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Cypermethrin-induced histopathological, ultrastructural and biochemical changes in liver of albino rats: The protective role of propolis and curcumin
Beni-Suef University Journal of Basic and Applied Sciences xxx (2017) xxx–xxx
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Cypermethrin-induced histopathological, ultrastructural and biochemical changes in liver of albino rats: The protective role of propolis and curcumin Manal Abdul-Hamid a,⇑, Nadia Moustafa a, Abd El Mawgoud Abd Alla Asran b, Lila Mowafy b a b
Department of Zoology, Faculty of Science, Beni-Suef University, Beni-Suef, Egypt Department of Plant Protection, Harmful Animals, Research Institute, Agricultural Research Center, Cairo, Egypt
a r t i c l e
i n f o
Article history: Received 26 November 2016 Received in revised form 3 March 2017 Accepted 3 March 2017 Available online xxxx Keywords: Cypermethrin Oxidative stress Histopathology Ultrastructure Propolis and curcumin
a b s t r a c t Cypermethrin (CYP), an insecticide belongs to a synthetic pyrethroid, is used for agriculture and household applications. The present study aimed to examine the toxic effects of CYP on rat liver and to clarify the hepatoprotective effects of propolis (PRO) and curcumin (CUR) against CYP. This study was assessed in male albino rats, each weighting (120–150 g). The rats were equally divided into six groups as follow; the 1st control group, 2nd PRO group (100 mg/kg/day) and 3rd CUR group (100 mg/kg/day). While 4th, 5th and 6th groups were orally treated with CYP (30 mg/kg/day), CYP plus PRO and CYP plus CUR, respectively for 28 days. The present study revealed that CYP-induced significant increase in hepatic markers enzymes (ALT, AST and ALP) and elevation in MDA concomitant with significant decrease of SOD and GPx levels. Histological and histochemical results revealed extensive vacuolar degeneration of hepatocytes, fatty change, blood vessel congestion and fibrosis in the liver of CYP- treated group and depletion of glycogen, protein and DNA. Moreover, ultrastructural observations showed vacuolation, damage of mitochondria and nuclear changes. On the other hand, treatment with PRO and CUR led to an obvious improvement of the injured liver tissues and ameliorating the damaging effects of CYP. In conclusion, PRO is markedly effective than CUR in protecting rats against CYP-induced histopathological, ultrastructural and biochemical changes. This protection may be due to its antioxidant properties and scavenging abilities against active free radicals. Ó 2017 Published by Elsevier B.V. on behalf of Beni-Suef University. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1. Introduction Pyrethroids are human- made of pyrethrins. Pyrethroids have two types which differentiate with their chemical structure and exposure symptoms (Saka et al., 2011). CYP, type II synthetic pyrethroid, have potent insecticidal property. It plays a role in agriculture, protection of food stuff, disease vector control and home pest control (Sankar et al., 2011). CYP is spreadly used as insecticide in developing countries to control many species of insects. It is reported that human exposure to CYP mainly occurred during application or from pyrethroids residues such as those detected in fruits, vegetables, cow’s milk and bread (Sankar et al., 2010). CYP is a hydrophobic molecule which can easily pass through the cell membrane and disturb its structure and cause leakage of cytoplasmic enzymes (Manna et al., 2004; Hussien et al., 2013). CYP
⇑ Corresponding author. E-mail addresses: [email protected], [email protected] (M. Abdul-Hamid).
produced ROS and damaged DNA directly proportional to the dose (Huang et al., 2016). There is a direct reaction between ROS and cellular biomolecules. It damages lipids, proteins and DNA in cells causing cell death (Ferrari, 2000). Sankar et al., 2011 realized that imbalance between amount of free radicals generated and antioxidant defenses in the body are one of the reasons for CYP toxicity. EL-Shemi et al. (2015) showed that the residue levels of CYP were in kidneys, spleen, muscles and liver in the descending order at levels ranging from 12.2 to 156.1 ng/g for CYP. The indicators of overall health status of an individual especially hepatocyte toxicity and related stress are serum enzyme levels (Khan et al., 2009). Antioxidants are the molecules that react with ROS to delay their function and to neutralize them, so they reduce oxidative stress and protect us from diseases state. (Majeed et al., 2016). Propolis (PRO), bee glue, is a sticky resinous substance applied by honey bees Apis mellifera L. as a building material in their hives and as a defensive substance against infections (Bankova et al., 2016). PRO is considered as a remedy in traditional medicine systems all over the world, mainly to treat burns, wounds, stomach
http://dx.doi.org/10.1016/j.bjbas.2017.03.002 2314-8535/Ó 2017 Published by Elsevier B.V. on behalf of Beni-Suef University. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Please cite this article in press as: Abdul-Hamid, M., et al. Cypermethrin-induced histopathological, ultrastructural and biochemical changes in liver of albino rats: The protective role of propolis and curcumin. Beni-Suef Univ. J. Basic Appl. Sci. (2017), http://dx.doi.org/10.1016/j.bjbas.2017.03.002
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Fig. 1. Activities of serum (a): ALT (b): AST and (c): ALP (U/L) of CYP, PRO, CUR, CYP plus PRO and CYP plus CUR in serum of male rats.
ulcer and sore throat. The chemical composition of PRO includes more than 200 compounds such as flavonoids, phenolics, vitamins, alcohols, aromatic acids and their derivatives, esters, minerals and trace elements (Bankova et al., 2000). PRO antioxidant activity is mainly resulted from its flavonoid content, which has the ability
of scavenging free radicals and thereby preservation against LPO (Yousef and Salama, 2009). Curcumin (CUR), is a polyphenolic compound, derives from turmeric (Curcuma longa). It is considerrd as a source for food additive or dietary pigment and in traditional medicine (Perrone et al.,
Please cite this article in press as: Abdul-Hamid, M., et al. Cypermethrin-induced histopathological, ultrastructural and biochemical changes in liver of albino rats: The protective role of propolis and curcumin. Beni-Suef Univ. J. Basic Appl. Sci. (2017), http://dx.doi.org/10.1016/j.bjbas.2017.03.002
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Fig. 2. Concentration of hepatic malondialdehyde (MDA) of CYP, PRO, CUR, CYP plus PRO and CYP plus CUR in liver homogenate of male rats.
(a):
(b):
Fig. 3. Concentrations of (a): hepatic superoxide dismutase (SOD) and (b): Glutathione peroxidase (GPx) of CYP, PRO, CUR, CYP plus PRO and CYP plus CUR in liver homogenate of male rats.
Please cite this article in press as: Abdul-Hamid, M., et al. Cypermethrin-induced histopathological, ultrastructural and biochemical changes in liver of albino rats: The protective role of propolis and curcumin. Beni-Suef Univ. J. Basic Appl. Sci. (2017), http://dx.doi.org/10.1016/j.bjbas.2017.03.002
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Fig. 4. a&b: Photomicrograph of a section in the liver of control rats showing radially arranged polyhedral hepatocytes forming cords around the central vein (CV) and sinusoids (S) (H&E, Scale bar = 100, 50 mm respectivelly).
Fig. 5. a&b: Photomicrograph of a liver section of PRO and CUR groups showing normal structure of liver with a central vein (CV) (H&E, Scale bar = 100 mm).
2015; Pattanayak et al., 2016). Elsayed (2016) clarified CUR as an antioxidant natural herb, a potent scavenger of many free radicals, as anti-carcinogenic, anti-tumoral, anti-inflammatory, antifungal, anti-viral, anti-parasitic, anti-mutagen, anti-infectious and antihepatotoxic natural substance. Moran et al. (2016) pointed that CUR is a powerful scavenger of a wide range of ROS and RNS including O2, H2O2 and NO and also has enhanced activities on different antioxidant systems such as CAT, SOD, GPx, GSH reduced glutathione and heme oxygenase-1. The present investigation aims to evaluate the protective effect of PRO and CUR against toxicity of CYP. Histopathological and ultrastructural changes of liver, biochemical as oxidative stress will be studied to evaluate the effects of treatment with PRO and CUR in the various control and tested groups. 2. Materials and methods 2.1. Chemicals Cypermethrin (CYP) was purchased from the pesticides store in Beni-Suef, Egypt. Propolis (PRO) was purchased from Sigma Pharmaceutical Industries (Cairo, Egypt). It is found in the form of Biopropolis capsules, each one containing 400 mg. Curcumin (CUR) was obtained from Merk Company, Germany.
2.2. Animals and experimental design 42 adult of laboratory males albino rats (Rattus norvegicus) of nearly the same age, weighing (120–150 g) bought from the Ophthalmology Research Institute, Giza, Egypt. Rats were kept under standard management conditions of temperature (25 °C). The animals were fed a standard commercials diet (ATMID Company, Giza, Egypt) and tap water ad libitum. All the rats were acclimatized for at least 15 days before the beginning of the experiment. In this study, animal care was carried out by following the European Community Directive (86/609/EEC) and national rules, this in accordance with the 8th edition of the NIH Guidelines for the care and use of Laboratory Animals. This was approved by the committee of the Zoology Department, Beni-Seuf University, Egypt. Rats were equally divided into six groups containing seven rats as follow: Group 1 serving as an untreated control group under the same laboratory conditions. Group 2 treated with PRO at a dose of 100 mg/kg/day according to Saleh (2012). Group 3 treated with CUR at a dose of 100 mg/kg/day according to Sankar et al. (2012) Group 4 received CYP at a dose of 30 mg/kg/day according to Inayat and Ilahi (2007).
Please cite this article in press as: Abdul-Hamid, M., et al. Cypermethrin-induced histopathological, ultrastructural and biochemical changes in liver of albino rats: The protective role of propolis and curcumin. Beni-Suef Univ. J. Basic Appl. Sci. (2017), http://dx.doi.org/10.1016/j.bjbas.2017.03.002
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Fig. 6. Photomicrograph of a cross section in liver of rat treated with CYP showing (a): Inflammatory cells (iF), extensive vacuolar degeneration of hepatocytes (short arrows), hyperemic sinusoid (arrow head) and congested blood vessel (long arrow) (H&E, Scale bar = 50 mm). b): Congested portal vein (CB) with thickened wall of portal vein (long arrow) and proliferating bile duct (short arrows) (H&E, Scale bar = 50 mm). c&d): The congestion of central vein (CB) and fibrosis (F) (H&E, Scale bar = 200 mm). (e): Oedema (O) and dissolution (d) (H&E, Scale bar = 50 mm). (f): Hepatocytes that undergo fatty change (F) and pyknotic nuclei (arrows) (H&E, Scale bar = 50 mm).
