6 propyl 2 thiouracil ptu

All experiments were conducted with prior approval from the United States Environmental Protection Agency’s (US EPA’s) Institutional Animal Care and Usage Committee and were carried out in an Association for Assessment and Accreditation of Laboratory Animal Care approved facility. N=16 timed pregnant Long Evans rats were purchased from Charles River (Morrisville, NC) and delivered on gestational day (GD) 2; sperm positive was considered GD0 and pup birth postnatal day 0 (PN0). Dams were single housed in polycarbonate cages, maintained a 12:12 light cycle and offered chow (Purina 5008) and deionized water ad libitum. Animals were weight ranked and then randomly allocated to two treatment groups. N=8 dams were exposed to 3 ppm (0.0003%) 6-propyl-2-thiouracil (PTU, purity ≥98%, Sigma) dissolved in deionized drinking water; N=8 control dams were administered vehicle only (deionized drinking water). The maternal exposure was initiated on GD6 and continued through PN14. This PTU exposure was not expected to induce overt toxicity in dams, but was sufficient to induce a periventricular heterotopia in their offspring (3 (link)). N=8 controls and N=7 PTU exposed dams gave birth.

Rats were randomly divided into five groups (n = 5): normal, hyperthyroidism-induced control, the MOK-pharmacopuncture (MOK group, 0.3 or 3 mg/kg, body weight; bw) group in hyperthyroidism rats, and the 6-Propyl-2-thiouracil (PTU, Sigma-Aldrich, CA, USA) pharmacopuncture (PTU group, 10 mg/kg, bw) group in hypothyroidism rats as a reference group. Hyperthyroidism was induced by intraperitoneal injection of LT4 (0.5 mg/kg, bw). In normal rats, only saline was intraperitoneally injected (Fig. 1). After 14 days, the rats were administrated MOK pharmacopuncture into the anterior neck near the thyroid gland at a volume of 0.05 mL/rat, dissolved in saline, once daily from days 15 to 28 in LT4-induced hyperthyroidism rats. As a reference drug, PTU (6-Propyl-2-thiouracil: Sigma-Aldrich, CA, USA) 10 mg/kg (body weight) was dissolved in 0.3 ml saline, and rats were given a daily subcutaneous injection of PTU into the dorsal neck.Fig. 1

 Experimental design in rat model of hyperthyroidism

Phosphate buffered saline (PBS), ethylenedyaminetetracetic sodium salt (EDTA), HEPES, bovine serum albumin (BSA), RPMI 1640 and Hank's balanced salt solution (HBSS) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The 6-propyl-2-thiouracil (PTU) and 2-ethylhexyl-4-methoxycinnamate (Octyl Methoxycinnamate—OMC) were purchased from Sigma-Aldrich (St. Louis, MO, USA). IgG anti-murine CD3 (clone 145-2C11), APC-conjugated hamster IgG anti-murine CD3, PE-conjugated hamster IgG anti-murine CD8, and FITC-conjugated rat IgG anti-murine CD4 were all obtained from EXBIO Praha (Vestec, Czech Republic). Fetal bovine serum was obtained from Hyclone (Logan, UT, USA). Carboxyfluorescein diacetate-succinimidyl ester (CFSE) was obtained from Invitrogen (Carlsbad, CA, USA). Cell Proliferation Kit I (MTT) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Thyroxine (T4) AccuBind® kit was purchased from Monobind Inc. (Lake Forest, CA, USA).