The Egyptian Journal of Histology:
Effect of bisphenol A on the myocardium of adult male albino rats and the possible role of lycopene: a histological and biochemical study
Abd El-Haleem, Manal Redaa; Abass, Marwa A.b
aDepartments of Histology and Cell Biology, Faculty of Medicine, Zagazig University, Zagazig, Egypt
bDepartments of Forensic Medicine and Clinical Toxicology, Faculty of Medicine, Zagazig University, Zagazig, Egypt
Correspondence to Manal Reda Abd El-Haleem, Department of Histology and Cell Biology, Faculty of Medicine, Zagazig University, Zagazig, Egypt. Tel: +20 122 642 2694; fax: +20 552 310294; e-mail: manal. firstname.lastname@example.org
Received November 27, 2011
Accepted February 9, 2012
Introduction: Despite the wide use of bisphenol A (BPA) in plastic and epoxy resin industries, its side effects have been a subject of controversy. Lycopene (a natural carotenoid) has a protective role in many cardiovascular diseases. This work aimed to study the biochemical and structural changes induced by BPA in the myocardium of adult rats and evaluate whether coadministration of lycopene could alter these effects.
Materials and methods: Twenty adult male albino rats were divided into four equal groups. Group I was the control. Group II received lycopene (4mg/kg body weight/day orally) for 8 weeks. Group III was given BPA (50mg/kg body weight/day orally) for 8 weeks. Group IV was given both BPA and lycopene in the same previous dose and for the same duration. At the end of the experiment, rats were anesthetized, and their hearts were taken and prepared for histological and biochemical studies. Area percentages of the collagen content and positive immune reaction for vimentin were morphometrically and statistically analyzed.
Results: Examination of group III revealed that some myocytes had a deeply acidophilic cytoplasm and were devoid of nuclei. Some myocytes appeared with pale vacuolated cytoplasm, and some had focal loss of myofibrils. Their sarcoplasm contained many distorted mitochondria and dilated T-tubules. Their nuclei were variable in shape. They were peripherally located, or deeply indented, or heterochromatic. Many interstitial cells, inflammatory cells, congested blood capillaries, and areas of edema were seen. A significant increase in collagen fibers and in the area percentage of positive immune reaction for vimentin compared with the control group was observed. Examination of group IV showed that the cardiac muscle cells had a normal architecture except for a few distorted muscle fibers and many congested blood capillaries. There was a significant decrease in the area percentage of positive immune reaction for vimentin in group IV compared with group III. The current study revealed significant increase in serum malondialdehyde, whereas tissue reduced glutathione and catalase showed significant decrease in group III compared with the control group. In contrast, in group IV, malondialdehyde showed significant decrease, and tissue reduced glutathione and catalase showed significant increase, compared with group III.
Conclusion: Long-term exposure to BPA could induce structural and biochemical changes in rat cardiac muscle. This could be partially minimized by concomitant administration of lycopene.
Bisphenol A (BPA) is an organic compound used primarily to manufacture polycarbonate plastics and epoxy resins incorporated in industrial processes such as manufacture of water pipes, in the lining of metal cans and containers for food and beverage, in toys, some dental sealants, and in bonding . BPA is also used in the coating of electrical, electronic, and sports safety equipment  and in toilet paper made from recycled paper .
BPA constitutes one of the largest volumes of chemicals produced worldwide, and over 100 tons are released into the environment annually . All the sources for human exposure have not been determined yet. Although dietary exposure is the main source of exposure to BPA , a contribution of alternative means of exposure, such as dermal absorption, has been proven .
Polymerized BPA molecules do not become a chemically bonded part of the plastic matrix and are linked by ester bonds that are subject to hydrolysis when exposed to high temperatures or to acidic or basic substances. Hence, they can migrate from the plastic product and are omnipresent in the environment . As widespread and continuous exposure of humans to BPA is evident, BPA has been detected in the urine and other body fluids of almost all human subjects studied .
Many concerns have been raised about BPA effects. The Health and Consumer Commissioner has stated that the European Food Safety Authority advice has thrown up ‘areas of uncertainty’, which meant that exposure to BPA should be minimized . Furthermore, it was reported that BPA causes alteration of liver enzyme levels , affection of reproductive function , and affection of the immune system . BPA was considered one of the endocrine disruptors (EDCs) as it is similar in structure to 17β-estradiol. Hence, it interferes with the endocrine signaling pathways [2,11]. In addition, BPA predispose to diabetes . Moreover, an association between BPA and cardiovascular diseases has been demonstrated . BPA has been known to induce oxidative stress in the liver , brain, and testes .
Lycopene is a carotenoid found in tomatoes, water melon, pink guava, pink grapefruit, and papaya. It has a structure similar to that of β-carotene, but its antioxidant activity in neutralizing singlet oxygen has been shown to be much stronger . The human body is unable to synthesize carotenoids. Hence, diet is the only source of these components in blood and tissues .
Vimentin is an intermediate filament protein that is predominantly expressed in cells of mesodermal origin, including fibroblasts, endothelial cells, and white blood cells, and that tends to form an irregular network in endomysial and perimysial sheaths of the myocardium [18,19]. Vimentin increases in association with other cytoskeletal proteins in the myocardium during stressful conditions such as heart failure [20,21].
There is only limited information on the effects of BPA on the heart structure. Oxidative stress is one of the main foci of research related to cardiovascular diseases . The relationship between lycopene and cardiovascular disease has been examined in several studies [23,24].
