Locomotive Activity as an Indicator of Acute Chlorpyrifos Toxicity in ...

Locomotive Activity as an Indicator of Acute Chlorpyrifos Toxicity in C. Elegans
Jennifer Korb
Presented at the annual international 2010 Society of Toxicology meeting. The sponsoring professor was Melinda J. Pomeroy-Black.
Introduction
Organophosphate esters are the most frequently utilized insecticide throughout the world. These compounds are
involved in more poisonings than any other class of pesticides (Sultatos, 1994). Chlorpyrifos is an organophosphate (OP)
insecticide that was first registered in the United States in 1965. Today, chlorpyrifos is incorporated into over 800 products
that are marketed for residential and agricultural pest control. It is available to the general public in dust or pellet form.
As a member of the OP family of insecticides, chlorpyrifos is neurotoxic. Clinical signs of toxicity due to chlorpyrifos
may be acute or delayed. Symptoms of acute toxicity, the predominant feature of OP compound exposure, include muscle
tremors, twitching, and in severe cases, paralysis and death (Davis and Richardson, 1980; Sultatos, 1994).
Upon normal firing, cholinergic neurons release the neurotransmitter acetylcholine (ACh) into the synaptic cleft.
The enzyme acetylcholinesterase (AChE) hydrolyzes ACh into acetate and choline such that ACh-induced synaptic
transmission ceases. Acetyl-CoA and choline are then removed from the synaptic cleft. The OP compounds inhibit AChE
by phosphorylating it, allowing ACh to remain in the synaptic cleft; thus, there is continuous stimulation of ACh receptors
on neighboring neurons (Ecobichon, 1996). This continuous stimulation of neurons results in early clinical signs, such as
muscle tremors. As neurons become desensitized to the presence of ACh in the synapse, paralysis and death may result.
The nematode Caenorhabditis elegans is the smallest known organism with a central nervous system (Anderson
et al., 2004). Several studies indicate that using C. elegans in toxicity testing are predictive of outcomes seen in higher
eukaryotes (Leung et al. 2008). In particular, the toxicity of several organophosphate compounds has been observed using
the behavior of C. elegans (Cole et al., 2003). Behavioral endpoints in toxicity testing using C. elegans include locomotion
and feeding (Anderson et al., 2004). Locomotion is evaluated by either the distance traveled or the number of body bends
of the worm. While documented in higher eukaryotes, the behavior of C. elegans upon chlorpyrifos exposure is not known.
We hypothesized that there would be a dose-dependent decrease in locomotive activity in C. elegans exposed
to chlorpyrifos. As the dose of chlorpyrifos to which the organism is exposed increases, the number of body bends will
decrease.
Materials and Methods
Bristol wildtype (WT) Caenorhabditis elegans strain N2 were maintained in an incubator at 20oC on nematode growth
medium (NGM) plates seeded with Escherichia coli strain OP50. Acetone used to solubilize chlorpyrifos powder (Sigma-
Aldrich, St. Louis, MO) and allowed to evaporate before the addition of K-medium (0.032 M KCl, 0.051 M NaCl in dH2O).
Each well of a 12-well sterile tissue culture plate contained K-medium with or without chlorpyrifos (0.01 mM, 0.005 mM and
0.001 mM chlorpyrifos). Using a platinum work pick, individual worms were collected from NGM plates and loaded into the
1-ml wells on the tissue culture plate. All plates were placed in an incubator at 20oC for 4 hours in the absence of food.
There were at least 7 replicates for each concentration and the control.
Locomotive activity was determined by counting the number of front body bends of the worm per minute using a
dissecting microscope. When the part of the worm just behind the pharynx reached a maximum bend in the opposite
direction from the last bend, the body bend count was advanced by one (Hart, 2006). The front body bends of each worm
were counted for two 3-minute periods.
Results
C. elegans treated with 0.01 mM chlorpyrifos exhibit significantly less movement per minute than C. elegans treated
with 0.005 mM chlorpyrifos (p = 0.001) or 0.001 mM chlorpyrifos (p = 0.001) (Figure 1).
The average number of body bends of worms exposed to K-medium only was 49.71 body bends per minute (n=8).
The worms were observed to be swimming vigorously throughout the well.
The average number of body bends of worms exposed to 0.001 mM chlorpyrifos was 35.12 body bends per minute
(n=7). At this concentration, worms were swimming near the center of the wells and making full body turns.
The average number of body bend of worms exposed to 0.005 mM chlorpyrifos was 18.25 body bends per minute
(n=8). At this concentration, the worms were found along the sides of the wells. They were moving in an S shape but were
not swimming.
The average number of body bends of worms exposed to 0.01 mM chlorpyrifos was 12.35 body bends per minute
(n=8). At this concentration, the worms were not swimming and were on wall of the well. Movement was drastically slowed;
the worms seemed to be balling up.
Discussion
The results of this study support the hypothesis that locomotive activity decreases in a dose-dependent manner in
C. elegans exposed to chlorpyrifos. Worms exposed to chlorpyrifos exhibited significantly less movement than did worms
exposed to 0.05 mM chlorpyrifos or 0.001 mM chlorpyrifos.
The data suggests that C. elegans are sensitive to chlorpyrifos at concentrations lower than those observed in

other animal models, including mammals and hens. It is more typical that in order to mimic the toxicant-induced behavior
of higher eukaryotes, one must use higher concentrations of the toxicant in C. elegans (Cole et al., 2003). It may be
that C. elegans are particularly susceptible to the effects of chlorpyrifos, perhaps because the compound penetrates
the exoskeleton of the C. elegans more effectively than other toxicants. Further studies altering the concentration of
chlorpyrifos to which C. elegans are exposed may yield an EC50 of the toxicant such that analysis of proteins affected upon
chlorpyrifos exposure can be performed.
Literature Cited
Anderson, GL, RD Cole, and PL Williams (2004). Assessing behavioral toxicity with Caenorhabditis elegans. Environ
Toxicol Chem 23(5): 1235-1240.
Cole, RD, GL Anderson and PL Williams (2003). The nematode Caenorhabditis elegans as a model of organophosphateinduced
mammalian neurotoxicity. Toxicol Appl Pharmacol 194: 248-256.
Davis, D and R Richardson (1980). Organophosphorous compounds. In: Experimental and Clinical Neurotoxicology. Eds:
P. Spencer and H. Schaumburg, Williams and Wilkins, Baltimore, pp. 527-544.
Ecobichon, D (1996). Toxic effect of pesticides. In: Casarett and Doull’s Toxicology: the basic science of poisons. Ed.: C.
Klaassen. McGraw-Hill, New York, pp. 643-689.
Hart, Anne C, ed. Behavior (July 3, 2006), WormBook, ed. The C. elegans Research Community, WormBook, doi/10.1895/
wormbook.1.87.1, http://www.wormbook.org.
Leung, MCK, PL Williams, A Benedetto, C Au, KJ Helmcke, M Aschner and JN Meyer (2008). Caenorhabditis elegans: An
Emerging Model in Biomedical and Environmental Toxicology. Tox Sci 106(1): 5-28.
Sultatos, L (1994). Mammalian toxicology of organophosphorous pesticides. J Toxicol Environ Health 43: 271-289.
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chlorpyrifos concentration (mM)
C. elegans treated with 0.01 mM chlorpyrifos exhibited significantly less movement per minute than C. elegans treated with
0.005 mM chlorpyrifos (p = 0.001) or 0.001 mM chlorpyrifos (p = 0.001).