Archs oral Bid
Vol.
2.
pp. 449 10 454.
Pergamon Press1977. Printed in GreatBritain
AUTORADIOGRAPHY OF 65ZINC IN DEVELOPING RAT TEETH AND BONES J. W.
BAWDEN
and L. E.
HAMMARSTRBM
The Dental Research Center, University of North Carolina. Chapel Hill,
North Carolina U.S.A., and the Department of Oral Histopathology. Faculty of Odontology, University of Lund, Malmii, Sweden Summary-The distribution of 65Zn in young rats was studied by whole-body autoradiography following single and repeated intraperitoneal injections of the isotope. The distribution of Zn after repeated injections was similar to that after a single injection. There was very slow release of the isotope from the site of injection. The liver showed a high concentration of 65Zn a few hours after injection. Zn concentration gradually increased in bone and dentine during the following days, eventually becoming the highest in the body. No 6’Zn was found in cartilage. Only traces, if any, were detected in developing enamel.
INTRODUCTION
Numerous investigations have demonstrated that zinc is a normally occurring element in mineralized tissues, including the dentine and enamel of teeth (Cruickshank, 1936, 1937; Lowater and Murray, 1937; Monsell and Hendershot, 1960; Haumont, 1961; Brudevold et al., 1963; Angmar, Carlstrom and Glas, 1963; Vincent, 1963; Lundberg, Soremark and Thilander, 1965; Babicky and Taylor, 1966; Nixon, Livingston and Smith, 1967; Baratieri, Miani and Picarelli. 1970; Bergman, 197Oa, b, c; Losee, Cutress and Brown, 1974). In bone, it is found most prominently in newly mineralizing areas (Haumont, 1961; Kinnamon, 1963 ; Vincent, 1963 ; Bergman, 197Oa, b, c). Distribution of zinc in developing teeth has been studied using histochemical methods (Fiore-Donno and Baume, 1966; Baratieri et al., 1970). Brudevold et al. (1963) presented evidence that, in man, most of the zinc in enamel is incorporated prior to eruption. Our aim was to obtain information on the distribution of Zn in developing rat teeth by autoradiography. MATERIALS AND
METHODS
activity = (specific from the Radiochemical Centre, Amersham, England. Three series of animals were used. In the first series, each of four litter mate. eight-day-old rats was given an intraperitoneal injection of 10 &i of 65Zn. The dose was contained in 0.1 ml of solution and delivered 1.3 pg of zinc/kg of body weight. The animals were killed with ether at 30 min, 4, 24 and 96 h respectively. A second series of four litter mate, 14&y-old rats were injected in the same manner with a similar dose of 6SZn and killed at 1, 4, 24 and 96 h respectively. In a third series, six litter mate rats were injected with 65Zn intraperitoneally twice a day starting on the fourth day of age. Each dose contained 2.5 PCi 65Zn in 0.05 ml saline. The animals were killed at 8. 9, 13, 14, 15 and 18 days of age. Thus the total dose per animal ranged from 20 to 70&i. [6”Zn]-zinc
chloride
0.4 mCi/pg) was obtained
All animals were embedded in carboxymethyl cellulose and frozen by immersion in hexane cooled with solid CO1 (-75°C). Sag&al 20 w thick frozen sections were taken at various levels, with particular attention directed to inclusion of the dental structures. Whole-body autoradiography was performed according to the method of Ullberg (1954, 1958). Structrix X-ray film (Agfa-Gevaert) was used with exposure times of 6 weeks in series one and three, and 8 weeks in series two. RESULTS
Acute experiments (series I and II) The general distribution of 6”Zn was characterized by a slow resorption from the site of injection. Most of the isotope then seemed to pass through the liver before appearing in other tissues such as bone and dentine. Concentration in the circulating blood as observed in the lumen of the heart was low throughout the period of the experiment. Autoradiographs obtained from animals injected at 8 days of age, and killed 30min post-injection, showed that the dose was still localized mainly in the intraperitoneal cavity. The concentration in the blood was low but exceeded that of most soft tissues. in which distribution was only marginally detectable. At four hours (Fig. 1), much of the dose remained in the intraperitoneal cavity, but a high concentration was also found in the liver and a moderate concentration in the kidney. Other soft tissues. such as muscle and epithelium, still showed low concentrations of 65Zn but exceeded that of the blood. In bone, particularly in areas of bone growth, uptake of the isotope was clearly demonstrable. No 65Zn was demonstrable in the epiphyseal cartilage. In the teeth. trace amounts of 6sZn were seen in newly formed dentine. At 1 day post-injection (9 days of age). the amount of 65Zn remaining in the intraperitoneal cavity was reduced but uptake in mineralizing tissues was more distinct. Also, in the liver the concentration was lower 1 day after injection than after 4 h. Muscle and epithelium continued to show higher concentrations than
