Protective Effects of a Red Grape Juice Extract against Bisphenol A-Induced Toxicity in Human Umbilical Vein Endothelial Cells

Abstract

Human exposure to bisphenol A (BPA) occurs through the ingestion of contaminated food and water, thus leading to endothelial dysfunction, the first signal of atherosclerosis. Vitis vinifera L. (grape) juice is well known for its health-promoting properties, due to its numerous bioactive compounds among which are polyphenols. The aim of this study was to evaluate the protective effect of a red grape juice extract (RGJe) against the endothelial damage induced by BPA in human umbilical vein endothelial cells (HUVECs) as an in vitro model of endothelial dysfunction. Our results showed that RGJe treatment counteracted BPA-induced cell death and apoptosis in HUVECs, blocking caspase 3 and modulating p53, Bax, and Bcl-2. Moreover, RGJe demonstrated antioxidant properties in abiotic tests and in vitro, where it reduced BPA-induced reactive oxygen species as well as restored mitochondrial membrane potential, DNA integrity, and nitric oxide levels. Furthermore, RGJe reduced the increase of chemokines (IL-8, IL-1β, and MCP-1) and adhesion molecules (VCAM-1, ICAM-1, and E-selectin), caused by BPA exposure, involved in the primary phase of atheromatous plaque formation. Overall, our results suggest that RGJe prevents BPA-induced vascular damage modulating specific intracellular mechanisms, along with protecting cells, owing to its antioxidant capability.

Keywords: HUVECs; atherosclerosis; bisphenol A; cytotoxicity; endothelial dysfunction; oxidative stress; red grape juice extract.

Figure 1

Protective effect of RGJe against BPA-induced cytotoxicity in HUVECs. Cell viability was evaluated by MTT test (A) and cell proliferation via BrdU incorporation test (B). Results of both assays are expressed as percentages ± SEM of the absorbance value detected in treated cells compared to untreated ones (control, CTRL). Three independent experiments in eight replicates were performed (n = 24). ** p < 0.01 and **** p < 0.0001 vs. CTRL; ° p < 0.05, °°° p < 0.001, and °°°° p < 0.0001 vs. BPA 300 µM.

Figure 2

RGJe reduced both ROS generation and fall of ∆Ψm induced by BPA in HUVECs. Cytofluorimetric evaluation of intracellular ROS (A) and mitochondrial membrane potential (B) was carried out employing DCFH-DA and R123 fluorescent probes, respectively. Representative plots of three independent experiments performed in triplicate (n = 9) are shown. For ROS detection (A) the histograms show the percentage ± SEM of healthy cells (DCF−, M1) and cells with increased intracellular ROS (DCF⁺, M2) of three separate experiments in triplicate (n = 9). For ∆Ψm evaluation the histograms represent the percentage ± SEM of healthy cells (R123⁺, M2) and the cells with impaired mitochondrial membrane potential (R123−, M1) of three separate experiments in triplicate (n = 9).

Figure 3

Protective effect of RGJe against the DNA oxidative damage induced by BPA in HUVECs. Levels of 8-oxo-dG were measured by flow cytometry, detecting the emission signals of fluorochrome FITC-labeled avidin. Representative plots of three different experimental sessions performed in triplicate (n = 9) are displayed (A). The histogram reports the percentage ± SEM of healthy (non-fluorescent, M1) and damaged (fluorescent, M2) cells (B), from three independent experiments performed in triplicate (n = 9).

Figure 4

Effect of the pre-treatment with RGJe on antioxidant defense systems reduced by BPA in HUVECs. The activity of SOD (A) and CAT (B), as well as GSH levels (C) are reported. Data are expressed as the mean ± SEM of three separate experiments performed in triplicate (n = 9). **** p < 0.0001 vs. CTRL; ° p < 0.05, °° p < 0.01, °°° p < 0.001 vs. BPA 300 µM.

Figure 5

Effect of RGJe on reduction of NO release induced by BPA in HUVECs. The NO levels were determined by a colorimetric assay. Data are expressed as the percentage value of NO detected in treated cells compared to untreated ones and are shown as mean ± SEM from three independent experiments performed in triplicate (n = 9). **** p < 0.0001 vs. CTRL; ° p < 0.05, °°° p < 0.001, and °°°° p < 0.0001 vs. BPA 300 µM.

Figure 6

RGJe reduced the BPA-induced apoptotic cell death and modulated Casp3 enzymatic activity and apoptotic-related genes in HUVECs. Evaluation of apoptosis was performed cytofluorimetrically by the Annexin V-FITC/PI test (A). Representative Annexin V vs. PI dot plots are shown where necrotic (Annexin V−/PI+), late apoptosis (Annexin V+/PI+), viable (Annexin V−/PI−), and early apoptosis (Annexin V+/PI−) cells are in Q4, Q3, Q1, and Q2, respectively. The histograms report the percentage of cells for each quadrant, expressed as the mean ± SEM of three experiments in triplicate (n = 9) (B). The mRNA levels of apoptosis-related genes BAX, BCL-2, P53, and CASP3 were quantified via RT-PCR using the 2^−ΔΔCT method. Data are expressed as n-fold change relative to the untreated cells, reporting the obtained values from three independent experiments performed in triplicate (n = 9) (C). Data of caspase 3 enzymatic activity are expressed as the percentage of the value detected in treated cells compared to the untreated ones and are displayed as the mean ± SEM of three experiments in triplicate (n = 9) (D). *** p < 0.001 and **** p < 0.0001 vs. CTRL; ° p < 0.05, °° p < 0.01, °°° p < 0.001, and °°°° p < 0.0001 vs. BPA 300 µM.

Figure 7

Effect of RGJe on IL-8 and IL-1β cytokines and MCP-1 chemokine gene expression in BPA-treated HUVECs. The results from RT-PCR of IL-8 (A), IL-1β (B), and MCP-1 (C) are expressed as an n-fold change compared to untreated cells after normalization against β-actin as endogenous control. Data represent the mean ± SEM of three different sets of experiments performed in triplicate (n = 9). **** p < 0.0001 vs. CTRL; ° p < 0.05, °° p < 0.01, °°° p < 0.001, and °°°° p < 0.0001 vs. BPA 300 µM.

Figure 8

RGJe reduced the BPA-induced mRNA increase of VCAM-1, ICAM-1, and E-selectin adhesion molecules in HUVECs. The results from RT-PCR are expressed as an n-fold change compared to untreated cells, after normalization against β-actin as endogenous control. Data represent the mean ± SEM of three different sets of experiments performed in triplicate (n = 9). ** p < 0.01, *** p < 0.001, and **** p < 0.0001 vs. CTRL; ° p < 0.05, °° p < 0.01, °°° p < 0.001, and °°°° p < 0.0001 vs. BPA 300 µM.

Figure 9

Heatmap of gene expression modulated by RGJe. The columns of the heatmap represent each treatment and the rows indicate the genes. Each cell is colored based on the amount of the gene found in that sample.