Pathological analysis of adipose in WHHL rabbits at 12 weeks.

<p>(A) The total mass of adipose tissue was measured and expressed as the ratio of adipose tissue weight to body weight. (B) Representative micrographs of adipose tissue (H&E-stained specimens), morphometric analysis of adipocyte cell size distribution and mean diameters in the vehicle and BPA-exposure. (400 µg/kg) groups. Data are expressed as the means ± SD, n = 6 for each group. *<i>p</i><0.05 vs. vehicles.</p

Serum pharmacokinetic parameters for unconjugated BPA in WHHL rabbits compared with monkey and mice.

a<p>C_max: maximum attained value; ^bTerminal t½: the terminal phase half-life;</p>c<p>AUC_0–24: the area under the curve at <i>t</i> = 24 hr after gavage.</p><p>Serum pharmacokinetic parameters for unconjugated BPA in WHHL rabbits compared with monkey and mice.</p

Rabbit aortic smooth muscle cells (SMCs) proliferation.

<p>SMCs were treated with either vehicle or BPA (0.5 and 5 ng/mL) for 24 h and 10% fetal bovine serum was used as a positive control. Cell proliferation was determined by CCK-8 assay. Five replicates were performed in each assay. Data are represented as % of control. *<i>p</i><0.05 vs. vehicles.</p

Body weights, serum lipids and glucose levels in WHHL rabbits over 12 weeks.

<p>These indexes were monitored every 2 weeks. Data are expressed as the means ± SD, n = 6 for each group.</p

Pathological changes and mRNA expression in the livers of WHHL rabbits at 12 weeks.

<p>(A) Representative micrographs stained with H&E showed steatosis (middle) and inflammatory cells infiltration (lower) in the BPA-exposure group. Arrows highlight steatosis and inflammatory cells. (B) RT-PCR analysis of mRNA expressions in the livers of WHHL rabbits after BPA exposure. The mRNA expression levels of genes related to the ER pathway, lipid metabolism, and liver inflammation in WHHL rabbits treated with 400 µg/kg BPA for 12 weeks were analyzed using real-time RT-PCR. Expression levels are expressed relative to the data obtained in the vehicle group. Data are expressed as the means ± SD, n = 6 for each group. *<i>p</i><0.05 vs. vehicles.</p

Quantitative analysis of aortic atherosclerosis in WHHL rabbits at 12 weeks.

<p>(A) Representative micrographs of the aortic trees stained with Sudan IV. (B) The sudanophilic en-face lesion areas in different parts of aortic trees were determined using an image analysis system as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110977#s2" target="_blank">Materials and Methods</a> section. Data are expressed as the means ± SD, n = 6 for each group. *<i>p</i><0.05 vs. vehicle.</p

Microscopic analysis of atherosclerosis in the aortic arches of WHHL rabbits at 12 weeks.

<p>(A) Representative micrographs stained with H&E and EVG for intimal lesion analysis are shown in the top two panels. Representative micrographs stained with HHF35 and RAM11 mAbs for immunohistochemical analysis of SMCs and macrophages are shown in the lower two panels. (B) Intimal lesions, advanced lesions, SMCs and Mφ-positive areas of the sections were quantified using an image analysis system as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110977#s2" target="_blank">Materials and Methods</a> section. Data are expressed as the means ± SD, n = 6 for each group. *<i>p</i><0.05 vs. vehicle. Original magnification: 10×.</p

Effects of BPA incubation on ER stress and inflammatory responses in HUVEC cells.

<p>Cultured HUVECs were incubated with either vehicle or BPA (0.5 and 5 ng/mL) for 24 hr, after which total RNA was extracted for RT-PCR analysis. The analysis was performed three times, and each assay was performed in triplicate. Representative data are shown as the means ± SD. *<i>p</i><0.05 vs. vehicles.</p

Pathological analyses of WHHL rabbit hearts at 12 weeks.

<p>(A) Heart specimens stained with H&E show disarrayed myocardiocytes along with (b) fatty degeneration (c) inflammatory cell infiltration, fibrosis, and (d) calcification in the BPA-exposure group. (B) The lesion areas in the vehicle and BPA-exposure groups were quantified using an image analysis system. Data are expressed as the means ± SD, n = 6 for each group. **<i>p</i><0.01 vs. vehicles.</p

Bisphenol A Exposure Enhances Atherosclerosis in WHHL Rabbits

<div><p>Bisphenol A (BPA) is an environmental endocrine disrupter. Excess exposure to BPA may increase susceptibility to many metabolic disorders, but it is unclear whether BPA exposure has any adverse effects on the development of atherosclerosis. To determine whether there are such effects, we investigated the response of Watanabe heritable hyperlipidemic (WHHL) rabbits to 400-µg/kg BPA per day, administered orally by gavage, over the course of 12 weeks and compared aortic and coronary atherosclerosis in these rabbits to the vehicle group using histological and morphometric methods. In addition, serum BPA, cytokines levels and plasma lipids as well as pathologic changes in liver, adipose and heart were analyzed. Moreover, we treated human umbilical cord vein endothelial cells (HUVECs) and rabbit aortic smooth muscle cells (SMCs) with different doses of BPA to investigate the underlying molecular mechanisms involved in BPA action(s). BPA treatment did not change the plasma lipids and body weights of the WHHL rabbits; however, the gross atherosclerotic lesion area in the aortic arch was increased by 57% compared to the vehicle group. Histological and immunohistochemical analyses revealed marked increases in advanced lesions (37%) accompanied by smooth muscle cells (60%) but no significant changes in the numbers of macrophages. With regard to coronary atherosclerosis, incidents of coronary stenosis increased by 11% and smooth muscle cells increased by 73% compared to the vehicle group. Furthermore, BPA-treated WHHL rabbits showed increased adipose accumulation and hepatic and myocardial injuries accompanied by up-regulation of endoplasmic reticulum (ER) stress and inflammatory and lipid metabolism markers in livers. Treatment with BPA also induced the expression of ER stress and inflammation related genes in cultured HUVECs. These results demonstrate for the first time that BPA exposure may increase susceptibility to atherosclerosis in WHHL rabbits.</p></div