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Exposure to high manganese (Mn) levels may damage the basal ganglia, leading to a syndrome analogous to Parkinson's disease, with motor and cognitive impairments. The molecular mechanisms underlying Mn neurotoxicity, particularly during development, still deserve further investigation. Herein, we addressed whether early-life Mn exposure affects motor coordination and cognitive function in adulthood and potential underlying mechanisms. Male Wistar rats were exposed intraperitoneally to saline (control) or MnCl2 (5, 10 or 20 mg/kg/day) from post-natal day (PND) 8-12. Behavioral tests were performed on PND 60-65 and biochemical analysis in the striatum and hippocampus were performed on PND14 or PND70. Rats exposed to Mn (10 and 20 mg/kg) performed significantly worse on the rotarod test than controls indicating motor coordination and balance impairments. The object and social recognition tasks were used to evaluate short-term memory. Rats exposed to the highest Mn dose failed to recognize a familiar object when replaced by a novel object as well as to recognize a familiar juvenile rat after a short period of time. However, Mn did not alter olfactory discrimination ability. In addition, Mn-treated rats displayed decreased levels of non-protein thiols (e.g. glutathione) and increased levels of glial fibrillary acidic protein (GFAP) in the striatum. Moreover, Mn significantly increased hippocampal glutathione peroxidase (GPx) activity. These findings demonstrate that acute low-level exposure to Mn during a critical neurodevelopmental period causes cognitive and motor dysfunctions that last into adulthood, that are accompanied by alterations in antioxidant defense system in both the hippocampus and striatum. (C) 2015 Elsevier Inc. All rights reserved.
Mycotoxins and pesticides regularly co-occur in agricultural products worldwide. Thus, humans can be exposed to both toxic contaminants and pesticides simultaneously, and multi-methods assessing the occurrence of various food contaminants and residues in a single method are necessary. A two-dimensional high performance liquid chromatography tandem mass spectrometry method for the analysis of 40 (modified) mycotoxins, two plant growth regulators, two tropane alkaloids, and 334 pesticides in cereals was developed. After an acetonitrile/water/formic acid (79:20:1, v/v/v) multi-analyte extraction procedure, extracts were injected into the two-dimensional setup, and an online clean-up was performed. The method was validated according to Commission Decision (EC) no. 657/2002 and document N° SANTE/12682/2019. Good linearity (R2 > 0.96), recovery data between 70-120%, repeatability and reproducibility values < 20%, and expanded measurement uncertainties < 50% were obtained for a wide range of analytes, including very polar substances like deoxynivalenol-3-glucoside and methamidophos. However, results for fumonisins, zearalenone-14,16-disulfate, acid-labile pesticides, and carbamates were unsatisfying. Limits of quantification meeting maximum (residue) limits were achieved for most analytes. Matrix effects varied highly (−85 to +1574%) and were mainly observed for analytes eluting in the first dimension and early-eluting analytes in the second dimension. The application of the method demonstrated the co-occurrence of different types of cereals with 28 toxins and pesticides. Overall, 86% of the samples showed positive findings with at least one mycotoxin, plant growth regulator, or pesticide.
Mycotoxins and pesticides regularly co-occur in agricultural products worldwide. Thus, humans can be exposed to both toxic contaminants and pesticides simultaneously, and multi-methods assessing the occurrence of various food contaminants and residues in a single method are necessary. A two-dimensional high performance liquid chromatography tandem mass spectrometry method for the analysis of 40 (modified) mycotoxins, two plant growth regulators, two tropane alkaloids, and 334 pesticides in cereals was developed. After an acetonitrile/water/formic acid (79:20:1, v/v/v) multi-analyte extraction procedure, extracts were injected into the two-dimensional setup, and an online clean-up was performed. The method was validated according to Commission Decision (EC) no. 657/2002 and document N° SANTE/12682/2019. Good linearity (R2 > 0.96), recovery data between 70-120%, repeatability and reproducibility values < 20%, and expanded measurement uncertainties < 50% were obtained for a wide range of analytes, including very polar substances like deoxynivalenol-3-glucoside and methamidophos. However, results for fumonisins, zearalenone-14,16-disulfate, acid-labile pesticides, and carbamates were unsatisfying. Limits of quantification meeting maximum (residue) limits were achieved for most analytes. Matrix effects varied highly (−85 to +1574%) and were mainly observed for analytes eluting in the first dimension and early-eluting analytes in the second dimension. The application of the method demonstrated the co-occurrence of different types of cereals with 28 toxins and pesticides. Overall, 86% of the samples showed positive findings with at least one mycotoxin, plant growth regulator, or pesticide.
