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32(1) 1999    
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Research Report

EFFECTS OF SUBTOXIC DOSES OF FLUORIDE ON SOME ENZYMES OF THE GLUCOSE METABOLISM IN SUBMANDIBULAR SALIVARY GLANDS OF FED AND OVERNIGHT-FASTED RATS

M V da Motta,* D N de Souza,* J Nicolau*
São Paulo, Brasil

SUMMARY: Effects of subtoxic doses of NaF on phosphofructokinase-1 (PFK-1), hexokinase (HK), pyruvate kinase (PK), glucose-6-phosphate dehydrogenase (G6PD) and lactate dehydrogenase (LDH) in the submandibular salivary glands of fed and overnight-fasted rats were studied. After NaF injection, fed animals showed increased activity of PFK-1 in the glands at 1 hour and reduced activity was observed at 3 and 6 hours. In overnight-fasted rats, PFK-1 exhibited increased activity in all groups. The activity of HK increased in fed animals at 12 hours after one dose and at 24 hours after two doses. One hour after NaF injection in fed animals, the activity of PK increased. The activity of G6PD increased in the fed group 12 hours after one injection and decreased in the fasted group after 3 and 6 hours. The activity of LDH increased only in the fed group 3 hours after NaF injection. We conclude that the effect of fluoride on these enzymes in the submandibular glands differs depending upon the nutritional state of the animals.

Key words: Fluoride injections, Fluoride in rats, Glucose metabolism, Rat saliva, Salivary enzymes, Submandibular glands.

*Oral Biology Research Center, Faculty of Dentistry USP,
Av. Prof Lineu Prestes 2227-05508-900, São Paulo, Brasil.

INTRODUCTION

An increase in dental fluorosis is occurring in developed countries due to excessive ingestion of fluoride, mainly from formulations intended for topical action.1-3 As with any pharmacological agent, the magnitude of metabolic effects of fluoride depends on various factors, such as the concentration in the blood and the time necessary for excretion. The plasma concentration 30 min. after ingestion reveals rapid absorption and generalized distribution.4

At the cellular level, as an enzymatic inhibitor, there are conflicting reports on the action of fluoride on adenyl cyclase.5-7 Increased salivation in fluoride-intoxicated individuals4,8 as well as secretion of fluoride ion by salivary glands,9 led some researchers to examine possible metabolic alterations in salivary glands, due to fluoride action.

Some reports point to a fluoride action on the salivary glands via adenyl cyclase.10,11 However, by examining the effect of fluoride on glycogen metabolism in the submandibular glands, we verified that the action of this element depends on the animal nutritional state,12 and does not necessarily stimulate this system.

The purpose of the present investigation was to extend our studies on the effect of sub-toxic doses of fluoride on submandibular salivary glands by examining enzymes of the carbohydrate metabolism of fed and overnight-fasted rats.

MATERIALS AND METHODS

Male Wistar rats weighing 175-220 g either fed ad libitum or denied food overnight (15 hours) were injected with 0.2 mL NaF solution, equivalent to 5 or 10 mg F-/kg body weight (EXP, experimental) or isovolumetric amounts of saline (C, control). After the animals were sacrificed by cranial traumatism, the submandibular salivary glands were immediately removed, clamped between aluminum tongs, pre-cooled in dry ice, weighed avoiding thawing, and homogenized at 10% (W/V) in a Potter type homogenizer with a teflon pestle. The homogenizing medium contained 50 mmoles/L imidazole buffer pH 7.0, 1 mmol/L beta-mercaptoethanol and 2 mmoles/L EDTA. The homogenates were centrifuged at 27,750 x g for 20 min. in a refrigerated centrifuge and the supernatants used for enzymatic and protein determinations.

