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Journal Article 2016; 37(Suppl 1): 118-122 PubMed PMID: 28263539 Citation  Keywords:  Acetylcholinesterase:drug effects, Celecoxib:pharmacology, Cholinesterase Inhibitors:pharmacology, Cyclooxygenase 2 Inhibitors:pharmacology, Dithionitrobenzoic Acid, Humans, Molecular Docking Simulation,.   

OBJECTIVES: Celecoxib is a nonsteroidal anti-inflammatory drug inhibiting enzyme cyclooxygenase-2 (COX-2). The drug was introduced in 1990s. In the work presented here, affinity of celecoxib to enzyme acetylcholinesterase (AChE) is inferred.

METHODS: Inhibition of human AChE by celecoxib was tested using standard spectrophotometric Ellman´s method and extrapolation of experimental data by Dixon plot. Interaction between AChE and celecoxib was also predicted by molecular docking using Swiss dock software.

RESULTS: A non-competitive mechanism of inhibition was revealed and equilibrium inhibitory constant equal to 313±40 µmol/l was determined. Comparing to AChE, celecoxib was not proved as an inhibitor of enzyme butyrylcholinesterase (BChE). The lowest ΔG was equal to -7.78 kcal/mol. In this case, celecoxib stacked sulfonamide moiety between TYR 337 and TYR 341 of alfa anionic subsite of active site. Cation-Π interactions appears to be responsible for the inhibition.

CONCLUSIONS: Though the here revealed and characterized inhibition has lower effect in real conditions than inhibition of COX-2, the inhibitory effect would be utilized in the next research and development of new AChE inhibitors.

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Journal Article 2016; 37(Suppl 1): 111-117 PubMed PMID: 28263538 Citation  Keywords:  Animals, Disease Models, Animal, Evoked Potentials, Hippocampus:drug effects, Lysosomes:drug effects, Male, Neurodegenerative Diseases:chemically induced, Neurons:physiology, Rats, Rats, Wistar, Trimethyltin Compounds:administration & dosage,.   

OBJECTIVES: Extensive effort has been made to identify early markers of neurodegeneration as late stages have no chance of treatment. Recently, many experimental models have been used to study hallmarks of neuronal injury. One of them is the model of trimethyltin (TMT)-induced damage associated with cognitive decline, thus called a model of Alzheimer-like disease.

OBJECTIVE AND METHODS: Our aim was to study neuronal transmission in hippocampal slices of male Wistar rats affected with a single dose of TMT (7.5 mg/kg, i.p.) during the first three weeks of its action. The monitored time periods after TMT administration were days 1-3; 8-10 and 15-17. At the same time periods, right hippocampi were collected for determination of changes in specific activities of two lysosomal enzymes. Electrophysiological measurements were based on stimulation of Schäffer collaterals and registration of evoked responses in the stratum pyramidale and the stratum radiatum at the CA3-CA1 synapse. Specific activities of N-acetyl-β-D-glucosaminidase (NAGA) and cathepsin D (Cat D) were determined spectrophotometrically.

RESULTS: During three weeks after i.p. TMT administration to rats, we found a time-dependent reduction of postsynaptic neuronal firing, expressed by diminished population spike (PoS) amplitude recorded in the stratum pyramidale accompanied with marked increase in specific activity of NAGA to respective 111%, 163% and 252% in the 1st, 2nd and 3rd week compared to unaffected rats. In the stratum radiatum, reduction of the slope of excitatory postsynaptic potential was not time-dependent but almost constantly reduced from the 1st to 3rd week after TMT administration (55-60%) compared to control rats. Specific activity of lysosomal enzyme Cat D was significantly increased in the 3rd week after TMT administration.

CONCLUSION: This work demonstrates a time-dependent reduction of somatic response in the hippocampus of TMT affected rats during the first three weeks. This reduction of neuronal firing was later accompanied with increase of specific activity of NAGA and Cat D, supporting evidence that lysosomal dysfunction may be one of the primary contributors to TMT-induced neurodegeneration.

