RESEARCH ARTICLE

Perinatal 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 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 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

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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 , the pregnane X offspring (48–51). To the best of our knowledge, how- receptor (PXR; also known as 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 receptors, PXR’s ligand- Animals and treatment • 2/2 binding domain is remarkably divergent, and PXR ex- huPXR ApoE mice were generated as previously de- scribed (35). In brief, PXR-humanized mice (mPXR knockout/ 2 2 hibits substantial differences in its pharmacology across hPXR transgenic) (52) were crossed with ApoE / mice to 2 2 2 2 2 2 species (29, 31, 34). BPA is a potent agonist for human generate huPXR•ApoE / (hPXRtg-PXR / ApoE / ) and 2 2 2 2 PXR (hPXR) but does not activate mouse PXR (mPXR) PXR / ApoE / mice (35). All the mice used in the present

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study had the same background (PXR and ApoE null alleles), embryos were dissected from the yolk sac, and embryonic heart 2 2 except for one allele of huPXR•ApoE / mice carrying the tubes were collected for analysis. The yolk sac was washed with hPXR gene. phosphate-buffered saline (PBS) three times and used for geno- 2 2 For the perinatal BPA exposure study, male huPXR•ApoE / typing. Images of the embryos were captured using a DFC420 5 2 2 2 2 mice were mated with female PXR / ApoE / mice (Fig. 1). The MPix digital camera (Leica Microsystems, Buffalo Grove, IL). day of vaginal plug detection was designated as embryonic day 2/2 2/2 (E)0.5. Pregnant PXR ApoE females were then housed Metabolic measurement separately and fed a modified semisynthetic diet containing 4.2% Body weight was measured weekly, and body composition fat and 0.02% cholesterol (control diet) or the same diet sup- was measured using nuclear magnetic resonance spectroscopy plemented with BPA (Sigma-Aldrich, St. Louis, MO) at a dose of (EchoMRI) (55). An intraperitoneal glucose tolerance test was 50 mg/kg (BPA diet) by Harland Laboratories, Inc. (Cumberland, performed as previously described (56). IN) (35). Dams continued consuming the control or BPA diet until their pups were weaned on postnatal day 21 (P21). The offspring were then fed with the control diet for 13 weeks until euthani- Plasma analysis and atherosclerosis quantification zation at 16 weeks of age (Fig. 1). The mice were housed in a Plasma total cholesterol and triglyceride concentrations were specific pathogen-free room with a 12-hour light/dark cycle in the determined enzymatically using colorimetric methods (Wako, University of Kentucky Division of Laboratory Animal Resources Mountain View, CA) as described previously (35). Lipoprotein under a protocol approved by the institutional animal care and fractions were also isolated by centrifuging at 70,000 rpm for 3 use committee. hours in a Beckman Optima TL-100 tabletop ultracentrifuge at its own density (1.006 g/mL). Next, the infranatant was ad- justed to a density of 1.063 g/mL with solid potassium bromide Embryo culture to harvest the high-density lipoprotein (HDL) fraction by Whole-embryo culture was performed as previously de- spinning at 70,000 rpm for 18 hours. The cholesterol content of 2 2 scribed (53, 54). In brief, male huPXR•ApoE / mice were HDL infranatant was measured enzymatically (Wako). To 2 2 2 2 mated with female PXR / ApoE / mice. Mouse embryos at quantify the lesion areas at the aortic root, optimal cutting E9.5 were dissected out of the uteri in Tyrode salt (Sigma- temperature compound–embedded hearts were sectioned at a Aldrich) (Fig. 2A). The parietal yolk sac was removed using 12-mm thickness, keeping all three valves of the aortic root in the forceps, and the visceral yolk sac was left intact. Embryos (two same plane, and stained with Oil Red O, as described previously per bottle) were cultured in 25% Tyrode salt solution and 75% (32, 35). rat serum that was freshly prepared from male rats. The em- bryos were cultured at 37°C in 30 revolutions per1 minute of rotation in the roller bottles, which were gassed with 5% Immunostaining /5% carbon dioxide/90% nitrogen for the first 12 hours Immunohistochemical staining of atherosclerotic lesions was and 20% oxygen/5% carbon dioxide/75% nitrogen for the last performed on 12-mm sections of aortic roots freshly embedded 12 hours. Embryos were cultured for a total of 24 hours in the in optimal cutting temperature compound, as previously de- presence or absence of 20 mM of BPA. At the end of culture, the scribed (56). The sections were first fixed in 100% ice-cold

2 2 2 2 2 2 Figure 1. Scheme of animal models and experimental design. Female PXR / ApoE / mice (F0) were mated with male huPXR•ApoE / mice. The pregnant female PXR2/2ApoE2/2 mice were fed a control diet or BPA diet until the pups were weaned on P21. The weaned offspring were fed a control diet until euthanization at 16 weeks of age. IPGTT, intraperitoneal glucose tolerance test.

