Synthesis of novel indole-thiazolidinone hybrid structures as promising scaffold with anticancer potential.

A series of novel indole-azolidinone hybrids has been synthesized via Knoevenagel reaction of 5-fluoro-3-formyl-1H-indole-2-carboxylic acid methyl ester and some azolidinones differing in heteroatoms in positions 1, 2 and 4. Their anticancer activity in vitro was screened towards MCF-7 (breast cancer), HCT116 (colon cancer), HepG2 (hepatoma), HeLa (cervical cancer), A549 (lung cancer), WM793 (melanoma) and THP-1 (leukemia) cell lines, and a highly active 5-fluoro-3-(4-oxo-2-thioxothiazolidin-5-ylidenemethyl)-1H-indole-2-carboxylic acid methyl ester (3a) was identified and subjected to in-depth investigation of cytotoxicity mechanisms. This compound was found to possess the highest cytotoxic action towards tumor cells comparing with the action of other derivatives (1, 3b, 3c, 3d, 3e). Compound 3a exhibited toxicity toward MCF-7, HCT116, and A549, HepG2 cancer cells, while the non-malignant cells (human keratinocytes of HaCaT line and murine embryonic fibroblasts of Balb/c 3T3 line) possessed moderate sensitivity to it. The compound 3a induced apoptosis in studied tumor cells via caspase 3-, PARP1-, and Bax-dependent mechanisms; however, it did not affect the G1/S transition in HepG2 cells. The compound 3a impaired nuclear DNA in HepG2, HCT116, and MCF-7 cells without intercalating this biomolecule, but much less DNA damage events were induced by 3a in normal Balb/c 3T3 fibroblasts compared with HepG2 carcinoma cells. Thus, 5-fluoro-3-(4-oxo-2-thioxothiazolidin-5-ylidenemethyl)-1H-indole-2-carboxylic acid methyl ester 3a was shown to trigger DNA damage and induce apoptosis of human tumor cells and it might be considered as an anticancer agent perspective for in-depth studies.

Multi-Class Procedure for Analysis of 50 Antibacterial Compounds in Eggshells Using Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometry.

In this work, for the first time, Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometry (UHPLC-MS/MS) method was developed for qualitative and quantitative analysis of veterinary antibiotics (cephalosporins, diaminopyrimidines, fluoro(quinolones), lincosamides, macrolides, penicillins, pleuromutilins, sulfonamides, tetracyclines, and sulfones) in hen eggshells. The sample preparation method is based on a liquid-liquid extraction with a mixture of metaphosphoric acid, ascorbic acid, EDTA disodium salt dihydrate, and acetonitrile. The chromatographic separation was performed on Luna® Omega Polar C18 10 column in gradient elution mode and quantitated in an 8 min run. Validation such as linearity, selectivity, precision, recovery, matrix effect, limit of quantification (LOQ), and limit of detection (LOD) was found to be within the acceptance criteria of the validation guidelines of the Commission Decision 2002/657/EC and EUR 28099 EN. Average recoveries ranged from 81-120%. The calculated LOQ values ranged from 1 to 10 µg/kg, the LOD values ranged from 0.3 to 4.0 µg/kg, depending on analyte. The developed method has been successfully applied to the determination of antibacterial compounds in hen eggshell samples obtained from different sources. The results revealed that enrofloxacin, lincomycin, doxycycline, and oxytetracycline were detected in hen eggshell samples.

Synthesis, Spectroscopy, Light Stability, Single-Crystal Analysis, and In Vitro Cytotoxic Activity on HepG2 Liver Cancer of Two Novel Silver(I) Complexes of Miconazole.

