Evaluation of the cyto- and genotoxic activity of yerba mate (Ilex paraguariensis) in human lymphocytes in vitro

Mutation Research 679 (2009) 18–23

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Mutation Research/Genetic Toxicology and Environmental Mutagenesis journal homepage: www.elsevier.com/locate/gentox Community address: www.elsevier.com/locate/mutres

Evaluation of the cyto- and genotoxic activity of yerba mate (Ilex paraguariensis) in human lymphocytes in vitro Maciej Wnuk a,∗ , Anna Lewinska b , Bernadetta Oklejewicz a , Monika Bugno c , Ewa Slota c , Grzegorz Bartosz b,d a

Department of Genetics, University of Rzeszow, Rejtana 16C, PL 35-959, Rzeszow, Poland Department of Biochemistry and Cell Biology, University of Rzeszow, Rzeszow, Poland Department of Immuno- and Cytogenetics, National Research Institute of Animal Production, Balice n. Cracow, Poland d Department of Molecular Biophysics, University of Lodz, Lodz, Poland b c

a r t i c l e

i n f o

Article history: Received 9 March 2009 Received in revised form 20 July 2009 Accepted 28 July 2009 Available online 4 September 2009 Keywords: Yerba mate Caffeine Micronuclei Silver-stained nucleolar organizer regions Fluorescence in situ hybridization

a b s t r a c t Despite its antioxidant capacity and well-known health benefits, yerba mate tea (Ilex paraguariensis) has been shown to possess some genotoxic and mutagenic activities and to increase incidence of some types of cancer. The aim of this study was to estimate the cyto- and genotoxicity of mate tea in human peripheral lymphocytes in vitro. We found that yerba mate extract induced a concentration-dependent, statistically significant increase in the level of apoptotic and necrotic cells and a decrease in the nuclear division index (NDI). Mate-exposed lymphocytes had a reduced transcriptional rDNA activity, which may be due to the stress conditions, and showed an elevated production of micronuclei. The FISH technique revealed the appearance of an acrocentric signal in mate-induced micronuclei, which suggests that under these conditions yerba mate extract may display aneugenic activity. Since caffeine is one of the most abundant compounds found in the dry mass of mate, we conducted additional experiments with caffeine alone. We showed that caffeine used at the same concentrations manifests a more potent cyto- and genotoxic effect that may account, at least in part, for the disadvantageous effects observed for yerba mate extract. © 2009 Elsevier B.V. All rights reserved.

1. Introduction Yerba mate (mate) extract, made from dried leaves of Ilex paraguariensis, is a tea-like beverage consumed in South America, mainly in Argentina, Brazil, Paraguay and Uruguay. Its popularity is widely increasing in Europe since it is a commercially available product sold as antirheumatic, diuretic and central nervous system stimulant. Mate tea is a known antioxidant [1–3], potent inhibitor of LDL oxidation [4] and nitrosative stress [5]. Many epidemiological papers have reported a correlation between mate drinking and cancer incidence, e.g., oral, oropharyngeal, laryngeal or bladder cancer [6–14]. On the other hand, in many cases it is difficult to estimate mate consumption as an independent cancer risk factor. Nevertheless, in Uruguay it was demonstrated that mate drinking was significantly associated with higher risk for esophageal cancer, independent of alcohol and tobacco consumption [15–18].

Abbreviations: AgNORs, silver-stained nucleolar organizer regions; BN cells, binucleated cells; CBMN assay, cytokinesis-block micronucleus assay; Cyt-B, cytochalasin B; FISH, fluorescence in situ hybridization; MN, micronucleus or micronuclei; NDI, nuclear division index; NORs, nucleolar organizer regions. ∗ Corresponding author. Tel.: +48 178723704; fax: +48 178723708. E-mail address: [email protected] (M. Wnuk). 1383-5718/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.mrgentox.2009.07.017

