Elicitation of lens formation from the 'ventral iris' epithelium of the newt by a carcinogen, iv-methyl-iv'-nitro-iv-nitrosoguanidine

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1 /. Embryo!, e.xp. Morpli. Vol. 3, 1, pp , Printed in Great Britain Elicitation of lens formation from the 'ventral iris' epithelium of the newt by a carcinogen, iv-methyl-iv'-nitro-iv-nitrosoguanidine By GORO EGUCHP AN KENJI WATANABE 1 From the Laboratory for ifferentiation and Morphogenesis, Institute of Biophysics, University of Kyoto SUMMARY A potent carcinogenic compound, iv-methyl-./v'-nitro-./v-nitrosoguanidine (NG), was administered to the lentectomized eyes of the newt, Triturus pyrrhogaster. Supernumerary lenses were formed from the dorsal iris and even from the ventral iris, which never transforms into lens following simple surgical lentectomy or the grafting of isolated iris pieces into lentectomized eyes. After the administration of NG the pigmented epithelial cells of the ventral iris were induced to gain the competence for transformation into lens cells. The induced competence of the iris epithelial cells was stably maintained for at least 12 months. The secondary regeneration of the lens 45 days or 12 months after the primary lentectomy took place at the same positions of the ventral area of the iris where the primary lens regeneration had been induced by NG. The effects of NG were also confirmed by implanting isolated pieces of iris treated with NG solution into lentectomized eyes. Five implants out of 42 available experiments on the ventral half of the iris were found to be transformed into lens. INTROUCTION The iris epithelium of newts is capable of transforming into lens. When a lens is surgically removed from the eye, the lens regeneration occurs always only from the dorsal marginal portion of the iris epithelium. Among a large number of papers so far published (for reviews see Reyer, 1954, 1962; Scheib, 1965; Yamada, 1967) there are none indicating the possibility of the transformation of ventral iris epithelium into lens. When isolated iris pieces are implanted in lentectomized host eyes, the pieces derived from the ventral half of the iris are incapable of transformation into a lens, but ones from the dorsal half are capable of transformation (Sato, 193, 1951; Mikami, 1941). At the cellular level, Eguchi & Shingai (1971) have recently suggested that there are two different cell populations, one being capable and the other incapable of transforming into lens, and cells of the former type are densely localized in the dorsal part of the iris. The question may then be asked if there may be any means of eliciting lens 1 Authors' address: Laboratory for ifferentiation and Morphogenesis, Institute of Biophysics, Faculty of Science, University of Kyoto, Kyoto 66, Japan.

2 64 G. EGUCHI AN K. WATANABE formation from the ventral iris, which mostly consists of cells apparently incapable of transformation. This paper deals with the finding that the administration of iv-methyl-iv'- nitro-jv-nitrosoguanidine (NG) to lentectomized newt eyes successfully elicits supernumerary lenses from the ventral or the lateral portions of the iris epithelium in addition to the usual dorsal portion. This drug, NG, is well known as a mutagen. Its very potent carcinogenic effect has also been shown (Sugimura, Fujimura & Baba, 197). MATERIALS AN METHOS Adult Japanese newts, Triturus pyrrhogaster, were used as materials for the present experiment. A small particle of NG was inserted into the lentectomized eyes. In another series of experiments pieces of iris were treated in the NG solution and were then implanted into the lentectomized eyes. The procedure for lentectomy was the same as given in Eguchi & Shingai (1971). The lens removal was performed on both eyes. Administration ofng crystal. A single particle of NG (crystallized NG, K & K Laboratories, Inc., Plainview, N.Y.) from 15 to 2 fig in weight was placed into the posterior eye chamber either immediately after or 5 days after the lentectomy. The inserted NG particle remained at least for 1 h within the eye chamber. Implantation of iris pieces treated with NG. The iris ring was obtained by cutting the irido-corneal junction of the isolated eyes in Kesselyak's saline. Then the iris ring was divided into six sectors as shown in Fig. 1. The dorsal () sector and the vental three (VN, VT and V) sectors were incubated in the NG solution (1/^g/ml) at a constant temperature of 2 C for an appropriate time, washed with Kesselyak's saline, and then implanted into the lentectomized host eyes. Histological examinations. Animals to be examined histologically were sacrificed at various time intervals after an administration of NG or the implantation of the iris pieces. For the fixed eyes sections were cut at 1 /an through the lens regenerates with the usual paraffin techniques and were stained by Mallory's staining method. RESULTS 1. Supernumerary lens formation from the ventral half of the iris Three series of experiments were achieved from 197 to 1972 (Table 1). In two series, A and C, the NG crystal was administered immediately after lentectomy. For series B an administration of NG was performed 5 days after lentectomy. If NG is incorporated into the NA of iris cells, its effect will be most pronounced by administration in the period of most active NA synthesis, which is around 5 days after lentectomy (Eguchi & Shingai, 1971). This trial,

