The Conflict between Regulatory Agencies over the 20,000-Fold Lowering of the Tolerable Daily Intake (TDI) for Bisphenol A (BPA) by the European Food Safety Authority (EFSA).

BACKGROUND: The European Food Safety Authority (EFSA) recommended lowering their estimated tolerable daily intake (TDI) for bisphenol A (BPA) 20,000-fold to 0.2 ng/kg body weight (BW)/day. BPA is an extensively studied high production volume endocrine disrupting chemical (EDC) associated with a vast array of diseases. Prior risk assessments of BPA by EFSA as well as the US Food and Drug Administration (FDA) have relied on industry-funded studies conducted under good laboratory practice protocols (GLP) requiring guideline end points and detailed record keeping, while also claiming to examine (but rejecting) thousands of published findings by academic scientists. Guideline protocols initially formalized in the mid-twentieth century are still used by many regulatory agencies. EFSA used a 21st century approach in its reassessment of BPA and conducted a transparent, but time-limited, systematic review that included both guideline and academic research. The German Federal Institute for Risk Assessment (BfR) opposed EFSA's revision of the TDI for BPA. OBJECTIVES: We identify the flaws in the assumptions that the German BfR, as well as the FDA, have used to justify maintaining the TDI for BPA at levels above what a vast amount of academic research shows to cause harm. We argue that regulatory agencies need to incorporate 21st century science into chemical hazard identifications using the CLARITY-BPA (Consortium Linking Academic and Regulatory Insights on BPA Toxicity) nonguideline academic studies in a collaborative government-academic program model. DISCUSSION: We strongly endorse EFSA's revised TDI for BPA and support the European Commission's (EC) apparent acceptance of this updated BPA risk assessment. We discuss challenges to current chemical risk assessment assumptions about EDCs that need to be addressed by regulatory agencies to, in our opinion, become truly protective of public health. Addressing these challenges will hopefully result in BPA, and eventually other structurally similar bisphenols (called regrettable substitutions) for which there are known adverse effects, being eliminated from all food-related and many other uses in the EU and elsewhere. https://doi.org/10.1289/EHP13812.

Modeling Mammary Organogenesis from Biological First Principles: A Systems Biology Approach.

Stromal-epithelial interactions mediate mammary gland development and the formation and progression of breast cancer. To study these interactions in vitro, 3D models are essential. We have successfully developed novel 3D in vitro models that allow the formation of mammary gland structures closely resembling those found in vivo and that respond to the hormonal cues that regulate mammary gland morphogenesis and function. Due to their simplicity when compared to in vivo studies, and to their accessibility to visualization in real time, these models are well suited to conceptual and mathematical modeling.

European Medicines Agency Conflicts With the European Food Safety Authority (EFSA) on Bisphenol A Regulation.

The European Food Safety Authority (EFSA) has revised their estimate of the toxicity of bisphenol A (BPA) and, as a result, have recommended reducing the tolerable daily intake (TDI) by 20 000-fold. This would essentially ban the use of BPA in food packaging such as can liners, plastic food containers, and in consumer products. To come to this conclusion, EFSA used a systematic approach according to a pre-established protocol and included all guideline and nonguideline studies in their analysis. They found that Th-17 immune cells increased with very low exposure to BPA and used this endpoint to revise the TDI to be human health protective. A number of regulatory agencies including the European Medicines Agency (EMA) have written formal disagreements with several elements of EFSA's proposal. The European Commission will now decide whether to accept EFSA's recommendation over the objections of EMA. If the Commission accepts EFSA's recommendation, it will be a landmark action using knowledge acquired through independent scientific studies focused on biomarkers of chronic disease to protect human health. The goal of this Perspective is to clearly articulate the monumental nature of this debate and decision and to explain what is at stake. Our perspective is that the weight of evidence clearly supports EFSA's proposal to reduce the TDI by 20 000-fold.

The cancer puzzle: Welcome to organicism.

