Showing papers in "Mutation Research in 2011"
TL;DR: The current status of knowledge and evidence on the mechanisms and involvement of intracellular oxidative stress and DNA damage in human malignancy evolution and possible use of these parameters as cancer biomarkers are presented and controversies related to specific methodologies used for the measurement of oxidatively induced DNA lesions in human cells or tissues are discussed.
Abstract: Cells in tissues and organs are continuously subjected to oxidative stress and free radicals on a daily basis. This free radical attack has exogenous or endogenous (intracellular) origin. The cells withstand and counteract this occurrence by the use of several and different defense mechanisms ranging from free radical scavengers like glutathione (GSH), vitamins C and E and antioxidant enzymes like catalase, superoxide dismutase and various peroxidases to sophisticated and elaborate DNA repair mechanisms. The outcome of this dynamic equilibrium is usually the induction of oxidatively induced DNA damage and a variety of lesions of small to high importance and dangerous for the cell i.e. isolated base lesions or single strand breaks (SSBs) to complex lesions like double strand breaks (DSBs) and other non-DSB oxidatively generated clustered DNA lesions (OCDLs). The accumulation of DNA damage through misrepair or incomplete repair may lead to mutagenesis and consequently transformation particularly if combined with a deficient apoptotic pathway. In this review, we present the current status of knowledge and evidence on the mechanisms and involvement of intracellular oxidative stress and DNA damage in human malignancy evolution and possible use of these parameters as cancer biomarkers. At the same time, we discuss controversies related to potential artifacts inherent to specific methodologies used for the measurement of oxidatively induced DNA lesions in human cells or tissues.
820 citations
TL;DR: Future concepts of metastasis intervention must simultaneously address the collective, mesenchymal and amoeboid mechanisms of cell invasion in order to advance in anti-metastatic strategies as these different types of movement can coexist and cooperate.
Abstract: Metastasis is the leading cause of cancer mortality. The metastatic cascade represents a multi-step process which includes local tumor cell invasion, entry into the vasculature followed by the exit of carcinoma cells from the circulation and colonization at the distal sites. At the earliest stage of successful cancer cell dissemination, the primary cancer adapts the secondary site of tumor colonization involving the tumor–stroma crosstalk. The migration and plasticity of cancer cells as well as the surrounding environment such as stromal and endothelial cells are mandatory. Consequently, the mechanisms of cell movement are of utmost relevance for targeted intervention of which three different types have been reported. Tumor cells can migrate either collectively, in a mesenchymal or in an amoeboid type of movement and intravasate the blood or lymph vasculature. Intravasation by the interaction of tumor cells with the vascular endothelium is mechanistically poorly understood. Changes in the epithelial plasticity enable carcinoma cells to switch between these types of motility. The types of migration may change depending on the intervention thereby increasing the velocity and aggressiveness of invading cancer cells. Interference with collective or mesenchymal cell invasion by targeting integrin expression or metalloproteinase activity, respectively, resulted in an amoeboid cell phenotype as the ultimate exit strategy of cancer cells. There are little mechanistic details reported in vivo showing that the amoeboid behavior can be either reversed or efficiently inhibited. Future concepts of metastasis intervention must simultaneously address the collective, mesenchymal and amoeboid mechanisms of cell invasion in order to advance in anti-metastatic strategies as these different types of movement can coexist and cooperate. Beyond the targeting of cell movements, the adhesion of cancer cells to the stroma in heterotypic circulating tumor cell emboli is of paramount relevance for anti-metastatic therapy.
659 citations
TL;DR: A brief overview of miRNA biogenesis and function, the identification and potential roles of circulating extracellular miRNAs, and the prospective uses of mi RNAs as clinical biomarkers are provided.
Abstract: MicroRNAs (miRNAs) are a recently discovered class of small, non-coding RNAs that regulate protein levels post-transcriptionally. miRNAs play important regulatory roles in many cellular processes, including differentiation, neoplastic transformation, and cell replication and regeneration. Because of these regulatory roles, it is not surprising that aberrant miRNA expression has been implicated in several diseases. Recent studies have reported significant levels of miRNAs in serum and other body fluids, raising the possibility that circulating miRNAs could serve as useful clinical biomarkers. Here, we provide a brief overview of miRNA biogenesis and function, the identification and potential roles of circulating extracellular miRNAs, and the prospective uses of miRNAs as clinical biomarkers. Finally, we address several issues associated with the accurate measurement of miRNAs from biological samples.
574 citations
TL;DR: The current state of knowledge on the role of ROS-induced oxidative stress in altering the genetic and epigenetic involvement during human carcinogenesis is reviewed.
Abstract: Cancer is a multistage and complex process characterized by molecular alterations that underlie all three phases of its development: (i) initiation, (ii) promotion and (iii) progression. Some of these molecular events include alterations in gene expression that are regulated by both genetic and epigenetic mechanisms. On the other hand, "oxidative stress" implies a cellular state where ROS production exceeds the cell's ability to metabolize them resulting in excessive accumulation of ROS that overwhelms cellular defenses. Such state has been shown to regulate both genetic and epigenetic cascades underlying altered gene expression in human disease including cancer. Throughout this manuscript, we review the current state of knowledge on the role of ROS-induced oxidative stress in altering the genetic and epigenetic involvement during human carcinogenesis.
