Effects of cadmium exposure on hormonal status and expression of metallothionein and glucose-6-phosphate dehydrogenase in silver sea bream, Sparus sarba.

Man, Ka Yan.Thesis (M.Phil.)--Chinese University of Hong Kong, 2008.Includes bibliographical references (leaves 101-126).Abstracts in English and Chinese.Chapter I. --- Title page --- p.iChapter II. --- Thesis committee --- p.iiChapter III. --- Acknowledgements --- p.iiiChapter IV. --- Abstract (English) --- p.ivChapter V. --- Abstract (Chinese) --- p.viChapter VI. --- Table of contents --- p.viiChapter VII. --- List of abbreviations --- p.xiiiChapter VIII. --- List of figures --- p.xvGeneral introduction --- p.1Chapter Chapter 1: --- Literature review --- p.4Chapter 1.1. --- Cadmium --- p.5Chapter 1.1.1. --- Cadmium - Ways of uptake in human and aquatic life --- p.5Chapter 1.1.2. --- Cadmium - Toxic effects in fish --- p.6Chapter 1.2. --- Cortisol --- p.11Chapter 1.2.1. --- Cortisol - General information and its regulations --- p.11Chapter 1.2.2. --- Cortisol - Functions --- p.12Chapter 1.3. --- Thyroid hormones --- p.14Chapter 1.3.1. --- THs - General information and its regulations --- p.14Chapter 1.3.2. --- THs - Functions --- p.15Chapter 1.4. --- Growth hormone --- p.18Chapter 1.4.1. --- GH - General information and its regulations --- p.18Chapter 1.4.2. --- GH - Functions --- p.20Chapter 1.5. --- Insulin-like growth factor --- p.22Chapter 1.5.1. --- IGF-I - General information and its regulations --- p.22Chapter 1.5.2. --- IGF-I - Functions --- p.24Chapter 1.6 --- Metallothioneins --- p.26Chapter 1.6.1. --- MTs - Definition and Classification --- p.26Chapter 1.6.2. --- MTs - Functions --- p.27Chapter 1.7. --- Glucose-6-phosphate dehydrogenase --- p.31Chapter 1.7.1. --- G6PDH - General information and its regulations --- p.31Chapter 1.7.2. --- G6PDH ´ؤ Functions --- p.32Chapter Chapter 2: --- "Effects of cadmium exposure on the endocrine status of silver sea bream, Sparus sarba" --- p.34Chapter 2.1. --- Introduction --- p.35Chapter 2.2. --- Materials and methods --- p.37Chapter 2.2.1. --- Overall experimental design --- p.37Chapter 2.2.2. --- In vivo exposure to waterborne cadmium --- p.37Chapter 2.2.2.1. --- Experimental animals --- p.37Chapter 2.2.2.2. --- Adaptation --- p.37Chapter 2.2.2.3. --- Tissue sampling --- p.38Chapter 2.2.2.4. --- Enzyme-linked immunosorbent assay (ELISA) --- p.38Chapter 2.2.2.4.1. --- Serum cortisol analysis --- p.39Chapter 2.2.2.4.2. --- Serum triiodothyronine analysis --- p.39Chapter 2.2.2.4.3. --- Serum thyroxine analysis --- p.39Chapter 2.2.2.5. --- Protein extraction and Protein quantification --- p.40Chapter 2.2.2.6. --- Protein gel electrophoresis and immunoblotting (Western blotting) --- p.40Chapter 2.2.2.7. --- RNA extraction --- p.41Chapter 2.2.2.8. --- Reverse transcription for first-strand cDNAs from total RNAs samples from liver --- p.42Chapter 2.2.2.9. --- Real-time quantitative PCR assays of IGF-I mRNA expression --- p.43Chapter 2.2.3. --- In vivo experiments involving cadmium injection --- p.44Chapter 2.2.3.1. --- Experimental animals --- p.44Chapter 2.2.3.2. --- Adaptation --- p.44Chapter 2.2.3.3. --- Tissue sampling --- p.45Chapter 2.2.3.4. --- Enzyme-linked immunosorbent assay (ELISA) --- p.45Chapter 2.2.3.4.1. --- Serum cortisol analysis --- p.45Chapter 2.2.3.4.2. --- Serum triiodothyronine analysis --- p.45Chapter 2.2.3.4.3. --- Serum thyroxine analysis --- p.46Chapter 2.2.3.5. --- Protein extraction and Protein quantification --- p.46Chapter 2.2.3.6. --- Protein gel electrophoresis and immunoblotting (Western blotting) --- p.46Chapter 2.2.3.7. --- RNA extraction --- p.46Chapter 2.2.3.8. --- Reverse transcription for the first-strand cDNAs from total RNAs samples from