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Toxicology Letters 213 (2012) 142– 150
Contents lists available at SciVerse ScienceDirect
Toxicology Letters
jou rn al h om epa ge: www.elsev ier .com/ locate / tox le t
ollow up studies on the respiratory pattern and total cholinesterase activities inichlorvos-poisoned rats
ania Duartea,∗, Chantal Martinb, Frédéric J. Baudc,d, Olivier Laprévotea, Pascal Houzéa,e
Laboratoire de Chimie-Toxicologie Analytique et Cellulaire (EA 4463), Université Paris Descartes, Sorbonne Paris Cité, Faculté de Pharmacie, 75006 Paris, FranceAnimalerie Centrale, Université Paris Descartes, Sorbonne Paris Cité, Faculté de Pharmacie, 75006 Paris, FranceINSERM U705, UMR 7157, Hôpital Fernand Widal, 75010 Paris, FranceMedical and Toxicological Critical Care Department, Assistance Publique- Hôpitaux de Paris, Groupe hospitalier Lariboisière-Saint Louis, University Paris Diderot, 75010 Paris, FranceBiochemistry Laboratory, Assistance Publique- Hôpitaux de Paris, Groupe hospitalier Lariboisière-Saint Louis, University Paris Diderot, 75010 Paris, France
i g h l i g h t s
Study was performed in Sprague-Dawley rats.Dichlorvos effect on ventilation and cholinesterase activities from 5 min to 72 h.Respiratory effects returned to control values within 90 min.Cholinesterase activities were significantly decreased until 24 h.No correlation between respiratory effects and residual cholinesterase activities.
r t i c l e i n f o
rticle history:eceived 6 September 2011eceived in revised form 11 June 2012ccepted 12 June 2012vailable online 19 June 2012
eywords:ichlorvosatsespiratory effects
a b s t r a c t
A human prospective study confirmed that the severity and time-course of organophosphate poisoningsdepend on the compound. Our purpose was to assess the ventilation at rest and cholinesterase activitiesfrom 5 min to 72 h in rats poisoned with dichlorvos at 40% of the MLD (5.12 mg/kg). Ventilation at restwas recorded by whole body plethysmography and core temperature by infrared telemetry (DSI system).Results are expressed as mean ± SEM. Statistical analyses used two-way ANOVA. Dichlorvos inducedthe onset of respiratory effects within 5 min and hypothermia which peaked at 15 min, both reversedwithin 90 min post-injection. Dichlorvos significantly decreased respiratory frequency, resulting froman increase in expiratory time and associated with increased tidal volume. Tissues and whole bloodcholinesterase activities were significantly decreased until the end of experiment. Our study showed
lethysmographyotal cholinesterase
that an inhibition of cholinesterase was correlated with an effect on respiratory functions at 15 min and60 min. However, 24 h post-poisoning, the increase in cholinesterase activity was not completed whileventilatory parameters were within the normal range.
Respiratory effects were both qualitatively and quantitatively similar to those induced by diethyl-paraoxon. However the effects strongly differed between diethylparaoxon lasting hours while dichlorvoslasted tens of minutes.
. Introduction
The early and intermediate phases of toxicity of OPs are relatedo the inhibition of cholinesterase activity, resulting in accumu-
ation of acetylcholine within the synapses throughout the bodyhich induces an overstimulation of the autonomic nervous sys-em (Takahashi et al., 1991; Tsai et al., 2007). Respiratory failure
∗ Corresponding author at: Chimie-Toxicologie Analytique et Cellulaire (EA 4463),niversité Paris Descartes, Sorbonne Paris Cité, Faculté de Pharmacie, 75 006 Paris,rance. Tel.: +33 1 42 49 92 99; fax: +33 1 42 49 92 47.
E-mail address: [email protected] (T. Duarte).
378-4274/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved.ttp://dx.doi.org/10.1016/j.toxlet.2012.06.010
© 2012 Elsevier Ireland Ltd. All rights reserved.
is considered as the primary cause of death (Durham and Hayes,1962; Lerman and Gutman, 1988; Yamashita et al., 1997). However,its mechanism remains unclear resulting from peripheral effects,including bronchorrhea, bronchoconstriction, and muscle weak-ness (Fryer et al., 2004; Gaspari and Paydarfar, 2009; Segura et al.,1999), and central apnea (Chang et al., 1990; Gaspari and Paydarfar,2007).
According to the World Health Organization, OP toxicity is clas-sified in four groups, (Ia, Ib, III, and IV) regarding the value of orally
LD50 of each compound in rats (WHO, 2001). A recent prospectivestudy on human poisonings resulting from chlorpyrifos, fen-thion, and dimethoate ingestion showed that the clinical findingsand severity of poisoning were significantly different from one
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T. Duarte et al. / Toxicolog
ompound to the other (Eddleston et al., 2005a) in terms of intuba-ion, odds of death, the mode of death and response to treatment.espite these data, human treatment of OP poisoning is similar forll pesticides (Tsai et al., 2007). In previous studies, we reportedentilatory effects in rats showing that a toxic but not lethal dosef diethylparaoxon, induced a decrease in respiratory frequencyssociated with an increase in the expiratory time and tidal vol-me (Houze et al., 2008; Villa et al., 2007). To test the hypothesishat the toxicity of OP is dependent on the nature of the compound,e studied the effects of dichlorvos on ventilation at rest in rats.
n WHO classification, dichlorvos is ranged among extremely haz-rdous compounds (group Ib) whereas diethylparathion and itsetabolite are in group Ia. Dichlorvos has been widely used as
arming or domestic pesticide in South Asia and Mediterraneanountries since 1955 (Brahmi et al., 2006; WHO, 2001; Yurumezt al., 2007). Regarding its high toxicity as well as its availability,ichlorvos is frequently involved in human poisonings (Eddlestont al., 2005b; Sungur and Guven, 2001).
