Colchicine으로 인한 기형발생에 관한 실험적연구

Abstract

[한글]

[영문]In recent years along with premature delivery congenital deformity has become one of the major causes of death in infants. This has led naturally to increasing interest in the field of congenital deformity. Many reports on the incidence of congenital deformity in various countries around the world have been accumulated (Neel, 1958; Burrman et al., 1958; Shapiro et al., 1958).

Congenital deformity was considered in the past to occur by a mutation of hereditary factors, but at present much stress has been laid on environmental effects, including other factors than hereditary, during the gestation period. Penrose (1951), Neel (1958) and Fraser (1959) reported that, in the formation of congenital deformity hereditary as well as environmental factors, plus their combined effects, should be considered.

Experimental data on the possible factors inducing congenital deformity have been accumulated: radiation by Yamazaki et al. (1954); X-ray irradiation by Hicks(1953), goldstein and Murphy(1929), Warkany and Roth(1947), Wilson et al.(1953a, 1953b); drug factors by Fraser and Fainstas(1951), Helen(1956), Warkany et al.(1959); vitamins and nutritional disturbances by Cohlan(1957), Nelson et al. (1952, 1955, 1956, 1957a, 1957b), Warkany and Roth (1948) and Warkany and Schraffenberger (1944); environmental factors by Fraser et al.(1957).

Among drug factors, the following varieties have been considered: azaserin(O-diazoacetyl-serin) by Dagg and Karnofski (1955); trypan-blue by Waddington and Carter (1952), Ferm (1958); busulfan by Diamond et al. (1960); diaminopyrimidine by Thiersch (1954); thalidomide by Helen (1962), Williamson et al. (1963).

Recently colchicine has been said to be a deformity inducing substance. In 1963 Ferm first observed that golden hamsters (Cricetus Auratus) which were intravenously injected with clochicine(10mg/kg) on the 8th day of gestation showed 51% of

mortality and 22% of congenital deformity. When the colchicine dosage doubled (20mg/kg), 98% of animals died and congenital deformity was seen in all cases. After being treated in the same manner with 50 mg/kg colchicine, all of the animals died. The observed congenital deformity consists of microphthalmia, anophthalmia, exencephaly and omphalocele.

However, clochicine action may have been variable because of possible protective and detoxifying actions from maternal side of the animal. For this reason, to clarify the exact fundamental studies of colchicine effects on congenital deformity, golden hamsters are not thought to be adequate. In contrast chick

embryos have several advantages; they are rapid in growth, easy to handle, respond well to deformity-inducing substances, and are convenient for morphological and histological studies of the developmental differentiation of each organ.

Regarding the appearance of congenital deformity in chick embryos from colchicine, only a few reports are yet available, and no studies of colchicine induced congenital deformity were found in the literature. Hence, the attempt has been made to shed light on the exact pattern of congenital deformity from clochicine, using chick embryos and rabbits, and also to study if there exists any common mechanism (s) of congenital deformity between avaians and mammals.

A total of 160 chick embryos, their original eggs weighing about 55 gm, obtained from pure blooded hens (white leghorn) raised on the same feed, and also 32 rabbits (angola), weighing over 2kg each, were used. Colchicine was procured from E.Merck

Co. Eggs were incubated in a ventilated incubator (38℃, relative humidity=72%). For gross observation colchicine, in various graded doses from 0.5γ to 4γ, was injected into the york sac on the 4th and 8th days of incubation, respectively, and embryos were examined on the 20th day. For histological examination 1γ colchicine was introduced into the york sa, embryos were taken from the eggs 48 hours later, and after 10% formalin fixation they were serially sectioned for microscopy with hematoxylin and eosin staining.

Rabbits were injected via an ear vein with 1mg, 2mg and 4mg per kg of body weight of colchicine, respectively, on the 8th to 12th day of gestation. With spontaneous delivery, enw-born rabbits were examined macroscopically.

Microscopic observations were conducted in the same manner as described above with tissue slices of heart, liver and kidney of both mateeernal and malformed new-born rabbits.

For control observations the same amount of saline was used instead of colchicine in both chick embryos and rabbits.

