# Ex vivo electrophysiological evaluation of peripheral nerve functioning following exposition of adult mice to the organophosphorus pesticide chlormephos

Robert Frangež, Evelyne Benoit

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Robert Frangež, Evelyne Benoit. Ex vivo electrophysiological evaluation of peripheral nerve functioning following exposition of adult mice to the organophosphorus pesticide chlormephos. Environmental Toxicology and Pharmacology, 2025, 114 (104645), pp.1-7. 10.1016/j.etap.2025.104645. hal-04922514

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Distributed under a Creative Commons Attribution 4.0 International License
*Ex vivo* electrophysiological evaluation of peripheral nerve functioning
following exposition of adult mice to the organophosphorus
pesticide chlormephos

Robert Frangez<sup>a,1</sup>, Evelyne Benoit<sup>b,*,2</sup>

ARTICLE INFO

Edited by Dr. M.D. Coleman

Keywords:

Chlormephos
Organophosphorus pesticide
Chronical oral administration
Mouse sciatic nerve
Diphasic compound action potential
*Ex vivo* electrophysiology

A B S T R A C T

The organophosphorus pesticide chlormephos was tested for its potential peripheral neurotoxicity by analyzing the diphasic compound action potential (CAP) of sciatic nerves isolated from adult mice chronically exposed to a sub-lethal dose of this pesticide, compared with control age-matched animals being only exposed to the vehicle. No significant modification was detected between chlormephos-exposed and control groups in their nerve responsiveness to stimulus. Furthermore, similar values of CAP kinetic variables were obtained from the two mouse groups. Finally, no abnormality concerning CAP refractory periods and responsiveness to high frequency stimulation (nerve fatigue) was exhibited by chlormephos-treated mice, when compared with control animals. These results complicate the screening for the potential neurotoxicity of organophosphates since they support the contention that the peripheral neuropathy induced by chronical exposition to sub-lethal doses of chlormephos is species dependents does not necessary correlate well with the clinical signs observed at the central nervous system level.

**1. Introduction**

Exposure to organophosphorus compounds occurs through inten-
tional or unintentional contamination and/or residua of food and water
sources. The primary mechanism of neurotoxic action of these com-
pounds is to covalently bind and inhibit serine hydrolases, mainly
acetylcholinesterase (AChE); this enzyme normally hydrolyzes the
neurotransmitter acetylcholine (ACh) by cleaving it into choline and
acetic acid in central and peripheral nervous systems (Aldridge, 1972).

Once AChE is inhibited, excessive ACh over-stimulates muscarinic and nicotinic receptors of the autonomic, central nervous systems and the neuromuscular junction of skeletal muscle fibers, leading to the acute signs and symptoms of organophosphate poisoning (Barthold and Schier, 2005; Vale and Lotti, 2015; Weinbroum, 2004). Certain organ- ophosphorus compounds directly inhibit the phospholipase activity of the neuropathy target esterase (NTE), showing more variability in NTE inhibition in mice than in rats and other sensitive species (Veronesi et al., 1991) although this esterase was detected in sciatic nerve axons,

**Abbreviations:** ACh, Acetylcholine; AChE, Acetylcholinesterase; Area, Area of the first phase of CAP; ARRIVE, Animal Research: Reporting of *In Vivo* Experiments; CAP, Compound action potential; DBCV, Di-n-butyl-2,2-dichlorovinyl phosphate; Decay Slope 90–10 %, Decay time between 90 % and 10 % Peak-1; Decay Time 90–10 %, Decay time between 90 % and 10 % Peak-1; Half-width, Width at 50 % Peak-1; HEPES, 2-[4–2-hydroxyethylpiperazin-1-yl]ethanesulfonic acid; Is, Stimulus intensity; IS₅₀, Stimulus intensity producing a CAP with 50 % maximal peak amplitude; LD₅₀, Lethal dose for 50 % of tested animals; NTE, Neuropathy target esterase; Peak Δtime, Interval between time-to-Peak-1 and time-to-Peak-2; Peak-1, Peak amplitude of the first phase of CAP; Peak-1max, Maximal peak amplitude of the first phase of CAP; Peak-1ss, Peak-1 values at infinite times; Peak-2, Peak amplitude of the second phase of CAP; PPDB, Pesticide Properties Data Base; R², Coefficient of determination; Rise Slope 10–90 %, Rise slope between 10 % and 90 % Peak-1; Rise Time 10–90 %, Rise time between 10 % and 90 % Peak-1; S, Curve slope; S.E.M., Standard error of the mean; T, Time; Tau τ, Time constant; Ti, Time-interval between twin pulses; Ti₅₀, Time-interval producing a CAP with 50 % maximal peak amplitude; TOCP, Tri-2-cresyl phosphate; WHO, World Health Organization.

