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Archives of Oral Biology

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Topical application of ointment containing 0.5% green tea catechinssuppresses tongue oxidative stress in 5-fluorouracil administered rats

Hisataka Miyaia, Takayuki Maruyamab,⁎, Takaaki Tomofujic, Toshiki Yonedaa, Tetsuji Azumaa,Hirofumi Mizunoa, Yoshio Sugiuraa, Terumasa Kobayashia, Daisuke Ekunia, Manabu Moritaa

a Department of Preventive Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1, Shikata-cho, Kita-ku, Okayama700-8525, Japanb Center for Innovative Clinical Medicine, Okayama University Hospital, 2-5-1, Shikata-cho, Kita-ku, Okayama 700-8558, Japanc Department of Community Oral Health, Asahi University School of Dentistry, 1851-1 Hozumi, Mizuho, Gifu 501-0296, Japan

A R T I C L E I N F O

Keywords:Antioxidant capacityChemotherapyGreen tea catechinOxidative stressTongue

A B S T R A C T

Objective: The purpose of this study was to investigate the preventive effects of topical application of green teacatechins on tongue oxidative stress induced by 5-fluorouracil (5-FU) administration in rats.Design: Male Wistar rats (n = 28, 8 weeks old) were divided into four groups of seven rats each: a negativecontrol group (saline administration and application of ointment without green tea catechins), a positive controlgroup (5-FU administration and application of ointment without green tea catechins), and two experimentalgroups (5-FU administration and application of ointment containing 0.1% or 0.5% green tea catechins). Topicalapplication of each ointment to the ventral surface of the tongue was performed once a day for 5 days. The levelof 8-hydroxydeoxyguanosine (8-OHdG) was determined to evaluate oxidative stress. Fluorescence staining wasalso performed to confirm nuclear factor erythroid 2-related factor 2 (Nrf2) translocation to the nucleus.Results: After the experimental period, the ratios of 8-OHdG-positive cells in the ventral tongue tissue werehigher in the positive control group than in the negative control group (P < 0.05). On the other hand, those inthe 0.5% green tea catechin group, but not in the 0.1% green tea catechin group, were lower than the positivecontrol group (P < 0.05). In addition, Nrf2 translocation to the nucleus was greater in the 0.5% green teacatechin group than in the positive control group (P < 0.05).Conclusions: Topical application of ointment containing 0.5% green tea catechins could prevent tongue oxidativestress in 5-FU administered rats, via up-regulation of the Nrf2 signaling pathway.

1. Introduction

Oral mucositis (OM) is one of the major side effects of cancertreatments such as chemotherapy (Villa & Sonis, 2015). The prevalenceof OM is approximately 40% in patients undergoing chemotherapy(Harris, 2006). OM is painful, impeding nutrient intake and causing anunexpected reduction of dosage or interruption during cancer treat-ment. Prevention of OM during chemotherapy is imperative in easingthe patient’s burden during cancer treatment.

The pathobiologic progression of OM in chemotherapy has beendescribed to follow four stages: initiation phase, signaling and ampli-fication phase, ulceration phase, and healing phase (Sonis, 2009).During the initiation phase, reactive oxygen species (ROS) are

generated in basal epithelial cells of the oral mucosa. When the balancebetween antioxidants and ROS is disrupted because of ROS accumula-tion, oxidative stress occurs (Perry et al., 2000). Then, during the sig-naling and amplification phase, oxidative stress activates transcriptionfactors such as nuclear factor-κB (NF-κB) (Sonis et al., 2007). Suchconditions further lead to the production of pro-inflammatory cytokines(i.e., interleukin [IL]-1β), which causes the initiation of OM. Therefore,reduction of oxidative stress following chemotherapy would be bene-ficial in preventing the initiation of OM.

Green tea contains several polyphenolic components includingepigallocatechin-3-gallate, epicatechin-3-gallate, epicatechin, and epi-gallocatechin (Yang & Landau, 2000). Green tea catechins have beenshown to possess potent antioxidant activity several times higher than

http://dx.doi.org/10.1016/j.archoralbio.2017.06.025Received 23 March 2017; Received in revised form 30 May 2017; Accepted 18 June 2017

⁎ Corresponding author.E-mail address: t-maru@md.okayama-u.ac.jp (T. Maruyama).

