- Email: [email protected]
Initial in vitro toxicity testing of functional foods rich in catechins and anthocyanins in human cells
Toxicology in Vitro 17 (2003) 723–729 www.elsevier.com/locate/toxinvit
Initial in vitro toxicity testing of functional foods rich in catechins and anthocyanins in human cells M. Gleia,*, M. Matuscheka, C. Steinera, V. Bo¨hmb, C. Persinc, B.L. Pool-Zobela a
Department of Nutritional Toxicology, Institute of Nutrition, Friedrich Schiller University Jena, Dornburger Str. 25, 07743 Jena, Germany b Department of Human Nutrition, Institute of Nutrition, Friedrich Schiller University Jena, Dornburger Str. 25, 07743 Jena, Germany c Kampffmeyer Food Service GmbH, Trettaustr. 32-34, 21107 Hamburg, Germany Accepted 30 May 2003
Abstract Functional foods need to be assessed for beneficial effects to support claims, but also for toxic effects. This report describes two examples of how complex food samples are initially characterized in human cells in vitro. Water extracts of green tea (GT) and black carrots (BC) were analyzed for key ingredients (catechins and anthocyanidins, respectively). Extracts, reconstituted mixtures of the major ingredients or individual compounds [()-epigallocatechin gallate or cyanidin, respectively] were evaluated in parallel using human colon cells (HT29 clone 19A). End points of cytotoxicity included determination of membrane integrity, proliferation inhibition, and genetic damage. Cells were pretreated with plant compounds at sub-toxic concentrations, and their resistance to toxicity of H2O2 was evaluated as a parameter of protection. The extracts reduced cell viability (BC) and cell growth (BC, GT) and caused DNA damage (BC, GT). They were more toxic than their key ingredients. Neither GT-samples nor BC protected against H2O2-induced DNA damage, whereas cyanidin did. In vitro analysis of extracts from functional foods firstly aims at defining the sub-toxic concentrations at which protective activities are then further characterized. It also allows comparing responses of complex samples and individual compounds, which is important since effects from protective food ingredients can be masked by accompanying toxic components. # 2003 Elsevier Ltd. All rights reserved. Keywords: In vitro toxicology; Green tea; Black carrot; DNA damage; HT29 clone 19A
1. Introduction Food safety policy must be based on a comprehensive and integrated approach of risk analysis throughout the food chain with three main components, risk assessment, risk management and risk communication (EC, 2000). The in vitro systems of toxicology are widely used for screening chemicals, for generating toxicological profiles and sporadically for risk assessment (Eisenbrand et al., 2002). The range of food-associated compounds which are testable includes natural ingredients, compounds formed endogenously after an exposure, permissible supplements, additives, residues and contaminants. An important application of in vitro systems is to obtain mechanism-derived information by studying different cells or tissue and target specific * Corresponding author. Tel.: +49-3641-949674; fax: +49-3641949672. E-mail address: [email protected] (M. Glei). 0887-2333/$ - see front matter # 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0887-2333(03)00099-7
effects. With in vitro systems it is possible to assess cytotoxicity, cellular responses, and to perform toxicokinetic modeling. They are valuable for characterizing hazards to humans through extrapolation of effects from the in vitro to the in vivo situation and from animals to men (Pool-Zobel et al., 1994). The end points of in vitro toxicology systems are numerous and a widely used approach is to determine genotoxicity for identifying carcinogenic potential of chemicals (Committee on Mutagenicity of Chemicals in Food, 2000, Ashby and Tennant, 1991). Additionally, in vitro systems are a powerful tool for biomarker development (Eisenbrand et al., 2002). Functional foods need to be evaluated from an additional point of view. In general, these are foods which provide a health benefit beyond the supply of traditional nutrients. They are expected to be beneficial because they contain components such as vitamins, polyphenols, dietary fibers or may not contain compounds with adverse effects. Polyphenols are classified as phytopro-
724
M. Glei et al. / Toxicology in Vitro 17 (2003) 723–729
numerous mechanisms, mainly derived from their high antioxidative properties (Seeram and Nair, 2002).
tectants on account of antioxidative properties and dietary fibers improve digestion and intestinal health (Glei and Pool-Zobel, 2001; Pool-Zobel et al., 1997, 2002). Moreover dietary fibre may counteract endogenous toxicity by scavenging intermediates, enhancing stool transit or by inducing chemoresistance (Ebert et al., 2001; Wollowski et al., 2001). Therefore, functional foods need to be assessed for beneficial properties, e.g. to find evidence which will support claims of functional effects. Also, for new functional foods which are processed to contain high levels of phytoprotectants it is necessary to exclude that these will act toxically when ingested in larger amounts. This is especially important since controversial results on the beneficial properties of phytoprotectants are also available. Examples are studies showing that polyphenols produce free radicals (Long et al., 2000), act toxic (Glei et al., 2002) and are prooxidative (Metodiewa et al., 1999). The experimental in vitro analysis of biological activities of whole foods will therefore serve several purposes. On the one hand, the determination of toxicity can be used as a tool to define concentrations at which chemoprotective effects can be further characterized. On the other hand, cells can be treated with sub-toxic concentrations of the compounds to find new cellular responses and among them mechanisms of potential chemoprotection. In addition, the knowledge of the balance of these activities induced by individual compounds and by complex mixtures from the foods will aid in disclosing the balance of beneficial and non beneficial properties. In this context, the aim of our studies reported below was to comparatively investigate two whole foods, a catechin-rich green tea (GT1) and an anthocyanin-rich plant juice [prepared from black carrots (BC)] for cytotoxicity, genotoxicity and protective effects in human colon cells. In line with our general strategy, we additionally investigated the pure compounds cyanidin and ()-epigallocatechin gallate (EGCG). Cyanidin is the aglycone of the main anthocyanins in BC; EGCG is the main green tea-catechin (Graham, 1992). Both groups of compounds are polyphenols and considered to be beneficial on account of
2. Materials and methods 2.1. Human tumour cell line HT29 clone 19A The human colon cell line HT29 was established in 1964 by J. Fogh (Memorial Sloan Kettering Cancer Centre, New York) (Rousset, 1986). The clone 19A was terminally differentiated with 5 mm sodium butyrate, and characterised by Augeron and Laboisse (Augeron and Laboisse, 1984). Our cells were a kind gift of Laboisse to G. Rechkemmer (Federal Research Centre, Karlsruhe), from whom we obtained them in passage 12. Cells were maintained in stocks at 140 C, thawed and grown in tissue culture flasks with DMEM (Dulbecco’s Modified Eagle Medium, Gibco BRL, Eggenstein, Germany) supplemented with 10% foetal calf serum and 1% penicillin/streptomycin at 37 C in a (95%) humidified incubator (5% CO2). This culture containing 45–60106 cells was trypsinized with 1–1.5 ml trypsin/versene (1:10 v/v) for a maximum of 10 min and sub-cultivated at a dilution of 1:8 in T75 flasks with supplemented DMEM. Two medium changes occurred on days 2 and 5 with 15 ml DMEM. 2.2. Treatment with chemicals The tea polyphenols [()-epicatechin (EC), ()-epigallocatechin (EGC), ()-epicatechin gallate (ECG), and ()-epigallocatechin gallate (EGCG)] were purchased from Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany. Cyanidin chloride was bought from ROTH GmbH Co., Karlsruhe, Germany. All chemicals were of analytical grade or compiled with the standards needed for tissue culture experiments. The green tea ethyl acetate extract (GT2) was from LKT Labs, St. Paul, USA. The GT1 and the plant juice prepared from BC were provided by Kampffmeyer Food Service GmbH, Hamburg, Germany. The aqueous extracts/
Table 1 Influence of different GT1-pretreatments (37 C, 30 min–48 h) on H2O2-induced (5 min, 4 C) DNA damage (strand breaks expressed as % fluorescence in tail) with and without a 30 min repair period in HT29 clone 19A cells Treatment
300 GT1 pretreatment, 2 mm, 37.5 mm H2O2
300 GT1 pretreatment, 2 mm, 150 mm H2O2
24 h GT1 pretreatment, 2 mm, 100 mm H2O2
24 h GT1 pretreatment, 20 mm, 100 mm H2O2
48 h GT1 pretreatment, 2 mm, 100 mm H2O2
n=3
n=3
n=13
n=3
n=3
Period 0 min
30 min
0 min
30 min
0 min A
30 min
0 min
30 min
0 min
30 min
A
Control Mean 44.21 S.D. 5.91
38.86 5.49
53.37 4.34
46.8 3.33
42.37 14.31
27.49 13.18
60.49 3.29
39.86 13.23
51.15 18.23
36.98 16.28
GT
36.06 9.82
48.77 1.21
43.11 4.45
42.15B 12.2
21.72B 9.15
57.44 15.98
44.74 15.57
38.83 14.25
28.58 8.27
Mean 44.81 S.D. 6.65
The same capital letter indicates significant differences between the groups: AP<0.05, BP<0.001 unpaired t-test.