3 Lens formation by a carcinogen 65 Fig. 1. A schematic drawing showing sectors of iris to be separated into pieces as implants., orsal; N, nasal, T, temporal; and V, ventral. however, resulted in no significant differences among the experimental series (cf. Table 1). The cornea became opaque within 5 days after an administration of NG, but in many cases it gradually recovered its transparency. Though depigmentation of the mid-dorsal iris epithelium was delayed more or less in many cases, the external and histological observation indicated that lens regeneration took place in all cases in the same manner as in the case of simple lentectomy. In the animals which were included in accompanying tables as unrecorded cases, eyes were so severely affected with NG as to render any external examination impossible. However, even in these animals lens regeneration took place, as confirmed by histological preparations. Supernumerary lens regeneration from the lateral or the ventral iris epithelium was found in considerable numbers of experiments through all series (Table 1, see Fig. 2). In one case, B-19 (cf. Table 1), three lenses were formed altogether; two additional lenses regenerated, one from the dorso-nasal (N) and one from ventro-nasal (VN) portions of the iris epithelium, besides the one from the dorsal portion. epigmentation of the iris epithelium was often observed in the ventral or lateral marginal portions of the iris in addition to the usual dorsal portion. The depigmented parts other than the dorsal iris, however, did not always form a lens rudiment. In the case in which the supernumerary lens regeneration took place, a small lens vesicle was formed from a restricted area of the depigmented part of the lateral or the ventral iris, and, moreover, repigmentation of once-depigmented cells was often observed. The results as a whole indicated that the iris epithelial cells of all portions around the pupil become capable of transformation into lens cells as a result of NG administration. 2. The secondary removal of Jens regenerates {secondary lentectomy from eyes with the supernumerary lens regeneration) In this series of experiments the lens regenerates were removed again in order to test the stability of the induced competence of the ventro-lateral iris epithelial 5 EMB 3

4 66 G. EGUCHI AN K. WATANABE Table 1. Effect of N-Methyl-N'-nitro-N-nitrosoguanidine (NG) on the regenerative transformation of the newt iris epithelium into lens The position of the secondary lens regeneration is indicated by a diagram for each individual case. The lens regenerates are shown with open circles. Experimental numbers are indicated to the upper left of each diagram (A-3, B-6, and so on). Experimental series A (Jan. 197) B (Feb. 197) C (Mar. 1972) Time of NG administration after lentectomy No. of eyes operated No. of available cases examined Lens regeneration from dorsal iris alone Supernumerary lenses formed from both dorsal and other portions of iris epithelium Immediately days Immediately A-3 B-6 C-l N T- N T etails of each case with supernumerary lens regeneration induced byng B-I2* -N T- C-4 -N T - B-19* C-l 8" T N B-27* C-2V N N * Used for the experiment of secondary lentectomy., dorsal; N, nasal; T, temporal; V, ventral.

5 Lens formation by a carcinogen 67 cells for transformation into lens cells. For this purpose, five animals (B-12, B-19, B-27, C-18 and C-21, listed in Table 1) were used. Series B. By the time of the secondary lentectomy (12 months after the primary lentectomy), the regenerated lenses in B-19 and B-27 were fused into a single large lens. In B-12, one dorsal and a ventro-nasal (VN) small lens were well separated. The recovery of iris was complete in these cases. Around 1 days after the secondary lentectomy the depigmentation of the iris epithelium started in two portions of the marginal iris; the mid-dorsal portions and the same position where the additional lens had been regenerated about 12 months previously (Fig. 3). Series C. In each of two cases of this series, C-18 and C-21, two lens regenerates, from dorsal () and ventro-temporal (VT) portions, had not yet become detached from the iris epithelium at the time of the secondary lentectomy 45 days after the primary lentectomy. After lentectomy with particular care a small number of depigmented cells still remained on the pigmented epithelium after the operation. Within 4 days of the secondary lentectomy depigmentation began to extend in the pigmented epithelium around the portions to which the primary lens regenerates had been connected. The lens vesicles were developed from the depigmented parts, and finally two secondary lens regenerates with the same size as the primary ones respectively were differentiated from the same positions where the two primary lenses had been situated (Fig. 4 A, B, C). The results obviously indicate that the induced competence of the iris epithelial cells for transformation into lens cells through an administration of NG has been stably maintained for at least 12 months. 3. Implantation of iris pieces treated with NG solution Following a number of preliminary tests, treatment of the iris pieces with 1 //g/ml NG for 6 min was chosen as optimal for this series of experiments. Four sectors (, VN, YT and V, shown in Fig. 1) from the isolated iris rings were respectively tested (Table 2). After the treatment a considerable number of implants were found to have disintegrated by the time of fixation at 25 days after implantation. Seventeen of 19 available dorsal implants () differentiated into lens. As to the pieces of the ventral half of the iris, lens formation was found in five implants (Fig. 5). In the control implants which were immersed in saline before implantation, lens formation occurred only from the dorsal sector (Table 2). 5-2

