RESUMEN
Exposure to ionizing radiation can induce genetic aberrations via unrepaired DNA strand breaks. To investigate quantitatively the dose-effect relationship at the molecular level, we irradiated dry pBR322 plasmid DNA with 3 MeV protons and assessed fragmentation yields at different radiation doses using long-read sequencing from Oxford Nanopore Technologies. This technology applied to a reference DNA model revealed dose-dependent fragmentation, as evidenced by read length distributions, showing no discernible radiation sensitivity in specific genetic sequences. In addition, we propose a method for directly measuring the single-strand break (SSB) yield. Furthermore, through a comparative study with a collection of previous works on dry DNA irradiation, we show that the irradiation protocol leads to biases in the definition of ionizing sources. We support this scenario by discussing the size distributions of nanopore sequencing reads in the light of Geant4 and Geant4-DNA simulation toolkit predictions. We show that integrating long-read sequencing technologies with advanced Monte Carlo simulations paves a promising path toward advancing our comprehension and prediction of radiation-induced DNA fragmentation.
Asunto(s)
Fragmentación del ADN , Método de Montecarlo , Plásmidos , Plásmidos/genética , Fragmentación del ADN/efectos de la radiación , Relación Dosis-Respuesta en la Radiación , Análisis de Secuencia de ADN/métodos , Roturas del ADN de Cadena Simple/efectos de la radiación , ADN/genéticaRESUMEN
The extremophilic bacterium Deinococcus radiodurans displays an extraordinary ability to withstand lethal radiation effects, due to its complex mechanisms for both proteome radiation protection and DNA repair. Published results obtained recently at this laboratory show that D. radiodurans submitted to ionizing radiation results in its DNA being shattered into small fragments which, when exposed to a "static electric field' (SEF), greatly decreases cell viability. These findings motivated the performing of D. radiodurans exposed to gamma radiation, yet exposed to a different exogenous physical agent, "static magnetic fields" (SMF). Cells of D. radiodurans [strain D.r. GY 9613 (R1)] in the exponential phase were submitted to 60Co gamma radiation from a gamma cell. Samples were exposed to doses in the interval 0.5-12.5 kGy, while the control samples were kept next to the irradiation setup. Exposures to SMF were carried out with intensities of 0.08 T and 0.8 T delivered by two settings: (a) a device built up at this laboratory with niobium magnets, delivering 0.08 T, and (b) an electromagnet (Walker Scientific) generating static magnetic fields with intensities from 0.1 to 0.8 T. All samples were placed in a bacteriological incubator at 30 °C for 48 h, and after incubation, a counting of colony forming units was performed. Two sets of cell surviving data were measured, each in triplicate, obtained in independent experiments. A remarkable similarity between the two data sets is revealed, underscoring reproducibility within the 5% range. Appraisal of raw data shows that exposure of irradiated cells to SMF substantially increases their viability. Data interpretation strongly suggests that the increase of D. radiodurans cell viability is a sole magnetic physical effect, driven by a stochastic process, improving the efficiency of the rejoining of DNA fragments, thus increasing cell viability. A type of cut-off dose is identified at 10 kGy, above which the irradiated cellular system loses recovery and the cell survival mechanism collapses.
Asunto(s)
Deinococcus/citología , Deinococcus/efectos de la radiación , Rayos gamma , Campos Magnéticos , Supervivencia Celular/efectos de la radiación , Fragmentación del ADN/efectos de la radiación , Deinococcus/genéticaRESUMEN
PURPOSE: Microgravity has severe effects on cellular and molecular structures as well as on metabolic interactions. The aim of this study is to investigate the effects of microgravity (µg) exposure on human frozen sperm samples. METHODS: Sibling samples from 15 normozoospermic healthy donors were frozen using glycerol as cryoprotectant and analyzed under microgravity and ground conditions. Microgravity was obtained by parabolic flights using a CAP10B plane. The plane executed 20 parabolic maneuvers with a mean of 8.5 s of microgravity for each parabola. RESULTS: Frozen sperm samples preserved in cryostraws and stored in a secure and specific nitrogen vapor cryoshipper do not suffer significant alterations after µg exposure. Comparing the study group (µg) and the control group (1 g), similar results were obtained in the main parameters studied: sperm motility (M/ml) 13.72 ± 12.57 vs 13.03 ± 12.13 (- 0.69 95% CI [- 2.9; 1.52]), progressive a + b sperm motility (%) 21.83 ± 11.69 vs 22.54 ± 12.83 (0.03 95% CI [- 0.08; 0.15]), sperm vitality (%) 46.42 ± 10.81 vs 44.62 ± 9.34 (- 0.04 95% CI [- 0.13; 0.05]), morphologically normal spermatozoa (%) 7.03 ± 2.61 vs 8.09 ± 3.61 (0.12 95% CI [0.01; 0.24]), DNA sperm fragmentation by SCD (%) 13.33 ± 5.12 vs 13.88 ± 6.14 (0.03 95% CI [- 0.09; 0.16]), and apoptotic spermatozoa by MACS (%) 15.47 ± 15.04 vs 23.80 ± 23.63 (- 0.20 95% CI [- 0.66; 1.05]). CONCLUSION: The lack of differences obtained between frozen samples exposed to µg and those maintained in ground conditions provides the possibility of considering the safe transport of human male gametes to space. Nevertheless, further research is needed to validate the results and to consider the possibility of creating a human sperm bank outside the Earth. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov: NCT03760783.
