Limb regeneration blastema cells in the adult newt, Notophthalmus viridescens, give aberrant response to colchicine

BRENDA L. COOMBER,' KARIN J. DAVIDSON, AND STEVEN R. SCADDING*

Department of Zoology, University of Guelph, Guelph, Ont., Canada NlG 2 Wl Received August 5, 1982

COOMBER, B. L., K. J. DAVIDSON, and S. R. SCADDING. 1983. Limb regeneration blastema cells in the adult newt, Notophthalmus viridescens, give aberrant response to colchicine. Can. J. Zool. 61: 553-559.

This study was designed to investigate the effect of colchicine on the mitotic index (number of mitotic figures per 1000 cells) in the newt limb regeneration blastema. Colchicine doses from 0.1 to 1OOO pg per gram body weight and treatment durations from 2 to 72 h were used to determine the dose of colchicine and treatment period resulting in optimum metaphase arrest. A preliminary experiment demonstrated that there was no circadian rhythm of mitosis in the newt limb regeneration blastema. A dose of 5 pg colchicine per gram body weight was found to give the maximum mitotic index; both higher and lower doses had less effect. Very high doses of colchicine resulted in a mitotic index the same as that of the controls. The mitotic index increased with increasing treatment time up to 36 h, after which there was no further increase. In other newt tissues such as duodenal mucosa and liver, a more typical dose-response relationship was observed in that increased colchicine dose led to increased mitotic indices up to the level of lethal doses of colchicine.

COOMBER, B. L., K. J. DAVIDSON et S. R. SCADDING. 1983. Limb regeneration blastema cells in the adult newt, Notophthalmus viridescens, give aberrant response to colchicine. Can. J. Zool. 61: 553-559.

Cette Ctude a CtC entreprise dans le but de determiner l'effet de la colchicine sur l'indice mitotique (nombre de formations rnitotiques par 1OOO cellules) dans la blastkme de regCnCration d'un membre chez le triton. Des doses de colchicine de 0,1 a 1000 pg par gramme de masse totale ont CtC administrkes aux tritons au cours de traitements d'une duke de 2 a 72 h, afin de dCterminer la dose et la duree de traitement qui entrainent l'arrlit optimal de la mCtaphase. Une experience preliminaire a permis d'Ctablir que la mitose ne suit pas un rythme circadien dans le blastkme. C'est une dose de 5 pg par gramme de masse totale qui donne le meilleur indice mitotique; des doses plus ClevCes et des doses plus faibles entrainent des effets moindres. A des doses trks ClevCes de colchicine, l'indice mitotique est semblable a celui des tritons tCmoins. L'indice mitotique augmente avec la duree du traitement jusqu'a une durCe de 36 h, aprks quoi il n'y a plus d'augmentation. Dans les autres tissus du triton, tels la muqueuse duodCnale et le foie, la rCaction est plus proportionnelle a la dose en ce sens que plus la dose de colchicine est ClevCe, plus l'indice mitotique est ClevC et cela est vrai pour toutes les doses non lCtales de colchicine.

[Traduit par le journal]

Introduction Studies of limb regeneration have frequently used

counts of mitotic figures as indicators of cell prolifera- tion, and many of these studies have used colchicine or related stathmokinetic substances (which arrest chromo- somal movement at metaphase) to collect mitotic figures and make counting easier. Colchicine, a plant alkaloid, halts mitosis at metaphase by disrupting the assembly of microtubules so that the mitotic spindle cannot be formed (Eigsti and Dustin 1955; Borisy and Taylor 1967). However, there is considerable variation in the dose of colchicine used, the timing of the injection, and in the mitotic indices (number of mitotic figures/ number of nuclei counted) obtained (Donaldson and Wilson 1975; Dunlap and Donaldson 1978; Scadding and Vinette 1978; Beach and Jacobson 1979; Wertz and Donaldson 1980; Gospodarowicz and Mescher 198 1 ;

'present address: Department of Anatomy, University of Toronto, Toronto, Ont .

2 ~ u t h o r to whom correspondence and reprint requests should be addressed.

Coomber and Scadding 1982). The results are confusing because when colchicine is injected, the maximum mitotic index is not always obtained with the highest dose. Dunlap and Donaldson (1978) used doses of 2 kg and 10 kg colchicine per gram body weight on newts and found that the lower dose collected more mitotic figures in epidermis than did the higher dose. S. R. Scadding and J. L. Vinette (unpublished observations) have made similar observations on Ambystoma larvae limb and tail regeneration blastemas.

