Summary Data for T47D-Kbluc Dose-Response Curves in Figure 2A

Figure 1. Names, acronyms, structures, and Chemical Abstracts Service (CAS) numbers of test chemicals.

Figure 2. Individual chemical in vitro (T47D-KBluc assay, panel a) and in vivo (ovariectomized rat uterotrophic, oral administration, panel b) dose response curves. Symbols and error bars represent mean ± standard error, dashed horizontal line indicates zero baseline. Data file for this figure follows on the next two pages.

Table 1.In vitro (T47D-KBluc) dose response curve parameters (slope, EC50 (M)) and relative potency factors (RPF; using EE2 as reference compound) for eight estrogenic compounds.

Chemical / Slope ± SE / EC50 (95% CI) / RPF
EE2 / 1.81 ± 0.19 / 9.98e-13 (8.81e-13 – 1.13e-12) / 1.00
E2 / 1.46 ± 0.09 / 1.68e-12 (1.52e-12 – 1.86e-12) / 0.594
BPC / 1.46 ± 0.11 / 5.71e-09 (5.03e-09 – 6.48e-09) / 0.000175
BPAF / 1.40 ± 0.11 / 2.06e-08 (1.81e-08 – 2.35e-08) / 0.0000485
BPA / 1.61 ± 0.18 / 1.27e-07 (1.08e-07 – 1.50e-07) / 0.00000784
BPS / 1.93 ± 0.27 / 6.43e-07 (5.30e-07 – 7.80e-07) / 0.00000155
BBP / 4.07 ± 0.87 / 1.31e-06 (1.12e-06 – 1.53e-06) / 0.000000763
MET / 2.49 ± 0.38 / 1.47e-06 (1.26e-06 – 1.72e-06) / 0.000000679
E2 / BPA / BPS / BPC / BPAF / MET / BBP / EE2
Log molar / Mean / SD / n / Mean / SD / n / Mean / SD / n / Mean / SD / n / Mean / SD / n / Mean / SD / n / Mean / SD / n / Mean / SD / n
-15.000 / 3.436 / 3.763 / 6
-14.523 / -1.206 / 3.367 / 6
-14.000 / -5.835 / 5.255 / 6
-13.523 / -9.476 / 9.991 / 35 / -9.790 / 2.923 / 6
-13.000 / 1.726 / 20.900 / 38 / -5.997 / 2.266 / 6
-12.523 / 8.335 / 10.276 / 44 / 12.177 / 4.894 / 6
-12.000 / 32.711 / 20.704 / 45 / 48.165 / 11.308 / 6
-11.523 / 68.899 / 17.844 / 45 / 82.708 / 7.827 / 6
-11.000 / 96.374 / 4.235 / 38 / 98.229 / 4.939 / 6
-10.523 / 100.074 / 4.313 / 41 / 96.174 / 5.183 / 6
-10.000 / 99.581 / 4.153 / 36 / -0.625 / 10.965 / 10 / 93.756 / 3.308 / 6
-9.523 / 98.286 / 4.381 / 28 / 3.800 / 10.389 / 10 / -2.197 / 11.123 / 6 / 93.826 / 0.272 / 2
-9.000 / 6.846 / 11.004 / 11 / 0.341 / 11.891 / 13
-8.523 / 33.781 / 9.563 / 11 / 3.064 / 8.047 / 13
-8.000 / -1.608 / 15.141 / 18 / 81.935 / 5.053 / 11 / 32.197 / 8.235 / 13 / -9.106 / 7.273 / 6
-7.523 / 9.214 / 16.721 / 18 / -8.148 / 16.957 / 17 / 107.990 / 5.225 / 11 / 68.375 / 10.655 / 13 / -4.818 / 3.812 / 4 / -9.246 / 9.372 / 5
-7.000 / 44.298 / 15.183 / 18 / -2.021 / 12.460 / 17 / 117.168 / 7.687 / 11 / 98.725 / 2.776 / 13 / -3.484 / 8.526 / 12 / -10.370 / 10.197 / 6
-6.523 / 84.232 / 10.810 / 18 / 18.940 / 10.673 / 17 / 116.425 / 5.979 / 3 / 109.206 / 4.810 / 9 / -1.615 / 6.861 / 12 / -8.784 / 5.360 / 6
-6.000 / 104.243 / 14.925 / 10 / 60.718 / 15.209 / 17 / 109.708 / 6.049 / 6 / 22.134 / 10.853 / 12 / 25.995 / 7.366 / 6
-5.699 / 32.608 / 5.842 / 4 / 86.187 / 6.538 / 6
-5.523 / 106.374 / 9.296 / 4 / 86.523 / 10.148 / 16 / 66.267 / 9.176 / 12 / 98.950 / 2.034 / 6
-5.222 / 65.831 / 10.438 / 8
-5.000 / 106.883 / 2.773 / 8 / 86.456 / 15.505 / 11 / 69.637 / 7.542 / 12

Summary data for T47D-KBluc dose-response curves in Figure 2a

Uterotrophic assay body mass and tissue mass (mean±SEM) for single chemical, oral gavage exposures to ovariectomized rats (*denotes significant difference from 0 dose control, p<0.05). Summary data for Figure 2b.

