IMPACT OF FLUORIDE AND SUPEROXIDE DISMUTASE ON THE GOLGI APPARATUS IN RAT RIB-CAGE CHONDROCYTES

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1 13 13 IMPACT OF FLUORIDE AND SUPEROXIDE DISMUTASE ON THE GOLGI APPARATUS IN RAT RIB-CAGE CHONDROCYTES Yanni Yu, a Binjie Meng a Guizhou, China SUMMARY: Chondrocytes of rib-cage cartilage from Wistar rats were cultured in vitro for 7 days to study the Golgi complex changes in the presence of various concentrations of fluoride (F) with or without the addition of superoxide dismutase (SOD). When the changes were observed by laser scanning confocal microscopy (LSCM), the average optical density (AOD) of the Golgi apparatus was progressively decreased at 40, 80, and 160 µmol NaF/L. On the other hand, the AOD of the Golgi apparatus was significantly increased by addition of SOD to the F groups. The results indicated: (1) the Golgi apparatus was adversely affected by F; and (2) SOD was able to protect the Golgi apparatus of chondrocytes from damage caused by F, especially at higher concentrations of F. Keywords: Fluoride and chondrocytes; Golgi apparatus; Laser scanning confocal microscopy (LSCM); Rat rib-cage chondrocytes; Superoxide dismutase (SOD). INTRODUCTION Endemic fluorosis is a chronic systemic disease, caused by excessive long-term fluoride (F) intake. Among its notable adverse effects are dental and skeletal fluorosis. Damage to articular cartilage is one of the symptoms in skeletal fluorosis and is often a main reason for joint pain and restriction of movement in joints. Cartilage tissue is composed of cartilage cells (chondrocytes) and cartilage matrix components. Cartilage matrix is synthesized and secreted by cartilage cells. 1-2 Chondrocytes are the only cellular components in cartilage. Their cytoplasm has a large complement of well-developed Golgi apparatus, rough endoplasmic reticulum (RER), and fewer mitochondria. The process of protein synthesis occurs in the endoplasmic reticulum (ER), and protein modification is present mainly in the Golgi apparatus. The structures of the ER and the Golgi apparatus in the cartilage cells, therefore, directly reflect the changes of the functional status of chondrocytes. The effect of F on RER of chondrocytes has been reported in literature. 3 Here the effect of F effect on the Golgi apparatus changes of the chondrocytes cultured from rat rib-cage cartilage is explored by laser scanning confocal microscopy (LSCM). RESEARCH MATERIAL: MATERIALS AND METHODS Main reagents: Dulbecco s Modified Eagle Medium (DMEM) was supplied by Gibco Company, USA. Fetal bovine serum (FBS) was supplied by Hangzhou Sijiqing Biological Company, China, and NaF was supplied by Shanghai Biochemistry Pharmacy. Type II collagenase was supplied by Sigma Corporation, USA, and FLc5-lactosylc was provided by Bodipy Invitrogn, USA. Superoxide a Corresponding author: Prof Yanni Yu, Department of Pathology of Guiyang Medical College , Guizhou, PR China. yuyanni5577@yahoo.com.cn.

