Cardiovasc Toxicol DOI 10.1007/s12012-013-9235-x
Chronic Exposure to Bisphenol A can Accelerate Atherosclerosis in High-Fat-Fed Apolipoprotein E Knockout Mice Min Joo Kim • Min Kyong Moon • Geun Hyung Kang • Kwan Jae Lee • Sung Hee Choi • Soo Lim • Byung-Chul Oh • Do Joon Park • Kyong Soo Park Hak Chul Jang • Young Joo Park
•
Ó Springer Science+Business Media New York 2013
Abstract In epidemiological studies, there is growing concern regarding the association between human exposure to bisphenol A (BPA) and an increased risk for cardiovascular disease. Therefore, we investigated whether BPA accelerates atherosclerosis in mouse model. Apolipoprotein E knockout (ApoE-/-) mice were fed a high-fat and highcholesterol diet with or without 50 lg/kg body weight/day BPA for 12 weeks. Atherosclerotic lesions of the aorta and aortic sinus were evaluated by Oil red O staining. After the 12-week BPA treatment, BPA significantly increased atherosclerotic lesions in the aortas of ApoE-/- mice by 1.7fold (p = 0.03). Non-high-density lipoprotein (HDL) cholesterol levels in the BPA group were significantly higher compared to those in the control group (1,035 ± 70 vs. 484 ± 48 mg/dL, p = 0.02) although body weight and blood glucose levels were not different between groups. Human umbilical vein endothelial cells (HUVECs) were treated with 0.1–10 nM BPA but BPA did not affect
Min Joo Kim and Min Kyong Moon have equally contributed to this work. M. J. Kim M. K. Moon S. H. Choi S. Lim D. J. Park K. S. Park H. C. Jang Y. J. Park (&) Department of Internal Medicine, Seoul National University College of Medicine, 28 Yongon-dong, Jongno-gu, Seoul 110-744, Republic of Korea e-mail: [email protected] M. J. Kim Department of Internal Medicine, Korea Cancer Center Hospital, Seoul 139-706, Republic of Korea M. K. Moon Department of Internal Medicine, Seoul Metropolitan Government Boramae Medical Center, Seoul 156-707, Republic of Korea
HUVEC proliferation or migration. BPA could accelerate atherosclerosis in ApoE-/- mice, which may have resulted from an increase in non-HDL cholesterol levels. Keywords Bisphenol A Atherosclerosis Non-HDL cholesterol Dyslipidemias
Introduction Mankind is ubiquitously exposed to bisphenol A (BPA) because it is a base material of various beverage and food containers [1]. BPA is reported to be detected in 92 % of the general population in USA [2]. In the initial stage, researchers focused on the estrogenic activity of BPA and its reproductive toxicity [3]. However, there is growing concern regarding the harmful effects of BPA exposure on human health besides reproductive toxicity [4]. In a large cross-sectional study using the United States National Health and Nutrition Examination Survey, it was found that a high urinary BPA concentration was associated with an
G. H. Kang Biomedical Research Institute, Seoul National University Hospital, Seoul 110-744, Republic of Korea K. J. Lee S. H. Choi S. Lim H. C. Jang Department of Internal Medicine, Seoul National University Bundang Hospital, Seongnam 463-707, Republic of Korea B.-C. Oh Department of Molecular Medicine, Lee Gil Ya Cancer and Diabetes Institute, Gachon University of Medicine and Science, Inchon 406-840, Republic of Korea
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increased risk for chronic medical disorders such as cardiovascular disease, diabetes, and abnormal liver function [4]. Subsequently, the relationship between higher BPA exposure and cardiovascular disease has been replicated in both cross-sectional and longitudinal studies [5, 6]. Exposure to BPA has also been reported as being associated with the severity of atherosclerosis in the coronary artery or carotid artery [7, 8], increased blood pressure, and reduced heart rate variability [9, 10]. Atherosclerosis is a fundamental process of cardiovascular disease. It is initiated by endothelial dysfunction and structural defect, and then, low-density lipoprotein (LDL) accumulates making early fatty streak lesion [11]. Increased circulating LDL cholesterol levels accelerate the development of atherosclerosis and cardiovascular disease. Oxidative stress such as reactive oxygen species modifies LDL [12]. Oxidized LDL triggers the expression of leukocyte adhesion molecules, and it recruits leukocytes such as monocyte-derived macrophages and lymphocytes, resulting in vascular inflammation. Consequently, atherosclerotic lesions consist of macrophage, lipids, and smooth muscle cells. Although many epidemiologic studies have established the association between BPA and atherosclerosis, a causal relationship remains uncertain. Previous epidemiological studies have reported that a high urinary BPA concentration was associated with insulin resistance [13], diabetes [4], and obesity [14, 15], and they have been considered possible links between BPA and atherosclerosis. In line with these data, perinatal exposure to BPA (10–100 lg/kg) has been shown to induce insulin resistance [16], weight gain [17], and dyslipidemia in rodents [18]. Although there is a paucity of research on adult rodents, it is reported that the short-term administration of BPA (10–100 lg/kg) to adult mice can at least induce insulin resistance [19, 20]. This evidence suggests that BPA can induce or accelerate atherosclerosis. However, no experimental study, not even animal or in vitro studies, has clarified that exposure to BPA can induce atherosclerosis. Therefore, we investigated whether BPA can accelerate atherosclerosis and the mechanism linking BPA to atherosclerosis using apolipoprotein E knockout (ApoE-/-) mice, an animal model of atherosclerosis.
