The possible alleviating effect of saffron on chlorpyrifos experimentally induced cardiotoxicity: Histological, immunohistochemical and biochemical study

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The possible alleviating effect of saffron on chlorpyrifos experimentally induced cardiotoxicity: Histological, immunohistochemical and biochemical study Hanaa Attia Khalafa,b,⁎, Ahmed Abd El-Rahman El-Mansya,b a b

Department of Medical Histology & Cell Biology, Mansoura Faculty of Medicine, Egypt Department of Histology, Hours University, Egypt

ARTICLE INFO

ABSTRACT

Keywords: Chlorpyrifos Saffron Cardiotoxicity Immunohistochemical study

Introduction: Pesticides are responsible for many occupational health hazards among farmers in developing countries. Chlorpyrifos (CPF) is one of the broad-spectrum organophosphorus (OP) insecticides used for agricultural, domestic and industrial purposes. Aim of the work: The present study was designed to examine the effects of CPF on cardiac muscles and to evaluate the possible protective role of crocin using biochemical and histological methods with the intention to recognize the molecular tools of its probable cardioprotective effects. Materials and methods: Thirty-six adult male albino rats were used in this study and were divided into 4 equal groups (9 rats each): negative control group, positive control group, CPF treated group and CPF & crocin treated group. The heart was removed for histological and immunohistochemical studies. Results: Stained sections of cardiac muscle fibers of group III with H&E revealed remarkable histological changes in the form of disorganization of the fibers with increase in the interstitial spaces between these fibers. Congested dilated blood capillaries could be observed with extravasation of the red blood cells leading to interstitial hemorrhage. Focal areas of mononuclear cellular infiltration could be seen in the interstitial tissue. A number of cardiac fibers achieved pale acidophilic vacuolated sarcoplasm while others achieved dark homogenous acidophilic sarcoplasm. Some nuclei were peripherally situated and pyknotic while others were centrally situated and encircled with halos. Apparently increased masses of collagen fibers among the cardiac muscle fibers and around the congested dilated blood vessels with the presence of focal parts of extensive collagen fiber deposition were noticed in Mallory-stained sections of group III. Strong positive immunoreactions in the endomysium and perimysium of the cardiac fibers, along with the walls of blood capillaries and in interstitial cells, could be detected in immunohistochemical staining sections of group III with vimentin antibody. Immunoreactivity to caspase 3 was higher in the sarcoplasm of the cardiac fibers of group III compared to that of control group. A highly significant decrease in the cardiac level of SOD and CAT; however, a highly significant increase in MDA level was noted between the control groups and CPF treated group. Additionally, there was a significant improvement of the chemical and histological representations of group IV, and these improvement pictures were toward the normal. Conclusion: The study concludes that crocin can alleviate the toxic effect of chlorpyrifos caused by oxidative stress on cardiac muscle.

1. Introduction Pesticides are responsible for many occupational health hazards among farmers in developing countries. Low educational levels, defective delivery of data, insufficient training on pesticide security, lowly spraying techniques, and improper safety of pesticide usage are the

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major causes of intoxication scenarios (Ahmed et al., 2017). Organophosphorus (OP) pesticides are the most frequently used in many regions of the world (Sharma et al., 2017). Chlorpyrifos (CPF) is one of the broad-spectrum OP insecticides used for cultivated, domestic and industrial purposes (El-Wakf et al., 2018). It is used to control Leptinotarsa sp., aphids, white fly, and termites (Sharma et al., 2017).

Corresponding author at: Department of Medical Histology & Cell Biology, Mansoura Faculty of Medicine, Egypt. E-mail addresses: [email protected] (H.A. Khalaf), [email protected] (A.A.E.-R. El-Mansy).

https://doi.org/10.1016/j.acthis.2019.03.003 Received 5 January 2019; Received in revised form 2 March 2019; Accepted 4 March 2019 0065-1281/ © 2019 Elsevier GmbH. All rights reserved.