The aims of the present study were to elucidate the effect of BPA on the histological structure and vimentin immunohistochemical features of the myocardium of adult male rats, investigate the role of BPA on oxidative markers such as malondialdehyde (MDA) and antioxidant markers such as reduced glutathione (GSH) and catalase (CAT), and evaluate whether coadministration of lycopene could alter these effects.
Materials and methods
BPA (CAS No. 80-05-7; purity of 99%) was purchased from Sigma–Aldrich Inc. (Germany). BPA was dissolved in corn oil (vehicle). Rats were treated with BPA at a dose of 50mg/kg body weight, which is equal to the lowest observed level leading to adverse effects, by oral gavage once daily throughout the period of the study . LD50 was administered to rats at a dose of 3250mg/kg orally .
Lycopene powder was purchased from Sigma Chemical Co. (CAS No. 502-65-8; St Louis, Missouri, USA). It was stored in dark glass containers at −70°C until use. It was then removed from the freezer, weighed, and immediately dissolved in corn oil in a dark room just before use. Lycopene was administered by oral gavage at doses of 4mg/kg. The dose of lycopene used in this study was selected on the basis of a previous study .
Kits purchased from Diagnostic and Research Reagents (Giza, Egypt) were used for estimation of MDA, GSH, and CAT.
All procedures in this study were performed in accordance with the medical research ethics committee of Zagazig University (Egypt). All rats were acclimatized to the laboratory environment for 8 days before the start of the experiment.
A total of 20 adult male albino rats (3–4 months old, 180–200g weight) were purchased from the animal house of the Faculty of Veterinary Medicine, Zagazig University. The rats were housed at 25–27°C. The animals had access to normal rat chow and water supplied from glass bottles ad libitum. Rats were randomized into four equal groups (five rats in each group). Group I served as the control group. Group II (the lycopene group) included rats that were given a daily dose of lycopene (4mg/kg body weight/day by oral gavage)  dissolved in corn oil for 8 weeks. Group III (the BPA group) included rats that were given BPA, dissolved in corn oil, at a dose of 50mg/kg body weight/day by oral gavage for 8 weeks . Group IV (the BPA+lycopene group) included rats treated with BPA along with lycopene at similar doses and for the same duration.
At the end of the experiment, all rats were anesthetized by an intraperitoneal injection of pentobarbital (50mg/kg) . Blood samples were taken for assessment of MDA as an indicator of systemic lipid peroxidation. Their hearts were then dissected out rapidly. The left ventricles were cut perpendicular to the long axis into rings of 1–2mm width  and used for assessment of tissue endogenous antioxidant markers, GSH and CAT, and for histological study, as described below.
Serum analysis of the malondialdehyde level
Blood samples were taken from the dorsal vein of the rats’ tails from all groups for assessment of MDA level. Blood samples were collected into tubes containing an anticoagulant (2% sodium oxalate). The samples were centrifuged at 200g for 5min at +4°C; the plasma was then removed immediately and stored at −20°C until it was analyzed for serum MDA according to the method of Draper and Hadley  and expressed as nmol/ml.
Assessment of tissue endogenous antioxidant markers
Heart specimens were wrapped with aluminum foil and embedded in liquid nitrogen for 1 h. They were kept frozen at −80°C until they were used for detection of GSH and CAT enzyme concentration levels. The homogenization of tissues was carried out in a Teflon-glass homogenizer with a buffer containing 1.15% KCl to obtain 1 : 10 (w/v) whole homogenate. The homogenates were centrifuged at 18000 × g for 30 minutes at 4°C to determine GSH concentration and CAT activities . The supernatant was stored at 4°C until assayed. Tissue GSH concentration was measured by an assay using the dithionitrobenzoic acid recycling method described by Ellman  and was expressed as μmol/g protein. CAT activity was determined according to the method of Aebi  and was expressed as U/g tissue protein.
Specimens for light microscopic examination were fixed in 10% formol saline for 24h and processed to prepare 5-μm-thick paraffin sections for H&E staining to verify the histological details  and for Mallory's trichrome staining to show collagen fibers .
In brief, the immunohistochemical staining for the vimentin was carried out by means of the avidin biotin–peroxidase complex (Dako company, Wiesentheid/Bavaria, Germany, Biotin Blocking System, Code X0590) method following the manufacturer's instructions. Paraffin sections of 4μm were deparaffinized in xylene and rehydrated in a descending series of ethanol. The specimens were subjected to antigen retrieval in a citrate buffered solution (pH 6.0) for 10min using a microwave. Endogenous peroxidase was eliminated by incubation in 10% H2O2 in phosphate-buffered saline (PBS; pH 7.4) for 10min. After washing, the specimens were blocked in ready-use normal goat serum for 20min at room temperature. The sections were incubated with vimentin antibody (VIM 3B4, mouse monoclonal antibody, catalog No. M7020; Dako Company, Wiesentheid/Bavaria). Primary antisera were diluted in antibody diluent (TA-125-UD; Labvision). Peroxidase activity was demonstrated using an AEC (3-amino-9-ethyl carbazole) substrate kit (TA-004-HAC; Labvision). The sections were rinsed in PBS. Negative control was obtained when the primary antibody was replaced with PBS .
