Exposure to a “safe” dose of environmental pollutant bisphenol A elevates oxidative stress and modulates vasoactive system in hypertensive rats

Abstract

Due to the prevalence of hypertension (one of the major risk factors of CVD) in the population, it is necessary to explore the adverse effects of daily tolerable and “safe” dose of bisphenol A (BPA) under hypertensive conditions. The current study exposed the Nω-nitro-l-arginine methyl ester (L-NAME, 40 mg/kg b.w/day) induced hypertensive Wistar rats to BPA (50 μg/kg b.w/day) by oral administration along with appropriate controls for 30 days period. The results illustrate that a ‘safe’ dose of BPA does not influence the systolic blood pressure (SBP) and levels of circulatory biomarkers of tissue damage. On the other hand, BPA exposure significantly (p < 0.05) elevates the thiobarbituric acid reactive substances (TBARS) content in plasma and tissues (heart, aorta, liver and kidney) in hypertensive rats when compared with respective control (BPA alone exposed) rats. Similarly, a significant modulation of ROS generation in RBC, plasma nitric oxide (NO) level and angiotensin-converting enzyme (ACE) activity was observed only under hypertensive milieu. In conclusion, the observed adverse effects during ‘safe’ dose of BPA exposure are specific to the hypertensive condition. Therefore, a precise investigation to explore the effects of BPA exposure in vulnerable hypertensive populations is highly suggested.

Keywords

Bisphenol A hypertension oxidative stress cardiovascular xenoestrogen

Introduction

For several years, there has been growing interest in studying the effects and mechanism of endocrine disruptors (including bisphenol A) and their relation with the environment and human health.¹ Over the last 10 years, many studies have examined the underlying molecular mechanisms of BPA toxicity and revealed links among BPA-induced oxidative stress, male and female reproductive defects, and human disease.² In addition, BPA exposure may elevate the risk of obesity, diabetes, and coronary heart diseases.³ Precisely, there is growing evidence regarding an association between BPA exposure, hypertension, and cardiovascular diseases (CVD).⁴ Also, recent epidemiological studies have shown that higher urinary BPA concentration in humans is associated with various types of CV diseases, including angina, hypertension, heart attack, and coronary and peripheral arterial disease.⁵

While focus on the tolerable daily intake (TDI, 50 μg/kg body weight [B.W]/day) of BPA, an earlier study showed that long-term exposure to a “safe” dose of BPA reduces histone acetylation in the male reproductive system, which may be related to the phenotypic paternal to-offspring transmission.⁶ In addition, an increased mild inflammatory cell infiltrate in epididymis was seen in adult rats exposed to 50 μg BPA/kg bw/day.⁷ In addition, a BPA intake 8 times lower than the European Food Safety Authority’s (EFSA’s) current tolerable daily intake (TDI) of 4 μg/kg bw/day of BPA delivered via drinking water during gestation and early development causes islet insulin hypersecretion in rat offspring up to 1 year after exposure.⁸ Moreover, orally administered BPA at the US EPA-approved safe dose of 50 μg/kg has immediate effects on indices of glucose metabolism in young, non-obese adults.⁹

At the mechanistic level, DNA damaging and apoptotic potentials of BPA and BPS in human bronchial epithelial cells indicate that BPA’s DNA damaging potential is mediated through an ATM/ATR/Chk1/p53-dependent pathway.¹⁰ Further, a recent exploration shows chronic exposure to BPA impairs carbohydrate and lipid metabolism by altering corresponding enzymatic and metabolic pathways.¹¹ In addition, exposure to BPA alone or in combination with phthalates decreases the cytokine release (TNFα, IL-6, IL-10, IFNγ, IL-4) from in vitro stimulated splenocytes and lymph node cells, indicating systemic changes in immune function.¹² Moreover, exposure of BPA to vascular cells (HUVEC cells) induced the expression of ER stress and inflammation-related genes.¹³ Furthermore, molecular impact on ERK/MAPK and PI3K/AKT pathways has been already highlighted.¹⁴

