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Screening of tea clones for inhibition of PhIP mutagenicity
Fundamental and Molecular Mechanisms of Mutagenesis
ELSEVIER
Mutation
Research
326 (1995) 219-225
Screening of tea clones for inhibition of PhIP mutagenicity Zenon Apostolides
a, John H. Weisburger b,*
aDepartment of Biochemistry, University of Pretoria, Pretoria 0002, South Africa b American Health Foundation, One Dana Rd, Valhalla, NY 10595, USA Received
8 September
1994; revision received
12 October
1994; accepted
24 October
1994
Abstract Standard black and green tea extracts have been known to inhibit mutagenicity caused by PhIP, in the Salmonella TA98 assay containing S9 fraction from the liver of rats induced with a-naphthoflavone and phenobarbital. Breeding and selection programs for high yielding tea clones have successfully increased yields in many tea producing areas. Six clonal teas and three seedling teas were obtained from a tea producing area in Southern Africa. Standard black and green teas were used as controls. Dose-dependent inhibition of the bacterial mutagenicity elicited by two concentrations of PhIP was found in the extracts of all the teas tested. This indicates that the clonal teas have not lost their anti-mutagenic properties. Small differences were found amongst the clonal teas in their ability to inhibit mutagenicity. This indicates that it may be possible to enhance this trait in future breeding and selection programs.
typhimutium
Keywords:
Tea; Camellia sinensis; Green tea; Black tea; Tea clones; Ames test; Cancer; 2-Amino-1-methyl-6-phenylimidazo[4,5b]pyridine; Heterocyclic amines
1. Introduction
Many heterocyclic amines (HCAs) form during the cooking of protein rich foods, especially meat and fish. About 19 HCAs have been isolated and identified (Becher et al., 1988; Adamson et al., 1995). One of these compounds, 2-amino-lmethyl-6-phenyl-imidazo[4,5-blpyridine (PhIP)
Abbreuiations: CTC, cut tear and curl; HCA, heterocyclic amine; HW, high yield variety; PhIP, 2-amino-1-methyl-6phenylimidazo[4,5-blpyridine; SEM, standard error of the mean. * Corresponding author. Fax (914) 592-6317; Internet [email protected] 0027-5107/95/$09.50 0 1995 Elsevier SSDI 0027-5107(94)00175-8
Science
Mutagenicity
testing;
(CAS No. 105650-23-5), is a major product. It is present at approximately 15 ppb of the original weight of uncooked beef. The concentration is a function of the mode of cooking. This accounts for 75% of the mass of genotoxic material and contributes 18% of the total mutagenicity of the fried beef (Felton et al., 1986). PhIP has a relatively low specific activity of 1950 revertants/pg in the Ames/Salmonella assay. Other compounds have higher specific activities (e.g. 2-amino-3,8-dimethylimidazo[4,5-f lquinoxaline (MeIQx), 58 000 revertants/pgg). PhIP is the most abundant mutagenic compound, by mass, in fried beef. PhIP is metabolized to N-OH-PhIP and 4’-OH-PhIP (Bounarati and Felton, 1990; Kaderlik et al.,
B.V. All rights reserved
220
Z. Apostolides, J. H. We&burger /Mutation Research 326 (1995) 219-225
1994). Synthetic and metabolically formed NOH-PhIP induce concentration-dependent mutagenesis in Salmonella typhimurium strain TA98. About 70% of ingested PhIP is excreted as NOH-PhIP in human urine. The formation of this product is inhibited by a CYPlA2 specific inhibitor, suggesting that CYPlA2 is the main enzyme system responsible for this conversion (Boobis et al., 1994). Earlier work in this Institute has indicated that green tea, black tea and individual tea polyphenols inhibit the activation of PhIP by murine liver enzymes to reactive metabolites as visualized by the Ames assay. Based on a number of such findings (Xu et al., 1992; Weisburger et al., 1995), moderate tea consumption (5 cups/day) has been encouraged as an additional means of reducing the risk of chronic diseases. Studies with large groups of people (805) over long periods (5 years) have correlated low incidence of coronary heart disease with high (30 mg/day) intake of flavonoids. The main source of flavonoids, in this study, was black tea (Hertog et al., 19931. Breast cancer was reported to have a lower incidence in women that were regular tea drinkers (Hunter et al., 1991). Gastric cancer was lower in people who were consuming 5 cups or more of tea (Yang and Wang, 1993). There are a number of types of black tea. They differ in production methods and genetic variety. Tea estates all over the world have selected varieties or clones that give high yields under their localized conditions. These high yield varieties (HYV) are selected on several traits, e.g. resistance to disease, drought, cold, etc. Good quality attributes, e.g. high theaflavin, or strong aroma have also been used as selection criteria (Banerjee, 1992). The possibility, thus, exists that the selection and breeding programs may have selected against anti-cancer properties. Another possibility is that manufacturing conditions and varietal differences may affect a given tea’s anticancer properties. The anti cancer properties of seven types of tea, which differed in varietal and manufacturing conditions, have been described (Chen, 1992). We investigated the possible anticancer properties of six clonal teas, by examining their effect on a genotoxin in cooked meat, PhIP.
These teas had been produced under similar agronomic, climatic and manufacturing conditions. For comparison, samples of seedling teas and standard black and green tea were studied.
