Multifunctional Silver(I) Complexes with Metronidazole Drug Reveal Antimicrobial Properties and Antitumor Activity against Human Hepatoma and Colorectal Adenocarcinoma Cells.

Silver salts and azole derivatives are well known for their antimicrobial properties. Recent evidence has demonstrated also their cytotoxic and genotoxic potential toward both normal and cancer cells. Still, little is known about the action of complexes of azoles with silver(I) salts. Thus, the goal of the study was to compare the chemical, cytotoxic and antimicrobial properties of metronidazole complexes with silver(I) nitrate and silver(I) sulfate to metronidazole and pure silver(I) salts. We synthetized a novel complex, [Ag(MTZ)2]2SO4, and confirmed its chemical structure and properties using 1H and 13C NMR spectroscopy and X-Ray, IR and elemental analysis. To establish the stability of complexes [Ag(MTZ)2NO3] and [Ag(MTZ)2]2SO4, they were exposed to daylight and UV-A rays and were visually assessed. Their cytotoxicity toward human cancer cells (HepG2, Caco-2) and mice normal fibroblasts (Balb/c 3T3 clone A31) was determined by MTT, NRU, TPC and LDH assays. The micro-dilution broth method was used to evaluate their antimicrobial properties against Gram-positive and Gram-negative bacteria. A biofilm eradication study was also performed using the crystal violet method and confocal laser scanning microscopy. The photo-stability of the complexes was higher than silver(I) salts. In human cancer cells, [Ag(MTZ)2]2SO4 was more cytotoxic than Ag2SO4 and, in turn, AgNO3 was more cytotoxic than [Ag(MTZ)2NO3]. For Balb/c 3T3 cells, Ag2SO4 was more cytotoxic than [Ag(MTZ)2]2SO4, while the cytotoxicity of AgNO3 and [Ag(MTZ)2NO3] was similar. Metronidazole in the tested concentration range was non-cytotoxic for both normal and cancer cells. The complexes showed increased bioactivity against aerobic and facultative anaerobic bacteria when compared to metronidazole. For the majority of the tested bacterial strains, the silver(I) salts and complexes showed a higher antibacterial activity than MTZ; however, some bacterial strains presented the reverse effect. Our results showed that silver(I) complexes present higher photo-stability, cytotoxicity and antimicrobial activity in comparison to MTZ and, to a certain extent, to silver(I) salts.

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.

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.

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.

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.

In Vivo Study of The Oestrogenic Activity of Milk.

INTRODUCTION: Milk has been suggested to be a possible source of oestrogenically active compounds. In order to assess the health risk for milk consumers and ensure the safety of this staple part of the human diet, it is important to study the effect of xenooestrogen mixtures present in milk. This investigation used the available in vivo model to learn to what extent such compounds may be endocrine disruptors. MATERIAL AND METHODS: The recommended immature golden hamster uterotrophic bioassay was chosen. A total of 132 animals were divided into nine groups of experimental animals and positive and negative control groups, each of 12 animals. The experimental females received ad libitum either one of five samples of raw cow's milk from individual animals or one of four samples of pasteurised or ultra-high temperature treated cow's milk as retail products. After 7 days, the animals were sacrificed and necropsied. Uterine weight increases were measured as the endpoint of oestrogenic activity in milk. RESULTS: The milk samples from individual cows and the retail milk samples did not show oestrogenic activity. However, in three groups, decreased uterine weights were observed. CONCLUSION: Considering that milk supplies are beneficial to health, contamination in this food should be avoided. There is a need for further animal experiments and epidemiological studies are warranted to evaluate any causative role of milk in human endocrinological disorders.

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.

Miniaturized multiresidue method for determination of 267 pesticides, their metabolites and polychlorinated biphenyls in low mass beebread samples by liquid and gas chromatography coupled with tandem mass spectrometry.

