Davor Zeljezic

This study investigated whether toothpastes containing different fluoride compounds influence cytotoxic and genotoxic alterations in buccal mucosal cells, with particular attention to the type of fluoride, the presence of fluoride itself, and the duration of exposure. Eighty-eight participants were randomly assigned to four parallel groups: a control group using fluoride-free toothpaste and three intervention groups using formulations containing sodium fluoride, sodium monofluorophosphate, or amine fluoride. Buccal cell samples were obtained at baseline (T0), after 30 days (T1), and after 45 days (T2), and evaluated using the buccal micronucleus cytome assay to quantify nuclear abnormalities and cytotoxic markers. All fluoride-containing toothpastes led to higher frequencies of micronuclei, nuclear buds, and “broken egg” cells at T1 and T2 compared with the control group (P ≤ 0.001). Amine fluoride and sodium monofluorophosphate produced sustained increases in cytogenetic markers, including nuclear buds (AmF: P = 0.013; NaMFP: P ≤ 0.001) and “broken egg“ cells (AmF: P ≤ 0.001; NaMFP: P = 0.004), while sodium fluoride demonstrated a slower, progressive increase in “broken egg“ cells (P = 0.036). The findings suggest that fluoride-based toothpastes may modulate cytogenetic responses in buccal epithelial cells, and that these effects differ according to the fluoride compound and exposure duration. The findings should be interpreted with caution due to study limitations, and further research is needed to clarify the long-term biological consequences of repeated fluoride exposure in oral hygiene.

Background: The primary objective of this study was to evaluate the cytotoxic and genotoxic effects of calcium silicate-based sealers (BioRoot RCS and MTA Fillapex) compared to an epoxy-based sealer (AH Plus). Materials and methods: The study was conducted in vitro with the cell lines HepG2 and V79 to evaluate cytotoxicity and genotoxicity using the comet and micronucleus assays. Eluates of the materials were tested at two different concentrations (3 cm²/mL and 0.5 cm²/mL) after an exposure time of 72 h. Data were analyzed using the Mann–Whitney and Kruskal–Wallis tests (p < 0.05). Results: At lower concentrations in both cell lines, MTA Fillapex showed no significant difference in the measured comet assay parameters compared to the negative control (p > 0.05). In addition, it showed significantly lower genotoxic effects compared to AH Plus for all comet assay parameters, concentrations, and cell lines (p ≤ 0.001). BioRoot RCS showed lower primary DNA damage (p ≤ 0.001) than AH Plus, only at higher concentrations and in the HepG2 cell line. Concerning the two tested bioceramic sealers, BioRoot RCS showed higher tail intensity values compared to MTA Fillapex (p < 0.05). In contrast to the results of the comet assay, BioRoot RCS significantly reduced the number of nuclear buds and nucleoplasmic bridges in the HepG2 cell line compared to MTA Fillapex, whereas reduction in the V79 cell line was only observed for nuclear buds (p < 0.05). Both materials increased the number of apoptotic cells compared to the negative control (p < 0.05). In comparison to AH Plus, BioRoot RCS and MTA Fillapex significantly reduced the number of cells with micronuclei and increased the number of cells with undamaged chromatin (p < 0.05). Conclusions: The findings suggest that MTA Fillapex and BioRoot RCS exhibit superior biocompatibility over AH Plus, as evidenced by their lower cytotoxic and genotoxic effects in vitro. These results support the use of calcium silicate-based sealers in clinical practice, highlighting the need for further studies to evaluate their performance in vivo and their implications for patient safety.

Chromosome instability in Alzheimer’s disease (AD) neurons has been previously reported. This pilot study aimed to establish a quantitative technique for assessing X chromosome centromere signals using fluorescence in situ hybridization (FISH). Hippocampal brain tissue was collected at autopsy from sporadic AD patients and age-and gender-matched controls. FISH was utilized to detect and measure the intensity of hybridization signals for X chromosome centromeres in the interphase nuclei of hippocampal brain cells. The premature centromere division (PCD) phenomenon, marked by a close bipartite signal appearing as two separated FISH spots, was examined to see if the hybridized DNA amount in each spot matched the expected centromere DNA amount. The technique effectively distinguished between PCD+ and PCD- signals. The average frequency of PCD,X in the AD group was 7 ± 1%, compared to 3.2 ± 0.84% in controls. This quantitative approach supports qualitative analyses of FISH centromere spots, reinforcing findings of chromosomal instability in AD. The presence of a double signal at the centromere of a single X chromosome indicates re-entered cell cycles, DNA replication, and PCD in hippocampal neurons. This technique provides a reliable method for identifying PCD + signals and contributes to understanding chromosomal instability in AD.

