RESUMO
Mitochondrial DNA (mtDNA) has been demonstrated to be a reliable biomarker of UV-induced genetic damage in both animal and human skin. Properties of the mitochondrial genome which allow for its use as a biomarker of damage include its presence in multiple copies within a cell, its limited repair mechanisms, and its lack of protective histones. To measure UV-induced mtDNA damage (particularly in the form of strand breaks), real-time quantitative PCR (qPCR) is used, based on the observation that PCR amplification efficiency is decreased in the presence of high levels of damage. Here, we describe the measurement of UV-induced mtDNA damage which includes the extraction of cellular DNA, qPCR to determine the relative amount of mtDNA, qPCR to determine UV-induced damage within a long strand of mtDNA, and the verification of the amplification process using gel electrophoresis.
Assuntos
DNA Mitocondrial/análise , DNA Mitocondrial/efeitos da radiação , Eletroforese em Gel de Ágar/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Pele/efeitos da radiação , Biomarcadores/análise , Dano ao DNA , DNA Mitocondrial/isolamento & purificação , Marcadores Genéticos , Humanos , Raios Ultravioleta/efeitos adversosRESUMO
Mitochondrial DNA double-strand breaks (mtDSBs) are toxic lesions that compromise the integrity of mitochondrial DNA (mtDNA) and alter mitochondrial function1. Communication between mitochondria and the nucleus is essential to maintain cellular homeostasis; however, the nuclear response to mtDSBs remains unknown2. Here, using mitochondrial-targeted transcription activator-like effector nucleases (TALENs)1,3,4, we show that mtDSBs activate a type-I interferon response that involves the phosphorylation of STAT1 and activation of interferon-stimulated genes. After the formation of breaks in the mtDNA, herniation5 mediated by BAX and BAK releases mitochondrial RNA into the cytoplasm and triggers a RIG-I-MAVS-dependent immune response. We further investigated the effect of mtDSBs on interferon signalling after treatment with ionizing radiation and found a reduction in the activation of interferon-stimulated genes when cells that lack mtDNA are exposed to gamma irradiation. We also show that mtDNA breaks synergize with nuclear DNA damage to mount a robust cellular immune response. Taken together, we conclude that cytoplasmic accumulation of mitochondrial RNA is an intrinsic immune surveillance mechanism for cells to cope with mtDSBs, including breaks produced by genotoxic agents.
Assuntos
Quebras de DNA de Cadeia Dupla , DNA Mitocondrial/imunologia , Imunidade Inata/imunologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Linhagem Celular , Células Cultivadas , Quebras de DNA de Cadeia Dupla/efeitos da radiação , DNA Mitocondrial/efeitos da radiação , Humanos , Mitocôndrias/imunologia , Mitocôndrias/efeitos da radiação , Comunicação Parácrina , Radiação Ionizante , Transcrição Gênica , Ubiquitina-Proteína Ligases/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismoRESUMO
REV3L encodes a catalytic subunit of DNA polymerase zeta (Pol zeta) which is essential for the tolerance of DNA damage by inducing translesion synthesis (TLS). So far, the only Mendelian disease associated with REV3L was Moebius syndrome (3 patients with dominant REV3L mutations causing monoallelic loss-of-function were reported). We describe a homozygous ultra-rare REV3L variant (T2753R) identified with whole exome sequencing in a child without Moebius syndrome but with developmental delay, hypotrophy, and dysmorphic features who was born to healthy parents (heterozygous carriers of the variant). The variant affects the amino acid adjacent to functionally important KKRY motif. By introducing an equivalent mutation (S1192R) into the REV3 gene in yeasts, we showed that, whereas it retained residual function, it caused clear dysfunction of TLS in the nucleus and instability of mitochondrial genetic information. In particular, the mutation increased UV sensitivity measured by cell survival, decreased both the spontaneous (P < 0.005) and UV-induced (P < 0.0001) mutagenesis rates of nuclear DNA and increased the UV-induced mutagenesis rates of mitochondrial DNA (P < 0.0005). We propose that our proband is the first reported case of a REV3L associated disease different from Moebius syndrome both in terms of clinical manifestations and inheritance (autosomal recessive rather than dominant). KEY MESSAGES: First description of a human recessive disorder associated with a REV3L variant. A study in yeast showed that the variant affected the enzymatic function of the protein. In particular, it caused increased UV sensitivity and abnormal mutagenesis rates.
