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Journal Cover   Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
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   Hybrid Journal Hybrid journal (It can contain Open Access articles)
   ISSN (Print) 0027-5107
   Published by Elsevier Homepage  [2812 journals]
  • Reduced repair capacity of a DNA clustered damage site comprised of
           8-oxo-7,8-dihydro-2′-deoxyguanosine and 2-deoxyribonolactone results
           in an increased mutagenic potential of these lesions
    • Abstract: Publication date: April 2014
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volume 762
      Author(s): Siobhan Cunniffe , Peter O’Neill , Marc M. Greenberg , Martine E. Lomax
      A signature of ionizing radiation is the induction of DNA clustered damaged sites. Non-double strand break (DSB) clustered damage has been shown to compromise the base excision repair pathway, extending the lifetimes of the lesions within the cluster, compared to isolated lesions. This increases the likelihood the lesions persist to replication and thus increasing the mutagenic potential of the lesions within the cluster. Lesions formed by ionizing radiation include 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) and 2-deoxyribonolactone (dL). dL poses an additional challenge to the cell as it is not repaired by the short-patch base excision repair pathway. Here we show recalcitrant dL repair is reflected in mutations observed when DNA containing it and a proximal 8-oxodGuo is replicated in Escherichia coli. 8-oxodGuo in close proximity to dL on the opposing DNA strand results in an enhanced frequency of mutation of the lesions within the cluster and a 20 base sequence flanking the clustered damage site in an E. coli based plasmid assay. In vitro repair of a dL lesion is reduced when compared to the repair of an abasic (AP) site and a tetrahydrofuran (THF), and this is due mainly to a reduction in the activity of polymerase β, leading to retarded FEN1 and ligase 1 activities. This study has given insights in to the biological effects of clusters containing dL.

      PubDate: 2015-05-26T23:34:47Z
  • Suppression of DNA-dependent protein kinase sensitize cells to radiation
           without affecting DSB repair
    • Abstract: Publication date: November 2014
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volume 769
      Author(s): Ann-Sofie Gustafsson , Andris Abramenkovs , Bo Stenerlöw
      Efficient and correct repair of DNA double-strand break (DSB) is critical for cell survival. Defects in the DNA repair may lead to cell death, genomic instability and development of cancer. The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is an essential component of the non-homologous end joining (NHEJ) which is the major DSB repair pathway in mammalian cells. In the present study, by using siRNA against DNA-PKcs in four human cell lines, we examined how low levels of DNA-PKcs affected cellular response to ionizing radiation. Decrease of DNA-PKcs levels by 80–95%, induced by siRNA treatment, lead to extreme radiosensitivity, similar to that seen in cells completely lacking DNA-PKcs and low levels of DNA-PKcs promoted cell accumulation in G2/M phase after irradiation and blocked progression of mitosis. Surprisingly, low levels of DNA-PKcs did not affect the repair capacity and the removal of 53BP1 or γ-H2AX foci and rejoining of DSB appeared normal. This was in strong contrast to cells completely lacking DNA-PKcs and cells treated with the DNA-PKcs inhibitor NU7441, in which DSB repair were severely compromised. This suggests that there are different mechanisms by which loss of DNA-PKcs functions can sensitize cells to ionizing radiation. Further, foci of phosphorylated DNA-PKcs (T2609 and S2056) co-localized with DSB and this was independent of the amount of DNA-PKcs but foci of DNA-PKcs was only seen in siRNA-treated cells. Our study emphasizes on the critical role of DNA-PKcs for maintaining survival after radiation exposure which is uncoupled from its essential function in DSB repair. This could have implications for the development of therapeutic strategies aiming to radiosensitize tumors by affecting the DNA-PKcs function.

