Breaks In Deoxyribonucleic Acid Research Articles

Base and Prime Editing in the Retina-From Preclinical Research toward Human Clinical Trials.

Inherited retinal diseases (IRDs) are a clinically and genetically heterogeneous group of diseases that are one of the leading causes of vision loss in young and aged individuals. IRDs are mainly caused by a loss of the post-mitotic photoreceptor neurons of the retina, or by the degeneration of the retinal pigment epithelium. Unfortunately, once these cells are damaged, it is irreversible and leads to permanent vision impairment. Thought to be previously incurable, gene therapy has been rapidly evolving to be a potential treatment to prevent further degeneration of the retina and preserve visual function. The development of clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) base and prime editors have increased the capabilities of the genome editing toolbox in recent years. Both base and prime editors evade the creation of double-stranded breaks in deoxyribonucleic acid (DNA) and the requirement of donor template of DNA for repair, which make them advantageous methods in developing clinical therapies. In addition, establishing a permanent edit within the genome could be better suited for patients with progressive degeneration. In this review, we will summarize published uses of successful base and prime editing in treating IRDs.

Exposure to Polybrominated Diphenyl Ether Flame Retardants Causes Deoxyribonucleic Acid Damage in Human Thyroid Cells In Vitro

IntroductionThe incidence of papillary thyroid cancer (PTC) in the United States has tripled in the past 30 y. Polybrominated diphenyl ethers (PBDEs) are flame retardants that were ubiquitously used over that time period, and exposure to PBDEs has been associated with PTC prevalence. They are potential carcinogens via their induction of reactive oxygen species (ROS) formation and resultant deoxyribonucleic acid (DNA) damage. We sought to determine the effects of PBDE and tris(2-chloroethyl) phosphate (TCEP), another flame retardant implicated in PTC incidence, on thyrocytes in vitro and measure PBDE levels in human thyroid tissue to determine their carcinogenic potential. MethodsNthy-Ori, an immortalized benign human thyroid follicular cell line was used as a model of normal human thyroid. MTT assays were used to measure cell viability after exposure to PBDEs and TCEP. ROS levels and double-stranded and single-stranded DNA breaks were measured to determine genotoxicity. DNA damage response protein levels were measured with immunoblotting. ResultsExposure to 20μM PBDE or TCEP for 48 h had minimal effects on thyrocyte viability. There was no significant increase in intracellular ROS up to 6 h following PBDE or TCEP exposure in thyrocytes; however, cells exposed to PBDE 47 showed evidence of DNA single-stranded and double-stranded breaks. There was a dose-dependent increase in γH2AX levels following exposure to PBDEs 47 and 209 in Nthy-Ori cells but not with TCEP treatment. ConclusionsPBDE 47 and 209 demonstrated genotoxicity but not cytotoxicity in follicular thyrocytes in vitro. Therefore, PBDE 47 and 209 may be carcinogenic in human thyroid cells.

An evaluation of DNA double strand break formation and excreted guanine species post whole body PET/CT procedure.

Ionizing radiation-induced oxidation and formation of deoxyribonucleic acid (DNA) double strand breaks (DSBs) are considered the exemplar of genetic lesions. Guanine bases are most prone to be oxidized when DNA and Ribonucleic acid (RNA) are damaged. The repair processes that are initiated to correct this damage release multiple oxidized guanine species into the urine. Hence, the excretion of guanine species can be related with the total repair process. Our study quantified the total DSBs formation and the amount of guanine species in urine to understand the DNA break and repair process after whole body (WB) exposure to 18F-FDG positron emission tomography/computed tomography (PET/CT). A total of 37 human participants were included with control and test groups and the average radiation dose was 27.50 ± 2.91 mSv. γ-H2AX foci assay in the collected blood samples was performed to assess the DSBs, and excreted guanine species in urine were analyzed by a competitive ELISA method. We observed a significant increase of DNA damage that correlated well with the increasing dose (p-value 0.009) and body weight (p-value 0.05). In the test group, excreted guanine species in urine sample significantly increased (from 24.29 ± 5.82 to 33.66 ± 7.20 mg/mmol creatinine). A minimum (r2 = 0.0488) correlation was observed between DSBs formation and excreted guanine species. A significant difference of DNA damage and 8-OHdG formation was seen in the test group compared to controls. Larger population studies are needed to confirm these observations, describe the fine-scale timing of changes in the biomarker levels after exposure, and further clarify any potential risks to patients from PET/CT procedures.

