- Email: [email protected]
Evaluation of the protective effect of Ilex paraguariensis and Camellia sinensis extracts on the prevention of oxidative damage caused by ultraviolet radiation
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
ScienceDirect journal homepage: www.elsevier.com/locate/etap
Evaluation of the protective effect of Ilex paraguariensis and Camellia sinensis extracts on the prevention of oxidative damage caused by ultraviolet radiation Marlon Barg a , Gislaine T. Rezin b , Daniela D. Leffa c , Fernanda Balbinot c , Lara M. Gomes a,d , Milena Carvalho-Silva a,d , Francieli Vuolo a,d , Fabricia Petronilho b , Felipe Dal-Pizzol a,d , Emilio L. Streck a,d , Vanessa M. Andrade c,∗ a
Laboratório de Bioenergética, Programa de Pós-Graduac¸ão em Ciências da Saúde, Universidade do Extremo Sul Catarinense, Criciúma, SC, Brazil b Laboratório de Fisiopatologia Clínica e Experimental, Programa de Pós-Graduac¸ão em Ciências da Saúde, Universidade do Sul de Santa Catarina, Tubarão, SC, Brazil c Laboratório de Biologia Celular e Molecular, Programa de Pós-Graduac¸ão em Ciências da Saúde, Universidade do Extremo Sul Catarinense, Criciúma, SC, Brazil d Instituto Nacional de Ciência e Tecnologia Translacional em Medicina, Hospital de Clínicas de Porto Alegre, Porto Alegre, RS, Brazil
a r t i c l e
i n f o
a b s t r a c t
Article history:
We evaluated the effects green and mate teas on oxidative and DNA damages in rats exposed
Received 26 March 2013
to ultraviolet radiation. Were utilized 70 adult male Wistar rats that received daily oral or
Received in revised form
topic green or mate tea treatment during exposed to radiation by seven days. After, animals
3 November 2013
were killed by decapitation. Thiobarbituric acid-reactive species levels, protein oxidative
Accepted 28 November 2013
damage were evaluated in skin and DNA damage in blood. Our results show that the rats
Available online 6 December 2013
exposed to ultraviolet radiation presented DNA damage in blood and increased protein car-
Keywords:
tea prevented lipid peroxidation, both treatments with mate tea also prevented DNA dam-
Skin cancer
age. However, only topic treatment with green tea and mate tea prevented increases in
bonylation and lipid peroxidation in skin. Oral and topic treatment with green tea and mate
Green tea
protein carbonylation. Our findings contribute to elucidate the beneficial effects of green
Mate tea
tea and mate tea, here in demonstrated by the antioxidant and antigenotoxic properties
DNA damage
presented by these teas.
Oxidative damage
© 2013 Elsevier B.V. All rights reserved.
∗ Corresponding author at: Laboratório de Biologia Celular e Molecular, Universidade do Extremo Sul Catarinense, UNESC, Avenida Universitária, 1105, 88806-000 Criciúma, SC, Brazil. Tel.: +55 48 3431 2757. E-mail address: [email protected] (V.M. Andrade). 1382-6689/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.etap.2013.11.028
196
1.
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
Introduction
Over the years, changes in lifestyle significantly increased human exposure to solar radiation, leading to a dramatic rise in the incidence of skin cancer (Gruijl, 1999). Photo-induced cancers are due to a complex multistage phenomenon, mediated by alterations in several cellular mechanisms (Sarasin, 1999). Increased ultraviolet radiation exposure is considered the main cause of the rising prevalence of skin cancer (Vries et al., 2005). In humans and animal models, both ultraviolet A and B radiations can cause gene mutations (Agar et al., 2004; Melnikova and Ananthaswamy, 2005) and immunity suppression (Halliday et al., 2004; Ullrich, 2005). These two biological events are related to skin cancer caused by ultraviolet radiation (Granstein and Matsui, 2004). Ultraviolet A and B radiations are responsible for irreversible genetic alterations, which ultimately lead to DNA mutations (Sarasin, 1999). The implication of ultraviolet A radiation to this genotoxic damage has been recently proven, although its accurate biological effects are still unclear (Bachelor and Bowden, 2004). The reactive oxygen species production by both ultraviolet A and B radiations also contributes to inflammation, immunosuppression, gene mutation and carcinogenesis (Halliday, 2005; Kvam and Tyrrell, 1997). In addition, studies have shown that oxidative stress also can cause DNA damage (Wang et al., 1998; Watt et al., 2007). DNA damage can include chemical and structural modifications to purine and pyrimidine bases and 2 -deoxyribose, apart from the formation of single- and double-strand breaks. Strand breaks within DNA can occur either directly, due to damage from reactive oxygen species exposure, or indirectly, due to cleavage of the DNA backbone during DNA base excision repair (El-Khamisy and Caldecott, 2006; Emerit, 1994). Previous studies have shown that polyphenols present in green tea (Camellia sinensis) can reduce ultraviolet radiationsinduced 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 water a process that, for the most part, prevents oxidation and polymerization of the plant’s polyphenols. It is these compounds that are thought to be the major chemopreventive mediators (Yusuf et al., 2007). Another tea presenting antioxidant properties and protection against DNA oxidation is mate tea or yerba mate (Ilex paraguariensis). Mate tea extract, made from dried leaves of I. 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 (Bracesco et al., 2003; Gugliucci, 1996). 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 (Bastos et al., 2006; Filip et al., 2000; Gugliucci, 1996; Ramirez-Mares et al., 2004; Schinella et al., 2000). Considering that ultraviolet radiation exposure induces gene mutations and leads to an increased reactive oxygen
species production, which enhances this harmful effect, and that green tea and mate tea possibly present antioxidant properties and protection against DNA damage, the present work evaluates the effect of green tea and mate tea on thiobarbituric acid-reactive species levels. Oxidative damage to proteins and DNA damage in rats exposed to ultraviolet radiation were also assessed.
2.
Materials and methods
2.1.
Animals
Adult male Wistar rats (250–300 g) obtained from Central Animal House of Universidade do Extremo Sul Catarinense were housed in seven groups of ten animals totaling 70 individuals with free access to food and water, and maintained on a 12-h light–dark cycle (lights on at 7:00 a.m.) at 22 ◦ C ± 1 ◦ C. All experimental procedures were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the Brazilian Society for Neuroscience and Behavior (SBNeC) recommendations for animal care, 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) base gel + ultraviolet radiation, (4) oral green tea + ultraviolet radiation, (5) green tea gel + ultraviolet radiation, (6) oral mate tea + ultraviolet radiation, (7) mate tea gel + ultraviolet radiation. Groups 2, 4, and 6 were treated for seven days orally with 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 were exposed to ultraviolet radiation for 1 h, once a day. Rats were killed 24 h after the last exposure session to ultraviolet radiation. Blood was collected to evaluate DNA damage and the whole skin, was removed of the animal to evaluate oxidative damage parameters.
2.3.
Oral administration of the extracts
Green and mate tea dry extracts was obtained from Embrafarma. The solutions were prepared daily in agreement with the Brazilian Pharmacopeia. Adding 15 mL of boiling water to 1.875 g of green tea extract and 20 g of mate tea extract to 100 mL of boiling water. After a 15-min extraction period, the solutions were filtered and administered to animals. The animals received 0.70 g/kg of mate tea and 0.45 g/kg green tea solutions daily by gavage, 30 min before exposure to ultraviolet radiation. 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 was applied daily on a 6 cm × 3 cm skin area previously shaven on each 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 using ethidium bromide (400×). The cells are assessed visually and received scores from 0 (undamaged) to 4 (maximally damaged), according to the size and shape of the tail.
2.5.
