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e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 7 ( 2 0 1 4 ) 195–201

Available online at www.sciencedirect.com

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valuation of the protective effect of Ilexaraguariensis and Camellia sinensis extractsn the prevention of oxidative damage causedy ultraviolet radiation

arlon Barga, Gislaine T. Rezinb, Daniela D. Leffac, Fernanda Balbinot c,ara M. Gomesa,d, Milena Carvalho-Silvaa,d, Francieli Vuoloa,d,abricia Petronilhob, Felipe Dal-Pizzola,d, Emilio L. Strecka,d,anessa M. Andradec,∗

Laboratório de Bioenergética, Programa de Pós-Graduacão em Ciências da Saúde, Universidade do Extremo Sulatarinense, Criciúma, SC, BrazilLaboratório de Fisiopatologia Clínica e Experimental, Programa de Pós-Graduacão em Ciências da Saúde,niversidade do Sul de Santa Catarina, Tubarão, SC, BrazilLaboratório de Biologia Celular e Molecular, Programa de Pós-Graduacão em Ciências da Saúde,niversidade do Extremo Sul Catarinense, Criciúma, SC, BrazilInstituto Nacional de Ciência e Tecnologia Translacional em Medicina, Hospital de Clínicas de Porto Alegre,orto Alegre, RS, Brazil

r t i c l e i n f o

rticle history:

eceived 26 March 2013

eceived in revised form

November 2013

ccepted 28 November 2013

vailable online 6 December 2013

eywords:

a b s t r a c t

We evaluated the effects green and mate teas on oxidative and DNA damages in rats exposed

to ultraviolet radiation. Were utilized 70 adult male Wistar rats that received daily oral or

topic green or mate tea treatment during exposed to radiation by seven days. After, animals

were killed by decapitation. Thiobarbituric acid-reactive species levels, protein oxidative

damage were evaluated in skin and DNA damage in blood. Our results show that the rats

exposed to ultraviolet radiation presented DNA damage in blood and increased protein car-

bonylation and lipid peroxidation in skin. Oral and topic treatment with green tea and mate

tea prevented lipid peroxidation, both treatments with mate tea also prevented DNA dam-

kin cancer

reen tea

ate tea

NA damage

xidative damage

age. However, only topic treatment with green tea and mate tea prevented increases in

protein carbonylation. Our findings contribute to elucidate the beneficial effects of green

tea and mate tea, here in demonstrated by the antioxidant and antigenotoxic properties

presented by these teas.

© 2013 Elsevier B.V. All rights reserved.

∗ Corresponding author at: Laboratório de Biologia Celular e Molecular,ersitária, 1105, 88806-000 Criciúma, SC, Brazil. Tel.: +55 48 3431 2757.

E-mail address: vmoraesdeandrade@yahoo.com.br (V.M. Andrade).382-6689/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.etap.2013.11.028

Universidade do Extremo Sul Catarinense, UNESC, Avenida Uni-

d p h

196 e n v i r o n m e n t a l t o x i c o l o g y a n

1. Introduction

Over the years, changes in lifestyle significantly increasedhuman exposure to solar radiation, leading to a dramatic risein the incidence of skin cancer (Gruijl, 1999). Photo-inducedcancers are due to a complex multistage phenomenon, medi-ated by alterations in several cellular mechanisms (Sarasin,1999).

Increased ultraviolet radiation exposure is considered themain cause of the rising prevalence of skin cancer (Vrieset al., 2005). In humans and animal models, both ultraviolet Aand B radiations can cause gene mutations (Agar et al., 2004;Melnikova and Ananthaswamy, 2005) and immunity suppres-sion (Halliday et al., 2004; Ullrich, 2005). These two biologicalevents are related to skin cancer caused by ultraviolet radia-tion (Granstein and Matsui, 2004).

Ultraviolet A and B radiations are responsible forirreversible genetic alterations, which ultimately lead toDNA mutations (Sarasin, 1999). The implication of ultravio-let A radiation to this genotoxic damage has been recentlyproven, although its accurate biological effects are still unclear(Bachelor and Bowden, 2004).

