Combined Exposure to Methylparaben and Butylparaben Elicits Distinct Epithelial Dysregulation in Prostate Organoids

다국어 초록 (Multilingual Abstract)

Parabens are widely used preservatives found in cosmetics, pharmaceuticals, and food products, and certain members of this chemical class have been reported to possess endocrine-disrupting properties. Human biomonitoring studies have detected multiple parabens, including methylparaben (MP) and butylparaben (BP), in biological samples; however, existing toxicological research has focused predominantly on single-compound exposure, leaving the biological effects of combined paraben exposure insufficiently characterized. The present study aimed to evaluate transcriptomic responses of prostate organoids to single MP exposure and combined MP/BP exposure across three concentration combinations, with the goal of determining whether mixture exposure induces responses that are not evident from single-compound exposure data. Mouse-derived prostate organoids were exposed to MP alone at three concentrations (1, 3.1, and 9.6 mg/L) or to MP and BP in combination (MP 9.6 + BP 0.18, MP 3.1 + BP 0.33, and MP 1 + BP 0.59 mg/L) for 30 days. Exposure concentrations were selected using toxicity-based reference values and further evaluated for long-term, non-cytotoxic biological responsiveness in prostate organoids. Transcriptomic changes were assessed by RNA sequencing, and key differentially expressed genes were validated by RT-qPCR, Western Blot analysis, and immunofluorescence. Single MP exposure did not induce significant cytotoxicity but produced a non- monotonic transcriptomic response, with the most pronounced changes observed at the middle concentration, 3.1 mg/L. Luminal identity markers, including Nkx3-1 and Ltf, were downregulated, whereas squamous- and keratinization-associated markers, including Krt13, Krt4, Slurp1, and Sprr2b, were upregulated, suggesting a shift in epithelial identity away from a luminal program. Under mixture exposure conditions, transcriptomic responses were dependent on concentration combination. The mid– mid combination (MP 3.1 mg/L + BP 0.33 mg/L; MMBM) produced no significant differentially expressed genes. The high–low combination (MP 9.6 mg/L+ BP 0.18mg/L;MHBL ) was associated with downregulation of luminal and barrier- associated markers, including Anpep, Adamts1, and Spink5, representing a response pattern distinct from that observed under low-concentration BP exposure alone. The low–high combination (MP 1 mg/L + BP 0.59 mg/L; MLBH) induced upregulation of Ube2c and Wif1 alongside downregulation of Palmd and Col4a3, with concurrent upregulation of Sprr2 family members, a pattern not observed under either single- compound exposure condition. These findings indicate that combined exposure to MP and BP induces concentration combination-dependent transcriptomic alterations in prostate organoids that are not evident from single-compound exposure data. This study highlights the importance of evaluating paraben mixture exposure and the limitations of single- chemical risk assessment frameworks in capturing mixture-specific epithelial responses.

목차 (Table of Contents)

• CHAPTER 1. Introduction 1
• 1. Parabens as Endocrine-Disrupting Chemicals 1
• 1.1 Overview and EDC Properties of Parabens 1
• 1.1.1 Definition and General Properties of Parabens 1
• 1.1.2 Human Exposure 3
• 1.1.3 Potential for Endocrine Disruption and Biological Effects 4
• 1.2 Methylparaben and Butylparaben as Representative Parabens 5
• 2. Mixture Toxicity of Parabens 6
• 2.1 Concepts of Mixture Toxicity and Limitations of Single-Chemical Assessment 7
• 2.2 Co-exposure Potential of Methylparaben and Butylparaben and Current Research Gaps 9
• 3. Organoids as an Experimental Model 10
• 3.1 Definition of Organoids 10
• 3.2 Rationale for Using Prostate Organoids in EDC Toxicity Assessment 12
• 3.2.1 The Prostate as a Hormone-Sensitive Target Organoid 12
• 3.2.2 Prostate Organoids as a Model for EDC Toxicity Assessment 12
• 4. Research Purpose 14
• CHAPTER 2. Materials and Methods 16
• 1. Reagents and antibodies 16
• 2. Prostate Organoid Establishment 18
• 3. Chemical Treatment 20
• 3.1 Selection of treatment concentrations 20
• 3.2 Exposure conditions 22
• 3.2.1 Single MP exposure 22
• 3.2.2 Combined MP and BP exposure 22
• 4. Cell Viability Assessment 23
• 5. Western Blot Analysis 24
• 6. RNA Extraction and Transcriptome Sequencing 25
• 7. RT-qPCR Analysis 26
• 8. Immunofluorescence Staining 28
• 9. Reactive Oxygen Species Measurement 29
• 10. Statistical Analysis 30
• CHAPTER 3. Results and Discussion 31
• 1. Long-term MP exposure preserves organoid viability and alters hormone receptor expression 31
• 2. MP induces a non-monotonic transcriptomic response centered on the mid concentration 33
• 3. MP attenuates luminal identity and promotes squamoid-like epithelial remodeling 37
• 4. MP elicits a mid-concentration-specific redox-associated response 40
• 5. MP/BP mixtures were designed to evaluate ratio-dependent responses under non-cytotoxic conditions 42
• 6. MP/BP mixtures induce ratio-dependent transcriptomic and pathway-level responses 44
• 7. MHBL mixture induces a mixture-specific transcriptomic response characterized by suppression of epithelial protective genes 48
• 8. MLBH mixture induces epithelial remodeling and proliferative activation 52
• CHAPTER 4. Conclusions 58
• References 60
• 국문초록 64