Exposure to radiation
Animals were exposed to ultraviolet A and B radiations for 7 days. Ultraviolet A and B lamps were used with emission spectrum of 280–320 nm for UVB, and 320–400 nm for UVA. The rats’ back were at a distance of 60 cm from the fluoresans lamps. Rats were irradiated for 1 h per day, all at the same time. In total, 70 rats were used in the present study. All animals were kept in cages throughout the experiment (5 animals per cage, 14 cages).
2.6.
Comet assay
A standard protocol for Comet assay preparation and analysis was adopted from Tice et al. (2000). The slides were prepared by mixing 5 L of whole blood with 95 L of low-melting-point agarose (0.75%). The mixture (cells/agarose) was added to a fully frosted microscope slide coated with a layer of 500 L of normally melting agarose (1%). After solidification, the coverslip was gently removed, and the slides were placed in lysis solution (2.5 M NaCl, 100 mM EDTA, and 10 mM Tris, pH 10.0–10.5, with freshly added 1% Triton X-100 and 10% DMSO) for 1 day. Subsequently, the slides were incubated in freshly prepared alkaline buffer (300 mM NaOH and 1 mM EDTA, pH 13) for 20 min. The DNA was electrophoresed for 15 min at 25 V (0.90 V/cm) and 300 mA. After electrophoresis, the slides were neutralized with Tris buffer (0.4 M; pH 7.5). Finally, the DNA was stained with ethidium bromide. Images of 100 randomly selected cells (50 cells from each of two replicate slides) from each animal were blindly analyzed using a fluorescence microscope equipped with an excitation filter of BP546/12 nm and a 590 nm barrier filter. Cells were scored from 0 (undamaged) to 4 (maximally damaged) according to the tail intensity (size and shape), resulting in a single DNA damage score (damage index) for each sample and, consequently, for each group. Thus, a damage index (DI) of the group could range from 0 (completely undamaged 100 = cells × 0) to 400 (maximum damage = 100 cells × 4) (Collins, 2004) (Fig. 1). The percentage damage frequency (DF) was calculated for each sample on the basis of the number of cells with a tail versus with no tail.
2.7.
Thiobarbituric acid reactive species (TBARS)
As an index of oxidative damage, we used the formation of TBARS during an acid-heating reaction, which is widely adapted as a sensitive method for measurement of lipid peroxidation, as previously described (Draper and Hadley, 1990). Briefly, samples were mixed with 1 mL of trichloroacetic acid 10% (TCA) and 1 mL of thiobarbituric acid 0.67% (TBA), and
Fig. 2 – Mean (±SD) values of damage index observed in peripheral blood of rats treated with green tea and mate tea, oral and topic, and submitted to radiation. *Data significant in relation to control at P < 0.05 (Kruskal–Wallis, Dunn).
then heated in a boiling water bath for 15 min. TBARS were determined by absorbance at 535 nm. Results are expressed as malondialdehyde (MDA) equivalents (pmol/mg protein).
2.8.
Measurement of protein carbonyls
The oxidative damage to proteins was assessed by the determination of carbonyl groups based on the reaction with dinitrophenylhydrazine (DNPH) as previously described (Levine et al., 1990). Briefly, proteins were precipitated by the addition of 20% trichloroacetic acid and redissolved in DNPH, and the absorbance read at 370 nm. Results are expressed as protein carbonyls (pmol/mg protein).
2.9.
Statistical analysis
For the analyses of DNA and oxidative damage parameters, all data were expressed as mean ± standard deviation (SD). Differences among experimental groups were determined by one-way analysis of variance (ANOVA) when normal distribution was observed. Multiple comparisons were performed by Duncan’ test. When non normal distribution was observed; comparisons were made using the Kruskal–Wallis test with Dunn’s test as post hoc. In all experiments, P < 0.05 was considered to indicate statistical significance. All analyses were performed using the 5.0 BioEstat software.
3.
Results
The present study showed that ultraviolet radiation exposure presented higher levels of DNA damage in peripheral blood 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 acidreactive species levels (Fig. 4), and protein carbonylation (Fig. 4) (P < 0.05; ANOVA – Duncan). Results for animals treated with both types of herbs in the two administration routes showed that only mate tea can significantly reverse DNA damage caused by UV radiation, unlike green tea, which does not present these protective effects for the genome, as observed in the high levels of DNA damage when 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 observed in peripheral blood of rats treated with green tea and mate tea, oral and topic, and submitted to radiation. *Data significant in relation to control at P < 0.05 (Kruskal–Wallis, Dunn).
(P < 0.05; Kruskal–Wallis, Dunn). However, both parameters of the comet assay (Figs. 2 and 3) revealed that this herb led to a similar decrease in DNA damage in animals topically treated with mate tea. Furthermore, we observed that oral and topic treatments with green tea or mate tea prevented the increased lipid peroxidation induced by ultraviolet radiation exposure (Fig. 4) (P < 0.05; ANOVA – Duncan). However, protein carbonylation was only prevented by topic treatment with green tea gel and mate tea gel. Oral treatment with green or mate teas did not prevent the increase in protein carbonylation caused by ultraviolet radiation exposure (Fig. 5) (P < 0.05; ANOVA – Duncan).
4.
Discussion
Exposure to solar ultraviolet radiation induces inflammatory responses, oxidative stress, immunosuppression, DNA damage and gene mutations, events related to skin diseases, including skin cancer (Katiyar et al., 2000, 2007; Katiyar, 2006). Multiple studies have demonstrated a relationship between ultraviolet exposure and increased risk of developing skin cancer. It was postulated that exposure of the skin to solar ultraviolet light is a major risk factor for the development of malignant melanoma and non-melanoma skin cancer, and
Fig. 4 – Mean (±SD) values of thiobarbituric acid reactive substances (TBARS) in skin of rats treated with green tea and mate tea, oral and topic, and submitted to radiation. *Data significant in relation to control at P < 0.05 (one-way ANOVA, Duncan test).
Fig. 5 – Mean (±SD) values of protein carbonyl content in skin of rats treated with green tea and mate tea, oral and topic, and submitted to radiation. *Data significant in relation to control at P < 0.05 (one-way ANOVA, Duncan test).
