Biological Trace Element Research

, Volume 176, Issue 2, pp 355–366 | Cite as

Effects of Different Levels of Calcium Intake on Brain Cell Apoptosis in Fluorosis Rat Offspring and Its Molecular Mechanism

  • Yan Sun
  • Lulu Ke
  • Xiangren Zheng
  • Tao Li
  • Wei Ouyang
  • Zigui Zhang


The purpose of the investigation is to reveal the influence of dietary calcium on fluorosis-induced brain cell apoptosis in rat offspring, as well as the underlying molecular mechanism. Sprague–Dawley (SD) female rats were randomly divided into five groups: control group, fluoride group, low calcium, low calcium fluoride group, and high calcium fluoride group. SD male rats were used for breeding only. After 3 months, male and female rats were mated in a 1:1 ratio. Subsequently, 18-day-old gestation rats and 14- and 28-day-old rats were used as experimental subjects. We determined the blood/urine fluoride, the blood/urine calcium, the apoptosis in the hippocampus, and the expression levels of apoptosis-related genes, namely Bcl-2, caspase 12, and JNK. Blood or blood/urine fluoride levels and apoptotic cells were found significantly increased in fluorosis rat offspring as compared to controls. Furthermore, the Bcl-2 messenger RNA (mRNA) expression levels significantly decreased, and caspase 12 mRNA levels significantly increased in each age group as compared to controls. Compared with the fluoride group, the blood/urine fluoride content and apoptotic cells evidently decreased in the high calcium fluoride group, Bcl-2 mRNA expression significantly increased and caspase 12 mRNA expression significantly decreased in each age group. All results showed no gender difference. Based on these results, the molecular mechanisms of fluorosis-induced brain cell apoptosis in rat offspring may include the decrease in Bcl-2 mRNA expression level and increase in caspase 12 mRNA expression signaling pathways. High calcium intake could reverse these gene expression trends. By contrast, low calcium intake intensified the toxic effects of fluoride on brain cells.


Fluorosis Calcium Apoptosis Bcl-2 Caspase 12 JNK 



This research was financed by the National Natural Science Foundation of China, grant number 81273015, and the National Natural Science Foundation of China, grant number 81573101.


