Persistence of micronuclei in human lymphocytes after fractionated irradiation in vitro

The study evaluate relationship between incidence of micronuclei and chromosomal aberrations after fractionated irradiation of human lymphocytes in vitro. Obtained results have shown that incidence of chromosome aberrations aberrations declined faster than micronuclei at all given doses if the time between two irradiationsis longer than 2 hours. The study confirmed that CBMN test is very sensitive assay for estimation of effects of ionizing radiation in the case of fractionated irradiation. This observation could be of interest for radiotherapy, particularly for applying micronucleus test as predictive test for hypersensitivity to ionizing radiation.Physical chemistry 2004 : 7th international conference on fundamental and applied aspects of physical chemistry; Belgrade (Serbia); 21-23 September 200

Positive correlation between micronuclei and necrosis of lymphocytes in medical personnel occupationally exposed to ionizing radiation

BACKGROUND: Current radiation protection standards are based on premise that any radiation dose may result in detrimental health effects. The aim of this study was to evaluate extent of the DNA damages (measured by induction of micronuclei) and interphase cell death in circulating lymphocytes of medical personnel exposed to ionizing radiation. METHODS: Baseline micronuclei were assessed using the cytokinesis-block micronucleus test. Cytotoxicity was analyzed by flow cytometry for human white blood cells to identify cells that displayed apoptosis-associated DNA condensation. Necrotic cells were analyzed simultaneously. All parameters were compared with corresponding controls. RESULTS: A statistically significant difference (t = 4.54, p = 0.002) was found between exposed and control group in the yield of baseline micronuclei. The level of baseline micronuclei correlated positively with necrosis of leucocytes (r=0.09, p=0.68 in exposed group, r=0.02, p=0.97 in controls). An inverse correlation between baseline micronuclei and apoptosis was noted in both groups of examinees (r = -0.26, p = 0.27 in exposed group, r = -0.09, p=0.80 in controls). The data obtained also suggested an inverse correlation between necrosis and apoptosis (r = -0.37, p = 0.11 in exposed group, r = -0.89, p = 0.001 in controls). CONCLUSION: Flow cytometry being a rapid, fast, and accurate method is strongly recommended in evaluation of radiation injuries. The integration of apoptosis and necrosis into micronucleus assay could be very important in the assessment of cumulative effects of ionizing radiation in occupationally exposed medical personnel

Radioprotective properties of nutraceutical Gonebazol: In vivo study

BACKGROUND: In vitro investigation of radioprotective properties of novel nutraceutical Gonebazol (Biofarm, Belgrade) has displayed its remarkable potential to reduce chromosomal aberrations and micronuclei induced by x rays. The goal of this study was to evaluate its protective properties in vivo. For this purpose, a group of medical staff performing invasive radiological diagnostics, that was identified to carry dicentrics in their lymphocytes, was selected for further monitoring. They consumed this nutraceutical for 3 weeks, 2 x 10 g per day (2 x 2 teaspoons dissolved in a glass of water). METHODS: Hematological parameters, chromosomal aberrations and micronucleus frequency were examined at the beginning of monitoring, on day 10 and 21 of the treatment. RESULTS: In the course of three weeks the incidence of chromosomal aberrations and micronuclei was significantly reduced (50%-80%), absolute number of granulocytes was lowered whereas the number of monocytes significantly was enhanced. This study has revealed that nutraceutic posses immunomodulatory properties seen as an improving monocyte-macrophage activity, which inversely correlates with incidence of lymphocyte micronuclei (r=0.75, p<0.05). CONCLUSION: Observed finding could be of particular importance in reducing cumulative effects of ionizing radiation in radiosensitive tissues and in preventing adverse health effects. No side effects were evidenced. Mechanisms of its immunomodulatory effects should be further examined

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

The radioprotective properties of polyphenols on human lymphocytes

The aim of this study was to evaluate radioprotective properties of medical plants Gentianella austriaca and Gentianella dinarica. For this purpose human lymphocytes were irradiated using 60Co γ rays and treated with different fractions of plant extracts, afterwards micronuclei (MN) and malondialdehyde (MDA) were measured. Polyphenols, fractions isolated from those plants have shown the protective effects, seen as significantly reduced incidence of micronuclei which was followed with reduced level of malondialdehyde. The results obtained in this study indicate that polyphenols isolated from Gentianella austriaca and Gentianella dinarica posses radioprotective properties possibly trough reduction of the lipid peroxidation.Physical chemistry 2004 : 7th international conference on fundamental and applied aspects of physical chemistry; Belgrade (Serbia); 21-23 September 200

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

Modulation of the ribavirin induced cytotoxicity in harvested human lymphocytes in vitro

The present investigation was undertaken to evaluate effect of vitamin B12 on ribavi- rin induced cytotoxicity in phytohemaglutinin-stimulated human lymphocytes. Blood samples, obtained from healthy volunteers (males, aged 31 years, non-smokers) are used for examination. Blood cells were treated with increasing doses of ribavirin: 0.05, 0.17, 0.32, 0.47 and 0.65µmol/ml at three different incubation times: 2, 4 and 17 hours. Duplicate ribavirin treated cultures were supplemented with 50 µl of B12 during the drug treatment. Supplementation with B12 lowers the MN frequency and slightly recovers the proliferation potential of treated cells in each treatment period of time except for the highest concentration and the longest treatment. Presence of ribavirin during entire S phase of the cell cycle strongly blocks cell cycle progression. This study have shown that B12 posses unique beneficial effects in reducing the toxic effects of ribavirin. Mechanism of beneficial effects of ribavirin should be examined further

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