Potential Effects of Bisphenol A on the Heart and Coronary Artery of Adult Male Rats and the Possible Role of L-Carnitine

Bisphenol A (BPA) is an environmental toxin utilized for the production of polycarbonate plastics and epoxy resins. Due to BPA's extensive production and environmental contamination, human exposure is unavoidable. The effects of low-dose of BPA on various body tissues and organs remain controversial. Our study investigated the potential of BPA to induce biochemical, histopathological, and immunohistochemical changes in the coronary artery and myocardium and the potential protective role of L-carnitine (LC). 24 adult Wistar albino male rats were divided equally into a control group, a BPA-treated group (40 mg/kg/d, by gavage for 4 weeks), and a BPA plus LC-treated group (received 40 mg/kg/d of BPA and 300 mg/kg/d of LC, by gavage for 4 weeks). BPA-exposed rats demonstrated structural anomalies in the coronary artery tissue including vacuolation of cells in the media and detachment of the endothelium of the intima. Congestion of blood vessels and infiltration by polynuclear cells were observed in the myocardium. There was an enhanced collagen deposition in both tissues indicating fibrosis. Immunohistochemical changes included enhanced eNOS and caspase-3 expression in the coronary artery and myocardium indicating vascular disease and apoptosis, respectively. Oxidative damage was evident in the coronary artery and the myocardium of BPA-treated rats, which was indicated by the reduced level of glutathione (GSH) and elevated malondydehyde (MDA) levels. The coadministration of LC significantly improved BPA-induced structural alterations and oxidative stress. In conclusion, BPA could potentially cause pathologic changes and oxidative damage in the coronary artery and myocardium, which could be improved by LC coadministration.


Introduction
Bisphenol A is one of the greatest volumes of chemicals produced globally, and more than one hundred tons are released into the environment every year [1]. It is used in the manufacturing of polycarbonate plastic products, which continuous exposure to BPA, this chemical has been detected in almost all humans studied [4].
Te health hazards related to BPA are mostly because of the partial polymerization leaving some unbound monomer BPA molecules in the product. Such monomers become released into food or beverages with time, particularly due to the efects of heat, acidic, or basic conditions [5]. It was stated that humans are exposed to BPA predominantly in their diet; however, there is evidence that exposure could also happen through inhalation of household dust or particles released during the BPA's industrial production [6].
Bisphenol A acts through several mechanisms. More specifcally, it interacts with multiple receptors such as estrogen, aryl hydrocarbon, androgen, thyroid hormone, and glucocorticoid receptors. BPA binds to classical nuclear estrogen receptors, classical and nonclassical membranebound ERs, and G protein-coupled receptors 30 [7]. Moreover, BPA can cause oxidative stress in diferent body tissues as reported in several studies [8,9]. In addition, BPA can act through epigenetic mechanisms and alterations in genomic methylation, including genes involved in immunological function, transport activity, metabolism, and chromosome X [10].
Tough the endocrine-disrupting efects of BPA have been thoroughly evaluated, there is still some uncertainty regarding its potential to harm the health. Much of such uncertainty is because of the controversies that surround the design and interpretation of results from hypothesis-driven BPA research [11].
Being an endocrine disruptor, BPA can trigger several disorders, including developmental and reproductive system abnormalities, impaired brain and neurologic functions, malignancy, cardiovascular disease (CVD), diabetes, early puberty, obesity, and immune dysregulation [12].
Cardiovascular diseases are the leading cause of mortality globally. Research has found that oxidative damage has a signifcant role in the development of coronary artery disease (CAD) [13]. A systematic review has demonstrated a correlation between increased BPA and the enhanced risk of CVD, obesity, diabetes, insulin resistance, and hypertension in humans [14].
Experimentally, it was shown that low BPA doses could afect estrogen signaling in cultured rodent cardiomyocytes [15]. Also, exposure to oral BPA (50 mg over 8 weeks) caused morphologic and structural alterations in the rat myocardial tissue, including vacuolation of myocytes, focal loss of myofbrils, and mitochondrial distortion [16]. However, the impact of low-dose BPA on rat myocardial tissue remains elusive. Kim and coworkers demonstrated that BPA (50 μg/ kg/d) accelerated atherosclerosis progression in a genetic mouse model prone to endothelial dysfunction and vasculitis [17].
BPA exposure was also associated with atherosclerosis progression in the coronary artery and aorta of rabbits [18], and other reports have shown a relationship of BPA with blood pressure in mice [19], and electrical contraction of rat heart extract [20].
In addition, Aboul Ezz and colleagues administered BPA to adult male rats by oral route for 6-10 weeks (25 or 10 mg/ kg/d) and assessed oxidative damage in the cardiac tissue. Tey reported enhanced lipid peroxidation, low GSH, and catalase levels. BPA was also associated with a reduction in nitric oxide, which may result in vasoconstriction and reduced blood supply to the cardiac tissue. Tere was also reduced acetylcholinesterase activity among the BPA-treated rats, which might cause a decrease in heart rate and contractility [21].
Te cardiotoxic efects of BPA could be triggered through its action on estrogenic receptors, modifcation of cardiac Ca 2+ -handling protein expression, ion channel inhibition/activation, oxidative damage, production of free radicals, and genome/transcriptome modifcations [22,23].
LC is a nonprotein amino acid, which is formed from lysine and methionine amino acids. It promotes the β-oxidation of fatty acids and has a role in the metabolism of branched-chain amino acids and the stabilization of cell membranes [24]. Also, several studies have shown that LC acts as a free radical scavenger and thus provides protection for the antioxidant enzymes against oxidative stress [25,26]. Compared to the wealth of data linking BPA to reproductive disorders, diabetes, and obesity, few studies have focused on the relationship of BPA with CVDs and in particular coronary artery pathology. Terefore, this work was conducted to assess the biochemical and histopathological alterations mediated by BPA in the myocardium and coronary artery of adult rats and to assess the efect of coadministration of LC against the BPA-induced alterations.

