In vitro safety assessment of the strawberry tree (Arbutus unedo L.) water leaf extract and arbutin in human peripheral blood lymphocytes.

Strawberry tree (Arbutus unedo L.) leaves have long been used in the traditional medicine of the Mediterranean region. One of their most bioactive constituents is the glycoside arbutin, whose presence makes A. unedo suitable as a potential substitute for bearberry [Arctostaphylos uva ursi (L.) Spreng] leaves, an herbal preparation widely used for treating urinary tract infections. The safety and biocompatibility of strawberry tree water leaf extract have not yet been documented well. This study estimated arbutin content in strawberry tree water leaf extract (STE) using high performance liquid chromatography. Furthermore, we performed an in vitro safety assessment of the 24 h exposure to three presumably non-toxic concentrations of standardized STE and arbutin in human peripheral blood lymphocytes using the apoptosis/necrosis assay, the alkaline comet assay, and the cytokinesis-block micronucleus cytome assay. The STE was also tested for total antioxidant capacity and lipid peroxidation. At a concentration corresponding to the maximum allowable daily intake of arbutin, the tested extract was not cytotoxic, had a negligible potential for causing primary DNA damage and even hindered micronuclei formation in lymphocytes. It also showed a valuable antioxidant capacity, and did not exert marked lipid peroxidation. These promising results represent a solid frame for further development of STE-based herbal preparations. Although arbutin generally had a low DNA damaging potential, the slowing down of lymphocyte proliferation observed after 24 h of exposure points to a cytostatic effect, which merits further research.

Evaluation of genotoxic effects of lead in pottery-glaze workers using micronucleus assay, alkaline comet assay and DNA diffusion assay.

PURPOSE: We investigated genotoxic effects of occupational exposure to lead acetate in pottery-glaze ceramic workers. METHODS: The study was carried out in 30 exposed workers and 30 matched controls, to whom several biochemical parameters-the blood lead (B-Pb; range: exposed, 41.68-404.77; controls, 12-52) and cadmium (B-Cd) level, the activity of delta-aminolevulinic acid dehydratase (ALAD), erythrocyte protoporphyrin (EP), the level of vitamin B(12) and folate in serum-were measured. The genotoxic effects were evaluated by the alkaline comet assay, the DNA diffusion assay and micronucleus test in peripheral blood lymphocytes. RESULTS: Subjects exposed to lead had significantly higher B-Pb level and, consequently, increased values of tail intensity (TI), frequency of apoptotic and necrotic cells, and frequency of micronuclei (MN). In contrast, their activity of ALAD, the level of vitamin B(12) and folate in serum were significantly lower compared to controls. Poisson regression analysis showed a significant correlation of profession, duration of exposure, smoking, level of cadmium in blood, ALAD and EP with primary DNA damage. A majority of primary damage repairs in a short period after exposure to a genotoxic agent. In addition, the influence of gender and level of vitamin B(12) and folate in serum MN frequency in exposed group was observed. CONCLUSIONS: In this study, DNA diffusion and micronucleus test showed higher influence of tested parameters to DNA damage. The results indicate a need for concomitant use of at least two different biomarkers of exposure when estimating a genetic risk of lead exposure.

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.

Antimicrobial activity of commercial zeolite A on Acinetobacter junii and Saccharomyces cerevisiae.

The influence of three samples of commercially produced zeolite A (named A, M and R) in water medium on the bacterium Acinetobacter junii and yeast Saccharomyces cerevisiae was investigated. These microorganisms were used in the bioassay and are not specifically related to the use of zeolite A. All zeolite samples showed the negative influence on the survival and physiological status of A. junii and S. cerevisiae. The EC(50) values for the inhibition of CFU of A. junii were 0.328, 0.138 and 0.139 g l(-1) for zeolite sample A, M and R, respectively. The EC(50) values of tested zeolites for S. cerevisiae, estimated by fermentation and fluorescence microscopy assay, ranged from 2.88 to 5.47 g l(-1). The genotoxic effect of three samples of zeolite to S. cerevisiae was shown by the alkaline comet assay. When assuming all the aspects of zeolite toxicity to bacterium and yeast, the zeolite sample R appeared to be less toxic than the samples A and M. The hydrolysis of zeolite crystals, amorphous aluminosilicate and unreacted gel fraction in water medium and consecutive dissolution and leaching of aluminium and silicon in the form of aluminosilicate molecules (700-1300 Da) was detected.

