Showing papers in "Mutation Research-reviews in Mutation Research in 2013"

γH2AX as a marker of DNA double strand breaks and genomic instability in human population studies

Abstract: DNA double strand breaks (DSB) are the gravest form of DNA damage in eukaryotic cells. Failure to detect DSB and activate appropriate DNA damage responses can cause genomic instability, leading to tumorigenesis and possibly accelerated aging. Phosphorylated histone H2AX (γH2AX) is used as a biomarker of cellular response to DSB and its potential for monitoring DNA damage and repair in human populations has been explored in this review. A systematic search was conducted in PubMed for articles, in English, on human studies reporting γH2AX as a biomarker of either DNA repair or DNA damage. A total of 68 publications were identified. Thirty-four studies (50.0%) evaluated the effect of medical procedures or treatments on γH2AX levels; 20 (29.4%) monitored γH2AX in specific pathological conditions with a case/control or case/case design; 5 studies (7.4%) evaluated the effect of environmental genotoxic exposures, and 9 (13.2%) were descriptive studies on cancer and aging. Peripheral blood lymphocytes (44.6%) or biopsies/tissue specimens (24.3%) were the most commonly used samples. γH2AX was scored by optical microscopy as immunostained foci (78%), or by flow cytometry (16%). Critical features affecting the reliability of the assay, including protocols heterogeneity, specimen, cell cycle, kinetics, study design, and statistical analysis, are hereby discussed. Because of its sensitivity, efficiency and mechanistic relevance, the γH2AX assay has great potential as a DNA damage biomarker; however, the technical and epidemiological heterogeneity highlighted in this review infer a necessity for experimental standardization of the assay.

Non-targeted effects of ionising radiation—Implications for low dose risk

Abstract: Non-DNA targeted effects of ionising radiation, which include genomic instability, and a variety of bystander effects including abscopal effects and bystander mediated adaptive response, have raised concerns about the magnitude of low-dose radiation risk. Genomic instability, bystander effects and adaptive responses are powered by fundamental, but not clearly understood systems that maintain tissue homeostasis. Despite excellent research in this field by various groups, there are still gaps in our understanding of the likely mechanisms associated with non-DNA targeted effects, particularly with respect to systemic (human health) consequences at low and intermediate doses of ionising radiation. Other outstanding questions include links between the different non-targeted responses and the variations in response observed between individuals and cell lines, possibly a function of genetic background. Furthermore, it is still not known what the initial target and early interactions in cells are that give rise to non-targeted responses in neighbouring or descendant cells. This paper provides a commentary on the current state of the field as a result of the non-targeted effects of ionising radiation (NOTE) Integrated Project funded by the European Union. Here we critically examine the evidence for non-targeted effects, discuss apparently contradictory results and consider implications for low-dose radiation health effects.

The HUMNxl scoring criteria for different cell types and nuclear anomalies in the buccal micronucleus cytome assay - an update and expanded photogallery.

Abstract: The buccal micronucleus cytome assay is a minimally invasive cytological and interphase cytogenetic technique for measuring DNA damage and cell death biomarkers in the oral epithelium. In this report we provide an updated and more comprehensive version of the cellular and nuclear scoring criteria used in the assay accompanied with a photogallery of the various cell types and nuclear anomalies. These detailed scoring criteria complement previous published protocols of this assay and form the basis for guiding intra- and inter-laboratory slide scoring comparisons. The scoring criteria update described in this paper is the outcome of ongoing efforts of the HUMN and HUMNxl projects (www.humn.org) to standardize the application of micronucleus assay for use in human biomonitoring and to update procedures as knowledge on mechanisms and technical capability improvements.

Matrix metalloproteinase and its drug targets therapy in solid and hematological malignancies: an overview.

