The Endocrine Society Centennial: No Longer a Surprise: Estrogenic Chemicals in a Multitude of Places

Abstract

Nowadays, we are bombarded with information on a large number of endocrine-disrupting chemicals. We hear and read about endocrine-disrupting chemicals on blogs, the web, news stories, television specials, advertisements, and of course scientific articles. Reports claim these ubiquitous compounds are responsible for increased rates of cancer, autism, obesity, hypospadias, and infertility, just to name a few. But it was not always this way. In fact, the scientific study of endocrine-disrupting chemicals is relatively new: a recent PubMed search found a total of 6184 hits for the term, 739 articles in 2015 as compared with 4, 20 years ago in 1995.

The field started in an unusual manner through the publication of Silent Spring by Rachel Carson (1), written for the lay public. Silent Spring was released in September of 1962, became a controversial bestseller, and sparked the creation of the Environmental Movement in the United States. Several decades later, Carson's observations of impaired fertility in wildlife were confirmed by scientific studies, conducted by field biologists who noticed and documented abnormalities in the breeding patterns, success, and reproductive physiology of their wild subjects (2–4).

The misguided treatment of pregnant women with the synthetic estrogen, diethylstilbestrol (DES), opened another chapter in the emergence of this field. Women prescribed DES in the 1940s, 1950s, and 1960s eventually developed a trend for higher incidence of breast cancer than nonusers (5). More chilling were the effects on daughters, including reproductive tract abnormalities that decreased their fertility and increased cases of cervical and breast cancers (6–8). These findings raised public awareness of human health risks from exposure to potent synthetic hormones. However, the connection between estrogens in our environment (as opposed to prescribed by a physician) and human health took more time to establish.

The article I selected to highlight was at the forefront of the work linking environmental estrogens to health. Published in 1993 in Endocrinology, Dr David Feldman's laboratory discovered biologically active estrogen-like compounds in commonly used laboratory flasks (9). Dr Feldman and his colleagues at Stanford University had been conducting endocrine research on yeast (Saccharomyces cerevisiae) and in this article made 2 seminal discoveries. First, they discovered that bisphenol A (BPA), in polycarbonate plastics, could have estrogenic actions at extremely low levels. Second, they discovered that BPA was released from polycarbonate plastics into water during the autoclaving process.

My commentary is not the first time Endocrinology has highlighted this important article. Then Editor-in-Chief, Dr Ken Korach, wrote an accompanying editorial titled “Surprising places of estrogenic activity” in the 1993 issue (10) and described the context of this discovery made by the David Feldman lab. Dr Korach stressed the implications of the findings both for public health as well as for scientific research.

The objective of the studies in Dr Feldman's laboratory was to determine if yeast, and other fungi, produced estrogens. Earlier the laboratory had found estrogen-binding proteins and an estrogen-like ligand in several species (11, 12) and considered the possibility these might represent early forms of hormone receptors (13). The estrogen binding protein in Candida albicans was eventually cloned and shown to be a flavin containing oxido-reductase enzyme (14). In the 1993 article highlighted here (9), Feldman's group extracted, isolated, and purified an estrogenic substance from their yeast culture medium, which they at first suspected might be an endogenous hormone synthesized by the yeast. However, they went on to identify the estrogenic substance to actually be BPA. Several control studies made it clear that the yeast cultures did not produce the estrogen-like substance. First, the unautoclaved medium without yeast did not contain the substance, but after autoclaving in “autoclavable” plastic flasks, estrogens were detected, and even autoclaved water without yeast or medium contained estrogenic activity. Second, the amount of the estrogen-like substance did not increase over time in growing yeast cultures. The estrogenic substances were extracted and purified by HPLC from medium and water autoclaved in the plastic flasks. Next, estrogenic binding activity was detected with a standard rat uterine cytosolic radio-receptor assay. At the time, sensitive estrogen immunoassays were not available and, in any case, would not have detected BPA, because BPA would not cross react with highly specific antibodies for estradiol. Estrogenic functional assays were conducted using a progesterone receptor induction bioassay performed with MCF-7 human breast cancer cells. Bioactive fractions were further resolved by a second round of HPLC. Nuclear magnetic resonance and mass spectrometry were performed and identified the estrogenic substance as BPA. Authentic BPA was obtained, and the estrogenic activity present in the autoclaved extracts was duplicated by BPA. The article is a wonderful example of both good detective work and excellent science converging with major implications for public health.

