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Polyaromatic hydrocarbons (PAHs) are a diverse group of environmental and dietary contaminants. PAHs are primarily metabolized through the aryl hydrocarbon receptor (AhR)-regulated enzymatic pathway. Both parent PAHs and their metabolites may exert various types of toxicity in target cells and tissues. In addition to their well-known genotoxic (and carcinogenic) effects, PAHs may alter numerous additional cellular events, including cell metabolism. In this project, we study whether strong AhR ligand benzo[k]fluoranthene (BkF) can modulate genes associated with glucose and lipid metabolism, as well as proteosynthesis, which is essential metabolic process performed at high energetic cost. We used an in vitro model of immortalized human hepatocytes, MIHA cell line. We observed a significant suppression of proteosynthesis induced by BkF in MIHA cells, as determined by the SUnSET method. We also observed a moderate deregulation of several genes associated with further evaluated metabolic processes. Based on this, we continue to evaluate possible impact of strong AhR ligands, such as BkF, on cellular metabolism, as well as functional role of this transcription factor in the complex effects of PAHs on cellular metabolism.

The ISO standard 10993 for medical device testing was accepted over three decades ago. During that period, ISO standard was changing and adapted based on the growing knowledge on adverse effects and regulatory needs. The term, medical devices, includes various products that are used in medicine, but their terapeutic effect is not acchieved by pharmacological, immunological or metabolic means. These products have different mechanical, physical and chemical properties. The procedures described in the ISO 10993 cover testing of raw materials as well as final products. However, composition of  medical devices can be quite complex, and it may sometimes be challenging to follow procedures laid down by the ISO 10993. The ISO 10993-12, reviewed in 2021, describes in detail the essentials of the sample preparation undergoing the in vivo or in vitro testing (i.e. extraction into polar and non-polar solvents). Unfortunately, there is only a little guidance for materials absorbing the extraction solvents  (ISO, 2018; ISO, 2021a; ISO, 2021b).
In our study, we tested medical devices used in dental/oral care. Among these products are adhesive pasted for dentures and cream for ulcer treatment. These formulations have shown to be highly solvent absorbing. We have performed several procedures for extracting such materials, using different ratios of the material/extractant and including/excluding the orbital shaking of the sample. The achieved extracts were subsequently tested in line with the ISO 10993-5 using 3T3 NRU assay and ISO 10993-23 using 3D reconstructed human tissue model of epidermis and another, non-keratinised epithelia, used to mimic the soft tissues in the oral cavity.
The results show that the exctract preparation procedure considerably influenced the results of the experiments. In the static conditions, the preparations showed significantly lower cytotoxic proppetries compared to the mixed extracts. Non-keratinised 3D epithelial tissue model was more sensitive to the preparations than 3D skin tissue, as expected. The testing battery helped us to predict possible side effects of the selected MD materials used in the oral cavity.

Oxime reactivators are causal antidotes commonly used against organophosphate (OP) poisoning. Although OPs are utilized in agriculture as pesticides, they can be misused by terroristic groups as nerve agents (NA). These compounds are fast acting inhibitors of human acetylcholinesterase (AChE) causing cholinergic crisis ultimately leading to death by respiratory failure. Treatment of OP intoxication consists of immediate administration of symptomatic drugs, namely, atropine and diazepam and antidotes in form of oxime reactivators of AChE. Undoubtedly commercially used reactivators are the best countermeasure for OP intoxications so far, however they have several downsides. First of all, there is no broad-spectrum reactivator effective against all NAs, secondly they cannot reactivate “aged” form of AChE and lastly they poorly cross the blood-brain barrier (BBB) due to low lipophilicity (1). For instance, widely used reactivator 2-PAM crosses the BBB approximately from only 10% (2). However, most of the techniques used for measuring the concentration of reactivator crossing BBB are relying on HPLC combined with MS or UV detector. BODIPY probe could be great alternative as it is a strongly fluorescing probe standardly used for imaging techniques in vitro (3). The aim of this work is in vitro evaluation and analysis of physicochemical properties of BODIPY labeled oxime reactivators of AChE.

Signal transducer and activator of transcription-3 (STAT3) is the most studied member from seven latent cytoplasmatic transcription factors (STATs 1-4, 5a, 5b and 6) that are closely associated with the occurrence of many types of cancer (breast, leukemia, lung, lymphoma, ovarian and prostate). STAT3 contains four functional domains, from which the Src homology 2 domain (SH2 domain) is the major target for inhibition of these proteins. Inhibition results in STAT3 tyrosine 705 (Y705) phosphorylation, dimerization, nuclear transport, DNA binding and transcription induction. The benzo[b]thiophene 1,1-dioxide (BTP) as a pharmacophore is part of many STAT3 inhibitors, such as Stattic, HJC0123, HJC0149 and HJC0416 (1,2,3). Stattic (6-nitro derivate of BTP) is a small molecule which selectively inhibited STAT3 SH2 domain function in vitro (3). HJC0123, HJC0149 and HJC0416 are carboxamides made from 6-amino derivate of BTP and substituted aromatic carboxylic acids (1,2). In this study we have in silico designed, synthesized and purificated 8 new Stattic-carboxamides. Their physical chemical properties were predicted in silico and then experimentally evaluated. Their biological activity was tested on murine and human cancer cell lines. Results were evaluated and compared to Stattic as a reference compound.

