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Exposure to Glyphosate- and/or Mn/Zn-Ethylene-bis-Dithiocarbamate-Containing Pesticides Leads to Degeneration of γ-Aminobutyric Acid and Dopamine Neurons in Caenorhabditis elegans

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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 (24 h) or acutely (30 min) 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 30 min, 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.
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Exposure to Glyphosate- and/or Mn/Zn-Ethylene-bis-
Dithiocarbamate-Containing Pesticides Leads to Degeneration
of γ-Aminobutyric Acid and Dopamine Neurons in
Caenorhabditis elegans
Rekek Negga*, J Andrew Stuart*, Morgan L Machen*, Joel Salva*, Amanda J Lizek*, S Jayne
Richardson*, Amanda S Osborne*, Oriol Mirallas*, Kenneth A McVey*, and Vanessa A
Fitsanakis*,†
*King College, Department of Biology, 1350 King College Road, Bristol, TN 37620 USA
Abstract
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 (24 hours) or acutely (30 min) exposed two Caenorhabditis elegans (C. elegans)
strains to varying concentrations (LC25, LC50 or LC75) of TouchDown® (TD) as per cent active
ingredient (glyphosate), or Mancozeb® (MZ) as per cent active ingredient (manganese/zinc
ethylene-bis-dithiocarbamate). Furthermore, to more precisely model environmental exposure,
worms were also exposed to TD for 30 min, 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.
Keywords
C. elegans; glyphosate; mancozeb; neurodegeneration; BZ555; EG1285
INTRODUCTION
Parkinson’s disease (PD) is typically associated with degeneration of the dopaminergic
(DAergic) neurons of the substantia nigra pars compacta (Weintraub et al. 2008). Thus,
many studies involving environmental toxicants, such as pesticides or heavy metals, which
may contribute to the etiology of PD, mainly focus on the ability of such chemicals to
damage DAergic neurons (Cicchetti et al. 2005; Kim et al. 2002; Li et al. 2005). More
Corresponding Author: Vanessa A Fitsanakis, PhD, King College, Department of Biology, 1350 King College Road, Bristol, TN
37620, Phone: (423) 652-6322, Fax: (423) 652-4833, vafitsan@king.edu.
NIH Public Access
Author Manuscript
Neurotox Res. Author manuscript; available in PMC 2013 April 1.
Published in final edited form as:
Neurotox Res
. 2012 April ; 21(3): 281–290. doi:10.1007/s12640-011-9274-7.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
recently, however, the importance of non-DAergic neurons, particularly those using γ-
aminobutyric acid (GABA), has been recognized in conjunction with non-motor symptoms
associated with the disease (Barone 2010; Langston 2006). Unfortunately, in vivo
determination of neuronal populations affected by pesticides is often complicated by the
complexity of human and mammalian nervous systems (Peterson et al. 2008). In vitro
experiments involving mixed cell or tissue cultures are often hampered by the inability to
recreate complex neural circuits. Model organisms with simple nervous systems (Danio
rerio, Periplaneta americana, Drosophila melanogaster) are being used with greater
frequency in toxicological studies to address these apparent discrepancies. We used the
nematode Caenorhabditis elegans (C. elegans) to examine the sensitivity of two neuronal
populations, specifically those containing dopamine or GABA, to two widely-used
pesticides, TouchDown® (TD) and Mancozeb® (MZ).
The glyphosate-containing herbicides, of which TD is a member, are some of the most
widely-used pesticides in the US (Donaldson et al. 2004; Kiely et al. 2004) and in the world
(Powles et al. 1997). Although the introduction of glyphosate-resistant crops in the late
1980s resulted in an exponential increase in their use (Fernandez-Cornejo 2010),
glyphosate-containing herbicides have steadily gained market shares since their introduction
in the 1970s (USGS 2002). While the active ingredient in these herbicides is glyphosate (2%
to 52.3%), the actual application mixture also contains what is referred to as “inert” or
“inactive” ingredients (Syngenta 2010). Although the formulation of glyphosate-containing
herbicides, but not glyphosate itself, are mitochondrial inhibitors (Olorunsogo et al. 1979;
Peixoto 2005), and mitochondrial inhibitors are known to induce parkinsonism in animal
models (Betarbet et al. 2006; Betarbet et al. 2000; Saravanan et al. 2005), few studies have
examined whether TD might actually lead to neurodegeneration (Axelrad et al. 2003;
Tierney et al. 2006; Tierney et al. 2007). Rather, pesticide research related to PD has
typically focused on organophosphate insecticides (Arima et al. 2003; Bhatt et al. 1999;
Manthripragada et al. 2010), due to their relatively long biological half-life and their
prolonged presence in lipophilic tissues. Since it is well-documented that PD patients have
decreased mitochondrial function (Mounsey and Teismann 2010; Zhu and Chu 2010), and it
is hypothesized that glyphosate pesticide formulations induce mitochondrial inhibition
(Olorunsogo et al. 1979; Peixoto 2005), we chose to examine whether C. elegans exposed to
TD show neurodegeneration in DAergic neurons similar to that observed in PD.
Additionally, we were interested in whether MZ, a manganese (Mn)/zinc (Zn) ethylene-bis-
dithiocarbamate fungicide closely related to maneb, would induce degeneration in either
DAergic or GABAergic neurons. While evidence suggests that exposure to Mn (and other
heavy metals) may be a risk factor for PD (Hozumi et al. 2011; Racette et al. 2005; Willis et
al. 2010), other studies using maneb suggest that DAergic neurons are vulnerable to its
exposure (Cicchetti et al. 2005; Costello et al. 2009; Domico et al. 2006; Ferraz et al. 1988;
Meco et al. 1994). Maneb, however, is currently being removed from the market (Billingslea
2009) due to recent rulings by the Environmental Protection Agency, and is being replaced
with MZ (Donaldson et al. 2004; Kiely et al. 2004). Thus, we wanted to determine whether
MZ is also capable of inducing changes in DAergic neurons similar to what has been
documented for maneb (Cicchetti et al. 2005; Domico et al. 2007; Filipov et al. 2005;
Thiruchelvam et al. 2000).
In order to determine whether exposure to TD and/or MZ could lead to DAergic or
GABAergic neurodegeneration, we treated two transgenic C. elegans strains either acutely
or chronically with these two agrochemicals. The highest concentration used in either
paradigm for TD was 10%, which is within the commercial recommendations for spot
spraying broad-leaf weeds (4–10% glyphosate; Syngenta 2010). Although the recommended
application concentration for MZ is 0.29 – 0.49% Mn/Zn-EBDC (Bonide 2010), is less than
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some of the concentrations used here, our concentrations are still well within the range of
those to which humans could be exposed, namely commercial formulations of up to 37%
Mn/Zn-EBDC (Bonide 2010). The third treatment paradigm involved a dual exposure of 2%
TD and varying concentrations of MZ (the approximate LC25, LC50 and LC75). This study
models agrochemical practices on farms, since weeds are initially killed with a herbicide
then, as crops mature, fungicides could be used.