Background: The safety of oral hygiene products is a growing concern, particularly regarding the toxicity of specific ingredients used in their formulations. The purpose of this study was to evaluate the knowledge and attitudes of dentists, physicians, pharmacists, and the general public regarding ingredients in oral hygiene products, especially fluoride. Additionally, this study aimed to identify which ingredients may exhibit potential toxicity based on historical records of any adverse effects being induced by a material/component. Methods: A self-administered questionnaire was used in an online cross-sectional observational study to collect data on sociodemographic characteristics, knowledge of fluoride in dental medicine, fluoride usage practices in oral hygiene products, opinions on ingredient toxicity in oral hygiene products, and personal experiences of adverse reactions to products and their components. The collected data underwent descriptive and regression analyses to reveal patterns and relationships within the dataset. Results: The study found a moderate overall knowledge level regarding fluoride usage in dentistry among participants (Md = 5.00, IQR 2.50–7.00). Healthcare professionals exhibited significantly higher knowledge scores compared to the general population (p ≤ 0.001), with dental professionals displaying the highest scores. Regarding concerns about the usage of fluoride, the majority of respondents (77.0%) did not express any concerns. Minor concerns included the risk of ingestion (6.0%) and dental fluorosis (4.6%). Among the other ingredients in oral hygiene products, respondents named alcohol as the most toxic ingredient (70.3%), followed by artificial colors (53.1%), artificial sweeteners (50.4%), and parabens (50.1%). It is noteworthy that the majority of participants (61.6%) stated that they had never experienced any side effects associated with the use of oral hygiene products. Conclusion: This study underscores disparities in fluoride knowledge between healthcare professionals and the general population in Croatia, with dental experts exhibiting a superior understanding. Despite lingering misconceptions about fluoride content and potential toxicity, the majority of participants acknowledge its oral health benefits and use fluoride products regularly.

Hydra is known for its natural occurrence, anatomical simplicity, intricate physiology, regenerative capacity, and ease of maintenance and manipulation in laboratory environments. It has proven to be a valuable model organism in various disciplines. Its applications range from developmental biology, stem cell research, animal physiology to environmental toxicology including ecotoxicology. The sensitivity of Hydra to a variety of environmental stressors and chemical agents such as metals, nanomaterials, and toxic organic compounds provides valuable insights into physiological mechanisms affected by environmental stressors and pollution, and Hydra can be of great use in environmental monitoring. Furthermore, since green Hydra lives in a symbiotic relationship with unicellular photoautotrophic algae, it is a suitable model organism for symbiosis research. Recently, it has become a popular model in holobiont research. The adaptability and importance of Hydra also extends to aquatic science and aquatic ecology, particularly in the context of monitoring and water pollution. Since the 1980s, Hydra has been increasingly used in various fields of research and has established itself as an important versatile model organism in numerous scientific studies. Hydra also represents an outstanding model in the fields of education and STEM. Hydra continues to be an important model in the 21st century, contributing significantly to our understanding of the biology of water and advancing freshwater research, and possibly finding its way to regenerative medicine and tumor pathobiology research.

Green agronomy promotes the implementation of natural and naturally derived substances in crop protection. In the present study, we evaluated the endocrine-disrupting potential of the allelopathic herbicide tembotrione in Wistar rats by studying the hormone status of offspring from the treated dams. Three doses of tembotrione (0.0004, 0.0007, and 4.0 mg/kg b.w./day) have been administered to dams during gestation and/or lactation. In the serum of newborn, weaning, and pubertal female and male offspring, 17β-estradiol and testosterone were determined using enzyme-linked immunosorbent assay. A decrease in 17β-estradiol and testosterone was observed in female and male weaning and pubertal offspring exposed to all doses of tembotrione during gestation and lactation. In weaning offspring exposed only during lactation, 17β-estradiol dropped significantly after exposure to the two lower doses and testosterone after exposure to the lowest dose of tembotrione. The greatest effect was observed at the lowest dose of tembotrione. In newborns, we observed increased 17β-estradiol after exposure to two lower doses of tembotrione and significantly increased testosterone after exposure to the lowest dose. The highest dose of tembotrione decreased 17β-estradiol significantly in newborn females. The obtained results suggest that tembotrione might be considered a pro-estrogenic or estrogen agonistic compound under the exposure conditions applied in this investigation.

