Effect of polyhexamethyleneguanidine on hepatic redox homeostasis through ER stress-mediated glutathione depletion = PHMG-P에 의한 소포체 스트레스 매개 글루타치온 고갈이 간의 산화/환원 항상성에 미치는 영향

다국어 초록 (Multilingual Abstract)

Polyhexamethyleneguanidine (PHMG) is a widely used polymeric antimicrobial agent to induce significant pulmonary toxicity. Several studies have reported that the liver also can be a target organ of PHMG toxicity, but the exact effect of this compound on liver cells is not well understood. To determine the effect of PHMG on the liver, 1.5 mg/kg PHMG-phosphate (PHMG-P) was intratracheally instilled to rats. Although lung injury was significantly induced by PHMG-P treatment, serum activity of alanine aminotransferase (ALT) and liver histological analysis showed no significant changes compared to control group. Interestingly, PHMG-P treatment induced oxidative stress, as evidenced by increased malondialdehyde (MDA) and protein oxidation, accompanied with endoplasmic reticulum (ER) stress in the liver. These results implicate that intratracheal instillation of PHMG-P affects liver homeostasis through both ER- and oxidative stress without liver damage.
To identify the effect of PHMG-P on ER stress in the liver, HepG2 liver cells were exposed to PHMG-P for 72 h. The increases observed in C/EBP homologous protein (CHOP), p-IRE, and p-JNK levels in PHMG-P-treated cells indicated the induction of ER stress. To verify the role of ER stress in PHMG-P-induced cytotoxicity, HepG2 cells were pretreated with the chemical chaperone, tauroursodeoxycholic acid (TUDCA) and then co-treated with TUDCA and PHMG-P for 24 h. TUDCA inhibited PHMG-P-induced ER stress and cytotoxicity in a dose-dependent manner. These results suggest that ER stress induced by PHMG-P treatment might be an important mechanism for mediating hepatic dysfunction.
To determine whether ER stress without tissue injury affects redox homeostasis in the liver, mice were treated with tunicamycin (TM) (2 mg/kg body weight) for 48 h to induce ER stress in the liver and examined the metabolic pathway that synthesizes the endogenous antioxidant, glutathione (GSH). TM treatment significantly increased mRNA levels of CHOP and GRP78 in the liver. Lipid peroxidation in the liver tissue also increased from TM treatment, which reflects an imbalance between generation of reactive substances and antioxidant capacity. To examine the involvement of GSH synthetic pathway, it was determined the metabolomic changes of sulfur amino acids in the liver. TM significantly decreased hepatic S-adenosylmethionine (SAM) concentration in the methionine cycle. The levels of cysteine in the liver were increased, while taurine concentration was maintained and GSH levels profoundly decreased. These results suggest that abnormal cysteine metabolism by TM treatment caused a decrease in GSH, followed by an induction of oxidative stress in the liver. In HepG2 cells, decreased GSH levels were observed by TM or PHMG-P treatment in a dose-dependent manner. Furthermore, pretreatment with TM or PHMG-P in HepG2 cells exacerbated the cytotoxic effects of tert-butyl hydroperoxide or hydroperoxide. In conclusion, PHMG-P-induced ER stress was accompanied by oxidative stress via reducing the GSH synthesis, which made the liver more susceptible to additional oxidative stress.

Keywords: PHMG, Lung, Liver, ER stress, Oxidative stress, Glutathione

목차 (Table of Contents)

• I. INTRODUCTION 1
• 1. Polyhexamethyleneguanidine (PHMG) 1
• 1.1. Characteristic of the PHMG . 1
• 1.2. Effect of PHMG exposure on the liver 2
• 2. Endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) 5
• 2.1. Role of ER in the liver 5
• 2.2. Differences between acute and chronic ER stress in the liver 7
• 3. Oxidative stress 7
• 3.1. Role of ROS in the liver 7
• 3.2. Antioxidant defense system 9
• 4. Sulfur-containing amino acid (SAA) metabolism in the liver 12
• 4.1. Regulation of SAA metabolism in the liver 12
• 4.2. GSH as a redox buffer 13
• 5. Purpose of the study 14
• II. MATERIALS AND METHODS 17
• 1. Animal experiments 17
• 2. Histological analysis 18
• 3. Examination of serum biochemical parameters 18
• 4. Determination of lipid peroxidation in the liver 18
• 5. Protein oxidation in the liver 19
• 6. Real-time reverse transcription-polymerase chain reaction (RT-PCR) 19
• 7. Tissue and cell lysis to extract protein 20
• 8. Western blotting 20
• 9. Examination of sulfur-containing substances 21
• 10. Cell culture 21
• 11. Cell viability assay and morphological study 22
• 12. Fluorescence activated cell sorting (FACS) analysis of apoptosis 22
• 13. FACS analysis of mitochondrial membrane potential 23
• 14. Luciferase reporter assay for ER stress response 23
• 15. Caspase-3 activity 24
• 16. ROS detection for oxidative stress response 24
• 17. Statistical analysis 24
• III. RESULTS 27
• 1. Increased ER- and oxidative stress in the rat liver by intratracheal instilled with PHMG-P 27
• 2. Cytotoxicity by PHMG-P in the liver cells 27
• 3. Apoptosis induced by PHMG-P in HepG2 cells . 31
• 4. ER stress induced by PHMG-P in HepG2 cells 35
• 5. Inhibition of PHMG-P-induced ER stress by TUDCA 35
• 6. Inhibition of PHMG-P-induced apoptosis by TUDCA 38
• 7. Tunicamycin (TM) induced ER stress in the liver 38
• 8. Increased hepatotoxicity accompanied with oxidative stress due to TM 44
• 9. Change in hepatic metabolism of sulfur containing substances due to TM treatment 44
• 10. Decreased levels of cellular GSH by TM treatment in a dose-dependent manner and potentiation of t-BHP-induced cell death by pre-exposure of TM in HepG2 liver cells 47
• 11. Involvement of mitochondrial dysfunction in the enhancement of t-BHP-induced mitochondrial dysfunction in the TM pre-exposed HepG2 liver cells 48
• 12. Enhancement of t-BHP-induced apoptotic cell death by pro-exposure of TM in HepG2 liver cells 48
• 13. Depletion of hepatic GSH in the rat liver by intratracheal instilled with PHMG-P 54
• 14. Increased level of cellular ROS and depletion of GSH by PHMG-P treatment in HepG2 and AML12 liver cells 54
• 15. Enhancement of H2O2-induced cytotoxicity by pre-exposure of PHMG-P in HepG2 and AML12 liver cells 56
• 16. Morphological changes of liver cells by treatment of PHMG-P and H2O2 56
• IV. DISCUSSION 60
• V. CONCLUSION 65
• VI. REFFERENCES 67
• ABSTRACT (in Korean) 79