니코틴이 임신 마우스 및 마우스 태자에 미치는 영향에 관한 세포유전학적 연구

Abstract

[한글]

최근 여성의 흡연인구 증가와 더불어 임산부의 흡연율이 증가되고 있어, 흡연이 모체, 태아 및 출산아에 미치는 영향에 대하여 많은 관심을 가지고 연구가 이루어지고 있다.

임신중 과다한 흡연은 조산율의 증가(Simpson, 1957), 자연유산율의 증가(Hudson Rucker, 1945), 저체중아 출산(Lowe, 1959: Becker와 King, 1966; Becker등, 1968)등과 유관하다고 하며, 이외에도 흡연산모에서 선친적 기형의 발생율이 증가하고 (Butler와 Alberman

, 1969), 태자가 질식하며 (Suzuki등, 1971), sudden infant death syndrom이 유발된다고 한다. 또한 태자의 뇌세포에 손상을 주는 세포독성을 나타낸다고 하는 등(Krouse, 1981), 흡연은 모체 및 태자에 유해하다는 임상적 연구가 많이 있다.

흡연시 발생하는 여러가지 화학물질에 대해서는 확실하게 알려져 있지 않으나, 지금까지 밝혀진 것으로는 carcinogen 및 cocarcinogen, irritant, 니코틴, 가스등이 있고 이런 유해물질들은 호흡기관을 통하여 흡수되는데, 이중 alkaloid인 니코틴은 흡연의 주요물

질로서, 체내에서 대사되어 cotinine, nicotine 1-oxidase 및 methylaminobutyric acid(MABA)로 변화하는데, 이중 cotinine과 nicotine 1-oxidase는 방광암을 유발하며 (Boyland, 1968), 임신 마우스에 투여한 니코틴이 마우스태자에 치사작용과 기형형성 작용을 한다(Nishimura와 Nakai, 1958)는 보고가 있는 반면 니코틴은 비발암성 유해물질로 알려지기도 하여 이에 대한 연구가 요망되고 있다. 약물의 효과를 세포유전학적으로 규명하기 위한 방법으로 자매염색분체교환(SCE) 및 미소핵 검사법을 이용한 유전학적 검증이 이루어지고 있는데, 흡연에 대한 세포유전학적 연구로는 사람의 백혈구 배양(Hollander등, 1978: Lambert등, 1978; Obe와 Herha, 1978; Crossen과 Morgan, 1980)과 실험동물의 조직배양(De Raat, 1979; Hopkin과 Evans; 1979)에서 다소 되어왔으나, 임신중 니코틴 투여시 in vivo 상태에서 모체와 태자의 DNA에 미치는 유전적 영향을 세포유전학적으로 밝힌 연구는 아직까지 찾아 볼 수 없다.

따라서 본 연구는 니코틴을 임신 마우스에 투여했을 때 모체와 태자에 미치는 유전적 영향을 SCE검사법과 미소핵 검사법을 이용하여 임신 시기별로 조사함으로서 임신중 니코틴이 미치는 유전적 영향의 가능성을 추정하고자 착수하였다.

실험재료 및 방법

체중 30g내외의 성숙한 ICR계통의 순종마우스를 본 실험실 내에서 임신시킨 후, 이를 임신0∼6일, 7∼13일, 14∼20일의 세군으로 대별하였다. 미소핵 검사는 니코틴을 1, 3, 5mg/kg의 용량별로 각 군의 마우스에 복강주사한 후 태자의 혈액세포와 모체의 골수세포를

채취하여 표본 제작하였고, May-Grunwald-Giemsa용액으로 염색하여 유발된 미소핵을 현미경하에 분석하였다.

SCE 검사는 5-fluorodeoxyuridine (FUdR) 0.4㎍/㎏을 주사하고, 5-bromodeoxyuridine (BU-dR) 0.04㎍/㎏을 1시간 간격으로 6회 주사한 후 니코틴 0.1, 0.5, 1.O㎎/㎏마우스에 복강 주사하였고, 도살전 colcemid로 처리하여 태자의 간세포와 모체의 골수세포를 채취

한 후 표본 제작하였으며, Hoechst 33258-Light-Giemsa 방법을 변형하여 염색한 후 현미경하에서 유발된 SCE를 분석하였다.

