Earlier experimental studies have demonstrated that: i) Cu,Zn-superoxide dismutase deficiency leads to oxidative stress and carcinogenesis; ii) dysregulation of NF-κB pathway can mediate a wide variety of diseases, including cancer. Therefore, we decided, for the first time, to examine the level of oxidative DNA damage and the DNA binding activity of NF-κB proteins in SOD1 knockout, heterozygous and wild-type mice. Two kinds of biomarkers of oxidatively damaged DNA: urinary excretion of 8-oxodG and 8-oxoGua, and the level of oxidatively damaged DNA were analysed using HPLC-GC-MS and HPLC-EC. The DNA binding activity of p50 and p65 proteins in a nuclear extracts was assessed using NF-κB p50/p65 EZ-TFA transcription factor assay. These parameters were determined in the brain, liver, kidney and urine of SOD1 knockout, heterozygous and wild-type mice. The level of 8-oxodG in DNA was higher in the liver and kidney of knockout mice than in wild type. No differences were found in urinary excretion of 8-oxoGua and 8-oxodG between wild type and the SOD1-deficient animals. The activity of the p50 protein was higher in the kidneys, but surprisingly not in the livers of SOD1-deficient mice, whereas p65 activity did not show any variability. Our results indicate that in Cu,Zn-SOD-deficient animals the level of oxidative DNA damage and NF-κB1 activity are elevated in certain organs only, which may provide some explanation for organ-specific ROS-induced carcinogenesis.
Abstract Large-scale radiation emergency scenarios involving protracted low dose rate radiation exposure (e.g. a hidden radioactive source in a train) necessitate the development of high throughput methods for providing rapid individual dose estimates. During the RENEB (Running the European Network of Biodosimetry) 2019 exercise, four EDTA-blood samples were exposed to an Iridium-192 source (1.36 TBq, Tech-Ops 880 Sentinal) at varying distances and geometries. This resulted in protracted doses ranging between 0.2 and 2.4 Gy using dose rates of 1.5–40 mGy/min and exposure times of 1 or 2.5 h. Blood samples were exposed in thermo bottles that maintained temperatures between 39 and 27.7 °C. After exposure, EDTA-blood samples were transferred into PAXGene tubes to preserve RNA. RNA was isolated in one laboratory and aliquots of four blinded RNA were sent to another five teams for dose estimation based on gene expression changes. Using an X-ray machine, samples for two calibration curves (first: constant dose rate of 8.3 mGy/min and 0.5–8 h varying exposure times; second: varying dose rates of 0.5–8.3 mGy/min and 4 h exposure time) were generated for distribution. Assays were run in each laboratory according to locally established protocols using either a microarray platform (one team) or quantitative real-time PCR (qRT-PCR, five teams). The qRT-PCR measurements were highly reproducible with coefficient of variation below 15% in ≥ 75% of measurements resulting in reported dose estimates ranging between 0 and 0.5 Gy in all samples and in all laboratories. Up to twofold reductions in RNA copy numbers per degree Celsius relative to 37 °C were observed. However, when irradiating independent samples equivalent to the blinded samples but increasing the combined exposure and incubation time to 4 h at 37 °C, expected gene expression changes corresponding to the absorbed doses were observed. Clearly, time and an optimal temperature of 37 °C must be allowed for the biological response to manifest as gene expression changes prior to running the gene expression assay. In conclusion, dose reconstructions based on gene expression measurements are highly reproducible across different techniques, protocols and laboratories. Even a radiation dose of 0.25 Gy protracted over 4 h (1 mGy/min) can be identified. These results demonstrate the importance of the incubation conditions and time span between radiation exposure and measurements of gene expression changes when using this method in a field exercise or real emergency situation.
This collaboration of five established European gene expression labs investigated the potential impact of culture conditions on the transcriptional response of peripheral blood to radiation exposure.Blood from one healthy donor was exposed ex vivo to a Cobalt 60 source to produce a calibration curve in addition to four unknown doses. After exposure, the blood samples were either diluted with RPMI medium or left untouched. After 24-h incubation at 37 °C the diluted blood samples were lysed, while the undiluted samples were mixed with the preservative RNALater and all samples were shipped frozen to the participating labs. Samples were processed by each lab using microarray (one lab) and QRT-PCR (four labs).We show that although culture conditions affect the total amount of RNA recovered (p < .0001) and its integrity (p < .0001), it does not significantly affect dose estimates (except for the true dose at 1.1 Gy). Most importantly, the different analysis approaches provide comparable mean absolute difference of estimated doses relative to the true doses (p = .9) and number of out of range (>0.5 Gy) measurements (p = .6).This study confirms the robustness of gene expression as a method for biological dosimetry.
