Ochratoxin A potentiates citrinin accumulation in kidney and liver of rats.

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.

Biomonitoring findings for occupational lead exposure in battery and ceramic tile workers using biochemical markers, alkaline comet assay, and micronucleus test coupled with fluorescence in situ hybridisation.

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.

Effects of the chloro-s-triazine herbicide terbuthylazine on DNA integrity in human and mouse cells.

Terbuthylazine belongs to the chloro-s-triazine group of herbicides and acts primarily as a photosynthesis inhibitor. The mechanisms of action related to its exposure, relevant both in animals and humans, are still insufficiently investigated. This comprehensive study focused on the outcomes of terbuthylazine exposure at cell level in vitro, and a mice model in vivo. Experiments in vitro were conducted on whole human peripheral blood, isolated lymphocytes, and HepG2 cells exposed for 4 h to terbuthylazine at 8.00, 0.80, and 0.58 ng/mL, which is comparable with current reference values set by the European Commission in 2011. Terbuthylazine cytotoxicity was evaluated using dual fluorescent staining with ethidium bromide and acridine orange on lymphocytes, and CCK-8 colorimetric assay on HepG2 cells. The levels of DNA damage were measured using alkaline and hOGG1-modified comet assays. The potency of terbuthlyazine regarding induction of oxidative stress in vitro was studied using a battery of standard oxidative stress biomarkers. The in vivo experiment was conducted on Swiss albino mice exposed to terbuthlyazine in the form of an active substance and its formulated commercial product Radazin TZ-50 at a daily dose of 0.0035 mg/kg bw for 14 days. Following exposure, the DNA damage levels in leukocytes, bone marrow, liver, and kidney cells of the treated mice were measured using an alkaline comet assay. In vitro results suggested low terbuthylazine cytotoxicity in non-target cells. The highest tested concentration (8.00 ng/mL) reduced lymphocyte viability by 15%, mostly due to apoptosis, while cytotoxic effects in HepG2 cells at the same concentration were negligible. Acute in vitro exposure of human lymphocytes and HepG2 cells to terbuthylazine resulted in low-level DNA instability, as detected by the alkaline comet assay. Further characterization of the mechanisms behind the DNA damage obtained using the hOGG1-modified comet assay indicated that oxidative DNA damage did not prevail in the overall damage. This was further confirmed by the measured levels of oxidative stress markers, which were mostly comparable to control. Results obtained in mice indicate that both the active substance and formulated commercial product of terbuthylazine produced DNA instability in all of the studied cell types. We found that DNA in liver and kidney cells was more prone to direct toxic effects of the parent compound and its metabolites than DNA in leukocytes and bone marrow cells. The overall findings suggest the formation of reactive terbuthylazine metabolites capable of inducing DNA cross-links, which hinder DNA migration. These effects were most pronounced in liver cells in vivo and HepG2 cells in vitro. To provide a more accurate explanation of the observed effects, additional research is needed. Nevertheless, the present study provides evidence that terbuthylazine at concentrations comparable with current reference values possesses toxicological risk because it caused low-level DNA instability, both at cellular and animal organism level, which should be further established in forthcoming studies.

Effects of combined treatment with ochratoxin A and citrinin on oxidative damage in kidneys and liver of rats.

A multimycotoxin analysis approach in grains results in frequent simultaneous findings of nephrotoxic mycotoxins ochratoxin A (OTA) and citrinin (CTN). The mechanism of CTN and OTA toxicities in biological systems is not fully understood but it is known that oxidative stress is involved. In this study, oxidative damage of DNA, lipids, and the concentration of glutathione (GSH), as well as possible antioxidative effects of resveratrol (RSV) were studied in vivo. Male adult Wistar rats were treated orally with OTA (0.125 and 0.250 mg kg-1 b.w.), RSV (20 mg kg-1 b.w.) for 21 days, CTN (20 mg kg-1 b.w.) for two days or with their combinations. The hOGG1 modified comet assay revealed kidneys and liver oxidative DNA damage in OTA + CTN treated animals, which was not reversed by RSV. CTN did not reduce glutathione (GSH) or increase malondialdehyde (MDA) concentration in any tissue, while OTA reduced kidneys GSH and increased kidneys and liver MDA. RSV increased GSH concentrations in all tissues and decreased MDA concentration in the liver only. Oxidative stress is involved in the toxicity of OTA and CTN but it seems that it differs significantly in organs. Most of the effects on GSH and MDA in combined toxicity may be attributed to the toxic effects of OTA. RSV was effective in restoring the depleted GSH in all tissues but had no effect on the MDA concentration and DNA damage.