449
J. W. Bawden and L. E. Hammarstriim
450
blood. A distinct line of the isotope was found in the pulpal part of the dentine of both the molars and incisors. At 4 days (Fig. l), the distribution of the isotope was similar to that after 1 day. The accumulation in bone and dentine was markedly visible against the low concentrations of isotope in the soft tissues, but no, or perhaps a very slight amount of, 65Zn was found in developing enamel. There was a very faint line of activity in the area of the ameloblasts. In the anterior part of the oral mucosa, apparently in the epithelium, a distinct uptake of 65Zn was noted. The concentration of 65Zn in this area was higher than in any other epithelium and in other soft tissues (Fig. 1). A distinct uptake was also noted in the growing parts of the tactile snout hairs and in the pancreatic islets. In the animals injected at 14 days, 65Zn distribution patterns were similar to those in animals injected at 8 days. Uptake in the dentine was obvious, but it was doubtful whether there was even a trace in the enamel. Chronic experiments (series III) The distribution of 65Zn in the rat after chronic administration was very similar to that after a single injection. The highest concentrations were in bone and dentine, followed by liver and kidney. Other major soft tissues, such as muscles and epithelium, showed low concentrations which, however, exceeded that of the blood. The concentration of 65Zn in the dentine was very high in the animal killed at 8 days of age (4 days after initiation of the twice-daily injections). It was questionable if any 65Zn was present in developing enamel (Fig. 2). The faint line of uptake in the zone of ameloblasts was visible. In the rats killed after longer periods of daily injections, the uptake of “Zn in bone and dentine progressively increased, but the content of 65Zn in the more mature enamel seemed to be even less than in the eight-day-old animal (Fig. 3). The concentration in the enamel organ was very low. The specific uptake of 65Zn in a certain area of the oral mucosa, noted in the acute series, was also apparent in this chronic experiment. DISCUSSION The general distribution patterns of 65Zn were in agreement with the findings of other investigators (Craig and Seigel, 1960; Haumont, 1961; Klnnamon, 1963; Vincent, 1963; Bergman, 197Oa, b, c). The slow distribution of the isotope from the site of injection, early appearance in the liver and subsequent distribution to other tissues suggests that zinc is metabolized in some way before general distribution, possibly being bound to a protein, as suggested by Craig and Seigel (1960). The uptake in mineralizing bone we noted is in agreement with other investigations (Haumont; Kinnamon; Vincent; Bergman). However, the rate of accumulation was relatively slow, as Rubini et al. (1961) also found. This is probably due to the protein-binding of the zinc, because Samachson et al. (1967) showed that the in vitro uptake of “‘Zn by powdered whole and inorganic bone is greater than that for
4sCa. They speculated that uptake of zinc by bone in uivo would be much higher if so much of the zinc in the extracellular fluids were not protein bound. Accumulation of zinc in the developing dentine has been shown by several investigators (Cruickshank, 1936; Lowater and Murray, 1937; Brudevold et al., 1963; Lundberg et al., 1965; Babicky and Taylor, 1966; Fiore-Donno and Baume, 1966; Huxley and Leaver, 1966; Baratieri et al., 1970). We expected to find uptake of 65Zn in developing enamel because zinc has been found in enamel in concentrations of 2&460 parts/lo’ (Cruickshank; Lowater and Murray; Monsell and Hendershot, 1960; Brudevold et al.; Babicky and Taylor, 1966; Huxley and Leaver; Nixon et al., 1967; Bergman, 197Oa).The larger value occurred in response to a high