Assuntos
Proteínas de Ligação a DNA/genética , DNA Polimerase Dirigida por DNA/genética , Deficiências do Desenvolvimento/genética , Mutação de Sentido Incorreto , Neoplasias Primárias Múltiplas/genética , Síndromes Neoplásicas Hereditárias/genética , Nevo Pigmentado/genética , Mutação Puntual , Neoplasias Cutâneas/genética , Aldose-Cetose Isomerases/genética , Domínio Catalítico/genética , Pré-Escolar , DNA/metabolismo , DNA Fúngico/genética , DNA Mitocondrial/genética , DNA Mitocondrial/efeitos da radiação , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/fisiologia , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/fisiologia , Deficiências do Desenvolvimento/patologia , Feminino , Homozigoto , Humanos , Masculino , Síndrome de Möbius/genética , Modelos Moleculares , Mutagênese/efeitos da radiação , Linhagem , Fenótipo , Conformação Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/efeitos da radiação , Proteínas de Saccharomyces cerevisiae/genética , Relação Estrutura-Atividade , Raios Ultravioleta/efeitos adversos , Sequenciamento do ExomaRESUMO
The mitochondrial mutation T414G (mtDNAT414G) has been shown to accumulate in aged and sun-exposed skin. The human eye is also exposed to solar harmful rays. More precisely, the anterior structures of the eye (cornea, iris) filter UV rays and the posterior portion of the eye (retina) is exposed to visible light. These rays can catalyse mutations in mitochondrial DNA such as the mtDNAT414G, but the latter has never been investigated in the human ocular structures. In this study, we have developed a technique to precisely assess the occurrence of mtDNAT414G. Using this technique, we have quantified mtDNAT414G in different human ocular structures. We found an age-dependent accumulation of mtDNAT414G in the corneal stroma, the cellular layer conferring transparency and rigidity to the human cornea, and in the iris. Since cornea and iris are two anterior ocular structures exposed to solar UV rays, this suggests that the mtDNAT414G mutation is resulting from cumulative solar exposure and this could make the mtDNAT414G a good marker of solar exposure. We have previously shown that the mtDNACD4977 and mtDNA3895 deletions accumulate over time in photo-exposed ocular structures. With the addition of mtDNAT414G mutation, it becomes feasible to combine the levels of these different mtDNA mutations to obtain an accurate assessment of the solar exposure that an individual has accumulated during his/her lifetime.
Assuntos
Biomarcadores , DNA Mitocondrial/genética , DNA Mitocondrial/efeitos da radiação , Olho/efeitos da radiação , Mitocôndrias/efeitos da radiação , Mutação , Reação em Cadeia da Polimerase/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Envelhecimento/psicologia , Córnea/efeitos da radiação , Substância Própria/efeitos da radiação , Humanos , Iris/efeitos da radiação , Pessoa de Meia-Idade , Pele/efeitos da radiação , Raios Ultravioleta/efeitos adversosRESUMO
Forensic genotyping can be impeded by γ-irradiation of biological evidence in the event of radiological crime; that is, criminal activity involving radioactive material. Oxidative effects within the mitochondria of living cells elicits greater damage to mitochondrial DNA (mtDNA) than nuclear DNA (nuDNA) at low doses. This study presents a novel approach for the assessment of nuDNA versus mtDNA damage from a comparison of genotype and quantity data, while exploring likely mechanisms for differential damage after high doses of γ-irradiation. Liquid (hydrated) and dried (dehydrated) whole blood samples were exposed to high doses of γ-radiation (1-50 kilogray, kGy). The GlobalFiler PCR Amplification Kit was used to evaluate short tandem repeat (STR) genotyping efficacy and nuDNA degradation; a comparison was made to mtDNA degradation measured using real-time PCR assays. Each assay was normalized before comparison by calculation of integrity indices relative to unirradiated controls. Full STR profiles were attainable up to the highest dose, although DNA degradation was noticeable after 10 and 25 kGy for hydrated and dehydrated blood, respectively. This was manifested by heterozygote imbalance more than allele dropout. Degradation was greater for mtDNA than nuDNA, as well as for hydrated than dehydrated cells, after equivalent doses. Oxidative effects due to water radiolysis and mitochondrial function are dominant mechanisms of differential damage to nuDNA versus mtDNA after high-dose γ-irradiation. While differential DNA damage was reduced by cell desiccation, its persistence after drying indicates innate differences between nuDNA and mtDNA radioresistance and/or continued oxidative effects within the mitochondria.
Assuntos
Degradação Necrótica do DNA/efeitos da radiação , DNA Mitocondrial/efeitos da radiação , Raios gama , Genótipo , Impressões Digitais de DNA , Relação Dose-Resposta à Radiação , Humanos , Repetições de Microssatélites , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Proton beam therapy is widely used for treating brain tumor. Despite the efficacy of treatment, the use of this therapy has met some limitations associated with possible damage to normal brain tissues located beyond the tumor site. In this context, the exploration of the harmful effects of protons on the normal brain tissues is of particular interest. We have investigated changes in the total mitochondrial DNA (mtDNA) copy number and identified mtDNA mutant copies in three brain regions (the hippocampus, cortex and cerebellum) of rats after irradiation their whole-head with 150 MeV protons at doses of 3 and 5 Gy. The study was performed in 2-months old male Spraque Dawley rats (n = 5 each group). The mtDNA copy numbers were determined by real-time PCR. The level of mtDNA heteroplasmy was estimated using Surveyor nuclease technology. Our results show that after head exposure to protons, levels of mtDNA copy number in three rat brain regions increase significantly as the levels of mtDNA mutant copies increase. The most significant elevation is observed in the hippocampus. In conclusion, an increase in mtDNA mutant copies may contribute to mitochondrial dysfunction accompanied by increased oxidative stress in different brain regions and promote the development of neurodegenerative diseases and the induction of carcinogenesis.
