Ameliorating Effect of Green Tea Aqueous Extract on Lead Acetate Induced Thyrotoxicity in Male Wistar Rats
Abdou, Khaled, A.1; Wala A Moselhy 1 and Salah Marwa2 1- Forensic medicine and Toxicology Department, Faculty of Veterinary Medicine, Beni- Suef University, Egypt 2- Biology Department, Faculty of Science, Beni- Suef University, Egypt
Abstract Green tea is known for its antioxidant and anti-carcinogenic effects. However, its effects on the thyroid gland have not been adequately investigated. The present investigation has been designed to evaluate effect of green tea extract (GTE) on the thyrotoxic effect of lead acetate (LA) in adult male Wistar rats. Four groups of rats, 10 animals each, were used in this study (control, LA, LA+GTE and GTE alone). Rats received a daily dose of LA (100 mg/kg) by stomach tube for 30 days. GTE at a dose of 5g/L was provided in the drinking water ad-libitum for the same time. Administration of LA induces marked effect on thyroid function in the treated group compared to control. Co-administration of LA and GTE attenuates the toxic and inhibitory effect of LA alone on serum levels of T3, T4 and TSH. These results were confirmed by microscopic examination of the cellular structure of the thyroid gland of treated rats. LA caused cystic dilatation with flattened lining epithelium in the follicles. Using a standard alkaline comet assay procedure, LA caused significant DNA damage as indicated by visible tail lengths. However, the thyroid damage was significantly reduced in animals received LA and GTE. In addition, GTE reduced DNA migration in of LA treated animals compared to LA alone. In conclusion, the present study suggests that green tea may be useful in combating the thyroid damage due to LA toxicity. However, this is limited study in concentration and duration. More investigation is needed to understand the effect of various concentration of GTE and the chronic effect of exposure. Keywords: green tea extract; thyroid toxicity; lead acetate;thyroid toxicity; Protective effect Corresponding author: E-mail address:
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1- Introduction Heavy metals rank among the most spread environmental contaminants. The lead consumption in the world was steadily increasing over the period 1965-1990 to about 5.6x106 metric tons in 1990 (OECD, 1993). In contemporary world, lead concentrations in the biosphere are 10,000 to 100,000 times above the natural levels (WHO, 1995). Lead (Pb) is considered one of the major environmental pollutants (Casas and Sordo, 2006). Lifestyle factors (e.g. cigarette smoking), certain occupations, proximity to industrial areas, lead mines or smelters, lead based paints, and leaded gasoline significantly contribute to lead pollution of the air, food, water and soil. Lead is absorbed mainly by ingestion and inhalation (WHO, 1995). Likewise, Lead can enter the environment through vehicle and Industry exhausts and sewage sledge application in agriculture (Vogiatizis, 2001). Environmental accumulation with lead has accelerated due to its dose relationship to industrialization 1
and its wide usage in paints and gasoline. The toxicity of many heavy metals is due to their ability to cause oxidative damage to tissues. Damage includes enhanced lipid peroxidation, DNA damage and the oxidation of protein sulfhydral groups (Stohs and Bagchi, 1995). Lead can cause profound hematological, neurological, gastrointestinal, renal, rheumatologic and endocrine manifestations even at levels previously considered safe and there are conflicting reports in the literature regarding the effect of occupational lead exposure on the thyroid functions in experimental animals and humans (Cullen et al., 1983; Pagliuca et al., 1990; Lyons and Pahwa, 2005; Singh et al., 2000; Mokhtari et al., 2007 and Badiei et al., 2009). In recent years, it has been pointed out that a few exogenous environmental pollutants have a disrupting effect on the endocrine system (Jun et al., 2005). Several endocrine effects of lead poisoning including impaired male reproductive function and depressed adrenal and pituitary function have been recognized also it causes functional impairment of pituitary-adrenal axis as well as the pituitary-thyroid axis (Cullen et al., 1984; Erfurth et al., 2001 and Singh and Dhawan, 1999). Lead is genotoxic itself or enhances the effect of other