toxic effects of fluoride on primary lymphoid organs and white blood cells … · 2014. 7. 8. ·...

Research reportFluoride 47(3)227–234July-September 2014

Toxic effects of fluoride on primary lymphoid organsand white blood cells in female mice

Zhao, Wang, Tian, Dong, Zhou

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TOXIC EFFECTS OF FLUORIDE ON PRIMARY LYMPHOID ORGANS AND WHITE BLOOD CELLS IN FEMALE MICE

Jing Zhao,a Hongwei Wang,a,b Erjie Tian,a Faming Dong,a Bianhua Zhoua,b

Luoyang, Henan, People’s Republic of China

SUMMARY: Our previous studies showed that excessive fluoride (F) induced thymusapoptosis in female rats. Here, we investigated the toxic effects of F on lymphocytesand the complete blood count in female mice. The results showed that excessive Freduced weight gain and induced ultrastructural changes and DNA damage in theimmunocytes. Compared to the control group, in the F group, in the thymus, bonemarrow, and blood lymphocytes, typical comet configurations were present and theratio of tailing and the tail length of the immunocytes were significantly increased.Decreases occurred in the blood in the total white blood cell (WBC) count (p<0.01),lymphocytes (p<0.05), middle cells (p<0.05), granulocytes (p<0.05) and thethrombocytocrit (p<0.05). In conclusion, in female mice, excessive F reduces weightgain, seriously damages the DNA and ultrastructure of immunocytes, and reducesblood total WBC count, lymphocytes, middle cells, granulocytes, and thethrombocytocrit.

Keywords: Blood lymphocytes; Bone marrow; DNA damage; Fluoride; Mice; Thymus

INTRODUCTION

Immunocytes, a class of cells involved in the immune response, includelymphocytes (T lymphocytes, B lymphocytes, K cells, and NK cells), accessorycells (mononuclear phagocytes and dendritic cells), and other immune cells likegranulocytes. With the exception of the T lymphocytes, whose stem cells maturein the thymus, the other immunocytes originate and mature in the bone marrow. Asan irreplaceable primary lymphoid organ, bone marrow replenishes the immunesystem using a pool of hematopoietic stem cells, and it is a critical site for thesustained production of common lymphoid progenitor cells.1 It is well known thatbone marrow serves as the primary site for development of B cells, NK cells,monocytes, and granulocytes, and that the thymus is the site that provides the mainvenue for the development and education of T cell progenitors. However,accumulated evidence also suggests that the bone marrow can also function as asecondary lymphoid organ for CD4 and CD8 cells, as well as a preferential homingsite for memory T cells.2 Damage to the two primary lymphoid organs can reducethe production of immunocytes and induce immune dysfunction.1,3-7

After differentiation in the bone marrow or thymus, immunocytes migrate to theblood and perform their functions. Lymphocytes are responsible for the specificimmune responses to infection, while the other immunocytes are considered to benon-specific. A complete blood count (routine or full blood screen) can indicatethe immune status of the body. Some studies have shown that a higher total whiteblood cell (WBC) count is an independent predictor of all-cause mortality in olderadults, and that the monocyte subtype provides even greater predictive ability.8

aCollege of Animal Science and Technology, Henan University of Science and Technology,Luoyang, Henan, PR of China. bFor correspondence: Professor Hongwei Wang and BianhuaZhou, College of Animal Science and Technology, Henan University of Science and Technology,70 Tianjin road, Luoyang, Henan 471003, PR of China; E-mail: [email protected].




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Disorders of the WBC subpopulations can also be indicative of some diseases.8-10

Moreover, some of the factors which are able to cause immune suppression canalso decrease the WBC count.11, 12

Fluoride (F) is not an essential trace element in the body and, when present in ahigh concentration, it can be harmful to teeth, bones, heart, liver, kidneys,gastrointestinal tract, lungs, brain, blood, hormones, the immune system, andvarious biochemical parameters in both experimental animals and humans.13-21

Our previous studies showed that excessive F ingestion can seriously damageimmune function and induce thymus apoptosis.22-24 However, the full effects of F-induced damage to the bone marrow and peripheral blood remained unclear. Theaim of this study was to further investigate, in female mice, the effects of F-induced immune system impairment on thymus ultrastructure, hematology, andthe immunocytes.

MATERIALS AND METHODSExperimental animals: A total of 24 healthy female Kunming mice (female:

male=3:1) were obtained from the Experimental Animal Center of ZhengzhouUniversity, and were kept in a standard animal house at 22–25ºC with ventilationand hygienic conditions.

Experimental design: The mice were randomly divided into two groups, of ninefemale and three male mice. The mice in the control group were reared on astandard diet and given ad libitum access to water. The mice in the F group weregiven a standard diet and drinking water containing 100 mg F/L. After threemonths of establishing the animal model, the female experimental animals wereallowed to become pregnant by natural mating. During and after nursing, theoffspring were reared in the same conditions as their parents. On the 21st day, thefemale mice were separated. On the 70th day, the separated female offspring micewere randomly selected for further study. The experimental design was approvedby the Institutional Animal Care and Use Committee of China.

