Bailey DC et al. (2017) Environ Toxicol Pharmacol "Chronic exposure to a glyphosate-containing pesticide leads to mitochondrial dysfunction and increased reactive oxygen species production in Caenorhabditis elegans."

Pressley AS, Denney RD, Snapp IB, Negga R, Burchfield SL, Traynor WL, Bailey DC, Fitsanakis VA, Todt CE

[Environ Toxicol Pharmacol, 2017]

Glyphosate-containing herbicides are among the most widely-used in the world. Although glyphosate itself is relatively non-toxic, growing evidence suggests that commercial herbicide formulations may lead to increased oxidative stress and mitochondrial inhibition. In order to assess these mechanisms in vivo, we chronically (24h) exposed Caenorhabditis elegans to various concentrations of the glyphosate-containing herbicide TouchDown (TD). Following TD exposure, we evaluated the function of specific mitochondrial electron transport chain complexes. Initial oxygen consumption studies demonstrated inhibition in mid- and high-TD concentration treatment groups compared to controls. Results from tetramethylrhodamine ethyl ester and ATP assays indicated reductions in the proton gradient and ATP levels, respectively. Additional studies were designed to determine whether TD exposure resulted in increased reactive oxygen species (ROS) production. Data from hydrogen peroxide, but not superoxide or hydroxyl radical, assays showed statistically significant increases in this specific ROS. Taken together, these data indicate that exposure of Caenorhabditis elegans to TD leads to mitochondrial inhibition and hydrogen peroxide production.

Negga R et al. (2011) Neurotoxicology "Exposure to Mn/Zn ethylene-bis-dithiocarbamate and glyphosate pesticides leads to neurodegeneration in Caenorhabditis elegans."

Fitsanakis VA, Negga R, Fields AS, Davis NS, Rudd DA, Hatfield HE, Valente AL, Justice AN

[Neurotoxicology, 2011]

Epidemiological evidence suggests positive correlations between pesticide usage and the incidence of Parkinson's disease (PD). To further explore this relationship, we used wild type (N2) Caenorhabditis elegans (C. elegans) to test the following hypothesis: Exposure to a glyphosate-containing herbicide (TD) and/or a manganese/zinc ethylene-bis-dithiocarbamate-containing fungicide (MZ) may lead to neurotoxicity. We exposed N2 worms to varying concentrations of TD or MZ for 30 min (acute) or 24h (chronic). To replicate agricultural usage, a third population was exposed to TD (acute) followed by MZ (acute). For acute TD exposure, the LC(50)=8.0% (r(2)=0.6890), while the chronic LC(50)=5.7% (r(2)=0.9433). Acute MZ exposure led to an LC(50)=0.22% (r(2)=0.5093), and chronic LC(50)=0.50% (r(2)=0.9733). The combined treatment for TD+MZ yielded an LC(50)=12.5% (r(2)=0.6367). Further studies in NW1229 worms, a pan-neuronally green fluorescent protein (GFP) tagged strain, indicated a statistically significant (p<0.05) and dose-dependent reduction in green pixel number in neurons of treated worms following each paradigm. This reduction of pixel number was accompanied by visual neurodegeneration in photomicrographs. For the dual treatment, Bliss analysis suggested synergistic interactions. Taken together, these data suggest neuronal degeneration occurs in C. elegans following treatment with environmentally relevant concentrations of TD or MZ.

McVey KA et al. (2016) Neurotoxicol Teratol "Exposure of C. elegans eggs to a glyphosate-containing herbicide leads to abnormal neuronal morphology."

Johnson MB, McVey KA, Negga R, Snapp IB, Fitsanakis VA, Pressley AS

[Neurotoxicol Teratol, 2016]

