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Effect of chlorophyllin on mercuric chloride-induced clastogenicity in mice
Fd Chem. Toxic. Vol. 29, No. 1I, pp. 777-779, 1991
0278-6915/91 $3.00+ 0.00 PergamonPress pie
Printed in Great Britain
E F F E C T OF C H L O R O P H Y L L I N ON M E R C U R I C C H L O R I D E - I N D U C E D C L A S T O G E N I C I T Y IN MICE A. K. GHOSH*, S. SENt, A. SHARMAand G. TALUKDER+ Genetic Toxicology Unit, Centre for Advanced Study in Cell and Chromosome Research, Department of Botany, University of Calcutta, 35 Ballygunge Circular Road, Calcutta 700019, India (Accepted 18 July 1991)
A~tract--The effect of chlorophyllin (1.5 mg/kg body weight) on the clastogenicity of mercuric chloride (HgC12) was studied in vivo in mouse bone marrow cells. HgCI2 (3.0, 6.0 and 12.0mg/kg body weight) administered by gavage induced chromosomal aberrations at frequencies directly proportional to the dose. Chlorophyllin was not clastogenic, and significantly reduced the mitotic index when given alone. Chlorophyllin administered simultaneously with HgCI2 significantly reduced the frequencies of chromosomal aberrations in a dose-dependent manner. When given simultaneously with the lowest HgCI2 concentration tested (3.0 mg/kg body weight), chlorophyllin provided total protection. A lower degree of protection was given by chlorophyllin administered 2 hr before HgCI2. The data demonstrate the potential of green plant components to modify the genotoxic activity of HgCI2 when administered orally.
INTRODUCTION Inorganic salts and organic forms of mercury are both environmental pollutants (Das et al., 1982) and potent clastogens (L6onard et al., 1983). Efforts have been made to counteract the toxic effects of these compounds using various metals (Das et al., 1985; Magos and Webb, 1979), vitamins (Ganther, 1980) and thiol compounds (Swensson and Ulfvarson, 1967). The constituents of green plants such as chlorophyll and its major water-soluble derivative, chiorophyllin, have been found to be antimutagenic against a variety of complex mutagens and N-nitroso compounds (Lai, 1979; Ong et al., 1986). The present work was undertaken to study the effects and modulatory role, if any, of chlorophyllin on the clastogenic action of the chloride salt of mercury in bone marrow cells of mice when administered orally in vivo.
MATERIALS AND METHODS
Chemicals. Mercuric chloride (HgCI2) was obtained from BDH Chemicals Ltd (India). Chlorophyllin (sodium-copper salt; 6.1% Na, 2.4% Cu) and eyclophosphamide were purchased from Sigma Chemical Co. (St Louis, MO, USA). All other chemicals were of Analar grade. Animals. The experiments were conducted on male inbred Swiss albino mice (8-10 wk old, 25-28 g) obtained from the Departmental animal house. The mice were housed in cages at a temperature of 28___2°C, relative humidity 60_+ 5% with a 12-hr
*To whom all correspondence should be addressed. tPresent address: Division Laboratory Animals, Central Drug Research Institute, Lucknow, India. :~Presentaddress: Vivekananda Institute of Medical Science, Calcutta, India. Abbreviation: HgCI2 = mercuric chloride.
