Experimental and Toxicologic Pathology 63 (2011) 407–411

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Experimental and Toxicologic Pathology

0940-29

doi:10.1

n Corr

E-m

journal homepage: www.elsevier.de/etp

Effect of fluoride on calcium ion concentration and expression of nucleartranscription factor kappa-B r65 in rat hippocampus

Jing Zhang, Wen-Jing Zhu, Xiao-Hong Xu, Zi-Gui Zhang n

College of Chemistry and Life Science, Zhejiang Normal University, Zhejiang 321004, PR China

a r t i c l e i n f o

Article history:

Received 22 November 2009

Accepted 28 February 2010

Keywords:

Fluoride toxicity

Hippocampus

Fura-2/AM

Calcium ions concentration

NF-kB r65

93/$ - see front matter Crown Copyright & 2

016/j.etp.2010.02.017

esponding author. Tel.: +86 057982291189;

ail address: zzg@zjnu.cn (Z.-G. Zhang).

a b s t r a c t

The study investigated the neurotoxicity of drinking water fluorosis in rat hippocampus. Just weaning

male Sprague-Dawley (SD) rats were given 15, 30, 60 mg/L NaF solution and tap water for 9 months.

The calcium ion concentration ([Ca2 +]) in synaptosomes was measured by double wavelength

fluorescence spectrophotometer and the expression level of nuclear transcription factor kappa-B r65

(NF-kB r65) in hippocampal CA3 region was measured by immunohistochemistry. The results showed

that [Ca2 +] significantly increased (F¼33.218, Po0.01) in moderate fluoride group compared with the

control group, and the expression level of NF-kB r65 in CA3 region presented an increasing trend as

fluoride concentration increased. These results indicate that increase of synaptosomes [Ca2 +] and NF-kB

r65 expression level may be the molecular basis of central nervous system damage caused by chronic

fluoride intoxication. NF-kB r65 in CA3 region is probably a target molecule for fluorosis.

Crown Copyright & 2010 Published by Elsevier GmbH. All rights reserved.

1. Introduction

Fluoride has a direct toxic effect on the central nervous system,but the specific mechanisms remained unknown. Hippocampushas been postulated to be one of the target sites attacked byfluoride (Hanen et al., 2007). Our previous study showed thatfluorosis may impair hippocampus synaptic interface structure(Zhang et al., 2008). And the changes in the synaptic interfacestructure would necessarily affect the transmission of neuralinformation (Shiyaraiashankara et al., 2002). A recent studyreported that fluoride can induce oxidative damage in hippocam-pal neurons in vitro (Zhang et al., 2007). Ca2 + in the nerve cells ofthe hippocampus was closely related to neural signal transduction(Concepcion et al., 2009). In the neurons, Ca2 + overload may causea series of damnification, including further promotion in thenumber of free radicals, damage in cell membrane and cytoske-leton, cell damage and even death. The nuclear transcriptionfactor kappa-B r65 (NF-kB r65) expression was activated by theincrease of [Ca2 +] and was sensitive to oxygen free radical(Thomas et al., 2004; Zhang et al., 2009). NF-kB regulates thepathological and physiological functions in nervous system as itcan both promote and protect against cell death (Thanos andMancatis, 1995; Denk et al., 2000; Mattson, 2005). Evidence hasproved a role of NF-kB in synaptic signaling and transcriptionalregulation mechanisms required for long-term plasticity (Meffertand Baltimore, 2005; Kaltschmidt et al., 2006).

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fax: +86 057982291190.

Hence in this study of fluoride neurotoxicology, we chose therat model of chronic fluorosis to explore the effects of differentdoses of fluoride on synaptosomes [Ca2 +] and hippocampusCA3 region NF-kB r65 expression level in order to provide atheoretical basis for chronic fluorosis-depth study on the centralnervous system toxicity mechanisms of molecular biology.Meanwhile, we also need to determine whether NF-kB r65 wasthe target molecule of fluorosis, and provided a scientific basis forearly diagnosis and early treatment of fluorosis.

2. Materials and methods

2.1. Chemical and biological reagents

Sodium fluoride (NaF molecular weight 41.99) was purchasedfrom Shantou Xilong Chemical (Guangdong, China). Rabbit anti-mouse antibodies against r65 were purchased from Santa CruzBiotechnology (Santa Cruz, CA). Fura-2/AM was purchased fromSigma (St. Louis, MO, USA). All other materials were purchasedfrom Booster (Booster, CHN).

