名古屋大学博士学位論文 学位の種類 : 博士(医学)(課程) 学位授与年月日:平成3年3月8日 葛谷雅文氏の博士論文として提出された

Lipid peroxidation is a key contributor to the pathophysiology of traumatic brain injury (TBI). Traditional antioxidant therapies are intended to scavenge the free radicals responsible for either the initiation or propagation of lipid peroxidation (LP). However, targeting free radicals after TBI is difficult as they rapidly react with other cellular macromolecules, and thus has a limited post-injury time window in which they may be intercepted by a radical scavenging agent. In contrast, our laboratory has begun testing an antioxidant approach that scavenges the final stages of LP i.e. formation of carbonyl-containing breakdown products. By scavenging breakdown products such as the highly reactive and neurotoxic aldehydes (often referred to as “carbonyls”) 4-hydroxynonenal (4-HNE) and acrolein (ACR), we are able to prevent the covalent modification of cellular proteins that are largely responsible for posttraumatic neurodegeneration. Without intervention, carbonyl additions render cellular proteins non-functional which initiates the loss of ionic homeostasis, mitochondrial failure, and subsequent neuronal death. Phenelzine (PZ) is an FDA-approved monoamine oxidase (MAO) inhibitor traditionally used for the treatment of depression. Phenelzine also possesses a hydrazine functional group capable of covalently binding neurotoxic carbonyls. The hypothesis of this dissertation is that carbonyl scavenging with PZ will exert an antioxidant neuroprotective effect in the traumatically injured rat brain mechanistically related to PZ’s hydrazine moiety reacting with the lipid peroxidation (LP)-derived reactive aldehydes 4-hydroxynonenal (4-HNE) and acrolein (ACR). Data from our ex vivo experiments demonstrate that the exogenous application of 4-HNE or ACR significantly reduced respiratory function and increased markers of oxidative damage in isolated non-injured rat cortical mitochondria, whereas PZ pre-treatment significantly prevented mitochondrial dysfunction and oxidative modification of mitochondrial proteins in a concentration-related manner. Additionally, PZ’s neuroprotective scavenging mechanism was confirmed to require the presence of a hydrazine moiety based on experiments with a structurally similar MAO inhibitor, pargyline, which lacks the hydrazine group and did not protect the isolated mitochondria from 4-HNE and ACR. Our in vivo work demonstrates that subcutaneous injections of PZ following TBI in the rat are able to significantly protect brain mitochondrial respiratory function, decrease markers of oxidative damage, protect mitochondrial calcium buffering capacity, and increase cortical tissue sparing without decreasing neuronal cytoskeletal spectrin degradation. These results confirm that PZ is capable of protecting mitochondrial function and providing neuroprotection after experimental TBI related to scavenging of neurotoxic LP degradation products.

Carbonyl and oxidative stress augment the development of diabetic complications. We evaluated the cytoprotectiveness of walnut and hazelnut extracts and catechins for decreasing cytotoxicity, lipid peroxidation, reactive oxygen species (ROS) formation, and protein carbonylation in cell death models of carbonyl and oxidative stress. Polar extracts (methanol or water) showed better cytoprotection than the non-polar (ethyl acetate) nut extracts against hydroperoxide-induced hepatocyte cell death and oxidative stress markers. Catechin flavonoids found in plants, including walnuts and hazelnuts, prevented serum albumin carbonylation in a carbonyl stress model (using glyoxal or methylglyoxal). Hepatocyte protein carbonylation and cell death were prevented and UV spectra data suggested a catechin:methylglyoxal adduct was formed. We conclude that (a) bioactive nut constituents in polar extracts were more protective than non-polar extracts against oxidative stress, and (b) catechins were effective under physiological temperature and pH, at preventing dicarbonyl induced cytotoxicity likely by trapping dicarbonyls or reversing early stage carbonylation.

Carbonyl and oxidative stress augment the development of diabetic complications. We evaluated the cytoprotectiveness of walnut and hazelnut extracts and catechins for decreasing cytotoxicity, lipid peroxidation, reactive oxygen species (ROS) formation, and protein carbonylation in cell death models of carbonyl and oxidative stress. Polar extracts (methanol or water) showed better cytoprotection than the non-polar (ethyl acetate) nut extracts against hydroperoxide-induced hepatocyte cell death and oxidative stress markers. Catechin flavonoids found in plants, including walnuts and hazelnuts, prevented serum albumin carbonylation in a carbonyl stress model (using glyoxal or methylglyoxal). Hepatocyte protein carbonylation and cell death were prevented and UV spectra data suggested a catechin:methylglyoxal adduct was formed. We conclude that (a) bioactive nut constituents in polar extracts were more protective than non-polar extracts against oxidative stress, and (b) catechins were effective under physiological temperature and pH, at preventing dicarbonyl induced cytotoxicity likely by trapping dicarbonyls or reversing early stage carbonylation.

