Antigenotoxic effect of apigenin on chromosomal damage induced by cyproterone acetate in human lymphocytes
Abstract
Cyproterone acetate (CPA) is not only a genotoxic agent but also a tumor initiating agent. It is used in oral contraceptives formulations and also in the treatment various sexual and metabolic disorders. Apigenin, a well known antioxidant and have number of properties that are beneficial in someway to humans. In this context, the antigenotoxic effect of apigenin was studied against the genotoxic doses of CPA using chromosomal aberrations, sister chromatid exchanges and cell cycle kinetics as parameters. The treatment of 20 and 30 M of CPA was given separately along with apigenin at the doses of 1, 5, 10 and 20 M of culture medium. A clear dose dependent decrease in the genotoxic damage of CPA was observed, suggesting a protective role of apigenin during CPA therapy. The results of the present study suggest that the apigenin can modulate the genotoxicity of CPA on human lymphocytes in vitro.
Introduction
Synthetic progestins are used in the treatment of sexual and metabolic disorders, and also in oral contraceptives either singly or in combination with estrogens 1 . Prolonged use of oral contraceptives has been shown to develop various types of malignancies in human and experimental animals 2,3 . Earlier studies reveal that synthetic progestins have DNA damaging potential 4,5 . The use of progestins cannot be ignored completely, but their genotoxic effects can be reduced by the use of antioxidants 6,7,8 and natural plants products 9,10,11,12. Cyproterone acetate (CPA) is a tumor initiating agent in the liver of female rats 13,14. It induced micronucleus in rat liver cells 14, chromosomal aberrations in V79 cells15, and human peripheral blood lymphocytes 16,17 , and also sister chromatid exchanges in human peripheral blood lymphocytes in vitro 17 .
Natural plant products and antioxidants have been reported to reduce the genotoxicity of estrogens and synthetic progestins, hence reducing the chances of developing the cancers during the therapy. Apigenin, is an active ingredient of many fruits and vegetables 18 . It is a member flavone family of the flavonoid. Apigenin is recognized in traditional or alternative medicine for its pharmacological activity 19 . It possess free radical scavenging, anticarcinogenic 20 , tumor inhibition 21 and antigenotoxic properties 22 . The present study was aimed to study the antigenotoxic effects of apigenin against the genotoxic effects of cyproterone acetate.
Materials and methods
Chemicals
Apigenin (CAS: 520-36-5; Sigma); cyproterone acetate (CAS:, 427-1-0,: Sigma); RPMI 1640, fetal calf serum; phytohaemagglutinin-M, antibiotic- antimycotic mixture (Gibco), dimethylsulphoxide, 5-bromo-2-deoxyuridine, colchicine (SRL, India); Giemsa stain (Merck); Mitomycin C.
Human lymphocyte culture
Duplicate peripheral blood cultures of two female donors were treated according to Carballo et al. 23. Briefly, heparinized, blood sample (0.5 ml), was obtained from a healthy female donor and was placed in a sterile culture tube containing 7 ml of RPMI 1640 medium, supplemented with fetal calf serum (1.0 ml), antibiotic-antimycotic mixture (1.0 ml) and phytohaemagglutinin (0.1 ml). The cultures tubes were placed in an incubator at 37 ° C for 24 h.
Chromosomal aberration analysis
After 24 h, 20 µM of CPA (dissolved in DMSO, 5 µg/ml) was given separately with 1, 5, 10 and 20 µM of apigenin. Similar treatment was given with 30 µM of CPA. After 47 h, an amount of 0.2 ml of colchicine (0.2 µg/ml) was added to culture tubes. Cells were centrifuged to 1000 rpm for 10 min. The supernatant was removed and 8 ml of prewarmed (37 ° C) 0.075M KCl (hypotonic solution) was added. Cells were resuspended and incubated at 37 ° C for 15 min. The supernatant was removed by centrifugation at 1000 rpm for 10 min, and subsequently 5 ml of chilled fixative was added. The fixative was removed by centrifugation and the procedure was repeated twice. The slides were stained in 3% Giemsa solution in phosphate buffer (pH 6.8) for 15 min. About, 300 metaphase were examine for the occurrence of different types of abnormality. Criteria to classify different types of aberrations were in accordance with the recommendation of Environmental Health Criteria 48 for Environmental Monitoring of Human Population 24 .
