- Email: [email protected]
Tea polyphenols protect against irradiation-induced injury in submandibular glands’ cells: A preliminary study
archives of oral biology 56 (2011) 738–743
available at www.sciencedirect.com
journal homepage: http://www.elsevier.com/locate/aob
Tea polyphenols protect against irradiation-induced injury in submandibular glands’ cells: A preliminary study Zhe Peng a,b, Zhi-wen Xu a,*, Wen-sheng Wen a, Ren-sheng Wang c a
Department of Otolaryngology and Head and Neck Surgery, The First Affiliated Hospital, Guangxi Medical University, Shuangyong Road, Nanning 530021, Guangxi, China b Department of Otorhinolaryngology Head and Neck Surgery, Beijing Tongren Hospital, Affiliated to Capital Medical University, Beijing 100013, China c Department of Radiotherapy, The First Affiliated Hospital, Guangxi Medical University, Nanning 530021, Guangxi, China
article info
summary
Article history:
Aim: To study the protective effect of tea polyphenols (TPs) on submandibular glands
Accepted 14 December 2010
affected by radiation injury. Methods: Sixty rats were randomly divided into radiation group (R-group, N = 30) and TP-
Keywords:
pre-treated-radiation group (TPR-group, N = 30). The rats were intragastrically administered
Radiation
with TP or normal sodium from 14 days before radiation, continuously daily, until the
Submandibular gland
experiment. All the rats in both groups were irradiated with a single exposure dose of 15 Gy
Tea polyphenols
gamma rays that were delivered to the head and neck areas. Ten rats of each group were
Apoptosis
anatomised on the 3rd, 6th and 30th day after irradiation, respectively. The submandibular glands of the rats were removed for the study. The morphologic changes of the submandibular glands were observed by transmission electron microscopy (TEM). The terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP)-biotin nick-end labelling (TUNEL) method was used to detect apoptosis of the submandibular glands’ cells. Results: Electron microscope observation of the submandibular glands showed that the lesions of the TPR-group were mild. Change in apoptosis of the cells was not obvious compared with the R-group. The cell apotosis was typical after irradiation in the R-group. Apoptosis index that was detected in the cells of submandibular glands of the TPR-group was statistically significantly decreased compared with the R-group (P < 0.01) on the 3rd, 6th and 30th day after irradiation. Conclusion: TP could protect submandibular glands from radiation injuries, and the protection mechanism may be realised by anti-apoptosis. # 2010 Elsevier Ltd. All rights reserved.
Radiotherapy plays an important role in the treatment of head and neck carcinomas. Conventional radiotherapy is given in daily fractions of 1.8–2.0 greys (Gy), up to total doses of 66– 70 Gy in 6–7 weeks as definitive treatment of head and neck cancers.1 The major salivary glands frequently receive a high radiation dose during the radiotherapy periods. A high dose of
radiation on the salivary glands has resulted in a reduction of salivary output and change in salivary composition, as the salivary glands are radiosensitive.2,3 Recent evidences suggested that alterations in the fractionation schedule and concomitant chemotherapy may significantly improve the result of therapy.4,5 However, the radiation-induced injury to
* Corresponding author. Tel.: +86 771 5356311. E-mail address: [email protected] (Z.-w. Xu). 0003–9969/$ – see front matter # 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.archoralbio.2010.12.009
archives of oral biology 56 (2011) 738–743
the salivary glands and the other oral sequelae of head and neck induced by radiotherapy are difficult to manage now. It has been shown that xerostomia resulted from decreasing saliva flow, and this change was irreversible.6 This significantly influenced patients’ quality of life.7 However, medical therapy cannot solve this problem now, and the mechanism of radiation-induced xerostomia remains to be elucidated.8 It is known that tea is the most widely consumed beverage in the world. Tea polyphenols (TPs), which are isolated from the leaves of green tea, have a number of beneficial effects on health, such as anticancer and other protection.9 The biological activities of TPs include antioxidation, modulation of enzyme systems for metabolising chemical carcinogens, scavenging of activated metabolites of chemical carcinogens and inhibition of tumour promotion.10–13 There were also some studies concerning the anti-radiation effect of TP.14–16 But there were few studies, which reported as to whether TP has a protective effect on submandibular gland injury induced by irradiation. This study aimed to investigate the effect of TP on submandibular gland injury induced by radiation.
1.
