Academic literature on the topic 'Cyp24a1 knockout'

Background: Cardiovascular events are the main cause of death in patients with chronic kidney disease. We hypothesize that the protective effects of renal cholesterol and vitamin D3 metabolism are lost under this condition. Nephropathy was induced by adenine in Apolipoprotein E knockout mice. The atherosclerotic phenotype was compared to mice with normal renal function. Methods: Mice were fed a western diet ±0.15% adenine. Urine and feces were collected to assess renal function and fecal output. Atherosclerosis, serum lipoprotein composition and functionality, hepatic lipids, and expression of genes involved in lipid metabolism, vitamin D3 and Na+ homeostasis, were assessed. Bones were analyzed by microCT. Results: Mice fed with adenine showed enhanced urinary Na+, Ca2+, and Pi excretion, reduced urinary pH, UreaUrine/UreaSerum, and CreatinineUrine/CreatinineSerum ratios. They developed less atherosclerosis. Lipoproteins in serum and hepatic lipids remained unchanged. Cholesterol efflux increased. Fecal output of cholesteryl ester and triglycerides increased. In the liver, mRNA levels of Cyp27a1, Cyp7a1, and Scarb1 increased; in the kidneys, Slc9a3, Slc12a3, Vdr, and Cyp24a1 decreased. Adenine increased cholesterol efflux in vitro. Tibias were shorter. Conclusion: Adenine induced tubular damage and was athero-protective because of enhanced cholesterol efflux and lipids elimination in feces. Bone growth was also affected.

1,25-Dihydroxvitamin D3 [1,25(OH)2D3] is the hormonally active form of vitamin D. The genomic mechanism of 1,25(OH)2D3 action involves the direct binding of the 1,25(OH)2D3 activated vitamin D receptor/retinoic X receptor (VDR/RXR) heterodimeric complex to specific DNA sequences. Numerous VDR co-regulatory proteins have been identified, and genome-wide studies have shown that the actions of 1,25(OH)2D3 involve regulation of gene activity at a range of locations many kilobases from the transcription start site. The structure of the liganded VDR/RXR complex was recently characterized using cryoelectron microscopy, X-ray scattering, and hydrogen deuterium exchange. These recent technological advances will result in a more complete understanding of VDR coactivator interactions, thus facilitating cell and gene specific clinical applications. Although the identification of mechanisms mediating VDR-regulated transcription has been one focus of recent research in the field, other topics of fundamental importance include the identification and functional significance of proteins involved in the metabolism of vitamin D. CYP2R1 has been identified as the most important 25-hydroxylase, and a critical role for CYP24A1 in humans was noted in studies showing that inactivating mutations in CYP24A1 are a probable cause of idiopathic infantile hypercalcemia. In addition, studies using knockout and transgenic mice have provided new insight on the physiological role of vitamin D in classical target tissues as well as evidence of extraskeletal effects of 1,25(OH)2D3 including inhibition of cancer progression, effects on the cardiovascular system, and immunomodulatory effects in certain autoimmune diseases. Some of the mechanistic findings in mouse models have also been observed in humans. The identification of similar pathways in humans could lead to the development of new therapies to prevent and treat disease.

Abstract Vitamin D deficiency is associated with a range of muscle disorders, including myalgia, muscle weakness, and falls. In humans, polymorphisms of the vitamin D receptor (VDR) gene are associated with variations in muscle strength, and in mice, genetic ablation of VDR results in muscle fiber atrophy and motor deficits. However, mechanisms by which VDR regulates muscle function and morphology remain unclear. A crucial question is whether VDR is expressed in skeletal muscle and directly alters muscle physiology. Using PCR, Western blotting, and immunohistochemistry (VDR-D6 antibody), we detected VDR in murine quadriceps muscle. Detection by Western blotting was dependent on the use of hyperosmolar lysis buffer. Levels of VDR in muscle were low compared with duodenum and dropped progressively with age. Two in vitro models, C2C12 and primary myotubes, displayed dose- and time-dependent increases in expression of both VDR and its target gene CYP24A1 after 1,25(OH)2D (1,25 dihydroxyvitamin D) treatment. Primary myotubes also expressed functional CYP27B1 as demonstrated by luciferase reporter studies, supporting an autoregulatory vitamin D-endocrine system in muscle. Myofibers isolated from mice retained tritiated 25-hydroxyvitamin D3, and this increased after 3 hours of pretreatment with 1,25(OH)2D (0.1nM). No such response was seen in myofibers from VDR knockout mice. In summary, VDR is expressed in skeletal muscle, and vitamin D regulates gene expression and modulates ligand-dependent uptake of 25-hydroxyvitamin D3 in primary myofibers.