Chemical / Dose
(mg/kg/d) / Sample size
(# rats) / Initial body weight (g) / Body weight gain (g)a / Uterus wet
(mg) / Uterus blotted
(mg) / Uterine fluid
(mg)
E2 / (+) control / 8 / 306.3±16.4 / 1.7±2.3* / 286.1±51.7* / 158.9±11.2* / 131.2±40.4*
0 / 8 / 312.8±16.2 / 11.7±2.6 / 41.7±5.2 / 35.7±4.9 / 6.0±1.0
0.001 / 5 / 273.8±6.6 / 15.4±2.2 / 43.7±4.2 / 38.0±3.6 / 5.7±1.8
0.003 / 5 / 278.3±4.9 / 15.1±1.3 / 42.9±3.3 / 36.8±3.4 / 6.0±1.0
0.01 / 5 / 268.9±8.5 / 14.9±2.2 / 48.7±7.5 / 42.0±6.6 / 6.6±1.2
0.03 / 5 / 263.7±7.7 / 13.0±1.8 / 66.7±2.4* / 59.1±2.5* / 7.6±0.9
0.1 / 7 / 294.4±17.2 / 7.9±2.6 / 81.5±9.4* / 70.0±6.9* / 11.6±2.6
0.3 / 7 / 280.6±21.8 / 1.1±2.9* / 131.0±10.6* / 106.5±6.9* / 24.5±4.3*
1 / 5 / 367.1±10.4 / -11.3±1.3* / 241.3±34.9* / 159.1±15.2* / 82.2±21.5*
3 / 5 / 356.5±11.2 / -11.5±1.6* / 234.6±18.4* / 150.1±9.0* / 84.5±10.2*
BBP / (+) control / 4 / 311.0±14.5 / -5.8±1.8* / 135.4±8.0* / 104.1±5.4* / 31.3±8.4*
0 / 4 / 326.4±2.7 / 5.4±1.0 / 31.6±3.8 / 27.1±3.2 / 4.4±0.9
400 / 4 / 341.7±10.8 / 5.5±0.7 / 31.7±2.6 / 25.9±1.9 / 5.9±0.9
600 / 4 / 348.0±9.6 / 4.6±1.7 / 34.8±2.2 / 31.0±1.7 / 3.8±0.9
800 / 4 / 347.8±9.3 / 3.7±1.6 / 32.5±1.8 / 26.9±2.1 / 5.7±0.5
BPA / (+) control / 5 / 362.7±13.1 / -7.6±2.3* / 238.0±34.7* / 153.3±5.8* / 84.7±30.6*
0 / 5 / 380.2±13.1 / 2.9±1.7 / 39.1±5.2 / 34.2±4.5 / 4.9±0.7
200 / 6 / 342.6±15.4 / -0.8±1.8 / 43.0±6.3 / 37.1±5.3 / 5.9±1.1
400 / 5 / 353.2±15.1 / -2.5±2.7 / 58.3±1.5 / 50.9±0.9 / 7.4±1.0
600 / 3 / 353.7±6.2 / -5.6±2.7* / 58.9±6.7 / 50.4±5.0 / 8.5±1.9
800 / 6 / 370.5±22.0 / -3.7±3.7 / 81.3±20.9* / 65.6±13.8* / 15.8±7.4
BPAF / (+) control / 6 / 322.9±3.1 / -29.3±3.1* / 974.9±65.6* / 341.3±17.9* / 633.6±66.6*
0 / 4 / 335.6±2.3 / 1.6±2.4 / 118.2±10.1 / 109.6±9.6 / 1.0±1.0
3.125 / 3 / 322.6±5.5 / -0.6±2.2 / 138.6±13.3 / 124.1±8.4 / 14.5±6.2
6.25 / 3 / 321.1±2.4 / 2.3±2.4 / 124.1±19.8 / 113.0±16.2 / 11.1±3.6
12.5 / 3 / 318.0±6.1 / -0.7±4.8 / 141.2±19.4 / 130.6±17.9 / 10.5±2.1
25 / 6 / 322.1±5.8 / -1.9±1.9 / 155.0±16.4 / 133.6±13.6 / 21.3±5.1
50 / 6 / 318.9±5.0 / -13.0±7.3 / 261.3±65.7* / 159.7±32.5 / 101.6±42.1
100 / 6 / 319.9±2.2 / -11.4±1.7 / 219.1±32.5* / 179.7±14.0* / 39.4±19.6
300 / 6 / 331.8±5.3 / -27.7±5.1* / 323.3±58.7* / 230.4±15.2* / 92.9±47.6
BPC / (+) control / 6 / 338.2±40.4 / -2.1±3.7 / 327.4±41.6* / 163.4±11.0* / 164.0±31.6*
0 / 6 / 304.3±25.4 / 6.2±2.8 / 42.7±4.7 / 35.9±4.0 / 6.8±0.9
1.2 / 3 / 252.6±9.3 / 14.7±1.0 / 31.7±5.0 / 27.3±4.3 / 4.4±1.2
4 / 6 / 305.3±22.9 / 7.3±3.9 / 34.0±4.5 / 30.6±3.5 / 3.5±1.2
13.5 / 6 / 338.0±36.9 / 4.8±5.0 / 47.3±4.9 / 40.8±3.9 / 6.5±1.1
45 / 6 / 327.8±40.4 / 1.4±5.9 / 113.3±14.0* / 93.1±10.0* / 20.2±4.2*
150 / 6 / 328.9±33.3 / 0.1±3.4 / 148.9±12.0* / 122.0±5.6* / 26.8±6.7*
500 / 6 / 327.9±33.7 / -2.7±3.4 / 176.1±12.1* / 134.7±9.8* / 41.3±7.9*

Uterotrophic assay body mass and tissue mass (mean±SEM) for single chemical, oral gavage exposures to ovariectomized rats (*denotes significant difference from 0 dose control, p<0.05).Summary data for Figure 2b.

Chemical / Dose
(mg/kg/d) / Sample size
(# rats) / Initial body weight (g) / Body weight
gain (g)a / Uterus wet
(mg) / Uterus blotted
(mg) / Uterine fluid
(mg)
BPS / (+) control / 6 / 338.2±9.9 / -10.4±1.4* / 287.3±24.9* / 162.4±7.8* / 124.9±22.8*
0 / 6 / 342.6±15.1 / 0.9±1.3 / 31.6±2.9 / 25.4±3.0 / 6.3±0.4
50 / 6 / 364.5±8.2 / -0.5±0.6 / 55.0±6.9* / 44.9±5.3* / 10.1±1.9
100 / 6 / 359.8±16.8 / -0.7±1.3 / 63.0±7.1* / 54.8±6.0* / 8.2±1.5
200 / 6 / 357.1±12.8 / -3.5±2.4 / 77.2±8.6* / 64.9±7.7* / 2.3±1.9
400 / 5 / 369.4±18.0 / -2.8±2.8 / 139.5±36.8* / 107.2±17.5* / 32.3±19.7*
800 / 6 / 376.2±13.2 / -4.2±1.3 / 156.7±13.4* / 121.4±7.6* / 35.3±6.8*
EE2 / (+) control / 3 / 348.7±7.2 / -13.3±1.0 / 1026.6±50.0* / 61.3±19.0* / 665.3±36.8*
0 / 3 / 344.7±17.5 / -1.0±1.3 / 103.0±7.8 / 89.8±7.1 / 13.2±2.3
0.0005 / 3 / 318.6±7.3 / -9.7±5.4 / 118.4±14.5 / 107.9±12.7 / 10.6±2.2
0.001 / 3 / 350.9±15.4 / -2.6±2.7 / 115.9±10.0 / 107.9±9.1 / 8.0±1.0
0.0025 / 3 / 356.2±4.7 / -5.4±1.5 / 142.3±37.0 / 128.7±35.6 / 13.6±2.3
0.005 / 3 / 345.7±2.5 / -6.0±1.6 / 132.3±17.4 / 118.8±15.2 / 13.5±3.1
0.01 / 3 / 353.9±3.2 / -12.3±6.5 / 244.5±64.6* / 175.3±12.1* / 69.2±52.6
0.05 / 2 / 313.5±19.2 / -8.5±3.6 / 526.6±24.4* / 206.2±4.9* / 320.5±9.6*
0.2 / 3 / 323.8±1.8 / -13.5±0.6 / 874.4±120.0* / 309.2±21.5* / 565.2±106.2*
MET / (+) control / 4 / 307.2±7.0 / -8.1±0.3* / 1107.2±236.5* / 395.8±39.6* / 711.4±207.7*
0 / 3 / 311.9±0.1 / 0.2±0.7 / 91.5±10.5 / 83.4±6.2 / 8.1±4.4
62.5 / 4 / 319.3±5.4 / 0.1±1.7 / 202.0±12.4* / 168.2±10.2* / 33.8±2.7*
125 / 4 / 316.8±2.5 / -1.5±0.2 / 319.7±37.7* / 229.4±22.8* / 90.3±26.9*
250 / 4 / 314.7±19.6 / -7.4±0.8* / 478.3±50.6* / 316.4±44.8* / 161.9±49.8*
500 / 4 / 310.1±6.7 / -9.7±2.0* / 574.2±62.1* / 307.0±9.0* / 267.2±67.9*

Table 2.In vivo (ovariectomized rat uterotrophic assay, oral gavage) dose response curve parameters (slope, ED50 (mg kg-1 d-1)) and relative potency factors (RPF; using EE2 as reference compound) across three endpoints (uterus blotted weight, uterus wet weight, and uterine fluid weight) for seven estrogenic compounds.