Sister chromatid exchange analysis
For sister chromatid exchange analysis, bromodeoxyuridine (10 µg/ml) was added at the beginning of the culture. After 24 h, similar treatment was given as described in chromosomal aberration analysis. Mitotic arrest was done 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 aberrations analysis. The sister chromatid exchange average was taken from an analysis of metaphase during second cycle of division 25.
Cell cycle kinetics
Cells undergoing, first (M1), second (M2) and third (M3) metaphase divisions were detected with BrdU-Harlequin technique for differential staining of metaphase chromosomes both in the presence as well as presence of metabolic activation (S9 mix) 26,27 . Treatments were similar as described earlier in the text. The replication index (RI), an indirect measure of studying cell cycle progression was calculated by applying the following formula 28 .
RI = (M1 + 2M2 + 3M3) / 100
where M1, M2 and M3 denote number of metaphases in the first, second and third cycle, respectively.
Statistical analysis
Student ‘t' test was used for analysis of CAs, SCEs and RI. Regression analysis was performed using Statistical Soft Inc.
Results
In chromosomal aberration analysis, treatment with apigenin resulted in a significant decrease of chromosomal aberrations. A dose dependent decrease in the number of abnormal cells was observed (Table 1). At the highest tested dose of apigenin i.e. 20 µM there was more than 50% decrease in the chromatid as well as chromosome breaks.
Table 1: Effect of apigenin on chromosomal aberrations (CAs) induced by cyproterone acetate
In sister chromatid exchange analysis, a significant increase in sister chromatid exchanges per cell was observed at 20 µM and 30µM of CPA (Table 2). Sister chromatid exchanges per cell decreased significantly when treated with 1, 5, 10 and 20 µM of apigenin, separately (Table 2). In cell cycle kinetics, treatment with 20µM and 30 µM of CPA results in a significant decrease in the replication index (Table 2).
Table 2: Effect of apigenin on cell cycle kinetics and sister chromatid exchanges (SCEs) induced by cyproterone acetate.
However, the treatment of CPA with different dosages of apigenin (i.e. 1, 5, 10 and 20 µM) results in a significant increase in the replication indices as compared to the CPA treatment alone (Table 2). Regression analysis was also performed to determine the dose effects of apigenin on CPA, for number of abnormal metaphases, SCEs/cell and replication index. A decrease in the slope of linear regression lines was observed as the dose of apigenin was increase in each of the treatment for abnormal metaphases and SCEs/cell analysis. For abnormal metaphases, the treatment of 20 µM of CPA (p<0.003) and 30 µM of CPA (p<0.0003) with 1, 5, 10 and 20 µM of apigenin an increase in the dosage of apigenin results in the decrease in slope of the linear regression line (Fig. 1 and 2).
Figure 1: Effect of apigenin on number of abnormal metaphases after treatment with 20µM of cyproterone acetate.
Figure 2: Effect of apigenin on number of abnormal metaphases after treatment with 30µM of cyproterone acetate.
For sister chromatid exchange analysis, the treatment of 20µM of CPA (p<0.0001) and 30µM of CPA (p< 0.0002) an increase in the dosage of apigenin results in the decrease in slope of the linear regression line (Fig. 3 and 4).
Figure 3: Effect of apigenin on sister chromatid exchanges/cell after treatment with 20µM of cyproterone acetate.
Figure 4: Effect of apigenin on sister chromatid exchanges/ cell after treatment with 30µM of cyproterone acetate.
