Chemicals and antibodies. BPA was purchased from Sigma Chemical Company (St. Louis, MO). Antibodies to epidermal growth factor receptor (EGFR), erbB2, phosphorylatederbB3, phosphorylatederbB3, insulin-like growth factor 1 receptor (IGF-1R), phosphorylated-IGF-1R, phosphorylated-Bad, PI3K, PTEN, Akt 1, Akt 3, phosphorylatedAkt, glycogen synthase kinase-3-beta (GSK-3β), and phosphorylatedGSK-3β were purchased from Cell Signaling Technologies (Danvers, MA). Antibodies to erbB3 and IGF-1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
Animal care and use. Animal care and use were conducted according to established guidelines approved by the Institutional Animal Care and Use Committee at the University of Alabama at Birmingham. All animals were housed in a temperature-controlled facility with a 12-hr light/dark cycle. All animals were treated humanely and with regard for alleviation of suffering. A colony of mouse mammary tumor virus (MMTV)-erbB2/neu transgenic mice [FVB/N-TgN(MMTV-neu202Mul)] was established through breeding pairs purchased from Jackson Laboratory (Bar Harbor, ME). Because progesterone and pregnancy have been reported to drive the MMTV promoter, only virgin female mice were used. Animals were fed AIN-76A diet (phytoestrogen-free; Dyets, Inc., Bethlehem, PA), housed in polypropylene cages, and provided glass water bottles.
Female MMTV-erbB2 mice were exposed to BPA via the drinking water, beginning at 56 days of age and continuing for the lifetime of the animals (252 days of age). Cagen et al. (1999)
and our own preliminary data have shown BPA to be stable in water for 1 week. Water with and without BPA was supplied fresh weekly. The following treatment groups were set up: 0 (control) and 2.5 (BPA2.5), 25 (BPA25), 250 (BPA250), and 2,500 (BPA2500) µg BPA/L drinking water (). Drinking water of all groups, including the control group, contained 0.05% by volume of the vehicle, ethanol.
Figure 1 Beginning at 56 days of age, female MMTV-erbB2 mice were provided 0, 2.5, 25, 250, or 2,500 µg BPA/L drinking water. Estimated daily intakes were calculated based on a 20-g mouse drinking 4 mL water daily. This is based on our preliminary study, (more ...)
All animals were palpated twice weekly. Mice were sacrificed at 252 days of age or when tumors exceeded 10% BW. At sacrifice, animals underwent complete necropsy. All tumors, gross lesions, and lung sections were dissected and blocked in paraffin. Tumor volume was calculated by measuring the length, width, and height of the dissected tumor. All dissected tumors were pathologically graded according to the consensus statement of the Annapolis meeting of medical and veterinary pathologists (Cardiff et al. 2000
). Only those tumors classified as invasive mammary adenocarcinoma were used in the final analysis of tumor latency (time to first, second, and third tumor), volume, and multiplicity. Quantification of lung metastases was determined through inflating the lungs with an India ink solution [15% vol/vol India ink, 0.5% (vol/vol) ammonia solution]. Excised lesions as well as whole-lung sections were evaluated. All pathological analyses were carried out under blinded conditions by a board-certified pathologist (I.E).
Cell proliferation index. We selected 112 days of age as the end point for mechanistic studies; at this age, no preneoplastic/neoplastic lesions were identified in the mammary glands of untreated mice in a preliminary ontogeny study (data not shown). At 112 days of age, the number four abdominal mammary glands were collected from MMTV-erbB2 mice treated with 0, 2.5, 25, 250, and 2,500 µg BPA/L drinking water. Tissue sections were deparaffinized and rehydrated through xylene and graded alcohol washes. Slides were boiled in citrate buffer, incubated in hydrogen peroxide, and blocked using serum. The slides were incubated in Ki-67 primary antibody (Dako, Glostrup, Denmark) overnight in a humidified chamber. After incubating the tissue sections in the appropriate conjugated secondary antibody, we employed the ImmPRESS kit (Vector Laboratories, Burlingame, CA). Positively stained cells were visualized by incubating the tissue sections with 3,3´-diamonobenzidine (DAB) and counterstained with hematoxylin. Tissue sections were dehydrated with graded alcohols, cleared with xylene, and mounted with a glass coverslip. A minimum of 500 mammary epithelial cells were counted from at least five different structures. All counts were performed in duplicate under blinded conditions, with the final index for each section being a mean of the duplicate counts.
Apoptotic index. The index of apoptosis was determined through the use of the ApopTag Plus Peroxidase In Situ Apoptosis Detection kit (Chemicon International, Billerica, MA) according to the manufacturer’s protocol. Cells that stained positively and exhibited morphologic characteristics of apoptosis were counted as positive. A minimum of 500 mammary epithelial cells were counted from at least five different structures. All counts were performed in duplicate under blinded conditions, with the final index for each section being a mean of the duplicate counts. A cell proliferation to apoptosis ratio was calculated for each individual gland.
Immunoblotting. Mammary glands were homogenized in RIPA lysis buffer (Pierce Biotechnolgy, Rockford, IL) with the use of a pestle and grinding kit (GE Healthcare Inc., Piscataway, NJ). Protein lysate was quantified using the Bradford assay (BioRad Laboratories, Hercules, CA). Equal protein content (40 μg) was loaded onto precast SDS Tris-HCl 4–20% polyacrylamide gels (BioRad Laboratories). Proteins were transferred to a nitrocellulose membrane overnight. The membrane was blocked and incubated in primary antibody. Secondary antibody and chemilume (Pierce Biotechnology) were added, and protein expression was visualized using film. Densitometry was assessed using Quantity One (BioRad Laboratories). Positive protein controls purchased from the supplier of the corresponding antibodies and Kaleidoscope Precision Plus Protein standards (BioRad Laboratories) were employed to identify the protein of interest.
The statistical method for evaluating tumor latency was performed as previously described (Betancourt et al. 2010
). Tumor multiplicity was analyzed using the Cochran–Armitage test. The incidence of pulmonary metastasis was compared using Fisher’s exact test. Normally distributed data were analyzed using analysis of variance followed by Bonferroni’s multiple comparison test (MCT). Data not normally distributed were analyzed using the Kruskal–Wallis test followed by Dunn’s MCT.