Graefes Arch Clin Exp Ophthalmol DOI 10.1007/s00417-014-2702-7

BASIC SCIENCE

Green tea extract suppresses N-methyl-N-nitrosourea-induced photoreceptor apoptosis in Sprague-Dawley rats Yuko Emoto & Katsuhiko Yoshizawa & Yuichi Kinoshita & Takashi Yuri & Michiko Yuki & Kazutoshi Sayama & Nobuaki Shikata & Airo Tsubura

Received: 16 April 2014 / Revised: 17 June 2014 / Accepted: 21 June 2014 # Springer-Verlag Berlin Heidelberg 2014

Abstract Background Retinitis pigmentosa (RP) is a group of inherited neurodegenerative human diseases characterized by the loss of photoreceptor cells by apoptosis and eventual blindness. A single intraperitoneal (ip) injection of N-methyl-N-nitrosourea (MNU) causes photoreceptor cell apoptosis within 7 days in rats. Green tea extract (THEA-FLAN 90S; GTE) is a common herbal supplement with pluripotent properties including antioxidant activity. The purpose of the present study was to evaluate the efficacy of GTE against photoreceptor apoptosis in 7-week-old female Sprague-Dawley rats that received a single ip injection of 40 mg/kg MNU. Methods The oral administration of 250 mg/kg/day GTE was initiated 3 days prior to MNU injection and continued once daily throughout the experiment. Rats were sacrificed at 12, 24, and 72 h and 7 days after MNU injection, and the eyes were examined morphologically and morphometrically. The photoreceptor cell ratio, retinal damage ratio, and retinal preservation ratio were used to determine the structural and functional alterations. The number of apoptotic photoreceptor cells per mm2 was determined in situ by TdT-mediated dUTPdigoxigenin nick end labeling (TUNEL). Our results indicated that oral administration of GTE significantly suppressed the Y. Emoto : K. Yoshizawa : Y. Kinoshita : T. Yuri : M. Yuki : A. Tsubura (*) Department of Pathology II, Kansai Medical University, Shinmachi 2-5-1, Hirakata, Osaka 573-1010, Japan e-mail: [email protected] Y. Kinoshita : N. Shikata Division of Diagnostic Cytopathology and Histopathology, Kansai Medical University Takii Hospital, Fumizono, Moriguchi, Osaka 570-8507, Japan K. Sayama Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, Ohya 836, Suruga, Shizuoka 422-8529, Japan

loss of photoreceptor cells morphometrically 7 days after MNU injection. The number of TUNEL-positive cells per mm2 in MNU-exposed rat central retina with or without GTE administration was 981 vs. 2056 at 24 h after MNU injection. Conclusions GTE structurally and functionally suppressed MNU-induced photoreceptor cell apoptosis. These findings indicate that GTE may help to ameliorate the onset and progression of human RP. Keywords Apoptosis . Epigallocatechin gallate (EGCG) . Green tea extract (GTE) . N-methyl-N-nitrosourea (MNU) . Retinal degeneration . Retinitis pigmentosa (RP)

Introduction Retinitis pigmentosa (RP) is one of the most common forms of inherited blindness worldwide [1, 2]. It is characterized by early night blindness followed by peripheral visual field alterations (known as tunnel vision) and eventual blindness [3]. Clinical signs typically start in the early teenage years, and severe visual impairment occurs by 40–50 years of age. The estimated incidence of RP is 1 in 4,000, which makes it one of the most common causes of severe visual impairment in humans [1]. Although more than 160 different mutations in genes that encode proteins with a variety of functions result in rod photoreceptor degeneration [4], the final common pathway is apoptotic cell death of the rod photoreceptors [5]. There are currently no effective treatments for RP; thus, the identification of chemicals that are effective against RP is desired. Animal models of RP are important for understanding human RP and are useful in the search for a treatment. Genetic mutant mice have been used as models for RP. rd10/rd10 mice carry a missense mutation in exon 13 of the beta subunit of the