To our knowledge, no studies previously compared the respi-atory toxicity in acute dichlorvos poisoning with cholinesterasectivities in whole blood and tissues. Therefore, we determined,rstly, the median lethal dose (MLD) of dichlorvos administeredubcutaneously; secondly, we studied the effects of 40% of the MLDf dichlorvos on ventilation at rest using whole body plethysmog-aphy and arterial blood gas analysis in awake rats between 5 mino 72 h post-injection. Finally, we studied the degree of inhibitionf whole blood and tissue cholinesterase activities.
. Materials and methods
All animal procedures used in this study were approved (P2.FB.071.09) by theegional Ethical Committee of Animal Experimentation (University René Descartes,aris Descartes).
.1. Animals
Male Sprague-Dawley rats (200 and 250 g) were purchased from JANVIER (Routees chênes secs BP5, FR-53940 Le Genest-St Isle, France). Animals were housed in a
ight-and temperature- controlled setting with access to food and water ad libitum.fter each experiment rats were euthanized using overdose of pentobarbital.
.2. Chemicals and drugs
Dichlorvos (2,2-dichlorovinyldimethylphosphate, purity greater than 98.8%,AS number 62-73-7), dimethylsulfoxyde, acetylcholinesterase of the electric eellectrophorus electricus (AchE; 200 UI/mg), dihydrated monosodium phosphate,ihydrated disodium phosphate, chloroacetic acid, chloridric acid, sodium chlo-ide, sodium hydroxide, triton X-100 and albumine bovine were obtained byigma–Aldrich (St Quentin Fallavier, France).
Dichlorvos was diluted in dimethylsulfoxide so as to obtain a mother solutionf 60 mg/ml. A daughter solution was prepared extemporaneously in isotonic salineolution (3.84 mg/ml) to facilitate the injection of doses equal to 40% of the MLD. Allolutions of dichlorvos were preserved at 4 ◦C in the dark for a maximum period of
weeks.Anaesthtic drugs, ketamine (Ketalar®) and xylazine (Rompum®) were obtained
rom Parke Davis (Paris, France) and Bayer (Paris, France), respectively. Hep-rine solution (Choay®) at 25000 UI/5 ml was obtained from Sanofi Winthrop,rance. [3H]Acetylcholine iodide (specific activity, 100 Ci/mmol) was obtained fromerkinElmer (Courtaboeuf, France) and was stored at −80 ◦C. Acetylcholine iodidemw: 273g/mol, C7H16INO2) was obtained from Fluka (Paris, France) and was storedt −80 ◦C. Precipath U (PPU) and Precinorm U (PNU) were obtained from RocheFrance). Isoamyl alcohol was obtained from Merck (Fontenay Sous Bois, France).istilled water (Frésenius FrancePharma, Louviers, France) was used for preparationf the various reagents.
.3. Safety precaution
All solutions of dichlorvos were prepared under fume hood using nitrile glovesnd goggles. Molar sodium carbonate solution was set under fume hood to neu-ralize immediately dichlorvos in the case of accidental spillage. Daily, after eachxperimental study, the same solution was used to decontaminate and clean up allaterials and areas.
ers 213 (2012) 142– 150 143
2.4. Methods
2.4.1. Clinical examinationThe animals were clinically observed at each time of the study and were simul-
taneously assessed by two observers. The following signs were noted: fasciculation,salivation, lacrimation, urination, defecation, ataxia, tonic–clonic seizures, pros-tration, coma, and death (De Candole et al., 1953; Tsao et al., 1990). Qualitativeassessment of each sign and symptom was made using a grading in severity, includ-ing, none (0), mild (1), moderate (2), and severe (3) according to that previouslyreported by De Candole. The central core temperature was simultaneously measuredby infra red telemetry (Houze et al., 2008).
2.4.2. Determination of the average lethal doses (MLD)The median lethal dose for 50% of rats (MLD) was determined using the up-
and-down method previously reported by Bruce and refined by Dixon (Bruce, 1985;Dixon, 1991).
2.4.3. Whole body plethysmographyRespiratory parameters were recorded in a whole-body plethysmography using
the barometric method validated in the rat by Bartlett and Tenney (1970), anddescribed in our previous studies with minor modifications (Houze et al., 2010, 2008;Villa et al., 2007). The following parameters were measured: barometric pressure,chamber temperature, core temperature, respiratory frequency (f), inspiratory time(TI), expiratory time (TE), total time (TTOT = TI + TE), tidal volume (VT), and minuteventilation (VE = VT × f).