연구성적 및 결론

1. 대조군에서 미소핵 발생율은 태자가 0.3%, 모체가 0.2%였고, SCE빈도는 태자가 3.5±1.49/cell, 모체가 3.1±0.39/cell로 나타났다.

2. Multiple micronuclei는 태자에서는 니코틴 3㎎/㎏, 5㎎/㎏투여시, 모체에서는 5㎎/㎏투여시 발생하였다.

3. 니코틴 투여군에서 미소핵 발생율과 자매염색분체 교환빈도는 태자가 모체보다 높았다.

4. 임신시기에 따라 니코틴을 용량별로 투여했을 때 미소핵 발생율과 자매염색분체 교환빈도는 용량이 증가함에 따라 태자와 모체 모두 증가하였다.

5. 니코틴 투여시기를 볼 때 임신 7∼13일에 니코틴을 투여한 경우 임신 0∼6일, 임신 14∼20일에 투여한 경우보다 태자의 미소핵 발생율 및 자매염색분체 교환빈도가 높았다.

Cytogenetic Studies on Pregnant Mice and Their Fetuses Exposed to Nicotine

Induction of Micronuclei and Sister Chromatid Exchanges

Dong Jae Cho

Department of Medical Science The Graduate School, Yonsei University

(Directed by Professor Hyun Mo Kwak, M.D.)

Extensive investigation has revealed that cigarette smoking during pregnancy

increases the fetal heart rate, incidence of premature birth, and the incidence of

spontaneous abortions among smokers as compared with non-smokers.

The active constituent of tobacco smoke is the alkaloid nicotine, which has

widespread effects and has been known for some time to be a mitotic poison among

other things.

Nishimura and Nakai(1958) reported that nicotine has a lethal effect upon embryos

of mice and a powerful teratogenic effect on their skeletal system when it is

administered during pregnancy.

Metabolites of nicotine, nicotine 1-oxidase and cotinine can induce urinary

bladder cancer (Boyland, 1968).

Further cytogenetic study of cigarette smoking has been reported but there has

been little cytogenetic information on the effects of nicotine during pregnancy.

The purpose of present investigation is to study the possible transplacental

genetic damage to a fetus when the pregnant mouse is administered nicotine as

measured by a cytogenetic study of the vivo micronucli and sister chromatid

exchange.

Materials and Methods

Male and female mice(ICR strain) bred in our institute were used in all

experiments. The dating of fetal developments was based on timed matings of male

and female in 3 groups on the 0∼6th, 7∼13th, 14∼20th day of pregnancy.

Micronuclei test

Nicotine was dissolved in saline to give final concentration of 1, 3, 5 ㎎/㎏ of

body weight. each one intraperitoneally injected. The animals were killed by

cervical dislocation, maternal bone marrow and fetal blood smears were prepared.

Groups of five mice were used for each dose and 2,000 polychromatic erythromates

per mouse were. analyzed for micronuclei.

The technique of staining and of preparation were performed according to the

method described by schmid (1975).

SCE analysis

FUdR at the dose of 0.4㎍/㎏ of body weight was injected intraperitoneally once.

BUdR at the dose of 0.04㎍/㎏ of body weight was injected six times at a one hour

interval. Nicotine at the dose of 0.1, 0.5, 1.0 ㎎/㎏ were injected i.p. Before 2

hours mice were injected with colcemid and killed later.

Maternal bone marrow and fetal liver cell were isolated from pregnant mice.

Vigorous pipetting eagles minimal essential medium (MEM) resulted in single

suspension.

Cell suspensions were centrifuged for 10 min, at 1,000 rpm : hypotonic treatment

was carried out with 0.75M KCI, fixed with methanol : acetic acid=3 : 1 and slides

were prepared by air drying, differential staining was performed according to

Hoechst 33258-Light·Giemsa method (Perry & Wolff, 1974).