Abstract: Matrix metalloproteinase (MMP) comprises a family of zinc-dependent endopeptidases that degrade various components of the extracellular matrix (ECM) and basement membrane. MMPs are involved in solid and hematological malignancy through modification of cell growth, activation of cancer cells and modulation of immune functions. Several polymorphisms of different MMPs such as MMP-1 (-1607 1G/2G), MMP-2 (-1306 C/T), MMP-3 (-1171 5A/6A) & MMP-9 (-1562 C/T) and their expression levels have been well documented in different types of solid cancer. These polymorphic variations were found to be associated with angiogenesis, cancer progression, invasion and metastasis. There is paucity of data available in the field of hematological malignancies. Hence the field of matrix biology of hematological malignancies is an area of active exploration. A number of MMP inhibitors (MMPIs) have been developed for the cancer treatment. The most extensively studied classes of MMP inhibitors include Batimastat, Marismastat, Salimatat, Prinomastat and Tanomastat. However, their efficacy and action have not been confirmed and more data is required. The application of one or more selective targeted MMPIs in combination with conventional anti-leukemic treatment may represent a positive approach in combat against hematopoietic malignancies. Balance of MMPs and TIMPs is altered in different malignancies and biochemical pathways. These alternations will add another dimension in the matrix biology of both solid tumor and leukemia. MMP and TIMP singly and in combination are increasingly being recognized as an important player in basic cellular biology. Exploration and exploitation of MMP and TIMP balance in various malignant and nonmalignant lesions is going to be one of the most interesting facets of future use of this system for human health care.

Environmental light and endogenous antioxidants as the main determinants of non-cancer ocular diseases.

Abstract: The human eye is constantly exposed to sunlight and artificial lighting. Exogenous sources of reactive oxygen species (ROS) such as UV light, visible light, ionizing radiation, chemotherapeutics, and environmental toxins contribute to oxidative damage in ocular tissues. Long-term exposure to these insults places the aging eye at considerable risk for pathological consequences of oxidative stress. Furthermore, in eye tissues, mitochondria are an important endogenous source of ROS. Over time, all ocular structures, from the tear film to the retina, undergo oxidative stress, and therefore, the antioxidant defenses of each tissue assume the role of a safeguard against degenerative ocular pathologies. The ocular surface and cornea protect the other ocular tissues and are significantly exposed to oxidative stress of environmental origin. Overwhelming of antioxidant defenses in these tissues clinically manifests as pathologies including pterygium, corneal dystrophies, and endothelial Fuch's dystrophy. The crystalline lens is highly susceptible to oxidative damage in aging because its cells and their intracellular proteins are not turned over or replaced, thus providing the basis for cataractogenesis. The trabecular meshwork, which is the anterior chamber tissue devoted to aqueous humor drainage, has a particular susceptibility to mitochondrial oxidative injury that affects its endothelium and leads to an intraocular pressure increase that marks the beginning of glaucoma. Photo-oxidative stress can cause acute or chronic retinal damage. The pathogenesis of age-related macular degeneration involves oxidative stress and death of the retinal pigment epithelium followed by death of the overlying photoreceptors. Accordingly, converging evidence indicates that mutagenic mechanisms of environmental and endogenous sources play a fundamental pathogenic role in degenerative eye diseases.

ITPA (inosine triphosphate pyrophosphatase): from surveillance of nucleotide pools to human disease and pharmacogenetics.

Abstract: Cellular nucleotide pools are often contaminated by base analog nucleotides which interfere with a plethora of biological reactions, from DNA and RNA synthesis to cellular signaling. An evolutionarily conserved inosine triphosphate pyrophosphatase (ITPA) removes the non-canonical purine (d)NTPs inosine triphosphate and xanthosine triphosphate by hydrolyzing them into their monophosphate form and pyrophosphate. Mutations in the ITPA orthologs in model organisms lead to genetic instability and, in mice, to severe developmental anomalies. In humans there is genetic polymorphism in ITPA. One allele leads to a proline to threonine substitution at amino acid 32 and causes varying degrees of ITPA deficiency in tissues and plays a role in patients' response to drugs. Structural analysis of this mutant protein reveals that the protein is destabilized by the formation of a cavity in its hydrophobic core. The Pro32Thr allele is thought to cause the observed dominant negative effect because the resulting active enzyme monomer targets both homo- and heterodimers to degradation.