Abstract Multiple myeloma (MM) is currently incurable, and its associated bone destruction is a major source of morbidity that significantly contributes to patient mortality. Development of novel treatments for MM is imperative, since myeloma patients continue to relapse and suffer the consequences of the bone disease, even when they are in remission. Thus new targets for treating and preventing MM induced bone destruction are needed. We found that TAF12 is highly expressed in CD138+ primary MM cells and bone marrow stromal cells (BMSC) from MM patients compared to their normal counterparts. TAF12 acts as a co-activator of VDR, that permits very low concentrations of 1,25-(OH)2D3 or analogs that bind VDR to enhance MM cell growth and bone destruction. The molecular responses of myeloma cells to vitamin D are unknown, as are its effects on the marrow microenvironment and myeloma bone disease. To assess the role of TAF12 in MM, we co-cultured JJN3 myeloma cell line with the human BMSC cell line (SAKA-T). The experiments showed that MM cells directly upregulates TAF12 in SAKA-T cells; this increase in TAF12 was mediated by IL-6 secreted by MM cells. Co-culture of MM cells with BMSC from TAF12 heterozygous knockout mice (TAF12+/-) blocked VCAM1 induction and the enhanced MM cell growth and osteoclast (OCL) formation in response to physiologic levels 1,25-(OH)2D3 (10-10 M) induced by BMSC. Thus, TAF12 plays an important role in increased cytokine production and adhesive interactions of MM cells/ BMSC induced by 1,25-(OH)2D3. We determined the role of TAF12 in the sensitivity of several MM cell lines to physiological concentrations of 1,25-(OH)2D3. TAF12 expression was tested in MM cell lines (MM1.S, JJN3, ANBL6, RPMI8226, U266 and OPM2). Five of 6 cell lines (except RPMI8226) expressed high levels of TAF12. In 3 of these five, 1,25-(OH)2D3 increasedRANKL. Interestingly, both MM1.S and JJN3 cells produced RANKL at very low concentrations of 1,25-(OH)2D3 (10-12-10-10 M), which also induced VEGF, DKK-1 and α4 integrin. To further investigate the mechanisms responsible the effects of low concentrations of 1,25-(OH)2D3 mediated by TAF12 in MM, we stably knocked-down (k/d) TAF12 in JJN3 myeloma cells using a TAF12 shRNA lentivirus (TAF12k/d-JJN3). The TAF12k/d-JJN3 cells demonstrated about 80% reduction in TAF12 protein. JJN3-Wt cells treated with 10-8M 1,25-(OH)2D3 increased CYP24A1 mRNA levels 60 fold compared to untreated cells. In contrast, CYP24A1 mRNA in TAF12k/d-JJN3 cells was not significantly induced even when treated with 10-8 M of 1,25-(OH)2D3. ChIP analysis using an anti-VDR or TAF12 antibody and primers flanking the two VDREs in the CYP24A1 promoter showed that 1,25-(OH)2D3 induced VDR and TAF12 binding to the CYP24A1 promoter in JJN3-Wt cells. In contrast, basal and induced levels of VDR binding were much lower in TAF12k/d-JJN3 cells. Further, VDR content in TAF12k/d-JJN3 cells treated with 1,25-(OH)2D3 (10-10-10-8M) was markedly decreased compared to JJN3-Wt cells. VDR content was quantified by Western blot. In addition, RANKL, VEGF, DKK1 and α4 integrin production by TAF12k/d-JJN3 cells treated with10-10 M of 1,25-(OH)2D3 were decreased 60% compared to JJN3-Wt cells. These results demonstrate that increased TAF12 in MM cells enhances transcriptional responses of a subset of VDR target genes, leading to hyper-responsivity to 1,25-(OH)2D3 in MM cells - suggesting that TAF12 is a potential therapeutic target for MM and MM bone disease. Disclosures Roodman: Eli Lilly and Co.: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees.

