Effects of a Carcinogen, N‐methyl‐N′‐nitro‐N‐nitrosoguanidine on Blastema Cells and Blastema Formation in Newt Limb Regeneration

Abstract

The effects of a potent ,carcinogen, N-methyl-N '-nitro-N-nitrosoguanidine (MNNG) on blastema formation and blastema cells were studied in the adult newt by means of scanning electron microscopy. By administration of MNNG to the blastema of the limbs the formation of the basement lamella, which lies between the epithelium and the mesenchyme, was effectively inhibited at least for a month. Basement lamella in treated limbs is formed 30 or 40 days after carcinogen administration. MNNG altered significantly cell surface morphology and cell motility of blastema cells isolated in vitro. Membrane's specializations such as the formation of filopodia were inhibited. Motility of the treated cells was much reduced compared to the control. The role of these alterations in the carcinogen-induced abnormal regeneration is discussed.The effects of carcinogens on limb regeneration have been recently defined (1,2). According to those studies, when a crystal of carcinogens is administered to the 7-day blastema a great retardation in the formation of the regeneration cone is cuased. The formation of the regeneration cone is completed 4-6 weeks after the administration of the carcinogens. Most of the cones receiving N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) develop to an abnormal limb with various malformations in the skeletal organization. During the retardation period mitosis in the stump is suppressed (1). It is very critical to know how the formation of the blastema is affected during the period of retardation. In an attempt to elucidate the possible cellular mechanisms involved in the induced abnormal regeneration, firstly we proceeded to the examination of cellular organization in the blastema by means of scanning electron microscopy, and secondly we examined the effects of MNNG on the isolated blastema cells in vitro searching for the action of this carcinogen to the cell behavior. MATERIALS AND METHODS Carcinogen administration to the blastemaSeven days after amputation through the mid-humerus, two crystals of MNNG (ca 10 pg) were inserted in the anterior and the posterior region of the blastemata, according to our previous study (1). In controls a needle was inserted in order to induce the same lesion as the insertion of carcinogens.Regenerating limbs of both experimentals and controls were dissected at 1-day, 2-day, 1-week, 2-week, 4-week, 5-week, and 6-week time intervals. CellsBlastemata were isolated 3 weeks after amputation of the limbs of the adult newts Triturus (Cynops) pyrrhogasrer, collected in Tagarasu, Fukui Prefecture, Japan. By the third week after amputation the blastema The day of administration was marked as 0 time.

The regeneration of newt limbs deformed in nature

“…As the table summarizes, of the 11 cases tested, the abnormal limbs consisted of some with mirror symmetrical reduplication of digit parts (case 4, 5, 7, 10, 11), some with supernumerary digits (cases 2,6,8), and some with partially deformed wrists (cases l, 3,9). Only the abnormal parts of these limbs were removed except in case 2, in which a part was left.…”

The mutant axolotl Short toes exhibits impaired limb regeneration and abnormal basement membrane formation.

“…Disruptions of these interactions by removal of the wound epithelium result in inhibition of regeneration (17)(18)(19)(20). In addition, in newt regenerates, the administration of potent chemical carcinogens retards formation of the basement membrane and eventually interferes with the normal regenerative process (21). Interestingly, the thick convoluted morphology ofthe basement membrane and excess extracellular material in the blastema resembles the one that can be formed in retinoic acid-treated nonregenerating double posterior zeugopodia and that seemed to have interfered with the normal epithelial-mesenchymal interactions as reported by Stocum and Crawford (22).…”

Formation of malformed pharynx and neoplasia in the planarian Bdellocephala brunnea following treatment with a carcinogen

“…Direct insertion of carcinogens into tissues can produce malformations; for instance, Breedis (1951Breedis ( , 1952 and Tsonis & Eguchi (1982, 1983 produced accessory limbs in newts by inserting a carcinogen into their forelimb. Benzo(a)pyrene is a well-known carcinogen occurring in crystalline form and thus easily manipulated in insertion into tissues.…”