_??_ 1991 by Cytologia, Tokyo Cytologi a 56: , Chromosomal Aberrations and Sister Chromatid Exchanges

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1 _??_ 1991 by Cytologia, Tokyo Cytologi a 56: , 1991 Chromosomal Aberrations and Sister Chromatid Exchanges in Cultured Human Lymphocytes lv. Concluding Remarks1 Ajay K.Jain and N. Sethi Division of Toxicology, Central Drug Research Institute, Chattar Manzil Palace, Post Box No. 173 Lucknow , (U. P.), India Accepted June 6, 1991 The strong correlation between carcinogenicity and mutagenicity (Mc Cann et al. 1975) has led a number of workers to identify environmental mutagens. The findings of Philips (1975) indicated that Seventh-Day Adventists who do not drink alcohol, coffee and tea show significantly lower incidences of all types of cancer than other American citizens. This sug gested that some of the food factors might be playing a significant role in the genesis of cancer. This encouraged a number of workers to identify mutagenic food products or constituents. Sugimura and Sato (1983) have reviewed the work progress on mutagens present in our food products and depending upon the mode of origin of mutagens they have classified them into 3 categories. (a) Natural mutagens Some mutagens may be naturally present as food constituents e. g. bracken fern which is still frequently eaten in Japan and other Asian countries. Sometimes food products may get contaminated with certain fungi and simultaneously with specific mycotoxin. Aflatoxin B1 is one of the most potent example of such mutagen and carcinogen. (b) Mutagens formed during the processing of food products These mutagens may form during storage, food processing and cooking, e. g. Trp-P-1 (3-amino-1, 4-dimethyl-5H-pyrido (4, 3-b) indole) and Trp-P-2 (3-amino-l-methyl-5H-Pyrido (4, 3-b) indole). These are formed during the cooking of proteinaceous foods, e. g. fish. (c) Added mutagens Agrochemicals (pesticides, fungicides etc) which are widely used for beneficial purposes may contaminate food products. Some of these chemicals are genotoxic (see overview, Jain 1989a). Besides, some flavouring and colouring agents in processed foods might also have mutagenic potentialities. Howeve, real genetic hazards of such mutagens is not only due to their presence but also considerably due to the lack of antimutagenic factors. Therefore, we are trying to identify mutagenic and antimutagenic factors in our food products which may be helpful to minimize the genetic hazards by adopting "Balanced Diet" if it is not possible to exclude mutagenic components completely. Earlier, we have reported that tea extracts decrease the mutagenicity of N-methyl-N Œ-nitro-N-nitrosoguanidine (MNNG), Benzo (a) pyrene (B(a)P) and 3-amino 1-methyl-5H-pyrido (4-3-b) indole (Trp-P-2) (Jain et al. 1987, 1989a) and promotion of trans formation induced by 12-0-tetradecanoyl-phorbol-13 acetate (TPA) (Jain et al. 1989b). The present study was planned to evaluate chromosomal aberrations and sister chromatid exchanges (SCE's) inducing effect of black and green tea and two of their constituents viz. epigallocatech -1 C. D. R. I. Communication number 4804.

2 550 Ajay K. Jain and N. Sethi Cytologia 56 ingallate (EGCG) and ascorbic acid (AA) (vitamin C) in cultured human lymphocyte system. It has been suggested that both in vitro and in vivo human lymphocytes are extremely sensitive indicators of radiation and chemically induced chromosomal aberrations (Bhatt 1982) and SCE's (Solomon and Bobrow 1975) which provide a direct evidence of damage to the genetic material. Another aim of the present study was to know the correlation between these two biological end points, i.e. chromosomal aberration and sister chromatid exchanges. See part I, II and III. Materials and methods Table 1. Constituting quantity of epigallocatechingallate (EGCG) and ascorbic acid (AA) in differentreatment doses of black and green tea Table 2. Correlation coefficient (r) value between aberrant metaphases and sister chromatid exchanges induced by black tea * Significant at 0.1 level Results and discussion Earlier, we have found that black tea (BT) extract contained 12mg/g epigallocatechingal late (EGCG) and 0.1mg/g ascorbic acid (AA) while green tea (GT) extract contained 103mg/g EGCG and 18.1mg/g AA (Jain et al. 1989a). On the basis of these values, the constituting quantities of EGCG and AA present in different treatment doses of BT and GT were calculated which have been summarized in Table 1. It is apparent from Tables 2 and 3 that 10.0ƒÊg/ml treatment 'A' of BT and GT enhanced the SCE's frequency considerably. 10.0ƒÊg of BT and GT contained EGCG 0.12ƒÊg and 1.03ƒÊg, respectively (Table 1). Treatment of these doses

