Materials
The ordinary/analytical grade chemicals and laboratory reagents required for experiments done in this study were purchased locally. Fine chemicals were purchased from reputed international firms and mentioned at the appropriate places in the manuscript.
Methods
In vivo treatment
The standard ethical guidelines were followed for experiments performed in the study. The experiments were executed by employing male Wistar rats. Protocols for performing animal experiments were approved by the institutional animal ethics committee. The experimental animals were housed appropriately under the optimum light/dark cycle, humidity and temperature. Male pups were treated with 1.5mg/kg cypermethrin (Thermo Fischer Scientific, Waltham, MA) during postnatal days 5-19, two times a week, intraperitoneally, along with respective control. The animals were left untreated for two months and then re-challenged with 15mg/kg cypermethrin, intraperitoneally, two times a week, for 4, 8 and 12 weeks. Experimental controls treated with vehicle were also developed in parallel [25].
Serum and brain isolation
At the end of the study (4weeks, 8weeks, 12weeks), animals were sedated under diethyl ether anaesthesia (Merck, Darmstadt, Germany). The peripheral blood serum and brain were isolated as described elsewhere [28]. Blood was drawn from descending aorta and was collected in heparinised tubes, centrifuged for 15 min at 1500xgat 4°C. After centrifugation, serum was collected and stored at -20°C until further use. For brain isolation, intra-cardiac perfusion was done with 0.9% normal saline at a fixed rate of 20 ml/min for 4 min. Subsequently, the brain was dissected out and kept at -20°C for further use.
Atomic absorption spectroscopy
The level of iron, copper, zinc and magnesium in the nigrostriatal region of rat's brain and serum was quantified spectroscopically as described elsewhere with minor changes [29]. In brief, the nigrostriatal region and serum were digested for 12 h in 2.5 ml digestion mixture containing nitric acid (HNO3) (Merck, Darmstadt, Germany) and perchloric acid (Thermo Fischer Scientific, Waltham, MA) in the ratio of 6:1 (v/v). Digested samples were kept on sand bath maintained at 60°C and re-digested until sample residue reached to 100μl. The final residue was dissolved in 3ml of 1% HNO3. Concentration of magnesium, zinc, copper and iron in the nigrostriatal region and serum was measured using AAS (PinAAcle 900F, PerkinElmer, Waltham, MA).
Cell culture and lysate preparation
Cell line and sub-culture were maintained as described elsewhere [24]. In summary, human neuroblastoma cells were indented from the National Centre for Cell Science, Pune, India for in vitro experiments. Human neuroblastoma cells, SH-SY5Y, were cultured in Dulbecco’s modified Eagle medium: nutrient mixture F-12 (Gibco Life Technologies, California, CA) media containing 10% fetal bovine serum (Gibco Life Technologies, California, CA) and 1% antibiotics/antimycotics (100 U/ml penicillin and 100 μg/ml streptomycin, Gibco Life Technologies, California, CA) and were maintained under humidified conditions at 37°C. Cells were seeded in T-25 flask for 24 h. The culture media was replaced with incomplete media containing 15 μM cypermethrin for 24 h along with respective control. After 24 h, media was decanted and T-25 flask containing cells was placed on ice. Cells were scraped using cell scraper in chilled lysis buffer containing cocktail of protease inhibitor (Thermo Fischer Scientific, Waltham, MA). Cells were collected in a tube, sonicated and kept at 4°Cfor 30 min. Subsequently, the cellular debris was then pellet down by spinning at 12,000xg at 4°C for 20 min. Supernatant was carefully taken out without disturbing the pellet. The concentration of protein was estimated employing Lowry’s method [30].
Protein extraction and estimation
Protein extraction was done as described somewhere else [26]. In brief, tissue homogenate (10% w/v)was made in the protein extraction buffer consisting of cocktail of protease inhibitor. Tissue in buffer was sonicated and subsequently kept for half an hour on ice. The content was centrifuged at 20,000xg, for 30 min at4°C. Concentration of proteins was estimated in the supernatant employing Lowry’s method [30].
