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Excessive levels of the essential metal manganese (Mn) may cause a syndrome similar to Parkinson’s disease. The model organism Caenorhabditis elegans mimics some of Mn effects in mammals, including dopaminergic neurodegeneration, oxidative stress, and increased levels of AKT. The evolutionarily conserved insulin/insulin-like growth factor-1 signaling pathway (IIS) modulates worm longevity, metabolism, and antioxidant responses by antagonizing the transcription factors DAF-16/FOXO and SKN-1/Nrf-2. AKT-1, AKT-2, and SGK-1 act upstream of these transcription factors. To study the role of these proteins in C. elegans response to Mn intoxication, wild-type N2 and loss-of-function mutants were exposed to Mn (2.5 to 100 mM) for 1 h at the L1 larval stage. Strains with loss-of-function in akt-1, akt-2, and sgk-1 had higher resistance to Mn compared to N2 in the survival test. All strains tested accumulated Mn similarly, as shown by ICP-MS. DAF-16 nuclear translocation was observed by fluorescence microscopy in WT and loss-of-function strains exposed to Mn. qRT-PCR data indicate increased expression of γ-glutamyl cysteine synthetase (GCS-1) antioxidant enzyme in akt-1 mutants. The expression of sod-3 (superoxide dismutase homologue) was increased in the akt-1 mutant worms, independent of Mn treatment. However, dopaminergic neurons degenerated even in the more resistant strains. Dopaminergic function was evaluated with the basal slowing response behavioral test and dopaminergic neuron integrity was evaluated using worms expressing green fluorescent protein (GFP) under the dopamine transporter (DAT-1) promoter. These results suggest that AKT-1/2 and SGK-1 play a role in C. elegans response to Mn intoxication. However, tissue-specific responses may occur in dopaminergic neurons, contributing to degeneration.
A novel method based on liquid-liquid extraction with subsequent gas chromatography separation and mass spectrometric detection (GC-MS) for the quantification of organic carbonates in cell culture materials is presented. Method parameters including the choice of extraction solvent, of extraction method and of extraction time were optimised and the method was validated. The setup allowed for determination within a linear range of more than two orders of magnitude. The limits of detection (LODs) were between 0.0002 and 0.002 mmol/L and the repeatability precisions were in the range of 1.5-12.9%. It could be shown that no matrix effects were present and recovery rates between 98 and 104% were achieved. The methodology was applied to cell culture models incubated with commercial lithium ion battery (LIB) electrolytes to gain more insight into the potential toxic effects of these compounds. The stability of the organic carbonates in cell culture medium after incubation was studied. In a porcine model of the blood-cerebrospinal fluid (CSF) barrier, it could be shown that a transfer of organic carbonates into the brain facing compartment took place.
Scope: Selenoneine (2-selenyl-N-alpha, N-alpha, N-alpha-trimethyl-L-histidine), the selenium (Se) analogue of the ubiquitous thiol compound and putative antioxidant ergothioneine, is the major organic selenium species in several marine fish species. Although its antioxidant efficacy has been proposed, selenoneine has been poorly characterized, preventing conclusions on its possible beneficial health effects. Methods and results: Treatment of Caenorhabditis elegans (C. elegans) with selenoneine for 18 h attenuated the induction of reactive oxygen and nitrogen species (RONS). However, the effect was not immediate, occurring 48 h post-treatment. Total Se and Se speciation analysis revealed that selenoneine was efficiently taken up and present in its original form directly after treatment, with no metabolic transformations observed. 48 h posttreatment, total Se in worms was slightly higher compared to controls and no selenoneine could be detected. Conclusion: The protective effect of selenoneine may not be attributed to the presence of the compound itself, but rather to the activation of molecular mechanisms with consequences at more protracted time points.
