Ethylene has long been considered as the main plant growth regulator that plays a key role in the regulation of leaf senescence. In sweet potato, ethephon, an ethylene releasing compound, promoted leaf senescence and H2O2 elevation. These ethephon-mediated effects were alleviated or attenuated by exogenous reduced glutathione and ascorbic acid. Ethephon treatment gradually increased endogenous total and reduced glutathione and ascorbic acid levels in sweet potato detached leaves 3 days after treatment. The H2O2 amount, however, was also increased at 72 h after treatment. Sweet potato detached leaves pretreated with reduced glutathione did significantly increased endogenous total and reduced glutathione levels at 24 h and remarkably decreased H2O2 amount at 72 h after ethephon application compared to that of ethephon alone control. Ethephon caused quick elevation of a small H2O2 peak at about 4 h after application, and the enhancement was eliminated by reduced glutathione pretreatment in treated sweet potato leaves. Pretreatment of diphenylene iodonium (DPI), an NADPH oxidase inhibitor, also repressed leaf senescence and H2O2 elevation at day 3 after ethephon treatment in sweet potato detached leaves, and the attenuation was effective within the first 4 h after ethephon treatment. For senescence-associated gene expression, ethephon and L-buthionine sulfoximine (BSO), an endogenous glutathione synthase inhibitor, did induced asparaginyl endopeptidase (SPAE) and cysteine proteases (SPCP1, SPCP2 and SPCP3) gene expression and the activation was repressed by reduced glutathione pretreatment. Based on these data we conclude that ethephon treatment may cause quick elevation of a small H2O2 peak likely via the NADPH oxidase, which may function as a signal component leading to leaf senescence, H2O2 elevation and senescence-associated gene expression in sweet potato detached leaves. The rate of endogenous antioxidant such as reduced glutathione elevation is also important and affects leaf senescence, H2O2 elevation and senescence-associated gene expression in sweet potato leaves.

Erythropoietin (EPO) is produced in the kidney and in fetal liver in response to hypoxia as well as to CoCl2. The EPO protein and mRNA can be induced in response to both stimuli in the human hepatoma cell (HCC) lines Hep 3B and Hep G2. An oxygen sensing mechanism in which a ligand dependent conformational change in the heme protein produces H2O2 in respone to either hypoxia or Cobalt has been demonstrated. However, an intriguing question can be raised as to why some HCC sublines, such as Hep G2 and Hep 3B are capable of expressing EPO gene, whereas in other HCC sublines, such as J5 and SK-Hep-I are completely devoid of the ability to express EPO gene. Along this line, does ¡§differentiation status¡¨ of these HCC cells play a pivotal role in regulating the expression of EPO gene? Next in line, how a differentiation-associated upregulation of g-glutemylcysteine synthetase (g-GCS), which tightly regulating the biosynthesis of endogenous glutathione(GSH) can modulate the expression of EPO. The objective of this research project was designed to address all these questions. Reported herein are several lines of evidence to demonstrate that endogenous GSH contents do play a pivotal role in the control and regulation of the expression of EPO gene. Firstly, using a group of five HCC lines with varying degrees of differentiation as the experimental model, we demonstrated that the endogenous GSH contents of these HCC cells were differentially upregulated depending on the degree of differentiation with an order of abundance being Hep G2> Hep 3B> J5> Mahlavu> SK-Hep-I. Coincidently, we also found that g-GCS heavy subunit activities as well as its mRNA correlated precisely with this order. Among these HCC cell lines tested, only two well-differentiated sublines, Hep G2 and Hep 3B expressed EPO gene implying that the latter process was dependent upon GSH and suggested a notion that a threshold level might be required for its optimal reactivation. Secondly, to further obtain the evidence to substantiate this possible role of GSH, we then supplemented to the cell culture media with an excessive quantity of nonlethal N-acetylcysteine for the purpose of reinforcing the endogenous GSH biosynthesis. Interestingly, we found that this manipulation could revert the reactivation of EPO gene in cell lines, such as J5 and SK-Hep-I, in which their EPO gene expressions were ortherwise shut down under a normal circumstance. Finally, we were able to demonstrated using RT-PCR and western blotting that the expression of EPO gene was reverted in GCS30, a SK-Hep-I subline that was permanently transfected with g-GCSh and is capable of overly expressing endogenous GSH. Taken together, we demonstrated herein for the first time that, besides hypoxia and CoCl2, endogenous GSH contents can also act as a positive regulator for the expression of EPO gene. The underlying mechanism of how GSH exerts its action in the regulation of EPO expression awaits further clarification.

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 72-81).

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 98-106).

Thesis (Ph. D.)--Institute of Paper Chemistry, 1980. / Bibliography: leaves 100-107.

