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Dose-response effects of colchicine and vinblastine on unimpeded eruption rates of the rat mandibular incisor
DOSE-RESPONSE EFFECTS OF COLCHICINE AND VINBLASTINE ON UNIMPEDED ERUPTION RATES OF THE RAT MANDIBULAR INCISOR M. Department
CHIRA,
K.
TAKIZAWA
and
S. OHSHIMA
of Pharmacology, School of Dental Medicine, Tsurumi 2-l-3 Tsurumi. Tsurumi-ku. Yokohama. Japan
University,
Summary-Dose-dependent inhibition of the eruption rate followed single injections of colchitine or vinblastine. 1.5 mg of colchicine/kg of body wt decelerated the eruption rate to 6 per cent of the control value in the initial 24 h after subcutaneous injection and 2.5 mg of vinblastine/kg of body wt to 60 per cent. Doses of colchicine and vinblastine which would reduce the eruption to zero were estimated to be 1.62 mg/kg and 14.07 mg/kg respectively. Toxicity developed slowly whereas inhibition of eruption appeared quickly. Colchicine toxicity was the less. The inhibitory action of these drugs may be related to disruption of microtubules in the cells which may produce the motive force of eruption. It has yet to be shown how microtubules could produce such a force.
INTRODUCTION
0.2 ml/100 g of body weight in all experiments. One control group of rats received a single subcutaneous injection of the same volume of saline. The time of injection ranged from lo:14 to 11:34 hours.
The effect of demecolcine, like colchicine a derivative of Colchicum, on the eruption of the rat incisor was examined by, for instance, Main and Adams (1966). The drug decelerated or even stopped the incisor eruption at a certain dose level (Chiba. Narraway and Ness, 1968; Berkovitz, 1972). The mechanism is not known although it is known that demecolcine arrests cells in metaphase in growing tissues (Chiba, Nakagawa and Mimura, 1966). It has been shown that Colchicurn alkaloids and Vinca alkaloids (vinblastine) bind specifically to tubulin and prevent its assembly into tubules and thus the function of cells in which microtubules are involved would be interrupted (see Taylor, 1973; Olmsted and Borisy, 1973; Snyder and McIntosh, 1976). In order to understand the mechanism of action of these drugs on the eruption of the rat incisor, it would be helpful to examine whether or not deceleration of eruption is produced by any inhibitor of microtubules. As a step in this direction. we undertook the present experiments.
MATERIALS
AND
Doses of cinhlastine Thirty-seven rats weighing 124g on the average were divided into 5 sub-groups of 7-8 animals. The animals of the 4 experimental groups received a single intraperitoneal injection of 1.0, 1.5, 2.0 or 2.5 mg of vinblastine/kg of body weight respectively. Vinblastine (Eli Lilly, “VELBE”, vinblastine sulphate, Basingstoke, England) was dissolved in physiological saline. The volume of injected solutions ranged from 0.1 to 0.25 ml/lOOg of body weight. One group control received a single intraperitoneal injection of 0.25 ml of saline/lOOg of body weight. The time of injection ranged from lo:07 to 11:36 hours. Meusurement
of the eruption rate
Eruption rates of the left mandibular incisors of all rats were measured at daily intervals from one day before the injection until the experiment was terminated 4 days later. A photographic method was used with the right incisor fixed to its jaw bone by a stainless steel pin to provide a stable reference tooth (Chiba et (I/., 1976). Photographs of the tooth surface were taken at magnification of x 3 or x 6.6. Prints were made from the negatives at final magnifications of x 82 and x 91. The jaws were radiographed at the end of the experimental period to examine the position of the pin fixing the incisor to its jaw bone. Under ether anaesthesia, all 4 incisors in each rat were shortened at the level of the inter-incisal crest or at the gingival margin one and 3 days before the injection and, when appropriate, repeatedly afterwards to maintain the unimpeded state. Fixation of the incisor to the bone was carried out one day before the in.jection of the drug.
METHODS
Seventy-two male rats of the Wistar strain, aged 5 to 6 weeks, were kept in metal cages, fed on a powdered diet during the experimental period and for at least one week beforehand and took water ad lihitum from the metal nozzle of a water delivery tube. Doses of colchicirle Thirty-five rats weighing 149 g on the average were divided into 4 sub-groups of 8-9 animals. The animals of the 3 experimental groups received a single subcutaneous injection of 0.5, 1.O or 1.5mg of colchicine/kg of body weight respectively. Colchicine (E. Merck, Darmstadt, West Germany) was dissolved in physiological saline. The volume of injected solutions was II5
116
M. Chiba.
Table I, Changes Control Saline
Day -1
148.7 147.0 146.2 145.8 145.3 146.9
0
1 2 3 4
k 21.4 k 20.9 _t 18.5 If: 18.1 f 16.0 + 12.7
II
9 9 9 9 9 9
K. Takirawa and S. Ohshima
in the body weight following injection
0.5 mg 154.3 149.5 146.7 149.2 148.8 149.4
* + + f + *
Il.3 9.5 9.4
II.8 Il.2 13.2
I1 9 9 9 9 9 9
of colchicine
Colchicine 1.0 mg 141.3 140.2 133.4 131.4 131.6 131.0
_+ 18.2 + 16.5 + 18.3* * 1x.4* + 1X.4* * 17.9t
I .5 mg
II 8 X 8 X 8 8
151.7 150.2 141.4 135.7 131.5 133.5
_+ 14.7 + 15.3 k 16.5’ * 13.9* + 13.6* f l3.3t
n 9 9 9 9 9 8
Mean + standard deviation of the population in each group of rats is shown. Incisors of all rats were shortened repeatedly during the experimental period. Saline, a group of rats given single injection of saline at day 0; colchicine, groups of rats given single injection of 0.5, 1.0 and 1.5 mg of colchicinefig of body weight respectively at day 0; n, number of rats in each group in each day. One rat in 1.5 mg group died between the 3rd and 4th day. Significant differences from day 0 values in each group, t p < 0.01, * p < 0.001. The method of paired comparisons was employed to estimate the r-values.
