Archives of Oral Biology

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Archives of Oral Biology

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Topical application of ointment containing 0.5% green tea catechins suppresses tongue oxidative stress in 5- ﬂ uorouracil administered rats

Hisataka Miyai

^a

, Takayuki Maruyama

^b,⁎

, Takaaki Tomofuji

^c

, Toshiki Yoneda

^a

, Tetsuji Azuma

^a

, Hirofumi Mizuno

^a

, Yoshio Sugiura

^a

, Terumasa Kobayashi

^a

, Daisuke Ekuni

^a

, Manabu Morita

^a

aDepartment of Preventive Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1, Shikata-cho, Kita-ku, Okayama 700-8525, Japan

bCenter for Innovative Clinical Medicine, Okayama University Hospital, 2-5-1, Shikata-cho, Kita-ku, Okayama 700-8558, Japan

cDepartment 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 capacity Chemotherapy Green tea catechin Oxidative stress Tongue

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 tea catechins 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 negative control group (saline administration and application of ointment without green tea catechins), a positive control group (5-FU administration and application of ointment without green tea catechins), and two experimental groups (5-FU administration and application of ointment containing 0.1% or 0.5% green tea catechins). Topical application of each ointment to the ventral surface of the tongue was performed once a day for 5 days. The level of 8-hydroxydeoxyguanosine (8-OHdG) was determined to evaluate oxidative stress. Fluorescence staining was also 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 were higher in the positive control group than in the negative control group (P< 0.05). On the other hand, those in the 0.5% green tea catechin group, but not in the 0.1% green tea catechin group, were lower than the positive control group (P< 0.05). In addition, Nrf2 translocation to the nucleus was greater in the 0.5% green tea catechin group than in the positive control group (P< 0.05).

Conclusions:Topical application of ointment containing 0.5% green tea catechins could prevent tongue oxidative stress 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 cancer treatments such as chemotherapy (Villa & Sonis, 2015). The prevalence of OM is approximately 40% in patients undergoing chemotherapy (Harris, 2006). OM is painful, impeding nutrient intake and causing an unexpected reduction of dosage or interruption during cancer treatment. Prevention of OM during chemotherapy is imperative in easing the patient’s burden during cancer treatment.

The pathobiologic progression of OM in chemotherapy has been described 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 balance between antioxidants and ROS is disrupted because of ROS accumula- tion, oxidative stress occurs (Perry et al., 2000). Then, during the signaling and amplification phase, oxidative stress activates transcription factors such as nuclear factor-κB (NF-κB) (Sonis et al., 2007). Such conditions 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 beneficial in preventing the initiation of OM.

Green tea contains several polyphenolic components including epigallocatechin-3-gallate, epicatechin-3-gallate, epicatechin, and epigallocatechin (Yang & Landau, 2000). Green tea catechins have been shown to possess potent antioxidant activity several times higher than

http://dx.doi.org/10.1016/j.archoralbio.2017.06.025

Received 23 March 2017; Received in revised form 30 May 2017; Accepted 18 June 2017

⁎Corresponding author.

E-mail address:[email protected](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 reaction array; WBC, white blood cell

MARK

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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 the oral mucosa may also act to suppress oxidative stress in basal epithelial cells of the oral mucosa. However, it remains unclear how green tea catechins aﬀect oxidative stress following chemotherapy.

In this study, we hypothesized that topical application of ointment containing green tea catechins to the oral mucosa might suppress oxidative 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 of the tongue is the major site of OM. In this context, the purpose of this study was to examine the preventive effects of topical application of green tea catechins on tongue oxidative stress in 5-FU administered rats.

2. Materials and methods 2.1. Animals

Experimental protocols were approved by the Animal Care and Use Committee of Okayama University (OKU-2014423). Twenty-eight male Wistar 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 powdered standard food (Oriental Yeast Co., Ltd., Osaka, Japan) and water ad libitum.

