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Knowledge about established breast carcinogens can support improved 21st-century toxicologic testing by identifying key mechanistic events. To clarify mechanisms, we developed adverse outcome pathways (AOPs) for two well-established breast carcinogens: ionizing radiation (IR) and the pharmaceutical hormone diethylstilbestrol (DES). The AOP for ionizing radiation includes these elements: IR increases production of reactive oxygen and nitrogen species (RONS), and directly and indirectly causes DNA damage. DNA damage leads to mutations and proliferation, leading to increased breast cancer. RONS also increases inflammation, contributing to direct and indirect effects (effects in cells not directly reached by IR) via positive feedback to RONS, and separately increases breast cancer risk through increased proliferation of cells and increased tumor growth and invasion. Estrogen and progesterone also influence the risk of breast cancer from ionizing radiation, as does developmental stage. For in utero estrogens like DES, the AOP links gestational estrogen exposure to transcriptional activity that promotes altered signaling between the epithelial and stromal tissue compartments, leading to disrupted development, including altered tensional homeostasis and tissue architecture, inflammation, and altered cellular differentiation. These key events lead to several adverse outcomes at the tissue and organ level that increase susceptibility to breast cancer, including altered density, structure, and hormone sensitivity along with hyperplasia. Epigenetic alterations are a cellular-level outcome that propagates gestational estrogen exposure to later-life risk through cellular memory that directs ER-mediated gene expression and altered mammary development. Increased susceptibility to breast cancer follows from these adverse outcomes. Dose-response and developmental timing of exposure are discussed in both AOPs. These AOPs provide a framework for evaluating the potential for mechanistically similar stressors—e.g., genotoxicants/RONS-inducers may act like IR, or estrogenic chemicals in utero may act like DES—to affect breast cancer risk. Citation Format: Jessica Helm, Andrea Hindman, Ruthann A. Rudel. Mechanistic pathways for breast carcinogens can highlight early effects as predictive endpoints for chemical safety testing: Case studies for ionizing radiation and for in utero exposure to estrogens such as DES [abstract]. In: Proceedings of the AACR Special Conference on Environmental Carcinogenesis: Potential Pathway to Cancer Prevention; 2019 Jun 22-24; Charlotte, NC. Philadelphia (PA): AACR; Can Prev Res 2020;13(7 Suppl): Abstract nr A51.

Over 200 chemicals have been associated with altered mammary gland development and cancer in rodent studies, yet the molecular mechanisms by which this diverse set of chemicals affect the breast remain poorly understood. We examined gene expression profiles of eight known and suspected mammary toxicants using the TempO-Seq platform and a targeted gene expression panel, Breast Carcinogen Screen (BCScreen). The BCScreen panel has 500 genes with at least 30 genes strongly associated with each of the 14 biologic processes relevant to breast cancer and mammary gland development. We compared gene expression profiles in MCF-7 cells treated for 24hrs with four concentrations (1 nM to 10 uM) of the following chemicals: three estrogenic chemicals (genistein, bisphenol A, butyl benzyl phthalate); the selective estrogen receptor modulator, tamoxifen (TAM); 1,4 benzoquinone (BQ), a genotoxic metabolite of the rodent mammary carcinogen benzene; perfluorooctanoic acid (PFOA), a persistent pollutant that alters mammary gland development; and two closely related perfluorinated chemicals that have not been evaluated for mammary toxicity/effects (PFHXA, PFNA). We identified expression patterns activated by mammary toxicants with known ER agonist activity (genistein, BPA, BBP). Some of these genes were suppressed by PFOA and BQ and TAM, mammary toxicants with antagonist or no known direct ER activity. The known weak estrogens genistein, BPA, and BBP had similar gene expression profiles and were enriched for genes associated with cell cycle and DNA repair, particularly at higher doses. Notably, PFOA, Tam, and BQ had greater gene expression changes at lowest (1nM) vs. higher doses and were enriched for genes associated with cell cycle. For context, serum PFOA levels in the general US population are generally five to ten times higher than the 1 nM dose where we observed these changes (i.e., > 0.4 micrograms/L). PFOA profiles were enriched for several transcription factors including E2F1 and E2F4, which regulate cell cycle genes and important processes in mammary development and tumor progression. Utilizing the BCScreen gene panel, we have identified mechanistic targets relevant to breast cancer that are perturbed by mammary toxicants. Citation Format: Vanessa Y. De La Rosa, Erik Lehnert, Marko Milanovic, Chris Vulpe, Ruthann A. Rudel. Targeted expression profiling identifies mechanisms of potential breast carcinogens [abstract]. In: Proceedings of the AACR Special Conference on Environmental Carcinogenesis: Potential Pathway to Cancer Prevention; 2019 Jun 22-24; Charlotte, NC. Philadelphia (PA): AACR; Can Prev Res 2020;13(7 Suppl): Abstract nr A52.

The recognition that biologic activities of carcinogens extend beyond “carcinogen = DNA-damaging agent” creates a roadmap for improved approaches to test chemicals and predict which ones are likely to increase cancer risk, so we can avoid them. For breast cancer, for instance, the established risks from hormone treatment in utero or later in life are examples of the powerful influence of receptor-mediated effects in carcinogenesis. Breast cancer research also implicates other important biologic activities including inflammation, oxidative stress, epigenetic alterations, and DNA repair, while the sensitivity of the breast to DNA-damaging agents is demonstrated by effects of ionizing radiation. In addition, over 200 diverse chemicals that cause mammary gland tumors in rodents provide insight into important structural features and biologic activities for breast carcinogenesis. For example, these chemicals include many halogenated hydrocarbons, aromatic amino/nitro compounds, and epoxide-forming chemicals, as well as hormones. Also, the increased risk associated with in utero hormone exposure highlights that new models are needed to detect chemicals that produce these effects and to predict effects on risk. To protect public health, it is necessary to develop and apply rapid, sensitive, and cost-effective chemical toxicity screening for the thousands of chemicals in commercial use. The characteristics of carcinogens provide a useful set of biologic targets for such testing. As an example that applies this concept, we developed a 500-gene panel called BCScreen that includes genes associated with 14 characteristics of breast carcinogens. In MCF-7, expression patterns distinguish known estrogenic chemicals like BPA and genistein from PFOA, a highly fluorinated pollutant that has been shown to alter mammary gland development. An important consideration is that responses in common in vitro models, most of which are cancer cell lines, may be different from normal tissues. Normal tissue models may provide more relevant output but also pose challenges. For example, expression profiling of estradiol response in normal breast epithelial organoids that maintain ER function shows different responses compared with MCF-7, and organoids from different individuals also had distinct responses. Assays that can detect chemicals that activate estrogen receptor or increase synthesis of estradiol or progesterone also highlight chemicals of concern for breast cancer. While human studies have not been especially helpful for detecting cancer risk associated with thousands of commercial chemicals, approaches that determine the biologic activities of the known human carcinogens and flag chemicals with similar activities may provide a pathway to prevention, and new methods are needed to do this. Risk predictions for environmental chemicals also must take into account the cofactors that influence background cancer risk, including host susceptibility and coexposures. Citation Format: Ruthann Rudel. High-throughput methods for screening for breast carcinogens [abstract]. In: Proceedings of the AACR Special Conference on Environmental Carcinogenesis: Potential Pathway to Cancer Prevention; 2019 Jun 22-24; Charlotte, NC. Philadelphia (PA): AACR; Can Prev Res 2020;13(7 Suppl): Abstract nr IA14.