2-Hydroxy-ethyl methacrylate (HEMA) is the major component released from resin-modified glass ionomer cements and dental adhesives. Human tissues mainly affected by HEMA are oral epithelium and dental pulp. We treated human gingival epithelial S-G cells and pulp fibroblasts (HPF) with various concentrations of HEMA, to evaluate its effects on cell growth, cell cycle progression, intracellular glutathione (GSH) level and reactive oxygen species (ROS) production. HEMA-induced growth inhibition in HPF and S-G cells in a dose-dependent manner, which may be partially explained by induction of cell cycle perturbation. G2/M phase arrest was noted after exposure of HPF to 5 and 10mM of HEMA, concomitant with glutathione depletion and ROS production. S-phase arrest occurred in S-G cells when treated with 2.5 and 5mM, while at 10mM a sub-G0/G1 peak was noted, indicating the potential induction of apoptosis. GSH depletion was marked in S-G cells only at concentrations of 5 and 10mM, but excessive ROS production was noted at concentration of 1mM and rose with dose increase between 1 and 5mM, then lessened at 10mM. This suggested that the increase of ROS in S-G cells was not mainly caused by GSH depletion. These results helped to define the mechanism of the cytotoxicity caused by HEMA.

Selenium is toxic at high doses, yet metabolically essential in trace amounts, and therefore provides an excellent illustration of the rule of paracelsus that "the dose alone determines the poison". The only mammalian selenoprotein of known function is glutathione peroxidase (GPx). This enzyme is expressed ubiquitously, and is responsible for detoxifying peroxides and hydroperoxides which, if left unchecked, may damage important biomolecules such as DNA and membrane fatty-acids. GPx is a homotetramer; each subunit contains one mole atom of selenium incorporated as a selenocysteine residue in the active site of the enzyme. Using oligonucleotides generated against the known bovine GPx amino-acid sequence, cDNA clones were isolated corresponding to the human GPx mRNA. Sequence analysis indicated that the selenocysteine in the active site of the enzyme was incorporated at an opal terminator (UGA) codon. Therefore the glutathione peroxidase mRNA constitutes the first example of natural suppression of a terminator codon in human cells. Regulation of human GPx gene expression by selenium was examined. Selenium replete HL-60 cells possessed approximately 30-fold more enzymatic GPx activity than selenium deficient cells. However steady-state GPx mRNA levels and rate of transcription of the GPx gene differed by less than 1.5-fold. Cycloheximide abolished the increase in enzymatic activity observed upon selenium replenishment. Cellular immunoreactive GPx protein levels correlated with enzymatic activity, indicating that the human GPx gene is regulated post-transcriptionally by selenium. The mechanism of this post-transcriptional regulation was investigated. A selenium labelled tRNA species was identified which exhibited features in common with a previously characterized tRNAUGA. This data suggested that selenium may be incorporated into GPx via a co-translational mechanism using a selenocysteinyl tRNA intermediate. Selenium did not alter cytoplasmic levels of the tRNAUGA, indicating that accumulation of cytoplasmic suppressor tRNA was not the point of regulation of GPx by selenium. A model is proposed for the co-translational insertion of selenocysteine into GPx mediated by a charged tRNA species present in selenium replete but absent from selenium deficient cells. Models are also proposed to explain the discrimination between the selenocysteine UGA codon and authentic UGA terminator codons. The regulation of the GPx gene was examined during mono-myelocytic differentiation of HL-60 cells in vitro and also during interferon-gamma activation of human peripheral blood macrophages and PMN. During phagocyte cell differentiation or activation, the ability to generate peroxide developed, however the peroxide-destroying capacities of GPx did not increase concomitantly. Complex regulatory patterns involving both transcriptional and translational controls were observed. The association of GPx gene expression with chronic granulomatous disease was explored. No correlation was found with either the autosomal or X-linked forms of the disease, a finding contradictory to previously published material.

Glutathione (GSH) is an important biological thiol, it performs significant biological functions such as serving an antioxidant which protect cells from oxidative stress by trapping free radicals which damage DNA and RNA. It is known that abnormal plasma levels of GSH have been linked to various human diseases. Therefore, the rapid, sensitive and highly selective detection of GSH is of great importance for investigating its functions in diseases diagnosis. Interestingly, we found in cetyl trimethylammonium bromide (CTAB) medium, the resorufin-based probe shows an extremely fast, highly selective response to GSH. The result indicates that this dye can be employed to detect GSH in biological samples such as human plasma. Cysteine (Cys) is another important biological thiol which is involved in a variety of significant cellur functions, including protein synthesis, detoxication, and metabolic process, etc. Abnormal levels of Cys are related to many diseases, such as slowed growth, Alzheimer's disease and cardiovascular disease. Thus, the detection and quantification of Cys in physiological media is of great importance. In this thesis, I am going to present two organic fluorescent probes (Resorufin-based probe and SNF probe) for the detection and quantification of Cys. In addition, we prove that they can directly quantify Cys in human plasma. The chemical mechanisms involved in the detection of Cys are discussed.

