식품첨가물 이산화티타늄과 매트릭스와의 상호작용에 따른 세포독성, 장관흡수 및 유전독성 연구 = Interaction effects between food additive TiO₂ and matrices on cytotoxicity, intestinal transport and genotoxicity

다국어 초록 (Multilingual Abstract)

Titanium dioxide (TiO_2) is widely used in various products, including pharmaceuticals, cosmetics, and paints. In particular, it is commonly used as a food coloring agent in a variety of commercial foods, such as candies, chewing gums, bakery products, soups, and creamers. Recently, the European Union (EU) raised safety concerns regarding the use of (TiO_2) in foods, specifically its genotoxicity potential. As a result, its use has been banned as a food additive in the EU. Indeed, many in vitro and in vivo studies have reported conflicting results about the genotoxicity of (TiO_2), making it difficult to draw definitive conclusions. In addition, existing research has mainly focused on lung models, so safety evaluations related to oral intake remain limited. During their addition to foods and subsequent ingestion, food additives may interact with food components or biological matrices, potentially altering their physicochemical properties and biological responses, including genotoxicity. Therefore, it is important to evaluate the toxicological behavior of (TiO_2) in the context of these interactions.
In this study, the interactions between two different sized food-grade (TiO_2) particles and food components or biological matrices, such as fetal bovine serum (FBS), albumin, and glucose, were investigated. Physicochemical properties were characterized by measuring constituent particle size, hydrodynamic diameter using scanning electron microscopy (SEM) and dynamic light scattering (DLS). Cell proliferation, cell membrane damage, reactive oxygen species (ROS) generation, and antioxidant enzyme activity were evaluated in human intestinal Caco-2 cells. The solubility, intestinal transport and absorption of (TiO_2) were assessed to determine the effects of interactions on biological responses. The evaluation of genotoxicity was performed using the comet assay and quantification of 8-hydroxy-2-deoxyguanosine (8-OHdg).
The results demonstrated that interactions with FBS and albumin led to increased hydrodynamic diameters compared to pristine (TiO_2). Regardless of particle type and size, the solubility, intestinal transport, and absorption of (TiO_2) remained extremely low. The presence of FBS or albumin reduced oxidative stress, intestinal transport, and absorption to untreated control levels, regardless of (TiO_2) particle size. This tendency was consistent in the genotoxicity assessment results. Furthermore, the cytotoxicity of (TiO_2) in Caco-2 cells was mainly associated with the generation of ROS. Intracellular ROS generation induced by (TiO_2) was closely linked to oxidative DNA damage.
In conclusion, interactions between (TiO_2) and food or biological matrices influenced physicochemical properties of (TiO_2), as well as intestinal transport and toxicological responses. Furthermore, the interaction with matrices was found to mitigate the toxicological responses of (TiO_2).These findings provide important data for assessing the safety of (TiO_2) in the food industry.

목차 (Table of Contents)

• 1. 서론 1
• 2. 재료 및 방법 5
• 2.1. 시험 물질 선정 및 샘플 준비 방법 5
• 2.2. (TiO_2)의 물리화학적 특성 분석 5
• 2.3. Microwave digestion system을 이용한 (TiO_2) 전처리법 6
• 2.4. ICP-AES를 이용한 (TiO_2) 정량분석 7
• 2.5. 세포주 및 세포 배양 조건 7
• 2.6. 단기 세포 성장 저해 시험 (WST-1 assay) 7
• 2.7. 세포막 손상 확인 시험 (LDH release assay) 8
• 2.8. 세포 내 활성산소종 정량 시험 (ROS generation assay) 9
• 2.9. 세포 내 항산화 효소 활성 측정 9
• 2.10. In vitro 3 step 연속 소화 모델에서의 용해도 분석 10
• 2.11. In vitro 위장관 흡수 메커니즘 비교 13
• 2.12. 실험동물 및 사육 조건 15
• 2.13. Ex vivo 장 내 흡수율 분석 15
• 2.14. In vitro DNA 손상 분석 (Comet assay) 18
• 2.15. In vitro 8-OHdg 정량 분석 19
• 2.16. 통계 분석 19
• 3. 결과 및 고찰 21
• 3.1. 식품첨가물 (TiO_2)의 물리화학적 특성 21
• 3.1.1. 구성 입자 크기 및 분포도 21
• 3.1.2. 수분산 입자크기 및 표면전하 23
• 3.2. 식품첨가물 (TiO_2)의 정량분석법 확립 25
• 3.3. 식품첨가물 (TiO_2)처리에 의한 in vitro 세포 독성 평가 28
• 3.3.1. 세포 성장 저해 시험 28
• 3.3.2. 세포막 손상 확인 시험 30
• 3.3.3. 세포 내 활성산소종 (ROS) 분석 시험 32
• 3.4. 세포 내 항산화 효소 활성 평가 34
• 3.5. In vitro 3 step 연속 소화모델에서의 용해도 분석 36
• 3.6. In vitro 위장관 흡수 메커니즘 비교 36
• 3.7. Ex vivo 장 내 흡수율 분석 39
• 3.8. 식품첨가물 (TiO_2) 처리에 의한 in vitro 유전독성평가 42
• 3.8.1. Comet assay를 통한 DNA 손상 분석 42
• 3.8.2. 8-OHdg 정량 분석 47
• 4. 결론 49
• 5. 참고문헌 53
• 영문요약 63