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

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

Biološki učinciI Echinacea purpurea na krvne stanice ljudi

The aim of this study was to investigate radioprotective properties of Echinacea purpurea tablets in vivo. We analysed lymphocyte chromosome aberrations (CA), micronuclei (MN), apoptosis of leukocytes and haematological parameters in a group of radiation workers who were identified as carrying dicentric chromosomes in their lymphocytes. All radiation workers were taking two 275 mg Echinacea tablets b.i.d., according to a pharmacist’s recommendation. All parameters were analysed before and after the two-week treatment. At the end of the treatment lymphocyte CA frequency dropped significantly, and the number of apoptotic cells increased. The inverse lymphocyte-to-granulocyte ratio at the beginning of the study changed to normal at its end. In conclusion, biological effects observed after administration of Echinacea purpurea preparation suggest that it may be beneficial for the prevention of adverse health effects in workers exposed to ionising radiation.Cilj ovog rada je utvrđivanje radioprotektivnih svojstava Echinacea purpurea (“Ehinacea”, Strong Nature, Srbija) in vivo. Analizirani su kromosomske aberacije, mikronukleusi, apoptoza leukocita i hematološki parametri u skupini ispitanika profesionalno izloženih ionizirajućem zračenju u čijim je limfocitima utvrđena prisutnost dicentričnih kromosoma. Sve osobe profesionalno izložene ionizirajućem zračenju su uzimale 4 “Echinacea” tablete na dan, prema preporučenoj ljekarničkoj dozi. Svi parametri su analizirani na početku i nakon dva tjedna tretmana. Na kraju studije učestalost kromosomskih aberacija u limfocitima značajno je smanjena, dok je apoptotski potencijal leukocita povećan. Omjer limfocita i granulocita utvrđen na početku studije nakon dva tjedna je dostigao normalne vrijednosti. Uočena svojstva Echinacea purpurea mogu biti važna u prevenciji i ublažavanju učinaka štetnih za zdravlje

Genotoxicity of the poly-D,L-lactide microparticles on the human lymphocytes

Particles generated of biodegradable material are extensively investigated as carriers for sustained drug delivery. Studies have been mainly focused on the monitoring of the drug release and of the rate of the particle degradation. However, the influence of the carrier particles on the human immune cells was only rarely addressed. In this work, the influence of the microparticles made of poly-D,L-lactide (PDLLA) on the function of human lymphocytes in a three-day culture was investigated. PDLLA microparticles have been prepared by a modified precipitation method and human lymphocytes were isolated from the blood of healthy volunteers by a Ficoll – density gradient centrifugation. Lymphocyte proliferation test and the cytochalasin B micronucleus test were used to assess the PDLLA particle effect on the lymphocytes. Results showed that PDLLA particles did not influence on the proliferation of the human lymphocytes. On the other hand, changes in the nuclei form, as well as nucleus buddings were observed. Moreover, the appearance of micronuclei could be detected. All together, these results might imply genotoxicity of the PDLLA particles, or some of the chemicals used for the particle preparation/stabilization on the human lymphocytes

Chemical and radiochemical fractionation of depleted uranium in contaminated soils

The results of the chemical and radiochemical characterization of depleted uranium present in the soils since it was used in Balkan intervention, 1999, are presented. The contamination levels and uranium fractionation in the soil substrates was examined using radiation spectrometry methods and by application of the five-step sequential extraction procedure. Alpha-spectrometric uranium isotopic analysis enabled to find out the recently appeared uranium in the environment mobility and/or fixation into stable forms in the soil, distinguishing depleted from naturally occurring uranium on the basis of 234U/238U and 235U/238U activities ratios.Physical chemistry 2006 : 8th international conference on fundamental and applied aspects of physical chemistry; Belgrade (Serbia); 26-29 September 200

Assessment of Single Nucleotide Polymorphisms in Screening 52 DNA Repair and Cell Cycle Control Genes in Fanconi Anemia Patients