Materials and methods
1.1. Animals were grouped randomly and given irradiation Sixty female Wistar rats aged 6–7 months were obtained from the Center of Animal Experiment of Guangxi Medical University. The rats were kept under standard housing conditions (laboratory rodent chow and water ad libidum and a 12-h light day cycle). All the rats were acclimated for a week. This animal experiment was carried out in accordance with Medical Faculty Polices and Guidelines for the Care and Use of Laboratory Animals. All efforts were made to minimise animals’ suffering.. All the rats were randomly divided into two groups: (1) Radiation group (R-group) (N = 30). Ten rats from the group were sacrificed and the experiment was performed on the 3rd, 6th and 30th day after irradiation (R-3d, R-6d and R-30d subgroups). (2) TP pre-treated group before radiation (TPR-group) (N = 30). Ten rats from this group were sacrificed and the experiment was also performed on the 3rd, 6th and 30th day after irradiation (TPR-3d, TPR-6d and TPR-30d subgroups). This random process was computer controlled. The TP was provided by the Tea Polyphenols Company of Zhejiang Province in China. The TP in the study contained total polyphenol no less than 90%, total catechins no less than 65%, epigallocatechin gallate (EGCg) no less than 40% and caffeine less than 12%. All the components were analysed by high-performance liquid chromatography (HPLC). In TPR-group, the TP was dissolved in saline at 0.2 g kg bodyweight 1. Intragastric administration with TP was initiated 14 days before the irradiation, continuously daily, until the experiment. The total time of administration with TP was 17 days in the TPR-3d subgroup, 20 days in TPR-6d subgroup and 44 days in TPR-30d subgroup. In the R-group,
739
vehicle saline (2 ml kg bodyweight 1) was administered in the same way and at the same times as in the TPR-group. The intragastric TP and vehicle saline were administered orally by a feeding tube daily. Irradiation was performed according to the study of Takeda et al. [17]. Briefly, all animals were anaesthetised with an intra-peritoneal injection with pelltobarbitalum natricum at 60 mg kg bodyweight 1, and were fixed in a plastic mould. Then, the rats were locally irradiated in the head and neck regions. Both groups were irradiated with a single dose of 15 Gy. Irradiation was performed by a standard source-tosurface distance (SSD) of 100 cm. Radiation field size was x = 15 cm, y1 = 0 cm and y2 = 3 cm, and the dose rate was 121 cGy min 1.
1.2. Tissue preparation and transmission electron microscopy The submandibular glands of rats that were removed surgically were fixed in 4% paraformaldehyde and processed for paraffin embedding, according to a standard procedure. Serial sections with a thickness of 3 mm were prepared and stained with haematoxylin–eosin (HE). Segments (1 mm 1 mm) of the rat submandibular gland were prefixed with 2.5% glutaraldehyde for 12 h, and then postfixed with 1% osmium tetroxide for 2 h. After dehydration in a graded series of ethanol, the tissue specimens were embedded in Epok 812. Ultrathin sections were cut with the Sorvall MT 5000 ultramicrotome equipped with a diamond knife, stained with uranyl acetate and lead citrate, and examined under an H-500 electron microscope at 75 kV accelerating voltage.
1.3. Terminal deoxynucleotidyl transferase (TdT)mediated deoxy uridine triphosphate (dUTP)-biotin nick-end labelling method (TUNEL) and quantification of apoptotic index The terminal deoxynucleotidyl transferase (TdT)-mediated deoxy uridine triphosphate (dUTP)-biotin nick-end labelling TUNEL method was performed to detect apoptotic cells. The In Situ Cell Death Detection Kit, POD (Roche Molecular Biochemicals, Catalogue Number 1684817) was used in this study. The sections were incubated with TdT and fluorescein dUTP with proteinase K (20 mg ml 1) pre-treatment. After phosphatebuffered saline (PBS) rinsing, anti-fluorescein-peroxidase antibody was applied, and the reaction was visualised by 3,3-diaminobenzidine (Fujian Maxin Company, China). The sections were counterstained with haematoxylin. Negative control sections were incubated with distilled water in the absence of TdT. The apoptotic index (AI) was determined by calculating the percentage of TUNEL-positive cells per animal using the following method. To ensure the objectivity of the analysis, the evaluation was carried out by two independent observers. Five sections were randomly chosen for each rat. Approximately 1000 cell nuclei from each cell population (acinar cells, granular convoluted tubule cells and intercalated duct cells) were counted for each section at 400 magnification, and the number of apoptotic nuclei was expressed as a percentage of
740
archives of oral biology 56 (2011) 738–743
the total. Striated ducts were omitted from the study, as too few of them were examined to provide useful information. Data are expressed as an average.
1.4.
Statistical analysis
The data values are presented as mean SD. The statistical significance of differences was checked by Student’s t-test. Differences were considered to be statistically significant at P < 0.05.
2.
Result
2.1.
Basic information of the animals
No rats died due to irradiation in both groups. The rats weighed 18 20 g in the initial study. There were no significant changes on the 3rd and 6th day after radiation. However, on the 30th day after radiation, the weights of the rats decreased significantly, and their activity also reduced obviously. Although the food intake seemingly decreased on the 30th day after irradiation, TP
was administrated intragastrically with a tube daily all the time to ensure adequate intake.
