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표제지 2

목차 7

1. 서론 12

2. 재료 및 방법 16

2.1. 물질 선정 및 샘플 준비 방법 16

2.2. TiO₂의 물리화학적 특성 분석 16

2.3. Microwave digestion system을 이용한 TiO₂ 전처리법 17

2.4. ICP-AES를 이용한 TiO₂ 정량분석 18

2.5. 세포주 및 세포 배양 조건 18

2.6. 단기 세포 성장 저해 시험 (WST-1 assay) 18

2.7. 세포막 손상 확인 시험 (LDH release assay) 19

2.8. 세포 내 활성 산소종 정량 시험 (ROS generation assay) 20

2.9. 세포 내 항산화 효소 활성 측정 20

2.10. In vitro 3 step 연속 소화 모델에서의 용해도 분석 21

2.11. In vitro 위장관 흡수 메커니즘 비교 24

2.12. 실험동물 및 사육 조건 26

2.13. Ex vivo 장 내 흡수율 분석 26

2.14. In vitro DNA 손상 분석(Comet assay) 29

2.15. In vitro 8-OHdg 정량 분석 30

2.16. 통계 분석 30

3. 결과 및 고찰 32

3.1. 식품첨가물 TiO₂의 물리화학적 특성 분석 32

3.1.1. 구성 입자 크기 및 분포도 32

3.1.2. 수분산 입자크기 및 표면전하 34

3.2. 식품첨가물 TiO₂의 정량 분석법 확립 36

3.3. 식품첨가물 TiO₂ 처리에 의한 in vitro 세포 독성 평가 39

3.3.1. 세포 성장 저해 시험 39

3.3.2. 세포막 손상 확인 시험 41

3.3.3. 세포 내 활성산소종 (ROS) 분석 시험 43

3.4. 세포 내 항산화 효소 활성평가 45

3.5. In vitro 3 step 연속 소화모델에서의 용해도 분석 47

3.6. In vitro 위장관 흡수 메커니즘 비교 47

3.7. Ex vivo 장 내 흡수율 분석 50

3.8. 식품첨가물 TiO₂ 처리에 의한 in vitro 유전독성평가 53

3.8.1. Comet assay를 통한 DNA 손상 분석 53

3.8.2. 8-OHdg 정량 분석 58

4. 결론 60

5. 참고문헌 64

ABSTRACT 74

표목차 11

Table 1. Composition of in vitro 3 step simulated digestion fluids 22

Table 2. Particle size distribution and hydrodynamic diameters of TiO₂ dispersed in different matrices 35

Table 3. Quantitative analysis of TiO₂ (T3) by ICP-AES analysis 37

Table 4. Quantitative analysis of TiO₂ (T4) by ICP-AES analysis 38

그림목차 9

Figure 1. Scheme for in vitro 3 step digestion model 23

Figure 2. Scheme for in vitro 2D/3D intestinal barrier models 25

Figure 3. Scheme for ex vivo everted small intestinal sac model 28

Figure 4. SEM image and size distribution of food additive TiO₂ (T3,... 33

Figure 5. Calibration curve for determination of TiO₂ (T3) 37

Figure 6. Calibration curve for determination of TiO₂ (T4) 38

Figure 7. Effect of interactions between TiO₂ particles and food or... 40

Figure 8. Effect of interactions between TiO₂ particles and food or... 42

Figure 9. Effect of interactions between TiO₂ particles and food or... 44

Figure 10. Effect of interactions between TiO₂ particles and food or... 46

Figure 11. Intestinal transports of TiO₂ particles using (A) in vitro... 49

Figure 12. Ex vivo intestinal absorption of TiO₂ particles (A, T3; B,... 52

Figure 13. Fluorescence images of Caco-2 cells after comet assay... 55

Figure 14. Fluorescence images of Caco-2 cells after comet assay... 56

Figure 15. Effect of interactions between TiO₂ particles (A, T3; B,... 57

Figure 16. Effect of interactions between TiO₂ particles and food or... 59

초록보기

 Titanium dioxide (TiO₂) 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 TiO₂ 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 TiO₂, 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 TiO₂ in the context of these interactions.

In this study, the interactions between two different sized food-grade TiO₂ 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 TiO₂ 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 TiO₂. Regardless of particle type and size, the solubility, intestinal transport, and absorption of TiO₂ remained extremely low. The presence of FBS or albumin reduced oxidative stress, intestinal transport, and absorption to untreated control levels, regardless of TiO₂ particle size. This tendency was consistent in the genotoxicity assessment results. Furthermore, the cytotoxicity of TiO₂ in Caco-2 cells was mainly associated with the generation of ROS. Intracellular ROS generation induced by TiO₂ was closely linked to oxidative DNA damage.

In conclusion, interactions between TiO₂ and food or biological matrices influenced physicochemical properties of TiO₂, as well as intestinal transport and toxicological responses. Furthermore, the interaction with matrices was found to mitigate the toxicological responses of TiO₂. These findings provide important data for assessing the safety of TiO₂ in the food industry.

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