For cell cycle kinetics, the treatment of 20 µM of CPA (p<0.0001) and 30µM (p<0.0001) with 1, 5, 10 and 20 µM of apigenin results in the increase in slope of linear regression line (Fig. 5 and 6).
Figure 5: Effect of apigenin on replication index after treatment with 20µM of cyproterone acetate.
Figure 6: Effect of apigenin on replication index after treatment with 30µM of cyproterone acetate.
Discussion
Our study clearly demonstrates the antigenotoxic potential of apigenin against the genotoxicity of CPA. The selected doses of apigenin were not genotoxic, however at 93 µM, an increase in micronucleus frequency was reported in human lymphocytes in vitro 29 . Apigenin showed clastogenic effects at 100 µM in Chinese hamster V79 cells, and proposed that the activity was due to the ability of apigenin to intercalate DNA molecule 30. Genotoxicity of steroids (synthetic estrogens and progestins) are of special significance due to the possibility that they may induce tumor formation as is evident in the experimental animals 1,2,3 . The genotoxicity testing provides human a risk assessment. An increase in the frequency of chromosomal aberrations in peripheral blood lymphocytes is associated with an increase overall risk of cancer 31,32 . Most of the chromosomal aberrations observed in the cells are lethal, but there are many other aberrations that are viable and cause genetic effects, either somatic or inherited 33 . The ready 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 this end point being used as indicator of DNA damage in blood lymphocytes of individuals exposed to genotoxic carcinogens 34. CPA produced a delay in the cell cycle resulting in the reduction of the number of cells in the second division and an increase of those in the first division, as is evident by the replication index. The treatment of apigenin resulted in the dose dependent increase of cells in the second division as is evident by the increase in the replication index. Apigenin hence reduces the cytotoxic effect of CPA.
The above genotoxic end points are well known markers of genotoxicity and any reduction in the frequency of these genotoxic end points gives an indication of the anti-genotoxicity of a particular compound 34 . In our earlier study with CPA the genotoxic damage was due to the generation of reactive oxygen species (ROS) in the test system 17 . In our present with CPA, apigenin reduce the genotoxic damage this is due to the possible scavenging of electrophiles/nucleophiles or it may enhance the DNA repair system or DNA synthesis 35 . Less information is available on the in vivo absorption, metabolism, and plasma concentration of apigenin 36. However, it has been reported that apigenin is absorb and metabolized by human after intake and its half life is about 12 h 37 . The International Agency of Research on Cancer (IARC), mainly on the basis of epidemiological studies classifies steroidal estrogens and estrogen-progestin combinations among agents carcinogenic to humans (Group 1), progestins as possibly carcinogenic (Group 2) and androgenic, anabolic steroids, as probably carcinogenic (Group 2A) 5 . Carcinogenicity to humans of sex steroids has been evaluated, and is reported that high dose of estrogen-progestin combinations can cause liver cancer to humans 38 . A study of the project on oral contraceptives and liver cancer reveal that oral contraceptives may enhance the risk of liver carcinomas 39 . Many plant products protect against xenobiotics either by inducing detoxifying enzymes or by inhibiting oxidative enzymes 40 . The treatment of apigenin reduced the frequency of SCEs/cell and chromosomal aberrations in the test system, thereby indicating the possibility of reducing the chances of carcinogenesis during the CPA therapy in patients. The identification and characterization of the possible mechanism involved in the reduction of the genotoxic damage of CPA will be the part of our future study, however at present it can be concluded from the present study that apigenin has the potential to reduce the genotoxic damage induced by CPA in cultured human lymphocytes.
Acknowledgement
Thanks are due to the DST, New Delhi for awarding the Project No: SR/FT/LS-003/2007 under SERC-Fast Track Scheme for young scientists to the author Yasir Hasan Siddique and to the Chairman, Department of Zoology, AMU, Aligarh, U.P., for laboratory facilities.