Additionally, we used BZ555 worms in which all DAergic neurons are tagged with green
fluorescent protein (GFP). The advantage to this model system is that C. elegans
hermaphrodites have only eight DAergic neurons: two anterior deirid (AD), four cephalic
(CEP) and two posterior deirid neurons (Chase and Koelle 2007). This is contrast to rodents,
which have 10,000–20,000 DAergic, or humans, which have greater than 40,000 DAergic
neurons (Cooper et al. 1996). Additionally, we used the EG1285 strain that has
GABAergic::GFP neurons. Of the 302 neurons in the hermaphrodite, 26 are GABAergic: 19
dorsal or ventral motor neurons (DDn, VDn) that innervate dorsal or ventral body muscles,
four motor neurons that innervate the head (RME), one interneuron (RIS), one mixed
anterior motor neuron/interneuron (AVL) and one mixed dorsal motor neuron/interneuron
(Jorgensen 2005). Considering their tremendously simplified nervous system, we wanted to
determine the vulnerability of DAergic and/or GABAergic neurons in C. elegans to
exposure to either acute or chronic TD and/or MZ.
MATERIALS AND METHODS
C. elegans and Escherichia coli strains
EG1285 and BZ555 worms, as well as NA22 Escherichia coli (E. coli) and OP50 E. coli (a
uracil auxotroph) used in this work were provided by the Caenorhabditis Genetics Center
(CGC; University of Minnesota), which is funded by the National Institutes of Health (NIH)
National Center for Research Resources (NCRR). In EG1285 worms
(oxIs12[Punc-47::GFP; lin-15(+)]), a green fluorescent protein gene (gfp) is fused with
unc-47, a transmembrane protein found in all GABAergic neurons and used to load GABA
into synaptic vesicles
(http://www.wormbase.org/db/gene/strain?name=EG1285;class=Strain). BZ555 worms
express GFP at the neuronal soma and processes due to the integration of egIs[Pdat-1::GFP],
resulting in the fusion of gfp with dat-1, a pre-synaptic DA reuptake protein found in all
DAergic neurons (http://www.wormbase.org/db/gene/strain?name=BZ555;class=Strain).
C. elegans maintenance and treatment
C. elegans were maintained at 20°C and synchronized according to standard protocols
(Brenner 1974), which are also consistent with previously published protocols (Negga et al.
2011). In order to maximize reproduction and growth, gravid worms were grown on 8P
plates (51.3 mM NaCl, 25.0 g bactoagar/L, 20.0 g bactopeptone/L, 1 mM CaCl2, 0.5 mM
potassium phosphate buffer (pH 6), 0.013 mM cholesterol (in 95% ethanol), 1 mM MgSO4)
with a lawn of NA22 E. coli (grown in 16 g tryptone/L, 10 g yeast extract/L, 85.5 mM
NaCl). They were then synchronized by exposure to a solution of sodium hypochlorite (4.0
mM) and sodium hydroxide (0.5 mM), and monitored using an ACCU-SCOPE light
microscope (4X) until worms released eggs. Approximately 18 h following isolation and
purification of eggs, 5000 synchronous L2 staged worms per treatment group were exposed
to the LC25, LC50 and LC75 concentrations of TD (Syngenta AG, Wilmington, DE), MZ
(Bonide Products, Inc., Oriskany, NY), or a sequential treatment, which reflects the actual
agricultural paradigm, of the two (TD/MZ; Table 1). In the combination treatment, all the
worms were exposed to 2% TD (application concentration recommended by the
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manufacturer) for 30 min (acute), followed by the LC25, LC50 and LC75 concentrations of
MZ for 30 min (acute).
For acute pesticide treatments, worms were exposed to the respective pesticide for 30 min,
washed at least 3X with sterile dH2O, then placed on fresh nematode growth media (NGM;
51.3 mM NaCl, 17.0 g bactoagar/L, 2.5g bactopeptone/L, 1 mM CaCl2, 1 mM MgSO4, 0.5
mM potassium phosphate buffer (pH 6.0), 12.9 mM cholesterol in 95% ethanol, 1.25 mL
nystatin/L, 0.2 g streptomycin/L) plates, with a lawn of OP50 E. coli (grown in 25 g Luria
broth/L, 200 mg streptomycin/L). Following a 24-h incubation period at 20°C, treatment
groups were counted and compared to controls under a dissecting microscope. Finally,
pictures were taken using a digital camera attached to a fluorescent microscope.
For chronic treatments, synchronous L2 staged worms were exposed to the respective
pesticide for 30 min, then, without additional washes, placed on clean NGM plates with the
respective pesticide still on the worms. After a 24-h incubation period (corresponding to an
equivalent human exposure of 10.5 years) at 20°C, the worms were counted, and the treated
were compared to controls under a dissecting microscope and photographed.
In agricultural settings, concentrated TD and MZ are diluted with water to the appropriate
application concentrations. As such, for all treatments paradigms (acute, chronic or dual),
control worms were treated with sterile dH2O.
Fluorescence Microscopy
Following the respective treatments, EG1285 worms from at least four separate
synchronizations were washed with sterile dH2O from plates and 10 µL of worms were
placed on 4% agarose pads on microscope slides. Fluorescence observations were performed
with an epifluorescence microscope (Leitz & Wetzlar, Halco Instruments, Inc) equipped
with a 50-W AC mercury source lamp (E. Leitz, Rockleigh, NJ) and various objective lenses
(Leitz & Wetzlar, Halco Instruments, Inc). The microscope was coupled to a digital camera
(Micrometrics, MilesCo Scientific, Princeton, MN) operated by Micrometrics software
(Micrometrics SE Premium, v2.7) for image acquisitions with consistent camera settings of
exposure time, saturation, light and color. All images are presented at a total magnification
of 400X. In Adobe Photoshop® 6.0.1 (Adobe Systems, Inc, San Jose, CA), the wand tool
(with a pre-assigned tolerance level of 18) was used for isolation of green pixels in the
photomicrographs. This allowed for selection of all pixels in a defined region that met the
minimum criteria. The histogram function in the program was used to assess the number of
green pixels. Data was entered into Graph Pad Prism® (version 4.00 GraphPad Software,
San Diego, CA). The same procedure was used for studies with BZ555 worms.