The continuous intragastric enteral feeding protocol in the rat was a major development in alcohol-induced liver injury (ALI) research. Much of what has been learned to date involves inhibitors or nutritional manipulations that may not be specific. Knockout technology avoids these potential problems. Therefore, we used long-term intragastric cannulation in mice to study early ALI. Reactive oxygen species are involved in mechanisms of early ALI; however, their key source remains unclear. Cytochrome P-450 (CYP)2E1 is induced predominantly in hepatocytes by ethanol and could be one source of reactive oxygen species leading to liver injury. We aimed to determine if CYP2E1 was involved in ALI by adapting the enteral alcohol (EA) feeding model to CYP2E1 knockout (−/−) mice. Female CYP2E1 wild-type (+/+) or −/− mice were given a high-fat liquid diet with either ethanol or isocaloric maltose-dextrin as control continuously for 4 wk. All mice gained weight steadily over 4 wk, and there were no significant differences between groups. There were also no differences in ethanol elimination rates between CYP2E1 +/+ and −/− mice after acute ethanol administration to naive mice or mice receiving EA for 4 wk. However, EA stimulated rates 1.4-fold in both groups. EA elevated serum aspartate aminotransferase levels threefold to similar levels over control in both CYP2E1 +/+ and −/− mice. Liver histology was normal in control groups. In contrast, mice given ethanol developed mild steatosis, slight inflammation, and necrosis; however, there were no differences between the CYP2E1 +/+ and −/− groups. Chronic EA induced other CYP families (CYP3A, CYP2A12, CYP1A, and CYP2B) to the same extent in CYP2E1 +/+ and −/− mice. Furthermore, POBN radical adducts were also similar in both groups. Data presented here are consistent with the hypothesis that oxidants from CYP2E1 play only a small role in mechanisms of early ALI in mice. Moreover, this new mouse model illustrates the utility of knockout technology in ALI research.

Conventional (whole body) CYP2E1 knockout mice displayed protection against high-fat diet-induced weight gain, obesity, and hyperlipidemia with increased energy expenditure despite normal food intake and spontaneous locomotor activity. In addition, the CYP2E1 knockout mice displayed a marked improvement in glucose tolerance on both normal chow and high-fat diets. Euglycemic-hyperinsulinemic clamps demonstrated a marked protection against high-fat diet-induced insulin resistance in CYP2E1 knockout mice, with enhanced adipose tissue glucose uptake and insulin suppression of hepatic glucose output. In parallel, adipose tissue was protected against high-fat diet-induced proinflammatory cytokine production. Taken together, these data demonstrate that the CYP2E1 deletion protects mice against high-fat diet-induced insulin resistance with improved glucose homeostasis in vivo.

In the kidney, progesterone is inactivated to 20α-dihydro-progesterone (20α-DH-progesterone) to protect the mineralocorticoid receptor from progesterone excess. In an attempt to clone the enzyme with 20α-hydroxysteroid activity using expression cloning in CHOP cells and a human kidney expression library, serendipitously cDNA encoding CYP27A1 was isolated. Overexpression of CYP27A1 in CHOP cells decreased progesterone conversion to 20α-DH-progesterone in a dose-dependent manner, an effect enhanced by cotransfection with adrenodoxin and adrenodoxin reductase. Incubation of CHOP cells with 27-hydroxycholesterol, a product of CYP27A1, increased the ratio of progesterone to 20α-DH-progesterone in a concentration-dependent manner, indicating that the effect of CYP27A1 overexpression was mediated by 27-hydroxycholesterol. To analyze whether these observations are relevant in vivo, progesterone and 20α-DH-progesterone were measured by gas chromatography-mass spectometry in 24-h urine of CYP27A1 gene knockout (ko) mice and their control wild-type and heterozygote littermates. In CYP27A1 ko mice, urinary progesterone concentrations were decreased, 20α-DH-progesterone increased, and the progesterone-to-20α-DH-progesterone ratio decreased threefold ( P < 0.001). Thus CYP27A1 modulates progesterone concentrations. The underlying mechanism is inhibition of 20α-hydroxysteroid dehydrogenase by 27-hydroxycholesterol.

