Influence of Chronic Fluorosis on Membrane Lipids in Rat Brain

Neurotoxicology and Teratology, Vol. 20, No. 5, pp. 537–542, 1998 © 1998 Elsevier Science Inc. Printed in the USA. All rights reserved 0892-0362/98 $19.00 ⫹ .00

PII S0892-0362(97)00136-0

Influence of Chronic Fluorosis on Membrane Lipids in Rat Brain ZHI-ZHONG GUAN,*† YA-NAN WANG,†§ KAI-QI XIAO,* DA-YING DAI,* YUAN-HUA CHEN,* JIA-LIU LIU,* PAVEL SINDELAR† AND GUSTAV DALLNER† *Department of Pathology and §Department of Scientific Research Administration, Guiyang Medical College, Guiyang 550004, Guizhou, China †Clinical Research Center, Karolinska Institutet, S-14186 Huddige, Sweden Received 6 August 1997; Accepted 2 December 1997 GUAN, Z.-Z., Y.-N. WANG, K.-Q. XIAO, D.-Y. DAI, Y.-H. CHEN, J.-L, LIU, P. SINDELAR AND G. DALLNER. Influence of chronic fluorosis on membrane lipids in rat brain. NEUROTOXICAOL TERATOL 20(5) 537–542, 1998.—Brain membrane lipid in rats were analyzed after being fed either 30 or 100 ppm fluoride for 3, 5, and 7 months. The protein content of brain with fluorosis decreased, whereas the DNA content remained stable during the entire period of investigation. After 7 months of fluoride treatment, the total brain phospholipid content decreased by 10% and 20% in the 30 and 100 ppm fluoride groups, respectively. The main species of phospholipid influenced by fluorosis were phosphatidylethanolamine, phosphatidylcholine, and phosphatidylserine. The fatty acid and aldehyde compositions of individual phospholipid classes were unchanged. No modifications could be detected in the amounts of cholesterol and dolichol. After 3 months of fluoride treatment, ubiquinone contents in brain were lower; however, at 7 months they were obviously increased in both groups of fluoride treatment. The results demonstrate that the contents of phospholipid and ubiquinone are modified in brains affected by chronic fluorosis and these changes of membrane lipids could be involved in the pathogenesis of this disease. © 1998 Elsevier Science Inc. Brain

Fatty acid

Fluorosis

Neutral lipid

Phospholipid

Most brain phospholipids and neutral lipids are associated with various membranes that are the structural basis for the normal function of brain. The lipid composition determines the fluidity, stability, and permeability of membranes and thereby influences function of enzymes, ion channels, and receptors (4). Various lipids are also important precursors for second messengers in many signal transduction mechanisms (2). A high intake of fluoride results in various biological changes but very few investigations have been performed on brain lipid composition (13). However, in one recent report on experimental animal fluorosis, it has been proposed that total phospholipid and cholesterol accumulate in the brain (21). In this study, we have investigated phospholipids and neutral lipids in the rat brain during chronic fluoride intoxication. The results show that the contents of phospholipid and ubiquinone are modified in brains affected by chronic fluorosis, which could be involved in the pathogenesis of the brain damage.

ENDEMIC fluorosis is related to a high concentration of fluoride present in drinking water or produced during coal-burning (15,26), and causes damage to the human body characterized by a vast array of symptoms and pathological changes in addition to the well-known effects on skeleton and teeth (5,27). The blood–brain barrier fails to exclude this ion from the nervous tissue and, consequently, fluoride accumulates in the brain (7,10). In fact, one of the main manifestations of fluorosis is injury to the central nervous system (19). In affected populations neurological symptoms have been reported such as partial paralysis of arms and legs, headache, spasticity in the extremities, visual disturbances, and mental retardation (25). Focal demyelination, decrease in the number of Purkinje cells, thickening and disappearance of dendrites, swelling of Nissl substance, and pyknosis of individual neurons are the most prominent histopathological changes following administration of large doses of fluoride (5). However, the mechanism of fluoride toxicity on the central nervous system has not been clarified.

