Antigenotoxic effect of apigenin against anti-cancerous drugs

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Toxicology in Vitro 22 (2008) 625–631 www.elsevier.com/locate/toxinvit

Antigenotoxic effect of apigenin against anti-cancerous drugs Yasir Hasan Siddique *, Tanveer Beg, Mohammad Afzal Human Genetics and Toxicology Lab, Section of Genetics, Department of Zoology, Faculty of Life Sciences, Aligarh Muslim University, Aligarh, U.P. 202002, India Received 7 September 2007; accepted 4 December 2007 Available online 21 February 2008

Abstract Mitomycin C and cyclophosphamide are well known anti-tumor drugs. Their genotoxic effects are well established in various test systems. The genotoxic effects in non-tumor cell are of special significance due to the possibility that they may induce secondary tumors in cancer patients. Apigenin is a well known anti-oxidant and possess number of properties that are beneficial in someway to humans. With this view, the present study deals with the effect of apigenin against the genotoxic doses of mitomycin C and cyclophosphamide using chromosomal aberrations, sister chromatid exchanges and cell cycle kinetics as a parameters. The treatment of apigenin results in a significant, dose dependent decrease in the genotoxic damage, induced by mitomycin C and cyclophosphamide. It is concluded that the apigenin is potent in reducing the genotoxic damage, induced by anti-cancerous drugs, thereby reducing the chances of developing secondary tumors during the therapy. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Apigenin; Chromosomal aberrations; Natural products; Sister chromatid exchanges; Cell cycle kinetics

1. Introduction Apigenin is one of the several active ingredients found naturally in many fruits and vegetables, including apples and cerlery. It is found in several popular spices, including basil, oregano, tarragon, cilantro and parsley. A high amount of apigenin is found in parsley, peppermint, lemon, berries and fruits (Peterson and Dwyer, 1998). It is a member of flavone family of the flavonoid. Apigenin is recognized in traditional or alternative medicine for its pharmacological activity (Hoftman, 2000). In the reverse mutation assay, apigenin was negative in Salmonella typhimurium strains TA97, TA98, TA100, TA102, TA1535 and TA1538 with and without metabolic activation by liver S9 microsomal fraction (NLM, 2000). The mutagenic activity of apigenin in Escherichia coli was examined and it weakly induced SOS repair system in E. coli K-12 strain PQ37 with and without metabolic activation (Czeczot and Bilbin, 1991).

*

Corresponding author. E-mail address: yasir_hasansiddique@rediffmail.com (Y.H. Siddique).

0887-2333/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2007.12.002

Apigenin possess anti-inflammatory effects, free radical scavenging and anti-carcinogenic effects (Kim et al., 1998; PHS-149, 1972). It has been shown to possess growth inhibitory properties in several cancer lines including breast, colon, skin, thyroid leukemia cells and pancreatic (Yin et al., 2001, 1999; Wang et al., 2000; Caltagirone et al., 2000; Taka-hashi et al., 1998; Ujiki et al., 2006), tumor inhibition (Wei et al., 1990; Li et al., 1996) and enzyme inhibitory properties (Wei et al., 1990; Jeong et al., 1992). There is much evidence for anti-carcinogenic activity of apigenin but less is known about its antigenotoxicity which is limited to mice (Khan et al., 2006). The present study was aimed to study the antigenotoxic effects of apigenin in both absence as well as presence of metabolic activation (S9 mix) system. Mitomycin C was used as positive control for the absence of metabolic activation experiments and cyclophosphamide act as a positive control for the metabolic activation experiments. The treatments with apigenin results in a significant, dose dependent decrease in the genotoxic damage, induced by mitomycin C and cyclophosphamide thereby, reducing the chances of developing secondary tumors during the chemotherapy.

