Iron (Fe) is a fundamental requirement for life since it is involved in many cellular processes critical for growth and proliferation. Indeed, studies which deplete Fe from neoplastic cells using specific chelating agents result in G1/S arrest and apoptosis. However, the precise role of Fe in the control of the cell cycle remains unclear. Chapter one of this thesis, discusses the potential mechanism(s) by which Fe chelators cause cell cycle arrest and apoptosis. Recent studies have shown that this process involves multiple molecules and is highly complex. Thus, the studies presented in this thesis assess the effects of specific high affinity Fe chelators on the expression of molecules that may play important roles in cellular arrest and apoptosis. The general methodology used in these studies is summarized in chapter 2. In previous investigations Fe chelators such as 2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone (311) were shown to be far more potent anti-tumor agents than the clinically used ligand, desferrioxamine (DFO). Studies detailed in chapter 3, examined the expression of the tumor suppressor protein p53 and the universal cyclindependent kinase inhibitor p21CIP1/WAF1 following Fe-deprivation. To further characterize the effects of chelators on cell cycle arrest, experiments compared their activity to the DNA-damaging agents, actinomycin D (Act D) and cisplatin (CP). The latter two compounds increase the expression of p53 and its target gene p21CIP1/WAF1. Incubation of normal and neoplastic cells with all agents resulted in increased accumulation of nuclear p53, with the effect being more pronounced for Act D and CP. As expected, both Act D and CP markedly increased nuclear p21CIP1/WAF1 protein levels, while DFO and 311 caused a significant (p &lt 0.0004) decrease. This result was surprising, since the WAF1 gene which encodes the universal cyclin dependent kinase inhibitor p21CIP1/WAF1 was markedly up-regulated at the mRNA level following Fe chelation. Proteasomal inhibition studies demonstrated the partial involvement of proteasomes in decreasing nuclear p21CIP1/WAF1 levels following DFO treatment. In contrast, the incubation of 311-treated cells with proteasomal inhibitors did not reverse the down-regulation of p21CIP1/WAF1 when compared to the control. Immunofluorescence experiments showed that only DNA-damaging agents and not Fe chelators increased the nuclear translocation of p21CIP1/WAF1. Collectively, this suggests that the chelators prevented translation of WAF1. Moreover, this decrease in nuclear p21CIP1/WAF1 protein expression did not appear to be due to a general effect in which Fe chelators inhibited mRNA translation since the transferrin receptor 1 was markedly up-regulated (15-21- fold) by DFO or 311. The combination of 311 with Act D or CP prevented the marked p21CIP1/WAF1 nuclear accumulation normally observed in response to these DNAdamaging agents. Significantly, the effect of chelation on reducing nuclear p21CIP1/WAF1 expression was reversed by the Fe-donor, ferric ammonium citrate (FAC), indicating that WAF1 translation was dependent on intracellular Fe levels. This study is the first to demonstrate that Fe chelators markedly up-regulate the mRNA expression of WAF1 but paradoxically inhibit its translation. The down-regulation of p21CIP1/WAF1 protein by chelators may be a novel mechanism by which these ligands cause G1/S arrest through cell cycle dysregulation. In chapter 4, gene arrays were used to assess the expression of molecules associated with cell cycle control and the p53 pathway following the incubation of cells with DFO, 311 or Act D. The N-myc downstream regulated gene 1 (Ndrg1) was the only molecule identified that was specifically up-regulated by Fe chelation and not DNA-damage. Although the exact function of the Ndrg1 protein is unclear, previous studies have shown that this molecule markedly slows down tumor growth (Kurdistani et al. Cancer Res. 1998:58;4439-44) and acts as a potent metastasis suppressor protein (Bandyopadhyay et al. Cancer Res. 2003:63;1731-6). Subsequent experiments examined the mRNA and protein expression of Ndrg1 using reverse-transcriptase PCR (RT-PCR) and Western blot analysis, respectively. Incubation of cells with DFO or 311 resulted in a marked increase in the expression of Ndrg1 mRNA and protein within 3 h and 6 h, respectively. This increase in Ndrg1 after incubation with chelators was not found with their Fe complexes nor when the Fe-binding site had been synthetically inactivated. In addition, the induction of Ndrg1 following Fe chelation was correlated with the ligands??? permeability and anti-proliferative activity. Indeed, the up-regulation of Ndrg1 by Fe chelators can be readily reversed by Fe-repletion. Collectively, this indicates that the induction of Ndrg1 was dependent on intracellular Fe levels. Previous studies demonstrated for the first time that the transactivation of Ndrg1 was dependent on HIF-1?? and p53 stabilization. In contrast, investigations using fibroblasts derived from a HIF-1?? knockout (HIF-1??-KO) mouse demonstrated that the transcriptional upregulation of Ndrg1 following Fe chelation was mediated by a HIF-1??-dependent and - independent mechanism. In addition, subsequent experiments using the p53-deficient H1299 lung carcinoma cell line revealed that the transactivation of Ndrg1 was not dependent on p53 status after Fe-depletion. Collectively, the presented studies suggest that Ndrg1 may be a novel link between Fe metabolism and the control of proliferation. The up-regulation of Ndrg1 by potent Fe chelators is significant and may be beneficial in the prevention of tumor metastasis. In conclusion, this thesis demonstrates that the molecular mechanisms behind the G1/S arrest and apoptosis involved multiple molecules following Fe chelation. Indeed, this study shows a potential mechanism by which Fe chelators may cause cell cycle dysregulation through the down-regulation of nuclear p21CIP1/WAF1 protein levels. In addition, the work presented identifies the up-regulation of the metastasis suppressor protein, Ndrg1, following Fe-deprivation. Collectively, the results indicate that potent Fe chelators can not only be used as anti-proliferative agents, but may also be beneficial at inhibiting tumor cell metastasis.

