Abd El Samad, Abeer A.; Raafat, Mona H.
For more than 20 years, anthracycline antineoplastic drugs, for example doxorubicin (DOXO), have been among the most effective and widely used anticancer chemotherapeutics in clinical practice. Its clinical utility is, however, largely limited by the adverse reactions that develop with its use. Besides the reversible and often easily manageable adverse effects typical of anticancer drugs (e.g. nausea, myelosuppression), there is a well-documented risk of a severe complication: cardiotoxicity . The exact mechanism of DOXO-induced cardiotoxicity remains unclear, but most studies support the view that oxidative stress plays an important role in the pathogenesis of DOXO cardiotoxicity [2–4].
Grape seeds (proanthocyanidin) are waste products of the grape juice industry. These seeds contain lipid, protein, carbohydrates, and 5–8% polyphenols depending on the variety . Some studies have been carried out on grape seed extracts (GSEs) and have showed excellent free radical scavenging and cardioprotective properties . GSE provided a unique protection against myocardial ischemia-reperfusion injury and myocardial infarction in rats [7–9]. However, the protective mechanisms underlying this effect remain elusive. In most cases, the activity of GSE is related to its antioxidative properties and can mainly be attributed to the phenolic compounds .
Moreover, the renin–angiotensin system is a central component of the physiological and pathological responses of the cardiovascular system. The activity of angiotensin II (Ang II), the main effector of renin–angiotensin system, is initiated by its interaction with at least two pharmacologically distinct subtypes of cell-surface receptors: AT-1 and AT-2. The major functions of Ang II in the cardiovascular system are mediated by the AT-1 receptor (AT-1R) . Previous studies have reported that Ang II plays a key role in the process of anthracycline-induced cardiotoxicity . However, studies have suggested that the angiotensin-converting enzyme inhibitor and the angiotensin receptor blocker play a protective role in DOXO-induced cardiotoxicity [13,14]. Recently, some investigators have shown that telmisartan, an angiotensin receptor blocker, also possesses anti-inflammatory and antioxidant properties .
Thus, the aim of this work was to determine the possible protective effect of GSE and telmisartan on DOXO-induced cardiotoxicity in male adult rats using light and electron microscopy.
Materials and methods
Thirty adult male albino rats weighing 220–250g were used in this study. They were housed in standard cages at the Medical Research Center in Ain Shams University. They had free access to water and a standard diet. Animals were kept for 1 week before the beginning of the experiment for acclimatization.
All animals were randomly divided into six groups, each including five rats:
Group I (control group): This group received a single intraperitoneal injection of 0.5ml of 0.9% physiologic saline.
Group II (DOXO group): This group received a single intraperitoneal injection of DOXO (Ebewe Pharma, Austria, Ges.m.b.H. Nfg.KG A-4866 Unterach, AUSTRIA) at a dose of 20mg/kg .
Group III (GSE+DOXO group): This group received GSE (Twinlab, USA, Utah, American fork) by a gavage daily for 12 days at a dose of 100mg/kg . On day 7, 2h after the administration of GSE, a single dose of 20 mg/kg DOXO was administered intraperitoneally.
Group IV (telmisartan+DOXO group): This group received telmisartan (Boehringer Ingelheim International, GmbH, Binger strasse, Germany) by a gavage daily for 12 days at a dose of 10mg/kg . On day 7, 2h after the administration of telmisartan, a single dose of 20mg/kg DOXO was administered intraperitoneally.
Group V (GSE group): This group received GSE alone daily for 12 days as in group III.
Group VI (telmisartan group): This group received telmisartan alone daily for 12 days as in group IV.
After 6 days, the rats of group II (DOXO) were sacrificed by decapitation. Meanwhile, the rest of the groups were sacrificed by decapitation after 12 days. The hearts were dissected through a chest incision. Specimens from the cardiac wall of the left ventricle were subjected to the following.
Light microscopic study
Half of the specimens from all groups were fixed immediately in a 10% neutral-buffered formalin solution and processed for light microscopic study to obtain paraffin sections of 5 μm thickness. They were stained with H&E and Masson's trichrome .