Specimens for electron microscopy were immediately fixed in 2.5% phosphate-buffered glutaraldehyde (pH 7.4). Thereafter, they were postfixed in 1% osmium tetroxide in the same buffer at 4°C, dehydrated, and embedded in epoxy resin . Semithin sections (1μm thick) were stained with 1% toluidine blue for light microscopic examination . Ultrathin sections were stained with uranyl acetate and lead citrate  and examined and photographed using a JEOL JEM 1010 electron microscope (Jeol Ltd, Tokyo, Japan) in the Electron Microscope Research Laboratory of the Histology and Cell Biology Department, Faculty of Medicine, Zagazig University (Egypt), and in the JEOL JEM 1200 EXII Electron Microscope (Jeol Ltd) Research Laboratory, Faculty of Science, Ain Shams University (Egypt).
The image analyzer computer system Leica Qwin 500 (Leica Ltd, Cambridge, UK) in the image analyzing unit of the Pathology department, Faculty of Dentists, Cairo University (Egypt), was used to evaluate the area percentage of the collagen content between cardiomyocytes and around blood vessels using Mallory's trichrome-stained sections and area percentage (area %) of positive immune reaction for vimentin using vimentin immunostained sections. It was measured using the interactive measure menu. The area percentage and standard measuring frame of a standard area equal to 118 476.6mm2 were chosen from the parameters measuring 10 readings from five sections from each rat of the randomly chosen five rats in each group. In each randomly chosen field, the section of the heart was enclosed inside the standard measuring frame; the areas where collagen fibers and brown positive immune reaction for vimentin were seen were then masked by blue binary color to be measured. These measurements were obtained by total magnification ×100 with the area % of collagen and total magnification ×400 with the area % of positive immune reaction for vimentin.
Data for all groups were expressed as mean ± SD (X ± SD). The data obtained from the image analyzer and the biochemical data were subjected to SPSS program version 14 (http://www.spss.com, Chicago, Illinois, USA). Statistical analysis using the one-way analysis of variance test was carried out. The results were considered statistically significant and nonsignificant when the P values were less than 0.001 and more than 0.001, respectively.
In the current study the statistical analysis of the serum MDA and tissue GSH and tissue CAT of groups I and II showed nonsignificant differences between them. Serum MDA showed significant increase in group III (the BPA group) in comparison with group I (control), whereas tissue GSH and tissue CAT showed significant decrease in group III in comparison with group I (Table 1 and Histogram 1). In contrast, in group IV (after concomitant use of BPA+lycopene) serum MDA showed significant decrease and tissue GSH and CAT showed significant increase in comparison with group III (Table 1 and Histogram 1).
The light and electron microscopic examinations of group I (the control group) and group II (the lycopene group) revealed similar morphological results.
Group I (the control group)
Light microscopic examination of the myocardium of the control adult male albino rats showed that it was formed of branching and anastomosing muscle fibers. Cardiomyocytes had central oval vesicular nuclei. Capillaries and fibroblasts were seen in the connective tissue endomysium between the cardiac muscle fibers (Fig. 1). Mallory's trichrome-stained sections of the left ventricle revealed few fine collagen fibers in the endomysium between the cardiac muscle fibers and around blood vessels (Fig. 2). Immunohistochemical stain for vimentin revealed brown positive immune reactions for vimentin in the wall of the blood capillaries and some interstitial cells, whereas the cardiac muscle appeared negative in reaction to vimentin (Fig. 3). Electron microscopic examination of the myocardium of the same group revealed cardiac muscle cells with oval euchromatic nuclei with prominent nucleoli and long parallel arrays of myofibrils. The myofibrils showed normal architecture with alternating dark and light bands and regular Z lines in the middle of I bands. Rows of mitochondria in between the myofibrils and in the perinuclear area were noticed. The diads were noticed at the level of the Z line. Each diad was formed of one T-tubule and small terminal cistern of smooth endoplasmic reticulum. Intact intercalated discs were observed. Each disc was formed of a transverse portion and a longitudinal portion (Figs 4 and 5).
Group III (the BPA group)
Light microscopic examination of the myocardium of BPA-treated rats showed that myocytes were separated from each other by wide intercellular spaces. Some of the cardiac muscle fibers attained pale acidophilic vacuolated sarcoplasm. Some fibers showed deeply stained homogenous acidophilic sarcoplasm devoid of nuclei. The nuclei of most of the cardiac muscle fibers were central vesicular nuclei, but some appeared small and deeply stained (pyknotic). Localized areas of pale acidophilic homogenous material representing edema were observed around congested blood capillaries. Extravasated red blood cells were also seen. Areas of focal aggregation of cells with dark flat nuclei, most probably fibroblasts and leukocytic cellular infiltration, were observed (Fig. 6). Mallory-stained sections showed an apparent increase in the amounts of collagen fibers between the cardiac muscle fibers and around blood vessels (Fig. 7a). Few focal areas of extensive collagen fiber deposition were also detected (Fig. 7b). Immunohistochemical stain for vimentin revealed extensive strong brown positive immune reactions for vimentin forming an irregular network in the endomysial and perimysial sheaths, in the walls of blood capillaries, and in some interstitial cells (Fig. 8). Electron microscopic examination of the myocardium of the same group revealed that myocytes contained many dilated and disorganized T-tubules, whereas a few T-tubules were still normal and of average diameter (Figs 9 and 10). Distorted mitochondria with heterogenous electron density were observed in rows between the myofibrils (Figs 10 and 11). Focal areas of loss of myofibrils were observed (Fig. 11). Distorted intercalated discs were observed (Figs 11 and 12). Cardiac muscle nuclei were variable in appearance. Some myocytes had deeply indented nuclei with clumps of heterochromatin. Their sarcoplasm had focal loss of the normal alignment pattern of myofibrils (Fig. 12). The nuclei of some myocytes were peripherally located. Many cardiac diads and T-tubules in the myocytes were dilated compared with those of the control group (Figs 13 and 14). Bundles of collagen fibers between cardiomyocytes were noticed (Fig. 14). Marked enfolding of the sarcolemma, especially at the level of Z lines in some myocytes, was seen. Some sarcomeres with loss of myofibrils were seen. Many interstitial and inflammatory cells were seen between cardiomyocytes in the endomysium. In addition, many fibroblast cells with flat nuclei and myofibroblast cells with irregular heterochromatic nuclei and cytoplasmic electron dense bodies were seen in the endomysium between cardiomyocytes and close to blood capillaries (Fig. 15).