Figure 1.

The Centennial Paper chosen was a milestone publication for research on endocrine disruptors. The authors discovered that Bisphenol A was released from standard laboratory flasks during the autoclaving process into water (Krishnan et al., 1993). The paper was highlighted by then editor of Dr. Ken Korach (1993) in a companion editorial.

While pondering the data, Dr Feldman contacted the plastic manufacturing company, Lexan, which at that time was owned by General Electric. The new plastic flasks the laboratory had used in the studies were marketed as autoclavable. A chemist from Lexan agreed to assay some of the autoclaved water. In contrast to Feldman's data, which found estrogen-like activity, which was confirmed and identified by mass spectrometry as BPA, the Lexan chemist failed to find any detectable BPA or estrogen-like compounds in the water using the company's routine assays.

When the article was published, it garnered substantial lay press and scientific attention. Dr Feldman was invited to present the findings at the Symposium on Estrogens in the Environment, III: Global Health Implications in early 1994 in Washington, DC where interest from the scientists in the emerging endocrine disruptor world as well as government agencies was high. He was interviewed by reporters from all over the world and wrote a commentary extending his findings in Environmental Health Perspectives (15). In this symposium and accompanying article (15), he and Dr Krishnan carefully reviewed their published data and the findings and provided additional data that directly compared estradiol, the autoclaved water, and authentic BPA for radio-receptor assay activity in a dose-response manner. The data were clear; the match for the autoclaved water curve was BPA. In what is likely the first direct comparison of BPA vs estradiol binding and functional activity in MCF-7 cells, BPA starting at 25nM had a significant effect, and in the same assay, 10pM estradiol gave an equivalent response. These same low concentrations of BPA and estradiol induced progestin receptors in MCF-7 cells. In their conclusions, the authors speculated that widespread use of polycarbonates may be problematic for both scientists using plastics in their lab work and the public, because exposure to BPA may occur from a myriad of sources where plastic is used, including baby bottles, food packaging, can linings and reusable beverage bottles among others. At the time, this was visionary.

Dr Feldman recounted to me that this publication was a potential turning point in his career. He thought long and hard about his research direction. Should he enter the new emerging field of Endocrine-Disruption Chemicals, or pursue his basic endocrinology program? His love for basic endocrinology swayed his decision and he continued on and had a highly successful career working on the mechanisms of vitamin D action especially its extraskeletal effects on cancer. Yet, he was concerned about estrogenic chemicals in the environment, and expressed these concerns to his children, who paid more attention to his warnings after they read about BPA in the popular press. He was also aware of the DES literature and worried about a seeming lack of concern by governmental agencies regarding potential transgenerational actions of BPA, because it has a very close molecular structure to DES. However, at that time, he made the assumption that now that the BPA data were clear and public, United States regulatory agencies (Food and Drug Administration and Environmental Protection Agency), along with the European Food Safety Authority, would do their jobs and protect the public. But to date, there has been little to no regulatory action.

Details and explanations for this surprising inaction are given in a new commentary (16). Implementation of Good Laboratory Practices set forth by the regulatory agencies disqualifies much of the collected data from consideration, because Good Laboratory Practices requirements are beyond the budget of most academic laboratories. Along with this traditional toxicology endpoints, lack of consideration of nonmonotonic dose responses, tactics used by the companies that produce BPA, and lack of governmental initiative to take on chemical companies all contribute to the stalemate. More than 20 years after Feldman's breakthrough article, we are still waiting for our regulatory agencies to “do the right thing.”