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Inhibitory effect of chlorophyllin on the frequency of sister chromatid exchanges produced by benzo[a]pyrene in vivo
Mutation Research 388 Ž1997. 79–83
Inhibitory effect of chlorophyllin on the frequency of sister chromatid exchanges produced by benzow axpyrene in vivo E. Madrigal-Bujaidar ) , N. Velazquez-Guadarrama, S. Dıaz-Barriga ´ ´ Laboratorio de Genetica. Escuela Nacional de Ciencias Biologicas, I.P.N. Carpio y Plan de Ayala. Mexico D.F. cp 11340, Mexico ´ ´ ´ ´ Received 21 September 1995; revised 20 September 1996; accepted 1 October 1996
Abstract The study was designed to determine the antigenotoxic potential of chlorophyllin ŽChl., against the frequency of sister-chromatid exchanges ŽSCE. produced by benzow axpyrene ŽBw axP. in vivo. We used the mouse bone marrow test system to measure the effect of a single injection of the compounds: 40 mgrkg of Bw axP, and 1 h later, 1.0, 2.0, 4.0 and 8.0 mgrkg of Chl. As controls we included both chemicals using the dosages mentioned above as well as mineral oil Ž0.25 mgrkg.. The results indicated the following: Ž1. Chl per se was not genotoxic, showing SCE values in the range of the control level; Ž2. Bw axP increased the rate of SCEs three times in relation to the basal level; Ž3. the SCE level produced with Bw axP was diminished by all 4 doses of Chl, but better results were obtained with 2–4 mgrkg, a range which induced Inhibition Indices of 80.9% and 77.5% respectively; Ž4. the Average Generation Time Index was not modified by the compounds used in the experiment; and Ž5. the Mitotic Index also showed no significant modification induced by the chemicals, with respect to the control value. Keywords: Antigenotoxicity; Sister chromatid exchange; Chlorophyllin; Benzow axpyrene
1. Introduction Chlorophyllin ŽChl. is a sodium or copper salt derived from chlorophyll w1x. This compound is obtained in a saponification process of crude extracts of green plants. A number of studies have shown that Chl possesses a strong antimutagenic capacity in microorganisms. Using the Ames-Salmonella typhimurium or the Salmonella arabinose-resistant assays, several investigators have reported an inhibitory effect of this chemical against the damage )
Corresponding author. Laboratorio de Genetica. Escuela Na´ cional de Ciencias Biologicas, I.P.N. Carpio y Plan de Ayala. Sto. ´ Tomas. D.F. C.P. 11340 ŽMexico ´ Mexico, ´ ´ ..
produced by other chemicals such as aflatoxin B 1 , heterocyclic amines, benzoŽa.pyrene, cigarette condensate, fried meat, red wine, tobacco snuff, airborne particles, coal dust and black pepper w2–6x. The effectiveness of Chl as an antimutagen in in vitro assays prompted the performance of studies to determine if this capacity is also expressed in vivo Žincluding mammalian organisms., since the final objective for the use of antimutagenic agents is their potential application in humans. In this area, the results in Drosophila, mouse, and Chinese hamsters were positive against the effects produced by chromium, cesium chloride, thio-tepa and ionizing radiation w7–12x, but negative results were obtained with respect to chlordane w10x.
1383-5718r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 1 2 1 8 Ž 9 6 . 0 0 1 3 9 - 5
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E. Madrigal-Bujaidar et al.r Mutation Research 388 (1997) 79–83
On the other hand, benzow axpyrene ŽBw axP. is an aromatic polycyclic hydrocarbon closely connected with human activities. It has been found in tobacco smoke, airborne particles and in an industrial environment mainly related to the production of coal tar and asphalt w13x; furthermore, it has been suggested that food resources may be contaminated with atmospheric Bw axP w14x. This chemical is a potent mutagen-carcinogen which requires metabolic activation to express these properties. Its genotoxicity has been determined in in vivo and in vitro studies and includes the induction of genic mutations and chromosome damage w15–18x. Man is exposed to a multitude of environmental poisons. The National Institute for Occupational Safety and Health ŽUSA. has estimated that millions of workers have been connected with potentially hazardous agents w19x, but the occupational environment is by no means the only source of mutagens. Other sources include water, soil, the atmosphere, food consumed by humans, and social habits. Bw axP is a potent genotoxicant which has been implicated as a causative agent in human cancer; therefore, reduction or elimination of its deleterious biological effects seems to be appropriate using different strategies which include the use of genotoxic modulators. Since Chl has shown a valuable antigenotoxic effect, in this report we determined its genotoxic modulation capacity in mice treated with Bw axP.
