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Journal Cover   Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
  [SJR: 0.245]   [H-I: 11]   [2 followers]  Follow
    
   Hybrid Journal Hybrid journal (It can contain Open Access articles)
   ISSN (Print) 0027-5107
   Published by Elsevier Homepage  [2811 journals]
  • Mitochondrial DNA mutations in single human blood cells
    • Abstract: Publication date: Available online 22 June 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
      Author(s): Yong-Gang Yao , Sachiko Kajigaya , Neal S. Young
      Determination mitochondrial DNA (mtDNA) sequences from extremely small amounts of DNA extracted from tissue of limited amounts and/or degraded samples is frequently employed in medical, forensic, and anthropologic studies. Polymerase chain reaction (PCR) amplification followed by DNA cloning is a routine method, especially to examine heteroplasmy of mtDNA mutations. In this review, we compare the mtDNA mutation patterns detected by three different sequencing strategies. Cloning and sequencing methods that are based on PCR amplification of DNA extracted from either single cells or pooled cells yield a high frequency of mutations, partly due to the artifacts introduced by PCR and/or the DNA cloning process. Direct sequencing of PCR product which has been amplified from DNA in individual cells is able to detect the low levels of mtDNA mutations present within a cell. We further summarize the findings in our recent studies that utilized this single cell method to assay mtDNA mutation patterns in different human blood cells. Our data show that many somatic mutations observed in the end-stage differentiated cells are found in hematopoietic stem cells (HSCs) and progenitors within the CD34+ cell compartment. Accumulation of mtDNA variations in the individual CD34+ cells is affected by both aging and family genetic background. Granulocytes harbor higher numbers of mutations compared with the other cells, such as CD34+ cells and lymphocytes. Serial assessment of mtDNA mutations in a population of single CD34+ cells obtained from the same donor over time suggests stability of some somatic mutations. CD34+ cell clones from a donor marked by specific mtDNA somatic mutations can be found in the recipient after transplantation. The significance of these findings is discussed in terms of the lineage tracing of HSCs, aging effect on accumulation of mtDNA mutations and the usage of mtDNA sequence in forensic identification.


      PubDate: 2015-06-26T13:58:47Z
       
  • APE1, the DNA base excision repair protein, regulates the removal of
           platinum adducts in sensory neuronal cultures by NER
    • Abstract: Publication date: Available online 26 June 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
      Author(s): Hyun-Suk Kim , Chunlu Guo , Eric L. Thompson , Yanlin Jiang , Mark R. Kelley , Michael R. Vasko , Suk-Hee Lee
      Peripheral neuropathy is one of the major side effects of treatment with the anticancer drug, cisplatin. One proposed mechanism for this neurotoxicity is the formation of platinum adducts in sensory neurons that could contribute to DNA damage. Although this damage is largely repaired by nuclear excision repair (NER), our previous findings suggest that augmenting the base excision repair pathway (BER) by overexpressing the repair protein APE1 protects sensory neurons from cisplatin-induced neurotoxicity. The question remains whether APE1 contributes to the ability of the NER pathway to repair platinum-damage in neuronal cells. To examine this, we manipulated APE1 expression in sensory neuronal cultures and measured Pt-removal after exposure to cisplatin. When neuronal cultures were treated with increasing concentrations of cisplatin for two or three hours, there was a concentration-dependent increase in Pt-damage that peaked at four hours and returned to near baseline levels after 24h. In cultures where APE1 expression was reduced by ∼80% using siRNA directed at APE1, there was a significant inhibition of Pt-removal over eight hours which was reversed by overexpressing APE1 using a lentiviral construct for human wtAPE1. Reduction in APE1 expression also altered the expression of the NER proteins RPA70 and XPA in sensory neuronal cultures. Overexpressing a mutant APE1 (C65 APE1), which only has DNA repair activity, but not its other significant redox-signaling function, mimicked the effects of wtAPE1. Overexpressing DNA repair activity mutant APE1 (226+177APE1), with only redox activity was ineffective suggesting it is the DNA repair function of APE1 and not its redox-signaling, that restores the Pt-damage removal. Together, these data provide the first evidence that a critical BER enzyme, APE1, helps regulate the NER pathway in the repair of cisplatin damage in sensory neurons.


      PubDate: 2015-06-26T13:58:47Z
       
  • MKP1 phosphatase mediates G1-specific dephosphorylation of H3Serine10P in
           response to DNA damage
    • Abstract: Publication date: August 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volume 778
      Author(s): Ajit K. Sharma , Shafqat A. Khan , Asmita Sharda , Divya V Reddy , Sanjay Gupta
      Histone mark, H3S10 phosphorylation plays a dual role in a cell by maintaining relaxed chromatin for active transcription in interphase and condensed chromatin state in mitosis. The level of H3S10P has also been shown to alter on DNA damage; however, its cell cycle specific behavior and regulation during DNA damage response is largely unexplored. In the present study, we demonstrate G1 cell cycle phase specific reversible loss of H3S10P in response to IR-induced DNA damage is mediated by opposing activities of phosphatase, MKP1 and kinase, MSK1 of the MAP kinase pathway. We also show that the MKP1 recruits to the chromatin in response to DNA damage and correlates with the decrease of H3S10P, whereas MKP1 is released from chromatin during recovery phase of DDR. Furthermore, blocking of H3S10 dephosphorylation by MKP1 inhibition impairs DNA repair process and results in poor survival of WRL68 cells. Collectively, our data proposes a pathway regulating G1 cell cycle phase specific reversible reduction of H3S10P on IR induced DNA damage and also raises the possibility of combinatorial modulation of H3S10P with specific inhibitors to target the cancer cells in G1-phase of cell cycle.


      PubDate: 2015-06-26T13:58:47Z
       
  • Effects of changes in intracellular iron pool on AlkB-dependent and
           AlkB-independent mechanisms protecting E.coli cells against mutagenic
           action of alkylating agent
    • Abstract: Publication date: August 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volume 778
      Author(s): Anna Sikora , Agnieszka M. Maciejewska , Jarosław Poznański , Tomasz Pilżys , Michał Marcinkowski , Małgorzata Dylewska , Jan Piwowarski , Wioletta Jakubczak , Katarzyna Pawlak , Elżbieta Grzesiuk
      An Escherichia coli hemH mutant accumulates protoporphyrin IX, causing photosensitivity of cells to visible light. Here, we have shown that intracellular free iron in hemH mutants is double that observed in hemH + strain. The aim of this study was to recognize the influence of this increased free iron concentration on AlkB-directed repair of alkylated DNA by analyzing survival and argE3 →Arg+ reversion induction after λ>320nm light irradiation and MMS-treatment in E. coli AB1157 hemH and alkB mutants. E.coli AlkB dioxygenase constitutes a direct single-protein repair system using non-hem Fe(II) and cofactors 2-oxoglutarate (2OG) and oxygen (O2) to initiate oxidative dealkylation of DNA/RNA bases. We have established that the frequency of MMS-induced Arg+ revertants in AB1157 alkB + hemH– /pMW1 strain was 40 and 26% reduced comparing to the alkB + hemH– and alkB + hemH+ /pMW1, respectively. It is noteworthy that the effect was observed only when bacteria were irradiated with λ >320nm light prior MMS-treatment. This finding indicates efficient repair of alkylated DNA in photosensibilized cells in the presence of higher free iron pool and AlkB concentrations. Interestingly, a 31% decrease in the level of Arg+ reversion was observed in irradiated and MMS-treated hemH – alkB – cells comparing to the hemH + alkB – strain. Also, the level of Arg+ revertants in the irradiated and MMS treated hemH– alkB– mutant was significantly lower (by 34%) in comparison to the same strain but MMS-treated only. These indicate AlkB-independent repair involving Fe ions and reactive oxygen species. According to our hypothesis it may be caused by non-enzymatic dealkylation of alkylated dNTPs in E. coli cells. In in vitro studies, the absence of AlkB protein in the presence of iron ions allowed etheno(ε) dATP and εdCTP to spontaneously convert to dAMP and dCMP, respectively. Thus, hemH – intra-cellular conditions may favor Fe-dependent dealkylation of modified dNTPs.


      PubDate: 2015-06-26T13:58:47Z
       
  • Nrf2 facilitates repair of radiation induced DNA damage through homologous
           recombination repair pathway in a ROS independent manner in cancer cells
    • Abstract: Publication date: Available online 22 June 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
      Author(s): Sundarraj Jayakumar , Debojyoti Pal , Santosh K. Sandur
      Nrf2 is a redox sensitive transcription factor that is involved in the co-ordinated transcription of genes involved in redox homeostasis. But the role of Nrf2 in DNA repair is not investigated in detail. We have employed A549 and MCF7 cells to study the role of Nrf2 on DNA repair by inhibiting Nrf2 using all-trans retinoic acid (ATRA) or by knock down approach prior to radiation exposure (4Gy). DNA damage and repair analysis was studied by γH2AX foci formation and comet assay. Results suggested that the inhibition of Nrf2 in A549 or MCF7 cells led to significant slowdown in DNA repair as compared to respective radiation controls. The persistence of residual DNA damage even in the presence of free radical scavenger N-acetyl cysteine, suggested that the influence of Nrf2 on DNA repair was not linked to its antioxidant functions. Further, its influence on non-homologous end joining repair pathway was studied by inhibiting both Nrf2 and DNA-PK together. This led to synergistic reduction of survival fraction, indicating that Nrf2 may not be influencing the NHEJ pathway. To investigate the role of homologous recombination repair (HR) pathway, RAD51 foci formation was monitored. There was a significant reduction in the foci formation in cells treated with ATRA or shRNA against Nrf2 as compared to their respective radiation controls. Further, Nrf2 inhibition led to significant reduction in mRNA levels of RAD51. BLAST analysis was also performed on upstream regions of DNA repair genes to identify antioxidant response element and found that many repair genes that are involved in HR pathway may be regulated by Nrf2. Together, these results suggest the involvement of Nrf2 in DNA repair, a hitherto unknown function of Nrf2, putatively through its influence on HR pathway.


