Calc. Tiss. Res. 2, 145--156 (1968)

Effect of Intraperitoneal Injections of Tetracycline Hydrochloride and Oxytetracycline on Forming Enamel of Rat Incisors* C.-G. LOFGREN, K . - A . OMNELL, a n d M. U. NYLON Oral Roentgendiagnostic Department, University of Lund, School of Dentistry, MalmS, Sweden, and the Laboratory of Histology and Pathology, National Institute of Dental Research, Bethesda, Md., USA Received February 29, 1968 The effect of tetracycline hydrochloride (TC) and oxytetracycline (OTC) on rat incisor enamel was compared in studies utilizing microroentgenography and fluorescence microscopy. Twenty animals received a course of 4 intraperitoneal injections, comprising first 2 low and subsequently 2 high equivalent dosages of the 2 antibiotics (Table 1). The low dosages had little if any effect on the enamel. In contrast, the high dosages of both TC and OTC resulted in developmental disturbances ranging from an incremental band only to one that also included a gross hypoplastic lesion. A comparison between the 2 drugs revealed a much higher incidence of hypoplasias among the defects caused by TC than those due the OTC (Table 2). Some of the hypoplastic defects exhibited tetracycline fluorescence while others were negative as were the incremental bands and the normal enamel. The labelled lesions included all of those caused by the third and none of those due the fourth injection (Table 2). I t is suggested that the fluorescent lesions were labelled by the administration subsequent to the one causing them. Key words: Tetracycline - - Rat Incisor - - Enamel Hypoplasia - - Enamel Fluorescence - Microroentgenography. L'action du chlorhydrate de t~tracycline (TC) et de la oxyt6tracycline (OTC) sur l'6mail de l'incisive du rat cst compar6e en des 6tudes utilisant la microroentg6nographie et la microscopie de fluorescence. Vingt animaux rccurent une s6rie de 4 injections intrap6riton6aux, comprenant d'abord 2 dosages faibles et, par la suite, 2 dosages d'6quivalence forte des 2 antibiotiques (Tableau 1). Les dosages faibles avaient peu ou prou d'aetion sur l'6mail. Au contraire, les dosages forts du TC et de I'OTC tons les deux aboutirent s troubles 6volutifs, ne variant que d'une bande incr6mentale jusqu'~ une bande que comprenait aussi une grosse 16sion hypoplastique. Une comparaison entre les deux medicaments r6v61a une incidence plus haute de hypoplasies entre les d6fauts provoqu6s par le TC que les hypoplasics caus6es par I'OTC (Tableau 2). Quelques-uns des d6fauts hypoplastiques manifestaient la fluorescence de la t6tracycline, tandis que d'autres 6taient n6gatifs comme l%taient les bandes incr~mentales et l'6mail normal. Les 16sions marqu6es comprenaient routes cclles provoqu6es par la troisi~me injection, et aucune d'elles provoqu6es par la quatri~me injection (Tableau 2). I1 est 6vident que les lesions fiuorescentes 6taient marqu6es par l'administration de l'injection qui les pr6cSde. Der Einflul3 von Tetracyclin Hydrochlorid (TC) und Oxytctracyclin (OTC) auf den Schmelz yon Rattenschneidezahnen wurde mit mikro-rSntgenologischen und fluorescenz-mikroskopischen Methoden untersucht. * This work was supported by the Swedish Medical Research Council, Project No. 12X-169. For reprints: Dr. C. G. LOFGREN, Oral Roentgendiagnostic Department, University of Lund, School of Dentistry, MalmS, Sweden.