      PubDate: 2015-05-26T23:34:47Z
  • Binding of MutS protein to oligonucleotides containing a methylated or an
           ethylated guanine residue, and correlation with mutation frequency
    • Abstract: Publication date: 2 April 2008
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volume 640, Issues 1–2
      Author(s): Kentaro Taira , Shintaro Nakamura , Khota Nakano , Daisuke Maehara , Keinosuke Okamoto , Sakae Arimoto , David Loakes , Leroy Worth Jr , Roel M. Schaaper , Kohji Seio , Mitsuo Sekine , Kazuo Negishi , Tomoe Negishi
      The MutS-based mismatch repair (MMR) system has been conserved from prokaryotes to humans, and plays important roles in maintaining the high fidelity of genomic DNA. MutS protein recognizes several different types of modified base pairs, including methylated guanine-containing base pairs. Here, we looked at the relationship between recognition and the effects of methylating versus ethylating agents on mutagenesis, using a MutS-deficient strain of E. coli. We find that while methylating agents induce mutations more effectively in a MutS-deficient strain than in wild-type, this genetic background does not affect mutagenicity by ethylating agents. Thus, the role of E. coli MMR with methylation-induced mutagenesis appears to be greater than ethylation-induced mutagenesis. To further understand this difference an early step of repair was examined with these alkylating agents. A comparison of binding affinities of MutS with O 6-alkylated guanine base paired with thymine, which could lead to transition mutations, versus cytosine which could not, was tested. Moreover, we compared binding of MutS to oligoduplexes containing different base pairs; namely, O 6-MeG:T, O 6-MeG:C, O 6-EtG:T, O 6-EtG:C, G:T and G:C. Dissociation constants (K d), which reflect the strength of binding, followed the order G:T-> O 6-MeG:T-> O 6-EtG:T-= O 6-EtG:C-≥ O 6-MeG:C->G:C. These results suggest that a thymine base paired with O 6-methyl guanine is specifically recognized by MutS and therefore should be removed more efficiently than a thymine opposite O 6-ethylated guanine. Taken together, the data suggest that in E. coli, the MMR system plays a more significant role in repair of methylation-induced lesions than those caused by ethylation.

      PubDate: 2015-05-26T23:34:47Z
  • Chronic inflammation and asthma
    • Abstract: Publication date: 7 August 2010
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volume 690, Issues 1–2
      Author(s): Jenna R. Murdoch , Clare M. Lloyd
      Allergic asthma is a complex and chronic inflammatory disorder which is associated with airway hyper-responsiveness and tissue remodelling of the airway structure. Although originally thought to be a Th2-driven inflammatory response to inhaled innocuous allergen, the immune response in asthma is now considered highly heterogeneous. There are now various in vivo systems which have been designed to examine the pathways leading to the development of this chronic immune response and reflect, in part this heterogeneity. Furthermore, the emergence of endogenous immunoregulatory pathways and active pro-resolving mediators hold great potential for future therapeutic intervention. In this review, the key cellular and molecular mediators relating to chronic allergic airway disease are discussed, as well as emerging players in the regulation of chronic allergic inflammation.

      PubDate: 2015-05-26T23:34:47Z
  • Complex germline and somatic mutation processes at a haploid human
           minisatellite shown by single-molecule analysis
    • Abstract: Publication date: 15 December 2008
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volume 648, Issues 1–2
      Author(s): Morag E. Shanks , Celia A. May , Yuri E. Dubrova , Patricia Balaresque , Zoë H. Rosser , Susan M. Adams , Mark A. Jobling
      Mutation at most human minisatellites is driven by complex interallelic processes that give rise to a high degree of length polymorphism and internal structural variation. MSY1, the only highly variable minisatellite on the non-recombining region of the Y chromosome, is constitutively haploid and therefore precluded from interallelic interactions, yet maintains high diversity in both length and structure. To investigate the basis of its mutation processes, an unbiased structural analysis of >500 single-molecule MSY1 PCR products from matched sperm and blood samples from a single donor was undertaken. The overall mutation frequencies in sperm and blood DNAs were not significantly different, at 2.68% and 1.88%, respectively. Sperm DNA showed significantly more length mutants than blood DNA, with mutants in both tissues involving small-scale (1–3 repeat units in a 77 repeat progenitor allele) increases or decreases in repeat block lengths, with no gain or loss bias. Isometric mutations altering structure but not length were found in both tissues, and involved either the apparent shift of a boundary between repeat unit blocks (a ‘boundary switch’) or the conversion of a repeat within a block to a different repeat type (‘modular structure’ mutant). There was a significant excess of boundary switch mutants and deficit of modular structure mutants in sperm. A comparison of mutant structures with phylogenetically matched alleles in population samples showed that alleles with structures resembling the blood mutants were unlikely to arise in populations. Mutation seems likely to involve gene conversion via synthesis-dependent strand annealing, and the blood-sperm differences may reflect more relaxed constraint on sister chromatid alignment in blood.

