Introduction

Epigallocatechin-3-gallate (EGCG), a compound closely related to Epicatechin gallate (ECG), is a catechin and polyphenolic antioxidant plant metabolite found in abundance in various types of tea, derived from the tea plant Camellia sinensis ₁ . It helps protect the skin from ultraviolet radiation-induced genotoxic damage and tumor formation ₂ . Methyl methanesulphonate is a monofunctional alkylating agent having neoplastic and mutagenic properties. It alkylates DNA at the N-7 position of guanine and N-3 position of adenine and these changes may result into abnormal base pairing at DNA replication ₃₄ . Methyl methanesulphonate has been shown to induce sister chromatid exchanges in human lymphocytes ₅₆₇₈ . Cyclophosphamide is also an alkylating agent and after metabolic activation it gives rise to active mutagenic metabolic phospharamide mustard ₉ . It reacts with electron-rich areas of vulnerable molecules like proteins and DNA ₁₀ . EGCG was studied for its antimutagenic effect on the chromosomal aberrations and sister chromatid exchanges induced by methyl methanesulphonate and cyclophosphamide (known mutagens), both in the presence and absence of metabolic activation system in human lymphocytes in vitro. The aim of this study was to assess the antigenotoxic effect of EGCG, a catechin, against anticancer drugs such as MMS and CP, in order to reduce the genotoxic effect of such useful drugs.

Methodology

Chemicals Used: Cyclophosphamide (Sigma-Aldrich, New Delhi); Methyl methanesulphonate (Sigma-Aldrich); Sodium phenobarbitone (Sigma-Aldrich); Colchicine (Microlab, USA); Dimethyl sulphoxide (Merck, New Delhi); Epigallocatechin-3-gallate (CAS No.: 989-51-5, Sigma-Aldrich); RPMI 1640 (GIBCO, Invitrogen, USA); Phytohaemagglutinin-M (GIBCO, Invitrogen); Antibiotic-antimycotic mixture (GIBCO, Invitrogen); Fetal serum - calf (GIBCO, Invitrogen); 5-bromo-2-deoxyuridine (Sigma-Aldrich); Hoechst 33258 stain (Sigma-Aldrich); Giemsa stain (Merck); Mitomycin-C (Sigma-Aldrich); NADP (SRL, New Delhi).

Human Lymphocyte Culture: Duplicate peripheral blood cultures were conducted according to Carballo et al. ₁₁ . First, 0.5 ml of heparinized blood sample was obtained from a healthy donor (with permission of the Human Ethical Committee of the department) and was placed in a sterile tube containing 7 ml of RPMI (medium) 1640, supplemented with 1.5 ml of fetal calf serum and 0.1 ml of phytohaemagglutinin. These tubes were placed in an incubator at 37 ° C for 24 hours. Untreated culture and also negative and positive controls were run simultaneously.

Chromosomal Aberration Analysis: Chromosomal aberration studies were conducted because an increase in the occurrence of aberrations in blood lymphocytes indicates the genotoxic potential of many drugs which are commonly used and also a greater risk of cancer. MMS at 60 µM concentration was dissolved in dimethylsulphoxide and was added later after 24 h. The cells were cultured for another 48 h at 37 ° C keeping them in an incubator. For metabolic activation experiments, 0.5 ml of S9 mix dose was added to the MMS treatment. S9 mix was prepared from the livers of healthy rats (with permission of the institutional Animal Ethical Committee to take liver from Wistar strain rats) as per standard procedure of Maron and Ames ₁₂ . The S9 fraction so obtained was enhanced by addition of 5 µM of NADP and 10 µM of glucose-6-phosphate just before being used to prepare the S9 mix. The S9 mix without NADP was also given with the tested dose of MMS. An amount of 0.2 ml of colchicine (0.2 µg/ml) was added to the culture tube, 1 h prior to harvesting. Cells were centrifuged at 1000 rpm for 10 min. The supernatant was removed and 5 ml of prewarmed (37 ° C) 0.075 M KCl (hypotonic solution) was added. Cells were resuspended and incubated at 37 ° C for 15 min. The supernatant was removed by centrifugation, and, subsequently 5 ml of chilled fixative (methanol: glacial acetic acid, 3:1) was added. The fixative was removed by centrifugation and this step was repeated two more times. To prepare slides, 3-5 drops of the fixed cell suspension were dropped on clean slides and air-dried. The slides were stained in 3% Giemsa solution in phosphate buffer (pH 6.8) for 15 min. About three hundred metaphases were examined for screening the presence of different types of abnormality. The criteria to classify different types of aberrations were in accordance with the recommendation of Environmental Health Committee (EHC) 46 for environmental monitoring of human populations ₁₃ .