2.2. Animals and administration

Just weaning male Sprague Dawley rats (60–80 g, n¼52) wereobtained from Experimental Animal Center of Zhejiang Province.Prior to dosing, rats were acclimatized for 7 days. Then based onpre-made in our laboratory’s acute and subchronic experimentalwork, rats were divided into control and experimental groups

All rights reserved.

Table 1The effects of fluoride in rats of blood F.

Group N Blood F (mg/L)

Control 7 0.11870.014

Low fluoride 7 0.27270.064n

Moderate fluoride 7 0.39870.033n

High fluoride 7 0.37970.037n

With the increasing concentration of fluoride, blood F increased in experimental

groups and had significant differences compared with the control (F¼18.053,nPo0.05). Data are presented as means7SE.

J. Zhang et al. / Experimental and Toxicologic Pathology 63 (2011) 407–411408

treated as follows (n¼13 per group): (1) control group: receivedtap water (fluoride concentrationo0.5 mg/L); (2) high fluoridegroup: received sodium fluoride (60 mg/L); (3) moderatefluoride group: received sodium fluoride (30 mg/L); and (4) lowfluoride group: received sodium fluoride (15 mg/L). Animals werehoused in plastic cage and allowed free access to standard ratchow and water in a temperature-controlled (23 1C) air-condi-tioned room, 50–60% humidity and with a 12 h light–dark cyclefor 9 months. All procedures were performed in accordance withthe Animal Care Guidelines published by the National Institutes ofHealth in USA (#85–23, revised in 1985), European Community(86/609/EEC) and the Institutional Animal Care and UseCommittee of China.

2.3. The measurements of conventional fluorosis poisoning

After feeding for 3 months, photographs were taken with a digitalcamera for rats (24 samples, 6 per group) to observe the degree ofdental fluorosis. Then fluoride ion electrode (VanLondon-pHoenix,US) was selected to determine the fluoride concentration in theblood (28 samples, 7 per group). The above two measurements ofconventional fluorosis poisoning are considered as the standard ofcopying poisoned animal models (Tamer et al., 2007).

2.4. Measurements of intracellular calcium ion concentration

2.4.1. Synaptosome preparation

After 9 months, fluoride exposure was stopped and a group ofrats (28 samples, 7 per group) were decapitated. Synaptosomes wereisolated essentially as described by Nagy and Delgado-Escueta(1984). The hippocampus were gently homogenized in 6 volumes ofan ice-cold medium (medium I) containing 320 mM sucrose, 2 mMEDTA and 20 mM HEPES, pH 7.4, in a motor driven Teflon–glasshomogenizer. The homogenate tissue was then centrifuged at 1500g

for 10 min. The supernatant was collected in another centrifugetube. The pellet was suspended in 4 volumes of an ice-cold medium(medium II) and then centrifuged at 1500g for 10 min. Wecomplicated the two supernatants and centrifuged at 15,000g for20 min. The pellet was resuspended in an isoosmotic solution to afinal protein concentration of 2–4 mg/mL. Synaptosomes wereprepared fresh daily and maintained at 0–4 1C throughout theprocedure. Protein concentration was determined by the BCAmethod using an enzyme mark instrument (Biotek InstrumentsPower Wave X microtiter plate UV–vis spectrophotometer, ThermoMultiskan spectrum, US).

2.4.2. Measurement of [Ca2 +]

After being rinsed with HEPES buffer solution (containing NaCl132 mM, KCl 3 mM, glucose 10 mM, HEPES 10 mM, and CaCl2

2 mM, pH 7.4), the synaptosomes were incubated with Fura-2/AM(final concentration 5 mM, Invitrogen) at 37 1C for 45 min under5% CO2 in incubator. Changes in the synaptosomes [Ca2 +] weremeasured using a fluorescence microscope by monitoring fluor-escence (Shimadzu RF5300, JP) at an emission wavelength of510 nm and excitation wavelengths alternating between 340 and380 nm. Alterations in synaptosomes [Ca2 +] were expressed asthe change in the fluorescence ratio of 340/380 nm, using theequation (Li et al., 2008)

Ca2þh i

¼ kdðR�RminÞ=ðRmax�RminÞ

where kd was the effective dissociation constant of Fura-2, valuewas 224 nmol/L; R was obtained from the observed fluorescenceratio 340/380 nm; Rmax and Rmin were measured after an additionof 10% TritonX-100 followed by 5 mM EGTA (Kuo, 2008),respectively.

