Marin Mladinić

In vitro genotoxicity of root canal sealers

AIM To evaluate the effect of leakage on differences in genotoxicity of root canal sealers ex vivo according to their main components using two different cytogenetic assays. METHODOLOGY Six materials of different composition (GuttaFlow, Epiphany, Diaket, IRM, SuperEBA and Hermetic) were tested on human peripheral blood lymphocytes using the comet assay and chromosomal aberration analysis. Prepared materials were eluted in physiological solution for 1 h, 1 day, 5 and 30 days. Thereafter cultures were treated with 8 microg, 4 microg and 2 microg of each sealer. Frequencies of chromatide and chromosome breaks and accentric fragments were determined. Comet assay was used to evaluate primary DNA damage by measuring tail length and tail intensity. Chi-square, Fishers PLSD (Protected Least Significant Difference) and Kruskall-Wallis non parametric tests were used for statistical analysis. RESULTS After 1-h elution only the highest dose of Diaket, Hermetic and SuperEBA significantly (P = 0.035, P = 0.048, P = 0.037 respectively) affected the measured cytogenetic parameters. The migration ability of DNA was more strongly affected than induction of chromosomal aberrations. After elutions longer than 24 h none of the tested sealers exhibited a genotoxic effect. CONCLUSION Under the conditions used in the study all sealers had acceptable biocompatibility in terms of genotoxicity.

Evaluation of genome damage and its relation to oxidative stress induced by glyphosate in human lymphocytes in vitro.

In the present study we evaluated the genotoxic and oxidative potential of glyphosate on human lymphocytes at concentrations likely to be encountered in residential and occupational exposure. Testing was done with and without metabolic activation (S9). Ferric‐reducing ability of plasma (FRAP), thiobarbituric acid reactive substances (TBARS) and the hOGG1 modified comet assay were used to measure glyphosates oxidative potential and its impact on DNA. Genotoxicity was evaluated by alkaline comet and analysis of micronuclei and other nuclear instabilities applying centromere probes. The alkaline comet assay showed significantly increased tail length (20.39 μm) and intensity (2.19%) for 580 μg/ml, and increased tail intensity (1.88%) at 92.8 μg/ml, compared to control values of 18.15 μm for tail length and 1.14% for tail intensity. With S9, tail length was significantly increased for all concentrations tested: 3.5, 92.8, and 580 μg/ml. Using the hOGG1 comet assay, a significant increase in tail intensity was observed at 2.91 μg/ml with S9 and 580 μg/ml without S9. Without S9, the frequency of micronuclei, nuclear buds and nucleoplasmic bridges slightly increased at concentrations 3.5 μg/ml and higher. The presence of S9 significantly elevated the frequency of nuclear instabilities only for 580 μg/ml. FRAP values slightly increased only at 580 μg/ml regardless of metabolic activation, while TBARS values increased significantly. Since for any of the assays applied, no clear dose‐dependent effect was observed, it indicates that glyphosate in concentrations relevant to human exposure do not pose significant health risk. Environ. Mol. Mutagen. 2009.

Characterization of chromatin instabilities induced by glyphosate, terbuthylazine and carbofuran using cytome FISH assay.

Possible clastogenic and aneugenic effects of pesticides on human lymphocytes at concentrations likely to be encountered in residential and occupational exposure were evaluated with (and without) the use of metabolic activation (S9). To get a better insight into the content of micronuclei (MN) and other chromatin instabilities, lymphocyte preparations were hybridized using pancentromeric DNA probes. Frequency of the MN, nuclear buds (NB) and nucleoplasmic bridges (NPB) in cultures treated with glyphosate slightly increased from 3.5microg/ml onward. Presence of S9 significantly elevated cytome assay parameters only at 580microg/ml. No concentration-related increase of centromere (C+) and DAPI signals (DAPI+) was observed for glyphosate treatment. Terbuthylazine treatment showed a dose dependent increase in the number of MN without S9 significant at 0.0008microg/ml and higher. At concentration lower than 1/16 LD50 occurrence of C+MN was significantly elevated regardless of S9, but not dose related, and in the presence of S9 only NBs containing centromere signals were observed. Carbofuran treatment showed concentration-dependent increase in the number of MN. The frequency of C+MN was significant from 0.008microg/ml onward regardless of S9. Results suggest that lower concentrations of glyphosate have no hazardous effects on DNA, while terbuthylazine and carbofuran revealed a predominant aneugenic potential.

