Ne u ro en do cri ne al te ra ti ons im pa ir ena mel mi ne ra li za ti on, to oth
erup ti on and sa li va in rats
Alte ra ções ne u ro en dó cri nas in ter fe rem com a mi ne ra li za ção do
es mal te, a erup ção den tá ria e a sa li va em ra tos
Ki kue Takebayashi Sas sa ki* Alber to Carlos Botazzo Del bem** Oto ni el Antonio Macedo dos San tos*** Car los Eduardo Shi ma bu co ro***
Ana Cláu dia de Melo Stevanato Na ka mu ne* João Cé sar Be dran-de-Cas tro****
Ri car do Martins Oli ve i ra-Fi lho*****
AB STRACT: Neo na tal ad min is tra tion of monosodium glu ta mate (MSG) in rats causes def i nite neuroendocrine dis tur -bances which lead to al ter ations in many or gan sys tems. The pos si bil ity that MSG could af fect tooth and sal i vary gland phys i ol ogy was ex am ined in this pa per. Male and fe male pups were in jected sub cu ta ne ously with MSG (4 mg/g BW) once a day at the 2nd, 4th, 6th, 8th and 10th day af ter birth. Con trol an i mals were in jected with sa line, fol low ing the same sched ule. Lower in ci sor erup tion was de ter mined be tween the 4th and the 10th postnatal days, and the erup tion rate was mea sured be tween the 43rd and the 67th days of age. Pilocarpine-stim u lated sal i vary flow was mea sured at 3 months of age; pro tein and am y lase con tents were thereby de ter mined. The an i mals treated with MSG showed sig nif i -cant re duc tions in the sal i vary flow (males, –27%; fe males, –40%) and in the weight of submandibular glands (about –12%). Body weight re duc tion was only about 7% for males, and did not vary in fe males. Sa liva of MSG-treated rats had in creased con cen tra tions of to tal pro teins and am y lase ac tiv ity. The erup tion of lower in ci sors oc curred ear lier in MSG-treated rats than in the con trol group, but on the other hand the erup tion rate was sig nif i cantly slowed down. The in ci sor micro hard ness was found to be lower than that of con trol rats. Our re sults show that neo na tal MSG treat -ment causes well-de fined oral dis tur bances in adult hood in rats, in clud ing sal i vary flow re duc tion, which co ex isted with un al tered pro tein syn the sis, and dis tur bances of den tal min er al iza tion and erup tion. These data sup port the view that some MSG-sen si tive hy po tha lamic nu clei have an im por tant modulatory ef fect on the fac tors which de ter mine car ies sus cep ti bil ity.
DES CRIP TORS: So di um glu ta ma te; Hypot ha la mic di se a ses; Den tal physi o logy; Sa li va; Rats.