Minibeam radiation therapy: A micro‐ and nano‐dosimetry Monte Carlo study

Minibeam radiation therapy (MBRT) is an innovative strategy based on a distinct dose delivery method that is administered using a series of narrow (submillimetric) parallel beams. To shed light on the biological effects of MBRT irradiation, we explored the micro- and nanodosimetric characteristics of three promising MBRT modalities (photon, electron, and proton) using Monte Carlo (MC) calculations. Irradiation with proton (100MeV), electron (300MeV), and photon (effective energy of 69keV) minibeams were simulated using Geant4 MC code and the Geant4-DNA extension, which allows the simulation of energy transfer points with nanometric accuracy. As the target of the simulations, cells containing spherical nuclei with or without a detailed description of the DNA (deoxyribonucleic acid) geometry were placed at different depths in peak and valley regions in a water phantom. The energy deposition and number of events in the cell nuclei were recorded in the microdosimetry study, and the number of DNA breaks and their complexity were determined in the nanodosimetric study, where a multi-scale simulation approach was used for the latter. For DNA damage assessment, an adapted DBSCAN clustering algorithm was used. To compare the photon MBRT (xMBRT), electron MBRT (eMBRT), and proton MBRT (pMBRT) approaches, we considered the treatment of a brain tumor located at a depth of 75mm. Both mean energy deposition at micrometric scale and DNA damage in the "valley" cell nuclei were very low as compared with these parameters in the peak region at all depths for xMBRT and at depths of 0 to 30mm and 0 to 50mm for eMBRT and pMBRT, respectively. Only the charged minibeams were favorable for tumor control by producing similar effects in peak and valley cells after 70mm. At the micrometer scale, the energy deposited per event pointed to a potential advantage of proton beams for tumor control, as more aggressive events could be expected at the end of their tracks. At the nanometer scale, all three MBRT modalities produced direct clustered DNA breaks, although the majority of damage (>93%) was composed of isolated single strand breaks. The pMBRT led to a significant increase in the proportion of clustered single strand breaks and double-strand breaks at the end of its range as compared to the entrance (7% at 75mm vs 3% at 10mm) in contrast to eMBRT and xMBRT. In the latter cases, the proportions of complex breaks remained constant, irrespective of the depth and region (peak or valley). Enhanced normal tissue sparing can be expected with these three MBRT techniques. Among the three modalities, pMBRT offers an additional gain for radioresistant tumors, as it resulted in a higher number of complex DNA damage clusters in the tumor region. These results can aid understanding of the biological mechanisms of MBRT.

Deoxyribonucleic Acid Damage and Repair: Capitalizing on Our Understanding of the Mechanisms of Maintaining Genomic Integrity for Therapeutic Purposes.

Deoxyribonucleic acid (DNA) is the self-replicating hereditary material that provides a blueprint which, in collaboration with environmental influences, produces a structural and functional phenotype. As DNA coordinates and directs differentiation, growth, survival, and reproduction, it is responsible for life and the continuation of our species. Genome integrity requires the maintenance of DNA stability for the correct preservation of genetic information. This is facilitated by accurate DNA replication and precise DNA repair. DNA damage may arise from a wide range of both endogenous and exogenous sources but may be repaired through highly specific mechanisms. The most common mechanisms include mismatch, base excision, nucleotide excision, and double-strand DNA (dsDNA) break repair. Concurrent with regulation of the cell cycle, these mechanisms are precisely executed to ensure full restoration of damaged DNA. Failure or inaccuracy in DNA repair contributes to genome instability and loss of genetic information which may lead to mutations resulting in disease or loss of life. A detailed understanding of the mechanisms of DNA damage and its repair provides insight into disease pathogeneses and may facilitate diagnosis and the development of targeted therapies.

