Manganese-Induced Toxicity in C. elegans: What Can We Learn from the Transcriptome?

Manganese (Mn) is an essential ubiquitous transition metal and, when occupationally or environmentally overexposed, a well-known risk factor for several neurological pathologies. However, the molecular mechanisms underlying Mn-induced neurotoxicity are largely unknown. In this study, addressing RNA-Seq analysis, bioavailability and survival assays, key pathways of transcriptional responses to Mn overexposure were investigated in the model organism Caenorhabditis elegans (C. elegans), providing insights into the Mn-induced cellular stress and damage response. Comparative transcriptome analyses identified a large number of differentially expressed genes (DEGs) in nematodes exposed to MnCl2, and functional annotation suggested oxidative nucleotide damage, unfolded protein response and innate immunity as major damage response pathways. Additionally, a time-dependent increase in the transcriptional response after MnCl2 exposure was identified by means of increased numbers of DEGs, indicating a time-dependent response and activation of the stress responses in Mn neurotoxicity. The data provided here represent a powerful transcriptomic resource in the field of Mn toxicity, and therefore, this study provides a useful basis for further planning of targeted mechanistic studies of Mn-induced neurotoxicity that are urgently needed in the face of increasing industrially caused environmental pollution with Mn.

Effects of Manganese on Genomic Integrity in the Multicellular Model Organism Caenorhabditis elegans.

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.

Maintaining Translational Relevance in Animal Models of Manganese Neurotoxicity.

Manganese is an essential metal, but elevated brain Mn concentrations produce a parkinsonian-like movement disorder in adults and fine motor, attentional, cognitive, and intellectual deficits in children. Human Mn neurotoxicity occurs owing to elevated exposure from occupational or environmental sources, defective excretion (e.g., due to cirrhosis), or loss-of-function mutations in the Mn transporters solute carrier family 30 member 10 or solute carrier family 39 member 14. Animal models are essential to study Mn neurotoxicity, but in order to be translationally relevant, such models should utilize environmentally relevant Mn exposure regimens that reproduce changes in brain Mn concentrations and neurological function evident in human patients. Here, we provide guidelines for Mn exposure in mice, rats, nematodes, and zebrafish so that brain Mn concentrations and neurobehavioral sequelae remain directly relatable to the human phenotype.

Simultaneous quantitation of oxidized and reduced glutathione via LC-MS/MS to study the redox state and drug-mediated modulation in cells, worms and animal tissue.

Alterations in reduced and oxidized glutathione (GSH/GSSG) levels represent an important marker for oxidative stress and potential disease progression in toxicological research. Since GSH can be oxidized rapidly, using a stable and reliable method for sample preparation and GSH/GSSG quantification is essential to obtain reproducible data. Here we describe an optimised sample processing combined with a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, validated for different biological matrices (lysates from HepG2 cells, C. elegans, and mouse liver tissue). To avoid autoxidation of GSH, samples were treated with the thiol-masking agent N-ethylmaleimide (NEM) and sulfosalicylic acid (SSA) in a single step. With an analysis time of 5 min, the developed LC-MS/MS method offers simultaneous determination of GSH and GSSG at high sample throughput with high sensitivity. This is especially interesting with respect of screening for oxidative and protective properties of substances in in vitro and in vivo models, e.g. C. elegans. In addition to method validation parameters (linearity, limit of detection (LOD), limit of quantification (LOQ), recovery, interday, intraday), we verified the method by using menadione and L-buthionine-(S,R)-sulfoximine (BSO) as well established modulators of cellular GSH and GSSG concentrations. Thereby menadione proved to be a reliable positive control also in C. elegans.

Mechanistic studies on the adverse effects of manganese overexposure in differentiated LUHMES cells.