2. Materials and methods A standard black and green tea was procured through the United States Tea Council from T.J. Lipton, Inc. (New Jersey). Seedling and clonal teas were obtained through the Tea Council of Southern Africa from Sapekoe Estates (Pty), Ltd. The seedling tea samples were from the Grenshoek Tea Estate, on three consecutive days of manufacture representing tea from different tea gardens. The clonal teas were from the Mambedi River Tea Estate. Both tea estates are situated in the Transvaal province of South Africa, about 300 meters above sea level. The HYV clones used in this study are used in commercial scale production and are well known among local tea brokers and blenders. All the teas from South Africa were manufactured during April 1994. The manufacturing conditions were 18 h wither, rolling by CTC (cut, tear and curl), enzymatic oxidation (formerly ‘fermentation’) for 60-90 min, fluid bed drying and sorting. Broken orange pekoe fannings, a large particle size grade, was used for all the tests. Tea is usually consumed by humans as a 2% (w/v) solution. Tea solutions (2% w/v) were prepared by adding 100 ml of boiled water to 2 g of each tea separately. The solutions were infused for 5 min and filtered while hot through Whatman number 42, folded 2 inch filter papers. The different concentration of tea extract, i.e. 0.25, 0.50, 1.00 and 2.00 mg/ml, were obtained by pipeting 12.5, 25, 50 or 100 ~1 aliquots of the 2% tea solution, sterilized by passage through a 0.25 pm Millipore filter. All water used in these tests was deionized with a Millipore Milli-Q system. S9 fractions were obtained from male Sprague-Dawley rats, induced with a-naphthoflavone and phenobarbital. The Ames tests, using Salmonella typhimutium TA98 + S9 with PhIP, were done in the standard method using the preincubation procedures (Maron and Ames,
Z. Apostolides, _I.H. Weisburger /Mutation Research 326 (1995) 219-225
1983; Gatehouse et al., 1994). Four concentrations of the tea solution were used (Table 1). The PhIP was added as 10 ~1 of a 1000 PM PhIP or 10 ~1 of a 5000 PM PhIP in DMSO solution to the 1000 ~1 total volume used in the assays. All tests were done in triplicate. The Fig.P (ver 6.0) software package (Sebaldt, 1991) was used for analysis of the data by the Student’s t-test procedure.
3. Results The positive control (10 PM PhIP alone) with no tea had 147 revertants/plate, while the negative control without PhIP (i.e. spontaneous revertants) was 47 revertants/plate. The mutagenicity caused by 10 PM PhIP in the Salmonella typhimurium TA98 + S9 assay was inhibited in a dose-dependent manner by all the tea extracts (Table 1). The standard black tea alone (no PhIP), in this concentration range, displayed no mutagenicity. This tea showed better inhibition of the mutagenicity caused by 10 PM PhIP, than green tea and all the other black tea samples in the 0.5-1.0 mg/ml range. Statistical analysis of the results of the three seedling teas, against each other, showed no significant differences at each of the four concentrations. Thus, the nine scores from the three seedling teas were pooled at each concentration. These combined scores formed the control group at each concentration. The scores of each of the six clones, the standard black and green teas were compared to the combined results of the three seedling teas at each concentration. The inhibition from the standard black tea was better than the seedling teas at three of the four tea extract concentrations, with 10 PM PhIP. This may be due to differences in particle size. Smaller particles release more soluble solids per unit weight due to larger surface area. Only clone 3 had statistically significant lower inhibition than the seedling teas at the two lowest concentrations. Although not statistically significant, clone 1 showed better inhibition than the seedling teas, while the other five clones showed poorer inhibition.
221
This series of experiments was repeated with 50 PM PhIP. The positive control (50 PM PhIP) with no tea had 668 revertants/plate, while the negative control without PhIP (i.e. spontaneous revertants) was 59 revertants/plate. Statistical analysis of the three seedling teas showed significant differences, and the p values were 0.05 or 0.02. These scores for the seedling teas were combined at each of the four tea extract concentrations. The commercial black and green teas and the six clones separately and together were compared to the control group of seedlings at each tea extract concentration. The difference was considered statistically significant only if the p value was lower than 0.02. These means, SEM and p values < 0.02 are shown in Table 1.
4. Discussion Earlier observations (Steele et al., 1985) have indicated that ( + )-catechin, a tea constituent, can inhibit mutagenesis of aromatic amines, as measured by the Ames/Salmonella typhimurium TA98 + S9 assay. This inhibition was due to inhibition of the S9 enzymes. Both benzphetamine N-demethylase (CYP2B) and ethoxyresorufin Odeethylase (CYPlAl) were inhibited by l-5 mM (0.3-1.5 mg/ml) concentrations. A concentration of 1.4 mM (0.42 mg/ml) gave 50% inhibition, The ( + )-catechin was also found to be a competitive inhibitor of NADPH cytochrome c reductase in this concentration range. However, these concentrations were regarded as too high for in vivo systems. Inhibition of mutagenicity in the Salmonella typhimurium assay by several genotoxins has been reported by Hayatsu et al. (1992). This was achieved with ( - )-epigallocatechin gallate (EGCG), the main constituent of green tea, and present in small amounts in black tea. Inhibition was observed in the 0.01-1.0 mM or 0.00426-0.426 mg/ml range, indicating that EGCG was a better inhibitor than (+)-catechin. Extracts of tea and coffee displayed mainly an antioxidant effect, although with some protocols, a prooxidant action was noted (Stadler et al., 1994). Humans normally consume tea at l-2% w/v
