Analytical studies on some pesticides with antifungal effects: Simultaneous determination by HPLC, investigation of interactions with DNA and DNA damages.

A simple, and fast method was developed for the simultaneous determination of five fungicides, namely thiram (THR), epoxiconazole (EPO), hexaconazole (HEX), tebuconazole (TEB), and diethofencarb (DIE), in different matrices by HPLC-UV. Parameters influencing the peak shape and resolution, such as the composition of mobile phase, pH and concentration of buffer solution, and column temperature, were examined and optimized. The proposed method was validated in terms of linearity, sensitivity, precision, and accuracy. Forced degradation studies were carried out for all analytes to demonstrate the specificity of the method and to evaluate the stability of analytes under different conditions. DNA interaction and DNA damage studies were conducted by HPLC and comet assay, respectively. All fungicides were found to bind DNA, except for DIE. While the binding coefficients for EPO, HEX, and TEB were of the order of 104, THR was found to interact more strongly with DNA with a binding coefficient of higher than 106. DIE did not induce DNA damage at any concentration tested. On the other hand, TEB, HEX, and EPO induced DNA damage up to 30 µg/mL. THR showed cytotoxic effects at 20 and 30 µg/mL and caused significant DNA damage at lower concentrations.

Three-dimensional (3D) cell culture studies: a review of the field of toxicology.

Traditional two-dimensional (2D) cell culture employed for centuries is extensively used in toxicological studies. There is no doubt that 2D cell culture has made significant contributions to toxicology. However, in today's world, it is necessary to develop more physiologically relevant models. Three-dimensional (3D) cell culture, which can recapitulate the cell's microenvironment, is, therefore, a more realistic model compared to traditional cell culture. In toxicology, 3D cell culture models are a powerful tool for studying different tissues and organs in similar environments and behave as if they are in in vivo conditions. In this review, we aimed to present 3D cell culture models that have been used in different organ toxicity studies. We reported the results and interpretations obtained from these studies. We aimed to highlight 3D models as the future of cell culture by reviewing 3D models used in different organ toxicity studies.

Bio-Functionalized Ultra-Thin, Large-Area and Waterproof Silicone Membranes for Biomechanical Cellular Loading and Compliance Experiments.

Biocompatibility, flexibility and durability make polydimethylsiloxane (PDMS) membranes top candidates in biomedical applications. CellDrum technology uses large area, <10 µm thin membranes as mechanical stress sensors of thin cell layers. For this to be successful, the properties (thickness, temperature, dust, wrinkles, etc.) must be precisely controlled. The following parameters of membrane fabrication by means of the Floating-on-Water (FoW) method were investigated: (1) PDMS volume, (2) ambient temperature, (3) membrane deflection and (4) membrane mechanical compliance. Significant differences were found between all PDMS volumes and thicknesses tested (p < 0.01). They also differed from the calculated values. At room temperatures between 22 and 26 °C, significant differences in average thickness values were found, as well as a continuous decrease in thicknesses within a 4 °C temperature elevation. No correlation was found between the membrane thickness groups (between 3−4 µm) in terms of deflection and compliance. We successfully present a fabrication method for thin bio-functionalized membranes in conjunction with a four-step quality management system. The results highlight the importance of tight regulation of production parameters through quality control. The use of membranes described here could also become the basis for material testing on thin, viscous layers such as polymers, dyes and adhesives, which goes far beyond biological applications.

Recombinant Activated Protein C (rhAPC) Affects Lipopolysaccharide-Induced Mechanical Compliance Changes and Beat Frequency of mESC-Derived Cardiomyocyte Monolayers.

BACKGROUND: Septic cardiomyopathy increases mortality by 70% to 90% and results in mechanical dysfunction of cells. METHODS: Here, we created a LPS-induced in-vitro sepsis model with mouse embryonic stem cell-derived cardiomyocytes (mESC-CM) using the CellDrum technology which simultaneously measures mechanical compliance and beat frequency of mESCs. Visualization of reactive oxygen species (ROS), actin stress fibers, and mRNA quantification of endothelial protein C receptor (EPCR) and protease-activated receptor 1 (PAR1) before/after LPS incubation were used for method validation. Since activated protein C (APC) has cardioprotective effects, samples were treated with human recombinant APC (rhAPC) with/-out LPS predamage to demonstrate the application in therapeutic studies. RESULTS: Twelve hours LPS treatment (5 µg/mL) increased ROS and decreased actin stress fiber density and significantly downregulated EPCR and PAR1 compared to control samples (0.26, 0.39-fold respectively). rhAPC application (5 µg/mL, 12 h) decreased ROS and recovered actin density, EPCR, and PAR1 levels were significantly upregulated compared to LPS predamaged samples (4.79, 3.49-fold respectively). The beat frequencies were significantly decreased after 6- (86%) and 12 h (73%) of LPS application. Mechanical compliance of monolayers significantly increased in a time-dependent manner, up to eight times upon 12-h LPS incubation compared to controls. rhAPC incubation increased the beat frequency by 127% (6h-LPS) and 123% (12h-LPS) and decreased mechanical compliance by 68% (12h-LPS) compared to LPS predamaged samples. CONCLUSION: LPS-induced contraction dysfunction and the reversal effects of rhAPC were successfully assessed by the mechanical properties of mESC-CMs. The CellDrum technology proved a decent tool to simulate sepsis in-vitro.