Manganese (Mn) is an essential trace element, but overexposure is associated with toxicity and neurological dysfunction. Accumulation of Mn can be observed in dopamine-rich regions of the brain in vivo and Mn-induced oxidative stress has been discussed extensively. Nevertheless, Mn-induced DNA damage, adverse effects of DNA repair, and possible resulting consequences for the neurite network are not yet characterized. For this, LUHMES cells were used, as they differentiate into dopaminergic-like neurons and form extensive neurite networks. Experiments were conducted to analyze Mn bioavailability and cytotoxicity of MnCl2, indicating a dose-dependent uptake and substantial cytotoxic effects. DNA damage, analyzed by means of 8-oxo-7,8-dihydro-2'-guanine (8oxodG) and single DNA strand break formation, showed significant dose- and time-dependent increase of DNA damage upon 48 h Mn exposure. Furthermore, the DNA damage response was increased which was assessed by analytical quantification of poly(ADP-ribosyl)ation (PARylation). Gene expression of the respective DNA repair genes was not significantly affected. Degradation of the neuronal network is significantly altered by 48 h Mn exposure. Altogether, this study contributes to the characterization of Mn-induced neurotoxicity, by analyzing the adverse effects of Mn on genome integrity in dopaminergic-like neurons and respective outcomes.

Caenorhabditis elegans as a Model to Study Manganese-Induced Neurotoxicity.

Caenorhabditis elegans (C. elegans) is a nematode present worldwide. The worm shows homology to mammalian systems and expresses approximately 40% of human disease-related genes. Since Dr. Sydney Brenner first proposed C. elegans as an advantageous experimental worm-model system for genetic approaches, increasing numbers of studies using C. elegans as a tool to investigate topics in several fields of biochemistry, neuroscience, pharmacology, and toxicology have been performed. In this regard, C. elegans has been used to characterize the molecular mechanisms and affected pathways caused by metals that lead to neurotoxicity, as well as the pathophysiological interrelationship between metal exposure and ongoing neurodegenerative disorders. Several toxic metals, such as lead, cadmium, and mercury, are recognized as important environmental contaminants, and their exposure is associated with toxic effects on the human body. Essential elements that are required to maintain cellular homeostasis and normal physiological functions may also be toxic when accumulated at higher concentrations. For instance, manganese (Mn) is a trace essential element that participates in numerous biological processes, such as enzymatic activities, energy metabolism, and maintenance of cell functions. However, Mn overexposure is associated with behavioral changes in C. elegans, which are consistent with the dopaminergic system being the primary target of Mn neurotoxicity. Caenorhabditis elegans has been shown to be an important tool that allows for studies on neuron morphology using fluorescent transgenic worms. Moreover, behavioral tests may be conducted using worms, and neurotransmitter determination and related gene expression are likely to change after Mn exposure. Likewise, mutant worms may be used to study molecular mechanisms in Mn toxicity, as well as the expression of proteins responsible for the biosynthesis, transport, storage, and uptake of dopamine. Furthermore, this review highlights some advantages and limitations of using the experimental model of C. elegans and provides guidance for potential future applications of this model in studies directed toward assessing for Mn neurotoxicity and related mechanisms.

Investigation of cardiolipin oxidation products as a new endpoint for oxidative stress in C. elegans by means of online two-dimensional liquid chromatography and high-resolution mass spectrometry.

The investigation of neurodegenerative and age-related diseases is a highly relevant topic in current research. Especially oxidative stress is thought to be the common underlying mechanism in diseases such as Parkinson's or Alzheimer's disease. The nematode Caenorhabditis elegans (C. elegans) is a prominent model organism, which is often used for such investigations and has gained extensive recognition in research regarding the linkage of reactive oxygen species (ROS) and neurodegeneration. Not only studies regarding genomics and proteomics have been increasingly conducted, also the number of studies based on the lipidome is rising. The phospholipid class of cardiolipin (CL) is a unique lipid class, which is exclusively located in mitochondria and is therefore of great relevance regarding oxidative stress and associated diseases. CL oxidation products have become a prominent marker for oxidative stress in various organisms. However, the CL distribution in the nematode C. elegans is still scarcely known on the molecular level and oxidation products have not yet been identified. In this work, we demonstrate the importance of CL distribution and the applicability of CL oxidation products as a sensitive marker for oxidative stress in C. elegans. For this reason, the CL distribution was determined by means of online two-dimensional liquid chromatography hyphenated with high-resolution mass spectrometry (2D-LC/HRMS). Subsequently, worms were treated with tert-butyl hydroperoxide (tBOOH) in order to provoke oxidative stress and induce the artificial formation of oxidized CL. We were able to detect increasing amounts of CL oxidation products of highly unsaturated CL species in a concentration-dependent manner. This finding emphasizes the great potential of CL oxidation products as a sensitive marker substance of oxidative stress in C. elegans, which is not only directly linked to mitochondria function but also favourable to other oxidative stress markers in terms of the needed sample material, relative substance stability and specificity of the oxidation site.

