Thesis (Ph.D. in Toxicology) -- University of Colorado at Denver and Health Sciences Center, 2006. / Typescript. Includes bibliographical references (leaves 119-138). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;

Thesis (Ph. D.)--University of Hong Kong, 2008. / Includes bibliographical references (leaf 112-132) Also available in print.

I hypothesise that cancer cells with high aldehyde dehydrogenase (ALDHhigh) activity present a new therapeutic target and will be selectively sensitive to 5-nitrofuran pro-drugs. Cancers are heterogeneous and contain subpopulations of ALDHhigh cells with tumour initiating potential. ALDH enzymes metabolize toxic aldehydes, and are highly expressed in somatic and cancer stem cells (CSCs), although their function in CSCs is not fully understood. In a small molecule screen coupled with target ID, Zhou et al. (2012) recently discovered that clinically active 5-nitrofurans (5-NFNs) are substrates of ALDH2. 5-NFNs are a class of pro-drug widely used to treat bacterial and parasitic infections, where their relative specificity is driven by nitroreductases, but little is known about the enzymes that bio-activate 5-NFNs in humans. Recent clinical cancer research has found that the 5-NFN, nifurtimox, has anti-cancer properties and it is currently in Phase 2 clinical trials for neuroblastoma and medulloblastoma (ClinicalTrials.gov Identifier: NCT00601003), however the mechanism underlying this anti-cancer activity is unknown. In melanoma and other cancers, ALDH1A1 and ALDH1A3 are highly expressed in CSCs. I demonstrate the anti-cancer activity of 5-NFNs in cancer cell lines, where they express high sensitivity to 5-NFNs in cell viability assays (A375 melanoma cells EC50 = 867nM). To test if ALDH1 enzymes are substrates of 5-NFNs, I performed in vitro activity assays by monitoring NADH production (λ = 340nm). I found that the clinically available 5-NFNs, nifuroxazide and nifurtimox, in addition to our own newly synthesised 5-NFNs, are competitive substrates for human ALDH1A3 activity in vitro (P < 0.05). Notably, nifuroxazide is not a substrate for ALDH2, suggesting that nifuroxazide may show selectivity toward ALDH1. Enzymatic assays with purified human ALDH2, demonstrate that ALDH2 requires NAD+ for bio-activation of 5-NFNs. Consistent with these assays, I found that 5-NFNs are competitive substrates for ALDH activity in melanoma cells by Aldefluor™, with 5-NFNs displaying a prolonged competitive inhibition of ALDH activity compared with the known inhibitor, DEAB. Importantly, no-nitro control compounds show no activity toward ALDH enzymes in vitro or in culture. Kinetic living-cell imaging (IncuCyte ZOOM®) reveals that a subpopulation of ALDH1A3 siRNA transfected A375 cells are protected from 5-NFN toxicity (P > 0.05) and cell death (DRAQ7™: P < 0.0001), demonstrating a functional role for ALDH1A3 in mediating 5-NFN activity in cancer cells. In contrast, A375 cells overexpressing ALDH1A3 by cDNA transient transfection were hypersensitive to 5-NFNs (P < 0.001), determined by Muse™ cell viability. Computational docking studies reveal that 5-NFNs have the potential to fit within the interior of the ALDH enzymatic cavity and interact with the catalytic cysteine, thereby offering a potential mechanism for 5-NFN bio-activation. Finally, in collaboration, we show a unique interaction between 5-NFNs and ALDH using mass spectrometry and have initiated protein crystallography trials. My work demonstrates a novel and biologically relevant 5-NFN-ALDH interaction in cancer cells. I propose 5-NFNs have the potential to target ALDHhigh CSCs within a tumour and advance the repurposing of clinical 5-NFN pro-drug antibiotics as anti-cancer therapeutics.

Indiana University-Purdue University Indianapolis (IUPUI) / The human aldehyde dehydrogenase (ALDH) superfamily consists of 19 isoenzymes that are critical for normal physiology as well as the removal of toxic aldehydes. Members of the ALDH1/2 family have vital roles in cell signaling during early development, ethanol metabolism, and the removal of aldehydes derived from oxidative stress. We sought to develop selective compounds toward ALDH2 to help determine its individual contribution to biological function, as many of the ALDH1/2 family possess overlapping substrate preferences. A high-throughput screen of over 100,000 compounds uncovered a class of aromatic lactones which inhibit the ALDH1/2 enzyme family. The lactones were then characterized using a combination of enzyme kinetics, X-ray crystallography, and cell culture experiments. We found that many of the lactones are over ten times more potent toward ALDH2 than daidzin, a previously described ALDH2 inhibitor. Our ability to produce many more ALDH isoenzymes allowed us to determine that daidzin is not as selective as previously believed, inhibiting ALDH2, ALDH1B1, and ALDH1A2 with equal potency. This inhibition pattern was seen with several of the aromatic lactones as well. Structural studies show that many of the lactones bind between key aromatic residues in the ALDH1/2 enzyme substrate-binding sites. One lactone in particular mimics the position of an aldehyde substrate and alters the position of the catalytic cysteine to interfere with the productive binding of NAD+ for enzyme catalysis. Further characterization of related compounds led to the realization that the mechanism of inhibition, potency, and selectivity differs amongst the lactones based off the substituents on the aromatic scaffold and its precise binding location. Two of these compounds were found to be selective for one of the ALDH1/2 family members, BUC22, selective for ALDH1A1, and BUC27, selective for ALDH2. BUC22 demonstrates ten-fold selectivity for ALDH1A1 over ALDH1A2 and does not inhibit the remaining ALDH1/2 enzymes. Additionally, treatment with BUC22 led to decreased growth of triple-negative breast cancer cells in culture. BUC27 inhibits ALDH2 with the same potency as daidzin. Both BUC22 and BUC27 could be further developed to use as chemical tools to better understand the functional roles of ALDH1A1 and ALDH2 in biological systems.