During the fifty years since President Nixon declared the "War on Cancer", those inside and outside the cancer community have witnessed the systematic moving of the goalposts attitude to accommodate evidence into an inadequate theory, that is, the Somatic Mutation Theory (SMT). This sorry state promoted a renewable yearly promise that at the end of the next 10-year period the promises uttered in 1971 would become reality. Each failure triggered calls to do more of the same research under the same theory, routinely using more and more sophisticated technology. Meanwhile, in the last few years, an unambiguous general consensus has emerged acknowledging that this overall long, intensive effort has failed, and that it is likely that the solution to the cancer problem resides elsewhere, namely, in alternative theoretical principles of biology. In this essay we concentrate, first, on the big picture, from the philosophical stance (reductionism versus organicism) to the need to adopt rigorous theories. From this novel perspective we conceptualize cancer as a disease of tissue organization akin to development gone awry. Finally, having identified both a promising stance and a useful theory, i.e., the tissue organization field theory (TOFT), we call for abandoning the SMT and for adopting the more promising TOFT.

Matrix Composition Modulates Vitamin D3's Effects on 3D Collagen Fiber Organization by MCF10A Cells.

Vitamin D3 (vitD3) has been implicated in various cellular functions affecting multiple tissue types. Epidemiological and laboratory studies suggest that vitD3 may be effective as a preventive or therapeutic option for breast cancer. However, randomized clinical trials have yet to confirm these suggestions. Breast neoplasias can arise from developmental alterations; based on this evidence, we seek to understand vitD3's role in normal breast development, particularly its role in epithelial morphogenetic processes such as ductal elongation, branching, and alveolar formation. These processes require extensive changes in the extracellular microenvironment, such as collagen fiber organization, and are largely influenced by hormones. Here, we build upon our past work to shed light on calcitriol's effects on collagen fiber organization by breast epithelial cells, and how such effects are modulated by extracellular matrix composition. We embedded MCF10A normal human breast epithelial cells in two different matrices-collagen type I and collagen type I + 10% Matrigel; treatment with calcitriol resulted in flatter epithelial structures. Next, using two-photon microscopy, we examined changes in collagen fiber organization and corresponding changes in epithelial structures. Applying a novel three-dimensional (3D) image analysis method, we show that increasing doses of calcitriol result in denser collagen fiber bundles in the localized area surrounding the epithelial structures, and that these bundles are aligned in a more parallel direction to epithelial structures when exposed to the highest vitD3 dose. Changed patterns in fiber organization may explain the flattening of epithelial structures; in turn, changes in biophysical forces in the matrix abutting these structures may be responsible for changes in the referred patterns. Addition of 10% Matrigel dampened the effects of calcitriol on both epithelial morphogenesis and fiber organization. Overall, we report novel functions of calcitriol in the breast epithelium and add to the growing body of evidence documenting how hormones affect biophysical processes. Impact statement In this study, we report novel functions of calcitriol in the breast epithelium and use a novel quantitative metric to parse the effects of calcitriol on collagen fiber organization that cannot be detected through conventional histological procedures. Despite the large body of literature on vitamin D3 (vitD3) and calcitriol's effects on cellular functions across tissue types, little is known about how they affect collagen fiber organization, an early critical step for breast epithelial development. This work provides further evidence that hormones affect morphogenesis by means of biophysical forces, with implications for a comprehensive view on vitD3's effects in breast development and neoplasia.

From Wingspread to CLARITY: a personal trajectory.

In the three decades since endocrine disruption was conceptualized at the Wingspread Conference, we have witnessed the growth of this multidisciplinary field and the accumulation of evidence showing the deleterious health effects of endocrine-disrupting chemicals. It is only within the past decade that, albeit slowly, some changes regarding regulatory measures have taken place. In this Perspective, we address some historical points regarding the advent of the endocrine disruption field and the conceptual changes that endocrine disruption brought about. We also provide our personal recollection of the events triggered by our serendipitous discovery of oestrogenic activity in plastic, a founder event in the field of endocrine disruption. This recollection ends with the CLARITY study as an example of a discordance between 'science for its own sake' and 'regulatory science' and leads us to offer a perspective that could be summarized by the motto attributed to Ludwig Boltzmann: "Nothing is more practical than a good theory".