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2. Materials and methods 2.1. Chemical Apigenin (CAS: 520-36-5; 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 (CAS No: 50-07-7) cyclophosphamide (CAS No: 6055-19-2). 2.2. Human lymphocyte culture Duplicate peripheral blood cultures of two female donors were treated according to Carballo et al. (1993). 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 culture tubes were placed in an incubator at 37 °C for 24 h. 2.3. Chromosomal aberration analysis After 24 h, 1, 5, 10, 20 lM of apigenin (dissolved in DMSO, 5 ll/ml) was given separately with 6 lM of mitomycin C. For metabolic activation experiments, 0.16 lg/ ml of cyclophosphamide was treated with 1, 5, 10 and 20 lM of apigenin separately, along with 0.5 ml of S9 mix. S9 mix was prepared according to the standard protocol of Maron and Ames (1983). The treatment of S9 mix was given for 6 h. The cells were collected by centrifugation and washed in prewarmed media to remove the traces of S9 mix and drugs. After 47 h, an amount of 0.2 ml of colchicine (0.2 lg/ml) was added to the culture tubes. Cells were centrifuged at 1000 rpm for 10 min. The supernatant was removed and 8 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, at 1000 rpm for 10 min, and subsequently 5 ml of chilled fixative (methanol:glacial acetic acid; 3:1) 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 examined 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 (IPCS, 1985). 2.4. Sister chromatid exchange analysis For sister chromatid exchange analysis, bromodeoxyuridine (10 lg/ml) was added at the beginning of the culture. After 24 h, 1, 5, 10 and 20 lM of apigenin (dissolved in

DMSO, 5 ll/ml) treatments were given separately with 6 lM of mitomycin C. Similar treatments were given with 0.16 lg/ml of cyclophosphamide in the presence of metabolic activation system (S9 mix). An amount of 0.5 ml of S9 mix was given with each treatment. The treatment of S9 mix was given for 6 h. The cells were collected by centrifugation and washed in prewarmed media to remove the traces of S9 mix and drugs. Mitotic arrest was attempted, 1 h prior to harvesting by adding 0.2 ml of colchicine (0.2 lg/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 (Perry and Wolff, 1974). 2.5. 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 absence as well as presence of metabolic activation (S9 mix) (Tice et al., 1976; Crossen and Morgan, 1977). 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 (Ivett and Tice, 1982) RI ¼

M 1 þ 2M 2 þ 3M 3 100

where M1, M2 and M3 denote number of metaphases in the first, second and third cycle, respectively. 2.6. Statistical analysis Student ‘t’ test was used for analysis of CAs, SCEs, and RI. Regression analysis was performed using Statistica Soft Inc. 3. Results In chromosomal aberration analysis, treatment with mitomycin C alone resulted in a significant increase of chromosomal aberrations. A significant decrease in number of abnormal cells were observed when 6 lM of mitomycin C was used separately with different dosages of apigenin, i.e., 1, 5, 10 and 20 lM (Table 1). Similar results were obtained for the cyclophosphamide (CP) treatment, with the different dosages of apigenin (Table 2). In sister chromatid exchange analysis, a significant increase in sister chromatid exchanges per cell was observed when treated with 6 lM of mitomycin C and 0.16 lg/ml of cyclophosphamide alone (Table 3). Sister chromatid exchanges per cell were decreased significantly when treated with 1, 5, 10 and 20 lM of apigenin, separately (Table 3). In cell cycle kinetics, treatment with mitomycin and cyclophosphamide alone resulted in a significant decrease in replication indices (Table 3). However, the treatment of mitomycin and cyclo-

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Table 1 Effect of apigenin on chromosomal aberrations (CAs) induced by mitomycin C Treatment

Cells scored

Abnormal metaphase

Chromosomal aberrations

Number

% ±S.E.

Gaps

CTB

CSB

CTE

DIC

MMC (lM) 6

300

34

11.33 ± 1.82a

17

22

16

4

3

Apigenin (lM) 1 5 10 20

300 300 300 300

2 3 3 4

0.67 ± 0.46 1.00 ± 0.57 1.00 ± 0.57 1.33 ± 0.66

1 2 2 2

2 2 2 2

 1 1 2

   

   

32 25 20 13 2

10.66 ± 1.78b 8.33 ± 1.59b 6.67 ± 1.44b 4.33 ± 1.17b 0.66 ± 0.46

14 12 10 8 2

16 14 12 8 2

13 11 8 5 

2    

1    

Apigenin (lM) + MMC (lM) 1+6 300 5+6 300 10 + 6 300 20 + 6 300 Untreated 300

MMC: mitomycin C; S.E.: standard error; CTB: chromatid break; CSB: chromosome break; CTE: chromatid exchange; DIC: dicentric. a P < 0.01 as compared to untreated. b P < 0.05 as compared to MMC.