476 citations
TL;DR: Mechanisms of DSB induction by IR are outlined and particular emphasis on backup pathways of NHEJ are placed on and summarized their increasing significance in various cellular processes, as well as their potential contribution to carcinogenesis.
Abstract: A defining characteristic of damage induced in the DNA by ionizing radiation (IR) is its clustered character that leads to the formation of complex lesions challenging the cellular repair mechanisms. The most widely investigated such complex lesion is the DNA double strand break (DSB). DSBs undermine chromatin stability and challenge the repair machinery because an intact template strand is lacking to assist restoration of integrity and sequence in the DNA molecule. Therefore, cells have evolved a sophisticated machinery to detect DSBs and coordinate a response on the basis of inputs from various sources. A central function of cellular responses to DSBs is the coordination of DSB repair. Two conceptually different mechanisms can in principle remove DSBs from the genome of cells of higher eukaryotes. Homologous recombination repair (HRR) uses as template a homologous DNA molecule and is therefore error-free; it functions preferentially in the S and G2 phases. Non-homologous end joining (NHEJ), on the other hand, simply restores DNA integrity by joining the two ends, is error prone as sequence is only fortuitously preserved and active throughout the cell cycle. The basis of DSB repair pathway choice remains unknown, but cells of higher eukaryotes appear programmed to utilize preferentially NHEJ. Recent work suggests that when the canonical DNA-PK dependent pathway of NHEJ (D-NHEJ), becomes compromised an alternative NHEJ pathway and not HRR substitutes in a quasi-backup function (B-NHEJ). Here, we outline aspects of DSB induction by IR and review the mechanisms of their processing in cells of higher eukaryotes. We place particular emphasis on backup pathways of NHEJ and summarize their increasing significance in various cellular processes, as well as their potential contribution to carcinogenesis.
394 citations
TL;DR: This review focuses on the main progress in this field and discusses the most important findings under a historical perspective, and examines microRNAs as markers of disease diagnosis and prognosis, and as new therapeutic targets.
Abstract: Since 1993, when the first small non-coding RNA was identified, our knowledge about microRNAs has grown exponentially. In this review, we focus on the main progress in this field and discuss the most important findings under a historical perspective. In addition, we examine microRNAs as markers of disease diagnosis and prognosis, and as new therapeutic targets.
371 citations
TL;DR: The PARTRAC suite of comprehensive Monte Carlo simulation tools for calculations of track structures of a variety of ionizing radiation qualities and their biological effects are described and essential results are recapitulated regarding the physical, physico-chemical and chemical stage of track structure development of radiation damage induction.
Abstract: This review describes the PARTRAC suite of comprehensive Monte Carlo simulation tools for calculations of track structures of a variety of ionizing radiation qualities and their biological effects. A multi-scale target model characterizes essential structures of the whole genomic DNA within human fibroblasts and lymphocytes in atomic resolution. Calculation methods and essential results are recapitulated regarding the physical, physico-chemical and chemical stage of track structure development of radiation damage induction. Recent model extension towards DNA repair processes extends the time dimension by about 12 orders of magnitude and paves the way for superior predictions of radiation risks.
310 citations
TL;DR: The current knowledge on DNA damage induced by endogenously produced reactive aldehydes in relation to the pathophysiology of human diseases is discussed.
Abstract: DNA damage plays a major role in various pathophysiological conditions including carcinogenesis, aging, inflammation, diabetes and neurodegenerative diseases. Oxidative stress and cell processes such as lipid peroxidation and glycation induce the formation of highly reactive endogenous aldehydes that react directly with DNA, form aldehyde-derived DNA adducts and lead to DNA damage. In occasion of persistent conditions that influence the formation and accumulation of aldehyde-derived DNA adducts the resulting unrepaired DNA damage causes deregulation of cell homeostasis and thus significantly contributes to disease phenotype. Some of the most highly reactive aldehydes produced endogenously are 4-hydroxy-2-nonenal, malondialdehyde, acrolein, crotonaldehyde and methylglyoxal. The mutagenic and carcinogenic effects associated with the elevated levels of these reactive aldehydes, especially, under conditions of stress, are attributed to their capability of causing directly modification of DNA bases or yielding promutagenic exocyclic adducts. In this review, we discuss the current knowledge on DNA damage induced by endogenously produced reactive aldehydes in relation to the pathophysiology of human diseases.
244 citations
TL;DR: Evaluated animal studies suggested positive associations between formaldehyde exposure and reproductive toxicity, mostly in males, and well-designed molecular epidemiologic studies, that include quantitative exposure assessment and diminish confounding factors, should examine both reproductive and developmental outcomes associated with exposure in males and females.