2. Materials and methods
Male mice ŽNIH. with a mean weight of 24 g were obtained from the National Institute of Hygiene. They were maintained in polypropylene cages at a mean temperature of 238C and permitted to freely consume water and standard pellets ŽPurina, Mexico City.. Ten lots with 5 animals each were used for the experiment. 2.2. Experimental procedure The basic experimental design has been previously published w20x. A subcutaneous implantation of a BrdU tablet was initially performed in slightly anesthetized animals, and 30 min later mice were intraperitoneally Ži.p.. injected as follows: lot 1 with mineral oil Ž0.25 mgrkg., lot 2 with Bw axP Ž40 mgrkg., lots 3, 4, 5 and 6 with Chl in the dosages 1.0, 2.0, 4.0 and 8.0 mgrkg, and lots 7, 8, 9 and 10 with Bw axP Ž40 mgrkg.; the last four groups of animals Žlots 7, 8, 9 and 10. one h later were i.p. injected with Chl with the dosages 1, 2, 4 and 8 mgrkg, respectively. At 21 h of BrdU implantation we injected colchicine ŽSigma, St. Louis Missouri, USA. Ž0.3 mlrkg. and 3 h later the animals were killed by cervical dislocation to obtain bone marrow cells in KCl 0.075 M, where they rested for 30 min at 378C. The next step was fixation with methanolracetic acid Ž3 : 1., and the preparation of slides by flaming. Chromatid differential staining was performed following the fluorescence-plus Giemsa technique initially described by Perry and Wolff w21x with slight modifications already reported w20x.
2.1. Chemicals and animals Chlorophyllin, 5-bromodeoxyuridine ŽBrdU., and benzow axpyrene were obtained from Sigma Chemicals ŽSt. Louis, MO, USA. and the fluorochrome Hoechst 33258 from Riedel de Haen ŽHamburg, Germany.. Chl was dissolved in distilled water and the following final dosages were established: 1.0, 2.0, 4.0 and 8.0 mgrkg of body weight. With respect to BrdU, a 50 mg tablet was made and partially coated with paraffin. Bw axP was dissolved in mineral oil and a final dose of 40 mgrkg of body weight was prepared. Finally, Hoechst 33258 was dissolved in distilled water to prepare a concentration of 100 mgrml.
2.3. Cytogenetic obserÕation and statistical eÕaluation Ž1. The frequency of SCEs was determined in 30 second division metaphases per animal. With the obtained data an Inhibition Index in % ŽII. was established according to the formula: II s X SCEsŽBw axP q Chl treated mice. y X SCEs ŽChl control.4 rX SCEsŽBw axP. y X SCEsŽsolvent control.4 . Ž2. The mitotic index ŽMI. was determined in 1000 cells per animal. Ž3. Cell proliferation kinetics ŽCPK. was evaluated by scoring 100 cells per animal to establish the
E. Madrigal-Bujaidar et al.r Mutation Research 388 (1997) 79–83
frequency of mitosis in first ŽM1., second ŽM2. and third ŽM3. cellular division. With these data the Average Generation Time ŽAGT. was obtained using the formula: AGT s
21
Ž M1 q 2M2 q 3M3.
= 100
Ž4. Statistical analyses were made with a two way Analysis of Variance and the Student’s t-test using an alpha s 0.01.
3. Results and discussion: Table 1 shows the results obtained in the SCE scoring. It was observed that SCE frequencies in all four Chl tested dosages were in the same range as the control group of animals treated with mineral oil with no statistical differences between them. On the contrary, the results obtained with Bw axP showed an SCE increase of approximately three times over the control level, a result which is in agreement with previous reports considering different endpoints and test systems w3,4,22,23x. With respect to the effect exerted by Chl on Bw axP genotoxicity, we observed that all four tested
Table 1 Effect of chlorophyllin ŽChl. on the SCE frequency produced by benzow axpyrene ŽBw axP. in mouse bone marrow cells Chemical
Dose Žmgrkg.
