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Thyroidectomy induced hepatic toxicity and possible amelioration by Ginkgo biloba leaf extract
Biomedicine & Preventive Nutrition 4 (2014) 391–397
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Original article
Thyroidectomy induced hepatic toxicity and possible amelioration by Ginkgo biloba leaf extract Ehab Tousson a,b,∗ , Areej J.M. Alghabban a , Heba Abou Harga c a b c
Biology Department, Faculty of Science, Tabuk University, Tabuk 71491, Saudi Arabia Zoology Department, Faculty of Science, Tanta University, Tanta 31527, Egypt Damenhour Fever Hospital, Damenhour, Egypt
a r t i c l e
i n f o
Article history: Received 12 April 2014 Accepted 6 June 2014 Keywords: Thyroidectomy Liver Toxicity Histopathology PCNA immunoreactivity Rat
a b s t r a c t The liver is a major target organ for thyroid hormone action and marked changes occur in liver functions in the case of hypo- or hyperthyroidism. This studied aimed at inverstigating the biochemical and histopathological changes in the liver after thyroidectomy and the ameliorating role of Ginkgo biloba leaf extract (GLE). A total of 50 male albino rats were equally divided into five groups; 1st to 3rd groups were control, sham operated and GLE groups while 4th group was thyroidectomized rat group and 5th group was treated thyrodectomized rat with GLE. Serum T3 and T4 levels after 3 weeks of thyroidectomy was significantly decreased when compared with the control group, while TSH significantly increased when compared with the control group increased. Serum ALT, AST, ALP and GGT showed significant (P < 0.05) increase in thyroidectomized group when compared with control, sham operated and sham operated with GLE groups. On the one hand, treatment of thyroidectomized rats with GLE improved this increase in serum ALT, AST, ALP and GGT in thyroidectomized rat group. Liver sections of thyroidectomy group showed marked positive reaction and increase number of PCNA staining of hepatocyte nuclei. On the other hand, liver in treated of thyroidectomized rat with GLE group showed a marked reduction in the number of PCNA-positive nuclei when compared with sections in thyroidectomy group. Treatment of thyroidectomized rat with GLE improves the biochemical, histopathological and immunohistochemical alternations and the intensity of PCNA immunoreactive cells demonstrating the recovery of some injury. © 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction Thyroid hormones regulate all metabolic activities, such as growth rate, sodium/potassium pump, cholesterol secretion in the bile, heart rate, blood pressure, respiration, oxygen consumption, digestion strength, lipid, carbohydrate and protein metabolism, central nervous system function, and the actions of other endocrine glands [1–12]. A thyroidectomy is an operation that involves the surgical removal of all or part of the thyroid gland. Surgeons often perform a thyroidectomy when a patient has thyroid cancer or some other condition of the thyroid gland (such as hyperthyroidism) or goiter. Hypothyroidism is caused by thyroid hormone deficiency. Hypothyroidism was induced in male rats by near-total thyroidectomy because this is a well-established model to study early vascular effects of hypothyroidism [1–11,13–15]. It is unclear
∗ Corresponding author at: Corresponding author. Biology Department, Faculty of Science, Tabuk University, Tabuk 71491, Saudi Arabia. Tel.: +966 53 65 19 175. E-mail addresses: [email protected], [email protected] (E. Tousson). http://dx.doi.org/10.1016/j.bionut.2014.06.001 2210-5239/© 2014 Elsevier Masson SAS. All rights reserved.
whether this is a direct thyroid effect on liver enzymes or secondary to altered intestinal handling of cholesterol and bile acids [16]. There is also evidence that hypothyroidism may directly affect the liver structure or function. Hypothyroidism has been associated in a few case reports with cholestatic jaundice attributed to reduced bilirubin and bile excretion. The triad of reduced bilirubin excretion, hypercholesterolemia and hypotonia of the gall bladder seen in hypothyroidism increases the incidence of gallstones [17]. Many traditional treatments have been recommended in the complementary and alternative treatment of various illnesses. They may prevent disease, reduce the risk of developing disease or enhance general health [18,19]. Herbal medicine is increasingly gaining acceptance from the public and medical professionals due to advances in the understanding of the mechanisms by which herbs positively influence health and quality of life [20,21]. Ginkgo biloba (maiden hair tree) is one of the oldest herbal medicines that have been used as a therapeutic agent in modern pharmacology. Standardized extracts from dried Ginkgo leaves take also important place in modern medicine [21]. G. biloba leaf extract (GLE) is standardized to contain approximately 24% flavone glycosides and 6% terpene lactones. These compounds are extracted from the tree’s
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healthy green leaves. The flavone glycosides in Ginkgo are primarily composed of quercetin, kaempferol, and isorhamnetin. These bioflavonoid constituents are thought to be the compounds that provide Ginkgo’s antioxidant effects. Ginkgo has been shown to be active against a wide variety of free radicals and free radical generating substances including nitric oxide, the superoxide, hydroxyl, oxoferryl and peroxyl radicals [22]. Popularly marketed to the general public, G. biloba extract is believed to provide beneficial effects in memory impairment, stroke, edema, inflammation, Alzheimer’s dementia, and vaso-occlusive disorders [23–30]. The liver plays an astonishing array of vital functions in the maintenance, performance and regulating homeostasis of the body. It is involved with almost all the biochemical pathways to growth, fight against disease, nutrient supply, energy provision and reproduction [31]. It functions as a center of metabolism of nutrients, such as carbohydrates, proteins and lipids and excretion of waste metabolites. Based on these evidences, the present study was conducted to examine the possible modifying effects of Ginkgo leaf extract (GLR) against hepatic toxicity induced by thyroidectomy in male rats. This could be fulfilled through the histological and biochemical analysis of liver tissues. 