2.4.4. Measurement of arterial blood gasesThe femoral catheterization and the measurement of arterial blood gases were
described in a previous study (Villa et al., 2007). Arterial blood samples were col-lected in heparinized syringes and immediately analyzed on a Rapidlab® 248, (BayerDiagnostics).
2.4.5. Measurement of whole blood and tissues cholinesterases (ChE) activity2.4.5.1. Blood sampling. Rats were anaesthetized with pentobarbital and blood col-lection was made by cardiac puncture (in heparinized syringes). All blood samplingwere collected in heparinized Eppendorf cups and stored at −80 ◦C until the assay.
2.4.5.2. Preparation of tissues homogenates. Before the collection of tissues rats wereanesthetized and thoracotomised. The right auricle of the heart was excised forexsanguination and all tissues were perfused simultaneously via the left ventriclewith 60 ml of heparinized saline solution at 37 ◦C while the heart was still beat-ing (Olivier et al., 1990). The volume of perfusion used in our study was necessaryto remove all circulating blood volume, plasma butyrylcholinesterase or residualhemoglobin. The circulating blood volume in rat was comprised between 50 and70 ml/kg (Morton et al., 1993). To check if the perfusion was correctly made weobserved the mucous membranes and the extremities. During the perfusion weobserved pale mucous membranes of the conjunctiva or inside the mouth, paletongue, and pale extremities and liver turned pale. After exsanguination and per-fusion, tissues (forebrain, brainstem, lungs, thigh and diaphragm muscles) werecollected and stored at −80 ◦C. Frozen tissues were diluted in 9 volumes of 0.1 Msodium phosphate buffer (1:10, w:v) (pH 7.4) with 1% Triton X-100, followed byhomogenization of the tissues for 30 s (Ultraturax IKA, T10 model, IMLAB, Lille,France). The supernatant was collected after centrifugation at 4 ◦C for 10 min at4000 rpm and the total protein concentration was measured by the method of Lowry(Lowry et al., 1951) on modular® (Roche, France) using bovine serum albumin asstandard and samples were frozen at −80 ◦C until radiometric assay.
2.4.5.3. Radiometric assay. Total cholinesterase (butyrylcholinesterase and acetyl-cholinesterase) activity was measured radiometrically by the method of Johnsonand Russell (1975) using [3H]acetylcholine iodide as substrate. This assay was basedon the selective extraction of the hydrolysis product, [3H]acetic acid, into a scintil-lation fluid. The incubation mixture was 0.12 ml containing 5 mM [3H]acetylcholineiodide and 50 mM phosphate buffer, pH 7.0, with 10 �l samples or control samples(Precipath: PPU and Precinorm: PNU) or acetylcholinesterase at 105 UI/l (hydrol-ysis total) for 15 min at room temperature. The reaction was stopped by adding0.1 ml of a solution with the composition 1.0 M monochloroacetic acid, 2 M sodiumchloride, and 0.5 M sodium hydroxide. Then 0.6 ml of isoamyl alcohol was added tothe mixture, shaken and centrifuged for 10 min at 4000 g. [3H]Acetic acid was selec-tively extracted from the acid solution into 3 ml of a scintillation fluid added to thereaction vial. Cholinesterase activities measured in whole blood were reported ascpm/min per/ml of erythrocytes and in homogenates tissues in cpm/min per/g pro-tein. Cholinesterase activities were expressed as the percent of control activity andplotted as a function of time after treatment.
2.4.5.4. Validation of the assay. The method was validate over a linear range of0–1.75 UI/ml. The limits of detection (LOD) and quantification (LOQ) were 0.03 UI/mland 0.09 UI/ml. Concerning the precision of the method, coefficients of variationhave been calculated on replicates of 10 samples at different enzymatic activity.
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Table 1Positive clinical signs and symptoms after dichlorvos poisoning.
Clinical Signs Time (min)
0 5 10 15 20 30 45 60 90 until the end
Fasciculation 0 1 2 2 3 3 3 2 0Salivation 0 1 1 1 1 1 0 0 0Lacrimation 0 1 1 1 1 1 0 0 0Urination 0 0 0 0 1 1 0 0 0Defecation 0 1 1 1 1 1 0 0 0
44 T. Duarte et al. / Toxicolog
he results showed ≤5% relative standard deviation. In order to study the selectiv-ty of the assays for acetylcholinesterase (AChE) and butyrylcholineterase (BChE) in
hole blood or tissues we would have assay in the presence of specific inhibitoro AChE and BChE. Like in the study of Grubic et al. (1981) specific inhibitors wereot used in our study and it was probably a limitation of our study. The methodf Johnson and Russell (1975) measured total cholinesterase (AChE and BChE). Inhole blood and tissues any differentiation can be made. In tissues, even removal
f the blood from circulation, however, does not guarantee a selectivity for acetyl-holinesterase. Moreover, the inhibition of butyrylcholinesterase by triton X-100 isot fully specific and at the concentration used probably not completed.
.5. Studies designs
Animals were randomized with regard to drug administration in non-blindedtudies designs.
.5.1. Study 1: determination of the median lethal doses (MLD) of dichlorvos inats
Approximately 18 h prior to experimentation, the animals were fasted, butllowed free access to water. Following drug administration, animals were placed inndividual cages, allowed to eat and drink, and maintained in the laboratory, which
as controlled for temperature and light. Every effort was made to reduce the num-er of animals required for the study. Accordingly, the up-and-down method asroposed by Dixon (Dixon, 1991) and refined by Bruce (Bruce, 1985, 1987), wasmployed.
