EVALUATING ENDOCRINE DISRUPTION BY PESTICIDES AND OTHER CONTAMINANTS OF THE FOOD SUPPLY

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: NEW
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1016772
• Project No.: CA-D-ETX-2233-H
• Project Start Date: Oct 1, 2018
• Project End Date: Sep 30, 2023

Project Director
La Merrill, MI.

Recipient Organization
UNIVERSITY OF CALIFORNIA, DAVIS
410 MRAK HALL
DAVIS,CA 95616-8671

Performing Department
Environmental Toxicology

Non Technical Summary
This project supports the mission of the Agricultural Experiment Station by addressing the Hatch Act area(s) of: processing, distribution, safety, marketing, and utilization of food and agricultural products.Chemicals that hurt our hormone systems are critically important hazards that represent pervasive public health problems in the United States such as infertility, obesity, diabetes, and breast cancer. For example obesity can speed up puberty, both of which can contribute to breast cancer risk. Many people are exposed to many chemicals through the food they eat, the water they drink, and the agricultural work they do. Unfortunately, most of these numerous chemicals have completely unknown effects on our health. The animals that we eat may be similarly affected which could negatively impact our food supply. This project focuses on learning more about how these chemicals may influence our hormone systems in order to lay a foundation for keeping us safe while living our daily lives.

Animal Health Component

100%

Research Effort Categories

Basic

85%

Applied

15%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
314	3840	1150	100%

Knowledge Area
314 - Toxic Chemicals, Poisonous Plants, Naturally Occurring Toxins, and Other Hazards Affecting Animals;

Subject Of Investigation
3840 - Laboratory animals;

Field Of Science
1150 - Toxicology;

Keywords

breast cancer

diabetes

endocrine disrupting chemical

food contaminant

obesity

pesticide

puberty

Goals / Objectives
There is a great need for high throughput assessment of endocrine disrupting activity, e.g. chemicals targeting the activity of receptors for estrogen, androgen, thyroid, and insulin. This will contribute to hazard assessment of chemicals of concern, namely those that are generated by agriculture, that have high production or waste volume, and that are found in the Californian food and drinking water supply. This project proposes to address that need. The goal of this project is two fold: 1) to develop an understanding of the endocrine disrupting properties of data-sparse chemicals in the environment (herein "chemicals of concern") and 2) to develop mechanistic insights into the hazards identified in the first goal.First, potential chemicals of concern will be screened in silico for their molecular docking to endocrine receptors. Second, candidate chemicals of concern will be examined for their dose-dependent effects on molecular and cellular receptor activities, such as receptor mediated- transcription and cell proliferation respectively. Third, a data-rich chemical of concern that was biologically active will be evaluated in vivo for endocrine disrupting effects on rodents, such as reproduction, puberty, metabolism, and mammary cancer. Lastly, molecular analyses will be conducted on chemicals of concern that produce endocrine disrupting effects on rodents to develop mechanistic insights into disease.

Project Methods
A. Evaluate molecular docking of chemicals of concern at endocrine receptorsi. Dock chemicals of concern at estrogen receptor alpha and beta, and the androgen receptor as the primary focusii. Pending successful development of docking programs, dock chemicals of concern at insulin- and thyroid hormone- receptorsB. Utilize cell-based in vitro assays to validate chemicals suspected to be EDCs based on their docking scoresi. Assess activity of chemicals of concern at endocrine receptorsa. Quantitate endocrine receptor agonism and antagonism by a subset of priority chemicals of concern (determined from agnostic docking screens) using receptor-specific luciferase assaysii. Determine whether a cellular function is impaired consistent with evidence of receptor action from abovea. Confirm disruption in estrogen and androgen receptor action by evaluating changes in receptor dependent- cellular proliferation caused by chemicals of concernb. Confirm disruption in insulin receptor action by evaluating changes in insulin-stimulated glucose uptake caused by chemicals of concernc. Confirm disruption in thyroid hormone receptor action by evaluating changes in uncoupled respiration caused by chemicals of concernd. Evaluate biological plausibility by further screen mechanistic pathways (e.g. qPCR) to further hazard characterization of toxicants with strongest dose-response and lowest threshold values.C. Conduct in vivo validation experiments exposing developing rodents to chemicals of concern identified above to ascertain the nature and degree of their endocrine disrupting effectsi. Evaluate dose-dependent pubertal and peri-pubertal metabolic outcomes in a data-rich priority chemical to anchor in silico and in vitro data to an in vivo experiment

