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Since 2002, no clinical candidate against Alzheimer’s disease has reached the market; hence, an effective therapy is urgently needed. We followed the so-called “multitarget directed ligand” approach and designed 36 novel tacrine phenothiazine heterodimers which were in vitro evaluated for their anticholinesterase properties. The assessment of the structure−activity relationships of such derivatives highlighted compound 1dC as a potent and selective acetylcholinesterase inhibitor with IC₅₀ = 8 nM and 1aA as a potent butyrylcholinesterase inhibitor with IC₅₀ = 15 nM. Selected hybrids, namely, 1aC, 1bC, 1cC, 1dC, and 2dC, showed a significant inhibitory activity toward τ(306−336) peptide aggregation with percent inhibition ranging from 50.5 to 62.1%. Likewise, 1dC and 2dC exerted a remarkable ability to inhibit self-induced Aβ₁₋₄₂ aggregation. Notwithstanding, in vitro studies displayed cytotoxicity toward HepG2 cells and cerebellar granule neurons; no pathophysiological abnormality was observed when 1dC was administered to mice at 14 mg/kg (i.p.). 1dC was also able to permeate to the CNS as shown by in vitro and in vivo models. The maximum brain concentration was close to the IC₅₀ value for acetylcholinesterase inhibition with a relatively slow elimination half-time. 1dC showed an acceptable safety and good pharmacokinetic properties and a multifunctional biological profile.

Monoterpenes, volatile molecules widely distributed in plants, are used in folk medicines, pharmaceutical and food industries, and cosmetics. Most terpenoids easily enter the human body by oral absorption, skin penetration, or inhalation leading to measurable blood concentrations. Numerous biological activities, including antitumor activity, of monoterpenes have been reported. On the other hand, some monoterpenes were reported to exhibit toxic effects in various organs of human organism, mostly in liver (1). The monoterpenes are also able to modulate the activity and/or expression of some drug-metabolizing enzymes. Present research was carried out to evaluate the effect of α-thujone (THU) or piperitone (PIP) on the activity and mRNA expression of the main detoxification enzymes in human liver. For this purpose, precision-cut liver slices (PCLS) were prepared from human liver samples (n=8) and incubated with THU or PIP (10 and 50 µM) for 24 h. These concentrations were non-toxic in primary rat hepatocytes. In human PCLS, THU and PIP in both concentrations caused significant increase in the specific activity of glutathione S-transferase (GST), however, the extent was dependent on basal GST activity. Regarding mRNA expression, induction of NAD(P)H:quinone oxidoreductase, cytochrome P450 2B6, glutathione peroxidase 3, and superoxide dismutase 1 was observed in samples treated with PIP 50 µM. The mRNA expression of GSTA and GSTP was induced by both compounds in several PCLS.

In this presentation, the first comprehensive toxicity study of Al₂O₃, SiO₂, ZrO₂, TiO₂ and WO₃ nanofibers effects in cultured epithelial A549 cells will be presented. The nanofibers were produced by centrifugal spinning from suitable spinning solutions and have an average diameter in the sub-micrometer range. At first, we characterized the nanofibers for their morphological, compositional and structural properties. Then, we estimated the biological effects of nanofibers in pulmonary epithelial A549 cells comparing them with biological effects of Al₂O₃, SiO₂, ZrO₂, TiO₂ and WO₃ nanoparticles. Multiwalled carbon nanotubes (MWCNT) were used as a positive control. The cells were treated with 1, 10 and 100 µg.mL-1 concentrations of a nanomaterial for 24 and 48 h. The dehydrogenase activity and glutathione levels were determined in cells as markers of cell injury. Experimental details and results of these investigations will be presented and discussed (1).

In recent years, the most mentioned nerve agents by the public are A–agents, so-called Novichoks. The official chemical structures of these new organophosphates have never been published. The possible structures were introduced by Vil Mirzayanov in 2009 (1), however, they do not correspond with structures published by Hoenig (2).
One type of Novichok was evaluated in our laboratory and its physical-chemical and biological properties were compared to well-known structures of nerve agents such as sarin or VX. Inhibition kinetics of human acetylcholinesterase and butyrylcholinesterase was determined. Further, the ability of standard oxime nerve agent antidotes to reactivate both inhibited cholinesterases was assessed. Cytotoxicity of Novichok compound was evaluated using human origin cell lines, including normal human lung fibroblasts (NHLF) and neuroblastoma cell line (SH-SY5Y), which undergo differentiation to mature neurons.
The research findings are initial dates in our research of fourth-generation nerve agents and should be useful towards the development of effective antidotes and possible subsequent therapy.

