Genotoxicity Assessment in C. carpio Cells Using the Comet Assay and Acteoside Mediated Restoration of Senescence Markers

다국어 초록 (Multilingual Abstract)

Genotoxins cause significant damage to the genetic material of aquatic organisms, requiring rapid and accurate assessment. Fish derived cells sensitive to genotoxins have proven to be a useful tool for measuring genotoxicity, but the long treatment times required for measurement limit their application in situations requiring rapid testing. Previous studies have shown that fish cells can be kept unstarved for up to 6 h using media containing 1% FBS. In this study, the 1% FBS/6 h parameter was used for genotoxicity assessment. Therefore, genotoxicity assessment was performed after only 6 h of genotoxin treatment in medium containing 1% FBS. The new genotoxicity assessment method provided faster and more accurate genotoxicity data for climbazole and metolachlor than the existing assessment system using the 15% FBS/96h parameter. Furthermore, these advantages in the new platform enabled the determination of genotoxicity of various genotoxins, such as dibenz[a,h]anthracene and ethoprophos. In summary, we have developed a genotoxicity assessment that can generate genotoxicity data rapidly and accurately. This new platform will serve as a foundation for rapid genotoxicity assessment of many genotoxins.

Damaged mitochondria generate high levels of reactive oxygen species (ROS), which worsen cellular senescence. Therefore, lowering ROS or removing damaged mitochondria is a promising strategy to relieve senescence. Using a small screen of natural products, we identified acteoside as a compound that clearly reduces intracellular ROS in senescent human dermal fibroblasts (HDFs). At 4 μM for 12 days, acteoside selectively induced apoptosis in senescent HDFs while reducing several senescence associated markers, including CDKN1A, RB1, MMP1, F2RL1 (PAR2), CXCL8, and IL1B. Acteoside also decreased lipofuscin and mitochondrial mass, consistent with the removal of damaged organelles, and increased basal oxygen consumption and ATP linked respiration, showing improved mitochondrial function. RNA-seq analysis showed strong induction of FOS; importantly, FOS overexpression alone reproduced reduced ROS, lower mitochondrial mass, higher respiration, and decreased CDKN1A expression, linking acteoside’s effects to an AP1-centered transcriptional program. Together, these findings suggest that acteoside is a senolytic candidate that improves mitochondrial quality and suppresses inflammatory signaling in vitro, supporting its potential as a natural lead compound for aging-related conditions.

목차 (Table of Contents)

• General Abstract i
• Table of Contents iii
• List of Tables vi
• List of Figures vii
• List of Supplementary Information viii
• General Introduction 1
• Chapter 1. Rapid and Accurate Genotoxicity Assessment Using the neutral comet assay in Cyprinus carpio cells 3
• Abstract 4
• 1.1. Introduction 5
• 1.2. Materials and Methods 8
• 1.2.1 Cells cultured from C. carpio 8
• 1.2.2 Ecotoxicological assessment 8
• 1.2.3 Neutral comet assay 8
• 1.2.4 Morphology analysis of cells 9
• 1.2.5 Statistical analyses 9
• 1.3. Results 10
• 1.3.1 Diagrammatic representation of the genotoxic assessment of traditional and novel techniques 10
• 1.3.2 Comparing traditional and new techniques for climbazole's genotoxicity in C. carpio cells 13
• 1.3.3 Comparing traditional and new techniques for metolachlor's genotoxicity in C. carpio cells 16
• 1.3.4 Genotoxicity assessment of dibenz[a,h]anthracene using the new method 19
• 1.3.5 Genotoxicity assessment of ethoprophos using the new method 22
• 1.4. Discussion 24
• 1.5. Conclusion 28
• Chapter 2. Acteoside reverses cellular senescence through senolytic activity and reductions in ROS and SASP markers 29
• Abstract 30
• 2.1. Introduction 31
• 2.2. Materials and Methods 34
• 2.2.1 Cell culture 34
• 2.2.2 Preparation of compounds 34
• 2.2.3 Flow cytometric analysis of reactive oxygen species (ROS) 34
• 2.2.4 Relative cell number assay 35
• 2.2.5 Apoptosis assay 35
• 2.2.6 Measurement of oxygen consumption rate (OCR) and ATP production 36
• 2.2.7 Immunofluorescence 36
• 2.2.8 qPCR analysis of senescence and inflammatory markers 37
• 2.2.9 Transcriptome expression profiling 37
• 2.2.10 Lentiviral production and infection 38
• 2.2.11 Statistical Analysis 38
• 2.3. Results 39
• 2.3.1 Acteoside Lowers ROS and Selectively Eliminates Senescent Fibroblasts Without Cytotoxicity 39
• 2.3.2 Acteoside selectively induces apoptosis in senescent fibroblasts. 42
• 2.3.3 Acteoside restores mitochondrial function in senescent fibroblasts, increasing basal oxygen consumption and ATP production. 44
• 2.3.4 Acteoside mitigates the inflammatory–senescent phenotype by regulating aging- and inflammation-related genes 46
• 2.3.5 Acteoside reduces lysosomal accumulation and is associated with improved autophagic flux in senescent fibroblasts 48
• 2.3.6 Acteoside induces FOS expression in senescent fibroblasts 51
• 2.3.7 FOS overexpression is associated with reduced ROS, mitochondrial regulation, and restoration of autophagic flux 53
• 2.3.8 FOS improves mitochondrial function and modulates senescence–related gene expression 55
• 2.4. Discussion 57
• 2.5. Conclusion 60
• References 61
• Supplementary Information 70
• 국문초록 96