Kathleen A. Bjornstad

Biomimetic Actinide Chelators: An Update on the Preclinical Development of the Orally Active Hydroxypyridonate Decorporation Agents 3,4,3-LI(1,2-HOPO) and 5-LIO(Me-3,2-HOPO)

The threat of a dirty bomb or other major radiological contamination presents a danger of large-scale radiation exposure of the population. Because major components of such contamination are likely to be actinides, actinide decorporation treatments that will reduce radiation exposure must be a priority. Current therapies for the treatment of radionuclide contamination are limited and extensive efforts must be dedicated to the development of therapeutic, orally bioavailable, actinide chelators for emergency medical use. Using a biomimetic approach based on the similar biochemical properties of plutonium(IV) and iron(III), siderophore-inspired multidentate hydroxypyridonate ligands have been designed and are unrivaled in terms of actinide-affinity, selectivity, and efficiency. A perspective on the preclinical development of two hydroxypyridonate actinide decorporation agents, 3,4,3-LI(1,2-HOPO) and 5-LIO(Me-3,2-HOPO), is presented. The chemical syntheses of both candidate compounds have been optimized for scale-up. Baseline preparation and analytical methods suitable for manufacturing large amounts have been established. Both ligands show much higher actinide-removal efficacy than the currently approved agent, diethylenetriaminepentaacetic acid (DTPA), with different selectivity for the tested isotopes of plutonium, americium, uranium and neptunium. No toxicity is observed in cells derived from three different human tissue sources treated in vitro up to ligand concentrations of 1 mM, and both ligands were well tolerated in rats when orally administered daily at high doses (>100 micromol kg d) over 28 d under good laboratory practice guidelines. Both compounds are on an accelerated development pathway towards clinical use.

Boronated protoporphyrin (BOPP): localization in lysosomes of the human glioma cell line SF-767 with uptake modulated by lipoprotein levels.

PURPOSE Boronated protoporphyrin (BOPP) is a candidate for use in both boron neutron capture therapy (BNCT) and photodynamic therapy (PDT) of glioblastoma multiforme (GBM). Our objectives are to identify factors that influence the uptake and retention of BOPP in vitro and to determine BOPP distribution in a human glioma cell line in vitro. This information will aid the development of compounds and treatment strategies that increase the effectiveness of BNCT therapy for GBM. METHODS AND MATERIALS The amount, distribution pattern, and site of internalization of BOPP were assessed using fluorescence microscopy. Living human glioma (SF-767) cells were imaged after a 24-h exposure to BOPP (20-135.6 microg/ml, normal serum). Dose-dependent uptake of BOPP was determined using both fluorescence microscopy of individual living cells and inductively-coupled plasma-atomic emission spectroscopy (ICP-AES) analysis of cell pellets. Lysosome- or mitochondria-specific fluorescent probes were used to identify the cellular compartment containing BOPP. Two human fibroblast cell lines, AG-1522 (LDL receptor-positive) and GM019-15C (LDL receptor-deficient), were used to investigate LDL receptor-dependent BOPP uptake. The dependence of BOPP uptake on lipoproteins in the media was determined by exposing each of the three cell types to BOPP in medium containing either normal (NS) or lipoprotein deficient serum (LPDS). RESULTS BOPP accumulated in the lysosomes of human glioma cells in vitro, and not in the mitochondria, as reported for C6 rat glioma cells in vitro. BOPP uptake was concentration-dependent and was also dependent on the amount of lipoproteins in the medium. Over the range of incubation concentrations studied and at the single exposure duration time point investigated (24 h), all cells retained a similar amount of BOPP. At the lowest incubation concentration (20 microg/ml, NS), the amount of boron retained was near 10(9) atoms per cell (15 microg B/g cells). Lysosomes containing high concentrations of BOPP were randomly distributed throughout the cytoplasm; however, larger lysosomes containing BOPP were concentrated around the cell nucleus. Little or no BOPP accumulated in the cell nucleus. At incubation concentrations of 20 and 40 microg/ml (24-h time point), BOPP uptake in SF-767 cells was reduced in LPDS compared with NS (66% reduction). A similar result was observed for normal human fibroblasts (AG-1522 cells, 40 microg/ml, 24 h). At 40 microg/ml, in both NS and LPDS at 24 h, BOPP accumulation in LDL receptor-deficient human fibroblasts (GM019-15C cells) was reduced relative to AG-1522 cells. BOPP accumulation in GM019-15C cells (40 microg/ml, 24 h) was not affected by serum lipoprotein levels. CONCLUSION In cell culture, BOPP is taken up by human glioma cells via the LDL pathway and is compartmentalized into cellular lysosomes. Knowledge of this mechanism of BOPP uptake and retention will be important in attempts to modify toxicity and efficacy of this drug.

