The effects of fluoridated water on rat urine and tissue cAMP levels

THE

EFFECTS

OF FLUORIDATED WATER ON RAT URINE AND TISSUE CAMP LEVELS H. S. KLEINER and D. W. ALLMANN

Department of Biochemistry. Indiana University School of Medicme Indianapolis. IN 46223. U.S.A Summary---Male Wistar rats were fed a fluoride deficient diet (co.5 parts/lob F). and either distilled water or fluoridated water (1.0 parts/106). By week 3. the control group had urinary excretions of 106 i 5 nmol CAMP/day (mean & SEM) whereas the experimental group excreted 129 + 6 nmol CAMP/day. After 1I I days, the control group excreted 270 + 26 nmol CAMP/day compared to 600 + 78 nmol CAMP/day for the experimental group. Body weight. food and water consumption, urine volume, and urinary creatinine and phosphate levels were not significantly different between the two groups. Tissue CAMP levels were determined after 4. 6 and 16 weeks. By week 4, the rats receiving the fluoridated water had significantly higher levels of CAMP in the liver (1 I3 per cent) tibia (130 per cent), femur (89 per cent) and heart (35 per cent). At week 6, the liver (119 per cent), tibia (296 per cent). heart (I 68 per cent), kidney (73 per cent) and submandibular gland (27 per cent) had significantly higher levels of CAMP. By week 16, the liver, femur, kidney and submandibular gland continued to have elevated levels of CAMP. Liver glycolytic metabolites were determined after 6 weeks, and the results suggested a decrease in pyruvate kinase activity. INTRODUCTION Fluoride inhibits a number of metabolic enzymes in citro (Wiseman, 1970). In broken-cell preparations (Sutherland, Rail and Menon. 1962) and in intact hepatocytes (Benac and Allmann, 1975; Shahed et al., 1979), fuoride causes an increase in CAMP production, presumably by stimulating adenylate cyclase (Martin et ~11..1980: Shahed, Chalker and Allmann, 1980). Because CAMP regulates a number of intracellular processes, any agent that stimulates CAMP production could alter intracellular metabolic and physiologic activity. Due to the ubiquitous nature of fluoride as well as our daily exposure to fluoride through oral hygiene products, fluoride additives to drinking water, and industrial fluoro-pollutants, it is important to determine if fluoride alters CAMP levels in ciao. There are conflicting reports about the effect of subtoxic doses of NaF on CAMP production in whole animals. Edgar, Jenkins and Prudhoe (1979) found a slight increase in urinary CAMP in human subjects as a result of an oral dose of 1 I mg NaF. Mornstad, Sundstrom and Hedner (1975) and Isenberg and Allmann (1976) found increases in urine CAMP levels as a result of subjects receiving oral doses of fluoride. In contrast, Wong. Singer and Ophaug (1979) and Ophaug, Wong and Singer (1979) fed fluoridated water (50 and 25 parts/IO6 respectively) to rats and found no change in either urine or tissue CAMP levels. Our purpose was to extend those experiments using a lower concentration of F in the water (1.0 parts/lo?.

MMTERIALSAND METHODS Sixteen male, Wistar rats were housed in metabolic cages in a constant temperature-humidity room with