Among active compounds found in mate samples, caffeine (1,3,7-trimetylxanthine) is one of the most abundant. Caffeine is thought to be antioxidant, anticarcinogen, energizer, diuretic, stimulant, vasodilator, antiobesity and antitumor agent, and topoisomerase-I and -II inhibitor [19]. However, the effects of caffeine on mammalian cells may be ambiguous and different in various cell types. It may stimulate cell proliferation or apoptosis, depending on the experimental model used, the duration of treatment and the concentration applied [20–22]. Mate extracts have already been shown to possess genotoxic and mutagenic activity in bacterial cells through lysogenic induction and occurrence of point mutations [23]. Additionally, mate extracts at concentrations ranging from 100 to 175 ␮g/ml have caused chromosomal aberrations in human peripheral lymphocytes in vitro [24]. However, using the same experimental model, it was shown that mate infusion (from 175 to 1400 ␮g/ml) did not induce a statistically significant increase in micronucleus formation [25]. Since experimental data concerning the genetic toxicity of mate are obscure and contradictory, we decided to re-evaluate the effects of mate on human peripheral lymphocytes in vitro. We chose the concentrations of mate extract ranging from 1 to 1000 ␮g/ml since lower concentrations were rarely taken into account. Here, we show with several methods that mate extract is cyto- and genotoxic

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in human peripheral lymphocytes in vitro. Additionally, we provide data concerning the impact on cells of caffeine alone, which demonstrate that caffeine may account, at least in part, for the effects seen for mate extract. 2. Materials and methods 2.1. Chemicals Giemsa stain solution was from Fluka (Buchs, Switzerland) (CAS registry number 51811-82-6) and DAPI II Counterstain was from Abbott Molecular (IL, USA). All other reagents, if not mentioned otherwise, were purchased from Sigma (Steinheim, Germany) and were of analytical grade.

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cellular rDNA transcriptional activity and is related to cell-proliferation activity [32,33]. 2.6. Cytokinesis-block micronucleus (CBMN) assay Chromosome damage was assessed with the cytokinesis-block micronucleus (CBMN) assay. A total of 500 binucleated cells (from three independent cultures for each concentration used) with a well-preserved cytoplasm on randomized and coded slides stained with Giemsa (5% in Sorensen Buffer) were analyzed for determination of micronucleus frequency (MN) and the nuclear division index (NDI) as previously described by Fenech et al. [34,35]. The NDI was calculated according to the method of Eastmond and Tucker [36] by scoring cells with 1, 2, 3 or 4 nuclei and using the formula: NDI = 1/N × [M1 + 2(M2) + 3(M3) + 4(M4)],