Inherited catalase deficiency: is it benign or a factor in various age related disorders?

Abstract: Hydrogen peroxide was - and is still - considered toxic for a wide range of living organisms. Oxidative stress occurs when there is an excess of pro-oxidants over antioxidants and it has been implicated in several diseases. Catalase is involved in hydrogen peroxide catabolism and is important in defense against oxidative stress. Acatalasemia means the inherited near-total deficiency of catalase activity, usually in reference to red cell catalase. Acatalasemia was thought at first to be an asymptotic disorder. In the absence of catalase, neither the Japanese, or Hungarian acatalasemics nor acatalasemic mice had significantly increased blood glutathione peroxidase activity. In animal models, catalase deficient tissues show much slower rates of removal of extracellular hydrogen peroxide. In catalase knock-out mice, a decreased hydrogen peroxide removing capacity and increased reactive oxygen species formation were reported. Hydrogen peroxide may cause methemoglobinemia in patients with catalase deficiency. During anesthesia for a Japanese acatalasemic patient the disinfection with hydrogen peroxide solution caused severe methemoglobinemia. Patients with inherited catalase deficiency, who are treated with uric acid oxidase (rasburicase) may experience very high concentrations of hydrogen peroxide and may suffer from methemoglobinemia and hemolysis. The high (18.5%) prevalence of diabetes mellitus in inherited catalase deficient individuals and the earlier (10 years) manifestation of the disease may be attributed to the oxidative damage of oxidant sensitive, insulin producing pancreatic beta-cells. Ninety-seven of 114 acatalasemics had diseases related to oxidative stress and aging. The oxidative stress due to catalase deficiency could contribute to the manifestation of diabetes while for the other diseases it may be one of the factors in their causations. In summary, inherited catalase deficiency is associated with clinical features, pathologic laboratory test results, age and oxidative stress related disorders. Rather than considering it a benign condition, it should be considered as a complicating condition for aging and oxidative stress.

Bacterial mutagenicity screening in the pharmaceutical industry.

Abstract: Genetic toxicity testing is used as an early surrogate for carcinogenicity testing Genetic toxicity testing is also required by regulatory agencies to be conducted prior to initiation of first in human clinical trials and subsequent marketing for most small molecule pharmaceutical compounds To reduce the chances of advancing mutagenic pharmaceutical candidates through the drug discovery and development processes, companies have focused on developing testing strategies to maximize hazard identification while minimizing resource expenditure due to late stage attrition With a large number of testing options, consensus has not been reached on the best mutagenicity platform to use or on the best time to use a specific test to aid in the selection of drug candidates for development Most companies use a process in which compounds are initially screened for mutagenicity early in drug development using tests that require only a few milligrams of compound and then follow those studies up with a more robust mutagenicity test prior to selecting a compound for full development This review summarizes the current applications of bacterial mutagenicity assays utilized by pharmaceutical companies in early and late discovery programs The initial impetus for this review was derived from a workshop on bacterial mutagenicity screening in the pharmaceutical industry presented at the 40th Annual Environmental Mutagen Society Meeting held in St Louis, MO in October, 2009 However, included in this review are succinct summaries of use and interpretation of genetic toxicity assays, several mutagenicity assays that were not presented at the meeting, and updates to testing strategies resulting in current state-of the art description of best practices In addition, here we discuss the advantages and liabilities of many broadly used mutagenicity screening platforms and strategies used by pharmaceutical companies The sensitivity and specificity of these early mutagenicity screening assays using proprietary compounds and their concordance (predictivity) with the regulatory bacterial mutation test are discussed