Thesis (Ph. D.)--University of New South Wales, 2004. / Also available online.

Background: Elevated levels of iron impair immune defence mechanisms specifically cell mediated immunity and macrophage function, favors infection with Mycobacterium tuberculosis (M.tb), its replication, progression to clinical disease and death form tuberculosis (TB). Chelation of iron in individuals with an excessive iron burden may restore host defence mechanisms, decrease M.tb viability and replication and could find application in the prevention and treatment strategies in a setting where both iron overload and TB are prevalent. Aim: The aim of this study was to investigate the effect of iron chelation on mycobacterial replication, host viability and defence mechanisms in iron-loaded monocyte-macrophages during M.tb infection. Materials and Methods: Mycobacterial replication was monitored using the microplate AlamarBlueTM assay (MABA) for M.tb strains H37Ra and H37Rv, or detection of green fluorescent protein (GFP) expression by BCG (GFP-BCG). Mitochondrial membrane potential (MMP), phosphatidylserine (PS) exposure and plasma membrane integrity of premonocytic U937 cells differentiated by vitamin D3 served as indicators of host cell viability after treatment with 500M FeSO4.7H2O alone or in combination with 500 M desferrioxamine (DFO) or silybin, and infection with M.tb at 1:1 infection ratio. Superoxide anion radical (O2-•) and total nitrate/nitrite generation was monitored as host defence mechanisms by absorption spectroscopy and fluorimetry respectively. Results: Iron supplementation enhanced intra- and extracellular growth of M.tb and BCG. Chelation of iron with DFO prevented the enhanced replication of mycobacteria promoted by iron. Iron overload increased host cell death of H37Ra-infected monocyte-macrophages through increased PS exposure and decreased MMP and plasma membrane integrity, while increasing O2-• production and decreasing NO production. DFO restored the iron-related increase in PS exposure, plasma membrane integrity, O2-• and NO production to levels similar in H37Ra-infected, iron-normal cells, while MMP remained suppressed. In contrast, infection of iron-loaded cells with H37Rv reduced the already suppressed monocyte-macrophage MMP while not affecting cell death or NO production. DFO restored the iron-related suppression of O2-• in H37Rv-infected cells, and induced the production of NO while not affecting host cell death. Conclusion: These results confirm the replication enhancing effect of iron-loading on M.tb. Its suppressive influence on macrophage viability and defence mechanisms by increasing cell death and decreasing NO production during infection, is also highlighted. The beneficial effect of iron chelation by DFO through the inhibition of mycobacterial replication and restoration of host viability and defence mechanisms are suggested. / Prof. Liza Bornman