Dichlorvos was administered in awake, unrestrained animals, via subcutaneousnjection in the neck at a dose corresponding to the MLD (13.83 mg/kg, SC) of theroduct cited in the literature (Stefanovic et al., 2006). Animals were examinedepeatedly during the first 4-h period after injection, then daily for 7 days, for evi-ence of toxic effects or death. At the end of the study period, the animals wereuthanized using pentobarbital overdose. Clinical observations were made beforenjection and thereafter at 30, 60, 90, 120, 150, 180 min, then at 4, 6 and 24 h, andhen daily up to 7 days post-injection.
.5.2. Study 2: whole body plethysmography in rats at restTwo groups of eight unrestrained animals were studied in order to investigate
he effects of dichlorvos at a dose corresponding to 40% MLD (5.12 mg/kg) on venti-ation at rest between 5 min and 72 h after administration. We chose this dose (40%f the MLD) due to a mortality rate of 100% when using 50% of the MLD in ani-als undergoing laparotomy to place the infrared telemetry sensor. Control group
eceived isotonic saline solution subcutaneously.
.5.2.1. Effects within 10 h post-injection. The animal was weighed and the teleme-ry transmitter activated. The animal was placed in the plethysmography chamber.he first plethysmography measurement was performed after a period of accom-odation of 30–60 min. Measurements were made three times to obtain baseline
alues. The temperature and clinical signs were recorded simultaneously.The animal was gently removed from the chamber for the subcutaneous injec-
ion of dichlorvos at a dose corresponding to 40% of the MLD, and replaced in thehamber for another session of respiratory recording. Ventilation was recordedvery 5 min during 20 min, then at 30, 45, 60 min and every 30 min during 270 min,ach record lasted about 60 s. After these first records, the animal returned to its boxn a temperature, light, and humidity controlled environment according to the inter-ational standards. The animals were given free access to water and a basal diet ad
ibidum. Animal was gently removed from their box and replaced in the plethysmog-aphy chamber, 30 min before the next session of respiratory recording at 300 minnd 330 min post-administration. After these records the animal returned to its boxn the same condition previously described. These procedures were performed at50 min and at 570 min post-injection.
.5.2.2. Effects after 10 h post-injection. The respiratory parameters were recordedt 24 h, 33 h, 48 h and 60 h post-administration. Between these recordings the animaleturned to its box under conditions of temperature and light adapted, water andoods were provided ad libidum.
The last respiratory recording was made at 72 h post-injection then the rats wereuthanized using a pentobarbital overdose.
.5.3. Study 3: arterial blood gases measurement
.5.3.1. Effects within 10 h post-injection. The day of the experimentation, rats were
laced individually in a Plexiglas cylinder (internal diameter: 6.5 cm, adjustableength up to 20 cm) (Harvard Apparatus, Inc, Massachusetts). Dichlorvos wasnjected subcutaneously in the neck and the dose was the same as that in study 2.lood samples were taken using the femoral arterial catheter. One hundred micro-
iters of blood was collected before dichlorvos or solvent injection, then at 5, 15, 30,5 and 60 min post-injection. Arterial blood gases measurements (pH, PaCO2, PaO2,CO3
− and SaO2) were sampled at the same time than that for recording clinicaligns and central core temperature.
Prostration 0 1 2 2 2 3 1 1 0
0: absent, 1: mild, 2: moderate, 3: severe.
2.5.3.2. Effects after 10 h post-injection. Arterial blood gases measurement were notstudied.
2.5.4. Study 4: effects of dichlorvos on cholinesterase activities2.5.4.1. Effects within 10 h post-injection. Rats were poisoned with dichlorvos at adose equal to 40% of the MLD. Rats were anaesthetized for blood collection at T0(before injection of dichlorvos), 15 min and 60 min post-injection by cardiac punc-ture (in heparinized syringes).
Tissues collections were made at the same time of blood sampling follow-ing the subcutaneous injection of dichlorvos. Exsanguined tissues were removedimmediately and stored at −80 ◦C until preparation of tissues homogenates andcholinesterase activity assay.
2.5.4.2. Effects after 10 h post-injection. Blood and tissues collections were made24 h post-injection. Cholinesterase activities were not determined after 24 h.
2.6. Statistical analysis
All measurements and calculations are expressed as mean ± SEM (standard errorof the mean). For each animal and ventilation parameters, the difference betweenthe value at each sampling time and its corresponding baseline value (at t = 0) wascalculated. For studies 2 and 3, analyses were performed using a two-way analysis ofvariance for repeated measurements. A p-value of less than 0.05 was considered sig-nificant. For parameters with a significant treatment × time interaction, the analysiswas followed by multiple Bonferroni comparison tests. Study 4 was performed usinga one-way analysis of variance. Graphs and statistical analysis were performed usingPrism version 5.0, GraphPad Software (San Diego, CA). All tests were two-tailed.
3. Results
3.1.1. Study 1: determination of the median lethal doses (MLD) ofdichlorvos in the rats
The MLD for dichlorvos was 12.8 mg/kg. The animals weresymptomatic: the most frequent clinical abnormalities were fas-ciculations, dyspnea, lacrimation and prostration.