Abbreviations: 5-FU, 5-fluorouracil; 8-OHdG, 8-hydroxydeoxyguanosine; HE, hematoxylin and eosin; IL, interleukin; Keap1, Kelch-like ECH-associated protein 1; NF-κB, nuclear factor-κB; Nrf2, nuclear factor erythroid 2-related factor 2; OM, oral mucositis; RBC, red blood cell; ROS, reactive oxygen species; RT-PCR array, reverse transcription-polymerase chain reactionarray; WBC, white blood cell

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that of vitamin E (Rice-Evans, Miller, Bolwell, Bramley, & Pridham,1995). In a previous study, γ-tocotrienol, which is a type of vitamin E,could effectively suppress oxidative stress in human oral keratinocytes(Takano et al., 2015). Topical application of green tea catechins to theoral mucosa may also act to suppress oxidative stress in basal epithelialcells of the oral mucosa. However, it remains unclear how green teacatechins affect oxidative stress following chemotherapy.

In this study, we hypothesized that topical application of ointmentcontaining green tea catechins to the oral mucosa might suppress oxi-dative stress and inflammation following chemotherapy. 5-fluorouracil(5-FU) is one of the chemotherapeutic drugs that have a mucosal-da-maging effect (Peterson et al., 2013). In addition, the ventral surface ofthe tongue is the major site of OM. In this context, the purpose of thisstudy was to examine the preventive effects of topical application ofgreen tea catechins on tongue oxidative stress in 5-FU administeredrats.

2. Materials and methods

2.1. Animals

Experimental protocols were approved by the Animal Care and UseCommittee of Okayama University (OKU-2014423). Twenty-eight maleWistar rats (8 weeks old) were bred in an air-conditioned room(23–25 °C) with a 12-h light-dark cycle. The animals were fed powderedstandard food (Oriental Yeast Co., Ltd., Osaka, Japan) and water adlibitum.

2.2. Experimental design

The rats were randomly divided into four groups of seven rats each.The first group (negative control group) received intraperitoneal (i.p.)injection of saline (20 mL/kg) and topical application of ointment [33%beeswax (33 g per 100 g ointment) and soybean oil] without anymedications. The second group (positive control group) received i.p.injection of 5-FU (Wako Pure Chemical Industries, Osaka, Japan)(20 mg/kg) and topical application of ointment without any medica-tions. The third group (0.1% catechin group) received i.p. injection of5-FU (20 mg/kg) and topical application of ointment containing 0.1%green tea catechins. The last group (0.5% catechin group) received i.p.injection of 5-FU (20 mg/kg) and topical application of ointment con-taining 0.5% green tea catechins. We prepared the ointment containinggreen tea catechins by combining the green tea extract (Wako PureChemical Industries) with the ointment and stirring until homogenous.The green tea extract was composed of 70.5% epigallocatechin-3-gal-late, 22.0% epicatechin-3-gallate, 5.5% epicatechin, and 1.0% epi-gallocatechin. Under general anesthesia (2–4% isoflurane delivered in100% oxygen gas), the ointment was applied to the ventral surface ofthe tongue with a cotton ball and wiped off after 5 min once a day forfive days. After 6 days, the animals were sacrificed under general an-esthesia with diethyl ether and tongues were resected en bloc. Bloodsamples were also collected via cardiac puncture to confirm the effect of5-FU on myelosuppression.

2.3. Histological and immunohistochemical analyses

For histological analysis, tongue samples were resected en bloc fromeach rat and immediately fixed in Bouin’s fluid for 1 day. Paraffin-embedded sections (2 μm) were stained with hematoxylin and eosin(HE) or other stains as described below.

