Alternativni pristup mjerenju koncentracije nanočestica ZnO u kulturama humanih limfocita - spoj elektrokemije i testova genotoksičnosti

Nanoparticle use has increased radically raising concern about possible adverse effects in humans. Zinc oxide nanoparticles (ZnO NPs) are among the most common nanomaterials in consumer and medical products. Several studies indicate problems with their safe use. The aim of our study was to see at which levels ZnO NPs start to produce adverse cytogenetic effects in human lymphocytes as an early attempt toward establishing safety limits for ZnO NP exposure in humans. We assessed the genotoxic effects of low ZnO NP concentrations (1.0, 2.5, 5, and 7.5 μg mL-1) in lymphocyte cultures over 14 days of exposure. We also tested whether low and high-density lymphocytes differed in their ability to accumulate ZnO NPs in these experimental conditions. Primary DNA damage (measured with the alkaline comet assay) increased with nanoparticle concentration in unseparated and high density lymphocytes. The same happened with the fragmentation of TP53 (measured with the comet-FISH). Nanoparticle accumulation was significant only with the two highest concentrations, regardless of lymphocyte density. High-density lymphocytes had significantly more intracellular Zn2+ than light-density ones. Our results suggest that exposure to ZnO NPs in concentrations above 5 μg mL-1 increases cytogenetic damage and intracellular Zn2+ levels in lymphocytes.S naglim porastom primjene nanočestica raste i mogućnost njihova štetna djelovanja u ljudi. Nanočestice cinkova oksida najčešći su oblik nanomaterijala u potrošačkim proizvodima i lijekovima. Nekoliko je istraživanja već upozorilo na probleme vezane uz njihovu sigurnu primjenu. Cilj je ovoga istraživanja bio utvrditi pri kojim razinama nanočestice ZnO počinju štetno citogenetski djelovati na humane limfocite i time otvoriti pitanje utvrđivanja graničnih razina za sigurnu primjenu nanočestica ZnO u ljudi. Stoga smo istražili genotoksične učinke niskih koncentracija nanočestica ZnO (1,0; 2,5; 5 i 7,5 μg mL-1) izloživši kulture humanih limfocita njihovu djelovanju tijekom 14 dana. Uz to smo izmjerili razlikuju li se limfociti niske gustoće od onih visoke gustoće u sposobnosti akumuliranja nanočestica ZnO pri istim eksperimentalnim uvjetima. Primarno oštećenje DNA (izmjereno alkalnim komet-testom) povećalo se s rastom koncentracije nanočestica u limfocitima koje nismo razdvojili po gustoći te u limfocitima visoke gustoće. Slično smo povećanje zamijetili s fragmentacijom tumorskoga supresorskoga gena TP53 (izmjereno komet-FISH testom). Nakupljanje Zn2+ iona u stanicama bilo je značajno samo kod primjene dviju najviših koncentracija nanočestica ZnO, bez obzira na gustoću limfocita. Osim toga, limfociti visoke gustoće iskazali su i značajno više razine unutarstaničnoga Zn2+ od limfocita niske gustoće. Naši rezultati upozoravaju na to da se izlaganjem razinama nanočestica ZnO višima od 5 μg mL-1 značajno povisuju razine Zn2+ u limfocitima te se povećava oštećenje tih stanica i njihova genoma

Effect of Electromagnetic Radiofrequency Radiation on the Rats’ Brain, Liver and Kidney Cells Measured by Comet Assay

The goal of study was to evaluate DNA damage in rat’s renal, liver and brain cells after in vivo exposure to radiofrequency/ microwave (Rf/Mw) radiation of cellular phone frequencies range. To determine DNA damage, a single cell gel electrophoresis/comet assay was used. Wistar rats (male, 12 week old, approximate body weight 350 g) (N=9) were exposed to the carrier frequency of 915 MHz with Global System Mobile signal modulation (GSM), power density of 2.4 W/m2, whole body average specific absorption rate SAR of 0.6 W/kg. The animals were irradiated for one hour/day, seven days/week during two weeks period. The exposure set-up was Gigahertz Transversal Electromagnetic Mode Cell (GTEM- -cell). Sham irradiated controls (N=9) were a part of the study. The body temperature was measured before and after exposure. There were no differences in temperature in between control and treated animals. Comet assay parameters such as the tail length and tail intensity were evaluated. In comparison with tail length in controls (13.5±0.7 mm), the tail was slightly elongated in brain cells of irradiated animals (14.0±0.3 mm). The tail length obtained for liver (14.5±0.3 mm) and kidney (13.9±0.5 mm) homogenates notably differs in comparison with matched sham controls (13.6±0.3 mm) and (12.9±0.9 mm). Differences in tail intensity between control and exposed animals were not significant. The results of this study suggest that, under the experimental conditions applied, repeated 915 MHz irradiation could be a cause of DNA breaks in renal and liver cells, but not affect the cell genome at the higher extent compared to the basal damage