No conclusive studies have been done to determine the effects of colchicine dose and treatment time on the mitotic index in the newt. However, colchicine is only a useful tool if its action is understood and predictable. Hence, this study is an attempt to provide a better understanding of the effects of colchicine on the newt limb regeneration blastema cells. Specifically, it is designed: (a) to determine the optimum dose of colchi- cine for collecting mitotic figures in the newt limb regeneration blastema; (b) to determine the optimum interval between colchicine injection and fixation, for the collecting of mitotic figures in the newt limb

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554 CAN. 1. ZOOL. VOL. 61. 1983

regeneration blastema; (c) to determine if the unusual response of the limb regeneration blastema to colchicine (low dose more effective than high dose) is consistent and repeatable; (d) to investigate why the previously published results using colchicine in regeneration stud- ies have been so variable; and (e) to determine if the unusual response of the limb regeneration blastema is observed in other newt tissues.

Since there is a circadian rhythm in mitotic rate in some animals (Bums 1978; Thorud et al. 1980), and since newt limb regeneration is known to be influenced by light (Maier and Singer 1977, 1982), a preliminary experiment was conducted to determine if there was any circadian variation in the mitotic index of the blastema cells in the regenerating newt limb.

Materials and methods Red-spotted newts, Notophthalmus viridescens (mean

weight 2 g), obtained from Charles D. Sullivan, Nashville, Tennessee, were maintained in the laboratory at 22OC and fed live Tubifex or Enchytraeus worms ad libitum. The newts were anaesthetized with 0.1% tricaine methane sulfonate and both forearms were amputated midway through the radius- ulna. Newts were then returned to water-filled aquaria, and limbs were allowed to regenerate for 23 to 28 days to the midcone stage of regeneration (Prichett and Dent 1972). The cone stage was used since it is characterized by the most active cell proliferation (Dresden and Moses 1973). For further details of maintenance and surgical procedures, see Scadding (1977, 198 1). Unamputated (intact) newts were used to study the effects of colchicine on mitotic counts in duodenal mucosa and liver. At the end of the experimental period the newts were reanaesthetized and both forelimbs were amputated at the shoulder and fixed in Bouin's fluid.

Colchicine was dissolved in saline to make injection solutions varying in concentration from 1 pg to 40mg colchicine/mL, allowing doses of 0.1 pg to 1000 pg of colchicine per gram body weight when approximately 0.05 to 0.1 mL was injected intraperitoneally into the experimental animals. All colchicine solutions were made up on the day prior to injection and refrigerated in the dark at 4°C.

This investigation involved five separate but related experi- ments designed to reach the objectives outlined in the introduction. The experiments were performed in the sequence listed since the results of some experiments influenced the design of the subsequent experiments.

Experiment I Objective: to determine if there is a circadian rhythm in the

mitotic cycle of the cells of the newt limb regeneration blastema. One hundred and ten newts were amputated as described above and then maintained on a light cycle of 12 h light (0700 to 1900) and 12 h dark over the entire period of regeneration. Once the midcone stage of regeneration was reached (day 26) four or five newts were reanaesthetized, and both forelimbs were amputated at the shoulder and fixed, at 2- or 3-h intervals over a period of 3 1 h from 0900 h one day to 1600 h the following day. Colchicine was not used in this experiment.

Experiment 2 Objective: to determine the number of mitotic arrests

occurring during 4 h of exposure to different doses of colchi- cine. Sixty-five newts with cone stage forelimb regenerates were randomly distributed into 11 treatment groups of five or six animals each. Animals in each treatment group received an injection of 0.5, 1, 2.5, 5, 10, 20, 50, 100, or 100Opg colchicine per gram body weight, one group received a saline injection, and one group received no injection. Limbs were removed and fixed 4 h after injection.

Experiment 3 Objective: to determine what effect the duration of exposure

to colchicine has on the number of mitotic arrests. Eighty newts with cone stage forelimb regenerates were divided into ten groups of eight animals each. All animals were injected with 5 pg colchicine per gram body weight at hour zero. Limbs were amputated and fixed as described above at 0 , 2 , 4 , 8, 12, 24, 36, 48, and 72 h postinjection.

Experiment 4 Objective: to determine the number of mitotic arrests

occurring during 36 h of exposure to different doses of colchicine. One hundred and ten newts with cone stage forelimb regenerates were divided into 11 treatment groups of 10 animals each. The colchicine doses of experiment 2 were repeated using a treatment time of 36 h.