Recent data demonstrate that chronic exposure of Caenorhabditis elegans (C. elegans) to a high-use glyphosate-containing herbicide, Touchdown (TD), potentially damages the adult nervous system. It is unknown, however, whether unhatched worms exposed to TD during the egg stage show abnormal neurodevelopment post-hatching. Therefore, we investigated whether early treatment with TD leads to aberrant neuronal or neurite development in C. elegans. Studies were completed in three different worm strains with green fluorescent protein (GFP)-tagged neurons to facilitate visual neuronal assessment. Initially, eggs from C. elegans with all neurons tagged with GFP were chronically exposed to TD. Visual inspection suggested decreased neurite projections associated with ventral nerve cord neurons. Data analysis showed a statistically significant decrease in overall green pixel numbers at the fourth larval (L4) stage (*p<0.05). We further investigated whether specific neuronal populations were preferentially vulnerable to TD by treating eggs from worms that had all dopaminergic (DAergic) or -aminobutyric acid (GABAergic) neurons tagged with GFP. As before, green pixel number associated with these discrete neuronal populations was analyzed at multiple larval stages. Data analysis indicated statistically significant decreases in pixel number associated with DAergic, but not GABAergic, neurons (***p<0.001) at all larval stages. Finally, statistically significant decreases (at the first larval stage, L1) or increases (at the fourth larval stage, L4) in superoxide levels, a developmental signaling molecule, were detected (*p<0.05). These data suggest that early exposure to TD may impair neuronal development, perhaps through superoxide perturbation. Since toxic insults during development may late render individuals more vulnerable to neurodegenerative diseases in adulthood, these studies provide some of the first evidence in this model organism that early exposure to TD may adversely affect the developing nervous system.

Burchfield SL et al. (2018) Environ Toxicol Pharmacol "Acute exposure to a glyphosate-containing herbicide formulation inhibits Complex II and increases hydrogen peroxide in the model organism Caenorhabditis elegans."

Fitsanakis VA, Todt CE, Burchfield SL, Denney RD, Negga R, Bailey DC

[Environ Toxicol Pharmacol, 2018]

Glyphosate-based herbicides, such as Touchdown (TD) and Roundup, are among the most heavily-used herbicides in the world. While the active ingredient is generally considered non-toxic, the toxicity resulting from exposure to commercially-sold formulations is less clear. In many cases, cell cultures or various model organisms exposed to glyphosate formulations show toxicity and, in some cases, lethality. Using Caenorhabditis elegans, we assessed potential toxic mechanisms through which a highly-concentrated commercial formulation of TD promotes neurodegeneration. Following a 30-min treatment, we assayed mitochondrial electron transport chain function and reactive oxygen species (ROS) production. Initial oxygen consumption studies indicated general mitochondrial inhibition compared to controls (^*p<0.05). When Complex II activity was further assessed, inhibition was observed in all TD-treated groups (^*p<0.05). Complex IV activity, however, was not adversely affected by TD. This electron transport chain inhibition also resulted in reduced ATP levels (^*p<0.05). Furthermore, hydrogen peroxide levels, but not other ROS, were increased (^*p<0.05). Taken together, these data indicate that commercially-available formulations of TD may exert neurotoxicity through Complex II (succinate dehydrogenase) inhibition, decreased ATP levels, and increased hydrogen peroxide production.

Kriegeskorte A et al. (2014) MBio "Inactivation of thyA in Staphylococcus aureus attenuates virulence and has a strong impact on metabolism and virulence gene expression."

Kahl BC, Becker K, Lore NI, Baum C, Seggewiss J, Peters G, Neumann C, Hertel P, Liebau E, Drescher M, Block D, Bragonzi A, Proctor RA, Windmuller N, Kriegeskorte A, Mellmann A

[MBio, 2014]