      PubDate: 2015-06-26T13:58:47Z
       
  • Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) interacts with
           apurinic/apyrimidinic sites in DNA
    • Abstract: Publication date: Available online 17 June 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
      Author(s): Anastasiya A. Kosova , Svetlana N. Khodyreva , Olga I. Lavrik
      Apurinic/apyrimidinic (AP) sites are some of the most frequent DNA damages and the key intermediates of base excision repair. Certain proteins can interact with the deoxyribose of the AP site to form a Schiff base, which can be stabilized by NaBH4 treatment. Several types of DNA containing the AP site were used to trap proteins in human cell extracts by this method. In the case of single-stranded AP DNA and AP DNA duplex with both 5ʹ and 3ʹ dangling ends, the major crosslinking product had apparent molecular mass of 45 kDa. Using peptide mass mapping based on mass spectrometry data, we identified the protein forming this adduct as an isoform of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) called “uracil-DNA glycosylase”. GAPDH is a glycolytic enzyme with many additional putative functions, which include interaction with nucleic acids, different DNA damages and DNA repair enzymes. We investigated interaction of GAPDH purified from HeLa cells and rabbit muscles with different AP DNAs. In spite of the ability to form a Schiff-base intermediate with the deoxyribose of the AP site, GAPDH does not display the AP lyase activity. In addition, along with borohydride-dependent adducts with AP DNA containing single-stranded regions, GAPDH was shown to form also stable borohydride-independent crosslinks with these DNA. GAPDH was proven to crosslink preferentially to AP DNA cleaved via β-elimination mechanism (spontaneously or by AP lyases) as compared to DNA containing the intact AP site. The level of GAPDH–AP DNA adduct formation depends on oxidation of the protein SH-groups; disulfide bond reduction in GAPDH leads to loss of its ability to form adducts with AP DNA. Possible role of formation of stable adducts with AP sites by GAPDH is discussed.
      Graphical abstract image

      PubDate: 2015-06-21T13:07:03Z
       
  • Increased Human AP Endonuclease 1 Level Confers Protection Against the
           Paternal Age Effect in Mice
    • Abstract: Publication date: Available online 20 June 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
      Author(s): Jamila R. Sanchez , Traci L. Reddick , Marissa Perez , Victoria E. Centonze , Sankar Mitra , Tadahide Izumi , C.Alex McMahan , Christi A. Walter.
      Increased paternal age is associated with a greater risk of producing children with genetic disorders originating from de novo germline mutations. Mice mimic the human condition by displaying an age-associated increase in spontaneous mutant frequency in spermatogenic cells. The observed increase in mutant frequency appears to be associated with a decrease in the DNA repair protein, AP endonuclease 1 (APEX1) and Apex1 heterozygous mice display an accelerated paternal age effect as young adults. In this study, we directly tested if APEX1 over-expression in cell lines and transgenic mice could prevent increases in mutagenesis. Cell lines with ectopic expression of APEX1 had increased APEX1 activity and lower spontaneous and induced mutations in the lacI reporter gene relative to the control. Spermatogenic cells obtained from mice transgenic for human APEX1 displayed increased APEX1 activity, were protected from the age-dependent increase in spontaneous germline mutagenesis, and exhibited increased apoptosis in the spermatogonial cell population. These results directly indicate that increases in APEX1 level confer protection against the murine paternal age effect, thus highlighting the role of APEX1 in preserving reproductive health with increasing age and in protection against genotoxin-induced mutagenesis in somatic cells.


      PubDate: 2015-06-21T13:07:03Z
       
  • The BAH domain of BAF180 is required for PCNA ubiquitination
    • Abstract: Publication date: Available online 17 June 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
      Author(s): Atsuko Niimi , Suzanna R Hopkins , Jessica A Downs , Chikahide Masutani
      Monoubiquitination of proliferating cell nuclear antigen (PCNA) is a critical regulator of Post Replication Repair (PRR). The depletion of BAF180, a unique subunit of the PBAF chromatin remodeling complex in human cells results in reduced PCNA ubiquitination leading to less efficient fork progression following DNA damage, but little is known about the mechanism. Here, we report that the expression of exogenous BAF180 in cells promotes PCNA ubiquitination during S-phase after UV irradiation and it persists for many hours. No correlation was observed between the protein level of ubiquitin-specific protease 1 (USP1) and ubiquitinated PCNA in BAF180 expressing cells. Analysis of cells expressing BAF180 deletion mutants showed that the bromo-adjacent homology (BAH) domains are responsible for this effect. Surprisingly, a deletion construct encoding only the BAH domain region is able to increase the level of ubiquitinated PCNA, even though it is unable to be assembled into the PBAF complex. These results suggest that the ATPase-dependent chromatin remodeling activity of PBAF is not necessary, but instead the BAH domains are sufficient to promote PCNA ubiquitination.


      PubDate: 2015-06-21T13:07:03Z
       
  • MWCNT uptake in Allium cepa root cells induces cytotoxic and genotoxic
           responses and results in DNA hyper-methylation
    • Abstract: Publication date: April 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volume 774
      Author(s): Manosij Ghosh , Sreetama Bhadra , Aremu Adegoke , Maumita Bandyopadhyay , Anita Mukherjee
      Advances in nanotechnology have led to the large-scale production of nanoparticles, which, in turn, increases the chances of environmental exposure. While humans (consumers/workers) are primarily at risk of being exposed to the adverse effect of nanoparticles, the effect on plants and other components of the environment cannot be ignored. The present work investigates the cytotoxic, genotoxic, and epigenetic (DNA methylation) effect of MWCNT on the plant system- Allium cepa. MWCNT uptake in root cells significantly altered cellular morphology. Membrane integrity and mitochondrial function were also compromised. The nanotubes induced significant DNA damage, micronucleus formation and chromosome aberration. DNA laddering assay revealed the formation of internucleosomal fragments, which is indicative of apoptotic cell death. This finding was confirmed by an accumulation of cells in the sub-G0 phase of the cell cycle. An increase in CpG methylation was observed using the isoschizomers MspI/HpaII. HPLC analysis of DNA samples revealed a significant increase in the levels of 5-methyl-deoxy-cytidine (5mdC). These results confirm the cyto-genotoxic effect of MWCNT in the plant system and simultaneously highlight the importance of this epigenetic study in nanoparticle toxicity.


      PubDate: 2015-06-14T11:42:17Z
       
  • T-cells enhance stem cell mutagenesis in the mouse colon
    • Abstract: Publication date: April 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volume 774
      Author(s): Ryan D. Whetstone , Barry Gold
      A role of inflammation in the etiology of cancer is attributed to the production of reactive oxygen/nitrogen species that can damage DNA. To test this hypothesis, we determined the mutation frequency (MF) in colonic stem cells in C57Bl/6 mice exposed to azoxymethane (AOM), dextran sulfate sodium (DSS) and a combination of AOM and DSS (AOM+DSS). AOM+DSS efficiently and rapidly produces colon tumors in B6 mice. AOM produces promutagenic O6-methylguanine lesions in DNA but does not induce colon tumors in C57Bl/6 mice as a single agent. DSS produces inflammation in the colon but does not produce tumors except upon multiple cycles of treatment in some DNA repair deficient mouse models. In addition, using TCRβ null mice we tested whether α/β T cells have any effect on the colonic stem cell MF in mice treated with AOM, DSS and AOM+DSS. The TCRβ−/− mice are devoid of the critical receptor required for normal cytolytic and regulatory α/β T-cell functions. The MF in the untreated and DSS treated WT and TCRβ−/− mice was the same (<10−5) indicating that DSS and subsequent inflammation does not generate stem cell mutations in mice that are WT for DNA repair. AOM yielded mutant crypts in WT and TCRβ−/− mice with MF's of ∼4×10−4 and 2×10−4, respectively, which represents a statistically significant decrease in the MF in the immune compromised mice. The combined treatment of AOM+DSS afforded fully mutated crypts in both strains with a statistically significant lower MF in the TCRβ−/− mice. In addition, the MF in both strains of mice after the combination of AOM+DSS is lower than observed with AOM alone indicating that DSS inflammation destroyed pre-existing AOM mutated crypts. Using the MF in WT mice, the efficiency for the conversion of promutagenic O6-methylguanine lesions into a stem cell mutations was calculated to be ∼0.4%.


      PubDate: 2015-06-14T11:42:17Z
       
  • Bisphenol A and congenital developmental defects in humans
    • Abstract: Publication date: April 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volume 774
      Author(s): Maurizio Guida , Jacopo Troisi , Carla Ciccone , Giovanni Granozio , Cosimo Cosimato , Attilio Di Spiezio Sardo , Cinzia Ferrara , Marco Guida , Carmine Nappi , Fulvio Zullo , Costantino Di Carlo
      Over 50% of the causes of fetal malformations in humans are still unknown. Recent evidence suggests the relationship between environmental exposure to endocrine disruptors and fetal malformations. Our study aims to establish the role of Bisphenol A (BPA), if any, in altering human reproduction. We enrolled 151 pregnant women who were divided into two groups: case group (CS, n =101), women with established diagnosis of developmental defect, and control group (CL, n =50), pregnant women with normally developed fetus. Total, free and conjugated BPA were measured in their blood using GC–MS with isotopic dilution. The results show a correlation between environmental exposure to BPA and the genesis of fetal malformations. Conjugated BPA, which was higher in the CL, casts light on the hypothesis that a reduced ability to metabolize the chemical in the mother can concur to the occurrence of malformation. In a more detailed manner, in case of chromosomal malformations, the average value of free BPA appears to be nearly three times greater than that of the controls. Similarly, in case of central and peripheral nervous system non-chromosomal malformations, the value of free BPA is nearly two times greater than that of the controls.


      PubDate: 2015-06-14T11:42:17Z
       
  • WRN translocation from nucleolus to nucleoplasm is regulated by SIRT1 and
           required for DNA repair and the development of chemoresistance
    • Abstract: Publication date: April 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volume 774
      Author(s): Sun-Young Lee , Hyunwoo Lee , Eun-Sun Kim , Sojin Park , Jiyoen Lee , Byungchan Ahn
      When defective or absent, Werner syndrome protein (WRN) causes a genetic premature aging disorder called Werner syndrome. Several studies have reported that defects in WRN function are responsible for not only progeria syndrome but also genomic instability via the deregulation of DNA repair, replication, recombination, and telomere stability. Given the importance of WRN in the repair process, we herein investigated the potential role of WRN in drug response by evaluating the DNA repair following exposure to cisplatin in human cancer cell lines. We found that the down-regulation of SIRT1 and inhibition of SIRT1 deacetylase activity blocked the translocation of WRN from the nucleolus to the nucleoplasm in response to genotoxic stresses. In addition, cells expressing low levels of WRN responded favorably to cisplatin, whereas cells expressing high levels responded poorly to cisplatin. The forced expression of WRN protein in chemosensitive cells resulted in an approximately two-fold increase in cell viability in response to cisplatin compared with vector controls and promoted DNA repair, while WRN-deficient cells accumulate unrepaired double-strand breaks following cisplatin exposure. These results suggest that WRN is regulated by SIRT1 and increased expression of WRN might be one of the determinants for the development of chemotherapeutic drug resistance.