Assuntos
Encéfalo/efeitos da radiação , DNA Mitocondrial/efeitos da radiação , Terapia com Prótons/efeitos adversos , Animais , Encéfalo/metabolismo , Cerebelo/efeitos da radiação , Córtex Cerebral/efeitos da radiação , Variações do Número de Cópias de DNA/genética , Hipocampo/efeitos da radiação , Masculino , Mitocôndrias/genética , Mutação/genética , Mutação/efeitos da radiação , Prótons/efeitos adversos , RatosRESUMO
This short review explores the utility and applications of CRISPR/Cas9 systems in radiobiology. Specifically, in the context of experimentally simulating genotoxic effects of Ionizing Radiation (IR) to determine the contributions from DNA targets and 'Complex Double-Stranded Breaks' (complex DSBs) to the IR response. To elucidate this objective, this review considers applications of CRISPR/Cas9 on nuclear DNA targets to recognize the respective 'nucleocentric' response. The article also highlights contributions from mitochondrial DNA (mtDNA) - an often under-recognized target in radiobiology. This objective requires accurate experimental simulation of IR-like effects and parameters with the CRISPR/Cas9 systems. Therefore, the role of anti-CRISPR proteins in modulating enzyme activity to simulate dose rate - an important factor in radiobiology experiments is an important topic of this review. The applications of auxiliary domains on the Cas9 nuclease to simulate oxidative base damage and multiple stressor experiments are also topics of discussion. Ultimately, incorporation of CRISPR/Cas9 experiments into computational parameters in radiobiology models of IR damage and shortcomings to the technology are discussed as well. Altogether, the simulation of IR parameters and lack of damage to non-DNA targets in the CRISPR/Cas9 system lends this rapidly emerging tool as an effective model of IR induced DNA damage. Therefore, this literature review ultimately considers the relevance of complex DSBs to radiobiology with respect to using the CRISPR/Cas9 system as an effective experimental tool in models of IR induced effects.
Assuntos
Sistemas CRISPR-Cas , Quebras de DNA de Cadeia Dupla , DNA/efeitos da radiação , Modelos Genéticos , Testes de Mutagenicidade/métodos , Proteína 9 Associada à CRISPR , DNA/metabolismo , Dano ao DNA , Reparo do DNA , DNA Mitocondrial/metabolismo , DNA Mitocondrial/efeitos da radiação , Humanos , Radiação IonizanteRESUMO
Radiobiology is moving towards a better understanding of the intercellular signaling that occurs upon radiation and how its effects relate to the dose applied. The mitochondrial role in orchestrating this biological response needs to be further explored. Cybrids (cytoplasmic hybrids) are useful cell models for studying the involvement of mitochondria in cellular processes. In the present study we used cybrid cell lines to investigate the role of mitochondria in the response to radiation exposure. Cybrid cell lines, derived from the osteosarcoma human cell line 143B, harboring, either wild-type mitochondrial DNA (Cy143Bwt), cells with mitochondria with mutated DNA that causes mitochondrial dysfunction (Cy143Bmut), as well as cells without mitochondrial DNA (mtDNA) (143B-Rho0), were irradiated with 0.2 Gy and 2.0 Gy. Evaluation of the non-targeted (or bystander) effects in non-irradiated cells were assessed by using conditioned media from the irradiated cells. DNA double stranded breaks were assessed with the γH2AX assay. Both directly irradiated cells and cells treated with the conditioned media, showed increased DNA damage. The effect of the irradiated cells media was different according to the cell line it derived from: from Cy143Bwt cells irradiated with 0.2 Gy (low dose) and from Cy143Bmut irradiated with 2.0 Gy (high dose) induced highest DNA damage. Notably, media obtained from cells without mtDNA, the143B-Rho0 cell line, produced no effect in DNA damage. These results point to a possible role of mitochondria in the radiation-induced non-targeted effects. Furthermore, it indicates that cybrid models are valuable tools for radiobiological studies.