RESUMO: A ad mi nis tra ção ne o na tal de glu ta ma to mo nos só di co (MSG) em ra tos pro vo ca dis túr b i os ne u ro en dó cri nos que acar re tam al te ra ções em vá ri os sis te mas or gâ ni cos. Nes te tra ba lho, ava li a mos as re per cus sões des se tra ta men to so bre den tes e glân du las sa li va res. Ra tos ma chos e fê me as re cém-nas ci dos fo ram in je ta dos com MSG (4 mg/g peso cor po ral, s.c.) uma vez ao dia nos 2º, 4º, 6º, 8º e 10º dias após o nas ci men to; o gru po con t ro le re ce beu so lu ção sa li na no mes mo es que ma. O mo men to da erup ção do in ci si vo in fe ri or foi de ter mi na do en tre o 4º e o 10º dia de vida, e o rit mo de erup ção foi me di do en tre o 43º e o 67º dia. O flu xo de sa li va e o con te ú do sa li var de pro te í na e ami la se fo ram de ter -mi na dos sob es ti mu la ção com pi lo car pi na aos 3 me ses de ida de. Os ani ma is tra ta dos com MSG mos tra ram re du ções sig ni fi ca ti vas do flu xo sa li var (ma chos: –27%; fê me as: –40%) e do peso das glân du las sub man di bu la res (cer ca de –12%). Ape nas em ma chos hou ve dis cre ta re du ção do peso cor po ral (7%). A sa li va dos ani m a is tra ta dos com MSG apre sen tou au men to na con cen tra ção de pro te í nas to ta is e na ati vi da de ami lá si ca. A erup ção dos in ci si vos in fe ri o res ocor reu mais pre co ce men te nos ra tos tra ta dos do que nos con tro les, po rém a taxa de erup ção apre sen touse sig ni fi ca ti va men te re du zi da. A mi cro du re za tam bém foi me nor nos ani ma is tra ta dos. Nos sos re sul ta dos mos tram que o tra ta men to de ra tos re cémnas ci dos com MSG ca u sa um qua dro de fi ni do de al te ra ções bucoden ta is no ani mal adul to, tra -du zi das por re -du ção do flu xo de sa li va (sem re -du ção da sín te se pro téi ca) e dis túr bi os de mi ne ra li za ção e erup ção den tá ri as. Esses da dos apon tam para o im por tan te pa pel mo du la dor que cer tos nú cle os hi po ta lâ mi cos sen sí ve is ao MSG exer cem so bre os fa to res que re gu lam a sus ce ti bi li da de à cá rie.
DESCRITORES: Glutamato de sódio; Doenças hipotalâmicas; Fisiologia dentária; Sa liva; Ratos.
* PhD, De part ment of Ba sic Sci en ces, Di vi si on of Bi o che mistry; **PhD, De part ment of So c i al and Pe di a tric Den tistry;
***Under gra du a te stu dent; ****PhD, De part ment of Ba sic Sci en ces, Di vi si on of Physi o l ogy – Scho ol of Den tistry of Ara ça tu ba, São Pa u lo Sta te Uni ver sity.
INTRODUCTION
Monosodium glu ta mate (MSG) is a widely used food stuff fla vour ing com pound, es pe cially in ori en tal food. In rats and mice, the neo na tal ad min is tra -tion of MSG leads to ex ten sive dam age of cer tain hy po tha lamic nu clei, thus caus ing se vere neuroendocrine dis tur bances in adult hood. The ab nor mal i ties, first ob served by Olney19
(1969), in -cluded growth im pair ment, marked obe sity (which can de velop with out hyperphagia) and re duc tion of or gan weights, among others1,3. Marked re pres sion
was ob served in the os si fi ca tion of de vel op ing endochondral bone, with the per sis tence of cartilagenous el e ments and chondrocytes9, re
-duced ra tio of min eral de po si tion, and slower bone maturation26.
The for ma tion, erup tion and growth of teeth are pro cesses un der the con certed and timely in flu ences of sev eral hor mones, such as growth hor -mone (GH)12,28,29 and thyroid14, sex24, and ad re nal
hormones25
. On the other hand, it is known that the de vel op ment and the func tion of ro dent sal i -vary glands de pend upon neurohormonal factors18,
and that the sal i vary se cre tion in rats is un der strong hy po tha lamic influences21.
Since the pro cess of tooth for ma tion and the den tal microenvironment are im por tant fac tors in -flu enc ing car ies sus cep ti bil ity, the pu ta tive modulatory role of hy po thal a mus in these pro cesses was eval u ated by study ing the tooth micro -hard ness, the sal i vary flow and the con cen tra tion of pro tein and am y lase in sa liva of con trol or neo -na tally MSG-in jected rats.
MATERIAL AND METHODS
Ani mals and tre at ments
Ne o nate male and fe male Wistar rats were treated with 5 sub cu ta ne ous in jec tions of monosodium glu ta mate (MSG, Sigma Co., 4 mg/g body weight) dis -solved in phys i o log i cal sa line. In jec tions were done once a day at the 2n d, 4th, 6th, 8th and 10th day of life.