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(57). In brief, the tissues were cut into small pieces and washed with cold PBS. The minced tissues were crosslinked with 1% (volume-to-volume ratio) formaldehyde for 10 minutes and then stopped by glycine for 5 minutes at room temperature. The samples containing chromatin were digested with micrococcal nuclease for 20 minutes at 37°C and stopped by EDTA. The nuclei pellet was collected by centrifugation at 16,000g for 1 minute at 4°C and sonicated to release the digested and cross-linked chromatin. For ChIP, the antibodies of H3K4me3 (Abcam, Cambridge, MA) or H3K27me3 (Abcam) were incubated with the cross-linked chromatin overnight at 4°C with shak- ing. The next day, the protein G magnetic beads were added into the immunopre- cipitation mixture and incubated for 4 hours at 4°C. Chromatin binding to the beads was then washed and reverse cross- linked by adding NaCl and proteinase K for 2 hours of incubation at 65°C. The DNA was purified using a spin column purifica- tion kit (Cell Signaling Technology), fol- lowed by quantitative polymerase chain reaction (qPCR) analysis with the primers targeting the PXR response element (2438 to 2424 nt) in the CD36 promoter (58). The forward primer (2487 to 2465 nt; 50-CTGAAAGTCTTCAGGTTCATGC-30) and reverse primer (2370 to 2349 nt; 50-ATGCTGGAGTGTAGCAGCAAG-30) were designed for ChIP-qPCR analysis. H3K4me3 or H3K27me3 enrichment in the CD36 promoter was calculated as Figure 2. BPA ex vivo treatment activates PXR in the heart tubes of PXR-humanized percentage of chromatin input. embryos. (A) Scheme of animal models and embryo genotypes. Female PXR2/2ApoE2/2 mice • 2/2 were mated with male huPXR ApoE mice. The embryos at E9.5 were isolated and RNA isolation and qPCR analysis cultured in control media or media supplemented with 20 mM BPA for 24 hours. (B) Representative images of cultured embryos. Scale bar = 2.5 mm. (C) The heart tubes of the Total RNA was isolated from mouse embryos were dissected for RNA extraction, and messenger RNA (mRNA) levels of PXR target tissues or cells using TRIzol Reagent genes were analyzed using qPCR (n = 4 to 7; **P , 0.01). (Thermo Fisher Scientific, Waltham, MA), and real-time qPCR was performed using acetone for 15 minutes and then washed with PBS for 20 minutes. gene-specific primers and the SYBR green PCR kit (Bio-Rad, The sections were permeabilized with PBS plus 0.1% Triton X- Hercules, CA), as described previously (59). 100 (PBST) for 10 minutes. Nonspecific binding was reduced by incubating slides in 10% rabbit sera diluted in PBST for 20 Statistical analysis minutes at room temperature. The sections were then incubated All data are presented as the mean 6 standard error of the with rat anti-mouse CD36 antibodies (1:100; AbD Serotec, mean. Individual pairwise comparisons were analyzed using the Hercules, CA) at 4°C overnight. The detailed information of the two-sample, two-tailed Student t test, unless otherwise noted, antibodies used in the present study is listed in Table 1. The next with P , 0.05 regarded as significant. day, the slides were rinsed with PBST and incubated with Texas Red-labeled goat anti-rat secondary antibodies (1:500; Vector Laboratories, Burlingame, CA). The nuclei were stained by Results mounting the slides with 40,6-diamidino-2-phenylindole me- dium (Vector Laboratories). Images were acquired with a Nikon BPA ex vivo exposure activates hPXR signaling in the fluorescence microscope (Nikon, Melville, NY). heart tubes of huPXR•ApoE2/2 embryos We previously reported that BPA can increase ath- Chromatin immunoprecipitation 2/2 Chromatin immunoprecipitation (ChIP) analysis was erosclerosis in ApoE mice in an hPXR-dependent performed via a SimpleChIP Enzymatic Chromatin IP Kit (Cell manner (35). To investigate whether exposure to BPA Signaling Technology, Danvers, MA), as previously described can also affect PXR signaling during the embryonic

