Mechanisms of aluminium neurotoxicity in oxidative stress-induced ...

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CHAPTER 1 followed by centrifugation at 13,000 rpm. The pellet was then discarded and the supernatant treated with trichloroacetic acid (1 M) followed consecutively by sonication and centrifugation in a microcentrifuge (model E, Beckman Instruments, Palo Alto, CA, USA) at 13,000 rpm for 5 min. The resulting pellet was reconstituted in NaOH (0.5 M) with vigorous vortexing for 3 min. Then, 10 mM 2,4-dinitrophenylhydrazine in 2 M chloric acid was added and the mixture incubated at room temperature for 1 h, in darkness, and with continuous agitation. After the addition of trichloroacetic acid (1 M), the resulting mixture was centrifuged at 13,000 rpm for 5 min. The resulting pellet was washed twice with ethyl acetate : ethanol (1:1, v/v). Then, the washed pellet was reconstituted with 6 M guanidine in a 20 mM KH2PO4 buffer (pH 2.3) and the absorbance of the solution measured at 370 nm. The PCC was calculated from the absorbance data using as absorption coefficient for dinitrophenylhydrazone ε = 22,000 M –1 cm –1 and expressing this parameter as nmol carbonyls/mg protein. Because of the numerous washing steps, protein content in the final pellet was estimated on an HCl blank pellet processed simultaneously using a BSA standard curve in 6 M guanidine, and reading the absorbance at 280 nm. Determination of protein thiol content (PTC) 90 The PTC of proteins was estimated spectrophotometrically using a modification introduced to a standard assay (Hermida-Ameijeiras et al. 2004). Briefly, an aliquot of the sample (200 μl) was treated with trichloroacetic acid (0.5 M) for protein precipitation. After vortexing and centrifugation at 13,000 rpm for 5 min, the resulting pellet was reconstituted in an 80 mM Na3PO4 and 2 mM EDTA buffer (pH 8.0) containing 70 mM sodium dodecylsulfate. Then, 100 μM 5,5‟-dithiobis-(2-nitrobenzoic acid) in a buffer 100 mM Na3PO4 (pH 8.0) were added and the mixture incubated at room temperature for 20 min with continuous mixing. After centrifugation at 13,000 rpm for 5 min, the absorbance of the resulting solution was measured at 412 nm and the PTC calculated from this data using as absorption coefficient for 2-nitro-5- mercaptobenzoic acid ε = 13,600 M –1 cm –1 and expressing the corresponding parameter as nmol thiols/mg protein.
Statistical analysis CHAPTER 1 Results were expressed as the mean ± SD from five animals. Statistical differences were tested using one-way ANOVA followed by LSD for multiple comparisons (p < 0.05). All statistical analyses were performed using Sigmastat 3.0 from Jandel Scientific (San Diego, CA, USA). 91
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Universidade de Santiago de Compost
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Don Ramón Soto Otero y Doña Estef
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Acknowledgements The work of this t
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Abbreviations AA ascorbic acid AD A
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Abbreviations (continuation) PCC pr
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List of figures (continuation) Chap
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TABLE OF CONTENTS INTRODUCTION ....
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CHAPTER 3 Brain oxidative stress an
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INTRODUCTION PARKINSON’S DISEASE
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INTRODUCTION PD is found in all eth
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Bradykinesia INTRODUCTION Bradykine
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INTRODUCTION predate the occurrence
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Table 1: Differential diagnostic in
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INTRODUCTION Table 3: National Inst
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Figure 4: Dopamine catabolism (Mén
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The basal ganglia INTRODUCTION The
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The death of SNpc DAergic neurons I
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INTRODUCTION the dorsomedial part o
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INTRODUCTION characterized as are l
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INTRODUCTION (Olanow et al. 2004).