Abstract: Formaldehyde, the recently classified carcinogen and ubiquitous environmental contaminant, has long been suspected of causing adverse reproductive and developmental effects, but previous reviews were inconclusive, due in part, to limitations in the design of many of the human population studies. In the current review, we systematically evaluated evidence of an association between formaldehyde exposure and adverse reproductive and developmental effects, in human populations and in vivo animal studies, in the peer-reviewed literature. The mostly retrospective human studies provided evidence of an association of maternal exposure with adverse reproductive and developmental effects. Further assessment of this association by meta-analysis revealed an increased risk of spontaneous abortion (1.76, 95% CI 1.20-2.59, p=0.002) and of all adverse pregnancy outcomes combined (1.54, 95% CI 1.27-1.88, p<0.001), in formaldehyde-exposed women, although differential recall, selection bias, or confounding cannot be ruled out. Evaluation of the animal studies including all routes of exposure, doses and dosing regimens studied, suggested positive associations between formaldehyde exposure and reproductive toxicity, mostly in males. Potential mechanisms underlying formaldehyde-induced reproductive and developmental toxicities, including chromosome and DNA damage (genotoxicity), oxidative stress, altered level and/or function of enzymes, hormones and proteins, apoptosis, toxicogenomic and epigenomic effects (such as DNA methylation), were identified. To clarify these associations, well-designed molecular epidemiologic studies, that include quantitative exposure assessment and diminish confounding factors, should examine both reproductive and developmental outcomes associated with exposure in males and females. Together with mechanistic and animal studies, this will allow us to better understand the systemic effect of formaldehyde exposure.
230 citations
TL;DR: This review focuses on the repair process and mutagenic consequences of clustered lesions in yeast and mammalian cells, and the effects of low vs high LET radiation on mammalian cells or tissues.
Abstract: A clustered DNA lesion, also known as a multiply damaged site, is defined as ≥ 2 damages in the DNA within 1-2 helical turns. Only ionizing radiation and certain chemicals introduce DNA damage in the genome in this non-random way. What is now clear is that the lethality of a damaging agent is not just related to the types of DNA lesions introduced, but also to how the damage is distributed in the DNA. Clustered DNA lesions were first hypothesized to exist in the 1990s, and work has progressed where these complex lesions have been characterized and measured in irradiated as well as in non-irradiated cells. A clustered lesion can consist of single as well as double strand breaks, base damage and abasic sites, and the damages can be situated on the same strand or opposing strands. They include tandem lesions, double strand break (DSB) clusters and non-DSB clusters, and base excision repair as well as the DSB repair pathways can be required to remove these complex lesions. Due to the plethora of oxidative damage induced by ionizing radiation, and the repair proteins involved in their removal from the DNA, it has been necessary to study how repair systems handle these lesions using synthetic DNA damage. This review focuses on the repair process and mutagenic consequences of clustered lesions in yeast and mammalian cells. By examining the studies on synthetic clustered lesions, and the effects of low vs high LET radiation on mammalian cells or tissues, it is possible to extrapolate the potential biological relevance of these clustered lesions to the killing of tumor cells by radiotherapy and chemotherapy, and to the risk of cancer in non-tumor cells, and this will be discussed.
205 citations
TL;DR: There is no convincing evidence that any genotoxic rodent carcinogens or in vivo genotoxins would remain undetected in an in vitro test battery consisting of Ames+MNvit.
Abstract: In vitro genotoxicity testing needs to include tests in both bacterial and mammalian cells, and be able to detect gene mutations, chromosomal damage and aneuploidy. This may be achieved by a combination of the Ames test (detects gene mutations) and the in vitro micronucleus test (MNvit), since the latter detects both chromosomal aberrations and aneuploidy. In this paper we therefore present an analysis of an existing database of rodent carcinogens and a new database of in vivo genotoxins in terms of the in vitro genotoxicity tests needed to detect their in vivo activity. Published in vitro data from at least one test system (most were from the Ames test) were available for 557 carcinogens and 405 in vivo genotoxins. Because there are fewer publications on the MNvit than for other mammalian cell tests, and because the concordance between the MNvit and the in vitro chromosomal aberration (CAvit) test is so high for clastogenic activity, positive results in the CAvit test were taken as indicative of a positive result in the MNvit where there were no, or only inadequate data for the latter. Also, because Hprt and Tk loci both detect gene-mutation activity, a positive Hprt test was taken as indicative of a mouse-lymphoma Tk assay (MLA)-positive, where there were no data for the latter. Almost all of the 962 rodent carcinogens and in vivo genotoxins were detected by an in vitro battery comprising Ames+MNvit. An additional 11 carcinogens and six in vivo genotoxins would apparently be detected by the MLA, but many of these had not been tested in the MNvit or CAvit tests. Only four chemicals emerge as potentially being more readily detected in MLA than in Ames+MNvit--benzyl acetate, toluene, morphine and thiabendazole--and none of these are convincing cases to argue for the inclusion of the MLA in addition to Ames+MNvit. Thus, there is no convincing evidence that any genotoxic rodent carcinogens or in vivo genotoxins would remain undetected in an in vitro test battery consisting of Ames+MNvit.
TL;DR: A major role of DNA damage in the modulation of longevity is suggested, possibly through effects on cell dysfunction and loss, although understanding how to modify DNA damage repair and response systems to delay ageing remains a crucial challenge.