      PubDate: 2015-06-14T11:42:17Z
       
  • Effect of phytochemical intervention on dibenzo[a,l]pyrene-induced DNA
           adduct formation
    • Abstract: Publication date: April 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volume 774
      Author(s): Gilandra K. Russell , Ramesh C. Gupta , Manicka V. Vadhanam
      Dibenzo[a,l]pyrene (DBP) has been found to be the most potent carcinogen of the polycyclic aromatic hydrocarbons (PAHs). Primary sources for DBP in the environment are combustion of wood and coal burning, gasoline and diesel exhaust, and tires. Given the likelihood of environmental exposure to DBP and strong experimental evidence of its potency, it is likely to contribute to lung cancer development. Intervention with compounds of natural origin (“phytochemicals”) is considered an effective means to prevent cancer development and favorably modulate the underlying mechanisms, including DNA adduct formation. In this study, several agents have been identified that inhibit environmental carcinogen-induced DNA adduct formation using a cell-free microsomal system. Of the ten agents tested, resveratrol (648±26 adducts/109 nucleotides), oltipraz (1007±348 adducts/109 nucleotides), delphinidin (1252±142 adducts/109 nucleotides), tanshinone I (1981±213 adducts/109 nucleotides), tanshinone IIA (2606±478 adducts/109 nucleotides) and diindoylmethane (3643±469 adducts/109 nucleotides) were the most effective compared to vehicle treatment (14,062±1097 adducts/109 nucleotides). DBP is metabolized by phase I metabolizing enzymes CYP1A1, CYP1A2, and CYP1B1. DBP-induced DNA adducts can be inhibited by several mechanisms. We found that all the test agents inhibited DNA adducts by inhibiting one or more of these enzymes. Oltipraz inhibited DNA adducts entirely by inhibiting the CYP450s, while resveratrol and delphinidin inhibited DNA adducts by also interacting directly with the carcinogenic metabolite, anti-dibenzo(a,l)pyrene-11,12-dihydrodiol-13,14-epoxide.


      PubDate: 2015-06-14T11:42:17Z
       
  • TITLE PAGE (EDI BOARD)
    • Abstract: Publication date: April 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volume 774




      PubDate: 2015-06-14T11:42:17Z
       
  • MYC impairs resolution of site-specific DNA double-strand breaks repair
    • Abstract: Publication date: April 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volume 774
      Author(s): Susanna Ambrosio , Stefano Amente , Giuliana Napolitano , Giacomo Di Palo , Luigi Lania , Barbara Majello
      Although it is established that when overexpressed, the MYC family proteins can cause DNA double-stand breaks (DSBs) and genome instability, the mechanisms involved remain unclear. MYC induced genetic instability may result from increased DNA damage and/or reduced DNA repair. Here we show that when overexpressed, MYC proteins induce a sustained DNA damage response (DDR) and reduce the wave of DSBs repair. We used a cell-based DSBs system whereby, upon induction of an inducible restriction enzyme AsiSI, hundreds of site-specific DSBs are generated across the genome to investigate the role of MYC proteins on DSB. We found that high levels of MYC do not block accumulation of γH2AX at AsiSI sites, but delay its clearance, indicating an inefficient repair, while the initial recognition of DNA damage is largely unaffected. Repair of both homologous and nonhomologous repair-prone segments, characterized by high or low levels of recruited RAD51, respectively, was delayed. Collectively, these data indicate that high levels of MYC proteins delay the resolution of DNA lesions engineered to occur in cell cultures.


      PubDate: 2015-06-14T11:42:17Z
       
  • Expression of human poly (ADP-ribose) polymerase 1 in Saccharomyces
           cerevisiae: Effect on survival, homologous recombination and
           identification of genes involved in intracellular localization
    • Abstract: Publication date: April 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volume 774
      Author(s): Marco La Ferla , Alberto Mercatanti , Giulia Rocchi , Samuele Lodovichi , Tiziana Cervelli , Luca Pignata , Maria Adelaide Caligo , Alvaro Galli
      The poly (ADP-ribose) polymerase 1 (PARP-1) actively participates in a series of functions within the cell that include: mitosis, intracellular signaling, cell cycle regulation, transcription and DNA damage repair. Therefore, inhibition of PARP1 has a great potential for use in cancer therapy. As resistance to PARP inhibitors is starting to be observed in patients, thus the function of PARP-1 needs to be studied in depth in order to find new therapeutic targets. To gain more information on the PARP-1 activity, we expressed PARP-1 in yeast and investigated its effect on cell growth and UV induced homologous recombination. To identify candidate genes affecting PARP-1 activity and cellular localization, we also developed a yeast genome wide genetic screen. We found that PARP-1 strongly inhibited yeast growth, but when yeast was exposed to the PARP-1 inhibitor 6(5-H) phenantridinone (PHE), it recovered from the growth suppression. Moreover, we showed that PARP-1 produced PAR products in yeast and we demonstrated that PARP-1 reduced UV-induced homologous recombination. By genome wide screening, we identified 99 mutants that suppressed PARP-1 growth inhibition. Orthologues of human genes were found for 41 of these yeast genes. We determined whether the PARP-1 protein level was altered in strains which are deleted for the transcription regulator GAL3, the histone H1 gene HHO1, the HUL4 gene, the deubiquitination enzyme gene OTU1, the nuclear pore protein POM152 and the SNT1 that encodes for the Set3C subunit of the histone deacetylase complex. In these strains the PARP-1 level was roughly the same as in the wild type. PARP-1 localized in the nucleus more in the snt1Δ than in the wild type strain; after UV radiation, PARP-1 localized in the nucleus more in hho1 and pom152 deletion strains than in the wild type indicating that these functions may have a role on regulating PARP-1 level and activity in the nucleus.


      PubDate: 2015-06-14T11:42:17Z
       
  • l-Carnitine supplementation decreases DNA damage in treated MSUD patients
    • Abstract: Publication date: May 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volume 775
      Author(s): Caroline Paula Mescka , Gilian Guerreiro , Tatiane Hammerschmidt , Jéssica Faverzani , Daniella de Moura Coelho , Vanusa Mandredini , Carlos Alberto Yasin Wayhs , Moacir Wajner , Carlos Severo Dutra-Filho , Carmen Regla Vargas
      Maple syrup urine disease (MSUD) is an inherited disorder caused by severe deficient activity of the branched-chain α-keto acid dehydrogenase complex involved in the degradation pathway of branched-chain amino acids (BCAAs) and their α-ketoacid derivatives. MSUD patients generally present ketoacidosis, poor feeding, ataxia, coma, psychomotor delay, mental retardation and brain abnormalites. Treatment consists of dietary restriction of the BCAA (low protein intake) supplemented by a BCAA-free amino acid mixture. Although the mechanisms of brain damage in MSUD are poorly known, previous studies have shown that oxidative stress may be involved in the neuropathology of this disorder. In this regard, it was recently reported that MSUD patients have deficiency of l-carnitine (l-car), a compound with antioxidant properties that is used as adjuvant therapy in various inborn errors of metabolism. In this work, we investigated DNA damage determined by the alkaline comet assay in peripheral whole blood leukocytes of MSUD patients submitted to a BCAA-restricted diet supplemented or not with l-car. We observed a significant increase of DNA damage index (DI) in leukocytes from MSUD patients under BCAA-restricted diet as compared to controls and that l-car supplementation significantly decreased DNA DI levels. It was also found a positive correlation between DI and MDA content, a marker of lipid peroxidation, and an inverse correlation between DI and l-car levels. Taken together, our present results suggest a role for reactive species and the involvement of oxidative stress in DNA damage in this disorder. Since l-car reduced DNA damage, it is presumed that dietary supplementation of this compound may serve as an adjuvant therapeutic strategy for MSUD patients in addition to other therapies.


      PubDate: 2015-06-14T11:42:17Z
       
  • High-throughput sequencing in mutation detection: A new generation of
           genotoxicity tests'
    • Abstract: Publication date: Available online 20 April 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
      Author(s): Alexander Y. Maslov , Wilber Quispe-Tintaya , Tatyana Gorbacheva , Ryan R. White , Jan Vijg
      The advent of next generation sequencing (NGS) technology has provided the means to directly analyze the genetic material in primary cells or tissues of any species in a high throughput manner for mutagenic effects of potential genotoxic agents. In principle, direct, genome-wide sequencing of human primary cells and/or tissue biopsies would open up opportunities to identify individuals possibly exposed to mutagenic agents, thereby replacing current risk assessment procedures based on surrogate markers and extrapolations from animal studies. NGS-based tests can also precisely characterize the mutation spectra induced by genotoxic agents, improving our knowledge of their mechanism of action. Thus far, NGS has not been widely employed in genetic toxicology due to the difficulties in measuring low-abundant somatic mutations. Here, we review different strategies to employ NGS for the detection of somatic mutations in a cost-effective manner and discuss the potential applicability of these methods in testing the mutagenicity of genotoxic agents.


      PubDate: 2015-06-14T11:42:17Z
       
  • Age dynamics of DNA damage and CpG methylation in the peripheral blood
           leukocytes of mice
    • Abstract: Publication date: May 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volume 775
      Author(s): Ilya Velegzhaninov , Vera Mezenceva , Olga Shostal , Ancha Baranova , Alexey Moskalev
      Certain types of DNA damage are known to accumulate with age. Here we present the quantitative data describing the extent of the spontaneous DNA damage in 144 SHK mice of various ages. In each animal, we assessed double strand breaks, single strand breaks and alkali-labile sites, as well as amounts of oxidized purines, oxidized pyrimidines and misincorporated uracils. In addition, overall levels of genome DNA methylation were evaluated. The amounts of oxidized pyrimidines were correlated with age in males only, while the amounts of double strand breaks (DSB) in DNA samples were correlated with age in females only (R =0.26; p <0.035). No age-related accumulation of single-strand breaks (SSB) was observed. The hypomethylation of DNA was significant in aging females, but not in aging males. Various types of DNA damage were correlated to each other. In attempt to develop more stable indicator of age-dependent alterations in DNA, the DNA Damage Level Differential (DDLD) indices was developed for comet assaying of peripheral blood leukocytes. As expected, DDLD index was shown to be better correlated with age than any single quantitative measure reflecting certain type of DNA damage. A variability of effectiveness of various kinds of DNA repair in individual animals was larger than expected. This conclusion may have a substantial impact on subsequent studies of the mutagens and other kinds of environmental stressors in animal populations. Nor DDLD neither individual quantitative measures of DNA damage were capable of prediction post-sampling survival time.