Vertical sleeve gastrectomy (VSG) is one of the most effective and durable therapies for morbid obesity and its related complications. Although bile acids (BAs) have been implicated as downstream mediators of VSG, the specific mechanisms through which BA changes contribute to the metabolic effects of VSG remain poorly understood. Here, we confirm that high fat diet-fed global farnesoid X receptor (Fxr) knockout mice are resistant to the beneficial metabolic effects of VSG. However, the beneficial effects of VSG were retained in high fat diet-fed intestine- or liver-specific Fxr knockouts, and VSG did not result in Fxr activation in the liver or intestine of control mice. Instead, VSG decreased expression of positive hepatic Fxr target genes, including the bile salt export pump (Bsep) that delivers BAs to the biliary pathway. This reduced small intestine BA levels in mice, leading to lower intestinal fat absorption. These findings were verified in sterol 27-hydroxylase (Cyp27a1) knockout mice, which exhibited low intestinal BAs and fat absorption and did not show metabolic improvements following VSG. In addition, restoring small intestinal BA levels by dietary supplementation with taurocholic acid (TCA) partially blocked the beneficial effects of VSG. Altogether, these findings suggest that reductions in intestinal BAs and lipid absorption contribute to the metabolic benefits of VSG.

Although the mortality rate of breast cancer has continued to decrease over the past several decades, it remains the most frequently diagnosed cancer and the leading cause of cancer death in women worldwide. Accumulating epidemiological and clinical evidence suggest that vitamin D insufficiency is associated with increased breast cancer incidence and poor clinical outcomes in patients with breast cancer, which makes vitamin D supplementation a potential preventive or therapeutic option for the management of breast cancer. However, the detailed mechanisms on how vitamin D protects against breast cancer remains largely unknown. This thesis mainly investigates the role of vitamin D signaling pathway in the prevention of breast cancer. There are two major themes. The first theme focuses on identifying target genes and molecular pathways of vitamin D in human breast cancer, while the second theme aims to characterize the role of the vitamin D inactivating enzyme Cyp24a1 in the development of mouse mammary gland morphogenesis. This thesis is in the publication format with Chapter III as a published article, Chapter IV, V, and VI as manuscripts in preparation, while the remaining parts of this thesis (Chapter I and II) are regarded as an introduction. Theme 1: Identification of vitamin D3 target genes in human breast cancer tissue: Chapter I summarizes the basic knowledge of vitamin D metabolism in breast tissue, our current understandings of the anti-tumor effects of vitamin D, and the role of vitamin D in breast cancer prevention based on epidemiological and clinical studies. Target genes of the vitamin D signaling pathway mainly generated from investigation of breast cancer cell lines are also reviewed. Chapter II presents the evidence from randomized clinical trials that vitamin D, plus or minus calcium supplementation, has the potential to prevent breast cancer. Our analyses indicate that the available data are insufficient to confirm any protective effect of vitamin D supplementation, with or without calcium, on the risk of breast cancer. More participants in future trials are required to make a reliable and conclusive assessment. Chapter III, published as an original research article in the Journal of Steroid Biochemistry and Molecular Biology, determines the target genes and molecular pathways influenced by 1,25(OH)2D in normal human breast and cancer tissues in