hTERT: Another brick in the wall of cancer cells

Abstract: In human cancer, expression of telomerase is positively correlated with tumour aggressiveness and metastatic potential. There is accumulating evidence that hTERT (the catalytic subunit of telomerase) favours an immortal phenotype by blocking programmed cell death (apoptosis) independently of its protective function at the telomere ends. This review summarized existing evidence for the anti-apoptotic role of hTERT in the context of tumour-cell resistance against DNA damage and aims to put hTERT in the context of cell-signal-transduction pathways leading either to survival or cell death. We found evidence that telomerase is cross-linked with many different signalling pathways that regulate cell proliferation, DNA damage repair, and also cell death. Thereby, hTERT survival function seems to occur at early stages of DNA damage recognition. We found some discrepancies in the published data though. Based on our findings, we suggest further exploration is needed of the interplay of the DNA damage response signalling network, including MAPK and p53 family activation, on telomerase regulation. This interaction is probably an important factor for fine tuning of the sensitivity of the cell to genotoxic stress. Using anti-neoplastic agents, further dose relationships on timing and extent of DNA damage, cellular repair and death should be established and correlated with hTERT expression/telomerase activation. Closing the data gaps identified here could profoundly improve our understanding of the relevance of telomerase for protecting the cell against anti-cancer agents and would contribute to developing new strategies for cancer therapy.

Shaping chromatin for repair.

Abstract: To counteract the adverse effects of various DNA lesions, cells have evolved an array of diverse repair pathways to restore DNA structure and to coordinate repair with cell cycle regulation. Chromatin changes are an integral part of the DNA damage response, particularly with regard to the types of repair that involve assembly of large multiprotein complexes such as those involved in double strand break (DSB) repair and nucleotide excision repair (NER). A number of phosphorylation, acetylation, methylation, ubiquitylation and chromatin remodeling events modulate chromatin structure at the lesion site. These changes demarcate chromatin neighboring the lesion, afford accessibility and binding surfaces to repair factors and provide on-the-spot means to coordinate repair and damage signaling. Thus, the hierarchical assembly of repair factors at a double strand break is mostly due to their regulated interactions with posttranslational modifications of histones. A large number of chromatin remodelers are required at different stages of DSB repair and NER. Remodelers physically interact with proteins involved in repair processes, suggesting that chromatin remodeling is a requisite for repair factors to access the damaged site. Together, recent findings define the roles of histone post-translational modifications and chromatin remodeling in the DNA damage response and underscore possible differences in the requirements for these events in relation to the chromatin context.

Harnessing genomics to identify environmental determinants of heritable disease

Abstract: Next-generation sequencing technologies can now be used to directly measure heritable de novo DNA sequence mutations in humans. However, these techniques have not been used to examine environmental factors that induce such mutations and their associated diseases. To address this issue, a working group on environmentally induced germline mutation analysis (ENIGMA) met in October 2011 to propose the necessary foundational studies, which include sequencing of parent–offspring trios from highly exposed human populations, and controlled dose–response experiments in animals. These studies will establish background levels of variability in germline mutation rates and identify environmental agents that influence these rates and heritable disease. Guidance for the types of exposures to examine come from rodent studies that have identified agents such as cancer chemotherapeutic drugs, ionizing radiation, cigarette smoke, and air pollution as germ-cell mutagens. Research is urgently needed to establish the health consequences of parental exposures on subsequent generations.

Ionizing radiation and genetic risks. XVII. Formation mechanisms underlying naturally occurring DNA deletions in the human genome and their potential relevance for bridging the gap between induced DNA double-strand breaks and deletions in irradiated germ cells