a 12 h light-l 2 h dark cycle. Rats were separated mto 2 groups with 8 rats in each group. They had been fed a commercial diet from weaning to being housed in our animal facility. The rats were then fed the fluoridedeficient diet (IU 500 diet) (Allmann, Mapes and Benac. 1975) which contains less than 0.5 parts/lo6 fluoride for 2 days before the start of the experiment. The control groups received distilled water and the experimental groups received fluoridated water (NaF) at a concentration of 1.0 parts/lOh fluoride. The data were obtained from 3 different sets of animals. The initial weight of the rats in the 4wk experiment was 67.7 + 3.4 g, for the 6 wk experiment the rats weighed 71.6 k 2.4g. and for the 16 wk experiment the rats weighed 85.4 f 5.2g. Urine was collected over a 24 h period beginning each morning. Concentrated HCI (20/t] per ml expected urine) was added to the collecting bottles prior to the collection to give a final concentration of about 0.1 M. The acid was found to stabilize the CAMP in the urine. presumably by preventing bacterial growth. The experimental periods lasted for 4. 6 or 16 wk. The rats were stunned, the abdominal cavity opened and their livers were then removed and freezeclamped in less than 8 s by the method of Wollenberger. Ristan and Schaffeer (1960). Other tissues were removed, frozen in liquid NZ, and stored at -20 C. Urine phosphate was determined according to the procedure of Fiske and Subbarow (1925). Urinary creatinine was determined by the method of Jaffe (1886) with some modifications of the procedure suggested by Lynch ef ul. (1963). Urine fluoride was assayed using an Orion fluoride specific electrode. Urine CAMP was determined by a CAMP binding protein assay described by Gilman (1970a. b) in a total assay volume of 501~1. The CAMP-binding protein (bovine heart) and protein-kinase inhibitor protein (bovine heart) were purchased from Sigma Chemical Company (St. Louis. MO.). 107

108

H. S. Kleiner

and D. W. Allmann

Perchloric acid extracts of liver were neutralized with KOH and assayed for 3-phosphoglycerate, 2-phosphoglycerate and phosphoenolpyruvate (Bergmeyer, 1963) and pyruvate and lactate (Hohorst, Kreutz and Bucher, 1959). Liver glycolytic metabolites were assayed the same day that the rats were killed. Tissue CAMP was purified and assayed as desribed by Shahed et al. (1979b). Statistical probability for all data was determined by the Student’s t-test which was used to determine the difference between means for 2 independent (unpaired) groups of data assuming homogeneity of population variance.

rate, and unlike some urine components (e.g. urea), daily creatinine is not influenced by urine volume or dietary protein (Davidson and Henry, 1974; Halsted, 1976). Thus, creatinine excretion is often used as a reference value for expressing other urinary constituents. We determined urine CAMP over several weeks, and while urine CAMP levels increased per day, urine creatinine also increased from an average of 1.9 mg creatinine per day at day 0 to an average of 6.4mg creatinine per day at day 33. Expression of the data in nmol cAMP/mg creatinine would result in an inverse relationship between urine CAMP and age. As the increase in creatinine excretion closely paralleled the increase in body wt. creatinine,/body wt provided a more consistant baseline from which to compare CAMP excretion. The nmol cAMP/mg creatine/kg body wt was determined for each rat. The results (Table 1) indicated that on day 20, there was a significant increase in urinary CAMP in the experimental group (4.47 nmol cAMP/mg creatinine/kg body wt) compared to the control group (3.66 nmol cAMP/mg creatine/kg body wt). Thus the rats receiving the fluoridated water exhibited a significant increase in urinary CAMP whether the CAMP levels were compared directly or expressed as cAMP/creatinine,body wt. The rats receiving the fluoridated water for up to 16 weeks showed no statistically significant differences in body weight, food consumption, water consumption, urine volume, urinary creatinine or urinary phosphate compared to the control group (data not presented). However, by the 3rd wk the rats receiving fluoridated water exhibited a significant increase (23 per cent) in urinary CAMP (Table 2). At the time of killing, the urinary CAMP levels in rats receiving the fluoridated water were 122 per cent higher than the control group. The data in Tables 1 and 2 suggested that consumption of 1.0 parts/lOh fluoridated water resulted in an increase in urinary CAMP excretion, but did not indicate the mechanism by which there is an increase in urine CAMP. Factors which may account for the increase in urinary CAMP include a decrease in phosphodiesterase activity, an increase in the glomerular