2.2. Pathohistology of submandibular glands post irradiation Pathohistological changes were observed in both groups. The changes were aggravated in the R-group, as discussed here: R-group (3 days after irradiation, R3d): Vacuolisation of some acinar cells and granular convoluted tubule cells and decrease of granules in the granular convoluted tubule cells. R-group (6 days after irradiation, R6d): Enlarged intercellular space, cellular atrophy and degranulation of granular convoluted tubule cells; enlarged tissue space. R-group (30 days after irradiation, R30d): Lysis of entire acini and granular convoluted tubule cells was observed, predominantly single cells were affected and lobules of the submandibular gland were disordered with a mass of deposition of fibrous tissue. Pathohistological changes of the TPR-groups (including TPR3d, TPR6d and TPR30d subgroups) were mild compared with the R-group, atrophy of
Fig. 1 – (A) R-group (day 6 post-irradiation): mitochondria swell, disorganization and reduction or vanish of the crista (arrow). (B) TPR-group (day 6 post-irradiation): rupture of the mitochondria (arrow), the rest are nearly normal. (C) R-group (day 30 post-irradiation): cell shrinkage (arrow), chromatin condensation. (D) TPR-group (day 30 post-irradiation): one cell showed the chromatin changes resembling apoptosis (arrow), another two cells were normal.
archives of oral biology 56 (2011) 738–743
Table 1 – Apoptotic index of 3 stages post-irradiation between R-group and TPR-group (mean W SD). Groups R-group TPR-group *
Day 3 (N = 10)
Day 6 (N = 10)
Day 30 (N = 10)
25.21 4.7 6.97 2.1*
17.46 2.1 5.36 1.6*
17.69 2.4 5.64 2.0*
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seen. Apoptotic activity was the greatest in granular convoluted tubule cells, whilst mucous acinar cells were relatively resistant to apoptosis. The AI of each group can be seen from Table 1 and Fig. 2. The difference was detected at the same time of post irradiation of the R-group and the TPR-group (P < 0.01).
P < 0.01.
3. gland was local and degranulation of granular convoluted tubule cells was alleviated.
2.3.
Transmission electron microscopy
In the R-group: We observed acinar cells, intercalated duct cells, and granular convoluted tubule cell that showed oedema, mitochondrial swelling, disorganisation and reduction or disappearance of the crista, and occasionally, rupture of the mitochondria. These cells also contained dilated Golgi apparatus. In addition, cell death resembling apoptosis was occasionally visible. These cells showed the chromatin changes seen in apoptosis; some cell showed chromatin condensation. In the TPR-group: Here, the changes of ultramicroscopic structure in the cells were lesser than in the R-group (Fig. 1).
2.4.
Apoptosis
In all gland compartments (acinar cells, intercalated duct cells and granular convoluted tubule cells), apoptotic activity was
Discussion
The pathophysiology mechanism of modifications in the salivary gland induced by radiation is not clear yet. In addition, currently, there are not adequate prevention and treatment measures available. There are convincing evidences that ionising radiation could induce apoptosis.2 It is believed that apoptosis played a major role only in acute post-irradiation damage.18–20 TP is a main component of tea. Further, research has confirmed the radioprotective effect of TP. Yang et al. [21] in their study, confirmed that the concentration of TP in saliva was 10-fold higher than serum concentration after administering TP to humans, and also indicated that catechins were absorbed through the oral mucosa. Hence, TP can be used to prevent oral diseases, such as xerostomia, induced by irradiation. There were convincing evidences that TP has a function in anti-irradiation.22–24 All of these factors make TP a good prospect for the prevention of some oral diseases. In our study, the morphological change of submandibular gland cells was significantly different in the two groups. In the R-group, the injury to the submandibular gland cells was
Fig. 2 – (A-1) R-group (day 3 post-irradiation), (A-2) TPR-group (day 3 post-irradiation); (B-1) R-group (day 30 post-irradiation), (B-2) TPR-group (day 30 post-irradiation). Introduction: Apoptotic activity was seen in all gland compartments (acinar cells, ID cells, and GCT cells). Apoptotic activity was the greatest in GCT cells (arrow). In the same stage post-irradiation, apoptotic activity of TPR-group was fewer than R-group.
