Long Neural Genes Harbor Recurrent DNA Break Clusters in Neural Stem/Progenitor Cells.

Repair of DNA double-strand breaks (DSBs) by non-homologous end joining is critical for neural development, and brain cells frequently contain somatic genomic variations that might involve DSB intermediates. We now use an unbiased, high-throughput approach to identify genomic regions harboring recurrent DSBs in primary neural stem/progenitor cells (NSPCs). We identify 27 recurrent DSB clusters (RDCs), and remarkably, all occur within gene bodies. Most of these NSPC RDCs were detected only upon mild, aphidicolin-induced replication stress, providing a nucleotide-resolution view of replication-associated genomic fragile sites. The vast majority of RDCs occur in long, transcribed, and late-replicating genes. Moreover, almost 90% of identified RDC-containing genes are involved in synapse function and/or neural cell adhesion, with a substantial fraction also implicated in tumor suppression and/or mental disorders. Our characterization of NSPC RDCs reveals a basis of gene fragility and suggests potential impacts of DNA breaks on neurodevelopment and neural functions.

High-resolution landscape of an antibiotic binding site.

Antibiotic binding sites are located in important domains of essential enzymes and have been extensively studied in the context of resistance mutations; however, their study is limited by positive selection. Using multiplex genome engineering1 to overcome this constraint, we generate and characterize a collection of 760 single-residue mutants encompassing the entire rifampicin binding site of Escherichia coli RNA polymerase (RNAP). By genetically mapping drug-enzyme interactions, we identify an alpha helix where mutations considerably enhance or disrupt rifampicin binding. We find mutations in this region that prolong antibiotic binding, converting rifampicin from a bacteriostatic to bactericidal drug by inducing lethal DNA breaks. The latter are replication dependent, indicating that rifampicin kills by causing detrimental transcription-replication conflicts at promoters. We also identify additional binding site mutations that greatly increase the speed of RNAP.Fast RNAP depletes the cell of nucleotides, alters cell sensitivity to different antibiotics and provides a cold growth advantage. Finally, by mapping natural rpoB sequence diversity, we discover that functional rifampicin binding site mutations that alter RNAP properties or confer drug resistance occur frequently in nature.

Oncometabolites suppress DNA repair by disrupting local chromatin signalling.

Deregulation of metabolism and disruption of genome integrity are hallmarks of cancer1. Increased levels of the metabolites 2-hydroxyglutarate, succinate and fumarate occur in human malignancies owing to somatic mutations in the isocitrate dehydrogenase-1 or -2 (IDH1 or IDH2) genes, or germline mutations in the fumarate hydratase (FH) and succinate dehydrogenase genes (SDHA, SDHB, SDHC and SDHD), respectively2-4. Recent work has made an unexpected connection between these metabolites and DNA repair by showing that they suppress the pathway of homology-dependent repair (HDR)5,6 and confer an exquisite sensitivity to inhibitors of poly (ADP-ribose) polymerase (PARP) that are being tested in clinical trials. However, the mechanism by which these oncometabolites inhibit HDR remains poorly understood. Here we determine the pathway by which these metabolites disrupt DNA repair. We show that oncometabolite-induced inhibition of the lysine demethylase KDM4B results in aberrant hypermethylation of histone 3 lysine 9 (H3K9) at loci surrounding DNA breaks, masking a local H3K9 trimethylation signal that is essential for the proper execution of HDR. Consequently, recruitment of TIP60 and ATM, two key proximal HDR factors, is substantially impaired at DNA breaks, with reduced end resection and diminished recruitment of downstream repair factors. These findings provide a mechanistic basis for oncometabolite-induced HDR suppression and may guide effective strategies to exploit these defects for therapeutic gain.

Mechanisms to Repair Stalled Topoisomerase II-DNA Covalent Complexes.

