Toxeminar-1, Feb 22, 2009 Biology and Medicine, 1 (2): 87-99, 2009 eISSN: 09748369, www.biolmedonline.com Protective role of green tea extract against genotoxic damage induced by anabolic steroids in cultured human lymphocytes Jyoti Gupta, Yasir Hasan Siddique, Tanveer Beg, Gulshan Ara, Mohammad Afzal* Human Genetics and Toxicology Laboratory, Section of Genetics, Department of Zoology, Aligarh Muslim University, Aligarh 202002 (UP), India. *Corresponding author: [email protected], [email protected] Abstract Tea (Camellia sinensis) is one of the most popular beverages consumed worldwide as an infusion of leaves and is valued for its medicinal properties. Tea is a rich source of polyphenols called flavonoids, effective antioxidants found throughout the plant kingdom. The slight astringent, bitter taste of green tea is attributed to polyphenols. A group of flavonoids in green tea are known as catechins, which are quickly absorbed into the body and are thought to contribute to some of the potential health benefits of tea. The fresh tea leaves contain four major catechins as colourless water soluble compounds. epicatechin (EC), epicatechin gallate (ECG), epigallocatechin (EGC) and epigallocatechin gallate (EGCG). Epidemiologic observations and laboratory studies have indicated that tea polyphenols act as antioxidants in vitro by scavenging reactive oxygen and nitrogen species and chelating redox- active transition metal ions and hence tea may reduce the risk of a variety of illnesses, including cancer and coronary heart disease. In this study we seen the antigenotoxic effect of green tea extract against genotoxic damage induced by two anabolic steroids Trenbolone and Methyltestosterone in cultured human lymphocytes, both in absence and presence of metabolic activation. The results prove the antigenotoxic potential of green tea extract. Because the epidemiologic studies and research findings in laboratory animals have shown the antigenotoxic potential of tea polyphenols, the usefulness of tea polyphenols for various human diseases like cancer and coronary heart disease etc should be evaluated in clinical trials. Keywords: Green tea extract (GTE); Tea polyphenols; Trenbolone; Methyltestosterone; Antigenotoxicity; Genotoxicity; Chromosomal aberrations; Sister chromatid exchanges. Introduction appears to be the most powerful of the catechins Tea is the second-most consumed beverage in – with antioxidant activity about 25-100 times the world (water is the first) and has been used more potent than vitamins C and E. Tea medicinally for centuries in India and China. The catechins and polyphenols are effective tea shrub (genus Camellia, family Theaceae) scavengers of reactive oxygen species in vitro [chromosome number (2n=30)] is a perennial and may also function indirectly as antioxidants evergreen with its natural habitat in the tropical through their effects on transcription factors and and sub tropical forests of the world. Cultivated enzyme activities (Higdon and Frei, 2003). varieties are grown widely in its home countries Trenbolone is a synthetic steroid used of South and South East Asia, as well as in parts frequently by veterinarians on livestock as a of Africa and the Middle East (Yamamoto et al., promoter of growth in animal husbandry 1997). Green tea is prepared by picking, lightly (Richold, 1988). Trenbolone is not used in an steaming and allowing the leaves to dry unrefined form, but is rather administered as (Werkhoven, 1978). Catechins are highly potent Trenbolone acetate. Trenbolone acetate is often flavonoids present in tea and serve perhaps as referred to as "Fina" by users, because injectible the best dietary source of natural antioxidants. trenbolone acetate is often prepared from Flavonoids are group of phenolic compounds