Requests for reprints should be addressed to Zhi-Zhong Guan, Department of Pathology, Guiyang Medical College, Guiyang 550004, Guizhou, P. R. China. Tel: ⫹86-851-6821432; Fax: ⫹86-851-6826734; E-mail: [email protected]

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GUAN ET AL. METHODS

Experimental Animals Seventy-two Wistar rats, weighing 100–120 g, were randomly divided into three groups with 24 animals in each. The animals in the control group were fed a standard diet containing less than 4 ppm fluoride and tap water containing less than 0.6 ppm. According to our previous experiment, the limited toxic dosage of fluoride to rats is the exposure to water containing 30 ppm fluoride. Therefore, the other two groups were fed the same diet but the drinking water contained 30 and 100 ppm fluoride (NaF), respectively. Animals were sacrificed at 3, 5, and 7 months and the dissected brains were kept at ⫺70⬚C until further analysis. General Examinations To determine whether an animal model with chronic fluorosis has been successfully produced, the following examinations were carried out. Fluoride concentration in the urine and brain tissue was measured (11) with CSB-F-I fluoride ion electrode (Changsa Analysis Instrumentation Co., China), and teeth were examined for dental fluorosis at each time point. The fresh brains were weighed. For light microscopy, brain tissue was embedded in paraffin after fixation in 10% neutral formalin for 24 h and then stained with hematoxylin-eosin (H & E). For transmission electron microscopic examination, fresh brain tissue was fixed in 2.5% glutaraldehyde, postfixed in 1% phosphate-buffered osmium tetroxide solution, dehydrated in ethanol, and embedded in Epon-618. Extrathin sections were doubled stained with uranyl acetate and lead citrate, and examined by 100 CX-2 electron microscope (Japan). Phospholipid Extraction and Separation The samples were homogenized (10%) in 0.25 M sucrose with an Ultra-Turrax blender (Janke & Kunkel, Germany) for 2 min (24,000 rpm). For lipid extraction (23), 6.6 ml chloroform and 6.6 ml methanol were added to 2 ml homogenate to obtain a final proportion of chloroformⲐmethanolⲐwater (CMW) 1:1:0.3 (vⲐv). Extraction was performed at 40⬚C for 60 min with magnetic stirring. The tubes were then centrifuged at 1200 ⫻ g for 15 min. The supernatant was transferred to a new tube. The pelleted residue was reextracted with 5 ml of chloroformⲐmethanol (CM) 2:1 (vⲐv), after which 1 ml of methanol was added and the mixture was centrifuged again at 1200 ⫻ g for 15 min. The second supernatant was combined with the first and the nonlipid residue was used for DNA quantification. The pooled supernatants were supplemented with 3.7 ml chloroform and 2.8 ml water to give a final CMW ratio of 3:2:1 (vⲐv). The mixture was centrifuged at 1000 ⫻ g for 15 min, and the lower organic phase was evaporated under nitrogen, redissolved in 2 ml chloroform, and placed onto a Silica-SepPak cartridge (Millipore, Waters Associates, Milford, MA) that had been equilibrated with chloroform. Neutral lipids were washed through with 25 ml chloroform and phospholipids were eluted with 30 ml methanol. The phospholipid fraction was evaporated under nitrogen, and the lipid residue was redissolved in CM and stored under nitrogen until further analysis. For separation of all major phospholipid classes, samples (4 ␮mol phospholipid) were dissolved in hexanⲐisopropanol (HP) 3:2 (vⲐv) and injected onto an HPLC system (Shimadzu LC10 AD, Japan) with a Zorbax SIL (25 cm ⫻ 9.2 mm ID, 5–6 mm particle size) column (Dupont, USA). The column was maintained at 34⬚C with a column heating block and the