The effects of temperature on the first and second acid dissociation constants of 8-hydroquinoline (oxine) have been measured and the following thermodynamic values assigned to solutions of 0.1M strength. For the process, H₂Ox⁺ ⇋ H⁺ + HOx at 25°C., pK₁ = 5.103, ΔH = 5.7 kcal/mole, and ΔS° = -4 e.u. For the second acid dissociation constant, HOx ⇋ H⁺ + Ox⁻ at 25°C., pK₂ = 9.76, ΔH = 6.3 kcal/mole, and ΔS° = -23 e.u. The values for the second acid dissociation constant agree with those previously published. The values for the first dissociation constant have not been previously determined but are consistent with the result expected for reactions of this charge and chemical type. The partition of 8-hydroxyquinoline was determined as a function of temperature for the following systems: water-benzene, water-nitrobenzene, water-toluene, and water-chloroform. The pertinent thermodynamic values for the partitions are as follows: a. Water-benzene: log P = 2.44; ΔH = -2.0 kcal/mole; ΔS° = 4.5 e.u. b. Water-nitrobenzene: log P = 2.67; ΔH = -1.7 kcal/mole; ΔS° = 6.6 e.u. c. Water-toluene: log P = 2.33; ΔH = -1.3 kcal/mole; ΔS° = 6.2 e.u. d. Water-chloroform log P = 2.703 ΔH = -2.5 kcal/mole; ΔS° = 3.9 e.u. The values obtained for the water-chloroform system agree well with tho literature and serve as a check on the rest of the data. The effect of temperature on the overall extraction constant of nickel oxinate and ferric oxinate was measured for the system water-chloroform. The thermodynamic values found for the process Me⁺ⁿ + nHOx ⇌ MeOx_n + nH⁺ are the following at 25°C. and an ionic strength of 0.1M: a. Nickel oxinate log Kₑ = -2.73; ΔH = -40.4 kcal/mole; ΔS° = -148 e.u. b. Ferric oxinate log Kₑ = -3.97; ΔH = -14.1 kcal/mole; ΔS° = -29 e.u. The effect of ionic strength on the partition of S-hydroxyquinoline between water and benzene was determined for four different uni-univalent salts: potassium chloride, sodium chloride, potassium nitrate and sodium perchlorate. The “salting out” of oxine observed is typical of non-electrolytes. / Doctor of Philosophy

Some asbestiform minerals may cause lung disease in humans such as asbestosis, mesothelioma, and lung cancer. Crocidolite, the asbestiform counterpart of the amphibole riebeckite, is particularly dangerous in cases of chronic exposure. Its pathogenic activity may result from the interaction of the fiber surfaces with physiological fluids. Fe removed from the fiber surface by molecular chelators present in the body can promote a series of reactions that yield the hydroxyl radicals (•OH) which are known to cause DNA damage. This breakdown of DNA may be part of the mechanism for crocidolite-induced pathogenesis. X-ray photoelectron spectroscopy (XPS) and solution chemistry were used to monitor the changes in surface composition of crocidolite fibers in a 50 mM NaCl solution at pH= 7.5 and 25°C in the presence of Fe chelators (citrate, EDTA, or desferrioxamine) for up to 30 days. The data show that the introduction of Fe chelators dramatically increases the rate at which Fe is released from the surface when compared to a control group where no chelators were added. In particular, XPS shows that Fe(III) is more effectively removed in the presence of the chelators. Past studies of the dissolution of Fe-containing silicates generally indicate that Fe removal is the rate-limiting step. Fe(III) is particularly insoluble under circumneutral conditions. However, our work suggests that crocidolite undergoes enhanced dissolution in the presence of a chelator. Therefore, based on our XPS and solution data, and assuming a typical fiber diameter, we can estimate that a crocidolite fiber will survive on the order of hundreds of years in lung-like conditions. This is at least two orders of magnitude longer than a chrysotile fiber of the same size, and corresponds well with the fiber content observed in human lung tissue. / Master of Science

A DISSERTATION SUBMITTED TO THE FACULTY OF MEDICINE, UNIVERSITY OF THE WITWATERSRAND, JOHANNESBURG, FR THE DEGREE OF MASTER OF SCIENCE IN MEDICINE. / The influence of iron on the outcome of various infections have been extensively reviewed. Clinical observations suggests that iron deficiency may be protective against malaria. Various researchers have shown that certain iron chelators blocked the proliferation of plasmodium falciparum in vitro and in vivo. (Abbreviation abstract) / Andrew Chakane 2018

Thesis (Ph. D.)--University of Missouri--Columbia, 1998. / Typescript. Vita. Includes bibliographical references (leaves: 157-166). Also available on the Internet.