Current work presents developed and validated miniaturized method for residue analysis of 261 pesticides and their metabolites as well as 6 congeners of non-dioxin like polychlorinated biphenyls (ndl-PCB) in a very low mass beebread sample. Sample preparation is based on modified QuEChERS protocol with all steps miniaturized to enable multiresidue analysis of sample with extremely low weight. Sample of beebread (0.3 g) was extracted with 1 mL of acetonitrile containing 5% formic acid and ammonium formate salt were added, then extract was subjected to clean-up by freezing and two-step dispersive solid phase extraction (dSPE) with a Supel QuE Verde sorbents (Supelclean ENVI-Carb Y; Supelclean PSA; Z-Sep+; magnesium sulfate). After 1st step dSPE a portion of extract was analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) for 200 pesticide residues. Remaining extract was subjected to 2nd step dSPE clean-up by another Supel QuE Verde and then after concentration and solvent exchange it was analyzed by gas chromatography tandem mass spectrometry (GC-MS/MS) for another 61 pesticide and 6 ndl-PCB residues. Method enables determination of residues of 101 insecticides, 72 herbicides, 67 fungicides, 10 acaricides, 6 growth regulators, 5 veterinary drugs and 6 ndl-PCB's. Particular attention was paid to the pesticides being active substances of plant protection products recommended for the protection of winter oilseed rape and apple orchards which during their blooming periods are one of the most attractive sources of food for pollinators and could serve as representatives of other economically important crops. Method was validated according to the Guidance document SANTE/12682/2019 at six concentration levels from 0.001 to 0.5 mg kg-1. The analysis of beebread samples spiked at the level of 0.01 mg kg-1showed mean recovery (trueness) value of about 98% and RSDr (precision) below 20%. The small weight of the sample did not adversely affect the limits of quantification and 75% of analytes could be quantified at least at concentration of 0.005 mg kg-1. Developed mini-method was tested in the analysis of beebread samples, each extracted from individual cell of honeycomb. It is the first time when analyses at single comb cell level were possible.

Transfer of enrofloxacin, ciprofloxacin, and lincomycin into eggshells and residue depletion in egg components after multiple oral administration to laying hens.

Regardless of whether antimicrobial drugs are administered to laying hens legally or illegally, residues of these drugs may be present in the eggs. Even if the eggs are not intended for human consumption, byproducts/biowaste, such as eggshells, may contain residues of the drugs used, which may pose a risk to human health and the environment. In the presented research, 2 different groups of laying hens received enrofloxacin (10 mg/kg body weight) and lincomycin (20 mg/kg body weight) once daily for 5 d. Eggs were collected daily and the concentration of enrofloxacin, its metabolite ciprofloxacin, and lincomycin residue in the eggshells, whole eggs, egg yolks, and egg whites were determined by ultra-high-performance liquid chromatography-tandem mass spectrometry. This study demonstrates the transfer of enrofloxacin, ciprofloxacin, and lincomycin into the eggshells and provides evidence for the distribution into the eggshells after administration of these drugs to laying hens. The enrofloxacin residues were detected in the eggshell for 10 d after cessation of treatment, ciprofloxacin and lincomycin were rapidly eliminated and 2 d after finish drugs administration they were no longer detected in the eggshell.

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.

Analysis of ß-agonists in Different Biological Matrices By Liquid Chromatography-tandem Mass Spectrometry.

INTRODUCTION: Wide use is made of ß-agonists in therapy due to their smooth muscle-relaxant properties. They also have a side effect of increasing muscle mass. Besides improving oxygen utilisation as bronchodilators, ß-agonists increase protein synthesis and promote fat burning. The growth- and performance-enhancing effects are often exploited in illegal use. The guiding objective of this study was to develop a procedure for the determination of ß-agonists by a single method in different types of matrices. MATERIAL AND METHODS: Five grams of homogenised samples were subjected to enzymatic hydrolysis with ß-glucuronidase in ammonium acetate, pH 5.2. Purification was performed by solid phase extraction. Analytes were eluted with 10% acetic acid in methanol. The eluted ß-agonists were analysed by high-performance liquid chromatography-tandem mass spectrometry. RESULTS: Validation results met the requirement of the confirmation criteria according to European Commission Decision 2002/657/EC in terms of apparent recoveries (93.2-112.0%), repeatability (3.1-7.1%) and intra-laboratory reproducibility (4.1-8.2%). CONCLUSION: The method can be successfully applied in the detection and determination of clenbuterol, salbutamol, mabuterol, mapenterol, terbutaline, brombuterol, zilpaterol, isoxsuprine and ractopamine in feed, drinking water, urine, muscle, lung and liver matrices.