146

C.-G. LOFGREIg,K.-A. OMNELLand M. U. I~YLEN:

Zwanzig Tiere erhielten zungchst 2 kleinere intraperitoneale Dosen, und danach 2 gr6Bere intraperitoneale Rosen der beiden Antibiotiea in gleichwertiger Menge (Tabelle 1). Die kleinen Dosen hatten wenig oder keinen Effekt am Zahnschmelz. Die groBen Dosen yon TC und OTC dagegen verursachten EntwicklungsstSrungen, die yon Ver~nderungen an den Wachstumsbgndern bis zu hypoplastischen Herden innerhalb dieser Bgnder schwankten. Die vergleichende Auswertung der beiden Medikamente zeigtc, dab hypoplastische Herde hgufiger nach Verabreichung yon TC vorkommen, als nach Verabreichung yon OTC. Manche hypoplastische Herde zeigten Tetracyclin-Fluorescenz; andere Herde, sowohl die Wachstumsb~nder a]s auch der normale Schmelz, waren negativ. Herde mit Fluorescenz waren durch die dritte Injektion verursacht, Herde die nach der vierten Injektion auftraten, zeigten keine Fluorescenz (Tabelle 2). Es ist anzunehmen, dag die Fluorescenz dieser Herde zustande kam, nachdem diese Herde durch die vorausgegangene Injektion verursacht worden waren. Introduction A number of clinical and experimental studies have shown t h a t tetracyclines administered during enamel formation m a y interfere with its normal development (BEvELA~D~ et al., 1961; WALL~AN and HILTOn, 1962a, b; HARCOURT, 1963; WITKOP and WOLF, 1963; HAXALA and MJ4KEL:~, 1963; STO~EY, 1963a, b; OWEN, 1963; ML~NI and B~USATI, 1963; KLr~E et al., 1964; NYLEN et al; 1964; LoJoDIc]~ et al., 1965; LU~IN and KLr~E, 1966; OM~ELL et al. 1966; KOWALEWSKA et al., 1966; I-IAMF, 1967). The effect of one of the tetraeylines, tetracycline hydroehloride (TC), on forming rat incisor enamel has been investigated in detail b y us in animals approximately seventy-five days old and the results will be reported elsewhere. I t was found t h a t the disturbances in structure and mineralization following intraperitoneal injections of the drug paralleled the dose level, i.e., the higher the dose, the more pronounced the enamel malformation. At doses below 30 mg/kg body weight, the enamel seemed normal most of the time, while at single dosages between 30 and 65 mg/kg, a hypomineralized incremental band was often found running from the dentino-enamel junction to the enamel surface. Following single and multiple injections with dosages of 130 mg/kg, enamel formation was much more severely affected. I n addition to incremental bands, the number of which corresponded to the number of injections, gross hypoplastie lesions were invariably present. The present paper reports on additional experiments in which the effect of another tetraeyline homoloque, oxytetraeycline (OTC), was compared to t h a t of TC. Both antibiotics are prescribed frequently to children in the age group when tooth formation occurs. I t has been suggested, however, t h a t OTC m a y not produce the same abnormality as TC (WALLMA~ and HrLTO~, 1962a; KoWXLEWSKA et al., 1966), but no experimental data are available to support this suggestion. Thus, the present investigation was undertaken to examine in more detail the possibility t h a t equivalent doses of the two homoloques m a y v a r y in their ability to interfere with enamel formation. Material and Methods Sprague-Dawley rats, approximately 85 days old, and maintained on a standard laboratory diet were used. Ten rats were given 25 mg/kg of TC on day zero and five days later an equivalent dose, tool per mol, of OTC 1 followed 1Commereia] samples of Tetradecin (TC) and Terramycin (OTC) were secured through the courtesy of Astra, Inc., SSdertglje, Sweden, and Pfizer, Inc., N~sbypark, Sweden, respectively

Effect of Tetracycline Hydrochloride and Oxytetracycline on Forming Enamel

147

by 150 mg/kg of TC on day 10 and an equivalent dosage of OTC on day 15. I n another series of 10 animals the sequence of administration was reversed, with a low-dose OTC injection starting the experiment (Table 1). Six animals serving as controls were given 4 injections of physiologic saline at comparable time intervals. The tetracyclines were dissolved in physiologic saline and all injections were made intraperitoneally under ether anesthesia. The injected volumes were approximately 1 ml. Table 1. Experimental procedure Series

