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International journal of molecular sciences, 21
February, 2020

Micronucleus Assay: The State of Art, and Future Directions.

Sommer, Sylwester, Buraczewska, Iwona, Kruszewski, Marcin

<p>During almost 40 years of use, the micronucleus assay (MN) has become one of the most popular methods to assess genotoxicity of different chemical and physical factors, including ionizing radiation-induced DNA damage. In this minireview, we focus on the position of MN among the other genotoxicity tests, its usefulness in different applications and visibility by international organizations, such as International Atomic Energy Agency, Organization for Economic Co-operation and Development and International Organization for Standardization. In addition, the mechanism of micronuclei formation is discussed. Finally, foreseen directions of the MN development are pointed, such as automation, buccal cells MN and chromothripsis phenomenon.</p>

Digital object identifier (DOI): 10.3390/ijms21041534

International journal of radiation biology, 96, 214--219
February, 2020

An alternative approach for the induction of premature chromosome condensation in human peripheral blood lymphocytes using mitotic Akodon cells.

Selvan Gnana Sekaran, Tamizh, Ricoul, Michelle, Brochard, Patricia, Herate, Cecile, Sabatier, Laure

<p>The premature chromosome condensation (PCC) technique is used to study exposure to external radiation through the determination of chromosome fragments observed in interphase cells. The presence of large telomeric signals in CHO cells interferes with the detection of PCC fragments and the identification of dicentric chromosomes. We present an improved method for the fusion of G0-lymphocytes with mitotic cells (few chromosomes and weakly-staining telomeric sequences) to induce PCC in combination with rapid quantification of dicentric chromosomes and centric rings as an alternative to the classical CHO cell fusion technique. Whole blood from three healthy volunteers was γ-irradiated with 0, 2, or 4 Gy. Following a 24 h incubation post-exposure at 37 °C, chromosome spreads of isolated lymphocytes were prepared by standard PCC procedures using mitotic cells. The percentage of scorable fusions, measured by telomere/centromere (T/C) staining, for -induced PCC was higher than that for CHO-induced PCC, irrespective of radiation exposure. Importantly, both techniques gave the same result for biodosimetry evaluation. The mitotic cell-induced PCC fusion assay, in combination with the scoring of dicentric chromosomes and rings by T/C staining of G0-lymphocytes is a suitable alternative for fast and reliable dose estimation after accidental radiation exposure.</p>

Digital object identifier (DOI): 10.1080/09553002.2019.1625493

Scientific reports, 10, 2899
February, 2020

A High Throughput Approach to Reconstruct Partial-Body and Neutron Radiation Exposures on an Individual Basis.

Shuryak, Igor, Turner, Helen C., Perrier, Jay R., Cunha, Lydia, Canadell, Monica Pujol, Durrani, Mohammad H., Harken, Andrew, Bertucci, Antonella, Taveras, Maria, Garty, Guy, Brenner, David J.

<p>Biodosimetry-based individualized reconstruction of complex irradiation scenarios (partial-body shielding and/or neutron + photon mixtures) can improve treatment decisions after mass-casualty radiation-related incidents. We used a high-throughput micronucleus assay with automated scanning and imaging software on ex-vivo irradiated human lymphocytes to: a) reconstruct partial-body and/or neutron exposure, and b) estimate separately the photon and neutron doses in a mixed exposure. The mechanistic background is that, compared with total-body photon irradiations, neutrons produce more heavily-damaged lymphocytes with multiple micronuclei/binucleated cell, whereas partial-body exposures produce fewer such lymphocytes. To utilize these differences for biodosimetry, we developed metrics that describe micronuclei distributions in binucleated cells and serve as predictors in machine learning or parametric analyses of the following scenarios: (A) Homogeneous gamma-irradiation, mimicking total-body exposures, vs. mixtures of irradiated blood with unirradiated blood, mimicking partial-body exposures. (B) X rays vs. various neutron + photon mixtures. The results showed high accuracies of scenario and dose reconstructions. Specifically, receiver operating characteristic curve areas (AUC) for sample classification by exposure type reached 0.931 and 0.916 in scenarios A and B, respectively. R for actual vs. reconstructed doses in these scenarios reached 0.87 and 0.77, respectively. These encouraging findings demonstrate a proof-of-principle for the proposed approach of high-throughput reconstruction of clinically-relevant complex radiation exposure scenarios.</p>

Digital object identifier (DOI): 10.1038/s41598-020-59695-9

Mutation research, 849, 503141
January, 2020

Premature chromosome condensation assay to study influence of high-level natural radiation on the initial DNA double strand break repair in human G, [email protected], lymphocytes.