京都大学 / 0048 / 新制・課程博士 / 博士(エネルギー科学) / 甲第21885号 / エネ博第386号 / 新制||エネ||75(附属図書館) / 京都大学大学院エネルギー科学研究科エネルギー基礎科学専攻 / (主査)教授 片平 正人, 教授 森井 孝, 教授 木下 正弘 / 学位規則第4条第1項該当 / Doctor of Energy Science / Kyoto University / DGAM

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One of the matchless ironies of the human body is its requirement for the highly reactive oxygen molecule, which has been clearly implicated in many diseases and the aging processes. Oxidants produced by metabolic processes damage cells by starting chemical chain reactions including oxidation of DNA and proteins as well as lipid peroxidation. Damage to DNA can cause mutations and lead to cancer if not reversed by DNA repair mechanisms. Damage to proteins causes enzyme inhibition, denaturation and protein degradation. Lipid peroxidation can cause cell lysis as well as creating mutagenic and carcinogenic by-products. The human body contains antioxidants and enzymes that together work to prevent oxidative damage to cellular components. By and large antioxidants either prevent these reactive oxygen species from being formed or remove them before they cause damage. There are many theories currently that tout the superior nature of diverse antioxidant combinations. One such theory is by Dr. Lester Packer of The University of California at Berkley. Dr. Packer puts forth the hypothesis that there is a superlative combination of five antioxidants that have the ability to "recharge" one another both in the blood plasma and intracellularly. This would result in a greater quality of antioxidant protection for an extended time. The current study evaluates Dr. Packer's theory of antioxidant combination from his book The Antioxidant Miracle. The decay rate of the antioxidants vitamin E, vitamin C, lipoic acid, glutathione, and coenzyme Q10 alone and in combination were determined using the ORAC (Oxygen Radical Absorbance Capacity) assay. The majority of the antioxidants retained activity for longer periods of time when tested alone, rather than in combination as Dr. Packer's theory would suggest. The assay was also preformed (using the same antioxidants and combinations) on oxidatively damaged Raji cancer cells. Cell viability and uptake of antioxidants into the cytoplasm were monitored. Finally, a variety of multivitamins were subjected to the ORAC assay and their antioxidant capacity compared to that of the "Packer Combination". The results suggest that multivitamins are superior antioxidants than the Packer ratio listed in The Antioxidant Miracle.

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Despite rapid advances in clinical oncology, acquired drug resistance still poses a significant threat to the long-term efficacy of current treatment regimens. Because most chemotherapy drugs aim to activate apoptosis in cancer cells, expansion of the pharmacopeia to include treatments targeting novel tumor cell death mechanisms is a promising anti-cancer strategy. Induction of ferroptosis, an iron-dependent form of regulated cell death, shows particular therapeutic potential as aggressive metastatic and drug-resistant cancer cell states have been demonstrated to possess an exquisite dependency on glutathione peroxidase 4 (GPX4), a key suppressor of the ferroptotic cell death pathway. However, current GPX4 inhibitors are limited by poor pharmacokinetic properties that preclude their clinical use. The development of novel drug-like GPX4 inhibitors would benefit from the discovery of new chemical scaffolds to both enhance our understanding of the structural basis of small molecule binding and inhibition as well as facilitate the rational design of future GPX4-targeted therapeutics. In this dissertation, we employed three high-throughput screening strategies to identify novel scaffolds of interest for GPX4 inhibitor development. First, a Lead-Optimized Compound (LOC) library was screened and we conducted further characterization and structure-activity relationship (SAR) studies on hit compound LOC880. Compared to the original hit, analogs QW-095 and QW-105 showed improved binding affinity and GPX4 inhibitory activity in vitro and also induced lipid peroxidation in cells suggestive of ferroptotic death. Further enhancement of the potency and ferroptosis specificity of this scaffold is still needed, but the potentially noncovalent and allosteric mechanism of action presents a novel approach for targeting GPX4. Second, we conducted extensive SAR analyses on another promising hit from the LOC library screen, LOC1886, which led to the identification of the lead compound QW-314. This analog showed significantly improved potency and ferroptosis specificity in multiple cancer cell contexts, including a drug-tolerant persister cell model of minimal residual disease. Characteristic markers of GPX4 inhibition and ferroptosis are also observed in cells treated with QW-314, including GPX4 protein degradation and induction of lipid peroxidation, and QW-314 exhibited excellent selectivity for GPX4 over another glutathione peroxidase family selenoprotein GPX1 in an in vitro assay using cell lysates. Moreover, we determined a baseline of pharmacokinetic measures including aqueous solubility and metabolic stability in human and mouse liver microsomes for further medicinal chemistry optimization. Lastly, we screened a DNA-encoded library (DEL) and an Enamine Diversity library, identifying 10 additional chemical starting points for future investigation.

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