Drought is a major environmental factor limiting crop productivity. Considering a significant area of crop production under water-limited rained conditions, there is a great need to develop production systems to sustain yield potentials under drought stress. Silicon has recently been recognized as an important element in plant nutrition. In this study, it was shown that supplying soybean with soluble silicon in the soil could improve vegetative growth and drought tolerance under water limiting conditions. In order to understand the molecular mechanism how silicon alleviates drought stress, the effects of silicon application on protein expression and antioxidant enzymes were examined. Soybean plants were grown in sand-containing pots supplied with 4 millimolar solutions of sodium silicate. To cancel the effect of sodium, the same amount of sodium chloride was used along with control plants. Soluble proteins were isolated from the leaves and roots of silicon-treated and control plants subjected to water deficit stress. Two-dimensional gel electrophoresis and mass spectrometry approaches were used to identify differentially expressed leaf and root proteins in response to silicon application under water deficit stress. Proteins that showed differential expression in response to silicon application included metabolic enzymes and proteins involved in the proteasome-dependent degradation pathway. These results indicate that silicon application could affect enzymes important for carbohydrate metabolism and stabilize aldehyde dehydrogenases and malic enzyme under water deficit stress, which may be attributable to drought tolerance.

No / Aromatic aldehydes are good substrates of aldehyde dehydrogenase activity but are relatively poor substrates of aldehyde oxidase and xanthine oxidase. However, the oxidation of xenobiotic-derived aromatic aldehydes by thelatter enzymes has not been studied to any great extent. The present investigation compares the relative contribution of aldehyde dehydrogenase, aldehyde oxidase and xanthine oxidase activities in the oxidation of isovanillin in separate preparations and also in freshly prepared and cryopreserved liver slices. The oxidation of isovanillin was also examined in the presence of specific inhibitors of each oxidizing enzyme. Minimal transformation of isovanillin to isovanillic acid was observed in partially purified aldehyde oxidase, which is thought to be due to residual xanthine oxidase activity. Isovanillin was rapidly metabolized to isovanillic acid by high amounts of purified xanthine oxidase, but only low amounts are present in guinea pig liver fraction. Thus the contribution of xanthine oxidase to isovanillin oxidation in guinea pig is very low. In contrast, isovanillin was rapidly catalyzed to isovanillic acid by guinea pig liver aldehyde dehydrogenase activity. The inhibitor studies revealed that isovanillin was predominantly metabolized by aldehyde dehydrogenase activity. The oxidation of xenobiotic-derived aromatic aldehydes with freshly prepared or cryopreserved liver slices has not been previously reported. In freshly prepared liver slices, isovanillin was rapidly converted to isovanillic acid, whereas the conversion was very slow in cryopreserved liver slices due to low aldehyde dehydrogenase activity. The formation of isovanillic acid was not altered by allopurinol, but considerably inhibited by disulfiram. It is therefore concluded that isovanillin is predominantly metabolized by aldehyde dehydrogenase activity, with minimal contribution from either aldehyde oxidase or xanthine oxidase.

The flavour or fragrance of Basmati rice is associated with the presence of 2-acetyl-1- pyrroline. This work shows that a gene with homology to betaine aldehyde dehydrogenase (BAD) has significant polymorphisms in the coding region of fragrant genotypes relative to non fragrant genotypes. Accumulation of 2-acetyl-1-pyrroline in fragrant rice genotypes may be explained by the presence of mutations resulting in loss of function of the fgr gene product. The fgr gene corresponds to the gene encoding BAD2 in rice while BAD1 is encoded by a gene on chromosome 4. Development of an allele specific amplification (ASA) based around the deletion in the gene encoding BAD2 allows, perfect, simple and low cost discrimination between fragrant and non-fragrant rice varieties and identifies homozygous fragrant, homozygous non-fragrant and heterozygous non-fragrant individuals in a population segregating for fragrance. The cDNAs transcribed from rice chromosomes 4 and 8, each encoding an enzyme with homology to betaine aldehyde dehydrogenase were cloned and expressed in E. coli. The enzyme responsible for fragrance, encoded from chromosome 8, had optimum activity at pH 10, showed low affinity towards betaine aldehyde (bet-ald) with Km value of approximately 63ìM but a higher affinity towards -aminobutyraldehyde (GABald) with a Km value of approximately 9ìM. The enzyme encoded from chromosome 4 had optimum activity at pH 9.5 and showed generally lower affinity towards most substrates compared to the enzyme encoded from chromosome 8, substrate specificities suggest that the enzymes have higher specificity to aminoaldehydes and as such both should be renamed as an aminoaldehyde dehydrogenase (AAD). The inactivation of AAD2 (BAD2) in fragrant rice varieties likely leads to accumulation of its main substrate GABald which then cyclises to 1-pyrroline the immediate precursor of 2AP.