      PubDate: 2015-05-26T23:34:47Z
  • Delayed repair of radiation induced clustered DNA damage: Friend or
    • Abstract: Publication date: 3 June 2011
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volume 711, Issues 1–2
      Author(s): Laura J. Eccles , Peter O’Neill , Martine E. Lomax
      A signature of ionizing radiation exposure is the induction of DNA clustered damaged sites, defined as two or more lesions within one to two helical turns of DNA by passage of a single radiation track. Clustered damage is made up of double strand breaks (DSB) with associated base lesions or abasic (AP) sites, and non-DSB clusters comprised of base lesions, AP sites and single strand breaks. This review will concentrate on the experimental findings of the processing of non-DSB clustered damaged sites. It has been shown that non-DSB clustered damaged sites compromise the base excision repair pathway leading to the lifetime extension of the lesions within the cluster, compared to isolated lesions, thus the likelihood that the lesions persist to replication and induce mutation is increased. In addition certain non-DSB clustered damaged sites are processed within the cell to form additional DSB. The use of E. coli to demonstrate that clustering of DNA lesions is the major cause of the detrimental consequences of ionizing radiation is also discussed. The delayed repair of non-DSB clustered damaged sites in humans can be seen as a “friend”, leading to cell killing in tumour cells or as a “foe”, resulting in the formation of mutations and genetic instability in normal tissue.

      PubDate: 2015-05-26T23:34:47Z
  • Satellite 2 demethylation induced by 5-azacytidine is associated with
           missegregation of chromosomes 1 and 16 in human somatic cells
    • Abstract: Publication date: 3 January 2012
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volume 729, Issues 1–2
      Author(s): Diddier Prada , Rodrigo González , Lisandro Sánchez , Clementina Castro , Eunice Fabián , Luis A. Herrera
      Satellite sequences are an important part of the pericentromeric regions in mammalian genomes; they play a relevant role in chromosome stability and DNA hypomethylation of these sequences has been reported in ICF syndrome and in some cancers that are closely associated with chromosomal abnormalities. Epigenetic modifications of satellite sequences and their consequences have not been extensively studied in human cells. In the present work, we evaluated satellite 2 methylation patterns in human lymphocytes exposed to 5-azacytidine (5-azaC) and assessed the relationship between these patterns and chromosome missegregation. Human lymphocytes were exposed to 10μM 5-azaC for 24, 48, and 72h. Segregation errors were evaluated in binucleate cells using FISH against pericentromeric regions of chromosomes 1, 9, and 16. DNA methylation patterns were evaluated by immunodetection, and by bisulfite plus urea conversion and sequencing. We have identified that 5-azaC induced missegregation of chromosomes 1 and 16, which have highly methylated satellite 2, after 72h of exposure. Chromosome methylation patterns showed a notable decrease in pericentromeric methylation. Bisulfite conversion and sequencing analysis demonstrated demethylation of satellite 2 associated to 5-azaC exposure, principally after 72h of treatment. This change occurred in a non-specific pattern. Our study demonstrates an association between loss of satellite 2 DNA methylation and chromosome loss in human lymphocytes.