Finaplix H pellets, an ear-implant used by cattle occurring abundantly in vegetables, fruits, and ranchers to maintain the weight of cattle during green plants that had attracted special attention shipping to slaughter. Trenbolone compounds as they showed high antioxidant property (Gupta have not yet been approved by the Food and et al., 2008).Several catechins are present in Drug Administration, USA for use by humans significant quantities; epicatechin (EC), due to their considerable negative side effects, epigallocatechin (EGC), epicatechin gallate although bodybuilders use the drug illegally to (ECG) and epigallocatechin gallate (EGCG) increase body mass and strength. Trenbolone (Zhu et al., 2000) [Figure 1]. EGCG makes up compounds increase nitrogen uptake by about 10-50% of the total catechin content and muscles after metabolization, leading to Toxeminar-1, Feb 22, 2009 Biology and Medicine, 1 (2): 87-99, 2009 increased rate of protein synthesis. Trenbolone affinity for the androgen receptor three times as is a very potent androgen with strong anabolic high as that of testosterone (Beg et al., 2007). activity. It is well suited for the rapid buildup of Cases of prostate and hepatic cancers have strength and muscle mass, usually providing the been associated with long term anabolic steroid user exceptional results in a relatively short time abuse (Roberts and Essenhigh ,1986; Overly et period. Trenbolone compounds have a binding al., 1984). Trenbolone C H O Molecular weight 270.366 18 22 2 17β-Hydroxyestra-4,9,11-trien-3-one Methyltestosterone is a 17-alpha-alkylated testosterone deficiency. It is also used in women anabolic steroid used to treat men with a to treat breast cancer, breast pain, swelling due Toxeminar-1, Feb 22, 2009 Biology and Medicine, 1 (2): 87-99, 2009 to pregnancy. Methyltestosterone capsules USP (Dewhurst and Gordon, 1984). Orally active (17- 10 mg are red capsules imprinted “ICN 0901” on alpha substituted) anabolic steroids can cause both sections. People who abuse abnormalities of hepatic function, manifest as Methyltestosterone could suffer from acute abnormally elevated hepatic enzyme activity in poisoning and may also be at risk of death from biochemical tests of liver function, and premature heart disease or cancer. A case of sometimes as overt jaundice. The histological night blindness was reported after continuous abnormality of peliosis hepatis has been use of Methyltestosterone (Nisbett et al., 1985). associated with anabolic steroid use (Soe et al., For women, long term effects include voice 1992). Angiosarcoma (Falk et al, 1979) and a changes and in children, fusion of the epiphyses case of hepatocellular carcinoma in an anabolic in children. Androgen ingestion by a pregnant steroid user has been reported (Overly et al., mother can cause virilization of a female fetus 1984). Methyltestosterone C H O , Molecular Weight. 302.46 20 30 2 17-β-hydroxy-17-methylandrost-4-en-3-one by soaking samples (30g of dry weight) in 300 Materials and Methods ml of acetone for 8-10 h at 40-60ºC in Soxhlet’s apparatus. After filtration, the excess of solvent Chemicals was removed by rotatory evaporator. The extract Methyltestosteone (CAS No. 58-18-4, Sigma- is labeled as Green tea extract (GTE). The Aldrich); Trenbolone (CAS No.: 10161-33- extract concentrations of 1.075X 10-4, 2.127 X 8,Sigma-Aldrich); Sodium Phenobarbitone 10-4 and 3.15 X 10-4 g/ml of culture medium (Sigma-Aldrich); Colchicine (Microlab); Dimethyl were established. sulphoxide (Merck); Methyl methane sulphonate(Sigma-Aldrich); RPMI 1640 (GIBCO, Human lymphocyte culture Invitrogen); Phytohaemagglutinin-M (GIBCO, Duplicate peripheral blood cultures were Invitrogen); Antibiotic-antimycotic mixture conducted according to Carballo et al., 1993. (GIBCO, Invitrogen); Fetal serum - calf (GIBCO, Briefly, 0.5 ml of the heparinized blood samples Invitrogen); 5-bromo-2-deoxyuridine (Sigma- was obtained from a healthy donor and was Aldrich); Hoechst 33258 stain (Sigma-Aldrich); placed subsequently in a sterile flask containing Giemsa stain (Merck). 