DNA topoisomerases regulate the topological state of DNA, relaxing DNA supercoils and resolving catenanes and knots that result from biologic processes, such as transcription and replication. DNA topoisomerase II (TOP2) enzymes achieve this by binding DNA and introducing an enzyme-bridged DNA double-strand break (DSB) where each protomer of the dimeric enzyme is covalently attached to the 5' end of the cleaved DNA via an active site tyrosine phosphodiester linkage. The enzyme then passes a second DNA duplex through the DNA break, before religation and release of the enzyme. However, this activity is potentially hazardous to the cell, as failure to complete religation leads to persistent TOP2 protein-DNA covalent complexes, which are cytotoxic. Indeed, this property of topoisomerase has been exploited in cancer therapy in the form of topoisomerase poisons which block the religation stage of the reaction cycle, leading to an accumulation of topoisomerase-DNA adducts. A number of parallel cellular processes have been identified that lead to removal of these covalent TOP2-DNA complexes, facilitating repair of the resulting protein-free DSB by standard DNA repair pathways. These pathways presumably arose to repair spontaneous stalled or poisoned TOP2-DNA complexes, but understanding their mechanisms also has implications for cancer therapy, particularly resistance to anti-cancer TOP2 poisons and the genotoxic side effects of these drugs. Here, we review recent progress in the understanding of the processing of TOP2 DNA covalent complexes, the basic components and mechanisms, as well as the additional layer of complexity posed by the post-translational modifications that modulate these pathways. SIGNIFICANCE STATEMENT: Multiple pathways have been reported for removal and repair of TOP2-DNA covalent complexes to ensure the timely and efficient repair of TOP2-DNA covalent adducts to protect the genome. Post-translational modifications, such as ubiquitination and SUMOylation, are involved in the regulation of TOP2-DNA complex repair. Small molecule inhibitors of these post-translational modifications may help to improve outcomes of TOP2 poison chemotherapy, for example by increasing TOP2 poison cytotoxicity and reducing genotoxicity, but this remains to be determined.

Emerging JWA-targeted Pt(IV) prodrugs conjugated with CX-4945 to overcome chemo-immune-resistance.

Two Pt(IV) prodrugs, Cx-platin-Cl and Cx-DN604-Cl, derived from the conjugation of cisplatin or DN604 with a CK2 inhibitor CX-4945, were constructed to suppress DNA damage repair-related elements. During in vitro biological studies, the Pt(IV) prodrugs had excellent cytotoxicity superior to cisplatin and DN604 to reverse drug resistance. Further mechanistic investigations revealed that the powerful anticancer activity of Cx-platin-Cl and Cx-DN604-Cl arisen from its suppression of JWA-XRCC1-mediated single-strand breaks repair. The emerging Pt(IV) prodrugs inhibited the growth of the xenografted tumors of C57BL6 and nude mice apart from JWA-/- mice. Between them, Cx-platin-Cl augmented the infiltration and proliferation of Teff cells, alleviated the recruitment of Treg cells. The results provided compelling preclinical support that Cx-platin-Cl and Cx-DN604-Cl could reverse chemo-immune resistance via decaying JWA-XRCC1-mediated SSBR and immunosuppression, improving the development of emerging Pt(IV) candidate as a potential immunotherapeutic agent for cancer resistant prevention.

Combined use of dbcAMP and IBMX minimizes the damage induced by a long-term artificial meiotic arrest in mouse germinal vesicle oocytes.