Asunto(s)
Criopreservación , Motilidad Espermática/genética , Espermatozoides/crecimiento & desarrollo , Ingravidez , Crioprotectores/farmacología , Fragmentación del ADN/efectos de la radiación , Congelación , Humanos , Masculino , Análisis de Semen , Preservación de Semen , Motilidad Espermática/efectos de la radiación , Espermatozoides/metabolismo , Espermatozoides/efectos de la radiaciónRESUMEN
Photobiomodulation (PBM) has been used to modulate the inflammatory and immune responses, pain relief, and to promote wound healing. PBM is widely used in dental practice and its cellular effects should be investigated. The aim was to evaluate if PBM changes proteins cell death-related, such as caspase-6 and Bcl-2, in periodontal ligament cells. Eighteen mice were divided in three groups (n = 6), i.e., (I) control, (II) 3 J cm-2, and (III) 30 J cm-2. Low power infrared laser (830 nm) parameters were power at 10 mW, energy densities at 3 and 30 J cm-2 in continuous emission mode, exposure time of 15 and 150 s, respectively for 4 days in a row. Twenty-four hours after last irradiation, the animals were euthanized, and their jaws were fixed and decalcified. Caspase-6 and Bcl-2 were analyzed by real-time polymerase chain reaction and immunocytochemical techniques, and DNA fragmentation was evaluated by TUNEL. Statistical differences were not significant to caspase-6 mRNA relative levels in tissues from jaws at both energy densities, but a significant increase of Bcl-2 mRNA relative levels was obtained at 30 J cm-2 group. Also, 30 J cm-2 group showed caspase-6 positive-labeled cells decreased and Bcl-2 positive-labeled cells significantly increased. TUNEL-labeled cells demonstrated DNA fragmentation decreased at 30 J cm-2. PBM can alter Bcl-2 mRNA relative level and both caspase-6 and Bcl-2 protein, modulating cell survival, as well as to reduce DNA fragmentation. More studies must be performed in order to obtain conclusive results about photobiostimulation effects using infrared low-level laser in apoptosis process as to achieve the optimum dosage.
Asunto(s)
Apoptosis/efectos de la radiación , Terapia por Luz de Baja Intensidad , Ligamento Periodontal/citología , Animales , Supervivencia Celular/efectos de la radiación , Fragmentación del ADN/efectos de la radiación , Regulación de la Expresión Génica/efectos de la radiación , Ratones , Proteínas Proto-Oncogénicas c-bcl-2/genética , ARN Mensajero/genética , Cicatrización de Heridas/efectos de la radiaciónRESUMEN
Background: Enhanced sperm motility is necessary for the successful journey of sperm inside the female genital tract, successful fertilization, and the increased chance of pregnancy. Objective: We investigated the impact of red and near-infrared (NIR) ranges of photobiomodulation (PBM) alone and together on fresh human sperm to validate an optimized PBM protocol that would maximize sperm motility and viability in vitro. Methods: We randomly divided 30 normal human semen samples into 3 different PBM protocols (red, NIR, and red+NIR lasers). Each sample was divided into four subparts, one control group sample and three experimental group samples. Each experimental group received one of the PBM protocols (red, NIR, or red+NIR). Each protocol was adjusted to three energy densities (0.6, 1.2, and 2.4 J/cm2). After exposure to the selected protocol, we determined the percentage of either viable or progressive sperm motility (PSM) and measured the DNA Fragmentation Index (DFI). Results: The NIR and red+NIR lasers at 2.4 J/cm2 energy density significantly increased PSM after 60 min compared with the control groups [least significant difference (LSD) test, p = 0.023 and p = 0.04, respectively]. Samples treated with the red laser at 0.6 J/cm2 had significantly decreased viability compared with the control group (LSD test, p = 0.003). Samples treated with the red+NIR lasers had significantly decreased viability at 0.6 J/cm2 (p = 0.003), 1.2 J/cm2 (p = 0.001), and 2.4 J/cm2 (p = 0.04) energy densities when compared with the control groups. The NIR laser resulted in no significant difference in sperm viability between the control and experimental groups. At 120 min after exposure, treatment with the red+NIR and red lasers at 2.4 J/cm2 density significantly increased DFI compared to the control groups (LSD test, p = 0.000, p = 0.007). Conclusions: In this study, sperm motility, viability, and DFI data confirmed the superiority of the NIR laser at 0.6 J/cm2 energy density compared with the red and red+NIR PBM protocols.
