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

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.


Introduction
Bisphenol A (BPA), 2,2-Bis(4-hydroxyphenyl) propane, a common component of polycarbonate plastics and epoxy resins, is one of the major chemicals produced by humans in the world. Due to BPA leaching from these polymers under heat and acidic or basic conditions, humans are exposed to this pollutant mainly via contaminated foods but also beverages, dental sealants, baby bottles, food cans, and containers [1]. Owing to its extensive use and large presence in the environment, exposure to BPA is common among general populations worldwide.
Several studies suggest that BPA has endocrine-disrupting properties and that it can be implied in the pathogenesis of a variety of human diseases mostly affecting reproductive systems [2,3], similar to other environmental pollutants [4], as well as cardiovascular systems [5]. In this frame, epidemiological studies reported that there is an association between exposure to BPA and an increased prevalence of cardiovascular diseases (CVDs), including atherosclerosis [6], coronary and peripheral artery disease [7,8], myocardial infarction [9], and angina pectoris [10], as well as their risk factors, such as hypertension [11] and diabetes [12]. At the basis of the pathogenesis of these illnesses, a pivotal role is played

Chemical Characterization of RGJe
The polyphenol profile of RGJe was investigated by reversed-phase ultrahigh-performance liquid chromatography coupled with mass spectrometry (RP-UHPLC-MS). A sample aliquot (50 mg) was suspended in 5 mL of water/acetonitrile (50:50, v/v), filtered through a 0.2 µm syringe PTFE filter, and directly injected into a Shimadzu Prominence UFLC XR system (Shimadzu, Kyoto, Japan). The latter consists of a CBM-20A controller, an LC-20AD-XR binary pump system, a DGU-20A3R degasser, a CTO-20AC column oven, and a SIL-20A XR autosampler and is interfaced through an electrospray ionization (ESI) source to an LCMS-8040 triple quadrupole mass spectrometer (Shimadzu, Kyoto, Japan), as reported elsewhere [21]. Chromatographic separations occurred on a UPLC ® BEH C18 analytical column (2.1 × 100 mm, 1.7 µm, Waters, Milford, MA, USA) protected by a guard column with the same stationary phase Acquity UPLC ® BEH C18 VanGuard column (2.1 × 5 mm, particle size 1.7 µm, Waters). The mobile phase was composed of water (phase A) and acetonitrile acidified with 0.1% formic acid (phase B), and the optimized gradient profile was as follows: 0-5 min, 5% B; 5-12 min 5-25% B; 12-15 min, 25-45% B; and 15-20 min, 75% B; this also included final column washing and re-equilibrating steps. Oven temperature and injection volume were set at 35 • C and 3 µL, respectively. The flow rate was 0.5 mL/min, and a stainless-steel splitting device (VICI AG International, Schenkon, Switzerland) assured that only 300 µL/min of the flow was directed from the LC system to the ESI interface. The ionization source was used in negative ionization mode according to the following parameters: capillary voltage, 4.5 kV; heat block temperature, 400 • C; desolvation line (DL) temperature and voltage, 250 • C and −35 V; drying gas flow (N2), 15 L/min; and nebulizing gas flow (N2), 3 L/min. Spectra acquisition occurred in full scan mode over an m/z range of 100-800.
The investigated compounds belonged to diverse polyphenol classes, and they were selected depending on standard availability and their occurrence in similar products [22,23]. For quantification purposes, an external calibration procedure was carried out by means of six-point calibration curves showing satisfactory linearity (r2 from 0.9986 (kaempferol-3glucoronide) to 0.9996 (luteolin)). Triplicate measurements along with analytical blanks (i.e., acetonitrile) were acquired and elaborated by the LCMS solution Ver. 3.30 software (Shimadzu, Kyoto, Japan).

Evaluation of RGJe Antioxidant Activity through Abiotic Assays
The total phenolic content of RGJe was evaluated through the Folin-Ciocalteu [24] assay, while its total antioxidant activity was through the oxygen radical absorbance capacity (ORAC) assay, as previously reported [25]. The 2,2-Diphenyl-1-picrylhydrazyl (DPPH • ) was employed to test the radical scavenging activity of our extract, in accordance with Lombardo and co-workers [26]. The reducing power of RGJe was determined, according to Ferlazzo and collaborators, through the potassium ferricyanide reducing power assay [27]. All tests were repeated three times, and the results are expressed as mean ± SEM. As standards, we employed gallic acid, Trolox, and ascorbic acid in relation to the assay performed.

