무기 나노물질의 세포 내 독성 메커니즘과 생체 내 거동 및 성상 연구

다국어 초록 (Multilingual Abstract)

Layered double hydroxide (LDH) nanoparticles, also known as anionic nanoclays, consist of positively charged metal hydorxide sheets and charge-compensating interlayer anions, which are in general hydrated with water molecules. This nanopaticles have been exploit as delivery carriers due to their high anion exchange capacity, pH-dependent solubility and controlled-release property. On the other hand, the high delivery efficiency of LDH nanoparticles with high reactivity and large surface area also raise concern about their toxicity potential in humans. Recently, LDH nanoparticles were determined to exhibit cytotoxicity in terms of oxidative stress and inflammation response in human lung A549 cells. However, the molecular mechanism related to LDH-caused toxicity has not been yet completely determined. Therefore, the aim of this study was to investigate a series of intracellular signaling pathway of LDH-induced oxidative stress and inflammation response.
First of all, LDH-induced oxidative stress was evaluated by analyzing reactive oxygen species (ROS) generation and antioxidant enzyme activities such as superoxide dismutase (SOD), glutathione reductase (GR), and catalase (CAT) as well as estimating expression level of inducible heme oxygenase-1 (HO-1). Subsequently, the expression levels of mitogen-activated protein (MAP) kinase including extracellular signal-regulated kinase-1 and -2 (ERK 1/2), the c-Jun-N-terminal kinases (JNKs), and p38 were investigated to determine toxicity mechanism-related to oxidative stress. Furthermore, Src family kinases (SFKs) that mediate MAPK activation in response to extracellular stimuli such as ROS were evaluated. Also nuclear factor-kappaB (NF-κB) and nuclear factor-E2-related factor 2 (Nrf2), oxidative stress-responding transcription factors, were investigated as downstream events. Induction of pro-inflammatory mediators and activation of MAPK or SFKs in LDH-treated cells, in the presence or absence of inhibitors of MAPK and SFKs, was measured by ELISA and western blot, respectively. Finally, antioxidant, ascorbic acid, was used to confirm LDH-induced oxidative stress and a series of inflammation response.
The results showed that activity of antioxidant enzymes increased in LDH-exposed cells, when LDH produced ROS. However, LDH did not inhibit cell proliferation, which may explain that toxicity of LDH in terms of oxidative stress was low. Pro-inflammatory mediators, such as IL-6 and IL-8, increased in cells treated with LDH, indicating induction of inflammation. Western blot analysis revealed that JNK and p38 were activated by SFKs as an upstream. Subsequently, NF-κB was activated by JNK and p38, leading to gene expression of antioxidant enzymes and cytokines. Expression of both JNK and p38 as well as cytokine induction were inhibited in the presence of each inhibitor and ascorbic acid in LDH-exposed cells. Taken together, LDH nanoparticles were determined to induce oxidative stress, which is mediated by JNK, p38, and c-Src pathway, finally resulting in inflammation response.
This research will provide practical and concrete information of toxicological effects of LDH nanoparticles, which will contribute to their wide application in nanofood fields.

Zinc oxide (ZnO) nanoparticles have been widely applied in products such as cosmetics, sunscreens, medicines, and food additives due to their catalytic, semi-conducting, magnetic, antimicrobial, and UV protection properties. Despite their diverse applications, toxicological effects of ZnO nanoparticles have not been clearly determined, particularly, with respects to particle size and surface charge. Furthermore, no information about the biological fate of ZnO nanoparticles at the systemic level is currently available. In this study, the effects of particle size (20 nm and 70 nm) and surface charge (negative and positive) on the pharmacokinetics, tissue distributions, and excretion of ZnO nanoparticles and their fate at the systemic level were examined following the administration of a single oral dose to rats.
Quantitative analysis of zinc in biological samples, such as blood, tissues, urine, and feces, was performed using inductively coupled plasma atomic emission spectroscopy (ICP-AES). The results showed that no significant effect of particle size or gender on pharmacokinetic profiles and biodistributions was found. However, ZnO (-) nanoparticles were markedly more absorbed by the systemic circulation than ZnO (+) nanoparticles. Furthermore, the kinetic behaviors of ZnO nanoparticles differed from those of zinc ions, which may indicate that ZnO nanoparticles seem to be absorbed into the systemic circulation in both particulate and zinc ion forms. ZnO nanoparticles were distributed to the main target organs, such as liver, kidney, and lungs. The major biological fate of ZnO nanoparticles in tissue appears to be in the ionic form, not the particulate form, and this was independent of exposure routes, particle size, gender, and surface charge. Elimination kinetics showed that small amount of ZnO nanoparticles were excreted via urine, while most of them were excreted via the fecal and biliary routes, which suggests that ZnO nanoparticles are eliminated in the same manner as zinc ions do. Furthermore, 20 nm particles were more rapidly eliminated than 70 nm particles, indicating that small particles could be more rapidly cleared from the body.
The overall results indicate that surface charge and particle size are critical modulators on biokinetic behaviors of ZnO nanoparticles. ZnO nanoparticles seems to be absorbed into the systemic circulation in both particulate and ionic forms, but mainly as a particulate form. Then, they are primarily localized in tissues as ionic forms and excreted as zinc ions do. These findings will be important to understand toxicokinetic behaviors and toxicity mechanism of ZnO nanoparticles and to predict their long-term chronic toxicity potential

