association with response to chemotherapy and DMFS. Results: Using recursive partitioning to develop a model of response using a training set (n=340), six of the 135 expression signatures stratify primary tumors into four groups based on signatures of proliferation, BRCA1 mutation, immune, luminal, Ras, and PI3K phenotypes (Table 1.). Response to NAC ranged from 11% to 61% pCR/RCB-I and results were highly concordant when applied to a validation set (n = 106, p = 0.006). The group that was highly proliferative but chemoresistant ('resistant' group) had a distinct transcriptional profile, including lower 'BRCA-ness' and DNA damage expression signatures with higher Ras and stem cell signatures. The 'resistant' group had the poorest DMFS (HR 2.48 [1.52-4.06]; log-rank p=0.002) and this poor survival was validated among chemotherapy-treated TNBCs in a separate dataset, METABRIC. Analyses of only patients with residual disease after NAC demonstrated that the 'resistant' group remained poorest prognosis, with median DMFS of only 31 months from diagnosis. Conclusions: Using a novel approach to categorize primary TNBC tumors based on six signatures, we can effectively distinguish subgroups with higher versus lower pCR rates. One specific group demonstrated high proliferation but low response to chemotherapy and particularly poor survival. This group demonstrates expression signatures implicating DNA damage repair, stemness, and Ras pathway activity as potential mediators of the phenotype. We identify specific molecular characteristics for investigation in patients within a poor prognosis subgroup of TNBC. Table 1. Proportion Pathologic Complete Response or RCB-I and Survival Low ProlifHigh Prolif / ResistantHigh Prolif / SensitiveHigh ImmuneSignature StratificationLow GGI + High LuminalHigh GGI + Low BRCA1mut or High RasHigh GGI + High PI3K or Low RasHigh TNBC ImmunepCR/RCB-I rate: Training Set11/105 (10.5%)26/127 (20.5%)42/81 (51.9%)16/27 (59.3%)pCR/RCB-I rate: Validation Set3/23 (13.0%)11/45 (24.4%)13/29 (44.8%)6/9 (66.7%)pCR/RCB-I rate: TOTAL14/128 (10.9%)37/172 (21.5%)55/110 (50.0%)22/36 (61.1%)Overall Survival (n=235)Hazard Ratio (95% CI)1.62 (0.99-2.64)2.48 (1.52-4.06)(ref.)0.47 (0.29-0.77)Signatures GGI (Sotiriou, JNCI 2006); Luminal (Lim, Nat Med 2009); BRCA1 mutation (van't Veer, Nature 2002); Ras (Pratilas, PNAS 2009); PI3K (Gatza, PNAS 2010), TNBC Immune (Lehmann, JCI 2011) Citation Format: Stover DG, Selfors LM, Winer EP, Partridge AH, Barry WT. Integrated transcriptional analysis of the triple negative 'proliferation paradox': High proliferation, chemosensitivity, and poor prognosis [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-07-05.

Abstract 3790: Targeting the one-carbon metabolism protein MTHFD2 for cancer therapy: Exploiting the unique redox status of cancer cells

Abstract Two well-established hallmarks of cancer are the need for nucleotides to support continuous cell proliferation and the requirement for cancer cell redox balance. In that respect cancer cells are characterized by an increased rate of reactive oxygen species (ROS) production which can be toxic to cancer cells. In this sense, cancer cells often display an increased ROS scavenging capacity, through antioxidants and NADPH that prevents ROS levels from reaching cytotoxic levels. Mitochondrial methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) is a critical component of one-carbon metabolism and promotes the reaction of 5, 10-methylene tetrahydrofolate to 10-formyl tetrahydrofolate which is coupled to purine synthesis and the production of NAD(P)H. In its role, MTHFD2 is required for maintaining nucleotide biosynthesis and cancer cell redox balance. Recent studies have shown that MTHFD2 expression is elevated in many cancers compared to normal tissue and expression is correlated with poor survival in breast cancer. In order to validate MTHFD2 as a potential cancer target we show that genetic knockdown of MTHFD2 led to impaired cancer cell survival and proliferation. In breast cancer cells MTHFD2 inhibition caused a decrease in NADPH, an increase in the levels of oxidized glutathione and also promoted the expression of DNA damage and death markers specifically in cancer cells with high MTHFD2 protein expression. Interestingly, ROS-scavenging antioxidants reversed these phenotypes in the presence of MTHFD2 knockdown. Normal cells and low MTHFD2 expressing breast cancer cells had a higher tolerance for the inhibition of the protein. Based on our data, MTHFD2 inhibition leads to the impairment of cancer cells’ antioxidant capacity and ROS-mediated cell death. Therefore, MTHFD2 inhibition may have clinical potential for the treatment of patients with breast cancer, and potentially various other cancers. Citation Format: Andrea Glasauer, Michael Steckel, Andrea Haegebarth, Marcus Bauser, Luisella Toschi. Targeting the one-carbon metabolism protein MTHFD2 for cancer therapy: Exploiting the unique redox status of cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3790.