Ethical Consideration.
In the current study, we followed the ethical guidelines according to the ethical norms approved by the Medical Research Ethics Committee, Mansoura University (Code No.: R/96).

2.2.
Chemicals. BPA and LC were purchased from Sigma--Aldrich Inc. Te required doses underwent dissolution in corn oil and were given to experimental animals by oral gavage.

Animals and Experimental Design.
Te study was performed on 24 male adult Wistar albino rats weighing about 200-250 g. Rats were housed and maintained under standard environmental conditions at a temperature of 22°C ± 2°C, 12 h of light/dark cycle, 41%-55% relative humidity, and provided with food and water throughout the experimental period. All animal work was carried out in accordance with the guidelines for the use of animals in research established by Mansoura University, Egypt. Also, the animals underwent handling according to the Guide for the Care and Use of Laboratory Animals [27].
Te study duration was 4 weeks. Te rats were randomly divided into three groups (8 rats each) as follows: Group 1: untreated rats and served as controls (received corn oil), Group 2 (BPA 40 mg/kg/d): rats were treated with 40 mg/kg/ d BPA dissolved in corn oil and given by oral gavage. Tis dose was chosen following the Chapel Hill BPA expert panel consensus statement, which defned the "low BPA dose" dose below the LOAEL (50 mg/kg/d) in animal models [28], and Group 3 (BPA 40 mg/kg/d + LC 300 mg/kg/d): rats received 40 mg/kg/d BPA along with the administration of LC 300 mg/kg/d [29], dissolved in corn oil and given by gavage by oral gavage.
At the end of the experimental work, rats were intraperitoneally injected with pentobarbital (50 mg/kg). Teir hearts were then dissected. Te left ventricles were cut perpendicular to the long axis into rings of 1 ̶ 2 mm width and utilized for the measurement of tissue GSH and MDA, and histopathological and immunohistochemical examination.