6 68 G. EGUCHI AN K. WATANABE 4B

7 Lens formation by a carcinogen 69 Table 2. Effects of N-methyl-N'-nitro-N-nitrosoguanidine (NG) on isolated iris pieces implanted into lentectomized host eyes (Symbols,, VN, VT, and V, in the first column correspond to those in Fig. 1.) Sectors of iris _ - -A. Treatment orsal NG Saline Ventral VN NG Saline VT NG Saline V NG Saline Total no. of implants Transformed into lens Lens vesicle * Some implants were partially depigmented. Implants dis- integrated Implants remained unchanged* ISCUSSION In the present experiment NG, a potent mutagenic as well as carcinogenic compound, was administered at rather high dosage into lentectomized newt eyes. Although severe toxic effects were brought about in a considerable FIGURES 2-5 Fig. 2. A histological section of a typical case, B-6, in which a supernumerary lens was regenerated from the ventro-nasal (VN) portion of the iris in addition to the dorsal lens regeneration. This case was administered with a piece of NG crystal 5 days after lentectomy and was fixed 35 days thereafter. One large lens {L) was formed from mid-dorsal iris, the other {VNL) from the ventro-nasal. x 9. Fig. 3. Regeneration of two lenses after secondary lentectomy at 12 months after the primary lentectomy in B-12. A photograph inserted in the right upper corner shows the inside view of the iris ring with two lens regenerates. This case was fixed 2 days after removal of the primarily regenerated lenses. A section showing the regeneration of two lenses; one {L) developed from the dorsal () iris and the other (VNL), from the ventro-nasal (VN) iris, x 9. Fig. 4. Regeneration of two lenses after secondary lentectomy at 45 days after the primary lentectomy in C-18. (A) A sketch of the eye at the time of the secondary lentectomy. (B) A sketch of the fixed (16 days after the secondary lentectomy) eye cleared in methylbenzoate. Each of two lens regenerates {L, dorsal, and VTL, ventro-temporal) is formed at the same position of the iris epithelium from which the primary lens regeneration had taken place. (C) A histological section through well-differentiated lens regenerates, x 4. A photograph inserted in the right lower corner shows another section through the ventro-temporal lens regenerate {VTL) at the site where the regenerate is continuous with the iris epithelium, m, Mitotic figure (x 15). Fig. 5. A lens formed from the ventral iris sector (V) which was treated with NG solution before the implantation, x 7.

8 7 G. EGUCHI AN K. WATANABE number of the experiments, no decisive carcinogenetic processes were found in the ocular tissues except hyperplasia of the cornea. It is a well-established fact that Wolffian lens regeneration in newts occurs only from the dorsal sector of the iris epithelium. This has been quite extensively confirmed through a number of investigations in the century since the discovery of this phenomenon in European, American and Japanese newts. In parallel with the present experiment, a simple lentectomy was made in about 1 eyes of Triturus pyrrhogaster obtained together with the materials used for the experiment of NG administration. No spontaneous supernumerary lens regeneration ever occurred. The results of the experiment suggests strongly that NG acts on the ventral half of the iris to alter it to be competent to transform into the lens without exhibiting carcinogenic effects. The effect of NG can be interpreted as more or less specific, not merely as a trigger. The fact that the induced competence of the ventral half of the iris by NG is stably maintained was well demonstrated through a secondary lentectomy from the eye with a supernumerary lens. The effect of NG on the iris epithelial cells was also confirmed by means of implantation of isolated iris pieces treated in NG solution immediately before implantation. A considerable number of implants derived from ventral iris differentiated into a typical lens with normal polarity. This suggests that NG is incorporated by the iris epithelial cells and it induces them to transform into lens cells. A direct modification of NA, such as by alkylation, is thought to be the means by which NG acts as a mutagen or carcinogen. The possibilities of modification of NA by this compound have been demonstrated by in vitro and in vivo studies (McCalla, 1968; Craddock, 1968; Lawley, 1968; Sugimura et al. 1969; Nagao et al. 1969). In the present experiment also, it is quite possible that the NA of some iris epithelial cells incapable of transformation can be modified by the action of NG to become capable of transformation, because the induced competence of these cells for transformation into lens is stable for at least as long as 12 months. However, in the present demonstration the ventral iris cells transformed only into a specific pathway; that is, into lens cells, and never into any other cell types. Furthermore, the iris epithelium conserves this specific metaplastic differentiative capacity. It therefore seems likely that the iris epithelial cells are already potentially canalized strictly towards the cell types of the lens. This appears to be a far more restricted change than that observed by Sugimura, Fujimura & Bab a (197), who have demonstrated that various kinds of tissues were formed, together with tumours, in the stomachs of mice which were recipients of NG solution. It is difficult to interpret the specific transformation of cell types in the present experiment by means of a non-specific action of NG on NA. The molecular analysis of the mode of action of NG in the present system can be expected to provide useful information for accessing the mechanism of cellular differentiation at molecular level in the higher organisms in general.