The flavour or fragrance of Basmati rice is associated with the presence of 2-acetyl-1- pyrroline. This work shows that a gene with homology to betaine aldehyde dehydrogenase (BAD) has significant polymorphisms in the coding region of fragrant genotypes relative to non fragrant genotypes. Accumulation of 2-acetyl-1-pyrroline in fragrant rice genotypes may be explained by the presence of mutations resulting in loss of function of the fgr gene product. The fgr gene corresponds to the gene encoding BAD2 in rice while BAD1 is encoded by a gene on chromosome 4. Development of an allele specific amplification (ASA) based around the deletion in the gene encoding BAD2 allows, perfect, simple and low cost discrimination between fragrant and non-fragrant rice varieties and identifies homozygous fragrant, homozygous non-fragrant and heterozygous non-fragrant individuals in a population segregating for fragrance. The cDNAs transcribed from rice chromosomes 4 and 8, each encoding an enzyme with homology to betaine aldehyde dehydrogenase were cloned and expressed in E. coli. The enzyme responsible for fragrance, encoded from chromosome 8, had optimum activity at pH 10, showed low affinity towards betaine aldehyde (bet-ald) with Km value of approximately 63ìM but a higher affinity towards -aminobutyraldehyde (GABald) with a Km value of approximately 9ìM. The enzyme encoded from chromosome 4 had optimum activity at pH 9.5 and showed generally lower affinity towards most substrates compared to the enzyme encoded from chromosome 8, substrate specificities suggest that the enzymes have higher specificity to aminoaldehydes and as such both should be renamed as an aminoaldehyde dehydrogenase (AAD). The inactivation of AAD2 (BAD2) in fragrant rice varieties likely leads to accumulation of its main substrate GABald which then cyclises to 1-pyrroline the immediate precursor of 2AP.

published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

Organic nitrates, such as glyceryl trinitrate (GTN), have been used clinically for more than a century. However optimal nitrate therapy is hindered by the development of tolerance, which is associated with a desensitized response to GTN, oxidative stress, and the inactivation of aldehyde dehydrogenase 2 (ALDH2). This thesis evaluated the ALDH2 inactivation hypothesis of GTN tolerance and investigated the role of oxidative stress in GTN tolerance mediated by the lipid peroxidation product, 4-hydroxynonenal (HNE). Evidence for a direct role of ALDH2 in nitrate action was sought using a stably transfected cell line that overexpressed ALDH2, or siRNA to deplete endogenous ALDH2. Neither manipulation altered GTN-induced cGMP formation, indicating that ALDH2 does not mediate GTN bioactivation and tolerance. In a second study using an in vivo GTN tolerance model and a cell culture model of nitrate action, a marked increase in HNE adduct formation was detected in GTN-tolerant tissues, and treatment with HNE reduced the cGMP and vasodilator responses to GTN, thus mimicking GTN-tolerance. Together, the results suggest a primary role for HNE in the development of GTN tolerance, and provide the framework for a unified hypothesis that accommodates the previous findings of sulfhydryl depletion, ALDH2 inactivation and oxidative stress that are associated with nitrate tolerance. Studies have implicated oxidative stress and increased HNE formation in the pathogenesis of Alzheimer’s disease (AD). It was hypothesized that the gene deletion of ALDH2 would result in increased HNE-adduct formation leading to impaired cognitive function, and AD-like pathological changes. We observed a marked increase in HNE-adduct formation in Aldh2-/- mouse hippocampi as well as hyperphosphorylated tau, activated caspases, age-related changes in hippocampal amyloid βeta1-42 (Aβ1-42), post-synaptic density protein 95 (PSD95) and phosphorylated cyclic adenosine monophosphate response element binding protein (pCREB) expression, endothelial dysfunction and other vascular pathologies. These data provide further evidence for the importance of HNE and oxidative stress in AD pathogenesis, and establish Aldh2-/- mice as a new, oxidative stress-based animal model of age-related cognitive impairment and AD. / Thesis (Ph.D, Pharmacology & Toxicology) -- Queen's University, 2013-05-31 11:10:58.145