Abstract: Given the central role of DNA in life, and how ageing can be seen as the gradual and irreversible breakdown of living systems, the idea that damage to the DNA is the crucial cause of ageing remains a powerful one. DNA damage and mutations of different types clearly accumulate with age in mammalian tissues. Human progeroid syndromes resulting in what appears to be accelerated ageing have been linked to defects in DNA repair or processing, suggesting that elevated levels of DNA damage can accelerate physiological decline and the development of age-related diseases not limited to cancer. Higher DNA damage may trigger cellular signalling pathways, such as apoptosis, that result in a faster depletion of stem cells, which in turn contributes to accelerated ageing. Genetic manipulations of DNA repair pathways in mice further strengthen this view and also indicate that disruption of specific pathways, such as nucleotide excision repair and non-homologous end joining, is more strongly associated with premature ageing phenotypes. Delaying ageing in mice by decreasing levels of DNA damage, however, has not been achieved yet, perhaps due to the complexity inherent to DNA repair and DNA damage response pathways. Another open question is whether DNA repair optimization is involved in the evolution of species longevity, and we suggest that the way cells from different organisms respond to DNA damage may be crucial in species differences in ageing. Taken together, the data suggest a major role of DNA damage in the modulation of longevity, possibly through effects on cell dysfunction and loss, although understanding how to modify DNA damage repair and response systems to delay ageing remains a crucial challenge.
TL;DR: 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.
Abstract: 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.
TL;DR: The integrative survey and analyses revealed candidate cancer-specific miRNA epigenetic signatures which provide the basis for new therapeutic strategies in cancer by targeting the epigenetic regulation of miRNAs.
Abstract: MicroRNAs (miRNAs) belong to the heterogeneous class of non-coding RNAs (ncRNAs) that regulate the translation and degradation of target mRNAs, and control approximately 30% of human genes. MiRNA genes might be silenced in human tumors (oncomiRs) by aberrant hypermethylation of CpG islands that encompass or lie adjacent to miRNA genes and/or by histone modifications. We performed literature search for research articles describing epigenetically regulated miRNAs in cancer and identified 45 studies that were published between 2006 and 7/2010. The data from those papers are fragmented and methodologically heterogeneous and our work represents first systematic review towards to integration of diverse sets of information. We reviewed the methods used for detection of miRNA epigenetic regulation, which comprise bisulfite genomic sequencing PCR (BSP), bisulfite pyrosequencing, methylation specific PCR (MSP), combined bisulfite restriction analysis (COBRA), methylation sensitive single nucleotide primer extension (Ms-SNuPE), MassARRAY technique and some modifications of those methods. This integrative study revealed 122 miRNAs that were reported to be epigenetically regulated in 23 cancer types. Compared to protein coding genes, human oncomiRs showed an order of magnitude higher methylation frequency (11.6%; 122/1048 known miRNAs). Nearly half, (45%; 55/122) epigenetically regulated miRNAs were associated with different cancer types, but other 55% (67/122) miRNAs were present in only one cancer type and therefore representing cancer-specific biomarker potential. The data integration revealed miRNA epigenomic hot spots on the chromosomes 1q, 7q, 11q, 14q and 19q. CpG island analysis of corresponding miRNA precursors (pre-miRNAs) revealed that 20% (26/133) of epigenetically regulated miRNAs had a CpG island within the range of 5kb upstream, among them 14% (19/133) of miRNAs resided within the CpG island. Our integrative survey and analyses revealed candidate cancer-specific miRNA epigenetic signatures which provide the basis for new therapeutic strategies in cancer by targeting the epigenetic regulation of miRNAs.
TL;DR: The results reveal that up-regulation of the key upstream signaling factor, PTEN, in MCF-7/DOX cells inhibited Akt phosphorylation, which ultimately causes increase in their regulatory p53 levels affecting the induction of G2/M cell cycle arrest and apoptosis.
Abstract: The use of innocuous naturally occurring compounds to overcome drug resistance and cancer recalcitrance is now in the forefront of cancer research. Thymoquinone (TQ) is a bioactive constituent of the volatile oil derived from seeds of Nigella sativa Linn. TQ has shown promising anti-carcinogenic and anti-tumor activities through different mechanisms. However, the effect of TQ on cell signaling and survival pathways in resistant cancer cells has not been fully delineated. Here, we report that TQ greatly inhibits doxorubicin-resistant human breast cancer MCF-7/DOX cell proliferation. TQ treatment increased cellular levels of PTEN proteins, resulting in a substantial decrease of phosphorylated Akt, a known regulator of cell survival. The PTEN expression was accompanied with elevation of PTEN mRNA. TQ arrested MCF-7/DOX cells at G2/M phase and increased cellular levels of p53 and p21 proteins. Flow cytometric analysis and agarose gel electrophoresis revealed a significant increase in Sub-G1 cell population and appearance of DNA ladders following TQ treatment, indicating cellular apoptosis. TQ-induced apoptosis was associated with disrupted mitochondrial membrane potential and activation of caspases and PARP cleavage in MCF-7/DOX cells. Moreover, TQ treatment increased Bax/Bcl2 ratio via up-regulating Bax and down-regulating Bcl2 proteins. More importantly, PTEN silencing by target specific siRNA enabled the suppression of TQ-induced apoptosis resulting in increased cell survival. Our results reveal that up-regulation of the key upstream signaling factor, PTEN, in MCF-7/DOX cells inhibited Akt phosphorylation, which ultimately causes increase in their regulatory p53 levels affecting the induction of G2/M cell cycle arrest and apoptosis. Overall results provide mechanistic insights for understanding the molecular basis and utility of the anti-tumor activity of TQ.