M. Glei et al. / Toxicology in Vitro 17 (2003) 723–729
solutions of GT1, GT2 and BC were prepared by mixing the samples with cell culture medium (10% w/v), ultrasonic treatment for 4 min and two centrifugation steps (10 min, 4000 g; 10 min, 16,000 g) to obtain clear, coloured solutions. The aqueous extracts/solutions were prepared fresh and stored at 4 C in the dark until utilization. HT29 clone 19A cells were incubated with cyanidin, catechins (dissolved in DMSO, Sigma, Taufkirchen, Germany) or GT and BC extracts at 37 C in suspension culture (short-term tests, 15–60 min) or for longer duration (24–72 h) using tissue culture protocols. H2O2 (obtained as a 30% aqueous solution from Merck, Darmstadt, Germany) was used in some experiments as genotoxic model substance. Cells were treated with H2O2 on freshly prepared slides on ice for 5 min (Pool-Zobel et al., 1999). Each experiment was performed independently at least three times. 2.3. Analytical characterisation of food extracts The catechin content of the tea extracts (Table 2) was assessed by using HPLC with UV detection (Schlesier et al., 2001). The quantity of GT extract investigated was related to its molar content of EGCG and the concentrations are therefore expressed as ‘‘EGCG-equivalents’’. The anthocyanin content of the BC extract was analysed by using HPLC with diode array detection. Three cyanidin-3-glycosides, bound to various other sugars, were identified (Harborne, 1976). On their basis (13.54 0.08 g/l) the quantities of BC extract are expressed as ‘‘cyanidin-3-glycoside-equivalents’’ (cy-3-gly-equivalent).
725
5 min, followed by addition of 40 ,6-diamidino-2-phenylindole dihydrochloride (DAPI) (Sigma-Aldrich, Taufkirchen, Germany). After 30 min, DNA content, reflecting the remaining cells, was detected by fluorimetrical analysis with Ex/Em 360/450 nm. Mean values (three determinations per experiment, at least three experiments) were recorded for final evaluation. 2.5. Detection of cytotoxicity and DNA damage Cell number and viability were determined with the trypan blue exclusion test, and/or with double staining using a mixture of fluoresceindiacetate (FDA) and ethidiumbromide (EB) solutions (5 mg FDA from SigmaAldrich Chemie GmbH, Taufkirchen dissolved in 1 ml acetone, Fisher Scientific, Schwerte; 200 mg EB from Sigma-Aldrich Chemie GmbH, Taufkirchen dissolved in 1 ml PBS). DNA damage was measured using single cell microgelelectrophoresis, also named ‘‘comet assay’’ on account of the comet like-images which are evaluated as result. For this, cells were embedded into agarose on microscopical slides, lysed and subjected to electrophoresis (Singh et al., 1988; Pool-Zobel et al., 1997). Images of damaged DNA (‘‘comets’’) are scored after staining with SYBR-Green (Sigma-Aldrich Chemie GmbH, Taufkirchen). The proportions and extent of DNA migration were determined for 50 DNA spots per slide. The intensity of fluorescence in the comet tail expressed as ‘‘% fluorescence in tail’’ is the evaluation criteria presented in the graphs. 2.6. Statistical evaluations
2.4. Determination of the cell proliferation Proliferation of colon cells were determined in 96-well microtiter plates. 24 h after seeding, the cells were treated by adding various concentrations of GT (0–120 mm EGCG equivalents) or BC (0–1400 mm cy-3-gly-equivalents) to the medium. To measure surviving cells as an indirect marker of cell proliferation, DNA was isolated by fixing and permeabilising the cells with methanol for Table 2 Composition of two different green tea sources—content of polyphenols (mg/g powder) in a normal green tea powder (real aqueous extract, GT1) and a commercial one (ethyl acetate extract powder, GT2, dissolved in medium) after the same preparation steps Polyphenol
()-Epigallocatechin-3-gallate, EGCG ()-Epigallocatechin, EGC ()-Epicatechin-3-gallate, ECG ()-Epicatechin, EC (+)-Catechin, C Cumulative
Data in the figures are mean values S.D. Unless otherwise stated, these means were calculated from the means of triplicate replicates obtained in at least three independent experiments. Statistical evaluation was performed with the GraphPad Prism Version 3.02 for Windows (GraphPad Software, San Diego, CA, USA, www.graphpad.com). Depending on sample size and type of experiment, one-way ANOVA was used to determine significance of the experimental variables. The significance of differences between individual treatment groups was determined with paired and unpaired t-tests, as appropriate.
GT1, aqueous GT2, ethyl extract acetate extract
3. Results
mg/g (%)
mg/g (%)
3.1. Cytotoxicity
98 (37) 70 (27) 40 (15) 37 (14) 19 (7) 264 (100)
335 (40) 95 (11) 95 (11) 295 (35) 20 (2) 840 (100)
The BC extract clearly reduced the viability of HT29 clone 19A cells in a dose related manner (dose range up to 2790 mm cy-3-gly-equivalents) and concentrations exceeding 45 mm cy-3-gly-equivalent were clearly cytotoxic (Fig. 1). Virtually identical results were obtained
726
M. Glei et al. / Toxicology in Vitro 17 (2003) 723–729
with the two staining procedures, one using the double labelling with FDA/EB (Fig. 1A) and the other with trypan blue (Fig. 1B). In contrast, the GT1-extract was not effective in the tested concentration range, since amounts up to 100 mM EGCG-equivalent GT1-extract had no effect on the cell viability (results not shown). Also cyanidin, the aglycone of the main anthocyanins in BC, was ineffective up to 250 mm (results not shown). 3.2. Proliferation Both extracts significantly inhibited proliferation of HT29 clone 19A cells (dose range up to 1400 mm cy-3gly equivalents, up to 120 mm ECGC equivalents, 24 and 48 h incubation), with an EC50 of about 30–50 mm for GT1 (Fig. 2A) and 45 mm for BC (Fig. 2B). The control compound cyanidin (up to 250 mm) did not inhibit the cell growth, whereas incubation with EGCG resulted in an EC50 of 60 mm (48 h) and 85 mm (24 h) (results not shown). 3.3. Genotoxicity Fig. 3 shows that BC and GT1 extracts are toxic by inducing genetic damage. After 24 h incubation with 88–350 mm cy-3-gly equivalents (Fig. 3A) and 40–100 mm
Fig. 1. Cell viability after 24 or 48 h incubation with BC: (a) by FDA/ EB double staining; (b) by trypan blue exclusion. BC decreased significantly the viable cell number: (a) *P <0.05, **P <0.01, ***P <0.001 paired t-test; (b) 24 h, *P <0.05, ***P <0.001 unpaired ttest, 48 h, *P<0.05 paired t-test, n=3.
