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Neonicotinoid insecticide acetamiprid is a part of relatively frequently used commercial formulations such as Mospilan 20SP or newly developed Carnadine, Kestrel and others. The popularity of these insecticides lies in their ease of handling and high efficacy in treating canola, corn, potatoes and apple trees. However, the possibility that pesticide products could also affect non-target organisms still needs to be taken into account. For this reason, the aim of our work was to assess the possible cytotoxic and genotoxic effects of acetamiprid-based insecticide Mospilan 20SP, which is one of the well-known commercially available formulations. Conventional tests such as chromosomal aberration assay and comet assays (alkaline and neutral) were employed under in vitro conditions using cultures of bovine whole blood cells and isolated bovine lymphocytes. The insecticide formulation was dissolved in pure water to prepare quantities of 5, 10, 25 and 50 μg.mL^-1 for analysis after 2 h, 24 h and 48 h of exposure, respectively. After 24 h of exposure, values of mitotic indices showed a decrease in mitotic activity of cells (p<0,05; p<0,01) depending on increasing concentrations. The 24 h exposure to pesticides also showed an increase in chromatide and chromosome breaks in a dose-dependent manner for the insecticide under study (p<0.05; p<0.01) compared to the negative control. Alkaline and neutral comet tests revealed the potential of acetamiprid-based formulation to induce DNA damage in bovine cells.

Taking into account the events of recent years, a risk of exposure to high toxic substances including chemical weapons is not negligible. Therefore the Department of Toxicology and Military Pharmacy, Faculty of Military Health Sciences, University of Defence introduced analytical and biological samples (plasma, urine) preparation methods for determination of biomarkers of exposure to nerve agent (sarin, cyclosarin, soman, tabun, VX and VR agent) and sulphur mustard. Evidence of chemical weapon use is based on detection of their metabolites (Alkyl methylphosphonic acids as metabolites of the organophosphorus nerve agents, thiodiglycol as a metabolite of sulphur mustard) and products of their reactions with biomolecules (tyrosine adducts of organophosphorus nerve agents, sulphur mustard-albumin adducts). Gas chromatography tandem mass spectrometry and liquid chromatography tandem mass spectrometry are used for determination of biomarkers. Functionality of described methods is validated and discussed participating in Biomedical proficiency test, which is organized by The Organisation for the Prohibition of Chemical Weapons every year.

Ministry of Defence resort has its own medical service to provide health care and occupational-medical care and also hygiene service for supervision in the field of public health protection of Czech Army members and employees of Ministry of Defence. The incidence of occupational diseases partly indicates the effectiveness of the preventive measures in place. Occupational diseases have been reported and recorded. In 2010 – 2019, a total of 190 occupational diseases (188 occupational diseases and 2 persons endangered by an occupational disease) were certified and recorded in the Ministry of Defence resort. Diseases caused by the biological agents dominated, it was about work-related infectious diseases. In addition to work in health care industry and forestry, the training of troops in field conditions and, above all, military deployment abroad also proved to be risky in terms of infection. Another important group of occupational diseases was the lesion of peripheral nerves of the upper limbs and damage of musculoskeletal system due to excessive exposure to vibration and local muscle strain. Mainly civilian employees in forestry and in repair companies were punished by these occupational diseases. Work at risk have been recorded. In the end of 2019, there was in total 428 different work at risk recorded in the Ministry of Defence resort. The number of professional soldiers working at risk was 2806 persons and 1904 other persons. The Decision about the categorization of military service was issued by the chief hygiene officer of the Czech Army in 2020. It classified military service in the Czech Army as work at risk and, as a result, the number of persons working at risk in the Ministry of Defence resort has increased remarkably. The system of occupational-medical service is undergoing reform.

Rivastigmine is a pseudo-irreversible cholinesterase inhibitor used for therapy of Alzheimer´s disease and non-Alzheimer dementia syndromes. In humans, rivastigmine can cause significant gastrointestinal side effects that can limit its clinical use. The aim of this study was to assess the impact of rivastigmine on gastric motor function by means of electrogastrography (EGG) in experimental pigs. Six experimental adult female pigs (3-month-old; mean weight 30.7±1.2 kg) were enrolled into the study twice and created two experimental groups. In group A, a single intragastric dose of 6 mg rivastigmine hydrogen tartrate was administered in the morning to fasting pigs before EGG recording. In group B, rivastigmine was administered to overnight fasting animals in a dietary bolus in the morning for 7 days (6 mg per day). On day 8, an intragastric dose of 12 mg rivastigmine was given in the morning to fasting pigs before EGG. EGG recording was accomplished by means of an EGG standalone system. Recordings from both groups were evaluated in dominant frequency and EGG power (areas of amplitudes). In total, 1,980 one-minute EGG intervals were evaluated. In group A, basal EGG power (median 1290.5; interquartile range 736.5-2330 μV²) was significantly higher in comparison with the power of intervals T6 (882; 577-1375; p=0.001) and T10 (992.5; 385-2859; p=0.032). In group B, the dominant frequency increased significantly from basal values (1.97±1.57 cycles per minute) to intervals T9 (3.26±2.16; p<0.001) and T10 (2.14±1.16; p=0.012), respectively. In group B, basal EGG power (median 1030.5; interquartile range 549-5093) was significantly higher in comparison with the power of intervals T7 (692.5; 434-1476; p=0.002) and T8 (799; 435-1463 μV²; p=0.004). In conclusion, both single as well as repeated intragastric administration of rivastigmine hydrogen tartrate caused a significant decrease of EGG power (areas of amplitudes) in experimental pigs. EGG power may serve as an indirect indicator of gastric motor competence. These findings provide a possible explanation of rivastigmine-associated dyspepsia in humans.

The nerve agents of the A-series are relatively recent chemical weapons with no antidote available yet (1). Once inside the human body, those chemicals act similarly to the classic nerve agents, by binding to the catalytic residue Serine 203 (Ser203) of human acetylcholinesterase (HssAChE) and thus preventing the proper function of this enzyme. However, there is no experimental evidence yet if the current antidotes for intoxication by nerve agents are also capable of restoring AChE inhibited by the nerve agents of the A-series. In order to launch some light on this issue, we used computational techniques (molecular docking, molecular dynamics and MM-PBSA interaction energy calculations) to assess the performances of the four currently available commercial oximes (2-PAM, HI-6, obidoxime and trimedoxime) when in contact with HssAChE inhibited by the agent A-242 (2). Based on the near-attack conformation (NAC) criterion, our results suggest that the commercial oximes would have limited efficacy to reactivate the enzyme since they are not able to properly approach the adduct Ser203-A-242. Among those oximes, trimedoxime seems to be the most promising, since it showed lower values of energy in the MM-PBSA calculations, a higher stability inside the catalytic anionic center (CAS) of HssAChE and was able to adopt a position closer to the NAC that could enable the reactivation mechanism.

The thyroid hormone regulation is a vital and complex process for proper organism function that involves multiple organs across species. A disruption of the thyroid hormone system (THS) by endocrine disruptors (EDs) can be linked with adverse effects such as developmental and autoimmune disorders. However, THS has been rather neglected, even though EDs have been gaining attention recently. Currently, the H2020 ERGO project aims to develop a battery of high-throughput in vitro bioassays based on molecular initiating events (MIEs) in the adverse outcome pathway network connected to THS disruption (tAOP) to narrow the knowledge gap.
The thyroid hormone (TH) activity may be disrupted on several levels including synthesis, transport, metabolism, and receptor (trans)activation. To evaluate the suitability of various in vitro models for studying different endpoints, these have been characterized regarding the expression of the set of genes corresponding to MIEs in tAOP. Moreover, we developed a set of in vitro transfected HEK-293T cell lines overexpressing genes of major interest, i.e., deiodinases type 1-3 (DIO1-3), natrium-iodide symporter (SLC5A5; NIS) and thyroid peroxidase (TPO). Next to this, we adopted and optimized bioassays for thyroxine/transthyretin (T4/TTR) displacement, aryl hydrocarbon receptor (AhR), and thyroid receptor (TR) activation and characterized the potential of selected human exposure-relevant compounds to disrupt prioritized MIEs.

