Cypermethrin

Essential oils (EOs) used in this bioassay are listed in Table 5. Six Myrtaceae plant EOs were obtained from Oshadhi Ltd. (Cambridge, England) and La Drome (Die, France). Wakogel C-200 (Wako Pure Chemical, Osaka, Japan) was used for chromatography. Dichlorvos (DDVP), and cypermethrin were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Non-polar and polar fractions of kanuka and manuka EOs were prepared as follows: a sample of oil (5 g) was loaded onto a column of activated silica gel (Wakogel C-200) and eluted with hexane and then with diethyl ether to yield non-polar and polar fractions. The triketone fraction was prepared by further fractionation of the polar fraction with 5% diethyl ether in hexane (Figure 1). The solvent was removed using a rotary evaporator and the fractions were dried and stored at 4 °C before analysis and testing.

Nafion, luminol (chemiluminescence reagent, ≥98.0%), BPA, cypermethrin (CYP), 17β-estradiol (E2), diethylstilbestrol (DES), phenol (PHE) and hexestrol (INN) were obtained from Sigma-Aldrich Trading Co., Ltd. (Shanghai, China). All other reagents were of analytical grade and the ultrapure water (resistivity ≥ 18.25 MΩ) was applied. The experiments were conducted at 25 °C.

All of the type-II pyrethroid standards (Pestanal grade), namely cypermethrin, deltamethrin, cyhalothrin, and fenvalerate were purchased from Sigma-Aldrich (St. Louis, MO, USA). Ninhydrin and polyvinylpyrrolidone (PVP) with an average molecular weight of 10,000 were supplied from Sigma-Aldrich (St. Louis, MO, USA). Ammonium acetate, sodium hydroxide, methanol, ethanol, Triton X-100, dichloromethane, and toluene were obtained from Merck (Darmstadt, Germany). Chromatography paper grade 1CHR was purchased from GE Healthcare (Pittsburgh, PA, USA). Parafilm was purchased from Bemis Company (Oshkosh, WI, USA). Laminating pouch film was purchased from DHA Siamwalla (Bangkok, Thailand).

Cypermethrin, cleaved-PARP antibody and N-acetylcysteine (NAC) were purchased from Sigma; γH2AX antibody was purchased from Millipore (Billerica, Bedford, MA, USA); Mouse- or rabbit-monoclonal antibodies specific for phospho-JNK1/2, JNK1/2, phospho-ERK1/2, ERK1/2, p53, p21, cyclin D1, cyclin E, and CDK4 were purchased from Cell Signaling Technology (Boston, MA, USA). 2, 7-dichlorofluorescin diacetate (DCFH-DA) was purchased from Sigma (St. Louis, MO, USA). The Annexin V-fluoresce isothiocyanate (FITC)/propidium iodide (PI) apoptosis detection kit, cell cycle staining kit and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody were obtained from Multisciences Biotechnology (Hangzhou, China).