Plastic pollution is a global problem caused by an excessive use of this material, its high resistance in the environment and poor waste management. In the environment, larger-sized plastic particles may be transformed into smaller particles, so-called microplastics (< 5 mm), which have been discovered in the bodies of various organisms, across all trophic levels of the aquatic environment (Provencher et al., 2017). Effects of microplastics on fish embryos are described as malformations, changes in behavior and swimming ability, early hatching, increased mortality and decreased heart rate (Bhagat et al., 2020). However, these negative effects must be interpreted based on environmental concentrations in water sources (0.0001 – 2 mg/L; Rodrigues et al., 2022), which leads to uncertainties when carrying out ecological risk assessment on these particles.
In our experiment, the effect of polystyrene microspheres (50 µm) on zebrafish embryos was tested in three concentrations – 0.001, 0.005 and 0.01% of solution for 96 hours with 18 eggs of zebrafish (Danio rerio) per each concentration. The plates were kept in a temperature-controlled test environment (26 °C) with a photoperiod of 12 h light/12 h dark. Testing solutions were replaced every day to make sure that the test concentrations remained their nominal values. During the test, each plate was checked for coagulation of fertilized eggs, lack of somite formation, lack of detachment of tail-bud from the yolk sack, lack of the heartbeat, hatching rate and developmental malformations. After 96 h exposure, 8 replicates were crated from each test group, with each sample containing 10 mg of embryos to subsequent PCR analysis of selected genes and proteomic to the systematic identification and quantification of selected proteins. The significant changes at tested concentrations were compared to control group, without microplastic addition.

Drug-mediated induction of the intestinal ABCB1 and CYP3A4 is a clinically relevant phenomenon associated with reduced drug bioavailability. Well-established human models to assess the induction are currently missing, so drug regulatory authorities provide no recommendations to test in vitro/ex vivo drugs' induction activity. Human precision-cut intestinal slices (hPCIS) contain cells in their natural environment and express physiological levels of nuclear factors required for induction. We recently found that hPCIS incubated for 48 h retained intact morphology, ATP content, and ABCB1 activity. We also confirmed that rifampicin (30 µM) induces gene expression and protein level of the ABCB1 over the 48-h incubation. Here, we aim to evaluate whether model ligands for glucocorticoid receptor (dexamethasone) and vitamin D receptor (vitamin D₃) induce ABCB1 and CYP3A4 expression in hPCIS over the 48-h incubation. Moreover, darunavir, a clinically used anti-HIV drug, was evaluated using this model. Dexamethasone (100 µM) increased the CYP3A4 and ABCB1 gene expression significantly after 48-h, 19.68- and 3.00-fold, respectively. Darunavir (50 µM) after 24-h significantly increased CYP3A4 and ABCB1 gene expression 7.42- and 2.07-fold, respectively. Vitamin D₃ (100 nM) increased CYP3A4 expression 2.29-fold after 48-h incubation; however, confirmation of inducibility requires multiple repeats. On ABCB1 expression, vitamin D₃ had no effect. To conclude, hPCIS is a promising model for investigating drug induction potential. The study was supported by the GAUK 364521 and SVV 260 549.

Three preservatives  used as ingredients in cosmetics and other consumer products (triclosan , triclocarban and resorcinol) were evaluated for their genotoxic/mutagenic potential by means of a set of in vitro alternative methods, namely Ames Test (MPF Test, Xenometrix, OECD TG 471) with strains TA 98, TA 100, TA 1535 and TA 1537, Comet assay on HaCat cell line (non-tumor human keratinocytes) and Mammalian chromosome aberration test (OECD TG 473) using human peripheral lymphocytes. In the chromosome aberration test all three chemicals were positive in the highest tested concentrations. Similarly, in the Comet assay  the percentage of DNA in tail was significantly increased in the highest concentrations. Moreover, the genotoxic effects  were clearly dependent on the increasing concentration and duration of exposure. The Ames Test revealed positivity only for resorcinol using strain TA 1537, detecting frameshift mutations. The positive results were recorded for extremely high concentrations which under foreseeable conditions could not be present in the human body, however, the results justify the need to regulate and limit the use of these preservatives in final products.

