항암제가 임신마우스 및 마우스태자에 미치는 영향에 관한 세포유전학적 연구

Abstract

[한글]

포유류의 태자는 모체의 태반을 통하여 여러가지 영양물을 교환하며 그 이외 여러 이물

질도 태반 장벽을 통과할 수 있다. 태반장벽은 이물질이 들어오는 것을 어느정도 경감시

키나, 이물질의 독성을 완전히 막아주는 것은 아니며, 간장에서의 gluculonidation과 신

장으로부터의 배설이 안되거나 약하여서 일부의 물질은 모체보다 고농도가 될 수 있고,

태자내에서 대사되면서 독성이 증가할수도 있다(King과 Wild, 1979). 이러한 이물질들이

DNA에 손상을 주는 경우 여기에 노출된 태자는 유산내지 사산, 기형을 포함한 암 및 유전

적 이상등을 초래하게 된다(Kram등, 1979).

항암제인 Cyclophosphamide(Cytoxan, Endexan)는 악성종양치료에 사용되고 있는 radiom

imetic alkylating agent로서 in vitro에서는 비교적 불활성이고 간에서 대사되어야 비로

서 활성적인 alkylating agent가 되는 바(Perry와 Evans, 1975), Gibson과 Becker(1968)

는 cyclophosphamide를 임신마우스에 투여하여 태자에서 골격 및 연조직의 기형이 유발됨

을 보고하였고, 이외에도 in vivo상태의 여러 실험동물의 체세포에서 미소핵형성, 염색체

절단, 염색분체교환등의 염색체 이상이 나타난다는 보고가 있었다(Matter 및 Grauwiler,

1974; perry 및 Evans, 1975; Maier 및 Schmid, 1976; Stetka 및 Wolff, 1976; Basler, 1

979; Schuler 및 Latt, 1979).

Adriamycin은 glycosidic anthracline항생제로 핵내의 DNA에 작용하여 RNA합성을 방해

함으로서 항암작용을 나타내는 것으로 알려져 있으며(Kim및 Kim, 1972; Silverstrini등,

1970), Oguro등(1973)은 adriamycin을 임신 rat와 마우스에 투여하였을 때 기형이 유발되

지 않았음을 보고하였으나, Thompson등(1978)은 임신 rat에서는 발기형물질(teratogen)로

, 토끼에서는 기형이 나타나는 대신 높은 유산율을 보였고, 이러한 현상은 약물의 양과

투여시기에 따라 다르다 하였다.

이와같이 종양에 현저한 항암작용을 나타내는 항암제가 세포독성(Cytotoxicity)으로 작

용할 수 있기 때문에 약물의 효과에 대한 연구가 중요한 비중을 차지하고 있고, 이를 규

명하기 위한 세포유전학적 방법으로 염색체이상 조사법, 미소핵 조사법, 자매염색분체교

환(Sister Chromatid Exchange, 이하 SCE로 표기) 조사법등이 이용되어 왔다. 특히 생체

내 SCE조사는 5-bromodeoxyuridine(BUdR)표지의 어려움 때문에 극히 소수에 불과하며 (Al

len등, 1977; Basler, 1979), 더구나 태반을 통하여 발생하는 태자의 염색체 이상을 관찰

하고, 약물의 효과를 규명한 연구는 아직 부진한 상태에 있다.

본 연구는 부인과영역에서 악성종양치료에 사용되는 항암제인 Cyclophosphamide(CP)와

adriamycin(ADM)을 임신 마우스에 투여하였을 때 태반을 통하여 태자에 미치는 영향을 추

정하기 위해 미소핵 검사법과 자매염색분체교환 검사법을 이용하여 in vivo 상태에서의

소견을 관찰함으로서 임신중 항암제 사용시 발생하는 태자의 유전적 손상을 규명하고자

본 실험에 착수하였다.

실험재료 및 방법

23∼26주된 ICR계통의 순종마우스를 본 실험실내에서 임신시켜 이를 임신 13일, 16일,

19일의 3군으로 대별하였다.

미소핵검사는 cyclophosphamide를 5, 10, 20, 50, 100 mg/kg씩, adriamycin을 0.1, 1,

10, 50 mg/kg씩 각 군의 마우스에 복강 주사하였고, 태자의 혈액세포와 모체의 골수세포

를 채취하여 표본 제작한 후 May-Grunwald-Giemsa용액으로 염색하고 현미경 하에서 분석

하였다.

SCE검사는 5-fluorodeoxyuridine(FUdR) 0.4㎍/kg을 주사하고, BUdR 0.04㎍/kg을 1시간

간격으로 6회 수사한 후 cyclophosphamide는 1, 5, 10mg/kg을, adriamycin은 0.1, 1, 5mg

/kg을 복강주사하여 도살전 colcermid로 처리하고, 태자의 간세포와 모체의 골수세포를

채취한 후 표본제작 하였으며 Fluorescent-Plus-Giemsa(FPG) 방법(Perry와 Wolff, 1974)

을 변형하여 염색한 후 현미경하에서 분석하였다.

연구성적 및 결론

1. 대조군에서의 미소핵 발생율은 태자가 평균 0.3%, 모체가 평균 0.2%였고, SCE빈도는

태자가 2.2∼3.7/cell, 모체가 3.4∼4.7/cell이었다.

2. Multiple micronuclei는 cyclophosphamide의 최고 농도인 100mg/kg 투여시 태자의

혈액세포에서, adriamycin은 10mg/kg과 50mg/kg 투여시 모체의 골수세포에서 발생하였다.