an ex vivo explant system. RNA-Seq revealed 523 genes that were differentially expressed in breast cancer tissues in response to 1,25(OH)2D treatment, and 127 genes with altered expression in normal breast tissues. The major finding of the present study is that exposure of both normal and malignant breast tissue to 1,25(OH)2D results in changes in cellular adhesion, metabolic pathways and tumor suppressor-like Chapter II presents the evidence from randomized clinical trials that vitamin D, plus or minus calcium supplementation, has the potential to prevent breast cancer. Our analyses indicate that the available data are insufficient to confirm any protective effect of vitamin D supplementation, with or without calcium, on the risk of breast cancer. More participants in future trials are required to make a reliable and conclusive assessment. Chapter III, published as an original research article in the Journal of Steroid Biochemistry and Molecular Biology, determines the target genes and molecular pathways influenced by 1,25(OH)2D in normal human breast and cancer tissues in an ex vivo explant system. RNA-Seq revealed 523 genes that were differentially expressed in breast cancer tissues in response to 1,25(OH)2D treatment, and 127 genes with altered expression in normal breast tissues. The major finding of the present study is that exposure of both normal and malignant breast tissue to 1,25(OH)2D results in changes in cellular adhesion, metabolic pathways and tumor suppressor-like pathways, which support epidemiological data suggesting that adequate vitamin D3 levels may improve breast cancer outcome. Theme 2: Characterization of the vitamin D-inactivating enzyme Cyp24a1 in pubertal mouse mammary gland morphogenesis. Chapter IV, presented as a manuscript in preparation, summarizes our current understanding from transgenic mouse models of the vitamin D receptor (Vdr) and vitamin D associated metabolic enzymes in mammary gland development, cancer initiation, and progression and discusses the implications of these findings for human breast cancer. An improved understanding of the mechanisms of action of vitamin D signaling pathway derived from these mouse models provides support that activation of vitamin D signaling pathway is a potential approach for human breast cancer prevention. Chapter V, also presented as a manuscript in preparation, characterizes the role of vitamin D-inactivating enzyme Cyp24a1 in mammary gland development. A novel mouse model was available with conditional knockout of the Cyp24a1 gene specifically in the mammary epithelium (MMTV-Cre x Cyp24a1lox/lox). Our major finding is that mammary glands from virgin Cyp24a1 knockout females display impaired ductal morphogenesis compared with age- and weight-matched wild-type mice, which is due to reduced proliferation of mammary epithelial cells with the ablation of Cyp24a1 activity. The final chapter (VI), presented as a manuscript in preparation, explores the regulatory network of RANKL expression in human breast cells. Although RANKL is well characterized as a target gene of vitamin D in bone tissue, it becomes a different story when it comes to breast tissue. Taken together, although the detailed mechanisms of how women with adequate levels of vitamin D have a lower incidence of breast cancer are yet to be defined, our findings definitely contribute to a better understanding of the role of vitamin D signaling pathway in the prevention of breast cancer. Our work focuses on analysing data from human breast cancer tissue and transgenic mouse models, which could facilitate translation from the bench to the bedside and ultimately benefit the welfare of people in general.
Thesis (Ph.D.) -- University of Adelaide, Adelaide Medical School, 2017

by Wong Wing-yee, Felice.