Medical devices (MDs) have an irreplaceable role in modern healthcare. The term 'medical device' covers a broad spectrum of products that are crucial in diagnosis and treatment, disease prevention and improving the quality of life of people suffering from disabilities or injuries. MDs used in the oral cavity are usually those helping in the treatment of aphthae or canker sores irritations and lesions of the oral mucosa by forming a barrier that adheres to the oral mucosa and promotes healing. Dental materials and dental prosthetic devices are also an important group of MDs with apparent contact with oral mucosa.
Most of the MDs bio-compatibility assessments is still conducted in animals. However, thanks to the advances in cell and 3D tissue engineering and due to the accelerated progress of validation of alternative methods, the MD regulations are also in vitro tests, as demonstrated recently by the adoption of the in vitro reconstructed epidermis test for intra-cutaneous testing into the ISO standard 10993-23. Biocompatibility testing of MDs is based on the toxicity assessment of extracts from MDs, that are in fact highly diluted solutions of potential irritants. Therefore any already validated in vitro tests and prediction models must be fine-tuned to achieve different levels of sensitivity for this specific type of materials.
The TraiN-SafeMD (i.e. Training Network for improving knowledge on safety of medical devices) project builds on the practical experiences gained in the validation study for intra-cutaneous testing of MDs in which the research teams from Slovakia and Czech republic participated between 2012-2018. The current project, with partners from Austria, Czech republic and Slovakia uses 3D reconstructed tissues of oral/buccal epithelia and cell cultures with the origin in the oral cavity with the aim to develop a highly sensitive testing strategy for local tolerance testing in vitro. The project also aims into the training of PhD students and early career scientist in the use of in vitro methods for the safety assessment of MDs. The TraiN-SafeMD project has 5 tasks and is divided into 7 stages over 72 months. The team's efforts aim in: 1. establishing an international collaboration focused on safety testing of medical devices (MD). 2. development of a training network for PhDs and young scientists engaged in methods in vitro for the safety assessment of medical devices. 3. identification of the most promising in vitro methods and strategies for in vitro safety assessment of MDs used in the oral cavity. 4. standardisation of selected in vitro methods and generation of first data for selected MD materials as a part of the preparation for a larger international collaborative project.
The presentation will summarise the activities in the final year of the project and will discuss the next steps toward validation and implementation of innovative testing strategies into the ISO standards.

The aim of this study is to evaluate and compare the physical, motoric and cardiovascular status of selected Czech professional and volunteer firefighters aged 25 to 35 years. Firefighters (especially professionals) were chosen as a template for Czech male population because they are considered the most physically fit among the adult population. Moreover, physical training is a part of the job description for professionals, their motoric performance is regularly checked, and a decline in fitness is grounds for termination of employment. Since volunteer firefighters assist professionals in emergencies, they tend to be under the same physical and mental stress and their motivation to maintain excellent physical condition is considerable. The somatic characteristics, body composition, motoric performance tests as an indicator of the level of speed, power and endurance abilities, blood pressure and heart rate measurements and the Ruffier test as an information about the status of the cardiovascular system were performed. This study used hand-grip dynamometry to determine muscle strength as well. The results of our investigation showed that professional and volunteer firefighters have good physical, motoric and cardiovascular status and do not differ significantly in body height and adiposity, in body composition, in motoric performance, in cardiovascular characteristics, but do differ significantly only in hand-grip dynamometry (higher mean values in professionals).

It is usual for information to be unavailable regarding the molecular composition of extracts from herbs or animal tissues that are popular in folk medicine. Here, we present analysis of the alcohol–ether extract from bovine tissue analogous to the basic substance used in such commercial products as Retisin, Imuregen, Actovegin, and Solcoseryl. The tested extract contains a whole spectrum of free amino acids, small proteins and oligopeptides of molecular weight up to 10 kDa, various nucleotides, and a small amount of phospholipids. Among the molecules that can explain some biological activities of the extract were identified those of taurine (2-aminoethanesulfonic acid, a derivative of the amino acid cysteine), several defensins, and bactericidal hemoglobin fragments known as hemocidins. All those molecules identified are natural components of bovine tissues, and a substantial number of them might be biologically active in vivo. Others are sources of readily available nutrients.

Acetylcholinesterase (AChE) oxime reactivators are used as antidotes to organophosphate (OP) poisoning, while butyrylcholinesterase (BChE) reactivators are suitable for pseudocatalytic uptake of OP. OP acts as irreversible inhibitors of AChE. Due to the inhibition of AChE OPs cause impairment of cholinergic functions, which can lead to the death of the organism (1). The modification of the already known structure of pralidoxime aims primarily at overcoming the problems associated with physicochemical properties. The disadvantage of quaternary pyridinium oximes, which have a permanently positive charge, is poor penetration across the blood-brain barrier (BBB), which makes them ineffective in the central nervous system (2). Introducing a substituent that could contribute to increased lipophilicity, may thus provide better penetration into BBB. At the same time, a substituent in a suitable position relative to the oxime group on the pyridinium ring can reduce its pKa value and thus lead to easier formation of the oximate anion, which is important for the ability to reactivate inhibited cholinesterase (3). The aim of this work is to synthesize oxime reactivators derived from the structure of pralidoxime, determine their stability and pK_a values. Finally, the new derivatives will be measured in vitro for their ability to reactivate OP-inhibited cholinesterases.

The aryl hydrocarbon receptor (AhR) is a well-known cellular sensor of xenobiotics and major transcription regulator of xenobiotics-metabolizing enzymes. Recent studies have indicated that AhR is also important for physiological immunological functions of barrier organs, such as skin, gut, and lung. Nevertheless, its functions in epithelial cells of barrier organs are far less explored. Alveolar epithelial type II cells (ATII), also known as type II pneumocytes, are important regulators of functions of alveolar epithelium, which contribute to its regeneration, and production of surfactant. Surfactant lipids and proteins, which cover alveolar epithelium, both reduce surface tension and provide protection to pneumocytes. Here, we studied potential role of the AhR in the production of surfactant in a human model of ATII cells, A549 cell line. We have used AhR wild type and AhR-deficient A549 cells, in order to compare their capacity to express surfactant proteins, synthesize surfactant phospholipids, and produce surfactant layer, when cultivated at air-liquid interface (ALI). We then evaluated presence of lamellar bodies, as functional markers of differentiated ATII cells. Our results show that cells lacking AhR have an altered pattern of ATII markers, as compared with wild-type cells. These results suggest that toxicants activating and/or inhibiting AhR might potentially contribute to disruption of ATII cell functions and thus alter their role in the maintenance of lung homeostasis.