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Etiology and pathogenesis of PD INT
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Ageing INTRODUCTION Ageing remains
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INTRODUCTION The finding of MPTP to
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INTRODUCTION may clarify why many n
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Misfolding and aggregation of prote
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INTRODUCTION (E3) (Figure 18A). Los
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INTRODUCTION in connection with Par
Page 63 and 64: INTRODUCTION Oxidative damage happe
Page 65 and 66: DA metabolism as a source of ROS IN
Page 67 and 68: Contribution of oxidative and nitro
Page 69 and 70: Excitotoxicity INTRODUCTION Glutama
Page 71 and 72: INTRODUCTION al. 1996, Cicchetti et
Page 73 and 74: Experimental models of PD INTRODUCT
Page 75 and 76: INTRODUCTION induce selective DAerg
Page 77 and 78: ALUMINIUM General features INTRODUC
Page 79 and 80: INTRODUCTION Varying amounts of alu
Page 81 and 82: INTRODUCTION and antiperspirants co
Page 83 and 84: INTRODUCTION approximately 3% of al
Page 85 and 86: Excretion INTRODUCTION As insoluble
Page 87 and 88: INTRODUCTION 3.5 mg may be increase
Page 89 and 90: INTRODUCTION Oteiza 1999), non-iron
Page 91 and 92: Aluminium as a cell membrane menace
Page 93 and 94: INTRODUCTION mobilization of Ca 2+
Page 95: Aluminium impairs neurotransmission
Page 99 and 100: AIMS OF THE PRESENT THESIS The main
Page 101: OBJETIVOS
Page 104 and 105: OBJETIVOS 3) Evaluar de forma preci
Page 107: CHAPTER 1 Time-course of brain oxid
Page 110 and 111: CHAPTER 1 INTRODUCTION 86 6-OHDA (2
Page 112 and 113: CHAPTER 1 MATERIALS AND METHODS Che
Page 116 and 117: CHAPTER 1 RESULTS Effects of 6-OHDA
Page 118 and 119: CHAPTER 1 DISCUSSION 94 As shown by
Page 120 and 121: CHAPTER 1 strategies or to study th
Page 122 and 123: CHAPTER 1 Fig. 2 Changes in PCC in
Page 125: CHAPTER 2
Page 129 and 130: ABSTRACT CHAPTER 2 In the present w
Page 131 and 132: MATERIALS AND METHODS Chemicals CHA
Page 133 and 134: CHAPTER 2 atomization used were 1,5
Page 135 and 136: DISCUSSION CHAPTER 2 Aluminium ente
Page 137: Table 1. Graphite furnace programme
Page 141: CHAPTER 3 Brain oxidative stress an
Page 144 and 145: CHAPTER 3 INTRODUCTION 120 The huma
Page 146 and 147: CHAPTER 3 MATERIALS AND METHODS Che
Page 148 and 149: CHAPTER 3 1:15 for hippocampus and
Page 150 and 151: CHAPTER 3 initially incorporated to
Page 152 and 153: CHAPTER 3 RESULTS In vivo effects o
Page 154 and 155: CHAPTER 3 Effects of aluminium admi
Page 156 and 157: CHAPTER 3 DISCUSSION 132 Aluminium
Page 158 and 159: CHAPTER 3 hippocampus, showed simil
Page 160 and 161: CHAPTER 3 assume that the distinct
Page 162 and 163: CHAPTER 3 Fig. 1 Levels of TBARS (A
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CHAPTER 3 Fig. 2 Enzyme activity of
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CHAPTER 3 Fig. 3 Microphotographs s
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CHAPTER 3 Fig. 5 Levels of TBARS (A
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CHAPTER 3 Fig. 6 In vitro effects o
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SUMMARY
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SUMMARY values were attained at 48
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SUMMARY neurodegeneration in the DA
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CONCLUSIONS The results obtained in
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13) Aluminium does affect neither M
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RESUMEN RESUMEN Desde el punto de v
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RESUMEN zonas cerebrales excepto en
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CONCLUSIONES
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CONCLUSIONES Capítulo 2 4) La admi
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CONCLUSIONES 172 En base a las conc
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Publications as a direct result fro
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