목차 (Table of Contents)

• Ⅰ. 층상형 이중층 수산화물의 독성 메커니즘 연구
• 1. 서론 2
• 2. 재료 및 방법 5
• 2.1. LDH 나노물질의 합성 5
• 2.2. 세포주 및 배양 조건 5
• 2.3. 물리화학적 특성 분석 7
• 2.4. 세포 성장 저해 시험(WST-1 assay) 7
• 2.5. 활성산소종 정량 분석(ROS assay) 7
• 2.6. 항산화 효소 분석 8
• 2.6.1. Catalase 효소 분석(CAT assay) 8
• 2.6.2. Glutathione reductase 효소 분석(GR assay) 9
• 2.6.3. Superoxide dismutase 효소 분석(SOD assay) 9
• 2.7. Immunoblotting 9
• 2.7.1. 산화스트레스 및 염증 관련 단백질 발현 정량 10
• 2.7.2. MAPKs와 SFKs 억제제 처리 실험 11
• 2.7.3. 항산화제 처리 실험 11
• 2.8. Enzyme-linked immunosorbent assay(ELISA) 11
• 2.8.1. 염증 전구체 정량 12
• 2.8.2. MAPKs와 SFKs 억제제 처리 실험 12
• 2.8.3. 항산화제 처리 실험 12
• 2.9. 통계분석 12
• 3. 결과 및 고찰 14
• 3.1. LDH 나노물질의 물리화학적 특성 14
• 3.2. 세포 증식에 미치는 영향 17
• 3.3. 활성산소종 생성 19
• 3.4. 항산화 효소 활성 21
• 3.4.1. CAT 효소 활성 21
• 3.4.2. GR 효소 활성 23
• 3.4.3. SOD 효소 활성 25
• 3.4.4. HO-1의 활성 27
• 3.5. 산화스트레스 관련 신호전달 단백질 발현 양상 29
• 3.5.1. MAPKs 및 전사인자의 활성화 29
• 3.5.2. Src의 활성화 36
• 3.5.3. MAPKs와 SFKs 억제제 존재 시 p38, JNK, c-Src의 활성화 정도 38
• 3.5.4. 항산화제 존재 시 p38, JNK, c-Src의 활성화 정도 40
• 3.6. 염증 관련 단백질 발현 양상 42
• 3.6.1. 염증 전구체(IL-6, IL-8, IL-1β, TNF-α) 유도 발현 42
• 3.6.2. MAPKs와 SFKs 억제제 존재 시 염증 전구체 발현 정도 44
• 3.6.3. 항산화제 존재 시 염증 전구체 발현 정도 46
• 4. 결론 48
• Ⅱ. 산화 아연 나노물질의 생체 내 거동 및 성상 연구
• 1. 서론 53
• 2. 재료 및 방법 56
• 2.1. ZnO 나노물질의 표면 전하 개질 56
• 2.2. ZnO 나노물질의 물리화학적 특성 분석 56
• 2.3. 실험 동물 모델 및 사육 조건 56
• 2.4. 투여 방법 및 농도 57
• 2.5. 혈류 시스템으로의 흡수 및 거동 연구 57
• 2.6. 체내 조직분포 연구 58
• 2.7. 체외 배출거동 연구 58
• 2.8. ZnO 나노물질의 용해도 분석 58
• 2.9. ZnO 나노물질과 ZnCl₂의 약동학적 비교 연구 59
• 2.10. ICP-AES 분석 59
• 2.11. Transmission electron microscopy(TEM)을 통한 조직 내 ZnO 나노물질의 성상 분석 60
• 2.12. 통계분석 60
• 3. 결과 및 고찰 62
• 3.1. ZnO 나노물질의 물리화학적 특성 62
• 3.2. 혈류 시스템으로의 흡수 및 거동 64
• 3.3. 체내 조직분포 69
• 3.4. 체외 배출거동 76
• 3.5. ZnO 나노물질의 용해도 83
• 3.6. ZnO 나노물질과 ZnCl₂의 약동학적 거동 비교 85
• 3.7. 조직 내 ZnO 나노물질의 성상 89
• 4. 결론 91
• Ⅲ. 참고문헌
• Ⅳ. 영문요약