3.1.2. Study 2: whole body plethysmography in the rats at rest
3.1.2.1. Comparison of baseline valuesThere were no significant differences of the clinical findings, the
temperature, the respiratory frequency (f), inspiratory time (TI),expiratory time (TE), total time TTOT, tidal volume (VT), and minuteventilation (VE) values between control and dichlorvos groups.
3.1.2.2. Clinical findingsEffects within 10 h post-injection. The animals in the control group
were free of symptoms. In the dichlorvos group, the most frequentclinical abnormalities were fasciculations, salivation, lacrimation,urination, prostration and defecation. Dichlorvos poisoning at 40%of the MLD did not induced ataxia, tonic–clonic seizures, comaand death during the study period. The severity of each sign andsymptom are shown in Table 1. There was a significant decrease in
the mean body temperature in the dichlorvos group in comparisonwith that in the control group (Fig. 1).Dichlorvos induced a transient but significant decrease in tem-perature from 37 ± 0.10 ◦C at baseline to 35 ± 0.31 ◦C 15 min after
T. Duarte et al. / Toxicology Letters 213 (2012) 142– 150 145
Fig. 1. Time-course of central core temperature in control rats (open squares) and dichlorvos poisoned rats (black squares) at a dose corresponding to 40% of the MLD(5.12 mg/kg). Each point is the mean of eight rats, and all results were expressed as mean ± SEM. D0 denote the part of the first day including controls values and post-injection values up to 10 h after injection (early phase). D1 denotes the period of time during the first day starting 10 h after injection up to the end of the day (24 h). D2
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enotes the period of time starting at 24 h after injection until 48 h. D3 represent a* p < 0.01; *** p < 0.001 Dichlorvos group versus Control group.
ichlorvos injection (Tmax). Sixty minutes post-injection, the tem-erature tends to return to control values until the end of thetudy.
Effects after 10 h post-injection. There were no significant differ-nces regarding clinical findings and temperature between controlnd poisoned rats from 10 h until the end of the study (Fig. 1).
.1.2.3. Whole body plethysmographyEffects within 10 h post-injection. After dichlorvos administra-
ion, there was a significant decrease in f from 114 ± 4 b/min beforeo 82 ± 7 b/min, 5 min after dichlorvos injection (Fig. 2A). Con-ersely, there was a significant increase in the total time (p < 0.001)f the respiratory cycle (TTOT) from 0.53 ± 0.02 s to 0.77 ± 0.07 s
min before and after dichlorvos injection, respectively (Fig. 2B.).hereafter, the decrease in f and the increase in TTOT transientlylateaued and remained significantly different from the values inhe control group until 60 min. The increase in TTOT (p < 0.001)esulted from an increase in TE (p < 0.001) with no significantffect on TI (Fig. 2C and D). There was a significant increase inT (p < 0.001) from 2027 ± 122 �l to 2989 ± 174 �l 10 min beforend after dichlorvos injection, respectively (Fig. 3A). No significantffects were observed on VE (Fig. 3B). For each respiratory param-ter the time-to-maximum effect (Tmax) and the maximum effectEmax) are presented in Table 2.
Effects after 10 h post-injection. There were no significant differ-
nces in any respiratory parameters between the two groups from4 h until the completion of the study.able 2ime-to-maximum effect (Tmax) and maximum effect (Emax) of respiratory parame-ers after dichlorvos poisoning.
Respiratory Parameters(units)
Tmax (min) Emax (mean ± SEM) Duration ofplateau
Frequency (f) (b/min) 10 69 ± 2 60TTOT (s) 10 0.88 ± 0.02 60TE (s) 10 0.70 ± 0.02 60TI (s) N/A N/A N/AVT (�l) 10 3308 ± 168 60VE (�l/min) N/A N/A N/A
/A: not applicable.
eriod of the study and it range from 48 h to 72 h. Statistical significance: *p < 0.05;
3.1.3. Study 3: arterial blood gases measurement
There were no significant differences in the pHa, PaCO2, PaO2,and blood bicarbonate between the control and the dichlorvosgroups at any time during the study period.
3.1.4. Study 4: effects of dichlorvos on cholinesterase activities
3.1.4.1. Effects within 10 h post-injectionThe 40% of the MLD dose induced a decrease in residual
total cholinesterase activity in whole blood to 5 ± 1% at 15 minafter dichlorvos poisoning. One hour after injection, there wasan increase in residual total cholinesterase activity up to 33 ± 3%(Table 3).
In tissues, the residual total cholinesterase activities at 15 minpost-injection were 12 ± 2% in the brainstem, 13 ± 4% in the thigh,and 12 ± 1% in the diaphragm muscles (Table 3). The lowest residualtotal cholinesterase activity (8 ± 3%) was observed in the forebrainwhile the highest residual total cholinesterase activity was mea-sured in the lungs (22 ± 2%).
In all tissues except for lungs, the mean residual totalcholinesterase activities increased approximately two-fold within1 h after poisoning. In contrast, the residual total cholinesteraseactivity in the lungs 1 h after poisoning was similar to that at 15 minpost-poisoning (Table 3).