The reactive oxygen species production by both ultravioletA and B radiations also contributes to inflammation, immuno-suppression, gene mutation and carcinogenesis (Halliday,2005; Kvam and Tyrrell, 1997). In addition, studies have shownthat oxidative stress also can cause DNA damage (Wang et al.,1998; Watt et al., 2007). DNA damage can include chemicaland structural modifications to purine and pyrimidine basesand 2′-deoxyribose, apart from the formation of single- anddouble-strand breaks. Strand breaks within DNA can occureither directly, due to damage from reactive oxygen speciesexposure, or indirectly, due to cleavage of the DNA backboneduring DNA base excision repair (El-Khamisy and Caldecott,2006; Emerit, 1994).

Previous studies have shown that polyphenols present ingreen tea (Camellia sinensis) can reduce ultraviolet radiations-induced skin cancer in animal model (Mantena et al., 2005).Green tea is manufactured from the fresh leaves of the plant C.sinensis. The leaves of this plant are immersed in boiling watera process that, for the most part, prevents oxidation and poly-merization of the plant’s polyphenols. It is these compoundsthat are thought to be the major chemopreventive mediators(Yusuf et al., 2007).

Another tea presenting antioxidant properties and protec-tion against DNA oxidation is mate tea or yerba mate (Ilexparaguariensis). Mate tea extract, made from dried leaves of I.paraguariensis, is a tea-like beverage consumed in South Amer-ica, mainly in Argentina, Brazil, Paraguay and Uruguay. Its pop-ularity is widely increasing in Europe since it is a commerciallyavailable product sold as antirheumatic, diuretic and centralnervous system stimulant (Bracesco et al., 2003; Gugliucci,1996). Yerba mate is rich in several bioactive compounds suchas caffeine, phenolic acids and saponins, which are absorbedby the body and may act as antioxidants or as free radical scav-

engers (Bastos et al., 2006; Filip et al., 2000; Gugliucci, 1996;Ramirez-Mares et al., 2004; Schinella et al., 2000).

Considering that ultraviolet radiation exposure inducesgene mutations and leads to an increased reactive oxygen

a r m a c o l o g y 3 7 ( 2 0 1 4 ) 195–201

species production, which enhances this harmful effect, andthat green tea and mate tea possibly present antioxidant prop-erties and protection against DNA damage, the present workevaluates the effect of green tea and mate tea on thiobarbituricacid-reactive species levels. Oxidative damage to proteins andDNA damage in rats exposed to ultraviolet radiation were alsoassessed.

2. Materials and methods

2.1. Animals

Adult male Wistar rats (250–300 g) obtained from Central Ani-mal House of Universidade do Extremo Sul Catarinense werehoused in seven groups of ten animals totaling 70 individ-uals with free access to food and water, and maintained ona 12-h light–dark cycle (lights on at 7:00 a.m.) at 22 ◦C ± 1 ◦C.All experimental procedures were carried out in accordancewith the National Institutes of Health Guide for the Care andUse of Laboratory Animals and the Brazilian Society for Neu-roscience and Behavior (SBNeC) recommendations for animalcare, with the approval of local Ethics Committee.

2.2. Experimental procedure

Animals were divided into seven groups, as follows: (1)control group, (2) oral saline + ultraviolet radiation, (3) basegel + ultraviolet radiation, (4) oral green tea + ultraviolet radi-ation, (5) green tea gel + ultraviolet radiation, (6) oral matetea + ultraviolet radiation, (7) mate tea gel + ultraviolet radia-tion. Groups 2, 4, and 6 were treated for seven days orallywith saline, green tea and mate tea (respectively). Groups 3,5, and 7 were treated for seven days topically with base gel,green tea gel and mate tea gel. After treatment, animals wereexposed to ultraviolet radiation for 1 h, once a day. Rats werekilled 24 h after the last exposure session to ultraviolet radi-ation. Blood was collected to evaluate DNA damage and thewhole skin, was removed of the animal to evaluate oxidativedamage parameters.

2.3. Oral administration of the extracts

Green and mate tea dry extracts was obtained from Embra-farma. The solutions were prepared daily in agreement withthe Brazilian Pharmacopeia. Adding 15 mL of boiling waterto 1.875 g of green tea extract and 20 g of mate tea extractto 100 mL of boiling water. After a 15-min extraction period,the solutions were filtered and administered to animals. Theanimals received 0.70 g/kg of mate tea and 0.45 g/kg green teasolutions daily by gavage, 30 min before exposure to ultravioletradiation. Control groups received saline (NaCl 0.9%).

