Suppression of Hydroperoxide-Induced Cytotoxicity by Polyphenols1
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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 hydrogen peroxide (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 ester, 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 phenol 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 esters, 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 phenols 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 radical 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 Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1994 American Association for Cancer Research. si'i'i'RissioN BYPOI.YIMII-:NOI.S 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