Western blot analysis
Sample containing 60-100 μg of protein was equally loaded on two parallel gels. The proteins depending upon the molecular weight were resolved on sodium dodecyl sulphate-polyacrylamide gel electrophoresis and electroblotted onto polyvinylidene difluoride membrane (PVDF) (Bio-Rad Laboratories, Inc., Hercules, CA)as described elsewhere [26]. To prevent non-specific binding, PVDF membrane was incubated with 5% skimmed milk (HiMedia Laboratories Pvt. Ltd., Mumbai, India) for 60 min. Incubation of membrane was done with the primary antibodies [anti-transferrin, anti-ferroportin, anti-DMT-1, anti-β-actin (Santa Cruz Biotechnology Inc., Dallas, TX), anti-ceruloplasmin or anti-hepcidin (Abcam, Cambridge, UK)] for overnight followed by 1 h incubation with anti-goat/anti-rabbit/anti-mouse (Santa Cruz Biotechnology Inc., Dallas, TX) secondary antibody conjugated with alkaline phosphatase (AP). Since the secondary antibody used in the experiment was AP-conjugated, one gel was processed for target protein and other for respective loading control. The protein bands were developed using AP specific chromogenic substrates, 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium chloride (Thermo Fischer Scientific, Waltham, MA). Band density ratio was calculated in relation to β-actin using Alpha Imager software.
Cryosectioning and DAB-enhanced Perl’s iron staining
Cryosectioning and 3,3′-diaminobenzidine (DAB)-enhanced Perl’s iron staining were performed as mentioned elsewhere [25,31]. In brief, rats were anesthetized using diethyl ether and perfused trans-cardially using 0.9% saline and 4% paraformaldehyde (PFA) (Sigma-Aldrich, St. Louis, MO). Brain was isolated and kept overnight in 10% PFA followed by incubation in increasing sucrose (Sigma-Aldrich, St. Louis, MO)gradient [10%, 20% and 30% w/v each in phosphate buffer saline (PBS)]. Brain tissue was sliced coronally across the SN into 20 μm thick sections in a cryotome (MEV cryostat, SLEE medical GmbH, Neider-Olm, Germany) maintained at -20°C. Brain sections were then fixed with 4% PFA for 5 min. After 2 washes in PBS, sections were incubated in freshly prepared solutions of 2% hydrochloric acid (Merck, Darmstadt, Germany) and 2% potassium ferrocyanide (Thermo Fischer Scientific, Waltham, MA) for 30 min. Sections were incubated with 50% methanol (Sisco Research Laboratories Pvt. Ltd., Mumbai, India) and 10% hydrogen peroxide (Merck, Darmstadt, Germany) in PBS to block endogenous peroxidase activity. Prior to washing with PBS, the iron staining was enhanced using DAB (Sigma-Aldrich, St. Louis, MO). The section were mounted and observed under microscope (DM6000, Leica Microsystems, Wetzlar, Germany). In the SN, number of iron positive cells was quantified using ImageJ software.
Molecular docking studies of cypermethrin with transition metal transporter proteins of human and rat
Molecular docking studies of cypermethrin were executed by employingAutoDockTools (ADT) version 1.6.6rc3 and AutoDock version 4.0 docking program[32] to analyse its potential interaction with iron metal transporter proteins of both human and rat origin. The pdb structures of human ceruloplasmin (1KCW) receptor, transferrin (4XID) receptor, ferroportin (6W4S) receptor, hepcidin (1M4E) receptor, DMT-1 (5F0L) receptor and the rat ceruloplasmin (5N0K) receptor were downloaded from the RCSB PDB database. The pdb structures of the other four receptors of rat (transferrin, ferroportin, hepcidin, DMT-1) were not found in PDB database. Therefore, these receptors were searched on AlphaFold. Using their AlphaFold id, these receptors [transferrin (AF-P12346-F1), ferroportin (AF-Q923U9-F1), hepcidin(AF-Q99MH3-F1), DMT-1 (AF-O54902-F)] were downloaded from the Uniprot database. The 3D structure of ligand (cypermethrin) was drawn using ACD/ChemSketch[33]. The 3D structure of the ligand was then converted into pdb format using OpenBabelGUI software[34]. Initially, these transporter receptors were prepared for docking followed by the addition of the polar hydrogens and Kollaman charges to respective receptors. The receptor and ligand structures were saved in pdb format using OpenBabelGUI. The ligands were subsequently saved in pdbqt format in ADT to carry out the docking studies. The AutoDockdefault parameters were considered for the docking studies. In each case, ten docked conformations were generated. The energy calculations were achieved by genetic algorithms and blind docking centered on the target. Binding energies, the hydrogen bonding with the receptor and receptor-ligand interactions were the parameters used for analysing the docked ligand conformations. Chimera 1.11[35] was used to visualize the receptor with the ligand binding site and Discovery Studio 2021[36] was used to analyse different types of interactions.
Statistical analysis
Unpaired student’s t-test or two-way analysis of variance followed by the Bonferroni post-test was employed for comparison between or among groups. The outcomes are expressed as mean ± standard error of the mean (SEM). The GraphPad Prism version 5.0 was employed to analyse the changes and plot the bar diagrams. Changes were judged as statistically significant only when the ‘p’value was less than 0.05.