Group 5 given CYP (30 mg/kg/day) parallel with PRO at 100 mg/ kg/day. Group 6 given CYP (30 mg/kg/day) parallel with CUR at 100 mg/ kg/day. Each of CYP, PRO and CUR was dissolved in distilled water and was given by gastric gavage daily for 28 days. At the end of experiment, rats were starved for 12 h and then sacrificed by anesthesia under light diethyl ether and blood samples were collected and allowed to coagulate at room temperature then centrifuged at 3000 r.p.m. for 30 min. The clear non-
haemolyzed supernatant sera was quickly removed and kept at 20 °C for subsequent biochemical analysis. Liver homogenate, obtained by grinding a small piece of freshly excised tissue in 10 volumes of 0.9% NaCl (10% w/v) using Teflon homogenizer (Glas-Col, Terre Haute, USA). The supernatants were kept at -20°C till use in the determination of LPO, SOD and GPx. Symptoms of CYP toxicity in laboratory animals include burrowing, salivation, tremors, writhing and seizures. Also, there were mortality; two rats died from eight rats in CYPtreated group.
Please cite this article in press as: Abdul-Hamid, M., et al. Cypermethrin-induced histopathological, ultrastructural and biochemical changes in liver of albino rats: The protective role of propolis and curcumin. Beni-Suef Univ. J. Basic Appl. Sci. (2017), http://dx.doi.org/10.1016/j.bjbas.2017.03.002
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Fig. 7. a&b: (a): Photomicrograph of a liver section of rat treated with CYP plus PRO group showing great recovery and nearly normal structure of the liver with nearly normal central vein (CV) and hepatocytes (H&E, Scale bar = 100 mm). (b): Photomicrograph of a section in the liver of CYP plus CUR group showing approximate regain of normal appearance of hepatocytes and central vein (CV) (H&E, Scale bar = 100 mm).
2.3. The assay of serum biochemical Alanine amino transferase (ALT) (EC 2.6.1.2) and aspartate amino transferase (AST) (EC 2.6.1.1) activity was according to the method of Schumann and Klauke (2003) using reagent kits purchased from ‘‘Human Diagnostics” (Germany). The determination of alkaline phosphatase (ALP) (EC 3.1.3.1) was spectrophotometrically according to the method of Wenger et al. (1984) and Rosalki et al. (1993) using reagent kits purchased from SPINREACT Company. 2.4. Assessment of oxidative stress and antioxidant enzyme activity The activity of lipid peroxidation (LPO) in liver homogenate was determined according to the method of Yagi (1987). The activity of superoxide dismutase (SOD) (EC 1.15.1.1) in liver homogenate was assayed according to the method of Marklund and Marklund (1974). Glutathione peroxidase (GPx) (EC 1.11.1.9) activity in liver homogenate was assayed according to the method suggested by Matkovics and Szabo (1998).
phate buffer, then followed by post fixation in 1% osmium tetroxide. The specimens were then dehydrated in a series of alcohols, cleared in propylene oxide and finally embedded in Epon epoxy resin. After that, the blocks were trimmed and sectioned with glass knives by an ultra-microtome. Semithin sections (1 mm) were stained with toluidine blue and examined on light microscope to select the suitable area for the ultrathin sections. Ultrathin sections (70–90 nm) were cut on the same ultramicrotome and stained with uranyl acetate and lead citrate (Bozzola and Russell, 1999). Examination of the stained sections was carried out by Joel CX 100 transmission electron microscope operated at an accelerating voltage of 60 kV.
2.8. Statistical analysis The data have been expressed as mean ± SEM. Package for the Social Sciences (SPSS for WINDOWS, version 20.0; SPSS Inc, Chicago) (IBM crop, 2011) was used for the statistical analysis of data. Data were analyzed by one way ANOVA test followed by Duncan’s multiple range tests post hoc analysis values. A value of P < 0.05 was considered statistically significant.
2.5. Histological preparations After 28 days of treatment, the animals were anesthetized under light diethyl and dissected to get the liver. After fixation in10% neutral buffered formalin for 24 h, they were dehydrated through ascending series of ethyl alcohol, cleared in xylene and embedded in paraffin wax. 5 mm thick sections were stained with haematoxylin and eosin for histopathological studies (Bancroft and Gamble, 2002). 2.6. Histochemical preparations Liver sections of all the groups were stained with periodic acid schiff (PAS) reaction (Hotchkiss, 1948), mercuric bromophenol blue method (Mazia et al., 1953) and Feulgen method (Feulgen and Rossenenbeck, 1924) for demonstration of polysaccharides, total proteins and DNA contents respectively. 2.7. Ultrastructural preparations Small pieces of liver of all groups were immediately fixed in 3% glutraldehyde-formaldehyde at 4 °C for 18–24 h, rinsed in phos-
3. Results 3.1. Effects of Cypermethrin, Propolis and Curcumin on serum biomarkers The present study showed that CYP adminstration displayed a significant (P < 0.05) increase of serum ALT activity compared to normal control, PRO and CUR values after 28 days. Treatment with PRO or CUR in concomitant with CYP decrease ALT activity compared to the CYP-treated group (Fig. 1a). Also, CYP-treated rats significantly (P0.05) increased AST activity compared to the control, PRO, CUR groups values. Administration of PRO or CUR in concomitant with CYP decreased AST activity (Fig 1b). Moreover, there was no significant difference in ALP level between control group value and PRO, CUR groups but there is a significant (P < 0.05) increase in ALP activity in CYP group value after 28 days. Treatment of PRO or CUR with CYP decrease ALP activity compared to CYP-treated group (Fig. 1c). Values were expressed as mean ± standard error.
Please cite this article in press as: Abdul-Hamid, M., et al. Cypermethrin-induced histopathological, ultrastructural and biochemical changes in liver of albino rats: The protective role of propolis and curcumin. Beni-Suef Univ. J. Basic Appl. Sci. (2017), http://dx.doi.org/10.1016/j.bjbas.2017.03.002
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Fig. 8. a–c: Photomicrograph of a section in the liver of control, PRO and CUR rats showing highly increased amounts of glycogen in hepatocytes (arrows) (d): Liver of CYP treated rat showing depletion of glycogen in hepatocytes (arrow), (e&f): Liver of CYP plus PRO and CYP plus CUR treated rat showing considerable amount of glycogen (arrows) (PAS reaction, Scale bar = 50 mm).
Means which share the same superscript symbol(s) are not significantly different. No. of samples in each group is six. (Propolis: (PRO), curcumin: (CUR), Cypermethrin: (CYP), Cypermethrin plus Propolis (CYP + PRO) and Cypermethrin plus Curcumin (CYP + CUR). 3.2. Effects of Cypermethrin, Propolis and Curcumin on lipid peroxidation Fig. 2 indicated that lipid peroxidation in the form of malonaldhyde (MDA) significantly (P < 0.05) increased in CYP treated group as compared with control, PRO, CUR, PRO + CYP and CUR + CYP groups.
Values were expressed as mean ± standard error. Means which share the same superscript symbol(s) are not significantly different. No. of samples in each group is six. Propolis: (PRO), curcumin: (CUR), Cypermethrin: (CYP), Cypermethrin plus Propolis (CYP + PRO) and Cypermethrin plus Curcumin (CYP + CUR). 3.3. Effects of Cypermethrin, Propolis and Curcumin on enzymatic antioxidant enzyme CYP adminstration induced a significant (P < 0.05) decrease in SOD levels and GPx when compared to the control, PRO, CUR groups. However, administration of PRO or CUR to CYP-treated rats
Please cite this article in press as: Abdul-Hamid, M., et al. Cypermethrin-induced histopathological, ultrastructural and biochemical changes in liver of albino rats: The protective role of propolis and curcumin. Beni-Suef Univ. J. Basic Appl. Sci. (2017), http://dx.doi.org/10.1016/j.bjbas.2017.03.002
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Fig. 9. a–c: Photomicrograph of a section in the liver of control, PRO and CUR rats showing intensive amounts of protein in the cytoplasm of the hepatocytes (arrows) (d): Liver of CYP treated rat showing reduced amount of protein (arrow), (e&f): Liver of CYP plus PRO and CYP plus CUR treated rat showing increased amount of protein (arrows) (Bromophenol blue, Scale bar = 50 mm).
revealed marked amelioration in the altered level of SOD and GPx activities when compared with CYP-treated group (Fig. 3a&b). Values were expressed as mean ± standard error. Means which share the same superscript symbol(s) are not significantly different. No. of samples in each group is six. Propolis: (PRO), curcumin: (CUR), Cypermethrin: (CYP), Cypermethrin plus Propolis (CYP + PRO) and Cypermethrin plus Curcumin (CYP + CUR). The most biochemical parameters revealed that PRO is markedly effective than CUR in ameliorating the biochemical alterations.
3.4. Histopathological observations The examination of light microscope of the control group (Fig. 4a&b), PRO and CUR groups (Fig. 5a&b) showed normal structure of the liver, each lobule has a central vein and hepatic cords in radiating shape. The administration of CYP showed an obvious signs of hepatic alterations; these include congested blood vessels, mononuclear inflammatory cell infiltration and extensive vacuolar degeneration of hepatocytes (Fig. 6a). Congested portal vein with thickened wall and proliferated bile duct were also detected (Fig. 6b). Congested blood vessel and fibrotic tissues were also noticed (Fig. 6c&d). Moreover, hyperemic sinusoids and oedema
Please cite this article in press as: Abdul-Hamid, M., et al. Cypermethrin-induced histopathological, ultrastructural and biochemical changes in liver of albino rats: The protective role of propolis and curcumin. Beni-Suef Univ. J. Basic Appl. Sci. (2017), http://dx.doi.org/10.1016/j.bjbas.2017.03.002
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Fig. 10. a–c: Photomicrograph of a section in the liver of control, PRO and CUR rats showing intensive amount of DNA in the nuclei of the hepatocytes (arrows) (d): Liver of CYP treated rat showing decreased DNA content (arrows) (e&f): Liver of CYP plus PRO and CYP plus CUR treated rat showing almost normal DNA content (Feulgen, Scale bar = 20mm).
(Fig. 6e), vacuolar degeneration of hepatocytes and fatty changes were seen (Fig. 6f). The liver sections of group treated with CYP plus PRO or CUR revealed marked recovery and restoration of almost normal hepatic configuration. (Fig. 7a&b).
3.5. Histochemical observations A strong PAS-positive reaction was showed in the hepatocytes of the control group, PRO and CUR- treated rats groups indicating the presence of large amount of glycogen (Fig. 8a–c). On the other hand, marked reduction of PAS reaction was observed in CYP treated group (Fig. 8d). However, after treatment with PRO and CUR,
the hepatocytes revealed an obvious increase of glycogen which approximated that of the control liver (Fig. 8e&f). Liver sections of control, PRO and CUR- treated groups stained with bromophenol blue revealed a dense blue staining. The proteins appeared in the form of bluish granules of various sizes randomly distributed in the ground cytoplasm of the hepatic cells (Fig. 9a–c). CYP-treated group showed marked depletion of proteins intensity in the cytoplasm of hepatocytes (Fig. 9d). Intake of PRO plus CYP and CUR plus CYP increased the intensity of blue color of protein than that of CYP treated group (Fig. 9e&f) respectively. Using Feulgen reaction, the hepatocytes of control group, PRO and CUR treated rats showed marked DNA amount in their nuclei
Please cite this article in press as: Abdul-Hamid, M., et al. Cypermethrin-induced histopathological, ultrastructural and biochemical changes in liver of albino rats: The protective role of propolis and curcumin. Beni-Suef Univ. J. Basic Appl. Sci. (2017), http://dx.doi.org/10.1016/j.bjbas.2017.03.002
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Fig. 11. a&b: Electron micrographs of hepatocytes of the control group showing normal nucleus (N), nucleolus (NU), mitochondria (M), rough endoplasmic reticulum (RER) and glycogen granules (G). (c): Normal bile ducts (BD). (d): Normal Kupffer cell with nucleus (N) and lysosome (L). Scale bar = 2mm.