Phosphodiesterase (PDE)-mediated reduction of cyclic adenosine monophosphate (cAMP) activity can initiate germinal vesicle (GV) breakdown in mammalian oocytes. It is crucial to maintain oocytes at the GV stage for a long period to analyze meiotic resumption in vitro. Meiotic resumption can be reversibly inhibited in isolated oocytes by cAMP modulator forskolin, cAMP analog dibutyryl cAMP (dbcAMP), or PDE inhibitors, milrinone (Mil), Cilostazol (CLZ), and 3-isobutyl-1-methylxanthine (IBMX). However, these chemicals negatively affect oocyte development and maturation when used independently. Here, we used ICR mice to develop a model that could maintain GV-stage arrest with minimal toxic effects on subsequent oocyte and embryonic development. We identified optimal concentrations of forskolin, dbcAMP, Mil, CLZ, IBMX, and their combinations for inhibiting oocyte meiotic resumption. Adverse effects were assessed according to subsequent development potential, including meiotic resumption after washout, first polar body extrusion, early apoptosis, double-strand DNA breaks, mitochondrial distribution, adenosine triphosphate levels, and embryonic development. Incubation with a combination of 50.0 µM dbcAMP and 10.0 µM IBMX efficiently inhibited meiotic resumption in GV-stage oocytes, with low toxicity on subsequent oocyte maturation and embryonic development. This work proposes a novel method with reduced toxicity to effectively arrest and maintain mouse oocytes at the GV stage.

Redox-Active Bis-Cyclometalated Iridium(III) Complex as a DNA Photo-Cleaving Agent.

The development of new photoactive metal complexes that can trigger oxidative damages to the genetic material is of great interest. In the present paper, we describe the detailed study of a highly photo-oxidant iridium(III) complex that triggers photoinduced electron transfer (PET) with purine DNA bases. The PET has been studied by luminescence and laser flash photolysis experiments. From plasmid DNA agarose gel electrophoresis experiments, we demonstrated the high ability of the iridium complex to induce strand breaks upon light irradiation. Reactive oxygen species (ROS)-specific scavengers and stabilizers were employed to identify that the photocleavage process, the results of which infer singlet oxygen and hydrogen peroxide as the predominant species. To the best of our knowledge, the present work represents one of the few study for highly photo-oxidant bis-cyclometalated iridium(III) complex toward DNA.

Effect of lansoprazole on DNA integrity of human spermatozoa and activity of seminal creatine kinase.

Although lansoprazole (brand name Prevacid) is a commonly used dug to manage various acid-related gastrointestinal diseases, little is known about its effects on human semen quality and sperm parameters. Here, we aimed to investigate the effect of lansoprazole on DNA integrity of human spermatozoa and activity of seminal creatine kinase. DNA integrity of human spermatozoa was assessed by the Apo-Direct™ kit followed by flow cytometry. The activity of creatine kinase was measured by kinetic spectrophotometric method using commercially available kits following the International Federation of Clinical Chemistry recommendations. Lansoprazole at 3 µg/ml, after 1-hr incubation period, did not show any significant increase in fluorescein isothiocyanate fluorescence (p > .05) and hence on the content of DNA breaks of human spermatozoa. In addition, there was no significant change (p = .8113) in the activity of seminal creatine kinase by the effect of lansoprazole. In conclusion, lansoprazole at 3 µg/ml did not alter DNA integrity of human spermatozoa or activity of seminal creatine kinase after 1-hr incubation period.

Comparative toxicity of selected PAHs in rainbow trout hepatocytes: genotoxicity, oxidative stress and cytotoxicity.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous contaminants in aquatic ecosystems, which may have potentially toxic effects on organisms. In this study occurrence of DNA strand breaks, oxidative stress, and cytotoxicity were investigated in rainbow trout hepatocytes following in vitro exposure for 24 h to four PAHs (0.01-10 µM): naphthalene, fluoranthene, pyrene, and benzo[a]pyrene (B[a]P). The exposed hepatocytes were analyzed for DNA strand breaks using the comet assay and for antioxidant status by measuring intracellular glutathione (GSH) content using the fluorescent probe mBCl. The cytotoxicity of PAHs was assessed using the fluorescent probe CFDA-AM. The results showed that fluoranthene, pyrene, and B[a]P were genotoxic at all exposure concentrations, whereas naphthalene was genotoxic at concentrations ≥0.1 µM. All treatments reduced the intracellular concentrations of GSH for all four PAHs, except 10 µM of B[a]P, suggesting that some level of oxidative stress was present. The cytotoxic effect was observed for naphthalene at concentrations ≥0.1 µM and pyrene at all exposure concentrations, whereas fluoranthene and B[a]P were not cytotoxic at the tested concentrations. The study shows that low-molecular-weight PAHs may cause DNA strand breaks as high-molecular-weight PAHs do in fish tissue. In addition, two- to five-ring PAHs can induce oxidative stress and cytotoxicity.

