REGULATION AND REVERSAL OF EFFECTS OF FLUORIDE ON CALCIUM HOMEOSTASIS IN RATS

This study was undertaken to determine the effects of fluoride on calcium homeostasis and gene expression together with their reversal by normal calcium and fluoride free water (FFW). In phase I lasting 6 months, 76 weanling, male rats were divided into equal four groups that received a normal calcium (0.5%) diet (NCD) + FFW (Group 1/control n = 12), a low calcium (0.25%) diet (LCD) + FFW (Group 2, n = 16), NCD + 100 ppm fluoride (Group 3, n = 24, NCD + F), and LCD + F (Group 4, n = 24). After 6 months, 6 animals from group 1 and 8 animals from all other groups were sacrificed by CO sub(2) asphyxiation. In phase II (reversal of phase I), lasting for 3 months, LCD of group 2 and 4 was replaced by NCD. In phase II, group 3 and 4 were divided into two subgroups (n = 8), i.e., 3a, 3b and 4a, 4b, respectively. Subgroups 3a and 4a received FFW whereas 3b and 4b were continued with fluoride water. Blood was collected by retro-orbital sinus puncture after overnight fasting at the interval of 90 days and serum was separated and stored at -80 degree C. At end of phase I and II, the remaining rats were sacrificed by CO sub(2) asphyxiation. Proximal small intestine (5 cm) was collected and immediately washed with cold saline. Duodenal mucosa was scraped off, snap frozen, and preserved at -80 degree C for gene expression studies. Serum calcium (SCa) was measured by atomic absorption spectroscopy and serum 25-(OH) vitamin D sub(3) 1,25-(OH) sub(2) vitamin D sub(3) and intact parathyroid hormone were assessed using kits. Quantitative PCR was conducted for calcium sensing receptor (Casr), vitamin D receptor (Vdr), and Calbindin D 9K (Calb3,). In phase I, SCa in groups 2, 3, and 4 was significantly lower than in group 1. In phase II (reversal phase), in spite of improvement in average SCa level, all experimental groups showed significantly lower SCa as compared to group 1. At day 90, serum PTH level in group 2 was higher than in group 1. As expected, LCD groups 2 and 4 had significantly lower serum 25-(OH) vitamin D sub(3) levels and significantly higher serum 1,25-(OH) sub(2) vitamin D sub(3) as compared to NCD groups 1 and 3 in phase I. In phase II, subgroup 4b showed significantly higher PTH as compared to subgroup 3b. Serum 25-(OH) vitamin D sub(3) and serum 1,25-(OH) sub(2) vitamin D sub(3) in all groups were comparable. Down regulation of any of the receptors studied was not observed in the presence of fluoride in phase I. However, in phase II, Calb3 expression of fl