Vivek Kumar, P. R., Karuppasamy, C. V., Ramachandran, E. N., Anil Kumar, V., Jaikrishan, G., Das, Birajalaxmi

<p>The inherent capacity of individuals to efficiently repair ionizing radiation induced DNA double strand breaks (DSBs) may be inherited, however, it is influenced by several epigenetic and environmental factors. A pilot study tested whether chronic low dose natural radiation exposure influences the rejoining of initial DNA DSBs induced by a 2 Gy γ-irradiation in 22 individuals from high (&gt;1.5 mGy/year) and normal (≤1.5 mGy/year) level natural radiation areas (H&amp;NLNRA) of Kerala. Rejoining of DSBs (during 1 h at 37 °C, immediately after irradiation) was evaluated at the chromosome level in the presence and absence of wortmannin (a potent inhibitor of DSB repair in normal human cells) using a cell fusion-induced premature chromosome condensation (PCC) assay. The PCC assay quantitates DSBs in the form of excess chromosome fragments in human G lymphocytes without the requirement for cell division. A quantitative difference was observed in the early rejoining of DNA DSBs between individuals from HLNRA and NLNRA, with HLNRA individuals showing a higher (P = 0.05) mean initial repair ratio. The results indicate an influence of chronic low dose natural radiation on initial DNA DSB repair in inhabitants of HLNRA of the Kerala coast.</p>

Digital object identifier (DOI): 10.1016/j.mrgentox.2020.503141

Mutation research, 849, 503086
January, 2020

Analysis of historical negative control group data from the rat in vivo micronucleus assay.

Lovell, D. P., Fellows, M., Saul, J., Whitwell, J., Custer, L., Dertinger, S., Escobar, P., Fiedler, R., Hemmann, U., Kenny, J., Smith, R., van der Leede, B. M., Zeller, A.

<p>A database of micronuclei counts for historical negative control data from rat in vivo micronuclei tests performed in 10 different laboratories was established. Data were available from over 4000 negative control rats from 10 laboratories. The mean frequency of micronucleated cells (MN)/1000 cells ranged from 0.44 to 2.22, a 5-fold range. Overall there were no major sex or strain differences in frequency, although there were some small but statistically significant differences within laboratories. There was appreciable variability between experiments compared with variability within experiments in some laboratories. No specific factor was identified which could explain this variability although it was noted that many different vehicles were used in the experiments. It is hoped that these data will help laboratories beginning studies with the rat micronucleus assay and those involved in the assessment of micronucleus assay results.</p>

Digital object identifier (DOI): 10.1016/j.mrgentox.2019.503086

Health physics, 117, 618--624
December, 2019

Biological Dosimetry Network in Africa: Establishment of a Dose-Response Curve Using Telomere and Centromere Staining.

Soumboundou, Mamadou, Nkengurutse, Innocent, Dossou, Julien, Colicchio, Bruno, Djebou, Catherine, Gadji, Macoura, Houenon, Germain, Dem, Ahmadou, Dedjan, Alexandre, Diarra, Mounibé, Adjibade, Rachad, Finot, Francis, Hempel, William, Dieterlen, Alain, Jeandidier, Eric, Rodriguez-Lafrasse, Claire, M'kacher, Radhia

<p>Biological dosimetry, based on the relationship between the absorbed dose after exposure to ionizing radiation and the frequency of scored aberrations, has been and continues to be an important tool for estimating the dose after exposure. Dicentric chromosomes are considered to be the most specific and sensitive aberration related to radiation exposure. Here, we established the dose-response curve following in vitro irradiation of circulating lymphocytes from healthy donors from three African countries after scoring unstable chromosomal aberrations. Blood samples from 16 African donors were exposed to various doses (0 to 4 Gy) using an X-RAD320 x-ray system with a maximum photon energy of 250 kV at a dose rate of 0.1 Gy min. Blood lymphocytes were cultured for 48 h, and chromosomal aberrations were scored during the first mitosis by telomere and centromere staining. The distribution of dicentric chromosomes was determined. No dicentric chromosomes were found after the analysis of 2,669 first-division metaphases before in vitro exposure. We established a linear-quadratic dose-response curve based on the frequency of dicentric and ring chromosomes and calculated double-strand breaks, taking into account all scored aberrations. The generation of a specific dose-response curve for African donors will allow the practice of precise biological dosimetry in these countries. This work is the first step towards realizing an African biodosimetry network and the establishment of a biological dosimetry laboratory, which could play a major role in the application of radioprotection norms.</p>