The Cytomegalovirus (CMV) pass through placenta is connected with consequences in prenatal as well as postnatal phase of development. Newborns can be asymptomatic, can have mild or even life-threating symptoms with serious long-term sequels. All CMV infected newborns are at risk of long-term neurodevelopmental problems and sensorineural hearing problems.
The aim of the work: To analyse prenatal and postnatal characteristics, clinical and laboratory findings, results of investigations in the group of 11 newborns with congenital CMV infection, who were hospitalized at Neonatal Department of Intensive Medicine between January 1st 2012 and March 31st, 2022 included.
Results: Prenatal foetal sonography revealed in patient 5 and 8 positive calcifications in brain; in patient 6, 9 and 11 isolated ventriculomegaly was found. Neurological examination was clinically negative in patient 1 and 10, changes of muscular tonicity and spontaneous activity was confirmed in the rest of group. In patient 5 and 10 one-sided positivity of otoacoustic emissions was confirmed. Chorioretinitis with bilateral negative otoacoustic emissions was confirmed in patient 5. Clinical status of patient 11 was complicated by pneumonitis. Three patients were treated with antiviral drugs orally, and 11 newborns had combination of intravenous and oral form of treatment.
Conclusion: The results of analysis will contribute to a society-wide solution of prevention. Monitoring of the frequency of CMV infection in population with education of population can decreased the number of affected newborns.

Bisphenol A (BPA), a widely used plastic additive with proven endocrine disrupting properties, has been previously linked to reproductive system disorders and tumors. Due to such harmful effects, its use has become restricted but other bisphenol (BP) analogs have been introduced as replacements for BPA usage in many applications. However, little is known about their biological actions or about similarities with the actions of the prototype BPA. The present study used the human prostate cell lines: androgen-unresponsive PC3 cells and androgen-responsive LNCaP cells, to examine effects of bisphenols AF, S, F and B, in comparison to BPA. We focused on BP effects on cell viability, proliferation, and oxidative stress (concentrations 10^-10–10^-4M) at several time points. We observed different sensitivity of both cell lines to toxic effects of the individual analogs. Although the highest concentration of each of the five BPs inhibited cell proliferation (BrdU incorporation) in both cell lines, this resulted in viability decrease only in LNCaP cells while in PC3 cells, the viability remained unaffected (MTS assay). The exception was observed in the presence of the BPAF analog with the profound cytotoxic effects, which seem to be caused by the early apopotic actions and increase in caspase activity (fluorescent assay). In addition, the highest concentration of each BP analog elevated reactive oxygen species production by the prostate cells. The results show that high concentrations of BPs can affect prostate cancer cell status and that androgen-responsive LNCaP cells are significantly more sensitive than the unresponsive PC3 cells, what indicates possible involvement of androgen receptors in the action of BP derivatives, and will be a subject of further elucidation.