Cell Culture
This study was conducted using the human umbilical vein endothelial cells (HUVECs) originally from American Type Culture Collection (ATCC; Rockville, MD, USA). HUVECs were cultured in uncoated polystyrene dishes in endothelial basal medium (EBM TM -2; Lonza, Walkersville, MD, USA) supplemented with Endothelial Cell Growth Medium-2 (EGM TM -2) BulletKit™ (Lonza) at 37 • C in a 5% CO 2 air humified atmosphere and used between the third and sixth passages.

Cell Viability Assays
Cell viability was evaluated by the 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) test [28]. HUVECs were seeded into 96-well plates at a density of 1 × 10 4 cells/well in a complete medium and left to attach for 24 h. Before evaluating the potential protective effect of RGJe against toxicity induced by BPA, we evaluated the effect of BPA treatment (ranging from 0 to 1000 µM) for 24 h and RGJe (ranging from 0 to 200 µg/mL) for 48 h in HUVECs, tracing the timings of the following experiments.
Then, cells were treated with RGJe at various concentrations (from 1 to 50 µg/mL) for 24 h and BPA 300 µM for an additional 24 h. Then, the plates were centrifuged at 1200 rpm for 10 min, supernatants were removed, and fresh media without phenol red containing 0.5 mg/mL of MTT (Sigma-Aldrich, Milan, Italy) was added to each well. Plates were put in the incubator for an additional 4 h. Afterwards, insoluble formazan crystals were dissolved in 100 µL HCl/isopropanol 0.1 N lysis solution. The absorbance was spectrophotometrically quantified through iMark™ microplate reader (Bio-Rad Laboratories, Milan, Italy) at a wavelength of 570 nm with reference at 630 nm. The viability was determined as the percentage of viable cells in treated cultures compared to those in untreated ones.
The protective effect of RGJe against BPA-induced cell death was also evaluated by bromo-2 -deoxyuridine (BrdU) cell proliferation assay (Roche Diagnostics GmbH, Mannheim, Germany), as reported by Giofrè and co-workers [29]. Briefly, 4 × 10 3 cells/well were plated in a 96-well plate for 24 h. Then, cells were treated as previously described for MTT assay. Afterwards, cells were incubated with BrdU labeling solution for another 24 h at 37 • C followed by fixation and incubation with anti-BrdU peroxidase conjugate for an additional 1.5 h at room temperature. Finally, after substrate reaction, color intensity was measured with iMark™ microplate reader (Bio-Rad Laboratories) at a wavelength of 450 nm with reference at 690 nm.
The viability was determined as the percentage of viable cells in treated cultures compared to those in untreated ones.

Determination of Reactive Oxygen Species and Mitochondrial Membrane Potential
Reactive oxygen species (ROS) and mitochondrial membrane potential (∆ψ m ) were evaluated by flow cytometry. HUVECs were plated on 6-well plates (8 × 10 4 cells/well) and treated as described for cell viability assays. Each sample was harvested, centrifuged at 1200 rpm for 5 min, washed with PBS, and resuspended in a serum-free medium. Aliquots of cell suspension were loaded with 10 µM DCFH-DA for 30 min at 37 • C and used to evaluate the intracellular ROS [30]. The signals of the highly fluorescent oxidized derivative 2 -7 -dichlorofluorescein (DCF), formed in the presence of ROS, were collected with Novocyte 2000 cytofluorimeter (Agilent, Santa Clara, CA, USA). To assess the trans-membrane potential (∆ψm), we stained the HUVECs cells with 10 µM rhodamine 123 (R123, Invitrogen Molecular Probes), a fluorescent probe that accumulates in the matrix of functional mitochondria, for 10 min at 37 • C [31]. Signals were collected cytofluorimetrically.