Effects of Iron Ions, Protons and X Rays on Human Lens Cell Differentiation

Abstract Chang, P. Y., Bjornstad, K. A., Rosen, C. J., McNamara, M. P., Mancini, R., Goldstein, L. E., Chylack, L. T. and Blakely, E. A. Effects of Iron Ions, Protons and X Rays on Human Lens Cell Differentiation. Radiat. Res. 164, 531–539 (2005). We have investigated molecular changes in cultured differentiating human lens epithelial cells exposed to high-energy accelerated iron-ion beams as well as to protons and X rays. In this paper, we present results on the effects of radiation on gene families that include or are related to DNA damage, cell cycle regulators, cell adhesion molecules, and cell cytoskeletal function. A limited microarray survey with a panel of cell cycle-regulated genes illustrates that irradiation with protons altered the gene expression pattern of human lens epithelial cells. A focus of our work is CDKN1A (p21CIP1/WAF1), a protein that we demonstrate here has a role in several pathways functionally related to LET-responsive radiation damage. We quantitatively assessed RNA and protein expression in a time course before and after single 4-Gy radiation doses and demonstrated that transcription and translation of CDKN1A are both temporally regulated after exposure. Furthermore, we show qualitative differences in the distribution of CDKN1A immunofluorescence signals after exposure to X rays, protons or iron ions, suggesting that LET effects likely play a role in the misregulation of gene function in these cells. A model of molecular and cellular events is proposed to account for precataractous changes in the human lens after exposure to low- or high-LET radiations.

Particle Irradiation Induces FGF2 Expression in Normal Human Lens Cells

Abstract Chang, P. Y., Bjornstad, K. A., Chang, E., McNamara, M., Barcellos-Hoff, M. H., Lin, S. P., Aragon, G., Polansky, J. R., Lui, G. M. and Blakely, E. A. Particle Irradiation Induces FGF2 Expression in Normal Human Lens Cells. Particle radiations, including both proton and helium-ion beams, have been used to successfully treat choroidal melanoma, but with the complication of radiation-induced cataract. We have investigated a role for radiation-induced changes in the expression of basic fibroblast growth factor (FGF2) gene expression as part of the mechanism(s) underlying lens cell injury associated with cataract. Normal human lens epithelial (HLE) cells were cultured in vitro on extracellular matrix (ECM) originated from bovine corneal endothelial cells. This study reports evidence for rapid but transient induction of FGF2 transcripts, an increase of between 5- and 8-fold, within 0.5 h after exposure to particle radiation, followed by another wave of increased transcription at 2–3 h postirradiation. Immunofluorescence results confirm the enhanced levels of FGF2 protein rapidly after exposure to protons or helium ions, followed by another wave of increased activity unique to helium at 6 h postirradiation. This second wave of increased immunoreactivity was not observed in the proton-irradiated samples. Total FGF2 protein analysis after helium-ion exposures shows induced expression of three FGF2 isoforms, with an increase of up to 2-fold in the 18-kDa low-molecular-weight species. Studies of the effects of protons on individual FGF2 protein isoforms are in progress. Several mechanisms involving a role for FGF2 in radiation-induced cataract are discussed.

Harderian Gland Tumorigenesis: Low-Dose and LET Response

Increased cancer risk remains a primary concern for travel into deep space and may preclude manned missions to Mars due to large uncertainties that currently exist in estimating cancer risk from the spectrum of radiations found in space with the very limited available human epidemiological radiation-induced cancer data. Existing data on human risk of cancer from X-ray and gamma-ray exposure must be scaled to the many types and fluences of radiations found in space using radiation quality factors and dose-rate modification factors, and assuming linearity of response since the shapes of the dose responses at low doses below 100 mSv are unknown. The goal of this work was to reduce uncertainties in the relative biological effect (RBE) and linear energy transfer (LET) relationship for space-relevant doses of charged-particle radiation-induced carcinogenesis. The historical data from the studies of Fry et al. and Alpen et al. for Harderian gland (HG) tumors in the female CB6F1 strain of mouse represent the most complete set of experimental observations, including dose dependence, available on a specific radiation-induced tumor in an experimental animal using heavy ion beams that are found in the cosmic radiation spectrum. However, these data lack complete information on low-dose responses below 0.1 Gy, and for chronic low-dose-rate exposures, and there are gaps in the LET region between 25 and 190 keV/μm. In this study, we used the historical HG tumorigenesis data as reference, and obtained HG tumor data for 260 MeV/u silicon (LET ∼70 keV/μm) and 1,000 MeV/u titanium (LET ∼100 keV/μm) to fill existing gaps of data in this LET range to improve our understanding of the dose-response curve at low doses, to test for deviations from linearity and to provide RBE estimates. Animals were also exposed to five daily fractions of 0.026 or 0.052 Gy of 1,000 MeV/u titanium ions to simulate chronic exposure, and HG tumorigenesis from this fractionated study were compared to the results from single 0.13 or 0.26 Gy acute titanium exposures. Theoretical modeling of the data show that a nontargeted effect model provides a better fit than the targeted effect model, providing important information at space-relevant doses of heavy ions.

