Hydrogen peroxide induces DNA single strand breaks in respiratory epithelial cells

The respiratory epithelium is often exposed to oxidant gases, including ozone from photochemical smog and toxic oxygen metabolites released from neutrophils recruited in conditions of airway inflammation. We evaluated DNA single strand break formation by alkaline elution as a measure of oxidant-induced DNA damage to bronchial epithelial cells. Human AdenoSV-40-transformed bronchial epithelial cells (BEAS), subclone R1.4 or nonhuman primate bronchial epithelial cells were cultured in growth factor supplemented Ham's F12 medium on polycarbonate filters. DNA was labeled by incubation with [³H]thymidine. Cells were incubated for 1 h in HBSS or HBSS and increasing concentrations of hydrogen peroxide (H₂O₂). Cells incubated in H₂O₂ demonstrated dose-dependent increases in strand break formation, and BEAS cells were more sensitive to H₂O₂-induced injury than primary bronchial epithelial cells. The addition of catalase or preincubation of cells with the iron chelator desferoxamine prevented H₂O₂-induced strand breakage. DNA strand break formation may be an important mechanism of oxidant injury in respiratory epithelial cells.This work was supported by NIEHS grant ES-00628 and California Primate Research Center Base grant. Portions of this work were presented at the American Thoracic Society annual meeting, May 1992, Miami, Florida.     

Clothianidin induces DNA damage and oxidative stress in bronchial epithelial cells

Clothianidin (CHN) is a member of the neonicotinoid group of insecticides. Its oxidative and DNA damage potential for human lung cells are not known. Therefore, the present study was designed to examine the effects of CHN on DNA damage and oxidative stress in human bronchial epithelial cells (BEAS-2B) treated with CHN for 24, 72, and 120 hr. Our results indicate that CHN decreased cell viability in a concentration-dependent manner. CHN induced DNA single-strand breaks because alkaline comet parameters such as tail intensity, DNA in the tail, tail moment, and tail length increased. All CHN concentrations also significantly induced the formation of DNA double-strand breaks (DSBs) because it increased phosphorylated H2AX protein foci for all treatment times and p53-binding protein 1 foci for all treatments except for the lowest concentration (0.15 mM) of 120-hr treatment. DNA damage caused by DNA DSBs was not repaired in a 24-hr recovery period. CHN also induced oxidative stress by decreasing reduced glutathione and increasing lipid peroxidation. These results make it necessary to conduct studies about the detailed carcinogenic potential of CHN in humans because it can induce both oxidative and DNA damage.     

The kinetics of repair of oxidative DNA damage (strand breaks and oxidised pyrimidines) in human cells

Single cell gel electrophoresis is a sensitive method for detecting DNA strand breaks. Cells embedded in agarose are converted to nucleoids by treating with detergent and high salt. DNA breaks render the nucleoid DNA susceptible to extension by electrophoresis, forming ‘comets’. We find that when DNA breakage resulting from H₂O₂ treatment is examined, freshly isolated normal human lymphocytes are relatively resistant compared with transformed human cells. When incubated after treatment with H₂O₂, HeLa cells repair most strand breaks within 1 h, and a substantial fraction of the oxidised pyrimidines (detected by converting them to DNA breaks with endonuclease III) within 4 h. However, lymphocytes are less proficient at repair; during incubation for 4 h after treatment with H₂O₂, no detectable removal of endonuclease III-sensitive sites is seen. While the addition of deoxyribonucleosides promotes completion of repair of UV damage by lymphocytes, it has no significant effect on repair of oxidative damage.     

Cigarette smoke extract induces DNA damage but not apoptosis in human bronchial epithelial cells

Whether DNA damage caused by cigarette smoke leads to repair or apoptosis has not been fully elucidated. The current study demonstrates that cigarette smoke induces single-strand DNA damage in human bronchial epithelial cells. Cigarette smoke also stimulated caspase 3 precursors as well as intact poly (ADP-ribose) polymerase (PARP) production, but did not activate caspase 3 or cleave PARP, while the alkaloid camptothecin did so. Neither apoptosis nor necrosis was induced by cigarette smoke when the insult was removed within a designated time period. In contrast, DNA damage following cigarette smoke exposure was repaired as evidenced by decreasing terminal dUTP-biotin nick-end labeling positivity. The PARP inhibitor, 3-aminobenzamide blocked this repair. Furthermore, cells subjected to DNA damage were able to survive and proliferate clonogenically when changed to smoke-free conditions. These results suggest that cigarette smoke-induced DNA damage in bronchial epithelial cells is not necessarily lethal, and that PARP functions in the repair process. Our data also suggest that the potency of cigarettes as a carcinogen may result from their ability to induce DNA damage while failing to trigger the apoptotic progression permitting survival of cells harboring potentially oncogenic mutations.     