RESULTS
Dose-response
effects
of colchicine
Changes in the body weight. Table 1 shows the changes in the body weight in each group. The mean body weights in the experimental groups tended to decrease after injection of the drug; the bigger the doses, the more the weight loss. Diarrhoea was sometimes present but was not severe even in a group of rats given 1.5 mg of colchicine/kg of body weight. The rats in this group were very ill, particularly on the 2nd day, but most were recovering from the 3rd day after the injection. One rat in this group died on the 4th day. Eruption rates. Figure 1 shows daily eruption rates from one day before and 4 days after the injection of various doses of colchicine. Mean values in the experimental groups were compared with the respective 1500
i
control ones by Student’s t-test. No significant differences were found before the injection of the drug in all experimental groups and also in OSmg group throughout the experimental period. In the I.0 mg group. the mean eruption rate fell to 41.3 per cent of the control value on the 1st day (p < 0.001); it was 63.3 per cent on the 2nd day after the injection @ < 0.002). The differences were still significant on the 3rd 0, < 0.05) but not on the 4th day. In the 1.5mg group, the mean eruption rates fell to 6.3 per cent of the control value on the 1st (p K 0.001) and 16.0 per cent on the 2nd day (p << O.OOl) respectively. Although the eruption rates recovered considerably on the 3rd and 4th days, the differences were still significant 0, < 0.001 and co.05 respectively). Dose-response rrlatiotisirip. Figure 2 shows the relationship betweeen the logarithm of the dose of colchitine and the percentage reduction of the eruption rates for initial 24 h after injection of the drug. The eruption rate for 24 h before the injection was used as the control value in each animal. The formula of the regression line was estimated as follows: J = 179.5
x + 62.5
where y is the percentage reduction of the eruption rate and x is the log dose of colchicine. The corre-
Fig. 1. Changes in the daily eruption rates of unimpeded mandibular incisors of rats following injection of various doses of colchicine. Open circles, solid circles, open squares and solid squares represent groups of rats given single subcutaneous injection of saline, 0.5, 1.0 or 1.5 mg of colchitine/kg of body weight respectively at day 0. Each point and vertical bar represent the mean + 1 SD of the population.
log
dose
of
colchiclne
Fig. 2. The relationship between the log dose of colchicine and the percentage reduction of the eruption rate for initial 24 h after injection of the drug. Each point and vertical bar represent the mean ) 1SD of the population.
Dose-response
effects of colchicine
and vinblastine
117
Table 2. Changes in the body weight following injection of vinblastine
Day -1 0 1 2 3 4
Control Saline 114.1 + 115.6 & 118.1 f 119.9 + 121.1 f 123.0 f
11.2 11.2 11.4* 12.4* 13.6t 14.8*
n 8 8 8 8 8 8
1.0 mg 124.1 k 125.6 + 120.7 + 116.9 + 116.2 + 115.1 k
4.9 5.6 8.1 12.7 16.6 19.6
n
1.5mg
7 7 7 7 6 6
124.7 + 15.8t 127.3 + 13.9 121.1 _+ 15.3t 119.2 _t 20.6 119.5 4 25.8 120.6 & 27.5
Vinblastine n 2.0mg 8 8 8 7 6 6
n
125.2 _+ 15.2? 128.1 + 15.7 121.1 f 13.47 116.7 + 15.6t 118.7 f 18.4 114.2f 19.7t
7 7 7 7 6 6
2.5 mg 132.8 + 135.1 + 128.6 k 127.6 f 125.0 _t 118.9 +
13.4 11.8 13.5* 17.lt 15.6-k 19.3
n 7 7 7 7 6 5
Mean f standard deviation of the population in each group of rats is shown. Incisors of all rats were shortened repeatedly during the experimental period. Saline, control group of rats given single injection of saline at day 0; Vinblastine, groups of rats given single injection of 1.0, 1.5, 2.0 and 2.5 mg of vinblastine/kg of body weight respectively at day 0; n, number of rats in each group in each day. Decreases of the number of rats indicate the death of the animals. Significant differences from day 0 values in each group, t p < 0.05, * p < 0.01. The method of paired comparisons was employed to estimate the t-vaiues.
lation coefficient was estimated to be +0.950 which was significantly different from zero (p 6 0.001, t = 14.90, degree of freedom = 24). Dose-response eflects qf vinblastine Changes in the body weight. Table 2 shows the changes in the body weight after injection of vinblastine in each group. The average body weights in the experimental groups tended to decrease after injection of the drug: the bigger the doses the more the weight loss. Diarrhoea was present from the 2nd day in 2.0 mg and 2.5 mg groups and was most prominent on the 3rd and 4th days in the same groups, indicating gastro-intestinal disturbance by the drug. Most rats in 1.Omg and 1.5 mg groups were not very ill during the experimental period and seemed to have recovered from the toxic effect of the drug on the 4th day. The
I
0
I
I
0
I
2
3
rats in 2.0mg and 2.5 mg groups were still very ill on the 4th day. Seven rats out of 29 in the experimental groups died by the end of the experimental period. Eruption rates. Figure 3 shows daily eruption rates from one day before and 4 days after the injection of various doses of vinblastine. Average values in the experimental groups were also compared with the respective control ones by the t-test. No significant differences were found before the injection of the drug in all experimental groups and in the l.Omg group throughout the experimental period. In the 1.5 mg group, significant decrease of the eruption rate was only found on the 1st day (JJ< 0.01). In the 2.0 mg group, the average eruption rate dropped to 70 per cent of the control value on the first day (p < 0.001). The differences between the experimental and the respective control values were still significant on the 2nd day (p < 0.01) but not on the 3rd and 4th days. In the 2.5 mg group, the mean eruption rate dropped to 60 per cent of the control value on the 1st day (p < 0.001). The differences were still significant on the 2nd and 3rd days (p < 0.01) but not on the 4th
J 4
Days
Fig. 3. Changes in the daily eruption rates of unimpeded mandibular incisors of rats following injection of various doses of vinblastine. Open circles, solid circles, triangles, open squares and solid squares represent groups of rats given single intraperitoneal injection of saline, 1.0, 1.5, 2.0 or 2.5 mg of vinblastinebg of body weight respectively at day 0. Each point and vertical bar represent the mean & 1 SD of the population.
