Experimental protocol
Forty male Wistar rats of two ages, young rats aged 3 weeks weighing 75 ± 5g, and adults aged 9 weeks weighing 220 ± 5g, were purchased from the Egyptian Institute for Serological and Vaccine Production, Helwan, Egypt. The animals were maintained in stainless steel cages at room temperature (25 ± 5℃) and humidity 50 ± 5% with a natural day and night cycle. Rats were provided food and water ad libitum under the approval of Animal Care Committee of Mansoura University, Egypt (No. Sci-Z-P-2022-82). After an acclimation period of a week, each age group was randomly subdivided into two groups, each of 10 rats: control young (CN-Y) and exposed young (EX-Y) groups, as well as control adult (CN-A) and exposed adult (EX-A) groups. Control groups inhaled ambient air, while exposed groups were allowed to inhale mosquito coil smoke in an exposure chamber of dimensions (1.5 x 0.9 x 2.1) m3 previously described in Abdrabouh (2021), for 6 hours (8 am-2 pm) /day, 6 days/week for 4 weeks.
Mosquito coil characters
Mosquito coil characters
A commercially available brand of mosquito repellent coil (Jinjiang Laojun Chemical Co., Ltd, China) with productive approved certificate number HNP35042-I2707 was purchased from retrial outlets at Aga villages, Mansoura, Dakahlia, Egypt, was used in this study. The repellent black coils were declared to contain 0.05% meperfluthrin as an active ingredient. Each coil was approximately 15 cm in diameter, weighed 24–32 g, and was alight for 7–10 hours. The same brand of repellent coils was used throughout the study period to avoid variations in commercial products.
Detection of indoor air quality
DI. ZENE portable air quality detector (model: DZ-8600, USA) was used to detect levels of particulate matter; PM2.5 and PM10 (µg/m3), total volatile organic compounds (TVOCs), and formaldehyde (HCHO) mg/m3 inside the exposure chamber at zero time and after each hour of the exposure period (6 hrs/day). Also, the quality of the ambient air in the control chamber was detected.
Blood sampling
After the experimental period, each animal group was sacrificed under anesthesia by intraperitoneal (I.P) injection by a mixture of ketamine (0.08 ml/g) and xylazine (0.008 ml/g) where each rat received 0.001 ml/g of this mixture.
Two blood samples were collected per rat. The first sample was pooled in ethylenediaminetetraacetic acid (EDTA) tubes for investigating hematological parameters, including red blood cells (RBCs) count, hemoglobin (Hb) content, hematocrit (HCT)%, platelets (PLTs) count and total white blood cells (WBCs) count, using a fully automatic hematological analyzer (Sysmex XE-2100, Corp. Kobe, Japan), as described by Dacie and Lewis (2001). The second blood sample was centrifuged for 15 minutes at 855 xg, and the separated serum was preserved at -20°C until analysis.
Serum biochemical studies
In serum samples of each group, erythropoietin hormone (EPO), interleukin-6 (IL-6) as an inflammatory marker, and serum levels of cardiac troponin-I (cTn-I) were determined using the enclosed methods of ELISA kit of Cusabio, Houston, USA, with catalog numbers CSB-E07323r, CSB-E04640r, and CSB-E08594r, respectively. However, the Spinreact Kit, Ctra Santa Coloma, Spain was used to detect serum levels of C-reactive protein (CRP), lipid profile including total cholesterol (TC), total triglycerides (TG), low- and high-density lipoprotein cholesterols (LDL) and (HDL), as well as aspartate aminotransferase (AST), lactate dehydrogenase (LDH), creatine kinase-MB (CK-MB) enzymes, in addition to Na+ and K+ ions according to the enclosed method of each parameter. Each with catalog number of MDTLIS40-P, TKBSIS48-E, MXBSIS49-I, MXBSIS51-I, MDBSIS37-I, MDBEIS46-I, TKBEIS43-I, MXBEIS31-I, BSIS54-I, BSIS53-I, respectively. However, serum myoglobin (MB) was determined according to the included method of BioVision (Milpitas, California, USA), catalog number E4328-100.