(Fig. 10a–c). In contrast, CYP-treated rats revealed severe decrease of DNA amount in the nuclei of the liver cells. Few cells appeared nucleated, while others showed vacuolated nuclei. (Fig. 10d). On the other hand, administration of PRO to CYP and CUR to CYP groups caused marked increase of DNA amount in the nuclei of the hepatocytes which approximately reached that of the control rats (Fig. 10e&f). 3.6. Ultrastructural observations Ultrastructural examination of liver sections of control rats displayed normal structure. The cytoplasm of the hepatocytes contains numerous mitochondria. They are round in shape with well-developed cristae. The rough endoplasmic reticulum consists
of closely pack parallel and flattened cisternae. The nuclei appear spherical with normal pattern of chromatin distribution. Numerous electron-dense glycogen rosettes were clearly detected. The nucleus of the hepatocytes appears spherical with a distinct nuclear envelope and one or more prominent nucleoli. The nucleoplasm showed a unique distribution of a small amount of heterochromatin at the peripheral regions and a central large amount of euchromatin (Fig. 11a&b). Normal bile duct with normal structure of microvilli was seen (Fig. 11c). Kupffer cell also appeared with normal nucleus, lysosomes and rough endoplasmic reticulum (Fig. 11d). However, marked ultrastructural changes were revealed in CYP-treated rats. In some hepatocytes, the mitochondria appeared swollen, vacuolated with destructed cristae, in addition to the presence of cytoplasmic vacuolations (Fig. 12a). Other cells
Please cite this article in press as: Abdul-Hamid, M., et al. Cypermethrin-induced histopathological, ultrastructural and biochemical changes in liver of albino rats: The protective role of propolis and curcumin. Beni-Suef Univ. J. Basic Appl. Sci. (2017), http://dx.doi.org/10.1016/j.bjbas.2017.03.002
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Fig. 12. a: Electron micrographs of hepatocytes of rat treated with CYP showing vacuolated mitochondria (M), vacuoles (V). (b): dissolution of some hepatocytes (D) and diltation of bile duct with destructed microvilli (BD). (c): Kupffer cell of CYP- treated rat showing elongated nucleus (N), cytoplasmic vacuolation (V), endocytic vesicles (EV) and lysosomes (L). (d): large fat droplets (FD) and many cytoplasmic vacuoles (V). Scale bar = 2mm.
showed electron-dense mitochondria, dissolution of some hepatocytes, completely lysed nucleus and dilatation of bile duct with destructed microvilli (Fig. 12b). Kupffer cell showed elongated nucleus, endocytic vesicles and marked increase of lysosomes number (Fig. 12c). In other hepatocytes, cytoplasmic vacuoles and fat droplets were also observed (Fig. 12d). On the other hand, the liver treated with PRO plus CYP revealed that the hepatocytes restored their normal structure (Fig. 13a) and bile duct also was regained to its normal structure (Fig. 13b). Kupffer cells displayed their normal structure except the presence of some fat droplets (Fig. 13c). The liver section treated with CUR plus CYP showed marked recovery of the cytoplasmic organelles, and hepatic cells except some vacuoles (Fig. 14a) and bile duct also restored its nor-
mal structure (Fig. 14b). Furthermore, Kupffer cells displayed their normal structure but some vacuoles were still observed (Fig. 14c).
4. Discussion The mode of action of CYP can be expected to have two ways: it may generate ROS that induce oxidative stress or it may accumulate in cell membrane and disturb membrane structure due to its hydrophobic nature (Saxena and Saxena, 2010). In the current study, CYP administration increased ALT, AST, ALP and MDA. The increasing of these parameters act as indicator to liver toxicity, oxidative stress and LPO (Bhushan et al., 2013; Soliman et al.,
Please cite this article in press as: Abdul-Hamid, M., et al. Cypermethrin-induced histopathological, ultrastructural and biochemical changes in liver of albino rats: The protective role of propolis and curcumin. Beni-Suef Univ. J. Basic Appl. Sci. (2017), http://dx.doi.org/10.1016/j.bjbas.2017.03.002
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Fig. 13. a–c: Electron micrographs of rat treated with CYP and PRO: (a): showing marked recovery of the hepatocyte nucleus (N) and cytoplasmic organelles; mitochondria (M), rough endoplasmic reticulum (RER). (b): showing bile duct (arrow). (c): Kupffer cell with normal structure and normal nucleus (N) and fat droplet (FD). Scale bar = 2mm.
2014). Oxidative stress contributes to initiation and progression of liver injury, so it is considered a main pathological mechanism (Li et al., 2015). Also, CYP has been demonstrated to cause a significant decrease in the activities of SOD and GPx in the rats of the present study. In consistent with these results, Gomaa et al. (2011) found that CYP induced a significant increase in the mean values of AST, ALT, ALP, MDA levels and a significant decrease in the mean values of antioxidant enzyme activities (CAT, SOD and GPx) in rats liver. The present histopathological observations of liver sections of rats in the current study showed congested blood vessels, mononuclear inflammatory cell infiltration, congested portal vein with thickened wall and proliferated bile duct was also detected. Moreover, hyperemic sinusoids, vacuolar degeneration of hepatocytes and oedema. In addition, many hepatocytes underwent fatty change and fibrosis. These results were lined with Soliman et al. (2014) and Mossa et al. (2015) who showed liver damage during CYP toxicity in rats. Also, Grewal et al. (2010) observed marked degenerative changes of hepatocytes and congestion after administration of CYP at 14.5 mg/kg for 30 consecutive days. These histopathological alterations in the hepatocytes because CYP has inhibitory effect on total adenine triphosphate activity in the liver, which may disorganize active transport of Na+, K+ and Ca2+ ions, causing hepatocytes injuring (Khan et al., 2009). Concerning the present ultrastructural study of hepatocytes, nuclear changes, cytoplasmic vacuolation, damage mitochondria and breakdown of RER were observed. In this respect, Aldana et al. (1998) reported that after CYP exposure, hepatocytes had
an ovoid nucleus with slightly irregular profile, appearing of many lipid droplets in cytoplasm and a lot of swelling mitochondria. Antioxidants may play an important role to protect the cells against oxidative stress and damage caused by free radicals (Bouayed and Bohn, 2010). El-Masry et al. (2011) showed that PRO has twelve different flavonoids, acacetin, pinocembrin, chrysin, rutin, catechin, naringenin, galangin, luteolin, a pigenin, kaemferol, myricetin and quercetin, two phenolic acids, cinnamic and caffeic acid are present in PRO. PRO is an excellent source of essential elements, including Mg2+, Zn2+, Fe2+, Cu2 +, Ca2+, Ni2+ and Mn2+ (Haro et al., 2002), which might also be responsible for reactivating antioxidant enzymes by providing optimum trace elements. The first mechanism of the effect of PRO may require the scavenging of free radicals that induce LPO. The second mechanism may inhibit xanthine oxidase, which is known to cause generation of free radicals (Kanbura et al., 2009). In the present study, PRO co-administration with CYP induced a significant decrease in AST, ALT, ALP, MDA levels and a significant increase in the mean values of antioxidant enzyme activities (CAT, SOD and GPx) as compared with CYP treated group. This result is in agreement with (Eraslan et al., 2008). PRO induced a reduction in the increased activity of AST and ALT in the serum of rats treated with doxorubicin (Omar et al., 2016). The present histopathological examination revealed marked recovery and restoration of almost normal hepatic cells by PRO therapy in contrast to CYP intoxicated animals. The normal hepatocytes were observed in most areas when propolis was co-administered with CYP, however, mild sinusoidal dilation with inflammatory cell infiltration was also present
Please cite this article in press as: Abdul-Hamid, M., et al. Cypermethrin-induced histopathological, ultrastructural and biochemical changes in liver of albino rats: The protective role of propolis and curcumin. Beni-Suef Univ. J. Basic Appl. Sci. (2017), http://dx.doi.org/10.1016/j.bjbas.2017.03.002
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Fig. 14. a–c: Electron micrographs of rat treated with CYP and CUR showing (a): marked recovery of the hepatocyte nucleus (N) and cytoplasmic organelles; mitochondria (M), rough endoplasmic reticulum (RER) and vacuoles (V). (b): bile duct (arrow). (c): Kupffer cell with normal structure and normal nucleus (N) and vacuoles (V). Scale bar = 2mm.
(Gomaa et al., 2011). Electron microscopic examination of the present study displayed a better preservation of the hepatic cells of rat administrated PRO with CYP compared to those treated with CYP alone. CUR has protective effects against oxidative damage and has antioxidant and anticonvulsant properties exerting powerful ROS scavenging effects and increased intracellular glutathione concentration, thereby protecting against LPO (Aboul Ezz et al., 2011; Du et al., 2012). Co-administration of CUR with CYP revealed a decrease in the concentrations of ALT, AST, ALP and MDA as compared to CYP alone treated rats in this study. These findings are in agreement with Sankar et al. (2012) and Hismiogullari et al. (2014). In addition, Salahshoor et al. (2016) exhibit that the mean values of ALT, AST, and ALP enzymes decreased significantly in CUR and CUR plus nicotine in all groups compared to the nicotine group. In the current study, the liver of CUR plus CYP group displayed a relatively normal architecture. Also, Sankar et al. (2012) showed a reduction in the degenerative changes in liver of CUR plus CYP group. In conclusion, the present study clarified that administration of PRO and CUR are beneficial to ameliorate the biochemical, histopathological and ultrastructural alterations in liver of rats induced by CYP due to their antioxidants effects. The protective effects of PRO are markedly effective than CUR against CYP.
Conflict of interest statement The authors declare that there are no conflicts of interest.
Funding This research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors.
