Critical Review

While organochlorine pesticides are a useful tool for the short term reduction of agricultural and domestic pests, they have been shown to contribute to neurological disorders such as
Parkinsons disease and autism spectrum disorders (Caudle, 2015). As more data is discovered
linking pesticides to functional and behavioral deficits, it is becoming more important to discover
the mechanisms by which they disrupt cell physiology. In particular, endosulfan has been shown
to both accumulate in high levels in the cord blood and breast milk of expecting mothers and effect GABAergic signaling (Cabaleiro et al., 2008, Jimenez Torres et al., 2006). Prior to the work
of Wilson et al there was a distinct lack of data on the remaining cellular and molecular processes affected by endosulfan (Wilson, 2014). As such, Wilson et al sought to use an IMR-32 neuroblastoma cell line, a primary cortical culture model, and an in vivo mouse model to uncover
changes to cell viability, neuron properties, and neurotransmitter circuits caused by endosulfan.
In order to test the cell mortality caused by exposure to endosulfan Wilson et al first utilized an in vitro IMR-32 neuroblastoma cell line, which was chosen for its known expression of
GABAergic receptors (Anderson et al., 1993; Noble et al., 1993; Sapp and Yeh, 2000). They
found that exposure to endosulfan dissolved in DMSO at six different concentrations reduced
cell viability significantly when compared to a DMSO control. Higher concentrations were
shown to cause greater reductions in cell viability (Figure 1a). The authors interpreted this data
as providing support for the neurotoxic role of endosulfan in the brain.
In order to test the effects of endosulfan on neuron growth, synaptic puncta, and neurite
length, Wilson et al generated primary cultures from the medial frontal cortex of post natal day
1-3 mice (Figure 1b). Following growth the cultures were split into a 96 well plate where they
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were treated with five different concentrations of endolsulfan, and a DMSO control. Following
the nine day treatment period the cultures were fixed in 4% PFA and incubated overnight in antiMAP2 and rabbit anti-synapsin at 4C. The following day fluorescent secondary antibodies were
added to the cultures at room temperature for one hour. The cells were then stained with DAPI
and stored in PBS until they were visualized using the 10x objective on the array scan. From the
images they found that there were significant reductions in neurite length at all endosulfan concentrations, significantly less synaptic puncta at concentrations higher than 2uM, and a significant increase in neurons per field at 1uM (Figure 1b). The authors believe the data suggest endosulfan can moderately damage neuronal processes before affecting the cell body. Additionally,
they posit that the synaptic puncta appear particularly sensitive to endosulfan exposure.
In the third part of their study, Wilson et al utilized eight-week-old mice to create a prenatal endosulfan exposure model. All mice were maintained on a 12:12 light dark cycle and given unrestricted access to food and water. Beginning two weeks prior to mating female mice were
given either plain peanut butter, or peanut butter containing 1 mg/kg endosulfan every two days
(Figure 1c). This protocol was continued throughout breeding, gestation, and lactation. Upon
birth, pups were split into 6-8 pup litters, and separated by sex at 12 weeks. At this time male
mice were sacrificed for immunohistochemistry and immunoblot analysis.
After homogenizing brain samples, they were subjected to gel electrophoresis and transferred to polyvinylidene difluoride membranes. The membranes were blocked in Tris-buffered
7.5% nonfat dry milk and incubated in primary overnight. The following day membranes were
incubated in a horseradish peroxidase secondary (1:10,000) followed by a chemiluminescent
reagent. Membranes were subsequently imaged and then stripped for 15 minutes at room tem!2

perature to be incubated in new primary antibodies. Analysis of the images revealed the following changes in protein expression: 23% reduction in VGAT protein expression; 21% reduction in
GAT1 protein expression; 91% increase in GABAa receptor protein expression; 178% increase
in vGlut protein expression; 32% reduction in GLuN 2B receptor expression; 53% reduction in
DAT protein expression; 44% reduction in TH protein expression; 39% reduction in VMAT2
protein expression; 90% increase in D2 receptor protein expression. The authors believe the data
suggest endosulfan exposure results in changes to the GABAergic, glutamatergic, and dopaminergic neurotransmission pathways.
The authors then fixed whole brain samples for control and endosulfan offspring in 4%
PFA and serially sectioned them at 40um. These sections were incubated overnight with rabbit
anti-GAT1, rabbit anti-vGAT, mouse anti-GABAA 2a receptor subunit, and rabbit anti-vGlut,
and the following day incubated in secondary for one hour. Brain slices were then stained with
DAB for three minutes, mounted on slides and visualized with the array scan to show the neurotransmitter related targets inside the cell.
A major strength of this paper lies in the justification for the study. The authors provide a
substantial amount of information supporting the need to study endosulfan as a neurotoxin, including its propensity to accumulate in major human organs (Moreno Frias et al., 2004). They
also provide a logical explanation for choosing the IMR-32 neuroblastoma cell line for their in
vitro experiment, namely its expression of GABAa receptors and response to GABAergic compounds (Anderson et al., 1993; Noble et al., 1993; Sapp and Yeh, 2000).