zinc diet after eruption of the teeth. Brudevold et al. observed that most of the zinc accumulated in human enamel was deposited before tooth emergence. Baratiere et al. identified zinc in the enamel of developing rat teeth by staining undecalcified sections with the dithizone technique. However, we found very low concentrations of 65Zn, of questionable significance, in our experiments, in spite of the high doses of 65Zn and long autoradiographic exposure times. Similar methods have easily demonstrated uptake of fluoride, cadmium, molybdenum, selenium and vanadium in developing rat molar tooth enamel and enamel organ (Hammarstriim, 1971; Bawden and Hammarstrom, 1975, 1976). These substances, with the exception of fluoride, are in lower concentrations in the mature human enamel than has been reported for zinc (Losee et al., 1974). It would seem that, in the rat, most of the zinc in mature enamel accumulates posteruptively. The uptake of zinc by oral mucosa is interesting because dietary zinc deficiency induces parakeratosis in orthokeratinized epithelia in a wide variety of species (Follis, Day and McCollum, 1947; Miller and Miller, 1962; Ott et al., 1964; Anderson, Cooper and Hoekstra, 1967; Barney et al., 1967). Rats, for example, develop parakeratosis of oral epithelia and these areas also show increased rates of mitosis and cellular hypertrophy (Alvarez and Meyer, 1968 ; Osmanski and Meyer, 1969; A. B. Sjogren, A. B. Larsson and L. E. Hammarstriim, unpublished) found high alkaline phosphatase activity in the same anterior region of the oral mucosa, where zinc accumulated in the present investigation. As alkaline phosphatase is a zinc-metallo enzyme (Vallee, 1974) the accumulation of zinc in the oral mucosa may be associated with the presence of this enzyme. On the other hand little or no 65Zn appeared in the enamel organ which also has high alkaline phosphatase activity (Reith and Butcher, 1967). Acknowledgement-This investigation was supported by a grant from the Swedish Medical Research Council (B76-24x-04549-02A), and by NIH grant number RR-05333 from the Division of Research Facilities and Resources.
REFERENCES
Alvarez 0. and Meyer J. 1968. Regional differences in parakeratotic response to mild zinc deficiency. Archs Derm. WI, 191-201.
Zinc in rat teeth and bones Anderson J.. Cooper G. A. and Hoekstra W. G. 1967. The histochemistry of the parakeratotic lesion of swine. J. invest. derm. 48, Q-530. Angmar B., Carlstrijm D. and Glas J. E. 1963. Studies on the ultrastructure of dental enamel. J. Ultrastrucc. Res. 8, 12-23. Babicky A. and Taylor D. M. 1966. Determination of zinc in human teeth using the activation analysis. Cs. Stomat. 66, 167-170. Baratieri A.. Miani C. and Picarelli A. 1970. Localisations du zinc dans les tissues dentaires en voie de developpement du rat. Bull. Grpmt int. Reck scient. Stomat. 13. 369.-38 I. Barney G. H., Macpinlac M. P., Pearson W. N. and Darby W. J. 1967. Parakeratosis of the tongue-a unique histopathologic lesion in the zinc-deficient squirrel monkey. J. Nutr. 93, 51 l-517. Bawden J. W. and Hammarstriim L. 1975. Distribution of cadmium in developing teeth and bone of young rats. Stand. J. dent. Res. 83, 179-186. Bawden J. W. and Hammarstriim L. 1976. Autoradiography of 99Mo in developing rat teeth and bone. Stand. J. dent. Res. 84. Bergman B. 1970a. Concentration of zinc in some hard and soft tissues of the rat, determined by neutron activation analysis. Acto Radio/. 9, 420-432. Bergman B. 1970b. Comparative study of distribution of injected zinc-65 in the mandibular condyle and other tissues in the rat as determined by gamma scintillation. Acta Radiol. 9, 577-595.
Bergman B. 1970~. The distribution of ‘j5Zn in the endochondral growth sites of the mandibular condyle and the proximal end of the tibiae in young rats-An autoradiographic and gamma scintillation study. Odont. Revy 21, 261-274. Brudevold F., Steadman L. T., Spinelli M. A., Amdur B. H. and Gron P. 1963. A studv of zinc in human teeth. Archs oral Biol. 8, 135-144. _ Craig F. A. and Siegel E. 1960. Distribution in blood and excretion of “Zn in man. Proc. Sot. exp. Biol. 104, 391-393. Cruickshank D. B. 1936. The natural occurrence of zinc in teeth. Preliminary experiments. Br. dent. J. 61, 530-531. Cruickshank D. B. 1937. The natural occurrence of zinc in teeth. II-Some general considerations. Br. dent. J. 63. 395-399.
