RESUMO
Endogenous electrophiles, ionizing and non-ionizing radiation, and hazardous chemicals present in the environment and diet can damage DNA by forming covalent adducts. DNA adducts can form in critical cancer driver genes and, if not repaired, may induce mutations during cell division, potentially leading to the onset of cancer. The detection and quantification of specific DNA adducts are some of the first steps in studying their role in carcinogenesis, the physiological conditions that lead to their production, and the risk assessment of exposure to specific genotoxic chemicals. Hundreds of different DNA adducts have been reported in the literature, and there is a critical need to establish a DNA adduct mass spectral database to facilitate the detection of previously observed DNA adducts and characterize newly discovered DNA adducts. We have collected synthetic DNA adduct standards from the research community, acquired MSn (n = 2, 3) fragmentation spectra using Orbitrap and Quadrupole-Time-of-Flight (Q-TOF) MS instrumentation, processed the spectral data and incorporated it into the MassBank of North America (MoNA) database, and created a DNA adduct portal Web site (https://sites.google.com/umn.edu/dnaadductportal) to serve as a central location for the DNA adduct mass spectra and metadata, including the spectral database downloadable in different formats. This spectral library should prove to be a valuable resource for the DNA adductomics community, accelerating research and improving our understanding of the role of DNA adducts in disease.
Assuntos
Adutos de DNA , DNA , Humanos , DNA/química , Espectrometria de Massas , Dano ao DNA , CarcinogêneseRESUMO
Exposure to the physicochemical agents that interact with nucleic acids (NA) may lead to modification of DNA and RNA (i.e., NA modifications), which have been associated with various diseases, including cancer. The emerging field of NA adductomics aims to identify both known and unknown NA modifications, some of which may also be associated with proteins. One of the main challenges for adductomics is the processing of massive and complex data generated by high-resolution tandem mass spectrometry (HR-MS/MS). To address this, we have developed a software called "FeatureHunter", which provides the automated extraction, annotation, and classification of different types of key NA modifications based on the MS and MS/MS spectra acquired by HR-MS/MS, using a user-defined feature list. The capability and effectiveness of FeatureHunter was demonstrated by analyzing various NA modifications induced by formaldehyde or chlorambucil in mixtures of calf thymus DNA, yeast RNA and proteins, and by analyzing the NA modifications present in the pooled urines of smokers and nonsmokers. The incorporation of FeatureHunter into the NA adductomics workflow offers a powerful tool for the identification and classification of various types of NA modifications induced by reactive chemicals in complex biological samples, providing a valuable resource for studying the exposome.
Assuntos
Expossoma , Ácidos Nucleicos , Espectrometria de Massas em Tandem/métodos , Adutos de DNA , Fluxo de Trabalho , Software , RNARESUMO
A comet assay is a trusted and widely used method for assessing DNA damage in individual eukaryotic cells. However, it is time-consuming and requires extensive monitoring and sample manipulation by the user. This limits the throughput of the assay, increases the risk of errors, and contributes to intra- and inter-laboratory variability. Here, we describe the development of a device which automates high throughput sample processing for a comet assay. This device is based upon our patented, high throughput, vertical comet assay electrophoresis tank, and incorporates our novel, patented combination of assay fluidics, temperature control, and a sliding electrophoresis tank to facilitate sample loading and removal. Additionally, we demonstrated that the automated device performs at least as well as our "manual" high throughput system, but with all the advantages of a fully "walkaway" device, such as a decreased need for human involvement and a decreased assay run time. Our automated device represents a valuable, high throughput approach for reliably assessing DNA damage with the minimal operator involvement, particularly if combined with the automated analysis of comets.