Two novel silver(I) complexes of the biologically active ligand miconazole in the form of Ag(MCZ)2X (MCZ = 1-[2-(2,4-dichlorobenzyloxy)-2-(2,4-dichlorophenyl)ethyl]-1H-imidazole]; X = NO3- (1), ClO4- (2)) were synthesized and fully characterized. The complexes were obtained by reactions of Ag(I) salts with miconazole (MCZ). Silver(I) complexes were characterized by elemental analysis, 1H-NMR and infrared (IR) spectroscopy, electrospray ionization (ESI)-MS spectrometry, and X-ray-crystallography. This work also presents a cytotoxicity study of the silver(I) complexes of miconazole and appropriate silver(I) salts using Balb/c 3T3 and HepG2 cell lines. The cytotoxicity of the compounds was assessed based on four biochemical endpoints: lysosomal activity (neutral red uptake (NRU) assay), mitochondrial activity (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay), total protein content (TPC assay), and cellular membrane integrity (lactate dehydrogenase (LDH) assay). The cancer HepG2 cells were more sensitive to the complexes tested, and the most affected endpoint was cellular membrane damage compared to Balb/c 3T3 fibroblasts. Moreover, study complexes inhibited the growth of cancer cells at submicromolecular concentrations (0.26-0.47 µM) lower than that required for the anticancer agent, cisplatin, in MTT, NRU, and TPC assays. Both complexes were characterized by higher toxicity to human cancer cells (HepG2) than silver(I) salts and the free ligand. Combination of Ag(I) salts with miconazole is associated with the marked improvement of cytotoxic activities that can be considered as the significant point in the construction of a new generation of antineoplastic agents.

Primary Human Hepatocytes, but Not HepG2 or Balb/c 3T3 Cells, Efficiently Metabolize Salinomycin and Are Resistant to Its Cytotoxicity.

Salinomycin is a polyether antibiotic showing anticancer activity. There are many reports of its toxicity to animals but little is known about the potential adverse effects in humans. The action of the drug may be connected to its metabolism. That is why we investigated the cytotoxicity of salinomycin and pathways of its biotransformation using human primary hepatocytes, human hepatoma cells (HepG2), and the mouse fibroblast cell line (Balb/c 3T3). The cytotoxicity of salinomycin was time-dependent, concentration-dependent, and cell-dependent with primary hepatocytes being the most resistant. Among the studied models, primary hepatocytes were the only ones to efficiently metabolize salinomycin but even they were saturated at higher concentrations. The main route of biotransformation was monooxygenation leading to the formation of monohydroxysalinomycin, dihydroxysalinomycin, and trihydroxysalinomycin. Tiamulin, which is a known inhibitor of CYP450 izoenzymes, synergistically induced cytotoxicity of salinomycin in all cell types, including non-metabolising fibroblasts. Therefore, the pharmacokinetic interaction cannot fully explain tiamulin impact on salinomycin toxicity.

Determination of Polypeptide Antibiotic Residues in Food of Animal Origin by Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometry.

A novel UHPLC-MS/MS method for the determination of polypeptide antibiotic residues in animal muscle, milk, and eggs was developed and validated. Bacitracin A, colistin A, colistin B, polymyxin B1, and polymyxin B2 were extracted from the samples with a mixture of acetonitrile/water/ammonia solution 25%, 80/10/10 (v/v/v), and put through further evaporation, reconstitution, and filtration steps. The chromatographic separation was performed on a C18 column in gradient elution mode. Mass spectral acquisitions were performed in selective multiple reaction monitoring mode by a triple quadrupole mass spectrometer. The method was validated according to the criteria of Commission Decision 2002/657/EC. The method quantifies polypeptides in a linear range from 10 to 1000 µg kg-1, where the lowest concentration on the calibration curve refers to the limit of quantification (LOQ). The recoveries ranged from 70 to 99%, the repeatability was below 13%, and within-laboratory reproducibility was lower than 15%. The decision limit (CCα) and detection capability (CCß) values were calculated, and ruggedness and stability studies were performed, to fulfill the criteria for confirmatory methods. Moreover, the developed method may also be used for screening purposes by its labor efficiency.