Immunostaining for 8-hydroxydeoxyguanosine (8-OHdG) (an in-dicator of oxidative stress), IL-1β, and NF-κB was performed using acommercial kit (Histofine Simple Stain MAX PO; Nichirei Co., Tokyo,Japan). Polyclonal antibodies against 8-OHdG (Chemicon International,Temecula, CA), IL-1β (Serotec, Oxford, UK), and NF-κB (Abcam,Cambridge, MA) were diluted in phosphate buffered saline at 1:200 (8-

OHdG), 1:200 (IL-1β), and 1:100 (NF-κB), respectively. Color devel-opment proceeded by placing sections in a solution of 3-3′-diamino-benzidine tetrahydrochloride. Sections were counterstained withMayer’s hematoxylin. Using an image analyzer (ImageJ; 1.50b softwareNational Institutes of Health-NIH, Bethesda, MD), 8-OHdG-positivecells, IL-1β-positive cells, NF-κB-positive cells, or total cells werecounted at a magnification of 200× in the center field of the ventraltongue tissue (Fig. 1). Also, 8-OHdG-positive cells were further countedat a magnification of 200× in the middle and dorsal areas of the tonguetissue to confirm the localization of effects of the applied green teacatechins on oxidative stress. We confirmed intra-examiner reproduci-bility by double-scoring 10 randomly selected sections at two-weekintervals. Intra-examiner agreement for 8-OHdG-positive cells, IL-1β-positive cells, and NF-κB positive cells was greater than 80%.

2.4. Gene expression analysis with real-time reverse transcription-polymerase chain reaction array (RT-PCR array)

Total RNA from pooled tongue samples (N = 3/group) was ex-tracted with TRI reagent (Molecular Research Center Inc., Montgomery,OH). All RNA samples were confirmed to be of sufficient purity (A260/A230 ratio > 1.8, A260/A280 ratio = 1.8–2.0) and concentration(> 100 ng/μL). Next, these RNAs were reverse transcribed. First-strandcomplementary DNA was composed of total RNA (0.8 μg) using the RT2First-Strand Kit (cat # 330401, Qiagen, Hilden, Germany). The reversetranscription reaction was performed at 37 °C. In brief, 0.8 μg of totalRNA was added to 2 μL of Buffer GE (5× gDNA Elimination Buffer),and the final volume was adjusted to 10 μL with RNase-free water. Themixture was denatured at 42 °C for 5 min. Subsequently, the mixturewas cooled on ice for 1 min. Reverse transcription was carried out afteradding 10 μL of reverse transcription mix to the solution. The reactionmixture was incubated at 42 °C for 15 min, after which it was termi-nated by heating at 95 °C for 5 min. The cDNA samples generated(20 μL) were then diluted with 91 μL RNase-free water and stored at−20 °C until further analysis.

Real-time PCR was performed by using a Rotor-Gene 6000 Real-Time PCR detection system (Qiagen). Gene expression was examinedusing the Rat Oxidative Stress and Antioxidant Defense RT2 Profiler™PCR Array (Qiagen). The expression of 84 different genes was targetedfor detection by real-time PCR. The RT2 Profiler™ PCR Array includesbuilt-in undercoating primers for 84 tested and 5 housekeeping genesand positive control elements to determine the efficiency of the reversetranscription reaction, capacity of the PCR reaction, and detection ofgenomic DNA contamination. The PCR mixture for 100 reactions con-tained 1150 μL of SYBR Green ROX FAST Mastermix (Qiagen), 102 μL

Fig. 1. Sagittal section of the tongue [ventral (A), middle (B), dorsal (C) areas]　(mag-nification: 20×).CT, connective tissue; MT, muscle tissue.

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of cDNA template, and 1048 μL of RNase-free water. The PCR reactionmix was added to the wells of the PCR plate in equal amounts (20 μL),and then the real-time PCR cycling program was run. The thermal cy-cling program recommended by the plate manufacturer for Rotor-Gene6000 was as follows: 10 min at 95 °C, followed by 40 cycles of dena-turation at 95 °C for 15 s, with 30 s annealing and elongation at 60 °C,followed by melting curve analysis. Rotor-Gene Q (version 2.1.0;Qiagen) software was used for analysis. We calculated the fold changevalues of the 0.5% catechin group for each gene in reference to the geneexpression of the positive control group.

2.5. Histological evaluation of nuclear factor erythroid 2-related factor 2(Nrf2) translocation

To confirm Nrf2 translocation to the nucleus, the staining procedureincluded double-fluorescence staining of the slides. Antigen retrievalwas performed using Histo VT One (Nacalai Tesque, Kyoto, Japan) at98 °C for 40 min followed by incubation for 20 min at room tempera-ture. A polyclonal antibody against Nrf2 (Santa Cruz BiotechnologyInc., Dallas, TX) was diluted to 1:500 in phosphate-buffered saline(Wang et al., 2013). Alexa Fluor 594-conjugated anti-rabbit IgG (1:250)

Fig. 2. 8-OHdG-positive cells in the ventral tonguetissue (A: negative control group, B: positive controlgroup, C: 0.1% catechin group, D: 0.5% catechingroup). The positive cells were stained dark brown.Using binarized images, black points were defined aspositive cells (E: negative control group, F: positivecontrol group, G: 0.1% catechin group, H: 0.5% ca-techin group) (magnification: 200×).