Group IV (the BPA+lycopene group)
Light microscopic examination of the myocardium of rats that concomitantly received BPA and lycopene showed that the cardiac muscle fibers preserved the normal architecture except for a few distorted muscle fibers, some wide interstitial spaces, and many congested blood capillaries (Fig. 16). Mallory trichrome-stained sections revealed minimal amount of collagen fibers between the cardiac muscle fibers and around blood capillaries (Fig. 17). Immunohistochemical stain for vimentin revealed brown positive immune reactions for vimentin in the walls of blood capillaries and in a few interstitial cells. Fine endomysial and perimysial brown positive immune reaction was observed, whereas the cardiac muscle appeared negative in reaction to vimentin (Fig. 18). Electron microscopic examination of the myocardium of the same group revealed cardiac muscle cells with elongated oval euchromatic nuclei with prominent nucleoli. Long parallel arrays of myofibrils with normal architecture with alternating dark and light bands and regular Z lines in the middle of I bands were observed. Rows of mitochondria in between the myofibrils and in the perinuclear area were detected. Intact intercalated discs with transverse and horizontal portions were observed (Fig. 19).
Statistical analysis of the mean values of the area percentages of the collagen fibers of the negative control group (group I) and the lycopene group (group II) showed nonsignificant differences between them. Area percentage of the collagen fibers in the BPA group (group III) showed significant increase in comparison with groups I, II, and IV and that of group IV (BPA+lycopene) showed significant increase compared with groups I and II and significant decrease compared with group III (Table 2 and Histogram 2). Statistical analysis of the mean values of the area percentage of the positive immune reaction for vimentin of groups I and II showed nonsignificant differences between them. Area percentage of the positive immune reaction for vimentin in the BPA group (group III) showed significant increase compared with groups I, II, and IV, whereas that of group IV (BPA+lycopene) showed significant decrease compared with group III but it still showed significant increase with group I and group II (Table 3 and Histogram 2).
People exposed to higher levels of BPA seem to be more prone to heart diseases .
In the current study, examination of the myocardium from the BPA group showed some myocytes with deeply homogenous acidophilic cytoplasm and devoid of nuclei. Focal areas of loss of myofibrils were seen. Similar results had been noticed in patients suffering from diabetic cardiomyopathy . BPA creates favorable conditions for the onset and progression of cardiovascular diseases by indirectly promoting diabetes  or by directly enhancing oxidant enzyme activity .
In the current study, examination of the myocardium from the BPA group showed many distorted mitochondria with heterogenous electron density, many dilated T-tubules in most of the muscle fibers, and marked enfolding of the sarcolemma of some myocytes. Similar degenerative findings were observed in the myocardium exposed to estrogenic contaminants, such as nonylphenol ethoxylates . BPA is similar in structure to 17β-estradiol and is hence considered one of the endocrine disruptors (EDCs) . BPA may activate intracellular signals through either of two pathways: genomic  or nongenomic . In genomic activation, BPA binds to both estrogen receptors α and β . In nongenomic mechanisms, BPA may affect the ion channel function as increasing the activity of Maxi-K channels .
It has been demonstrated that certain EDCs such as estrogenic contaminants are able to degrade the layer-by-layer assembly because of their ability to penetrate into the cell membranes by their lipophilic nature and disrupt phospholipids within the membraneous organelles and in T-tubules [37,42]. Alterations in these membranes lead to defects in calcium transport . Many of the proteins involved in cellular Ca2+ cycling appear to be concentrated at the T-tubule. Changes in T-tubule structure occur during heart failure . Hence, we suggested that destabilization of phospholipids within membranes of the T-tubules and in membranous organelles within myocytes may be one of the potential ways by which BPA exerts its toxicity on the myocardium.
In the current study, disorganized intercalated discs were observed while examining the myocardium from the BPA group. Deformed intercalated discs lead to loss of effective contraction force in most of the affected areas .
In the current study, the nuclei of the myocytes from the BPA group were variable in shape. Some of them were peripherally located. Deeply indented and dark heterochromatic pyknotic nuclei were observed. BPA caused DNA damage through estrogenic activity . Myocyte necrosis and apoptosis have been demonstrated to be linked to elevated proinflammatory cytokine levels, such as interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and interferon-γ . During the progression of cardiac hypertrophy to heart failure, the nuclear shape of the myocytes became bizarre with an irregular nuclear envelope . Increased nuclear enfolding could have been due to increased synthesis of the nuclear membrane in excess of that needed to accommodate the increase in volume during cardiac hypertrophy [48,49].