that this effect is mediated by a chain of events, including DNA damage by ultraviolet light and subsequent formation of mutations (Runger, 2003). In the life of a cell, DNA damage represents a great threat to genome stability, leading to loss or amplification of chromosomal activity, which may result in carcinogenesis or tissue aging (Nakanishi et al., 2009). Genome integrity is continuously threatened by both endogenous (e.g. reactive oxygen species produced by normal metabolism, DNA replication ‘errors’) and exogenous (ultraviolet light, ionizing radiations and chemical carcinogens) factors (Mocellin et al., 2009). In the present study we observed the occurrence of DNA damage in peripheral blood and oxidative damage in the skin of rats exposed to radiation. On the other hand, we showed that green and mate teas administered through two different routes (oral and topic gel) have prevented radiation-induced lipid peroxidation. However, protein carbonylation was prevented only by green and mate teas used as gel. Also, only topically administered mate tea prevented the harmful effects of UV radiation to genetic material. ˘ et al. (2010) showed that no UV-induced The study Türkoglu erythema was observed at the black and green tea gel sites in any of the subjects. UV-induced erythema was consistently present in various grades at caffeine gel, carbomer gel, and control sites. Therefore, tea extracts were found to be promising candidates for their ability to protect against the harmful effects of UV radiation, such as erythema and premature aging of the skin. Studies showed that consumption of green tea polyphenols in drinking water prevents photocarcinogenesis in mice; however, the molecular mechanisms underlying this effect have not been fully elucidated (Meeran et al., 2009). Study of Xu et al. (2010) showed that green tea polyphenols effectively suppressed the decrease in viability of the UVB stressed retinal pigment epithelial cells and the UVB suppression of surviving gene expression level. Green tea polyphenols alleviated mitochondria dysfunction and DNA fragmentation induced by UVB. Polyphenols isolated from the leaves of green tea have a number of beneficial healthy effects, including anticarcinogenic activity, which has been demonstrated in various
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
tumor models (Katiyar and Mukhtar, 1996; Yang et al., 2002). It has been also shown that oral administration of an aqueous extract of green tea or green tea polyphenols (a mixture of polyphenols) in drinking water inhibits ultraviolet radiation-induced skin carcinogenesis in mice in terms of tumor incidence, tumor multiplicity and tumor growth/size (Mantena et al., 2005; Wang et al., 1992). In addition, UV exposure causes physical changes to the skin due to alterations that occur in the connective tissue via the formation of lipid peroxides, cell contents and enzymes and reactive oxygen species (Benaiges et al., 1998; Kaur et al., 2006). Other studies showed that green tea also exerts anti-oxidant activity (Mantena et al., 2005; Okayasu et al., 2003). Flavonoids and phenolic compounds were proved to be present in mate tea leaves, and the consumption of these compounds has been associated with the prevention of age-related chronic conditions, including cancer (McKay and Blumberg, 2002; Weisburger, 2002). Various phytochemical constituents, such as flavonoids, have been isolated from mate plants and related species, and some of these compounds may be responsible for the observed antioxidant activity (Schinella et al., 2000). Furthermore, data from in vitro and in vivo studies suggest a potential beneficial effect of tea polyphenols against cancer at most stages of development (Hirose et al., 2002; Su and Arab, 2002; Tapiero et al., 2002). In this scenery, study of Chandra and De Mejia (2004) showed that the concentration of polyphenols in green tea is major when compared with mate tea. Thus, the concentration of green and mate tea used in the present study result in amounts polyphenols similar in both teas. Conversely, many studies in cell culture models as well as in animals seem to converge to show an antimutagenic and DNA protecting effect for I. paraguariensis extracts and its individual components, chlorogenic acid, rutin and quercetin (Bracesco et al., 2003; Miranda et al., 2008). Study of Leonard et al. (2010) indicated that mate scavenged hydroxyl radicals and superoxide radicals. This study indicates that mate possesses potent antioxidant effects against hydroxyl and superoxide radicals in chemical and cell culture systems, as well as DNA-protective properties. Besides, study of Miranda et al. (2008) showed that mate tea is not genotoxic in liver, kidney and bladder cells. The regular ingestion of mate tea increased the resistance of DNA to H2 O2 -induced DNA strand breaks and improved the DNA repair after H2 O2 challenge in liver cells. These results suggest that mate tea could protect against DNA damage and enhance the DNA repair activity. Protection may be attributed to the antioxidant activity of the mate bioactive compounds cited above (Bracesco et al., 2003; Miranda et al., 2008). However, study of Wnuk et al. (2009) in vitro cultured human lymphocytes indicates that mate infusion may cause both cytotoxic and genotoxic effects. Differences concentrations of mate from 1 to 1000 g/mL was evaluated and found that 10 g/mL of mate significantly increased the frequency of micronuclei and decreased the nuclear division index 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. Moreover, no genotoxic effect of mate
199
(175–1400 g/mL) on human lymphocytes was reported by Alves et al. (2008). In conclusion, we showed that the exposure of rats to radiation led to DNA and oxidative damage, and that green tea and mate tea have prevented these effects. Our data are in line with previous works that demonstrated the anti-carcinogenic and 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 do Extremo 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.
references
Agar, N.S., Halliday, G.M., Barnetson, R.S., Ananthaswamy, H.N., Wheeler, M., Jones, A.M., 2004. The basal layer in human squamous tumors harbors more UVA than UVB fingerprint mutations: a role for UVA in human skin carcinogenesis. Proc. Natl. Acad. Sci. U.S.A. 101, 4954–4959. Alves, R.J., Jotz, G.P., do Amaral, V.S., Montes, T.M., Menezes, H.S., de Andrade, H.H., 2008. The evaluation of maté (Ilex paraguariensis) genetic toxicity in human lymphocytes by the cytokinesis-block in the micronucleus assay. Toxicol. In Vitro 22, 695–698. Bachelor, M.A., Bowden, G.T., 2004. UVA-mediated activation of signaling pathways involved in skin tumor promotion and progression. Semin. Cancer Biol. 14, 131–138. Bastos, D.H., Ishimoto, E.Y., Marques, M.O.M., Ferri, A.F., Torres, E.A.F.S., 2006. Essential oil and antioxidant activity of green mate and mate tea (Ilex paraguariensis) infusions. J. Food Compos. Anal. 19, 538–543. Benaiges, A., Marcet, P., Armengol, R., Betes, C., Gironés, E.A., 1998. Study of the refirming effect of a plant complex. Int. J. Cosmet. Sci. 20, 223–233. Bracesco, N., Dell, M., Rocha, A., Behtash, S., Menini, T., Gugliucci, A., Nunes, E., 2003. Antioxidant activity of a botanical extract preparation of Ilex paraguariensis: prevention of DNA double-strand breaks in Saccharomyces cerevisiae and human low-density lipoprotein oxidation. J. Altern. Complement Med. 9, 379–387. Chandra, S., De Mejia, G.E., 2004. Polyphenolic compounds, antioxidant capacity, and quinone reductase activity of an aqueous extract of Ardisia compressa in comparison to mate (Ilex paraguariensis) and green (Camellia sinensis) teas. J. Agric. Food Chem. 52, 3583–3589. Collins, A.R., 2004. The comet assay for DNA damage and repair: principles, applications, and limitations. Mol. Biotechnol. 26, 249–326. Draper, H.H., Hadley, M., 1990. Malondialdehyde determination as index of lipid peroxidation. Methods Enzymol. 186, 421–431.
