199 results on '"Aschner, Michael"'
Search Results
2. Identification of the bacterial metabolite aerugine as potential trigger of human dopaminergic neurodegeneration.
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Ückert AK, Rütschlin S, Gutbier S, Wörz NC, Miah MR, Martins AC, Hauer I, Holzer AK, Meyburg B, Mix AK, Hauck C, Aschner M, Böttcher T, and Leist M
- Subjects
- Animals, Humans, Animals, Genetically Modified, Antioxidants metabolism, Neurons, Caenorhabditis elegans metabolism, Parkinson Disease
- Abstract
The causes of nigrostriatal cell death in idiopathic Parkinson's disease are unknown, but exposure to toxic chemicals may play some role. We followed up here on suggestions that bacterial secondary metabolites might be selectively cytotoxic to dopaminergic neurons. Extracts from Streptomyces venezuelae were found to kill human dopaminergic neurons (LUHMES cells). Utilizing this model system as a bioassay, we identified a bacterial metabolite known as aerugine (C
10 H11 NO2 S; 2-[4-(hydroxymethyl)-4,5-dihydro-1,3-thiazol-2-yl]phenol) and confirmed this finding by chemical re-synthesis. This 2-hydroxyphenyl-thiazoline compound was previously shown to be a product of a wide-spread biosynthetic cluster also found in the human microbiome and in several pathogens. Aerugine triggered half-maximal dopaminergic neurotoxicity at 3-4 µM. It was less toxic for other neurons (10-20 µM), and non-toxic (at <100 µM) for common human cell lines. Neurotoxicity was completely prevented by several iron chelators, by distinct anti-oxidants and by a caspase inhibitor. In the Caenorhabditis elegans model organism, general survival was not affected by aerugine concentrations up to 100 µM. When transgenic worms, expressing green fluorescent protein only in their dopamine neurons, were exposed to aerugine, specific neurodegeneration was observed. The toxicant also exerted functional dopaminergic toxicity in nematodes as determined by the "basal slowing response" assay. Thus, our research has unveiled a bacterial metabolite with a remarkably selective toxicity toward human dopaminergic neurons in vitro and for the dopaminergic nervous system of Caenorhabditis elegans in vivo. These findings suggest that microbe-derived environmental chemicals should be further investigated for their role in the pathogenesis of Parkinson's disease., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)- Published
- 2023
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3. Mitochondria in the Spotlight: C. elegans as a Model Organism to Evaluate Xenobiotic-Induced Dysfunction.
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Martins AC, Virgolini MB, Ávila DS, Scharf P, Li J, Tinkov AA, Skalny AV, Bowman AB, Rocha JBT, and Aschner M
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- Humans, Animals, Mitochondria, Cell Respiration, Apoptosis, Mammals, Caenorhabditis elegans, Xenobiotics
- Abstract
Mitochondria play a crucial role in cellular respiration, ATP production, and the regulation of various cellular processes. Mitochondrial dysfunctions have been directly linked to pathophysiological conditions, making them a significant target of interest in toxicological research. In recent years, there has been a growing need to understand the intricate effects of xenobiotics on human health, necessitating the use of effective scientific research tools. Caenorhabditis elegans ( C. elegans ), a nonpathogenic nematode, has emerged as a powerful tool for investigating toxic mechanisms and mitochondrial dysfunction. With remarkable genetic homology to mammals, C. elegans has been used in studies to elucidate the impact of contaminants and drugs on mitochondrial function. This review focuses on the effects of several toxic metals and metalloids, drugs of abuse and pesticides on mitochondria, highlighting the utility of C. elegans as a model organism to investigate mitochondrial dysfunction induced by xenobiotics. Mitochondrial structure, function, and dynamics are discussed, emphasizing their essential role in cellular viability and the regulation of processes such as autophagy, apoptosis, and calcium homeostasis. Additionally, specific toxins and toxicants, such as arsenic, cadmium, and manganese are examined in the context of their impact on mitochondrial function and the utility of C. elegans in elucidating the underlying mechanisms. Furthermore, we demonstrate the utilization of C. elegans as an experimental model providing a promising platform for investigating the intricate relationships between xenobiotics and mitochondrial dysfunction. This knowledge could contribute to the development of strategies to mitigate the adverse effects of contaminants and drugs of abuse, ultimately enhancing our understanding of these complex processes and promoting human health.
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- 2023
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4. A Reliable Method Based on Liquid Chromatography-Tandem Mass Spectrometry for the Simultaneous Quantification of Neurotransmitters in Caenorhabditis elegans .
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Weishaupt AK, Kubens L, Ruecker L, Schwerdtle T, Aschner M, and Bornhorst J
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- Animals, Chromatography, Liquid methods, Dopamine analysis, Acetylcholine, Neurotransmitter Agents chemistry, gamma-Aminobutyric Acid, Chromatography, High Pressure Liquid methods, Mammals, Caenorhabditis elegans, Tandem Mass Spectrometry methods
- Abstract
Neurotransmitters like dopamine (DA), serotonin (SRT), γ-aminobutyric acid (GABA) and acetylcholine (ACh) are messenger molecules that play a pivotal role in transmitting excitation between neurons across chemical synapses, thus enabling complex processes in the central nervous system (CNS). Balance in neurotransmitter homeostasis is essential, and altered neurotransmitter levels are associated with various neurological disorders, e.g., loss of dopaminergic neurons (Parkinson's disease) or altered ACh synthesis (Alzheimer's disease). Therefore, it is crucial to possess adequate tools to assess precise neurotransmitter levels, and to apply targeted therapies. An established in vivo model to study neurotoxicity is the model organism Caenorhabditis elegans ( C. elegans ), as its neurons have been well characterized and functionally are analogous to mammals. We have developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method including a sample preparation assuring neurotransmitter stability, which allows a simultaneous neurotransmitter quantification of DA, SRT, GABA and ACh in C. elegans , but can easily be applied to other matrices. LC-MS/MS combined with isotope-labeled standards is the tool of choice, due to its otherwise unattainable sensitivity and specificity. Using C. elegans together with our analytically validated and verified method provides a powerful tool to evaluate mechanisms of neurotoxicity, and furthermore to identify possible therapeutic approaches.
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- 2023
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5. The Human LRRK2 Modulates the Age-Dependent Effects of Developmental Methylmercury Exposure in Caenorhabditis elegans.
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Ke T, Tinkov AA, Skalny AV, Santamaria A, Farina M, Rocha JBT, Bowman AB, and Aschner M
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- Aged, Animals, Dopamine metabolism, Dopamine Plasma Membrane Transport Proteins genetics, Dopamine Plasma Membrane Transport Proteins metabolism, Dopaminergic Neurons, Humans, Leucine pharmacology, Leucine-Rich Repeat Serine-Threonine Protein Kinase-2 genetics, Leucine-Rich Repeat Serine-Threonine Protein Kinase-2 metabolism, Mutation genetics, Tyrosine 3-Monooxygenase metabolism, Caenorhabditis elegans, Methylmercury Compounds toxicity
- Abstract
Methylmercury (MeHg) neurotoxicity exhibits age-dependent effects with a latent and/or persistent neurotoxic effect on aged animals. Individual susceptibility to MeHg neurotoxicity is governed by both exposure duration and genetic factors that can magnify or mitigate the pathologic processes associated with this exposure. We previously showed the G2019S mutation of leucine-rich repeat kinase 2 (LRRK2) modulates the response of worms to high levels of MeHg, mitigating its effect on neuronal morphology in pre-vesicles in cephalic (CEP) dopaminergic neurons. Here we sought to better understand the long-term effects of MeHg exposure at low levels (100-fold lower than that in our previous report) and the modulatory role of the LRRK2 mutation. Worms exposed to MeHg (10 or 50 nM) at the larval stage (L1 stage) were compared at adult stages (young age: day 1 adult; middle age: day 5 adult; old age: day 10 adult) for the swimming speeds in M9 buffer, moving speeds during locomotion on an OP50-seeded plate, and the numbers of CEP dopaminergic pre-vesicles, vesicular structures originating from the dendrites of CEP for exportation of cellular content. In addition, the expression levels of Caenorhabditis elegans homologs of dopamine transporter (dat-1) and tyrosine hydroxylase (cat-2) were also analyzed at these adult stages. Our data showed that swimming speeds were reduced in wild-type worms at the day 10 adult stage at 50 nM MeHg level; yet, reduced swimming speeds were noted in the G2019S LRRK2 transgenic line upon MeHg exposures as low as 10 nM. Compared to locomotor speeds, swimming speeds appear to be more sensitive to the behavioral effects of developmental MeHg exposures, as the locomotor speeds were largely intact and indistinguishable from controls following MeHg exposures. Furthermore, we showed an age-dependent modulation of dat-1 and cat-2 expressions, which could also be modified by the LRRK2 mutation. Although MeHg exposures did not change the number of pre-vesicles, the LRRK2 mutation was associated with increased numbers of pre-vesicles in aged worms. Our data suggest that the latent behavioral effects of MeHg are sensitized by the G2019S LRRK2 mutation, and the underlying mechanism likely involves age-dependent changes in dopaminergic signaling., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)
- Published
- 2022
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6. Methylmercury exposure-induced reproductive effects are mediated by dopamine in Caenorhabditis elegans.
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Ke T, Santamaria A, Junior FB, Rocha JBT, Bowman AB, and Aschner M
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- Animals, Dopamine metabolism, Humans, Male, Reproduction, Semen metabolism, Caenorhabditis elegans metabolism, Methylmercury Compounds toxicity
- Abstract
Methylmercury (MeHg) is a neurotoxicant that exists in the natural environment, which level can be greatly increased because of human activity. MeHg exposures have the risk of being detrimental to the development of the nervous system. Studies on MeHg toxicity have largely focused on the mechanisms of its neurotoxicity following developmental exposures. Additionally, reproductive toxicity of developmental MeHg exposures has been noted in rodent models. The model organism Caenorhabditis elegans (C. elegans) is a self-fertilizing animal which has a short lifespan around 20 days. Most C. elegans are hermaphrodites that can generate both sperm and oocytes. To investigate the effects of developmental MeHg exposures on the reproduction in C. elegans, larvae stage 1 worms were exposed to MeHg (0, 0.01 or 0.05 μM) for 24 h. The laid eggs and oocytes were compared during each day at adult stages for 6 days. We showed that MeHg exposure significantly induced an increased number of eggs in day 1 adults without an effect on the timing of egg laying or the total number of eggs or oocytes over the 6-day period. The expression of dat-1 and cat-2 and dopamine levels were increased in worms exposed to MeHg. Supplementation with 100 μM dopamine recapitulated the effect of MeHg on the number of eggs present in day 1 adults. Furthermore, the effect of MeHg on the number of eggs was abrogated in the cat-2 mutant worms CB1112. The number of oocytes in the 6-day adult stages was decreased by MeHg in the dat-1 mutant RM2702. MeHg exposures did not change the mating rate or the number of offspring from mating. Combined, these novel findings show that developmental exposure to low levels of MeHg has limited effects on the reproduction in C. elegans. Furthermore, our data support a modulatory role of dopamine in MeHg-induced effects on reproduction in this model system., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)
- Published
- 2022
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7. Developmental lead exposure affects dopaminergic neuron morphology and modifies basal slowing response in Caenorhabditis elegans: Effects of ethanol.
- Author
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Albrecht PA, Fernandez-Hubeid LE, Deza-Ponzio R, Martins AC, Aschner M, and Virgolini MB
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- Animals, Dopaminergic Neurons, Ethanol toxicity, Lead toxicity, Tyrosine 3-Monooxygenase, Caenorhabditis elegans, Caenorhabditis elegans Proteins genetics
- Abstract
Lead (Pb) and ethanol (EtOH) are neurotoxicants that affect the dopaminergic (DAergic) system. We first sought to assess the morphology of the DAergic neurons in the Caenorhabditis elegans BY200 strain. The results demonstrated dose-dependent damage in these neurons induced by developmental Pb exposure. Secondly, transgenic worms exposed to 24 μM Pb and administered with 200 mM EtOH were evaluated in the basal slowing response (BSR). Pb induced impairment in the BSR in the wild-type strain that did not improve in response to EtOH, an effect also observed in strains that lack the DOP-1, DOP-2, and DOP-3 receptors. The animals that overexpress tyrosine hydroxylase (TH), or lack the vesicular transport (VMAT) showed a Pb-induced impairment in the BSR that seemed to improve after EtOH. Interestingly, a dramatic impairment in the BSR was observed in the Pb group in strains lacking the DOP-4 receptor, resembling the response of the TH-deficient strain, an effect that in both cases showed a non-significant reversal by EtOH. These results suggest that the facilitatory effect of EtOH on the impaired BSR observed in Pb-exposed null mutant strains may be the result of a compensatory effect in the altered DAergic synapse present in these animals., (Copyright © 2022 Elsevier B.V. All rights reserved.)
