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The mutagenic potentiator effect of chlorophyllin by the HGPRT assay
Toxicology in Vitro 18 (2004) 147–149 www.elsevier.com/locate/toxinvit
Short Communication
The mutagenic potentiator effect of chlorophyllin by the HGPRT assay Priscilla Davidson Negraesa, Berenice Quinzani Jorda˜oa, Veronica Elisa Pimenta Vicentinib, Ma´rio Se´rgio Mantovania,* a
Universidade Estadual de Londrina, Departamento de Biologia Geral, Campus Universitario CP 6001, Laborato´rio de Mutageˆnese in vitro, Londrina, Parana 86051 990, Brazil b Universidade Estadual de Maringa´, Departamento de Biologia Celular e Gene´tica, Laborato´rio de Gene´tica, Maringa´, PR, Brazil Received 19 May 2003; received in revised form 12 June 2003; accepted 7 July 2003
Abstract Chlorophyllin, a sodium-copper salt derivative of chlorophyll-a and -b, was evaluated for antimutagenic activity against ethyl methane sulfonate by the hypoxanthin-guanine-phosphoribosyl transferase (HGPRT) assay. The results obtained suggest that this chlorophyllin can potentiate the mutagenicity of an alkylating agent which induces DNA damage. # 2003 Elsevier Ltd. All rights reserved. Keywords: Chlorophyllin; CHO-K1 cells; hgprt; Mutagenicity; Potentiator effect
1. Introduction Chlorophyll-a and -b found in green plants, as well as chlorophyllin (Chln), a sodium–copper salt derivative of chlorophyll, has been studied for its protective action against the carcinogenic effects of many physical and chemical agents and for its relation to the mutagenic and clastogenic activities of genotoxic agents. The mechanisms by which chlorophyllin acts include bioantimutagenic and desmutagenic activities (Bez et al., 2001a,b; Rampazo et al., 2002). However, other studies have shown that both chlorophyll and Chln can display ambiguous genotoxic and carcinogenic effects (Sarkar et al., 1994; Sarkar et al., 1996; Romert et al., 1992), acting as a free radical. In this context, the mutagenicity of such compounds could be investigated by using assays in mammalian cells in vitro, including such test systems as testing for mutations at the hypoxanthine-guaninephosphoribosyl transferase locus in Chinese hamster ovary cells (CHO), which has been extensively used. HGPRT is an enzyme that is not essential for cell growth in culture, since purines can be obtained by de novo synthesis. Thus, HGPRT mutants lose their capa* Corresponding author. Fax: +55-43-3371-4207. E-mail address: [email protected] (M.S. Mantovani). 0887-2333/$ - see front matter # 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2003.07.001
city to utilize purine analogues that are lethal only to wild-type cells, and are easily identified when grown in medium containing 6-thioguanine, for example (Li et al., 1987). The present study examined the effect of Chln on mutations induced at the hgprt locus by ethyl methane sulfonate (EMS), in Chinese hamster ovary cells (CHO-K1).
2. Material and methods 2.1. Cell culture and treatments CHO-K1 cells (Chinese hamster ovary), furnished by the Mutagenesis Laboratory of USP—Ribeira˜o Preto (SP)/Brazil, were grown as monolayers in 25-cm2 culture flasks, in Dulbecco’s modified Eagle’s medium (DMEM)/Ham’s F12, supplemented with 10% fetal bovine serum and 0.1% of antibiotic-antimycotic solution, in a BOD chamber at 37 C. Under these conditions, the cell cycle was 12 h. After growing the cells for two cell cycles (24 h), cultures containing approximately 6106 cells were treated for a period of 3 h (without fetal bovine serum) with: (a) PBS, pH 7.4, negative control; (b) 620 mg/ml EMS, positive control; (c) 25 mg/ ml Chln; (d) EMS+6.25 mg/ml Chln; (e) EMS+12.5
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P.D. Negraes et al. / Toxicology in Vitro 18 (2004) 147–149
mg/ml Chln; and (f) EMS+25 mg/ml Chln. Afterward, the cells were washed, trypsinized (0.025% trypsin) and transferred to new culture flasks before assaying for cytotoxicity and effect on mutagenicity. Cytotoxicity (absolute cloning efficiency, ACE1) was determined after plating approximately 125 cells in three replicate flasks and allowing them to grow for 7 days. The colonies formed were fixed with methanol, stained with 10% Giemsa and counted. Effect on mutagenicity was determined by seeding a minimum of 2106 cells in flasks maintained for 9 days, and replating every 48 h for the expression of the mutant phenotype. Subsequently, 105 cells were transferred to five flasks/treatment each containing selective medium (10 mg/ml 6-thioguanine). At the moment of replating to selective medium, cloning efficiency (ACE2) was determined in non-selective medium (three replicate flasks; 125 cells each). After 9 days, colonies resistant to 6-thioguanine were fixed, stained and counted, and the mutation frequency (MF) was calculated in relation to non-selective cell survival (ACE2). The assay was performed according to Li et al. (1987). 2.2. Statistical analysis The data were analyzed by the conditional test for rare event detection (Pereira, 1991) at =0.05 level of significance.