Histopathological Examination.
Te myocardial and coronary artery tissues of the sacrifced rats were dissected immediately, and part of it was fxed in 10% neutral bufered formalin for histopathologic examination. Te parafnembedded sections were prepared to be 5 μm thicknesses, and deparafnized using 100% xylene, followed by rehydration with 100% and then 70% ethyl alcohol. Transverse sections underwent staining with hematoxylin and eosin (HE) for visualization of general tissue morphology and Masson trichrome to detect fbrotic areas. Stained sections were examined under light microscopy [30,31].

2.5.
Immunohistochemistry. For the immunohistochemical study, sections were cut at 3 μm thickness. Tey were collected on poly-L-lysine coated slides, dried in a thermostat at 37°C for 24 h to achieve an appropriate adhesion of the biologic material to the surface of the slide, and then stained with the appropriate antibodies (anti-eNOS and anticaspase-3). Immunohistochemistry examination by eNOS was performed according to Felaco et al. [32], while examination by caspase-3 was performed according to Hamed et al. [33].

Biochemical Tests.
Te myocardial and coronary artery specimens from the sacrifced rats were homogenized in phosphate bufer solution (0.01 M sodium phosphate bufer, pH 7.4, containing 0.14 M NaCl) at 1 ml volume/g tissue wet weight ratio of 4 : 1. Homogenates underwent centrifugation at 13, 000 × g for 20 minutes, and the resultant supernatant was used for oxidative activity analysis. GSH and MDA concentrations were measured in the tissue homogenates utilizing commercially available kits.

Statistical Analysis.
Te data were analyzed using IBM SPSS Statistics for Windows, v 22.0. Armonk, NY: IBM Corp. Qualitative data were presented as numbers and percentages. Quantitative data were presented as means and standard deviations for parametric data following testing normality by the Kolmogorov ̶ Smirnov test. Te signifcance of a result was judged at the (0.05) level. Te one-way ANOVA test was utilized to compare more than 2 independent groups with the post hoc Tukey test to detect pair-wise comparison.

Results
Tere were no reported mortalities among the experimental animals. In each group, the same area of each section was selected for hematoxylin and eosin, Masson trichome, eNOS, and caspase-3 stainings. Te current study revealed the following fndings.

Hematoxylin and Eosin.
To investigate BPA's efects on the coronary artery, sections were stained using HE and examined using a light microscope. Unlike the control rats, which demonstrated the normal histological structure of the artery, sections from the BPA group show vacuolation of cells in the media and detachment of the endothelium of the intima of the artery. In contrast, sections taken from rats that received the BPA + LC demonstrated a near-normal structure to control rats ( Figure 1). Regarding the myocardium, the control group demonstrated branching and anastomosing muscle fbers. Cardiomyocytes show central oval nuclei. Capillaries and fbroblasts were observed in the connective tissue endomysium in between muscle fbers. On the other hand, the hearts of BPA-treated rats showed congested blood vessels and infltration by polynuclear cells in comparison to control rats. Coadministration of LC improved these BPA-induced changes ( Figure 2).

Masson Trichrome Stain.
Te Masson Trichrome stain stains the collagen-rich fbrotic areas in a bluish color. As demonstrated in Figure 3, the expression of the bluish color is notably enhanced in rats receiving BPA in comparison with rats in the BPA + LC group, which demonstrated reduced bluish color expression, indicating that BPA resulted in signifcant fbrosis in the wall of the coronary artery (P < 0.001) ( Table 1). On the other hand, the Masson trichrome stain of the hearts demonstrated that BPA signifcantly enhanced the collagen fber content in the myocardium in comparison with the control group (P < 0.001). However, LC coadministration was associated with a signifcant reduction in collagen deposition between muscle fbers ( Figure 4) (P < 0.001). Moreover, as shown in Figure 5, the quantitative analysis of the interstitial collagen fber area percentage in the heart revealed that BPA increased the fbrous tissue content compared to the control group (47.34 ± 3.60% versus 11.11 ± 1.54%). However, coadministration of LC and BPA caused a signifcant reduction in collagen deposition in comparison with the BPA-treated rats (30.02 ± 4.09% versus 47.34 ± 3.60%) (P < 0.001).

