The study was undertaken to know the effect of fluoride (with low and normal calcium) on calcium homeostasis and expression of genes involved in calcium homeostasis and its reversal by normal calcium and fluoride free water (FFW).

Methodology. The animal experiment was done in two phases. In phase I (duration-6 months), 76 weanling, male rats were divided to four groups and received normal calcium (0.5%) diet (NCD) +FFW (Group 1/ control n=12) or low calcium (0.25%) diet (LCD)+ FFW (Group 2, n=16) or NCD + 100 ppm fluoride (Group 3, n=24, NCD+F) or  LCD+F (Group 4, n=24). After 6 months of this treatment, 6 animals from group 1 and 8 animals from all other groups were killed. In phase II (reversal Phase), 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 had been continued with fluoride water. Duration of phase II was 3 months.  Blood was collected by retro-orbital sinus puncture after overnight fast at the interval of 90 days. Serum was separated and stored at -80º C till further investigations. At end of phase I and II of the experiment, rats were killed by CO₂ asphyxiation. Proximal small intestine (5 cm) was collected and immediately washed with cold saline. Duodenal mucosa was scraped, snap frozen and preserved at -80°C for gene expression studies. Serum calcium (SCa) was measured by atomic absorption spectroscopy and Serum 25(OH) vitamin D_3, 1,25(OH)₂ vitamin D₃ and intact parathyroid hormone were assessed using kits. Quantitative PCR was carried out for calcium sensing receptor (Casr), vitamin D receptor (Vdr) and Calbindin D 9K (Calb3).

Results. In phase I, SCa group 2, 3 and 4 was significantly lower as compared 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 group1.  Serum PTH level in group 2 was higher as compared to group 1 at day 90.  As expected, LCD groups (group 2 and 4) had significantly lower serum 25(OH) vitamin D₃ levels and significantly higher serum 1,25(OH)₂ vitamin D₃ as compared to NCD groups (group 1 and 3) in phase I. In phase II of the study, subgroup 4b showed significantly higher PTH as compared to subgroup 3b. Serum 25(OH) vitamin D₃ and serum 1,25(OH)₂ vitamin D₃ of all groups were comparable. Downregulation of any of the receptor studied was not observed in presence of fluoride in phase I. However, in phase II, Calb3 expression of fluoride treated subgroups 3b (2.5 fold) and 4b (2 fold) was downregulated as compared to subgroup 3b.

Summary and Conclusion. It is concluded that adverse effects of chronic fluoride toxicity on calcium homeostasis is more pronounced in presence of calcium deficiency. These effects may be reversible with adequate calcium nutrition and safe drinking water. Chronic fluoride toxicity may affect calcium absorption in duodenum due to inhibitory effect on expression of calb3- a key molecule involved in calcium absorption.

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1. Nutritional, Clinical and Biochemical status in fluoride (F=8 ppm) affected  village, Nalgonda District, Andhra Pradesh, India.