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.

In this study, we aimed to document the freshwater symbiotic interactions along with the impact of the abiotic environment and anthropogenic effects on the functionality of freshwater organisms. Symbiotic green hydra (Z) and free-living brown hydra (S), either separately or both species together, were treated with the herbicide norflurazon in concentrations of 2 × 10⁻⁶ mol/L (N6) and 2 × 10⁻⁷ mol/L (N7) for 72 h. Also, hydras were treated with both norflurazon and UV radiation at a wavelength of 254 nm for 2 min or were irradiated only. The next part of the experiment was performed in the same way but with added suspensions of isolated endosymbiotic alga, free-living alga, or both algae together. Mortality, migration, tentacle and tissue damage, changes in the thickness of the mesoglea of hydras, and clustering of algae were monitored. Green hydra generally showed lower rates of migration, and mortality was observed only in green hydra exposed to UV radiation. Tentacle damage was more pronounced in green hydra and included a specific fork-like structure. The use of cryofixation and TEM enabled us to partly elucidate the effect of clustering of algae. In summary, our study provides new insights into the influence of different environmental stressors and their combination on symbiotic and free-living freshwater hydras and algae and a better understanding of interactions in freshwater ecosystems.

The freshwater water flea (Daphnia magna Straus, 1820) is prey for numerous predators. Yet it possesses a wide range of strategies to defend itself against predation. The aim of this work is to investigate the defensive mechanisms employed by D. magna to reduce predation by the coelenterate Hydra viridissima, and two planarians, Polycelis felina and Dugesia gonocephala. To do this, we used a freshwater microcosm. An additional aim is to investigate interactions with the presence of the isolated endosymbiotic algae from green hydra, thus combining and observing the interaction of the zooplankton and microalgal component. Each experiment included five replicates (13.5 °C, 25 °C), in crystallizing glass containers (60 mL volume, 60 mm diameter, 35 mm height), including satiated (fed with larvae of Artemia salina) and starved predators, respectively (one or five individuals of a particular predator species in one microcosm). As the isolated microalgae are unique, we tracked the following three mechanisms of Daphnia defense for the first time including precisely this microalgal component: (i) grouping (visual magnification), i.e., two or more Daphnia holding together; (ii) the phenomenon of overproduction, i.e., any number of Daphnia in one container above the 10 initially added individuals; and (iii) accelerated movement (“bullet movement”), i.e., high-speed movements in particular microcosms. The results provide new information for a better understanding of the interspecific relationships in systems that include both zooplankton and microalgal components.

The microcosm is a laboratory method frequently used in ecological studies related to population and food web interactions and environmental dynamics. It simultaneously brings into interaction different species in the same controlled laboratory experimental area and provides an opportunity for modeling and reconstruction of relationships in the natural biocenoses and ecosystems. We applied that approach to determine and improve our understanding of predator–prey interactions in different freshwater environments. The inhabitants of the microcosms were isolated endosymbiotic microalga Desmodesmus subspicatus (Chlorophyceae) (Chodat) Hegewald et Schmidt (CZ), green hydras, freshwater turbellarians, and large water fleas. Experiments were performed in five replicates, at 25 °C and 13.5 °C, with fed and hungry predators, respectively. Herein, we proposed a mechanism for microalgal hunting net formation in the freshwater microcosm. Ultrastructural visualization of the endosymbiotic microalgae revealed rod-like structures on the cell wall surface, structures that could possibly fit together and interconnect, suggesting the possibility of microalgal hunting net formation. Interspecific cooperation between isolated microalgae and turbellarians resulted in stronger hunting net formation in preying upon water fleas. This study contributes to the diversity of species interactions and shows the producers as a top link, as opposed to what is generally considered as a basic link in the food web, and presents the microalgae as triggers of the dynamics in the freshwater microcosm.