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Mutagenesis Advance Access originally published online on February 27, 2008
Mutagenesis 2008 23(4):261-265; doi:10.1093/mutage/gen011
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© The Author 2008. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org.

Protective effects of mate tea (Ilex paraguariensis) on H2O2-induced DNA damage and DNA repair in mice

Daniel D. C. Miranda1, Demétrius P. Arçari1, José Pedrazzoli, Jr1, Patrícia de O. Carvalho2, Suzete M. Cerutti3, Deborah H. M. Bastos4 and Marcelo L. Ribeiro1,*

1Unidade Integrada de Farmacologia e Gastroenterologia, Universidade São Francisco, Av. São Francisco de Assis, 218. Jd. São José, Bragança Paulista, SP, Brazil 2Laboratório Multidisciplinar de Pesquisa, Universidade São Francisco 3Laboratório de Plasticidade Neural e Fitoterápicos, Universidade São Francisco 4Departamento de Nutrição, Escola de Saúde Publica, Universidade de São Paulo, São Paulo, SP, Brazil

Yerba mate (Ilex paraguariensis) is rich in several bioactive compounds that can act as free radical scavengers. Since oxidative DNA damage is involved in various pathological states such as cancer, the aim of this study was to evaluate the antioxidant activity of mate tea as well as the ability to influence DNA repair in male Swiss mice. Forty animals were randomly assigned to four groups. The animals received three different doses of mate tea aqueous extract, 0.5, 1.0 or 2.0 g/kg, for 60 days. After intervention, the liver, kidney and bladder cells were isolated and the DNA damage induced by H2O2 was investigated by the comet assay. The DNA repair process was also investigated for its potential to protect the cells from damage by the same methodology. The data presented here show that mate tea is not genotoxic in liver, kidney and bladder cells. The regular ingestion of mate tea increased the resistance of DNA to H2O2-induced DNA strand breaks and improved the DNA repair after H2O2 challenge in liver cells, irrespective of the dose ingested. These results suggest that mate tea could protect against DNA damage and enhance the DNA repair activity. Protection may be afforded by the antioxidant activity of the mate tea's bioactive compounds.


   Introduction
Top
 Introduction
Materials and methods
 Results
 Discussion
 Funding
 References
 
Yerba mate (Ilex paraguariensis) is one of the most widely consumed plants in South America. It grows naturally or is cultivated in Argentina, Brazil, Uruguay and Paraguay. The leaves are used to prepare different beverages, such as chimarrão (green dried leaves brewed with hot water in a vessel called a ‘cuia’), tererê (green dried leaves brewed with cold water in the same kind of vessels) and the mate tea (roasted leaves brewed with hot water and drank as any other herbal tea). Yerba mate is rich in several bioactive compounds such as caffeine, phenolic acids and saponins, which are absorbed by the body and may act as antioxidants or as free radical scavengers (1Go–10Go). The chlorogenic acids (CGAs), which seem to have antitumor activity as well as the ability to inhibit carcinogenesis, are the main polyphenols in yerba mate; CGAs have revealed properties that may be beneficial during the use of nutraceuticals in cancer therapy (11Go–13Go).

Recent studies in rats fed a diet supplemented with CGA showed that minor hydrolysis of CGA (<1%) occurred in the stomach and small intestine, whereas 15–32% of ingested CGA was hydrolysed into caffeic acid in the cecum. Both compounds appeared early in plasma and urine, suggesting absorption of CGA into the upper part of the gastrointestinal tract. In this study it was also shown that CGA was quickly absorbed in the rat stomach in its intact form (14Go). Additionally, in a model system using HepG2 cells, as a hepatic model system, Mateos et al. (15Go) observed moderate uptake and metabolism of caffeic acid, which underwent methylation, glucuronidation and sulphation. Conversely, CGA showed null metabolism but effective, although limited, uptake by this cell line as a model system of the human liver.

Currently, it is well established that oxidative stress is involved in various pathological states such as cancer, cardiovascular disorders, diabetes, arthritis, inflammation and liver diseases (16Go,17Go). The eukaryotic cells are continuously attacked by reactive oxygen species (ROS), which arise as natural by-products of normal cellular energy production or are generated in large amounts by exhaustive exercise or by chemical agents in the environment (18Go). The oxidative damage to genomic DNA by ROS results in DNA base modifications, single- and double-strand breaks and the formation of apurinic/apyrimidinic lesions (19Go,20Go). Since the oxidized adducts of DNA are pro-mutagenic lesions, if they are not repaired, they can result in mutations (21Go).