Multiple mycotoxins analysis in animal feed with LC-MS/MS: Comparison of extract dilution and immunoaffinity clean-up.

The aim of this study was a performance comparison of two clean-up procedures (dilutions versus immunoaffinity columns) in the simultaneous determination of eight mycotoxins (aflatoxin B1, deoxynivalenol, fumonisin B1 & B2, ochratoxin A, toxin T-2 & HT-2 and zearalenone) in the animal feed. After extraction the analytes were separated on a Kinetex Biphenyl column with a gradient elution using methanol/0.01 M ammonium acetate as a mobile phase and analyzed with the LC-MS/MS technique. Both of the procedures were validated by analysis of a series of spiked feed samples (n = 6) at three different concentration levels. Better signal to noise ratios were observed for immunoaffinity clean-up. The recoveries of analyses were in the range 88-110% for the dilution procedure and 78-120% for the immunoaffinity clean-up. The dilution procedure was more precise (coefficient of variation of the within-laboratory reproducibility for it was 7.8-22.4% in comparison to 12-35.5% for the immunoaffinity clean-up. The results show that both procedures fulfilled the requirements for mycotoxin analysis and can be used successfully in multi-analyte determination. Although the dilution procedure shows better precision and trueness, the immunoaffinity clean-up procedure can have advantages in more complex feed samples thanks to lower matrix effect and limits of detections.

Fast analysis of 19 anabolic steroids in bovine tissues by high performance liquid chromatography with tandem mass spectrometry.

For the detection of 19 steroid hormones in bovine muscle, a fast and sensitive liquid chromatography with electrospray ionization tandem mass spectrometry method was developed using both positive and negative ionization mode. Chromatographic separation on Poroshell 120-EC C18 column was achieved in less than 10 min using isocratic elution of mobile phase of acetonitrile/methanol/water. The compounds were extracted from muscle tissue using ethyl acetate and quick, easy, cheap, effective, rugged, and safe technique. The purification of the obtained extract was performed by dispersive solid-phase extraction with sorbents C18, primary secondary amine and magnesium sulphate. The method was validated in accordance with the Commission Decision 2002/657/EC. For all steroids tested good recoveries were obtained (from 51.2 to 121.4%) in the concentration range from decision limits until 5 µg/kg. The values of decision limits and the detection capabilities for individual compounds were in the range 0.10-0.48 and 0.17-0.95 µg/kg, respectively. The method was characterized by satisfactory linearity for most compounds (correlation coefficients   > 0.99) and the reproducibility was lower than 35%. The elaborated procedure has met the criteria for confirmatory methods and is currently used in the official control of hormones.

The Usefulness of MS3 to Confirm Poisoning on the Example of Dog Poisoning with Strychnine.

Strychnine is an alkaloid with strong toxic properties. Poisoning results in muscular contractions and death through asphyxiation. Intentional or accidental poisonings with strychnine occur mainly in small animals, especially dogs and occasionally cats. Strychnine can be detected in the liver or stomach contents. Unfortunately, the determination of strychnine in these matrices, especially in postmortem examination, is subject to a significant matrix effect that makes it difficult to confirm the presence of the substance being determined. Therefore, we developed a new liquid chromatography method combined with mass spectrometry. One-gram homogenized samples were extracted and partitioned after adding acetonitrile and 5-mol solution of ammonium acetate. After extraction, the samples were analyzed using high-pressure liquid chromatography-MS/MS/MS. The results of validation fulfil the requirement of the confirmatory criteria according to SANTE/11945/2015 regarding apparent recoveries (98.97% to 104.0%), repeatability (2.9%-4.1%), and within-laboratory reproducibility (3.3%-4.6%). The method can be successfully applied to confirm strychnine poisoning cases.

QuEChERS and HPLC-MS/MS Combination for the Determination of Chloramphenicol in Twenty Two Different Matrices.