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archives of oral biology 56 (2011) 738–743
serious, and cell apoptosis as seen by electron microscopy was widespread. However, in the TPR-group, the extent of cell injury and the number of cell apoptosis events was significantly reduced compared with the R-group. Transmission electron microscopic studies revealed mitochondrial swelling, and cristae appeared indistinct in the cells. Hence, we infer that the mechanism of cell change in the submandibular gland after irradiation was induced by apoptosis. Cell apoptosis was further assessed by the TUNEL method. AI in the TPR-group was significantly reduced compared with the R-group. The result confirmed that TP can protect submandibular gland cells from being injured by radiation through anti-apoptosis. Furthermore, the peak of apoptosis after irradiation was in the early post-irradiation phase18,20; but we had observed significant AI in irradiated cells that persisted to the late post-irradiation (30 days post-irradiation) period yet. We speculated that the anti-apoptosis function of TP continued its existence beyond the early stage. Compared with the same phase post irradiation, we observed that the AIs of the submandibular gland in the Rgroups (including the 3rd day, 6th day and 30th day subgroup) were obviously higher than for the TPR-groups. This may prove that TP protected the submandibular gland by influencing the cell cycle. The Yamamoto et al. [25,26] in vitro studies confirmed that TP could decrease cell apoptosis after irradiation. TP plays different roles in submandibular gland cells and tumour cells according to the concentration. At some certain physiological concentrations (0–50 mmol l 1), TP has a protective effect both on submandibular gland cells and tumour cells. However, a high concentration of TP (>200 mmol l 1) had a protective effect on normal submandibular gland cells yet, whilst it still suppressed the tumour cells at the same time. Further, it may be able to protect salivary gland cells from damage caused by chemotherapeutic drug and radiation therapy. Hence, in our study, we only focus on the high dose concentration (>200 mmol l 1) to investigate the protective effect of TP on normal submandibular gland cells; in this study, we used 0.2 g kg bodyweight 1 (436 mmol l 1) considering both the high concentration and the median lethal dose (LD50). In conclusion, TP can protect submandibular glands from radiation injuries by anti-apoptosis. It proves that TP can be used in the clinic to prevent irradiation-induced salivary gland injury. However, this is an initial study and further studies will be needed to confirm the modulated mechanic pathway of the anti-apoptosis function of TP in the future.
Funding This work was supported by the Emphasis Research Funding of Health Department of Guangxi Zhuang Autonomous Region (Emphasis Healthy of Guangxi, No. 200634).
Conflict of interest statement None of the authors have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.
Ethical approval This project was approved by the Animal Ethical Committee of the First Affiliated Hospital of Guangxi Medical University.
Acknowledgement We thank Jie Liang assistant professor, Institute of Foreign Languages of Guangxi Medical University, for all the efforts and advice on the writing and grammar in the revised article.
references
1. Dirix P, Nuyts S, Van den Bogaert W. Radiation-induced xerostomia in patients with head and neck cancer: a literature review. Cancer 2006;107(11):2525–34. 2. Konings AW, Coppes RP, Vissink A. On the mechanism of salivary gland radiosensitivity. Int J Radiat Oncol Biol Phys 2005;62(4):1187–94. 3. Harrison LB, Zelefsky MJ, Pfister DG, Carper E, Raben A, Kraus DH, et al. Detailed quality of life assessment in patients treated with primary radiotherapy for squamous cell cancer of the base of the tongue. Head Neck 1997;19(3):169–75. 4. Rosenthal DI, Blanco AI. Head and neck squamous cell carcinoma: optimizing the therapeutic index. Expert Rev Anticancer Ther 2005;5(3):501–14. 5. Krstevska V, Crvenkova S. Altered and conventional fractionated radiotherapy in locoregional control and survival of patients with squamous cell carcinoma of the larynx, oropharynx, and hypopharynx. Croat Med J 2006;47(1):42–52. 6. Sasaki T, Yamamoto M, Takeda M. Function of parotid gland following irradiation and its relation to biological parameters. Radiat Res 1980;83(3):579–91. 7. Dirix P, Nuyts S, Vander Poorten V, Delaere P, Van den Bogaert W. The influence of xerostomia after radiotherapy on quality of life: results of a questionnaire in head and neck cancer. Support Care Cancer 2008;16(2):171–9. 8. Nagler RM. Effects of head and neck radiotherapy on major salivary glands—animal studies and human implications. In Vivo 2003;17(4):369–75. 9. Stangl V, Dreger H, Stangl K, Lorenz M. Molecular targets of tea polyphenols in the cardiovascular system. Cardiovasc Res 2007;73(2):348–58. 10. Yang CS, Wang ZY. Tea and cancer. J Natl Cancer Inst 1993;85(13):1038–49. 11. Stoner GD, Mukhtar H. Polyphenols as cancer chemopreventive agents. J Cell Biochem Suppl 1995;22:169–80. 12. Mukhtar H, Wang ZY, Katiyar SK, Agarwal R. Tea components: antimutagenic and anticarcinogenic effects. Prev Med 1992;21(3):351–60. 13. Mukhtar H, Katiyar SK, Agarwal R. Green tea and skin—anticarcinogenic effects. J Invest Dermatol 1994;102(1):3–7. 14. Lee HJ, Kim JS, Moon C, Kim JC, Lee YS, Jang JS, et al. Modification of gamma-radiation response in mice by green tea polyphenols. Phytother Res 2008;22(10):1380–3. 15. Liu ML, Yu LC. Potential protection of green tea polyphenols against ultraviolet irradiation-induced injury on rat cortical neurons. Neurosci Lett 2008;444(3):236–9.