200
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
El-Khamisy, S.F., Caldecott, K.W., 2006. TDP1-dependent DNA single-strand break repair and neurodegeneration. Mutagenesis 21, 219–224. Emerit, I., 1994. Reactive oxygen species, chromosome mutation, and cancer: possible role of clastogenic factors in carcinogenesis. Free Radic. Biol. Med. 16, 99–109. Filip, R., Lotito, S., Ferraco, G., Raga, C., 2000. Antioxidant activity of Ilex paraguariensis and related species. Nutr. Res. 20, 1437–1446. Granstein, R.D., Matsui, M.S., 2004. UV radiation-induced immunosuppression and skin cancer. Cutis 74, 4–9. Gruijl, F.R., 1999. Skin cancer and solar UV radiation. Eur. J. Cancer 35, 2003–2009. Gugliucci, A., 1996. Antioxidant effects of Ilex paraguariensis: induction of decreased oxidability of human LDL in vivo. Biochem. Biophys. Res. Commun. 224, 338–344. Halliday, G.M., Byrne, S.N., Kuchel, J.M., Poon, T.S., Barnetson, R.S., 2004. The suppression of immunity by ultraviolet radiation: UVA, nitric oxide and DNA damage. Photochem. Photobiol. Sci. 3, 736–740. Halliday, G.M., 2005. Inflammation, gene mutation and photoimmunosuppression in response to UVR-induced oxidative damage contributes to photocarcinogenesis. Mutat. Res. 571, 107–120. Hirose, M., Nishikawa, A., Shibutani, M., Imai, T., Shirai, T., 2002. Chemoprevention of heterocyclic amine-induced mammary carcinogenesis in rats. Environ. Mol. Mutagen. 39, 271–278. Katiyar, S., Elmets, C.A., Katiyar, S.K., 2007. Green tea and skin cancer: photoimmunology, angiogenesis and DNA repair. J. Nutr. Biochem. 18, 287–296. Katiyar, S.K., Matsui, M.S., Mukhtar, H., 2000. Kinetics of UV light-induced cyclobutane pyrimidine dimers in human skin in vivo: an immunohistochemical analysis of both epidermis and dermis. Photochem. Photobiol. 72, 788–793. Katiyar, S., Mukhtar, H., 1996. Tea in chemoprevention of cancer. Int. J. Oncol. 8, 221–238. Katiyar, S.K., 2006. Oxidative stress and photocarcinogenesis: strategies for prevention. In: Singh, K.K. (Ed.), Oxidative Stress Disease and Cancer. Imperial College Press, London, pp. 933–964. Kaur, G., Jabbar, Z., Athar, M., Alam, M.S., 2006. Punica granatum (pomegranate) flower extract possesses potent antioxidant activity and abrogates Fe-NTA induced hepatotoxicity in mice. Food Chem. Toxicol. 44, 984–993. Kvam, E., Tyrrell, R.M., 1997. Induction of oxidative DNA base damage in human skin cells by UV and near visible radiation. Carcinogenesis 18, 2379–2384. Leonard, S.S., Hogans, V.J., Coppes-Petricorena, Z., Peer, C.J., Vining, T.A., Fleming, D.W., Harris, G.K., 2010. Analysis of free-radical scavenging of Yerba Mate (Ilex paraguariensis) using electron spin resonance and radical-induced DNA damage. J. Food Sci. 75, 14–20. Levine, R.L., Garland, D., Oliver, C.N., Amici, A., Climent, I., Lenz, A.G., Ahn, B.W., Shaltie, l.S., Stadtman, E.R., 1990. Determination of carbonyl content in oxidatively modified proteins. Methods Enzymol. 186, 464–478. Mantena, S.K., Meeran, S.M., Elmets, C.A., Katiyar, S.K., 2005. Orally administered green tea polyphenols prevent ultraviolet radiation-induced skin cancer in mice through activation of cytotoxic T cells and inhibition of angiogenesis in tumors. J. Nutr. 135, 2871–2877. McKay, D.L., Blumberg, J.B., 2002. The role of tea in human health: an update. J. Am. Coll. Nutr. 21, 1–13. Meeran, S.M., Akhtar, S., Katiyar, S.K., 2009. Inhibition of UVB-induced skin tumor development by drinking green tea polyphenols is mediated through DNA repair and subsequent inhibition of inflammation. J. Invest. Dermatol. 129, 258–1270.
Melnikova, V.O., Ananthaswamy, H.N., 2005. Cellular and molecular events leading to the development of skin cancer. Mutat. Res. 571, 91–106. Miranda, D.D., Arc¸ari, D.P., Pedrazzoli Jr., J., Carvalho Pde, O., Cerutti, S.M., Bastos, D.H., Ribeiro, M.L., 2008. Protective effects of mate tea (Ilex paraguariensis) on H2 O2 -induced DNA damage and DNA repair in mice. Mutagenesis 23, 261–265. Mocellin, S., Verdi, D., Nitti, D., 2009. DNA repair gene polymorphisms and risk of cutaneous melanoma: a systematic review and meta-analysis. Carcinogenesis 30, 1735–1743. Nakanishi, M., Niida, H., Murakami, H., Shimada, M., 2009. DNA damage responses in skin biology – implications in tumor prevention and aging acceleration. J. Dermatol. Sci. 56, 76–81. Okayasu, H., Suzuki, F., Satoh, K., Shioda, S., Dohi, K., Ikeda, Y., Nakashima, H., Komatsu, N., Fujimaki, M., Hashimoto, K., Maki, J., Sakagami, H., 2003. Comparison of cytotoxicity and radical scavenging activity between tea extracts and Chinese medicines. In Vivo 17, 577–581. Ramirez-Mares, M.V., Chandra, S., de Mejia, E.G., 2004. In vitro chemopreventive activity of Camellia sinensis, Ilex paraguariensis and Ardisia compressa tea extracts and selected polyphenols. Mutat. Res. 4, 53–65. Runger, T.M., 2003. Ultraviolet light. In: Bolognia, J.L., Jorizzo, J.L., Rapini, R.P. (Eds.), Dermatology. Mosby Press, London, pp. 1353–1363. Sarasin, A., 1999. The molecular pathways of ultraviolet-induced carcinogenesis. Mutat. Res. 428, 5–10. Schinella, G.R., Troiani, G., Dávila, V., de Buschiazzo, P.M., Tournier, H.A., 2000. Antioxidant effects of an aqueous extract of Ilex paraguariensis. Biochem. Biophys. Res. Commun. 269, 357–360. Su, L.J., Arab, L., 2002. Tea consumption and the reduced risk of colon cancer – results from a national prospective cohort study. Public Health Nutr. 5, 419–426. Tapiero, H., Tew, K.D., Ba, G.N., Mathé, G., 2002. Polyphenols: do they play a role in the prevention of human pathologies? Biomed. Pharmacother. 56, 200–207. Tice, R.R., Agurell, E., Anderson, D., Burlinson, B., Hartmann, A., Kobayashi, H., Miyamae, Y., Rojas, E., Ryu, J.C., Sasaki, Y.F., 2000. Single CellGel/Comet Assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ. Mol. Mutagen. 35, 206–221. ˘ ˘ Türkoglu, M., Ugurlu, T., Gedik, G., Yılmaz, A.M., Süha Yalc¸in, A., 2010. In vivo evaluation of black and green tea dermal products against UV radiation. Drug Discov. Ther. 4, 362–367. Ullrich, S.E., 2005. Mechanisms underlying UV-induced immune suppression. Mutat. Res. 571, 185–205. Vries, de E., Van de Poll-Franse, L.V., Louwman, W.J., de Gruijl, F.R., Coebergh, J.W., 2005. Predictions of skin cancer incidence in the Netherlands up to 2015. Br. J. Dermatol. 152, 481–488. Wang, D., Kreutzer, D.A., Essigmann, J.M., 1998. Mutagenicity and repair of oxidative DNA damage: insights from studies using defined lesions. Mutat. Res. 400, 1155–1199. Wang, Z.Y., Huang, M.T., Ferraro, T., Wong, C.Q., Lou, Y.R., Reuhl, K., Iatropoulos, M., Yang, C.S., Conney, A.H., 1992. Inhibitory effect of green tea in the drinking water on tumorigenesis by ultraviolet light and 12-O-tetradecanoylphorbol-13-acetate in the skin of SKH-1 mice. Cancer Res. 52, 1162–1170. Watt, N.T., Routledge, M.N., Wild, C.P., Hooper, N.M., 2007. Cellular prion protein protects against reactive-oxygen-species-induced DNA damage. Free Radic. Biol. Med. 43, 959–967. Weisburger, J.H., 2002. Lifestyle health and disease prevention: the underlying mechanisms. Eur. J. Cancer Prev. 11, 1–7. Wnuk, M., Lewinska, A., Oklejewicz, B., Bugno, M., Slota, E., Bartosz, G., 2009. Evaluation of the cyto- and genotoxic
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
activity of yerba mate (Ilex paraguariensis) in human lymphocytes in vitro. Mutat. Res. 679, 18–23. Xu, J.Y., Wu, L.Y., Zheng, X.Q., Lu, J.L., Wu, M.Y., Liang, Y.R., 2010. Green tea polyphenols attenuating ultraviolet B-induced damage to human retinal pigment epithelial cells in vitro. Invest. Ophthalmol. Vis. Sci. 51, 6665–6670.
Yang, C.S., Maliakal, P., Meng, X., 2002. Inhibition of carcinogenesis by tea. Annu. Rev. Pharmacol. Toxicol. 42, 25–54. Yusuf, N., Irby, C., Katiyar, S.K., Elmets, C.A., 2007. Photoprotective effects of green tea polyphenols. Photodermatol. Photoimmunol. Photomed. 23, 48–56.