3. Cyclophosphamide 투여군에서의 미소핵발생율과 SCE빈도는 태자가 모체보다 높았다.

4. Adriamycin 투여군에서의 미소핵발생율과 SCE빈도는 태자가 모체보다 낮았다.

5. 임신시기별로 항암제의 용량이 증가함에 따라 미소핵 발생율과 SCE빈도가 태자와 모

체 모두 증가하였다.

6. 약물투여시기별로 볼 때 임신 13일에서 태자의 미소핵 발생율 및 SCE빈도가 16, 19

일에서 보다 높았다.

[영문]

The mammalian fetus exchanges, electrolytes, amino acids, carbohydrates, fats,

and other metabolic products through the placenta with the maternal organism. In

addition to these essential nutrients, xenobiotic compounds can also cross the

"placental barrier." Although this barrier function of the placenta can be

recognized by a delayed entrance of xenobiotics into the fetus and by lower maximal

concentrations on the fetal than the maternal side, it by no means renders the

fetus resistant to toxic agents. Absence or low efficiency in the fetus of

elimination reactions such as glucuronidation and renal excretion can cause higher

concentration of certain xenobiotics and their metabolites in fetal organs as

compared to the maternal blood, and a more prolonged action (King & Wild, 1979).

The fetus is not only sensitive to toxic influence from the maternal organism

across the placenta, but it may itself increase the risk from potentially toxic

agents. Fetal exposure to agents which damage DNA can result in abortion, fetal

death, congenital anomalies, neoplasia, and heritable mutations in the germ line.

An antineoplastic agent is cytotoxic in vitro and has in vivo antitumor activity

against various slow-growing transplantable tumors.

The antitumor agent, cyclophosphamide is an indirect mutagen and a bifunctional

nitrogen mustard derivative, which is metabolized in mammalian liver to alkylating

agents (Brock et al., 1971; Perry & Evans, 1975).

Gibson and Becker(1968) reported that cp administered intraperitoneally to

timedated pregnant mice on gestational days 9 through 14 in a dosage of 20 mg/kg,

resulted in inc reased numbers of resorptionsand a variety of teratogenic effects.

Besides, cp has been reported to be induced chromosomal aberrations in laboratory

animals(Matter & Gratwiler, 1974; Stetka & Wolff, 1976).

Adriamycin is a glycosidic, anthracline antibiotics isolated from Streptomyces

peucetius which has significant antitumor activity against variety of malignancies

including sarcomas, lymphomas, leukemia, neuroblastoma and breast cancer(Tan et

al., 1973). One mechanism of action for this drug lies in it's ability to inhibit

nucleic acid synthesis through intercalation with DNA (Kim and Kim, 1972:

Silverstrini et al., 1972).

Thompson et al. (1978) reported that ADM was teratogenic in the rat and

nonteratogenic when given i.v. to rabbits at doses up to and including 0.6

mg/kg/day on days 6-18 of gestation, but a high incidence of abortion occured in

rabbits treated with adriamycin.

On the other hand, Oguro et al. (1973) reported adriamycin was considered to be

nonteratogenic in experiments using rats and mice.

In general, compounds possessing mutagenic carcinogen and teratogen activities

show teratogenesis early in development, whereas carcinogenic activity tends to

occur later in gestation. The majority of available therapeutic agents are both

effective anticancer drugs as well as potent toxic agents.

The purpose of the present investigation was to study the transplacental genetic

damage with the use of antitumor agents during pregnancy through the observation in

an in vivo state of micronuclei and sister chromatid exchange as occurs in a mouse

fetus when the pregnant mother is administered cyclophosphamide and adriamyoin,

used in the treatment of malignancies in the medical field.

Materials and methods

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

experiments. Animals were between 23 and 26 weeks of age the dating of fetal

development was based on timed matings of male and fewmale mice in 3 groups on the

13th, 16th, and 19th day of pregnancy.

Micronuclei test

Mice treated with cyclophosphamide at the rate of 5, 10, 20, 50 and 100 mg/kg of

body weight and adriamycin at the rate of 0.1, 1, 10 and 50 mg/kg of body weight

each one intraperitoneally injection; 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 2000 polychromatic erythromatic erythrocytes

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 rate of 0.4㎍/kg of boby weight was injected intraperitoneally once.

BUdR at the rate of 0.04 ㎍/kg of body weight was injected six times at a 1-hour

interval. Both cyclophosphamide and adriamycin at the rate of 1, 5 and 10 mg/kg of

body weight were injected separately i.p. After 24 hours mice were injected with

Colcemid and killedlater.

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

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

suspensions. Cell suspensions were centrifuged for 10 min, at 1000 rpm; hypotonic

treatment was carried out with 0.075M KCI, fixed(methanol/acetic acid, 3 : 1) and

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

to Hoechst 33258-Light-Giemsa methed (Prrry and 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 2.2∼3.7 in the progeny,

3.4∼4.7 in the mother.

2. After treatment with high dose of antitumor agents a concomitant increase in

micronuclei formation was observed. Multiple micronuclei were visible at the

highest dose of cyclosphosphamide(100 mg/kg, in fetal blood and at doses of

adriamycin (10, 50 mg/kg) in maternal bone marrow.

3. At each dose of cyclophosphamide, micronuclei incidence and SCEs frequency

were higher in the progeny than in the mother at equal dose.

4. At each dose of adriamycin, micronuclei ineidence and SCEs frequency were

lower in the progeny than in the mother at equal dose.

5. At various gestational ages, cyclophosphamide and adriamycin produced

significant and dose-related increases in micronuclei incidence and SCEs frequency.

6. The micronuclei anti SCEs induction by chemicais injected on day 13 of

development were higher than days 16 and 19 of gestation.