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16. Meeran SM, Akhtar S, Katiyar SK. 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 2009;129(5):1258–70. 17. Takeda I, Kizu Y, Yoshitaka O, Saito I, Yamane GY. Possible role of nitric oxide in radiation-induced salivary gland dysfunction. Radiat Res 2003;159(4):465–70. 18. Belka C, Jendrossek V, Pruschy M, Vink S, Verheij M, Budach W. Apoptosis-modulating agents in combination with radiotherapy—current status and outlook. Int J Radiat Oncol Biol Phys 2004;58(2):542–54. 19. Ross GM. Induction of cell death by radiotherapy. Endocr Relat Cancer 1999;6(1):41–4. 20. Bump EA, Braunhut SJ, Palayoor ST, Medeiros D, Lai LL, Cerce BA, et al. Novel concepts in modification of radiation sensitivity. Int J Radiat Oncol Biol Phys 1994;29(2):249–53. 21. Yang CS, Lee MJ, Chen L. Human salivary tea catechin levels and catechin esterase activities: implication in human cancer prevention studies. Cancer Epidemiol Biomarkers Prev 1999;8(1):83–9.
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22. Mnich CD, Hoek KS, Virkki LV, Farkas A, Dudli C, Laine E, et al. Green tea extract reduces induction of p53 and apoptosis in UVB-irradiated human skin independent of transcriptional controls. Exp Dermatol 2009;18(1): 69–77. 23. Uchida S, Ozaki M, Suzuki K, Shikita M. Radioprotective effects of ( )-epigallocatechin 3-O-gallate (green-tea tannin) in mice. Life Sci 1992;50(2):147–52. 24. Arora R, Gupta D, Chawla R, Sagar R, Sharma A, Kumar R, et al. Radioprotection by plant products: present status and future prospects. Phytother Res 2005;19(1):1–22. 25. Yamamoto T, Hsu S, Lewis J, Wataha J, Dickinson D, Singh B, et al. Green tea polyphenol causes differential oxidative environments in tumor versus normal epithelial cells. J Pharmacol Exp Ther 2003;307(1):230–6. 26. Yamamoto T, Staples J, Wataha J, Lewis J, Lockwood P, Schoenlein P, et al. Protective effects of EGCG on salivary gland cells treated with gamma-radiation or cisplatinum(II)diammine dichloride. Anticancer Res 2004;24(5A):3065–73.
available at www.sciencedirect.com
journal homepage: http://www.elsevier.com/locate/aob
Tea polyphenols protect against irradiation-induced injury in submandibular glands’ cells: A preliminary study Zhe Peng a,b, Zhi-wen Xu a,*, Wen-sheng Wen a, Ren-sheng Wang c a
Department of Otolaryngology and Head and Neck Surgery, The First Affiliated Hospital, Guangxi Medical University, Shuangyong Road, Nanning 530021, Guangxi, China b Department of Otorhinolaryngology Head and Neck Surgery, Beijing Tongren Hospital, Affiliated to Capital Medical University, Beijing 100013, China c Department of Radiotherapy, The First Affiliated Hospital, Guangxi Medical University, Nanning 530021, Guangxi, China
article info
summary
Article history:
Aim: To study the protective effect of tea polyphenols (TPs) on submandibular glands
Accepted 14 December 2010
affected by radiation injury. Methods: Sixty rats were randomly divided into radiation group (R-group, N = 30) and TP-
Keywords:
pre-treated-radiation group (TPR-group, N = 30). The rats were intragastrically administered
Radiation
with TP or normal sodium from 14 days before radiation, continuously daily, until the
Submandibular gland
experiment. All the rats in both groups were irradiated with a single exposure dose of 15 Gy
Tea polyphenols
gamma rays that were delivered to the head and neck areas. Ten rats of each group were
Apoptosis
anatomised on the 3rd, 6th and 30th day after irradiation, respectively. The submandibular glands of the rats were removed for the study. The morphologic changes of the submandibular glands were observed by transmission electron microscopy (TEM). The terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP)-biotin nick-end labelling (TUNEL) method was used to detect apoptosis of the submandibular glands’ cells. Results: Electron microscope observation of the submandibular glands showed that the lesions of the TPR-group were mild. Change in apoptosis of the cells was not obvious compared with the R-group. The cell apotosis was typical after irradiation in the R-group. Apoptosis index that was detected in the cells of submandibular glands of the TPR-group was statistically significantly decreased compared with the R-group (P < 0.01) on the 3rd, 6th and 30th day after irradiation. Conclusion: TP could protect submandibular glands from radiation injuries, and the protection mechanism may be realised by anti-apoptosis. # 2010 Elsevier Ltd. All rights reserved.
