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ICANCERRESEARCHISUPPI..)54.I99is-i993s.Aprii1.1994] Suppression of Hydroperoxide-induced Cytotoxicity by Polyphenols1

Tsutomu Nakayama Sellout of Food and Nutritional Sciences, University of Shiziioka, 52-1 Yada, Sliizitoka 422, Japan

Abstract HBS for 30 min. After the cells were cultured in MEM supplemented with 10% FBS for 5 days, the number of colonies was counted. The A variety of synthetic and dietary polyphenols protect mammalian and survival (% of control) was calculated by dividing the number of bacterial cells from cytotoxicity induced by hydropernxides, especially colonies of the cells treated with polyphenols and/or H,O2 by the (H2O,). Cytotoxicity of H2O, on Chinese hamster V79 cells was assessed with a colony formation assay. Cytotoxicity and mula- number of colonies of untreated control cells. First, we determined the genicity of H2O, on Salmonella TA104 were assessed with the Ames test. dose dependence of the cytotoxic effects of polyphenols. Then, we SOS response induced by II..O, was investigated in the SOS chromotest adjusted their concentrations so that each polyphenol was itself not with Kscherichia coli PQ37. The polyphenol-bearing o-dihydroxy (cate- toxic. chol) structure, i.e., nordihydroguaiaretic acid, caffeic acid , gallic NDGA, a plant polyphenol derived from Lama divaricata, is a acid ester, quercetin, and catechin, were effective for suppression of lipid-solublc antioxidant and an inhibitor of lipoxygenases from a II .o.-indiimi cytotoxicity in these assay systems. In contrast, neither wide variety of sources (9) (Fig. 1). Various biological effects of ferulic acid ester-bearing o-methoxyphenol structure nor a-tocopherol NDGA have been reported. It was found to suppress 12-O-tetrade- were effective, indicating that o-dihydroxy or its equivalent structure in canoylphorbol-13-acetate-induced chromosomal aberration in human flavonoids is essential for the protection. There are many reports describ ing that polyphenols act as prooxidants in the presence of metal ions. Our lymphocytes (10) and ^-O-tetradecanoylphorbol-LVacetate-stimu- results suggest, however, that they act as antioxidants in the cells, when no lated protein kinase C activity in LLC-PK1 cells (11). Antimutagenic metal ions are added to the medium. activity in the Ames test and antitumorigenic activity in Sencar mice were also reported (12). We found that NDGA effectively suppressed Introduction H,O, and /-butylhydroperoxide-induced cytotoxicity (7, 13). To Hydroperoxides such as H2O2 and lipid hydroperoxides have been the best of our knowledge, this is the first evidence that a plant poly directly inhibits hydroperoxide-induced cytotoxicity. Tetrame- implicated as mediators of cellular injuries in a variety of clinical conditions including cancer. In particular, there are many reports thoxynordihydroguaiaretic acid, an NDGA analogue bearing no phe nolic OH (Fig. 1). showed no preventive effect against H2O2-induced describing the cytotoxic effects of H2O2 on mammalian cells, includ ing viability, DNA lesions, mutation, chromosomal aberration, and cytotoxicity, indicating that phenolic OH is essential for its activity. morphological transformation (1-7). Occurrence of such oxidative When the cells were treated with same amount of caffeic acid, ferulic cellular injuries itself indicates deficiency of cellular antioxidants acid, and their methyl , only caffeic acid methyl ester prevented such as a-tocophenol, ascorbic acid, glutathione, catalase, Superoxide the cytotoxicity of H2O2 (14) (Fig. 1). This result indicates that dismutase, and glutathione peroxidase in the