TL;DR: Evidence is analyzed that supports a role for autophagy as an integral part of the DNA damage response, a cellular catabolic process that maintains a balance between synthesis, degradation, and recycling of cellular components.
Abstract: Both exogenous and endogenous agents are a threat to DNA integrity. Exogenous environmental agents such as ultraviolet (UV) and ionizing radiation, genotoxic chemicals and endogenous byproducts of metabolism including reactive oxygen species can cause alterations in DNA structure (DNA damage). Unrepaired DNA damage has been linked to a variety of human disorders including cancer and neurodegenerative disease. Thus, efficient mechanisms to detect DNA lesions, signal their presence and promote their repair have been evolved in cells. If DNA is effectively repaired, DNA damage response is inactivated and normal cell functioning resumes. In contrast, when DNA lesions cannot be removed, chronic DNA damage triggers specific cell responses such as cell death and senescence. Recently, DNA damage has been shown to induce autophagy, a cellular catabolic process that maintains a balance between synthesis, degradation, and recycling of cellular components. But the exact mechanisms by which DNA damage triggers autophagy are unclear. More importantly, the role of autophagy in the DNA damage response and cellular fate is unknown. In this review we analyze evidence that supports a role for autophagy as an integral part of the DNA damage response.
TL;DR: The published data on miRNAs in relation to the exposure to several environmental chemicals are reviewed, and the potential mechanisms that may link environmental chemicals to miRNA alterations are discussed.
Abstract: MicroRNAs (miRNAs) are short single-stranded non-coding molecules that function as negative regulators to silence or suppress gene expression. Aberrant miRNA expression has been implicated in a several cellular processes and pathogenic pathways of a number of diseases. Evidence is rapidly growing that miRNA regulation of gene expression may be affected by environmental chemicals. These environmental exposures include those that have frequently been associated with chronic diseases, such as heavy metals, air pollution, bisphenol A, and cigarette smoking. In this article, we review the published data on miRNAs in relation to the exposure to several environmental chemicals, and discuss the potential mechanisms that may link environmental chemicals to miRNA alterations. We further discuss the challenges in environmental-miRNA research and possible future directions. The accumulating evidence linking miRNAs to environmental chemicals, coupled with the unique regulatory role of miRNAs in gene expression, makes miRNAs potential biomarkers for better understanding the mechanisms of environmental diseases.
TL;DR: It is hypothesized that the induced mitotic arrest and following cell death was due to a premature chromosome condensation caused by a combination of DNA, mitochondrial and spindle damage.
Abstract: Airborne particulate matter (PM) is considered to be an important contributor to lung diseases. In the present study we report that Milan winter-PM2.5 inhibited proliferation in human bronchial epithelial cells (BEAS-2B) by inducing mitotic arrest. The cell cycle arrest was followed by an increase in mitotic-apoptotic cells, mitotic slippage and finally an increase in "classical" apoptotic cells. Exposure to winter-PM10 induced only a slight effect which may be due to the presence of PM2.5 in this fraction while pure combustion particles failed to disturb mitosis. Fewer cells expressing the mitosis marker phospho-histone H3 compared to cells with condensed chromosomes, suggest that PM2.5 induced premature mitosis. PM2.5 was internalized into the cells and often localized in laminar organelles, although particles without apparent plasma membrane covering were also seen. In PM-containing cells mitochondria and lysosomes were often damaged, and in mitotic cells fragmented chromosomes often appeared. PM2.5 induced DNA strands breaks and triggered a DNA-damage response characterized by increased phosphorylation of ATM, Chk2 and H2AX; as well as induced a marked increase in expression of the aryl hydrocarbon receptor (AhR)-regulated genes, CYP1A1, CYP1B1 and AhRR. Furthermore, some disturbance of the organization of microtubules was indicated. It is hypothesized that the induced mitotic arrest and following cell death was due to a premature chromosome condensation caused by a combination of DNA, mitochondrial and spindle damage.
TL;DR: The potential of a new technology allowing direct visualization of different types of DNA lesions in human cells and the emerging significance of live-cell imaging for elucidation of the spatio-temporal characterization of complex DNA damage are highlighted.
Abstract: Low-linear energy transfer (LET) radiation (i.e., γ- and X-rays) induces DNA double-strand breaks (DSBs) that are rapidly repaired (rejoined). In contrast, DNA damage induced by the dense ionizing track of high-atomic number and energy (HZE) particles is slowly repaired or is irreparable. These unrepaired and/or misrepaired DNA lesions may contribute to the observed higher relative biological effectiveness for cell killing, chromosomal aberrations, mutagenesis, and carcinogenesis in HZE particle irradiated cells compared to those treated with low-LET radiation. The types of DNA lesions induced by HZE particles have been characterized in vitro and usually consist of two or more closely spaced strand breaks, abasic sites, or oxidized bases on opposing strands. It is unclear why these lesions are difficult to repair. In this review, we highlight the potential of a new technology allowing direct visualization of different types of DNA lesions in human cells and document the emerging significance of live-cell imaging for elucidation of the spatio-temporal characterization of complex DNA damage. We focus on the recent insights into the molecular pathways that participate in the repair of HZE particle-induced DSBs. We also discuss recent advances in our understanding of how different end-processing nucleases aid in repair of DSBs with complicated ends generated by HZE particles. Understanding the mechanism underlying the repair of DNA damage induced by HZE particles will have important implications for estimating the risks to human health associated with HZE particle exposure.
