Following conjugation with glutathione, xenobiotics are converted into cysteinylglycine conjugates, cysteine conjugates, and, finally, mercapturic acids. The structural factors determining the activities of dipeptidases for the metabolism of toxicologically-relevant cysteinylglycine conjugates are not well understood. I purified porcine kidney cortex membrane dipeptidase (MDP) to homogeneity, via phosphatidylinositol-specific phospholipase C-mediated cleavage of the protein’s membrane anchor and cilastatin affinity chromatography. The homodimeric structure of the MDP protein was confirmed by mass spectrometry. To test the enzyme activity of purified MDP, the cysteinylglycine conjugates of 1-(chloromethyl)naphthalene, 4-nitrobenzyl chloride, and 1-chloro-2,4-dinitrobenzene were synthesized and HPLC separation methods for their quantitation were developed. MDP catalyzed the hydrolysis of all three conjugates, but the rate of this activity was strongly dependent on the nature of the substituent on the cysteine sulfur atom.

To examine the effects of fat composition and supplemental vitamin E (Vit E) and selenium (Se) on in vivo lipid peroxidation, diet-induced hypercholesterolemia, and glutathione (GSH) metabolism, male Syrian hamsters were fed for three weeks butter fat (BF-) or fish oil- (FO-)based diets supplemented with Vit E and/or Se. The effect of supplemental Vit E and Se on tissue lipid peroxidation (LPO), glutathione peroxidase (GSH-Px) activity and GSH concentrations differed between heart and liver and also was affected by dietary fat. The reduced glutathione/oxidized glutathione (GSH/GSSG) ratio was more consistently associated with tissue lipid peroxidation than was tissue Vit E content. Plasma lipids were lowered with supplemental Se and Vit E. Se supplementation, however, exerted a more potent hypolipidemic effect than Vit E. A pro-oxidative action of Se in hearts of FO-fed hamsters was noted, which was inhibited by supplemental Vit E. Hence, the combination of Vit E and Se may offer the most benefit against diet-induced oxidative stress and hyperlipidemia.

Life as we know it would be impossible in the absence of oxygen. However, too much oxygen can be toxic to the aerobic organisms which depend on it for their very existence. This apparent paradox arises as a result of oxygen's ability to accept electrons, forming highly reactive (reduced) oxygen species such as superoxide, hydrogen peroxide and the hydroxyl radical. The toxicity of oxygen is greatly enhanced in illuminated plants, due to the photosynthetic reactions which produce both oxygen and highly energetic electrons in close proximity to one another. These problems are further exacerbated when plants are exposed to a variety of stress conditions, since these conditions reduce the ability of plants to utilise excess electrons. As a result of the danger posed by· these reactive oxygen species, plants have· evolved a complex antioxidant system for their scavenging. Research has shown that plants with naturally elevated levels of the components of the antioxidant system are better equipped to deal with stress conditions which enhance the production of reactive oxygen species. A considerable amount of research has thus been dedicated to the elucidation of the antioxidant system. Almost as much research has been dedicated to enhancing the antioxidant system, with the aim of improving plant productivity under stress conditions. This study sought to evaluate plants carrying elevated levels of two of the enzymes of the antioxidant system. For these purposes, tobacco was transformed with the gene for E. coli glutathione reductase (GR), an enzyme believed to catalyse the rate limiting reaction in the scavenging of hydrogen peroxide. This gene was fused to the gene for the RUBISCO small subunit transit peptide - a peptide capable of targeting proteins to the chloroplast. Due to the presence of this peptide the transformed plants exhibited high chloroplastic levels of GR activity. These plants were crossed with a second tobacco transformant carrying high levels of chloroplastic tomato superoxide dismutase (SOD) - an enzyme responsible for the scavenging of superoxide. These hybrid plants were shown to exhibit high GR and SOD activities in the chloroplast .- the subcellular compartment most susceptible to damage caused by reactive oxygen species. The transgenic hybrids were evaluated for their ability to tolerate oxidative stress by treating them with paraquat - a herbicide whose mode of action involves the production of large quantities of activated oxygen. Under stress conditions, plants carrying just E. coli GR showed a slight improvement in their ability to deal with oxidative stress. In contrast to this, the SOD transformants showed more cellular damage than untransformed control plants. This was attributed to the inability of other enzymes in the antioxidant pathway to deal with the increased flow of metabolites through the pathway. The hybrid transformants showed enhanced stress tolerance in the initial stages of oxidative stress, but this declined with ongoing exposure to stress conditions. As with the SOD transformants, this decline in protection was . ascribed to the relatively low activities of the other enzymes in the antioxidant pathway. It was concluded that elevated levels of the two enzymes conferred greater stress tolerance than just one of the enzymes, but for true stress tolerance it will be necessary to evaluate the antioxidant system and enhance the activity of further enzymes in the pathway. It may also be necessary to improve the regulation of transgene expression, ensuring that none of the enzymes are overwhelmed by the increased flow of metabolites through the system. / Thesis (M.Sc.)-University of Natal, 1996

