RESEARCH ARTICLE Perinatal Bisphenol A Exposure Increases Atherosclerosis in Adult Male PXR-Humanized Mice Yipeng Sui,1 Se-Hyung Park,1 Fang Wang,1 and Changcheng Zhou1 1Department of Pharmacology and Nutritional Sciences, University of Kentucky, Lexington, Kentucky 40536 Bisphenol A (BPA) is a base chemical used extensively in numerous consumer products, and human exposure to BPA is ubiquitous. Higher BPA exposure has been associated with an increased risk of atherosclerosis and cardiovascular disease (CVD) in multiple human population-based studies. However, the underlying mechanisms responsible for the associations remain elusive. We previously reported that BPA activates the xenobiotic receptor pregnane X receptor (PXR), which has proa- therogenic effects in animal models. Because BPA is a potent agonist for human PXR but does not 2 2 affect rodent PXR activity, a suitable PXR-humanized apolipoprotein E–deficient (huPXR•ApoE / ) mouse model was developed to study BPA’s atherogenic effects. Chronic BPA exposure increased atherosclerosis in the huPXR•ApoE2/2 mice. We report that BPA exposure can also activate human PXR signaling in the heart tubes of huPXR•ApoE2/2 embryos, and perinatal BPA exposure exac- erbated atherosclerosis in adult male huPXR•ApoE2/2 offspring. However, atherosclerosis devel- opment in female offspring was not affected by perinatal BPA exposure. Perinatal BPA exposure did not affect plasma lipid levels but increased aortic and atherosclerotic lesional fatty acid transporter 2 2 CD36 expression in male huPXR•ApoE / offspring. Mechanistically, PXR epigenetically regulated CD36 expression by increasing H3K4me3 levels and decreasing H3K27me3 levels in the CD36 promoter in response to perinatal BPA exposure. The findings from the present study contribute to our understanding of the association between BPA exposure and increased atherosclerosis or CVD risk in humans, and activation of human PXR should be considered for future BPA risk assessment. (Endocrinology 159: 1595–1608, 2018) espite major advances in diagnosis and treatment, EDC exposure on human health have become the subject Datherosclerotic cardiovascular disease (CVD) is still of intense interest (6–11). However, the mechanisms of the leading cause of mortality and morbidity world- how exposure to EDCs contributes to the development of wide (1). Atherosclerosis is a complex chronic disease various chronic diseases, including atherosclerosis, are involving the interaction of genetic and environ- still poorly understood. mental factors over multiple years. In addition to the One EDC in particular, bisphenol A (BPA), has obvious contributions of diet and lifestyle, the attracted considerable attention and controversy. BPA chemical environment to which we are exposed has is a base chemical used extensively in polycarbonate significantly changed in the past few decades and has plastics in many consumer products. Numerous bio- recently been implicated in the etiology of CVD (2–6). monitoring studies have indicated that human exposure Mounting evidence has suggested that many chemicals to BPA is ubiquitous and .95% of the U.S. population such as endocrine-disrupting chemicals (EDCs) can in- has been exposed to BPA (12, 13). Although research on terfere with organisms’ complex endocrine signaling and the adverse effects of BPA initially focused primarily on result in adverse consequences. Thus, the influences of reproduction and development, recent findings have ISSN Online 1945-7170 Abbreviations: ApoE2/2, apolipoprotein E–deficient; BPA, bisphenol A; CAR, constitutive Copyright © 2018 Endocrine Society androstane receptor; ChIP, chromatin immunoprecipitation; CVD, cardiovascular disease; Received 20 December 2017. Accepted 31 January 2018. EDC, endocrine-disrupting chemical; ER, estrogen receptor; E, embryonic day; HDL, high- First Published Online 7 February 2018 density lipoprotein; hPXR, human pregnane X receptor; huPXR•ApoE2/2, pregnane X receptor–humanized apolipoprotein E–deficient; P21, postnatal day 21; mPXR, mouse pregnane X receptor; PBS, phosphate-buffered saline; PBST, phosphate-buffered saline plus 0.1% Triton X-100; PXR, pregnane X receptor; qPCR, quantitative polymerase chain reaction. doi: 10.1210/en.2017-03250 Endocrinology, April 2018, 159(4):1595–1608 https://academic.oup.com/endo 1595 Downloaded from https://academic.oup.com/endo/article-abstract/159/4/1595/4841949 by University of Kentucky user on 08 March 2018 1596 Sui et al Perinatal BPA Exposure Increases Atherosclerosis Endocrinology, April 2018, 159(4):1595–1608 linked BPA exposure to CVD (2–5). Several large and or rat PXR (27). Because BPA is an hPXR-selective ligand, well-conducted cross-sectional and longitudinal studies one of the key challenges to studying