Frequency of cell treatment with cold microwave argon plasma is important for the final outcome

The purpose of this work was to establish the influence of a regime of cold microwave argon plasma treatments on the physiological characteristics of human fibroblasts and keratinocytes. We used three regimes of plasma application: a single treatment, double treatment with a 48 h interval, and daily treatments for 3 d. Cell proliferation after plasma application was quantified in real time, and immunohistochemistry was used to establish the viability of the cells and determine changes in their physiology.It was established that the frequency of cell treatments is important for the outcome. In the samples treated with single plasma application and double plasma applications with a 48 h interval, a 42.6% and 32.0% increase was observed in the number of cells, respectively. In addition, there were no signs of deoxyribonucleic acid breaks immediately after plasma application. In contrast, plasma application increased the accumulation of cells in the active phases of the cell cycle. The activation of proliferation correlated with a decrease in the level of β-galactosidase, a senescence marker. This could be due to cell renovation after plasma application. Daily treatment decreased cell proliferation up to 29.1% in comparison with the control after 3 d.

Nanotetrac targets integrin αvβ3 on tumor cells to disorder cell defense pathways and block angiogenesis

The extracellular domain of integrin αvβ3 contains a receptor for thyroid hormone and hormone analogs. The integrin is amply expressed by tumor cells and dividing blood vessel cells. The proangiogenic properties of thyroid hormone and the capacity of the hormone to promote cancer cell proliferation are functions regulated nongenomically by the hormone receptor on αvβ3. An L-thyroxine (T4) analog, tetraiodothyroacetic acid (tetrac), blocks binding of T4 and 3,5,3′-triiodo-L-thyronine (T3) by αvβ3 and inhibits angiogenic activity of thyroid hormone. Covalently bound to a 200 nm nanoparticle that limits its activity to the cell exterior, tetrac reformulated as Nanotetrac has additional effects mediated by αvβ3 beyond the inhibition of binding of T4 and T3 to the integrin. These actions of Nanotetrac include disruption of transcription of cell survival pathway genes, promotion of apoptosis by multiple mechanisms, and interruption of repair of double-strand deoxyribonucleic acid breaks caused by irradiation of cells. Among the genes whose expression is suppressed by Nanotetrac are EGFR, VEGFA, multiple cyclins, catenins, and multiple cytokines. Nanotetrac has been effective as a chemotherapeutic agent in preclinical studies of human cancer xenografts. The low concentrations of αvβ3 on the surface of quiescent nonmalignant cells have minimized toxicity of the agent in animal studies.

The comet assay a method to measure DNA damage in oral submucous fibrosis patients: A case-control study

Context: Oral submucous fibrosis (OSF) is a chronic, progressive condition and rise in incidence of this disease has been attributed to an increased uptake of betel nut and its products, especially among young people. Due to its potential for malignant transformation, easy and reliable techniques for its early detection are needed. Comet assay is a simple technique to detect and quantify single strand breaks, double strand breaks and alkali labile damages. Aims: The present pilot study was undertaken to assess the deoxyribonucleic acid (DNA) damage in OSF and healthy groups using comet assay. Settings and Design: The hospital-based study was conducted by evaluating buccal mucosal cells of 50 individuals in the age range of 16-47 years and comet assay was carried out for all the two groups under alkali conditions. Materials and Methods: Oral epithelial cells were obtained from buccal mucosa of each 25 cases of healthy and OSF groups were subjected to comet assay. DNA damage was evaluated by measuring the tail length. Statistical Analysis Used: Student's t-test and Turkeys-multiple post-hoc procedures were used to statistically analyze the obtained data. Results: Increase in the tail length in buccal epithelial cells of OSF group when compared with the healthy group was noted. Furthermore, there was a significant increase in the DNA damage with duration of habits. Conclusions: We conclude that increase in the tail length formation in OSF groups when compared to healthy groups; this indicates DNA damage in the oral epithelial cells. The comet assay is a relatively simple, but sensitive and well-validated tool for measuring strand breaks in DNA in single cells.