2. Materials and methods The experiments were performed on 50 male Wistar rats weighing 130 ± 10 g. They were obtained from the farm of Helwan, Egypt. The rats were kept in the laboratory for one week before the experimental work and fed a standardized diet (Barley) ad libitum. The temperature in the animal room was maintained at 23 ± 2 ◦ C. The laboratory cycle was 12:12-h light-dark cycle. Rats were anesthetized by intraperitoneal injection with thiopental sodium (50 mg/kg; EIPICO Co., Egypt) for sham operation or thyroidectomy and after surgery, the animals were given Bivatracin (Neomycin, Bacitracin: Topical antibiotic aerosol powder spray; ECAP Co., Egypt) two times/day for 6 days. The experimental protocol was approved by Local Ethics Committee and Animals Research. 2.1. Animal treatments The rats were randomly and equally divided into the following groups: • group I: (GI ) control group – thirty rats were fed a standardized diet ad libitum and dissecting after 3 weeks; • group 2: (G2 ) sham operated group – rats were subjected to sham operation and did not received any treatment and dissecting after 3 weeks; • group 3: (G3 ) sham operated and G. biloba leaf extract treated group (GLE) – rats were subjected to sham operation and from 3rd day orally treated with GLE (0.11 g/kg body weight) for 3 weeks rats; • group 4 (G4 ) thyroidectomized group – rats were surgically subjected to thyroidectomy [10,32] and dissecting after 3 weeks; • group 5: (G5 ) treated thyroidectomized rats with GLE – rats were subjected to thyroidectomy and from 3rd day orally treated with GLE for 3 weeks rats. 2.2. Thyroidectomy Thyroidectomy was performed on rats anesthetized with intraperitoneal injection with sodium pentobarbital and subjected to a complete necropsy according to the method of Tousson et al. [10] and Francisco et al. [32]. Briefly, by using a stereomicroscope (Zeiss, Germany) for better observation, a midline skin incision was made along the length of the neck. The underlying tissues were
removed, and the salivary glands were retracted laterally. The two halves of the sternohyoid muscle were separated and retracted laterally. The thyroid muscle was separated from the lobes of the thyroid gland and retracted along with the sternohyoid muscle. A midline cut was made in the isthmus, and the thyroid glands were excised bilaterally. Extreme care was taken not to damage the laryngeal nerve. Sham (euthyroid/control) operated animals underwent the same surgical procedures without removal of the thyroid gland. After surgery, ketorolac (Sintex-Mexico) (50 mg/kg im) and gentamicin (Shering Plough-Mexico) (10 mg/kg) were administered over 5 days to alleviate pain and prevent infection. 2.3. Extract preparation The extraction procedure for G. biloba leaf extract (GLE) was carried out as reported by Lichtblau et al. [51]. 2.4. Sample preparation Animals were fasted overnight and for clinical chemistry blood samples from each rat were collected from the eyes by retro-orbital puncture using blood capillary tubes (with and without heparin as per requirement) under mild ether anesthesia. Blood samples that collected without heparin was incubated at room temperature for 10 min and left to clot then centrifuged at 3000 rpm for 10 min and the serum were collected, serum was separated and kept in clean stopper plastic vial at –80 ◦ C until the analysis of serum parameters. 2.5. Biochemical analysis Estimation of serum tri-iodothyronine (T3) was assayed by using commercial test supplied by the Diagnostic systems Laboratories (Taxes, USA) according to the method of Chopra et al. [33]. Estimation of serum thyroxine (T4 ) was assayed by using kits for these hormones were obtained from Calbiotech INC (CBI), USA according to the methods of Thakur et al. [34]. Estimation of serum thyrotropin (TSH) was assayed by using commercial Kit supplied by Coat-A-Count TSH IRMA (Los Angeles, USA) according to the method of Engall et al. [35]. Estimation of liver enzymes, alanine transaminase (ALT) and aspartate transaminase (AST) activities in serum were assayed by using commercial kit that was supplied by Randox (Egypt) according to the method of Reitman and Frankel [36]. Concentration of total and direct bilirubin was spectrophotometrically determined using commercial diagnostic kits supplied by Human (German) according to the method of Pearlman and Lee [37]. Determination of alkaline phosphatase (ALP) and gamma-glutamyltranspeptidase (GGT) was according to the method of Bessey et al. [38] and Rosalki [39], respectively. 2.5.1. Histopathological examination Immediately after decapitation animals were dissected, liver from different groups were quickly removed, washed using chilled saline solution and fixed in 10% neutral buffered formalin. After fixation, specimens were dehydrated in an ascending series of alcohol, cleared in two changes of xylene and embedded in molten paraffin (mp 50–58 ◦ C). Sections of 5 microns thickness were cut using rotary microtome and mounted on gelatin chromalum-coated glass slides. Some of liver sections were stained with Ehrlich’s haematoxylin and counterstained with eosin as a routine method after Bancroft and Stevens [40]. The rest of the sections were stored at room temperature until further processing. 2.5.2. Immunohistochemical detection of PCNA The rest of paraffin-embedded rat liver sections were deparaffinized and hydrated for PCNA immunoreactivity [6]. Endogenous
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Table 1 Changes in the serum tri-iodothyronine (T3 ), thyroxine (T4) and thyrotropin (TSH) levels in the different groups under study.
T3 level (ng/dL) T4 (ng/dL) TSH level (IU/mL)
G1
G2
G3
G4
G5
46.4 ± 2.50a
45.8 ± 3.13a
44.0 ± 2.75a
25.31 ± 1.75b
44.3 ± 1.05a
2.85 ± 0.046a
2.54 ± 0.04a
2.63 ± 0.19a
1.33 ± 0.24b
2.06 ± 0.09a,b
1.33 ± 0.19a
1.30 ± 0.29a
1.33 ± 0.14b
4.56 ± 0.32b
2.24 ± 0.27a
Data are expressed as mean ± SEM for 10 observations. a Significant difference from the thyroidectomized group (G4 ) at P < 0.05, where G1 , control group; G2 , sham operated group; G3 , sham operated and treated with GLE group; G4 , thyroidectomized rats group; G5 , treated thyroidectomized rats with GLE group. b Significant difference from the control group (G1 ) at P < 0.05. Table 2 Body weight gain (BWG) and relative liver weight (RLW) in different groups under study.