Alleviation of Aflatoxin B1-Induced Genomic Damage by Proanthocyanidins via Modulation of DNA Repair.

In order to study the mechanisms underlying the alleviation of aflatoxin B1-induced genomic damage by proanthocyanidins (PAs), we examined the modulation of oxidative DNA damage induced by aflatoxin B1 in PAs-pretreated animals. The effects of PAs on changes in the expression of DNA damage and repair genes induced by aflatoxin B1 were also evaluated in rat marrow cells. Administration of PAs before aflatoxin B1 significantly mitigated aflatoxin B1-induced oxidative DNA damage in a dose-dependent manner. Aflatoxin B1 treatment induced significant alterations in the expression of specific DNA repair genes, and the pre-treatment of rats with PAs ameliorated the altered expression of these genes. Conclusively, PAs protect against aflatoxin B1-induced oxidative DNA damage in rats. These protective effects are attributed to the antioxidant effects of PA and enhanced DNA repair through modulation of DNA repair gene expression. Therefore, PAs are a promising chemoprotective agent for averting genotoxic risks associated with aflatoxin B1 exposure.

Imatinib enchances the sensitivity of gastrointestinal stromal tumors to topoisomerase II inhibitors

Objective: to study the sensitivity of gastrointestinal stromal tumors (GISTs) to the topoisomerases type II inhibitors and ability of imatinib to enhance GISTs sensitivity to the chemotherapeutic drugs indicated above. Subjects and Methods. We studied the sensitivity of gastrointestinal stromal tumors (GISTs) to the topoisomerases II inhibitors and ability of imatinib to enhance GISTs sensitivity to these chemotherapeutic agents. The expression of DNA damage and repair (DDR) markers was examined by western-blotting. Cleaved forms of poly (ADP-rybose) polymerase and caspase-3 were served as an apoptotic markers measured by western blotting. Amount of apoptotic cells was counted by flow cytometry analysis by using a propidium iodide DNA staining procedure and counting the numbers of hypodiploid cells. Results. We observed the sensitivity of GISTs to topoisomerase II inhibitors – doxorubicine and etoposide inducing DNA double-strand breaks and apoptotic cell death. Imatinib enhances GISTs sensitivity to topoisomerase II inhibitors. This might be due to reduced ability of GISTs to repair DNA damage by homologous recombination. Imatinib-induced reduction of Rad51 recombinase might be due to increased proteasome-dependent degradation. Conclusion. GIST cells are sensitive to topoisomerase II inhibitors (etoposide and doxorubicin) in vitro. Imatinib enhances GISTs sensitivity to the chemotherapeutic agents indicated above.

Effects of Cryopreservation on the Molecular Integrity of Ovarian Grafts

INTRODUCTION: The objective of this study was to compare the effects of slow freezing and vitrification on the molecular integrity of ovarian grafts. METHODS: Fresh bovine ovaries were processed and divided into three groups (fresh control, slow freezing, and vitrification) before transplantation. For slow freezing and vitrification, 1.5 M dimethylsulfoxide-based cryoprotectant and a two-step vitrification method were used, respectively. Two pieces of ovarian tissue were transplanted onto the back muscle of the severe combined immunodeficient mouse (six animals for each group). Ovarian grafts were recovered on day 7 and day 28 after transplantation to assess DNA damage, apoptosis, and autophagy by histology, immunohistochemistry, and Western blot analysis. RESULTS: The slow freezing group showed significantly higher normal follicle count compared with the vitrification group. There was no significant difference in apoptosis (terminal deoxynucleotide transferase-mediated dUTP nick-end labeling, caspase 3) among three groups; however, autophagy (LC3B) was decreased in both slow freezing and vitrification groups. On day 28, a significant decrease in LC3B in the vitrification group was noted compared with the slow freezing group. There was no difference in expression of DNA damage and repair markers (γH2AX, Rad51, PCNA) and vascular density makers (CD31). CONCLUSION: The expression pattern of biochemical markers for DNA damage and repair and apoptosis in ovarian grafts was similar between three groups. There is not yet strong evidence that vitrification is better than slow freezing. Therefore, further improved vitrification protocols are needed.