Every medical device that is in contact with human body must be subjected to series of biological tests during the risk assessment process. Cytotoxicity testing belongs to the group of endpoints for biological evaluation of medical devices and its whole process is defined in ISO 10993-5 “Biological evaluation of medical devices – Part 5: Tests for in vitro cytotoxicity”. In a short time, it provides an initial information about toxicity, which serves as a good indicator of general toxic properties and thus it can reduce the number of in vivo models required for subsequent toxicity analyzes. There are different approaches to cytotoxicity testing, either direct contact toxicity evaluation or elution methods. In our study, we focused on the latter approach, namely on optimizing the preparation of the material extracts in accordance with recommendations from ISO 10993-12 “Biological evaluation of medical devices – Part 12: Sample preparation and reference materials”. Based on the specific properties of tested materials, we selected volume of solvent used per surface area of test samples. Material samples were extracted in appropriate cell culture medium supplemented with serum. The materials were extracted at 37 °C for 24 hours with continuous circulation of sterile material immersed in the extraction medium in sealed tubes. In addition, we further extended the analyzes to dose ranging cytotoxicity evaluation to determine the level at which cytotoxicity no longer occurs.

Harman is a heterocyclic aromatic amine discovered in coffee, cigarette smoke, roasted meat, or fish (1). However, carcinogenic properties of harmane were proved - partly explained by its interaction via the AhR receptor and induction of CYP1A1 (2). Cytochrome P450 (CYP) enzymes are responsible for the metabolism of 75 % of drugs used in clinical practice (3). Our study aimed to determine the effects of harman on the most important CYP variants in a preclinical experiment.
Harman was administered to Wistar Albino rats intragastrically at the doses of 25, 40, and 64 mg/kg/day for 8 days (control group - 66% propylene-glycol). Microsomes were prepared from liver samples via differential ultracentrifugation. The content of total protein and CYP was measured in isolated microsomes. To evaluate the metabolic activity, the microsomes were incubated in vitro with CYP specific substrates: diclofenac (CYP2C6), dextromethorphan (CYP2D1/2), phenacetin (CYP1A2), testosterone (CYP2A, CYP3A, CYP2C).
Harman significantly decreased the metabolic activity of rat CYP2B, CYP3A, CYP2D1/2 (40 and 64 mg/kg/day) and CYP2C11 (25, 40 and 64 mg/kg/day). The metabolic activity of CYP1A2, CYP2A or CYP2C6 was not affected. Our results did not confirm the potential of harmane to induce liver CYP450 in rat. Nevertheless, significant inhibition of various CYP enzymes was proved. To exclude the risk of serious interactions, the effect of harmane on CYP in humans should be studied.

Non-alcoholic Fatty Liver Disease (NAFLD) promotes to Non-alcoholic Steatosis Hepatitis (NASH), liver cirrhosis and cancer. However, there is no specific clinical treatment for NASH. Previously, we screened in vitro model for novel drug candidates towards NAFLD/NASH. The mTOR inhibitor was found to significantly alleviate palmitic acid-induced lipotoxicity in hepatocyte culture. Therefore, the aim of presented study was to investigate the effect of non-specific mTOR inhibitor (KU-0063794) when given orally in dietary model of NAFLD/NASH. To develop NAFLD and NASH in vivo, male mice C57Bl6J were fed with Atherogenic High Fat Diet (AHFD), and fructose/glucose in drinking water for 12 and 16 weeks, respectively. KU-0063794 treatment for 17 days displayed a trend towards decreasing serum glucose, inflammation (e.g. serum TNF-α), hepatocyte oxidative stress (e.g. conjugated DENES) and an improvement in expression of metabolic genes in liver homogenates. However, KU-0063794 had no effect on liver NASH morphology (e.g. NAS or fibrosis score). In conclusion, oral KU-0063794 treatment for an acute period displayed a trend to improve the highly progressed NASH with no signs of toxicity and therefore, chronic treatment should follow.