SCE"
SD
Mineral oil. Chl.
0.25 1.0 2.0 4.0 8.0
2.23 a 2.40 a 2.90 a 2.43 a 2.61 a
0.9 0.38 0.44 0.34 0.28
Bw axP Bw axPqChl.
40.0 40q1.0 40q2.0 40q4.0 40q8.0
6.88 4.39 a 3.81 a 3.64 a 4.90 a
0.61 0.42 0.84 0.30 0.6
Inhibition index Ž%.
57.26 80.94 77.50 50.64
To determine the SCE frequency we scored 30 cells per animal Ž5 mice per dose.. a Significant difference with respect to the positive control ŽBw axP.. ANOVA and Student’s t-tests Ž ps 0.01. Inhibition Index s ŽX SCEs ŽBw axPqChl treated mice.-X SCEs ŽChl control..r X SCEs ŽBw axP.-X SCEs Žsolvent control..=100
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Table 2 Average generation time ŽATG. and mitotic index ŽMI. in mouse bone marrow cells treated with Bw axP and Chl Chemical
Dose Žmgrkg.
AGT Žh.
MI Ž%.
Mineral oil Chl
0.25 1.0 2.0 4.0 8.0
12.09 12.14 12.09 12.29 11.0
6.06 5.92 6.62 6.64 5.30
Bw axP Bw axPqChl
40.0 40q1.0 40q2.0 40q4.0 40q8.0
11.17 13.10 12.48 13.12 11.24
4.88 4.96 7.36 6.36 5.54
AGT was established in 100 cells per animal Ž5 animals per dose. scoring the frequency of M1, M2 and M3 cells and using the formula AGT s 21rŽM1q2M2q3M3.=100. MI was established in 1000 cells per animal. ANOVA and Student’s t-test showed no statistical differences in both parameters Ž ps 0.01..
doses significantly reduced the SCE frequency induced by Bw axP alone. In comparison with the control level, the Inhibition Index values indicated an SCE reduction which varies from 50.6% ŽChl s 8.0 mgrkg. to 80.9% ŽChl s 2.0 mgrkg. with a mean of 66.5%. Our results also suggest a better antigenotoxic response in the Chl range of 2.0–4.0 mgrkg. If the observation were confirmed with a high Chl dose, this effect may be related to the determination of the effective dose in mouse, which is about 2.0 mgrkg w10x. Chl doses higher than 4.0 mgrkg may represent an excess of the chemical with respect to the constant amount of Bw axP, which could destabilize the reversible interaction between both compounds. The analyses of cell proliferation kinetics expressed in the Average Generation Time values ŽAGT. are indicated in Table 2. A homogeneous behavior was observed in all tested doses of both Chl and Bw axP, administered alone or together, although in the last form a mild non-significant AGT increase was detected. Concerning the evaluation of mitotic Index data, Žshown in the same Table 2., there were no statistical differences between dosages and compounds used in the experiment. As higher doses of Bw axP may induce a marked cytotoxicity w13x, our data suggest that we basically measured the geno-
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E. Madrigal-Bujaidar et al.r Mutation Research 388 (1997) 79–83
toxic and the antigenotoxic capacity of Chl with no interference from other type of cellular damage. The widespread development of mutagens and their potentially dangerous effects in human health have promoted the development of strategies to cope with these agents. One of these approaches is the introduction of mutagen modulators. Among chemicals with this property, Chl appears to be one of the most promising antimutagens, because of its high response against a number of substances as well as its lack of important toxic effects in studies measuring gene or chromosome damage, features that we confirmed in this investigation with respect to Bw axP genotoxicity. Chl is believed to operate as interceptor molecules, and in the case of Bw axP the involved mechanism may be to accelerate the degradation of the ultimate carcinogen, benzow a xpyrene-7,8-dihydrodiol-9,10epoxide, along with inhibition of carcinogen activation w5,24x. It has also been determined that different epoxides may form a complex with Chl, including the one produced