Berberine attenuates XRCC1-mediated base excision repair and sensitizes breast cancer cells to the chemotherapeutic drugs.

Berberine (BBR) is a natural isoquinoline alkaloid, which is used in traditional medicine for its anti-microbial, anti-protozoal, anti-diarrhoeal activities. Berberine interacts with DNA and displays anti-cancer activities, yet its effects on cellular DNA repair and on synthetic treatments with chemotherapeutic drugs remain unclear. In this study, we investigated the effects of BBR on DNA repair and on sensitization of breast cancer cells to different types of DNA damage anti-tumoural drugs. We found BBR arrested cells in the cell cycle S phase and induced DNA breaks. Cell growth analysis showed BBR sensitized MDA-MB-231 cells to cisplatin, camptothecin and methyl methanesulfonate; however, BBR had no synergistic effects with hydroxurea and olaparib. These results suggest BBR only affects specific DNA repair pathways. Western blot showed BBR down-regulated XRCC1 expressions, and the rescued XRCC1 recovered the resistance of cancer cells to BBR. Therefore, we conclude that BBR interferes with XRCC1-mediated base excision repair to sensitize cancer cells to chemotherapeutic drugs. These finding can contribute to understanding the effects of BBR on cellular DNA repair and the clinical employment of BBR in treatment of breast cancer.

Oxidation of phosphorothioate DNA modifications leads to lethal genomic instability.

Genomic modification by sulfur in the form of phosphorothioate (PT) is widespread among prokaryotes, including human pathogens. Apart from its physiological functions, PT sulfur has redox and nucleophilic properties that suggest effects on bacterial fitness in stressful environments. Here we show that PTs are dynamic and labile DNA modifications that cause genomic instability during oxidative stress. In experiments involving isotopic labeling coupled with mass spectrometry, we observed sulfur replacement in PTs at a rate of ∼2% h-1 in unstressed Escherichia coli and Salmonella enterica. Whereas PT levels were unaffected by exposure to hydrogen peroxide (H2O2) or hypochlorous acid (HOCl), PT turnover increased to 3.8-10% h-1 after HOCl treatment and was unchanged by H2O2, consistent with the repair of HOCl-induced sulfur damage. PT-dependent sensitivity to HOCl extended to cytotoxicity and DNA strand breaks, which occurred at HOCl doses that were orders of magnitude lower than the corresponding doses of H2O2. The genotoxicity of HOCl in PT-containing bacteria suggests reduced fitness in competition with HOCl-producing organisms and during infections in humans.

Famotidine does not affect human sperm fertility characteristics (motility, viability and DNA integrity) in vitro.

Famotidine, a histamine-2 receptor antagonist, is commonly used to relieve the acid-related gastrointestinal diseases; however, its effect on human sperm parameters, and hence on sperm function, is still undetermined. Here, we intended to measure human sperm motility, viability, and DNA integrity of ejaculated human sperm in the presence of famotidine at 0, 0.1, 1 and 10 mM concentrations in vitro. Forty-nine semen samples of normal count, motility, and morphology were included in this study. Sperm motility was assessed using Makler counting chamber and a phase contrast optics (200× magnification), whereas sperm viability was assessed using eosin-nigrosin staining procedure. The effect of famotidine on sperm DNA integrity was measured using flow cytometry. Famotidine at 0.1, 1 or 10 mM had insignificant effect on human sperm motility (progressive, p = .9594; and total, p = .8420), sperm viability (p = .6471), and content of DNA breaks in sperm (p > .05) compared with the control. In conclusion, famotidine at 0.1, 1 or 10 mM did not alter human sperm motility, viability or DNA integrity in vitro. Although, these findings indicate safety of famotidine in human sperm, further in vivo studies are required to establish the drug's safety.