- Published
- 2022
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8. Effects of Manganese on Genomic Integrity in the Multicellular Model Organism Caenorhabditis elegans .
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Nicolai MM, Weishaupt AK, Baesler J, Brinkmann V, Wellenberg A, Winkelbeiner N, Gremme A, Aschner M, Fritz G, Schwerdtle T, and Bornhorst J
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- Animals, Caenorhabditis elegans drug effects, Caenorhabditis elegans genetics, Caenorhabditis elegans Proteins genetics, DNA Repair, Dose-Response Relationship, Drug, Models, Animal, Mortality, Oxidative Stress drug effects, Poly(ADP-ribose) Polymerases genetics, Time Factors, Caenorhabditis elegans growth & development, DNA Damage, GTPase-Activating Proteins genetics, Manganese toxicity
- Abstract
Although manganese (Mn) is an essential trace element, overexposure is associated with Mn-induced toxicity and neurological dysfunction. Even though Mn-induced oxidative stress is discussed extensively, neither the underlying mechanisms of the potential consequences of Mn-induced oxidative stress on DNA damage and DNA repair, nor the possibly resulting toxicity are characterized yet. In this study, we use the model organism Caenorhabditis elegans to investigate the mode of action of Mn toxicity, focusing on genomic integrity by means of DNA damage and DNA damage response. Experiments were conducted to analyze Mn bioavailability, lethality, and induction of DNA damage. Different deletion mutant strains were then used to investigate the role of base excision repair (BER) and dePARylation (DNA damage response) proteins in Mn-induced toxicity. The results indicate a dose- and time-dependent uptake of Mn, resulting in increased lethality. Excessive exposure to Mn decreases genomic integrity and activates BER. Altogether, this study characterizes the consequences of Mn exposure on genomic integrity and therefore broadens the molecular understanding of pathways underlying Mn-induced toxicity. Additionally, studying the basal poly(ADP-ribosylation) (PARylation) of worms lacking poly(ADP-ribose) glycohydrolase (PARG) parg-1 or parg-2 (two orthologue of PARG), indicates that parg-1 accounts for most of the glycohydrolase activity in worms.
- Published
- 2021
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9. Caenorhabditis elegans as a model for studies on quinolinic acid-induced NMDAR-dependent glutamatergic disorders.
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Limana da Silveira T, Lopes Machado M, Bicca Obetine Baptista F, Farina Gonçalves D, Duarte Hartmann D, Marafiga Cordeiro L, Franzen da Silva A, Lenz Dalla Corte C, Aschner M, and Antunes Soares FA
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- 1-Octanol pharmacology, Adenosine Triphosphate biosynthesis, Animals, Animals, Genetically Modified, Citrate (si)-Synthase metabolism, Disease Models, Animal, Glutamic Acid metabolism, Humans, Kynurenine metabolism, Motor Activity drug effects, Neurodegenerative Diseases chemically induced, Neurodegenerative Diseases genetics, Signal Transduction drug effects, Synaptic Transmission, Amino Acid Metabolism, Inborn Errors chemically induced, Caenorhabditis elegans physiology, Quinolinic Acid, Receptors, N-Methyl-D-Aspartate drug effects, Receptors, N-Methyl-D-Aspartate genetics
- Abstract
Quinolinic acid (QUIN) is an agonist of the neurotransmitter glutamate (Glu) capable of binding to N-methyl-D-aspartate receptors (NMDAR) increasing glutamatergic signaling. QUIN is known for being an endogenous neurotoxin, able to induce neurodegeneration. In Caenorhabditis elegans, the mechanism by which QUIN induces behavioral and metabolic toxicity has not been fully elucidated. The effects of QUIN on behavioral and metabolic parameters in nmr-1 and nmr-2 NMDA receptors in transgenic and wild-type (WT) worms were performed to decipher the pathway by which QUIN exerts its toxicity. QUIN increased locomotion parameters such as wavelength and movement amplitude medium, as well as speed and displacement, without modifying the number of body bends in an NMDAR-dependent-manner. QUIN increased the response time to the chemical stimulant 1-octanol, which is modulated by glutamatergic neurotransmission in the ASH neuron. Brood size increased after exposure to QUIN, dependent upon nmr-2/NMDA-receptor, with no change in lifespan. Oxygen consumption, mitochondrial membrane potential, and the flow of coupled and unbound electrons to ATP production were reduced by QUIN in wild-type animals, but did not alter citrate synthase activity, altering the functionality but the mitochondrial viability. Notably, QUIN modified fine locomotor and chemosensory behavioral parameters, as well as metabolic parameters, analogous to previously reported effects in mammals. Our results indicate that QUIN can be used as a neurotoxin to elicit glutamatergic dysfunction in C. elegans in a way analogous to other animal models., (Copyright © 2021 Elsevier Inc. All rights reserved.)
- Published
- 2021
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10. Latent alterations in swimming behavior by developmental methylmercury exposure are modulated by the homolog of tyrosine hydroxylase in Caenorhabditis elegans.
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Ke T, Prince LM, Bowman AB, and Aschner M
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- Animals, Caenorhabditis elegans embryology, Caenorhabditis elegans physiology, Dose-Response Relationship, Drug, Larva drug effects, Methylmercury Compounds administration & dosage, Swimming, Tyrosine 3-Monooxygenase metabolism, Caenorhabditis elegans drug effects, Methylmercury Compounds toxicity, Tyrosine 3-Monooxygenase drug effects
- Abstract
Methylmercury (MeHg) is a persistent environmental neurotoxicant that may cause adverse neurodevelopmental effects. Previous studies showed that developmental MeHg exposure caused damage to brain functions that were unmasked after a silent period of years or decades. However, the underlying mechanisms of the latent neurotoxicity associated with MeHg exposure from earlier developmental stages have yet to be fully understood. Herein, we established a Caenorhabditis elegans (C. elegans) model of developmental MeHg latent toxicity. Synchronized L1 stage worms were exposed to MeHg (0, 0.05, 0.5 and 5 μM) for 48 h. Swimming moving speeds at adulthood were analyzed in worms exposed to MeHg exposure at early larvae stages. Worms developmentally exposed to MeHg had a significant decline in swimming moving speed on day 10 adult stage, but not on day 1 or 5 adult stage, even though the mercury level in the worms exposed to 0.05 or 0.5 μM MeHg were below the quantification limit on day 10 adult. Day 10 adult worms treated with MeHg showed a significant decrease in bending angle and bending frequency during swimming. Furthermore, their reduced moving speeds tended to increase during the 300-s swimming experiment. Dopamine signaling is known to be involved in the regulation of worms' moving speed. Accordingly, the moving speed of worms with cat-2 (mammalian tyrosine hydroxylase homolog) mutation or dat-1 deletion were assayed on day 10 adult. The cat-2 mutant worms did not show a decline in moving speeds, body bends or bending angles during swimming on day 10 adult stage. Analyses of moving speeds of worms with dat-1 deletion showed that the moving speeds were further reduced after MeHg exposure. However, the effects of MeHg and dat-1 deletion were not synergistic, as the interaction between these parameters did not attain statistical significance. Altogether, our results suggest that developmental MeHg exposure reduced moving speed, and this latent toxicity was less pronounced in the context of deficient production of dopamine synthesis. Tyrosine hydroxylase plays an important role in regulating dopamine-mediated modulation of neurobehavioral functions. These findings uncovered a pivotal role of dopamine and its metabolism in the latent neurotoxic effects of MeHg., (Copyright © 2021 Elsevier Inc. All rights reserved.)
- Published
- 2021
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11. Overview of Chemotaxis Behavior Assays in Caenorhabditis elegans.
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Queirós L, Marques C, Pereira JL, Gonçalves FJM, Aschner M, and Pereira P
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- Animals, Cues, Ecosystem, Humans, Neurons, Caenorhabditis elegans, Chemotaxis
- Abstract
Environmental pollution related to anthropogenic pressures, and the associated repercussions on public health, represent a worldwide problem. Thus, the study of the effects that environmental contaminants can pose to natural ecosystems and human health is of vital importance. Laboratory model organisms such as Caenorhabditis elegans have played a significant role in clarifying multilevel effects of those agents. Although the evaluation of contaminant effects at the behavioral level of organisms is an emerging approach in ecotoxicology, studies assessing chemotaxis behavior in C. elegans within the ecotoxicological research context are still scarce. Chemotaxis studies in C. elegans have contributed to the understanding of both the neuronal mechanisms involved in the behavioral effects triggered by environmental cues and the impact of contaminants on natural ecosystems. Its compact and well-characterized nervous system, as well as the availability of transgenic strains and molecular tools, allows a detailed examination of behavioral, molecular, and genetic chemosensation mechanisms. This overview provides a summary and general comparison of methods used to measure chemotaxis behavior in C. elegans, with the aim of helping researchers select the most suitable approach in their chemotaxis studies. We compare methods based on the type of chemical tested, advantages and drawbacks of the different approaches, and specific experimental goals. Lastly, we hope to encourage the evaluation of C. elegans chemotaxis behavior in ecotoxicology studies, as well as its potential integration in standardized protocols assessing environmental quality. © 2021 Wiley Periodicals LLC., (© 2021 Wiley Periodicals LLC.)
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- 2021
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12. Conjugates of desferrioxamine and aromatic amines improve markers of iron-dependent neurotoxicity.
- Author
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Carvalho RRV, Peres TV, Liria CW, Machini MT, Aschner M, and Espósito BP
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- Acetylcholinesterase metabolism, Amines chemistry, Amyloid beta-Peptides antagonists & inhibitors, Amyloid beta-Peptides metabolism, Animals, Antioxidants chemical synthesis, Antioxidants chemistry, Biphenyl Compounds antagonists & inhibitors, Caenorhabditis elegans enzymology, Cholinesterase Inhibitors chemical synthesis, Cholinesterase Inhibitors chemistry, Deferoxamine chemistry, Humans, Iron Chelating Agents chemical synthesis, Iron Chelating Agents chemistry, Molecular Structure, Picrates antagonists & inhibitors, Protein Aggregates drug effects, Amines pharmacology, Antioxidants pharmacology, Caenorhabditis elegans drug effects, Cholinesterase Inhibitors pharmacology, Deferoxamine pharmacology, Iron Chelating Agents pharmacology
- Abstract
Alzheimer's Disease (AD) is a complex neurodegenerative disorder associated in some instances with dyshomeostasis of redox-active metal ions, such as copper and iron. In this work, we investigated whether the conjugation of various aromatic amines would improve the pharmacological efficacy of the iron chelator desferrioxamine (DFO). Conjugates of DFO with aniline (DFOANI), benzosulfanylamide (DFOBAN), 2-naphthalenamine (DFONAF) and 6-quinolinamine (DFOQUN) were obtained and their properties examined. DFOQUN had good chelating activity, promoted a significant increase in the inhibition of β-amyloid peptide aggregation when compared to DFO, and also inhibited acetylcholinesterase (AChE) activity both in vitro and in vivo (Caenorhabditis elegans). These data indicate that the covalent conjugation of a strong iron chelator to an AChE inhibitor offers a powerful approach for the amelioration of iron-induced neurotoxicity symptoms.
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- 2021
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13. Nutritive Manganese and Zinc Overdosing in Aging C. elegans Result in a Metallothionein-Mediated Alteration in Metal Homeostasis.