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Another strength of the study was the large range of cell and neurotransmitter effects that
were covered. Although previous studies have researched the neurological effects of endosulfan,
they did not include in vitro models (Cabaleiro et al., 2008; Lakshmana and Raju, 1994). While
the previous studies did cover a significant number of neurotransmitter targets, Wilson et al takes
a more in depth look into the neurotransmitter pathways, testing protein expression levels for
multiple targets including transporters, vesicular transporters, and receptors. Other possible targets for analysis could have been the components of the cholinergic system, including the vesicular acetylcholine transporter, the nicotinic acetylcholine receptor, and the muscarinic acetylcholine receptor. Any effects of endosulfan on the cholinergic system could assist in the research
of endosulfan as a contributor to Alzheimers, as the reduction of cholinergic neurons is a major
phenotype of Alzheimers disease.
A major flaw lies in the authors propensity towards omitting data. Offering explanations
based on absent data reduced my ability to judge the validity of the statements, and completely
eliminated the option to form an independent hypothesis (Nat. Chem. Biol. 4, 575, 2008). For
example, the author states that endosulfan did not have adverse effects on the growth rates or
general health of pups, yet they omit the data to support this. Similarly, the authors show almost
exclusively positive results, omitting most negative data. While positive results are alluring, the
omission of negative data reduced my ability to form my own connections between the results
provided (Matosin, 2014). Put simply, when everything is shown to change it is impossible to
figure out exactly why.
Experimentally, the authors neglect to provide the necessary information for me to easily
replicate their experiments. While they explain where they bought their primary antibodies, they
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do not provide the concentrations that were used. They also neglect to disclose their IMR-32 cell
supplier, or how they thawed their cells once they purchased them. Incorrectly thawing the cells,
such as thawing them too rapidly, could result in osmotic damage and cell death, impairing my
ability to replicate their in vitro neuroblastoma experiments (Mazur, 1984).
A major claim of the paper is that endosulfan exposure during critical periods of brain
development causes neurological deficits. However, endosulfan was given to pregnant dams prior to and during the entirety of their pregnancy. If the authors wanted to conclude that endosulfan
exposure during critical stages of development caused the observed changes in frontal cortex
protein expression, I believe they should have begun exposing the dams to endosulfan during
known periods of neocortex neurogenesis. It has been experimentally determined that rats begin
to develop their neocortex at gestational day 14, so it would have been reasonable to begin exposure at this time (Rice, 2000).
The purpose of this study was to discover unknown effects of endosulfan on brain development, and in this it succeeded. The authors provided new data detailing the effects of endosulfan across multiple neurotransmission systems, with multiple targets per system. The impact of
these finding has yet to be seen, as very few papers have utilized the data of this relatively recent
study. Despite omitting some negative data, the authors did contribute new and significant information towards discovering the effects of endosulfan pesticide exposure, and thus this paper
deserves to be published.

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a.

b.

c.