I II

No. of rats

Dosein mg/kg body weight day 0

day 5

day 10

day 15

day 25

10 10

TC 25.0 OTC 25.8

OTC 25.8 TC 25.0

TC 150.0 OTC 154.8

OTC 154.8 TC 1 5 0 . 0

Sacrifice Sacrifice

The rats were sacrificed by ether inhalation 10 days after the last injection. Immediately following sacrifice, the upper incisors were removed en bloc with surrounding bone. The specimens were fixed in 70 per cent alcohol and embedded in Ward's Bioplastic. Planoparallel midsagittal sections, approximately 50 ~m thick were prepared by manual grinding. Contact microroentgenography was carried out utilizing a Phihps P W 1010 roentgen generator equipped with a tube operated at 24 kV providing Ni-filtered CuK-radiation (8.1 keV) and a Philips CMR-5 unit operated at 4.8 kV giving continuous radiation. Several microroentgenograms were obtained of each section so that all areas were imaged within a range of photographic densities where minor differences in mineral distribution could be discovered by direct microscopy. The sections were examined for fluorescence using a Leitz Ortholux microscope equipped with a light field condenser for general surveys, or a dry dark field condenser for higher magnifications. An Osram HBO 200 W type L 1 lamp served as light source. Ultraviolet light at wavelenghts between 300 and 400 ~m was obtained by passing the light through a 2 mm UG-1, a 3 mm UG-5 and a 4 mm BG-38 filter. A 2.5 mm OG-1 filter or an UV absorption filter served as barrier filter. Results

All animals survived the full length of the experimental period although some of the rats lost weight and/or exhibited an abdominal swelling following administration of the large doses of the antibiotics. I n the experimental animals 2 distinct hypomineralized bands were observed in the apical third of the enamel in all but one specimen. In this one specimen only the most apically located band was seen. The relationship of the 2 bands was such that the more apically-located band reached the outer surface approximately where the other band began at the dentino-enamel junction. Because the rodent upper incisor is a continuously-growing tooth, an incremental band which forms along the formative front of the enamel at the time of the drug administration subsequently moves incisally. In the meantime, new enamel is laid down outside and apical to it. I t was concluded because of the relative position of the 2 bands 11

Calc. Tiss. Res., Vol. 2

148

C.-G. L6FGtCEN, K.-A. O~r

and M. U. NYLEN:

Figs. 1 and 2

Effect of Tetracycline Hydrochloride and Oxytetracycline on Forming Enamel

149

and of their limited incisal movement that they were caused by the 2 injections with high dosages of the drugs, received 10 and 15 days prior to sacrifice. I n 3 rats a single, extremely faint hypomineralized line, which could be attributed to one of the injections with the low dosage of the drugs was present in the enamel incisal to the apical pair. I n all the other animals this portion of the enamel appeared completely normal. No pathologic changes were observed in the control specimens. The incremental band was the one manifestation common to all the defects in the enamel, yet it was not the only disturbance. Many of the apically-located lesions comprised additional changes which ranged from purely internal disturbances to gross hypop]astic lesions. In spite of considerable variation, the lesions shared common features which made it possible to group them according to the severity of the disturbance. Group 1 comprised defects where a hypomineralized incremental band was the only major disturbance (Fig. 1). Group 2 included lesions in which additional and marked irregularities were found outside or apical to the incremental band (Figs. 2 and 3). I n both groups, however, the enamel layer was of normal thickness and its outer contour also seemed normal. The presence of gross hypoplastie lesions was considered the most severe response, and all these lesions were placed in group 3 (Figs. 4 and 5). A comparison of the effect of OTC and TC on enamel formation is shown in Table 2. Only one tooth is listed from each rat since the type of defect was found to be identical in contralateral teeth (Fig. 6B and C). The 3 scores available for the low doses are not included in the table. Of the three, all of which belonged to group 1, one resulted from a TC and two from an OTC injection. Table 2 reveals that while only 1 group-3 lesion occurred following an OTC injection, 15 of a total of 20 animals developed this type of malformation as a result of the TC administration. Of the remaining 5 TC induced lesions, 4 belonged to group 2 and only 1 to group 1. I n contrast, 17 of the OTC lesions were classed in group 1. The microroentgenographic studies also provided detailed information about the morphology of the defects. While a thorough discussion will be presented elsewhere, a few details are included in the present paper. I n most specimens, for example, the hypomineralized bands were associated with a bending of the prism layers as evidenced by the changed orientation of the normal line pattern (Fig. 1 C). In some specimens an abrupt shift in roentgen absorption was observed between Fig. 1. A Part of upper incisor from rat which received IP injection of 154.8 mg/kg body weight of oxytetracycline (OTC) 15 days prior to sacrifice. Hypomineralized band indicates location of forming enamel surface at time of drug administration. In this and all subsequent illustrations incisal-apical direction is from left to right. Contact microroentgenogram, • 50. B Schematic drawing of the lesion depicted in A. This type of developmental defect was classed in group 1. Main characteristic: hypomineralized incremental band. C Detail from contact microroentgenogram of lesion shown in A. Prism layers outside band form a larger incisal angle with dentino-enamel junction than those inside, • 360 Fig. 2. A Enamel disturbance caused by injection of 154.8 mg/kg OTC 10 days before sacrifice. Zone of irregular enamel is found outside and along apical half of hypomineralizcd incremental band. Contact microroentgenogram, • 55. B Schematic drawing of enamel lesion depicted in A. This type of disturbance together with that shown in Fig. 3B, was classed in group 2. Main characteristics: hypomineralized incremental band and irregular enamel outside and along its apical part. C Higher magnification of irregular enamel from A, • 350 11"

150

C.-G. L6FGEEI,~, K.-A. OMNELL and M. U. NYLEN:

Figs. 3 and 4

Effect of Tetracycline Hydrochloride and Oxytetracycline on Forming Enamel

151

Table 2. Severity o/enamel de/ects caused by high dosages o] TC or OTC Series I, l0 animals

Series II, 10 animals

Injection

No. 3

No. 4

No. 3

:No. 4

Dosage

150.0 mg/ kg bw TC

154.8 mg/ kg bw OTC

154.8 mg/ kg bw OTC

150.0 rag/ kg bw TC

Age of defect

15 days

10 days

15 days

10 days

Sex

male

male

male

male

Severity of enamel defect

1 3'

female

female

female

3' 2 3' Sex

female

Severity of enamel defect

3l

2 3~ 3'

1 1 1

1

3~

1 = incremental band only; 2 incremental band with additional marked disturbances; 3 ~ incremental band and gross hypoplastic lesion; ' ~ fluorescent defects. the enamel layers on either side of the i n c r e m e n t a l b a n d (Fig. 4A). This difference, which was more m a r k e d the more severe the lesion, was m u c h more p r o n o u n c e d i n 10 t h a n in 15 d a y old lesions of identical severity. Fig. 4 also shows the only specimen in which a n increase was f o u n d of a v e r y small p o r t i o n of the enamel n e a r the base of the i n c r e m e n t a l band. All the other lesions classed as group 3 were characterized b y a considerable r e d u c t i o n of the e n a m e l layer coupled with the presence of calcified bodies peripheral to the defects (Figs. 5 a n d 6). The gross hypoplastic defects were located a t or n e a r the base of the i n c r e m e n t a l b a n d a n d i n v o l v e d a p p r o x i m a t e l y 0.5 m m of the e n a m e l in a n apical-incisal direction. I n a d d i t i o n to the reduced enamel volume, the bulk of the defective area was hypomineralized, including in most instances the p o r t i o n below the i n c r e m e n t a l b a n d (Fig. 5C). E x a m i n a t i o n of the g r o u n d sections b y ultraviolet light revealed t h a t the 4 injections were recorded i n the d e n t i n as 4 fluorescent lines, the relative position Fig. 3. A Enamel malformation due to injection of 150.0 mg/kg tetracycline hydrochloride (TC) 15 days prior to sacrifice. In addition to disturbances similar to those shown in Fig. 2A, another area of irregular enamel is found at apical end of band. Contact microroentgenogram. • 55. B See legend Fig. 2B. C Higher magnification showing apical disturbance in A in more detail, • 450 Fig. 4. A Enamel lesion resulting from injection of 150.0 mg/kg TC 10 days before sacrifice. Enamel at apical end of incremental band exhibits localized increase in thickness. Note abrupt shift in roentgen absorption between enamel inside and outside incremental band. Contact microroentgenogram, • 55. B Schematic drawing of lesion depicted in A. This type of malformation together with that shown in Fig. 5 B represent group 3. Main characteristics: enamel disturbance includes abnormalities in surface contour in addition to some or all of those associated with groups 1 and 2. C Higher magnification of defective area seen in A, • 225