Doxorubicin (DOX) is a broad-spectrum and effective chemotherapeutic agent, but its use in oncologic practice is limited by dose-dependent cumulative cardiotoxicity. DOX-induced cardiotoxicity is in large part due to its ability to cause oxidative stress. Multidrug resistance associated protein 1 (MRP1/ABCC1) is a member of the ATP-binding cassette (ABC) transporter superfamily. By effluxing a wide variety of endogenous and exogenous substrates, Mrp1 plays important physiological roles in multiple tissues and also protects normal tissues against toxicants. However, the role of MRP1 in heart is largely unknown. The role of Mrp1 in DOX-induced cardiotoxicity was investigated in Mrp1 null (Mrp1-/-) and their C57BL (WT) littermates. Chronic DOX caused body weight loss and hemotoxicity, and these adverse effects were significantly exacerbated in Mrp1-/- vs WT mice. Importantly, loss of Mrp1 potentiated DOX-induced cardiotoxicity, presenting as worsened cardiac function and more cellular apoptosis in DOX treated Mrp1-/- mice. Mrp1 also protected neonatal mouse cardiomyocytes (CM) and cardiac fibroblasts (CF) culture against DOX cytotoxicity in vitro. This was demonstrated by the decreased cell survival, more apoptosis and more DNA damage in DOX treated Mrp1-/- vs WT cells. In addition, the effects of deletion of Mrp1 was studied on glutathione (GSH)/glutathione disulfide (GSSG) homeostasis, glutathione conjugate of 4-hydroxy-2-nonenal (GS-HNE) accumulation, protein oxidative damage and expression of antioxidant enzymes. Loss of Mrp1 led to significantly higher GSH and GSSG basal levels in heart. Following DOX treatment, Mrp1-/- CM and CF showed increased GSH and GSSG levels vs WT cells. Meanwhile, DOX increased expression of the GSH synthesis enzymes in Mrp1-/- but not WT cells. Thus, increased GSH synthesis may contribute to the further increase in the GSH pool in DOX-treated Mrp1-/- cells. DOX induced comparable increases of GS-HNE concentration in WT and Mrp1-/- mice hearts. Finally, expression of extracellular superoxide dismutase (ECSOD/SOD3) was significantly lower in Mrp1-/- vs. WT CM treated with either saline or DOX. In summary, this study is the first to document a protective role of Mrp1 in DOX-induced cardiotoxicity. It gives critical information regarding the potential adverse sequelae of introduction of MRP1 inhibitors as adjuncts to clinical chemotherapy of multidrug resistant tumors.

published_or_final_version / abstract / toc / Pathology / Master / Master of Philosophy

The modification of proteins by xenobiotic and endogenous electrophilic species produced in cells undergoing oxidative stress contributes to cellular toxicity and disease processes. Many xenobiotics are themselves reactive electrophiles; however non-reactive compounds may become reactive towards proteins and DNA following metabolism. Identifying actual sites of adduction on target proteins is critical for determining the structural and functional consequences associated with the modification. 1,4-benzoquinone (BQ) is a reactive quinone and environmental toxicant, formed from oxidative metabolism of benzene, an aromatic hydrocarbon found in gasoline and other fuels. Although environmental and occupational exposure to benzene is associated with the development of aplastic anemia and leukemia, the mechanism of toxicity remains elusive. Due to the electrophilic nature of BQ, it reacts with glutathione to form quinol-thioether (QT) conjugates that retain the ability to redox cycle between the reduced (HQ) and oxidized (BQ) forms. BQ and its QT metabolites are reactive, and can produce cellular necrosis through oxidative stress and protein modification. One further consequence of oxidative stress is the elevation of cellular membrane lipid peroxidation, resulting in the formation of reactive lipid-aldehydes such as 4-hydroxynonenal (4HNE). Adduction of critical amino acid residues in target bone marrow proteins by 4HNE and QTs following exposure to benzene could contribute to its hematotoxic effects. This dissertation builds upon the foundation of proteins targeted by electrophilic adduction by outlining techniques to pinpoint the specific amino acids targeted and furthermore predict the functional releavance of adduction. For the first time, protein targets of reactive endogenous lipid aldehydes are reported in the bone marrow of chemically treated rats. Furthermore, novel sites of adduction by aldehydes and benzene-glutathione conjugates are reported within functional regions of topoisomerase II. Inhibition of bone marrow DNA topoisomerase II by benzene metabolites is implicated as a potential mechanism of benzene-induced hematotoxicity and acute-myeloid leukemia. The strong inhibitory effect of these compounds on topoisomerase II activity suggests that their presence in the bone marrow may play a role in benzene-induced myelotoxicity.

