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The effect of ultraviolet radiation of pancreatic exocrine cells in mole rats: An ultrastructural study
J o u r n a l o f R a d i a t i o n R e s e a r c h a n d A p p l i e d S c i e n c e s 8 ( 2 0 1 5 ) 4 9 e5 4
Available online at www.sciencedirect.com
H O S T E D BY
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Journal of Radiation Research and Applied Sciences journal homepage: http://www.elsevier.com/locate/jrras
The effect of ultraviolet radiation of pancreatic exocrine cells in mole rats: An ultrastructural study Hu¨seyin Tu¨rker* Ankara University, Science Faculty, Department of Biology, 06500 Ankara, Turkey
article info
abstract
Article history:
The purpose of this study was to investigate the ultrastructural effects of ultraviolet C
Received 16 September 2014
(UVC) radiation on the pancreatic exocrine cells of mole rats. The mole rats were divided
Accepted 24 October 2014
into two groups as control and test groups. Control group did not receive any radiation. The
Available online 29 November 2014
other group was irradiated with UV radiation for 14 and 28 days. The pancreatic tissue samples were prepared and then analyzed through transmission electron microscope.
Keywords:
Depending on the radiation exposure, it is likely to say that the zymogen granules
Ultraviolet radiation
decreased to more than 70 per cent for the control group and the dilation of rough endo-
Pancreatic exocrine cells
plasmic reticulum and vacuolization of mitochondria increased in the pancreatic exocrine
Ultrastructure
cells at the 14 days of radiation as compared with the control group; however, they were
Mole rats
easily observed in the 28 days of radiation exposure. Particularly in the 28 days of radiation, the zymogen granules decreased and vacuolated, and the rough endoplasmic reticulum was frequently shortened and dilated. These findings clearly demonstrated the effects of UVC radiation on pancreatic exocrine cells in an exposure-period dependent manner. Copyright © 2014, The Egyptian Society of Radiation Sciences and Applications. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).
1.
Introduction
Living organisms are continually exposed to radiation in nature as well as from nuclear weapons testing, occupations, consumer products and medical procedures (Hall & Giaccia, 2006; Nias, 1998; Sharma, Parmar, Sharma, Verma, & Goyal, 2011). Radiation effects on the living beings are generally divided into two categories: deterministic and stochastic effects. Deterministic effects are those whose severity increases as dose increases. The level of damage on the cell structure particularly depends on the radiation dose received. Stochastic effects of radiation are independent of absorbed dose and under certain exposure conditions, the effects may or
may not occur. There is no threshold and the probability of having the effects is not proportional to the dose absorbed. Curability of the effect has little to do with the dosage of radiation received. Stochastic effects modify a limited number of cells after the radiation exposure (Alexandra, Merriline, Shilpa, & Thomas, 2006; Buckley, Bines, Kemball, & Williams, 1998; Chodick et al., 2008; NCRP, 2009). Solar ultraviolet radiation is a part of the spectrum of electromagnetic radiation emitted by the sun. It is arbitrarily divided into 3 categories of different wavelength: Ultraviolet A (UVA) 400e320 nm, Ultraviolet B (UVB) 320e290 nm and Ultraviolet C (UVC) 290e200 nm (Dong et al., 2007; WHO, 1994). UVA rays cause light brown tan in a short time; the subsequent darkening is due to melanin, which accumulates in
* Tel.: þ90 3122805153; GSM: þ90 05323603741. E-mail address: [email protected]. Peer review under responsibility of The Egyptian Society of Radiation Sciences and Applications. http://dx.doi.org/10.1016/j.jrras.2014.10.006 1687-8507/Copyright © 2014, The Egyptian Society of Radiation Sciences and Applications. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).
50
J o u r n a l o f R a d i a t i o n R e s e a r c h a n d A p p l i e d S c i e n c e s 8 ( 2 0 1 5 ) 4 9 e5 4
the skin. UVB rays cause is delayed but long-term tan mostly resulting in melanin synthesis in the skin. It causes serious sunburn, associated with intensified erythema and oedema, ache, and blister formation in less than one day of exposure. UVC rays, which have sterilization and biocidal properties and are especially important in terms of harmful effects to living beings. Fortunately, majority of this emission is filtered by the ozone (O3) layer (Steel, 2002; Stolarski et al., 1992; WHO, 1994). Decrease in ozone layer leads to increase in the amount of dangerous UV radiation that reaches the earth's surface (Thiele, Dreher, Maibach, & Packer, 2003). As the thickness of this layer has been reduced, it is estimated that skin cancer, cataract and immune deficiency syndrome cases will be € rn, Bais, & increased in near future (Mayer, 1992; McKenzie, Bjo Ilyas, 2003; Verma et al., 2011). The effects of UVC radiation as a noxious environmental agent on the pancreatic exocrine cells have not been studied despite the fact that it comprises the main component of solar ultraviolet radiation. For this reason, the effects of UVC radiation on the pancreatic exocrine cells of mole rats were examined by the electron microscope. For this reason, the mole rats were selected for this study as these animals live in underground galleries and have no UV exposure in their habitat. That's why, they exposed to artificially produced UVC radiation in lab and pancreatic cells changes were compared to control group.