152

C.-G. L 6 F G ~ r , K.-A. 0MNELL and M. U. NYLE3r:

Figs. 5 and 6

Effect of Tetracycline Hydrochloride and Oxytetracycline on Forming Enamel

153

of which also reflected t h e continuous g r o w t h of t h e t o o t h (Fig. 6 A). N o c o m p a r a b l e fluorescent lines were seen in t h e enamel, n o t even corresponding to t h e location of t h e h y p o m i n e r a l i z e d i n c r e m e n t a l bands. The color of t h e d e n t i n lines varied, w i t h t h e OTC b a n d s yielding a greenish yellow a n d t h e TC b a n d s a golden yellow fluorescence. Comparison of t h e m i c r o r o e n t g e n o g r a p h i c a n d fluorescent images r e v e a l e d a consistent r e l a t i o n s h i p b e t w e e n t h e 2 m o s t apical d e n t i n lines a n d t h e 2 d i s t i n c t i n c r e m e n t a l b a n d s in t h e enamel. I t was f o u n d t h a t t h e fluorescent d e n t i n lines t e r m i n a t e d a t t h e d e n t i n o . e n a m e l j u n c t i o n s l i g h t l y apical to t h e enamel b a n d s w h e t h e r or n o t t h e l a t t e r were associated w i t h gross lesions. A l t h o u g h , as said, t h e i n c r e m e n t a I b a n d s in the e n a m e l were n o t labelled, nor a n y p o r t i o n of t h e n o r m a l enamel, i t was f o u n d t h a t all t h e gross h y p o p l a s t i c defects associated w i t h t h e first high dose injection fluoresced. I n c o n t r a s t those caused b y t h e second a d m i n i s t r a t i o n 5 d a y s l a t e r were non-fluorescent or v e r y w e a k l y fluorescent (Fig. 6A). I n t h e labelled lesions t h e fluorescence was n o t r e s t r i c t e d to a certain n a r r o w zone. I n s t e a d t h e entire h y p o m i n e r a l i z e d e n a m e l b o t h inside a n d outside t h e i n c r e m e n t a l b a n d was labelled. A l t h o u g h all t h e s t r o n g l y fluorescent lesions r e s u l t e d from a TC i n j e c t i o n (Table 2), the color of t h e fluorescence a p p e a r e d greenish r a t h e r t h a n golden yellow.