Assuntos
Efeito Espectador , Quebras de DNA de Cadeia Dupla , Mitocôndrias/efeitos da radiação , Radiação Ionizante , Linhagem Celular Tumoral , DNA Mitocondrial/genética , DNA Mitocondrial/efeitos da radiação , Humanos , Mitocôndrias/genéticaRESUMO
The ability of solar ultraviolet (UV) to induce skin cancer and photoaging is well recognized. The effect of the infrared (IR) and visible light (Vis) components of solar radiation on skin and their interaction with UV is less well known. This study compared the effects of physiologically relevant doses of complete (UV + Vis + IR) solar-simulated light and its individual components on matched primary dermal fibroblasts and epidermal keratinocytes from human donors on three biomarkers of cellular damage (reactive oxygen species (ROS) generation, mitochondrial DNA (mtDNA), and nuclear DNA (nDNA) damage). There was a greater induction of ROS, mtDNA, and nDNA damage with the inclusion of the visible and IR components of solar-simulated light in primary fibroblast cells compared to primary keratinocytes (P < .001). Experiments using exposure to specific components of solar light alone or in combination showed that the UV, Vis, and IR components of solar light synergistically increased ROS generation in primary fibroblasts but not primary keratinocytes (P < .001). Skin cell lines were used to confirm these findings. These observations have important implications for different skin cell type responses to the individual and interacting components of solar light and therefore photodamage mechanisms and photoprotection interventions.
Assuntos
Biomarcadores/metabolismo , Raios Infravermelhos , Queratinócitos/efeitos da radiação , Luz , Pele/citologia , Raios Ultravioleta , Células Cultivadas , Ensaio Cometa , DNA/metabolismo , DNA Mitocondrial/efeitos da radiação , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Humanos , Queratinócitos/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Purpose: Our aim was to evaluate whether mitochondrial DNA (mtDNA) damage in hair bulbs could be a suitable biomarker for the detection of local exposure to ionizing radiation.Materials and methods: Mouse hair was collected 4 and 24 hours, 3 and 10 days after single whole-body exposure to 0, 0.1, and 2 Gy radiation. Pubic hair (treated area) and scalp hair (control area) were collected from 13 prostate cancer patients before and after fractioned radiotherapy with an average total dose of 2.7 Gy to follicles after five fractions. Unspecified lesion frequency of mtDNA was analyzed with long PCR, large mtDNA deletion levels were tested with real-time PCR.Results: Unspecified lesion frequency of mtDNA significantly increased in mouse hair 24 hours after irradiation with 2 Gy, but variance among samples was high. No increase in lesion frequency could be detected after 0.1 Gy irradiation. In prostate cancer patients, there was no significant change in either the unspecified lesion frequency or in the proportion of 4934-bp deleted mtDNA in pubic hair after radiotherapy. The proportions of murine 3860-bp common deletion, human 4977-bp common deletion and 7455-bp deleted mtDNA were too low to be analyzed reliably.Conclusions: Our results suggest that the unspecified lesion frequency and proportion of large deletions of mtDNA in hair bulbs are not suitable biomarkers of exposure to ionizing radiation.
Assuntos
Dano ao DNA , DNA Mitocondrial/efeitos da radiação , Folículo Piloso/efeitos da radiação , Idoso , Animais , Biomarcadores , Feminino , Humanos , Transferência Linear de Energia , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Fuchs endothelial corneal dystrophy (FECD) is a leading cause of corneal endothelial (CE) degeneration resulting in impaired visual acuity. It is a genetically complex and age-related disorder, with higher incidence in females. In this study, we established a nongenetic FECD animal model based on the physiologic outcome of CE susceptibility to oxidative stress by demonstrating that corneal exposure to ultraviolet A (UVA) recapitulates the morphological and molecular changes of FECD. Targeted irradiation of mouse corneas with UVA induced reactive oxygen species (ROS) production in the aqueous humor, and caused greater CE cell loss, including loss of ZO-1 junctional contacts and corneal edema, in female than male mice, characteristic of late-onset FECD. UVA irradiation caused greater mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) damage in female mice, indicative of the sex-driven differential response of the CE to UVA, thus accounting for more severe phenotype in females. The sex-dependent effect of UVA was driven by the activation of estrogen-metabolizing enzyme CYP1B1 and formation of reactive estrogen metabolites and estrogen-DNA adducts in female but not male mice. Supplementation of N-acetylcysteine (NAC), a scavenger of reactive oxygen species (ROS), diminished the morphological and molecular changes induced by UVA in vivo. This study investigates the molecular mechanisms of environmental factors in FECD pathogenesis and demonstrates a strong link between UVA-induced estrogen metabolism and increased susceptibility of females for FECD development.