Bis(2-ethylhexyl) phthalate induces DNA strand breaks and gene expression alterations in larval zebrafish Danio rerio.

Contamination of the aquatic environment by plastic industrial products and their by-products is remarkable. Because of their physical, chemical, and biological degradation resistance, plasticizers can enter the food chain of living organisms, accumulate in the body and generate toxic effects. Here we determined the potential toxic effects of bis(2-ethylhexyl) phthalate (DEHP) plasticizer to larval (72 h post fertilization) zebrafish (Danio rerio) by analyzing changes in expression levels of stress-related genes (p53, rad51, and xrcc5) by the quantitative real-time polymerase chain reaction. Also, possible DNA damage by DEHP in larvae was determined. The concentration of DEHP (0-160 mg/l) that killed 50% of the larval zebrafish within 96 h was 54.02 mg/l. There was a concentration-related increase in DNA damage in cells from larvae exposed (96 h) to DEHP. DNA damage of 31.13% (mean ± standard error of the mean) was observed in larvae at the highest sublethal DEHP concentration (10 mg/l). Some significant differences in the induction of stress-related genes were also observed in larvae exposed to DEHP relative to control (p < 0.05). The conclusion drawn from this ecotoxicological risk assessment is that, under present use and exposure patterns, DEHP presents a small hazard to zebrafish larvae.

Diazinon negatively affects the integrity of environmental DNA stability: a case study with common carp (Cyprinus carpio).

Environmental DNA (eDNA) has been used to detect the presence of various species in aquatic ecosystems, but its degradation by several environmental factors can influence the correct identification of aquatic organisms. The present study examined the effects of a pesticide, diazinon, on breakage of Cyprinus carpio eDNA. The specimens were exposed to 0 (control), 0.06, 0.1, and 1 ppm of diazinon for 9 days. Water samples were collected at three time points (3, 6, and 9 days postexposure, dpe), and eDNA was extracted. The cytochrome oxidase I (COI) gene was successfully amplified by PCR, and a fuzzy inference system was used to convert DNA smears and breakage to numerical values. eDNA breakage percentage increased with diazinon concentration at all sampling times. At 3 dpe, the maximum eDNA breakage percentage occurred at 0.06 and 0.1 ppm of diazinon; whereas at 6 and 9 dpe, the maximum breakage was found at 1 ppm of diazinon, while exposure time had no significant effect. To the best of our knowledge, this is the first study to demonstrate that eDNA integrity can be compromised by a diazinon in surface waters. Hence, it is recommended that future eDNA studies take into account pesticide pollution when detecting aquatic species.

Oxidative stress in metabolic syndrome & its association with DNA-strand break.

BACKGROUND & OBJECTIVES: Oxidative stress (OS) is associated with numerous components of metabolic syndrome (MetS). This study was aimed to investigate if hydrogen peroxide (H2O2) as the reactive oxygen species was capable of depicting OS in MetS, and If MetS patients showed DNA damage in the form of DNA strand breaks (DSB). METHODS: A total of 160 participants (90 males, 70 females) ≥20 yr of age were categorized into four groups based on the number of MetS risk parameters (n=40 in each group). Sugar and lipid profile, H2O2concentration in blood and DNA-strand breaks were measured. RESULTS: DSB was significantly more in those with MetS (n=40) than those without (n=120) whereas H2O2levels were the same in both the study groups. The number of DSB differed significantly between the control and 3 risk factor groups. DSB was also higher in groups with 2 and 1 risk factors compared to 0 risk but the difference was not significant. H2O2 level was higher in groups with 3, 2 and 1 risk factors compared to 0 risk group but the difference was not significant. The H2O2level correlated positively with triglyceride values but not with other MetS risk parameters. There was no significant correlation between DSB and MetS risk parameters. INTERPRETATION & CONCLUSIONS: Our findings showed a cumulative and synergistic effect of the risk factors of MetS on DSB. Individuals with three risk parameters had a greater effect on DNA damage than in those with two or one risk parameter. Although plasma H2O2level increased with an increase in the fat depots, use of H2O2to depict OS in MetS should be coupled with an adjunct and estimation of DSB in peripheral blood lymphocytes may be used as indicator of OS in MetS patients.