TL;DR: Each of these three pathways for repairing DNA double-strand breaks in mammalian cells is reviewed with an emphasis on the role of the DNA-dependent protein kinase, a critical component of the non-homologous end joining pathway, in influencing which pathway is ultimately utilized for repair.
Abstract: DNA double-strand breaks are extremely harmful lesions that can lead to genomic instability and cell death if not properly repaired. There are at least three pathways that are responsible for repairing DNA double-strand breaks in mammalian cells: non-homologous end joining, homologous recombination and alternative non-homologous end joining. Here we review each of these three pathways with an emphasis on the role of the DNA-dependent protein kinase, a critical component of the non-homologous end joining pathway, in influencing which pathway is ultimately utilized for repair.
TL;DR: Results demonstrated that MWCNTs could induce cytotoxic and genotoxic effects in HUVECs, probably through oxidative damage pathways.
Abstract: Carbon nanomaterials have multiple applications in various areas. However, it has been suggested that exposure to nanoparticles may be a risk for the development of vascular diseases due to injury and dysfunction of the vascular endothelium. Therefore, in the present study, the cytotoxic and genotoxic effects of multi-wall carbon nanotubes (MWCNTs) on human umbilical vein endothelial cells (HUVECs) were evaluated. Optical and transmission electronic microscopy (TEM) study showed that MWCNTs were able to enter cells rapidly, distribute in the cytoplasm and intracellular vesicles and induce morphological changes. Exposure to MWCNTs reduced the viability of HUVECs, and induced apoptosis in HUVECs. Furthermore, MWCNTs could cause DNA damage as indicated by the formation of γH2AX foci. MWCNTs also affected cellular redox status, e.g., increasing intracellular reactive oxygen species (ROS) and malondialdehyde (MDA) levels, as well as altering superoxide dismutase (SOD) activity and glutathione peroxidase (GSH-Px) levels. On the other hand, the free radical scavenger N-acetyl-l-cysteine (NAC) preincubation can inhibit the cytotoxic and genotoxic effects of MWCNTs. Taken together, these results demonstrated that MWCNTs could induce cytotoxic and genotoxic effects in HUVECs, probably through oxidative damage pathways.
TL;DR: The results clearly indicate that lead induced DNA fragmentation in a dose-dependant manner with a maximum effect at 10μM, which reveals the major role of reactive oxygen species (ROS) in the genotoxicity of lead.
Abstract: Genotoxic effects of lead (0-20μM) were investigated in whole-plant roots of Vicia faba L., grown hydroponically under controlled conditions. Lead-induced DNA damage in V. faba roots was evaluated by use of the comet assay, which allowed the detection of DNA strand-breakage and with the V. faba micronucleus test, which revealed chromosome aberrations. The results clearly indicate that lead induced DNA fragmentation in a dose-dependant manner with a maximum effect at 10μM. In addition, at this concentration, DNA damage time-dependently increased until 12h. Then, a decrease in DNA damages was recorded. The significant induction of micronucleus formation also reinforced the genotoxic character of this metal. Direct interaction of lead with DNA was also evaluated with the a-cellular comet assay. The data showed that DNA breakages were not associated with a direct effect of lead on DNA. In order to investigate the relationship between lead genotoxicity and oxidative stress, V. faba were exposed to lead in the presence or absence of the antioxidant Vitamin E, or the NADPH-oxidase inhibitor dephenylene iodonium (DPI). The total inhibition of the genotoxic effects of lead (DNA breakage and micronucleus formation) by these compounds reveals the major role of reactive oxygen species (ROS) in the genotoxicity of lead. These results highlight, for the first time in vivo and in whole-plant roots, the relationship between ROS, DNA strand-breaks and chromosome aberrations induced by lead.
TL;DR: These effects are caused by a secondary genotoxic mechanism associated with inflammation and/or oxidative stress, and indicate a potential health hazard associated with exposure to TiO(2) particles.
Abstract: Titanium dioxide is manufactured worldwide in large quantities for use in a wide range of applications including as food additives, in cosmetics and pigments for coloring ingested and externally applied drugs. Although TiO(2) is chemically inert it can cause negative health effects, such as lung cancer in rats. However, the mechanisms involved in TiO(2)-induced genotoxicity and carcinogenicity have not been clearly defined and are poorly studied in vivo. In the present research genotoxicity and carcinogenicity of titanium dioxide were studied in a mouse model. We treated CBAB6F1 mice by oral gavage with titanium dioxide particles (microsized, TDM, 160nm; nanosized, TDN, 33nm) in doses of 40, 200 and 1000mg/kg bw, daily for seven days. Genotoxic effects were analyzed in the cells of brain, liver and bone marrow by means of the Comet assay and in the cells of bone marrow, forestomach, colon and testis with a poly-organ karyological assay (analysis of micronuclei, nuclear protrusions, atypical nuclei, multinucleated cells, mitotic and/or apoptotic index). TDM induced DNA-damage and micronuclei in bone-marrow cells and TDN induced DNA-damage in the cells of bone marrow and liver. TDM and TDN increased the mitotic index in forestomach and colon epithelia, the frequency of spermatids with two and more nuclei, and apoptosis in forestomach (only TDN) and testis. This is one of the first poly-organ studies of TDM- and TDN-induced genotoxicity in vivo in mice. These effects are caused by a secondary genotoxic mechanism associated with inflammation and/or oxidative stress. Given the increasing use of TiO(2) nanoparticles, these findings indicate a potential health hazard associated with exposure to TiO(2) particles.