Digital object identifier (DOI): 10.1097/HP.0000000000001102

European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences, 129, 181–189
March, 2019

Multi- and unilamellar liposomal encapsulation of ciprofloxacin as ways to modify its phototoxicity and photodegradation.

Zgadzaj, A, Giebułtowicz, J, Gubernator, J, Podbielska, M, Sommer, S, Zaremba-Czogalla, M, Nałęcz-Jaweckia, G

<p>Liposomes are vesicular preparations that improve bioavailability of many pharmaceuticals, used even in ocular therapy. In addition, it is well documented that vesicular carriers could affect the photodegradation of molecules encapsulated inside, which is especially important for drugs that may exhibit phototoxicity when they are applied topically on sensitive light-exposed tissues. In this study, we investigated the effect of ciprofloxacin encapsulation into liposomes on its photodegradation, phototoxicity and photogenotoxicity in vitro at the concentration ranges applied in ophthalmology. We tested two variants of liposomes: large unilamellar vesicles (LUV) and multilamellar vesicles (MLV) in comparison to antibiotic solutions without phospholipids (CPX). On the basis of our research, the kinetics of ciprofloxacin photolysis was the fastest in formulations with vesicles with low drug-to-lipid ratio. Depending on vesicles type (drug-to-lipid ratio, MLV or LUV) and time of irradiation different degradants were produced. We proposed structures of the novel ciprofloxacin photolysis products characteristic for vesicles. We did not notice any photoprotective effect of application of ciprofloxacin encapsulation into liposomes, but it significantly affected the photodegradation product profile of the drug and the Photo-Irritation-Factor of the vesicular preparations. In the MTT and micronucleus assays impact of encapsulation was not as clearly visible.</p>

Digital object identifier (DOI): 10.1016/j.ejps.2019.01.006

Cells, 8
January, 2019

Oxidative Stress Induces Telomere Dysfunction and Senescence by Replication Fork Arrest.

Coluzzi, Elisa, Leone, Stefano, Sgura, Antonella

<p>Oxidative DNA damage, particularly 8-oxoguanine, represents the most frequent DNA damage in human cells, especially at the telomeric level. The presence of oxidative lesions in the DNA can hinder the replication fork and is able to activate the DNA damage response. In this study, we wanted to understand the mechanisms by which oxidative damage causes telomere dysfunction and senescence in human primary fibroblasts. After acute oxidative stress at telomeres, our data demonstrated a reduction in TRF1 and TRF2, which are involved in proper telomere replication and T-loop formation, respectively. Furthermore, we observed a higher level of γH2AX with respect to 53BP1 at telomeres, suggesting a telomeric replication fork stall rather than double-strand breaks. To confirm this finding, we studied the replication of telomeres by Chromosome Orientation-FISH (CO-FISH). The data obtained show an increase in unreplicated telomeres after hydrogen peroxide treatment, corroborating the idea that the presence of 8-oxoG can induce replication fork arrest at telomeres. Lastly, we analyzed the H3K9me3 histone mark after oxidative stress at telomeres, and our results showed an increase of this marker, most likely inducing the heterochromatinization of telomeres. These results suggest that 8-oxoG is fundamental in oxidative stress-induced telomeric damage, principally causing replication fork arrest.</p>

Digital object identifier (DOI): 10.3390/cells8010019

Annals of laboratory medicine, 39, 91–95
January, 2019

Dose Estimation Curves Following In Vitro X-ray Irradiation Using Blood From Four Healthy Korean Individuals.