2.5. Histology and immunochemistry

At the same time, the other rats (24 samples, 6 per group) weresacrificed and their brains were quickly removed from the cranialcavity. The hippocampus was dissected and CA3 region sliceswere cut, fixed in 4% paraformaldehyde and embedded in paraffinblocks. Tissue sections were prepared as described (Clauss et al.,1990; Iijima et al., 1993; Su et al., 2006) and stained withhematoxylin and eosin (HE). The slides were washed andincubated with biotinylated, affinity-purified goat-anti-rabbitIgG. After avidin–biotin amplification, the slides were incubatedwith diaminobenzidine.

2.6. Quantitative analysis

Analysis was performed blinded, by assessing 25 consecutivehigh-power fields (�400 magnification) for each section ofimmunostaining (Media Cybernetics Image-Pro V5.1, US).

2.7. Statistical analysis

Data values given are means7standard error (SEM). Compar-ison of means were conducted using one way analysis of variance(ANOVA) followed by least significant difference post-hoc test tocompare means between the different groups. Differences wereconsidered as significant when Po0.05 or Po0.01. All analyseswere performed using the SPSS 15.0 software.

3. Results

3.1. Conventional fluorosis indices

Table 1 shows that as the concentration of fluoride increased,blood F increased in experimental groups and had significantdifferences (F¼18.053, Po0.05) compared with the controlgroup. In Fig. 1, as the concentration of fluoride increased, thelevel and occurrence probability of dental fluorosis in ratsincreased. The teeth of high fluoride group rats becamedeformed and defected to some degree. Both indices also showthat the fluorosis model worked in the research.

3.2. Effect of fluoride on [Ca2 +] in synaptosome

Table 2 shows that synaptosomes [Ca2 +] increased in theexperimental groups with the increase in fluoride. Compared withthe control group, [Ca2 +] in the moderate fluoride group had aremarkable increase (F¼33.218, Po0.01).

3.3. Effect of fluoride on HE staining in hippocampus CA3 region

HE staining in hippocampus CA3 region (Fig. 2) shows thatobserved under a light microscope (�400), pyramidal cells in

Fig. 1. The results of dental fluorosis. A: control group; B: high fluoride group. Control group: the enamel represents the usual translucent semivitriform type of structure,

and the surface is smooth, glossy. High fluoride group: the enamel surfaces of the teeth are subject to attrition show wear and the teeth became deformed. The prevalence

of dental fluorosis was nearly 100%, with the prevalence of defect dental fluorosis increasing with increase in fluoride intake.

Table 2Effect of fluoride on Ca2 + concentration in synaptosome in rats.

Group N [Ca2 +] (nmol/L)

Control 7 297.59733.91

Low fluoride 7 318.12755.56

Moderate fluoride 7 375.47759.80nn

High fluoride 7 342.22731.02

The synaptosomes Ca2+ concentration increased in the experimental group with

the increase in fluoride. Compared with the control group, Ca2+ concentration in

the moderate fluoride group had a remarkable increase (F¼33.218, nnPo0.01).

Data are presented as means7SE.

J. Zhang et al. / Experimental and Toxicologic Pathology 63 (2011) 407–411 409

hippocampus CA3 region of the control group had a regulararrangement and morphology. In contrast, the low fluoride groupcells with vacuole were sparse and arranged in a disorderedmanner, and it got worse in the moderate fluoride group and highfluoride group. The results indicated that fluorosis could injurethe pyramidal cells in the hippocampus CA3 region to somedegree.

3.4. Effect of fluoride on the NF-kB r65 expression level in CA3

region

Fig. 3 shows that on immunohistochemical staining under alight microscope we observed that NF-kB r65 positive cellsshowed brown particles located in the cytoplasm. When theNF-kB r65 subunit moved into the nucleus, positive cells wereexpressed as nuclei stained brown or brown yellow particledeposition. In the rat hippocampus CA3 region of the controlgroup, on immunohistochemical staining we could observe asmall amount of NF-kB r65 subunit positive neurons in thecytoplasm and the NF-kB r65 subunit existed as an inactive form.In the experimental groups, NF-kB r65 subunit was activated andthere was multi-expression in the nucleus. And with the increaseddose of fluoride, NF-kB r65 subunit positive cells were increased.Synchronously, the determination of integral optical density (IOD)

showed that with the concentration of fluoride increasing, IODincreased (Table 3).