Pesticides guarantee us high productivity in agriculture, but the long-term costs have proved too high. Acute and chronic intoxication of humans and animals, contamination of soil, water and food are the consequences of the current demand and sales of these products. In addition, pesticides such as glyphosate are sold in commercial formulations which have inert ingredients, substances with unknown composition and proportion. Facing this scenario, toxicological studies that investigate the interaction between the active principle and the inert ingredients are necessary. The following work proposed comparative toxicology studies between glyphosate and its commercial formulation using the alternative model Caenorhabditis elegans. Worms were exposed to different concentrations of the active ingredient (glyphosate in monoisopropylamine salt) and its commercial formulation. Reproductive capacity was evaluated through brood size, morphological analysis of oocytes and through the MD701 strain (bcIs39), which allows the visualization of germ cells in apoptosis. In addition, the metal composition in the commercial formulation was analyzed by ICP-MS. Only the commercial formulation of glyphosate showed significant negative effects on brood size, body length, oocyte size, and the number of apoptotic cells. Metal analysis showed the presence of Hg, Fe, Mn, Cu, Zn, As, Cd and Pb in the commercial formulation, which did not cause reprotoxicity at the concentrations found. However, metals can bio-accumulate in soil and water and cause environmental impacts. Finally, we demonstrated that the addition of inert ingredients increased the toxic profile of the active ingredient glyphosate in C. elegans, which reinforces the need of components description in the product labels. (C) 2019 Elsevier Ltd. All rights reserved.
Methylmercury (MeHg) is an environmental pollutant linked to many neurological defects, especially in developing individuals. The thioredoxin (TRX) system is a key redox regulator affected by MeHg toxicity, however the mechanisms and consequences of MeHg-induced dysfunction are not completely understood. This study evaluated the role of the TRX system in C. elegans susceptibility to MeHg during development. Worms lacking or overexpressing proteins from the TRX family were exposed to MeHg for 1 h at different developmental stage: L1, L4 and adult. Worms without cytoplasmic thioredoxin system exhibited age-specific susceptibility to MeHg when compared to wild-type (wt). This susceptibility corresponded partially to decreased total glutathione (GSH) levels and enhanced degeneration of dopaminergic neurons. In contrast, the overexpression of the cytoplasmic system TRX-1/TRXR-1 did not provide substantial protection against MeHg. Moreover, transgenic worms exhibited decreased protein expression for cytoplasmic thioredoxin reductase (TRXR-1). Both mitochondrial thioredoxin system TRX-2/TRXR-2, as well as other thioredoxin-like proteins: TRX-3, TRX-4, TRX-5 did not show significant role in C. elegans resistance to MeHg. Based on the current findings, the cytoplasmic thioredoxin system TRX-1/TRXR-1 emerges as an important age-sensitive protectant against MeHg toxicity in C. elegans.
Arsenic-containing hydrocarbons (AsHCs), a subgroup of arsenolipids (AsLs) occurring in fish and edible algae, possess a substantial neurotoxic potential in fully differentiated human brain cells. Previous in vivo studies indicating that AsHCs cross the blood–brain barrier of the fruit fly Drosophila melanogaster raised the question whether AsLs could also cross the vertebrate blood–brain barrier (BBB). In the present study, we investigated the impact of several representatives of AsLs (AsHC 332, AsHC 360, AsHC 444, and two arsenic-containing fatty acids, AsFA 362 and AsFA 388) as well as of their metabolites (thio/oxo-dimethylpropionic acid, dimethylarsinic acid) on porcine brain capillary endothelial cells (PBCECs, in vitro model for the blood–brain barrier). AsHCs exerted the strongest cytotoxic effects of all investigated arsenicals as they were up to fivefold more potent than the toxic reference species arsenite (iAsIII). In our in vitro BBB-model, we observed a slight transfer of AsHC 332 across the BBB after 6 h at concentrations that do not affect the barrier integrity. Furthermore, incubation with AsHCs for 72 h led to a disruption of the barrier at sub-cytotoxic concentrations. The subsequent immunocytochemical staining of three tight junction proteins revealed a significant impact on the cell membrane. Because AsHCs enhance the permeability of the in vitro blood–brain barrier, a similar behavior in an in vivo system cannot be excluded. Consequently, AsHCs might facilitate the transfer of accompanying foodborne toxicants into the brain.