Discussion A n u m b e r of i n v e s t i g a t o r s h a v e suggested differences in t h e effect of t h e various t e t r a c y c l i n e homologues on developing bones a n d teeth. S A X ~ (1966 a, b) f o u n d t h a t OTC h a d a less p r o n o u n c e d i n h i b i t o r y effect on calcification t h a n TC. On t h e o t h e r h a n d , THANIK a n d McMu•PHY (1966) concluded t h a t OTC caused a g r e a t e r decrease in f e m u r length, volume, a n d d e n s i t y a t high dosages t h a n did TC. Most of t h e studies involving t e e t h h a v e been l i m i t e d to consideration of v a r i a t i o n s in discoloration (W~YMAN, 1965; OW~N, 1963; KIE~TITZ, 1965 a n d IBSEN et al., 1965). T h e only a t t e m p t a t c o m p a r i n g t h e effect of different t e t r a eyclines on t o o t h s t r u c t u r e was m a d e b y WALLMA:N a n d HILTON (1962b). These i n v e s t i g a t o r s suggested, on t h e basis of clinical d a t a , t h a t OTC was less likely to p r o d u c e a b n o r m a l i t i e s t h a n TC when either was a d m i n i s t e r e d to children in t h e n e o n a t a l period. Fig. 5. A Enamel defect caused by injection of 150.0 mg/kg of TC 15 days before sacrifice. Gross hypoplastic defect approximately 0.5 mm in apical-incisal direction is found peripheral to apical third of incremental band. Calcified bodies are located outside defect. The bulk of enamel involved in the defect is hypomineralized, including the portion below the incremental line. Contact microroentgcnogram, x60. B See legend Fig. 4A. C Higher magnification showing in more detail incisal portion of gross hypoplastic defect in A, X 270 Fig. 6. A Fluorescence photomicrograph of upper incisor from rat given 150.0 mg/kg of TC and 154.8 mg/kg of OTC 15 and 10 days, respectively, prior to sacrifice. Left defect, corresponding to first injection is strongly fluorescent, while right defect due to second administration does not seem to fluoresce. Strongly fluorescent dentin lines terminate immediately apical to respective enamel defects, 50 x . B and C Contact microroentgenograms: B from section shown in A, and C from contralateral incisor. High degree of similarity between defects from contralateral teeth was consistent finding. In both sections, second incremental band terminates at the enamel surface approximately at point where first band begins at dentino-enamel junction, X 50

154

C.-G. LSFGR~, K.-A. OMNELLand M. U. ~YLE~:

I n the present study on the effect of TC and OTC on rodent incisor enamel formation, high dosages of TC resulted in a much higher incidence of hypop]astie lesions than did equivalent dosages of OTC administered under identical conditions, thus affirming the suggestion b y WALL~AN and HILTON (1962a). I t should be emphasized, however, t h a t OTC was not harmless as evidenced by the consistent presence of a hypomineralized incremental band and an occasional more severe lesion. The changes in enamel formation following administration of low doses of TC and OTC were so few and diffuse t h a t no conclusion could be made regarding possible differences in the action of the 2 antibiotics at this level. Experiments are presently aimed at comparing the effect of OTC and TC on enamel formation, utilizing dosages between those listed in the present investigation. The shift in roentgen absorption across the incremental band was found frequently in all groups, but was most pronounced in the group-3 lesions resulting from the last high dose injection. Whether the mineral content was increased in the inner enamel as found after sodium fluoride injections (WEBwR and u 1964) and/or decreased in the outer enamel in comparison to normal levels for this stage of development was not determined. However, as observed by WEBER and YEAGER (1964) the difference seemed to decrease or disappear as the enamel matured. Published data concerning labelling of enamel by tetracycline have been rather contradictory, particularly with regard to the permanence of the label. STO~EY (1963, a, b), BoYDE (1964), SUGA and MURAYAMA (1965), BEVELANDE~ and 57AKA~A~A (1966), and HAMMARSTRSM (1967) found experimentally that tetracycline was taken up very rapidly b y the entire layer of immature enamel. The fluorescence faded, however, as the labelled enamel matured and disappeared completely 5 - - 7 days after administration of the antibiotic. This loss of fluorescence was interpreted as due to either removal of the tetracycline or loss of its ability to fluoresce during maturation of the enamel (BoYDE, 1964). BEVELANDER et al. (1961) and A~TALOVSK/~ (1966) observed persistence of weakly fluorescent bands in the enamel of rat incisors. I n a later study, BEVELANDER and NAKAHARA (1966) demonstrated the presence of a diffusely fluorescent enamel layer in incisors of rats which had received high dosages of TC subcutaneously 5--7 days prior to sacrifice. EGE~ et al. (1965), who also used rat incisors in their experiments, found t h a t the enamel was not labelled following either single or multiple injections intravenously with low dosages. HARCOURT (1963) detected tetracycline in hypoplastic areas within the enamel of deciduous human teeth whereas the fully calcified matrix did not fluoresce. I n view of this latter finding it was interesting to discover in the present material t h a t of the 16 gross hypoplastic defects only the 7 which were exposed to a subsequent administration fluoresced strongly (Table 2). In contrast, the other 9 were non-fluorescent or fluoresced very weakly. In the labelled lesions the entire gross defect fluoresced, including portions which, judging from their position relative to the 2 incremental bands, were laid down after the first, but prior to the second injection (Fig. 6A and B). Secondly, the color of the fluorescence was greenish rather than golden yellow, matching that of the OTC bands in the dentin. These findings together with the fact t h a t all but one of the 16 lesions were caused b y identical dosages of TC strongly suggest that the fluorescence