Assuntos
Dano ao DNA , Células Eucarióticas , Humanos , Ensaio Cometa/métodosRESUMO
Exposures from the external and internal environments lead to the modification of genomic DNA, which is implicated in the cause of numerous diseases, including cancer, cardiovascular, pulmonary and neurodegenerative diseases, together with ageing. However, the precise mechanism(s) linking the presence of damage, to impact upon cellular function and pathogenesis, is far from clear. Genomic location of specific forms of damage is likely to be highly informative in understanding this process, as the impact of downstream events (e.g. mutation, microsatellite instability, altered methylation and gene expression) on cellular function will be positional-events at key locations will have the greatest impact. However, until recently, methods for assessing DNA damage determined the totality of damage in the genomic location, with no positional information. The technique of "mapping DNA adductomics" describes the molecular approaches that map a variety of forms of DNA damage, to specific locations across the nuclear and mitochondrial genomes. We propose that integrated comparison of this information with other genome-wide data, such as mutational hotspots for specific genotoxins, tumour-specific mutation patterns and chromatin organisation and transcriptional activity in non-cancerous lesions (such as nevi), pre-cancerous conditions (such as polyps) and tumours, will improve our understanding of how environmental toxins lead to cancer. Adopting an analogous approach for non-cancer diseases, including the development of genome-wide assays for other cellular outcomes of DNA damage, will improve our understanding of the role of DNA damage in pathogenesis more generally.
Assuntos
Dano ao DNA/genética , DNA/genética , Genoma/genética , Animais , Mapeamento Cromossômico/métodos , Estudo de Associação Genômica Ampla/métodos , Genômica/métodos , Humanos , Mutação/genética , Neoplasias/genéticaRESUMO
The DNA of all living cells undergoes continuous structural and chemical alteration, which may be derived from exogenous sources, or endogenous, metabolic pathways, such as cellular respiration, replication and DNA demethylation. It has been estimated that approximately 70,000 DNA lesions may be generated per day in a single cell, and this has been linked to a wide variety of diseases, including cancer. However, it is puzzling why potentially mutagenic DNA modifications, occurring at a similar level in different organs/tissue, may lead to organ/tissue specific cancers, or indeed non-malignant disease - what is the basis for this differential response? We suggest that it is perhaps the precise location of damage, within the genome, that is a key factor. Finally, we draw attention to the requirement for reliable methods for identification and quantification of DNA adducts/modifications, and stress the need for these assays to be fully validated. Once these prerequisites are satisfied, measurement of DNA modifications may be helpful as a clinical parameter for treatment monitoring, risk group identification and development of prevention strategies.
Assuntos
Antineoplásicos/uso terapêutico , Biomarcadores Tumorais , Transformação Celular Neoplásica , DNA/metabolismo , Mutagênese/genética , Neoplasias/terapia , Animais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/genética , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/genética , Predisposição Genética para Doença , Humanos , Mutagênese/efeitos dos fármacos , Neoplasias/genética , Neoplasias/patologia , Fatores de RiscoRESUMO
Mass spectrometry-based DNA adductomics is an emerging approach for the human biomonitoring of hazardous chemicals. A mass spectral database of DNA adducts will be created for the scientific community to investigate the associations between chemical exposures, DNA damage, and disease risk.
Assuntos
Adutos de DNA/efeitos dos fármacos , Bases de Dados de Compostos Químicos , Poluentes Ambientais/farmacologia , Compostos Orgânicos/farmacologia , Dano ao DNA , Poluentes Ambientais/química , Humanos , Espectrometria de Massas , Compostos Orgânicos/químicaRESUMO
DNA-DNA crosslinks, especially interstrand crosslinks (ICLs), cause cytotoxicity via blocking replication and transcription. Most measurements of ICLs lack sensitivity and structural information. Here, a high resolution, accurate mass spectrometry (HRMS) method was developed to comprehensively determine the untargeted, totality of DNA crosslinks, a.k.a. DNA crosslinkomics. Two novel features were introduced into this method: the accurate mass neutral losses of both two 2-deoxyribose (dR) and one dR groups will screen for ICLs as modified dinucleosides; the accurate mass neutral losses of both of the two nucleobases and one nucleobase will detect unstable DNA crosslinks, that could undergo depurination. Our crosslinkomics approach was tested by screening for crosslinks in formaldehyde- and chlorambucil-treated calf thymus DNA. The results showed that all expected drug-bridged crosslinks were detected successfully, along with various unexpected crosslinks. Using HRMS, the molecular formula and chemical structures of these unexpected crosslinks were determined. The formation of apurinic/apyrimidinic (AP) site-derived crosslinks, at levels comparable to those for drug-bridged crosslinks, highlighted their novel, potential role in cytotoxicity. Our new crosslinkomics approach can detect expected and unexpected environmental and drug-induced crosslinks in biological samples. This broadens the existing cellular DNA adductome and offers the potential to become a powerful tool in precision medicine.
