Molecular-cytogenetic characterization of chromosomal breakages in peripheral blood lymphocytes of Fanconi anemia patients in different stages of the disease

Česta fragilna mesta (CFS) su hromozomski regioni skloni lezijama, suženjima i prekidima u uslovima replikativnog stresa in vitro. Najčešće se mogu naći kod sindroma hromozomskih nestabilnosti kao što je Fankonijeva anemija (FA), retka nasledna bolest koju karakteriše sklonost ka razvoju maligniteta i progresivna insuficijencija koštane srži, povećan broj hromozomskih prekida i radijala i telomerna fragilnost. Cilj ovog istraživanja je molekularno-citogenetička karakterizacija hromozomskih prekida u limfocitima periferne krvi, ispitivanje zajedničkog mesta javljanja - kolokalizacije prekida sa CFS i analiza mutacija u FANCD2 genu kod FA-D2 pacijenata iz Srbije kako bi se utvrdilo da li CFS zavise od tipa mutacija u FANCD2 genu. Navedeni parametri su analizirani kod pacijenata u različitim fazama bolesti kako bi se identifikovali prognostički parametri bolesti. Rezultati su pokazali da FA-D2 hromozomski prekidi kolokalizuju sa CFS, karakteristični su za komplementacionu grupu i distribucija im se menja sa progresijom bolesti. Učestalost radijala i telomernih fuzija značajno je viša kod pacijenata sa teškom insuficijencijom koštane srži i može predstavljati prognostički parametar bolesti. Radijali se formiraju između nehomologih hromozoma, uključujući i polne i autozome. Otkriveno je novo fragilno mesto u regionu 1q42.2. Sangerovim sekvenciranjem je otkriveno 10 varijanti FANCD2 gena, uglavnom dubokih intronskih, od kojih su tri novootkrivene (c.2396 C>A, c.206-246delG i c.2715+573 C>T). In-silico analizom identifikovane su tri patogene varijante kod više pacijenata koji nisu u srodstvu, koje mogu biti specifične za populaciju u Srbiji. Poređenje rezultata molekularno-citogenetičke i analize mutacija pokazalo je da ne postoji povezanost CFS i varijanti FANCD2 gena.Common fragile sites (CFSs) are chromosomal regions prone to gaps, constrictions and breaks under conditions of replication stress in vitro. They are mostly found in chromosomal instability syndromes such as Fanconi anemia (FA). FA is rare inherited disease characterized by cancer predisposition, progressive bone marrow failure, increased level of chromosomal breakages, radial figures and marked telomere fragility. The aim of this study was to perform molecular-cytogenetic characterization of chromosomal breakages and co-localization with CFSs in peripheral blood lymphocytes, as well as to analyze FANCD2 gene mutations in FA-D2 patients from Serbia. These parameters were monitored in patients at different stages of the disease in order to identify the prognostic parameters of the disease. The results of this study showed that FA-D2 chromosomal breakages co-localize with CFS, they are specific for the complementation group and their distribution pattern changes with the disease progression. The frequency of radials and telomere fusions is significantly higher in patients with severe bone marrow failure and could be of predictive value. Radials are composed of non-homologous chromosomes, including sex chromosomes as well as autosomes. One novel fragile site is found in region 1q42.2. Ten variants of FANCD2 gene are detected by Sanger sequencing, mostly in deep intronic regions, among which three are novel (c.2396 C>A, c.206-246delG i c.2715+573 C>T). In-silico analysis revealed three pathogenic variants in several unrelated patients, indicating their possible association to Serbian population. Matching the results of molecular-cytogenetic characterization and mutation analysis showed that there is no relationship between CFS and FANCD2 variants

Lomljivost telomera u bolničkih radnika profesionalno izloženih niskim dozama ionizirajućega zračenja