In a synergistic aspect with cardiovascular risk factors, a previous study has shown that BPA exposure enhances atherosclerosis.¹³ In this connection, chronic exposure to BPA (50 μg/kg/day BPA) was shown to accelerate atherosclerosis in high-fat–fed apolipoprotein E knockout mice.¹⁵ Also, with the inflammatory perspective, low doses of BPA have pro-inflammatory and pro-oxidant effects, stimulate lipid peroxidation, and increase the cardiotoxicity of doxorubicin in cardiomyoblasts.¹⁶ Further, oral exposure to low-dose BPA aggravates allergic airway inflammation in mice.¹⁷ On the other hand, studies on diabetic models illustrate that BPA exposure aggravates multiple low-dose streptozotocin-induced type 1 diabetes in C57BL/6 mice.¹⁸ Mechanistically, a previous observation indicates that BPA exposure may increase diabetes risk due to the exacerbated toxic aggregation of human islet amyloid polypeptide.¹⁹

Based on the above evidence, we hypothesized that the safe dose of BPA may aggravate hypertension-associated pathogenic events including oxidative stress. To begin addressing this question, the aim of this present study was to investigate the adverse effect of the safe dose of BPA (50 μg/kg bw/day) under normal and hypertensive milieu. Here, the Nω-nitro-l-arginine methyl ester (L-NAME)–induced pharmacological model of hypertension in Wistar rats has been employed to study the effects under hypertensive milieu.

Materials and method

Animal and ethical approval

Male albino Wistar rats of 8–10 weeks old (weighing 180–220 g) were procured from the Laboratory Animal Medicine Unit, TANUVAS, Chennai, India. The animals were housed in a group of five per polypropylene cage and fed (feed and water) ad libitum at a controlled room temperature of 25 ± 3°C and 40–55% of humidity with an alternating 12 h light/12 h dark cycle to acclimatize the conditions. All animal protocols were approved by the Animal Ethical Committee of Bharathiar University (Reg No. 722/Go/Re/S/02/CPCSEA, Proposal number: 06) following the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals, New Delhi, India.

Chemicals and reagents

Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME) and bisphenol A (BPA) were purchased from Sigma-Aldrich, USA. All other chemicals used in this study were of analytical grade obtained from Merck, SRL, and HiMedia, India.

Experimental design

Bisphenol A at the safe dose of 50 μg/kg b.w/day was used in the current study since it has been already reported that BPA at the dose of 50 μg/kg b.w/day is considered a safe dose.⁶ BPA was administrated orally by dissolving in 0.3% of carboxymethylcellulose (CMC). For the induction of hypertension, here we employed the pharmacological model of hypertension using L-NAME at the dose of 40 mg/kg b.w/day dissolved appropriately in drinking water as mentioned early.²⁰ The total experimental period was 30 days. Totally, 24 animals were used and each group contains six animals (n = 6).

Animals were grouped into four groups consisting of

Group I (Control)—(Vehicle alone—0.3% of carboxymethylcellulose).

Group II (BPA)—50 μg/kg bw/day dissolved in 0.3% of carboxymethylcellulose

Group III (L—NAME)—40 mg/kg bw/day dissolved in drinking water

Group IV (Combined exposure)—BPA (50 μg/kg) + L—NAME (40 mg/kg)

Blood pressure measurement

After acclimatization, the animals were trained with the instrument for blood pressure measurement. The systolic blood pressure (SBP) was measured noninvasively using the tail-cuff method (IITC, model 31, USA) on the first and last day of the experimental period. The average of three values is recorded and analyzed using a computerized data acquisition system and software.²¹

Serum biochemistry

At the end of the treatment period, the animals were euthanized by giving 100 mg/kg of ketamine (Intra Peritoneal). Blood (0.5 mL) was collected by the cardiac puncture method using sterile syringes. The blood was then transferred into a sterile tube and allowed to coagulate at room temperature for 45 min to separate serum. Then, the blood samples were centrifuged at 490 x g speed for 10 min and the upper serum layer was transferred into sterile tubes for the biochemical analysis. The plasma was separated from blood samples by centrifuging at 1000 × g speed for 10 min with an anticoagulant (EDTA) to evaluate the nitric oxide (NO) metabolite level and angiotensin-converting enzyme (ACE) activity.

The blood glucose and cholesterol levels were determined by the enzymatic method using the commercially available kit (Agappe Diagnostics, India). The tissue/cellular damage was evaluated by assessing the activity of enzymatic markers in serum. The activity of Cardiac marker enzymes lactate dehydrogenase (LDH) and creatine kinase (CK-MB); hepatic marker enzymes such as alkaline phosphatase (ALP), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) were determined in the serum samples according to the manufacturer’s instructions (Agappe Diagnostics, India). The kidney function was evaluated by quantifying the serum level of urea, uric acid, and creatinine. For all above assays, the absorbance was measured using a UV–Vis spectrophotometer (Shimadzu, Japan).