Assuntos
Reagentes de Ligações Cruzadas/química , DNA/química , Animais , Bovinos , Clorambucila/química , Cromatografia Líquida , Formaldeído/química , Espectrometria de MassasRESUMO
Single cell gel electrophoresis, also known as the comet assay, has become a widespread DNA damage assessment tool due to its sensitivity, adaptability, low cost, ease of use, and reliability. Despite these benefits, this assay has shortcomings, such as long assay running time, the manipulation of multiple slides, individually, through numerous process steps, the challenge of working in a darkened environment, and reportedly considerable inter- and intra-laboratory variation. All researchers typically perform the comet assay based upon a common core approach; however, it appears that some steps in this core have little proven basis, and may exist, partly, out of convenience, or dogma. The aim of this study was to critically re-evaluate key steps in the comet assay, using our laboratory's protocol as a model, firstly to understand the scientific basis for why certain steps in the protocol are performed in a particular manner, and secondly to simplify the assay, and decrease the cost and run time. Here, the shelf life of the lysis and neutralization buffers, the effect of temperature and incubation period during the lysis step, the necessity for drying the slides between the electrophoresis and staining step, and the need to perform the sample workup and electrophoresis steps under subdued light were all evaluated.
Assuntos
Ensaio Cometa/métodos , Monitoramento Ambiental/métodos , Análise de Célula Única/métodos , Dano ao DNA/genética , Humanos , Laboratórios/normas , TemperaturaRESUMO
The distribution of DNA damage and repair is considered to occur heterogeneously across the genome. However, commonly available techniques, such as the alkaline comet assay or HPLC-MS/MS, measure global genome levels of DNA damage, and do not reflect potentially significant events occurring at the gene/sequence-specific level, in the nuclear or mitochondrial genomes. We developed a method, which comprises a combination of Damaged DNA Immunoprecipitation and next generation sequencing (DDIP-seq), to assess the induction and repair of DNA damage induced by 0.1 J/cm2 solar-simulated radiation at the sequence-specific level, across both the entire nuclear and mitochondrial genomes. DDIP-seq generated a genome-wide, high-resolution map of cyclobutane thymine dimer (T<>T) location and intensity. In addition to being a straightforward approach, our results demonstrated a clear differential distribution of T<>T induction and loss, across both the nuclear and mitochondrial genomes. For nuclear DNA, this differential distribution existed at both the sequence and chromosome level. Levels of T<>T were much higher in the mitochondrial DNA, compared to nuclear DNA, and decreased with time, confirmed by qPCR, despite no reported mechanisms for their repair in this organelle. These data indicate the existence of regions of sensitivity and resistance to damage formation, together with regions that are fully repaired, and those for which > 90% of damage remains, after 24 h. This approach offers a simple, yet more detailed approach to studying cellular DNA damage and repair, which will aid our understanding of the link between DNA damage and disease.