AbstractBiološki učinci ionizirajućega zračenja (IZ) pripisuju se oštećenjima DNA i indirektnim učincima kroz povećanu proizvodnju reaktivnih vrsta kisika. Iako se telomere rabe kao pokazatelji radioosjetljivosti, o njihovu ponašanju kao odgovoru na ionizirajuće zračenje u uvjetima profesionalne izloženosti i dalje se raspravlja. U ovom radu željeli smo istražiti duljinu i strukturu telomera u bolničkih radnika koji su profesionalno izloženi ionizirajućem zračenju te povezati te nalaze s oksidacijskim biomolekulama i kromosomskim aberacijama. Uzorci krvi izloženih ispitanika i zdravih kontrola uzeti su za analizu tijekom rutinskoga godišnjeg zdravstvenog pregleda. Osim kromosomskih aberacija, u uzorcima plazme izmjereni su i parametri oksidacijskoga stresa [prooksidacijska/antioksidacijska ravnoteža (PAB), lipidna peroksidacija i 8-okso-dG], a procjena duljine i strukture telomera provedena je metodom Q-FISH na metafaznim kromosomima. Analiza kromosomskih aberacija pokazala je da od 34 ispitanika njih 14 ima kromosomske aberacije (skupina 1), a 20 nije imalo aberacije (skupina 2). Nije bilo značajne razlike u spolu ili dobi ni u duljini telomera između skupina. Međutim, incidencija lomljivih telomera bila je značajno veća u objema skupinama ispitanika izloženih IZ-u u usporedbi s kontrolnim ispitanicima. Produkti peroksidacije lipida i 8-okso-dG također su bili značajno viši u objema skupinama. Učestalost lomljivih telomera u pozitivnoj je korelaciji (statistički značajna) s razinama 8-okso-dG

Biological properties of nitinol archwires coated with titanium nitride- copper films

Objectives: The main purpose of orthodontic treatment (OT) is to accomplish an optimal occlusal relationship in order to obtain adequate oral function and aesthetic appearance. Under optimal economic conditions, demand for OT reaches at least 35%, but in higher socioeconomic areas in US more than 50% of children are receiving orthodontic care. Acceptance of OT in Europe, like in western population and Scandinavian countries, is at similar levels. Very desirable mechanical characteristics, such as shape memory effect and superelasticity, expanded the use of nitinol (NiTi) archwires in orthodontics significantly. The complex conditions present in the oral cavity, including biofilm formation on the exposed surfaces, substantially alter the surface and structural properties of the NiTi archwires compromising the safety of OT. The necessity for developing novel material coating that would decrease Ni release and improve biological properties is of a great importance. Materials-Methods: Copper doped titanium nitride films (TiN-Cu) on the surface of NiTi archwires were obtained by combination of the cathodic arc evaporation and DC magnetron sputtering. The physicochemical characterization was performed using energy-dispersive X-ray spectroscopy (EDS) and X-ray diffraction (XRD). Ion release was assessed using inductively coupled plasma optical emission spectrometry (ICP-OES). The cytotoxicity of NiTi archwires, stainless steel (SS) archwires and TiN-Cu coated archwires was investigated using 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyl tetrazolium bromide (MTT) test. In order to evaluate bacterial adhesion and biofilm formation, the following strains were used: Streptococcus mutans and Streptococcus mitis. Results: Physicochemical characterization revealed well-designed coatings with the presence of TiN phase with incorporated Cu. The release of Ni was the lowest regarding TiN-Cunanocoated archwires (p < 0.05) and increased in acidic conditions, while the release of Ti was constant. Contrary, the release of Cu was higher in neutral conditions and decreased during the observation time (p < 0.05). The cytotoxicity was the lowest in 28-day eluates of TiN-Cu-nanocoated archwires (p < 0.05). The coating inhibited the adhesion and growth of bacteria such as Streptococcus mitis and Streptococcus mutans (p < 0.05). Conclusion: Taking into account the results of cytotoxicity test and biofilm formation, TiN-Cunanocoated archwires may be considered as a good candidate for further clinical investigations.27th BaSS : November 9-11, Istanbul, Turkey, 2023

Optimizacija metode za izolaciju epitelnih stanica iz nežljezdanog dijela želuca štakora za protočnu citometriju

