In vitro Study on Role of Hsp70 Expression in DNA Damage of Human Embryonic Lung Cells Exposed to Benzo[a]pyrene

Objective Benzo[a]pyrene (B[a]P), a ubiquitous environmental pollutant, is a potent procarcinogen and mutagen that can elicit tumors, leading to malignancy. Heat shock proteins (Hsp) have been shown to protect cells against damages caused by various stresses including exposure to numerous chemicals. Whether Hsps, or more specifically Hsp70, are involved in repair of B[a]P-induced DNA damage is currently unknown. Methods We assessed the potential role of the inducible form of Hsp70 in B[a]P-induced DNA damage of human embryonic lung (HEL) cells using immunoblot and the comet assay (i.e., the single cell gel electrophoresis assay). Results Exposure to B[a]P induced a dose-dependent decrease in the level of Hsp70, but a dose-dependent +-increase in DNA damage both in untreated (control) HEL cells and in cells preconditioned by a heat treatment. Heat preconditioning prior to B[a]P exposure potentiated the effect of B[a]P at a low dose (10 μmol/L), but appeared to be protective at higher doses. There was a negative correlation between Hsp70 level and DNA damage in the non-preheated as well as in the preconditioned cells. Conclusion These data suggest that exposure of HEL cells to B[a]P may induce a dose-dependent reduction in the levels of the inducible Hsp70. The detailed mechanisms for the reduction of Hsp70 levels by B[a]P and the role of Hsp70 in DNA damage under different concentrations of B[a]P remains to be determined.

The DNA damage repair complex MoMMS21-MoSMC5 is required for infection-related development and pathogenicity of Magnaporthe oryzae

The conserved DNA damage repair complex,MMS21-SMC5/6(Methyl methane sulfonate 21-Structural maintenance of chromosomes 5/6),has been extensively studied in yeast,animals,and plants.However,its role in phytopathogenic fungi,particularly in the highly destructive rice blast fungus Magnaporthe oryzae,remains unknown.In this study,we functionally characterized the homologues of this complex,MoMMS21 and MoSMC5,in M.oryzae.We first demonstrated the importance of DNA damage repair in M.oryzae by showing that the DNA damage inducer phleomycin inhibited vegetative growth,infection-related development and pathogenicity in this fungus.Additionally,we discovered that MoMMS21 and MoSMC5 interacted in the nuclei,suggesting that they also function as a complex in M.oryzae.Gene deletion experiments revealed that both MoMMS21 and MoSMC5 are required for infection-related development and pathogenicity in M.oryzae,while only MoMMS21 deletion affected growth and sensitivity to phleomycin,indicating its specific involvement in DNA damage repair.Overall,our results provide insights into the roles of MoMMS21 and MoSMC5 in M.oryzae,highlighting their functions beyond DNA damage repair.

LncRNA HOTAIR promotes DNA damage repair and radioresistance by targeting ATR in colorectal cancer

Long non-coding RNAs(lncRNAs)have been implicated in cancer progression and drug resistance development.Moreover,there is evidence that lncRNA HOX transcript antisense intergenic RNA(HOTAIR)is involved in colorectal cancer(CRC)progression.The present study aimed to examine the functional role of lncRNA HOTAIR in conferring radiotherapy resistance in CRC cells,as well as the underlying mechanism.The relative expression levels of HOTAIR were examined in 70 pairs of CRC tumor and para-cancerous tissues,as well as in radiosensitive and radioresistant samples.The correlations between HOTAIR expression levels and clinical features of patients with CRC were assessed using the Chi-square test.Functional assays such as cell proliferation,colony formation and apoptosis assays were conducted to determine the radiosensitivity in CRC cells with HOTAIR silencing after treatment with different doses of radiation.RNA pull-down assay andfluorescence in situ hybridization(FISH)were used to determine the interaction between HOTAIR and DNA damage response mediator ataxia-telangiectasia mutated-and Rad3-related(ATR).HOTAIR was significantly upregulated in CRC tumor tissues,especially in radioresistant tumor samples.The elevated expression of HOTAIR was correlated with more advanced histological grades,distance metastasis and the poor prognosis in patients with CRC.Silencing HOTAIR suppressed the proliferation and promoted apoptosis and radiosensitivity in CRC cells.HOTAIR knockdown also inhibited the tumorigenesis of CRC cells and enhanced the sensitivity to radiotherapy in a mouse xenograft model.Moreover,the data showed that HOTAIR could interact with ATR to regulate the DNA damage repair signaling pathway.Silencing HOTAIR impaired the ATR-ATR interacting protein(ATRIP)complex and signaling in cell cycle progression.Collectively,the present results indicate that lncRNA HOTAIR facilitates the DNA damage response pathway and promotes radioresistance in CRC cells by targeting ATR.