Graefes Arch Clin Exp Ophthalmol

Pde6b gene, which results in a defect in the beta subunit of cyclic guanosine monophosphate phosphodiesterase; rd10/ rd10 mice provide a model of recessive RP that is more slowly progressive than rd1/rd1 mice, which carry a nonsense mutation in exon 7 of the Pde6b gene [6]. RP in rd2 mice is caused by a mutation in the gene coding for peripherin/rds (Prph2) [7]. Transgenic rats carrying a P23H mutation of the rhodopsin-encoding gene, which is the most prevalent cause of human RP, exhibit photoreceptor cell death by apoptosis [8]. In addition to inherited RP models, there are chemically induced retinal degeneration models. N-methyl-N-nitrosourea (MNU), an alkylating agent, exhibits its cytotoxicity by transferring its methyl group to nucleobases in nucleic acid. MNU selectively damages photoreceptor cells and is a good candidate for the induction of photoreceptor degeneration. MNUinduced photoreceptor degeneration is due to selective 7methyldeoxyguanosine DNA adduct formation in photoreceptor nuclei followed by photoreceptor cell death via an apoptotic mechanism, similar to the mechanism of human RP [3]. MNU-induced retinal degeneration can be induced in various animal species [3, 9, 10]. Oxidative stress contributes to the pathogenesis of neurodegenerative disorders [11]. Oxidative damage contributes to photoreceptor cell death in RP, and antioxidant treatment suppresses photoreceptor damage in animal models including the MNU model [6, 9, 12–14]. Recently, we reported that the natural product curcumin suppresses MNU-induced photoreceptor apoptosis in Sprague-Dawley rats [15]. Curcumin is the yellow pigment of turmeric or curry powder that is isolated from the rhizomes of Curcuma longa. Curcumin has beneficial properties that include antioxidant activity [16–18]. Green tea is one of the most popular beverages worldwide. Its major components include (-)-epicatechin (EC), (-)-epicatechin-3-galllate (ECG), (-)-epigallocatechin (EGC) and (-)epigallocatechin-3-gallate (EGCG). Green tea has demonstrated various effects including strong antioxidant activity [19].The major component of green tea is EGCG, which acts as an antioxidant; EGCG increases endogenous antioxidant defenses and down-regulates pro-apoptotic genes [20]. EGCG has the most potent effect among all of the catechins in green tea [21]. Theoretically, EGCG-containing green tea extract (GTE) may ameliorate photoreceptor cell damage caused by MNU. To date, suppression by GTE has not been reported in the MNU-induced RP model. The present study was designed to explore the efficacy of GTE against photoreceptor cell damage caused by MNU in Sprague-Dawley rats.

with paper bedding (Paper Clean, SLC, Hamamatsu, Japan) in a specific pathogen-free environment maintained at 22±2 °C and 60±10 % relative humidity, with a 12-h light/dark cycle (lights on from 8:00 am to 8:00 pm; illumination intensity less than 60 lux at the cage level). Animals were maintained on a commercial pellet diet (CMF 30 kGy, Oriental Yeast, Chiba, Japan) and had free access to water. After a 1-week acclimatization period, experiments were begun when rats were 7 weeks of age. The study protocol and animal procedures were approved by the animal care and use committee of Kansai Medical University (Permit Number: 13-007). Throughout the experiments, animals were cared for in accordance with the Guidelines for Animal Experimentation of Kansai Medical University. Chemicals MNU in powder form was obtained from SigmaAldrich (St. Louis, MO, USA) and stored at 4 °C in the dark. A 5 mg/ml solution was prepared by dissolving MNU in physiologic saline containing 0.1 % acetic acid immediately before use. The GTE used was THEA-FLAN 90S (ITO EN, Ltd., Tokyo, Japan), a decaffeinated product. Approximately 70 % of the polyphenols in GTE are tea catechins with a galloyl moiety [22]; these include epigallocatechin gallate (54 %, EGCG), epicatechin (12.4 %), gallocatechin gallate (2.8 %), catechin gallate (0.4 %), epigallocatechin (0.3 %), gallocatechin (0.1 %), caffeine (0.0 %), and others (0.3 %). GTE was dissolved at 40 mg/ml in sterile distilled water just prior to use.

Materials and Methods

Experimental Procedures Female rats received a single intraperitoneal (ip) injection of 40 mg/kg MNU at 7 weeks of age. A daily dose of 250 mg/kg GTE was orally administered by gastric intubation starting 3 days prior to MNU administration, and the administration was continued once daily until the termination of the experiment (Fig. 1a). Doses of 250 mg/kg GTE were selected based on previous reports [20, 23]. Control rats received an equivalent volume of saline instead of MNU and sterile distilled water instead of GTE at the same time points. Rats were divided into the following groups: control (MNU−/GTE−); MNU-unexposed and GTE-treated (MNU−/ GTE+), MNU-exposed and GTE-untreated (MNU+/GTE−), and MNU-exposed and GTE-treated (MNU+/GTE+). On the day of MNU exposure, GTE was administered 2 h prior to MNU injection. GTE was not given on the day of sacrifice. All rats were observed daily for clinical signs of toxicity and were weighed on the day of MNU administration and on the day of sacrifice. Rats were sacrificed 12, 24, and 72 h and 7 days after MNU administration. At the time of sacrifice, both eyes were quickly removed from each animal.

Animals Six-week-old female Sprague-Dawley rats [Crl:CD (SD)] were purchased from Charles River Japan (Osaka, Japan). Animals were housed in groups of 5 in a plastic cage

Serum and Retinal EGCG Concentrations In addition to the above experiment depicted in Fig. 1a, the concentration of EGCG was determined by LC-MS/MS in MNU−/GTE− and

Graefes Arch Clin Exp Ophthalmol

transitions were m/z 459.1→139.0 for EGCG (positive ion), 457.1→169.0 for EGCG (negative ion), and 197.2→124.1 for EG (IS, negative ion).