Titanium is one of the most widely used materials for medical and dental implants due to its resistance to body fluid effects, low ion release, great tensile strength, flexibility, and high corrosion resistance. Despite its promising properties, titanium implants tend to be encapsulated by fibrous tissue in vivo and show a lack of osseointegration, which can lead to infections and implant failure (Hansson; 1983).
To improve the surface properties of materials, the atomic layer deposition method is used, where the surface of the material is modified with one or more uniform layers of metal oxide, which directly correlates with the improvement of metal surface properties. The atomic layer deposition is the only technique that enables the deposition of very uniform and conformal layers of various materials, regardless its shape – planar as well as porous substrates (Dvorak; 2019).
In the present work, titanium sheets and titanium sheets with TiO₂ nanotube layers and their surface modifications were used to evaluate the adhesion, growth and proliferation of different cells. Cell adhesion and growth were investigated using fluorescence staining and cell counting. The presented results showed that surface modification of titanium sheets by atomic layer deposition has a significant effect on the biocompatibility of materials and is promising for application in implant materials.

The broad use of nanomaterials in material science, medicine or industry has implied the request to evaluate their biological effects. In vitro cellular models provide a suitable approach to study those effects, but it can be difficult to ensure valid and repeatable experimental conditions for proper setting of biological testing. Indeed, a number of physicochemical parameters (e.g. type of dispersion, aggregation, colloid stability, agglomeration) can influence the obtained results. Thus, the topical goal of our study was to provide a complete view on testing of commonly used nanomaterial, i.e. titanium dioxide nanoparticles, in cultured cells.
We used several advanced methods to characterize the nanomaterials (SEM, XRD, Raman) and to determine their behavior at experimental conditions. Thus, we focused on determining optimal conditions of dispersion (DLS, AFM). To evaluate the biological effects in cultured cells, we used routine biochemical assays (dehydrogenase activity, glutathione levels) and microscopy. We found that the experimental parameters were capable to influence obtained results on cellular effects significantly. In contrast, we were allowed to obtain repeatable and valid results when optimal experimental conditions were guaranteed. Our study provided information on limitations and challenges in testing of nanotoxicity in vitro, especially in nanoparticles.

Oxysterols are 27-carbon derivatives of cholesterol formed by enzymatic, as well as non-enzymatic, oxidation of cholesterol. They participate in cholesterol metabolism and influence many cellular processes, but they are also involved in the etiology of different diseases, including cancer. Previous studies found that oxysterols influence anti-cancer treatment in vitro, e.g., the presence of different oxysterols modulates the activity of doxorubicin, 5-fluorouracil, docetaxel, or cisplatin.
The aim of this study is the analysis of the role of nine oxysterols in pancreatic cancer in vitro. Two human pancreatic cell lines, Paca-44 (mutated in KRAS gene) and BxPC3 (wild-type) are included in this study. To study the effect of different oxysterols, both cell lines were seeded on a 96-well plate and incubated with a medium containing one of the oxysterols. After 72 hours, the cell viability was measured using a CellTiter-Blue^® Cell Viability Assay, and the IC₅₀ of each oxysterol was counted.
The IC₅₀ of some oxysterols was very similar in both cell lines, yet 25-hydroxycholesterol and 5α,6α-epoxycholestanol efficacy varied between Paca-44 and BxPC3 cells. Moreover, two oxysterols, 27-hydroxycholesterol and 4β-hydroxycholesterol, showed no or very low effect on cell viability in both cell lines. In future studies, we would like to analyze the role of oxysterols on the effect of gemcitabine in pancreatic cancer in vitro.

Depression is becoming the most common psychiatric illness worldwide. Its etiology is not fully understood, but the monoamine theory is supported by antidepressant mechanism of action that modulates monoaminergic systems. Known antidepressants have various side effects, so there is a need to search for new therapeutics. Our previous study revealed an antidepressant effect after acute pyridoindole derivative SMe1EC2M3 treatment (1).
We studied the effect of chronic administration of the SMe1EC2M3 under stressed conditions induced by chronic mild stress (CMS) procedure in Sprague-Dawley male rats (n=72). From day 8th of the CMS, we intraperitoneally treated the animals by 5 or 25 mg/kg/day dose. We evaluated changes in behavior in sucrose preference test (SPT), open field test (OF) and forced swim test (FST). Potential neurotoxicity was investigated using primary hippocampal neurons cultures from Wistar neonates. Cells were treated without or with SMe1EC2M3 (0.25; 0.50; 1.00; 1.50 µM) or all-trans retinoic acid (ATRA). We evaluated 3 coverslips/group and 7 areas of interest/ coverslip. Using Sholl analysis, we counted dendrites intersection by concentric circles from the soma to 200 µm and the length of the longest neurite from the nucleus to the apical end.
Higher immobility in FST, lower consumption in SPT and shorter distance traveled in OF confirmed the depression-like behavior. Both doses reversed the effect of CMS by reducing immobility and prolonging the swimming. No neurotoxicity of the SMe1EC2M3 was observed. In group 1.50 µM SMe1EC2M3, neurites length was stimulated and we found more neurons with the longest neurite over 200 µm. No significant changes in the number of neurite branches were found between groups.