3 1991 Chromosomal Aberrations and Sister Chromatid Exchanges in C ultured Human Lymphocytes IV 551 of EGCG also increased SCE's highly significantly (Table 4). However, the enhancement in SCE's frequency due to treatment 'A' of BT and GT was not exactly of that magnitude as had been induced by the treatment of EGCG. Treatment 'A' of AA (100.0ƒÊg/ml) significantly reduced the SCE's value but lower doses than this were not found to be effecti ve in lowering the SCE's (Table 5). 10.0ƒÊg BT and GT contained AA 0.01ƒÊg and 0.181ƒÊg (Table 1), re - Table 3. Correlation coefficient (r) value between aberrant metaphases and sister chromatid exchanges induced by green tea Table 4. Correlation coefficient (r) value between aberrant metaphases and sister chromatid exchanges induced by epigallocatechingallate *** Significant at.001 level. spectively, which seems not to be potent to decrease the SCE's frequency. Thus it appears that SCE enhancing effect of crude extract of BT and GT is not entirely due to their EGCG constituent. There seems to present some other factor (s) which also interact with EGCG, resulting in decreasing the DNA damaging potentiality of EGCG. Contrary to the SCE's finding the magnitude of aberrant metaphases after treatment 'A' of BT, 10.0ƒÊg/ml (6.0%)

4 552 Ajay K. Jain and N. Sethi Cytologia 56 and GT 10.0ƒÊg/ml (7.0%) (Tables 2, 3) appears to be almost of similar magnitude as induced by their corresponding constituent values of EGCG in BT and GT: 5.47% (0.1ƒÊg in 10.0ƒÊg BT) and 5.12% (1.03ƒÊg in 10.0ƒÊg GT) (Tables 1-4). Similar conclusions are apparent from treatment 'B' of GT which significantly decreased the SCE's frequency like EGCG (Tables 2, 4). Aberrant metaphases were also in similar ranges (Tables 2, 4). On the contrary, treatment 'B' of BT significantly elevatatd SCE frequency and aberrant metaphases irrespective of the EGCG fraction. This led to presume that BT might contain some other potent factor (s) which suppress (es) DNA repair inducing potentiality of EGCG. From these observations, it can be deduced that net genotoxicity of a single pure food component can not be directly extrapolated to human population in propor tion to the in vitro outcome of a pure compound, because food products are consumed in complex form due to which their genotoxic potentiality may get enhanced or inhibited or diminished. Therefore, it seems of most significance to have possible combinational and complex interaction studies of various food components before drawing a final conclusion. Table 5. Correlation coefficient (r) value between aberrant metaphases and sister chromatid exchanges induced by ascorbic acid Chromosomal aberrations and SCE's analysis have been widely accepted to identify mu tagens and carcinogens. It has been found that most of the mutagens produce both SCE's and chromosomal changes (Latt 1974, Perry and Evans 1975). Furakawa et al. (1978) and Abe and Sasaki (1977) noticed enhanced SCE's but not chromosomal aberrations, while some other workers (Kihlman 1975, Abe and Sasaki 1977, Jain 1989b) found significant elevation of aberrant metaphases but not SCE's. Thus correlation between them is obscure and it seems of much significance to know the interrelationship between these biological end points (CA and SCE's). It is evident from the present study that significant correlation exist between aberrant metaphases and SCE's after treatment 'B' of BT ('r'=0.92 with chromatid gap; 'r'=0.99, without chromatid gap) (Table 2) and treatment 'A' of EGCG (r=0.84, with chro matid gap) (Table 4). This supports our earlier view that positive/negative correlation between these biological end points seems entirely specific and may depend upon the type of mutagens and their mode of action (Jain 1989c). Thus it can be deduced that CA and SCE may or may not originate from the identical loci. Besides, the absence of significant 'r' values after treat ment 'A' of BT and treatment 'B' of EGCG in the present study suggests that the cell cycle's

