Assessment of multiple anthropogenic contaminants and their potential genotoxicity in the aquatic environment of Plitvice Lakes National Park, Croatia.

In this study, the influence of anthropogenic pollution on the aquatic environment of Plitvice Lakes National Park (PLNP) was investigated during 2011-2012 using a combination of chemical and cytogenetic analyses. Four groups of major contaminants [(volatile organic compounds: benzene, toluene, ethylbenzene, and xylenes (BTEX); persistent organochlorine pollutants: organochlorine pesticides (OCPs) and polychlorinated biphenyls (PCBs); major and trace elements; anthropogenic radionuclides (90Sr, 134Cs, and 137Cs)] were determined in three aquatic compartments (water, sediment, fish). Mass fractions of inorganic constituents in different compartments reflected the geological background of the area, indicating their origin from predominantly natural sources. Levels of volatile and persistent organic compounds in water and fish, respectively, were very low, at levels typical for remote pristine areas. Analysis of anthropogenic radionuclides in water and sediment revealed elevated activity concentrations of 137Cs in water, and measurable 134Cs in the upper sediment layers from April 2011, possibly as a consequence of the Fukushima nuclear accident in March 2011. The potential genotoxicity of river and lake water and lake sediment was assessed under laboratory conditions using the alkaline comet assay on human peripheral blood lymphocytes, and measured levels of primary DNA damage were within acceptable boundaries. The results showed that despite the protected status of the park, anthropogenic impact exists in both its terrestrial and aquatic components. Although contaminant levels were low, further monitoring is recommended to make sure that they will not rise and cause potentially hazardous anthropogenic impacts.

In vitro assessment of the cytotoxic, DNA damaging, and cytogenetic effects of hydroquinone in human peripheral blood lymphocytes.

This study investigated the mechanisms of hydroquinone toxicity and assessed the relationships between its cytotoxic, genotoxic, and cytogenetic effects tested at 8, 140, and 280 µg mL-1 in human peripheral blood lymphocytes exposed for 24 h. The outcomes of the treatments were evaluated using the apoptosis/necrosis assay, the alkaline comet assay, and the cytokinesis-block micronucleus (CBMN) cytome assay. The tested hydroquinone concentrations produced relatively weak cytotoxicity in resting lymphocytes, which mostly died via apoptosis. Hydroquinone's marked genotoxic effects were detected using the alkaline comet assay. Significantly decreased values of all comet parameters compared to controls indicated specific mechanisms of hydroquinone-DNA interactions. Our results suggest that the two higher hydroquinone concentrations possibly led to cross-linking and adduct formation. Increased levels of DNA breakage measured following exposure to the lowest concentration suggested mechanisms related to oxidative stress and inhibition of topoisomerase II. At 8 µg mL-1, hydroquinone did not significantly affect MN formation. At 140 and 280 µg mL-1, it completely blocked lymphocyte division. The two latter concentrations also led to erythrocyte stabilization and prevented their lysis. At least two facts contribute to this study's relevance: (I) this is the first study that quantifies the degree of reduction in total comet area measured in lymphocyte DNA after hydroquinone treatment, (II) it is also the first one on a lymphocyte model that adopted the "cytome" protocol in an MN assay and found that lymphocytes exposure even to low hydroquinone concentration resulted in a significant increase of nuclear bud frequency. Considering the limitations of the lymphocyte model, which does not possess intrinsic metabolic activation, in order to unequivocally prove the obtained results further studies using other appropriate cell lines are advised.

Assessment of oxidative stress responses and the cytotoxic and genotoxic potential of the herbicide tembotrione in HepG2 cells.

Tembotrione is a triketone herbicide, usually used for post-emergence weed control in corn. Currently, there is little or no published data on its genotoxicity to human cells either in vitro or in vivo. This study evaluated the impact of acute (4 and 24 h) exposure to low concentrations of tembotrione [corresponding to the acceptable daily intake (0.17 µg/mL), residential exposure level (0.002 µg/mL) and acceptable operator exposure level (0.0012 µg/mL)] on human hepatocellular carcinoma cell line HepG2, using biomarkers of oxidative stress, CCK-8 colorimetric assay for cell viability, alkaline comet assay, and cytokinesis-block micronucleus "cytome" assay. Tembotrione applied at concentrations likely to be encountered in occupational and residential exposures induced cytogenetic outcomes in non-target cells despite non-significant changes in the values of oxidative stress biomarkers. We assume that the observed effects were mainly the consequence of impaired metabolic pathways in HepG2 cells due to the inhibition of the enzyme 4-hydroxyphenyl-pyruvate-dioxygenase by tembotrione, which possibly caused a depletion of folate levels leading to excess formation of nuclear buds in the affected cells. Regardless of the fact that tembotrione was previously reported negative for mutations and chromosome aberrations in vitro, our findings call for more precaution in its use.