      PubDate: 2015-05-26T23:34:47Z
  • Telomerase and idiopathic pulmonary fibrosis
    • Abstract: Publication date: 1 February 2012
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volume 730, Issues 1–2
      Author(s): Mary Armanios
      Idiopathic pulmonary fibrosis (IPF) is the most common manifestation of telomere-mediated disorders. Germline mutations in the essential telomerase genes, hTERT and hTR, are the causal genetic defect in up to one-sixth of pulmonary fibrosis families. The presence of telomerase mutations in this subset is significant for clinical decisions as affected individuals can develop extra-pulmonary complications related to telomere shortening such as bone marrow failure and cryptogenic liver cirrhosis. There is also evidence that IPF is an ancestral manifestation of autosomal dominant telomere syndromes where, with successive generations, the disease evolves from pulmonary fibrosis into a bone marrow failure-predominant disorder, defining a unique form of genetic anticipation. Here I review the significance of telomere defects for understanding the genetics, disease patterns and pathophysiology of IPF. The importance of this diagnosis for patient care decisions will also be discussed.

      PubDate: 2015-05-26T23:34:47Z
  • Investigating the effects of dietary folic acid on sperm count, DNA damage
           and mutation in Balb/c mice
    • Abstract: Publication date: 1 September 2012
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volume 737, Issues 1–2
      Author(s): Breanne G. Swayne , Alice Kawata , Nathalie A. Behan , Andrew Williams , Mike G. Wade , Amanda J. MacFarlane , Carole L. Yauk
      To date, fewer than 50 mutagens have been studied for their ability to cause heritable mutations. The majority of those studied are classical mutagens like radiation and anti-cancer drugs. Very little is known about the dietary variables influencing germline mutation rates. Folate is essential for DNA synthesis and methylation and can impact chromatin structure. We therefore determined the effects of folic acid-deficient (0mg/kg), control (2mg/kg) and supplemented (6mg/kg) diets in early development and during lactation or post-weaning on mutation rates and chromatin quality in sperm of adult male Balb/c mice. The sperm chromatin structure assay and mutation frequencies at expanded simple tandem repeats (ESTRs) were used to evaluate germline DNA integrity. Treatment of a subset of mice fed the control diet with the mutagen ethylnitrosourea (ENU) at 8 weeks of age was included as a positive control. ENU treated mice exhibited decreased cauda sperm counts, increased DNA fragmentation and increased ESTR mutation frequencies relative to non-ENU treated mice fed the control diet. Male mice weaned to the folic acid deficient diet had decreased cauda sperm numbers, increased DNA fragmentation index, and increased ESTR mutation frequency. Folic acid deficiency in early development did not lead to changes in sperm counts or chromatin integrity in adult mice. Folic acid supplementation in early development or post-weaning did not affect germ cell measures. Therefore, adequate folic acid intake in adulthood is important for preventing chromatin damage and mutation in the male germline. Folic acid supplementation at the level achieved in this study does not improve nor is it detrimental to male germline chromatin integrity.
      Graphical abstract image Highlights ► We examine the effects of dietary folic acid on germ cell integrity. ► Sperm counts, chromatin damage and tandem repeat mutation frequencies were measured. ► Folic acid deficiency increased germline chromatin damage and mutation frequencies. ► Folic acid supplementation does not affect germline integrity in mice.