2.
Materials and methods
Ten adult Mole rats (Spalax leucodon) of both sexes, weighing 180e200 g were used in this study. All rats were caught within the rural areas of Ankara in Turkey. They were kept at the laboratory for 10 days at a stable temperature (20 ± 2 C) in order to obtain adaptation of the new environment. The mole rats were housed individually in special cages called terrarium and a constant UVC dosage was applied from the upper. All animals were fed with carrot, potato, plant roots and no special diet was given. A “Mazda TG” ultraviolet lamp in 30 W powers and in 90 cm length was placed to the upper cover of the terrarium. The intensity of the UV emitted from the lamp was measured to be 254 nm in wavelength and the energy in 1 s was found to be 0.0014 J/cm2. The mole rats were divided into control and test groups. Group I was separated as the control, and was not received any radiation. Taking into account of sunlight period, the other groups were exposed to artificial UVC radiation for 8 h daily (between 08.00 and 17.00 h). A feeding interval was given at midday for 1 h. A timer was used to standardize UVC exposure times. Group II was irradiated for 14 days (Total dosage was 564,48 J/cm2) and Group III was irradiated for 28 days (Total dosage was 1.128,96 J/cm2). At the end of the experiments, the animals were anesthetized using ether inhalation and were sacrificed. The pancreas tissues were dissected out, and cut into small pieces that were taken and prepared for electron microscopic studies. The small pancreas pieces were fixed in glutaraldehyde, then washed in buffer and post-fixed in 1% osmium tetroxide, dehydrated in ethanol, cleared in propylene oxide
and embedded in Araldite CY-212. Ultrathin sections were prepared, stained with uranyl acetate and lead citrate and then examined on Jeol JEM 100 CX-II electron microscope. All experiments were carried out in accordance with the Ankara University guidelines for the care of experimental animals. Also, guiding principles for experimental procedures found in Declaration of Helsinki of the World Medical Association regarding animal experimentation were followed in the study.
3.
Results
3.1. Group I (control group): pancreatic exocrine cells from controlled mole rats The pancreatic exocrine cells were formed of serous cells which were pyramidal in shape and they were lined around a central lumen forming an acinus. Each pancreatic acinar cell was enveloped by a plasma membrane which showed distinct regional variation. The basal and lateral plasma membrane was straight and unfolded. The acinar lumen, the surface of which formed varying numbers of microvilli, often contained secretory material (Fig. 1). The nuclei were smoothly rounded in general and tended to be slightly eccentric toward its basal portion. Each nucleus contained a nucleolus. The pancreatic exocrine cells were characterized by numerous zymogen granules (smoothly rounded formations with a homogeneous content of high density) in the apical region of the cytoplasm. They consisted of electron dense material surrounded by a limiting membrane. Some condensing vacuoles containing secretory material of low density were seen in the zymogen granules. Oval and rod-like mitochondria were present in both the apical and basal parts of the acinar cells (Fig. 1). A well developed rough endoplasmic reticulum which was distributed basally and laterally to the nucleus. The rough
Fig. 1 e Electron micrograph of pancreatic exocrine cells from control mole rats. Showing normal architecture of pancreatic exocrine cells. Nucleus (N), zymogen granules (Zg), condensing vacuole (Cv), mitochondria (m), acinar lumen (L) and rough endoplasmic reticulum (rER). £5000.
J o u r n a l o f R a d i a t i o n R e s e a r c h a n d A p p l i e d S c i e n c e s 8 ( 2 0 1 5 ) 4 9 e5 4
endoplasmic reticulum consisted of parallel cisterns together with many ribosomes attached to the outer surface of the membranes. But, the rough endoplasmic reticulum was sacculus or cisterns in some cells (Fig. 1).
3.2. Group II. Pancreatic exocrine cells from mole rats exposed to UVC radiation for 14 days The most prominent ultrastructural change in the pancreatic exocrine cells of this group was a reduction in the number of zymogen granules and occasional absence of them in the apical portion of the cell. No obvious changes were seen in the zymogen granules as compared with the control group (Figs. 2 and 3). Acinar cells were severely damaged and their cytoplasms showed open electron density (Figs. 2e5). The acini cells began to destroy and most of the exocrine cells were smaller in size than those of the previous groups. Conversely, some immature zymogen granules were observed among the zymogen granules. The rough endoplasmic reticulum and mitochondria were severely vacuolated in some cells. Some nuclei shapes were changed (Figs. 2e4). Condensing vacuoles were markedly present among the zymogen granules (Fig. 3).
51
remained apparently unchanged, the nuclei shown significant changes in appearance (Figs. 6e8). The rough endoplasmic reticulum was increased in amount and length, the cisterns were irregularly dilated (Figs. 7e9). The zymogen granules in the 28-day irradiated mole rats were decreased and vacuolated according to the 14-day irradiated mole rats cells (Figs. 6, 8 and 9). The condensing vacuole was observed in the apical area of acinar cells (Figs. 8 and 9). The acinar lumen was full of secretary materials (Figs. 6 and 9).
4.