Statistical Analysis
Fluorescence data from photomicrograph analysis are presented as mean green pixel number
± SD. Data are from at least four separate synchronizations (N 4), with at least eight to ten
worms per intra-experimental replication (n 8), for each treatment paradigm. Differences
in pixel number were determined by one-way analysis of variance (ANOVA), followed by a
post-hoc Bonferroni test. Data was considered to be statistically significantly different from
controls when *p < 0.05.
RESULTS
Decreased GFP Pixel Number in DAergic Neurons Following Acute (30-min) Treatment
In order to determine whether acute exposure to TD or MZ leads to neurotoxicity in
DAergic neurons in C. elegans, BZ555 worms were acutely treated with varying
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concentrations of either TD or MZ (Figure 1). Number of green pixels was analyzed in the
nerve ring, which contains GFP-tagged DAergic neurons (Rand and Nonet 1997). Analysis
indicated that acute treatments with 10% glyphosate (LC75; Figure 1a) resulted in a
statistically significant decrease in pixel number compared to controls (*p < 0.01).
Computer analysis of photomicrograph indicated a decrease in the soma of DAergic neurons
of treated worms (Figure 1c) when compared to controls (Figure 1b). Similarly, following
acute treatment with MZ (Figure 1d), a statistically significant reduction in green pixel
number was observed at 30% Mn/Zn-EBDC (LC75) compared to either controls (*p < 0.05)
or worms treated with 7.5% Mn/Zn-EBDC (LC50; ^^^p < 0.001). This decrease in pixel
number corresponded to a shrinkage of the soma of DAergic neurons (Figure 1e) compared
to those in control worms (Figure 1b). These data confirmed what was detected during
visual inspection of the same photomicrographs.
Decreased GFP Pixel Number in DAergic Neurons Following Chronic (24-h) Treatment
In order to determine whether chronic exposure to TD or MZ might lead to DAergic
neurodegeneration, BZ555 worms were chronically treated with either TD or MZ (Figure 2).
Analysis of green pixel number associated with DAergic neurons showed a statistically
significant decrease (Figure 2a) in 9.8% glyphosate treatment (LC75) compared to controls
(**p < 0.01) or 2.7% glyphosate (^p < 0.05). As with the acute treatments, the reduction in
pixel number corresponded with an apparent shrinkage of the soma of neurons in the worms
treated with 9.8% glyphosate (Figure 2c) compared to control worms (Figure 2b). Chronic
treatment with MZ (Figure 2d) showed a statistically significant reduction in green pixel
number, accompanied by decreased somal size (Figure 2e), at 1.5% Mn/Zn-EBDC (LC75)
compared to control (*p < 0.05). The computer analysis again reinforced what was observed
visually following examination of the photomicrographs.
GFP Pixel Number in DAergic Neurons Following Dual Treatment
Dual exposure to TD and MZ was used in order to determine neurotoxicity in DAergic
neurons in C. elegans. BZ555 worms were acutely (30-min) treated with TD (2%
glyphosate) followed by acute (30-min) treatment of the approximate LC25, LC50 or LC75 of
MZ (as Mn/Zn-EBDC). Visual inspection was confirmed by analysis of green pixel number
in the nerve ring showing no statistically significant differences compared to control worms
(data not shown).
Decreased GFP Pixel Number in GABAergic Neurons Following Acute (30-min) Treatment
In order to determine whether acute exposure to TD or MZ might be neurotoxic to
GABAergic neurons in C. elegans, EG1285 worms were acutely treated with varying
concentrations of either TD or MZ (Figure 3). The number of green pixels was analyzed in
GABAergic neurons of the ventral nerve cord. Pixel analysis of acute treatments with TD
(Figure 3a) demonstrated a statistically significant decrease in pixel number at 7%
glyphosate (LC50) and 10% glyphosate (LC75) compared to controls (*p < 0.05). Compared
to control worms (Figure 3b), the somas of GABAergic neurons from worms treated with
10% glyphosate were much smaller (Figure 3c). Acute treatment with MZ (Figure 3d) lead
to a significant reduction in green pixel number when comparing 30% Mn/Zn-EBDC (LC75)
with controls (***p < 0.001), 0.1% Mn/Zn-EBDC (^^p < 0.01) or with 7.5% Mn/Zn-EBDC
(##p < 0.01). The somas of the GABAergic neurons from treated worms (30% Mn/Zn-
EBDC) were observed to be smaller (Figure 3e), when compared to worms treated only with
water (Figure 3b).
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Decreased GFP Pixel Number in GABAergic Neurons Following Chronic (24-h) Treatment
In order to assess the effect of chronic exposure to TD or MZ on GABAergic neurons,
EG1285 worms were chronically treated with either TD or MZ (Figure 4). Analysis of the
green pixels in the ventral nerve cord showed a statistically significant decrease in green
pixel number at 2.7% (LC25), 5.5% glyphosate (LC50; **p < 0.01) and 9.8% (LC75; ***p <
0.001) compared to controls (Figure 4a). In addition a significant reduction in green pixel
number was seen at 9.8% glyphosate compared to 5.5% glyphosate (^p < 0.05). Compared
to control worms (Figure 4b), this decrease in pixel numbers was accompanied by a
concomitant shrinkage of the somas for all concentrations (data only shown for 2.7%
glyphosate in Figure 4c). Following chronic MZ treatment (Figure 4d), a statistically
significant reduction in green pixel number was observed at 1.0% (LC50) and 1.5% Mn/Zn-
EBDC (LC75; ***p < 0.001) in comparison to controls. Also a significant reduction in green
pixel number was observed at 1.0% and 1.5% Mn/Zn-EBDC (^^p < 0.01) compared to 0.1%
Mn/Zn-EBDC (LC25). Similar to the chronic treatment with TD, the somas of GABAergic
neurons from worms treated with Mn/Zn-EBDC were smaller (data shown only for 1.5%
Mn/Zn-EBDC in Figure 4e) than controls (Figure 4b).
GFP Pixel Number in GABAergic Neurons Following Dual Treatment
The dual treatment paradigm exposure to TD and MZ was used in order to determine
neurotoxicity in GABAeric neurons in C. elegans. EG1285 worms were acutely treated with
TD (2% glyphosate) followed by acute treatment with varying concentrations of MZ.
Analysis of number of green pixels in the ventral nerve cord showed no significant reduction
in green pixel number at any concentrations (data not shown). This was confirmed by visual
inspection of the neurons as well, as no difference in soma size or projections was observed.
DISCUSSION
Although there is a well-documented genetic contribution to early onset PD (Bekris et al.