Radioprotective effects of propolis and quercetin in gamma-irradiated mice evaluated by the alkaline comet assay.

The radioprotective effects of ethanolic extract of propolis (EEP) and quercetin on the white blood cells of the whole-body irradiated CBA mice were investigated. Irradiation was performed using a gamma-ray source ((60)Co), and absorbed dose was 9 Gy. The efficiency of test components was evaluated when given intraperitoneally (ip) at a dose of 100 mg kg(-1) for 3 consecutive days before and/or after irradiation. Moreover, possible genotoxic effects of test components were also assessed on non-irradiated animals. For each experimental group leukocyte count was determined and the primary DNA damage in leukocytes was assessed using the alkaline comet assay. The higher efficiency of EEP and quercetin was observed when given preventively. The results suggest that propolis and quercetin given to mice before irradiation protect their white blood cells from lethal effects of irradiation and diminish primary DNA damage as confirmed by the alkaline comet assay. Positive results obtained on gamma-irradiated mice given EEP and quercetin, complementary with our earlier observations on survival of irradiated mice, indicate that these compounds could be considered effective non-toxic radioprotectors. The exact mechanisms of radioprotection by these compounds and their effects on DNA repair processes are still to be elucidated.

Alkaline comet assay study with breast cancer patients: evaluation of baseline and chemotherapy-induced DNA damage in non-target cells.

The sensitivity of the alkaline comet assay for the evaluation of baseline and treatment-induced DNA damage in white blood cells of breast cancer patients receiving adjuvant chemotherapy according to three conventional anthracycline- and cyclophosphamide-containing protocols was investigated. Additionally, baseline DNA damage in cancer patients was compared with the levels of DNA damage recorded in healthy women. Altogether 30 patients with diagnosed breast cancer and 30 female blood donors with no known familial history of breast cancer participated in the study. Alkaline comet assay was performed according to standard protocol and DNA migration in peripheral blood leukocytes was measured by a computer-based image analysis system. For each subject the frequency of "damaged" cells, i.e., long-tailed nuclei (LTN) with tail length exceeding 95th percentile for the considered parameter among controls, is also reported. Breast cancer patients had significantly increased background levels of DNA damage in their peripheral blood leukocytes as compared to healthy women. Prior to the chemotherapy, a majority of patients showed a statistically significant increase in the number of LTN compared to healthy blood donors. Marked interindividual variations in baseline DNA damage among patients were recorded, some of them related to the disease stage and status. The present study confirmed the alkaline comet assay as a sensitive technique able to detect significantly elevated DNA migration in blood cells of patients already one hour after completion of the first cycle of chemotherapy. Administration of antineoplastic drugs in three chemotherapy protocols studied induced a similar increase of primary DNA damage in nontarget cells. The evaluation of the LTN frequencies indicates the best response to the protocol containing cyclophosphamide, methotrexate and 5-fluorouracil (CMF). Our results point to the significance of simultaneous evaluation of DNA migration and frequency of LTN in the same subject and approved the use of alkaline comet assay as a suitable method for the routine detection of critical DNA lesions produced after administration of antineoplastic drugs in the clinical settings.

Evaluation of antidotal effects of adamantyl derivative Tamorf in soman poisoning.