Please cite this article as: Hanaa Attia Khalaf and Ahmed Abd El-Rahman El-Mansy, Acta Histochemica, https://doi.org/10.1016/j.acthis.2019.03.003

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The chemical properties of CPF provided flexibility for use in multiple delivery systems (Gao et al., 2017). Unfortunately, CPF has a high affinity to organic and inorganic components of the soil, leading to its accumulation and slow steady discharge into living organisms and plants (Sharma et al., 2017). CPF-induced Toxicity led to the restriction of some of its domestic uses, but it is still considered one of the most widely used insecticides (Ambali et al., 2010). Although CPF toxicity is considered moderate compared to many other OPs, many health hazards were recorded especially the neurodevelopmental effects (Gao et al., 2017). CPF is also postulated to affect the cardiovascular system as well as respiratory system of fish (Sharma et al., 2017). The adverse effect of lipid peroxidation is counteracted by free radical scavenging mechanism provided by both enzymatic antioxidants, like superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) and non-enzymatic ones like melatonin and many vitamins as E, C (Bas and Kalender, 2011). Oxidative stress results when the endogenous antioxidants fail to oppose free radical generation. Therefore, exogenous supplementation of antioxidants improves the ability of the tissue to cope with high antioxidant levels (Ambali et al., 2010). Over the last decade, scientists have begun to focus on the potentiality of using natural antioxidants to protect against oxidative stress (Pohl and Kong Thoo Lin, 2018). Saffron (Crocus sativus Linn) is one of the most frequently used as a dietary ingredient, for hundreds of years, all over the world. It has also been applied, for many centuries, in traditional medicine (Abedimanesh et al., 2017). It was also used as anti-inflammatory, anti-spasmodic, expectorant and aphrodisiac (Heidari et al., 2017). Saffron contains about 300 volatile and nonvolatile constituents, including safranal, crocin, picrocrocin and some other carotenoids. The therapeutic effects of saffron are mainly owing to the presence of crocin (Javandoost et al., 2017). Crocin has free radical scavenging activity hence it is known as a powerful herbal antioxidative agent contrary to oxidative stress encouraged by chronic stress, cisplatin and carbon tetrachloride (Yaribeygi et al., 2018). It may be used as a new treatment, due to its antitumor and anti-inflammatory effects. It was also supposed that crocin can protect against cardiotoxicity, hepatotoxicity and DNA damage (Mohammadi et al., 2018). So, the present study was planned to study the effects of CPF on cardiac muscles and to evaluate the possible protective role of crocin by biochemical and histological methods with the intention of recognizing the molecular tools of its probable cardioprotective effects.

Blvd, Rocklin, CA 95677, USA]. This antibody was used in rats before according to Abd El-Haleem and Abass (2012). 4 Anti-caspase 3/CPP32 rabbit polyclonal antibodies [(Catalog No., RP096 as 1 ml concentration), Diagnostic Biosystem, 6616 Owens Drive, Pleasanton, CA 9458, USA]. This antibody was used in rats before according to Black et al. (1998). 2.2. Animals Thirty-six adult male albino rats, weighing 180–200 g m each, were purchased from Mansoura Experimental Research Center (MERC), and were used with consideration of the Animal Welfare Act and Guide for Care Use of MERC. Before the study, animals were housed for one week in a quite non-stressful environment with ad libitum feeding and free access to water throughout the study. 2.3. Experimental design Animals were classified into 4 equal groups (9 rats each): Group I: Each animal received distilled water via intragastric tube and via intraperitoneal injection for 4 weeks and served as a negative control group. Group II: Each animal received crocin dissolved in distilled water through intraperitoneal injection at a dose of 40 mg/kg/ day for 4 weeks (Yaribeygi et al., 2018) and served as a positive control group. It was found that treatment of animals with crocin at this dose (40 mg/kg) decreased lipid peroxidation and elevated glutathione (GSH) in the liver and brain tissues. Serum levels of tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) were also decreased (Mohammadi et al., 2018). Group III: Each animal received CPF (% 99 purity-powders) dissolved in distilled water through intra gastric tube at a dose 5.4 mg/kg/ day [1/25 of the calculated median lethal dose (LD50)] for 4 weeks (Bas and Kalender, 2011). This dose was selected according to former researches that postulated CPF at 1/25 LD50 induced biochemical changes in rats without morbidity and the dose was prepared by putting CPF in room temperature then dissolves it in distilled water immediately before daily use (Mansour and Mossa, 2009). Group IV: Each animal was given both crocin (at the same dose of group II) via intraperitoneal injection and CPF (at a dose similar to that of group III) via intragastric tube for 4 weeks. All animals were sacrificed at the end of experiment, dissected and the heart was removed for histological and immunohistochemical studies.