* Corresponding author.

E-mail addresses: Robert.Frangez@vf.uni-lj.si (R. Frangež), evelyne.benoit@cea.fr (E. Benoit).

¹ ORCID: 0000-0002-0636-9277.

² ORCID: 0000-0001-5501-0888.

<footnote><sup>a</sup> Institute of Preclinical Sciences, Veterinary Faculty, University of Ljubljana, Gerbičeva 60, Ljubljana 1000, Slovenia</footnote>

<footnote><sup>b</sup> Université Paris-Saclay, CEA, Institut des sciences du vivant Frédéric Joliot, Département Médicaments et Technologies pour la Santé (DMTS), Service d'Ingénierie Moléculaire pour la Santé (SIMoS), EMR CNRS 9004, Gif-sur-Yvette 91191, France</footnote>
in non-myelinated Schwann cells and, in less extend, in myelinating
Schwann cells of mice (McFerrin et al., 2017).

Chlormephos [(S-(chloromethyl) O,O-diethyl phosphorodithioate; Fig. 1) is a soil organophosphorus pesticide used to prevent infestations of wireworms and white grubs following sod in crop areas (Roberts and Hutson, 1999). The degradation products that may be formed from this pesticide, under a variety of conditions, are also of significance with respect to environmental safety (Hudson et al., 1991). The pesticide, as O,O-diethyl phosphorothioate and diethyl phosphate degrading compounds, is almost completely eliminated in the urine within 24 h after oral administration to rats (Terence and Hutson, 1999). According to the World Health Organization (WHO) classification, chlormephos is an extremely hazardous chemical since the pesticide lethal dose for 50 % of tested animals (LD₅₀) is approximately 7 mg/kg when acutely and orally administered to rats (Lewis et al., 2016). Hence, for more than ten years now, the use of chlormephos and/or pesticides containing chlormephos as an active ingredient is limited, if not prohibited, to the composition of preparations benefiting from a marketing authorization, in particular in the United States and in the European Union countries listed in the Pesticide Properties Data Base (PPDB<fnref n="3" />). However, there remains a dearth of reports on the neurotoxicity of chronic exposure to sub-lethal doses of this organophosphorus pesticide through food and/or water sources. In addition, although the cholinergic molecular targets of organophosphorus compounds are well known, there is still debate as to whether other non-cholinergic targets may, or may not be relevant in this agent's neurotoxicity.

For these reasons, the aim of the present work was to evaluate the
organophosphorus pesticide chlormephos for its potential peripheral
neurotoxicity. Therefore, the diphasic compound action potential (CAP)
of sciatic nerves isolated from adult mice chronically exposed to a sub-
lethal dose of this pesticide (i.e. 3.5 µg per mL of drinking water for
30 days), was analyzed and compared with control age-matched animals
being only exposed to the vehicle. Some important variables concerned
with the physiology of mouse peripheral nerve fibers were evaluated,
which may contribute to better understanding the long-term develop-
ment of peripheral nerve disfunction in mammals exposed chronically to
sub-lethal doses of organophosphorus compounds. The choice of a
3.5 µg/mL dose of chlormephos in drinking water for 30 days was based
on a previous study by Ceh et al. (2012). In that study, mice exposed
neonatally to a high dose of chlormephos (3.5 µg/mL of drinking water)
spent less time in both the open and closed arms of an elevated plus
maze, compared to those exposed to a lower dose (0.35 µg/mL of
drinking water) or to the animal control group. To our knowledge, no
information is available on the lethal dose of chlormephos when
chronically and orally administered to mice, despite an LD₅₀ of 7 mg/kg
being reported for acute oral administration in rats (Lewis et al., 2016).
Therefore, the dose of 3.5 µg/mL of drinking water for 30 days was
assumed in this study to be sub-lethal, as no animal deaths occurred at
this dose (Ceh et al., 2012). The choice of sciatic nerve preparations was
primarily based on several previous studies. Commonly used pesticides,
such as chlorpyrifos, dichlorvos and cypermethrin, have been associated