2.2. Experimental design

The rats were randomly divided into four groups of seven rats each.

Theﬁrst 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 any medications. 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 medications. The third group (0.1% catechin group) received i.p. injection of 5-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 containing 0.5% green tea catechins. We prepared the ointment containing green tea catechins by combining the green tea extract (Wako Pure Chemical Industries) with the ointment and stirring until homogenous.

The green tea extract was composed of 70.5% epigallocatechin-3-gallate, 22.0% epicatechin-3-gallate, 5.5% epicatechin, and 1.0% epigallocatechin. Under general anesthesia (2–4% isoflurane delivered in 100% oxygen gas), the ointment was applied to the ventral surface of the tongue with a cotton ball and wiped offafter 5 min once a day for five days. After 6 days, the animals were sacrificed under general anesthesia with diethyl ether and tongues were resected en bloc. Blood samples were also collected via cardiac puncture to confirm the effect of 5-FU on myelosuppression.

2.3. Histological and immunohistochemical analyses

For histological analysis, tongue samples were resected en bloc from each rat and immediately fixed in Bouin’s fluid for 1 day. Paraffin- embedded sections (2μm) 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 a commercial 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 development proceeded by placing sections in a solution of 3-3′-diamino- benzidine tetrahydrochloride. Sections were counterstained with Mayer’s hematoxylin. Using an image analyzer (ImageJ; 1.50b software National Institutes of Health-NIH, Bethesda, MD), 8-OHdG-positive cells, IL-1β-positive cells, NF-κB-positive cells, or total cells were counted at a magnification of 200× in the centerfield of the ventral tongue tissue (Fig. 1). Also, 8-OHdG-positive cells were further counted at a magnification of 200× in the middle and dorsal areas of the tongue tissue to confirm the localization of effects of the applied green tea catechins on oxidative stress. We confirmed intra-examiner reproduci- bility by double-scoring 10 randomly selected sections at two-week intervals. 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-strand complementary DNA was composed of total RNA (0.8μg) using the RT2 First-Strand Kit (cat # 330401, Qiagen, Hilden, Germany). The reverse transcription reaction was performed at 37 °C. In brief, 0.8μg of total RNA was added to 2μL of Buffer GE (5× gDNA Elimination Buffer), and thefinal volume was adjusted to 10μL with RNase-free water. The mixture was denatured at 42 °C for 5 min. Subsequently, the mixture was cooled on ice for 1 min. Reverse transcription was carried out after adding 10μL of reverse transcription mix to the solution. The reaction mixture 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 (20μL) were then diluted with 91μL 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 examined using the Rat Oxidative Stress and Antioxidant Defense RT2 Proﬁler™

PCR Array (Qiagen). The expression of 84 different genes was targeted for detection by real-time PCR. The RT2 Profiler™PCR Array includes built-in undercoating primers for 84 tested and 5 housekeeping genes and positive control elements to determine the efficiency of the reverse transcription reaction, capacity of the PCR reaction, and detection of genomic DNA contamination. The PCR mixture for 100 reactions con- tained 1150μL of SYBR Green ROX FAST Mastermix (Qiagen), 102μL Fig. 1.Sagittal section of the tongue [ventral (A), middle (B), dorsal (C) areas]　(magnification: 20×).

CT, connective tissue; MT, muscle tissue.

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of cDNA template, and 1048μL of RNase-free water. The PCR reaction mix was added to the wells of the PCR plate in equal amounts (20μL), and then the real-time PCR cycling program was run. The thermal cycling program recommended by the plate manufacturer for Rotor-Gene 6000 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 change values of the 0.5% catechin group for each gene in reference to the gene expression 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 procedure included double-fluorescence staining of the slides. Antigen retrieval was performed using Histo VT One (Nacalai Tesque, Kyoto, Japan) at 98 °C for 40 min followed by incubation for 20 min at room tempera- ture. A polyclonal antibody against Nrf2 (Santa Cruz Biotechnology Inc., 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 tongue tissue (A: negative control group, B: positive control group, C: 0.1% catechin group, D: 0.5% catechin group). The positive cells were stained dark brown.