A simple method for the determination of chloramphenicol in 22 matrices was prepared based on the QuEChERS and HPLC-MS/MS combination. Following a hydrolysis step, the homogenized samples were extracted and partitioned after adding sodium chloride with acetonitrile. Chloramphenicol was analysed by HPLC-MS/MS in negative electrospray mode by monitoring the daughter ions m/z: 321→194 and 321→152. The limit of decision (CCα) was calculated at the range of 0.10 µg kg-1 to 0.15 µg kg-1 and detection capability (CCß) from 0.12 µg kg-1 to 0.18 µg kg-1. Validation results showed that this method is suitable for the determination and confirmation of chloramphenicol in various matrices.

Silver(I) Complexes of the Pharmaceutical Agents Metronidazole and 4-Hydroxymethylpyridine: Comparison of Cytotoxic Profile for Potential Clinical Application.

In previous papers, we have reported on the high antifungal and significant antibacterial activity against Gram-positive and Gram-negative bacteria of the water-soluble silver(I) complexes of metronidazole and derivatives of pyridine compared to silver nitrate. In the present study, the cytotoxic activity of the silver(I) complexes of metronidazole and 4-hydroxymethylpyridine was compared with that of silver nitrate. Metronidazole and 4-hydroxymethylpyridine were investigated using Balb/c 3T3 and HepG2 cell lines in order to evaluate the potential clinical application of silver(I) complexes. The cells were exposed for 72 h to compounds at eight concentrations. The cytotoxic concentrations (IC50) of the study compounds were assessed within four biochemical endpoints: mitochondrial activity, lysosomal activity, cellular membrane integrity, and total protein content. The investigated silver(I) complexes displayed comparable cytotoxicity to that of silver nitrate used in clinics. Mean cytotoxic concentrations calculated for investigated silver(I) complexes from concentration-response curves ranged from 2.13 to 26.5 µM. HepG2 cells were less sensitive to the tested complexes compared to fibroblasts (Balb/c 3T3). However, the most affected endpoint for HepG2 cells was cellular membrane damage. The cytotoxicity of both silver complexes was comparable for Balb/c 3T3 cells. The cytotoxic potential of the new silver(I) compounds compared to that of silver nitrate used in medicine indicates that they are safe and could be used in clinical practice. The presented results are yet more stimulating to further studies that evaluate the therapeutic use of silver complexes.

Concentration of Mercury in the Livers of Small Terrestrial Rodents from Rural Areas in Poland.

Small terrestrial mammals could be used as accumulative biomonitors of different environmental contaminants, but the knowledge of the level of Hg in their bodies is scant. The aim of our research was to verify the factors influencing Hg bioaccumulation and to analyze the concentration of total mercury (Hg) in the livers of four species of wild terrestrial rodents from different rural areas of Poland: the yellow-necked mouse (Apodemus flavicollis), striped field mouse (Apodemus agrarius), common vole (Microtus arvalis), and bank vole (Myodes glareolus). The concentration of total Hg was analyzed in liver tissue by atomic absorption spectrometry using a direct mercury analyzer. The concentration of Hg found in the livers of rodents ranged from <1 to 36.4 µg/kg of wet weight, differed between study sites, species, and sexes, and was related to body weight. We addressed feeding habits as potential causes of differences in liver Hg concentration among species.

Development, validation and application to real samples of a liquid chromatography with tandem mass spectrometry method for the determination of tetracyclines in beeswax.