Experiment 5 Objective: to determine if the unusual dose-response rela-

tionship (low dose more effective than high dose) between colchicine and mitotic index, as seen in the limb regeneration blastema, is observed in other newt tissues. Eighty intact newts were divided into 11 treatment groups of six to eight animals each. Newts in each treatment group received an injection of 0.1, 0.3, 1.0, 2, 5, 10, 30, 100, or 1000pg colchicine per gram body weight, saline alone, or no injection. Forty-eight hours later the animals were anaesthetized, the abdominal cavity was opened with a midventral incision, and the entire carcass was fixed in Bouin's fluid. Subsequently, portions of the duodenum and liver were dissected out for further analysis.

The amputated limbs were fixed in Bouin's fluid, decalcified in Jenkin's solution (Humason 1979), dehydrated in graded alcohols, cleared in methyl salicylate, and embedded in paraffin. Serial longitudinal sections were cut, then stained with Delafield's haematoxylin and Putt's eosin (Humason 1979). Duodenum and liver were processed similarly except that the decalcification step was omitted.

Mitotic figures and pyknotic nuclei were counted from alternate sections through the central portion of the limb regeneration blastema. In each forelimb 1000 cells were counted and the mitotic index or pyknotic index was expressed as number of mitotic figures or pyknotic nuclei per 1000 cells for each animal. Similar procedures were followed for duodenal and liver sections. Results were subjected to analysis of variance using the general linear models procedure for unbalanced ANOVA, and least squares means procedure, with a 99% confidence level.

Results Experiment 1 (circadian rhythm)

Figure 1 illustrates the mitotic indices observed in the

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COOMBER ET AL.

0900 1500 2100 0300 0900 1500 1 day1 day 2

TIME (hours )

FIG. 1 . This graph shows the relationship between the mitotic index in the limb regeneration blastema and time of fixation over a 31-h period from 0900 one day to 1600 the following day. The daily light cycle over the entire period of regeneration was 12 h light (0700 to 1900) and 12 h dark. Bars indicate standard deviations.

midcone blastema cells at 2- to 3-h intervals over a 3 1-h period. There are no significant differences among the different times of fixation. All of the mean mitotic indices lie in the range from 2.5 to 5.0 mitotic figures per 1000 cells.

Experiment 2 (4 h of exposure to different doses of colchicine)

Uninjected controls had a mean mitotic index of 9.33 k 1.87 (mean + SD) and a mean pyknotic index of 6.56 + 0.38. Saline injected controls had a mean mitotic index of 8.75 + 3.14 and a mean pyknotic index of 7.13 k 3.23. Figure 2 shows the mitotic and pyknotic indices from the blastemas of colchicine-injected animals. The maximal number of mitotic figures was obtained with a dose of 5 kg colchicine per gram body weight. The mitotic index of control animals and those receiving 1000 kg colchicine were significantly lower than the mitotic indices of middose animals (2.5- 10 kg colchi- cine) ( p < 0.01). The mitotic index of animals receiving a dose of 5 kg colchicine was significantly higher than all other mitotic indices ( p < 0.01). The pyknotic indices obtained with low doses (0-5 kg colchicine) were significantly lower than those obtained with high doses (20- 1000 kg) (p < 0.01). Thus, a dose of 5 kg colchicine per gram body weight best maximizes num- bers of mitotic figures and minimizes cytotoxicity as reflected by pyknotic nuclei.

DOSE (pg colchlc~ne/g body wl.)

1 I0 100 1000 DOSE ( p g colch~c~ne/g body wl.1

FIG. 2 . This figure shows the effects of varying doses of colchicine on the mitotic index (upper graph) and pyknotic index (lower graph) in the limb regeneration blastema, when the blastema was fixed 4 h after colchicine injection. Unin- jected controls had a mean mitotic index of 9 . 3 + 1.9 and a mean pyknotic index of 6 . 6 + 0 . 4 . Saline-injected controls had a mean mitotic index of 8 .8 + 3.1 and a mean pyknotic index of 7.1 + 3.2 . Bars indicate standard deviations.

with long colchicine treatment (24-72 h) ( p < 0.01). The curve seems to plateau at 36 h, after which there was no significant increase in mitotic index with further treatment time. The pyknotic indices for 36 and 48 h of treatment were significantly higher than all other pyk- notic indices ( p < 0.01). While a treatment time of 36 h yielded one of the highest pyknotic indices, it also yielded one of the highest mitotic indices.

