Prenatal Bisphenol A Exposure in Mice Induces Multitissue Multiomics Disruptions Linking to Cardiometabolic Disorders

The health impacts of endocrine-disrupting chemicals (EDCs) remain debated, and their tissue and molecular targets are poorly understood. In this study, we leveraged systems biology approaches to assess the target tissues, molecular pathways, and gene regulatory networks associated with prenatal exposure to the model EDC bisphenol A (BPA). Prenatal BPA exposure at 5 mg/kg/d, a dose below most reported no-observed-adverse-effect levels, led to tens to thousands of transcriptomic and methylomic alterations in the adipose, hypothalamus, and liver tissues in male offspring in mice, with cross-tissue perturbations in lipid metabolism as well as tissue-specific alterations in histone subunits, glucose metabolism, and extracellular matrix. Network modeling prioritized main molecular targets of BPA, including Pparg, Hnf4a, Esr1, Srebf1, and Fasn as well as numerous less studied targets such as Cyp51 and long noncoding RNAs across tissues, Fa2h in hypothalamus, and Nfya in adipose tissue. Lastly, integrative analyses identified the association of BPA molecular signatures with cardiometabolic phenotypes in mouse and human. Our multitissue, multiomics investigation provides strong evidence that BPA perturbs diverse molecular networks in central and peripheral tissues and offers insights into the molecular targets that link BPA to human cardiometabolic disorders. A central concept in the Developmental Origins of Health and Disease states that adverse environmental exposure during early developmental stages is an important determinant for later-onset adverse health outcomes, even in the absence of continuous exposure in adulthood (1–3). Bisphenol A (BPA) is one of the most prevalent environmental metabolic disruptors identified to date, with widespread exposure in human populations, and likely plays a role in Developmental Origins of Health and Disease (3–5). BPA is used in the production of synthetic polymers, including epoxy resins and polycarbonates and, with its advantageous mechanical properties, is ubiquitously found in everyday goods such as plastic bottles and inner coating of canned foods (6, 7). The ability of BPA to leach from these everyday products is a primary route of human exposure (8). BPA has been shown to have the ability to disrupt endocrine signaling important for many biological functions and has been linked to body weight, obesity, insulin resistance, diabetes, metabolic syndrome (MetS), and cardiovascular diseases in both human epidemiologic and animal studies (9–17). Importantly, it has been suggested that the developing fetus is particularly vulnerable to BPA exposure (9, 18). Intrauterine growth retardation has been consistently observed after developmental BPA exposure at intake doses below the suggested human safety level and has been associated with low birth weight, elevated adult fat weight, and altered glucose homeostasis (9, 19–22). As a precaution, BPA has been banned from baby products in Europe, Canada, and the United States. However, BPA is still in use in nonbaby products, renewing concerns about the continuous exposure of populations in addition to the description of its ability to influence health outcomes, including obesity and MetS, over several generations (23–26). Together, these lines of evidence support an intriguing hypothesis that BPA may have been a contributing factor to the rise of MetS and cardiometabolic diseases worldwide in the past decades (27–29). Despite numerous studies connecting BPA with adverse health outcomes, there remain ample conflicting findings, as summarized by the European Food Safety Agency (30), the BPA Joint Emerging Science Working Group of the US Food and Drug Administration (31), and the recent National Toxicology Program report, CLARITY-BPA, in which functions of multiple organs were examined (32). Although inconsistencies across studies might be attributable to nonmonotonic dose response, exposure window difference, and varying susceptibility among testing models (14, 33), there are also several additional layers of complexity and challenges hindering the full dissection of the biological effects of BPA. First, previous studies examining BPA in various cell types and tissues suggest a broad impact on biological systems (25, 34–36). Second, BPA has been found to modulate multidimensional molecular events, such as gene expression and epigenetic changes, which are functionally important for processes such as metabolism and immune response (37–42). However, due to most studies being designed to focus on one factor at a time as well as noncomparable study designs, it is difficult to directly compare effects across 1/26/21, 7(35 AM Prenatal Bisphenol A Exposure in Mice Induces Multitissue Multiomics Disruptions Linking to Cardiometabolic Disorders Page 2 of 23 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6349005/ tissues or types of molecular data to derive the molecular rules of sensitivity