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Clastogenic effects produced by black pepper in mitotic cells of Vicia faba
Mutation Research, 224 (1989) 281-285
281
Elsevier MUTGEN 01481
Clastogenic effects produced by black pepper in mitotic cells of Vicia faba Susan Abraham
a n d A n n i e T. J o h n
Department of Botany, Unioersity of Kerala, Kariaoattom, Trivandrum (India) (Received 1 September 1988) (Revision received 14 April 1989) (Accepted 29 May 1989)
Keywords: Black pepper; Mitotic cells; Chromosome aberrations
Summary Black pepper, as is well known, is an important spice widely used in the cooking and processing of meat and fish. The aim of the present investigation was to evaluate the clastogenic potential of black pepper. This was accomplished by treating root meristems of Vicia faba with aqueous extracts of black pepper. Examination of the treated roots showed the presence of chromatid breaks, chromosome breaks, gaps and exchanges. Statistically significant differences from controls were observed. Experiments to evaluate its clastogenic potential in mouse systems are in progress and the results will be published elsewhere.
Extensive knowledge has accumulated during the past few years from the testing of mutagenic activity of chemicals and other agents as an index of a potential hazard to man. Several investigators have focussed attention on food, water and other beverages as sources of human exposure to environmental mutagens (Stitch et al., 1982; Sugimura, 1982a,b; Dean and Vos, 1986). Various methods have been proposed for the evaluation of genotoxicity of drugs, chemicals and biologicals (Preston et al., 1987; Sobels, 1987). The present study was designed to evaluate the clastogenic potential of black pepper (Piper nigrum). This was done by treating root tip cells of Vicia faba with extracts of black pepper. The Vicia faba root tip system is considered suitable for the screening of chemicals and environmental mutagens and a good
Correspondence: Dr. S. Abraham, Department of Botany, University of Kerala, Kariavattom, Trivandrum (India).
number of chemicals have been effectively tested by this system (Ma, 1982). Black pepper, known as the 'king of spices', is one of the most ancient Indian spices. Its value as an essential preservative for meat and other perishable foods has been known for centuries. In many parts of India, people get accustomed to the pungent taste of chillies and pepper and relish food containing them. Some people can tolerate extreme quantities of pepper in their diet and the degree of tolerance varies in the population. Even in countries where spices and condiments are restricted in use, pepper and salt are offered with most meals. Pepper is also used in the manufacture of various commercially prepared foods such as pickles, soup, meat and even baked products. Pepper is the pungent aromatic condiment obtained from the dried mature berries of Pipet nigrum. The different alkaloids present in pepper have been identified and reported by different scientists. Pepper is known to contain piperine, chavicine, piperettine and myristicine (Ott and
0165-1218/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
282
Eichler, 1922; Marion, 1950; Spring and Stark, 1950). Pepper also contains small amounts of the known carcinogen safrole (Richard and Jennings, 1971) and various terpenes (Roe and Field, 1965) which are considered tumour promotors (Russel and Jennings, 1969; Schoental, 1976). Due to the widespread use of pepper as an ingredient in food items it was thought relevant to study its action on cell division. Evidence of carcinogenicity of black pepper was provided by Concon et al. (i979). In this paper we report the clastogenic effects produced by extracts of black pepper in root tip cells of Vicia faba. Materials and methods
Black pepper, free from mould or insect infestation and carefully collected from climbing vines, was used for the studies. Sun-dried pepper was ground in a micromill with stainless steel cup and blade. Required weights of finely powdered pepper were taken and made into a fine paste in a mortar using a few drops of distilled water. Concentrations ranging from 1 /~g/ml to 200 /~g/ml were made using distilled water. The extraction was done at room temperature and the insoluble traces were filtered away using W h a t m a n filter paper before treatment.
Test system The clastogenic effects produced by extracts of black pepper were studied in root tip cells of Vicia faba. The rationale for selecting Vicia faba root tip cells was the fact that the chromosomes are few in number (2n = 12) and large in size. It is generally accepted that the Vicia faba root tip system has enough advantages to make use of it in conjunction with or as a complement to other test systems (Ma, 1962; Kihlman, 1982).