Sister Chromatid Exchange Analysis: Bromo-deoxyuridine (BrDU, 10 µg/ml) was added at the beginning of the culture. After 24 h, MMS at the final concentration of 60 µM, earlier dissolved in dimethylsulphoxide, was added and kept for another 48 h at 37 ° C in an incubator. For metabolic activation experiments, 0.5 ml of S9 mix with and without NADP was given along with the tested dose. Mitotic arrest was attempted 1 h prior to harvesting by adding 0.2 ml of colchicine (0.2 µg/ml). Hypotonic treatment and fixation were done in the same way as described for chromosomal aberration analysis. The slides were processed according to Perry and Wolff ₁₄ , and Afzal and Azfer ₁₅ . The sister chromatid exchange induction was analysed by observing at least 50 plates of second division mitoses.

A similar method was used for CA and SCE analysis using CP at 0.16 µg/ml in a separate experiment.

For chromosomal aberration analysis in human lymphocytes treated with MMS in the presence of EGCG, after 24 h of incubation of human lymphocyte culture, MMS (60 µM) was administered, with 10, 20, and 30 µM of EGCG respectively and kept for 48 h at 38 ° C in the incubator. Prior to 1 h of harvesting, 0.2 ml of colchicine (0.2 µg/ml) was added to the culture flasks. Hypotonic treatment, fixation and processing of slides were done as described earlier in the text. At least three hundred metaphases were examined for the occurrence of different types of abnormality i.e. gaps, breaks and exchanges, as recommended by the EHC 46 for environmental monitoring of human populations ₁₃ .

For sister chromatid exchange analysis in human lymphocytes treated with MMS in the presence of EGCG, bromodeoxyuridine (BrdU, 10 µg/ml) was added at the beginning of the culture. After 24 h of the initiation of culture, 60 µM of MMS separately and along with 10, 20, and 30 µM of EGCG were treated and kept for 48 h at 37 ° C in the incubator. Mitotic arrest was done one hour prior to harvesting by adding 0.2 ml of colchicines (0.2 µg/ml). Hypotonic treatment and fixation were performed in the same way as described earlier in the text. The slides were processed according to Perry and Wolff ₁₄ . Sister chromatid exchange average was taken from an analysis of 50 second division metaphases.

A similar method was followed for CA and SCE analysis in human lymphocytes treated with CP (at 0.16 µg/ml along with EGCG at 10, 20, and 30 µM) in a separate experiment.

Statistical Analysis: Student’s two tailed t-test was used for the analysis of chromosomal aberrations and sister chromatid exchanges. The level of significance was tested from standard statistical tables of Fisher and Yates ₁₆ .

Results

Epigallocatechin-3-gallate proved its worth as an antimutagenic agent by substantially reducing the chromosomal aberrations induced by methyl methanesulphonate in the absence of metabolic activation and also those induced by cyclophosphamide in the presence of metabolic activation, but epigallocatechin-3-gallate was more effective in ameliorating the genotoxic damage in the case of cyclophosphamide when metabolic activation system was used (Table 1 and 2).

Figure 1

Table 1: Effect of EGCG on CAs induced by MMS in human lymphocytes without S9 mix.

Significant difference: ^a P<0.01 with respect to untreated, ^b P<0.05 with respect to MMS. SE: Standard Error.

Figure 2

Table 2: Effect of EGCG on CAs induced by CP in human lymphocytes with S9 mix.

Significant difference: ^a P<0.01 with respect to untreated, ^b P<0.05 with respect to CP.

A similar pattern was observed for EGCG when SCEs were induced by MMS without S9 mix and by CP with S9 mix (Table 3 and 4).

Figure 3

Table 3: Effect of EGCG on SCEs induced by MMS in human lymphocytes without S9 mix.

Significant difference: ^a P<0.01 with respect to untreated, ^b P<0.05 with respect to MMS.

Figure 4

Table 4: Effect of EGCG on SCEs induced by CP in human lymphocytes with S9 mix.