Assessing Cellular DNA Damage from A Helium Plasma Needle

The aim of the present study is to determine the deoxyribonucleic acid (DNA) damage by cells exposed to atmospheric pressure non-thermal plasma (APNTP). Mouse leukocytes embedded in agarose were exposed to the plasma at two different distances from a helium plasma needle outlet and during three different exposure periods. Damage was assessed by the single cell gel electrophoresis assay. The results indicate that, at 0.1 cm from the plasma needle, the exposure caused complete DNA fragmentation determined by the presence of so called "clouds". Samples exposed at 0.5 cm from the slide sample surface presented damage proportional to the exposure periods in terms of tail intensity, tail moment and "clouds" frequency. Studies performed with alkaline single cell gel electrophoresis assay to determine DNA breaks and alkali-labile sites, indicates that DNA damage produced by exposure to APNTP was caused mainly by oxidative radicals, rather than by UV light which causes cyclobutane pyrimidine dimers. These results allow us to conclude that plasma needle induced DNA breaks in mice leukocytes proportionally to exposure time.

Changes in γ-H2AX expression in irradiated feline sarcoma cells: An indicator of double strand DNA breaks

Feline injection site sarcoma (ISS) is a highly invasive soft tissue tumor that is commonly treated with radiation. Cellular deoxyribonucleic acid (DNA) is the principal target for the biologic effects of radiation with cell killing correlating to the number of double stranded DNA breaks (DSBs). The objective of this study was to determine if radiation-induced damage to feline ISS cells could be detected using a commercially available DNA DSB detection kit. Feline ISS cells were irradiated and evaluated for extent of DSB induction with a γ-H2AX chemiluminescent kit; results were validated by Western Blot analysis. Irradiated cells showed a significant increase in double strand break induction compared to control cells, which was supported by Western Blot. DNA damage in feline sarcoma cells following single exposure of radiation can be indirectly detected using a commercially available mouse anti-human monoclonal antibody for γ-H2AX.

Single-strand DNA breaks in human hair root cells exposed to mobile phone radiation

Purpose: To analyze the short-term effects of radiofrequency radiation (RFR) exposure on genomic deoxyribonucleic acid (DNA) of human hair root cells.Subjects and methods: Hair samples were collected from eight healthy human subjects immediately before and after using a 900-MHz GSM (Global System for Mobile Communications) mobile phone for 15 and 30 min. Single-strand DNA breaks of hair root cells from the samples were determined using the ‘comet assay’.Results: The data showed that talking on a mobile phone for 15 or 30 min significantly increased (p < 0.05) single-strand DNA breaks in cells of hair roots close to the phone. Comparing the 15-min and 30-min data using the paired t-test also showed that significantly more damages resulted after 30 min than after 15 min of phone use.Conclusions: A short-term exposure (15 and 30 min) to RFR (900-MHz) from a mobile phone caused a significant increase in DNA single-strand breaks in human hair root cells located around the ear which is used for the phone calls.

To live or to die: a matter of processing damaged DNA termini in neurons

Defects in the repair of deoxyribonucleic acid (DNA) damage underpin several hereditary neurological diseases in humans. Of the different activities that repair chromosomal DNA breaks, defects in resolving damaged DNA termini are among the most common causes of neuronal cell death. Here, the molecular mechanisms of some of the DNA end processing activities are reviewed and the association with human neurodegenerative disease is discussed.

New evidence on iron, copper accumulation and zinc depletion and its correlation with DNA integrity in aging human brain regions.