2. Materials and methods

2.4. Histological study

The protocol of this study was accepted (Code Number: R/ 18. 3. 109) by the Institutional Research Board (IRB) of the Faculty of Medicine, Mansoura University, Mansoura City, Egypt on 2/4/2018. This experimental study was carried out in the laboratory of Medical Histology and Cell Biology Department, Faculty of Medicine, Mansoura University

Specimens from heart were fixed, dehydrated, cleared and embedded in paraffin. Paraffin blocks were sectioned at 5 μm thickness and put on clean glass slides. These sections were stained with hematoxylin and eosin (H&E) according to (Bancroft and Layton, 2013), and Mallory trichrome according to (Kiernan, 2008).

2.1. Chemicals

2.5. Immunohistochemical (IHC) study

1 CPF (Dursban) was obtained from Kafr El-Zyat Agricultural Pesticides Company, Kafr El-Zyat city, Egypt. It was dissolved in distilled water. 2 Crocin was obtained from Sigma Aldrich Co. (USA). It was liquefied in distilled water. 3 Anti-vimentin mouse monoclonal antibodies [(Catalog No., 347M14 as 0.1 ml concentration) V9, Cell Marque, 6600 Sierra College

Paraffin sections from the heart specimens were picked up on positive slides. Avidin-biotin technique immunostaining was performed. After deparaffinization and rehydration, endogenous peroxidase activity was blocked with 0.01% hydrogen peroxide (H2O2). Antigenic site unmasking was done by buffering the sections in 0.01 M citrate buffer at pH 6 for 30 min and in ethanol for 10 min. Antigen retrieval was made in the microwave for 20 min. Incubation of sections was done

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Fig. 1. A: The cardiac muscle fibers of control groups are longitudinally settled with oval central vesicular nuclei (arrows) and acidophilic faintly striated sarcoplasm (curved arrows). The fibers are disconnected by tiny interstitial space containing flat fibroblasts with flat nuclei (zigzag arrows). B–F of CPF treated group, Separation of cardiac fibers with increased interstitial spaces between them is seen (asterisk). Congested dilated blood capillaries (BV) could be observed with extravasation of red blood cells (tailed arrows) and interstitial hemorrhage (H). Mononuclear cellular infiltration could be also seen in the interstitial tissue (thick arrows). Some cardiac fibers achieved pale acidophilic vacuolated sarcoplasm (V) whereas others achieved dark homogenous acidophilic sarcoplasm (double arrows). Some nuclei were peripherally situated, small and deeply stained (crossed arrows) while others were centrally situated and were encircled with halos (arrows head). (G): The cardiac muscle fibers of CPF & crocin treated group are organized approximately parallel to those of the control groups. The fibers are longitudinally settled with oval central vesicular nuclei (arrows) and acidophilic faintly striated sarcoplasm (curved arrows). The fibers are disconnected by tiny interstitial space containing flat fibroblasts with flat nuclei (zigzag arrows). (H): Few cardiac muscle fibers are slightly disorganized (F) with slight increase in interstitial spaces between them (asterisk). Mild congestion of blood vessels (BV) could also be seen. (B& C × 100 and A, D, E, F, G&H ×400)