RESULTS

In the first set of experiments, rats received a fluoride deficient diet and either distilled water or 1.0 parts/lo6 fluoridated water for 33 days. The rats in both the control and experimental group gained about 150 g and showed an increase in both food and water consumption with no significant difference between the two groups, suggesting that they were healthy; thus any differences observed between the two groups were not the result of malnutrition. At the beginning of the study, the rats receiving fluoridated water consumed about 22pg fluoride per day. This increased to roughly 26pg per day by day 33. The difference in fluoride consumption of the control and the experimental group was evident by a difference in urinary fluoride (Table 1). By day 20, the experimental group began to exhibit a significantly higher level of urinary fluoride (8.8 + 0.4 pg/day) compared to the control (6.2 f 0.3 pg/day). At the same time, urinary CAMP levels also began to differ. By day 20, a significant increase (26 per cent) in urinary CAMP was observed in rats receiving the fluoridated water as compared to the controls (Table 1). The experimental group continued to show significantly elevated levels of urinary CAMP until they were killed. The differences in CAMP excretion was further substantiated by the comparison of the CAMP excreted to the creatinine/body wt. Creatinine is normally excreted in the urine at a relatively constant

Table 1. Effect of fluoridated

Day 1 6 13 20 23 27 33

Fluoride? - NaF + NaF 8.6 + 0.4

8.0 + 0.3

8.4 6.2 5.8 3.6 4.4

8.1 8.8 9.0 9.3 9.5

f f + f +

0.5 0.4 0.4 0.3 0.4

f f f f k

0.5 0.5* 0.5* 0.6* 0.6*

water on urine, fluoride, creatinine

Creatininex - NaF + NaF 23 20 22 23 24 24 23

t_ * + + + k &

2 2 2 2 2 2 2

23 20 22 23 23 24 22

k + * f f k +

The results represent the mean + SEM for 6-7 rats. *p < 0.05. tpg Fluoride/day. Smg Creatinine/day/kg body wt. §nmol CAMP/day. jlnmol cAMP/mg creatinine/kg body wt.

2 2 2 2 2 2 2

and CAMP

cAMP$ -NaF 52 59 80 84 82 80 107

* f + k & + +

+ NaF 3 1 1 5 6 2 5

41 71 83 106 112 92 127

+ 4 *5 $ 4 f 5* * 9* + 6* + I*

cAMP/creatininell -NaF + NaF 2.2 2.8 3.7 3.7 3.2 3.4 3.7

_t 0.1 _+ 0.5 & 0.1 + 0.2 * 0.2 _+ 0.1 f 0.2

2.0 3.6 3.9 4.5 4.5 4.0 4.3

* f * * f * +

0.1 0.3 0.2 0.2* 0.2* 0.2* 0.1*

109

Fluoridated water and tissue CAMP

in the heart (40 per cent), femur (90 per cent), liver (110 per cent) and tibia (130 per cent) (Table 3). The kidney was the only tissue examined that showed no significant increase in CAMP at 4 wk. However, by the 6th wk. the kidney (70 per cent), submandibular gland (30 per cent), liver (120 per cent), heart (170 per cent) and tibia (300 per cent) had significantly elevated levels of CAMP. After 16 wk of fluoridated water, the experimental group continued to show significantly elevated levels of CAMP in the liver, femur, kidney and submandibular gland compared to the control group. With evidence of an increase in liver CAMP levels, liver glycolytic metabolites were assayed to determine possible changes in glucose metabolism. After 4 wk. the rats fed fluoridated water had significantly higher levels of liver 3-phosphoglycerate (47 per cent) and 2-phosphoglycerate (75 per cent). At 6wk (Table 4) the rats receiving NaF had significantly higher levels of 3-phosphoglycerate (57 per cent). 2-phosphoglycerate (18 per cent) and phosphoenolpyurvate (62 per cent), and significantly lower levels of pyruvate (18 per cent) and lactate (32 per cent). The data from Table 4 was plotted (Fig. 1) as a percentage of the control for the rats receiving NaF which showed that there was a cross-over between phosphoenolpyruvate and pyruvate. The cross-over is an indication that the activity of pyruvate kinase was decreased in those rats receiving NaF.