Jang, Mi Ae, Han, Eun Ae, Lee, Jin Kyung, Cho, Kwang Hwan, Shin, Hee Bong, Lee, You Kyoung

<p>Cytogenetic dosimetry is useful for evaluating the absorbed dose of ionizing radiation based on analysis of radiation-induced chromosomal aberrations. We created two types of dose-response calibration curves for dicentric chromosomes (DC) and translocations (TR) induced by X-ray irradiation, using an electron linear accelerator, which is the most frequently used medical device in radiotherapy. We irradiated samples from four healthy Korean individuals and compared the resultant curves between individuals. Aberration yields were studied in a total of 31,800 and 31,725 metaphases for DC and TR, respectively, obtained from 11 X-ray irradiation dose-points (0, 0.05, 0.1, 0.25, 0.5, 0.75, 1, 2, 3, 4, and 5 Gy). The dose-response relationship followed a linear-quadratic equation, Y=C+αD+βD², with the coefficients C=0.0011 for DC and 0.0015 for TR, α =0.0119 for DC and 0.0048 for TR, and β=0.0617 for DC and 0.0237 for TR. Correlation coefficients between irradiation doses and chromosomal aberrations were 0.971 for DC and 0.6 for TR, indicating a very strong and a moderate correlation, respectively. This is the first study implementing cytogenetic dosimetry following exposure to ionizing X-radiation.</p>

Digital object identifier (DOI): 10.3343/alm.2019.39.1.91

Chemosphere, 215, 703–709
January, 2019

Nanomaterials induce DNA-protein crosslink and DNA oxidation: A mechanistic study with RTG-2 fish cell line and Comet assay modifications.

Klingelfus, T, Disner, G R, Voigt, C L, Alle, L F, Cestari, M M, Leme, D M

<p>Genotoxic effects of nanomaterials (NMs) have been controversially reported in literature, and the mode of action (MoA) via DNA oxidation is cited as the main damage caused by them. Evidence of nano-silver as a crosslinker has been previously reported by the present research team in an in vivo fish genotoxicity study. Thus, aiming to confirm the evidence about NMs as crosslinker agent, the present investigation elucidated the genotoxic potential of NMs and their genotoxic MoA through in vitro assay with RTG-2 cells line (rainbow trout gonadal) by exposure to nano-silver (PVP-coated) and nano-titanium. The types and levels of DNA damage were assessed by the Comet assay (standard alkaline, hOGG1-modified alkaline, and two crosslink-modified alkaline versions). It was demonstrated that the use of the standard alkaline Comet assay alone may inaccurately predict the genotoxicity of NMs since oxidative and crosslink DNA damages were also verified in RTG-2 cells when assessed by the modified versions of the alkaline protocol. More importantly, it was confirmed that both nano-silver and nano-titanium acted as DNA-protein crosslinkers through the Comet assay version with proteinase K. As both nano-silver and nano-titanium present a great risk to aquatic life, these findings reinforce the need of genotoxicity testing strategies that encompass the assessment of different types of DNA damage, in order to ensure an accurate prediction of the genotoxic potential of NMs.</p>

Digital object identifier (DOI): 10.1016/j.chemosphere.2018.10.118

Mutation Research/Genetic Toxicology and Environmental Mutagenesis, 847, 503087
2019

Optimization and validation of automated dicentric chromosome analysis for radiological/nuclear triage applications

Ryana, Terri L., Escalonaa, Maria B., Smith, Tammy L., Albanese, Joseph, Iddinsa, Carol J., Balajee, Adayabalam S.

<p>Dicentric Chromosome Assay (DCA) is the most preferred cytogenetic technique for absorbed radiation dose assessment in exposed humans. However, DCA is somewhat impractical for triage application owing to its labor intensive and time consuming nature. Although lymphocyte culture for 48 h <em>in vitro</em> is inevitable for DCA, manual scoring of dicentric chromosomes (DCs) requires an additional time of 24–48 h, making the overall turnaround time of 72–96 h for dose estimation. To accelerate the speed of DC analysis for dose estimation, an automated tool was optimized and validated for triage mode of scoring. Several image training files were created to improve the specificity of automated DC analysis algorithm. Accuracy and efficiency of the automated (unsupervised) DC scoring was compared with the semi-automated scoring that involved human verification and correction of DCs (elimination of false positives and inclusion of true positives). DC scoring was performed by both automated and semi-automated modes for different doses of X-rays and γ-rays (0 Gy–5 Gy). Biodoses estimated from the frequencies of DCs detected by both automated (unsupervised) and semi-automated (supervised) scoring modes were grossly similar to the actual delivered doses in the range of 0.5 to 3 Gy of low LET radiation. We suggest that the automated DC tool can be effectively used for large scale radiological/nuclear incidents where a rapid segregation is essential for prioritizing moderately or severely exposed humans to receive appropriate medical countermeasures.</p>

Digital object identifier (DOI): https://doi.org/10.1016/j.mrgentox.2019.503087

Radiation protection dosimetry, 182, 139–145
December, 2018

DEVELOPMENT OF A MINIATURIZED VERSION OF DICENTRIC CHROMOSOME ASSAY TOOL FOR RADIOLOGICAL TRIAGE.