Procjena in vitro toksičnosti stakleno-ionomernih cemenata primjenom mikronukleus testa, alkalnog komet testa i komet testa modificiranog hOGG1 enzimom

The purpose of this study was to evaluate the genotoxic potential of components leached from two conventional self-curing glass-ionomer cements (Fuji IX and Ketac Molar), and light-curing, resin modified glass-ionomer cements (Vitrebond, Fuji II LC). Evaluation was performed on human lymphocytes using alkaline and hOGG1 modified comet, and micronucleus assays. Each material, polymerised and unpolymerised, was eluted in extracellular saline (1 cm2 mL-1) for 1 h, 1 day, and 5 days. Cultures were treated with eluates using final dilutions of 10-2, 10-3, and 10-4. Alkaline comet assay did not detect changes in DNA migration of treated cells regardless of the ionomer tested, polymerisation state, and elution duration. Glass ionomers failed to significantly influence micronucleus frequency. No oxidative DNA damage in treated lymphocytes was observed using hOGG1 modified comet assay. Obtained results indicate high biocompatibility of all tested materials used in the study under experimental conditions.Svrha istraživanja bila je procijeniti genotoksični potencijal komponenata koje izlučuju dva konvencionalna samopolimerizirajuća stakleno-ionomerna cementa (Fuji IX i Ketac Molar) te svjetlosno polimerizirajući i smolom modificirani stakleno-ionomerni cementi (Vitrebond, Fuji II LC). Istraživanje je provedeno na ljudskim limfocitima primjenom alkalnog komet testa, komet testa modificiranog hOGG1 enzimom te mikronukleus testa. Svaki materijal, polimerizirani i nepolimerizirani, eluiran je u fiziološkoj otopini (1 cm2 mL-1) tijekom jednog sata, jednog dana i tijekom 5 dana. Kulture limfocita tretirane su eluatima u razrjeđenjima 10-2, 10-3 i 10-4. Alkalnim komet testom nisu zabilježene promjene u migraciji DNA iz tretiranih stanica bez obzira na ispitani ionomer, vrstu polimerizacije i trajanje elucije. Izloženost staklenim ionomerima nije značajno utjecala na učestalost mikronukleusa. Primjenom hOGG1 modificiranog komet testa nije zamijećeno oksidativno oštećenje DNA u tretiranim limfocitima. Dobiveni rezultati upućuju na visoki stupanj biokompatibilnosti svih testiranih materijala koji su se koristili u eksperimentalnim uvjetima