EGCG equivalents (Fig. 3B) a significant strand breakinduction was observed in HT29 clone 19A cells. 3.4. Protective effects H2O2, an endogenous genotoxic risk factor, causes DNA damage by decomposing to the hydroxyl radical (OH.). This may result in DNA instability, mutagenesis and ultimately carcinogenesis. Catechins and anthocyanins are efficient antioxidants, which could also prevent H2O2-induced DNA damage. Table 1 shows the influence of different GT1 pre-treatments (2, 20 mm, 30 min, 24, 48 h) on H2O2 (37,5, 100, 150 mm)-induced DNA damage and on the persistence of damage for 30 min post-treatment. We specifically chose a short incubation period (30 min) during which damage by relatively high doses of H2O2 ( 100 mm) is only marginally repaired to allow detecting the additional GT effect. However, it is obvious that GT1 did not protect against the genotoxicity of H2O2. Damage level and persistence were only modified insignificantly. To assess the relative protective efficacy of our aqueous GT-extract (GT1) we compared it with EGCG, the
Fig. 2. Cell proliferation, measured as a function of DNA content, after 24 and 48 h incubation with GT1 (a) and BC extract (b). GT1 and BC decreased significantly the cell number (*P< 0.05, **P<0.01, ***P <0.001 t-test against control).
M. Glei et al. / Toxicology in Vitro 17 (2003) 723–729
727
Fig. 4. Influence of different polyphenol sources (GT1—aqueous extract, Syn-GT—mixture of the most abundant polyphenols of GT1, GT2—commercial ethyl acetate extract, EGCG—main green tea catechin) (37 C, 24 h, 2 mm EGCG equivalent) on H2O2-induced (5 min, 4 C) DNA damage (strand breaks expressed as % fluorescence in tail) with and without a 30 min repair period in HT29 clone 19A cells. The same capital letter indicates significant differences between the groups: A,CP <0.05, BP<0.001 unpaired t-test.
Fig. 3. DNA damage after 24 h treatment with BC (a) and GT1 extract (b). BC (P <0.01, one way ANOVA, *P <0.05, ***P <0.001 Dunnett’s multiple comparison test against control, n=3) and GT1 (*P<0.05 unpaired t-test against control, n=4) induced significantly DNA strand breaks.
main catechin, with a comparable mixture (Syn-GT) of the 5 most abundant polyphenols of GT1 (Table 2), and with a commercial green tea extract (GT2) (Fig. 4). In fact, none of the four tested samples (standardised to 2 mm EGCG-equivalent) were able to reduce the genotoxicity of H2O2 per se. However, when regarding the impact of GT on persistence of H2O2-induced damage, it appears as if the complex samples GT1 and GT2 enhance repair, since they caused a significant reduction of DNA damage during a 30 min post incubation period. The pre-treatment of HT29 clone 19A cells with BCextract and cyanidin had quite different effects on H2O2induced DNA damage (Fig. 5). Short term incubation (15-30 min) with cyanidin (100 mm) significantly decreased the genotoxicity of H2O2 (18.75–150 mm). However, the similar treatment with the complete BC extract (BC: 45 mm cy-3-gly-equivalent) enhanced the DNA-damaging effect.
4. Discussion Anthocyanins are a group of polyphenolic flavonoids which occur ubiquitously in foods of plant origin (Hollman and Katan, 1999). They are implicated in
Fig. 5. Effect of 15–30 min preincubation with BC (45 mm cy-3-glyequivalent) and cyanidin (100 mm) on H2O2-induced (0–150 mm) DNA damage. There was a clear H2O2 dose–effect relationship (one-way ANOVA, P <0.001). Cyanidin pretreatment reduced the level of induced fluorescence in tail (BC: *P <0.05, **P <0.01, unpaired t-test, n=6, Cyanidin: P<0.05, P <0.01, paired-test, n=8).
preventing diseases related to oxidative stress, such as coronary heart disease (Hertog et al., 1993) and cancer (Stoner and Mukhtar, 1995) maybe due to their antioxidant properties (Kandaswami and Middleton 1994; Sellappan and Akoh, 2002). Pool-Zobel et al. (1999) demonstrated that isolated compounds and complex samples of Aronia melanocarpa are powerful antioxidants in vitro. Also, they acted intracellularly by reducing H2O2 induced DNA strand breaks in human colon cells. Knekt et al. (2002) used a dietary history method with over 10,000 men and women and showed, that a high intake of different flavonoids was associated with lower mortality from ischemic heart disease, lower lung and prostata cancer as well as lower asthma incidence. The plant extract we investigated was prepared from BC at GNT International, Vaals, Netherlands. BC have been cultivated and eaten for more than 3000
728
M. Glei et al. / Toxicology in Vitro 17 (2003) 723–729
years. They are considered to be rich in anthocyanins. Harborne (1976) described their anthocyanin species, which were identified to be the cyanidin 3-sinapoylxylosylglucosylgalactoside (Harborne et al., 1983). Tea is one of the most popular beverages consumed worldwide. Among all tea produced in the world, 20% is green tea, which is commonly consumed in Asian countries where the tea is a major beverage (Yang, 1999). The catechins, (EGCG, EGC, ECG, EC, C) are major active constituents of green tea. These compounds have been shown to be highly active as anticarcinogens in vivo using several rodent test systems (Katiyar and Mukhtar, 1996) and epidemiological studies on the consumption of green tea indicate a protection against stomach (Yu et al., 1995) and colon (Kato et al., 1990) cancers. Several mechanisms have been identified by which catechins can operate as anticarcinogens, including inhibition of cytochrome P-450 activation of carcinogen-precursors (Wang et al., 1988), inhibition of angiogenesis (Cao and Cao, 1999), scavenging of reactive oxygen species (Rice-Evans et al., 1996) or electron transfer from catechins to ROSinduced radical sites on the DNA (Anderson et al., 2001). Besides, Benelli et al. (2002) concluded that green tea polyphenols appear to be non-conventional inhibitors of metallo and serine proteases involved in matrix degradation. In this study, we observed distinctly different effects between the complex extracts and of their isolated ingredients on several parameters of in vitro toxicology (proliferation, cytotoxicity and genotoxicity). The major differences are apparent when comparing the effects of cyanidin to those of BC. Whereas the aglycone cyanidin was protective as expected, on the basis of previous studies, the anthocyanin-rich BC extract did not protect from H2O2-induced DNA damage. Instead, BC clearly enhanced H2O2-induced genotoxicity. Apparently the complex plant extract contains additional components, which add on to the H2O2-mediated activities. At the investigated in vitro concentrations (there is no information available on the actual concentration in the colon), the protective activity of cyanidin was not sufficient to counteract these toxic responses. It will be important in the future to identify potential genotoxic candidates and to evaluate these for their relative contribution to the observed genotoxic effects. Furthermore, we are aware that to discern between the effects of the original compounds and their metabolites, the stability of test compounds must be considered in further investigations (Hong et al., 2002). In conclusion, these findings clearly point to the necessity of performing safety evaluations for individual complex plant extracts, before using them as food supplements. It is not sufficient to rely on former findings of beneficial effects by key ingredients, without toxicological assessment of the complex plant product
itself. Methods of in vitro toxicology will be useful in this context. By using the methods, it is possible to define the sub-toxic concentrations of extracts from functional foods at which protective activities are then further characterized. Additionally, it is possible to compare responses of complex samples and of individual compounds, which is important since effects from protective food ingredients can be masked by accompanying toxic components.