<figure><img src="image_2.png" /><figcaption>Fig. 1. Chemical structure of chlormephos (C<sub>5</sub>H<sub>12</sub>ClO<sub>2</sub>PS<sub>2</sub>).</figcaption></figure>

with neuropsychiatric disorders and impaired locomotor activity in
humans (Ommati et al., 2024). Certain organophosphates have been
shown to cause damage to the afferent fibers of peripheral nerves,
leading to symptoms such as weakness, paralysis and paresthesia in the
legs, which can persist for weeks to years in humans (Roberts and Rei-
gart, 2013). In dogs, overt toxic signs like muscle fasciculations and
high-stepping gait with incoordination of the hind limbs were observed
after oral administration of chlormephos (RHONE-POULENC INC;
Chlormephos: Minimal Lethal Dose Study in Beagle Dogs; 09/01/75;
EPA Doc No. 88-920008851; Fiche No. OTS0570896 - https://pubchem.
ncbi.nlm.nih.gov/source/hsdb/6456#section=TSCA-Test-Submissions
-%28Complete%29&fullscreen=true). Additionally, symptoms such as
motor weakness have been consistently reported in exposed individuals
(Holmes and Gaon, 1957; Metcalf and Holmes, 1969).

**2. Materials and methods**

Experiments are reported in line with the ARRIVE (Animal Research:
Reporting of *In Vivo* Experiments) guidelines developed in consultation
with the scientific community as part of an NC3Rs initiative to improve
standards of reporting the results of animal experiments, maximizing
information published and minimizing unnecessary studies (Kilkenny
et al., 2010; Schulz et al., 2010).

**2.1. Chemicals and drugs**

All chemicals and drugs were purchased from Sigma Aldrich (St.
Louis, MO, USA). Due to its low solubility in water, chlormephos (mo-
lecular mass of 234.70) was dissolved in ethanol as a 3.5 mg/mL
pesticide stock solution that was stored at 4°C.

**2.2. *Experimental animals***

Experiments were carried out on 16-week-old male Balb/C mice (*Mus musculus*, weighting 24–26 g) obtained from the animal breeding facility of the Veterinary Faculty (University of Ljubljana, Slovenia). The animals were treated in strict adherence with the European Community guidelines for laboratory animal handling (EEC86/609 Council Directive). In particular, the mice were housed in a room with controlled temperature and a 12-h light/12-h darkness cycle, in standard laboratory cages with environmental enrichment, and were allowed to free access to water and food. All animal experimental procedures followed ethical standards and were approved by the administration of the Republic of Slovenia for food safety as well as for veterinary and plant protection (permit #3440-176/2006).

Six mice were chronically exposed for 30 days to a sub-lethal dose of
chlormephos in drinking water, i.e. at a final concentration of 3.5 µg/
mL. The control group of animals thus included six age-matched mice
exposed for 30 days to 0.1 % ethanol (i.e. the pesticide vehicle) in
drinking water. All mice were daily observed for clinical signs (mainly
diarrhea and behavioral impairments such as hyperactivity, muscle
twitching, flaccid paralysis, loss of equilibrium...) by an observer who
was blinded to the treatment.

**2.3. Electrophysiological recordings**

Electrophysiological recordings were performed ex vivo on the
mouse isolated right and left sciatic nerves, the day following the end of
animal exposition to chlormephos and/or ethanol, at room temperature
(20-21 °C) and using a conventional electrophysiological technique, as
previously described (Benoit et al., 2000; Boullot et al., 2021). Briefly,
the sciatic nerves were dissected from mice beforehand sacrificed by
cervical dislocation, followed by immediate exsanguination, and placed
into a glass silicon-lined Petri dish filled with a standard physiological
(Krebs-Ringer) solution, bubbled with pure oxygen and containing
154 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 5 mM

<footnote n="3">³ https://sitem.herts.ac.uk/aeru/ppdb/en/Reports/142.htm</footnote>
2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES), and 11 mM D-glucose (pH 7.4), to recover and equilibrate for at least 20–30 min before experiments.

A given nerve was then transferred to the recording chamber which consisted of a custom-designed temperature-controlled Plexiglas chamber with a ladder comprising seven platinum hook-shaped stimulating and recording electrodes. The nerve was gently placed in such a way so as to touch all electrodes without being stretched. It was kept moist with the Krebs-Ringer solution, and the chamber was covered with a lid to prevent the nerve from drying out. The nerve was neither bathed nor superfused during the recording period (which lasted no more than 10 min) to avoid shunting effects of the medium. Two (stimulating) electrodes were connected to a stimulator (ITIS d.o.o., Ljubljana, Republic of Slovenia) delivering square-wave current pulses. Two other (recording) electrodes were connected to the input of a dual channel differential amplifier (ITIS d.o.o.) to amplify the CAP, and a data acquisition system (Digidata® 1440A; Molecular Devices, Sunnyvale, CA, USA) was used to acquire data which were stored on a computer and then analyzed by means of the AxoScope 11.3 software (Molecular Devices). Finally, one electrode, located between the stimulating and recording electrode pairs, was connected to the ground to reduce the stimulation artifact and the signal noise.