Fiore-Donno P. C. and Baume L. J. 1966. Etude histochimique de la dentinogenkse humaine. Helu. odont. Acta (Suppl. IV), 141-185. Follis R. H., Day H. G. and McCollum E. V. 1947. Histochemical studies of tissues of rats fed a diet extremely low in zinc. J. Nutr. 22, 223-233. Hammarstriim L. 1971. Distribution in developing rat enamel of simultaneously injected sodium fluoride and calcium. Stand. J. dent. Res. 70, 369-376.
Haumont S. 1961. Distribution
451 of zinc in bone tissue. J.
Histochem. 9, 141-145.
Huxley H. G. and Leaver A. G. 1966. The effect of different levels of dietary zinc and calcium upon the zinc concentration of the rat femur and incisor. Archs oral Biof. 11, 1337-1344. Kinnamon K. F. 1963. Some independent and combined effects of copper, molybdenum, and zinc on the placental transfer of zinc-65 in the rat. J. Nutr. 81, 312-320. iosee F. L., Cutress T. W. and Brown R. 1974. Natural elements of the periodic table in human dental enamel. Caries Res. 8, 123-134.
Lowater F. and Murray M. M. 1937. Chemical composition of teeth. V. Spectrographic analysis. Biochem. J. 31, 837-841.
Lundberg M., SSremark R. and Thilander H. 1965. Gamma-ray spectrometric analysis of some elements in coronal dentine of unerupted (impacted) human teeth. Odont. Reuy 16, 97-100. Miller J. K. and Miller W. J. 1962. Experimental zinc deficiency and recovery of calves. J. N&r. 76, 467473. Monsell R. E. and Hendershot L. C. 1960. The snectrochemica1 analysis of metals in rat molar enamel, femurs, and incisors. Archs oral Biol. 2, 31-37. Nixon G. S., Livingston H. D. and Smith H. 1967. Estimation of zinc in human enamel by activation analysis. Archs oral Biol. 12, 411416. Osmanski C. P. and Meyer J. 1969. Ultrastructural changes in buccal and nalatinal mucosa of zinc-deficient rats. 2. invest. Derm. Sj, 14-28. Ott E. A.. Smith W. H.. Stab M. and Beeson W. M. 1964. Zinc-deficiency syndrome in the young lamb. J. nutr. 82, 41-50.
Reith E. J. and Butcher E. 0. 1967. Microanatomy and histochemistry of amelogenesis In: Structural and Chemical Organization of Teeth. (Edited by Miles A. E. W.). pp. 317-397. Academic Press, New York. Rubini M. E., Moutalvo G., Lockhart C. P. and Johnson C. R. 1961. Metabolism of 65Zn. Am. J. Physiol. 200, 1345-1348. Samachson J., Dennis J., Fowler R. and Schmitz A. 1967. The reaction of ‘sZn with the surfaces of bone and bone mineral. Biochem. biophys. Acta 148, 767-773. Ullberg S. 1954. Studies on the distribution and fate of “S labeled benzylpenicillin in the body. Acta Radiol. (Suppl. I 18). Ullberg S. 1958. Autoradiographic studies on the distribution of labeled drugs in the body. Second V.N. Int. Conf: Peaceful Uses of Atomic Energy, pp. 248-254. Vallee B. L. 1974. Metalloenzymes. In: Enzymology in the Practice of Laboratory Medicine. (Edited by Blume P. and Freier E. F.), pp. 95-126. Academic Press, New York. Vincent J. 1963. Microscopic aspects of mineral metabolism in bone tissue with special reference to calcium, lead, and zinc. Clin. Orthop. 26. 161-175.