Assuntos
Ciclobutanos/química , Heterogeneidade Genética , Genoma Mitocondrial , Estudo de Associação Genômica Ampla , Genoma , Dímeros de Pirimidina/química , Sobrevivência Celular/genética , Dano ao DNA , Reparo do DNA , Sequenciamento de Nucleotídeos em Larga EscalaRESUMO
Adductomics is expected to be useful in the characterization of the exposome, which is a new paradigm for studying the sum of environmental causes of diseases. DNA adductomics is emerging as a powerful method for detecting DNA adducts, but reliable assays for its widespread, routine use are currently lacking. We propose a novel integrated strategy for the establishment of a DNA adductomic approach, using liquid chromatography-triple quadrupole tandem mass spectrometry (LC-QqQ-MS/MS), operating in constant neutral loss scan mode, screening for both known and unknown DNA adducts in a single injection. The LC-QqQ-MS/MS was optimized using a representative sample of 23 modified 2'-deoxyribonucleosides reflecting a range of biologically relevant DNA lesions. Six internal standards (ISTDs) were evaluated for their ability to normalize, and hence correct, possible variation in peak intensities arising from matrix effects, and the quantities of DNA injected. The results revealed that, with appropriate ISTDs adjustment, any bias can be dramatically reduced from 370 to 8.4%. Identification of the informative DNA adducts was achieved by triggering fragmentation spectra of target ions. The LC-QqQ-MS/MS method was successfully applied to in vitro and in vivo studies to screen for DNA adducts formed following representative environmental exposures: methyl methanesulfonate (MMS) and five N-nitrosamines. Interestingly, five new DNA adducts, induced by MMS, were discovered using our adductomic approach-an added strength. The proposed integrated strategy provides a path forward for DNA adductomics to become a standard method to discover differences in DNA adduct fingerprints between populations exposed to genotoxins, and facilitate the field of exposomics.
Assuntos
Cromatografia Líquida/métodos , Adutos de DNA/análise , Exposição Ambiental/análise , Espectrometria de Massas em Tandem/métodos , Animais , Adutos de DNA/química , Adutos de DNA/urina , Masculino , Metanossulfonato de Metila/toxicidade , Camundongos Endogâmicos ICR , Nitrosaminas/toxicidadeRESUMO
8-Oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) is the most investigated product of oxidatively damaged DNA lesion that has been associated with the development of aging, cancer and some degenerative diseases. Here, we present the first liquid chromatography-tandem mass spectrometry method that enables the simultaneous measurement of its repair products in plasma and saliva, namely 8-oxo-7,8-dihydroguanine (8-oxoGua) and 8-oxodGuo. Using this method, we investigated the underlying transport mechanism of the repair products of oxidatively damaged DNA between cellular compartments and biological matrices. Plasma, saliva and urine samples were collected concurrently from 57 healthy subjects. Various deproteinization methods were evaluated, and the precipitants acetonitrile and sodium hydroxide-methanol were, respectively, selected for plasma and saliva samples due to their effect on recovery efficiencies and chromatography. The mean baseline concentrations of 8-oxoGua and 8-oxodGuo in plasma were demonstrated to be 0.21 and 0.016 ng/mL, respectively, while in saliva they were 0.85 and 0.010 ng/mL, respectively. A relatively high concentration of 8-oxoGua was found in saliva with a concentration factor (CF, concentration ratio of saliva to plasma) of 4 as compared to that of 8-oxodGuo (CF: 0.6), implying that 8-oxoGua in plasma may be actively transported to saliva, whereas 8-oxodGuo was most dependent on a passive diffusion. Good correlations between urine and plasma concentrations were observed for 8-oxoGua and 8-oxodGuo, suggesting that blood was a suitable matrix in addition to urine. Significant correlation between 8-oxoGua and 8-oxodGuo in urine was only observed when the concentrations were not corrected for urinary creatinine, raising the issue of applicability of urinary creatinine to adjust 8-oxoGua concentrations.
