Absence of detrimental effects of fluoride exposure in diabetic rats

Archs oral Biol. Vol. 41, No. 2, pp. 191-203, 1996 Copyright © 1996. Published by ElsevierScience Ltd. All rights reserved Printed in Great Britain 0003-9969(95)00118-2 0003-9969/96 $15.00 + 0.00

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ABSENCE OF DETRIMENTAL EFFECTS OF FLUORIDE EXPOSURE IN DIABETIC RATS A. J. D U N I P A C E , l* C. A. W I L S O N , 1 M. E. W I L S O N , 1 W. Z H A N G , l A. H. K A F R A W Y , 2 E. J. B R I Z E N D I N E , 3 L. L. M I L L E R , 1 B. P. K A T Z , 1'3 J. M. W A R R I C K 1 and G. K. S T O O K E Y l ~Oral Health Research Institute, 2Department of Stomatology, School of Dentistry and 3Division of Biostafistics, School of Medicine, Indiana University, Indianapolis, IN 46202-2876, U.S.A. (Accepted 11 September 1995)

Summary--This study is part of a comprehensive programme to investigate fluoride toxicity and the hypothesis that fluoride ingested by 'medically compromised' animals will result in altered physiological function. Its objectives were to monitor fluoride retention, tissue fluoride concentrations and genetic variables in diabetic and control rats chronically exposed to fluoride, and to determine whether or not adverse effects occurred. Male, Zucker fatty diabetic rats and Zucker age-matched lean controls were fed a low-fluoride diet (< 1.2 parts/106 F - ) ad libitum and received 0, 5, 15 or 50 parts/106 fluoride in their drinking waler for 3 or 6 months. Fluoride metabolic balance was determined for 4 days before the end of each study phase. Plasma and urine were analysed for biochemical markers of tissue function, and plasma, urine, faeces and tissues were analysed for fluoride. Bone marrow cells from animals killed after 6 months of treatment were examined for frequency of sister chromatid exchange, a marker of genetic damage. The diabetic rats consumed, excreted and retained significantly greater amounts of fluoride than the controls (p < 0.05). There were dose-related increases in fluoride excretion, retention and tissue concentrations in both classes of animals, which were significantly greater in the diabetic rats. In spite of greater amounts of fluoride in the tissues of diabetic animals, there was no evidence, under these experimental conditions, that any of the fluoride exposures tested caused measurable adverse effects on the physiological, biochemical or genetic variables that were monitored. Key words: fluoride, diabetes, animals.

INTRODUCTION The public-health use of fluoride as a cariostatic agent is, under conditions of optimal water fluoridation (1.0 mg/l), considered highly beneficial and safe (NRC, 1993). Fluoride can cause fluorosis, or mottling, in developing dental enamel, but whether moderate or severe dental fluorosis is an adverse effect on health is still controversial (Kaminsky et al., 1990). While there continue to be concerns about a possible association of fluoride with bone fractures, induced mutations and cancer, there is, to date, no well-established, credible evidence that chronic, low-level exposure to fluoride presents significanl health risks in normal, healthy individuals (Kaminsky et al., 1990; PHS, 1991). On the other hand, for medically compromised persons in whom the metabc,lism of fluoride may be altered, chronic exposure might result in increased fluoride retention and a manifestation of potentially toxic side-effects if such effects of fluoride do occur. Diabetes mellitus is a c o m m o n chronic systemic disease, characterized by insulin deficiency and ab*To whom all correspondence should be addressed. Abbreviation: ANOVA, analysis of variance.

normally high blood-glucose levels, which result in altered metabolism with subsequent vascular and tissue damage. If diabetes is inadequately controlled by exogenous insulin and/or dietary restriction, as is frequently the case, physiological changes occur which have the potential to affect fluoride metabolism. Spillage of glucose into the urine causes osmotic diuresis and, therefore, diabetic individuals consume increased quantities of water. Ketosis resulting from fatty acid breakdown may occur, causing metabolic acidosis, and a decrease in extracellular pH can drive the movement of fluoride into tissue cells (Whitford, 1989). If metabolic acidosis subsequently leads to a fall in urinary pH, increased reabsorption of fluoride will occur in the kidney tubules (Whitford, Pashley and Stringer, 1976). Secondary to metabolic changes, diabetic individuals are subject to a number of chronic complications, the most notable of which are vascular lesions that manifest, in large part, in the kidney as nephrosclerosis. Because excretion of fluoride by the kidneys is one of the two primary mechanisms in the body responsible for its clearance from the blood (Whitford, 1989), renal impairment may also cause increased fluoride retention. Because of resulting changes in fluid intake and urine output and 191

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A.J. Dunipace et al.

disturbances in acid-base balance, diabetes mellitus is a disease that can affect the metabolism and biological impact of fluoride (Whitford, 1990). There have been only a few studies of fluoride metabolism in diabetics. In an investigation of plasma fluoride concentration and fluoride excretion under various pathological conditions in man, Hanhijarvi (1954) reported a significant increase in mean plasma fluoride concentration in diabetic individuals compared to that of age-matched controls. This same investigator also reported reduced fluoride excretion in two diabetic patients in whom the serum creatinine was elevated, indicating concomitant renal insufficiency. Two studies have been conducted on animals in which diabetes was chemically induced with alloxan or streptozotocin. In 1962, Sweeney et al. observed greater fluoride deposition in femurs from diabetic rats than from control animals similarly treated with fluoride; however, they did not monitor fluoride intake and excretion, or measure plasma or soft-tissue fluoride concentrations. Allmann et al. (1984) reported greater fluoride retention and higher tissue fluoride levels in diabetic rats receiving drinking water fluoridated at 1.0 parts/106 for 9-16 days than in similarly maintained control animals. None of these studies, however, monitored clinical toxicity, which may have been affected by the increase in tissue fluoride concentrations. Our objective now was to investigate the effect of chronic fluoride exposure on a number of variables in diabetic rats in order to determine the clinical significance of increased fluoride retention in these animals. The first specific aim was to determine whether the tissues in which fluoride concentrations increased will continue normal physiological function, by monitoring standard clinical markers of tissue integrity and 'wellness'. The second aim was to observe whether an increase in the body burden of fluoride had detrimental genotoxic effects, manifested by a rise in the frequency of bone-marrow sister chromatid exchange. Did increased fluoride retention in diabetic rats cause adverse effects not ordinarily observed in metabolically healthy animals, and was the margin of safety for fluoride exposure narrowed in these 'medically compromised' animals? In order to avoid the possibility of side-effects that may occur when diabetes is chemically induced, genetic Zucker diabetic fatty male rats (ZDF/Drt-fa) were chosen for this study. This rat model, first described by Clark, Palmer and Shaw (1983), has been successfully developed by generations of inbreeding fatty diabetic males with their sibling females. The model expresses non-insulin-dependent diabetes meilitus with characteristics similar to those of human, type II, adult-onset diabetes (Davidson and DiGirilamo, 1991). Their level of diabetes and neuropathic changes makes the Z D F rat a good model for investigating the complications of noninsulin-dependent diabetes (Peterson et al., 1990a). The control animals were lean Zucker male rats

which are genetically similar to the obese animals except for the obesity trait. MATERIALS AND METHODS