Measurement of Superoxide Dismutase and Catalase Activities and Glutathione Levels
HUVECs were seeded at a density of 8 × 10 4 cells/well in 6-well plates and were treated as previously described. Then, superoxide dismutase (SOD) and catalase (CAT) activities, along with glutathione (GSH) content, were measured using commercial assay kits (AbCam, Cambridge, MA, USA) according to the manufacturer's protocols. The absorbance was recorded by an iMark™ microplate reader (Bio-Rad Laboratories) at 450 nm for SOD, 570 nm for CAT, and 405 nm for GSH [25].

Determination of NO Accumulation in HUVECs Culture Supernatant
The nitric oxide (NO) production was evaluated by a colorimetric commercial kit (Sigma-Aldrich). Supernatants were collected and processed as described by Ferlazzo and colleagues [32]. Absorbance was recorded at a wavelength of 540 nm by iMark™ microplate reader (Bio-Rad Laboratories).

Detection of Apoptotic Cell Death and Caspase-3 Enzymatic Activity
The protective effect of RGJe against BPA-induced cell death was evaluated cytofluorimetrically by the Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining [33,34]. The treated HUVECs were collected and processed as reported above. Finally, samples were run on a cytofluorimeter. Caspase 3 enzymatic activity was assessed using a commercial kit (AbCam) according to the manufacturer's instructions. Absorbance was detected at 405 nm by iMark™ microplate reader (Bio-Rad Laboratories) [32].

Real Time-PCR
The HUVECs were treated as previously described. Then, total RNA of treated or untreated cells was extracted and reverse transcribed following the previously described procedures [35,36]. Messenger RNA levels were analyzed by real-time PCR, using SYBR green as a fluorescent probe. The analysis was carried out on a 7300 Real-Time PCR System (Applied Biosystems, Waltham, MA, USA). The β-actin was used as housekeeping control, and a standard dissociation stage was included to assess primer specificity. Data were collected and analyzed using the 2 −∆∆CT relative quantification method. Values are presented as fold change relative to untreated cells. The primer sequences used for real-time PCR are listed in Table 1.

Statistical Analysis
One-way analysis of variance (ANOVA) was used to interpret the data. Multiple comparisons of the means of the groups were performed using Dunnett's multiple comparison test (GraphPad Prism 8 Software, San Diego, CA, USA). p-values less than or equal to 0.05 were considered significant.

Polyphenolic Profile of RGJe
Based on the available reference standards, a total of 20 polyphenols from diverse chemical classes, including phenolic acids, hydroxycinnamates, flavonols, flavanols, resveratrols, dihydroflavonols, dihydrochalcones, and flavones, were reliably determined in RGJe. The most abundant polyphenols were the flavonols kaempferol-3-glucoronide (1241.64 mg/kg) and quercetin-3-glucoronide (8341.89 mg/kg) and the cis isomer of resveratrol (3821.14 mg/kg). Other compounds determined in relevant amounts were the ellagic acid (1124.32 mg/kg) and the caffeic acid (1400.65 mg/kg). On the other hand, the least concentrated polyphenols were certain flavonols, such as rutin (14.90 mg/kg) and luteolin (39.49 mg/kg), as well as phenolic acids, such as methyl-gallate (14.55 mg/kg) and p-hydroxybenzoic acid (29.74 mg/kg). The quantitative determination of polyphenols identified in RGJe is listed in Table 2.

Antioxidant Activity of RGJe in Cell-Free Models
The antioxidant and radical scavenging properties of RGJe were proved by a range of tests, as described in the Materials and Methods. The total phenolic content, expressed as milligrams of gallic acid equivalents (GAE) per gram of RGJe and evaluated by the Folin-Ciocalteu method, was 80.0634 ± 1.4295. The valuable antiradical activity of RGJe, shown in the DPPH test and expressed as milligrams of Trolox equivalents (TE) per gram of extract (mg TE/g), was 69.7117 ± 9.3961, while in the Reducing Power test, it was 82.5972 ± 4.3769, expressed as milligrams of ascorbic acid equivalent (AAE) per gram of RGJe (mg AAE/g) (Table 3). Moreover, the obtained values of 130.7420 ± 9.2355 mol TE/g of RGJe demonstrated a high antioxidant capacity of RGJe against peroxyl radicals (Table 3).