References Augeron, C., Laboisse, C.L., 1984. Emergence of permanently differentiated cell clones in a human colonic cancer cell line in culture after treatment with sodium butyrate. Cancer Research 44, 3961– 3969. Anderson, R.F., Fisher, L.J., Hara, Y., Harris, T., Mak, W.B., Melton, L.D., Packer, J.E., 2001. Green tea partially protect DNA from OH radical-induced strand breaks and base damage through fast chemical repair of DNA radicals. Carcinogenesis 22, 1189–1193. Ashby, J., Tennant, R.W., 1991. Definitive relationships among chemical structure, carcinogenicity and mutagenicity for 301 chemicals tested by the U.S. NTP. Mutation Research 257, 229–306. Benelli, R., Vene, R., Bisacchi, D., Garbisa, S., Albini, A., 2002. Antiinvasive effects of green tea polyphenol epigallocatechin-3-gallate (EGCG), a natural inhibitor of metallo and serine proteases. Journal of Biological Chemistry 383, 101–105. Cao, Y., Cao, R., 1999. Angiogenesis inhibited by drinking tea. Nature 398, 381. Committee on Mutagenicity of Chemicals in Food, Consumer Products and the Environment COM UK. Guidelines on a Strategy for Testing of Chemicals for Mutagenicity. December 2000. Ebert, M.N., Beyer-Sehlmeyer, G., Liegibel, U.M., Kautenburger, T., Becker, T.W., Pool-Zobel, B.L., 2001. Butyrate-induced activation of glutathione S-transferases protects human colon cells from genetic damage by 4-hydroxynonenal. Nutrition and Cancer 41, 156–164. EC (2000) EC White paper on food safety COM (1999) 719 final, January 2000. Eisenbrand, G., Pool-Zobel, B.L., Baker, V., Balls, M., Blaauboer, B.J., Boobis, A., Carere, A., Kevekordes, S., Lhuguenot, J.-C., Pieters, R., Kleiner, J., 2002. Methods of in vitro toxicology. Food and Chemical Toxicology 40, 193–236. Glei, M., Latunde-Dada, G.O., Klinder, A., Becker, T.W., Hermann, U., Voigt, K., Pool-Zobel, B.L., 2002. Iron-overload induces oxidative DNA damage in the human colon carcinoma cell line HT29 clone 19A. Mutation Research 519, 151–161. Glei, M., Pool-Zobel, B.L., 2001. Krebspra¨vention und Erna¨hrung: Riskoverminderung durch eine lebenslange gesunde Erna¨hrung. In: Friedenthal-Haase, M., Meinhold, G., Schneider, K., Zwiener, U. (Eds.), Studien zur Pa¨dagogik, Andragogik und Gerontagonik, Alt werden-alt sein, Band 50. Peter Lang, Europa¨ischer Verlag der Wissenschaften, pp. 99–110. Graham, H.N., 1992. Green tea composition, consumption, and polyphenol chemistry. Preventive Medicine 21, 334–350. Harborne, J.B., 1976. A unique pattern of anthocyanins in Daucus carota and other Umbelliferae. Biochemical Systemics and Ecology 4, 31–35. Harborne, J.B., Mayer, A.M., Bar-Nun, N., 1983. Identification of the major anthocyanin of carrot cells in tissue cultures as cyanidin 3(sinapoylxylosylglucosylgalactoside). Zeitschrift Naturforschung 38c, 1055–1056. Hertog, M.G.L., Feskens, E.J.M., Hollman, P.C.H., Katan, M.B.,
M. Glei et al. / Toxicology in Vitro 17 (2003) 723–729 1993. Dietary antioxidant flavonoids and risk of coronary heart disease: the Zutphen Elderly Study. The Lancet 342, 1007–1011. Hollman, P.C.H., Katan, M.B., 1999. Dietary flavonoids: intake, health effects and bioavailability. Food and Chemical Toxicology 37, 937–942. Hong, J., Lu, H., Meng, X., Ryu, J.-H., Hara, Y., Yang, C.S., 2002. Stability, cellular uptake, biotransformation, and efflux of tea polyphenol ()-epigallocatechin-3-gallate in HT-29 human colon adenocarcinoma cells. Cancer Research 62, 7241–7246. Kandaswami, C., Middleton, E., 1994. Free radical scavenging and antioxidant activity of plant flavonoids. Advances in Experimental Medicine and Biology 366, 351–376. Katiyar, S.K., Mukhtar, H., 1996. Tea in chemoprevention of cancer: epidemiologic and experimental studies (review). International Journal of Oncology 8, 221–238. Kato, I., Tominaga, S., Matsuura, A., Yoshii, Y., Shirai, M.E.A., 1990. A comparative case-control study of colorectal cancer and adenoma. Japanese Journal of Cancer Research 81, 1101–1108. Knekt, P., Kumpulainen, J., Ja¨rvinen, R., Rissanen, H., Helio¨vaara, M., Reunanen, A., Hakulinen, T., Aromaa, A., 2002. Flavonoid intake and risk of chronic diseases. American Journal of Clinical Nutrition 76, 560–568. Long, L.H., Clement, M.V., Halliwell, B., 2000. Artifacts in cell culture: rapid generation of hydrogen peroxide on addition of ()-epigallocatechin, ()-epigallocatechin gallate, (+)-catechin, and quercetin to commonly used cell culture media. Biochemical and Biophysical Research Communications 273, 50–53. Metodiewa, D., Jaiswal, A.K., Cenas, N., Dickancaite´, E., SeguraAguilar, J., 1999. Quercetin may act as a cytotoxic prooxidant after its metabolic activtion to semiquinone and quinoidal product. Free Radical Biology and Medicine 26, 107–116. Pool-Zobel, B.L., Lotzmann, N., Knoll, M., Kuchenmeister, F., Lambertz, R., Leucht, U., Schro¨der, H.-G., Schmezer, P., 1994. Detection of genotoxic effects in human gastric and nasal mucosa cells isolated from biopsy samples. Environmental and Molecular Mutagenesis 24, 23–45. Pool-Zobel, B.L., Bub, A., Mu¨ller, H., Wollowski, I., Rechkemmer, G., 1997. Consumption of vegetables reduces genetic damage in humans: first results of a human intervention trial with carotenoidrich foods. Carcinogenesis 18, 1847–1850.
729
Pool-Zobel, B.L., Bub, A., Schro¨der, N., Rechkemmer, G., 1999. Anthocyanins are potent antioxidants in vitro but do not reduce oxidative DNA damage within human colon cells. European Journal of Nutrition 38, 227–234. Pool-Zobel, B.L., Van Loo, J., Rowland, I.R., Roberfroid, M.B., 2002. Experimental evidences on the potential of prebiotic fructans to reduce the risk of colon cancer. British Journal of Nutrition 87, S273–S281. Rice-Evans, C.A., Miller, N.J., Paganga, G., 1996. structure-antioxidant activity relationships of flavonoids and phenolic acids. Free Radical Biology and Medicine 20, 933–956. Rousset, M., 1986. The human colon carcinoma cell lines HT-29 and Caco-2: two in vitro models for the study of intestinal differentiation. Biochimie 68, 1035–1040. Schlesier, K., Bo¨hm, V., Bitsch, R., 2001. HPLC-Methode zur Detektion von Flavonoiden und Phenolcarbonsa¨uren in Obst und Gemu¨se. Lebensmittelchemie 55, 38–39. Seeram, N.P., Nair, M.G., 2002. Inhibition of lipid peroxidation and structure-activity-related studies of the dietary constituents anthocyanins, anthocyanidins, and catechins. Journal of Agricultural and Food Chemistry 50, 5308–5312. Sellappan, S., Akoh, C.C., 2002. Flavonoids and antioxidant capacity of georgia-grown vidalia onions. Journal of Agricultural and Food Chemistry 50, 5338–5342. Singh, N.P., McCoy, M.T., Tice, R.R., Schneider, E.L., 1988. A simple technique for quantitation of low levels of DNA damage in individual cells. Experimental Cell Research 175, 184–191. Stoner, G.D., Mukhtar, H., 1995. Polyphenols as cancer chemopreventive agents. Journal of Cellular Biochemistry 22, 169–180. Wang, Z.Y., Das, M., Bickers, D.R. Mukhtar, H, 1988. Interaction of epicatechins derived from green tea with rat hepatic cytochrome P450. Drug Metabolism and Disposition 16, 98–103. Wollowski, I., Rechkemmer, G., Pool-Zobel, B.L., 2001. Protective role of probiotics and prebiotics in colon cancer. American Journal of Clinical Nutrition 73, 451s–545s. Yang, C.S., 1999. Tea and health. Nutrition 15, 946–948. Yu, G., Hsieh, C., Wang, L., Yu, S., Li, X.E.A., 1995. Green-tea consumption and risk of stomach cancer: a population-based casecontrol study in Shanghai, China. Cancer Causes Control 3, 137– 143.