DNA Damage-driven Inflammatory Cytokines:Reprogramming of Tumor Immune Microenvironment and Application of Oncotherapy

DNA damage occurs across tumorigenesis and tumor development.Tumor intrinsic DNA damage can not only increase the risk of mutations responsible for tumor generation but also initiate a cellular stress response to orchestrate the tumor immune microenvironment(TIME)and dominate tumor progression.Accumulating evidence documents that multiple signaling pathways,including cyclic GMP-AMP synthase-stimulator of interferon genes(cGAS-STING)and ataxia telangiectasia-mutated protein/ataxia telangiectasia and Rad3-related protein(ATM/ATR),are activated downstream of DNA damage and they are associated with the secretion of diverse cytokines.These cytokines possess multifaced functions in the anti-tumor immune response.Thus,it is necessary to deeply interpret the complex TIME reshaped by damaged DNA and tumor-derived cytokines,critical for the development of effective tumor therapies.This manuscript comprehensively reviews the relationship between the DNA damage response and related cytokines in tumors and depicts the dual immunoregulatory roles of these cytokines.We also summarize clinical trials targeting signaling pathways and cytokines associated with DNA damage and provide future perspectives on emerging technologies.

Enhancing the radiosensitivity of colorectal cancer cells by reducing spermine synthase through promoting autophagy and DNA damage

BACKGROUND Colorectal cancer(CRC),the third most common cancer worldwide,has increasingly detrimental effects on human health.Radiotherapy resistance diminishes treatment efficacy.Studies suggest that spermine synthase(SMS)may serve as a potential target to enhance the radiosensitivity.AIM To investigate the association between SMS and radiosensitivity in CRC cells,along with a detailed elucidation of the underlying mechanisms.METHODS Western blot was adopted to assess SMS expression in normal colonic epithelial cells and CRC cell lines.HCT116 cells were transfected with control/SMS-specific shRNA or control/pcDNA3.1-SMS plasmids.Assessments included cell viability,colony formation,and apoptosis via MTT assays,colony formation assays,and flow cytometry.Radiosensitivity was studied in SMS-specific shRNA-transfected HCT116 cells post-4 Gy radiation,evaluating cell viability,colony formation,apoptosis,DNA damage(comet assays),autophagy(immunofluorescence),and mammalian target of rapamycin(mTOR)pathway protein expression(western blot).RESULTS Significant up-regulation of SMS expression levels was observed in the CRC cell lines.Upon down-regulation of SMS expression,cellular viability and colonyforming ability were markedly suppressed,concomitant with a notable increase in apoptotic indices.Furthermore,attenuation of SMS expression significantly augmented the sensitivity of HCT116 cells to radiation therapy,evidenced by a pronounced elevation in levels of cellular DNA damage and autophagy.Impor tantly,down-regulation of SMS corresponded with a marked reduction in the expression levels of proteins associated with the mTOR signaling pathway.CONCLUSION Knocking down SMS attenuates the mTOR signaling pathway,thereby promoting cellular autophagy and DNA damage to enhance the radiosensitivity of CRC cells.