RGJe Prevents BPA-Induced HUVECs Cell Death and Counteracts the Reduction of Cell Proliferation
Firstly, we evaluated the effect of BPA and RGJe alone on HUVECs viability for 24 and 48 h, respectively. The IC50 value for BPA was 303.91 µM; therefore, for further experiments, we decided to use BPA at a concentration of 300 µM ( Figure 1A, left). Moreover, we observed that RGJe at all the tested concentrations (1-200 µg/mL) did not affect HUVECs cell death ( Figure 1A, center). Therefore, we evaluated the potential protective effect of RGJe against BPA-induced cell death. The incubation of HUVECs with BPA (300 µM) reduced cell viability by 51 ± 3.05% compared to that of control cells (p < 0.0001; Figure 1A, right), while pre-treatment with RGJe at concentrations of 2.5, 5, 10, 25, and 50 µg/mL restored cell viability up to 66 ± 3.07%, 78 ± 5.06%, 91 ± 4.85%, 94 ± 5.12%, and 92 ± 4.53%, respectively (p < 0.05, p < 0.001 and p < 0.0001 vs. BPA; Figure 1A, right). On the contrary, pre-treatment of HUVECs with RGJe 1 µg/mL did not improve cell viability ( Figure 1A, right). Data from the BrdU assay followed the same pattern as those of the MTT test ( Figure 1B).

RGJe Counteracts BPA-Induced ROS Production and Restore the Loss of ∆Ψm in HUVECs
The incubation of HUVECs with BPA (300 µM) caused a remarkable growth of intracellular ROS compared to untreated cells (Figure 2A). In details, we observed that 66.55 ± 1.5% of cells showed enhanced intracellular ROS after BPA exposure. Contrariwise, the pretreatment with RGJe at 2.5, 5 and 10 µg/mL counteracted this effect ( Figure 2A); indeed, the percentage of fluorescent cells were 55.73 ± 1.7%, 47.41 ± 2.1% and 28.14 ± 2.88%, respectively ( Figure 2A). RGJe at concentration of 1 µg/mL did not evoke any significant reduction of ROS generation by BPA (Figure 2A). significant reduction of ROS generation by BPA (Figure 2A).

RGJe Protective Effects on BPA-Induced DNA Damage in HUVECs
To study the effectiveness of RGJe in attenuating DNA oxidative damage, we detected the levels of 8-oxo-dG by a FITC-conjugated avidin probe. As shown in Figure 3, the exposure of HUVECs to BPA 300 µM for 24 h induced DNA oxidation of 64.61 ± 2.3% Moreover, the exposure of HUVECs to BPA 300 µM for 24 h significantly affected the ∆Ψm ( Figure 2B). In particular, we observed that 33.93 ± 2.2% of cells are R123 + compared to untreated cells (99.79 ± 1.3%), showing a significant fall of ∆Ψm. This effect was prevented by RGJe at 2.5, 5 and 10 µg/mL (43.64 ± 1.7%, 51.63 ± 2.9% and 69.42 ± 2% of R123 + cells, respectively). Also, the lowest concentration of RGJe (1 µg/mL) used restored, even with less extent, the loss of ∆Ψm evoked by BPA ( Figure 2B).

RGJe Protective Effects on BPA-Induced DNA Damage in HUVECs
To study the effectiveness of RGJe in attenuating DNA oxidative damage, we detected the levels of 8-oxo-dG by a FITC-conjugated avidin probe. As shown in Figure 3, the exposure of HUVECs to BPA 300 µM for 24 h induced DNA oxidation of 64.61 ± 2.3% compared to untreated cells. The pre-treatment with 5 and 10 µg/mL of RGJe for 24 h before the exposure to BPA decreased oxidative DNA damage by about 29.5 ± 2% and 36.4 ± 2.8%, relative to BPA alone-treated cells, respectively (Figure 3). The lowest concentrations of RGJe employed (1 and 2.5 µg/mL) restored, even with less extent, DNA damage elicited by BPA (Figure 3). The treatment with RGJe alone did not cause DNA oxidation, since the emission values almost overlapped those detected in untreated cells.