Enzyme Assays

Enzyme activities of various supernatants were assayed by changes in absorption at 340 nm due to reduction of NADP or oxidation of NADH. The reaction was initiated by adding aliquots of homogenate supernatant to the reaction mixture in the cuvette, and the absorbance was monitored for 10 min. at 25°C in a Beckman DU 68 spectrophotometer, using a 1-mL cuvette with 1-cm path length. One unit of enzyme activity corresponds to the amount of enzyme that converts 1 µmol of the substrate per min.; specific activity is expressed in U/mg of protein. Enzyme activities (hexokinase, HK; phosphofructokinase-1, PFK-1; pyruvate kinase, PK; lactate dehydrogenase, LDH, and glucose-6-phosphate dehydrogenase, G6PD) were measured according to the methods described elsewhere.13

RESULTS

The results obtained in this investigation are expressed in Tables 1 to 6. When fluoride was injected into fed animals, observations included an increased activity of PFK-1 at 1 hour and a reduced activity in the glands of rats sacrificed at 3 and 6 hours after injection (Tables 1 and 2). The activity of HK increased at 12 hours after one dose and 24 hours after two doses of fluoride (Tables 1 and 2). The activity of PK increased only 1 hour after one injection. The activity of the other two enzymes, G6PD and LDH, increased at 12 hours for G6PD and at 3 hours for LDH, after one injection of fluoride (Table 3).

With overnight-fasted rats a different pattern was observed. Only PFK-1 (Table 4) and G6PD (Table 5) showed variation. The activity of PFK-1 increased in all groups studied, and the activity of G6PD decreased in the groups examined 3 and 6 hours after the injection of 10 mg F-/kg body weight. Table 6 shows that blood glucose increased in the groups studied 1 and 3 hours after one injection of fluoride.

Tables 1 and 2.  Specific activity of hexokinase (HK), phosphofructokinase-1 (PFK-1) and pyruvate kinase (PK) in submandibular glands of fed rats injected with a single dose and two doses of fluoride (5 or 10 mg F-/kg body weight) (EXP) or NaCl solution (C), and sacrificed at the indicated times after injection.


Table 1.  5 mg F-/kg body weight
Groups HK (mU/mg protein) PFK-1 (mU/mg protein) PK (U/mg protein)
  (Hours) C EXP C EXP C EXP
1 1 H (12) 13.11
± 1.34		 (10) 13.26
± 1.70		 (6) 16.63
± 0.71		 (7) 23.20
± 1.90*		 (13) 0.22
± 0.03		 (14) 0.27
± 0.03*
  3 H (11) 13.13
± 2.13		 (8) 13.51
± 2.69		 (11) 16.11
± 1.69		 (19) 13.90
± 2.61*		 (9) 0.22
± 0.03		 (15) 0.22
± 0.04
  6 H (12) 13.75
± 1.78		 (16) 14.63
± 1.57		 (8) 16.60
± 2.03		 (17) 13.94
± 2.52*		 (15) 0.22
± 0.04		 (10) 0.23
± 0.03
  12 H (9) 14.89
± 2.21		 (11) 18.34
± 2.70*		 (12) 16.25
± 1.22		 (15) 15.55
± 2.19		 (10) 0.22
± 0.04		 (11) 0.24
± 0.04
2 24 H (12) 13.04
± 2.61		 (11) 16.58
± 1.49*		 (7) 15.88
± 1.98		 (13) 15.91
± 2.95		 (8) 0.22
± 0.03		 (9) 0.21
± 0.03
Values are mean ± S.D. for the number of samples indicated in parenthesis. The asterisks mean statistically significant by the Student's "t" test (p < 0.01).


Table 2.  10 mg F-/kg body weight
Groups HK (mU/mg protein) PFK-1 (mU/mg protein) PK (U/mg protein)
  (Hours) C EXP C EXP C EXP
1 1 H (13) 13.11
± 1.34		 (14) 13.72
± 2.99		 (6) 16.63
± 0.71		 (7) 21.79
± 3.17*		 (14) 0.22
± 0.03		 (13) 0.25
± 0.03*
  3 H (10) 13.14
± 2.13		 (8) 12.79
± 0.99		 (11) 16.11
± 1.69		 (16) 13.54
± 2.92*		 (9) 0.22
± 0.03		 (17) 0.23
± 0.03
  6 H (12) 13.74
± 1.78		 (14) 13.72
± 2.11		 (8) 16.60
± 2.03		 (15) 13.95
± 2.94*		 (16) 0.22
± 0.04		 (13) 0.22
± 0.04
  12 H (9) 14.89
± 2.21		 (7) 18.77
± 1.67*		 (14) 16.25
± 1.22		 (17) 17.59
± 2.18		 (9) 0.22
± 0.03		 (20) 0.23
± 0.02
2 24 H (12) 13.06
± 2.61		 (12) 17.23
± 1.36*		 (7) 15.88
± 2.95		 (12) 15.91
± 2.95		 (8) 0.22
± 0.03		 (12) 0.21
± 0.02
Values are mean ± S.D. for the number of samples indicated in parenthesis. The asterisks mean statistically significant by the Student's "t" test (p < 0.01).