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Table 1. Antibody Table

Peptide/ Antigen Species Raised in; Protein Sequence Manufacturer, Monoclonal or Dilution Target (if Known) Name of Antibody Catalog No. Polyclonal Used RRID Histone 3 Proprietary Anti-H3K4me3 Abcam, ab8580 Rabbit; polyclonal 2.5 mg/5 mg AB_306649 lysine 4 DNA (ChIP) trimethyl Histone 3 Proprietary Anti-H3K27me3 Abcam, ab6002 Mouse; monoclonal 2.5 mg/5 mg AB_305237 lysine 27 DNA (ChIP) trimethyl CD36 Proprietary Anti-mouse CD36 Bio-Rad/AbD Serotec, Rat; monoclonal 1:100 AB_2072640 MCA2748 Rat IgG Whole IgG Texas Red goat Vector Laboratories, Goat antibody 1:500 AB_2336204 anti-rat IgG (H+L) TI-9400

Abbreviations: AB, antibody; H, heavy (chain); IgG, immunoglobulin G; L, light (chain); RRID, Research Resource Identifier.

period, we adapted a whole-embryo culture system indicated that this dose will likely provide circulating serum (53, 54) and cultured the E9.5 embryos from the concentrations similar to that observed in humans (62). We 2 2 2 2 PXR / ApoE / female dams that were mated with male also reported that exposure of mice to 50 mg/kg BPA feed 2 2 huPXR•ApoE / mice (Fig. 2A). Valvulogenesis during weight provided urinary BPA concentrations similar to that embryonic development is the formation of endocardial observed in humans (35). Furthermore, BPA exposure also cushions in the atrioventricular canal and outflow tract of stimulated expression of the prototypic PXR target genes in 2 2 2 2 2 2 the primitive looped heart tubes, and cardiac valves form huPXR•ApoE / mice but not in PXR / ApoE / mice 2 2 between E9.5 and E14.5 (maturation of leaflets) in (35), indicating that exposure huPXR•ApoE / mice to mice (60, 61). All the embryos we collected carried the 50 mg BPA/kg feed weight can efficiently activate hPXR in same mPXR and mApoE null alleles, and approxi- vivo. mately one half of them carried the hPXR gene. The To determine whether perinatal BPA exposure affects embryos were incubated with control media or media weight gain and body composition of the offspring, supplemented with BPA for 24 hours (Fig. 2B). As the body weight was measured weekly and the body expected, BPA treatment can stimulate the expression composition was measured by EchoMRI. Perinatal of known PXR target genes, including CYP3A11, BPA exposure did not affect the growth curve (Fig. 3A) or the GSTA1, MDR1a,andCD36, in the heart tube of body composition, including fat and lean mass of both 2 2 2 2 2 2 2 2 2 2 2 2 huPXR•ApoE / embryos but not in PXR / ApoE / huPXR•ApoE / and PXR / ApoE / offspring (Fig. 3B). embryos (Fig. 2C). These results suggest that BPA In addition, plasma glucose levels were not affected by exposure can activate hPXR signaling in PXR- perinatal BPA exposure in those mice (Fig. 4A). Glucose humanized embryos. tolerance tests also demonstrated that perinatal BPA exposure did not alter the glucose tolerance in either Perinatal BPA exposure does not affect the genotype (Fig. 4B). metabolic phenotypes of offspring The main route of human exposure to BPA is oral and Perinatal exposure to BPA increases atherosclerosis pharmacokinetic studies have demonstrated that exposure in adult male huPXR•ApoE2/2 mice without altering via diet is a more natural exposure route than other methods plasma lipid levels commonly used in exposed animals (62). To determine the The effects of perinatal BPA exposure on plasma effect of perinatal BPA exposure on atherosclerosis de- lipid and lipoprotein levels were next determined, 2 2 2 2 velopment of offspring, female PXR / ApoE / mice and we found that perinatal BPA exposure did not 2 2 were mated with male huPXR•ApoE / mice, and the alter the plasma triglyceride, total cholesterol, or 2 2 pregnant dams were then fed a control diet or a diet HDL cholesterol levels in either huPXR•ApoE / or 2 2 2 2 supplemented with BPA at a dose of 50 mg/kg until the PXR / ApoE / offspring (Fig. 5). The atheroscle- pups were weaned on P21 (Fig. 1). Previous studies have rotic lesion areas were then determined in the aortic 2 2 demonstrated that exposure to 50 mg BPA/kg feed weight root (Fig. 6). Adult male huPXR•ApoE / offspring can induce epigenetic changes in laboratory rodents, from BPA-exposed dams had lesion areas that were 2 2 and this dose is also well below the diet-administered increased by 36% compared with huPXR•ApoE / maximum nontoxic dose for rodents (200 mg/kg body mice from control dams (Fig. 6A). However, perinatal weight daily) (62–65). BPA pharmacokinetic studies have BPA exposure did not affect atherosclerosis in male