References Aboul Ezz, H.S., Khadrawy, Y.A., Noor, N.A., 2011. The neuroprotective effect of curcumin and Nigella sativa oil against oxidative stress in the pilocarpine model of epilepsy: a comparison with valproate. Neurochem. Res. 36, 2195–2204. Aldana, L., Gonz0 alez de Mej0 ıa, E., Craigmill, A., Tsutsumi, V., Armendariz-Borunda, J., Panduro, A., Rinc0 on, A.R., 1998. Cypermethrin increases apo A-1 and apo B mRNA but not hyperlipidemia in rats. Toxicol. Lett. 16, 31–39. Bancroft, J.D., Gamble, M., 2002. Theory and Practice of Histological Techniques. Churichill Livingstone, Edinburgh, London. Bankova, V.S., De Castro, S.L., Marcucci, M., 2000. Propolis: recent advances in research on chemistry and plant origin. Apidologie 31, 3–15. Bankova, V., Popova, M., Trusheva, B., 2016. New emerging fields of application of propolis. Maced. J. Chem. Chem. Eng. 35 (1), 1–11. Bhushan, B., Saxena, P.N., Saxena, N., 2013. Biochemical and histological changes in rat liver caused by cypermethrin and beta-cyfluthrin. Arh. Hig. Rada Toksikol. 64, 57–67. Bouayed, J., Bohn, T., 2010. Exogenous antioxidants – double-edged swords in cellular redox state: health beneficial effects at physiologic doses versus deleterious effects at high doses. Oxid. Med. Cell Longev. 3 (4), 228–237. Bozzola, J.J., Russell, L.D., 1999. Electron Microscopy: Principles and Techniques for Biologists. Jones and Bartlett Publishes, Sudbury, MA, USA. Du, P., Tang, H.Y., Li, X., Lin, H.J., Peng, W.F., Ma, Y., Fan, W., Wang, X., 2012. Anticonvulsive and antioxidant effects of curcumin on pilocarpine-induced seizures in rats. Chin. Med. J. 125, 1975–1979. El-Masry, T.A., Emara, A.M., El-Shitany, N.A., 2011. Possible protective effect of propolis against lead induced neurotoxicity in animal model. J. Evol. Biol. Res. 3 (1), 4–11.
Please cite this article in press as: Abdul-Hamid, M., et al. Cypermethrin-induced histopathological, ultrastructural and biochemical changes in liver of albino rats: The protective role of propolis and curcumin. Beni-Suef Univ. J. Basic Appl. Sci. (2017), http://dx.doi.org/10.1016/j.bjbas.2017.03.002
14
M. Abdul-Hamid et al. / Beni-Suef Univ. J. Basic Appl. Sci. xxx (2017) xxx–xxx
Elsayed, A.S.I., 2016. The curcumin as an antioxidant natural herb, with emphasize on its effects against some diseases. Int. J. App. Biol. Pharmacol. Technol. 7 (1), 26–40. EL-Shemi A G, Abou El-Ella G A. Hepatic and neural toxicity and tissue residue of cypermethrin in male Somali sheep (Berbera blackhead). Assiut Vet. Med. J. 2015; 61 (146):52–59. Eraslan G, Kanbur M, Silici S, Altinordulu S, Karabacak M. Effects of cypermethrin on some biochemical changes in rats: the protective role of propolis, Exp. Anim. 2008; 57 453–460. Ferrari, C.K.B., 2000. Free radicals, lipid peroxidation and antioxidants in apoptosis: implications in cancer, cardiovascular and neurological diseases. Biologia 55, 581–590. Feulgen, R., Rossenenbeck, H., 1924. Mikroskopisch chemischer nach weis einen nucleinsaure von typus der thymonucleinsaure und de darauf berhende elective farbung von Zelkernen in microskopischen preparaten. Zeitschrift Physiol. Chem. 135, 203. Gomaa M S, Abd Alla M A, Sameer M M. The possible protective effect of propolis (Bee glue) on cypermethrin-induced hepatotoxicity in adult albino rats. Mansoura J. Forensic Med. Clin. Toxicol. 2011; Vol. XIX, No. 1: 17–32. Grewal, K.K., Sandhu, G.S., RanjitKaur Brar, R.S., Sandhu, H.S., 2010. Toxic impacts of cypermethrin on behavior and histology of certain tissues of albino rats. Toxicol. Int. 17, 94–98. Haro, A., Lopez-Aliaga, I., Lisbona, F., Barrionuevo, M., Alferez, M.J.M., Campos, M.S., 2002. Beneficial effect of pollen and/or propolis on the metabolism of iron, calcium, phosphorus, and magnesium in rats with nutritional ferropenic anemia. J. Agric. Food Chem. 48, 5715–5722. Huang, F., Liu, Q., Xie, S., Xu, J., Huang, B., Wu, Y., Xia, D., 2016. Cypermethrin induces macrophages death through cell cycle arrest and oxidative stressmediated JNK/ERK signaling regulated apoptosis. Int. J. Mol. Sci. 17, 885. Hismiogullari, S.E., Hismiogullari, A.A., Sunay, F.B., Paksoy, S., Can, M., Aksit, H., Karaca, O., Yavuz, O., 2014. The protective effect of curcumin on carbon tetrachloride induced liver damage. Revue Méd. Vét. 165 (7–8), 194–200. Hotchkiss, R.D.A., 1948. Microchemical reaction resulting in the staining of polysaccharide structures in fixed tissue preparations. Arch. Biochem. 16, 131–141. Hussien, H.M., Abdou, H.M., Yousef, M.I., 2013. Cypermethrin induced damage in genomic DNA and histopathological changes in brain and haematotoxicity in rats: the protective effect of sesame oil. Brain Res. Bull. 92, 76–83. IBM crop (2011): IBM SPSS Statistic for windows, Version 20.0 Armonk, N.Y., IBM Crop. Inayat, Q., Ilahi, M., Khan, J., 2007. A morphometric and histological study of the kidney of mice after dermal application of cypermethrin. J. Pak. Med. Assoc. 57, 587–591. Kanbura, M., Eraslan, G., Silici, S., 2009. Antioxidant effect of propolis against exposure to propetamphos in rats. Ecotoxicol. Environ. Safety 72, 909–915. Khan, A., Faridi, H.A.M., Ali, M., Khan, M.Z., Siddique, M., Hussain, I., Ahmad, M., 2009. Effects of cypermethrin on some clinico-hematobiochemical and pathological parameters in male dwarf goats (Capra hircus). Exp. Toxicol. Pathol. 61, 151–160. Li, S., Tan, H., Wang, N., Zhang, Z., Lao, L., Wong, C., Feng, Y., 2015. The role of oxidative stress and antioxidants in liver diseases. Int. J. Mol. Sci. 16 (11), 26087–26124. Majeed M, Nagabhushanam K, Choudhury AK. Antioxidants: Balancing the Good, the Bad and the Ugly. Nutr. Food Technol. 2016; 2(2): doi http://dx.doi. org/ 10.16966/2470-6086.123. Manna, S., Bhattacharyya, D., Mandal, T.K., Das, S., 2004. Repeated dose toxicity of alfa-cypermethrin in rats. J. Vet. Sci. 5, 241–245. Marklund, S., Marklund, G., 1974. Involvement of superoxide anion radical in the autooxidation of pyrogallol and convenient assay for superoxide dismutase. Eur. J. Biochem. 47, 469–474.
Matkovics, B., Szabo, L., 1998. Varga IS Determination of enzyme activities in lipid peroxidation and glutathione pathways (in Hungarian). Laboratoriumi Diagnosztika 15, 248–249. Mazia, D., Brewer, P.A., Alfert, M., 1953. The cytochemical staining and measurement of protein with mercuric bromophenol blue. Biol. Bull. 104, 57– 67. Moran, M.P., Fernandez, J.M., Tortosa, C.R., Tortosa, M.C.R., 2016. Curcumin and health. Molecules 21, 264. Mossa, A.H., Heikal, T.M., Belaiba, M., Raoelison, E.G., Ferhout, H., Bouajila, J., 2015. Antioxidant activity and hepatoprotective potential of Cedrelopsis grevei on cypermethrin induced oxidative stress and liver damage in male mice. BMC Complement. Alternat. Med. 15, 251. Omar, N.A.A., Allithy, A.N.A., Baghdadi, H., Zolaly, M., Abdel-haleem, M., Helmy, M. M., Ayat, M.M.A., El Sayed, S.M., 2016. Hepatoprotective effects exerted by propolis against doxorubicin-induced rat liver toxicity: a biochemical and histopathological study. Am. J. Cancer Prevention 4 (3), 36–40. Pattanayak, R., Basak, P., Sen, S., Bhattacharyya, M., 2016. Interaction of kras gquadruplex with natural polyphenols: a spectroscopic analysis with molecular modeling. Int. J. Biol. Macromol. 89, 228–237. Perrone, D., Ardito, F., Giannatempo, G., Dioguardi, M., Troiano, G., Lo Russo, L., Laino, L., Lo, Muzio.L., 2015. Biological and therapeutic activities, and anticancer properties of curcumin. Exp. Ther. Med. 10, 1615–1623. Rosalki, S.B., Foo, A.Y., Burlina, A., Prellwitz, W., Stieber, P., Neumeier, D., Klein, G., Poppe, W.A., Bodenmüller, H., 1993. Multicenter evaluation of Iso-ALP test kit for measurement of bone alkaline phosphatase activity in serum and plasma. Clin. Chem. 39 (4), 648–652. Saka, W.A., Akhigbe, R.E., Azeez, O.M., Babatunde, T.R., 2011. Effect of pyrethroid exposure on haematological and haemostatic profiles in rats. Pak. J. Biol. Sci. 14, 1024–1027. Salahshoor, M., Mohamadian, S., Kakabaraei, S., Roshankhah, S., Jalili, C., 2016. Curcumin improves liver damage in male mice exposed to nicotine. J. Traditional Complement. Med. 6 (2), 176–183. Saleh, E.M., 2012. Antioxidant effect of aqueous extract of propolis on hepatotoxicity induced by octylphenol in male rats. Acta Toxicol. Argent. 20, 68–81. Sankar, P., Telang, A.G., Manimaran, A., 2010. Curcumin protects against cypermethrin induced genotoxicity in rats. Environ. Toxicol. Pharmacol. 30, 289–291. Sankar, P., Telang, A.G., Manimaran, A., 2011. Effect of piperine on cypermethrininduced oxidative damage in rats. J. Veterinar. Sci. Technol. 2, 105. Sankar, P., Telang, A.G., Manimaran, A., 2012. Protective effect of curcumin on cypermethrin-induced oxidative stress in Wistar rats. Exp. Toxicol. Pathol. 64 (5), 487–493. Saxena, P., Saxena, A.K., 2010. Cypermethrin induced biochemical alterations in the blood of albino rats. Jordan J. Biol. Sci. 3, 111–114. Schumann, G., Klauke, R., 2003. New IFCC reference procedures for the determination of catalytic activity concentrations of five enzymes in serum: preliminary upper reference limits obtained in hospitalized subjects. Clin. Chim. Acta 327 (1–2), 69–79. Soliman M M, Attia H F, Abou El-Ella GA. Genetic and histopathological alterations induced by cypermethrin in rat kidney and liver: Protection by sesame oil. Int. J. Immunopathol. Pharmacol. 2014; 1–13. Wenger C, Kaplan A, Rubaltelli FF, Hammerman C. Alkaline phosphatase. Clin. Chem. The C.V. Mosby Co., St. Louis. Toronto. Princeton. 1984; 1094–1098. Yagi, K., 1987. Lipid peroxides and human diseases. Chem. Phys. Lipids 45, 337–351. Yousef, M.I., Salama, A.F., 2009. Propolis protection from reproductive toxicity caused by aluminium chloride in male rats. Food Chem. Toxicol. 47, 1168–1175.