Hydrogen peroxide induces oxidative DNA damage in rat type II pulmonary epithelial cells.

Type II epithelial cells, which line the alveolar surface of the lung, are exposed to a variety of potentially mutagenic and carcinogenic insults. The purpose of this study was to determine if type II cells are susceptible to oxidative DNA damage in vitro. Treatment of cultured rat type II lung epithelial cells with hydrogen peroxide led to increased concentrations (nmol/mg DNA) of 12 of 14 monitored DNA base modifications, suggesting oxidative damage by the hydroxyl radical. These base modifications are typically associated with oxidative stress, and elevated levels have been correlated with mutagenesis and carcinogenesis. These data demonstrate that type II cells are indeed vulnerable to oxidative DNA damage.     

Gossypol induces DNA strand breaks in human fibroblasts and sister chromatid exchanges in human lymphocytes in vitro.

The male contraceptive agent gossypol was found to induce a dose related increase of DNA strand breaks in human fibroblasts in vitro at concentrations of 5 to 40 micrograms/ml. The effect was reduced in the presence of 2% fetal calf serum. A weak but reproducible increase in the SCE frequency was found in human lymphocytes treated for 1 hour in serum-free medium with 0.04 to 4 micrograms/ml of gossypol.     

Radiation-induced double strand breaks and subsequent apoptotic DNA fragmentation in human peripheral blood mononuclear cells

In case of accidental radiation exposure or a nuclear incident, physical dosimetry is not always complete. Therefore, it is important to develop tools that allow dose estimates and determination that are based on biological markers of radiation exposure. Exposure to ionizing radiation triggers a large-scale activation of specific DNA signaling and repair mechanisms. This includes the phosphorylation of γH2AX in the vicinity of a double-strand break (DSB). A DNA DSB is a cytotoxic form of DNA damage, and if not correctly repaired can initiate genomic instability, chromosome aberrations, mutations or apoptosis. Measurements of DNA DSBs and their subsequent repair after in?vitro irradiation has been suggested to be of potential use to monitor cellular responses. The bone marrow and the blood are known to be the most radiosensitive tissues of the human body and can therefore be of particular importance to find radiation-induced biological markers. In the present study, changes in H2AX phosphorylation and apoptosis of irradiated human peripheral blood mononuclear cells (PBMCs) were analyzed. Freshly isolated PBMCs from healthy donors were irradiated with X-rays (0.1, 0.25, 0.5, 1, 2 and 4?Gy). The phosphorylation of γH2AX was measured at different time points (0, 0.25, 1, 2, 4, 6 and 24?h) after irradiation. We detected a linear dose-dependency of γH2AX phosphorylation measured by γH2AX foci scoring using immunofluorescence microscopy as well as by γH2AX fluorescence detection using flow cytometry. Apoptosis was detected by measuring DNA fragmentation at different time points (0, 24, 48, 72, 96?h) after X-irradiation using DNA ladder gel electrophoresis. The apoptotic DNA fragmentation increased in a dose-dependent manner. In conclusion, DNA DSBs and subsequent apoptotic DNA fragmentation monitoring have potential as biomarkers for assessing human exposure in radiation biodosimetry.     

DNA damage response and MCL-1 destruction initiate apoptosis in adenovirus-infected cells

Expression of adenovirus E1A deregulates cell proliferation to facilitate viral DNA replication, prompting the initiation of apoptosis signaled primarily through proapoptotic BAK in productively infected cells. We demonstrate here that in uninfected cells, BAK is complexed with the anti-apoptotic BCL-2 family member Myeloid Cell Leukemia 1 (MCL-1). E1A expression during infection resulted in the specific down-regulation of MCL-1 through destabilization of the protein and loss of the mRNA. Upon loss of the MCL-1-BAK complex, BAK complexed with either BAX in proapoptotic E1B mutant adenovirus-infected cells, or with the adenovirus BCL-2 homolog E1B 19K in cells infected with the wild-type virus in which apoptosis is inhibited. Loss of MCL-1 was required to initiate the apoptotic pathway in infected cells as restoration of MCL-1 expression rescued infected cells from E1A-induced apoptosis. Analogous to E1A expression, DNA damage down-regulates MCL-1, and adenovirus infection resulted in the accumulation of phosphorylated H2AX and ataxia-telangiectasia mutant protein (ATM), hallmarks of DNA double-strand breaks. Thus, MCL-1 may function by maintaining BAK in an inactive state, and the loss of MCL-1 upon activation of the DNA damage response, perhaps through replication stress induced in virus infected cells, may be required to initiate the apoptotic response.     