201
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.elsevier.com/locate/etap
Evaluation of the protective effect of Ilex paraguariensis and Camellia sinensis extracts on the prevention of oxidative damage caused by ultraviolet radiation Marlon Barg a , Gislaine T. Rezin b , Daniela D. Leffa c , Fernanda Balbinot c , Lara M. Gomes a,d , Milena Carvalho-Silva a,d , Francieli Vuolo a,d , Fabricia Petronilho b , Felipe Dal-Pizzol a,d , Emilio L. Streck a,d , Vanessa M. Andrade c,∗ a
Laboratório de Bioenergética, Programa de Pós-Graduac¸ão em Ciências da Saúde, Universidade do Extremo Sul Catarinense, Criciúma, SC, Brazil b Laboratório de Fisiopatologia Clínica e Experimental, Programa de Pós-Graduac¸ão em Ciências da Saúde, Universidade do Sul de Santa Catarina, Tubarão, SC, Brazil c Laboratório de Biologia Celular e Molecular, Programa de Pós-Graduac¸ão em Ciências da Saúde, Universidade do Extremo Sul Catarinense, Criciúma, SC, Brazil d Instituto Nacional de Ciência e Tecnologia Translacional em Medicina, Hospital de Clínicas de Porto Alegre, Porto Alegre, RS, Brazil
a r t i c l e
i n f o
a b s t r a c t
Article history:
We evaluated the effects green and mate teas on oxidative and DNA damages in rats exposed
Received 26 March 2013
to ultraviolet radiation. Were utilized 70 adult male Wistar rats that received daily oral or
Received in revised form
topic green or mate tea treatment during exposed to radiation by seven days. After, animals
3 November 2013
were killed by decapitation. Thiobarbituric acid-reactive species levels, protein oxidative
Accepted 28 November 2013
damage were evaluated in skin and DNA damage in blood. Our results show that the rats
Available online 6 December 2013
exposed to ultraviolet radiation presented DNA damage in blood and increased protein car-
Keywords:
tea prevented lipid peroxidation, both treatments with mate tea also prevented DNA dam-
Skin cancer
age. However, only topic treatment with green tea and mate tea prevented increases in
bonylation and lipid peroxidation in skin. Oral and topic treatment with green tea and mate
Green tea
protein carbonylation. Our findings contribute to elucidate the beneficial effects of green
Mate tea
tea and mate tea, here in demonstrated by the antioxidant and antigenotoxic properties
DNA damage
presented by these teas.
Oxidative damage
© 2013 Elsevier B.V. All rights reserved.
∗ Corresponding author at: Laboratório de Biologia Celular e Molecular, Universidade do Extremo Sul Catarinense, UNESC, Avenida Universitária, 1105, 88806-000 Criciúma, SC, Brazil. Tel.: +55 48 3431 2757. E-mail address: [email protected] (V.M. Andrade). 1382-6689/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.etap.2013.11.028
196
1.
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
Introduction
Over the years, changes in lifestyle significantly increased human exposure to solar radiation, leading to a dramatic rise in the incidence of skin cancer (Gruijl, 1999). Photo-induced cancers are due to a complex multistage phenomenon, mediated by alterations in several cellular mechanisms (Sarasin, 1999). Increased ultraviolet radiation exposure is considered the main cause of the rising prevalence of skin cancer (Vries et al., 2005). In humans and animal models, both ultraviolet A and B radiations can cause gene mutations (Agar et al., 2004; Melnikova and Ananthaswamy, 2005) and immunity suppression (Halliday et al., 2004; Ullrich, 2005). These two biological events are related to skin cancer caused by ultraviolet radiation (Granstein and Matsui, 2004). Ultraviolet A and B radiations are responsible for irreversible genetic alterations, which ultimately lead to DNA mutations (Sarasin, 1999). The implication of ultraviolet A radiation to this genotoxic damage has been recently proven, although its accurate biological effects are still unclear (Bachelor and Bowden, 2004). The reactive oxygen species production by both ultraviolet A and B radiations also contributes to inflammation, immunosuppression, gene mutation and carcinogenesis (Halliday, 2005; Kvam and Tyrrell, 1997). In addition, studies have shown that oxidative stress also can cause DNA damage (Wang et al., 1998; Watt et al., 2007). DNA damage can include chemical and structural modifications to purine and pyrimidine bases and 2 -deoxyribose, apart from the formation of single- and double-strand breaks. Strand breaks within DNA can occur either directly, due to damage from reactive oxygen species exposure, or indirectly, due to cleavage of the DNA backbone during DNA base excision repair (El-Khamisy and Caldecott, 2006; Emerit, 1994). Previous studies have shown that polyphenols present in green tea (Camellia sinensis) can reduce ultraviolet radiationsinduced 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 water a process that, for the most part, prevents oxidation and polymerization of the plant’s polyphenols. It is these compounds that are thought to be the major chemopreventive mediators (Yusuf et al., 2007). Another tea presenting antioxidant properties and protection against DNA oxidation is mate tea or yerba mate (Ilex paraguariensis). Mate tea extract, made from dried leaves of I. 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 (Bracesco et al., 2003; Gugliucci, 1996). 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 (Bastos et al., 2006; Filip et al., 2000; Gugliucci, 1996; Ramirez-Mares et al., 2004; Schinella et al., 2000). Considering that ultraviolet radiation exposure induces gene mutations and leads to an increased reactive oxygen
species production, which enhances this harmful effect, and that green tea and mate tea possibly present antioxidant properties and protection against DNA damage, the present work evaluates the effect of green tea and mate tea on thiobarbituric acid-reactive species levels. Oxidative damage to proteins and DNA damage in rats exposed to ultraviolet radiation were also assessed.
2.
Materials and methods
2.1.
Animals
Adult male Wistar rats (250–300 g) obtained from Central Animal House of Universidade do Extremo Sul Catarinense were housed in seven groups of ten animals totaling 70 individuals with free access to food and water, and maintained on a 12-h light–dark cycle (lights on at 7:00 a.m.) at 22 ◦ C ± 1 ◦ C. All experimental procedures were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the Brazilian Society for Neuroscience and Behavior (SBNeC) recommendations for animal care, 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) base gel + ultraviolet radiation, (4) oral green tea + ultraviolet radiation, (5) green tea gel + ultraviolet radiation, (6) oral mate tea + ultraviolet radiation, (7) mate tea gel + ultraviolet radiation. Groups 2, 4, and 6 were treated for seven days orally with 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 were exposed to ultraviolet radiation for 1 h, once a day. Rats were killed 24 h after the last exposure session to ultraviolet radiation. Blood was collected to evaluate DNA damage and the whole skin, was removed of the animal to evaluate oxidative damage parameters.
2.3.
Oral administration of the extracts
Green and mate tea dry extracts was obtained from Embrafarma. The solutions were prepared daily in agreement with the Brazilian Pharmacopeia. Adding 15 mL of boiling water to 1.875 g of green tea extract and 20 g of mate tea extract to 100 mL of boiling water. After a 15-min extraction period, the solutions were filtered and administered to animals. The animals received 0.70 g/kg of mate tea and 0.45 g/kg green tea solutions daily by gavage, 30 min before exposure to ultraviolet radiation. 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 was applied daily on a 6 cm × 3 cm skin area previously shaven on each 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 using ethidium bromide (400×). The cells are assessed visually and received scores from 0 (undamaged) to 4 (maximally damaged), according to the size and shape of the tail.
2.5.