Plates l-2 overleaf
Zinc
in rat teeth and
bones
453
‘4 hour&
Plate 1 Fig. 1. Autoradiograms showing the distribution of 65Z in rats injected with the isotope at 8 days of age and killed 4 h and 4 days later. Above: at 4 h. Most isotope is still at the site of injection in the intraperitoneal cavity, but there is a marked accumulation in the liver. Bone and kidney show a Ion concentration. In developing teeth the concentration is very low. Below: 4 days. High concentration of isotope in bone and dentine. Low concentrations in kidney and liver and most soft tissues. There is notable uptake in the anterior oral mucosa. C_I 2)
4s4
J. W. Bawden and L. E. HammarsrrGm
Plate 2 Fig. 2. Autoradiogram (left) and the corresponding stained section (right), showing distribution of “Zn in developing molar teeth of rat injected twice daily with the isotope beginning at age of 4 days and killed 10 days later. High concentration of the isotope in bone and as distinct line in dentine. Low concentration in enamel organ and gingival epithelium, and none in enamel. B bone; D dentine; E = enamel: P = pulp: GE gingival epithelium. ( :$ 30) Fig. 3. Autoradiograph from a rat injected twice daily with ??In beginning at 4 days of age and killed at 14 days of age. High concentration of Zn in bone and dentine. Uptake in enamel is doubtful. There is a concentration in part of the oral mucosa (white arrows) and in the tactile hairs (black lower incisor. (7 7) arrows). B bone: M first molar; L
Vol.
2.
pp. 449 10 454.
Pergamon Press1977. Printed in GreatBritain
AUTORADIOGRAPHY OF 65ZINC IN DEVELOPING RAT TEETH AND BONES J. W.
BAWDEN
and L. E.
HAMMARSTRBM
The Dental Research Center, University of North Carolina. Chapel Hill,
North Carolina U.S.A., and the Department of Oral Histopathology. Faculty of Odontology, University of Lund, Malmii, Sweden Summary-The distribution of 65Zn in young rats was studied by whole-body autoradiography following single and repeated intraperitoneal injections of the isotope. The distribution of Zn after repeated injections was similar to that after a single injection. There was very slow release of the isotope from the site of injection. The liver showed a high concentration of 65Zn a few hours after injection. Zn concentration gradually increased in bone and dentine during the following days, eventually becoming the highest in the body. No 6’Zn was found in cartilage. Only traces, if any, were detected in developing enamel.
INTRODUCTION
Numerous investigations have demonstrated that zinc is a normally occurring element in mineralized tissues, including the dentine and enamel of teeth (Cruickshank, 1936, 1937; Lowater and Murray, 1937; Monsell and Hendershot, 1960; Haumont, 1961; Brudevold et al., 1963; Angmar, Carlstrom and Glas, 1963; Vincent, 1963; Lundberg, Soremark and Thilander, 1965; Babicky and Taylor, 1966; Nixon, Livingston and Smith, 1967; Baratieri, Miani and Picarelli. 1970; Bergman, 197Oa, b, c; Losee, Cutress and Brown, 1974). In bone, it is found most prominently in newly mineralizing areas (Haumont, 1961; Kinnamon, 1963 ; Vincent, 1963 ; Bergman, 197Oa, b, c). Distribution of zinc in developing teeth has been studied using histochemical methods (Fiore-Donno and Baume, 1966; Baratieri et al., 1970). Brudevold et al. (1963) presented evidence that, in man, most of the zinc in enamel is incorporated prior to eruption. Our aim was to obtain information on the distribution of Zn in developing rat teeth by autoradiography. MATERIALS AND
METHODS
activity = (specific from the Radiochemical Centre, Amersham, England. Three series of animals were used. In the first series, each of four litter mate. eight-day-old rats was given an intraperitoneal injection of 10 &i of 65Zn. The dose was contained in 0.1 ml of solution and delivered 1.3 pg of zinc/kg of body weight. The animals were killed with ether at 30 min, 4, 24 and 96 h respectively. A second series of four litter mate, 14&y-old rats were injected in the same manner with a similar dose of 6SZn and killed at 1, 4, 24 and 96 h respectively. In a third series, six litter mate rats were injected with 65Zn intraperitoneally twice a day starting on the fourth day of age. Each dose contained 2.5 PCi 65Zn in 0.05 ml saline. The animals were killed at 8. 9, 13, 14, 15 and 18 days of age. Thus the total dose per animal ranged from 20 to 70&i. [6”Zn]-zinc
chloride
0.4 mCi/pg) was obtained