7 ml of RPMI 1640, supplemented with 1.5 ml of fetal calf serum, 1.0 ml antibiotic-antimycotic Preparation of leaf extract mixture and 0.1 ml of phytohaemagglutinin. Camellia sinensis L. leaves were collected from These flasks were placed in an incubator at the nursery of Forest Research Institute (FRI), 37oC for 24 hours. Untreated culture, negative Dehradun (U.A.) and were air dried and and positive controls were run simultaneously. grounded to fine powder. Extraction was done Duplicate peripheral blood cultures were done Toxeminar-1, Feb 22, 2009 Biology and Medicine, 1 (2): 87-99, 2009 and placed in the incubator at 37ºC for 24 hr and Statistical analysis then Green tea extract (GTE) (tested doses Student’s two tailed “t” test was used to were 1.075X 10-4, 2.127 X 10-4 and 3.15 X 10-4 calculate the statistical significance in CAs and g/ml), Trenbolone (tested doses were 40 and 60 SCEs for antigenotoxicity experiment of tea μM) and Methyltestosterone (tested doses were polyphenol EGCG. Kruskall Wallis test was used 40 and 60 μM) were added, separately (Both the for the analysis of the means of frequencies of steroids were dissolved in DMSO). Then, all the SCEs induced by Trenbolone and Docetaxel tested doses of Green tea extract (GTE) were and cell cycle kinetics was analysed by chi- treated with both of the tested doses of square test. Student’s‘t’ test were also Trenbolone and Methyltestosterone, separately. performed. The level of significance was tested For metabolic activation experiments, 0.5 ml of from standard statistical table of Fisher and S9 mix was given with each of the tested dose Yates (1963). for 6 h. S9 mix was prepared according to standard protocol of Maron and Ames (1983). Results Genotoxic effect of a genotoxic steroid Sister chromatid exchange (SCE) analysis Trenbolone and Methyltestosteone was studied For SCE analysis, bromodeoxyuridine (BrdU, 10 using sister chromatid exchanges (SCEs) and μg/ml) was added at the beginning of the Replication Index as genotoxic end points. A culture. After 24 hr of the initiation of culture, dose dependent increase in frequencies of treatments were given similarly as described SCEs and decrease in cell cycle kinetics is above. The cells were collected by centrifugation observed for both Trenbolone and and washed in prewarmed media to remove Methyltestosteone both in presence as well as traces of S9 mix and drugs. One hour before absence of S9 mix (Table 1 & 2). There has harvesting i.e. after 46 h, 0.2 ml of colchicines been an increment in M1 cells and decrease of (0.2 μg/ml) was added to the culture flask for M2 and M3 cells as the doses of Trenbolone mitotic arrest. Cells were centrifuged at 1000 and Docetaxel increase. rpm for 10 min. The supernatant was removed We found that the genotoxicity induced and 8ml of prewarmed (37oC) 0.075 M KCl by Trenbolone and Methyltestosteone, (hypotonic solution) was added. Cells were separately can be countered with 1.075X 10-4, resuspended and incubated at 37oC for 15 min. 2.127 X 10-4 and 3.15 X 10-4 g/ml of Green tea The supernatant was removed by centrifugation, extract (GTE). Frequencies of SCEs were at 1000 rpm for 10 min, and subsequently 5ml reduced when cultures expose to 40 and 60 M chilled fixative (methanol: glacial acetic acid, of Trenbolone, were treated with Green tea 3:1) was added. The fixative was removed by extract (GTE), both in presence as well as centrifugation and the procedure was repeated absence of S9 mix (Table 3 & 4; Fig 1 & 