Con trol an i mals were treated with the drug ve hi cle. Vol ume in jected was al ways 0.02 ml/g BW.
Rats were weaned at 21 days and put there af ter on reg u lar Purina rat chow and wa ter ad li bi tum. The an i mals were main tained on rou tine lab o ra tory care con di tions (12 h dark/light cy cle, lights on 08:00 a.m., 24 ± 2ºC). The ex per i men tal pro to col was ap proved by the Eth ics Com mit tee on An i mal Ex per -i men ta t-ion, UNESP School of Den t-istry, Araçatuba.
To oth erup ti on
In fe rior in ci sors erup tion day was de ter mined upon daily ex am i na tion from the 4t h up to the 10th
day of age. The rate of normofunctional tooth erup -tion was de ter mined by ex am i na -tion ev ery 2 days of the su pe rior in ci sors in the pe riod from 43 to 67 days of age. A start ing mark at the gingival limit level was made un der light ether an es the sia in the tooth enamel with a cy lin dri cal bur and mensurations were then car ried out fol low ing the method de scribed by Gerlach et al.1 0 (2000).
Sa li vary flow
At 90 days of age, af ter 12 h fast ing, the rats were an es the tized with so dium pentobarbital (Hypnol®, Cristalia, 40 mg/kg BW, IP). Sal i vary se
-cre tion was stim u lated by pilocarpine ni trate (Sigma, 5 mg/kg BW, IP). Whole sa liva was then collected4
into preweighed ves sels and main tained on crushed ice dur ing 20 min af ter the first drop had fallen. Vol umes were es ti mated by weight, as -sum ing the spe cific grav ity of sa liva to be 1.0 g/ml. Af ter col lec tion, the an i mals were killed by ex cess pentobarbital an es the sia, and the parotid, submandibular and sublingual sal i vary glands were care fully dis sected out and weighed.
Pro te in and amy la se de ter mi na ti ons in sa li va
To tal pro tein in sa liva was de ter mined by the method of Low ry et al.15 (1951) and the sal i vary am y
-lase ac tiv ity by that of Caraway7
(1959). One unit of am y lase ac tiv ity is re ferred to as the amount of en -zyme needed to hy dro lyze 10 mg of starch in 30 min at 37ºC.
Mi cro hard ness
The up per and lower in ci sors were re moved and dis sected free from any for eign a d her ent tis sue. The right teeth were em bed ded and lon gi tu di nally placed into acrylic resin; the left teeth were sec -tioned and placed trans versely into the resin23
. In both sec tions (lon gi tu di nal and transversal), two in den ta tions were made, one on the crown and the other on the root, at the me dian por tion of enamel thick ness. A micro hard ness tester Shimadzu HMV-2000®
cou pled to a Knoop-like penetrator set was used with a 50 g load. Micro hard ness re sults are given in terms of kgf/mm2 x 10–3.
Sta tis ti cal analy sis
-par i sons tests. A 2.01 ver sion of the GraphPad InStatÒ
soft ware was used for this pur pose.
RESULTS
Ponderal data are seen in Graph 1. The in sert shows that neo na tal MSG treat ment caused a sig -nif i cant im pair ment of body weight gain of male rats, but this ef fect was not so clearly ev i dent in fe -males. The weights of sal i vary glands rel a tive to the body weight were dif fer ently in flu enced by MSG treat ment, as it caused an about 12% re duc -tion of both male and fe male submandibular gland weights, a 21% re duc tion of male (but not of fe -male) parotid glands, and did not in ter fere with the weights of sublingual glands of ei ther sex.
Sal i vary func tion stud ies are sum ma rized i n Ta ble 1. MSG caused re mark able re duc tions in the pilocarpine-stim u lated sal i vary flow, both in male (–27%) and in fe male rats (– 40%). On the other hand, sal i vary con tents of to tal pro tein and am y -lase ac tiv ity rose strongly, both in males (mean rise 31%) and in fe males (mean rise 49%).