Materials and Methods Animals ApoE-/- male mice were purchased from Jackson Laboratories (Sacramento, CA, USA). All mice were fed ad libitum laboratory chow diet (Purina irradiated laboratory chow 38057, Purina Korea, Seoul, Korea), followed by
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a high-fat and high-cholesterol diet (Research Diets Inc., New Brunswick, NJ, USA) containing 21 % fat and 0.21 % cholesterol when they were 8 weeks old. The mice were assigned to the control or BPA group when they started the high-fat and high-cholesterol diet. BPA (purity [99 %) was purchased from Sigma Aldrich (St. Louis, MO, USA). The BPA group received 50 lg/kg body weight (bw)/ day BPA via drinking water for 12 weeks. This BPA dose is below the no observed adverse effect level (NOAEL) of 5 mg/kg bw/day set by the United States Food and Drug Administration (US FDA) [21]. All mice were housed in conventional plastic cages with free access to feed and water at 23 ± 2 °C, 60 ± 10 % humidity, and a 12-h light/ dark photoperiod. At the end of the 12 weeks, the mice were anesthetized and then killed. The experiment was repeated 5 times with 3 or 4 mice in each group. All procedures involving the use of laboratory animals were in accordance with the Guide for Standard Operation Procedures and were performed after receiving the approval of the Institutional Animal Care and Use Committee (IACUC) of the Clinical Research Institute, Seoul National University Bundang Hospital (IACUC Approval No. BA1012-074/068-01). Measurement of Body Weight and Blood Glucose Levels Body weight was monitored before and after the experiments. Lean body mass (LBM) and percent body fat content were measured using a dual energy X-ray absorptiometer (GE Lunar PIXImus, Fitchburg, WI, USA). After each 12-week experiments, an intraperitoneal glucose tolerance test (IPGTT) was performed after a 6-h fast by administering an intraperitoneal injection of 2 g/kg bw glucose. Blood glucose levels from tail vein blood were determined using an Accu-Chek Active glucometer (Roche, Mannheim, Germany) before and 15, 30, 90, and 120 min after glucose injection. Measurement of BPA Concentration, Lipid Profiles, and Cytokines Serum total BPA concentrations were measured using the enzyme-linked immunosorbent assay (EcologienaÒ, Osaka, Japan). Total cholesterol, triglyceride, and high-density lipoprotein (HDL) cholesterol were determined using a Beckman Coulter AU480 automatic biochemistry analysis system (Japan) with reagent kits provided by the manufacturer. Tumor necrosis factor-a (TNF-a) and interleukin6 (IL-6) were measured using a radioimmunoassay kit (Diaclone, Besanc¸on, France) following the protocol provided by the manufacturer.
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Quantitative Real-Time Polymerase Chain Reaction (PCR) Total RNA was isolated from aorta using TRizol reagent (Ambion, Foster City, CA, USA), and M-MLV reverse transcriptase (Invitrogen, Carlsbad, CA, USA) was used for cDNA synthesis. Quantitative real-time PCR was performed using the ABI 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA), and the SYBR Premix Ex Taq polymerase (Takara, Otsu, Japan) was used for amplification. The primer sequences were as follows: for TNF-a, forward primer sequence was 50 CCTCCCTCTCATCAGTTCTA-30 , and the reverse primer sequence was 50 -CAGCATCGTTTGGTGGTTCA0 ; for IL6, the forward primer sequence was 50 -ACCACGGCCT TCCCTACTTC-30 , and the reverse primer sequence was 50 -GACGAGTTCACTACCCTATGG-30 . Data were analyzed using ABI 7500 software version 2.0.5 (Applied Biosystems, Foster City, CA, USA). Measurement of Atherosclerotic Lesions in the Aorta and Aortic Sinus To measure the extent of atherosclerosis in the aorta, the entire aorta was removed, placed in 4 % formaldehyde, and prepared using the ‘‘en face’’ method. Each aorta was opened longitudinally from the heart to the iliac arteries, dissected, and pinned on a plate. The fixed aortas were stained with Oil red O to delineate the atherosclerotic lesions, and images were obtained with a light microscope (Carl Zeiss, Germany). The Oil red O-positive area and total aortic surface area were measured using Adobe Photoshop software version 5.0.1 (Adobe Systems, San Jose, CA, USA). Atherosclerotic lesion of the aorta is defined as the percentage of Oil red O-positive area to total aortic surface area and presented as fold changes compared to the control group in each set. To measure the extent of atherosclerosis in the aortic sinus, tissues of the heart and ascending aorta were embedded in OCT compound (Sakura Finetek, Torrance, CA, USA). Frozen tissue was sectioned into 30-lm-thick slices. We selected sections that contained the cusps of 3 aortic valves at the junction of the aorta and the left ventricle. The sections were stained with Oil red O to determine areas containing atherosclerotic lesions. Sections were examined under a light microscope (Carl Zeiss, Germany), and at least 2 images were obtained from each mice. The Oil red O-positive red color spot contained area was measured to define the area of atheroma in aortic sinus using Adobe Photoshop software version 5.0.1 (Adobe Systems, San Jose, CA, USA). In some cases (3 control and 3 BPA group), corresponding sections were also stained with hematoxylin and eosin (H&E) to measure the area of
atherosclerotic lesions, and the area quantified by H&E staining was well correlated with that by Oil red O staining (data not shown). Atherosclerotic lesions of the aortic sinus are presented as fold changes compared to the control group in each set. Macrophage Infiltration into Atherosclerotic Lesions To stain macrophages, tissue sections were incubated with an anti-F4/80 antibody (eBioscience, San Diego, CA, USA) and subsequently with biotinylated anti-rabbit IgG antibody. The positively stained areas were measured by Axiovision software version 4.8 (Carl Zeiss, Germany). The extent of macrophage infiltration is presented as the percentage of anti-F4/80 antibody-positive stained area to the Oil red O-positive area. Cell Culture Primary human umbilical vein endothelial cells (HUVECs) were purchased from Cambrex (East Rutherford, NJ, USA) and cultured in endothelial basal medium (EBM-2) containing endothelial growth factor (EGM-2 Bullet Kit, Cambrex, East Rutherford, NJ, USA) and 2 % fetal bovine serum (FBS). For the experiments, HUVECs were starved in EBM-2 medium with 0.4 % FBS. Cell Proliferation Cell proliferation was evaluated using a bromodeoxyuridine (BrdU) cell proliferation assay kit (Cell Signaling Technology Inc., Danvers, MA, USA) according to the manufacturer’s protocol. In brief, HUVECs were cultured in 98-well plates. Each well was seeded with 2,500 cells and incubated with vehicle or 0.1, 1, or 10 nM BPA for 24 h. BrdU was then added to the medium and the cells were reincubated for 4 h. After the medium was removed, the HUVECs were fixed and the DNA was denatured. An anti-BrdU mouse antibody was added to detect the incorporated BrdU. Anti-mouse IgG, a horseradish peroxidase (HRP)-linked antibody, was then used to identify the bound detection antibody. The HRP substrate tetramethylbenzidine was added to develop color. The magnitude of the absorbance of the developed color is proportional to the quantity of BrdU incorporated into cells, which is a direct indication of cell proliferation. The experiment was repeated 3 times with 6 wells per each condition. Wound Healing Assay HUVECs were cultured in 6-well plates and grown in a monolayer. Each well was scratched with a pipette tip to create a wound [22]. Next, HUVECs were treated with
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vehicle or 0.1, 1, or 10 nM BPA for 24 h. After 24 h, the images were captured and the distance covered by the cells was measured at least 3 distance points to estimate cell migration. The experiment was repeated 3 times. Transwell Migration Assay The modified Boyden chamber assay was performed using a Transwell system (Corning, Rochester, NY, USA). The chamber insert contained a polycarbonate membrane with 8-lm pores that permitted cells to migrate. Each insert was coated with 10 lg gelatin solution, and 104 HUVECs in medium containing 0.4 % FBS were loaded. Each well was filled with 500 lL starvation medium with or without BPA, and the inserts were placed into 24-well plates. After 24 h, the upper surface of inserts was swabbed with a cottontipped applicator to remove non-migrated cells. The inserts were fixed in methanol for 10 min and stained with 1 % crystal violet for 2 h. The number of cells that had moved was counted to estimate cell migration. The experiment was repeated 5 times with 2 wells per each condition.