      PubDate: 2015-05-26T23:34:47Z
  • Molecular analysis of point mutations in a barley genome exposed to MNU
           and gamma rays
    • Abstract: Publication date: October–November 2012
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volumes 738–739
      Author(s): Marzena Kurowska , Anna Labocha-Pawłowska , Dominika Gnizda , Miroslaw Maluszynski , Iwona Szarejko
      We present studies aimed at determining the types and frequencies of mutations induced in the barley genome after treatment with chemical (N-methyl-N-nitrosourea, MNU) and physical (gamma rays) mutagens. We created M2 populations of a doubled haploid line and used them for the analysis of mutations in targeted DNA sequences and over an entire barley genome using TILLING (Targeting Induced Local Lesions in Genomes) and AFLP (Amplified Fragment Length Polymorphism) technique, respectively. Based on the TILLING analysis of the total DNA sequence of 4,537,117bp in the MNU population, the average mutation density was estimated as 1/504kb. Only one nucleotide change was found after an analysis of 3,207,444bp derived from the highest dose of gamma rays applied. MNU was clearly a more efficient mutagen than gamma rays in inducing point mutations in barley. The majority (63.6%) of the MNU-induced nucleotide changes were transitions, with a similar number of G>A and C>T substitutions. The similar share of G>A and C>T transitions indicates a lack of bias in the repair of O6-methylguanine lesions between DNA strands. There was, however, a strong specificity of the nucleotide surrounding the O6-meG at the −1 position. Purines formed 81% of nucleotides observed at the −1 site. Scanning the barley genome with AFLP markers revealed ca. a three times higher level of AFLP polymorphism in MNU-treated as compared to the gamma-irradiated population. In order to check whether AFLP markers can really scan the whole barley genome for mutagen-induced polymorphism, 114 different AFLP products, were cloned and sequenced. 94% of bands were heterogenic, with some bands containing up to 8 different amplicons. The polymorphic AFLP products were characterised in terms of their similarity to the records deposited in a GenBank database. The types of sequences present in the polymorphic bands reflected the organisation of the barley genome.
      Highlights ► MNU and γ rays revealed 2.2 and 0.3 mutations per 1Mb of gene sequences. ► The majority (63.6%) of the MNU-induced nucleotide changes were transitions. ► The nucleotide composition surrounding the methylated guanine was checked. ► We obtained higher level of AFLP polymorphism in MNU-treated population. ► The types of identified sequences reflected the organisation of the barley genome.

      PubDate: 2015-05-26T23:34:47Z
  • No evidence for transgenerational genomic instability in the F1 or F2
           descendants of Muta™Mouse males exposed to N-ethyl-N-nitrosourea
    • Abstract: Publication date: January–February 2013
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volumes 741–742
      Author(s): Jason M. O’Brien , Andrew Williams , John Gingerich , George R. Douglas , Francesco Marchetti , Carole L. Yauk
      Exposure of male mice to genotoxic agents can increase mutation frequencies in their unexposed descendants. This phenomenon, known as transgenerational genomic instability (TGI), can persist for several generations. However, little is known about the underlying mechanisms. Chemically-induced TGI has been demonstrated in non-coding unstable tandem repeat DNA regions, but it is unclear whether it extends to other genetic endpoints. We investigated whether exposure of Muta™Mouse males to a single dose of 75mg/kg N-ethyl-N-nitrosourea (ENU) increased the spontaneous frequency of gene mutations or chromosome damage in their offspring. Treated males were mated with untreated females 3 days, 6 weeks or 10 weeks post-exposure to produce the F1 generation. Offspring were thus conceived from germ cells exposed to ENU as mature spermatozoa, dividing spermatogonia, or spermatogonial stem cells, respectively. F2 mice were generated by mating F1 descendants with untreated partners. Mutations in the lacZ transgene were quantified in bone marrow and micronucleus frequencies were evaluated in red blood cells by flow-cytometry for all F0 and their descendants. LacZ mutant frequencies were also determined in sperm for all exposed males and their male descendants. In F0 males, lacZ mutant frequencies were significantly increased in bone marrow at least 10-fold at all three time points investigated. In sperm, lacZ mutant frequency was significantly increased 7–11-fold after exposure of dividing and stem cell spermatogonia, but not in replication-deficient haploid sperm. Micronucleus frequencies assessed two days after ENU treatment were increased 5-fold in F0 males, but returned to control levels after 10 weeks. Despite the strong mutagenic response in F0 males, pre- and post-meiotic ENU exposure did not significantly increase lacZ mutant or micronucleus frequencies in F1 or F2 offspring. These findings suggest that TGI may not extend to all genetic endpoints and that further investigation of this phenomenon and its health relevance will require multiple measures of genomic damage.