DNA-damaging agents and carcinogenic (Miadokova et al., 1999 ; Vilena et al., 2004; Yedjou et al., 2010 and Tripti and Santosh, 2011). Nowadays attention has been paid to the protective effect of natural antioxidants against chemically induced toxicities (Frei and Higdon, 2003). Green tea is the water extract of the dry leaves of the plant camellia sinensis, and an evergreen shrub of the theaceae family, is a popular beverage commonly known as tea. Tea has been consumed by some human populations for many generations and in some parts of the world, has been considered to have health – promoting potentials. A drink contains many compounds, including a mixture of polyphenols (GTP). The main components of green tea used worldwide as a popular beverage that exert a wide range of biochemical and pharmacological effects, were numerously reported that have significant anti-oxidative anti-mutagenic and anti-carcinogenic activities in the human population (Lung et al., 2002; Yang et al., 1998; Weisburger et al., 1997 and Zowail et al., 2009). The main components of green tea powder (GTP) are catechins, which have a polyphenol structure, including epigallocatechin-3-gallate (EGCG), epigallocatechin (EGC), picatechin-3- gallate (ECG) and epicatechin (EC), all these catechins have strong antioxidant activity (Rice-Evans et al., 1996; Higdon and Frei et al., 2008 and Chuan et al., 2007). Considering the toxic effects of lead acetate and the possible protective role of green tea extract on different types of heavy metals toxicity, we therefore undertook the present study to demonstrate the chelating property of green tea extract against the lead acetate induced on thyroid tissues of male rats.
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2- Materials and Methods 2.1. Chemicals All chemicals used for the present study were of analytical grade and were purchased from Sigma, USA. Lead acetate (C4H6O4pb.H2o) with molecular weight 379.33 in the form of pure crystals, obtained from Riedel-De Haen AG-SeelzeHannover Germany. Green tea was dissolved in the drinking water at a concentration of 5g/L. Tea was freshly prepared three times per week and stored at 4C. the content of drinking vessels was renewed daily. 2.2. Animals Healthy forty mature Wistar male albino rats weighing 150±10 g were obtained from the Breeding unit of the Veterinary Hygiene and Management Department, Faculty of Veterinary Medicine, Cairo University. All rats were housed in standard polypropylene clean cages with dust free wood husk as bedding and kept in wellventilated room with free access to water and standard pellet diet. 2.3. Experimental protocol All animals received humane care and were allowed to acclimatize for 7 days prior to initiation of the experiments. The rats were randomly divided into 4 groups (10 rats/group, 5 rat/cage). The rats of group I received no treatment and kept as control group, rats in the second group received lead acetate treatment (LA-treated group) using stomach tube in a dose of 100mg/kg. b.wt / day for one month according to Zhang et al., (2003); rats in the third group received lead acetate using stomach tube in a dose of 100mg/kg. b.wt / day plus green tea in their drinking water in a concentration of 5g/L for one month and rats in the fourth group received Green Tea extract (GTE) in their drinking water in a concentration of 5g/L for one month (Skrzydlewska et al., 2002). 2.4. Samples collection and preparation To obtain serum, blood samples were collected from the retro-orbital plexus, were placed at room temperature for approximately 30 minutes then the tubes were centrifuged at 3000 r.p.m. for 15 minutes at 4C°, then stored at- 20C° till used for determination of triiodothyronine (T3), tetraiodothronine (T4) and thyroid stimulating hormone (TSH) . Thyroid glands were removed and divided in to two parts one of them was immediately fixed in 10% formalin for histopathological examination and the other one was washed with ice cold saline then immediately immersed in liquid nitrogen and stored at -80C°for DNA fragmentation. 2.5. Measurement of thyroid hormones level: Serum concentration of triiodothyronine (T3), tetraiodothronine (T4) and thyroid stimulating hormone (TSH) were estimated by radioimmunoassay technique, using