Assuntos
Desoxiadenosinas/análise , Guanina/análogos & derivados , Adulto , Cromatografia Líquida , Desoxiadenosinas/sangue , Desoxiadenosinas/urina , Guanina/análise , Guanina/sangue , Guanina/urina , Humanos , Saliva/química , Extração em Fase Sólida , Espectrometria de Massas em TandemRESUMO
This study investigated the levels of DNA strand breaks and formamidopyrimidine DNA glycosylase (FPG) sensitive sites, as assessed by the comet assay, in peripheral blood mononuclear cells (PBMC) from healthy women from five different countries in Europe. The laboratory in each country (referred to as 'centre') collected and cryopreserved PBMC samples from three donors, using a standardised cell isolation protocol. The samples were analysed in 13 different laboratories for DNA damage, which is measured by the comet assay. The study aim was to assess variation in DNA damage in PBMC samples that were collected in the same way and processed using the same blood isolation procedure. The inter-laboratory variation was the prominent contributor to the overall variation. The inter-laboratory coefficient of variation decreased for both DNA strand breaks (from 68 to 26%) and FPG sensitive sites (from 57 to 12%) by standardisation of the primary comet assay endpoint with calibration curve samples. The level of DNA strand breaks in the samples from two of the centres (0.56-0.61 lesions/10(6) bp) was significantly higher compared with the other three centres (0.41-0.45 lesions/10(6) bp). In contrast, there was no difference between the levels of FPG sensitive sites in PBMC samples from healthy donors in the different centres (0.41-0.52 lesion/10(6) bp).
Assuntos
Separação Celular/métodos , Dano ao DNA , Laboratórios , Leucócitos Mononucleares/metabolismo , Adulto , Calibragem , Ensaio Cometa , Quebras de DNA de Cadeia Dupla , DNA-Formamidopirimidina Glicosilase/metabolismo , Feminino , Humanos , Pessoa de Meia-Idade , Testes de Mutagenicidade , Análise de RegressãoRESUMO
Elevated genomic instability in cancer cells suggests a possible model-scenario for their selective killing via the therapeutic delivery of well-defined levels of further DNA damage. To examine this scenario, this study investigated the potential for redox modulation of oxidatively-induced DNA damage by ascorbate in malignant melanoma (MM) cancer cells, to selectively enhance both DNA damage and MM cell killing. DNA damage was assessed by Comet and ɣH2AX assays, intracellular oxidising species by dichlorofluorescein fluorescence, a key antioxidant enzymatic defence by assessment of catalase activity and cell survival was determined by clonogenic assay. Comet revealed that MM cells had higher endogenous DNA damage levels than normal keratinocytes (HaCaT cells); this correlated MM cells having higher intracellular oxidising species and lower catalase activity, and ranked with MM cell melanin pigmentation. Comet also showed MM cells more sensitive towards the DNA damaging effects of exogenous H2O2, and that ascorbate further enhanced this H2O2-induced damage in MM cells; again, with MM cell sensitivity to induced damage ranking with degree of cell pigmentation. Furthermore, cell survival data indicated that ascorbate enhanced H2O2-induced clonogenic cell death selectively in MM cells whilst protecting HaCaT cells. Finally, we show that ascorbate serves to enhance the oxidising effects of the MM therapeutic drug Elesclomol in both established MM cells in vitro and primary cell cultures ex vivo. Together, these results suggest that ascorbate selectively enhances DNA damage and cell-killing in MM cells. This raises the option of incorporating ascorbate into clinical oxidative therapies to treat MM.
Assuntos
Melanoma , Estresse Oxidativo , Humanos , Catalase/metabolismo , Peróxido de Hidrogênio/farmacologia , Peróxido de Hidrogênio/metabolismo , Melanoma/tratamento farmacológico , Melanoma/genética , Ácido Ascórbico/farmacologia , Antioxidantes/farmacologia , Antioxidantes/metabolismo , Oxirredução , Morte Celular , Dano ao DNARESUMO
The concept of the exposome is the encompassing of all the environmental exposures, both exogenous and endogenous, across the life course. Many, if not all, of these exposures can result in the generation of reactive species, and/or the modulation of cellular processes, that can lead to a breadth of modifications of DNA, the nature of which may be used to infer their origin. Because of their role in cell function, such modifications have been associated with various major human diseases, including cancer, and so their assessment is crucial. Historically, most methods have been able to only measure one or a few DNA modifications at a time, limiting the information available. With the development of DNA adductomics, which aims to determine the totality of DNA modifications, a far more comprehensive picture of the DNA adduct burden can be gained. Importantly, DNA adductomics can facilitate a "top-down" investigative approach whereby patterns of adducts may be used to trace and identify the originating exposure source. This, together with other 'omic approaches, represents a major tool for unraveling the complexities of the exposome and hence allow a better a understanding of the environmental origins of disease.