Traditional methods in cell proliferation studies are based on immunohistochemical detection of proliferating cells in the target tissue. Since they are time consuming, optimization of novel, more efficient methods is important for large scale proliferation studies. In this study, we aimed to optimize the isolation of single epithelial rat forestomach cells for flow cytometry. As a marker of cellular proliferation we used the Ki-67 antibody to detect this nuclear protein expressed in proliferating cells. We also performed immunohistochemical detection of Ki-67 positive cells and propidium iodide staining to validate the results. 3-tert- butyl -4-hydroxyanisole was used as the positive control to ensure cellular proliferation. The results showed that isolation of epithelial cells with collagenase, trypsin and cell strainer ensures great cell viability (>95%) and the purity of the samples. Flow cytometry and immunostaining with the Ki-67 antibody indicated that 3-tert- butyl-4-hydroxyanisole treatment leads to a significant increase in proliferation. A significant positive correlation was observed between the results obtained by immunohistochemistry and flow cytometry, but the flow cytometric data had a smaller measurement error, suggesting the equal sensitivity and greater accuracy of this method. Propidium iodide staining showed that the percentage of cells in the G2+S phase of the cell cycle correlated positively with the percentage of Ki-67 positive cells assessed by flow cytometry, indicating that Ki-67 positive cells reflect an active dividing cell pool. We conclude that the isolation of forestomach epithelial cells described is a simple and reliable method for obtaining viable cells for use in flow cytometry. Compared to immunohistochemistry, flow cytometric detection of the Ki-67 antigen is equally sensitive, but much faster and provides more accurate results.Tradicionalne metode u ispitivanju stanične proliferacije temelje se na imunohistokemijskom otkrivanju proliferacijskih stanica u ciljanom tkivu. Kako su dugotrajne, optimizacija novih i učinkovitijih metoda važna je za velika istraživanja o proliferaciji. U ovom smo radu željeli optimizirati izolaciju epitelnih stanica prednjeg želuca štakora za protočnu citometriju. Kao marker stanične proliferacije koristili smo Ki-67 protutijelo za otkrivanje ovoga nuklearnog proteina izraženog u proliferacijskim stanicama. Također smo učinili imunohistokemijsku detekciju Ki- 67 pozitivnih stanica i bojenje propidij-jodidom kako bismo potvrdili rezultate. Butil-hidroksianizol korišten je kao pozitivna kontrola da se osigura stanična proliferacija. Rezultati su pokazali da izolacija epitelnih stanica s kolagenazom, tripsinom i staničnim cjedilom osigurava veliku vijabilnost stanica (> 95 %) i čistoću uzoraka. Protočna citometrija i Ki-67 bojenje pokazali su da tretman butil-hidroksianizolom dovodi do znakovitog porasta proliferacije. Primijećena je znakovita pozitivna korelacija između rezultata dobivenih imunohistokemijom i protočnom citometrijom, dok su protočni citometrijski podaci imali manju pogrešku mjerenja, što upućuje na jednaku osjetljivost i veću točnost ove metode. Bojenje propidij-jodidom pokazalo je da postotak stanica u G2+S fazi staničnog ciklusa pozitivno korelira s postotkom Ki-67 pozitivnih stanica procijenjenih protočnom citometrijom, što upućuje na to da Ki-67 oslikava stanice u aktivnoj diobi. Zaključujemo da je opisana izolacija epitelnih stanica prednjeg želuca štakora jednostavna i pouzdana metoda za dobivanje održivih stanica za upotrebu u protočnoj citometriji. U usporedbi s imunohistokemijom, protočna citometrijska detekcija antigena Ki-67 jednako je osjetljiva, ali mnogo brža i daje točnije rezultate

Vodeni ekstrakt kaduljina lišća umanjuje upalu i oksidacijsku genotoksičnost u ljudskim mononuklearnim stanicama periferne krvi