Electron microscopic study
The other half of the specimens were cut into small pieces of about 1mm3 and rapidly fixed in 2.5% glutaraldehyde for 24h. They were washed in 0.1mol/l phosphate buffer at 4°C and then postfixed in 1% osmium tetroxide at room temperature. Specimens were dehydrated in ascending grades of ethyl alcohol and then embedded in Epon resin. Semithin sections of 1μm were cut and stained with 1% toluidine blue in borax and examined with a light microscope. Ultrathin sections of 50nm were cut, mounted on copper grids, and stained with uranyl acetate and lead citrate . Specimens were examined and photographed using a JEM 1200 EXII transmission electron microscope, JEOL, Tokyo, Japan (TEM) at the Faculty of Science, Ain Shams University.
Morphometric and statistical study
The cardiac wall of the left ventricle in five different-stained sections from all the rats in each group (five high-power fields per section) were examined to determine cardiomyocytes’ diameters of H&E-stained slides and the area percentage of collagen fibers (green colored) in Masson's trichrome-stained slides. Quantitative measurements were performed using the image analyzer (Olympus Image J, NIH, 1.41b; Melville, NY, USA) at the Oral Pathology Department, Faculty of Dentistry, Ain Shams University, which was calibrated for distance and area before its use. Mean ± SDs were calculated. The differences among all groups in all the data parameters measured were statistically analyzed using one-way analysis of variance and a post-hoc test using the SSPS program version 17 (IBM Corporation, Somers, New York, USA). The calculations were considered significant if P value was less than 0.05.
Light microscopic results
In group I (control group), the H&E staining sections of the left ventricle wall showed branching and anastomosing cardiac muscle fibers in different directions. Connective tissue cells and fibers were found between the cardiac muscle fibers (Fig. 1). The semithin sections stained by toluidine blue showed longitudinally cut fibers with transverse striations and intercalated discs. The nuclei appeared central, oval, and vesicular (Fig. 2). In Masson's trichrome-stained sections of the same group, few and fine collagen fibers were observed between the cardiac muscle fibers (Fig. 3).
The H&E-stained sections of group II (DOXO) showed degenerated and fragmented cardiac muscle fibers with an inflammatory cellular infiltration in between the muscle fibers (Fig. 4). Congested blood vessels with extravasated red blood cells were observed between the cardiac muscle fibers (Fig. 5). Examination of the semithin sections indicated that the cardiac muscle fibers had disrupted transverse striations and pale-stained nuclei. Large and congested blood vessels were observed between the muscle fibers (Fig. 6). In Masson's trichrome-stained sections of the same group, the contents of collagen fibers were increased and replaced some areas of degenerated cardiac muscle fibers (Fig. 7).
Examination of sections stained by H&E of group III (GSE+DOXO) showed preservation of the normal pattern of cardiac muscle fibers with vesicular nuclei, almost similar to that of group I, but with some inflammatory cellular infiltration between the cardiac muscle fibers (Fig. 8). Most of the longitudinally cut fibers of group III in semithin sections showed normally arranged transverse striations with intercalated discs between fibers and oval vesicular nuclei, whereas some muscle fibers showed disrupted striations (Fig. 9). The Masson's trichrome-stained sections showed an apparent decrease in collagen fibers compared with group II, but slightly more than that of group I (Fig. 10).
Group IV (telmisartan+DOXO) showed almost normal cardiac muscle fibers with apparent hypertrophy in H&E-stained sections compared with the previous groups. Meanwhile, congested blood vessels were observed between the cardiac muscle fibers (Fig. 11). Semithin sections showed a more or less similar pattern of longitudinally cut muscle fibers and intercalated discs, with some congested blood vessels between the cardiac muscle fibers (Fig. 12). Sections stained with Masson's trichrome showed few collagen fibers compared with group II and were almost similar to group I (Fig. 13).