The ef fects of neo na tal MSG ad min is tra tion on the erup tion of rat in ci sors are seen in Ta ble 2. The to tal and daily rates of male rat tooth erup tion were faster than those of fe males, and this dif fer ence was main tained or even some what ac cen tu ated as an ef fect of MSG. On the other hand, erup -tion it self oc curred sig nif i cantly ear lier in male or fe male rats treated with MSG than in their sex-matched con trols.
Ta ble 3 shows the micro hard ness anal y sis of rat teeth. Over all, micro hard ness was sig nif i cantly higher for male than for fe male teeth, what ever the sec tion or the an a tom i cal re gion con sid ered. Micro hard ness val ues ob tained in tooth lon gi tu di -nal sec tions were higher than those in trans verse sec tions, and also higher for the crowns when com pared to the roots. We ob served that neo na tal MSG treat ment caused, in adult hood, an evenly low ered tooth micro hard ness, thus main tain ing not only that sex di mor phism but also the dif fer -ences pre vi ously seen re gard ing the tooth sec tions and an a tom i cal re gions.
DISCUSSION
The early postnatal ad min is tra tion of monosodium glu ta mate (MSG) to rats is known to per ma nently dam age neu rons in the hy po tha lamic ar cu ate nu cleus. The en su ing in ap pro pri ate brainneuroendocrineim mune reg u la tion was re cently dem on strated to in flu ence periodontal dis -ease sus cep ti bil ity and progression5
. B e ing so, in this pa per we ex am ined the pre sum able con se -quences of MSG treat ment on sal i vary func tional char ac ter is tics and on den tal min er al iza tion, which could con trib ute to den tal de cay.
Our re sults sho wed that male rats ne o na tally tre a ted with MSG not only had body we ight gain re duc ti on but also lo wer sub man di bu lar gland (SMG) we -ights (Graph 1). Espe ci ally in ro dents, it is well es ta blis hed that SMG de ve lop ment and dif fe ren ti a ti on are un der the in flu en ce of a mul ti hor mo nal con -trol18
, which plays a de ci si ve role on its se xu al di -morp hism. Sin ce 70% of to tal sa li va are from the SMG11
, the hor mo nal im ba lan ce trig ge red by MSG could ex pla in the re duc ti on of SMG we ight (Graph 1) and the im pa i red sa li vary res pon se to pi lo car pi ne sti mu la ti on (Ta ble 1). In ad di ti on, it is con ce i va ble
TABLE 1 - Sa li vary func ti ons of male and fe ma le rats. Re sults for con trols and rats ne o na tally tre a ted with mo no so di -um glu ta ma te (MSG). Re sults are mean ± stan dard er ror of the mean (SEM) of 16 ob ser va ti ons throug hout.
Groups Salivary flow
(ml/min per 100 g BW)
Total protein content (mg/ml)
Amylase activity (U/ml ´ 1 0– 2)
Con trol Ma les 17.60 ± 0.50
a
8.39 ± 0.28a
14.70 ± 0.47a
Fe ma les 23.40 ± 0.60c
8.13 ± 0.34a
15.03 ± 0.38a
MSG-tre a ted Ma les 12.80 ± 0.50
b
10.32 ± 0.41b
20.39 ± 0.60b
Fe ma les 14.10 ± 0.60b
12.18 ± 0.24c
22.29 ± 0.26c
Me ans fol lo wed by dis tinct su pers cript let ters are sig ni fi cantly dif fe rent from each ot her (ANOVA, p < 0.05).
TABLE 2 - Erup ti on of the in ci sors of male and fe ma le rats. Re sults for con trols and rats ne o na tally tre a ted with mo no -so di um glu ta ma te (MSG). Re sults are mean ± stan dard er ror of the mean (SEM).