A significant global problem of contamination of the aquatic ecosystem is the abundance of pharmaceuticals entering the aquatic environment as a result of their widespread use. An important group of drugs commonly detected in surface waters are non-steroidal anti-inflammatory drugs (e.g. ibuprofen and diclofenac). The aim of the present study was to evaluate the potentially negative effects of diclofenac and ibuprofen administered in feed on selected biochemical and haematological parameters of common carp (Cyprinus carpio). The test organisms were exposed to various concentrations of these non-steroidal anti-inflammatory drugs at concentrations of 20 and 2000 μg/kg for six weeks. The test substances were administered in the feed at a dose of 3 % of body weight. After the end of the exposure, blood was drawn with a sterile syringe from the tail vein into Eppendorf tube containing sodium heparin. One part of heparinized blood was used for analysis of selected haematological indices (red blood count, white blood count, haematocrit, haemoglobin). The second part of the heparinized blood was centrifuged (4 ⁰C, 10 min, 3,000 rpm) and the separated plasma was used for analysis of basic biochemical indices (glucose, albumin, total protein, cholesterol, LDH, ALT, AST, ALP, calcium, phosphorus, ammonium) using biochemical analyser Konelab 20i. Our results confirm that the substances tested pose a risk to aquatic organisms.

Benchmark dose (BMD) approach, as an advanced statistical methodology for dose-effect analysis in toxicological research (1) was used to quantitatively characterize in vivo efficacy of two experimental bispyridinium oximes K203 and K027, following promising findings on their low acute toxicity and potential to reactivate acetylcholinesterase (AChE) inhibited by organophosphorus (OP) pesticides and nerve agents in vitro and in vivo (2). Immediately after DDVP challenge (75% LD₅₀, s.c.), male Wistar rats were treated with oxime (0/1.25/2.5/5/25/50% LD₅₀, i.m.). Erythrocyte and diaphragm AChE activity was determined by Ellman's method 60 min after the treatment. Benchmark analysis was done in PROAST software ver. 65.5 (RIVM, The Netherlands). Derived BMD_er were K203 = 194 (153, 243) and K027 = 100 (81, 125) µmol/kg bw, BMD_diaph were K203 = 117 (56, 209) and K027 = 21 (10, 37) µmol/kg bw, indicating that oxime K027 induces the same effect size with 2 and 5.5-times lower dose compared to oxime K203 in erythrocites and diaphragm, respectively. Quantification of equieffective doses of oxime reactivators would enable more reliable definition of their therapeutic widths, which further contributes to better determination of therapeutic dosage regimens and, finally, increases the relevance of results obtained in animal models for the human population.

Uridine diphosphate sugar-utilizing glycosyltransferases (UGTs) are an enzyme superfamily that catalyzes glycosyl residues transfer from activated nucleotide sugars to acceptor molecules. In addition to various endogenous compounds, numerous xenobiotics are substrates of UGTs. As the glycosides formed are generally less active/toxic and more hydrophilic than aglycones, UGTs effectively protect organisms from potentially harmful xenobiotics. Therefore, increased UGTs expression and/or activity improves the protection of the organism and may contribute to the development of individuals that become more resistant to certain xenobiotics. While the function of UGTs in the resistance of human cancer cells to chemotherapy is now well known, other organisms and other xenobiotics have attracted much less attention. UGTs play an important role in defense against xenobiotics not only in humans, but in countless other organisms such as parasites, insects, and plants. Moreover, many recent studies clearly show the participation of UGTs in the resistance of nematodes to anthelmintics, insects to insecticides, weeds to herbicides as well as humans to various drugs (not only those used in cancer therapy but also in the treatment of epilepsy, psychiatric disorders, hypertension, hypercholesterolemia, and HIV infection). Nevertheless, although the contribution of UGTs to xenobiotic resistance in diverse organisms has become obvious, many pieces of information remain missing, for example with regard to the mechanisms of UGTs regulation.

The aim of the presented work was to evaluate the decontamination efficacy of newly developed decontamination agents based on surfactants with hydroxyl functional group.
Sarin and VX were selected as representative nerve agents (NA). The ability to accelerate the catalytic hydrolysis of the NA was assessed by the pH value measurement using an automatic burette. The hydrolysis of NA in a contaminated solution leads to a decline of pH value due to the formation of O-alkyl methylphosphonic acids (MPAs). The ability of tested surfactants to penetrate through the skin and their rinsing effectivity was observed using modified Franz-type of glass diffusion cells and ex vivo dermatomed porcine skin as a model membrane. The amounts of surfactants penetrated into the receptor fluid after application on the skin surface were determined by HPLC with fluorescent detection. The solutions of surfactants were used to rinse the contaminated skin. Simultaneously, the same solution was contaminated by corresponding amount of NA to simulate the situation when 100 % of a contaminant is rinsed off. The MPAs in the rinsing fluid and NA penetrated into the receptor fluid were determined by HPLC-MS. The decontamination efficacy of the tested solutions was expressed in % of an agent washed away by the rinsing fluid.
All of tested newly synthesized compounds had better hydrolytic activity compared to the commercially available surfactant. Their penetration through the skin was not observed. The rinsing efficacy of all tested solutions was comparable.