Deoxyribonucleic acid (DNA) conformation and stability play an important role in brain function. Earlier studies reported alterations in DNA integrity in the brain regions of neurological disorders like Parkinson’s and Alzheimer’s diseases. However, there are only limited studies on DNA stability in an aging brain and the factors responsible for genomic instability are still not clear. In this study, we assess the levels of Copper (Cu), Iron (Fe) and Zinc (Zn) in three age groups (Group I: below 40 years), Group II: between 41-60 years) and Group III: above 61 years) in hippocampus and frontal cortex regions of normal brains. The number of samples in each group was eight. Genomic DNA was isolated and DNA integrity was studied by nick translation studies and presented as single and double strand breaks. The number of single strand breaks correspondingly increased with aging compared to double strand breaks. The strand breaks were more in frontal cortex compared to hippocampus. We observed that the levels of Cu and Fe are significantly elevated while Zn is significantly depleted as one progresses from Group I to Group III, indicating changes with aging in frontal cortex and hippocampus. But the elevation of metals was more in frontal cortical region compared to hippocampal region. There was a clear correlation between Cu and Fe levels versus strand breaks in aging brain regions. This indicates that genomic instability is progressive with aging and this will alter the gene expressions. To our knowledge, this is a new comprehensive database to date, looking at the levels of redox metals and corresponding strand breaks in DNA in two brain regions of the aging brain. The biological significance of these findings with relevance to mental health will be discussed.

Monitoring the (photo)genotoxicity of photosensitizer drugs: Direct quantitation of single-strand breaks in deoxyribonucleic acid using an oligonucleotide chip

Oligonucleotide chip-based assays can be a sample-thrifty, time-saving, routine tool for evaluation of chemical-induced DNA strand breaks. This article describes a novel approach using an oligonucleotide chip to determine photosensitizer-induced DNA single-strand breaks. Surface coverage of fluorophore-labeled oligonucleotides on silicon dioxide chip surfaces was determined on alkaline phosphatase digestion. Fluorescence maxima (at 520 nm) of the solutions were converted to molar concentrations of the fluorescein-modified oligonucleotide by interpolation from a predetermined standard linear calibration curve. The photosensitizing activity of chlorpromazine and triflupromazine toward DNA single-strand breaks was then studied at different drug doses and also as a function of photoirradiation time. Photoinduced single-strand breaks calculated using the method described here agreed with values predicted by theoretical extrapolation of the single-strand breaks obtained for plasmid DNAs from agarose gel electrophoresis, and thereby indirectly validated the chip-based assays. Under UV irradiation (⩾93.6 kJ/m 2) chlorpromazine (⩾0.08 mM) was found to have significant photogenotoxicity. However, triflupromazine did not exhibit any (photo)genotoxicity over the concentration range studied (0.04–0.20 mM). The method developed will be useful for quantitative screening of drug genotoxicity in terms of induction of breaks in DNA.

Triplex configuration in the nick-free DNAs that constitute the chromosomal scaffolds in grasshopper spermatids

After applying proper deoxyribonucleic acid (DNA) probes, fluorescence in situ hybridization (FISH) showed that the 8/9 centromeres-one per chromatid of the male haploid complement (X0) of Pyrgomorpha conica grasshopper-colocalized at the spermatid blunt end, where the spermatozoa flagellum inserts. A bundle of aligned 4',6-diamidino-2-phenylindole-positive chromatid scaffolds, which formed the central spermatid core, was observed after DNA breakage detection followed by FISH. Modular nature of scaffold DNA was occasionally evident. The technique also showed that in the early spermatid, the chromatid scaffolds lacked any DNA nick, whereas abundant breaks accumulated in the surrounding loops. Moreover, immunodetection showed that scaffold DNA participated in the formation of triplex DNA, while this configuration was absent from the loops. During spermatid maturation, triplex DNA disappeared from the scaffold in parallel with loop retraction, while protamines replace histones. Thus, the presence of triplex DNA in the chromatid scaffold correlates with the anchoring of expanded DNA loops to it. After loop retraction, the scaffolds of all chromatids coiled as a single unit in the spermatid head. This cooperative coiling produced enlargement and tilting of the distal telomeric signals, which were distributed along the spermatid head according to the length of each chromosome. We propose that specific DNA sequences dispersed throughout the whole chromatid fold forward and backward coaxially to chromatid length, forming individual scaffold modules whose linear assembly accounts for the minimum length of each individual chromatid. Finally, the core of the grasshopper male spermatid should be considered as a single chromosome in which the DNA scaffolds of the whole set of the nonhomologous chromosomes of the haploid complement are interconnected. This pattern of chromatin organization applies probably to other elongated spermatids.

