• 한국환경보건학회지
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• 제29권2호
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• pp.50-55
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• 2003

3,3'-디클로로벤지딘(DCB)는 실험동물에 발암물질로 밝혀졌고, 사람에게 암을 유발시킬 수 있는 발암물질로 의심되고있다. 많은 연구자들이 사업장에서 DCB에 폭로된 근로자들을 대상으로 뇨중에 배설된 대사물질, 헤모글로빈 부가체, 그리고 암 발생율 등에 대하여 연구를 하고 있다. 이러한 연구를 하기 위해서는 표준물질로 되어 있는 DCB의 대사물질이 꼭 필요하다. 따라서 본 연구의 목적은 DCB를 이용하여 이들의 대사물질을 합성하여 표준물질로 사용코자 합니다. DCB는 벤젠. 에테르, 에탄올, 메탄올 등에 부분적으로 용해되지만, 구연산이 1 % 이하로 함유된 70% 아세트산, 피리딘, 0.1N NaOH와 톨로엔이 1:2로 섞인 혼합물, 20 mM TRIZA염으로 포화된 페놀 등에는 완전히 용해되기 때문에 본 연구에서는 DCB를 피리딘에 녹여서 사용하였다. DCB와 대사물질인 mono-acetyl-DCB 및 diacetyl-DCB는 가스크로마노그래피(GC/MS)로 분석하였고, 검출기는 NPD와 SIM를 사용하였다. DCB의 기본피크는 252 m/z이었고, mono-acetyl-DCB의 기본피크는 252와 294 m/z로 구성되어 있었으며. diacetyl-DCB의 기본피크는 252, 294, 336 m/z로 구성되어 있었다. Discetyl-DCB는 피리딘에 용해된 DCB에 염소아세틸를 충분히 적정하여 합성하였다. 이렇게 얻은 diacetyl-DCB의 순도는 98.7%이었다. 침전물위에 있는 용해물질 속에는 DCB. mono-acetyl-DCB, diacetyl-DCB가 함유되어 있었는데, 아세트산을 아세틸화를 조절하는 물질로 사용하여 DCB를 모두 아세틸화시키었고, diacetyl-DCB로부터 mono-acetyl-DCB를 분리하여 추출하였다. 추출된 mono-acetyl-DCB는 아세톤으로 세척하여 98.8%의 순도를 얻었다.

• 한국환경보건학회지
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• 제28권4호
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• pp.6-11
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• 2002

3.3'-Dichlorobenzidine(DCB) has be shown carcinogenic in several animals, and the development of non-invasive biomonitoring method in workers exposed with it is a very important subject. DNA adduct is a good biomarker for biomonitoring about carcinogens exposure, and lymphocytes is a good non-invasive samples. So we studied to analyze metabolites in blood lymphocytes of female Sprague-Dawley rats exposed orally with DCB(20, 30, and 40 mg/kg wt.) for 3 weeks. For analysis of them, we isolated DNA adducts from blood lymphocytes by using the enzymes method in /sup 32/P-postlabeling, and measured them by using gas chromatography/mass spectrometry-selected ion monitoring(GC/MS-SIM). 4-aminobiphenyl and phenanthrene-d/sub 10/ were added as internal standard for blank sample. Standard metabolites of DCB were synthesized with using pyridine and acetic acid which were promoter and controller in acetylation of DCB. And they were used for calibration curve. Our results showed two kinds of metabolites in DNA adducts of blood lymphocytes. They were N-acetyl 3,3'-dichlorobenzidine(acDCB) and N,N'-diacetyl 3,3'-dichiorobenzidine(di-acDCB ). They were combined with DNA at the same time as an acetyl of it was removed. So we measured DCB and acDCB for two kinds of metabolites in DNA adducts of blood lymphocytes. Our results showed the levels of DCB were 1.46∼2.26 times more than that of acDCB. And also the levels of metabolites in 20, 30 and 40 mg/kg wt. were gradually increased with going days from 1st to 3rd week. They are 1.66, 1.38 and 0.90 times in total metabolites, 1.76, 1.49 and 1.02 times in DCB, and 1.51, 1.22 and 1.28 times in acDCB. In conclusion, the results of this study showed DCB exposed to rats formed DNA adduct in blood lymphocytes after acetylated to N-acetyl 3.3'-dichloro benzidine(acDCB) and N,N'-diacetyl 3,3'-dichlorobenzidine(di-acDCB), and they could be analyzed by us ing gas chromatography/mass spectrometry-selected ion monitoring(GC/MS-SIM).

• 한국환경보건학회지
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• 제28권1호
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• pp.21-29
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• 2002

To identify and evaluate the dichlorobenzidine(DCB)-DNA adducts in liver cell and bladder epithelial cells by $^{32}$ P-postlabeling and GC/MS-SIM, we orally exposed the dichlorobenzidine(20mg/kh body wt./day) to male Sprague-Dawley rats(l85$\pm$10g) for 14 days. Two kinds of DCB-DNA adduct(A1 and A2) were found at the same site of thin layer chromatogram of $^{32}$ P-postlabeling method in liver cells and bladder epithelial cells. In liver cells, relative adduct labeling(RAL) $\times$ 10$^{12}$ of DCB-DNA adduct A1 were 34.1$\pm$3.71 and 69.9$\pm$5.02, that of adduct A2 were 74.1$\pm$10.1 and 105.1$\pm$10.1 on 10 and 14 days after treatment, respectively. And in bladder epithelia cells, RAL $\times$ 10$^{12}$ of DCB-DNA adduct A1 were 5.92$\pm$1.60 and 15.9$\pm$1.31, that of adduct A2 were 9.81$\pm$2.81 and 22.8$\pm$1.79 on 10 and 14 days after treatment, respectively. DCB metabolites formed DNA adducts were monoacetyl-dichlorobenzidine(acDCB) and diacetyl-dichlorobenzidine(di-acDCB), which was identify by gas chromatography/mass spectrometry-scan ionization mode(GC/MS-SIM), after hydrolysis of DCB-DNA adducts isolated from live cells and bladder epithelial cells. The base peak of acDCB were 252 and 294 m/z, and that of di-acDCB were 252, 294 and 336 m/z. In conclusion, the exposed DCB formed two kinds of DCB-DNA adduct, the proximate materials of that were acDCB and di-acDCB in liver and bladder epithelial cells. And the above GC/MS-SIM method was found the DCB-DNA adducts could be monitoring by gas chromatography.