Asunto(s)
Terapia por Luz de Baja Intensidad , Motilidad Espermática/efectos de la radiación , Espermatozoides/efectos de la radiación , Adulto , Técnicas de Cultivo de Célula , Supervivencia Celular/efectos de la radiación , Fragmentación del ADN/efectos de la radiación , Humanos , Irán , MasculinoRESUMEN
trans-Fatty acids (TFAs) are unsaturated fatty acids that contain one or more carbon-carbon double bonds in trans configuration. Epidemiological evidence has linked TFA consumption with various disorders, including cardiovascular diseases. However, the underlying pathological mechanisms are largely unknown. Here, we show a novel toxic mechanism of TFAs triggered by DNA damage. We found that elaidic acid (EA) and linoelaidic acid, major TFAs produced during industrial food manufacturing (so-called as industrial TFAs), but not their corresponding cis isomers, facilitated apoptosis induced by doxorubicin. Consistently, EA enhanced UV-induced embryonic lethality in C. elegans worms. The pro-apoptotic action of EA was blocked by knocking down Sab, a c-Jun N-terminal kinase (JNK)-interacting protein localizing at mitochondrial outer membrane, which mediates mutual amplification of mitochondrial reactive oxygen species (ROS) generation and JNK activation. EA enhanced doxorubicin-induced mitochondrial ROS generation and JNK activation, both of which were suppressed by Sab knockdown and pharmacological inhibition of either mitochondrial ROS generation, JNK, or Src-homology 2 domain-containing protein tyrosine phosphatase 1 (SHP1) as a Sab-associated protein. These results demonstrate that in response to DNA damage, TFAs drive the mitochondrial JNK-Sab-ROS positive feedback loop and ultimately apoptosis, which may provide insight into the common pathogenetic mechanisms of diverse TFA-related disorders.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Fragmentación del ADN/efectos de los fármacos , Ácido Linoleico/farmacología , Mitocondrias/efectos de los fármacos , Ácidos Oléicos/farmacología , Especies Reactivas de Oxígeno/metabolismo , Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Apoptosis/efectos de la radiación , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/antagonistas & inhibidores , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Línea Celular Tumoral , Fragmentación del ADN/efectos de la radiación , Doxorrubicina/farmacología , Embrión no Mamífero , Retroalimentación Fisiológica , Regulación de la Expresión Génica , Factores de Intercambio de Guanina Nucleótido/antagonistas & inhibidores , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Células HEK293 , Células HeLa , Células Endoteliales de la Vena Umbilical Humana/citología , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Ratones , Mitocondrias/metabolismo , Osteoblastos/citología , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 6/genética , Proteína Tirosina Fosfatasa no Receptora Tipo 6/metabolismo , Células RAW 264.7 , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Especies Reactivas de Oxígeno/agonistas , Rayos UltravioletaRESUMEN
Rapid sample preparation is one of the leading bottlenecks to low-cost and efficient sample component detection. To overcome this setback, a technology known as Lyse-It has been developed to rapidly (less than 60 seconds) lyse Gram-positive and-negative bacteria alike, while simultaneously fragmenting DNA/RNA and proteins into tunable sizes. This technology has been used with a variety of organisms, but the underlying mechanism behind how the technology actually works to fragment DNA/RNA and proteins has hitherto been studied. It is generally understood how temperature affects cellular lysing, but for DNA/RNA and protein degradation, the temperature and amount of energy introduced by microwave irradiation of the sample, cannot explain the degradation of the biomolecules to the extent that was being observed. Thus, an investigation into the microwave generation of reactive oxygen species, in particular singlet oxygen, hydroxyl radicals, and superoxide anion radicals, was undertaken. Herein, we probe one aspect, the generation of reactive oxygen species (ROS), which is thought to contribute to a non-thermal mechanism behind biomolecule fragmentation with the Lyse-It technology. By utilizing off/on (Photoinduced electron transfer) PET fluorescent-based probes highly specific for reactive oxygen species, it was found that as oxygen concentration in the sample and/or microwave irradiation power increases, more reactive oxygen species are generated and ultimately, more oxidation and biomolecule fragmentation occurs within the microwave cavity.