peculiar conditions. One esterification made caffeic acid effective for protection and that sub possible way to prevent oxidative cell damage might be to take dietary stitution of one methoxy for the corresponding phenolic OH of caffeic acid methyl ester diminishes the activity. In other words, o-dihydroxy antioxidants that are effective inside of cells. Plant foods contain a large amount of polyphenols whose antioxidative activities have been (catechol) structure seems to be essential for prevention, and it seems proved in various chemical assay systems. There have been, however, difficult for polyphenols bearing free carboxyl groups to be incorpo a limited number of reports indicating that these polyphenols directly rated into the cells or the cell membranes because of electrostatic suppress oxidative damage in biological assay systems. We tried to repulsion owing to the negative charge of the carboxyl group and the find suppressive effects of dietary polyphenols against mammalian membrane phospholipids. The same observation of difference in the and bacterial cytotoxicity induced by H2O2. suppressive effects applies to gallic acid and its three esters (Fig. 2). The protective activity of methyl, propyl, and lauryl gallate increased Mammalian Assay Systems in that order (Fig. 3), and gallic acid showed no protective effects, indicating that the affinity of each polyphenol for the cell membrane Cytotoxic effects of H2O2 and protective effects of polyphenols is critical for protection (14). Since caffeic acid ester and ferulic acid against H2O,-induced cytotoxicity were assessed by a colony forma ester have inhibitory effects on lipoxygenase at the same degree (15). tion assay, one of the most reliable methods of assessing cytotoxic it is concluded that suppression of H2O2-induced mammalian cyto effects (8). In our experiments we avoided direct reactions of poly toxicity cannot to be ascribed to lipoxygenase inhibition. Flavonoids with H2O2 in the medium in order to evaluate the real effects are another series of polyphenols found in a wide variety of plant of the polyphenols in the cell or the cell membrane. Chinese hamster sources as vegetables, herbs, nuts, and teas. Various chemical and lung fibroblast V79 cells were seeded in 60-mm Petri dishes (200 biochemical effects of flavonoids such as free scavengers (16) cells/dish) and incubated in 5 ml of MEM2 supplemented with 10% and inhibitors of lipoxygenase, cyclooxygenase, and lipid peroxida- heat-inactivated FBS in a humidified atmosphere of 5% CO2 in air at tion (17, 18) have been reported. But there have been few reports that 37°C.After the medium was changed to 5 ml of MEM free of FBS, flavonoids directly suppress H2O,-induced mammalian cytotoxicity. a polyphenol solution in ethanol (up to 40 fj.1) was added to the In our assay system the flavonoids quercetin, kaempferol, catechin, medium, and the cells were incubated for 4 h. After being washed and taxifolin (Fig. 2) suppressed cytotoxicity of H2O2 (19). Quercetin with 4-(2-hydroxyethyl)-l-pipcrazineethanesulfonic acid-buffered sa and kaempferol showed protective effects at concentrations >5 /¿M. line (pH 7.3), the cells were treated with H2O2 (Its concentration, On the other hand, much higher concentrations of catechin and taxi usually 40-80 JU.M,depended on the conditions of the cells) in 5 ml of folin were necessary to prevent the cytotoxicity of H2O2. This differ ence might be ascribed to their structures depicted in Fig. 2 and 1 Presented al Ihe 4lh International Conference on Anticarcinogcnesis & Radiation affinity for cell membrane. Actually, quercetin is soluble in ethyl Protection. April 18-23, 1993, Baltimore, MD. -The abbreviations used are: MEM. minimum essential medium; FBS, fetal bovine acetate. On the other hand, catechin is water soluble. Bors et al, (16) scrum; NDGA, nordihydroguaiaretic acid; RMA. relative mutagenic activity. indicated that flavonoids bear three structural groups for radical- IWls