高糖对THP-1细胞及平滑肌细胞肿瘤坏死因子相关凋亡诱导配体受体4表达的影响

目的：探讨高糖对单核/巨噬细胞系THP-1细胞及人主动脉平滑肌细胞肿瘤坏死因子相关凋亡诱导配体受体4(TRAIL-R4)表达的影响。方法：用PMA孵育THP-1细胞，诱导其分化成为巨噬细胞。流式细胞仪检测成功诱导的巨噬细胞粘附分子CD11b及CD11c。采用不同糖浓度干预THP-1细胞，用Western blotting技术检测TRAIL-R4的表达。用25 mmol/L 高糖培养液在不同时点孵育人主动脉平滑肌细胞，观察TRAIL-R4蛋白表达的变化。用PKC激动剂干预THP-1细胞后，用Western blotting技术检测TRAIL-R4的表达。结果：佛波酯(PMA)孵育THP-1细胞48 h可诱导其分化成为巨噬细胞。20 mmol/L 高糖明显上调人THP-1细胞TRAIL-R4的表达。高糖对人主动脉平滑肌细胞TRAIL-R4表达上调的影响随着刺激时间的增加而增加。PKC激活后THP-1细胞TRAIL-R4的表达明显上调。结论：高糖上调TRAIL-R4蛋白表达，TRAIL-R4通过抑制巨噬细胞及平滑肌细胞的凋亡促进动脉粥样硬化。     

CUDC-907诱导胶质瘤细胞DNA损伤与自噬的实验研究

目的:探讨新型组蛋白去乙酰化酶(histone deacetylase,HDAC)/磷脂酰肌醇3-激酶(phosphatidyl-inositol 3-kinase,PI3K)双靶点抑制剂CUDC-907对人胶质瘤U251细胞DNA损伤、细胞周期及自噬的影响。方法:采用不同浓度的CUDC-907处理U251细胞24 h,MTT法检测细胞活力的变化;激光共聚焦显微镜观察DNA损伤标志物γ-H2AX在细胞内的分布;流式细胞术分析CUDC-907对细胞周期的影响;Western blot实验检测细胞内相关蛋白表达水平的变化。结果:CUDC-907能够抑制U251细胞活力。在CUDC-907处理的细胞中,蛋白激酶B(PKB)/Akt和p70核糖体蛋白S6激酶(p70s6K)的磷酸化水平降低,γ-H2AX的焦点数量与蛋白表达显著升高(P 0.05);U251细胞经CUDC-907作用后,G2/M期细胞数量增多;Western blot实验结果表明,CUDC-907促进p21的表达,同时抑制细胞周期素B1(cyclin B1)的蛋白表达和细胞分裂周期蛋白2(Cdc2)的磷酸化水平(P 0.05);另外,CUDC-907能够诱导细胞自噬,抑制自噬可促进CUDC-907诱导的DNA损伤。结论:CUDC-907能够显著抑制PI3K/Akt信号通路,诱导胶质瘤细胞发生DNA损伤,并阻滞细胞于G_2/M期,同时可诱导胶质瘤细胞发生保护性自噬。     

p38MAPK介导高糖下调肾小管上皮细胞表达BMP-7

目的:观察高糖环境中培养的原代肾小管上皮细胞骨形态发生蛋白-7(BMP-7)表达及p38丝裂原活化蛋白激酶(p38MAPK)信号通路的作用。方法:原代培养大鼠肾小管上皮细胞,随机分为正常对照组、高糖组、p38MAPK阻断剂SB202190+高糖组和高渗组,处理72h后收集贴壁细胞,免疫细胞化学检测BMP-7和纤维连接蛋白(FN)表达;Westernblotting检测p38MAPK和磷酸化p38MAPK(p-p38MAPK)的表达;RT-PCR法检测BMP-7和FNmRNA水平。结果:正常对照组肾小管上皮细胞BMP-7主要表达于细胞浆,有少量总p38MAPK与FN表达,未见p-p38MAPK;高糖状态激活了p38MAPK,p-p38MAPK蛋白表达明显增加,FN的表达也明显增多而BMP-7的表达显著减少;与SB202190共同培养72h后,p-p38MAPK的表达较高糖组减少约80%,BMP-7的表达却被显著上调,而FN的表达减少。高渗组与正常对照组比较无明显差异。结论:高糖状态下肾小管上皮细胞BMP-7蛋白和mRNA均减少,阻断p38MAPK信号通路可促进内源性BMP-7增多,提示p38MAPK可能参与高糖下调肾小管上皮细胞BMP-7的表达。     