      PubDate: 2015-06-14T11:42:17Z
       
  • Effect of chronic low dose natural radiation in human peripheral blood
           mononuclear cells: Evaluation of DNA damage and repair using the alkaline
           comet assay
    • Abstract: Publication date: May 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volume 775
      Author(s): P.R. Vivek Kumar , M. Seshadri , G. Jaikrishan , Birajalaxmi Das
      This study investigates whether peripheral blood mononuclear cells (PBMCs) from inhabitants of Kerala in southwest India, exposed to chronic low dose natural radiation in vivo (>1mSvyear−1), respond with a radioadaptive response to a challenging dose of gamma radiation. Toward this goal, PBMCs isolated from 77 subjects from high-level natural radiation areas (HLNRA) and 37 subjects from a nearby normal level natural radiation area (NLNRA) were challenged with 2Gy and 4Gy gamma radiation. Subjects from HLNRA were classified based on the mean annual effective dose received, into low dose group (LDG) and high dose group (HDG) with mean annual effective doses of 2.69mSv (N =43, range 1.07mSvyear−1 to 5.55mSvyear−1) and 9.62mSv (N =34, range 6.07mSvyear−1 to17.41mSvyear−1), respectively. DNA strand breaks and repair kinetics (at 7min, 15min and 30min after 4Gy) were evaluated using the alkaline single cell gel electrophoresis (comet) assay. Initial levels of DNA strand breaks observed after either a 2Gy or a 4Gy challenging dose were significantly lower in subjects of the HDG from HLNRA compared to subjects of NLNRA (2Gy, P =0.01; 4Gy, P =0.02) and LDG (2Gy P =0.01; 4Gy, P =0.05). Subjects of HDG from HLNRA showed enhanced rejoining of DNA strand breaks (HDG/NLNRA, P =0.06) during the early stage of repair (within 7min). However at later times a similar rate of rejoining of strand breaks was observed across the groups (HDG, LDG and NLNRA). Preliminary results from our study suggest in vivo chronic low-level natural radiation provides an initial exposure that allows an adaptation to a subsequent higher radiation exposure, perhaps through improving DNA repair via an unknown mechanism. Therefore, further investigations would be necessary in this population to understand the biological and health effects of chronic low-level natural radiation exposures.


      PubDate: 2015-06-14T11:42:17Z
       
  • Paternal irradiation perturbs the expression of circadian genes in
           offspring
    • Abstract: Publication date: May 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volume 775
      Author(s): Andre M.G.F. Gomes , Ruth C. Barber , Yuri E. Dubrova
      The circadian system represents a complex network which influences the timing of many biological processes. Recent studies have established that circadian alterations play an important role in the susceptibility to many human diseases, including cancer. Here we report that paternal irradiation in mice significantly affects the expression of genes involved in rhythmic processes in their first-generation offspring. Using microarrays, the patterns of gene expression were established for brain, kidney, liver and spleen samples from the non-exposed offspring of irradiated CBA/Ca and BALB/c male mice. The most over-represented categories among the genes differentially expressed in the offspring of control and irradiated males were those involved in rhythmic process, circadian rhythm and DNA-dependent regulation of transcription. The results of our study therefore provide a plausible explanation for the transgenerational effects of paternal irradiation, including increased transgenerational carcinogenesis described in other studies.


      PubDate: 2015-06-14T11:42:17Z
       
  • A novel method for monitoring functional lesion-specific recruitment of
           repair proteins in live cells
    • Abstract: Publication date: May 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volume 775
      Author(s): Jordan Woodrick , Suhani Gupta , Pooja Khatkar , Kalpana Dave , Darya Levashova , Sujata Choudhury , Hadi Elias , Tapas Saha , Susette Mueller , Rabindra Roy
      DNA–protein relationships have been studied by numerous methods, but a particular gap in methodology lies in the study of DNA adduct-specific interactions with proteins in vivo, which particularly affects the field of DNA repair. Using the repair of a well-characterized and ubiquitous adduct, the abasic (AP) site, as a model, we have developed a comprehensive method of monitoring DNA lesion-specific recruitment of proteins in vivo over time. We utilized a surrogate system in which a Cy3-labeled plasmid containing a single AP-site was transfected into cells, and the interaction of the labeled DNA with BER enzymes, including APE1, Polβ, LIG1, and FEN1, was monitored by immunofluorescent staining of the enzymes by Alexafluor-488-conjugated secondary antibody. The recruitment of enzymes was characterized by quantification of Cy3-Alexafluor-488 co-localization. To validate the microscopy-based method, repair of the transfected AP-site DNA was also quantified at various time points post-transfection using a real time PCR-based method. Notably, the recruitment time kinetics for each enzyme were consistent with AP-site repair time kinetics. This microscopy-based methodology is reliable in detecting the recruitment of proteins to specific DNA substrates and can be extended to study other in vivo DNA–protein relationships in any DNA sequence and in the context of any DNA structure in transfectable proliferating or quiescent cells. The method may be applied to a variety of disciplines of nucleic acid transaction pathways, including repair, replication, transcription, and recombination.


      PubDate: 2015-06-14T11:42:17Z
       
  • Conservation of CFTR codon frequency through primates suggests synonymous
           mutations could have a functional effect
    • Abstract: Publication date: May 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volume 775
      Author(s): Lucilla Pizzo , Andrés Iriarte , Fernando Alvarez-Valin , Mónica Marín
      Cystic fibrosis is an inherited chronic disease that affects the lungs and digestive system, with a prevalence of about 1:3000 people. Cystic fibrosis is caused by mutations in CFTR gene, which lead to a defective function of the chloride channel, the cystic fibrosis transmembrane conductance regulator (CFTR). Up-to-date, more than 1900 mutations have been reported in CFTR. However for an important proportion of them, their functional effects and the relation to disease are still not understood. Many of these mutations are silent (or synonymous), namely they do not alter the encoded amino acid. These synonymous mutations have been considered as neutral to protein function. However, more recent evidence in bacterial and human proteins has put this concept under revision. With the aim of understanding possible functional effects of synonymous mutations in CFTR, we analyzed human and primates CFTR codon usage and divergence patterns. We report the presence of regions enriched in rare and frequent codons. This spatial pattern of codon preferences is conserved in primates, but this cannot be explained by sequence conservation alone. In sum, the results presented herein suggest a functional implication of these regions of the gene that may be maintained by purifying selection acting to preserve a particular codon usage pattern along the sequence. Overall these results support the idea that several synonymous mutations in CFTR may have functional importance, and could be involved in the disease.
      Graphical abstract image

      PubDate: 2015-06-14T11:42:17Z
       
  • HUMAN CIRCULATING PLASMA DNA SIGNIFICANTLY DECREASES WHILE LYMPHOCYTE DNA
           DAMAGE INCREASES UNDER CHRONIC OCCUPATIONAL EXPOSURE TO LOW-DOSE
           GAMMA-NEUTRON AND TRITIUM β-RADIATION
    • Abstract: Publication date: Available online 15 May 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
      Author(s): Inna B. Korzeneva , Svetlana V. Kostuyk , Liza S. Ershova , Andrian N. Osipov , Veronika F. Zhuravleva , Galina V. Pankratova , Lev N. Porokhovnik , Natalia N. Veiko
      The blood plasma of healthy people contains cell-fee (circulating) DNA (cfDNA). Apoptotic cells are the main source of the cfDNA. The cfDNA concentration increases in case of the organism's cell death rate increase, for example in case of exposure to high-dose ionizing radiation (IR). The objects of the present research are the blood plasma and blood lymphocytes of people, who contacted occupationally with the sources of external gamma/neutron radiation or internal β-radiation of tritium (N=176). As the controls (references), blood samples of people, who had never been occupationally subjected to the IR sources, were used (N=109). With respect to the plasma samples of each donor there were defined: the cfDNA concentration (the cfDNA index), DNase1 activity (the DNase1 index) and titre of antibodies to DNA (the Ab DNA index). The general DNA damage in the cells was defined (using the Comet assay, the tail moment (TM) index). A chronic effect of the low-dose ionizing radiation on a human being is accompanied by the enhancement of the DNA damage in lymphocytes along with a considerable cfDNA content reduction, while the DNase1 content and concentration of antibodies to DNA (Ab DNA) increase. All the aforementioned changes were also observed in people, who had not worked with the IR sources for more than a year. The ratio cfDNA/(DNase1×Ab DNA×TM) is proposed to be used as a marker of the chronic exposure of a person to the external low-dose IR. It was formulated the assumption that the joint analysis of the cfDNA, DNase1, Ab DNA and TM values may provide the information about the human organism's cell resistivity to chronic exposure to the low-dose IR and about the development of the adaptive response in the organism that is aimed, firstly, at the effective cfDNA elimination from the blood circulation, and, secondly – at survival of the cells, including the cells with the damaged DNA.


      PubDate: 2015-06-14T11:42:17Z
       
  • TITLE PAGE (EDI BOARD)
    • Abstract: Publication date: May 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volume 775




      PubDate: 2015-06-14T11:42:17Z
       
  • The effect of low dose ionizing radiation on homeostasis and functional
           integrity in an organotypic human skin model
    • Abstract: Publication date: May 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volume 775
      Author(s): Claere von Neubeck , Matthew J. Geniza , Paula M. Kauer , R. Joe Robinson , William B. Chrisler , Marianne B. Sowa
      Outside the protection of Earth's atmosphere, astronauts are exposed to low doses of high linear energy transfer (LET) radiation. Future NASA plans for deep space missions or a permanent settlement on the moon are limited by the health risks associated with space radiation exposures. There is a paucity of direct epidemiological data for low dose exposures to space radiation-relevant high LET ions. Health risk models are used to estimate the risk for such exposures, though these models are based on high dose experiments. There is increasing evidence, however, that low and high dose exposures result in different signaling events at the molecular level, and may involve different response mechanisms. Further, despite their low abundance, high LET particles have been identified as the major contributor to health risk during manned space flight. The human skin is exposed in every external radiation scenario, making it an ideal epithelial tissue model in which to study radiation induced effects. Here, we exposed an in vitro three dimensional (3-D) human organotypic skin tissue model to low doses of high LET oxygen (O), silicon (Si) and iron (Fe) ions. We measured proliferation and differentiation profiles in the skin tissue and examined the integrity of the skin's barrier function. We discuss the role of secondary particles in changing the proportion of cells receiving a radiation dose, emphasizing the possible impact on radiation-induced health issues in astronauts.