Effect of RGJe on Biomarkers of BPA-Induced Oxidative Stress in HUVECs
We measured the amount of SOD and CAT activities, as well as GSH content to determine the level of oxidative stress in HUVECs after BPA treatment with or without RGJe. As shown in Figure 4, the treatment of HUVECs with BPA caused a significant reduction of the levels of these biomarkers in comparison to untreated cells (45 ± 2.65%, 55 ± 2.8% and 40 ± 2.11% for SOD, CAT, and GSH, respectively, p < 0.0001).
compared to untreated cells. The pre-treatment with 5 and 10 µg/mL of RGJe for 24 h before the exposure to BPA decreased oxidative DNA damage by about 29.5 ± 2% and 36.4 ± 2.8%, relative to BPA alone-treated cells, respectively (Figure 3). The lowest concentrations of RGJe employed (1 and 2.5 µg/mL) restored, even with less extent, DNA damage elicited by BPA (Figure 3). The treatment with RGJe alone did not cause DNA oxidation, since the emission values almost overlapped those detected in untreated cells .

Effect of RGJe on Biomarkers of BPA-Induced Oxidative Stress in HUVECs
We measured the amount of SOD and CAT activities, as well as GSH content to determine the level of oxidative stress in HUVECs after BPA treatment with or without RGJe. As shown in Figure 4, the treatment of HUVECs with BPA caused a significant reduction of the levels of these biomarkers in comparison to untreated cells (45 ± 2.65%, 55 ± 2.8% and 40 ± 2.11% for SOD, CAT, and GSH, respectively, p < 0.0001).
Pre-treatment with RGJe at concentration of 2.5 µg/mL (68 ± 2.75 and 59 ± 3 for SOD and CAT, respectively; p < 0.05), 5 µg/mL (76 ± 3.46 and 72 ± 4.4 for SOD and CAT, respectively; p < 0.01 and p < 0.001) and 10 µg/mL (83 ± 3.35 and 77 ± 5 for SOD and CAT, respectively; p < 0.001) for 24 h prior BPA exposure for further 24 h, significantly augmented the activity of both SOD and CAT ( Figure 4A,B). The lowest concentration of RGJe tested in this study (62 ± 3.53 and 50 ± 2.13 for SOD and CAT, respectively) did not cause any significant improvement in these biomarkers ( Figure 4A,B). The data obtained from the assessment of GSH content showed a similar trend even with lesser extent ( Figure 4C). In fact, only the pre-treatment with RGJe at 5 and 10 µg/mL (80 ± 4.05 and 81 ± 5.11, respectively; p < 0.01) was able to significantly restore the GSH content affected by BPA ( Figure 4C). Concentrations 1 and 2.5 µg/mL of RGJe did not exert any significant effect ( Figure 4C).

Effect of RGJe on NO Release Modulated by BPA in HUVECs
The incubation of HUVECs with BPA for 24 h caused about a 60 ± 2.76% reduction of NO production (p < 0.0001; Figure 5) that was significantly counteracted by pre-treatment with RGJe at 2.5, 5, and 10 µg/mL (42.5 ± 3.8%, 26.35 ± 3.7%, and 15.24 ± 5.86%, respectively; p < 0.05, p < 0.001, and p < 0.0001) compared to BPA-treated cells. Pretreatment with the lowest concentration used in this study (1 µg/mL) did not induce any significant increase in NO levels. Pre-treatment with RGJe at concentration of 2.5 µg/mL (68 ± 2.75 and 59 ± 3 for SOD and CAT, respectively; p < 0.05), 5 µg/mL (76 ± 3.46 and 72 ± 4.4 for SOD and CAT, respectively; p < 0.01 and p < 0.001) and 10 µg/mL (83 ± 3.35 and 77 ± 5 for SOD and CAT, respectively; p < 0.001) for 24 h prior BPA exposure for further 24 h, significantly augmented the activity of both SOD and CAT ( Figure 4A,B). The lowest concentration of RGJe tested in this study (62 ± 3.53 and 50 ± 2.13 for SOD and CAT, respectively) did not cause any significant improvement in these biomarkers ( Figure 4A,B). The data obtained from the assessment of GSH content showed a similar trend even with lesser extent ( Figure 4C). In fact, only the pre-treatment with RGJe at 5 and 10 µg/mL (80 ± 4.05 and 81 ± 5.11, respectively; p < 0.01) was able to significantly restore the GSH content affected by BPA ( Figure 4C). Concentrations 1 and 2.5 µg/mL of RGJe did not exert any significant effect ( Figure 4C).

