Haematology and blood chemistry changes in mice treated with terbuthylazine and its formulation Radazin TZ-50.

TBA is an herbicide in general low acute toxicity and placed into a third category of toxicity. The aim of this study was to determine the effect of TBA and its formulation Radazin TZ-50 in doses of ADI values and 1/100 LD 50 on haematological and biochemical blood parameters in mice. The number of leukocytes was significantly decreased (p < 0.05) in all treated groups compared to non-treated mice (8.81 ± 3.23 × 10(9)/L). The lowest value 3.90 ± 0.74 × 10(9)/L was observed in group treated with TBA (1/100 LD 50) followed by TBA (ADI) 4.49 ± 0.98 × 10(9)/L, Radazin TZ-50 (1/100 LD 50) 4.67 ± 1.24 × 10(9)/L and Radazin TZ-50 (ADI) 4.73 ± 1.15 × 10(9)/L. The values of the enzyme AST was increased from 190.00 ± 26.46-270.00 ± 147.30 U/L in serum of all treated groups as compared to non-treated mice (110.00 ± 20.00). LDH values showed significant increase (3236.67 ± 56.86-4054.33.5 ± 837.16 U/L) as compared to non-treated mice (1010.00 ± 222.71 U/L). Total protein value was significantly (p < 0.05) increased in TBA 1/100 LD50 (63.00 ± 7.48 g/L) and Radazin TZ-50 1/100 LD50 (60.00 ± 2.00 g/L) compared to non-treated mice 52.00 ± 4.00 g/L. Increased serum concentrations of urea and creatinine obtained in mice treated with TBA and Radazin TZ-50 indicates a greater degree of dysfunction of the nephron. TBA and its formulation of Radazin TZ-50 in applied doses demonstrate changes in the number of leukocytes and limited hepatotoxic effects.

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.

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, Fisher's 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.

Early toxic effects of fumonisin B1 in rat liver.

Mycotoxin fumonisin B(1) (FB(1)) is hepatotoxic and carcinogenic in experimental animals. It is known that long-term exposure of experimental animals to FB(1) causes apoptosis and lipid peroxidation. In this study, male adult Wistar rats were treated with single FB(1) doses (5, 50, and 500 microg/kg b.w.) and sacrificed 4, 24, and 48 hours after treatment. Parameters of oxidative stress, histopathological changes, and DNA damage were monitored in the liver of treated and control animals. Parameters of oxidative stress were not affected by such treatment. A significant increase in apoptotic cells appeared in animals when 5 microg/kg b.w. dose was given and sacrificed after 24 hours with further increase at higher doses. In contrast to the number of mitotic figures and karyomegaly seen mostly at lower FB(1) doses, necrosis was the prominent feature at higher doses. Significant increase in liver cells DNA mobility was observed 48 hours following treatment with 50 and 500 microg/kg b.w. as compared to control (tail length 15.2 +/- 0.3, 16.4 +/- 0.5, and 13.5 +/- 0.1 mum, respectively). Tail intensity appeared to be more sensitive parameter for detecting DNA damage even at 5 microg/kg b.w. after 48 hours (1.69 +/- 0.27% DNA; control 0.59 +/- 0.11% DNA). This study proved that FB(1)-induced DNA damage is time- and dose-dependent, and that it could be caused in Wistar rats by a single dose.

DNA damage by ochratoxin A in rat kidney assessed by the alkaline comet assay.

There are few studies of ochratoxin A (OTA) genotoxicity in experimental animals and the results obtained with cell cultures are inconsistent, although the carcinogenic potential of OTA for the kidney of experimental animals has been well established. We studied the genotoxic potential of OTA in the kidney of adult female Wistar rats (5 in each group) treated intraperitoneally with OTA (0.5 mg kg body weight-1 day-1 for 7, 14, and 21 days) measuring DNA mobility on agarose gel stained with ethidium-bromide using standard alkaline single-cell gel electrophoresis (comet assay). Negative control animals were treated with solvent (Tris buffer, 1.0 mg/kg) and positive control animals were treated with methyl methanesulfonate (40 mg/kg) according to the same schedule. OTA concentrations in plasma and kidney homogenates in 7-, 14-, and 21-day treated animals were 4.86 +/- 0.53, 7.52 +/- 3.32, 7.85 +/- 2.24 microg/mL, and 0.87 +/- 0.09, 0.99 +/- 0.06, 1.09 +/- 0.15 microg/g, respectively. In all OTA-treated groups, the tail length, tail intensity, and tail moment in kidney tissue were significantly higher than in controls (P < 0.05). The tail length and tail moment were higher after 14 days than after 7 days of treatment (P < 0.05), and still higher after 21 days (P < 0.05). The highest tail intensity was observed in animals treated for 21 days, and it differed significantly from animals treated for 7 and 14 days (P < 0.05). OTA concentrations in plasma and kidney tissue increased steadily and OTA concentration in kidney tissue strongly correlated with tail intensity and tail moment values. These results confirm the genotoxic potential of OTA, and show that the severity of DNA lesions in kidney correlates with OTA concentration.

