Does the Symbiotic Relationship Between Hydra Viridissima and Photoautotrophic Alga Provide an Evolutionary Advantage in Protecting DNA against Damage by the Cytotoxic or Genotoxic Mode of Action of Environmental Stressors?

The aim of this paper was to evaluate whether symbiotic cooperation between green hydra (Hydra viridissima) and photoautotrophic alga gives higher resistance of the preservation of DNA integrity compared to brown hydra (Hydra oligactis). Norflurazon concentrations were 0.061 or 0.61 mg/L and UV-B light 254 nm, 0.023mWcm- 2 applied separately or simultaneously. By alkaline comet assay primary DNA damage was assessed and cytotoxicity by fluorescent staining. Norflurazon at 0.61 mg L- 1 significantly increased DNA damage in brown hydras compared to the control (6.17 ± 0.6 µm, 5.2 ± 1.7% vs. 2.9 ± 0.2 µm, 1.2 ± 0.2%). Cytotoxicity was significantly elevated, being higher in brown hydras (25.7 ± 3.5% vs. 8.2 ± 0.2%). UV-B irradiation induced significant DNA damage in brown hydras (13.5 ± 1.0 µm, 4.1 ± 1.0%). Simultaneous exposure to UV-B and norflurazon led to a synergistic DNA damaging. The frequency of cytotoxicity and hedgehog nucleoids was more pronounced in brown (78.3 ± 9.4%; 56.4 ± 6.0%) than in green hydras (34.7 ± 2.5%; 24.2 ± 0.6%). Evolutionary established symbiotic cooperation proved to provide resistance against cyto/genotoxicity.

ASSESSMENT OF CYTOGENETIC DAMAGE TO EXFOLIATED GINGIVAL CELLS IN PATIENTS WITH CHRONIC GENERALIZED PERIODONTITIS.

The aim of this cross-sectional study was to investigate the occurrence of chromosomal abnormalities through the frequency of micronuclei and other genomic damage markers in patients with chronic generalized periodontitis and without periodontal disease. Micronucleus assay was performed in exfoliated gingival epithelial cells of 35 patients with generalized chronic periodontitis and 30 control subjects with healthy periodontium. Full mouth clinical examination was performed to define periodontal condition. The mean number of cells with micronuclei observed in chronic periodontitis and control groups was 1.8 (±1.49) and 2.0 (±1.34), respectively. Differences between the groups were not significant (p=0.574). Compared to control subjects, patients with chronic periodontitis showed a significant increase in the number of binucleated cells (p≤0.001) and number of cells with nucleoplasmic bridges (p=0.042). Study results indicated that chronic periodontitis was not associated with higher occurrence of chromosomal damage in gingival cells compared to individuals with healthy periodontium.

Nuclear morphological changes in gingival epithelial cells of patients with periodontitis.

OBJECTIVES: This study assessed the frequency of nuclear morphological changes in gingival epithelial cells, as a biomarker for DNA damage, in individuals with periodontitis, before and after implementation of periodontal therapy, and compared the morphology to those with healthy periodontal tissues. MATERIALS AND METHODS: Exfoliated gingival cells were taken from 30 participants without periodontal destruction in any teeth and 30 participants with periodontitis before and after 45 and 90 days following treatment. Nuclear morphological changes were analyzed using the micronucleus test. RESULTS: Compared with the healthy volunteers, those with periodontitis had a significant increase in the number of cells with nuclear broken eggs (P = 0.048), condensed chromatin (P = 0.015), karyolysis (P < 0.001), or binuclei (P < 0.001). In the periodontitis group, the pretreatment frequencies of cells with micronuclei (P = 0.008), binuclei (P < 0.001), karyolysis (P = 0.038), nuclear buds (P = 0.005), and condensed chromatin (P = 0.015) were significantly higher than 90 days after treatment. CONCLUSION: Our observations suggest that periodontal disease increases the frequency of nuclear morphological changes in gingival epithelial cells and that the implementation of periodontal therapy was associated with a reduction of that number. CLINICAL RELEVANCE: The micronucleus test could serve as a tool for estimating genotoxic damage in assessing the success of periodontal therapy.