3. Results and discussion Chlorophylls and their derivatives have been extensively investigated for their potential as antigenotoxic, anticlastogenic, antimutagenic and anticarcinogenic agents in many test systems (Renner, 1990; Gosh et al., 1991; Sen et al., 1991; Abraham et al., 1994; Sarkar et al., 1994; Edenharder et al., 1995; Negishi et al., 1997; Pimentel et al., 1999; Bez et al., 2001a, 2001b; Rampazo
et al., 2002). However, other studies have demonstrated that both chlorophylls and Chln can show genotoxic, clastogenic and carcinogenic effects (Romert et al., 1992; Sarkar et al., 1994, 1996). Table 1 presents the absolute and relative cloning efficiencies and the mutation frequency obtained in the evaluation of Chln by the HGPRT assay. This first set of experiments, Chln acts as a potentiator of the mutagenicity of the alkylating agent EMS, since the number of mutant colonies observed with combined treatment was significantly higher than that for the positive control. The principal effect of chlorophylls and chlorophyllin reported in the literature is its capacity to protect against DNA damage caused by many chemical and physical agents. However, Sarkar et al. (1993) studying the effect of Chln (1.5–2.0 mg/Kg b.w.) on the clastogenicity of chromium (IV) oxide and chordane in vivo, observed an anticlastogenic effect only with first of these latter two chemicals, demonstrating that the protective activity of compounds such as Chln cannot be generalized and depends directly on the type or class of DNA damage-inducing agent utilized. Moreover, some studies have suggested that the effect of these compounds correlate with the concentrations used, low or high, but do not appear be dose-dependent, as shown by Romert et al. (1992). These authors found that low concentrations of Chln potentiated twofold the mutagenicity of the tobacco-specific nitrosamines, NNN and NNK in Salmonella and the V79 assay, but high concentrations blocked the mutagenicity of the nitrosamines. This led to the proposal that Chln is capable of acting as a positive or negative modifier of mutagenicity. We have tested other higher concentrations of Chln in CHO-K1 cells, and they did not show any antimutagenic activity, but a potentiating effect in the mutagenicity of EMS. This suggests that the concentrations used may modify results in the assays, mainly with respect to the analysis of various parameters (e.g. chromosome aberration, micronucleus, comet, gene
Table 1 Mean cloning efficiency and mutation frequency observed in cells treated with chlorophyllin with or without ethyl methane sulfonate, and submitted to the in vitro HGPRT mutagenicity assaya Treatments
Cytotoxicity
Mutant selection
CHO-K1
ACE1 (%)
RCE (%)
n. mutant colonies
ACE2 (%)
MF (%)
Control EMS Chln 25 mg/ml Chln+EMS 6.25 mg/ml 12.5 mg/ml 25 mg/ml
50.64 37.84
– 74.72
0.6 5.8*
33.84 26.40
1.42 17.57
60.24
118.96
1.4
29.84
3.75
44.80 52.00 53.84
88.47 102.68 106.32
10.2* 12* 13.8*
42.08 24.80 46.40
19.39 38.71 23.79
a EMS, ethyl methane sulfonate 620 mg/ml; Chln, chlorophyllin; ACE, absolute cloning efficiency; RCE, relative cloning efficiency; MF, mutation frequency. * Statistically significant, 0.001
P.D. Negraes et al. / Toxicology in Vitro 18 (2004) 147–149
mutation—hgprt) and inducing agent utilized (e.g. EMS, MMS, and MMC, among others). Therefore, our results suggest that at the concentrations utilized, Chln acts as a positive modifier responsible for the increase in the number of mutations induced at the hgprt locus in the cell line employed. There was apparent evidence of a dose–response type relation, since increasing concentrations of Chln tested caused a gradual increase in the number of mutant colonies observed.