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Baesler J, Michaelis V, Stiboller M, Haase H, Aschner M, Schwerdtle T, Sturzenbaum SR, and Bornhorst J
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- Aging physiology, Animals, Animals, Genetically Modified, Biological Availability, Caenorhabditis elegans physiology, Caenorhabditis elegans Proteins genetics, Dopaminergic Neurons drug effects, Dopaminergic Neurons pathology, Drug Overdose metabolism, Homeostasis drug effects, Homeostasis genetics, Manganese administration & dosage, Manganese pharmacokinetics, Metallothionein genetics, Mutation, Toxicity Tests, Chronic, Ubiquitin-Protein Ligases genetics, Zinc administration & dosage, Zinc pharmacokinetics, Aging drug effects, Caenorhabditis elegans drug effects, Caenorhabditis elegans Proteins metabolism, Manganese adverse effects, Metallothionein metabolism, Zinc adverse effects
- Abstract
Scope: Manganese (Mn) and zinc (Zn) are not only essential trace elements, but also potential exogenous risk factors for various diseases. Since the disturbed homeostasis of single metals can result in detrimental health effects, concerns have emerged regarding the consequences of excessive exposures to multiple metals, either via nutritional supplementation or parenteral nutrition. This study focuses on Mn-Zn-interactions in the nematode Caenorhabditis elegans (C. elegans) model, taking into account aspects related to aging and age-dependent neurodegeneration., Methods and Results: Chronic co-exposure of C. elegans to Mn and Zn increases metal uptake, exceeding levels of single metal exposures. Supplementation with Mn and/or Zn also leads to an age-dependent increase in metal content, a decline in overall mRNA expression, and metal co-supplementation induced expression of target genes involved in Mn and Zn homeostasis, in particular metallothionein 1 (mtl-1). Studies in transgenic worms reveal that mtl-1 played a prominent role in mediating age- and diet-dependent alterations in metal homeostasis. Metal dyshomeostasis is further induced in parkin-deficient nematodes (Parkinson's disease (PD) model), but this did not accelerate the age-dependent dopaminergic neurodegeneration., Conclusions: A nutritive overdose of Mn and Zn can alter interactions between essential metals in an aging organism, and metallothionein 1 acts as a potential protective modulator in regulating homeostasis., (© 2021 The Authors. Molecular Nutrition & Food Research published by Wiley-VCH GmbH.)
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- 2021
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14. Evaluations of Environmental Pollutant-Induced Mitochondrial Toxicity Using Caenorhabditis elegans as a Model System.
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Zheng F, Aschner M, and Li H
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- Adenosine Triphosphate metabolism, Animals, Caenorhabditis elegans genetics, Caenorhabditis elegans metabolism, Gene Expression drug effects, Mitochondria genetics, Mitochondria metabolism, Mitochondria ultrastructure, Oxygen Consumption drug effects, Reactive Oxygen Species metabolism, Toxicity Tests methods, Caenorhabditis elegans drug effects, Environmental Pollutants toxicity, Mitochondria drug effects
- Abstract
Environmental pollutants inevitably exert adverse effects on humans and other species. Quick identification and in-depth characterization of the pollutants are requisite objectives for clinicians and environmental health scientists. The nematode Caenorhabditis elegans has been utilized as a model organism for toxicity evaluation of environmental pollutants, due to its transparency, short lifespan, entire genome sequencing, and economical characteristics. However, few researchers have systematically addressed mitochondrial toxicity in response to toxicants, despite the critical role mitochondria play in energy production and respiration, as well as the generation of reactive oxygen species. Mitochondria are vulnerable to environmental pollutants, and their dysfunction contributes to cellular damage and toxicity in plethora of diseases. Here, we describe methods in step-by-step for mitochondrial toxicity evaluation in response to pollutants, including exposure of C. elegans to toxicants, mitochondrial ROS detection, mitochondrial morphology analysis, mitochondrial function analysis, such as ATP production and oxygen consumption, and gene expression studies, with the application of corresponding genetically modified strains.
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- 2021
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15. Haloperidol Interactions with the dop-3 Receptor in Caenorhabditis elegans.
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Krum BN, Martins AC Jr, Queirós L, Ferrer B, Milne GL, Soares FAA, Fachinetto R, and Aschner M
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- Animals, Caenorhabditis elegans drug effects, Caenorhabditis elegans genetics, Dopamine metabolism, Dopaminergic Neurons drug effects, Dopaminergic Neurons metabolism, Gene Expression Regulation drug effects, Haloperidol pharmacology, Locomotion drug effects, Longevity drug effects, Models, Biological, Mutation genetics, Nerve Degeneration pathology, RNA, Messenger genetics, RNA, Messenger metabolism, Caenorhabditis elegans metabolism, Caenorhabditis elegans Proteins metabolism, Haloperidol metabolism, Receptors, Dopamine D2 metabolism
- Abstract
Haloperidol is a typical antipsychotic drug commonly used to treat a broad range of psychiatric disorders related to dysregulations in the neurotransmitter dopamine (DA). DA modulates important physiologic functions and perturbations in Caenorhabditis elegans (C. elegans) and, its signaling have been associated with alterations in behavioral, molecular, and morphologic properties in C. elegans. Here, we evaluated the possible involvement of dopaminergic receptors in the onset of these alterations followed by haloperidol exposure. Haloperidol increased lifespan and decreased locomotor behavior (basal slowing response, BSR, and locomotion speed via forward speed) of the worms. Moreover, locomotion speed recovered to basal conditions upon haloperidol withdrawal. Haloperidol also decreased DA levels, but it did not alter neither dop-1, dop-2, and dop-3 gene expression, nor CEP dopaminergic neurons' morphology. These effects are likely due to haloperidol's antagonism of the D2-type DA receptor, dop-3. Furthermore, this antagonism appears to affect mechanistic pathways involved in the modulation and signaling of neurotransmitters such as octopamine, acetylcholine, and GABA, which may underlie at least in part haloperidol's effects. These pathways are conserved in vertebrates and have been implicated in a range of disorders. Our novel findings demonstrate that the dop-3 receptor plays an important role in the effects of haloperidol.
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- 2021
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16. Cephalic Neuronal Vesicle Formation is Developmentally Dependent and Modified by Methylmercury and sti-1 in Caenorhabditis elegans.
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Ke T, Santamaria A, Rocha JBT, Tinkov A, Bornhorst J, Bowman AB, and Aschner M
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- Animals, Animals, Genetically Modified, Caenorhabditis elegans genetics, Dendrites pathology, Dendrites ultrastructure, Dopaminergic Neurons pathology, Dopaminergic Neurons ultrastructure, Gene Knockout Techniques, Head innervation, Male, Caenorhabditis elegans drug effects, Caenorhabditis elegans Proteins genetics, Dendrites drug effects, Dopaminergic Neurons drug effects, Heat-Shock Proteins genetics, Methylmercury Compounds toxicity
- Abstract
Methylmercury (MeHg) is a potent neurotoxicant. The mechanisms underlying MeHg-induced neurotoxicity are not fully understood. Several studies have shown that protein chaperones are involved in MeHg toxicity. The protein co-chaperone, stress inducible protein 1 (STI-1), has important functions in protein quality control of the chaperone pathway. In the current study, dopaminergic (DAergic) cephalic (CEP) neuronal morphology was evaluated in the Caenorhabditis elegans (C. elegans) sti-1 knockout strain. In the control OH7193 strain (dat-1::mCherry + ttx-3::mCherry), we characterized the morphology of CEP neurons by checking the presence of attached vesicles and unattached vesicles to the CEP dendrites. We showed that the attached vesicles were only present in adult stage worms; whereas they were absent in the younger L3 stage worms. In the sti-1 knockout strain, MeHg treatment significantly altered the structures of CEP dendrites with discontinuation of mCherry fluorescence and shrinkage of CEP soma, as compared to the control. 12 h post treatment on MeHg-free OP50-seeded plates, the discontinuation of mCherry fluorescence of CEP dendrites in worms treated with 0.05 or 0.5 µM MeHg returned to levels statistically indistinguishable from control, while in worms treated with 5 µM MeHg a higher percentage of discontinuation of mCherry fluorescence persisted. Despite this strong effect by 5 µM MeHg, CEP attached vesicles were increased upon 0.05 or 0.5 µM MeHg treatment, yet unaffected by 5 µM MeHg. The CEP attached vesicles of sti-1 knockout strain were significantly increased shortly after MeHg treatment, but were unaffected 48 h post treatment. In addition, there was a significant interactive effect of MeHg and sti-1 on the number of attached vesicles. Knock down sti-1 via RNAi did not alter the number of CEP attached vesicles. Taking together, our data suggests that the increased occurrence of attached vesicles in adult stage worms could initiate a substantial loss of membrane components of CEP dendrites following release of vesicles, leading to the discontinuation of mCherry fluorescence, and the formation of CEP attached vesicles could be regulated by sti-1 to remove cellular debris for detoxification.
- Published
- 2020
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17. Drp-1-Dependent Mitochondrial Fragmentation Contributes to Cobalt Chloride-Induced Toxicity in Caenorhabditis elegans.
- Author
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Zheng F, Chen P, Li H, and Aschner M
- Subjects
- Animals, Chlorides, Dynamins metabolism, Mitochondria metabolism, Oxidative Stress, Caenorhabditis elegans metabolism, Caenorhabditis elegans Proteins metabolism, Cobalt toxicity
- Abstract
Excess cobalt may lead to metallosis, characterized by sensorineural hearing loss, visual, and cognitive impairment, and peripheral neuropathy. In the present study, we sought to address the molecular mechanisms of cobalt-induced neurotoxicity, using Caenorhabditis elegans as an experimental model. Exposure to cobalt chloride for 2 h significantly decreased the survival rate and lifespan in nematodes. Cobalt chloride exposure led to increased oxidative stress and upregulation of glutathione S-transferase 4. Consistently, its upstream regulator skn-1, a mammalian homolog of the nuclear factor erythroid 2-related factor 2, was activated. Among the mRNAs examined by quantitative real-time polymerase chain reactions, apoptotic activator egl-1, proapoptotic gene ced-9, autophagic (bec-1 and lgg-1), and mitochondrial fission regulator drp-1 were significantly upregulated upon cobalt exposure, concomitant with mitochondrial fragmentation, as determined by confocal microscopy. Moreover, drp-1 inhibition suppressed the cobalt chloride-induced reactive oxygen species generation, growth defects, and reduced mitochondrial fragmentation. Our novel findings suggest that the acute toxicity of cobalt is mediated by mitochondrial fragmentation and drp-1 upregulation., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)
- Published
- 2020
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18. Metal-induced neurotoxicity in a RAGE-expressing C. elegans model.
- Author
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Lawes M, Pinkas A, Frohlich BA, Iroegbu JD, Ijomone OM, and Aschner M
- Subjects
- Animals, Animals, Genetically Modified, Cadmium Chloride, Cadmium Poisoning etiology, Cadmium Poisoning genetics, Cadmium Poisoning pathology, Caenorhabditis elegans genetics, Chlorides, Dopaminergic Neurons drug effects, Dopaminergic Neurons pathology, Glucose toxicity, Manganese Compounds, Manganese Poisoning etiology, Manganese Poisoning genetics, Manganese Poisoning pathology, Receptor for Advanced Glycation End Products genetics, Serotonergic Neurons drug effects, Serotonergic Neurons pathology, Cadmium Poisoning metabolism, Caenorhabditis elegans metabolism, Dopaminergic Neurons metabolism, Manganese Poisoning metabolism, Nerve Degeneration, Receptor for Advanced Glycation End Products metabolism, Serotonergic Neurons metabolism
- Abstract
Environmental and occupational metal exposure poses serious global concerns. Metal exposure have severally been associated with neurotoxicity and brain damage. Furthermore, receptor for advanced glycation end products (RAGE) is also implicated in neurological disorders, particularly those with altered glucose metabolism. Here, we examine potential compounding effect of metal exposure and RAGE expression on dopamine (DA) and serotonin (SER) neurons in C. elegans. In addition, we evaluate the effect of RAGE expression on DA and SER neurons in hyperglycemic conditions. Newly generated RAGE-expressing C. elegans tagged with green fluorescent proteins (GFP) in DAergic and SERergic neurons were treated with cadmium (Cd) or manganese (Mn). Additionally, the RAGE-expressing worms were also exposed to high glucose conditions. Results showed metals induced neurodegeneration both in the presence and absence of RAGE expression, but the manner of degeneration differed between Cd and Mn treated nematodes. Furthermore, RAGE-expressing worms showed significant neurodegeneration in both DAergic and SERergic neurons. Our results indicate co-occurrence of metal exposure and RAGE expression can induce neurodegeneration. Additionally, we show that RAGE expression can exacerbate hyperglycemic induced neurodegeneration., (Copyright © 2020. Published by Elsevier B.V.)