The nerve agent soman, a powerful inhibitor of acetylcholinesterase (AChE; EC 3.1.1.7), causes an array of toxic effects in the central nervous system. Adamantyl tenocyclidine derivative Tamorf (1-[2-(2-thienyl)-2-adamantyl] morpholine), a compound with potential activity at the N-methyl-D-aspartate (NMDA) receptors and with neuroprotective properties, is effective against convulsions and brain lesions related to soman poisoning. The objective of this study was to evaluate the antidotal potency of Tamorf (2.5 mg kg(-1)), which was tested alone as a pretreatment or in combination with atropine (10.0 mg kg(-1)) as a therapy in rats poisoned with two different sub-lethal doses of soman (1/4 and 1/2 of LD50). The effect of Tamorf was compared with carbamate physostigmine (0.1 mg kg(-1)). The study also determined the possible genotoxic effects of Tamorf and physostigmine, especially primary DNA damage in white blood cells, liver and brain tissue. Tamorf administered 5 min before poisoning stopped soman-induced seizures, was successful against sub-lethal doses of soman and protected AChE activity in the brain (P = 0.0014, P = 0.0019), and in plasma (P = 0.0464, P = 0.0405). Compared with Tamorf, physostigmine was slightly effective in the elimination of soman-induced poisoning in rats. The pharmacological effect of Tamorf and atropine was less effective as therapy, but did not increase soman toxicity (P > 0.05 for all interactions). The results obtained indicate that Tamorf and physostigmine are not genotoxic to rats in the concentrations tested. Treatment with Tamorf seems to be a good alternative for current pretreatment in soman poisoning. Its antidotal mechanism is complex and is based on combined biochemical and receptor properties.

Toxicity testing of herbicide norflurazon on an aquatic bioindicator species--the planarian Polycelis felina (Daly.).

Norflurazon is a bleaching, preemergence herbicide. Due to its mobility and long half-life it presents a potential for groundwater contamination. The aim of our study was to investigate toxic effects of norflurazon on non-targeted aquatic bioindicator organism, the planarian Polycelis felina (Daly.). Animals were exposed to water solutions of norflurazon in concentrations 200, 20, 2 and 0.2 microM. Mortality, locomotive and morphological changes were monitored. Histological changes were studied both on treated and control animals with light microscopy. The primary DNA damage on single planarian cells was studied using the alkaline comet assay. Three comet parameters were studied: tail length, percentage of DNA in comet tail and tail moment. The results showed that norflurazon caused mortality, locomotive, morphological and histological changes in treated animals compared to corresponding controls. The most prominent histological changes were damage of the outer mucous layer, lack of rhabdites, damage to epidermis and extensive damage to parenchyma cells. The results of alkaline comet assay indicated that norflurazon in concentrations of 2 and 0.2 microM induces significant increase of primary DNA damage in planarian cells compared to the corresponding control animals. The mean values of all three measured parameters were significantly elevated on the fourth day of the treatment compared with the first and the seventh day. Based on the results of mortality and locomotive observations, we conclude that the fourth day of the treatment represents a certain threshold within planarian metabolism followed by the beginning of detoxification and recovery. However, histological preparations and comet data statistics show results indicating that high toxicity on the seventh day of the treatment gave the results of decrease of DNA damage due to the tissue/cell damage (apoptosis) and not recovery. The present study showed the ability of norflurazon to induce a wide range of different toxicological responses in freshwater planarian Polycelis felina (Daly.).

The impairments of neoblast division in regenerating planarian Polycelis felina (Daly.) caused by in vitro treatment with cadmium sulfate.

The effects of cadmium sulfate on the neoblast mitotic activity in regenerating planarian Polycelis felina (Daly.) were investigated. Mitotic abnormalities and chromosomal aberrations were evaluated after 6-h treatment and 24-h recovery period. The blastema were fixed, and examined cytologically through routine lactoorceine squash preparations. Mitotic indices were also determined. Cadmium sulfate induced a dose-dependent decrease in neoblast mitotic activity, accompanied with disturbances in distribution of cells over mitotic phases. Different cytological abnormalities with varying frequency were observed. Marked mitotic depression was concentration-dependent. Toxic effects of cadmium in regenerating planarian were mainly associated with mitotic spindle disturbances. Immediately after treatment mitotic abnormalities were prevalent over chromosomal and C-mitosis was the most prominent one. After 24-h recovery period a prevalence of mitotic over chromosomal aberrations was still present in animals treated with two higher concentrations of cadmium sulfate. However, the proportions of cells with chromosome stickiness in all treated animals were significantly increased compared to their post-treatment values. Observed mitotic impairments could be related to mitotic arrest contributing to retardations and delays, especially in animals treated with the highest concentration tested. The results obtained indicated usefulness of short term invertebrate assays as an alternative to in vitro pre-screening of toxic chemicals.