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Cypermethrin-induced histopathological, ultrastructural and biochemical changes in liver of albino rats: The protective role of propolis and curcumin Manal Abdul-Hamid a,⇑, Nadia Moustafa a, Abd El Mawgoud Abd Alla Asran b, Lila Mowafy b a b
Department of Zoology, Faculty of Science, Beni-Suef University, Beni-Suef, Egypt Department of Plant Protection, Harmful Animals, Research Institute, Agricultural Research Center, Cairo, Egypt
a r t i c l e
i n f o
Article history: Received 26 November 2016 Received in revised form 3 March 2017 Accepted 3 March 2017 Available online xxxx Keywords: Cypermethrin Oxidative stress Histopathology Ultrastructure Propolis and curcumin
a b s t r a c t Cypermethrin (CYP), an insecticide belongs to a synthetic pyrethroid, is used for agriculture and household applications. The present study aimed to examine the toxic effects of CYP on rat liver and to clarify the hepatoprotective effects of propolis (PRO) and curcumin (CUR) against CYP. This study was assessed in male albino rats, each weighting (120–150 g). The rats were equally divided into six groups as follow; the 1st control group, 2nd PRO group (100 mg/kg/day) and 3rd CUR group (100 mg/kg/day). While 4th, 5th and 6th groups were orally treated with CYP (30 mg/kg/day), CYP plus PRO and CYP plus CUR, respectively for 28 days. The present study revealed that CYP-induced significant increase in hepatic markers enzymes (ALT, AST and ALP) and elevation in MDA concomitant with significant decrease of SOD and GPx levels. Histological and histochemical results revealed extensive vacuolar degeneration of hepatocytes, fatty change, blood vessel congestion and fibrosis in the liver of CYP- treated group and depletion of glycogen, protein and DNA. Moreover, ultrastructural observations showed vacuolation, damage of mitochondria and nuclear changes. On the other hand, treatment with PRO and CUR led to an obvious improvement of the injured liver tissues and ameliorating the damaging effects of CYP. In conclusion, PRO is markedly effective than CUR in protecting rats against CYP-induced histopathological, ultrastructural and biochemical changes. This protection may be due to its antioxidant properties and scavenging abilities against active free radicals. Ó 2017 Published by Elsevier B.V. on behalf of Beni-Suef University. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1. Introduction Pyrethroids are human- made of pyrethrins. Pyrethroids have two types which differentiate with their chemical structure and exposure symptoms (Saka et al., 2011). CYP, type II synthetic pyrethroid, have potent insecticidal property. It plays a role in agriculture, protection of food stuff, disease vector control and home pest control (Sankar et al., 2011). CYP is spreadly used as insecticide in developing countries to control many species of insects. It is reported that human exposure to CYP mainly occurred during application or from pyrethroids residues such as those detected in fruits, vegetables, cow’s milk and bread (Sankar et al., 2010). CYP is a hydrophobic molecule which can easily pass through the cell membrane and disturb its structure and cause leakage of cytoplasmic enzymes (Manna et al., 2004; Hussien et al., 2013). CYP
⇑ Corresponding author. E-mail addresses: [email protected], [email protected] (M. Abdul-Hamid).
produced ROS and damaged DNA directly proportional to the dose (Huang et al., 2016). There is a direct reaction between ROS and cellular biomolecules. It damages lipids, proteins and DNA in cells causing cell death (Ferrari, 2000). Sankar et al., 2011 realized that imbalance between amount of free radicals generated and antioxidant defenses in the body are one of the reasons for CYP toxicity. EL-Shemi et al. (2015) showed that the residue levels of CYP were in kidneys, spleen, muscles and liver in the descending order at levels ranging from 12.2 to 156.1 ng/g for CYP. The indicators of overall health status of an individual especially hepatocyte toxicity and related stress are serum enzyme levels (Khan et al., 2009). Antioxidants are the molecules that react with ROS to delay their function and to neutralize them, so they reduce oxidative stress and protect us from diseases state. (Majeed et al., 2016). Propolis (PRO), bee glue, is a sticky resinous substance applied by honey bees Apis mellifera L. as a building material in their hives and as a defensive substance against infections (Bankova et al., 2016). PRO is considered as a remedy in traditional medicine systems all over the world, mainly to treat burns, wounds, stomach
http://dx.doi.org/10.1016/j.bjbas.2017.03.002 2314-8535/Ó 2017 Published by Elsevier B.V. on behalf of Beni-Suef University. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Please cite this article in press as: Abdul-Hamid, M., et al. Cypermethrin-induced histopathological, ultrastructural and biochemical changes in liver of albino rats: The protective role of propolis and curcumin. Beni-Suef Univ. J. Basic Appl. Sci. (2017), http://dx.doi.org/10.1016/j.bjbas.2017.03.002
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Fig. 1. Activities of serum (a): ALT (b): AST and (c): ALP (U/L) of CYP, PRO, CUR, CYP plus PRO and CYP plus CUR in serum of male rats.
ulcer and sore throat. The chemical composition of PRO includes more than 200 compounds such as flavonoids, phenolics, vitamins, alcohols, aromatic acids and their derivatives, esters, minerals and trace elements (Bankova et al., 2000). PRO antioxidant activity is mainly resulted from its flavonoid content, which has the ability
of scavenging free radicals and thereby preservation against LPO (Yousef and Salama, 2009). Curcumin (CUR), is a polyphenolic compound, derives from turmeric (Curcuma longa). It is considerrd as a source for food additive or dietary pigment and in traditional medicine (Perrone et al.,
Please cite this article in press as: Abdul-Hamid, M., et al. Cypermethrin-induced histopathological, ultrastructural and biochemical changes in liver of albino rats: The protective role of propolis and curcumin. Beni-Suef Univ. J. Basic Appl. Sci. (2017), http://dx.doi.org/10.1016/j.bjbas.2017.03.002
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Fig. 2. Concentration of hepatic malondialdehyde (MDA) of CYP, PRO, CUR, CYP plus PRO and CYP plus CUR in liver homogenate of male rats.
(a):
(b):
Fig. 3. Concentrations of (a): hepatic superoxide dismutase (SOD) and (b): Glutathione peroxidase (GPx) of CYP, PRO, CUR, CYP plus PRO and CYP plus CUR in liver homogenate of male rats.
Please cite this article in press as: Abdul-Hamid, M., et al. Cypermethrin-induced histopathological, ultrastructural and biochemical changes in liver of albino rats: The protective role of propolis and curcumin. Beni-Suef Univ. J. Basic Appl. Sci. (2017), http://dx.doi.org/10.1016/j.bjbas.2017.03.002
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Fig. 4. a&b: Photomicrograph of a section in the liver of control rats showing radially arranged polyhedral hepatocytes forming cords around the central vein (CV) and sinusoids (S) (H&E, Scale bar = 100, 50 mm respectivelly).
Fig. 5. a&b: Photomicrograph of a liver section of PRO and CUR groups showing normal structure of liver with a central vein (CV) (H&E, Scale bar = 100 mm).
2015; Pattanayak et al., 2016). Elsayed (2016) clarified CUR as an antioxidant natural herb, a potent scavenger of many free radicals, as anti-carcinogenic, anti-tumoral, anti-inflammatory, antifungal, anti-viral, anti-parasitic, anti-mutagen, anti-infectious and antihepatotoxic natural substance. Moran et al. (2016) pointed that CUR is a powerful scavenger of a wide range of ROS and RNS including O2, H2O2 and NO and also has enhanced activities on different antioxidant systems such as CAT, SOD, GPx, GSH reduced glutathione and heme oxygenase-1. The present investigation aims to evaluate the protective effect of PRO and CUR against toxicity of CYP. Histopathological and ultrastructural changes of liver, biochemical as oxidative stress will be studied to evaluate the effects of treatment with PRO and CUR in the various control and tested groups. 2. Materials and methods 2.1. Chemicals Cypermethrin (CYP) was purchased from the pesticides store in Beni-Suef, Egypt. Propolis (PRO) was purchased from Sigma Pharmaceutical Industries (Cairo, Egypt). It is found in the form of Biopropolis capsules, each one containing 400 mg. Curcumin (CUR) was obtained from Merk Company, Germany.
2.2. Animals and experimental design 42 adult of laboratory males albino rats (Rattus norvegicus) of nearly the same age, weighing (120–150 g) bought from the Ophthalmology Research Institute, Giza, Egypt. Rats were kept under standard management conditions of temperature (25 °C). The animals were fed a standard commercials diet (ATMID Company, Giza, Egypt) and tap water ad libitum. All the rats were acclimatized for at least 15 days before the beginning of the experiment. In this study, animal care was carried out by following the European Community Directive (86/609/EEC) and national rules, this in accordance with the 8th edition of the NIH Guidelines for the care and use of Laboratory Animals. This was approved by the committee of the Zoology Department, Beni-Seuf University, Egypt. Rats were equally divided into six groups containing seven rats as follow: Group 1 serving as an untreated control group under the same laboratory conditions. Group 2 treated with PRO at a dose of 100 mg/kg/day according to Saleh (2012). Group 3 treated with CUR at a dose of 100 mg/kg/day according to Sankar et al. (2012) Group 4 received CYP at a dose of 30 mg/kg/day according to Inayat and Ilahi (2007).
Please cite this article in press as: Abdul-Hamid, M., et al. Cypermethrin-induced histopathological, ultrastructural and biochemical changes in liver of albino rats: The protective role of propolis and curcumin. Beni-Suef Univ. J. Basic Appl. Sci. (2017), http://dx.doi.org/10.1016/j.bjbas.2017.03.002
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Fig. 6. Photomicrograph of a cross section in liver of rat treated with CYP showing (a): Inflammatory cells (iF), extensive vacuolar degeneration of hepatocytes (short arrows), hyperemic sinusoid (arrow head) and congested blood vessel (long arrow) (H&E, Scale bar = 50 mm). b): Congested portal vein (CB) with thickened wall of portal vein (long arrow) and proliferating bile duct (short arrows) (H&E, Scale bar = 50 mm). c&d): The congestion of central vein (CB) and fibrosis (F) (H&E, Scale bar = 200 mm). (e): Oedema (O) and dissolution (d) (H&E, Scale bar = 50 mm). (f): Hepatocytes that undergo fatty change (F) and pyknotic nuclei (arrows) (H&E, Scale bar = 50 mm).