Cells have evolved a complex network of defence barriers to counteract the generation of ROS and protect against the oxidation of macromolecules by scavenging ROS. The dietary intake of antioxidants is thought to play a major role in this network (22Go). The beneficial effects of dietary polyphenols on human health have been widely assumed to act through various biological effects, such as free radical scavenging, metal chelation, modulation of enzymatic activity and alterations in the signal transduction pathway (23Go–25Go). Epidemiological studies have also highlighted the association between the consumption of polyphenol-rich food and beverages and the prevention of various human diseases such as cancer, coronary heart disease and inflammation (26Go–29Go).

The integrity of DNA is vital to cell division and oxidative alterations can disrupt transcription, translation, DNA replication and can give rise to mutations, cell senescence and death. DNA damage may either cause cell death or initiate several error-free or -prone repair pathways (30Go). Thus, the aim of the present investigation was to study the effects of mate tea on oxidative DNA damage and the resistance of DNA against H2O2-induced DNA strand breaks in liver, kidney and bladder cells from Swiss mice ex vivo. The DNA repair process was also investigated for its potential to protect the cells from damage using single-cell microgel electrophoresis (comet assay) (31Go)


   Materials and methods
Top
 Introduction
 Materials and methods
Results
 Discussion
 Funding
 References
 
Mate tea preparation and administration
The mate tea used in this study contained 350 mg/g of phenolic compounds, determined by the Folin–Ciocalteau methodology, using 5-caffeoylquinic acid as standard for the calibration curve.

The roasted I. paraguariensis mate tea beverage was prepared by dissolving lyophilized instant mate tea (Leao Jr, Curitiba-PR, Brazil) in water using a homogenizer and was prepared fresh each day. The aqueous mate tea and vehicle (pure water) were administered by intragastric gavage, to guarantee total ingestion.

Animals
Forty male Swiss mice (30–35 g) free of specific pathogens were obtained from Centro Multidisciplinar de Bioterismo (UNICAMP, Campinas, SP, Brazil) and were kept in groups of five animals per cage. Throughout the experiment, animals were housed in a room under a 12:12 h dark:light cycle (lights turned on at 6:00 a.m.) with controlled temperature (22 ± 2°C) and relative humidity (53 ± 2), with free access to food and water.

The mice were randomly assigned to four groups in accordance to the intervention and dose used. The animals were treated for 60 consecutive days and received three different doses of aqueous extract of roasted mate: 0.5 g/kg (n = 10), 1.0 g/kg (n = 10) or 2.0 g/kg (n = 10). Other animals were used as a control group and received water (control group; n = 10). This is equal to that found in 0.75, 1.5 and 3 l/day of mate tea, respectively. After intervention, mice were deeply anaesthetized (1:1 xylazine:ketamine) and sacrificed by a transcardiac perfusion with 70 ml isotonic saline solution (4°C) over a period of 6 min. The livers, kidneys and bladders were removed and maintained in a fixative solution, described below.

The procedures used for the manipulation of animals were in agreement with the Ethical Principles in Animal Research, adopted by the Brazilian College for Animal Experimentation according to the American Psychological Association Guidelines for Ethical Conduct in the Care and Use of Animals.

Comet assay
The comet assay detects DNA damage (strand breaks and alkali-labile sites) at the individual cell level. Cells from liver, kidney and bladder biopsies were isolated as described below. The biopsies were pooled and incubated with 5.5 mg proteinase K (Sigma–Aldrich, St Louis, MO, USA) and 3 mg collagenase (Invitrogen Life Technologies, Grand Island, NY, USA) in 3 ml of Hank's balanced salt solution (HBSS; Invitrogen Life Technologies) for 45 min at 37°C to liberate the cells; the cells were then re-suspended in 10 ml of HBSS. The resulting suspensions were centrifuged at 750 x g for 5 min and the supernatant was discarded.

Since a high leukocyte content could lead to a bias in the levels of DNA damage from liver, kidney and bladder cells, leukocyte contamination was assessed in the cell suspensions. Aliquots of 100 µl were dropped onto a slide, fixed with acetone and stained with haematoxylin and eosin. The slides were analysed by blinded examiners for leukocyte levels.