BWG (g) RLW (g/100 g)
G1
G2
G3
G4
G5
39.7 ± 2.4a 3.5 ± 0.53
38.3 ± 1.55a 3.4 ± 0.18
39.1 ± 2.24a 3.4 ± 0.42
34.7 ± 2.3b 3.4 ± 0.19
36.8 ± 2.15a,b 3.5 ± 0.06
RLW = (liver weight/final body weight) × 100, data are expressed as mean ± SEM for 10 observations. a Significant difference from the thyroidectomized group (G4 ) at P < 0.05, where G1 , control group; G2 , sham operated group; G3 , sham operated and treated with GLE group; G4 , thyroidectomized rats group; G5 , treated thyroidectomized rats with GLE group. b Significant difference from the control group (G1 ) at P < 0.05.
peroxidase activity was blocked by incubation using 3% H2 O2 for 5 min. The tissue sections were incubated over night with proliferating cell nuclear antigen (PCNA) monoclonal antibody (Dako Corporation, Carpentaria, CA, USA) and washed with phosphate buffer saline (PBS) for 5 min. The monoclonal antibody was then linked with biotinylated goat anti-mouse IgG antibody (Daco, LASB Universal Kit) for 30 min. After being washed with PBS for 5 min, the sections were incubated with streptavidin-conjugated peroxidase for 30 min. A brown coloured reaction was developed by exposing sections to 3,3-diaminobenzidine (DAB) tetrahydrochloride solution for 5 min and washed in distilled water. Sections were counterstained with haematoxylin and eosin [41]. Cells were considered positive for PCNA if there was brown nuclear staining of the cells and negative (nuclei not stained) appeared blue. The number of PCNA-positive cells was counted in 10 randomly selected sections and nonoverlapping fields and expressed as the number of PCNA-positive cells/mm2 . All stained slides were viewed by using Olympus microscope and images were captured by a digital camera (Cannon 620). Brightness and contrast were adjusted using Adobe Photoshop software. Image analysis was adjusted using PAXit image analysis software. The data was statistically analyzed using SPSS. 2.5.3. Statistical analysis Data were expressed as mean values ± SE and statistical analysis was performed using one way ANOVA to assess significant differences among treatment groups. The criterion for statistical significance was set at P < 0.05 for the biochemical data. All
statistical analyses were performed using SPSS statistical version 16 software package (SPSS® Inc., USA). 3. Results 3.1. Toxicity The animals under practice appeared healthy and did not show clinical signs of disease and no mortality was recorded during the experiment duration. The dose of Ginkgo leaf extract (GLE) did not initiate any side effects for the animals, whereas various side effects were observed in thyroidectomized animals, such as loosing of loss of activity, body weight, and weakness. 3.2. Changes in thyroid hormones Table 1 showed that no changes in the serum T3 , T4 and TSH levels in sham operated (G2 ) and sham operated and GLE (G3 ) groups as compared with the control (G1). Serum T3 and T4 levels in thyroidectomized rats (G4 ) were significantly (P < 0.05) decreased as compared with the control (G1), sham operated (G2 ) and sham operated and GLE (G3 ) groups (Table 1). On the other hand, serum TSH levels were significantly (P < 0.05) increased in thyroidectomized rats (G4 ) when compared with the control (G1), sham operated (G2 ) and sham operated and GLE (G3 ) groups. Treatment of thyroidectomized rats with GLE (G5 ) significantly (P < 0.05) increased T3 and T4 and significantly (P < 0.05) decreased TSH levels when compared with the thyroidectomized rats (Table 1).
Table 3 Changes in ALT (U/L), AST (U/L), total bilirubin (mg/dL), ALP (U/L) and GGT (U/L) in different groups under study. G1 ALT AST Total bilirubin ALP GGT
28.16 31.52 0.16 39.3 55
G2 ± ± ± ± ±
a
2.44 1.15a 0.06 1.55a 1.2a
31.55 32.44 0.19 37.5 55.8
G3 ± ± ± ± ±
a
1.84 2.05a 0.06 2.16a 2.91a
29.2 33.65 0.17 45.5 50.4
G4 ± ± ± ± ±
a
2.07 2.11a 0.06 2.75a 2.32a
58.52 63.45 0.23 109.8 100
G5 ± ± ± ± ±
b
2.42 2.47b 0.02 4.16b 2.42b
39.41 38.29 0.21 68.1 76.5
± ± ± ± ±
2.61a 3.06a 0.03 4.07a 1.53a,b
Data are expressed as mean ± SEM for 10 observations. a Significant difference from the thyroidectomized group (G4 ) at P < 0.05, where G1 , control group; G2 , sham operated group; G3 , sham operated and treated with GLE group; G4 , thyroidectomized rats group; G5 , treated thyroidectomized rats with GLE group. b Significant difference from the control group (G1 ) at P < 0.05.
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Fig. 1. A–F. Photomicrographs of rat liver sections of different experimental groups stained with haematoxylin and eosin. A–C. Liver sections of control, sham operated and sham operated treated with GLE groups. D and E. Liver sections of thyroidectomized rats group. F. Liver sections of treated thyroidectomized rats with GLE group (CV, central vein; PV, portal vein).
3.3. Body weight gain and relative weight of liver The data summarized in Table 1 indicates that a significant (P < 0.05) decrease in body weight gain in thyroidectomized rats group (G4 ) when compared with control (G1 ), sham operated (G2 ) and sham operated with GLE (G3 ) groups. However, treatment of thyroidectomized rats with GLE improved this decrease in body weight gain in thyroidectomized rat group (G4 ). On the other hand, no significant change in the relative liver weight (RLW) were observed in the different groups under study (Table 2). 3.4. Changes in liver enzymes Table 3 showed that serum ALT, AST, ALP and GGT showed significant (P < 0.05) increase in thyroidectomized group (G4 ) when
compared with control (G1 ), sham operated (G2 ) and sham operated with GLE (G3 ) groups. On the other hand, treatment of thyroidectomized rats with GLE improved this increase in serum ALT, AST, ALP and GGT in thyroidectomized rat group (Table 3). In contrast, no significant change in the total bilirubin was detected in the different groups under study (Table 3). 3.5. Effect of GLE on histopathology Liver section in rats is divided into hepatic lobules formed of radially arranged strands of hepatocytes that extend from the central vein to the periphery of the lobule. Liver sections in control (G1 ), sham operated (G2 ) and sham operated and GLE (G3 ) groups showed normal structure of hepatocytes where the hepatocytes are polygonal in shape with eosinophilic granular cytoplasm and
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Fig. 2. A–F. Photomicrographs of rat liver sections stained by PCNA immunoreactivity in different group under study. A–C. Control, sham operated and sham operated treated with GLE groups showed the negative immune reaction indicated by blue stains due to normal central hepatic vein and hepatocytes. D and E. Liver sections in thyroidectomized rats group showed strong positive reaction for PCNA in most of hepatocytes nuclei (black arrows). F. Liver sections in treated thyroidectomized rat with GLE group showed a marked reduction in the number of PCNA-positive nuclei when compared with sections in of thyroidectomy group.