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Journal Article 2016; 37(Suppl 1): 95-102 PubMed PMID: 28263536 Citation  Keywords:  Antineoplastic Agents, Phytogenic:metabolism, Cytochrome P-450 CYP3A:metabolism, DNA Adducts:metabolism, Ellipticines:metabolism, Humans, Liposomes, Microsomes,.   

OBJECTIVES: Ellipticine is an anticancer agent that functions through multiple mechanisms participating in cell cycle arrest and initiation of apoptosis. This drug forms covalent DNA adducts after its enzymatic activation with cytochrome P450 (CYP), which is one of the most important ellipticine DNA-damaging mechanisms of its cytotoxic effects. The improvements of cancer treatment are the major challenge in oncology research. Nanotransporters (nanoparticles) are promising approaches to target tumor cells, frequently leading to improve drug therapeutic index. Ellipticine has already been prepared in nanoparticle forms. However, since its anticancer efficiency depends on the CYP3A4-mediated metabolism in cancer cells, the aim of our research is to develop nanoparticles containing this enzyme that can be transported to tumor cells, thereby potentiating ellipticine cytotoxicity.

METHODS: The CYP3A4 enzyme encapsulated into two nanoparticle forms, liposomes and microsomes, was tested to activate ellipticine to its reactive species forming covalent DNA adducts. Ellipticine-derived DNA adducts were determined by the 32P-postlabeling method.

RESULTS: The CYP3A4 enzyme both in the liposome and microsome nanoparticle forms was efficient to activate ellipticine to species forming DNA adducts. Two DNA adducts, which are formed from ellipticine metabolites 12-hydroxy- and 13-hydroxyellipticine generated by its oxidation by CYP3A4, were formed by both CYP3A4 nanoparticle systems. A higher effectiveness of CYP3A4 in microsomal than in liposomal nanoparticles to form ellipticine-DNA adducts was found.

CONCLUSION: Further testing in a suitable cancer cell model is encouraged to investigate whether the DNA-damaging effects of ellipticine after its activation by CYP3A4 nanoparticle forms are appropriate for active targeting of this enzyme to specific cancer cells.

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Journal Article 2016; 37(Suppl 1): 84-94 PubMed PMID: 28263535 Citation  Keywords:  Animals, Benzo(a)pyrene:pharmacology, Cytochrome P-450 Enzyme System:metabolism, Endocrine Disruptors:pharmacology, Estradiol:pharmacology, Estrogens:pharmacology, Ethinyl Estradiol:pharmacology, Male, Microsomes, Liver:metabolism, Rats, Rats, Wistar,.   

OBJECTIVES: The term "endocrine disruptor" (ED) is used for compounds that mimic or antagonize the effects of endogenous hormones. Synthetic estrogen 17α-ethinylestradiol (EE2) and a human carcinogen benzo[a]pyrene (BaP) are assigned as exogenous endocrine disruptors and an estrogenic hormone estradiol is a natural endogenous disruptor. Here, the potency of these three disruptors administered to rats individually and in combination to induce expression of cytochrome P450 (CYP) enzymes involved in their own metabolism (CYP1A1, 2C and 3A) in vivo was investigated.

METHODS: Changes in CYP protein expression after exposure of rats to BaP, EE2 or estradiol were analyzed by Western blotting. Using the HPLC method, CYP1A1, 2C and 3A specific activities in hepatic microsomes isolated from exposed rats were analyzed.