      PubDate: 2015-06-14T11:42:17Z
       
  • A combinatorial role for MutY and Fpg DNA glycosylases in mutation
           avoidance in Mycobacterium smegmatis
    • Abstract: Publication date: Available online 9 June 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
      Author(s): Farzanah Hassim , Andrea O. Papadopoulos , Bavesh D. Kana , Bhavna G. Gordhan
      Hydroxyl radical (OH) among reactive oxygen species cause damage to nucleobases with thymine being the most susceptible, whilst in contrast, the singlet oxygen (102) targets only guanine bases. The high GC content of mycobacterial genomes predisposes these organisms to oxidative damage of guanine. The exposure of cellular DNA to OH and one-electron oxidants results in the formation of two main degradation products, the pro-mutagenic 8-oxo-7,8-dihydroguanine (8-oxoGua) and the cytotoxic 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua). These lesions are repaired through the base excision repair (BER) pathway and we previously, demonstrated a combinatorial role for the mycobacterial Endonuclease III (Nth) and the Nei family of DNA glycosylases in mutagenesis. In addition, the Formamidopyrimidine (Fpg/MutM) and MutY DNA glycosylases have also been implicated in mutation avoidance and BER in mycobacteria. In this study, we further investigate the combined role of MutY and the Fpg/Nei DNA glycosylases in Mycobacterium smegmatis and demonstrate that deletion of mutY resulted in enhanced sensitivity to oxidative stress, an effect which was not exacerbated in Δfpg1 Δfpg2 or Δnei1 Δnei2 double mutant backgrounds. However, combinatorial loss of the mutY, fpg1 and fpg2 genes resulted in a significant increase in mutation rates suggesting interplay between these enzymes. Consistent with this, there was a significant increase in C → A mutations with a corresponding change in cell morphology of rifampicin resistant mutants in the Δfpg1 Δfpg2 ΔmutY deletion mutant. In contrast, deletion of mutY together with the nei homologues did not result in any growth/survival defects or changes in mutation rates. Taken together these data indicate that the mycobacterial MutY, in combination with the Fpg DNA N-glycosylases, plays an important role in controlling mutagenesis under oxidative stress.


      PubDate: 2015-06-14T11:42:17Z
       
  • The different radiation response and radiation-induced bystander effects
           in colorectal carcinoma cells differing in p53 status
    • Abstract: Publication date: Available online 10 June 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
      Author(s): Maria Widel , Anna Lalik , Aleksandra Krzywon , Jan Poleszczuk , Krzysztof Fujarewicz , Joanna Rzeszowska-Wolny
      Radiation-induced bystander effect, appearing as different biological changes in cells that are not directly exposed to ionizing radiation but are under the influence of molecular signals secreted by irradiated neighbors, have recently attracted considerable interest due to their possible implication for radiotherapy. However, various cells present diverse radiosensitivity and bystander responses that depend, inter alia, on genetic status including TP53, the gene controlling the cell cycle, DNA repair and apoptosis. Here we compared the ionizing radiation and bystander responses of human colorectal carcinoma HCT116 cells with wild type or knockout TP53 using a transwell co-culture system. The viability of exposed to X-rays (0-8Gy) and bystander cells of both lines showed a roughly comparable decline with increasing dose. The frequency of micronuclei was also comparable at lower doses but at higher increased considerably, especially in bystander TP53-/- cells. Moreover, the TP53-/- cells showed a significantly elevated frequency of apoptosis, while TP53+/+ counterparts expressed high level of senescence. The cross-matched experiments where irradiated cells of one line were co-cultured with non-irradiated cells of opposite line show that both cell lines were also able to induce bystander effects in their counterparts, however different endpoints revealed with different strength. Potential mediators of bystander effects, IL-6 and IL-8, were also generated differently in both lines. The knockout cells secreted IL-6 at lower doses whereas wild type cells only at higher doses. Secretion of IL-8 by TP53-/- control cells was many times lower than that by TP53+/+ but increased significantly after irradiation. Transcription of the NFκBIA was induced in irradiated TP53+/+ mainly, but in bystanders a higher level was observed in TP53-/- cells, suggesting that TP53 is required for induction of NFκB pathway after irradiation but another mechanism of activation must operate in bystander cells.


      PubDate: 2015-06-14T11:42:17Z
       
  • A eudesmane-type sesquiterpene isolated from Pluchea odorata (L.) Cass.
           combats three hallmarks of cancer cells: Unrestricted proliferation,
           escape from apoptosis and early metastatic outgrowth in vitro
    • Abstract: Publication date: July 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volume 777
      Author(s): Michael Blaschke , Ruxandra McKinnon , Chi Huu Nguyen , Silvio Holzner , Martin Zehl , Atanas Georgiev Atanasov , Karin Schelch , Sigurd Krieger , Rene Diaz , Richard Frisch , Björn Feistel , Walter Jäger , Gerhard F. Ecker , Verena M. Dirsch , Michael Grusch , Istvan Zupko , Ernst Urban , Brigitte Kopp , Georg Krupitza
      Pluchea odorata is ethno pharmaceutically used to treat inflammation-associated disorders. The dichloromethane extract (DME) was tested in the carrageenan-induced rat paw oedema assay investigating its effect on inflammation that was inhibited by 37%. Also an in vitro anti-neoplastic potential was reported. However, rather limited information about the bio-activity of purified compounds and their cellular mechanisms are available. Therefore, two of the most abundant eudesmanes in P. odorata were isolated and their anti-neoplastic and anti-intravasative activities were studied. HL-60 cells were treated with P. odorata compounds and metabolic activity, cell number reduction, cell cycle progression and apoptosis induction were correlated with relevant protein expression. Tumour cell intravasation through lymph endothelial monolayers was measured and potential causal mechanisms were analyzed by Western blotting. Compound PO-1 decreased the metabolic activity of HL-60 cells (IC50 =8.9μM after 72h) and 10μM PO-1 induced apoptosis, while PO-2 showed just weak anti-neoplastic activities at concentrations beyond 100μM. PO-1 arrested the cell cycle in G1 and this correlated with induction of JunB expression. Independent of this mechanism 25μM PO-1 decreased MCF-7 spheroid intravasation through the lymph endothelial barrier. Hence, PO-1 inhibits an early step of metastasis, impairs unrestricted proliferation and induces apoptosis at low micromolar concentrations. These results warrant further testing in vivo to challenge the potential of PO-1 as novel lead compound.


      PubDate: 2015-06-14T11:42:17Z
       
  • The effect of age at exposure on the inactivating mechanisms and relative
           contributions of key tumor suppressor genes in radiation-induced mouse
           T-cell lymphomas
    • Abstract: Publication date: Available online 10 June 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
      Author(s): Masaaki Sunaoshi , Yoshiko Amasaki , Shinobu Hirano-Sakairi , Benjamin J. Blyth , Takamitsu Morioka , Mutsumi Kaminishi , Yi Shang , Mayumi Nishimura , Yoshiya Shimada , Akira Tachibana , Shizuko Kakinuma
      Children are considered more sensitive to radiation-induced cancer than adults, yet any differences in genomic alterations associated with age-at-exposure and their underlying mechanisms remain unclear. We assessed genome-wide DNA copy number and mutation of key tumor suppressor genes in T-cell lymphomas arising after weekly irradiation of female B6C3F1 mice with 1.2Gy X-rays for 4 consecutive weeks starting during infancy (1 week old), adolescence (4 weeks old) or as young adults (8 weeks old). Although T-cell lymphoma incidence was similar, loss of heterozygosity at Cdkn2a on chromosome 4 and at Ikaros on chromosome 11 was more frequent in the two older groups, while loss at the Pten locus on chromosome 19 was more frequent in the infant-irradiated group. Cdkn2a and Ikaros mutation/loss was a common feature of the young adult–irradiation group, with Ikaros frequently (60%) incurring multiple independent hits (including deletions and mutations) or suffering a single hit predicted to result in a dominant negative protein (such as those lacking exon 4, an isoform we have designated Ik12, which lacks two DNA binding zinc–finger domains). Conversely, Pten mutations were more frequent after early irradiation (60%) than after young adult–irradiation (30%). Homozygous Pten mutations occurred without DNA copy number change after irradiation starting in infancy, suggesting duplication of the mutated allele by chromosome mis-segregation or mitotic recombination. Our findings demonstrate that while deletions on chromosomes 4 and 11 affecting Cdkn2a and Ikaros are a prominent feature of young adult irradiation–induced T-cell lymphoma, tumors arising after irradiation from infancy suffer a second hit in Pten by mis-segregation or recombination. This is the first report showing an influence of age-at-exposure on genomic alterations of tumor suppressor genes and their relative involvement in radiation-induced T-cell lymphoma. These data are important for considering the risks associated with childhood exposure to radiation.


      PubDate: 2015-06-14T11:42:17Z
       
  • MicroRNA transcriptome analysis identifies miR-365 as a novel negative
           regulator of cell proliferation in Zmpste24-deficient mouse embryonic
           fibroblasts
    • Abstract: Publication date: July 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volume 777
      Author(s): Xing-dong Xiong , Hwa Jin Jung , Saurabh Gombar , Jung Yoon Park , Chun-long Zhang , Huiling Zheng , Jie Ruan , Jiang-bin Li , Matt Kaeberlein , Brian K. Kennedy , Zhongjun Zhou , Xinguang Liu , Yousin Suh
      Zmpste24 is a metalloproteinase responsible for the posttranslational processing and cleavage of prelamin A into mature laminA. Zmpste24 −/− mice display a range of progeroid phenotypes overlapping with mice expressing progerin, an altered version of lamin A associated with Hutchinson-Gilford progeria syndrome (HGPS). Increasing evidence has demonstrated that miRNAs contribute to the regulation of normal aging process, but their roles in progeroid disorders remain poorly understood. Here we report the miRNA transcriptomes of mouse embryonic fibroblasts (MEFs) established from wild type (WT) and Zmpste24 −/− progeroid mice using a massively parallel sequencing technology. With data from 19.5×106 reads from WT MEFs and 16.5×106 reads from Zmpste24 −/− MEFs, we discovered a total of 306 known miRNAs expressed in MEFs with a wide dynamic range of read counts ranging from 10 to over 1 million. A total of 8 miRNAs were found to be significantly down-regulated, with only 2 miRNAs upregulated, in Zmpste24 −/− MEFs as compared to WT MEFs. Functional studies revealed that miR-365, a significantly down-regulated miRNA in Zmpste24 −/− MEFs, modulates cellular growth phenotypes in MEFs. Overexpression of miR-365 in Zmpste24 −/− MEFs increased cellular proliferation and decreased the percentage of SA-β-gal-positive cells, while inhibition of miR-365 function led to an increase of SA-β-gal-positive cells in WT MEFs. Furthermore, we identified Rasd1, a member of the Ras superfamily of small GTPases, as a functional target of miR-365. While expression of miR-365 suppressed Rasd1 3′ UTR luciferase-reporter activity, this effect was lost with mutations in the putative 3′ UTR target-site. Consistently, expression levels of miR-365 were found to inversely correlate with endogenous Rasd1 levels. These findings suggest that miR-365 is down-regulated in Zmpste24 −/− MEFs and acts as a novel negative regulator of Rasd1. Our comprehensive miRNA data provide a resource to study gene regulatory networks in MEFs.