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Z. Apostolides, J.H. Wekburger/Mutation
Table 1 The inhibition Tea
of the mutagenicity Dose
(m&late)
Seedling
Mutagenicity
and black tea infusions (revertants/plate)
10 /LM PhIP
(mean f SEM) a
Percent
inhibition
50/.~M PhIP
10 /.LM PhIP ’
50 /.LM PhIP ’
0.25 0.50 1.00 2.00
130* 80+ 49+ 34*
7 6 2 2
1113+53 860 + 44 516+39 130+ 7
12 46 67 77
0 0 23 81
Clone 1 BB35
0.25 0.50 1.00 2.00
134* 63+ 46+ 32i_
1 5 1 1
1110 763 408 121
9 57 69 78
0 0 39 82
Clone 2 MT12
0.25 0.50 1.00 2.00
148 f 12 86k 6 43f 3 37f 5
750 578 361 131
+ 55 (0.005) + 13 (0.001) + f 20 (0.010) + + 8
0 41 71 75
0 13 46 80
Clone 3 PC1
0.25 0.50 1.oo 2.00
169 f 104* 57+ 38k
715 f 44 (0.001) 601 k 18 (0.001) + 360 k 15 (0.005) + 119+ 6
0 29 61 74
0 10 46 82
Clone 4 SFSlSO
0.25 0.50 1.00 2.00
173 + 20 100 * 10 64f 6 40& 3
666 552 289 96
f 49 (0.0011 f 17 (0.001) + 2 (0.001) + 4 (0.001)
+ + + +
0 32 56 73
0 17 57 86
Clone 5 SFS204
0.25 0.50 1.00 2.00
151+ 87f 55* 33+
10 6 7 2
836 667 366 123
f f + +
+ +
0 41 63 78
0 0 45 74
Clone 6 TRI 6/8
0.25 0.50 1.00 2.00
137 _+ 14 105 f 10 53+ 6 40+ 6
828 594 260 77
* 17 (0.001) * 33 (0.0021 + + 28 (0.001) + i 1(0.0001, +
7 29 64 73
0 11 61 88
Clones all
0.25 0.50 1.00 2.00
152 k 91f 53f 37f
5 (0.050) 4 2 1
820 626 341 111
f 38 (0.001) * 19 (0.002) + * 14 (0.002) + f 5 (0.050)
0 38 64 73
0 6 49 83
Standard black
0.25 0.50 1.00 2.00
92 f 76+ 40+ 45 +
4 (0.002) + 7 2(0.0501 + 3 (0.0501 +
936 + 32 (0.020) 691 + 32 (0.020) 368 + 22 (0.010) 90* 3(0.001)
37 48 73 69
0 0 45 87
20 46 59 78
0 0 26 78
Standard green
b
of PhIP by green
Research 326 (1995) 219-225
0.25 0.50 1.00 2.00
l(O.001) 4(0.010) 6 2
118 f 10 79f 4 60f 4 32f 1
+
1054 715 497 150
* f f *
+ f * k
27 39 ll(O.050) 10
39 (0.005, 14 (0.005) 20 (0.010) 14
42 49 14 16
+ +
a His+ revertant numbers. b This represents the pooled data from all three seedling teas, i.e. nine plates. ’ The positive control with 10 PM PhIP and no tea had 147 rev/plate. ’ The positive control with 50 FM PhIP and no tea had 668 rev/plate. + This indicates statistically significant inhibition relative to the control group (i.e. all seedlings) at this concentration. This indicates statistically significant stimulation relative to the control group (i.e. all seedlings) at this concentration. Note: In the 50 PM PhIP experiment, some of the seedlings showed statistically significant differences at the p = 0.05 and p = 0.02 level. Thus, only p < 0.01 values are marked with + to denote inhbition or - to denote no inhibition.
concentration. When tea is extracted with hot water, about 35% of the dry weight is solubilized. Thus a 2% w/v tea extract would contain 700 mg/lOO ml of soluble solids, or 7 mg/ml. The soluble solids are characterized by carbohydrates, amino acids, methylxanthines, minerals and several fractions of phenolic compounds (Graham, 1992; Balentine, 1991; Hara, 1992). The phenolic compounds in black tea are catechins (10% or 0.7 mg/ml), theaflavins (6% or 0.42 mg/ml), thearubigins (18% or 1.26 mg/ml) and flavonols (8% or 0.56 mg/ml). All the phenolic compounds contribute 42% of the soluble solids. Thus, the phenolic compounds are 2.9 mg/ml in a 2% tea extract. During the Ames test, 100 ~1 of the 2% tea solution is added to 900 ~1 preincubation assay mixture. Thus, the above concentrations are diluted lo-fold, i.e. 0.29 mg/ml of phenolic compounds and 0.042 mg/ml of theaflavins. The concentration of theaflavin in the Ames test would be about 10 PM. This may also be achieved under physiological conditions. Furthermore, since two or more theaflavins can act synergistically, better inhibition could be obtained with whole extracts than with single compounds at equal concentrations. Tea solutions (2% w/v> were not toxic to the bacteria in the Ames test. This agrees with earlier work of Stich (1992). Our results show that the mutagenicity caused by 10 and 50 PM PhIP in Salmonella typhimurium TA98 + S9 is inhibited by green and black tea extracts in a dose-dependent manner. This was achieved in the 0.25-2.0 mg/ml range, with tea extracts. The Salmonella ~ph~mu~urn TA98 assay has been used widely as a qualitative test for many mutagens. The inclusion of the S9 enzymes enables this method to be used for promutagens. Inhibition in this assay system by many compounds has been interpreted as indicative of the anti-mutagenic properties of such compounds. Many compounds are able to inhibit the S9 cytochrome c isoenzymes, in this assay, at high enough concentrations that are not toxic to the bacteria. Such high concentrations may not be achievable with in vivo systems (Steele et al., 1985). This may be due to absorption, transportation, dilution, metabolism and excretion (Klaas-
sen and Rozman, 1991). Thus, compounds that test positive with respect to inhibition of mutagenicity in this assay may not show anti-cancer properties in mammals due to inhibition of the S9 (phase I detoxification) enzymes. While the limitations of this assay are appreciated, it was used as a preliminary screening of our samples. Lower concentrations of carcinogen lead to higher inhibition by equal amounts of tea. This phenomenon cannot be fully explained at present. Similar results have been found in dose-response tests with BHA and BHT in several models of carcinogenesis (Cohen et al., 1986). With less carcinogen, the ratio of inhibitor/carcinogen is more favorable. The positive results obtained in these experiments suggest that an extension of this research is warranted. In any case, 2% tea solutions have modified phase I and phase II rat liver enzyme systems (Sohn et al., 1994).