Table 3.  Specific activity of glucose-6-phosphate dehydrogenase (G6PD) and lactate dehydrogenase (LDH) in the submandibular glands of fed rats injected with 5 mg F-/kg body weight (EXP5), 10 mg F-/kg body weight (EXP10) or NaCl solution (C) and sacrificed at the indicated times following the injection.
Groups G6PD (mU/mg protein) LDH (U/mg protein)
  (Hours) C EXP5 EXP10 C EXP5 EXP10
1 1 H (11) 17.95
± 2.38		 (11) 17.45
± 3.76		 (11) 18.71
± 2.84		 (12) 0.27
± 0.02		 (11) 0.26
± 0.03		 (8) 0.26
± 0.03
  3 H (9) 18.48
± 3.18		 (10) 18.08
± 2.19		 (10) 18.13
± 2.66		 (10) 0.26
± 0.03		 (11) 0.21
± 0.03*		 (8) 0.23
± 0.03*
  6 H (12) 17.18
± 3.06		 (15) 18.38
± 2.86		 (15) 18.43
± 2.42		 (16) 0.26
± 0.03		 (17) 0.25
± 0.04		 (17) 0.27
± 0.02
  12 H (12) 18.42
± 2.11		 (17) 21.89
± 2.16*		 (20) 21.90
± 2.19*		 (6) 0.26
± 0.02		 (6) 0.26
± 0.04		 (7) 0.27
± 0.02
2 24 H (12) 18.87
± 2.84		 (14) 20.61
± 1.44		 (14) 20.08
± 1.72		 (9) 0.27
± 0.02		 (7) 0.25
± 0.03		 (7) 0.26
± 0.03
Values are mean ± S.D. for the number of samples indicated in parenthesis. The asterisks mean statistically significant by the Student's "t" test (p < 0.001).


Table 4.  Specific activity of hexokinase (HK), phosphofructokinase (PFK-1) and pyruvate kinase (PK) in the submandibular glands of overnight fasted (15 hours) rats injected with 10 mg F-/kg body weight (EXP) or NaCl solution (C) and sacrificed at the indicated times after injection.
Groups HK (mU/mg protein) PFK-1 (mU/mg protein) PK (U/mg protein)
(Hours) C EXP C EXP C EXP
1 (12) 20.08
± 1.85		 (12) 19.74
± 2.36		 (9) 20.86
± 2.28		 (11) 25.34
± 2.97*		 (6) 0.29
± 0.02		 (10) 0.29
± 0.03
3 (9) 21.68
± 1.82		 (12) 22.25
± 1.75		 (9) 20.23
± 2.07		 (9) 26.17
± 3.00*		 (7) 0.31
± 0.02		 (10) 0.31
± 0.04
6 (10) 20.79
± 1.93		 (11) 20.79
± 2.16		 (9) 20.18
± 2.61		 (12) 24.27
± 3.48*		 (9) 0.30
± 0.03		 (10) 0.33
± 0.04
12 (10) 21.23
± 1.19		 (10) 22.03
± 2.56		 (9) 22.54
± 2.69		 (11) 28.91
± 3.19*		 (10) 0.30
± 0.03		 (4) 0.29
± 0.02
Values are mean ± S.D. for the number of samples indicated in parenthesis. The asterisks mean statistically significant by the Student's "t" test (p < 0.001).