Asunto(s)
Métodos Analíticos de la Preparación de la Muestra/métodos , Técnicas Bacteriológicas/métodos , Fragmentación del ADN/efectos de los fármacos , Detergentes/farmacología , Estabilidad del ARN/efectos de los fármacos , Fragmentación del ADN/efectos de la radiación , ADN Bacteriano/química , ADN Bacteriano/efectos de los fármacos , ADN Bacteriano/efectos de la radiación , Hidrólisis/efectos de la radiación , Listeria monocytogenes/efectos de los fármacos , Listeria monocytogenes/genética , Listeria monocytogenes/efectos de la radiación , Microondas , Oxidación-Reducción/efectos de los fármacos , Oxidación-Reducción/efectos de la radiación , Oxígeno/análisis , Oxígeno/metabolismo , Proteolisis/efectos de los fármacos , Proteolisis/efectos de la radiación , Estabilidad del ARN/efectos de la radiación , ARN Bacteriano/química , ARN Bacteriano/efectos de los fármacos , ARN Bacteriano/efectos de la radiación , Especies Reactivas de Oxígeno/análisis , Especies Reactivas de Oxígeno/metabolismo , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/genética , Staphylococcus aureus/efectos de la radiación , Temperatura , Factores de Tiempo , Vibrio cholerae/efectos de los fármacos , Vibrio cholerae/genética , Vibrio cholerae/efectos de la radiaciónRESUMEN
This study examines the ability of arabinoxylan rice bran (MGN-3/Biobran) to enhance the anti-cancer effects of fractionated X-ray irradiation of Ehrlich solid tumor-bearing mice. Swiss albino mice bearing tumors were exposed to the following: (i) Biobran treatment (40 mg/kg/day, intraperitoneal injections) beginning on day 11 post-tumor cell inoculation until day 30; (ii) ionizing radiation (Rad) 2 Gy at three consecutive doses on days 12, 14 and 16; or (iii) Biobran + Rad. Final tumor weight was suppressed by 46% for Biobran, 31% for Rad and 57% for the combined treatment (Biobran + Rad) relative to control untreated mice. Biobran and Rad also arrested the hypodiploid cells in the sub-G1-phase, signifying apoptosis by +102% and +85%, respectively, while the combined treatment induced apoptosis by +123%, with similar results in the degree of DNA fragmentation. Furthermore, Biobran + Rad upregulated the relative gene expression and protein level of p53 and Bax in tumor cells, down-regulated Bcl-2 expression, and increased the Bax/Bcl-2 ratio and caspase-3 activity, with the combined treatment greater than for either treatment alone. Additionally, the combined treatment modulated the decrease in body weight, the increase in liver and spleen weight, and the elevation of liver enzymes aspartate aminotransferase, alanine aminotransferase and gamma-glutamyl transferase to be within normal values. We conclude that Biobran enhances radiation therapy-induced tumor regression by potentiating apoptosis and minimizing toxicities related to radiation therapy, suggesting that Biobran may be useful in human cancer patients undergoing radiotherapy and warranting clinical trials.
Asunto(s)
Carcinoma de Ehrlich/tratamiento farmacológico , Carcinoma de Ehrlich/radioterapia , Xilanos/uso terapéutico , Animales , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Peso Corporal/efectos de los fármacos , Peso Corporal/efectos de la radiación , Carcinoma de Ehrlich/genética , Ciclo Celular/efectos de los fármacos , Ciclo Celular/efectos de la radiación , Proliferación Celular/efectos de los fármacos , Proliferación Celular/efectos de la radiación , Terapia Combinada , Daño del ADN , Fragmentación del ADN/efectos de los fármacos , Fragmentación del ADN/efectos de la radiación , Femenino , Regulación de la Expresión Génica , Hígado/efectos de los fármacos , Hígado/enzimología , Hígado/patología , Hígado/efectos de la radiación , Ratones , Tamaño de los Órganos/efectos de los fármacos , Tamaño de los Órganos/efectos de la radiación , Bazo/efectos de los fármacos , Bazo/patología , Bazo/efectos de la radiación , Carga Tumoral/efectos de los fármacos , Carga Tumoral/efectos de la radiación , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Rayos X , Xilanos/farmacología , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Purpose: Gamma-rays and carbon ions are frequently used for mutation breeding in diverse plant species, whereas proton ions have been rarely used for this purpose. This study assessed the potential of proton ions for plant mutation breeding. Materials and methods: We compared the effects of radiation on creeping bentgrass seeds with γ-rays, proton ions, and carbon ions on seed germination, plant growth parameters, and DNA fragmentation. Results and conclusions: The lethal dose 50 (LD50) doses based on seed germinability were 115.9 Gy (γ-rays), 225.1 Gy (proton ions), and 57.7 Gy (carbon ions). Threshold doses for survival were 150 Gy (γ-rays), 150 Gy (proton ions), and 25 Gy (carbon ions). Suppression of plant growth was displayed at 100 Gy (γ-rays), 25 Gy (proton ions), and 25 Gy (carbon ions). Similar patterns of decreasing head DNA percentage were observed for γ-rays and proton ions. Carbon ions induced the lowest frequency of DNA fragmentation. The biological effects of the ionizing radiation types on creeping bentgrass are summarizable as follows: germination, carbon ions (C)>γ-rays (G)>proton ions (P); survival, C > P = G; growth, C ≥ P > G; DNA fragmentation, G ≥ P > C. These results indicate that proton ions are useful as a physical mutagen in plant mutation breeding.