- Published
- 2020
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19. Generating Bacterial Foods in Toxicology Studies with Caenorhabditis elegans.
- Author
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Ke T, Santamaría A, Tinkov AA, Bornhorst J, and Aschner M
- Subjects
- Animals, Caenorhabditis elegans drug effects, Ecosystem, Feeding Behavior, Microbial Viability, Nutritive Value, Caenorhabditis elegans metabolism, Diet, Escherichia coli metabolism, Toxicity Tests
- Abstract
Caenorhabditis elegans is a free-living animal that is used as a powerful experimental model in biological sciences. The natural habitat of the animal are areas rich in material from rotting plants or fruits being decomposed by a growing number of microorganisms. The ecology of the natural habitat of C. elegans is a complex interactive network involving many species, including numerous types of bacteria, viruses, fungi, slugs, snails, and isopods, among which bacteria play multifaceted roles in the natural history of C. elegans. Under laboratory conditions, C. elegans is routinely cultured in a petri dish filled with solidified agar and seeded with Escherichia coli strain OP50, the latter offering an alternative model to study the interaction between bacteria and host. Because of the clear advantages of generating specific bacterial foods for mechanistic studies in C. elegans, it is important to develop a robust protocol to generate high-quality bacterial foods commensurate with experimental requirements. Based on previous work by us and others, herein we present a protocol on how to generate these optimal bacterial food-based research tools. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Preparing concentrated E. coli OP50 Basic Protocol 2: Titrating bacteria concentration Basic Protocol 3: Generating dead bacterial food by heating Basic Protocol 4: Generating dead bacterial food by antibiotics Basic Protocol 5: Feeding C. elegans with bacterial foods in liquid., (© 2020 John Wiley & Sons, Inc.)
- Published
- 2020
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20. Toxicity of three types of arsenolipids: species-specific effects in Caenorhabditis elegans.
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Bornhorst J, Ebert F, Meyer S, Ziemann V, Xiong C, Guttenberger N, Raab A, Baesler J, Aschner M, Feldmann J, Francesconi K, Raber G, and Schwerdtle T
- Subjects
- Animals, Caenorhabditis elegans drug effects, Arsenic toxicity, Caenorhabditis elegans growth & development, Fatty Acids toxicity, Hydrocarbons toxicity, Triglycerides toxicity
- Abstract
Although fish and seafood are well known for their nutritional benefits, they contain contaminants that might affect human health. Organic lipid-soluble arsenic species, so called arsenolipids, belong to the emerging contaminants in these food items; their toxicity has yet to be systematically studied. Here, we apply the in vivo model Caenorhabditis elegans to assess the effects of two arsenic-containing hydrocarbons (AsHC), a saturated arsenic-containing fatty acid (AsFA), and an arsenic-containing triacylglyceride (AsTAG) in a whole organism. Although all arsenolipids were highly bioavailable in Caenorhabditis elegans, only the AsHCs were substantially metabolized to thioxylated or shortened metabolic products and induced significant toxicity, affecting both survival and development. Furthermore, the AsHCs were several fold more potent as compared to the toxic reference arsenite. This study clearly indicates the need for a full hazard identification of subclasses of arsenolipids to assess whether they pose a risk to human health.
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- 2020
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21. Methylmercury Induces Metabolic Alterations in Caenorhabditis elegans: Role for C/EBP Transcription Factor.
- Author
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Caito SW, Newell-Caito J, Martell M, Crawford N, and Aschner M
- Subjects
- Adipogenesis drug effects, Adipogenesis genetics, Animals, Animals, Genetically Modified, CCAAT-Enhancer-Binding Proteins genetics, Caenorhabditis elegans genetics, Caenorhabditis elegans metabolism, Caenorhabditis elegans Proteins genetics, Energy Metabolism genetics, Feeding Behavior drug effects, Gene Expression Regulation, Lipid Metabolism genetics, Locomotion drug effects, Mutation, CCAAT-Enhancer-Binding Proteins metabolism, Caenorhabditis elegans drug effects, Caenorhabditis elegans Proteins metabolism, Energy Metabolism drug effects, Lipid Metabolism drug effects, Methylmercury Compounds toxicity
- Abstract
Methylmercury (MeHg) is a well-known neurotoxicant; however, its role in metabolic diseases has been gaining wider attention. We have previously shown that MeHg causes metabolic alterations in Caenorhabditis elegans, leading to decreased nicotinamide adenine dinucleotide cofactor, mitochondrial dysfunction, and oxidative stress. We were, therefore, interested in whether MeHg also affects nutrient metabolism, particularly lipid homeostasis, which may contribute to the development of metabolic conditions such as obesity or metabolic syndrome (MS). RNA from wild-type worms exposed to MeHg was collected immediately after treatment and used for gene expression analysis by DNA microarray. MeHg differentially regulated 215 genes, 17 genes involved in lipid homeostasis, and 12 genes involved in carbohydrate homeostasis. Of particular interest was cebp-1, the worm ortholog to human C/EBP, a pro-adipogenic transcription factor implicated in MS. MeHg increased the expression of cebp-1 as well as pro-adipogenic transcription factors sbp-1 and nhr-49, triglyceride synthesis enzyme acl-6, and lipid transport proteins vit-2 and vit-6. Concurrent with the altered gene expression, MeHg increased triglyceride levels, lipid storage, and feeding behaviors. Worms expressing mutant cebp-1 were protected from MeHg-induced alterations in lipid content, feeding behaviors, and gene expression, highlighting the importance of this transcription factor in the worm's response to MeHg. Taken together, our data demonstrate that MeHg induces biochemical, metabolic, and behavioral changes in C. elegans that can lead to metabolic dysfunction., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)
- Published
- 2020
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22. The role of poly(ADP-ribose) polymerases in manganese exposed Caenorhabditis elegans.
- Author
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Neumann C, Baesler J, Steffen G, Nicolai MM, Zubel T, Aschner M, Bürkle A, Mangerich A, Schwerdtle T, and Bornhorst J
- Subjects
- Animals, Chromatography, Liquid, Glutathione metabolism, Tandem Mass Spectrometry, Caenorhabditis elegans drug effects, Caenorhabditis elegans enzymology, Caenorhabditis elegans Proteins metabolism, Manganese pharmacology, Poly(ADP-ribose) Polymerases metabolism
- Abstract
Background and Aim: When exceeding the homeostatic range, manganese (Mn) might cause neurotoxicity, characteristic of the pathophysiology of several neurological diseases. Although the underlying mechanism of its neurotoxicity remains unclear, Mn-induced oxidative stress contributes to disease etiology. DNA damage caused by oxidative stress may further trigger dysregulation of DNA-damage-induced poly(ADP-ribosyl)ation (PARylation), which is of central importance especially for neuronal homeostasis. Accordingly, this study was designed to assess in the genetically traceable in vivo model Caenorhabditis elegans the role of PARylation as well as the consequences of loss of pme-1 or pme-2 (orthologues of PARP1 and PARP2) in Mn-induced toxicity., Methods: A specific and sensitive isotope-dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed to quantify PARylation in worms. Next to monitoring the PAR level, pme-1 and pme-2 gene expression as well as Mn-induced oxidative stress was studied in wildtype worms and the pme deletion mutants., Results and Conclusion: While Mn failed to induce PARylation in wildtype worms, toxic doses of Mn led to PAR-induction in pme-1-deficient worms, due to an increased gene expression of pme-2 in the pme-1 deletion mutants. However, this effect could not be observed at sub-toxic Mn doses as well as upon longer incubation times. Regarding Mn-induced oxidative stress, the deletion mutants did not show hypersensitivity. Taken together, this study characterizes worms to model PAR inhibition and addresses the consequences for Mn-induced oxidative stress in genetically manipulated worms., (Copyright © 2019 Elsevier GmbH. All rights reserved.)
- Published
- 2020
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23. Bacteria affect Caenorhabditis elegans responses to MeHg toxicity.
- Author
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Ke T and Aschner M
- Subjects
- Animals, Caenorhabditis elegans growth & development, Caenorhabditis elegans microbiology, Dose-Response Relationship, Drug, Food, Oxidative Stress drug effects, Bacteria metabolism, Caenorhabditis elegans drug effects, Methylmercury Compounds toxicity
- Abstract
The organic form of mercurial complex, methylmercury (MeHg), is a neurotoxin that bioaccumulates in the food web. Studies in model organisms, such as Caenorhabditis elegans (C. elegans), provide powerful insights on the role of genetic factors in MeHg-induced toxicity. C. elegans is a free living worm that is commonly cultured in nematode growth medium (NGM) agar plates seeded with bacteria. The bacteria have broad impact on C. elegans biology, including development, reproduction and lifespan, as well as metabolism of experimental chemicals. We hypothesized that MeHg toxicity in C. elegans could be modified by the bacterial food. Using a C. elegans wild-type (WT) strain and transgenic reporter strains, we found that bacterial food reduced the chronic toxicity of MeHg in C. elegans in a dose- and live-status-dependent manner. The MeHg-induced death rate in C. elegans was highest in fasted worms, followed by dehydrated dead bacteria, dead bacteria and live bacteria fed worms. Among the different bacterial foods, dehydrated dead bacteria fed worms were most sensitive to the toxicity of MeHg. The distinct bacteria food (dehydrated dead bacteria food) attenuated oxidative stress and development delay in C. elegans exposed to MeHg. The FOXO transcriptional factor DAF16 was not changed by MeHg but modified by the distinct bacteria food. Independent of MeHg treatment, daf-16 expression in fed worms migrated from the intestine to muscle. We conclude that, in chronic exposure studies in C. elegans, the effects of bacteria on toxicological outcomes should be considered., (Copyright © 2019 Elsevier B.V. All rights reserved.)
- Published
- 2019
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24. Zn homeostasis in genetic models of Parkinson's disease in Caenorhabditis elegans.
- Author
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Baesler J, Kopp JF, Pohl G, Aschner M, Haase H, Schwerdtle T, and Bornhorst J
- Subjects
- Animals, Biological Availability, Biomarkers analysis, Ethylenediamines analysis, Ethylenediamines metabolism, Ethylenediamines pharmacokinetics, Zinc Sulfate analysis, Zinc Sulfate metabolism, Caenorhabditis elegans genetics, Caenorhabditis elegans metabolism, Homeostasis, Models, Genetic, Parkinson Disease genetics, Parkinson Disease metabolism, Zinc Sulfate pharmacokinetics
- Abstract
While the underlying mechanisms of Parkinson's disease (PD) are still insufficiently studied, a complex interaction between genetic and environmental factors is emphasized. Nevertheless, the role of the essential trace element zinc (Zn) in this regard remains controversial. In this study we altered Zn balance within PD models of the versatile model organism Caenorhabditis elegans (C. elegans) in order to examine whether a genetic predisposition in selected genes with relevance for PD affects Zn homeostasis. Protein-bound and labile Zn species act in various areas, such as enzymatic catalysis, protein stabilization pathways and cell signaling. Therefore, total Zn and labile Zn were quantitatively determined in living nematodes as individual biomarkers of Zn uptake and bioavailability with inductively coupled plasma tandem mass spectrometry (ICP-MS/MS) or a multi-well method using the fluorescent probe ZinPyr-1. Young and middle-aged deletion mutants of catp-6 and pdr-1, which are orthologues of mammalian ATP13A2 (PARK9) and parkin (PARK2), showed altered Zn homeostasis following Zn exposure compared to wildtype worms. Furthermore, age-specific differences in Zn uptake were observed in wildtype worms for total as well as labile Zn species. These data emphasize the importance of differentiation between Zn species as meaningful biomarkers of Zn uptake as well as the need for further studies investigating the role of dysregulated Zn homeostasis in the etiology of PD., (Copyright © 2019 Elsevier GmbH. All rights reserved.)