Thesis (M.Phil.)--Chinese University of Hong Kong, 1998.
Includes bibliographical references (leaves 144-166).
Abstract also in Chinese.
Acknowledgements --- p.i
List of Abbreviations --- p.ii
Abstract --- p.iv
Abstract (Chinese Version) --- p.vi
Table of Contents --- p.viii
List of Tables --- p.xii
List of Figures --- p.xiv
List of Appendices --- p.xvi
Chapter Chapter I --- Literature Review
Chapter 1. --- Introduction --- p.1
Chapter 2. --- Background of Cytochrome P450 --- p.3
Chapter 2.1 --- Discovery --- p.3
Chapter 2.2 --- Tissue Distribution --- p.3
Chapter 2.3 --- Structure and Functions --- p.7
Chapter 2.4 --- Nomenclature of the P450 Superfamily --- p.10
Chapter 3. --- Cytochrome P450 2E1 (CYP2E1) --- p.11
Chapter 3.1 --- Discovery --- p.11
Chapter 3.2 --- Tissue Distribution --- p.12
Chapter 3.3 --- Substrates and Inducers --- p.13
Chapter 3.4 --- Toxicological Role of CYP2E1 --- p.15
Chapter 4. --- CYP2E1-knockout Mouse Model --- p.17
Chapter Chapter II --- Carbon Tetrachloride (CC14) Study
Chapter 1. --- Introduction --- p.19
Chapter 1.1 --- General Properties and Usage of CC14 --- p.19
Chapter 1.2 --- Toxicological Aspects of CC14 --- p.19
Chapter 1.3 --- Mechanism of CCl4-induced Hepatotoxicity --- p.20
Chapter 1.4 --- Role of CYP2E1 in CCl4-induced Hepatotoxicity --- p.23
Chapter 1.5 --- Objectives of the Study --- p.27
Chapter 2. --- Materials and Methods --- p.29
Chapter 2.1 --- Chemicals and Materials --- p.29
Chapter 2.2 --- Animals --- p.29
Chapter 2.3 --- Acute CC14 Treatment --- p.29
Chapter 2.4 --- Preparation of Microsomal Fractions --- p.30
Chapter 2.5 --- Determination of Microsomal Protein Concentration --- p.31
Chapter 2.6 --- Determination of Serum Aminotransferase Activities --- p.31
Chapter 2.7 --- Liver Histology --- p.32
Chapter 2.8 --- Hepatic Microsomal CYP2E1 Activity -p-nitrophenol Assay --- p.34
Chapter 2.9 --- SDS-PAGE and Western Blot Analysis --- p.35
Chapter 2.10 --- Detection of Lipid Peroxidation in vitro and in vivo --- p.35
Chapter 2.10.1 --- In vitro Lipid Peroxidation - 2-Thiobarbituric Acid (TBA) assay --- p.35
Chapter 2.10.2 --- In vivo Lipid Peroxidation - Microsomal Conjugated Dienes Detection --- p.36
Chapter 2.11 --- Hepatic Lipid Fatty Acid Composition Analysis --- p.39
Chapter 2.11.1 --- Lipid Extraction --- p.39
Chapter 2.11.2 --- Thin Layer Chromatography --- p.39
Chapter 2.11.3 --- Methylation --- p.40
Chapter 2.11.4 --- Gas Chromatography --- p.40
Chapter 2.12 --- Statistical Analysis --- p.41
Chapter 3. --- Results --- p.42
Chapter 3.1 --- "Mortality, Liver Weight and Liver Color" --- p.42
Chapter 3.2 --- Hepatotoxicity --- p.42
Chapter 3.2.1 --- Serum ALT and AST activities --- p.42
Chapter 3.2.2 --- Liver Histology --- p.45
Chapter 3.3 --- CYP2E1-catalysed PNP Activities and CYP2E1 Protein Levels --- p.49
Chapter 3.3.1 --- CYP2El-catalyzed PNP Activities --- p.49
Chapter 3.3.2 --- CYP2E1 Protein Levels --- p.52
Chapter 3.4 --- Lipid Peroxidation --- p.52
Chapter 3.4.1 --- In vitro Lipid Peroxidation --- p.52
Chapter 3.4.2 --- In vivo Lipid Peroxidation --- p.54
Chapter 3.5 --- Hepatic Lipid Fatty Acid Composition --- p.56
Chapter 3.5.1 --- Fatty Acid Composition in Hepatic Phospholipid --- p.56
Chapter 3.5.2 --- Fatty Acid Composition in Hepatic Microsomal Phospholipid --- p.59
Chapter 3.5.3 --- Fatty Acid Composition in Hepatic Triglyceride --- p.61
Chapter 4. --- Discussion --- p.63
Chapter 4.1 --- CYP2E1 is Required in CCl4-mediated Hepatotoxicity --- p.63
Chapter 4.2 --- CYP2E1 is Degraded following CC14 Exposure --- p.65
Chapter 4.3 --- CYP2E1 is Required in CCl4-induced Lipid Peroxidation --- p.67
Chapter 4.4 --- CYP2E1 is Required in CCl4-induced Hepatic Phospholipid Depletion --- p.70
Chapter 4.5 --- CYP2E1 is Required in CCl4-induced Hepatic Triglyceride Accumulation --- p.72