Progress 10/01/19 to 09/30/20

Outputs
Target Audience:The target audience includes the research community, physicians, concerned community members, and the risk assessors, chemists and others at the California Environmental Protection Agency (CalEPA) and DTSC. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Graduate student Ms. Fecilia Castriota was trained in how to conduct a mouse study with arsenic in drinking water and phenotyping using state of the art metabolic and transcriptome methods. Postdoctoral fellow Dr. Phum Tachachartvanich contributed in vitro PFAS to androgen receptor binding data to refine our in silico androgen receptor model. He tested the androgen receptor in silico predictions about hydraulic fracturing chemicals in vitro. Dr. Tachachartvanich submitted this hydraulic fracturing chemicals research to ENDO 2020, the annual meeting of the Endocrine Society. His abstract was considered so high impact that ENDO featured it in a press release: https://www.endocrine.org/news-and-advocacy/news-room/2020/fracking-chemical-may-interfere-with-male-sex-hormone-receptor How have the results been disseminated to communities of interest?Epidemiology and mechanisms of chemical obesogens in California presented 10/19/19 to Department of Toxic Substances Control (DTSC) of CalEPA, Oakland CA Global health impacts of exposure to endocrine-disrupting chemicals presented 10/24/19 to Collegium Ramazzini (physicians and scientists), Carpi Italy Consensus on the key characteristics of endocrine-disrupting chemicals as a basis for hazard identification presented 12/10/19 to the Collaborative on Health and the Environment (CHE), webinar to citizens and other community members https://www.healthandenvironment.org/webinars/96502 Key characteristics of endocrine-disrupting chemicals with a focus on obesogens presented 2/24/20 to Safer Consumer Products Program at DTSC of CalEPA, Sacramento CA What do you plan to do during the next reporting period to accomplish the goals?We will continue on both Goals and the four objectives as is deemed a priority in our two-way conversations with CalEPA. We specifically expect to publish our in vitro confirmation of PFAS which bind the androgen receptor. We recognize that an Endocrinologist will see patients for diseases such as obesity and diabetes yet the etiology of these disease need not exclusively lay in the endocrine system. Hence future efforts may examine non-endocrine mechanisms of endocrine diseases.

Impacts
What was accomplished under these goals? Goal 1. CalEPA approached our team about the unknown hazard of two classes of chemicals that were contaminating California water, chemicals reported to be used in hydraulic fracturing processes in California and chemicals called PFAS (perfluoroalkyl and polyfluoroalkyl substances). Virtually nothing was known about the potential toxicity of these chemicals when CalEPA approached us. Because there were about 100 hydraulic fracturing chemicals and over 5,000 PFAS chemicals that could be contaminating the water that Californians drink and use in agriculture, the need for toxic hazard assessment was urgent. In our previous reporting period, we developed an in silico model of the androgen receptor. In the current reporting period, we applied this in silico model to two classes of chemicals of concern. We were able to identify a few hydraulic fracturing and PFAS chemicals that bound the androgen receptor in silico (objective 1). For the hydraulic fracturing chemical, Genapol X, we showed that it was causing a dose-dependent inhibition of this endocrine receptor (objective 2). We revealed this with inhibition and activation of androgen receptor-dependent luciferase reporter gene expression, and depressed expression of androgen receptor-regulated genes (objective 2). This was critical information to provide to state risk assessors given that the androgen receptor is essential to male reproduction and also involved in prostate cancer development. CalEPA is utilizing this information as they further evaluate these chemical classes. Goal 2. CalEPA needs us to demonstrate that when our predictive methods are applied to chemicals of unknown hazard, that our predictions are biologically plausible. We have jointly agreed that the best way to address that need is to conduct in depth in vivo and/or molecular analyses with data-rich chemicals of concern. There are two chemicals that continue to demand for the attention of the CalEPA which are data-rich: the pesticides arsenic and DDT. We exposed mice to arsenic in their drinking water at levels of relevance to Californians. We demonstrated that metabolic consequences of experimental arsenic were consistent with human disease risk associated with arsenic (objective 3). We also conducted transcriptomics analysis of mouse adipose to substantiate the biological plausibility of these metabolic toxicities from arsenic (objective 4). We simultaneously conducted a systematic review of the experimental literature on metabolic toxicities from arsenic which revealed insulin (endocrine) disruption. This formal systematic review included dose response analysis to better inform risk assessment of arsenic by CalEPA. We approached the validation of endocrine disruption by DDT and its metabolite DDE with a focus on humans given we have previously elucidated mechanisms with cell- and animal- based approaches. For example, in our previous reporting, our systematic analysis of the DDT and DDE experimental literature demonstrated that DDT and DDE consistently contributed to increased obesity and excess lipids in the liver. Steatosis, a disease defined by fatty liver and its progressive harms, is believed to be common in people because of its association with obesity and insulin resistance. Steatosis is very difficult to diagnose without expensive imaging or invasive biopsy. We leveraged a cohort of immigrants from India, where DDT continues to be manufactured, to California in whom fatty liver was diagnosed with computed tomography (CT) clinical imaging. We found that DDT exposure increased the odds of obesity, insulin resistance and fatty liver among these immigrants to California (objective 3). We leveraged another cohort in California, comprised of people born in the 1960s at Oakland Kaiser Permanente, to show that prenatal DDT exposure during its peak use here in California is associated with increased risk of obesity in middle-age (objective 3). We then pursued evidence for a molecular basis of these metabolic problems in humans in a cohort of morbidly obese adolescents, where we measured DDE and metabolome levels in biopsied fat to show that DDE was associated with metabolic pathways underlying obesity, insulin resistance and fatty liver (objective 4). CalEPA is utilizing this demonstration that DDT and DDE are associated with metabolic diseases similarly occuring in rodents and people with similar underlying molecular mechanisms across these mammalian species.