Study design

Four groups of control rats and four groups of diabetics (n = 36-40/group) were treated with 0, 5, 15 or 50 parts/106 fluoride (as sodium fluoride, NaF) delivered in their drinking water. Treatment began when the animals were 8 weeks of age, and rats from each treatment group were killed after 3 or 6 months of treatment. Four animals were included in each of the 6-month treatment groups for the assay of sister chromatid exchange for genotoxicity. The plasma glucose was monitored in all animals at intervals throughout the treatment periods to document diabetic status. During the 4 days preceding the end of the study, animals from each group were placed in metabolism cages. Water consumption and urine volume were measured, and urine and faeces were saved for fluoride analyses. At death, plasma and tissues were saved for fluoride determination; plasma and urine were obtained for clinical chemistry analyses of physiological wellness markers; and, from designated 6-month treated animals, tissues were taken for histological evaluation. Femur bonemarrow cells were harvested from the animals chosen for scoring the frequency of sister chromatid exchange. The study protocol was reviewed and approved by the Indiana Animal Care and Use Committee, and all animal care and treatment procedures complied with guidelines established by the NIH, F D A and the USDA. Animals

Male, Zucker diabetic fatty rats and their lean controls (Genetic Models, Inc., Indianapolis, IN) were used. The animals were received, at approx. 5 weeks of age, as 150 matched pairs of one obese diabetic and one age-matched control. The animals were maintained in the Bioresearch Facility at the Indiana University School of Dentistry until they were 8 weeks of age, at which time the study began. At the time of receipt the animals were each given an identification number by means of ear punch and a cage card. They were weighed upon receipt, at the time of stratification, bimonthly during the treatment period, and before death. Just before the study began the rats were assigned to treatment groups. Groups within each class of animals (i.e. diabetic or non-diabetic) were balanced on the basis of initial mean body weight and blood glucose values. Because of the large number of animals used (n = 300), the study was done in three phases (A, B, C), with 100 animals/phase, initiated at monthly intervals and with animals in each phase distributed evenly across all treatment groups. The animals were housed individually in polypropylene box cages in the AAALAC-accredited Bioresearch Facility. During

Chronic fluoride exposure in diabetic rats metabolic periods the animals were housed individually in metabolism cages.

Diet From the time of receipt and for the duration of the study all animals were fed a pelleted, lowfluoride (F ~< 1.2 parts/106), high-fat diet (modified Formulab 5751C-K, Purina Mills, Inc., Richmond, IN), ad libitum. This diet was a custom modification of the Purina 5008 Formulab chow ordinarily fed to Z D F rats (Peterson et al., 1990b) and contained the following ingredients: protein, 24.6%; carbohydrate, 50.0%; fat 6.5%; fibre 3.5%; mineral ash, 5.0%; vitamins 0.4%; and water, 10%. Each purchased batch of diet was analysed for fluoride before use and met the study requirement of F ~< 1.2 parts/106.

Treatment Fluoride was provided to all animals ad libitum in their drinking water, which was prepared by dissolving N a F in deionized water. All prepared water was analysed for fluoride content before use; samples from individual water bottles were randomly tested during the course of the study to ensure treatment as designated.

Monitoring the plasma glucose To ensure the diabetic status of the Z D F animals, the fed plasma glucose concentrations of all animals were monitored before study began, midway through each treatment phase, and 1 week before the end of the study. Blood samples for these analyses were obtained from all animals between 8:00 and 11:00 a.m. Blood from each animal was collected in heparinized capillary tubes by tail clipping, and the animals were tested in random order to control for the variations in blood glucose related to the time of day or amount of food ingested.

Metabolic balance periods For 4 days just before each phase was ended, 12 of the animals from each group scheduled for killing were placed individually in metabolism cages (Nalgene No. 4650-0350, Fisher Scientific, Cincinnati, OH). Each day during this period, water and food consumption and ~otai urine volume were recorded. Some of the urine and all of the faecal output from each day were saved for determination of fluoride content. A portion of the final 24-h urine sample was also saved for urea and creatinine analyses. Because caging was limited, animals designated for the sister chromatid assay were not placed in metabolism cages.

Study termination Animals from the eight treatment groups were killed in random order between 8:00 a.m. and 12:00 noon. The animals were anaesthetized with a mixture of ketamine (100mg/ml) and xylazine (20 mg/ml). These agents were m ~ e d 10.0 ml to 5.5 ml, respect-

193

ively and injected intramuscularly at 0.14ml/100g body wt. Blood was drawn by cardiac puncture and death was procured by bilateral pneumothorax. Blood, liver, kidneys, one femur and a section of lumbar vertebrae (L2-L4) were obtained from each animal, and carcasses from the 12 animals in each group that had been placed in metabolism cages were saved for fluoride analyses. From representative, randomly selected, 6-month animals (n = 6-7/group), a lobe of the liver, one kidney and also a tibia were saved for histological analyses. Four animals from each 6-month treatment group were used to determine sister chromatid exchange. Metabolic balance and tissue fluoride data were not obtained from these animals. The analytical procedure for sister chromatids followed that of Perry and Thomson (1984). Twenty-four hours before death the animals were anaesthetized with the ketamine: xylazine mixture, and two 5-bromo-2' desoxyuridine tablets (50-60mg) were implanted subcutaneously in the lateral abdominal region. Positive control animals that had received no fluoride were given 10mg/kg of the known mutagen cyclophosphamide (Sigma Chemical Co., St. Louis, MO), intraperitoneally, 16h before death, and all animals were treated with 0.6mg colchicine/kg, intraperitoneally, 2 h before death to arrest cell division. After death, both femurs were removed and bone marrow cells were harvested for scoring sister chromatid exchange.