Staphylococcus aureus thymidine-dependent small-colony variants (TD-SCVs) are frequently isolated from patients with chronic S. aureus infections after long-term treatment with trimethoprim-sulfamethoxazole (TMP-SMX). While it has been shown that TD-SCVs were associated with mutations in thymidylate synthase (TS; thyA), the impact of such mutations on protein function is lacking. In this study, we showed that mutations in thyA were leading to inactivity of TS proteins, and TS inactivity led to tremendous impact on S. aureus physiology and virulence. Whole DNA microarray analysis of the constructed thyA mutant identified severe alterations compared to the wild type. Important virulence regulators (agr, arlRS, sarA) and major virulence determinants (hla, hlb, sspAB, and geh) were downregulated, while genes important for colonization (fnbA, fnbB, spa, clfB, sdrC, and sdrD) were upregulated. The expression of genes involved in pyrimidine and purine metabolism and nucleotide interconversion changed significantly. NupC was identified as a major nucleoside transporter, which supported growth of the mutant during TMP-SMX exposure by uptake of extracellular thymidine. The thyA mutant was strongly attenuated in virulence models, including a Caenorhabditis elegans killing model and an acute pneumonia mouse model. This study identified inactivation of TS as the molecular basis of clinical TD-SCV and showed that thyA activity has a major role for S. aureus virulence and physiology. Importance: Thymidine-dependent small-colony variants (TD-SCVs) of Staphylococcus aureus carry mutations in the thymidylate synthase (TS) gene (thyA) responsible for de novo synthesis of thymidylate, which is essential for DNA synthesis. TD-SCVs have been isolated from patients treated for long periods with trimethoprim-sulfamethoxazole (TMP-SMX) and are associated with chronic and recurrent infections. In the era of community-associated methicillin-resistant S. aureus, the therapeutic use of TMP-SMX is increasing. Today, the emergence of TD-SCVs is still underestimated due to misidentification in the diagnostic laboratory. This study showed for the first time that mutational inactivation of TS is the molecular basis for the TD-SCV phenotype and that TS inactivation has a strong impact on S. aureus virulence and physiology. Our study helps to understand the clinical nature of TD-SCVs, which emerge frequently once patients are treated with TMP-SMX.

Zuryn S et al. (2014) Science "Transdifferentiation. Sequential histone-modifying activities determine the robustness of transdifferentiation."

White ER, Margueron R, Zuryn S, Morin MC, Jarriault S, Ahier A, Portoso M

[Science, 2014]

Natural interconversions between distinct somatic cell types have been reported in species as diverse as jellyfish and mice. The efficiency and reproducibility of some reprogramming events represent unexploited avenues in which to probe mechanisms that ensure robust cell conversion. We report that a conserved H3K27me3/me2 demethylase, JMJD-3.1, and the H3K4 methyltransferase Set1 complex cooperate to ensure invariant transdifferentiation (Td) of postmitotic Caenorhabditis elegans hindgut cells into motor neurons. At single-cell resolution, robust conversion requires stepwise histone-modifying activities, functionally partitioned into discrete phases of Td through nuclear degradation of JMJD-3.1 and phase-specific interactions with transcription factors that have conserved roles in cell plasticity and terminal fate selection. Our results draw parallels between epigenetic mechanisms underlying robust Td in nature and efficient cell reprogramming in vitro.

Biswas SN et al. (1996) Res Commun Mol Pathol Pharmacol "A phenomenological model describing pharyngeal pulsing in the nematode Caenorhabditis elegans anesthetized by alcohol."

Okada H, Shingai R, Biswas SN, Ebina Y

[Res Commun Mol Pathol Pharmacol, 1996]

The recovery process of the suppressed pharyngeal pulsation in the nematode has been investigated for several concentrations of a homologous primary alcohol series (CnH2n-1OH, n = 1,2,3). A mathematical model describing the time course of the recovery process is phenomenologically constructed by using two time constants of delay time tD and recovery time tau. The values of tD and tau are obtained by fitting the equation to experimental data. The obtained values increase with increasing alcohol concentration. To observe the characteristics of tD and tau against the alcohol of order n, the inverse of these time constants are computed at 25 v/v% concentration and plotted on a semi-logarithmic scale. The plot curves decrease non-linearly and are dissimilar to the well-known curves illustrating the importance of lipid solubility in the cell membrane in anesthetic phenomena.

Yang YF et al. (2017) Int J Nanomedicine "Toxicity-based toxicokinetic/toxicodynamic assessment of bioaccumulation and nanotoxicity of zerovalent iron nanoparticles in Caenorhabditis elegans."