Groups Eruption rate (mm) Eruption observed (% of the litter)
Total Per day At the 8th
day At the 9th
day
Con trol
Ma les 11.859 ± 0.100a
0.566 ± 0.0050a
25 75
Fe ma les 11.488 ± 0.141b
0.549 ± 0.0068b 44 56
MSG-tre a ted Ma les 11.416 ± 0.085
b
0.542 ± 0.0045b 53 47
Fe ma les 10.841 ± 0.071c 0.517 ± 0.0036c
67 33
Me ans fol lo wed by dis tinct su pers cript let ters are sig ni fi cantly dif fe rent from each ot her (ANOVA, p< 0.05). For the erup ti on rate and erup ti on day, data are from 16 ob ser va ti ons for every group.
TABLE 3 - Mi cro hard ness of in ci sor ena mel of male and fe ma le rats. Re sults for con trols and rats ne o na tally tre a ted with mo no so di um glu ta ma te (MSG). Re sults are mean ± stan dard er ror of the mean (SEM) of de ter mi na ti ons car ri ed out in 56 te eth for every group.
Groups
Microhardness (kgf/mm2´ 1 0– 3)
Longitudinal section
Transverse
section Crown Root
Con trol Ma les 271.84 ± 2.26
a
259.98 ± 3.39d
273.98 ± 2.07a
257.84 ± 3.43d
Fe ma les 262.62 ± 2.02b 253.85 ± 2.75e 263.44 ± 2.07b 253.03 ± 2.69e
MSG-tre a ted
Ma les 262.78 ± 2.12b 253.79 ± 2.49e 265.24 ± 1.76b 251.33 ± 2.50e
Fe ma les 257.52 ± 1.12c 245.08 ± 2.23f 256.01 ± 1.99c 246.59 ± 2.51f
that some re duc ti on in the den sity and/or res pon si -ve ness of au to no mic mus ca ri nic re cep tors of the gland could con tri bu te to the al te red res pon se.
As a part of the histomorphological and bio chem i cal sex ual di mor phism of the sal i vary glands in var i -ous mam mal species22
, the num ber of muscarinic and b-adrenergic re cep tors in the SMG can be 25-51% higher in fe male than in male rats6, and this could ex plain the higher sal i vary flow of our con trol fe males (Ta ble 1). On the other hand, this sex dif fer ence dis ap peared in our MSGtreated an i mals, pre sum ably as a func tion of the drugin duced al ter -ations of cir cu lat ing lev els of go nadal steroids17.
Con cen tra tions of to tal pro teins and of am y lase ac tiv ity in sa liva of MSGtreated rats were sig nif i -cantly higher than those found in con trols (Ta ble 1). How ever, if these val ues are taken as a func tion of the sal i vary flow, such dif fer ences are blunted (for ex am ple, con trol males = 0.148 ± 0.004 and MSGtreated males = 0.132 ± 0.005 mg pro tein se -creted/min per 100 g BW; p > 0.1). These re sults sug gest that the net syn the sis and/or re lease of sal i vary pro teins were not af fected by the drug, and only the fluid pro duc tion was in fact re duced. On the other hand, the mag ni tude of pro tein in -crease in sa liva was undistinguishable from that showed by am y lase, thus sug gest ing that the spe -cific pro tein which in creased as an ef fect of MSG treat ment was largely am y lase.
Re gard ing enamel min er al iza tion, as ob served in other tis sues and func tions, a rea son able de gree of sex di mor phism ex ists in the micro hard ness of the tooth enamel, that of males be ing higher than that of fe males. Though MSG treat ment was able to sig nif i cantly di min ish both crown and root micro hard ness, those sex dif fer ences were not abol ished (Ta ble 3).