Analytical procedures Sample were identified only by animal number, and all analyses were made blindly without knowledge of treatment or animal class. Plasma glucose analyses. Capillary amounts of blood obtained from each animal by tail clipping were centrifuged and the plasma recovered. A 10-#1 portion of each plasma sample was analysed for glucose [Glucose Analyses II instrument (Beckman Instruments, Fullerton, CA) in the Department of Anatomy at the Indiana University School of Medicine (Indianapolis, IN)]. Fluoride analyses. All faecal and soft-tissue samples were prepared for fluoride analyses by homogenization: total daily faecal output, both kidneys (except where one was used for histology) and a preweighed amount of liver were individually homogenized in known volumes of deionized water, and portions were analysed for fluoride. Blood was centrifuged immediately after collection, and plasma was saved for analysis. Fluoride was measured in a centrifuged portion of each urine sample after recording the total sample volume. Femur samples were placed in open, porcelain crucibles and ashed in a muffle furnace at 600°C for 6 h. Carcass specimens, which included all remaining tissues that had not been removed for individual analyses, were burned over bunsen burners to a black ash and were then reduced to a white ash in the muffle furnace. Ashed samples were then

194

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weighed and pulverized before analysing approx. 10 mg of each sample for fluoride content. Pelleted diet samples were pulverized before analysis. The drinking water was analysed for fluoride using a combination fluoride ion-specific electrode (Orion No. 96-09-00) and a pH-ion meter (Accumet 950, Fisher Scientific, Cincinnati, OH). One millilitre of each sample was diluted 1: 1 with total ionic-strength adjustment buffer (Tisab II: Orion Research, Cambridge, MA) and placed directly under the electrode. Fluoride content was determined by comparison with a series of standards analysed in a similar manner at the same time. All diet, soft and mineralized tissues, urine and faecal samples were analysed using a modification of the hexamethyldisiloxane (Sigma) microdiffusion method of Taves (1968) as modified by Dunipace et al. (1995). After overnight diffusion of fluoride from a known volume or preweighed amount of each sample into a 0.05 or 0.075 M NaOH trap, the trap was buffered to pH5.2 with 0.15 or 0.20M acetic acid. Each buffered solution was adjusted to a fixed volume (100/~1) with distilled, deionized water and placed under the electrode. Sample fluoride concentrations were determined by comparison to a series of fluoride standards simultaneously diffused in the same manner. Clinical chemistry analyses. Biochemical markers of tissue integrity and function in plasma and urine were analysed using Ektachem 700 multichannel (Kodak, Rochester, NY) and Dimension (Dupont, Wilmington, DE) autoanalysers in the Department of Pathology, Wishard Memorial Hospital, Indianapolis, IN). The plasma constituents that were analysed included: urea, glucose, creatinine, calcium, phosphorus, uric acid, cholesterol, total protein, albumin, total bilirubin, alkaline phosphatase, and glutamate oxaloacetate transaminase, and were all part of a Chem 12 SMA profile. In addition, urine urea and creatinine were also analysed, and creatinine clearance, a measure of the volume of plasma cleared of creatinine each minute and a marker of renal function, was calculated [clearance (ml/min) = [urine flow (ml/min) × urine creatinine (mg/dl)] plasma creatinine (mg/dl)]. Histological evaluations (6-month animals only). At the time of death, a portion of the liver and one kidney were obtained from randomly selected animals in each 6-month group and fixed in 10% formalin. The coded specimens were then processed for paraffin embedding and sections (approximately 7#m) were prepared. After staining with haemotoxylin and eosin, sections were examined for pathological changes without knowledge of animal class or treatment regimen. Tibia samples obtained from the same animals were fixed in 10% formalin and were then demineralized in 5°/'o formic acid, embedded in paraffin and sectioned. After staining, thickness measurements were made using a micrometer eyepiece, and specimens were examined for skeletal

fluorosis as evidenced by exuberant periosteal bone formation and osteosclerosis (Fejerskov, Kragstrup and Richards, 1988). Bone-marrow sister chromatid exchange (6-month animals only). Procedures for harvesting and fixing the bone marrow cells, for staining the chromosomes, and for preparing and scoring the slides have been described by Li et al. (1987). Bone-marrow cells were flushed from both femurs of an animal with warm (37°C), hypotonic KCI (0.075 M) and pooled. After gentle mixing and incubation for 30 min at 37°C, the cells were centrifuged at 1000 rev/min for 5 min and were then fixed by drop-wise addition of 5 ml of a fresh solution of methanol and acetic acid (3 : 1). The fixing procedure was repeated two more times. Fixed cells were then dropped on a clean, refrigerated slide and air-dried overnight. The fluorescence plus Giema method (Goto et al., 1978) was used to stain the chromosomes. All slides were coded so that scoring was done without knowledge of treatment, and 25 M2 cells from each animal were examined for frequency of sister chromatid exchange.

Data analysis ANOVA was used to analyse the outcome measures of the study. The ANOVA model contained three factors: class, treatment and age, as well as their interaction. For all fluoride data, logarithmic transformation was required before analysis to correct for unequal variances. When significant main effects were detected, means were compared using a least significant-difference procedure. Within classes, pair-wise comparisons were made at the different age and treatment levels using the Ryan Enot Gabriel Welch Q multiple comparison test, which controls for types 1 and 2 experimental error. Mortality and qualitative histological data on periosteal osteoid accumulation were analysed using the Mantel-Haenszel X2 procedure. Results for sister chromatid exchange were analysed by two-way ANOVA, and the least significant difference analysis was used to determine the significance of differences among the treatment-group means. The level of significance for all statistical analyses was set at p < 0.05.

Results Characteristic differences between the diabetic and control animals are summarized in Table 1. Although animals were the same age when the study began, the mean initial body weights of the two classes were significantly different, as were the initial plasma glucose concentrations. As expected, the diabetic rats, irrespective of treatment, consumed significantly greater amounts of water and excreted significantly larger volumes of urine. Data for the individual treatment groups in each animal class are not presented in Table 1, but statistical analyses showed that, among the control animals, daily water intake and urine output were not affected by fluoride treatment. However, the diabetic rats exposed to 50 parts/106

195

Chronic fluoride exposure in diabetic rats Table 1. Animal characteristics a (mean 4- SEM) b Animal class

Initial weight (g)

Initial plasma glucose (mg/dl)

Water intake (ml/day)

Urine output (ml/day)

Corttrols (C) Diabetics (D)

282 __+1.8 351 _+ 1.4

121.9 4- 1.7 453.5 _+ 8.2

27.3 _+0.4 162.4 + 3.9

12.4 _+0.2 134.0 _+ 3.7

aAll treatment and age groups combined within each class. bn: for initial weight and glucose, C = 148, D = 136; for water intake and urine output, C=95, D=88.

fluoride consumed significantly less water and excreted significantly less urine than did the other diabetic animals.

between fluoride treatment either animal class.