The incidence of ischemic heart disease (IHD) in patients with OSAS is estimated at around 20%. This greatly affect a common risk factors for both diseases: male gender, obesity, age and diabetes and hypertension. Attention is drawn to the possibility of genetic determinants of IHD. The aim of study was to answer the question whether the presence of polymorphisms of selected genes possibly related to IHD may be useful to isolate the group of patients with OSAS, especially vulnerable as a complication of IHD. Materials and methods. The study included 600 people with OSAS, which was isolated in patients with IHD (127 people). The remaining 473 individuals were observed as a control group. The polymorphism of three genes were evaluated to find possible influence on the occurrence of IHD or myocardial infarction as follows: SREBF1 (sterol regulatory element binding transcription factor 1), REBF2 (sterol regulatory element binding transcription factor 2) and HIF1 (hypoxia inducible factor 1, alpha subunit). Results. Analysis of relationship between polymorphisms of selected genes and the diagnosis of IHD in the whole group of patients with OSAS showed a relationship only for the gene SREBF1 finding the lowest frequency of its occurrence in AA homozygotes (at 13.6%) and twice with GG homozygotes (26.1%). Conclusions. Rating polymorphisms studied genes did not reveal their relationship to the occurrence of IHD in patients with OSAS, both in the whole group as well as separate subgroups.
OSA increases the risk of arterial hypertension (AH) and coronary heart disease (CHD). One of the directions for the research aimed at the identification of patients particularly susceptible to serious cardiovascular complications is the assessment gene polymorphism of the genes associated with an increased risk of cardiovascular diseases. Aim: Evaluation of some genetic polymorphisms that may affect the incidence of cardiovascular disease in patients with obstructive sleep apnea. Material and Methods: We enrolled 600 patients (449M, 151F) aged 54.4±10.3 yrs with OSA. The following gene polymorphisms were evaluated by real-time PCR in peripheral blood: ALOX5AP (arachidonate 5- lipoxygenase-activating protein), LPL (lipoprotein lipase), SREBF1 (sterol regulatory element binding transcription factor 1), SREBF2 (sterol regulatory element binding transcription factor 2), APOA5 (apolipoprotein A-V) and FTO (fat mass and obesity associated protein). Correlations between these polymorphisms and the diagnosis of CHD, myocardial infarction and AH were evaluated. Results: AH was diagnosed in 427 (71.2%), CHD in 127 (21.2%) and myocardial infarction in 26 (4.3%). We observed a statistically significant correlation only for SREBF1 homozygous GG gene and coronary heart disease (in 72 out of 127 patients were homozygous GG, p <0.05) in the whole group. We found a higher incidence heterozygous AG for FTO gene in women with CHD (16.7% vs 6.3%, p <0.05) and in women with myocardial infarction (60% vs. 6.1%, p <0.0001). Conclusions: Determination of polymorphism of SREBF1 and FTO genes may be useful in assessing the risk of cardiovascular complications in patients with OSA.
Introduction: Obstructive sleep apnoea (OSA) in an independent risk factor for arterial hypertension development. Both OSA and arterial hypertension frequently coexist, thus common genetic background can be suspected.
Material and methods: We enrolled 600 participants with diagnosis of OSA in all stages of severity from 2 tertiary care outpatient clinics in Warsaw, Poland. All patients undergone polysomnography and were evaluated for the diagnosis of hypertension. Gene polymorphisms for genes encoding ALOX5AP, SREBF2, ADRA2A, APOA5 and TCF7L2 were analyzed in blood samples. Chisquare and Mann-Whitney tests were used for comparisons.
Results: Data of 449 (74.8%) men and 151 (25.2%) women were analyzed. Arterial hypertension was diagnosed in 427 (71.2%) participants. In the hypertensive patients with OSA median age was 59 (53–64) years, BMI 32.8 (28.9– 37.3) kg/m2, AHI 39 (24–60)/hour. Among tested genes only TCF7L2 polymorphism ([C/T] rs7903146) was linked to increased incidence of arterial hypertension.
Conclusions: Results of our analysis suggest potential relationship of TCF7L2 polymorphism ([C/T] rs7903146) and development of hypertension in patients with OSA, although further studies regarding impact of genetic in comparison with traditional risk factor for hypertension are needed.
