Data on dysfunctional muscle contraction and genes contractile expression associated with chlorpyrifos exposure in slow twitch skeletal muscle☆

Chlorpyrifos (CPF) is a toxic organophosphate commonly used worldwide. Its residues are being detected in different environmental matrixes and hence in the food chain. Repeated CPF exposure might pose health risk for the general population on long term. This data article contains the data of contractility impairment further to dietary exposure to CPF on a hind limb skeletal muscle; soleus, a typical slow twitch skeletal muscle. Thirty adult male rats Sprague Dawley are divided into three groups receiving the following daily diet for 6 weeks: Group 1 (vehicle), Group 2: CPF1 (CPF 1mg/kg/day) and Group 3: CPF5 (CPF 5 mg/kg/day). Soleus twitch tension and fatigability index are determined at the end of the treatment. The activity of acteylcholinesterase enzyme is assessed in the tissues homogenate. Additionally, we examined the expression levels of ryanodine type 1 receptor (RyR1), ATPase Sarcoplasmic/Endoplasmic Reticulum Ca2+ Transporting 1 (Atp2a1), ATPase Sarcoplasmic/Endoplasmic Reticulum Ca2+ Transporting 2 (Atp2a2) and nicotinic acetylcholine receptor (nAChR) in CPF-exposed skeletal muscle tissue using quantitative real time polymerase chain reaction. CPF exposure at two different doses induced an increase in twitch contraction in soleus muscle along with an increase in fatigability index. These increases are accompanied by low level of acetylcholinesterase enzyme activity as well as modification in genes level expression of nAChR, RyR1, Atp2a1 and Atp2a2 involved in contractility.


a b s t r a c t
Chlorpyrifos (CPF) is a toxic organophosphate commonly used worldwide. Its residues are being detected in different environmental matrixes and hence in the food chain. Repeated CPF exposure might pose health risk for the general population on long term. This data article contains the data of contractility impairment further to dietary exposure to CPF on a hind limb skeletal muscle; soleus, a typical slow twitch skeletal muscle. Thirty adult male rats Sprague Dawley are divided into three groups receiving the following daily diet for 6 weeks: Group 1 (vehicle), Group 2: CPF1 (CPF 1mg/kg/day) and Group 3: CPF5 (CPF 5 mg/kg/day). Soleus twitch tension and fatigability index are determined at the end of the treatment. The activity of acteylcholinesterase enzyme is assessed in the tissues homogenate. Additionally, we examined the expression levels of ryanodine type 1 receptor (RyR1), ATPase Sarcoplasmic/Endoplasmic Reticulum Ca 2þ Transporting 1 (Atp2a1), ATPase Sarcoplasmic/Endoplasmic Reticulum Ca 2þ Transporting 2 (Atp2a2) and nicotinic acetylcholine receptor (nAChR) in CPF-exposed skeletal muscle tissue using quantitative real time polymerase chain reaction. CPF exposure at two different doses induced an increase in twitch contraction in soleus muscle along with an increase in fatigability index. These increases are accompanied by low level of acetylcholinesterase enzyme activity as well as modification in genes level expression of nAChR, RyR1, Atp2a1 and Atp2a2 involved in contractility.  Value of the Data The data reveals the impairment of soleus contractions in CPF-treated rats at two doses which may be interesting for researchers investigating the physiology of skeletal muscles further to organophosphate intoxication. The data reveals the expression levels of some genes involved in muscle contraction and responsible of calcium flux. The data may be useful in suggesting additional mechanism of action for CPF other than the acetlycholinesterase inhibition.
The data provides the basis for deeper investigation to understand the modification of the contractile machinery of skeletal muscle further to subchronic intoxication with CPF.
contraction time of 87.13 ± 6.21 ms and half relaxation time of 99.32 ± 4.40 ms (n ¼ 10). A significant increase in the amplitude of twitch tension by 28% and 67% in CPF1 and CPF5 groups is recorded respectively (p < 0.05). The other parameters, contraction time and half relaxation time are not significantly modified in exposed rats compared to control.

Effect of CPF on soleus fatigability index
A significant effect of CPF exposure on fatigability index is observed in soleus muscle. The fatigability index is higher by 21% in CPF1 and CPF5 compared to the control group (p < 0.05) (Fig. 1). There is no dose related difference in soleus fatigability index.

