Introduction

EGCG makes up about 10-50% of the total catechin content and appears to be the most powerful of the catechins. In green tea catechin levels ranged from 51.5 - 84.3 mg/g, with epigallocatechin gallate (EGCG) being the main catechin in Chinese and Indian green teas. (EGCG) epigallocatechin gallate, a major catechin of found in green tea, has possible role in chemoprevention and chemotherapy of various types of cancers mainly prostate cancer (₁, ₂) and colon cancer (₃, ₄). EGCG is found to be the major and most photoprotective polyphenolic component of green tea (₅). EGCG also induces growth arrest and apoptosis through multiple mechanisms, and can be used for cancer prevention, mainly pancreatic (₆). Tea catechins and polyphenols are effective scavengers of reactive oxygen species in vitro and may also function indirectly as antioxidants through their effects on transcription factors and enzyme activities (₇).

Trenbolone is a synthetic steroid used frequently by veterinarians on livestock as a promoter of growth in animal husbandry (₈). Trenbolone compounds have not yet been approved by the Food and Drug Administration, USA for use by humans due to their considerable negative side effects, although bodybuilders use the drug illegally to increase body mass and strength. Cases of prostate and hepatic cancers have been associated with long term anabolic steroid abuse (₉,₁₀). Trenbolone compounds increase nitrogen uptake by muscles after metabolization, leading to increased rate of protein synthesis.

Docetaxel is a clinically well established anti-mitotic chemotherapy medication used mainly for the treatment of breast, ovarian, and non-small cell lung cancer (₁₁) Docetaxel has an approved claim for treatment of patients who have locally advanced, or metastatic breast or non small-cell lung cancer who have undergone anthracycline-based chemotherapy and failed to stop cancer progression or relapsed. Docetaxel is marketed worldwide under the name Taxotere by Sanofi-Aventis. Evaluation of docetaxel pharmacokinetics in phase II and III clinical studies were with 100 mg/m² dosages given over one-hour infusions every three weeks Docetaxel is a chemotherapeutic agent and is a cytotoxic compound and so is effectively a biologically damaging drug (₁₂). Because docetaxel is a cell cycle specific agent, it is cytotoxic to all dividing cells in the body.

EGCG was studied for its antimutagenic effect on the CAs and SCEs induced by Trenbolone and Docetaxel, both in the presence and absence of metabolic activation system in human lymphocytes in vitro. The readily quantifiable nature of sister chromatid exchanges with high sensitivity for revealing toxicant-DNA interaction and the demonstrated ability of genotoxic chemicals to induce significant increase in sister chromatid exchanges in cultured cells has resulted in this endpoint being used as indicator of DNA damage in blood lymphocytes of individuals exposed to genotoxic carcinogens (₁₃). The above genotoxic endpoints are well known markers of genotoxicity and any reduction in the frequency of these genotoxic endpoints gives us indication of the antigenotoxicity of a particular compound (₁₄). Many products protect against xenobiotics either by inducing detoxifying enzymes or by inhibiting oxidative enzymes (₁₅).

Materials and Methods

Chemicals

Human lymphocyte culture

Sister chromatid exchange (SCEs) analysis

Replication Index (RI)

Statistical analysis

Results

Genotoxic effect of a genotoxic steroid Trenbolone and an anticancer drug Docetaxel was studied using sister chromatid exchanges (SCE's) and Replication Index as genotoxic end points.A dose dependent increase infrequencies of SCEs and cell cycle kinetics is observed for both Trenbolone and Docetaxel, both in presence as well as absence of S9 mix (Table 1 & 2; Fig 1 & 2). A significant increase in the value of SCEs/cell was observed at 30 µM (P<0.03) of Trenbolone, in presence of metabolic activation. The cell proliferation kinetics which is the average number of cells that have undergone replication showed a significant differences between the cultures exposed to 30 µM of Docetaxel and the normal control (P<0.03). There has been an increment in M1 cells and decrease of M2 and M3 cells as the doses of Trenbolone and Docetaxel increase.

We found that the genotoxicity induced by Trenbolone and Docetaxel, separately can be countered with 20 and 30 µM of Epigallo catechin gallate (EGCG), a major tea polyphenol. Frequencies of SCEs were reduced when cultures expose to 45 and 55 µM of Trenbolone, were treated with EGCG (20 and 30 µM ), both in presence as well as absence of S9 mix (Table 3 & 4; Fig 3 & 4). Similar results were obtained when cultures expose to 3 and 6 µM of Docetaxel, were treated with EGCG (20 and 30 µM ), both in presence as well as absence of S9 mix (Table 5 & 6; Fig 5 & 6).

Figure 1

Table 1: Sister chromatid exchanges (SCEs) and Replication Index (RI) in human lymphocytes treated with Trenbolone and Docetaxel, each, in absence of S9 mix.

Figure 2

Table 2: Sister chromatid exchanges (SCEs) and Replication Index (RI) in human lymphocytes treated with Trenbolone and Docetaxel, each, in presence of S9 mix.

Figure 3

Table 3: Antimutagenic effect of EGCG on SCEs induced by Trenbolone in cultured human lymphocytes without S9 mix.