Initial in vitro toxicity testing of functional foods rich in catechins and anthocyanins in human cells M. Gleia,*, M. Matuscheka, C. Steinera, V. Bo¨hmb, C. Persinc, B.L. Pool-Zobela a
Department of Nutritional Toxicology, Institute of Nutrition, Friedrich Schiller University Jena, Dornburger Str. 25, 07743 Jena, Germany b Department of Human Nutrition, Institute of Nutrition, Friedrich Schiller University Jena, Dornburger Str. 25, 07743 Jena, Germany c Kampffmeyer Food Service GmbH, Trettaustr. 32-34, 21107 Hamburg, Germany Accepted 30 May 2003
Abstract Functional foods need to be assessed for beneficial effects to support claims, but also for toxic effects. This report describes two examples of how complex food samples are initially characterized in human cells in vitro. Water extracts of green tea (GT) and black carrots (BC) were analyzed for key ingredients (catechins and anthocyanidins, respectively). Extracts, reconstituted mixtures of the major ingredients or individual compounds [()-epigallocatechin gallate or cyanidin, respectively] were evaluated in parallel using human colon cells (HT29 clone 19A). End points of cytotoxicity included determination of membrane integrity, proliferation inhibition, and genetic damage. Cells were pretreated with plant compounds at sub-toxic concentrations, and their resistance to toxicity of H2O2 was evaluated as a parameter of protection. The extracts reduced cell viability (BC) and cell growth (BC, GT) and caused DNA damage (BC, GT). They were more toxic than their key ingredients. Neither GT-samples nor BC protected against H2O2-induced DNA damage, whereas cyanidin did. In vitro analysis of extracts from functional foods firstly aims at defining the sub-toxic concentrations at which protective activities are then further characterized. It also allows comparing responses of complex samples and individual compounds, which is important since effects from protective food ingredients can be masked by accompanying toxic components. # 2003 Elsevier Ltd. All rights reserved. Keywords: In vitro toxicology; Green tea; Black carrot; DNA damage; HT29 clone 19A
1. Introduction Food safety policy must be based on a comprehensive and integrated approach of risk analysis throughout the food chain with three main components, risk assessment, risk management and risk communication (EC, 2000). The in vitro systems of toxicology are widely used for screening chemicals, for generating toxicological profiles and sporadically for risk assessment (Eisenbrand et al., 2002). The range of food-associated compounds which are testable includes natural ingredients, compounds formed endogenously after an exposure, permissible supplements, additives, residues and contaminants. An important application of in vitro systems is to obtain mechanism-derived information by studying different cells or tissue and target specific * Corresponding author. Tel.: +49-3641-949674; fax: +49-3641949672. E-mail address: [email protected] (M. Glei). 0887-2333/$ - see front matter # 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0887-2333(03)00099-7
effects. With in vitro systems it is possible to assess cytotoxicity, cellular responses, and to perform toxicokinetic modeling. They are valuable for characterizing hazards to humans through extrapolation of effects from the in vitro to the in vivo situation and from animals to men (Pool-Zobel et al., 1994). The end points of in vitro toxicology systems are numerous and a widely used approach is to determine genotoxicity for identifying carcinogenic potential of chemicals (Committee on Mutagenicity of Chemicals in Food, 2000, Ashby and Tennant, 1991). Additionally, in vitro systems are a powerful tool for biomarker development (Eisenbrand et al., 2002). Functional foods need to be evaluated from an additional point of view. In general, these are foods which provide a health benefit beyond the supply of traditional nutrients. They are expected to be beneficial because they contain components such as vitamins, polyphenols, dietary fibers or may not contain compounds with adverse effects. Polyphenols are classified as phytopro-
724
M. Glei et al. / Toxicology in Vitro 17 (2003) 723–729
numerous mechanisms, mainly derived from their high antioxidative properties (Seeram and Nair, 2002).
tectants on account of antioxidative properties and dietary fibers improve digestion and intestinal health (Glei and Pool-Zobel, 2001; Pool-Zobel et al., 1997, 2002). Moreover dietary fibre may counteract endogenous toxicity by scavenging intermediates, enhancing stool transit or by inducing chemoresistance (Ebert et al., 2001; Wollowski et al., 2001). Therefore, functional foods need to be assessed for beneficial properties, e.g. to find evidence which will support claims of functional effects. Also, for new functional foods which are processed to contain high levels of phytoprotectants it is necessary to exclude that these will act toxically when ingested in larger amounts. This is especially important since controversial results on the beneficial properties of phytoprotectants are also available. Examples are studies showing that polyphenols produce free radicals (Long et al., 2000), act toxic (Glei et al., 2002) and are prooxidative (Metodiewa et al., 1999). The experimental in vitro analysis of biological activities of whole foods will therefore serve several purposes. On the one hand, the determination of toxicity can be used as a tool to define concentrations at which chemoprotective effects can be further characterized. On the other hand, cells can be treated with sub-toxic concentrations of the compounds to find new cellular responses and among them mechanisms of potential chemoprotection. In addition, the knowledge of the balance of these activities induced by individual compounds and by complex mixtures from the foods will aid in disclosing the balance of beneficial and non beneficial properties. In this context, the aim of our studies reported below was to comparatively investigate two whole foods, a catechin-rich green tea (GT1) and an anthocyanin-rich plant juice [prepared from black carrots (BC)] for cytotoxicity, genotoxicity and protective effects in human colon cells. In line with our general strategy, we additionally investigated the pure compounds cyanidin and ()-epigallocatechin gallate (EGCG). Cyanidin is the aglycone of the main anthocyanins in BC; EGCG is the main green tea-catechin (Graham, 1992). Both groups of compounds are polyphenols and considered to be beneficial on account of
2. Materials and methods 2.1. Human tumour cell line HT29 clone 19A The human colon cell line HT29 was established in 1964 by J. Fogh (Memorial Sloan Kettering Cancer Centre, New York) (Rousset, 1986). The clone 19A was terminally differentiated with 5 mm sodium butyrate, and characterised by Augeron and Laboisse (Augeron and Laboisse, 1984). Our cells were a kind gift of Laboisse to G. Rechkemmer (Federal Research Centre, Karlsruhe), from whom we obtained them in passage 12. Cells were maintained in stocks at 140 C, thawed and grown in tissue culture flasks with DMEM (Dulbecco’s Modified Eagle Medium, Gibco BRL, Eggenstein, Germany) supplemented with 10% foetal calf serum and 1% penicillin/streptomycin at 37 C in a (95%) humidified incubator (5% CO2). This culture containing 45–60106 cells was trypsinized with 1–1.5 ml trypsin/versene (1:10 v/v) for a maximum of 10 min and sub-cultivated at a dilution of 1:8 in T75 flasks with supplemented DMEM. Two medium changes occurred on days 2 and 5 with 15 ml DMEM. 2.2. Treatment with chemicals The tea polyphenols [()-epicatechin (EC), ()-epigallocatechin (EGC), ()-epicatechin gallate (ECG), and ()-epigallocatechin gallate (EGCG)] were purchased from Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany. Cyanidin chloride was bought from ROTH GmbH Co., Karlsruhe, Germany. All chemicals were of analytical grade or compiled with the standards needed for tissue culture experiments. The green tea ethyl acetate extract (GT2) was from LKT Labs, St. Paul, USA. The GT1 and the plant juice prepared from BC were provided by Kampffmeyer Food Service GmbH, Hamburg, Germany. The aqueous extracts/
Table 1 Influence of different GT1-pretreatments (37 C, 30 min–48 h) on H2O2-induced (5 min, 4 C) DNA damage (strand breaks expressed as % fluorescence in tail) with and without a 30 min repair period in HT29 clone 19A cells Treatment
300 GT1 pretreatment, 2 mm, 37.5 mm H2O2
300 GT1 pretreatment, 2 mm, 150 mm H2O2
24 h GT1 pretreatment, 2 mm, 100 mm H2O2
24 h GT1 pretreatment, 20 mm, 100 mm H2O2
48 h GT1 pretreatment, 2 mm, 100 mm H2O2
n=3
n=3
n=13
n=3
n=3
Period 0 min
30 min
0 min
30 min
0 min A
30 min
0 min
30 min
0 min
30 min
A
Control Mean 44.21 S.D. 5.91
38.86 5.49
53.37 4.34
46.8 3.33
42.37 14.31
27.49 13.18
60.49 3.29
39.86 13.23
51.15 18.23
36.98 16.28
GT
36.06 9.82
48.77 1.21
43.11 4.45
42.15B 12.2
21.72B 9.15
57.44 15.98
44.74 15.57
38.83 14.25
28.58 8.27
Mean 44.81 S.D. 6.65
The same capital letter indicates significant differences between the groups: AP<0.05, BP<0.001 unpaired t-test.