Beeswax is a valuable honeybee product, which finds applications in the food and pharmaceutical industries, as well as in cosmetic production. However, some substances used in apiculture, like tetracyclines, can be delivered to hives where they cause contamination of honeybee products such as beeswax. Tetracyclines are commonly used for the treatment of American and European foulbrood diseases, but in the European Union their usage by beekeepers is forbidden. Thus, a sensitive method for the analysis of tetracyclines in beeswax is an important analytical tool. A new liquid chromatography with tandem mass spectrometry method for the analysis of tetracyclines including oxytetracycline, 4-epioxytetracycline, tetracycline, 4-epitetracycline, chlorotetracycline, 4-epichlorotetracycline, and doxycycline in beeswax was developed. The method involved dilution of beeswax in n-hexane after a melting step, liquid-liquid extraction with oxalic acid and clean-up using a weak cation exchange phase. Satisfactory separation was performed on an octadecyl column with 0.1% formic acid and acetonitrile in a total run time of 5 min. The application of this method was evaluated by the analysis of real beeswax samples. The presence of oxytetracycline was confirmed in 5 out of 48 tested beeswax samples, which shows the method can be successfully used to determine the tetracyclines in beeswax.

Analysis of thyreostats in bovine feces using ultra high performance liquid chromatography with tandem mass spectrometry.

A method based on ultra-high performance liquid chromatography was developed and validated to detect six thyreostatic compounds: tapazole, thiouracil, methylthiouracil, dimethylthiouracil, propylthiouracil, and phenylthiouracil in faeces of bovine. Thyreostats were extracted from the matrix with a mixture of methanol and buffer (pH = 8). Next step was derivatization of analytes with 3-iodobenzylbromide. The liquid chromatographic separation of derivatives was obtained on a SB-C18 column (50 × 2.1 mm; 1.8 µm, Agilent) with gradient elution using a mobile phase consisting of acetonitrile/0.1% acetic acid within 7.5 min. The analysis was performed on a Shimadzu NEXERA X2 ultra-high performance liquid chromatograph with triple quadrupole MS 8050 instrument operating in positive electrospray ionization mode. Depending on the target compound, two or three diagnostic signals (selected reaction monitoring transitions) were monitored. The procedure was validated according to the Commission Decision 2002/657/EC. Recovery and repeatability met the performance criteria specified by this document for banned compounds. The recovery ranged from 97.5 to 110.5%, and repeatability did not exceed 14.1%. Decision limits and detection capabilities were below 10 µg/kg. The highest decision limits and detection capabilities concentrations were observed for phenylthiouracil of 3.48 and 6.96 µg/kg, respectively.

Imidacloprid slows the development of preference for rewarding food sources in bumblebees (Bombus impatiens).

Bee pollination is economically and ecologically vital and recent declines in bee populations are therefore a concern. One possible cause of bee declines is pesticide use. Bumblebees exposed to imidacloprid, a neonicotinoid pesticide, have been shown to be less efficient foragers and collect less pollen on foraging trips than unexposed bees. We investigated whether bumblebees (Bombus impatiens) chronically exposed to imidacloprid at field-realistic levels of 2.6 and 10 ppb showed learning deficits that could affect foraging. Bumblebees were tested for their ability to associate flower colour with reward value in a simulated foraging environment. Bumblebees completed 10 foraging trips in which they collected sucrose solution from artificial flowers that varied in sucrose concentration. The reward quality of each artificial flower was predicted by corolla colour. Unexposed bumblebees acquired a preference for feeding on the most rewarding flower colour on the second foraging trip, while bumblebees exposed at 2.6 and 10 ppb did not until their third and fifth trip, respectively. The delay in preference acquisition in exposed bumblebees may be due to reduced flower sampling and shorter foraging trips. These results show that bumblebees exposed to imidacloprid are slow to learn the reward value of flowers and this may explain previously observed foraging inefficiencies associated with pesticide exposure.

Expression of metallothionein in the liver and kidneys of the red deer (Cervus elaphus L.) from an industrial metal smelting area of Poland.