Figure 1. Experimental Protocol and Data

a.
IMR-32 Neuroblastoma cells were cultured and split into a 96 well plate at 40,000 cells/well at 100uL. Cells were then treated with endosulfan dissolved in DMSO at 0uM (DMSO only), 25uM, 50uM, 100uM, 200uM, 300uM, and 400uM concentrations. Following 24 hour treatment, 10uL of
WST-1 was added per well and the cells were left to incubate at 37C for 3 hours. Spectral absorbance was measured at 450nM and reductions in cell
viability were calculated. N=3
b.
Post natal day 1-3 mice were sacrificed and their brains removed. The medial section of their frontal cortex was removed and cultured until the
mixed glial culture could be split into a 96 well plate at 40,000 cells/well. Cells were then treated daily with endosulfan dissolved in DMSO at 0uM,
1uM, 2uM, 3uM, 4uM, or 5uM. 17 wells/plate were used per treatment group, and three total plates were tested. Following 9 days of treatment the
cells were fixed, incubated in anti-MAP2 and rabbit anti-synapsin overnight at 4C, and treated with fluorescent secondary antibodies the following
day for 1 hour at room temperature. Cells were then stained with DAPI and visualized on the Array scan. Neurons/field, neurite length, and synaptic
puncta were counted and compared to DMSO control.
c.
Eight week old females were given endosulfan dissolved in peanut butter at 1mg/kg or a peanut butter vehicle every 2 days for two weeks prior to
mating. Treatment was continued throughout gestation and lactation, and at birth mice were split into litters of 6-8. At 12 weeks male mice were separated and sacrificed. The frontal cortex was isolated, homogenized, and probed for GAD67, GAT1, vGAT, GABAA 2a receptor subunit, vGlut,
GluN2B receptor subunit, DAT, TH, VMAT2, and dopamine D2 receptor. Whole brains were immersion fixed and sliced to 40um diameter. Slices
were incubated in rabbit anti-GAT1, rabbit anti-vGAT, mouse anti-GABAA 2a receptor subunit, and rabbit anti-vGlut primary overnight. The following day they were incubated in secondary for 1 hour at room temperature, stained with DAB for 3 minutes, mounted on slides, dehydrated, and cover
slipped. N=6-8 group

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References
Anderson SM, De Souza RJ, Cross AJ. 1993. The human neuroblastoma cell line, IMR-32 possesses a
GABAA receptor lacking the benzodiazepine modulatory site. Neuropharmacology 32:455460.
Cabaleiro T, Caride A, Romero A, Lafuente A. 2008. Effects of in utero and lactational exposure to
endosulfan in prefrontal cortex of male rats. Toxicol Lett 176:5867.
Caudle, W. (2015). Vulnerability of synapses in the frontal cortex of mice developmentally exposed to an
insecticide: Potential contribution to neuropsychiatric disease. Neurotransmitter, 2(1), E514E514. doi:10.14800/nt.514
Data shown. (2008). Nature Chemical Biology, 5(10), 575-575. doi:10.1038/nchembio1008-575
Jimenez Torres M, Campoy Folgoso C, Canabate Reche F, Rivas Velasco A, Cerrillo Garcia I, Mariscal
Arcas M, Olea-Serrano F. 2006. Organochlorine pesticides in serum and adipose tissue of
pregnant women in Southern Spain giving birth by cesarean section. Sci Total Environ 372:32
38.
Lakshmana MK, Raju TR. 1994. Endosulfan induces small but significant changes in the levels of
noradrenaline, dopamine and serotonin in the developing rat brain and deficits in the operant
learning performance. Toxicology 91:139150.
Matosin, N., Frank, E., Engel, M., Lum, J. S., & Newell, K. A. (2014). Negativity towards negative
results: a discussion of the disconnect between scientific worth and scientific culture. Disease
Models & Mechanisms, 7(2), 171173. http://doi.org/10.1242/dmm.015123
Mazur, P. (1984). Freezing of living cells: Mechanisms and implications. American Journal of Physiology,
C125-C142. Retrieved October 26, 2015, from http://ajpcell.physiology.org/content/247/3/
C125.article-info
Moreno Frias M, Jimenez Torres M, Garrido Frenich A, Martinez Vidal JL, Olea-Serrano F, Olea N. 2004.
Determination of organochlorine compounds in human biological samples by GC-MS/MS.
Biomed Chromatogr 18:102111.
Noble PJ, Anderson SM, De Souza RJ, Cross AJ, Stephenson FA. 1993. Identification of the GABAA
receptor alpha 3 subunit in the IMR-32 neuroblastoma cell line. J Neurochem 61:752755.
Rice, D., & Barone, S. (2000). Critical periods of vulnerability for the developing nervous system:
evidence from humans and animal models. Environmental Health Perspectives, 108(Suppl 3),
511533.
Sapp DW, Yeh HH. 2000. Heterogeneity of GABA(A) receptor- mediated responses in the human
IMR-32 neuroblastoma cell line. J Neurosci Res 60:504510.
Wilson, W., Onyenwe, W., Bradner, J., Nennig, S., & Caudle, W. (2014). Developmental exposure to the
organochlorine insecticide endosulfan alters expression of proteins associated with
neurotransmission in the frontal cortex. Synapse, 485-497.

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