Table 5.  Specific activity of glucose-6-phosphate dehydrogenase (G6PD) and lactate dehydrogenase (LDH) in the submandibular glands of overnight-fasted (15 hours) rats injected with 10 mg F-/kg body weight (EXP) or NaCl solution (C) and sacrificed at the indicated times after injection.
Groups G6PD (mU/mg protein) LDH (U/mg protein)
(Hours) C EXP C EXP
1 (8) 2.31 ± 0.31 (10) 2.55 ± 0.36 (7) 0.56 ± 0.04 (8) 0.55 ± 0.05
3 (9) 2.31 ± 0.22 (8) 2.04 ± 0.42* (7) 0.55 ± 0.04 (8) 0.55 ± 0.07
6 (11) 2.39 ± 0.29 (9) 2.17 ± 0.45* (7) 0.55 ± 0.03 (9) 0.55 ± 0.08
12 (10) 2.40 ± 0.31 (9) 2.51 ± 0.23 (6) 0.54 ± 0.03 (10) 0.54 ± 0.06
Values are mean ± S.D. for the number of samples indicated in parenthesis. The asterisks mean statistically significant by the Student's "t" test (p < 0.01).


Table 6.  Blood glucose concentration of overnight-fasted (15 hours) rats injected with 10 mg F-/kg body weight (EXP) or NaCl solution (C) and sacrificed at the indicated times after injection.
Groups Glucose (mg/100 ml blood)
C EXP
1 (5) 74.82 ± 6.38 (6) 108.42 ± 8.40*
3 (5) 72.75 ± 4.89 (5) 101.38 ± 11.51*
6 (4) 74.00 ± 4.89 (4) 78.00 ± 5.16
12 (4) 65.98 ± 1.03 (4) 67.78 ± 3.97
Values are mean ± S.D. for the number of samples indicated in parenthesis. The asterisks mean statistically significant by the Student's "t" test (p < 0.01).


DISCUSSION

Reports reveal that for the liver, fluoride has an inhibitory effect on some enzymes, such as PFK-1, PK, G6PD, enolase and phosphoglyceromutase.11,14-17 Under the same experimental conditions (excess vitamin A), we have shown that the response of the submandibular gland is different from the liver.18

In fluoride liver studies, other authors report a reduction of the glycogen content.16,19,20 However, we found that administration of fluoride to rats increased the glycogen content of the submandibular gland,12 and that the glycogenolysis depends upon the nutritional state of the animals.

Our results clearly show that the activities of both HK and PFK-1 in the submandibular gland are influenced by fluoride. Animals injected with fluoride showed increased HK activity from one dose after 12 hours, and from 2 doses after 24 hours. HK is located in a crucial point for glucose utilization by the cell, since it promotes the phosphorylation of glucose for subsequent use in glycolysis. When fluoride was injected into overnight-fasted rats, no difference in the activity of this enzyme was found compared with the control.

On the other hand, the results obtained for PFK-1 were more evident. In fed animals there was an increase in the activity of this enzyme in the groups sacrificed after 1 hour and reduction in the groups sacrificed 3 and 6 hours after fluoride injection. However, the administration of fluoride to overnight-fasted rats showed increased activity of this enzyme in all groups sacrificed after one injection. In overnight-fasted rats the injection of NaF causes a transitory hyperglycemia, thereby increasing the supply of glucose substrate for the glands. This result led us to conclude that fluoride acts on the submandibular gland of fasting animals through different mechanisms from those observed in the activation of adenyl cyclase system.

Our data agree with the hypothesis21 that fluoride acts on the membrane proton transport system through inhibition of ATPase/ATP synthesis. In this condition, only a small amount of ATP would be formed at this level, requiring, therefore, a greater action by the glycolytic pathway.

The results obtained for G6PD showed an increased activity in the submandibular glands of fed animals sacrificed 12 hours after administration of fluoride. As there was a reduction in PFK-1 activity in the groups sacrificed at 3 and 6 hours after injection of fluoride, we can conclude that G6P is being diverted to the pentose phosphate pathway. On the other hand, in overnight-fasted rats sacrificed at 3 and 6 hours following fluoride injection, there was a reduction in the G6PD activity, indicating a prevalence of the glycolytic pathway in this nutritional condition.

Concerning LDH, a bifunctional enzyme, a reduction in its activity is seen only in fed animals, sacrificed 3 and 6 hours after fluoride injection.

In conclusion, under the experimental conditions of this study, we find that fluoride acts differently on the enzyme activities of the submandibular glands, depending upon the nutritional status of the animals.


ACKNOWLEDGEMENTS

This work was partially supported by CNPq. J. Nicolau holds a fellowship from CNPq.