Lin YJ, Liao CM, Yang YF

[Int J Nanomedicine, 2017]

Elucidating the relationships between the toxicity-based-toxicokinetic (TBTK)/toxicodynamic (TD) properties of engineered nanomaterials and their nanotoxicity is crucial for human health-risk analysis. Zerovalent iron (Fe(0)) nanoparticles (NPs) are one of the most prominent NPs applied in remediating contaminated soils and groundwater. However, there are concerns that Fe(0)NP application contributes to long-term environmental and human health impacts. The nematode Caenorhabditis elegans is a surrogate in vivo model that has been successfully applied to assess the potential nanotoxicity of these nanomaterials. Here we present a TBTK/TD approach to appraise bioaccumulation and nanotoxicity of Fe(0)NPs in C. elegans. Built on a present C. elegans bioassay with estimated TBTK/TD parameters, we found that average bioconcentration factors in C. elegans exposed to waterborne and food-borne Fe(0)NPs were ~50 and ~5x10(-3), respectively, whereas 10% inhibition concentrations for fertility, locomotion, and development, were 1.26 (95% CI 0.19-5.2), 3.84 (0.38-42), and 6.78 (2.58-21) g-g(-1), respectively, implicating that fertility is the most sensitive endpoint in C. elegans. Our results also showed that biomagnification effects were not observed in waterborne or food-borne Fe(0)NP-exposed worms. We suggest that the TBTK/TD assessment for predicting NP-induced toxicity at different concentrations and conditions in C. elegans could enable rapid selection of nanomaterials that are more likely to be nontoxic in larger animals. We conclude that the use of the TBTK/TD scheme manipulating C. elegans could be used for rapid evaluation of in vivo toxicity of NPs or for drug screening in the field of nanomedicine.

Negga R et al. (2012) Neurotox Res "Exposure to glyphosate- and/or Mn/Zn-ethylene-bis-dithiocarbamate-containing pesticides leads to degeneration of -aminobutyric acid and dopamine neurons in Caenorhabditis elegans."

Richardson SJ, Negga R, McVey KA, Fitsanakis VA, Lizek AJ, Stuart JA, Salva J, Machen ML, Osborne AS, Mirallas O

[Neurotox Res, 2012]

Previous studies demonstrate a positive correlation between pesticide usage and Parkinson's disease (PD), which preferentially targets dopaminergic (DAergic) neurons. In order to examine the potential relationship between two common pesticides and specific neurodegeneration, we chronically (24h) or acutely (30min) exposed two Caenorhabditis elegans (C. elegans) strains to varying concentrations (LC(25), LC(50) or LC(75)) of TouchDown() (TD) as percent active ingredient (glyphosate), or Mancozeb() (MZ) as percent active ingredient (manganese/zinc ethylene-bis-dithiocarbamate). Furthermore, to more precisely model environmental exposure, worms were also exposed to TD for 30min, followed by 30-min incubation with varying MZ concentrations. Previous data from out lab suggested general neuronal degeneration using the worm strain NW1229 (pan-neuronal//green fluorescent protein (GFP) construct). To determine whether distinct neuronal groups were preferentially affected, we specifically used EG1285 (GABAergic neurons//GFP construct) and BZ555 (DAergic neurons//GFP construct) worms to verify GABAergic and DAergic neurodegeneration, respectively. Results indicated a statistically significant decrease, when compared to controls (CN), in number of green pixels associated with GABAergic neurons in both chronic (*P<0.05) and acute (*P<0.05) treatment paradigms. Analysis of the BZ555 worms indicated a statistically significant decrease (*P<0.05) in number of green pixels associated with DAergic neurons in both treatment paradigms (chronic and acute) when compared to CN. Taken together, our data suggest that exposure to TD and/or MZ promotes neurodegeneration in both GABAergic and DAergic neurons in the model organism C. elegans.

Matyash V et al. (2008) J Lipid Res "Lipid extraction by methyl-tert-butyl ether for high-throughput lipidomics."

Schwudke D, Matyash V, Liebisch G, Shevchenko A, Kurzchalia TV

[J Lipid Res, 2008]

Accurate profiling of lipidomes relies upon the quantitative and unbiased recovery of lipid species from analyzed cells, fluids, or tissues and is usually achieved by two-phase extraction with chloroform. We demonstrated that methyl-tert-butyl ether (MTBE) extraction allows faster and cleaner lipid recovery and is well suited for automated shotgun profiling. Because of MTBE's low density, lipid-containing organic phase forms the upper layer during phase separation, which simplifies its collection and minimizes dripping losses. Nonextractable matrix forms a dense pellet at the bottom of the extraction tube and is easily removed by centrifugation. Rigorous testing demonstrated that the MTBE protocol delivers similar or better recoveries of species of most all major lipid classes compared with the "gold-standard" Folch or Bligh and Dyer recipes.