2010), multiple studies strongly suggest that the interplay of environmental toxicants and
potential susceptibility genes play a much greater role in the later onset of the disease
(Brown et al. 2006; Tanner 2010). In general, numerous pesticides are recognized as playing
a role in neurodegeneration in studies involving rodents (Bahrami et al. 2009; Binukumar
and Gill 2010; Jiang et al. 2010; Slotkin et al. 2009). This degeneration may or may not
strictly involve the DAergic system, which contributes to the cardinal symptomatology
associated with PD. Interestingly, few studies exist in the literature related to glyphosate-
containing herbicides even though they are some of the most ubiquitously applied pesticides
in the world (Woodburn 2000). While it is recognized that glyphosate is relatively non-toxic,
with an oral LD50 = 4320 mg/kg in rats (Birch 1993), several reports suggest that the actual
formulations of glyphosate-containing herbicides, such as RoundUp® or TD, may be
mitochondrial inhibitors (Olorunsogo et al. 1979; Peixoto 2005). In light of data potentially
delineating differences between the active ingredient and commercial formulations, we
sought to determine what effect the actual formulations to which humans are exposed had on
DAergic and GABAergic neurons in C. elegans.
Previous work from our lab (Negga et al. 2011) demonstrated that neurons in C. elegans are
vulnerable to treatment with varying concentrations of commercially available TD or MZ,
which has been linked previously to DAergic degeneration (Domico et al. 2007; Domico et
al. 2006) and mitochondrial inhibition (Zhang et al. 2003). Those studies (Negga et al.
2011) were limited in their specificity by the fact that all neurons in the worm strain used
(NW1229) were tagged with GFP. Although this provided evidence of general
neurodegeneration, it did not allow in-depth analysis of the effect of TD and/or MZ on
specific neuronal populations. In the current study, we used strains that had GFP selectively
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attached to proteins found in either DAergic (BZ555) or GABAergic (EG1285) neuronal
populations. Both strains have been used to examine sensory neuron cilia formation (Senti
and Swoboda 2008), neuronal degeneration (Samara et al. 2010), and DAergic
neurodegeneration (Benedetto et al. 2010). By comparing data from our first study (Negga
et al. 2011) with the present information, it is possible to better assess the vulnerability of
discrete neuronal populations.
It is important to note that the pesticide concentrations used in the present investigation,
while at the LC25, LC50 and LC75 for the worms (Table 1) are within the environmentally
relevant concentrations available and applied. For example, the recommended application
concentration for TD for fields with weeds over 15 cm tall is 0.6 – 0.9% glyphosate,
although the concentration can be as high as 4 – 10% glyphosate for spot spraying (Syngenta
2010). Typically, however, glyphosate products for use by the general public are sold with
2% glyphosate, a general use concentration that does not require dilution. On the other hand,
the recommended application concentration for MZ is 0.29 – 0.49% Mn/Zn-EBDC (Bonide
2010). When considering the concentrations for the dual treatment, we considered the
following: (1) in a growing season, herbicides (in this case TD) would be applied prior to
any fungicides (Randall et al. 2008a); (2) because of the time difference during a growing
season when herbicides or fungicides were used do not generally overlap, the pesticides
should not be applied as a true mixture (which would also violate the application
instructions provided for agricultural workers; Randall et al. 2008b)); (3) since the most
common concentration of TD used is 2% glyphosate, this was the concentration used in the
dual treatment studies (Negga et al, 2011).
When BZ555 worms were treated acutely with either TD (Figure 1A) or MZ (Figure 1D),
changes in green pixel numbers and neuronal morphology were only observable at the LC75
for each pesticide concentration (10% glyphosate or 30% Mn/Zn-EBDC, respectively). This
was in contrast to previous data (Negga et al, 2011), in which morphological and pixel
changes in the nerve ring and head region were also observed at the LC25 and LC50 for TD
concentrations. In addition to DAergic neurons, which were the only neurons labeled in the
BZ555 strain, the NW1229 worms also had glutamatergic (Glu) interneurons, serotonergic
(5-HT) amphid and interneurons, and cholinergic (ACh) ring interneurons and motor
neurons labeled. As the previous data demonstrated noticeable neurodegeneration at lower
concentrations of TD or MZ, the current data suggest that other populations (Glu, 5-HT and
ACh) are more vulnerable than DAergic neurons following an acute exposure to TD when
considered in light of our previous data (Table 2). This is similar to the acute treatment with
MZ, in which statistically significant changes in pixel number and morphology were only
noted at the LC75 for MZ in BZ555 worms, rather than the lower concentrations (Figure
1D).
When BZ555 worms were treated chronically with MZ (Figure 2D), DAergic neurons were
affected only at the highest concentration of Mn/Zn-EBDC (1.5%), suggesting that previous
data (Negga et al. 2011) indicating morphological changes more likely reflected
degeneration of Glu, 5-HT and ACh neurons (Table 2). The data from MZ-treated worms
(BZ555) were in contrast to the data from worms chronically treated with TD (Figure 2A).
Here the TD data closely paralleled that generated from NW1229 worms, suggesting that in
a chronic paradigm, DAergic neurons are more vulnerable than other neuronal populations
in C. elegans.
In general, GABAergic neuronal degeneration was more evident in EG1285 worms (Figure
3 and 4) at lower concentrations of pesticides than that observed in NW1229 worms. Data
from our current study confirmed that, for acute TD and chronic MZ treatments, the neurons
that are apparently the most vulnerable are those of the GABAergic system. In addition to
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GABAergic neurons in the ventral nerve cord of C. elegans, there are also ACh ventral
motor neurons, and 5-HT motor neurons (Rand and Nonet 1997). As no measureable
degeneration was observed in NW1229 worms treated either acutely with MZ or chronically
with TD, our interpretation is that, because of the multiple populations tagged in NW1229
worms, the GABAergic degeneration observed in the current study was overshadowed by
the presence of the other neuronal populations. Thus, by focusing solely on the GABAergic
neurons in this study, we were able to increase the specificity and sensitivity of our
evaluation of these neurons (Table 2).
In complex in vivo model systems, it is often difficult to examine the effect of various
toxicants on the nervous system. In the case of C. elegans, with its 302 neurons (Chalfie and
White 1988; Thomas and Lockery 1999), it is much easier to determine the fate of distinct
populations than in mammals, which have millions of neurons. This is evident in the current
study where we were able to determine the differential vulnerability of various neuronal
populations in the nematode following exposure to environmental toxicants. While a large
body of work involving pesticide exposure has focused on DA, greater recognition of the
role of other neurotransmitters has become the focus of more recent studies (Lester et al.
2010; Wang et al. 2010; Yamamoto and Soghomonian 2009). In the context of the
expanding interest of neurotransmitters other than DA involved in PD, it is timely that our
research has shown a potentially increased vulnerability of GABAergic neurons to a widely-
used glyphosate-containing herbicide (TD), and a popular Mn-containing fungicide, MZ.