The primary objective of this research was to determine if changes in iron availability influence the periodicity of blue-green algal growth. A secondary goal was to resolve how iron availability was related to events such as calcite (calcium carbonate) precipitation and sediment nutrient release. The biogeochemical regulation of blue-green algal succession was studied in three eutrophic hardwater lakes located upon the Thompson Plateau in south-central British Columbia. The experimental approaches included iri situ bottle and limnocorral experiments, sediment core analysis, monitoring of seasonal changes in water chemistry, and whole-lake manipulation by hypolimnetic aeration, or calcium hydroxide addition. Growth and primary production bioassays were used to evaluate iron availability. Microbial chelators were isolated from algal cultures and lake water, quantified by a chelation assay, and used to determine their in situ effects on algal productivity and bacterial heterotrophy. Microbes were able to regulate the bioavailability of iron. Algal siderophore isolates were rapidly assimilated in lake water and they were highly specific for iron chelation. Moreover, chelator concentrations in Black Lake usually exceeded the dissolved iron concentration. Algae excreted chelators that could suppress growth of some other species of algae by 90%, enhance the primary production of some other algal species by 30%, or suppress the heterotrophic activity of bacteria by 14-98%. The degree of iron limitation varied greatly during the summer. In Black Lake, iron limitation was more than ten-fold more intense in early summer than in late summer. Dense blooms of blue-green algae occurred in Black Lake only after the iron content of the lake increased from 20 to more than 100 ug/L. An increase in iron concentration in the water column of the three lakes was caused by a midsummer sediment release of iron. Although sediment pyrite formation converted available iron into refractory iron in both Chain and Frisken lakes, the degree of iron limitation varied greatly among the lakes. Unlike in Black Lake, the algae in Chain Lake were not limited by iron availability. Phosphorus solubility was a good index of iron availability. Black and Frisken lakes had too little iron for iron phosphate to precipitate, but the higher iron concentration in Chain Lake regulated phosphorus solubility. The differences among lakes was primarily a function of external iron loading, not sediment iron release. Chain Lake received 10³ more iron per m² than Frisken or Black lakes. Carbonate equilibria integrated the microbial responses to iron enrichment. When iron availability was increased in the epilimnion of Black Lake, algal productivity was enhanced which resulted in an increase in pH and the coprecipitation of more calcite and phosphorus than in control treatments. The precipitation of calcite could sediment as much as 90% of the algae and 97% of the phosphorus from the epilimnion. The hypolimnia of the iron-enriched limnocorrals had the lowest pH and highest dissolution of precipitated phosphorus. Three reactions, iron chelation, sediment iron release, and calcite precipitation, can regulate much of the periodicity of blue-green algal growth in hardwater lakes. / Science, Faculty of / Zoology, Department of / Graduate

Eastern North Dakota has received excessive rainfall events since 1995, and soils are prone to waterlogging. This research evaluated the effects of subsurface tile drainage, raised beds, and iron-chelate (Fe-EDDHA) seed-application on iron-deficiency chlorosis (IDC) incidence in soybean [Glycine max (L.) Merr.], soybean growth, and yield, across six environments during 2013 and 2014. Tile drainage without beds increased soybean yield and reduced IDC by 11%. Beds resulted in more vigorous plants with 9% more biomass and increased soybean yield by 6%. There was no yield advantage to using both tile and raised beds within the same field. The Fe-EDDHA reduced plant population and IDC expression, increased plant biomass, but did not result in a yield increase. Farmers are encouraged to consider utilizing raised beds as a means to mitigate excess water. Additional research is needed to determine the cause of lower established plant density after seed application with Fe-EDDHA. / Minnesota Soybean Research and Promotion Council / North Dakota Soybean Council / DuPont Pioneer

A series of studies were initiated to investigate growth responses of Kentucky bluegrass (Poa praetensis L.) and creeping bentgrass (Agrostis palustris Huds.) to foliar applications of two plant growth regulators (PGR) and/or chelated Fe (Na Fe diethylene triamine pentaacetate). Environmental variables considered were N levels, soil moisture regimes, and saline irrigations. The two materials investigated for PGR properties were a commercial product called Roots (a cold-water extract of seaweed and peat humus fortified with "intermediate metabolites" and thiamine) and the systemic fungicide propiconazole, trade name: (Banner) and chemical name: [1- {(2-(2,4-dichlorophenyl)-4-propyl-l,3-dioxolan-2yl}methyl-l H-1,2,4-triazole]. Fortified seaweed extract (FSE) was applied at 9.3 L/ha, and propiconazole (PPC) was applied at 0.93 L a.i/ha. Each was applied alone or in conjunction with chelated Fe at 0.11 kg a.i/ha. Kentucky bluegrass foliage height, root and shoot dry weight, and several foliar nutrients increased following PGR treatments when grown under either limited soil moisture or saline irrigation. Foliar applications of PGR and/or chelated Fe to creeping bentgrass reduced wilting and evapotranspiration, and increased leaf water status, root strength, and shoot dry matter at two levels of N during and after drought stress. / Ph. D.