- Published
- 2019
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25. Selenoneine ameliorates peroxide-induced oxidative stress in C. elegans.
- Author
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Rohn I, Kroepfl N, Aschner M, Bornhorst J, Kuehnelt D, and Schwerdtle T
- Subjects
- Animals, Antioxidants chemistry, Dose-Response Relationship, Drug, Histidine chemistry, Histidine pharmacology, Molecular Structure, Organoselenium Compounds chemistry, Peroxides pharmacology, Protective Agents chemistry, Structure-Activity Relationship, Antioxidants pharmacology, Caenorhabditis elegans drug effects, Caenorhabditis elegans metabolism, Histidine analogs & derivatives, Organoselenium Compounds pharmacology, Oxidative Stress drug effects, Peroxides antagonists & inhibitors, Protective Agents pharmacology
- Abstract
Scope: Selenoneine (2-selenyl-N
α , Nα , Nα -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 post-treatment, 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., (Copyright © 2019. Published by Elsevier GmbH.)- Published
- 2019
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26. Treatment of Caenorhabditis elegans with Small Selenium Species Enhances Antioxidant Defense Systems.
- Author
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Rohn I, Raschke S, Aschner M, Tuck S, Kuehnelt D, Kipp A, Schwerdtle T, and Bornhorst J
- Subjects
- Animals, Caenorhabditis elegans Proteins genetics, Caenorhabditis elegans Proteins metabolism, Gene Expression Regulation drug effects, Mutation, Selenious Acid pharmacology, Selenocysteine analogs & derivatives, Selenocysteine pharmacology, Selenomethionine pharmacology, Thioredoxin Reductase 1 genetics, Thioredoxin Reductase 1 metabolism, tert-Butylhydroperoxide toxicity, Antioxidants metabolism, Caenorhabditis elegans drug effects, Caenorhabditis elegans physiology, Selenium pharmacokinetics, Selenium Compounds pharmacology
- Abstract
Scope: Small selenium (Se) species play a key role in Se metabolism and act as dietary sources of the essential trace element. However, they are redox-active and trigger pro- and antioxidant responses. As health outcomes are strongly species-dependent, species-specific characteristics of Se compounds are tested in vivo., Methods and Results: In the model organism Caenorhabditis elegans (C. elegans), immediate and sustained effects of selenite, selenomethionine (SeMet), and Se-methylselenocysteine (MeSeCys) are studied regarding their bioavailability, incorporation into proteins, as well as modulation of the cellular redox status. While all tested Se compounds are bioavailable, only SeMet persistently accumulates and is non-specifically incorporated into proteins. However, the protection toward chemically-induced formation of reactive species is independent of the applied Se compound. Increased thioredoxin reductase (TXNRD) activity and changes in mRNA expression levels of antioxidant proteins indicate the activation of cellular defense mechanisms. However, in txnrd-1 deletion mutants, no protective effects of the Se species are observed anymore, which is also reflected by differential gene expression data., Conclusion: Se species protect against chemically-induced reactive species formation. The identified immediate and sustained systemic effects of Se species give rise to speculations on possible benefits facing subsequent periods of inadequate Se intake., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)
- Published
- 2019
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27. Combined exposure to methylmercury and manganese during L1 larval stage causes motor dysfunction, cholinergic and monoaminergic up-regulation and oxidative stress in L4 Caenorhabditis elegans.
- Author
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Schetinger MRC, Peres TV, Arantes LP, Carvalho F, Dressler V, Heidrich G, Bowman AB, and Aschner M
- Subjects
- Animals, Caenorhabditis elegans Proteins biosynthesis, Caenorhabditis elegans Proteins genetics, Female, Larva drug effects, Lethal Dose 50, Male, Manganese pharmacokinetics, Methylmercury Compounds pharmacokinetics, Nerve Degeneration chemically induced, Nerve Degeneration pathology, Reproduction, Up-Regulation drug effects, Biogenic Monoamines biosynthesis, Caenorhabditis elegans, Manganese toxicity, Methylmercury Compounds toxicity, Movement Disorders, Oxidative Stress drug effects, Parasympathetic Nervous System drug effects
- Abstract
Humans are exposed simultaneously to a variety of neurotoxic agents, including manganese (Mn) and methylmercury (MeHg). Therefore, the study of combined exposures to toxicants is timely. This work aimed to study changes in cholinergic system focusing on acetylcholinesterase (ace-2), monoaminergic system focusing on vesicular monoamine transporter (VMAT, cat-1) expression, to address changes in antioxidant enzymatic systems, namely, the expression of superoxide dismutase (sod-3 and sod-4) and catalase (ctl-3), as well as worm reproduction and locomotion. C. elegans in the L1 larval stage were exposed to Mn, MeHg or both. All analyses were done 24 h after the end of exposure, except for behavior and reproduction tests that were assessed in L4 larval stage worms. The values obtained for lethal dose 50% (LD
50 ) were 17.78 mM for Mn and 30.63 μM for MeHg. It was observed that body bends, pharyngeal pumping and brood size decreased in worms exposed to metals when undergoing combined exposures. Relative mRNA content of ace-2, cat-1, sod-3, sod-4 and ctl-3 was increased at the highest concentration of the interaction (50 mM Mn + 50 μM MeHg). Cholinergic degeneration was observed in all groups co-exposed to both metals. Notably, combined exposure to metals was more toxic to the worms than when exposed to a single metal., (Copyright © 2018 Elsevier B.V. All rights reserved.)- Published
- 2019
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28. A C. elegans Model for the Study of RAGE-Related Neurodegeneration.
- Author
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Pinkas A, Lee KH, Chen P, and Aschner M
- Subjects
- Animals, Animals, Genetically Modified, Central Nervous System Depressants pharmacology, Dopamine metabolism, Ethanol pharmacology, Heat-Shock Response physiology, Locomotion drug effects, Locomotion physiology, Neurons metabolism, Receptor for Advanced Glycation End Products genetics, Reproduction physiology, Caenorhabditis elegans, Disease Models, Animal, Neurodegenerative Diseases metabolism, Receptor for Advanced Glycation End Products metabolism
- Abstract
The receptor for advanced glycation products (RAGE) is a cell surface, multi-ligand receptor belonging to the immunoglobulin superfamily; this receptor is implicated in a variety of maladies, via inflammatory pathways and induction of oxidative stress. Currently, RAGE is being studied using a limited number of mammalian in vivo, and some complementary in vitro, models. Here, we present a Caenorhabditis elegans model for the study of RAGE-related pathology: a transgenic strain, expressing RAGE in all neurons, was generated and subsequently tested behaviorally, developmentally, and morphologically. In addition to RAGE expression being associated with a significantly shorter lifespan, the following behavioral observations were made when RAGE-expressing worms were compared to the wild type: RAGE-expressing worms showed an impaired dopaminergic system, evaluated by measuring the fluorescent signal of GFP tagging; these worms exhibited decreased locomotion-both general and following ethanol exposure-as measured by counting body bends in adult worms; RAGE expression was also associated with impaired recovery of quiescence and pharyngeal pumping secondary to heat shock, as a significantly smaller fraction of RAGE-expressing worms engaged in these behaviors in the 2 h immediately following the heat shock. Finally, significant developmental differences were also found between the two strains: RAGE expression leads to a significantly smaller fraction of hatched eggs 24 h after laying and also to a significantly slower developmental speed overall. As evidence for the role of RAGE in a variety of neuropathologies accumulates, the use of this novel and expedient model should facilitate the elucidation of relevant underlying mechanisms and also the development of efficient therapeutic strategies.
- Published
- 2019
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29. Caenorhabditis elegans and its applicability to studies on restless legs syndrome.
- Author
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Chen P, Ijomone OM, Lee KH, and Aschner M
- Subjects
- Animals, Caenorhabditis elegans Proteins metabolism, Dopamine metabolism, Humans, Iron metabolism, Neurons metabolism, Neurons pathology, Restless Legs Syndrome diagnosis, Restless Legs Syndrome genetics, Restless Legs Syndrome therapy, Caenorhabditis elegans physiology, Restless Legs Syndrome pathology
- Abstract
Restless legs syndrome (RLS) is a common neurological disorder in the United States. This disorder is characterized by an irresistible urge to move the legs, although the symptoms vary in a wide range. The pathobiology of RLS has been linked to iron (Fe) deficiency and dopaminergic (DAergic) dysfunction. Several genetic factors have been reported to increase the risk of RLS. Caenorhabditis elegans (C. elegans) is a well-established animal model with a fully sequenced genome, which is highly conserved with mammals. Given the detailed knowledge of its genomic architecture, ease of genetic manipulation and conserved biosynthetic and metabolic pathways, as well as its small size, ease of maintenance, speedy generation time and large brood size, C. elegans provides numerous advantages in studying RLS-associated gene-environment interactions. Here we will review current knowledge about RLS symptoms, pathology and treatments, and discuss the application of C. elegans in RLS study, including the worm homologous genes and methods that could be performed to advance the pathophysiology RLS., (© 2019 Elsevier Inc. All rights reserved.)
- Published
- 2019
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30. C. elegans as a model in developmental neurotoxicology.
- Author
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Ruszkiewicz JA, Pinkas A, Miah MR, Weitz RL, Lawes MJA, Akinyemi AJ, Ijomone OM, and Aschner M
- Subjects
- Animals, Caenorhabditis elegans growth & development, Caenorhabditis elegans metabolism, Humans, Models, Animal, Neurons metabolism, Neurons pathology, Neurotoxicity Syndromes metabolism, Neurotoxicity Syndromes pathology, Neurotoxicity Syndromes physiopathology, Risk Assessment, Species Specificity, Caenorhabditis elegans drug effects, Neurons drug effects, Neurotoxicity Syndromes etiology, Toxicity Tests methods
- Abstract
Due to many advantages Caenorhabditis elegans (C. elegans) has become a preferred model of choice in many fields, including neurodevelopmental toxicity studies. This review discusses the benefits of using C. elegans as an alternative to mammalian systems and gives examples of the uses of the nematode in evaluating the effects of major known neurodevelopmental toxins, including manganese, mercury, lead, fluoride, arsenic and organophosphorus pesticides. Reviewed data indicates numerous similarities with mammals in response to these toxins. Thus, C. elegans studies have the potential to predict possible effects of developmental neurotoxicants in higher animals, and may be used to identify new molecular pathways behind neurodevelopmental disruptions, as well as new toxicants., (Copyright © 2018 Elsevier Inc. All rights reserved.)
- Published
- 2018
- Full Text
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31. C. elegans -An Emerging Model to Study Metal-Induced RAGE-Related Pathologies.
- Author
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Pinkas A, Cunha Martins A Jr, and Aschner M
- Subjects
- Animals, Humans, Inflammation etiology, Models, Animal, Caenorhabditis elegans physiology, Glycation End Products, Advanced toxicity, Inflammation physiopathology, Metals toxicity, Oxidative Stress physiology, Receptor for Advanced Glycation End Products physiology
- Abstract
The receptor for advanced glycation end products (RAGE), a multi-ligand receptor, is mostly associated with promoting inflammation and oxidative stress. In addition to advanced glycation end products (AGEs), its ligands include High mobility group box 1 protein (HMGB-1), S-100 proteins and beta-sheet fibrils. The effects of several metals and metalloids on RAGE expression and activation have been recently studied: in vivo and in vitro exposure to methylmercury, selenium, zinc, manganese, and arsenic was associated with a variety of RAGE-related alterations and behavioral impairments, which are mostly dependent upon the administration procedure (local vs. systemic) and age during exposure. Recently, C. elegans has been proposed as a potential novel model for studying RAGE-related pathologies; preliminary data regarding such model and its potential contribution to the study of metal-induced RAGE-related pathologies are discussed.
- Published
- 2018
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32. Selenium species-dependent toxicity, bioavailability and metabolic transformations in Caenorhabditis elegans.