Interestingly, however, our dual treatment paradigm, based on current commercial
application procedures continues to demonstrate a decreased, rather than increased, neuronal
vulnerability (see also Negga et al, 2011). In light of the visual differences between treated
and control neuronal populations in NW1229 worms, and the measured loss of green pixel
number, we had anticipated that the dual treatment would lead to greater degeneration in
both the BZ555 and EG1285 worms. This was obviously not the case, and requires further
mechanistic investigations.
CONCLUSION
This work is significant because it represents one of the first studies in the literature to
systematically identify potentially vulnerable neuronal populations in C. elegans following
exposure to the glyphosate-containing herbicide TD, a representative of a ubiquitous
herbicide class, and a commonly- and highly-used fungicide (MZ) similar to maneb, which
has already been linked to DAergic neurodegeneration. It also demonstrates that chronic, but
perhaps not acute, exposure to TD leads to DAergic degeneration in C. elegans.
Furthermore, it highlights the sensitivity of GABAergic neurons to these pesticides, whether
an acute or chronic exposure paradigm is used, suggesting that future work with to these two
pesticide classes examine GABAergic degeneration as a potentially early marker of neuronal
vulnerability.
Acknowledgments
FUNDING
This work was supported by the National Institute of Environmental Health Sciences [R15 ES015628 to VF] and by
the Appalachian College Association Colonel Lee B. Ledford Endowment Fund [to RN, AJ and VF].
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Fig 1.
Acute treatments of BZ555 nematodes. Following analysis of photomicrographs from
worms acutely exposed to TD or MZ, the number of green pixels was determined as
described in the methods. Data in (A) and (D) are presented as mean pixel number ± SD of
the DAergic neurons in the nerve ring. *p < 0.05 and **p < 0.01 compared to nerve ring of
controls and ^^^ p < 0.001 compared to the nerve ring of 0.7% Mn/Zn-EBDC. The blue
arrows point to soma in the region of the nerve ring. Blue arrows in photomicrographs of
worms treated with 10% (C) glyphosate and 30% (E) Mn/Zn-EBDC emphasize the
decreased sizes of the soma compared to the soma of CN worms (B).
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Fig 2.
Chronic treatments of BZ555 nematodes. Following analysis of photomicrographs of worms
chronically exposed to TD or MZ, the number of green pixels was determined as described
in the methods. Data in (A) and (D) are presented as mean pixel number ± SD of the
DAergic neurons in the nerve ring. *p < 0.05 and **p < 0.01 compared to nerve ring of
controls and ^p < 0.05 compared to nerve ring of 2.7% glyphosate. The blue arrows point to
soma in the region of the nerve ring. Blue arrows in photomicrographs of worms treated
with 9.8% (C) glyphosate and 1.5% (E) Mn/Zn-EBDC emphasizes the decreased sizes of
the soma compared to the soma of CN worms (B).
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Fig 3.
Acute treatments of EG1285 nematodes. Following analysis of photomicrographs of worms
chronically exposed to TD or MZ, the number of green pixels was determined as described
in the methods. Data in (A) and (D) are presented as mean pixel number ± SD of
GABAergic neurons in the ventral nerve cord. *p < 0.05, ***p < 0.001 compared to ventral
nerve cord of controls, ^^p < 0.01 compared to ventral nerve cord of 0.1% Mn/Zn-EBDC
and ##p < 0.01 compared to ventral nerve cord of 7.5% Mn/Zn-EBDC. The blue arrows
point to soma along the ventral nerve cord. Blue arrows in photomicrographs of worms
treated with 10% (C) glyphosate and 0.5% (E) Mn/Zn-EBDC emphasize the decreased sizes
of the soma compared to the soma of CN worms (A).
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Fig 4.
Chronic treatments of EG1285 nematodes. Following analysis of photomicrographs of
worms chronically exposed to TD or MZ, the number of green pixels was determined as
described in the methods. Data in (A) and (D) are presented as mean pixel number ± SD of
GABAergic neurons in the ventral nerve cord. **p < 0.01 and ***p < 0.001 compared to
ventral nerve cord of controls, ^p < 0.05 compared to 2.7% glyphosate and ^^p < 0.01
compared to 0.1% Mn/Zn-EBDC. The blue arrows point to soma along the ventral nerve
cord. Blue arrows in photomicrographs of worms treated with 2.7% (C) glyphosate and
1.5% (E) Mn/Zn EBDC emphasize the decreased sizes of the soma compared to the soma of
CN worms (B).
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Table 1
Comparison of lethal concentrations among paradigms
Pesticides Active
Ingredient Recommended
Application
Concentration
^Acute
LC25, LC50,
LC75
^Chronic
LC25, LC50,
LC75
^Dual
LC25, LC50,
LC75
TouchDown® (TD) Glyphosate 0.6–10% 3.0%, 7.0%, 10.0% 2.7%, 5.5%, 9.8% *2%
Mancozeb® (MZ) (Mn)/zinc (Zn) ethylene-bis-dithiocarbamate 0.29–0.49% 0.7%, 7.5%, 30% 0.1%, 1.0%, 1.5% 10%, 12%,20%
*2% is the typically available application concentration
^All the experiments have a value of (N 4) and (n 12).
Table summarizes relevant pesticide application concentrations, and compares the lethal concentrations used for the various study paradigms.
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Table 2
Summary of Vulnerability of Neuronal Populations
Treatment
Group DAergic Sensitivity
(BZ555) GABAergic Sensitivity
(EG1285) Other Neuronal Populations
(Based on Negga et al, 2001)
Acute TD No Yes Yes (head)
Acute MZ No Yes Yes (head)
Chronic TD Yes Yes No
Chronic MZ No Yes Yes (head)
Dual Treatment No No Likely
Differences in data between the current study and Negga et al. 2011 indicate that GABAergic neurons C. elegans may be more sensitive to TD or
MZ exposure than DAergic neurons, regardless of single exposure paradigm.
Neurotox Res. Author manuscript; available in PMC 2013 April 1.
... The 0.1 mM concentration of glyphosate seems to be a critical dosage for the effects observed in our electroshock assay, both for convulsion duration and non-recovery observations. To build upon previously reported findings of neurotoxicity and stress in C. elegans 22,23,32,33 , we provided some critical insights into prolonged locomotive abnormalities that result from exposure to glyphosate and Roundup. To our knowledge, Roundup and glyphosate have never been tested at our experimental concentration of 0.1 mM. ...