6
0.17610
!?a0
03979
Log dose of vinblastine Fig. 4. The relationship between the log dose of vinblastine and the percentage reduction of the eruption rate for initial 24 h after injection of the drug. Each point and vertical bar represent the mean + 1 SD of the population.
IIX
M. Chiba.
K. Takizawa and S. Ohshima
Dosc-rc,spor~\c, r&tionship. Figure 4 shows the relationship between the logarithm of the dose of vinblastine and the percentage reduction of the eruption rates for initial 2411 after injection of the drug. The eruption rate for 24 h heforc the injection was used as the control vatuc in each animal. The formula of the regression tine is estimated as follows: I‘ = 91.x
Y -- 5.4
where J is the percentage reduction of the eruption rate and Y is the log dose of vinblastine. The correlation coetficlcnr was estimated to be +O.%O which was significant11 different from zero (p < 0.01. I : 3.51. degree of flreedom = 37).
Single injections of both cotchicine and vinblastine produced dose-dependent inhibition of the eruption rate of the rat mandibular incisor. However. the effect of colchicine was much stronger than that of vinblastine, A dose of 1.5mg of colchicine/kg of body weight (3.76 x 10 ” mol;kg) decelerated the eruption rate to 6 per cent of the control value in the initial 24 h after the injection: and 2.5 mg of vmblastinekg of body weight (3.08 x IO ” mo;kg) to 60 per cent. By the extrapolation of the dose response lines (Figs 2 and 4). it is possible to estimate the expected doses of colchicine and vinblastine to reduce the eruption rate to 7cro for ;I day or so: that is. 1.62 mg/kg and An expected dose of 14.07 mg; kg respectively. demecolcine to stop the eruption of the rat incisor for about a da) would perhaps bc about 12 mg,‘kg (Chiba C( trl.. 1%X). It seems that unlike colchicine, vinbtastine and demecolcinc do not stop the eruption at below their lethal doses. The toxicity of cotchicine derivatives has been examined extensively (Goldberg cutul.. 1950: Santavy. Lang and Malinsky. 1950: Streicher, 1951: SchL. Loustalot and Gross, 1954). Ferguson (1952) showed that acute actlons of colchicine are characterized by a delayed appearance of effects, severe gastro-intestinal disturbances and eventually death. His findings were in agreement with ours for colchicine or vinblastine and also with those previously for demecolcine (Chiba t’t nl.. t 968). At doses that stop incisor eruption. the toxicity 01 colchicine seemed to be less than vinblastine and demecolcine: its inhibition of incisor eruption at such small doses suggests that its action may be specific. ft is worth mentioning that toxicity and anti-neoplastic activity do not change concomitantly or equally with modification of the chemical structure of colchicine derivatives (Goldberg et al.. 1950). In our experiments. delayed appearance of the toxicity and the almost immediate inhibition of incisor eruption by both drugs used suggest that they acted directly on eruption and indirectly by affecting the animals’ general condition. Pharmacological actions of colchicine are diverse and it is not possible to attribute the inhibition of the incisor eruption confidently to any single mode of action. Hc,wever. some important actions of colchitine, demecotclnc and vinblastine have been shown to be related to their attachment to tubulin and prevention of asscmhlg into tubules followed by the dissolution and disappcarancc of microtubules (Borisy and
Taylor, 1967; Wilson and Friedkin. 1967; Wilson et (I/.. 1970; Makdwista. Sato and Bensch, 1968). Cellular functions such as mitosis (InouC, 1952; Inoui and Sato. 1967: McIntosh. Hepler and Van Wie, 1969), migration of fibroblasts (Weimer. Fellman and Davis, 1969; Waddell. Robson and Edwards, 1974) and monocytes (Crispe. 1976). ciliary and flagellar movement (Satir. 1965; Mooseker and Tilney, 1973), movement of intracellular particles (Malawista, 1965; Schmitt, 1968: Obika et al.. 1978) and the intracellular production of substances such as collagen (Diegelmann and Peterkofsky. 1972; Kudo, 1975) have been related to microtubule function. Ness I 1967) postulated that the periodontal connective fibroblasts may produce a motive force to pull the tooth forwards. In fact. the presence of microtubules which may be related with the motility of these fibroblasts has been demonstrated by Beertsen. Evcrts and Van den Hooff (1974). Nevertheless, it is not yet known how microtubules in fibroblasts produce a motive force nor how colchicine interferes with the eruptive machine. REFERENCES