Fatty liver index as an indicator of metabolic syndrome

OBJECTIVE The aim of this study was to find an early indicator of metabolic syndrome (MetS). DESIGN AND METHODS We measured several anthropometric, biochemical, haematological, and oxidative damage parameters in 128 middle-aged Caucasian men divided into two groups: patients with MetS (n=69) and healthy controls (n=59), and used Weka REPTree and SimpleCART algorithms to identify the most reliable predictor of MetS. RESULTS Oxidative damage parameters did not differ between the groups, suggesting that oxidative damage is less prominent at the early stage of MetS. The algorithms singled out fatty liver index (FLI) as the best variable for discriminating between healthy and MetS subjects. This finding was confirmed by the receiver-operating characteristic (ROC) curve analysis, which set FLI 68.53 as the threshold value for MetS diagnosis. CONCLUSIONS FLI is the most reliable tool for diagnosing MetS. The absence of oxidative damage does not rule out oxidative stress but may indicate that MetS is at an early stage.

Irinotecan Side Effects Relieved by the Use of HI-6 Oxime: In Vivo Experimental Approach

Some compounds, although not primarily designed as supportive drugs in chemotherapy, are promising candidates for clinical use. The ability of HI-6 oxime to relieve the side effects of irinotecan was recently determined in vitro. In this animal study, we investigated the efficacy of HI-6 in vivo, when given as a pre-treatment and concomitantly with irinotecan. We evaluated the cholinesterase (ChE)/acetylcholinesterase (AChE) activity, the levels of oxidative stress markers, DNA damage and the radical scavenging capacity of HI-6. Both HI-6 and irinotecan inhibited ChE/AChE activity but showed different levels of ChE inhibition in plasma and AChE inhibition in the liver and brain tissue. We also observed a weak antioxidant capacity of HI-6, undiscovered until now, and found an acceptable genotoxicity profile in three types of somatic cells in rats. The in vivo erythrocyte micronucleus assay showed that HI-6 did not significantly change either the frequency of micronuclei or the ratio of polychromatic and normorchromatic erythrocytes. Taken together, our results provide a good argument in favour of HI-6 as a promising molecule for further studies and eventual use in humans.

Evaluation of cytotoxic and genotoxic effects of two resin-based root-canal sealers and their components on human leucocytes in vitro

AIM To evaluate the in vitro genotoxicity and cytotoxicity of two resin-based root canal sealers and to determine the type of cell death they induce. METHODOLOGY The sealers tested were Epiphany and RealSeal. Each component of the material (Epiphany Primer, Epiphany Thinning Resin, Epiphany Sealant, RealSeal Primer, RealSeal Thinning Resin and RealSeal Root Canal Sealant), components in permutual combinations and all components mixed together were tested on human peripheral blood leucocytes using ethidium bromide/acridine orange viability staining and comet assay. Simultaneously, untreated negative control cultures were analysed in the same manner. DNA damage was evaluated following 4 h of treatment and after 24 h in the absence of the components of the materials. RESULTS After 4 h of treatment, except thinning resin, each individual component and the different combinations of components induced a significant increase in DNA migration ability (P < 0.05). After 24 h, combination of primer, thinning resin and sealant of both materials caused cell death inducing intense apoptosis. After 24 h, cells exposed to Epiphany Sealant and RealSeal Root Canal Sealant, both in polymerized and unpolymerized form, exhibited a level of DNA damage that was similar to the control. CONCLUSIONS Primer and thinning resin of both resin-based root canal sealers and their combinations were cytotoxic and induced apoptosis. Both sealants had no significant effect on the viability of the human leucocytes.

Assessment of genotoxic potency of sulfate-rich surface waters on medicinal leech and human leukocytes using different versions of the Comet assay.

