Bisphenol af

Bisphenol A (≥99%), Bisphenol AF (≥99%), Bisphenol AP (≥99%), bisphenol B (≥98%), bisphenol BP (≥98%), bisphenol C (≥98%), bisphenol E (≥98%), bisphenol F (≥98%), bisphenol G (≥98%), bisphenol M (≥99%), bisphenol P (≥99%), bisphenol PH (≥99%), bisphenol S (≥98%), bisphenol TMC (≥97%), and bisphenol Z (≥99%) were purchased from Sigma-Aldrich. Stock standard solutions (100 mg/L) were individually prepared by dissolving standard compounds in acetonitrile. A stock standard mixture containing all the individual standards (100 mg/L) was also prepared and stored at −20 °C. Working solutions for calibrations were prepared by serial dilution of the stock standard mixture with acetonitrile (Sigma-Aldrich). Beta-glucuronidase from E. coli (200 units/mL) was purchased from La Roche AG group. Bis(trimethylsilyl)trifluoroacetamide (BSTFA) with 1% of trimethylchlorosilane (TMCS) solution was used for BPs derivatization and was purchased from Sigma-Aldrich. Ammonium acetate (98%), calcium chloride (CaCl₂) (≥99%), creatinine hydrochloride (≥97%), ammonium phosphate dibasic (≥99%), magnesium chloride hexahydrate (≥99%), potassium chloride (≥99%), sodium sulphate (≥99%), urea (≥98%), acetone (99.9%), formic acid (≥95%), and methanol (99.9%) were purchased from Sigma-Aldrich. Nanopure water was provided by an ultrapure water system (ariumPro, Sartorius, Germany).

Standards of bisphenol A, bisphenol F, bisphenol S, bisphenol AF, bisphenol E, and bisphenol B (Fig. 1) were purchased from Sigma-Aldrich (St. Louis, MO, USA) and were each of > 99% purity. MS-grade methanol was purchased from Sigma-Aldrich, and MS-grade acetonitrile was from POCh (Gliwice, Poland). As an additive to HPLC, mobile phase ammonium acetate ≥ 99% (Fluka Analytical, Switzerland) was used. The HPLC-grade water was prepared by reverse osmosis in a Demiwa system from Watek (Ledec nad Sazavou, Czech Republic), followed by double distillation from a quartz apparatus. The yellow tetrazole dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) used in the MTT assay was purchased from Sigma-Aldrich. All salts used for preparation of media were purchased from Avantor (Gliwice, Poland).

Mice were divided into four treatment groups per pollutant (5 to 8 animals/group randomly assigned), as follows: (1) a control group, (2) a group exposed to the pollutant at 50 mg/kg/day (Bisphenol AF or Dibutyl Phthalate, Sigma-Aldrich, Saint Louis, MO, USA), (3) a group exposed to an oral antioxidant supplementation (Fertlix^®, CellOxess, Ewing, NJ, USA), and (4) a group co-exposed to the pollutant and oral antioxidant supplementation simultaneously. The pollutants and antioxidant supplementations were administered via dilution in drinking water and the mice were exposed for 14 days. At the end of the exposure, the mice were anesthetized with isoflurane and a cardiac puncture was performed to collect blood plasma prior to euthanasia via cervical dislocation for tissue collection. Preliminary studies (not shown) were conducted upstream comparing several low doses (2, 10 and 50 mg/kg/day) of pollutants. We chose the 50 mg/kg/day dose for the present study because of the appearance of significant effects, specifically with this dose, on sperm DNA oxidation. We also tested the effect of both pollutants, under the same exposure conditions, on CD1 mice. However, as these mice were less sensitive to sperm DNA oxidation than C57/BL6 mice, we decided to use the more sensitive mice for the final study.

Bisphenol S (BPS) (purity ≥ 98%), Bisphenol A (BPA) (purity ≥ 99%), Bisphenol E (BPE) (purity ≥ 98%), 2,2-Bis(4-hydroxy-3-methylphenyl)propane (3-3BPA) (purity ≥ 97%), Bisphenol B (BPB) (purity ≥ 98%), Bis(4-hydroxyphenyl)-2,2-dichloroethylene (BPC) (purity ≥ 98%), Bisphenol BP (BPBP)(purity ≥ 98%), Bisphenol F (BPF) (purity ≥ 98%), Bisphenol FL (BPFL) (purity ≥ 97%), Bisphenol Z (BPZ) (purity ≥ 98%), 4,4′-Dihydroxybiphenyl (BP4-4) (purity ≥ 97%), Bisphenol AP (BPAP) (purity ≥ 99%), Bisphenol AF (BPAF) (purity ≥ 97%), Bisphenol P (BPP) (purity ≥ 98%), Bisphenol M (BPM) (purity ≥ 99%) were purchased from Sigma-Aldrich (Saint Louis, MO, USA).