Group 5 given CYP (30 mg/kg/day) parallel with PRO at 100 mg/ kg/day. Group 6 given CYP (30 mg/kg/day) parallel with CUR at 100 mg/ kg/day. Each of CYP, PRO and CUR was dissolved in distilled water and was given by gastric gavage daily for 28 days. At the end of experiment, rats were starved for 12 h and then sacrificed by anesthesia under light diethyl ether and blood samples were collected and allowed to coagulate at room temperature then centrifuged at 3000 r.p.m. for 30 min. The clear non-
haemolyzed supernatant sera was quickly removed and kept at 20 °C for subsequent biochemical analysis. Liver homogenate, obtained by grinding a small piece of freshly excised tissue in 10 volumes of 0.9% NaCl (10% w/v) using Teflon homogenizer (Glas-Col, Terre Haute, USA). The supernatants were kept at -20°C till use in the determination of LPO, SOD and GPx. Symptoms of CYP toxicity in laboratory animals include burrowing, salivation, tremors, writhing and seizures. Also, there were mortality; two rats died from eight rats in CYPtreated group.
Please cite this article in press as: Abdul-Hamid, M., et al. Cypermethrin-induced histopathological, ultrastructural and biochemical changes in liver of albino rats: The protective role of propolis and curcumin. Beni-Suef Univ. J. Basic Appl. Sci. (2017), http://dx.doi.org/10.1016/j.bjbas.2017.03.002
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Fig. 7. a&b: (a): Photomicrograph of a liver section of rat treated with CYP plus PRO group showing great recovery and nearly normal structure of the liver with nearly normal central vein (CV) and hepatocytes (H&E, Scale bar = 100 mm). (b): Photomicrograph of a section in the liver of CYP plus CUR group showing approximate regain of normal appearance of hepatocytes and central vein (CV) (H&E, Scale bar = 100 mm).
2.3. The assay of serum biochemical Alanine amino transferase (ALT) (EC 2.6.1.2) and aspartate amino transferase (AST) (EC 2.6.1.1) activity was according to the method of Schumann and Klauke (2003) using reagent kits purchased from ‘‘Human Diagnostics” (Germany). The determination of alkaline phosphatase (ALP) (EC 3.1.3.1) was spectrophotometrically according to the method of Wenger et al. (1984) and Rosalki et al. (1993) using reagent kits purchased from SPINREACT Company. 2.4. Assessment of oxidative stress and antioxidant enzyme activity The activity of lipid peroxidation (LPO) in liver homogenate was determined according to the method of Yagi (1987). The activity of superoxide dismutase (SOD) (EC 1.15.1.1) in liver homogenate was assayed according to the method of Marklund and Marklund (1974). Glutathione peroxidase (GPx) (EC 1.11.1.9) activity in liver homogenate was assayed according to the method suggested by Matkovics and Szabo (1998).
phate buffer, then followed by post fixation in 1% osmium tetroxide. The specimens were then dehydrated in a series of alcohols, cleared in propylene oxide and finally embedded in Epon epoxy resin. After that, the blocks were trimmed and sectioned with glass knives by an ultra-microtome. Semithin sections (1 mm) were stained with toluidine blue and examined on light microscope to select the suitable area for the ultrathin sections. Ultrathin sections (70–90 nm) were cut on the same ultramicrotome and stained with uranyl acetate and lead citrate (Bozzola and Russell, 1999). Examination of the stained sections was carried out by Joel CX 100 transmission electron microscope operated at an accelerating voltage of 60 kV.
2.8. Statistical analysis The data have been expressed as mean ± SEM. Package for the Social Sciences (SPSS for WINDOWS, version 20.0; SPSS Inc, Chicago) (IBM crop, 2011) was used for the statistical analysis of data. Data were analyzed by one way ANOVA test followed by Duncan’s multiple range tests post hoc analysis values. A value of P < 0.05 was considered statistically significant.
2.5. Histological preparations After 28 days of treatment, the animals were anesthetized under light diethyl and dissected to get the liver. After fixation in10% neutral buffered formalin for 24 h, they were dehydrated through ascending series of ethyl alcohol, cleared in xylene and embedded in paraffin wax. 5 mm thick sections were stained with haematoxylin and eosin for histopathological studies (Bancroft and Gamble, 2002). 2.6. Histochemical preparations Liver sections of all the groups were stained with periodic acid schiff (PAS) reaction (Hotchkiss, 1948), mercuric bromophenol blue method (Mazia et al., 1953) and Feulgen method (Feulgen and Rossenenbeck, 1924) for demonstration of polysaccharides, total proteins and DNA contents respectively. 2.7. Ultrastructural preparations Small pieces of liver of all groups were immediately fixed in 3% glutraldehyde-formaldehyde at 4 °C for 18–24 h, rinsed in phos-
3. Results 3.1. Effects of Cypermethrin, Propolis and Curcumin on serum biomarkers The present study showed that CYP adminstration displayed a significant (P < 0.05) increase of serum ALT activity compared to normal control, PRO and CUR values after 28 days. Treatment with PRO or CUR in concomitant with CYP decrease ALT activity compared to the CYP-treated group (Fig. 1a). Also, CYP-treated rats significantly (P0.05) increased AST activity compared to the control, PRO, CUR groups values. Administration of PRO or CUR in concomitant with CYP decreased AST activity (Fig 1b). Moreover, there was no significant difference in ALP level between control group value and PRO, CUR groups but there is a significant (P < 0.05) increase in ALP activity in CYP group value after 28 days. Treatment of PRO or CUR with CYP decrease ALP activity compared to CYP-treated group (Fig. 1c). Values were expressed as mean ± standard error.
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Fig. 8. a–c: Photomicrograph of a section in the liver of control, PRO and CUR rats showing highly increased amounts of glycogen in hepatocytes (arrows) (d): Liver of CYP treated rat showing depletion of glycogen in hepatocytes (arrow), (e&f): Liver of CYP plus PRO and CYP plus CUR treated rat showing considerable amount of glycogen (arrows) (PAS reaction, Scale bar = 50 mm).
Means which share the same superscript symbol(s) are not significantly different. No. of samples in each group is six. (Propolis: (PRO), curcumin: (CUR), Cypermethrin: (CYP), Cypermethrin plus Propolis (CYP + PRO) and Cypermethrin plus Curcumin (CYP + CUR). 3.2. Effects of Cypermethrin, Propolis and Curcumin on lipid peroxidation Fig. 2 indicated that lipid peroxidation in the form of malonaldhyde (MDA) significantly (P < 0.05) increased in CYP treated group as compared with control, PRO, CUR, PRO + CYP and CUR + CYP groups.
Values were expressed as mean ± standard error. Means which share the same superscript symbol(s) are not significantly different. No. of samples in each group is six. Propolis: (PRO), curcumin: (CUR), Cypermethrin: (CYP), Cypermethrin plus Propolis (CYP + PRO) and Cypermethrin plus Curcumin (CYP + CUR). 3.3. Effects of Cypermethrin, Propolis and Curcumin on enzymatic antioxidant enzyme CYP adminstration induced a significant (P < 0.05) decrease in SOD levels and GPx when compared to the control, PRO, CUR groups. However, administration of PRO or CUR to CYP-treated rats
Please cite this article in press as: Abdul-Hamid, M., et al. Cypermethrin-induced histopathological, ultrastructural and biochemical changes in liver of albino rats: The protective role of propolis and curcumin. Beni-Suef Univ. J. Basic Appl. Sci. (2017), http://dx.doi.org/10.1016/j.bjbas.2017.03.002
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Fig. 9. a–c: Photomicrograph of a section in the liver of control, PRO and CUR rats showing intensive amounts of protein in the cytoplasm of the hepatocytes (arrows) (d): Liver of CYP treated rat showing reduced amount of protein (arrow), (e&f): Liver of CYP plus PRO and CYP plus CUR treated rat showing increased amount of protein (arrows) (Bromophenol blue, Scale bar = 50 mm).
revealed marked amelioration in the altered level of SOD and GPx activities when compared with CYP-treated group (Fig. 3a&b). Values were expressed as mean ± standard error. Means which share the same superscript symbol(s) are not significantly different. No. of samples in each group is six. Propolis: (PRO), curcumin: (CUR), Cypermethrin: (CYP), Cypermethrin plus Propolis (CYP + PRO) and Cypermethrin plus Curcumin (CYP + CUR). The most biochemical parameters revealed that PRO is markedly effective than CUR in ameliorating the biochemical alterations.
3.4. Histopathological observations The examination of light microscope of the control group (Fig. 4a&b), PRO and CUR groups (Fig. 5a&b) showed normal structure of the liver, each lobule has a central vein and hepatic cords in radiating shape. The administration of CYP showed an obvious signs of hepatic alterations; these include congested blood vessels, mononuclear inflammatory cell infiltration and extensive vacuolar degeneration of hepatocytes (Fig. 6a). Congested portal vein with thickened wall and proliferated bile duct were also detected (Fig. 6b). Congested blood vessel and fibrotic tissues were also noticed (Fig. 6c&d). Moreover, hyperemic sinusoids and oedema
Please cite this article in press as: Abdul-Hamid, M., et al. Cypermethrin-induced histopathological, ultrastructural and biochemical changes in liver of albino rats: The protective role of propolis and curcumin. Beni-Suef Univ. J. Basic Appl. Sci. (2017), http://dx.doi.org/10.1016/j.bjbas.2017.03.002
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Fig. 10. a–c: Photomicrograph of a section in the liver of control, PRO and CUR rats showing intensive amount of DNA in the nuclei of the hepatocytes (arrows) (d): Liver of CYP treated rat showing decreased DNA content (arrows) (e&f): Liver of CYP plus PRO and CYP plus CUR treated rat showing almost normal DNA content (Feulgen, Scale bar = 20mm).
(Fig. 6e), vacuolar degeneration of hepatocytes and fatty changes were seen (Fig. 6f). The liver sections of group treated with CYP plus PRO or CUR revealed marked recovery and restoration of almost normal hepatic configuration. (Fig. 7a&b).
3.5. Histochemical observations A strong PAS-positive reaction was showed in the hepatocytes of the control group, PRO and CUR- treated rats groups indicating the presence of large amount of glycogen (Fig. 8a–c). On the other hand, marked reduction of PAS reaction was observed in CYP treated group (Fig. 8d). However, after treatment with PRO and CUR,
the hepatocytes revealed an obvious increase of glycogen which approximated that of the control liver (Fig. 8e&f). Liver sections of control, PRO and CUR- treated groups stained with bromophenol blue revealed a dense blue staining. The proteins appeared in the form of bluish granules of various sizes randomly distributed in the ground cytoplasm of the hepatic cells (Fig. 9a–c). CYP-treated group showed marked depletion of proteins intensity in the cytoplasm of hepatocytes (Fig. 9d). Intake of PRO plus CYP and CUR plus CYP increased the intensity of blue color of protein than that of CYP treated group (Fig. 9e&f) respectively. Using Feulgen reaction, the hepatocytes of control group, PRO and CUR treated rats showed marked DNA amount in their nuclei
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Fig. 11. a&b: Electron micrographs of hepatocytes of the control group showing normal nucleus (N), nucleolus (NU), mitochondria (M), rough endoplasmic reticulum (RER) and glycogen granules (G). (c): Normal bile ducts (BD). (d): Normal Kupffer cell with nucleus (N) and lysosome (L). Scale bar = 2mm.
