Montgomery K et al. (2018) Neurotoxicol Teratol "Transport of a manganese/zinc ethylene-bis-dithiocarbamate fungicide may involve pre-synaptic dopaminergic transporters."

Corona C, Frye R, Fitsanakis VA, Montgomery K, Bailey A, Barnett R

[Neurotoxicol Teratol, 2018]

Mancozeb (MZ), an organic-metal fungicide used predominantly on vegetables and fruits, has been linked to neurodegeneration and behavioral disruptions in a variety of organisms, including humans. Both -aminobutyric acid and dopamine neurons appear to be more vulnerable to MZ exposure than other neuronal populations. Based on these observations, we hypothesized that MZ may be differentially transported into these cells through their presynaptic neurotransmitter transporters. To test this, we pretreated Caenorhabditis elegans with transporter antagonists followed by exposure to various concentrations of MZ. Potential neuroprotection was monitored via green fluorescence associated with various neuron populations in transgenic worm strains. Neurodegeneration associated with subacute MZ treatment (30min) was not altered by transporter antagonist pretreatment. On the other hand, pretreatment with a dopamine transporter antagonist (GBR12909) appeared to protect dopaminergic neurons from chronic (24h) MZ treatment. These results are consistent with other reports that dopamine transporter levels or activity may modulate toxicity for neurotoxicants.

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.

Todt CE et al. (2016) Neurotoxicology "Acute Exposure to a Mn/Zn Ethylene-bis-Dithiocarbamate Fungicide Leads to Mitochondrial Dysfunction and Increased Reactive Oxygen Species Production in Caenorhabditis elegans."

Traynor WL, Snapp IB, Todt CE, Orfield SE, Bailey AC, Bailey DC, Pressley AS, Denney RD, Montgomery KM, Fitsanakis VA, Negga R

[Neurotoxicology, 2016]

Mn/Zn ethylene-bis-dithiocarbamate (Mn/Zn-EBDC) fungicides are among some the most widely-used fungicides in the world. Although they have been available for over 50 years, little is known about their mechanism of action in fungi, or their potentially toxic mechanisms in humans. To determine if exposure of Caenorhabditis elegans (C. elegans) to a representative fungicide (Manzate; MZ) from this group inhibits mitochondria or produces reactive oxygen species (ROS), we acutely (30min) exposed worms to various MZ concentrations. Initial oxygen consumption studies showed an overall statistically significant decrease in oxygen consumption associated with addition of Complex I- and/or II-substrate in treatment groups compared to controls (*p<0.05). In order to better characterize the individual complex activity, further studies were completed that specifically assessed Complex II or Complex IV. Data indicated that neither of these two complexes were targets of MZ treatment. Results from tetramethylrhodamine ethyl ester (proton gradient) and ATP assays showed statistically significant reductions in both endpoints (*p<0.05, **p<0.01, respectively). Additional studies were completed to determine if MZ treatment also resulted in increased ROS production. These assays provided evidence that hydrogen peroxide, but not superoxide or hydroxyl radical levels were statistically significantly increased (*p<0.05). Taken together, these data indicate exposure of C. elegans to MZ concentrations to which humans are exposed leads to mitochondrial inhibition and concomitant hydrogen peroxide production. Since mitochondrial inhibition and increased ROS are associated with numerous neurodegenerative diseases, we suggest further studies to determine if MZ catalyzes similar toxic processes in mammals.

Bailey DC et al. (2016) Neurotoxicology "Caenorhabditis elegans chronically exposed to a Mn/Zn ethylene-bis-dithiocarbamate fungicide show mitochondrial Complex I inhibition and increased reactive oxygen species."

Bailey AC, Pressley AS, Snapp IB, Fitsanakis VA, Orfield SE, Denney RD, Negga R, Bailey DC, Montgomery KM, Todt CE, Traynor WL

[Neurotoxicology, 2016]