Table 1 Metabolic parameters and inflammatory cytokines in ApoE-/- mice Control (n = 15)
BPA (n = 19)
p value
Body weight (g)
31.7 ± 1.0
30.4 ± 1.0
0.38
LBM (g)
25.7 ± 0.8
23.5 ± 1.0
0.09
Percentage of body fat Fasting blood glucose (mg/dL)
17.3 ± 0.6 153 ± 10
20.7 ± 1.3 134 ± 6
0.09 0.08
Total cholesterol (mg/dL)
522 ± 46
1,088 ± 70
Triglyceride (mg/dL)
104 ± 24
HDL cholesterol (mg/dL)
38 ± 1
Non-HDL cholesterol (mg/dL)
484 ± 48
TNF-a (pg/mL) IL-6 (pg/mL) BPA (ng/mL)
94 ± 9 53 ± 2
0.02 0.90 0.02
1,035 ± 70
0.02
195 ± 195
121 ± 64
0.83
19 ± 7 0.15 ± 0.04
27 ± 12 1.26 ± 0.62
0.83 0.02
Statistical Analysis Results are presented as mean ± standard error. The Mann–Whitney test was used to compare the BPA and control groups. The IPGTT results were analyzed with repeated measures analysis of variance. The Spearman’s correlation test was used to assess the relationship between several metabolic parameters and atherosclerosis. All statistical analyses were performed using SPSS software (Chicago, IL, USA). Statistical significance was assumed at p \ 0.05.
Fig. 1 Blood glucose levels during IPGTT in ApoE-/- mice
Results Effects of BPA on Metabolic Profiles in ApoE-/- Mice After the 12-week BPA treatment, serum BPA concentrations of the BPA group were significantly higher than those of the control group (1.26 ± 0.62 vs. 0.15 ± 0.04 ng/mL, p = 0.02). The body weights of the BPA group were not different from those of the control group (Table 1). The BPA group had a higher percentage of body fat and lower LBM, but there was no statistical significance (p = 0.09; Table 1). Blood glucose levels during the IPGTT were not different between the 2 groups (p = 0.25; Fig. 1). BPA significantly increased the levels of both non-HDL and HDL cholesterol levels (p = 0.02; Table 1). Non-HDL cholesterol levels in the BPA group were twice as high as those in the control group (1,035 ± 70 vs. 484 ± 48 mg/ dL, p = 0.02; Table 1).
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Fig. 2 Expression of TNF-a and IL-6 in the aorta using real-time PCR. *p \ 0.05
To evaluate the effect of BPA on inflammation, we evaluated the RNA expression of TNF-a and IL-6 in the tissues containing whole aortic wall using quantitative realtime PCR (Fig. 2). BPA significantly increased IL-6 by 2.1-fold (p = 0.02), and TNF-a showed the tendency to increase (p = 0.05). However, the serum TNF-a and IL-6 levels were not different between the groups (Table 1).
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BPA Accelerated Atherosclerotic Lesions in ApoE-/Mice To determine the effect of BPA on the formation of atherosclerotic lesions in mice, we stained their aortas and aortic sinuses with Oil red O. BPA significantly increased atherosclerotic lesions in the aorta by 1.7-fold (p = 0.03; Fig. 3a, b). However, atherosclerotic lesions in the aortic sinus did not show the difference between the groups (Fig. 3c, d). As vulnerable plaques contain more active inflammation, resulting in a larger number of macrophages, determining the amount of macrophages in plaques can be an
indirect indicator of unstable plaques [23]. We stained the macrophages in the aortic sinuses and evaluated the extent of macrophage infiltration. The percentage of macrophagepositive area to total atherosclerotic plaque in the aortic sinus in the BPA group was not different from that in the control group (39 ± 3 vs. 34 ± 6 %, p = 0.24; Fig. 3e, f). Non-HDL Cholesterol Concentrations Correlated with Atherosclerotic Lesions in ApoE-/- Mice Among several parameters, there was a significant association between serum cholesterol levels and atherosclerotic
Fig. 3 Atherosclerotic lesions in the aorta and aortic sinus of ApoE-/- mice. a Representative photographs of aortas stained with Oil red O. Magnification 940. b Relative ratio of atheroma in aortas. *p \ 0.05. c Representative photographs of aortic sinuses stained with Oil red O. Scale bars represent 1 mm. d Relative ratio of atheroma in aortic sinuses. e Representative photographs of aortic sinuses stained with an anti-F4/80 antibody. Scale bars represent 1 mm. f Percentage of macrophages in an atherosclerotic plaque of aortic sinuses
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lesions of the aorta (Table 2). Total cholesterol and nonHDL cholesterol levels were significantly correlated with atherosclerotic lesions of the aorta (r = 0.89, p = 0.02). There was no association between atherosclerotic lesions and body weight, blood glucose levels, or serum inflammatory markers (Table 2). BPA did not Affect HUVEC Proliferation and Migration As the BPA concentration detected in humans has been reported to range 0.7–20 nM [24, 25] and that of ApoE-/mice in our experiments was 0.4–14 nM. HUVECs were treated with 0.1, 1, or 10 nM BPA for 24 h. We assessed cell proliferation using a BrdU assay kit and found that the BPA treatment did not affect cell proliferation (Fig. 4a). To evaluate the effect of BPA on cell migration, we performed a wound healing assay and Transwell migration assay. HUVECs grew into the wounds after HUVEC monolayer was scratched, and the distance the cells covered was measured. BPA treatment did not alter the distance covered by the cells (Fig. 4b). In the Transwell migration assay, BPA treatment did not affect the number of cells that passed through the chamber either (Fig. 4c).