Controls Vicia faba seed paste prepared at the same concentrations in a similar manner, using distilled water, was used as a control. The aberration frequency induced by pepper extract was tested against control values to determine whether or not it can be considered clastogenic.
Cytogenetic assay Germinating seeds of Vicia faba with primary roots about 2 - 3 cm long were treated with different concentrations of black pepper and fixed 5 h after treatment. The minimum and m a x i m u m concentrations selected after conducting test experiments were those producing a clastogenic effect detectable within the sample.
Endpoints of study (i) Mitotic index (MI) In order to examine whether cell divisions were inhibited by the treatment, mitotic indices were measured in the different treatments. The mitotic index is expressed as the percentage of cells in division.
(ii) Clastogenic effects The amount of chromosome damage produced was obtained by scoring cells in metaphase. Chromatid breaks, chromosome breaks, exchanges and gaps or achromatic lesions were considered as clastogenic manifestations.
Sampling, scoring and presentation of data In each experiment, 3 roots from 3 different seedlings were examined per treatment to score the aberrations present. The experiment was repeated thrice and the data were pooled and presented in tables. For determining the mitotic index (MI), 500 cells were scored from each slide. For
TA B LE 1 M I T O T I C I N D I C E S IN T R E A T M E N T S U S I N G P E P P E R O R Vicia faba SEED PASTE Concentration (/t g / m l )
Treatments using pepper
Treatments using Viciafaba seed paste
1 2 10 20 50 100 150 200
4.15 3.72 3.05 2.61 2.38 2.02 * 1.94 * 1.77 *
4.81 3.45 3.31 3.11 3.10 3.31 3.06 3.23
* Significant at P < 0.05.
283 assessing clastogenic effects, a minimum of 200 metaphases were analysed per treatment. In order to avoid scorer-to-scorer variation, scoring was done by a single individual. Statistical analysis to determine the significance of the response was done by making comparisons of aberrations per treatment and controls.
Results Table 1 indicates the MI values for each substance and concentration. A fall in mitotic index can be clearly observed in pepper treatments. The
maximum reduction in mitotic index was observed in the 200-/~g/ml treatment. N o evidence of mitotic arrest was observed in higher concentrations of Vicia faba seed paste treatments used as controls. Chromosome aberrations observed in the different treatments are recorded in Table 2. As may be observed from the Table, clastogenic effects were observed at low levels, even at 1 t t g / m l of pepper treatment. Chromatid breaks, chromosome breaks, gaps and exchanges were the major aberrations noticed (Fig. 1). Of these, chromatid breaks were seen only occasionally. The frequent gaps
Fig. 1. Chromosomeaberrations i'n pepper treatments. (a) Chromatid break. (b) Gaps, giving the appearance of a banded pattern. (c) Fragments. (d) Numerous fragments and gaps.
284 TABLE 2 CHROMOSOME Concentration (/t g / m l )
ABERRATION
YIELDS AFTER TREATMENT
WITH PEPPER OR WITH
Vicia faba
T r e a t m e n t using p e p p e r
T r e a t m e n t using seed p a s t e
Cells with
Cells with
Chromosomal aberrations p e r 100 cells
aber-
B
ration
G
aber-
E
ration
(%)
SEED PASTE
Chromosomal aberrations per 100 cells B
G
E
(%)
1
2.30
3.45
0.00
0.00
1.39
1.39
0.00
0.00
2
3.14
5.38 *
0.00
0.00
1.61
1.61
0.00
0.00
10 20 50 100 150 200
9.62 * * 9.82 11.59 13.79 18.32 25.84
* ** ** ** **
14.43 * * 12.50 17.39 25.62 24.05 22.01
** ** ** ** **
6.53 * * 3.57 5.80 9.85 27.10 66.03
* ** ** ** **
0.00
2.78
3.70
0.00
0.00
0.00 1.81 3.45 * * 3.05 * * 1.44 *
2.54 2.47 2.60 2.30 1.83
3.39 3.09 2.60 2.30 1.83
0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00
B, b r e a k s ; G , gaps; E, e x c h a n g e s . * Significant at P < 0.05. * * Significant at P < 0.01.