5. Conclusion It was found that under the conditions of these in vitro tests, all the teas examined inhibited the mutagenicity of PhIP. These results also indicate that clonal teas selected as HYV have not lost their ability to inhibit the activation of the procarcinogen PhIP. Small clonal differences were found in the efficacy of inhibition of mutagenicity. About 50% inhibition was found at 0.5 mg/ml. The mechanism was probably the inhibition of the S9 enzymes by the tea polyphenolic compounds (Steele et al., 1985). It is unlikely that the same mechanism prevails in vivo. Drinking tea enhanced the levels of rat liver detoxification enzymes. Both phase I and phase II enzymes are induced by tea (Sohn et al., 1985). Tea drinking may also induce higher levels of glutathione (Prestera, 19931, so that detoxification of ingested PhIP may occur more efficiently. Other biochemical mechanisms have been proposed for the anti-cancer properties of tea, e.g. induction of DNA repair, binding with activated procarcinogen (Hayatsu et al., 1992; Nagab-
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Z. Apostolides, J.H. We&burger /Mutation Research 326 (1995) 219-225
hushan et al., 1988). These need further investigation. As explained above, small varietal differences were found in these HYV tea clones. If these differences can be repeated through in vivo approaches, they may represent genetic traits that could be enhanced by breeding and selection of tea clones in the future.
Acknowledgements Parts of this research were supported by PHSNIH Grant CA-42381 from the National Cancer Institute, and the Tea Health Research Group. Z.A. is indebted to the Tea Council of Southern Africa and the Office of International Affairs, U.S. National Cancer Institute, NIH, for financial assistance. We would like to thank the U.S. Tea Council, T.J. Lipton (USA) and Sapekoe (South Africa) for the supply of tea samples. We are indebted to F.Q. Luo for performing the Ames tests and B.A. McKinney for preparing the manuscript.
References Adamson, R.H., J.A. Gustafsson, N. Ito, M. Nagao, T. Sugimura, K. Wakabayashi and Y. Yamazoe (1995) Heterocyclic amines in cooked foods, in: Proceedings of the 23rd International Symposium of the Princess Takamatsu Cancer Research Fund, Princeton Scientific Publication. Princeton, NJ, in press. Balentine, D.A. (1991) Manufacturing and chemistry of tea, in: C.T. Ho, C.Y. Lee and M.T. Huang (Ed%), Phenolic Compounds in Food and Their Effects on Health I, American Chemical Society, Washington, DC, pp. 34-50. Banerjee. B. (1992) Selection and breeding of tea, in: K.C. Wilson and M.N. Clifford (Eds.), Tea: Cultivation to consumption, Chapman and Hall, London, pp. 53-86. Becher, G., M.G. Knize, I.F. Nes and J.S. Felton (1988) Isolation and identification of mutagens from a fried Norwegian meat product, Carcinogenesis, 9, 247-53. Boobis, A.R., A.M. Lynch, S. Murray, R. de la Torre, A. Solans, M. Farre, J. Segura, N.J. Gooderham and D.S. Davies (1994) CYPlA%catalysed conversion of dietary heterocyclic amines to their proximate carcinogens is their major route of metabolism in humans, Cancer Res., 54, 89-94. Buonarati, M.H. and J.S. Felton (1990) Activation of 2-aminol-methyl-6-phenylimidazo[4,5-blpyridine (PhIP) to mutagenic metabolites, Carcinogenesis, 11, 1133-1138.
Chen, J. (1992) The effects of Chinese tea on the occurrence of esophageal tumors induced by N-nitrosomethylbenzylamine in rats, Prev. Med., 21, 385-391. Cohen, L.A., K. Choi, S. Numoto, M. Reddy, B. Berke and J.H. Weisburger (1986) Inhibition of chemically induced mammary carcinogenesis in rats by long-term exposure to buytlated hydroxytoluene (BHT): Interrelation among BHT concentration, carcinogen dose, and diet, J. Natl. Cancer Inst., 76, 721-729. Felton, J.S., M.G. Knize, N.H. Shen, P.R. Lewis, B.D. Andresen, J. Happe and F.T. Hatch (1986) The isolation and identification of a new mutagen from fried ground beef: 2-amino-I-methyl-6-phenylimidazo[4,5-blpyridine (PhIP), Carcinogenesis, 7, 1081-1086. Gatehouse, D., S. Haworth, T. Cebula, E. Gocke, L. Kier, T. Matsushima, C. Melcion, T. Nohmi. T. Ohta, S. Vennitt and E. Zeiger (1994) Recommendations for the performance of bacterial mutation assays. Mutation Res., 312, 217-233. Graham, H.N. (1992) Green tea composition, consumption and polyphenol chemistry, Prev. Med., 21, 334-350. Hara, Y. (1994) Prophylactic functions of tea polyphenols, in: CT. Ho, T. Osawa, M.T. Huang and T.R. Rosen (Eds.), Food Phytochemicals for Cancer Prevention II, Teas, Spices and Herbs, ACS Symposium Series 547, Washington, DC, pp. 34-50. Hayatsu, H., N. Inada, T. Kakutani, S. Arimoto, T. Negishi. K. Mori, T. Okuda and I. Sakata (1992) Suppression of genotoxicity of carcinogenesis by (- )-epigallocatechin gallate, Prev. Med., 21, 370-376. Hertog, M.G.L., E.J.M. Feskens, P.C.H. Hollman, M.B. Katan and D. Kromhout (1993) Dietary antioxidant flavonoids and risk of coronary heart disease: the Zutphen elderly study, Lancet, 342, 1007-1011. Horiba, N., Y. Maekawa, M. Ito, T. Matsumoto and H. Nakamura (1991) A pilot study of Lalanese green tea as a medicament: Antibacterial and bactericidal effects. J. Endodontics, 17, 122-124. Hunter, O.J., J.E. Manson, M.J. Stampfer, G.A. Colditz, B. Rosner, C.H. Hennekens, F.E. Speizer and W.C. Willett (1992) A prospective study of caffeine, coffee, tea and breast cancer, Am. J. Epidemiol., 136, 1000-1001. Kaderlik, K.R., R.F. Minchin, G.J. Mulder, K.F. Ilett, M. Daugaard-Jenson, C.H. Teitel and F.K. Kadlubar (1994) Metabolic activation pathway for the formation of DNA adducts of the carcinogen 2-amino-l-methyl-6-phenylimidazo[4,5-blpyridine (PhIP) in rat extrahepatic tissues, Carcinogenesis, 15. 1703-1709. Klaassen, C.D. and K. Rozman (1991) Principles of toxicology, in: M.O. Amdur, J. Doull and CD. Klaassen (Eds.), Casarett and Doull’s Toxicology, 4th edn., Pergamon Press, New York, pp. 12-49. Maron, D.M. and B.N. Ames (1983) Revised methods for the Salmonella mutagenicity test, Mutation Res., 113, 173-215. Nagabushan, M.. A.J. Amonkar, U.J. Nair, U. Santhanam, N. Ammigan, A.V. D’Souza and S.V. Bhide (1988) Catechin as an antimutagen: its mode of action, J. Cancer Res. Clin. Oncol.. 114, 177-182.