... These findings lend themselves to two critical distinctions: there is evidence to further investigate how chronic exposure and accumulation may lead to neurodegenerative disease such as Parkinson's Disease, but, critical to this study, there is also a sub-neurodegenerative threshold that may dramatically impact dysregulation of neurotransmission. In C. elegans, there is a concentration where exposure to a glyphosate-based herbicide is neurotoxic 22 , but GABAergic dysregulation, specifically deficits in GABAergic signaling, is a common pathophysiology in humans and rodent models with mood disorders such as anxiety disorder and depression [41][42][43][44] . Acute exposure at 0.002% glyphosate results in a significant behavioral and physiological effect. ...
... Finding neurological effects at very dilute concentrations may also support the concern that herbicide exposure affects GABA-mediated mental health symptoms, particularly depression, in humans 34,43 . While high concentrations of 7% and 10% glyphosate may be necessary to induce acute toxicity 22,23 , an acute exposure at a concentration of 0.002% confers equally concerning and physiologically relevant behavioral effects. Our findings provide novel insight into the GABA-A receptor mediated proconvulsant effects of glyphosate and Roundup and generate concern over how herbicide use might affect soil-dwelling organisms like C. elegans. ...
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Aging is associated with the increased risk of most age-related diseases in humans. Complanatoside A (CA) is a flavonoid compound isolated from the herbal medicine Semen Astragali Complanati. CA was reported to have potential anti-inflammatory and anti-oxidative activities. In this study, we investigated whether CA could increase the stress resistance capability and life span of Caenorhabditis elegans. Our results showed that CA could extend the longevity of C. elegans in a dosage-dependent manner, while 50 μM of CA has the best effect and increased the life span of C. elegans by about 16.87%. CA also improved the physiological functions in aging worms, such as enhanced locomotor capacity, and reduced the accumulation of the aging pigment. CA could also reduce the accumulation of toxic proteins (α-synuclein and β-amyloid) and delay the onset of neurodegenerative disorders, such as Alzheimer’s disease and Parkinson’s disease, in models of C. elegans. Further investigation has revealed that CA requires DAF-16/FOXO, SKN-1, and HSF-1 to extend the life span of C. elegans. CA could increase the antioxidation and detoxification activities regulated by transcription factor SKN-1 and the heat resistance by activating HSF-1 that mediated the expression of the chaperone heat shock proteins. Our results suggest that CA is a potential antiaging agent worth further research for its pharmacological mechanism and development for pharmaceutical applications.
... Studies with the nematode C. elegans revealed median lethal concentration values of 0.22% to 0.50% v/v (formulated mancozeb, corresponding to 814-1850 mg/L a.i. [Mn/Zn EBDC]; Negga et al., 2011), while sublethal doses of mancozeb were able to induce neuronal degradation (Negga et al., 2011(Negga et al., , 2012, induced a heat shock response at 100 mg a.i./L, inhibited growth at 400 mg a.i./L (Easton et al., 2001), and caused behavioral deficits at 1850 mg a.i./L (Brody et al., 2013). Assuming a comparable bioavailability of mancozeb in soil (present study) and agar (Brody et al., 2013;Easton et al., 2001), the soil test according to ISO 10872 (ISO, 2020) was considerably more sensitive even if using the nominal soil concentrations (EC10, 32-54 mg a.i./kg dry soil; EC50, 128-155 mg a.i./kg dry soil). ...
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Fungicides make up the largest part of the total pesticide use, with the dithiocarbamate mancozeb being widely applied as a non‐systemic contact pesticide to protect a wide range of field crops against fungal diseases. Although nematodes are key drivers of soil functioning, data on effects of fungicides, and especially mancozeb, on these non‐target organisms are scarce. Therefore, the effects of mancozeb on a soil nematode community from a natural grassland was assessed in small‐scale soil microcosms. Nematodes were exposed to mancozeb‐spiked soil in six nominal concentrations (7 – 133 mg/kg dry soil) and analyzed after 14, 56 and 84 days in terms of densities, genus composition and functional traits. As this fungicide is known to quickly degrade in soils (DT50 < 1 day), mancozeb concentrations were analyzed for all sampling occasions. Chemical analysis revealed considerably lower measured concentrations as compared to the aimed nominal soil concentrations at the begin of the exposure (1 to 18 mg/kg dry soil), suggesting a fast degradation during the spiking process. Nevertheless, the native nematode community responded sensitively to the fungicide mancozeb, revealing lower NOEC and EC10 values than reported for other soil invertebrates such as springtails and earthworms. Using the EC10 for the most sensitive nematode community endpoint (% predators and omnivores: 1.2 mg/kg dry soil), the risk assessment exhibited a toxicity exposure ratio of 0.66 and thus a high risk of mancozeb for soil nematodes. Keeping in mind their abundance and their central roles in soil food web functioning, the demonstrated sensitivity to a widely applied fungicide underscores the relevance of the inclusion of nematodes into routine risk assessment programs for pesticides. This article is protected by copyright. All rights reserved.
... The neurotoxic effects of GBH have already been shown in many animal models. Experimental evidence has shown that GBHs induce oxidative stress, apoptosis, and disbalance in neurotransmitter systems [8][9][10][11]. Gallegos and colleagues have demonstrated that RUp ingestion during pregnancy and lactation in rats was also shown to impact the structure and function of the brains of the offspring [12]. However, the molecular mechanisms behind these neural injuries remain unknown. ...
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RoundUp® (RUp) is a comercial formulation containing glyphosate (N-(phosphono-methyl) glycine), and is the world’s leading wide-spectrum herbicide used in agriculture. Supporters of the broad use of glyphosate-based herbicides (GBH) claim they are innocuous to humans, since the active compound acts on the inhibition of enzymes which are absent in human cells. However, the neurotoxic effects of GBH have already been shown in many animal models. Further, these formulations were shown to disrupt the microbiome of different species. Here, we investigated the effects of a lifelong exposure to low doses of the GBH-RUp on the gut environment, including morphological and microbiome changes. We also aimed to determine whether exposure to GBH-RUp could harm the developing brain and lead to behavioral changes in adult mice. To this end, animals were exposed to GBH-RUp in drinking water from pregnancy to adulthood. GBH-RUp-exposed mice had no changes in cognitive function, but developed impaired social behavior and increased repetitive behavior. GBH-Rup-exposed mice also showed an activation of phagocytic cells (Iba-1–positive) in the cortical brain tissue. GBH-RUp exposure caused increased mucus production and the infiltration of plama cells (CD138-positive), with a reduction in phagocytic cells. Long-term exposure to GBH-RUp also induced changes in intestinal integrity, as demonstrated by the altered expression of tight junction effector proteins (ZO-1 and ZO-2) and a change in the distribution of syndecan-1 proteoglycan. The herbicide also led to changes in the gut microbiome composition, which is also crucial for the establishment of the intestinal barrier. Altogether, our findings suggest that long-term GBH-RUp exposure leads to morphological and functional changes in the gut, which correlate with behavioral changes that are similar to those observed in patients with neurodevelopmental disorders.