observed at higher concentrations often gave the appearance of a banded pattern. Fragments of different sizes were observed and occasionally cells showing many fragments and gaps were observed. An analysis of the results shows that the percentage of cells showing aberrations increased significantly in pepper treatments. Treatments using Vicia faba seed paste did not elevate the frequency of chromosome aberrations in higher concentrations. Discussion
The present study shows that black pepper is clastogenic in mitotic cells of Vicia faba. The capacity of a large number of xenobiotics to which man is exposed to cause adverse effects on the immune system is well established (Dean and Vos, 1986). Mitodepressive activity exerted by black pepper has been reported in treatments using a variety of agents (Kabarity and Malallah, 1980; Alam et al., 1987). During the present study the clastogenic property of black pepper was clearly manifested in all the treatments. No detectable response was observed in concentrations below 1 /~g/ml. Hence 1 # g / m l can be considered as the 'no effect' level. Osawa et al. (1981) in their studies on the reaction of sodium nitrite with black or white
pepper observed mutagenicity in both cases, using Salmonella typhimurium. When extract of black pepper was applied cutaneously to albino Swiss mice of both sexes, all pepper-treated mice surviving after 17 months developed tumours, particularly in the lung, liver and skin (Concon et al., 1979). An earlier study has shown that extracts of green chillies produced severe chromosome breakage and other abnormalities in cell divisions in Allium cepa (Abraham and Koshy, 1979). The evidence presented here suggests that black pepper can penetrate cells and act directly or indirectly on the DNA, thus producing visible effects on chromosomes. It is now well recognised that diet and nutrition play an important role in the aetiology and prevention of cancer (McMichael, 1979; Sabine, 1979; Ames, 1986). Hirayama (1970) in his review on the effects of diet on cancers of the lung, oesophagus, stomach, colon, breast and cervix has stressed the need for testing the mutagenicity of each food item. During the present study the clastogenic properties of black pepper were established from studies on the root tip cells of Vicia faba. As a method for detecting agents that may be of genetic risk to man, the plant root tip technique has the disadvantage of not allowing for mammalian metabolism. But it has been pointed out by many scientists that when root tio cells and cultured
285 m a m m a l i a n cells are c o m p a r e d as test s y s t e m s , t h e r e a p p e a r s to b e a g o o d c o r r e l a t i o n b e t w e e n t h e r e s u l t s o b t a i n e d w i t h t h e 2 s y s t e m s . It is g e n e r a l l y noticed that an agent which produces chrom o s o m e a b e r r a t i o n s in o n e o f t h e 2 s y s t e m s d o e s t h e s a m e in the o t h e r . It is k n o w n t h a t s o m e g e n o t o x i c c h e m i c a l s c a n p r o d u c e t h e i r e f f e c t s dir e c t l y w h e r e a s o t h e r s r e q u i r e m e t a b o l i s m to m u t a g e n i c a l l y a c t i v e i n t e r m e d i a t e s . T h e n e e d for p e r f o r m i n g in v i v o r a t h e r t h a n in v i t r o tests in the e v a l u a t i o n o f g e n o t o x i c i t y has b e e n s t r e s s e d b y S o b e l s (1987). M o r e p e r t i n e n t e x p e r i m e n t s o n the genotoxicity of black pepper using the mouse mic r o n u c l e u s test a n d g e r m cell m u t a g e n i c i t y a s s a y a r e in p r o g r e s s .
Acknowledgements T h e a u t h o r s are g r a t e f u l to t h e H e a d o f the Department of Botany for providing laboratory facilities. T h e a w a r d o f a J u n i o r R e s e a r c h F e l l o w s h i p to o n e o f us (A.J.) b y t h e C o u n c i l o f S c i e n tific a n d I n d u s t r i a l R e s e a r c h , G o v e r n m e n t o f India, is g r a t e f u l l y a c k n o w l e d g e d .