Plasma nitric oxide metabolites (nitrite/nitrate) level

The quantitative analysis of nitrite and nitrate was determined in the plasma by the Griess method. The principle of this assay is reduction of nitrate by vanadium (III) combined with detection by the acidic Griess reaction.²² Briefly, plasma samples were mixed with the Griess reagent (sulfanilamide 1% w/v, naphthylethylenediaminedihydrochloride 0.1% w/v, and orthophosphoric acid 2.5% v/v) and incubated for 10 min at room temperature. The absorbance was measured spectrophotometrically at 540 nm. The level of stable nitric oxide metabolites (nitrite/nitrate) was determined from the standard curve.

Plasma angiotensin-converting enzyme activity

The activity of ACE in the blood plasma was determined by quantifying the cleavage of the substrate Hip-His-Leu into hippuric acid and His-Leu by ACE. After incubation, ethyl acetate was used to extract hippuric acid and measured at 228 nm in a UV–Vis spectrophotometer as mentioned in our earlier study.²³ The units were expressed as mU per mg protein.

Fatty acid composition—gas chromatography analysis

Total plasma fatty acids content was evaluated according to the method developed by Glaser.²⁴ Briefly, 100 μl of plasma with 100 μl of heptadecanoic acid (internal standard) was added to a tube containing 1.5 mL of methanolic HCl and shaken for 30 s. Then, the tube was kept in a water bath at 85°C for 45 min and allowed to cool. Then, 0.5 mL of hexane was added to the tube and shaken well for 30 s. The tube was allowed for phase separation at room temperature for 2 min. An aliquot of the upper hexane phase was transferred into a new tube. About 2.0 μl of hexane phase was taken for fatty acid methyl ester (FAME) quantification. The FAME was quantified by gas chromatography (GC) with flame ionization detection (FID). GC analysis was performed on a Perkin Elmer (Clarus® 590 GC, USA) using a capillary column (Elite 5, 30 m × 0.25 mm I.D, 25 μm film, Perkin Elmer, USA). Helium was used as carrier gas with a set point of 1.0 mL/min, and the GC conditions were optimized with oven initial temperature of 180°C with injector temperature of 250°C. The total analysis time was 50 min. Peaks were identified using FAME standards (Sigma-Aldrich, USA), and the relative peak area of each major peak was quantified by comparing it with the peak area of the internal standard.

RBC Oxidative stress—Fluorimetric assay

Reactive oxygen species were determined according to a microplate-based method as described in the previous study.²⁵ Briefly, erythrocytes (995 μL of 10%, v/v suspension in PBS) were incubated with 5 μL of dichlorodihydrofluorescein-diacetate (DCFDA, 10 mol/L) at 37°C for 30 min. Under these conditions, oxidation by RBC-derived reactive oxygen species produces fluorescent 2,7-dichlorofluorescein (DCF). Fluorimetric quantification of fluorescence was determined at 530 nm after excitation at 495 nm using a multimode microplate reader (BioTek, USA). ROS formation was expressed as an arbitrary unit of fluorescence.

Scanning electron microscopy studies on red blood cells

Blood was collected into EDTA-coated tubes (1.6 mg/mL). RBC sample preparation and SEM analysis was performed as mentioned previously.²⁶ Briefly, 100 μl of red blood cells (RBC) was isolated and washed three times with phosphate buffer saline (PBS at pH 7.4) through centrifugation at 1000 rpm for 10 min and re-suspended in PBS. Specimens were fixed overnight at 4°C by adding one drop of each sample to plastic tubes containing 500 μl of 2.5% glutaraldehyde in Milli-Q water. After fixation, RBC samples were washed twice in Milli-Q water by centrifuged at 1000 rpm for 10 min. About 20 μl of each sample was placed on glass-covered stubs, air-dried at room temperature, gold-coated in a sputtering device for 3 min at 13.3 Pa (Quorum q150RS), and examined under field emission scanning electron microscopy (FEI Quanta-250). The abundance of normal (discocytes) and abnormal (echinocytes) RBCs were estimated from 20 random cell images and expressed as a relative percentage.