      PubDate: 2015-06-14T11:42:17Z
       
  • Cytogenetic heterogeneity and their serial dynamic changes during
           
    • Abstract: Publication date: July 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volume 777
      Author(s): Jung-Ah Kim , Kyong Ok Im , Si Nae Park , Ji Seok Kwon , Seon Young Kim , Keunhee Oh , Dong-Sup Lee , Min Kyung Kim , Seong Who Kim , Mi Jang , Gene Lee , Yeon-Mok Oh , Sang Do Lee , Dong Soon Lee
      To minimize the risk of tumorigenesis in mesenchymal stem cells (MSCs), G-banding analysis is widely used to detect chromosomal aberrations in MSCs. However, a critical limitation of G-banding is that it only reflects the status of metaphase cells, which can represent as few as 0.01% of tested cells. During routine cytogenetic testing in MSCs, we often detect chromosomal aberrations in minor cell populations. Therefore, we aimed to investigate whether such a minority of cells can expand over time or if they ultimately disappear during MSC passaging. We passaged MSCs serially while monitoring quantitative changes for each aberrant clone among heterogeneous MSCs. To investigate the cytogenetic status of interphase cells, which represent the main population, we also performed interphase FISH analysis, in combination with G-banding and telomere length determination. In human adipose tissue-derived MSCs, 4 types of chromosomal aberrations were found during culturing, and in umbilical cord MSCs, 2 types of chromosomal aberrations were observed. Sequential dynamic changes among heterogeneous aberrant clones during passaging were similar to the dynamic changes observed in cancer stem cells during disease progression. Throughout all passages, the quantitative G-banding results were inconsistent with those of the interphase FISH analysis. Interphase FISH revealed hidden aberrations in stem cell populations with normal karyotypes by G-banding analysis. We found that telomere length gradually decreased during passaging until the point at which cytogenetic aberrations appeared. The present study demonstrates that rare aberrant clones at earlier passages can become predominant clones during later passages. Considering the risk of tumorigenesis due to aberrant MSCs, we believe that our results will help to establish proper safety guidelines for MSC use. In particular, we believe it is critical to test for chromosomal aberrations using both G-banding and FISH to ensure the safety of human stem cells that are manufactured in vitro for clinical applications.


      PubDate: 2015-06-14T11:42:17Z
       
  • Role of chromatin structure modulation by the histone deacetylase
           inhibitor trichostatin A on the radio-sensitivity of ataxia telangiectasia
           
    • Abstract: Publication date: July 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volume 777
      Author(s): Roberta Meschini , Elisa Morucci , Andrea Berni , Wilner Lopez-Martinez , Fabrizio Palitti
      At present, a lot is known about biochemical aspects of double strand breaks (DBS) repair but how chromatin structure affects this process and the sensitivity of DNA to DSB induction is still an unresolved question. Ataxia telangiectasia (A-T) patients are characterised by very high sensitivity to DSB-inducing agents such as ionising radiation. This radiosensitivity is revealed with an enhancement of chromosomal instability as a consequence of defective DNA repair for a small fraction of breaks located in the heterochromatin, where they are less accessible. Besides, recently it has been reported that Ataxia Telangiectasia Mutated (ATM) mediated signalling modifies chromatin structure. In order to study the impact of chromatin compaction on the chromosomal instability of A-T cells, the response to trichostatin-A, an histone deacetylase inhibitor, in normal and A-T lymphoblastoid cell lines was investigated testing its effect on chromosomal aberrations, cell cycle progression, DNA damage and repair after exposure to X-rays. The results suggest that the response to both trichostatin-A pre- and continuous treatments is independent of the presence of either functional or mutated ATM protein, as the reduction of chromosomal damage was found also in the wild-type cell line. The presence of trichostatin-A before exposure to X-rays could give rise to prompt DNA repair functioning on chromatin structure already in an open conformation. Differently, trichostatin-A post-treatment causing hyperacetylation of histone tails and reducing the heterochromatic DNA content might diminish the requirement for ATM and favour DSBs repair reducing chromosomal damage only in A-T cells. This fact could suggest that trichostatin-A post-treatment is favouring the slow component of DSB repair pathway, the one impaired in absence of a functionally ATM protein. Data obtained suggest a fundamental role of chromatin compaction on chromosomal instability in A-T cells.


      PubDate: 2015-06-14T11:42:17Z
       
  • Transmission of clonal chromosomal abnormalities in human hematopoietic
           stem and progenitor cells surviving radiation exposure
    • Abstract: Publication date: July 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volume 777
      Author(s): Daniela Kraft , Sylvia Ritter , Marco Durante , Erhard Seifried , Claudia Fournier , Torsten Tonn
      In radiation-induced acute myeloid leukemia (rAML), clonal chromosomal abnormalities are often observed in bone marrow cells of patients, suggesting that their formation is crucial in the development of the disease. Since rAML is considered to originate from hematopoietic stem and progenitor cells (HSPC), we investigated the frequency and spectrum of radiation-induced chromosomal abnormalities in human CD34+ cells. We then measured stable chromosomal abnormalities, a possible biomarker of leukemia risk, in clonally expanded cell populations which were grown for 14 days in a 3D-matrix (CFU-assay). We compared two radiation qualities used in radiotherapy, sparsely ionizing X-rays and densely ionizing carbon ions (29 and 60–85keV/μm, doses between 0.5 and 4Gy). Only a negligible number of de novo arising, unstable aberrations (≤0.05 aberrations/cell, 97% breaks) were measured in the descendants of irradiated HSPC. However, stable aberrations were detected in colonies formed by irradiated HSPC. All cells of the affected colonies exhibited one or more identical aberrations, indicating their clonal origin. The majority of the clonal rearrangements (92%) were simple exchanges such as translocations (77%) and pericentric inversions (15%), which are known to contribute to the development of rAML. Carbon ions were more efficient in inducing cell killing (maximum of ∼30–35% apoptotic cells for 2Gy carbon ions compared to ∼25% for X-rays) and chromosomal aberrations in the first cell-cycle after exposure (∼70% and ∼40% for 1Gy of carbon ions and X-rays, respectively), with a higher fraction of non-transmissible aberrations. In contrast, for both radiation qualities the percentage of clones with chromosomal abnormalities was similar (40%). Using the frequency of colonies with clonal aberrations as a surrogate marker for the leukemia risk following radiotherapy of solid tumors, charged particle therapy is not expected to lead to an increased risk of leukemia in patients.


      PubDate: 2015-06-14T11:42:17Z
       
  • γ-H2AX induced by linear alkylbenzene sulfonates is due to
           deoxyribonuclease-1 translocation to the nucleus via actin disruption
    • Abstract: Publication date: July 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volume 777
      Author(s): Xiaoxu Zhao , Tatsushi Toyooka , Toru Kubota , Guang Yang , Yuko Ibuki
      Phosphorylation of histone H2AX (γ-H2AX) occurs following formation of DNA double strand breaks (DSBs). Other types of DNA damage also generate DSBs through DNA replication and repair, leading to the production of γ-H2AX. In the present study, we demonstrated that linear alkylbenzene sulfonates (LAS), the most widely used and non-genotoxic anionic surfactants, could generate γ-H2AX via a novel pathway. Breast adenocarcinoma MCF-7 cells were treated with five kinds of LAS with alkyl chains ranging from 10 to 14 carbon units (C10–C14LAS). The generation of DSBs and subsequent production of γ-H2AX increased in a manner that depended on the number of carbon units in LAS. γ-H2AX could also be generated with non-cytotoxic doses of LAS and was independent of the cell cycle, indicating the non-apoptotic and DNA replication-independent formation of DSBs. The generation of γ-H2AX could be attenuated by EGTA and ZnCl2, deoxyribonuclease-1 (DNase I) inhibitors, as well as by the knockdown of DNase I. LAS weakened the interaction between DNase I and actin, and the enhanced release of DNase I was dependent on the number of carbon units in LAS. DNase I released by the LAS treatment translocated to the nucleus, in which DNase I attacked DNA and generated γ-H2AX. These results suggested that the LAS-induced generation of γ-H2AX could be attributed to the translocation of DNase I to the nucleus through the disruption of actin, and not to LAS-induced DNA damage.
      Graphical abstract image

      PubDate: 2015-06-14T11:42:17Z
       
  • Methods for comparing mutation rates using fluctuation assay data
    • Abstract: Publication date: July 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volume 777
      Author(s): Qi Zheng
      Comparing microbial mutation rates via the fluctuation assay protocol is an important routine task in the laboratory. However, methods for the comparison of mutation rates are still scarce and not well understood. This paper proposes new methods to address this situation. First, it provides a likelihood ratio test. Second, it explores the use of confidence intervals that can be readily computed. Third, it uses simulations to assess the new and existing methods. Both the likelihood ratio test and the use of confidence intervals were found to be superior to the general-purpose Mann–Whitney test, while the t-test was found to be unsuitable for fluctuation assay data. In addition, the method based on confidence intervals is suitable for comparison of experiments in which the terminal cell population sizes differ.


      PubDate: 2015-06-14T11:42:17Z
       
  • Inhibition of Bcl-2 or IAP proteins does not provoke mutations in
           surviving cells
    • Abstract: Publication date: July 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volume 777
      Author(s): Tanmay M. Shekhar , Maja M. Green , David M. Rayner , Mark A. Miles , Suzanne M. Cutts , Christine J. Hawkins
      Chemotherapy and radiotherapy can cause permanent damage to the genomes of surviving cells, provoking severe side effects such as second malignancies in some cancer survivors. Drugs that mimic the activity of death ligands, or antagonise pro-survival proteins of the Bcl-2 or IAP families have yielded encouraging results in animal experiments and early phase clinical trials. Because these agents directly engage apoptosis pathways, rather than damaging DNA to indirectly provoke tumour cell death, we reasoned that they may offer another important advantage over conventional therapies: minimisation or elimination of side effects such as second cancers that result from mutation of surviving normal cells. Disappointingly, however, we previously found that concentrations of death receptor agonists like TRAIL that would be present in vivo in clinical settings provoked DNA damage in surviving cells. In this study, we used cell line model systems to investigate the mutagenic capacity of drugs from two other classes of direct apoptosis-inducing agents: the BH3-mimetic ABT-737 and the IAP antagonists LCL161 and AT-406. Encouragingly, our data suggest that IAP antagonists possess negligible genotoxic activity. Doses of ABT-737 that were required to damage DNA stimulated Bax/Bak-independent signalling and exceeded concentrations detected in the plasma of animals treated with this drug. These findings provide hope that cancer patients treated by BH3-mimetics or IAP antagonists may avoid mutation-related illnesses that afflict some cancer survivors treated with conventional DNA-damaging anti-cancer therapies.
      Graphical abstract image

      PubDate: 2015-06-14T11:42:17Z
       
  • Electrophysiologic and cellular characteristics of cardiomyocytes after
           X-ray irradiation
    • Abstract: Publication date: July 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volume 777
      Author(s): Johannes L. Frieß , Anja Heselich , Sylvia Ritter , Angelina Haber , Nicole Kaiser , Paul G. Layer , Christiane Thielemann
      The aim of this study was to investigate possible effects of ionizing irradiation on the electrophysiological functionality of cardiac myocytes in vitro. Primary chicken cardiomyocytes with spontaneous beating activity were irradiated with X-rays (dose range of 0.5–7Gy). Functional alterations of cardiac cell cultures were evaluated up to 7 days after irradiation using microelectrode arrays. As examined endpoints, cell proliferation, apoptosis, reactive oxygen species (ROS) and DNA damage were evaluated. The beat rate of the cardiac networks increased in a dose-dependent manner over one week. The duration of single action potentials was slightly shortened. Additionally, we observed lower numbers of mitotic and S-phase cells at certain time points after irradiation. Also, the number of cells with γH2AX foci increased as a function of the dose. No significant changes in the level of ROS were detected. Induction of apoptosis was generally negligibly low. This is the first report to directly show alterations in cardiac electrophysiology caused by ionizing radiation, which were detectable up to one week after irradiation.