Transmission electron microscopic observation of thymus cells: To study theeffect of F on thymus ultrastructure, the fresh thymus in mice was removed andfixed in 0.1% sodium arsenate dimethyl (pH 7.4) for observation with atransmission electron microscope (TEM). The sections were treated with 1%osmic acid and embedded in Araldite resin. About 50 nm thick sections wereprocessed, stained with uranyl acetate and lead citrate, and examined with an H-7650 TEM.

Analysis of DNA damage in bone marrow cells, thymus cells, and bloodlymphocytes: To analysis the effect of F on DNA damage, the fresh thymus inmice was removed and treated as reported in our earlier study.22 Using single cellgel electrophoresis (SCGE), the ratio of tailing was calculated by counting thepercentage of cells showing tailing. The DNA damage was assessed from thelength of DNA migration derived by subtracting the diameter of the nucleus fromthe total length of the image.

Complete blood count examination (routine or full blood screen): On the 70thday of the treatment, 6 female mice were selected randomly from each group and




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were deprived of food for 12 hr. Blood was collected from the peri-orbital sinusand the complete blood counts performed with a blood cell analyzer.

Statistical analysis: Data are expressed as mean±SD. Statistical analyses wereperformed by Student’s t test. Values of p< 0.05 were considered significant.

RESULTSBody weight of mice: Compared with the control group, the growth of the mice

was significantly inhibited by the excessive F ingestion in the F group after 42days of treatment (Figure 1).

TEM observation of thymus cell: In the control group, the thymus cells showednormal ultrastructural features under TEM while, in the F-exposed groups,significantly more apoptotic thymus cells were present (Figure 2).

The apoptotic cells had morphological changes with the nuclear chromatinslessened, condensed, marginalized against the nuclear envelope, and aggregated

Body weight (g)

45

40

35

30

25

20

15

10

5

0

Control group

Fluoride group

14 21 28 35 42 49 56 63 70 Time (days)

*† †

† †

Figure 1. Effect of fluoride on body weight in mice. Compared to the control group: *p<0.05, †p<0.01.

Figure 2. Effect of fluoride on ultrastructural changes in thymus of mice. a: Normal thymus cells of mice in the control group, b: Damaged thymus cells of mice in the fluoride group.




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into large dark, compact masses. The cytoplasm was concentrated and associatedwith multiple cytoplasm vacuolization. The bilayer structure of the nuclearmembrane of the apoptotic thymus cells was less distinct and interrupted in places.The mitochondria showed irregularities and swelling with dissolution anddisappearance of the cristae (Figure 2b).

Analysis of DNA damage: In comparison with the control group, F-inducedDNA damage occurred in the blood lymphocytes and the thymus and bonemarrow immunocytes with the F-group cells showing typical comet configurationsand significant increases in both the ratio of tailing and the tail length. Theseresults indicate that F induced DNA damage in the mice immunocytes. (Tables 1and 2).

Complete blood count examination: Compared with the control group, in the Fgroup the blood counts were reduced for the total WBCs, lymphocytes, middlecells, and granulocytes (Figure 3). The lymphocyte proportion increased from46% to 51% while that for granulocytes decreased from 39% to 34% (Figure 4). Incomparison with the control group, in the F group MCHC, MCV, MCH, RDWHCT, MPV, and HGB did not change significantly while the PCT decreased(p<0.05). The results indicate that F induced WBC changes in the blood of mice(Tables 3 and 4).

Table 1. Ratio of tailing (% of cells showing tailing) and tail length in mice cells induced by fluoride (Values are mean±SD; n = 4)

Tissue Group Ratio of tailing (%) Tail length (µm)

Control group 13.21±0.75 3.24±0.85 Bone Marrow

Fluoride group 27.37±1.38† 16.26±2.93†

Control group 18.22±1.31 3.48±0.74 Blood lymphocytes

Fluoride group 38.67±3.07† 18.26±3.49†

Control group 15.63±1.26 3.39±0.73

Thymus Fluoride group 32.71±2.46† 18.97±2.93†

Compared to the control group: *p<0.05, †p<0.01

Table 2. Undamaged and migrated cells according to severity by grade (%)

Tissue Group Normal Grade I Grade II Grade III

Control group 86.79 11.78 1.19 0.24 Bone Marrow

Fluoride group 72.63 9.02 10.42 7.92

Control group 81.78 15.28 2.31 0.63 Blood Lymphocytes

Fluoride group 61.33 11.71 12.42 14.54

Control group 84.37 13.39 1.83 0.41 Thymus

Fluoride group 67.29 8.97 10.98 12.76




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Control group

Fluoride group

White blood Lymphocytes Middle cells Granulocytes cells

14

12

10

8

6

4

2

0

Blood white blood cell count (×109/L)

†

*

*

*

b

Gran34%

Mid15%

Lymph51%

Granulocytes 34%

Lymphocytes 51%

Middle cells 15%

Figure 4. Effect of fluoride on the differential white blood cell count in mice. a: control group, b: fluoride group.