Assuntos
Citocromo P-450 CYP1B1/metabolismo , Adutos de DNA/efeitos da radiação , Dano ao DNA/efeitos da radiação , Estrogênios/metabolismo , Distrofia Endotelial de Fuchs/etiologia , Raios Ultravioleta/efeitos adversos , Acetilcisteína/administração & dosagem , Animais , Humor Aquoso/efeitos dos fármacos , Humor Aquoso/metabolismo , Humor Aquoso/efeitos da radiação , Adutos de DNA/metabolismo , Dano ao DNA/efeitos dos fármacos , DNA Mitocondrial/metabolismo , DNA Mitocondrial/efeitos da radiação , Modelos Animais de Doenças , Endotélio Corneano/efeitos dos fármacos , Endotélio Corneano/patologia , Endotélio Corneano/efeitos da radiação , Feminino , Sequestradores de Radicais Livres/administração & dosagem , Distrofia Endotelial de Fuchs/diagnóstico , Distrofia Endotelial de Fuchs/tratamento farmacológico , Distrofia Endotelial de Fuchs/patologia , Humanos , Masculino , Camundongos , Estresse Oxidativo/efeitos da radiação , Espécies Reativas de Oxigênio/metabolismo , Índice de Gravidade de DoençaRESUMO
BACKGROUND: Despite increased understanding of the genetic events underlying pediatric high-grade gliomas (pHGGs), therapeutic progress is static, with poor understanding of nongenomic drivers. We therefore investigated the role of alterations in mitochondrial function and developed an effective combination therapy against pHGGs. METHODS: Mitochondrial DNA (mtDNA) copy number was measured in a cohort of 60 pHGGs. The implication of mtDNA alteration in pHGG tumorigenesis was studied and followed by an efficacy investigation using patient-derived cultures and orthotopic xenografts. RESULTS: Average mtDNA content was significantly lower in tumors versus normal brains. Decreasing mtDNA copy number in normal human astrocytes led to a markedly increased tumorigenicity in vivo. Depletion of mtDNA in pHGG cells promoted cell migration and invasion and therapeutic resistance. Shifting glucose metabolism from glycolysis to mitochondrial oxidation with the adenosine monophosphate-activated protein kinase activator AICAR (5-aminoimidazole-4-carboxamide ribonucleotide) or the pyruvate dehydrogenase kinase inhibitor dichloroacetate (DCA) significantly inhibited pHGG viability. Using DCA to shift glucose metabolism to mitochondrial oxidation and then metformin to simultaneously target mitochondrial function disrupted energy homeostasis of tumor cells, increasing DNA damage and apoptosis. The triple combination with radiation therapy, DCA and metformin led to a more potent therapeutic effect in vitro and in vivo. CONCLUSIONS: Our results suggest metabolic alterations as an onco-requisite factor of pHGG tumorigenesis. Targeting reduced mtDNA quantity represents a promising therapeutic strategy for pHGG.
Assuntos
Aminoimidazol Carboxamida/análogos & derivados , Neoplasias Encefálicas/metabolismo , DNA Mitocondrial/metabolismo , Ácido Dicloroacético/farmacologia , Metabolismo Energético/fisiologia , Glioma/metabolismo , Ribonucleotídeos/farmacologia , Aminoimidazol Carboxamida/farmacologia , Animais , Neoplasias Encefálicas/genética , Criança , DNA Mitocondrial/efeitos dos fármacos , DNA Mitocondrial/efeitos da radiação , Metabolismo Energético/efeitos dos fármacos , Metabolismo Energético/efeitos da radiação , Dosagem de Genes , Glioma/genética , Glicólise/efeitos dos fármacos , Glicólise/efeitos da radiação , Humanos , Camundongos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
AIMS: Ionizing radiation may lead to mitochondrial DNA (mtDNA) mutations and changes in mtDNA content in cells, major driving mechanisms for carcinogenesis, vascular aging and neurodegeneration. The aim of this study was to investigate the possible induction of common mitochondrial deletion (mtDNA4977) and mtDNA copy number (mtDNA-CN) changes in peripheral blood of personnel working in high-volume cardiac catheterization laboratories (Cath Labs). METHODS: A group of 147 Cath Lab workers (median individual effective dose = 16.8 mSv, for the 41 with lifetime dosimetric record) and 74 unexposed individuals were evaluated. The occupational radiological risk score was computed for each subject on the basis of the length of employment, individual caseload and proximity to the radiation source. mtDNA4977 deletion and mtDNA-CN were assessed by using quantitative real-time polymerase chain reaction. RESULTS: Increased levels of mtDNA4977 deletion were observed in high-exposure Cath Lab workers compared with unexposed individuals ( p < 0.0001). Conversely, mtDNA-CN was significantly greater in the low-exposure workers ( p = 0.003). Occupational radiological risk score was positively correlated with mtDNA4977 deletion (Spearman's r = 0.172, p = 0.03) and inversely correlated with mtDNA-CN (Spearman's r = -0.202, p = 0.01). In multiple regression model, occupational radiological risk score emerged as significant predictor of high levels of mtDNA4977 deletion (ß coefficient = 0.236, p = 0.04). CONCLUSION: mtDNA4977 deletion is significantly high in Cath Lab personnel. Beyond the well-recognized nuclear DNA, mtDNA damage might deserve attention as a pathogenetic molecular pathway and a potential therapeutic target of ionizing radiation damage.