Multi-element determination in human samples is very challenging. Especially in human intervention studies sample volumes are often limited to a few microliters and due to the high number of samples a high-throughput is indispensable. Here, we present a state-of-the-art ICP-MS/MS-based method for the analysis of essential (trace) elements, namely Mg, Ca, Fe, Cu, Zn, Mo, Se and I, as well as food-relevant toxic elements such as As and Cd. The developed method was validated regarding linearity of the calibration curves, method LODs and LOQs, selectivity and trueness as well as precision. The established reliable method was applied to quantify the element serum concentrations of participants of a human intervention study (LeguAN). The participants received isocaloric diets, either rich in plant protein or in animal protein. While the serum concentrations of Mg and Mo increased in participants receiving the plant protein-based diet (above all legumes), the Se concentration in serum decreased. In contrast, the animal protein-based diet, rich in meat and dairy products, resulted in an increased Se concentration in serum.
Here, a reliable and sensitive method for the determination of 38 (modified) mycotoxins was developed. Using a QuEChERS-based extraction method [acetonitrile/water/formic acid (75:20:5, v/v/v)], followed by two runs of high performance liquid chromatography tandem mass spectrometry with different conditions, relevant mycotoxins in cereals were analyzed. The method was validated according to the performance criteria defined by the European Commission (EC) in Commission Decision no. 657/2002. Limits of quantification ranged from 0.05 to 150 μg/kg. Good linearity (R2 > 0.99), recovery (61–120%), repeatability (RSDr < 15%), and reproducibility (RSDR < 20%) were obtained for most mycotoxins. However, validation results for Alternaria toxins and fumonisins were unsatisfying. Matrix effects (−69 to +59%) were compensated for using standard addition. Application on reference materials gave correct results while analysis of samples from local retailers revealed contamination, especially with deoxynivalenol, deoxynivalenol-3-glucoside, fumonisins, and zearalenone, in concentrations up to 369, 58, 1002, and 21 μg/kg, respectively.
A fast high performance liquid chromatography tandem mass spectrometry multi-method based on an ACN-precipitation extraction was developed for the analysis of 41 (modified) mycotoxins in beer. Validation according to the performance criteria defined by the European Commission (EC) in Commission Decision no. 657/2002 revealed good linearity (R2 > 0.99), repeatability (RSDr < 15%), reproducibility (RSDR < 15%), and recovery (79–100%). Limits of quantification ranging from 0.04 to 75 µg/L were obtained. Matrix effects varied from −67 to +319% and were compensated for using standard addition. In total, 87 beer samples, produced worldwide, were analyzed for the presence of mycotoxins with a focus on modified mycotoxins, whereof 76% of the samples were contaminated with at least one mycotoxin. The most prevalent mycotoxins were deoxynivalenol-3-glucoside (63%), HT-2 toxin (15%), and tenuazonic acid (13%). Exposure estimates of deoxynivalenol and its metabolites for German beer revealed no significant contribution to intake of deoxynivalenol.
The Caenorhabditis elegans (C. elegans) is a model organism that has been increasingly used in health and environmental toxicity assessments. The quantification of such elements in vivo can assist in studies that seek to relate the exposure concentration to possible biological effects.
Therefore, this study is the first to propose a method of quantitative analysis of 21 ions by ion chromatography (IC), which can be applied in different toxicity studies in C. elegans.
The developed method was validated for 12 anionic species (fluoride, acetate, chloride, nitrite, bromide, nitrate, sulfate, oxalate, molybdate, dichromate, phosphate, and perchlorate), and 9 cationic species (lithium, sodium, ammonium, thallium, potassium, magnesium, manganese, calcium, and barium).
The method did not present the presence of interfering species, with R2 varying between 0.9991 and 0.9999, with a linear range from 1 to 100 mu g L-1.
Limits of detection (LOD) and limits of quantification (LOQ) values ranged from 0.2319 mu g L-1 to 1.7160 mu g L-1 and 0.7028 mu g L-1 to 5.1999 mu g L-1, respectively.
The intraday and interday precision tests showed an Relative Standard Deviation (RSD) below 10.0 % and recovery ranging from 71.0 % to 118.0 % with a maximum RSD of 5.5 %.