vesicular basophilic nuclei (Fig. 1A–C). Some of the hepatocytes are binucleated and the liver cords are separated from each other by narrow blood sinusoids lined with endothelial cells and von Kupffer cells (Fig. 1A–C). Liver sections of thyroidectomy group (G4 ) showed mild changes in liver hepatocytes as dilation or congestion in central veins, atrophied and vacuolated hepatocytes, degeneration with focal area, focal necrosis and dilated sinusoid with proliferated Kupffer cells (Fig. 1D and E). Liver sections in the treatment of thyroidectomized rat with GLE group (G5 ) shows a moderate degree of improvement in hepatocytes where a few atrophied and/or vacuolated hepatocytes, dilated congested sinusoids and no interlobular hemorrhage were observed (Fig. 1F). 3.6. Effect of GLE on PCNA-ir Liver sections of normal control, sham operated (G2 ) and sham operated and GLE (G3 ) groups showed a few hepatocytes nuclei
that displayed faint stain of PCNA (Fig. 2A–C). Liver sections of thyroidectomy group (G4 ) showed marked positive reaction and increase number of PCNA staining of hepatocyte nuclei (Fig. 2D and E). On the other hand, liver sections in treatment of thyroidectomized rat with GLE group (G5 ) showed a marked reduction in the number of PCNA-positive nuclei especially in peripheral zones compared with sections in of thyroidectomy group (Fig. 2F). 4. Discussion Thyroid hormones are involved in the regulation of numerous body functions, including lipid and carbohydrate metabolism, oxygen consumption and several physiological functions, such as development, reproduction, and growth [1–12]. The present study was designed to investigate the possible modifying effects of Ginkgo leaf extract (GLR) against hepatic toxicity induced by thyroidectomy in male rats. In order to ensure the thyroidectomy that leads
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to hyperthyroid state, we regularly determined the serum T3 , T4 and TSH through the dose period where serum T3 and T4 levels is increased and serum TSH levels is depressed in thyroidectomized rats, indicating the induction of hyperthyroid state. In the present study, Serum T3 and T4 levels in treated thyroidectomized rats with GLE were significantly decreased when compared with the thyroidectomized rats group while TSH levels in treated thyroidectomized rats with GLE were significantly increased when compared with the hyperthyroid group. Treatment of thyroidectomized rats with GLE improved these changes in thyroid hormones and leads to euthyroid state. This finding is compatible with Tenorio-Velasquez et al. [13], Tousson et al. [10], Ali et al. [15] and Francisco et al. [32] that used thyroidectomy to achieve hypothyroid state and hypothyroidism was confirmed biochemically in thyroidectomized rats by a pronounced elevation in serum TSH. The increase in TSH level can be explained by a decreased production of T3 from the thyroid gland that minimizes TSH feedback inhibition; resulting in an increase in its secretion by the anterior pituitary gland [42]. The present results compatible with Yatvin et al. [43] who reported that thyroidectomy reduce food intake, decrease body weight gain, liver weight, total protein and relative liver protein in rats. It is unclear whether this is a direct thyroid effect on liver enzymes or secondary to altered intestinal handling of cholesterol and bile acids [16]. Clinical diagnosis of disease and damage to the structural integrity of liver is commonly assessed by monitoring the status of serum AST and ALT activities. In the present study, the liver enzymes (AST, ALT, ALP and GGT) elevation may be attributed to myopathies, which are usually associated with hypothyroidism [44]. The result of serum total bilirubin concentration in the present study was unexpected, where there are no significant changes of its level in thyroidectomized group when compared with control or sham operated or treated with GLE groups. This finding is not compatible with Malik and Hodgson [44]. Our histopathological results shown that the liver in thyroidectomized rats revealed a variety of histopathological lesions when compared with control or sham operating groups. Our results are in agreement with a number of studies which provided evidence that hypothyroidism causes an adverse effect on the human health [5,8,45,46]. Our results are in not agreement with Cano-Europa et al. [14] who reported that thyroidectomy is not effected on the structures of some organs as spleen, liver, lung, kidney and heart. Numerous reports suggest that hypothyroidism may have features that mimic liver disease, such as myalgias, fatigue and muscle cramps in the presence of an elevated AST from myopathy [47]. An obvious sign of hepatic injury is the leakage of cellular enzymes into the plasma due to the disturbance in the transport function of hepatocytes. When liver cell membrane is damaged, a variety of enzymes located normally in cytosol is released into blood stream, causing increased enzyme level in the serum. The estimation of these enzymes in the serum is a useful quantitative marker for the extent and type of hepatocellular damage [48]. Our histological observations basically supported the results obtained from serum enzyme assays. Immunohistochemical observations of the liver tissues showed a significant increase of the PCNA immunoreactivity after thyrodectomy. The expression of the nucleic PCNA were very low in the control liver and sham sections. A significant increase in the expression of the nucleic PCNA was observed in the liver sections in thyroidectomized rats, comparing with the control one, this may attributed to response of hepatocytes to liver damage. Also, treatment of thyroidectomized rats with GLE significantly decreased PCNA immunoreactivity when compared with the thyroidectomized rat group. This suggests that liver cells of rats after GLE treatment have stronger replicative activity. This result is in agreement with Sakr et al. [49] who reported that proliferating cell nuclear antigen (PCNA) elevated in hepatocytes