Blotted / Wet / Fluid
Chem / Slope ± SE / ED50
(95% CI) / RPF / Slope ± SE / ED50
(95% CI) / RPF / Slope ± SE / ED50
(95% CI) / RPF
EE2 / 0.69
± 0.14 / 0.0179
(0.00922 – 0.0346) / 1.00 / 1.08
± 0.17 / 0.0208
(0.0138 – 0.0315) / 1.00 / 1.89
± 0.56 / 0.0216
(0.0123 – 0.0379) / 1.00
E2 / 1.09
± 0.17 / 0.103
(0.0756 – 0.138) / 0.173 / 1.17
± 0.18 / 0.166
(0.127 – 0.216) / 0.126 / 1.48
± 0.29 / 0.249
(0.190 – 0.328) / 0.0866
BPC / 1.70
± 0.34 / 39.8
(30.6 – 51.7) / 0.000449 / 0.92
± 0.15 / 95.4
(66.1 – 137.7) / 0.000218 / 0.85
± 0.25 / 286.8
(148.7 – 553.3) / 0.0000753
BPAF / 0.75
± 0.19 / 209.1
(111.0 – 394.0) / 0.0000854 / 0.62
± 0.15 / 465.2
(198.0 – 1093.0) / 0.0000448 / 0.47
± 0.26 / 872.9
(49.7 – 15346.0) / 0.0000247
BPA / 1.40
± 0.70 / 1118.0
(529.1 – 2360.0) / 0.0000160 / 1.34
± 0.72 / 1286.0
(504.8 – 3277.0) / 0.0000162 / 1.26
± 1.13 / 1737.0
(225.0 – 13418.0) / 0.0000124
BPS / 0.91
± 0.14 / 150.0
(113.8 – 197.5) / 0.000119 / 0.83
± 0.13 / 259.2
(194.2 – 345.9) / 0.0000804 / 1.05
± 0.28 / 676.1
(412.7 – 1108.0) / 0.0000319
MET / 1.08
± 0.22 / 71.4
(49.6 – 102.8) / 0.000250 / 0.90
± 0.13 / 126.7
(98.9 – 162.3) / 0.000164 / 0.83
± 0.20 / 104.4
(67.9 – 160.5) / 0.000207

No values reported for BBP because it was negative in the oral uterotrophic assay

Table 3. Predicted ED50 (mg kg-1 d-1) values for individual chemicals in the ovariectomized rat uterotrophic assay across three endpoints (uterus blotted weight, uterus wet weight, and uterine fluid weight). Predictions were calculated using in vitro (T47D-KBluc) relative potency factors with EE2 as the reference compound. Predicted to observed ratios (P:O) indicate accuracy of prediction (P:O = 1.0 indicates perfect prediction).

Blotted / Wet / Fluid
Chem. / Predicted
ED50 / P:O / Predicted
ED50 / P:O / Predicted
ED50 / P:O
EE2 / - / - / - / - / - / -
E2 / 0.030 / 0.3 / 0.035 / 0.2 / 0.036 / 0.1
BPC / 102.1 / 2.6 / 119.1 / 1.2 / 123.5 / 0.4
BPAF / 368.3 / 1.8 / 429.8 / 0.9 / 445.5 / 0.5
BPA / 2276.2 / 2.0 / 2656.2 / 2.1 / 2753.1 / 1.6
BPS / 11497.1 / 76.6 / 13416.5 / 51.8 / 13906.0 / 20.6
BBP / 23387.6 / - / 27292.0 / - / 28287.8 / -
MET / 26284.2 / 368.3 / 30672.2 / 242.1 / 31791.3 / 304.5

Figure 3. Predicted (black dashed lines) versus observed (grey circles and black solid lines except panel b where E2 is represented by grey squares) dose response curves for uterus blotted weight in the ovariectomized rat uterotrophic assay (oral gavage). Predicted lines were generated using in vitro (T47D-KBluc) relative potency factors with 17α-ethinyl estradiol as a reference compound. Panel (a) includes additional data estimated from prior studies on the uterotrophic response to BBP (*data from Brady et al. 2000 represents uterine wet weight).

Figure 4. Observed data (gray circles) versus dose addition (DA) models of binary (BPAF+MET; BPS+MET) and multi-chemical (BPAF+BPC+BPS+EE2+MET) mixtures in the ovariectomized rat uterotrophic assay (oral gavage). DA models were generated using individual compound in vitro (T74D-Kbluc, black dashed lines) and in vivo (black solid lines) response data. Observed data represent mean ± 95% CI; statistical significance based on model predictions overlapping the observed 95% CI. Data file follows on the next page

Uterotrophic assay body mass and tissue mass (mean±SEM) for binary and multi-chemical mixtures via oral gavage exposures to ovariectomized rats (*denotes significant difference from 0 dose control, p<0.05).Summary data for Figure 4.

Chemical / Dose
(mg/kg/d) / Sample size
(# rats) / Initial body weight (g) / Body weight
gain (g)a / Uterus wet
(mg) / Uterus blotted
(mg) / Uterine fluid
(mg)
BPAF+MET / (+) control / 10 / 336.5±9.2 / -14.8±1.0* / 935.2±71.2* / 333.3±28.9* / 593.0±51.5*
0 / 9 / 338.8±7.9 / -2.6±1.5 / 100.4±6.8 / 85.1±4.7 / 15.2±3.2
62.5 / 8 / 342.5±4.1 / -3.4±1.4 / 179.4±11.5* / 159.3±10.0* / 20.1±4.0
125 / 8 / 320.1±8.8 / -7.6±3.5 / 229.5±13.3* / 193.1±13.2* / 36.4±7.7*
250 / 9 / 337.2±5.0 / -7.4±1.1 / 316.1±17.8* / 210.1±7.2* / 102.5±15.4*
375 / 9 / 326.1±8.2 / -20.8±3.0* / 410.7±47.8* / 227.4±13.9* / 176.2±42.9*
500 / 8 / 336.5±8.5 / -26.7±3.8* / 669.3±47.0* / 244.8±16.6* / 415.5±53.5*
BPS+MET / (+) control / 5 / 355.5±9.0 / -6.9±1.0* / 167±10.6* / 127.2±5.9* / 39.8±6.2*
0 / 5 / 341.4±10.0 / 7.1±1.7 / 31.6±2.7 / 26.6±2.2 / 5.0±0.6
15 / 5 / 346.4±12.3 / 3.1±1.3 / 27.8±2.0 / 24.4±1.6 / 3.4±1.1
45 / 5 / 331.6±3.7 / 2.1±1.1 / 39.5±2.2 / 32.9±2.0 / 6.6±0.4
135 / 6 / 366.7±7.1 / -1.4±2.0* / 65.8±4.2* / 57.3±3.5* / 8.4±0.8
405 / 6 / 371.8±5.2 / -0.3±1.1* / 97.9±10.7* / 82.5±8.1* / 15.4±3.0*
1000 / 5 / 349.9±4.2 / -4.8±3.5* / 145.4±11.2* / 119.3±10.2* / 26.1±2.9*
BPAF+BPC+
BPS+EE2+MET / (+) control / 5 / 376.0±10.0 / -6.5±2.2* / 245.2±29.7* / 156.4±8.9* / 88.7±27.1*
0 / 4 / 356.1±9.9 / 3.8±0.9 / 37.8±1.5 / 33.1±2.1 / 4.7±0.7
29 / 5 / 326.3±7.0 / 2.2±0.8 / 43.6±3.6 / 38.2±2.9 / 5.4±0.9
58 / 5 / 362.2±13.1 / -2.9±2.5 / 81.3±4.2* / 71.5±4.0* / 9.8±1.1
117 / 5 / 331.9±9.3 / -5.8±2.2* / 92.6±4.3* / 79.2±3.0* / 13.3±1.8*
235 / 4 / 323.1±7.6 / -6.1±1.9* / 114.7±19.3* / 96.7±16.1* / 18.0±3.4*
470 / 5 / 370.7±13.1 / -3.3±3.0 / 156.0±10.4* / 122.9±9.1* / 33.0±3.9*

1

Observed and predicted (Pred.) ED50 values (mg kg-1 d-1) for binary (BPAF+MET, BPS+MET) and multi-chemical (BPAF+BPC+BPS+EE2+MET) mixtures in the ovariectomized rat uterotrophic assay across three endpoints (blotted uterus weight, wet uterus weight, uterine fluid weight). Predicted values were calculated using individual chemical response data from in vivo (uterotrophic) and in vitro (T47D-KBluc) experiments and the dose addition model. Predicted to observed ratios (P:O) indicate the accuracy of the prediction (P:O=1 represents perfect prediction).