Beer&on W.. Everts V. and Van den Hooff A. 1974. Fine structure of fibroblasts in the periodontal ligament of the rat incisor and their possible role in tooth eruption. ./ltchs oral Biol. 19. 1087m 1098. Bcrkovitz B. K. B. 1972. The effect of democolcine and of triethanomelamine on the unimpeded eruption rate of normal and root resected incisor teeth in rats. Archs oral Hiol. 17. 937-947. Borisy C. G. and Taylor E. W. 1967. The mechanism of action of colchicine. Binding of colchlcine-3H to cellular protein. J. Ctpll Bioi. 34, 525 533. Chiba M., Nakagawa K. and Mimura T. 1966. Estimation of mitotic rate and mitotic duration in the internal enamel epithelium of the rat maxillary incisor using a colchlcine technique. .3r&s orul Biol. I I. 803-814. C’hiba M.. Narraway .I. M. and Ness A. R. 1968. Impeded and unimpeded eruptlon of the mandibular incisor of the adult male rat and its stoppage by demecolcine. J. dent. Ra. 47. YX6 (Abstr.1. (‘hiha M.. Tashiro T.. Tsuruta M. and Eto K. 1976. Acceleration and circadian rhythm of eruption rates in the rat incisor. .,lr-~/IS,,vrr/ 8101. 21. 269%27 I. C‘rispe I. h. 1976. The effect of vinblastine, colchicine and hexylene glycol on migration of human monocytes. Expl. Cell Res. 100, 443447. Diegelmann R. F. and Peterkofsky B. 1972. Inhibition of collagen secretion from hone and cultured fibroblasts by microtubular disruptive drugs. Proc. *mm. Atad. Sci. 69, X97 X96. Ferguson F. C. 1952. Colchicinc~~l. General pharmacology. J. f’llurmuc~. c’.~p. Tltrr. 106, 261-270. Goldb&g B., Ortega L. G.. Goldin A., Ullyot G. E. and Schoenbach E. B. 1950. Studies on colchicine derivatlves--1. Toxicity in mice and effects on mouse sarcoma 1x0.Cmcer 3, 12&l 29. InouL: S. 1952. The effect of colchlcine on the microscopic and submicroscopic structure of the mitotic spindle. i. \-1’1Cc,// R?S. (SuppI.) 2. 305m 3 18. InouL: S. and Sato H. 1967. Cell motility by labile associatton of molecules. The nature of mitotic spindle fibers and their role in chromosome movement. J. grn. Physiol. 50, 759 292. Kudo N. 1975. Effect of colchicine on the secretion of matrices of dentine and enamel in the rat incisor: an autoradiographic study using [‘HI-proline. Culcij Tiss. Rc.\. IS. 37 46.
Dose-response
effects of colchicine
Main J. H. P. and Adams D. 1966. Experiments on the rat incisor into the cellular proliferation and blood-pressure theories of the tooth eruption. Archs oral Bid/. 11, 163-l 78. Malawista S. E. 1965. On the action of colchicine. The melanocyte model. J. e.rp. Med. 122, 361-384. Malawista S. E., Sato H. and Bensch K. G. 1968. Vinblastine and griseofulvin reversibly disrupt the living mitotic spindle. Science 160, 77@772. McIntosh J. R., Hepler P. K. and Van Wie D. G. 1969. Model for mitosis. Nature 224. 6599663. Mooseker M. S. and Tilney L. G. 1973. Isolation and reactivation of the axostyle. Evidence for a dynein-like ATPase in the axostyle. J. Cell Biol. 56, 13-26. Ness A. R. 1967. Eruption-a review. In: The Mechanisms of Tooth Support. A Symposium, pp. 84-88. John Wright, Bristol. Obika M., Turner W. A.. Negishi S., Menter D. G., Tchen T. T. and Taylor J. D. 1978. The effects of lumicolchitine, colchicine and vinblastine on pigment migration in fish chromatophores. J. exp. Zoo/. 205, 95-l 10. Olmsted J. B. and Borisy G. G. 1973. Microtubules. A. Reo. Biochrm. 42. 5077540. Santavy F., Lang B. et Malinsky J. 1950. L’action mitotique et la toxicite des nouvelles substances isolees du colchique (Colchicum autumnale L.). Archs int. Pharmacodyn. ThPr. 84, 257-268. Satir P. 1965. Studies on cilia-II. Examination of the distal region of the ciliary shaft and the role of the filaments in motility. J. Ce// Bio/. 26. 805-834.
and vinblastine
119
Schar B., Loustalot P. und Gross F. 1954. Demecolcin (Substanz F), ein neues, aus Colchicum autumnale isoliertes Alkaloid mit starker antimitotischer Wirkung. Klin. Wschs. 32. 49- 57. Schmitt F. 0. 1968. Fibrous proteins- neuronal orpanelles. Proc. nufn. Acad. Sci. 60, 1092- 1101. Snyder J. A. and McIntosh J. R. 1976. Biochemistry and physiology of microtubules. A. Rev. Biochtm. 45, 699m720. Streicher E. 1951. Toxicity of colchicine. di-isopropyl fluorophosphate, intocostrin. and potassium cyanide in mice at 4°C. Proc. Sot. e.up. Biol. Med. 76, 536-538. Taylor E. W. 1973. Macromolecular assembly inhibitors and their action on the cell cycle. In: Drugs and the Cell Cycle (Edited by Zimmerman A. M., Padilla G. M. and Cameron I. L.), pp. 11-24. Academic Press. New York and London. Waddell A. W.. Robson R. T. and Edwards J. G. 1974. Colchicine and vinblastine inhibit fibroblast aggregation. Nature 248, 239-241. Weimar V. L.. Fellman M. J. and Davis M. 1969. Inhibitory action of colchicine on cell movement in organ culture. Proc. Sot. cup. Biol. Med. 131, 1457 1459. Wilson L. and Friedkin M. 1967. The biochemical events of mitosis--II. The in VIVOand in vitro binding of colchitine in grasshopper embryos and its possible relation to inhibition of mitosis, Biochemistry 6. 3 126~ 3 135. Wilson L.. Bryan J.. Ruby A. and Maria D. 1970. Precipitation of proteins by vmblastine and calcium ions I+)<,. nuts. Acad. Sci. 66. 807 8 14.