Fig. 1 Experimental design. Female rats received a single intraperitoneal (ip) injection of 40 mg/kg MNU at 7 weeks of age, and 250 mg/kg GTE was orally administered each day from 3 days prior to MNU injection until the termination of the experiment. Retinas were examined 12, 24, and 72 h and 7 days after MNU injection (a) or 1 and 2 h after the 4th daily administration of GTE (b)

MNU−/GTE+ rats 1 and 2 h after the administration of the 4th daily dose of GTE (Fig. 1b). To determine the total amount of EGCG, including free and conjugated forms, a serum sample (400 μL) was incubated with a mixture of β-glucuronidase (1000 units) at 37 °C for 45 min. Immediately after incubation, 1 mL of methanol and 50 μL of ethyl gallate solution (0.1 μg/mL) in methanol as an internal standard were added to the reaction mixture, which was then vigorously mixed for 5 min. The mixture was centrifuged at 10,000 x g for 10 min at 4 °C to precipitate proteins. The resulting supernatant was transferred to another tube and then evaporated to dryness in a vacuum. The retina sample (15–28 mg) was homogenized with 0.8 mL of saline and then centrifuged at 10,000 x g for 10 min at 4 °C. The resulting supernatant (400 μL) was treated in the same manner as the serum sample. After precipitating the proteins, the supernatant was transferred to another tube and then evaporated to dryness in a vacuum. Each residue was redissolved in 1 mL of 0.5 % formic acid aqueous solution and then applied to a Bond Elute cartridge (C18, size 50 mg/1 mL, Agilent, Santa Clara, CA, USA) that was preconditioned by rinsing with 2 mL of methanol and then with 2 mL of water. EGCG in the cartridge was eluted with 1 mL of 50 % methanol-50 % acetonitrile. The resulting eluents were evaporated to dryness in a vacuum. The residue was redissolved in 200 μL of mobile phase HPLC (20 % acetonitrile/water, 0.1 % folic acid), filtered through a 0.45-μm filter, and then applied to the LC-MS/MS system. LC-MS/MS Conditions Liquid chromatography was performed in a Shimadzu LC-20 AD apparatus (Kyoto, Japan) with an HPLC column, Inertsil ODS-3 (3 x 150 mm, 5 μm, GL Sciences Inc, Tokyo). HPLC separation was performed at 40 °C with a mobile phase 20 % acetonitrile/water-0.1 % folic acid solution, and the flow rate was 0.4 ml/min. Detection was performed with a Shimadzu LCMS 8040 LC-ESI-MS/MS tandem-mass spectrometer. The instrument was operated in the positive/negative ionization mode. Multiple reaction monitoring mode was used for the quantification. The selected

Tissue Fixation and Processing One eye was fixed overnight in 10 % neutral buffered formalin and the other eye was fixed overnight in methacarn (60 % methanol, 30 % chloroform, and 10 % acetic acid). Formalin- and methacarn-fixed samples were embedded in paraffin, sectioned at 4 μm, and stained with hematoxylin and eosin (HE). Sections including the ora serrata and optic nerve were used for the morphological and morphometrical evaluation. Morphometric analysis of photoreceptor cell ratio, retinal damage ratio, and retinal preservation ratio Methacarn-fixed and HE-stained ocular sections were obtained at 12, 24, and 72 h and 7 days after MNU exposure or from age-matched MNU-unexposed rats. To evaluate the structurally damaged retinas, the photoreceptor cell ratio [(photoreceptor thickness/ total retinal thickness) x100] and retinal damage ratio [(length of damaged retina, which equals the length of retina consisting less than four rows of photoreceptor nuclei/whole retinal length) x100] were calculated; the measurement was in accordance with our previous report [15]. In brief, measurements were made from the central retina (approximately 400 μm from the optic nerve) and the peripheral retina (approximately 400 μm from both sides of the ciliary body). To evaluate the functionally preserved retinas, anti-phosphodiesterase (PDE) 6β antibody (× 500; Abcam, Cambridge, UK) and antirhodopsin monoclonal antibody (×100; Millipore, Temecula, CA, USA) were used to detect the functional preservation of photoreceptor cells. Sections were deparaffinized, hydrated,

Fig. 2 Body weight change. When the body weight changes from the day of MNU administration to the termination of the experiment were compared, MNU exposure diminished body weight gain while GTE did not affect body weight change. Bar indicates mean ± S.E. **p

Green tea extract suppresses N-methyl-N-nitrosourea-induced photoreceptor apoptosis in Sprague-Dawley rats.

Retinitis pigmentosa (RP) is a group of inherited neurodegenerative human diseases characterized by the loss of photoreceptor cells by apoptosis and e...
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