Methylglyoxal (MG), a highly reactive dicarbonyl, interacts with proteins to form advanced glycation end products (AGEs). AGEs include a variety of compounds which were shown to have damaging potential and to accumulate in the course of different conditions such as diabetes mellitus and aging. After confirming collagen as a main target for MG modifications in vivo within the extracellular matrix, we show here that MG-collagen disrupts fibroblast redox homeostasis and induces endoplasmic reticulum (ER) stress and apoptosis. In particular, MG-collagen-induced apoptosis is associated with the activation of the PERK-eIF2 alpha pathway and caspase-12. MG-collagen contributes to altered redox homeostasis by directly generating hydrogen peroxide and oxygen-derived free radicals. The induction of ER stress in human fibroblasts was confirmed using collagen extracts isolated from old mice in which MG-derived AGEs were enriched. In conclusion, MG-derived AGEs represent one factor contributing to diminished fibroblast function during aging.
The toxicologically most relevant mercury (Hg) species for human exposure is methylmercury (MeHg). Thiomersal is a common preservative used in some vaccine formulations. The aim of this study is to get further mechanistic insight into the yet not fully understood neurotoxic modes of action of organic Hg species. Mercury species investigated include MeHgCl and thiomersal. Additionally HgCl2 was studied, since in the brain mercuric Hg can be formed by dealkylation of the organic species. As a cellular system astrocytes were used. In vivo astrocytes provide the environment necessary for neuronal function. In the present study, cytotoxic effects of the respective mercuricals increased with rising alkylation level and correlated with their cellular bioavailability. Further experiments revealed for all species at subcytotoxic concentrations no induction of DNA strand breaks, whereas all species massively increased H2O2-induced DNA strand breaks. This co-genotoxic effect is likely due to a disturbance of the cellular DNA damage response. Thus, at nanomolar, sub-cytotoxic concentrations, all three mercury species strongly disturbed poly(ADP-ribosyl)ation, a signalling reaction induced by DNA strand breaks. Interestingly, the molecular mechanism behind this inhibition seems to be different for the species. Since chronic PARP-1 inhibition is also discussed to sacrifice neurogenesis and learning abilities, further experiments on neurons and in vivo studies could be helpful to clarify whether the inhibition of poly(ADP-ribosyl)ation contributes to organic Hg induced neurotoxicity.
cis-Diamminedichloroplatinum(II) (Cisplatin) is one of the most important and frequently used cytostatic drugs for the treatment of various solid tumors. Herein, a laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) method incorporating a fast and simple sample preparation protocol was developed for the elemental mapping of Cisplatin in the model organism Caenorhabditis elegans (C. elegans). The method allows imaging of the spatially-resolved elemental distribution of platinum in the whole organism with respect to the anatomic structure in L4 stage worms at a lateral resolution of 5 μm. In addition, a dose- and time-dependent Cisplatin uptake was corroborated quantitatively by a total reflection X-ray fluorescence spectroscopy (TXRF) method, and the elemental mapping indicated that Cisplatin is located in the intestine and in the head of the worms. Better understanding of the distribution of Cisplatin in this well-established model organism will be instrumental in deciphering Cisplatin toxicity and pharmacokinetics. Since the cytostatic effect of Cisplatin is based on binding the DNA by forming intra- and interstrand crosslinks, the response of poly(ADP-ribose)metabolism enzyme 1 (pme-1) deletion mutants to Cisplatin was also examined. Loss of pme-1, which is the C. elegans ortholog of human poly(ADP-ribose) polymerase 1 (PARP-1) led to disturbed DNA damage response. With respect to survival and brood size, pme-1 deletion mutants were more sensitive to Cisplatin as compared to wildtype worms, while Cisplatin uptake was indistinguishable.
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.