Studies were conducted using cultured immortalized wild type (non-CF) and mutant (CF) DeltaF508 cystic fibrosis transmembrane conductance regulator (CFTR) tracheal epithelial cells on the anti-inflammatory impact of agents that may alter ceramide and glutathione (GSH) metabolism. The CF cells demonstrated abnormally high levels of GSH and glutathione disulfide (GSSG), which could diminish intracellular production of ceramide, a key modulator of inflammation and apoptosis. Hence, additional cell culture studies were carried out with a known inducer of in situ ceramide synthesis, N-4(4-hydroxyphenyl) retinamide (fenretinide) on interleukin (IL)-8 release, intracellular ceramide content, and cellular proliferation in both the basal state and following the inflammatory stimuli of tumor necrosis factor (TNF) -alpha. Fenretinide treatment was associated with a dose-dependent increase in the cellular content of ceramide in both CF and non CF cells. Also, an inhibition of IL-8 release in the inflammatory condition of TNF-alpha treatment was observed following fenretinide treatment in the CF cells. As hyperbaric treatment of whey proteins was previously associated with improved survivability and higher GSH content in a Pseudomonas aeruginosa murine model of cystic fibrosis (CF), the anti-inflammatory role of low molecular weight peptides (< 1kDa) generated from enzymatic hydrolysis of native and pressurized whey protein isolates (WPI) was examined. Pressure treatment of WPI was associated with an enhanced protein digestibility and an altered peptide profile following in vitro digestion. The whey peptides were tested CF and non-CF lung epithelial cells to identify for their effects on GSH metabolism. The impact of the combined treatment of fenretinide and WPH was also tested in terms of apoptosis and cytokine release in cell culture. As opposed to non-CF cells, CF cells showed a strong downtrend in release of IL-8 following the combined fenretinide and whey peptide treatment. In addition, whey peptides protected wild type epithelial cells from the apoptotic effect of fenretinide. Our results suggest the usefulness of these agents as a pharmacological treatment in CF.

As intestinal epithelial cells mature, they continuously transition from proliferation to differentiation to apoptosis under the influence of cell signalling pathways including c-Jun N-terminal kinase (JNK). Glutathione S-transferases (GSTs) are cytoprotective detoxification enzymes, some of which, including GSTA1, also sequester and inhibit JNK through complex formation. Thus, GSTA1 may be a key sensor of cellular state and regulator of responses to cell stress stimuli. The focus of this research study was to investigate the functional importance of GSTA1 in two contexts: 1) modulating complex integrity with JNK and activation of JNK by oxidative stress, 2) controlling cellular transitioning between proliferation, differentiation and apoptosis. In the first study, the impact of GSTA1 levels on dissociation of GSTA1-JNK complexes and JNK activation in response to cellular stress was investigated in the human colonic adenocarinoma Caco-2 cells. The pro-oxidant menadione caused GSTA1-JNK complex dissociation in preconfluent Caco-2 cells, whereas postconfluent cells were relatively resistant to this effect. Preconfluent cells were more sensitive than postconfluent cells to menadione-induced cytotoxicity. Additionally, menadione-induced JNK activation was transient since removal of the stimulus resulted in re-association of GSTA1 with JNK and significantly reduced cytotoxicity. Over-expression and knockdown of GSTA1 affected the degree of GSTA1-JNK complex association without altering the JNK activation. However, enhanced GSH levels by N-acetyl cysteine blocked menadione-induced complex dissociation and JNK activation in Caco-2 cells. The results suggest that the mechanism of menadione-mediated JNK activation involves the production of reactive oxygen species, likely superoxide anion, and that the level of intracellular GSH plays an important role in preventing menadione-induced GSTA1-JNK complex dissociation and subsequent JNK activation. The functional importance of GSTA1 in controlling cellular proliferation, differentiation and apoptosis was investigated. Sodium butyrate (NaB) is a short-chain fatty acid, physiologically present in the human large intestine and modulates transitioning of cell states in colon cancer cell lines. GSTA1 levels increased in association with differentiation markers in postconfluent Caco-2 cells. Forced expression of GSTA1 significantly reduced cellular proliferation and siRNA-mediated down-regulation of GSTA1 significantly increased cells in S-phase and associated cell proliferation. NaB (1 mM) reduced Caco-2 cell proliferation, increased differentiation and up-regulated GSTA1 activity. In contrast, higher dose of NaB (10 mM) caused toxicity in preconfluent cells via apoptosis through caspase-3 activation in association with reduced GSTA1 activity. GSTA1 down-regulation by siRNA did not alter NaB-induced differentiation or the sensitivity of Caco-2 cells to NaB-induced apoptosis. Furthermore, NaB (10 mM) caused GSTA1-JNK complex dissociation but did not affect JNK activation. These findings suggest that GSTA1 levels may play a role in modulating enterocyte proliferation but do not influence differentiation or apoptosis.