BPA–Aniline Co-exposure in Liver Organoids: Antioxidant Defense Impairment and Inflammatory Signaling Activation

다국어 초록 (Multilingual Abstract)

Environmental co-exposure to multiple chemicals is increasingly recognized as a critical determinant of toxicological outcomes, yet most studies continue to evaluate compounds in isolation. Bisphenol A (BPA), a ubiquitous endocrine-disrupting chemical, and aniline, an industrial aromatic amine with environmental and dietary exposure relevance, represent a realistic co-exposure scenario whose combined hepatotoxic effects remain poorly characterized. This study investigated mixture- specific toxicity in mouse-derived liver organoids chronically exposed to BPA (0.3 μM) combined with aniline at three concentrations (2.56, 6.4, and 16 μg/L) for 15 days. LDH assays indicated no overt cytotoxicity, while DCFDA revealed concentration-dependent intracellular ROS accumulation. Transcriptomic profiling identified a substantial number of co-exposure-specific differentially expressed genes, the majority of which were not observed under single-compound conditions, indicating emergent mixture-specific transcriptomic responses. ClueGO pathway analysis implicated lipid storage, hypoxia response, prostanoid metabolism, and ATP metabolic processes. qPCR and Western blot validated downregulation of Gbe1 and Hilpda, and reduced expression of Hmox1/HO-1 and NQO1, indicating an attenuated Nrf2-mediated antioxidant response despite elevated ROS. COX-2 exhibited a directional reversal under co-exposure, consistent with NF-κB pathway activation, and HNF4α downregulation suggested hepatocellular functional decline. Collectively, these findings demonstrate emergent, mixture-specific sublethal hepatotoxicity converging on three interconnected pathological axes, namely hepatic metabolic dysregulation, oxidative stress defense insufficiency, and inflammatory signaling activation, each corresponding to the core components of MASH pathology including steatosis, hepatocellular injury, and lobular inflammation. These co-exposure-specific alterations are consistent with an early MASH-like pathology signature that is not predictable from single-compound exposure data alone, underscoring the necessity of mixture-based approaches in environmental hepatotoxicity research.

목차 (Table of Contents)

• CHAPTER 1. Introduction 1
• 1.1 Organoids as Toxicological Models 1
• 1.1.1 Definition and Biological Features of Organoids 1
• 1.1.2 Liver Organoids in Hepatotoxicity Assessment 3
• 1.2 Environmental Chemicals and Hepatotoxicity 4
• 1.2.1 Bisphenol A 4
• 1.2.2 Aniline 5
• 1.3 Mixture Toxicity and Hepatic Stress Responses 6
• 1.3.1 Concepts of Mixture Toxicity 6
• 1.3.2 Oxidative Stress and Nrf2–Keap1 Signaling 7
• 1.3.3Chemical-induced Metabolic Liver Pathology 8
• 1.4 Research objectives 9
• CHAPTER 2. Materials and methods 10
• 2.1 Mouse liver organoid formation 10
• 2.2 Hepatic differentiation of liver organoids 13
• 2.3 Treatment of liver organoids with BPA and aniline 15
• 2.4 LDH assay 16
• 2.5Measurement of intracellular ROS 17
• 2.6 RNA extraction and cDNA synthesis 18
• 2.7 RT-qPCR 19
• 2.8 Next-generation sequencing (NGS) analysis 21
• 2.9 Western blot 22
• 2.10 Immunofluorescence staining 23
• 2.11 Statistical analysis 24
• CHAPTER 3. Results and Discussion 25
• 3.1 Establishment of liver organoid co-exposure model and confirmation of hepatic identity 25
• 3.2 BPA–aniline co-exposure induces intracellular ROS accumulation without overt cytotoxicity 27
• 3.3 Transcriptomic analysis reveals co-exposure-specific gene expression changes 30
• 3.4 Pathway enrichment analysis identifies functionally interconnected hepatic stress pathways 36
• 3.5 qPCR validation of energy storage-related gene expression 39
• 3.6 Antioxidant defense impairment under co-exposure conditions 41
• 3.7 Inflammatory signaling activation and hepatocyte functional impairment 43
• CHAPTER 4. Conclusion 47
• References 49
• 국문초록 54