Discussion
Under long-term radiation, the pancreatic exocrine cells and most of the cellular organelles underwent degenerative changes to some extent. Some differences were in the extent of degeneration of the cells; naturally, the damage to the cell in irradiated group was generally greater than the one in the 14 day irradiated group. Some acinar cells were severely damaged, the cytoplasm of cells which had become coarse and showed open electron density (Fig. 6). The mitochondria
All living organisms on Earth are being perpetually exposed to some amount of radiation originating from a variety of sources. The deformation in the cells could develop depending on the dosage and the exposal period of the radiation (Gridley, Pecaut, Miller, Moyers, & Nelson, 2001; Mansoub & Sarvestani, 2011; Sharma & Saini, 2003; Sharma et al., 2011). At high doses, radiation destroys the cells structure. At low doses, radiation is no longer considered to be as harmful as once though. Because the pathological changes can be prevent by some antioxidants (Arulselvan & Subramanian, 2007; Jagetia & Reddy, 2005). Low-dose radiation significantly increases endogenous antioxidants in different tissues including liver, spleen, brain and testes (Avti et al., 2005; Cai, 1999; Yamaoka, Edamatsu, & Mori, 1991). The harmful effects of UV radiation on living organisms occur indirectly. Free radicals are released as a result of radiolysis of water molecules when they are exposed to radiation and these radicals lead to changes in the structures and functions of various molecules by interacting with them (Barcellos-Hoff, 1998; Jagetia & Reddy, 2005; Zare, Hashemi, & Salari, 2007). In the present study, the first detectable changes in pancreatic exocrine cells appeared to develop after 14 day of
Fig. 2 e Electron micrograph of pancreatic exocrine cells from mole rats exposed to UVC radiation for 14 days showing the nuclei (N), zymogen granules (Zg), immature zymogen granules (arrow), condensing vacuole (Cv), mitochondria (m) and rough endoplasmic reticulum (rER). £5000.
Fig. 3 e Electron micrograph of pancreatic exocrine cells from mole rats exposed to UVC radiation for 14 days showing the nucleus (N), zymogen granules (Zg), immature zymogen granules (arrow), condensing vacuole (Cv), mitochondria (m) and rough endoplasmic reticulum (rER). £7000.
3.3. Group III. Pancreatic exocrine cells from mole rats exposed to UVC radiation for 28 days
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J o u r n a l o f R a d i a t i o n R e s e a r c h a n d A p p l i e d S c i e n c e s 8 ( 2 0 1 5 ) 4 9 e5 4
Fig. 4 e Electron micrograph of pancreatic exocrine cells from mole rats exposed to UVC radiation for 14 days showing destruction in nuclei (N), zymogen granules (Zg), condensing vacuole (Cv), mitochondria (m) and severely dilated rough endoplasmic reticulum (rER). £5000.
UVC radiation exposure. Electron microscopic examination revealed that radiation exposure caused severe degenerative changes, such as decreased in zymogen granules, dilatation of rough endoplasmic reticulum, vacuolation of mitochondria, condensation of chromatin onto the nuclear lamina of pancreatic acinar cells. Microvilli of acinar cells showed changes in length, frequency and alteration of bordering membrane. Some acinar cells were severely damaged and the cytoplasm of cells showed open electron density. These data were corroborated by previous studies done on some animals by investigators (Arulselvan & Subramanian, 2007; Carr et al., 1992; Du Toit et al., 1987; Somosy, 2000).
Fig. 5 e Electron micrograph of pancreatic exocrine cells from mole rats exposed to UVC radiation for 14 days showing the nuclei (N), zymogen granules (Zg), immature zymogen granules (arrow), vacuolated mitochondria (m) and dilated rough endoplasmic reticulum (rER). £5000.
Fig. 6 e Electron micrograph of pancreatic exocrine cells from mole rats exposed to UVC radiation for 28 days showing the acinar cells, cell nuclei (N), mitochondria (m), rough endoplasmic reticulum (rER) and acinar lumen (L). The lumen was full of secretory material and some zymogen granules were vacuolated. £5000.
After 28 day of UVC radiation exposure, the pancreatic exocrine cells and most of the cellular organelles underwent degenerative changes to some extent. There were some differences in the extent of degeneration of the cells; naturally, the damage to the cell in irradiated group was generally greater than that in the 14 day irradiated group. Some acinar cells were damaged severely and their cytoplasm showed an open electron density. The nuclei of acinar cells showed significant changes in appearance. The rough endoplasmic reticulum was increased in amount and length, the cisterns were irregularly dilated. The zymogen granules were decreased and generally vacuolated. Some condensing
Fig. 7 e Electron micrograph of pancreatic exocrine cells from mole rats exposed to UVC radiation for 28 days showing the nuclei (N), zymogen granules (Zg), condensing vacuole (Cv), mitochondria (m) and dilated rough endoplasmic reticulum (rER). £7000.