Exposure to radiation
Animals were exposed to ultraviolet A and B radiations for 7 days. Ultraviolet A and B lamps were used with emission spectrum of 280–320 nm for UVB, and 320–400 nm for UVA. The rats’ back were at a distance of 60 cm from the fluoresans lamps. Rats were irradiated for 1 h per day, all at the same time. In total, 70 rats were used in the present study. All animals were kept in cages throughout the experiment (5 animals per cage, 14 cages).
2.6.
Comet assay
A standard protocol for Comet assay preparation and analysis was adopted from Tice et al. (2000). The slides were prepared by mixing 5 L of whole blood with 95 L of low-melting-point agarose (0.75%). The mixture (cells/agarose) was added to a fully frosted microscope slide coated with a layer of 500 L of normally melting agarose (1%). After solidification, the coverslip was gently removed, and the slides were placed in lysis solution (2.5 M NaCl, 100 mM EDTA, and 10 mM Tris, pH 10.0–10.5, with freshly added 1% Triton X-100 and 10% DMSO) for 1 day. Subsequently, the slides were incubated in freshly prepared alkaline buffer (300 mM NaOH and 1 mM EDTA, pH 13) for 20 min. The DNA was electrophoresed for 15 min at 25 V (0.90 V/cm) and 300 mA. After electrophoresis, the slides were neutralized with Tris buffer (0.4 M; pH 7.5). Finally, the DNA was stained with ethidium bromide. Images of 100 randomly selected cells (50 cells from each of two replicate slides) from each animal were blindly analyzed using a fluorescence microscope equipped with an excitation filter of BP546/12 nm and a 590 nm barrier filter. Cells were scored from 0 (undamaged) to 4 (maximally damaged) according to the tail intensity (size and shape), resulting in a single DNA damage score (damage index) for each sample and, consequently, for each group. Thus, a damage index (DI) of the group could range from 0 (completely undamaged 100 = cells × 0) to 400 (maximum damage = 100 cells × 4) (Collins, 2004) (Fig. 1). The percentage damage frequency (DF) was calculated for each sample on the basis of the number of cells with a tail versus with no tail.
2.7.
Thiobarbituric acid reactive species (TBARS)
As an index of oxidative damage, we used the formation of TBARS during an acid-heating reaction, which is widely adapted as a sensitive method for measurement of lipid peroxidation, as previously described (Draper and Hadley, 1990). Briefly, samples were mixed with 1 mL of trichloroacetic acid 10% (TCA) and 1 mL of thiobarbituric acid 0.67% (TBA), and
Fig. 2 – Mean (±SD) values of damage index observed in peripheral blood of rats treated with green tea and mate tea, oral and topic, and submitted to radiation. *Data significant in relation to control at P < 0.05 (Kruskal–Wallis, Dunn).
then heated in a boiling water bath for 15 min. TBARS were determined by absorbance at 535 nm. Results are expressed as malondialdehyde (MDA) equivalents (pmol/mg protein).
2.8.
Measurement of protein carbonyls
The oxidative damage to proteins was assessed by the determination of carbonyl groups based on the reaction with dinitrophenylhydrazine (DNPH) as previously described (Levine et al., 1990). Briefly, proteins were precipitated by the addition of 20% trichloroacetic acid and redissolved in DNPH, and the absorbance read at 370 nm. Results are expressed as protein carbonyls (pmol/mg protein).
2.9.
Statistical analysis
For the analyses of DNA and oxidative damage parameters, all data were expressed as mean ± standard deviation (SD). Differences among experimental groups were determined by one-way analysis of variance (ANOVA) when normal distribution was observed. Multiple comparisons were performed by Duncan’ test. When non normal distribution was observed; comparisons were made using the Kruskal–Wallis test with Dunn’s test as post hoc. In all experiments, P < 0.05 was considered to indicate statistical significance. All analyses were performed using the 5.0 BioEstat software.
3.
Results
The present study showed that ultraviolet radiation exposure presented higher levels of DNA damage in peripheral blood 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 acidreactive species levels (Fig. 4), and protein carbonylation (Fig. 4) (P < 0.05; ANOVA – Duncan). Results for animals treated with both types of herbs in the two administration routes showed that only mate tea can significantly reverse DNA damage caused by UV radiation, unlike green tea, which does not present these protective effects for the genome, as observed in the high levels of DNA damage when 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 observed in peripheral blood of rats treated with green tea and mate tea, oral and topic, and submitted to radiation. *Data significant in relation to control at P < 0.05 (Kruskal–Wallis, Dunn).
(P < 0.05; Kruskal–Wallis, Dunn). However, both parameters of the comet assay (Figs. 2 and 3) revealed that this herb led to a similar decrease in DNA damage in animals topically treated with mate tea. Furthermore, we observed that oral and topic treatments with green tea or mate tea prevented the increased lipid peroxidation induced by ultraviolet radiation exposure (Fig. 4) (P < 0.05; ANOVA – Duncan). However, protein carbonylation was only prevented by topic treatment with green tea gel and mate tea gel. Oral treatment with green or mate teas did not prevent the increase in protein carbonylation caused by ultraviolet radiation exposure (Fig. 5) (P < 0.05; ANOVA – Duncan).
4.
Discussion
Exposure to solar ultraviolet radiation induces inflammatory responses, oxidative stress, immunosuppression, DNA damage and gene mutations, events related to skin diseases, including skin cancer (Katiyar et al., 2000, 2007; Katiyar, 2006). Multiple studies have demonstrated a relationship between ultraviolet exposure and increased risk of developing skin cancer. It was postulated that exposure of the skin to solar ultraviolet light is a major risk factor for the development of malignant melanoma and non-melanoma skin cancer, and
Fig. 4 – Mean (±SD) values of thiobarbituric acid reactive substances (TBARS) in skin of rats treated with green tea and mate tea, oral and topic, and submitted to radiation. *Data significant in relation to control at P < 0.05 (one-way ANOVA, Duncan test).
Fig. 5 – Mean (±SD) values of protein carbonyl content in skin of rats treated with green tea and mate tea, oral and topic, and submitted to radiation. *Data significant in relation to control at P < 0.05 (one-way ANOVA, Duncan test).
