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.

Using the Key Characteristics of Carcinogens to Develop Research on Chemical Mixtures and Cancer.

BACKGROUND: People are exposed to numerous chemicals throughout their lifetimes. Many of these chemicals display one or more of the key characteristics of carcinogens or interact with processes described in the hallmarks of cancer. Therefore, evaluating the effects of chemical mixtures on cancer development is an important pursuit. Challenges involved in designing research studies to evaluate the joint action of chemicals on cancer risk include the time taken to perform the experiments because of the long latency and choosing an appropriate experimental design. OBJECTIVES: The objectives of this work are to present the case for developing a research program on mixtures of environmental chemicals and cancer risk and describe recommended approaches. METHODS: A working group comprising the coauthors focused attention on the design of mixtures studies to inform cancer risk assessment as part of a larger effort to refine the key characteristics of carcinogens and explore their application. Working group members reviewed the key characteristics of carcinogens, hallmarks of cancer, and mixtures research for other disease end points. The group discussed options for developing tractable projects to evaluate the joint effects of environmental chemicals on cancer development. RESULTS AND DISCUSSION: Three approaches for developing a research program to evaluate the effects of mixtures on cancer development were proposed: a chemical screening approach, a transgenic model-based approach, and a disease-centered approach. Advantages and disadvantages of each are discussed. https://doi.org/10.1289/EHP8525.

Testing the low dose mixtures hypothesis from the Halifax project.

In 2013, 60 scientists, representing a larger group of 174 scientists from 26 nations, met in Halifax, Nova Scotia to consider whether - using published research - it was logical to anticipate that a mixture of chemicals, each thought to be non-carcinogenic, might act together in that mixture as a virtual carcinogen. The group identified 89 such chemicals, each one affecting one or more Hallmark(s) - collectively covering all Hallmarks of Cancer - confirming the possibility that a chemical mixture could induce all the Hallmarks and function as a virtual carcinogen, thereby supporting the concern that chemical safety research that does not evaluate mixtures, is incomplete. Based on these observations, the Halifax Project developed the Low-Dose Carcinogenesis Hypothesis which posits "…that low-dose exposures to [mixtures of] disruptive chemicals that are not individually carcinogenic may be capable of instigating and/or enabling carcinogenesis." Although testing all possible combinations of over 80,000 chemicals of commerce would be impractical, prudence requires designing a methodology to test whether low-dose chemical mixtures might be carcinogenic. As an initial step toward testing this hypothesis, we conducted a mini review of published empirical observations of biological exposures to chemical mixtures to assess what empirical data exists on which to base future research. We reviewed studies on chemical mixtures with the criteria that the studies reported both different concentrations of chemicals and mixtures composed of different chemicals. We found a paucity of research on this important question. The majority of studies reported hormone related processes and used chemical concentrations selected to facilitate studying how mixtures behave in experiments that were often removed from clinical relevance, i.e., chemicals were not studied at human-relevant concentrations. New research programs must be envisioned to enable study of how mixtures of small doses of chemicals affect human health, starting, when at all possible, from non-malignant specimens when studies are done in vitro. This research should use human relevant concentrations of chemicals, expand research beyond the historic focus on endocrine endpoints and endocrine related cancers, and specifically seek effects that arise uniquely from exposure to chemical mixtures at human-relevant concentrations.

The Key Characteristics of Carcinogens: Relationship to the Hallmarks of Cancer, Relevant Biomarkers, and Assays to Measure Them.

The key characteristics (KC) of human carcinogens provide a uniform approach to evaluating mechanistic evidence in cancer hazard identification. Refinements to the approach were requested by organizations and individuals applying the KCs. We assembled an expert committee with knowledge of carcinogenesis and experience in applying the KCs in cancer hazard identification. We leveraged this expertise and examined the literature to more clearly describe each KC, identify current and emerging assays and in vivo biomarkers that can be used to measure them, and make recommendations for future assay development. We found that the KCs are clearly distinct from the Hallmarks of Cancer, that interrelationships among the KCs can be leveraged to strengthen the KC approach (and an understanding of environmental carcinogenesis), and that the KC approach is applicable to the systematic evaluation of a broad range of potential cancer hazards in vivo and in vitro We identified gaps in coverage of the KCs by current assays. Future efforts should expand the breadth, specificity, and sensitivity of validated assays and biomarkers that can measure the 10 KCs. Refinement of the KC approach will enhance and accelerate carcinogen identification, a first step in cancer prevention.See all articles in this CEBP Focus section, "Environmental Carcinogenesis: Pathways to Prevention."