3.1.4.2. Effects after 10 h post-injectionThe mean residual total cholinesterase activity in whole blood
at 24 h post-injection was 66 ± 8% (Table 3).
Table 3Residual activity of whole cholinesterase following dichlorvos (40% of the MLD)poisoning in rats (n = 8 per group).
Parameters Residual cholinesterase activity (% ± SEM)
Time 15 min 60 min 24 h
Whole blood 5 ± 1 33 ± 3 66 ± 8Forebrain 8 ± 3 17 ± 2 33 ± 2Brainstem 12 ± 2 25 ± 2 45 ± 2Diaphragm 12 ± 1 19 ± 1 24 ± 2Lungs 22 ± 2 23 ± 2 36 ± 3Thigh muscle 13 ± 4 35 ± 6 60 ± 2
146 T. Duarte et al. / Toxicology Letters 213 (2012) 142– 150
Fig. 2. Time-course of f (A), TTOT (B), TE (C), TI (D) in control rats (open squares) and dichlorvos poisoned rats (black squares) at a dose corresponding to 40% of the MLD(5.12 mg/kg). Each point is the mean of eight rats, and all results were expressed as mean ± SEM. D0 denote the part of the first day including controls values and post-injectionvalues up to 10 h after injection (early phase). D1 denotes the period of time during the first day starting 10 h after injection up to the end of the day (24 h). D2 denotes theperiod of time starting at 24 h after injection until 48 h. D3 represent a final period of the study and it range from 48 h to 72 h. Statistical significance: *p < 0.05; ** p < 0.01; ***p < 0.001 Dichlorvos group versus Control group.
T. Duarte et al. / Toxicology Letters 213 (2012) 142– 150 147
Fig. 3. Time-course of VT (A) and VE (B) in control rats (open squares) and dichlorvos poisoned rats (black squares) at a dose corresponding to 40% of the MLD (5.12 mg/kg).Each point is the mean of eight rats, and all results were expressed as mean ± SEM. D0 denote the part of the first day including controls values and post-injection values upto 10 h after injection (early phase). D1 denotes the period of time during the first day starting 10 h after injection up to the end of the day (24 h). D2 denotes the period oft y andD
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ime starting at 24 h after injection until 48 h. D3 represent a final period of the studichlorvos group versus Control group.
The tissular residual total cholinesterase activities measured at4 h post-injection were the lowest in the diaphragm (24 ± 2%) andhe highest in the thigh muscle (60 ± 2%) (Table 3).
. Discussion
The mechanism of respiratory failure induced by OPs remainsnclear despite many studies. Most of the studies were performedsing chemical weapons (Aas et al., 1987; De Candole et al.,953; Johnson and Wilcox, 1975; Nambiar et al., 2007). Only aew reported respiratory effects of pesticides (Eddleston et al.,005a,b,c,d). In previous studies, we reported the respiratory effectf 50% of the MLD of diethylparaoxon determined in our laboratoryn rats. The results were highly reproducible (Houze et al., 2005,010, 2008; Villa et al., 2007). We aimed at determining thereforehe effects of dichlorvos, frequently reported in human poisonings,nd exhibiting significant differences in its pharmacokinetics inomparison with diethylparaoxon (Askar et al., 2011; Gajewski andatkiewicz, 1981; Li et al., 1989).
In the present study, we administrated subcutaneously a dosef dichlorvos equal to 40% of the MLD. Indeed, in rats having hadbdominal placing of the telemetry probe, the administration of0% of the MLD of dichlorvos death eventually ensued, in spite of theour days of recovery, time recommended prior to experimentation
Houze et al., 2008).Dichlorvos administration induced signs and symptoms, includ-ng hypothermia, that were similar to those previously reportedy Villa et al. (2007). However, hypothermia and alteration of
it range from 48 h to 72 h. Statistical significance: *p < 0.05; ** p < 0.01; *** p < 0.001
ventilation at rest returned to normal values within 90 min afterdichlorvos injection instead of 4 h in diethylparaoxon poisoned rats.