In vivo evaluation of fluoride and sodium lauryl sulphate in toothpaste on buccal epithelial cells toxicity.

OBJECTIVES: The present study addresses the effect of fluoride and sodium lauryl sulphate content of toothpaste on oral epithelial cells in vivo conditions. SUBJECTS AND METHOD: Forty volunteers were assigned into two experimental groups, each of them applying the different brand of toothpaste. Every group has been using three different types of toothpaste (non-fluoride and non-SLS, fluoride and non-SLS, and the fluoride and SLS) of the same brand for 6 months, each for 2 months. The buccal epithelial cells were sampled at baseline and 30, 60, 90, 120, 150 and 180 days after the beginning of the research. Effect on DNA damage was analyzed by micronucleus assay Results: After 60 days of use, for both tested kinds of toothpaste with fluoride and without SLS, all studied parameters were not significantly different from the results obtained at the time when the participants used a non-fluoride toothpaste. While, after 60 days of use, for one kind of toothpaste with SLS and fluoride, was observed significantly higher incidence of pyknotic cells (2.20 ± 0.95, 0.00 ± 0.00 vs. 0.05 ± 0.22, respectively; p = .001), cells with karyorrhexis (2.35 ± 1.14, 0.85 ± 0.93 vs. 0.40 ± 0.68, respectively; p = .001), and nuclear buds (1.35 ± 0.68, 0.45 ± 0.51 vs. 0.45 ± 0.60, respectively; p = .001), compared to toothpastes of the same brand with fluoride and without SLS, and without fluoride and without SLS, for the same period. CONCLUSIONS: Based on the results, can be concluded that there is no fluorine-dependent cytotoxic or genotoxic effect, while SLS dentifrice increases the number of nuclear morphological changes in buccal epithelial cells.

In vivo assessment of genotoxicity in buccal cells of children undergoing tooth restoration.

OBJECTIVES: Whereas dental materials came in direct or close contact with oral tissue, it is a great concern about the biocompatibility of those materials. This study was performed to evaluate possible DNA damage to buccal cells exposed to dental materials. METHODS: This prospective, longitudinal clinical study was conducted over a three months period. Class II restorations were placed in 60 young patients with no previous filling using one of three tested dental materials (two glass ionomers; Ketac Molar and Ionofil Molar and one compomer material; Twinky Star). DNA damage was analysed by micronucleus assays, in buccal exfoliated epithelial cells. RESULTS: In patients treated with Ketac Molar, a significant frequency of micronuclei (p = 0.027) and binucleated cells in samples taken 30 days following restoration (p = 0.029) was confirmed. In patients treated with Twinky Star, a statistically significant increase in the number of binucleated cells in samples taken after 7 and 30 days following restoration (p = 0.001 and p < 0.001, respectively) was observed. In all samples collected 90 days after treatment, there was no statistical difference in the number of any cell changes. CONCLUSION: In this study long-term biocompatibility of tested materials was confirmed. Glass ionomers and compomers are widely used materials in paediatric dentistry, and this study has proved their safety for usage in children.

Cytogenetic Biomonitoring of Dental Technicians: A Cross-Sectional Study.

PURPOSE: This is a cross-sectional study to assess the incidence of micronuclei and other nuclear morphological changes in buccal epithelial cells of dental technicians. MATERIALS AND METHODS: The study was conducted on 45 dental technicians versus 2 control groups: 50 dentists and 50 dental assistants. DNA damage was analyzed in exfoliated buccal mucosa cells by micronucleus assay. The differences in the frequency of detected types of cytogenetic damage between experimental groups were analyzed by applying 2-way ANOVA with Tukey's HSD post-hoc test. RESULTS: Dentists and dental assistants have significantly lower incidence of micronucleated cells than technicians (mean ± SD: 0.68 ± 0.74, 0.58 ± 0.81, and 1.58 ± 2.07; p = 0.031 and p = 0.015, respectively), and this trend also holds for karyolitic cells (0.10 ± 0.30, 0.20 ± 0.49, and 1.42 ± 1.25; p < 0.001 and p < 0.001, respectively), condensed chromatin (0.16 ± 0.37, 0.14 ± 0.35, and 0.76 ± 0.98; p < 0.001 and p < 0.001, respectively), and pyknotic cells (0.04 ± 0.20, 0.08 ± 0.27, and 0.96 ± 1.24; p < 0.001 and p < 0.001, respectively). CONCLUSION: Cytogenetic biomarkers in dental technician buccal mucosa are increased compared with control groups. This increase may be associated with more extensive exposure to potentially harmful components of the materials used in everyday dental practice.