Experiment 4 (36 h of exposure to different doses of colchicine)

Uninjected controls had a mean mitotic index of 12.9 + 0.72 and a mean pyknotic index of 8.79 + 0.57. Saline injected controls had a mean mitotic index of 13.65 + 1.35 and a mean pyknotic index of 8.50 +

Experiment 3 (various lengths of treatment with col- 1.59. Figure 4 shows the mitotic and pyknotic indices chicine at a constant dose) from the blastemas of colchicine-injected newts. The

Figure 3 shows the mitotic and pyknotic indices from mitotic indices obtained from blastemas of animals experiment 3. Mitotic indices obtained for short colchi- treated with low colchicine doses (0-2.5 kg colchicine) cine treatment (0-4 h), at a constant dose of 5 kg/g body were significantly higher than the mitotic indices ob- weight, were significantly lower than those obtained tained from animals treated with high colchicine doses

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L

O0 10 20 30 LO 50 60 70 80

TIME ( hours

FIG. 3. This graph shows the effect of differing colchicine treatment duration on the mitotic index ( 0 , upper line) and the pyknotic index ( 0 , lower line) in limb regeneration blastemas of newts injected with 5 kg of colchicine per gram body weight. Bars indicate standard deviations.

O L - - I 1 10 100 1000

D O S E ( p g c o l c h ~ c ~ n e / g b o d y w t

2 0 ' I .I 1 10 100 1000

DOSE OJg c o l c h ~ c l n e / g body w t )

FIG. 4. This figure shows the effects of varying doses of colchicine on the mitotic index (upper graph) and pyknotic index (lower graph) in the limb regeneration blastema, when the blastema was fixed 36 h after colchicine injection. Unin- jected controls had a mean mitotic index of 12.9 + 0.7 and a mean pyknotic index of 8 .8 + 0.6. Saline-injected controls had a mean mitotic index of 13.7 +- 1.4 and a mean pyknotic index of 8.5 + 1.6. Bars indicate standard deviations.

" 1 10 10 100 1000

DOSE (pg colch~c~ne/g body wt.)

FIG. 5 . This graph shows the relationship between colchi- cine dose and mitotic index in duodenal mucosa ( 0 ) and liver ( 0 ) . Bars indicate standard deviations.

(10- 1000 pg colchicine) ( p < 0.01). The mitotic index from blastemas of animals treated with 5 pg colchicine per gram body weight were significantly higher than mitotic indices from all other groups ( p < 0.01).

Pyknotic indices from adjacent sampling times were not significantly different from each other ( p < 0.0 1 ) in most cases. Animals treated with 1000 pg colchicine per gram body weight had a pyknotic index which was significantly higher than all other pyknotic indices ( p < 0.0 1). The colchicine dose which resulted in the highest mitotic index while still maintaining a low pyknotic index was 5 pg colchicine per gram body weight. Thus, this dose level was optimum at both 4 h and 36 h of treatment.

Experiment 5 (effect of different doses of colchicine on mitotic indices in duodenal mucosa and liver)

Figure 5 shows the dose-response relationship be- tween colchicine dose and mitotic index in the duodenal mucosa and liver. The dose-response curve here in- creases and reaches a maximum at 100-1000 pg per gram body weight. The higher dose levels are often lethal to newts. At 1000 pg per gram body weight 50% of the newts (418) died within 48 h of injection and hence could not be included in these results.

Discussion There is no apparent circadian rhythm in the mitotic

index of the limb regeneration blastema, at least at the cone stage used in this study. Figure 1 illustrates that over the 31-h period of the experiment there was no significant variation in the mitotic index. Thus, the subsequent experiments could be conducted with no fear that a circadian rhythm of mitosis would interfere with the- results.

Whether the treatment time with colchicine was 4 h or

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COOMBER ET AL. 557

36 h, the colchicine dose yielding the maximum mitotic index was 5 pg per gram body weight. Doses higher than this constantly resulted in lower mitotic indices. Thus, the aberrant response of the regeneration blastema to variable doses of colchicine seems to be consistent and repeatable. The pyknotic nuclei counts continue to increase as the dose increases, indicating that the colchicine is present and active in the cells.

When the dose of colchicine was fixed at 5 pg per gram body weight, and the interval between injection and fixation was varied, the mitotic index continued to increase to 36 h of exposure at which point it did not increase further (Fig. 3). This could be due to either a decrease in the circulatory level of colchicine to a noneffective level, or to a loss of the metaphase arrested cells due to cell lysis. Thus, subsequent experiments used an exposure time of 36 or 48 h to maximize the observed mitotic index.

When tissues such as duodenum and liver from intact newts were exposed to different doses of colchicine, a more typical dose-response relationship was observed in which the mitotic index continued to increase as the colchicine dose increased. The mitotic index only decreased at 1000 pg colchicine per gram body weight for the counts from the duodenal mucosa, and at this dose level the colchicine killed 50% of the newts within 48h. Gospodorwicz and Mescher (1981) have also reported similar toxicity of high doses of colchicine in newts. Consequently, the results from newts which survived the colchicine dose of 1000 pg per gram body weight may be misleading since these animals may also have been dying.