Among other fac tors pos si bly in volved, the hor monal im bal ances due to MSG treat ment con ceiv -ably play a very im por tant role. Pre- or postnatal hypothyroidism slows down den tal de vel op ment, lead ing to de fects in the enamel which are ob -served later in life14. In ter fer ence with the growth hor mone (GH) could also im pair the for ma tion and min er al iza tion of the teeth, since GH re cep tors are pres ent in di vid ing cells, preameloblasts, dif fer en ti at ing preodontoblasts, and in se cret ing amelo -blasts and odon to -blasts of 45-day rat in ci sors and molars28
. In ad di tion, GH de fi ciency re duces rRNA ex pres sion in preameloblasts and preodonto -blasts29
and the syn the sis of two proteo glycans, decorin and biglycan, thus im pair ing the cor rect tooth for ma tion and mi nera lization30.
Over all, the hor monal al ter ations caused by neo -na tal treat ment of rats with MSG most pre sum ably in ter fered with sev eral steps of tooth for ma tion, in -clud ing the in tes ti nal ab sorp tion of Ca2+, and the syn
-the sis of pro teins and proteoglycans which built up the extracellular ma trix and play a fur ther role in the pro cess of den tal min er al iza tion. Also, the en zymes in volved in amelogenesis could also be af fected.
In fe rior in ci sors erup tion both in male and fe male rats oc curred at the 9th postnatal day in con trols and
at the 8th
day in MSG-treated an i mals (Ta ble 2). The lim it ing fac tor to den tal erup tion is the re sorp tion of al ve o lar bone, which forms a path for erup tion and de pends upon the for ma tion and ac tiv ity of osteoclast cells2 7. The pro cess as a whole, me di ated
through the ex pres sion of osteoprotegerin, is reg u lated by a del i cate hor monal bal ance be tween syn er -gis tic (parathyroid hor mone, glucocorticoids)13,27
and an tag o nis tic in flu ences (estrogens, GH)2. Al though
the ex act mech a nism by which neo na tal MSG caused an ac cel er ated rat in ci sors erup tion is at pres ent an un re solved ques tion, this might be the re sult of multifactorialde pend ent, in creased os -teoclastic ac tiv ity.
The rates of in ci sor erup tion were found to be slower in fe male than in male rats; neo na tal MSG treat ment caused a global re duc tion in these rates but did not in ter fere with the ob served sex dif fer -ence (Ta ble 2). It is known that erup tion reg u la tion in rats de pends upon the bal anced ac tiv ity of cementoblasts and of periodontal lig a ment cells, which in turn are stim u lated by GH and in hib ited by parathyroid hor mone (PTH)20. Even though in
this ex per i men tal model the se rum lev els of PTH have not been stud ied yet, and the par tic i pa tion of corticoid hor mones can not be discarded16, our re -sults can be par tially ex plained by the re duc tion of GH cir cu lat ing lev els caused by MSG8
.
CONCLUSION
In con clu sion, neo na tal MSG treat ment causes a se ries of oral dis tur bances in adult hood in rats, in clud ing sal i vary flow re duc tion and in ci sors min -er al iza tion and -erup tion dis tur bances. Our data sup port the view that the co hort of hor monal im -bal ances caused by hy po tha lamic mal func tion ing can be ac counted for by many (if not all) of the al -ter ations re ported herein, and may cul mi nate in higher car ies sus cep ti bil ity.
ACKNOW LED GE MENT
REFERENCES
1. Ali MM, Ba wa ri M, Mis ra UK, Babu GN. Lo co mo tor and learn ing de fi cits in adult rats ex po sed to mo no so di umLglu -ta ma te du ring early life. Ne u ros ci Lett 2000;284:57-60. 2. Au bin JE, Bon nel ye E. Oste o pro te ge rin and its li gand: a
new pa ra digm for re gu la ti on of os te o clas to ge ne sis and bone re sorp ti on. Oste o po ros Int 2000;11:905-13.