Asunto(s)
Agrostis/efectos de la radiación , Carbono , Rayos gamma , Protones , Agrostis/genética , Agrostis/crecimiento & desarrollo , Fragmentación del ADN/efectos de la radiación , Relación Dosis-Respuesta en la Radiación , Germinación/efectos de la radiación , Semillas/crecimiento & desarrollo , Semillas/efectos de la radiaciónRESUMEN
Photobiomodulation (PBM) by low-level laser has demonstrated excellent results for inflammatory treatments, promoting repair of injured tissues. Knowledge regarding the molecular mechanisms involved in this process has been increasing, but its effect on cell death/survival-related gene expression after laser irradiation with different doses is not well understood. So, it is important to know these effects in order to guarantee the safety of therapeutic protocols based on PBM. This study aimed to investigate the mRNA levels of genes related to proteins involved in cell death/survival pathways of healthy tissues from talocrural joint of mice after PBM. Mice were divided into three groups: control, PBM at 3 J cm-2, and PBM at 30 J cm-2. Laser irradiation was performed on talocrural joint during four consecutive days. Morphological analyses, immunocytochemistry, FasL, Fas, Bax, Apaf1, Caspase9, Caspase3, Caspase6, Bcl2 mRNA levels, and DNA fragmentation were performed to verify cell death induction after laser irradiation. PBM can increase mRNA levels of almost genes pro-apoptotic. On the other hand, mRNA level of anti-apoptotic protein Bcl-2 gene was not significantly altered. Bcl-2/Bax ratio (indicator of protective molecular response) was decreased after PBM at 30 J cm-2, trending to DNA fragmentation. Results obtained in this study indicate that PBM by low-level infrared laser alters mRNA relative levels of genes involved in cell death pathways. However, these molecular alterations were not able to cause DNA fragmentation in cells in talocrural joint tissues, indicating that infrared laser was not enough to cause cell death.
Asunto(s)
Apoptosis/genética , Apoptosis/efectos de la radiación , Regulación de la Expresión Génica/efectos de la radiación , Terapia por Luz de Baja Intensidad , Animales , Fragmentación del ADN/efectos de la radiación , Masculino , Ratones Endogámicos C57BL , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Oligonucleosomal DNA fragmentation and chromatin condensation are two hallmarks of apoptosis. However, their generation mechanisms are not entirely understood. Histone H1, a positively charged nuclear protein located in the linker region of chromatin, is involved in higher-order chromatin structures and tight chromatin packing. On the basis of the physical and biochemical characteristics of histone H1, we hypothesized that histone H1 plays a role in determining the efficiencies of apoptotic DNA fragmentation and chromatin condensation. Therefore, we examined histone H1 quantity in five human leukemia cell lines and compared the efficiencies. The cell lines were categorized into two groups according to their origins: (i) Ramos and Molt-4 cells of lymphoid origin and (ii) U937, ML-1, and HL60 cells of myeloid origin. Compared to the lymphoid-origin group, the myeloid-origin group had lower levels of histone H1 but more open chromatin. Furthermore, the myeloid-origin group showed marked DNA fragmentation but less chromatin condensation during apoptosis. These results suggested that histone H1 determined chromatin structure and that its quantity affected the efficiencies of DNA fragmentation and chromatin condensation in apoptosis.
Asunto(s)
Apoptosis , Cromatina/genética , Cromatina/metabolismo , Fragmentación del ADN , Histonas/metabolismo , Apoptosis/genética , Apoptosis/efectos de la radiación , Línea Celular Tumoral , Fragmentación del ADN/efectos de la radiación , Humanos , Rayos UltravioletaRESUMEN
Photopolymerization provides a favorable method for hydrogel formation due to its simplicity, convenience, and versatility. However, the light exposure required to initiate photopolymerization is known to have a cytotoxic effect on encapsulated cells. Here, a 3D in vitro model of the nervous system microenvironment, micropatterned through the use of digital projection photolithography using a single hydrogel formulation that cross-links similarly under ultraviolet A (UVA, 315-400 nm) and visible light (400-700 nm) exposure, is presented. This setup allowed for the investigation of neuronal responses to different light wavelengths and exposure times during photoencapsulation, while ruling out effects due to the hydrogel formulation or photoinitiators used. Cellular studies-including neurite viability, DNA fragmentation, and neurite outgrowth for both UVA and visible light irradiation, the most common spectra used in biological photomicropatterning applications-were performed to assess the effect of light source on neuronal cultures. These studies indicated that while cell death occurs after exposure to either spectrum, visible light was less phototoxic than UVA, when using comparable levels of irradiation, and interestingly, glial cells were more susceptible to phototoxicity than neuronal cells. Thus, while utilizing visible light for micropatterning and cell encapsulation for nervous system applications is beneficial, it is helpful to keep the light exposure low to ensure optimal neuronal survival and growth. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 134-144, 2019.