The aim of the present study was to investigate how exposure to sulfate-rich surface waters affects the level of primary DNA damage in hemocytes of leech Hirudo medicinalis. Samples of surface water were collected at two sites near a gypsum factory (Knin, Croatia) and two reference sites. In the laboratory, samples were subjected to detailed chemical analysis and used in toxicity testing. For that purpose, previously acclimatized individuals of H. medicinalis were sub-chronically exposed (for 28 days) to tested water samples. Levels of primary DNA damage were evaluated using the alkaline Comet assay in hemocytes collected on days 7, 14, 21 and 28 of exposure and compared with their baseline values. Genotoxic potency of the water sample with the highest sulfate concentration was further evaluated using the alkaline, neutral and hOGG1-modified Comet assay on human peripheral blood leukocytes exposed ex vivo for 30 min. The purpose was to explore which mechanisms are responsible for DNA damage. Chemical analysis revealed that sulfate concentrations in two water samples collected in Mali Kukar Lake (1630 mg/L SO₄) and Kosovčica River (823.3 mg/L SO₄) exceeded the WHO and US EPA defined limits for sulfate in drinking water. Increased levels of metals were found only in the water sample collected in Mali Kukar Lake. However, of the 65 elements analyzed, only nickel and titanium exceed the value legally accepted in Croatia for drinking water. The levels of DNA damage, estimated by the alkaline Comet assay in hemocytes of medicinal leech, increased with the duration of exposure to two sulfate-rich water samples. Since hemocytes responded sensitively to treatment, they could be used for biomonitoring purposes. As observed on treated human peripheral blood leukocytes, all versions of the Comet assay were effective in detecting DNA damage, which was measured in samples with sulfate concentrations equal to or higher than the legally accepted levels for drinking water. Based on the obtained results, it can be assumed that genotoxicity was a consequence both of direct (single- and double-strand DNA breaks) and indirect effects (oxidative damage) caused by the combined effects of all contaminants present in the tested water samples. Our results indicate the need for in situ monitoring and purification of gypsum mine water prior to its release in the natural environment.

The Effects of Hyaluronic Acid, Calcium Hydroxide, and Dentin Adhesive on Rat Odontoblasts and Fibroblasts

The Effects of Hyaluronic Acid, Calcium Hydroxide, and Dentin Adhesive on Rat Odontoblasts and Fibroblasts 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. Utjecaj hijaluronske kiseline, kalcijeva hidroksida i dentinskih adheziva na odontoblaste i fibroblaste štakora 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].

Normalne i granične vrijednosti mikronukleus-testa na limfocitima periferne krvi u ispitanika opće populacije Republike Hrvatske 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. Normal and Cut-Off Values of the Cytokinesis-Block Micronucleus Assay on Peripheral Blood Lymphocytes in the Croatian General Population 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 know 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 influences 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 influenced by sex, age, and smoking. Women had higher MN levels than men. However, only age and smoking significantly 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 influence 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.

Evaluation of the mechanism of nucleoplasmic bridge formation due to premature telomere shortening in agricultural workers exposed to mixed pesticides: indication for further studies.

Agricultural workers are often exposed to high levels of pesticides over prolonged periods of time. We attempted to determine whether exposure to multiple pesticides shortens relative telomere length (RTL) and causes nucleoplasmic bridge (NPB) formation via the mechanism of telomere-end fusion in the lymphocytes of agricultural workers. For measuring RTL, we used quantitative fluorescent in situ hybridization, while NPB frequency was measured as part of the cytome assay. Multivariate analysis of variances taking into account confounding factors (age, gender, years of exposure, smoking, and alcohol intake) did not show a decrease, but rather an increase of RTL in agricultural workers compared to control individuals. In the exposed population, NPB frequency was significantly higher compared to controls (6 times, p<0.05). Multiple regression between NPB, RTL, and confounding factors was not significant. Using Spearman correlation, we did not find proof for our initial hypothesis. Our hypothesis that telomere shortening is a mechanism of NPB origin was not proven, indicating that telomere-end fusion is not a mechanism of NPB formation under our experimental conditions for agricultural workers.