Neurotoxicity is commonly associated not only with neurodegenerative damage but also with organophosphate intoxication. Neuronal death may be associated with acute and life-threatening symptoms and subsequent long-term secondary disorders. This study focuses on the development and validation of a cellular model of mature human neurons. A model was obtained by differentiating the neuroblastoma cell line SH-SY5Y. In vitro cell model is suitable for studying neurotoxicity and its potential countermeasures. The protocol involved stimulation of the cell line with retinoic acid and brain-derived neurotrophic factor for 9-12 days. Observation of morphological signs of neurons (characteristic synaptic connections), using lighting microscopy and a detection of specific neuronal markers (tau protein, microtubulle-associated protein (MAP), synaptophysin (SYN), post-synaptic density protein (PSD-95)) using fluorescence microscopy was used for validating this model. Another experiment was focused on the quantification of acetylcholinesterase in differentiated and undifferentiated cells. As a result, it has been shown that the level of enzyme in the differentiated cells is significantly higher than in the original undifferentiated cells. Another planned use of the model is in vitro testing of the neurotoxic effects of organophosphates and screening of potential prophylactics or drugs for the treatment of neurodegenerative injuries.

Nowadays, pyrethroids (e.g., flumethrin and tau-fluvalinate) are commonly used in apiculture as an active ingredient of several authorised veterinary medicinal products (acaricides) licensed worldwide. The beekeepers who regularly use synthetic acaricides throughout the year may potentially put the bee colonies at risk. Pyrethroids are frequently related to long half-life inside the hive matrices, which may adversely affect the health of bee colony. In this project we assessed potential harmful lethal and sublethal effects of synthetic acaricide tau-fluvalinate (tech.) on winter adult honeybees according to OECD 245 (2017). In vitro reared winter honeybees showed no dose-dependent mortality after the oral 10-days exposure to sucrose solution (50% w/w) spiked with a maximum concentration of 750 μg a.i./kg diet and its 1/10 value. The No Observed Effect Concentration (NOEC) appears to be higher than or equal 750 μg a.i./kg diet. The results of tau-fluvalinate testing for the potential sublethal genotoxic effects showed that that pyrethroid tau-fluvalinate at tested concentrations induced significant levels of DNA damage in bees, which may result in a potential genotoxic effect.

The aim of this project is heterologous expression of chosen carbonyl-reducing enzymes from Haemonchus contortus, a gastrointestinal nematode of small ruminants. Carbonyl-reducing enzymes (such as SDR and AKR) catalyze the first phase of xenobiotics biotransformation and thus participate in drug metabolism. Increased elimination leads to  decreased toxicity and reduced efficacy of drugs in Barber´s pole worm. One of the proven mechanisms of  deactivation of anthelmintics (e.g., flubendazole) is the reduction of the carbonyl group by these enzymes.
In addition, the previous metabolism analysis has demonstrated a higher ability of resistant strain of H. contortus to reduce flubendazole more effectively than the sensitive strain. The genome of H. contortus contains approximately 70 SDR genes and 24 AKR genes; however, information about expression and function is  not yet known. Furthermore, previous quantitative analysis of gene expression in H. contortus, have shown that the most highly expressed genes were SDR1, SDR3, SDR12 and SDR18. Also, expression of SDR12 was significantly higher in all life stages of the resistant strain.
These genes have been selected for heterologous expression, including cloning and expression in two systems: in E. coli typical expression system for soluble enzymes, and in eucaryotic cell lines, an  expression system enabling a higher level of posttranslational modifications. Expression of selected SDR or AKR, in a suitable system will allow us to characterize them, determine their enzyme activity, even test new potential inhibitors in the future.