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: La Merrill MA, Johnson CL, Smith MT, Kandula NR, Macherone A, Pennell KD, Kanaya AM. Exposure to persistent organic pollutants (POPs) and their relationship to hepatic fat and insulin insensitivity among Asian Indian immigrants in the United States. Environmental Science and Technology, 2019, 53,13906-13918, doi:10.1021/acs.est.9b03373.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Azhagiya Singam ER, Tachachartvanich P, Fourches D, Soshilov A, Hsieh JCY, La Merrill MA, Smith MT, Durkin KA. Structure-Based Virtual Screening of Perfluoroalkyl and Polyfluoroalkyl Substances (PFASs) as Endocrine Disruptors of Androgen Receptor Activity Using Molecular Docking and Machine Learning. Environmental Research, 2020.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Tachachartvanich P, Azhagiya Singam,ER, Durkin KA, Smith MT, La Merrill MA. Structure-based discovery of the endocrine disrupting effects of hydraulic fracturing chemicals as novel androgen receptor antagonist. Chemosphere, 2020, 257,127178, doi: 10.1016/j.chemosphere.2020.127178.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: La Merrill MA, Krigbaum NY, Cirillo PM, Cohn BA. Association between maternal exposure to the pesticide dichlorodiphenyltrichloroethane (DDT) and risk of obesity. International Journal of Obesity, 2020, doi: 10.1038/s41366-020-0586-7, PMID 32415255.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Castriota F, Rieswijk L, Dahlberg S, La Merrill MA, Steinmaus C, Smith MT, Wang J-C. A state-of-the-science review of arsenics effects on glucose homeostasis in experimental models. Environmental Health Perspectives, 2020, doi:10:12.89/EHP4517.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Castriota F, Zushin PJ, Sanchez SS, Phillips RV, Hubbard A, Stahl A, Smith MT, Wang JC, La Merrill MA. Chronic arsenic exposure impairs adaptive thermogenesis in male C57BL/6J mice. American Journal of Physiology-Endocrinology and Metabolism, 2020, E667-E677, doi:10.1152/ajpendo.00282.2019.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Valvi D, Walker D, Inge T, Bartell S, Jenkins T, Helmrath M, Ziegler T, La Merrill MA, Eckel S, Conti D, Liang Y, McConnell R, Chatzi L. Environmental chemical burden in metabolic tissues and systemic biological pathways in adolescent bariatric surgery patients: A pilot untargeted metabolomic approach. Environmental International, 2020, 143:105957, doi:10.1016/j.envint.2020.105957, PMID 32683211.