4. Discussion

Ca2+ was an important intracellular messenger enabling severalphysiological processes such as learning-memory. Calcium signalsinitiated many neuromodulation responses (Ehrich et al., 2009),including axonal growth, secretion of neurotransmitters andsynaptic plasticity. Neurons Ca2+ overload was considered thecommon pathways of neurons damage induced by variousstimulations (Sobczak et al., 2005). NF-kB is ubiquitously detectedin various regions of the central nerve system, particularly inhippocampus, cortex, granular layer of cerebellum and pontinenuclei (Freudenthal et al., 2004; Gutierrez et al., 2005). During thedevelopment of the nervous system, NF-kB is activated by someneurotrophic factors and can induce the expression of genesinvolved in cell differentiation and survival (Mattson and Meffert,2006). In the mature nervous system, NF-kB is also activated insynapses in response to excitatory synaptic transmission (Meffertand Baltimore, 2005).

Our results showed that the hippocampus synaptosomes [Ca2+]were overloaded and the expression level of CA3 region NF-kB r65subunit presented an increasing trend in experimental groups. Sowe speculated the nervous toxic mechanism of fluorosis could bethe fluorine attacked oxygen and interfered with oxygen metabo-lism when the body ingested excessive fluoride (Freudenthal et al.,2004; Memet, 2006; Shrum and Meffert, 2008). Then nerve cellmembrane could be damaged by mass free radicals, and the [Ca2+]increased by damaged nerve cell membrane. Ca2 + overload led toan abnormal control of CaM and CaMK II signaling pathways (Shenet al., 2008). It could set off a complex chain of signal transductionin nerve cells (Jeonga et al., 2005) and activate nuclear transcrip-tion factor NF-kB (Torricelli et al., 2008).

Besides, we found that compared with moderate fluoridegroup, [Ca2 +] and NF-kB expression levels presented a decrease inhigh fluoride group. It suggested that fluoride-induced toxicity is

Fig. 2. Effects of fluoride acting on hippocampus CA3 pyramidal cells in rats (�400). Group A: control group; Group B: low fluoride group; Group C: moderate fluoride

group; Group D: high fluoride group. Control group: the pyramidal cells in hippocampus CA3 region had regular arrangement and morphology. Experimental groups: the

low fluoride group cells with vacuole were sparse and arranged in a disordered manner, and it got worse in the moderate fluoride group and high fluoride group compared

with the control group.

Fig. 3. Immunoreactive products of NF-kb r65 in hippocampus CA3 region (�400). Group A: control group; Group B: low fluoride group; Group C: moderate fluoride

group; Group D: high fluoride group. As the arrow point shows, in the rat hippocampus CA3 region of the control group, immunohistochemical staining can be seen a small

amount of NF-kB r65 subunit positive neurons in the cytoplasm and the NF-kB r65 subunit exists as an inactive form. In the experimental groups, NF-kB r65 subunit was

activated and there was multi-expression in the nucleus. And with the increased dose of fluoride, NF-kB r65 subunit positive cells were increased.

J. Zhang et al. / Experimental and Toxicologic Pathology 63 (2011) 407–411410

mediated through oxidative stress, particularly at a certain levelof exposure. If it was exceeded, the body may start a certainprotective mechanism coupled with body damage.

In summary, our data showed the enhancement of synapto-somes [Ca2 +] and expression level of NF-kB r65 in the CA3 region

of hippocampus, which may be the molecular basis of centralnervous system damage by fluorosis. The results supported theidea that fluorosis can generate neurotoxicity effect by changingthe [Ca2 +] in nerve cells. And NF-kB r65 may be one of the relatedmolecular target factors in central nervous system damage by

Table 3

Optical density of immunoreactive products of NF-kb r65 in the hippocampus

CA3 region.

Group N NF-kB q65 IOD

Control 6 0.1470.02

Low fluoride 6 0.1770.02

Moderate fluoride 6 0.1970.01

High fluoride 6 0.1870.02

With the concentration of fluoride increasing, IOD of the experimental groups

increased compared with the control group. Data are presented as means7SE.

J. Zhang et al. / Experimental and Toxicologic Pathology 63 (2011) 407–411 411

fluorosis. However, the detailed nervous toxic mechanism offluorosis is very complex and requires further exploration.

Acknowledgments

This study was supported by the National Natural ScienceFoundation of China [grant number, 30871295]; and ProvinceNatural Science Foundation of Zhe Jiang Province [grant number,Y207751].

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