in the Bw axP metabolism w24x; therefore, our antigenotoxic results may be due to this type of complex formation of Chl with the mutagen and its degradation to inactive tetrols. Nevertheless, the effectiveness of Chl, as usually happens with other antimutagens, seems to depend on specific factors and experimental conditions; one of them is the dose-range used and another may be the time of administration w4,8,11x, a factor connected with the route, concentration, type of cell and species used in the experiment. The antigenotoxicity of Chl observed in different test systems has encouraged its potential use in humans, but the results in the dimethylhydrazine colorectal animal model w25x, showing the ability of Chl to potentiate tumor induction caused by other carcinogens, suggest the desirability of developing further in vivo studies to detect the real extent of anticarcinogenic potential of this chemical. It is difficult to compare the results obtained by Nelson w25x with ours, because the Chl administration used by the mentioned investigator lasted at least 20 weeks with a daily concentration higher than that used in the present study, and also because the studied endpoint was different. Nevertheless, it is convenient to note that in a previous study w26x the authors observed a decrease in the amount of nuclear damage
induced by dimethylhydrazine when the animals were treated with Chl; these two apparently contradictory results led to the suggestion that the carcinogenic effect of Chl may be due to a non-genotoxic activity, such as an increase in the mucosal proliferative rate w25x.
Acknowledgements The authors deeply appreciate the economic support provided by CONACYT.
References w1x Kephart, J.C. Ž1955. Chlorophyll derivatives, their chemistry, commercial preparation and use, Econ. Bot., 9, 3–38. w2x Dashwood, R.H., U. Breinholt and G.S. Bailey Ž1991. Chemopreventive properties of chlorophyllin: inhibition of aflatoxin B1 ŽAFB1 .-DNA binding in vivo and antimutagenic activity against AFB1 , and two heterocyclic amines in Salmonella mutagenicity assay, Carcinogenesis, 12, 939–942. w3x Terwel, L. and J.C. Van der Hoeven Ž1985. Antimutagenic activity of some naturally occurring compounds towards cigarette-smoke condensate and benzo w a xpyrene in Salmonellarmicrosome assay, Mutation Res., 152, 1–4. w4x Warner, J.R., J. Nath and T.M. Ong Ž1991. Antimutagenicity studies of chlorophyllin using the Salmonella arabinose resistant assay system, Mutation Res., 262, 25–30. w5x Whong, W.Z., J. Stewart and T.M. Ong Ž1988. Comparative antimutagenicity of chlorophyllin and five other agents against Aflatoxin B1 induced reversion in Salmonella typhimurium strain TA98, Teratogen. Carcinogen. Mutagen., 8, 215–224. w6x Ong, T.M. and W.Z. Whong Ž1986. Chlorophyllin: a potent antimutagen against environmental and dietary complex mixtures, Mutation Res., 173, 111–115. w7x Zimmering, S. and O. Olvera Ž1990. Evidence for a radioprotective effect of chlorophyllin in Drosophila, Mutation Res., 245, 47–49. w8x Ghosh, A. and S. Sen Ž1991. Inhibition of clastogenic effects of cesium chloride in mice in vivo by chlorophyllin, Toxicol. Lett., 57, 11–17. w9x Olvera, O., S. Zimmering, C. Arceo and M. Cruces Ž1993. The protective effects of chlorophyllin in treatment with chromium ŽVI. oxide in somatic cells of Drosophila, Mutation Res., 301, 33–38. w10x Sarkar, D., A. Sharma and G. Talukder Ž1993. Differential protection of chlorophyllin against clastogenic effect of chromium and chlordane in mouse bone marrow in vivo, Mutation Res., 244, 185–188. w11x Rener, H.W. Ž1990. In vivo effects of single or combined dietary antimutagens on mutagen induced chromosomal aberrations, Mutation Res., 244, 185–188.