REFERENCES

  1. Morgan L, Allred E, Tavares M, Bellinger D, Needleman H. Investigations of the possible associations between fluorosis, fluoride exposure, and childhood behavior problems. Pediatric Dentistry 20 244-252 1998.
  2. Heller KE, Eklund SA, Burt BA. Dental caries and dental fluorosis at varying water fluoride concentrations. Journal of Public Health Dentistry 57 136-143 1997.
  3. Mascarenhas AK, Burt BA, Fluorosis risk from early exposure to fluoride toothpaste. Community Dental Oral Epidemiology 26 241-248 1998. (for abstract, see p 35 of this issue of Fluoride).
  4. Whitford GM. The metabolism and toxicity of fluoride. Myers HM editor Monographs in Oral Science. 13, Karger, Basel, 1989; 2nd revised edition (as Vol. 16), 1996.
  5. Edgar WM, Jenkins GN, Prudhoe K. Urinary cAMP excretion in human subjects following single and divided doses of sodium fluoride [abstract 35]. Journal of Dental Research 58 1229 1979.
  6. Holland RI. Cytotoxicity of fluoride. Acta Odontologica Scandinavica 38 69-79 1980.
  7. Hongslo CF, Hongslo JK, Ekstrand J, Holland RI. Cyclic AMP in urine, kidney and liver following long-term administration of fluoride to rats. Acta Pharmacologica et Toxicologica 52 276-280 1983.
  8. Duxbury AJ, Leach FN, Duxbury JT. Acute fluoride toxicity. British Dental Journal 153 64-66 1982.
  9. Oliveby A, Lagerlöf F, Ekstrand J, Dawes C. Studies on fluoride concentrations in human submandibular/sublingual saliva and their relation to flow rate and plasma fluoride levels. Journal of Dental Research 68 146-149 1989.
  10. Allmann DW, Shahed AR. The effect of NaF on salivary gland function. Deutsche Zahnarztliche Zeitschrift 42 (10 Suppl 1) S95-S98 1987.
  11. Kleiner HS, Allmann DW. The effects of fluoridated water on rat urine and tissue cAMP levels. Archives of Oral Biology 27 107-112 1982.
  12. Nicolau J, Ribeiro DM. Metabolism of glycogen in submandibular glands of rats. Alteration by NaF. Journal de Biologie Buccale 20 97-102 1992.
  13. Ferreira FD, Nicolau J. Changes in glucose metabolism in submandibular salivary glands of rats after isoproterenol or incisor-tooth amputation. Archives of Oral Biology 32 499-503 1987.
  14. Carlson JR, Suttie JW. Pentose phosphate pathway enzymes and glucose oxidation in fluoride-fed rats. American Journal of Physiology 210 79-83 1966.
  15. Shearer TR, Suttie JW. Effect of fluoride on glycolytic and citric acid cycle metabolites in rat liver. Journal of Nutrition 100 749-756, 1970.
  16. Shahed AR, Miller AR, Allmann DW. Influence of NaF and Na2PO3F (MFP) on glucose metabolism in rat hepatocytes. Biochemical Biophysical Research Communications 91 583-591 1979.
  17. Allmann DW, Kleiner HS. Effect of NaF on rat tissue cAMP levels in vivo. Pharmacol Ther Dent 5 73-78 1980.
  18. Ferreira FD, Nicolau J. Different responses of the submandibular salivary glands and liver of rats subjected to excess vitamin A: study on glycogen metabolism. International Journal for Vitamin and Nutrition Research 55 239-244 1985.
  19. Allmann DW, Benac M. Effect of chronic feeding of NaF on 3’5’cyclic AMP and glucose metabolism [IADR abstract L32]. Journal of Dental Research 54 (special issue A) L8 1975.
  20. Sashi, Singh JP, Tapar SP. Changes in glycogen content in some tissues during fluorosis: an experimental study on rabbits. Fluoride 21 82-86 1988.
  21. Lunardi J, Dupuis A, Garin J, Issartel J-P, Michel L, Chabre M, Vignais PV. Inhibition of H+-transporting ATPase by formation of a tight nucleoside diphosphate-fluoroluminate complex at the catalytic site. Proceedings of the National Academy of Sciences of the United States of America 85 8958-8962 1988.
FLUORIDE 32 (1)
1999 pp 20-26 		International Society for Fluoride Researchup
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