Progress 10/01/18 to 09/30/19

Outputs
Target Audience:The target audience is the California EPA (CalEPA). Dr La Merrill gave three presentations to the CalEPA Office of Environmental Health Hazard Assessment (OEHHA) and one presentation to the CalEPA Department of Toxic Substances Control (DTSC). Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Postdoctoral fellow Dr. Annalise vonderEmbse has conducted immunohistochemical staining and ran the analysis scripts she wrote for high content image analysis of stellate and brown adipose immunohistochemistry of adult mice (Aim 2a). She is currently working with Drs. La Merrill and Lein to revise this manuscript for publication. She identified that mice have impaired thermogenesis as early as parturition (Aim 1a), and conducted and interpreted omics analyses of this neonatal mouse tissue (Aim 1b). She is currently conducting immunohistochemical staining of these neonatal mouse tissues (Aim 2a). Integrated Genetics and Genomics Graduate student Kyle Jackson drafted two manuscripts related to brown adipose DNA methylation: in one he has identified candidate genes that persist across different ages of mice and humans (we integrated human epigenome data funded elsewhere). In the second manuscript he demonstrates persistent molecular signals in the brown adipose of young and adult mice. Pharmacology and Toxicology Graduate student Julianne Jugan has been validating various genes for their DNA methylation changes in brown adipocytes treated with DDT in vitro. She presented her research at a local retreat, of the UCSF Diabetes Center. Postdoctoral fellow Dr. Phum Tachachartvanich has been validating various genes for their DNA methylation changes in brown adipocytes treated with DDT in vitro. He has also been exploring the effects of DDT upstream of thermogenesis after treatment with DDT in vitro. All of these trainees attended weekly lab meetings where they presented their data and trouble-shot experiments. They also attended weekly journal paper reading club to learn more about the biology and methods underlying our research area. Kyle met with Dr. La Merrill weekly and attends a bimonthly oral exams practice meeting to provide further mentoring and training opportunities. How have the results been disseminated to communities of interest?Dr La Merrill gave three presentations to the CalEPA Office of Environmental Health Hazard Assessment (OEHHA) and one presentation to the CalEPA Department of Toxic Substances Control (DTSC). What do you plan to do during the next reporting period to accomplish the goals?Dr. La Merrill plans to continue to evaluate the mechanism of metabolic toxicity caused by DDT and DDE. Dr. La Merrill is also working closely with the CalEPA OEHHA to screen per- and poly- fluoroalkyl acids (PFAAs) for their endocrine disruption. There are over 7000 PFAAs and they are ubiquitously found in water supplies in the United States including California. Most of these PFAAs have not had any toxicity testing but we know they can be taken up by plants and animals and thus may adversely impact agriculture.

Impacts
What was accomplished under these goals? Our publications on the metabolic toxicity of the pesticide DDT and its persistent metabolite DDE highlight additional concerns about ongoing contamination of the California food supply by DDE and DDT. Another endocrine disruptor, tributyl tin, was used as a marine anti-foulant and can contaminant the products of the offcoast California seafood industry. We also used our androgen receptor model to screen for androgenic activity of California hydraulic fracturing chemicals. These chemicals are of concern to the CalEPA due to their potential to contaminant ground and drinking water sources. We found one chemical inhibited androgen receptor activity and this research is currently under review at a journal.

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: 1. Cohn BA, Cirillo PM, La Merrill MA. Correlation of body mass index with serum DDTs predicts lower risk of breast cancer before the age of 50: prospective evidence in the Child Health and Development Studies. Journal of Exposure Science & Environmental Epidemiology, 2019, doi:10.1038/s41370-018-0072-7, PMID 30224754.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: 1. da Costa CS, Miranda-Alves L, La Merrill MA, Silva IV, Graceli JB. The tributyltin leads to obesogenic mammary gland abnormalities in adult female rats. Toxicology Letters, 2019, 307: 59-71, doi:10.1016/j.toxlet.2019.02.016.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: 1. Hu X, Li S, Cirrilo P, Krigbaum N, Tran V, Ishikawa T, La Merrill MA, Jones DP, Cohn B. Metabolome Wide Association Study of serum DDT and DDE in Pregnancy and Early Postpartum, Reproductive Toxicology, 2019, doi:10.1016/j.reprotox.2019.05.059.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: 1. Azhagiya Singam ER, Tachachartvanich P, La Merrill MA, Smith MT, Durkin KA. Structural Dynamics of Agonist and Antagonist Binding to the Androgen Receptor, Journal of Physical Chemistry B, 2019, 123, 7657-7666, doi:/10.1021/acs.jpcb.9b05654.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: 1. Elmore SE, La Merrill MA*. Oxidative phosphorylation impairment by DDT and DDE, Frontiers in Endocrinology, 2019, 10:1-8, doi:10.3389/fendo.2019.00122.