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Figure 3. Perinatal BPA exposure did not affect body weight and body composition of offspring. (A) Growth curves of huPXR•ApoE2/2 and PXR2/2ApoE2/2 mice from control or BPA-exposed dams (n = 12 to 16). (B) Fat mass and lean mass of 14-week-old huPXR•ApoE2/2 and PXR2/2ApoE2/2 mice from control or BPA-exposed dams (n = 9 to 14).

2 2 2 2 PXR / ApoE / mice (Fig. 6A), suggesting that perina- Perinatal BPA exposure increases aortic and tal BPA exposure increased atherosclerosis in male mice atherosclerotic lesional CD36 expression in adult in an hPXR-dependent manner. Furthermore, perinatal male huPXR•ApoE2/2 mice BPA exposure did not alter the atherosclerotic lesion We previously reported that chronic BPA exposure 2 2 2 2 2 2 areas in female huPXR•ApoE / and PXR / ApoE / activated hPXR to increase the expression of the fatty mice (Fig. 6B). Collectively, these results have demon- acid transporter CD36, leading to increased atheroscle- 2 2 strated the hPXR- and sex-dependent effect of perinatal rosis in huPXR•ApoE / mice (35). CD36 is a scavenger BPA exposure on atherosclerosis development in the receptor that plays an important role in mediating lipid offspring. uptake and foam cell formation (34, 66, 67). We next

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Figure 4. Perinatal BPA exposure did not affect the plasma glucose levels or glucose tolerance of offspring. (A) The fasting glucose levels of huPXR•ApoE2/2 and PXR2/2ApoE2/2 mice from control or BPA-exposed dams (n = 8 to 14). (B) Intraperitoneal (I.P.) glucose tolerance test of huPXR•ApoE2/2 and PXR2/2ApoE2/2 mice from control or BPA-exposed dams (n = 6 to 14).

measured the messenger RNA levels of CD36 in the aorta CD36 protein levels in atherosclerotic lesions of 2 2 of male offspring and found that perinatal BPA exposure huPXR•ApoE / mice from BPA-exposed dams com- 2 2 increased CD36 expression in huPXR•ApoE / mice pared with mice from control dams (Fig. 7B). Consis- 2 2 2 2 but not in PXR / ApoE / mice (Fig. 7A). Furthermore, tently, perinatal BPA exposure did not affect lesional 2 2 2 2 immunofluorescence staining also showed increased CD36 expression in PXR / ApoE / mice.

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Figure 5. Perinatal BPA exposure had no effects on the plasma lipid levels of offspring. The plasma triglyceride, total cholesterol, and HDL cholesterol levels were measured in (A) male and (B) female huPXR•ApoE2/2 and PXR2/2ApoE2/2 mice from control or BPA-exposed dams (n = 10).

Perinatal BPA exposure leads to PXR-mediated analyzed by ChIP assays using the aortas from male 2 2 2 2 2 2 epigenetic regulation of CD36 expression huPXR•ApoE / and PXR / ApoE / mice (Fig. 8A). Although CD36 is a prototypic PXR-activated gene Perinatal BPA exposure significantly increased the levels 2 2 (58), it is unlikely that the increased CD36 expression of H3K4me3 in the CD36 promoter of huPXR•ApoE / resulted from the BPA-mediated hPXR transactivation, mice (Fig. 8B). In contrast, the levels of H3K27me3 because BPA has been withdrawn from the food after were found to be significantly decreased in CD36 pro- 2 2 weaning, and those mice had not been subsequently moter in huPXR•ApoE / mice from BPA-exposed dams exposed to BPA. Furthermore, the expression levels of the (Fig. 8C). Deficiency of hPXR abolished the effect of other PXR target genes, including GSTA1 and MDR1a, perinatal BPA exposure on the changes in H3K4me3 were similar in mice from control and BPA-exposed dams and H3K27me3 levels in the CD36 promoter of 2 2 2 2 (Fig. 7A). In addition to direct transactivation of the PXR / ApoE / mice. Taken together, these results sug- target genes, PXR has been demonstrated to epigeneti- gestanimportantroleofPXRintheepigeneticregulationof cally modulate gene expression by altering histone genes that influence CVD risk. methylation, and PXR ligands can increase the levels of H3K4me3, an epigenetic mark for gene activation, but Discussion decrease the levels of H3K27me3, an epigenetic mark for gene suppression, in the promoter of its target genes (6, Although considerable progress has been achieved in 68, 69). To reveal the influence of perinatal BPA exposure identifying the risk factors that contribute to athero- on histone modifications of the CD36 promoter, the sclerotic CVD, the role played by “gene–environment enriched levels of H3K4me3 and H3K27me3 around interactions” in predisposing individuals to atheroscle- the PXR response element of CD36 promoter were rosis remains relatively unexplored. To sense and