The efficacy of prophylactic use of memantine alone or in combination with clinically used reversible acetylcholinesterase inhibitors (pyridostigmine, donepezil, or rivastigmine) against soman toxicity and their influence on post-exposure therapy consisting of atropine and HI-6 was studied. The effectiveness was assessed by comparison of LD₅₀ values over 24 h after soman poisoning. Pyridostigmine failed to decrease the acute toxicity of soman. But memantine, donepezil and rivastigmine reduced the acute toxicity of soman, with donepezil showing the best efficacy. Combination of memantine with pyridostigmine or one of the centrally-acting reversible acetylcholinesterase inhibitors attenuated soman acute toxicity significantly. The pharmacological pretreatment influenced the efficacy of post-exposure treatment in a similar fashion; i) pyridostigmine or memantine did not affect the therapeutic effectiveness of antidotal treatment ii) centrally acting reversible acetylcholinesterase inhibitors slightly increased the efficacy of antidotal treatment, iii) combination of memantine with reversible acetylcholinesterase inhibitors increased the effectiveness of antidotal treatment more markedly. In conclusion, memantine alone slightly decrease the acute toxicity of soman and failed to increase post-exposure antidotal treatment efficacy. On the other hand, the combination of memantine with donepezil significantly decrease the acute toxicity of soman and increased post-exposure effectiveness. Both drugs, when applied together, mitigate soman toxicity and boost post-exposure treatment.

Helenalin (HEL) is a sesquiterpene lactone used as an antiphlogistic in European and Chinese folk medicine. Its characteristic anti-inflammatory activity is mediated by direct alkylation of Cys38 within the DNA binding domain of NF-κB subunit p65/RelA. In addition, HEL is a broad-spectrum active compound, featuring an antitumor, antibacterial, and antiprotozoal activity. Recently, a new interest in the biological and pharmacological activities of HEL or its synthetic analogs has been observed (1). HEL has been found to undergo oxidative as well as reductive biotransformation in human liver. In addition, HEL acted as a mechanism-based inhibitor of human cytochrome P450 enzymes (2). Yet, information concerning its potential hepatotoxicity in human is limited. To address this issue, the hepatotoxic effect of HEL was studied in differentiated HepaRG cells that represent a human hepatocytes-like model. Firstly, the cytotoxic effect of HEL was determined using neutral red uptake (NRU) and MTT assay. After 72-hour incubation, the half-maximal inhibitory concentration (IC₅₀) of HEL was 13 µM and 11 µM using NRU and MTT assay, respectively. Secondly, the pro-oxidant activity of HEL was assessed using oxidant-sensitive probe. HEL was found to increase the formation of reactive oxygen species in a time- and concentration-dependent manner. Thirdly, a comprehensive proteomic analysis using isobaric labeling was performed to study the changes in hepatic proteome upon HEL treatment.

Despite widespread use of metal nanoparticles (NPs) in diagnostic and therapeutic applications for neurodegenerative disorders, there is a lack of reliable neurotoxicological studies on the exact molecular mechanisms of NPs action on neuronal cells (1). The aim of the present study was to evaluate neurotoxic potential of silver NPs (AgNPs) of different sizes (10, 100 nm) in rat pheochromocytoma cells PC12. The cells were cultured in the presence of AgNPs (0.1–10 µg/ml) for different time periods (3–72 h) depending on the used analysis. Cell viability was assessed by detection of mitochondrial activity based on the MTT reduction and membrane integrity measuring LDH release. Intracellular ROS levels were measured using fluorescent probe DCF-DA. Levels of IL-6 in the culture media were measured by ELISA kit. Differentiation of PC12 cell was induced by recombinant human β-NGF (100 ng/ml), average length of neurites was evaluated based on microscopic images on days 1, 3, 5 of treatment (2). The exposure of PC12 cells to AgNPs induced concentration- and time-dependent inhibition of cell viability and increase of LDH release. The value IC₅₀ ranged from 40 to 4 µg/ml depending on NP size and time of exposure. AgNPs (10 µg/ml) elevated ROS levels at all times monitored. Treatment of cells with AgNPs had no effect on IL-6 levels. Inhibition of neurite outgrowth induced by rhNGF in PC12 cells was observed after AgNPs treatment. In-depth evaluation of the neurotoxicity of NPs is crucial for designing safer nanosystems.

Graphene has been used increasingly in recent years in many fields for its unique physical, chemical and optical properties such as optical sensitivity, electronic and thermal conductivity. (1, 2). For this reason, it is important to verify its biocompatibility and the effect of graphene cell accumulation on metabolism, cell proliferation and motility. We tested a stable lung epithelial cell line A549 treated with chronic doses of nanographene platelets PlasmaChem GmbH (Berlin, Germany, product number PL-P-G750) in non-toxic concentrations of 5, 15 and 30 μg/ml graphene for 8 weeks. After this long-term cultivation the cells were analysed for metabolism, proliferation, cell cycle and motility. In this presentation, we focused on the study of cell migration, motility and microscopic imaging of the cytoskeleton and graphene accumulation in cells.
Our results demonstrate that accumulation of graphene platelets significantly decreased motility of cells. The motility was studied as chemotaxis in transwell assays and spontaneous cell motility (3). Chemotaxis migration was limited to 25% and spontaneous motility to 54% compared to control.
We consider the findings obtained in this pilot study important, since cellular motility is a key component of vital biological processes and survival of cells with accumulated graphene platelets could potentially impair regeneration of tissues.