This study covers five separate experiments. Since they were performed at different times, direct compari- sons of the actual mitotic indices observed need to be treated with caution. Minor differences in season, temperature, light cycles, stage of regeneration, or the fact that newts were obtained at different times, could have contributed to the observed differences in the control level of the mitotic index.

Our dose-response curves for the effectiveness of colchicine in causing mitotic arrest in the regeneration blastema verify and explain the previous reports that low doses are often more effective than high doses (Dunlap and Donaldson 1978; Beach and Jacobson 1979). Thus, the assumption made by several researchers that a high dose of colchicine will result in more metaphase arrests than a low or zero dose is not justified (Donaldson and Wilson 1975; Mescher and Gospodarowicz 1979; Gos- podarowicz and Mescher 1981). It appears that each tissue may respond differently to colchicine, and the level at which a maximal response is obtained needs to be determined for each specific set of circumstances.

Why a low dose of colchicine should result in more metaphase arrests than a high dose is not clear. In the

case of the limb regeneration process, one possible explanation is that since mitosis in the limb regeneration blastema is nerve dependent (Tassava and McCullough 1978; Globus 1978) and since this neurotrophic activity probably requires axoplasmic transport (Singer 1974; Scadding and Liversage 1979) which is blocked by colchicine (Aguilar et al. 1973; Hanson and Edstrom 1978; Davis and Benloucif 1981), then colchicine may be blocking mitosis by blocking the neurotrophic effect of the nerve. Thus, in the blastema colchicine may be blocking the cell cycle prior to mitosis, for example in G2, by indirect action via the nerves. This hypothesis could also explain why the mitotic index of the blastema can only be increased slightly by colchicine (Figs. 2 and 4), since above a certain dose level (5 pg per gram body weight) some cells may be inhibited from entering mitosis.

Colchicine blocks mitosis at metaphase by inhibiting the formation of the microtubules of the mitotic spindle apparatus (Dustin 1978). The colchicine binds to the cytoplasmic tubulin to form a colchicine-tubulin com- plex which when added to the growing microtubule prevents further assembly of the microtubule (Margolis et al. 1980; Thyberg et al . 1980). The uptake of colchicine is time dependent, and sufficient colchicine- tubulin complex must be formed before mitotic spindle formation is disrupted (Friedkin et al. 1980). In our study, the number of mitotic figures collected increased until a maximum was reached after approximately 36 h of treatment with colchicine. Since time is required for colchicine to enter the cell (Serpinskaya et al. 198 1) and for the colchicine-tubulin complex to form (Friedkin et al. 1980), these results are not unexpected. Other workers have found time-related, colchicine-induced metaphase arrest (Stevens-Hopper 196 1 ; Thorud et al. 1980). The observation that the mitotic count does not continue to increase beyond 36 h (Fig. 3) could be due to a secondary action of colchicine which blocks cells from entering mitosis as described above, or to a loss or degeneration of colchicine-blocked cells in metaphase which had already formed, or to a decrease in the concentration of colchicine in the tissues to ineffective levels.

The detrimental effects of colchicine are not restrict- ed to the mitotic spindle. Colchicine may also disrupt any cellular processes which require microtubules, such as pseudopodial formation and cell locomotion (Ivanova et al. 1980), endocytosis and lysosomal activity (Fried- kin et al. 1980; Galivan 1981). and Golgi apparatus function (Thyberg et al . 1980). Cellular activities which do not involve microtubules, such as nucleoside trans- port (Galivan 198 1) and membrane-mediated transport of proteins and lipids (Friedkin et al. 1980) may also be affected. These disruptions in cellular functions may be cytotoxic and manifest themselves as pyknotic nuclei. In

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this study, the number of pyknotic nuclei increased with increasing doses of colchicine, for both 4 and 36 h of treatment (Figs. 2 and 4). With higher doses of colchicine, more colchicine enters the cell to complex with tubulin, and more colchicine-tubulin complex is available to disrupt the cellular functions which require microtubules (Serpinskaya et al. 198 1). In many cases, this disruption is sufficient to cause pyknosis and cell death. Galivan (1981) confirms that the cytotoxic activity of colchicine is dose dependent.

Acknowledgement This investigation was supported by a grant from the

Natural Sciences and Engineering Research Council of Canada to S . R. Scadding.

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