Persistent Organochlorine Pollutants in Placentas Sampled from Women in Croatia and an Evaluation of Their DNA Damaging Potential In Vitro.

This study investigated the levels and distribution of 17 polychlorinated biphenyls (PCBs) and organochlorine pesticides (HCB, α-HCH, ß-HCH, γ-HCH, p,p'-DDE, p,p'-DDD, and p,p'-DDT) in placenta samples from women living in the coastal area of Croatia. During November 2012 to February 2013, 51 placenta samples were collected from healthy mothers. This study presents the first report about Croatian placenta samples. Each of the analysed compounds were found in all of the samples; all of the maximum values were < 1 ng g-1 w.w., and the highest median value found for PCB-28 was 11.2 pg g-1 w.w. PCBs and organochlorine pesticide (OCPs) present in placenta samples were tested for their genotoxic potential using the alkaline comet assay. The alkaline comet assay is one of the most reliable methods in assessing the DNA lesions that occurs in direct interaction of a chemical and the genome. The detected levels of PCBs and OCPs in the placenta did not pose a significant risk to the children's DNA during embryonic and foetal growth following short-term exposure. PCB and OCP concentrations in the placenta samples did not induce any significant primary damage to DNA in terms of DNA strand breaks and changes in the primary chemical structure, which could be detected by the alkaline comet assay.

Assessment of cytotoxic and genotoxic effects of conventional and whitening kinds of toothpaste on oral mucosa cells.

OBJECTIVE: This study aimed to evaluate possible DNA damages to oral epithelial cells exposed to whitening kinds of toothpaste considering the effect of conventional non-whitening toothpaste. MATERIALS AND METHODS: Sixty volunteers were assigned into three experimental groups, each of them using a different regular toothpaste for the initial 2 months, followed by the use of whitening kind of toothpaste of the same brand for next 2 months. The oral epithelial cells were sampled prior and 30, 60, 90 and 120 days after the beginning of the use of tested kinds of toothpaste. Chromosomal damages were analyzed by micronucleus assay. RESULTS: For just one kind of tested whitening toothpaste was observed the significant increase in the number of micronucleated cells after 60 days of use compared values obtained 60 days of usage of conventional non-whitening toothpaste (6.35 ± 3.67 and 2.8 ± 1.91; p < .05). There was no statistically significant difference in other micronucleus assay endpoints between tested types of toothpaste at either of the sampling times during the period of toothpaste application. CONCLUSIONS: Based on the results, it can be concluded that the use of certain whitening kinds of toothpaste may cause a limited biologically insignificant genotoxic effect on buccal epithelial cells.

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.

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.

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.

Composite-induced toxicity in human gingival and pulp fibroblast cells.

