Pulmonary toxicity of polytetrafluoroethylene (PTFE) particles

다국어 초록 (Multilingual Abstract)

PTFE (Polytetrafluoroethylene), widely used in various industries such as cookware coatings, has been reported to have human toxicity due to thermal decomposition products at high temperatures, but the pulmonary toxicity of the PTFE particles at low temperatures has rarely been reported. However, recently, cases of fibrotic lung diseases such as pulmonary granuloma and pneumoconiosis have been reported among workers handling PTFE-containing coatings at low temperatures, indicating the need to evaluate the pulmonary toxicity of PTFE particles themselves. The objective of this study was to investigate the mechanisms of toxicity and fibrosis in lung cells exposed to PTFE particles and to assess the potential pulmonary toxicity in rats following a single intratracheal instillation of PTFE particles. In vitro results showed that both lung epithelial cells (A549) and lung fibroblasts (MRC-5) exhibited decreased cell viability and increased lactate dehydrogenase (LDH) levels. Lung epithelial cells underwent a mesenchymal-like transformation, with changes observed in epithelial-mesenchymal transition (EMT)-associated proteins, including E-cadherin. In lung fibroblasts, the expression levels of cytokines and proteins, including Transforming growth factor-beta1 (TGF-β1) and α-SMA, associated with fibroblast-myofibroblast transition (FMT), increased, along with those of the fibrosis-associated protein YKL-40. The in vivo results indicate that no toxicological or inflammatory changes were observed in the exposed group after 28 days. Additionally, there was no evidence of collagen deposition or expression of proteins associated with EMT or FMT in lung tissues. However, residual PTFE particles were observed in the lungs up to 28 days post-exposure. Histopathological examination revealed macrophage infiltration in the lung tissue of both the high and medium concentration groups, with granulomatous inflammation accompanied by foreign body reaction observed specifically in the high concentration group. These results confirmed that PTFE particles induce pulmonary cell toxicity and trigger EMT, FMT responses associated with fibrosis, and pulmonary granulomatous inflammatory reactions. Considering that these results are similar to lung tissue findings reported in workers handling PTFE-containing coating liquids, the risk of developing fibrotic lung diseases such as pneumoconiosis cannot be underestimated. Despite the limitations of this study regarding lung toxicity studies, such as exposure methods, it provides important experimental evidence and guides future research directions investigating lung toxicity induced by PTFE particles.

국문 초록 (Abstract)

조리용품 코팅제 등 다양한 산업에 널리 사용되고 있는PTFE (Polytetrafluoroethylene)는 고온에서 발생하는 열분해 산물로 인한 인체 유해성이 보고되고 있지만, 입자 자체의 폐 독성은 거의 보고되지 않았다. 그러나 최근 저온에서 PTFE 함유 코팅제를 취급하는 작업자들에서 폐 육아종, 진폐증 등 섬유화성 폐질환 발병이 보고되고 있어 PTFE 입자 자체의 폐 독성 평가가 필요하다. 본 연구에서는 PTFE 입자에 노출된 폐 세포에서 독성 및 섬유화 관련 기전을 확인하고, 단회 기관내주입(intra tracheal instillation)으로 PTFE 입자에 노출된 랫드의 폐 독성 가능성을 평가하고자 하였다. 세포실험 결과, 폐 상피세포(A549) 및 폐 섬유아세포(MRC5)에서 모두 세포생존율 감소 및 젖산탈수소효소(Lactate Dehydrogenase)가 증가하였고, 폐 상피세포의 경우 중간엽과 유사한 형태로 변환이 관찰되었으며, β-catenin 등 상피간엽이행(epithelial mesechymal transition, EMT) 단백질의 변화가 확인되었다. 폐 섬유아세포에서는 근섬유아세포전환(fibroblast myofibroblast transition, FMT) 관련 사이토카인과 단백질인 Transforming growth factor-beta1 (TGF-β1), a-SMA 및 섬유화 관련 단백질 YKL-40의 발현량 증가하였다. 동물실험 결과, 노출 28일 후 노출군에서 독성학적 변화 및 폐에서 염증성 변화는 없었고, 폐조직에서 콜라겐 침착 및 EMT, FMT 관련 단백질의 발현 또한 관찰되지 않았다. 그러나, 노출군에서PTFE 입자가 노출 28일 후까지 폐 잔류가 관찰되었고, 중농도 및 고농도군의 폐 조직에서 대식세포 침윤 및 고농도군에서 이물(foreign body)을 동반한 육아종성 염증 병변이 관찰되었다. 본 실험결과를 통해 PTFE 입자는 폐 세포 독성, 섬유화와 관련된 EMT, FMT 반응 및 폐 육아종성 염증반응을 유도할 수 있음이 확인되었다. 이러한 결과는 PTFE 함유 코팅액 취급 작업자에서 보고된 폐조직 소견과 유사함에 따라 진폐증과 같은 섬유화성 폐질환으로 진행될 위험성은 간과할 수 없다. 본 연구는 PTFE 입자에 대한 폐 독성 연구로써 노출방법 등의 제한점이 있지만, PTFE 입자의 폐 독성연구에 대한 미래 연구 방향을 제시하는 데 중요한 실험적 증거를 제공할 수 있을 것이다.

