흰쥐 적출간 관류법을 이용한 벤지딘계 염료 Direct Black 38의 대사

Abstract

[한글]

Direct Black 38은 벤지딘계 직접 염료의 일종으로 직물, 가죽, 면화, 셀룰로스성 천연섬유와 재생섬유, 종이, 울 , 비단, 플라스틱 등의 다양한 물질의 염색에 이용되는 염료이다. 1970년대 초반부터 벤지딘계 염료의 발암 가능성이 문제되어, 현재에는 다른 물질로 대체 사용하거나 가능한 최소량으로 폭로시킬 것을 권장하고 있다

본 연구에서는 Direct Black 38이 간에서 대사되는 양상을 보기 위하여 흰쥐의 간을 적출하여 생리적 버퍼 용액을 관류시키면서 염료를 각각 8.0μM, 30.5μM, 63.3μM 투여하여 대사되는 양상을 관찰하였다. 관류 시스템에는 산소와 온도를 일정하게 유지하였으며,

Direct Black 38을 투여한 후 각각 0, 10, 20, 30, 60, 90, 120분에 시료를 채취하였다.

채취한 시료는 sep-pak과 메탄올을 이용하여 전처리하였으며 파장 287nm의 자외선 검출기를 이용한 고성능액체크로마토그래피로 분석하였다. 관류액과 간에 남아 있는 미반응 염료는 미국산업안전보건연구소 방법 5013에 따라 벤지딘으로 환원시켜 분석하였다.

실험 결과 검출된 Direct Black 38의 대사물은 벤지딘, 아세틸벤지딘, 디아세틸벤지딘이었으며, 관류를 시작한지 10분 후부터 검출되었다. 아세틸벤지딘의 검출량이 가장 많았고, 벤지딘과 디아세틸벤지딘 순이었다. 벤지딘으로 대사된 양은 평균 0.1μM이었으며 이는 염료의 투여 농도와 시간에 따라 거의 일정한 양상을 보였다. 아세틸벤지딘의 양은 0.22μM이었으며 이것도 투여 농도와 시간에 따라 큰 차이를 보이지 않았다. 디아세틸벤지딘은 30.5μM을 투여한 실험에서 가장 많이 검출되었으며 시간에 따라 증가하는 양상을 보였지만 다른 두 개의 투여 농도 군에서는 시간에 따라 일정한 양상을 보였다.

위와 같은 결과로 Direct Black 38은 간에서 투여 농도에 관계없이 일정한 양만이 대사 된다고 가정할 수 있다. 그러나 이런 결과를 뒷받침 할만한 기존의 연구가 없으므로 앞으로 이를 증명하기 위한 연구가 계속되어야할 것으로 생각된다.

The metabolism of benzidine-based ye Direct Black 38 in the isolated perfused rat

liver

Jong Uk Won

Department of Public Health, The Graduate School, Yonsei University

(Directed by Professor Jaehoon Roh, M.D., Ph.D. )

Direct Black 38, a kind of benzidine-based azo dye, is widely used as a dye for

fabric, leather, cotton, cellulosic material, paper, wool, silk, and soon.

Benzidine-based ago dyes are proven mutagen and linked to bladder cancer. In 1978,

National Institute for Occupational Safety and Health recommended that three widely

used benzidine-based dyes(Direct Black 38, Direct Blue 6, and Direct Brown 95)

should be treated as carcinogens.

In this experiment, metabolism of the benzidine-based dye Direct Black38 was

examined by using an isolated liver perfusion system. To measure the metabolites of

Direct Black 38, 8.0μM, 30.5μM and 63.3μM of Direct Black 38 was added into the

recirculating perfusate of the isolated perfused rat liver. Samples were collected

at 0, 10, 20, 30, 60, 90, 120 minute. They were treated with sep-pak and methanol,

and the metabolites were detected and quantified with high performance liquid

chromatography(HPLC). Residual non-reactive dye in the perfusate and liver was

reduced to benzidine and then analyzed by HPLC.

Detected metabolites of Direct Black 38 were benzidine, N-acetylbenzidine, and

N,N'-diacetylbenzidine. The average concentration of benzidine was 0.1μM and this

concentration was maintained throughout the experimental Period. The average

concentration of N-acetylbenzidine was 0.22μM and took the same pattern of

benzidine. When 30.5μM of Direct Black 38 was added to the perfusate, only

N,N'-diacetylbenzidine increased slightly with time.

From the above results we suggest that only small amount of Direct Black 38 might

be metabolized to benzidine regardless of the Direct Black38. There are some

possible explanations. The liver was damaged during the preparation. And the

function of the perfused liver decreased rapidly because adsorbing of dye. The

benzidine, acetylbenzidine and diacetylbenzidine were metabolized to other

metabolites not detected in this experiment.

[영문]

Direct Black 38, a kind of benzidine-based azo dye, is widely used as a dye for fabric, leather, cotton, cellulosic material, paper, wool, silk, and soon.

Benzidine-based ago dyes are proven mutagen and linked to bladder cancer. In 1978, National Institute for Occupational Safety and Health recommended that three widely used benzidine-based dyes(Direct Black 38, Direct Blue 6, and Direct Brown 95)

should be treated as carcinogens.

In this experiment, metabolism of the benzidine-based dye Direct Black38 was examined by using an isolated liver perfusion system. To measure the metabolites of Direct Black 38, 8.0μM, 30.5μM and 63.3μM of Direct Black 38 was added into the recirculating perfusate of the isolated perfused rat liver. Samples were collected at 0, 10, 20, 30, 60, 90, 120 minute. They were treated with sep-pak and methanol, and the metabolites were detected and quantified with high performance liquid chromatography(HPLC). Residual non-reactive dye in the perfusate and liver was reduced to benzidine and then analyzed by HPLC.

Detected metabolites of Direct Black 38 were benzidine, N-acetylbenzidine, and N,N'-diacetylbenzidine. The average concentration of benzidine was 0.1μM and this concentration was maintained throughout the experimental Period. The average

concentration of N-acetylbenzidine was 0.22μM and took the same pattern of benzidine. When 30.5μM of Direct Black 38 was added to the perfusate, only N,N'-diacetylbenzidine increased slightly with time.

From the above results we suggest that only small amount of Direct Black 38 might be metabolized to benzidine regardless of the Direct Black38. There are some possible explanations. The liver was damaged during the preparation. And the function of the perfused liver decreased rapidly because adsorbing of dye. The

benzidine, acetylbenzidine and diacetylbenzidine were metabolized to other metabolites not detected in this experiment.