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(Thermo Fisher Scientific K. K., Kanagawa, Japan), which produces redfluorescence at an excitation maximum of 561 nm and emission ofabout 594 nm, was used as a secondary antibody (Hjelmeland,Fujikawa, Oltjen, Smit-McBride, & Braunschweig, 2010). Next, amounting medium with 4′,6-diamidino-2-phenylindole (DAPI) (Im-munoSelect Antifading Mounting Medium; Dianova, Hamburg, Ger-many), which produces blue fluorescence at an excitation maximum of365 nm and emission of about 460 nm, was used to cover the slides

(Neria et al., 2009).

2.6. Hematological analysis

White blood cell (WBC) counts, red blood cell (RBC) counts, he-moglobin levels and platelet counts were determined using a hemo-cytometer and the microscopic count method.

Fig. 3. Ratios of 8-OHdG-positive cells to total cells in the ventral (A), middle (B), and dorsal areas (C) of the tongue tissue. Bars represent means ± SD (N = 7). *P < 0.05, according toone-way ANOVA followed by Tukey’s method.

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2.7. Statistical analysis

All data are expressed as the mean ± standard deviation. One-wayANOVA followed by Tukey’s method or Student’s t-test was used toevaluate the significance of differences between the groups using astatistical software package (SPSS version 22.0; IBM, Tokyo, Japan).Values of P < 0.05 were considered to be statistically significant.

3. Results

There were no significant differences among the four groups interms of body weight and food consumption during the experimental

period (data not shown). At the end of the experimental period,minimal redness or ulcer lesions on the tongue surface were observed inall groups. Also, HE staining findings showed that there was minimalinflammatory cell infiltration and epithelial basal cell breakdown in allgroups.

8-OHdG-positive cells were detected in the ventral tongue tissue(Fig. 2). The ratios of 8-OHdG-positive cells to total cells in the ventraltongue tissue were higher in the positive control group than in thenegative control group (P= 0.015) (Fig. 3). 8-OHdG-positive cells inthe ventral tongue tissue were lower in the 0.5% catechin group than inthe positive control group (P = 0.029); however, there was no sig-nificant difference between the positive control and 0.1% catechin

Fig. 4. NF-κB-positive cells (arrow, brown-stained nucleus) in the ventral tongue tissue (A: negative control group, B: positive control group, C: 0.1% catechin group, D: 0.5% catechingroup) (magnification: 200×). Ratios of NF-κB-positive cells to total cells in the ventral tongue tissue (E). Bars represent means ± SD (N = 7). *P < 0.05, according to one-way ANOVAfollowed by Tukey’s method.

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groups. The number of 8-OHdG positive cells in the middle area of thetongue tissue was higher in the positive control group than in the ne-gative control group, and lower in the 0.1% and 0.5% catechin groupsthan in the positive control group; however, these differences were notsignificant. Furthermore, the number of 8-OHdG-positive cells in thedorsal area of the tongue tissue was also significantly higher in thepositive control group than in the negative control group (P = 0.011);however, there was no significant difference between the positivecontrol and 0.1% catechin groups or between the positive control and0.5% catechin groups.

NF-κB- and IL-1β-positive cells were also detected in the ventral

tongue tissue (Figs. 4 and 5). The ratios of NF-κB and IL-1β-positivecells to total cells in the ventral tongue tissue were higher in the posi-tive control group than in the negative control group (P = 0.011 and0.001, respectively). Furthermore, the 0.5% catechin group showedsignificantly lower ratios compared to the positive control group(P = 0.003 and 0.014, respectively). However, there were no sig-nificant differences in NF-κB and IL-1β expression between the positivecontrol and 0.1% catechin groups.