References Abraham, S., and M.P. Koshy (1979) Mutagenic potential of green chillies, Cytologia, 44, 221-225. Alam, S., G. Kabir, M.N. Amin and M. Islam (1987) Mitotic effect of leaf extracts of lpomoea carnea on Allium cepa, Cytologia, 52, 721-724. Ames, B.N. (1986) Food constituents as a source of mutagens, carcinogens and anticarcinogens, in: J.B. Kundsen (Ed.), Genetic Toxicology of the Diet, Alan R. Liss Inc., New York, pp. 3-32. Ashby, J. (1986) The prospects for a simplified and internationally harmonized approach to the detection of possible human carcinogens and mutagens, Mutagenesis, 1, 3-16. Concon J.M., D.S. Newburg and T.W. Swerczet (1979) Black pepper (Piper nigrum). Evidence of carcinogenicity, Nutr. Cancer, 1, 22-26. Dean, J.H., and J.G. Vos (1986) An introduction to immunotoxicology assessment, in: J. Descotes (Ed.), Immunotoxicology of Drugs and Chemicals, Elsevier, Oxford, 400 pp. Hirayama, T. (1979) Diet and cancer, Nutr. Cancer, 1, 67-81. Kabirity, A., and G. Malallah (1980) Mitotic depression effect of khat extract in the meristematic region of Allium cepa root tips, Cytologia, 45, 733-738.
Kihlman, B.A. (1982) Root tips of Vicia faba as a material for studying the induction of chromosomal aberrations and sister chromatid exchanges, in: T.C. Hsu (Ed.), Cytogenetic Assays of Environmental Mutagens, IBH Publishing Co., Oxford, pp. 81-105. Ma, T.H. (1962) Vicia cytogenetic tests for environmental mutagens. A report of the U.S. Environmental Protection Agency, Mutation Res., 99, 257-271. Marion, L. (1950) The pyridine alkaloids, in: R.H.F. Manske and H.L. Holmes (Eds.), The Alkaloids, Academic Press, New York, pp. 165-260. McMichael, A.J. (1979) Alimentary tract cancer in Australia in relation to diet and alcohol, Nutr. Cancer, 1, 82-88. Osawa, T., H. Ishibashi, M. Namiki, M. Yamanaka and K. Namiki (1981) Formation of mutagens by pepper nitrite reaction, Mutation Res., 91,291-295. Ott, E., and R. Eichler (1922) Natural and artificial pepper substances. II. The chavicine of pepper resin, the especially active constituent of black pepper, Ber. Dtsch. Bot. Ges., 55B, 2653-2663. Preston, J.R., J.R.S. Sebastian and A.F. McFee (1987) The in vitro human lymphocyte assay for assessing the clastogenicity of chemical agents, Mutation Res., 189, 175-183. Richard, H.M., and W.G. Jennings (1971) Volatile composition of black pepper, J. Food Sci., 36, 584-589. Roe, F.J.C., and W.E.H. Field (1965) Chronic toxicity of essential oil and certain other products of natural origin, Food Cosmet. Toxicol., 3, 311-324. Russel, G.J., and W.C. Jennings (1969) Constituents of black pepper: some oxygenated compounds. J. Agr. Fqod Chem., 17, 1107-1112. Sabine, J.R. (1979) Susceptibility to cancer and the influence of nutrition, Nutr. Cancer, 1, 52-57. Schoental, R. (1976) Carcinogens in plants and microorganisms, in: G.E. Searle (Ed.), Chemical Carcinogenesis, American Chemical Society, Washington, DC, pp. 626-689. Sobels, F.H. (1987) In vitro testing as a step in the evaluation of in vivo genotoxicity, Mutation Res., 189, 7-10. Spring, F.S., and J. Stark (1950) Piperettine from Piper nigrum: its isolation, identification and synthesis, J. Chem. Soc., 1177-1180. Stitch, H.F., C. Wu and W. Powric (1982) Enhancement and suppression of genotoxicity of food by naturally occurring components in these products, in: T. Sugimura, S. Kondo and H. Takebe (Eds.), Environmental Mutagens and Carcinogens, University of Tokyo Press, Tokyo, pp. 347-353. Sugimura, T. (1982a) Mutagens, carcinogens and tumor promotors in our daily food, Cancer, 49, 1970-1984. Sugimura, T. (1982b) A view of a cancer researcher on environmental mutagens, in: T. Sugimura, S. Kondo and H. Takebe (Eds.), Environmental Mutagens and Carcinogens, University of Tokyo Press, Tokyo, pp. 3-20.