Mycotoxic nephropathy of pigs is mainly associated with ochratoxin A exposure, so it is a chronic poisoning. Ochratoxin A is a mycotoxin produced by several species of fungi from the genera Aspergillus and Penicillium on a wide range of agricultural commodities used for feed production. The severity of the disease is determined by the amount of ochratoxin ingested and the duration of its action. Lower concentrations of ochratoxin A in the feed may not cause obvious clinical signs, but lead to findings during pathological-anatomical examination of the kidneys of slaughtered animals. In addition to nephrotoxicity, which can lead to kidney failure and animal death, it can also cause immunosuppression in pigs and increase susceptibility to secondary infections. In pigs, the highest concentrations of ochratoxin A are usually found in the blood, followed by the kidneys, liver, muscles and fat. The concentrations depend on the amount of ochratoxin A in the feed and, as it cumulates in the body due to enterohepatic cycle, also on the duration of feeding the contaminated feed. The aim of this paper is to evaluate the content of ochratoxin A in pig kidneys, which were examined in the Czech Republic during the regular monitoring of the safety and quality of food of animal origin during the years 2012-2021 and assess the risk of chronic poisoning by this mycotoxin in pigs in the Czech Republic.

Despite its unprecedented efficacy against some cancers, anthracycline cardiotoxicity represents the main limitation of its clinical use. Its’ mechanisms are elusive, but quite recently TOP2B was addressed as a possible target in cardiomyocytes (1). This study introduces the putative TOP2B selective inhibitor XK469 (2) as a potential cardioprotective agent in the management of anthracycline cardiotoxicity. Its’ potential was compared with the cardioprotection of the only approved cardioprotective drug dexrazoxane (DEX, ICRF-187) (3). Initially, the selectivity of XK469 and the character of TOP2 inhibition were re-examined in vitro using isolated TOP2A and TOP2B isoforms and the detection of DNA-TOP2 covalent complexes. Contrary to XK469 original presentation, we found it rather non-selectively targets both enzyme isoforms and does induce only very little covalent complexes. Consequently, in vitro pilot results suggested that XK469 was protective against daunorubicin (DAU)-induced cardiotoxicity in vitro in a slightly higher concentration than DEX. Nevertheless, in vivo XK469 was not cardioprotective in both acute and chronic settings. This can be partly explained by marked differences in the pharmacokinetics of the two agents. Modified incubation protocol in neonatal cardiomyocytes reflecting the longer half-life of XK469 revealed an increased trend in its toxicity. Thus, despite the promising characteristics of XK469, the cardioprotective ability of XK469 was not confirmed.

Current symptomatic pharmacotherapy for Alzheimer’s disease is primarily focused on acetylcholinesterase inhibitors and blocking of the NMDA receptor (N-methyl-D-aspartate). Tacrine, a molecule with both of the mechanisms of action, was withdrawn from the market in 2013 after 20 years of use due to the hepatotoxicity probably caused by its 7-hydroxytacrine metabolite. A rationale substitution of the tacrine molecule can potentially hinder the formation of a toxic species. The introduction of the methoxy or phenoxy group to position 7 led to 7-phenoxytacrine (7-PhO-THA) which we hypothesize to bypass the toxic metabolization via 7-OH tacrine and quinon methid. Furthermore, 7-PhO-THA was confirmed being of dual potency, i.e. potent and balanced inhibition of both AChE and NMDARs. We discovered that it selectively inhibits the GluN1/GluN2B subtype of NMDARs via an ifenprodil-binding site, in addition to its voltage-dependent inhibitory effect at both GluN1/GluN2A and GluN1/GluN2B subtypes of NMDARs. Furthermore, whereas NMDA-induced lesion of the dorsal hippocampus confirmed potent anti-excitotoxic and neuroprotective efficacy, behavioral observations showed that 7-PhO-THA manages to avoid side effects, symptoms of schizophrenia typical for NMDA antagonists.

Reproductive health is challenged by contemporary lifestyles. Health issues such as menstrual problems, cancers, infertility, and sexual dysfunction have been rising (1-3). For example, nowadays, more than 20% of couples experience infertility problems (1). Recently, a growing piece of evidence supports an association between chemical exposure and reproductive disorders (2,3). Traditionally, animal models have been used to elucidate human reproductive health development, disorders, and pathologies, including mechanistic insight. Reproductive toxicity testing of chemicals for regulatory purposes also relies on them. Therefore, it is vital to address the challenges concerning the use of animal-based models for reproductive toxicology and biomedicine and provide an overview of currently available 3Rs strategies. This talk will acknowledge the urgent need for animal-free models focusing on testes and ovaries, as their proper development and functionality are critical for a lifelong healthy reproductive system and due to their sensitivity to chemicals (2,3), with some specific examples from our current studies. Then, the adverse outcome pathway (AOP) concept will be applied to summarize currently available mechanistic knowledge covering key events at all levels of the biological organization and some potential early biomarkers of reproductive impairments will be proposed. Finally, chemically-induced disruption leading to reproductive disorders and dysfunctions will be discussed.