Toxic airborne S, PAH, and trace element legacy of the superhigh-organic-sulphur Rasa coal combustion: Cytotoxicity and genotoxicity assessment of soil and ash.

This paper presents the levels of sulphur, polycyclic aromatic hydrocarbons (PAHs), and potentially toxic trace elements in soils surrounding the Plomin coal-fired power plant (Croatia). It used domestic superhigh-organic-sulphur Rasa coal from 1970 until 2000. Rasa coal was characterised by exceptionally high values of S, up to 14%, making the downwind southwest (SW) area surrounding the power plant a significant hotspot. The analytical results show that the SW soil locations are severely polluted with S (up to 4%), and PAHs (up to 13,535ng/g), while moderately with Se (up to 6.8mg/kg), and Cd (up to 4.7mg/kg). The composition and distribution pattern of PAHs in the polluted soils indicate that their main source could be airborne unburnt coal particles. The atmospheric dispersion processes of SO2 and ash particles have influenced the composition and distribution patterns of sulphur and potentially toxic trace elements in studied soils, respectively. A possible adverse impact of analysed soil on the local karstic environment was evaluated by cytotoxic and genotoxic methods. The cytotoxicity effects of soil and ash water extracts on the channel catfish ovary (CCO) cell line were found to be statistically significant in the case of the most polluted soil and ash samples. However, the primary DNA-damaging potential of the most polluted soil samples on the CCO cells was found to be within acceptable boundaries.

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.

In vitro cytotoxicity assessment of imidazolium ionic liquids: biological effects in fish Channel Catfish Ovary (CCO) cell line.

Increasing interest in the application of ionic liquids as green replacement for volatile organic solvents emphasized the need for the evaluation of their toxic effects at different biological systems in order to reduce the risk for human health and environment. To our knowledge, effects of imidazolium ionic liquids on cellular level of fish cell lines have not been studied yet. The cytotoxicity of imidazolium ionic liquids containing different anions and alkyl chain lengths as the substituent at the cation ring towards the fish CCO cell line was determined by WST-1 proliferation assay. Morphological alterations were examined by fluorescent microscopy using acridine orange/ethidium bromide staining and flow cytometry analysis was also performed. The results showed concentration-dependent cytotoxicity of ionic liquids in CCO cells, related to the type of anion and alkyl chain length, while EC50 values showed moderate to high cytotoxicity of tested imidazolium ionic liquids. Distinct morphological changes observed under fluorescence microscope and data obtained by flow cytometry suggest that the toxicity of imidazolium ionic liquids with longer alkyl chains could be related to necrosis. Results presented in here may be helpful for filling existing gaps of knowledge about ionic liquids toxicity and their impact on aquatic environment.

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.

Assessment of tryptophol genotoxicity in four cell lines in vitro: a pilot study with alkaline comet assay.

Tryptophol is an aromatic alcohol and secondary metabolite of the opportunistic fungus Candida albicans. Although its toxicity profile at cell level has been poorly investigated, recent data point to cytotoxic, cytostatic, and genotoxic effects in lymphocytes and the induction of apoptosis in leukaemic blood monocytes. In this pilot study we evaluated the genotoxicity of tryptophol in vitro on four permanent cell lines of animal and human origin: ovary cells, alveolar epithelium, liver cells, and blood monocytes using the alkaline comet assay. We selected cells that might be principal targets of tryptophol and other low-molecular geno(toxins) secreted by Candida albicans during host invasion. Our results suggest that tryptophol applied in vitro at 2 mmol L(-1) for 24 h damages DNA in HepG2, A549 and THP-1 cells, obviously due to bioactivation and/or decomposition of the parent compound, which results in the formation of more genotoxic compound(s) and production of reactive species that additionally damage DNA. On the other hand, notably lower levels of primary DNA damage were recorded in CHO cells, which lack metabolic activity. Future studies with tryptophol should look further into mechanisms involved in its toxic action and should focus on other cell types prone to infection with Candida spp. such as vaginal epithelial cells or keratinocytes of human origin.

The assessment of genotoxic effects of wastewater from a fertilizer factory.