Application of the alkaline comet assay in biodosimetry: assessment of in vivo DNA damage in human peripheral leukocytes after a gamma radiation incident.

The alkaline comet assay was employed in the assessment of DNA damage in leukocytes of a worker incidently exposed to gamma radiation (221 mSv, 60Co source). The comet tail lengths and tail moments were studied. By using the alkaline comet assay immediately after accidental exposure a high level of DNA damage was recorded. The highest levels of DNA damage were recorded one day and one week after the radiation incident. Later on, a decrease in both comet parameters was observed. Although the level of DNA damage was diminished during a one year period, it was still elevated compared to normal values recorded in leukocytes of a healthy, unexposed person. The results obtained indicate that the alkaline comet assay is a rapid and sensitive microdosimetric technique and is suitable for in vivo human biomonitoring, especially in cases of incidental exposure to ionising radiation.

Application of the alkaline comet assay in human biomonitoring for genotoxicity: a study on Croatian medical personnel handling antineoplastic drugs.

The alkaline comet assay was used to evaluate the genotoxicity towards peripheral blood lymphocytes of medical personnel regularly handling various antineoplastic drugs with different safety precautions. The study population consisted of 50 exposed subjects working in the oncology, pulmonology, gynaecology and haematology units of nine Croatian hospitals and 20 unexposed control subjects. Peripheral blood lymphocytes from the subjects were embedded in agarose on a microscope slide and lysed; the DNA was unwound and subjected to electrophoresis at pH 13. Staining with a fluorescent dye was used to identify cells with DNA damage, as judged by increased migration of genetic material from the cell nucleus. DNA damage was quantified by measuring the displacement between the genetic material of the nucleus and the resulting tail using an image analysis system. Three parameters were used as indicators of DNA damage: i.e. tail length, percentage of DNA in the tail and tail moment. Statistically significant differences in all three parameters were observed between the exposed and control groups. Within the exposed group, there were marked differences between individuals in the comet tail parameters. In the majority of exposed subjects an effect on DNA damage of age or duration of occupational exposure could be excluded. In the exposed group, the highest level of DNA damage was recorded in subjects who used only latex gloves in their work with antineoplastic drugs. The observed DNA damage was lower in exposed subjects who used more than one type of protective equipment and who worked in a well-ventilated safety cabinet. No statistically significant differences were found between the mean values of comet tail parameters for smoking and non-smoking subpopulations from the exposed group. In view of the results obtained, the alkaline comet assay, as a simple, rapid and sensitive method, appears to be a promising additional test for biomonitoring purposes in human populations.

Application of cytogenetic endpoints and Comet assay on human lymphocytes treated with vincristine in vitro.

The genotoxic potential of vincristine is assessed on human peripheral blood lymphocytes following administration of the drug at a dose 0.0875 microg/ml by use of single cell gel electrophoresis - Comet assay (SCGE), analysis of structural chromosome aberrations (CA), micronucleus assay (MN) and sister chromatid exchange (SCE) analysis. In vitro treatment of human lymphocytes with vincristine was performed on cells in G0 phase, as well on lymphocyte cultures 24 hours after stimulation with mitogen phytohemagglutinine. For the Comet assay at 24, 48 and 72 h the treated cells were embedded in agarose on slides, lysed with alkaline lysis solution and exposed to an electric field. DNA migrated within the agarose and formed comets whose length depends on the amount of DNA damage. For the analysis of structural CA cells were grown on F-10 medium for 48 hours, and for MN and SCE analysis for 72 hours. The results on SCGE showed an increase in tail length compared to control both in cells treated in G0 and in cells treated 24 h after mitogen stimulation. The amount of DNA damage was higher in cells treated with vincristine 24 h after mitogen stimulation. Administered concentration of drug caused total inhibition of lymphocytes growth in 72-h cultures for MN and SCE analysis indicating strong microtubule distruptive effects of vincristine. Analysis of structural CA reveals chromatid breaks and acentric fragments as the main aberration types both in cells treated in G0 and in cells treated 24 h after mitogen stimulation. Number of these aberrations was higher in cells treated in G0 phase. Results obtained in this study by use of different cytogenetic endpoints confirmed that vincristine exhibits both aneugenic and clastogenic effects on human lymphocytes.