Genotoxicity of Corrosion Eluates Obtained From Endosseous Implants

Commercially pure titanium alloys are currently used as metallic biomaterials in implantology. Corrosion phenomena appear to play a decisive role in metallic implant long-term behavior. Thus, the goal of this study was to examine the genotoxic potential of corrosion eluates obtained from dental implants using Chinese ovary hamster cells in vitro by the single-cell gel (comet) assay. This technique detects deoxyribonucleic acid strand breaks in individual cells in alkaline conditions. The materials tested included 3 dental implants commercially available. Each of the tested materials was corroded in a solution consisting of equal amounts of acetic acid and sodium chloride (0.1 M) for 1, 3, 7, 14, and 21 days. The Chinese ovary hamster cultures were then exposed to all corrosion eluates obtained from endosseous dental implants for 30 minutes at 37 degrees C. None of the eluates was found to exhibit genotoxicity, regardless of the type of dental implant used. The results suggest that all dental implants tested in this study did not induce deoxyribonucleic acid breakage as depicted by the single-cell gel (comet) assay.

Transient and reversible deoxyribonucleic acid damage in human left ventricle under controlled ischemia and reperfusion

ObjectivesWe sought to describe the sequence of molecular events during ischemia and reperfusion of the human heart and to determine the activation of stress kinases and deoxyribonucleic acid (DNA) damage response elements on apoptosis in ischemia or reperfusion of the human heart. BackgroundBrief ischemia is tolerated by cardiac myocytes, but it determines immediate metabolic changes and block of contraction. Prompt restoration of coronary blood flow is inexorably associated with a slow recovery of myocardial contractile function. The prolonged, postischemic contractile dysfunction in the viable tissue is called myocardial stunning. The molecular mechanisms underlying myocardial stunning and ischemia-reperfusion injury are still poorly understood. Their elucidation would be valuable in order to identify novel therapeutic strategies. MethodsWe examined human left ventricular samples taken from 20 patients undergoing elective valve surgery before aortic cross-clamping, 20 ± 2 min (brief ischemia), 58 ± 5 min after the cross-clamping period (prolonged ischemia), and 21 ± 4 min after reconstitution of coronary blood flow (reperfusion). Stress kinases and DNA damage sensor proteins (ATM, H2AX, p53) were determined by immunoblotting with specific antibodies. Electron microscopy analysis was carried out on ischemic and reperfused samples. ATP content, reactive oxygen species (ROS) levels, and cytochrome oxidase activity were determined by biochemical assays. ResultsIschemia caused accumulation of ROS, reduction of cytochrome C oxidase and ATP, and activation of stress kinases p38 and Jun terminal kinase. Electron microscopy showed significant mitochondrial swelling in the majority of cells, but no appreciable apoptosis of cardiomyocytes. During ischemia, myocytes were intensely stained by TUNEL, and many cells showed proliferative cell nuclear antigen-positive nuclei. Finally, we found in ischemic tissues increased p53/p21WAFlevels and phosphorylation of histone H2AX, a substrate of ATM kinase, which marks double-strand DNA breaks. Reperfusion caused a robust extracellular signal-regulated kinase-1/2 activation, a marked reduction of TUNEL staining, and persistent activation of ATM checkpoint. ConclusionsThese data indicate that ischemia induces extensive DNA damage and activation of ATM checkpoint. Reperfusion allows the repair of the DNA lesions and salvage of ischemic cells.

The comet assay for DNA damage and repair: principles, applications, and limitations.