and mortality within

Mean metabolic variables Growth and mortality Fluoride treatment had no significant effect on weight gain in either the control or diabetic rats during the course of the study. After 3 months of treatment the control and diabetic animals had gained 171.3 + 2.2 g and 86.0 + 3.9 g, respectively. By the end of the study the controls had gained 2 2 4 . 7 + 2 . 5 g for a mean final body weight of 508.8 + 4.2 g, while the weight gain of the diabetic rats was 94.8 + 3.6 g and their mean final weight was 445.4 + 4.1 g. The differences in weight gain and final body weight between the classes were significant but these differences were disease-related rather than treatment-related. Mortality data were calculated individually for each class of animals. During the first 3 months of treatment, none of the control animals died. Mortality among the diabetic rats during this period was 4.3%, with 6.3, 0, 6.3 and 4.8% mortality occurring in the groups receiving 0, 5, 15 and 50 parts/106 fluoride, respectively. Between 3 and 6 months, one control animal (0 parts/106) died (control group mortality 1.2% ), and 12.8% of the diabetic animals did not survive. O f the diabetic animals that died, 20, 5, 10 and 15.4% were from the groups treated with 0, 5, 15 and 50 parts/106 fluoride, respectively. Statistical analysis indicated that there was no association

Because statistical analyses indicated that mean metabolic data were not significantly affected by the duration of fluoride treatment, 3- and 6-month results were combined for presentation in Table 2. F o o d intake was estimated by periodically monitoring consumption by representative animals in each class and treatment group. F o o d consumption did not change with fluoride treatment so average food intake was calculated for each class of animals. Because the diabetic animals consumed greater amounts of water than the controls (Table 1), their fluoride intake was significantly greater than that of similarly treated control rats. There were highly significant (p <0.001) doserelated differences in both urine and faecal fluoride excretion among all four treatment groups in both animal classes (Table 2B and C), and total fluoride excretion was significantly greater for the diabetic animals than for their corresponding controls. When faecal fluoride data were expressed as a percentage of ingested fluoride by calculating the percentage for each fluoride-treated rat individually, and then determining the group average, control animals excreted 37.1 + 1.1% of ingested fluoride in their faeces; diabetic faecal excretion accounted for significantly less, 24.8 + 0.8%, of their fluoride intake. Animals that were not exposed to fluoride (0 parts/106 F groups) were not included in these calculations. In both

Table 2. Mean metabolic data a (mean + SEM) b Fluoride treatment (parts/106) Animal class

0

A. Fluoride intake (l~g/day) Controls 23.0 _+0.9 c Diabetics 42.0 + 1.6c B. Urine fluoride (pg/day) Com:rols 11.2 _ 0.5 Diabetics 24.2 _ 1.4 C. Faecalfluoride (#g/day) Controls 24.3 ___1.5 Diabetics 43.5 4- 3.7 D. Fluoride retention (#g/day) Controls -- 12.5 4- 1.8 Diabetics --25.7+4.2

5

15

50

157.2 _ 4.0 893.9 4- 34.9

435.0 _+ 11.9 2678.1 _+ 111.6

1420.0 _ 37.7 6902.1 + 430.8

41.7 _+ 2.3 281.1 4- 17.1

111.2 __+5.2 922.0 4- 47.2

386.8 + 15.6 2632.2 _ 255.6

72.7 4- 3.2 260.7 + 14.8

165.0 + 6.6 632.5 4- 34.5

383.9 4- 17.0 1410.7 +__75.8

42.8 _+ 3.1 352.1 _+23.9

158.9 _+ 10.6 1123.6+83.2

643.9 4- 37.1 2859.24-215.1

a3- and 6-month data combined. bn = 22-24. CFood intake averaged from periodic measurements.

196

A. J. Dunipace et al. Table 3. Plasma and tissue fluoride (mean ± SEM)a

Animal class

Treatment duration (months)

Fluoride treatment (parts/106) 0

5

15

50

0.54 ± 0.06 0.42 ± 0.04 0.51 +0.05 0.65 _ 0.06

0.88 + 0.10 0.77 _ 0.07 1.88 +0.18 2.37 ± 0.23

1.46 ± 0.15 1.49 __+0.11 5.51 +0.41 6.06 ± 0.49

4.28 + 0.31 3.77 ± 0.23 13.60+ 1.27 19.18 __+2.64

0.74 ± 0.13 0.43 + 0.04 1.00 _+0.09 0.59 _ 0.09

0.97 _ 1.26 _ 1.62 ± 1.62 ±

2.16 + 1.03 _ 4.75 _ 6.06 _

3.44 ± 2.76 ± 12.03 _ 16.07 ±

0.30 0.37 1.32 1.84

A. Plasma (#tool F/l) Controls Diabetics

3 6 3 6

B. Liver (ltmol F/kg wet wt) Controls Diabetics

3 6 3 6

0.12 0.57 0.16 0.21

0.55 0.11 0.42 1.14

C. Kidneys (#mol F/kg wet wt) Controls Diabetics

3 6 3 6

1.78 + 2.67 + 3.21 _ 2.27 _

0.27 0.72 1.89 0.29

2.60 ± 0.36 5.51 + 1.85 6.15±0.50 10.52 ± 2.99

8.83 _ 2.20 8.32 + 1.77 37.44___ 16.13 36.40 + 9.13

26.77 + 31.82 + 71.70 ± 73.54 ±

6.06 10.55 15.13 10.53

171.2 + 192.9 ± 199.8 + 255.0 ±

7.8 6.4 8.3 10.5

409.5 + 6.8 486.7 + 11.9 999.2 _+20.2 1483.4 + 53.9

871.9 ± 23.4 1120.9 _ 18.0 2724.6 ± 97.2 3735.3 +_ 122.3

2523.8 + 3078.8 _ 6843.2 + 8453.1 +

42.9 62.5 167.6 308.7

432.9 + 559.0_ 1193.3 + 1953.4 +

962.5 -}-28.7 1330.4 + 24.3 3129.5 ± 161.4 4648.5 + 128.1

2864.7 __+81.9 3580.1 + 71.0 6914.6 __+260.5 9449.9 _+ 346.1

D. Femur (#g F/g ash) Controls Diabetics

3 6 3 6

E. Vertebra (ttg F/g ash) Controls Diabetics

3 6 3 6

175.6 + 12.4 194.1 + 3.5 226.3 __+14.0 309.2 + 13.0

10.7 14.2 47.1 71.0

F. Carcass (total mg F) Controls Diabetics

3 6 3 6

1.77 ± 2.16 + 1.58 + 2.03 ±

0.17 0.07 0.07 0.10

4.11 -}-0.17 5.58 _ 0.20 7.94 + 0.34 11.50 ± 0.55

8.98 + 12.85 _ 21.32 + 31.94 _

0.51 0.38 0.95 0.72

24.00 + 33.71 _ 51.85 + 74.36 ±

1.02 1.22 1.46 4.71

an = 13-19;for carcass, n = 8-12.