This thesis describes the results obtained from the phytochemical analysis of Matricaria chamomilla, and the microbial transformation of oxymatrine (85) and harmine (87), as summarized below. 1. Chemical investigation of the crude methanolic extract of Matricaria chamomilla resulted in the isolation of a new natural product, matriisobenzofuran (72), along with four known compounds: apigenin (73), apigenin-7-O-β-glucopyranoside (74), scopoletin (75), and fraxidin (76). The structures of compounds 72-76 were elucidated with the aid of extensive NMR and mass spectroscopic studies. All of the aforementioned compounds showed moderate to good inhibitory activities against glutathione S-transferase, an enzyme which has been implicated in the resistance of cancer cells to chemotherapeutic agents. These compounds were also evaluated for antioxidant activity and displayed moderate to good free radical scavenging activity. Additionally, compounds 72-76 were screened for anti-leishmanial activity. Compounds 75 and 76 were significantly active in this assay, while the remaining compounds were weakly active. In the antibacterial and antifungal assays, compounds 72-76 were not active. 2. The second part of this thesis deals with the biotransformation studies on oxymatrine (85) and harmine (87). Oxymatrine (85) was metabolized to the deoxy analogue, matrine (84) by Penicillum chrysogeneum (ATCC 9480), Cunninghamella bainieri (ATCC 9244), Cunninghamella blakesleena (ATCC 9245 and 8688A), Curvularia lunata (ATCC 12017), and Fusarium sp. In the time-based analysis of this transformation, the metabolism of oxymatrine (85) could be detected after 48 hours of incubation. Additionally, incubation of harmine (87) with Mucor plumbeus (ATCC 4740) resulted in the isolation of harmine-N-oxide (94). The biotransformed products (84 and 94) were identified using IR, UV, NMR, and mass spectroscopic techniques. Compound 94 was evaluated for its ability to inhibit the enzyme acetylcholinestrase, whose overexpression has been linked to Alzheimer’s disease, and was found to possess weaker activity than harmine (87).