Significant difference: ^a P<0.01 with respect to untreated, ^b P<0.05 with respect to CP.

Discussion

Methyl methanesulphonate and cyclophosphamide are potentially genotoxic in human lymphocytes in vitro at higher dosage in the presence and absence of metabolic activation. The change in chromosome structure due to a break or an exchange of chromosomal material is known as Chromosomal Aberration (CA). Most of the chromosomal aberrations are lethal in the cells, but still many of them are viable and capable of genetic effects, either somatic or inherited ₁₇ .These events can lead to the loss of chromosomal material at mitosis or to the inhibition of exact chromosome segregation at anaphase. The result of these changes is cell lethality ₁₈ . In our experiment, we came across significant differences compared with control in the CA frequency at 60 µM and 0.16 µg/ml of MMS and CP respectively, with and without S9 mix. SCE is usually a more sensitive indicator of genotoxic effects than chromosomal aberration ₁₈ . There is a correlation between the carcinogenicity and SCE inducing ability of many chemicals. Moreover, the SCE induction mechanism is heterogeneous and very different from the mechanism of chromosomal aberration induction ₁₉ . There is little information available on the exact reasons for the genotoxic behavior of MMS and CP. The outcome of most investigative experiments shows that MMS and CP have the potential to be genotoxic and cytotoxic, especially at 60 µM and 0.16 µg/ml respectively, with and without metabolic activation, in cultured human lymphocytes. The evaluation of these genotoxicity tests is a useful tool for determining the toxic effects of potentially genotoxic chemicals, leading to identification of such carcinogenic agents. It is advisable to use the known mutagens studied here at their lowest effective dosage so that the risk to public health could be minimized. The risk of damage to human genetic material is a likely outcome at higher doses of these drugs.

An increase in the frequency of chromosomal aberrations in peripheral blood lymphocytes is associated with an increased overall risk of cancer ₂₀₂₁ . The readily quantifiable nature of SCEs with high sensitivity for revealing toxicant-DNA interaction and the demonstrated ability of genotoxic chemicals to induce significant increase in SCEs in cultured cells has resulted in this endpoint being used as indicator of DNA damage in blood lymphocytes of individuals exposed to genotoxic carcinogens ₂₂ . The above genotoxic endpoints are well known markers of genotoxicity and any reduction in the frequency of these genotoxic endpoints gives us an indication of the antigenotoxicity of a particular compound ₂₂ .

Numerous plant products protect against xenobiotics either by inducing detoxifying enzymes or by inhibiting oxidative enzymes ₂₃ . Natural plant products have been reported to reduce genotoxic effect of anticancer drugs in various in vitro and in vivo models _24252627 . Epigallocatechin gallate, a major catechin found in green tea, has possible role in chemoprevention and chemotherapy of various types of cancers, mainly prostate cancer ₂₈₂₉ and colon cancer ₃₀₃₁ . EGCG inhibits the growth of gastric cancer by reducing VEGF production and angiogenesis, and is a promising candidate for anti-angiogenic treatment of gastric cancer ₃₂ . EGCG is now acknowledged as a cancer preventive in Japan and has established the concept of a cancer preventive beverage ₃₃ . All these studies indicate the strong antigenotoxicity potential of EGCG against mutagenic chemicals. In our study, EGCG reduces genotoxicity induced by known mutagens i.e. MMS and CP, in the presence as well as absence of metabolic activation system, in human lymphocytes. EGCG was more effective in reducing genotoxic damage in the presence of metabolic activation. The reduction in genotoxic damage may be due to the possibility of the prevention of metabolic activation of MMS and CP by EGCG.

Conclusion

The selected dosage of EGCG is potent enough to reduce genotoxicity. The dosage is the lowest possible, selected after reviewing the literature regarding the earlier studies done with EGCG. The concentrations studied here are higher than the concentrations commonly used for such anticancer drugs. The higher concentration may be reached in some clinical conditions and this higher concentration may lead to genotoxic damage and may further increase the possibility of the development of various types of cancers. EGCG reduced the genotoxic damage induced by the mutagens significantly at 20 and 30 µM clearly indicating its protective role.

Acknowledgements

Correspondence to

Prof. Mohammad Afzal Human Genetics and Toxicology Lab Department of Zoology Aligarh Muslim University (AMU) Aligarh 202002 (UP), India. Email: afzal_amu@yahoo.com