      PubDate: 2015-05-26T23:34:47Z
  • Investigating the mechanisms of ribonucleotide excision repair in
           Escherichia coli
    • Abstract: Publication date: March 2014
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volume 761
      Author(s): Alexandra Vaisman , John P. McDonald , Stephan Noll , Donald Huston , Gregory Loeb , Myron F. Goodman , Roger Woodgate
      Low fidelity Escherichia coli DNA polymerase V (pol V/UmuD′2C) is best characterized for its ability to perform translesion synthesis (TLS). However, in recA730 lexA(Def) strains, the enzyme is expressed under optimal conditions allowing it to compete with the cell's replicase for access to undamaged chromosomal DNA and leads to a substantial increase in spontaneous mutagenesis. We have recently shown that a Y11A substitution in the “steric gate” residue of UmuC reduces both base and sugar selectivity of pol V, but instead of generating an increased number of spontaneous mutations, strains expressing umuC_Y11A are poorly mutable in vivo. This phenotype is attributed to efficient RNase HII-initiated repair of the misincorporated ribonucleotides that concomitantly removes adjacent misincorporated deoxyribonucleotides. We have utilized the ability of the pol V steric gate mutant to promote incorporation of large numbers of errant ribonucleotides into the E. coli genome to investigate the fundamental mechanisms underlying ribonucleotide excision repair (RER). Here, we demonstrate that RER is normally facilitated by DNA polymerase I (pol I) via classical “nick translation”. In vitro, pol I displaces 1–3 nucleotides of the RNA/DNA hybrid and through its 5′→3′ (exo/endo) nuclease activity releases ribo- and deoxyribonucleotides from DNA. In vivo, umuC_Y11A-dependent mutagenesis changes significantly in polymerase-deficient, or proofreading-deficient polA strains, indicating a pivotal role for pol I in ribonucleotide excision repair (RER). However, there is also considerable redundancy in the RER pathway in E. coli. Pol I's strand displacement and FLAP-exo/endonuclease activities can be facilitated by alternate enzymes, while the DNA polymerization step can be assumed by high-fidelity pol III. We conclude that RNase HII and pol I normally act to minimize the genomic instability that is generated through errant ribonucleotide incorporation, but that the “nick-translation” activities encoded by the single pol I polypeptide can be undertaken by a variety of back-up enzymes.

      PubDate: 2015-05-26T23:34:47Z
  • Single-cell transcriptogenomics reveals transcriptional exclusion of
           ENU-mutated alleles
    • Abstract: Publication date: February 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volume 772
      Author(s): Wenge Li , R. Brent Calder , Jessica C. Mar , Jan Vijg
      Recently, great progress has been made in single cell genomics and transcriptomics. Here, we present an integrative method, termed single-cell transcriptogenomics (SCTG), in which whole exome sequencing and RNA-seq is performed concurrently on single cells. This methodology enables one to track germline and somatic variants directly from the genome to the transcriptome in individual cells. Mouse embryonic fibroblasts were treated with the powerful mutagen ethylnitrosourea (ENU) and subjected to SCTG. Interestingly, while germline variants were found to be transcribed in an allelically balanced fashion, a significantly different pattern of allelic exclusion was observed for ENU-mutant variants. These results suggest that the adverse effects of induced mutations, in contrast to germline variants, may be mitigated by allelically biased transcription. They also illustrate how SCTG can be instrumental in the direct assessment of phenotypic consequences of genomic variants.