separation was performed as previously described (8). Solvent A was HP (3:2, vⲐv) and solvent B was hexaneⲐisopropanolⲐ water (56.7:37.8:5.5, vⲐv) with 2.5 mM ammonium sulfate (pH 7.4) in water. A stepwise gradient was used from 50% to 100% B, and the total separation time was 50 min. Absorption of the eluate was determined at 205 nm and the flow rate was 6 mlⲐmin. The major individual phospholipid classes, including phosphatidylethanolamine (PE), phosphatidylcholine (PC), cardiolipid (CL), phosphatidylinositol (PI), phosphatidylserine (PS), and sphingomyelin (SH), were collected and quantified by measuring lipid phosphorus. Analysis of Fatty Acids and Aldehydes For analysis of fatty acids and aldehydes of phospholipids, an aliquot of lipid solution was dried and 1 ml 14% boron trifluoride in methanol was added (18). The tubes were flushed with nitrogen, closed with screw caps, and heated at 90⬚C for 30 min. After cooling the tubes to 0⬚C, the resultant fatty acyl methyl esters (FAME) and dimethyl acetals (DMA) were extracted by adding 4 ml pentane and 1 ml 5 N sodium hydroxide. Complete phase separation was obtained by centrifugation, and the methylated products in the upper phase were analyzed by gas chromatography on a Shimadzu GC-9A (Kyoto, Japan) with a flame ionization detector. The column was a fused silica capillary with cyanosiloxane 60 as the stationary phase (30 m ⫻ 0.25 mm id., df ⫽ 0.18 mm). The temperature was programmed from 130⬚C to 190⬚C and the total time per run was 45 min. Quantification was achieved by integrating the peaks on a Shimadzu Chromatopac C-R3A. Neutral Lipid Extraction and Separation One milliliter of 10% brain homogenate was mixed with 18 ml methanol and 12 ml petroleum ether (b.p. 40–60⬚ C). and ubiquinone-6 and dolichol-23 were added as internal standards. After intensive vortexing, the phases were separated by centrifugation and the upper organic phase was collected and dried under nitrogen.

TABLE 1 WEIGHT, AND PROTEIN AND DNA CONTENTS OF BRAIN FROM FLUORIDE-TREATED RATS

3 Months Control 30 ppm fluoride 100 ppm fluoride 5 Months Control 30 ppm fluoride 100 ppm fluoride 7 Months Control 30 ppm fluoride 100 ppm fluoride

Brain weight (g)

Protein (mg/g wet wt.)

DNA (mg/g wet wt.)

1.28 ⫾ 0.12 1.29 ⫾ 0.13 1.27 ⫾ 0.09

118.5 ⫾ 6.5 119.7 ⫾ 5.8 107.4 ⫾ 4.2*

1.18 ⫾ 0.05 1.19 ⫾ 0.04 1.16 ⫾ 0.04

1.32 ⫾ 0.10 1.28 ⫾ 0.13 1.27 ⫾ 0.08

122.5 ⫾ 6.7 109.6 ⫾ 10.5* 106.9 ⫾ 3.9†

1.16 ⫾ 0.06 1.15 ⫾ 0.14 1.16 ⫾ 0.10

1.30 ⫾ 0.08 1.28 ⫾ 0.11 1.26 ⫾ 0.12

118.3 ⫾ 4.8 103.4 ⫾ 5.7† 101.5 ⫾ 3.8†

1.11 ⫾ 0.05 1.11 ⫾ 0.06 1.13 ⫾ 0.07

The rat brains were analyzed after being fed either 30 or 100 ppm fluoride for 3, 5, and 7 months. The fresh brains were weighed. The brain tissues were homogeneted and used for protein and DNA measurements. The values are the means ⫾ SD for eight cases. Significant difference (*p ⬍ 0.05; †p ⬍ 0.01) compared with controls, using oneway ANOVA.