RESULTS: Whereas exposure to BaP induces expression of CYP1A1 protein and its marker activity (Sudan I oxidation) in liver, kidney and lung of rats, no significant induction of this CYP and its enzyme activity was produced by EE2 and estradiol. Treatment of BaP in combination with EE2 and/or estradiol decreased the BaP-mediated CYP1A1 induction in liver of exposed rats. BaP also induces CYP2C11 protein in rat liver and kidney, but does not increase its enzyme activity measured as testosterone 16α-hydroxylation. The enzyme activity of another enzyme of the 2C subfamily, CYP2C6, diclofenac 4'-hydroxylation, is even decreased by BaP. The CYP2C11 protein expression and/or its activity are also increased in liver of rats treated with EE2 and estradiol, but its expression is significantly decreased in lung. The CYP2C6 activity is also elevated by treatment of rats with EE2 and estradiol administered individually as well as in their combination. Whereas only a slight increase in CYP3A protein expression was found by BaP in rat liver, its enzyme activity, testosterone 6β-hydroxyalation, increased significantly in this organ. In contrast, no effect or even a decrease in CYP3A expression and its enzyme activity was produced by EE2 and estradiol in rats exposed to these compounds....

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Journal Article 2016; 37(Suppl 1): 78-83 PubMed PMID: 28263534 Citation  Keywords:  Analgesics:toxicity, Animals, Cytotoxins:toxicity, Deer, Heart:drug effects, Ketamine:analogs & derivatives, Kidney:cytology, Liver:cytology, Toxicity Tests:methods, Xylazine:analogs & derivatives,.   

OBJECTIVES: Chemical restraint of wild animals is practiced to accomplish intended procedures such as capture, clinical examination, collection of diagnostic samples, treatment and/or transport. Extra-label use of animal medicinal drugs is often necessary in wildlife because most approved therapeutics do not list wild species on the labelling. Here, we used cellular in vitro models, a cutting-edge tool of biomedical research, to examine cytotoxicity of anaesthetic agents in fallow deer and extrapolate these data for anaesthetic risks in wildlife.

METHODS: We examined the cytotoxic effects of ketamine, xylazine, and ketamine-xylazine, i.e. the Hellabrunn mixture, on liver-, heart- and kidney-derived cell cultures prepared from a fallow deer (Dama dama) specimen. In line with preliminary studies we exposed cells to 10 µM, 50 µM, 100 µM, 1 mM, and 10 mM ketamine or xylazine. The combination of ketamine-xylazine was dosed at 0.025+0.02 mg/ml, 0.05+0.04 mg/ml, 0.75+0.06 mg/ml, 0.1+0.08 mg/ml, and 0.125+0.1 mg/ml per one well containing 10 000 cells. The quantification of cytotoxicity was based on lactate dehydrogenase activity released from damaged cells.

RESULTS: Liver-derived cells show higher sensitivity to the cytotoxic effects of both ketamine and xylazine administered as single drugs when compared with cells cultured from the heart and kidney. The Hellabrunn mixture induced significantly higher cytotoxicity for kidney-derived cells ranging from 16.78% to 35.6%. Single and combined exposures to ketamine and xylazine resulted only in high-dose cytotoxicity in the heart-derived cells.

CONCLUSIONS: Our results indicate that immobilization drugs significantly differ in their cytotoxic effects on cells derived from various organs of the fallow deer.

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Journal Article 2016; 37(Suppl 1): 67-77 PubMed PMID: 28263533 Citation  Keywords:  Animals, Carps, Disinfectants:toxicity, Formaldehyde:toxicity, Toxicity Tests:methods,.   

OBJECTIVES: The aim of this study is to assess the effects of a treatment bath in a formalin solution on fish, focusing on haematological, biochemical and histopathological profiles.

METHODS: A total of 96 common carps (Cyprinus carpio) were randomly assigned to eight groups. Four experimental groups were placed in the test solution for 60 minutes. The concentration of the formalin bath was 0.17 ml/l (38% formaldehyde), with the water temperature of 20°C. The effects of the bath were monitored immediately (E0) and 24 hours (E24h), 48 hours (E48h) and 10 days (E10d) after the bath. There was a control group (C0, C24h, C48h, C10d) for each of the experimental groups.

