Procarbamate계 살충제인 Benfuracarb의 활성화 과정을 통한 독성 발현 기작에 관한 연구

다국어 초록 (Multilingual Abstract)

It has been proposed that benfuracarb must be converted to a toxic metabolite. The purpose of this study was to understand a role of cytochrome P_(450) and GST/GSH which was assummed to participate in the bioactivation of benfuracarb.
1. The bimolecular inhibition rate constants(k_(i) of benfuracarb and the carbofuran to acetylcholinesterase(AChE) were l.lx10^(3)M^(-1) · min^(-1) and 7.7x10^(5)M^(-1) · min^(-1) and those to butyrylcholinesterase (BuChE) were 3.2x10^(5)M^(-1) · min^(-1) and 3.4x10^(4)'M^(-1) · min^(-1) , respectively.
Benfuracarb inhibited AChE about seven hundred times slower than carbofuran did. However, benfuracarb was more effective than carbofuran to the inhibition of BuChE.
2. The potency of benfuracarb as an inhibitor of AChE was in creased in 10 fold when the benfuracarb and AChE were incubated with microsomes fortified with NADPH compared with microsome alone. Piperonyl butoxide(PB) addition to these coupled systems reduced the inhibition of target enzymes by blocking bioactivation process. However, this bioactivation effect couldn't be observed to BuChE. When the benfuracarb, glutathione-s-transferase(GST) and glutathione(GSH) were incubated together, the potency of benfura-carb as an inhibitor of AChE was increased more than 50 % compared with GST alone or without GST and GSH.
3. I_(50) of benfuracarb to the mouse brain AChE was 22.7mg/kg. When PB was pretreated, the inhibition of brain AChE was decreased significantly. This result showed that cytochrome Pwo. must be an important enzyme for the toxicity of benfuracarb in a biological system.
4. In the MFO system, the conversion of benfuracarb to carbofuran was increased more than 50% compared with that in microsome alone, while decreased when PB was treated. In the GST/GSH system, the amount of carbofuran was increased more than 80% compared with that in no GST/GSH system. In MFO and GST/GSH combination systems, the amount of carbofuran was mostly increased compared with the others.
5. In vitro inhibition of benfuracarb to the mouse liver cytosolic esterases was not observed in a native polyacrylamide gel electrophoresis.
6. When benfuracarb was treated intraperitoneally to mice at 0, 25, 50, 75 and 100mg/kg, mouse of liver cytosolic esterase and α,β-esterase were ininhibited largely in over 50mg/kg treatments and slightly even in 100mg/kg, respectively.
7. This study showed that the bioactivation processes of benfuracarb by the action of cytochrone P_(450) and GST/GSH system must be essential for showing its toxicity. And it has been confirmed that carbofuran could be a major toxic metabolite in the biological systems.

목차 (Table of Contents)

• Ⅰ. 서론 = 1
• Ⅱ. 연구사 = 7
• Ⅲ. 재료 및 방법 = 11
• 1. 재료 = 11
• (1) 공시약제 = 11
• (2) 공시동물 = 11
• (3) 사용시약 = 11
• (4) 사용기기 = 13
• 2. 실험방법 = 13
• (1) 공시약제의 조제 = 13
• 1) 공시약제의 정제 = 13
• 2) 순도확인 = 14
• 3) 표준용액조제 = 14
• (2) Metabolic Oxidation Systems = 14
• 2.1. In vitro에서 Cholinesterase에 대한 benfuracarb의 독성효과 = 14
• 1) Enzymes preparation = 14
• 1.1) AChE, BuChE 용액조제 = 14
• 1.2) Microsome과 Cytosolic fraction의 조제 = 15
• 1.3) 단백질 정량 = 15
• 2) ChE에 대한 이분자 저해 속도상수 (ki) 측정 = 15
• 3) ChE/MFO coupling system에서 benfuracarb의 microsomal oxidative activation 효과 = 18
• 3.1) Ellman's Assay = 18
• 3.2) Oxidase Assay = 19
• 4) GST/GSH System에서 benfuracarb의 activation 효과 = 20
• 2.2 In vivo에서 생쥐 brain AChE에 대한 독성발현 = 20
• 1) 약제처리 = 20
• 2) 뇌 AChE의 활성 검정 = 21
• (3) Benfuracarb의 독성 대사물 분석 = 21
• 1) 분석조건 및 검량선 작성 = 21
• 2) MFO system에 의한 독성대사물의 생성 = 24
• 3) GST/GSH system에 의한 독성대사물의 생성 = 25
• 4) MFO와 GST/GSH Combination system에 의한 독성대사물의 생성 = 26
• (4) Cytosolic esterase의 활성저해 = 28
• 4.1 Mouse의 간 cytosolic esterase의 활성저해 = 28
• 1) in vitro와 in vivo에서의 electrophoretic Assay = 28
• 4.2 α 및 β-esterase의 활성저해 = 29
• 1) in vitro와 in vivo에서의 Riskllash's Assay = 29
• Ⅳ. 결과 = 31
• 1. AChE 및 BuChE에 대한 이분자 저해 속도상수(ki)측정 = 31
• 2. ChE/MFO coupling system을 이용한 microsomal oxidative activation 효과 = 31
• (1) Incubation 시간에 따른 bioactivation 효과 = 31
• (2) Benfuracarb의 bioactivation 효과 = 34
• 1) Ellman's Assay = 34
• 2) Oxidase Assay = 35
• 3. GST/GSH에 의한 benfuracarb의 bioactivation 효과 = 35
• 4. Benfuracarb의 생쥐 brain AChE 독성 = 35
• 5. Benfuracarb의 독성대사물 생성 = 40
• (1) 검량선 결과 = 40
• (2) 회수율 및 검출한계 = 40
• (3) MFO system에서의 독성대사물 생성 = 40
• (4) GST/GSH system에서의 독성대사물 생성 = 47
• (5) MFO+GST/GSH combination system에서의 독성대사물 생성 = 51
• 6. In vivo 및 In vitro에서의 cytosolic esterase의 활성 저해효과 = 61
• (1) Electrophoretic Assay = 61
• 7. α 및 β-esterase 활성저해 = 64
• (1) Riskllah's Assay = 64
• Ⅴ. 고찰 = 66
• 적요 = 72
• 참고문헌 = 74
• Summary = 80