Asunto(s)
Técnicas de Cultivo de Célula , Fragmentación del ADN/efectos de la radiación , Hidrogeles/química , Neuritas/metabolismo , Rayos Ultravioleta/efectos adversos , Animales , Ratas , Ratas Long-EvansRESUMEN
Fucoidan from brown alga Fucus evanescens and its product of enzymatic hydrolysis have precisely established structure and possess significant biological activities. The aim of present study was to determine radiosensitizing activity of fucoidan from brown alga F. evanescens and its derivative in human melanoma, breast adenocarcinoma, and colorectal carcinoma cell lines and elucidate mechanism of their action. The fucoidan from F. evanescens and its derivative had a comparable radiosensitizing activity and increased the inhibiting effect of X-ray radiation on proliferation and colony formation of human cancer cells, with significant inhibition of melanoma cells. The molecular mechanism of this action was associated with the induction of apoptosis by activating the initiator and effector caspases, suppressing the expression of the anti-apoptotic protein, and enhancing the fragmentation of DNA. The obtained data confirm the prospects of using fucoidan's derivative in combination with radiation therapy for the improvement of the schemes of cancer therapy.
Asunto(s)
Antineoplásicos/farmacología , Polisacáridos/farmacología , Fármacos Sensibilizantes a Radiaciones/farmacología , Antineoplásicos/aislamiento & purificación , Antineoplásicos/efectos de la radiación , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , ADN/química , ADN/efectos de la radiación , Fragmentación del ADN/efectos de los fármacos , Fragmentación del ADN/efectos de la radiación , Fucus/química , Humanos , Polisacáridos/aislamiento & purificación , Polisacáridos/efectos de la radiación , Fármacos Sensibilizantes a Radiaciones/aislamiento & purificación , Fármacos Sensibilizantes a Radiaciones/efectos de la radiación , Rayos XRESUMEN
The development of UV-B protective mechanisms in aquacultural species is essential for the sustainable production of healthy aqua crop. Freshwater carp Catla catla larvae (13.5 ± 1.12 mg) were fed with a diet containing 0.5% vitamin C (D1) and a control diet (D2) for 40 days. Each group was exposed to two doses of UV-B irradiation: 360 (5 min, D15 min and D25 min) and 720 mJ cm-2 (10 min, D110 min and D210 min) for 15 days. Significantly (p < 0.05) higher survival and average weight were recorded in D1 compared to D2 exposed to the same dose. Also, significantly (p < 0.001) higher nitric oxide synthase and lower thiobarbituric acid reactive substances and heat shock protein 70 levels were recorded in D15 min compared to the other groups. A direct relationship was found between the dose of UV-B and DNA fragmentation in muscles. DNA damage indices such as tail DNA, tail extent moment and olive tail moment were significantly (p < 0.01) lower in D15 min. Thus, supplementation of vitamin C in the diet provides UV-B protection to larvae.
Asunto(s)
Ácido Ascórbico/farmacología , Carpas/crecimiento & desarrollo , Suplementos Dietéticos , Protección Radiológica , Protectores Solares/farmacología , Rayos Ultravioleta/efectos adversos , Animales , Antioxidantes/farmacología , Fragmentación del ADN/efectos de los fármacos , Fragmentación del ADN/efectos de la radiación , Proteínas HSP70 de Choque Térmico/metabolismo , Larva/efectos de la radiación , Modelos Animales , Óxido Nítrico Sintasa/metabolismo , Tiobarbitúricos/metabolismoRESUMEN
The use of artificial insemination in cattle breeding has evolved to global extent, and insemination doses are often shipped via air transport which requires strict radiation-based examinations. For the determination of effect of non-ionizing radiation (NIR), to which are beings frequently exposed due to protection of airport or cultural event security, freshly ejaculated and cryopreserved bovine spermatozoa were used as experimental model. Following radiation with hand-held metal detector in various exposition times (0, 10 s, 15, 30 and 60 min-groups FR, FR10, FR15, FR30 and FR60) the spermatozoa underwent motility and DNA fragmentation analyses. Study on cryoconserved semen treated with NIR was performed in time intervals 0, 10 s, 1 and 5 min (insemination doses radiated before cryoconservation-CB, CB10, CB1, CB5; samples radiated after freezing-CA, CA10, CA1 and CA5). Fresh semen and insemination doses radiated after cryoconservation showed significantly lower total and progressive motility. No effect on motility parameters was detected in semen extended with cryopreservative medium and radiated prior to freezing. Surprisingly, NIR showed a potential to stimulate spermatozoa velocity; however, the effect was modulated throughout the post-thawing incubation. Based on the DNA fragmentation assay, sperm DNA stayed intact. Present study underlines the potential harm of NIR, which is frequently used in everyday life, with overall adverse impact on human and animal reproduction. Current study also points out on interesting short-term spermatozoa stimulation induced by NIR.