In the RT-PCR array analysis, of the 84 genes tested, 8 showed amore than two-fold increase in expression between the positive controland 0.5% catechin groups (Table 1). The elevated mRNAs included

Fig. 5. IL-1β-positive cells (arrow, brown-stained cytoplasm) in the ventral tongue tissue (A: negative control group, B: positive control group, C: 0.1% catechin group, D: 0.5% catechingroup) (magnification: 200×). Ratios of IL-1β-positive cells to total cells in the ventral tongue tissue (E). Bars represent means ± SD (N = 7). *P < 0.05, according to one-way ANOVAfollowed by Tukey’s method.

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ferritin, thioredoxin, superoxide dismutase 1, peroxiredoxin, seleno-protein P, myoglobin, cathepsin B and glutamate cysteine ligasemodifier subunit.

Using an Nrf2 nuclear translocation assay, the nuclear level of Nrf2in the 0.5% catechin group was observed to be significantly higher thanin the positive control group (P < 0.05).

The WBC count was significantly lower in the positive control(P = 0.002) and 0.5% catechin (P= 0.003) groups than in the negativecontrol group. Also, the RBC and platelet counts, and hemoglobin levelswere lower in the positive control and 0.5% catechin groups than in thenegative control group; however, the differences were not significant(Table 2).

4. Discussion

In this study, the positive control showed a higher ratio of 8-OHdG-positive cells in the ventral tongue tissue compared with the negativecontrol (Fig. 3). Among the markers of oxidative stress, 8-OHdG isgenerally accepted as a reliable marker (Kasai, 2002). This indicatesthat i.p. injection of 5-FU induced oxidative stress in the ventral tonguetissue. On the other hand, the ratio of 8-OHdG positive cells in the 0.5%catechin group was lower than the positive control group. Thus, it issuggested that topical application of 0.5% green tea catechins sup-pressed 5-FU-induced ventral tongue oxidative stress.

In contrast, the ratios of 8-OHdG-positive cells in the middle anddorsal areas of the tongue were not significantly different in the 0.5%catechin group compared to the positive control. In this study, theointment containing green tea catechins was applied only to the ventraltongue surface. Therefore, the applied green tea catechins might notpenetrate to the middle and dorsal areas of the tongue. It seems rea-sonable that the preventive effects of green tea catechins on oxidativestress were limited to the applied areas in our model.

The present study showed that NF-κB protein expression was sup-pressed by 0.5% green tea catechins (Fig. 4). NF-κB is redox-sensitiveand is a potential target for ROS in endothelial and vascular smoothmuscle cells, and has been widely implicated as a pro-inflammatorynuclear transcription factor (Lawrence, 2009; Mittal, Siddiqui, Tran,Reddy, &Malik, 2014). The results suggest that suppressed expressionof NF-κB with topical application of 0.5% green tea catechins could

ameliorate oxidative stress-induced inflammatory responses followingchemotherapy. Also, there was a significant reduction in IL-1β geneexpression between the positive control and the 0.5% catechin group(Fig. 5). These findings indicate that the 0.5% green tea catechins in-hibited inflammatory cytokines in this study. The preventive effects ofgreen tea catechins against oxidative stress as well as inflammatorycytokines would be beneficial for maintaining ventral tongue healthduring chemotherapy.

In the RT-PCR array analysis, 8 antioxidant-related genes werehighly up-regulated in the 0.5% catechin group (Table 1). Among these,thioredoxin-1 is a redox sensitive antioxidant protein that has an anti-apoptotic function via the inhibition of apoptosis signal-regulating-ki-nase 1 (Saitoh et al., 1998). Superoxide dismutase is an antioxidantenzyme that is involved in the scavenging of superoxide radicals. Ad-ditionally, peroxiredoxin 1 is an antioxidant enzyme and suppresses celldeath by directly associating with NF-κB (Hansen, Moriarty-Craige, & Jones, 2007). These results suggest that topical application ofgreen tea catechins enhanced the antioxidant capacity of the tonguetissue, and such a condition could contribute to decreasing ventraltongue oxidative stress following 5-FU administration.