E. Madrigal-Bujaidar et al.r Mutation Research 388 (1997) 79–83 w12x Morales Ramırez, Ž1994. In P. and M.C. Garcıa-Rodrıguez ´ ´ ´ vivo effect of chlorophyllin on X-ray induced sister chromatid exchange in murine bone marrow cells, Mutation Res., 320, 329–334. w13x International Agency for Research on Cancer Ž1993. Monographs on the evaluation of the carcinogenic risk of chemicals to humans. World Health Organization, Lyon, 32, 211– 234. w14x Lijinsky, W. Ž1991. The formation and occurrence of polynuclear aromatic hydrocarbons associated with food, Mutation Res., 259, 251–261. w15x Pal, K., P.L. Grover and P. Sims Ž1980. The induction of sister chromatid exchanges in Chinese hamster ovary cells by some epoxides and phenolic derivatives of benzow axpyrene, Mutation Res., 78, 193–199. w16x Kliesch, U. and J. Roupova Ž1982. Induction of chromosome damage in mouse bone marrow by benzow axpyrene diol epoxides in CHO cells, Mutation Res., 102, 263–273. w17x Mersch-Sundermann, V., S. Mochayedi and J. Kevekordes Ž1992. Genotoxicity of polycyclic aromatic hydrocarbons in Escherichia coli PQ37, Mutation Res., 278, 1–9.. w18x MacLeod, M.C., A. Daylong and G. Adair Ž1991. Differences in the rate of DNA adduct removal and the efficiency of mutagenesis for two benzowaxpyrene diol epoxides in CHO cells, Mutation Res., 261, 267–279. w19x National Institute for Occupational Safety and Health ŽNIOSH., Centers for Disease Control Ž1982. Proposed hazard communication standard 47 Fed. Reg. 12092, 12108.
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w20x Rivas-Olmedo, G., S. Dıaz-Barriga and E. Madrigal-Bujaidar ´ Ž1992. Inhibition of mitomycin C-induced sister chromatid exchanges by vitamin C in vivo, J. Toxicol. Environ. Health, 35, 107–113. w21x Perry, P. and S. Wolff Ž1974. New Giemsa method for the differential staining of sister chromatids, Nature, 261, 156– 158. w22x Katoh, Y., N. Nemoto, M. Tanaka and S. Takayama Ž1983. Inhibition of benzow axpyrene induced mutagenesis in Chinese hamster V79 cells by hemin and related compounds, Mutation Res., 121, 153–157. w23x Romert, L., L. Curvall and D. Jensen Ž1992. Chloropyllin is both a positive and negative modifier of mutagenicity, Mutagenesis, 7, 349–355. w24x Tachino, N., D. Guo, W.M. Dashwood, S. Yamane, R. Larsen and R. Dashwood Ž1994. Mechanisms of the in vitro antimutagenic action of chlorophyllin against benzowaxpyrene: Studies of enzyme inhibition molecular complex formation and segregation of the ultimate carcinogen, Mutation Res., 308, 191–203. w25x Nelson, R.L. Ž1992. Chlorophyllin, an antimutagen, acts as a tumor promoter in the rat-dimethylhydrazine colon carcinogenesis model, Anticancer Res., 12, 737–740. w26x Robins, E., R.L. Nelson Ž1989. Inhibition of 1,2 dimethylhidrazine induced nuclear damage in the rat colon by chlorophyllin, Anticancer, 9, 981–986.