FLUORIDE AND BRAIN LIPIDS

539 TABLE 2

PHOSPHOLIPID CONTENTS IN BRAIN FROM THE RATS WITH CHRONIC FLUOROSIS

3 Months Control 30 ppm fluoride 100 ppm fluoride 5 Months Control 30 ppm fluoride 100 ppm fluoride 7 Months Control 30 ppm fluoride 100 ppm fluoride

Total PL

PE

CL

PI

PS

40.83 ⫾ 2.56 40.81 ⫾ 2.17 37.54 ⫾ 2.42*

16.6 ⫾ 1.9 16.7 ⫾ 2.0 15.4 ⫾ 1.8*

38.77 ⫾ 1.52 36.26 ⫾ 2.12* 36.02 ⫾ 1.84* 38.48 ⫾ 2.78 34.95 ⫾ 2.04* 31.02 ⫾ 2.22†

PC

SPH

0.10 ⫾ 0.02 0.87 ⫾ 0.01 0.76 ⫾ 0.03

1.09 ⫾ 0.01 1.16 ⫾ 0.03 0.99 ⫾ 0.01

4.15 ⫾ 0.06 4.37 ⫾ 0.07 3.76 ⫾ 0.06*

16.5 ⫾ 2.3 16.4 ⫾ 2.2 15.4 ⫾ 3.8

1.46 ⫾ 0.01 1.34 ⫾ 0.01 1.23 ⫾ 0.02

15.5 ⫾ 2.0 14.1 ⫾ 1.8* 14.4 ⫾ 1.9*

1.29 ⫾ 0.01 1.19 ⫾ 0.04 1.45 ⫾ 0.02

0.99 ⫾ 0.02 1.10 ⫾ 0.04 1.03 ⫾ 0.03

3.83 ⫾ 0.04 3.90 ⫾ 0.05 3.58 ⫾ 0.04*

15.6 ⫾ 1.6 14.3 ⫾ 1.4* 14.8 ⫾ 1.4*

1.59 ⫾ 0.03 1.66 ⫾ 0.02 1.44 ⫾ 0.03

16.0 ⫾ 2.1 14.0 ⫾ 2.1* 12.8 ⫾ 1.9†

0.82 ⫾ 0.01 0.86 ⫾ 0.02 0.58 ⫾ 0.02*

0.94 ⫾ 0.01 1.06 ⫾ 0.01 0.79 ⫾ 0.02

3.64 ⫾ 0.04 2.81 ⫾ 0.04* 2.32 ⫾ 0.03†

15.8 ⫾ 1.8 14.7 ⫾ 1.7* 13.0 ⫾ 1.8*

1.29 ⫾ 0.02 1.49 ⫾ 0.02 1.43 ⫾ 0.01

Values are ␮mol/mg DNA. Lipids were extracted from brain homogenate and phospholipids were separated by HPLC on a silica column. Quantification was performed by determining lipid phosphorous. The values are the means ⫾ SD for eight cases. Significant difference (*p ⬍ 0.05; †p ⬍ 0.01) compared with controls, using one-way ANOVA.

For determination of cholesterol, total dolichol, ubiquinone-9 and -10, the neutral lipid fraction was dissolved in 100 ␮l of CM (2:1, vⲐv) and injected onto on an HPLC system equipped with a reverse-phase column (Hewlett–Packard Hypersil ODS 3 ␮m). A convex gradient with methanolⲐwater (9:1, vⲐv) as solvent A and methanolⲐisopropanolⲐhexane (2:1:1, vⲐv) as solvent B was used with a program time of 46 min. The flow rate was 1.5 mlⲐmin and the absorption of the eluate at 210 nm was monitored (6). Other Methods For determination of lipid phosphorus, the solutions of total phospholipid or individual classes were evaporated under nitrogen, dissolved in 0.3 ml of 70% perchloric acid, and heated at 180⬚C for 60 min. Each sample was then supplemented with 400 ␮l of 1.25% ammonium molybdate, 400 ␮l of 5% ascorbic acid, and water to a final volume of 1.8 ml. This

mixture was heated in boiling water for 5 min and the absorption was measured at 797 nm (24). DNA quantification was performed by the method of Burton (3). Protein content in whole homogenate was determined according to Lowry (16) with bovine serum albumin as standard. Chemicals Solvents for HPLC were obtained from Merck (Merck Ltd., Darmstadt, Germany). Various phospholipids, fatty acid methyl esters, ubiquinone-6, dolichol-23, and other chemicals were obtained from Sigma (St. Louis, MO). RESULTS