[4–6]. However, no report has demonstrated a direct relationship between BPA and atherosclerosis, and some speculation exists for that relationship. To our knowledge, this is the first report revealing that BPA can induce further atherosclerosis in animals. As this result suggests that BPA can cause cardiovascular disease in humans, we consider it noteworthy. The primary cause of BPA-induced atherosclerosis in this study might have been high non-HDL cholesterol levels. BPA administration significantly increased nonHDL cholesterol levels and they were significantly correlated with atherosclerotic lesions in the aorta. Dyslipidemia plays a pivotal role in the development of atherosclerosis [11], and the accumulated evidence suggests that BPA can affect lipid metabolism [26–28]. BPA could stimulate adipocyte differentiation [26], inhibit adiponectin release
Discussion In this study, BPA accelerated atherosclerosis in ApoE-/mice. BPA increased non-HDL cholesterol levels and these correlated with atherosclerotic lesions in the aorta. These results suggest that an increase in non-HDL cholesterol levels may be a major contributing factor to BPA-induced atherosclerosis. The results from epidemiological studies leave little doubt that BPA is associated with cardiovascular disease
Table 2 Correlation between metabolic parameters and atherosclerotic lesions in the aorta of ApoE-/- mice Spearman correlations coefficients Body weight LBM
0.22 -0.3
p value 0.47 0.93
Percentage of body fat
0.62
0.05
Fasting blood glucose
0.49
0.09
Total cholesterol
0.89
0.02
Triglyceride
0.26
0.62
HDL cholesterol
0.71
0.11
Non-HDL cholesterol
0.89
0.02
0.09
0.76
-0.35
0.24
TNF-a IL-6
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Fig. 4 HUVEC proliferation and migration. HUVECs were treated with vehicle or 0.1–10 nM BPA for 24 h. a Cell proliferation determined by BrdU assay. b Cell migration determined by wound healing assay. The distance covered by cells with or without BPA was measured. Data are represented as % of control. c Cell migration determined by Transwell migration assay. The number of cells with or without BPA was counted. Data are represented as % of control
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from human adipose tissue [27], and increase the expression of lipogenic genes in mouse liver [28]. Exposure to BPA during perinatal period increased serum cholesterol levels and body weight in mice [18]. However, most epidemiologic studies have found no association between BPA and non-HDL cholesterol levels in humans [4, 13]. We unexpectedly observed that BPA increased HDL cholesterol levels in ApoE-/- mice. An increase in HDL cholesterol levels can be due to the estrogenic effect of BPA because estrogen can increase HDL cholesterol levels [29]. In this study, BPA accelerated atherosclerosis despite of an increase in HDL cholesterol levels. One possible explanation for that may be that an increase in non-HDL cholesterol levels was larger than that of HDL cholesterol levels (2.1-fold vs. 1.4-fold). In addition, the HDL composition and function such as cholesterol efflux, paroxonase activity, and antioxidative property has been suggested to be more important than HDL cholesterol level itself [30]. Therefore, it might be possible that the compositional or functional difference in HDL cholesterol could exist in our experiments. Inflammation is a key feature of atherosclerosis [31]. In this study, the expression of TNF-a and IL-6 in the aorta increased although serum levels of those inflammatory cytokines were not different among the groups. It is consistent with previous reports that BPA could increase the expression of IL-6 in the mouse liver [32] or the expression of TNF-a and IL-6 release in human adipose tissue [33]. All these findings suggest that inflammation could be one of the possible mechanisms of BPA-induced atherosclerosis; however, we filed to show the increments of macrophages infiltration to the atherosclerotic lesions. Therefore, further studies about the pathogenic role of inflammation or inflammatory cytokines are required. Previously, insulin resistance [13, 19, 20] and obesity [14, 15] were also suggested as possible links between BPA and atherosclerosis. However, in this study, BPA did not increase blood glucose levels and body weights in ApoE-/- mice. Therefore, in this animal model at least, insulin resistance and obesity were not the main mechanism behind BPA-induced atherosclerosis. We investigated the effect of BPA on endothelial cell proliferation and migration to elucidate the molecular mechanisms of BPA-induced atherosclerosis. In this study, BPA did not affect endothelial cell proliferation or migration in vitro. BPA did not appear to have a direct effect on endothelial cells, which play a pivotal role in the development of atherosclerosis [11]. Therefore, we suspect that BPA did not accelerate atherosclerosis directly, but indirectly by increasing non-HDL cholesterol levels. The strength of this study is that a low dose of BPA was chronically (12 weeks) administered to the mice through drinking water, similar to actual conditions in everyday
life. When BPA was administered with diet, slightly decreased but similar absorption rate (81 %) was shown [34]. Therefore, it is speculated that the result of this study will not be different if BPA is administered with diet. Regulatory agencies such as the US FDA have documented that exposure to the low doses of BPA currently on the market is safe [21]. However, several recent studies investigating BPA have reported adverse effects at concentrations below the NOAEL, substantiating growing concerns [35]. The BPA doses administered to the mice in this study were below the NOAEL, and serum BPA concentration in mice (0.4–14 nM) was comparable to those reported in human studies (0.7–20 nM). However, even in these low doses of BPA, some pro-atherosclerotic effects were observed in our study, and this is the first study demonstrated the harmful effects of BPA on atherosclerosis in in vivo models. These results are consistent with epidemiologic data from human [5]. Therefore, our study supports the need for the regulation strengthening for BPA. In addition, BPA was administered to the adult mice in this study. It suggests that exposure to BPA not only during the perinatal period but also in adulthood can affect atherosclerosis and the restriction of BPA intake may be needed even in adulthood. This study has some limitations. First, changes in estrogenic activity after BPA administration were not assessed. BPA binds and activates the estrogen receptor [36], and estrogenic activity can affect atherosclerosis. However, whether exogenous estrogen protects or harms blood vessels remains still controversial, and the results from ApoE-/- mice treated with estrogen are also conflicting [37–39]. Second, we did not examine other potential mechanism such as oxidative stress and mitochondrial dysfunction. Oxidative stress has received attention as an important mechanism of BPA action [32, 40, 41], and it can contribute to the development of atherosclerosis through lipid oxidation which in turn increases vascular inflammation [12]. Last, although ApoE-/- mice are widely used in atherosclerosis experiments as a model of familial combined hypercholesterolemia, the lipid metabolism of rodents is not same as that of human; thus, these results from ApoE-/- mice could not be generalized to human and should be interpreted with caution. Studies in another animal model or human beings are required. In conclusion, this study demonstrated the possibility that chronic exposure to low-dose BPA accelerates atherosclerosis in ApoE-/- mice and suggested that the increase in non-HDL cholesterol levels is a primary factor contributing to BPA-induced atherosclerosis. Acknowledgments This work was supported by National Research Foundation of Korea (NRF) Grant funded by the Korean Government (2010-0023068).