The Markers of Stress in Swine Oral Fluid.

INTRODUCTION: The study measured the hormonal and protein markers of acute stress, those of oxidative stress and total antioxidant capacity (TAC) in swine oral fluid, determined which of these parameters would be the most appropriate for future livestock welfare assessment and established the time when the samples should be taken. MATERIAL AND METHODS: Stress was induced in 7 out of 14 castrated six-week-old Danbred×Duroc pigs by immobilisation on a nasal snare at 8 a.m., 1 p.m., and 6 p.m. and samples were taken both directly after the stressor was applied and 30 min later. The remaining pigs were the control group, which were not immobilised; their samples were taken at the same times. The concentrations of hormones and malondialdehyde (MDA) were measured using liquid chromatography with tandem mass spectrometry, while those of alpha-amylase and TAC were measured using spectrophotometry. RESULTS: The levels of cortisol and cortisone increased with statistical significance immediately after the acute stress response and 30 min later. A cut-off value set at 0.25 ng/mL cortisol concentration was capable of distinguishing between the stressed and control groups with 100% accuracy in evening samples and 95% accuracy overall. Prednisolone was not present, and the levels of testosterone and corticosterone were low and not distinctive. Alpha-amylase became significantly more concentrated during stress induction and 30 min later. The TAC and MDA levels rose after the stress but without statistical significance. CONCLUSION: The most suitable markers of acute stress were cortisol, cortisone and alpha-amylase. Oral fluid is a reliable material for monitoring the level of pigs' stress and should be collected in the evening.

An assessment of the estrogenic and androgenic properties of tetra- and hexachloronaphthalene by YES/YAS in vitro assays.

Many persistent organic pollutants (POPs) exhibit endocrine disrupting activity but studies on some POPs, e.g., polychlorinated naphthalenes (PCNs), are very scarce. The present study investigates the (anti)estrogenic and (anti)androgenic activities of 1,2,3,5,6,7-hexachloronaphthalane (PCN67) and 1,3,5,8-tetrachloronaphthalene (PCN43) using the yeast estrogen and androgen reporter bioassays. Among the tested substances, antiestrogenic response was only shown by PCN67. The strongest inhibition of estrogenic activity (up to 17.4%) was observed in the low concentration ranges (5 pM - 0.5 nM) in the presence of 1.5 nM 17ß-estradiol. Both tested compounds showed partial estrogenic activity with a hormetic-type response. However, both studied chemicals showed strong antiandrogenic effects: their potency in the presence of 100 nM 17ß-testosterone for PCN43 (IC50 = 2.59 µM) and PCN67 (IC50 = 3.14 µM) was approximately twice that of the reference antiandrogen flutamide (IC50 = 6.14 µM). It cannot be excluded that exposure to PCNs, together with other endocrine disrupting chemicals (EDCs), may contribute to the deregulation of sex steroid hormone signaling.

Determination of selected testosterone esters in blood serum of slaughter animals by liquid chromatography with tandem mass spectrometry.

Anabolic hormones, which cause muscle growth, have been banned for anabolic purposes in animal husbandry in Europe since the 1980s. Control of hormones from the list of Annex I to Directive 96/23/EC is mandatory in the European Union. The presence of hormones in samples of animal origin may be due to their endogeneous nature or illegal use. One way to distinguish their origin is to study hormones, particularly steroids in the form of ester derivatives. In the body synthetic hormone esters could be only exogenous therefore their detection in animal tissues is the undisputed evidence of illegal administration. The analytical procedure involves the extraction of esters from serum with organic solvents, derivatisation with methoxyamine and detection by liquid chromatography tandem mass spectrometry. The method was approved in accordance with the applicable legislative criteria and its effectiveness was verified in the proficiency test. The research material consisted of bovine serum samples officially taken. During the validation good apparent recovery, precision, decision limits and detection capabilities in the range 0.006-0.012 µg L-1 and 0.010-0.020 µg L-1 respectively were obtained. The developed method met the criteria for confirmation set out in Commission Decision 2002/657/EC. Since the inclusion of serum in 2018 for testing for testosterone esters in the National Residue Control Program, 130 samples have been examined. In none of the serum samples, esters above the decision limits were found. The control of animals and food of animal origin for hormone esters will be continued to ensure the health and safety of consumers.