      PubDate: 2015-06-14T11:42:17Z
       
  • Functional interaction between hMYH and hTRADD in the TNF-α-mediated
           survival and death pathways of HeLa cells
    • Abstract: Publication date: July 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volume 777
      Author(s): An Hue Vy Tran , Soo-Hyun Hahm , Se Hee Han , Ji Hyung Chung , Geon Tae Park , Ye Sun Han
      The tumor necrosis factor (TNF) signaling pathway is a classical immune system pathway that plays a key role in regulating cell survival and apoptosis. The TNF receptor-associated death domain (TRADD) protein is recruited to the death domain of TNF receptor 1 (TNFR1), where it interacts with TNF receptor-associated factor 2 (TRAF2) and receptor-interacting protein (RIP) for the induction of apoptosis, necrosis, nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB), and mitogen-activated protein (MAP) kinase activation. In this study, we found that the human MutY homolog (hMYH) interacted with human TRADD (hTRADD) via the C-terminal domain of hMYH. Moreover, under conditions promoting TNF-α-induced cell death or survival in HeLa cells, this interaction was weakened or enhanced, respectively. The interaction between hMYH and hTRADD was important for signaling pathways mediated by TNF-α. Our results also suggested that the hTRADD–hMYH association was involved in the nuclear translocation of NFκB and formation of the TNFR1–TRADD complex. Thus, this study identified a novel mechanism through which the hMYH–hTRADD interaction may affect the TNF-α signaling pathway. Implications In HeLa cells, the hTRADD–hMYH interaction functioned in both cell survival and apoptosis pathways following TNF-α stimulation.


      PubDate: 2015-06-14T11:42:17Z
       
  • TITLE PAGE (EDI BOARD)
    • Abstract: Publication date: July 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volume 777




      PubDate: 2015-06-14T11:42:17Z
       
  • The genetic consequences of paternal acrylamide exposure and potential for
           amelioration
    • Abstract: Publication date: July 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volume 777
      Author(s): Aimee L. Katen , Shaun D. Roman
      Acrylamide is a toxin that humans are readily exposed to due to its formation in many carbohydrate rich foods cooked at high temperatures. Acrylamide is carcinogenic, neurotoxic and causes reproductive toxicity when high levels of exposure are reached in mice and rats. Acrylamide induced effects on fertility occur predominantly in males. Acrylamide exerts its reproductive toxicity via its metabolite glycidamide, a product which is only formed via the cytochrome P450 detoxifying enzyme CYP2E1. Glycidamide is highly reactive and forms adducts with DNA. Chronic low dose acrylamide exposure in mice relevant to human exposure levels results in significantly increased levels of DNA damage in terms of glycidamide adducts in spermatocytes, the specific germ cell stage where Cyp2e1 is expressed. Since cells in the later stages of spermatogenesis are unable to undergo DNA repair, and this level of acrylamide exposure causes no reduction in fertility, there is potential for this damage to persist until sperm maturation and fertilisation. Cyp2e1 is also present within epididymal cells, allowing for transiting spermatozoa to be exposed to glycidamide. This could have consequences for future generations in terms of predisposition to diseases such as cancer, with growing indications that paternal DNA damage can be propagated across multiple generations. Since glycidamide is the major contributor to DNA damage, a mechanism for preventing these effects is inhibiting the function of Cyp2e1. Resveratrol is an example of an inhibitor of Cyp2e1 which has shown success in reducing damage caused by acrylamide treatment in mice.


      PubDate: 2015-06-14T11:42:17Z
       
  • Prognostic role of APC and RASSF1A promoter methylation status in cell
           free circulating DNA of operable gastric cancer patients
    • Abstract: Publication date: August 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volume 778
      Author(s): I. Balgkouranidou , D. Matthaios , A. Karayiannakis , H. Bolanaki , P. Michailidis , N. Xenidis , K. Amarantidis , L. Chelis , G. Trypsianis , E. Chatzaki , E.S. Lianidou , S. Kakolyris
      Gastric carcinogenesis is a multistep process including not only genetic mutations but also epigenetic alterations. The best known and more frequent epigenetic alteration is DNA methylation affecting tumor suppressor genes that may be involved in various carcinogenetic pathways. The aim of the present study was to investigate the methylation status of APC promoter 1A and RASSF1A promoter in cell free DNA of operable gastric cancer patients. Using methylation specific PCR, we examined the methylation status of APC promoter 1A and RASSF1A promoter in 73 blood samples obtained from patients with gastric cancer. APC and RASSF1A promoters were found to be methylated in 61 (83.6%) and 50 (68.5%) of the 73 gastric cancer samples examined, but in none of the healthy control samples (p <0.001). A significant association between methylated RASSF1A promoter status and lymph node positivity was observed (p =0.005). Additionally, a significant correlation between a methylated APC promoter and elevated CEA (p =0.033) as well as CA-19.9 (p =0.032) levels, was noticed. The Kaplan–Meier estimates of survival, significantly favored patients with a non-methylated APC promoter status (p =0.008). No other significant correlations between APC and RASSF1A methylation status and different tumor variables examined was observed. Serum RASSF1A and APC promoter hypermethylation is a frequent epigenetic event in patients with early operable gastric cancer. The observed correlations between APC promoter methylation status and survival as well as between a hypermethylated RASSF1A promoter and nodal positivity may be indicative of a prognostic role for those genes in early operable gastric cancer. Additional studies, in a larger cohort of patients are required to further explore whether these findings could serve as potential molecular biomarkers of survival and/or response to specific treatments.


      PubDate: 2015-06-14T11:42:17Z
       
  • The role of base excision repair in the development of primary open angle
           glaucoma in the Polish population
    • Abstract: Publication date: August 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volume 778
      Author(s): Magda Cuchra , Lukasz Markiewicz , Bartosz Mucha , Dariusz Pytel , Katarzyna Szymanek , Janusz Szemraj , Jerzy Szaflik , Jacek P. Szaflik , Ireneusz Majsterek
      Glaucoma is a leading cause of irreversible blindness in developing countries. Previous data have shown that progressive loss of human TM cells may be connected with chronic exposure to oxidative stress. This hypothesis may suggest a role of the base excision repair (BER) pathway of oxidative DNA damage in primary open angle glaucoma (POAG) patients. The aim of our study was to evaluate an association of BER gene polymorphism with a risk of POAG. Moreover, an association of clinical parameters was examined including cup disk ratio (c/d), rim area (RA) and retinal nerve fiber layer (RNFL) with glaucoma progression according to BER gene polymorphisms. Our research included 412 patients with POAG and 454 healthy controls. Gene polymorphisms were analyzed by PCR-RFLP. Heidelberg Retinal Tomography (HRT) clinical parameters were also analyzed. The 399Arg/Gln genotype of the XRCC1 gene (OR 1.38; 95% CI 1.02–1.89 p =0.03) was associated with an increased risk of POAG occurrence. It was indicated that the 399Gln/Gln XRCC1 genotype might increase the risk of POAG progression according to the c/d ratio (OR 1.67; 95% CI 1.07–2.61 P =0.02) clinical parameter. Moreover, the association of VF factor with 148Asp/Glu of APE1 genotype distribution and POAG progression (OR 2.25; 95% CI 1.30–3.89) was also found. Additionally, the analysis of the 324Gln/His MUTYH polymorphism gene distribution in the patient group according to RNFL factor showed that it might decrease the progression of POAG (OR 0.47; 95% CI 0.30–0.82 P =0.005). We suggest that the 399Arg/Gln polymorphism of the XRCC1 gene may serve as a predictive risk factor of POAG.


      PubDate: 2015-06-14T11:42:17Z
       
  • Genetic variants of H2AX gene were associated with PM2.5-modulated DNA
           damage levels in Chinese Han populations
    • Abstract: Publication date: August 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volume 778
      Author(s): Chongqi Sun , Minjie Chu , Weihong Chen , Guangfu Jin , Jianhang Gong , Meng Zhu , Jing Yuan , Juncheng Dai , Meilin Wang , Yun Pan , Yuanchao Song , Xiaojie Ding , Mulong Du , Jing Dong , Zhengdong Zhang , Zhibin Hu , Tangchun Wu , Hongbing Shen
      Exposure to particulate matter 2.5 (PM2.5) may result in DNA damage. Histone variant H2AX phosphorylation plays a central role in the response to damaged chromatin. In the current study, we investigated whether H2AX gene polymorphisms account for PM2.5-modulated DNA damage levels. A total of 307 healthy urban residents were collected from three cities in southern, central, and northern China, Zhuhai, Wuhan, and Tianjin, respectively. The dust mass concentrations of PM2.5 were detected by Gilian 5000 pumps, and the DNA damage levels were measured using comet assay. Seven potentially functional single nucleotide polymorphisms (SNPs) of H2AX gene were selected and genotyped by Illumina Infinium® BeadChip. We found that three SNPs (rs10790283 G>A, rs604714 C>A and rs7759 A>G) were significantly associated with DNA damage levels (adjusted P =0.002, 0.018 and 0.027, respectively). Significant interactions (P <0.05) were observed between certain genetic polymorphisms and PM2.5-modulated DNA damage levels. These results suggested that genetic variations of H2AX might be associated with the DNA damage levels in urban residents with different exposure to PM2.5. Further studies with large sample size in independent populations merit validating these findings.