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38. Cann, J. A., Register, T. C., Adams, M. R., St Clair, R. W., Espeland, M. A., & Williams, J. K. (2008). Timing of estrogen replacement influences atherosclerosis progression and plaque leukocyte populations in ApoE-/- mice. Atherosclerosis, 201, 43–52. 39. Freudenberger, T., Oppermann, M., Heim, H. K., Mayer, P., Kojda, G., Schror, K., et al. (2010). Proatherogenic effects of estradiol in a model of accelerated atherosclerosis in ovariectomized ApoE-deficient mice. Basic Research in Cardiology, 105, 479–486. 40. Bindhumol, V., Chitra, K. C., & Mathur, P. P. (2003). Bisphenol A induces reactive oxygen species generation in the liver of male rats. Toxicology, 188, 117–124. 41. Hassan, Z. K., Elobeid, M. A., Virk, P., Omer, S. A., Elamin, M., Daghestani, M. H., et al. (2012). Bisphenol A induces hepatotoxicity through oxidative stress in rat model. Oxidative Medicine and Cellular Longevity, 2012, 194829.
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Chronic Exposure to Bisphenol A can Accelerate Atherosclerosis in High-Fat-Fed Apolipoprotein E Knockout Mice Min Joo Kim • Min Kyong Moon • Geun Hyung Kang • Kwan Jae Lee • Sung Hee Choi • Soo Lim • Byung-Chul Oh • Do Joon Park • Kyong Soo Park Hak Chul Jang • Young Joo Park
•
Ó Springer Science+Business Media New York 2013
Abstract In epidemiological studies, there is growing concern regarding the association between human exposure to bisphenol A (BPA) and an increased risk for cardiovascular disease. Therefore, we investigated whether BPA accelerates atherosclerosis in mouse model. Apolipoprotein E knockout (ApoE-/-) mice were fed a high-fat and highcholesterol diet with or without 50 lg/kg body weight/day BPA for 12 weeks. Atherosclerotic lesions of the aorta and aortic sinus were evaluated by Oil red O staining. After the 12-week BPA treatment, BPA significantly increased atherosclerotic lesions in the aortas of ApoE-/- mice by 1.7fold (p = 0.03). Non-high-density lipoprotein (HDL) cholesterol levels in the BPA group were significantly higher compared to those in the control group (1,035 ± 70 vs. 484 ± 48 mg/dL, p = 0.02) although body weight and blood glucose levels were not different between groups. Human umbilical vein endothelial cells (HUVECs) were treated with 0.1–10 nM BPA but BPA did not affect
Min Joo Kim and Min Kyong Moon have equally contributed to this work. M. J. Kim M. K. Moon S. H. Choi S. Lim D. J. Park K. S. Park H. C. Jang Y. J. Park (&) Department of Internal Medicine, Seoul National University College of Medicine, 28 Yongon-dong, Jongno-gu, Seoul 110-744, Republic of Korea e-mail: [email protected] M. J. Kim Department of Internal Medicine, Korea Cancer Center Hospital, Seoul 139-706, Republic of Korea M. K. Moon Department of Internal Medicine, Seoul Metropolitan Government Boramae Medical Center, Seoul 156-707, Republic of Korea
HUVEC proliferation or migration. BPA could accelerate atherosclerosis in ApoE-/- mice, which may have resulted from an increase in non-HDL cholesterol levels. Keywords Bisphenol A Atherosclerosis Non-HDL cholesterol Dyslipidemias
Introduction Mankind is ubiquitously exposed to bisphenol A (BPA) because it is a base material of various beverage and food containers [1]. BPA is reported to be detected in 92 % of the general population in USA [2]. In the initial stage, researchers focused on the estrogenic activity of BPA and its reproductive toxicity [3]. However, there is growing concern regarding the harmful effects of BPA exposure on human health besides reproductive toxicity [4]. In a large cross-sectional study using the United States National Health and Nutrition Examination Survey, it was found that a high urinary BPA concentration was associated with an
G. H. Kang Biomedical Research Institute, Seoul National University Hospital, Seoul 110-744, Republic of Korea K. J. Lee S. H. Choi S. Lim H. C. Jang Department of Internal Medicine, Seoul National University Bundang Hospital, Seongnam 463-707, Republic of Korea B.-C. Oh Department of Molecular Medicine, Lee Gil Ya Cancer and Diabetes Institute, Gachon University of Medicine and Science, Inchon 406-840, Republic of Korea
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increased risk for chronic medical disorders such as cardiovascular disease, diabetes, and abnormal liver function [4]. Subsequently, the relationship between higher BPA exposure and cardiovascular disease has been replicated in both cross-sectional and longitudinal studies [5, 6]. Exposure to BPA has also been reported as being associated with the severity of atherosclerosis in the coronary artery or carotid artery [7, 8], increased blood pressure, and reduced heart rate variability [9, 10]. Atherosclerosis is a fundamental process of cardiovascular disease. It is initiated by endothelial dysfunction and structural defect, and then, low-density lipoprotein (LDL) accumulates making early fatty streak lesion [11]. Increased circulating LDL cholesterol levels accelerate the development of atherosclerosis and cardiovascular disease. Oxidative stress such as reactive oxygen species modifies LDL [12]. Oxidized LDL triggers the expression of leukocyte adhesion molecules, and it recruits leukocytes such as monocyte-derived macrophages and lymphocytes, resulting in vascular inflammation. Consequently, atherosclerotic lesions consist of macrophage, lipids, and smooth muscle cells. Although many epidemiologic studies have established the association between BPA and atherosclerosis, a causal relationship remains uncertain. Previous epidemiological studies have reported that a high urinary BPA concentration was associated with insulin resistance [13], diabetes [4], and obesity [14, 15], and they have been considered possible links between BPA and atherosclerosis. In line with these data, perinatal exposure to BPA (10–100 lg/kg) has been shown to induce insulin resistance [16], weight gain [17], and dyslipidemia in rodents [18]. Although there is a paucity of research on adult rodents, it is reported that the short-term administration of BPA (10–100 lg/kg) to adult mice can at least induce insulin resistance [19, 20]. This evidence suggests that BPA can induce or accelerate atherosclerosis. However, no experimental study, not even animal or in vitro studies, has clarified that exposure to BPA can induce atherosclerosis. Therefore, we investigated whether BPA can accelerate atherosclerosis and the mechanism linking BPA to atherosclerosis using apolipoprotein E knockout (ApoE-/-) mice, an animal model of atherosclerosis.