General Manifestations Fluoride concentration in urine and brain tissue increased in all fluoride-treated animals in comparison with the control,

TABLE 3 FAME AND DMA COMPOSITIONS OF PE IN BRAIN FROM FLUORIDE-TREATED RATS % of Total FAME

3 Months Control 30 ppm fluoride 100 ppm fluoride 5 Months Control 30 ppm fluoride 100 ppm fluoride 7 Months Control 30 ppm fluoride 100 ppm fluoride

% of Total DMA

16:0

18:0

18:1

20:1

20:4

22:4

22:6

16:0

18:0

18:1

6.63 6.28 5.64

20.16 19.98 19.52

21.00 21.29 21.71

2.89 3.23 3.79

15.26 15.28 15.47

7.59 7.73 8.19

26.19 26.23 25.71

24.81 22.49 23.04

40.98 42.01 42.43

34.21 35.51 34.54

6.59 7.08 6.52

19.47 19.52 19.75

23.37 23.52 23.61

3.87 3.52 3.82

14.62 14.85 14.47

7.39 7.14 7.43

24.69 24.16 24.41

25.16 25.60 23.19

38.70 38.79 39.53

36.14 35.61 37.28

6.32 6.35 5.88

20.52 20.82 19.95

20.39 17.85 19.89

3.10 2.55 3.00

15.90 16.92 15.89

7.81 7.60 8.22

25.98 27.92 27.19

22.89 22.62 23.39

40.97 45.21 42.02

36.16 32.18 34.60

PE isolated from rat brain was treated with boron trifluorde in methanol in order to prepare fatty acyl methyl esters and dimethyl acetals. Separation and quantification of the individual components were achieved by gas chromatography. The values are the means from eight cases. SDs were 3–8% of these means. No significant difference compared with controls, using one-way ANOVA.

540

GUAN ET AL. TABLE 4 CHOLESTEROL AND DOLICHOL CONTENTS IN BRAIN FROM FLUORIDE-TREATED RATS

3 Months Control 30 ppm fluoride 100 ppm fluoride 5 Months Control 30 ppm fluoride 100 ppm fluoride 7 Months Control 30 ppm fluoride 100 ppm fluoride

Cholesterol (mg/mg DNA)

Dolichol (␮g/mg DNA)

8.60 ⫾ 0.75 9.15 ⫾ 0.64 9.22 ⫾ 0.94

30.23 ⫾ 1.71 32.51 ⫾ 1.27 28.46 ⫾ 1.98

8.53 ⫾ 0.72 8.51 ⫾ 1.10 8.85 ⫾ 0.59

33.67 ⫾ 1.68 33.92 ⫾ 4.23 35.07 ⫾ 3.85

7.99 ⫾ 1.22 8.17 ⫾ 1.55 8.45 ⫾ 2.16

36.03 ⫾ 1.75 35.82 ⫾ 2.88 38.17 ⫾ 2.99

Cholesterol and dolichol extracted from brain homogenate were separated by reversed-phase HPLC. The values are the means ⫾ SD for eight cases. No significant difference compared with controls, using one-way ANOVA.

especially in the group treated with the high amount of fluoride at 7 months (data not shown). Dental fluorosis was mainly observed in the high fluoride group, but some cases were also identified in rat treated with the lower fluoride-containing diet. Light microscopic examination of brains showed neurons and glia cells with regular shape and homogeneous staining in all groups. No histopathological changes were found in any rat brain with fluorosis. Upon electron microscopy, mitochondrial swelling and endoplasmic reticulum dilation of neurons were present in some areas of the brain at 5 and 7 months of fluoride treatment, especially in the 100 ppm fluoride group. These results proved that the animal model with chronic fluorosis has been successfully produced, in which the changes are in accordance with previous reports (7,10,22).