Radiotherapy plays an important role in the treatment of head and neck carcinomas. Conventional radiotherapy is given in daily fractions of 1.8–2.0 greys (Gy), up to total doses of 66– 70 Gy in 6–7 weeks as definitive treatment of head and neck cancers.1 The major salivary glands frequently receive a high radiation dose during the radiotherapy periods. A high dose of
radiation on the salivary glands has resulted in a reduction of salivary output and change in salivary composition, as the salivary glands are radiosensitive.2,3 Recent evidences suggested that alterations in the fractionation schedule and concomitant chemotherapy may significantly improve the result of therapy.4,5 However, the radiation-induced injury to
* Corresponding author. Tel.: +86 771 5356311. E-mail address: [email protected] (Z.-w. Xu). 0003–9969/$ – see front matter # 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.archoralbio.2010.12.009
archives of oral biology 56 (2011) 738–743
the salivary glands and the other oral sequelae of head and neck induced by radiotherapy are difficult to manage now. It has been shown that xerostomia resulted from decreasing saliva flow, and this change was irreversible.6 This significantly influenced patients’ quality of life.7 However, medical therapy cannot solve this problem now, and the mechanism of radiation-induced xerostomia remains to be elucidated.8 It is known that tea is the most widely consumed beverage in the world. Tea polyphenols (TPs), which are isolated from the leaves of green tea, have a number of beneficial effects on health, such as anticancer and other protection.9 The biological activities of TPs include antioxidation, modulation of enzyme systems for metabolising chemical carcinogens, scavenging of activated metabolites of chemical carcinogens and inhibition of tumour promotion.10–13 There were also some studies concerning the anti-radiation effect of TP.14–16 But there were few studies, which reported as to whether TP has a protective effect on submandibular gland injury induced by irradiation. This study aimed to investigate the effect of TP on submandibular gland injury induced by radiation.
1.
Materials and methods
1.1. Animals were grouped randomly and given irradiation Sixty female Wistar rats aged 6–7 months were obtained from the Center of Animal Experiment of Guangxi Medical University. The rats were kept under standard housing conditions (laboratory rodent chow and water ad libidum and a 12-h light day cycle). All the rats were acclimated for a week. This animal experiment was carried out in accordance with Medical Faculty Polices and Guidelines for the Care and Use of Laboratory Animals. All efforts were made to minimise animals’ suffering.. All the rats were randomly divided into two groups: (1) Radiation group (R-group) (N = 30). Ten rats from the group were sacrificed and the experiment was performed on the 3rd, 6th and 30th day after irradiation (R-3d, R-6d and R-30d subgroups). (2) TP pre-treated group before radiation (TPR-group) (N = 30). Ten rats from this group were sacrificed and the experiment was also performed on the 3rd, 6th and 30th day after irradiation (TPR-3d, TPR-6d and TPR-30d subgroups). This random process was computer controlled. The TP was provided by the Tea Polyphenols Company of Zhejiang Province in China. The TP in the study contained total polyphenol no less than 90%, total catechins no less than 65%, epigallocatechin gallate (EGCg) no less than 40% and caffeine less than 12%. All the components were analysed by high-performance liquid chromatography (HPLC). In TPR-group, the TP was dissolved in saline at 0.2 g kg bodyweight 1. Intragastric administration with TP was initiated 14 days before the irradiation, continuously daily, until the experiment. The total time of administration with TP was 17 days in the TPR-3d subgroup, 20 days in TPR-6d subgroup and 44 days in TPR-30d subgroup. In the R-group,
739
vehicle saline (2 ml kg bodyweight 1) was administered in the same way and at the same times as in the TPR-group. The intragastric TP and vehicle saline were administered orally by a feeding tube daily. Irradiation was performed according to the study of Takeda et al. [17]. Briefly, all animals were anaesthetised with an intra-peritoneal injection with pelltobarbitalum natricum at 60 mg kg bodyweight 1, and were fixed in a plastic mould. Then, the rats were locally irradiated in the head and neck regions. Both groups were irradiated with a single dose of 15 Gy. Irradiation was performed by a standard source-tosurface distance (SSD) of 100 cm. Radiation field size was x = 15 cm, y1 = 0 cm and y2 = 3 cm, and the dose rate was 121 cGy min 1.