Acknowledgements This study has been supported by UEL, CAPES and Fundac¸a˜o Arauca´ria.
References Abraham, S.K., Sarma, L., Kesavan, P.C., 1994. Role of chlorophyllin as an in vivo anticlastogens: protection against gamma-radiation and chemical clastogens. Mutat. Res. 322, 209–212. Bez, G.C., Jorda˜o, B.Q., Vicentini, V.E.P., Mantovani, M.S., 2001a. Investigation of genotoxic and antigenotoxic activities of chlorophylls and chlorophyllin in cultured V79 cells. Mutat. Res. 497, 139–145. Bez, G.C., Jorda˜o, B.Q., Vicentini, V.E.P., Mantovani, M.S., 2001b. Differential protection of A and B chlorophyll in MMC-mediated induction of chromatid and isochromatid breaks in human lumphocytes culture. Cytologia 66, 313–318. Edenharder, R., Leopold, C.H., Kries, M., 1995. Modifying actions of solvent extracts from fruit and vegetable residues on 2-amino3methylimidazo[4,5]quinoline (IQ) and 2-amino-3,4-dimethylimidazo[4,5]quinxaline (MeIQx) induced mutagenesis in Salmonella typhimurium TA 98. Mutat. Res. 341, 302–318.
149
Ghosh, A.K., Sen, S., Sharma, A., Talukder, G., 1991. Effect of chlorophyllin on mercuric chloride-induced clastogenicity in mice. Food Chem Toxic 29, 777–779. Li, A.P., Carver, J.H., Choy, W.N., Hsie, A.W., Gupta, R.S., Loveday, K.S., O’Neill, J.P., Riddle, J.C., Stankowski Jr., L.F., Yang, L.L., 1987. A guide for the performance of the Chinese hamster ovary cell/hipoxanthine-guanine phosphoribosyl transferase gene mutation assay. Mutat. Res. 189, 135–141. Negishi, T., Ray, H., Hayatsu, H., 1997. Antigenotoxicity of natural chlorophylls. Mutat. Res. 376, 97–100. Pereira, A.A.B., 1991. Testes estatı´sticos para comparar proporc¸o˜es em problemas de citogene´tica. In: Rabello-Gay, M.N., Rodrigues, M.A.L.R., Monteleone-Neto, R. (Eds.), Mutageˆnese, Carcinogeˆnese e Teratogeˆnese: Me´todos e Crite´rios de Avaliac¸a˜o. Revista Brasileira de Gene´tica, Ribeira˜o Preto, SP, Brasil, pp. 113–121. Pimentel, E., Cruces, M.P., Zimmering, S., 1999. On the persistence of the radioprotective effect of chlorophyllin (CHLN) in somatic cells of Drosophyla. Mutat. Res. 446, 189–192. Rampazo, L.G.L., Jorda˜o, B.Q., Vicentini, V.E.P., Mantovani, M.S., 2002. Chlorophyllin antimutagenesis mechanisms under different treatment conditions in the micronucleus assay in V79 cells. Cytologia 67, 323–327. Renner, H.W., 1990. In vivo effects of single or combined dietary antimutagens on mutagen-induced chromosomal aberrations. Mutat. Res. 244, 185–188. Romert, L., Curvall, M., Jenssen, D., 1992. Chlorophyllin is both a positive and negative modifier of mutagenicity. Mutagenesis 7, 349– 355. Sarkar, D., Sharma, A., Talukder, G., 1993. Differential protection of chlorophyllin against clastogenic effects of chromium and chlordane in mouse bone marrow in vivo. Mutat. Res. 301, 33–38. Sarkar, D., Sharma, A., Talukder, G., 1994. Chlorophyll and chlorophyllin as modifiers of genotoxic effects. Mutat. Res. 318, 239–247. Sarkar, D., Sharma, A., Talukder, G., 1996. Chlorophyll and chromosome breakage. Mutat. Res. 360, 187–191. Sen, S., Sharma, A., Talukder, G., 1991. Inhibition of clastogenic effects of nicotine by chlorophyllin in mice bone marrow cells in vivo. Phytother Res. 5, 130–133.