2.2. Yerba mate preparation The commercially available yerba mate (Ilex paraguariensis) (Argentina) was prepared into a mate infusion (200 g/l, brewed up for 10 min after boiling) which was filtered (0.22 mm Millipore filter) and immediately used. 2.3. Cell culture and growth conditions To avoid the impact of inter-individual variation in AgNOR and micronuclei on our results [26,27] we used human blood from one healthy donor (lab volunteer, 28-year-old non-smoking male). Heparinized blood was added at a volume ratio of 1:9 to RPMI 1640 medium with l-glutamine, HCO3 − and Phenol Red containing 10% (v/v) fetal calf serum (FCS) (Biomed, Lublin, Poland), penicillin G potassium salt (100 U/ml) (Sigma, Deisenhofen Germany) (CAS registry number 113-98-4), streptomycin sulfate salt (100 ␮g/ml) (Sigma, Deisenhofen, Germany) (CAS registry number 3810-74-0), lectin from pokeweed Phytolacca americana (5 ␮g/ml) (Sigma, St. Louis, USA), with/without yerba mate or caffeine (1–1000 ␮g/ml) and incubated at 37 ◦ C for 72 h. For cytotoxicity test lymphocytes were isolated with HISTOPAQUE® -1077 with the standard protocol according to the manufacturer’s instructions (Sigma, St. Louis, USA) and were cultured with/without yerba mate or caffeine (CAS registry number 58-08-2) (1–1000 ␮g/ml) in a NuncTM Lab-Tek® Chamber SlideTM System (Thermo Fisher Scientific, Germany) at a concentration of 106 cells/ml at 37 ◦ C for 24 h. For the cytokinesis-block micronucleus (CBMN) assay cytochalasin B (Cyt-B) from Drechslera dematioidea (Sigma, St. Louis, USA) (CAS registry number 1493092-2) (6 ␮g/ml) was added to the cultures 28 h prior to the cell harvest [28]. All stock solutions of the test agents were freshly prepared before adding them to the medium. After 72 h, lymphocytes were harvested by use of a 30-s treatment with 75 mM KCl and immediately fixed with a mixture of methanol:glacial acetic acid (3:1). For silver staining, interphase lymphocytes were obtained from routine 72-h lymphocyte cultures and after 10 min treated with 75 mM KCl at 37 ◦ C and fixed in a methanol:glacial acetic acid (3:1). 2.4. Cytotoxicity assay with acridine orange-ethidium bromide staining Lymphocytes after 24 h of culture with or without test agent on the Chamber SlideTM System were washed twice with phosphate-buffered saline (PBS) (Gibco, Invitrogen Corporation, Grand Island, NY, USA). Then, a mixture of acridine orange (100 ␮g/ml in PBS) and ethidium bromide (100 ␮g/ml in PBS) at a volume ratio of 1:1 was added to the cells, which were then analyzed with an Axiophot ZeissOpton fluorescence microscope (Carl Zeiss, Germany) equipped with a Cohu HighPerformance CCD camera and a LUCIA computer image-analysis system. Cells with a light green nucleus without chromatin condensation were scored as live, cells with a light green nucleus with dark green chromatin inclusion bodies were characterized as early apoptotic, cells with orange nucleus with dark orange chromatin inclusion bodies were scored as apoptotic, and cells with an orange nucleus without chromatin condensation were classified as necrotic. A total of 100 cells from each of three independent cultures per concentration used were counted. 2.5. Silver staining (AgNOR) Silver staining was performed according to Howell and Black [29], by incubation of the slides with a colloidal developer containing 50% AgNO3 (Sigma, St. Louis, USA) (CAS registry number 7762-88-8) at 37 ◦ C in the dark for 15 min. After a wash in tap water the preparations were stained with 5% Giemsa for 10 s. 2.5.1. AgNOR analysis The analysis of interphase AgNOR areas of 100 lymphocytes was conducted by use of the morphometric method according to Derenzini and Trere [30] and following the guidelines of the Committee on AgNOR Quantification [31], using a MultiScan 6.08 computer image-analysis system. We measured the area occupied by AgNOR proteins exclusively in the lymphocytes that contained only one AgNOR dot. Interphase NORs activity was expressed as total size of silver deposit areas, which reflects

where M1 to M4 stands for the number of cells with 1–4 nuclei and N is used to denote the total number of viable cells examined. 2.7. Fluorescence in situ hybridization (FISH) After the CBMN assay, the slides with lymphocytes from control cultures and from cells exposed to selected concentrations of yerba mate or caffeine were discolored by a 1-h incubation in 70% ethanol, washed with water and air-dried. Then, the slides were treated with 100 ␮g/ml RNase (CAS registry number 9001-99-4) in 2× saline sodium citrate (SSC) buffer (Sigma, Saint Louis, USA) in a humidified chamber at 37 ◦ C for 1 h for better results. Next, the slides were washed three times with 2× SSC buffer and treated with 1% pepsin (CAS registry number 9001-75-6) in 10 mM HCl in a Coplin jar at 37 ◦ C for 10 min. The slides were washed twice with PBS, once with PBS supplemented with 50 mM MgCl2 (CAS registry number 7786-30-3) and passed through a series of ethanol solutions (70, 80 and 95%) at room temperature for 3 min. For DNA denaturation, we treated the cells with 70% formamide (CAS registry number 75-12-7) with 2× SSC solution at 72 ◦ C for 10 min. Then, the slides were passed through a series of ethanol solutions (70, 80 and 95%) at −20 ◦ C for 3 min and air-dried. The Acro p-arm spectrum-orange (SO) probe is fairly specific for the region of p12–p13 of acrocentric chromosomes (Vysis, Bergisch-Gladbach, Germany). The probe was denaturated (at 72 ◦ C for 5 min) and applied on the slides (10 ␮l per slide) with a coverslip and sealed with fixogum to prevent evaporation. Hybridization was performed in a humidified chamber with 70% formamide at 37 ◦ C for 18 h. Next, the slides were washed three times with 50% formamide in 2× SSC at 45 ◦ C for 5 min and twice with 2× SSC containing 0.1% Tergitol® Type NP-40 (CAS registry number 127087-87-0) at 45 ◦ C for 5 min (post-hybridization). The slides were left to air-dry in the dark, counterstained with a drop of the mounting medium with DAPI II Counterstain and analyzed with an Axiophot Zeiss-Opton fluorescence microscope (Carl Zeiss, Germany) equipped with a Cohu High-Performance CCD camera and a LUCIA computer image-analysis system. 2.8. Statistical analysis The results represent the mean ± SD from at least three independent experiments. Differences between (a) control vs yerba mate-treated cultures and (b) control vs caffeine-treated cultures were assessed with the one-way analysis of variance (ANOVA) with post hoc testing using a Dunnett’s multiple comparison test. A p-value of less than 0.05 was considered significant. Statistical analysis of the data was performed using StatSoft, Inc. (2005), STATISTICA, version 7.0 (www.statsoft.com).