of male albino rats injected intraperitioneally with a 12-fold dose range of thioacetamide fungicide. Also, this result is in accordance with that of He et al. [50] who reported an increase in PCNA-positive cells in the liver of mice treated with fumonisin B1 (FB1) and the number of PCNA-positive cells increased five-fold in silymarin plus FB1-treated mice compared to FB1 only group. The present results suggested that fenugreek extract both decreased the cellular damage and increased the regeneration of liver when co-administered with ADR. Treatment of thyroidectomized rats with GLE improved this changes in liver structure and functions, where it decreased the cellular damage and increased the regeneration of liver after thyroidectomy. References [1] Toshihiro I. Thyroid hormone and atherosclerosis. Vasc Pharmacol 2010;52: 151–6. [2] Ibrahim W, Tousson E, Ali EM, Mansour MA. 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Original article
Thyroidectomy induced hepatic toxicity and possible amelioration by Ginkgo biloba leaf extract Ehab Tousson a,b,∗ , Areej J.M. Alghabban a , Heba Abou Harga c a b c
Biology Department, Faculty of Science, Tabuk University, Tabuk 71491, Saudi Arabia Zoology Department, Faculty of Science, Tanta University, Tanta 31527, Egypt Damenhour Fever Hospital, Damenhour, Egypt
a r t i c l e
i n f o
Article history: Received 12 April 2014 Accepted 6 June 2014 Keywords: Thyroidectomy Liver Toxicity Histopathology PCNA immunoreactivity Rat
a b s t r a c t The liver is a major target organ for thyroid hormone action and marked changes occur in liver functions in the case of hypo- or hyperthyroidism. This studied aimed at inverstigating the biochemical and histopathological changes in the liver after thyroidectomy and the ameliorating role of Ginkgo biloba leaf extract (GLE). A total of 50 male albino rats were equally divided into five groups; 1st to 3rd groups were control, sham operated and GLE groups while 4th group was thyroidectomized rat group and 5th group was treated thyrodectomized rat with GLE. Serum T3 and T4 levels after 3 weeks of thyroidectomy was significantly decreased when compared with the control group, while TSH significantly increased when compared with the control group increased. Serum ALT, AST, ALP and GGT showed significant (P < 0.05) increase in thyroidectomized group when compared with control, sham operated and sham operated with GLE groups. On the one hand, treatment of thyroidectomized rats with GLE improved this increase in serum ALT, AST, ALP and GGT in thyroidectomized rat group. Liver sections of thyroidectomy group showed marked positive reaction and increase number of PCNA staining of hepatocyte nuclei. On the other hand, liver in treated of thyroidectomized rat with GLE group showed a marked reduction in the number of PCNA-positive nuclei when compared with sections in thyroidectomy group. Treatment of thyroidectomized rat with GLE improves the biochemical, histopathological and immunohistochemical alternations and the intensity of PCNA immunoreactive cells demonstrating the recovery of some injury. © 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction Thyroid hormones regulate all metabolic activities, such as growth rate, sodium/potassium pump, cholesterol secretion in the bile, heart rate, blood pressure, respiration, oxygen consumption, digestion strength, lipid, carbohydrate and protein metabolism, central nervous system function, and the actions of other endocrine glands [1–12]. A thyroidectomy is an operation that involves the surgical removal of all or part of the thyroid gland. Surgeons often perform a thyroidectomy when a patient has thyroid cancer or some other condition of the thyroid gland (such as hyperthyroidism) or goiter. Hypothyroidism is caused by thyroid hormone deficiency. Hypothyroidism was induced in male rats by near-total thyroidectomy because this is a well-established model to study early vascular effects of hypothyroidism [1–11,13–15]. It is unclear
∗ Corresponding author at: Corresponding author. Biology Department, Faculty of Science, Tabuk University, Tabuk 71491, Saudi Arabia. Tel.: +966 53 65 19 175. E-mail addresses: [email protected], [email protected] (E. Tousson). http://dx.doi.org/10.1016/j.bionut.2014.06.001 2210-5239/© 2014 Elsevier Masson SAS. All rights reserved.
whether this is a direct thyroid effect on liver enzymes or secondary to altered intestinal handling of cholesterol and bile acids [16]. There is also evidence that hypothyroidism may directly affect the liver structure or function. Hypothyroidism has been associated in a few case reports with cholestatic jaundice attributed to reduced bilirubin and bile excretion. The triad of reduced bilirubin excretion, hypercholesterolemia and hypotonia of the gall bladder seen in hypothyroidism increases the incidence of gallstones [17]. Many traditional treatments have been recommended in the complementary and alternative treatment of various illnesses. They may prevent disease, reduce the risk of developing disease or enhance general health [18,19]. Herbal medicine is increasingly gaining acceptance from the public and medical professionals due to advances in the understanding of the mechanisms by which herbs positively influence health and quality of life [20,21]. Ginkgo biloba (maiden hair tree) is one of the oldest herbal medicines that have been used as a therapeutic agent in modern pharmacology. Standardized extracts from dried Ginkgo leaves take also important place in modern medicine [21]. G. biloba leaf extract (GLE) is standardized to contain approximately 24% flavone glycosides and 6% terpene lactones. These compounds are extracted from the tree’s
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healthy green leaves. The flavone glycosides in Ginkgo are primarily composed of quercetin, kaempferol, and isorhamnetin. These bioflavonoid constituents are thought to be the compounds that provide Ginkgo’s antioxidant effects. Ginkgo has been shown to be active against a wide variety of free radicals and free radical generating substances including nitric oxide, the superoxide, hydroxyl, oxoferryl and peroxyl radicals [22]. Popularly marketed to the general public, G. biloba extract is believed to provide beneficial effects in memory impairment, stroke, edema, inflammation, Alzheimer’s dementia, and vaso-occlusive disorders [23–30]. The liver plays an astonishing array of vital functions in the maintenance, performance and regulating homeostasis of the body. It is involved with almost all the biochemical pathways to growth, fight against disease, nutrient supply, energy provision and reproduction [31]. It functions as a center of metabolism of nutrients, such as carbohydrates, proteins and lipids and excretion of waste metabolites. Based on these evidences, the present study was conducted to examine the possible modifying effects of Ginkgo leaf extract (GLR) against hepatic toxicity induced by thyroidectomy in male rats. This could be fulfilled through the histological and biochemical analysis of liver tissues. 