(Fig. 10a–c). In contrast, CYP-treated rats revealed severe decrease of DNA amount in the nuclei of the liver cells. Few cells appeared nucleated, while others showed vacuolated nuclei. (Fig. 10d). On the other hand, administration of PRO to CYP and CUR to CYP groups caused marked increase of DNA amount in the nuclei of the hepatocytes which approximately reached that of the control rats (Fig. 10e&f). 3.6. Ultrastructural observations Ultrastructural examination of liver sections of control rats displayed normal structure. The cytoplasm of the hepatocytes contains numerous mitochondria. They are round in shape with well-developed cristae. The rough endoplasmic reticulum consists
of closely pack parallel and flattened cisternae. The nuclei appear spherical with normal pattern of chromatin distribution. Numerous electron-dense glycogen rosettes were clearly detected. The nucleus of the hepatocytes appears spherical with a distinct nuclear envelope and one or more prominent nucleoli. The nucleoplasm showed a unique distribution of a small amount of heterochromatin at the peripheral regions and a central large amount of euchromatin (Fig. 11a&b). Normal bile duct with normal structure of microvilli was seen (Fig. 11c). Kupffer cell also appeared with normal nucleus, lysosomes and rough endoplasmic reticulum (Fig. 11d). However, marked ultrastructural changes were revealed in CYP-treated rats. In some hepatocytes, the mitochondria appeared swollen, vacuolated with destructed cristae, in addition to the presence of cytoplasmic vacuolations (Fig. 12a). Other cells
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Fig. 12. a: Electron micrographs of hepatocytes of rat treated with CYP showing vacuolated mitochondria (M), vacuoles (V). (b): dissolution of some hepatocytes (D) and diltation of bile duct with destructed microvilli (BD). (c): Kupffer cell of CYP- treated rat showing elongated nucleus (N), cytoplasmic vacuolation (V), endocytic vesicles (EV) and lysosomes (L). (d): large fat droplets (FD) and many cytoplasmic vacuoles (V). Scale bar = 2mm.
showed electron-dense mitochondria, dissolution of some hepatocytes, completely lysed nucleus and dilatation of bile duct with destructed microvilli (Fig. 12b). Kupffer cell showed elongated nucleus, endocytic vesicles and marked increase of lysosomes number (Fig. 12c). In other hepatocytes, cytoplasmic vacuoles and fat droplets were also observed (Fig. 12d). On the other hand, the liver treated with PRO plus CYP revealed that the hepatocytes restored their normal structure (Fig. 13a) and bile duct also was regained to its normal structure (Fig. 13b). Kupffer cells displayed their normal structure except the presence of some fat droplets (Fig. 13c). The liver section treated with CUR plus CYP showed marked recovery of the cytoplasmic organelles, and hepatic cells except some vacuoles (Fig. 14a) and bile duct also restored its nor-
mal structure (Fig. 14b). Furthermore, Kupffer cells displayed their normal structure but some vacuoles were still observed (Fig. 14c).
4. Discussion The mode of action of CYP can be expected to have two ways: it may generate ROS that induce oxidative stress or it may accumulate in cell membrane and disturb membrane structure due to its hydrophobic nature (Saxena and Saxena, 2010). In the current study, CYP administration increased ALT, AST, ALP and MDA. The increasing of these parameters act as indicator to liver toxicity, oxidative stress and LPO (Bhushan et al., 2013; Soliman et al.,
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Fig. 13. a–c: Electron micrographs of rat treated with CYP and PRO: (a): showing marked recovery of the hepatocyte nucleus (N) and cytoplasmic organelles; mitochondria (M), rough endoplasmic reticulum (RER). (b): showing bile duct (arrow). (c): Kupffer cell with normal structure and normal nucleus (N) and fat droplet (FD). Scale bar = 2mm.
2014). Oxidative stress contributes to initiation and progression of liver injury, so it is considered a main pathological mechanism (Li et al., 2015). Also, CYP has been demonstrated to cause a significant decrease in the activities of SOD and GPx in the rats of the present study. In consistent with these results, Gomaa et al. (2011) found that CYP induced a significant increase in the mean values of AST, ALT, ALP, MDA levels and a significant decrease in the mean values of antioxidant enzyme activities (CAT, SOD and GPx) in rats liver. The present histopathological observations of liver sections of rats in the current study showed congested blood vessels, mononuclear inflammatory cell infiltration, congested portal vein with thickened wall and proliferated bile duct was also detected. Moreover, hyperemic sinusoids, vacuolar degeneration of hepatocytes and oedema. In addition, many hepatocytes underwent fatty change and fibrosis. These results were lined with Soliman et al. (2014) and Mossa et al. (2015) who showed liver damage during CYP toxicity in rats. Also, Grewal et al. (2010) observed marked degenerative changes of hepatocytes and congestion after administration of CYP at 14.5 mg/kg for 30 consecutive days. These histopathological alterations in the hepatocytes because CYP has inhibitory effect on total adenine triphosphate activity in the liver, which may disorganize active transport of Na+, K+ and Ca2+ ions, causing hepatocytes injuring (Khan et al., 2009). Concerning the present ultrastructural study of hepatocytes, nuclear changes, cytoplasmic vacuolation, damage mitochondria and breakdown of RER were observed. In this respect, Aldana et al. (1998) reported that after CYP exposure, hepatocytes had
an ovoid nucleus with slightly irregular profile, appearing of many lipid droplets in cytoplasm and a lot of swelling mitochondria. Antioxidants may play an important role to protect the cells against oxidative stress and damage caused by free radicals (Bouayed and Bohn, 2010). El-Masry et al. (2011) showed that PRO has twelve different flavonoids, acacetin, pinocembrin, chrysin, rutin, catechin, naringenin, galangin, luteolin, a pigenin, kaemferol, myricetin and quercetin, two phenolic acids, cinnamic and caffeic acid are present in PRO. PRO is an excellent source of essential elements, including Mg2+, Zn2+, Fe2+, Cu2 +, Ca2+, Ni2+ and Mn2+ (Haro et al., 2002), which might also be responsible for reactivating antioxidant enzymes by providing optimum trace elements. The first mechanism of the effect of PRO may require the scavenging of free radicals that induce LPO. The second mechanism may inhibit xanthine oxidase, which is known to cause generation of free radicals (Kanbura et al., 2009). In the present study, PRO co-administration with CYP induced a significant decrease in AST, ALT, ALP, MDA levels and a significant increase in the mean values of antioxidant enzyme activities (CAT, SOD and GPx) as compared with CYP treated group. This result is in agreement with (Eraslan et al., 2008). PRO induced a reduction in the increased activity of AST and ALT in the serum of rats treated with doxorubicin (Omar et al., 2016). The present histopathological examination revealed marked recovery and restoration of almost normal hepatic cells by PRO therapy in contrast to CYP intoxicated animals. The normal hepatocytes were observed in most areas when propolis was co-administered with CYP, however, mild sinusoidal dilation with inflammatory cell infiltration was also present
Please cite this article in press as: Abdul-Hamid, M., et al. Cypermethrin-induced histopathological, ultrastructural and biochemical changes in liver of albino rats: The protective role of propolis and curcumin. Beni-Suef Univ. J. Basic Appl. Sci. (2017), http://dx.doi.org/10.1016/j.bjbas.2017.03.002
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Fig. 14. a–c: Electron micrographs of rat treated with CYP and CUR showing (a): marked recovery of the hepatocyte nucleus (N) and cytoplasmic organelles; mitochondria (M), rough endoplasmic reticulum (RER) and vacuoles (V). (b): bile duct (arrow). (c): Kupffer cell with normal structure and normal nucleus (N) and vacuoles (V). Scale bar = 2mm.
(Gomaa et al., 2011). Electron microscopic examination of the present study displayed a better preservation of the hepatic cells of rat administrated PRO with CYP compared to those treated with CYP alone. CUR has protective effects against oxidative damage and has antioxidant and anticonvulsant properties exerting powerful ROS scavenging effects and increased intracellular glutathione concentration, thereby protecting against LPO (Aboul Ezz et al., 2011; Du et al., 2012). Co-administration of CUR with CYP revealed a decrease in the concentrations of ALT, AST, ALP and MDA as compared to CYP alone treated rats in this study. These findings are in agreement with Sankar et al. (2012) and Hismiogullari et al. (2014). In addition, Salahshoor et al. (2016) exhibit that the mean values of ALT, AST, and ALP enzymes decreased significantly in CUR and CUR plus nicotine in all groups compared to the nicotine group. In the current study, the liver of CUR plus CYP group displayed a relatively normal architecture. Also, Sankar et al. (2012) showed a reduction in the degenerative changes in liver of CUR plus CYP group. In conclusion, the present study clarified that administration of PRO and CUR are beneficial to ameliorate the biochemical, histopathological and ultrastructural alterations in liver of rats induced by CYP due to their antioxidants effects. The protective effects of PRO are markedly effective than CUR against CYP.
Conflict of interest statement The authors declare that there are no conflicts of interest.
Funding This research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors.
References Aboul Ezz, H.S., Khadrawy, Y.A., Noor, N.A., 2011. The neuroprotective effect of curcumin and Nigella sativa oil against oxidative stress in the pilocarpine model of epilepsy: a comparison with valproate. Neurochem. Res. 36, 2195–2204. Aldana, L., Gonz0 alez de Mej0 ıa, E., Craigmill, A., Tsutsumi, V., Armendariz-Borunda, J., Panduro, A., Rinc0 on, A.R., 1998. Cypermethrin increases apo A-1 and apo B mRNA but not hyperlipidemia in rats. Toxicol. Lett. 16, 31–39. Bancroft, J.D., Gamble, M., 2002. Theory and Practice of Histological Techniques. Churichill Livingstone, Edinburgh, London. Bankova, V.S., De Castro, S.L., Marcucci, M., 2000. Propolis: recent advances in research on chemistry and plant origin. Apidologie 31, 3–15. Bankova, V., Popova, M., Trusheva, B., 2016. New emerging fields of application of propolis. Maced. J. Chem. Chem. Eng. 35 (1), 1–11. Bhushan, B., Saxena, P.N., Saxena, N., 2013. Biochemical and histological changes in rat liver caused by cypermethrin and beta-cyfluthrin. Arh. Hig. Rada Toksikol. 64, 57–67. Bouayed, J., Bohn, T., 2010. Exogenous antioxidants – double-edged swords in cellular redox state: health beneficial effects at physiologic doses versus deleterious effects at high doses. Oxid. Med. Cell Longev. 3 (4), 228–237. Bozzola, J.J., Russell, L.D., 1999. Electron Microscopy: Principles and Techniques for Biologists. Jones and Bartlett Publishes, Sudbury, MA, USA. Du, P., Tang, H.Y., Li, X., Lin, H.J., Peng, W.F., Ma, Y., Fan, W., Wang, X., 2012. Anticonvulsive and antioxidant effects of curcumin on pilocarpine-induced seizures in rats. Chin. Med. J. 125, 1975–1979. El-Masry, T.A., Emara, A.M., El-Shitany, N.A., 2011. Possible protective effect of propolis against lead induced neurotoxicity in animal model. J. Evol. Biol. Res. 3 (1), 4–11.