3. Results Yerba mate extract caused a concentration-dependent, statistically significant, increase in the rate of cell death (Fig. 1A). Yerba mate extract at a concentration of 10 ␮g/ml induced a 3.2-fold increase in the level of early apoptotic cells. At higher concentrations it induced a 3- and 4.6-fold augmentation in the percentage of apoptotic cells, respectively, as compared with control (Fig. 1A). Moreover, at the concentrations of 100 and 1000 ␮g/ml the extract caused a 7- and 12.2-fold increase in the level of necrotic cells in comparison with standard growth conditions, respectively (Fig. 1A). Since caffeine is found at the highest concentration among xanthines examined in the dry mass of mate (1–2%) [19], we decided to check the impact of caffeine alone on the viability of lymphocytes. We used the same concentrations of caffeine as was present in the yerba mate extract. We recorded a statistically significant, early apoptotic event at the lowest concentration of caffeine applied (1 ␮g/ml); at a concentration of 1000 ␮g/ml only necrotic

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Fig. 2. Growth impairment estimated as NDI value of human lymphocytes exposed to yerba mate extract (A) and caffeine (B) and inspected after 72 h. Mean values ± SD, n = 3, *p < 0.05, **p < 0.01, ***p < 0.001 as compared with controls (one-way ANOVA and Dunnett’s multiple comparison test). Fig. 1. (A) Yerba mate (YM) extract-induced and (B) caffeine (C)-induced cell death. Human peripheral lymphocytes were incubated with the appropriate concentrations of the test agents and inspected after 24 h. Bars indicate SD, n = 3, # p < 0.05, ## p < 0.01, ### p < 0.001 as compared with live control cells, ˆ p < 0.05 as compared with early apoptotic control cells, *p < 0.05 as compared with apoptotic control cells, + p < 0.05, ++ p < 0.01, +++ p < 0.001 as compared with necrotic control cells (one-way ANOVA and Dunnett’s multiple comparison test).

cells were seen (Fig. 1B). This extremely toxic concentration of caffeine was excluded from further analysis. Additionally, yerba mate extract and caffeine caused a reduction of the value of NDI, which can be considered as a parameter of the proliferative status of the viable cell fraction and simultaneously as an indicator of the cytostatic effects of the agents examined in this study [37] (Fig. 2). Since the transcriptional rDNA activity, reflecting the physiological state of the cell, may be diminished under stress conditions [38], we have examined the impact of mate and caffeine on the value of the mean AgNOR area of interphase lymphocytes. After exposure to 100 and 1000 ␮g/ml mate, the mean transcriptional activity of cellular rRNA dropped significantly, by 3.2- and 3.9-fold, respectively (Fig. 3A). For caffeine treatment we recorded an even more pronounced decrease in the mean AgNOR area: a 2-, 2.8- and 6.1-fold decrease for 1, 10 and 100 ␮g/ml, respectively (Fig. 3B). In parallel, we checked an ability of mate and caffeine to induce genotoxicity estimated with the cytokinesis-block micronucleus (CBMN) assay. We found a 3.3- and 6.2-fold elevation, respectively, of the micronucleus frequency after exposure of lymphocytes to mate or caffeine at a concentration of 10 ␮g/ml (Fig. 4). Simultaneously, we recorded a diminution in the formation of micronuclei when a higher concentration of caffeine (100 ␮g/ml) was used, apparently due to massive apoptosis and necrosis under

Fig. 3. Impact of (A) yerba mate extract and (B) caffeine on mean transcriptional activity of cellular rRNA expressed as mean AgNOR area of interphase lymphocytes assessed after a 72-h treatment. Mean ± SD, n = 3, **p < 0.01, ***p < 0.001 as compared with controls (one-way ANOVA and Dunnett’s multiple comparison test).