Blotted / Wet / Fluid
Mix. / Observed
ED50
(95% CI) / In vivo / In vitro / Observed
ED50
(95% CI) / In vivo / In vitro / Observed
ED50
(95% CI) / In vivo / In vitro
Pred. / P:O / Pred. / P:O / Pred. / P:O / Pred. / P:O / Pred. / P:O / Pred. / P:O
BPAF+MET / 79.3
(54.7-115.0) / 118.0 / 1.5 / 709.8 / 7.7 / 197.4
(172.8-225.5) / 224.9 / 1.1 / 608.2 / 3.6 / 249.4
(213.0-291.9) / 221.3 / 0.9 / 735.6 / 2.9
BPS+
MET / 162.6
(128.5-205.7) / 110.1 / 0.7 / 14108.0 / 86.8 / 191.6
(151.0-243.1) / 192.9 / 1.0 / 16463.0 / 85.9 / 337.6
(243.8-467.5) / 241.3 / 0.7 / 17064.0 / 50.5
BPAF+
BPC+
BPS+
EE2+
MET / 95.5
(72.1-126.4) / 93.2 / 1.0 / 237.3 / 2.5 / 135.7
(104.0-176.9) / 164.1 / 1.2 / 278.0 / 2.0 / 211.5
(161.0-277.9) / 222.8 / 1.1 / 288.2 / 1.4

1

In vitro gene expression methods with data sheets following this

T47D-KBluc gene expression

T47D-KBluc cells were harvested in duplicate from maintenance media (T47DS-1 and T47DS-2; see Materials and Methods for media composition), withdrawal media (T47DD-1 and T47DD-2; see Materials and Methods for media composition), and following ~24hr exposure to withdrawal media containing 30 pM 17β-estradiol (E2-1 and E2-2), 300 pM 1,3,5-tris(4-hydroxyphenyl)-4-propyl-1H pyrazole (PPT-1 and PPT-2; ERα agonist, CAS# 263717-53-9), or 30 nM 2,3-bis(4-hydroxyphenyl) propionitrile (DPN-1 and DPN-2; ERβ agonist, CAS# 1428-67-7). Cells were homogenized, mRNA was extracted and converted to cDNA and run on Qiagen Breast Cancer qPCR plates (CAT# 330231 PAHS-131A) in a BioRadiCycler. Table S1 below reports the CT values with ERα (ESR1) and ERβ (ESR2) genes highlighted in bold. It is apparent from the qPCR results that ERβ mRNA was minimally expressed, even following exposure to the ERβ specific agonist DPN. CV-1 cells were also homogenized and analyzed in duplicate in order to assess the capacity of the gene arrays to discriminate between cell types.

T47D-KBluc transcriptional activation

Experiments were conducted identical to those described in the Materials and Methods. Cells were exposed to a reference compound (E2), ERα specific agonist (PPT, identified above), and ERβ specific agonist (DPN, identified above) across a range of concentrations. Luciferase activity was normalized to % of control using saturating concentrations of E2 and dose response curves were graphed using GraphPad Prism. The ERα agonist (PPT) was ~1000-fold more potent than the ERβ agonist (DPN) indicating the dominance of the ERα receptor subtype in the T47D-KBluc assay response for the culture of this cell line currently maintained in our laboratory (Figure S1). DPN is an ERβ agonist, however there is some degree of cross-reactivity with ERα.

1

Expression (CT) of genes in CV-1 cells and in T47D-KBluc cells under culture conditions and exposed to three estrogen receptor agonists with varying receptor subtype specificity.