A Ternary Mixture of Common Chemicals Perturbs Benign Human Breast Epithelial Cells More Than the Same Chemicals Do Individually.

As a continuous source of hormonal stimulation, environmentally ubiquitous estrogenic chemicals, ie, xenoestrogens (XEs), are a potential risk factor for breast carcinogenesis. Given their wide distribution in the environment and the fact that bisphenol-A (BPA), methylparaben (MP), and perfluorooctanoic acid (PFOA) are uniformly detected in unselected body fluid samples, it must be assumed that humans are simultaneously exposed to these chemicals almost daily. We studied the effects of a ternary mixture of BPA, MP, and PFOA on benign breast epithelial cells at the range of concentrations observed for single chemicals in human samples. Measurements of exposure impact relevant to the breast were based on endpoints associated with "hallmarks" of cancer and "key characteristics" of carcinogens. These included modulation of total estrogen receptor (ER)α, phosphorylated ERα (pERα), total ERß, S-phase induction, and apoptotic evasion. Data from live cell measurements were fit to a log-linear dose-response model. Concentration-dependent reduction of ERß and apoptosis evasion was observed concurrently with the induction of ERα, pERα, and S-phase fraction, and an increased rate of cell proliferation. Beyond additive effects predicted by the sum of individual test XEs, mixture treatment demonstrated synergism for the ERß and apoptosis suppression phenotypes (p > .001). Nonmalignant breast cells were more sensitive than commonly used breast cancer lines to XE treatment in 3 of 5 endpoints. All observations were validated with cells isolated from the normal breast tissue of 14 individuals. At relatively low concentrations, a chemical mixture has striking effects on normal cell function that are missed by evaluation of single components.

Low-Dose Mixture Hypothesis of Carcinogenesis Workshop: Scientific Underpinnings and Research Recommendations.

BACKGROUND: The current single-chemical-as-carcinogen risk assessment paradigm might underestimate or miss the cumulative effects of exposure to chemical mixtures, as highlighted in recent work from the Halifax Project. This is particularly important for chemical exposures in the low-dose range that may be affecting crucial cancer hallmark mechanisms that serve to enable carcinogenesis. OBJECTIVE: Could ongoing low-dose exposures to a mixture of commonly encountered environmental chemicals produce effects in concert that lead to carcinogenesis? A workshop held at the NIEHS in August 2015 evaluated the scientific support for the low-dose mixture hypothesis of carcinogenesis and developed a research agenda. Here we describe the science that supports this novel theory, identify knowledge gaps, recommend future methodologies, and explore preventative risk assessment and policy decision-making that incorporates cancer biology, environmental health science, translational toxicology, and clinical epidemiology. DISCUSSION AND CONCLUSIONS: The theoretical merits of the low-dose carcinogenesis hypothesis are well founded with clear biological relevance, and therefore, the premise warrants further investigation. Expert recommendations include the need for better insights into the ways in which noncarcinogenic constituents might combine to uniquely affect the process of cellular transformation (in vitro) and environmental carcinogenesis (in vivo), including investigations of the role of key defense mechanisms in maintaining transformed cells in a dormant state. The scientific community will need to acknowledge limitations of animal-based models in predicting human responses; evaluate biological events leading to carcinogenesis both spatially and temporally; examine the overlap between measurable cancer hallmarks and characteristics of carcinogens; incorporate epigenetic biomarkers, in silico modelling, high-performance computing and high-resolution imaging, microbiome, metabolomics, and transcriptomics into future research efforts; and build molecular annotations of network perturbations. The restructuring of many existing regulatory frameworks will require adequate testing of relevant environmental mixtures to build a critical mass of evidence on which to base policy decisions. Citation: Miller MF, Goodson WH III, Manjili MH, Kleinstreuer N, Bisson WH, Lowe L. 2017. Low-Dose Mixture Hypothesis of Carcinogenesis Workshop: scientific underpinnings and research recommendations. Environ Health Perspect 125:163-169; http://dx.doi.org/10.1289/EHP411.

Assessing the carcinogenic potential of low-dose exposures to chemical mixtures in the environment: the challenge ahead.