Reports have linked human exposure to Mn/Zn ethylene-bis-dithiocarbamate (Mn/Zn-EBDC) fungicides with multiple pathologies, from dermatitis to central nervous system dysfunction. Although members of this family of agrochemicals have been available for over 50 years, their mechanism of toxicity in humans is still unclear. Since mitochondrial inhibition and oxidative stress are implicated in a wide variety of diseases, we hypothesized that Caenorhabditis elegans (C. elegans) exposed to a commercially-available formulation of an Mn/Zn-EBDC-containing fungicide (Manzate; MZ) would also show these endpoints. Thus, worms were treated chronically (24h) with various MZ concentrations and assayed for reduced mitochondrial function and increased levels of reactive oxygen species (ROS). Oxygen consumption studies suggested Complex I inhibition in all treatment groups compared to controls ((**)p<0.01). In order to verify these findings, assays specific for Complex II or Complex IV activity were also completed. Data analysis from these studies indicated that neither complex was adversely affected by MZ treatment. Additional data from ATP assays indicated a statistically significant decrease ((***)p<0.001) in ATP levels in all treatment groups when compared to control worms. Further studies were completed to determine if exposure of C. elegans to MZ also resulted in increased ROS concentrations. Studies demonstrated that hydrogen peroxide, but not superoxide or hydroxyl radical, levels were statistically significantly increased (*p<0.05). Since hydrogen peroxide is known to up-regulate glutathione-S-transferase (GST), we used a GST:green fluorescent protein transgenic worm strain to test this hypothesis. Results from these studies indicated a statistically significant increase ((***)p<0.001) in green pixel number following MZ exposure. Taken together, these data indicate that C. elegans treated with MZ concentrations to which humans are exposed show mitochondrial Complex I inhibition with concomitant hydrogen peroxide production. Since these mechanisms are associated with numerous human diseases, we suggest further studies to determine if MZ exposure induces similar toxic mechanisms in mammals.

Huang L et al. (2021) Light Sci Appl "Recurrent neural network-based volumetric fluorescence microscopy."

Chen H, Rivenson Y, Ozcan A, Luo Y, Huang L

[Light Sci Appl, 2021]

Volumetric imaging of samples using fluorescence microscopy plays an important role in various fields including physical, medical and life sciences. Here we report a deep learning-based volumetric image inference framework that uses 2D images that are sparsely captured by a standard wide-field fluorescence microscope at arbitrary axial positions within the sample volume. Through a recurrent convolutional neural network, which we term as Recurrent-MZ, 2D fluorescence information from a few axial planes within the sample is explicitly incorporated to digitally reconstruct the sample volume over an extended depth-of-field. Using experiments on C. elegans and nanobead samples, Recurrent-MZ is demonstrated to significantly increase the depth-of-field of a 63x/1.4NA objective lens, also providing a 30-fold reduction in the number of axial scans required to image the same sample volume. We further illustrated the generalization of this recurrent network for 3D imaging by showing its resilience to varying imaging conditions, including e.g., different sequences of input images, covering various axial permutations and unknown axial positioning errors. We also demonstrated wide-field to confocal cross-modality image transformations using Recurrent-MZ framework and performed 3D image reconstruction of a sample using a few wide-field 2D fluorescence images as input, matching confocal microscopy images of the same sample volume. Recurrent-MZ demonstrates the first application of recurrent neural networks in microscopic image reconstruction and provides a flexible and rapid volumetric imaging framework, overcoming the limitations of current 3D scanning microscopy tools.

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.

Shintoku Y et al. (2005) J Parasitol "Strongyloides ratti infection in the large intestine of wild rats, Rattus norvegicus."

Kimura E, Hasegawa H, Shintoku Y, Kondo S, Islam MZ, Kadosaka T, Itoh M

[J Parasitol, 2005]

The large intestine of a rat has been neglected almost completely as a site of Strongyloides sp. infection. We reported that adult Strongyloides ratti remained in the large intestine for more than 80 days, producing more number of infective larvae than small intestine adults, and therefore hypothesized that parasitism in this site could be a survival strategy. In wild rats, however, no study has focused on large intestine infections of Strongyloides. The present study revealed that 32.4% of 68 wild rats, Rattus norvegicus, had the infection of S. ratti in the large intestine, with an average of 4.7 worms. These worms harbored normal eggs in the uterus. In a laboratory experiment with S. ratti and Wister rats, daily output of infective larvae by 4.7 females in the large intestine was estimated to be 4,638.4, suggesting that a few parasites could play a role in the parasite transmission. Five species of nematode found in the wild rats showed seasonality in infection intensity, with highest intensities in March-May. The number of S. ratti in the large intestine was also highest in these months.