Table 2 Effect of apigenin on chromosomal aberrations induced by cyclophosphamide in the presence of metabolic activation Treatment

Cells scored

CP (lg/ml) 0.16 300 Apigenin (lM) 1 300 5 300 10 300 20 300 Apigenin (lM) + CP (lg/ml) 1 + 0.16 300 5 + 0.16 300 10 + 0.16 300 20 + 0.16 300 Untreated 300

Abnormal metaphase

Chromosomal aberrations

Number

% ±S.E.

Gaps

CTB

CSB

CTE

DIC

38

12.67 ± 1.92a

18

25

18

5

4

2 2 3 3

0.66 ± 0.46 0.66 ± 0.46 1.00 ± 0.57 1.00 ± 0.57

1 1 2 2

2 2 2 2

– – 1 1

– – – –

– – – –

33 22 17 12 2

11.0 ± 1.80b 7.33 ± 1.50b 5.67 ± 1.33b 1.00 ± 1.13b 0.66 ± 0.46

16 13 12 9 1

21 14 11 8 2

15 8 6 4 –

3 – – – –

2 – – – –

MMC: mitomycin C; S.E.: standard error; CTB: chromatid break; CSB: chromosome break; CTE: chromatid exchange; DIC: dicentric. a P < 0.01 as compared to untreated. b P < 0.05 as compared to CP.

phosphamide with different dosages of apigenin (i.e., 1, 5, 10, 20 lM) results in a significant increase in the replication indices as compared to mitomycin and cyclophosphamide treatments alone (Table 3). Regression analysis was also performed to determine the dose effects of apigenin on mitomycin and cyclophosphamide, for a number of abnormal metaphases, SCEs and RI. A decrease in the slope of linear regression lines was observed as the dose of the apigenin was increase, in each of the treatment. For abnormal metaphases, the treatment of 6 lM of mitomycin C (F = 42.88; P < 0.002) and 0.16 lg/ml of cyclophosphamide (F = 10.73; P < 0.01), the increase in the dosages of apigenin results in the decrease in slope of the linear regression lines (Figs. 1 and 2). For sister chromatid exchange analysis, the treat-

ment of 6 lM of mitomycin C (F = 10.36; P < 0.006) and 0.16 lg/ml of cyclophosphamide (F = 6.25; P < 0.01), the increase in the dosages of apigenin results in the decrease in slope of the linear regression lines (Figs. 3 and 4). For cell cycle kinetics, the treatment of 6 lM of mitomycin C (F = 3.40; P < 0.003) and 0.16 lg/ml of cyclophosphamide (F = 2.81; P < 0.002) with the different dosages of apigenin, results in the decrease in the slope of the linear regression lines (Figs. 5 and 6). 4. Discussion Our study clearly demonstrates the antigenotoxic potential of apigenin both in the absence as well as presence of metabolic activation (S9 mix) systems. The selected doses

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Table 3 Effect of apigenin on cell cycle kinetics and sister chromatid exchanges in the absence as well as presence of metabolic activation Without S9 mix

With S9 mix

Treatments

Cells scored

MMC (lM) 6

200

Apigenin (lM) 1 5 10 20

200 200 200 200

RI

Apigenin (lM) + MMC (lM) 1+6 200 5+6 200 10 + 6 200 20 + 6 200 Untreated 200

SCEs/cell ± SE

Treatments

Cells scored

RI

SCEs/cell ± SE

1.41a

18.62 ± 0.18

CP (lg/ml) 0.16

200

1.46a

17.84 ± 0.92a

1.82 1.80 1.78 1.79

1.58 ± 0.18 1.62 ± 0.24 1.66 ± 0.28 1.72 ± 0.29

Apigenin (lM) 1 5 10 20

200 200 200 200

1.80 1.79 1.76 1.75

1.62 ± 0.25 1.68 ± 0.30 1.74 ± 0.33 1.78 ± 0.38

1.44b 1.70b 1.74b 1.78b 1.88

17.61 ± 0.90b 13.68 ± 0.76b 11.42 ± 0.64b 9.82 ± 0.54b 1.44 ± 0.12

1.48c 1.72c 1.73c 1.77c 1.84

16.80 ± 0.86c 12.32 ± 0.74c 10.24 ± 0.55c 9.22 ± 0.53c 1.64 ± 0.26

Apigenin (lM) + CP (lg/ml) 1 + 0.16 200 5 + 0.16 200 10 + 1.6 200 20 + 1.6 200 Untreated 200