CHIRA,
K.
TAKIZAWA
and
S. OHSHIMA
of Pharmacology, School of Dental Medicine, Tsurumi 2-l-3 Tsurumi. Tsurumi-ku. Yokohama. Japan
University,
Summary-Dose-dependent inhibition of the eruption rate followed single injections of colchitine or vinblastine. 1.5 mg of colchicine/kg of body wt decelerated the eruption rate to 6 per cent of the control value in the initial 24 h after subcutaneous injection and 2.5 mg of vinblastine/kg of body wt to 60 per cent. Doses of colchicine and vinblastine which would reduce the eruption to zero were estimated to be 1.62 mg/kg and 14.07 mg/kg respectively. Toxicity developed slowly whereas inhibition of eruption appeared quickly. Colchicine toxicity was the less. The inhibitory action of these drugs may be related to disruption of microtubules in the cells which may produce the motive force of eruption. It has yet to be shown how microtubules could produce such a force.
INTRODUCTION
0.2 ml/100 g of body weight in all experiments. One control group of rats received a single subcutaneous injection of the same volume of saline. The time of injection ranged from lo:14 to 11:34 hours.
The effect of demecolcine, like colchicine a derivative of Colchicum, on the eruption of the rat incisor was examined by, for instance, Main and Adams (1966). The drug decelerated or even stopped the incisor eruption at a certain dose level (Chiba. Narraway and Ness, 1968; Berkovitz, 1972). The mechanism is not known although it is known that demecolcine arrests cells in metaphase in growing tissues (Chiba, Nakagawa and Mimura, 1966). It has been shown that Colchicurn alkaloids and Vinca alkaloids (vinblastine) bind specifically to tubulin and prevent its assembly into tubules and thus the function of cells in which microtubules are involved would be interrupted (see Taylor, 1973; Olmsted and Borisy, 1973; Snyder and McIntosh, 1976). In order to understand the mechanism of action of these drugs on the eruption of the rat incisor, it would be helpful to examine whether or not deceleration of eruption is produced by any inhibitor of microtubules. As a step in this direction. we undertook the present experiments.
MATERIALS
AND
Doses of cinhlastine Thirty-seven rats weighing 124g on the average were divided into 5 sub-groups of 7-8 animals. The animals of the 4 experimental groups received a single intraperitoneal injection of 1.0, 1.5, 2.0 or 2.5 mg of vinblastine/kg of body weight respectively. Vinblastine (Eli Lilly, “VELBE”, vinblastine sulphate, Basingstoke, England) was dissolved in physiological saline. The volume of injected solutions ranged from 0.1 to 0.25 ml/lOOg of body weight. One group control received a single intraperitoneal injection of 0.25 ml of saline/lOOg of body weight. The time of injection ranged from lo:07 to 11:36 hours. Meusurement
of the eruption rate
Eruption rates of the left mandibular incisors of all rats were measured at daily intervals from one day before the injection until the experiment was terminated 4 days later. A photographic method was used with the right incisor fixed to its jaw bone by a stainless steel pin to provide a stable reference tooth (Chiba et (I/., 1976). Photographs of the tooth surface were taken at magnification of x 3 or x 6.6. Prints were made from the negatives at final magnifications of x 82 and x 91. The jaws were radiographed at the end of the experimental period to examine the position of the pin fixing the incisor to its jaw bone. Under ether anaesthesia, all 4 incisors in each rat were shortened at the level of the inter-incisal crest or at the gingival margin one and 3 days before the injection and, when appropriate, repeatedly afterwards to maintain the unimpeded state. Fixation of the incisor to the bone was carried out one day before the in.jection of the drug.
METHODS
Seventy-two male rats of the Wistar strain, aged 5 to 6 weeks, were kept in metal cages, fed on a powdered diet during the experimental period and for at least one week beforehand and took water ad lihitum from the metal nozzle of a water delivery tube. Doses of colchicirle Thirty-five rats weighing 149 g on the average were divided into 4 sub-groups of 8-9 animals. The animals of the 3 experimental groups received a single subcutaneous injection of 0.5, 1.O or 1.5mg of colchicine/kg of body weight respectively. Colchicine (E. Merck, Darmstadt, West Germany) was dissolved in physiological saline. The volume of injected solutions was II5
116
M. Chiba.
Table I, Changes Control Saline
Day -1
148.7 147.0 146.2 145.8 145.3 146.9
0
1 2 3 4
k 21.4 k 20.9 _t 18.5 If: 18.1 f 16.0 + 12.7
II
9 9 9 9 9 9
K. Takirawa and S. Ohshima
in the body weight following injection
0.5 mg 154.3 149.5 146.7 149.2 148.8 149.4
* + + f + *
Il.3 9.5 9.4
II.8 Il.2 13.2
I1 9 9 9 9 9 9
of colchicine
Colchicine 1.0 mg 141.3 140.2 133.4 131.4 131.6 131.0
_+ 18.2 + 16.5 + 18.3* * 1x.4* + 1X.4* * 17.9t
I .5 mg
II 8 X 8 X 8 8
151.7 150.2 141.4 135.7 131.5 133.5
_+ 14.7 + 15.3 k 16.5’ * 13.9* + 13.6* f l3.3t
n 9 9 9 9 9 8
Mean + standard deviation of the population in each group of rats is shown. Incisors of all rats were shortened repeatedly during the experimental period. Saline, a group of rats given single injection of saline at day 0; colchicine, groups of rats given single injection of 0.5, 1.0 and 1.5 mg of colchicinefig of body weight respectively at day 0; n, number of rats in each group in each day. One rat in 1.5 mg group died between the 3rd and 4th day. Significant differences from day 0 values in each group, t p < 0.01, * p < 0.001. The method of paired comparisons was employed to estimate the r-values.