light/dark cycle. The animals were fed standard diet (Lipton, India) and unlimited water was given. E x p e r i m e n t a l p r o t o c o l . The protocol and guidelines for in vivo cytogenetic studies were followed strictly (Preston et al., 1987). Five mice were used for each dose of HgCI 2 tested. HgC12 in distilled water was administered in three doses: 3.0, 6.0 and 12.0 mg/kg body weight equivalent to fractions of 1/40, 1/20 and 1/10 of the LDs0 determined in the strain of mice used. Chlorophyllin was dissolved in distilled water. A single concentration of 1.5mg/kg body weight was used. All chemicals were administered orally by gavage. Animals were killed 24 hr after exposure (Preston et al., 1987). The modulatory effect of chlorophyllin on the clastogenicity of HgC12 was studied. Chlorophyllin was given 2 hr before HgCI 2 in one set of experiments, and simultaneously with the toxic compound in a second set. Detailed treatment protocols are shown in Table 1. Positive a n d negative control. Cyclophosphamide (25 mg/kg body weight) administered ip served as the positive control (Preston et al., 1987). Double glassdistilled water (10 g/kg body weight) was administered by gavage as the negative (vehicle) control. Mice were killed 24 hr after exposure. C h r o m o s o m e preparations. Bone-marrow chromosomes were prepared by the standard protocol of Sharma and Sharma (1984): colchicine (4 mg/kg body weight, ip)--hypotonic (1% sodium citrate)--fixative (1:3, glacial acetic acid-ethanol)--flame drying. The slides were stained in diluted Giemsa stain. 50 perfect metaphase plates were scored randomly per animal for chromosomal aberrations, and 1000 cells were scored for the mitotic index. The types of aberrations were scored strictly in accordance with the method of Tice et al. (1987). All aberrations (chromatid gap, chromosome gap, chromatid break, chromosome break and rearrangement) were considered equal, irrespective of the number of events involved. The results are expressed as percentages of aberrant
777
778
A.K. GHOSH et al.
metaphase cells excluding gaps (% DC), and numbers of aberrations per cell excluding gaps (CA/cell). S t a t i s t i c a l analysis. For all statistical analyses, the level of significance was established at P ~<0.05. A one-tailed trend test (Margolin et al., 1986) was performed to observe the effects of HgClz. A one-way ANOVA test followed by Duncan's multiple range test (Sokal and Rohlf, 1981) was carried out to detect significant differences among different treatment protocols. Student's t-test was used to compare the mitotic index in the different sets of experiments.
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RESULTS Table l summarizes the different aberrations recorded, frequencies of chromosomal aberrations per cell (excluding gaps), numbers of damaged cell and mitotic indexes. A one-way ANOVA test followed by Duncan's multiple range test showed that in all sets the pretreatment with chlorophyllin protected only partially against the clastogenic action of HgCL (Table 2). The number of aberrations was reduced ai all HgC1, doses but the degree of protection was lower than that obtained with simultaneous administration of chlorophyllin and HgCI,. Simultaneous treatment of mice with the two compounds protected partially against the highest dose of HgC12 tested (12 mg/kg body weight). The number of chromosome aberrations induced by the middle dose (6 mg HgCl2/kg body weight) was significantly reduced, while the effect of the lowest dose (3 mg HgCl:/kg body weight) was totally counteracted by chlorophyllin. Mitotic indexes were inversely related to the doses of HgCI: used (Table 1). Chlorophyllin was not clastogenic and significantly reduced the mitotic index when given alone. Simultaneous administration of HgC12 and chlorophyllin reduced partially the mitostatic action of higher doses of HgC12 and totally counteracted that of the lowest dose of HgCI2 (3 mg/kg body weight).
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Chlorophyll and chlorophyllin are universally present in green plants and have been shown to be non-toxic and antimutagenic (Arimoto et al., 1980; Hayatsu et al., 1988; Lai et al., 1980; Ong et al., 1989). The data from our laboratory demonstrated a modulatory action of fresh green leaves on the cytotoxic effects of betel-quid ingredients (Sen et al., 1989) and a protective effect of chlorophyllin against the clastogenicity of nicotine (Sen et al., 1991). In the present paper, the results demonstrate that chlorophyUin administered before HgCI~ protected only partially against the clastogenicity of mercury in mouse bone marrow cells. However, when administered simultaneously with HgCI,,, chlorophyllin inhibited significantly the clastogenicity of the toxic compound: a partial protection was obtained at the highest dose of HgCl2 (12 mg/kg body weight), while the frequencies of aberrations were greatly reduced at the middle dose of HgCI2 (6 mg/kg body weight) and reduced to the level of the negative control at the lowest dose of HgCI= (3 mg/kg body weight).