Tools for radiation exposure reconstruction are required to support the medical management of radiation victims in radiological or nuclear incidents. Different biological and physical dosimetry assays can be used for various exposure scenarios to estimate the dose of ionizing radiation a person has absorbed. Regular validation of the techniques through inter-laboratory comparisons (ILC) is essential to guarantee high quality results. In the current RENEB inter-laboratory comparison, the performance quality of established cytogenetic assays [dicentric chromosome assay (DCA), cytokinesis-block micronucleus assay (CBMN), stable chromosomal translocation assay (FISH) and premature chromosome condensation assay (PCC)] was tested in comparison to molecular biological assays [gamma-H2AX foci (gH2AX), gene expression (GE)] and physical dosimetry-based assays [electron paramagnetic resonance (EPR), optically or thermally stimulated luminescence (LUM)]. Three blinded coded samples (e.g., blood, enamel or mobiles) were exposed to 0, 1.2 or 3.5 Gy X-ray reference doses (240 kVp, 1 Gy/min). These doses roughly correspond to clinically relevant groups of unexposed to low exposed (0-1 Gy), moderately exposed (1-2 Gy, no severe acute health effects expected) and highly exposed individuals (>2 Gy, requiring early intensive medical care). In the frame of the current RENEB inter-laboratory comparison, samples were sent to 86 specialized teams in 46 organizations from 27 nations for dose estimation and identification of three clinically relevant groups. The time for sending early crude reports and more precise reports was documented for each laboratory and assay where possible. The quality of dose estimates was analyzed with three different levels of granularity, 1. by calculating the frequency of correctly reported clinically relevant dose categories, 2. by determining the number of dose estimates within the uncertainty intervals recommended for triage dosimetry (±0.5 Gy or ±1.0 Gy for doses <2.5 Gy or >2.5 Gy), and 3. by calculating the absolute difference (AD) of estimated doses relative to the reference doses. In total, 554 dose estimates were submitted within the 6-week period given before the exercise was closed. For samples processed with the highest priority, earliest dose estimates/categories were reported within 5-10 h of receipt for GE, gH2AX, LUM, EPR, 2-3 days for DCA, CBMN and within 6-7 days for the FISH assay. For the unirradiated control sample, the categorization in the correct clinically relevant group (0-1 Gy) as well as the allocation to the triage uncertainty interval was, with the exception of a few outliers, successfully performed for all assays. For the 3.5 Gy sample the percentage of correct classifications to the clinically relevant group (≥2 Gy) was between 89-100% for all assays, with the exception of gH2AX. For the 1.2 Gy sample, an exact allocation to the clinically relevant group was more difficult and 0-50% or 0-48% of the estimates were wrongly classified into the lowest or highest dose categories, respectively. For the irradiated samples, the correct allocation to the triage uncertainty intervals varied considerably between assays for the 1.2 Gy (29-76%) and 3.5 Gy (17-100%) samples. While a systematic shift towards higher doses was observed for the cytogenetic-based assays, extreme outliers exceeding the reference doses 2-6 fold were observed for EPR, FISH and GE assays. These outliers were related to a particular material examined (tooth enamel for EPR assay, reported as kerma in enamel, but when converted into the proper quantity, i.e. to kerma in air, expected dose estimates could be recalculated in most cases), the level of experience of the teams (FISH) and methodological uncertainties (GE). This was the first RENEB ILC where everything, from blood sampling to irradiation and shipment of the samples, was organized and realized at the same institution, for several biological and physical retrospective dosimetry assays. Almost all assays appeared comparably applicable for the identification of unexposed and highly exposed individuals and the allocation of medical relevant groups, with the latter requiring medical support for the acute radiation scenario simulated in this exercise. However, extreme outliers or a systematic shift of dose estimates have been observed for some assays. Possible reasons will be discussed in the assay specific papers of this special issue. In summary, this ILC clearly demonstrates the need to conduct regular exercises to identify research needs, but also to identify technical problems and to optimize the design of future ILCs.
Nuclear factor kappa B (NF-κB) signalling pathway plays a central role in the regulation of cellular response to stress. The aim of the study was to investigate the ability of silver nanoparticles (AgNPs), gold nanoparticles (AuNPs), CdTe quantum dots (CdTeQDs) or their binary mixtures to stimulate NF-κB binding in HepG2 cells. A dual luciferase reporter system was used to investigate NF-κB binding.Cells were transiently transfected with a firefly luciferase reporter system and Renilla luciferase expression plasmid as a transfection efficiency control. Twenty- four hours after transfection, the cells were treated with nanoparticles (10 μg/cm3 AgNPs, 10 μg/cm3 AuNPs, 3 μg/cm3 CdTeQDs) or with 10 ng/cm3 TNFα as a positive control. Six hours later, the cells were lysed and the activities of the luminescence of firefly and Renilla luciferases were measured using the Dual-Luciferase Reporter Assay System.AuNPs and CdTeQDs alone significantly inhibited NF-κB binding activity. Co-treatment with AgNPs and CdTeQDs resulted in an additive effect, whereas the presence of AgNPs diminished the inhibitory effect of AuNPs. Interestingly, significant antagonism was observed between AuNPs and CdTeQDs, suggesting a similar mode of action.Comparison of the NF-κB binding activity induced by the mixtures of NPs suggests that in some cases NF-κB binding activity might differ from that observed for the NPs alone.
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