Hypochlorous acid (HOCl) is a powerful oxidant produced by activated phagocytes at sites of inflammation to kill a wide range of pathogens. Yet, it may also damage and kill the neighbouring host cells. The abundance of dead macrophages in atherosclerotic plaques and their colocalization with HOCl-modified proteins implicate HOCl may play a role in killing macrophages, contributing to disease progression. The first part of this research was to investigate the cytotoxic effect and cell death mechanism(s) of HOCl on macrophages. Macrophages require efficient defense mechanism(s) against HOCl to function properly at inflammatory sites. The second part of the thesis was to examine the antioxidative effects of glutathione (GSH) and 7,8-dihydroneopterin (7,8-NP) on HOCl-induced cellular damage in macrophages. GSH is an efficient scavenger of HOCl and a major intracellular antioxidant against oxidative stress, whereas 7,8-NP is secreted by human macrophages upon interferon-γ (IFN-γ) induction during inflammation and can also scavenge HOCl. HOCl caused concentration-dependent cell viability loss in human monocyte derived macrophage (HMDM) cells above a specific concentration threshold. HOCl reacted with HMDMs to cause viability loss within the first 10 minutes of treatment, and it posed no latent effect on the cells afterwards regardless of the HOCl concentrations. The lack of caspase-3 activation, rapid influx of propidium iodide (PI) dye, rapid loss of intracellular ATP and cell morphological changes (cell swelling, cell membrane integrity loss and rupture) were observed in HMDM cells treated with HOCl. These results indicate that HOCl caused HMDM cells to undergo necrotic cell death. In addition to the loss of intracellular ATP, HOCl also caused rapid loss of GAPDH enzymatic activity and mitochondrial membrane potential, indicating impairment of the metabolic energy production. Loss of the mitochondrial membrane potential was mediated by mitochondrial permeability transition (MPT), as blocking MPT pore formation using cyclosporin A (CSA) prevented mitochondrial membrane potential loss. HOCl caused an increase in cytosolic calcium ion (Ca2+) level, which was due to both intra- and extra-cellular sources. However, extracellular sources only contributed significantly above a certain HOCl concentration. Preventing cytosolic Ca2+ increase significantly inhibited HOCl-induced cell viability loss. This suggests that cytosolic Ca2+ increase was associated with HOCl-induced necrotic cell death in HMDM cells, possibly via the activation of Ca2+-dependent calpain cysteine proteases. Calpain inhibitors prevented HOCl-induced lysosomal destabilisation and cell viability loss in HMDM cells. Calpains induced HOCl-induced necrotic cell death possibly by degrading cytoskeletal and other cellular proteins, or causing the release of cathepsin proteases from ruptured lysosomes that also degraded cellular components. The HOCl-induced cytosolic Ca2+ increase also caused mitochondrial Ca2+ accumulation and MPT activation-mediated mitochondrial membrane potential loss. MPT activation, like calpain activation, was also associated with the HOCl-induced necrotic cell death, as preventing MPT activation completely inhibited HOCl-induced cell viability loss. The involvement of both calpain activation and MPT activation in HOCl-induced necrotic cell death in HMDM cells implies a cause and effect relationship between these two events. HMDM cells depleted of intracellular GSH using diethyl maleate showed increased susceptibility towards HOCl insult compared to HMDM cells with intact intracellular GSH levels, indicating that intracellular GSH played an important role in protecting HMDM cells against HOCl exposure. Intracellular GSH level in each HMDM cell preparation directly correlated with HOCl concentration required to kill 50% of population for each cell preparation, indicating intracellular GSH concentrations determine the efficiency of GSH in preventing HOCl-induced damage to HMDM cells. Intracellular GSH and cell viability loss induced by 400 μM HOCl were significantly prevented by 300 μM extracellular 7,8-NP, indicating that added 7,8-NP is an efficient scavenger of HOCl and out-competed intracellular GSH for HOCl. The amount of 7,8-NP synthesized by HMDM cells upon IFN-γ induction was too low to efficiently prevent HOCl-mediated intracellular GSH and cell viability loss. HOCl clearly causes HMDM cells to undergo necrosis when the concentration exceeds the intracellular GSH concentrations. Above this concentration HOCl causes oxidative damage to the Ca2+ ion channels on cell and ER membranes, resulting in an influx of Ca2+ ions into the cytosol and possibly the mitochondria. The rise in Ca2+ ions triggers calpain activation, resulting in the MPT-mediated loss of mitochondrial membrane potential, lysosomal instability and cellular necrosis.

Multidrug resistance-associated protein 1 (MRP1) is a member of the adenosine 5’-triphosphate (ATP)-binding cassette (ABC) transporters. MRP1 actively effluxes a variety of endogenous and exogenous substrates from cells, ultimately, working to remove these compounds from the body. MRP1 was initially discovered based on its ability to confer resistance against a variety of chemotherapeutics when overexpressed in cancer cells lines. MRP1 function is important for a number of physiological processes, including regulating cellular and extracellular levels of the anti-inflammatory leukotriene C4 (LTC4) and the antioxidant glutathione (GSH). Our studies have focused on the role of MRP1 in regulating hematopoietic stem cell (HSC) self-renewal and differentiation and the role of CK2 as a regulator of MRP1 function. Reactive Oxygen Species (ROS) cellular levels are tightly regulated and fluctuations in ROS levels affect many cellular processes, including the self-renewal and differentiation of hematopoietic stem cells and kinase signaling pathways. MRP1 regulates ROS through the transport of reduced and oxidized GSH. MRP1 is highly expressed in HSCs, therefore we hypothesized that MRP1 regulates ROS levels in HSCs via efflux of GSH. We have shown that MRP1 regulates HSC self-renewal by modulating cellular ROS via the efflux of GSH. The decrease in ROS results in downregulation of p38 activity and altered expression of a number of redox response genes. CK2 is a master regulator of the cell and controls cell growth, proliferation, death and survival. Yeast studies from our lab using Ycf1p (a homologue of MRP1) and Cka1p (a homologue of CK2) have found that Cka1p regulates Ycf1p function. This result suggests that CK2 regulates MRP1 function via phosphorylation. We have found that CK2 does regulate MRP1 function via phosphorylation of the N-terminal extension at Thr249. Using A549, H460, and HeLa cancer cell lines, we found that inhibition of CK2 with tetrabromobenzimidazole (TBBz) reduces MRP1 function and increases cellular toxicity to known MRP1 substrates.