CP: cyclophosphamide; MMC: mitomycin C; S.E.: standard error. a P < 0.01 as compared to untreated. b P < 0.05 as compared to MMC. c P < 0.05 as compared to CP.

Y = 31.099 -. 9554 X

Y = 30.045 -1.005 X

Correlation: r = -.9775

Correlation: r = -.9181 38 Regression 95% confid.

30

Regression 95% confid.

34 Number of abnormal metaphases

Number of abnormal metaphases

34

26

22

18

14

30 26 22 18 14

10 -2

2

6

10

14

18

22

Concentration of apigenin

10 -2

2

6

10

14

18

22

Concentration of apigenin

Fig. 1. Regression analysis for the dose effect of apigenin on abnormal metaphases induced by mitomycin C (6 lM).

Fig. 2. Regression analysis for the dose effect of apigenin on abnormal metaphases induced by cyclophosphamide (0.16 lg/ml).

of apigenin were not genotoxic, however at 93 lM, an increase in micronucleus frequency was reported in human lymphocytes in vitro (Rithidech et al., 2005). A study conducted by Snyder and Gillies (2002) also showed clastogenic activity of apigenin at 100 lM in Chinese hamster V79 cells, and proposed that clastogenic activity of apigenin was due to the ability of apigenin to intercalate DNA molecules. Genotoxic effects of anti-cancer drugs in nontumor cells are of special significance due to the possibility that they may induce secondary tumors in cancer patients. Furthermore, the mutagenic and carcinogenic effects of antineoplastic agents on the health-care persons handling these drugs also need to be considered carefully (Aydemir et al., 2005). It is quite possible that uptake of complex plant – derived mixtures may modulate the genotoxicity of anti-cancer drugs and thus may reduced the chances of

developing secondary tumors in cancer patients. Therefore, the effect of apigenin was studied against the genotoxic damage induced by mitomycin C and cyclophosphamide. Mitomycin C is an anti-tumor, antibiotic compound that has a range of genotoxic effects including the inhibition of DNA synthesis, mutagenesis and clastogenesis (Tomasz, 1995). Mitomycin C is a direct acting clastogen (Grisolia, 2002), requiring only intra-cellular reductive activation to initiate its potent DNA crosslinking action (Tomasz, 1995). Cyclophosphamide is an alkylating agent and after metabolic activation it produces phospharamide mustard (Ghaskadbi et al., 1992). It reacts with electron rich area of susceptible molecules such as DNA and proteins (Krishna et al., 1986). It is predominantly metabolized by CYP2B6, along with minor contributions from CYP3A4, CYP2C19 and CYP2C9 (Winter et al., 2007). The genotox-

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629

Y = 1.5313 + .01485 X

Y = 1.5645 + .01228 X

Correlation: r = .79385

Correlation: r = .76446 1.80

1.85 1.80

1.74

1.70

Replication index

Replication index

1.75

1.65 1.60 1.55

1.68

1.62

1.56

1.50 1.50

Regression 95% confid.

1.45

Regression 95% confid. 1.44

1.40 -2

2

6

10

14

18

-2

22

2

6

10

14

Fig. 3. Regression analysis for the dose effect of apigenin on replication index induced by mitomycin C (6 lM).

22

Fig. 5. Regression analysis for the dose effect of apigenin on replication index induced by cyclophosphamide (0.16 lg/ml).

Y = 16.526 -.3770 X

Y = 15.354 - .3565 X

Correlation: r = -.9156

Correlation: r = -.8705

19

18 Regression 95% confid.

Regression 95% confid. 17

Sister chromatid exchanges / cell

Sister chromatid exchanges / cell

18

Concentration of apigenin

Concentration of apigenin

15

13

11

9

16

14

12

10

8

-2

2

6

10

14

18

22

Concentration of apigenin

-2

2

6

10

14

18

22

Concentration of apigenin

Fig. 4. Regression analysis for the dose effect of apigenin on sister chromatid exchanges/cell induced by mitomycin C (6 lM).