Assuntos
Biomarcadores , Adutos de DNA , Exposição Ambiental , Expossoma , Humanos , Animais , DNARESUMO
ELISA is widely used for urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) analysis. It is the method of choice for laboratories that lack specialized chromatographic instrumentation. It allows fast, high-throughput sample analysis without a need for extensive samples processing. However, a lack of agreement between ELISA and chromatographic methods confines its application to the assessment of relative urinary 8-oxodG levels. We investigated various ELISA modifications, seeking optimal conditions that would yield a good agreement between ELISA and high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS /MS). Purification of urine by solid phase extraction (SPE), then incubation with the anti-8-oxodG antibody at 4 °C overnight and subsequent normalization of 8-oxodG levels per urinary creatinine resulted in a near-perfect correlation and agreement in mean levels between ELISA and HPLC-MS /MS (r=0.917, p<0.001; and paired t-test p=0.803, respectively). Our data show that, after introduction of a simple modification, ELISA quantification urinary 8-oxodG substantially improves. Although more sample manipulation is required, the method retains its key advantages over chromatography (high-throughput analysis that does not require expensive instrumentation). This represents a significant advance for the ELISA, and encouraging its use in more studies adding to our knowledge of the role of this biomarker of oxidative stress in health and disease.
Assuntos
Desoxiguanosina/análogos & derivados , Ensaio de Imunoadsorção Enzimática/normas , 8-Hidroxi-2'-Desoxiguanosina , Adulto , Biomarcadores/urina , Cromatografia Líquida de Alta Pressão/normas , Desoxiguanosina/urina , Humanos , Masculino , Pessoa de Meia-Idade , Estresse Oxidativo , Sensibilidade e Especificidade , Extração em Fase Sólida , Espectrometria de Massas em Tandem/normasRESUMO
The alkaline comet assay is an established, sensitive method extensively used in biomonitoring studies. This method can be modified to measure a range of different types of DNA damage. However, considerable differences in the protocols used by different research groups affect the inter-laboratory comparisons of results. The aim of this study was to assess the inter-laboratory, intra-laboratory, sample and residual (unexplained) variations in DNA strand breaks and formamidopyrimidine DNA glycosylase (FPG)-sensitive sites measured by the comet assay by using a balanced Latin square design. Fourteen participating laboratories used their own comet assay protocols to measure the level of DNA strand breaks and FPG-sensitive sites in coded samples containing peripheral blood mononuclear cells (PBMC) and the level of DNA strand breaks in coded calibration curve samples (cells exposed to different doses of ionising radiation) on three different days of analysis. Eleven laboratories found dose-response relationships in the coded calibration curve samples on two or three days of analysis, whereas three laboratories had technical problems in their assay. In the coded calibration curve samples, the dose of ionising radiation, inter-laboratory variation, intra-laboratory variation and residual variation contributed to 60.9, 19.4, 0.1 and 19.5%, respectively, of the total variation. In the coded PBMC samples, the inter-laboratory variation explained the largest fraction of the overall variation of DNA strand breaks (79.2%) and the residual variation (19.9%) was much larger than the intra-laboratory (0.3%) and inter-subject (0.5%) variation. The same partitioning of the overall variation of FPG-sensitive sites in the PBMC samples indicated that the inter-laboratory variation was the strongest contributor (56.7%), whereas the residual (42.9%), intra-laboratory (0.2%) and inter-subject (0.3%) variations again contributed less to the overall variation. The results suggest that the variation in DNA damage, measured by comet assay, in PBMC from healthy subjects is assay variation rather than variation between subjects.