2. Materials and methods The experiments were performed on 50 male Wistar rats weighing 130 ± 10 g. They were obtained from the farm of Helwan, Egypt. The rats were kept in the laboratory for one week before the experimental work and fed a standardized diet (Barley) ad libitum. The temperature in the animal room was maintained at 23 ± 2 ◦ C. The laboratory cycle was 12:12-h light-dark cycle. Rats were anesthetized by intraperitoneal injection with thiopental sodium (50 mg/kg; EIPICO Co., Egypt) for sham operation or thyroidectomy and after surgery, the animals were given Bivatracin (Neomycin, Bacitracin: Topical antibiotic aerosol powder spray; ECAP Co., Egypt) two times/day for 6 days. The experimental protocol was approved by Local Ethics Committee and Animals Research. 2.1. Animal treatments The rats were randomly and equally divided into the following groups: • group I: (GI ) control group – thirty rats were fed a standardized diet ad libitum and dissecting after 3 weeks; • group 2: (G2 ) sham operated group – rats were subjected to sham operation and did not received any treatment and dissecting after 3 weeks; • group 3: (G3 ) sham operated and G. biloba leaf extract treated group (GLE) – rats were subjected to sham operation and from 3rd day orally treated with GLE (0.11 g/kg body weight) for 3 weeks rats; • group 4 (G4 ) thyroidectomized group – rats were surgically subjected to thyroidectomy [10,32] and dissecting after 3 weeks; • group 5: (G5 ) treated thyroidectomized rats with GLE – rats were subjected to thyroidectomy and from 3rd day orally treated with GLE for 3 weeks rats. 2.2. Thyroidectomy Thyroidectomy was performed on rats anesthetized with intraperitoneal injection with sodium pentobarbital and subjected to a complete necropsy according to the method of Tousson et al. [10] and Francisco et al. [32]. Briefly, by using a stereomicroscope (Zeiss, Germany) for better observation, a midline skin incision was made along the length of the neck. The underlying tissues were
removed, and the salivary glands were retracted laterally. The two halves of the sternohyoid muscle were separated and retracted laterally. The thyroid muscle was separated from the lobes of the thyroid gland and retracted along with the sternohyoid muscle. A midline cut was made in the isthmus, and the thyroid glands were excised bilaterally. Extreme care was taken not to damage the laryngeal nerve. Sham (euthyroid/control) operated animals underwent the same surgical procedures without removal of the thyroid gland. After surgery, ketorolac (Sintex-Mexico) (50 mg/kg im) and gentamicin (Shering Plough-Mexico) (10 mg/kg) were administered over 5 days to alleviate pain and prevent infection. 2.3. Extract preparation The extraction procedure for G. biloba leaf extract (GLE) was carried out as reported by Lichtblau et al. [51]. 2.4. Sample preparation Animals were fasted overnight and for clinical chemistry blood samples from each rat were collected from the eyes by retro-orbital puncture using blood capillary tubes (with and without heparin as per requirement) under mild ether anesthesia. Blood samples that collected without heparin was incubated at room temperature for 10 min and left to clot then centrifuged at 3000 rpm for 10 min and the serum were collected, serum was separated and kept in clean stopper plastic vial at –80 ◦ C until the analysis of serum parameters. 2.5. Biochemical analysis Estimation of serum tri-iodothyronine (T3) was assayed by using commercial test supplied by the Diagnostic systems Laboratories (Taxes, USA) according to the method of Chopra et al. [33]. Estimation of serum thyroxine (T4 ) was assayed by using kits for these hormones were obtained from Calbiotech INC (CBI), USA according to the methods of Thakur et al. [34]. Estimation of serum thyrotropin (TSH) was assayed by using commercial Kit supplied by Coat-A-Count TSH IRMA (Los Angeles, USA) according to the method of Engall et al. [35]. Estimation of liver enzymes, alanine transaminase (ALT) and aspartate transaminase (AST) activities in serum were assayed by using commercial kit that was supplied by Randox (Egypt) according to the method of Reitman and Frankel [36]. Concentration of total and direct bilirubin was spectrophotometrically determined using commercial diagnostic kits supplied by Human (German) according to the method of Pearlman and Lee [37]. Determination of alkaline phosphatase (ALP) and gamma-glutamyltranspeptidase (GGT) was according to the method of Bessey et al. [38] and Rosalki [39], respectively. 2.5.1. Histopathological examination Immediately after decapitation animals were dissected, liver from different groups were quickly removed, washed using chilled saline solution and fixed in 10% neutral buffered formalin. After fixation, specimens were dehydrated in an ascending series of alcohol, cleared in two changes of xylene and embedded in molten paraffin (mp 50–58 ◦ C). Sections of 5 microns thickness were cut using rotary microtome and mounted on gelatin chromalum-coated glass slides. Some of liver sections were stained with Ehrlich’s haematoxylin and counterstained with eosin as a routine method after Bancroft and Stevens [40]. The rest of the sections were stored at room temperature until further processing. 2.5.2. Immunohistochemical detection of PCNA The rest of paraffin-embedded rat liver sections were deparaffinized and hydrated for PCNA immunoreactivity [6]. Endogenous
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Table 1 Changes in the serum tri-iodothyronine (T3 ), thyroxine (T4) and thyrotropin (TSH) levels in the different groups under study.
T3 level (ng/dL) T4 (ng/dL) TSH level (IU/mL)
G1
G2
G3
G4
G5
46.4 ± 2.50a
45.8 ± 3.13a
44.0 ± 2.75a
25.31 ± 1.75b
44.3 ± 1.05a
2.85 ± 0.046a
2.54 ± 0.04a
2.63 ± 0.19a
1.33 ± 0.24b
2.06 ± 0.09a,b
1.33 ± 0.19a
1.30 ± 0.29a
1.33 ± 0.14b
4.56 ± 0.32b
2.24 ± 0.27a
Data are expressed as mean ± SEM for 10 observations. a Significant difference from the thyroidectomized group (G4 ) at P < 0.05, where G1 , control group; G2 , sham operated group; G3 , sham operated and treated with GLE group; G4 , thyroidectomized rats group; G5 , treated thyroidectomized rats with GLE group. b Significant difference from the control group (G1 ) at P < 0.05. Table 2 Body weight gain (BWG) and relative liver weight (RLW) in different groups under study.