Effect of RGJe on NO Release Modulated by BPA in HUVECs
The incubation of HUVECs with BPA for 24 h caused about a 60 ± 2.76% reduction of NO production (p < 0.0001; Figure 5) that was significantly counteracted by pre-treatment with RGJe at 2.5, 5, and 10 µg/mL (42.5 ± 3.8%, 26.35 ± 3.7%, and 15.24 ± 5.86%, respectively; p < 0.05, p < 0.001, and p < 0.0001) compared to BPA-treated cells. Pre-treatment with the lowest concentration used in this study (1 µg/mL) did not induce any significant increase in NO levels. 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.

Effect of RGJe on NO Release Modulated by BPA in HUVECs
The incubation of HUVECs with BPA for 24 h caused about a 60 ± 2.76% reduction of NO production (p < 0.0001; Figure 5) that was significantly counteracted by pre-treatment with RGJe at 2.5, 5, and 10 µg/mL (42.5 ± 3.8%, 26.35 ± 3.7%, and 15.24 ± 5.86%, respectively; p < 0.05, p < 0.001, and p < 0.0001) compared to BPA-treated cells. Pretreatment with the lowest concentration used in this study (1 µg/mL) did not induce any significant increase in NO levels. 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.

RGJe Reduces the Apoptotic Cell Death, Modulates Both Caspase 3 Activity and Apoptotic-Related Genes
The protective effect induced by RGJe was also evaluated cytofluorimetrically by Annexin V-FITC/PI assay. As shown in Figure 6A,B, the incubation of HUVECs with BPA for 24 h increased the percentage of cells in early (22.26 ± 1.9%, Annexin V+/PI−) and late (40.69 ± 2.1%, Annexin V+/PI+) apoptosis compared to untreated cells ( Figure 6A,B). The

RGJe Reduces the Apoptotic Cell Death, Modulates Both Caspase 3 Activity and Apoptotic-Related Genes
The protective effect induced by RGJe was also evaluated cytofluorimetrically by Annexin V-FITC/PI assay. As shown in Figure 6A,B, the incubation of HUVECs with BPA for 24 h increased the percentage of cells in early (22.26 ± 1.9%, Annexin V+/PI−) and late (40.69 ± 2.1%, Annexin V+/PI+) apoptosis compared to untreated cells ( Figure 6A,B). The pre-treatment with RGJe for 24 h prior to the incubation with BPA 300 µM reduced the number of cells undergoing apoptosis. In particular, the RGJe 5 and 10 µg/mL decreased the percentage of early apoptosis by 3.58 ± 1.9% and 16.82 ± 2%, respectively, while that of late apoptosis was reduced to 14.4 ± 2.2% for 5 µg/mL and 27.03 ± 2.7 for 10 µg/mL, in comparison to BPA-treated cells ( Figure 6A,B). The two lowest concentrations of RGJe tested in this study only partially reduced BPA-induced apoptosis in HUVECs ( Figure 6A,B).

Discussion
Grapes are one of the most widely grown fruits worldwide, with Europe, followed by Asia and the Americas, leading the world s producers index of the winemaking history of the various countries [37]. Apart from wine, grapes are acknowledged for the wide plethora of biological activities, such as anticancer [38], anti-obesity [39], and antimicrobial [40], as well as the most relevant one, namely, antioxidant [41]. These effects are due to the richness of bioactive compounds, mostly polyphenols, present in the various part of the grape (i.e., skin, pulp, and seeds). Indeed, grapes major polyphenols include flavan-3-ols, flavonols, and anthocyanins, whereas other non-flavonoids typically consist of phenolic acids and stilbenes. Proanthocyanidins, which account for the majority of flavan-3-ols in grapes, are a family of chemicals formed by the fusion of flavanol monomers [42]. Anthocyanins are hydrophilic pigments mainly concentrated in the outer part of colored grape cultivars (i.e., red or purple grapes) [43]. In the RGJe employed in our study, kaempferol-3-glucoronide, quercetin-3-glucoronide, and the cis-isomer of resveratrol were the most abundant flavonoids found, followed by ellagic and caffeic acids, whose composition trace those already published [44].
As stated, polyphenols of grapes are well-established natural antioxidants. Indeed, it has been proven that they have radical scavenging, ion chelating, and reducing activities [41]. In our study, we found that RGJe possesses noteworthy antioxidant effects, as demonstrated by several abiotic assays, thus reinforcing the value of our phytocomplex. This is because oxidative stress, defined as an imbalance in the body s oxidation and antioxidation condition, plays a significant role in accelerating cellular degeneration, leading to a variety of illnesses [45]. Environmental contaminants, such as BPA, strongly impair the body s defenses due to an overwhelming increase in oxidative stress. This leads to a loss of the physiological functionality of various organs, with the cardiovascular system being one of the most affected [5]. In our study, BPA impaired the viability of