Abstract: While much is known about radiation-induced DNA double-strand breaks (DSBs) and their repair, the question of how deletions of different sizes arise as a result of the processing of DSBs by the cell's repair systems has not been fully answered. In order to bridge this gap between DSBs and deletions, we critically reviewed published data on mechanisms pertaining to: (a) repair of DNA DSBs (from basic studies in this area); (b) formation of naturally occurring structural variation (SV) - especially of deletions - in the human genome (from genomic studies) and (c) radiation-induced mutations and structural chromosomal aberrations in mammalian somatic cells (from radiation mutagenesis and radiation cytogenetic studies). The specific aim was to assess the relative importance of the postulated mechanisms in generating deletions in the human genome and examine whether empirical data on radiation-induced deletions in mouse germ cells are consistent with predictions of these mechanisms. The mechanisms include (a) NHEJ, a DSB repair process that does not require any homology and which functions in all stages of the cell cycle (and is of particular relevance in G0/G1); (b) MMEJ, also a DSB repair process but which requires microhomology and which presumably functions in all cell cycle stages; (c) NAHR, a recombination-based DSB repair mechanism which operates in prophase I of meiosis in germ cells; (d) MMBIR, a microhomology-mediated, replication-based mechanism which operates in the S phase of the cell cycle, and (e) strand slippage during replication (involved in the origin of small insertions and deletions (INDELs). Our analysis permits the inference that, between them, these five mechanisms can explain nearly all naturally occurring deletions of different sizes identified in the human genome, NAHR and MMBIR being potentially more versatile in this regard. With respect to radiation-induced deletions, the basic studies suggest that those arising as a result of the operation of NHEJ/MMEJ processes, as currently formulated, are expected to be relatively small. However, data on induced mutations in mouse spermatogonial stem cells (irradiation in G0/G1 phase of the cell cycle and DSB repair presumed to be via NHEJ predominantly) show that most are associated with deletions of different sizes, some in the megabase range. There is thus a 'discrepancy' between what the basic studies suggest and the empirical observations in mutagenesis studies. This discrepancy, however, is only an apparent but not a real one. It can be resolved by considering the issue of deletions in the broader context of and in conjunction with the organization of chromatin in chromosomes and nuclear architecture, the conceptual framework for which already exists in studies carried out during the past fifteen years or so. In this paper, we specifically hypothesize that repair of DSBs induced in chromatin loops may offer a basis to explain the induction of deletions of different sizes and suggest an approach to test the hypothesis. We emphasize that the bridging of the gap between induced DSB and resulting deletions of different sizes is critical for current efforts in computational modeling of genetic risks.

How fruit flies came to launch the chromosome theory of heredity.

Abstract: Fruit flies were used by several laboratories between 1901 and 1910 for studies of experimental evolution at Harvard, Indiana University, and Cold Spring Harbor before Thomas Hunt Morgan found his white-eyed mutation that we associate with the beginnings of the fly lab at Columbia University. The major players prior to Morgan were William Castle and his students at Harvard University, Frank Lutz at Cold Spring Harbor, and Fernandus Payne whose ideas for working with fruit flies were shaped by his studies of blind cave fauna at Indiana University. Payne's interests were stimulated by the work of Carl Eigenmann, an authority on blind cave fauna, and William Moenkhaus, who introduced Payne to fruit flies at Indiana University before Payne moved to Columbia to pursue graduate work with Morgan and Edmund Wilson. The motivations of the laboratories differed in the theories used for their work. Castle spread the word about the utility of fruit flies for research, but Payne gave Morgan his first fruit flies for research leading to the discovery of the white-eye mutation.

H.J. Muller's contributions to mutation research.

Abstract: H. J. Muller is best known for his Nobel Prize work on the induction of mutations by ionizing radiation. Geneticists are less familiar with his contributions to mutation and how he related the process of mutagenesis to the gene and distinguished gene mutations from other genetic and epigenetic events such as polyploidy, chromosome rearrangements, and position effects. The hallmark of Muller's contributions is his design of genetic stocks to solve genetic problems and allow experimentation to reveal new phenomena. In this review I relate Muller's personality to his teaching and research and present a history of Muller's ideas on mutation from his first days in Morgan's fly lab to his final thoughts on what became called "Muller's ratchet", a term he did not get to enjoy because it was coined seven years after his death.

The rise and fall of photomutagenesis.