3. Bel lis le F. Glu ta ma te and the UMAMI tas te: sen sory, me ta -bo lic, nu tri ti o nal and be ha vi ou ral con si de ra ti ons. A re vi ew of the li te ra tu re pu blis hed in the last 10 ye ars. Ne u ros ci Bi -o be hav Rev 1999;23:423-38.
4. Be nar de MA, Fa bi an FW, Ro sen S, Hop pert CA, Hunt HR. A met hod for the col lec ti on of lar ge quan ti ti es of rat sa li va. J Dent Res 1956;35:326-7.
5. Bre i vik T, Thra ne PS, Gjer mo P, Fon num F. Post na tal glu ta ma tein du ced cen tral ner vous system le si ons al ter pe ri o don tal di se a se sus cep ti bi lity in adult Wis tar rats. J Clin Pe -ri o don tol 2001;28:904-9.
6. Bylund DB, Mar ti nez JR, Pi er ce DL. Re gu la ti on of au to no -mic re cep tors in rat sub man di bu lar gland. Mol Phar ma col 1982;21:27-35.
7. Ca ra way WT. A sta ble starch subs tra te for the determina -tion of amy la se in se rum and ot her body flu ids. Am J Clin Pat hol 1959;32:97-9.
8. Dada MO, Camp bell GT, Bla ke CA. Effects of ne o na tal ad -mi nis tra ti on of mo no so di um glu ta ma te on so ma to trophs and growth hor mo ne se cre ti on in pre pu ber tal male and fe -ma le rats. Endo cri no logy 1984;115:996-1003.
9. Dhind sa KS, Omran RG, Bhup R. Effect of mo no so di um glu ta ma te on the his to ge ne sis of bone mar row in mice. Acta Anat 1978;101:212-7.
10. Ger lach RF, To le do DB, No va es PD, Mer zel J, Line SRP. The ef fect of lead on the erup ti on ra tes of in ci sor te eth in rats. Archs Oral Biol 2000;45:951-5.
11. Hol lo way PJ, Wil li ams RAD. A study of the oral se cre ti on of rats sti mu la ted by pi lo car pi ne. Archs Oral Biol 1965;10:237-344.
12. Jo seph BK, Sa va ge NW, Yonny WG, Wa ters MJ. Pre na tal ex pres si on of growth hor mo ne re cep tor/bin ding pro te in and in su linlike growth, fac torI (IGFI) in the ena mel or gan. Role for growth hor mo ne and IGFI in cel lu lar dif fe ren -ti a -ti on du ring early to oth for ma -ti on? Anat Embryol 1994;189:489-94.
13. Kan za wa M, Su gi mo to T, Ka na ta ni M, Chi ha ra K. Invol ve ment of os te o pro te ge rin/os te o clas to ge ne sis inhi bi tory fac tor in the sti mu la ti on of os te o clast for ma ti on by pa rath yro -id hor mo ne in mou s e bone cells. Eur J Endo cri nol 2000;142:661-4.
14. Kel ler EE, Sat her AH, Hay les AB. Den tal and ske le tal de ve -lop ment in va ri ous en do cri ne and me ta bo lic di se a ses. J Am Dent Assoc 1970;81:415-9.
15. Lowry OH, Ro se brough NS, Farr AL, Ran dall RJ. Pro te in me a su re ment with the Fo lin phe nol re a gent. J Biol Chem 1951;193:275-82.
16. Ma cho L, Je zo va D, Zo rad S, Fic ko va M. Post na tal mo no so di um glu ta ma te tre at ment re sults in at te nu a ti on of cor ti
-cos te ro ne m e ta bo lic rate in adult rats. Endocr Re gul 1999;33:61-7.