DNA damage by ochratoxin A in rat kidney assessed by the alkaline comet assay

There are few studies of ochratoxin A (OTA) genotoxicity in experimental animals and the results obtained with cell cultures are inconsistent, although the carcinogenic potential of OTA for the kidney of experimental animals has been well established. We studied the genotoxic potential of OTA in the kidney of adult female Wistar rats (5 in each group) treated intraperitoneally with OTA (0.5 mg kg body weight-1 day-1 for 7, 14, and 21 days) measuring DNA mobility on agarose gel stained with ethidium-bromide using standard alkaline single-cell gel electrophoresis (comet assay). Negative control animals were treated with solvent (Tris buffer, 1.0 mg/kg) and positive control animals were treated with methyl methanesulfonate (40 mg/kg) according to the same schedule. OTA concentrations in plasma and kidney homogenates in 7-, 14-, and 21-day treated animals were 4.86 ± 0.53, 7.52 ± 3.32, 7.85 ± 2.24 æg/mL, and 0.87 ± 0.09, 0.99 ± 0.06, 1.09 ± 0.15 æg/g, respectively. In all OTA-treated groups, the tail length, tail intensity, and tail moment in kidney tissue were significantly higher than in controls (P < 0.05). The tail length and tail moment were higher after 14 days than after 7 days of treatment (P < 0.05), and still higher after 21 days (P < 0.05). The highest tail intensity was observed in animals treated for 21 days, and it differed significantly from animals treated for 7 and 14 days (P < 0.05). OTA concentrations in plasma and kidney tissue increased steadily and OTA concentration in kidney tissue strongly correlated with tail intensity and tail moment values. These results confirm the genotoxic potential of OTA, and show that the severity of DNA lesions in kidney correlates with OTA concentration.

Genotoxicity evaluation of five different dentin bonding agents by chromosomal aberration analysis.

Dentin bonding agents became unavoidable in today's aesthetic restorative dentistry. Nevertheless, more and more evidences on their possible cytotoxicity and/or genotoxicity emerge. Still, only limited number of studies has been published on that issue. In our work we evaluated possible genotoxicity of five different adhesives: Adper Single Bond, Adper Single Bond 2 with nanofiller, Excite, OptiBond Solo Plus and Prompt L-pop. Genotoxicity assessment was carried out on human lymphocytes in vitro, using chromosomal aberration analysis. Polymerized adhesives were tested at three different dilutions of the 0.5 g mL(-1) eluate stock (2.5 x 1:10(6), 1:10(6) and 1:10(5)) after 1 h, 24 h and 5 days of elution. Slight but significant increase in the number of chromatid breaks was observed after 24-h elution period, for adhesives Adper Single Bond 2, Excite, and OptiBond Solo Plus at dilutions of 1:10(6) and 1:10(5), and for other two only at dilution of 1:10(5). First three adhesives also appeared to be slightly genotoxic after 1 h of elution but only at 1:10(5). As a bonding agent remains in close contact with living dental tissue over a long period of time, information on their possible genotoxicity and carcinogenicity should be more clearly clarified in the near future.

Genotoxicity evaluation of pesticide formulations containing alachlor and atrazine in multiple mouse tissues (blood, kidney, liver, bone marrow, spleen) by comet assay.

Every year, in the European countries more than 2 million tons of pesticides are released into the environment. More than 60% of those substances appear to be herbicides. Due to extensive production and application of this chemical their putative detrimental effect on life should be known and minimized. In this study we applied the comet assay on blood and 4 mouse organs (kidney, liver, bone marrow, and spleen) to evaluate possible genome damage caused by two pesticide formulations (Bravo and Gesaprim) containing alachlor and atrazine as active ingredients. Five male CBA mice were assigned to each of 4 treatment groups and control group. Bravo and Gesaprim were injected intraperitoneally once. Two different doses of Bravo were used: 0.031 ml/kg and 0.021 microl/kg, so that doses of alachlor mice received within the pesticide formulation given were 15 mg/kg and 0.01 mg/kg. Also Gesaprim was given at two different doses: 1.08 ml/kg and 0.07 microl/kg so that the doses of atrazine contained within the pesticide formulation given were 540 mg/kg and 3.5 x 10(-2) mg/kg. Mice were sacrificed 24 hours after treatment. Alkaline comet assay on the blood samples, kidney, liver, bone marrow and spleen was performed. Statistically significant (p<0.01) increase of tail length for all 5 tissues examined in mice treated with both Bravo and Gesaprim compared to the control was found. For both pesticides DNA of kidney and liver showed largest increase in migration. Also, distribution of tail length values for Bravo and Gesaprim for all mouse tissues examined showed a shift to the right when compared to the controls.

Examination of cytotoxicity and mutagenicity of AH26 and AH Plus sealers.