Heart Na-K ATPase
Half of the heart tissue from each rat was detached to estimate heart Na-K ATPase activity. Approximately 0.5 g of heart tissue was homogenized in phosphate buffer (0.1 M, pH 7.4) and centrifuged at 855xg for 10 minutes. Then clear supernatants were collected and frozen at -80°C until detection using the method enclosed in the ELISA kit (MyBiosource, Inc., San Diego, USA), catalog number: MBS7245054.
Heart histopathological examinations
The other half of the detached heart tissue was washed through a saline solution, fixed in 10% formaldehyde for 72 hours, then dehydrated through an alcohol series, cleared in xylene, embedded in paraffin wax, and cut into sections of 5µm thickness to be used in histological, histochemical and immunohistochemical assays.
Histological studies
a-Hematoxylin and eosin (H&E) staining for evaluation of the heart histopathological alterations. Paraffin sections were cleared in xylene for 2 min., three times, then hydrated by three changes of ethanol (100%, 95%, 70%), 2 min. for each concentration. Slides were rinsed by running tap water for 2 min. Heart sections were stained with hematoxylin solution for 3 min., then rinsed with tap water for 5 min. After that, a working eosin solution was applied for 2 min. Dehydration of heart sections was achieved by dipping slides in 95% ethanol for 5 min., then transferred to 100% ethanol twice for 2 min per each. Consequently, heart sections were cleared in xylene 3 times, 2 min. per each and a drop of mounting medium was placed over the slide and covered by a coverslip (Cardiff et al. 2014).
Histopathological evaluation and semiquantitative scoring
For H&E heart sections in both young and adult groups, the extent of cardiac tissue injury was evaluated via a semi-quantitative scoring system; five randomly selected fields were evaluated for each section in each group. The scoring of cardiac lesion severity depended on the percentage of tissue involvement, as described by Khafaga and El-Sayed (2018): none (0): no involvement of evaluated field; mild (1): involvement of 0–25% of evaluated field; moderate (2): involvement of 25–50% of evaluated field; and severe (3): involvement of 50–100% of the evaluated field.
b-Masson’s trichrome (MTC) stain was used to selectively stain collagen fibers in order to study cardiac pathologies related to fibrosis. The stain consists of three stains (Weigert’s Hematoxylin, Biebrich scarlet-acid fuschin solution, and Aniline blue). The method of using this stain was documented by Kiernan (2008). After slides deparaffinization and hydration processes, as mentioned above, Weigert’s Hematoxylin solution was applied to heart sections for 10 min., then rinsed with tap water for 10 min. Biebrich scarlet-acid fuschin solution was applied for 10–15 min. Differentiation in phosphomolybdic acid solution was achieved for 10–15 min. Then slides were transferred to aniline blue solution for 5–10 min., rinsed and differentiated in 1% acetic acid solution for 2–5 min, washed in distilled water, quickly dehydrated in 95% ethyl alcohol, cleared in xylene, and mounted with a mounting medium.
Histochemical studies
a-Periodic acid Schiff’s (PAS) reagent was used for the detection of glycogen in cardiomyocytes. The heart slides were deparaffinized and placed in xylene several times, then hydrated through descending series of ethyl alcohol and water. Sections were placed in periodic acid solution (1%) for 5–10 minutes, washed 2 times with distilled water, covered with Schiff’s reagent for 20–30 minutes, then rinsed in running tap water for 5–10 minutes. Hematoxylin solution was used as a counterstain for 3–5 minutes. Heart sections were then dehydrated in ascending concentrations of ethyl alcohol, cleared by xylene, and mounted by DPX (Alkhamiss 2020).
b-Bromophenol blue (BPB) staining was employed for the localization of total proteins. After deparaffinization, heart sections were brought to absolute alcohol and incubated for 15 minutes in mercuric bromophenol. Then, sections were rinsed in 0.5% acetic acid for 20 minutes with three changes, washed in tap water until the sections appears blue, rinsed in phosphate-buffered saline (PBS) for 3 minutes and left to dry in air. After that, heart sections were dehydrated through ascending concentrations of ethyl alcohol, cleared in xylene, and mounted with DPX (Mazia et al. 1953).