Short Communication
The mutagenic potentiator effect of chlorophyllin by the HGPRT assay Priscilla Davidson Negraesa, Berenice Quinzani Jorda˜oa, Veronica Elisa Pimenta Vicentinib, Ma´rio Se´rgio Mantovania,* a
Universidade Estadual de Londrina, Departamento de Biologia Geral, Campus Universitario CP 6001, Laborato´rio de Mutageˆnese in vitro, Londrina, Parana 86051 990, Brazil b Universidade Estadual de Maringa´, Departamento de Biologia Celular e Gene´tica, Laborato´rio de Gene´tica, Maringa´, PR, Brazil Received 19 May 2003; received in revised form 12 June 2003; accepted 7 July 2003
Abstract Chlorophyllin, a sodium-copper salt derivative of chlorophyll-a and -b, was evaluated for antimutagenic activity against ethyl methane sulfonate by the hypoxanthin-guanine-phosphoribosyl transferase (HGPRT) assay. The results obtained suggest that this chlorophyllin can potentiate the mutagenicity of an alkylating agent which induces DNA damage. # 2003 Elsevier Ltd. All rights reserved. Keywords: Chlorophyllin; CHO-K1 cells; hgprt; Mutagenicity; Potentiator effect
1. Introduction Chlorophyll-a and -b found in green plants, as well as chlorophyllin (Chln), a sodium–copper salt derivative of chlorophyll, has been studied for its protective action against the carcinogenic effects of many physical and chemical agents and for its relation to the mutagenic and clastogenic activities of genotoxic agents. The mechanisms by which chlorophyllin acts include bioantimutagenic and desmutagenic activities (Bez et al., 2001a,b; Rampazo et al., 2002). However, other studies have shown that both chlorophyll and Chln can display ambiguous genotoxic and carcinogenic effects (Sarkar et al., 1994; Sarkar et al., 1996; Romert et al., 1992), acting as a free radical. In this context, the mutagenicity of such compounds could be investigated by using assays in mammalian cells in vitro, including such test systems as testing for mutations at the hypoxanthine-guaninephosphoribosyl transferase locus in Chinese hamster ovary cells (CHO), which has been extensively used. HGPRT is an enzyme that is not essential for cell growth in culture, since purines can be obtained by de novo synthesis. Thus, HGPRT mutants lose their capa* Corresponding author. Fax: +55-43-3371-4207. E-mail address: [email protected] (M.S. Mantovani). 0887-2333/$ - see front matter # 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2003.07.001
city to utilize purine analogues that are lethal only to wild-type cells, and are easily identified when grown in medium containing 6-thioguanine, for example (Li et al., 1987). The present study examined the effect of Chln on mutations induced at the hgprt locus by ethyl methane sulfonate (EMS), in Chinese hamster ovary cells (CHO-K1).
2. Material and methods 2.1. Cell culture and treatments CHO-K1 cells (Chinese hamster ovary), furnished by the Mutagenesis Laboratory of USP—Ribeira˜o Preto (SP)/Brazil, were grown as monolayers in 25-cm2 culture flasks, in Dulbecco’s modified Eagle’s medium (DMEM)/Ham’s F12, supplemented with 10% fetal bovine serum and 0.1% of antibiotic-antimycotic solution, in a BOD chamber at 37 C. Under these conditions, the cell cycle was 12 h. After growing the cells for two cell cycles (24 h), cultures containing approximately 6106 cells were treated for a period of 3 h (without fetal bovine serum) with: (a) PBS, pH 7.4, negative control; (b) 620 mg/ml EMS, positive control; (c) 25 mg/ ml Chln; (d) EMS+6.25 mg/ml Chln; (e) EMS+12.5
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P.D. Negraes et al. / Toxicology in Vitro 18 (2004) 147–149
mg/ml Chln; and (f) EMS+25 mg/ml Chln. Afterward, the cells were washed, trypsinized (0.025% trypsin) and transferred to new culture flasks before assaying for cytotoxicity and effect on mutagenicity. Cytotoxicity (absolute cloning efficiency, ACE1) was determined after plating approximately 125 cells in three replicate flasks and allowing them to grow for 7 days. The colonies formed were fixed with methanol, stained with 10% Giemsa and counted. Effect on mutagenicity was determined by seeding a minimum of 2106 cells in flasks maintained for 9 days, and replating every 48 h for the expression of the mutant phenotype. Subsequently, 105 cells were transferred to five flasks/treatment each containing selective medium (10 mg/ml 6-thioguanine). At the moment of replating to selective medium, cloning efficiency (ACE2) was determined in non-selective medium (three replicate flasks; 125 cells each). After 9 days, colonies resistant to 6-thioguanine were fixed, stained and counted, and the mutation frequency (MF) was calculated in relation to non-selective cell survival (ACE2). The assay was performed according to Li et al. (1987). 2.2. Statistical analysis The data were analyzed by the conditional test for rare event detection (Pereira, 1991) at =0.05 level of significance.