Lifestyle factors are responsible for a considerable portion of cancer incidence worldwide, but credible estimates from the World Health Organization and the International Agency for Research on Cancer (IARC) suggest that the fraction of cancers attributable to toxic environmental exposures is between 7% and 19%. To explore the hypothesis that low-dose exposures to mixtures of chemicals in the environment may be combining to contribute to environmental carcinogenesis, we reviewed 11 hallmark phenotypes of cancer, multiple priority target sites for disruption in each area and prototypical chemical disruptors for all targets, this included dose-response characterizations, evidence of low-dose effects and cross-hallmark effects for all targets and chemicals. In total, 85 examples of chemicals were reviewed for actions on key pathways/mechanisms related to carcinogenesis. Only 15% (13/85) were found to have evidence of a dose-response threshold, whereas 59% (50/85) exerted low-dose effects. No dose-response information was found for the remaining 26% (22/85). Our analysis suggests that the cumulative effects of individual (non-carcinogenic) chemicals acting on different pathways, and a variety of related systems, organs, tissues and cells could plausibly conspire to produce carcinogenic synergies. Additional basic research on carcinogenesis and research focused on low-dose effects of chemical mixtures needs to be rigorously pursued before the merits of this hypothesis can be further advanced. However, the structure of the World Health Organization International Programme on Chemical Safety 'Mode of Action' framework should be revisited as it has inherent weaknesses that are not fully aligned with our current understanding of cancer biology.

Exposure to the polyester PET precursor--terephthalic acid induces and perpetuates DNA damage-harboring non-malignant human breast cells.

Identification of early perturbations induced in cells from non-cancerous breast tissue is critical for understanding possible breast cancer risk from chemical exposure. We have demonstrated previously that exposure to the ubiquitous xenoestrogens, bisphenol A (BPA) and methyl paraben, promotes the hallmarks of cancer in non-malignant human high-risk donor breast epithelial cells (HRBECs) isolated from several donors. Here we show that terephthalic acid (TPA), a major chemical precursor of polyethylene terephthalate (PET) containers used for the storage of food and beverages, increased the ERα: ERß ratio in multiple HRBEC samples, suggesting an estrogenic effect. Although, like BPA and methyl paraben, TPA also promoted resistance to tamoxifen-induced apoptosis, unlike these chemicals instead of inducing an increased S-phase fraction, TPA treatment arrested cell proliferation. DNA-PK, ATM and members of the MRN complex, known to be involved in DNA damage sensor and effector proteins, were elevated indicating induction of DNA strand breaks. Early DNA damage checkpoint response, mediated through p53/p21, led to G1 arrest in TPA-exposed cells. Removal of TPA from the growth medium resulted in the rapid induction of BCL2, increasing the ratio of anti-: pro-apoptotic proteins, together with overexpression of Cyclin A/CDK2 proteins. Consequently, despite elevated p53(pSer15) and H2AX(pSer139), indicating sustained DNA damage, TPA exposed cells resumed robust growth rates seen prior to TPA exposure. The propensity for the perpetuation of DNA aberrations that activate DNA damage pathways in non-malignant breast cells justifies careful consideration of human exposure to TPA, particularly at vulnerable life stages.

Bisphenol-A-induced inactivation of the p53 axis underlying deregulation of proliferation kinetics, and cell death in non-malignant human breast epithelial cells.

Widespread distribution of bisphenol-A (BPA) complicates epidemiological studies of possible carcinogenic effects on the breast because there are few unexposed controls. To address this challenge, we previously developed non-cancerous human high-risk donor breast epithelial cell (HRBEC) cultures, wherein BPA exposure could be controlled experimentally. BPA consistently induced activation of the mammalian target of rapamycin (mTOR) pathway--accompanied by dose-dependent evasion of apoptosis and increased proliferation--in HRBECs from multiple donors. Here, we demonstrate key molecular changes underlying BPA-induced cellular reprogramming. In 3/3 BPA-exposed HRBEC cell lines, and in T47D breast cancer cells, proapoptotic negative regulators of the cell cycle (p53, p21(WAF1) and BAX) were markedly reduced, with concomitant increases in proliferation-initiating gene products (proliferating cell nuclear antigen, cyclins, CDKs and phosphorylated pRb). However, simultaneous exposure to BPA and the polyphenol, curcumin, partially or fully reduced the spectrum of effects associated with BPA alone, including mTOR pathway proteins (AKT1, RPS6, pRPS6 and 4EBP1). BPA exposure induced an increase in the ERα (Estrogen Receptor): ERß ratio--an effect also reversed by curcumin (analysis of variance, P < 0.02 for all test proteins). At the functional level, concurrent curcumin exposure reduced BPA-induced apoptosis evasion and rapid growth kinetics in all cell lines to varying degrees. Moreover, BPA extended the proliferation potential of 6/6 primary finite-life HRBEC cultures--another effect reduced by curcumin. Even after removal of BPA, 1/6 samples maintained continuous growth--a hallmark of cancer. We show that BPA exposure induces aberrant expression of multiple checkpoints that regulate cell survival, proliferation and apoptosis and that such changes can be effectively ameliorated.