2 14 14 dismutase (SOD) was purchased from the Xian Guoan Biological Company, China. Chondrocyte separation and culture: Three male and three female Wistar rats, two months old, were provided by the Laboratory Animal Center of Guiyang Medical College. Primary chondrocyte cultures were obtained from the parietal bones of calvaria of the rats. The cells were adjusted to 10 6 /ml and cultured in 25- ml tissue culture flasks plated with DMEM supplemented with 10% FBS. When the cells had grown to confluence, 0.25% trypsin was used for the secondary culture. The cell number was adjusted to a density of cells/ml. Experimental steps and sub-groups: After 7 days of cell culture, the original culture medium was discarded. The cells cultured in DMEM culture medium containing 15% FBS were divided into 7 groups as follows: (1) control group: addition of normal saline without fluoride (0 mg NaF/L); (2) low fluoride group: addition of 40 µmol NaF/L; (3) middle fluoride group: 80 µmol NaF/L; (4) high fluoride group: 160 µmol NaF/L; (5) low fluoride group plus SOD group: 40 µmol NaF/L and SOD (6I U/mL); (6) middle fluoride group: 80 µmol NaF/L and SOD (61 U/mL); (7) high fluoride group: 160 µmol NaF/L and SOD (61 U/mL). Handling of cultured cells: After 24 hr, the culture medium was discarded, the cultured cells were washed 3 times with PBS, fixed with 5% paraformaldehyde for 10 min, and the paraformaldehyde removed. The cells were then washed 3 times with PBS after checking the following experiments. MORPHOLOGY: Monitoring changes in the cellular ultrastructure: After washing the climbing film from cells with PBS, Bodipy flc5 (1:50) and Mito tracker (1:50) were added to the film at 37ºC, which was then kept in a dark incubator for 1 hr. Afterward it was washed 5 times with PBS, and the films were observed under laser scanning confocal microscopy (LSCM) in the West China Hospital of Sichuan University, Transplantation Immunology Laboratory. By excitation with 588-nm red laser light, green light at 522 nm was seen in the Golgi apparatus. Images were obtained by Mias 2001 image analysis and recorded at an average of 50 optical density scans. Statistical analysis: An independent sample T-test was performed to analyze differences in changes of Golgi apparatus between the F-treated group and the control group. Results are expressed as Mean±SEM. Differences with p<0.05 are considered statistically significant. RESULTS Morphological changes of cultured chondrocytes: The active chondrocytes that adhered to the walls of the flasks for 7 days had a spindly or cobblestone appearance as described in the literature. 4 Compared with the control group, there were no obvious pathological changes in the experimental F and F + SOD groups. The green fluorescence of the Golgi apparatus in the chondrocytes was observed after excitation with 588-nm laser light. As seen in the photos of Figures 1 and 2,

15 15 scattering in the cytoplasm was relatively more intense around the nucleus.

middle, and high F groups showed a marked decrease (see Figure 1). A B C D Figure 1.

after laser excitation at 588 nm, with scattering in the cytoplasm being relatively more intensive around the nucleus.

3 15 15 scattering in the cytoplasm was relatively more intense around the nucleus. Compared with the control, the average optical density (AOD) of fluorescence readings of the Golgi apparatus in the low, middle, and high F groups showed a marked decrease (see Figure 1). A B C D Figure 1. The release of green fluorescence at 522 nm in the control (A) from the Golgi apparatus in the chondrocytes was observed after laser excitation at 588 nm, with scattering in the cytoplasm being relatively more intensive around the nucleus. Compared with the control (A), the average optical density (AOD) of fluorescence intensities of the Golgi apparatus in the low, middle, and high F groups showed a marked decrease (B, C, and D).

16 16 Changes in the Golgi apparatus after SOD intervention: Compared with the control,

groups treated with SOD showed a marked increase (see Figure 2). A E F G Figure 2.

the chondrocytes was observed after laser excitation at 588 nm, with scattering in the

Compared with the control (A), the average optical density (AOD) of fluorescence

4 16 16 Changes in the Golgi apparatus after SOD intervention: Compared with the control, the AOD of fluorescence intensities of Golgi apparatus in the low, middle, and high F groups treated with SOD showed a marked increase (see Figure 2). A E F G Figure 2. The release of green fluorescence at 522 nm in the control (A) from the Golgi apparatus in the chondrocytes was observed after laser excitation at 588 nm, with scattering in the cytoplasm being relatively more intensive around the nucleus. Compared with the control (A), the average optical density (AOD) of fluorescence intensities of the Golgi apparatus in the low F plus SOD, the middle F plus SOD, and high F plus SOD groups showed a marked increase (E, F, and G).