Fanconi anemia (FA) is a rare genetically heterogeneous disorder associated with bone marrow failure, birth defects and cancer susceptibility. Apart from the disease-causing mutations in FANC genes, the identification of specific DNA variations, such as single nucleotide polymorphisms (SNPs), in other candidate genes may lead to a better clinical description of this condition enabling individualized treatment with improvement of the prognosis. In this study, we have assessed 95 SNPs located in 52 key genes involved in base excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR), double strand break (DSB) repair and cell cycle control using a DNA repair chip (Asper Biotech, Estonia) which includes most of the common variants for the candidate genes. The SNP genotyping was performed in five FA-D2 patients and in one FA-A patient. The polymorphisms studied were synonymous (n=10), nonsynonymous (missense) (n=52) and in non-coding regions of the genome (introns and 5 and 3 untranslated regions (UTR)) (n=33). Polymorphisms found at the homozygous state are selected for further analysis. Our results have shown a significant inter-individual variability among patients in the type and the frequency of SNPs and also elucidate the need for further studies of polymorphisms located in ATM, APEX APE 1, XRCC1, ERCC2, MSH3, PARP4, NBS1, BARD1, CDKN1B, TP53 and TP53BP1 which may be of great importance for better clinical description of FA. In addition, the present report recommends the use of SNPs as predictive and prognostic genetic markers to individualize therapy of FA patients

Kombinovano prisustvo genskih polimorfizma faktora koagulacije XIII V34L i inhibitora plazminogen aktivatora 1 4G/5G značajno utiče na rizik od spontanog pobačaja u srpskoj populaciji

Background:Recurrent pregnancy loss (RPL) is a heterogeneous condition affecting up to 5% of women of reproductive age. Inherited thrombophilia have been postulated as one of the causes of RPL. Here we examined the prevalence of nine thrombophilic gene polymorphisms among women with history of recurrent miscarriages and fertile controls.Methods:The study included 70 women with history of at least three early pregnancy losses and 31 fertile controls with no miscarriages. We investigated mutations in genes responsible for clotting and fibrinolysis, including factor V(FV) Leiden, FV H1299R, factor II (FII) G20210A, methyl-ene tetrahydrofolate reductase (MTHFR) C677T and A1298C, factor XIII (FXIII) V34L, plasminogen activator inhibitor-1 (PAI-1) 4G/5G and endothelial protein C receptor (EPCR) H1 and H3 haplotypes using reverse polymerase chain reaction ViennaLab cardiovascular disease StrippAssays. Results:Our results showed no significant increase inprevalence of tested polymorphisms in women with RPL. However, relative risk for PRL among women heterozygousfor FXIII V34L was 2.81 times increased (OR 2.81, 95% CI1.15–6.87, P=0.023). Haplotype analysis showed that combined presence of high-risk genotypes for FXIII andPAI-1 significantly increases risk for RPL (OR 13.98, CI95% 1.11–17.46, P=0.044).Conclusions:This is the first study in Serbian population that investigated prevalence of FVR2, A1298C, FXIII V34Land EPCR gene variants. Compound heterozygosity forFXIII V34L and PAI-1 4G is significant risk factor for recur-rent miscarriage. Our results should be viewed in context of small case-control study, so further large prospective studies are need for confirmation of our findings.Uvod: Ponavljani spontani pobačaji (PSP) su etiološki heterogeni i javljaju se kod 5% parova u reproduktivnom period. Jedan od mogućih uzroka PSP su i nasledne trombofilije. U okviru ove studije analizirali smo učestalost devet trombofilnih polimorfizama kod pacijentkinja sa ponavljanim spontanim pobačajima. Metode: Ispitanici su u studiji podeljeni u dve grupe na osnovu anamnestičkih podataka o broju spontanih pobačaja (70 u grupi sa PSP i 31 u kontrolnoj grupi). Ispitivani su sledeći genski polimorfizmi: faktor V Lajden (FVL), FVR2, faktor II (FII) G21210A, metilentetrahidrofolat reduktaza (MTHFR) C677T i A1298C polimorfizmi, inhibitor aktivatora plazminogena 1 (PAI-1) 4G/5G, faktor XIII (FXIII) V34L i endotelni protein C receptor (EPRC) H1, H2 i H3 haplotipovi. Za detekciju navedenih polimorfizama je korišćena metoda multipleks reakcije lančanog umnožavanja i reverzne hibridizacije na ViennaLab stripovima. Rezultati: Dobijeni rezultati nisu pokazali povećanu učestalost ispitivanih polimorfizama u grupi sa PSP. Posmatrajući uticaj pojedinačnih polimorfizama na ishod trudnoće pokazano je da polimorfizam FXIII V34L povećava rizik za ponavljane spontane pobačaje (OR 2,81, 95%CI 1,15-6,87, P=0,023). Analizom haplotipova ustanovljeno je da kombinovano prisustvo V34L i PAI-1 4G varijanti značajno povećava rizik za PSP (OR 13,98, CI 95% 1,11-17,46, P=0,044). Zaključak: Ovo je prva studija koja je ispitivala prevalencu FVR2, A1298C, FXIII V34L and EPCR polimorfizama u populaciji žena iz Srbije. Složeni heterozigoti za FXIII V34L i PAI-1 4G polimorfizme imaju značajno povišen rizik sa ponavljane gubitke trudnoće. Radi potvrde dobijenih rezultata potrebne su veće prospektivne studije

