Background: Chemicals having estrogenic activity (EA) reportedly cause many adverse health effects, especially at low (picomolar to nanomolar) doses in fetal and juvenile mammals.Objectives: We sought to determine whether commercially available plastic resins and products, including baby bottles and other products advertised as bisphenol A (BPA) free, release chemicals having EA.Methods: We used a roboticized MCF-7 cell proliferation assay, which is very sensitive, accurate, and repeatable, to quantify the EA of chemicals leached into saline or ethanol extracts of many types of commercially available plastic materials, some exposed to common-use stresses (microwaving, ultraviolet radiation, and/or autoclaving).Results: Almost all commercially available plastic products we sampled—independent of the type of resin, product, or retail source—leached chemicals having reliably detectable EA, including those advertised as BPA free. In some cases, BPA-free products released chemicals having more EA than did BPA-containing products.Conclusions: Many plastic products are mischaracterized as being EA free if extracted with only one solvent and not exposed to common-use stresses. However, we can identify existing compounds, or have developed, monomers, additives, or processing agents that have no detectable EA and have similar costs. Hence, our data suggest that EA-free plastic products exposed to common-use stresses and extracted by saline and ethanol solvents could be cost-effectively made on a commercial scale and thereby eliminate a potential health risk posed by most currently available plastic products that leach chemicals having EA into food products.

<p>E2 suppressed the expression of PPARγ target gene ACOX3 in three breast cancer cell lines. MCF-7 cells were transferred from phenol red containing medium to E2-free medium for 3 days. Next, (A) MCF-7, (B) MCF-7:5C, and (C) MCF-7:2A cells were seeded in six-well plates. After 24 hours, cells were treated with a vehicle control (0.1% EtOH) or E2 (1 nM) for 24, 48, 72 hours. Cells were then harvested in TRIzol reagent. ACOX3 expression levels were quantitated using RT-PCR. *P<0.05 compared with control; **P<0.001 compared with control.</p>

<p>Expression of PPARγ and its function in breast cancer cell lines. A, MCF-7 cells were transferred from phenol red containing medium to E2-free medium for 3 days. Cells were then harvested similarly to that performed for MCF-7:5C and MCF-7:2A cells in TRIzol reagent. PPARγ expression levels were quantitated using RT-PCR. **P<0.001 compared with control. B, MCF-7 cells were seeded in 24-well plates. Then, cells were treated with a vehicle control (0.1% DMSO) or different concentrations (from 50 nM to 5µM) of T0070907 for 7 days. Cells were harvested for DNA proliferation assay. *P<0.05, **P<0.001. C, MCF-7:5C and MCF-7:2A cells were seeded in 24-well plates. Then, cells were treated with a vehicle control (0.1% DMSO) or T0070907 (5µM) for 7 days. Cells were harvested for DNA proliferation assay. **P<0.001. D, Growth response to T0070907 in T47D-derived cells. T47D cells were transferred to E2-free medium for 3 days. Then, T47D and T47D:C42 were treated with a vehicle control (0.1% DMSO) or T0070907 (5µM) in 24-well plates for 7 days. Cells were then harvested for DNA proliferation assay. *P<0.05, **P<0.001. E, ZR-75-1 cells were transferred from phenol red containing medium to E2-free medium for 3 days and seeded in a 24-well plate. After 24 hours, cells were treated with a vehicle control (0.1% DMSO) or T0070907 (5µM) for 7 days. Cells were then harvested for use in a DNA proliferation assay. **P<0.001 compared with control. F, BT-474 were seeded in 24-well plates. Then, cells were treated with a vehicle control (0.1% DMSO) or different concentrations (from 1 µM to 10µM) of T0070907 for 7 days. Cells were harvested for DNA proliferation assay. **P<0.001. G, Sk-Br-3 were seeded in 24-well plates. Then, cells were treated with a vehicle control (0.1% DMSO) or different concentrations (from 1 µM to 10µM) of T0070907 for 7 days. Cells were harvested for DNA proliferation assay. *P<0.05, **P<0.001. H, MDA-MB-231 cells were seeded in 24-well plates. Then, cells were treated with a vehicle control (0.1% DMSO) or different concentrations (from 2 µM to 10µM) of T0070907 for 7 days. Cells were harvested for DNA proliferation assay.</p>

<p>Validation of cell lines</p>

<p>Supplementary Figure 1: Western blot showing ERalpha expression in T47D:A18/neo, T47D:A18/PKCalpha, T47D:A18, T47D:A18-TAM1, MCF-7:WS8 and MCF-7 5C cell lines.</p>

<p>(A) The JNK inhibitor effectively blocked phosphorylation of JNK. MCF-7:2A cells were treated with vehicle (0.1% DMSO) or SP600125 (10-5 mol/L) for 24 hours. p-JNK was examined by Western blot. Total JNK was measured as loading control. (B) The JNK inhibitor could not block E2-induced apoptosis in MCF-7:2A cells. MCF-7:2A cells were treated with vehicle (0.1% DMSO; con), E2 (10-9 mol/L), SP600125 (10-5 mol/L), and E2 (10-9 mol/L) plus SP600125 (10-5 mol/L) for 6 days. Annexin V binding assay was used to detect apoptosis. p<0.001, ** compared with control. (C) Cell viability after E2 combination with the JNK inhibitor. MCF-7:2A cells were treated with vehicle (0.1% DMSO), E2 (10-9 mol/L), SP600125 (10-5 mol/L), and E2 (10-9 mol/L) plus SP600125 (10-5 mol/L). Cells were harvested after 7 days treatment and cell viability was quantitated by determination of total DNA. p<0.001, ** compared with control.</p>

No abstract

<p>Supplementary Figure 6. A. MCF-7:WS8 cells were transfected with an ERE-luciferase reporter construct for 24 hours, then treated with vehicle, 1µM ICI, 1µM R5020, 1µM RU486, 1nM E2, 1µM MPA, 1µM NETA, or combinations for 24 hours. ERE activity was measured by luciferase assay and normalized to vehicle control. Means represent samples in triplicate. B. MCF-7:WS8 cells were transfected with an ERE-luciferase reporter construct for 24 hours, then treated with vehicle, 1µM 4-OHT, 1µM R5020, 1µM RU486, 1nM E2, 1µM MPA, 1µM NETA, or combinations for 24 hours. ERE activity was measured by luciferase assay and normalized to vehicle control. Means represent samples in triplicate. Error bars too small to visualize.</p>