Using binarized images, black points were deﬁned as positive cells (E: negative control group, F: positive control group, G: 0.1% catechin group, H: 0.5% catechin group) (magniﬁcation: 200×).

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(Thermo Fisher Scientific K. K., Kanagawa, Japan), which produces red fluorescence at an excitation maximum of 561 nm and emission of about 594 nm, was used as a secondary antibody (Hjelmeland, Fujikawa, Oltjen, Smit-McBride, & Braunschweig, 2010). Next, a mounting medium with 4′,6-diamidino-2-phenylindole (DAPI) (Im- munoSelect Antifading Mounting Medium; Dianova, Hamburg, Ger- many), which produces bluefluorescence at an excitation maximum of 365 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, hemoglobin 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 to one-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-way ANOVA followed by Tukey’s method or Student’st-test was used to evaluate the signiﬁcance of diﬀerences between the groups using a statistical software package (SPSS version 22.0; IBM, Tokyo, Japan).

Values ofP< 0.05 were considered to be statistically signiﬁcant.

3. Results

There were no signiﬁcant diﬀerences among the four groups in terms 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 in all groups. Also, HE stainingfindings showed that there was minimal inflammatory cell infiltration and epithelial basal cell breakdown in all groups.

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 ventral tongue tissue were higher in the positive control group than in the negative control group (P= 0.015) (Fig. 3). 8-OHdG-positive cells in the ventral tongue tissue were lower in the 0.5% catechin group than in the positive control group (P= 0.029); however, there was no significant 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% catechin group) (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 ANOVA followed by Tukey’s method.

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groups. The number of 8-OHdG positive cells in the middle area of the tongue tissue was higher in the positive control group than in the negative control group, and lower in the 0.1% and 0.5% catechin groups than in the positive control group; however, these differences were not significant. Furthermore, the number of 8-OHdG-positive cells in the dorsal area of the tongue tissue was also significantly higher in the positive control group than in the negative control group (P= 0.011);

however, there was no signiﬁcant diﬀerence between the positive control and 0.1% catechin groups or between the positive control and 0.5% 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-positive cells to total cells in the ventral tongue tissue were higher in the positive control group than in the negative control group (P= 0.011 and 0.001, respectively). Furthermore, the 0.5% catechin group showed significantly lower ratios compared to the positive control group (P= 0.003 and 0.014, respectively). However, there were no significant differences in NF-κB and IL-1βexpression between the positive control and 0.1% catechin groups.

In the RT-PCR array analysis, of the 84 genes tested, 8 showed a more than two-fold increase in expression between the positive control and 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% catechin group) (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 ANOVA followed by Tukey’s method.

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ferritin, thioredoxin, superoxide dismutase 1, peroxiredoxin, selenoprotein P, myoglobin, cathepsin B and glutamate cysteine ligase modiﬁer subunit.

Using an Nrf2 nuclear translocation assay, the nuclear level of Nrf2 in the 0.5% catechin group was observed to be signiﬁcantly higher than in 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 negative control group. Also, the RBC and platelet counts, and hemoglobin levels were lower in the positive control and 0.5% catechin groups than in the negative 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 negative control (Fig. 3). Among the markers of oxidative stress, 8-OHdG is generally accepted as a reliable marker (Kasai, 2002). This indicates that i.p. injection of 5-FU induced oxidative stress in the ventral tongue tissue. 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 is suggested that topical application of 0.5% green tea catechins suppressed 5-FU-induced ventral tongue oxidative stress.

In contrast, the ratios of 8-OHdG-positive cells in the middle and dorsal areas of the tongue were not signiﬁcantly diﬀerent in the 0.5%

catechin group compared to the positive control. In this study, the ointment containing green tea catechins was applied only to the ventral tongue surface. Therefore, the applied green tea catechins might not penetrate to the middle and dorsal areas of the tongue. It seems rea- sonable that the preventive eﬀects of green tea catechins on oxidative stress were limited to the applied areas in our model.