Interactive effects of ultraviolet-B radiation and pesticide exposure on DNA photo-adduct accumulation and expression of DNA damage and repair genes in Xenopus laevis embryos

Pesticide use and ultraviolet-B (UVB) radiation have both been suggested to adversely affect amphibians; however, little is known about their interactive effects. One potential adverse interaction could involve pesticide-induced dysregulation of DNA repair pathways, resulting in greater numbers of DNA photo-adducts from UVB exposure. In the present study, we investigated the interactive effects of UVB radiation and two common pesticides (endosulfan and α-cypermethrin) on induction of DNA photo-adducts and expression of DNA damage and repair related genes in African clawed frog (Xenopus laevis) embryos. We examined 13 genes that are, collectively, involved in stress defense, cell cycle arrest, nucleotide excision repair (NER), base excision repair, mismatch repair, DNA repair regulation, and apoptosis. We exposed X. laevis embryos to 0, 25, and 50μg/L endosulfan or 0, 2.5, and 5.0μg/L α-cypermethrin for 96h, with environmentally relevant exposures of UVB radiation during the last 7h of the 96h exposure. We measured the amount of cyclobutane pyrimidine dimers (CPDs) and mRNA abundance of the 13 genes among treatments including control, pesticide only, UVB only, and UVB and pesticide co-exposures. Each of the co-exposure scenarios resulted in elevated CPD levels compared to UVB exposure alone, suggesting an inhibitory effect of endosulfan and α-cypermethrin on CPD repair. This is attributed to results indicating that α-cypermethrin and endosulfan reduced mRNA abundance of XPA and HR23B, respectively, to levels that may affect the initial recognition of DNA lesions. In contrast, both pesticides increased transcript abundance of CSA and MUTL. In addition, mRNA abundance of HSP70 and GADD45α were increased by endosulfan and mRNA abundance of XPG was increased by α-cypermethrin. XPC, HR23B, XPG, and GADD45α exhibited elevated mRNA concentrations whereas there was a reduction in MUTL transcript concentrations in UVB-alone treatments. It appeared that even though expression of XPC and CSA were induced by exposure to UVB or pesticides, XPA was the limiting factor in the NER pathway. Our results suggest that pesticides may increase the accumulation of UVB-induced DNA photo-adducts and one likely mechanism is the alteration of critical NER gene expression. The present study provides important implications for evaluating the combined risks of pesticide usage and potentially increasing UVB radiation in aquatic ecosystems.

The effect of shore location on biomarker expression in wild Mytilus spp. and its comparison with long line cultivated mussels

Biomarkers are a common tool in the assessment of potential effects of contaminants in aquatic organisms. In order to identify the effects of anthropogenic pollution it is essential to identify background levels and to know the range of natural variability in the biomarker response. In this study, we examined various biomarkers of stress (glutathione S-transferase and metallothionein), damage (lipid peroxidation (LPO) and DNA damage (DNA)) and reproduction (vitellin-like proteins) in marine mussels (Mytilus spp.) from four locations along a vertical transect from high to low shore and compared them with cultivated long line mussels. High shore and cultivated mussels showed significantly higher LPO and DNA damage expression than the low shore mussels indicating a level of oxidative stress resulting from mussel location. Significant effects in physiological endpoints were also found. This study highlights the need to consider the diversity of natural environmental stress factors when using biomarkers in environmental assessment.