TiO2 nanoparticles induce DNA double strand breaks and cell cycle arrest in human alveolar cells

TiO₂ nanoparticles (NPs) have the second highest global annual production (～3000 tons) among the metal‐containing NPs. These NPs are used as photocatalysts for bacterial disinfection, and in various other consumer products including sunscreen, food packaging, therapeutics, biosensors, surface cleaning agents, and others. Humans are exposed to these NPs during synthesis (laboratory), manufacture (industry), and use (consumer products, devices, medicines, etc.), as well as through environmental exposures (disposal). Hence, there is great concern regarding the health effects caused by exposure to NPs and, in particular, to TiO₂ NPs. In the present study, the genotoxic potential of TiO₂ NPs in A549 cells was examined, focusing on their potential to induce ROS, different types of DNA damage, and cell cycle arrest. We show that TiO₂ NPs can induce DNA damage and a corresponding increase in micronucleus frequency, as evident from the comet and cytokinesis‐block micronucleus assays. We demonstrate that DNA damage may be attributed to increased oxidative stress and ROS generation. Furthermore, genomic and proteomic analyses showed increased expression of ATM, P53, and CdC‐2 and decreased expression of ATR, H2AX, and Cyclin B1 in A549 cells, suggesting induction of DNA double strand breaks. The occurrence of double strand breaks was correlated with cell cycle arrest in G2/M phase. Overall, the results indicate the potential for genotoxicity following exposure to these TiO₂ NPs, suggesting that use should be carefully monitored. Environ. Mol. Mutagen. 56:204–217, 2015. © 2014 Wiley Periodicals, Inc.     

CtIP‐ and ATR‐dependent FANCJ phosphorylation in response to DNA strand breaks mediated by DNA replication

FANCJ, also called BACH1/BRIP1, is a 5′‐3′ DEAH helicase, whose mutations are known as a risk factor for Fanconi anemia and also breast and ovarian cancer. FANCJ is thought to contribute to DNA double‐strand break (DSB) repair and S‐phase checkpoint through binding to multiple partner proteins, such as BRCA1 and TopBP1, but its molecular regulation remains unclear. We focused on DNA damage‐induced phosphorylation of FANCJ and found that reagents that cause DSB or replication fork stalling induce FANCJ hyperphosphorylation. In particular, camptothecin (CPT) induced rapid and efficient FANCJ hyperphosphorylation that was largely dependent on TopBP1 and ATM‐Rad3 related (ATR) kinase. Furthermore, DNA end resection that exposes single‐strand DNA at the DSB site was required for hyperphosphorylation. Interestingly, upon CPT treatment, a dramatic increase in the FANCJ–TopBP1 complex was observed, and this increase was not alleviated even when ATR‐dependent hyperphosphorylation was suppressed. These results suggest that FANCJ function may be modulated by hyperphosphorylation in a DNA end resection‐ and ATR‐dependent manner and by FANCJ–TopBP1 complex formation in response to replication‐coupled DSBs.     

A Leptospira borgpetersenii serovar Hardjo vaccine induces a Th1 response, activates NK cells, and reduces renal colonization

Chronic infection of cattle with Leptospira borgpetersenii serovar Hardjo reduces animal production through reproductive failure and presents a persistent health threat to workers in the animal industry. Cattle are maintenance hosts for serovar Hardjo, and development of vaccines that establish long-term protective immunity has been problematic; induction of high titers of anti-serovar Hardjo antibody does not appear to be protective. Rather, development of an antigen-specific Th1 response appears to be critical for limiting renal colonization and urinary shedding of bacteria. In this study we compared two monovalent killed bacterial cell vaccines to assess long-term (12 months) protection against live serovar Hardjo challenge. Although neither vaccine prevented infection, renal colonization and urinary shedding of bacteria were reduced compared to those of control animals. Increased proliferation of CD4(+), CD8(+), and γδ T cells from vaccinated, but not control, animals was detected. In addition, NK cells from vaccinated animals and from all animals following infection, when exposed to antigen ex vivo, demonstrated a gamma interferon (IFN-γ) recall response. We propose that programming NK cells to respond quickly to L. borgpetersenii serovar Hardjo infection may be an important step toward developing protective immunity.     