classes o f animals, the percentage o f ingested fluoride excreted in the faeces, and, therefore, not absorbed, increased with treatment d u r a t i o n and was inversely related to the level o f fluoride exposure. Similar calculations determined that fluoride-treated control rats a b s o r b e d 62.9 -}- 1.1%0 o f their fluoride intake, while 75.2 + 0.8% o f their ingested fluoride was absorbed by the fluoride-treated diabetics. In contrast to faecal fluoride excretion, the percentage o f absorbed fluoride increased significantly as fluoride-treatment levels increased and tended to decrease between 3 and 6 months. Excluding the 0 parts/106 fluoride-treated rats, the percentages o f absorbed fluoride that were subsequently eliminated in the urine were 43.7 + 1.6% and 45.8 + 1.4% for the fluoride-treated control and diabetic animals, respectively, and were not significantly different. Neither treatment level nor duration had a significant effect on the percentage o f absorbed fluoride eliminated in the urine. Table 2(D) presents m e a n daily fluoride retention (intake-excretion), which increased significantly in b o t h classes o f animals as fluoride exposure increased, and indicated that the 'medically c o m p r o mised' diabetic animals retained significantly greater a m o u n t s o f fluoride. F o r the control animals exposed to 5, 15 and 50 parts/106 fluoride, fluoride retention accounted for 27.4 + 1.9, 36.3 _ 1.9 and 44.9 ± 2.2% o f their fluoride intake, respectively; these percent-

ages for the diabetic rats were 39.2 + 2.0, 41.6 ___2.0 and 42.0 _ 2.2%. The differences in these percentages between animal classes were significant only in the case o f 5 parts/106 fluoride-treated groups.

Tissue fluoride Fluoride concentrations in plasma and representative soft and mineralized tissues are presented in Table 3. In plasma and all m o n i t o r e d b o d y tissues there were significant, dose-related differences in fluoride concentration in both animal classes. At each fluoride treatment level, the diabetics retained significantly larger a m o u n t s o f fluoride in all m o n i t o r e d tissues than did the corresponding controls, and this difference became greater as fluoride treatment increased. While analyses indicated that duration of exposure was a significant factor in determining tissue fluoride levels, pair-wise c o m p a r i s o n s indicated that its significance was not consistent across all treatment groups in b o t h animal classes. In general, the tissue fluoride level in diabetic animals increased as the animals aged, while there was a reduction, or more gradual increase, in tissue fluoride levels in the control animals between 3 and 6 m o n t h s o f treatment.

Clinical chemistry There were significant differences between control and diabetic animals for m o s t o f the m o n i t o r e d blood

Chronic fluoride exposure in diabetic rats

197

Table 4. Clinical chemistry variables unaffected by fluoride treatment a (mean + SEM) b Animal class

L Blood Urea nitrogen (mg/dl) Glucose (mg/dl)c Creatinine (mg/dl) Calcium (mg/dl) Phosphorus (mg/dl) Uric acid (mg/dl) albumin (g/dl) SGOT (U/l) II. Urine Creatinine (mg/dl) Creatinine clearance (ml/min) Urea (mg/dl)

Controls (C)

Diabetics (D)

18.16 _ 0.19 310.51 +__6.01 0.62 -+ 0.01 10.12 -+ 0.02 5.66 -+ 0.07 0.90 _ 0.04 3.21 + 0.01 253.02 + 9.71

22.52 +__0.74** 695.37 -+ 7.58** 0.52 -+ 0.01"* 10.62 _+0.04** 7.05 _ 0.10"* 1.05 -+ 0.07 d 3.03 + 0.02** 380.48 _ 18.13"*

141.79 _ 2.16 1.98 + 0.06 3516.12 -+ 47.40

12.68 + 0.48** 2.13 _ 0.09 698.82 + 13.06"*

SGOT, glutamine oxaloacetate transaminase. aAge and treatment groups combined. bn: for blood variables, C = 129, D = 120; for urine variables, C = 92, D = 88. **Diabetic and control values are significantly different (p < 0.05) as determined by ANOVA. CThese glucose values are post-anaesthetic. dp = 0.0612.

and urine variables. Where fluoride had no effect on these 'wellness' markers, data for the age and treatment groups were combined within each class and are presented in Table 4. Total bilirubin and alkaline phosphatase were not included in Table 4 because the A N O V A indicated a significant three-way interaction among class, fluoride and treatment duration for these two variables. F o r total bilirubin, there were no differences a m o n g the control treatment groups at either 3 or 6 months, or in the diabetic rats at 3 months. At 6 months there were isolated differences among the diabetics, but the means did not shown a fluoride dose-response relation (0 parts/106: 0.21; 5 parts/106: 0.29; 15 parts/106; 0.26; 50 parts/106: 0.19mg/dl). Similarly, there were no significant differences in the alkaline phosphatase values among the control group,;. There were scattered differences in alkaline phosphatase among the diabetic rats at both times, but no fluoride dose-response effect was evident (3 months: 0 parts/106: 326; 5 parts/106: 395; 15 parts/106: 361; 50 parts/106: 286; 6 months: 0 parts/106: 395; 5 parts/106: 317; 15 parts/106: 300; 50 parts/106:349 units/l).

The glucose values presented in Table 4(B) were determined on cardiac blood samples obtained after the animals had been anaesthetized at the termination of the study. For both classes of animals, these data were higher than terminal pre-anaesthesia results from tail-clipped blood samples, which were 129.6 + 1.7 and 453.5 _ 8.2 mg/dl for the control and diabetic rats, respectively. Two of the clinical blood variables were affected by fluoride in the diabetic animals (Table 5). Fluoride had no significant effect on either cholesterol or total protein concentrations in control rats. However, cholesterol was significantly lower and total protein was significantly higher in the 50 parts/106 fluoridetreated diabetic animals than in other diabetic treatment groups. In the case of cholesterol, the effect of 50 parts/106 fluoride was directly and significantly increased by treatment duration (data not shown). Sister chromatid exchange Table 6 presents the results of sister chromatid analyses conducted in representative control and diabetic animals after 6 months of chronic fluoride

Table 5. Clinical chemistry variables affected by fluoridea (mean -+ SEM) b Cholesterol (mg/dl)

Total protein (g/dl)

Treatment (parts/106)

Control (C)

Diabetic (D)

Control (C)

Diabetic (D)

0 5 15 50

82.7+ 1.29¢ 1 79.9_~ 1.51c | 80.4 __+0.99¢ | 82.0+ 1.27c J

186.1_+7.79] 182.6+7.13 / 185.3__+7.36J 153.5__+4.73

5.89--+0.14] 5.96-+0.03 / 5.85 + 0.04 / 5.90 _ 0.04 J

5.94-+0.09] 6.06-+0.07 / 6.08 -+ 0.08 J 6.28+0.08

a3- and 6-month data combined. bn: C = 29-37; D = 28-33. CValues within brackets are not significantly different (p > 0.05) as determined by pair-wise least significant difference analyses.

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A.J. Dunipace et al.