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Fig. 5. (A) Upper panel: typical feature of a BN cell with (arrowhead, red) and without an acrocentric signal in two micronuclei. After the CBMN assay, the FISH technique was applied to the same slides with fixed control lymphocytes and cells exposed to 10 ␮g/ml mate or caffeine. The slides were stained with 10 ␮l of antifading solution containing DAPI II Counterstain (blue). The slides were analyzed with a fluorescence microscope equipped with a CCD camera and LUCIA software. (B) Lower panel: comparison of the aneugenic events in the different cell culture conditions. FISH discrimination between clastogenic/aneugenic activity of test agents was performed for the same number of BN cells as for the CBMN assay. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.) Fig. 4. Micronucleus induction by exposure to (A) yerba mate extract and (B) caffeine, assessed after 72 h. Mean values ± SD, n = 3, *p < 0.05 as compared with controls (one-way ANOVA and Dunnett’s multiple comparison test).

these conditions (Figs. 1 and 4). Since the level of live cells exposed to a concentration of 100 ␮g/ml caffeine dropped 3.7-fold, it is not surprising that micronucleus formation also decreased almost to the control level. The same effect was seen for mate (100 and 1000 ␮g/ml), but it was slightly less pronounced since the level of live cells at a concentration of 100 ␮g/ml mate decreased 1.5-fold. We have chosen a concentration of 10 ␮g/ml mate and caffeine as inducing the highest detectable genotoxicity, and have performed FISH discrimination between the clastogenic/aneugenic activity of mate and caffeine. We have used a probe for the p-arm of acrocentric chromosomes since these chromosomes are frequently involved in non-disjunction events. We were able to record an acrocentric signal in micronuclei formed after treatment with mate and caffeine (Fig. 5). After complete slide examination, the frequencies of aneugenic activity of 10 ␮g/ml mate and caffeine were determined to be 12.5% and 20%, respectively. These values were obtained as the ratio of the aneugenic signals in control micronuclei and agent-treated micronuclei. 4. Discussion Mate tea is well-known for its high antioxidant activity due to the high polyphenol content, contributed mainly by caffeoyl derivatives. Moreover, mate extracts have shown to possess anticancer potential involving cytotoxicity and inhibitory activity for TPA-induced ornithine decarboxylase (ODC), quinine reductase and topoisomerase [39]. On the other hand, many epidemiological studies have reported a correlation between mate drinking and increased risk for cancer [6–14]. Additionally, the data concerning in vitro effects of mate extracts are scarce and conflicting [23–25]. The present study on in vitro cultured human lymphocytes indicates that mate infusion may cause both cytotoxic and genotoxic