Immunohistochemical studies
5µm thick positively charged slides were first processed with xylene and alcohol as in histopathological procedures. Antigen retrieval was performed by boiling the samples in 9 mmol/L citrate buffer (PH 6) (Invitrogen, CA, USA) for 30 min. Heart sections were examined for expression of caspase 3, P53, cytochrome C (1:100 dilution; Genemed), and anti-SVV polyclonal antibody (1:75 dilution; Santa Cruz, CA) by overnight incubation with different antibodies at 4°C. Afterward, slides were incubated with HRP conjugated secondary antibody (1:500 dilution, Santa Cruz, CA) for one hour (Magaki et al. 2019). Staining with DAB chromogen was conducted using the Vector lab detection kit according to the manufacturers' instructions (Vector Lab, CA, USA).
For antigen retrieval of the detoxifying enzyme, cytochrome P450 (CYP450), heart tissue slides were placed in antigen retrieval solution (Citrate buffer solution, pH 6), then slides were microwaved at power 10 for 5 min. two times with adding water if necessary to avoid dryness. Slides were allowed to cool for 15 min., then were washed in deionized water 5 times, and incubated with an endogenous peroxidase blocking reagent containing hydrogen peroxide and sodium azide (DAKO peroxidase blocking reagent, Cat. No.S 2001). One to two drops of the supersensitive primary monoclonal antibody against cytochrome P450 (CYP450) were then put on the sections. Slides were incubated horizontally in humid chamber at room temperature for 60 min. Excess reagent was thrown off and slides were rinsed twice for 5 min. in phosphate buffer solution. After blotting excess buffer, 1–2 drops of the ready-to-use DAKO EnVision + system were applied for 20 minutes at room temperature. Chromogen used was DAB (diaminobenzidine), 1–2 drops for 10–20 min. until a desirable brown color was obtained, the slides were then washed in buffer. Sections were taken to distilled water then nuclear counter staining was done using Mayer's hematoxylin (Hx) solution for 3–5 min. according to degree of nuclear staining. Then, sections were washed in tap water, differentiated in acid-alcohol, and washed again in water. Slides were left to dry in air, then mounted in Canada balsam (Abdraboh et al. 2011).
Quantitative morphometric measurements
Quantitative morphometric measurements by Leica Quin 500^ image analyzer computer system (Leica image system Ltd.; Cambridge, England) were used to detect the percent of area occupied by collagen fibers, glycogen, and protein contents, as well as expressions of caspase 3, P53, cytochrome C, and CYP450 in heart sections in each group at a magnification of X400, according to Shi et al. (2007).
Molecular docking study of meperfluthrin
The crystal structures were obtained from Protein Data Bank (https://www.rcsb.org/) and meperfluthrin structure from PubChem (https://pubchem.ncbi.nlm.nih.gov/). The docking study was performed using MOE software 2022.02(1), the protein structures were prepared, optimized as well as the ligand structure and the docking was set to perform 100 runs keeping the top 10 poses for visual inspection.
Statistical analysis
One-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was used with GraphPad Prism software (v 5.04, GraphPad Software Inc., La Jolla, CA, USA) to statistically analyze the obtained data. The results are expressed as the mean ± standard deviation (SD), and significant values were recorded at p < 0.05. The semi-quantitative scoring of cardiac injury parameters was analyzed using Kruskal–Wallis test followed by Dunn's test to assess the significance between mean scores.