Activation of the mTOR pathway by low levels of xenoestrogens in breast epithelial cells from high-risk women.

Breast cancer is an estrogen-driven disease. Consequently, hormone replacement therapy correlates with disease incidence. However, increasing male breast cancer rates over the past three decades implicate additional sources of estrogenic exposure including wide spread estrogen-mimicking chemicals or xenoestrogens (XEs), such as bisphenol-A (BPA). By exposing renewable, human, high-risk donor breast epithelial cells (HRBECs) to BPA at concentrations that are detectable in human blood, placenta and milk, we previously identified gene expression profile changes associated with activation of mammalian target of rapamycin (mTOR) pathway genesets likely to trigger prosurvival changes in human breast cells. We now provide functional validation of mTOR activation using pairwise comparisons of 16 independent HRBEC samples with and without BPA exposure. We demonstrate induction of key genes and proteins in the PI3K-mTOR pathway--AKT1, RPS6 and 4EBP1 and a concurrent reduction in the tumor suppressor, phosphatase and tensin homolog gene protein. Altered regulation of mTOR pathway proteins in BPA-treated HRBECs led to marked resistance to rapamycin, the defining mTOR inhibitor. Moreover, HRBECs pretreated with BPA, or the XE, methylparaben (MP), surmounted antiestrogenic effects of tamoxifen showing dose-dependent apoptosis evasion and induction of cell cycling. Overall, XEs, when tested in benign breast cells from multiple human subjects, consistently initiated specific functional changes of the kind that are attributed to malignant onset in breast tissue. Our observations demonstrate the feasibility of studying renewable human samples as surrogates and reinforce the concern that BPA and MP, at low concentrations detected in humans, can have adverse health consequences.

Distinctive responsiveness to stromal signaling accompanies histologic grade programming of cancer cells.

Whether stromal components facilitate growth, invasion, and dissemination of cancer cells or suppress neoplastic lesions from further malignant progression is a continuing conundrum in tumor biology. Conceptualizing a dynamic picture of tumorigenesis is complicated by inter-individual heterogeneity. In the post genomic era, unraveling such complexity remains a challenge for the cancer biologist. Towards establishing a functional association between cellular crosstalk and differential cancer aggressiveness, we identified a signature of malignant breast epithelial response to stromal signaling. Proximity to fibroblasts resulted in gene transcript alterations of >2-fold for 107 probes, collectively designated as Fibroblast Triggered Gene Expression in Tumor (FTExT). The hazard ratio predicted by the FTExT classifier for distant relapse in patients with intermediate and high grade breast tumors was significant compared to routine clinical variables (dataset 1, n = 258, HR--2.11, 95% CI 1.17-3.80, p-value 0.01; dataset 2, n = 171, HR--3.07, 95% CI 1.21-7.83, p-value 0.01). Biofunctions represented by FTExT included inflammatory signaling, free radical scavenging, cell death, and cell proliferation. Unlike genes of the 'proliferation cluster', which are overexpressed in aggressive primary tumors, FTExT genes were uniquely repressed in such cases. As proof of concept for our correlative findings, which link stromal-epithelial crosstalk and tumor behavior, we show a distinctive differential in stromal impact on prognosis-defining functional endpoints of cell cycle progression, and resistance to therapy-induced growth arrest and apoptosis in low vs. high grade cancer cells. Our experimental data thus reveal aspects of 'paracrine cooperativity' that are exclusively contingent upon the histopathologically defined grade of interacting tumor epithelium, and demonstrate that epithelial responsiveness to the tumor microenvironment is a deterministic factor underlying clinical outcome. In this light, early attenuation of epithelial-stromal crosstalk could improve the management of cases prone to be clinically challenging.

Optimization of clinical breast examination.