      PubDate: 2015-06-14T11:42:17Z
       
  • Are effects of common ragwort in the Ames test caused by pyrrolizidine
           alkaloids'
    • Abstract: Publication date: August 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volume 778
      Author(s): Toine F.H. Bovee , Richard J.R. Helsdingen , Ron L.A.P. Hoogenboom , Monique W.C.M. de Nijs , Xiaojie Liu , Klaas Vrieling , Peter G.L. Klinkhamer , Ad A.C.M. Peijnenburg , Patrick P.J. Mulder
      It has previously been demonstrated by others that acetone extracts of Senecio jacobaea (syn. Jacobaea vulgaris, common or tansy ragwort) test positive in the Salmonella/microsome mutagenicity test (Ames test). Pyrrolizidine alkaloids (PAs) are thought to be responsible for these mutagenic effects. However, it was also observed that the major PA present in common ragwort, jacobine, produced a negative response (with and without the addition of rat liver S9) in Salmonella test strains TA98, TA100, TA1535 and TA1537. To investigate which compounds in the plant extracts were responsible for the positive outcome, the present study investigated the contents and mutagenic effects of methanol and acetone extracts prepared from dried ground S. jacobaea and Senecio inaequidens (narrow-leafed ragwort). Subsequently, a fractionation approach was set up in combination with LC-MS/MS analysis of the fractions. It was shown that the positive Ames test outcomes of S. jacobaea extracts are unlikely to be caused by PAs, but rather by the flavonoid quercetin. This study also demonstrates the importance of identifying compounds responsible for positive test results in bioassays.


      PubDate: 2015-06-14T11:42:17Z
       
  • Genetic determinants of quantitative traits associated with cardiovascular
           disease risk
    • Abstract: Publication date: August 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volume 778
      Author(s): Božena Smolková , Stefano Bonassi , Verona Buociková , Mária Dušinská , Alexandra Horská , Daniel Kuba , Zuzana Džupinková , Katarína Rašlová , Juraj Gašparovič , Ivan Slíž , Marcello Ceppi , Branislav Vohnout , Ladislava Wsólová , Katarína Volkovová
      Established risk factors for cardiovascular diseases (CVD) may be moderated by genetic variants. In 2403 unrelated individuals from general practice (mean age 40.5 years), we evaluated the influence of 15 variants in 12 candidate genes on quantitative traits (QT) associated with CVD (body mass index, abdominal obesity, glucose, serum lipids, and blood pressure). Prior to multiple testing correction, univariate analysis associated APOE rs429358, rs7412 and ATG16L1 rs2241880 variants with serum lipid levels, while LEPR rs1137100 and ATG16L1 rs2241880 variants were linked to obesity related QTs. After taking into account confounding factors and correcting for multiple comparisons only APOE rs429358 and rs7412 variants remained significantly associated with risk of dyslipidemia. APOE rs429358 variant almost tripled the risk in homozygous subjects (OR=2.97; 95% CI 1.09–8.10, p <0.03) and had a lesser but still highly significant association also in heterozygous individuals (OR=1.67; 95% CI 1.24–2.10; p <0.001). Associations with hypertension, diabetes mellitus, and metabolic syndrome were not significant after Bonferroni correction. The influence of genetic variation is more evident in dyslipidemia than in other analyzed QTs. These results may contribute to strategic research aimed at including genetic variation in the set of data required to identify subjects at high risk of CVD.


      PubDate: 2015-06-14T11:42:17Z
       
  • Why the DNA self-depurination mechanism operates in HB-β but not in
           β-globin paralogs HB-δ, HB-ɛ1, HB-γ1 and HB-γ2
    • Abstract: Publication date: August 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volume 778
      Author(s): Olga Amosova , Juan R. Alvarez-Dominguez , Jacques R. Fresco
      The human β-globin, δ-globin and ɛ-globin genes contain almost identical coding strand sequences centered about codon 6 having potential to form a stem-loop with a 5′GAGG loop. Provided with a sufficiently stable stem, such a structure can self-catalyze depurination of the loop 5′G residue, leading to a potential mutation hotspot. Previously, we showed that such a hotspot exists about codon 6 of β-globin, with by far the highest incidence of mutations across the gene, including those responsible for 6 anemias (notably Sickle Cell Anemia) and β-thalassemias. In contrast, we show here that despite identical loop sequences, there is no mutational hotspot in the δ- or ɛ1-globin potential self-depurination sites, which differ by only one or two base pairs in the stem region from that of the β-globin gene. These differences result in either one or two additional mismatches in the potential 7-base pair-forming stem region, thereby weakening its stability, so that either DNA cruciform extrusion from the duplex is rendered ineffective or the lifetime of the stem-loop becomes too short to permit self-catalysis to occur. Having that same loop sequence, paralogs HB-γ1 and HB-γ2 totally lack stem-forming potential. Hence the absence in δ- and ɛ1-globin genes of a mutational hotspot in what must now be viewed as non-functional homologs of the self-depurination site in β-globin. Such stem-destabilizing variants appeared early among vertebrates and remained conserved among mammals and primates. Thus, this study has revealed conserved sequence determinants of self-catalytic DNA depurination associated with variability of mutation incidence among human β-globin paralogs.


      PubDate: 2015-06-14T11:42:17Z
       
  • Controlled exposure to diesel exhaust and traffic noise – Effects on
           oxidative stress and activation in mononuclear blood cells
    • Abstract: Publication date: Available online 28 March 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
      Author(s): Jette Gjerke Hemmingsen , Peter Møller , Kim Jantzen , Bo A.G. Jönsson , Maria Albin , Aneta Wierzbicka , Anders Gudmundsson , Steffen Loft , Jenny Rissler
      Particulate air pollution increases risk of cancer and cardiopulmonary disease, partly through oxidative stress. Traffic-related noise increases risk of cardiovascular disease and may cause oxidative stress. In this controlled random sequence study, 18 healthy subjects were exposed for 3h to diesel exhaust (DE) at 276μg/m3 from a passenger car or filtered air, with co-exposure to traffic noise at 48 or 75dB(A). Gene expression markers of inflammation, (interleukin-8 and tumor necrosis factor), oxidative stress (heme oxygenase (decycling-1)) and DNA repair (8-oxoguanine DNA glycosylase (OGG1)) were unaltered in peripheral blood mononuclear cells (PBMCs). No significant differences in DNA damage levels, measured by the comet assay, were observed after DE exposure, whereas exposure to high noise levels was associated with significantly increased levels of hOGG1-sensitive sites in PBMCs. Urinary levels of 8-oxo-7,8-dihydro-2′-deoxyguanosine were unaltered. In auxiliary ex vivo experiments whole blood was incubated with particles from the exposure chamber for 3h without effects on DNA damage in PBMCs or intracellular reactive oxygen species production and expression of CD11b and CD62L adhesion molecules in leukocyte subtypes. Conclusion 3-h exposure to DE caused no genotoxicity, oxidative stress or inflammation in PBMCs, whereas exposure to noise might cause oxidatively damaged DNA.


      PubDate: 2015-04-04T03:03:13Z
       
  • Single-molecule PCR analysis of an unstable microsatellite for detecting
           mutations in sperm of mice exposed to chemical mutagens
    • Abstract: Publication date: Available online 30 March 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
      Author(s): Marc A. Beal , Andrea Rowan-Carroll , Caleigh Campbell , Andrew Williams , Christopher M. Somers , Francesco Marchetti , Carole L. Yauk
      Single-molecule PCR (SM-PCR) analysis of long and repetitive DNA sequences, known as expanded simple tandem repeats (ESTRs), has been the most efficient method for studying germline mutation induction in endogenous sequences to date. However, the long length of these sequences makes mutation detection imprecise and laborious, and they have been characterized only in mice. Here, we explore the use of unstable microsatellite sequences that can be typed with high precision by capillary electrophoresis as alternative loci for detecting germline mutations. We screened 24 microsatellite loci across inbred mouse strains and identified Mm2.2.1 as the most polymorphic microsatellite locus. We then optimized SM-PCR of Mm2.2.1 to detect mutations in sperm. SM-PCR analysis of sperm from untreated B6C3F1 and Muta™Mouse samples revealed mutation frequencies that are consistent with rates derived from family pedigree analysis (∼5×10−3). To determine whether this locus can be used to detect chemically induced germline mutations, Muta™Mouse males were exposed by oral gavage to a single dose of 100mg/kg of N-ethyl-N-nitrosourea (ENU) or to 100mg/kg of benzo(a)pyrene (BaP) for 28 days alongside vehicle treated controls. Sperm were collected 10 weeks post-ENU exposure to sample sperm exposed as spermatogonial stem cells and 6 weeks post-BaP exposure to sample sperm that were dividing spermatogonia when the exposure was terminated. Both treatments resulted in a significant (approximately 2-fold) increase in mutation frequency in sperm compared to the control animals. The work establishes the utility of this microsatellite for studying mutation induction in the germ cells of mice. Because microsatellites are found in virtually every species, this approach holds promise for other organisms, including humans.


      PubDate: 2015-04-04T03:03:13Z
       
  • Breast cancer risk and possible mechanisms of radiation-induced genomic
           instability in the Swedish hemangioma cohort after reanalyzed dosimetry
    • Abstract: Publication date: Available online 11 March 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
      Author(s): Markus Eidemüller , Erik Holmberg , Peter Jacob , Marie Lundell , Per Karlsson
      The cohort of 17,200 female Swedish hemangioma patients, who had been exposed to ionizing radiation because of skin hemangioma, was analyzed for breast cancer incidence with descriptive excess relative risk models and mechanistic models of carcinogenesis. The dosimetry system has recently been updated, leading to substantially reduced doses for the most highly exposed part of the Stockholm cohort. The follow-up includes persons until December 2009 with 877 breast cancer cases. All models agree on the risk estimates. The excess relative and excess absolute risk at the age of 50 years are 0.48 Gy−1 (95% CI 0.28; 0.69) and 10.4 ( 10 4 PYR Gy ) − 1 (95% CI 6.1; 14.4), respectively. These risk estimates are about a factor of two higher than previous analyses of this cohort as a consequence of the re-evaluation of the dosimetry system. Explicit models incorporating effects of genomic instability were developed and applied to the hemangioma cohort. It was found that a radiation-induced transition towards genomic instability was highly significant. The models indicate that the main effect of radiation-induced genomic instability is to increase the rate of transition of non-initiated cells to initiated cells with a proliferative advantage. The magnitude of such an acceleration cannot be inferred from epidemiological data alone, but must be complemented by radiobiological measurements.


      PubDate: 2015-03-15T04:20:08Z
       
 
 
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