Z. Apostolides, J.H. Weisburger /Mutation Research 326 (1995) 219-225 Prestera, T., Y. Zhang, S.R. Spencer, CA. Wilczak and P. Talalay (1993) The electrophile counterattack response: protection against neoplasia and toxicity, Adv. Enzyme Regul., 33, 281-96. Sebaldt, R.J. (1991) Figure Processor, User’s manual, Version 6.0, Biosoft, Cambridge. Sohn, OS., A. Surace, E.S. Fiala, J.P. Richie, S. Colosimo, E. Zang and J.H. Weisburger (1994) Effects of green and black tea on hepatic xenobiotic metabolising systems in the male F344 rat, Xenobiotica, 24, 119-127. Stadler, R.H., R.J. Turesky, 0. Miiller, J. Markovic and P.-M. Leong-Morgenthaler (1994) The inhibitory effects of coffee on radical-mediated oxidation and mutagenicity, Mutation Res., 308, 177-190. Steele, C.M., M. Lalies and C. Ioannides (1985) Inhibition of the mutagenicity of aromatic amines by the plant flavonoid (+ J-catechin, Cancer Res., 45, 3573-3577.
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Weisburger, J.H., A. Rivenson, D.G.I. Kingston, T.D. Wilkins, R.L. Van Tassell, M. Nagao, T. Sugimura and Y. Hara (1995) Dietary modulation of the carcinogenicity of the heterocyclic amines, in: R.H. Adamson, J.A. Gustafsson, N. Ito, M. Nagao, M. Sugimura, K. Wakabayashi and Y. Yamazoe (Eds.), Proceedings, 23rd International Symposium of the Princess Takamatsu Cancer Research Fund, Princeton Scientific Publishing Co., Princeton, NJ. Wohlleb, J.C., C.F. Hunter, B. Blass, F. Kadlubar, D.Z.J. Chu and N.P. Lang (1990) Aromatic amine acetyltransferase as a marker for colorectal cancer: environmental and demographic associations, Int. J. Cancer, 46. 22-30. Xu, Y., C.T. Ho, S.G. Amin, C. Han and F.L. Chung (1992) Inhibition of tobacco-specific nitrosamine-induced lung tumorigenesis in A/J mice by green tea and its major polyphenol as antioxidants, Cancer Res., 52, 3875-3879.
ELSEVIER
Mutation
Research
326 (1995) 219-225
Screening of tea clones for inhibition of PhIP mutagenicity Zenon Apostolides
a, John H. Weisburger b,*
aDepartment of Biochemistry, University of Pretoria, Pretoria 0002, South Africa b American Health Foundation, One Dana Rd, Valhalla, NY 10595, USA Received
8 September
1994; revision received
12 October
1994; accepted
24 October
1994
Abstract Standard black and green tea extracts have been known to inhibit mutagenicity caused by PhIP, in the Salmonella TA98 assay containing S9 fraction from the liver of rats induced with a-naphthoflavone and phenobarbital. Breeding and selection programs for high yielding tea clones have successfully increased yields in many tea producing areas. Six clonal teas and three seedling teas were obtained from a tea producing area in Southern Africa. Standard black and green teas were used as controls. Dose-dependent inhibition of the bacterial mutagenicity elicited by two concentrations of PhIP was found in the extracts of all the teas tested. This indicates that the clonal teas have not lost their anti-mutagenic properties. Small differences were found amongst the clonal teas in their ability to inhibit mutagenicity. This indicates that it may be possible to enhance this trait in future breeding and selection programs.