Inhibitory effect of chlorophyllin on the frequency of sister chromatid exchanges produced by benzow axpyrene in vivo E. Madrigal-Bujaidar ) , N. Velazquez-Guadarrama, S. Dıaz-Barriga ´ ´ Laboratorio de Genetica. Escuela Nacional de Ciencias Biologicas, I.P.N. Carpio y Plan de Ayala. Mexico D.F. cp 11340, Mexico ´ ´ ´ ´ Received 21 September 1995; revised 20 September 1996; accepted 1 October 1996
Abstract The study was designed to determine the antigenotoxic potential of chlorophyllin ŽChl., against the frequency of sister-chromatid exchanges ŽSCE. produced by benzow axpyrene ŽBw axP. in vivo. We used the mouse bone marrow test system to measure the effect of a single injection of the compounds: 40 mgrkg of Bw axP, and 1 h later, 1.0, 2.0, 4.0 and 8.0 mgrkg of Chl. As controls we included both chemicals using the dosages mentioned above as well as mineral oil Ž0.25 mgrkg.. The results indicated the following: Ž1. Chl per se was not genotoxic, showing SCE values in the range of the control level; Ž2. Bw axP increased the rate of SCEs three times in relation to the basal level; Ž3. the SCE level produced with Bw axP was diminished by all 4 doses of Chl, but better results were obtained with 2–4 mgrkg, a range which induced Inhibition Indices of 80.9% and 77.5% respectively; Ž4. the Average Generation Time Index was not modified by the compounds used in the experiment; and Ž5. the Mitotic Index also showed no significant modification induced by the chemicals, with respect to the control value. Keywords: Antigenotoxicity; Sister chromatid exchange; Chlorophyllin; Benzow axpyrene
1. Introduction Chlorophyllin ŽChl. is a sodium or copper salt derived from chlorophyll w1x. This compound is obtained in a saponification process of crude extracts of green plants. A number of studies have shown that Chl possesses a strong antimutagenic capacity in microorganisms. Using the Ames-Salmonella typhimurium or the Salmonella arabinose-resistant assays, several investigators have reported an inhibitory effect of this chemical against the damage )
Corresponding author. Laboratorio de Genetica. Escuela Na´ cional de Ciencias Biologicas, I.P.N. Carpio y Plan de Ayala. Sto. ´ Tomas. D.F. C.P. 11340 ŽMexico ´ Mexico, ´ ´ ..
produced by other chemicals such as aflatoxin B 1 , heterocyclic amines, benzoŽa.pyrene, cigarette condensate, fried meat, red wine, tobacco snuff, airborne particles, coal dust and black pepper w2–6x. The effectiveness of Chl as an antimutagen in in vitro assays prompted the performance of studies to determine if this capacity is also expressed in vivo Žincluding mammalian organisms., since the final objective for the use of antimutagenic agents is their potential application in humans. In this area, the results in Drosophila, mouse, and Chinese hamsters were positive against the effects produced by chromium, cesium chloride, thio-tepa and ionizing radiation w7–12x, but negative results were obtained with respect to chlordane w10x.
1383-5718r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 1 2 1 8 Ž 9 6 . 0 0 1 3 9 - 5
80
E. Madrigal-Bujaidar et al.r Mutation Research 388 (1997) 79–83
On the other hand, benzow axpyrene ŽBw axP. is an aromatic polycyclic hydrocarbon closely connected with human activities. It has been found in tobacco smoke, airborne particles and in an industrial environment mainly related to the production of coal tar and asphalt w13x; furthermore, it has been suggested that food resources may be contaminated with atmospheric Bw axP w14x. This chemical is a potent mutagen-carcinogen which requires metabolic activation to express these properties. Its genotoxicity has been determined in in vivo and in vitro studies and includes the induction of genic mutations and chromosome damage w15–18x. Man is exposed to a multitude of environmental poisons. The National Institute for Occupational Safety and Health ŽUSA. has estimated that millions of workers have been connected with potentially hazardous agents w19x, but the occupational environment is by no means the only source of mutagens. Other sources include water, soil, the atmosphere, food consumed by humans, and social habits. Bw axP is a potent genotoxicant which has been implicated as a causative agent in human cancer; therefore, reduction or elimination of its deleterious biological effects seems to be appropriate using different strategies which include the use of genotoxic modulators. Since Chl has shown a valuable antigenotoxic effect, in this report we determined its genotoxic modulation capacity in mice treated with Bw axP.
2. Materials and methods
Male mice ŽNIH. with a mean weight of 24 g were obtained from the National Institute of Hygiene. They were maintained in polypropylene cages at a mean temperature of 238C and permitted to freely consume water and standard pellets ŽPurina, Mexico City.. Ten lots with 5 animals each were used for the experiment. 2.2. Experimental procedure The basic experimental design has been previously published w20x. A subcutaneous implantation of a BrdU tablet was initially performed in slightly anesthetized animals, and 30 min later mice were intraperitoneally Ži.p.. injected as follows: lot 1 with mineral oil Ž0.25 mgrkg., lot 2 with Bw axP Ž40 mgrkg., lots 3, 4, 5 and 6 with Chl in the dosages 1.0, 2.0, 4.0 and 8.0 mgrkg, and lots 7, 8, 9 and 10 with Bw axP Ž40 mgrkg.; the last four groups of animals Žlots 7, 8, 9 and 10. one h later were i.p. injected with Chl with the dosages 1, 2, 4 and 8 mgrkg, respectively. At 21 h of BrdU implantation we injected colchicine ŽSigma, St. Louis Missouri, USA. Ž0.3 mlrkg. and 3 h later the animals were killed by cervical dislocation to obtain bone marrow cells in KCl 0.075 M, where they rested for 30 min at 378C. The next step was fixation with methanolracetic acid Ž3 : 1., and the preparation of slides by flaming. Chromatid differential staining was performed following the fluorescence-plus Giemsa technique initially described by Perry and Wolff w21x with slight modifications already reported w20x.