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

ameliorate oxidative stress-induced inflammatory responses following chemotherapy. Also, there was a significant reduction in IL-1β gene expression between the positive control and the 0.5% catechin group (Fig. 5). Thesefindings indicate that the 0.5% green tea catechins in- hibited inflammatory cytokines in this study. The preventive effects of green tea catechins against oxidative stress as well as inflammatory cytokines would be beneficial for maintaining ventral tongue health during chemotherapy.

In the RT-PCR array analysis, 8 antioxidant-related genes were highly 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-kinase 1 (Saitoh et al., 1998). Superoxide dismutase is an antioxidant enzyme that is involved in the scavenging of superoxide radicals. Ad- ditionally, peroxiredoxin 1 is an antioxidant enzyme and suppresses cell death by directly associating with NF-κB (Hansen, Moriarty- Craige, & Jones, 2007). These results suggest that topical application of green tea catechins enhanced the antioxidant capacity of the tongue tissue, and such a condition could contribute to decreasing ventral tongue oxidative stress following 5-FU administration.

Nrf2, which is a redox-sensitive transcription factor, plays an important role in antioxidant defense in tissues (Ishii et al., 2000). Nrf2 is suppressed by Kelch-like ECH-associated protein 1 (Keap1) in a state without oxidative stress (Itoh et al., 1999). However, under an oxidative stress condition, Nrf2 is released from Keap1-suppression and is then transferred into the nucleus to bind antioxidant response elements (Takagi et al., 2014). In this study, the nuclear translocation of Nrf2 occurred more frequently in the 0.5% catechin group than in the positive control group (Fig. 6). This suggests that green tea catechins re- inforced ventral tongue antioxidants through up-regulation of the Nrf2 signaling pathway. This is consistent with previous ﬁndings, which concluded that epigallocatechin-3-gallate could promote angiogenesis in middle cerebral artery occlusion mice, possibly via upregulation of the Nrf2 signaling pathway (Bai et al., 2017).

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

Green tea catechins are a natural agent and possess potent antioxidant capacity. From previous studies, the antioxidantsα-lipoic acid (Ahmed, Selim, & El-Sayed, 2017) and aloe vera (de Freitas Cuba, Braga Filho, Cherubini, Salum, & Figueiredo, 2016) have been validated to reduce the severity of oral mucositis following chemotherapy. Taken together, the previous and present studies support the concept that antioxidants can be applied to control chemotherapy-induced oxidative stress. However, some of the literature has shown that antioxidants have 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 demonstrated the preventive effects of green tea catechins on tongue oxidative stress, we could not elucidate whether green tea catechins actually affect the progression of OM. Further studies are needed to clarify the relationship between the preventive effects of green tea catechins on tongue oxidative stress and the progression of OM during chemotherapy. Second, we evaluated oxidative stress and inflammatory responses only in Table 1

List of the diﬀerentially expressed genes between the 0.5% catechin group and the positive control group.

Genes Fold change

Ferritin 9.92

Thioredoxin 9.69

Superoxide dismutase 1 6.81

Peroxiredoxin 1 5.7

Selenoprotein P 5.54

Myoglobin 2.39

Cathepsin B 2.06

Glutamate cysteine ligase, modiﬁer subunit 2.04

Table 2

Results of hematological analysis.

Negative control group

Positive control group

0.5% catechin group WBC (×10²/μL) 90.7 ± 24.2 49.1 ± 11.8^* 45.8 ± 15.3^*

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

hemoglobin (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'st-test).

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tongue tissue. The responses of the buccal mucosa and lips to green tea catechins 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 administered 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 of Education, Culture, Sports, Science and Technology, Tokyo, Japan.

Ethical approval

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

Acknowledgements Not applicable.

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