typhimutium
Keywords:
Tea; Camellia sinensis; Green tea; Black tea; Tea clones; Ames test; Cancer; 2-Amino-1-methyl-6-phenylimidazo[4,5b]pyridine; Heterocyclic amines
1. Introduction
Many heterocyclic amines (HCAs) form during the cooking of protein rich foods, especially meat and fish. About 19 HCAs have been isolated and identified (Becher et al., 1988; Adamson et al., 1995). One of these compounds, 2-amino-lmethyl-6-phenyl-imidazo[4,5-blpyridine (PhIP)
Abbreuiations: CTC, cut tear and curl; HCA, heterocyclic amine; HW, high yield variety; PhIP, 2-amino-1-methyl-6phenylimidazo[4,5-blpyridine; SEM, standard error of the mean. * Corresponding author. Fax (914) 592-6317; Internet [email protected] 0027-5107/95/$09.50 0 1995 Elsevier SSDI 0027-5107(94)00175-8
Science
Mutagenicity
testing;
(CAS No. 105650-23-5), is a major product. It is present at approximately 15 ppb of the original weight of uncooked beef. The concentration is a function of the mode of cooking. This accounts for 75% of the mass of genotoxic material and contributes 18% of the total mutagenicity of the fried beef (Felton et al., 1986). PhIP has a relatively low specific activity of 1950 revertants/pg in the Ames/Salmonella assay. Other compounds have higher specific activities (e.g. 2-amino-3,8-dimethylimidazo[4,5-f lquinoxaline (MeIQx), 58 000 revertants/pgg). PhIP is the most abundant mutagenic compound, by mass, in fried beef. PhIP is metabolized to N-OH-PhIP and 4’-OH-PhIP (Bounarati and Felton, 1990; Kaderlik et al.,
B.V. All rights reserved
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Z. Apostolides, J. H. We&burger /Mutation Research 326 (1995) 219-225
1994). Synthetic and metabolically formed NOH-PhIP induce concentration-dependent mutagenesis in Salmonella typhimurium strain TA98. About 70% of ingested PhIP is excreted as NOH-PhIP in human urine. The formation of this product is inhibited by a CYPlA2 specific inhibitor, suggesting that CYPlA2 is the main enzyme system responsible for this conversion (Boobis et al., 1994). Earlier work in this Institute has indicated that green tea, black tea and individual tea polyphenols inhibit the activation of PhIP by murine liver enzymes to reactive metabolites as visualized by the Ames assay. Based on a number of such findings (Xu et al., 1992; Weisburger et al., 1995), moderate tea consumption (5 cups/day) has been encouraged as an additional means of reducing the risk of chronic diseases. Studies with large groups of people (805) over long periods (5 years) have correlated low incidence of coronary heart disease with high (30 mg/day) intake of flavonoids. The main source of flavonoids, in this study, was black tea (Hertog et al., 19931. Breast cancer was reported to have a lower incidence in women that were regular tea drinkers (Hunter et al., 1991). Gastric cancer was lower in people who were consuming 5 cups or more of tea (Yang and Wang, 1993). There are a number of types of black tea. They differ in production methods and genetic variety. Tea estates all over the world have selected varieties or clones that give high yields under their localized conditions. These high yield varieties (HYV) are selected on several traits, e.g. resistance to disease, drought, cold, etc. Good quality attributes, e.g. high theaflavin, or strong aroma have also been used as selection criteria (Banerjee, 1992). The possibility, thus, exists that the selection and breeding programs may have selected against anti-cancer properties. Another possibility is that manufacturing conditions and varietal differences may affect a given tea’s anticancer properties. The anti cancer properties of seven types of tea, which differed in varietal and manufacturing conditions, have been described (Chen, 1992). We investigated the possible anticancer properties of six clonal teas, by examining their effect on a genotoxin in cooked meat, PhIP.
These teas had been produced under similar agronomic, climatic and manufacturing conditions. For comparison, samples of seedling teas and standard black and green tea were studied.
2. Materials and methods A standard black and green tea was procured through the United States Tea Council from T.J. Lipton, Inc. (New Jersey). Seedling and clonal teas were obtained through the Tea Council of Southern Africa from Sapekoe Estates (Pty), Ltd. The seedling tea samples were from the Grenshoek Tea Estate, on three consecutive days of manufacture representing tea from different tea gardens. The clonal teas were from the Mambedi River Tea Estate. Both tea estates are situated in the Transvaal province of South Africa, about 300 meters above sea level. The HYV clones used in this study are used in commercial scale production and are well known among local tea brokers and blenders. All the teas from South Africa were manufactured during April 1994. The manufacturing conditions were 18 h wither, rolling by CTC (cut, tear and curl), enzymatic oxidation (formerly ‘fermentation’) for 60-90 min, fluid bed drying and sorting. Broken orange pekoe fannings, a large particle size grade, was used for all the tests. Tea is usually consumed by humans as a 2% (w/v) solution. Tea solutions (2% w/v) were prepared by adding 100 ml of boiled water to 2 g of each tea separately. The solutions were infused for 5 min and filtered while hot through Whatman number 42, folded 2 inch filter papers. The different concentration of tea extract, i.e. 0.25, 0.50, 1.00 and 2.00 mg/ml, were obtained by pipeting 12.5, 25, 50 or 100 ~1 aliquots of the 2% tea solution, sterilized by passage through a 0.25 pm Millipore filter. All water used in these tests was deionized with a Millipore Milli-Q system. S9 fractions were obtained from male Sprague-Dawley rats, induced with a-naphthoflavone and phenobarbital. The Ames tests, using Salmonella typhimutium TA98 + S9 with PhIP, were done in the standard method using the preincubation procedures (Maron and Ames,
Z. Apostolides, _I.H. Weisburger /Mutation Research 326 (1995) 219-225
1983; Gatehouse et al., 1994). Four concentrations of the tea solution were used (Table 1). The PhIP was added as 10 ~1 of a 1000 PM PhIP or 10 ~1 of a 5000 PM PhIP in DMSO solution to the 1000 ~1 total volume used in the assays. All tests were done in triplicate. The Fig.P (ver 6.0) software package (Sebaldt, 1991) was used for analysis of the data by the Student’s t-test procedure.
