impact of fluoride and superoxide dismutase on …the golgi apparatus has a key modification and...

Research reportFluoride 43(1)13–18January-March 2010

Impact of F and SOD on the Golgi apparatus in rib-cage chondrocytesYu, Meng 1313

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

Yanni Yu,a Binjie Menga

Guizhou, China

SUMMARY: Chondrocytes of rib-cage cartilage from Wistar rats were cultured in vitrofor 7 days to study the Golgi complex changes in the presence of variousconcentrations 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 progressivelydecreased at 40, 80, and 160 µmol NaF/L. On the other hand, the AOD of the Golgiapparatus was significantly increased by addition of SOD to the F groups. The resultsindicated: (1) the Golgi apparatus was adversely affected by F; and (2) SOD was ableto protect the Golgi apparatus of chondrocytes from damage caused by F, especiallyat 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-termfluoride (F) intake. Among its notable adverse effects are dental and skeletalfluorosis. Damage to articular cartilage is one of the symptoms in skeletal fluorosisand is often a main reason for joint pain and restriction of movement in joints.

Cartilage tissue is composed of cartilage cells (chondrocytes) and cartilagematrix components. Cartilage matrix is synthesized and secreted by cartilagecells.1-2 Chondrocytes are the only cellular components in cartilage. Theircytoplasm has a large complement of well-developed Golgi apparatus, roughendoplasmic reticulum (RER), and fewer mitochondria. The process of proteinsynthesis occurs in the endoplasmic reticulum (ER), and protein modification ispresent mainly in the Golgi apparatus. The structures of the ER and the Golgiapparatus in the cartilage cells, therefore, directly reflect the changes of thefunctional status of chondrocytes.

The effect of F on RER of chondrocytes has been reported in literature.3 Herethe effect of F effect on the Golgi apparatus changes of the chondrocytes culturedfrom rat rib-cage cartilage is explored by laser scanning confocal microscopy(LSCM).

MATERIALS AND METHODS

RESEARCH MATERIAL: Main reagents: Dulbecco’s Modified Eagle Medium (DMEM) was supplied by

Gibco Company, USA. Fetal bovine serum (FBS) was supplied by HangzhouSijiqing Biological Company, China, and NaF was supplied by ShanghaiBiochemistry Pharmacy. Type II collagenase was supplied by Sigma Corporation,USA, and FLc5-lactosylc was provided by Bodipy Invitrogn, USA. Superoxide

aCorresponding author: Prof Yanni Yu, Department of Pathology of Guiyang Medical College550001, Guizhou, PR China. E-mail: [email protected].



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 GuiyangMedical College. Primary chondrocyte cultures were obtained from the parietalbones of calvaria of the rats. The cells were adjusted to 106/mL and cultured in 25-mL tissue culture flasks plated with DMEM supplemented with 10% FBS. Whenthe cells had grown to confluence, 0.25% trypsin was used for the secondaryculture. The cell number was adjusted to a density of 2×105 cells/mL.

Experimental steps and sub-groups: After 7 days of cell culture, the originalculture medium was discarded. The cells cultured in DMEM culture mediumcontaining 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) highfluoride group: 160 µmol NaF/L; (5) low fluoride group plus SOD group: 40 µmolNaF/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, thecultured cells were washed 3 times with PBS, fixed with 5% paraformaldehyde for10 min, and the paraformaldehyde removed. The cells were then washed 3 timeswith 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 addedto the film at 37ºC, which was then kept in a dark incubator for 1 hr. Afterward itwas washed 5 times with PBS, and the films were observed under laser scanningconfocal microscopy (LSCM) in the West China Hospital of Sichuan University,Transplantation Immunology Laboratory. By excitation with 588-nm red laserlight, green light at 522 nm was seen in the Golgi apparatus. Images were obtainedby Mias 2001 image analysis and recorded at an average of 50 optical densityscans.

Statistical analysis: An independent sample T-test was performed to analyzedifferences in changes of Golgi apparatus between the F-treated group and thecontrol group. Results are expressed as Mean±SEM. Differences with p<0.05 areconsidered statistically significant.

RESULTS

Morphological changes of cultured chondrocytes: The active chondrocytes thatadhered to the walls of the flasks for 7 days had a spindly or cobblestoneappearance as described in the literature.4 Compared with the control group, therewere no obvious pathological changes in the experimental F and F + SOD groups.