BACKGROUND: Breast examination is necessary for evaluation of the 8% to 17% of cancers missed by mammograms, but it is being done less often and less effectively. To improve the use of breast examination, we tested whether a technique to focus attention could improve the call rate (the percent of examinations leading to further evaluation), a measure of quality, without retraining in examination technique. METHODS: Clinicians were randomized to complete 1 of 2 dedicated, de-identified forms after routine breast examination: a long form intended to focus attention by requesting general breast descriptors along with clinical information and breast examination findings (10 clinicians recorded 964 examinations) or a short form recording only clinical information and examination findings (11 clinicians recorded 558 examinations). There was no technique retraining. Study call rates were compared with historical controls (298 breast examinations by 16 clinicians). RESULTS: The call rates by the study groups of clinicians were similar, but the call rate using either form (8.3%) was significantly higher than the call rate in the preceding year when no dedicated form was used (4.7%; P=.031), suggesting a Hawthorne effect in which altering conditions of data collection (using the dedicated forms) functioned as an independent variable. Surveillance, Epidemiology, and End Results data predicted 3.4 cancers in all 1822 patients; 4 cancers were found. CONCLUSION: Breast examination call rate doubled when attention was focused on examination results using a dedicated form, and we found the anticipated cancers. Breast examination quality can be improved by focusing clinician attention without retraining in technique.

Bisphenol A induces a profile of tumor aggressiveness in high-risk cells from breast cancer patients.

Breast cancer outcome is highly variable. Whether inadvertent exposure to environmental xenobiotics evokes a biological response promoting cancer aggressiveness and a higher probability of tumor recurrence remains unknown. To determine specific molecular alterations which arise in high-risk breast tissue in the presence of the ubiquitous xenoestrogen, bisphenol A (BPA), we used nonmalignant random periareolar fine-needle aspirates in a novel functional assay. Early events induced by BPA in epithelial-stromal cocultures derived from the contralateral tissue of patients with breast cancer included gene expression patterns which facilitate apoptosis evasion, endurance of microenvironmental stress, and cell cycle deregulation without a detectable increase in cell numbers. This BPA response profile was significantly associated with breast tumors characterized by high histologic grade (P < 0.001) and large tumor size (P = 0.002), resulting in decreased recurrence-free patient survival (P < 0.001). Our assays show a biological "fingerprint" of probable prior exposure to endocrine-disrupting agents, and suggest a scenario in which their presence in the microenvironmental milieu of high-risk breast tissue could play a deterministic role in establishing and maintaining tumor aggressiveness and poor patient outcome.

New models and online calculator for predicting non-sentinel lymph node status in sentinel lymph node positive breast cancer patients.

BACKGROUND: Current practice is to perform a completion axillary lymph node dissection (ALND) for breast cancer patients with tumor-involved sentinel lymph nodes (SLNs), although fewer than half will have non-sentinel node (NSLN) metastasis. Our goal was to develop new models to quantify the risk of NSLN metastasis in SLN-positive patients and to compare predictive capabilities to another widely used model. METHODS: We constructed three models to predict NSLN status: recursive partitioning with receiver operating characteristic curves (RP-ROC), boosted Classification and Regression Trees (CART), and multivariate logistic regression (MLR) informed by CART. Data were compiled from a multicenter Northern California and Oregon database of 784 patients who prospectively underwent SLN biopsy and completion ALND. We compared the predictive abilities of our best model and the Memorial Sloan-Kettering Breast Cancer Nomogram (Nomogram) in our dataset and an independent dataset from Northwestern University. RESULTS: 285 patients had positive SLNs, of which 213 had known angiolymphatic invasion status and 171 had complete pathologic data including hormone receptor status. 264 (93%) patients had limited SLN disease (micrometastasis, 70%, or isolated tumor cells, 23%). 101 (35%) of all SLN-positive patients had tumor-involved NSLNs. Three variables (tumor size, angiolymphatic invasion, and SLN metastasis size) predicted risk in all our models. RP-ROC and boosted CART stratified patients into four risk levels. MLR informed by CART was most accurate. Using two composite predictors calculated from three variables, MLR informed by CART was more accurate than the Nomogram computed using eight predictors. In our dataset, area under ROC curve (AUC) was 0.83/0.85 for MLR (n = 213/n = 171) and 0.77 for Nomogram (n = 171). When applied to an independent dataset (n = 77), AUC was 0.74 for our model and 0.62 for Nomogram. The composite predictors in our model were the product of angiolymphatic invasion and size of SLN metastasis, and the product of tumor size and square of SLN metastasis size. CONCLUSION: We present a new model developed from a community-based SLN database that uses only three rather than eight variables to achieve higher accuracy than the Nomogram for predicting NSLN status in two different datasets.

Closure of partial mastectomy.

A longstanding controversy surrounds whether to close breast parenchyma at the completion of a partial mastectomy for breast cancer. Modification of a technique described 80 years ago finds the middle ground between various opinions and simultaneously addresses 2 issues: (1) approximation of the deep and superficial surfaces of the parenchyma, without sutures within the parenchyma, minimizes "dents;" and (2) a radial suture line preserves the distance from the nipple to the periphery of the breast which minimizes traction on the nipple.