Depressive disorder is a serious mental illness whose incidence is constantly increasing in developed countries. About 20% of pregnant women suffer from maternal depression. Both untreated and treated maternal depression represents risk factor for development of fetus and newborn. The maternal hormones, such glucocorticoids, have been reported to be altered in response to a maternal challenging environment. These molecules cross the placenta and reach the fetus, altering the neurodevelopmental pathways in the fetus that may affect the proper brain functioning, leading to an increased risk for neurodevelopmental disorders. Antidepressants used to treat maternal depression represent also a risk factor for fetal and neonatal development. The results of our experimental studies have been shown beneficial effects of selected antidepressants on neurogenesis, synaptic plasticity and behavioral variables on rat offspring. However, there are many controversy on effects of maternal depression and antidepressants on fetal and early postnatal development. Therefore, it is highly topical to investigate their potential unfavorable as well as beneficiary effects on healthy development of the offspring.

Multidrug resistance-associated protein 2 (Mrp2) is the crucial transporter for the biliary secretion of anionic compounds. The genetically determined absence of this transporter may occur in humans, causing conjugated hyperbilirubinemia and increased risk of intrahepatic cholestasis of pregnancy (ICP). The ICP threatens fetuses with adverse pregnancy outcomes due to increased bile acids (BAs) plasma concentrations. This study aimed to characterize BAs metabolomics in Mrp2 deficiency and ICP. Cholestasis was induced in Mrp2-deficient and wild-type rats by ethinylestradiol as a model of ICP. BAs were analyzed in plasma, bile, and stool to describe their metabolomics together with liver and intestinal enzymes and transporters responsible for BAs enterohepatic recycling. Mrp2-deficiency reduced the biliary secretion of BAs and increased their plasma concentrations in part due to increased BAs efflux from hepatocytes to the blood via upregulated Mrp3 and Mrp4 transporters. The intestinal BAs reabsorption was also reduced in these rats due to downregulated ileal sodium/bile acid cotransporter. The activation of constitutive androstane receptor-nuclear factor erythroid 2 related factor 2 pathway by accumulating bilirubin might be responsible for observed changes in BAs metabolomics in Mrp2-deficient rats. The plasma concentrations of BAs were further increased by ethinylestradiol administration in Mrp2-negative rats due to reduced BAs uptake and increased hepatocyte efflux via reduced Slco1a1 and upregulated Mrp4 transporters. Our results confirmed the hypothesis that impaired Mrp2 transporter predisposes to increased plasma concentrations in estrogen-induced cholestasis due to complex changes in liver transporting proteins. We, therefore, recommend regular monitoring of BAs in the plasma of pregnant women with conjugated hyperbilirubinemia.

The goal of this project is to create algorithm and software solution for prediction of drugs’ side effects. The algorithm is based on calculations of interactions between active substances and known macromolecules.
Methods of molecular docking and reverse molecular docking are used there. Each exanimated drug substance is docked into binding site of each macromolecule of sc-PDB database while the interaction between them is calculated. A set of decoys is selected from the ZINC database based on chemical properties. Decoys are docked into binding site of each macromolecule as well as exanimated drug substances. The docking results of exanimated substances and decoys are compared to determine the specificity of interactions. These methods are implemented using AutoDock Vina. All of docking interactions are analyzed by R software and the list of resulting macromolecules are released. The active drugs substances can be a substrate, inhibitor, or inducer of resulting macromolecules and they are suitable for in vitro testing.
The software solution is designed as a public service accessible by a web browser.

Tuberculosis (TB) is one of the top 10 causes of death worldwide from a single infectious agent. The World Health Organization (WHO) estimated 10 million new cases and 1.5 million deaths from TB in 2020 (1). Some strains of mycobacteria causing TB show numerous resistances to first-line drugs (isoniazid /INH/ and rifampicin) and to second-line drugs (fluoroquinolones, amikacin, bedaquilin, etc.). The development of new anti-TB drugs with new mechanism of action is necessary to improve TB therapy and to fight against resistant TB as well.
We screened our in-house library of small molecules for their therapeutic potential and identified compound K1297 with good anti-TB activity with minimum inhibitory concentration MIC₉₉ = 4 μM against H₃₇R_v strain (for comparison, MIC₉₉ (INH) = 0.5 μM). The main structural motif of this molecule is purine scaffold, which was modified and functionalized to elucidate the structure-activity relationships (SAR) and to identify derivatives with low toxicity and higher efficiency than the initial hit K1297. The effect of individual structural fragments on in vitro antimycobacterial activity, toxicity and selectivity of action have been evaluated. Finally, derivatives with optimized activity/toxicity ratio have been found and their pharmacokinetic profile and in vivo efficacy will be evaluated.