In the current study, examination of the BPA group revealed focal areas of cellular infiltration, as well as fibroblast cells, myofibroblast cells, and inflammatory cells. Extravasated red blood cells, acidophilic material (edema), and congested blood vessels were observed. We suggest that these findings reflect the inflammatory process associated with BPA administration. Similar findings were detected in the hearts taken from rats with diabetes or hyperglycemia . Lang et al.  linked BPA to diabetes. Proinflammatory cytokines such as TNF-α and IL-1β contribute not only to depression of the left ventricular function and cardiomyocyte loss by apoptosis but also to maintaining the balance in extracellular matrix remodeling [46,51]. Proinflammatory cytokines can trigger neurohumoral activation and increase in oxidative stress, leading to activation of p38-MAP kinase and nuclear factor-κB. These induce cardiomyocytolysis and reduce calcium uptake by the sarcoplasmatic reticulum and hence induce negative inotropic effects [52,53].
In the present study, area percentage of the collagen fibers in the myocardium of the BPA group showed significant increase in comparison with the control. Similar findings were detected in the hearts taken from diabetic rats . Lang et al.  linked BPA to diabetes. Collagen accumulation in cases of diabetic cardiomyopathy was due to impaired collagen degradation rather than to overproduction . Hence, cardiac fibrosis could be explained by the indirect effect of hyperglycemia caused by BPA. On the other hand, it was reported that BPA can induce fibroblast hyperplasia in other organs . Fibroblasts may respond to both mechanical loading  and transforming growth factor-β1 stimulation  by a switch to a myofibroblastic phenotype that is accompanied by stimulation of collagen production. Accumulation of collagen in the cardiac interstitium or ‘reactive interstitial fibrosis’ is accompanied by loss of matrix cross-link integrity  and by a change in the ratio of collagens I and III . This may contribute to increased myocardial stiffness with impaired systolic and diastolic functions [57,58].
In the current study, examination of the BPA group revealed focal areas of extensive collagen fibers, numerous congested blood vessels, many fibroblasts, myofibroblast cells, inflammatory cells, and degenerated muscle fibers. Similar changes had been detected in chronic myocardial infarction . BPA exposure has been linked to increase in the risk for coronary artery disease . Hence, structural changes in the myocardium of the BPA group could have been due to the occurrence of minute segmental infarction.
In the current work, significant increase in the area percentage of positive immune reaction for vimentin in the BPA group compared with the control was detected. Similar results were previously demonstrated in cases of dilated cardiomyopathy in humans . The same researchers demonstrated a negative correlation between myocardial vimentin content and the actin–myosin sliding rate. This led to reduced myocyte contraction compared with that in normal individuals . Derangements of cytoskeletal proteins such as vimentin contribute to alterations in intracellular signaling, myocyte function, and coupling of myocytes to the extracellular matrix during cardiac hypertrophy and failure [20,21,61]. Moreover, the myocardial cytoskeleton maintains the internal organization of cellular organelles and transmits the mechanical forces within the cells to and from the adjacent cells and extracellular matrix . The alteration in vimentin expression may be caused by changes in intracellular calcium concentrations . In contrast, the study by Rastogi et al.  showed no changes in vimentin expression in the myocardium of dogs with heart failure. The different species used in the previous studies and different methods of vimentin detection could explain this discrepancy in their results.
The present study showed a positive relation between the level of area percentage of positive immune reaction for vimentin and that of collagen. Increase in the area percentage of positive immune reaction for vimentin could be linked to an increase in interstitial heart tissue . Moreover, the same researchers found proliferation of T-tubules associated with overexpression of vimentin in cardiomyopathy. A positive correlation between vimentin-positive cells and fibrosis was detected, indicating an increase in interstitial tissue in this area .
The current study revealed oxidative damage manifested by significant increase in serum MDA, whereas tissue GSH and CAT showed significant decrease in the BPA group compared with control. BPA induces oxidative stress by decreasing antioxidant enzymes and increasing H2O2 and lipid peroxidation in many organs [14,15]. MDA has been considered a biomarker for lipid peroxidation and oxidative stress . Reduced GSH enzyme is increased in cells exposed to oxidative stress as an adaptive process to maintaining cell integrity because of its reducing properties and participation in cell metabolism . Catalase enzyme, which hydrolyzes H2O2, has been found to increase in cardiotoxicity and aims to improve cardiac function . The heart tissue is very sensitive to free radical damage because of its highly oxidative metabolism and because of its fewer antioxidant defenses . Reactive oxygen species are cytotoxic agents that lead to oxidative damage by attacking biomolecules such as membrane lipids and DNA in cells . Hence, oxidative damage could be the cause of the ultrastructural changes seen in the myofibrils of the BPA group.
In the current study, examination of the myocardiums of the rats in group IV, which received BPA along with lycopene, revealed that the myocardium had an almost normal architecture with the exception of many congested blood capillaries and a few distorted myocytes. Biochemical evaluation of group IV showed that serum MDA showed significant decrease, whereas tissue GSH and CAT showed significant increase in comparison with group III. Lycopene is highly lipophilic and hence has the maximum effect on the scavenging function of the reactive oxygen species in cell lipophilic compartments . Lycopene is most likely involved in the scavenging of two of the reactive oxygen species, singlet molecular oxygen (1O2) and peroxyl radicals. Thus, administration of lycopene reduces lipid peroxidation [17,23] and hence protects the membranous organelles of the cardiac cells. Endogenous antioxidative enzymes can also be stimulated by lycopene . Moreover, lycopene upregulates the gene connexin 43, which allows direct intercellular gap junction communication, allowing small molecules and ions to pass between cells .