Materials and Methods Animals ApoE-/- male mice were purchased from Jackson Laboratories (Sacramento, CA, USA). All mice were fed ad libitum laboratory chow diet (Purina irradiated laboratory chow 38057, Purina Korea, Seoul, Korea), followed by
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a high-fat and high-cholesterol diet (Research Diets Inc., New Brunswick, NJ, USA) containing 21 % fat and 0.21 % cholesterol when they were 8 weeks old. The mice were assigned to the control or BPA group when they started the high-fat and high-cholesterol diet. BPA (purity [99 %) was purchased from Sigma Aldrich (St. Louis, MO, USA). The BPA group received 50 lg/kg body weight (bw)/ day BPA via drinking water for 12 weeks. This BPA dose is below the no observed adverse effect level (NOAEL) of 5 mg/kg bw/day set by the United States Food and Drug Administration (US FDA) [21]. All mice were housed in conventional plastic cages with free access to feed and water at 23 ± 2 °C, 60 ± 10 % humidity, and a 12-h light/ dark photoperiod. At the end of the 12 weeks, the mice were anesthetized and then killed. The experiment was repeated 5 times with 3 or 4 mice in each group. All procedures involving the use of laboratory animals were in accordance with the Guide for Standard Operation Procedures and were performed after receiving the approval of the Institutional Animal Care and Use Committee (IACUC) of the Clinical Research Institute, Seoul National University Bundang Hospital (IACUC Approval No. BA1012-074/068-01). Measurement of Body Weight and Blood Glucose Levels Body weight was monitored before and after the experiments. Lean body mass (LBM) and percent body fat content were measured using a dual energy X-ray absorptiometer (GE Lunar PIXImus, Fitchburg, WI, USA). After each 12-week experiments, an intraperitoneal glucose tolerance test (IPGTT) was performed after a 6-h fast by administering an intraperitoneal injection of 2 g/kg bw glucose. Blood glucose levels from tail vein blood were determined using an Accu-Chek Active glucometer (Roche, Mannheim, Germany) before and 15, 30, 90, and 120 min after glucose injection. Measurement of BPA Concentration, Lipid Profiles, and Cytokines Serum total BPA concentrations were measured using the enzyme-linked immunosorbent assay (EcologienaÒ, Osaka, Japan). Total cholesterol, triglyceride, and high-density lipoprotein (HDL) cholesterol were determined using a Beckman Coulter AU480 automatic biochemistry analysis system (Japan) with reagent kits provided by the manufacturer. Tumor necrosis factor-a (TNF-a) and interleukin6 (IL-6) were measured using a radioimmunoassay kit (Diaclone, Besanc¸on, France) following the protocol provided by the manufacturer.
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Quantitative Real-Time Polymerase Chain Reaction (PCR) Total RNA was isolated from aorta using TRizol reagent (Ambion, Foster City, CA, USA), and M-MLV reverse transcriptase (Invitrogen, Carlsbad, CA, USA) was used for cDNA synthesis. Quantitative real-time PCR was performed using the ABI 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA), and the SYBR Premix Ex Taq polymerase (Takara, Otsu, Japan) was used for amplification. The primer sequences were as follows: for TNF-a, forward primer sequence was 50 CCTCCCTCTCATCAGTTCTA-30 , and the reverse primer sequence was 50 -CAGCATCGTTTGGTGGTTCA0 ; for IL6, the forward primer sequence was 50 -ACCACGGCCT TCCCTACTTC-30 , and the reverse primer sequence was 50 -GACGAGTTCACTACCCTATGG-30 . Data were analyzed using ABI 7500 software version 2.0.5 (Applied Biosystems, Foster City, CA, USA). Measurement of Atherosclerotic Lesions in the Aorta and Aortic Sinus To measure the extent of atherosclerosis in the aorta, the entire aorta was removed, placed in 4 % formaldehyde, and prepared using the ‘‘en face’’ method. Each aorta was opened longitudinally from the heart to the iliac arteries, dissected, and pinned on a plate. The fixed aortas were stained with Oil red O to delineate the atherosclerotic lesions, and images were obtained with a light microscope (Carl Zeiss, Germany). The Oil red O-positive area and total aortic surface area were measured using Adobe Photoshop software version 5.0.1 (Adobe Systems, San Jose, CA, USA). Atherosclerotic lesion of the aorta is defined as the percentage of Oil red O-positive area to total aortic surface area and presented as fold changes compared to the control group in each set. To measure the extent of atherosclerosis in the aortic sinus, tissues of the heart and ascending aorta were embedded in OCT compound (Sakura Finetek, Torrance, CA, USA). Frozen tissue was sectioned into 30-lm-thick slices. We selected sections that contained the cusps of 3 aortic valves at the junction of the aorta and the left ventricle. The sections were stained with Oil red O to determine areas containing atherosclerotic lesions. Sections were examined under a light microscope (Carl Zeiss, Germany), and at least 2 images were obtained from each mice. The Oil red O-positive red color spot contained area was measured to define the area of atheroma in aortic sinus using Adobe Photoshop software version 5.0.1 (Adobe Systems, San Jose, CA, USA). In some cases (3 control and 3 BPA group), corresponding sections were also stained with hematoxylin and eosin (H&E) to measure the area of
atherosclerotic lesions, and the area quantified by H&E staining was well correlated with that by Oil red O staining (data not shown). Atherosclerotic lesions of the aortic sinus are presented as fold changes compared to the control group in each set. Macrophage Infiltration into Atherosclerotic Lesions To stain macrophages, tissue sections were incubated with an anti-F4/80 antibody (eBioscience, San Diego, CA, USA) and subsequently with biotinylated anti-rabbit IgG antibody. The positively stained areas were measured by Axiovision software version 4.8 (Carl Zeiss, Germany). The extent of macrophage infiltration is presented as the percentage of anti-F4/80 antibody-positive stained area to the Oil red O-positive area. Cell Culture Primary human umbilical vein endothelial cells (HUVECs) were purchased from Cambrex (East Rutherford, NJ, USA) and cultured in endothelial basal medium (EBM-2) containing endothelial growth factor (EGM-2 Bullet Kit, Cambrex, East Rutherford, NJ, USA) and 2 % fetal bovine serum (FBS). For the experiments, HUVECs were starved in EBM-2 medium with 0.4 % FBS. Cell Proliferation Cell proliferation was evaluated using a bromodeoxyuridine (BrdU) cell proliferation assay kit (Cell Signaling Technology Inc., Danvers, MA, USA) according to the manufacturer’s protocol. In brief, HUVECs were cultured in 98-well plates. Each well was seeded with 2,500 cells and incubated with vehicle or 0.1, 1, or 10 nM BPA for 24 h. BrdU was then added to the medium and the cells were reincubated for 4 h. After the medium was removed, the HUVECs were fixed and the DNA was denatured. An anti-BrdU mouse antibody was added to detect the incorporated BrdU. Anti-mouse IgG, a horseradish peroxidase (HRP)-linked antibody, was then used to identify the bound detection antibody. The HRP substrate tetramethylbenzidine was added to develop color. The magnitude of the absorbance of the developed color is proportional to the quantity of BrdU incorporated into cells, which is a direct indication of cell proliferation. The experiment was repeated 3 times with 6 wells per each condition. Wound Healing Assay HUVECs were cultured in 6-well plates and grown in a monolayer. Each well was scratched with a pipette tip to create a wound [22]. Next, HUVECs were treated with
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vehicle or 0.1, 1, or 10 nM BPA for 24 h. After 24 h, the images were captured and the distance covered by the cells was measured at least 3 distance points to estimate cell migration. The experiment was repeated 3 times. Transwell Migration Assay The modified Boyden chamber assay was performed using a Transwell system (Corning, Rochester, NY, USA). The chamber insert contained a polycarbonate membrane with 8-lm pores that permitted cells to migrate. Each insert was coated with 10 lg gelatin solution, and 104 HUVECs in medium containing 0.4 % FBS were loaded. Each well was filled with 500 lL starvation medium with or without BPA, and the inserts were placed into 24-well plates. After 24 h, the upper surface of inserts was swabbed with a cottontipped applicator to remove non-migrated cells. The inserts were fixed in methanol for 10 min and stained with 1 % crystal violet for 2 h. The number of cells that had moved was counted to estimate cell migration. The experiment was repeated 5 times with 2 wells per each condition.