Mercury exists in the environment as elemental, metallic mercury, inorganic mercury and organic mercury.  Elemental and inorganic mercury released into the atmosphere from combustion of fossil fuels, mining, smelting and various industrial activities. In the aquatic environment is mercury deposited to sediments where is transformed into the main organic form methylmercury. Mercury methylation occures in the aquatic environment due to microorganisms metabolism as sulfite reducing bacteria.  The toxicity of mercury in humans or animals depends on its chemical form. Elemental mercury is volatile, gastrointestinal absorption of inorganic mercury is between 10 - 30% range. Methylmercury is the most toxic form of mercury. In comparison with the inorganic form, methylmercury is absorbed from more than 80 % in the gastrointestinal tract, than is widely distributed to all tissues, although the largest deposition occurs in the kidney. The enterohepatic cycle results in a long half-life for this compound compared to inorganic mercury. Methylmercury is able to cross the blood-brain and the placental barriers,  mainly affects the central nervous system and is harmful to the nervous system development of the fetus.
The greatest risk of human exposure to mercury comes from food chain. Various authorities, such as United States Environmental Protection Agency (USEPA),  the Joint Expert Committee on Food additives (JECFA) of the Food Agriculture Organization (FAO), World Health Organization (WHO) and European Food Safety Authority (EFSA),  have established a reference dose for human consumption, such as Provisional Tolerable Weekly Intake (PTWI) for methylmercury.
Our study were focused on two year report from Rapid Alert System for Food and Feed (RASFF) and evaluation of  various fish species from market chain exceeded established tresholds by the Commission Regulation (EC) No. 1881/2006 of 19^th December 2006 setting maximum levels for certain contaminants in foodstuffs. According to this study, in 2017 and 2018 y., 113 cases of exceeding the Hg content limit in fish and seafood products were reported. The most commonly reported fish is swordfish (Xiphias gladius), blue shark (Prionace glauca) and mako shark (Isurus oxyrinchus).

Coronavirus disease COVID-19 is highly infectious disease caused by SARS-CoV-2 virus as a novel coronavirus led to pandemic. Due to fast transmission COVID-19 has become a global problem. The virus is spread by aerosol from infected people and persists in the air for a long period. SARS-CoV-2 affects the lungs, but it can also affect digestiv or cardiovascular systems, can attact the brain, damage vessels and can lead to neurological manifestation. COVID-19 has several clinical symptoms and can lead to multiorgan dysfunction and death. On the other hand, in some infected persons COVID-19 can take place asymptomatic. Due to the risk of rapid spread of COVID-19, the early prediction of the onset of the epidemic is important. The Water Based Epidemiology (WBE) is an effective tool for monitoring the number of infections and can serve as a tool to monitoring various human activity including health status of population in given area. One of the advantage of WBE in last pandemic is their capability to reveal the outbreaks at an early stage, including presymptomatic or asymptomatic transmission of SARS-CoV-2. The use of WBE is based on the principle of viral shedding in stool samples and on the detection of SARS-CoV-2 viral mRNA by PCR method in wastewater samples. On the other hand, some non-specific markers from COVID-19 infected persons can also be used in WBE. Viral infections are associated with inflammation. Neopterin, a pteridine derivative, is produced in the metabolism after some stimulus. Neopterin was found in urine and other body fluids, as blood serum, cerebral spinal fluid in COVID-19 infected persons in high levels. Through urinary excretion, neopterin was detected in wastewater samples. The other markers, as toxin-like peptides, similar or identical to toxic components of venoms from animals, such as conotoxins, phospholipases, phosphodiesterases, zinc metal proteinases, and bradykinins, were identified in blood plasma, urine and faecal samples from COVID-19 patients. These markers, including neopterin, can be potencially useful in WBE as markers of rapid prediction of the onset of the epidemic. The early detection of the presence of SARS-CoV-2 within communities can also give healthcare authorities time to prepare for potential outbreaks and time to prepare measures to protect population.

Cadmium is a toxic transition heavy metal commonly found in the environment and its occurrence is mainly conditioned by agricultural and industrial production. Increased exposure to cadmium, especially because of its accumulation in organisms, causes damage of kidney, liver, and bones. Intracellularly, there are proteins, metallothionines, that are actively involved in the detoxification of cadmium by its uptake. In response to increased ROS production after exposure to cadmium, the Nrf2 protein, which modulates glutathione synthesis, is activated (1). The activation rate of Nrf2 protein depends on the concentration of cadmium in a certain time interval (2).
In our work, we focused on testing selected concentrations of cadmium chloride (5-100 µM) in SH-SY5Y neuronal cells in vitro. To describe the cellular toxicity, we used a spectrofluorimetric method to determine the intracellular concentration of glutathione (GSH) using monochlorobimane (3) in selected time intervals (1-24 hours). The results suggested that especially at concentrations under 50 µM CdCl₂, there is a transient increase in intracellular GSH levels relatively to untreated cells. A significant increase in GSH concentration up to 150% was detected in 5-50 µM CdCl₂ treated cells after 24 hours.