Mammary Gland as a Sensitive End Point to Effects of Endocrine Disruptors

National Institute Of Environmental Health Sciences

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Abstract

Our research is focused on understanding the effects of high priority environmental chemicals on mammary gland (MG) development, function, and cancer susceptibility. We have expanded this focus area in recent years to include the effect of environmental chemicals on breast cancer risk factors such as pregnancy-related birth outcomes, puberty timing, obesity, breast density and lactation. The use of animal models for human disease allows us to evaluate different routes of exposure, internal dose of the test chemical, as well as a variety of effects in the rodent that are relevant to human health. During the last several years, we have produced numerous important documents that are being used in the risk regulation of brominated flame retardants (DE-71), perfluorooctanoic acid (PFOA) and atrazine (ATR). Recently we added GenX (HFPO-DA), a replacement chemical for PFOA, to that list. Flame retardants and perfluorinated chemicals, such as PFOA and GenX, are currently regulated at the state level and federal risk assessments are beginning. In the female rodent offspring, the mammary gland is one of the most endocrine-sensitive end points that we have evaluated following prenatal chemical exposures. Our studies using volatile organic chemicals (VOCs), bisphenol analogues, PFOA, GenX, other PFAS mixtures, tetrabromobisphenol A (TBBPA), and arsenic all demonstrate this finding. We have conducted studies evaluating mammary effects of arsenic in mice, human relevant VOC mixtures in rats, PFOA and GenX mechanism of action and strain differences in mice, and two BPA replacement chemicals in mice. BPS and BPAF demonstrated low dose effects of bisphenol analogues on mammary lesions following exposure in utero. In 2020, we published in Environmental Health Perspectives our evaluation and analyses of the mammary samples from the BPA-CLARITY study and a manuscript comparing health effects of PFOA and GenX in pregnant mice and their developing fetuses. The latter work was cited more than 30 times in the first year. We have made major progress on our work involving investigation of VOCs and their developmental exposure effects on the mammary gland, specifically in males. These compounds are theorized to have a role in male breast cancer diagnoses in men who were born or grew up at the Camp Lejeune military base in NC, USA. We have nearly finished dosimetry studies in the pregnant and lactating rat and her offspring in conjunction with our CDC collaborators. We have evaluated mammary gland development and carcinogen-induced mammary tumors following prenatal exposure and are finalizing manuscripts on the effects of these compounds in male and female rats. With this study and our mammary gland atlas of Harlan Sprague Dawley rats that was published two years ago, we have added significantly to what is known about male rodent mammary gland development, susceptibility to endocrine disruption, and carcinogen-induced mammary cancer. We are working with Melissa Troester, an epidemiologist at UNC, to document normal aging of the male and female mammary gland of mice and rats, in comparison to her human samples. She has shown that in women with breast cancer there is an interruption of the normal aging process, and by using chemically-exposed rodent tissues, we may determine which types of chemicals may predispose populations to increased breast cancer risk via that mode of action. Due to COVID-19, this work was not finished this year, as we had planned. In late 2018, we published a study describing susceptibility to mammary gland hyperplasia in female mice exposed to bisphenol A (BPA), BPAF and BPS (fluorinated and sulfonated forms) in early life. These studies cover important susceptible tissues not evaluated in other contracted NTP studies and add to information garnered in the BPA-CLARITY study and other DNTP bisphenol work. Disposition study manuscripts are written and will be put into clearance soon. These studies have been delayed due to low access to mass spec time in the NIEHS core facility and in NTPL. Our recent studies involving TBBPA and Firemaster are large collaborative studies where we either conducted the study for many PIs to get target tissues of interest (TBBPA) or we are providing a service of isolating and evaluating mammary tissue (Firemaster). Three papers were published thus far and three remain, but have been delayed due to COVID-19 and access to the lab. Both of these flame retardants seem to act as endocrine disruptors, affecting multiple reproductive tissues, fat cells, and hormone levels. We finalized our lab-based work on a round robin global validation effort assessing the methods and 3T3-L1 cell sources used to assess adipogenesis and lipogenesis. The Fenton lab was one of many providing data, interpretation and written comments on the resulting manuscript. This work was coordinated by Chris Kassotis, of Wayne State University, and was accepted for publication in August 2021. Other projects having to do with obesogen testing have been slow to finish, as we changed imaging equipment in mid-stream and COVID-19 left some employees critical to this project with little access to lab. Since 2018, there has been an emergent need to evaluate the effects of uncharacterized perfluoroalkyl acids (PFAS) and related compounds for health effects and mechanisms of action. We have developed a set of in vitro assays (complimented by our colleagues in the in silico and in vivo areas) that will provide us an our collaborators at the EPA with a rapid understanding of which groups of PFAS are targeting tissues known to be affected by the legacy compounds before them that are no longer produced in the U.S. We are testing nearly 50 of the most common compounds in numerous cell types to determine bench mark doses across the group. We have co-authored a paper with Rebecca Fry's lab describing gene changes in human placental cells following serum plus/minus culture conditions. Three other papers describing the effects of PFAS on human placental, liver, and ovarian cancer cells are in final stages and moving toward clearance. These important contributions will move the field forward in high throughput testing of the PFAS. Our in vitro work testing contemporary AFFF mixtures in the liver has produced a paper, with our EPA and Harvard collaborators, that identified 14 PFAS compounds in the AFFF, most of which there is no health data published. Our work defining the potential hepatic effects of AFFF and the PFAS it contains is in final stages of being written. Studies are being planned to test the primary PFAS in AFFF for health effects in mice. Our work in mice comparing GenX and PFOA effects in the offspring exposed in utero is under review at the journal and shows significant potential for GenX to cause liver and metabolic disease, in a sex-dependent manner. Additional studies addressing mechanisms of action for mammary related effects of PFOA and GenX were finished in 2020 and are in final stages of author review for clearance. Finally, three invited reviews on PFAS health effects were written in 2020. Two were published in early 2021 and one is undergoing final editorial changes for the journal. One of the reviews was so contemporary and thorough that it is being used to guide database updates of the PFAS-TOX Database developed by Dr. Katie Pelch and colleagues.