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Bacterial Assay Systems

Mutagenicity and cytotoxicity of H2O2 to Salmonella typhiimiriiim TA 104, which was developed to detect peroxides and other oxidants H3CO' (20), were investigated using the preincubation method of the Ames NDGA TMNDGA test (21, 22). The bacterial culture (1 ml) and a specific amount of gallic acid ester were incubated at 37°Cfor 30 min. After being HO HO. washed by centrifugion, the bacterial solution was incubated with H20-, at 37°Cfor 30 min. After being washed by centrifugion again, HO -¿ V-CH=CH—COOCH3 HO -CH=CH—COOH 0.1 ml of this bacterial solution was mixed with 2.5 ml of top agar and caltele acid methyl ester catfeic acid poured on agar plates. Colonies of histidine prototrophs (His ' ) were counted after incubation at 37°Cfor 2 days. Viable cells of 10s-fold H3CO diluted bacterial solution were also counted after incubation at 37°C HO-{~y-CH=CH—COOCH3 HO CH=CH— COOH for 2 days. When the culture of 5. typhimurium TA104 was pretreated with gallic acid esters before challenge with H2O2, the number of ferulic acid methyl ester ferulic acid surviving colonies increased with the dose of gallic acid esters, Fig. I. Structures of polyphcnols. The compounds with an underline show suppressive indicating that these polyphenols suppress H2O2-induced bacterial effects against H2O2-induced cytotoxicily. cytotoxicity (23). Lauryl gallate was especially effective even at 0.5 /xg/plate. In contrast to the esters, gallic acid itself showed no obvious protective effect against the cytotoxicity of H,O2. The effects of gallic acid esters on the numbers of revertants are not as obvious as the effects on survival. If we calculate the RMA by dividing the number of revertants by the number of viable cells, the values of RMA decrease with the dose of gallic acid esters. But the decrease in RMA lauryl gallate simply reflects an increase in the number of viable cells rather than a decrease in the number of revertants (23). Induction of the SOS response by H2O2 was measured in E. coli HO-/)-COOH PQ37, whose ß-galactosidaseexpression is strictly dependent on SOS )=/ response in spite of constitutive synthesis of alkaline phosphatase (24, HO 25). After treatment with polyphenols and H2O2, the culture was gallic acid incubated at 37°Cfor 2 h, and activities of both ß-galactosidase and alkaline phosphatase of each culture were measured. The SOS induc tion factor is calculated as the ratio of the activities of ß-galactosidase and alkaline phosphatase at each measuring point divided by the value of a control culture. The SOS response caused by H2O2-induced DNA lesions in E. coli mutants decreased with the addition of gallic acid esters (23). Also in this assay, lauryl gallate was most effective among the three gallic acid esters. In contrast, gallic acid itself showed no R: OH quercatin R: CH2 catechin effect. Consequently, the protective effects of gallic acid esters against R: H kaempferol R: C=O taxifolin cytotoxicity to S. typhimurium TA 104 and SOS responses in E. coli PQ37 induced by H2O2 supports the idea that the protection of the polyphenols against H2O2-induced cytotoxicity would be common in various assay systems with prokaryote in addition to eukaryote. (CH2CH2CH2CH)3CH3 CH, a-tocopherol