Fig. 6. Regression analysis for the dose effect of apigenin on replication index induced by cyclophosphamide (0.16 lg/ml).

icity testing provides human a risk assessment. An increase in the frequency of chromosomal aberrations in peripheral blood lymphocytes is associated with an increased overall risk of cancer (Hagmar et al., 1994, 1998). 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 (Swierenga et al., 1919). 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 endpoint being used as indicator of DNA damage in blood lymphocytes of individuals exposed to genotoxic carcinogens (Albertini et al., 2000). The above genotoxic endpoints are well known markers of genotoxicity and any reduction in the frequency of these genotoxic endpoints gives an indication of the antigenotoxicity of a particular compound

(Albertini et al., 2000). In our present study with cyclophosphamide, apigenin reduced the genotoxic damage, this is due to the possible prevention of metabolic activation of cyclophosphamide by apigenin or may also scavenge electrophiles/nucleophiles or enhance the DNA repair system or DNA synthesis (Noel et al., 2006). The concentrations of apigenin used in the present study were not genotoxic itself and reduced the genotoxic effects of mitomycin C and cyclophosphamide. Information on plasma or serum concentrations of apigenin is lacking and studies on in vivo absorption, metabolism and potential genotoxicity of apigenin are warranted (Rithidech et al., 2005). Higher doses of apigenin have been reported to induce micronucleus in cultured human lymphocytes (Noel et al., 2006). The genotoxic damage by flavonoids at higher concentration are considered to be due to DNA intercalation, poisoning of DNA topoisomerase II, generation of reactive metabolites and inhibition of key enzymes (Stopper et al.,

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2005; Fimognari et al., 2004; Snyder and Gillies, 2002; Popp and Schimmer, 1991). Furthermore, it is quite possible that a particular flavonoid may exhibit genotoxic damage in isolation (Noel et al., 2006) but may modulate the genotoxic effect when associated with other compound, as is evident from the present study. Less information is available on the in vivo absorption, metabolism, and plasma concentration of apigenin (Manach et al., 2004). However, it has been reported that apigenin is absorb and metabolized by human after intake and its half life is about 12 h (Nielsen et al., 1999). It has been well established that secondary cancers are a complication of traditional treatments with chemotherapy (Meadows et al., 1985). For these reasons, utilization of anti-carcinogenic nutrients could play a vital role in protecting those exposed to chemotherapeutic agents. Several in vitro studies have shown that when antioxidants such as ascorbate, vitamin A, carotenoids and their metabolites are combined with chemotherapeutic drugs, they can all enhance the growth inhibitory effects of most of the currently used chemotherapeutic agents on selected cancers types (Blaylock, 2000; Prasad, 1980; Ripoli et al., 1986). The extent of this enhance effectiveness was dependent on the dose and form of the vitamin, the dose and the type of chemotherapeutic agent and the tumor type (Prasad et al., 1994). Seifter et al. (1984) reported an enhanced killing of a transplanted adenocarcinoma of the breast when either vitamin A or synthetic beta carotene was used in combination with X-irradiation or cyclophosphamide. The vitamins do not protect the melanoma cells from the chemotherapeutic agents, but rather enhanced their killing power (Prasad et al., 1994). Cancer cells can accumulate higher inter-cellular concentrations of vitamins/anti-oxidants than normal cells due to loss of homeostatic controls. The high concentration of vitamins/antioxidants can alter cancer cell metabolism and cell signaling (Prasad et al., 1994). Because of the aforementioned differences in normal verses cancer cells, vitamin/flavonoids complementary therapy can protect the normal tissues from adverse effects of chemotherapeutic agents with out negating therapeutic efficiency (Blaylock, 2000). The lower doses of chemotherapeutic agents when combined with selected anti-oxidants, could be used to obtain the same killing power as higher doses of the agent. Unlike, using higher dose of chemotherapeutic agents the enhanced efficiency mixture would be expected to reduce significantly the complications associated with chemotherapeutic agents (Blaylock, 2000). Flavonoids are a complex group of aromatic compounds that include chalcones, anthocyanidins and numerous other derivatives. In several studies, the flavonoids have been found to be very protective against chemotherapy toxicity. For example, quercetin has been found to protect renal tubular cells against cisplatin toxicity, and glutathione has been shown to reduce the toxicity of alkylating agents on cardiac and skeletal muscle, and neurotoxicity of cisplatin in vitro (Kuhlmann et al., 1998). The results of the present study showed that the selected doses are potent enough to reduce the genotoxic damage