DNA damage response-related immune activation signature predicts the response to immune checkpoint inhibitors: from gastrointestinal cancer analysis to pan-cancer validation

Objective: DNA damage response(DDR) deficiency has emerged as a prominent determinant of tumor immunogenicity. This study aimed to construct a DDR-related immune activation(DRIA) signature and evaluate the predictive accuracy of the DRIA signature for response to immune checkpoint inhibitor(ICI) therapy in gastrointestinal(GI) cancer.Methods: A DRIA signature was established based on two previously reported DNA damage immune response assays. Clinical and gene expression data from two published GI cancer cohorts were used to assess and validate the association between the DRIA score and response to ICI therapy. The predictive accuracy of the DRIA score was validated based on one ICI-treated melanoma and three pan-cancer published cohorts.Results: The DRIA signature includes three genes(CXCL10, IDO1, and IFI44L). In the discovery cancer cohort, DRIA-high patients with gastric cancer achieved a higher response rate to ICI therapy than DRIA-low patients(81.8% vs. 8.8%;P < 0.001), and the predictive accuracy of the DRIA score [area under the receiver operating characteristic curve(AUC) = 0.845] was superior to the predictive accuracy of PD-L1 expression, tumor mutational burden, microsatellite instability, and Epstein–Barr virus status. The validation cohort demonstrated that the DRIA score identified responders with microsatellite-stable colorectal and pancreatic adenocarcinoma who received dual PD-1 and CTLA-4 blockade with radiation therapy. Furthermore, the predictive performance of the DRIA score was shown to be robust through an extended validation in melanoma, urothelial cancer, and pan-cancer.Conclusions: The DRIA signature has superior and robust predictive accuracy for the efficacy of ICI therapy in GI cancer and pancancer, indicating that the DRIA signature may serve as a powerful biomarker for guiding ICI therapy decisions.

TOPK Inhibition Enhances the Sensitivity of Colorectal Cancer Cells to Radiotherapy by Reducing the DNA Damage Response

Objective Abnormal expression of T-lymphokine-activated killer cell-originated protein kinase(TOPK)was reported to be closely related to the resistance of prostate cancer to radiotherapy and to targeted drug resistance in lung cancer.However,the role of TOPK inhibition in enhancing radiosensitivity of colorectal cancer(CRC)cells is unclear.This study aimed to evaluate the radiosensitization of TOPK knockdown in CRC cells.Methods The expression of TOPK was detected in CRC tissues by immunohistochemistry,and the effect of TOPK knockdown was detected in CRC cells by Western blotting.CCK-8 and clonogenic assays were used to detect the growth and clonogenic ability of CRC cells after TOPK knockdown combined with radiotherapy in CRC cells.Furthermore,proteomic analysis showed that the phosphorylation of TOPK downstream proteins changed after radiotherapy.DNA damage was detected by the comet assay.Changes in the DNA damage response signaling pathway were analyzed by Western blotting,and apoptosis was detected by flow cytometry.Results The expression of TOPK was significantly greater in CRC tissues at grades 2–4 than in those at grade 1.After irradiation,CRC cells with genetically silenced TOPK had shorter comet tails and reduced expression levels of DNA damage response-associated proteins,including phospho-cyclin-dependent kinase 1(p-CDK1),phospho-ataxia telangiectasia-mutated(p-ATM),poly ADP-ribose polymerase(PARP),and meiotic recombination 11 homolog 1(MRE11).Conclusions TOPK was overexpressed in patients with moderately to poorly differentiated CRC.Moreover,TOPK knockdown significantly enhanced the radiosensitivity of CRC cells by reducing the DNA damage response.