Table 2. Effect of fluoridated water on ruine CAMP excretion for 16 weeks Days

+ NaF

- NaF

nmol CAM P/day I

5 16 20 26 49 70 85 111

65 69 76 106 92 340 282 331 270

+ + ? & + + * * +

6 10 9 5 9 76 12 38 26

77 * II 71*4 127 & 22 129 +_ 6* 142 + 12* 613 k 14* 640 k 64* 895 -+_120* 600 + 78*

The results represent SEM for 7-8 rats. * p < 0.05.

the mean If:

filtration rate. or an increase in a tissue adenylate cyclase activity. To determine if the elevated levels of urinary CAMP were associated with increased intracellular levels of CAMP, tissue CAMP was also measured. Rats receiving 1.0 parts/lo’ fluoridated water for 4 wk had significantly elevated CAMP levels

Table 3. Effect of fluoridated

water on rat tissue CAMP levels

4 Weeks Tissue

-- NaF

16 Weeks

6 Weeks +NaF

+ NaF

- NaF

- NaF

+NaF

567 + 22

893 k 64*

pmol CAMP/g wet wt Liver Tibia Femur Heart Kidney Submandibular Gland

530 1070 565 2480 2320

The results represent * p < 0.05.

+ 52 _t 90 + 55 -+_100 + 110

1130 2450 1070 3440 2500

_t k + + +

60* 250* 15tY 80* 80

the mean + SEM for 68

Table 4. Effect of fluoridated

Metabolite

515 f 35 1140 + 340 -1890 +_ 350 1930 * 200 1930 + 170

1130 * 140* 4510 & 930*

322 1020 955 1290

5070 f 450* 3330 + 540* 2460 _t 40*

(3PG) (2PG) (PEP)

63 30 38 90

rats.

water on rat liver metabolites weeks - NaF

89 f 10056 77 * 390 + 2100 *

4 5 16 170

The rats are the same as the 6 wk experiment * p < 0.05.

after 6

+NaF

nmol metabolite/g 3-Phosphoglycerate 2-Phosphoglycerate Phosphoenolpyruvate Pyruvate (Pyr) Lactate (Lac)

I * * k

140 118 125 320 1440

wet wt + 5 + + &

5* 3* ll* 32* 150*

listed in Table 3.

5770 994 1060 2050

f k + k

1320* 64 lO* 1 lO*

110

H. S. Kleiner and D. W. Allmann

-6

o-

3

2

:

:

P E P

WEEKS

P Y R

L A C

Fig. 1. The liver metabolite data in Table 4 is plotted percentage of the control (distilled water group). abbreviations are described in Table 4.

as a The

DISCUSSION

One part/lo’ was selected because communities fluoridate their water adjust the final fluoride concentration from 0.8 to 1.2 parts/lo6 (Katz, McDonald and Stookey, 1979). The fluoride-deficient diet was selected because Weddle and Muhler (1954) reported that commercial diets may contain up to 50 parts/lo6 fluoride. By using a diet of known composition, it is more likely that the biochemical changes observed are due to the effects of the fluoride supplement. Also, by using a fluoride-deficient diet, the amount of fluoride exposure can be controlled by regulating the amount of added fluoride. Although there was an increase in urinary excretion of phosphate, creatinine and urine volume with age, there was no significant difference between the control and experimental group (data not presented). Polyuria (Franscino, 1972; Roman et al.. 1977) and phosphaturia (Suketa, Mikami and Yammoto, 1976; Suketa and Mikami, 1977) which are characteristic signs of fluorosis, were not evident. Thus, the significant increase in urinary fluoride and urinary CAMP in the rats receiving the fluoridated water was not a result of kidney malfunction. All the rats used had been fed a commercial chow diet from the time of weaning until the rats were placed on the fluoride deficient-diet, so that while on the fluoride-deficient diet, fluoride would be released from the skeleton. After about 2 wk the control rats were excreting less fluoride than on day 1 and continued to excrete less fluoride throughout the experiment. The experimental group excreted the same amount of fluoride throughout the length of the experiment since they were consuming fluoride. The rats receiving fluoridated water began excreting significantly more urine CAMP (Tables 1 and 2) after about 3 wk. The increase in urinary CAMP occurred at about the same time as when the rats receiving fluoridated water were excreting significantly more fluoride. The increase in urinary CAMP excretion was evident