- Author
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Rohn I, Marschall TA, Kroepfl N, Jensen KB, Aschner M, Tuck S, Kuehnelt D, Schwerdtle T, and Bornhorst J
- Subjects
- Animals, Biological Availability, Caenorhabditis elegans drug effects, Caenorhabditis elegans growth & development, Selenious Acid toxicity, Selenocysteine metabolism, Selenocysteine toxicity, Selenomethionine metabolism, Selenomethionine toxicity, Thioredoxin Reductase 1 metabolism, Caenorhabditis elegans metabolism, Selenious Acid metabolism, Selenocysteine analogs & derivatives, Selenomethionine analogs & derivatives
- Abstract
The essential micronutrient selenium (Se) is required for various systemic functions, but its beneficial range is narrow and overexposure may result in adverse health effects. Additionally, the chemical form of the ingested selenium contributes crucially to its health effects. While small Se species play a major role in Se metabolism, their toxicological effects, bioavailability and metabolic transformations following elevated uptake are poorly understood. Utilizing the tractable invertebrate Caenorhabditis elegans allowed for an alternative approach to study species-specific characteristics of organic and inorganic Se forms in vivo, revealing remarkable species-dependent differences in the toxicity and bioavailability of selenite, selenomethionine (SeMet) and Se-methylselenocysteine (MeSeCys). An inverse relationship was found between toxicity and bioavailability of the Se species, with the organic species displaying a higher bioavailability than the inorganic form, yet being less toxic. Quantitative Se speciation analysis with HPLC/mass spectrometry revealed a partial metabolism of SeMet and MeSeCys. In SeMet exposed worms, identified metabolites were Se-adenosylselenomethionine (AdoSeMet) and Se-adenosylselenohomocysteine (AdoSeHcy), while worms exposed to MeSeCys produced Se-methylselenoglutathione (MeSeGSH) and γ-glutamyl-MeSeCys (γ-Glu-MeSeCys). Moreover, the possible role of the sole selenoprotein in the nematode, thioredoxin reductase-1 (TrxR-1), was studied comparing wildtype and trxr-1 deletion mutants. Although a lower basal Se level was detected in trxr-1 mutants, Se toxicity and bioavailability following acute exposure was indistinguishable from wildtype worms. Altogether, the current study demonstrates the suitability of C. elegans as a model for Se species dependent toxicity and metabolism, while further research is needed to elucidate TrxR-1 function in the nematode.
- Published
- 2018
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33. Insights into the differential toxicological and antioxidant effects of 4-phenylchalcogenil-7-chloroquinolines in Caenorhabditis elegans.
- Author
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Salgueiro WG, Goldani BS, Peres TV, Miranda-Vizuete A, Aschner M, da Rocha JBT, Alves D, and Ávila DS
- Subjects
- Animals, Antioxidants chemical synthesis, Caenorhabditis elegans genetics, Caenorhabditis elegans growth & development, Caenorhabditis elegans metabolism, Caenorhabditis elegans Proteins genetics, Caenorhabditis elegans Proteins metabolism, Chalcogens chemical synthesis, DNA-Binding Proteins genetics, DNA-Binding Proteins metabolism, Forkhead Transcription Factors genetics, Forkhead Transcription Factors metabolism, Gene Expression Regulation, Longevity drug effects, NF-E2-Related Factor 2 genetics, NF-E2-Related Factor 2 metabolism, Organometallic Compounds chemical synthesis, Organoselenium Compounds chemical synthesis, Oxidants antagonists & inhibitors, Oxidants toxicity, Oxidative Stress, Paraquat antagonists & inhibitors, Paraquat toxicity, Quinolines chemical synthesis, Signal Transduction, Superoxide Dismutase genetics, Superoxide Dismutase metabolism, Transcription Factors genetics, Transcription Factors metabolism, Antioxidants pharmacology, Caenorhabditis elegans drug effects, Chalcogens pharmacology, Organometallic Compounds pharmacology, Organoselenium Compounds pharmacology, Quinolines pharmacology, Tellurium pharmacology
- Abstract
Organic selenium and tellurium compounds are known for their broad-spectrum effects in a variety of experimental disease models. However, these compounds commonly display high toxicity and the molecular mechanisms underlying these deleterious effects have yet to be elucidated. Thus, the need for an animal model that is inexpensive, amenable to high-throughput analyses, and feasible for molecular studies is highly desirable to improve organochalcogen pharmacological and toxicological characterization. Herein, we use Caenorhabdtis elegans (C. elegans) as a model for the assessment of pharmacological and toxicological parameters following exposure to two 4-phenylchalcogenil-7-chloroquinolines derivatives (PSQ for selenium and PTQ for tellurium-containing compounds). While non-lethal concentrations (NLC) of PTQ and PSQ attenuated paraquat-induced effects on survival, lifespan and oxidative stress parameters, lethal concentrations (LC) of PTQ and PSQ alone are able to impair these parameters in C. elegans. We also demonstrate that DAF-16/FOXO and SKN-1/Nrf2 transcription factors underlie the mechanism of action of these compounds, as their targets sod-3, gst-4 and gcs-1 were modulated following exposures in a daf-16- and skn-1-dependent manner. Finally, in accordance with a disturbed thiol metabolism in both LC and NLC, we found higher sensitivity of trxr-1 worm mutants (lacking the selenoprotein thioredoxin reductase 1) when exposed to PSQ. Finally, our study suggests new targets for the investigation of organochalcogen pharmacological effects, reinforcing the use of C. elegans as a powerful platform for preclinical approaches., (Copyright © 2017 Elsevier Inc. All rights reserved.)
- Published
- 2017
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34. Scavengers of reactive γ-ketoaldehydes extend Caenorhabditis elegans lifespan and healthspan through protein-level interactions with SIR-2.1 and ETS-7.
- Author
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Nguyen TT, Caito SW, Zackert WE, West JD, Zhu S, Aschner M, Fessel JP, and Roberts LJ 2nd
- Subjects
- Aging physiology, Animals, Animals, Genetically Modified, Caenorhabditis elegans Proteins genetics, Proto-Oncogene Proteins c-ets genetics, Sirtuins genetics, Caenorhabditis elegans metabolism, Caenorhabditis elegans Proteins metabolism, Lipid Peroxidation physiology, Longevity physiology, Proto-Oncogene Proteins c-ets metabolism, Sirtuins metabolism
- Abstract
Isoketals (IsoKs) are highly reactive γ-ketoaldehyde products of lipid peroxidation that covalently adduct lysine side chains in proteins, impairing their function. Using C. elegans as a model organism, we sought to test the hypothesis that IsoKs contribute to molecular aging through adduction and inactivation of specific protein targets, and that this process can be abrogated using salicylamine (SA), a selective IsoK scavenger. Treatment with SA extends adult nematode longevity by nearly 56% and prevents multiple deleterious age-related biochemical and functional changes. Testing of a variety of molecular targets for SA's action revealed the sirtuin SIR-2.1 as the leading candidate. When SA was administered to a SIR-2.1 knockout strain, the effects on lifespan and healthspan extension were abolished. The SIR-2.1-dependent effects of SA were not mediated by large changes in gene expression programs or by significant changes in mitochondrial function. However, expression array analysis did show SA-dependent regulation of the transcription factor ets-7 and associated genes. In ets-7 knockout worms, SA's longevity effects were abolished, similar to sir-2.1 knockouts. However, SA dose-dependently increases ets-7 mRNA levels in non-functional SIR-2.1 mutant, suggesting that both are necessary for SA's complete lifespan and healthspan extension., Competing Interests: No conflict of interest could be disclosed for any author.
- Published
- 2016
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35. NAD+ Supplementation Attenuates Methylmercury Dopaminergic and Mitochondrial Toxicity in Caenorhabditis Elegans.
- Author
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Caito SW and Aschner M
- Subjects
- Animals, Animals, Genetically Modified, Behavior, Animal drug effects, Caenorhabditis elegans genetics, Caenorhabditis elegans metabolism, Cytoprotection, Dopaminergic Neurons metabolism, Dopaminergic Neurons pathology, Dose-Response Relationship, Drug, Genotype, Mitochondria metabolism, Mitochondria pathology, Mutation, Nerve Degeneration, Oxidation-Reduction, Phenotype, Caenorhabditis elegans drug effects, Dopaminergic Neurons drug effects, Methylmercury Compounds toxicity, Mitochondria drug effects, NAD pharmacology, Oxidative Stress drug effects
- Abstract
Methylmercury (MeHg) is a neurotoxic contaminant of our fish supply that has been linked to dopaminergic (DAergic) dysfunction that characterizes Parkinson's disease. We have previously shown that MeHg causes both morphological and behavioral changes in the Caenorhabditis elegans DAergic neurons that are associated with oxidative stress. We were therefore interested in whether the redox sensitive cofactor nicotinamide adenine dinucleotide (NAD(+)) may be affected by MeHg and whether supplementation of NAD( + )may prevent MeHg-induced toxicities. Worms treated with MeHg showed depletion in cellular NAD( + )levels, which was prevented by NAD( + )supplementation prior to MeHg treatment. NAD( + )supplementation also prevented DAergic neurodegeneration and deficits in DAergic-dependent behavior upon MeHg exposure. In a mutant worm line that cannot synthesize NAD( + )from nicotinamide, MeHg lethality and DAergic behavioral deficits were more sensitive to MeHg than wildtype worms, demonstrating the importance of NAD( + )in MeHg toxicity. In wildtype worms, NAD( + )supplementation provided protection from MeHg-induced oxidative stress and mitochondrial dysfunction. These data show the importance of NAD( + )levels in the response to MeHg exposure. NAD( + )supplementation may be beneficial for MeHg-induced toxicities and preventing cellular damage involved in Parkinson's disease., (© The Author 2016. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)
- Published
- 2016
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36. Reversible reprotoxic effects of manganese through DAF-16 transcription factor activation and vitellogenin downregulation in Caenorhabditis elegans.
- Author
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Gubert P, Puntel B, Lehmen T, Bornhorst J, Avila DS, Aschner M, and Soares FAA
- Subjects
- Animals, Caenorhabditis elegans genetics, Ovum drug effects, Vitellogenins deficiency, Caenorhabditis elegans drug effects, Caenorhabditis elegans metabolism, Caenorhabditis elegans Proteins metabolism, Down-Regulation drug effects, Forkhead Transcription Factors metabolism, Manganese toxicity, Vitellogenins metabolism
- Abstract
Aims: Vitellogenesis is the yolk production process which provides the essential nutrients for the developing embryos. Yolk is a lipoprotein particle that presents lipids and lipid-binding proteins, referred to as vitellogenins (VIT). The Caenorhabditis elegans nematode has six genes encoding VIT lipoproteins. Several pathways are known to regulate vitellogenesis, including the DAF-16 transcription factor. Some reports have shown that heavy metals, such as manganese (Mn), impair brood size in C. elegans; however the mechanisms associated with this effect have yet to be identified. Our aim was to evaluate Mn's effects on C. elegans reproduction and better understand the pathways related to these effects., Main Methods: Young adult larval stage worms were treated for 4h with Mn in 85mM NaCl and Escherichia coli OP50 medium., Key Findings: Mn reduced egg-production and egg-laying during the first 24h after the treatment, although the total number of progenies were indistinguishable from the control group levels. This delay may have occurred due to DAF-16 activation, which was noted only after the treatment and was not apparent 24h later. Moreover, the expression, protein levels and green fluorescent protein (GFP) fluorescence associated with VIT were decreased soon after Mn treatment and recovered after 24h., Significance: Combined, these data suggest that the delay in egg-production is likely regulated by DAF-16 and followed by the inhibition of VIT transport activity. Further studies are needed to clarify the mechanisms associated with Mn-induced DAF-16 activation., (Copyright © 2016 Elsevier Inc. All rights reserved.)
- Published
- 2016
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37. Extracellular dopamine and alterations on dopamine transporter are related to reserpine toxicity in Caenorhabditis elegans.