to BPA exposures. These research gaps in our understanding of the pleiotropy of endocrine-disrupting chemicals and toxicant biological actions necessitated the establishment of the National Institute for Environmental Health Sciences (NIEHS) TaRGET consortium (43) and a more recent call for the research community to systemically interrogate multiple -omics in multiple tissues to accelerate the discovery of key biological fingerprints of environmental exposure (44). Here, we address some of the aforementioned limitations of past studies by using a highly integrative approach. We conducted a multitissue, multiomics systems biology study to examine the systems-level influence of prenatal BPA exposure using modern integrative genomics and network modeling approaches in a mouse model (Fig. 1A). We first used next-generation sequencing technologies to characterize perturbations in both the transcriptome and the epigenome across three tissues (white adipose tissue, hypothalamus, and liver) in mouse offspring that had experienced in utero exposure to BPA. These tissues were chosen due to their important role in energy and metabolic homeostasis. The hypothalamus is the central regulator of endocrine and metabolic systems and plays a critical role in the regulation of nutrient and energy sensing, feeding, and energy expenditure (45); liver is critical for glucose and cholesterol homeostasis (46); and white adipose tissue is essential for energy and lipid storage, serves as a endocrine organ secreting numerous hormones related to metabolic regulation, and contributes to inflammatory processes by releasing various cytokines (47, 48). These tissues interact with one another to coordinately regulate metabolism and energy balance. Based on mounting evidence that genes operate in highly complex tissue-specific regulatory networks, we hypothesized that prenatal BPA exposure induces genomic and epigenomic reprogramming in the offspring by affecting the organization and functions of tissue-specific gene networks (49–52). Using both transcription factor (TF) and Bayesian networks (BNs), we modeled the dynamics of transcriptomic and epigenomic signatures and predicted potential regulators that govern the actions of BPA. Furthermore, the transcriptome, epigenome, and network information were layered upon metabolic phenotypes such as body weight, adiposity, circulating lipids, and glucose levels in the mouse offspring to evaluate disease association. Lastly, to assess the relevance of the BPA molecular targets identified in our mouse model for human diseases, we applied integrative genomics to bridge the mouse molecular signatures and genetic disease association data from human studies. Our study represents a comprehensive data-driven, systemslevel investigation of the molecular and health impact of BPA. Figure 1. Overall study design and the measurements of metabolic traits in male and female offspring. (A) Framework of multiomics approaches to investigate the impact of prenatal BPA exposure. (B and C) Comparison of body weight, serum lipids, and glucose level . Materials and Methods Ethics statement All animal experiments were performed in accordance with the Institutional Animal Care and Use Committee guidelines. Animal studies and procedures were approved by the Chancellor’s Animal Research Committee of the University of California, Los Angeles. Mouse model of prenatal BPA exposure Inbred C57BL/6J mice were maintained on a special diet (5V01; LabDiet, St. Louis, MO), certified to contain <150 ppm estrogenic isoflavones, and housed under standard housing conditions (room temperature 22°C to 24°C) with a 12:12-hour light/dark cycle before mating at 8 to 10 weeks of age. Upon mating, female mice were randomly assigned to either the BPA treatment group or the control group. From 1-day postconception to 20 days postconception, BPA (Sigma-Aldrich, St. Louis, MO) dissolved in corn oil was administered to pregnant female mice via oral gavage (mimicking the common exposure route in humans) at 5 mg/kg/d on a daily basis. The dosage is situated below most reported no-observed-adverse-effect levels according to toxicity testing (https://comptox.epa.gov/dashboard/dsstoxdb/results? search=Bisphenol+A) and was typically used in previous studies (25, 53–55). We chose this dosage as a proof-of-concept for our systems biology study design and to facilitate comparison with previous studies. Control mice were fed the same amount of corn oil as vehicle. We chose corn oil over other solvents for BPA because BPA is water insoluble, and corn oil was 1/26/21, 7(35 AM Prenatal Bisphenol A Exposure in Mice Induces Multitissue Multiomics Disruptions Linking to Cardiometabolic Disorders Page 3 of 23 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6349005/ found to be the least toxic compared with other commonly used solvents (56), but cannot exclude potential confounding from the combined effects of corn oil and BPA. Polycarbonate-free water bottles and cages were used to minimize any unintended exposure to BPA.