3. Results The positive control (10 PM PhIP alone) with no tea had 147 revertants/plate, while the negative control without PhIP (i.e. spontaneous revertants) was 47 revertants/plate. The mutagenicity caused by 10 PM PhIP in the Salmonella typhimurium TA98 + S9 assay was inhibited in a dose-dependent manner by all the tea extracts (Table 1). The standard black tea alone (no PhIP), in this concentration range, displayed no mutagenicity. This tea showed better inhibition of the mutagenicity caused by 10 PM PhIP, than green tea and all the other black tea samples in the 0.5-1.0 mg/ml range. Statistical analysis of the results of the three seedling teas, against each other, showed no significant differences at each of the four concentrations. Thus, the nine scores from the three seedling teas were pooled at each concentration. These combined scores formed the control group at each concentration. The scores of each of the six clones, the standard black and green teas were compared to the combined results of the three seedling teas at each concentration. The inhibition from the standard black tea was better than the seedling teas at three of the four tea extract concentrations, with 10 PM PhIP. This may be due to differences in particle size. Smaller particles release more soluble solids per unit weight due to larger surface area. Only clone 3 had statistically significant lower inhibition than the seedling teas at the two lowest concentrations. Although not statistically significant, clone 1 showed better inhibition than the seedling teas, while the other five clones showed poorer inhibition.
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This series of experiments was repeated with 50 PM PhIP. The positive control (50 PM PhIP) with no tea had 668 revertants/plate, while the negative control without PhIP (i.e. spontaneous revertants) was 59 revertants/plate. Statistical analysis of the three seedling teas showed significant differences, and the p values were 0.05 or 0.02. These scores for the seedling teas were combined at each of the four tea extract concentrations. The commercial black and green teas and the six clones separately and together were compared to the control group of seedlings at each tea extract concentration. The difference was considered statistically significant only if the p value was lower than 0.02. These means, SEM and p values < 0.02 are shown in Table 1.
4. Discussion Earlier observations (Steele et al., 1985) have indicated that ( + )-catechin, a tea constituent, can inhibit mutagenesis of aromatic amines, as measured by the Ames/Salmonella typhimurium TA98 + S9 assay. This inhibition was due to inhibition of the S9 enzymes. Both benzphetamine N-demethylase (CYP2B) and ethoxyresorufin Odeethylase (CYPlAl) were inhibited by l-5 mM (0.3-1.5 mg/ml) concentrations. A concentration of 1.4 mM (0.42 mg/ml) gave 50% inhibition, The ( + )-catechin was also found to be a competitive inhibitor of NADPH cytochrome c reductase in this concentration range. However, these concentrations were regarded as too high for in vivo systems. Inhibition of mutagenicity in the Salmonella typhimurium assay by several genotoxins has been reported by Hayatsu et al. (1992). This was achieved with ( - )-epigallocatechin gallate (EGCG), the main constituent of green tea, and present in small amounts in black tea. Inhibition was observed in the 0.01-1.0 mM or 0.00426-0.426 mg/ml range, indicating that EGCG was a better inhibitor than (+)-catechin. Extracts of tea and coffee displayed mainly an antioxidant effect, although with some protocols, a prooxidant action was noted (Stadler et al., 1994). Humans normally consume tea at l-2% w/v
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Z. Apostolides, J.H. Wekburger/Mutation
Table 1 The inhibition Tea
of the mutagenicity Dose
(m&late)
Seedling
Mutagenicity
and black tea infusions (revertants/plate)
10 /LM PhIP
(mean f SEM) a
Percent
inhibition
50/.~M PhIP
10 /.LM PhIP ’
50 /.LM PhIP ’
0.25 0.50 1.00 2.00
130* 80+ 49+ 34*
7 6 2 2
1113+53 860 + 44 516+39 130+ 7
12 46 67 77
0 0 23 81
Clone 1 BB35
0.25 0.50 1.00 2.00
134* 63+ 46+ 32i_
1 5 1 1
1110 763 408 121
9 57 69 78
0 0 39 82
Clone 2 MT12
0.25 0.50 1.00 2.00
148 f 12 86k 6 43f 3 37f 5
750 578 361 131
+ 55 (0.005) + 13 (0.001) + f 20 (0.010) + + 8
0 41 71 75
0 13 46 80
Clone 3 PC1
0.25 0.50 1.oo 2.00
169 f 104* 57+ 38k
715 f 44 (0.001) 601 k 18 (0.001) + 360 k 15 (0.005) + 119+ 6
0 29 61 74
0 10 46 82
Clone 4 SFSlSO
0.25 0.50 1.00 2.00
173 + 20 100 * 10 64f 6 40& 3
666 552 289 96
f 49 (0.0011 f 17 (0.001) + 2 (0.001) + 4 (0.001)
+ + + +
0 32 56 73
0 17 57 86
Clone 5 SFS204
0.25 0.50 1.00 2.00
151+ 87f 55* 33+
10 6 7 2
836 667 366 123
f f + +
+ +
0 41 63 78
0 0 45 74
Clone 6 TRI 6/8
0.25 0.50 1.00 2.00
137 _+ 14 105 f 10 53+ 6 40+ 6
828 594 260 77
* 17 (0.001) * 33 (0.0021 + + 28 (0.001) + i 1(0.0001, +
7 29 64 73
0 11 61 88
Clones all
0.25 0.50 1.00 2.00
152 k 91f 53f 37f
5 (0.050) 4 2 1
820 626 341 111
f 38 (0.001) * 19 (0.002) + * 14 (0.002) + f 5 (0.050)
0 38 64 73
0 6 49 83
Standard black
0.25 0.50 1.00 2.00
92 f 76+ 40+ 45 +
4 (0.002) + 7 2(0.0501 + 3 (0.0501 +
936 + 32 (0.020) 691 + 32 (0.020) 368 + 22 (0.010) 90* 3(0.001)
37 48 73 69
0 0 45 87
20 46 59 78
0 0 26 78
Standard green
b
of PhIP by green
Research 326 (1995) 219-225
0.25 0.50 1.00 2.00
l(O.001) 4(0.010) 6 2
118 f 10 79f 4 60f 4 32f 1
+
1054 715 497 150
* f f *
+ f * k
27 39 ll(O.050) 10
39 (0.005, 14 (0.005) 20 (0.010) 14
42 49 14 16
+ +
a His+ revertant numbers. b This represents the pooled data from all three seedling teas, i.e. nine plates. ’ The positive control with 10 PM PhIP and no tea had 147 rev/plate. ’ The positive control with 50 FM PhIP and no tea had 668 rev/plate. + This indicates statistically significant inhibition relative to the control group (i.e. all seedlings) at this concentration. This indicates statistically significant stimulation relative to the control group (i.e. all seedlings) at this concentration. Note: In the 50 PM PhIP experiment, some of the seedlings showed statistically significant differences at the p = 0.05 and p = 0.02 level. Thus, only p < 0.01 values are marked with + to denote inhbition or - to denote no inhibition.