Traditional medicine has used sage (Salvia officinalis L.) preparations for centuries to prevent and treat various inflammatory and oxidative stress-induced conditions. The aim of this in vitro study was to determine the bioactive properties of a sage leave extract obtained with environmentally friendly aqueous extraction and lyophilisation in primary human peripheral blood cells. To that end we measured the total phenolic and flavonoid content (TPC and TFC, respectively) with gas chromatography-mass spectrometry (GC-MS). Non-cytotoxic concentrations determined with the trypan blue assay were used to assess the antioxidant (DPPH, ABTS, and PAB assay), antigenotoxic (CBMN assay), immunomodulatory (IL-1β and TNF-α), and neuroprotective effects (AChE inhibition). The extract contained high TPC (162 mg GAE/g of dry extract) and TFC (39.47 mg QE/g of dry extract) concentrations, while β-thujone content was unexpectedly low (below 0.9 %). Strong radical-scavenging activity combined with glutathione reductase activation led to a decrease in basal and H2O2-induced oxidative stress and DNA damage. A decrease in TNF-α and increase in IL-1β levels suggest complex immunomodulatory response that could contribute to antioxidant and, together with mild AChE inhibition, neuroprotective effects. Overall, this study has demonstrated that aqueous sage leave extract reduces the levels of thujone, 1,8-cineole, pinene, and terpene ketones that could be toxic in high concentrations, while maintaining high concentrations of biologically active protective compounds which have a potential to prevent and/or treat inflammatory and oxidative stress-related conditions.Salvia officinalis L. stoljećima se koristi u tradicionalnoj medicini za prevenciju i liječenje raznih upalnih i oksidacijskim stresom izazvanih poremećaja. U ovoj studiji željeli smo ekstrahirati kaduljino lišće korištenjem ekološki prihvatljivog, “zelenog” pristupa vodenom ekstrakcijom i liofilizacijom te odrediti njegova bioaktivna svojstva u primarnim ljudskim perifernim krvnim stanicama. Ukupni sadržaj fenola i flavonoida i GC-MS korišteni su za karakterizaciju ekstrakta. Necitotoksične koncentracije, određene metodom bojenja s bojom tripan plavo, analizirane su za procjenu antioksidacijskih (DPPH, ABTS i PAB test), antigenotoksičnih (CBMN test), imunomodulacijskih (IL-1β i TNF-α) i neuroprotektivnih učinaka (AChE inhibicija). Ekstrakt je sadržavao visoku koncentraciju ukupnih fenola (162 mg GAE/g liofilizata) i flavonoida (39,47 mg QE/g liofilizata), dok je sadržaj β-tujona bio neočekivano nizak (niži od 0,9 %). Snažna aktivnost hvatanja radikala u kombinaciji s aktivacijom glutation reduktaze dovela je do smanjenja bazalnog i H2O2 induciranog oksidacijskog stresa i oštećenja DNA. Smanjenje TNF-α i povišenje razine IL-1β sugeriraju kompleksan imunomodulatorni odgovor koji bi mogao pridonijeti antioksidacijskim i, zajedno s blagom inhibicijom AChE, neuroprotektivnim učincima. Sveukupno, ova je studija pokazala da vodena ekstrakcija kaduljina lišća smanjuje toksične spojeve kao što su tujon, 1,8-cineol, pinen i terpenski ketoni, a održava visoku koncentraciju biološki aktivnih zaštitnih spojeva u ekstraktu, što bi moglo imati potencijal za prevenciju i/ili liječenje oksidacijskih i upalnih poremećaja

Nanotechnology approach for diminishing quercetin toxicity toward peripheral blood mononuclear cells

Quercetin (Q) is one of the most common and well researched antioxidant flavonoids, which usually occurs in plant-based foods, and medicinal plants. It was shown that quercetin exerts many beneficial effects on human health, including prevention of cancer and heart diseases. Quercetin was found to be toxic toward various types of cancer cell, still, at higher concentrations it was also shown toxic toward normal human cells [1,2]. One of the approaches to overcome this shortcoming offers nanotechnology which enables the novel perspective of phytochemical usage in contemporary medicine [3]. The strategy of binding quercetin to the gold nanoparticles during their synthesis was used, which resulted in quercetin capped gold nanoparticles (NPQ) [4]. Trypan blue exclusion test [5] was used to evaluate peripheral blood mononuclear cells (PBMC) viability after their exposure to either NPQ or free Q during 24, 48 and 72 h, at 37 °C, in the range of quercetin concentrations from 5 to 50 μg/mL. A significant reduction in the cell count was observed in PBMC cultures treated with 10, 20, and 50 μg/mL of free Q, for all exposure times. The treatments of increasing concentrations and exposure times lowered the cells viability, resulting in 63% of the viable cells, following 72 h of the treatment with 50 μg/mL of free Q. Although NPQ treatments affected the cells viability in a concentration- and time-dependent manner the treatment with 50 μg/mL of NPQ for 72 h, had a milder effect on PBMC cultures than free Q, resulting in 81% of the viable cells (Figure 1). According to the obtained results, NPQ were shown less toxic toward PBMC than free Q.1-EuSPMF - 1st European Symposium on Phytochemicals in Medicine and Food; 7-9 September, 2022; Belgrad

Factors effecting the induction of rat forestomach hyperplasia induced by Swedish oral smokeless tobacco (snus)