      PubDate: 2015-05-26T23:34:47Z
  • New tool for biological dosimetry: Reevaluation and automation of the gold
           standard method following telomere and centromere staining
    • Abstract: Publication date: December 2014
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volume 770
      Author(s): Radhia M’kacher , Elie E.L. Maalouf , Michelle Ricoul , Leonhard Heidingsfelder , Eric Laplagne , Corina Cuceu , William M. Hempel , Bruno Colicchio , Alain Dieterlen , Laure Sabatier
      Purpose The dicentric chromosome (dicentric) assay is the international gold-standard method for biological dosimetry and classification of genotoxic agents. The introduction of telomere and centromere (TC) staining offers the potential to render dicentric scoring more efficient and robust. In this study, we improved the detection of dicentrics and all unstable chromosomal aberrations (CA) leading to a significant reevaluation of the dose–effect curve and developed an automated approach following TC staining. Material and methods Blood samples from 16 healthy donors were exposed to 137Cs at 8 doses from 0.1 to 6Gy. CA were manually and automatically scored following uniform (Giemsa) or TC staining. The detection of centromeric regions and telomeric sequences using PNA probes allowed the detection of all unstable CA: dicentrics, centric and acentric rings, and all acentric fragments (with 2, 4 or no telomeres) leading to the precise quantification of estimated double strand breaks (DSB). Results Manual scoring following TC staining revealed a significantly higher frequency of dicentrics (p <10−3) (up to 30%) and estimated DSB (p <10−4) compared to uniform staining due to improved detection of dicentrics with centromeres juxtaposed with other centromeres or telomeres. This improvement permitted the development of the software, TCScore, that detected 95% of manually scored dicentrics compared to 50% for the best currently available software (DCScore™). Conclusion The use of TC staining has permitted a reevaluation of the dose–response curve and the highly efficient automation of the scoring process, marking a new step in the management and follow-up of populations exposed to genotoxic agents including ionizing radiation.
      Graphical abstract image

      PubDate: 2015-05-26T23:34:47Z
  • 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
  • TP53 mutations induced by BPDE in Xpa-WT and Xpa-Null human TP53 knock-in
           (Hupki) mouse embryo fibroblasts
    • Abstract: Publication date: Available online 30 January 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
      Author(s): Jill E. Kucab , Harry van Steeg , Mirjam Luijten , Heinz H. Schmeiser , Paul A. White , David H. Phillips , Volker M. Arlt
      Somatic mutations in the tumour suppressor gene TP53 occur in more than 50% of human tumours; in some instances exposure to environmental carcinogens can be linked to characteristic mutational signatures. The Hupki (human TP53 knock-in) mouse embryo fibroblast (HUF) immortalisation assay (HIMA) is a useful model for studying the impact of environmental carcinogens on TP53 mutagenesis. In an effort to increase the frequency of TP53-mutated clones achievable in the HIMA, we generated nucleotide excision repair (NER)-deficient HUFs by crossing the Hupki mouse with an Xpa-knockout (Xpa-Null) mouse. We hypothesized that carcinogen-induced DNA adducts would persist in the TP53 sequence of Xpa-Null HUFs leading to an increased propensity for mismatched base pairing and mutation during replication of adducted DNA. We found that Xpa-Null Hupki mice, and HUFs derived from them, were more sensitive to the environmental carcinogen benzo[a]pyrene (BaP) than their wild-type (Xpa-WT) counterparts. Following treatment with the reactive metabolite of BaP, benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE), Xpa-WT and Xpa-Null HUF cultures were subjected to the HIMA. A significant increase in TP53 mutations on the transcribed strand was detected in Xpa-Null HUFs compared to Xpa-WT HUFs, but the TP53-mutant frequency overall was not significantly different between the two genotypes. BPDE induced mutations primarily at G:C base pairs, with approximately half occurring at CpG sites, and the predominant mutation type was G:C>T:A in both Xpa-WT and Xpa-Null cells. Further, several of the TP53 mutation hotspots identified in smokers’ lung cancer were mutated by BPDE in HUFs (codons 157, 158, 245, 248, 249, 273). Therefore, the pattern and spectrum of BPDE-induced TP53 mutations in the HIMA are consistent with TP53 mutations detected in lung tumours of smokers. While Xpa-Null HUFs exhibited increased sensitivity to BPDE-induced damage on the transcribed strand, NER-deficiency did not enhance TP53 mutagenesis resulting from damage on the non-transcribed strand in this model.

      PubDate: 2015-02-03T08:37:16Z
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