Di n butyl phthalate

Following deep anesthesia, the brain of each mouse was removed and immediately placed in a glass tube. The brains were immediately immersed in 15 mL of undiluted Golgi solution and stored in the dark at room temperature (Das et al., 2015 (link)). The solution was replaced on the second day and returned to the dark at room temperature for an additional 9 days. Finally, the brains were incubated for 3 days at 4°C in 30% sucrose solution (Sigma-Aldrich) for dehydration. Each brain was firmly mounted on the vibratome platform using superglue and sliced into 150 μm thick sections at room temperature. Sections were put on gelatinized slides and coated with 30% sucrose and air-dried in a dark room for 3 days. The slides were immersed in a diluted developing solution for 7–10 min and rinsed in double distilled H₂O. Water was removed by soaking the slides in increasingly higher concentrations of ethanol(s) for 10 min each. The slides were coverslipped using di-n-butyl phthalate in xylene (Sigma-Aldrich) as mounting medium and kept in 100% xylene (for detailed protocol see Das et al., 2015 (link)).

Phthalates: di-n-butyl phthalate (DBP), butylbenzyl phthalate (BBP), phthalate metabolites: mono-n-butylphthalate (MBP), mono-benzylphthalate (MBzP) (99–99.5% purity) were bought from Sigma-Aldrich, USA. Fetal bovine serum (FBS), penicylin-streptomycin, low-melting point (LMP) and normal melting point (NMP) agarose and plasmid pUC19 were bought from Sigma-Aldrich (USA). RPMI 1640 medium with L-glutamine and lymphocyte separation medium (LSM) (1.077 g/cm³) were purchased in Cytogen (Germany), while other chemicals were bought from Roth (Germany) and POCh (Poland) and were of analytical grade^{1 (link)}.

4-nonylphenol (NP) was purchased from Tokyo Kasei Kogyo Co. (Tokyo, Japan). Di-n-butyl phthalate (DBP), Mono-butyl phthalate (MBP) and corn oil were purchased from Sigma-Aldrich Inc. (St. Louis, Mo, USA). Dulbecco's modified Eagle's medium-Ham's F-12 medium (DMEM-F12 medium), penicillin, streptomycin sulfate, trypsin and collagenase I were purchased from Sigma–Aldrich Inc. (St. Louis, MO, USA). C8H17N2O4SNa (HEPES sodium salt) was obtained from Amresco Inc. (Solon, OH, USA).

LC-MS-grade acetonitrile (MeCN) and H₂O were purchased from Fluka (Steinheim, Germany). A Milli-Q purification device (Millipore Direct-QR 3, Millipore International, Merck KGaA, Darmstadt, Germany) was used for obtaining the water used in the analyses. Acetone (>99%) and hexane (HPLC grade) were obtained from Centralchem (Bratislava, Slovakia) and VWR Chemicals (Radnor, PA, USA), respectively. Magnesium sulphate (MgSO₄), graphitized carbon black (GCB), C18 sorbents, and primary secondary amine (PSA) were supplied by Agilent Technologies (Santa Clara, CA, USA). The aniline standards, namely 3,4-dichloroaniline (3,4-DCA) and 3,5-dichloroaniline (3,5-DCA), were obtained from Riedel de Haën (Seelze, Germany), with purity higher than 99.9%. Phthalate analytical standards, such as dimethyl phthalate (DMP), di-n-butyl phthalate (DnBP), mono-butyl phthalate (mBP), benzyl butyl phthalate (BBP), di-n-octyl phthalate (DnOP), and di-(2-ethyl hexyl) phthalate (DEHP)), were supplied by Sigma Aldrich (Steinheim, Germany).

The chemicals CuSO₄, MnSO₄, benzyl butyl phthalate (BBP), di-n-butyl phthalate (DBP), dicyclohexyl phthalate (DCP), diethyl phthalate (DEP), di-(2-ethylhexyl) phthalate (DEHP), acetosyringone (AS), syringaldehyde (SA), 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), vanillin (VA), N-hydroxyphthalimide (HPI), (2,2,6,6-tetramethylpiperidin-1-yl) oxyl (TEMPO), and hexane, along with other solvents (analytical grade, 99.0% purity), were purchased from Sigma-Aldrich (Merck/Millipore Sigma, St. Louis, Missouri, USA). The chemical structures of the compounds used in this study are shown in Figure 1A and Supplementary Table S1. Laccases from three fungi (Aspergillus sp., Rhus vernicifera, and Trametes versicolor) were purchased from Sigma-Aldrich (Merck/Millipore Sigma, St. Louis, Missouri, USA).

All the chemicals, such as tizanidine powder (>98% purity) (TZN), flaxseed oil, coriander seed oil and Anhydrous sodium hydroxide NaOH (>96 wt%) were procured from Leads Pharma (Leads Pharma, Islamabad, Islamabad Capital Territory, Pakistan). Eudragit RL100, ethyl cellulose (99%), polyvinyl alcohol-1750 (PVA1750), polyvinyl pyrrolidone-K30 (PVAK30), low average molecular weight chitosan (100,000 g/mol) with degree of deacetylation (75–85%) and viscosity of 20–300 cP and di-n-butylphthalate were purchased from Sigma-Aldrich (Sigma-Aldrich Chemie GmbH. Kappelweg 1 91,625 Schnelldorf, Germany), potassium dihydrogen phosphate, methanol, and ethanol were obtained from Dow Chemical Co (E Patrick St, Midland, MI, USA).