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Figure 6. Perinatal BPA exposure increased atherosclerosis in aortic roots of adult male huPXR•ApoE2/2 mice. Quantitative analysis of atherosclerotic lesion sizes in the aortic root of (A) male and (B) female huPXR•ApoE2/2 and PXR2/2ApoE2/2 mice from control or BPA-exposed dams (n = 9 to 12; *P , 0.05). The representative Oil red O-stained sections are shown as indicated.

respond to environmental chemicals, mammals have mechanisms for these associations are still poorly un- evolved a defensive network governed by xenobiotic derstood and contribute to the hampered risk assessment receptors such as PXR (31, 34). The role of PXR as a of BPA. We previously reported that BPA is an hPXR- xenobiotic sensor has been well-studied, and PXR has selective ligand, and chronic BPA exposure can increase 2 2 been considered a master regulator of xenobiotic meta- atherosclerosis development in huPXR•ApoE / mice bolism (31, 34). The identification of PXR as a xenobiotic (27, 35). In the present study, we found that perinatal sensor has also provided an important tool for the study BPA exposure also exacerbated atherosclerosis in adult 2 2 of new mechanisms through which chemical exposure male huPXR•ApoE / offspring. Perinatal BPA expo- affects diseases (34). Numerous studies have reported sure led to increased expression of CD36 in these mice, that exposure to the ubiquitous EDC BPA could cause likely through PXR-dependent epigenetic regulation. adverse health effects in humans (10–12, 70–72), and CD36 plays a key role in foam cell formation and ath- recent studies have also associated BPA exposure with the erosclerosis development, which could contribute to the 2 2 increased CVD risk (2–5, 14). However, the underlying increased atherosclerosis in huPXR•ApoE / mice from

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2 2 Figure 7. Perinatal BPA exposure increases aortic and atherosclerotic lesional CD36 expression in adult male huPXR•ApoE / mice. (A) Messenger RNA (mRNA) levels of PXR target genes were measured in aorta of male huPXR•ApoE2/2 and PXR2/2ApoE2/2 offspring by qPCR (n = 5to6;*P , 0.05). (B) Sections of atherosclerotic lesions in the aortic root of male huPXR•ApoE2/2 and PXR2/2ApoE2/2 offspring were stained with anti-CD36 primary antibodies, followed by fluorescein-labeled secondary antibodies. Nuclei were stained with 40,6-diamidino-2-phenylindole (DAPI). Scale bar = 100 mm.

BPA-exposed dams. To the best of our knowledge, ours incidence of metabolic disorders in both humans and is the first study to demonstrate the effect of perinatal animal models (10, 48, 63, 73). Higher urinary con- BPA exposure on atherosclerosis development in adult centrations of BPA in prepubescent girls have been as- offspring. sociated with decreased global methylation, which has In addition to activating ER and hPXR (27, 73), BPA been corresponded to changes in expression of genes has been shown to activate another nuclear receptor, involved in inflammation and metabolism (76). In utero constitutive androstane receptor (CAR) (74). However, it BPA exposure altered methylation within the carnitine is unlikely that BPA-mediated CAR activation contrib- palmitoyltransferase 1a gene, leading to hepatic de- uted to the increased atherosclerosis in our model, be- position of triglycerides in rats fed a high-fat diet (77). cause studies have demonstrated that activation of CAR Several well-controlled animal studies were conducted to can decrease the plasma lipid levels and atherosclerotic investigate the effect of maternal exposure to BPA during lesions in animal models (75). Furthermore, BPA expo- pregnancy on body weight, glucose homeostasis, and sure should have similar effects on CAR activity in both gene expression in adult offspring (49–51). The in- 2 2 2 2 2 2 huPXR•ApoE / and PXR / ApoE / mice, which vestigators found that in utero exposure to BPA led to have the same background except for one allele of hyperglycemia, glucose intolerance, increased plasma 2 2 huPXR•ApoE / mice carrying the hPXR gene. In ad- nonesterified fatty acids and hepatic triglyceride levels in dition to nuclear receptor signaling, BPA has been shown adult male offspring (50). In utero BPA exposure influ- to cause epigenetic changes that might increase the enced the expression of genes involved in b-cell

