Archr oral Biol. Vol. 32. No. 6. pp. 433437. 1987 ooo3-9969187 53.00 + 0.00 Printed in Great Britain. All rights reserved Copyright c 1987Pergamon zyxwvutsrqpon Journals Ltd A RADIOAUTOGRAPHIC STUDY OF THE EFFECTS OF VINBLASTINE ON THE FATE OF INJECTED 4SCALCIUM AND [‘2SI]-INSULIN IN THE RAT INCISOR T. UCHIDA,’ M. D. MCKEE* and H. WARSHAWSKY~‘• ‘Department of Anatomy, Yamanashi Medical College, Tamaho, Yamanashi 409-38, Japan and *Department of Anatomy. McGill University, 3640 University Street, Montreal. Quebec, Canada H3A 2B2 Summary-To examine the effect of vinblastine on the movement of calcium and macromolecules through the enamel organ in the secretion zone and through the odontoblast layer, ‘JCaCl, and [ ‘*sIl_insulin were used as radioautographic tracers. Vinblastine did not alter the localization of either labelled Ca or insulin in the enamel organ and underlying enamel, but eliminated both labels in the pulp, odontoblasts and dentine. It is concluded that vinblastine has no effect on the passage of Ca and macromolecules in the enamel organ and secretory ameloblast layers, whereas its effect on the pulp and odontoblasts prevents passage of these tracers into the predentine and dentine. INTRODUCTION Ameloblasts and odontoblasts are directly re- sponsible for forming the organic matrix of the enamel and dentine, respectively. However, these cells have only been indirectly implicated in the mineral- ization of the matrices. The effect of anti- microtubular agents such as colchicine, colcemid and vinblastine on these cells has been studied both ultrastructurally and functionally in relation to the synthesis and secretion of the organic matrix (Moe and Mikkelsen, 1977; Karim and Warshawsky, 1979; Miake, Yanagisawa and Takuma, 1982; Takuma, Sawada and Yanagisawa, 1982; McKee and War- shawsky, 1986). We have traced no report of vin- blastine’s effect on the passage of ions or molecules into the enamel and dentine. It is known that *%Za enters the enamel and dentine within seconds after injection (Munhoz and Leblond, 1974), and that [ lZI]-insulin enters the dentine and predentine within minutes (Martineau-Doii et al., 1986). so we exam- ined the effects of vinblastine on the passage of Ca and large molecules such as insulin through the cellular layers overlying enamel and dentine. Radio- autography was used to visualize these tracers after vinblastine-treated rats were injected with 4sCaCl, and ‘2’I-labelled insulin. MATERIALS AND METHODS Male Sherman rats weighing 100 +- 10 g were used; experimental animals were anaesthetized and injected with 5 mg of vinblastine sulphate (Sigma) in 0.3 ml of physiological saline via the external jugular vein. Controls were injected with 0.3 ml of normal saline. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONM 45Caexperiment One hour after vinblastine injection, experimental and control animals were injected with 10 rCi/g body *TO whom correspondence should be addressed. weight of 45 Ca (as CaCI, sp. act. 10 pCi/pg, New England Nuclear) via the contralateral external jug- ular vein. They were perfused through the left ventri- cle 2 min later with Ringer’s lactate for 30 s to rinse out free tracer, followed by 5 per cent glutaraldehyde in 0.05 M cacodylate buffer, pH 7.3, for about 10 min. The upper and lower incisors were dissected out, post-fixed with 1 per cent osmium tetroxide in 0.1 M cacodylate buffer @H 7.3) for 1 h, dehydrated through graded acetones, and embedded in Epon 8 12. Throughout these procedures, experimental and control teeth were treated identically to equalize any loss of water-soluble labelled Ca. Experimental and control incisors were embedded together in one block (see Fig. 1) and l-pm-thick sections were cut, floated on ethylene glycol and mounted on glass slides; they were then processed for light-microscope radio- autography (Kopriwa and Leblond, 1962). After radioautographic exposure, the slides were developed and stained with 1 per cent toltidine blue. [ ‘251j-insulin experiment One hour after vinblastine injection, another group of experimental and control animals were injected with 0.15 ml of freshly-prepared [ *SI]-porcine insu- line (2.5 c(g/lOO g body weight, sp. act. 165 pCi/pg) via the contralateral external jugular vein. The [‘Zr]-insulin was prepared by the chloramine-T method as described by Posner, Josef&erg and Bergeron (1978). Five minutes after injection of the labelled insulin, the rats were killed by perfusion of rinsing agent and then fixative as described above. The upper and lower jaws were removed and im- mersed in the same fixative for 2 h. After demin- eralization with 4.13 per cent isotonic disodium EDTA (Warshawsky and Moore 1967) for 16 days at 4”C, the jaws were cut into cross-sectional segments which were post-fixed in osmium, dehydrated and embedded in Epon. One-micrometre-thick sections mounted on glass slides were stained with iron hae- matoxylin and processed for light-microscope radio- autography. 433