2.4. Topic administration of the extracts

The aqueous extracts were obtained as described above and

were incorporated to base gel (5%, w:w). One gram of gel wasapplied daily on a 6 cm × 3 cm skin area previously shaven oneach rat’s back 30 min before exposure to ultraviolet radiation.Control groups received base gel without any extract.

e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 7 ( 2 0 1 4 ) 195–201 197

Fig. 1 – Comet assay. Evaluation of DNA damage usingethidium bromide (400×). The cells are assessed visuallyand received scores from 0 (undamaged) to 4 (maximallyd

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2

Awbafnel1fp12wDdfmaa(aT0ddb

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Fig. 2 – Mean (±SD) values of damage index observed inperipheral blood of rats treated with green tea and mate tea,oral and topic, and submitted to radiation. *Data significant

amaged), according to the size and shape of the tail.

.5. Exposure to radiation

nimals were exposed to ultraviolet A and B radiations for 7ays. Ultraviolet A and B lamps were used with emission spec-rum of 280–320 nm for UVB, and 320–400 nm for UVA. The rats’ack were at a distance of 60 cm from the fluoresans lamps.ats were irradiated for 1 h per day, all at the same time. Inotal, 70 rats were used in the present study. All animals wereept in cages throughout the experiment (5 animals per cage,4 cages).

.6. Comet assay

standard protocol for Comet assay preparation and analysisas adopted from Tice et al. (2000). The slides were prepared

y mixing 5 �L of whole blood with 95 �L of low-melting-pointgarose (0.75%). The mixture (cells/agarose) was added to aully frosted microscope slide coated with a layer of 500 �L oformally melting agarose (1%). After solidification, the cov-rslip was gently removed, and the slides were placed inysis solution (2.5 M NaCl, 100 mM EDTA, and 10 mM Tris, pH0.0–10.5, with freshly added 1% Triton X-100 and 10% DMSO)or 1 day. Subsequently, the slides were incubated in freshlyrepared alkaline buffer (300 mM NaOH and 1 mM EDTA, pH3) for 20 min. The DNA was electrophoresed for 15 min at5 V (0.90 V/cm) and 300 mA. After electrophoresis, the slidesere neutralized with Tris buffer (0.4 M; pH 7.5). Finally, theNA was stained with ethidium bromide. Images of 100 ran-omly selected cells (50 cells from each of two replicate slides)rom each animal were blindly analyzed using a fluorescence

icroscope equipped with an excitation filter of BP546/12 nmnd a 590 nm barrier filter. Cells were scored from 0 (undam-ged) to 4 (maximally damaged) according to the tail intensitysize and shape), resulting in a single DNA damage score (dam-ge index) for each sample and, consequently, for each group.hus, a damage index (DI) of the group could range from

(completely undamaged 100 = cells × 0) to 400 (maximumamage = 100 cells × 4) (Collins, 2004) (Fig. 1). The percentageamage frequency (DF) was calculated for each sample on theasis of the number of cells with a tail versus with no tail.

.7. Thiobarbituric acid reactive species (TBARS)

s an index of oxidative damage, we used the formationf TBARS during an acid-heating reaction, which is widely

dapted as a sensitive method for measurement of lipid per-xidation, as previously described (Draper and Hadley, 1990).riefly, samples were mixed with 1 mL of trichloroacetic acid0% (TCA) and 1 mL of thiobarbituric acid 0.67% (TBA), and

in relation to control at P < 0.05 (Kruskal–Wallis, Dunn).

then heated in a boiling water bath for 15 min. TBARS weredetermined by absorbance at 535 nm. Results are expressedas malondialdehyde (MDA) equivalents (pmol/mg protein).

2.8. Measurement of protein carbonyls

The oxidative damage to proteins was assessed by thedetermination of carbonyl groups based on the reactionwith dinitrophenylhydrazine (DNPH) as previously described(Levine et al., 1990). Briefly, proteins were precipitated by theaddition of 20% trichloroacetic acid and redissolved in DNPH,and the absorbance read at 370 nm. Results are expressed asprotein carbonyls (pmol/mg protein).

2.9. Statistical analysis

For the analyses of DNA and oxidative damage parameters, alldata were expressed as mean ± standard deviation (SD).

Differences among experimental groups were determinedby one-way analysis of variance (ANOVA) when normal distri-bution was observed. Multiple comparisons were performedby Duncan’ test. When non normal distribution was observed;comparisons were made using the Kruskal–Wallis test withDunn’s test as post hoc.