Okratoksin A pospješuje nakupljanje citrinina u bubrezima i jetri štakora

Ochratoxin A (OTA) and citrinin (CTN) are nephrotoxic mycotoxins often found together in grain. The aim of this study was to measure their accumulation in the kidney and liver of adult male Wistar rats, see how it would be affected by combined treatment, and to determine if resveratrol (RSV) would decrease their levels in these organs. The rats received 125 or 250 mg/kg bw of OTA by gavage every day for 21 days and/or 20 mg/kg bw of CTN a day for two days. Two groups of rats treated with OTA+CTN were also receiving 20 mg/kg bw of RSV a day for 21 days. In animals receiving OTA alone, its accumulation in both organs was dose-dependent. OTA+CTN treatment resulted in lower OTA but higher CTN accumulation in both organs at both OTA doses. RSV treatment increased OTA levels in the kidney and liver and decreased CTN levels in the kidney. Our findings point to the competition between CTN and OTA for organic anion transporters 1 and 3.Okratoksin A(OTA) i citrinin (CTN) nefrotoksični su mikotoksini koji zajednički kontaminiraju žitarice. Cilj ovoga istraživanja bio je izmjeriti koncentraciju OTA-e i CTN-a u bubrezima i jetri štakora, tretiranih tim mikotoksinima, te provjeriti hoće li tretman resveratrolom (RSV) smanjiti koncentraciju mikotoksina u tkivima. Istraživanje je provedeno na mužjacima štakora soja Wistar, koji su 21 dan bili tretirani OTA-om (0,125 i 0,250 mg/kg t. m.), a dva dana CTN-om (20 mg/kg t. m.) ili kombinacijama tih mikotoksina. Dvije skupine štakora koje su tretirane mikotoksinima OTA+CTN dobivale su 21 dan RSV (20 mg/kg t. m.). Povećanje koncentracija OTA-e u bubrezima i jetri bio je u skladu s povećanjem doze. Tretman mikotoksinima OTA+CTN smanjio je nakupljanje OTA-e u bubrezima i jetri, a povećao je koncentraciju CTN-a. Tretman RSV-om povećao je koncentraciju OTA-e u bubrezima i jetri, ali je smanjio koncentraciju CTN-a u bubrezima tretiranih štakora. Koncentracija OTA-e značajno se smanjila u prisutnosti CTN-a, vjerojatno zbog kompeticije CTN-a i OTA-e za prijenosnike OAT1 i 3, koji služe za prijenos tih toksina kroz membrane u bubrezima

Rezultati biomonitoringa radnika profesionalno izloženih olovu u industriji izrade baterija i keramičkih pločica ...

Manufacture of lead-containing products has long been associated with various health risks. To get an insight into the related genotoxic risks, we conducted a biomonitoring study in 50 exposed workers and 48 matched controls using a battery of endpoints that sensitively detect the extent of genome instability in peripheral blood lymphocytes. The levels of primary DNA damage were estimated with the alkaline comet assay, while cytogenetic abnormalities were determined with the cytokinesis-block micronucleus (CBMN) cytome assay. Additionally, CBMN slides of 20 exposed and 16 control participants were subjected to fluorescence in situ hybridisation (FISH), coupled with pancentromeric probes to establish the incidence of centromere-positive micronuclei, nuclear buds, and nucleoplasmic bridges. Blood lead levels (B-Pb) were measured with atomic absorption spectrometry. To further characterise cumulative effects of occupational exposure, we measured erythrocyte protoporphyrin (EP) concentrations and delta-aminolevulinic acid dehydratase (ALAD) activity in blood. We also assessed the influence of serum folate (S-folate) and vitamin B12 (S-B12) on genome stability. Compared to controls, occupationally exposed workers demonstrated significantly higher B-Pb (298.36±162.07 vs 41.58±23.02), MN frequency (18.71±11.06 vs 8.98±7.50), centromere positive MN (C+ MN) (8.15±1.8 vs 3.69±0.47), and centromere negative MN (C- MN) (14.55±1.80 vs 4.56±0.89). Exposed women had significantly higher comet tail intensity (TI) and length (TL) than control women. Furthermore, workers showed a positive correlation between age and nuclear buds and MN, between MN and years of exposure, and between S-B12 levels and TI and ALAD activity, while a negative correlation was found between TI and B-Pb. These findings suggest that occupational settings in the manufacture of lead-containing products pose significant genotoxic risks, which calls for developing more effective work safety programmes, including periodical monitoring of B-Pb and genetic endpoints.Profesionalna izloženost u industriji olova štetno utječe na zdravlje radnika. Ovo je istraživanje provedeno na 50 radnika izloženih olovu te 48 ispitanika kontrolne skupine primjenom testova za procjenu genomske nestabilnosti u limfocitima periferne krvi. Razine primarnoga oštećenja DNA procijenjene su alkalnim komet-testom, a citogenetičke abnormalnosti utvrđene su citohalazin blokiranim mikronukleus-testom (CBMN). Na podskupini od 20 izloženih i 16 kontrolnih ispitanika dodatno je proveden mikronukleus-test u kombinaciji s fluorescencijskom in situ hibridizacijom (MN-FISH). Primjenom pancentromernih sonda istražili smo učestalost mikronukleusa pozitivnih na centromere, nuklearnih pupova I nukleoplazmatskih mostova. Razine olova u krvi (B-Pb) izmjerene su atomskom apsorpcijskom spektrometrijom. Kako bismo utvrdili kumulativne učinke profesionalne izloženosti, izmjerili smo koncentracije eritrocitnoga protoporfirina (EP) i aktivnost dehidrataze delta-aminolevulinske kiseline (ALAD) u krvi. Također smo procijenili utjecaj serumskoga folata (S-folata) i vitamina B12 (S-B12) na stabilnost genoma. U usporedbi s podudarnim kontrolama, profesionalno izloženi radnici imali su značajno višu razinu B-Pb (298,36±162,07 vs. 41,58±23,02), učestalost MN-a (18,71±11,06 vs. 8,98±7,50), mikronukleusa pozitivnih na centromere (C+ MN) (8,15±1,8 vs. 3,69±0,47) i mikronukleusa negativnih na centromere (C- MN) (14,55±1,80 vs. 4,56±0,89). Izložene radnice imale su značajno veći intenzitet (TI) i duljinu (TL) repa u komettestu od ženskih kontrola. Nadalje, u radnika je utvrđena pozitivna korelacija između učestalosti jezgrinih pupova i MN-a s dobi, između MN-a i godina izloženosti te između TI-ja i aktivnosti ALAD-a i razina S-B12. Negativna korelacija utvrđena je između TI-ja i B-Pb. Dobiveni rezultati upućuju na to da profesionalna izloženost olovu predstavlja značajan genotoksični rizik, što zahtijeva razvoj učinkovitijih programa zaštite na radu, uključujući povremeno praćenje B-Pb i genetičkih markera