- Author
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Reckziegel P, Chen P, Caito S, Gubert P, Soares FA, Fachinetto R, and Aschner M
- Subjects
- Animals, Animals, Genetically Modified, Caenorhabditis elegans genetics, Caenorhabditis elegans metabolism, Caenorhabditis elegans Proteins metabolism, Defecation drug effects, Disease Models, Animal, Dopamine pharmacology, Dopamine Plasma Membrane Transport Proteins metabolism, Female, Gene Expression Regulation drug effects, Green Fluorescent Proteins genetics, Green Fluorescent Proteins metabolism, Ovum drug effects, Parkinson Disease physiopathology, Caenorhabditis elegans drug effects, Caenorhabditis elegans Proteins genetics, Dopamine metabolism, Dopamine Plasma Membrane Transport Proteins genetics, Reserpine toxicity
- Abstract
Reserpine is used as an animal model of parkinsonism. We hypothesized that the involuntary movements induced by reserpine in rodents are induced by dopaminergic toxicity caused by extracellular dopamine accumulation. The present study tested the effects of reserpine on the dopaminergic system in Caenorhabditis elegans. Reserpine was toxic to worms (decreased the survival, food intake, development and changed egg laying and defecation cycles). In addition, reserpine increased the worms' locomotor rate on food and decreased dopamine levels. Morphological evaluations of dopaminergic CEP neurons confirmed neurodegeneration characterized by decreased fluorescence intensity and the number of worms with intact CEP neurons, and increased number of shrunken somas per worm. These effects were unrelated to reserpine's effect on decreased expression of the dopamine transporter, dat-1. Interestingly, the locomotor rate on food and the neurodegenerative parameters fully recovered to basal conditions upon reserpine withdrawal. Furthermore, reserpine decreased survival in vesicular monoamine transporter and dat-1 loss-of-function mutant worms. In addition, worms pre-exposed to dopamine followed by exposure to reserpine had decreased survival. Reserpine activated gst-4, which controls a phase II detoxification enzymes downstream of nuclear factor (erythroid-derived-2)-like 2. Our findings establish that the dopamine transporter, dat-1, plays an important role in reserpine toxicity, likely by increasing extracellular dopamine concentrations.
- Published
- 2016
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38. A Simple Light Stimulation of Caenorhabditis elegans.
- Author
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Lee KH and Aschner M
- Subjects
- Animals, Behavior, Animal physiology, Microscopy, Fluorescence, Sensory Thresholds physiology, Caenorhabditis elegans physiology, Cilia physiology, Light, Photic Stimulation, Sensory Receptor Cells physiology
- Abstract
Response via noxious stimulus can be an important indicator of sensory neuron function and overall health of an organism. If the stimulation is quick and simple, and the animal can be rescued afterwards, such a method not only allows for assays pertaining to changed sensory ability after various treatments, but also increases the reliability of the statistical relationships that are established. This protocol demonstrates a stimulation assay in Caenorhabditis elegans, using blue light from common laboratory equipment: the fluorescent microscope. The nematode detects blue light using a set of amphid ciliary sensory neurons, and blue light is detrimental to its overall health after a prolonged exposure. However, under brief exposure, blue light stimulation provides a rapid and easy method for quantifying sensory functions and health without harming the animal., (Copyright © 2016 John Wiley & Sons, Inc.)
- Published
- 2016
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39. Highly sensitive isotope-dilution liquid-chromatography-electrospray ionization-tandem-mass spectrometry approach to study the drug-mediated modulation of dopamine and serotonin levels in Caenorhabditis elegans.
- Author
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Schumacher F, Chakraborty S, Kleuser B, Gulbins E, Schwerdtle T, Aschner M, and Bornhorst J
- Subjects
- Animals, Benzophenones pharmacology, Catechol O-Methyltransferase Inhibitors pharmacology, Deuterium chemistry, Limit of Detection, Monoamine Oxidase Inhibitors pharmacology, Nitrophenols pharmacology, Selegiline pharmacology, Time Factors, Tolcapone, Caenorhabditis elegans drug effects, Caenorhabditis elegans metabolism, Chromatography, High Pressure Liquid methods, Dopamine metabolism, Serotonin metabolism, Spectrometry, Mass, Electrospray Ionization methods, Tandem Mass Spectrometry methods
- Abstract
Dopamine (DA) and serotonin (SRT) are monoamine neurotransmitters that play a key role in regulating the central and peripheral nervous system. Their impaired metabolism has been implicated in several neurological disorders, such as Parkinson's disease and depression. Consequently, it is imperative to monitor changes in levels of these low-abundant neurotransmitters and their role in mediating disease. For the first time, a rapid, specific and sensitive isotope-dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantification of DA and SRT in the nematode Caenorhabditis elegans (C. elegans). This model organism offers a unique approach for studying the effect of various drugs and environmental conditions on neurotransmitter levels, given by the conserved DA and SRT biology, including synaptic release, trafficking and formation. We introduce a novel sample preparation protocol incorporating the usage of sodium thiosulfate in perchloric acid as extraction medium that assures high recovery of the relatively unstable neurotransmitters monitored. Moreover, the use of both deuterated internal standards and the multiple reaction monitoring (MRM) technique allows for unequivocal quantification. Thereby, to the best of our knowledge, we achieve a detection sensitivity that clearly exceeds those of published DA and SRT quantification methods in various matrices. We are the first to show that exposure of C. elegans to the monoamine oxidase B (MAO-B) inhibitor selegiline or the catechol-O-methyltransferase (COMT) inhibitor tolcapone, in order to block DA and SRT degradation, resulted in accumulation of the respective neurotransmitter. Assessment of a behavioral output of the dopaminergic system (basal slowing response) corroborated the analytical LC-MS/MS data. Thus, utilization of the C. elegans model system in conjunction with our analytical method is well-suited to investigate drug-mediated modulation of the DA and SRT system in order to identify compounds with neuroprotective or regenerative properties., (Copyright © 2015 Elsevier B.V. All rights reserved.)
- Published
- 2015
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40. RNASeq in C. elegans Following Manganese Exposure.
- Author
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Parmalee NL, Maqbool SB, Ye B, Calder B, Bowman AB, and Aschner M
- Subjects
- Animals, Base Sequence, RNA genetics, Caenorhabditis elegans metabolism, Gene Expression Regulation drug effects, Manganese toxicity, RNA metabolism
- Abstract
Manganese is a metal that is required for optimal biological functioning of organisms. Absorption, cellular import and export, and excretion of manganese are all tightly regulated. While some genes involved in regulation, such as DMT-1 and ferroportin, are known, it is presumed that many more are involved and as yet unknown. Excessive exposure to manganese, usually in industrial settings such as mining or welding, can lead to neurotoxicity and a condition known as manganism that closely resembles Parkinson's disease. Elucidating transcriptional changes following manganese exposure could lead to the development of biomarkers for exposure. This unit presents a protocol for RNA sequencing in the worm Caenorhabditis elegans to assay for transcriptional changes following exposure to manganese. This protocol is adaptable to any environmental exposure in C. elegans. The protocol results in counts of gene transcripts in control versus exposed conditions and a ranked list of differentially expressed genes for further study., (Copyright © 2015 John Wiley & Sons, Inc.)
- Published
- 2015
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41. Elemental bioimaging of Cisplatin in Caenorhabditis elegans by LA-ICP-MS.
- Author
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Crone B, Aschner M, Schwerdtle T, Karst U, and Bornhorst J
- Subjects
- Animals, Reproduction drug effects, Antineoplastic Agents pharmacokinetics, Antineoplastic Agents toxicity, Caenorhabditis elegans drug effects, Caenorhabditis elegans physiology, Cisplatin pharmacokinetics, Cisplatin toxicity, Mass Spectrometry methods
- Abstract
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.
- Published
- 2015
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42. High-Resolution Multi-Photon Imaging of Morphological Structures of Caenorhabditis elegans.
- Author
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Bixel GM, Fretham SJ, and Aschner M
- Subjects
- Animals, Caenorhabditis elegans ultrastructure, Nonlinear Dynamics, Caenorhabditis elegans anatomy & histology, Imaging, Three-Dimensional methods, Microscopy, Fluorescence, Multiphoton methods, Muscles anatomy & histology, Neurons ultrastructure, Pharynx anatomy & histology
- Abstract
In this protocol, we combine two-photon excitation fluorescence with nonlinear optical measurements to reconstruct the three-dimensional architecture of the pharyngeal region and the muscular system of the anterior and mid-body region of Caenorhabditis elegans (C. elegans). Femto-second laser pulses excite second-harmonic generation (SHG) and third-harmonic generation (THG) signals, which show detailed structural information regarding the organization of myofibrils that are arranged around the central pharynx region. The combination of two-photon excitation with SHG and THG imaging is a very powerful tool to study cell morphology, microarchitecture, and tissue arrangement in C. elegans., (Copyright © 2013 John Wiley & Sons, Inc. All rights reserved.)
- Published
- 2015
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43. Loss of pdr-1/parkin influences Mn homeostasis through altered ferroportin expression in C. elegans.
- Author
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Chakraborty S, Chen P, Bornhorst J, Schwerdtle T, Schumacher F, Kleuser B, Bowman AB, and Aschner M
- Subjects
- Animals, Caenorhabditis elegans metabolism, Down-Regulation, Gene Deletion, Homeostasis, RNA, Messenger genetics, Ubiquitin-Protein Ligases metabolism, Caenorhabditis elegans genetics, Caenorhabditis elegans Proteins genetics, Caenorhabditis elegans Proteins metabolism, Cation Transport Proteins genetics, Manganese metabolism, Ubiquitin-Protein Ligases genetics
- Abstract
Overexposure to the essential metal manganese (Mn) can result in an irreversible condition known as manganism that shares similar pathophysiology with Parkinson's disease (PD), including dopaminergic (DAergic) cell loss that leads to motor and cognitive impairments. However, the mechanisms behind this neurotoxicity and its relationship with PD remain unclear. Many genes confer risk for autosomal recessive, early-onset PD, including the parkin/PARK2 gene that encodes for the E3 ubiquitin ligase Parkin. Using Caenorhabditis elegans (C. elegans) as an invertebrate model that conserves the DAergic system, we previously reported significantly increased Mn accumulation in pdr-1/parkin mutants compared to wildtype (WT) animals. For the current study, we hypothesize that this enhanced accumulation is due to alterations in Mn transport in the pdr-1 mutants. While no change in mRNA expression of the major Mn importer proteins (smf-1-3) was found in pdr-1 mutants, significant downregulation in mRNA levels of the putative Mn exporter ferroportin (fpn-1.1) was observed. Using a strain overexpressing fpn-1.1 in worms lacking pdr-1, we show evidence for attenuation of several endpoints of Mn-induced toxicity, including survival, metal accumulation, mitochondrial copy number and DAergic integrity, compared to pdr-1 mutants alone. These changes suggest a novel role of pdr-1 in modulating Mn export through altered transporter expression, and provides further support of metal dyshomeostasis as a component of Parkinsonism pathophysiology.
- Published
- 2015
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44. Age- and manganese-dependent modulation of dopaminergic phenotypes in a C. elegans DJ-1 genetic model of Parkinson's disease.
- Author
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Chen P, DeWitt MR, Bornhorst J, Soares FA, Mukhopadhyay S, Bowman AB, and Aschner M
- Subjects
- Animals, Caenorhabditis elegans drug effects, Disease Models, Animal, Dopaminergic Neurons drug effects, Dopaminergic Neurons metabolism, Forkhead Transcription Factors metabolism, Gene Deletion, Green Fluorescent Proteins metabolism, Longevity drug effects, Models, Biological, Movement drug effects, Parkinson Disease genetics, Phenotype, Signal Transduction drug effects, Survival Analysis, Aging pathology, Aldehyde Oxidoreductases metabolism, Caenorhabditis elegans metabolism, Caenorhabditis elegans Proteins metabolism, Dopaminergic Neurons pathology, Manganese pharmacology, Models, Genetic, Parkinson Disease pathology
- Abstract
Parkinson's disease (PD) is the second most common neurodegenerative disease, yet its etiology and pathogenesis are poorly understood. PD is characterized by selective dopaminergic (DAergic) degeneration and progressive hypokinetic motor impairment. Mutations in dj-1 cause autosomal recessive early-onset PD. DJ-1 is thought to protect DAergic neurons via an antioxidant mechanism, but the precise basis of this protection has not yet been resolved. Aging and manganese (Mn) exposure are significant non-genetic risk factors for PD. Caenorhabditis elegans (C. elegans) is an optimal model for PD and aging studies because of its simple nervous system, conserved DAergic machinery, and short 20-day lifespan. Here we tested the hypothesis that C. elegans DJ-1 homologues were protective against Mn-induced DAergic toxicity in an age-dependent manner. We showed that the deletion of C. elegans DJ-1 related (djr) genes, djr-1.2, decreased survival after Mn exposure. djr-1.2, the DJ-1 homologue was expressed in DAergic neurons and its deletion decreased lifespan and dopamine (DA)-dependent dauer movement behavior after Mn exposure. We also tested the role of DAF-16 as a regulator of dj-1.2 interaction with Mn toxicity. Lifespan defects resulting from djr-1.2 deletion could be restored to normal by overexpression of either DJR-1.2 or DAF-16. Furthermore, dauer movement alterations after djr-1.2 deletion were abolished by constitutive activation of DAF-16 through mutation of its inhibitor, DAF-2 insulin receptor. Taken together, our results reveal PD-relevant interactions between aging, the PD environmental risk factor manganese, and homologues of the established PD genetic risk factor DJ-1. Our data demonstrate a novel role for the DJ-1 homologue, djr-1.2, in mitigating Mn-dependent lifespan reduction and DA signaling alterations, involving DAF-2/DAF-16 signaling.