OBJECTIVE: The most important requirement for a material to be used in medical applications is its biocompatibility. Dental composite materials come into direct contact with oral tissues, especially gingival and pulpal cells. This study was performed to evaluate possible DNA damage in cells of human origin exposed to dental composites in vitro using a cytogenetic assay. MATERIALS AND METHODS: Two composite resins (Vertise Flow, Kalore) were tested on human gingival and pulp fibroblasts using the acridine orange/ethidium bromide viability staining and alkaline comet assay. Cultures were treated with polymerized composites in two different concentrations (20 mg/ml, 40 mg/ml) for 14 days. Chi-square and Kruskall-Wallis non-parametric test were used for the statistical analysis (p < 0.05). RESULTS: Significant cytotoxicity was observed for 40 mg/ml of Vertise Flow in both cultures, while Kalore (40 mg/ml) showed cytotoxic effect only on human pulp fibroblasts. A significant level of DNA damage was detected for both materials and concentrations, in both cell cultures. CONCLUSION: If the two cell cultures are compared, the pulp cells were more sensitive to the cyto/genotoxic effects of dental composites. Based on the results, one can conclude that the use of tested materials may cause cellular damage in gingival and pulp fibroblasts in vitro.

Evaluation of Cytotoxic and Genotoxic Effects in Buccal Mucosal Cells in Non-Smokers and Users of Traditional Combustible Tobacco Products and Non-Combustible Alternatives.

AIMS/OBJECTIVES: The aim of this cross-sectional observational study was to investigate cytogenetic damage to the buccal mucosa in non-smokers and consumers of traditional combustible tobacco products and non-combustible alternatives. METHODS: A total of 160 participants were divided into four groups according to the type of product used, including non-smokers, users of conventional combustible tobacco (cigarettes), heated tobacco, and electronic, tobacco-free vapor products (e-cigarettes). Buccal mucosa samples were analyzed using the micronucleus cytome assay to assess cytotoxic and genotoxic damage. RESULTS: E-cigarette users showed significantly higher values for all tested parameters in the micronucleus test compared to non-smokers (p < 0.05). Similarly, users of tobacco heating products showed an increase in all parameters (p < 0.05), with the exception of the number of cells with micronuclei. Conventional cigarette smokers showed a notable increase in the number of binucleated cells and cells with karyorrhexis and karyolysis (p ≤ 0.05). When assessing the differences between users of traditional combustible tobacco products and non-combustible alternatives, these did not appear to be significant, except for e-cigarette users, who had significantly more cells with condensed chromatin (p ≤ 0.001), while users of tobacco heating products had more pyknotic cells (p ≤ 0.001). CONCLUSION: The results of this study underscore the heightened occurrence of cytotoxic and genotoxic damage in users of both conventional combustible tobacco products and non-combustible alternatives compared to non-smokers, emphasizing the detrimental impact of these products on the oral mucosa.

Genotoxic biomonitoring of flowable and non-flowable composite resins in peripheral blood leukocytes.

OBJECTIVE: Composite restorative materials represent one of the most important groups of materials in contemporary dental practice. However, their incomplete polymerization may lead to monomer-induced genotoxicity. The objective of this study was to evaluate the genotoxicity of three flowable (Filtek Supreme XT Flow, Tetric EvoFlow, Gradia Direct Flo) and three non-flowable dental composite materials (Filtek Z250, Tetric EvoCeram, Gradia Direct Posterior). MATERIALS AND METHODS: Genotoxicity assessment of composite materials was carried out in vitro in human peripheral blood leukocytes using the alkaline single-cell gel electrophoresis technique (comet assay). Prepared materials were eluted in saline solution for 1 h, 1 day and 5 days. Thereafter leukocyte cultures were treated with different concentrations of eluates obtained from each of the tested dental composite materials. Kruskall-Wallis non-parametric test was used for statistical analysis (p < 0.05). RESULTS: The tested materials did not show genotoxic effects after exposure of leucocytes to 1 h eluates. Culture treated with 1 day eluates of all tested materials, only at a highest concentration (10(-2)), affected the measured cytogenetic parameters. Of all tested materials, only Filtek Z250 and Filtek Supreme XT Flow did not exhibit a genotoxic effect in cultures that were under the influence of 5 day eluates. CONCLUSION: Tested materials exhibited limited genotoxic activity in peripheral blood leukocytes. Since the effect was observed only in leukocyte cultures treated by 1-day eluates at the highest concentration (10(-2)) and it decreases in cultures exposed to 5 day eluates, it should not pose a significant risk to the human genome.