Effects of low doses of glyphosate on DNA damage, cell proliferation and oxidative stress in the HepG2 cell line.

We studied the toxic effects of glyphosate in vitro on HepG2 cells exposed for 4 and 24 h to low glyphosate concentrations likely to be encountered in occupational and residential exposures [the acceptable daily intake (ADI; 0.5 µg/mL), residential exposure level (REL; 2.91 µg/mL) and occupational exposure level (OEL; 3.5 µg/mL)]. The assessments were performed using biomarkers of oxidative stress, CCK-8 colorimetric assay for cell proliferation, alkaline comet assay and cytokinesis-block micronucleus (CBMN) cytome assay. The results obtained indicated effects on cell proliferation, both at 4 and 24 h. The levels of primary DNA damage after 4-h exposure were lower in treated vs. control samples, but were not significantly changed after 24 h. Using the CBMN assay, we found a significantly higher number of MN and nuclear buds at ADI and REL after 4 h and a lower number of MN after 24 h. The obtained results revealed significant oxidative damage. Four-hour exposure resulted in significant decrease at ADI [lipid peroxidation and glutathione peroxidase (GSH-Px)] and OEL [lipid peroxidation and level of total antioxidant capacity (TAC)], and 24-h exposure in significant decrease at OEL (TAC and GSH-Px). No significant effects were observed for the level of reactive oxygen species (ROS) and glutathione (GSH) for both treatment, and for 24 h for lipid peroxidation. Taken together, the elevated levels of cytogenetic damage found by the CBMN assay and the mechanisms of primary DNA damage should be further clarified, considering that the comet assay results indicate possible cross-linking or DNA adduct formation.

The effect of insecticides chlorpyrifos, α-cypermethrin and imidacloprid on primary DNA damage, TP 53 and c-Myc structural integrity by comet-FISH assay.

In parallel with the continuous use of conventional insecticides, introduction of more environmentally friendly substances continues to grow in modern agriculture. In the present study, we evaluated chlorpyrifos, and imidacloprid and α-cypermethrin as two representatives of green insecticides for their genotoxic activity. We conducted a 14-day treatment in extended human lymphocytes cultures using real life exposure relevant concentrations. An alkaline comet assay was used to detect primary DNA damage. Simultaneously, the effect on the specific action towards the TP 53 and c-Myc genes in terms of fragmentation and copy number were determined. Both genes are responsible for cell cycle regulation; thus playing an active role in carcinogenesis. Contrary to what was expected, imidacloprid showed the highest genotoxicity potential, irrespective of the fact that none of the insecticides induced a significant level of primary DNA damage at all tested concentrations. Similar, no significant effect towards the TP 53 and c-Myc gene was recorded. The present study indicates that low level use of chlorpyrifos as a conventional insecticide and imidacloprid and α-cypermethrin as green insecticides does not pose a risk to DNA in general, nor to the TP 53 and c-Myc gene structural integrity.

An alternative approach to studying the effects of ZnO nanoparticles in cultured human lymphocytes: combining electrochemistry and genotoxicity tests.

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.

Cytotoxic, genotoxic and biochemical markers of insecticide toxicity evaluated in human peripheral blood lymphocytes and an HepG2 cell line.

This study evaluated the cyto- and genotoxic effects of three pesticides: α-cypermethrin, chlorpyrifos and imidacloprid applied in vitro to human lymphocytes and HepG2 cells for exposure times of 4 and 24 h at concentrations corresponding to OEL, ADI and REL. Assessments were made using oxidative stress biomarkers and the alkaline comet, cytokinesis-block micronucleus cytome and cell viability assays. Low doses of all three pesticides displayed DNA damaging potential, both in lymphocytes and HepG2 cells. At the tested concentrations, all three compounds induced lymphocyte apoptosis, though α-cypermethrin and chlorpyrifos were generally more cyto- and genotoxic than imidacloprid. At the tested concentrations, oxidative stress biomarkers were not significantly altered, and the effects mediated indirectly through free radicals may not have a key role in the formation of DNA damage. It is likely that the DNA damaging effects were caused by direct interactions between the tested compounds and/or their metabolites that destabilized the DNA structure. The tested pesticides had the potential for MN, NB and NPB formation and to disturb cell cycle kinetics in both cell types. There were also indications that exposure to α-cypermethrin led to the formation of crosslinks in DNA, though this would require more detailed study in the future.