I conducted electrophysiological work in C57BL/6J (B6) and PWD/PhJ (PWD) mice, with the goal of providing insight into the genetic and physiological controls of calcium intake. Prior behavioral preference tests indicated that PWD mice have higher preferences for calcium compounds compared to B6 mice, though several mechanisms could underlie this observation. I therefore measured taste-evoked chorda tympani (CT) responses in B6 and PWD mice, in order to investigate the specific role of taste sensation. A second experiment was conducted to investigate the role of the calciumsensing receptor (CaSR) is in gustatory transduction of calcium ions, using the CaSR agonist glutathione. In experiment 1, responses were significantly larger in PWD than B6 mice for CaCl2, MgCl2, citric acid and quinine, but did not differ between the strains for sucrose, KCl and NaCl. These strain differences in CT responses were especially large for tonic, rather than phasic, responding. These data suggest that differences in peripheral events, such as taste transduction, contribute to differences between B6 and PWD mice in preferences for taste solutions such as CaCl2. In experiment 2, glutathione at 100 μM had negligible effects on taste-evoked CT responses, which does not support a role for CaSR in mediating taste transduction of calcium ions. / Department of Physiology and Health Science

The present study determined whether dietary supplementation with reduced glutathione (GSH), glutathione ester (GSHE) or N-acetylcysteine (NAC) increased the mitochondrial level of GSH, the major antioxidant inside cells, in rat kidney cortex and medulla. Nine month-old female Lewis rats were given daily intraperitoneal injections of isotonic saline (n=6), or saline containing GSH (250mg or 0.81mmol/Kg of body wt; n=7), GSHE (12mg or 0.03mmol/Kg; n=8), or NAC (200mg or 1.22mmol/Kg; n=8) for four weeks. At the end of the injection period, the rats were anesthetized and the kidneys removed. The kidneys were separated into cortical and medullary sections, weighed, and homogenized. The sections were separated into cytosolic and mitochondrial fractions by differential centrifugation. The GSH levels were determined by a colorimetric assay. Cortical and medullary mitochondrial GSH levels were significantly increased by all three supplements. Cytosolic GSH levels were also significantly increased in both cortical and medullary sections. Thus, dietary supplementation can significantly increase the mitochondrial pool of GSH in the rat kidney. / Department of Physiology and Health Science

This study investigated whether exogenous supplementation with reduced glutathione (GSH) increased kidney mitochondrial and cytosolic GSH levels in young and old female Lewis rats. The young rats were 3 months of age and old rats were 22 months old. The rats were divided into a young control group (n=8), an old control group (n=5), a young experimental group (n=7), and an old experimental group (n=7). Rats in the young and old control groups did not receive any treatment, while rats in both the young and old experimental groups were injected with GSH (250 mg/Kg of body weight) into the peritoneal cavity once a day for a week. At the end of the injection period, the rats were anesthetized and kidneys were harvested. The mitochondrial and cytosolic fractions were separated from rat cortex and medulla by differential centrifugation. GSH concentrations were measured using a spectrophotometric assay. Both mitochondrial and cytosolic GSH levels in kidneys from young and old female Lewis rats were significantly increased with GSH supplementation. The results indicate that kidneys from both young and old rats respond to exogenous dietary supplementation with GSH. / Access to thesis permanently restricted to Ball State community only. / Department of Physiology and Health Science

UCP1 is a member of the mitochondrial transmembrane anion carrier protein superfamily and is required to mediate adaptive thermogenesis in brown adipose tissue (BAT). Once activated, UCP1 uncouples mitochondrial respiration from ATP synthesis, thereby wasting the protonmotive force formed across the mitochondrial inner membrane as heat. It is hypothesized that proton leaks through UCP1 could be a molecular target to combat certain forms of obesity. Although it is well established that UCP1 is regulated by allosteric mechanisms, alternative methods such as post-translational modification still remain to be explored. The aims of the present study were to confirm the phosphorylation of UCP1 and the physiological relevance of this modification. Using isoelectric focusing, we confirmed that UCP1 displayed acidic shifts consistent with phosphorylation in BAT mitochondria isolated from cold exposed versus warm acclimated mice. A mouse model that ectopically expressed UCP1 in skeletal muscle was used to explore the link between the mitochondrial redox status and UCP1 function. Our results show that the expression of UCP1 in skeletal muscle led to decreases in body and tissues weights. In contrast, glucose uptake into skeletal muscle, food intake and energy expenditure was increased with the expression of UCP1. Finally, proton leaks through UCP1 were determined to be increased in isolated mitochondria from transgenic versus wild-type mice. Taken together these results indicate a complex interplay between mitochondrial redox status, post-translational modification and UCP1 function. Elucidation of novel mechanisms regulating UCP1 offers alternatives strategies that can be explored in order to modulate BAT thermogenesis.