In all experiments, P < 0.05 was considered to indicate sta-tistical significance. All analyses were performed using the 5.0BioEstat software.

3. Results

The present study showed that ultraviolet radiation expo-sure presented higher levels of DNA damage in peripheralblood in both parameters of the comet assay, damage index(Fig. 2) and damage frequency (Fig. 3) (P < 0.05; Kruskal–Wallis,Dunn). We also showed that was increased thiobarbituric acid-reactive species levels (Fig. 4), and protein carbonylation (Fig. 4)(P < 0.05; ANOVA – Duncan).

Results for animals treated with both types of herbs in thetwo administration routes showed that only mate tea can sig-nificantly reverse DNA damage caused by UV radiation, unlike

green tea, which does not present these protective effects forthe genome, as observed in the high levels of DNA damagewhen orally administered, in comparison to the control group

198 e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 7 ( 2 0 1 4 ) 195–201

Fig. 3 – Mean (±SD) values of damage frequency observedin peripheral blood of rats treated with green tea and matetea, oral and topic, and submitted to radiation. *Datasignificant in relation to control at P < 0.05 (Kruskal–Wallis,

Fig. 5 – Mean (±SD) values of protein carbonyl content inskin of rats treated with green tea and mate tea, oral andtopic, and submitted to radiation. *Data significant in

Dunn).

(P < 0.05; Kruskal–Wallis, Dunn). However, both parameters ofthe comet assay (Figs. 2 and 3) revealed that this herb led to asimilar decrease in DNA damage in animals topically treatedwith mate tea.

Furthermore, we observed that oral and topic treatmentswith green tea or mate tea prevented the increased lipid per-oxidation induced by ultraviolet radiation exposure (Fig. 4)(P < 0.05; ANOVA – Duncan). However, protein carbonylationwas only prevented by topic treatment with green tea gel andmate tea gel. Oral treatment with green or mate teas did notprevent the increase in protein carbonylation caused by ultra-violet radiation exposure (Fig. 5) (P < 0.05; ANOVA – Duncan).

4. Discussion

Exposure to solar ultraviolet radiation induces inflammatoryresponses, oxidative stress, immunosuppression, DNA dam-age and gene mutations, events related to skin diseases,including skin cancer (Katiyar et al., 2000, 2007; Katiyar, 2006).Multiple studies have demonstrated a relationship betweenultraviolet exposure and increased risk of developing skin

cancer. It was postulated that exposure of the skin to solarultraviolet light is a major risk factor for the development ofmalignant melanoma and non-melanoma skin cancer, and

Fig. 4 – Mean (±SD) values of thiobarbituric acid reactivesubstances (TBARS) in skin of rats treated with green teaand mate tea, oral and topic, and submitted to radiation.*Data significant in relation to control at P < 0.05 (one-wayANOVA, Duncan test).

relation to control at P < 0.05 (one-way ANOVA, Duncan test).

that this effect is mediated by a chain of events, includingDNA damage by ultraviolet light and subsequent formationof mutations (Runger, 2003).

In the life of a cell, DNA damage represents a greatthreat to genome stability, leading to loss or amplificationof chromosomal activity, which may result in carcinogene-sis or tissue aging (Nakanishi et al., 2009). Genome integrityis continuously threatened by both endogenous (e.g. reac-tive oxygen species produced by normal metabolism, DNAreplication ‘errors’) and exogenous (ultraviolet light, ionizingradiations and chemical carcinogens) factors (Mocellin et al.,2009).

In the present study we observed the occurrence of DNAdamage in peripheral blood and oxidative damage in the skinof rats exposed to radiation. On the other hand, we showedthat green and mate teas administered through two differentroutes (oral and topic gel) have prevented radiation-inducedlipid peroxidation. However, protein carbonylation was pre-vented only by green and mate teas used as gel. Also, onlytopically administered mate tea prevented the harmful effectsof UV radiation to genetic material.

The study Türkoglu et al. (2010) showed that no UV-inducederythema was observed at the black and green tea gel sites inany of the subjects. UV-induced erythema was consistentlypresent in various grades at caffeine gel, carbomer gel, andcontrol sites. Therefore, tea extracts were found to be promis-ing candidates for their ability to protect against the harmfuleffects of UV radiation, such as erythema and premature agingof the skin.