On ventilation at rest, dichlorvos induced a significant decreasein respiratory rate (f) (−38%) resulting for an increase in expira-tory time (TE) (+150%) without significant effects on the inspiratorytime (TI). These alterations on the ventilation at rest induced bydichlorvos were within the same range of magnitude in compar-ison with those induced by diethylparaoxon (Houze et al., 2008;Villa et al., 2007). Only few studies reported the respiratory effectsof dichlorvos in rodents. Our data are in agreement with thestudies of Gaspari and Paydarfar (Gaspari and Paydarfar, 2007,2009). Indeed, the subcutaneous administration of dichlorvos ata dose of 100 mg/kg induced a decrease in respiratory rate. Cen-tral apnea was reported; however, dichlorvos was administeredat a dose of 2.5 or 10 mg/kg intravenously in rats anaesthetizedwith urethane (Takahashi et al., 1991). In contrast with the previ-ous study (Takahashi et al., 1991), we did not observe any centralapnea. However, the previous study was performed in anaes-thetized rats. Indeed, conflicting results on respiratory toxicity ofOPs in anaesthetized and awake animals were reported (Foutz et al.,1987; Takahashi et al., 1991). In the present study, dichlorvos-induced respiratory toxicity at rest was similar to that described byVilla et al. (2007) for diethylparaoxon at nearly equipotent dosesuntil 240 min. However, dichlorvos-induced respiratory toxicityresumed within 90 min while diethylparaoxon-induced respira-
tory toxicity persisted until 4 h. We recorded dichlorvos-inducedrespiratory toxicity till 72 h post-injection to detect any delayedeffects as those reported in humans with the intermediate syn-drome (Parajuli et al., 2005; Yang and Deng, 2007). In the present
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tudy, during this late period of time after injection, respiratoryarameters were not significantly different from those in the con-rol group from 10 h to 72 h post-dichlorvos poisoning. Differencesn the time-course of respiratory effects induced by toxic, but notethal, doses of diethylparaoxon and dichlorvos could be explainedy differences in the pharmacokinetics. Indeed, dimethyldichlorvos
s rapidly absorbed, distributed, and metabolized in both animalsnd humans (Nordgren et al., 1981; Ringman and Cummings, 1999;nni et al., 1994). In rats the Tmax was observed at 0.17 h with Cmax
f 5.36 ± 0.63 �g/ml after a 10 mg/kg dose of dichlorvos adminis-ered intraperitoneally (Wang et al., 2010). Protein binding waseported to be 50% for dichlorvos (Ringman and Cummings, 1999).t was also reported that the elimination half-life from plasma
as of about 10 min after inhalation of dichlorvos (RTECS, 2005).owever, the mean terminal half-life of dichlorvos was different
o that reported for parathion (7.2 h) administered at a 10 mg/kgose intravenously (Braeckman et al., 1980). Our study showedhat dichlorvos significantly impaired ventilation at rest withoutignificantly altering arterial blood gases. Our results are in agree-ent with those previously reported by Bakima et al. (1989) who
dministered intravenously dichlorvos (2 mg/kg) to French alpineoats. However, our results are in contrast with those reportedy Stefanovic et al. (2006) who showed that awake rats receiv-
ng dichlorvos at a 10.64 mg/kg dose administered subcutaneouslyesulted in severe respiratory acidosis (Stefanovic et al., 2006).ifferences between the results of the present study and those
eported in the study of Stefanovic et al. could be related, at leastn part, to a greater dose used in the previous study in comparison
ith the present.For the radiochemical determination of cholinesterase activi-
ies in whole blood and tissues we used the method describedy Johnson and Russell (1975). However, the cited methodas only validated for pure electric eel acetylcholinesterase (noatrix). However, when looking at the literature on this topic,
holinesterase activities were reported in various tissues, includinghose of interest in our study. In a number of papers, the authorssed the spectrophotometric assay reported previously by Ellmant al. (1961) without any specification regarding the use of specificatrices for validation in the tissues of interest (Li et al., 2000).ther authors, used tissue blanks with the Ellman method while
hey used the method of Johnson and Russell for the erythrocytesPadilla et al., 1994). Carr and Chambers used the Ellman methodor measurement of AChE activity in the brain (Carr and Chambers,991). Pope et al. used the method of Johnson and Russell in differ-nt matrices: plasma, erythrocyte samples, and whole brain (Popet al., 1991). According to the results from the literature we can con-lude that matrix effects appear to play a minor role. However, itill be important to investigate this issue in the future. Indeed, oneajor limitation of our study results from the fact that we did not
se the different matrices for the calibration curve. Consequently,hese limitations preclude any definitive conclusion regarding thectual enzymatic activities in the various tissues. Activities wereeasured at each T0, before dichlorvos administration. Further-ore, in the control group the activities were measured at each
ime of the experiment. Therefore, the observed, rather than thectual activities in the different tissues were assessed and com-ared at each time to the value in each tissue in the control group.
n our study, the method was validated over a linear activity rangef 0–1.75 UI/ml. The limits of detection (LOD) and quantificationLOQ) were 0.03 UI/ml and 0.09 UI/ml, respectively.
In our study, dichlorvos induced a significant decrease in totalholinesterase activities in various tissues, including whole blood.
ur findings agree with those previously reported by Taylor et al.2008) who showed that the acute inhalation of sublethal dichlor-os concentrations (35–75 mg/m3) resulted in rapid inhibition oflood acetylcholinesterase at 20 min after exposure. Our study
ers 213 (2012) 142– 150
showed that dichlorvos administered at a dose equal to 40%of the MLD resulted in residual total cholinesterase activity inwhole blood of about 5 ± 1% at 15 min and did not return to con-trol values at T24 h (66 ± 8%) while the ventilatory parameterswere within the normal range. Our results are also in agreementwith those of Hinz et al. A dose of 10 mg/kg administered bythe oral route induced the inhibition of cholinesterase activitiesthat reached a maximum of inhibition in blood 20–45 min afteradministration (Hinz et al., 1996). Independently of the route ofadministration and doses, dichlorvos induced a maximum inhi-bition of cholinesterase activity in whole blood approximately atthe same time to that reported by Taylor et al. and Hinz et al.We showed that a dose of dichlorvos equal to 40% of the MLDinduced the inhibition of cholinesterase activities, in whole bloodand tissues. To our knowledge, no previous studies comparedthe cholinesterase activities in various tissues and the ventila-tory effects at rest. In our study we observed that residual totalcholinesterase activities in the diaphragm (24 ± 2%) and the lungs(36 ± 3%) remained low until 24 h post-injection while the ventila-tory parameters were within the normal range at the same times.The residual total cholinesterase activity in whole blood (5 ± 1%)and forebrain (8 ± 3%) at 15 min were close together. Thereafter,the residual cholinesterase activities were greater in forebrain at60 min (17 ± 2%) and 24 h (33 ± 2%) post-poisoning, which wereabout half those measured in whole blood at the same times. Theseresults might be, at least in part, explained by the pharmacokineticsof dichlorvos which is rapidly degraded in whole blood. In tissues,the highest residual total cholinesterase activity compared withcontrols was observed in the thigh (13 ± 4% at 15 min increased to60 ± 2% at 24 h post-poisoning). Our results agree with the study ofGajewski and Katkiewicz (1981) who showed the highest residualactivity of acetylcholinesterase compared with control, was foundin the tibialis muscle (about 80%).