2). twice. The slides were processed according to Replication Index showed an increase when Perry and Wolff (1974), with some modification. cultures expose to 40 and 60 M of Trenbolone, The slides were stained in 3% Giemsa solution were treated with Green tea extract (GTE), both in phosphate buffer (pH 6.8) for 15 min. 200 in presence as well as absence of S9 mix (Table second divisions metaphases per dose were 3 & 4; Fig 3 & 4). Similar results were obtained analysed. when cultures expose to 40 and 60 M of Methyltestosteone, were treated with Green tea Replication Index (RI) extract (GTE), both in presence as well as 100 metaphases per culture were examined. absence of S9 mix (Table 5 & 6; Fig 1, 2, 3, 4). Each metaphase was classified as being in the first (M1), second (M2), or third (M3) division Discussion (Schneider et al., 1981). The replication index Anabolic steroids are a class of steroid (RI) was calculated by formula (Ivett and Tice, hormones related to the hormone testosterone. 1982) as follows: Anabolic steroids have been used by physicians for many purposes as for hypoplastic anemias RI = [(% of cells in M ) + 2(% of cells in M ) + due to leukemia or kidney failure, especially 1 2 3(% of cells in M )] / 100 aplastic anemia,(Basaria et al., 2001) Growth 3 stimulation, to increase lean body mass and prevent bone loss in elderly men etc. (Kenny et al., 2001; Baum and Crespi, 2007; Francis, 2007) They increase protein synthesis within Toxeminar-1, Feb 22, 2009 Biology and Medicine, 1 (2): 87-99, 2009 cells, which results in the buildup of cellular demonstrated ability of genotoxic chemicals to tissue (anabolism), especially in muscles. The induce significant increase in sister chromatid main way in which steroid hormones interact exchanges in cultured cells has resulted in this with cells is by binding to proteins called steroid endpoint being used as indicator of DNA receptors. When steroids bind to these damage in blood lymphocytes of individuals receptors, the proteins move into the cell exposed to genotoxic carcinogens (Albertini et nucleus and either alter the expression of genes al., 2000). The above genotoxic endpoints are (Lavery and McEwan, 2005) or activate well known markers of genotoxicity and any processes that send signals to other parts of the reduction in the frequency of these genotoxic cell (Cheskis, 2004). In the case of anabolic endpoints gives us indication of the steroids, the receptors involved are called the antigenotoxicity of a particular compound (Many androgen receptors. The mechanisms of action products protect against xenobiotics either by differ depending on the specific anabolic steroid. inducing detoxifying enzymes or by inhibiting Different types of anabolic steroids bind to the oxidative enzymes (Morse and Stoner, 1993). androgen receptor with different affinities, The antigenotoxic potential of the plant extracts depending on their chemical structure (Hartgens have been attributed to their total phenolic and Kuipers, 2004). The earlier studies have content (Maurich et al., 2004). It has been shown that various plant extracts and natural shown that, through several mechanisms, tea plant products possess protective role against polyphenols present antioxidant and the genotoxic effects of certain estrogens, anticarcinogenic activities, thus affording several synthetic progestins and anticancerous drugs in health benefits (González de Mejía, 2003; Afzal cultured human lymphocytes (Siddique and et al., 2008).The health benefits of catechins Afzal, 2004; Siddique and Afzal, 2005 a,b; Beg have been studied extensively in humans and in et al., 2007a,b; Siddique et al., 2006 a,b; animal models. The anticarcinogenic potential of Siddique et al., 2007 a-c; Siddique et al.,2008 a- green tea catechins have