M. Glei et al. / Toxicology in Vitro 17 (2003) 723–729
solutions of GT1, GT2 and BC were prepared by mixing the samples with cell culture medium (10% w/v), ultrasonic treatment for 4 min and two centrifugation steps (10 min, 4000 g; 10 min, 16,000 g) to obtain clear, coloured solutions. The aqueous extracts/solutions were prepared fresh and stored at 4 C in the dark until utilization. HT29 clone 19A cells were incubated with cyanidin, catechins (dissolved in DMSO, Sigma, Taufkirchen, Germany) or GT and BC extracts at 37 C in suspension culture (short-term tests, 15–60 min) or for longer duration (24–72 h) using tissue culture protocols. H2O2 (obtained as a 30% aqueous solution from Merck, Darmstadt, Germany) was used in some experiments as genotoxic model substance. Cells were treated with H2O2 on freshly prepared slides on ice for 5 min (Pool-Zobel et al., 1999). Each experiment was performed independently at least three times. 2.3. Analytical characterisation of food extracts The catechin content of the tea extracts (Table 2) was assessed by using HPLC with UV detection (Schlesier et al., 2001). The quantity of GT extract investigated was related to its molar content of EGCG and the concentrations are therefore expressed as ‘‘EGCG-equivalents’’. The anthocyanin content of the BC extract was analysed by using HPLC with diode array detection. Three cyanidin-3-glycosides, bound to various other sugars, were identified (Harborne, 1976). On their basis (13.54 0.08 g/l) the quantities of BC extract are expressed as ‘‘cyanidin-3-glycoside-equivalents’’ (cy-3-gly-equivalent).
725
5 min, followed by addition of 40 ,6-diamidino-2-phenylindole dihydrochloride (DAPI) (Sigma-Aldrich, Taufkirchen, Germany). After 30 min, DNA content, reflecting the remaining cells, was detected by fluorimetrical analysis with Ex/Em 360/450 nm. Mean values (three determinations per experiment, at least three experiments) were recorded for final evaluation. 2.5. Detection of cytotoxicity and DNA damage Cell number and viability were determined with the trypan blue exclusion test, and/or with double staining using a mixture of fluoresceindiacetate (FDA) and ethidiumbromide (EB) solutions (5 mg FDA from SigmaAldrich Chemie GmbH, Taufkirchen dissolved in 1 ml acetone, Fisher Scientific, Schwerte; 200 mg EB from Sigma-Aldrich Chemie GmbH, Taufkirchen dissolved in 1 ml PBS). DNA damage was measured using single cell microgelelectrophoresis, also named ‘‘comet assay’’ on account of the comet like-images which are evaluated as result. For this, cells were embedded into agarose on microscopical slides, lysed and subjected to electrophoresis (Singh et al., 1988; Pool-Zobel et al., 1997). Images of damaged DNA (‘‘comets’’) are scored after staining with SYBR-Green (Sigma-Aldrich Chemie GmbH, Taufkirchen). The proportions and extent of DNA migration were determined for 50 DNA spots per slide. The intensity of fluorescence in the comet tail expressed as ‘‘% fluorescence in tail’’ is the evaluation criteria presented in the graphs. 2.6. Statistical evaluations
2.4. Determination of the cell proliferation Proliferation of colon cells were determined in 96-well microtiter plates. 24 h after seeding, the cells were treated by adding various concentrations of GT (0–120 mm EGCG equivalents) or BC (0–1400 mm cy-3-gly-equivalents) to the medium. To measure surviving cells as an indirect marker of cell proliferation, DNA was isolated by fixing and permeabilising the cells with methanol for Table 2 Composition of two different green tea sources—content of polyphenols (mg/g powder) in a normal green tea powder (real aqueous extract, GT1) and a commercial one (ethyl acetate extract powder, GT2, dissolved in medium) after the same preparation steps Polyphenol
()-Epigallocatechin-3-gallate, EGCG ()-Epigallocatechin, EGC ()-Epicatechin-3-gallate, ECG ()-Epicatechin, EC (+)-Catechin, C Cumulative
Data in the figures are mean values S.D. Unless otherwise stated, these means were calculated from the means of triplicate replicates obtained in at least three independent experiments. Statistical evaluation was performed with the GraphPad Prism Version 3.02 for Windows (GraphPad Software, San Diego, CA, USA, www.graphpad.com). Depending on sample size and type of experiment, one-way ANOVA was used to determine significance of the experimental variables. The significance of differences between individual treatment groups was determined with paired and unpaired t-tests, as appropriate.
GT1, aqueous GT2, ethyl extract acetate extract
3. Results
mg/g (%)
mg/g (%)
3.1. Cytotoxicity
98 (37) 70 (27) 40 (15) 37 (14) 19 (7) 264 (100)
335 (40) 95 (11) 95 (11) 295 (35) 20 (2) 840 (100)
The BC extract clearly reduced the viability of HT29 clone 19A cells in a dose related manner (dose range up to 2790 mm cy-3-gly-equivalents) and concentrations exceeding 45 mm cy-3-gly-equivalent were clearly cytotoxic (Fig. 1). Virtually identical results were obtained
726
M. Glei et al. / Toxicology in Vitro 17 (2003) 723–729
with the two staining procedures, one using the double labelling with FDA/EB (Fig. 1A) and the other with trypan blue (Fig. 1B). In contrast, the GT1-extract was not effective in the tested concentration range, since amounts up to 100 mM EGCG-equivalent GT1-extract had no effect on the cell viability (results not shown). Also cyanidin, the aglycone of the main anthocyanins in BC, was ineffective up to 250 mm (results not shown). 3.2. Proliferation Both extracts significantly inhibited proliferation of HT29 clone 19A cells (dose range up to 1400 mm cy-3gly equivalents, up to 120 mm ECGC equivalents, 24 and 48 h incubation), with an EC50 of about 30–50 mm for GT1 (Fig. 2A) and 45 mm for BC (Fig. 2B). The control compound cyanidin (up to 250 mm) did not inhibit the cell growth, whereas incubation with EGCG resulted in an EC50 of 60 mm (48 h) and 85 mm (24 h) (results not shown). 3.3. Genotoxicity Fig. 3 shows that BC and GT1 extracts are toxic by inducing genetic damage. After 24 h incubation with 88–350 mm cy-3-gly equivalents (Fig. 3A) and 40–100 mm
Fig. 1. Cell viability after 24 or 48 h incubation with BC: (a) by FDA/ EB double staining; (b) by trypan blue exclusion. BC decreased significantly the viable cell number: (a) *P <0.05, **P <0.01, ***P <0.001 paired t-test; (b) 24 h, *P <0.05, ***P <0.001 unpaired ttest, 48 h, *P<0.05 paired t-test, n=3.