BWG (g) RLW (g/100 g)
G1
G2
G3
G4
G5
39.7 ± 2.4a 3.5 ± 0.53
38.3 ± 1.55a 3.4 ± 0.18
39.1 ± 2.24a 3.4 ± 0.42
34.7 ± 2.3b 3.4 ± 0.19
36.8 ± 2.15a,b 3.5 ± 0.06
RLW = (liver weight/final body weight) × 100, data are expressed as mean ± SEM for 10 observations. a Significant difference from the thyroidectomized group (G4 ) at P < 0.05, where G1 , control group; G2 , sham operated group; G3 , sham operated and treated with GLE group; G4 , thyroidectomized rats group; G5 , treated thyroidectomized rats with GLE group. b Significant difference from the control group (G1 ) at P < 0.05.
peroxidase activity was blocked by incubation using 3% H2 O2 for 5 min. The tissue sections were incubated over night with proliferating cell nuclear antigen (PCNA) monoclonal antibody (Dako Corporation, Carpentaria, CA, USA) and washed with phosphate buffer saline (PBS) for 5 min. The monoclonal antibody was then linked with biotinylated goat anti-mouse IgG antibody (Daco, LASB Universal Kit) for 30 min. After being washed with PBS for 5 min, the sections were incubated with streptavidin-conjugated peroxidase for 30 min. A brown coloured reaction was developed by exposing sections to 3,3-diaminobenzidine (DAB) tetrahydrochloride solution for 5 min and washed in distilled water. Sections were counterstained with haematoxylin and eosin [41]. Cells were considered positive for PCNA if there was brown nuclear staining of the cells and negative (nuclei not stained) appeared blue. The number of PCNA-positive cells was counted in 10 randomly selected sections and nonoverlapping fields and expressed as the number of PCNA-positive cells/mm2 . All stained slides were viewed by using Olympus microscope and images were captured by a digital camera (Cannon 620). Brightness and contrast were adjusted using Adobe Photoshop software. Image analysis was adjusted using PAXit image analysis software. The data was statistically analyzed using SPSS. 2.5.3. Statistical analysis Data were expressed as mean values ± SE and statistical analysis was performed using one way ANOVA to assess significant differences among treatment groups. The criterion for statistical significance was set at P < 0.05 for the biochemical data. All
statistical analyses were performed using SPSS statistical version 16 software package (SPSS® Inc., USA). 3. Results 3.1. Toxicity The animals under practice appeared healthy and did not show clinical signs of disease and no mortality was recorded during the experiment duration. The dose of Ginkgo leaf extract (GLE) did not initiate any side effects for the animals, whereas various side effects were observed in thyroidectomized animals, such as loosing of loss of activity, body weight, and weakness. 3.2. Changes in thyroid hormones Table 1 showed that no changes in the serum T3 , T4 and TSH levels in sham operated (G2 ) and sham operated and GLE (G3 ) groups as compared with the control (G1). Serum T3 and T4 levels in thyroidectomized rats (G4 ) were significantly (P < 0.05) decreased as compared with the control (G1), sham operated (G2 ) and sham operated and GLE (G3 ) groups (Table 1). On the other hand, serum TSH levels were significantly (P < 0.05) increased in thyroidectomized rats (G4 ) when compared with the control (G1), sham operated (G2 ) and sham operated and GLE (G3 ) groups. Treatment of thyroidectomized rats with GLE (G5 ) significantly (P < 0.05) increased T3 and T4 and significantly (P < 0.05) decreased TSH levels when compared with the thyroidectomized rats (Table 1).
Table 3 Changes in ALT (U/L), AST (U/L), total bilirubin (mg/dL), ALP (U/L) and GGT (U/L) in different groups under study. G1 ALT AST Total bilirubin ALP GGT
28.16 31.52 0.16 39.3 55
G2 ± ± ± ± ±
a
2.44 1.15a 0.06 1.55a 1.2a
31.55 32.44 0.19 37.5 55.8
G3 ± ± ± ± ±
a
1.84 2.05a 0.06 2.16a 2.91a
29.2 33.65 0.17 45.5 50.4
G4 ± ± ± ± ±
a
2.07 2.11a 0.06 2.75a 2.32a
58.52 63.45 0.23 109.8 100
G5 ± ± ± ± ±
b
2.42 2.47b 0.02 4.16b 2.42b
39.41 38.29 0.21 68.1 76.5
± ± ± ± ±
2.61a 3.06a 0.03 4.07a 1.53a,b
Data are expressed as mean ± SEM for 10 observations. a Significant difference from the thyroidectomized group (G4 ) at P < 0.05, where G1 , control group; G2 , sham operated group; G3 , sham operated and treated with GLE group; G4 , thyroidectomized rats group; G5 , treated thyroidectomized rats with GLE group. b Significant difference from the control group (G1 ) at P < 0.05.
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Fig. 1. A–F. Photomicrographs of rat liver sections of different experimental groups stained with haematoxylin and eosin. A–C. Liver sections of control, sham operated and sham operated treated with GLE groups. D and E. Liver sections of thyroidectomized rats group. F. Liver sections of treated thyroidectomized rats with GLE group (CV, central vein; PV, portal vein).
3.3. Body weight gain and relative weight of liver The data summarized in Table 1 indicates that a significant (P < 0.05) decrease in body weight gain in thyroidectomized rats group (G4 ) when compared with control (G1 ), sham operated (G2 ) and sham operated with GLE (G3 ) groups. However, treatment of thyroidectomized rats with GLE improved this decrease in body weight gain in thyroidectomized rat group (G4 ). On the other hand, no significant change in the relative liver weight (RLW) were observed in the different groups under study (Table 2). 3.4. Changes in liver enzymes Table 3 showed that serum ALT, AST, ALP and GGT showed significant (P < 0.05) increase in thyroidectomized group (G4 ) when
compared with control (G1 ), sham operated (G2 ) and sham operated with GLE (G3 ) groups. On the other hand, treatment of thyroidectomized rats with GLE improved this increase in serum ALT, AST, ALP and GGT in thyroidectomized rat group (Table 3). In contrast, no significant change in the total bilirubin was detected in the different groups under study (Table 3). 3.5. Effect of GLE on histopathology Liver section in rats is divided into hepatic lobules formed of radially arranged strands of hepatocytes that extend from the central vein to the periphery of the lobule. Liver sections in control (G1 ), sham operated (G2 ) and sham operated and GLE (G3 ) groups showed normal structure of hepatocytes where the hepatocytes are polygonal in shape with eosinophilic granular cytoplasm and
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Fig. 2. A–F. Photomicrographs of rat liver sections stained by PCNA immunoreactivity in different group under study. A–C. Control, sham operated and sham operated treated with GLE groups showed the negative immune reaction indicated by blue stains due to normal central hepatic vein and hepatocytes. D and E. Liver sections in thyroidectomized rats group showed strong positive reaction for PCNA in most of hepatocytes nuclei (black arrows). F. Liver sections in treated thyroidectomized rat with GLE group showed a marked reduction in the number of PCNA-positive nuclei when compared with sections in of thyroidectomy group.