DNA damage and cell necrosis induced by naphthalene due to the modulation of biotransformation enzymes in an estuarine crab Scylla serrata

The sublethal effect of naphthalene (2.5, 5, and 10 mg L(-1)) was studied in an estuarine crab Scylla serrata with reference to macromolecular changes. Biotransformation enzymes such as cytochrome P450, cytochrome b(5), NADPH cytochrome P450 reductase, aryl hydrocarbon hydroxylase, glutathione-S-transferase, and UDP-glucuronyl transferase were elevated in the hepatopancreas of naphthalene-exposed crabs in comparison with control. Remarkable amount of DNA damage and cell necrosis was observed in hepatopancreas, hemolymph, and ovary of the crabs exposed to naphthalene, when compared with control. For all the parameters studied, a concentration-dependent gradient of the changes was observed. The expression of DNA damage and cell necrosis suggests an increased production of oxidants during naphthalene metabolism.

Abstract 780: Atorvastatin Activates A Novel Nbs1-dependent Mechanism Of Accelerating Dna Repair In Atherosclerosis

There is increasing evidence that reactive oxidant species (ROS) and DNA damage promote the development and complications of atherosclerosis. Although statin therapy reduces both ROS and DNA damage in atherosclerosis, the mechanism of this effect is unknown. We first examined expression of DNA damage and repair markers in vascular smooth muscle cells (VSMCs) of human atherosclerotic plaques. With increasing disease severity, there was increased VSMC expression of the DNA repair markers P-ATM/ATR substrate and P-H2AX from 2.7%±2.2 and 0.5±0.71 [mean±SEM] (AHA Grade I/II), to 21%±3.5 and 36.5±2.1 (Grade III) lesions, and 86.5%±0.7 and 69.3±7.6 (Grade IV/V). Cultured plaque VSMCs also showed a 1.5 fold increased oxidant stress; a 4.4 fold increased double-stranded DNA breaks, and expression of P-H2AX by Western blots. ROS analogues induced a robust DNA damage response in VSMCs, characterised by lengthening of tails on COMET assay, and activation of ATM and P-H2AX, with completion of repair by 6 hours. Atorvastatin pre-treatment accelerated DNA repair by approximately 2 hours without inhibiting ROS induction or DNA damage, and markedly accelerated the kinetics of nibrin (NBS-1) and P-H2AX activation, both proteins recruited to sites of DNA damage, by preventing degradation of NBS-1. Atorvastatin induced phosphorylation of HDM2, an E3 ligase and putative regulator of NBS-1 stability, and siRNA knockdown of HDM2 replicated the effect of atorvastatin on NBS-1. The ability of atorvastatin to accelerate repair was completely dependent upon NBS-1, as atorvastatin was ineffective in cells either null or expressing constitutively active NBS-I. In summary, we have demonstrated a novel NBS-1-dependent mechanism by which statins accelerate DNA repair in atherosclerosis, through HDM2 phosphorylation and stabilisation of NBS-1. We believe that both NBS-1 and HDM2 are critical to DNA repair in atherosclerosis.

Partial volume rat lung irradiation: Temporal fluctuations of in-field and out-of-field DNA damage and inflammatory cytokines following irradiation

Purpose: The current study investigated the early activation of inflammatory cytokines and macrophages in different regions of the lung following partial volume irradiation. We examined temporal fluctuations in DNA damage, cytokine expression and macrophage activation during 16 weeks post-irradiation.Materials and methods: We irradiated the lower lung of Sprague-Dawley rats with 10 Gy. A micronucleus assay was used to examine DNA damage. Real-time Reverse Transcription-Polymerase Chain Reaction (RT-PCR) was used to analyse the RNA expression of Interleukin-1 alpha (IL-1α), Interleukin-1 beta (IL-1β), Interleukin-6 (IL-6), Tumour Necrosis Factor alpha (TNF-α) and Transforming Growth Factor beta (TGF-β) relative to Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH). The activation of macrophages was determined using the antibody ED-1 for immunohistochemical analysis.Results: The expression of DNA damage, the activation of macrophages and the expression of inflammatory cytokines all fluctuated in a cyclic pattern. The initial induction of cytokine expression and the activation of macrophages occurred at very early times (1 h) following irradiation. Waves of cytokine expression and macrophage activation were also seen at later times (up to 16 weeks) following irradiation. DNA damage also occurred in a cyclic pattern though this was less pronounced out-of-field. The levels of cytokines and activated macrophages were elevated to a similar degree both in- and out-of-field, whereas there was a greater micronuclei yield in-field than out-of-field.Conclusions: An inflammatory response triggered by the partial volume irradiation occurs in the whole rat lung at very early times following irradiation and is maintained in a cyclic pattern to later times when the onset of functional symptoms is expected. We hypothesize that Reactive Oxygen Species (ROS) induced by this response play an important role in the induction of both in-field and out-of-field DNA damage.