Assuntos
Cateterismo Cardíaco/efeitos adversos , DNA Mitocondrial/efeitos da radiação , Exposição Ocupacional/efeitos adversos , Saúde Ocupacional , Doses de Radiação , Exposição à Radiação/efeitos adversos , Radiografia Intervencionista/efeitos adversos , Deleção de Sequência , Adulto , Cardiologistas , Estudos Transversais , Variações do Número de Cópias de DNA , DNA Mitocondrial/genética , Feminino , Dosagem de Genes , Humanos , Masculino , Pessoa de Meia-Idade , Enfermeiras e Enfermeiros , Medição de Risco , Fatores de RiscoRESUMO
PURPOSE: Mitochondria have been implicated in initiating and/or amplifying the biological effects of ionizing radiation not mediated via damage to nuclear DNA. To help elucidate the underlying mechanisms, energy deposition patterns to mitochondria and radiation damage to their DNA have been modelled. METHODS: Track-structure simulations have been performed with PARTRAC biophysical tool for 60Co γ-rays and 5 MeV α-particles. Energy deposition to the cell's mitochondria has been analyzed. A model of mitochondrial DNA reflecting experimental information on its structure has been developed and used to assess its radiation-induced damage. RESULTS: Energy deposition to mitochondria is highly inhomogeneous, especially at low doses. Although a dose-dependent fraction of mitochondria sees no energy deposition at all, the hit ones receive rather high amounts of energy. Nevertheless, only little damage to mitochondrial DNA occurs, even at large doses. CONCLUSION: Mitochondrial DNA does not represent a critical target for radiation effects. Likely, the key role of mitochondria in radiation-induced biological effects arises from the communication between mitochondria and/or with the nucleus. Through this signaling, initial modifications in a few heavily hit mitochondria seem to be amplified to a massive long-term effect manifested in the whole cell or even tissue.
Assuntos
Dano ao DNA , DNA Mitocondrial/genética , DNA Mitocondrial/efeitos da radiação , Modelos Biológicos , Transferência Linear de Energia , RadiobiologiaRESUMO
The increasing risk of acute large-scale radiological/nuclear exposures of population underlines the necessity of developing new, rapid and high throughput biodosimetric tools for estimation of received dose and initial triage. We aimed to compare the induction and persistence of different radiation exposure biomarkers in human peripheral blood in vivo. Blood samples of patients with indicated radiotherapy (RT) undergoing partial body irradiation (PBI) were obtained soon before the first treatment and then after 24 h, 48 h, and 5 weeks; i.e. after 1, 2, and 25 fractionated RT procedures. We collected circulating peripheral blood from ten patients with tumor of endometrium (1.8 Gy per fraction) and eight patients with tumor of head and neck (2.0-2.121 Gy per fraction). Incidence of dicentrics and micronuclei was monitored as well as determination of apoptosis and the transcription level of selected radiation-responsive genes. Since mitochondrial DNA (mtDNA) has been reported to be a potential indicator of radiation damage in vitro, we also assessed mtDNA content and deletions by novel multiplex quantitative PCR. Cytogenetic data confirmed linear dose-dependent increase in dicentrics (p < 0.01) and micronuclei (p < 0.001) in peripheral blood mononuclear cells after PBI. Significant up-regulations of five previously identified transcriptional biomarkers of radiation exposure (PHPT1, CCNG1, CDKN1A, GADD45, and SESN1) were also found (p < 0.01). No statistical change in mtDNA deletion levels was detected; however, our data indicate that the total mtDNA content decreased with increasing number of RT fractions. Interestingly, the number of micronuclei appears to correlate with late radiation toxicity (r2 = 0.9025) in endometrial patients suggesting the possibility of predicting the severity of RT-related toxicity by monitoring this parameter. Overall, these data represent, to our best knowledge, the first study providing a multiparametric comparison of radiation biomarkers in human blood in vivo, which have potential for improving biological dosimetry.