120 Fig. 2. Slructures of polyphcnols. The compounds with an underline show suppressive effects against H,O2-induced cytotoxicity. 100

80 scavenging potential and antioxidative potential: (a) the o-dihydroxy structure in the B ring; (b) the 2,3-double bond in conjugation with the 4-oxo function in the C ring; (c) the 3- and 5-hydroxyl groups with the 60 4-oxo function in the A and C rings (Fig. 2). Since kaempferol lacks the a group, its preventive effects against the cytotoxicity of active 40 oxygen species can be ascribed to the structural properties of b and c lauryl (C12)gallate group. On the other hand, the «-dihydroxy moiety in the B ring seems 20 propyl (C3) gallate essential for the protective effects of catechin, which lack the b and c methyl (CI(gallate groups. Since neither a-tocopherol (Fig. 2) nor ascorbic acid showed any protective effects in our assay systems, radical-scavenging activ .01 .1 1 10 100 ity is not directly related to the protection. In other words, we can Concentration of Gallic Acid Esters (,M) expect new biological activities other than those of the antioxidative vitamins. Fig. 3. Typical dose effects of gallic acid esters against H2O2-induced cytotoxicity. The V79 cells were treated with each polyphenol for 4 h and with H,O2 (60 /J.M)for 30 min. 1992s

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Inhibitory Effects against H2O2-induced DNA Strand Breakage killing and clastogenicity induced hy H,O,. Mutai. Res.. 22«:221-228. 1990. 7. Nakayama. T., Mori. K.. Tcrazawa. K., and Kawakishi. S. Comparison of the cyto in a Model System toxicity of different hydroperoxides to V79 cell. Free Radical Res. Commun.. 14: 173-178. 1991. The results obtained from the SOS chromotest described above S. Mitchell. J. B-. and Cook. J. A. Viability measurements in mammalian cell systems. suggests that polyphenols suppress H,O,-induced DNA damage in the Anal. Biochem., 179: 1-7, 1989. cells. It is well known, however, that some polyphenols show geno- 9. Kemal. C.. Louis-Flamber. P.. Krupinski-Olsen. R.. and Shorter. A. L. Reductive toxic activities in the presence of transition metal ions such as Fe'1* inactivation of soybean lipoxygenase I hy eatechols: a possible mechanism for regulation of lipoxygenase activity. Biochemistry. 26: 7064-7072, 1987. and Cu" ' in the medium and that these activities are ascribed to H2O2 10. Emeril, I., Levy, A., and Cerotti. P. Suppression of tumor promoter phorbolmyristate formed during of the polyphenols (26, 27). It is also acetate-induced chromosome breakage hy antioxidants and inhibitors of arachidonic acid metabolism. Mutât.Res.. 110: 327-335, 1983. reported that caffeic acid causes cellular and isolated DNA damage 11. Rondeau. F... Guide!. B.. Lacave. R.. Bens. M., Sraer. J.. Nagamine. Y.. Ardaillou. R.. through HjO, formation in the presence Cu"* (28, 29). Consequently, and Sraer. J-D. Nordihydroguaiaretic acid inhibits urokinase synthesis hy phorhol these polyphenols have opposite effects on the cytotoxicity of H2O2 in myristate acetate-simulated LLC-PKI cells. Biochim. Biophys. Acta. /055: 165-172. 1990. different assay systems. To determine the factors leading to the 12. Wang, Z. Y., Agarwal. R., Zhou, Z. C., Bickers. I). R.. and Mukhtar. H. Antimula- opposite results, we investigated the effects of states of Fe ions on genic and antitumorigenic activities of nordihydroguaiaretic acid. Mutât.Res., 267: H,O-,-induced DNA single-strand breakage by observing the change 153-162, 1991. 