Cytotoxicity, genotoxicity, oxidative stress, apoptosis, and cell cycle arrest in human Sertoli cells exposed to boric acid.

BACKGROUND: Boron, which is used in a range of industries worldwide, is an essential micronutrient for plants and "probably essential" for humans. Conflicting reports have been published regarding the toxicity of boron compounds. Moreover, boric acid and sodium borates are classified as toxic to development and reproduction in the European Union-Classification, Labelling and Packaging Regulation (EU-CLP Regulation). The scope of our study was to ascertain whether boric acid caused the cytotoxic and genotoxic effects, as well as oxidative stress, apoptosis, and cell cycle profiles on human Sertoli cells. METHODS: The possible toxic effects of boric acid on human Sertoli cells were investigated by in vitro methods. The cellular viability and DNA damage were examined by neutral red uptake and alkaline comet assay, respectively. Oxidative stress, apoptosis, and cell cycle arrest profiles were analyzed by flow cytometry. RESULTS: Boric acid was neither cytotoxic nor genotoxic in a wide concentration range (0.5-1000 µM) on human Sertoli cells. No significant difference in the DNA damage was observed between boric acid-treated and control groups (p > 0.05). Boric acid did not stimulate oxidative stress, apoptosis, and cell cycle arrest within the tested concentrations. CONCLUSION: Our study provides novel insights into the influences of boric acid on human Sertoli cells which are used as a model in male reproductive toxicity studies. The concentrations tested in our study were extremely higher than the blood and semen boron levels reported in epidemiological studies.

A Novel In Vitro Wound Healing Assay Using Free-Standing, Ultra-Thin PDMS Membranes.

Advances in polymer science have significantly increased polymer applications in life sciences. We report the use of free-standing, ultra-thin polydimethylsiloxane (PDMS) membranes, called CellDrum, as cell culture substrates for an in vitro wound model. Dermal fibroblast monolayers from 28- and 88-year-old donors were cultured on CellDrums. By using stainless steel balls, circular cell-free areas were created in the cell layer (wounding). Sinusoidal strain of 1 Hz, 5% strain, was applied to membranes for 30 min in 4 sessions. The gap circumference and closure rate of un-stretched samples (controls) and stretched samples were monitored over 4 days to investigate the effects of donor age and mechanical strain on wound closure. A significant decrease in gap circumference and an increase in gap closure rate were observed in trained samples from younger donors and control samples from older donors. In contrast, a significant decrease in gap closure rate and an increase in wound circumference were observed in the trained samples from older donors. Through these results, we propose the model of a cell monolayer on stretchable CellDrums as a practical tool for wound healing research. The combination of biomechanical cell loading in conjunction with analyses such as gene/protein expression seems promising beyond the scope published here.

Evaluation of the dislodgement resistance of bioceramic reparative cements placed in a retrograde cavity using a different technique

Aim: Calcium silicate-based fillings have been widely used in surgical endodontic treatment because of hard-tissue conductive and inductive properties. The aim of present study is to investigate the bond strength of different calcium silicate-based fillings in retrograde cavities. Methods: Forty-four maxillary single rooted teeth were endodontically treated. The apical portions of the teeth were removed and root-end cavities were prepared using an ultrasonic tip. The roots were randomly divided into four experimental groups (n = 11) according to the material used; (1) MTA-FILLAPEX, (2) MTA Repair HP, (3) MTA-FILLAPEX+ MTA Repair HP, and (4) MTA Plus. Two horizontal cross sections (1±0.1 mm thick) from each specimen were resected from the apices. These sections were placed in a universal testing machine to evaluate the push-out bond strength force required for dislodgement of the root end filling was recorded. The failure type was also evaluated by using a stereomicroscope. The differences in bond strength were analyzed using the two-way analysis of variance (ANOVA). Results: MTA-FILLAPEX and MTA Plus displayed the lowest and highest dislocation resistance, respectively (P < 0.05). In the apical level, bond strength was significantly higher than the coronal level in all groups except for MTA-FILLAPEX. Mixed failure was prevalent in all groups, except for MTA-FILLAPEX, which showed purely cohesive failures. Conclusions: Investigated calcium silicate-based filling materials showed different bond strength to the root-end cavity. The bond strength was significantly decreased when the prior application of MTA-FILLAPEX before delivery of MTA Repair HP