Utjecaj hijaluronske kiseline, kalcijeva hidroksida i dentinskih adheziva na odontoblaste i fibroblaste štakora

The aim of this study was to investigate the effects and efficiency of pulp capping preparations based on hyaluronic acid, calcium hydroxide, and dentin adhesive on the pulp tissue of Sprague-Dawley rats. The rats were killed and extracted teeth sectioned transversely through the pulp. The slices were placed in a RPMI 1640 cell culture medium supplemented with 10 % foetal calf serum. During 14 days of cultivation cultures were treated with preparations that contained hyaluronic acid (Gengigel Prof®), and calcium hydroxide (ApexCal®), or with dentin adhesive (Excite®). Cellularity and viability of fibroblasts and odontoblasts was analysed using a haemocytometer. Hyaluronic acid proved most efficient and the least toxic for direct pulp capping. Even though calcium hydroxide and dentin adhesive demonstrated a higher degree of cytotoxicity, their effects were still acceptable in terms of biocompatibility.Cilj ovog rada bio je istražiti djelovanje preparata na bazi hijaluronske kiseline i kalcijeva hidroksida te dentinskog adheziva na pulpno tkivo Sprague-Dawley štakora u svrhu procjene učinkovitosti navedenih materijala kod direktnog prekrivanja pulpe. Izvađeni zubi transverzalno su podijeljeni kroz pulpu. Naresci su uzgajani u RPMI 1640 staničnom mediju obogaćenom s 10 % fetalnoga telećeg seruma u plastičnim bočicama za staničnu kulturu. Kulture su tijekom 14 dana tretirane preparatima s hijaluronskom kiselinom (Gengigel Prof®), kalcijevim hidroksidom (ApexCal®) i dentinskim adhezivom (Excite®). Nakon 14 dana pristupilo se analizi staničnosti i vijabilnosti s pomoću hemocitometra. Iako su preparati na bazi kalcijeva hidroksida i dentinski adheziv pokazali nešto viši stupanj citotoksičnosti, dobiveni su rezultati u granicama biokompatibilnosti. Primjena preparata na bazi hijaluronske kiseline postigla je najbolje rezultate te se ovaj materijal pokazao najboljim za direktno prekrivanje pulpe između tri ispitivana preparata

Normal and Cut-Off Values of the Cytokinesis-Block Micronucleus Assay on Peripheral Blood Lymphocytes in the Croatian General Population