AIM: To study in vitro the cytotoxic and mutagenic effects of AH26 and AH Plus. METHODOLOGY: Cytotoxic effects on Chinese hamster V79 cells were determined by counting viable cells following incubation with eluations of AH26 and AH Plus. In one set of experiments, the materials were mixed, set for 1 h and then eluted with dimethyl sulphoxide (DMSO) for 1 h, 24 h and 7 days. In the other set, AH26 and AH Plus were mixed and set for 1 h, 24 h and 7 days in physiological saline then crushed and eluted in DMSO for 24 h. The cytotoxic effects of these eluates were evaluated. Three concentrations were chosen to examine the mutagenic effects of AH26 and AH Plus: 5.57, 16.7 and 55.7 microg mL(-1). The structural chromosomal aberration analysis and micronucleus test were performed on human lymphocytes according to standard procedures. RESULTS: Dose-response curves of cell survival were obtained. Both materials were shown to be cytotoxic in doses larger than 55.7 microg mL(-1), except for AH26, after 7 days setting time. AH Plus was also shown to be toxic in concentrations of 16.7 microg mL(-1), except after 7 days setting time. Neither AH26 nor AH Plus induced a significant increase of chromosomal aberrations or micronuclei induction at any setting time or concentration. CONCLUSION: There was no mutagenicity found for AH26 and AH Plus on human lymphocytes in highly controlled conditions in vitro.

Cytogenetic monitoring of croatian population occupationally exposed to a complex mixture of pesticides.

This paper describes a longitudinal study of possible genetic damage in Croatian workers occupationally exposed to a complex mixture of pesticides. The methods of choice were chromosomal aberration analysis, sister chromatid exchange analysis (SCE), micronucleus assay and comet assay. In order to determine primary genotoxic effects in workers, blood samples were taken after the workers spent 8 months in the production of pesticides. During the production all subjects were simultaneously exposed to a complex mixture of pesticides containing atrazine, alachlor, cyanazine, 2,4-dichlorophenoxyacetic acid, and malathion. To detect DNA repair in lymphocytes of the same subjects the second series of blood samples was taken 8 months after the workers were removed from production. Regardless of the time sampling time the exposed workers showed an increased number of chromosomal aberrations, SCE frequency, micronucleus (MN) frequency, and values of comet assay parameters. After 8 months of non-exposure the workers showed a significantly decreased number of chromosomal aberrations, MN frequency, and DNA migration compared to the results of the first sampling, but it was still significantly higher than in controls. Furthermore, the SCE frequency in the exposed subjects did not drop after the 8 months of non-exposure, which indicates long-term exposure to a mixture of pesticides.

Chromosomal aberration and single cell gel electrophoresis (Comet) assay in the longitudinal risk assessment of occupational exposure to pesticides.

In recent years the use of pesticides in agriculture has been increasing steadily. At present there are more than 1000 chemicals classified as pesticides. Therefore, the widespread use of pesticides and their potential genetic hazard suggests that evaluation of their genotoxicity should be extended using the newer assays now available. In the present study chromosomal aberration analysis and the alkaline single cell gel electrophoresis (Comet) assay were used to evaluate the extent of DNA damage and DNA repair in peripheral blood lymphocytes of subjects employed in pesticide production. In order to determine possible primary genotoxic effects in workers blood samples were taken after an 8 month long period of exposure to a complex mixture of pesticides. To detect the possible occurrence of DNA repair in lymphocytes of the same subjects the second blood sample was taken after an 8 month long period of absence from the pesticide exposure zone. Regardless of the period of sampling, in the exposed group statistically significantly increased numbers of aberrant cells, chromatid and chromosome breaks, acentric fragments and dicentric chromosomes compared with the controls were found. After the workers had spent 8 months out of the pesticide exposure zone the number of aberrant cells and all types of chromatid and chromosome aberrations decreased significantly compared with sampling after the high exposure period, but it still remained significantly higher in comparison with the control group. After the period of high exposure to a mixture of pesticides statistically significantly increased levels of DNA damage in the Comet assay in terms of tail length and tail moment were found. After the workers were removed from production for 8 months both Comet assay end-points decreased significantly compared with the first sampling point, but they remained increased compared with the control.

Evaluation of DNA damage in workers occupationally exposed to pesticides using single-cell gel electrophoresis (SCGE) assay. Pesticide genotoxicity revealed by comet assay.

The comet assay, also called the single-cell gel electrophoresis (SCGE) assay, is a rapid and sensitive method for the detection of DNA damage (strand breaks and alkali-labile sites) in individual cells. The assay is based on the embedding of cells in agarose, their lysis in alkaline buffer and finally subjection to an electric current. In the present study, alkaline SCGE was used to evaluate the extent of primary DNA damage and DNA repair in peripheral blood lymphocytes of workers employed in pesticide production. After the period of high pesticide exposure, lymphocytes of the occupationally exposed workers manifested increased tail length and tail moment compared to the control group. After the workers spent 6 months out of the pesticide exposure zone, both endpoints were still above that of the control but significantly decreased as compared to the results of the first analysis.