Gene symbol / CV1-1 / CV1-2 / T47DS-1 / T47DS-2 / T47DD-1 / T47DD-2 / E2-1 / E2-2 / PPT-1 / PPT-2 / DPN-1 / DPN-2
ABCB1 / 23.3 / 22.2 / 36.8 / - / - / - / - / - / - / - / - / -
ABCG2 / - / - / 35.3 / 35.6 / 35.6 / - / 33.5 / 35.8 / 36.6 / - / 35.7 / -
ADAM23 / - / 34.6 / - / - / - / 35.9 / - / 35.8 / - / - / - / -
AKT1 / 25.9 / 24.8 / 24.1 / 24.0 / 23.8 / 22.7 / 22.8 / 23.5 / 24.1 / 24.3 / 24.6 / 23.7
APC / - / - / 29.6 / 29.8 / 28.8 / 27.7 / 28.1 / 28.9 / 29.1 / 29.6 / 29.8 / 28.9
AR / 35.9 / - / 26.1 / 26.8 / 24.9 / 24.0 / 24.7 / 25.2 / 25.0 / 24.6 / 25.7 / 24.8
ATM / 36.7 / 35.4 / 27.7 / 27.7 / 27.7 / 26.5 / 26.9 / 27.8 / 27.9 / 28.0 / 28.7 / 27.8
BAD / 29.1 / 27.9 / 26.1 / 26.2 / 25.5 / 24.4 / 24.9 / 25.7 / 26.4 / 26.1 / 26.9 / 25.8
BCL2 / 26.0 / 24.7 / 29.7 / 29.2 / 29.7 / 29.7 / 28.4 / 30.1 / 32.5 / 30.8 / 33.5 / 32.3
BIRC5 / 30.0 / 29.4 / 29.6 / 30.1 / 30.9 / 30.3 / 28.4 / 30.5 / 31.6 / 31.8 / 31.7 / 31.1
BRCA1 / 26.7 / 25.6 / 26.4 / 25.8 / 27.7 / 25.9 / 25.5 / 26.2 / 27.0 / 28.0 / 27.8 / 26.9
BRCA2 / - / - / 28.3 / 27.5 / 29.6 / 29.0 / 27.5 / 28.7 / 28.8 / 29.6 / 29.5 / 28.7
CCNA1 / 28.9 / 27.8 / 36.5 / - / - / - / - / - / - / - / - / -
CCND1 / 24.6 / 23.5 / 25.7 / 25.2 / 26.6 / 25.5 / 25.2 / 25.6 / 26.6 / 27.5 / 27.6 / 26.8
CCND2 / 34.5 / 34.0 / 34.2 / 37.1 / 35.7 / 33.6 / 35.9 / 36.1 / 36.1 / 35.8 / 35.4 / 37.0
CCNE1 / 25.9 / 24.6 / 28.1 / 28.1 / 27.9 / 27.3 / 26.3 / 27.1 / 27.9 / 28.4 / 28.6 / 27.8
CDH1 / 35.6 / 37.2 / 22.9 / 23.1 / 22.0 / 20.8 / 21.2 / 22.1 / 22.5 / 22.8 / 23.1 / 21.8
CDH13 / 27.7 / 26.2 / 29.6 / 29.7 / 29.0 / 28.2 / 28.7 / 29.5 / 29.2 / 30.4 / 30.3 / 29.2
CDK2 / 25.7 / 24.1 / 25.6 / 25.4 / 25.8 / 24.5 / 23.7 / 24.6 / 26.0 / 26.3 / 26.4 / 25.6
CDKN1A / 32.3 / 31.4 / 24.8 / 24.9 / 25.7 / 24.4 / 24.8 / 25.4 / 26.4 / 26.0 / 27.1 / 25.7
CDKN1C / 27.6 / 26.8 / 33.0 / 32.4 / 31.8 / 30.8 / 30.6 / 32.4 / 31.9 / 31.3 / 31.7 / 30.9
CDKN2A / 37.9 / - / 27.5 / 27.5 / 26.4 / 25.1 / 25.5 / 26.7 / 27.6 / 27.2 / 28.1 / 27.1
CSF1 / 25.6 / 24.6 / 30.6 / 30.5 / 32.9 / 31.2 / 30.0 / 31.4 / 31.8 / 32.2 / 32.1 / 31.3
CST6 / 36.6 / 38.0 / 35.5 / 33.1 / 30.8 / 29.6 / 28.6 / 30.9 / 32.6 / 32.2 / 32.8 / 32.1
CTNNB1 / 25.0 / 23.5 / 23.0 / 22.8 / 22.9 / 21.4 / 22.1 / 22.7 / 23.5 / 23.6 / 23.9 / 22.8
CTSD / 23.9 / 22.7 / 22.7 / 22.6 / 23.8 / 22.7 / 20.9 / 21.6 / 21.0 / 22.4 / 21.9 / 21.0
EGF / 36.7 / 34.5 / 28.0 / 28.1 / 28.8 / 27.7 / 27.8 / 28.2 / 27.1 / 27.0 / 27.8 / 26.6
EGFR / 26.2 / 25.5 / 27.1 / 27.6 / 27.1 / 26.6 / 26.3 / 27.6 / 28.6 / 28.5 / 28.7 / 28.3
ERBB2 / 27.2 / 26.0 / 26.7 / 26.7 / 24.1 / 23.4 / 23.9 / 24.7 / 25.1 / 25.4 / 25.6 / 24.6
ESR1 / 36.0 / 34.8 / 25.7 / 26.1 / 25.8 / 25.0 / 24.1 / 25.3 / 25.1 / 25.5 / 25.5 / 24.8
ESR2 / 32.7 / 31.3 / 32.7 / 32.6 / 34.6 / 33.9 / 30.6 / 31.8 / 31.9 / 35.1 / 32.0 / 33.0
FOXA1 / 28.5 / 27.5 / 24.7 / 24.5 / 24.5 / 23.4 / 24.0 / 24.5 / 25.0 / 25.0 / 25.3 / 24.2
GATA3 / 31.1 / 29.6 / 24.6 / 24.1 / 24.6 / 22.9 / 23.2 / 24.0 / 24.6 / 25.0 / 25.2 / 24.3
GLI1 / 33.4 / 33.6 / 33.8 / 34.1 / 35.5 / 34.1 / 34.8 / 35.3 / 34.7 / 36.5 / 35.1 / 35.4
GRB7 / 26.5 / 25.9 / 28.0 / 27.3 / 27.3 / 25.7 / 26.1 / 26.9 / 26.9 / 26.6 / 27.4 / 26.3
GSTP1 / - / 36.7 / 30.0 / 29.9 / 32.5 / 30.7 / 32.0 / 32.9 / 30.5 / 31.5 / 30.9 / 29.8
HIC1 / 29.5 / 28.8 / 34.0 / 34.5 / 35.3 / 33.9 / 33.7 / 35.0 / 35.0 / 33.0 / 35.5 / 34.8
ID1 / 23.3 / 22.3 / 25.1 / 25.1 / 23.5 / 22.4 / 25.7 / 25.8 / 25.7 / 26.3 / 26.1 / 25.3
IGF1 / 33.6 / 34.1 / 30.9 / 31.3 / 31.7 / 30.9 / 29.9 / 30.5 / 30.0 / 30.8 / 30.8 / 30.1
IGF1R / 30.4 / 29.4 / 24.5 / 25.0 / 25.6 / 24.7 / 24.8 / 25.5 / 25.9 / 26.8 / 26.4 / 25.4
IGFBP3 / - / - / 32.9 / 35.4 / 30.5 / 29.6 / 29.8 / 30.7 / 32.0 / 33.4 / 32.6 / 32.1
IL6 / 34.6 / 33.6 / - / - / - / 34.1 / - / - / - / 36.2 / - / -
JUN / 24.7 / 23.4 / 25.8 / 27.5 / 24.4 / 23.2 / 25.2 / 25.5 / 26.3 / 26.8 / 26.7 / 25.7
KRT18 / 23.3 / 22.3 / 21.2 / 22.1 / 19.4 / 18.4 / 18.1 / 18.5 / 18.7 / 19.4 / 19.7 / 18.7
KRT19 / - / - / 19.8 / 19.8 / 19.1 / 17.9 / 18.2 / 18.8 / 19.0 / 19.9 / 19.9 / 18.8
KRT5 / - / 36.5 / - / - / - / - / - / - / - / - / - / -
KRT8 / 32.7 / 33.1 / 22.6 / 22.3 / 21.1 / 19.6 / 19.1 / 19.9 / 20.3 / 21.0 / 21.1 / 19.9
MAPK1 / 23.9 / 23.0 / 24.2 / 23.6 / 24.5 / 23.0 / 23.6 / 24.3 / 25.0 / 25.0 / 25.8 / 24.6