The metallothionein 1 (MT1) coding sequence of red deer was identified and compared to orthologous sequences from other mammals. Over 90% identity was observed between red deer MT1 amino acid sequence and MT1 sequences of other ruminants. Liver and kidney samples of red deer were collected from the industrial zinc smelting site of Miasteczko Slaskie and from the Masuria Lake District serving as a pollution-free control site. The concentrations of cadmium (Cd), lead (Pb), copper (Cu) and zinc (Zn) were analyzed by the atomic absorption spectrometry technique (AAS). The levels of Cd in the liver of red deer from the metal smelting region was about 8 times higher than for the reference control site. Next, the expression of MT1 mRNA in the liver of red deer was quantified by the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and the expression of MT1/2 protein in the liver and kidneys was analyzed by immunohistochemistry. Positive correlations were found between expression levels for MT1 mRNA and the concentrations of Cu and Zn in liver of red deer, and with the age of animals. Immunohistochemical staining demonstrated the nuclear and cytoplasmatic expression in both liver and kidney tissues, but with no obvious relationship shown for the expression of MT1/2 protein and tissue metal levels. Our results showed that the analysis of MT expression levels in the red deer could not be used independently as a biomarker for identifying exposure to Cd, but could be co-analyzed with tissue metal levels to give better prognosis for environmental exposure to metals.

Comparison of the Multiple Reaction Monitoring and Enhanced Product Ion Scan Modes for Confirmation of Stilbenes in Bovine Urine Samples Using LC-MS/MS QTRAP® System.

In accordance with Commission Decision 2002/657/EC, confirmatory methods for the detection of prohibited substances should comply with specific requirements, including the criteria for confirmation. Two strategies: multiple reaction monitoring (MRM) and enhanced product ion (EPI) scanning functions were compared for confirming the anabolic compounds from synthetic stilbenes group in bovine urine samples. In the research, twenty samples fortified at the Recommended Concentration (RC) of 1 µg L-1 with diethylstilbestrol, dienestrol and hexestrol were analyzed by liquid chromatography-tandem mass spectrometry on a QTRAP 5500 instrument. The analytical procedure, validated in accordance with the Commission Decision 2002/657/EC, used in the official control of hormones in Poland was applied. The validation parameters were in agreement with 2002/657/EC performance criteria. The effectiveness of MRM and EPI scanning modes for confirmation purposes was evaluated based on the percentage of the results confirmed. In all urine samples recorded in the MRM mode, the confirmation criteria (retention time, relative intensities between transitions) have been fulfilled. The presence of stilbenes in all urine samples using EPI scan mode was confirmed too as evidenced by a good matching of stilbenes spectra in the samples to the reference spectra with critical match factor above 0.7. The results of the research show that EPI scanning function provides the same effectiveness for confirmation of banned compounds as the mostly used MRM scan mode and can be an additional tool to confirm the doubtful case results in the analysis of hormones residues, even at such low concentration levels.

Simple and reliable determination of total arsenic and its species in seafood by ICP-MS and HPLC-ICP-MS.

Quantitative, rapid, selective and sensitive methods for the determination of total arsenic (tAs) and six arsenic compounds (arsenite (As(III)), arsenate (As(V)), arsenobetaine (AsB), arsenocholine (AsC), monomethylarsonic acid (MMA), dimethylarsonic acid (DMA)) in seafood were developed. The measurement of the tAs concentration was performed using quadrupole inductively-coupled plasma mass spectrometry (ICP-MS). Microwave-assisted extraction was used for the isolation of arsenic species. The separation and quantification of analysed compounds were performed by ion-exchange chromatography coupled with ICP-MS in one chromatographic run using ammonium carbonate-based buffers, which has little effect on ICP-MS sensitivity compared to commonly used phosphate buffers. The results of validation and proficiency tests confirmed the reliability, robustness, and applicability of the developed procedures to various types of matrices. The proposed methods are relatively simple, time- and cost-efficient, therefore could be used to routinely analyse tAs content and arsenic species in different types of seafood at trace and ultra-trace levels.

White-Tailed Eagles' (Haliaeetus albicilla) Exposure to Anticoagulant Rodenticides and Causes of Poisoning in Poland (2018-2020).