Table 6. Frequency of sister chromatid exchange in rats treated with fluoride for 6-months (mean __+SEM) a Animal class Controls

Diabetics

Positive control

Treatment

Frequency (n/cell)

0 parts/106 F 5 parts/106 F 15 parts/106 F 50 parts/106 F 0 parts/106 F 5 parts/106 F 15 parts/106 F 50 parts/106 F 10 mg/kg CP b

2.50 + 0.12c 2.55 ___0.2F 2.18 + 0.13¢ 2.53 + 0.15c 2.27 + 0.22c 2.83 _ 0.28~ 2.53 __+0.46c 2.20 __+0.29c 48.88 + 8.83

an 3-4; CP, n = 2; 25 M: cells scored/animal. bCp: cyclophosphamide. CValues within brackets are not significantly different (p > 0.05) as determined by ANOVA. =

The mean value for sister chromatid exchange for the 0 parts/106 fluoride-treated control animals was typical of baseline values reported previously (Tice, Chaillet and Schneider, 1976; K r a m et al., 1978; Dunipace et al., 1995). Animals in the positive control, cyclophosphamide-treated group had a significantly greater (p < 0.001) number of sister chromatid exchange/cell than did the animals in the negative control, 0 parts/106 fluoride-treated group, thereby validating the method. However, none of the fluoride-treated rats, in either class of animals, had a frequency of sister chromatid exchange significantly greater than that of the negative controls, even though there were significantly greater amounts of fluoride in the femur tissues of those animals (Table 3E). Histology Liver, kidney and tibia from representative animals in each of the 6-month treatment groups were examined histologically. Qualitative pathological changes were evident in the kidneys of all diabetic animals examined, with no apparent relation to the level of fluoride treatment. Hyaline casts indicative of proteinuria were evident. Enlargement of the renal pelvis and hydronephrosis indicative of obstruction at some level of the urinary tract were also commonly found. Areas of chronic interstitial inflammation were also frequently observed in the kidneys of the diabetic animals. These changes were seldom observed in the kidneys of the control animals, again with no apparent relation to the fluoride level. Liver samples from both diabetic and control animals

receiving different fluoride levels were microscopically similar, with the c o m m o n finding being foci of small hepatocytes suggestive of glycogen depletion. The tibias were evaluated at different sites. The width of the epiphyseal plate was measured at two peripheral sites and near the middle. The mean thickness of the epiphyseal plate was not markedly different between the control and the diabetic animals or in animals of either class receiving different fluoride levels. The metaphyseal region was examined qualitatively for evidence of accumulation or depletion of trabeculae. Accumulation was evident in the control animals receiving different levels of fluoride; less metaphyseal bone was evident in the diabetic animals that had received fluoride at 0, 5 or 15 parts/106. While three of the diabetic rats treated with 50 parts/106 fluoride also had less metaphyseal bone accumulation, four out of the seven diabetic animals that were examined from this group showed accumulation that was similar to that of the control animals exposed to 50 parts/106 fluoride. The thickness of the diaphysis near the middle of the shaft was measured on each side of the medullary cavity; the mean thickness was computed; and the width of the medullary cavity was also measured. The diaphysis data are presented in Table 7. In the animals treated with 0, 5 or 15 parts/106 fluoride, the thickness of the mid-shaft diaphyseal bone was significantly less in the diabetic animals than in the controls. However, in diabetic animals receiving fluoride at 50 parts/106, the thickness of the diaphyseal cortex near the middle of the shaft was no longer significantly less than that of the control animals receiving the same fluoride treatment. The width of the medullary cavity was similar across animal class and treatment groups but was numerically larger in the diabetic animals exposed to 50 parts/106 fluoride. Tibias were also qualitatively examined for periosteal bone accumulation and osteosclerosis as evidence of skeletal fluorosis. Periosteat osteoid accumulation was scored as none (0), slight ( + ) , moderate ( + + ) or exuberant ( + + +). Slight to moderate accumulation was evident in one of the controls and two of the diabetic animals treated with 0 parts/106 fluoride. This was considered to be indicative of periosteal osteogenesis, probably due to a minor injury. In general, the incidence and severity of periosteal accumulation were directly proportional to the level of fluoride exposure within both animal classes, and were not significantly different between

Table 7. Tibia diaphyseal width (#m) (mean + SEM) a Fluoride treatment (parts/106) Animal class Controls Diabetics

0 668 _ 32 531 + 23

5

15

50

720 __+50 540 + 28

663 _ 21 572 + 20

648 + 16b] 630 __+38bJ

an = 6 or 7. bValues within brackets are not significantly different (p >0.05) as determined by pair-wise least significant difference analysis.

Chronic fluoride exposure in diabetic rats the diabetic and control rats. None of the examined specimens showed evidence of osteosclerosis. DISCUSSION

The purposes of this study were (1) to test whether diabetic rats, becau,;e of disease-induced changes in fluid intake, metabolism and urine output, retain a greater amount of the fluoride they ingest than healthy control animals, and (2) if so, to determine whether the elevation in tissue fluoride results in detrimental physiological, biochemical or genetic effects. The diabetic, ZDF/Drt-fa model exhibits complications similar to those observed in human diabetics and in other long-term diabetic animals, and the level of diabetes and neuropathic changes make the Z D F rat a good model for studying non-insulin-dependent diabetes (Peterson et al., 1990a). Our diabetic animals had developed hyperglycaemia in excess of 400 mg% by the time they were 8 weeks of age, and remained diabetic for the duration of the study. While the body weight of the diabetic animals was initially significantly greater than that of their age-matched controis, these animals grew less than the controls during the study and at the end weighed significantly less. Also, as originally described for this model, the blood cholesterol concentrations of the diabetic rats were significantly higher than those of the controls. Treatment of the animals with fluoride did not, therefore, alter the model as originally characterized (Peterson et al., 1990b). The levels of water fluoride tested were greater than those ordinarily encountered by human populations. Rats are reportedly less susceptible than humans to the effects of fluoride (Angmar-Mansson and Whitford, 1984) and it has been suggested that fluoride exposure must be four to five times greater in rats in order to elicit concentrations of serum fluoride similar to those in man (Angmar-Mansson and Whitford, 1982; Dunipace et al., 1995). We estimate that water fluoride levels tested here correspond to fluoride concentrations of 1, 3 and 10 parts/106 in human populations. Although both animal classes were treated with the same concentration's of fluoride, fluoride exposure was much greater figr the diabetic rats. Mean daily fluoride intake based on body weight was 0.33 _+ 0.01 mg F/kg per day for control rats exposed to 5 parts/106 fluoride and 3.04_ 0.16 mg F/kg per day for the control animals receiving 50 parts/106 fluoride. These data were similar to those reported in a comparable study (Dunipace et al., 1995). However, because the water intake of the diabetic rats was so much greater than that of the controls, diabetic animals receiving similar chronic fluoride treatment ingested 1.99 +_ 0.15 and 16.26_+ 1.42 mg F/kg per day. The fluoride exposure of the diabetic animals was also increased, relative to that of the controls, because a greater percentage of the fluoride ingested