Effect of Tetracycline Hydrochloride and Oxytetracycline on Forming Enamel

155

r e m a i n i n g in t h e more incisally l o c a t e d defects o r i g i n a t e d f r o m t h e s u b s e q u e n t injection. The o b s e r v a t i o n t h a t the fluorescent areas were h y p o m i n e r a l i z e d as were those seen b y HARCOURT (1963) m a y be one i m p o r t a n t factor. Thus Sg~A a n d KATAGIRI (1967) f o u n d t h a t h y p o m i n e r a l i z e d defects i n d u c e d b y N a F a n d SrC12 could be labelled s u b s e q u e n t l y b y t e t r a c y c l i n e . I t is n o t surprising if t h e t e t r a c y c l i n e p e r m e a t e s h y p o m i n e r a l i z e d enamel. T h e large i n t e r c r y s t a l l i n e spaces in these areas (NYL]~N et al., u n p u b l i s h e d d a t a ) would m a k e this enamel as accessible to t h e fluorophore as t h e i m m a t u r e t i s s u e . H o w e v e r , t h e lack of label in t h e i n c r e m e n t a l b a n d s a n d its presence in t h e gross defects suggest t h a t t h e a n t i b i o t i c o n l y g a i n e d access where t h e h y p o m i n e r a l i z e d enamel b o r d e r e d d i r e c t l y on t h e ameloblasts. Since it is n o t u n d e r s t o o d w h y n o r m a l enamel loses its fluorescence d u r i n g m a t u r a t i o n , i t is difficult to e x p l a i n w h y t h e label should be r e t a i n e d in p r e e x i s t i n g h y p o m i n e r a l i z e d lesions. The d a t a does i n d i c a t e one thing, however, i.e. t h e n o r m a l loss of fluorescence is p r o b a b l y n o t due solely to space restrictions (BoYDE, 1964). If this h a d been the case one would e x p e c t t h a t the apical lesions h a d r e t a i n e d the label below the i n c r e m e n t a l line since t h e spaces here a p p a r e n t l y were sufficiently large to allow r e - e n t r y of t h e a n t i b i o t i c l a t e r on (Fig. 6A). I n considering t h e origin of an e n a m e l lesion, t h e findings of SUGA a n d KATAGIRI (1967) show t h a t e n a m e l defects which fluoresce are n o t necessarily caused b y t h e fluorophore. On t h e o t h e r hand, as d e m o n s t r a t e d in t h e p r e s e n t m a t e r i a l , nonfluorescent lesions m a y be due to t h e antibiotic. W h e t h e r fluorescent or not, only defects t h a t are associated with m a t c h i n g fluorescent d e n t i n lines as illust r a t e d in Fig. 6 A a n d B, should be a t t r i b u t e d to t h e antibiotic. These findings also suggest t h a t where e n a m e l is concerned i n c o r p o r a t i o n of a t e t r a c y c l i n e is n o t l i m i t e d to the f o r m a t i v e stage, b u t m a y occur p o s s i b l y during t h e entire p r e - e r u p t i v e phase p r o v i d e d defective zones are present. References

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