The stimulation protocols consisted in delivering, through the stimulating electrodes, (1) series of square-wave pulses of increasing intensity (0.10, 0.25, 0.50, 1.00, 1.50, 2.00, 2.50, 3.00, 4.00, 5.00, 6.00, 7.00, 8.00, 9.00 and 10.00 V) and duration (10, 30, 50 and 100 µs), every 3 s, to establish the CAP peak amplitude - stimulus intensity relationships, (2) supramaximal square-wave twin pulses (10-V intensity) of 10-µs duration, every 3 s, with increasing time-intervals (from 0.5 to 20 ms) between the first and second pulses (i.e. 0.50, 1.00, 1.11, 1.25, 1.42, 1.66, 2.00, 2.50, 2.85, 3.30, 4.00, 5.00, 6.66, 8.00, 10.00, 13.33 and 20.00 ms), to access to CAP absolute and relative refractory periods, and (3) supramaximal square-wave pulses (10-V intensity) of 10-µs duration, at a high stimulation frequency (100-Hz) for 300 s, to evaluate the nerve fatigue.

## 2.4. Data analyzes and statistics

The kinetics of a given recorded diphasic CAP were characterized by nine measured variables (Fig. 2): (i) the peak amplitude of the first (Peak-1) and second (Peak-2) phases (in mV), (ii) the interval between time-to-Peak-1 and time-to-Peak-2 (Peak Δtime, in ms), correlated with CAP conduction velocity, (iii) the area of the first phase (Area, in mV·ms), (iv) the width at 50 % Peak-1 (Half-width, in ms), (v) the rise and decay times between 10 % and 90 % Peak-1 (Rise Time 10–90 % and Decay Time 90–10 %, respectively, in ms), and (vi) the rise and decay slopes between 10 % and 90 % Peak-1 (Rise Slope 10–90 % and Decay Slope 90–10 %, respectively, in mV/ms).

The CAP peak amplitude - stimulus intensity relationship was established by plotting Peak-1, expressed relatively to its maximal amplitude (Peak-1max) recorded in response to large stimulus intensities, as a function of the stimulus intensity (Is). The theoretical curve corresponds to non-linear regression analysis of data points

(coefficient of determination R²), according to the following equation (GraphPad Prism 5 software): Peak-1/Peak-1max = Is/(Is₅₀ + Is), where Is₅₀ is the stimulus intensity producing a CAP with 50 % Peak-1max (Fig. 3A).

The CAP absolute and relative refractory periods were accessed using supramaximal square-wave twin pulses and by plotting Peak-1, recorded in response to the second pulse and expressed relatively to Peak-1max recorded in response to the first pulse, as a function of the time-interval between the first and second pulses (Ti). The theoretical curve corresponds to non-linear regression analysis of data points (R²), according to the following equation (GraphPad Prism 5 software): Peak-1/Peak-1max = 1/[1 + exp ((Ti₅₀ – Ti)/S)], where Ti₅₀ is the time-interval producing a CAP with 50 % Peak-1max and S is the slope of the curve (Fig. 3B).

The curve of nerve fatigue was established by plotting Peak-1, recorded in response to a 100-Hz stimulation frequency and expressed relatively to its maximal amplitude (Peak-1max) recorded at time zero, as a function of time (T). The theoretical curve corresponds to non-linear regression analysis of data points (R²), according to the following equation (GraphPad Prism 5 software): Peak-1/Peak-1max = (1 – Peak-1ss) exp (-T/τ) + Peak-1ss, where Peak-1ss is the Peak-1 value at infinite times, relative to Peak-1max, and τ is the time constant (Fig. 3C).

Data are presented as the mean ± standard error of the mean (S.E. M.) of 12 sciatic nerves isolated from 6 mice. Statistical analysis of data was executed with the Sigma Plot software for Windows (version 12.5; Systa Software Inc., USA). Comparison of data between the sciatic nerves isolated from chlormephos- exposed and control groups of mice, and submitted to similar experimental conditions, was performed using the Student's t-test (two-tailed). Data were considered significant at *P* < 0.05.