RESULTS
Dose-response
effects
of colchicine
Changes in the body weight. Table 1 shows the changes in the body weight in each group. The mean body weights in the experimental groups tended to decrease after injection of the drug; the bigger the doses, the more the weight loss. Diarrhoea was sometimes present but was not severe even in a group of rats given 1.5 mg of colchicine/kg of body weight. The rats in this group were very ill, particularly on the 2nd day, but most were recovering from the 3rd day after the injection. One rat in this group died on the 4th day. Eruption rates. Figure 1 shows daily eruption rates from one day before and 4 days after the injection of various doses of colchicine. Mean values in the experimental groups were compared with the respective 1500
i
control ones by Student’s t-test. No significant differences were found before the injection of the drug in all experimental groups and also in OSmg group throughout the experimental period. In the I.0 mg group. the mean eruption rate fell to 41.3 per cent of the control value on the 1st day (p < 0.001); it was 63.3 per cent on the 2nd day after the injection @ < 0.002). The differences were still significant on the 3rd 0, < 0.05) but not on the 4th day. In the 1.5mg group, the mean eruption rates fell to 6.3 per cent of the control value on the 1st (p K 0.001) and 16.0 per cent on the 2nd day (p << O.OOl) respectively. Although the eruption rates recovered considerably on the 3rd and 4th days, the differences were still significant 0, < 0.001 and co.05 respectively). Dose-response rrlatiotisirip. Figure 2 shows the relationship betweeen the logarithm of the dose of colchitine and the percentage reduction of the eruption rates for initial 24 h after injection of the drug. The eruption rate for 24 h before the injection was used as the control value in each animal. The formula of the regression line was estimated as follows: J = 179.5
x + 62.5
where y is the percentage reduction of the eruption rate and x is the log dose of colchicine. The corre-
Fig. 1. Changes in the daily eruption rates of unimpeded mandibular incisors of rats following injection of various doses of colchicine. Open circles, solid circles, open squares and solid squares represent groups of rats given single subcutaneous injection of saline, 0.5, 1.0 or 1.5 mg of colchitine/kg of body weight respectively at day 0. Each point and vertical bar represent the mean + 1 SD of the population.
log
dose
of
colchiclne
Fig. 2. The relationship between the log dose of colchicine and the percentage reduction of the eruption rate for initial 24 h after injection of the drug. Each point and vertical bar represent the mean ) 1SD of the population.
Dose-response
effects of colchicine
and vinblastine
117
Table 2. Changes in the body weight following injection of vinblastine
Day -1 0 1 2 3 4
Control Saline 114.1 + 115.6 & 118.1 f 119.9 + 121.1 f 123.0 f
11.2 11.2 11.4* 12.4* 13.6t 14.8*
n 8 8 8 8 8 8
1.0 mg 124.1 k 125.6 + 120.7 + 116.9 + 116.2 + 115.1 k
4.9 5.6 8.1 12.7 16.6 19.6
n
1.5mg
7 7 7 7 6 6
124.7 + 15.8t 127.3 + 13.9 121.1 _+ 15.3t 119.2 _t 20.6 119.5 4 25.8 120.6 & 27.5
Vinblastine n 2.0mg 8 8 8 7 6 6
n
125.2 _+ 15.2? 128.1 + 15.7 121.1 f 13.47 116.7 + 15.6t 118.7 f 18.4 114.2f 19.7t
7 7 7 7 6 6
2.5 mg 132.8 + 135.1 + 128.6 k 127.6 f 125.0 _t 118.9 +
13.4 11.8 13.5* 17.lt 15.6-k 19.3
n 7 7 7 7 6 5
Mean f standard deviation of the population in each group of rats is shown. Incisors of all rats were shortened repeatedly during the experimental period. Saline, control group of rats given single injection of saline at day 0; Vinblastine, groups of rats given single injection of 1.0, 1.5, 2.0 and 2.5 mg of vinblastine/kg of body weight respectively at day 0; n, number of rats in each group in each day. Decreases of the number of rats indicate the death of the animals. Significant differences from day 0 values in each group, t p < 0.05, * p < 0.01. The method of paired comparisons was employed to estimate the t-vaiues.
lation coefficient was estimated to be +0.950 which was significantly different from zero (p 6 0.001, t = 14.90, degree of freedom = 24). Dose-response eflects qf vinblastine Changes in the body weight. Table 2 shows the changes in the body weight after injection of vinblastine in each group. The average body weights in the experimental groups tended to decrease after injection of the drug: the bigger the doses the more the weight loss. Diarrhoea was present from the 2nd day in 2.0 mg and 2.5 mg groups and was most prominent on the 3rd and 4th days in the same groups, indicating gastro-intestinal disturbance by the drug. Most rats in 1.Omg and 1.5 mg groups were not very ill during the experimental period and seemed to have recovered from the toxic effect of the drug on the 4th day. The
I
0
I
I
0
I
2
3
rats in 2.0mg and 2.5 mg groups were still very ill on the 4th day. Seven rats out of 29 in the experimental groups died by the end of the experimental period. Eruption rates. Figure 3 shows daily eruption rates from one day before and 4 days after the injection of various doses of vinblastine. Average values in the experimental groups were also compared with the respective control ones by the t-test. No significant differences were found before the injection of the drug in all experimental groups and in the l.Omg group throughout the experimental period. In the 1.5 mg group, significant decrease of the eruption rate was only found on the 1st day (JJ< 0.01). In the 2.0 mg group, the average eruption rate dropped to 70 per cent of the control value on the first day (p < 0.001). The differences between the experimental and the respective control values were still significant on the 2nd day (p < 0.01) but not on the 3rd and 4th days. In the 2.5 mg group, the mean eruption rate dropped to 60 per cent of the control value on the 1st day (p < 0.001). The differences were still significant on the 2nd and 3rd days (p < 0.01) but not on the 4th
J 4
Days
Fig. 3. Changes in the daily eruption rates of unimpeded mandibular incisors of rats following injection of various doses of vinblastine. Open circles, solid circles, triangles, open squares and solid squares represent groups of rats given single intraperitoneal injection of saline, 1.0, 1.5, 2.0 or 2.5 mg of vinblastinebg of body weight respectively at day 0. Each point and vertical bar represent the mean & 1 SD of the population.
