6-aminonicotinamide투여로 인한 흰쥐의 염색체변화에 관한 실험적 연구

Abstract

[한글]

Chromosome Aberrations in 6-Aminonicotinamide induced Teratogenesis in Rats

Jin Man Wang, M.D.

Department of Medical Science The Graduate School, Yonsei University

(Directed by Professors: In Hee Chung and Tai Sun Shin)

With the advance of science, the frequency of diagnosis of congenital anomaly in

human beings has progressively increased and congenital anomaly constitutes a great

task in the field of medical science and also a major social problem.

Experimental work on induced congenital anomaly in various animals has been

performed with various agents including chemicals, physical factors, drugs, food

and environment (Fraser and Fainstat 1951, eraser et al. 1957; Jurand 1961: Warkany

and Kalter 1961: Ingalls et at.1964; Kury and Craig 1866: Saxon 1966).

It is now well known that several human congenital syndromes are accompanied by

chromosomal anomalies. A number of aborted human embryos have also been reported to

be characterized by abnormal karyotypes(Nance et al. 1965; Szulman, 1965: Kerr and

Nabil Rashed, 1966: Duthie and Townes 1967; Nance and Engel 1967). Now, chromosomal

anomalis can also be induced by several teratogenic agents in various species of

animals when exposed to these agents at certain stages of embryonic development

(Ingalls et al. 1963; Soukup et al. 1965: Joneja and Ungthavorn 1968: Philip and

Jensen 1970; Roux and Taillemite 1970).

The association of chromosome changes with certain syndromes of congenital

malformations has raised new questions as to the possible role of abnormal

chromosome complements in terategenesis. The main purpose of this investigation is

to determine whether 6-Aminonicotinamide which induces congenital anomalies in rat

embryos also induces chromosomal aberrations in the embryos and in the mothers.

Sprague-Dawley strain healthy female rats earth weighing about 700gm were used

for the experimental study. For intraperitoneal use, 6-Aminonicotinamide (Sigma)

was suspended in carboxylmethylcellulose with saline.

The animals were divided into the following four groups.

Group A: Control

Group B: Experimental

Ⅰ 4mg/kg 6-Aminonicotinamide treatment

Ⅱ. 6mg/kg 6-Aminonicotinamide treatment

Ⅲ. 8mg/kg 6-Aminonicotinamide treatment

The female rats were examined for the presence of vaginal plugs twice a day and

the day in which sperm were found in the vagina was counted as the first day of

pregnancy. On the 10-11th day, 6-Aminonicotinamide suspended in

carboxylmethylcellulese was injected intraperitoneally into pregnant females in

single doses of 4mg/kg, 6m7/kg or 8mg/kg body weight. The control group was

injected with saline in a dose of 6mg/kg body weight.

For embryologic purposes, the animals were killed on the IS-19 day of gestation.

Total implantation, resorption site, survival and gross appearance of the fetus

were studied. The control rats were studied with the same procedures.

Cytologic examinations were made by the fellowing procedures. To search for

cytogenetic effects in maternal tissues in control and treated groups, both femurs

and iliac bones of rats were used for the study of bone marrow by a culture method

of Roux and Taillemite(1970). To search for cytogenetic effects in fetal tissues in

control and treated groups, embryos were placed in a liver tissues of embryos were

processed a direct method of Ford and Woollam(1963), after preliminary in vivo

injection of colchicine (1-2 hours before killing).

From the data obtained, results were as follows:

Ⅰ Gross findings

1. The average of implantations in the 4mg/kg treated group was 6.5 fetuses, with

no dead fetuses. The body weight of treated mothers was reduced on an average of

20gm in spite of pregnancy. There was moderate developmental retardation, compared

with control fetuses.

2. The average of implantations in the 6mg/kg treated group was 4 fetuses. There

was moderate developmental retardation compared with control fetuses, and fecal

hemorrhages, amelia, hydrocephalus, shortening of the limb and lobster foot.

3. There was marked developmental retardation in the 8mg/kg treated group.

4. The development was normal in the control group of rat fetuses. There were no

gross abnormalities in control rat fetuses. The average of implantations and

survivals of fetuses in control rats was 9, with no dead fetuses.

Ⅱ. Chromosomal analysis

1. A total of 506 metaphase figures were counted from treated mothers. Among

these, normal metaphase figures were 376(74.3%) and abnormal metaphase figures were

130(25.7%). The majority of abnormal metaphase figurers was gap and break 82(16.2%)

and deletion 31 (6.1%).

2. A total of 411 metaphase figures were counted from treated fetuses. Among

these, normal metaphase figures were 302(73.7%) and abnormal metaphase figures were

109(36.9%). The majority of abnormal metaphase figures was gap and break 70(17%)

and deletion 9(4.6%).

3. A total of 211 metaphase figures were counted from the control mothers. Among

these, normal metaphase figures were 207 (98.1%) and abnormal metaphase figures

were 4(1.9%).