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Chlorophyllin and toxicity of mercuric chloride Table 2. Significantdifferencesbetween different experimental sets used to study the clastogenicityof HgC12in mousebone marrow cells (Duncan's multiple range test)* Dose Set no. Results 3 mg HgCl2/kg body weight
Neg C 0.40"1" CPY 0.40t CPY + HgCI2 0.605" CPY -~ HgCI2 1.40 HgCI2 2.00 6 mg HgCI2/kg body weight Neg C 0.40t CPY 0.40"1" CPY + HgCI2 1.20 CPY ---,HgCI2 1.80 HgC12 2.60 12 mg HgCI2/kg body weight Neg C 0.40t CPY 0.40"t CPY + HgCI2 2.40 CPY --*HgCI2 3.20t HgCI2 3.60"t" *Neg C = negative control; CPY = chlorophyllin 1.5mg/kg body weight; CPY+ HgCI2= both chemicals administered simultaneously; CPY ~HgCl: = chlorophyllin given 2 hr before HgCl2 administration. ~'No significantdifferencesbetweentwo experimentalsets at the level of P = 0.05.
The mechanism involved in the protective action o f chlorophyllin is still not fully u n d e r s t o o d (Hayatsu et al., 1988). Chlorophyllin may exert its protective action by absorbing (or adsorbing) toxic c o m p o u n d s ; it could also act by forming complexes or by scavenging cytotoxic free radicals (Dhir, 1989; Sharma, 1990). One o f these mechanisms may be responsible for the protection against the clastogenicity o f HgC12.
Acknowledgements--The authors are grateful to Professor A. K. Sharma, programme co-ordinator, for the facilities provided, and to the University Grants Commission and Council for Scientific and Industrial Research for their financial assistance.
REFERENCES Arimoto S. Y., Ohara Y., Namba T., Negishi T. and Hayatsu H. (1980) Inhibition of the mutagenicity of amino acid pyrolysis products by hemin and other biological pyrole pigments. Biochemical and Biophysical Research Communications 92, 662-668. Das S. K., Giri A. K., Sharma A. and Talukder G. (1985) Effects of mercury selenium antagonism on mammalian cell division. Cytobios 42, 271-277. Das S, K., Sharma A. and Talukder G. (1982) Effects of mercury on cellular systems in mammals--a review. Nucleus 25, 193--230. Dhir H. (1989) Plant extracts as desmutagens, some aspects. Acta Botanica Indica 17, 1-11.
779
Ganther H. E. (1980) Interaction of Vit. E and selenium with mercury and silver. Annals of the New York Academy of Sciences 355, 212. Hayatsu H., Arimoto S. and Negishi T. (1988) Dietary inhibitions of mutagenesis and carcinogenesis. Mutation Research 202, 429-446. Lai C. N. (1979) The active factor in wheat sprout extract inhibiting the metabolic activation of carcinogens in vitro. Nutrition and Cancer 1, 19-21. Lai C. N., Butter M. A. and Matney T. S. (1980) Antimutagenic activities of common vegetables and their chlorophyll content. Mutation Research 77, 245-250. L6onard A., Jacquet P. and Lauwerys R. R. (1983) Mutagenicity and teratogenicity of mercury compounds. Mutation Research 114, 1-18. Magos L. and Webb M. (1979) Synergism and antagonism in the toxicology of mercury. In The Biogeochemistry of Mercury in the Environment. Edited by J. O. Nriagu. p. 581. North Holland Biomedical, Amsterdam. Margolin B. H., Resmick N. A., Rimpo J, Y., Archer P., Galloway S. M., Bloom A. D. and Zeiger E. (1986) Statistical analysis for in vitro cytogenetic assay using Chinese hamster ovary cells. Environmental Mutagenesis 8, 183-204. Ong T. M., Whong W. Z., Stewart J. and Brockman H. E. (1986) Chlorophyllin: a potent antimutagen against environmental and dietary complex mixture. Mutation Research 173, 111-115. Ong T. M., Whong W. Z., Stewart J. and Brockman H. E. (1989) Comparative antimutagenicity of 5 