All animals were embedded in carboxymethyl cellulose and frozen by immersion in hexane cooled with solid CO1 (-75°C). Sag&al 20 w thick frozen sections were taken at various levels, with particular attention directed to inclusion of the dental structures. Whole-body autoradiography was performed according to the method of Ullberg (1954, 1958). Structrix X-ray film (Agfa-Gevaert) was used with exposure times of 6 weeks in series one and three, and 8 weeks in series two. RESULTS
Acute experiments (series I and II) The general distribution of 6”Zn was characterized by a slow resorption from the site of injection. Most of the isotope then seemed to pass through the liver before appearing in other tissues such as bone and dentine. Concentration in the circulating blood as observed in the lumen of the heart was low throughout the period of the experiment. Autoradiographs obtained from animals injected at 8 days of age, and killed 30min post-injection, showed that the dose was still localized mainly in the intraperitoneal cavity. The concentration in the blood was low but exceeded that of most soft tissues. in which distribution was only marginally detectable. At four hours (Fig. 1), much of the dose remained in the intraperitoneal cavity, but a high concentration was also found in the liver and a moderate concentration in the kidney. Other soft tissues. such as muscle and epithelium, still showed low concentrations of 65Zn but exceeded that of the blood. In bone, particularly in areas of bone growth, uptake of the isotope was clearly demonstrable. No 65Zn was demonstrable in the epiphyseal cartilage. In the teeth. trace amounts of 6sZn were seen in newly formed dentine. At 1 day post-injection (9 days of age). the amount of 65Zn remaining in the intraperitoneal cavity was reduced but uptake in mineralizing tissues was more distinct. Also, in the liver the concentration was lower 1 day after injection than after 4 h. Muscle and epithelium continued to show higher concentrations than
449
J. W. Bawden and L. E. Hammarstriim
450
blood. A distinct line of the isotope was found in the pulpal part of the dentine of both the molars and incisors. At 4 days (Fig. l), the distribution of the isotope was similar to that after 1 day. The accumulation in bone and dentine was markedly visible against the low concentrations of isotope in the soft tissues, but no, or perhaps a very slight amount of, 65Zn was found in developing enamel. There was a very faint line of activity in the area of the ameloblasts. In the anterior part of the oral mucosa, apparently in the epithelium, a distinct uptake of 65Zn was noted. The concentration of 65Zn in this area was higher than in any other epithelium and in other soft tissues (Fig. 1). A distinct uptake was also noted in the growing parts of the tactile snout hairs and in the pancreatic islets. In the animals injected at 14 days, 65Zn distribution patterns were similar to those in animals injected at 8 days. Uptake in the dentine was obvious, but it was doubtful whether there was even a trace in the enamel. Chronic experiments (series III) The distribution of 65Zn in the rat after chronic administration was very similar to that after a single injection. The highest concentrations were in bone and dentine, followed by liver and kidney. Other major soft tissues, such as muscles and epithelium, showed low concentrations which, however, exceeded that of the blood. The concentration of 65Zn in the dentine was very high in the animal killed at 8 days of age (4 days after initiation of the twice-daily injections). It was questionable if any 65Zn was present in developing enamel (Fig. 2). The faint line of uptake in the zone of ameloblasts was visible. In the rats killed after longer periods of daily injections, the uptake of “Zn in bone and dentine progressively increased, but the content of 65Zn in the more mature enamel seemed to be even less than in the eight-day-old animal (Fig. 3). The concentration in the enamel organ was very low. The specific uptake of 65Zn in a certain area of the oral mucosa, noted in the acute series, was also apparent in this chronic experiment. DISCUSSION The general distribution patterns of 65Zn were in agreement with the findings of other investigators (Craig and Seigel, 1960; Haumont, 1961; Klnnamon, 1963; Vincent, 1963; Bergman, 197Oa, b, c). The slow distribution of the isotope from the site of injection, early appearance in the liver and subsequent distribution to other tissues suggests that zinc is metabolized in some way before general distribution, possibly being bound to a protein, as suggested by Craig and Seigel (1960). The uptake in mineralizing bone we noted is in agreement with other investigations (Haumont; Kinnamon; Vincent; Bergman). However, the rate of accumulation was relatively slow, as Rubini et al. (1961) also found. This is probably due to the protein-binding of the zinc, because Samachson et al. (1967) showed that the in vitro uptake of “‘Zn by powdered whole and inorganic bone is greater than that for