DNA damage in a human population affected by chronic psychogenic stress

The effects of chronic psychogenic stress on the expression of DNA damage and cellular response to the damage were investigated. Using the comet assay, basal DNA damage was found to be similar in lymphocytes of both affected and non-affected populations (n=30 in both groups). The induction of DNA damage in lymphocytes by external factors (H2O2 and γ-irradiation), was also investigated. In these studies, cells were treated with 50, 100 and 150 μM H2O2 for 5 minutes or with 0.8, 2.5 and 4.2 Gy γ-rays. A significant difference was found between the chronically stressed and the control populations, indicating the enhanced sensitivity of the former population. Cells were also held for 2 hours after the treatment, allowing time for the cells to deal with the induced DNA damage. Based on the level of residual DNA strand breaks, cells from the stressed population had more breaks than the controls. Gender does not alter these findings. In conclusion, our data indicate that cells from the stressed population were more sensitive to the induction of DNA damage and had higher level of residual damage. Therefore, stress conditions may cause the affected individuals to be susceptible to environmental mutagenic agents.

EFFECTS OF SIMULATED PULMONARY SURFACTANT ON THE CYTOTOXICITY AND DNA-DAMAGING ACTIVITY OF RESPIRABLE QUARTZ AND KAOLIN

Respirable-sized quartz and kaolin dusts were pretreated with simulated pulmonary surfactant dispersions of dipalmitoyl phosphatidylcholine (DPPC) in saline to model the conditioning of particles depositing in alveolar regions of the lung. DPPC-treated and untreated dusts were used to challenge lavaged rat pulmonary alveolar macrophages in vitro. Cytotoxicity was determined over a 5-d period using both total and viable cell counts from a fluorescence-based viability assay. DNA damage, as an indication of genotoxicity, was determined over a 7-d period by the single-cell gel electrophoresis assay. Untreated quartz and kaolin both expressed a significant and potent cytotoxicity, which increased with concentration and time. DPPC-surfactant pretreatment delayed significant expression of this cytotoxicity until 3 to 5 d after challenge. Untreated quartz also caused DNA damage, which increased with concentration and time. DPPC-surfactant treatment of quartz delayed most DNA damage expression to 5 and 7 d. Untreated kaolin expressed weaker activity for DNA damage, significant at the highest concentration through 5 d, and at the higher concentrations on d 7. Surfactant treatment delayed most kaolin activity for DNA damage to 7 d after challenge.

Influence of sunlight on DNA damage in mussels exposed to polycyclic aromatic hydrocarbons

DNA single-strand break levels were compared in mussels deployed in San Diego Bay. Mussels were deployed at two locations, a reference site and at a location known to have high concentrations of PAHs. Two vertically oriented lines of mussels were deployed at each location for a period of 32 days. Mussels were attached to the lines at depths of 0.3, 1 and 3 m. One line was fully exposed to ambient sunlight, the other was shaded from sunlight to examine the role of PAH light activation on the expression of DNA damage in hemocytes and whole organism growth. Damage and growth measurements were made on days 0, 5, 14, and 32. Temperatures at the stations varied only by ≈0.5 °C. Hydrocarbon levels at the high PAH station were on average six times higher than the reference station. Slower growth and higher DNA damage was observed in cells from light exposed mussels at the PAH contaminated site. These effects decreased with depth. High levels of DNA damage could be induced with low intensity ultraviolet light exposure of cells from depurated or shaded mussels exposed to PAHs. DNA damage in mussels from the high impact site could be reduced to baseline levels after 24 h depuration in the dark. These results confirm previous findings showing increased DNA damage in PAH and sunlight exposed mussels, and demonstrate the significant impact of photoactivation on the health of these organisms.