The method was applied to real samples of C. elegans treated with 200 uM of thallium acetate solution, determining the uptake and bioaccumulated Tl+ content during acute exposure.
Nanomaterials play an important role in mimicking the biochemical and biophysical cues of the extracellular matrix in human mesenchymal stem cells (MSCs). Increasing studies have demonstrated the crucial impact of functional groups on MSCs, while limited research is available on how the functional group's density on nanoparticles regulates MSC behavior. Herein, the effects of dendritic polyglycerol (dPG)-conjugated gold nanostars (GNSs) with different densities of functional groups on the osteogenesis of MSCs are systematically investigated. dPG@GNS nanocomposites have good biocompatibility and the uptake by MSCs is in a functional group density-dependent manner. The osteogenic differentiation of MSCs is promoted by all dPG@GNS nanocomposites, in terms of alkaline phosphatase activity, calcium deposition, and expression of osteogenic protein and genes. Interestingly, the dPGOH@GNSs exhibit a slight upregulation in the expression of osteogenic markers, while the different charged densities of sulfate and amino groups show more efficacy in the promotion of osteogenesis. Meanwhile, the sulfated nanostars dPGS20@GNSs show the highest enhancement. Furthermore, various dPG@GNS nanocomposites exerted their effects by regulating the activation of Yes-associated protein (YAP) to affect osteogenic differentiation. These results indicate that dPG@GNS nanocomposites have functional group density-dependent influence on the osteogenesis of MSCs, which may provide a new insight into regulating stem cell fate.
Trace elements, like Cu, Zn, Fe, or Se, are important for the proper functioning of antioxidant enzymes. However, in excessive amounts, they can also act as pro-oxidants. Accordingly, trace elements influence redox-modulated signaling pathways, such as the Nrf2 pathway. Vice versa, Nrf2 target genes belong to the group of transport and metal binding proteins. In order to investigate whether Nrf2 directly regulates the systemic trace element status, we used mice to study the effect of a constitutive, whole-body Nrf2 knockout on the systemic status of Cu, Zn, Fe, and Se. As the loss of selenoproteins under Se-deprived conditions has been described to further enhance Nrf2 activity, we additionally analyzed the combination of Nrf2 knockout with feeding diets that provide either suboptimal, adequate, or supplemented amounts of Se. Experiments revealed that the Nrf2 knockout partially affected the trace element concentrations of Cu, Zn, Fe, or Se in the intestine, liver, and/or plasma. However, aside from Fe, the other three trace elements were only marginally modulated in an Nrf2-dependent manner. Selenium deficiency mainly resulted in increased plasma Zn levels. One putative mediator could be the metal regulatory transcription factor 1, which was up-regulated with an increasing Se supply and downregulated in Se-supplemented Nrf2 knockout mice.
Trace elements, like Cu, Zn, Fe, or Se, are important for the proper functioning of antioxidant enzymes. However, in excessive amounts, they can also act as pro-oxidants. Accordingly, trace elements influence redox-modulated signaling pathways, such as the Nrf2 pathway. Vice versa, Nrf2 target genes belong to the group of transport and metal binding proteins. In order to investigate whether Nrf2 directly regulates the systemic trace element status, we used mice to study the effect of a constitutive, whole-body Nrf2 knockout on the systemic status of Cu, Zn, Fe, and Se. As the loss of selenoproteins under Se-deprived conditions has been described to further enhance Nrf2 activity, we additionally analyzed the combination of Nrf2 knockout with feeding diets that provide either suboptimal, adequate, or supplemented amounts of Se. Experiments revealed that the Nrf2 knockout partially affected the trace element concentrations of Cu, Zn, Fe, or Se in the intestine, liver, and/or plasma. However, aside from Fe, the other three trace elements were only marginally modulated in an Nrf2-dependent manner. Selenium deficiency mainly resulted in increased plasma Zn levels. One putative mediator could be the metal regulatory transcription factor 1, which was up-regulated with an increasing Se supply and downregulated in Se-supplemented Nrf2 knockout mice.
A novel surface coating with durable broad-spectrum antibacterial ability was prepared based on mussel inspired dendritic polyglycerol (MI-dPG) embedded with copper nanoparticles (Cu NPs). The functional surface coating is fabricated via a facile dip-coating process followed by in situ reduction of copper ions with a MI-dPG coating to introduce Cu NPs into the coating matrix. This coating has been demonstrated to possess efficient long-term antibacterial properties against Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), and kanamycin-resistant E. coli through an "attract-kill-release" strategy. The synergistic antibacterial activity of the coating was shown by the combination of two functions of the contact killing, reactive oxygen species production and Cu ions released from the coating. Furthermore, this coating inhibited biofilm formation and showed good compatibility to eukaryotic cells. Thus, this newly developed Cu NP-incorporated MI-dPG surface coating may find potential application in the design of antimicrobial coating, such as implantable devices.