2.1. Chemicals and animals Chlorophyllin, 5-bromodeoxyuridine ŽBrdU., and benzow axpyrene were obtained from Sigma Chemicals ŽSt. Louis, MO, USA. and the fluorochrome Hoechst 33258 from Riedel de Haen ŽHamburg, Germany.. Chl was dissolved in distilled water and the following final dosages were established: 1.0, 2.0, 4.0 and 8.0 mgrkg of body weight. With respect to BrdU, a 50 mg tablet was made and partially coated with paraffin. Bw axP was dissolved in mineral oil and a final dose of 40 mgrkg of body weight was prepared. Finally, Hoechst 33258 was dissolved in distilled water to prepare a concentration of 100 mgrml.
2.3. Cytogenetic obserÕation and statistical eÕaluation Ž1. The frequency of SCEs was determined in 30 second division metaphases per animal. With the obtained data an Inhibition Index in % ŽII. was established according to the formula: II s X SCEsŽBw axP q Chl treated mice. y X SCEs ŽChl control.4 rX SCEsŽBw axP. y X SCEsŽsolvent control.4 . Ž2. The mitotic index ŽMI. was determined in 1000 cells per animal. Ž3. Cell proliferation kinetics ŽCPK. was evaluated by scoring 100 cells per animal to establish the
E. Madrigal-Bujaidar et al.r Mutation Research 388 (1997) 79–83
frequency of mitosis in first ŽM1., second ŽM2. and third ŽM3. cellular division. With these data the Average Generation Time ŽAGT. was obtained using the formula: AGT s
21
Ž M1 q 2M2 q 3M3.
= 100
Ž4. Statistical analyses were made with a two way Analysis of Variance and the Student’s t-test using an alpha s 0.01.
3. Results and discussion: Table 1 shows the results obtained in the SCE scoring. It was observed that SCE frequencies in all four Chl tested dosages were in the same range as the control group of animals treated with mineral oil with no statistical differences between them. On the contrary, the results obtained with Bw axP showed an SCE increase of approximately three times over the control level, a result which is in agreement with previous reports considering different endpoints and test systems w3,4,22,23x. With respect to the effect exerted by Chl on Bw axP genotoxicity, we observed that all four tested
Table 1 Effect of chlorophyllin ŽChl. on the SCE frequency produced by benzow axpyrene ŽBw axP. in mouse bone marrow cells Chemical
Dose Žmgrkg.
SCE"
SD
Mineral oil. Chl.
0.25 1.0 2.0 4.0 8.0
2.23 a 2.40 a 2.90 a 2.43 a 2.61 a
0.9 0.38 0.44 0.34 0.28
Bw axP Bw axPqChl.
40.0 40q1.0 40q2.0 40q4.0 40q8.0
6.88 4.39 a 3.81 a 3.64 a 4.90 a
0.61 0.42 0.84 0.30 0.6
Inhibition index Ž%.
57.26 80.94 77.50 50.64
To determine the SCE frequency we scored 30 cells per animal Ž5 mice per dose.. a Significant difference with respect to the positive control ŽBw axP.. ANOVA and Student’s t-tests Ž ps 0.01. Inhibition Index s ŽX SCEs ŽBw axPqChl treated mice.-X SCEs ŽChl control..r X SCEs ŽBw axP.-X SCEs Žsolvent control..=100
81
Table 2 Average generation time ŽATG. and mitotic index ŽMI. in mouse bone marrow cells treated with Bw axP and Chl Chemical
Dose Žmgrkg.