281
Elsevier MUTGEN 01481
Clastogenic effects produced by black pepper in mitotic cells of Vicia faba Susan Abraham
a n d A n n i e T. J o h n
Department of Botany, Unioersity of Kerala, Kariaoattom, Trivandrum (India) (Received 1 September 1988) (Revision received 14 April 1989) (Accepted 29 May 1989)
Keywords: Black pepper; Mitotic cells; Chromosome aberrations
Summary Black pepper, as is well known, is an important spice widely used in the cooking and processing of meat and fish. The aim of the present investigation was to evaluate the clastogenic potential of black pepper. This was accomplished by treating root meristems of Vicia faba with aqueous extracts of black pepper. Examination of the treated roots showed the presence of chromatid breaks, chromosome breaks, gaps and exchanges. Statistically significant differences from controls were observed. Experiments to evaluate its clastogenic potential in mouse systems are in progress and the results will be published elsewhere.
Extensive knowledge has accumulated during the past few years from the testing of mutagenic activity of chemicals and other agents as an index of a potential hazard to man. Several investigators have focussed attention on food, water and other beverages as sources of human exposure to environmental mutagens (Stitch et al., 1982; Sugimura, 1982a,b; Dean and Vos, 1986). Various methods have been proposed for the evaluation of genotoxicity of drugs, chemicals and biologicals (Preston et al., 1987; Sobels, 1987). The present study was designed to evaluate the clastogenic potential of black pepper (Piper nigrum). This was done by treating root tip cells of Vicia faba with extracts of black pepper. The Vicia faba root tip system is considered suitable for the screening of chemicals and environmental mutagens and a good
Correspondence: Dr. S. Abraham, Department of Botany, University of Kerala, Kariavattom, Trivandrum (India).
number of chemicals have been effectively tested by this system (Ma, 1982). Black pepper, known as the 'king of spices', is one of the most ancient Indian spices. Its value as an essential preservative for meat and other perishable foods has been known for centuries. In many parts of India, people get accustomed to the pungent taste of chillies and pepper and relish food containing them. Some people can tolerate extreme quantities of pepper in their diet and the degree of tolerance varies in the population. Even in countries where spices and condiments are restricted in use, pepper and salt are offered with most meals. Pepper is also used in the manufacture of various commercially prepared foods such as pickles, soup, meat and even baked products. Pepper is the pungent aromatic condiment obtained from the dried mature berries of Pipet nigrum. The different alkaloids present in pepper have been identified and reported by different scientists. Pepper is known to contain piperine, chavicine, piperettine and myristicine (Ott and
0165-1218/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
282
Eichler, 1922; Marion, 1950; Spring and Stark, 1950). Pepper also contains small amounts of the known carcinogen safrole (Richard and Jennings, 1971) and various terpenes (Roe and Field, 1965) which are considered tumour promotors (Russel and Jennings, 1969; Schoental, 1976). Due to the widespread use of pepper as an ingredient in food items it was thought relevant to study its action on cell division. Evidence of carcinogenicity of black pepper was provided by Concon et al. (i979). In this paper we report the clastogenic effects produced by extracts of black pepper in root tip cells of Vicia faba. Materials and methods
Black pepper, free from mould or insect infestation and carefully collected from climbing vines, was used for the studies. Sun-dried pepper was ground in a micromill with stainless steel cup and blade. Required weights of finely powdered pepper were taken and made into a fine paste in a mortar using a few drops of distilled water. Concentrations ranging from 1 /~g/ml to 200 /~g/ml were made using distilled water. The extraction was done at room temperature and the insoluble traces were filtered away using W h a t m a n filter paper before treatment.