Information, programme, signal: dead metaphors that negate the agency of organisms.

The metaphorical adoption of the concepts of information, program and signal introduced into biology the logic and implicit causal structure of the mathematical theories of information; this is inimical to biology. In turn, those metaphors have hindered the development of a theory of organisms by transferring the agency of organisms to natural selection and to DNA. Moreover, those metaphors introduced into biology the dualism software-hardware and a Laplacian causal structure. Instead, we propose to uphold the agency of the living by adopting three foundational principles for a theory of organisms: namely, 1) the principle of biological inertia (i.e., the default state of cells is proliferation and motility), 2) the principle of variation, and 3) the principle of organization.

Over a century of cancer research: Inconvenient truths and promising leads.

Despite over a century of intensive efforts, the great gains promised by the War on Cancer nearly 50 years ago have not materialized. Since 1999, we have analyzed the lack of progress in explaining and "curing" cancer by examining the merits of the premises that determine how cancer is understood and treated. Our ongoing critical analyses have aimed at clarifying the sources of misunderstandings at the root of the cancer puzzle while providing a plausible and comprehensive biomedical perspective as well as a new theory of carcinogenesis that is compatible with evolutionary theory. In this essay, we explain how this new theory, the tissue organization field theory (TOFT), can help chart a path to progress for cancer researchers by explaining features of cancer that remain unexplainable from the perspective of the still hegemonic somatic mutation theory (SMT) and its variants. Of equal significance, the premises underlying the TOFT offer new perspectives on basic biological phenomena.

Vitamin D3 constrains estrogen's effects and influences mammary epithelial organization in 3D cultures.

Vitamin D3 (vitD3) and its active metabolite, calcitriol (1,25-(OH)2D3), affect multiple tissue types by interacting with the vitamin D receptor (VDR). Although vitD3 deficiency has been correlated with increased incidence of breast cancer and less favorable outcomes, randomized clinical trials have yet to provide conclusive evidence on the efficacy of vitD3 in preventing or treating breast cancer. Additionally, experimental studies are needed to assess the biological plausibility of these outcomes. The mammary gland of VDR KO mice shows a florid phenotype revealing alterations of developmental processes that are largely regulated by mammotropic hormones. However, most research conducted on vitD3's effects used 2D cell cultures and supra-physiological doses of vitD3, conditions that spare the microenvironment in which morphogenesis takes place. We investigated the role of vitD3 in mammary epithelial morphogenesis using two 3D culture models. VitD3 interfered with estrogen's actions on T47D human breast cancer cells in 3D differently at different doses, and recapitulated what is observed in vivo. Also, vitD3 can act autonomously and affected the organization of estrogen-insensitive MCF10A cells in 3D collagen matrix by influencing collagen fiber organization. Thus, vitD3 modulates mammary tissue organization independent of its effects on cell proliferation.

3D organizational mapping of collagen fibers elucidates matrix remodeling in a hormone-sensitive 3D breast tissue model.

Hormones play an important role in normal and diseased breast tissue development. However, they can also disrupt cell-matrix interactions and their role in extracellular matrix reorganization during epithelial morphogenesis remains poorly understood, partly due to a lack of sensitive approaches for matrix characterization. Here, we assess the hormonal regulation of matrix reorganization in a three-dimensional (3D) breast tissue culture model using a novel metric, i.e., 3D directional variance, to characterize the 3D organization of collagen fibers visualized via high-resolution, second harmonic generation imaging. This metric enables resolving and quantifying patterns of spatial organization throughout the matrix surrounding epithelial structures treated with 17ß-estradiol (E2) alone, and E2 in combination with either promegestone, a progestogen, or prolactin. Addition of promegestone results in the most disorganized fibers, while the E2 alone treatment leads to the most organized ones. Location-dependent organization mapping indicates that only the prolactin treatment leads to significant heterogeneities in the regional organization of collagen fibers, with higher levels of alignment observed at the end of the elongated epithelial structures. The observed collagen organization patterns for all groups persist for tens of micrometers. In addition, a comparison between 3D directional variance and typical 2D analysis approaches reveals an improved sensitivity of the 3D metric to identify organizational heterogeneities and differences among treatment groups. These results demonstrate that 3D directional variance is sensitive to subtle changes in the extracellular micro-environment and has the potential to elucidate reciprocal cell-matrix interactions in the context of numerous applications involving the study of normal and diseased tissue morphogenesis.