Mikronukleus (MN) test na limfocitima periferne krvi jedna je od najvažnijih metoda koje se primjenjuju u citogenetičkom nadzoru. Osnovni preduvjet za primjenu nekog testa u svrhu nadzora profesionalno izloženih populacija jest poznavanje normalnih vrijednosti promatranoga biološkog pokazatelja (biomarkera) u kontrolnoj populaciji. Baze podataka na razini opće populacije moraju se redovito obnavljati novim podacima. Cilj ovog istraživanja bio je utvrditi normalne i granične vrijednosti MN-testa na limfocitima periferne krvi 200 zdravih ispitanika obaju spolova iz opće populacije Republike Hrvatske te ispitati koji čimbenici pridonose spontanom nastanku MN. Na razini istražene populacije utvrđeno je prosječno (6,90±3,32) MN (medijan 7 MN), dok je raspon pojedinačnih vrijednosti iznosio 0 do 18 MN u 1000 binuklearnih stanica. Gornja granična vrijednost dobivena izračunavanjem 95. percentila za cjelokupnu promatranu populaciju iznosi 12,5 MN na 1000 limfocita. Utvrđeno je da na spontani nastanak MN utječu spol, dob i navika pušenja. Žene u prosjeku imaju više vrijednosti svih parametara MN-testa od muškaraca, a u njih je bio i naglašeniji porast vrijednosti citogenetičkog nalaza zbog navike pušenja. Kako su literaturni podaci o utjecaju pušenja cigareta na nastanak MN kontradiktorni, planiran je nastavak istraživanja radi razjašnjavanja utjecaja dnevno utrošenog broja cigareta i ukupnog trajanja pušačkog staža na vrijednosti parametara MN-testa. Usporedba rezultata s literaturnim podacima potvrdila je da su dobivene vrijednosti u skladu s vrijednostima MN-testa zabilježenim na općoj populaciji u drugim svjetskim laboratorijima. Normalne i granične vrijednosti MN-testa utvrđene u ovome istraživanju poslužit će kao osnova za usporedbu i tumačenje nalaza MN-testa u ispitanika izloženih populacija te daljnju nadogradnju laboratorijske baze podataka.The cytokinesis-block micronucleus (CBMN) assay on peripheral blood lymphocytes is one of the most important methods employed in cytogenetic biomonitoring. For the purposes of biological dosimetry, it is important to kno the spontaneous frequency of a biomarker and its normal values in general population. These values are used for population databases, which should be updated regularly. In this study, MN levels were investigated in cytokinesis-blocked lymphocytes of 200 healthy male and female blood donors selected at random from the general population of Croatia. The aim was to assess the variability and determine possible infl uences of external and/or internal factors on the background levels of MN and to establish the cut-off value for the CBMN assay. The background frequency of MN was (6.90±3.32) MN (median 7 MN) and the range was 0 to 18 MN per 1000 binuclear lymphocytes. The cut-off value, which corresponds to 95th percentile of the distribution of 200 individual values, was 12.5 MN. Spontaneous formation of MN was infl uenced by sex, age, and smoking. Women had higher MN levels than men. However, only age and smoking signifi cantly increased the values of all parameters evaluated by the CBMN assay. Since the existing literature data on smoking-related formation of MN are contradictory, we will continue these investigations to resolve how the number of cigarettes smoked per day and the duration of smoking in years infl uence the results of the CBMN assay. Our results are consistent with the background MN frequencies reported by other cytogenetic laboratories worldwide. Normal and cut-off values estimated in this study will be used to update the current general population data and as reference for occupationally or accidental exposure

Tenocyclidine treatment in soman-poisoned rats - intriguing results on genotoxicity versus protection

This study aimed to evaluate the antidotal potency of tenocyclidine (TCP) that probably might protect acetylcholinesterase (AChE) in the case of organophosphate poisoning. TCP was tested alone as a pretreatment or in combination with atropine as a therapy in rats poisoned with ¼ and ½ of LD50 of soman. Possible genotoxic effects of TCP in white blood cells and brain tissue were also studied. Results were compared with previous findings on the adamantyl tenocyclidine derivative TAMORF. TCP given alone as pretreatment, 5 min before soman, seems to be superior in the protection of cholinesterase (ChE) catalytic activity in the plasma than in brain, especially after administration of the lower dose of soman. Plasma activities of the enzyme after a joint treatment with TCP and soman were significantly increased at 30 min (P < 0.001) and 24 h (P = 0.0043), as compared to soman alone. TCP and atropine, given as therapy, were more effective than TCP administered alone as a pretreatment. The above therapy significantly increased activities of the enzyme at 30 min (P = 0.046) and 24 h (P < 0.001), as compared to controls treated with ¼ LD50 of soman alone. Using the alkaline comet assay, acceptable genotoxicity of TCP was observed. However, the controversial role of TCP in brain protection of soman-poisoned rats should be studied further