Antimicrobial drugs are chemical substances (of natural or synthetic origin) that suppress the growth of (or destroy) microorganisms (e. g. antibiotics act primarily against bacteria). Copper complexes ([Cu₂(pmdien)₂(H₂O)₂(μ-fu)](ClO₄)₂ – complex No. 5; [Cu₂(pmdien)₂(H₂O)₂(μ-dtdp)](ClO₄)₂ – complex No. 6), on which this study is focused, show antibacterial activity (1). As with every promising compound, these copper complexes were tested for their potential to inhibit activities of liver microsomal cytochromes P450 (CYP) in vitro. Porcine liver microsomes served as a model system. In the first step, possible effect of these copper complexes on enzyme activity of CYP2D (bufuralol 1´ hydroxylation) was determined. Copper complexes decreased enzyme activity of CYP2D to 1 % (IC_{50 complex No. 5} = 3.4 μmol.l^-1), 4 % (IC5_{0 complex No. 6} = 24.9 μmol.l^-1), respectively, at 50 μmol.l^-1 concentration of individual complexes in the reaction mixture. The Dixon plots and Lineweaver–Burk plots indicate most probably a partially noncompetitive inhibition in both cases. Verification of this interaction was confirmed with human liver microsomal CYP2D6. Enzyme activity of human CYP2D6 was affected too (decrease to 0 % of activity in both cases at 50 μmol.l^-1 concentration of individual complexes in the reaction mixture). IC_{50 complex No. 5} = 12.4 μmol.l^-1 and IC_{50 complex No. 6} = 6.3 μmol.l^-1 for human CYP2D6 were determined. Potential adverse drug interactions could occur in patients taking e. g. antidepressants (amitriptyline, paroxetine) or analgesics (codeine, tramadol) which are known to be metabolized by the CYP2D6 enzyme (2). However, determination of interaction of this copper complexes with another important liver microsomal drug metabolizing CYP should be studied in further experiments in vitro.

Anthracycline (ANT) anticancer drugs (e.g., doxorubicin and daunorubicin) are known for their cardiotoxic effects which are associated with degenerative changes in cardiomyocytes, programmed and non-programmed cell death, pathological remodeling of the myocardium and cardiomyopathy development. Historically, the predominant hypothesis of ANT cardiotoxicity development emphasized redox cycling of ANT molecule in the heart with or without participation of free iron resulting to ROS production and direct oxidative damage to the heart. However, different antioxidants and selective biocompatible iron chelators largely failed in clinically relevant experimental models and few clinical trials performed so far. Although many theories were proposed over decades none of them yielded a druggable target for effective cardioprotection against ANT cardiotoxicity. This has been changed in last decade when ANT cardiotoxicity has been proposed to be topoisomerase II beta (TOP2B)-dependent with subsequent induction of DNA damage with apoptosis and failure of mitochondrial biogenesis. Our network of laboratories in Hradec Kralove has brought several important experimental and translational insights into pharmacological cardioprotection against ANT cardiotoxicity which further develop this paradigm changing hypothesis. In this contribution, the complicated pathway to new TOP2B targeting cardioprotective drugs will be described together with possible future research directions.

Drug-metabolizing enzymes represent the main defense system against xenobiotics in all organisms. Long-term exposure to drugs can lead to changes in the expression of specific enzymes and to development of drug resistance. The previous studies have shown that increased anthelmintics inactivation via increased expression of certain drug-metabolizing enzymes belongs to a significant mechanism of drug resistance in Haemonchus contortus.
H. contortus, a gastrointestinal parasite of ruminants, has developed resistance to all used anthelmintics. As short-chain dehydrogenases/reductases (SDRs) catalyze the deactivation of carbonyl-containing anthelmintics (e.g. flubendazole, mebendazole), the increased expression of these SDRs could promote decreased susceptibility of H. contortus to these anthelmintics.
For these reasons, the present study was designed to follow the changes in the relative expression of selected SDR genes in adults of H. contortus exposed to different concentrations of flubendazole (FLU). FLU-mediated responses in adults from drug-susceptible strain (ISE) and drug-resistant strain (IRE) were compared. The adult nematodes were incubated ex vivo with or without FLU (0.01 µM, 0.1 µM, 1 µM, and 5 µM) in a culture medium for 4 h or 12 h. After incubation, total RNA was isolated and the expression level of individual SDRs were analyzed using qPCR. The results have shown the variability in the expression of individual SDR genes in H. contortus adults after contact with sub lethal doses of FLU.