Our study clearly showed that an inhibition of totalcholinesterase was correlated with an effect on respiratoryfunctions at 15 min and 60 min. However, 24 h post-poisoning,the increase in cholinesterase activity was not completed whileventilatory parameters were within the normal range at the sametime. The lack of correlation between respiratory effects at rest andresidual total cholinesterase activities observed at 24 h could beexplained by findings cited in the literature. Acetylcholinesteraseactivities are present in functional excess, a phenomenon that issimilar in various species and various cholinergic synapses, so thatimpairment of muscle force generation is observed only when thecritical level falls below about 25% (Heffron and Hobbiger, 1979).Barnes and Duff (1953) showed that fasciculations and enhancedresponse to single stimuli take place while the cholinesterase activ-ities were reduced from 50% down to 10% of normal. The ability tosustain tetanic contractions in rat diaphragms and anterior tibialmuscles did not subside at AChE activity >10%. The conclusiontherefore must be either that cholinesterase is not essential for thefunctioning of the myoneural junction, or that some very smallfraction of the enzyme at the myoneural junction is protected fromthe action of the inhibitor (Barnes and Duff, 1953). Meeter andWolthuis (1968) showed that only 2% of cholinesterase activity inthe brain is sufficient to preserve spontaneous respiration in rats.In contrast, other authors found that in order to be able to sustaina tetanic contraction, about 50% of the cholinesterase activities inthe unhomogenized diaphragm must still be active (van der Meerand Wolthuis, 1965).
The time-course of recovery of cholinesterase activity dependson the nature of the inhibitor (Hobbiger, 1951). The stability of
dimethyl phosphorylated acetylcholinesterase, which is generatedduring a poisoning with dichlorvos, is quite low, compared tothe diethyl species, which is generated during a poisoning withdiethylparaoxon. This fast spontaneous reactivation during the
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arly stages of poisoning with dimethylated insecticides allows percentage of the enzyme to be active even in the presence of
high concentration of the insecticide. On the other hand, agingf dimethyl phosphorylated acetylcholinesterase is considerablyaster than that of diethyl phosphorylated acetylcholinesteraseEddleston et al., 2002; Hobbiger, 1951; Mason et al., 1993). Lotti2001) showed that the half time for the spontaneous reactiva-ion (ks) and the aging of acetylcholinesterase in human poisonedith dichlorvos were respectively 0.85 h and 3.9 h while in diethyl
pecies (chlorpyrifos-oxon) rate of spontaneous reactivation andging were much higher. The rate of ks to inhibited erythrocyteChE by dichlorvos was observed to be 0.347 h for rat (Askart al., 2011). The degree of AChE inhibition and its duration in vivoepend on the rate of aging and spontaneous reactivation. Whenhe rate of spontaneous reactivation is higher than that of theging, almost complete recovery of activity is expected. On theontrary, if the rate of aging is higher than that of spontaneouseactivation, then irreversible inhibition takes place (Lotti, 2001).hese facts may explain the lack of correlation between respiratoryffects at rest and residual cholinesterase activities observed in ouronditions.
Our study suffers from a number of limitations. Firstly, wetudied the effects of dichlorvos only. Therefore, we cannotssume that these results can be extended without any cau-ion to other organophosphate compounds. The second limitationf our study was the lack of measurement of the time-coursef dichlorvos blood concentrations that precludes any study ofhe pharmacokinetic–pharmacodynamic correlations. Finally, weested only one dose of dichlorvos. Therefore, we cannot extentithout caution the present results to those that might be obtainedsing different doses.
. Conclusions
Respiratory effects induced by dichlorvos at a dose equal to 40%f the MLD administered subcutaneously were both qualitativelynd quantitatively similar to those induced by diethylparaoxondministered at a dose equal to 50% of the MLD (SC). How-ver, dichlorvos induced the rapid onset of respiratory effectsTmax = 10 min) and returned to control values quickly (<90 min)hile diethylparaoxon-induced maximum effects occurred at
0 min post-injection and respiratory toxicity persisted until 4 h.oteworthy, the dichlorvos-induced respiratory effects occurrednd resolved while cholinesterase activities did not return to con-rol values.
onflict of interest
The authors declare no conflict of interest.
cknowledgment
This study was supported by SERB Laboratories (Paris, France).ERB laboratories did not have any control over the resulting pub-ication.
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