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CD36 expression and exacerbate atherosclerosis in 2 2 huPXR•ApoE / mice (35). In the present study, how- ever, the adult offspring had not been exposed to BPA after P21; thus, the increased CD36 expression could not be explained by direct PXR transactivation. If the in- creased CD36 expression was due to the BPA-mediated direct PXR transactivation, the expression levels of other PXR target genes should also have been elevated. However, the expression of GSTA1 and MDR1a was not affected by perinatal BPA exposure. In addition to binding to its responsive motifs and transcriptionally regulating target gene expression, recent studies have indicated a po- tential role of PXR in epigenetic regulation of downstream targets. For example, PXR can directly interact with arginine methyltransferase 1, the primary methyltransferase that deposits the asymmetric dimethylarginine mark (69). ChIP analysis revealed a ligand-dependent recruitment of arginine methyltransferase 1 to PXR regulatory regions within the CYP3A4 promoter (69). A more recent study demonstrated that hPXR ligand rifampicin treatment can increase levels of H3K4me3 and decrease levels of H3K27me3 in the CYP3A4 promoter in human intestinal cells, and knock- down of PXR abolished those epigenetic changes in the promoter (68). The levels of H3K4me3 and H3K27me3 in the CD36 promoter were respectively increased and de- 2 2 creased by perinatal BPA exposure in huPXR•ApoE / mice. These changes were apparently mediated by hPXR, Figure 8. Perinatal BPA exposure alters histone modification in because deficiency of hPXR abolished these effects elicited CD36 promoter. (A) Scheme of ChIP-qPCR primers that cover the by perinatal BPA exposure. Our findings have not only CD36 PXR element (PXRE) in promoter. ChIP-qPCR analysis of confirmed the role of PXR in the epigenetic regulation of (B) H3K4me3 and (C) H3K27me3 enrichment of CD36 promoter in aorta of male huPXR•ApoE2/2 and PXR2/2ApoE2/2 offspring gene expression but have also demonstrated that EDC- (n = 3; *P , 0.05 and **P , 0.01). mediated modulation of PXR activity during early life can lead to adverse health effects in late life. In addition to the hPXR-dependent impact on development, which might contribute to the impaired atherosclerosis, perinatal BPA exposure also had sex- glucose tolerance observed in adult offspring (51). They dependent atherogenic effects in the offspring. Perina- also found that in utero BPA exposure affected the ex- tal BPA exposure only increased atherosclerosis in male pression of several key genes involved in lipid and fatty PXR-humanized mice but not in female mice. Human and acid metabolism, including SREBP-1c and PPARa in the animal studies have also demonstrated that BPA expo- offspring (50). Although the epigenetic status such as sure has sex-dependent effects on metabolic diseases and DNA methylation of those genes was not investigated, it cardiac functions. For example, higher urinary BPA ex- is plausible that the effect of in utero BPA exposure on posure might be associated with an elevated lean body the expression of those genes was through epigenetic mass in boys but not in girls (78). In contrast, higher BPA regulation. exposure might be associated with an elevated fat mass in In the present study, we found that perinatal BPA girls but not in boys (78). Bhandari et al. (79) also found a exposure increased the expression of the fatty acid significantly positive association between urinary BPA transporter CD36 in adult male offspring in an hPXR- levels and obesity in boys but not in girls. Another study dependent manner. CD36 is a direct PXR target gene of Chinese school-age children found that girls with (58) that plays an important role in atherosclerosis de- higher urinary BPA levels had twice the risk of becoming velopment (32, 34, 66, 67). We previously demonstrated overweight than did those with lower BPA levels; that activation of PXR increases CD36 levels and foam however, a similar association was not observed in boys 2 2 cell formation in ApoE / mice (32). Chronic BPA (80). Animal studies also showed the sex-dependent effect exposure-mediated hPXR activation can also increase of BPA exposure on metabolic diseases and cardiac

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