5 17 17 Optometry indicators: The pictures of cells were observed using LSCM and were recorded by the Mias 2001 image analysis system. The AOD of the Golgi apparatus in the cultured chondrocytes with low, middle, and high F concentrations with and without SOD present are shown in the Table below. A significant decrease in the AOD in the presence of F occurred (p<0.01), but with SOD the effect of F was reversed as seen by the marked increased in the AOD (p<0.01). Table. Effect of fluoride and SOD on the Golgi apparatus complexes of cultured rat rib-cage chondrocytes Group Average optical density (AOD) (number of readings) 0 mg/l NaF (control) 39.33±17.09 (36) 40 µmol/l NaF 18.13±10.12 * (26) 80 µmol/l NaF 17.03±9.12 * (30) 160 µmol/l NaF 15.16±8.54 * (34) 40 µmol/l NaF + SOD (61 U/mL) 52.13±15.12 * (26) 80 µmol/l NaF + SOD (61 U/mL) 54.98±17.42 * (30) 160 µmol/l NaF + SOD (61 U/mL) ±33.27 * (34) *p<0.01 compared with the control. p<0.01 compared with the same concentration of NaF ± SOD. p<0.01 compared with 40 µmol/l NaF + SOD group. DISCUSION Owing to close contact of the endoplasmic reticulum (ER) and the Golgi apparatus with each other, protein synthesis in cartilage cells occurs mainly in the rough endoplasmic reticulum (RER). According to our previous experiment, 3 the ER in cultured cartilage cells showed injury mainly with a the high concentration of F (p<0.01).the synthesis of proteins is affected by damage to the ER. Thus injury to ER is imparted to cartilage cells and is bound to affect the synthesis of proteins like proteoglycan and collagen and ultimately the function of cartilage tissue. Since the ER may be the main target of F damage to the sub-cellular structure of the cartilage cells, the function of chondrocytes for protein synthesis may be impeded by damage to the ER in cartilage cells. The Golgi apparatus has a key modification and transition role in protein synthesis. However, changes in the Golgi apparatus in fluorosis have rarely been reported. Our present study showed that with increasing F concentration, the numbers and function of the Golgi apparatus were decreased. The AOD of the Golgi apparatus in cultured chondrocytes of rat was significantly reduced

6 18 18 especially in the high F groups under LSCM (p<0.01). The Golgi apparatus therefore clearly appeared to be injured by F in cultured chondrocytes. Thus the Golgi apparatus may be one of the main target organelles of cartilage cells affected by sub-cellular injury by F and thus interfere with protein synthesis in chondrocytes. A large number of clinical observations and animal experiments show that F poisons animals with increased levels of free radicals and lipid peroxidation. In the human body, the activity of free radical scavengers such as glutathione peroxidase (GSH-Px) and SOD decreases. Antioxidants like SOD promote urinary F excretion, maintain antioxidant enzyme activity, and repair a variety of liver and kidney tissue free radical metabolic disorders. 5-7 Here the fluorescence intensity of the Golgi apparatus showed a marked increase in the various F groups after SOD intervention, thus demonstrating that SOD has a protective effect on injury to the Golgi apparatus caused by F. Although not obvious under conditions of low concentrations of F, SOD antagonism was very obvious at high concentrations of F as seen in plate G of Figure 2 and in the Table. ACKNOWLEDGEMENTS This research was funded by the Guizhou Science Research Fund [No (2007) and No (2008)], the Guiyang Science Research Fund (No. T2009-3), and Ministry of Science and Technology Research Fund (No. 2010DFB30530). REFERENCES 1 Bayliss MT. Proteoglycan structure in normal and osteoarthritic human cartilage. In: Kuettner KE, Schleyerbach R. Hascall VC, editor. Articular cartilage biochemistry. New York: Raven Press; p Rosenberg DC, Buckwalter JA. Cartilage proteoglycans. In: Kuettner KE, Schleyerbach R. Hascall VC, editor. Articular cartilage biochemistry. New York: Raven Press; p Yu YN, Meng BJ. Influence of fluoride on the endoplasmic reticulum of cultured chondrocytes and antagonizing effect of superoxide dismutase. Chin J Endemiol 2006;25(5): Wang Y, Yang ZM, Xie HQ, et al. The biological characteristics of human fetal articular chondrocytes cultured in vitro. Chinese Journal of Experimental Surgery 2000; 17(4): Rzeuski R, Chlubek D, Machoy Z. Interaction between fluoride and biological free radical reactions. Fluoride 1998;31(1): Chlubek D. Fluoride and oxidative stress [editorial review]. Fluoride 2003;36(4): Inkielewicz I, Krechniak J. Fluoride effects on glutathione peroxidase and lipid peroxidation in rats. Fluoride 2004;37(1):7-12. Copyright 2010 The International Society for Fluoride Research Inc Editorial Office: 727 Brighton Road, Ocean View, Dunedin 9035, New Zealand.

IN VITRO HORMESIS EFFECTS OF SODIUM FLUORIDE ON KIDNEY CELLS OF THREE-DAY-OLD MALE RATS

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