There were no significant modifications of the average wet weights of rat brains among these different groups (Table 1). The brain protein content decreased in both groups of fluoride-treated rats compared with controls (Table 1). At 7 months of treatment the decrease was about 15% on weight basis in both groups. In contrast, the concentration of total DNA in brain remained stable at about 1.11 to 1.19 mgⲐg wet weight of tissue during the entire period of investigation. Thus, only DNA content could be used as the basis for expression of lipid content. Phospholipids The total phospholipid content in rat brain from controls ranged from 40.83 to 38.48 ␮molⲐmg DNA (Table 2). However, there was a significant decrease in brain phospholipid by 10% and 20% in the 30 and 100 ppm fluoride groups, respectively, after 7 months of fluoride administration. This decrease of phospholipid could already be detected in the 30 ppm fluoride group at 5 months and in the 100 ppm fluoride group at 3 months of fluoride treatment. The phospholipid fraction of normal rat brain contained about 40% PE and 40% PC. The remaining 20% consisted of PS, SPH, PI, and CL in decreasing order of amount. Similar to the total phospholipid, the major individual species of phospholipid, such as PE, PC, and PS, were decreased upon fluoride treatment when their amounts were calculated on a DNA basis (Table 2). Fatty Acid and Aldehyde Composition of Individual Phospholipid Class The fatty acid and aldehyde compositions of total PE for all time points investigated are presented in Table 3. No statistically significant changes were observed in any saturated or unsaturated fatty acids in fluoride-treated animals. The aldehydes of PE showed no difference between normal and fluoride-intoxicated animals. The fatty acid compositions of PC, PS, SPH, PI, and CL were also investigated (data not shown). No modifications were found.

FIG. 1. Ubiquinone contents in brain from fluoride-treated rat. Ubiquinone extracted from brain homogenate was separated by reverse-phase HPLC. The values given are the sums of the oxidized and reduced forms. (A) Ubiquinone-9; (B) ubiquinone-10. Black bars represent controls; white bars, 30 ppm fluoride group; striped bars, 100 ppm fluoride group. The data are the means ⫾ SD for eight cases. Significant difference (p ⬍ 0.05) compared with controls, using one-way ANOVA.

FLUORIDE AND BRAIN LIPIDS

541

Neutral Lipids The mevalonate pathway lipids, including cholesterol, dolichol, and ubiquinone, were measured by HPLC at all time points. The cholesterol concentration in control animals remained unchanged during the entire 7 months of investigation (Table 4). No changes were found in brain cholesterol content in rats with fluorosis. In agreement with previous findings, dolichol content in the brain increases during the whole life period. On the other hand, no statistical difference between fluoride-treated and control rats could be established in the various groups (Table 4). In contrast to cholesterol and dolichol, the brain ubiquinone content was clearly modified by fluoride intoxication (Fig. 1). After 3 months of fluoride treatment, ubiquinone-9 content in brain was 11.5% and 13.5% lower in the 30 and 100 ppm fluoride groups, respectively, compared with control animals. In the 30 ppm fluoride group, ubiquinone-9 then increased to 3.6% above controls at 5 months and to 21.3% at 7 months. In the 100 ppm fluoride group, ubiquinone-9 was still 10.1% lower than controls at 5 months but then increased to 19.6% above controls at 7 months. Similar changes were observed for ubiquinone-10, although not as pronounced as for ubiquinone-9. The ratio of reduced ubiquinone to total ubiquinone was about 0.15 in control animals during the entire investigation. No changes were observed in this ratio in rats with fluorosis (data not shown). DISCUSSION

Fluoride is an essential trace element but the beneficial range is so narrow that health may be influenced adversely if excessive fluoride is supplied. In this investigation rats were treated 7 months with 30 and 100 ppm fluoride in drinking water to study the effects of chronic fluorosis on brain membrane lipids. The increased amounts of fluoride detected in the urine and brain from these animals indicate that this ion accumulates in the body, including the brain. The specific dental lesions confirmed that the animals suffered from chronic fluoride intoxication. Some degenerative changes in subcellular organelles of neurons were detected by electron microscopy. These consisted mainly of mitochondrial swelling and endoplasmic reticulum dilation and were especially obvious in rats treated with 100 ppm fluoride for 7 months. All of these changes showed that the animal model with chronic fluorosis was successfully produced by feeding higher concentration of fluoride in water. On weight and DNA bases both protein and total phospholipid amounts were decreased upon fluoride treatment. These two main membrane components were modified in a parallel fashion and the changes were dependent on both the concentration and the duration of the fluoride feeding. Some reports pointed out that protein synthesis is affected in various ways by fluoride ions (22). The main parts of membrane phospholipid influenced by fluorosis were PE, PC, and PS and, significantly, all of them decreased gradually during the treatment period with fluoride. Taking these facts together, it is quite clear that the changes are elicited by a decrease in membrane content. It is possible that fluoride intoxication af-

fects specific organelle(s), causing cytological modifications of certain cell types. The patterns of the fatty acid and aldehyde compositions of the individual phospholipid classes remained unchanged during the whole period investigated. These results indicate that there is a specific effect of toxicity with the results of a total membrane area decrease without any significant structural modification of the individual phospholipid components. Three end products of the mevalonate pathway were analyzed, namely cholesterol, dolichol and ubiquinone. Some reports have suggested modifications of cholesterol in erythrocytes and liver after chronic exposure to fluoride (14,17). In our experiments, the cholesterol level in rat brain was quite stable, and no significant changes were found in the different groups. Because the synthesis and catabolism pathways of cholesterol are very complex, further investigations are needed to get more detail explanation for the cholesterol level. Total dolichol concentration of brain increased slightly during the lifetime of the rat, but no modifications could be seen in rat brain with fluorosis compared with normal animals. Thus, it appears that cholesterol and dolichol contents in rat brain are not influenced to any great extent when the rats are treated with fluoride in various amounts and time periods. The major form of ubiquinone in rat brain contains 9 isoprene residues in its side chain, but relatively high levels of the lipid; about one third have 10 isoprene units. The total ubiquinone concentration in rat brain was found to be decreased after 3 months of fluoride treatment. Thereafter the content of the lipid was substantially elevated, particularly after 7 months of fluoride treatment. It has been reported that fluoride intoxication decreases the activities of superoxide dismutase and glutathione peroxidase, which could result in heavy increase of free radicals (9,20). The imbalance between production and elimination of free radicals can induce a wide range of damages, including membrane lipid peroxidation (12). Ubiquinone has a broad distribution, being present in all tissues and membranes. The high concentration of this compound in the inner mitochondrial membranes is a prerequisite for an efficient respiratory chain activity and oxidative phosphorylation. In recent years it has established that ubiquinone is present in all cellular membranes and its main function at these locations is to serve as an endogenous, lipid soluble antioxidant (1). There are a number of studies demonstrating the role of this lipid in the brain as one of the main antioxidant preventing the effects of free radicals and reactive oxygen metabolites. It is therefore plausible that the initial decrease in ubiquinone in fluoride intoxication is caused by peroxidative stress and that the later increase of this lipid is a compensatory reaction of the cells to protect themselves against free radicals. Furthermore, the changes in ubiquinone suggest that free radicals could be an important part in the pathological mechanisms of fluorosis. ACKNOWLEDGEMENTS

This work was supported by the National Natural Science Foundation of China and the Swedish Medical Research Council.

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