correlated their c) and mice bone marrow cells (Siddique et cytotoxic effects with the induction of apoptosis, al.,2005; Siddique et al., 2006b; Siddique et al., activation of caspases, inhibition of protein 2008a).Green tea extract (GTE) was studied for kinase, modulation of cell cycle regulation and its antimutagenic effect on the SCEs and RI inhibition of cell proliferation (Yang, 1999). For induced by Trenbolone and Methyltestosterone, cancer prevention, evidence is so overwhelming both in the presence and absence of metabolic that the Chemoprevention Branch of the activation system in human lymphocytes in vitro. National Cancer Institute has initiated a plan for The readily quantifiable nature of sister developing tea compounds as cancer- chromatid exchanges with high sensitivity for chemopreventive agents in human trials revealing toxicant-DNA interaction and the (Siddiqui et al., 2004; Gupta et al., 2008). Table 1. Sister chromatid exchanges (SCEs) and Replication Index (RI) in human lymphocytes treated with Trenbolone and Methyltestosterone, each, in absence of S9 mix. Treatment Cells Scored SCEs/cell RI (M) (mean) Trenbolone 40 200 4.22+ 0.31b 1.72 60 200 5.84  0.46 b 1.69 Methyltestosterone 40 200 4.42  0.32 b 1.69 60 200 5.34  1.29 b 1.67 Untreated 200 2.04  0.12 1.91 Negative control 200 2.52  0.15 1.88 (DMSO, 5 l/ml) Positive control 200 16.62  0.68a 1.65 MMS (6 M) Significant at aP<0.03 Vs Normal Kruskall-Wallis test bP<0.05 with respect to Untreated. SE: Standard Error, DMSO: Dimethylsulphoxide, MMS: Methylmethane sulphonate Toxeminar-1, Feb 22, 2009 Biology and Medicine, 1 (2): 87-99, 2009 Table 2. Sister chromatid exchanges (SCEs) and Replication Index (RI) in human lymphocytes treated with Trenbolone and Methyltestosterone, each, in presence of S9 mix. Treatment Cells Scored SCEs/cell RI (M) (mean) Trenbolone 40 200 5.64 + 0.42 b 1.71 60 200 8.22  0.82 b 1.67 Methyltestosterone 40 200 4.74  0.36 b 1.68 60 200 9.56  0.49 b 1.62 Untreated 200 2.46  0.14 1.89 Negative control (DMSO, 5 l/ml) 200 3.02  0.17 1.87 Positive control 200 22.22  0.95a 1.45 MMS (6 M) Significant at aP< 0.01 Vs Normal Kruskall-Wallis test. bP<0.05 with respect to Untreated. DMSO: Dimethylsulphoxide, MMS: Methylmethane sulphonate Table 3: Effect of GTE on Sister chromatid exchanges (SCEs) and Replication Index (RI) induced by Trenbolone in human lymphocytes without S9 mix. Treatment (M) Cells scored SCEs/cell (Mean  SE) RI GTE (g/ml) 1.075X10-4 200 2.08  0.21 1.82 2.127X10-4 200 2.28  0.23 1.80 3.15X10-4 200 2.60  0.25 1.78 T1(M)+ GTE (g/ml) 40 + 1.075X10-4 200 4.06  0.42 a b 1.73 40 + 2.127X10-4 200 3.12  0.33 a b 1.74 40 + 3.15X10-4 200 2.84  0.31a b 1.76 T2(M) + GTE (g/ml) 60 + 1.075X10-4 200 4.52  0.48 a b 1.70 60 + 2.127X10-4 200 4.44  0.46 a b 1.72 60 + 3.15X10-4 200 4.14  0.42 a b 1.74 Untreated 200 2.04  0.12 1.91 Negative control 200 2.52  0.15 1.88 (DMSO, 5 l/ml) Significant difference: aP<0.01 with respect to untreated bP<0.05 with respect to Trenbolone (Values given in Table1) T1: 40 M Trenbolone; T2: 60 M Trenbolone. GTE: Green tea extract; DMSO:Dimethylsulphoxide; SE: Standard Error. Toxeminar-1, Feb 22, 2009 Biology and Medicine, 1 (2): 87-99, 2009 Acknowledgements Prof. Mohammad Afzal and to the Chairman, Thanks are due to the UGC, New Delhi for Department of Zoology, A.M.U., Aligarh, for awarding the project No.-32-482/2006 (SR) to providing laboratory facilities. ------------------------------------------------------------------------ References Fisher RA, Yates Y,1963. Statistical table for Afzal M, Siddique YH, Beg T, 2008. Mutagen induced biological, agricultural and medical research, 6th genotoxicity and action of tea polyphenolics in edition, Oliver and Boyd, Edinburg, pp 138. mammalian test system: in vitro and in vivo models and methodological considerations. In: Recent Trends Francis RM, 2001. Androgen replacement in aging in Toxicology. Ed. Siddique YH. Transworld Research men. Calcified Tissue International, 69 (4): 235–8. Network, 37/661 (2), fort P.O., Trivandrum, Kerala, India González de Mejía E, 2003. 