Single Cell Gel Electrophoresis Assay of Porcine Leydig Cell DNA Damage Induced by Zearalenone

[Objective] This study aimed to investigate the effect of zearalenone (ZEN) on DNA damage of porcine leydig cells. [Method] Porcine leydig cells cultured in vitro were collected to determine the median lethal dose (LD50) of ZEN with tetrazolium-based colorimetric assay (MTT assay). Comet assay was carried out to detect the DNA damage of porcine leydig cells exposed to at 0 (negative group), 1, 5, 10, 20, 40 μmol/L of ZEN. [Result] The percentage of cell tail was 16.67%, 34.00%, 40.67%, 52.00% and 64.67% under 0, 1, 5, 10 and 20 μmol/L of ZEN, respectively; the differences between the percentages of cell tail in various experimental groups had extremely significant statistical significance compared with the negative group (P<0.01), showing a significant dose-effect relationship; Tail length in various groups was 57.60±4.78, 57.75±6.25, 78.97±5.83, 100.50±6.94 and 146.83±12.31 μm, respectively; Tail DNA % in various groups was 21.29±2.25%, 22.24±2.43%, 31.21±6.27%, 37.45±4.33% and 60.68±9.83%, respectively; Tail length and Tail DNA % in experimental groups with ZEN concentration above 5 μmol/L showed significant differences (P<0.05) compared with the negative group, which showed an upward trend with the increase of ZEN concentration. [Conclusion] ZEN has genotoxic effect on porcine leydig cells, which can cause DNA damage, with a significant dose-effect relationship.

DNA Damage and Repair of Two Ecotypes of Phragmites communis Subjected to Water Stress

In order to thoroughly understand the mechanism Of drought resistance in plants at DNA level, the DNA damage of two ecotypes of reeds (Phragmites communis T.) stressed by PEG 6000 was analyzed by means of fluorescence analysis of DNA unwinding (FADU). The results showed that the residual double strand DNA percentages (dsDNA%) in dune reed (DR) were significantly higher than those in swamp reed (SR) treated with either 20% or 30% PEG 6000. This meant that the DNA of DR was less damaged in comparison with SR. Similarly, DR resisted DNA damage more strongly than SR as reactive oxygen species (ROS) increased by adding ROS producers diethyldithio carbamate (DDC), H2O2 and Fe2+ of different concentrations. Meanwhile, treating PEG stressed SR with ROS scavengers such as dimethyl sulphoxide (DMSO) and ascorbic acid (Vc) resulted in the reduction of DNA damage, suggesting that ROS could cause DNA damage. In addition, the DNA repair for water-stressed reeds indicated that DR repaired DNA damage much faster and more completely. This might be the first indication that drought stress led to DNA damage in plants and that drought resistance of plants was closely related to DNA damage and repair.

Effect of Farnesyl Caffeate-Induced Apoptosis of Lung Carcinoma Cell Line from Damage to DNA

Farnesyl caffeate, synthesis of propolis and polar bud excretion, has been reported to exhibit anti-allergic effects in mice. However, little is known about anti-tumor effects. In this study, we investigated the effect of Farnesyl caffeate in cell proliferation of lung carcinoma cell line (H157). Antiproliferative effect and apoptosis on H157 cell were evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay (MTT) and DNA fragmentation assay, respectively. Farnesyl caffeate inhibited the growth of H157 cell in dose-dependent manner. Morphological changes of nuclei by staining Hoechst 33258 and DNA fragmentation suggested that Farnesyl caffeate induced death involved in a mechanism of apoptosis. Moreover, caspase-3, caspase-7 and caspase-9 were activated by Farnesyl caffeate on H157 cell. The protein expressions of Bax and Bcl-2 were down-regulated by Farnesyl caffeate, resulting in cytochrome c release from the mitochondria. Farnesyl caffeate significantly increased the expression of p53 proteins which indicates that p53 plays a pivotal role in the initiation phase of Farnesyl caffeate-induced HepG2 cell apoptosis. These results demonstrated Farnesyl caffeate-induced apoptosis in human lung carcinoma cell line. More detailed mechanism of ?Farnesyl caffeate-induced H157 apoptosis remains to be elucidated.