Background: Being an essential trace element, copper is involved in diverse physiological processes. However, excess levels might lead to adverse effects. Disrupted copper homeostasis, particularly in the brain, has been associated with human diseases including the neurodegenerative disorders Wilson and Alzheimer?s disease. In this context, astrocytes play an important role in the regulation of the copper homeostasis in the brain and likely in the prevention against neuronal toxicity, consequently pointing them out as a potential target for the neurotoxicity of copper. Major toxic mechanisms are discussed to be directed against mitochondria probably via oxidative stress. However, the toxic potential and mode of action of copper in astrocytes is poorly understood, so far. Methods: In this study, excess copper levels affecting human astrocytic cell model and their involvement in the neurotoxic mode of action of copper, as well as, effects on the homeostasis of other trace elements (Mn, Fe, Ca and Mg) were investigated. Results: Copper induced substantial cytotoxic effects in the human astrocytic cell line following 48 h incubation (EC30: 250 ?M) and affected mitochondrial function, as observed via reduction of mitochondrial membrane potential and increased ROS production, likely originating from mitochondria. Moreover, cellular GSH metabolism was altered as well. Interestingly, not only cellular copper levels were affected, but also the homeostasis of other elements (Ca, Fe and Mn) were disrupted. Conclusion: One potential toxic mode of action of copper seems to be effects on the mitochondria along with induction of oxidative stress in the human astrocytic cell model. Moreover, excess copper levels seem to interact with the homeostasis of other essential elements such as Ca, Fe and Mn. Disrupted element homeostasis might also contribute to the induction of oxidative stress, likely involved in the onset and progression of neurodegenerative disorders. These insights in the toxic mechanisms will help to develop ideas and approaches for therapeutic strategies against copper-mediated diseases.
Characterization of freeze-fractured epithelial plasma membranes on nanometer scale with ToF-SIMS
(2015)
Time-of-flight secondary ion mass spectrometry (ToF-SIMS) was used to characterize the freeze-fracturing process of human epithelial PANC-1 and UROtsa cells. For this purpose, phosphatidylcholine, sphingomyelin, phosphatidylethanolamine, and phosphatidylserine standard samples were investigated to find specific signals with both high specificity and signal intensity. The results were used to investigate single cells of subconfluent cell layers prepared with a special silicon wafer sandwich preparation technique. This freeze-fracturing technique strips cell membranes off the cells, isolating them on opposing silicon wafer substrates. Criteria were found for defining regions with stripped off cell membranes and, on the opposing wafer, complementary regions with the remaining cells. Measured ethanolamine/choline and serine/choline ratios in these regions clearly showed that in the freeze-fracturing process, the lipid bilayer of the plasma membrane is split along its central zone. Accordingly, only the outer lipid monolayer is stripped off the cell, while the inner lipid monolayer remains attached to the cell on the opposing wafer, thus allowing detailed analysis of a single lipid monolayer. Furthermore, it could be shown that using different washing procedures did not influence the transmembrane lipid distribution. Under optimized preparation conditions, it became feasible to detect lipids with a lateral resolution of approximately 100 nm. The data indicate that ToF-SIMS would be a very useful technique to study with very high lateral resolution changes in lipid composition caused, for example, by lipid storage diseases or pharmaceuticals that interfere with the lipid metabolism.
Arsenolipids, especially arsenic-containing hydrocarbons (AsHC), are an emerging class of seafood originating contaminants. Here we toxicologically characterize a recently identified oxo-AsHC 332 metabolite, thioxo-AsHC 348 in cultured human liver (HepG2) cells. Compared to results of previous studies of the parent compound oxo-AsHC 332, thioxo-AsHC 348 substantially affected cell viability in the same concentration range but exerted about 10-fold lower cellular bioavailability. Similar to oxo-AsHC 332, thioxo-AsHC 348 did not substantially induce oxidative stress nor DNA damage. Moreover, in contrast to oxo-AsHC 332 mitochondria seem not to be a primary subcellular toxicity target for thioxo-AsHC 348. This study indicates that thioxo-AsHC 348 is at least as toxic as its parent compound oxo-AsHC 332 but very likely acts via a different mode of toxic action, which still needs to be identified.