9 Lens formation by a carcinogen 71 The authors wish to express their cordial gratitude to Professor T. S. Okada for his valuable advice in planning the present study and kind help in revising the manuscript, and to Professor T. Sugimura, National Institute for Cancer Research, Tokyo, who kindly gave us the NG used in the present study. They also thank Mr M. Okamoto for his kind help in preparation of the text figures, and Miss H. Takada who took trouble in preparing the manuscript. G. Eguchi expresses his gratitude to Mrs Ruth M. Clayton for her critical reading of the manuscript and valuable discussions during his stay in the Institute of Animal Genetics, University of Edinburgh, and also to Mr A. Gillies for his kind help in revising the illustrations. The present work was supported by the Grants for Basic Cancer Research nos and from the Japan Ministry of Education. REFERENCES CRAOCK, V. M. (1968). The reaction of iv-methyl-n'-nitro-n-nitrosoguanidine with deoxyribonucleic acid. Biochem. J. 16, EGUCHI, G. & SHJNGAI, R. (1971). Cellular analysis on localization of lens forming potency in the newt iris epithelium. ev. Growth, iff. 13, LAWLEY, P.. (1968). Methylation of NA by Af-Methyl-./V-nitroso-./V'-nitroguanidine. Nature, Lond. 218, MCCALLA,. R. (1968). Reaction of iv-methyl-w-nitro-jv-nitrosoguanidine and TV-MethyliV-nitroso-P-toluenesulfonamide with NA in vitro. Biochem. biophys. Acta 155, MIKAMI, Y. (1941). Experimental analysis of the Wolffian lens-regeneration in adult newt, Triturus pyrrhogaster. Jap. J. Zool. 9, NAGAO, M., HOSOI, H., FUJIMURA, S., KOGURE, K., YOKOSHIMA, T. & SUGIMURA, T. (1969). In vitro and in vivo interaction of N-Methyl-iV'-nitro-7V-nitrosoguanidine with cellular components (in Japanese). Symposia for Cellular Chemistry 2, REYER, R. W. (1954). Regeneration of the lens in the amphibian eye. Q. Rev. Biol. 29, REYER, R. W. (1962). Regeneration in the amphibian eye. In Regeneration (ed.. Rudnick), pp New York: Ronald Press. SATO, T. (193). Beitrage zur Analyse der Wolffschen Linsen-regeneration. I. Wilhelm Roux Arch. EntwMech. Org. All, SATO, T. (1951). Uber die linsenbildende Fahigkeit des Pigmentepithels bei iemyctylus pyrrhogaster. I. Pigmentepithel aus dorsalem Augenbereich. Embryologia 1, SCHEJB,. (1965). Recherches recentes sur la regeneration due cristallin chez les vertebres. Evolution du probleme entre 1931 et Ergebn. Anat. EntwGesch. 38, SuGrMURA, T., FUJIMURA, S., NAGAO, M., YOKOSHIMA, T. & HASEGAWA, M. (1969). Reaction of iv-methyl-iv'-nitro-iv-nitrosoguanidine with protein. Biochem. biophys. Acta 17, SUGIMURA, T., FUJIMURA, S. & BABA, T. (197). Tumor production in the glandular stomach and alimentary tract of the rat by Af-methyl-iV'-nitro-JV-nitrosoguanidine. Cancer Res. 3, YAMAA, T. (1967). Cellular and subcellular events in Wolffian lens regeneration. In Current Topics in evelopmental Biology, vol. 2 (ed. A. A. Moscona and A. Monroy), pp New York: Academic Press. (Received 23 November 1972)

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