# 3. Results

## 3.1. Effects of chlormephos on CAP kinetics

The CAP of sciatic nerves isolated from chlormephos-exposed and control groups of mice was recorded in response to stimuli of 10-V intensity and increasing duration (10, 30, 50 and 100 µs). No marked change in kinetics was detected on CAP trace recordings, whatever the stimulus duration was between 10 and 100 µs, as illustrated by representative CAP traces in Fig. 4A. In addition, the nine measured variables of chlormephos-exposed group CAP, recorded in response to stimuli of 10-V intensity and 10-µs duration, were not significantly different (*P* ≥ 0.332) from those of control group CAP recorded under similar conditions (Fig. 4B).

These results indicate that the nine variables representative of CAP kinetics were not relevant to point out a potential effect of chlormephos on peripheral nerves of mice chronically exposed for 30 days to a sub-lethal dose of this organophosphorus pesticide (i.e. 3.5 µg/mL in drinking water).

## 3.2. Effects of chlormephos on CAP peak amplitude - stimulus intensity relationships

To appraise the effects of chlormephos on the membrane excitability level, the CAP of sciatic nerves isolated from chlormephos-exposed and control groups of mice was recorded in response to stimuli of increasing intensity (from 0.1 to 10 V) and duration (from 10 to 100 µs) to establish CAP peak amplitude - stimulus intensity relationships (Fig. 5).

For both groups, the peak amplitude of the first phase of CAP was dependent on the stimulus intensity and behaved similarly in response to stimuli of increased duration. Hence, in response to stimuli of a given duration and increasing intensities, the peak amplitude increased, reached a plateau value, and then remained constant despite further intensity increases. The mean plateau values were found to be 12–14 mV, whatever the stimulus duration was between 10 and 100 µs,

<figure><img src="image_3.png" /><figcaption>Fig. 2. Variables characterizing the CAP kinetics.</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>Fig. 3. Theoretical curves used to analyze data. (A) CAP peak amplitude - stimulus intensity relationship: Peak-1/Peak-1max = Is/(Is50 + Is). (B) CAP absolute and relative refractory periods: Peak-1/Peak-1max = 1/[1 + exp ((Ti50 – Ti)/S)]. (C) Nerve fatigue: Peak-1/Peak-1max = (1 – Peak-1ss) exp (-T/τ) + Peak-1ss. See more details in the text.</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>Fig. 4. Effects of chlormephos on CAP kinetics. (A) Superimposed CAP traces recorded from sciatic nerves of chlormephos-exposed (in red) and control (in black) groups of mice, stimulated with pulse amplitude of 10 V and indicated duration (10, 30, 50 or 100 µs). (B) Histogram of variables characterising the CAP kinetics of sciatic nerves isolated from chlormephos-exposed (in red) and control (in black) groups of animals, stimulated with pulse amplitude of 10 V and duration of 10 µs. Each value represents the mean ± S.E.M. of data obtained from 12 sciatic nerves (i.e. 6 mice).</figcaption></figure>

and were not significantly different (P ≥ 0.129) between sciatic nerves isolated from chlormephos-exposed and control groups of animals (Table 1). It is worth noting that the fact that the peak amplitude attained a plateau value indicates that all the nerve fibers which could be electrically stimulated, under our experimental conditions, were recruited by stimuli of at least 6-V intensity and 10-µs duration. Therefore, for the following experiments, the CAP was recorded in response to stimuli of 10-V intensity and 10-µs duration.

The mean values of the stimulus intensity producing a CAP with 50 % maximal peak amplitude (Is50) were found to be 0.08–0.12 V for a stimulus duration between 30 and 100 µs, values that were significantly lower (P < 0.001) than those obtained for a stimulus duration of 10 µs, i. e. 0.31–0.36 (Fig. 5 and Table 1). However, here again, the Is50 values were not significantly different (P ≥ 0.086) between sciatic nerves isolated from chlormephos-exposed and control groups of mice, whatever the stimulus duration was between 10 and 100 µs (Table 1).

These results strongly suggest that the CAP peak amplitude - stimulus intensity relationships were not a distinguished criterion of a potential

effect of chlormephos on peripheral nerves of mice chronically exposed for 30 days to a sub-lethal dose of this organophosphorus pesticide (i.e. 3.5 µg/mL in drinking water).

### 3.3. Effects of chlormephos on CAP absolute and relative refractory periods

The effects of chlormephos on the membrane excitability level was also appraised by studying the CAP absolute and relative refractory periods of sciatic nerves isolated from chlormephos-exposed and control groups of mice, and determining the corresponding variables. It is worth noting that the refractory periods represent another important determinant of axonal function. As a measure of this property, two identical stimuli of 10 V and 10 µs were applied at various time-intervals, and the peak amplitude of the first phase of the second CAP, relative to that of the first CAP, was plotted as a function of the time-interval between the first and second stimuli (Fig. 6).