compounds against 5 mutagenic complex mixtures in Salmonella typhimurium strain TA98. Mutation Research 222, 19-25. Preston R. J., Dean B. D., Galloway S., Holden H., McFee A. F. and Shelby M. (1987) Mammalian in vivo cytogenetic assays. Analysis of chromosome aberrations in bone marrow cells. Mutation Research 189, 157-165. Sen S., Sharma A. and Talukder G. (1991) Inhibition of clastogenic effects of nicotine by chlorophyllin in mice bone marrow cells in vivo. Phytotherapy Research 5, 130-133, Sen S., Talukder G. and Sharma A. (1989) Betel cytotoxicity: a review. Journal of Ethnopharmacology 26, 217-247. Sharma A. (1990) Plant products as modulators of mutagenesis. Santanu Memorial Lecture Science Cult. Calcutta. 1-30. Sharma A. K. and Sharma A. (1980) In Chromosome Techniques: Theory and Practice. 3rd Ed. Butterworths, London. Sokal R. R. and Rohlf F. J. (1981) In Biometry. 3rd Ed. Freeman, San Francisco. Swensson A. and Ulfvarson U. (1967) Experiments with different antidotes in acute poisoning by different mercury compounds. Effects on survival and on the distribution and excretion of mercury. Arehivefu'r Gewerbepathologie und Gewerbehygiene 24, 12. Tice R. R., Boucher R., Luke C. A. and Shelby M. D. (1987) Comparative cytogenetic analysis of bone marrow damage induced in male B6C3F mice by multiple exposure to gaseous 1,3-butadiene. Environmental Mutagenesis 9, 235-250.
0278-6915/91 $3.00+ 0.00 PergamonPress pie
Printed in Great Britain
E F F E C T OF C H L O R O P H Y L L I N ON M E R C U R I C C H L O R I D E - I N D U C E D C L A S T O G E N I C I T Y IN MICE A. K. GHOSH*, S. SENt, A. SHARMAand G. TALUKDER+ Genetic Toxicology Unit, Centre for Advanced Study in Cell and Chromosome Research, Department of Botany, University of Calcutta, 35 Ballygunge Circular Road, Calcutta 700019, India (Accepted 18 July 1991)
A~tract--The effect of chlorophyllin (1.5 mg/kg body weight) on the clastogenicity of mercuric chloride (HgC12) was studied in vivo in mouse bone marrow cells. HgCI2 (3.0, 6.0 and 12.0mg/kg body weight) administered by gavage induced chromosomal aberrations at frequencies directly proportional to the dose. Chlorophyllin was not clastogenic, and significantly reduced the mitotic index when given alone. Chlorophyllin administered simultaneously with HgCI2 significantly reduced the frequencies of chromosomal aberrations in a dose-dependent manner. When given simultaneously with the lowest HgCI2 concentration tested (3.0 mg/kg body weight), chlorophyllin provided total protection. A lower degree of protection was given by chlorophyllin administered 2 hr before HgCI2. The data demonstrate the potential of green plant components to modify the genotoxic activity of HgCI2 when administered orally.
INTRODUCTION Inorganic salts and organic forms of mercury are both environmental pollutants (Das et al., 1982) and potent clastogens (L6onard et al., 1983). Efforts have been made to counteract the toxic effects of these compounds using various metals (Das et al., 1985; Magos and Webb, 1979), vitamins (Ganther, 1980) and thiol compounds (Swensson and Ulfvarson, 1967). The constituents of green plants such as chlorophyll and its major water-soluble derivative, chiorophyllin, have been found to be antimutagenic against a variety of complex mutagens and N-nitroso compounds (Lai, 1979; Ong et al., 1986). The present work was undertaken to study the effects and modulatory role, if any, of chlorophyllin on the clastogenic action of the chloride salt of mercury in bone marrow cells of mice when administered orally in vivo.