Studies showed that consumption of green tea polyphe-nols in drinking water prevents photocarcinogenesis in mice;however, the molecular mechanisms underlying this effecthave not been fully elucidated (Meeran et al., 2009). Study ofXu et al. (2010) showed that green tea polyphenols effectivelysuppressed the decrease in viability of the UVB stressedretinal pigment epithelial cells and the UVB suppressionof surviving gene expression level. Green tea polyphenolsalleviated mitochondria dysfunction and DNA fragmentationinduced by UVB.

Polyphenols isolated from the leaves of green tea havea number of beneficial healthy effects, including anti-carcinogenic activity, which has been demonstrated in various

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umor models (Katiyar and Mukhtar, 1996; Yang et al.,002). It has been also shown that oral administration ofn aqueous extract of green tea or green tea polyphe-ols (a mixture of polyphenols) in drinking water inhibitsltraviolet radiation-induced skin carcinogenesis in mice inerms of tumor incidence, tumor multiplicity and tumorrowth/size (Mantena et al., 2005; Wang et al., 1992). Inddition, UV exposure causes physical changes to the skinue to alterations that occur in the connective tissue viahe formation of lipid peroxides, cell contents and enzymesnd reactive oxygen species (Benaiges et al., 1998; Kaurt al., 2006). Other studies showed that green tea also exertsnti-oxidant activity (Mantena et al., 2005; Okayasu et al.,003).

Flavonoids and phenolic compounds were proved to beresent in mate tea leaves, and the consumption of these com-ounds has been associated with the prevention of age-relatedhronic conditions, including cancer (McKay and Blumberg,002; Weisburger, 2002). Various phytochemical constituents,uch as flavonoids, have been isolated from mate plants andelated species, and some of these compounds may be respon-ible for the observed antioxidant activity (Schinella et al.,000). Furthermore, data from in vitro and in vivo studies sug-est a potential beneficial effect of tea polyphenols againstancer at most stages of development (Hirose et al., 2002; Sund Arab, 2002; Tapiero et al., 2002).

In this scenery, study of Chandra and De Mejia (2004)howed that the concentration of polyphenols in green teas major when compared with mate tea. Thus, the concentra-ion of green and mate tea used in the present study result inmounts polyphenols similar in both teas.

Conversely, many studies in cell culture models as wells in animals seem to converge to show an antimutagenicnd DNA protecting effect for I. paraguariensis extracts and itsndividual components, chlorogenic acid, rutin and quercetinBracesco et al., 2003; Miranda et al., 2008). Study of Leonardt al. (2010) indicated that mate scavenged hydroxyl radi-als and superoxide radicals. This study indicates that mateossesses potent antioxidant effects against hydroxyl anduperoxide radicals in chemical and cell culture systems, asell as DNA-protective properties.

Besides, study of Miranda et al. (2008) showed that mateea is not genotoxic in liver, kidney and bladder cells. Theegular ingestion of mate tea increased the resistance ofNA to H2O2-induced DNA strand breaks and improved theNA repair after H2O2 challenge in liver cells. These resultsuggest that mate tea could protect against DNA damagend enhance the DNA repair activity. Protection may bettributed to the antioxidant activity of the mate bioactiveompounds cited above (Bracesco et al., 2003; Miranda et al.,008).

However, study of Wnuk et al. (2009) in vitro cultured humanymphocytes indicates that mate infusion may cause bothytotoxic and genotoxic effects. Differences concentrationsf mate from 1 to 1000 �g/mL was evaluated and found that0 �g/mL of mate significantly increased the frequency oficronuclei and decreased the nuclear division index value.

t the higher concentrations of 100 and 1000 �g/mL, mateas responsible for an augmentation in the level of apop-

otic and necrotic cells. Moreover, no genotoxic effect of mate

m a c o l o g y 3 7 ( 2 0 1 4 ) 195–201 199

(175–1400 �g/mL) on human lymphocytes was reported byAlves et al. (2008).

In conclusion, we showed that the exposure of rats to radia-tion led to DNA and oxidative damage, and that green tea andmate tea have prevented these effects. Our data are in linewith previous works that demonstrated the anti-carcinogenicand antioxidant activities of green tea and mate tea.

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Acknowledgment

This research was supported by grants from Universidade doExtremo Sul Catarinense – UNESC (Brazil).

Appendix A. Supplementary data

Supplementary data associated with this article can be found,in the online version, at http://dx.doi.org/10.1016/j.etap.2013.11.028.

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