that this effect is mediated by a chain of events, including DNA damage by ultraviolet light and subsequent formation of mutations (Runger, 2003). In the life of a cell, DNA damage represents a great threat to genome stability, leading to loss or amplification of chromosomal activity, which may result in carcinogenesis or tissue aging (Nakanishi et al., 2009). Genome integrity is continuously threatened by both endogenous (e.g. reactive oxygen species produced by normal metabolism, DNA replication ‘errors’) and exogenous (ultraviolet light, ionizing radiations and chemical carcinogens) factors (Mocellin et al., 2009). In the present study we observed the occurrence of DNA damage in peripheral blood and oxidative damage in the skin of rats exposed to radiation. On the other hand, we showed that green and mate teas administered through two different routes (oral and topic gel) have prevented radiation-induced lipid peroxidation. However, protein carbonylation was prevented only by green and mate teas used as gel. Also, only topically administered mate tea prevented the harmful effects of UV radiation to genetic material. ˘ et al. (2010) showed that no UV-induced The study Türkoglu erythema was observed at the black and green tea gel sites in any of the subjects. UV-induced erythema was consistently present in various grades at caffeine gel, carbomer gel, and control sites. Therefore, tea extracts were found to be promising candidates for their ability to protect against the harmful effects of UV radiation, such as erythema and premature aging of the skin. Studies showed that consumption of green tea polyphenols in drinking water prevents photocarcinogenesis in mice; however, the molecular mechanisms underlying this effect have not been fully elucidated (Meeran et al., 2009). Study of Xu et al. (2010) showed that green tea polyphenols effectively suppressed the decrease in viability of the UVB stressed retinal pigment epithelial cells and the UVB suppression of surviving gene expression level. Green tea polyphenols alleviated mitochondria dysfunction and DNA fragmentation induced by UVB. Polyphenols isolated from the leaves of green tea have a number of beneficial healthy effects, including anticarcinogenic activity, which has been demonstrated in various
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
tumor models (Katiyar and Mukhtar, 1996; Yang et al., 2002). It has been also shown that oral administration of an aqueous extract of green tea or green tea polyphenols (a mixture of polyphenols) in drinking water inhibits ultraviolet radiation-induced skin carcinogenesis in mice in terms of tumor incidence, tumor multiplicity and tumor growth/size (Mantena et al., 2005; Wang et al., 1992). In addition, UV exposure causes physical changes to the skin due to alterations that occur in the connective tissue via the formation of lipid peroxides, cell contents and enzymes and reactive oxygen species (Benaiges et al., 1998; Kaur et al., 2006). Other studies showed that green tea also exerts anti-oxidant activity (Mantena et al., 2005; Okayasu et al., 2003). Flavonoids and phenolic compounds were proved to be present in mate tea leaves, and the consumption of these compounds has been associated with the prevention of age-related chronic conditions, including cancer (McKay and Blumberg, 2002; Weisburger, 2002). Various phytochemical constituents, such as flavonoids, have been isolated from mate plants and related species, and some of these compounds may be responsible for the observed antioxidant activity (Schinella et al., 2000). Furthermore, data from in vitro and in vivo studies suggest a potential beneficial effect of tea polyphenols against cancer at most stages of development (Hirose et al., 2002; Su and Arab, 2002; Tapiero et al., 2002). In this scenery, study of Chandra and De Mejia (2004) showed that the concentration of polyphenols in green tea is major when compared with mate tea. Thus, the concentration of green and mate tea used in the present study result in amounts polyphenols similar in both teas. Conversely, many studies in cell culture models as well as in animals seem to converge to show an antimutagenic and DNA protecting effect for I. paraguariensis extracts and its individual components, chlorogenic acid, rutin and quercetin (Bracesco et al., 2003; Miranda et al., 2008). Study of Leonard et al. (2010) indicated that mate scavenged hydroxyl radicals and superoxide radicals. This study indicates that mate possesses potent antioxidant effects against hydroxyl and superoxide radicals in chemical and cell culture systems, as well as DNA-protective properties. Besides, study of Miranda et al. (2008) showed that mate tea is not genotoxic in liver, kidney and bladder cells. The regular ingestion of mate tea increased the resistance of DNA to H2 O2 -induced DNA strand breaks and improved the DNA repair after H2 O2 challenge in liver cells. These results suggest that mate tea could protect against DNA damage and enhance the DNA repair activity. Protection may be attributed to the antioxidant activity of the mate bioactive compounds cited above (Bracesco et al., 2003; Miranda et al., 2008). However, study of Wnuk et al. (2009) in vitro cultured human lymphocytes indicates that mate infusion may cause both cytotoxic and genotoxic effects. Differences concentrations of mate from 1 to 1000 g/mL was evaluated and found that 10 g/mL of mate significantly increased the frequency of micronuclei and decreased the nuclear division index 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. Moreover, no genotoxic effect of mate
199
(175–1400 g/mL) on human lymphocytes was reported by Alves et al. (2008). In conclusion, we showed that the exposure of rats to radiation led to DNA and oxidative damage, and that green tea and mate tea have prevented these effects. Our data are in line with previous works that demonstrated the anti-carcinogenic and 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 do Extremo 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.
references
Agar, N.S., Halliday, G.M., Barnetson, R.S., Ananthaswamy, H.N., Wheeler, M., Jones, A.M., 2004. The basal layer in human squamous tumors harbors more UVA than UVB fingerprint mutations: a role for UVA in human skin carcinogenesis. Proc. Natl. Acad. Sci. U.S.A. 101, 4954–4959. Alves, R.J., Jotz, G.P., do Amaral, V.S., Montes, T.M., Menezes, H.S., de Andrade, H.H., 2008. The evaluation of maté (Ilex paraguariensis) genetic toxicity in human lymphocytes by the cytokinesis-block in the micronucleus assay. Toxicol. In Vitro 22, 695–698. Bachelor, M.A., Bowden, G.T., 2004. UVA-mediated activation of signaling pathways involved in skin tumor promotion and progression. Semin. Cancer Biol. 14, 131–138. Bastos, D.H., Ishimoto, E.Y., Marques, M.O.M., Ferri, A.F., Torres, E.A.F.S., 2006. Essential oil and antioxidant activity of green mate and mate tea (Ilex paraguariensis) infusions. J. Food Compos. Anal. 19, 538–543. Benaiges, A., Marcet, P., Armengol, R., Betes, C., Gironés, E.A., 1998. Study of the refirming effect of a plant complex. Int. J. Cosmet. Sci. 20, 223–233. Bracesco, N., Dell, M., Rocha, A., Behtash, S., Menini, T., Gugliucci, A., Nunes, E., 2003. Antioxidant activity of a botanical extract preparation of Ilex paraguariensis: prevention of DNA double-strand breaks in Saccharomyces cerevisiae and human low-density lipoprotein oxidation. J. Altern. Complement Med. 9, 379–387. Chandra, S., De Mejia, G.E., 2004. Polyphenolic compounds, antioxidant capacity, and quinone reductase activity of an aqueous extract of Ardisia compressa in comparison to mate (Ilex paraguariensis) and green (Camellia sinensis) teas. J. Agric. Food Chem. 52, 3583–3589. Collins, A.R., 2004. The comet assay for DNA damage and repair: principles, applications, and limitations. Mol. Biotechnol. 26, 249–326. Draper, H.H., Hadley, M., 1990. Malondialdehyde determination as index of lipid peroxidation. Methods Enzymol. 186, 421–431.