Assuntos
Leucócitos/efeitos da radiação , Exposição à Radiação , Radiometria/métodos , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/sangue , Aberrações Cromossômicas , DNA Mitocondrial/efeitos da radiação , Relação Dose-Resposta à Radiação , Neoplasias do Endométrio/sangue , Neoplasias do Endométrio/radioterapia , Feminino , Neoplasias de Cabeça e Pescoço/sangue , Neoplasias de Cabeça e Pescoço/radioterapia , Humanos , Leucócitos/patologia , Masculino , Micronúcleos com Defeito Cromossômico , Pessoa de Meia-Idade , Radioterapia/efeitos adversos , Dosagem Radioterapêutica , Transcrição Gênica/efeitos da radiaçãoRESUMO
Mitochondria play a major role in aging. Over time, mutations accumulate in mitochondrial DNA leading to reduced adenosine triphosphate (ATP) production and increased production of damaging reactive oxygen species. If cells fail to cope, they die. Reduced ATP will result in declining cellular membrane potentials leading to reduced central nervous system function. However, aged mitochondrial function is improved by long wavelength light (670 nm) absorbed by cytochrome c oxidase in mitochondrial respiration. In Drosophila, lifelong 670-nm exposure extends lifespan and improves aged mobility. Here, we ask if improved mitochondrial metabolism can reduce functional senescence in metabolism, sensory, locomotor, and cognitive abilities in old flies exposed to 670 nm daily for 1 week. Exposure significantly increased cytochrome c oxidase activity, whole body energy storage, ATP and mitochondrial DNA content, and reduced reactive oxygen species. Retinal function and memory were also significantly improved to levels found in 2-week-old flies. Mobility improved by 60%. The mode of action is likely related to improved energy homeostasis increasing ATP availability for ionic ATPases critical for maintenance of neuronal membrane potentials. 670-nm light exposure may be a simple route for resolving problems of aging.
Assuntos
Envelhecimento/fisiologia , Envelhecimento/efeitos da radiação , Cognição/fisiologia , Drosophila melanogaster/fisiologia , Drosophila melanogaster/efeitos da radiação , Metabolismo Energético/efeitos da radiação , Raios Infravermelhos , Mitocôndrias/metabolismo , Mitocôndrias/efeitos da radiação , Atividade Motora/fisiologia , Acuidade Visual/fisiologia , Trifosfato de Adenosina/metabolismo , Envelhecimento/psicologia , Animais , DNA Mitocondrial/metabolismo , DNA Mitocondrial/efeitos da radiação , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/efeitos da radiação , Potenciais da Membrana , Memória/fisiologia , Memória/efeitos da radiação , Espécies Reativas de Oxigênio/metabolismo , Retina/fisiologia , Retina/efeitos da radiaçãoRESUMO
The process of human cardiac development can be faithfully recapitulated in a culture dish with human pluripotent stem cells, where the impact of environmental stressors can be evaluated. The consequences of ionizing radiation exposure on human cardiac differentiation are largely unknown. In this study, human-induced pluripotent stem cell cultures (hiPSCs) were subjected to an external beam of 3.7 MeV α-particles at low mean absorbed doses of 0.5, 3, and 10 cGy. Subsequently, the hiPSCs were differentiated into beating cardiac myocytes (hiPSC-CMs). Pluripotent and cardiac markers and morphology did not reveal differences between the irradiated and nonirradiated groups. While cell number was not affected during CM differentiation, cell number of differentiated CMs was severely reduced by ionizing radiation in a dose-responsive manner. ß-adrenergic stimulation causes calcium (Ca2+) overload and oxidative stress. Although no significant increase in Ca2+ transient amplitude was observed in any group after treatment with 1 µmol/L isoproterenol, the incidence of spontaneous Ca2+ waves/releases was more frequent in hiPSC-CMs of the irradiated groups, indicating arrhythmogenic activities at the single cell level. Increased transcript expression of mitochondrial biomarkers (LONP1, TFAM) and mtDNA-encoded genes (MT-CYB, MT-RNR1) was detected upon differentiation of hiPSC-CMs suggesting increased organelle biogenesis. Exposure of hiPSC-CM cultures to 10 cGy significantly upregulated MT-CYB and MT-RNR1 expression, which may reflect an adaptive response to ionizing radiation. Our results indicate that important aspects of differentiation of hiPSCs into cardiac myocytes may be affected by low fluences of densely ionizing radiations such as α-particles.