PKCα and HMGB1 antagonistically control hydrogen peroxide-induced poly-ADP-ribose formation.

Harmful oxidation of proteins, lipids and nucleic acids is observed when reactive oxygen species (ROS) are produced excessively and/or the antioxidant capacity is reduced, causing 'oxidative stress'. Nuclear poly-ADP-ribose (PAR) formation is thought to be induced in response to oxidative DNA damage and to promote cell death under sustained oxidative stress conditions. However, what exactly triggers PAR induction in response to oxidative stress is incompletely understood. Using reverse phase protein array (RPPA) and in-depth analysis of key stress signaling components, we observed that PAR formation induced by H2O2 was mediated by the PLC/IP3R/Ca(2+)/PKCα signaling axis. Mechanistically, H2O2-induced PAR formation correlated with Ca(2+)-dependent DNA damage, which, however, was PKCα-independent. In contrast, PAR formation was completely lost upon knockdown of PKCα, suggesting that DNA damage alone was not sufficient for inducing PAR formation, but required a PKCα-dependent process. Intriguingly, the loss of PAR formation observed upon PKCα depletion was overcome when the chromatin structure-modifying protein HMGB1 was co-depleted with PKCα, suggesting that activation and nuclear translocation of PKCα releases the inhibitory effect of HMGB1 on PAR formation. Together, these results identify PKCα and HMGB1 as important co-regulators involved in H2O2-induced PAR formation, a finding that may have important relevance for oxidative stress-associated pathophysiological conditions.

Platinum(IV)-nitroxyl complexes as possible candidates to circumvent cisplatin resistance in RT112 bladder cancer cells.

The therapeutic efficacy of the anticancer drug cisplatin is limited by the development of resistance. We therefore investigated newly synthesized platinum-nitroxyl complexes (PNCs) for their potential to circumvent cisplatin resistance. The complexes used were PNCs with bivalent cis-PtII(R·NH2)(NH3)Cl2 and cis-PtII(DAPO)Ox and four-valent platinum cis,trans,cis-PtIV(R·NH2)(NH3)(OR)2Cl2 and cis,trans,cis-PtIV(DAPO)(OR)2Ox, where R· are TEMPO or proxyl nitroxyl radicals, DAPO is trans-3,4-diamino-2,2,6,6-tetramethylpiperidine-1-oxyl, and OR and Ox are carboxylato and oxalato ligands, respectively. The complexes were characterized by spectroscopic methods, HPLC, log P ow data and elemental analysis. We studied intracellular platinum accumulation, DNA platination and cytotoxicity upon treatment with the PNCs in a model system of the bladder cancer cell line RT112 and its cisplatin-resistant subline RT112-CP. Platinum accumulation and DNA platination were similar in RT112 and RT112-CP cells for both bivalent and four-valent PNCs, in contrast to cisplatin for which a reduction in intracellular accumulation and DNA platination was observed in the resistant subline. The PNCs were found to platinate DNA in relation to the length of their axial RO-ligands. Furthermore, the PNCs were increasingly toxic in relation to the elongation of their axial RO-ligands, with similar toxicities in RT112 and its cisplatin-resistant subline. Using a cell-free assay, we observed induction of oxidative DNA damage by cisplatin but not PNCs suggesting that cisplatin exerts its toxic action by platination and oxidative DNA damage, while cells treated with PNCs are protected against oxidatively induced lesions. Altogether, our study suggests that PNCs may provide a more effective treatment for tumors which have developed resistance toward cisplatin.

The Brassica-derived phytochemical indolo[3,2-b]carbazole protects against oxidative DNA damage by aryl hydrocarbon receptor activation.