Cell viability. The comet assay should be performed only on samples having a cell viability of >75%. Therefore, cell viability (liver: 82–96%, with a mean of 92%; kidney: 78–90%, with a mean of 82% and bladder: 80–91%, with a mean of 84%; after H2O2-treatment mean of 87%, 76% and 77%, for liver, kidney and bladder cells, respectively) was determined using the fluorescein diacetate (FDA)/ethidium bromide (EtBr; Sigma–Aldrich) assay. Briefly, a fresh staining solution was prepared containing 30 µl FDA in acetone (5 mg/ml), 200 µl EtBr in phosphate-buffered saline (PBS, 200 µg/ml) and 4.8 ml PBS (Invitrogen Life Technologies). The single-cell suspension (25 µl) was then mixed with 25 µl of the staining solution, spread onto a slide and covered with a coverslip. Viable cells appeared fluorescent green, whereas red-stained nuclei indicated dead cells. At least 200 cells were counted per sample.

Determination of DNA damage. The alkaline comet assay was performed on the single-cell suspensions, according to Singh et al. (32Go), but with some modifications. Briefly, 15 µl of the single-cell suspension (~2 x 104 cells) were mixed with molten 0.5% low-melting-point agarose (Promega Co., Madison, WI, USA) and spread on agarose-precoated microscope slides. DNA strand breaks were introduced ex vivo by exposing the cells to 100 µM H2O2 in salt-free PBS (Invitrogen Life Technologies) on ice for 30 min prior to embedding in agarose. The slides were immersed overnight at 4°C in freshly prepared cold lysing solution (2.5 M NaCl, 100 mM ethylenediaminetetraacetic acid (EDTA), 10 mM Tris, 2% sodium salt N-lauryl sarcosine, pH 10, with 1% Triton X-100 and 10% dimethyl sulphoxide; all from Sigma–Aldrich). Subsequently, the cells were exposed to alkaline buffer (1 mM EDTA and 300 mM NaOH, pH ~= 13.4) at 4°C for 40 min to allow DNA unwinding and expression of alkali-labile sites. Electrophoresis was then conducted in the same solution at 4°C for 20 min using 25 V and 300 mA.

To assess DNA repair the single-cell suspension was mixed with molten 0.5% low-melting-point agarose and spread on agarose-precoated microscope slides, which were placed in tubes with PBS (pH 7.4) before lysis and incubated for 1 h at 37°C. Additionally, DNA strand breaks were also introduced ex vivo by exposing the cells to 100 µM H2O2 on ice for 30 min prior to embedding in agarose. After that the slides were placed in tubes with PBS (pH 7.4) before lysis and incubated for 1 h at 37°C. After incubation, the slides were washed in cold PBS; lysis, denaturation and electrophoresis were then performed in the same manner as described above. After electrophoresis, the slides were neutralized (0.4 M Tris, pH 7.5), stained with 40 µl EtBr (20 µg/ml) and analysed with a fluorescence microscope (Eclipse E400; Nikon, Melville, NY, USA), using an image analysis system (Komet 5.5; Kinetic Imaging, Nottingham, UK). Two hundred randomly selected cells (100 from each of two replicate slides) were evaluated from each sample and the mean of the olive tail moment DNA was determined. Tail moment (TM) is defined as the product of DNA in the tail and the mean distance of migration in the tail and is calculated by multiplying tail intensity/sum comet intensity by the tail's centre of gravity – peak position. A higher percentage of tail DNA signifies a higher level of DNA damage.

Statistical analysis
All data are expressed as the mean ± standard deviation. The statistical analysis consisted of the application of one-way analysis of variance followed by Bonferroni's post hoc test for multiple comparisons. The dose–response effect was evaluated by means of a simple regression linear model. Statistical tests were performed using BioEstat 1.0 and an associated probability (P-value) <5% was considered significant.