Result and Summary

1. Control values of micronuclei were found to be 0.3% in the progeny, 0.2% in

the mother and of the SCEs per cell was found to be 3.5±1.49 in the progny,

3.1±0.39/cell in the mother.

2. Multiple micronuclei w ere visible at doses of nicotine 3㎎/㎏, 5㎎/㎏ in

fetal blood and at dose of 5 ㎎/㎏ in maternal bone marrow.

3. At each dose of nicotine, micronuclei incidence and SCEs frequency were higher

in the progeny than in the mother at equal dose.

4. At various gestational ages, nicotine produced significantly dose-related

increases in micronuclei and SCEs frequency.

5. The micronuclei and SCEs induction by nicotine injected on days 7∼13 of

development were higher than those on days 0∼6 and 14∼20 of gestation.

[영문]

Extensive investigation has revealed that cigarette smoking during pregnancy increases the fetal heart rate, incidence of premature birth, and the incidence of spontaneous abortions among smokers as compared with non-smokers.

The active constituent of tobacco smoke is the alkaloid nicotine, which has widespread effects and has been known for some time to be a mitotic poison among other things.

Nishimura and Nakai(1958) reported that nicotine has a lethal effect upon embryos of mice and a powerful teratogenic effect on their skeletal system when it is administered during pregnancy.

Metabolites of nicotine, nicotine 1-oxidase and cotinine can induce urinary bladder cancer (Boyland, 1968).

Further cytogenetic study of cigarette smoking has been reported but there has been little cytogenetic information on the effects of nicotine during pregnancy.

The purpose of present investigation is to study the possible transplacental genetic damage to a fetus when the pregnant mouse is administered nicotine as measured by a cytogenetic study of the vivo micronucli and sister chromatid exchange.

Materials and Methods

Male and female mice(ICR strain) bred in our institute were used in all experiments. The dating of fetal developments was based on timed matings of male and female in 3 groups on the 0∼6th, 7∼13th, 14∼20th day of pregnancy.

Micronuclei test

Nicotine was dissolved in saline to give final concentration of 1, 3, 5 ㎎/㎏ of body weight. each one intraperitoneally injected. The animals were killed by cervical dislocation, maternal bone marrow and fetal blood smears were prepared.

Groups of five mice were used for each dose and 2,000 polychromatic erythromates per mouse were. analyzed for micronuclei.

The technique of staining and of preparation were performed according to the method described by schmid (1975).

SCE analysis

FUdR at the dose of 0.4㎍/㎏ of body weight was injected intraperitoneally once.

BUdR at the dose of 0.04㎍/㎏ of body weight was injected six times at a one hourinterval. Nicotine at the dose of 0.1, 0.5, 1.0 ㎎/㎏ were injected i.p. Before 2 hours mice were injected with colcemid and killed later.

Maternal bone marrow and fetal liver cell were isolated from pregnant mice.

Vigorous pipetting eagles minimal essential medium (MEM) resulted in single suspension.

Cell suspensions were centrifuged for 10 min, at 1,000 rpm : hypotonic treatment was carried out with 0.75M KCI, fixed with methanol : acetic acid=3 : 1 and slides were prepared by air drying, differential staining was performed according to

Hoechst 33258-Light·Giemsa method (Perry & Wolff, 1974).

Result and Summary

1. Control values of micronuclei were found to be 0.3% in the progeny, 0.2% in the mother and of the SCEs per cell was found to be 3.5±1.49 in the progny, 3.1±0.39/cell in the mother.

2. Multiple micronuclei w ere visible at doses of nicotine 3㎎/㎏, 5㎎/㎏ in fetal blood and at dose of 5 ㎎/㎏ in maternal bone marrow.

3. At each dose of nicotine, micronuclei incidence and SCEs frequency were higher in the progeny than in the mother at equal dose.

4. At various gestational ages, nicotine produced significantly dose-related increases in micronuclei and SCEs frequency.

5. The micronuclei and SCEs induction by nicotine injected on days 7∼13 of development were higher than those on days 0∼6 and 14∼20 of gestation.