3. Results and discussion Chlorophylls and their derivatives have been extensively investigated for their potential as antigenotoxic, anticlastogenic, antimutagenic and anticarcinogenic agents in many test systems (Renner, 1990; Gosh et al., 1991; Sen et al., 1991; Abraham et al., 1994; Sarkar et al., 1994; Edenharder et al., 1995; Negishi et al., 1997; Pimentel et al., 1999; Bez et al., 2001a, 2001b; Rampazo
et al., 2002). However, other studies have demonstrated that both chlorophylls and Chln can show genotoxic, clastogenic and carcinogenic effects (Romert et al., 1992; Sarkar et al., 1994, 1996). Table 1 presents the absolute and relative cloning efficiencies and the mutation frequency obtained in the evaluation of Chln by the HGPRT assay. This first set of experiments, Chln acts as a potentiator of the mutagenicity of the alkylating agent EMS, since the number of mutant colonies observed with combined treatment was significantly higher than that for the positive control. The principal effect of chlorophylls and chlorophyllin reported in the literature is its capacity to protect against DNA damage caused by many chemical and physical agents. However, Sarkar et al. (1993) studying the effect of Chln (1.5–2.0 mg/Kg b.w.) on the clastogenicity of chromium (IV) oxide and chordane in vivo, observed an anticlastogenic effect only with first of these latter two chemicals, demonstrating that the protective activity of compounds such as Chln cannot be generalized and depends directly on the type or class of DNA damage-inducing agent utilized. Moreover, some studies have suggested that the effect of these compounds correlate with the concentrations used, low or high, but do not appear be dose-dependent, as shown by Romert et al. (1992). These authors found that low concentrations of Chln potentiated twofold the mutagenicity of the tobacco-specific nitrosamines, NNN and NNK in Salmonella and the V79 assay, but high concentrations blocked the mutagenicity of the nitrosamines. This led to the proposal that Chln is capable of acting as a positive or negative modifier of mutagenicity. We have tested other higher concentrations of Chln in CHO-K1 cells, and they did not show any antimutagenic activity, but a potentiating effect in the mutagenicity of EMS. This suggests that the concentrations used may modify results in the assays, mainly with respect to the analysis of various parameters (e.g. chromosome aberration, micronucleus, comet, gene
Table 1 Mean cloning efficiency and mutation frequency observed in cells treated with chlorophyllin with or without ethyl methane sulfonate, and submitted to the in vitro HGPRT mutagenicity assaya Treatments
Cytotoxicity
Mutant selection
CHO-K1
ACE1 (%)
RCE (%)
n. mutant colonies
ACE2 (%)
MF (%)
Control EMS Chln 25 mg/ml Chln+EMS 6.25 mg/ml 12.5 mg/ml 25 mg/ml
50.64 37.84
– 74.72
0.6 5.8*
33.84 26.40
1.42 17.57
60.24
118.96
1.4
29.84
3.75
44.80 52.00 53.84
88.47 102.68 106.32
10.2* 12* 13.8*
42.08 24.80 46.40
19.39 38.71 23.79
a EMS, ethyl methane sulfonate 620 mg/ml; Chln, chlorophyllin; ACE, absolute cloning efficiency; RCE, relative cloning efficiency; MF, mutation frequency. * Statistically significant, 0.001
P.D. Negraes et al. / Toxicology in Vitro 18 (2004) 147–149
mutation—hgprt) and inducing agent utilized (e.g. EMS, MMS, and MMC, among others). Therefore, our results suggest that at the concentrations utilized, Chln acts as a positive modifier responsible for the increase in the number of mutations induced at the hgprt locus in the cell line employed. There was apparent evidence of a dose–response type relation, since increasing concentrations of Chln tested caused a gradual increase in the number of mutant colonies observed.