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.

Simultaneous Determination of Deoxynivalenol, Its Modified Forms, Nivalenol and Fusarenone-X in Feedstuffs by the Liquid Hromatography-Tandem Mass Spectrometry Method.

A liquid chromatography-tandem mass spectrometry method was developed for simultaneous determination of deoxynivalenol (DON), 3-acetyldeoxynivalenol (3Ac-DON), 15-acetyldeoxynivalenol (15Ac-DON), DON-3-glucoside (DON-3Glc) nivalenol and fusarenone-X in feedstuffs. Different techniques of sample preparation were tested: solid-liquid-extraction, QuEChERS, solid phase extraction with OASIS HLB columns or immunoaffinity columns and a Mycosep 225 Trich column. None of the six immunoaffinity columns tested showed cross-reactivity to all of the mycotoxins. Surprisingly, the results show that if the immunoaffinity columns bound 3Ac-DON, then they did not bind 15Ac-DON. The most efficient sample preparation was achieved with a Mycosep 225 Trich column clean-up. The chromatography was optimised to obtain full separation of all analytes (including 3Ac-DON and 15Ac-DON isomeric form). The validation results show the relative standard deviations for repeatability and reproducibility varied from 4% to 24%. The apparent recovery ranged between 92% and 97%, and the limit of quantification described a 1.30 to 50 µg/kg range. The method trueness was satisfactory, as assessed by a proficiency test and analysis of reference material. A total of 99 feed samples were analysed by the developed method, revealing the presence of DON and DON-3Glc in 85% and 86% of examined animal feeds, respectively at concentrations between 1.70 and 1709 µg/kg. The ratios DON-3Glc to DON in the surveyed feedstuffs were from a low of 3% to high of 59%.

Total mercury levels in the muscle and liver of livestock and game animals in Poland, 2009-2018.

The bioaccumulation of mercury (Hg) in the food chain may pose a threat to human health. The risk of dietary Hg intake is mostly caused by the consumption of fish and seafood, therefore the knowledge on the exposure from land animal products is limited. In our article, we summarized the results of analyses of Hg in muscle tissue and liver of different livestock and game animals obtained during ten years of official monitoring that was carried out in Poland from 2009 to 2018. The majority of the results in muscle tissue were below the limits of quantification (LOQs). The mean Hg concentrations in muscle tissue ranged from 0.6 to 5.6 µg kg-1 of wet weight and the mean liver Hg concentrations were within the range of 0.8-16.4 µg kg-1 of wet weight, with lowest levels in chickens and highest in wild boars. The results revealed decreasing trends in liver Hg in cattle and cervids over the years, which was congruous with decreasing emission of Hg in Europe. Our results showed that the consumption of meat and liver of livestock and game animals in Poland may be considered to be safe for human health, which was confirmed by the low number of noncompliant samples relative to the applicable legal limits, as well as by estimated dietary exposure.

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.

Doxycycline transfer from substrate to white button mushroom (Agaricus bisporus) and assessment of the potential consumer exposure.

The presence of antibiotic residues in the food chain may pose a serious risk to human health. Locating and evaluating new sources of consumer exposure to antibiotic residues in food is a very important element of health protection. The possibility of doxycycline uptake from the substrate for mushroom cultivation by the white button mushroom (Agaricus bisporus) fruit body was investigated. Mushrooms were experimentally cultivated on substrate contaminated with 8 different doxycycline concentrations in substrate and analyte concentrations in mushroom samples were measured using ultra-high performance liquid chromatography - triple quadrupole tandem mass spectrometry (UHPLC-MS/MS) The obtained results clearly indicated that doxycycline transfers from contaminated substrate to mushrooms at concentrations ranging from 0.87 to 72.3 µg/kg, depending on substrate contamination concentration level and order of harvesting.

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.