J o u r n a l o f R a d i a t i o n R e s e a r c h a n d A p p l i e d S c i e n c e s 8 ( 2 0 1 5 ) 4 9 e5 4
53
irradiation or after high dose radiation (Gridley et al., 2001; Jagetia & Reddy, 2005; Santra & Mannal, 2009), and also some structural studies carried out on living organisms by some researchers (Barcellos-Hoff, 1998; Du Toit et al., 1987; Mansoub & Sarvestani, 2011; Somosy, 2000). The results clearly indicated the effects of UVC radiation on the pancreatic exocrine cells. It was found that these effects occurred depending on the dosage and the exposal period of the radiation. Further experimental studies may be needed to confirm the relation between the exposure time, dosage and cell deformation in the pancreatic acinar cells.
Conflict of interest None. Fig. 8 e Electron micrograph of pancreatic exocrine cells from mole rats exposed to UVC radiation for 28 days showing destructed nuclei (N), vacuolated zymogen granules (Zg), dilated rough endoplasmic reticulum (rER) Mitochondria (m). £5000.
Financial support There was no financial support taken for this manuscript.
The contribution manner of authors vacuoles and immature granules with low electron density were observed in the apical part of the acinar cells. These histological damage might result from an increase in the process of lipid peroxidation and a decrease in the activity of antioxidant enzymes of the body with the consequent damage of cellular membranes (Saada & Azab, 2001; Sugiyama, Kahyama, Hidaka, Kumano, & Ogura, 1984; Yamaoka et al., 1991). Such degenerative changes that occurred in cytoplasm and organelles resulting in significant functions in each phase of radiation in order that cell could carry on its crucial activities would certainly decrease its process and distort its structure. Our results were in agreement with studies done on some animals tissues due to local
Fig. 9 e Electron micrograph of pancreatic exocrine cells from mole rats exposed to UVC radiation for 28 days showing the nucleus (N), vacuolated zymogen granules (Zg), dilated rough endoplasmic reticulum (rER) and mitochondria (m). £5000.
These authors contributed equally to this work.
Acknowledgements The authors thank Prof. Dr. Turan Gu¨ven and Prof. Dr. Mustafa Yel (Gazi University, Gazi Education Faculty, Department of € Biology, Ankara, Turkey) and Prof. Dr. Emin Ozat (Histology Department in GATA University, Ankara, Turkey) for assisting the experiment and proofreading of the manuscript.
references
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Cai, L. (1999). Research on the adaptive response induced by lowdose radiation: where have we been and where should we go? Human & Experimental Toxicology, 18, 419e425. Carr, K. E., McCullough, J. S., Nelson, A. C., Hume, S. P., Nunn, S., & Kamel, H. M. (1992). Relationship between villous shape and mural structure in neutron irradiated small intestine. Scanning Microscopy, 6, 561e572. PMID: 1462140 [PubMed e indexed for MEDLINE]. Chodick, G., Bekiroglu, N., Hauptmann, M., Alexander, B. H., Freedman, D. M., Doody, M. M., et al. (2008). Risk of cataract after exposure to low doses of ionizing radiation: a 20-year prospective cohort study among US radiologic technologists. American Journal of Epidemiology, 168(6), 620e631. http:// dx.doi.org/10.1093/aje/kwn171. Dong, P. D., Munson, C. A., Norton, W., Crosnier, C., Pan, X., Gong, Z., et al. (2007). Fgf10 regulates hepatopancreatic ductal system patterning and differentiation. Nature Genetics, 39, 397e402. PMID: 17259985 [PubMed e indexed for MEDLINE]. Du Toit, D. F., Heydenrych, J. J., Smit, B., Zuurmond, T., Louw, G., Laker, L., et al. (1987). The effect of ionizing radiation on the primate pancreas: an endocrine and morphologic study. Journal of Surgical Oncology, 34(1), 43e52. http://dx.doi.org/ 10.1002/jso.2930340113. Gridley, D. S., Pecaut, M. J., Miller, G. M., Moyers, M. F., & Nelson, G. A. (2001). Dose and dose rate effects of whole-body gamma-irradiation: II. Hematological variables and cytokines. In Vivo, 15(3), 209e216. PMID: 11491015. Hall, E. J., & Giaccia, A. J. (2006). Radiobiology for the radiologist (6th ed.). Philadelphia, PA: Lippincott, Williams & Wilkins. Jagetia, G. C., & Reddy, T. K. (2005). Modulation of radiation induced alteration in the antioxidant status of mice by naringin. Life Sciences, 77(7), 780e794. http://dx.doi.org/10.1016/ j.lfs.2005.01.015. Mansoub, N. H., & Sarvestani, A. H. (2011). Effects of gamma irradiation on histomorphology of different organs in rats. Scholars Research Library Annals of Biological Research, 2(6), 431e436. www.scholarsresearchlibrary.com. Mayer, S. J. (1992). Stratospheric ozone depletion and animal health. Veterinary Record, 131, 120e122. http://dx.doi.org/ 10.1136/vr.131.6.120. € rn, L. O., Bais, A., & Ilyas, M. (2003). Changes in McKenzie, R. L., Bjo biologically active ultraviolet radiation reaching the earth's surface. Photochemical & Photobiological Sciences, 5e15. http:// dx.doi.org/10.1039/b211155. National Council on Radiation Protection and Measurements. (2009). Ionizing radiation exposure of the population of the United States. Bethesda, MD: NCRP. Report 160 www.ncrponline.org/ Publications/Press_Releases/160press.html. Nias, A. H. W. (1998). An introduction to radiobiology (2nd ed.). New York, NY: Wiley. Saada, H. N., & Azab, K. S. (2001). Role of lycopene in recovery of radiation induced injury to mammalian cellular organelles. Europe PMC (Die Pharmazie), 56(3), 239e241. PMID: 11265592.