Assessment of Cytotoxic and Genotoxic Effect of Modern Dental Materials in vivo.

Objectives: The aim of the study was to assess the biocompatibility of modern composite and amalgam dental fillings. Material and Methods: The research was conducted on 150 healthy patients between the ages of 10 and 20 who had amalgam and composite fillings between 6 and 12 months. Under in vivo conditions, a swab of buccal cells near the fillings was taken, and the cytotoxic and genotoxic impact of composite and amalgam fillings on these cells was analyzed using the extended micronucleus test (cytomeassay). Results: The results showed statistically significant differences between the groups of subjects with amalgam and composite fillings and subjects without fillings for the following parameters: number of micronuclei (p=0.006), number of buds (p<0.001), number of binuclear cells (p<0.001), number of nucleoplasmic bridges (p<0.001).The number of micronuclei was statistically significantly higher in the group of subjects with amalgam and composite fillings compared to the group without fillings. The results for nuclear buds, for the number of binuclear cells and the number of nucleoplasmic bridges showed that the group with amalgam fillings had a statistically significantly higher number of these changes compared to other groups.The results of the analysis of the relationship between the parameters of the micronucleus test and the number of amalgam and composite surfaces did not show statistically significant values. Parameters indicating cell cytotoxicity were not statistically significantly elevated in subjects with fillings. The results of the analysis of the influence of the patients' lifestyle on the results of the micronucleus test showed statistically significant results for certain predictors (diagnostic X-ray radiation, coffee consumption, consumption of cooked, dried meat and baked food). Conclusion: Based on the results, it can be concluded that the buccal cells of subjects with amalgam fillings showed the highest degree of genotoxic changes, followed by those with composite fillings and the least buccal cells of patients without fillings.

Safety and efficacy of the feed additive consisting of 6-phytase (produced by Aspergillus oryzaeDSM 33699) (RONOZYME® Hiphos GT/L) for poultry, pigs for fattening, weaned piglets and sows (DSM Nutritional Products Ltd).

The additive RONOZYME® Hiphos (GT/L) contains 6-phytase produced with a genetically modified strain of the filamentous fungus Aspergillus oryzae, it is currently authorised for poultry, pigs for fattening, weaned piglets and sows. The applicant has requested to change the production strain, substituting strain A. oryzae DSM 22594 for A. oryzae DSM 33699. RONOZYME® Hiphos (GT/L), manufactured with the production strain A. oryzae DSM 33699, did not give rise to safety concerns with regard to the genetic modification of the production strain. No viable cells of the production strain nor its recombinant DNA were detected in an intermediate product representative of both final forms of the additive. RONOZYME® Hiphos (GT/L) was considered safe for poultry, pigs for fattening, weaned piglets and sows at the recommended inclusion levels of 500-4,000 FYT/kg complete feed. The use of RONOZYME® Hiphos GT and L manufactured with the production strain A. oryzae DSM 33699 raised no concerns for consumers. In the absence of data on the final formulations, the Panel could not conclude on the potential of the additive to be irritant to eyes or skin, or a skin sensitiser. Due to the proteinaceous nature of the active substance, the additive was considered a respiratory sensitiser. The additive manufactured by A. oryzae DSM 33699 raises no safety concerns for the environment. The additive has the potential to be efficacious in poultry, pigs for fattening, weaned piglets and sows at 500 FYT/kg complete feed.

Safety and efficacy of a feed additive consisting of 25-hydroxycholecalciferol (produced by Pseudonocardia autotrophicaDSM 32858) for all pigs, all poultry for fattening and ornamental birds and other poultry species (Huvepharma NV).