EGCG equivalents (Fig. 3B) a significant strand breakinduction was observed in HT29 clone 19A cells. 3.4. Protective effects H2O2, an endogenous genotoxic risk factor, causes DNA damage by decomposing to the hydroxyl radical (OH.). This may result in DNA instability, mutagenesis and ultimately carcinogenesis. Catechins and anthocyanins are efficient antioxidants, which could also prevent H2O2-induced DNA damage. Table 1 shows the influence of different GT1 pre-treatments (2, 20 mm, 30 min, 24, 48 h) on H2O2 (37,5, 100, 150 mm)-induced DNA damage and on the persistence of damage for 30 min post-treatment. We specifically chose a short incubation period (30 min) during which damage by relatively high doses of H2O2 ( 100 mm) is only marginally repaired to allow detecting the additional GT effect. However, it is obvious that GT1 did not protect against the genotoxicity of H2O2. Damage level and persistence were only modified insignificantly. To assess the relative protective efficacy of our aqueous GT-extract (GT1) we compared it with EGCG, the
Fig. 2. Cell proliferation, measured as a function of DNA content, after 24 and 48 h incubation with GT1 (a) and BC extract (b). GT1 and BC decreased significantly the cell number (*P< 0.05, **P<0.01, ***P <0.001 t-test against control).
M. Glei et al. / Toxicology in Vitro 17 (2003) 723–729
727
Fig. 4. Influence of different polyphenol sources (GT1—aqueous extract, Syn-GT—mixture of the most abundant polyphenols of GT1, GT2—commercial ethyl acetate extract, EGCG—main green tea catechin) (37 C, 24 h, 2 mm EGCG equivalent) on H2O2-induced (5 min, 4 C) DNA damage (strand breaks expressed as % fluorescence in tail) with and without a 30 min repair period in HT29 clone 19A cells. The same capital letter indicates significant differences between the groups: A,CP <0.05, BP<0.001 unpaired t-test.
Fig. 3. DNA damage after 24 h treatment with BC (a) and GT1 extract (b). BC (P <0.01, one way ANOVA, *P <0.05, ***P <0.001 Dunnett’s multiple comparison test against control, n=3) and GT1 (*P<0.05 unpaired t-test against control, n=4) induced significantly DNA strand breaks.
main catechin, with a comparable mixture (Syn-GT) of the 5 most abundant polyphenols of GT1 (Table 2), and with a commercial green tea extract (GT2) (Fig. 4). In fact, none of the four tested samples (standardised to 2 mm EGCG-equivalent) were able to reduce the genotoxicity of H2O2 per se. However, when regarding the impact of GT on persistence of H2O2-induced damage, it appears as if the complex samples GT1 and GT2 enhance repair, since they caused a significant reduction of DNA damage during a 30 min post incubation period. The pre-treatment of HT29 clone 19A cells with BCextract and cyanidin had quite different effects on H2O2induced DNA damage (Fig. 5). Short term incubation (15-30 min) with cyanidin (100 mm) significantly decreased the genotoxicity of H2O2 (18.75–150 mm). However, the similar treatment with the complete BC extract (BC: 45 mm cy-3-gly-equivalent) enhanced the DNA-damaging effect.
4. Discussion Anthocyanins are a group of polyphenolic flavonoids which occur ubiquitously in foods of plant origin (Hollman and Katan, 1999). They are implicated in
Fig. 5. Effect of 15–30 min preincubation with BC (45 mm cy-3-glyequivalent) and cyanidin (100 mm) on H2O2-induced (0–150 mm) DNA damage. There was a clear H2O2 dose–effect relationship (one-way ANOVA, P <0.001). Cyanidin pretreatment reduced the level of induced fluorescence in tail (BC: *P <0.05, **P <0.01, unpaired t-test, n=6, Cyanidin: P<0.05, P <0.01, paired-test, n=8).
preventing diseases related to oxidative stress, such as coronary heart disease (Hertog et al., 1993) and cancer (Stoner and Mukhtar, 1995) maybe due to their antioxidant properties (Kandaswami and Middleton 1994; Sellappan and Akoh, 2002). Pool-Zobel et al. (1999) demonstrated that isolated compounds and complex samples of Aronia melanocarpa are powerful antioxidants in vitro. Also, they acted intracellularly by reducing H2O2 induced DNA strand breaks in human colon cells. Knekt et al. (2002) used a dietary history method with over 10,000 men and women and showed, that a high intake of different flavonoids was associated with lower mortality from ischemic heart disease, lower lung and prostata cancer as well as lower asthma incidence. The plant extract we investigated was prepared from BC at GNT International, Vaals, Netherlands. BC have been cultivated and eaten for more than 3000
728
M. Glei et al. / Toxicology in Vitro 17 (2003) 723–729
years. They are considered to be rich in anthocyanins. Harborne (1976) described their anthocyanin species, which were identified to be the cyanidin 3-sinapoylxylosylglucosylgalactoside (Harborne et al., 1983). Tea is one of the most popular beverages consumed worldwide. Among all tea produced in the world, 20% is green tea, which is commonly consumed in Asian countries where the tea is a major beverage (Yang, 1999). The catechins, (EGCG, EGC, ECG, EC, C) are major active constituents of green tea. These compounds have been shown to be highly active as anticarcinogens in vivo using several rodent test systems (Katiyar and Mukhtar, 1996) and epidemiological studies on the consumption of green tea indicate a protection against stomach (Yu et al., 1995) and colon (Kato et al., 1990) cancers. Several mechanisms have been identified by which catechins can operate as anticarcinogens, including inhibition of cytochrome P-450 activation of carcinogen-precursors (Wang et al., 1988), inhibition of angiogenesis (Cao and Cao, 1999), scavenging of reactive oxygen species (Rice-Evans et al., 1996) or electron transfer from catechins to ROSinduced radical sites on the DNA (Anderson et al., 2001). Besides, Benelli et al. (2002) concluded that green tea polyphenols appear to be non-conventional inhibitors of metallo and serine proteases involved in matrix degradation. In this study, we observed distinctly different effects between the complex extracts and of their isolated ingredients on several parameters of in vitro toxicology (proliferation, cytotoxicity and genotoxicity). The major differences are apparent when comparing the effects of cyanidin to those of BC. Whereas the aglycone cyanidin was protective as expected, on the basis of previous studies, the anthocyanin-rich BC extract did not protect from H2O2-induced DNA damage. Instead, BC clearly enhanced H2O2-induced genotoxicity. Apparently the complex plant extract contains additional components, which add on to the H2O2-mediated activities. At the investigated in vitro concentrations (there is no information available on the actual concentration in the colon), the protective activity of cyanidin was not sufficient to counteract these toxic responses. It will be important in the future to identify potential genotoxic candidates and to evaluate these for their relative contribution to the observed genotoxic effects. Furthermore, we are aware that to discern between the effects of the original compounds and their metabolites, the stability of test compounds must be considered in further investigations (Hong et al., 2002). In conclusion, these findings clearly point to the necessity of performing safety evaluations for individual complex plant extracts, before using them as food supplements. It is not sufficient to rely on former findings of beneficial effects by key ingredients, without toxicological assessment of the complex plant product
itself. Methods of in vitro toxicology will be useful in this context. By using the methods, it is possible to define the sub-toxic concentrations of extracts from functional foods at which protective activities are then further characterized. Additionally, it is possible to compare responses of complex samples and of individual compounds, which is important since effects from protective food ingredients can be masked by accompanying toxic components.