- Published
- 2015
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45. Quantification of Glutathione in Caenorhabditis elegans.
- Author
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Caito SW and Aschner M
- Subjects
- Animals, Caenorhabditis elegans drug effects, Glutathione metabolism, Oxidative Stress drug effects, Sensitivity and Specificity, Spectrophotometry, Caenorhabditis elegans metabolism, Dithionitrobenzoic Acid toxicity, Glutathione analysis, Toxicity Tests methods
- Abstract
Glutathione (GSH) is the most abundant intracellular thiol with diverse functions from redox signaling, xenobiotic detoxification, and apoptosis. The quantification of GSH is an important measure for redox capacity and oxidative stress. This protocol quantifies total GSH from Caenorhabditis elegans, an emerging model organism for toxicology studies. GSH is measured using the 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) cycling method originally created for cell and tissue samples but optimized for whole worm extracts. DTNB reacts with GSH to from a 5'-thio-2-nitrobenzoic acid (TNB) chromophore with maximum absorbance of 412 nm. This method is both rapid and sensitive, making it ideal for studies involving a large number of transgenic nematode strains.
- Published
- 2015
- Full Text
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46. F3-Isoprostanes as a Measure of in vivo Oxidative Damage in Caenorhabditis elegans.
- Author
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Nguyen TT and Aschner M
- Subjects
- Animals, Caenorhabditis elegans metabolism, Isoprostanes metabolism, Oxidative Stress
- Abstract
Oxidative stress has been implicated in the development of a wide variety of disease processes, including cardiovascular disease, cancer, and neurodegenerative diseases, as well as progressive and normal aging processes. Isoprostanes (IsoPs) are prostaglandin-like compounds that are generated in vivo from lipid peroxidation of arachidonic acid (AA, C20:4, ω-6) and other polyunsaturated fatty acids (PUFA). Since the discovery of IsoPs by Morrow and Roberts in 1990, quantification of IsoPs has been shown to be an excellent source of biomarkers of in vivo oxidative damage. Eicosapentaenoic acid (EPA, C20:5, ω-3) is the most abundant PUFA in Caenorhabditis elegans and gives rise to F3-IsoPs upon nonenzymatic free-radical-catalyzed lipid peroxidation. The protocol presented is the current methodology that our laboratory uses to quantify F3-IsoPs in C. elegans using gas chromatography/mass spectrometry (GC/MS). The methods described herein have been optimized and validated to provide the best sensitivity and selectivity for quantification of F3-IsoPs from C. elegans lysates., (Copyright © 2014 John Wiley & Sons, Inc.)
- Published
- 2014
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47. The effects of pdr1, djr1.1 and pink1 loss in manganese-induced toxicity and the role of α-synuclein in C. elegans.
- Author
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Bornhorst J, Chakraborty S, Meyer S, Lohren H, Brinkhaus SG, Knight AL, Caldwell KA, Caldwell GA, Karst U, Schwerdtle T, Bowman A, and Aschner M
- Subjects
- Aldehyde Oxidoreductases metabolism, Animals, Caenorhabditis elegans metabolism, Caenorhabditis elegans Proteins metabolism, Disease Models, Animal, Gene Deletion, Gene Expression Regulation, Humans, Mutation, Oxidative Stress, Parkinson Disease metabolism, Parkinson Disease pathology, Protein Serine-Threonine Kinases metabolism, Ubiquitin-Protein Ligases metabolism, alpha-Synuclein genetics, Aldehyde Oxidoreductases genetics, Caenorhabditis elegans genetics, Caenorhabditis elegans Proteins genetics, Manganese metabolism, Parkinson Disease genetics, Protein Serine-Threonine Kinases genetics, Ubiquitin-Protein Ligases genetics, alpha-Synuclein metabolism
- Abstract
Parkinson's disease (PD) is a neurodegenerative brain disorder characterized by selective dopaminergic (DAergic) cell loss that results in overt motor and cognitive deficits. Current treatment options exist to combat PD symptomatology, but are unable to directly target its pathogenesis due to a lack of knowledge concerning its etiology. Several genes have been linked to PD, including three genes associated with an early-onset familial form: parkin, pink1 and dj1. All three genes are implicated in regulating oxidative stress pathways. Another hallmark of PD pathophysiology is Lewy body deposition, associated with the gain-of-function genetic risk factor α-synuclein. The function of α-synuclein is poorly understood, as it shows both neurotoxic and neuroprotective activities in PD. Using the genetically tractable invertebrate Caenorhabditis elegans (C. elegans) model system, the neurotoxic or neuroprotective role of α-synuclein upon acute Mn exposure in the background of mutated pdr1, pink1 or djr1.1 was examined. The pdr1 and djr1.1 mutants showed enhanced Mn accumulation and oxidative stress that was reduced by α-synuclein. Moreover, DAergic neurodegeneration, while unchanged with Mn exposure, returned to wild-type (WT) levels for pdr1, but not djr1.1 mutants expressing α-synuclein. Taken together, this study uncovers a novel, neuroprotective role for WT human α-synuclein in attenuating Mn-induced toxicity in the background of PD-associated genes, and further supports the role of extracellular dopamine in exacerbating Mn neurotoxicity.
- Published
- 2014
- Full Text
- View/download PDF
48. Elemental bioimaging of manganese uptake in C. elegans.
- Author
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Brinkhaus SG, Bornhorst J, Chakraborty S, Wehe CA, Niehaus R, Reifschneider O, Aschner M, and Karst U
- Subjects
- Animals, Biological Transport, Caenorhabditis elegans chemistry, Laser Therapy, Manganese analysis, Mass Spectrometry, Caenorhabditis elegans metabolism, Manganese metabolism
- Abstract
A new method for elemental bioimaging with laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) was developed and applied to study the uptake of manganese (Mn) in Caenorhabditis elegans (C. elegans). C. elegans is a well-established model organism in neuroscience, genetics and genomics, which has been extensively studied to decipher mechanisms of heavy metal induced toxicity. Knowledge about the distribution of manganese (Mn) and other metals in this organism will be helpful in elucidating pathways and mechanisms of transport, distribution and excretion. The LA-ICP-MS method requires limited sample preparation and can be used rapidly and easily to visualize the Mn distribution in C. elegans. Due to thorough optimization of the analytical parameters, intense Mn signals in C. elegans wild-type (WT) and mutants were obtained at a spatial resolution as small as 4 μm, thus proving the suitability of LA-ICP-MS to study the uptake of metals in C. elegans.
- Published
- 2014
- Full Text
- View/download PDF
49. Seleno- and telluro-xylofuranosides attenuate Mn-induced toxicity in C. elegans via the DAF-16/FOXO pathway.
- Author
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Wollenhaupt SG, Soares AT, Salgueiro WG, Noremberg S, Reis G, Viana C, Gubert P, Soares FA, Affeldt RF, Lüdtke DS, Santos FW, Denardin CC, Aschner M, and Avila DS
- Subjects
- Animals, Caenorhabditis elegans metabolism, Dose-Response Relationship, Drug, Forkhead Transcription Factors, Reactive Oxygen Species metabolism, Caenorhabditis elegans drug effects, Caenorhabditis elegans Proteins metabolism, Manganese toxicity, Selenium Compounds pharmacology, Tellurium pharmacology, Transcription Factors metabolism
- Abstract
Organochalcogens are promising pharmacological agents that possess significant biological activities. Nevertheless, because of the complexity of mammalian models, it has been difficult to determine the molecular pathways and specific proteins that are modulated in response to treatments with these compounds. The nematode worm Caenorhabditis elegans is an alternative experimental model that affords easy genetic manipulations, green fluorescent protein tagging and in vivo live analysis of toxicity. Abundant evidence points to oxidative stress in mediating manganese (Mn)-induced toxicity. In this study we challenged worms with Mn, and investigated the efficacy of inedited selenium- and tellurium-xylofuranosides in reversing and/or protecting the worms from Mn-induced toxicity. In addition, we investigated their putative mechanism of action. First, we determined the lethal dose 50% (LD50) and the effects of the xylofuranosides on various toxic parameters. This was followed by studies on the ability of xylofuranosides to afford protection against Mn-induced toxicity. Both Se- and Te-xylofuranosides increased the expression of superoxide dismutase (SOD-3). Furthermore, we observed that the xylofuranosides induced nuclear translocation of the transcription factor DAF-16/FOXO, which in the worm is known to regulate stress responsiveness, aging and metabolism. These findings suggest that xylofuranosides attenuate toxicity Mn-induced, by regulating the DAF-16/FOXO signaling pathway., (Copyright © 2013 Elsevier Ltd. All rights reserved.)
- Published
- 2014
- Full Text
- View/download PDF
50. The Role of skn-1 in methylmercury-induced latent dopaminergic neurodegeneration.
- Author
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Martinez-Finley EJ, Caito S, Slaughter JC, and Aschner M
- Subjects
- Animals, Base Sequence, Behavior, Animal, Caenorhabditis elegans metabolism, Chromatography, High Pressure Liquid, DNA Primers, Mass Spectrometry, Nervous System metabolism, Caenorhabditis elegans drug effects, Caenorhabditis elegans Proteins physiology, DNA-Binding Proteins physiology, Dopamine metabolism, Methylmercury Compounds pharmacology, Nervous System drug effects, Transcription Factors physiology
- Abstract
Mercury (Hg) is a persistent environmental bioaccumulative metal, with developmental exposure to methylmercury (MeHg) resulting in long-term health effects. We examined the impact of early-life exposure to MeHg and knockdown of skn-1 on dopaminergic (DAergic) neurodegeneration in the nematode Caenorhabditis elegans. SKN-1, a the major stress-activated cytoprotective transcription factors, promotes the transcription of enzymes that scavenge free radicals, synthesizes glutathione and catalyzes reactions that increase xenobiotic excretion. Deletions or mutations in this gene suppress stress resistance. Thus, we hypothesized that the extent of MeHg's toxicity is dependent on intact skn-1 response; therefore skn-1 knockout (KO) worms would show heightened sensitivity to MeHg-induced toxicity compared to wildtype worms. In this study we identified the impact of early-life MeHg exposure on Hg content, stress reactivity and DAergic neurodegeneration in wildtype, and skn-1KO C. elegans. Hg content, measured by Inductively Coupled Plasma Mass Spectrometry, showed no strain-dependent differences. Reactive oxygen species generation was dramatically increased in skn-1KO compared to wildtype worms. Structural integrity of DAergic neurons was microscopically assessed by visualization of fluorescently-labeled neurons, and revealed loss of neurons in skn-1KO and MeHg exposed worms compared to wildtype controls. Dopamine levels detected by High-performance liquid chromatography, were decreased in response to MeHg exposure and decreased in skn-1KO worms, and functional behavioral assays showed similar findings. Combined, these studies suggest that knockdown of skn-1 in the nematode increases DAergic sensitivity to MeHg exposure following a period of latency.
- Published
- 2013
- Full Text
- View/download PDF
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