Evaluation of genome damage in subjects occupationally exposed to possible carcinogens.

In occupational exposures, populations are simultaneously exposed to a mixture of chemicals. We aimed to evaluate DNA damage due to possible carcinogen exposure (phenylhydrazine, ethylene oxide, dichloromethane, and 1,2-dichloroethane) in lymphocytes of pharmaceutical industry workers from the same production line. Population comprised 16 subjects (9 females and 7 males) who were exposed to multiple chemicals for 8 months. Genome damage was assessed using alkaline comet assay, micronucleus assay, and comet assay coupled with fluorescent in situ hybridization (comet-FISH). After 8 months of exposure, the issue of irregular use of all available personal protective equipment (PPE) came into light. To decrease the risk of exposure, strict use of PPE was enforced. After 8 months of strict PPE use, micronuclei frequency and comet assay parameters in lymphocytes of pharmaceutical workers significantly decreased compared with prior period of irregular PPE use. Comet-FISH results indicated a significant shift in distribution of signals for the TP 53 gene toward a more frequent occurrence in the comet tail. Prolonged exposure to possible carcinogens may hinder DNA repair mechanisms and affect structural integrity of TP 53 Two indicators of loss of TP 53 gene integrity have risen, namely, TP 53 fragmentation rate in lymphocytes with persistently elevated primary damage and incidence of TP 53 deletions in undamaged lymphocytes.

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.

In vitro detection of DNA damage in human leukocytes induced by combined effect of resin composites and adhesive systems.

PURPOSE: To simultaneously evaluate the genotoxicity of dental composites and adhesive systems in vitro using a cytogenetic assay, with respect to the influence of composite shade. METHODS: Genotoxicity assessment was carried out in human peripheral blood leukocytes using the comet assay. Three resin composite materials, two microhybrids and one nano-hybrid, in shade A1 and A3.5 were used with manufacturer-recommended four adhesive systems. Cultures were treated for 48 hours with samples after elusion for 1 hour, 1 day, 7 days or 30 days, in two different concentrations (4.16 mg/mL, 8.33 mg/mL). Kruskall-Wallis test was used for the statistical analysis (alpha = 0.05). RESULTS: For combinations of micro-hybrid composite (A3.5) with two self-etch adhesives (16.1 +/- 5.50 and 16.2 +/- 9.52) after exposure to samples eluted for 1 day, the incidence of primary DNA damage was significantly higher than for the corresponding negative control (14.7 +/- 2.85). Genotoxicity was also higher after treatment with samples eluted for 1 hour (15.3 +/- 4.70) and 1 day (15.3 +/- 9.10), comprised of nano-hybrid composite (A1) with self-etch adhesive in relation to the control (13.1 +/- 1.70). There was no clear trend of increased DNA damage in material combinations with darker shades of composites. Material composition and higher material concentrations showed greater influence on the genotoxicity.

Micronucleus, alkaline, and human 8-oxoguanine glycosylase 1 modified comet assays evaluation of glass-ionomer cements - in vitro.

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.

Genotoxicity in gingival cells of patients undergoing tooth restoration with two different dental composite materials.

OBJECTIVES: Dental composite materials come into direct contact with oral tissue, especially gingival cells. This study was performed to evaluate possible DNA damage to gingival cells exposed to resin composite dental materials. MATERIALS AND METHODS: Class V restorations were placed in 30 adult patients using two different composite resins. The epithelial cells of the gingival area along the composite restoration were sampled prior to and after 7, 30, and 180 days following the restoration of the tooth. DNA damage was analysed by comet and micronucleus assays in gingival exfoliated epithelial cells. RESULTS: The results showed significantly higher comet assay parameters (tail length and % DNA in the tail) within periods of 30 and 180 days. The micronucleus test for the same exposure time demonstrated a higher number of cells with micronuclei, karyolysis, and nuclear buds. Results did not reveal any difference between the two composite materials for the same duration of exposure. CONCLUSION: Based on the results, we can conclude that the use of composite resins causes cellular damage. As dental composite resins remain in intimate contact with oral tissue over a long period of time, further research on their possible genotoxicity is advisable. CLINICAL RELEVANCE: Long-term exposure of gingival cells to two different composite materials demonstrated certain DNA damage. However, considering the significant decline in micronuclei frequency after 180 days and efficiency in the repair of primary DNA damage, the observed effects could not be indicated as biologically relevant.