References Augeron, C., Laboisse, C.L., 1984. Emergence of permanently differentiated cell clones in a human colonic cancer cell line in culture after treatment with sodium butyrate. Cancer Research 44, 3961– 3969. Anderson, R.F., Fisher, L.J., Hara, Y., Harris, T., Mak, W.B., Melton, L.D., Packer, J.E., 2001. Green tea partially protect DNA from OH radical-induced strand breaks and base damage through fast chemical repair of DNA radicals. Carcinogenesis 22, 1189–1193. Ashby, J., Tennant, R.W., 1991. Definitive relationships among chemical structure, carcinogenicity and mutagenicity for 301 chemicals tested by the U.S. NTP. Mutation Research 257, 229–306. Benelli, R., Vene, R., Bisacchi, D., Garbisa, S., Albini, A., 2002. Antiinvasive effects of green tea polyphenol epigallocatechin-3-gallate (EGCG), a natural inhibitor of metallo and serine proteases. Journal of Biological Chemistry 383, 101–105. Cao, Y., Cao, R., 1999. Angiogenesis inhibited by drinking tea. Nature 398, 381. Committee on Mutagenicity of Chemicals in Food, Consumer Products and the Environment COM UK. Guidelines on a Strategy for Testing of Chemicals for Mutagenicity. December 2000. Ebert, M.N., Beyer-Sehlmeyer, G., Liegibel, U.M., Kautenburger, T., Becker, T.W., Pool-Zobel, B.L., 2001. Butyrate-induced activation of glutathione S-transferases protects human colon cells from genetic damage by 4-hydroxynonenal. Nutrition and Cancer 41, 156–164. EC (2000) EC White paper on food safety COM (1999) 719 final, January 2000. Eisenbrand, G., Pool-Zobel, B.L., Baker, V., Balls, M., Blaauboer, B.J., Boobis, A., Carere, A., Kevekordes, S., Lhuguenot, J.-C., Pieters, R., Kleiner, J., 2002. Methods of in vitro toxicology. Food and Chemical Toxicology 40, 193–236. Glei, M., Latunde-Dada, G.O., Klinder, A., Becker, T.W., Hermann, U., Voigt, K., Pool-Zobel, B.L., 2002. Iron-overload induces oxidative DNA damage in the human colon carcinoma cell line HT29 clone 19A. Mutation Research 519, 151–161. Glei, M., Pool-Zobel, B.L., 2001. Krebspra¨vention und Erna¨hrung: Riskoverminderung durch eine lebenslange gesunde Erna¨hrung. In: Friedenthal-Haase, M., Meinhold, G., Schneider, K., Zwiener, U. (Eds.), Studien zur Pa¨dagogik, Andragogik und Gerontagonik, Alt werden-alt sein, Band 50. Peter Lang, Europa¨ischer Verlag der Wissenschaften, pp. 99–110. Graham, H.N., 1992. Green tea composition, consumption, and polyphenol chemistry. Preventive Medicine 21, 334–350. Harborne, J.B., 1976. A unique pattern of anthocyanins in Daucus carota and other Umbelliferae. Biochemical Systemics and Ecology 4, 31–35. Harborne, J.B., Mayer, A.M., Bar-Nun, N., 1983. Identification of the major anthocyanin of carrot cells in tissue cultures as cyanidin 3(sinapoylxylosylglucosylgalactoside). Zeitschrift Naturforschung 38c, 1055–1056. Hertog, M.G.L., Feskens, E.J.M., Hollman, P.C.H., Katan, M.B.,
M. Glei et al. / Toxicology in Vitro 17 (2003) 723–729 1993. Dietary antioxidant flavonoids and risk of coronary heart disease: the Zutphen Elderly Study. The Lancet 342, 1007–1011. Hollman, P.C.H., Katan, M.B., 1999. Dietary flavonoids: intake, health effects and bioavailability. Food and Chemical Toxicology 37, 937–942. Hong, J., Lu, H., Meng, X., Ryu, J.-H., Hara, Y., Yang, C.S., 2002. Stability, cellular uptake, biotransformation, and efflux of tea polyphenol ()-epigallocatechin-3-gallate in HT-29 human colon adenocarcinoma cells. Cancer Research 62, 7241–7246. Kandaswami, C., Middleton, E., 1994. Free radical scavenging and antioxidant activity of plant flavonoids. Advances in Experimental Medicine and Biology 366, 351–376. Katiyar, S.K., Mukhtar, H., 1996. Tea in chemoprevention of cancer: epidemiologic and experimental studies (review). International Journal of Oncology 8, 221–238. Kato, I., Tominaga, S., Matsuura, A., Yoshii, Y., Shirai, M.E.A., 1990. A comparative case-control study of colorectal cancer and adenoma. Japanese Journal of Cancer Research 81, 1101–1108. Knekt, P., Kumpulainen, J., Ja¨rvinen, R., Rissanen, H., Helio¨vaara, M., Reunanen, A., Hakulinen, T., Aromaa, A., 2002. Flavonoid intake and risk of chronic diseases. American Journal of Clinical Nutrition 76, 560–568. Long, L.H., Clement, M.V., Halliwell, B., 2000. Artifacts in cell culture: rapid generation of hydrogen peroxide on addition of ()-epigallocatechin, ()-epigallocatechin gallate, (+)-catechin, and quercetin to commonly used cell culture media. Biochemical and Biophysical Research Communications 273, 50–53. Metodiewa, D., Jaiswal, A.K., Cenas, N., Dickancaite´, E., SeguraAguilar, J., 1999. Quercetin may act as a cytotoxic prooxidant after its metabolic activtion to semiquinone and quinoidal product. Free Radical Biology and Medicine 26, 107–116. Pool-Zobel, B.L., Lotzmann, N., Knoll, M., Kuchenmeister, F., Lambertz, R., Leucht, U., Schro¨der, H.-G., Schmezer, P., 1994. Detection of genotoxic effects in human gastric and nasal mucosa cells isolated from biopsy samples. Environmental and Molecular Mutagenesis 24, 23–45. Pool-Zobel, B.L., Bub, A., Mu¨ller, H., Wollowski, I., Rechkemmer, G., 1997. Consumption of vegetables reduces genetic damage in humans: first results of a human intervention trial with carotenoidrich foods. Carcinogenesis 18, 1847–1850.
729
Pool-Zobel, B.L., Bub, A., Schro¨der, N., Rechkemmer, G., 1999. Anthocyanins are potent antioxidants in vitro but do not reduce oxidative DNA damage within human colon cells. European Journal of Nutrition 38, 227–234. Pool-Zobel, B.L., Van Loo, J., Rowland, I.R., Roberfroid, M.B., 2002. Experimental evidences on the potential of prebiotic fructans to reduce the risk of colon cancer. British Journal of Nutrition 87, S273–S281. Rice-Evans, C.A., Miller, N.J., Paganga, G., 1996. structure-antioxidant activity relationships of flavonoids and phenolic acids. Free Radical Biology and Medicine 20, 933–956. Rousset, M., 1986. The human colon carcinoma cell lines HT-29 and Caco-2: two in vitro models for the study of intestinal differentiation. Biochimie 68, 1035–1040. Schlesier, K., Bo¨hm, V., Bitsch, R., 2001. HPLC-Methode zur Detektion von Flavonoiden und Phenolcarbonsa¨uren in Obst und Gemu¨se. Lebensmittelchemie 55, 38–39. Seeram, N.P., Nair, M.G., 2002. Inhibition of lipid peroxidation and structure-activity-related studies of the dietary constituents anthocyanins, anthocyanidins, and catechins. Journal of Agricultural and Food Chemistry 50, 5308–5312. Sellappan, S., Akoh, C.C., 2002. Flavonoids and antioxidant capacity of georgia-grown vidalia onions. Journal of Agricultural and Food Chemistry 50, 5338–5342. Singh, N.P., McCoy, M.T., Tice, R.R., Schneider, E.L., 1988. A simple technique for quantitation of low levels of DNA damage in individual cells. Experimental Cell Research 175, 184–191. Stoner, G.D., Mukhtar, H., 1995. Polyphenols as cancer chemopreventive agents. Journal of Cellular Biochemistry 22, 169–180. Wang, Z.Y., Das, M., Bickers, D.R. Mukhtar, H, 1988. Interaction of epicatechins derived from green tea with rat hepatic cytochrome P450. Drug Metabolism and Disposition 16, 98–103. Wollowski, I., Rechkemmer, G., Pool-Zobel, B.L., 2001. Protective role of probiotics and prebiotics in colon cancer. American Journal of Clinical Nutrition 73, 451s–545s. Yang, C.S., 1999. Tea and health. Nutrition 15, 946–948. Yu, G., Hsieh, C., Wang, L., Yu, S., Li, X.E.A., 1995. Green-tea consumption and risk of stomach cancer: a population-based casecontrol study in Shanghai, China. Cancer Causes Control 3, 137– 143.