   Results
Top
 Introduction
 Materials and methods
 Results
Discussion
 Funding
 References
 
The levels of DNA damage in the liver, kidney and bladder cells in the control group did not differ from those observed in animals treated with mate tea at 0.5, 1.0 and 2.0 g/kg (Table I). The resistance of liver, kidney and bladder cell DNA to oxidative attack ex vivo was assessed as an indicator of antioxidant status. We chose a high concentration of H2O2 (100 µM) to exacerbate DNA damage and pinpoint cells able to protect themselves from oxidative stress. Our results demonstrate that mate tea was able to decrease H2O2-induced DNA breakage after intervention only in liver cells (Table I). The detected DNA damage levels, in the liver cells, were significantly lower in animals treated with 0.5 g/kg (TM = 1.72 ± 0.23), 1.0 g/kg (TM = 1.66 ± 0.22) and 2.0 g/kg mate tea (TM = 1.27 ± 0.10) than those of the control group (TM = 2.44 ± 0.32; Table I). Although we found an increase in the resistance of DNA to H2O2-induced DNA strand breaks, results did not show a dose–response effect ({rho} = 0.13).


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  		Table I. Effect of mate tea intervention (0.5, 1.0 and 2.0 g/kg) on DNA damage levels in cells isolated from liver, kidney and bladder before and after 100 µM H2O2 challenge

 
Since the lower steady-state level of DNA oxidation could result from an increased rate of DNA repair, as well as from enhanced antioxidant status, we studied the DNA repair process ex vivo in liver cells. The results shown in Figure 1 demonstrate that intervention with 2.0 g/kg of mate tea was able to significantly increase the DNA repair activity ({rho} = 0.04).


Figure 1
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  		Fig. 1. DNA damage and DNA repair activity in liver cells from mice after the intervention with mate tea (0.5, 1.0 and 2.0 g/kg). The columns represent the means ± standard deviations of 10 mice; *P < 0.05, compared to DNA damage at 2.0 g/kg.

 
We also checked the DNA repair after H2O2 challenge (Figure 2). The data in Figure 2 indicate that the intervention with mate tea significantly increased the ability to remove the DNA damage generated by H2O2 in liver cells isolated from animals treated with 0.5 g/kg (TM = 0.71 ± 0.14), 1.0 g/kg (TM = 0.70 ± 0.12) and 2.0 g/kg of mate tea (TM = 0.59 ± 0.10), when compared to the control group (TM = 1.58 ± 0.30, Figure 2). The results did not show a dose–response effect ({rho} = 0.15).


Figure 2
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  		Fig. 2. DNA damage and DNA repair activity induced by challenge with 100 µM H2O2 in liver cells from mice pretreated with mate tea (0.5, 1.0 and 2.0 g/kg). The columns represent the means ± standard deviations of 10 mice; *P < 0.05, compared to the control group.

 

   Discussion
Top
 Introduction
 Materials and methods
 Results
 Discussion
Funding
 References
 
There is evidence that polyphenol compounds may have beneficial effects on human health and that some may exert antioxidant activity. Thus, it is expected that individuals who consume diets with a high content of phenolic antioxidant should be better protected against oxidative cellular damage than individuals who do not (23Go–25Go). Polyphenols have been proven to act as potent antioxidants, protecting the body's tissues from oxidative stress. In this sense, several studies in animal models have shown that tea polyphenols can prevent against chemically induced carcinogenesis in lung, stomach, oesophagus, duodenum, pancreas, breast, colon and liver (33Go–36Go).

In this study, mate tea (I. paraguariensis) was tested for its antioxidant activity in liver, kidney and bladder cells. The putative antioxidant effect of mate tea was investigated in mice liver, kidney and bladder cells, ex vivo, before and after a H2O2 challenge by the comet assay; this assay has been explored as a potential tool for detecting the non-genotoxic and antioxidant effects of food or nutrients (37Go). Hydrogen peroxide causes DNA strand breakage by generating hydroxyl radicals close to the DNA molecule, via the Fenton reaction (38Go). Thus, antioxidant activity is assessed as the decreased induction of DNA breaks. Additionally, the DNA repair activity was evaluated by its potential to protect the cells from DNA damage.

Phenolic compounds present in roasted mate are mainly CGAs [monocaffeoylquinic acid (CQA) and dicaffeoylquinic acid (diCQA)] and hydroxycinnamic acids (caffeic acid, quinic acid). Earlier studies showed that rutin, a flavonoid present in the green yerba mate leaves, is lost after the roasting process (39Go). Although the biological properties of CGA depend on its absorption in the gut and on its metabolism, little is known about the bioavailability of CGA. Lafay et al. (14Go) showed an absorption of CGA into the upper part of the gastrointestinal tract. In this study it was shown also that CGA was quickly absorbed in the rat stomach in its intact form. In humans Monteiro et al. (40Go) showed that CGA compounds are differentially absorbed and/or metabolized in humans, and, despite the large inter-individual variation, CQA isomers and diCQA isomers were identified in the plasma of all subjects after coffee consumption. In a model system using HepG2 cells, as a hepatic model system, Mateos et al. (15Go) observed moderate uptake and metabolism of caffeic acid. This behaviour might explain the antioxidant activity observed in the liver after mate tea (rich in CGA and caffeic acid) ingestion.