Caspase-3.
To measure the apoptosis in the coronary artery, sections were stained with the caspase-3 immune stain ( Figure 6). BPA-exposed rats showed an increased expression of caspase-3 as compared with control rats. In contrast, the BPA + LC group demonstrated a decrease in caspase-3 expression in comparison to BPA-treated rats (P < 0.001) ( Table 1). Te apoptosis in the heart was also assessed in this study. Heart sections of BPA-exposed rats showed an increased caspase-3 expression (evidenced by excess brownish staining) as compared with control rats (P < 0.001). In contrast, the BPA + LC group showed a decrease in caspase-3 expression in comparison to the BPA group ( Figure 7) (P < 0.001). Te quantitative analysis of caspase-3 expression area percentage of the heart demonstrated that BPA enhanced caspase-3 expression in comparison with control rats (240.69 ± 4.84% versus 182.92 ± 6.44%) and that coadministration of LC and BPA was associated with a signifcant reduction in caspase-3

eNOS.
In our study, the endothelial function was assessed by staining coronary artery sections with the eNOS immune stain (Figure 9). BPA caused an enhanced expression of eNOS when compared with the control group (P < 0.001). Conversely, coadministration of LC caused a signifcant reduction in eNOS expression in the coronary artery (P < 0.001) ( Table 1). Regarding the myocardium, Figure 10 demonstrated that the cardiac muscle showed an increased expression of the eNOS in rats exposed to the BPA group (P < 0.001). On the other hand, there was a decreased expression of the stain in the BPA + LC group (P < 0.001). Te quantitative analysis of the eNOS expression area % of the heart (Figure 11) revealed increased eNOS expression in BPA-treated rats in comparison with control rats (114.84 ± 6.21% versus 52.39 ± 4.05%). However, coadministration of LC and BPA was associated with a signifcant decrease in eNOS expression in comparison to the BPA group (81.86 ± 5.94% versus 114.84 ± 6.21%) (P < 0.001).

Tissue Glutathione and Malondialdehyde.
Te statistical analysis of tissue MDA and GSH demonstrated a highly statistically signifcant diference (P < 0.001) between BPAtreated rats and control rats (approximately 3-fold for MDA and 2.5-fold for GSH). In the group of BPA + LC, the MDA demonstrated a statistically signifcant decrease (P < 0.001) of the mean MDA level (20.75 nmol/g versus 32.82 nmol/g) while the GSH demonstrated a statistically signifcant rise (P < 0.001) of mean GSH level (1.22 mmol/g versus 0.82 mmol/g) ( Table 2).