TL;DR: Enzymatic methods that are based on the use of DNA repair glycosylases in order to convert oxidized bases into strand breaks are suitable, even if they are far less specific than HPLC methods, to deal with low levels of single modifications.
Abstract: This survey focuses on the critical evaluation of the main methods that are currently available for monitoring single and complex oxidatively generated damage to cellular DNA. Among chromatographic methods, HPLC-ESI-MS/MS and to a lesser extent HPLC-ECD which is restricted to a few electroactive nucleobases and nucleosides are appropriate for measuring the formation of single and clustered DNA lesions. Such methods that require optimized protocols for DNA extraction and digestion are sensitive enough for measuring base lesions formed under conditions of severe oxidative stress including exposure to ionizing radiation, UVA light and high intensity UVC laser pulses. In contrast application of GC-MS and HPLC-MS methods that are subject to major drawbacks have been shown to lead to overestimated values of DNA damage. Enzymatic methods that are based on the use of DNA repair glycosylases in order to convert oxidized bases into strand breaks are suitable, even if they are far less specific than HPLC methods, to deal with low levels of single modifications. Several other methods including immunoassays and (32)P-postlabeling methods that are still used suffer from drawbacks and therefore are not recommended. Another difficult topic is the measurement of oxidatively generated clustered DNA lesions that is currently achieved using enzymatic approaches and that would necessitate further investigations.
TL;DR: Results of validation studies that demonstrated correlation of manual focus counting with results obtained using the computational algorithm for mouse jejunum touch prints, mouse tongue sections and human blood lymphocytes as well as radiation dose response of γH2AX focus induction for these biological specimens are reported.
Abstract: The γH2AX focus assay represents a fast and sensitive approach for the detection of one of the critical types of DNA damage - double-strand breaks (DSB) induced by various cytotoxic agents including ionising radiation. Apart from research applications, the assay has a potential in clinical medicine/pathology, such as assessment of individual radiosensitivity, response to cancer therapies, as well as in biodosimetry. Given that generally there is a direct relationship between numbers of microscopically visualised γH2AX foci and DNA DSB in a cell, the number of foci per nucleus represents the most efficient and informative parameter of the assay. Although computational approaches have been developed for automatic focus counting, the tedious and time consuming manual focus counting still remains the most reliable way due to limitations of computational approaches. We suggest a computational approach and associated software for automatic focus counting that minimises these limitations. Our approach, while using standard image processing algorithms, maximises the automation of identification of nuclei/cells in complex images, offers an efficient way to optimise parameters used in the image analysis and counting procedures, optionally invokes additional procedures to deal with variations in intensity of the signal and background in individual images, and provides automatic batch processing of a series of images. We report results of validation studies that demonstrated correlation of manual focus counting with results obtained using our computational algorithm for mouse jejunum touch prints, mouse tongue sections and human blood lymphocytes as well as radiation dose response of γH2AX focus induction for these biological specimens.
TL;DR: The range of B ER capacity among individuals and the functional consequences of BER genetic variants are described and studies that associate BER deficiency with disease risk are discussed, as well as the current state of BERN capacity measurement assays.
Abstract: The major DNA repair pathway for coping with spontaneous forms of DNA damage, such as natural hydrolytic products or oxidative lesions, is base excision repair (BER). In particular, BER processes mutagenic and cytotoxic DNA lesions such as non-bulky base modifications, abasic sites, and a range of chemically distinct single-strand breaks. Defects in BER have been linked to cancer predisposition, neurodegenerative disorders, and immunodeficiency. Recent data indicate a large degree of sequence variability in DNA repair genes and several studies have associated BER gene polymorphisms with disease risk, including cancer of several sites. The intent of this review is to describe the range of BER capacity among individuals and the functional consequences of BER genetic variants. We also discuss studies that associate BER deficiency with disease risk and the current state of BER capacity measurement assays.
TL;DR: This review summarizes the current knowledge regarding the repair and biochemical effects of the most ubiquitous form of DPCs, which are associated with no flanked DNA strand breaks.
Abstract: Genomic DNA is associated with various structural, regulatory, and transaction proteins. The dynamic and reversible association between proteins and DNA ensures the accurate expression and propagation of genetic information. However, various endogenous, environmental, and chemotherapeutic agents induce DNA-protein crosslinks (DPCs), and hence covalently trap proteins on DNA. Since DPCs are extremely large compared to conventional DNA lesions, they probably impair many aspects of DNA transactions such as replication, transcription, and repair due to steric hindrance. Recent genetic and biochemical studies have shed light on the elaborate molecular mechanism by which cells repair or tolerate DPCs. This review summarizes the current knowledge regarding the repair and biochemical effects of the most ubiquitous form of DPCs, which are associated with no flanked DNA strand breaks. In bacteria small DPCs are eliminated by nucleotide excision repair (NER), whereas oversized DPCs are processed by RecBCD-dependent homologous recombination (HR). NER does not participate in the repair of DPCs in mammalian cells, since the upper size limit of DPCs amenable to mammalian NER is smaller than that of bacterial NER. Thus, DPCs are processed exclusively by HR. The reactivation of the stalled replication fork at DPCs by HR seems to involve fork breakage in mammalian cells but not in bacterial cells. In addition, recent proteomic studies have identified the numbers of proteins in DPCs induced by environmental and chemotherapeutic agents. However, it remains largely elusive how DPCs affect replication and transcription at the molecular level. Considering the extremely large nature of DPCs, it is possible that they impede the progression of replication and transcription machineries by mechanisms different from those for conventional DNA lesions. This might also be true for the DNA damage response and signaling mechanism.