Epidemiological studies suggest that a high intake of Brassica vegetables protects against colon carcinogenesis. Brassica vegetables are rich in glucosinolates which are hydrolysed during digestion to various products including indole-3-carbinol. In animal studies, a protective effect of indole-3-carbinol has been demonstrated in colon carcinogenesis. Indole-3-carbinol is highly unstable and, therefore, the observed protection likely results from condensation products of indole-3-carbinol, e.g. diindolylmethane or indolo[3,2-b]carbazole (ICZ). Interestingly, ICZ is a potent activator of the aryl hydrocarbon receptor (AhR), a transcription factor known to mediate toxic effects of environmental pollutants, such as dioxin and polycyclic aromatic hydrocarbons. Here, we show that ICZ protects against oxidative DNA damage in various cell lines including the colon carcinoma cell line Caco-2. When preincubated for 24 h, ICZ decreases DNA single-strand break (SSB) and 8-oxo-dG formation induced by tertiary-butylhydroperoxide (t-BOOH), hydrogen peroxide or benzo[a]pyrene. Simultaneous addition of ICZ does not protect against t-BOOH-induced SSB formation, which disproves a direct radical scavenging effect. The repair of SSBs was not enhanced, but the data indicate that ICZ attenuates the ROS level following t-BOOH. The antioxidant response factor Nrf2 was not activated following ICZ. Functional inhibition of the AhR and AhR-/ARNT-defective cell lines demonstrate that the AhR/ARNT pathway is mandatory for the observed ROS defence caused by ICZ, supporting the hypothesis that AhR-mediated regulation of defence genes is involved. The data point to a hitherto unknown protective function of ICZ and a novel role of the AhR in the defence against oxidative DNA damage.

Synthesis and Functional Investigations of Computer Designed Novel Cladribine-Like Compounds for the Treatment of Multiple Sclerosis.

Cladribine (2-CdA) is used as an anti-cancer drug but is currently studied as a potential treatment for use in relapsing-remitting multiple sclerosis (MS). In this study, we computer designed, synthesized, and characterized two novel derivatives of 2-CdA, K1-5d and K2-4c, and investigated their underlying mechanism of beneficial effect using the CCRF-CEM and RAJI cell lines. For this purpose, we first determined their effect on MS and DNA damage and repair-related gene expression profiles using custom arrays along with 2-CdA treatment at non-toxic doses. Then, we determined whether cells underwent apoptosis after treatment with 2-CdA, K1-5d, and K2-4c in CCRF-CEM and RAJI cells, using the DNA fragmentation assay. It was found that both derivatives modulated the expression of the pathway-related genes that are important in inflammatory signaling, apoptosis, ATM/ATR, double-strand break repair, and the cell cycle. Furthermore, 2-CdA, K1-5d, and K2-4c significantly activated apoptosis in both cell lines. In summary, our data demonstrate that although both derivatives act as anti-inflammatory and apoptotic agents, inducing the accumulation of DNA strand breaks and activating the ultimate tumor suppressor p53 in T and B lymphocytes, the K1-5d derivative has shown more promising activities for further studies.

Protective Effect of Juglans regia L. Walnut Extract Against Oxidative DNA Damage.

Walnuts (Juglans regia L.) are relevant components of the Mediterranean diet providing important macronutrients, micronutrients and other bioactive constituents including unsaturated fatty acids, proteins, fiber, vitamins, minerals, phytosterols and polyphenols. Although the walnut beneficial effects in human health are widely recognized by a lot of epidemiologic studies very little is known regarding its effect on damaged DNA. The aim of the present study was to investigate the effect of Juglans regia L. ethanolic extract from kernel on the induction of DNA strand breaks by thiol/Fe3+/O2 mixed function oxidase, tert-butyl hydroperoxide or UVC radiations in acellular and cellular models. Plasmid DNA cleavage and fast Halo assay were used to monitor oxidative damage to DNA. Both approaches showed protection of oxidatively injured DNA. These results agree with a lot of scientific proofs which recommend walnut as dietary adjunct in health promotion and prevention as well as in treatment of lifestyle-related oxidative diseases.