Table 1 Metabolic parameters and inflammatory cytokines in ApoE-/- mice Control (n = 15)
BPA (n = 19)
p value
Body weight (g)
31.7 ± 1.0
30.4 ± 1.0
0.38
LBM (g)
25.7 ± 0.8
23.5 ± 1.0
0.09
Percentage of body fat Fasting blood glucose (mg/dL)
17.3 ± 0.6 153 ± 10
20.7 ± 1.3 134 ± 6
0.09 0.08
Total cholesterol (mg/dL)
522 ± 46
1,088 ± 70
Triglyceride (mg/dL)
104 ± 24
HDL cholesterol (mg/dL)
38 ± 1
Non-HDL cholesterol (mg/dL)
484 ± 48
TNF-a (pg/mL) IL-6 (pg/mL) BPA (ng/mL)
94 ± 9 53 ± 2
0.02 0.90 0.02
1,035 ± 70
0.02
195 ± 195
121 ± 64
0.83
19 ± 7 0.15 ± 0.04
27 ± 12 1.26 ± 0.62
0.83 0.02
Statistical Analysis Results are presented as mean ± standard error. The Mann–Whitney test was used to compare the BPA and control groups. The IPGTT results were analyzed with repeated measures analysis of variance. The Spearman’s correlation test was used to assess the relationship between several metabolic parameters and atherosclerosis. All statistical analyses were performed using SPSS software (Chicago, IL, USA). Statistical significance was assumed at p \ 0.05.
Fig. 1 Blood glucose levels during IPGTT in ApoE-/- mice
Results Effects of BPA on Metabolic Profiles in ApoE-/- Mice After the 12-week BPA treatment, serum BPA concentrations of the BPA group were significantly higher than those of the control group (1.26 ± 0.62 vs. 0.15 ± 0.04 ng/mL, p = 0.02). The body weights of the BPA group were not different from those of the control group (Table 1). The BPA group had a higher percentage of body fat and lower LBM, but there was no statistical significance (p = 0.09; Table 1). Blood glucose levels during the IPGTT were not different between the 2 groups (p = 0.25; Fig. 1). BPA significantly increased the levels of both non-HDL and HDL cholesterol levels (p = 0.02; Table 1). Non-HDL cholesterol levels in the BPA group were twice as high as those in the control group (1,035 ± 70 vs. 484 ± 48 mg/ dL, p = 0.02; Table 1).
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Fig. 2 Expression of TNF-a and IL-6 in the aorta using real-time PCR. *p \ 0.05
To evaluate the effect of BPA on inflammation, we evaluated the RNA expression of TNF-a and IL-6 in the tissues containing whole aortic wall using quantitative realtime PCR (Fig. 2). BPA significantly increased IL-6 by 2.1-fold (p = 0.02), and TNF-a showed the tendency to increase (p = 0.05). However, the serum TNF-a and IL-6 levels were not different between the groups (Table 1).
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BPA Accelerated Atherosclerotic Lesions in ApoE-/Mice To determine the effect of BPA on the formation of atherosclerotic lesions in mice, we stained their aortas and aortic sinuses with Oil red O. BPA significantly increased atherosclerotic lesions in the aorta by 1.7-fold (p = 0.03; Fig. 3a, b). However, atherosclerotic lesions in the aortic sinus did not show the difference between the groups (Fig. 3c, d). As vulnerable plaques contain more active inflammation, resulting in a larger number of macrophages, determining the amount of macrophages in plaques can be an
indirect indicator of unstable plaques [23]. We stained the macrophages in the aortic sinuses and evaluated the extent of macrophage infiltration. The percentage of macrophagepositive area to total atherosclerotic plaque in the aortic sinus in the BPA group was not different from that in the control group (39 ± 3 vs. 34 ± 6 %, p = 0.24; Fig. 3e, f). Non-HDL Cholesterol Concentrations Correlated with Atherosclerotic Lesions in ApoE-/- Mice Among several parameters, there was a significant association between serum cholesterol levels and atherosclerotic
Fig. 3 Atherosclerotic lesions in the aorta and aortic sinus of ApoE-/- mice. a Representative photographs of aortas stained with Oil red O. Magnification 940. b Relative ratio of atheroma in aortas. *p \ 0.05. c Representative photographs of aortic sinuses stained with Oil red O. Scale bars represent 1 mm. d Relative ratio of atheroma in aortic sinuses. e Representative photographs of aortic sinuses stained with an anti-F4/80 antibody. Scale bars represent 1 mm. f Percentage of macrophages in an atherosclerotic plaque of aortic sinuses
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lesions of the aorta (Table 2). Total cholesterol and nonHDL cholesterol levels were significantly correlated with atherosclerotic lesions of the aorta (r = 0.89, p = 0.02). There was no association between atherosclerotic lesions and body weight, blood glucose levels, or serum inflammatory markers (Table 2). BPA did not Affect HUVEC Proliferation and Migration As the BPA concentration detected in humans has been reported to range 0.7–20 nM [24, 25] and that of ApoE-/mice in our experiments was 0.4–14 nM. HUVECs were treated with 0.1, 1, or 10 nM BPA for 24 h. We assessed cell proliferation using a BrdU assay kit and found that the BPA treatment did not affect cell proliferation (Fig. 4a). To evaluate the effect of BPA on cell migration, we performed a wound healing assay and Transwell migration assay. HUVECs grew into the wounds after HUVEC monolayer was scratched, and the distance the cells covered was measured. BPA treatment did not alter the distance covered by the cells (Fig. 4b). In the Transwell migration assay, BPA treatment did not affect the number of cells that passed through the chamber either (Fig. 4c).