200
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
El-Khamisy, S.F., Caldecott, K.W., 2006. TDP1-dependent DNA single-strand break repair and neurodegeneration. Mutagenesis 21, 219–224. Emerit, I., 1994. Reactive oxygen species, chromosome mutation, and cancer: possible role of clastogenic factors in carcinogenesis. Free Radic. Biol. Med. 16, 99–109. Filip, R., Lotito, S., Ferraco, G., Raga, C., 2000. Antioxidant activity of Ilex paraguariensis and related species. Nutr. Res. 20, 1437–1446. Granstein, R.D., Matsui, M.S., 2004. UV radiation-induced immunosuppression and skin cancer. Cutis 74, 4–9. Gruijl, F.R., 1999. Skin cancer and solar UV radiation. Eur. J. Cancer 35, 2003–2009. Gugliucci, A., 1996. Antioxidant effects of Ilex paraguariensis: induction of decreased oxidability of human LDL in vivo. Biochem. Biophys. Res. Commun. 224, 338–344. Halliday, G.M., Byrne, S.N., Kuchel, J.M., Poon, T.S., Barnetson, R.S., 2004. The suppression of immunity by ultraviolet radiation: UVA, nitric oxide and DNA damage. Photochem. Photobiol. Sci. 3, 736–740. Halliday, G.M., 2005. Inflammation, gene mutation and photoimmunosuppression in response to UVR-induced oxidative damage contributes to photocarcinogenesis. Mutat. Res. 571, 107–120. Hirose, M., Nishikawa, A., Shibutani, M., Imai, T., Shirai, T., 2002. Chemoprevention of heterocyclic amine-induced mammary carcinogenesis in rats. Environ. Mol. Mutagen. 39, 271–278. Katiyar, S., Elmets, C.A., Katiyar, S.K., 2007. Green tea and skin cancer: photoimmunology, angiogenesis and DNA repair. J. Nutr. Biochem. 18, 287–296. Katiyar, S.K., Matsui, M.S., Mukhtar, H., 2000. Kinetics of UV light-induced cyclobutane pyrimidine dimers in human skin in vivo: an immunohistochemical analysis of both epidermis and dermis. Photochem. Photobiol. 72, 788–793. Katiyar, S., Mukhtar, H., 1996. Tea in chemoprevention of cancer. Int. J. Oncol. 8, 221–238. Katiyar, S.K., 2006. Oxidative stress and photocarcinogenesis: strategies for prevention. In: Singh, K.K. (Ed.), Oxidative Stress Disease and Cancer. Imperial College Press, London, pp. 933–964. Kaur, G., Jabbar, Z., Athar, M., Alam, M.S., 2006. Punica granatum (pomegranate) flower extract possesses potent antioxidant activity and abrogates Fe-NTA induced hepatotoxicity in mice. Food Chem. Toxicol. 44, 984–993. Kvam, E., Tyrrell, R.M., 1997. Induction of oxidative DNA base damage in human skin cells by UV and near visible radiation. Carcinogenesis 18, 2379–2384. Leonard, S.S., Hogans, V.J., Coppes-Petricorena, Z., Peer, C.J., Vining, T.A., Fleming, D.W., Harris, G.K., 2010. Analysis of free-radical scavenging of Yerba Mate (Ilex paraguariensis) using electron spin resonance and radical-induced DNA damage. J. Food Sci. 75, 14–20. Levine, R.L., Garland, D., Oliver, C.N., Amici, A., Climent, I., Lenz, A.G., Ahn, B.W., Shaltie, l.S., Stadtman, E.R., 1990. Determination of carbonyl content in oxidatively modified proteins. Methods Enzymol. 186, 464–478. Mantena, S.K., Meeran, S.M., Elmets, C.A., Katiyar, S.K., 2005. Orally administered green tea polyphenols prevent ultraviolet radiation-induced skin cancer in mice through activation of cytotoxic T cells and inhibition of angiogenesis in tumors. J. Nutr. 135, 2871–2877. McKay, D.L., Blumberg, J.B., 2002. The role of tea in human health: an update. J. Am. Coll. Nutr. 21, 1–13. Meeran, S.M., Akhtar, S., Katiyar, S.K., 2009. Inhibition of UVB-induced skin tumor development by drinking green tea polyphenols is mediated through DNA repair and subsequent inhibition of inflammation. J. Invest. Dermatol. 129, 258–1270.
Melnikova, V.O., Ananthaswamy, H.N., 2005. Cellular and molecular events leading to the development of skin cancer. Mutat. Res. 571, 91–106. Miranda, D.D., Arc¸ari, D.P., Pedrazzoli Jr., J., Carvalho Pde, O., Cerutti, S.M., Bastos, D.H., Ribeiro, M.L., 2008. Protective effects of mate tea (Ilex paraguariensis) on H2 O2 -induced DNA damage and DNA repair in mice. Mutagenesis 23, 261–265. Mocellin, S., Verdi, D., Nitti, D., 2009. DNA repair gene polymorphisms and risk of cutaneous melanoma: a systematic review and meta-analysis. Carcinogenesis 30, 1735–1743. Nakanishi, M., Niida, H., Murakami, H., Shimada, M., 2009. DNA damage responses in skin biology – implications in tumor prevention and aging acceleration. J. Dermatol. Sci. 56, 76–81. Okayasu, H., Suzuki, F., Satoh, K., Shioda, S., Dohi, K., Ikeda, Y., Nakashima, H., Komatsu, N., Fujimaki, M., Hashimoto, K., Maki, J., Sakagami, H., 2003. Comparison of cytotoxicity and radical scavenging activity between tea extracts and Chinese medicines. In Vivo 17, 577–581. Ramirez-Mares, M.V., Chandra, S., de Mejia, E.G., 2004. In vitro chemopreventive activity of Camellia sinensis, Ilex paraguariensis and Ardisia compressa tea extracts and selected polyphenols. Mutat. Res. 4, 53–65. Runger, T.M., 2003. Ultraviolet light. In: Bolognia, J.L., Jorizzo, J.L., Rapini, R.P. (Eds.), Dermatology. Mosby Press, London, pp. 1353–1363. Sarasin, A., 1999. The molecular pathways of ultraviolet-induced carcinogenesis. Mutat. Res. 428, 5–10. Schinella, G.R., Troiani, G., Dávila, V., de Buschiazzo, P.M., Tournier, H.A., 2000. Antioxidant effects of an aqueous extract of Ilex paraguariensis. Biochem. Biophys. Res. Commun. 269, 357–360. Su, L.J., Arab, L., 2002. Tea consumption and the reduced risk of colon cancer – results from a national prospective cohort study. Public Health Nutr. 5, 419–426. Tapiero, H., Tew, K.D., Ba, G.N., Mathé, G., 2002. Polyphenols: do they play a role in the prevention of human pathologies? Biomed. Pharmacother. 56, 200–207. Tice, R.R., Agurell, E., Anderson, D., Burlinson, B., Hartmann, A., Kobayashi, H., Miyamae, Y., Rojas, E., Ryu, J.C., Sasaki, Y.F., 2000. Single CellGel/Comet Assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ. Mol. Mutagen. 35, 206–221. ˘ ˘ Türkoglu, M., Ugurlu, T., Gedik, G., Yılmaz, A.M., Süha Yalc¸in, A., 2010. In vivo evaluation of black and green tea dermal products against UV radiation. Drug Discov. Ther. 4, 362–367. Ullrich, S.E., 2005. Mechanisms underlying UV-induced immune suppression. Mutat. Res. 571, 185–205. Vries, de E., Van de Poll-Franse, L.V., Louwman, W.J., de Gruijl, F.R., Coebergh, J.W., 2005. Predictions of skin cancer incidence in the Netherlands up to 2015. Br. J. Dermatol. 152, 481–488. Wang, D., Kreutzer, D.A., Essigmann, J.M., 1998. Mutagenicity and repair of oxidative DNA damage: insights from studies using defined lesions. Mutat. Res. 400, 1155–1199. Wang, Z.Y., Huang, M.T., Ferraro, T., Wong, C.Q., Lou, Y.R., Reuhl, K., Iatropoulos, M., Yang, C.S., Conney, A.H., 1992. Inhibitory effect of green tea in the drinking water on tumorigenesis by ultraviolet light and 12-O-tetradecanoylphorbol-13-acetate in the skin of SKH-1 mice. Cancer Res. 52, 1162–1170. Watt, N.T., Routledge, M.N., Wild, C.P., Hooper, N.M., 2007. Cellular prion protein protects against reactive-oxygen-species-induced DNA damage. Free Radic. Biol. Med. 43, 959–967. Weisburger, J.H., 2002. Lifestyle health and disease prevention: the underlying mechanisms. Eur. J. Cancer Prev. 11, 1–7. Wnuk, M., Lewinska, A., Oklejewicz, B., Bugno, M., Slota, E., Bartosz, G., 2009. Evaluation of the cyto- and genotoxic
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
activity of yerba mate (Ilex paraguariensis) in human lymphocytes in vitro. Mutat. Res. 679, 18–23. Xu, J.Y., Wu, L.Y., Zheng, X.Q., Lu, J.L., Wu, M.Y., Liang, Y.R., 2010. Green tea polyphenols attenuating ultraviolet B-induced damage to human retinal pigment epithelial cells in vitro. Invest. Ophthalmol. Vis. Sci. 51, 6665–6670.
Yang, C.S., Maliakal, P., Meng, X., 2002. Inhibition of carcinogenesis by tea. Annu. Rev. Pharmacol. Toxicol. 42, 25–54. Yusuf, N., Irby, C., Katiyar, S.K., Elmets, C.A., 2007. Photoprotective effects of green tea polyphenols. Photodermatol. Photoimmunol. Photomed. 23, 48–56.
201