Preclinical studies in experimental pigs are carried out mostly under general anaesthesia. Ketamine is commonly used for introduction to anaesthesia. However, concerns exist, whether ketamine, an NMDA-receptor antagonist, influences gastric motor function. The aim of this study was to investigate porcine gastric myoelectric activity by means of electrogastrography (EGG). Seventeen female animals (mean weight 36.2±3.8 kg) were enrolled. Drugs used as an introduction to anaesthesia were: Group A (n=5): medetomidine 0.1 mg/kg i.m.; butorphanol 0.3 mg/kg i.m.; midazolam 0.3 mg/kg i.m.; Group B (n=6): azaperon 2.2 mg/kg i.m.; Group C (n=6): ketamine 20 mg/kg i.m.; azaperon 2.2 mg/kg i.m., all groups followed by i.v. propofol (repeated one-mL-boluses, 10-12 mL in total). EGG recording started 15 min. after the introduction administration and lasted 30 min. Results were evaluated as dominant frequency of gastric slow waves (DF) and EGG power (areas of amplitudes). In total, 510 one-minute EGG intervals were assessed. DFs were (mean ± standard deviation): 1.4±0.4 (Group A), 1.3±0.3 (Group B) and 0.2±0.1 cycles per min. (Group C). The difference between group C and groups A and B was statistically significant (p<0.001). Median power (IQR) was 0.13 (0.02-0.44; Group A), 0.13 (0.03-0.54; Group B) and 0.30 V^2 (0.07-1.44; Group C). The difference between groups A and C was of borderline significance (p=0.066; type 2 error beta 0.295). In conclusion, ketamine, even in a single low-dose, affected myoelectric function of the porcine stomach. Therefore, it should be avoided in gastrointestinal motility studies in experimental pigs.

Atopic dermatitis (AD) is a chronic inflammatory skin disease, which is still not fully understood. Crucial roles in the pathogenesis play Th2 immune response dysregulation and epidermal barrier alterations. Defects of this skin barrier have been considered in the initial step of AD development. Inflammatory skin conditions AD have a negative wide-ranging impact on a patient’s life quality. However, testing methods and/or treatment options for this disease are unsatisfactory nowadays.
To study the anti-inflammatory effect of substances on AD pathogenesis we established inflammatory models by stimulating HaCaT cells with tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ). We designed two different treatment models (pre- and post-treatment) to mimic keratinocytes ̓ inflammatory conditions of AD that simulate relapse and therapy. HaCaT cells are a suitable model to follow the release of inflammatory mediators interleukin-6 (IL-6) and interleukin-8 (IL-8) in response to TNF-α and IFN-γ treatment. The AD-like models were verified by using natural flavonoid quercetin and synthetic glucocorticoid dexamethasone. Both substances should be associated with the inhibition of inflammatory cytokines production in the skin. Based on the data obtained, these models could open the way for screening of new preventive or therapeutic AD agents. The details of the experiments will be discussed in our poster contribution.

Melanins are heterogeneous polymeric dark pigments colored in black with relatively diverse properties, functions and rather non-defined structure. Many of commercially available melanins used in scientific studies have been produced synthetically or isolated from natural sources (1, 2).
We studied two types of melanin, i.e. commercially available synthetic melanin (SM) and biotechnologically obtained bacterial melanin (BM) from Bacillus thuringiensis. The topical aim of our study was to estimate the biological effects of both melanins in the neuroblastoma SH-SY5Y cell line. After melanin treatment (0-200 μg/mL), cell viability, glutathione levels (3) and cell respiration were assessed.
Both melanins were structurally characterized in detail and BM was more hydrophilic. After melanin treatment of SH-SY5Y cells, we found that SM at similar dosage caused always larger cell impairment compared to BM. In addition, more severe toxic effect of SM was found in mitochondria. In general, we conclude that more hydrophilic BM induces lower cell impairment in comparison to SM. Although additional elucidation of the toxicity mechanism is needed, it seems that the difference in level of toxicity induced by melanins can be dependent on their water solubility. Our findings can be usable for neuroscientific studies estimating the potential use for study of neuroprotection or neuromodulation.

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.

As in everyday life, it was necessary to respond to the ongoing COVID-19 pandemic at the campus of the Faculty of Military Health Sciences, University of Defence, as well. The management of the faculty took a number of measures, but for the academic sphere, the most important of these was the restriction of contact teaching. The way, in which teaching and training would proceed in the limited conditions, has been delegated to the heads of departments and guarantors of individual subjects. The Department of Military Medical Service Organization tested a teaching model which brought new knowledge that can be worked with in the future.

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).

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.