vesicular basophilic nuclei (Fig. 1A–C). Some of the hepatocytes are binucleated and the liver cords are separated from each other by narrow blood sinusoids lined with endothelial cells and von Kupffer cells (Fig. 1A–C). Liver sections of thyroidectomy group (G4 ) showed mild changes in liver hepatocytes as dilation or congestion in central veins, atrophied and vacuolated hepatocytes, degeneration with focal area, focal necrosis and dilated sinusoid with proliferated Kupffer cells (Fig. 1D and E). Liver sections in the treatment of thyroidectomized rat with GLE group (G5 ) shows a moderate degree of improvement in hepatocytes where a few atrophied and/or vacuolated hepatocytes, dilated congested sinusoids and no interlobular hemorrhage were observed (Fig. 1F). 3.6. Effect of GLE on PCNA-ir Liver sections of normal control, sham operated (G2 ) and sham operated and GLE (G3 ) groups showed a few hepatocytes nuclei
that displayed faint stain of PCNA (Fig. 2A–C). Liver sections of thyroidectomy group (G4 ) showed marked positive reaction and increase number of PCNA staining of hepatocyte nuclei (Fig. 2D and E). On the other hand, liver sections in treatment of thyroidectomized rat with GLE group (G5 ) showed a marked reduction in the number of PCNA-positive nuclei especially in peripheral zones compared with sections in of thyroidectomy group (Fig. 2F). 4. Discussion Thyroid hormones are involved in the regulation of numerous body functions, including lipid and carbohydrate metabolism, oxygen consumption and several physiological functions, such as development, reproduction, and growth [1–12]. The present study was designed to investigate the possible modifying effects of Ginkgo leaf extract (GLR) against hepatic toxicity induced by thyroidectomy in male rats. In order to ensure the thyroidectomy that leads
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to hyperthyroid state, we regularly determined the serum T3 , T4 and TSH through the dose period where serum T3 and T4 levels is increased and serum TSH levels is depressed in thyroidectomized rats, indicating the induction of hyperthyroid state. In the present study, Serum T3 and T4 levels in treated thyroidectomized rats with GLE were significantly decreased when compared with the thyroidectomized rats group while TSH levels in treated thyroidectomized rats with GLE were significantly increased when compared with the hyperthyroid group. Treatment of thyroidectomized rats with GLE improved these changes in thyroid hormones and leads to euthyroid state. This finding is compatible with Tenorio-Velasquez et al. [13], Tousson et al. [10], Ali et al. [15] and Francisco et al. [32] that used thyroidectomy to achieve hypothyroid state and hypothyroidism was confirmed biochemically in thyroidectomized rats by a pronounced elevation in serum TSH. The increase in TSH level can be explained by a decreased production of T3 from the thyroid gland that minimizes TSH feedback inhibition; resulting in an increase in its secretion by the anterior pituitary gland [42]. The present results compatible with Yatvin et al. [43] who reported that thyroidectomy reduce food intake, decrease body weight gain, liver weight, total protein and relative liver protein in rats. It is unclear whether this is a direct thyroid effect on liver enzymes or secondary to altered intestinal handling of cholesterol and bile acids [16]. Clinical diagnosis of disease and damage to the structural integrity of liver is commonly assessed by monitoring the status of serum AST and ALT activities. In the present study, the liver enzymes (AST, ALT, ALP and GGT) elevation may be attributed to myopathies, which are usually associated with hypothyroidism [44]. The result of serum total bilirubin concentration in the present study was unexpected, where there are no significant changes of its level in thyroidectomized group when compared with control or sham operated or treated with GLE groups. This finding is not compatible with Malik and Hodgson [44]. Our histopathological results shown that the liver in thyroidectomized rats revealed a variety of histopathological lesions when compared with control or sham operating groups. Our results are in agreement with a number of studies which provided evidence that hypothyroidism causes an adverse effect on the human health [5,8,45,46]. Our results are in not agreement with Cano-Europa et al. [14] who reported that thyroidectomy is not effected on the structures of some organs as spleen, liver, lung, kidney and heart. Numerous reports suggest that hypothyroidism may have features that mimic liver disease, such as myalgias, fatigue and muscle cramps in the presence of an elevated AST from myopathy [47]. An obvious sign of hepatic injury is the leakage of cellular enzymes into the plasma due to the disturbance in the transport function of hepatocytes. When liver cell membrane is damaged, a variety of enzymes located normally in cytosol is released into blood stream, causing increased enzyme level in the serum. The estimation of these enzymes in the serum is a useful quantitative marker for the extent and type of hepatocellular damage [48]. Our histological observations basically supported the results obtained from serum enzyme assays. Immunohistochemical observations of the liver tissues showed a significant increase of the PCNA immunoreactivity after thyrodectomy. The expression of the nucleic PCNA were very low in the control liver and sham sections. A significant increase in the expression of the nucleic PCNA was observed in the liver sections in thyroidectomized rats, comparing with the control one, this may attributed to response of hepatocytes to liver damage. Also, treatment of thyroidectomized rats with GLE significantly decreased PCNA immunoreactivity when compared with the thyroidectomized rat group. This suggests that liver cells of rats after GLE treatment have stronger replicative activity. This result is in agreement with Sakr et al. [49] who reported that proliferating cell nuclear antigen (PCNA) elevated in hepatocytes
of male albino rats injected intraperitioneally with a 12-fold dose range of thioacetamide fungicide. Also, this result is in accordance with that of He et al. [50] who reported an increase in PCNA-positive cells in the liver of mice treated with fumonisin B1 (FB1) and the number of PCNA-positive cells increased five-fold in silymarin plus FB1-treated mice compared to FB1 only group. The present results suggested that fenugreek extract both decreased the cellular damage and increased the regeneration of liver when co-administered with ADR. Treatment of thyroidectomized rats with GLE improved this changes in liver structure and functions, where it decreased the cellular damage and increased the regeneration of liver after thyroidectomy. References [1] Toshihiro I. Thyroid hormone and atherosclerosis. Vasc Pharmacol 2010;52: 151–6. [2] Ibrahim W, Tousson E, Ali EM, Mansour MA. 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