Figure 4

Table 4: Antimutagenic effect of EGCG on SCEs induced by Trenbolone in cultured human lymphocytes with S9 mix.

Figure 5

Table 5: Antimutagenic effect of EGCG on SCEs induced by Docetaxel in cultured human lymphocytes without S9 mix.

Figure 6

Table 6: Antimutagenic effect of EGCG on SCEs induced by Docetaxel in cultured human lymphocytes with S9 mix.

Figure 7

Figure 1: Sister chromatid exchange (SCEs/cell)(mean) in human lymphocytes treated with Trenbolone and Docetaxel, each, in

N- Normal; NC- Negative control; PC- Positive control MMS (6 µM)

T1- Trenbolone 10 µM; T2- Trenbolone 20 µM; T3- Trenbolone 30 µM;

D1- Docetaxel 10 µM; D2- Docetaxel 10 µM; D3- Docetaxel 30 µM

Figure 8

Figure 2: Sister chromatid exchange (SCEs/cell)(mean) in human lymphocytes treated with Trenbolone and Docetaxel, each, in

N- Normal; NC- Negative control; PC- Positive control MMS (6 µM)

T1- Trenbolone 10 µM; T2- Trenbolone 20 µM; T3- Trenbolone 30 µM;

D1- Docetaxel 10 µM; D2- Docetaxel 10 µM; D3- Docetaxel 30 µM.

Figure 9

Figure 3: Effect of EGCG on SCEs induced by Trenbolone in cultured human lymphocytes

N- Normal; NC-Negative control; T1- Trenbolone 45 µM;

T2- Trenbolone 55 µM; EGCG1- Epigallocatechin gallate 20 µM;

EGCG2- Epigallocatechin gallate 30 µM.

Figure 10

Figure 4: Effect of EGCG on SCEs induced by Trenbolone in cultured human lymphocytes

N- Normal; NC-Negative control; T1- Trenbolone 45 µM;

T2- Trenbolone 55 µM; EGCG1- Epigallocatechin gallate 20 µM;

EGCG2- Epigallocatechin gallate 30 µM.

Figure 11

Figure 5: Effect of EGCG on SCEs induced by Docetaxel in cultured human lymphocytes

N- Normal; NC-Negative control; D1- Docetaxel 3 µM;

D2- Docetaxel 6 µM; EGCG1- Epigallocatechin gallate 20 µM ;

EGCG2- Epigallocatechin gallate 30 µM.

Figure 12

Figure 6: Effect of EGCG on SCEs induced by Docetaxel in cultured human lymphocytes

N- Normal; NC-Negative control; D1- Docetaxel 3 µM;

D2- Docetaxel 6 µM; EGCG1- Epigallocatechin gallate 20 µM ;

EGCG2- Epigallocatechin gallate 30 µM.

Discussions

Plants are the essential source of medicines. Through the advances in pharmacology and synthetic organic chemistry, the dependence on natural products, remain unchanged (₂₁). In India, the majority of populations use traditional natural prepartion derived from the plant material for the treatment of various diseases (₂₃). The earlier studies have shown that various plant extacts and natural plant products possess protective role against the genotoxic effects of certain estrogens, synthetic progestins and anticancerous drugs in cultured human lymphocytes (₁₉, ₂₀, ₂₁, ₂₂, ₂₄, ₂₅, ₂₆, ₂₇, ₂₈). The antigenotoxic potential of the plant extracts have been attributed to their total phenolic content (₂₉). It has been shown that, through several mechanisms, tea polyphenols present antioxidant and anticarcinogenic activities, thus affording several health benefits (₃₀, ₃₁).The health benefits of catechins have been studied extensively in humans and in animal models. The anticarcinogenic potential of green tea catechins have correlated their cyotoxic effects with the induction of apoptosis, activation of caspases, inhibition of protein kinase, modulation of cell cycle regulation and inhibition of cell proliferation (₃₂). For cancer prevention, evidence is so overwhelming that the Chemoprevention Branch of the National Cancer Institute has initiated a plan for developing tea compounds as cancer-chemopreventive agents in human trials (₃₃). In this study we have found that a major tea polyphenol, EGCG, can modulte the genotoxic effects of Trebolone and Docetaxel, indicating its protective role. EGCG is widely accepted as an antioxidant. EGCG scavenges superoxide anion radicals (O₂·–), hydrogen peroxide (H₂O₂), hydroxy radicals (HO·), peroxyl radicals, singlet oxygen, and peroxynitrite (₃₄, _{35 36}, ₃₇, ₃₈, ₃₉). The reactive species liberated by genotoxic steroids and anticancer drugs, before or after metabolic activation, may be responsible for their genotoxic action. Formation of endogenous adducts and certain neoplastic change have also been reported earlier (₄₀, ₄₁). The suggested mechanism of action of tea polyphenols includes the following (₄₂):

• Antioxidant activity and scavenging free radicals.

• Modulating enzymes implicated in the carcinogenic process.

• Modifying the pathways of signal transduction, thereby positively altering the expression of genes involved in cell proliferation, angiogenesis, and apoptosis, all important stages of cancer progression.

• Antimicrobial actions (association between Helicobacter pylori and gastric cancer).

Acknowledgements