The comparison between chlormephos-exposed and control groups
<figure><img src="image_6.png" /><figcaption>**Fig. 5.** Effects of chlormephos on CAP peak amplitude - stimulus intensity relationships of sciatic nerves isolated from chlormephos-exposed (in red) and control (in black) groups of mice. For a given indicated stimulus duration (10, 30, 50 or 100 µs), the peak amplitude of the first phase of CAP was measured in response to increasing stimulus intensity (from 0.1 to 10 V) and expressed, as a function of the stimulus intensity, relatively to its maximal amplitude recorded in response to large stimulus intensities (Peak-1max). Each value represents the mean ± S.E.M. of data obtained from 12 sciatic nerves (i.e. 6 mice). The curves correspond to data fits (R² ≥ 0.801), as detailed in Materials and methods, with the Peak-1max and Is₅₀ mean values indicated in Table 1.</figcaption></figure>

<caption>**Table 1**</caption>

Variables of CAP peak amplitude - stimulus intensity relationships determined from chlormephos-exposed and control groups of mice. Mean ± S.E.M. of data obtained from 12 sciatic nerves (i.e. 6 mice).

<table><thead><tr><th rowspan="2"></th><th rowspan="2"></th><th colspan="4">Stimulus duration</th></tr><tr><th>10 µs</th><th>30 µs</th><th>50 µs</th><th>100 µs</th></tr></thead><tbody><tr><td rowspan="3">Control group</td><td>Peak-1max (mV)<sup>a</sup></td><td>12.45 ± 0.66</td><td>12.68 ± 0.71</td><td>12.64 ± 0.73</td><td>12.76 ± 0.76</td></tr><tr><td>Is<sub>50</sub> (V)<sup>b</sup></td><td>0.31 ± 0.06<sup>*</sup></td><td>0.12 ± 0.02</td><td>0.08 ± 0.01</td><td>0.08 ± 0.02</td></tr><tr><td>Chlormephos-exposed group</td><td>Peak-1max (mV)<sup>a</sup></td><td>12.53 ± 1.23</td><td>13.16 ± 1.17</td><td>13.23 ± 1.13</td><td>14.43 ± 0.59</td></tr><tr><td></td><td>Is<sub>50</sub> (V)<sup>b</sup></td><td>0.36 ± 0.01<sup>*</sup></td><td>0.11 ± 0.01</td><td>0.12 ± 0.02</td><td>0.12 ± 0.01</td></tr></tbody></table>

of animals revealed no marked change in CAP trace recordings,

whatever the time-interval was between 1 and 10 ms (Fig. 6A), and in the curves and corresponding variables reflecting the CAP refractory periods (Fig. 6B). Hence, the curve slopes and the time-intervals at which the first phase of second CAP recovered to 50 % of that of initial CAP were not significantly different (P ≥ 0.678) between chlormephos-exposed (0.50 ± 0.10 ms⁻¹ and 2.93 ± 0.34 ms, respectively) and control (0.55 ± 0.04 ms⁻¹ and 2.94 ± 0.18 ms, respectively) groups of mice.

In essence, the exposure to chlormephos at the given sub-lethal dose did not significantly alter the CAP absolute and relative refractory periods of peripheral nerves in the mice, suggesting that this particular aspect of nerve function is not a key factor in the pesticide's impact under the experimental conditions used.

### 3.4. Effects of chlormephos on the nerve fatigue

Finally, the effects of chlormephos were investigated on the nerve fatigue by studying the time evolution of the peak amplitude of the first phase of CAP of sciatic nerves, isolated from chlormephos-exposed and

<figure><img src="image_7.png" /><figcaption>**Fig. 6.** Effects of chlormephos on CAP absolute and relative refractory periods of sciatic nerves isolated from chlormephos-exposed (in red) and control (in black) groups of mice. (A) Superimposed traces of CAP recorded in response to supramaximal square-wave twin pulses of 10-V intensity and 10-µs duration, applied at the indicated time-intervals (1, 2.5 or 10 ms). (B) Peak amplitude of the first phase of the second CAP, relative to that of the first CAP, recorded in response to supramaximal square-wave twin pulses of 10-V intensity and 10-µs duration, as a function of the time-interval (from 0.5 to 20 ms) between the first and second pulses. Each value represents the mean ± S.E.M. of data obtained from 12 sciatic nerves (i.e. 6 mice). The curves correspond to data fits (R² ≥ 0.917), as detailed in Materials and methods, with the Ti₅₀ (dashed lines) and S (curve slope) mean values indicated in the text.</figcaption></figure>