Tacrine was the first drug to be approved for Alzheimer´s disease (AD) treatment, acting as a cholinesterase inhibitor. The neuropathological hallmarks of AD are amyloid-rich senile plaques, neurofibrillary tangles, and neuronal degeneration. Squaramides (SQ) are derivatives of squaric acid that are widely used in a variety fields of experties. Examples of small molecules with incorporated squaramide scaffold are Perzinfotel or Navarixin. Considering the relatively simple synthesis approach and other interesting properties (rigidity, aromatic character, H-bond formation) of squaramide motif, we developed 21 novel dimers amalgamating squaric acid with either tacrine, 6-chlorotacrine or 7-methoxytacrine representing various acetylcholinesterase inhibitors (AChEIs). All new derivatives were evaluated for their anti-cholinesterase activities, hepatotoxicity and screened to predict their ability to cross the blood-brain barrier. In ongoing study, we also demonstrate that a common butyrylcholinesterase variant confers resistance to tacrine, which can be overcome by using derivatives from tacrine-squaramaide family. These findings underscore the importance of genetic drug target variability for personalized medicine.

Non-alcoholic fatty liver disease (NAFLD) is a multifactorial, complex, and chronic liver disease with increasing worldwide prevalence. Metabolic syndrome and obesity are the main risk factors associated with NAFLD. High-fat diet causes an excessive fat accumulation in the liver referred to as simple steatosis, which can further progress to more severe stages of NAFLD including non-alcoholic steatohepatitis (NASH), fibrosis, cirrhosis, and hepatocellular carcinoma. As NAFLD has been reported to cause changes in the liver homeostasis and metabolism of xenobiotics (1), the objective of this study was to investigate NAFLD-induced changes in the expression and/or activity of selected phase I drug-metabolizing liver enzymes. For our study, liver samples from wild type mice fed with standard or high-fat diet were used. The mRNA and protein expression as well as the specific activity of several cytochrome P450 (CYP), carbonyl reductase (CBR), and aldo-keto reductase (AKR) enzymes were determined by using real-time quantitative PCR, western blotting, and spectrophotometric methods, respectively. High-fat diet caused significant induction of mRNA as well as protein expression and specific activity of CYP1A1/2. The mRNA expression of CYP3A11 and CYP3A13 (orthologs of human CYP3A4) was decreased, while its protein expression remained unchanged. Induction was observed also for AKR1C6 and AKR1C20 (orthologs of human AKR1C1) at mRNA and protein level.

Non-steroidal anti-inflammatory drugs (NSAIDs) are drugs with analgesic, antipyretic and anti-inflammatory effects. They are widely used in human and veterinary medicine. The most used NSAIDs include ibuprofen, diclofenac, naproxen. Due to their excessive use surface water pollution occurs. A total of 65 samples of surface water from the Elbe river basin were taken during August 2018 when the weather was constant without any significant fluctuations. The analysis was performed by means of liquid chromatography with tandem mass spectrometry (LC-MS/MS). A statistically significant negative correlation between the river flow rate in the monitored locations and the residue concentration was found for ibuprofen, naproxen, diclofenac. The most significant findings of the monitored drug residues were mostly determined in samples from small streams below larger urban settlements with a hospital or other health facilities. It was found that non-steroidal anti-inflammatory drugs can in increased concentration cause oxidative stress, damage to the liver, gills and kidneys and cause reproductive disorders in fish. They also negatively affect aquatic invertebrates, representatives of producers (phytoplankton) and consumers (zooplankton, zoobenthos) and destruents (bacteria).

The solution of high-molar-mass hyaluronan (HA) was subjected to oxidative degradation by ascorbate and cupric ions in the absence and presence of one of three ortho isomeric Mn(III) porphyrins or para isomeric Mn(III) porphyrin. Of methods, rotational viscometry was used, by which time-dependent changes in the dynamic viscosity of the HA solutions were measured. The ortho isomers MnTE-2-PyP⁵⁺, MnTnBuOE-2-PyP⁵⁺ and MnTnHex-2-PyP⁵⁺ interact with ascorbate whereby producing H₂O₂, which subsequently reacts with Cu(I) to initiate the hydroxyl radical-induced HA degradation. In contrast, when examining the para isomer, MnTM-4-PyP⁵⁺, HA degradation was inhibited, due to its inertness towards redox cycling with ascorbate. The results of rotational viscometry were confirmed by using electron paramagnetic resonance (EPR). These findings could provide further insight into the anticancer potential of redox-active ortho isomeric Mn(III) porphyrins.