4sCa. They speculated that uptake of zinc by bone in uivo would be much higher if so much of the zinc in the extracellular fluids were not protein bound. Accumulation of zinc in the developing dentine has been shown by several investigators (Cruickshank, 1936; Lowater and Murray, 1937; Brudevold et al., 1963; Lundberg et al., 1965; Babicky and Taylor, 1966; Fiore-Donno and Baume, 1966; Huxley and Leaver, 1966; Baratieri et al., 1970). We expected to find uptake of 65Zn in developing enamel because zinc has been found in enamel in concentrations of 2&460 parts/lo’ (Cruickshank; Lowater and Murray; Monsell and Hendershot, 1960; Brudevold et al.; Babicky and Taylor, 1966; Huxley and Leaver; Nixon et al., 1967; Bergman, 197Oa).The larger value occurred in response to a high zinc diet after eruption of the teeth. Brudevold et al. observed that most of the zinc accumulated in human enamel was deposited before tooth emergence. Baratiere et al. identified zinc in the enamel of developing rat teeth by staining undecalcified sections with the dithizone technique. However, we found very low concentrations of 65Zn, of questionable significance, in our experiments, in spite of the high doses of 65Zn and long autoradiographic exposure times. Similar methods have easily demonstrated uptake of fluoride, cadmium, molybdenum, selenium and vanadium in developing rat molar tooth enamel and enamel organ (Hammarstriim, 1971; Bawden and Hammarstrom, 1975, 1976). These substances, with the exception of fluoride, are in lower concentrations in the mature human enamel than has been reported for zinc (Losee et al., 1974). It would seem that, in the rat, most of the zinc in mature enamel accumulates posteruptively. The uptake of zinc by oral mucosa is interesting because dietary zinc deficiency induces parakeratosis in orthokeratinized epithelia in a wide variety of species (Follis, Day and McCollum, 1947; Miller and Miller, 1962; Ott et al., 1964; Anderson, Cooper and Hoekstra, 1967; Barney et al., 1967). Rats, for example, develop parakeratosis of oral epithelia and these areas also show increased rates of mitosis and cellular hypertrophy (Alvarez and Meyer, 1968 ; Osmanski and Meyer, 1969; A. B. Sjogren, A. B. Larsson and L. E. Hammarstriim, unpublished) found high alkaline phosphatase activity in the same anterior region of the oral mucosa, where zinc accumulated in the present investigation. As alkaline phosphatase is a zinc-metallo enzyme (Vallee, 1974) the accumulation of zinc in the oral mucosa may be associated with the presence of this enzyme. On the other hand little or no 65Zn appeared in the enamel organ which also has high alkaline phosphatase activity (Reith and Butcher, 1967). Acknowledgement-This investigation was supported by a grant from the Swedish Medical Research Council (B76-24x-04549-02A), and by NIH grant number RR-05333 from the Division of Research Facilities and Resources.
REFERENCES
Alvarez 0. and Meyer J. 1968. Regional differences in parakeratotic response to mild zinc deficiency. Archs Derm. WI, 191-201.
Zinc in rat teeth and bones Anderson J.. Cooper G. A. and Hoekstra W. G. 1967. The histochemistry of the parakeratotic lesion of swine. J. invest. derm. 48, Q-530. Angmar B., Carlstrijm D. and Glas J. E. 1963. Studies on the ultrastructure of dental enamel. J. Ultrastrucc. Res. 8, 12-23. Babicky A. and Taylor D. M. 1966. Determination of zinc in human teeth using the activation analysis. Cs. Stomat. 66, 167-170. Baratieri A.. Miani C. and Picarelli A. 1970. Localisations du zinc dans les tissues dentaires en voie de developpement du rat. Bull. Grpmt int. Reck scient. Stomat. 13. 369.-38 I. Barney G. H., Macpinlac M. P., Pearson W. N. and Darby W. J. 1967. Parakeratosis of the tongue-a unique histopathologic lesion in the zinc-deficient squirrel monkey. J. Nutr. 93, 51 l-517. Bawden J. W. and Hammarstriim L. 1975. Distribution of cadmium in developing teeth and bone of young rats. Stand. J. dent. Res. 83, 179-186. Bawden J. W. and Hammarstriim L. 1976. Autoradiography of 99Mo in developing rat teeth and bone. Stand. J. dent. Res. 84. Bergman B. 1970a. Concentration of zinc in some hard and soft tissues of the rat, determined by neutron activation analysis. Acto Radio/. 9, 420-432. Bergman B. 1970b. Comparative study of distribution of injected zinc-65 in the mandibular condyle and other tissues in the rat as determined by gamma scintillation. Acta Radiol. 9, 577-595.