<footnote>a Maximal peak amplitude of the first phase of CAP.</footnote>

<footnote>b Stimulus intensity producing a CAP with 50 % maximal peak amplitude.</footnote>

<footnote>* P ≤ 0.005 (versus 100 µs).</footnote>
control groups of mice and submitted to a 100-Hz stimulation frequency (Fig. 7).

The comparison between chlormephos-exposed and control groups of animals revealed no marked change in CAP trace recordings in response to a 100-Hz stimulation frequency for 300 s (Fig. 7A), and in the curves and corresponding variables reflecting the nerve fatigue (Fig. 6B). Hence, the value of the first phase of CAP at infinite times expressed relatively to its maximal amplitude recorded at time zero (Peak-1ss) and the time constant (τ) were not significantly different (P ≥ 0.134) between chlormephos-exposed (0.79 ± 0.03 and 9.42 ± 1.25 s, respectively) and control (0.78 ± 0.05 and 12.29 ± 2.36 s, respectively) groups of mice.

These results indicate that the nerve fatigue was also not a distinguished criterion of a potential effect of chlormephos on peripheral nerves of mice chronically exposed for 30 days to a sub-lethal dose of this organophosphorus pesticide (i.e. 3.5 µg/mL in drinking water).

## 4. Discussion

Organophosphorus compounds are well-known to cause four main neurotoxic disorders in humans: the cholinergic syndrome, the intermediate syndrome, organophosphate-induced delayed polyneuropathy and chronic organophosphate-induced neuropsychiatric disorder. The delayed or chronic toxicity, including the neuropathic effects, merits to be further studied since this toxicity contributes substantially to the long-term morbidity after severe acute or chronic exposition to organophosphorus compounds. Because only a few electrophysiological studies have detailed the organophosphate-induced neuropathy at the peripheral nervous system level, we evaluated the potential peripheral action of the organophosphorus pesticide chlormephos, knowing that peripheral nerve conduction measurement is an important tool for the detection of neurotoxicity and different kinds of nerve disorders.

Chickens are well-known to be the sentinel species for the study of organophosphate-induced neuropathies because these domesticated birds have a high accumulation rate of organophosphate insecticides, contributing thus to their neurotoxicity. However, exaggerated effects in chickens overestimate often the neurotoxic risk for humans (Abou-Donia, 2003). On the other hand, rodents have the great advantage to belong to the family of mammals and, as such, are frequently used in toxicologic or medical research. In addition, they have been described to acquire the characteristic neurotoxic signs and typical biochemical changes of organophosphate-induced neuropathies without ataxia. Hence, some organophosphorus compounds have been reported to inhibit NTE and AChE in both the rat and chicken brains, but with different sensitivities (Ehrich et al., 1995).

Electrophysiological evaluation of mammal peripheral nerves has primarily and largely focused on the measurement of conduction velocity which was observed to be unchanged following administration of

<figure><img src="image_8.png" /><figcaption>**Fig. 7.** Effects of chlormephos on the fatigue of sciatic nerves isolated from chlormephos-exposed (in red) and control (in black) groups of mice, and submitted to a 100-Hz stimulation frequency for 300 s. (A) Representative traces of CAP. (B) Peak amplitude of the first phase of CAP, expressed relatively to its maximal amplitude recorded at time zero, as a function of the stimulation time. Each value represents the mean ± S.E.M. (in grey) of data obtained from 12 sciatic nerves (i.e. 6 mice). The curves correspond to data fits (R² ≥ 0.844), as detailed in Materials and methods, with the Peak-1ss and τ mean values indicated in the text.</figcaption></figure>

organophosphorus compounds to cats, rats and hens (for references, see Robertson et al., 1987). These results are corroborated by the absence of chlormephos effect on the interval between time-to-Peak-1 and time-to-Peak-2 (Peak Δtime), correlated with CAP conduction velocity (see Fig. 4). Occasionally, other functional measurements, such as the relative refractory period and sensory afferent activity, were previously performed but, to the best of our knowledge, the effects of organophosphorus compounds on a large variety of electrophysiological nerve parameters were not systematically analyzed in mammals, except in both the hen tibial and sciatic nerves (Robertson et al., 1987) and, now, in the mouse sciatic nerve (see Figs. 4–7).