TL;DR: It is demonstrated that miRNA expression levels can be altered after X-ray irradiation and that these miRNA are involved in chromatin remodeling and DNA methylation, suggesting that epigenetic aberrations may arise in the cell without initiating chromosomal instability.
Abstract: Epigenetics, including DNA methylation and microRNA (miRNA) expression, could be the missing link in understanding radiation-induced genomic instability (RIGI). This study tests the hypothesis that irradiation induces epigenetic aberrations, which could eventually lead to RIGI, and that the epigenetic aberrations induced by low linear energy transfer (LET) irradiation are different than those induced by high LET irradiations. GM10115 cells were irradiated with low LET X-rays and high LET iron (Fe) ions and evaluated for DNA damage, cell survival and chromosomal instability. The cells were also evaluated for specific locus methylation of nuclear factor-kappa B (NFκB), tumor suppressor in lung cancer 1 (TSLC1) and cadherin 1 (CDH1) gene promoter regions, long interspersed nuclear element 1 (LINE-1) and Alu repeat element methylation, CpG and non-CpG global methylation and miRNA expression levels. Irradiated cells showed increased micronucleus induction and cell killing immediately following exposure, but were chromosomally stable at delayed times post-irradiation. At this same delayed time, alterations in repeat element and global DNA methylation and miRNA expression were observed. Analyses of DNA methylation predominantly showed hypomethylation, however hypermethylation was also observed. We demonstrate that miRNA expression levels can be altered after X-ray irradiation and that these miRNA are involved in chromatin remodeling and DNA methylation. A higher incidence of epigenetic changes was observed after exposure to X-rays than Fe ions even though Fe ions elicited more chromosomal damage and cell killing. This distinction is apparent at miRNA analyses at which only three miRNA involved in two major pathways were altered after high LET irradiations while six miRNA involved in five major pathways were altered after low LET irradiations. This study also shows that the irradiated cells acquire epigenetic changes suggesting that epigenetic aberrations may arise in the cell without initiating chromosomal instability.
TL;DR: It is demonstrated how small changes in comet-assay variables may significantly affect the results, by use of two cell types, viz. human peripheral blood lymphocytes and the lymphoblastoid cell line TK-6.
Abstract: The comet assay is now the method of choice for measuring most kinds of DNA damage in cells. However, due to the lack of a standardised protocol inter-laboratory comparisons are of limited value. The aim of this paper is to demonstrate how small changes in comet-assay variables may significantly affect the results. We examined the effect of varying agarose concentrations, alkaline unwinding time, electrophoresis time, voltage and current, by use of two cell types, viz. human peripheral blood lymphocytes and the lymphoblastoid cell line TK-6. All these variables have marked effects on assay performance and, therefore, on the determination of DNA damage. Here we identify factors of particular importance.
TL;DR: It appears that microRNAs mainly behave as biomarkers of effect and that exposure to high-dose, lasting for an adequate period of time, is needed to trigger the CS-related carcinogenesis process in the experimental animal model used.
Abstract: Our previous studies demonstrated that exposure to cigarette smoke (CS), either mainstream or environmental, results in a remarkable downregulation of microRNA expression in the lung of both mice and rats. The goals of the present study were to evaluate the dose responsiveness to CS and the persistence of microRNA alterations after smoking cessation. ICR (CD-1) neonatal mice were exposed whole-body to mainstream CS, at the doses of 119, 292, 438, and 631mg/m(3) of total particulate matter. Exposure started within 12h after birth and continued daily for 4 weeks. The levels of bulky DNA adducts and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) were measured by (32)P postlabeling procedures, and the expression of 697 mouse microRNAs was analyzed by microarray. The highest CS dose was lethal. Exposure to CS caused a dose-dependent increase of DNA alterations. DNA adducts and, even more sharply, 8-oxodGuo were reverted 1 and 4 weeks after smoking cessation. Exposure to CS resulted in an evident dysregulation of microRNA expression profiles, mainly in the sense of downregulation. The two lowest doses were not particularly effective, while the highest nonlethal dose produced extensive microRNA alterations. The expression of most downregulated microRNAs, including among others 7 members of the let-7 family, was restored one week after smoking cessation. However, the recovery was incomplete for a limited array of microRNAs, including mir-34b, mir-345, mir-421, mir-450b, mir-466, and mir-469. Thus, it appears that microRNAs mainly behave as biomarkers of effect and that exposure to high-dose, lasting for an adequate period of time, is needed to trigger the CS-related carcinogenesis process in the experimental animal model used.