199

was absorbed. For each of the three fluoride concentrations tested, effective exposure to fluoride, as assessed by the plasma fluoride concentration, was five to six times greater for the diabetic rats than for their similarly treated controls. The ingestion of fluoride by human adults has been estimated at 1.2 to 2.2 mg/day (Whitford, 1994), so the treatment doses of fluoride tested were all higher, in both classes of animals, than the estimated daily exposure of 0.0160.040 mg F/kg per day for healthy, 75- and 55-kg humans consuming optimally fluoridated water. Fluoride in the plasma and tissues of the Zucker lean control rats did not cause any detrimental physiological, biochemical or genetic effects. If fluoride had caused toxicological changes, significant dose-related changes would have occurred in some of the clinical 'wellness' markers, and genetic effects would have manifested as an increase in the frequency of sister chromatid exchange. None of this happened. These results are similar to those reported from a recent investigation of fluoride effects in ageing Sprague-Dawley rats, which concluded that fluoride in the tissues of those animals chronically exposed to 5, 15 or 50 parts/106 fluoride for as long as 18 months had no adverse effects (Dunipace et al., 1995). Our metabolic data showed marked differences between the control and diabetic rats, with the diabetics ingesting, absorbing and retaining significantly greater amounts of fluoride than their similarly treated controls. Why a faecal fluoride excretion accounted for a lower percentage of ingested fluoride in the diabetic than in the control animals is unclear, but this might be related to the gastroparesis and delayed gastric emptying that occur in diabetes (Jurado and Walker, 1991) which would increase the time during which gastric absorption of fluoride could occur. This premise could be challenged by findings in fasted rats that acutely administered fluoride was absorbed primarily in the small intestine (Messer and Ophaug, 1991), but it is supported by the work of other investigators, who have determined that appreciable amounts of fluoride are absorbed from the stomach (Whitford and Pashley, 1984) and that, in fed rats, where gastric emptying was delayed by the presence of dietary fat, fluoride absorption was increased (McGown and Suttie, 1974). While there was a disease-related increase in fluoride retention in the diabetic rats exposed to 5 parts/106 fluoride, the physiological handling of fluoride by the control and diabetic rats treated with 15 or 50 parts/106 fluoride was not markedly different. However, because of their increased fluoride ingestion, there were significantly greater amounts of fluoride in all monitored body tissues of the fatty diabetic rats than in their corresponding lean controls. The faecal fluoride excreted by the control and diabetic rats that were given fluoride-free drinking water was not significantly different from the amount

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A.J. Dunipace et al.

of fluoride they were estimated to have ingested in their food. These data indicate that fluoride in the food was not bioavailable. Urinary excretion of fluoride by these rats was most likely due to the slow mobilization and elimination of endogenous fluoride that had accumulated in their developing skeleton during gestation and before weaning (Weddle and Muhler, 1955). Before these animals were received at 5 weeks of age and placed on the low-fluoride diet, they had been exposed to the high-fluoride (30.0 parts/106) Purina 5008 Formulab Chow diet routinely fed by the supplier (Peterson et al., 1990b). For the fluoride-treated animals, the soft tissue: plasma fluoride concentration ratios ranged from 0.79 + 0.06 to 1.69 + 0.54 for the liver, and were between 3.98 + 0.55 and 7.81 _ 1.76 for the kidney. These ratios were not significantly affected by animal class, fluoride exposure or treatment duration. For both tissues, these data were somewhat higher than previously reported (Whitford, 1990; Dunipace et al., 1995). We have no explanation for these differences other than that they may relate to analytical variation or to the unusual strain of rats used here. Comparison of our control data and those from a recent investigation of chronic fluoride effects in ageing Sprague-Dawley rats (Dunipace et al., 1995) revealed that, even though fluoride intake, excretion and balance were generally similar in both studies, there were strain-specific differences in fluoride sensitivity. In spite of the substantial increase in their body burden of fluoride, there was no evidence that the higher plasma and tissue fluoride concentrations had any detrimental effects in the diabetic rats. Fluoride was not a factor in their growth or mortality. In autopsy reports prepared for each of the 14 diabetic animals that died during this study, death was primarily attributed to general washing, sepsis and/or kidney complications with subsequent heart failure. These conditions are all characteristic complications of diabetes (Daniels and Goetz, 1991; Davidson and DiGirolamo, 1991; McGowan, 1991). As tissues are damaged by disease or toxic conditions, cellular alterations occur that result in detectable changes of various constituents in blood and urine, and assay of these components is used as a means of diagnosing changes in tissue physiology or function. Our analysis of a series of 'wellness' markers was based on the premise that if fluoride in the body was causing toxic effects, significant, fluoride dose-related changes would occur in some of these variables; individually or in combination, changes in their values could indicate tissue or cellular damage and altered metabolic or physiological status. Cellular damage, particularly in the heart or liver, can result in elevated concentrations of glutamate oxaloacetate transaminase, while altered liver status can manifest as an elevated serum bilirubin or hypoalbuminaemia. Chronic renal failure is routinely indicated by an increased blood urea nitrogen and can result in reduced blood protein concentrations as well as a

decrease in urinary creatinine excretion resulting from a reduction in glomerular filtration rate. While alkaline phosphatase is present in numerous tissues, an increase in its concentration in the blood is frequently indicative of rapid bone regeneration or may occur in cases of altered liver function. Most of the clinical variables monitored were significantly different in the diabetic and control animals, but these differences reflected the disease characteristics of diabetes rather than detrimental effects of fluoride. An elevation of the blood glucose is the defining condition of diabetes. The increase in glutamate oxaloacetate transaminase and bilirubin, and the hypoalbuminaemia, in the diabetic rats probably reflected altered heart and liver function. While impaired renal function is a common complication in diabetes (Daniels and Goetz, 1991), creatinine clearance was not significantly different between the two animal classes, indicating that the diabetic rats did not experience renal insufficiency. Rather, the fall in urine urea concentration (mg/dl) in the diabetic animals was a result of osmotic diuresis; when urine volume was taken into account, total urea excretion averaged 433.1 ___6.9 and 914.0 + 21.8 mg/day for the control and diabetic rats, respectively. Osmotic diuresis also results in a reduction in plasma volume, which explains the significant increase in blood urea nitrogen concentration in the diabetic animals. Plasma alkaline phosphatase, which was significantly elevated in the diabetic rats, might reflect the sustained rate of bone resorption reported to occur in diabetes (Khokler, 1993). In spite of these disease-related changes in most of the measured clinical variables, fluoride, even at the high tissue concentrations achieved in the diabetic animals, failed to have a significant effect on any of the markers of physiological 'wellness' listed in Table 4. 'There also were no dose-response effects of fluoride on total bilirubin or alkaline phosphatase in either animal class. Two of the clinical markers were affected by fluoride in the diabetic but not in the control animals: blood cholesterol and total protein levels were significantly decreased and increased, respectively, in diabetic animals exposed to 50 parts/106 fluoride. The reason for the reduced cholesterol levels is unclear, but this effect of fluoride has also been observed recently in individuals living for long periods of time in a high-fluoride (4.8 parts/106) area of China (Li et al., 1995). The mechanism by which fluoride influenced the increase in total protein concentration is also unknown. While the plasma albumin decreases in diabetes, as was observed here, lipoproteins, globulins and a number of additional blood proteins increase, and these elevations are markedly aggrevated by poor control of the blood glucose (McMillan, 1991). It is possible that the high plasma fluoride concentrations obtained in diabetic animals exposed to 50 parts/106 fluoride may in some way have intensified the metabolic disturbances that occur in