overnight at 40 °C with the diluted primary antibody at dilution 1/100 1/500 monoclonal mouse antibodies for vimentin and 1/50 - 1/100 polyclonal rabbit antibodies for caspase 3. Sections were incubated with the avidin-biotin complex (ABC) element for 60 min then in peroxidase solution for 6–10 minutes. Finally, hematoxylin counter staining was performed and reactivity was visualized in the cytoplasm for both antibodies. Specific primary antibody was swapped by phosphate buffer saline for negative control slide. Tonsil slides were used as positive control for both anti-vimentin assays (McCluggage, 2002), and anti-caspase 3 assays (Krajewska et al., 1997).

2.6. Biochemical study Tissue samples from the heart were rapidly isolated and immediately homogenized in phosphate buffer solutions then centrifuge for 20 min and the supernatants were obtained for biochemical study. The activities of superoxide dismutase (SOD) (Beauchamp and Fridovich, 1971), catalase (CAT) (Sinha, 1972), and contents of malondialdehyde (MDA) (Ohkawa et al., 1979) in heart homogenates were determined by the assay kits.

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Fig. 2. (A: control group): Very little collagen fibers (crossed arrows) can be detected in the interstitial tissues of cardiac muscle fibers. (B: group III): Increased amounts of collagen fibers among the cardiac muscle fibers (crossed arrows) with broad parts of highly collagen fiber deposition (asterisk) are noticed. (C: group III): Extensive amounts of collagen fibers among the cardiac muscle fibers (crossed arrows) and around the congested dilated blood vessels (curved arrows) are noticed. (D: group IV): Few collagen fibers (crossed arrows) can be detected in the interstitial tissues of cardiac muscle fibers. (Mallory trichrome-stain, × 400)

2.7. Morphometric study

obtained images were analyzed on an Intel Core 13 computer via Video Test Morphology software (Saint Petersburg, Russia) with a specific built-in routine for determining area % of the following measures:

The slides were photographed by an Olympus digital camera (E2410 mega pixel- China) fit on an Olympus microscope with a ×0.5 photo adaptor by objective lens ×40. Five non-overlapping fields were examined/ section and three sections were assessed/ animal. The

1 Collagen fibers in Mallory trichrome stained sections. 2 Positive reaction in immunostained sections. Fig. 3. (A: control group): Positive immunoreactivity to vimentin antibody could be seen along with the wall of blood capillaries (arrows head) and interstitial cells (crossed arrows) with negative immunoreaction in the cardiac fibers (F). (B& C: group III): Strong brown positive reactions could be seen in the endomysium and perimysium of the cardiac fibers (arrows head) as well as along with the wall of blood capillaries (arrows head) and in interstitial cells (crossed arrows). (D: group IV): Minimal positive immunoreaction approximately parallel to that of the control groups could be seen in the walls of blood capillaries (arrows head) and interstitial cells (crossed arrows) with negative immunoreaction in the cardiac fibers. (Anti vimentin immunostain, × 400)

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Fig. 4. (A: control group): Negative immunoexpression for caspase-3 antibody could be exhibited in cardiac fibers (F). (B: group III): Strong Immunoreactivity to caspase 3 was detected in the cardiac fibers (crossed arrows). (C: group IV): Few fibers exhibit weak immunoexpression for caspase-3 antibody (crossed arrows) while remaining fibers show negative reaction (F). (Anti caspase-3 immunostain, × 400).

2.8. Statistical analysis

Student’s t-test and expressed as mean value ± standard deviation. A probability value of P < 0.05 will be considered significant and P < 0.01 highly significant (Wilcox, 2009).

Statistical analyses were done by means of Statistical Package for Social Sciences (SPSS) software version 15.0 (SPSS, Inc., Chicago, IL, USA). The morphometric and biochemical data were studied using the

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vimentin antibody showed minimal positive immunoreaction approximately parallel to that of the control groups along with the wall of blood capillaries and interstitial cells with negative immunoreaction in the cardiac fibers (Fig. 3D).