1.2. Tissue preparation and transmission electron microscopy The submandibular glands of rats that were removed surgically were fixed in 4% paraformaldehyde and processed for paraffin embedding, according to a standard procedure. Serial sections with a thickness of 3 mm were prepared and stained with haematoxylin–eosin (HE). Segments (1 mm 1 mm) of the rat submandibular gland were prefixed with 2.5% glutaraldehyde for 12 h, and then postfixed with 1% osmium tetroxide for 2 h. After dehydration in a graded series of ethanol, the tissue specimens were embedded in Epok 812. Ultrathin sections were cut with the Sorvall MT 5000 ultramicrotome equipped with a diamond knife, stained with uranyl acetate and lead citrate, and examined under an H-500 electron microscope at 75 kV accelerating voltage.
1.3. Terminal deoxynucleotidyl transferase (TdT)mediated deoxy uridine triphosphate (dUTP)-biotin nick-end labelling method (TUNEL) and quantification of apoptotic index The terminal deoxynucleotidyl transferase (TdT)-mediated deoxy uridine triphosphate (dUTP)-biotin nick-end labelling TUNEL method was performed to detect apoptotic cells. The In Situ Cell Death Detection Kit, POD (Roche Molecular Biochemicals, Catalogue Number 1684817) was used in this study. The sections were incubated with TdT and fluorescein dUTP with proteinase K (20 mg ml 1) pre-treatment. After phosphatebuffered saline (PBS) rinsing, anti-fluorescein-peroxidase antibody was applied, and the reaction was visualised by 3,3-diaminobenzidine (Fujian Maxin Company, China). The sections were counterstained with haematoxylin. Negative control sections were incubated with distilled water in the absence of TdT. The apoptotic index (AI) was determined by calculating the percentage of TUNEL-positive cells per animal using the following method. To ensure the objectivity of the analysis, the evaluation was carried out by two independent observers. Five sections were randomly chosen for each rat. Approximately 1000 cell nuclei from each cell population (acinar cells, granular convoluted tubule cells and intercalated duct cells) were counted for each section at 400 magnification, and the number of apoptotic nuclei was expressed as a percentage of
740
archives of oral biology 56 (2011) 738–743
the total. Striated ducts were omitted from the study, as too few of them were examined to provide useful information. Data are expressed as an average.
1.4.
Statistical analysis
The data values are presented as mean SD. The statistical significance of differences was checked by Student’s t-test. Differences were considered to be statistically significant at P < 0.05.
2.
Result
2.1.
Basic information of the animals
No rats died due to irradiation in both groups. The rats weighed 18 20 g in the initial study. There were no significant changes on the 3rd and 6th day after radiation. However, on the 30th day after radiation, the weights of the rats decreased significantly, and their activity also reduced obviously. Although the food intake seemingly decreased on the 30th day after irradiation, TP
was administrated intragastrically with a tube daily all the time to ensure adequate intake.
2.2. Pathohistology of submandibular glands post irradiation Pathohistological changes were observed in both groups. The changes were aggravated in the R-group, as discussed here: R-group (3 days after irradiation, R3d): Vacuolisation of some acinar cells and granular convoluted tubule cells and decrease of granules in the granular convoluted tubule cells. R-group (6 days after irradiation, R6d): Enlarged intercellular space, cellular atrophy and degranulation of granular convoluted tubule cells; enlarged tissue space. R-group (30 days after irradiation, R30d): Lysis of entire acini and granular convoluted tubule cells was observed, predominantly single cells were affected and lobules of the submandibular gland were disordered with a mass of deposition of fibrous tissue. Pathohistological changes of the TPR-groups (including TPR3d, TPR6d and TPR30d subgroups) were mild compared with the R-group, atrophy of
Fig. 1 – (A) R-group (day 6 post-irradiation): mitochondria swell, disorganization and reduction or vanish of the crista (arrow). (B) TPR-group (day 6 post-irradiation): rupture of the mitochondria (arrow), the rest are nearly normal. (C) R-group (day 30 post-irradiation): cell shrinkage (arrow), chromatin condensation. (D) TPR-group (day 30 post-irradiation): one cell showed the chromatin changes resembling apoptosis (arrow), another two cells were normal.
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Table 1 – Apoptotic index of 3 stages post-irradiation between R-group and TPR-group (mean W SD). Groups R-group TPR-group *
Day 3 (N = 10)
Day 6 (N = 10)
Day 30 (N = 10)
25.21 4.7 6.97 2.1*
17.46 2.1 5.36 1.6*
17.69 2.4 5.64 2.0*
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seen. Apoptotic activity was the greatest in granular convoluted tubule cells, whilst mucous acinar cells were relatively resistant to apoptosis. The AI of each group can be seen from Table 1 and Fig. 2. The difference was detected at the same time of post irradiation of the R-group and the TPR-group (P < 0.01).
P < 0.01.
3. gland was local and degranulation of granular convoluted tubule cells was alleviated.
2.3.