  1. 1.
    Xing DL, Xu SY, Zhang WL, Li GS (2006) The effect of excessive fluoride on oxidative stress and apoptosis in rat brain. Chin J Ctrl Endem Dis 21:7–9Google Scholar
  2. 2.
    Ge YM, Ning HM, Liu JW, Chen JJ, Zhong H, Cui YH (2010) Apoptosis in brain cells of different generations in rats exposed to fluoride. China Animal Husbandry & Veterinary Medicine 37(7):44–46Google Scholar
  3. 3.
    Yu J, Shi YX, Wang W, Wu Y, Sun DJ (2008) Effects of calcium on the growth of femur distal epiphyseal plate cartilage in fluorosis rats. Chin J Endemiol 27:43–46Google Scholar
  4. 4.
    Wang LF (2007) Several kinds of dental fluorosis of Dean's taxonomy analysis. Endem Dis Bull 22:71–73Google Scholar
  5. 5.
    Liu YZ, Chen SQ, Li ZP (2010) Variation and clinical application value of fluoride concentrations in the serum and urine in patients with acute organic fluoride poisoning by inhaling. Chin. J Emerg Med 19:1078–1081Google Scholar
  6. 6.
    Yue XT, Xu HL, Zhai M, Yang BF (1999) A survey on the relationship between the contents of fluoride and calcium in drinking water and the endemic dental fluorosis. J Environ Health 16:12–163Google Scholar
  7. 7.
    Teotia M, Teotia SPS, Singh KP (1998) Endemic chronic fluoride toxicity and dietary calcium deficiency interaction syndromes of metabolic bone diease and deformities in India: year 2000. Indian J Pediatr 65:371–381CrossRefPubMedGoogle Scholar
  8. 8.
    Fujita T, Palmieri GMA (2000a) Calcium paradox disease: calcium deficiency prompting secondary hyperparathyroidism and cellular calcium overload. J Bone Miner Metab 18(3):109–125CrossRefPubMedGoogle Scholar
  9. 9.
    Fujita T (2000b) Calcium paradox: consequences of calcium deficiency manifested by a wide variety of diseases. J Bone Miner Metab 18:234–236CrossRefPubMedGoogle Scholar
  10. 10.
    Zhang J, Zhu WJ, Zhang ZG (2013) Effect of chronic fluorosis on CaMKIIɑ/c-fos/Bax:Bcl-2 channel signal in rat hippocampus. Fluoride 46:135–141Google Scholar
  11. 11.
    Zhang J, Zhu WJ, Zhang ZG (2009) Influence of fluorosis on learning memory of pup rats and activities of SOD and content of MDA in brain. Chin J Public Health 25:1347–1348Google Scholar
  12. 12.
    Zhang WL, Zhao H, Xue LJ, Zhang XY, Li GS (2006) Intracellular ionized calcium level and oxidative stress in renal of Wistar rats after intaking of excessive fluoride. Chin J Endemiol 23:25–27Google Scholar
  13. 13.
    Bu LS, Zhang WL, Wen L, Bo LH, Yin WJ, Li GS (2003) The effect of excessive fluoride on ionized calcium level and apoptosis in liver cells. Chin J Ctrl Endem Dis 18:257–259Google Scholar
  14. 14.
    Lv XH, Li GS, Sun B (2000) Study of the mechanism of neurone apoptosis in rats from the chronic fluorosis. Chin J Endemiol 19:96–98Google Scholar
  15. 15.
    Yuan CQ, Ding ZH (2002) Activation and function of caspases in apoptosis. Progress in Physiological Sciences 33:220–224Google Scholar
  16. 16.
    Wei W, Gao YH, Wang C, Zhao LJ, Sun DJ (2013) Excessive fluoride induces endoplasmic reticulum stress and interferes enamel proteinases secretion. Environ Toxicol 28:332–341CrossRefPubMedGoogle Scholar
  17. 17.
    Zhang YL, Sun XN, Li T, Feng SM, Liao LB, Chen L, et al. (2014) Effects on expression of osteogenesisgene in the osteoblast with endoplasmic reticulum stress induced by fluoride. Wei Sheng Yan Jiu 43:967–971PubMedGoogle Scholar
  18. 18.
    Danial NN, Korsmeyer SJ (2004) Cell death: critical control points. Cell 116:205–219CrossRefPubMedGoogle Scholar
  19. 19.
    Zhang K (2010) Integration of ER stress, oxidative and the inflammatory response and disease. Int J Clin Exp Med 2:33–40Google Scholar
  20. 20.
    Szegezdi E, Logue SE, Gorman AM, Samali A (2006) Mediators of endoplasmic reticulum stress-induced apoptosis. EMBO Rep 7:880–885CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Gordon PM, Marjelo AM, Elonore B, Tae-Y E, Ling S, Anna AZ, et al. (2011) Glycogen synthase kinase-3 regulates endoplasmic reticulum (ER) stress-induced CHOP expression in neuronal cells. Exp Cell Res 317:1621–1628CrossRefGoogle Scholar
  22. 22.
    Yasuhiro K, Yaeko T, Touru I, Kumiko I, Yoshihisa I (2011) Neuroprotective effect of mithramycin against endoplasmic reticulum stress-induced neurotoxicity in organotypic hippocampal slice cultures. Neuropharmacology 61:252–261CrossRefGoogle Scholar
  23. 23.
    Gu HF, Zhang Y, Zhang KQ, Liu L, Ma L, Cheng RB, et al. (2014) Effect of fluoride on the expression of endoplasmic reticulum chaperone GRP78 and caspase-12 in osteoblasts of rat bone. Chin J Practical Stomatology 7:665–672Google Scholar
  24. 24.
    Zhu ZJ, Yu YN, Tao XL, Zhao LN (2015) Expression of Janus kinase/signal transduction and transcriptional activation (JAK1 and STAT3) in liver of fluorosis rats. Chin J Endem 34:733–738Google Scholar
  25. 25.
    Liu YJ, Gao Q, Wu CX, Guan ZZ (2010) Changes of the c-Jun N-terminal kinase in the brains of rats with chronicfluorosis. Chin J Endemiol 29:608–612Google Scholar
  26. 26.
    Wei LS, Yu WF, Zhan ZL, Zhou BF, Wu ZQ, Guan ZZ (2015) Role of apoptosis-inducing factor in neuron apoptosis in brain tissue of rats with chronic fluorosis. Chin J Endemiol 34:655–659Google Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Yan Sun
    • 1
  • Lulu Ke
    • 1
  • Xiangren Zheng
    • 1
  • Tao Li
    • 1
  • Wei Ouyang
    • 2
  • Zigui Zhang
    • 1
    • 3
  1. 1.College of Chemistry and Life ScienceZhejiang Normal UniversityJinhuaPeople’s Republic of China
  2. 2.College of Sports and Health ScienceZhejiang Normal UniversityJinhuaPeople’s Republic of China
  3. 3.College of Xing ZhiZhejiang Normal UniversityJinhuaPeople’s Republic of China

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