3. Result 3.1. Histological results 3.1.1. H&E stain Examination of cardiac muscle fibers of H&E stained sections of control groups (group I&II) designated that both groups displayed normal histological pattern of the myocardium. The cardiac muscle fibers were built of branching and anastomosing cylinder-shaped fibers. The fibers had oval central vesicular nuclei and acidophilic sarcoplasm. The endomysium in between the cardiac fibers exhibited blood capillaries and flat fibroblasts with flat nuclei (Fig. 1A). The H&E stained sections of cardiac muscle fibers of CPF treated group (group III) revealed remarkable histological changes in the form of disorganization of the fibers with increase in the interstitial spaces between these fibers (Fig. 1B). Congested dilated blood capillaries could be observed with extravasation of the red blood cells leading to interstitial hemorrhage (Fig. 1B–D). Restricted areas of mononuclear cellular infiltration could be seen in the interstitial tissue (Fig. 1E). A number of cardiac fibers achieved pale acidophilic vacuolated sarcoplasm while others achieved dark homogenous acidophilic sarcoplasm (Fig. 1F). Some nuclei were peripherally situated, small and deeply stained (pyknotic) while others were centrally situated and were encircled with halos (Fig. 1E & F). Examination of H&E stained sections of cardiac muscle fibers of CPF plus crocin treated group (group IV) revealed organized fibers approximately parallel to that of the control groups (Fig. 1G). However, few fibers seemed to be slightly disorganized with slight increase in interstitial spaces between them. Mild congestion of blood vessels could also be seen (Fig. 1H).

3.1.4. Caspase-3 antibody stain Negative immunoexpression of caspase-3 could be exhibited in cardiac sections of the control groups (Fig. 4A). Immunoreactivity to caspase 3 was higher in the sarcoplasm of the cardiac fibers of group III compared to that of control group (Fig. 4B). Negative to weak immunoexpression of caspase-3 parallel to that of the control groups was seen in cardiac muscle fibers of group IV (Fig. 4C). 3.2. Morphometric study As regard the mean area% of collagen fibers, no significant alteration could be detected between the area% of collagen fibers between of the control groups (group I& II) and also with that of group IV. Highly significant increase and decrease in area% of collagen fibers of group III was detected when compared with that of control groups or when compared with that of group IV respectively (Histogram 1). As regard the mean area% of vimentin immunoexpression, no significant alteration could be detected between the area% of vimentin immunoreaction between the control groups (group I& II) and also with that of group IV. Highly significant increase and decrease in area% of vimentin immunoexpression of group III was detected when compared with that of control groups or when compared with that of group IV respectively (Histogram 2). As regard the mean area% of caspase-3 immunoexpression, no significant alteration could be detected between the area% of caspase-3 immunoreaction between the control groups (group I& II) and also with that of group IV. Highly significant increase and decrease in area% of caspase-3 immunoexpression of group III was detected when compared with that of control groups or when compared with that of group IV respectively (Histogram 3).

3.1.2. Mallory's trichrome stain Very little collagen fibers could be detected in the interstitial tissues of cardiac muscle fibers of control groups stained with Mallory's trichrome-stain (Fig. 2A). Mallory-stained sections of group III showed apparently increased masses of collagen fibers among the cardiac muscle fibers and around the congested dilated blood vessels (Fig. 2B & C). Focal parts of extensive collagen fiber deposition were noticed (Fig. 2B). Mallory-stained sections of group IV displayed an obvious reduction in collagen fibers among the systematized arranged cardiac muscle compared with group III, but faintly more than that of group I (Fig. 2D).