In this study we investigated cytotoxic, mutagenic and genotoxic effects of different concentrations of wastewater from the phosphoric gypsum depot near the factory for fertilizing agents 'INA Petrokemija' (Kutina, Croatia). The Ames test was performed on Salmonella typhimurium TA98 and TA100 strains, in the presence of S9 mix, glutathione and buffer, respectively. Cytotoxicity was studied on human laryngeal carcinoma cells (HEp2) and human cervical cells (HeLa). The level of lipid peroxidation in these two cell lines was evaluated in parallel. To establish the levels of primary DNA damage, the alkaline comet assay was performed on treated human peripheral blood leukocytes. No mutagenic effects of phosphoric gypsum on Salmonella typhimurium strains in the presence of S9 mix, GSH or PBS were observed. However, strong cytotoxic effect was observed on both human cell lines when they were treated with different concentrations of wastewater. Lipid peroxidation was induced and increased by prolonged time of incubation, highlighting that the damage was not repaired, but increased with the time of incubation. The results of the alkaline comet assay indicate significant DNA damaging potential of wastewater for human leukocytes. Since phosphoric gypsum transport water in its present composition and acidity is highly toxic and acts as prooxidant, causing free radicals formation and DNA damage, urgent neutralization/purification of the wastewater to a level acceptable for disposal into the environment is mandatory.

Assessment of genotoxic risks in Croatian health care workers occupationally exposed to cytotoxic drugs: a multi-biomarker approach.

The aim of the present study was to evaluate genome damage induced in peripheral blood lymphocytes of Croatian health care workers occupationally exposed to cytotoxic drugs. A comprehensive multi-biomarker approach using the alkaline comet assay and cytogenetic endpoints (analysis of structural chromosome aberrations, SCE assay, lymphocyte proliferation kinetics and cytokinesis-block micronucleus assay) was employed. The study included two populations of subjects: 50 health care workers occupationally exposed to cytotoxic drugs and 50 control subjects matched in age, gender and smoking habit. An investigation regarding the handling practice with cytotoxic drugs was conducted in parallel. Results obtained indicate high exposure levels at workplace that should be reduced. The values recorded among the occupationally exposed subjects were as follows: mean comet tail length: 17.46+/-0.08 microm; the incidence of long-tailed nuclei: 54.68+/-3.93%; 4.48+/-0.33 structural chromosome aberrations per 200 cells; 5.81+/-0.04 SCE per 50 cells; 29.28+/-2.21% of high-frequency cells; proliferation rate index: 1.97+/-0.12; and 16.32+/-0.85 micronuclei per 1000 binuclear cells. All these values indicated higher levels of DNA and cytogenetic damage compared to the general population. Obtained results also confirmed that the frequency of long-tailed nuclei in the alkaline comet assay represents a helpful complement to other well-established comet parameters. The age of subjects and smoking habit significantly influenced the values of both comet and cytogenetic endpoints. Overall results of this study confirmed that handling cytotoxic drugs without appropriate safety precautions involves a potential genotoxic risk for exposed subjects. Before a strict monitoring of exposure levels on each workplace becomes a standard practice in Croatian hospitals, cytogenetic surveillance of exposed workers is also recommended, at least in cases of accidental exposure.

Assessment by survival analysis of the radioprotective properties of propolis and its polyphenolic compounds.

The radioprotective effects of propolis and polyphenolic compounds from propolis on the radiation-induced mortality of mice exposed to 9 Gy of gamma-irradiation were studied. Intraperitoneal (i.p.) treatment of mice at doses of 100 mg kg(-1) body weight of propolis (water or ethanolic extract; WSDP or EEP) or its polyphenolic compounds (quercetin, naringin caffeic acid, chrysin) consecutively for 3 d before irradiation, delayed the onset of mortality and reduced the symptoms of radiation sickness. All test compounds provided protection against hematopoietic death (death within 30 d after irradiation). The greatest protection was achieved with quercetin; the number of survivors at the termination of the experiment was 63%. According to statistical analyses by the Kaplan-Meier method and the log-rank test, a significant difference between test components and control was found (p<0.001). Treatment with test components after lethal irradiation was ineffective. These results suggest that propolis and its polyphenolic compounds given to mice before irradiation protect mice from the lethal effects of whole-body irradiation.

Assessment of the radioprotective effects of amifostine and melatonin on human lymphocytes irradiated with gamma-rays in vitro.

Radioprotective effects of amifostine and melatonin as well as their ability to modulate the level of spontaneous and gamma-irradiation-induced genetic changes on human peripheral blood lymphocytes were investigated using the cytokinesis-block micronucleus (CBMN) assay and sister chromatid exchange (SCE). Parallel blood samples were pre-treated with amifostine, melatonin and their combination for 30 minutes. Negative controls were also included. After the treatment with radioprotectors, one blood sample of each experimental group was exposed to gamma-rays from a 60Co source. The radiation dose absorbed was 2 Gy. Our research confirmed the radioprotective effects of both chemicals in vitro, with no significant genotoxicity. Pre-treated irradiated blood samples showed a decrease in the total number of micronuclei (MN) and in the number of cells with more than one MN. They also showed significantly lower mean SCE values. This study shows that it is possible combine these radioprotectors by adjusting the doses of amifostine to achieve the best radioprotective effect with as few side effects as possible. However, further in vitro and clinical studies are needed to clarify their mechanisms of action and possible interactions.