Identification of DNA Damage Caused by Heavy Metal Ions with Small Molecular DNA

[Objective] The aim was to establish a convenient and effective method to evaluate the toxicity of heavy metal ions by using small molecular DNA. [Method] pUC18 DNA which had exposed to the four heavy metal ions of Hg2＋, Cr6＋, Pb2＋, Cd2＋ was used to study the bioactivity of DNA; simultaneously, gel electrophoresis and hyperchromic effect were employed to detect the mechanism of DNA damage. [Result] The bioactivity of the exposed DNA was decreased and the influence degree was Hg2＋Cr6＋Pb2＋Cd2＋; the gel electrophoresis and hyperchromic effect proved that the main reason leading to reduce the bioactivity was DNA cross link, in the order pf Hg2＋Cr6＋Pb2＋Cd2＋. [Conclusion] The study indicated that pUC18 DNA could be used to assay the damage of DNA causing by heavy mental ions, which may be a potential, simple and effective tool to evaluate toxicity of heavy metal ions to DNA.

Apoptosis and DNA damage in human spermatozoa

DNA damage is frequently encountered in spermatozoa of subfertile males and is correlated with a range of adverse clinical outcomes including impaired fertilization, disrupted preimplantation embryonic development, increased rates of miscarriage and an enhanced risk of disease in the progeny. The etiology of DNA fragmentation in human spermatozoa is closely correlated with the appearance of oxidative base adducts and evidence of impaired spermiogenesis. We hypothesize that oxidative stress impedes spermiogenesis, resulting in the generation of spermatozoa with poorly remodelled chromatin. These defective cells have a tendency to default to an apeptotic pathway associated with motility loss, caspase activation, phosphatidylserine exteriorization and the activation of free radical generation by the mitochondria. The latter induces lipid peroxidation and oxidative DNA damage, which then leads to DNA fragmentation and cell death. The physical architecture of spermatozoa prevents any nucleases activated as a result of this apoptotic process from gaining access to the nuclear DNA and inducing its fragmentation. It is for this reason that a majority of the DNA damage encountered in human spermatozoa seems to be oxidative. Given the important role that oxidative stress seems to have in the etiology of DNA damage, there should be an important role for antioxidants in the treatment of this condition. If oxidative DNA damage in spermatozoa is providing a sensitive readout of systemic oxidative stress, the implications of these findings could stretch beyond our immediate goal of trying to minimize DNA damage in spermatozoa as a prelude to assisted conception therapy.

Oxidative stress and DNA damages induced by cadmium accumulation

Experimental evidence shows that cadmium （Cd） could induce oxidative stress and then causes DNA damage in animal cells, however, whether such effect exists in plants is still unclear. In the present study, Vicia faba plants was exposed to 5 and 10 mg/L Cd for 4 d to investigate the distribution of Cd in plant, the metal effects on the cell lipids, antioxidative enzymes and DNA damages in leaves. Cd induced an increase in Cd concentrations in plants. An enhanced level of lipid peroxidation in leaves and an enhanced concentration of H2O2 in root tissues suggested that Cd caused oxidative stress in Vicia faba. Compared with control, Cd-induced enhancement in superoxide dismutase activity was significant at 5 mg/L than at 10 mg/kg in leaves, by contrast, catalase and peroxidaseactivities were significantly suppressed by Cd addition. DNA damage was detected by neutral/neutral, alkaline/neutral and alkaline/alkaline Comet assay. Increased levels of DNA damages induced by Cd occurred with reference to oxidative stress in leaves, therefore, oxidative stress induced by Cd accumulation in plants contributed to DNA damages and was possibly an important mechanism of Cd-phytotoxicity in Vicia faba plants.