Assuntos
Diferenciação Celular/efeitos da radiação , Miócitos Cardíacos/fisiologia , Miócitos Cardíacos/efeitos da radiação , Proliferação de Células/efeitos da radiação , Células Cultivadas , DNA Mitocondrial/metabolismo , DNA Mitocondrial/efeitos da radiação , Humanos , Células-Tronco Pluripotentes Induzidas , Contração Miocárdica/efeitos da radiação , Radiação Ionizante , Estresse Fisiológico/efeitos da radiaçãoRESUMO
Proton beam therapy has recently been used to improve local control of tumor growth and reduce side-effects by decreasing the global dose to normal tissue. However, the regulatory mechanisms underlying the physiological role of proton beam radiation are not well understood, and many studies are still being conducted regarding these mechanisms. To determine the effects of proton beams on mitochondrial biogenesis, we investigated: mitochondrial DNA (mtDNA) mass; the gene expression of mitochondrial transcription factors, functional regulators, and dynamic-related regulators; and the phosphorylation of the signaling molecules that participate in mitochondrial biogenesis. Both the mtDNA/nuclear DNA (nDNA) ratio and the mitochondria staining assays showed that proton beam irradiation increases mitochondrial biogenesis in 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced aggressive HT-29 cells. Simultaneously, proton beam irradiation increases the gene expression of the mitochondrial transcription factors PGC-1α, NRF1, ERRα, and mtTFA, the dynamic regulators DRP1, OPA1, TIMM44, and TOM40, and the functional regulators CytC, ATP5B and CPT1-α. Furthermore, proton beam irradiation increases the phosphorylation of AMPK, an important molecule involved in mitochondrial biogenesis that is an energy sensor and is regulated by the AMP/ATP ratio. Based on these findings, we suggest that proton beam irradiation inhibits metastatic potential by increasing mitochondrial biogenesis and function in TPA-induced aggressive HT-29 cells.
Assuntos
Adenocarcinoma/radioterapia , Neoplasias Colorretais/radioterapia , DNA Mitocondrial/efeitos da radiação , Biogênese de Organelas , Adenocarcinoma/genética , Adenocarcinoma/patologia , Monofosfato de Adenosina/metabolismo , Monofosfato de Adenosina/efeitos da radiação , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/efeitos da radiação , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Células HT29 , Humanos , Proteínas de Neoplasias/efeitos da radiação , Fosforilação/efeitos da radiação , Terapia com Prótons , Acetato de Tetradecanoilforbol/toxicidadeRESUMO
HL-60 cells, derived from human promyelocytic leukemia, were exposed to continuous wave 900MHz radiofrequency fields (RF) at 120µW/cm2 power intensity for 4h/day for 5 consecutive days to examine whether such exposure is capable damaging the mitochondrial DNA (mtDNA) mediated through the production of reactive oxygen species (ROS). In addition, the effect of RF exposure was examined on 8-hydroxy-2'-dexoyguanosine (8-OHdG) which is a biomarker for oxidative damage and on the mitochondrial synthesis of adenosine triphosphate (ATP) which is the energy required for cellular functions. The results indicated a significant increase in ROS and significant decreases in mitochondrial transcription factor A, mtDNA polymerase gamma, mtDNA transcripts and mtDNA copy number in RF-exposed cells compared with those in sham-exposed control cells. In addition, there was a significant increase in 8-OHdG and a significant decrease in ATP in RF-exposed cells. The response in positive control cells exposed to gamma radiation (GR, which is also known to induce ROS) was similar to those in RF-exposed cells. Thus, the overall data indicated that RF exposure was capable of inducing mtDNA damage mediated through ROS pathway which also induced oxidative damage. Prior-treatment of RF- and GR-exposed the cells with melatonin, a well-known free radical scavenger, reversed the effects observed in RF-exposed cells.
Assuntos
Dano ao DNA , DNA Mitocondrial , Estresse Oxidativo , Ondas de Rádio/efeitos adversos , 8-Hidroxi-2'-Desoxiguanosina , Trifosfato de Adenosina/metabolismo , Técnicas de Cultura de Células , DNA Mitocondrial/genética , DNA Mitocondrial/efeitos da radiação , Desoxiguanosina/análogos & derivados , Desoxiguanosina/metabolismo , Relação Dose-Resposta à Radiação , Células HL-60 , Humanos , Estresse Oxidativo/genética , Estresse Oxidativo/efeitos da radiação , Espécies Reativas de Oxigênio/metabolismoRESUMO
The retinal pigment epithelium (RPE) is a highly metabolic layer of postmitotic cells lining Bruch's membrane in the retina. While these cells contain endogenous photosensitizers that mediate blue light-induced damage, it has also been shown that blue light exposure damages mitochondrial DNA in RPE cells resulting in mitochondrial dysfunction and unregulated generation of reactive oxygen species (ROS). As RPE cells are postmitotic, it is imperative to decrease oxidative stress to these cells and preserve function. Dietary plant-derived antioxidants such as anthocyanins offer a simple and accessible solution for decreasing oxidative stress. The anthocyanins malvidin-3-O-glucoside (oenin) and pelargonidin-3-O-glucoside (callistephin) were tested for their ability and efficacy in decreasing ROS generation and preserving mitochondrial redox activity in blue light-irradiated ARPE-19 cells. A significant decrease in intracellular ROS with concurrent increase in mitochondrial redox activity was observed for tested concentrations of oenin, while callistephin was beneficial to stressed cells at higher concentrations. These findings suggest anthocyanins are effective antioxidants in blue light-stressed RPE cells in vitro. Additionally, oxidation products of these anthocyanins were examined using LC/MS and findings suggest the possibility of multiple oxidation sites for these compounds.