Genotyping Fanconi Anemia Patients from Serbia Reveals Three Novel Fancd2 Variants

Fanconi anemia is rare inherited disease characterized by wide spectrum of congenital anomalies, progressive pancytopenia, and predisposition to hematological malignancies and solid tumors. Molecular genetic analysis of mutations in FANC genes is of a great importance for diagnosis confirmation, prenatal and carrier testing, as well as for prediction of chemotherapy outcome and disease complications. In this study we performed screening of frequently affected regions of FANCD2 gene for sequence variants in six unrelated FA-D2 patients in Serbia. This is the first molecular analysis of FANCD2 gene in Serbian FA-D2 patients. A total of 10 sequence variants were detected, one in homozygous, and nine in heterozygous state. Two variants were found within exons, and eight within introns, in deep intronic regions. In-silico analysis showed that among all detected variants one exon variant and three intron variants might have impact on splicing mechanism. Heterozygous variants found in intron 3, c. 206-246delG; exon 26, c. 2396 C GT A and intron 28, c. 2715+573 C GT T were not previously reported. In-silico analysis revealed that among them, two (intron 3, c. 206-246 delG and exon 26, c. 2396 C GT A) could be novel disease-causing mutations. Many variants were found in more than one patient, including those unreported, indicating their possible ethnic association. Great number of variants in some patients suggests their non-random emergence in Fanconi anemia pathway

First molecular-cytogenetic characterization of Fanconi anemia fragile sites in primary lymphocytes of FA-D2 patients in different stages of the disease

Background: Fanconi anemia (FA) is a chromosomal instability syndrome characterized by increased frequency of chromosomal breakages, chromosomal radial figures and accelerated telomere shortening. In this work we performed detailed molecular-cytogenetic characterization of breakpoints in primary lymphocytes of FA-D2 patients in different stages of the disease using fluorescent in situ hybridization. Results: We found that chromosomal breakpoints co-localize on the molecular level with common fragile sites, whereas their distribution pattern depends on the severity of the disease. Telomere quantitative fluorescent in situ hybridization revealed that telomere fusions and radial figures, especially radials which involve telomere sequences are the consequence of critically shortened telomeres that increase with the disease progression and could be considered as a predictive parameter during the course of the disease. Sex chromosomes in FA cells are also involved in radial formation indicating that specific X chromosome regions share homology with autosomes and also could serve as repair templates in resolving DNA damage. Conclusions: FA-D2 chromosomal breakpoints co-localize with common fragile sites, but their distribution pattern depends on the disease stage. Telomere fusions and radials figures which involve telomere sequences are the consequence of shortened telomeres, increase with disease progression and could be of predictive value

Enhanced Frequency of Sister Chromatid Exchanges Induced By Diepoxybutane Is Specific Characteristic of Fanconi Anemia Cellular Phenotype

Fanconi anemia (FA) is a rare genetically heterogeneous disease characterized by developmental abnormalities, progressive bone marrow failure, and cancer susceptibility. We examined spontaneous, diepoxybutane (DEB)-induced and radiation-induced sister chromatid exchanges (SCEs) in wholeblood lymphocyte cultures of bone marrow failure (BMF) patients including Fanconi anemia, mothers of affected individuals, and healthy controls. The baseline frequency of SCE in FA cells was similar to that observed in controls. However, in response to DEB SCE frequencies in FA patients and their mothers were significantly increased compared to both non-FA BMF families and healthy controls. In response to ionizing radiation, cells displayed increased frequency of SCE, but no differences between FA patients and non-FA BMF patients were seen. Our data confirm and expand previous findings by showing that SCE induced by DEB can be used as an adjunct diagnostic test not only for FA patients, but also for female heterozygous carriers, at least for complementation groups FANCA and FANCD2