In the current work, the myocardium from rats of group IV (BPA+lycopene) showed significant decrease in the area percentage of collagen and vimentin compared with group III. No inflammatory cellular infiltration was detected in the myocardium of the same group. Lycopene can partially reduce the extent of pulmonary fibrosis in rats through the suppression of oxidative stress . Moreover, lycopene has been proven to exert anti-inflammatory properties .
Long-term exposure to BPA could induce biochemical and structural changes in rat cardiac muscles. Moreover, these results suggest that generation of reactive oxygen species plays an important role in the mechanisms of BPA toxicity. These deleterious changes could be partially minimized by concomitant treatment with lycopene. We recommended re-examination of the safety profile of BPA. Consumption of naturally occurring lycopene-rich foods is also recommended for prevention of oxidative stress associated with environmental BPA exposure and with heart diseases. However, further studies are needed before the complete effects of lycopene are properly understood.
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Conflicts of interest
No conflict of interest to declare.
Vandenberg LN, Hauser R, Marcus M, Olea N, Welshons WV. Human exposure to bisphenol A (BPA). Reprod Toxicol. 2007;24:139–177
Rubin BS. Bisphenol A: an endocrine disruptor with widespread exposure and multiple effects. J Steroid Biochem Mol Biol. 2001;127:27–34
Gehring M, Vogel D, Tennhardt L, Weltin D, Bilitewski BBrebbia CA, Kungolos S, Popov V, Itoh H. Bisphenol A contamination of wastepaper, cellulose and recycled paper products Waste management and the environment II. 2004 WIT Press:293–300 In: , pp.
Vandenberg LN, Chahoud I, Heindel JJ, Padmanabhan V, Paumgartten FJ, Schoenfelder G. Urinary, circulating and tissue biomonitoring studies indicate widespread exposure to bisphenol A. Environ Health Perspect. 2010;118:1055–1070
Geens T, Goeyens L, Covaci A. Are potential sources for human exposure to bisphenol A overlooked? Int J Hyg Environ Health. 2011;214:339–347
Zalko D, Jacques C, Duplan H, Bruel S, Perdu E. Viable skin efficiently absorbs and metabolizes bisphenol A. Chemosphere. 2011;82:424–430
Mercea P. Physicochemical processes involved in migration of bisphenol A from polycarbonate. J Appl Polym Sci. 2009;112:579–593
Korkmaz A, Ahbab MA, Kolankaya D, Barlas N. Influence of vitamin C on bisphenol A, nonylphenol and octylphenol induced oxidative damages in liver of male rats. Food Chem Toxicol. 2010;48:2865–2871
Salian S, Doshi T, Vanage G. Perinatal exposure of rats to bisphenol A affects the fertility of male offspring. Life Sci. 2009;85:742–752
Alizadeh M, Ota F, Hosoi K, Kato M, Sakai T, Satter MA. Altered allergic cytokine and antibody response in mice treated with bisphenol A. J Med Invest. 2006;53:70–80
Wetherill YB, Akingbemi BT, Kanno J, McLachlan JA, Nadal A, Sonnenschein C, et al. In vitro molecular mechanisms of bisphenol A action. Reprod Toxicol. 2007;24:178–198
Alonso Magdalena P, Ropero AB, Soriano S, Quesada I, Nadal A. Bisphenol A: a new diabetogenic factor? Hormones. 2010;9:118–126
Melzer D, Rice NE, Lewis C, Henley WE, Galloway TS. Association of urinary bisphenol A concentration with heart disease: evidence from NHANES 2003/06. PLoS One. 2010;5:e8673
Bindhumol V, Chitra KC, Mathur PP. Bisphenol A induces reactive oxygen species generation in the liver of male rats. Toxicology. 2003;188:117–124
Kabuto H, Amakawa M, Shishibori T. Exposure to bisphenol A during embryonic/fetal life and infancy increases oxidative injury and causes underdevelopment of the brain and testis in mice. Life Sci. 2004;74:2931–2940
Stahl W, Sies H. Antioxidant activity of carotenoids. Mol Aspects Med. 2003;24:345–351
Böhm F, Edge R, Burke M, Truscott TG. Dietary uptake of lycopene protects human cells from singlet oxygen and nitrogen dioxide-ROS components from cigarette smoke. J Photochem Photobiol B. 2001;64:176–178
Osborn MKreis T, Vale R. Vimentin Guidebook to the cytoskeletal and motor proteins: a Sambrook and Tooze publication. 1993 Oxford, UK Oxford University Press:169–171 In: , pp.