Although it has been reported that mate tea has several beneficial effects, there are some studies reporting an increased risk of bladder cancer and renal cell carcinoma associated with mate consumption in humans (41Go–44Go). Hypothetically, this may be attributed to the presence of some carcinogens in mate's constitution. Experimental studies have shown that caffeic acid (a metabolite of CGA) has a carcinogenic effect on the kidney in mice (45Go). Additionally, Fonseca et al. (46Go) suggested that mate tea displays mutagenic and clastogenic activities in cell culture. The data presented herein show that mate tea is not genotoxic in liver, kidney and bladder cells, since the levels of DNA damage remained unaltered after the intervention period.

Our results demonstrate that regular ingestion of mate tea increase the resistance of DNA to H2O2-induced DNA strand breaks in liver cells, irrespective of the dose ingested. The observed protection may be related to the presence of polyphenolic compounds; CGA (the main phenolic compound in mate leaves) are potent antioxidant compounds and may act as hydrogen or electron donors as well as transition metal ion chelators (47Go). Several authors have reported increased antioxidant and anticarcinogenic activities due to these compounds (48Go–52Go).

Compared with normal cells, cancer cells constitutively generate large but non-lethal amounts of ROS that apparently function as signalling molecules, constantly activating redox-sensitive transcription factors and responsive genes. These gene products are involved in the survival of cancer cells, as well as their proliferation (53Go). Thus, the reduction in oxidative stress may suppress the proliferation of tumour cells (52Go,53Go). Feng et al. (51Go) reported molecular evidence for the anticarcinogenic potential of CGA, showing that it displayed a stronger antioxidant activity than ascorbic acid. Additionally, these authors showed an in vitro inhibitory effect of CGA on the proliferation of cancer cells, probably due to its antioxidant properties, via the up-regulation of cellular antioxidant enzymes, disturbing the favourable redox condition in cancer cells.

The consumption of mate tea may be an effective way to protect against the DNA damage that has been shown to cause mutations through miscoding and, therefore, responsible for initiating carcinogenesis. However, further studies are necessary to evaluate the role of mate tea in the modulation of the expression of key genes involved in carcinogenesis, as well as in the organism's endogenous antioxidant defences.

DNA damage is countered in cells by DNA repair, which is a basic and universal process to protect the genetic integrity of organisms. The DNA repair enzymes continuously monitor chromosomes to correct DNA damage. If left unrepaired, DNA damage can lead to biological consequences in cells, including cell death, mutations and transformation of cells to malignant ones. Therefore, DNA repair is regarded as one of the essential events in all life forms (54Go). The lower steady-state level of DNA oxidation could result from an increased rate of DNA repair, as well as from enhanced antioxidant status, indicating the potential of mate tea to enhance the DNA repair process. Furthermore, after H2O2 challenge, mate tea intervention did improve the DNA repair, irrespective of the dose ingested.

Although our data demonstrate that the intervention of mate tea was able to increase the DNA repair activity in liver cells, Torbergsen and Collins (55Go) reported that the capacity of lymphocytes, isolated from healthy volunteers supplemented with carotenoids, to recover the DNA damage seems to be an antioxidant effect against the additional damage induced by atmospheric oxygen rather than a stimulation on DNA repair enzymes. Therefore, expression assays are necessary to evaluate the effects of mate tea on DNA repair activity without the atmospheric oxygen bias.


   Funding
Top
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
References
 
Funding by Leão Junior S/A and FAPESP (2006/61797-0)


   Acknowledgments
 
Conflict of interest statement: None declared.


   Notes
 
* To whom correspondence should be addressed. Tel: +55 11 4034 8135; Fax: +55 11 40341825; Email: marcelo.ribeiro{at}saofrancisco.edu.br


   References
Top
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 

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Received on October 26, 2007; revised on December 5, 2007; accepted on January 31, 2008.


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