The comet assay (single-cell gel electrophoresis) is a simple method for measuring deoxyribonucleic acid (DNA) strand breaks in eukaryotic cells. Cells embedded in agarose on a microscope slide are lysed with detergent and high salt to form nucleoids containing supercoiled loops of DNA linked to the nuclear matrix. Electrophoresis at high pH results in structures resembling comets, observed by fluorescence microscopy; the intensity of the comet tail relative to the head reflects the number of DNA breaks. The likely basis for this is that loops containing a break lose their supercoiling and become free to extend toward the anode. The assay has applications in testing novel chemicals for genotoxicity, monitoring environmental contamination with genotoxins, human biomonitoring and molecular epidemiology, and fundamental research in DNA damage and repair. The sensitivity and specificity of the assay are greatly enhanced if the nucleoids are incubated with bacterial repair endonucleases that recognize specific kinds of damage in the DNA and convert lesions to DNA breaks, increasing the amount of DNA in the comet tail. DNA repair can be monitored by incubating cells after treatment with damaging agent and measuring the damage remaining at intervals. Alternatively, the repair activity in a cell extract can be measured by incubating it with nucleoids containing specific damage.

Functional and ultrastructural features of DNA-fragmented human sperm.

The functional significance of deoxyribonucleic acid (DNA) fragmentation in ejaculated human sperm is unclear. In this study the extent of DNA strand breakage in swim-up selected spermatozoa was evaluated by terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick end labeling (TUNEL)-coupled flow cytometry and correlated with several functional and morphological sperm parameters. The extent of DNA fragmentation (mean = 11.07%+/-8.00%, range = 0.79%-42.64%, n = 140) was positively related to abnormal morphology and associated with defects of the sperm tail. A negative correlation was found between DNA breakage and progressive motility. When a stepwise multiple linear regression model was used to analyze the relationship between DNA fragmentation and the aforementioned parameters, only motility results were included in the model. The presence of spermatozoa showing submicroscopic characteristics resembling those of somatic apoptosis has been reported in human ejaculate. To verify whether sperm DNA fragmentation was associated with the presence of such apoptotic-like cells, we performed electron microscopy and TUNEL-coupled flow cytometry in a limited number of sperm samples (n = 24). Although we did not observe any significant relationship between DNA breakage and the characteristics that are suggestive of apoptosis, an association was found with several ultrastructural features, indicating an impaired motility. Hence, we conclude that in ejaculated sperm, DNA fragmentation does not correspond to the apoptosis-like phenomenon and that it is associated with defects of motility.

Enhanced ADP-ribosylation and its diminution by lipoamide after ischemia-reperfusion in perfused rat heart

Poly-ADP-ribose polymerase (PARP) is considered to play an important role in oxidative cell damage. We assumed that ischemia-reperfusion resulting from the increasing reactive oxygen species (ROS) can lead to the activation of endogenous mono- and poly-ADP-ribosylation reactions and that the reduction of ROS level by lipoamide, a less known antioxidant, can reverse these unfavorable processes. Experiments were performed on isolated Langendorff hearts subjected to 60-min ischemia followed by reperfusion. ROS, malondialdehyde, deoxyribonucleic acid (DNA) breaks, and NAD + content were assayed in the hearts, and the ADP-ribosylation of cytoplasmic and nuclear proteins were determined by Western blot assay. Ischemia-reperfusion caused a moderate (30.2 ± 8%) increase in ROS production determined by the dihydrorhodamine123 method and significantly increased the malondialdehyde production (from <1 to 23 ± 2.7 nmol/ml), DNA damage (undamaged DNA decreased from 71 ± 7% to 23.1 ± 5%), and NAD + catabolism. In addition, ischemia-reperfusion activated the mono-ADP-ribosylation of GRP78 and the self-ADP-ribosylation of the nuclear PARP. The perfusion of hearts with lipoamide significantly decreased the ischemia-reperfusion-induced cell membrane damage determined by enzyme release (LDH, CK, and GOT), decreased the ROS production, reduced the malondialdehyde production to 5.5 ± 2.4 nmol/ml, abolished DNA damage, and reduced NAD + catabolism. The ischemia-reperfusion-induced activation of poly- and mono-ADP-ribosylation reactions were also reverted by lipoamide. In isolated rat heart mitochondria, dihydrolipoamide was found to be a better antioxidant than dihydrolipoic acid. Ischemia-reperfusion by ROS overproduction and increasing DNA breaks activates PARP leading to accelerated NAD + catabolism, impaired energy metabolism, and cell damage. Lipoamide by reducing ROS levels halts PARP activation and membrane damage and improves the recovery of postischemic myocardium.