Kim HD. Expression of intermediate filament desmin and vimentin in the human fetal heart. Anat Rec. 1996;246:271–278
Heling A, Zimmermann R, Kostin S, Maeno Y, Hein S, Devaux B, et al. Increased expression of cytoskeletal, linkage and extracellular proteins in failing human myocardium. Circ Res. 2000;86:846–853
Sharov VG, Kostin S, Todor A, Schaper J, Sabbah HN. Expression of cytoskeletal, linkage and extracellular proteins in failing dog myocardium. Heart Fail Rev. 2005;10:297–303
Dhalla NS, Temsah RM, Netticadan T. Role of oxidative stress in cardiovascular diseases. J Hypertens. 2000;18:655–673
Ferreira ALA, Yeum KJ, Matsubara LS, Matsubara BB, Correa CR, Pereira EJ, et al. Doxorubicin as an antioxidant: maintenance of myocardial levels of lycopene under doxorubicin treatment. Free Radic Biol Med. 2007;43:740–751
Yilmaz S, Atessahin A, Sahna E, Karahan I, Ozer S. Protective effect of lycopene on adriamycin-induced cardiotoxicity and nephrotoxicity. Toxicology. 2006;218:164–171
Richter CA, Birnbaum LS, Farabollini F, Newbold RR, Rubin BS, Talsness CE, et al. In vivo
effects of bisphenol A in laboratory rodent studies. Reprod Toxicol. 2007;24:199–224
Chapin RE, Adams J, Boekelheide K, Gray LE Jr, Hayward SW, Lees PS, et al. NTP-CERHR expert panel report on the reproductive and developmental toxicity of bisphenol A. Birth Defects Res B Dev Reprod Toxicol. 2008;83:157–395
Shen X, Zheng S, Thongboonkerd V, Xu M, Pierce WM Jr, Klein JB, Epstein PN. Cardiac mitochondrial damage and biogenesis in a chronic model of type 1 diabetes. Am J Physiol Endocrinol Metab. 2004;287:E896–E905
Draper HH, Hadley M. Malondialdehyde determination as index of lipid peroxidation. Methods Enzymol. 1990;186:421–431
Ellman GL. Tissue sulphydryl groups. Arch Biochem Biophys. 1959;82:70–77
Aebi HBergmeyer HU. Catalase Methods of enzymatic analysis. 1974 New York and London Verlag Chemie Academic Press:673–678 In: , pp.
Bancroft JD, Gamble M Theory and practice of histological techniques. 20025th ed. Philadelphia Churchill Livingstone
Drury RA, Wallington EA Carlton's histological techniques. 19805th ed. Oxford Oxford University Press
Ramos Vara JA, Kiupel M, Baszler T, Bliven L, Brodersen B, Chelack B, et al. .; American Association of Veterinary Laboratory Diagnosticians Subcommittee on Standardization of Immunohistochemistry. Suggested guidelines for immunohistochemical techniques in veterinary diagnostic laboratories. J Vet Diagn Invest. 2008;20:393–413
Glauert AM, Lewis PR Biological specimen preparation for transmission electron microscopy. 19981st ed. London Portland Press
Ayaz M, Can B, Ozdemir S, Turan B. Protective effect of selenium treatment on diabetes-induced myocardial structural alterations. Biol Trace Elem Res. 2002;89:215–226
Lang IA, Galloway TS, Scarlett A, Henley WE, Depledge M, Wallace RB, Melzer D. Association of urinary bisphenol-A concentration with medical disorders and laboratory abnormalities in adults. JAMA. 2008;300:1303–1310
Perrotta I, Tripepi S. Ultrastructural alterations in the ventricular myocardium of the adult Italian newt (Lissotriton italicus) following exposure to nonylphenol ethoxylate. Micron. 2012;43:183–191
Kurosawa T, Hiroi H, Tsutsumi O, Ishikawa T, Osuga Y, Fujiwara T, et al. The activity of bisphenol A depends on both the estrogen receptor subtype and the cell type. Endocr J. 2002;49:465–471
Watson CS, Alyea RA, Jeng YJ, Kochukov MY. Non-genomic actions of low concentration estrogens and xenoestrogens on multiple tissues. Mol Cell Endocrinol. 2007;274:1–7
Asano S, Tune JD, Dick GM. Bisphenol A activates Maxi-K (K Ca1.1) channels in coronary smooth muscle. Br J Pharmacol. 2010;160:160–170
Yan S, Chen Y, Dong M, Song W, Belcher SM, Wang HS. Bisphenol A and 17β-estradiol promote arrhythmia in the female heart via alteration of calcium handling. PLoS One. 2011;6:e25455
Nakane Y, Kubo I. Layer-by-layer of liposomes and membrane protein as a recognition element of biosensor. Thin Solid Films. 2009;518:678–681
Brette F, Orchard C. T-tubule function in mammalian cardiac myocytes. Circ Res. 2003;92:1182–1192
Hu LW, Liberti EA, Barreto Chaves ML. Myocardial ultrastructure in cardiac hypertrophy induced by thyroid hormone – an acute study in rats. Virchows Arch. 2005;446:265–269
Iso T, Watanabe T, Iwamoto T, Shimamoto A, Furuichi Y. DNA damage caused by bisphenol A and estradiol through estrogenic activity. Biol Pharm Bull. 2006;29:206–210
Rutschow S, Li J, Schultheiss HP, Pauschinger M. Myocardial proteases and matrix remodeling in inflammatory heart disease. Cardiovasc Res. 2006;69:646–656
Gupta A, Gupta S, Young D, Das B, McMahon J, Sen S. Impairment of ultrastructure and cytoskeleton during progression of cardiac hypertrophy to heart failure. Lab Invest. 2010;90:520–530
Ferrans VJ, Jones M, Maron BJ, Roberts WC. The nuclear membranes in hypertrophied human cardiac muscle cells. Am J Pathol. 1975;78:427–460