Sperm DNA damage in men from infertile couples

Aim： To investigate the prevalence of high levels of sperm DNA damage among men from infertile couples with both normal and abnormal standard semen parameters. Methods： A total of 350 men from infertile couples were assessed. Standard semen analysis and sperm chromatin structure assay （SCSA） were carried out. Results： Ninety-seven men （28% of the whole study group） had a DNA fragmentation index （DFI） 〉 20%, and 43 men （12%） had a DFI 〉 30%. In the group of men with abnormal semen parameters （n = 224）, 35% had a DFI 〉 20%, and 16% had a DFI 〉 30%, whereas these numbers were 15% and 5%, respectively, in the group of men with normal semen parameters （n = 126）. Men with low sperm motility and abnormal morphology had significantly higher odds ratios （ORs） for having a DFI 〉 20% （4.0 for motility and 1.9 for morphology） and DFI 〉 30% （6.2 for motility and 2.8 for morphology） compared with men with normal sperm motility and morphology. Conclusion： In almost one-third of unselected men from infertile couples, the DFI exceeded the level of 20% above which, according to previous studies, the in vivo fertility is reduced. A significant proportion of men with otherwise normal semen parameters also had high sperm DNA damage levels. Thus, the SCSA test could add to explaining causes of infertility in cases where semen analysis has not shown any deviation from the norm. We also recommend running the SCSA test to choose the appropriate assisted reproductive technique （ART）.

Effects of Fluoride on Lipid Peroxidation, DNA Damage and Apoptosis in Human Embryo Hepatocytes

Objective To investigate the effects of fluoride on lipid peroxidation, DNA damage and apoptosis in human embryo hepatocyte L-02 cells. Methods Lipid peroxide (LPO) level, reduced glutathione (GSH) content, DNA damage, apoptosis, and cell cycle analysis were measured after in vitro cultured L-02 cells were exposed to sodium fluoride at different doses (40 μg/mL, 80 μg/mL, and 160 μg/mL) for 24 hours. Results Fluoride caused an increase of LPO levels and a decrease of GSH content in L-02 cells. There appeared to be an obvious dose-effect relationship between the fluoride concentration and the observed changes. Fluoride also caused DNA damage and apoptosis and increased the cell number in S phase of cell cycle in the cells tested. There was a statistically significant difference in DNA damage and apoptosis when comparing the high dose of fluoride treated cells with the low dose of fluoride treated cells. Conclusion Fluoride can cause lipid peroxidation, DNA damage, and apoptosis in the L-02 cell experimental model and there is a significant positive correlation between fluoride concentration and these pathological changes.

An Investigation of Oxidative DNA Damage in Pharmacy Technicians Exposed to Antineoplastic Drugs in Two Chinese Hospitals Using The Urinary 8-OHdG Assay

Objective To investigate oxidative DNA damage in pharmacy technicians preparing antineoplastic drugs at the PIVAS （Pharmacy Intravenous Admixture Service） in two Chinese hospitals. Methods Urinary 8-OHdG served as a biomarker. 5-Fluorouracil （5-FU） concentrations in air, masks and gloves were determined. The spill exposure of each PIVAS technician to antineoplastic drugs was investigated. Eighty subjects were divided into exposed group t, II, and control group I, II. Results 5-FU concentration ratios for gloves and masks in exposed group I were significantly higher than those in exposed group II （P〈0.05 or P〈0.01）. The average urinary 8-OHdG concentrations in exposed group I, control group I, exposed group II, and control group II were 24.69＋0.93, 20.68＋1.07, 20.57＋0.55, and 12.96_＋0.73 ng/mg Cr, respectively. Urinary 8-OHdG concentration in exposed group I was significantly higher than that in control group I or that in exposed group 11 （P〈0.02）. There was a significant correlation between urinary 8-OHdG concentrations and spill frequencies per technician （P〈0.01）. Conclusion There was detectable oxidative DNA damage in PIVAS technicians exposed to antineoplastic drugs. This oxidative DNA damage may be associated with their spill exposure experience and contamination of their personal protective equipment.