Nrf2, which is a redox-sensitive transcription factor, plays an im-portant role in antioxidant defense in tissues (Ishii et al., 2000). Nrf2 issuppressed by Kelch-like ECH-associated protein 1 (Keap1) in a statewithout oxidative stress (Itoh et al., 1999). However, under an oxida-tive stress condition, Nrf2 is released from Keap1-suppression and isthen transferred into the nucleus to bind antioxidant response elements(Takagi et al., 2014). In this study, the nuclear translocation of Nrf2occurred more frequently in the 0.5% catechin group than in the po-sitive control group (Fig. 6). This suggests that green tea catechins re-inforced ventral tongue antioxidants through up-regulation of the Nrf2signaling pathway. This is consistent with previous findings, whichconcluded that epigallocatechin-3-gallate could promote angiogenesisin middle cerebral artery occlusion mice, possibly via upregulation ofthe Nrf2 signaling pathway (Bai et al., 2017).

In this study, the WBC count was significantly lower in the positivecontrol and 0.5% catechin groups than in the negative control(Table 2). Hemoglobin content, RBC and platelet counts also showedthe tendency to be lower in the positive control and 0.5% catechingroups than in the negative control. These indicate that 5-FU admin-istration induced systemic immunosuppression in this study. Moreover,such a condition easily initiates OM. However, our model was not ableto replicate the clinical and histological characteristics of OM. In aprevious study, mucositis was reported to be induced via daily i.p. in-jection of 50 mg/kg 5-FU for 4 days (Koizumi et al., 2017). On the otherhand, the present model involved i.p. injection of 20 mg/kg 5-FU for5 days, which is representative of the concentration and duration ty-pically used in routine cancer chemotherapy. However, the con-centration of 5-FU used in this study may not be sufficient to initiatemucositis.

Green tea catechins are a natural agent and possess potent anti-oxidant capacity. From previous studies, the antioxidants α-lipoic acid(Ahmed, Selim, & El-Sayed, 2017) and aloe vera (de Freitas Cuba, BragaFilho, Cherubini, Salum, & Figueiredo, 2016) have been validated toreduce the severity of oral mucositis following chemotherapy. Takentogether, the previous and present studies support the concept thatantioxidants can be applied to control chemotherapy-induced oxidativestress. However, some of the literature has shown that antioxidantshave little effect on oral mucositis in cancer therapy (Yarom et al.,2013). Further studies are needed to resolve these conflicting results.

This study has some limitations. First, although we demonstratedthe preventive effects of green tea catechins on tongue oxidative stress,we could not elucidate whether green tea catechins actually affect theprogression of OM. Further studies are needed to clarify the relationshipbetween the preventive effects of green tea catechins on tongue oxi-dative stress and the progression of OM during chemotherapy. Second,we evaluated oxidative stress and inflammatory responses only in

Table 1List of the differentially expressed genes between the 0.5% catechin group and the po-sitive control group.

Genes Fold change

Ferritin 9.92Thioredoxin 9.69Superoxide dismutase 1 6.81Peroxiredoxin 1 5.7Selenoprotein P 5.54Myoglobin 2.39Cathepsin B 2.06Glutamate cysteine ligase, modifier subunit 2.04

Table 2Results of hematological analysis.

Negative controlgroup

Positive controlgroup

0.5% catechingroup

WBC (×102/μL) 90.7 ± 24.2 49.1 ± 11.8* 45.8 ± 15.3*

RBC (×104/μL) 808 ± 42 796 ± 32 712 ± 78hemoglobin (g/

dL)17.1 ± 0.8 16.6 ± 0.9 15.2 ± 1.7

platelet count(×104/μL)

97.6 ± 27.8 97.2 ± 21.7 67.1 ± 23.7

mean ± SD.* P < 0.05 compared with Negative control group (Student's t-test).

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tongue tissue. The responses of the buccal mucosa and lips to green teacatechins may differ from those of tongue tissue.

In conclusion, topical application of ointment containing 0.5%green tea catechins could prevent tongue oxidative stress in 5-FU ad-ministered rats via up-regulation of the Nrf2 signaling pathway.

Conflict of interest

There is no conflict of interest.

Funding

This work was supported by Grants-in-Aid for Scientific Research(Nos. 26861830, 16K11855 and 16K20693) from the Ministry ofEducation, Culture, Sports, Science and Technology, Tokyo, Japan.

Ethical approval

Experimental protocols were approved by the Animal Care and UseCommittee of Okayama University (OKU-2014423).

Acknowledgements

Not applicable.

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