3.3. Biochemical study No significant change was noted in the cardiac levels of SOD and CAT between both control groups. A highly significant decrease (P < 0.001) in the cardiac levels of SOD and CAT was noted in CPF treated group compared to the control groups. The cardiac levels of SOD and CAT of CPF and crocin treated group were nearly close to that of normal while it showed highly significant increase (P < 0.001) compared with those of CPF treated group (Histogram 4&5). Histogram (6) showed non-significant change between the level of cardiac MDA in both control groups, while there was a highly significant increase (P < 0.001) in this level in CPF treated group compared to the control groups. However, the cardiac level of MDA of CPF and crocin treated group was nearly close to normal and it was highly significant decreased (P < 0.001) compared with that of CPF treated group.

3.1.3. Vimentin antibody stain Immunohistochemical staining sections with anti-vimentin antibody revealed positive immunoreactivity in the wall of blood capillaries and interstitial cells with negative immunoreaction in the cardiac fibers of rats of control groups (Fig. 3A). Immunohistochemical staining sections of group III with anti-vimentin antibody showed strong positive immunoreactions in the endomysium and perimysium of the cardiac fibers, along with the wall of blood capillaries and in interstitial cells (Fig. 3B & C). Immunohistochemical staining sections of group IV with anti-

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Histogram (1): Area % of Mallory trichrome (mean ± SD) of control and experimental groups:

Histogram (2): Area % of anti-Vimintin antibody positive reaction (mean ± SD) of control and experimental groups:

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Histogram (3): Area % of anti-caspase-3 antibody positive reaction (mean ± SD) of control and experimental groups:

Histogram (4): SOD level in heart tissue (mean ± SD) of control and experimental groups:

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Histogram (5): CAT level in heart tissue (mean ± SD) of control and experimental groups:

Histogram (6): MDA level in heart tissue (mean ± SD) of control and experimental groups:

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4. Discussion

Vimentin is one of the five intermediate filaments present in our bodies. Overall, utmost mesenchymal cells like fibrocytes, smooth muscle cells, vascular endothelial cells, in addition to myoepithelial cells of sweat and salivary glands were clearly stained with vimentin (Azumi and Battifora, 1987). Therefore, it signifies as a pointer of the interstitial cells (Heling et al., 2000). Accordingly, the extensive vimentin immunoexpression in this work was mainly due to an increase in interstitial heart tissue as well as fibrosis. This has been previously linked to degeneration of the cardiac muscle fiber replaced by fibrosis, and might clarify the role of fibroblasts in myocardial fibrosis (Di Somma et al., 2000; Rastogi et al., 2008). Cardiac fibroblasts play an important role in myocardial fibrosis. They may change into myofibroblasts to secrete extracellular matrix components like collagen, fibronectin, and laminin, and finally leading to fibrosis. As a source for fibroblasts, endothelial cells lose their endothelial phenotypes as CD31 & VE-cadherin and gain myofibroblastic properties such as vimentin and α-SMA expression during endothelial–mesenchymal transition (Cai et al., 2017). In the present work, immunohistochemical stained sections with anti-caspase-3 antibody showed strong immunoreactivity in the cardiac fibers of group III compared with that of the control group. These obtained results are parallel to those achieved by Kapoor and Kakkar (2014) and El-Wakf et al. (2018), who found that CPF treatment initiated notable enhancement in the proteins of apoptosis; Bax, caspase-3 and p53. They postulated that CPF induces apoptosis mainly by ROS that lead to peroxidation of the mitochondrial membrane, destruction of cellular integrity and DNA damage that finally lead to cell demise. Moreover, CPF initiates apoptosis in different organs via activating the intracellular caspase 3 pathways, as reported by former researches (Abolaji et al., 2017; Chougule et al., 2013; Li et al., 2009; Yu et al., 2008). Generally, there are two pathways for apoptosis: extrinsic and intrinsic pathway. The extrinsic one is related to caspase 3 activities. For instance, caspase-3 is considered a symbol of apoptosis; hence, it used in assays of cellular death (Leist and Jäättelä, 2001). There was a significant rise, in this study, in cardiac content of MDA parallel with significant decrease in cardiac SOD and CAT in the CPF group (III) compared with control group. These results hypothesized the occurrence of oxidative stress in CPF treated group. The obtained results are in harmony with the previous ones (Abolaji et al., 2017; Ahmed et al., 2017; El-Wakf et al., 2018; Imbaby et al., 2014). They demonstrated that CPF leads to excessive production of mitochondrial ROS that initiates oxidative stress. Also, Georgiadisa and his colleague stated that oxidative stress is the main mode of action of organophosphorus leading to its side effect on myocardial tissue (Georgiadis et al., 2018). Oxidative stress is convoluted in the occurrence of several pathophysiological conditions (Poprac et al., 2017). These conditions were mainly due to overproduction of free radicals which exceed the antioxidant defense system (ADS). This discrepancy was linked with various cardiovascular illnesses (Klein et al., 2017). The cardiac muscle is actually sensitive to these free radicals since it is subjected to highly oxidative breakdown and had fewer ADS (Yilmaz et al., 2006). MDA is reported as a sign of oxidative stress and lipid peroxidation (Yang et al., 2009). CAT and SOD enzymes are the main stage of the ADS. SOD catalyzes the superoxide radicals and forms H2O2 hydrolyzed by CAT into H2O (Gupta, 2011). Examination of H&E stained sections of cardiac muscle fibers of CPF plus crocin treated group (group IV), in this study, revealed remarkable improvement in the histological changes caused by CPF. There were organized fibers approximately parallel to those of the control groups, despite the presence of few fibers seemed to be slightly disorganized with slight increase in the interstitial spaces between them and mild extravasation of RBCs. These modifications may point to the ameliorative effect of crocin in contrast to CPF-induced cardiac fibrosis. Moreover, these results were settled by both histochemical and