13. Nakayama. T.. Hori, K.. Osawa. T.. and Kawakishi. S. Suppression of hydrogen of the supercoiled DNA of phage xl74into the open circular form peroxide-induced mammalian cytoloxicity by nordihydroguaiaretic acid. Biosci. in a model system (30). Concerning the amount of transition metals in Biotcchnol. Biochem., 5ft: 1162-1163, 1992. living cells, such protein-bound forms as heme proteins, ferritin, and 14. Nakayama, T.. Numi. T.. Osawa. T., and Kawakishi. S. The protective role of transferin are more common than the "free" forms chelated by low- polyphenols in cytotoxicity of hydrogen peroxide. Mutât.Res., 2N1: 77-80, 1992. 15. Koshihara. Y., Neichi. T.. Murota. S.. Lao. A.. Fujimoto. Y.. and Tatsuno. T. Caffeic molecular-weight acids such as citric acid and ADP (31). There are acid is a selective inhibitor for leukotriene biosynthesis. Biochim. Biophys. Acta, 792: reports that mitochondria are major intracellular targets inactivated by 92-97, 1984. 16. Bors. W.. Heller. W.. Michael. C., and Sarán.M. Flavonoids as antioxidants: deter H2O-, (32) and that mitochondrial DNA is modified by prooxidants in mination of radical-scavenging efficiencies. Methods F.nzymol., lHf>:343-355. 1990. the aging process (33, 34). Cytochromes in mitochondria are sug 17. Uiughton. M. J.. Evans, P. J.. Moroncy. M. A.. Moult. J. R. S.. and Halliwell, B. gested as possible catalysts in these oxidative damages (35). Thus, we Inhibition of mammalian 5-lipoxygcnase and cyclo-oxygcnase by flavonoids and phenolic dietary additives. Relationship to anlioxidanl activity and lo iron ion- compared the effects of free Fe ion and cytochrome c on H2O2- reducing ability. Biochem. Pharmacol.. 42: 1673-1681. 1991. induced DNA damage. First, we found that H,O, induced DNA strand 18. DcWhalley, C V.. Rankin. S. M.. Houli. J. R. S.. Jessup. W., and Leakc. I). S. break in the presence of cytochrome c. Then, we found that caffeic Flavonoids inhibit the oxidative modification of low density lipoproteins by macro phages. Biochcm. Pharmacol., 39: 1743-1750. 1990. acid dose dependently inhibited H2O,-induced DNA strand breakage 19. Nakayama. T.. Yamada. M.. Osawa. T., and Kawakishi. S. Suppression of active in the presence of cytochrome c. In contrast, caffeic acid caused DNA oxygen-induced cytotoxicity by flavonoids. Biochcm. Pharmacol.. 44: 265-267, strand breakage in the presence of free Fe ion even without addition 1993. 20. Levin, D. E., Hollstein. M., Christman, M. F., Schwiers. E. A., and Ames. B. N. A of H2O2. Inhibition by catalase in the latter case indicates that the new SaímíHiflltitesterstrain (TAI02) with AT base pairs at the site of mutation DNA strand breakage is ascribed to H2O2 formed during autoxidation defects oxidative mutagens. Proc. Nati. Acad. Sci. USA. 79: 7445-7449. 1982. 21. Ames. B. N. Methods for detecting carcinogens and mutagens with the Stiltnont'lla: of caffeic acid. That is, antioxidative and prooxidative activity of mammalian-microsome mutagenicity lest. Muta!. Res.. .?/: 347-364. 1975. caffeic acid toward H,O,-induced DNA strand breakage depends on 22. Yahagi, T.. Nagao. M.. Scino. Y.. Matsushima. T.. Sugimura. T.. and Okada. M. the state of the Fe ion in the medium. The protective effects of the Mutagenicilies of W-nitrosoamines on Sulnumcllii. Mutât.Res.. 4M: 121-130. 1977. polyphenols against H,O,-induced cytotoxicity in several assays de 23. Nakayama. T., Hiramitsu. M.. Osawa, T.. and Kawakishi. S. The protective role of gallic acid esters in bacterial cytotoxicity and SOS responses induced hy hydrogen scribed in this manuscript suggest, at least, that catalytic roles of free peroxide. Mutât.Res.. 303: 29-34. 1993. 24. Ouillardet. P.. Huisman. R.. D'ari. R.. and Hofnung. M. SOS chromolcst. a direct metal ions in autoxidation of polyphenols would not be critical in the assay of induction of an SOS function in kscìicrìchiacoliK-12 to measure genotox- cells. icity. Proc. Nati. Acad. Sci. USA, 79: 5971-5975, 1982. 