Cytogenetic monitoring of cardiology unit hospital workers exposed to Doppler ultrasound.

We studied the effects of ultrasound on the peripheral blood lymphocytes of medical personnel from a cardiology unit working with colour Doppler ultrasonic equipment. Cytogenetic risks from ultrasound exposure were assessed by analysis of chromosome aberrations, sister chromatid exchanges (SCE), study of cell-cycle kinetics and micronucleus assay. We found significant increases (P < 0.001) in the total number of chromosome aberrations, mainly due to chromatid breaks and acentric fragments, increases in the total number of micronuclei and SCE and disturbances in cell-cycle kinetics in the exposed group compared to the control. In spite of their limitations, the results of the present investigation indicate that individuals occupationally exposed to ultrasound may experience an increased genotoxic risk and strongly emphasize the need for more research into the nature and extent of the biological consequences to medical personnel working with Doppler ultrasound.

Induction of micronuclei in human lymphocytes after occupational exposure to ultrasound.

Micronucleus assay combined with Giemsa and DAPI staining was performed on blood samples of subjects occupationally exposed to ultrasound. Lymphocytes were cultivated in vitro for 72 h. At 44h cytochalasin-B was added in cultures. Frequencies of micronuclei in exposed subjects statistically significant increased compared to control. The frequency of micronucleated cells and micronuclei in exposed subjects shows interindividual variability. Using DAPI staining we observed signal-positive and signal-negative micronuclei. Percentage of signal-positive micronuclei varies between 0 and 66.7% and signal-negative micronuclei between 33.3% and 100%. This study indicate harmful effects of ultrasound on human genome, but further investigations are necessary.

Incidence of micronuclei in cytokinesis-blocked lymphocytes of medical personnel occupationally exposed to ultrasound.

In order to investigate possible DNA damaging effects of ultrasound, the micronucleus assay on cytokinesis blocked human lymphocytes was performed. Preparations were stained by conventional Giemsa staining technique combined with additional staining techniques using fluorescent dye DAPI and silver nitrate. Blood samples were taken from medical personnel employed on ultrasonic scanning in medical diagnosis and unexposed control subjects from general population. Lymphocytes were cultivated in vitro at 37 degrees C. Cytochalasin-B in final concentration of 6 microg/ml was added 44 h after mitogen stimulation and cultures were harvested 28 h thereafter. Staining with both additional techniques can be used to distinguish micronuclei originating from breakage or mitotic loss of certain human chromosomes bearing DAPI-positively stained or silver-positively stained regions. The results obtained indicate statistically significant increases in total number of micronuclei and changes in their distribution in exposed subjects compared to control. Based on different intensity of DAPI staining signal-positive and signal-negative "type" of micronuclei are distinguished, while silver staining has revealed Ag-NOR+ and Ag-NOR- micronuclei. In exposed subjects a prevalence in number of Ag-NOR+ micronuclei over Ag-NOR-micronuclei compared to control was observed, indicating greater susceptibility of chromosomes from D and G groups to damage caused by continuous occupationally exposure to ultrasound. In spite of their limitations, our results indicate that combination of conventional Giemsa staining of micronuclei with fluorescent dye DAPI and silver nitrate staining techniques can be valuable complement to the standard micronucleus assay.