6
0.17610
!?a0
03979
Log dose of vinblastine Fig. 4. The relationship between the log dose of vinblastine and the percentage reduction of the eruption rate for initial 24 h after injection of the drug. Each point and vertical bar represent the mean + 1 SD of the population.
IIX
M. Chiba.
K. Takizawa and S. Ohshima
Dosc-rc,spor~\c, r&tionship. Figure 4 shows the relationship between the logarithm of the dose of vinblastine and the percentage reduction of the eruption rates for initial 2411 after injection of the drug. The eruption rate for 24 h heforc the injection was used as the control vatuc in each animal. The formula of the regression tine is estimated as follows: I‘ = 91.x
Y -- 5.4
where J is the percentage reduction of the eruption rate and Y is the log dose of vinblastine. The correlation coetficlcnr was estimated to be +O.%O which was significant11 different from zero (p < 0.01. I : 3.51. degree of flreedom = 37).
Single injections of both cotchicine and vinblastine produced dose-dependent inhibition of the eruption rate of the rat mandibular incisor. However. the effect of colchicine was much stronger than that of vinblastine, A dose of 1.5mg of colchicine/kg of body weight (3.76 x 10 ” mol;kg) decelerated the eruption rate to 6 per cent of the control value in the initial 24 h after the injection: and 2.5 mg of vmblastinekg of body weight (3.08 x IO ” mo;kg) to 60 per cent. By the extrapolation of the dose response lines (Figs 2 and 4). it is possible to estimate the expected doses of colchicine and vinblastine to reduce the eruption rate to 7cro for ;I day or so: that is. 1.62 mg/kg and An expected dose of 14.07 mg; kg respectively. demecolcine to stop the eruption of the rat incisor for about a da) would perhaps bc about 12 mg,‘kg (Chiba C( trl.. 1%X). It seems that unlike colchicine, vinbtastine and demecolcinc do not stop the eruption at below their lethal doses. The toxicity of cotchicine derivatives has been examined extensively (Goldberg cutul.. 1950: Santavy. Lang and Malinsky. 1950: Streicher, 1951: SchL. Loustalot and Gross, 1954). Ferguson (1952) showed that acute actlons of colchicine are characterized by a delayed appearance of effects, severe gastro-intestinal disturbances and eventually death. His findings were in agreement with ours for colchicine or vinblastine and also with those previously for demecolcine (Chiba t’t nl.. t 968). At doses that stop incisor eruption. the toxicity 01 colchicine seemed to be less than vinblastine and demecolcine: its inhibition of incisor eruption at such small doses suggests that its action may be specific. ft is worth mentioning that toxicity and anti-neoplastic activity do not change concomitantly or equally with modification of the chemical structure of colchicine derivatives (Goldberg et al.. 1950). In our experiments. delayed appearance of the toxicity and the almost immediate inhibition of incisor eruption by both drugs used suggest that they acted directly on eruption and indirectly by affecting the animals’ general condition. Pharmacological actions of colchicine are diverse and it is not possible to attribute the inhibition of the incisor eruption confidently to any single mode of action. Hc,wever. some important actions of colchitine, demecotclnc and vinblastine have been shown to be related to their attachment to tubulin and prevention of asscmhlg into tubules followed by the dissolution and disappcarancc of microtubules (Borisy and
Taylor, 1967; Wilson and Friedkin. 1967; Wilson et (I/.. 1970; Makdwista. Sato and Bensch, 1968). Cellular functions such as mitosis (InouC, 1952; Inoui and Sato. 1967: McIntosh. Hepler and Van Wie, 1969), migration of fibroblasts (Weimer. Fellman and Davis, 1969; Waddell. Robson and Edwards, 1974) and monocytes (Crispe. 1976). ciliary and flagellar movement (Satir. 1965; Mooseker and Tilney, 1973), movement of intracellular particles (Malawista, 1965; Schmitt, 1968: Obika et al.. 1978) and the intracellular production of substances such as collagen (Diegelmann and Peterkofsky. 1972; Kudo, 1975) have been related to microtubule function. Ness I 1967) postulated that the periodontal connective fibroblasts may produce a motive force to pull the tooth forwards. In fact. the presence of microtubules which may be related with the motility of these fibroblasts has been demonstrated by Beertsen. Evcrts and Van den Hooff (1974). Nevertheless, it is not yet known how microtubules in fibroblasts produce a motive force nor how colchicine interferes with the eruptive machine. REFERENCES