Transmission electron microscopy
In the R-group: We observed acinar cells, intercalated duct cells, and granular convoluted tubule cell that showed oedema, mitochondrial swelling, disorganisation and reduction or disappearance of the crista, and occasionally, rupture of the mitochondria. These cells also contained dilated Golgi apparatus. In addition, cell death resembling apoptosis was occasionally visible. These cells showed the chromatin changes seen in apoptosis; some cell showed chromatin condensation. In the TPR-group: Here, the changes of ultramicroscopic structure in the cells were lesser than in the R-group (Fig. 1).
2.4.
Apoptosis
In all gland compartments (acinar cells, intercalated duct cells and granular convoluted tubule cells), apoptotic activity was
Discussion
The pathophysiology mechanism of modifications in the salivary gland induced by radiation is not clear yet. In addition, currently, there are not adequate prevention and treatment measures available. There are convincing evidences that ionising radiation could induce apoptosis.2 It is believed that apoptosis played a major role only in acute post-irradiation damage.18–20 TP is a main component of tea. Further, research has confirmed the radioprotective effect of TP. Yang et al. [21] in their study, confirmed that the concentration of TP in saliva was 10-fold higher than serum concentration after administering TP to humans, and also indicated that catechins were absorbed through the oral mucosa. Hence, TP can be used to prevent oral diseases, such as xerostomia, induced by irradiation. There were convincing evidences that TP has a function in anti-irradiation.22–24 All of these factors make TP a good prospect for the prevention of some oral diseases. In our study, the morphological change of submandibular gland cells was significantly different in the two groups. In the R-group, the injury to the submandibular gland cells was
Fig. 2 – (A-1) R-group (day 3 post-irradiation), (A-2) TPR-group (day 3 post-irradiation); (B-1) R-group (day 30 post-irradiation), (B-2) TPR-group (day 30 post-irradiation). Introduction: Apoptotic activity was seen in all gland compartments (acinar cells, ID cells, and GCT cells). Apoptotic activity was the greatest in GCT cells (arrow). In the same stage post-irradiation, apoptotic activity of TPR-group was fewer than R-group.
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serious, and cell apoptosis as seen by electron microscopy was widespread. However, in the TPR-group, the extent of cell injury and the number of cell apoptosis events was significantly reduced compared with the R-group. Transmission electron microscopic studies revealed mitochondrial swelling, and cristae appeared indistinct in the cells. Hence, we infer that the mechanism of cell change in the submandibular gland after irradiation was induced by apoptosis. Cell apoptosis was further assessed by the TUNEL method. AI in the TPR-group was significantly reduced compared with the R-group. The result confirmed that TP can protect submandibular gland cells from being injured by radiation through anti-apoptosis. Furthermore, the peak of apoptosis after irradiation was in the early post-irradiation phase18,20; but we had observed significant AI in irradiated cells that persisted to the late post-irradiation (30 days post-irradiation) period yet. We speculated that the anti-apoptosis function of TP continued its existence beyond the early stage. Compared with the same phase post irradiation, we observed that the AIs of the submandibular gland in the Rgroups (including the 3rd day, 6th day and 30th day subgroup) were obviously higher than for the TPR-groups. This may prove that TP protected the submandibular gland by influencing the cell cycle. The Yamamoto et al. [25,26] in vitro studies confirmed that TP could decrease cell apoptosis after irradiation. TP plays different roles in submandibular gland cells and tumour cells according to the concentration. At some certain physiological concentrations (0–50 mmol l 1), TP has a protective effect both on submandibular gland cells and tumour cells. However, a high concentration of TP (>200 mmol l 1) had a protective effect on normal submandibular gland cells yet, whilst it still suppressed the tumour cells at the same time. Further, it may be able to protect salivary gland cells from damage caused by chemotherapeutic drug and radiation therapy. Hence, in our study, we only focus on the high dose concentration (>200 mmol l 1) to investigate the protective effect of TP on normal submandibular gland cells; in this study, we used 0.2 g kg bodyweight 1 (436 mmol l 1) considering both the high concentration and the median lethal dose (LD50). In conclusion, TP can protect submandibular glands from radiation injuries by anti-apoptosis. It proves that TP can be used in the clinic to prevent irradiation-induced salivary gland injury. However, this is an initial study and further studies will be needed to confirm the modulated mechanic pathway of the anti-apoptosis function of TP in the future.
Funding This work was supported by the Emphasis Research Funding of Health Department of Guangxi Zhuang Autonomous Region (Emphasis Healthy of Guangxi, No. 200634).
Conflict of interest statement None of the authors have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.
Ethical approval This project was approved by the Animal Ethical Committee of the First Affiliated Hospital of Guangxi Medical University.
Acknowledgement We thank Jie Liang assistant professor, Institute of Foreign Languages of Guangxi Medical University, for all the efforts and advice on the writing and grammar in the revised article.
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