THE ANATOMICAL RECORD 211:17-23 (1985) The Effect of Adriamycin on Rat Incisor One Day After Administration A.C. KARIM Department ofAnatomy, University of Manitoba, Winnipeg, Manitoba, Canada R3E OW3 ABSTRACT The effect of adriamycin on rat incisor was investigated 1 day after administration. Rats were injected intravenously with adriamycin a t a dose of 5 mgl kg body weight and sacrificed by perfusion with a 2.5% phosphate buffered glutaraldehyde solution. The principal effect of the drug on the incisor was the production of cell degeneration. This was extensive in the apical region, being present along the entire periphery of the dental papilla. In sections examined more incisally, cell degeneration gradually disappeared from the labial portion of the incisor but was present in the lingual portion. This degeneration of cells was not present a t the site where mature odontoblasts had differentiated on the lingual surface of the pulp chamber. It appears that the affected cells were early preodontoblasts and the precursors of preodontoblasts. However, as these cells became more differentiated they apparently became more resistant to the drug’s effect. Osteodentin resembles bone because of the cellular inclusions present within the matrix. The latter appears less dense than that of normal dentin. This may be due to the large amount of ground substance normally found in osteodentin (Takuma et al., 1967),and the presence of fewer collagen fibers (Takuma et al., 1967; Karim and Eddy, 1984). Unlike dentin, osteodentin is atubular (Takuma et al., 1967)and is deposited continuously until the pulp is completely obliterated (Karim and Eddy, 1984). The cells that are responsible for normal dentin production differentiate into odontoblasts from the mesenchymal cells of the dental papilla under the organizing influence of the inner dental epithelium (Bhussry, 1976).Radioautographic studies have demonstrated that the odontoblasts participate actively in the formation of the dentin matrix (Weinstock and Leblond, 1974; Josephesen and Warshawsky, 1982). These studies have demonstrated protein synthesis in the odontoblasts after 3H-proline administration. This protein synthesis was first observed in the rough endoplasmic reticulum and in the Golgi apparatus. It then appeared to migrate from the Golgi zone to the odontoblastic process in the form of dense granules, which then emptied into the extracellular collagenous matrix. It has been proposed that osteodentin formation in the rat incisor, seen after the administration of cyclophosphamide (Koppang, 1973) and vincristine (Stene, 19781, was the result of a n injury to preodontoblasts and their precursors. Although this had never been demonstrated, it was further suggested that abnormally differentiated pulp mesenchymal cells would replace these damaged cells and begin the production of osteodentin (Stene and Koppang, 1980; Koppang, 1981). Recently, adriamycin was shown to cause osteodentin formation in rat incisors (Karim and Eddy, 1984). The present study was undertaken to determine whether injury to preodontoblasts and precursor cells in the lesion was present in the rat 0 1985 ALAN R. LISS, INC incisors after administration of adriamycin. This effect is of significant interest in view of the hypothesis that osteodentin formation may be the result of such a lesion, and would provide a basis for future studies on osteodentin formation in this animal model. MATERIALS AND METHODS Ten male Sprague-Dawley rats (100 g rt 5) were given a n intravenous injection of adriamycin at a dose of 5 m g k g body weight. The adriamycin was obtained from Adria Laboratories of Canada Limited. One day later the rats were sacrificed by perfusion with a 2.5% phosphate buffered glutaraldehyde solution. Control animals injected with only physiological saline were treated in the same manner. The incisors were removed with the jaws and demineralized in a 4.13% isotonic EDTA solution which was continuously agitated. After demineralization the incisors were cut into 1=mm =thick cross =sectional segments. Longitudinal segments were also prepared. All segments were washed overnight in cold phosphate buffer, postfixed in 1% osmium tetroxide in distilled water at 4°C for 3 hours, dehydrated in graded concentrations of acetone, infiltrated with acetone-Epon mixtures, and embedded in Epon. One=pm =thick sections were routinely prepared and examined with the light microscope. Those segments in which the lesion was found were further trimmed, and ultrathin sections were prepared. These sections were placed on formvar=coated slotted grids and stained with 4% uranyl acetate for 10 min and with lead citrate (Reynolds, 1963) for 15 min. Observations were made with a Hitachi HU-12 electron microscope operated a t 75 kv. Received December 5, 1983; accepted July 20, 1984. 