for up to 16 wk in rats receiving fluoridated water, at which time they were excreting 122 per cent more CAMP than the controls. It is possible that had we used weaning rats, i.e. rats that had not received any dietary fluoride, the experimental group might have excreted significantly higher urinary CAMP in less than 3 wk. A fluoride-induced stimulation of adenylate cyclase could cause an increase in CAMP production with a concomitant increase in urinary CAMP. Similar effects are well documented with glucagon (Broadus et ul., 1970a; Taylor et al., 1970). According to Broadus ef ul. (1970b), about two-thirds of urine CAMP is derived from glomerular filtration and one-third from the kidney. If fluoride stimulates an increase in tissue CAMP, this might produce an increase in plasma CAMP which would result in an increase in the filter load. We selected the liver and kidney because Allmann and Kleiner (1980) reported that these tissues have increased CAMP levels after experimental animals received intraperitoneal injections of NaF. We selected the tibia and femur because over 95 per cent of stored fluoride is sequestered in skeletal tissue (Hodge, 1961). and the heart because fluoride toxicity can produce heart damage (Jansen and Thompson, 1974). The submandibular gland was assayed because CAMP is suspected of stimulating amylase secretion (Schramm and Naim, 1970). By week 6, the rats receiving fluoridated water had significantly higher levels of CAMP in all tissues than the controls. The femur, which was not assayed for CAMP on week 6, showed high CAMP levels at weeks 4 and 16. It is to be noted that Sundstrom (1972) described morphological alterations in the femoral cortices in rats raised on 1.0 and 5.0 parts/lo6 fluoridated water. The higher urinary and tissue CAMP levels suggest that NaF stimulates CAMP production in ciao. The mechanism of action by which low, physiologic doses of fluoride might stimulate adenylate cyclase is not known. However, it is possible that low concentrations of fluoride bind to the enzyme and produce an irreversible activation as is seen in intact hepatocytes (Shahed et ul., 1980a). The cross over observed between phosphoenolpyruvate and pyruvate (Fig. 1) at week 6 suggests a decrease in the activity of pyruvate kinase. These data do not rule out a possible inhibition of enolase because the enzyme activities were not measured. Fluoride added to isolated hepatocytes causes inhibition of pyruvate kinase and enolase (Shahed, Miller and Allmann, 1979a; 1980b). Shearer and Suttie (1969) report finding a decrease in pyruvate kinase activity with rats fed 450 or 600 parts/lo’ fluoride in their diet but attribute the decrease to low food consumption. According to Seubert and Schooner (1971). liver pyruvate kinase is regulated by the nutritional state of the animal, a fed-state enhancing activity. The rats in our study, however, showed no reduction in body weight, food consumption, or water consumption, suggesting that the low pyruvate kinase activity was not a result of starvation, but more probably the effect of higher tissue CAMP levels which can stimulate phosphorylation of the enzyme. Evidence for inhibition of pyruvate kinase by CAMP is reported by a number of investigatiors in in vitro studies (Harris, 1975; Pilkis et ul., 1975; Blair et a/., 1976). Our inves-

Fluoridated

111

water and tissue CAMP

tigation showed that fluoride altered the pyruvate kinase activity in uivo. The exact mechanisms by which NaF alters glucose metabolism still remains to be elucidated. Acknowlengrmenrs~This investigation was supported in part by the Grace M. Showalter Trust and NIDR grant DE-04387.

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