Discussion
Grapes are one of the most widely grown fruits worldwide, with Europe, followed by Asia and the Americas, leading the world's producers index of the winemaking history of the various countries [37]. Apart from wine, grapes are acknowledged for the wide plethora of biological activities, such as anticancer [38], anti-obesity [39], and anti-microbial [40], as well as the most relevant one, namely, antioxidant [41]. These effects are due to the richness of bioactive compounds, mostly polyphenols, present in the various part of the grape (i.e., skin, pulp, and seeds). Indeed, grapes' major polyphenols include flavan-3-ols, flavonols, and anthocyanins, whereas other non-flavonoids typically consist of phenolic acids and stilbenes. Proanthocyanidins, which account for the majority of flavan-3-ols in grapes, are a family of chemicals formed by the fusion of flavanol monomers [42]. Anthocyanins are hydrophilic pigments mainly concentrated in the outer part of colored grape cultivars (i.e., red or purple grapes) [43]. In the RGJe employed in our study, kaempferol-3-glucoronide, quercetin-3-glucoronide, and the cis-isomer of resveratrol were the most abundant flavonoids found, followed by ellagic and caffeic acids, whose composition trace those already published [44].
As stated, polyphenols of grapes are well-established natural antioxidants. Indeed, it has been proven that they have radical scavenging, ion chelating, and reducing activities [41]. In our study, we found that RGJe possesses noteworthy antioxidant effects, as demonstrated by several abiotic assays, thus reinforcing the value of our phytocomplex. This is because oxidative stress, defined as an imbalance in the body's oxidation and antioxidation condition, plays a significant role in accelerating cellular degeneration, leading to a variety of illnesses [45]. Environmental contaminants, such as BPA, strongly impair the body's defenses due to an overwhelming increase in oxidative stress. This leads to a loss of the physiological functionality of various organs, with the cardiovascular system being one of the most affected [5]. In our study, BPA impaired the viability of HUVECs, employed as an in vitro model of vascular architecture, whereas pre-treatment with RGJe was able to hamper the harm induced by the toxicant, following a concentration-dependent fashion. Our results are in line with other studies reporting the potentiality of both grape seed and green tea extracts, rich in polyphenols, to hinder the toxicity of BPA in both HUVECs and isolated rat aorta [46,47].
These protective effects have been claimed to be principally due to polyphenols' antioxidant potential. Indeed, we observed that pre-treatment with RGJe was able to lower intracellular ROS, as well as restore mitochondrial functionality after exposure to BPA. Moreover, RGJe effectively counteracted BPA-induced DNA oxidative damage, as showed by the evaluation of oxidized nucleosides. Notably, grape juice has already displayed protective effects against oxidative damage towards mitochondria and nucleic acids in cadmium-induced redox stress in rats, another alarming environmental contaminant under the spotlight of scientific community [48], thus corroborating grape juice antigenotoxic potential [49]. Furthermore, we proved that RGJe was able to boost two of the most powerful antioxidant enzymes, namely SOD and CAT, which are responsible for the detoxification of intracellular ROS over-production and were both hampered by the exposure of BPA. Moreover, it caused a depletion of GSH content, due to HUVECs' demand to counteract the witnessed abnormal oxidative stress, an effect reverted by the pre-treatment with RGJe. BPA also decreased NO levels, since it is known that vascular oxidative stress lowers NO availability, thus stimulating endothelial dysfunction [50], an event that was significantly reverted by RGJe. Proanthocyanidins of grape peels and seeds have been studied for these activities in other experimental models, showing their ability to counteract different pro-oxidant stimuli via the increase of antioxidant enzymes [51,52].
Endothelial dysfunction caused by the imbalance between ROS production and removal by nonenzymatic and enzymatic antioxidant systems is claimed to cause apoptosis and necrosis in endothelial cells, promote the accumulation of macrophage-derived cytokines, and increase endothelial permeability [53]. In our study, exposure to BPA unleashed apoptosis of HUVECs, the index of cellular distress. This outcome was reverted by RGJe pre-treatment, which also modulated the whole apoptotic machinery via the increase of anti-apoptotic genes, such as BCL-2, and lowered the pro-apoptotic ones, such as BAX, P53, and CASP3, as well as the latter's enzymatic activity.
Regarding the effect on cytokine production induced by BPA exposure in HUVECs, we witnessed a massive release of IL-8, IL-1β, and MCP-1, whose over-expression induces cell migration and adhesion, angiogenesis, and vascular permeability exacerbating atherosclerosis. RGJe was able to block this inflammatory-driven process, lowering the gene expression of these three chemokines. Our results are in line with previous reports suggesting that an anthocyanin-rich extract of Aronia meranocalpa attenuated the expressions of inflammatory cytokines in HUVECs stimulated with TNF-α [54].
In human atherosclerotic lesions, endothelial cells have been shown to over-express intracellular VCAM-1, ICAM-1, and E-selectin in order to promote leukocyte adhesion and eventually stimulate endothelial dysfunction [55]. In this study, BPA expectedly enhanced the expression of the above-mentioned adhesion molecules, which is proof of its pro-inflammatory effect in HUVECs and its atherogenic activity. Notably, pre-treatment with RGJe thwarted this effect, restoring the physiologic levels of VCAM-1, ICAM-1, and E-selectin. In this field, others have discussed the protective effects of polyphenols from grapes, such as ellagic acid and anthocyanidins, in hampering the over-expression of adhesion molecules in endothelial cells after pro-inflammatory stimuli [56,57]. The heatmap of the genes regulated by RGJe in HUVECs exposed to BPA is shown in Figure 9.