Santra, K. B., & Mannal, C. K. (2009). X-rays induced changes in biochemical and histochemical parameters in the testis of male wild Indian house rat, Rattus rattus. Ceylon Journal of Science (Biological Sciences), 38(2), 39e49. http://dx.doi.org/ 10.4038/cjsbs.v38i2.1857. Sharma, P., Parmar, J., Sharma, P., Verma, P., & Goyal, P. K. (2011). Radiation-induced testicular injury and its amelioration by Tinospora cordifolia (an Indian medicinal plant) extract. Evidence-Based Complementary and Alternative Medicine, 1e9. http://dx.doi.org/10.1155/2011/643847, 643847. Sharma, P., & Saini, M. (2003). Modification of radiation induced prenatal mortality by cysteamine, MPG and their combination in Swiss albino mice. Indian Journal of Nuclear Medicine, 18(1e2), 12e18. Somosy, Z. (2000). Radiation response of cell organelles. Micron, 31(2), 165e181. http://dx.doi.org/10.1016/s0968-4328(99)000839. Steel, G. G. (2002). Basic clinical radiobiology (3rd ed.). London, England: Arnold www.amazon.com/Basic-ClinicalRadiobiology...Steel/.../0340807830. Stolarski, R., Bojkov, R., Bishop, L., Zeferos, C., Staehelin, J., & Zawodny, J. (1992). Measured trends in stratospheric ozone. Science, 256(5655), 342e349. http://dx.doi.org/10.1126/ science.256.5055.342. Sugiyama, M., Kahyama, K., Hidaka, T., Kumano, S., & Ogura, R. (1984). Lipid peroxidation and radical formation in methyl linoleate following ultraviolet light exposure. Journal of Dermatology, 11, 455e459. PubMed/ChemPort. Thiele, J. J., Dreher, F., Maibach, H. I., & Packer, L. (2003). Impact of ultraviolet radiation and ozone on the transepidermal water loss as a function of skin temperature in hairless mice. Skin Pharmacology and Applied Skin Physiology, 16, 283e290. http:// dx.doi.org/10.1159/000072068. Verma, P., Sharma, P., Parmar, J., Sharma, P., Agrawal, A., & Goyal, P. K. (2011). Amelioration of radiation-induced hematological and biochemical alterations in Swiss albino mice by Panax ginseng extract. Integrative Cancer Therapies, 10(1), 77e84. http://dx.doi.org/10.1177/1534735410375098. WHO. (1994). Environmental health criteria (EHC) 160: Ultraviolet radiation. Geneva http://www.who.int/mediacentre/ multimedia/en/. Yamaoka, K., Edamatsu, R., & Mori, A. (1991). Increased SOD activities and decreased lipid peroxide levels induced by low dose X irradiation in rat organs. Free Radical Biology and Medicine, 11(3), 299e306. http://dx.doi.org/10.1016/08915849(91)90127-. Zare, M. A., Hashemi, H., & Salari, M. R. (2007). Intracorneal ring segment implantation for the management of keratoconus: safety and efficacy. Journal of Cataract & Refractive Surgery, 33(11), 1886e1891. http://dx.doi.org/ 10.1016/j.jcrs.2007.06.055.
Available online at www.sciencedirect.com
H O S T E D BY
ScienceDirect
Journal of Radiation Research and Applied Sciences journal homepage: http://www.elsevier.com/locate/jrras
The effect of ultraviolet radiation of pancreatic exocrine cells in mole rats: An ultrastructural study Hu¨seyin Tu¨rker* Ankara University, Science Faculty, Department of Biology, 06500 Ankara, Turkey
article info
abstract
Article history:
The purpose of this study was to investigate the ultrastructural effects of ultraviolet C
Received 16 September 2014
(UVC) radiation on the pancreatic exocrine cells of mole rats. The mole rats were divided
Accepted 24 October 2014
into two groups as control and test groups. Control group did not receive any radiation. The
Available online 29 November 2014
other group was irradiated with UV radiation for 14 and 28 days. The pancreatic tissue samples were prepared and then analyzed through transmission electron microscope.
Keywords:
Depending on the radiation exposure, it is likely to say that the zymogen granules
Ultraviolet radiation
decreased to more than 70 per cent for the control group and the dilation of rough endo-
Pancreatic exocrine cells
plasmic reticulum and vacuolization of mitochondria increased in the pancreatic exocrine
Ultrastructure
cells at the 14 days of radiation as compared with the control group; however, they were
Mole rats
easily observed in the 28 days of radiation exposure. Particularly in the 28 days of radiation, the zymogen granules decreased and vacuolated, and the rough endoplasmic reticulum was frequently shortened and dilated. These findings clearly demonstrated the effects of UVC radiation on pancreatic exocrine cells in an exposure-period dependent manner. Copyright © 2014, The Egyptian Society of Radiation Sciences and Applications. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).