High blood glucose levels induce metabolic disorders that initiate a sequence of events including renal, arterial, cardiac and retinal disorders. Diabetes mellitus increases oxidative stress in tissues of animals including humans. The resulting oxidative stress might play role in the development of diabetic complications. In the present study, 36 male Wistar rats (250-300g) were divided into 5 groups as Control (n=6), Diabetic (n=7), Diabetic + Vit C (n=7), Diabetic + &amp / #945 / -Lipoic acid (n=6) and Diabetic + Combination of Vit C and &amp / #945 / -Lipoic acid (n=10). From the livers of all groups cytoplasmic and microsomal membrane fractions were prepared from liver and antioxidant enzymes namely, superoxide dismutase, glutathione peroxidase, catalase and glutathione S-transferase activities were measured. Microsomal lipid peroxidation, total lipid, total protein, reduced glutathione levels of each group was determined and compared. Microsomal fractions were also analyzed by FT-IR spectroscopy. The total protein levels of diabetic rats were found to be decreased significantly (p&lt / 0.05) compared to controls and the &amp / #945 / -lipoic acid and vitamin C supplemented groups tend to compensate the decreased levels of total proteins. Decreased catalase activity in diabetic group compared to control was restored by &amp / #945 / -lipoic acid, vitamin C treatment and/or combination of both. Increased glutathione peroxidase activity was decreased to control levels by the treatement of both &amp / #945 / -lipoic acid and vitamin C. Superoxide dismutase activities of diabetic rats were increased (p&lt / 0.05) compared to control group. Whereas glutathione S-transferase activities though showed some fluctuations, the results were not statistically significant. Total glutathione levels decreased in all groups significantly (p&lt / 0.0.5) compared to control group but any of the agent failed to compensate the reduced levels of glutathione. As an index of lipid peroxidation, TBA-reactivity (MDA) levels increased significantly in all diabetic groups and only combination group&rsquo / s TBARS levels decreased significantly compared to diabetic group. FT-IR study of rat liver microsomal membranes was carried out in order to understand the effects of diabetes on membrane order, dynamics and lipid peroxidation status. For this purpose CH2 symmetric wavenumber, CH2 antisymmetric bandwidth, =CH olefinic band area were compared. In temperature dependent FT-IR studies microsomal membrane phase behavior, order and dynamics were analyzed. Diabetic samples showed apparent decrease in both frequency and bandwidth. =CH olefinic band integrated area was increased for diabetic samples compared to controls. Alpha-lipoic acid and vitamin C supplemented groups showed similar effects. They tend to restore decreased levels of band frequency and bandwidth. Additive effect between &amp / #945 / -lipoic acid and vitamin C was seen in some cases that only the combination group achieved to restore control values while &amp / #945 / -lipoic acid and vitamin C were failed to restore alone. In conclusion, STZ-induced diabetes mainly caused an increase in antioxidant enzyme activities. Also, increase in lipid peroxidation caused a decrease in the fluidity and order of the membrane resulting in more rigid membrane structures. The loss of cooperation between the antioxidant network may play a role in the secondary complications of diabetes.

Free radicals are the compounds having one or more unpaired electrons in their outer orbital and this unpaired electron make these compounds very reactive. Especially as their concentration increases, they initiate a chain oxidation reaction of lipids, proteins and nucleic acids. The condition, in which the production of free radicals exceeds their elimination or tissue defense mechanism decrease against them or both occur together, is called oxidative stress. In diabetes mellitus which is a glucose metabolism disorder, there occurs excessive non-enzymatic protein oxidation, glucose autoxidation and enhanced activity of polyol pathway enzymes, which are the possible sources of the oxidative stress in this disease. In this study, the conditions of the activity measurements of major antioxidant enzymes, namely superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), glutathione peroxidase (GPx, 1.11.1.9) and glutathione S-transferase (GST, EC 2.5.1.18) were studied and the optimum conditions (pH, temperature and substrate concentrations) for each assay were determined. Further objectives of the study were to characterize the enzymatic antioxidant systems (catalase, superoxide dismutase, glutathione peroxidase and glutathione S-transferase), tissue oxidation status (concentrations of TBARS, protein carbonylation, and lipid/protein ratios) and nonenzymatic antioxidant (reduced glutathione) levels of the diabetic rat liver tissues. According to our results, the hepatic SOD and GPx activities significantly increased whereas CAT activity markedly decreased in diabetic rats compared to control group. Also, GST activities did not change in diabetes. As a result of oxidative stress, TBARS concentration, lipid/protein ratios and protein carbonylation increased and GSH levels decreased in diabetic rats compared to control rats. This increase in tissue damage, in spite of the increase in antioxidant enzyme activities, could have been due to the overproduction of reactive oxygen species that exceeded the capacity of the antioxidant enzymes during the eight week of diabetes.

Thesis (Ph. D.)--Case Western Reserve University, 2005. / [School of Medicine] Department of Physiology and Biophysics. Includes bibliographical references. Available online via OhioLINK's ETD Center.

Thesis (Ph.D. in Toxicology) -- University of Colorado Denver, 2008. / Typescript. Includes bibliographical references (leaves 111-122). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;

Thesis (Ph.D. in Toxicology) -- University of Colorado, 2005. / Typescript. Includes bibliographical references (leaves 141-156). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;

Thesis (Ph.D. in Toxicology) -- University of Colorado at Denver and Health Sciences Center, 2005. / Typescript. Includes bibliographical references (leaves 128-138).