Preparation of tissue homogenates

To prepare tissue homogenates, major organs (aorta, heart, kidney, liver, lungs, pancreas, spleen, and brain) were excised and rinsed with 0.9% of physiological saline solution to remove excessive blood. The tissues were homogenized by adding 0.1 M Tris-HCl in cold condition (pH 7.0) to obtain 20% (w/v) homogenate. The homogenate was centrifuged at 560 × g for 10 min at 4°C, and the supernatant was used for various biochemical estimations as mentioned previously.²⁷

Estimation of thiobarbituric acid reactive substances level

Lipid peroxidation level in plasma and tissues were estimated spectrophotometrically by measuring thiobarbituric acid reactive substances (TBARS). To estimate TBARS, 250 μl of serum or tissue homogenate was diluted with 500 μl of ultra-pure water and mixed well. Then, 2 mL of thiobarbituric acid (TBA)-trichloroacetic acid (TCA)-hydrochloric acid (HCL) reagent was added and placed in a water bath for 15 min. The tubes were cooled at room temperature and centrifuged for 10 min. The supernatant was collected and the absorbance was measured at 535 nm against the reagent and blank using a UV–Vis spectrophotometer (Shimadzu, Japan).

Statistical analysis

Data were analyzed by one-way analysis of variance (ANOVA) followed by Duncan’s multiple range test (DMRT) using SPSS software, version 16.0 (SPSS, Chicago, IL). The values were expressed in mean ± SD in each group and considered significant when p < 0.05.

Results

Systolic blood pressure

The effect of BPA on L-NAME–induced hypertension was illustrated in terms of systolic blood pressure (SBP) for the first and last day of exposure (Figure 1). Results indicate that BPA (50 μg/kg) exposure to normal rats did not significantly elevate the SBP when compared with the control group, whereas significant (p < 0.05) elevation of SBP was observed in L-NAME-induced hypertensive rats which do not increase further due to combined exposure of BPA (BPA + L-NAME). This illustrates that BPA does not influence blood pressure under hypertensive milieu.

Figure 1.

Effect of BPA on systolic blood pressure. Values are expressed in mean ± SD for six rats in each group (0th day and 30th day). The values denoted with distinct letters indicate statistically significant difference (p < 0.05) with each other.

Serum biochemistry

While a focus on serum biochemical parameters, results show there was no significant (p < 0.05) modulation of serum glucose level among all the treatment groups (Figure 2(a)). In the case of cholesterol, there was an increased serum level observed in L-NAME treated animals when compared with control, but the combined exposure group (BPA + L-NAME) does not show any significant changes compared with L-NAME hypertensive rats (Figure 2(b)). Cardiac enzyme markers (CK-MB and LDH) do not show any significant (p < 0.05) alteration inactivity (Figure 2(c) and Figure 2(d)). In addition, the liver marker enzyme activity remained unchanged and no significant (p < 0.05) effect was observed among groups (Figures. 3(a–c)). In the case of renal function markers, significantly (p < 0.05) elevated serum level of urea, uric acid, and creatinine was observed in L-NAME–treated hypertensive animals compared with control and BPA alone treated animals. No further elevation was observed during BPA+L-NAME combined exposure (Figures 3(d–f)).

Figure 2.

Effect of BPA on glucose, cholesterol, and cardiotoxic markers under hypertensive milieu. (a) Serum glucose level in different experimental groups. (b) Serum cholesterol level in different experimental groups. (c) Creatine kinase myocardial band (CK-MB) level in the serum of different experimental groups. (d) Lactate dehydrogenase (LDH) level in the serum of different experimental groups. Values are expressed in mean ± SD for six rats in each group. The values denoted with distinct letters indicate statistically significant difference (p < 0.05) with each other.

Figure 3.

Effect of BPA on hepatic and renal function markers under hypertensive milieu. Hepatic function markers: (a) Alkaline phosphatase (ALP), (b) aspartate aminotransferase (AST), (c) alanine aminotransferase (ALT). Renal function markers: (d) Urea, (e) uric acid, (f) creatinine. Values are expressed in mean ± SD for six rats in each group. The values denoted with distinct letters indicate statistically significant difference (p < 0.05) with each other.