CPF is the utmost frequently used OP pesticide despite its toxicity (Heilmair et al., 2008). CPF, resembling other OP, applies its noxious effects mainly by depressing the action of enzyme acetylcholinesterase (Kaur and Sandhu, 2008). In light of this, the current work was planned for examining the cardiotoxicity of CPF in adult male albino rats and the possible protective role of crocin on these changes. In this work, we perceived that CPF at a dose of (5.4 mg/kg/ day) creates remarkable histological changes in the form of disorganization of the cardiac muscle fibers with increase in the interstitial spaces between these fibers. A number of cardiac fibers achieved pale acidophilic vacuolated sarcoplasm with some peripherally situated pyknotic nuclei was appeared. These data come in accordance with other work (Bas and Kalender, 2011; Bayır et al., 2013; El-Wakf et al., 2018; Georgiadis et al., 2018). They noted that CPF caused disorganization, degenerative changes in myocardial fibers and edema in connective tissue. They postulated that these changes may be due to increased secretion of mitochondrial reactive oxygen species (ROS) in cardiac tissues and induction of oxidative stress. This led to diminution of mitochondrial energy, secretion of proteolytic enzymes and DNA disintegration causing apoptosis (Yu et al., 2008). Nevertheless, lethal effect of CPF on the myocardium appeared in this work as extensive part of necrotic myocardium with the existence of perinuclear halos (myolysis). Investigators designated myolysis as relative loss of myofibrils around the nuclei of the cardiac fibers (perinuclear) (Canty and Suzuki, 2002). It also may be related to the increased proinflammatory cytokines, like interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and interferon-γ which trigger cardiomyocytolysis (Rutschow et al., 2006). Conferring to earlier researches, CPF led to cardiotoxicity through a direct myocardial endothelial injury and damage of cardiac muscle fibers (Shanmugarajan et al., 2008). In the existing work, we noticed congested dilated blood capillaries with extravasation of red blood cells leading to interstitial hemorrhage. Focal areas of mononuclear cellular infiltration could be also detected in the interstitial tissue. These data are parallel to that achieved by other researchers (Ahmed et al., 2017; Jalili et al., 2007; Mondal et al., 2014; Razavi et al., 2015). The injury detected in the heart muscle might be due to the effect of disruption of oxidative phosphorylation. The OP components led to destruction in vascular endothelium with increased permeability, which resulted in extravasation of the red blood cells and interstitial hemorrhage (Imbaby et al., 2014). In the existing work, cardiac sections of the CPF treated groups (III) showed obvious fibroblast proliferation and deposition of collagen fibers among the cardiac muscle fibers and around the congested dilated blood vessels. Moreover, focal parts of extensive collagen fiber were detected in sections stained with Mallory trichrome. Such findings coincide with those obtained by Bas and Kalender (2011) and Ahmed et al. (2017), who stated that CPF treatment leads to extensive creation of ROS with consequences of inflammatory reaction introducing cardiac toxicity, fibroblasts activation and succeeding fibrosis. Under stress situations, the heart produces transforming growth factor (TGF)-β1 which plays a serious role in cardiac restoration. It stimulated the transformation of cardiac myofibroblasts into fibroblasts as well as proliferating them (Lucas et al., 2009). Furthermore, as a result of cardiac fiber necrosis, the myofibroblasts are proliferated and activated. These activated myofibroblasts produce angiotensin I & II, and fibrogenic growth factors (as TGF-β1), altogether leading to collagen synthesis and hence fibrosis (Weber et al., 2013). Immunohistochemical stained sections with anti-vimentin antibody of group III in this work, showed strong brown positive reactions, in the endomysium and perimysium of the cardiac fibers, along with the wall of blood capillaries and in the interstitial cells representing increased myofibroblast activity and further fibrosis. 10