MAPK3 / 30.0 / 29.6 / 26.6 / 26.5 / 26.6 / 25.9 / 25.9 / 26.9 / 27.4 / 27.7 / 28.1 / 26.9
MAPK8 / 26.4 / 25.5 / 26.1 / 25.7 / 26.3 / 25.6 / 25.7 / 26.3 / 26.6 / 26.9 / 27.1 / 26.7
MGMT / 26.5 / 25.5 / 25.0 / 24.8 / 25.0 / 24.0 / 24.6 / 25.0 / 25.1 / 25.7 / 25.7 / 24.8
MKI67 / 28.0 / 26.3 / 24.7 / 24.5 / 25.8 / 24.7 / 23.4 / 24.0 / 25.3 / 27.0 / 26.1 / 25.3
MLH1 / 27.3 / 26.0 / 24.6 / 24.7 / 24.7 / 23.6 / 23.8 / 24.3 / 24.7 / 25.5 / 25.5 / 24.2
MMP2 / 27.7 / 26.9 / - / - / - / - / - / - / - / - / - / -
MMP9 / 35.6 / 33.6 / 30.6 / 31.1 / 30.9 / 30.1 / 29.6 / 30.7 / 30.5 / 30.9 / 32.1 / 30.6
MUC1 / - / - / 22.6 / 22.7 / 21.8 / 20.6 / 21.3 / 22.1 / 21.9 / 22.2 / 22.5 / 21.4
MYC / 29.1 / 27.7 / 27.0 / 26.7 / 30.8 / 29.0 / 27.8 / 28.4 / 29.1 / 30.3 / 30.2 / 29.4
NME1 / 22.9 / 21.9 / 22.0 / 21.6 / 23.1 / 21.2 / 21.2 / 21.7 / 22.3 / 22.8 / 23.0 / 22.1
NOTCH1 / - / - / 29.6 / 28.8 / 30.3 / 28.7 / 27.9 / 28.6 / 30.2 / 30.4 / 30.6 / 29.6
NR3C1 / 25.7 / 24.5 / 28.3 / 27.8 / 27.7 / 26.5 / 26.7 / 28.1 / 28.1 / 27.5 / 28.6 / 27.6
PGR / - / - / 20.7 / 20.2 / 22.9 / 21.9 / 21.0 / 21.7 / 22.3 / 23.7 / 23.2 / 22.5
PLAU / 34.5 / 34.2 / 34.8 / 35.1 / 32.2 / 30.4 / 31.4 / 31.9 / 32.6 / 32.5 / 32.0 / 31.1
PRDM2 / - / 34.9 / 26.4 / 26.0 / 25.5 / 24.6 / 25.3 / 25.7 / 26.3 / 26.2 / 26.7 / 25.6
PTEN / 25.1 / 23.9 / 23.5 / 23.1 / 23.4 / 22.5 / 22.6 / 23.8 / 23.9 / 24.3 / 24.6 / 23.7
PTGS2 / 36.5 / 35.8 / 35.6 / 35.6 / 35.7 / 34.8 / - / 35.4 / 35.9 / - / 36.2 / -
PYCARD / 29.4 / 28.6 / 28.7 / 28.5 / 27.7 / 27.0 / 26.8 / 28.3 / 28.2 / 27.9 / 28.7 / 27.7
RARB / 27.9 / 26.8 / 30.7 / 30.7 / 30.2 / 29.3 / 28.8 / 30.7 / 29.9 / 30.6 / 30.7 / 29.8
RASSF1 / 27.9 / 27.3 / 28.7 / 28.0 / 28.8 / 27.8 / 27.1 / 27.5 / 28.7 / 28.6 / 28.5 / 28.2
RB1 / 25.5 / 24.5 / 25.6 / 25.1 / 26.1 / 24.5 / 24.9 / 25.6 / 26.1 / 26.8 / 26.7 / 25.6
SERPINE1 / 21.5 / 20.8 / 36.2 / 35.1 / 34.9 / 33.6 / 35.7 / 35.2 / 34.4 / 34.0 / 34.7 / 33.9
SFN / 29.6 / 28.6 / 25.9 / 25.7 / 26.5 / 24.8 / 23.8 / 24.6 / 25.9 / 26.1 / 26.5 / 25.6
SFRP1 / 27.0 / 26.7 / - / 37.7 / - / - / - / - / 38.2 / - / - / -
SLC39A6 / 26.0 / 24.9 / 24.6 / 24.1 / 24.5 / 23.3 / 23.8 / 24.4 / 24.8 / 25.1 / 25.6 / 24.7
SLIT2 / 32.6 / 31.8 / - / - / - / - / - / 36.1 / - / - / - / -
SNAI2 / 25.1 / 24.0 / - / - / 35.4 / 33.8 / - / - / 33.4 / 34.9 / - / 34.3
SRC / 27.9 / 26.8 / 27.0 / 26.3 / 27.1 / 26.5 / 26.6 / 27.3 / 27.7 / 27.9 / 28.2 / 27.6
TFF3 / 30.9 / 29.8 / 21.3 / 20.8 / 24.7 / 23.7 / 22.1 / 22.9 / 23.1 / 25.0 / 23.9 / 23.0
TGFB1 / 24.3 / 23.0 / 28.9 / 28.4 / 30.2 / 29.6 / 27.5 / 28.8 / 30.3 / 30.6 / 31.1 / 30.2
THBS1 / 31.3 / 30.8 / 22.5 / 22.5 / 24.1 / 22.9 / 22.3 / 23.1 / 24.3 / 24.0 / 24.6 / 23.5
TP53 / - / - / 24.2 / 23.8 / 25.3 / 23.9 / 23.8 / 24.8 / 25.7 / 25.4 / 25.9 / 25.3
TP73 / 31.7 / 30.7 / 30.7 / 30.5 / 30.9 / 30.1 / 29.0 / 30.9 / 30.6 / 30.6 / 30.8 / 30.3
TWIST1 / 32.3 / 32.1 / 29.6 / 29.7 / 28.5 / 27.0 / 27.6 / 28.6 / 31.9 / 31.4 / 31.2 / 30.6
VEGFA / 22.5 / 21.4 / 25.4 / 25.0 / 26.6 / 25.2 / 25.0 / 25.8 / 26.7 / 26.6 / 27.1 / 26.5
XBP1 / 22.7 / 21.9 / 19.8 / 19.7 / 21.5 / 20.6 / 20.0 / 20.7 / 20.4 / 21.0 / 20.9 / 20.0
B2M / 35.9 / 34.9 / 21.5 / 21.0 / 22.2 / 20.8 / 21.2 / 21.9 / 21.9 / 22.3 / 22.7 / 21.5
HPRT1 / 24.2 / 22.9 / 25.9 / 25.7 / 26.6 / 25.3 / 25.5 / 25.4 / 26.4 / 27.0 / 27.2 / 26.1
RPL13A / 23.2 / 22.3 / 21.4 / 21.1 / 22.3 / 20.9 / 21.7 / 22.5 / 22.8 / 22.9 / 23.4 / 22.5
GAPDH / 24.4 / 23.0 / 20.5 / 20.7 / 20.7 / 19.5 / 20.0 / 20.7 / 21.2 / 21.7 / 21.8 / 20.9
ACTB / 18.8 / 17.8 / 19.0 / 18.7 / 18.2 / 16.7 / 17.4 / 17.9 / 18.5 / 19.5 / 19.2 / 18.0
HGDC / 37.9 / - / - / 37.7 / 38.4 / 38.1 / 32.9 / - / - / - / - / 36.9
RTC / 22.7 / 22.3 / 22.7 / 23.0 / 22.4 / 22.5 / 21.8 / 23.1 / 22.8 / 22.7 / 22.4 / 23.2
RTC / 23.3 / 22.4 / 23.1 / 22.9 / 21.3 / 22.9 / 22.1 / 23.6 / 23.3 / 22.9 / 23.3 / 23.6
RTC / 23.6 / 23.5 / 23.6 / 23.5 / 21.8 / 23.3 / 22.6 / 23.9 / 23.6 / 22.9 / 23.6 / 23.8
PPC / 18.3 / 18.5 / 18.9 / 18.4 / 16.0 / 18.7 / 18.5 / 18.7 / 18.6 / 19.3 / 18.6 / 18.7
PPC / 18.7 / 19.0 / 18.5 / 18.8 / 18.7 / 19.0 / 19.0 / 19.0 / 18.9 / 19.6 / 19.0 / 19.0
PPC / 18.6 / 18.8 / 18.5 / 18.8 / 17.5 / 18.7 / 17.2 / 18.9 / 18.6 / 19.5 / 18.8 / 18.9