the effects of BPA- have found that greater BPA exposure is consistently mediated hPXR activation on atherosclerosis is the associated with increased CVD risk in the general pop- development of a mouse model that recapitulates the ulation (2–4). Lang et al. (2) first reported that higher human response to PXR ligands. To address this issue, we 2 2 urinary BPA levels were significantly associated with an developed a PXR-humanized ApoE / mouse model 2 2 increased incidence of CVD, including coronary heart (huPXR•ApoE / ) to investigate BPA’s atherogenic ef- disease, myocardial infarction, and angina, all of which fects (35). Chronic exposure to BPA increased athero- 2 2 can be caused by atherosclerosis. Melzer et al. (3) rep- sclerosis in huPXR•ApoE / mice but not in their control licated the early association between urinary BPA con- littermates (35). BPA exposure did not affect plasma lipid centrations and coronary heart disease using a separate levels but increased fatty acid transporter CD36 ex- 2 2 database. The association between BPA exposure and pression and foam cell formation in huPXR•ApoE / incident coronary artery disease has also been confirmed mice (35). After we first demonstrated that chronic BPA in a longitudinal study (4). These associations are in- exposure can increase atherosclerosis development in the 2 2 dependent of traditional CVD risk factors, including huPXR•ApoE / mouse model, other studies have also body mass index, blood pressure, lipid concentrations, found that BPA can increase atherosclerosis in hyper- and levels of physical activity (2, 4). Several indepen- lipidemic rabbit models (36, 37). Although the differences dent studies have also directly associated BPA exposure between human and rodent PXR pharmacology are clear, with coronary atherosclerosis (14), carotid atheroscle- the activation profiles of the human and rabbit PXR are rosis (5), and peripheral arterial disease (15), suggesting very similar (31, 38). Therefore, it is plausible that PXR that BPA exposure might directly affect atherosclerosis signaling also contributes to BPA’s atherogenic effects in development. those rabbit models. BPA is a weak agonist of the estrogen receptor (ER) The progression of atherosclerosis is considered to (16, 17), and studies have found similar adverse effects of begin during adolescence in humans; however, athero- exposure to BPA or estrogen on cardiac functions in sclerotic lesions can be detected in young children and, isolated rodent hearts (18, 19). Chronic or lifelong BPA even, in fetuses (39, 40). The prenatal period is known to exposure has also been demonstrated to affect cardiac be a susceptible period for adverse health effects of en- functions and cardiac remodeling after myocardial in- vironmental exposure, and studies have demonstrated farction in mice (20–22). However, the estrogenic effects that environmental factors can contribute to the CVD of BPA might not be sufficient to explain the link between risk of the exposed individuals’ offspring (41–43). BPA BPA exposure and increased atherosclerosis in humans, can cross the placenta (44), and the associations between because numerous animal and human studies have prenatal exposure to BPA and adverse health effects in identified protective effects of estrogen against athero- early childhood have been reported (45–47). Animal sclerosis (23–26). studies have also demonstrated that in utero or perinatal In addition to ER, we previously reported that BPA BPA exposure can increase metabolic disorders in the can activate another nuclear receptor, the pregnane X offspring (48–51). To the best of our knowledge, how- receptor (PXR; also known as steroid and xenobiotic ever, no studies have reported on the effect of perinatal receptoror SXR) (27, 28). PXR functions as a xenobi- BPA exposure on atherosclerosis development in the otic sensor that regulates many genes involved in xe- offspring. We report that perinatal BPA exposure exac- 2 2 nobiotic metabolism (29–31). Although the function of erbated atherosclerosis in adult male huPXR•ApoE / PXR in the regulation of xenobiotic metabolism has been offspring but had no effects on their control littermates. extensively studied, we recently reported PXR’sproathero- Perinatal BPA exposure did not alter plasma lipid levels genic effects in animal models and found that activation but did increase aortic and atherosclerotic lesional CD36 of PXR can increase atherosclerosis in atherosclerosis-prone expression, potentially through PXR-dependent epige- 2 2 apolipoprotein E–deficient (ApoE / )mice(32–34). netic regulation. Therefore, BPA-mediated PXR activation could potentially accelerate atherosclerosis development and increase CVD Materials and Methods risk in humans. Unlike many other nuclear