[4–6]. However, no report has demonstrated a direct relationship between BPA and atherosclerosis, and some speculation exists for that relationship. To our knowledge, this is the first report revealing that BPA can induce further atherosclerosis in animals. As this result suggests that BPA can cause cardiovascular disease in humans, we consider it noteworthy. The primary cause of BPA-induced atherosclerosis in this study might have been high non-HDL cholesterol levels. BPA administration significantly increased nonHDL cholesterol levels and they were significantly correlated with atherosclerotic lesions in the aorta. Dyslipidemia plays a pivotal role in the development of atherosclerosis [11], and the accumulated evidence suggests that BPA can affect lipid metabolism [26–28]. BPA could stimulate adipocyte differentiation [26], inhibit adiponectin release
Discussion In this study, BPA accelerated atherosclerosis in ApoE-/mice. BPA increased non-HDL cholesterol levels and these correlated with atherosclerotic lesions in the aorta. These results suggest that an increase in non-HDL cholesterol levels may be a major contributing factor to BPA-induced atherosclerosis. The results from epidemiological studies leave little doubt that BPA is associated with cardiovascular disease
Table 2 Correlation between metabolic parameters and atherosclerotic lesions in the aorta of ApoE-/- mice Spearman correlations coefficients Body weight LBM
0.22 -0.3
p value 0.47 0.93
Percentage of body fat
0.62
0.05
Fasting blood glucose
0.49
0.09
Total cholesterol
0.89
0.02
Triglyceride
0.26
0.62
HDL cholesterol
0.71
0.11
Non-HDL cholesterol
0.89
0.02
0.09
0.76
-0.35
0.24
TNF-a IL-6
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Fig. 4 HUVEC proliferation and migration. HUVECs were treated with vehicle or 0.1–10 nM BPA for 24 h. a Cell proliferation determined by BrdU assay. b Cell migration determined by wound healing assay. The distance covered by cells with or without BPA was measured. Data are represented as % of control. c Cell migration determined by Transwell migration assay. The number of cells with or without BPA was counted. Data are represented as % of control
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from human adipose tissue [27], and increase the expression of lipogenic genes in mouse liver [28]. Exposure to BPA during perinatal period increased serum cholesterol levels and body weight in mice [18]. However, most epidemiologic studies have found no association between BPA and non-HDL cholesterol levels in humans [4, 13]. We unexpectedly observed that BPA increased HDL cholesterol levels in ApoE-/- mice. An increase in HDL cholesterol levels can be due to the estrogenic effect of BPA because estrogen can increase HDL cholesterol levels [29]. In this study, BPA accelerated atherosclerosis despite of an increase in HDL cholesterol levels. One possible explanation for that may be that an increase in non-HDL cholesterol levels was larger than that of HDL cholesterol levels (2.1-fold vs. 1.4-fold). In addition, the HDL composition and function such as cholesterol efflux, paroxonase activity, and antioxidative property has been suggested to be more important than HDL cholesterol level itself [30]. Therefore, it might be possible that the compositional or functional difference in HDL cholesterol could exist in our experiments. Inflammation is a key feature of atherosclerosis [31]. In this study, the expression of TNF-a and IL-6 in the aorta increased although serum levels of those inflammatory cytokines were not different among the groups. It is consistent with previous reports that BPA could increase the expression of IL-6 in the mouse liver [32] or the expression of TNF-a and IL-6 release in human adipose tissue [33]. All these findings suggest that inflammation could be one of the possible mechanisms of BPA-induced atherosclerosis; however, we filed to show the increments of macrophages infiltration to the atherosclerotic lesions. Therefore, further studies about the pathogenic role of inflammation or inflammatory cytokines are required. Previously, insulin resistance [13, 19, 20] and obesity [14, 15] were also suggested as possible links between BPA and atherosclerosis. However, in this study, BPA did not increase blood glucose levels and body weights in ApoE-/- mice. Therefore, in this animal model at least, insulin resistance and obesity were not the main mechanism behind BPA-induced atherosclerosis. We investigated the effect of BPA on endothelial cell proliferation and migration to elucidate the molecular mechanisms of BPA-induced atherosclerosis. In this study, BPA did not affect endothelial cell proliferation or migration in vitro. BPA did not appear to have a direct effect on endothelial cells, which play a pivotal role in the development of atherosclerosis [11]. Therefore, we suspect that BPA did not accelerate atherosclerosis directly, but indirectly by increasing non-HDL cholesterol levels. The strength of this study is that a low dose of BPA was chronically (12 weeks) administered to the mice through drinking water, similar to actual conditions in everyday
life. When BPA was administered with diet, slightly decreased but similar absorption rate (81 %) was shown [34]. Therefore, it is speculated that the result of this study will not be different if BPA is administered with diet. Regulatory agencies such as the US FDA have documented that exposure to the low doses of BPA currently on the market is safe [21]. However, several recent studies investigating BPA have reported adverse effects at concentrations below the NOAEL, substantiating growing concerns [35]. The BPA doses administered to the mice in this study were below the NOAEL, and serum BPA concentration in mice (0.4–14 nM) was comparable to those reported in human studies (0.7–20 nM). However, even in these low doses of BPA, some pro-atherosclerotic effects were observed in our study, and this is the first study demonstrated the harmful effects of BPA on atherosclerosis in in vivo models. These results are consistent with epidemiologic data from human [5]. Therefore, our study supports the need for the regulation strengthening for BPA. In addition, BPA was administered to the adult mice in this study. It suggests that exposure to BPA not only during the perinatal period but also in adulthood can affect atherosclerosis and the restriction of BPA intake may be needed even in adulthood. This study has some limitations. First, changes in estrogenic activity after BPA administration were not assessed. BPA binds and activates the estrogen receptor [36], and estrogenic activity can affect atherosclerosis. However, whether exogenous estrogen protects or harms blood vessels remains still controversial, and the results from ApoE-/- mice treated with estrogen are also conflicting [37–39]. Second, we did not examine other potential mechanism such as oxidative stress and mitochondrial dysfunction. Oxidative stress has received attention as an important mechanism of BPA action [32, 40, 41], and it can contribute to the development of atherosclerosis through lipid oxidation which in turn increases vascular inflammation [12]. Last, although ApoE-/- mice are widely used in atherosclerosis experiments as a model of familial combined hypercholesterolemia, the lipid metabolism of rodents is not same as that of human; thus, these results from ApoE-/- mice could not be generalized to human and should be interpreted with caution. Studies in another animal model or human beings are required. In conclusion, this study demonstrated the possibility that chronic exposure to low-dose BPA accelerates atherosclerosis in ApoE-/- mice and suggested that the increase in non-HDL cholesterol levels is a primary factor contributing to BPA-induced atherosclerosis. Acknowledgments This work was supported by National Research Foundation of Korea (NRF) Grant funded by the Korean Government (2010-0023068).
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