The aryl hydrocarbon receptor (AhR) is well known for its detoxification/bioactivation role. However, it has been suggested to participate also in the control of inflammatory responses. Diesel exhaust particles (DEP) are known to induce inflammation in respiratory tract. Here, we used standard reference mixture of diesel exhaust particles (SRM1650b) in order to evaluate the functional role of the AhR (using wild-type and AhR-deficient A549 lung epithelial cells) in the control of inflammatory responses towards DEP-associated pollutants. We compared their effects with those of pro-inflammatory cytokines, such as IL-1β. We found that the induction of cyclooxygenase-2 (and secretion of prostaglandins), as well as expression of pro-inflammatory cytokines, were notably higher in the AhR-deficient A549 cells, when exposed to inflammatory cytokines. Both crude organic extract of SRM1650b and its polar fraction induced an increased inflammatory response in AhR KO cells, including the induction of COX-2, TNFα, CXCL8 and IL-6 mRNAs. Our further experiments have identified increased nuclear factor-κB (NF-κB) activity to contribute to the exacerbated inflammatory response in the AhR-deficient A549 cells. Thus, the interplay of AhR and NF-κB signaling may modulate responses of lung epithelial cells towards complex mixtures of organic pollutants, such as those associated with DEP.

Activation of the aryl hydrocarbon receptor (AhR) represents a key toxic event elicited by numerous persistent organic pollutants and other environmental contaminants, such as polycyclic aromatic hydrocarbons (PAHs). Ar present, unlike so-called priority PAHs, the AhR-mediated activities of numerous other PAHs, substituted (e.g. methylated or halogenated PAHs) remain poorly defined. Importantly, these contaminants are often neglected, when toxicities of complex mixtures of environmental polyaromatic pollutants are being evaluated. Our past work has revealed that such compounds can indeed contribute significantly to overall AhR-mediated toxicities of mixtures of organic pollutants derived from freshwater sediments, airborne particles or direct products of combustion engines, such as diesel exhaust particles. This presentation is intended to provide an overview of principle groups of  polycyclic aromatic contaminants, their AhR-mediated activities and strategies necessary to fill the gaps in our current knowledge of their toxicity, including their potential roles in endocrine and metabolic disruption.

Haemonchus contortus, a widely distributed parasitic nematode of ruminants, has become resistant to most anthelmintic classes. Therefore, there has been a major demand for new compounds effective against H. contortus and related nematodes. Previous phenotypic screening and further testing have revealed one compound, substituted benzamide BLK127, active against H. contortus larvae and adults (1,2). Based on these results, 13 derivatives of this compound were designed and synthetized. The aim of the present study was to assess the toxicity of these derivatives in H. contortus eggs and adults and their potential hepatotoxicity in sheep in vitro. Isolated eggs and adults from sheep experimentally infective with H. contortus of three strains (one drug-sensitive and two drug-resistant) and precise cut ovine liver slices were used. Only one compound significantly inhibited the egg hatching. On the other hand, four compounds significantly decreased the viability of H. contortus adults at micromolar concentrations. Some of them were effective not only in the drug-sensitive strain but also in the drug-resistant strains. None of the derivatives exhibited a hepatotoxic effect, even at the highest concentration tested (100 µM). Based on these findings, the two most potent compounds were selected for further testing in vivo.

Since cancer is one of the most common medical causes of death worldwide, its prevention remains the most promising strategy for reducing its incidence and mortality. The study of the potential protective effects of natural compounds, that could be used in health protection, is therefore very important. Plants represent a source of secondary metabolites possessing such properties and curcumin isolated from plant Curcuma longa L. with many beneficial biomedical effects is one of them.
The objectives of our study were (I) to investigate the potential DNA-damaging/DNA-protective effects of curcumin using DNA topology assay; (II) to determine the possible mechanism of its protective activity using cell-free methods and (III) to evaluate its potential anti/genotoxic effect by comet assay on human lymphocytes.
Curcumin displayed DNA-protective effects against ROS induced by ferrous ions on plasmid DNA that increased in a concentration-dependent manner. Higher doses showed significant reducing power and DPPH radicals scavenging activity. Curcumin did not exhibit any chelating activity. Moreover, genotoxic effects of curcumin were observed in human lymphocytes. On the other hand, using the same concentration range, curcumin exhibited ability to protect human cells against H₂O₂ mostly in the higher concentrations. Our results represent basis in examining the effects of curcumin on biological systems, and therefore further research is needed to explain its dual effects.