The results may be summarized as follows:

1. Chick embryo experiments:

In the grop with 0.5γ colchicine on the 4th day, 60% of embryos died and 66.6% of deformity was observed. When colchicine doses were doubled, mortality increased(80%) with a decreased deformity occurrence (50%). The observed congenital deformity involved microphthalmai, exencephaly, retared growth and abnormal growth. With saline controls 46.7% of mortality and 37.5% of deformity were induced, the deformity being microphthalmia and retarded growth.

When the 8th day embryos were injected with 0.5γ colchicine, embryos showed 44.4% of mortality and 50% of congenital deformity. A doubled amount of colchicine gave 50% mortality and 55% deformity. At 2γ of colchicine, both mortality and deformity occurrence increased by 55.5% and 62.5%, respectively. By 4γ of

colchicine all of the embryos died. In the control experiments 30% of cases died in course of incubation and deformity was observed in 28.6% of cases. Microphthalmia, exencephaly, retarded growth, abnormal growth and syndactylia were observed in the experimental group whereas in the control group only microphthalmia was seen.

2. Rabbit experiments:

On the 8th day of gestation 1 mg of colchicine per kg of body weight induced 26% of congenital deformity, and doubled colchicine doses gave 37.5%, the deformity consisting of microphthalmia, anophthalmia, omphalocele and retarded growth. Such a severe deformity as anophthalmia appeared only when a large amount of colchicine (over 2mg per kg) was used.

On the 9th day of gestation 2mg per kg of colchicine was followed by 30% of congenital deformity (anophthalmia, omphalocele, retared growth). No fatal cases were observed with up to 2 mg/kg of colchicine. From 4mg of colchicine/kg abortion

or death of mother rabbits resulted.

When 2mg per kg of colchicine were used on the 10th day of gestation 27.2% of rabbits showed congenital deformity consisting of anophthalmia, omphalocele and retarded growth. Two mg colchicine on the 11th day of gestation resulted in retarded growth in 28.6% of cases with the same retarded growth in 25% of cases received 2mg/kg of colchicine on the 12th day of gestation.

No control experiments showed any kind of congenital deformity. Usually delivery was prolonged on an average by 2days in the colchicine rabbits.

3. Histological observations:

Chick embryos pre-treatedwith colchicine at an early stage showed in general a poorer eosin staining on slide specimens than the controls. Such embryo tissues as somites and liver were so undifferentiated as to be distinguishable rarely from the neighbouring mesenchymal tissues and showed a poor eosin staining. Cell arrangement in the central nervous system, particularly in the neural tube, and in such sensory organs as eyes and retinae, was completely disrupted, showing irregular cell clumps and cell distribution, i.e., fragmented neural tube well and retina, and in some instances a complete defect of lens.

As for rabbits no changes from the controls appeared.

On the basis of the above results the following conclusions may be made:

1. Colchicine injected into the york sac of chick embryos induced microphtalmia, exencephaly, abnormal growth, syndactylia and retarded growth.

2. In the 4th day chick embryos mortality increased and deformity decreased with a rising amount of colchicine. In the 8 th day chick embryos, with increasing doses of colchicine, both mortality and deformity occurrence tended to increase.

3. When colchicine was given (i.v.) to pregnant rabbits, anophthalmia, microphthalmia, omphalocele and retarded growth were observed in their offsprings.

4. The greatest possibility of congenital deformity from colchicine was observed in chick embryos on the 4th to 8th day of incubation and in rabbits on the 8th to 12th bay of gestation.

5. In chick embryos at early stages of incubation colchicine gave rise to poor staining properties of tissue slices for eosin and to abnormal cell arrangement in tissues and to fragmentation of tissues, leading to abnormality in tissue development and in organ formation. However, in rabbits no histological abnormalities occurred from colchicine.

6. Colchicine proved to act as a teratogenic agent for avians and mammals. Moreover, in view of the literature and of the obtained observations, there appeared to exist some discrepancies in the quantity of colchicine required to induce congenital deformity and in the variety of colchicine deformity from animal

to animal.