목차 (Table of Contents)

• CONTENTS
• LIST OF FIGURES-------------------------------------------------------- ⅳ
• LIST OF TABLES -------------------------------------------------------- ⅶ
• ABBREVIATIONS -------------------------------------------------------- ⅸ
• ABSTRACT IN ENGLISH --------------------------------------------- ⅹⅲ
• I. INTRODUCTION ------------------------------------------------------ 1
• II. MATERIALS AND METHODS ----------------------------------- 9
• 1. Materials ---------------------------------------------------------------------------- 9
• 2. Cell lines and cell culture --------------------------------------------------------- 10
• 3. Animals ----------------------------------------------------------------------------- 11
• 4. Characterization of PTFE --------------------------------------------------------- 14
• 5. Cell viability and cytotoxicity --------------------------------------------------- 15
• 6. Analysis of cytokine and fibronectin concentrations -------------------------- 16
• 7. Western blot analysis ------------------------------------------------------------- 17
• 8. Intratracheal Instillation ---------------------------------------------------------- 20
• 9. General symptoms, body weight, food intake, and lung weight ------------- 22
• 10. Hematological analysis ---------------------------------------------------------- 23
• 11. Clinical biochemistry analysis -------------------------------------------------- 24
• 12. Coagulation test ------------------------------------------------------------------ 24
• 13. Collection of bronchoalveolar lavage (BAL) fluid and cell counting ----- 25
• 14. Analysis of oxidative stress markers in BAL fluids ------------------------- 26
• 15 The dilution factor for BAL fluids analytes correction ----------------------- 29
• 16. High-resolution optical image analysis ---------------------------------------- 31
• 17. Histological analysis ------------------------------------------------------------- 33
• 18. Immunohistochemistry ---------------------------------------------------------- 34
• 19. Statistical analysis ---------------------------------------------------------------- 35
• Ⅲ. RESULTS
• 1. Characterization of PTFE particles ---------------------------------------------- 36
• 2. The effects of PTFE particles on lung fibroblast ------------------------------- 42
• 2.1. In vitro cytotoxicity of PTFE particles in MRC-5 cells ------------------ 42
• 2.2. TGF-β1 concentration of MRC-5 cells ------------------------------------- 44
• 2.3. FMT-related protein expression changes in MRC-5 cells --------------- 47
• 3. The effects of PTFE particles on lung epithelial cells
• 3.1. In vitro cytotoxicity of PTFE particles in A549 cells -------------------- 49
• 3.2. Mesenchymal-like morphological changes of A549 cells --------------- 51
• 3.3. Changes in EMT associated protein expression in A549 cells ---------- 53
• 4. Body weight and feed intake of rats exposed to PTFE particles ------------- 57
• 5. Hematological results of rats exposed to PTFE particles --------------------- 59
• 6. Biochemistry results of rats exposed to PTFE particles ----------------------- 63
• 7. Blood coagulation results of rats exposed to PTFE particles ----------------- 68
• 8. Relative lung weight of rats exposed to PTFE particles ---------------------- 71
• 9. High-resolution optical images of lung tissue----------------------------------- 74
• 10. Analysis of BAL cells in rats exposed to PTFE particles ------------------- 79
• 11. Inflammatory markers in BAL fluids of rats exposed to PTFE particles - 82
• 12. Concentration of cytokines in BAL fluids of rats exposed to PTFE particles
• --------------------------------------------------------------------------------------- 85
• 13. Oxidative stress markers in BAL fluids of rats exposed to PTFE particles -
• --------------------------------------------------------------------------------------- 88
• 14. Histopathological toxicity assessment of PTFE particles in lung tissue
• of rats exposed to PTFE particles ---------------------------------------------- 81
• 15. Collagen fiber assessment in lungs of rats exposed to PTFE particles ---- 96
• 16. Fibrosis associated protein expression in lungs of rats exposed to PTFE
• particles -------------------------------------------------------------------------- 99
• Ⅳ. DISCUSSION ------------------------------------------------------ 102
• V. CONCLUSION ----------------------------------------------------- 114
• Ⅵ. REFERENCES ---------------------------------------------------- 116
• ABSTRACT IN KOREAN ------------------------------------------- 129