Typical Cell Signaling Response to Ionizing Radiation: DNA Damage and Extranuclear Damage

To treat many types of cancer, ionizing radiation （IR） is primarily used as external-beam radiotherapy, brachytherapy, and targeted radionuclide therapy. Exposure of tumor cells to JR can induce DNA damage as well as generation of reactive oxygen species （ROS） and reactive nitrogen species （RNS） which can cause non-DNA lesions or extracellular damage like lipid perioxidation. The initial radiation-induced cell responses to DNA damage and ROS like the proteolytic processing, as well as synthesis and releasing ligands （such as growth factors, cytokines, and hormone） can cause the delayed secondary responses in irradiated and unirradiated bystander cells through paracrine and autocrine pathways.

Oxidative Damage to Lung Tissue and Peripheral Blood in Endotracheal PM_(2.5)-treated Rats

Objective To investigate the oxidative damage to lung tissue and peripherial blood in PM2.5-treated rats. Methods PM2.5 samples were collected using an auto-sampling instrument in summer and winter. Treated samples were endotracheally instilled into rats. Activity of reduced glutathione peroxidase （GSH-Px） and concentration of malondialdehyde （MDA） were used as oxidative damage biomarkers of lung tissue and peripheral blood detected with the biochemical method. DNA migration length （μm） and rate of tail were used as DNA damage biomarkers of lung tissue and peripheral blood detected with the biochemical method. Results The activity of GSH-Px and the concentration of MDA in lung tissue significantly decreased after exposure to PM2.5 for 7-14 days. In peripheral blood, the concentration of MDA decreased, but the activity of GSH-Px increased 7 and 14 days after experiments. The two indicators had a dose-effect relation and similar changing tendency in lung tissue and peripheral blood. The DNA migration length （μm） and rate of tail in lung tissue and peripheral blood significantly increased 7 and 14 days after exposure to PM2.5. The two indicators had a dose-effect relation and similar changing tendency in lung tissue and peripheral blood. Conclusion PM2.5 has a definite oxidative effect on lung tissue and peripheral blood. The activity of GSH-Px and the concentration of MDA are valuable biomarkers of oxidative lung tissue damage induced by PM2.5. The DNA migration length （μm） and rate of tail are simple and valuable biomarkers of PM2 5-induced DNA damage in lung tissues and peripheral blood. The degree of DNA damage in peripheral blood can predict the degree of DNA damage in lung tissue.

Sperm DNA damage and its clinical relevance in assessingreproductive outcome

The routine examination of semen, which assesses sperm concentration, percentage motility and morphology, does not identify subtle defects in sperm chromatin architecture. The focus on the genomic integrity of the male gamete has intensified recently due to the growing concern that genetic diseases may be transmitted via assisted reproductive techniques (ART). Accordingly, the intent of this review is to describe the details of the information pertaining to mitochondrial/nuclear sperm DNA damage with an emphasis on its clinical significance and its relation ship with male infertility. Assessment of sperm DNA damage appears to be a potential tool for evaluating semen samples prior to their use in ART. Testing DNA integrity may help select spermatozoa with intact DNA or with the least amount of DNA damage for use in assisted conception. In turn, this may alleviate the financial, social and emotional problems associated with failed ART attempts.

Randomized Terminal Linker-dependent PCR: A Versatile and Sensitive Method for Detection of DNA Damage

Objective To design and develop a novel, sensitive and versatile method for in vivo foot printing and studies of DNA damage, such as DNA adducts and strand breaks. Methods Starting with mammalian genomic DNA, single-stranded products were made by repeated primer extension, these products were ligated to a double-stranded linker having a randomized 3 overhang, and used for PCR. DNA breaks in p53 gene produced by restriction endonuclease AfaI were detected by using this new method followed by Southern hybridization with DIG-labeled probe. Results This randomized terminal linker-dependent PCR (RDPCR) method could generate band signals many-fold stronger than conventional ligation-mediated PCR (LMPCR), and it was more rapid, convenient and accurate than the terminal transferase-dependent PCR (TDPCR). Conclusion DNA strand breakage can be detected sensitively in the gene level by RDPCR. Any lesion that blocks primer extension should be detectable.