5 1991 Chromosomal Aberrations and Sister Chromatid Exchanges in Cultured Human Lymphocytes IV 553 stage in which cells are treated or exposed to a mutagen appears to play a significant role in sharing identical DNA lesions for the manifestation of chromosomal aberrations and SCE's. Our interpretation further gets supports from the studies of Heddle et al. (1969) who noted a high correlation between the location of chromosome breaks and exchanges and of Ikushima (1977), Shiriashi and Sandberg (1970, 1977), Sano and Sakaguchi (1979) and Galloway and Wolff (1979) who reported the occurrence of chromosomal aberrations and SCE's at different loci. Significant 'r' value between SCE's per cell and aberrant metaphases (including chromatid gap) and negative insignificant 'r' value (after excluding chromatid gap) between them after treatment 'A' of EGCG (Table 4) suggest to assume that in certain cases chromatid gaps may be closely associated with SCE's. Hence, chromatid gaps should not be excluded in scoring chromosomal aberrations. Summary It has been widely recognized that food factors play a significant role in carcinogenesis. This has led us to investigate chromosomal aberrations (CA) and SCE's inducing potentiality of black tea (BT), green tea (GT), epigallocatechingallate (EGCG) and ascorbic acid (AA) (See part I, II and III). On the basis of quantitative ratio of EGCG in BT and GT it seems that SCE enhancing effect of BT and GT after treatment 'A' is not entirely due to the presence of EGCG. They may contain some other factor (s) which diminish the DNA damaging poten tiality of EGCG. BT and GT seems to contain different active factor (s) which may have different mode of actions because treatment 'B' of GT decreased the SCE frequency like EGCG, while treatment 'B' of BT significantly enhanced the SCE's and aberrant metaphases. An effort was made to know correlation between CA and SCE's. Various studies have been discussed. The results of present study support our earlier view that positive correlation between these biological end points seems specific and may depend upon the type of mutagens and their mode of action. The present study further suggests that the cell cycle's stage in which cells are treated or exposed to a mutagen also play a significant role in sharing common DNA lesions for the manifestation of CA and SCE's. On the basis of significant correlation coefficient 'r' value between aberrant metaphases (including chromatid gap) and mean SCE/cell and negative 'r' value between them (after dele ting chromatid gap) after treatment 'A' of EGCG, it has been suggested that chromatid gap should not be excluded in scoring chromosomal aberrations. Acknowledgement Thanks are due to C. S. I. R., New Delhi, India for providing financial assistance. References Abe, S. and Sasaki, M Chromosome aberrations and sister chromatid exchanges in Chinese hamster cells exposed to various chemicals. J. Natl. Cancer Inst. 58: Bhatt, B The study of chromosome aberrations in human lymphocytes in: Evaluation of Mutagenic and Carcinogenic Potential of Environmental Agents. Environmental Mutagen Society of India, pp Furakawa, M., Shiriashi, S. K., Tan, J. C. and Huang, C. C Sister chromatid exchanges and growth inhibition induced by the flame retardant Tris(2, 3-dibromopropyl phosphate) in Chinese hamster cells. J. Natl. Cancer Inst. 60: Galloway, S. M. and Wolff, S The relationship between chemically induced sister chromatid exchanges and chromatid breakage. Mutat. Res. 61:

6 554 Ajay K. Jain and N. Sethi Cytologia 56 Heddle, J. A., Whissel, D. and Bodycote, J. D Changes in chromosome structure induced by radiation: a test of the two chief hypotheses. Nature 221: Ikushima, T Role of sister chromatid exchanges in chromatid aberration formation. Nature 268: Jain, A. K. 1989a. Are pesticides genotoxic? An overview. Biol. Mem. 14: b. 6-thioguanine (6TG) resistant mutation, chromosomal aberrations and sister chromatid exchanges (SCE's) in V79 cells I. Induced by Safrole. Cytologia 54: (1989c). 6-thioguanine (6TG) resistant mutation chromosomal aberrations and sister chromatid exchanges (SCE's) in V79 cells III. Concluding remarks. Cytologia 54: , Shimoi, K., Nakamura, Y., Tomita, I. and Kada, T Preliminary study on the desmutagenic and - - antimutagenic effect of some natural products. Curr. Sc. 56: , -, -, Kada, T., Hara, Y. and Tomita, I. 1989a. Crude tea extracts decrease the mutagenic activity of N - methyl-n Œ-Nitro-N-nitrosoguanidine in vitro and in intragastric tract of rats. Mutat. Res. 210: 1-8., -, -, Sano, M. and Tomita, I. 1989b. Effect of tea on 12-o-tetradecanoyl-phorbol-13 acetate (TPA) in duced promotion of transformation in JB6 mouse epidermal cells. Ind. J. Cancer 26: Kihlman, B. A Sister chromatid exchanges in Vicia faba II. Effects of thiotepa, caffein and 8-ethoxy caffein on the frequency of SCE's. Chromosoma 51: Latt, S. A Sister chromatid exchanges, indices of human chromosome damage and repair, detection by fluorescence and induction by mitomycin C. Proc. Natl. Acad. Sci. (USA) 71: McCann, J., Choi, E., Yamasaki, E. and Ames, B. N Detection of carcinogens as mutagens in the Salmonella/microsome test: Assay of 300 chemicals. Proc. Natl. Acad. Sci. (USA) 72: Perry, P. and Evans, H. J Cytological detection of mutagens-carcinogen exposure by sister chromatid exchanges. Nature 258: Phillips, R. L Role of life style and dietary habits in Seventh-Day Adventists. Cancer Res. 35: Sasaki, M. S Sister chromatid exchanges and chromatid interchange on possible manifestation of dif ferent DNA repair processes. Nature 269: Shiriashi, Y. and Sandberg, A. A The relationship between sister chromatid exchanges and chromo some aberration in Bloom's syndrome. Cytogenet. Cell Genet. 18: and Effects of caffein induced DNA replication on SCE and chromosome aberrations produced by alkylating agents. Mutat. Res. 72: Solomon, E. and Bobrow, M Sister chromatid exchanges-a sensitive assay of agents damaging human chromosomes. M utat. Res. 30: Sono, A. and Sakaguchi, K Effects of inhibitors of nucleic acid and protein synthesis on ethyl methane sulfonate induced sister chromatid exchanges in Chinese hamster cells. Cell Struct. Funct. 4: Sugimura, T. and Sato, S Mutagen-carcinogens in foods. Cancer Res. 43: