116 Research report 116 Fluoride 40(2)116–127 Stawiarska ... · Fluoride 40(2)116–127 April-June 2007 Influence of fluoride on rabbit organ morphology in atheromatosis Stawiarska-Pięta,

Research reportFluoride 40(2)116–127April-June 2007

Influence of fluoride on rabbit organ morphology in atheromatosisStawiarska-Pięta, Birkner, Stojko, Grucka-Mamczar,

116116

Szaflarska-Stojko, Maj, Birkner, Wyszyńska

INFLUENCE OF FLUORIDE ON RABBIT ORGAN MORPHOLOGY IN ATHEROMATOSIS

Barbara Stawiarska-Pięta,a Ewa Birkner,b Rafał Stojko,a Ewa Grucka-Mamczar,b

Ewa Szaflarska-Stojko,a Anna Maj,a Beata Birkner,b Magdalena Wyszyńskaa

Sosnowiec and Zabrze, Poland

SUMMARY: A three-month study was conducted on the influence of fluoride on thedevelopment of pathomorphological changes in the inner organs of New Zealandmale rabbits in experimental atherosclerosis. Five groups of six rabbits were used: acontrol group and four study groups. Animals in study groups I and II were fed acholesterol-supplemented diet providing 0.5 g cholesterol/rabbit/day. Additionally,animals in study group II were supplemented with 3 mg F/kg bw/day in their drinkingwater. Animals in study group III were fed a higher cholesterol-supplemented dietproviding 2.0 g of cholesterol/rabbit/day. Animals in study group IV were fed thesame high-cholesterol diet and were also given 3 mg F/kg bw/day) in their drinkingwater. Plasma lipids were monitored monthly, and after three months the experimentwas terminated. At autopsy, blood was collected for biochemical analyses, and theheart, kidney, liver, and aorta were examined and collected for histopathologicaltests. Pathomorphological changes in these organs were assessed with slides madeby the normal paraffin method stained with hematoxilin and eosin, and also by thefreezing microtome method stained with Sudan III for detecting neutral fats. At 3 mg/kg bw/day, fluoride did not have a significant preventive influence from the high-cholesterol diet on the atherogenesis processes in the aorta and heart arterialvessels or on the regressive changes (steatosis) occurring in the liver and kidneys.However, this daily dosage of fluoride was found to inhibit the atherogenesisprocesses caused by 0.5 g of cholesterol/rabbit/day to occur in the aorta and arterialvessels in the heart as well as the regressive changes (fatty degeneration) in the liverand kidneys.Keywords: Aorta; Atheromatosis; Cholesterol in rabbits; Fluoride and atheromatosis; Heart; Histopathology; Kidney; Liver; Rabbits and cholesterol.

INTRODUCTION

Atherosclerosis is a very common ailment of the cardiovascular system,especially in highly developed countries. It is characterized by regressive andprogressive changes in the intima and tunica of arterial vessels.1 Cholesterol is awell-documented pathogenic factor in atherosclerosis. Recent studies indicate thatoxidative processes play an important role in the pathogenesis of atherosclerosis.Oxidative free radicals oxidize low density lipoproteins (LDL) that then damageblood vessel endothelium. These free radicals also affect lamella adhesion, smoothmuscle cell proliferation, immunological responses, and the influx ofmacrophages into inner tunica. And evidently this is how favorable conditions foratherosclerosis are created.2-5 Changes usually occur in the aorta and coronaryarteries and later in brain and kidney arteries.6,7

Enzymes are the first line of defense against oxidative free radicals: superoxidedismutase, glutathione peroxidase, and catalase. Moreover, vitamins A, E, and C,

aFor Correspondence: Dr Barbara Stawiarska-Pięta, Department of Pathology, Faculty ofPharmacy in Sosnowiec, Silesian Medical School in Katowice, Poland; E-mail: [email protected] of General Biochemistry, Faculty of Medicine in Zabrze, Silesian Medical School inKatowice, Poland.



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as well as methionine, cysteine, and co-enzyme Q, help protect the organism fromfree radical oxidation.8-10 Recent studies show that fluoride (F) ions play a role inthe free-radical processes11,12 as well as in the metabolism of lipids.13-16 F maydecrease lipid levels,14-17 increase them,13,18,19 or even have no effect onthem.20,21

F ions inhibit lipid peroxidation as well as the development of oxidatively-modified LDL (oxy-LDL) and therefore prevent its entrapment by scavengerreceptors found in macrophages and monocytes. In this way, F decreases theaccumulation of cholesterol and its esters in the above-mentioned cells.22 The roleF therefore plays in lipid metabolism and free radical processes suggests that itmay be an important link in the atherosclerosis process.

The aim of this study was to assess the influence of alimentary administration ofF on the morphology of the aorta, liver, kidney, and heart organs of rabbits inexperimental atherosclerosis produced by a cholesterol (CH) supplemented diet.

MATERIALS AND METHODS

The study was performed on 30 male New Zealand rabbits with an initial bodyweight of 3000g±50g. The animals were provided by the Central ExperimentalAnimal Quarters of the Silesian Medical School in Katowice, Poland. They weredivided into five equal groups of six as follows. Animals in the control group (C)were fed standard rabbit ration. Animals in the study Group I (0.5 g CH) were onan atherosclerosis diet and received 0.5 g of cholesterol/rabbit/24 hr, whileanimals in study Group II (0.5 g CH+F) were fed the same cholesterol-supplemented diet but were also given 3 mg F/kg bw/24 hr in their drinking waterby receiving 9 mg F (=19.9 mg NaF) in 100 mL distilled water every morning andafterward being given distilled water without F ad libitum until the next morning.Animals in study groups III (2.0 g CH) and IV (2.0 g CH+F) were fed acholesterol-supplemented diet providing 2.0 g cholesterol/rabbit/24 hr.Additionally, as in study Group II, animals in study Group IV were alsosupplemented with 3 mg F/kg bw/24 hr in their drinking water. The experimentlasted 3 months with the animals being kept in wooden cages. Once a month,blood was collected for biochemical analyses from the marginal vein of the ear.The concentration of LDL cholesterol in plasma was determined by an enzymaticmethod using a BioMerieux kit (France) while the level of HDL cholesterol andtriglycerides was assessed with an Alpha Diagnostics kit (Germany).

Results were analyzed statistically with Statistica PL software. The U MannWhitney test was used to compare differences between particular groups.Statistical significance was restricted at p≤0.05.

Methodology of the histopathological studyDuring postmortem examination the following were collected for

histopathological tests: aorta, liver, heart, and kidneys. The organs were preservedin aqueous formaldehyde for pathomorphological assessment, and changes wereassessed on the basis of paraffin preparations stained with hematoxilin and eosin(H-E). The presence of fats in the atheromatous plaques of the aortas and vessels



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as well as in the organ tissues was confirmed by examination of preparationsobtained on a freezing microtome and stained with Sudan III for neutral fats.23

Microphotographs were taken with the Docuval microscope equipped with a CarlZeiss Jena photo device.

This study was approved by the Bioethical Committee for Animal Testing of theSilesian Medical University.

RESULTS

Figures 1–3 show changes in the lipid concentrations in plasma of the rabbitsafter three months in the four groups on a cholesterol diet: Group I (0.5 g CH);Group II (0.5 g CH+F); Group III (2.0 g CH) and Group IV (2.0 g CH+F). Theconcentration of triglycerides increased significantly in all the study groupscompared with the control group; p<0.01 (Figure 1).

Moreover, there was also a significant increase in the concentration of LDLcholesterol in these groups compared with the control group; p=0.004 (Figure 2).

Administration of F along with 0.5 g cholesterol/kg bw/day (Group II) caused adownward tendency in the LDL concentration when compared to Group I without

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cent

ratio

n TG

[mm

ol /

L]

Control 0.5 g CH 0.5 g CH + F 2.0 g CH 2.0 g CH + F

*p < 0.01 *p < 0.01

*p < 0.01*p < 0.01

Figure 1. Triglycerides (TG) concentrations in plasma of animals on a cholesterol diet after 3 months of experiment (mean values and SEM, *p<0.01 when compared to control group).

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n LD

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/ L]


*p = 0.004

*p = 0.004

*p = 0.004

*p = 0.004

Figure 2. LDL cholesterol concentration in plasma of animals on a cholesterol diet after 3 months of experiment (mean values and SEM, *p=0.004 when compared to control group).



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F. No statistically significant change in the HDL cholesterol concentration wasobserved in these two groups. In Group IV with the higher level of cholesterol andF (2.0 g CH+F) there was a statistically significant increase in HDL after the thirdmonth when compared to group III (2.0 g CH) without F and to the control group(Figure 3).

Histopathological findingsMacroscopic assessment:The presence of creamy atheromatous plaque was noticed in the arch area and

abdominal part of aortas in studied groups. In groups I, III, and IV the foci ofchanges were large and covered the entire circumference, while in Group II (0.5 gCH+F) they were much less intense. There was a color change—xanthochromia—in the liver of all the study groups. However, there was no macroscopic change inthe kidney or heart.

Microscopic assessment:Aorta. In contrast to the condition of the aorta in the control group C (Figure

4A), the study groups I (0.5 g CH) and III (2.0 g CH) exhibit a focal hyperplasia oftunica interna in the form of a large atheromatous plaque. There were also changesin the tunica media of animals in this group in the form of a focal proliferation ofmacrophages in the proximity of membrana elastica interna. There were numerousfoam-like cells in the atheromatous plaque, and fat was also noticed inintercellular spaces. There were also changes in the tunica media of animals fromthis group in the form of a focal thickening with no signs of fibrosis as well as afocal proliferation of macrophages in the proximity of membrana elastica interna.There were also calcification foci in interna membrana (in 2 animals) and focallosses of elastica lamella. The atherogenic changes in aortas of Group II (0.5 gCH+F) (Figure 4C) were much less intense than in Group I (0.5g CH) (Figure 4B).On the other hand, the morphology of aortas of rabbits in Group IV (2.0 g CH+F)(Figure 4E) was similar to that of animals in Group III (2.0 g CH) (Figure 4D).

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n H

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]


* p < 0.05** p < 0.01

Figure 3. HDL cholesterol concentration in plasma of animals on a cholesterol diet after 3 months of experiment (mean values and SEM, *p<0.05 when compared to 2.0 CH, **p<0.01 when compared to control group).



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Liver. Unlike the liver of the control group C (Figure 5A), there was a distinctsteatosis of hepatocytes in the liver of all study groups. It was intensified aroundthe central veins of the liver lobules (Figures 5B, 5C, 5D, and 5E). In Group II(0.5g CH+F), the steatosis of hepatocytes was of lesser intensity (Figure 5C).Steatosis of hepatocytes in Group IV (2.0 g CH+F) was similar to that in Group III(2.0 g CH) (Figures 5E and 5D, respectively).

Figure 4. Aorta.

Figure 4B. Group I (0.5g CH). Aorta. Atheromatous plaque. Foam cells in the intima. H-E. 130×.

Figure 4C. Group II (0.5g CH+F). Aorta. Atheromatous plaque. Foam cells in the intima. H-E. 150×

Figure 4D. Group III (2.0g CH). Aorta. Atheromatous plaque. Foam cells in the intima. H-E. 140×.

Figure 4E. Group IV (2.0g CH+F). Aorta. Atheromatous plaque. Foam cells in the intima. H-E. 180×

Figure 4A. Control group (C). Aorta. Normal pattern. H-E. 180×.



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Kidney. In contrast to the kidneys of the control group C (Figure 6A), there wasa focal steatosis in the tubule epithelium cells noticed only in kidneys of the 0.5 gCH and 2.0 g CH groups I and III (Figures 6B and 6D, respectively). Alsoobserved in the kidneys of Group III were areas of local parenchyma fibrosis.There were also foci of renal tubule cell steatosis in Group IV (2.0 g CH+F)(Figure 6E), but no regressive changes were seen in kidneys of the 0.5 g CH+FGroup II (Figure 6C).

Figure 5B. Group I (0.5g CH). Liver. Steatosis of hepatocytes around the central vein. H-E. 280×.

Figure 5C. Group II (0.5g CH+F). Liver. Steatosis of hepatocytes around the central vein. H-E. 140×.

Figure 5D. Group III (2.0 g CH). Liver. Steatosis of hepatocytes around the central vein. H-E. 140×.

Figure 5E. Group IV (2.0g CH + F). Liver. Steatosis of hepatocytes around the central vein. H-E. 140×.

Figure 5. Liver.

Figure 5A. Control group (C).Liver. Normal pattern. H-E. 140×



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Heart. Differing from the appearance of the arterial vessels of the control groupC (Figure 7A), there was a focal proliferation and steatosis of membrana interna(atheromatous plaque) in the arterial vessels of the heart in all groups (Figures 7B,7C, 7D, and 7E). In the 0.5 g CH Group I the above-described changes wereintense and applied to arterial vessels of different size (Figure 7B), whereas in the0.5 g CH+F Group II smaller atheromatous plaques were present (Figure 7C). InGroup III (2.0 g CH) atheromatous plaques were large and covered the entire

Figure 6. Kidney.

Figure 6B. Group I (0.5g CH). Kidney. Steatosis of tubulus epithelium. H-E. 160×.

Figure 6C. Group II (0.5g CH + F). Kidney. Normal pattern. H-E. 150×.

Figure 6D. Group III (2.0g CH). Kidney. Steatosis of tubulus epithelium. H-E. 140×.

Figure 6E. Group IV (2.0g CH+F). Kidney. Steatosis of tubulus epithelium. H-E. 180×.

Figure 6A. Control group (C). Kidney. Normal pattern. H-E. 280×.



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circumference of the vessel (Figure 7D). However, atheromatous plaques inarterial vessels of the heart were not so numerous and slightly smaller in the GroupIV (2.0 g CH+F) (Figure 7E).

The presence of fatty deposits in the atheromatosis laminas as well as in therenal tubule cells and hepatocytes was confirmed by histochemical studies. Fatswere stained with Sudan III into yellow-orange color.

Figure 7B. Group I (0.5g CH). Heart. Atheromatous plaque in arteriole. H-E. 150×.

Figure 7C. Group II (0.5g CH + F). Heart. Atheromatous plaque in arteriole. H-E. 280×.

Figure 7D. Group III (2.0g CH). Heart. Atheromatous plaque in arteriole. H-E. 200×.

Figure 7E. Group IV (2.0g CH + F). Heart. Atheromatous plaque in arteriole. H-E. 140×.

Figure 7. Heart.

Figure 7A. Control group (C). Heart. Normal pattern. H-E. 280×.



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DISCUSSION

Our study shows that an increased serum lipid level is not only an atherogenicfactor,1,24 but it also leads to pathomorphological changes in the internal rabbitorgans in the form of steatosis of both the renal tubule epithelium and hepatocytesin liver. The unquestionable regressive changes in the parenchymatous cells inliver and kidney disturb the regular functioning of an organism, including normalcholesterol metabolism. Administration of ionic fluoride (F) with the highercholesterol diet (Group IV) did not significantly influence the development ofatherosclerosis or regressive changes in organs. The morphological picture of therabbit organs in the study groups correlated with the biochemical assays results forthese animals.

The administration of F, together with the cholesterol diet (0.5g CH/kg bw/day)had an inhibitory effect on the atherogenesis and the development of the regressivechanges in organs. Our results indicate that the early atheromatous changes (lowcholesterol dose) were accompanied by the preventive activity of F ions on theobserved histopathological changes resulting from the cholesterol burden. Recentstudies indicate that in the pathogenesis of atheromatosis, oxidative processes playa significant role, especially the oxidation of low-density lipoproteins.25 Otherstudies are concerned with the role of F in oxidative stress. For example, deFerreyra et al.26 suggested a rise in reduced glutathione levels caused by F withsubsequent removal of hydrogen peroxide and oxygen free radicals by glutathioneperoxidase, and, in effect inhibition of peroxidation. There are also studiesdisclosing the activation27 or inhibition10,28 of F activity on lipid peroxidation andthe formation of malondialdehyde (MDA). Chlubek et al.10 has suggested that F atrelatively low concentrations stimulates lipid peroxidation, but at high and veryhigh concentrations may act as inhibitor of MDA generation. Findings by Gardnerand Fridovich29 indicate that F ions stabilize dehydratase and block the effect ofoxidants. In contrast, a number of studies indicate that generation of ROS andformation of MDA can be directly induced by F.30,31 There are also recent reportsindicating that F ions can inhibit the activity of enzymes responsible for oxygenfree radical metabolism (glutathione peroxidase, superoxide dismutase).12,32

According to Chlubek et al,11 a decrease in SOD activity can be attributed to adirect action of F on the enzyme rather than to increased generation of freeradicals induced by F intoxication. Reddy et al.33 reported finding no changes inlipid peroxide and GSH levels or in GSH-Px, SOD, and CAT (catalase) activitiesin red blood cells of F-intoxicated rabbits and fluorotic humans.

Recently, Birkner9 reported that cholesterol added to the diet of male rabbitscaused a statistically significant increase of MDA in blood plasma in comparisonto the control group. In our Group II rabbits (0.5 g CH+F) the MDA concentrationin plasma was considerably lower after three months in comparison to Group I(0.5 g CH). The lower concentration of MDA—an oxidation stress marker5—probably prevents dysfunction of vascular epithelia. Also, as Birkner9 noted,biochemical studies of male rabbits fed a 0.5% cholesterol diet supplemented withF for three months revealed a statistically significant increase of SOD in plasmawhen compared to the cholesterol control group without F. Thus Birkner’s results



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showing a positive effect of F on the rate of oxidation processes are in agreementwith the pathomorphological findings in our research.

As already noted, administration of F with cholesterol (0.5g CH+F) caused adownward tendency in the LDL plasma concentration when compared to the 0.5 gCH group. In the biochemical studies, no influence of F on the concentration oftriglycerides and HDL cholesterol in plasma was noticed when compared toGroup I (0.5 g CH). In our experiments, we observed a statistically significantincrease in HDL cholesterol levels in animals of Group IV (2.0g CH+F) comparedto Group III (2.0g CH) without F. Other lipid parameters (LDL and triglycerides)did not change significantly in both study groups compared to the control group.However, recent studies indicate that F may play a significant role inatherogenesis through its influence on the lipid profile in plasma. For example,Luoma et al.15,16 found that F causes a reduction in the levels very low densitylipoproteins (VLDL), LDL cholesterol, and LDL phospholipids in rats, as Singh etal.17 had reported earlier. Likewise, Kessabi et al.14 record similar findings insheep.

On the other hand, other studies on the influence of F on the serum level of lipidsare ambiguous. An increase in lipid concentration caused by F has been reportedin earlier studies,13,19 but a later report indicated no influence at all by F on thelevel of lipids in blood.34

The observed steatosis of hepatocytes in the liver and renal tubule epitheliumcells resulting from a cholesterol burden indicates a malfunction in lipidmetabolism and lipoprotein metabolism in the liver and kidney.35 In animalexperiments it has also been shown that F causes regressive changes in the liver.36

The results we have observed indicate that although the influence of F on lipidmanagement and free radical processes is controversial, lipid disorders are knownto be a major risk factor in atherosclerosis. Lipid metabolism (lipogenesis orlipolysis) in the organism may be affected by F due to repression of the activity ofa number of enzymes for lipid transformation: some nonspecific esterases such aslipase.37

In our studies we have seen that regulation of the metabolism of cholesterol inthe liver was disturbed. Fluoride ions administered together with a lowercholesterol atherogenic diet inhibited the atheromatosic changes in the aorta andarterial vessels as well as the regressive changes in the liver and kidneys.

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116 Research report 116 Fluoride 40(2)116–127 Stawiarska ... · Fluoride 40(2)116–127 April-June 2007 Influence of fluoride on rabbit organ morphology in atheromatosis Stawiarska-Pięta,