Sections stained with H&E and toluidine blue of group V (receiving GSE alone) showed an almost similar pattern of cardiac muscle fibers as that of the control group (Figs 14 and 15), whereas group VI (which received only telmisartan) showed apparent hypertrophy of muscle fibers compared with group I in H&E-stained sections (Fig. 16). Semithin sections of group VI showed cardiac muscle fibers that were more or less similar to those of the control group (Fig. 17). Sections stained with Masson's trichrome of both groups V and VI appeared similar to those of the control group.
Electron microscopic results
Group I showed regularly arranged myofilaments between successive Z lines in the sarcomeres. Their nuclei appeared with dispersed nuclear chromatin. Mitochondria, with apparent cristae, were arranged in rows between the myofibrils (Fig. 18). Intercalated discs between two adjacent cardiac muscle fibers appeared step like, with both the transverse and the longitudinal part. Adherent junctions could be observed in the transverse part by their cytoplasmic densities as anchoring sites for microfilaments (Fig. 19).
Group II showed cardiac muscle fiber nuclei with irregular nuclear membranes. Mitochondria appeared distorted into different shapes and sizes around the nuclei and between the myofibrils. The intercellular spaces between adjacent muscle fibers appeared wide (Fig. 20). Areas of degenerated myofibrils with irregularly arranged mitochondria were observed. The matrix of the mitochondria appeared dense with unapparent cristae (Figs 21 and 22). Widening of the intercalated discs was observed between the cardiac muscle fibers (Fig. 22).
Group III showed more regular nuclear membranes of cardiac muscle fiber nuclei. Mitochondria appeared with a dense matrix between the myofibrils (Fig. 23). Apparently normal intercalated discs were observed between cardiac muscle fibers, whereas some vacuolations were detected in between the myofibrils (Fig. 24).
Group IV showed an almost normal appearance of cardiac muscle fiber nuclei and the mitochondria appeared regularly arranged between the myofibrils with apparent cristae (Figs 25 and 26). The intercalated discs retained their normal appearance between cardiac muscle fibers (Fig. 26).
Groups V and VI showed regularly arranged myofibrils in the sarcomeres. Mitochondria were observed to be arranged in rows between the myofibrils with apparent cristae (Figs 27 and 29). The intercalated discs appeared almost similar to those of group I (Figs 28 and 30).
Morphometric and statistical results
Table 1 and Histogram (1A) show the mean ± SD of the cardiomyocyte diameter of different groups. The table shows that group II (DOXO) showed a significant (P < 0.05) decrease in the mean cardiomyocyte diameter as compared with all the groups. However, all the other groups showed a nonsignificant (P > 0.05) change in the mean cardiomyocyte diameter as compared with the control group. Meanwhile, group VI (telmisartan) showed a significant (P < 0.05) increase in the mean cardiomyocyte diameter as compared with the control group.
Area percentage of collagen detected by Masson's trichrome stain
Table 1 and Histogram (1B) show the mean ± SD of area percentage of collagen of different groups. It can be seen from the table that group II (DOXO) showed a significant (P < 0.05) increase in the mean area percentage of collagen as compared with all the groups, whereas all other groups showed a nonsignificant (P > 0.05) change in the mean area percentage of collagen compared with the control group.
Histogram (1B). Mean...Image Tools
DOXO is a powerful anthracycline widely used as an anticancer drug in the clinic, but it is known to induce cardiotoxicity . Most of the DOXO-induced cardiotoxicity can be attributed to the formation of reactive oxygen species (ROS) . Moreover, it has been reported that DOXO produces oxygen free radicals, which cause damage in proteins and DNA by lipid peroxidation as well as interference with the cytoskeleton structure [4,22]. In addition, it has been reported that DOXO causes oxidative stress and depletes antioxidants, which might damage the myocardial cell membrane, making it more permeable and ruptured .
In the current study, the H&E and semithin sections of the DOXO group (group II) showed degenerated and fragmented cardiac muscle fibers with disrupted transverse striations and a significant decrease in the mean cardiomyocyte diameter as compared with all the groups. Some investigators [16,24] have reported results similar to those of the current study. Congested blood vessels were observed with inflammatory cellular infiltration and extravasated red blood cells in between the degenerated cardiac muscle fibers in the present study. These findings are in agreement with those of some authors , who have reported marked granular blood cells between the degenerated cardiac muscle fibers.