RESULTS: Histopathological indices were strongly affected. Extensive changes were found on gill and skin immediately and also after 24 h, 48 h and 10 d of the formalin treatment bath. A plurality of mucinous elements was observed on the skin. Moreover, structural devastation of lamellas and numerous mucinous cells were observed on the gill. Changes were also found in haematological and biochemical indices.

CONCLUSIONS: It can be concluded that after applying the treatment bath in the formalin solution, the monitored profiles were affected immediately after the bath and also after 10 days (histopathological changes of skin and gill). Such significant impact of therapeutic formalin bath should be considered in treatment of fish.

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Journal Article 2016; 37(Suppl 1): 60-66 PubMed PMID: 28263532 Citation  Keywords:  Animals, Carps:metabolism, Insecticides:toxicity, Pyrethrins:toxicity, Toxicity Tests:methods,.   

OBJECTIVES: The present study aimed to assess the acute effect of cypermethrin on common carp (Cyprinus carpio). Evaluation of toxicity effects of cypermethrin on carp was performed on based assessment of hematological profile, antioxidant and oxidative biomarkers and histopathology.

METHODS: For testing of toxicity was used the commercial product Cyperkill 25 EC - CY, which contains 250 g.L-1 of cypermethrin. Carp were exposed to two concentrations (1CY - 4.57 µg.L-1 and 2CY - 45.7 µg.L-1) of CY and a control for 96 hours.

RESULTS: Significant reduction (p<0.05) in leukocyte count were in fish exposed to concentration 45.7 µg.L-1 (2CY). The both exposure of CY lead to significant differences (p<0.01) in antioxidants biomarkers (superoxide dismutase, catalase, glutathione reductase, reduced glutathione and glutathione S-transferase) and in lipid peroxidation in carp tissues. Many of these changes were observed in liver, gills, muscle, intestine, brain and kidney. Additionally, exposure to CY caused many histological changes in gills, liver and caudal kidney.

CONCLUSION: Exposure to CY caused hematological, biochemical and histopathological changes in carp. This study provides and complements other important results for evaluating the toxicity effect of pyrethroids on fish.

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Journal Article 2016; 37(Suppl 1): 53-59 PubMed PMID: 28263531 Citation  Keywords:  Animals, Antioxidants:metabolism, Astacoidea:metabolism, Biomarkers, Insecticides:toxicity, Oxidative Stress, Pyrethrins:toxicity, Toxicity Tests:methods,.   

OBJECTIVES: Pyrethroid insecticides are known to be highly toxic to non-target aquatic organisms. Toxic effects of different types of pyrethroids to fish has been well described but there is a dearth of data on their effects to aquatic crustaceans. The aim of this study was to assess the effects of a commercial product Cyperkill 25 EC (25% of cypermethrin as an active substance) on oxidative stress and antioxidants activity in marbled crayfish.

DESIGN: The juvenile crayfish were exposed to two concentrations of Cyperkill 25 EC: 0.02 µg.L-1 (10% 96hLC50) and 0.05 µg.L-1 (30% 96hLC50) for 72 hours. Activity of superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), glutathione-S-transpherase (GST) and reduced glutathione (GSH), as well as levels of thiobarbituric acid reactive substances (TBARS) were assessed in the whole-body homogenate.

RESULTS: Cyperkill 25 EC in both tested concentrations caused significant (p<0.01) decreased in levels of TBARS. Changes in the antioxidants activity were observed in every examined parameters except for the catalase. Cyperkill 25 EC in concentration 0.05 µg.L-1 caused significant increase (p<0.05) of SOD activity and significant decrease of GST (p<0.01) and GSH (p<0.05) activity. The activity of GR was significantly (p<0.01) decreased in both treated groups.

CONCLUSIONS: This study suggests that Cyperkill 25 EC causes oxidative stress and also disruption of antioxidant systems in crayfish. Expanding to our previous knowledge, obtained results describe effects of cypermethrin also to non-target aquatic invertebrates and suggest a need of deeper understanding of mechanisms involved in this process.

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