AGT Žh.
MI Ž%.
Mineral oil Chl
0.25 1.0 2.0 4.0 8.0
12.09 12.14 12.09 12.29 11.0
6.06 5.92 6.62 6.64 5.30
Bw axP Bw axPqChl
40.0 40q1.0 40q2.0 40q4.0 40q8.0
11.17 13.10 12.48 13.12 11.24
4.88 4.96 7.36 6.36 5.54
AGT was established in 100 cells per animal Ž5 animals per dose. scoring the frequency of M1, M2 and M3 cells and using the formula AGT s 21rŽM1q2M2q3M3.=100. MI was established in 1000 cells per animal. ANOVA and Student’s t-test showed no statistical differences in both parameters Ž ps 0.01..
doses significantly reduced the SCE frequency induced by Bw axP alone. In comparison with the control level, the Inhibition Index values indicated an SCE reduction which varies from 50.6% ŽChl s 8.0 mgrkg. to 80.9% ŽChl s 2.0 mgrkg. with a mean of 66.5%. Our results also suggest a better antigenotoxic response in the Chl range of 2.0–4.0 mgrkg. If the observation were confirmed with a high Chl dose, this effect may be related to the determination of the effective dose in mouse, which is about 2.0 mgrkg w10x. Chl doses higher than 4.0 mgrkg may represent an excess of the chemical with respect to the constant amount of Bw axP, which could destabilize the reversible interaction between both compounds. The analyses of cell proliferation kinetics expressed in the Average Generation Time values ŽAGT. are indicated in Table 2. A homogeneous behavior was observed in all tested doses of both Chl and Bw axP, administered alone or together, although in the last form a mild non-significant AGT increase was detected. Concerning the evaluation of mitotic Index data, Žshown in the same Table 2., there were no statistical differences between dosages and compounds used in the experiment. As higher doses of Bw axP may induce a marked cytotoxicity w13x, our data suggest that we basically measured the geno-
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toxic and the antigenotoxic capacity of Chl with no interference from other type of cellular damage. The widespread development of mutagens and their potentially dangerous effects in human health have promoted the development of strategies to cope with these agents. One of these approaches is the introduction of mutagen modulators. Among chemicals with this property, Chl appears to be one of the most promising antimutagens, because of its high response against a number of substances as well as its lack of important toxic effects in studies measuring gene or chromosome damage, features that we confirmed in this investigation with respect to Bw axP genotoxicity. Chl is believed to operate as interceptor molecules, and in the case of Bw axP the involved mechanism may be to accelerate the degradation of the ultimate carcinogen, benzow a xpyrene-7,8-dihydrodiol-9,10epoxide, along with inhibition of carcinogen activation w5,24x. It has also been determined that different epoxides may form a complex with Chl, including the one produced in the Bw axP metabolism w24x; therefore, our antigenotoxic results may be due to this type of complex formation of Chl with the mutagen and its degradation to inactive tetrols. Nevertheless, the effectiveness of Chl, as usually happens with other antimutagens, seems to depend on specific factors and experimental conditions; one of them is the dose-range used and another may be the time of administration w4,8,11x, a factor connected with the route, concentration, type of cell and species used in the experiment. The antigenotoxicity of Chl observed in different test systems has encouraged its potential use in humans, but the results in the dimethylhydrazine colorectal animal model w25x, showing the ability of Chl to potentiate tumor induction caused by other carcinogens, suggest the desirability of developing further in vivo studies to detect the real extent of anticarcinogenic potential of this chemical. It is difficult to compare the results obtained by Nelson w25x with ours, because the Chl administration used by the mentioned investigator lasted at least 20 weeks with a daily concentration higher than that used in the present study, and also because the studied endpoint was different. Nevertheless, it is convenient to note that in a previous study w26x the authors observed a decrease in the amount of nuclear damage
induced by dimethylhydrazine when the animals were treated with Chl; these two apparently contradictory results led to the suggestion that the carcinogenic effect of Chl may be due to a non-genotoxic activity, such as an increase in the mucosal proliferative rate w25x.
Acknowledgements The authors deeply appreciate the economic support provided by CONACYT.
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