Hence, the results obtained in the present study reveal no significant effect of chlormephos on peripheral nerves of mice chronically exposed for 30 days to a sub-lethal dose of this organophosphorus pesticide (i.e. 3.5 µg/mL in drinking water), whatever the studied criterion of CAP kinetics was: area, time-to-peak, half-width, rise and decay slopes and times (see Fig. 4). In contrast, significant peripheral nerve abnormalities, i.e. increased duration of tibial nerve action potential and decreased onset conduction velocity of sciatic nerve action potential, were detected 21 days after treatment of hens with a single oral dose of 30–750 mg/kg of tri-2-cresyl phosphate (TOCP, Robertson et al., 1987). In addition, we observed no change in the CAP peak amplitude - stimulus intensity relationships (see Fig. 5 and Table 1), whereas elevated threshold excitability of both tibial and sciatic nerves was reported 21 days after treatment of hens with a single oral dose of TOCP or 4 mg/kg of di-n-butyl-2,2-dichlorovinyl phosphate (DBCV, Robertson et al., 1987). Finally, no alteration occurred in CAP absolute and relative refractory periods (see Fig. 6), and thus in the responsiveness to high-frequency stimulation (nerve fatigue, see Fig. 7), in contrast to the pronounced effects produced by TOCP and DBCV on the relative refractory period of both tibial and sciatic nerve action potentials, indicating impairment of the nerve ability to carry high-frequency stimulations (Robertson et al., 1987).

One explanation that can be proposed to take into account the above result discrepancies is that the cumulative dose of chlormephos after 30 days may not be enough to cause organophosphate-induced neuropathies since, in peripheral nerves, a NTE inhibition threshold of at least 70 % was reported to be necessary to achieve such neuropathies after repeated expositions to organophosphorus compounds (Lotti and Johnson, 1980). Under our experimental conditions, each mouse of the chlormephos-exposed group was in contact with 3.5 µg/mL of this organophosphorus pesticide in drinking water, for 30 days. Assuming an average water consumption of 6 mL per day, each animal was therefore exposed to a cumulative dose of 21 µg of chlormephos per day and thus of 630 µg after 30 days, i.e. 0.84 and 25.2 mg/kg, respectively, for a mouse weighing 25 g (see Materials and methods). It is worth noting that the organophosphorus pesticide LD₅₀ was reported to be approximately 7 mg/kg when acutely and orally administered to rats (Lewis et al., 2016). Therefore, it is unlikely that a NTE inhibition under the threshold for neuropathy development may explain the absence of chlormephos effects described in the present work. In addition, the alterations of peripheral nerve functioning produced by some organophosphorus compounds (such as TOCP) were shown to be not necessarily correlated with NTE inhibition and even with manifestation of organophosphate-induced neuropathies in hens (Robertson et al., 1987). These observations question the validity of a NTE inhibition threshold necessary for the production of organophosphate-induced neuropathies and strongly suggest that NTE is not the sole enzyme involved in the clinical these neuropathies. Indeed, pesticides are well-known to be far better inhibitors of AChE than NTE (Lotti and Moretto, 2005) and thus, may eventually lead to peripheral neuropathy as a result of cholinergic toxicity.

In conclusion, and to the best of our knowledge, this is the first report of chlormephos effects on peripheral motor, but also sensory, nerve conduction of mammals exposed chronically to a low dose of this organophosphorus pesticide. Due to the fact that toxicity data are very
limited for chlormephos and although further experiments are needed to definitively conclude, it is likely that the present study contributes to better understanding the long-term development of toxicity and health concern of this organophosphorus pesticide.

## Funding information

This work was supported by the Slovenian Research and Innovation Agency Programs (P4-0053) and the Bilateral Slovenian/French Proteus Program (BI-FR-21-22-PROTEUS-002/46143VH).

## CRediT authorship contribution statement

**Evelyne Benoit:** Writing – review & editing, Writing – original draft, Validation, Resources, Formal analysis. **Robert Frangez:** Writing – review & editing, Writing – original draft, Investigation, Funding acquisition, Conceptualization.

## Declaration of Competing Interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Robert Frangez reports financial support was provided by Slovenian Research and Innovation Agency programs (P4-0053). Evelyne Benoit reports travel was provided by Protéus. Robert Frangez reports travel was provided by Protéus. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Acknowledgments

The authors would like to thank Boštjan Drolc and Katarina Babnik for excellent technical support.

## Data availability

All data are included in the manuscript

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