Characterization of MCF-12A cell phenotype, response to estrogens, and growth in 3D.

BACKGROUND: Three-dimensional cultures of mammary epithelial cells allow for biologically-relevant studies of the development of the mammary gland in rodents and humans under normal and pathological conditions, like carcinogenesis. Under these conditions, mammotropic hormones play significant roles in tissue morphogenesis. Therefore, a system that recreates the normal, hormonally responsive epithelium would be a valuable tool to study the normal state and its transition to carcinogenesis. MCF-12A cells have been claimed to be non-tumorigenic mammary epithelial cells with reported sensitivity to estrogens. In this study, we aimed at characterizing MCF-12A cells for use in a hormone-responsive 3D culture system to determine their usefulness as a tool to identify normal and abnormal microenvironmental cues. METHODS: MCF-12A cells were single-cell cloned in order to investigate their heterogeneous makeup. The parental cells were then treated with estradiol to investigate proliferative and transcriptional responses through the estrogen receptor alpha. Finally, parental cells and epithelial-like cell-derived clones were seeded in rat-tail collagen I to profile the morphogenesis of multicellular 3D structures. The resultant structures were then analyzed using unsupervised morphometric analysis. RESULTS: MCF-12A cells consist of epithelial-like colonies which shed elongated, freely growing cells on the colony's edges. The cells express E-cadherin as well as mesenchymal vimentin but do not express markers associated with myoepithelial cells or fibroblasts. Treatment with estradiol does not affect either the proliferation rate or the induction of gene expression in MCF-12A cells. Parental MCF-12A cells form acini, solid spheres and elongated branching ducts when grown in rat-tail collagen type I matrix, the geometries and distribution of which are altered following the removal of fibroblast-like cells. CONCLUSIONS: MCF-12A cells are a heterogeneous pseudo-epithelial cell line capable of forming a variety of multicellular structures in 3D culture. We found no indication that the cells display estrogen-responsive characteristics, thus refuting previous studies which reported estrogen responsiveness. We report that MCF-12A cells are not suited for use in studies in which differential behaviors of "normal" and "cancerous" estrogen-responsive cells are to be compared.

An Integrative Approach Toward Biology, Organisms, and Cancer.

Over the last two decades, we have challenged the hegemony of the somatic mutation theory of carcinogenesis (SMT) based on the lack of theoretical coherence of the premises adopted by its followers. We offered instead a theoretical alternative, the tissue organization field theory (TOFT), that is based on the premises that cancer is a tissue-based disease and that proliferation and motility is the default state of all cells. We went on to use a theory-neutral experimental protocol that simultaneously tested the TOFT and the SMT. The results of this test favored adopting the TOFT and rejecting the SMT. Recently, an analysis of the differences between the Physics of the inanimate and that of the living matter has led us to propose principles for the construction of a much needed theory of organisms. The three biological principles are (a) a default state, (b) a principle of variation, and (c) one of organization. The TOFT, defined as "development gone awry," fits well within the principles that we propose for a theory of organisms. This radical conceptual change opened up the possibility of anchoring mathematical modeling on genuine biological principles. By identifying constraints to the default state, multilevel biomechanical explanations become as legitimate as the molecular ones on which other modelers that adopt the SMT rely. Expanding research based on the premises of our theory of organisms will enrich a comprehensive understanding of normal development and of the one that goes awry.

Reductionism, Organicism, and Causality in the Biomedical Sciences: A Critique.