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Published Siddique YH, Ara G, Beg T, Afzal M, 2006b. Effect of by CRC Press, 1997. ISBN 0849340063, vitamin C on cyproterone acetate induced genotoxic 9780849340062 damage in mice. Research Journal of Biological Sciences, 1, 69-73. Yang CS, 1999. Tea and health. Nutrition. 15(11- 12):946-9. Siddique YH., Ara G, Beg T, Shahi MH, Afzal M, 2006c. Protective role of Ocimum sanctum L. infusion Zhu M, Chen Y ,Li RC, 2000. Oral absorption and against norethynodrel induced genotoxic damage in bioavailability of tea catechins. Planta Medica. cultured human peripheral blood lymphocytes. 66(5):444-7. Journal of Indian Society of Toxicology, 2, 10-14. Toxeminar-1, Feb 22, 2009 Biology and Medicine, 1 (2): 87-99, 2009 Table 4: Effect of GTE on Sister chromatid exchanges (SCEs) and Replication Index (RI) induced by Trenbolone in human lymphocytes with S9 mix. Treatment (M) Cells scored SCEs/cell (Mean  SE) RI GTE (g/ml) 1.075X10-4 200 2.32  0.22 1.80 2.127X10-4 200 2.44  0.24 1.79 3.15X10-4 200 2.70  0.28 1.76 T1 (M)+ GTE (g/ml) 40+1.075X10-4 200 4.48  0.45 a b 1.71 40+2.127X10-4 200 4.44  0.43 a b 1.73 40+3.15X10-4 200 4.28  0.35 a b 1.75 T2 (M)+ GTE (g/ml) 60+1.075X10-4 200 7.32  0.64 a b 1.68 60+2.127X10-4 200 6.52  0.61 a b 1.70 60+3.15X10-4 200 6.02  0.54 a b 1.71 Untreated 200 2.46  0.14 1.89 Negative control 200 3.02  0.17 1.87 (DMSO, 5 l/ml) Significant difference: aP<0.01 with respect to untreated, bP<0.05 with respect to Trenbolone ( Values given in Table2). T1: 40 M Trenbolone; T2: 60 M Trenbolone. GTE: Green tea extract; DMSO:Dimethylsulphoxide; SE: Standard Error. Table 5: Effect of GTE on Sister chromatid exchanges (SCEs) and Replication Index (RI) induced by Methyltestosterone in human lymphocytes without S9 mix. Treatment (M) Cells scored SCEs/cell (Mean  SE) RI GTE (g/ml) 1.075X10-4 200 2.08  0.21 1.82 2.127X10-4 200 2.28  0.23 1.80 3.15X10-4 200 2.60  0.25 1.78 M1 (M)+ GTE (g/ml) 40+1.075X10-4 200 3.14  0.30 a b 1.70 40+2.127X10-4 200 2.36  0.21 a b 1.73 40+3.15X10-4 200 2.24  0.17 a b 1.75 M2 (M)+ GTE (g/ml) 60+1.075X10-4 200 4.44  0.43 a b 1.68 60+2.127X10-4 200 4.22+ 0.31 a b 1.70 60+3.15X10-4 200 3.10  0.31 a b 1.73 Toxeminar-1, Feb 22, 2009 Biology and Medicine, 1 (2): 87-99, 2009 Untreated 200 2.04 0.12 1.91 Negative control 200 2.52  0.15 1.88 (DMSO, 5 l/ml) Significant difference: aP<0.01 with respect to untreated bP<0.05 with respect to Methyltestosterone (Values given in Table1). M1: 40 M Methyltestosterone; M2: 60 M Methyltestosterone. GTE: Green tea extract; DMSO:Dimethylsulphoxide; SE: Standard Error. Table 6: Effect of GTE on Sister chromatid exchanges (SCEs) and Replication Index (RI) induced by Methyltestosterone in human lymphocytes with S9 mix. Treatment (M) Cells scored SCEs/cell (Mean  SE) RI GTE (g/ml) 1.075X10-4 200 2.32  0.22 1.80 2.127X10-4 200 2.44  0.24 1.79 3.15X10-4 200 2.70  0.28 1.76 M1 (M)+ GTE (g/ml) 40+1.075X10-4 200 4.06  0.42 a b 1.70 40+2.127X10-4 200 2.67  0.29 a b 1.72 40+3.15X10-4 200 2.28  0.23 a b 1.74 M2 (M)+ GTE (g/ml) 60+1.075X10-4 200 7.32  0.64 a b 1.63 60+2.127X10-4 200 6.52  0.61 a b 1.64 60+3.15X10-4 200 6.34  0.59 a b 1.66 Untreated 200 2.45  0.14 1.89 Negative control 200 3.01  0.17 1.87 (DMSO, 5 l/ml) Significant difference: aP<0.01 with respect to untreated bP<0.05 with respect to Methyltestosterone (Values given in Table 2). M1:40 M Methyltestosterone; M2: 60 M Methyltestosterone. GTE: Green tea extract; DMSO:Dimethylsulphoxide; SE: Standard Error.