Test system The clastogenic effects produced by extracts of black pepper were studied in root tip cells of Vicia faba. The rationale for selecting Vicia faba root tip cells was the fact that the chromosomes are few in number (2n = 12) and large in size. It is generally accepted that the Vicia faba root tip system has enough advantages to make use of it in conjunction with or as a complement to other test systems (Ma, 1962; Kihlman, 1982).
Controls Vicia faba seed paste prepared at the same concentrations in a similar manner, using distilled water, was used as a control. The aberration frequency induced by pepper extract was tested against control values to determine whether or not it can be considered clastogenic.
Cytogenetic assay Germinating seeds of Vicia faba with primary roots about 2 - 3 cm long were treated with different concentrations of black pepper and fixed 5 h after treatment. The minimum and m a x i m u m concentrations selected after conducting test experiments were those producing a clastogenic effect detectable within the sample.
Endpoints of study (i) Mitotic index (MI) In order to examine whether cell divisions were inhibited by the treatment, mitotic indices were measured in the different treatments. The mitotic index is expressed as the percentage of cells in division.
(ii) Clastogenic effects The amount of chromosome damage produced was obtained by scoring cells in metaphase. Chromatid breaks, chromosome breaks, exchanges and gaps or achromatic lesions were considered as clastogenic manifestations.
Sampling, scoring and presentation of data In each experiment, 3 roots from 3 different seedlings were examined per treatment to score the aberrations present. The experiment was repeated thrice and the data were pooled and presented in tables. For determining the mitotic index (MI), 500 cells were scored from each slide. For
TA B LE 1 M I T O T I C I N D I C E S IN T R E A T M E N T S U S I N G P E P P E R O R Vicia faba SEED PASTE Concentration (/t g / m l )
Treatments using pepper
Treatments using Viciafaba seed paste
1 2 10 20 50 100 150 200
4.15 3.72 3.05 2.61 2.38 2.02 * 1.94 * 1.77 *
4.81 3.45 3.31 3.11 3.10 3.31 3.06 3.23
* Significant at P < 0.05.
283 assessing clastogenic effects, a minimum of 200 metaphases were analysed per treatment. In order to avoid scorer-to-scorer variation, scoring was done by a single individual. Statistical analysis to determine the significance of the response was done by making comparisons of aberrations per treatment and controls.
Results Table 1 indicates the MI values for each substance and concentration. A fall in mitotic index can be clearly observed in pepper treatments. The
maximum reduction in mitotic index was observed in the 200-/~g/ml treatment. N o evidence of mitotic arrest was observed in higher concentrations of Vicia faba seed paste treatments used as controls. Chromosome aberrations observed in the different treatments are recorded in Table 2. As may be observed from the Table, clastogenic effects were observed at low levels, even at 1 t t g / m l of pepper treatment. Chromatid breaks, chromosome breaks, gaps and exchanges were the major aberrations noticed (Fig. 1). Of these, chromatid breaks were seen only occasionally. The frequent gaps
Fig. 1. Chromosomeaberrations i'n pepper treatments. (a) Chromatid break. (b) Gaps, giving the appearance of a banded pattern. (c) Fragments. (d) Numerous fragments and gaps.
284 TABLE 2 CHROMOSOME Concentration (/t g / m l )
ABERRATION
YIELDS AFTER TREATMENT
WITH PEPPER OR WITH
Vicia faba
T r e a t m e n t using p e p p e r
T r e a t m e n t using seed p a s t e
Cells with
Cells with
Chromosomal aberrations p e r 100 cells
aber-
B
ration
G
aber-
E
ration
(%)
SEED PASTE
Chromosomal aberrations per 100 cells B
G
E
(%)
1
2.30
3.45
0.00
0.00
1.39
1.39
0.00
0.00
2
3.14
5.38 *
0.00
0.00
1.61
1.61
0.00
0.00
10 20 50 100 150 200
9.62 * * 9.82 11.59 13.79 18.32 25.84
* ** ** ** **
14.43 * * 12.50 17.39 25.62 24.05 22.01
** ** ** ** **
6.53 * * 3.57 5.80 9.85 27.10 66.03
* ** ** ** **
0.00
2.78
3.70
0.00
0.00
0.00 1.81 3.45 * * 3.05 * * 1.44 *
2.54 2.47 2.60 2.30 1.83
3.39 3.09 2.60 2.30 1.83
0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00
B, b r e a k s ; G , gaps; E, e x c h a n g e s . * Significant at P < 0.05. * * Significant at P < 0.01.