MATERIALS AND METHODS
Chemicals. Mercuric chloride (HgCI2) was obtained from BDH Chemicals Ltd (India). Chlorophyllin (sodium-copper salt; 6.1% Na, 2.4% Cu) and eyclophosphamide were purchased from Sigma Chemical Co. (St Louis, MO, USA). All other chemicals were of Analar grade. Animals. The experiments were conducted on male inbred Swiss albino mice (8-10 wk old, 25-28 g) obtained from the Departmental animal house. The mice were housed in cages at a temperature of 28___2°C, relative humidity 60_+ 5% with a 12-hr
*To whom all correspondence should be addressed. tPresent address: Division Laboratory Animals, Central Drug Research Institute, Lucknow, India. :~Presentaddress: Vivekananda Institute of Medical Science, Calcutta, India. Abbreviation: HgCI2 = mercuric chloride.
light/dark cycle. The animals were fed standard diet (Lipton, India) and unlimited water was given. E x p e r i m e n t a l p r o t o c o l . The protocol and guidelines for in vivo cytogenetic studies were followed strictly (Preston et al., 1987). Five mice were used for each dose of HgCI 2 tested. HgC12 in distilled water was administered in three doses: 3.0, 6.0 and 12.0 mg/kg body weight equivalent to fractions of 1/40, 1/20 and 1/10 of the LDs0 determined in the strain of mice used. Chlorophyllin was dissolved in distilled water. A single concentration of 1.5mg/kg body weight was used. All chemicals were administered orally by gavage. Animals were killed 24 hr after exposure (Preston et al., 1987). The modulatory effect of chlorophyllin on the clastogenicity of HgC12 was studied. Chlorophyllin was given 2 hr before HgCI 2 in one set of experiments, and simultaneously with the toxic compound in a second set. Detailed treatment protocols are shown in Table 1. Positive a n d negative control. Cyclophosphamide (25 mg/kg body weight) administered ip served as the positive control (Preston et al., 1987). Double glassdistilled water (10 g/kg body weight) was administered by gavage as the negative (vehicle) control. Mice were killed 24 hr after exposure. C h r o m o s o m e preparations. Bone-marrow chromosomes were prepared by the standard protocol of Sharma and Sharma (1984): colchicine (4 mg/kg body weight, ip)--hypotonic (1% sodium citrate)--fixative (1:3, glacial acetic acid-ethanol)--flame drying. The slides were stained in diluted Giemsa stain. 50 perfect metaphase plates were scored randomly per animal for chromosomal aberrations, and 1000 cells were scored for the mitotic index. The types of aberrations were scored strictly in accordance with the method of Tice et al. (1987). All aberrations (chromatid gap, chromosome gap, chromatid break, chromosome break and rearrangement) were considered equal, irrespective of the number of events involved. The results are expressed as percentages of aberrant
777
778
A.K. GHOSH et al.
metaphase cells excluding gaps (% DC), and numbers of aberrations per cell excluding gaps (CA/cell). S t a t i s t i c a l analysis. For all statistical analyses, the level of significance was established at P ~<0.05. A one-tailed trend test (Margolin et al., 1986) was performed to observe the effects of HgClz. A one-way ANOVA test followed by Duncan's multiple range test (Sokal and Rohlf, 1981) was carried out to detect significant differences among different treatment protocols. Student's t-test was used to compare the mitotic index in the different sets of experiments.