Other circulatory pathogenic markers

Apart from tissue toxicity assessment markers, the current study explores important pathogenic circulatory indicators such as nitric oxide (NO) level, ACE activity, RBC generated ROS, and TBARS level. In the current observation, L-NAME–induced hypertension rats show a significant (p < 0.05) reduction in the level of stable nitric oxide metabolites (nitrite/nitrate) in plasma when compared with control and BPA alone treated animals. Notably, a significant (p < 0.05) decrease in NO level was observed in the combined group (L-NAME + BPA) when compared with L-NAME treated and other groups (Figure 4(a)). Similarly, BPA exposure under hypertensive milieu significantly (p < 0.05) elevates ACE activity when compared with L-NAME and BPA alone treated group (Figure 4(b)). Further, BPA exposure under hypertensive milieu significantly (p < 0.05) elevates RBC-derived ROS generation (arbitrary fluorescence unit) when compared with L-NAME and BPA alone treated group (Figure 4(c)). In this connection, the current study also observed that, BPA exposure under hypertensive milieu significantly (p < 0.05) elevates serum TBARS level when compared with L-NAME and BPA alone treated group (Figure 4(d)).

Figure 4.

Effect of BPA on circulatory nitric oxide, ACE activity, RBC-ROS generation and serum TBARS under hypertensive milieu. (a) Nitric oxide metabolites level, (b) angiotensin-converting enzyme activity, (c) relative ROS generation level in RBC, (d) level of thiobarbituric acid reactive substances (TBARS) in serum. Values are expressed in mean ± SD for six rats in each group. The values denoted with distinct letters indicate statistically significant difference (p < 0.05) with each other.

Fatty acid composition

Plasma fatty acid composition is illustrated in Figure 5. The results indicate that only the saturated fatty acids (C16:0 and C18:0) were significantly (p < 0.05) elevated due to L-NAME treatment (hypertensive animals) when compared with control and BPA alone treated animals, whereas BPA exposure does not show any significant (p < 0.05) impact on it.

Figure 5.

Effect of BPA on RBC shape under hypertensive milieu (Scanning electron microscopic study). (a) Control. (b) BPA-treated, (c) L-NAME–induced hypertensive animals, (d) combined exposure (L-NAME + BPA) group. (e) Percentage distribution of rat erythrocytes (normal and abnormal) in different experimental groups.

Erythrocyte shape and composition

The effect of BPA on erythrocytes shape was evaluated through scanning electron microscopy (SEM) analysis, and its relative composition is illustrated in Figure 6a and Figure 6b. The resulting images have shown that the normal flat, discocytes (biconcave disc shape) erythrocytes were abundantly observed in the control and BPA treated group, whereas L-NAME–induced hypertension causes a slight reduction of relative composition of normal discocytes and elevates the level of echinocytes (abnormal/thorny projection shape). In addition, BPA exposure under the hypertensive milieu does not show any deviation in the relative composition compared with the L-NAME hypertensive group.

Figure 6.

Effect of BPA on plasma fatty acid level under hypertensive milieu. Relative fold change of fatty acids level between groups. Values are expressed in mean ± SD for three rats in each group. The values denoted with * indicate a statistically significant difference with control. Significance (S) and Not-significance (N.S).

Estimation of lipid peroxidation level

The tissue oxidative stress (lipid peroxidation) in terms of TBARS level for multiple organs is illustrated in Figure 7. Among the various organs, only the aorta, heart, liver, and kidney tissues of the combined exposure group (L-NAME + BPA) show an increase in the TBARS level drastically (p < 0.05) when compared to L-NAME induced group.

Figure 7.

Effect of BPA on tissue oxidative stress marker—TBARS under hypertensive milieu. Values are expressed in mean ± SD for six rats in each group. The values denoted with distinct letters indicate statistically significant difference (p < 0.05) with each other.

Discussion

Cardiovascular diseases (CVDs) are by far the leading cause of death in the world with an estimated 31% of all deaths worldwide.²⁸ Adverse health effects may be exacerbated in vulnerable populations including those with pre-existing cardiovascular diseases/risk factors (including hypertension) when they are exposed to environmental pollutants. Therefore, adequate awareness about the “safe dose” of BPA exposure may help us to reduce the susceptible hypertensive population’s exposure to BPA thereby contributing to the prevention of CVD-related pathogenic events. As a whole, a consensus about BPA’s safety and its role in human disease including CVDs has not been reached.