observed at higher concentrations often gave the appearance of a banded pattern. Fragments of different sizes were observed and occasionally cells showing many fragments and gaps were observed. An analysis of the results shows that the percentage of cells showing aberrations increased significantly in pepper treatments. Treatments using Vicia faba seed paste did not elevate the frequency of chromosome aberrations in higher concentrations. Discussion
The present study shows that black pepper is clastogenic in mitotic cells of Vicia faba. The capacity of a large number of xenobiotics to which man is exposed to cause adverse effects on the immune system is well established (Dean and Vos, 1986). Mitodepressive activity exerted by black pepper has been reported in treatments using a variety of agents (Kabarity and Malallah, 1980; Alam et al., 1987). During the present study the clastogenic property of black pepper was clearly manifested in all the treatments. No detectable response was observed in concentrations below 1 /~g/ml. Hence 1 # g / m l can be considered as the 'no effect' level. Osawa et al. (1981) in their studies on the reaction of sodium nitrite with black or white
pepper observed mutagenicity in both cases, using Salmonella typhimurium. When extract of black pepper was applied cutaneously to albino Swiss mice of both sexes, all pepper-treated mice surviving after 17 months developed tumours, particularly in the lung, liver and skin (Concon et al., 1979). An earlier study has shown that extracts of green chillies produced severe chromosome breakage and other abnormalities in cell divisions in Allium cepa (Abraham and Koshy, 1979). The evidence presented here suggests that black pepper can penetrate cells and act directly or indirectly on the DNA, thus producing visible effects on chromosomes. It is now well recognised that diet and nutrition play an important role in the aetiology and prevention of cancer (McMichael, 1979; Sabine, 1979; Ames, 1986). Hirayama (1970) in his review on the effects of diet on cancers of the lung, oesophagus, stomach, colon, breast and cervix has stressed the need for testing the mutagenicity of each food item. During the present study the clastogenic properties of black pepper were established from studies on the root tip cells of Vicia faba. As a method for detecting agents that may be of genetic risk to man, the plant root tip technique has the disadvantage of not allowing for mammalian metabolism. But it has been pointed out by many scientists that when root tio cells and cultured
285 m a m m a l i a n cells are c o m p a r e d as test s y s t e m s , t h e r e a p p e a r s to b e a g o o d c o r r e l a t i o n b e t w e e n t h e r e s u l t s o b t a i n e d w i t h t h e 2 s y s t e m s . It is g e n e r a l l y noticed that an agent which produces chrom o s o m e a b e r r a t i o n s in o n e o f t h e 2 s y s t e m s d o e s t h e s a m e in the o t h e r . It is k n o w n t h a t s o m e g e n o t o x i c c h e m i c a l s c a n p r o d u c e t h e i r e f f e c t s dir e c t l y w h e r e a s o t h e r s r e q u i r e m e t a b o l i s m to m u t a g e n i c a l l y a c t i v e i n t e r m e d i a t e s . T h e n e e d for p e r f o r m i n g in v i v o r a t h e r t h a n in v i t r o tests in the e v a l u a t i o n o f g e n o t o x i c i t y has b e e n s t r e s s e d b y S o b e l s (1987). M o r e p e r t i n e n t e x p e r i m e n t s o n the genotoxicity of black pepper using the mouse mic r o n u c l e u s test a n d g e r m cell m u t a g e n i c i t y a s s a y a r e in p r o g r e s s .
Acknowledgements T h e a u t h o r s are g r a t e f u l to t h e H e a d o f the Department of Botany for providing laboratory facilities. T h e a w a r d o f a J u n i o r R e s e a r c h F e l l o w s h i p to o n e o f us (A.J.) b y t h e C o u n c i l o f S c i e n tific a n d I n d u s t r i a l R e s e a r c h , G o v e r n m e n t o f India, is g r a t e f u l l y a c k n o w l e d g e d .
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