1.
Introduction
Living organisms are continually exposed to radiation in nature as well as from nuclear weapons testing, occupations, consumer products and medical procedures (Hall & Giaccia, 2006; Nias, 1998; Sharma, Parmar, Sharma, Verma, & Goyal, 2011). Radiation effects on the living beings are generally divided into two categories: deterministic and stochastic effects. Deterministic effects are those whose severity increases as dose increases. The level of damage on the cell structure particularly depends on the radiation dose received. Stochastic effects of radiation are independent of absorbed dose and under certain exposure conditions, the effects may or
may not occur. There is no threshold and the probability of having the effects is not proportional to the dose absorbed. Curability of the effect has little to do with the dosage of radiation received. Stochastic effects modify a limited number of cells after the radiation exposure (Alexandra, Merriline, Shilpa, & Thomas, 2006; Buckley, Bines, Kemball, & Williams, 1998; Chodick et al., 2008; NCRP, 2009). Solar ultraviolet radiation is a part of the spectrum of electromagnetic radiation emitted by the sun. It is arbitrarily divided into 3 categories of different wavelength: Ultraviolet A (UVA) 400e320 nm, Ultraviolet B (UVB) 320e290 nm and Ultraviolet C (UVC) 290e200 nm (Dong et al., 2007; WHO, 1994). UVA rays cause light brown tan in a short time; the subsequent darkening is due to melanin, which accumulates in
* Tel.: þ90 3122805153; GSM: þ90 05323603741. E-mail address: [email protected]. Peer review under responsibility of The Egyptian Society of Radiation Sciences and Applications. http://dx.doi.org/10.1016/j.jrras.2014.10.006 1687-8507/Copyright © 2014, The Egyptian Society of Radiation Sciences and Applications. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).
50
J o u r n a l o f R a d i a t i o n R e s e a r c h a n d A p p l i e d S c i e n c e s 8 ( 2 0 1 5 ) 4 9 e5 4
the skin. UVB rays cause is delayed but long-term tan mostly resulting in melanin synthesis in the skin. It causes serious sunburn, associated with intensified erythema and oedema, ache, and blister formation in less than one day of exposure. UVC rays, which have sterilization and biocidal properties and are especially important in terms of harmful effects to living beings. Fortunately, majority of this emission is filtered by the ozone (O3) layer (Steel, 2002; Stolarski et al., 1992; WHO, 1994). Decrease in ozone layer leads to increase in the amount of dangerous UV radiation that reaches the earth's surface (Thiele, Dreher, Maibach, & Packer, 2003). As the thickness of this layer has been reduced, it is estimated that skin cancer, cataract and immune deficiency syndrome cases will be € rn, Bais, & increased in near future (Mayer, 1992; McKenzie, Bjo Ilyas, 2003; Verma et al., 2011). The effects of UVC radiation as a noxious environmental agent on the pancreatic exocrine cells have not been studied despite the fact that it comprises the main component of solar ultraviolet radiation. For this reason, the effects of UVC radiation on the pancreatic exocrine cells of mole rats were examined by the electron microscope. For this reason, the mole rats were selected for this study as these animals live in underground galleries and have no UV exposure in their habitat. That's why, they exposed to artificially produced UVC radiation in lab and pancreatic cells changes were compared to control group.
2.
Materials and methods
Ten adult Mole rats (Spalax leucodon) of both sexes, weighing 180e200 g were used in this study. All rats were caught within the rural areas of Ankara in Turkey. They were kept at the laboratory for 10 days at a stable temperature (20 ± 2 C) in order to obtain adaptation of the new environment. The mole rats were housed individually in special cages called terrarium and a constant UVC dosage was applied from the upper. All animals were fed with carrot, potato, plant roots and no special diet was given. A “Mazda TG” ultraviolet lamp in 30 W powers and in 90 cm length was placed to the upper cover of the terrarium. The intensity of the UV emitted from the lamp was measured to be 254 nm in wavelength and the energy in 1 s was found to be 0.0014 J/cm2. The mole rats were divided into control and test groups. Group I was separated as the control, and was not received any radiation. Taking into account of sunlight period, the other groups were exposed to artificial UVC radiation for 8 h daily (between 08.00 and 17.00 h). A feeding interval was given at midday for 1 h. A timer was used to standardize UVC exposure times. Group II was irradiated for 14 days (Total dosage was 564,48 J/cm2) and Group III was irradiated for 28 days (Total dosage was 1.128,96 J/cm2). At the end of the experiments, the animals were anesthetized using ether inhalation and were sacrificed. The pancreas tissues were dissected out, and cut into small pieces that were taken and prepared for electron microscopic studies. The small pancreas pieces were fixed in glutaraldehyde, then washed in buffer and post-fixed in 1% osmium tetroxide, dehydrated in ethanol, cleared in propylene oxide
and embedded in Araldite CY-212. Ultrathin sections were prepared, stained with uranyl acetate and lead citrate and then examined on Jeol JEM 100 CX-II electron microscope. All experiments were carried out in accordance with the Ankara University guidelines for the care of experimental animals. Also, guiding principles for experimental procedures found in Declaration of Helsinki of the World Medical Association regarding animal experimentation were followed in the study.