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reagent kits purchased from Bayer Corporation (USA), according to the method of Maxon, et al., (1987). 2.6. Histological preparations: Tissue samples of thyroid tissues were fixed in 10% neutral buffered formalin for 24 hours, then specimens were embedded in paraffin wax using conventional method and stained by Harris Haematoxyline and Eosin (Gamble and Bancroft, 2008) for histopathological studies. 2.7. Comet assay: Single cell gel electrophoresis and scoring (Comet assay) was carried out in thyroid tissues according to the protocol described by Singh et al., (1988) with slight modification. Tissues were collected and pressed through a screen in homogenization buffer (0.075 M NaCl and 0.024 M EDTA, pH 7.5) in a ratio of 1g of tissue to 1ml, of buffer then cooled to 4C using Potter-type homogenizer. 6 l of thyroid tissues suspended in 100 l of 0.5 % low-melting agarose (LMA) (Sigma, USA), were pipetted immediately into pre-cleaned a glass microscopic slides having a layer of 300l of 0.6% NMP agarose. After solidification on ice for 10 min, the slides were covered with another layer of o.5 percentage LMP agarose. Then, the slides were immersed in ice-cold lysis solution, consisting of 100m M Na2EDTA, 2.5 Ml NaCl, 10mM Tris-Hcl, and 1% sodium sarcosinate, adjusted to pH 10 with 1% Triton X100 and 10% DMSO, added just prior to use for 1 hour to remove cellular membranes, proteins and so forth before electrophoresis. Slides were then placed in a single row (near the anode) in a horizontal electrophoresis unit filled with an alkaline buffer (1mM Na2EDTA and 300mM NaOH, pH 13) at 4C° for 20 minutes for DNA unwinding. After 20 minutes, the current was switched on and electrophoresis was carried out in freshly prepared alkaline solution at 25 V, 300 mA for 20 minutes at the same temperature (4C). Electrophoresis at high pH results in structures resembling comets, as observed by fluorescence microscopy; the intensity of the comet tail relative to the head reflects the number of DNA breaks. Following removal of slides from lysis solution, the slides were neutralized by adding Tris-buffer (pH 7.5), stained with 200l of ethidium bromide (2 LmL-1), covered and stored in sealed boxes at 4C for analyzing. All preparation steps were conducted under dimmed light to minimize the possibility of cellular DNA damage. Slides were analyzed microscopically by using a Leitz Orthoplan epifluorescence microscope (magnification 250 X) equipped with an excitation filter of 515-560 nm and a barrier filter of 590 nm. The microscope was connected through a camera to a computerbased image analysis system (Comet Assay IV software, Perspective Instruments). Images of 100 randomly selected cells (50 counts on each duplicate slide) were analyzed for each sample. A total of 500 cells of the same kind from each group were analyzed. Comet was randomly captured at a constant depth of gel, avoiding the edges of the gel, occasional dead cells and superimposed comets. DNA damage was measured as tail length (TL= distance of DNA migration from the Centre of the body of the nuclear core) and % of DNA in the tail that migrated during the electrophoresis 4
from the nuclear core to the tail). The comet assay was applied to determine the percentage of damaged DNA concentration in the comet tail by measuring total intensity of ethidium bromide fluorescence in cells. 2.8. Statistical analysis Means, standard deviations and standard errors for experimental parameters were calculated using Microsoft Excell software. Data were presented as mean ± S.E. of 10 animals per group. The software package SPSS 11.0 was used for statistical analysis. The statistically significant differences between various groups were assessed by oneway analysis of variance (ANOVA) followed by multiple mean comparison by Student-Newman-Keul's test. 3- Results 3.1. General observations No mortality associated with LA administration was observed throughout the experimental period and the behavior of the LA-intoxicated rats not be distinguished from that of the controls. 3.2. Thyroid hormones level: Effects of green tea extract (GTE) on serum concentrations of triiodothyronine (T3), tetraiodothronine (T4) and thyroid stimulating hormone (TSH) on normal and lead acetate (LA) intoxicated male Wistar rats are shown in Table 1, Figure, 1 & 2. Serum T3, T4 and TSH concentrations were decreased significantly in LA intoxicated rats compared with the control values (P