EdU induces DNA damage response and cell death in mESC in culture

Recently, a novel DNA replication precursor analogue called 5-ethynyl-2′-deoxyuridine (EdU) has been widely used to monitor DNA synthesis as an alternative to bromodeoxyuridine. Use of EdU benefits from simplicity and reproducibility and the simple chemical detection systems allows excellent preservation of nuclear structure. However, the alkyne moiety is highly reactive, raising the possibility that incorporation might compromise genome stability. To assess the extent of possible DNA damage, we have analysed the effect of EdU incorporation into DNA during short- and long-term cell culture using a variety of cell lines. We show that EdU incorporation has no measurable impact on the rate of elongation of replication forks during synthesis. However, using different cell lines we find that during long-term cell culture variable responses to EdU incorporation are seen, which range from delayed cell cycle progression to complete cell cycle arrest. The most profound phenotypes were seen in mouse embryonic stem cells, which following incorporation of EdU accumulated in the G2/M-phase of the cell cycle before undergoing apoptosis. In long-term cell culture, EdU incorporation also triggered a DNA damage response in all cell types analysed. Our study shows that while EdU is extremely useful to tag sites of on-going replication, for long-term studies (i.e. beyond the cell cycle in which labelling is performed), a careful analysis of cell cycle perturbations must be performed in order to ensure that any conclusions made after EdU treatment are not a direct consequence of EdU-dependent activation of cell stress responses.     

Copper impairs biliary epithelial cells and induces protein oxidation and oxidative DNA damage in the isolated perfused rat liver

Copper is one of the major metals causing environmental contamination. Previous studies showed that copper induced toxic effects in isolated perfused rat liver models and these effects were associated with lipid peroxidation. Here we investigated whether effects of copper (at concentrations of 0.01, 0.03, and 0.1 mM of Cu(2+) in Krebs-Henseleit buffer perfusing the isolated rat liver for 60 min), were associated with biliary epithelial cell injury, as well as protein oxidation and oxidative DNA damage. The highest concentration of copper in perfusate (0.1 mM) did not allow complete evaluation of all parameters because it blocked portal flow within 30 min of perfusion, indicating severe microcirculatory disturbances. Further, copper decreased secretion of bile and it increased lactate dehydrogenase, aspartate transaminase, and alanine transaminase leakage into perfusate as well as liver weight in a dose-dependent manner. Biliary gamma-glutamyltransferase, an index of biliary epithelial cell integrity increased similarly at 0.01 and 0.03 mM copper concentrations in perfusate. Compared to controls, 0.01 and 0.03 mM concentrations of copper increased the amount of thiobarbituric acid reacting substances, a marker of lipid peroxidation, tissue protein carbonyl groups, an index of protein oxidation, and 8-oxo-7,8-dihydro-2'-deoxyguanosine, a marker of oxidative DNA damage. The results suggest that toxic effects of copper in the isolated perfused rat liver may involve biliary epithelial cells and they are associated with lipid peroxidation, protein oxidation, and oxidative DNA damage.     

Induction of DNA strand breaks and oxidative stress in HeLa cells by ethanol is dependent on CYP2E1 expression

Induction of cytochrome P4502E1 (CYP2E1) is considered to be an important mechanism by which ethanol can cause toxicity related to oxidative stress both in vivo and in vitro. In the current study, we used HeLa cells with doxycycline-regulated CYP2E1 expression to test the hypothesis that induction of CYP2E1 could lead to secondary DNA oxidation that could potentially contribute to the carcinogenicity of ethanol in vivo. Overexpression of CYP2E1 protein was not associated with oxidative stress per se as assessed by markers of lipid peroxidation (cis-parinaric acid oxidation), glutathione depletion and elevation of intracellular reactive oxygen species (dichlorofluoroscin oxidation) in the presence or absence of ethanol substrate (10 mM, 24 h). Furthermore, there was no evidence of elevation of frequency of DNA strand breaks as assessed by the comet assay. In contrast, however, after pre-incubation of cells with L-buthionine-(S,R)-sulphoximine (BSO, 10 microM) which caused a 75% reduction in intracellular reduced glutathione (GSH) levels, CYP2E1 expression resulted in oxidative stress as assessed by all of these markers and DNA strand breaks but only in the presence of ethanol (10 mM). No effect was observed under these conditions in control cells not expressing CYP2E1. Furthermore, these effects could be attenuated by co-incubation with 1-aminobenzotriazole (0.5 mM), a suicide inhibitor of P450 activity. In conclusion, in this in vitro model CYP2E1-mediated interaction with ethanol results in the intracellular oxidative stress and the formation of DNA strand breaks which are detectable in cells pre-sensitized by depletion of intracellular levels of GSH.