Beer&on W.. Everts V. and Van den Hooff A. 1974. Fine structure of fibroblasts in the periodontal ligament of the rat incisor and their possible role in tooth eruption. ./ltchs oral Biol. 19. 1087m 1098. Bcrkovitz B. K. B. 1972. The effect of democolcine and of triethanomelamine on the unimpeded eruption rate of normal and root resected incisor teeth in rats. Archs oral Hiol. 17. 937-947. Borisy C. G. and Taylor E. W. 1967. The mechanism of action of colchicine. Binding of colchlcine-3H to cellular protein. J. Ctpll Bioi. 34, 525 533. Chiba M., Nakagawa K. and Mimura T. 1966. Estimation of mitotic rate and mitotic duration in the internal enamel epithelium of the rat maxillary incisor using a colchlcine technique. .3r&s orul Biol. I I. 803-814. C’hiba M.. Narraway .I. M. and Ness A. R. 1968. Impeded and unimpeded eruptlon of the mandibular incisor of the adult male rat and its stoppage by demecolcine. J. dent. Ra. 47. YX6 (Abstr.1. (‘hiha M.. Tashiro T.. Tsuruta M. and Eto K. 1976. Acceleration and circadian rhythm of eruption rates in the rat incisor. .,lr-~/IS,,vrr/ 8101. 21. 269%27 I. C‘rispe I. h. 1976. The effect of vinblastine, colchicine and hexylene glycol on migration of human monocytes. Expl. Cell Res. 100, 443447. Diegelmann R. F. and Peterkofsky B. 1972. Inhibition of collagen secretion from hone and cultured fibroblasts by microtubular disruptive drugs. Proc. *mm. Atad. Sci. 69, X97 X96. Ferguson F. C. 1952. Colchicinc~~l. General pharmacology. J. f’llurmuc~. c’.~p. Tltrr. 106, 261-270. Goldb&g B., Ortega L. G.. Goldin A., Ullyot G. E. and Schoenbach E. B. 1950. Studies on colchicine derivatlves--1. Toxicity in mice and effects on mouse sarcoma 1x0.Cmcer 3, 12&l 29. InouL: S. 1952. The effect of colchlcine on the microscopic and submicroscopic structure of the mitotic spindle. i. \-1’1Cc,// R?S. (SuppI.) 2. 305m 3 18. InouL: S. and Sato H. 1967. Cell motility by labile associatton of molecules. The nature of mitotic spindle fibers and their role in chromosome movement. J. grn. Physiol. 50, 759 292. Kudo N. 1975. Effect of colchicine on the secretion of matrices of dentine and enamel in the rat incisor: an autoradiographic study using [‘HI-proline. Culcij Tiss. Rc.\. IS. 37 46.
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Main J. H. P. and Adams D. 1966. Experiments on the rat incisor into the cellular proliferation and blood-pressure theories of the tooth eruption. Archs oral Bid/. 11, 163-l 78. Malawista S. E. 1965. On the action of colchicine. The melanocyte model. J. e.rp. Med. 122, 361-384. Malawista S. E., Sato H. and Bensch K. G. 1968. Vinblastine and griseofulvin reversibly disrupt the living mitotic spindle. Science 160, 77@772. McIntosh J. R., Hepler P. K. and Van Wie D. G. 1969. Model for mitosis. Nature 224. 6599663. Mooseker M. S. and Tilney L. G. 1973. Isolation and reactivation of the axostyle. Evidence for a dynein-like ATPase in the axostyle. J. Cell Biol. 56, 13-26. Ness A. R. 1967. Eruption-a review. In: The Mechanisms of Tooth Support. A Symposium, pp. 84-88. John Wright, Bristol. Obika M., Turner W. A.. Negishi S., Menter D. G., Tchen T. T. and Taylor J. D. 1978. The effects of lumicolchitine, colchicine and vinblastine on pigment migration in fish chromatophores. J. exp. Zoo/. 205, 95-l 10. Olmsted J. B. and Borisy G. G. 1973. Microtubules. A. Reo. Biochrm. 42. 5077540. Santavy F., Lang B. et Malinsky J. 1950. L’action mitotique et la toxicite des nouvelles substances isolees du colchique (Colchicum autumnale L.). Archs int. Pharmacodyn. ThPr. 84, 257-268. Satir P. 1965. Studies on cilia-II. Examination of the distal region of the ciliary shaft and the role of the filaments in motility. J. Ce// Bio/. 26. 805-834.
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Schar B., Loustalot P. und Gross F. 1954. Demecolcin (Substanz F), ein neues, aus Colchicum autumnale isoliertes Alkaloid mit starker antimitotischer Wirkung. Klin. Wschs. 32. 49- 57. Schmitt F. 0. 1968. Fibrous proteins- neuronal orpanelles. Proc. nufn. Acad. Sci. 60, 1092- 1101. Snyder J. A. and McIntosh J. R. 1976. Biochemistry and physiology of microtubules. A. Rev. Biochtm. 45, 699m720. Streicher E. 1951. Toxicity of colchicine. di-isopropyl fluorophosphate, intocostrin. and potassium cyanide in mice at 4°C. Proc. Sot. e.up. Biol. Med. 76, 536-538. Taylor E. W. 1973. Macromolecular assembly inhibitors and their action on the cell cycle. In: Drugs and the Cell Cycle (Edited by Zimmerman A. M., Padilla G. M. and Cameron I. L.), pp. 11-24. Academic Press. New York and London. Waddell A. W.. Robson R. T. and Edwards J. G. 1974. Colchicine and vinblastine inhibit fibroblast aggregation. Nature 248, 239-241. Weimar V. L.. Fellman M. J. and Davis M. 1969. Inhibitory action of colchicine on cell movement in organ culture. Proc. Sot. cup. Biol. Med. 131, 1457 1459. Wilson L. and Friedkin M. 1967. The biochemical events of mitosis--II. The in VIVOand in vitro binding of colchitine in grasshopper embryos and its possible relation to inhibition of mitosis, Biochemistry 6. 3 126~ 3 135. Wilson L.. Bryan J.. Ruby A. and Maria D. 1970. Precipitation of proteins by vmblastine and calcium ions I+)<,. nuts. Acad. Sci. 66. 807 8 14.