Asunto(s)
Criopreservación/métodos , Campos Electromagnéticos/efectos adversos , Preservación de Semen/métodos , Espermatozoides/fisiología , Espermatozoides/efectos de la radiación , Animales , Bovinos , Criopreservación/veterinaria , Fragmentación del ADN/efectos de la radiación , Inseminación Artificial/veterinaria , Masculino , Radiación no Ionizante , Semen/fisiología , Preservación de Semen/veterinaria , Motilidad Espermática/efectos de la radiaciónRESUMEN
A compromised detection of radiation-induced plasmid DNA fragments results in underestimation of calculated damage yields. Electrophoretic methods are easy and cheap, but they can only detect a part of the fragments, neglecting the shortest ones. These can be detected with atomic force microscopy, but at the expense of time and price. Both methods were used to investigate their capabilities to detect the DNA fragments induced by high-energetic heavy ions. The results were taken into account in calculations of radiation-induced yields of single and double strand breaks. It was estimated that the double strand break yield is twice as high when the fragments are at least partially detected with the agarose electrophoresis, compared to when they were completely omitted. Further increase by 13% was observed when the measured fragments were corrected for the fraction of the shortest fragments up to 300 base pairs, as detected with the atomic force microscopy. The effect of fragment detection on the single strand break yield was diminished.
Asunto(s)
Roturas del ADN/efectos de la radiación , Fragmentación del ADN/efectos de la radiación , Electroforesis/métodos , Microscopía de Fuerza Atómica/métodos , Iones Pesados , Transferencia Lineal de Energía , PlásmidosRESUMEN
Sulfasalazine (SSZ) is a well-known anti-inflammatory drug and also an inhibitor of the cystine-glutamate antiporter that is known to reduce intracellular glutathione (GSH) level and increase cellular oxidative stress, indicating its anti-tumor potential. However, the combination of SSZ with other physical modalities remains unexplored. Here, the effects of SSZ on cold atmospheric helium plasma (He-CAP), which produces approximately 24 x higher concentration of hydroxyl radicals (. OH) compared to X-irradiation (IR) in aqueous solution, and on IR-induced apoptosis in human leukemia Molt-4 cells were studied to elucidate the mechanism of apoptosis enhancement. Both the Annexin V-FITC/PI and DNA fragmentation assay revealed that pre-treatment of cells with SSZ significantly enhanced He-CAP and IR-induced apoptosis. Similar enhancement was observed during the loss of mitochondrial membrane potential, intracellular Ca2+ ions, and mitochondria- and endoplasmic reticulum-related proteins. The concentration of intracellular reactive oxygen species (ROS) was much higher in He-CAP treated cells than in X-irradiated cells. On the other hand, strong enhancement of Fas expression and caspase-8 and -3 activities were only observed in X-irradiated cells. It might be possible that the higher concentration of intracellular and extracellular ROS suppressed caspase activities and Fas expression in He-CAP-treated cells. Notably, pretreating the cells with an antioxidant N-acetyl-L-cysteine (NAC) dramatically decreased apoptosis in cells treated by He-CAP, but not by IR. These results suggest that IR-induced apoptosis is due to specific and effective ROS distribution since intracellular ROS formation is marginal and the high production of ROS inside and outside of cells plays unique roles in He-CAP induced apoptosis. We conclude that our data provides efficacy and mechanistic insights for SSZ, which might be helpful for establishing SSZ as a future sensitizer in He-CAP or IR therapy for cancer.
Asunto(s)
Radical Hidroxilo/metabolismo , Oxidantes/farmacología , Gases em Plasma/farmacología , Sulfasalazina/farmacología , Linfocitos T/metabolismo , Acetilcisteína/farmacología , Antioxidantes/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Calcio/metabolismo , Caspasa 3/genética , Caspasa 3/metabolismo , Caspasa 8/genética , Caspasa 8/metabolismo , Cationes Bivalentes , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Fragmentación del ADN/efectos de los fármacos , Fragmentación del ADN/efectos de la radiación , Regulación de la Expresión Génica , Células HCT116 , Helio/química , Humanos , Radical Hidroxilo/agonistas , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Potencial de la Membrana Mitocondrial/efectos de la radiación , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/efectos de la radiación , Estrés Oxidativo , Transducción de Señal , Sulfasalazina/antagonistas & inhibidores , Linfocitos T/efectos de los fármacos , Linfocitos T/patología , Linfocitos T/efectos de la radiación , Rayos X , Receptor fas/genética , Receptor fas/metabolismoRESUMEN
Bone pain is the major manifestation of skeletal metastases. Although various treatment modalities are available for bone pain palliation, use of radiolabeled phosphonates is documented to be more effective. Among radionuclides available for this purpose, lutetium-177 is gaining popularity due to its moderate beta energy, theranostic capability, favorable half-life and convenient production logistics. 177 Lu-DOTMP has shown considerable promise as a metastatic bone pain palliating agent in preliminary evaluations and recent clinical studies. Therefore, an attempt was made to elucidate the possible mechanism of in vitro cell death induced by 177 Lu-DOTMP in MG63 cells. 177 Lu-DOTMP binding studies were carried out in mineralized bone of MG63 cells and around 50% binding was observed. Skeletons of Wistar rats showed 1.78 ± 0.5% IA/g at a 3 h time period which was almost constant up to 7 days. MG63 cells were incubated with 3.7 and 37 MBq of 177 Lu-DOTMP for 48 h prior to perform assays. An increase in the magnitude of cell toxicity and apoptotic DNA fragmentation was observed. Enhancement of G2/M phase cell cycle arrest and apoptosis were documented which were dose-dependent. Thus, 177 Lu-DOTMP induced apoptotic cell death in MG63 cells, which might be one of the primary causes of pain relief in osseous metastases.