Assessment of DNA damage in nuclear medicine personnel--comparative study with the alkaline comet assay and the chromosome aberration test.

Despite much research over the last few decades, there still remains considerable uncertainty as to the genetic impact of ionizing radiation on human populations, particularly at low levels. The aim of the present study was to provide data on the genetic hazards due to occupational exposure of low doses of ionizing radiation in nuclear medicine departments. The assessment of primary DNA damage in peripheral blood leukocytes of medical staff was performed using the alkaline comet assay and the data obtained were compared with the results of conventional cytogenetic biodosimetry using the chromosome aberration (CA) test. Altogether 120 subjects (60 exposed and 60 controls) participated in the study. Statistically significant increases in primary DNA damage and increased frequencies of CAs compared to controls were observed. Within the exposed population, significant inter-individual differences in DNA damage were found, indicating differences in genome sensitivity. Age and gender were not confounding factors, while smoking enhanced the levels of primary DNA damage only in control subjects, as revealed by both biomarkers studied. The present study suggests that genotoxic damage results from exposure to chronic low doses of ionizing radiation in nuclear medicine departments. Therefore, the exposed medical personnel should carefully comply with the radiation protection procedures and should minimize radiation exposure where possible to avoid potential genotoxic effects. The results obtained in this study point to the significance of biological indicators providing information on the actual risk to the radiation exposed individuals. According to our results, the alkaline comet assay and CA test are sensitive biomarkers that can be used as additional complements to physical dosimetry for assessing exposure to radiation in nuclear medicine personnel.

The alkaline Comet assay as biomarker in assessment of DNA damage in medical personnel occupationally exposed to ionizing radiation.

The alkaline Comet assay was selected as a biomarker of exposure to evaluate the ongoing exposure to ionizing radiation of 50 medical workers occupationally exposed to ionizing radiation and 50 corresponding unexposed control subjects. The primary DNA damage was evaluated by measuring the extent of DNA migration in peripheral blood leukocytes. The inter-individual differences in DNA damage between exposed subjects were compared with their dosimeter readings and occupation. It was found that medical workers who were occupationally exposed to ionizing radiation for different periods of time showed highly significant increases in levels of DNA damage compared with controls. However, influences of the different occupational settings and doses absorbed on the levels of DNA damage, assessed by use of the Comet assay, might be excluded in the majority of subjects. Differences in comet parameters measured due to smoking and gender were not statistically significant in either exposed or control subjects. The results obtained have confirmed the usefulness of the alkaline Comet assay as an additional complement to standard biodosimetric methods. By detection of momentary DNA damage and/or repair activity, it reflects the concurrent exposure and the actual levels of DNA damage present in peripheral blood leukocytes of the radiological workers at the moment of blood sampling.

Assessment of chemotherapy-induced DNA damage in peripheral blood leukocytes of cancer patients using the alkaline comet assay.

The alkaline comet assay was employed to assess the pre- and post-treatment levels of in vivo DNA damage in peripheral blood leukocytes of cancer patients. During the study all patients were given antineoplastic drugs, mainly as polychemotherapy. To quantify the DNA damage, two different comet parameters were evaluated: the tail length and the tail moment. Our results indicate marked interindividual variations between baseline DNA damage in peripheral blood leukocytes recorded among cancer patients prior to the chemotherapy. After intravenous administration of various antineoplastic drugs, a significantly increased level of DNA damage in all cancer patients compared to their pre-treatment values was recorded The highest level of DNA damage was seen following administration of 5-fluorouracil, adriamycin, and cisplatin (FAP protocol). The results indicate that administration of antineoplastic drugs in standard protocols is accompanied by significant DNA damage in peripheral blood leukocytes. In order to diminish the potential risks of developing second neoplasms, a continuous biomonitoring of cancer patients after the ending of chemotherapy becomes important. Despite their limitations, present results confirm the usefulness of the alkaline comet assay as a sensitive biomarker of exposure that enables rapid and simple detection of primary DNA damage in peripheral blood leukocytes of cancer patients. Together with standard cytogenetic endpoints, the comet assay provides a powerful technique for the routine detection of critical DNA lesions produced after administration of antineoplastic drugs in the clinical settings.