Abstract: UV is the most abundant human carcinogen, and protection from extensive exposure to it is a widespread human health issue. The use of chemicals (sunscreens) for protection is intuitive and efficacious. However, these chemicals may become activated to reactive intermediates when absorbing energy from UV, thus producing damage themselves, which may manifest itself in phototoxic, photoallergenic or photocarcinogenic reactions in humans. The development of safe sunscreens for humans is of high interest. Similar issues have been observed for some therapeutically used principles such as PUVA therapy for psoriasis or porphyrins for phototherapy of human cancers. Photoactivation has also been reported as a side effect of various pharmaceuticals such as the antibacterial fluoroquinolones. In this context, the authors have been involved over more than 20 years in the development and refinement of assays to test for photomutagenicity as an unwanted side effect of UV-mediated activation of such chemicals for cosmetic or pharmaceutical use. The initial years of great hopes for simple mammalian cell-based assays for photomutagenicity to screen out substances of concern for human use were followed by many years of collaborative trials to achieve standardization. However, it is now realized that this topic, albeit of human safety relevance, is highly complex and subject to many artificial modifiers, especially in vitro in mammalian cell culture. Thus, it is not really suitable for being engineered into a general testing framework within cosmetic or pharmaceutical testing guidelines. Much knowledge has been generated over the years to arrive at the conclusion that yes, photomutagenicity does exist with the use of chemicals, but how to best test for it will require a sophisticated case-by-case approach. Moreover, in comparison to the properties and risks of exposure to UV itself, it remains a comparatively minor human safety risk to address. In considering risks and benefits, we should also acknowledge beneficial effects of UV on human health, including an essential role in the production of Vitamin D. Thus, the interrelationships between UV, chemicals and human health remain a fascinating topic of research.

Kinetic models reveal the in vivo mechanisms of mutagenesis in microbes and man

Abstract: This review summarizes the evidence indicating that mutagenic mechanisms in vivo are essentially the same in all living cells. Unique metabolic reactions to a particular environmental stress apparently target specific genes for increased rates of transcription and mutation, resulting in higher mutation rates for those genes most likely to solve the problem. Kinetic models which have demonstrated predictive value are described and are shown to simulate mutagenesis in vivo in Escherichia coli, the p53 tumor suppressor gene, and somatic hypermutation. In all three models, direct correlations are seen between mutation frequencies and transcription rates. G and C nucleosides in single-stranded DNA (ssDNA) are intrinsically mutable, and G and C silent mutations in p53 and in VH framework regions provide compelling evidence for intrinsic mechanisms of mutability, since mutation outcomes are neutral and are not selected. During transcription, the availability of unpaired bases in the ssDNA of secondary structures is rate-limiting for, and determines the frequency of mutations in vivo. In vitro analyses also verify the conclusion that intrinsically mutable bases are in fact located in ssDNA loops of predicted stem-loop structures (SLSs).

Corrigendum to "Bacterial mutagenicity screening in the pharmaceutical industry" [Mutat. Res. 752(2) (2013) 99-118].

Abstract: P.A. Escobar *, R.A. Kemper , J. Tarca , J. Nicolette , M. Kenyon , S. Glowienke , S.G. Sawant , J. Christensen , T.E. Johnson , C. McKnight , G. Ward , S.M. Galloway , L. Custer , E. Gocke , M.R. O’Donovan , K. Braun , R.D. Snyder , B. Mahadevan b,1,2 Boehringer Ingelheim Pharmaceuticals Inc., Ridgefield, CT 06877-0368, USA Merck Research Laboratories West Point, PA 19486, USA AbbVie, Abbott Park, IL 60064-3500, USA d Pfizer Global Research and Development, Groton, CT, USA Novartis Pharma AG, Werk Klybeck Klybeckstrasse 141, Basel CH-4057, Switzerland Amgen Inc., Thousand Oaks, CA 91320-1799, USA Bristol Myers-Squib, New Brunswick, NJ 08903, USA Hoffman-La Roche Ltd., Basel, Switzerland AstraZeneca R&D, Alderley Park, Macclesfield, Cheshire SK10 4TG, UK j Sanofi-Aventis Deutschland GmbH, D-65926 Frankfurt, Germany RDS Consulting Services, Maineville, OH 45039, USA