Discussion
BPA is a pollutant that induces diferent health problems including metabolic disorders, hormonal-based tumors, and cardiovascular diseases [34]. According to Melzer et al. [35], humans with greater urinary BPA concentrations seemed to be more susceptible to heart disease. Data do exist about BPA's toxic efects on the heart; however, there is little data regarding such an efect on the coronary artery. Hence, in this research, BPA's efects on the heart and coronary artery were investigated. A BPA dose of 40 mg/kg/d was used in our study. Tis dose was selected from recently published studies that used a BPA dose of 40 mg/kg/d [36,37]. Te current study demonstrated signifcant histopathological and immunohistochemical changes in the coronary arteries and the hearts of the rats. Starting with coronary artery changes, the current BPA-associated histopathological alterations in the coronary artery, which included vacuolation of cells in the media and detachment of the endothelium of the intima of the artery, and increased collagen fbers in the arterial wall, are suggestive of coronary artery vasculitis and atherosclerosis. Tese changes can be elucidated by the infuence of BPA on the endothelial cells,  resulting in elevated levels of surface adhesion molecules that increase permeability and induce immune cell infltration [38]. BPA could also enhance the infammatory response by increasing the serum levels of proinfammatory cytokines, ending by apoptosis of coronary artery smooth muscle cells [38], which was also evident in our study by immunohistochemistry. Moreover, BPA-induced endothelial cell changes could lead to coronary vascular weakness and ventricular hemorrhage, which appear as cardiac dysfunction [39]. Melzer et al. [40] stated that BPA exposure was linked to an enhanced risk of CAD. Te latter has been suggested to be related to BPA by diferent receptormediated toxicity such as estrogenic and antiandrogenic receptors, which fnally afect the cardiovascular tissues. On the other hand, the myocardium of BPA-exposed rats showed congestion of the blood vessels and infltration by polynuclear cells in the myocardium. Tese fndings are suggestive of the infammation linked to BPA administration. Proinfammatory cytokines such as TNF-α and IL-1β can reduce the left ventricular function and cause loss of cardiomyocytes via apoptosis. Tese cytokines also have the ability to induce neurohumoral activation and cause oxidative damage, resulting in the initiation of the p38-MAP kinase and nuclear factor-κB. Collectively, this induces cardiomyocytolysis and reduces Ca +2 uptake by the sarcoplasmic reticulum, and therefore impairs cardiac inotropy [16]. Te congested and dilated blood vessels could explain the enhanced cellular infltrates in-between muscle fbers in the BPA-treated group. Such fndings are consistent with Klint and coworkers [41], who reported that BPA increased vascular endothelial growth factor and eNOS that controls vascular tone in cardiac tissues.
In addition, the myocardial changes were suggestive of increased fbrosis as indicated by a signifcant increase in collagen fbers. Similar fndings in the myocardium have been reported previously after oral administration of BPA (50 mg/kg) to rats, where the myocardium of BPA-exposed rats showed a signifcantly increased collagen fber deposition in comparison to control rats [16]. Also, Bahey et al. [42] reported that BPA-exposed animals showed structural myocardial abnormalities, which included disarrangement of myofbers, hypertrophy of myocytes, and fbrosis. Myocardial fbrosis was reported previously as well after the exposure of rats to a higher BPA dose [43]. It commonly occurs in many cardiac diseases including heart infarction, failure, and hypertension [44]. BPA-induced myocardial  fbrosis is mostly promoted by the cardiac fbroblasts' proliferation, collagen production, and mast cell activation [45]. In our work, the apoptosis caused by BPA was evaluated by the immunohistochemical study of the proapoptotic marker caspase-3. BPA caused apoptosis in the coronary artery and the cardiac tissue as indicated by enhanced caspase-3 expressions. Te immunohistochemistry examination of caspase-3 in cardiac slices revealed that caspase-3 expression was minimal in the control group, while it is signifcantly elevated in the BPA group and decreased in the BPA + LC group. Indeed, treatment of rats with LC improved the apoptotic efects of BPA, as evidenced by downregulatingcaspase-3 expressions. Myocyte apoptosis is associated with high concentrations of proinfammatory cytokines including IL-1β, NF-α, and interferon-c [46].
Due to the importance of eNOS activity to keep the vascular dilatation, eNOS expression was investigated in this study. Nitric oxide (NO) is produced by the endothelial nitric oxide synthase (eNOS) enzyme by converting the Larginine to NO in the vasculature [47]. Te eNOS is expressed in endocardial cells, endothelial cells, myocytes, and other myocardial cells [48]. NO underlies smooth muscles and acts as an endothelium-dependent vasodilator [49]. Te reduced NO level results in vasoconstriction, endothelial dysfunction, and hypertension [50]. Also, it was revealed that a defciency of eNOS accelerated atherosclerotic lesion formation in eNOS knockout mice [51].