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RESULTS Table l summarizes the different aberrations recorded, frequencies of chromosomal aberrations per cell (excluding gaps), numbers of damaged cell and mitotic indexes. A one-way ANOVA test followed by Duncan's multiple range test showed that in all sets the pretreatment with chlorophyllin protected only partially against the clastogenic action of HgCL (Table 2). The number of aberrations was reduced ai all HgC1, doses but the degree of protection was lower than that obtained with simultaneous administration of chlorophyllin and HgCI,. Simultaneous treatment of mice with the two compounds protected partially against the highest dose of HgC12 tested (12 mg/kg body weight). The number of chromosome aberrations induced by the middle dose (6 mg HgCl2/kg body weight) was significantly reduced, while the effect of the lowest dose (3 mg HgCl:/kg body weight) was totally counteracted by chlorophyllin. Mitotic indexes were inversely related to the doses of HgCI: used (Table 1). Chlorophyllin was not clastogenic and significantly reduced the mitotic index when given alone. Simultaneous administration of HgC12 and chlorophyllin reduced partially the mitostatic action of higher doses of HgC12 and totally counteracted that of the lowest dose of HgCI2 (3 mg/kg body weight).
2 41
41
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,'.'
Chlorophyll and chlorophyllin are universally present in green plants and have been shown to be non-toxic and antimutagenic (Arimoto et al., 1980; Hayatsu et al., 1988; Lai et al., 1980; Ong et al., 1989). The data from our laboratory demonstrated a modulatory action of fresh green leaves on the cytotoxic effects of betel-quid ingredients (Sen et al., 1989) and a protective effect of chlorophyllin against the clastogenicity of nicotine (Sen et al., 1991). In the present paper, the results demonstrate that chlorophyUin administered before HgCI~ protected only partially against the clastogenicity of mercury in mouse bone marrow cells. However, when administered simultaneously with HgCI,,, chlorophyllin inhibited significantly the clastogenicity of the toxic compound: a partial protection was obtained at the highest dose of HgCl2 (12 mg/kg body weight), while the frequencies of aberrations were greatly reduced at the middle dose of HgCI2 (6 mg/kg body weight) and reduced to the level of the negative control at the lowest dose of HgCI= (3 mg/kg body weight).
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8ca DISCUSSION
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Chlorophyllin and toxicity of mercuric chloride Table 2. Significantdifferencesbetween different experimental sets used to study the clastogenicityof HgC12in mousebone marrow cells (Duncan's multiple range test)* Dose Set no. Results 3 mg HgCl2/kg body weight
Neg C 0.40"1" CPY 0.40t CPY + HgCI2 0.605" CPY -~ HgCI2 1.40 HgCI2 2.00 6 mg HgCI2/kg body weight Neg C 0.40t CPY 0.40"1" CPY + HgCI2 1.20 CPY ---,HgCI2 1.80 HgC12 2.60 12 mg HgCI2/kg body weight Neg C 0.40t CPY 0.40"t CPY + HgCI2 2.40 CPY --*HgCI2 3.20t HgCI2 3.60"t" *Neg C = negative control; CPY = chlorophyllin 1.5mg/kg body weight; CPY+ HgCI2= both chemicals administered simultaneously; CPY ~HgCl: = chlorophyllin given 2 hr before HgCl2 administration. ~'No significantdifferencesbetweentwo experimentalsets at the level of P = 0.05.
The mechanism involved in the protective action o f chlorophyllin is still not fully u n d e r s t o o d (Hayatsu et al., 1988). Chlorophyllin may exert its protective action by absorbing (or adsorbing) toxic c o m p o u n d s ; it could also act by forming complexes or by scavenging cytotoxic free radicals (Dhir, 1989; Sharma, 1990). One o f these mechanisms may be responsible for the protection against the clastogenicity o f HgC12.
Acknowledgements--The authors are grateful to Professor A. K. Sharma, programme co-ordinator, for the facilities provided, and to the University Grants Commission and Council for Scientific and Industrial Research for their financial assistance.