Modification of comet-FISH technique by using temperature instead of chemical denaturation.

Comet-FISH technique is an extension of commonly used comet assay. Its purpose is to determine whether primary DNA damage which comet assay detects occurred within a sequence of interest that is visualized by hybridization of fluorescent probe. Presence of the signal in comet tail indicates impaired structural integrity of sequence. Our modifications to the original comet-FISH technique described by Rapp et al. (2000) [1] include:•increase in probe binding specificity,•increased rate of successful hybridization,•simultaneous temperature denaturation of both, slide and probe.

The use of FISH-comet to detect c-Myc and TP 53 damage in extended-term lymphocyte cultures treated with terbuthylazine and carbofuran.

Terbuthylazine and carbofuran are suspected to cause non-Hodgkin's lymphoma and lung cancer. We evaluated the effects of prolonged exposure to low concentrations on primary DNA damage by comet assay, and on the structural integrity of c-Myc and TP 53 genes by FISH-comet. Another novelty in studying these pesticides' genotoxicity is the use of 14-day extended-term human lymphocyte cultures. Concentrations corresponded to values of ADI and OEL: for terbuthylazine 0.58 ng/ml and 8 ng/ml; for carbofuran 8 ng/ml and 21.6 ng/ml, respectively. A possible effect of metabolic activation (S9) was also considered. Carbofuran treatment induced a significant migration of DNA into the tail in a concentration-dependent manner, while for terbuthylazine the effect was significant only at the higher concentration. Terbuthylazine caused migration of both c-Myc signals into the comet tail. A significant occurrence of TP 53 signals in the tail was observed at 8 ng/ml. Prolonged carbofuran treatment significantly elevated the migration of a single c-Myc signal into the tail in a concentration-dependent manner. With S9, distribution of signals shifted toward increased presence of both signals in tail. Our results showed impaired structural integrity of c-Myc and TP 53 due to prolonged exposure to terbuthylazine and carbofuran.

In vivo assessment of DNA damage induced in oral mucosa cells by fixed and removable metal prosthodontic appliances.

Given long-term effect on oral tissues due to contact with dental appliances, the biocompatibility studies of casting alloys are of great importance. It has been previously documented that metal dental appliances, due to corrosion, might induce genotoxic and mutagenic effects in cells. Therefore, the aim of presented study was to examine the genotoxicity of two dental casting alloys (Co-Cr-Mo and Ni-Cr) commonly used in fixed and removable prosthodontic appliances that are in contact with the oral epithelium for 5 years or more. For that purpose, 55 age-matched subjects were included in the study; 30 wearers of prosthodontic appliances and 25 controls. Buccal cells of oral mucosa were collected and processed for further analysis. The cell viability has been assessed by trypan blue exclusion test, while genotoxic effect of metal ions on DNA in oral mucosa cells was studied by use of alkaline comet assay. Results have shown significantly higher comet assay parameters (tail length and percentage DNA in the tail) in the group wearing metal appliances. Both subjects with Co-Cr-Mo alloy and Ni-Cr alloy showed significantly higher comet assay parameters when compared with controls. It has been confirmed that metal ions released by the two base metal dental casting alloys examined in this study, might be responsible for DNA damage of oral mucosa cells. Therefore, the results of this study emphasize the importance of the in vivo evaluation of dental materials with respect to their genotoxicity, which is of major importance to ensure long-term biocompatibility.

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.

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.

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 Kosovcica 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.

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 apart 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 microm), the tail was slightly elongated in brain cells of irradiated animals (14.0 +/- 0.3 microm). The tail length obtained for liver (14.5 +/- 0.3 microm) and kidney (13.9 +/- 0.5 microm) homogenates notably differs in comparison with matched sham controls (13.6 +/- 0.3 microm) and (12.9 +/- 0.9 microm). 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.