Balajee, Adayabalam S, Smith, Tammy, Ryan, Terri, Escalona, Maria, Dainiak, Nicholas

Use of ionizing radiation (IR) in various industrial, medical and other applications can potentially increase the risk of medical, occupational or accidental human exposure. Additionally, in the event of a radiological or nuclear (R/N) incident, several tens of hundreds and thousands of people are likely to be exposed to IR. IR causes serious health effects including mortality from acute radiation syndrome and therefore it is imperative to determine the absorbed radiation dose, which will enable physicians in making an appropriate clinical 'life-saving' decision. The 'Dicentric Chromosome Assay (DCA)' is the gold standard for estimating the absorbed radiation dose but its performance is time consuming and laborious. Further, timely evaluation of dicentric chromosomes (DCs) for dose estimation in a large number of samples provides a bottleneck because of a limited number of trained personnel and a prolonged time for manual analysis. To circumvent some of these technical issues, we developed and optimized a miniaturized high throughput version of DCA (mini-DCA) in a 96-microtube matrix with bar-coded 1.4 ml tubes to enable the processing of a large number of samples. To increase the speed of DC analysis for radiation dose estimation, a semi-automated scoring was optimized using the Metafer DCScore algorithm. The accuracy of mini-DCA in dose estimation was verified and validated though comparison with conventional DCA performed in 15 ml conical tubes. The mini-DCA considerably reduced the sample processing time by a factor of 4 when compared to the conventional DCA. Further, the radiation doses estimated by mini-DCA using the triage mode of scoring (50 cells or 30 DCs) were similar to that of conventional DCA using 300-500 cells. The mini-DCA coupled with semi-automated DC scoring not only reduced the sample processing and analysis times by a factor of 4 but also enabled the processing of a large number of samples at once. Our mini-DCA method, once automated for high throughput robotic platforms, will be an effective radiological triage tool for mass casualty incidents.

Digital object identifier (DOI): 10.1093/rpd/ncy127

Mutation research, 834, 35–41
October, 2018

Reprint of: A three-dimensional in vitro HepG2 cells liver spheroid model for genotoxicity studies.

Shah, Ume-Kulsoom, Mallia, Jefferson de Oliveira, Singh, Neenu, Chapman, Katherine E, Doak, Shareen H, Jenkins, Gareth J S

<p>The liver's role in metabolism of chemicals makes it an appropriate tissue for toxicity testing. Current testing protocols, such as animal testing and two-dimensional liver cell systems, offer limited resemblance to in vivo liver cell behaviour, in terms of gene expression profiles and metabolic competence; thus, they do not always accurately predict human toxicology. In vitro three-dimensional liver cell models offer an attractive alternative. This study reports on the development of a 3D liver model, using HepG2 cells, by a hanging-drop technique, with a focus on evaluating spheroid growth characteristics and suitability for genotoxicity testing. The cytokinesis-blocked micronucleus assay protocol was adapted to enable micronucleus (MN) detection in the 3D spheroid models. This involved evaluating the difference between hanging vs non-hanging drop positions for dosing of the test agents and comparison of automated Metafer scoring with manual scoring for MN detection in HepG2 spheroids. The initial seeding density, used for all experiments, was 5000 cells/20 μl drop hanging spheroids, harvested on day 4, with &gt;75% cell viability. Albumin secretion (7.8 g/l) and both CYP1A1 and CYP1A2 gene expression were highest in the 3D environment at day 4. Exposure to metabolically activated genotoxicants for 24 h resulted in a 6-fold increase in CYP1A1 enzyme activity (3 μM B[<em>a</em>]P) and a 30-fold increase in CYP1A2 enzyme activity (5 μM PhIP) in 3D hanging spheroids. MN inductions in response to B[a]P or PhIP were 2-fold and 3-fold, respectively, and were greater in 3D hanging spheroids than in 2D format, showing that hanging spheroids are more sensitive to genotoxic agents. HepG2 hanging-drop spheroids are an exciting new alternative system for genotoxicity studies, due to their improved structural and physiological properties, relative to 2D cultures.</p>

Digital object identifier (DOI): 10.1016/j.mrgentox.2018.06.020

Radiology, 288, 529--535
August, 2018

Abdominopelvic 1.5-T and 3.0-T MR Imaging in Healthy Volunteers: Relationship to Formation of DNA Double-Strand Breaks.