REFERENCES Arimoto S. Y., Ohara Y., Namba T., Negishi T. and Hayatsu H. (1980) Inhibition of the mutagenicity of amino acid pyrolysis products by hemin and other biological pyrole pigments. Biochemical and Biophysical Research Communications 92, 662-668. Das S. K., Giri A. K., Sharma A. and Talukder G. (1985) Effects of mercury selenium antagonism on mammalian cell division. Cytobios 42, 271-277. Das S, K., Sharma A. and Talukder G. (1982) Effects of mercury on cellular systems in mammals--a review. Nucleus 25, 193--230. Dhir H. (1989) Plant extracts as desmutagens, some aspects. Acta Botanica Indica 17, 1-11.
779
Ganther H. E. (1980) Interaction of Vit. E and selenium with mercury and silver. Annals of the New York Academy of Sciences 355, 212. Hayatsu H., Arimoto S. and Negishi T. (1988) Dietary inhibitions of mutagenesis and carcinogenesis. Mutation Research 202, 429-446. Lai C. N. (1979) The active factor in wheat sprout extract inhibiting the metabolic activation of carcinogens in vitro. Nutrition and Cancer 1, 19-21. Lai C. N., Butter M. A. and Matney T. S. (1980) Antimutagenic activities of common vegetables and their chlorophyll content. Mutation Research 77, 245-250. L6onard A., Jacquet P. and Lauwerys R. R. (1983) Mutagenicity and teratogenicity of mercury compounds. Mutation Research 114, 1-18. Magos L. and Webb M. (1979) Synergism and antagonism in the toxicology of mercury. In The Biogeochemistry of Mercury in the Environment. Edited by J. O. Nriagu. p. 581. North Holland Biomedical, Amsterdam. Margolin B. H., Resmick N. A., Rimpo J, Y., Archer P., Galloway S. M., Bloom A. D. and Zeiger E. (1986) Statistical analysis for in vitro cytogenetic assay using Chinese hamster ovary cells. Environmental Mutagenesis 8, 183-204. Ong T. M., Whong W. Z., Stewart J. and Brockman H. E. (1986) Chlorophyllin: a potent antimutagen against environmental and dietary complex mixture. Mutation Research 173, 111-115. Ong T. M., Whong W. Z., Stewart J. and Brockman H. E. (1989) Comparative antimutagenicity of 5 compounds against 5 mutagenic complex mixtures in Salmonella typhimurium strain TA98. Mutation Research 222, 19-25. Preston R. J., Dean B. D., Galloway S., Holden H., McFee A. F. and Shelby M. (1987) Mammalian in vivo cytogenetic assays. Analysis of chromosome aberrations in bone marrow cells. Mutation Research 189, 157-165. Sen S., Sharma A. and Talukder G. (1991) Inhibition of clastogenic effects of nicotine by chlorophyllin in mice bone marrow cells in vivo. Phytotherapy Research 5, 130-133, Sen S., Talukder G. and Sharma A. (1989) Betel cytotoxicity: a review. Journal of Ethnopharmacology 26, 217-247. Sharma A. (1990) Plant products as modulators of mutagenesis. Santanu Memorial Lecture Science Cult. Calcutta. 1-30. Sharma A. K. and Sharma A. (1980) In Chromosome Techniques: Theory and Practice. 3rd Ed. Butterworths, London. Sokal R. R. and Rohlf F. J. (1981) In Biometry. 3rd Ed. Freeman, San Francisco. Swensson A. and Ulfvarson U. (1967) Experiments with different antidotes in acute poisoning by different mercury compounds. Effects on survival and on the distribution and excretion of mercury. Arehivefu'r Gewerbepathologie und Gewerbehygiene 24, 12. Tice R. R., Boucher R., Luke C. A. and Shelby M. D. (1987) Comparative cytogenetic analysis of bone marrow damage induced in male B6C3F mice by multiple exposure to gaseous 1,3-butadiene. Environmental Mutagenesis 9, 235-250.