3.
Results
3.1. Group I (control group): pancreatic exocrine cells from controlled mole rats The pancreatic exocrine cells were formed of serous cells which were pyramidal in shape and they were lined around a central lumen forming an acinus. Each pancreatic acinar cell was enveloped by a plasma membrane which showed distinct regional variation. The basal and lateral plasma membrane was straight and unfolded. The acinar lumen, the surface of which formed varying numbers of microvilli, often contained secretory material (Fig. 1). The nuclei were smoothly rounded in general and tended to be slightly eccentric toward its basal portion. Each nucleus contained a nucleolus. The pancreatic exocrine cells were characterized by numerous zymogen granules (smoothly rounded formations with a homogeneous content of high density) in the apical region of the cytoplasm. They consisted of electron dense material surrounded by a limiting membrane. Some condensing vacuoles containing secretory material of low density were seen in the zymogen granules. Oval and rod-like mitochondria were present in both the apical and basal parts of the acinar cells (Fig. 1). A well developed rough endoplasmic reticulum which was distributed basally and laterally to the nucleus. The rough
Fig. 1 e Electron micrograph of pancreatic exocrine cells from control mole rats. Showing normal architecture of pancreatic exocrine cells. Nucleus (N), zymogen granules (Zg), condensing vacuole (Cv), mitochondria (m), acinar lumen (L) and rough endoplasmic reticulum (rER). £5000.
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endoplasmic reticulum consisted of parallel cisterns together with many ribosomes attached to the outer surface of the membranes. But, the rough endoplasmic reticulum was sacculus or cisterns in some cells (Fig. 1).
3.2. Group II. Pancreatic exocrine cells from mole rats exposed to UVC radiation for 14 days The most prominent ultrastructural change in the pancreatic exocrine cells of this group was a reduction in the number of zymogen granules and occasional absence of them in the apical portion of the cell. No obvious changes were seen in the zymogen granules as compared with the control group (Figs. 2 and 3). Acinar cells were severely damaged and their cytoplasms showed open electron density (Figs. 2e5). The acini cells began to destroy and most of the exocrine cells were smaller in size than those of the previous groups. Conversely, some immature zymogen granules were observed among the zymogen granules. The rough endoplasmic reticulum and mitochondria were severely vacuolated in some cells. Some nuclei shapes were changed (Figs. 2e4). Condensing vacuoles were markedly present among the zymogen granules (Fig. 3).
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remained apparently unchanged, the nuclei shown significant changes in appearance (Figs. 6e8). The rough endoplasmic reticulum was increased in amount and length, the cisterns were irregularly dilated (Figs. 7e9). The zymogen granules in the 28-day irradiated mole rats were decreased and vacuolated according to the 14-day irradiated mole rats cells (Figs. 6, 8 and 9). The condensing vacuole was observed in the apical area of acinar cells (Figs. 8 and 9). The acinar lumen was full of secretary materials (Figs. 6 and 9).
4.
Discussion
Under long-term radiation, the pancreatic exocrine cells and most of the cellular organelles underwent degenerative changes to some extent. Some differences were in the extent of degeneration of the cells; naturally, the damage to the cell in irradiated group was generally greater than the one in the 14 day irradiated group. Some acinar cells were severely damaged, the cytoplasm of cells which had become coarse and showed open electron density (Fig. 6). The mitochondria
All living organisms on Earth are being perpetually exposed to some amount of radiation originating from a variety of sources. The deformation in the cells could develop depending on the dosage and the exposal period of the radiation (Gridley, Pecaut, Miller, Moyers, & Nelson, 2001; Mansoub & Sarvestani, 2011; Sharma & Saini, 2003; Sharma et al., 2011). At high doses, radiation destroys the cells structure. At low doses, radiation is no longer considered to be as harmful as once though. Because the pathological changes can be prevent by some antioxidants (Arulselvan & Subramanian, 2007; Jagetia & Reddy, 2005). Low-dose radiation significantly increases endogenous antioxidants in different tissues including liver, spleen, brain and testes (Avti et al., 2005; Cai, 1999; Yamaoka, Edamatsu, & Mori, 1991). The harmful effects of UV radiation on living organisms occur indirectly. Free radicals are released as a result of radiolysis of water molecules when they are exposed to radiation and these radicals lead to changes in the structures and functions of various molecules by interacting with them (Barcellos-Hoff, 1998; Jagetia & Reddy, 2005; Zare, Hashemi, & Salari, 2007). In the present study, the first detectable changes in pancreatic exocrine cells appeared to develop after 14 day of
Fig. 2 e Electron micrograph of pancreatic exocrine cells from mole rats exposed to UVC radiation for 14 days showing the nuclei (N), zymogen granules (Zg), immature zymogen granules (arrow), condensing vacuole (Cv), mitochondria (m) and rough endoplasmic reticulum (rER). £5000.