... It is noteworthy that neurotoxic effects were mediated by both organic moiety and Mn2+ (75). Correspondingly, DA and GABAergic neurons were characterized by degeneration in MCZ-exposed C. elegans (76). These findings corroborate earlier indications of Mn2+ toxicity (77) with a special focus on dopaminergic and GABAergic neurons (78). ...
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The objective of the present study was to review the existing data on the mechanisms involved in the endocrine disrupting activity of mancozeb (MCZ) in its main targets, including thyroid and gonads, as well as other endocrine tissues that may be potentially affected by MCZ. MCZ exposure was shown to interfere with thyroid functioning through impairment of thyroid hormone synthesis due to inhibition of sodium-iodine symporter (NIS) and thyroid peroxidase (TPO) activity, as well as thyroglobulin expression. Direct thyrotoxic effect may also contribute to thyroid pathology upon MCZ exposure. Gonadal effects of MCZ involve inhibition of sex steroid synthesis due to inhibition of P450scc (CYP11A1), as well as 3β-HSD and 17β-HSD. In parallel with altered hormone synthesis, MCZ was shown to down-regulate androgen and estrogen receptor signaling. Taken together, these gonad-specific effects result in development of both male and female reproductive dysfunction. In parallel with clearly estimated targets for MCZ endocrine disturbing activity, namely thyroid and gonads, other endocrine tissues may be also involved. Specifically, the fungicide was shown to affect cortisol synthesis that may be mediated by modulation of CYP11B1 activity. Moreover, MCZ exposure was shown to interfere with PPARγ signaling, being a key regulator of adipogenesis. The existing data also propose that endocrine-disrupting effects of MCZ exposure may be mediated by modulation of hypothalamus-pituitary-target axis. It is proposed that MCZ neurotoxicity may at least partially affect central mechanisms of endocrine system functioning. However, further studies are required to unravel the mechanisms of MCZ endocrine disrupting activity and overall toxicity.
... The non-parasitic nematode Caenorhabditis elegans is a great model for ecotoxicological predictions since its habitat is mostly in the soil, where it feeds in organic matter. Taking advantage of strains that have green fluorescent protein (GFP) tagged to a protein of interest, the Fitsanakis group demonstrated that acute (30 min) and chronic (24 h) exposure to the Touchdown formulation causes significant GABAergic and dopaminergic neurodegeneration (Negga et al. 2012;McVey et al. 2016). A proposed mechanism for this neurotoxic effect is the mitochondrial dysfunction, since it has been evidenced that this GBH reduces oxygen consumption, inhibits complex II and reduced ATP production (McVey et al. 2016). ...
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Glyphosate [N-(phosphonomethyl)glycine] is one of the organophosphate herbicides which is most frequently used in agriculture, forestry, green public areas, and gardens for elimination of annual and perennial weeds. High efficiency in weed and pest control was achieved by using glyphosate, thus harvesting of clean and healthy final products was possible. However, extensive long-term application of glyphosate resulted in its accumulation in various environments. That accumulation poses a great concern for public health, suggesting the hazardous potential of this herbicide to various non-target organisms.
... Glyphosate [N-(phosphonomethyl) glycine] is an organophosphate and one of the most popular herbicides in the world and the active ingredient of the plant protection product Roundup®. As previous investigations performed on various organs have revealed, glyphosate causes hepatotoxicity (El-Shenawy, 2009), neurologic disorders (Negga et al., 2012;Rebai et al.,2017), and nephrotoxicity (Astiz et al., 2009). Importantly, detection of glyphosate residues in human urine has proposed that early evidence of kidney injury could be used to predict the risk of glyphosate toxicity (Valcke et al., 2017). ...
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Glyphosate, the active substance in Roundup®, is the most widely used pesticide in the world and may be present as a residue in derived foods and drinking water. Previous reports have confirmed that extracts from leaves of Morus alba exert many pharmacological activities. However, renoprotective effects of M. alba extract and its underling molecular mechanism is still unknown. Wistar rats (180-200 g) were used in this study (n=5-6). A control group received 0.2 ml normal saline intraperitoneally (i.p) once daily for two weeks. Control animals received standard diet. Treated groups received either polyphenolic extract (100 mg/kg,i.p) or glyphosate (100 mg/kg, i.p), or co-administration (extract μg ml−1 kg b.w. and glyphosate 100 mg kg−1 b.w, i.p), daily until the 15thday of treatment. Lactate deshydrogenase LDH, serum concentrations of blood urea, creatinine and nitric oxide were measured using standard coloromertic methods. Renal oxidative stress, evidenced by increased malondialdehyde (MDA) and protein carbonyl levels and decline in superoxide dismutase (SOD) activity, was significantly alleviated by mulberry leaves extract (MLE) administration. MLE also appears to be able to modulate altered biochemical parametres by maintaining free iron and Ca2+ homeostasis, and regulate the endogenous antioxidant enzymes system. It seems that concurrent use of the aqueous acetonic fraction of M. alba, rich in chlorogenic acid and its isomeres, can protect kidneys from glyphosate-induced nephrotoxicity. Overall, MLE may possess protective activity against glyphosate-induced toxicity, which may be attributed to chlorogenic acid and its isomers, the most abundant phenolic acids present in its extracts. Mulberry leaves are a source of phenolic compounds and can be a good start towards discovering a new chemical compound which may lead to a new drug. A mulberry extract supplement could serve as a candidate for developing a safe, and promising nutraceutical product for the management of nephrotoxicity. Keywords: glyphosate, oxidative stress, nephrotoxicity, mulberry, nephroprotective effect
Chapter
While the model organism Caenorhabditis elegans has been used by cell biologists since the 1970s, it has only recently been more widely adopted by the toxicology community. Although the worm's genome was published in 1998, the development of cheaper and more rapid sequencing technology has been instrumental in confirming that the human and C. elegans genomes share approximately 80% homology. This homology reinforces further supports using this model organism as an in vivo model for human toxicant exposure. The goal of this chapter is to provide information about the worm's development and reproductive cycles, outline pathways common to worms and humans, present endpoints for assessing development and reproduction, and provide examples of developmental or reproductive toxicants previously studied in C. elegans. This approach is designed to help scientists familiar with C. elegans gain a better understanding of how to incorporate toxicology into their research, and provide toxicologists with tools to enhance their use of these worms in developmental and reproductive toxicity studies.