Effect of CPF on AChE activity
AChE activity data revealed that CPF inhibited the enzyme activity in soleus of treated rats when compared to control group. The inhibition of AChE was about 41% in the two doses tested CPF1 and CPF5. (p < 0.05) (Fig. 2).
1.4. Real-time PCR detection for nAChR, RyR1, Atp2a1 and Atp2a2 mRNA expression A significant decrease is observed in mRNA expression levels of nAChR and RyR1 after CPF5 and CPF1 treatments respectively. On the other hand, a significant increase is detected in mRNA expression levels of Atp2a1 after CPF1 and CPF5 treatments and Atp2a2 at CPF1 treatment. (See Fig. 3). The raw data related to Fig. 1, Fig. 2 and Fig. 3 were shown in Appendix A. supplementary data.

Animals' treatment
Animal use protocol is approved and performed in accordance with the guidelines for animal care issued by the institutional review board (IRB) at Beirut Arab University (Approval code:2019H-0036-S-P-0295).
Male Sprague Dawley rats, approximately weighing 200 g at the beginning of the study, were habituated for one week to the local conditions (temperature 23 C, 12 h:12h light/darkness cycle) prior to the start of the experimentation. The rats were divided randomly into three groups. Control, CPF1 and CPF5 groups received daily standardized diet mixed with corn oil only, 1 mg/kg of CPF and 5 mg/kg of CPF respectively for six consecutive weeks. Each experimental group had free access to water. CPF (O, O 0 -diethyl-O-3, 5, 6-trichloro-2-pyridylphosphorothioate) was purchased from Sigma eAldrich and dissolved in corn oil.

Animals sacrifice and muscles dissection
After six weeks of exposure, the animals were euthanized with an intraperitoneal overdose of sodium pentobarbital (1 ml/kg; 200 mg/ml solution) and sacrificed. Soleus was immediately collected from both hind limbs. One soleus was deep frozen and stored at À80 C for acetylcholinesterase activity assay and genes expression level. The other soleus dissected was examined immediately for its contractility parameters.

Soleus contractility and fatigue resistance
The entire soleus dissected was directly transferred to oxygenated room filled with mammalian physiological solution (140 mM NaCl, 6 mM KCl, 5 mM HEPES, 3 mM CaCl 2 adjusted to pH 7.35). It was then appended at the distal and proximal tendons to a force transducer, stretched to optimal length (L 0 ), and electrically stimulated (12 V, 2 Hz for 5 minutes) through platinum electrodes positioned near the muscle. Contractions were recorded using a bridge amplifier and data acquisition system (Digidata 1200, Axon Instruments) controlled by custom-made software.
The peaks twitch tension (g/cm 2 ), contraction time (ms), half-relaxation time (ms) were determined. Force after 5 minutes of continuous stimulation was calculated as percentage of maximum force and was termed fatigability index (%) as described elsewhere [2].

Acetylcholinesterase activity
Acetylcholinestarase assay kit (ab138871, abcam, UK) was used for the detection of acetylcholinesterase activity. The kit provided a colorimetric one step assay for the detection of acetylcholinesterase activity by applying a modified Ellman method [3]. The procedure was done according to the manufacturer's instructions. Briefly, 20 mg of soleus were washed with cold phosphate buffer saline and homogenized with mammalian cell lysis buffer 5Â (ab179835, abcam, UK). The homogenates were centrifuged at 2500 rpm for 10 min. and the supernatant was used for acetylcholinesterase assay. 50 ml of the crude enzyme extracts were added to 96 well plates containing 50 ml of the reaction mixture (20 Â 5, 5 0 dithio-bis-2-nitrobenzoic acid and 20Â acetylthiocholine). The absorbance was measured spectrophotometrically at 414 nm with a microplate reader (MR-96A, Mindray). The protein concentrations of the isolated acetylcholinestrase were measured spectrophotometrically according to Bradford protein assay with bovine serum albumin as standard. All PCR reactions were performed in triplicate. Data, expressed as relative fold change in studied gene expression, was calculated using 2 -DDCq after being normalized to reference gene GADPH.

Data analysis
The data are expressed as the means ± S.E.M. Statistical comparisons using one-way analysis of variance (ANOVA) were used to determine statistical significance. The significance level for the main treatment effects was set at p < 0.05.