18 A.C. KARIM Light Microscopic Observations RESULTS At the apical end, where the inner dental epithelium The administration of adriamycin produced a lesion in which preodontoblasts and pulp mesenchymal cells were reflects a t the cervical loop to form the outer dental destroyed. This lesion was not localized, but extended epithelium (Fig. 2), cell necrosis was observed along the from the apical end beyond the point where the initial entire peripheral surface of the dental papilla. This cell layer of enamel was being deposited incisally. The fol- degeneration manifested itself as dark globules within lowing results describe this lesion as observed at differ- the sections. In the control sample (Fig. 11, cell degenerent sites along the incisor. Serial and longitudinal ation was not present. Instead, preodontoblasts were sections through many incisors have failed to show the observed along the labial, lateral, and mesial surfaces of the dental papilla. Near the apical foramen, mesenchyformation of osteodentin in this study. ma1 cells were closely apposed to the inner dental epithelium. Further incisally (Fig. 3a), cell degeneration was minimal within the labial portion of the tooth. At this site, there was no degeneration in the layer of cells in close apposition to the inner dental epithelium. Within the lingual portion (Fig. 3b), this cell layer was completely removed by necrosis. Necrosis was also seen within the mesenchymal cells on the periphery of the dental papilla. However, no necrosis was seen in the control sample from these sites. Within the labial portion of the incisor (Fig. 4a), odontoblasts were seen lining the periphery of the dental papilla. On the other hand, within the lingual portion (Fig. 4b), preodontoblasts were seen lining the periphery. Near the cervical loop, mesenchymal cells were closely apposed to the inner dental epithelium. However, at a site where the apical foramen was closed by the root sheath formation (Fig. 51, cell degeneration was seen along the periphery of the pulp chamber, and in the preodontoblast layer within the lingual portion of the incisors. Within the labial portion no degeneration was observed (Fig. 6). At this site secretory odontoblasts were present. Therefore, the cell degeneration was observed at the site of the future cemento-enamel junction (Fig. 6) and along the entire lingual portion of the pulp chamber. Examination of control tissue taken a t a n equivalent site (Fig. 7) has shown no cell degeneration within either the pulp or the preodontoblast layer. Further incisally, where secretory odontoblasts were present on the periphery of the lingual portion of the pulp chamber, a small region of cell degeneration was observed. This was located on the periphery of the pulp chamber and not within the odontoblast layer. When sections were examined incisal to this point, no degeneration was observed. Fig. 1. Cross-section through the apical foramen. In this control sample the inner dental epithelium (IDE) reflects at the cervical loop as the outer dental epithelium (ODE). The dental papilla (DP) is compact. The preodontoblasts (PO) are located on the periphery of the papilla and lining the inner surface of the inner dental epithelium. They do not line the entire surface at this level, but are located mainly within the labial half of the section. In the lingual half containing the apical foramen, the cells that are related to the inner surface of the inner dental epithelium are still immature undifferentiated mesenchyma1 cells of the dental papilla. Toluidine blue. x 160. Fig. 2. Cross-section through the apical foramen. In this experimental sample, note the extent of the cell degeneration (asterisks) within the dental papilla (DP). Some necrosis is also seen within the inner dental epithelium (IDE) and within the stratum intermedium (arrowhead).The effect of adriamycin at this level removed the entire population of preodontoblasts. ODE, outer dental epithelium. Toluidine blue. x 160. EFFECT OF ADRIAMYCIN ON RAT INCISOR Fig. 3. Cross-section through the apical foramen. One day after adriamycin treatment. In the labial portion (a), note that the necrosis (asterisks) is confined to the mesenchymal cells on the periphery of the dental papilla. At this site the differentiated preodontoblasts (arrowhead) associated with the inner dental epithelium (IDE)are unaffected. 0, odontoblasts; PO, preodontoblasts. In the lingual portion, (b), the preodontoblast layer is absent, thereby leaving a space adjacent to the inner dental epithelium. The necrosis (asterisks) is seen within the rnesenchymal cells on the periphery of the dental papilla. ODE, outer dental epithelium. Toluidine blue. x 640. 19 Fig. 4. Cross-section through the apical foramen. In these control samples, the labial half of the section (a)shows preodontoblasts (arrowliead) at the bottom and odontoblasts (0)a t the top, both lining the inner surface of the inner dental epithelium (IDE). The dental papilla (DP) has closely packed mesenchymal cells. In the lingual half of the section (b), note that the preodontoblasts (arrowheads) are lining the inner surface of the inner dental epithelium (IDE). However, at the level of the apical foramen, mesenchymal cells are associated with the inner dental epithelium. ODE, outer dental epithelium. Toluidine blue. x 200. 