Identification of genes from Qiagen Breast Cancer array

Symbol / Description / Gname
ABCB1 / ATP-binding cassette, sub-family B (MDR/TAP), member 1 / ABC20/CD243/CLCS/GP170/MDR1/MGC163296/P-GP/PGY1
ABCG2 / ATP-binding cassette, sub-family G (WHITE), member 2 / ABC15/ABCP/BCRP/BCRP1/BMDP/CD338/CDw338/EST157481/MGC102821/MRX/MXR/MXR1
ADAM23 / ADAM metallopeptidase domain 23 / MDC3
AKT1 / V-akt murine thymoma viral oncogene homolog 1 / AKT/MGC99656/PKB/PKB-ALPHA/PRKBA/RAC/RAC-ALPHA
APC / Adenomatous polyposis coli / BTPS2/DP2/DP2.5/DP3/GS
AR / Androgen receptor / AIS/DHTR/HUMARA/HYSP1/KD/NR3C4/SBMA/SMAX1/TFM
ATM / Ataxia telangiectasia mutated / AT1/ATA/ATC/ATD/ATDC/ATE/DKFZp781A0353/MGC74674/TEL1/TELO1
BAD / BCL2-associated agonist of cell death / BBC2/BCL2L8
BCL2 / B-cell CLL/lymphoma 2 / Bcl-2
BIRC5 / Baculoviral IAP repeat containing 5 / API4/EPR-1
BRCA1 / Breast cancer 1, early onset / BRCAI/BRCC1/BROVCA1/IRIS/PNCA4/PSCP/RNF53
BRCA2 / Breast cancer 2, early onset / BRCC2/BROVCA2/FACD/FAD/FAD1/FANCB/FANCD/FANCD1/GLM3/PNCA2
CCNA1 / Cyclin A1 / -
CCND1 / Cyclin D1 / BCL1/D11S287E/PRAD1/U21B31
CCND2 / Cyclin D2 / KIAK0002/MGC102758
CCNE1 / Cyclin E1 / CCNE
CDH1 / Cadherin 1, type 1, E-cadherin (epithelial) / Arc-1/CD324/CDHE/ECAD/LCAM/UVO
CDH13 / Cadherin 13, H-cadherin (heart) / CDHH/P105
CDK2 / Cyclin-dependent kinase 2 / p33(CDK2)
CDKN1A / Cyclin-dependent kinase inhibitor 1A (p21, Cip1) / CAP20/CDKN1/CIP1/MDA-6/P21/SDI1/WAF1/p21CIP1
CDKN1C / Cyclin-dependent kinase inhibitor 1C (p57, Kip2) / BWCR/BWS/KIP2/WBS/p57
CDKN2A / Cyclin-dependent kinase inhibitor 2A (melanoma, p16, inhibits CDK4) / ARF/CDK4I/CDKN2/CMM2/INK4/INK4A/MLM/MTS-1/MTS1/P14/P14ARF/P16/P16-INK4A/P16INK4/P16INK4A/P19/P19ARF/TP16
CSF1 / Colony stimulating factor 1 (macrophage) / MCSF/MGC31930
CST6 / Cystatin E/M / -
CTNNB1 / Catenin (cadherin-associated protein), beta 1, 88kDa / CTNNB/DKFZp686D02253/FLJ25606/FLJ37923
CTSD / Cathepsin D / CLN10/CPSD/MGC2311
EGF / Epidermal growth factor / HOMG4/URG
EGFR / Epidermal growth factor receptor / ERBB/ERBB1/HER1/PIG61/mENA
ERBB2 / V-erb-b2 erythroblastic leukemia viral oncogene homolog 2, neuro/glioblastoma derived oncogene homolog (avian) / CD340/HER-2/HER-2/neu/HER2/MLN 19/NEU/NGL/TKR1
ESR1 / Estrogen receptor 1 / DKFZp686N23123/ER/ESR/ESRA/Era/NR3A1
ESR2 / Estrogen receptor 2 (ER beta) / ER-BETA/ESR-BETA/ESRB/ESTRB/Erb/NR3A2
FOXA1 / Forkhead box A1 / HNF3A/MGC33105/TCF3A
GATA3 / GATA binding protein 3 / HDR/MGC2346/MGC5199/MGC5445
GLI1 / GLI family zinc finger 1 / GLI
GRB7 / Growth factor receptor-bound protein 7 / -
GSTP1 / Glutathione S-transferase pi 1 / DFN7/FAEES3/GST3/GSTP/PI
HIC1 / Hypermethylated in cancer 1 / ZBTB29/ZNF901/hic-1
ID1 / Inhibitor of DNA binding 1, dominant negative helix-loop-helix protein / ID/bHLHb24
IGF1 / Insulin-like growth factor 1 (somatomedin C) / IGF-I/IGF1A/IGFI
IGF1R / Insulin-like growth factor 1 receptor / CD221/IGFIR/IGFR/JTK13/MGC142170/MGC142172/MGC18216
IGFBP3 / Insulin-like growth factor binding protein 3 / BP-53/IBP3
IL6 / Interleukin 6 (interferon, beta 2) / BSF2/HGF/HSF/IFNB2/IL-6
JUN / Jun proto-oncogene / AP-1/AP1/c-Jun
KRT18 / Keratin 18 / CYK18/K18
KRT19 / Keratin 19 / CK19/K19/K1CS/MGC15366
KRT5 / Keratin 5 / CK5/DDD/EBS2/K5/KRT5A
KRT8 / Keratin 8 / CARD2/CK8/CYK8/K2C8/K8/KO
MAPK1 / Mitogen-activated protein kinase 1 / ERK/ERK2/ERT1/MAPK2/P42MAPK/PRKM1/PRKM2/p38/p40/p41/p41mapk
MAPK3 / Mitogen-activated protein kinase 3 / ERK1/HS44KDAP/HUMKER1A/MGC20180/P44ERK1/P44MAPK/PRKM3
MAPK8 / Mitogen-activated protein kinase 8 / JNK/JNK1/JNK1A2/JNK21B1/2/PRKM8/SAPK1
MGMT / O-6-methylguanine-DNA methyltransferase / -
MKI67 / Antigen identified by monoclonal antibody Ki-67 / KIA
MLH1 / MutL homolog 1, colon cancer, nonpolyposis type 2 (E. coli) / COCA2/FCC2/HNPCC/HNPCC2/MGC5172/hMLH1
MMP2 / Matrix metallopeptidase 2 (gelatinase A, 72kDa gelatinase, 72kDa type IV collagenase) / CLG4/CLG4A/MMP-II/MONA/TBE-1
MMP9 / Matrix metallopeptidase 9 (gelatinase B, 92kDa gelatinase, 92kDa type IV collagenase) / CLG4B/GELB/MANDP2/MMP-9
MUC1 / Mucin 1, cell surface associated / CD227/EMA/H23AG/KL-6/MAM6/MUC-1/MUC-1/SEC/MUC-1/X/MUC1/ZD/PEM/PEMT/PUM
MYC / V-mycmyelocytomatosis viral oncogene homolog (avian) / MRTL/bHLHe39/c-Myc
NME1 / Non-metastatic cells 1, protein (NM23A) expressed in / AWD/GAAD/NB/NBS/NDKA/NDPK-A/NDPKA/NM23/NM23-H1
NOTCH1 / Notch 1 / TAN1/hN1
NR3C1 / Nuclear receptor subfamily 3, group C, member 1 (glucocorticoid receptor) / GCCR/GCR/GR/GRL
PGR / Progesterone receptor / NR3C3/PR
PLAU / Plasminogen activator, urokinase / ATF/UPA/URK/u-PA
PRDM2 / PR domain containing 2, with ZNF domain / HUMHOXY1/KMT8/MTB-ZF/RIZ/RIZ1/RIZ2
PTEN / Phosphatase and tensin homolog / 10q23del/BZS/DEC/GLM2/MGC11227/MHAM/MMAC1/PTEN1/TEP1
PTGS2 / Prostaglandin-endoperoxide synthase 2 (prostaglandin G/H synthase and cyclooxygenase) / COX-2/COX2/GRIPGHS/PGG/HS/PGHS-2/PHS-2/hCox-2
PYCARD / PYD and CARD domain containing / ASC/CARD5/MGC10332/TMS/TMS-1/TMS1
RARB / Retinoic acid receptor, beta / HAP/NR1B2/RRB2
RASSF1 / Ras association (RalGDS/AF-6) domain family member 1 / 123F2/NORE2A/RASSF1A/RDA32/REH3P21
RB1 / Retinoblastoma 1 / OSRC/RB/p105-Rb/pRb/pp110
SERPINE1 / Serpin peptidase inhibitor, clade E (nexin, plasminogen activator inhibitor type 1), member 1 / PAI/PAI-1/PAI1/PLANH1
SFN / Stratifin / YWHAS
SFRP1 / Secreted frizzled-related protein 1 / FRP/FRP-1/FRP1/FrzA/SARP2
SLC39A6 / Solute carrier family 39 (zinc transporter), member 6 / LIV-1
SLIT2 / Slit homolog 2 (Drosophila) / FLJ14420/SLIL3/Slit-2
SNAI2 / Snail homolog 2 (Drosophila) / MGC10182/SLUG/SLUGH1/SNAIL2/WS2D
SRC / V-src sarcoma (Schmidt-Ruppin A-2) viral oncogene homolog (avian) / ASV/SRC1/c-SRC/p60-Src
TFF3 / Trefoil factor 3 (intestinal) / ITF/P1B/TFI
TGFB1 / Transforming growth factor, beta 1 / CED/DPD1/LAP/TGFB/TGFbeta
THBS1 / Thrombospondin 1 / THBS/THBS-1/TSP/TSP-1/TSP1
TP53 / Tumor protein p53 / FLJ92943/LFS1/P53/TRP53
TP73 / Tumor protein p73 / P73
TWIST1 / Twist homolog 1 (Drosophila) / ACS3/BPES2/BPES3/CRS1/SCS/TWIST/bHLHa38
VEGFA / Vascular endothelial growth factor A / MGC70609/MVCD1/VEGF/VPF
XBP1 / X-box binding protein 1 / TREB5/XBP2
B2M / Beta-2-microglobulin / -
HPRT1 / Hypoxanthine phosphoribosyltransferase 1 / HGPRT/HPRT
RPL13A / Ribosomal protein L13a / L13A/TSTA1
GAPDH / Glyceraldehyde-3-phosphate dehydrogenase / G3PD/GAPD/MGC88685
ACTB / Actin, beta / PS1TP5BP1
HGDC / Human Genomic DNA Contamination / HIGX1A
RTC / Reverse Transcription Control / RTC
RTC / Reverse Transcription Control / RTC
RTC / Reverse Transcription Control / RTC
PPC / Positive PCR Control / PPC
PPC / Positive PCR Control / PPC
PPC / Positive PCR Control / PPC