Suntharalingam, Saravanabavaan, Mladenov, Emil, Sarabhai, Theresia, Wetter, Axel, Kraff, Oliver, Quick, Harald H., Forsting, Michael, Iliakis, Georg, Nassenstein, Kai

Purpose To investigate the relationship between abdominopelvic magnetic resonance (MR) imaging and formation of DNA double-strand breaks (DSBs) in peripheral blood lymphocytes among a cohort of healthy volunteers. Materials and Methods Blood samples were obtained from 40 healthy volunteers (23 women and 17 men; mean age, 27.2 years [range, 21-37 years]) directly before and 5 and 30 minutes after abdominopelvic MR imaging performed at 1.5 T (n = 20) or 3.0 T (n = 20). The number of DNA DSBs in isolated blood lymphocytes was quantified after indirect immunofluorescent staining of a generally accepted DSB marker, γ-H2AX, by means of high-throughput automated microscopy. As a positive control of DSB induction, blood lymphocytes from six volunteers were irradiated in vitro with x-rays at a dose of 1 Gy (70-90 keV). Statistical analysis was performed by using a Friedman test. Results No significant alteration in the frequency of DNA DSB induction was observed after MR imaging (before imaging: 0.22 foci per cell, interquartile range [IQR] = 0.54 foci per cell; 5 minutes after MR imaging: 0.08 foci per cell, IQR = 0.39 foci per cell; 30 minutes after MR imaging: 0.09 foci per cell, IQR = 0.63 foci per cell; P = .057). In vitro radiation of lymphocytes with 1 Gy led to a significant increase in DSBs (0.22 vs 3.43 foci per cell; P = .0312). The frequency of DSBs did not differ between imaging at 1.5 T and at 3.0 T (5 minutes after MR imaging: 0.23 vs 0.06 foci per cell, respectively [P = .57]; 30 minutes after MR imaging: 0.12 vs 0.08 foci per cell [P = .76]). Conclusion Abdominopelvic MR imaging performed at 1.5 T or 3.0 T does not affect the formation of DNA DSBs in peripheral blood lymphocytes.

Digital object identifier (DOI): 10.1148/radiol.2018172453

International journal of radiation biology, 94, 664–670
July, 2018

Evaluation of chromosomal aberrations induced by 188Re-dendrimer nanosystem on B16f1 melanoma cells.

Tassano, Marcos, Oddone, Natalia, Fernández, Marcelo, Porcal, Williams, García, MF, Martínez-López, Wilner, Benech, Juan Claudio, Cabral, Pablo

<p>To study the rhenium-188 labeling of polyamidoamine (PAMAM) generation 4 (G4) dendrimer and its evaluation on biodistribution and chromosomal aberrations in melanoma cells induced by ionizing radiation as potential treatment agent. Dendrimers were first conjugated with Suc-HYNIC (succinimidyl 6-hydrazinopyridine-3-carboxylic acid hydrochloride). Dendrimer-HYNIC was then incubated with ReO . Biodistribution was performed administrating Re-dendrimer to normal (NM) or melanoma-bearing mice (MBM). Chromosome aberration test was conducted in order to measure treatment capacity of Re-dendrimer in melanoma cells. Radiolabeling yield of dendrimer was approx. 70%. Biodistribution studies in NM showed blood clearance with hepatic and renal depuration. MBM showed a similar pattern of biodistribution with tumor uptake of 6% of injected dose. Aberrant metaphases quantified in control cells were 7%, increasing to 29.5% in cells treated with 15μCi (0.555 MBq) of Re-dendrimer for 24 h. Re-dendrimer can produce double-stranded breaks in DNA induced by ionizing radiation in melanoma cells in vitro.</p>

Digital object identifier (DOI): 10.1080/09553002.2018.1478161

British journal of pharmacology
March, 2018

CO-EXPRESSION OF μ AND δ OPIOID RECEPTORS BY MOUSE COLONIC NOCICEPTORS.