Following a request from the European Commission, EFSA was asked to deliver a scientific opinion on the safety and efficacy of a feed additive consisting of 25-hydroxycholecalciferol (produced by Pseudonocardia autotrophica DSM 32858) for all pigs, all poultry for fattening and ornamental birds and other poultry species. The production strain P. autotrophica DSM 32858 is not genetically modified however, uncertainties remain on the possible presence of its viable cells in the final product. Due to the lack of adequate safety data and uncertainty on the presence of nano particles, the FEEDAP Panel cannot conclude on the safety of the additive for the target species and the consumer. The additive was shown not to be irritant to skin or eyes and it is not a skin sensitiser. Considering the low dusting potential of the additive, the FEEDAP Panel concluded that the exposure through inhalation is unlikely. However, the FEEDAP Panel considered that uncertainties remain on genotoxicity and on the possible presence of viable cells of P. autotrophica DSM 32858 in the final product which might have an impact on the safety for the users. The use of the feed additive is considered safe for the environment. The Panel concluded that the additive has a potential to be efficacious under the proposed conditions of use.

A potential role of calcium in apoptosis and aberrant chromatin forms in porcine kidney PK15 cells induced by individual and combined ochratoxin A and citrinin.

The aim of this study was to establish the involvement of calcium signalling in genotoxicity, apoptosis and necrosis evoked by ochratoxin A (OTA) and citrinin (CTN) alone or in combination in porcine kidney PK15 cells. Cell proliferation test (MTT) and trypan blue assays (24 h) demonstrated that CTN (IC(50) = 73.5 ± 1.0, 75.4 ± 1.4 µM, respectively) was less toxic than OTA (IC(50) = 14.0 ± 2.4, 20.5 ± 1.0 µM, respectively). To test their cytotoxic interactions, two doses of single OTA (6 and 10 µM) and CTN (30 and 50 µM) and their combinations were applied. Combined treatment showed additive cytotoxic effects. OTA and CTN induced dose-dependent increase in cytosolic calcium level (assessed with Fura-2 AM). However, combined treatment did not provoke additional increase in calcium signal. The rate of apoptosis and necrosis (DAPI-antifade staining) was significantly higher after 12 h than 24 h, while the frequencies of micronuclei (MNs) and nuclear buds (NBs) were higher after 24 h than 12 h treatment. Combined exposure resulted in apoptotic and necrotic synergism, while genotoxic effects of OTA + CTN were noted as antagonistic or additive. Co-exposure of cells to calcium chelator BAPTA-AM significantly reduced CTN and OTA + CTN-evoked apoptosis. Twenty-four hour after co-exposure to BAPTA-AM and a single OTA and CTN, MNs significantly decreased while NBs dropped significantly after co-treatment with BAPTA-AM and OTA + CTN. In conclusion, disturbance of Ca(2+) homeostasis caused by OTA and CTN plays a significant role in cell genotoxicity and death.

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.

The effect of low doses of chlorpyrifos on blood and bone marrow cells in Wistar rats.

The aim of this study was to investigate the genotoxic potential of low doses of chlorpyrifos (CPF) on blood and bone marrow cells in adult male Wistar rats. CPF was administered by oral gavage at daily doses of 0.010, 0.015, and 0.160 mg/kg of body weight (bw) for 28 consecutive days. Positive control (PC) was administered 300 mg/kg bw/day of ethyl methane sulphonate (EMS) for the final three days of the experiment. Toxic outcomes of exposure were determined with the in vivo micronucleus (MN) assay and alkaline comet assay. The 28-day exposure to the 0.015 mg/kg CPF dose, which was three times higher than the current value of acute reference dose (ARfD), reduced body weight gain in rats the most. The in vivo MN assay showed significant differences in number of reticulocytes per 1000 erythrocytes between PC and negative control (NC) and between all control groups and the groups exposed to 0.015 and 0.160 mg/kg bw/day of CPF. The number of micronucleated polychromatic erythrocytes per 2000 erythrocytes was significantly higher in the PC than the NC group or group exposed to 0.015 mg/kg bw/day of CPF. CPF treatment did not significantly increase primary DNA damage in bone marrow cells compared to the NC group. However, the damage in bone marrow cells of CPF-exposed rats was much higher than the one recorded in leukocytes, established in the previous research. Both assays proved to be successful for the assessment of CPFinduced genome instability in Wistar rats. However, the exact mechanisms of damage have to be further investigated and confirmed by other, more sensitive methods.