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Supplementary Figure S1. T47D-KBluc dose response curves and EC50 values for 17β-estradiol (E2), the ERα agonist 1,3,5-tris(4-hydroxyphenyl)-4-propyl-1H pyrazole (PPT), and the ERβ agonist 2,3-bis(4-hydroxyphenyl) propionitrile (DPN).

Supplementary Table S4. Histopathological evaluation results of rat uteri from individual chemical uterotrophic assays using oral gavage administration. Evaluation was conducted by an American Board of Pathology certified pathologist at Experimental Pathology Laboratories, Inc. (Durham, NC). Description of evaluation parameters on following pages.

Chemical / Dose
(mg/kg/d) / Sample size
(# rats) / Increased cell height
Grade 1 / Increased cell height
Grade 2 / Columnar differentiation Grade 1 / Columnar differentiation Grade 2
BBP / (+) control / 4 / - / 4 / - / 4
0 / 4 / - / - / - / -
400 / 4 / - / - / - / -
600 / 4 / - / - / - / -
800 / 4 / - / - / - / -
BPA / (+) control / 5 / - / 5 / - / 5
0 / 5 / - / - / - / -
200 / 6 / - / - / - / -
400 / 5 / - / - / - / -
600 / 3 / - / - / - / -
800 / 6 / 1 / - / 1 / -
BPAF / (+) control / 6 / - / 6 / - / 6
0 / 4 / - / - / - / -
3.125 / 3 / - / - / - / -
6.25 / 3 / - / - / - / -
12.5 / 3 / - / - / - / -
25 / 6 / - / - / - / -
50 / 6 / 4 / - / 4 / -
100 / 6 / 5 / 1 / 5 / 1
300 / 5 / 3 / 2 / 3 / 2
BPC / (+) control / 6 / - / 6 / - / 6
0 / 6 / - / - / - / -
1.2 / 3 / - / - / - / -
4 / 6 / - / - / - / -
13.5 / 6 / - / - / - / -
45 / 6 / 1 / - / 1 / -
150 / 6 / 6 / - / 6 / -
500 / 6 / 3 / 3 / 3 / 3
BPS / (+) control / 6 / - / 6 / - / 6
0 / 6 / - / - / - / -
50 / 6 / - / - / - / -
100 / 6 / - / - / - / -
200 / 6 / 4 / - / 4 / -
400 / 5 / 5 / - / 5 / -
800 / 6 / 5 / 1 / 5 / 1

Supplementary Table S4 cont.Histopathological evaluation results of rat uteri from individual chemical uterotrophic assays using oral gavage administration. Evaluation was conducted by an American Board of Pathology certified pathologist at Experimental Pathology Laboratories, Inc. (Durham, NC). Description of evaluation parameters on following pages.

Chemical / Dose
(mg/kg/d) / Sample size
(# rats) / Increased cell height
Grade 1 / Increased cell height
Grade 2 / Columnar differentiation Grade 1 / Columnar differentiation Grade 2
EE2 / (+) control / 3 / - / 3 / - / 3
0 / 3 / - / - / - / -
0.0005 / 3 / - / - / - / -
0.001 / 3 / - / - / - / -
0.0025 / 3 / - / - / - / -
0.005 / 3 / - / - / - / -
0.01 / 3 / 1 / - / 1 / -
0.05 / 2 / 2 / - / 2 / -
0.2 / 3 / - / 3 / - / 3
MET / (+) control / 4 / - / 4 / - / 4
0 / 3 / - / - / - / -
62.5 / 4 / - / - / - / -
125 / 4 / 2 / 2 / 2 / 2
250 / 4 / 1 / 3 / 1 / 3
500 / 5 / 2 / 3 / 2 / 3

Description of histopathological evaluation of uteri

Uterine sections from ovariectomized female Sprague Dawley rats were examined by light microscopy for the presence of findings, including: 1) changes in epithelial cell height and 2) luminal epithelium change from cuboidal to columnar differentiation. Findings were graded one to five depending on severity. The severity grading scale was minimal = 1, mild = 2, moderate = 3, moderately severe = 4 or severe = 5. For all groups, the “baseline” for uterine epithelial changes was a simple appearing epithelial lining consisting of low cuboidal to low columnar epithelium. The nucleus of epithelial cells occupied more cell space than the cytoplasm.

For all groups, changes in uterine epithelial cell height was diagnosed as greater when increases in cytoplasm were recognized; grade 1 or minimal change represented a ratio of 1:1 (nucleus:cytoplasm), grade 2 or mild change represented a ratio of 1:2 (nucleus:cytoplasm). Uterine epithelial change from cuboidal to columnar differentiation was diagnosed when most epithelial cells were clearly more columnar in shape than controls.

Grade 1 (Minimal): This corresponds to a histopathologic change ranging from inconspicuous to barely noticeable but so minor, small, or infrequent as to warrant no more than the least assignable grade. For multifocal or diffusely-distributed lesions, this grade was used for processes where less than approximately10% of the tissue in an average high-power field was involved. For focal or diffuse hyperplastic/hypoplastic/ atrophic lesions, this grade was used when the affected structure or tissue had undergone a less than approximately 10% increase or decrease in volume.

Grade 2 (Mild): This corresponds to a histopathologic change that is a noticeable but not a prominent feature of the tissue. For multifocal or diffusely-distributed lesions, this grade was used for processes where between approximately 10% and 25% of the tissue in an average high-power field was involved. For focal or diffuse hyperplastic/hypoplastic/atrophic lesions, this grade was used when the affected structure or tissue had undergone between an approximately 10% to 25% increase or decrease in volume.

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