Please cite this article in press as: Abdul-Hamid, M., et al. Cypermethrin-induced histopathological, ultrastructural and biochemical changes in liver of albino rats: The protective role of propolis and curcumin. Beni-Suef Univ. J. Basic Appl. Sci. (2017), http://dx.doi.org/10.1016/j.bjbas.2017.03.002
14
M. Abdul-Hamid et al. / Beni-Suef Univ. J. Basic Appl. Sci. xxx (2017) xxx–xxx
Elsayed, A.S.I., 2016. The curcumin as an antioxidant natural herb, with emphasize on its effects against some diseases. Int. J. App. Biol. Pharmacol. Technol. 7 (1), 26–40. EL-Shemi A G, Abou El-Ella G A. Hepatic and neural toxicity and tissue residue of cypermethrin in male Somali sheep (Berbera blackhead). Assiut Vet. Med. J. 2015; 61 (146):52–59. Eraslan G, Kanbur M, Silici S, Altinordulu S, Karabacak M. Effects of cypermethrin on some biochemical changes in rats: the protective role of propolis, Exp. Anim. 2008; 57 453–460. Ferrari, C.K.B., 2000. Free radicals, lipid peroxidation and antioxidants in apoptosis: implications in cancer, cardiovascular and neurological diseases. Biologia 55, 581–590. Feulgen, R., Rossenenbeck, H., 1924. Mikroskopisch chemischer nach weis einen nucleinsaure von typus der thymonucleinsaure und de darauf berhende elective farbung von Zelkernen in microskopischen preparaten. Zeitschrift Physiol. Chem. 135, 203. Gomaa M S, Abd Alla M A, Sameer M M. The possible protective effect of propolis (Bee glue) on cypermethrin-induced hepatotoxicity in adult albino rats. Mansoura J. Forensic Med. Clin. Toxicol. 2011; Vol. XIX, No. 1: 17–32. Grewal, K.K., Sandhu, G.S., RanjitKaur Brar, R.S., Sandhu, H.S., 2010. Toxic impacts of cypermethrin on behavior and histology of certain tissues of albino rats. Toxicol. Int. 17, 94–98. Haro, A., Lopez-Aliaga, I., Lisbona, F., Barrionuevo, M., Alferez, M.J.M., Campos, M.S., 2002. Beneficial effect of pollen and/or propolis on the metabolism of iron, calcium, phosphorus, and magnesium in rats with nutritional ferropenic anemia. J. Agric. Food Chem. 48, 5715–5722. Huang, F., Liu, Q., Xie, S., Xu, J., Huang, B., Wu, Y., Xia, D., 2016. Cypermethrin induces macrophages death through cell cycle arrest and oxidative stressmediated JNK/ERK signaling regulated apoptosis. Int. J. Mol. Sci. 17, 885. Hismiogullari, S.E., Hismiogullari, A.A., Sunay, F.B., Paksoy, S., Can, M., Aksit, H., Karaca, O., Yavuz, O., 2014. The protective effect of curcumin on carbon tetrachloride induced liver damage. Revue Méd. Vét. 165 (7–8), 194–200. Hotchkiss, R.D.A., 1948. Microchemical reaction resulting in the staining of polysaccharide structures in fixed tissue preparations. Arch. Biochem. 16, 131–141. Hussien, H.M., Abdou, H.M., Yousef, M.I., 2013. Cypermethrin induced damage in genomic DNA and histopathological changes in brain and haematotoxicity in rats: the protective effect of sesame oil. Brain Res. Bull. 92, 76–83. IBM crop (2011): IBM SPSS Statistic for windows, Version 20.0 Armonk, N.Y., IBM Crop. Inayat, Q., Ilahi, M., Khan, J., 2007. A morphometric and histological study of the kidney of mice after dermal application of cypermethrin. J. Pak. Med. Assoc. 57, 587–591. Kanbura, M., Eraslan, G., Silici, S., 2009. Antioxidant effect of propolis against exposure to propetamphos in rats. Ecotoxicol. Environ. Safety 72, 909–915. Khan, A., Faridi, H.A.M., Ali, M., Khan, M.Z., Siddique, M., Hussain, I., Ahmad, M., 2009. Effects of cypermethrin on some clinico-hematobiochemical and pathological parameters in male dwarf goats (Capra hircus). Exp. Toxicol. Pathol. 61, 151–160. Li, S., Tan, H., Wang, N., Zhang, Z., Lao, L., Wong, C., Feng, Y., 2015. The role of oxidative stress and antioxidants in liver diseases. Int. J. Mol. Sci. 16 (11), 26087–26124. Majeed M, Nagabhushanam K, Choudhury AK. Antioxidants: Balancing the Good, the Bad and the Ugly. Nutr. Food Technol. 2016; 2(2): doi http://dx.doi. org/ 10.16966/2470-6086.123. Manna, S., Bhattacharyya, D., Mandal, T.K., Das, S., 2004. Repeated dose toxicity of alfa-cypermethrin in rats. J. Vet. Sci. 5, 241–245. Marklund, S., Marklund, G., 1974. Involvement of superoxide anion radical in the autooxidation of pyrogallol and convenient assay for superoxide dismutase. Eur. J. Biochem. 47, 469–474.
Matkovics, B., Szabo, L., 1998. Varga IS Determination of enzyme activities in lipid peroxidation and glutathione pathways (in Hungarian). Laboratoriumi Diagnosztika 15, 248–249. Mazia, D., Brewer, P.A., Alfert, M., 1953. The cytochemical staining and measurement of protein with mercuric bromophenol blue. Biol. Bull. 104, 57– 67. Moran, M.P., Fernandez, J.M., Tortosa, C.R., Tortosa, M.C.R., 2016. Curcumin and health. Molecules 21, 264. Mossa, A.H., Heikal, T.M., Belaiba, M., Raoelison, E.G., Ferhout, H., Bouajila, J., 2015. Antioxidant activity and hepatoprotective potential of Cedrelopsis grevei on cypermethrin induced oxidative stress and liver damage in male mice. BMC Complement. Alternat. Med. 15, 251. Omar, N.A.A., Allithy, A.N.A., Baghdadi, H., Zolaly, M., Abdel-haleem, M., Helmy, M. M., Ayat, M.M.A., El Sayed, S.M., 2016. Hepatoprotective effects exerted by propolis against doxorubicin-induced rat liver toxicity: a biochemical and histopathological study. Am. J. Cancer Prevention 4 (3), 36–40. Pattanayak, R., Basak, P., Sen, S., Bhattacharyya, M., 2016. Interaction of kras gquadruplex with natural polyphenols: a spectroscopic analysis with molecular modeling. Int. J. Biol. Macromol. 89, 228–237. Perrone, D., Ardito, F., Giannatempo, G., Dioguardi, M., Troiano, G., Lo Russo, L., Laino, L., Lo, Muzio.L., 2015. Biological and therapeutic activities, and anticancer properties of curcumin. Exp. Ther. Med. 10, 1615–1623. Rosalki, S.B., Foo, A.Y., Burlina, A., Prellwitz, W., Stieber, P., Neumeier, D., Klein, G., Poppe, W.A., Bodenmüller, H., 1993. Multicenter evaluation of Iso-ALP test kit for measurement of bone alkaline phosphatase activity in serum and plasma. Clin. Chem. 39 (4), 648–652. Saka, W.A., Akhigbe, R.E., Azeez, O.M., Babatunde, T.R., 2011. Effect of pyrethroid exposure on haematological and haemostatic profiles in rats. Pak. J. Biol. Sci. 14, 1024–1027. Salahshoor, M., Mohamadian, S., Kakabaraei, S., Roshankhah, S., Jalili, C., 2016. Curcumin improves liver damage in male mice exposed to nicotine. J. Traditional Complement. Med. 6 (2), 176–183. Saleh, E.M., 2012. Antioxidant effect of aqueous extract of propolis on hepatotoxicity induced by octylphenol in male rats. Acta Toxicol. Argent. 20, 68–81. Sankar, P., Telang, A.G., Manimaran, A., 2010. Curcumin protects against cypermethrin induced genotoxicity in rats. Environ. Toxicol. Pharmacol. 30, 289–291. Sankar, P., Telang, A.G., Manimaran, A., 2011. Effect of piperine on cypermethrininduced oxidative damage in rats. J. Veterinar. Sci. Technol. 2, 105. Sankar, P., Telang, A.G., Manimaran, A., 2012. Protective effect of curcumin on cypermethrin-induced oxidative stress in Wistar rats. Exp. Toxicol. Pathol. 64 (5), 487–493. Saxena, P., Saxena, A.K., 2010. Cypermethrin induced biochemical alterations in the blood of albino rats. Jordan J. Biol. Sci. 3, 111–114. Schumann, G., Klauke, R., 2003. New IFCC reference procedures for the determination of catalytic activity concentrations of five enzymes in serum: preliminary upper reference limits obtained in hospitalized subjects. Clin. Chim. Acta 327 (1–2), 69–79. Soliman M M, Attia H F, Abou El-Ella GA. Genetic and histopathological alterations induced by cypermethrin in rat kidney and liver: Protection by sesame oil. Int. J. Immunopathol. Pharmacol. 2014; 1–13. Wenger C, Kaplan A, Rubaltelli FF, Hammerman C. Alkaline phosphatase. Clin. Chem. The C.V. Mosby Co., St. Louis. Toronto. Princeton. 1984; 1094–1098. Yagi, K., 1987. Lipid peroxides and human diseases. Chem. Phys. Lipids 45, 337–351. Yousef, M.I., Salama, A.F., 2009. Propolis protection from reproductive toxicity caused by aluminium chloride in male rats. Food Chem. Toxicol. 47, 1168–1175.
Please cite this article in press as: Abdul-Hamid, M., et al. Cypermethrin-induced histopathological, ultrastructural and biochemical changes in liver of albino rats: The protective role of propolis and curcumin. Beni-Suef Univ. J. Basic Appl. Sci. (2017), http://dx.doi.org/10.1016/j.bjbas.2017.03.002