17-β-Estradiol (E2) and 4-OH-Tamoxifen (Tam) were purchased from Sigma Biochemicals (St. Louis, MO). β-hexachlorocyclohexane (β-HCH) was purchased from Chem Service (Westchester, PA). E2 and pesticides were kept as high concentration stock solutions in ethanol. Human recombinant EGF other media supplies were obtained from Gibco BRL (Gaithersburg, MD).
Source and Maintenance
MCF10AT1 cells were obtained from the Michigan Cancer Foundation and maintained in phenol red free Dulbecco's modified Eagle's medium with F12 nutrient mixtures (DMEM/F12; Gibco, BRL) and supplemented with 2.5% heat-treated equine serum, 20 ng/mL EGF, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37°C under 5% CO2 conditions.
For each subline, cells were grown in culture media containing either E2 (1 nM); Tam (1 nM), β-HCH (1 μM), or just the 0.1% ethanol vehicle (control). Media and chemicals were refreshed twice a week and the plates were reseeded when plates reached 80–90 percent confluency (on average every 10–14 days). For each treatment, three separate plates were prepared and passaged independently. Cell passaging was accomplished by trypsinzation and reseeding of 1 × 106 cells (approximately 2.5% of a near confluent plate) into a 100 mm plate containing the above media and chemicals. For the quantification of our long-term studies, each reseeding event constituted one passage.
Fixation and staining of selected MCF10AT1 cells
In a six-well plate, 1,000,000 cells from the selected sublines were seeded (without selection chemicals). After reaching confluence (normally 3–4 days), media was refreshed twice (again without selection chemicals) for an additional week. Afterwards, cells were fixed by 10% Formalin (in PBS) for 30 minutes and stained with Giemsa for 1 minute. After which plates were washed of extra dye solution with tap water repeatedly. Cells were the observed under a compound microscope for morphology.
Proliferation test assay
After 20 passages in our test chemicals, cells were reseeded for cell proliferation assays. Cells were trypsinized from a starter plate and seeded at a concentration of approximately 100,000 cells per well in a 12 well plate (Hemocytometer counting) or 10,000 per well in a 96 well plate (Promega-AQueous one cell proliferation assay). In most cases, 2 samples from an independent triplicate of a similarly treated selected cell line were chosen (i.e. N = 6). It must be noted that none of the selection chemicals (i.e. E2, Tam or β-HCH) were added to the cell culture media used during this assay. At 24, 48 and 72 hours after plating, cells were trypsinized (hemocytometer counting) or analyzed directly for MTS reduction (AQueous assay). Trypan blue exclusion (0.08% dye) was used to determine viable cells (normally >96% of total cells).
mRNA analysis of gene expression
Cells that have been selected for 20 passages in our test chemicals were trypsinized and reseeded (500,000) into 60 mm dishes in our culture media. Note: no additional selection chemicals (i.e. E2, Tamoxifen, β-HCH) were added to the media during this analysis. After 24 hours, the media was refreshed. After an additional 24 hours, mRNA was extracted from the cells using the Qiagen (Valencia, CA) RNAeasy kit. cDNA was prepared from the extracted mRNA using the Omniscript Reverse Transcriptase Kit (Qiagen) and analyzed by real-time PCR on a Roche lightcycler.
Real-time PCR was performed on a Roche Molecular biochemical "Light Cycler". Samples were prepared using the Light cycler DNA Master SYBR green I kit. After sample preparation, cDNA was analyzed with the following primer pairs:
c-Neu : 5'-AGC TGG TGA CAC AGC TTA-3' FP 
5'-TGG TTG GGA CTC TTG AC-3' RP
ERα 5' CAA GCC CGC TCA TGA TCA A-3' FP
(228 bp) 5'-CTG ATC ATG GAG GGT CAA ATC CAC-3' RP
Cyclin D1 5' GGA TGC TGG AGG TCT GCG A-3' FP
(146 bp) 5'-AGA GGC CAC GAA CAT GCA AG-3' RP
p21 5'-ATT AGC AGC GGA ACA AAG AGT CAG ACA T-3' FP
(318 bp) 5'-CTG TGA AAG ACA CAG AAC AGT ACA GGG T-3' RP
p27 5'-AAC GTG CGA GTG TCT AAC GG-3' FP
(164 bp) 5'-CTT CCA TGT CTC TGC AGT GC-3' RP
VEGF 5'-AAG GAG GAG GGC AGA ATC AT-3' FP
(226 bp) 5'-ATC TGC ATG GTG ATG TTG GA-3' RP
IGF-IR 5'-ACA GAG AAC CCC AAG ACT GAG G-3' FP
(285 bp) 5'-TGA TGT TGT AGG TGT CTG CGG C-3' RP
c-met 5'-CAG GCA GTG CAG CAT GTA GT-3' FP
(201 bp) 5'-GAT GAT TCC CTC GGT CAG AA-3' RP
CK19 5'-CTA CAG CCA CTA CTA CAC GAC-3' FP
(148 bp) 5'-CAG AGC CTG TTC CGT CTC AAA-3' RP
MMP-12 5'-ACA CAT TTC GCC TCT CTG CT-3' FP
(192 bp) 5'-CCT TCA GCC AGA AGA ACC TG-3' RP
MMP-13 5'-AAC ATC CAA AAA CGC CAG AC-3' FP
(166 bp) 5'-GGA AGT TCT GGC CAA AAT GA-3' RP
NFκB 5'-CAC TTA TGG ACA ACT ATG AGG TCT CTG G-3' FP
(406 bp) 5'-CTG TCT TGT GGA CAA CGC AGT GGA ATT TTA GG-3' RP
β-Actin 5'-GGA CTT CGA GCA AGA GAT GG-3' FP
(234 bp) 5'-AGCACT GTG TTG GCT TAC AG-3' RP
The PCR cycling conditions were 95°C for 15 s, 60°C for 20 seconds and 72°C for 10 seconds
MMTV mice treatments
MMTV-neu mice were obtained from Jackson Laboratories (West Sacramento, CA) and separated into control and β-HCH treated groups. Each group contained 37–39 virgin female mice. Mice were injected with 33 mg/kg β-HCH (in corn oil:acetone; 90:10 mixture) or just the vehicle solution at 6, 10, 14 and 18 weeks. Mice were housed at normal temperature, food, and water conditions and examined twice weekly for tumor formation in the mammary regions. First palpable tumor time was used to calculate tumor latency in animals.
For proliferation and mRNA expression experiments
Several independently prepared batches of cells for each treatment group were assessed simultaneously to obtain means and standard deviations among different batches (i.e. "N" values shown in figure captions). ANOVA and Fisher's least-significant differences test (SYSTAT 11 Computer Software) were used to determine the significance of difference between control and chemically treated groups. A probability value of less than 0.05 was regarded as significant. Error bars in the figures indicate standard deviations from the mean.
For MMTV mouse tumor studies
The tumor free interval for mice in the β-HCH and corn-oil only treat groups were compared using the Cox proportional hazard model and the log rank test for comparison of significance between survival curves (MedCalc computer software).