Profiling of sphingolipids in Caenorhabditis elegans by two-dimensional multiple heart-cut liquid chromatography - mass spectrometry.

Sphingolipids exert important functions in cells, ranging from stabilising the cell membrane to bioactive signalling in signal transduction pathways. Changed concentrations of sphingolipids are associated with, among others, neurodegenerative and cardiovascular diseases. In this work, we present a novel two-dimensional liquid chromatography method (2D-LC) coupled to tandem mass spectrometry (MS/MS) for the identification of ceramides, hexosylceramides and sphingomyelins in the model organism Caenorhabditis elegans (C. elegans). The method utilises a multiple heart-cut approach with a hydrophilic interaction liquid chromatography (HILIC) separation in the first dimension. The fractions of the sphingolipid classes were cut out and thereby separated from the abundant glycerolipids, which offers a simplified sample preparation and a high degree of automation as it compensates the alkaline depletion step usually conducted prior to the chromatographic analysis. The fractions were stored in a sample loop and transferred onto the second column with the combination of two six port valves. A reversed phase liquid chromatography was performed as the second dimension and allowed for a separation of the species within a sphingolipid class and according to the fatty acid moiety of the sphingolipid. The segregation of the abundant glycerolipids and the reduced matrix effects allowed for better identification of low abundant species, especially dihydro-sphingolipids with a saturated sphingoid base. In addition, the separation of the three fractions was carried out parallel to the separation and equilibration in the first dimension, which leads to no extension of the analysis time for the 2D-LC compared to the one-dimensional HILIC method. In total 45 sphingolipids were detected in the C. elegans lipid extract and identified via accurate mass and MS/MS fragments.

Ageing-associated effects of a long-term dietary modulation of four trace elements in mice.

Trace elements (TEs) are essential for diverse processes maintaining body function and health status. The complex regulation of the TE homeostasis depends among others on age, sex, and nutritional status. If the TE homeostasis is disturbed, negative health consequences can result, e.g., caused by impaired redox homeostasis and genome stability maintenance. Based on age-related shifts in TEs which have been described in mice well-supplied with TEs, we aimed to understand effects of a long-term feeding with adequate or suboptimal amounts of four TEs in parallel. As an additional intervention, we studied mice which received an age-adapted diet with higher concentrations of selenium and zinc to counteract the age-related decline of both TEs. We conducted comprehensive analysis of diverse endpoints indicative for the TE and redox status, complemented by analysis of DNA (hydroxy)methylation and markers denoting genomic stability maintenance. TE concentrations showed age-specific alterations which were relatively stable and independent of their nutritional supply. In addition, hepatic DNA hydroxymethylation was significantly increased in the elderly mice and markers indicative for the redox status were modulated. The reduced nutritional supply with TEs inconsistently affected their status, with most severe effects regarding Fe deficiency. This may have contributed to the sex-specific differences observed in the alterations related to the redox status and DNA repair activity. Overall, our results highlight the complexity of factors impacting on the TE status and its physiological consequences. Alterations in TE supply, age, and sex proved to be important determinants that need to be taken into account when considering TE interventions for improving general health and supporting convalescence in the clinics.