Assuntos
Ensaio Cometa , Quebras de DNA , DNA-Formamidopirimidina Glicosilase/metabolismo , Leucócitos Mononucleares/metabolismo , Adulto , Ensaio Cometa/métodos , Quebras de DNA/efeitos da radiação , Relação Dose-Resposta à Radiação , Feminino , Raios gama/efeitos adversos , Humanos , Leucócitos Mononucleares/efeitos da radiação , Pessoa de Meia-Idade , Reprodutibilidade dos TestesRESUMO
RATIONALE: Asthma is characterized by disordered airway physiology as a consequence of increased airway smooth muscle contractility. The underlying cause of this hypercontractility is poorly understood. OBJECTIVES: We sought to investigate whether the burden of oxidative stress in airway smooth muscle in asthma is heightened and mediated by an intrinsic abnormality promoting hypercontractility. METHODS: We examined the oxidative stress burden of airway smooth muscle in bronchial biopsies and primary cells from subjects with asthma and healthy controls. We determined the expression of targets implicated in the control of oxidative stress in airway smooth muscle and their role in contractility. MEASUREMENTS AND MAIN RESULTS: We found that the oxidative stress burden in the airway smooth muscle in individuals with asthma is heightened and related to the degree of airflow obstruction and airway hyperresponsiveness. This was independent of the asthmatic environment as in vitro primary airway smooth muscle from individuals with asthma compared with healthy controls demonstrated increased oxidative stress-induced DNA damage together with an increased production of reactive oxygen species. Genome-wide microarray of primary airway smooth muscle identified increased messenger RNA expression in asthma of NADPH oxidase (NOX) subtype 4. This NOX4 overexpression in asthma was supported by quantitative polymerase chain reaction, confirmed at the protein level. Airway smooth muscle from individuals with asthma exhibited increased agonist-induced contraction. This was abrogated by NOX4 small interfering RNA knockdown and the pharmacological inhibitors diphenyleneiodonium and apocynin. CONCLUSIONS: Our findings support a critical role for NOX4 overexpression in asthma in the promotion of oxidative stress and consequent airway smooth muscle hypercontractility. This implicates NOX4 as a potential novel target for asthma therapy.
Assuntos
Asma/enzimologia , Brônquios/fisiopatologia , Contração Muscular/fisiologia , Músculo Liso/fisiopatologia , NADPH Oxidases/metabolismo , Adulto , Biomarcadores/metabolismo , Western Blotting , Brônquios/enzimologia , Estudos de Casos e Controles , Dano ao DNA , Feminino , Citometria de Fluxo , Imunofluorescência , Humanos , Masculino , Pessoa de Meia-Idade , Músculo Liso/enzimologia , NADPH Oxidase 4 , Análise de Sequência com Séries de Oligonucleotídeos , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Superóxido Dismutase/metabolismoRESUMO
This Discussion article aims to explore the potential for a new generation of assay to emerge from cellular and urinary DNA adductomics which brings together DNA-RNA- and, to some extent, protein adductomics, to better understand the role of the exposome in environmental health. Components of the exposome have been linked to an increased risk of various, major diseases, and to identify the precise nature, and size, of risk, in this complex mixture of exposures, powerful tools are needed. Modification of nucleic acids (NA) is a key consequence of environmental exposures, and a goal of cellular DNA adductomics is to evaluate the totality of DNA modifications in the genome, on the basis that this will be most informative. Consequently, an approach which encompasses modifications of all nucleic acids (NA) would be potentially yet more informative. This article focuses on NA adductomics, which brings together the assessment of both DNA and RNA modifications, including modified (2'-deoxy)ribonucleosides (2'-dN/rN), modified nucleobases (nB), plus: DNA-DNA, RNA-RNA, DNA-RNA, DNA-protein, and RNA-protein crosslinks (DDCL, RRCL, DRCL, DPCL, and RPCL, respectively). We discuss the need for NA adductomics, plus the pros and cons of cellular vs. urinary NA adductomics, and present some evidence for the feasibility of this approach. We propose that NA adductomics provides a more comprehensive approach to the study of nucleic acid modifications, which will facilitate a range of advances, including the identification of novel, unexpected modifications e.g., RNA-RNA, and DNA-RNA crosslinks; key modifications associated with mutagenesis; agent-specific mechanisms; and adductome signatures of key environmental agents, leading to the dissection of the exposome, and its role in human health/disease, across the life course.