of anti-cancerous drugs, therapy reducing the chances of developing secondary tumors in the cancerous patients, but care should be taken with regard to its concentration, if consumed in isolation. There are also considerable evidences that the flavonoids enhance the cytotoxic activity of chemotherapeutic agents against multidrug-resistant tumors. For example, quercetin has been shown to increase the efficacy of cisplatin as well as other agents both in vitro and in vivo in animal studies (Schmbia et al., 1994). Since the plants contain a complex mixture of flavonoid, it may be possible that the genotoxicity of one flavonoid may be modulated by the antigenotoxic ability of another flavonoid and vice-versa (Virgilio et al., 2004). Acknowledgements Thanks are due to the CSIR, New Delhi for the fellowship No. 9/112(355)/2003-EMR to the author (Y.H.S.) and to the Chairman, Department of Zoology, A.M.U., Aligarh for the laboratory facilities. References Albertini, R.J., Anderson, D., Douglas, G.R., Hagmar, L., Heminiki, K., Merelo, F., Natrajan, A.T., Norppa, H., Shuker, D.E.G., Tice, R., Walters, M.D., Aitio, A., 2000. IPCS guidelines for the monitoring of genotoxic effects of carcinogens in humans. Mutation Research 463, 111–172. Aydemir, N., Celikler, S., Bilalouglu, R., 2005. In vitro genotoxic effects of the anti-cancer drug gemcitabine in human lymphocytes. Mutation Research 582, 35–41. Blaylock, R.L., 2000. A review of conventional cancer prevention and treatment and the adjunctive use of nutraceutical supplements and antioxidants: is there a danger or a significant benefit. Journal of American Nutraceutical Association 3, 75–95. Caltagirone, S., Rossi, C., Poggi, A., Ranelletti, F.O., Natali, P.G., Brunetti, M., Aiello, F.B., Piantelli, M., 2000. Flavonoids apigenin and quercertin inhibit melanoma growth and metastatic potential. International Journal of Cancer 87, 595–600. Carballo, M.A., Alvarez, S., Boveris, A., 1993. Cellular stress by light and Rose Bengal in human lymphocytes. Mutation Research 288, 215– 222. Crossen, P.E., Morgan, W.F., 1977. Analysis of human lymphocyte cell cycle time in culture measured by sister chromatid differentiated staining. Experimental Cell Research 104, 453–457. Czeczot, H., Bilbin, M., 1991. Effect of flavones and their metabolites on induction of SOS repair in the strain PQ37 – E. coli K-12. Acta Biochimica Polonica 38, 71–74. Fimognari, C., Berti, F., Cantelli-Forti, G., Hrelia, P., 2004. Effect of cyanidin 3-o-betaglucopyranoside on micronucleus induction in cultured human lymphocytes by four different mutagens. Environmental and Molecular Mutagenesis 43, 45–52. Ghaskadbi, G., Rajamachikar, S., Agate, C., Kapadi, A.H., Vaidya, V.G., 1992. Modulation of cyclophosphamide mutagenicity by vitamin C in the in vivo rodent micronuclei assay. Teratogenesis Carcinogenesis and Mutagenesis 12, 11–17. Grisolia, C.K., 2002. A comparison between mouse and fish micronucleus test using cyclophosphamide, mitomycin C and various pesticides. Mutation Research 518, 145–150. Hagmar, L., Brogger, A., Hansteen, I.L., Heims, S., Hoggstedt, B., Knudsen, L., Lambert, B., Linnaimaa, K., Mitelman, F., Nordenson, I., Reuterwall, C., Salomaa, S., Skerfving, S., Sorsa, M., 1994. Cancer risk in humans predicted by increased level of chromosomal aberrations

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