Supplemental Tables I. a Analysis of 2-OHE2 Data Or B 4-OHE2 Data from Mice Given a Control

Supplemental Tables I. a Analysis of 2-OHE2 data or b 4-OHE2 data from mice given a control implant, 30 mg TMS implants or 60 mg TMS implants for eight weeks. Mammary glands were dissected and then analysis of 2-hydroxy c and 4-hydroxy d estrogen metabolites were determined by UPLC coupled to a tandem mass spectrometer (See Materials & Methods).

Supplemental Tables II. a Low dose, 30 mg TMS, is not toxic. Aromatase-transgenic mice were divided into three groups: intact control, mice from which the ovaries were removed, mice that were given 30 mg TMS silastic capsules. Mice were treated for eight weeks and the body weight at the start of treatment was compared with the end of treatment. The weight of the uterus and ovaries was determined at the end of treatment. b High dose, 50 mg/kg TMS, does not change body weight, but uterine weight is slightly increased. Aromatase-transgenic mice were divided into three groups: intact control; mice from which the ovaries were removed; mice that were given 50 mg/kg TMS via subcutaneous injection. Mice were treated for eight weeks and the body weight from the start of treatment was compared with the end of treatment. The weight of the uterus and ovaries was determined at the end of treatment.

Supplemental Table III. Blood was harvested at the end of the eight-week treatment period and sent for whole cell blood analysis. Known normal mouse ranges are shown in the right hand column.

Supplemental Fig. 1. Release rate of TMS from silastic capsules a Optical density readings (OD260, gray diamonds; OD280 gray squares; OD310 open triangles) with increasing TMS concentrations. b Two silastic capsules containing 15 mg of TMS each were placed in a beaker of 0.8% saline with constant stirring for 15 days. The OD310 nm was determined by spectrophotometer measurements daily.

Supplemental Fig. 2. Whole mounts demonstrate that 30 mg of TMS given for 3 months caused reductions of ductal structures in young mice, which formed after the addition of estrogen and in the presence of 4-OHE2. (1) At the beginning of the experiment the left mammary gland, #4 was removed and whole mounted. (2) Mice were ovariectomized and after one month, the right gland #5 was removed and whole mounted. (3) E2 implants were placed into the back of the mice to deliver 130 pg/ml E2 for one month after which the right mammary gland #3 was removed and whole mounted. (4) Mice were given 4-OHE2 and 30 mg TMS implants for one month and then the left mammary gland #3 was removed and whole mounted.

Supplemental Fig. 3. Evaluation of additional mouse organs after treatment with high dose, 50 mg/kg, TMS. Photomicrographs of hematoxylin and eosin stained formalin fixed, paraffin embedded slides of aromatase mouse organs following control injection or injection with 50 mg/kg TMS for eight weeks.