liver --- p.46Chapter 2.2.3.9. --- Real-time quantitative PCR of IGF-I mRNA expression --- p.46Chapter 2.2.4. --- In vitro part of the project (Primary cell culture: hepatocytes exposed to cadmium medium) --- p.47Chapter 2.2.4.1. --- Experimental animals --- p.47Chapter 2.2.4.2. --- Primary hepatocytes culture preparation --- p.47Chapter 2.2.4.3. --- Cadmium treatment and cell harvest --- p.48Chapter 2.2.4.4. --- "RNA extraction, reverse transcription for the first-strand cDNAs from total RNAs samples from lysed cells and real-time quantitative PCR of IGF-I mRNA expression" --- p.48Chapter 2.2.5. --- Statistical analysis --- p.48Chapter 2.3. --- Results --- p.49Chapter 2.3.1. --- Serum cortisol level --- p.49Chapter 2.3.2. --- Serum triiodothyronine level --- p.49Chapter 2.3.3. --- Serum thyroxine level --- p.49Chapter 2.3.4. --- Pituitary growth hormone level --- p.50Chapter 2.3.5. --- Hepatic insulin-like growth factor mRNA expression --- p.50Chapter 2.4. --- Discussion --- p.58Chapter 2.4.1. --- Serum cortisol level --- p.58Chapter 2.4.2. --- Thyroid hormones --- p.61Chapter 2.4.3. --- Growth hormone --- p.64Chapter 2.4.4. --- Insulin-like growth factor-I --- p.67Chapter 2.5. --- Conclusion --- p.70Chapter Chapter 3: --- "Effects of cadmium exposure on MT and G6PDH mRNA expressions of silver sea bream, Sparus sarba" --- p.71Chapter 3.1. --- Introduction --- p.72Chapter 3.2. --- Materials and methods --- p.74Chapter 3.2.1. --- Overall experimental design --- p.74Chapter 3.2.2. --- In vivo experiments involving exposure to waterborne cadmium --- p.74Chapter 3.2.2.1. --- Experimental animals --- p.74Chapter 3.2.2.2. --- Adaptation --- p.74Chapter 3.2.2.3. --- Tissue sampling --- p.74Chapter 3.2.2.4. --- RNA extraction --- p.74Chapter 3.2.2.5. --- Reverse transcription for first-strand cDNAs from total RNAs samples from gill and liver --- p.75Chapter 3.2.2.6. --- Amplification of partial fragments of metallothionein (MT) --- p.75Chapter 3.2.2.7. --- Rapid amplification of 5´ة and 3´ة cDNA ends of metallothionein (MT) --- p.76Chapter 3.2.2.7.1. --- Amplification of 5´ة cDNA end --- p.76Chapter 3.2.2.7.2. --- Amplification of 3´ة cDNA end --- p.78Chapter 3.2.2.8. --- Real-time quantitative PCR of MT and G6PDH mRNA expressions --- p.79Chapter 3.2.3. --- In vivo injection of cadmium --- p.80Chapter 3.2.3.1. --- "Experimental animals, adaptation and tissue sampling" --- p.80Chapter 3.2.3.2. --- RNA extraction --- p.80Chapter 3.2.3.3. --- Reverse transcription for first-strand cDNAs from total RNAs samples from gill and liver --- p.81Chapter 3.2.3.4. --- Real-time quantitative PCR of MT and G6PDH mRNA expression --- p.81Chapter 3.2.4. --- In vitro exposure of primary hepatocyte culture to cadmium --- p.81Chapter 3.2.4.1. --- Experimental animals --- p.81Chapter 3.2.4.2. --- Preparation of the hepatocytes for cell culture --- p.81Chapter 3.2.4.3. --- Cadmium treatment and cell harvest --- p.81Chapter 3.2.4.4. --- "RNA extraction, reverse transcription for first-strand cDNAs from total RNAs samples from lysed cells and real-time quantitative PCR of MT and G6PDH mRNA expression" --- p.82Chapter 3.2.5. --- Statistical analysis --- p.82Chapter 3.3. --- Results --- p.83Chapter 3.3.1. --- MT cloning and characterization --- p.83Chapter 3.3.2. --- Metallothionein mRNA expression --- p.83Chapter 3.3.3. --- Hepatic glucose-6-phosphate dehydrogenase mRNA expression --- p.84Chapter 3.4. --- Discussion --- p.91Chapter 3.4.1. --- MT cloning and characterization --- p.91Chapter 3.4.2. --- Metallothioneins mRNA expression --- p.92Chapter 3.4.3. --- Hepatic glucose-6-phosphate dehydrogenase mRNA expression --- p.95Chapter 3.5. --- Conclusion --- p.98General conclusion --- p.99References --- p.10