Guerrero-Alba, Raquel, Valdez-Morales, Eduardo Emmanuel, Jiménez-Vargas, Nestor Nivardo, Bron, Romke, Poole, Daniel, Reed, David, Castro, Joel, Campaniello, Melissa, Hughes, Patrick A, Brierley, Stuart M, Bunnett, Nigel, Lomax, Alan E, Vanner, Stephen

<p>To better understand opioid signaling in visceral nociceptors, we examined the expression and selective activation of mu (MOR) and delta opioid receptors (DOR) by dorsal root ganglia (DRG) neurons innervating the mouse colon. DRG neurons projecting to the colon were identified by retrograde tracing. DOR-GFP reporter mice, in situ hybridization, single-cell RT-PCR, and MOR-specific antibodies were used to characterize expression of MOR and DOR. Voltage-gated Ca currents and neuronal excitability were recorded in small diameter nociceptive neurons (capacitance &lt; 30pF) by patch clamp and ex vivo single-unit afferent recordings were obtained from the colon. In situ hybridization of oprm1 expression in Fast Blue-labeled DRG neurons was observed in 61% of neurons. MOR and DOR were expressed by 36-46% of colon DRG neurons, and co-expressed by ~25 % of neurons. MOR and DOR agonists inhibited Ca currents in DRG and these effects were blocked by opioid antagonists. One or both agonists inhibited action potential firing by colonic afferent endings. Incubation of neurons with supernatants from inflamed colon segments inhibited Ca currents and neuronal excitability. The MOR but not the DOR antagonist, inhibited the supernatant effects on Ca currents, whereas both antagonists inhibited their actions on neuronal excitability. A significant number of small diameter colonic nociceptors co-express MOR and DOR and are inhibited by agonists and endogenous opioids in inflamed tissues. Thus, opioids that act at MOR or DOR or their heterodimers may be effective in the treatment of visceral pain.</p>

Digital object identifier (DOI): 10.1111/bph.14222

Nanoscale, 10, 4320–4331
March, 2018

Super-resolution localization microscopy of radiation-induced histone H2AX-phosphorylation in relation to H3K9-trimethylation in HeLa cells.

Hausmann, Michael, Wagner, Emma, Lee, Jin-Ho, Schrock, Gerrit, Schaufler, Wladimir, Krufczik, Matthias, Papenfuß, Franziska, Port, Matthias, Bestvater, Felix, Scherthan, Harry

<p>Ionizing radiation (IR)-induced damage confers functional and conformational changes to nuclear chromatin associated with DNA single and double strand breaks. This leads to the activation of complex DNA repair machineries that aim to preserve the integrity of the DNA molecule. Since hetero- and euchromatin are differentially accessible to DNA repair pathways, local chromatin re-arrangements and structural changes are among the consequences of an activated DNA damage response. Using super-resolution localization microscopy (SRLM), we investigated the X-ray-induced repositioning of γ-H2AX and histone H3K9me3 heterochromatin marks in the nuclei of HeLa cells. Aliquots of cells exposed to different IR doses (0.5, 1 and 2 Gy) were fixed at certain repair times for SRLM imaging. The number and size of nano-scale γ-H2AX molecule signal clusters detected increased with rising irradiation doses, with the number and size being the highest 0.5 h after irradiation. With growing repair time both the number and size of γ-H2AX nano-clusters decreased. Eight hours after irradiation, the number of clusters reached control levels, in agreement with the disappearance of most IR-induced foci seen by conventional microscopy. SRLM investigation of heterochromatin marks in spatial relation to γ-H2AX clusters showed that on average the heterochromatin density was high in the vicinity of γ-H2AX, which is in agreement with the observation that DSBs seem to relocate to the surface of heterochromatin clusters for DNA repair. The data demonstrate the potential of pointillist images obtained by SRLM for quantitative investigations of chromatin conformation changes and repair-protein recruitment on the nanoscale as measures for a radiation response.</p>

Digital object identifier (DOI): 10.1039/c7nr08145f

Scientific reports, 8, 2286
February, 2018

DNA damage in leukocytes after internal ex-vivo irradiation of blood with the α-emitter Ra-223.