17. Ne me roff CB, La mar ti ni e ri CA, Ma son GA, Squilb RE, Hong JS, Bondy SC. Mar ked re duc ti on in go na dal ste ro id hor mo ne le vels in rats tre a ted ne o na tally with mo no so di umLglu ta ma te: fur -ther evi den ce for dis rup ti on of hypot ha la mic-pi tu i tary-go na dal axis re gu la ti on. Ne u ro en do cri no logy 1981;33:265-7.
18. Oli ve i ra-Fi lho RM, Fava-de-Mo ra es F, Mi net ti C, Mou ra NM, To le do MI. Mul ti hor mo nal con trol of the mu ri ne sub man di bu lar gland. US/La tin Ame ri can Work shop on Sa li -vary Re se arch. Pro ce e dings of a Con fe ren ce held at the Pan Ame ri can He alth Orga ni za ti on. Was hing ton: C&A Press; 1992. p.109-30.
19. Olney JW. Bra in le si ons, obe sity and ot her dis tur ban ces in mice tre a ted with mo no so di um glu ta ma te. Sci en ce 1969;164:719-21.
20. Ou yang H, McCa u ley LK, Berry JE, D’Errico JA, Stryhorn CL, So mer man, MJ. Res pon se of im mor ta li zed mu ri ne ce -men to blasts/pe ri o don tal li ga -ment cells to pa rath yro id hor mo ne and pa rath yro id hor mo ne-re la ted pro te in in vi tro. Arch Oral Biol 2000; 45:293-303.
21. Ren zi A, Lo pes RA, Sala MA, Ca mar go LAA, Me na ni JV, Saad WA, et al. Morp ho lo gi cal, morp ho me tric and ste re o lo -gi cal study of sub man di bu lar glands in rats with le si on of the an te ro ven tral re gi on of the third ven tri cle (AV3V). Exp Pat hol 1990;38:177-87.
22. Sa wa da K, Nou mu ra T. Effects of cas tra ti on and sex ste -roids on se xu ally di morp hic de ve lop ment of the mou se sub man di bu lar gland. Acta Anat 1991;140:97-103. 23. Shi no da H. Effect of long-term ad mi nis tra ti on of flu o ri de
on physi co-che mi cal pro per ti es of the rat in ci sor ena mel. Cal cif Tiss Res 1975;18:91-100.
24. Spi e gel R, Sat her H, Hay les A. Cep ha lo me tric study of chil -dren with va ri ous en do cri ne di se a ses. Am J Orthod 1971;59:362-75.
25. Teng CM, Sob bows ki J, John ston L Jr. The ef fect of cor ti so -ne on the erup ti on rate of root re sec ted in ci sors in the rat. Am J Orthod Den to fac Orthop 1989;95:67-71.
26. Thomp son MC, Nor ton NS, Ro dri guez-Si er ra JF, Lip pi el lo L. Growth hor mo nere le a sing hor mo ne de ple ti on in the fe -ma le rat, si mi la ri ti es to aging. J Ge ron tol A Biol Sci Med Sci 1996;51:B83-90.
27. Wise GE, Gri er RL, Lump kin SJ, Zhang Q. Effects of de xa -met ha so ne on to oth erup ti on in rats: dif fe ren ces in in ci sor and mo lar erup ti on. Clin Anat 2001;14:204-9.
28. Zhang CZ, Young WG, Wa ters MJ. Immu nocy to che mi cal lo ca li za ti on of growth hor mo ne re cep tor in rat ma xil lary teeth . Archs Oral Biol 1992;37:77-84.
29. Zhang CZ, Young WG, Li H, Ro bin son S, Wa ters MJ. Growth hor mo ne re gu la tes nu cle o lar or ga ni zer re gi ons du -ring odon to ge ne sis in the rat. J Oral Pat hol Med 1992;21:395-400.
30. Zhang CZ, Li H, Bar told PM, Young WG, Wa ters MJ. Effect of growth hor mo ne on the dis tri bu ti on of de co rin and bi -glycan du ring odon to ge ne sis in the rat in ci sor. J Dent Res 1995;74:1636-43.