Asunto(s)
Apoptosis/efectos de la radiación , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de la radiación , Lutecio/uso terapéutico , Puntos de Control de la Fase M del Ciclo Celular/efectos de la radiación , Compuestos Organofosforados/farmacología , Osteosarcoma/patología , Radioisótopos/uso terapéutico , Animales , Densidad Ósea/efectos de la radiación , Línea Celular Tumoral , Fragmentación del ADN/efectos de la radiación , Humanos , Compuestos Organofosforados/farmacocinética , Ratas Wistar , Distribución TisularRESUMEN
Acute respiratory distress syndrome (ARDS) and acute lung injury (ALI) are defined as pulmonary inflammation that could occur from sepsis and lead to pulmonary permeability and alveolar edema making them life-threatening diseases. Photobiomodulation (PBM) properties have been widely described in the literature in several inflammatory diseases; although the mechanisms of action are not always clear, this could be a possible treatment for ARDS/ALI. Thus, the aim of this study was to evaluate the mRNA levels from caspase-3 and BCL-2 genes and DNA fragmentation in lung tissue from Wistar rats affected by ALI and subjected to photobiomodulation by exposure to a low power infrared laser (808 nm; 100 mW; 3.571 W cm-2; four points per lung). Adult male Wistar rats were randomized into 6 groups (n = 5, for each group): control, PBM10 (10 J cm-2, 2 J and 2 seconds), PBM20 (20 J cm-2, 5 J and 5 seconds), ALI, ALI + PBM10 and ALI + PBM20. ALI was induced by intraperitoneal Escherichia coli lipopolysaccharide injection. Lung samples were collected and divided for mRNA expression of caspase-3 and Bcl-2 and DNA fragmentation quantifications. Data show that caspase-3 mRNA levels are reduced and Bcl-2 mRNA levels increased in ALI after low power infrared laser exposure when compared to the non-exposed ALI group. DNA fragmentation increased in inflammatory infiltrate cells and reduced in alveolar cells. Our research shows that photobiomodulation can alter relative mRNA levels in genes involved in the apoptotic process and DNA fragmentation in inflammatory and alveolar cells after lipopolysaccharide-induced acute lung injury. Also, inflammatory cell apoptosis is part of the photobiomodulation effects induced by exposure to a low power infrared laser.
Asunto(s)
Lesión Pulmonar Aguda/terapia , Caspasa 3/genética , Fragmentación del ADN/efectos de la radiación , Genes bcl-2/efectos de la radiación , Terapia por Luz de Baja Intensidad , Pulmón/patología , ARN Mensajero/genética , Lesión Pulmonar Aguda/genética , Lesión Pulmonar Aguda/patología , Animales , Apoptosis/efectos de la radiación , Regulación de la Expresión Génica/efectos de la radiación , Rayos Infrarrojos/uso terapéutico , Pulmón/metabolismo , Pulmón/efectos de la radiación , Masculino , Ratas WistarRESUMEN
Radiation therapy is critical for the control of many tumors and lung is an important dose-limiting organ that impacts radiation dose prescribed to avoid irreversible pulmonary fibrosis in cancer survivors. Idiopathic pulmonary fibrosis (IPF) is a chronic, irreversible lung disease caused by aberrantly activated lung (myo)fibroblasts. The presence of pro-fibrotic, apoptosis-resistant fibroblasts in IPF promotes progressive fibrosis and may have a role in other diseases, if these resistant cells are selected for as a consequence of treatment. However, the pathological response of IPF fibroblasts to radiation compared to non-IPF lung fibroblasts is not known. To address this, we examined fibroblast viability following radiation in lung fibroblasts from IPF and non-IPF patients and the underlying mechanism that protects IPF fibroblasts from radiation-induced death. IPF fibroblasts are significantly more resistant to apoptosis compared to non-IPF lung fibroblasts, suggesting that resistance to radiation-induced cell death is a predominant mechanism leading to lung fibrosis. Analysis of γH2AX induction demonstrated that radiation-induced DNA damage is reduced in IPF fibroblasts and correlates to the activation of the transcription factor forkhead box M1 (FoxM1) and subsequent upregulation of DNA repair proteins RAD51 and BRCA2. FoxM1 activation occurs secondary to FoxO3a suppression in IPF fibroblasts while restoration of FoxO3a function sensitizes IPF fibroblasts to radiation-induced cell death and downregulates FoxM1, RAD51, and BRCA2. Our findings support that increased FoxO3a/FoxM1-dependent DNA repair may be integral to the preservation of death-resistant fibrotic fibroblasts after radiation and that selective targeting of radioresistant fibroblasts may mitigate fibrosis.