Biology is undergoing a crisis whereby technical innovations designed to overcome the difficulties encountered in explaining complex biological phenomena have not delivered the expected results. To overcome this problem, mainstream biomedical researchers favor adopting new technological wonders without considering that what hinders their research could be due to their philosophical stance, theoretical frame, or looking into the wrong level of biological organization. We address the conceptual problems underlying the scientific crisis by examining the philosophical stances that have illuminated biological thought for the last 200 years and their evolution into the conceptual frames now known as reductionism and organicism. We also analyze how these stances seek to establish causality. The reductionist fixation on bottom-up causation is based on physicalism, a stance that does not allow for emergent phenomena. Organicism, by contrast, allows for emergence and asserts that biological entities are defined by the relation between what they are and what they do; thus, they generate novel qualities and structures. Also, only organisms experience illness and health. Organicism recognizes the role of agency and normativity in determining biological phenomena. Based on these premises, we favor adopting organicism to resolve the current crisis in the biomedical sciences.

New insights into fetal mammary gland morphogenesis: differential effects of natural and environmental estrogens.

An increased breast cancer risk during adulthood has been linked to estrogen exposure during fetal life. However, the impossibility of removing estrogens from the feto-maternal unit has hindered the testing of estrogen's direct effect on mammary gland organogenesis. To overcome this limitation, we developed an ex vivo culture method of the mammary gland where the direct action of estrogens can be tested during embryonic days (E)14 to 19. Mouse mammary buds dissected at E14 and cultured for 5 days showed that estrogens directly altered fetal mammary gland development. Exposure to 0.1 pM, 10 pM, and 1 nM 17 ß-estradiol (E2) resulted in monotonic inhibition of mammary buds ductal growth. In contrast, Bisphenol-A (BPA) elicited a non-monotonic response. At environmentally relevant doses (1 nM), BPA significantly increased ductal growth, as previously observed in vivo, while 1 µM BPA significantly inhibited ductal growth. Ductal branching followed the same pattern. This effect of BPA was blocked by Fulvestrant, a full estrogen antagonist, while the effect of estradiol was not. This method may be used to study the hormonal regulation of mammary gland development, and to test newly synthesized chemicals that are released into the environment without proper assessment of their hormonal action on critical targets like the mammary gland.

Evidence of Absence: Estrogenicity Assessment of a New Food-Contact Coating and the Bisphenol Used in Its Synthesis.

Consumer concerns about exposure to substances found in food contact materials with estrogenic activity (EA) have created substantial demand for alternatives. We assessed the potential EA of both a new bisphenol monomer used to synthesize polymeric coatings for metal food-contact applications and the nonintentionally added substances (NIAS) that may migrate into food. We evaluated tetramethyl bisphenol F (TMBPF) using in vitro and in vivo assays. We extracted the polymeric coating using food simulants ethanol (50% v/v) and acetic acid (3% w/v) and measured migration using tandem liquid chromatography (LC)/mass spectrometry (MS) and LC time-of-flight MS for TMBPF and NIAS, respectively. We also tested migrants for EA using the E-SCREEN assay. TMBPF did not show estrogenic activity in the uterotrophic assay and did not alter puberty in male and female rats or mammary gland development in female rats. Neither TMBPF nor the migrants from the final polymeric coating increased proliferation of estrogen-sensitive MCF7 cells. TMBPF did not show estrogen-agonist or antagonist activity in the estrogen receptor-transactivation assay. TMBPF migration was below the 0.2 parts per billion detection limit. Our findings provide compelling evidence for the absence of EA by TMBPF and the polymeric coating derived from it and that human exposure to TMBPF would be negligible.

Minireview: Endocrine Disruptors: Past Lessons and Future Directions.

Within the past few decades, the concept of endocrine-disrupting chemicals (EDCs) has risen from a position of total obscurity to become a focus of dialogue, debate, and concern among scientists, physicians, regulators, and the public. The emergence and development of this field of study has not always followed a smooth path, and researchers continue to wrestle with questions about the low-dose effects and nonmonotonic dose responses seen with EDCs, their biological mechanisms of action, the true pervasiveness of these chemicals in our environment and in our bodies, and the extent of their effects on human and wildlife health. This review chronicles the development of the unique, multidisciplinary field of endocrine disruption, highlighting what we have learned about the threat of EDCs and lessons that could be relevant to other fields. It also offers perspectives on the future of the field and opportunities to better protect human health.