20 A.C. KARIM Fig. 5. Cross-section through the lingual portion of the incisor. One day after adriamycin treatment. At this site the apical foramen is closed by the formation of the root sheath (RS). Note that the necrosis (asterisks) is present within the preodontoblast layer on the inner surface of the root sheath, and within the peripheral region of the pulp chamber (PI.Toluidine blue. x 320. Fig. 6. Cross-section through the incisor. One day after adriamycin treatment. The necrosis (asterisks) is seen a t the level of the future cementoenamel junction and along the lingual portion of the incisor, as seen in Figure 5. At this site, where the initial layer of dentin is present (arrowheads),there is no necrosis within the labial portion of the incisor. PO, preodontoblasts; RS, root sheath; 0, odontoblasts; A, ameloblasts; P, pulp chamber. Toluidine blue. X 320. Fig. 7. Cross-section through the incisor. In this control sample, taken at the same level as Figures 5 and 6 , note that there is no necrosis within the pulp chamber (P).A, ameloblasts; 0, odontoblasts. Toluidine blue. x 100. dental epithelium and the preodontoblasts (Fig. 10). Nearer the labial surface, the preodontoblasts had difThese observations were made from sections that are ferentiated further (Fig. 11)and eventually attained the fully differentiated state of odontoblasts on the labial represented in Figures 3a and 3b. In the lingual portion of the tooth near the cervical surface of the dental papilla (Fig. 12). Collagen fibers loop, the cells that were degenerating contained large were present between the inner dental epithelium and electron-dense globules (Fig. 8).These cells had few pro- the odontoblast layer. These fibers also formed large files of rough endoplasmic reticulum, but there was an bundles between odontoblasts. Examination of control abundance of free ribosomes within their cytoplasm. At samples taken through the apical foramen has shown this site a wide space, containing few collagen fibers and immature mesenchymal cells a t the cervical loop. Differa fine flocculent material, separated the degenerating entiated preodontoblasts were near the labial surface of cells from the inner dental epithelium. Further from the the dental papilla, and fully differentiated odontoblasts cervical loop, nearer the labial surface (Fig. 9), cell de- were seen on the labial surface. There was no necrosis generation within the dental papilla was still present. within the pulp or the preodontoblast layer. Nevertheless, cells were present in close apposition to DISCUSSION the inner dental epithelium. Irregularly arranged collaThe presence of necrotic cells in a confined region of gen fibers were present among these cells. At the site where there was no cell degeneration, a the apical end of the pulp, as demonstrated in the presfine fibrillar material was present between the inner ent study, appears to be within the area of proliferating Electron Microscopic Observations EFFECT OF ADRIAMYCIN ON RAT INCISOR 21 Figs. 8 and 9.Electron micrographs of sections through the lesion near the apical foramen as shown in Figures 3a and 3b. One day after adriamycin treatment. In the vicinity of the apical foramen (Fig. 8), the necrosis (asterisks) is seen in cells containing a n abundance of free ribosomes. Note also the space between the necrotic cells and the inner dental epithelium (IDE). Nearer the labial surface (Fig. 9) there are fewer necrotic cells (asterisks) within the layer of cells associated with the inner dental epithelium (IDE). Many collagen fibers (arrowheads) are seen among the cells at this site. DP, dental papilla. X 14,400. cells, a s seen in the 3H-thymidine studies of Smith and Warshawsky (1976). These investigators have shown, through serial sections, that as the cells are carried by eruption from the apical to the incisal end of the rat incisor, those on the labial surface mature earlier than those on the lingual surface. This indicated that imma- ture cells were present on the lingual surface for a longer distance from the apical end. In light of the extent of the proliferating zone in the apical end of the rat incisor (Smith and Warshawsky, 1976) and the toxic effect of adriamycin on proliferating cells (Barranco, 1975), the present results indicate that mainly imma- 22 A.C. KARIM Figs. 10 and 11. Electron micrographs of cross-sections through preodontoblasts and inner dental epithelium, as seen in Figure 3a. One day after adriamycin treatment. There is no necrosis in the preodontoblast layer (PO), which is separated from the inner dental epithelium (IDE) by a fine fibrillar matrix. rer, rough endoplasmic reticulum. x 14,400. Fig. 12. Electron micrograph of cross-section through the odontoblasts and inner dental epithelium, as seen in Figure 3a. One day after ndriomycin trcntmcnt. On the lnbinl surfncc tho odontobhttn (0)nrc unaffected by the adriamycin. Large bundles of Van Korff's fibers (arrowheads) are seen passing between the odontoblasts and fanning out into the initial layer of dentin. IDE, inner dental epithelium; rer, rough endoplasmic reticulum. x 10,800. 