25. Ouiltardet. P.. and Hofnung. M. The SOS chromotest, a colorimetrie bacterial assay Acknowledgments for genotoxins: procedures. Mutai. Res.. 147: 65-78. 1985. 26. Stich, H. F. The beneficial and hazardous effects of simple phenolic compounds. All of our experiments described in this paper were carried out in Labora Mutât.Res., 259: 307-324. 1991. tory of Food Chcmislry, Department of Food Science and Technology, Nagoya 27. llanham. A. F., Dunn. B. P., and Stich, II. F. Claslogcnic activity of caffeic acid and its relationship to hydrogen peroxide generated during autcxixidalion. Mutai. Res.. University. I would like to acknowledge Drs. Shunro Kawakishi and Toshihiko //6: 333-339, 1983. Osawa in the laboratory for support of this research and valuable suggestions. 28. Yamada, K.. Shirahata, S.. Murakami. H.. Nishiyama. K.. Shinohara. K.. and Omura. H. DNA breakage hy phenyl compounds. Agrie. Biol. Chcm., 49: 1423-1428, 1985. References 29. Inoue. S.. Ito, K.. Yamamoto. K.. and Kawanishi, S. Caffcic acid causes metal- dependent damage to cellular and isolated DNA through H,O, formation. Carcino 1. Mello Filho, A. ('., and Meneghini. R. In viva formation of single-strand breaks hy genesis (Lond.). 13: 1497-1502. 1992. hydrogen peroxide is mediated hy the Haber-Weiss reaction. Biochim. Biophys. Ada. 30. Nakayama. T.. Kuno. T., Hiramitsu. M.. Osawa. T.. and Kawakishi. S. Antioxidative 781: 56-63, 1984. and prooxidative activity of caffeic acid toward H,O:-induccd DNA strand breakage 2. Mello l;ilho. A. ('.. and Meneghini. R. Protection of mammalian cells hy o-phcnan- dependent on the state of the Fe ¡oninthe medium. Biosci. Biotechnol. Biochcm., 57: throline from lethal and DNA-damaging effects produced hy active oxygen species. 174-176, 1993. Biochim. Biophys. Acta. 847: 82-X9. 1985. 31. Halliwell. B.. and (iulteridge. J. M. C. Role of free radicals and catalytic metal ions 3. Cantoni. O.. Sestili. P.. Cattahcni. F.. Bellomo, G., Pou, S., Cohen. M., and Cerotti, in human disease: an overview. Methods F.n/.ymol., AS6: 1-85. 1990. P. Calcium chelalor (Juin 2 prevents hydrogen-peroxide-induced DNA breakage and 32. Hyslop, P. A.. Hinshaw, D. B., Halsey. W. A.. Jr., Schraufstatter. I. U., Saucrhehcr, cytotoxicity. Eur. i. Biochem., /«2:2(19-212, 1989. R. D., Spragg, R. G., Jackson, J. H.. and Cochrane. C. Ci. Mechanisms of oxidant- 4. Nassi-Calo, L., Mello Filho, A. C., and Meneghini. R. o-Phenanthroline protects medialcd cell injury. J. Biol. Chem.. 26.?: 1665-1675, 1988. mammalian cells from hydrogen peroxide-induced gene mutation and morphological 33. Richter. C.. Park. J-W.. and Ames. B. N. Normal oxidative damage to mitochondrial transformation. Carcinogenesis (I,ond.), If): 1055-1057. 1989. and nuclear DNA is extensive. Proc. Nati. Acad. Sci. USA, «5:6465-6467, 1988. 5. Shacter. H., l.opcz. R. L.. Bcecham. lì.J.. and Janz. S. DNA damage induced hy 34. Hayakawa. M.. Torii. K.. Sugiyama. S.. Tanaka. M.. and Ozawa. Biochcm. Biophys. phorhot ester-stimulated neutrophils is augmented hy extracellular cofactors. Role of Res. Commun., 179: 1023-1029, 1991. histidine and metals. J. Biol. C'hem.. 265: 6693-6699, 1990. 35. Radi. R.. Turrens. J. F., and Freeman. B. A. Cvlochrome c-catalvzed membrane lipid 6. Tachón. P. Intracellular iron mediates the enhancing effect of histidine on the cellular peroxidalion by hydrogen peroxide. Arch. Biochem. Biophys.. 2Wi: 118-125, 1991.

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Tsutomu Nakayama

Cancer Res 1994;54:1991s-1993s.

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