Primarily, the systolic blood pressure (SBP) level illustrates that a safe dose of BPA does not increase the systolic blood pressure under normal and hypertensive milieu. A previous observation showed that the lowest BPA dosage that can induce both high blood pressure and vascular dysfunction in CD1 mice is 0.1 μg/kg/day.²⁹ However, a deviated result was observed in the current study using Wistar rats (50 μg/kg/day) when compared with CD1 mice which require further investigation with the context of inter-species or genetic variations.³⁰ Further, assessing the activity of various enzymatic biomarkers is necessary for the precise understanding of toxicity during experimental exposure.^31,32 In this connection, elevated levels of serum LDH and CK-MB are known to indicate the degree of cardiotoxicity.³³ Also, serum activities of alanine and aspartate aminotransferases (ALT and AST) are used as gold standard biomarkers for the diagnosis of hepatocellular injury.³⁴ Serum levels of urea, uric acid, and creatinine were also found to be useful for the evaluation of renal toxicity under various treatment conditions.³⁵ In the current observation, there were no significant changes of cardiotoxic and hepatotoxic markers in both L-NAME and BPA alone treated and combined exposure group, whereas kidney function markers were found to be elevated in hypertensive animals, notably which does not increase further due to BPA exposure. In addition, the unchanged glucose level and an elevated level of cholesterol were also observed in hypertensive rats which were not be influenced by BPA treatment. Although the current observation does not intend to explore the serum pharmacokinetics of BPA, the elevated serum level of kidney function markers under hypertensive milieu indicates the need of studying the blood concentration of BPA (BPA accumulation/pharmacokinetics) under hypertensive milieu since BPA is excreted in urine and builds up when the glomerular filtration rate decreases.³⁶

It is well known that oxidative stress and nitric oxide (NO) levels have been modulated in addition to suppression of antioxidant enzymes activity in L-NAME–treated nitric oxide inhibited hypertension models.^20,21 Notably, in the current study, the elevated level of serum TBARS has further increased in BPA-exposed animals. On the other hand, plasma nitric oxide (nitrite/nitrate) levels decreased further by BPA exposure when compared with the hypertensive animals. Apart from this, the serum ACE activity also increased in BPA administered group under hypertensive milieu. In this regard, BPA exposure and the induction of reactive oxygen species (ROS) or oxidative stress have been highlighted in various studies.³⁷ Notably, a previous study has shown that a low dose range of BPA (0.2, 2.0, and 20 μg/kg/day) induces ROS generation in the liver of male rats³⁸ and elicits the depletion of the antioxidant defence system and induces oxidative stress in epididymal sperm of rats.³⁹ In our study, the safe dose of BPA (50 μg/kg/day) did not increase the serum TBARS level in normal rats; instead, its generation is more under hypertensive condition. While considering the impact on NO level, long-term dietary nitrite and nitrate deficiency causes metabolic syndrome, endothelial dysfunction, and cardiovascular death in mice.⁴⁰ Moreover, BPA induces endothelial cell necroptosis and promotes the weakening of the coronary vascular wall that ultimately leads to cardiac dysfunction.⁴¹ Precisely, oral administration of BPA induces high blood pressure through angiotensin II/CaMKII–dependent uncoupling of eNOS.²⁹ Although there is no elevation of blood pressure observed during BPA treatment in the current study, the decreasing level of serum nitric oxide (nitrite/nitrate) was observed in BPA-treated under hypertensive milieu when compared with BPA exposure only. The elevated oxidative stress level may be one of the reasons behind reduced NO level since its bioavailability is highly dependent on the redox status.⁴² Moreover, the current study observed a notable impact of BPA on ACE activity under hypertensive milieu. ACE, which transforms angiotensin I into the pressor angiotensin II, is one of the main regulators of the vascular tone and blood pressure where previous studies show that the age of rats and increasing time of L-NAME treatment increase the ACE activity in the aorta.⁴³ An earlier study also indicates that anti-hypertensive and renoprotective effects of protective agents in nitric oxide (NO)–deficient rats might be mediated via its ACE inhibitory activity.⁴⁴ Strikingly, the adverse effect of the safe dose of BPA on oxidative stress, nitric oxide metabolism, and vasoactive factors that played crucial role in cardiovascular physiology was observed only under hypertensive milieu which requires further consideration.