23 EFFECT OF ADRIAMYCIN ON RAT INCISOR ture dividing cells may be affected. This conclusion is also supported by our electron microscopic results, which have shown that morphologically the necrotic cells had few profiles of rough endoplasmic reticulum and a n abundance of free ribosomes. They have also shown that once these immature cells (preodontoblasts and their precursors) have differentiated to the stage where they have begun to secrete the first layer of dentin, they are presumably insensitive to the cytotoxic effect of the drug. The necrosis seen within the lesion may be due to a n effect of the drug at the nuclear level (Di Marco et al., 1971; Wang et al., 1972). On the other hand, since macrophage activity was not observed within the lesion, the dead cells are probably not removed from the lesion, and instead, may undergo complete autodigestion through the activation of hydrolytic enzyme within them (Singal et al., 1984). The destruction of preodontoblasts may not be an effect unique to adriamycin. Although it was not previously demonstrated, it has been suggested that the formation of osteodentin in rat incisor after treatment with cyclophosphamide (Koppang, 1973) and vincristine (Stene, 1978) was due to a n injury to preodontoblasts and possibly their precursors. In the present study, the lesion extended from the apical end to some point incisally, beyond the closure of the apical foramen. However, one cannot ascertain from this data whether the bulbous portion of the odontogenic organ (Smith and Warshawsky, 1976) was affected. Nevertheless, the destruction of the preodontoblasts and precursor cells has significant functional implications. By way of speculation, if the preodontoblast cell population is not replaced, one would expect a failure in dental hard tissue formation and in closure of the apical foramen. However, since the incisor is erupting continuously, this defect eventually would be eliminated, provided that the stem cell compartment in the bulbous portion of the odontogenic organ was not affected. On the other hand, if these destroyed cells are replaced by abnormally differentiated cells, a s was proposed earlier (Koppang, 19731, this would be of signifcant interest in the mechanism of osteodentin formation. In conclusion, this study has shown that immature pulp cells a t the apical end of the rat incisor are selectively destroyed after adriamycin administration. The functional significance of these morphological findings is presently being investigated. ACKNOWLEDGMENTS The author would like to thank Mrs. S.P. Pylypas for her technical assistance, and Mr. R. Simpson for the photographic illustrations. This work was supported by grants from The Manitoba Health Research Council and The Medical Research Council of Canada. LITERATURE CITED Barranco, S.C. (1975) Review of the survival and cell kinetic effects of adriamycin (NSC-123127)on mammalian cells. Cancer Chemother. Rep. 6(3):147-152. Bhussry, B.R. (1976) Development and growth of teeth. In: Orban's Oral Histology and Embryology. S.N. Bhaskar. ed. C.V. Mosbv Company, Sayit Louis, pp. 53-4;. Di Marco, A., F. Zunio, R. Silvestrini, C. Gambarucci, and R.A. Gambetta (1971) Interaction of some daunomycin derivatives with deoxyribonucleic acid and their biological activity. Biochem. Pharmacol., 20: 1323-1328. Josephesen, K., and H. Warshawsky (1982) Radioautography of r a t incisor dentin as a continuous record of the incorporation of a single dose of 'H-labeled proline and tyrosine. Am. J. Anat., 164:4556. Karim, A.C., and E. Eddy (1984) A light and electron microscopic study of osteodentin formation in the rat incisor after adriamycin administration. Am. J. Anat., 169207-219. Koppang, H.S. (1973) Autoradiographic investigation of the effect of cyclophosphamide on dentinogenesis of the r a t incisor. Scand. J. Dent. Res., 81:397-405. Koppang, H.S. (1981) Effect of cyclophosphamide on dentinogenesis in the rat incisor: Fluorescence microscopic and microradiographic investigations. Scand. J. Dent. Res., 8959-70. Reynolds, E.S. 11963)The use of lead citrate a t high pH as a n electron opaque stain in electron microscopy. J. Cell Biol., 17208-212. Singal, P.K., R.J. Segstro, M. Kutry, and R. Singh (1984) Lysosomal hydrolases in adriamycin induced cardiomyopathy. Fed. Proc., 43:701 (Abstract). Smith, C.E., and H. Warshawsky (1976) Movement of entire cell populations during renewal of the r a t incisor as shown by radioautography after labeling with 'H-thymidine. Am. J. Anat., 145:225260. Stene, T. (1978) Vincristine's effect on dentinogenesis in r a t incisor. Scand. J. Dent. Res., 87:39-49. Stene, T.,and H.S. Koppang (1980) Autoradiographic investigation of proliferative responses in the r a t incisor pulp after vincristine administration. Scand. J. Dent. Res., 88:96-103. Wang, J.J., D.S. Chervinsky, and J.M. Rosen (1972) Comparative biochemical studies of adriamycin and daunomycin in leukemic cells. Cancer Res., 32511-5 15. Weinstock, M. and C.P. Leblond (1974) Synthesis, migration, and release of precursor collagen by odontoblasts as visualized by radioautography after 'H-proline administration. J. Cell Biol., 60:92127.