Chronic fluoride exposure in diabetic rats diabetes and are responsible for an increase in total plasma protein. The potential genotoxic effect of chronic fluoride exposure was investigated by analyses of sister chromatid exchange: the reciprocal interchange of D N A between sister chrornatids as a result of chromosome damage, easily visuailized in metaphase chromosomes (M2 ceils) in which sister chromatids have been differentially labelled~ with 5-bromo-2'-desoxyuridine. There is reported to be a direct relation between the mutagenicity of an a~gent and its effect on increasing the incidence of sister chromatid exchange, and this exchange is a much more sensitive index of chromosome changes than are gross chromosomal aberrations (Latt, 1974; Perry and Evans, 1975). Our data showed that chronic exposure to fluoride, at concentrations as high as 50 parts/106 in drinking water, had no effect on the frequency of bone-marrow sister chromatid exchange in either the control or diabetic animals. There are reports that fluoride is mutagenic and causes an increase in the frequency of sister chromatid exchange when tested in a variety of cell systems (He et al., 1983; Tsutsui, Susuki and Ohmori, 1984; Caspary et al., 1987). However, our data support the results of other cell-culture studies (Thomson, Kilanowski and Perry, 1985; Li et al., 1987) and also those of the majority of in vivo studies, including four studies of chronic fluoride exposure, which have found that fluoride does not increase the frequency of sister chromatid exchange (Kram et al., 1978; Thomson, Kilanowski and Perry, 1985; Tong et al., 1988; Li et al., 1989; Dunipace et al., 1995). Our results also agree with data from two recent studies of the effects of chronic fluoride exposure on the frequency of sister chromatid exchange in human blood lymphocytes. These studies concluded that neither long-term exposure to 4-5 parts/106 fluoride in drinking water ,(Li et al., 1995) nor chronic, therapeutic treatment of osteoporosis with high (23 mg F) daily doses of fluoride (Jackson et al., 1994) had genotoxic effects in humans. Even though our diabetic animals rel~ained far greater amounts of fluoride in their tissues, they experienced no genotoxic risk as a result of fluoride exposure. For the most part, the histological changes in tissue specimens from representative 6-month animals were also unrelated to fluoride exposure. Enlargement of the kidneys, proteinuria and the chronic, renal interstitial inflammation observed in the diabetic animals are all hallmarks of diabetic nephropathy (Daniels and Goetz, 1991). Hepatic glycogen depletion, also observed in the diabetic animals, characteristically occurs in diabetes where insulin deficiency drastically reduces glucose entry into insulin-sensitive cells and glycogenolysis occurs in the liver, converting glycogen to glucose (Lilly, 1980). The one histological variable on which fluoride did exert an effect in the diabetic animals was the width of the cortex in the tibia. Loss of bone mass and density are characteristic of both experimental and

201

human diabetes (Khokher, 1993) and were evident in our diabetic animals in which the diaphyseal bone width was significantly less than in controls treated with 0, 5 or 15 parts/106 fluoride. However, in diabetic rats exposed to 50 parts/106 fluoride, the width of the tibial cortex was similar to that of the corresponding control group, indicating that, at high concentration, fluoride stimulated bone deposition in these animals. While there was evidence of skeletal fluorosis in both classes of animals, the diabetic rats were not at increased risk of fluorosis, in spite of retaining greater amounts of fluoride in mineralized tissue. In 1990, the National Toxicology Program reported the occurrence of bone osteosarcomas in four male rats (4/130 for 3.1%) exposed for 2 yr to 45 or 79 parts/106 fluoride in their drinking water (NTP, 1990). While this incidence of osteosarcomas was not significantly different (p > 0.05) from that in the concurrent control animals in that study, a Review Panel concluded that the NTP study data provided equivocal, or uncertain, findings on the carcinogenic activity of fluoride (PHS, 1991). Three of the bone neoplasms detected in the NTP study were vertebral in origin, and the fourth occurred in a proximal humerus. Three of the tumours were initially observed upon gross examination, and all four were visible on radiographs. The maximum mean fluoride concentration in bone samples from those fluoridetreated rats was reported as 6200/~g/g ash. By comparison, bone fluoride in our diabetic rats treated with 50 parts/106 fluoride for 6 months reached mean maximum concentrations in excess of 8000 and 9000/1 g/g ash in femur and vertebra, respectively. We did not make gross or microscopic examinations of the vertebral specimens, but radiographs of femur samples from all control and diabetic rats, and histological examination of representative tibial specimens, found no neoplastic lesions. While the results of animal studies must be applied to human populations with caution, animal models are a valuable tool for studying the beneficial and toxic effects of fluoride in man (Richards, 1990). Our results support the original hypothesis that, for any given level of fluoride exposure, rats 'medically compromised' by diabetes will retain significantly greater amounts of fluoride. However, while treatment of the diabetic animals with 50 parts/106 fluoride significantly altered their blood cholesterol and total protein levels, and increased cortical bone formation in the tibia, there was no evidence that any of the levels of fluoride exposure tested had clinically relevant effects on the physiological, biochemical or genetic variables measured here. Acknowledgements--We thank Dr Kenneth W. Ryder, Pro-

fessor of Pathology, IU School of Medicine, and his staff for doing the clinical chemistry assays. We also gratefully acknowledge Dr Richard G. Peterson, Professor of Anatomy, IU School of Medicine, for valuable consultation on the animal model; Mr Mark D. Roth for conducting blood

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glucose analyses; Ms Shirley E. Shazer for processing the histological specimens; and Ms Marilyn J. Richards for her patient assistance during the development of this manuscript. This investigation was supported by USPHS Research Grant P01 DE-09835-01 from the National Institute of Dental Research, National Institutes of Health, Bethesda, MD 20892, U.S.A. REFERENCES

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