effects (Figs. 1A, 2A and 4A). We used a range of concentrations of mate from 1 to 1000 ␮g/ml and found that 10 ␮g/ml of mate significantly increased the frequency of micronuclei and decreased the NDI value. At the higher concentrations of 100 and 1000 ␮g/ml, mate was responsible for an augmentation in the level of apoptotic and necrotic cells. The ability of mate extract (100–175 ␮g/ml) to induce chromosomal aberrations in human lymphocytes has already been shown [24]. Moreover, in a prokaryotic model it was demonstrated that mate may cause lysogenic induction and point mutations [23]. Simultaneously, no genotoxic effect of mate (175–1400 ␮g/ml) on human lymphocytes was reported [25]. However, Alves et al. [25] presented the data concerning micronucleus frequencies with no impact of mate on cell viability. As we have shown, the number of micronuclei dropped when higher concentrations of mate were applied (100 and 1000 ␮g/ml) which was due to elevated occurrence of apoptosis and necrosis (Figs. 1A and 4A). Additionally, to check if mate treatment may mimic stress conditions, we used the interphase AgNOR staining for assessment of transcriptional activity of cellular rDNA [38]. We found a decrease in the expression of nucleolar organizer regions at the higher test concentrations of mate (100 and 1000 ␮g/ml) (Fig. 3A). Since mate extract was found to be genotoxic in human lymphocytes (Fig. 4A), we employed the FISH technique with a commercial probe for the most incompletely segregated acrocentric chromosomes in order to distinguish between the clastogenic or aneugenic effects of the extract. We showed an aneugenic activity of 10 ␮g/ml mate infusion which simultaneously was a potent inducer of micronucleus formation (Fig. 5A). Caffeine, the most abundant xanthine found in mate tea, may exert different effects on mammalian cells depending on its concentration used and cell type applied. The mechanisms of these effects are mostly unknown. It was shown, mainly on cancer cells, that caffeine at higher concentrations (4–10 mM) may induce apoptosis or cell-cycle arrest, hence anticarcinogenic effects [40–43]. The beneficial effects of caffeine in anticancer therapy may be due,

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at least in part, to the inhibitory activity on topoisomerases of this compound, leading to inhibition of cell division and simultaneously to its inhibitory activity on DNA repair, enhancing the cytotoxic potential of anticancer drugs and radiation. Caffeine, inhibiting ATM and ATR kinases and disrupting cell-cycle checkpoints, may also sensitize tumor cells to antitumor drugs [44]. Additionally, the effects of caffeine on anticancer therapy may involve modulation of immune responses via inhibition of cyclic adenosine monophosphate (cAMP)-phosphodiesterase, or indirect regulation of neovascularization of human tumors via inhibition of adenosine receptors [44]. It was reported that caffeine-potentiated chemotherapy may be of benefit for metastatic carcinoma, lymphoma or high-grade soft-tissue sarcoma, prolonging overall survival of the patients at all stages [45–47]. The effects of caffeine were also studied in non-tumor cells and it was found that caffeine may have antioxidant activity or may be an inducer of apoptosis depending on the concentration applied [20,21]. According to data presented above, showing that mate tea and caffeine alone may display some similar biological activities, we decided to check the effect of caffeine alone on human lymphocytes. We used the same concentrations of caffeine as for mate infusion (1–1000 ␮g/ml), which corresponds to 5, 50, 500 and 5000 ␮M, respectively, and found an even more pronounced cytoand genotoxic effect of caffeine compared with the mate extract (Figs. 1B, 2B, 3B, 4B and 5B). Our experimental data are in agreement with previous findings concerning in vitro cultured Chinese hamster cells and rat lung alveolar macrophages [21,48]. The cyto- and genotoxic activity of caffeine reported here may be, at least in part, responsible for total effects seen for mate infusion. However, complex relationships in the mixture of substances in mate extract may also be involved in the overall disadvantageous effects of mate extract. Many reports showed that mate drinking may be correlated with the development of several cancer types [6–15]. However, some of these studies were done on populations simultaneously consuming alcohol and/or tobacco and it would be difficult to conclude on this basis that mate consumption is an independent cancer risk factor. Additionally, discrimination between hot/cold mate drinking is important since thermal injury may also account for increased cancer incidence [16,49]. In summary, our data suggest cyto- and genotoxic activity of mate tea and caffeine alone in human lymphocytes cultured in vitro. At present, according to available literature data it is hard to predict whether and how the effects of mate may be extrapolated for in vivo conditions. It is known that coffee or tea drinkers have an average plasma concentration of caffeine of about 10 ␮M. In our hands, under in vitro conditions, caffeine showed an evident deleterious effect with all methods applied, starting from a concentration of 50 ␮M (which corresponds to 10 ␮g/ml). To make it more complex, there is no human epidemiological evidence that caffeine is a carcinogen while it has been shown that coffee drinking may be associated with bladder cancer development [50]. Contradictory outcomes reported concerning the effects of caffeine may be partly due to different in vivo conditions in different studies. Taken together, further studies are required regarding the effects of mate and caffeine alone in vitro and in vivo involving different experimental models. Conflict of interest

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