Since the discovery of penicillin, antibiotic use and misuse lead to the development of bacterial strains resistant to not only antibiotics, but to common disinfecting agents as well. Monomeric quaternary ammonium compounds (QACs) – common disinfecting agents – are already rendered inefficient due to the acquired resistance. The development of novel agents is now a major component in the fight against the spread of diseases. New designs should enhance the advantages of existing substances (high efficacy, low price), while embracing biodegradability, improved solubility and lower cytotoxicity.
In recent years new, yet related class of disinfectants is on the rice. Diammonium quaternary salts – or so-called ‘Gemini’ QACs, which contain in contrast to their monomeric cousin two polar heads. In total, the molecule contains two positively charged nitrogen atoms and two alkyl chains. From this group; octenidine emerged. Despite its promising results against a broad range of resistant microorganisms, it has many shortcomings: low solubility and mild cytotoxicity. These properties need to be overcome.
In this report, we prepared novel series of octenidine derivates, which have been tested against common nosocomial bacterial strains and against biofilms, which possess unique properties in comparison to single cells. The antimicrobial activity has been evaluated against Gram-positive and Gram-negative bacterial strains. Furthermore, the substances have been tested for cytotoxicity on a mammalian cell line to evaluate one of the key safety parameters.

In this study, it has been investigated the oxidative status and morphological alterations in the brain of Wistar rats induced by repeated application of low doses of selected acetylcholinesterase reactivators - asoxime, obidoxime, K027, K048, K074, and K075. Each oxime (0.1 of LD50/kg im) was given 2 times per week for 4 weeks. The animals’ whole-body, organ weight, oxidative status, as well as microscopic examination of the brain, were done on day 35 of the study. Markers of oxidative stress, lipid peroxidation (malondialdehyde (MDA) and advanced oxidation protein products (AOPP)), as well as the activity of antioxidant enzymes (catalase (CAT) and superoxide dismutase (SOD)), were measured in the brain homogenates (1,2). Brain alterations were quantified by semiquantitative grading scales – brain damage score (BDS) (3). Oxidative stress parameters MDA and AOPP were significantly elevated in the K075-treated group compared to the control group (p < 0.001). SOD activity was significantly higher in the K075-treated group (p < 0.001), without differences in CAT activity. In the same groups of rats, brain injuries were significantly more severe than those observed in animals receiving only asoxime or K027 (p < 0.001). Our results can help to predict likely adverse systemic toxic effects, and target organ systems, which are crucial for establishing risk categories, as well as in dose selection of K-oximes as drug candidates. (MFVMA01/23-25).

Anthracyclines (ANT) are very effective anticancer drugs, but they are feared for their cardiotoxicity. The only drug approved to counteract this severe side effect in clinical settings is a bisdioxopiperazine dexrazoxane (DEX). However, another bisdioxopiperazine agent ICRF-193 was recently shown to be more effective than DEX in vitro. Its poor water-solubility was solved by design of its prodrugs, from which GK-667 was selected for in vivo studies. The aim of this study was to examine cardioprotective effects of GK-667 on a rabbit model of chronic ANT cardiotoxicity in vivo. Cardiotoxicity was induced with daunorubicin (DAU; 3 mg/kg, i.v., weekly, 10 weeks) and GK-667 (1 or 5 mg/kg, i.v.) was administered 30 min before each DAU dose. DAU-induced mortality, blood congestion and left ventricular (LV) dysfunction were completely prevented with GK-667. Dose-dependency of the effects was visible on molecular markers of cardiac damage and dysfunction. GK-667 prevented p53-mediated DNA damage response induced in LV by the chronic, as well as acute DAU administration (single dose). This was attributable to topoisomerase IIβ inhibition provided by active metabolite of GK-667 (ICRF-193). In addition, the plasma pharmacokinetics of DAU and its main metabolite was not altered by GK-667 administration in vivo and it also did not reduce anticancer effect of DAU in vitro. Therefore, GK-667 is a promising drug candidate for further research and development.

Anthracycline (ANT) anticancer drugs (e.g., doxorubicin and daunorubicin) are known for their cardiotoxic effects which are associated with degenerative changes in cardiomyocytes, programmed and non-programmed cell death, pathological remodeling of the myocardium and cardiomyopathy development. Historically, the predominant hypothesis of ANT cardiotoxicity development emphasized redox cycling of ANT molecule in the heart with or without participation of free iron resulting to ROS production and direct oxidative damage to the heart. However, different antioxidants and selective biocompatible iron chelators largely failed in clinically relevant experimental models and few clinical trials performed so far. Although many theories were proposed over decades none of them yielded a druggable target for effective cardioprotection against ANT cardiotoxicity. This has been changed in last decade when ANT cardiotoxicity has been proposed to be topoisomerase II beta (TOP2B)-dependent with subsequent induction of DNA damage with apoptosis and failure of mitochondrial biogenesis. Our network of laboratories in Hradec Kralove has brought several important experimental and translational insights into pharmacological cardioprotection against ANT cardiotoxicity which further develop this paradigm changing hypothesis. In this contribution, the complicated pathway to new TOP2B targeting cardioprotective drugs will be described together with possible future research directions.