Kuruvilla S, Balakrishnan KR, Parvathy U. Right ventricular myocardium in Fallot's tetralogy: a light microscopic, morphometric and ultrastructural study. Images Paediatr Cardiol. 2004;6:1–30
Fiordaliso F, Leri A, Cesselli D, Limana F, Safai B, Nadal Ginard B, et al. Hyperglycemia activates p53 and p53-regulated genes leading to myocyte cell death. Diabetes. 2001;50:2363–2375
Torre Amione G, Kapadia S, Lee J, Durand JB, Bies RD, Young JB, Mann DL. Tumor necrosis factor-α and tumor necrosis factor receptors in the failing human heart. Circulation. 1996;93:704–711
Liao F, Andalibi A, Qiao JH, Allayee H, Fogelman AM, Lusis AJ. Genetic evidence for a common pathway mediating oxidative stress, inflammatory gene induction and aortic fatty streak formation in mice. J Clin Invest. 1994;94:877–884
Barnes PJ, Karin M. Nuclear factor-κB-A pivotal transcription factor in chronic inflammatory diseases. N Engl J Med. 1997;336:1066–1071
Ramos JG, Varayoud J, Sonnenschein C, Soto AM, Muñoz de Toro M, Luque EH. Prenatal exposure to low doses of bisphenol A alters the periductal stroma and glandular cell function in the rat ventral prostate. Biol Reprod. 2001;65:1271–1277
Wang J, Chen H, Seth A, McCulloch CA. Mechanical force regulation of myofibroblast differentiation in cardiac fibroblasts. Am J Physiol Heart Circ Physiol. 2003;285:H1871–H1881
Petrov VV, Fagard RH, Lijnen PJ. Stimulation of collagen production by transforming growth factor-beta1 during differentiation of cardiac fibroblasts to myofibroblasts. Hypertension. 2002;39:258–263
Sabbah HN, Sharov VG, Lesch M, Goldstein S. Progression of heart failure: a role for interstitial fibrosis. Mol Cell Biochem. 1995;147:29–34
Pauschinger M, Knopf D, Petschauer S, Doerner A, Poller W, Schwimmbeck PL, et al. Dilated cardiomyopathy is associated with significant changes in collagen type I/III ratio. Circulation. 1999;99:2750–2756
Manikandan P, Sumitra M, Nayeem M, Manohar BM, Lokanadam B, Vairamuthu S, et al. Time course studies on the functional evaluation of experimental chronic myocardial infarction in rats. Mol Cell Biochem. 2004;267:47–58
Melzer D, Osborne NJ, Henley WE, Cipelli R, Young A, Money C, et al. Urinary bisphenol A concentration and risk of future coronary artery disease in apparently healthy men and women. Circulation. 2012;125:1482–1490
Di S, Marotta M, Salvatore G, Cudemo G, Cuda G, de Vivo F, et al. Changes in myocardial cytoskeletal intermediate filaments and myocyte contractile dysfunction in dilated cardiomyopathy: an in vivo
study in humans. Heart. 2000;84:659–667
Ganote C, Armstrong S. Ischaemia and the myocyte cytoskeleton: review and speculation. Cardiovasc Res. 1993;27:1387–1403
Selmin O, Thorne PA, Caldwell PT, Johnson PD, Runyan RB. Effects of trichloroethylene and its metabolite trichloroacetic acid on the expression of vimentin in the rat H9c2 cell line. Cell Biol Toxicol. 2005;21:83–95
Rastogi S, Mishra S, Zacá V, Alesh I, Gupta RC, Goldstein S, Sabbah HN. Effect of long-term monotherapy with the aldosterone receptor blocker eplerenone on cytoskeletal proteins and matrix metalloproteinases in dogs with heart failure. Cardiovasc Drugs Ther. 2007;21:415–422
Schaper J, Froede R, Hein S, Hashizume H, Speiser B, Friedl A, Bleese N. Impairment of the myocardial ultrastructure and changes of the cytoskeleton in dilated cardiomyopathy. Circulation. 1991;83:504–514
Yang YJ, Hong YC, Oh SY, Park MS, Kim H, Leem JH, Ha EH. Bisphenol A exposure is associated with oxidative stress and inflammation in postmenopausal women. Environ Res. 2009;109:797–801
Salvemini F, Franzé A, Iervolino A, Filosa S, Salzano S, Ursini MV. Enhanced glutathione levels and oxidoresistance mediated by increased glucose-6-phosphate dehydrogenase expression. J Biol Chem. 1999;274:2750–2757
Xu MF, Tang PL, Qian ZM, Ashraf M. Effects by doxorubicin on the myocardium are mediated by oxygen free radicals. Life Sci. 2001;68:889–901
Kabuto H, Hasuike S, Minagawa N, Shishibori T. Effects of bisphenol A on the metabolisms of active oxygen species in mouse tissues. Environ Res. 2003;93:31–35
Rao AV, Agarwal S. Role of antioxidant lycopene in cancer and heart disease. J Am Coll Nutr. 2000;19:563–569
Bose KSC, Agrawal BK. Effect of short term supplementation of tomatoes on antioxidant enzymes and lipid peroxidation in type-II diabetes. Indian J Clin Biochem. 2007;22:95–98
Heber D, Lu QY. Overview of mechanisms of action of lycopene. Exp Biol Med. 2002;227:920–923
Zhou C, Han W, Zhang P, Cai M, Wei D, Zhang C. Lycopene from tomatoes partially alleviates the bleomycin-induced experimental pulmonary fibrosis in rats. Nutr Res. 2008;28:122–130
Yaping Z, Wenli Y, Weile H, Ying Y. Anti-inflammatory and anticoagulant activities of lycopene in mice. Nutr Res. 2003;23:1591–1595
bisphenol A; catalase; reduced glutathione; heart structure; lycopene; malondialdehyde; vimentin
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