4. A total of 237 metaphase figures were counted from control fetuses. Among

these, normal metaphase figures were 228(96.2%) and abnormal metaphase figures were

9(3.8%).

5. The 6-Aminonicotinamide, which induced some kind of anomaly in the rat

embryos, also induced chromosomal aberrations in the embryos and mothers.

[영문]

With the advance of science, the frequency of diagnosis of congenital anomaly in human beings has progressively increased and congenital anomaly constitutes a great task in the field of medical science and also a major social problem.

Experimental work on induced congenital anomaly in various animals has been performed with various agents including chemicals, physical factors, drugs, food and environment (Fraser and Fainstat 1951, eraser et al. 1957; Jurand 1961: Warkany

and Kalter 1961: Ingalls et at.1964; Kury and Craig 1866: Saxon 1966).

It is now well known that several human congenital syndromes are accompanied by chromosomal anomalies. A number of aborted human embryos have also been reported to be characterized by abnormal karyotypes(Nance et al. 1965; Szulman, 1965: Kerr and

Nabil Rashed, 1966: Duthie and Townes 1967; Nance and Engel 1967). Now, chromosomal anomalis can also be induced by several teratogenic agents in various species of animals when exposed to these agents at certain stages of embryonic development (Ingalls et al. 1963; Soukup et al. 1965: Joneja and Ungthavorn 1968: Philip and Jensen 1970; Roux and Taillemite 1970).

The association of chromosome changes with certain syndromes of congenital malformations has raised new questions as to the possible role of abnormal chromosome complements in terategenesis. The main purpose of this investigation is to determine whether 6-Aminonicotinamide which induces congenital anomalies in rat embryos also induces chromosomal aberrations in the embryos and in the mothers.

Sprague-Dawley strain healthy female rats earth weighing about 700gm were used for the experimental study. For intraperitoneal use, 6-Aminonicotinamide (Sigma) was suspended in carboxylmethylcellulose with saline.

The animals were divided into the following four groups.

Group A: Control

Group B: Experimental

Ⅰ 4mg/kg 6-Aminonicotinamide treatment

Ⅱ. 6mg/kg 6-Aminonicotinamide treatment

Ⅲ. 8mg/kg 6-Aminonicotinamide treatment

The female rats were examined for the presence of vaginal plugs twice a day and the day in which sperm were found in the vagina was counted as the first day of pregnancy. On the 10-11th day, 6-Aminonicotinamide suspended in carboxylmethylcellulese was injected intraperitoneally into pregnant females in single doses of 4mg/kg, 6m7/kg or 8mg/kg body weight. The control group was

injected with saline in a dose of 6mg/kg body weight.

For embryologic purposes, the animals were killed on the IS-19 day of gestation. Total implantation, resorption site, survival and gross appearance of the fetus were studied. The control rats were studied with the same procedures.

Cytologic examinations were made by the fellowing procedures. To search for cytogenetic effects in maternal tissues in control and treated groups, both femurs and iliac bones of rats were used for the study of bone marrow by a culture method of Roux and Taillemite(1970). To search for cytogenetic effects in fetal tissues in control and treated groups, embryos were placed in a liver tissues of embryos were processed a direct method of Ford and Woollam(1963), after preliminary in vivo injection of colchicine (1-2 hours before killing).

From the data obtained, results were as follows:

Ⅰ Gross findings

1. The average of implantations in the 4mg/kg treated group was 6.5 fetuses, with no dead fetuses. The body weight of treated mothers was reduced on an average of 20gm in spite of pregnancy. There was moderate developmental retardation, compared with control fetuses.

2. The average of implantations in the 6mg/kg treated group was 4 fetuses. There was moderate developmental retardation compared with control fetuses, and fecal hemorrhages, amelia, hydrocephalus, shortening of the limb and lobster foot.

3. There was marked developmental retardation in the 8mg/kg treated group.

4. The development was normal in the control group of rat fetuses. There were no gross abnormalities in control rat fetuses. The average of implantations and survivals of fetuses in control rats was 9, with no dead fetuses.

Ⅱ. Chromosomal analysis

1. A total of 506 metaphase figures were counted from treated mothers. Among these, normal metaphase figures were 376(74.3%) and abnormal metaphase figures were 130(25.7%). The majority of abnormal metaphase figurers was gap and break 82(16.2%) and deletion 31 (6.1%).

2. A total of 411 metaphase figures were counted from treated fetuses. Among these, normal metaphase figures were 302(73.7%) and abnormal metaphase figures were 109(36.9%). The majority of abnormal metaphase figures was gap and break 70(17%) and deletion 9(4.6%).

3. A total of 211 metaphase figures were counted from the control mothers. Among these, normal metaphase figures were 207 (98.1%) and abnormal metaphase figures were 4(1.9%).

4. A total of 237 metaphase figures were counted from control fetuses. Among these, normal metaphase figures were 228(96.2%) and abnormal metaphase figures were 9(3.8%).

5. The 6-Aminonicotinamide, which induced some kind of anomaly in the rat embryos, also induced chromosomal aberrations in the embryos and mothers.