Fig. 3 e Electron micrograph of pancreatic exocrine cells from mole rats exposed to UVC radiation for 14 days showing the nucleus (N), zymogen granules (Zg), immature zymogen granules (arrow), condensing vacuole (Cv), mitochondria (m) and rough endoplasmic reticulum (rER). £7000.
3.3. Group III. Pancreatic exocrine cells from mole rats exposed to UVC radiation for 28 days
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Fig. 4 e Electron micrograph of pancreatic exocrine cells from mole rats exposed to UVC radiation for 14 days showing destruction in nuclei (N), zymogen granules (Zg), condensing vacuole (Cv), mitochondria (m) and severely dilated rough endoplasmic reticulum (rER). £5000.
UVC radiation exposure. Electron microscopic examination revealed that radiation exposure caused severe degenerative changes, such as decreased in zymogen granules, dilatation of rough endoplasmic reticulum, vacuolation of mitochondria, condensation of chromatin onto the nuclear lamina of pancreatic acinar cells. Microvilli of acinar cells showed changes in length, frequency and alteration of bordering membrane. Some acinar cells were severely damaged and the cytoplasm of cells showed open electron density. These data were corroborated by previous studies done on some animals by investigators (Arulselvan & Subramanian, 2007; Carr et al., 1992; Du Toit et al., 1987; Somosy, 2000).
Fig. 5 e Electron micrograph of pancreatic exocrine cells from mole rats exposed to UVC radiation for 14 days showing the nuclei (N), zymogen granules (Zg), immature zymogen granules (arrow), vacuolated mitochondria (m) and dilated rough endoplasmic reticulum (rER). £5000.
Fig. 6 e Electron micrograph of pancreatic exocrine cells from mole rats exposed to UVC radiation for 28 days showing the acinar cells, cell nuclei (N), mitochondria (m), rough endoplasmic reticulum (rER) and acinar lumen (L). The lumen was full of secretory material and some zymogen granules were vacuolated. £5000.
After 28 day of UVC radiation exposure, the pancreatic exocrine cells and most of the cellular organelles underwent degenerative changes to some extent. There were some differences in the extent of degeneration of the cells; naturally, the damage to the cell in irradiated group was generally greater than that in the 14 day irradiated group. Some acinar cells were damaged severely and their cytoplasm showed an open electron density. The nuclei of acinar cells showed significant changes in appearance. The rough endoplasmic reticulum was increased in amount and length, the cisterns were irregularly dilated. The zymogen granules were decreased and generally vacuolated. Some condensing
Fig. 7 e Electron micrograph of pancreatic exocrine cells from mole rats exposed to UVC radiation for 28 days showing the nuclei (N), zymogen granules (Zg), condensing vacuole (Cv), mitochondria (m) and dilated rough endoplasmic reticulum (rER). £7000.
J o u r n a l o f R a d i a t i o n R e s e a r c h a n d A p p l i e d S c i e n c e s 8 ( 2 0 1 5 ) 4 9 e5 4
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irradiation or after high dose radiation (Gridley et al., 2001; Jagetia & Reddy, 2005; Santra & Mannal, 2009), and also some structural studies carried out on living organisms by some researchers (Barcellos-Hoff, 1998; Du Toit et al., 1987; Mansoub & Sarvestani, 2011; Somosy, 2000). The results clearly indicated the effects of UVC radiation on the pancreatic exocrine cells. It was found that these effects occurred depending on the dosage and the exposal period of the radiation. Further experimental studies may be needed to confirm the relation between the exposure time, dosage and cell deformation in the pancreatic acinar cells.
Conflict of interest None. Fig. 8 e Electron micrograph of pancreatic exocrine cells from mole rats exposed to UVC radiation for 28 days showing destructed nuclei (N), vacuolated zymogen granules (Zg), dilated rough endoplasmic reticulum (rER) Mitochondria (m). £5000.
Financial support There was no financial support taken for this manuscript.
The contribution manner of authors vacuoles and immature granules with low electron density were observed in the apical part of the acinar cells. These histological damage might result from an increase in the process of lipid peroxidation and a decrease in the activity of antioxidant enzymes of the body with the consequent damage of cellular membranes (Saada & Azab, 2001; Sugiyama, Kahyama, Hidaka, Kumano, & Ogura, 1984; Yamaoka et al., 1991). Such degenerative changes that occurred in cytoplasm and organelles resulting in significant functions in each phase of radiation in order that cell could carry on its crucial activities would certainly decrease its process and distort its structure. Our results were in agreement with studies done on some animals tissues due to local
Fig. 9 e Electron micrograph of pancreatic exocrine cells from mole rats exposed to UVC radiation for 28 days showing the nucleus (N), vacuolated zymogen granules (Zg), dilated rough endoplasmic reticulum (rER) and mitochondria (m). £5000.
These authors contributed equally to this work.
Acknowledgements The authors thank Prof. Dr. Turan Gu¨ven and Prof. Dr. Mustafa Yel (Gazi University, Gazi Education Faculty, Department of € Biology, Ankara, Turkey) and Prof. Dr. Emin Ozat (Histology Department in GATA University, Ankara, Turkey) for assisting the experiment and proofreading of the manuscript.
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