Acknowledgements This study has been supported by UEL, CAPES and Fundac¸a˜o Arauca´ria.
References Abraham, S.K., Sarma, L., Kesavan, P.C., 1994. Role of chlorophyllin as an in vivo anticlastogens: protection against gamma-radiation and chemical clastogens. Mutat. Res. 322, 209–212. Bez, G.C., Jorda˜o, B.Q., Vicentini, V.E.P., Mantovani, M.S., 2001a. Investigation of genotoxic and antigenotoxic activities of chlorophylls and chlorophyllin in cultured V79 cells. Mutat. Res. 497, 139–145. Bez, G.C., Jorda˜o, B.Q., Vicentini, V.E.P., Mantovani, M.S., 2001b. Differential protection of A and B chlorophyll in MMC-mediated induction of chromatid and isochromatid breaks in human lumphocytes culture. Cytologia 66, 313–318. Edenharder, R., Leopold, C.H., Kries, M., 1995. Modifying actions of solvent extracts from fruit and vegetable residues on 2-amino3methylimidazo[4,5]quinoline (IQ) and 2-amino-3,4-dimethylimidazo[4,5]quinxaline (MeIQx) induced mutagenesis in Salmonella typhimurium TA 98. Mutat. Res. 341, 302–318.
149
Ghosh, A.K., Sen, S., Sharma, A., Talukder, G., 1991. Effect of chlorophyllin on mercuric chloride-induced clastogenicity in mice. Food Chem Toxic 29, 777–779. Li, A.P., Carver, J.H., Choy, W.N., Hsie, A.W., Gupta, R.S., Loveday, K.S., O’Neill, J.P., Riddle, J.C., Stankowski Jr., L.F., Yang, L.L., 1987. A guide for the performance of the Chinese hamster ovary cell/hipoxanthine-guanine phosphoribosyl transferase gene mutation assay. Mutat. Res. 189, 135–141. Negishi, T., Ray, H., Hayatsu, H., 1997. Antigenotoxicity of natural chlorophylls. Mutat. Res. 376, 97–100. Pereira, A.A.B., 1991. Testes estatı´sticos para comparar proporc¸o˜es em problemas de citogene´tica. In: Rabello-Gay, M.N., Rodrigues, M.A.L.R., Monteleone-Neto, R. (Eds.), Mutageˆnese, Carcinogeˆnese e Teratogeˆnese: Me´todos e Crite´rios de Avaliac¸a˜o. Revista Brasileira de Gene´tica, Ribeira˜o Preto, SP, Brasil, pp. 113–121. Pimentel, E., Cruces, M.P., Zimmering, S., 1999. On the persistence of the radioprotective effect of chlorophyllin (CHLN) in somatic cells of Drosophyla. Mutat. Res. 446, 189–192. Rampazo, L.G.L., Jorda˜o, B.Q., Vicentini, V.E.P., Mantovani, M.S., 2002. Chlorophyllin antimutagenesis mechanisms under different treatment conditions in the micronucleus assay in V79 cells. Cytologia 67, 323–327. Renner, H.W., 1990. In vivo effects of single or combined dietary antimutagens on mutagen-induced chromosomal aberrations. Mutat. Res. 244, 185–188. Romert, L., Curvall, M., Jenssen, D., 1992. Chlorophyllin is both a positive and negative modifier of mutagenicity. Mutagenesis 7, 349– 355. Sarkar, D., Sharma, A., Talukder, G., 1993. Differential protection of chlorophyllin against clastogenic effects of chromium and chlordane in mouse bone marrow in vivo. Mutat. Res. 301, 33–38. Sarkar, D., Sharma, A., Talukder, G., 1994. Chlorophyll and chlorophyllin as modifiers of genotoxic effects. Mutat. Res. 318, 239–247. Sarkar, D., Sharma, A., Talukder, G., 1996. Chlorophyll and chromosome breakage. Mutat. Res. 360, 187–191. Sen, S., Sharma, A., Talukder, G., 1991. Inhibition of clastogenic effects of nicotine by chlorophyllin in mice bone marrow cells in vivo. Phytother Res. 5, 130–133.