Researchers soon realized the estrogenic effect was due to a contaminant rather than a hormone that was causing the breast cancer cells to rapidly multiply. They determined that the contaminant was bisphenol-A – BPA and that the source of the contamination was the polycarbonate lab flasks used to sterilize the water used in the experiments….
In a 1993 paper, the Stanford team reported their discovery and their discussions with the manufacturer of polycarbonate, GE Plastics Company. Apparently aware that polycarbonate will leach, particularly if exposed to high temperatures and caustic cleaners, the company had developed a special washing regimen that they thought had eliminated the problem.
In working with the company, however, the researchers discovered that GE could not detect bisphenol-A in samples sent by the Stanford lab-samples that were causing proliferation in estrogen-responsive breast cancer cells. The problem proved to be the detection limit in GE’s chemical assay-a limit of ten parts per billion. The Stanford team found that two to five parts per billion of bisphenol-A was enough to prompt an estrogenic response in cells in the lab. pages 130 – 131
Bisphenol-A: an estrogenic substance is released from polycarbonate flasks during autoclaving.
AV Krishnan, P Stathis, SF Permuth, L Tokes and D Feldman
Division of Endocrinology, Stanford University School of Medicine, California 94305
Endocrinology, Vol 132, 2279-2286
In studies to determine whether Saccharomyces cerevisiae produced estrogens, the organism was grown in culture media prepared using distilled water autoclaved in polycarbonate flasks. The yeast- conditioned media showed the presence of a substance that competed with [3H]estradiol for binding to estrogen receptors (ER) from rat uterus. However, it soon became clear that the estrogenic substance in the conditioned media was not a product of the yeast grown in culture, but was leached out of the polycarbonate flasks during the autoclaving procedure. [3H]Estradiol displacement activity was monitored by ER RRA, and the active substance was purified from autoclaved medium using a series of HPLC steps. The final purified product was identified as bisphenol-A (BPA) by nuclear magnetic resonance spectroscopy and mass spectrometry. BPA could also be identified in distilled water autoclaved in polycarbonate flasks without the requirement of either the organism or the constituents of the culture medium. Authentic BPA was active in competitive RRAs, demonstrating an affinity approximately 1:2000 that of estradiol for ER. In functional assays, BPA (10-25 nM) induced progesterone receptors in cultured human mammary cancer cells (MCF-7) at a potency of approximately 1:5000 compared to that of estradiol. The BPA effect on PR induction was blocked by tamoxifen. In addition, BPA (25 nM) increased the rate of proliferation of MCF-7 cells assessed by [3H]thymidine incorporation. Thus, BPA exhibited estrogenic activity by both RRA and two functional bioresponse assays. Finally, MCF-7 cells grown in media prepared with water autoclaved in polycarbonate exhibited higher progesterone receptor levels than cells.grown in media prepared with water autoclaved in glass, suggesting an estrogenic effect of the water autoclaved in polycarbonate. Our findings raise the possibility that unsuspected estrogenic activity in the form of BPA may have an impact on experiments employing media autoclaved in polycarbonate flasks. It remains to be determined whether BPA derived from consumer products manufactured from polycarbonate could significantly contribute to the pool of estrogenic substances in the environment.