Long term exposure to oral smokeless tobacco may induce lesions in the oral cavity characterized by a hyperplastic epithelium. The possible role of nicotine and the physical properties of oral tobacco for developing these lesions, as well as of dysplasia and neoplasia is unclear. Low nitrosamine Swedish snus as well as non-genotoxic butylated hydroxyanisole induces increased cellular proliferation in the rat forestomach epithelia. Using this model, we report here on the effects of nicotine, pH, and particle size. Snus with different properties had no impact on oxidative stress as determined by 8-oxo-7,8-dihydro-2′-deoxyguanosine, or on interleukin IL-1b. Whereas BHA boosted IL-6, probably due to the presence of nicotine. there was no significant enhancement of cell divisions with increasing particle size, although in individual samples the variations in proliferation rates increased greatly with increasing particle size. Conforming to human experience, the enhanced cell proliferation caused by snus was found to be completely reversible. A cacao bean extract had a protective action similar to that previously found for blueberries. The main cause of the observed tobacco induced cell proliferation could be mechanical irritation, possibly in combination with nicotine, whereas within the studied range, pH did not affect the rate of cell division. © 201

Prolonged exposure to mesoporous silica decrease cell viability in vitro

„Contemporary Materials 2021“ : XIV International Scientific Conference : programme and book of abstracts : September 9-10, 2021; Banja Luka, Bosnia and Herzegovin

Toxicity assessment of Gentiana lutea L. root extract and its monoterpene compounds

Root of Gentiana lutea commercially available as gentian root, a natural antidote for different types of poisons, possess antioxidative, immunomodulatory, cytoprotective and anti-inflammatory, and adverse, genotoxic and mutagenic effects. It has monoterpenes loganic acid, swertiamarin, gentiopicroside and sweroside as most abundant constituents. In this study, we assessed the toxicity of monoterpenes’ reactive molecular fragments using in silico prediction by VEGA-QSAR platform. Further, we compared the data obtained with in vitro geno- and cyto- toxicity testing of the above monoterpenes and the G. lutea root extract (GE), on human primary unstimulated and mitogen-stimulated peripheral blood mononuclear cells (PBMCs). Viability was assessed by TB and XTT tests after 48 h treatment. DNA damage was evaluated by alkaline comet assay on unstimulated cells, whereas cytokinesis-block micronucleus assay was employed on mitogen-stimulated PBMCs. Stability of compounds throughout treatment was monitored by UPLC. The observed in vitro results had highest compliance with in silico IRFMN/ISSCAN-CGX prediction model. Compounds showed high stability during experiment while treatment with single compounds reduced number of viable cells and increased DNA damage. GE treatment had toxic impact on unstimulated PBMCs but no significant genotoxic influence on mitogen-stimulated PBMCs. In summary, the mild GE effect suggests that the complexity of crude GE extract chemical composition may attenuate the toxicity of the tested monoterpenes loganic acid, swertiamarin, gentiopicroside and sweroside

Sweroside displays a cytotoxic effect by activating apoptosis in human peripheral blood mononuclear cells

Sweroside (Sw) is a secondary metabolite commonly found in plants belonging to the Gentianaceae family [1]. This iridoid compound is well-known for its anti-inflammatory [2], antidiabetic [3] and antitumor properties [4], which have been studied in pathological model systems. In transformed cell lines, Sw displays an antitumor effect by apoptosis activation [5]. Since healthy cells are also exposed to cancer therapy applied in vivo, our goal was to determine Sw’s cytotoxic concentration in primary human peripheral blood mononuclear cells (PBMCs) after 48 h of treatment with a range of concentration from 20 μM to 130 μM Sw in vitro, and to analyze whether the cytotoxic effect was due to activated apoptosis. According to the obtained results of the trypan blue dye exclusion test, 48 h of treatment with 50 μM Sw and higher concentrations led to a significant decrease in cell number. The DNA fragmentation assay indicated that following 50 μM Sw treatment, cells are dying in an apoptosis-like manner since the level of DNA fragments was 3.5 times higher than in the untreated control. The type of cell death was confirmed by immunoblot analysis of apoptosis- specific protein markers, which revealed the elevation of cleaved caspase-3 and PARP1 89 kDa fragments. Our findings showed that like in transformed cell lines, Sw in healthy cells can also activate apoptosis. A potential difference in sensitivity to Sw treatment between healthy and transformed cells could justify Sw treatment in anticancer therapy.1-EuSPMF - 1st European Symposium on Phytochemicals in Medicine and Food; 7-9 September, 2022; Belgrad