Chapter 5. --- Conclusion --- p.76
Chapter Chapter III --- Chronic Ethanol Consumption Study
Chapter 1. --- Introduction --- p.77
Chapter 1.1 --- Multiple Metabolic Pathways for Ethanol Metabolism --- p.77
Chapter 1.2 --- Metabolism of Ethanol by the Microsomal Ethanol Oxidizing System --- p.79
Chapter 1.3 --- Role of CYP2E1 in Ethanol Metabolism --- p.82
Chapter 1.4 --- Role of CYP2E1 in Alcoholic Liver Disease and Associated Oxidative Stress --- p.84
Chapter 1.5 --- Objectives of the Study --- p.89
Chapter 2. --- Materials and Methods --- p.90
Chapter 2.1 --- Chemicals and Materials --- p.90
Chapter 2.2 --- Animals --- p.90
Chapter 2.3 --- Chronic Ethanol Treatment --- p.90
Chapter 2.3.1 --- Ethanol Diet Composition --- p.90
Chapter 2.3.2 --- Ethanol Feeding --- p.90
Chapter 2.4 --- Monitoring of Blood Ethanol Levels --- p.96
Chapter 2.5 --- Preparation of Microsomal Fractions --- p.96
Chapter 2.6 --- Determination of Microsomal Protein Concentration --- p.97
Chapter 2.7 --- Determination of Serum Aminotransferase Activities --- p.98
Chapter 2.8 --- Liver Histology --- p.98
Chapter 2.9 --- SDS-PAGE and Western Blot Analysis --- p.99
Chapter 2.10 --- Hepatic Fatty Acid Composition Analysis --- p.100
Chapter 2.10.1 --- Lipid Extraction --- p.100
Chapter 2.10.2 --- Thin Layer Chromatography --- p.101
Chapter 2.10.3 --- Methylation --- p.101
Chapter 2.10.4 --- Gas Chromatography --- p.102
Chapter 2.11 --- Statistical Analysis --- p.103
Chapter 3. --- Results --- p.104
Chapter 3.1 --- Average Food Consumption --- p.104
Chapter 3.2 --- Average Ethanol Consumption for Ethanol Liquid Diet Feeding Group --- p.104
Chapter 3.3 --- Body Weight Gain --- p.104
Chapter 3.4 --- Blood Ethanol Levels --- p.108
Chapter 3.5 --- "Mortality, Liver Weight and Liver Color" --- p.108
Chapter 3.6 --- Serum ALT and AST Activities --- p.110
Chapter 3.7 --- Liver Histology --- p.114
Chapter 3.8 --- Western Blot Analysis --- p.119
Chapter 3.9 --- Hepatic Lipid Fatty Acid Composition --- p.119
Chapter 3.9.1 --- Fatty Acid Composition in Hepatic Phospholipid --- p.119
Chapter 3.9.2 --- Fatty Acid Composition in Hepatic Triglyceride --- p.123
Chapter 4. --- Discussion --- p.126
Chapter 4.1 --- Nutrients Displacement after Chronic Ethanol Consumption --- p.126
Chapter 4.2 --- Varied Blood Ethanol Levels after Chronic Ethanol Consumption --- p.127
Chapter 4.3 --- Increase in CYP2E1 Levels after Chronic Feeding of Ethanolin WT mice --- p.127
Chapter 4.4 --- Lack of Evidence Indicating the Development of Ethanol- Induced Liver Injury --- p.129
Chapter 4.4.1 --- No Elevations in Serum ALT and AST Activities --- p.129
Chapter 4.4.2 --- Normal Liver Histology --- p.130
Chapter 4.4.3 --- Lack of Triglyceride Accumulation --- p.131
Chapter 4.4.4 --- Elevations in Hepatic PL --- p.132
Chapter 4.5 --- Possible Reasons for the Absence of Liver Damage after Chronic Ethanol Consumption in our Mouse Model --- p.134
Chapter 5. --- Conclusion --- p.137
Chapter Chapter IV --- Concluding Remarks
Chapter 1. --- A Comparison between Acute CC14 Study and Chronic Ethanol Consumption Study --- p.139
Chapter 1.1 --- Regulation of CYP2E1 Expression --- p.139
Chapter 1.2 --- Free Radical Production Involved in CC14- and Chronic Ethanol Consumption-Mediated Liver Injury --- p.140
Chapter 1.3 --- An Overall Comparison between CC14 study and Chronic Ethanol Consumption Study --- p.140
Chapter 2. --- Future Studies --- p.142
Chapter 2.1 --- Acute CC14 Study --- p.142
Chapter 2.1.1 --- Calcium Homeostasis Studies --- p.142
Chapter 2.1.2 --- Spin Trapping Studies --- p.142
Chapter 2.2 --- Chronic Ethanol Study --- p.142
Chapter 2.2.1 --- "Generation of a Heterozygous ""Ethanol-Sensitive"" Mouse Strain (SV/129/ter x C57BL/6)" --- p.143
Chapter 3. --- Concluding Remarks --- p.143
References --- p.144
Appendix --- p.167