The visceral protein transthyretin (TTR) is frequently affected by oxidative post-translational protein modifications (PTPMs) in various diseases. Thus, better insight into structure-function relationships due to oxidative PTPMs of TTR should contribute to the understanding of pathophysiologic mechanisms. While the in vivo analysis of TTR in mammalian models is complex, time-and resource-consuming, transgenic Caenorhabditis elegans expressing hTTR provide an optimal model for the in vivo identification and characterization of drug-mediated oxidative PTPMs of hTTR by means of matrix assisted laser desorption/ionization - time of flight - mass spectrometry (MALDI-TOF-MS). Herein, we demonstrated that hTTR is expressed in all developmental stages of Caenorhabditis elegans, enabling the analysis of hTTR metabolism during the whole life-cycle. The suitability of the applied model was verified by exposing worms to D-penicillamine and menadione. Both drugs induced substantial changes in the oxidative PTPM pattern of hTTR. Additionally, for the first time a covalent binding of both drugs with hTTR was identified and verified by molecular modelling.
The visceral protein transthyretin (TTR) is frequently affected by oxidative post-translational protein modifications (PTPMs) in various diseases. Thus, better insight into structure-function relationships due to oxidative PTPMs of TTR should contribute to the understanding of pathophysiologic mechanisms. While the in vivo analysis of TTR in mammalian models is complex, time- and resource-consuming, transgenic Caenorhabditis elegans expressing hTTR provide an optimal model for the in vivo identification and characterization of drug-mediated oxidative PTPMs of hTTR by means of matrix assisted laser desorption/ionization – time of flight – mass spectrometry (MALDI-TOF-MS). Herein, we demonstrated that hTTR is expressed in all developmental stages of Caenorhabditis elegans, enabling the analysis of hTTR metabolism during the whole life-cycle. The suitability of the applied model was verified by exposing worms to D-penicillamine and menadione. Both drugs induced substantial changes in the oxidative PTPM pattern of hTTR. Additionally, for the first time a covalent binding of both drugs with hTTR was identified and verified by molecular modelling.
The visceral protein transthyretin (TTR) is frequently affected by oxidative post-translational protein modifications (PTPMs) in various diseases. Thus, better insight into structure-function relationships due to oxidative PTPMs of TTR should contribute to the understanding of pathophysiologic mechanisms. While the in vivo analysis of TTR in mammalian models is complex, time- and resource-consuming, transgenic Caenorhabditis elegans expressing hTTR provide an optimal model for the in vivo identification and characterization of drug-mediated oxidative PTPMs of hTTR by means of matrix assisted laser desorption/ionization – time of flight – mass spectrometry (MALDI-TOF-MS). Herein, we demonstrated that hTTR is expressed in all developmental stages of Caenorhabditis elegans, enabling the analysis of hTTR metabolism during the whole life-cycle. The suitability of the applied model was verified by exposing worms to D-penicillamine and menadione. Both drugs induced substantial changes in the oxidative PTPM pattern of hTTR. Additionally, for the first time a covalent binding of both drugs with hTTR was identified and verified by molecular modelling.
Brain capillary pericytes contribute to the immune defense in response to cytokines or LPS in vitro
(2014)
The prevention of an inflammation in the brain is one of the most important goals the body has to achieve. As pericytes are located on the abluminal side of the capillaries in the brain, their role in fighting against invading pathogens has been investigated in some points, mostly in their ability to behave like macrophages. Here we studied the potential of pericytes to react as immune cells under inflammatory conditions, especially regarding the expression of the inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), major histocompatibility complex II (MHC II) molecules, CD68, as well as the generation of reactive oxygen and nitrogen species (RONS), and their ability in phagocytosis. Quantitative real time PCR and western blot analysis showed that pericytes are able to increase the expression of typical inflammatory marker proteins after the stimulation with tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1 beta), interferon-gamma (IFN-gamma), or lipopolysaccharides (LPS). Depending on the different specific pro-inflammatory factors pericytes changed the expression of alpha smooth muscle actin (alpha SMA), the most predominant pericyte marker. We conclude that the role of the pericytes within the immune system is regulated and fine-tuned by different cytokines strongly depending on the time when the cytokines are released and their concentration. The present results will help to understand the pericyte mediated defense mechanisms in the brain.