The white-tailed eagle (Haliaeetus albicilla) is strictly protected in Poland due to its threat of extinction. This study's main goal was to assess their exposure to indirect poisoning by anticoagulant rodenticides (AR). This study presents the investigation results of 40 white-tailed eagles' suspected poisoning cases in the years 2018-2020 in Poland. In all tested liver samples, using a liquid chromatography-mass spectrometry method, at least one of the AR (bromadiolone, brodifacoum, difenacoum, flocoumafen) was detected and confirmed. The other tested AR compounds (chlorophacinone, coumachlor, coumatetralyl, difethialone, diphacinone, warfarin) were not detected. The mean concentration of the sum of rodenticides was 174.4 µg/kg (from 2.5 to 1225.0 µg/kg). In 20 cases, the sum concentration was above 100 µg/kg and in 10 cases it was above 200 µg/kg. Interpretation of cases of AR poisonings should take into account their concentration in the liver, anatomopathological lesions, circumstances of death/finding of the animal, and elimination of other possible causes of poisoning. Based on this study, AR was the direct cause of death in 10% of incidents. Extensive use of rodenticides generates a high risk of poisonings of white-tailed eagles in Poland.

Bee Venom Effect on Glioblastoma Cells Viability and Gelatinase Secretion.

BACKGROUND: The involvement of MMP-2 and MMP-9 in the pathogenesis of various kinds of cancers including glioblastoma is well documented. The evaluation of the anticancer potential of honey bee (Apis mellifera) venom (BV) consisting of the inhibition of MMP-2 and MMP-9 secretion in a glioblastoma cell culture model was the aim of the study. METHODS: 8-MG-BA and GAMG human primary glioblastoma cell lines vs. HT-22 mouse hippocampal neuronal cells were applied for the study. The BV dose (0.5, 1.0, 1.25, 1.5, 1.75, 2.0, 2.5, and 5.0 µg/ml) and time-dependent (24, 48, 72 h) cytotoxicity was evaluated with the tetrazolium-based colorimetric assay (MTT test). MMP-2 and MMP-9 activities in the cell culture medium under different BV concentrations were determined by gelatin zymography. RESULTS: A dose and time-dependent BV effect on cytotoxicity of both glioblastoma cell lines and hippocampus line was observed. The weakest, but statistically important effect was exerted by BV on HT-22 cells. The greatest cytotoxic effect of BV was observed on the 8-MG-BA line, where a statistically significant reduction in viability was observed at the lowest BV dose and the shortest incubation time. The reduction of both gelatinases secretion was observed at 8-MG-BA and GAMG lines without significant effect of HT-22 cell line. CONCLUSION: In vitro studies indicate that BV has both cytotoxic and inhibitory effects on the secretion of MMP-2 and MMP-9 in selected lines of glioma, suggesting anticancer properties of BV.

A Preliminary Study of the Poultry Body Weight Effect of Carvacrol in Litter and Of Carvacrol Residue in Organ Tissue of Exposed Chickens.

Introduction: Carvacrol is an essential oil extracted from oregano which can be used as a natural additive in poultry litter and could have a positive impact not only on production rates but also on the quality of poultry meat. The aim of this study was to evaluate the effect of the addition of carvacrol to litter on weight gain and the occurrence of residues in chicken tissues. Material and Methods: One-day-old Ross 308 chicks were used for the study and were randomly divided into two experimental groups. For 42 days, one group was kept in a room with litter enriched with carvacrol and the second group was kept in a room with litter without carvacrol. After 42 days, the birds were sacrificed and necropsied. Carvacrol content was determined in homogenised organ tissue samples by liquid chromatography-mass spectrometry. Results: Weekly weighing results showed that exposure to carvacrol in litter had no impact on chicken body weight. The analysis of plasma, muscle, liver and lung tissue after 42 days' exposure clearly indicated that there were residues of carvacrol in the analysed matrices. Conclusion: Exposure of chickens to carvacrol left residues but did not affect body weight.