Our fndings revealed an increased expression of eNOS in BPA-exposed rats, which was in agreement with Klint et al. [41]. It should be mentioned that this fnding is not against the previously mentioned concept that eNOS defciency is associated with atherogenesis. To clarify, eNOS may have two faces in the pathophysiology of atherosclerosis. Tis depends on the vascular tissue levels of tetrahydrobiopterin. For example, eNOS overexpression may promote atherogenesis through increased free radical production from dysfunctional eNOS, i.e., eNOS-mediated superoxide generation [51]. Interestingly and supporting our results, it was postulated that oxidative stress (which was evident to be induced by BPA in our study) can enhance the eNOS expression, and, indeed, eNOS expression is enhanced in most types of vascular disease. Tis might be an attempt of the organism to compensate for the decreased NO activity. However, such compensation is usually inefective, as the eNOS becomes or remains uncoupled under pathologic conditions. Te upregulation of eNOS expression makes the condition even worse as the uncoupled eNOS worsens  Figure 11: Graph demonstrating the quantitative analysis of eNOS expression area percentage of the heart. Data are expressed as means ± SDs. # : signifcantly diferent from the control group, while $ signifcantly diferent from the BPA group (P < 0.001).
oxidative damage [48]. Moreover, other diferent mechanisms could modulate the BPA-induced eNOS expression including the Ca 2+ /calmodulin-dependent mechanism [52], and the angiotensin II pathway [53]. At the biochemical level, BPA-induced oxidative stress was investigated. Te biochemical analysis showed the occurrence of oxidative damage in BPA-treated rats that was indicated by the reduced level of the antioxidant GSH and the elevated level of the lipid peroxidation marker (MDA). Te heart is signifcantly sensitive to free radicals due to its high oxidative metabolism and due to its few antioxidant defenses. ROS are cytotoxic agents, which cause oxidative stress by targeting biomolecules like membrane lipids and cellular DNA [16]. Terefore, oxidative stress could be the cause of the structural alterations observed in the hearts of BPA-treated rats. Oxidative stress could also trigger cardiac fbrosis via diferent mechanisms, such as stimulation of transforming growth factor-beta1 expression [54] and the JAK/STAT signaling pathway [55]. Tus, the current data may indicate a possible correlation between BPA-mediated oxidative stress and cardiac fbrosis. Other studies have reported BPA-induced oxidative damage in the myocardial tissue of adult male albino rats. For instance, the study by Abd El-Haleem and Abass [16] revealed a signifcant rise in serum MDA and a signifcant reduction in tissue-reduced GSH and catalase in the myocardium of BPA-treated animals. Similarly, it was found that BPA caused a signifcant rise in MDA, and a signifcantly decreased catalase [21].
Te increased evidence of BPA-related negative efects on health has encouraged researchers to search for a drug or natural substance that protects against these efects, especially for those at high exposure risk [42]. Tis study reports, for the frst time, that LC has a signifcant protective efect against BPA-related coronary and myocardial toxicity. It was clear that LC could reduce the BPA-induced lipid peroxidation and elevate the antioxidant GSH levels as well. Similarly, LC showed protective efects against isoproterenol-induced myocardial infarction by reducing oxidative damage markers and infammatory cell infltration [56]. In our study, the BPA-induced histopathological alterations were reduced in the LC-treated group, which was indicated by the lower cells' infltration, normal myocardial fber structure, and normal coronary artery architecture. Tese fndings are explaining the ability of LC to reduce cardiac fbrosis pathogenesis and myocardial infarction, which are consistent with many previous reports [56][57][58].
Moreover; LC could attenuate the BPA-induced eNOS expression revealing its protective efect against the probable PBA-induced coronary artery vasoconstriction. It is known that the LC role is enhanced by diferent mechanisms, such as calcium channel regulation, endothelial integrity maintenance, and cellular homeostasis control [59]. Tus, LC can protect against the myocardial and coronary artery damage that commonly occurs in cardiovascular diseases.

Conclusion
Exposure to BPA has been reported to be linked to CVD. However, few studies have focused on its association with coronary artery pathology. Tis study concluded that BPA exposure can result in biochemical and pathologic alterations in the myocardium and coronary artery. Te results suggest that oxidative stress, fbrosis, and apoptosis play important roles in BPA-related toxicity. Importantly, the harmful alterations caused by BPA could be partially improved by the coadministration of LC. However, more studies are required to evaluate the efects of prolonged exposure to diferent BPA doses on the cardiovascular system. Also, it is recommended to regularly administer LC to help reduce the deleterious efects of exposure to BPAcontaining products.

Data Availability
All data used to support the fndings of the current study are included within the article.

Conflicts of Interest
Te authors declare that they have no conficts of interest.