Other than biochemical parameters, circulatory factors including RBC and fatty acid profile also been illustrated. In the current study, at the RBC morphology level normal RBCs were observed as predominant ones in all treatment conditions, whereas abnormal RBCs (echinocytes and other types) were occasionally encountered in hypertensive animals which were not increased further by BPA treatment. On the other hand, the elevated level of ROS generation in RBCs was observed in BPA-exposed animals under hypertensive milieu. An earlier case-control study comparing RBCs from healthy and hypertensive participants found that RBC deformability was decreased, and levels of ROS were increased in RBCs from hypertensive patients as compared to RBCs from aged-matched healthy controls.⁴⁵ In another axis, endothelial dysfunction induced by RBCs obtained from type 2 diabetes mellitus (T2DM) patients was prevented by inhibition of arginase and ROS generation.⁴⁶ While a focus on the serum total fatty acid composition, the elevated level of two notable saturated fatty acids (C16:0 and C18:0) was observed in the hypertensive group without any impact by BPA exposure. An earlier study indicates an increased level of saturated fatty acids in RBCs obtained from hypertensive animals which indirectly illustrate the circulating fatty acid level.⁴⁷

While a focus on tissues, an increasing body of evidence suggests that oxidative stress, which results in an excessive generation of ROS, has a key role in the pathogenesis of hypertension and associated consequences.⁴⁸ The modulation of the vasomotor system involves ROS as mediators of vasoconstriction induced by angiotensin II, endothelin-1, and urotensin-II, among others.⁴² While a focus on oxidative stress in multiple tissues, only the aorta, heart, kidney, and liver have shown an elevated level of TABRS due to BPA exposure under hypertensive milieu. The TBARS level is widely used as a generic metric of lipid peroxidation in biological fluids and is often considered a good indicator of the levels of oxidative stress within a biological sample.⁴⁹ A previous study indicates that BPA (50 μg/kg) exposure significantly increased atherosclerotic lesions in the aortas of ApoE (−/−) mice.¹⁵ Further, exposure to the low dose of BPA at 5 μg/kg/day plus fructose increased mRNA expression of genes controlling angiogenesis and vascular tone (Vegf, Vegfr2, eNos, and Ace1) in rat cardiac tissues.⁵⁰ Precisely, earlier results suggest that BPA has cardiotoxic effects mediated by oxidative stress resulting from the overproduction of free radicals, the deficiency of NO, and the inhibition of AchE leading to cholinergic activation.⁵¹ Another study has shown that BPA exposure alters the transcription of genes that are recognized for their role in cardiac pathophysiology. Importantly, myosin heavy chain, cardiac isoform alpha (Myh6) was down-regulated in the left ventricle, and “A Disintegrin and Metalloprotease 12,” long isoform (Adam12-l) was up-regulated in both ventricles and the right atrium of the heart in BPA exposed fetuses.⁵² Moreover, oral BPA (50 μg/kg) exposure worsens liver immune-metabolic and mitochondrial dysfunction induced by high-fat diet in adult mice through cross-talk between oxidative stress and inflammasome pathway.⁵³ Further, an induction effect of BPA on gene expression (HO-1) involving hepatic oxidative stress was also observed in the rat model at the dose of 25 μg/kg/day.⁵⁴ Mechanistically, bisphenol A induces apoptosis, oxidative stress, and inflammatory response in colon and liver of mice in a mitochondria-dependent manner.⁵⁵ In addition, BPA exposure is associated with low-grade urinary albumin excretion in children.⁵⁶ Therefore, the elevation of oxidative stress (lipid peroxidation) in the aorta, heart, liver, and kidney tissues provoked by BPA exposure at the safe dose (50 μg/kg/day) under hypertensive milieu needs to be considered due to its pivotal role in pathophysiology.

Conclusion

The results of the present study demonstrate that low-dose (safe dose) exposure of BPA in normal animals may not elicit any notable changes (parameters studied), whereas its adverse effects were profound under hypertensive milieu. Primarily, the oxidative stress-related impacts were observed in animals exposed to BPA under hypertensive condition. Therefore, deeper investigations on the effects of BPA exposure to susceptible hypertensive population are highly recommended.

Footnotes

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

ORCID iD

Jeganathan Manivannan

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