concentration. When tea is extracted with hot water, about 35% of the dry weight is solubilized. Thus a 2% w/v tea extract would contain 700 mg/lOO ml of soluble solids, or 7 mg/ml. The soluble solids are characterized by carbohydrates, amino acids, methylxanthines, minerals and several fractions of phenolic compounds (Graham, 1992; Balentine, 1991; Hara, 1992). The phenolic compounds in black tea are catechins (10% or 0.7 mg/ml), theaflavins (6% or 0.42 mg/ml), thearubigins (18% or 1.26 mg/ml) and flavonols (8% or 0.56 mg/ml). All the phenolic compounds contribute 42% of the soluble solids. Thus, the phenolic compounds are 2.9 mg/ml in a 2% tea extract. During the Ames test, 100 ~1 of the 2% tea solution is added to 900 ~1 preincubation assay mixture. Thus, the above concentrations are diluted lo-fold, i.e. 0.29 mg/ml of phenolic compounds and 0.042 mg/ml of theaflavins. The concentration of theaflavin in the Ames test would be about 10 PM. This may also be achieved under physiological conditions. Furthermore, since two or more theaflavins can act synergistically, better inhibition could be obtained with whole extracts than with single compounds at equal concentrations. Tea solutions (2% w/v> were not toxic to the bacteria in the Ames test. This agrees with earlier work of Stich (1992). Our results show that the mutagenicity caused by 10 and 50 PM PhIP in Salmonella typhimurium TA98 + S9 is inhibited by green and black tea extracts in a dose-dependent manner. This was achieved in the 0.25-2.0 mg/ml range, with tea extracts. The Salmonella ~ph~mu~urn TA98 assay has been used widely as a qualitative test for many mutagens. The inclusion of the S9 enzymes enables this method to be used for promutagens. Inhibition in this assay system by many compounds has been interpreted as indicative of the anti-mutagenic properties of such compounds. Many compounds are able to inhibit the S9 cytochrome c isoenzymes, in this assay, at high enough concentrations that are not toxic to the bacteria. Such high concentrations may not be achievable with in vivo systems (Steele et al., 1985). This may be due to absorption, transportation, dilution, metabolism and excretion (Klaas-
sen and Rozman, 1991). Thus, compounds that test positive with respect to inhibition of mutagenicity in this assay may not show anti-cancer properties in mammals due to inhibition of the S9 (phase I detoxification) enzymes. While the limitations of this assay are appreciated, it was used as a preliminary screening of our samples. Lower concentrations of carcinogen lead to higher inhibition by equal amounts of tea. This phenomenon cannot be fully explained at present. Similar results have been found in dose-response tests with BHA and BHT in several models of carcinogenesis (Cohen et al., 1986). With less carcinogen, the ratio of inhibitor/carcinogen is more favorable. The positive results obtained in these experiments suggest that an extension of this research is warranted. In any case, 2% tea solutions have modified phase I and phase II rat liver enzyme systems (Sohn et al., 1994).
5. Conclusion It was found that under the conditions of these in vitro tests, all the teas examined inhibited the mutagenicity of PhIP. These results also indicate that clonal teas selected as HYV have not lost their ability to inhibit the activation of the procarcinogen PhIP. Small clonal differences were found in the efficacy of inhibition of mutagenicity. About 50% inhibition was found at 0.5 mg/ml. The mechanism was probably the inhibition of the S9 enzymes by the tea polyphenolic compounds (Steele et al., 1985). It is unlikely that the same mechanism prevails in vivo. Drinking tea enhanced the levels of rat liver detoxification enzymes. Both phase I and phase II enzymes are induced by tea (Sohn et al., 1985). Tea drinking may also induce higher levels of glutathione (Prestera, 19931, so that detoxification of ingested PhIP may occur more efficiently. Other biochemical mechanisms have been proposed for the anti-cancer properties of tea, e.g. induction of DNA repair, binding with activated procarcinogen (Hayatsu et al., 1992; Nagab-
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hushan et al., 1988). These need further investigation. As explained above, small varietal differences were found in these HYV tea clones. If these differences can be repeated through in vivo approaches, they may represent genetic traits that could be enhanced by breeding and selection of tea clones in the future.
Acknowledgements Parts of this research were supported by PHSNIH Grant CA-42381 from the National Cancer Institute, and the Tea Health Research Group. Z.A. is indebted to the Tea Council of Southern Africa and the Office of International Affairs, U.S. National Cancer Institute, NIH, for financial assistance. We would like to thank the U.S. Tea Council, T.J. Lipton (USA) and Sapekoe (South Africa) for the supply of tea samples. We are indebted to F.Q. Luo for performing the Ames tests and B.A. McKinney for preparing the manuscript.
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