Table 3. Effect of fluoride on mean corpuscular hemoglobin concentration (MCHC), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and red cell distribution width (RDW) in mice

(Values are mean±SD)

Group MCHC (g/L) MCV (fL) MCH (pg) RDW (%)

Control group 340.00±7.04 51.05±1.26 17.32±0.45 15.08±1.07

Fluoride group 338.00±6.84 51.88±0.48 17.48±0.48 14.43±0.59

Table 4. Effect of f luoride on hematocrit (HCT), mean platelet volume (MPV), hemoglobin (HGB), and thromobcytocrit (PCT) in mice. (Values are mean±SD)

Group HCT (%) MPV (fL) HGB (g/L) PCT (%)

Control group 40.20±3.99 9.62±0.43 136.83±13.59 0.24±0.04

Fluoride group 39.10±1.61 9.62±0.74 132±33±3.78 0.17±0.05*

Compared to the control group: *p<0.05

Figure 3. Effect of fluoride on white blood cell count in mice. Compared to the control group: *p<0.05, †p<0.01.

a

Gran39%

Mid15%

Lymph46%

Lymphocytes 46%

Granulocytes 39%

Middle cells 15%




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DISCUSSIONThe development, differentiation, and metabolism of almost every tissue in the

body of mammals requires not only nutrients but also a variety of hormones. Inthis study, growth in mice was significantly inhibited by 100 mg/L F in drinkingwater, which is consistent with the results of our previous studies in rabbits.23 TheF-induced decrease in body weight gain may be due to a reduction in food intaketogether with a depletion of soft tissue protein.25 Protein and calciumsupplementation can alleviate these F-induced body weight changes23,26 Thyroidhormone plays an important role in regulating body growth,27 and excessive F caninduce thyroid dysfunction with decreased serum thyroid hormone levels.26 ThusF-induced inhibition of weight gain may be due to a decreased food intake, adepletion of protein in soft tissues and thyroid dysfunction.

Caspases are a class of apoptosis-related proteins, which can be divided intoapoptotic initiators, including caspase-9, and apoptotic effectors, such as caspase-3.28 In the present study, the F-treated mice had ultrastructural changes in thethymus with a significant increase in apoptotic thymus cells. This may due to a F-induced up-regulation in the thymus in the relative expression levels of caspase-3and caspase-9.22 Numerous studies have shown that F can induce apoptosis indifferent tissues. Matsui et al. demonstrated that NaF may induce apoptosis-likenecrosis in rat thymocytes with increases in the population of shrunken cells andcells positive to annexin V, both of which are associated with an early stage ofapoptosis. 21 Miao et al. and Cheng et al. found that F-induced apoptosis wascorrelated with increased levels of Fas/FasL and the high expression of Bcl-2 andBax.29,30 These mechanisms may account for the occurrence of F-inducedapoptosis in thymocytes.

As is well known, DNA is the main genetic material of life, and DNA damagemay alter cell functions, disrupt protein generation, and lead to carcinogenesis andmutagenesis. Our study showed that excessive F can lead to DNA damage inimmune cells. The comet assay images showed that the DNA damage in bonemarrow cells, blood lymphocytes, and thymocytes in F-treated mice was moreserious than in the control group. F-induced apoptosis is apparently associatedwith the DNA damage. It is also well documented that oxidative stress is one ofaccepted mechanisms of F-induced toxicity.31,32 Moreover, a large number ofstudies have shown that DNA damage and oxidative stress are inextricablylinked.33,34 Thus we can safely conclude that F-induced DNA damage in bonemarrow cells, blood lymphocytes, and thymocytes is closely related to oxidativestress.

The total and differential WBC count is an important auxiliary diagnostic test aschanges occur with systemic inflammation and other diseases. An increasedgranulocyte count has been associated with an increased risk of heart failure inapparently healthy men, while a decreased risk has been associated with anincreased monocyte count.35 Since the mature immunocytes migrate fromlymphoid organs to the peripheral blood and then exert their effects, a completeblood count is necessary to assess the immune status of the body. Huyan et al.showed that the immunosuppressive effect of cyclophosphamide can decrease the




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counts of WBC and lymphocyte in Balb/c mice.11 Moreover, systemic lupuserythematosus patients have decreased peripheral WBC numbers with lowlymphocytes, and there is evidence that these reductions are caused byautoantibody-related apoptosis.36 In the present study, the total blood WBC count,together with the subpopulations of lymphocytes, middle cells, and granulocytes,were significantly decreased in the F group which is consistent with a F-inducedapoptosis of immune cells.

In conclusion, excessive F can inhibit the growth and general health in mice andimpair their immune functions by destroying the ultrastructure of the thymus,damaging the DNA in the thymus, bone marrow, and blood lymphocytes, and bydecreasing the total and differential counts of WBC in female mice.

ACKNOWLEDGEMENTThis research was sponsored by the China National Natural Science Foundation

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toxic effects of fluoride on primary lymphoid organs and white blood cells … · 2014. 7. 8. ·...

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