Determinants of bilirubin neurotoxicity by an in vitro molecular approach

3 INTRODUCTION . 4 1. BILIRUBIN PHYSIOLOGY AND METABOLISM . 5 2. NEONATAL HYPERBILIRUBINEMIA . 7 2.1. The bilirubin in newborns . 7 2.2. Bilirubin encephalophaties and Kernicterus . 7 3. BILIRUBIN NEUROTOXICITY . 8 4. MOLECULAR BASIS OF NEUROTOXICITY . 8 5. BRAIN NEUROPROTECTIVE MECHANISMS . 11 5.1. The blood brain barrier (BBB) . 11 5.2. Bilirubin transport . 12 5.3. Bilirubin metabolism by CYPs . 15 5.4. Antioxidant players in neurons . 15 5.4.1. The Glutathione protection against the oxidative environment . 15 5.4.2. The Cystine transporter System Xc . 17 5.4. 3. Cysteine providers for glutathione synthesis in neurons . 18 5.4. 4. Other antioxidant and detoxifying players . 19 6. THE SH-SY5Y CELL LINE AS A NEURONAL MODEL . 20 7. AIMS OF THE STUDY . 22 MATERIALS AND METHODS . 23 1. CHEMICALS . 24 2. BASIC PROCEDURES AND TECHNIQUES . 24 2.1. SH-SY5Y Cell Culture . 24 2.2. Bilirubin preparation . 24 2.3. Viability determination by MTT test . 25 2.4. RNA extraction and quantification . 25 2.5. cDNA Preparation . 25 2.6. Quantitative Real-Time PCR . 25 2.7. Protein extraction and Quantification . 27 2.8. Western Blot analysis . 27 3. MODEL DEFINITION . 28 3.1. Sensitivity of SH-SY5Y cells to free bilirubin (Bf) . 28 3.2. Cellular growth curve of SH-SY5Y cells after bilirubin treatment . 28 3.3. Response of SH cells to a second treatment with bilirubin during 4 hours . 28 4. SH-SY5Y CELL MODEL TO STUDY BILIRUBIN RESISTANCE . 29 4.1. The dynamic of the bilirubin . 29 4.1.1. Bilirubin entrance analysis by 3H-UCB uptake analysis . 29 4.1.2. Bilirubin accumulation by HPLC analysis . 29 4.1.3. Bilirubin entrance mediated by OATP transporters . 29 4.1.4. Bilirubin Extrusion mediated by ABC transporters . 30 4.2. Metabolic changes inside the cells . 30 4.2.1. The Cystine Transporter System Xc . 30 4.2.1.1. Effect of bilirubin on mRNA expression levels of genes involved in cystine uptake. 30 4.2.1.2. L-[C]-Cystine uptake by bilirubin treated and untreated SH-SY5Y cells . 30 4.2.1.3. Uptake contribution of neuronal L-Cystine transporters: System Xc, XAG and GGT. 31 4.2.1.4. Glutathione determinations after bilirubin treatment . 31 4.2.1.5. Cell viability of SH-SY5Y cells pre-treated with UCB after hydrogen peroxide stress . 32 4.2.1.6. Small interference RNA-mediated System Xc (SLC7A11) gene silencing . 32 4.2.1.7. xCT Protein expression and glutathione content after DEM treatment . 33 4.2.1.8. SH-SY5Y cell viability after DEM and bilirubin treatment . 33 4.2.1.9. SH-SY5Y cell viability after DEM and hydrogen peroxide treatment . 33 4.2.2. Other possible targets involved in Bilirubin SH-SY5Y resistance . 33 4.2.2.1. HO-1, HO-2, NQO1 and GCLC mRNA expression . 33 4.2.2.2. Bilirubin metabolism by CYP1A1, CYP1A2, CYP2A6 and UGT1A1 . 34 4.2.3. SH-SY5Y Cell line population. Possible different susceptibilities to bilirubin treatment . 34 4.2.3.1. Morphologic SH-SY5Y separation and bilirubin treatment . 34 4.2.3.2. Specific markers mRNA expression for the “S” and “N” SH-SY5Y subpopulations . 34 4.2.3.3. Vimentin expression in SH-SY5Y cells after bilirubin treatment . 34 5. SH-SY5Y MODEL TO STUDY BILIRUBIN NEUROTOXICITY . 35 5.1. Oxidative stress generation by UCB . 35 5.1.1. Intracellular ROS accumulation after bilirubin treatment . 35 5.2. Glutamate excitotoxicity . 35 5.2.1. Glutamate release by SH-SY5Y cells exposed to UCB . 35 5.2.2. nNOS, iNOS and eNOS mRNA expression after bilirubin treatment . 35 6. STATISTICAL ANALYSIS . 36 RESULTS . 37 1. MODEL DEFINITION .