The previous changes were confirmed by an electron microscopic examination, which showed cardiac muscle fiber nuclei with an irregular nuclear membrane. Areas of degenerated myofibrils and irregularly arranged mitochondria with a dense matrix were observed. Widening of the intercalated discs was also observed. These results have been reported by some authors, who reported that this myofibrillar injury might be a secondary event after mitochondrial dysfunction, which could lead to imbalance in calcium uptake and loss of ATP production. These factors are known to be important in normal myofibrillar function [25,26]. The collagen fiber content was significantly increased in the DOXO group (group II) compared with group I, a finding that has been reported by other authors .
Examination of sections of group III (GSE+DOXO) of the current work showed preservation of the normal pattern of cardiac muscle fibers almost similar to that of group I, but with some inflammatory cellular infiltration between the cardiac muscle fibers, and some muscle fibers appeared to have disrupted striations. Some authors  have reported similar results and attributed this protective effect to scavenging of free radicals formed by DOXO.
Proanthocyanidins have been shown to exert a novel range of biological, pharmacological, therapeutic, and chemoprotective properties against oxygen free radicals and oxidative stress [6,8,28]. Grape seed proanthocyanidin extract attenuated exogenous H2O2 and oxidant stress, improving cell survival and allowing the recovery of contractile function in cultured cardiomyocytes. Its protective effects were because of its ability to scavenge ROS directly and possibly chelate iron and copper involved in the generation of ROS .
In addition to its antioxidant properties, procyanidin has been shown to modulate the activity of antioxidant enzymes such as cyclooxygenase and lipoxygenase to limit the production of free radicals [29,30]. Moreover, grape seeds could prevent DNA fragmentation and inhibit apoptotic signaling pathways . The improvement in cardiac myofibrils observed on electron microscopic examination in this study was in agreement with the observations of some authors . The collagen fibers showed a significant decrease compared with group II in the current study. This result is in agreement with other works .
It is noteworthy that some authors have reported that procyanidin (GSE) did not attenuate the antitumor activity of DOXO, whereas treatment with procyanidin induced cytotoxicity in the carcinoma cells. This suggests that it may have antitumor activity . The potential cytotoxicity of procyanidins to tumor cells makes it more promising to examine its use as an adjunct in DOXO chemotherapy .
In the present study, H&E and semithin sections of group IV (telmisartan+DOXO) showed almost normal cardiac muscle fibers compared with the control group. Meanwhile, congested blood vessels were observed between the cardiac muscle fibers. The cardioprotective effect of telmisartan could be attributed to the marked suppression of inflammation, endoplasmic reticulum stress, and myocardial apoptosis [32,33]. Moreover, some investigators  have reported that DOXO-induced cardiotoxicity is associated with a marked increase in malondialdehyde, which is a marker of lipid peroxidation and an indicator of oxidative injury. These elevated levels of malondialdehyde were significantly decreased in the heart tissue of DOXO-treated rats that were also treated with telmisartan, which suggested that it protected the myocardium against DOXO-induced lipid peroxidation. In addition, some authors  have suggested that the activity of GPx, which is an important enzyme that protects against oxidative stress, was also decreased in DOXO-treated rats, and the combination of DOXO with telmisartan counteracted the depletion of this enzyme. Group IV showed a significant decrease in collagen fibers compared with group II in this study. This result was in agreement with the work of other investigators .
DOXO affected the structure of the rat's heart, which can be protected by GSE and telmisartan. However, the protective effects of GSE on DOXO-induced cardiac injury should be studied for a longer duration of time to obtain more definitive results. In addition, natural antioxidant food such as GSE is safer than chemically prepared drugs such as telmisartan. So we recommend further study with different time schedule (long period) of GSE as it is a well-known natural antioxidant food.
Table. No title avai...Image Tools
Conflicts of interest
There are no conflicts of interest.
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