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immunohistochemical techniques, as there was notable fall in the mean area% of collagen and vimentin immunoexpression compared with those of groups III. Even so, there was noticeable reduction in the area% of caspase-3 immunoreactivity of cardiac muscle of group IV compared with that of CPF treated group. At present, there was a strong impression on supplementation of antioxidant as it is capable of suppressing DNA fragmentation and hence inhibit apoptosis (Galang et al., 2000). The alleviating effect of various antioxidants on the histological and biochemical changes in the cardiac muscle induced by CPF was previously studied by many researchers (Ahmed et al., 2017; Bas and Kalender, 2011; Bayır et al., 2013; El-Wakf et al., 2018). They concluded that the harmful effect of CPF is mainly mediated by oxidative stress; hence, administration of antioxidants could ameliorate these dangerous effects via its effect as a free radical scavenger. It is important to take into consideration that the effects of crocin on CPF induced cardiotoxicity have not been investigated yet. Crocin is a yellow carotenoid commonly pulled out from Crocus sativus (saffron) and Gardenia jasminoides fruits (Bandegi et al., 2014). It was reported that crocin has many therapeutical properties, as anticancer, anti-hyperlipidemic, antidepressant, anti- inflammatory, antioxidant (Alavizadeh and Hosseinzadeh, 2014; Jam et al., 2017) and hepatoprotective (Yousefsani et al., 2018) effects. Moreover, crocin could improve the oxidative stress in cardiac tissue caused by many drugs. It has been reported that Crocin improved the histopathological alterations in the cardiac tissue, ventricular pressure and ejection fraction caused by doxorubicin (Razmaraii et al., 2016) and also in cardiotoxicity induced by diazinon (Razavi et al., 2012). Similarly, it has been found that crocin has the ability to avert isoproterenol cardiotoxicity (Goyal et al., 2010). Our findings were in a close agreement with those presented by Bandegi et al. (2014), who observed that crocin could alleviate the oxidative stress induced by chronic stress via its antioxidative effects. The antioxidative property of saffron was created mainly via removal of the free radicals or through enhancement of ADS such as CAT and SOD enzymes (Assimopoulou et al., 2005; Bandegi et al., 2014; Yaribeygi et al., 2018). This was represented in our work by significant decrease in MDA parallel with rise in both SOD and CAT activities in cardiac tissue of group IV compared with those of CPF treated group.

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