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The rise of various neurodegenerative disorders are somewhat correlating with the worldwide application of multiple anthropogenic toxicants. Though different possible targets were revealed to date, for example, for organophosphorus compounds (OPs), plenty of questions remain. Several decarboxylases (aromatic amino acid decarboxylase, AADC; histidine decarboxylase, HDC; glutamate decarboxylase, GAD) catalyze the biosynthesis of neurotransmitters and neuromodulators and contain pyridoxal phosphate (PLP) as a cofactor. In the current work, 18 OPs which have different neurotoxicity (chemical warfare agents and pesticides) and can penetrate through the blood–brain barrier, were selected. Then, their possible interaction with these decarboxylases in both apo- and holoforms was revealed using computer modeling methods (molecular docking and dynamics). The main amino acid residues of the enzymes responsible for binding OPs have been identified. Individual substances that are most dangerous from the point of view of a possible negative effect on the activity of several decarboxylases were revealed among studied OPs. Glyphosate should be of special interest, since it is not highly toxic towards serine hydrolases, but may prove to be a strong inhibitor for decarboxylases. Holo-AADC could be the most inhibition-prone enzyme among all those investigated.
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Background: Environmental exposures are implicated in the etiology of amyotrophic lateral sclerosis (ALS). Application of insecticides, herbicides, and fungicides with neurotoxic properties to crops is permitted in the U.S., however reporting of the quantities is government mandated. Objective: To identify pesticides that may be associated with ALS etiology for future study. Methods: We geospatially estimated exposure to crop-applied pesticides as risk factors for ALS in a large de-identified medical claims database, the SYMPHONY Integrated Dataverse®. We extracted residence at diagnosis of ∼26,000 nationally distributed ALS patients, and matched non-ALS controls. We mapped county-level U.S. Geological Survey data on applications of 423 pesticides to estimate local residential exposure. We randomly broke the SYMPHONY dataset into two groups to form independent discovery and validation cohorts, then confirmed top hits using residential history information from a study of NH, VT, and OH. Results: Pesticides with the largest positive statistically significant associations in both the discovery and the validation studies and evidence of neurotoxicity in the literature were the herbicides 2,4-D (OR 1.25 95% CI 1.17-1.34) and glyphosate (OR 1.29 95%CI 1.19-1.39), and the insecticides carbaryl (OR 1.32 95%CI 1.23-1.42) and chlorpyrifos (OR 1.25 95%CI 1.17-1.33). Significance: Our geospatial analysis results support potential neurotoxic pesticide exposures as risk factors for sporadic ALS. Focused studies to assess these identified potential relationships are warranted.
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Glyphosate herbicide is the largest-selling single crop-protection chemical product in the market today. This non-selective weedkiller was initially targeted at the non-crop areas in agriculture and for industrial applications but, with the continuing development of minimum- and no-tillage agricultural practices, glyphosate also found usage in a number of crop outlets. Most recently, glyphosate has found direct crop usage on plant varieties that have been genetically modified to be tolerant of glyphosate applications. Such has been the continuing success of the product that its annual volume consumption growth has averaged in excess of 20% in recent years in agricultural use. (C) 2000 Society of Chemical Industry.
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This chapter focuses on the threat to the productivity of world agriculture imposed by the evolution of herbicide-resistant weed populations. Implicit in this chapter is that herbicides should and will continue to be a major tool for weed control. It is believed that modern herbicides are a cost-effective, efficient, and environmentally benign means for obtaining weed control. Proponents rightly identify the benefits of herbicides in achieving weed control as well as positive environmental influences in substituting for soil cultivation in weed management. Reliance on herbicides for weed control is expected to continue because there is no attractive superior technology available. However, for sustainable weed management to be achieved, changes to current herbicide use patterns are required. Multiple-resistant populations of weeds, such as L. rigidum and A . myosuroides, are current indicators of potential worst-case weed problems of the future. Resistance will continue to increase if present herbicide use patterns are not altered.
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Parkinsonism is a rare complication in patients with organophosphate poisoning. To date there have been two cases of transient parkinsonism after acute and severe cholinergic crisis, both of which were successfully treated using amantadine, an anti-parkinsonism drug. We report on an 81-year-old woman who was admitted for the treatment of acute severe organophosphate poisoning. Although acute cholinergic crisis was treated successfully with large doses of atropine and 2-pyridine aldoxime methiodide (PAM), extrapyramidal manifestations were noticed on hospital day 6. The neurological symptoms worsened, and the diagnosis of parkinsonism was made by a neurologist on hospital day 9. Immediately, biperiden (5 mg), an anti-parkinsonism drug, was administered intravenously, and her symptoms markedly improved. From the following day, biperiden (5 mg/day) was given intramuscularly for eight days. Subsequently, neurological symptoms did not relapse, and no drugs were required. Our patient is the third case of parkinsonism developing after an acute severe cholinergic crisis and the first case successfully treated with biperiden. Patients should be carefully observed for the presence of neurological signs in this kind of poisoning. If present, an anti-parkinsonism drug should be considered.
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Manganese (Mn) exposure can cause parkinsonism. Pathological changes occur mostly in the pallidum and striatum. Two patients with a long history of occupational Mn exposure presented with Mn-induced parkinsonism. In one patient, magnetic resonance imaging (MRI) showed findings consistent with Mn exposure, and Mn concentration was increased in the blood and urine. However, this patient's clinical features were typical of idiopathic Parkinson disease (PD). Previous pathological and positron emission tomography studies indicate that striatal dopamine transporter density is normal in Mn-induced parkinsonism, whereas it is decreased in PD. Therefore, we performed [123I]-(1r)-2β-carboxymethoxy-3β-(4-iodophenyl)tropane ([123I]-β-CIT) single-photon emission computed tomography. Severe reduction of striatal β-CIT binding was indicated, which is consistent with PD. We propose three interpretations: (1) the patients have PD, and Mn exposure is incidental; (2) Mn induces selective degeneration of presynaptic dopaminergic nerve terminals, thereby causing parkinsonism; or (3) Mn exposure acts as a risk of PD in these patients. Our results and careful review of previous studies indicate that the axiom that Mn causes parkinsonism by pallidal lesion may be over-simplified; Mn exposure and parkinsonism may be more complex than previously thought. Further studies are required to elucidate the relationship between Mn and various forms of parkinsonism. © 2002 Movement Disorder Society
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Parkinson's disease (PD) is likely due to the combined effects of environment and genes in most cases. Environmental factors inversely associated with PD (or, putative protective factors) include cigarette smoking, use of coffee/caffeine, higher uric acid levels, and anti-inflammatory drug use. Less well-established inverse associations with PD include higher cholesterol levels, statin use, higher dietary vitamin B6, and night shift work. Putative risk factors are pesticide exposure, head trauma, certain occupations, and milk consumption. The pathogenesis of PD may begin decades before motor symptoms. PD may have shared determinants with other neurodegenerative disorders involving abnormal protein aggregation. © 2010 Movement Disorder Society