The green fluorescence of the Golgi apparatus in the chondrocytes was observedafter excitation with 588-nm laser light. As seen in the photos of Figures 1 and 2,



scattering in the cytoplasm was relatively more intense around the nucleus.Compared with the control, the average optical density (AOD) of fluorescencereadings of the Golgi apparatus in the low, middle, and high F groups showed amarked 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).



Changes in the Golgi apparatus after SOD intervention: Compared with thecontrol, 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).



Optometry indicators: The pictures of cells were observed using LSCM andwere recorded by the Mias 2001 image analysis system. The AOD of the Golgiapparatus in the cultured chondrocytes with low, middle, and high Fconcentrations with and without SOD present are shown in the Table below. Asignificant decrease in the AOD in the presence of F occurred (p<0.01), but withSOD the effect of F was reversed as seen by the marked increased in the AOD(p<0.01).

DISCUSION

Owing to close contact of the endoplasmic reticulum (ER) and the Golgiapparatus with each other, protein synthesis in cartilage cells occurs mainly in therough endoplasmic reticulum (RER). According to our previous experiment,3 theER in cultured cartilage cells showed injury mainly with a the high concentrationof F (p<0.01).The synthesis of proteins is affected by damage to the ER. Thusinjury to ER is imparted to cartilage cells and is bound to affect the synthesis ofproteins like proteoglycan and collagen and ultimately the function of cartilagetissue. Since the ER may be the main target of F damage to the sub-cellularstructure of the cartilage cells, the function of chondrocytes for protein synthesismay be impeded by damage to the ER in cartilage cells.

The Golgi apparatus has a key modification and transition role in proteinsynthesis. However, changes in the Golgi apparatus in fluorosis have rarely beenreported. Our present study showed that with increasing F concentration, thenumbers and function of the Golgi apparatus were decreased. The AOD of theGolgi apparatus in cultured chondrocytes of rat was significantly reduced

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) 131.91±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.



especially in the high F groups under LSCM (p<0.01). The Golgi apparatustherefore clearly appeared to be injured by F in cultured chondrocytes. Thus theGolgi apparatus may be one of the main target organelles of cartilage cells affectedby sub-cellular injury by F and thus interfere with protein synthesis inchondrocytes.

A large number of clinical observations and animal experiments show that Fpoisons animals with increased levels of free radicals and lipid peroxidation. In thehuman body, the activity of free radical scavengers such as glutathione peroxidase(GSH-Px) and SOD decreases. Antioxidants like SOD promote urinary Fexcretion, maintain antioxidant enzyme activity, and repair a variety of liver andkidney tissue free radical metabolic disorders.5-7 Here the fluorescence intensity ofthe Golgi apparatus showed a marked increase in the various F groups after SODintervention, thus demonstrating that SOD has a protective effect on injury to theGolgi apparatus caused by F. Although not obvious under conditions of lowconcentrations of F, SOD antagonism was very obvious at high concentrations of Fas seen in plate G of Figure 2 and in the Table.

ACKNOWLEDGEMENTS

This research was funded by the Guizhou Science Research Fund [No. 400119(2007) and No. 3054 (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. NewYork: Raven Press; 1986. p.295.

2 Rosenberg DC, Buckwalter JA. Cartilage proteoglycans. In: Kuettner KE, Schleyerbach R.Hascall VC, editor. Articular cartilage biochemistry. New York: Raven Press; 1986. p.39.

3 Yu YN, Meng BJ. Influence of fluoride on the endoplasmic reticulum of culturedchondrocytes and antagonizing effect of superoxide dismutase. Chin J Endemiol2006;25(5):507-10.

4 Wang Y, Yang ZM, Xie HQ, et al. The biological characteristics of human fetal articularchondrocytes cultured in vitro. Chinese Journal of Experimental Surgery 2000; 17(4): 319-321.

5 Rzeuski R, Chlubek D, Machoy Z. Interaction between fluoride and biological free radicalreactions. Fluoride 1998;31(1):43-5.

6 Chlubek D. Fluoride and oxidative stress [editorial review]. Fluoride 2003;36(4):217-28.7 Inkielewicz I, Krechniak J. Fluoride effects on glutathione peroxidase and lipid peroxidation

in rats. Fluoride 2004;37(1):7-12.

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