Bergman B. 1970~. The distribution of ‘j5Zn in the endochondral growth sites of the mandibular condyle and the proximal end of the tibiae in young rats-An autoradiographic and gamma scintillation study. Odont. Revy 21, 261-274. Brudevold F., Steadman L. T., Spinelli M. A., Amdur B. H. and Gron P. 1963. A studv of zinc in human teeth. Archs oral Biol. 8, 135-144. _ Craig F. A. and Siegel E. 1960. Distribution in blood and excretion of “Zn in man. Proc. Sot. exp. Biol. 104, 391-393. Cruickshank D. B. 1936. The natural occurrence of zinc in teeth. Preliminary experiments. Br. dent. J. 61, 530-531. Cruickshank D. B. 1937. The natural occurrence of zinc in teeth. II-Some general considerations. Br. dent. J. 63. 395-399.
Fiore-Donno P. C. and Baume L. J. 1966. Etude histochimique de la dentinogenkse humaine. Helu. odont. Acta (Suppl. IV), 141-185. Follis R. H., Day H. G. and McCollum E. V. 1947. Histochemical studies of tissues of rats fed a diet extremely low in zinc. J. Nutr. 22, 223-233. Hammarstriim L. 1971. Distribution in developing rat enamel of simultaneously injected sodium fluoride and calcium. Stand. J. dent. Res. 70, 369-376.
Haumont S. 1961. Distribution
451 of zinc in bone tissue. J.
Histochem. 9, 141-145.
Huxley H. G. and Leaver A. G. 1966. The effect of different levels of dietary zinc and calcium upon the zinc concentration of the rat femur and incisor. Archs oral Biof. 11, 1337-1344. Kinnamon K. F. 1963. Some independent and combined effects of copper, molybdenum, and zinc on the placental transfer of zinc-65 in the rat. J. Nutr. 81, 312-320. iosee F. L., Cutress T. W. and Brown R. 1974. Natural elements of the periodic table in human dental enamel. Caries Res. 8, 123-134.
Lowater F. and Murray M. M. 1937. Chemical composition of teeth. V. Spectrographic analysis. Biochem. J. 31, 837-841.
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Plates l-2 overleaf
Zinc
in rat teeth and
bones
453
‘4 hour&
Plate 1 Fig. 1. Autoradiograms showing the distribution of 65Z in rats injected with the isotope at 8 days of age and killed 4 h and 4 days later. Above: at 4 h. Most isotope is still at the site of injection in the intraperitoneal cavity, but there is a marked accumulation in the liver. Bone and kidney show a Ion concentration. In developing teeth the concentration is very low. Below: 4 days. High concentration of isotope in bone and dentine. Low concentrations in kidney and liver and most soft tissues. There is notable uptake in the anterior oral mucosa. C_I 2)
4s4
J. W. Bawden and L. E. HammarsrrGm
Plate 2 Fig. 2. Autoradiogram (left) and the corresponding stained section (right), showing distribution of “Zn in developing molar teeth of rat injected twice daily with the isotope beginning at age of 4 days and killed 10 days later. High concentration of the isotope in bone and as distinct line in dentine. Low concentration in enamel organ and gingival epithelium, and none in enamel. B bone; D dentine; E = enamel: P = pulp: GE gingival epithelium. ( :$ 30) Fig. 3. Autoradiograph from a rat injected twice daily with ??In beginning at 4 days of age and killed at 14 days of age. High concentration of Zn in bone and dentine. Uptake in enamel is doubtful. There is a concentration in part of the oral mucosa (white arrows) and in the tactile hairs (black lower incisor. (7 7) arrows). B bone: M first molar; L