Schumann, Sarah, Eberlein, Uta, Muhtadi, Razan, Lassmann, Michael, Scherthan, Harry

<p>Irradiation with high linear energy transfer α-emitters, like the clinically used Ra-223 dichloride, severely damages cells and induces complex DNA damage including closely spaced double-strand breaks (DSBs). As the hematopoietic system is an organ-at-risk for the treatment, knowledge about Ra-223-induced DNA damage in blood leukocytes is highly desirable. Therefore, 36 blood samples from six healthy volunteers were exposed ex-vivo (in solution) to different concentrations of Ra-223. Absorbed doses to the blood were calculated assuming local energy deposition of all α- and β-particles of the decay, ranging from 0 to 142 mGy. γ-H2AX+53BP1 co-staining and analysis was performed in leukocytes isolated from the irradiated blood samples. For DNA damage quantification, leukocyte samples were screened for occurrence of α-induced DNA damage tracks and small γ-H2AX+53BP1 DSB foci. This revealed a linear relationship between the frequency of γ-induced γ-H2AX damage tracks and the absorbed dose to the blood, while the frequency of small γ-H2AX+53BP1 DSB foci indicative of β-irradiation was similar to baseline values, being in agreement with a negligible β-contribution (3.7%) to the total absorbed dose to the blood. Our calibration curve will contribute to the biodosimetry of Ra-223-treated patients and early after incorporation of α-emitters.</p>

Digital object identifier (DOI): 10.1038/s41598-018-20364-7

Environmental science and pollution research international
2018

Toxicological evaluation of nail polish waste discarded in the environment.

Felzenszwalb, Israel, Fernandes, Andreia da Silva, Brito, Lara Barroso, Oliveira, Gisele Augusto Rodrigues, Silva, Paula Aquino Soeiro, Arcanjo, Maria Elena, Marques, Monica Regina da Costa, Vicari, Taynah, Leme, Daniela Morais, Cestari, Marta Margarete, Ferraz, Elisa Raquel Anastacio

Nail polish has been widely used around the world. However, the hazards of nail polishes discarded in the environment are still poorly investigated. Thus, the toxicogenetic effects of solubilized (SE) and leached (LE) extracts from nail polishes were investigated, simulating their disposal on water and landfill, respectively, and identifying their physicochemical properties and chemical constituents. Organic compounds and metals were detected in both extracts. SE and LE only induced mutagenic effects in TA98 Salmonella strain in the presence and absence of exogenous metabolic activation. Although both extracts did not significantly increase the frequency of micronucleated HepG2 cells, the cell viability was affected by 24-h exposure. No DNA damage was observed in gonad fish cells (RTG-2) exposed to both extracts; however, the highest SE and LE concentrations induced significant lethal and sublethal effects on zebrafish early-life stages during 96-h exposure. Based on our findings, it can be concluded that if nail polishes enter aquatic systems, it may cause negative impacts to the environment.

Digital object identifier (DOI): 10.1007/s11356-018-1880-y

Scientific Reports, 8(1), 1141
2018

First experimental proof of Proton Boron Capture Therapy (PBCT) to enhance protontherapy effectiveness

Cirrone, GAP, Manti, L, Margarone, D, Petringa, G, Giuffrida, L, Minopoli, A, Picciotto, A, Russo, G, Cammarata, F, Pisciotta, P, others

Protontherapy is hadrontherapy’s fastest-growing modality and a pillar in the battle against cancer. Hadrontherapy’s superiority lies in its inverted depth-dose profile, hence tumour-confined irradiation. Protons, however, lack distinct radiobiological advantages over photons or electrons. Higher LET (Linear Energy Transfer) 12C-ions can overcome cancer radioresistance: DNA lesion complexity increases with LET, resulting in efficient cell killing, i.e. higher Relative Biological Effectiveness (RBE). However, economic and radiobiological issues hamper 12C-ion clinical amenability. Thus, enhancing proton RBE is desirable. To this end, we exploited the p + 11B → 3α reaction to generate high-LET alpha particles with a clinical proton beam. To maximize the reaction rate, we used sodium borocaptate (BSH) with natural boron content. Boron-Neutron Capture Therapy (BNCT) uses 10B-enriched BSH for neutron irradiation-triggered alpha particles. We recorded significantly increased cellular lethality and chromosome aberration complexity. A strategy combining protontherapy’s ballistic precision with the higher RBE promised by BNCT and 12C-ion therapy is thus demonstrated.