Conclusions
Environmental pollutants represent an alarming issue for industrialized countries, given the resulting impairment of human health and the consequent socio-economic costs. Nutraceutical approaches may represent a hope for maintaining and defending the wellbeing of subjects exposed to these toxicants. In this study, we have shed light on the potentiality of RGJe as a tool for boosting one self s antioxidant defense, severely hampered by environmental pollutants. This is particularly relevant for the cardiovascular system, whose damaged functionality, by BPA for example, may lead to atherosclerosis. In addition to the remarkable antioxidant properties, RGJe blocked apoptosis of endothelial cells as well as chemokine release and adhesion molecules increase induced by BPA. Our results sustain the value of RGJe in counteracting endothelial damage induced by environmental pollutants, laying the foundation for its possible employment in this field.
Author Contributions: C.R. and A.M. performed the experiments and analyzed the data; A.A. and G.D. performed the chemical characterization; M.N. conceived the study and critically revised the paper; S.C. designed the study and drafted the paper. All authors have read and agreed to the published version of the manuscript.

Conclusions
Environmental pollutants represent an alarming issue for industrialized countries, given the resulting impairment of human health and the consequent socio-economic costs. Nutraceutical approaches may represent a hope for maintaining and defending the wellbeing of subjects exposed to these toxicants. In this study, we have shed light on the potentiality of RGJe as a tool for boosting one self's antioxidant defense, severely hampered by environmental pollutants. This is particularly relevant for the cardiovascular system, whose damaged functionality, by BPA for example, may lead to atherosclerosis. In addition to the remarkable antioxidant properties, RGJe blocked apoptosis of endothelial cells as well as chemokine release and adhesion molecules increase induced by BPA. Our results sustain the value of RGJe in counteracting endothelial damage induced by environmental pollutants, laying the foundation for its possible employment in this field.
Author Contributions: C.R. and A.M. performed the experiments and analyzed the data; A.A. and G.D. performed the chemical characterization; M.N. conceived the study and critically revised the paper; S.C. designed the study and drafted the paper. All authors have read and agreed to the published version of the manuscript.