238Pu elimination profiles after delayed treatment with 3,4,3LI(1,2HOPO) in female and male Swiss-Webster mice

Abstract Purpose: To characterize the dose-dependent and sex-related efficacy of the hydroxypyridinonate decorporation agent 3,4,3-LI(1,2-HOPO) at enhancing plutonium elimination when post-exposure treatment is delayed. Materials and methods: Six parenteral dose levels of 3,4,3-LI(1,2-HOPO) from 1–300 μmol/kg were evaluated for decorporating plutonium in female and male Swiss-Webster mice administered a soluble citrate complex of 238Pu and treated 24 hours later. Necropsies were scheduled at four time-points (2, 4, 8, and 15 days post-contamination) for the female groups and at three time-points (2, 4, and 8 days post-contamination) for the male groups. Results: Elimination enhancement was dose-dependent in the 1–100 μmol/kg dose range at all necropsy time-points, with some significant reductions in full body and tissue content for both female and male animals. The highest dose level resulted in slight toxicity, with a short recovery period, which delayed excretion of the radionuclide. Conclusions: While differences were noted between the female and male cohorts in efficacy range and recovery times, all groups displayed sustained dose-dependent 238Pu elimination enhancement after delayed parenteral treatment with 3,4,3-LI(1,2-HOPO), the actinide decorporation agent under development.

Demand for Zn2+ in Acid-Secreting Gastric Mucosa and Its Requirement for Intracellular Ca2+

Background and Aims Recent work has suggested that Zn2+ plays a critical role in regulating acidity within the secretory compartments of isolated gastric glands. Here, we investigate the content, distribution and demand for Zn2+ in gastric mucosa under baseline conditions and its regulation during secretory stimulation. Methods and Findings Content and distribution of zinc were evaluated in sections of whole gastric mucosa using X-ray fluorescence microscopy. Significant stores of Zn2+ were identified in neural elements of the muscularis, glandular areas enriched in parietal cells, and apical regions of the surface epithelium. In in vivo studies, extraction of the low abundance isotope, 70Zn2+, from the circulation was demonstrated in samples of mucosal tissue 24 hours or 72 hours after infusion (250 µg/kg). In in vitro studies, uptake of 70Zn2+ from media was demonstrated in isolated rabbit gastric glands following exposure to concentrations as low as 10 nM. In additional studies, demand of individual gastric parietal cells for Zn2+ was monitored using the fluorescent zinc reporter, fluozin-3, by measuring increases in free intracellular concentrations of Zn2+ {[Zn2+]i} during exposure to standard extracellular concentrations of Zn2+ (10 µM) for standard intervals of time. Under resting conditions, demand for extracellular Zn2+ increased with exposure to secretagogues (forskolin, carbachol/histamine) and under conditions associated with increased intracellular Ca2+ {[Ca2+]i}. Uptake of Zn2+ was abolished following removal of extracellular Ca2+ or depletion of intracellular Ca2+ stores, suggesting that demand for extracellular Zn2+ increases and depends on influx of extracellular Ca2+. Conclusions This study is the first to characterize the content and distribution of Zn2+ in an organ of the gastrointestinal tract. Our findings offer the novel interpretation, that Ca2+ integrates basolateral demand for Zn2+ with stimulation of secretion of HCl into the lumen of the gastric gland. Similar connections may be detectable in other secretory cells and tissues.

Involvement of metals in Aβ aggregation in Alzheimer's disease brain and lens using X-ray Fluorescent Microscopy (XRFM) and Quasi-Elastic Light Scattering (QLS)

enriched tau proteins from the control and transgenic mice were immunoprecipitated with a monoclonal antibody against tau. The immuno-precipitants were then digested with trypsin. The phosphorylated peptides in the control and transgenic mouse samples were enriched once again by the CHT-based method. The phosphopeptides were then analyzed by MALDI-TOF mass spectrometry and the peptide mass profiles of tau protein were compared between the age-matched wild type and transgenic animals to examine the changes in tau protein phosphorylation. Conclusions: Our study demonstrated that CHT-based fractionation is an easy-to-use, fast and convenient method for phosphoprotein/peptide enrichment with high binding capacity. It could potentially be used to enrich highly-phophorylated proteins and facilitate the biochemical study of hyperphosphorylated tau in Alzheimer’s disease.

LDL RECEPTOR STATUS OF HUMAN GLIOBLASTOMA CELLS

Porphyrins are known to associate with plasma lipoproteins, particularly low density lipoproteins (LDLs), and are thus likely to be taken up into cells by the high affinity saturable LDL receptor mechanism.1 We have previously reported that the boronated protoporphyrin, BOPP, is incorporated into the human glioblastoma cell line, SF 767 (derived from a glioblastoma multiforme biopsy specimen obtained from a grade 4 malignant glioma), and requires the presence of lipoproteins for maximum uptake into cells.2 We concluded from these studies that, like other porphyrins, BOPP forms a complex with LDL before it is taken up into the SF 767 cells via the LDL receptor; however, the presence of saturable LDL receptors on SF 767 cells has not been previously demonstrated. We have therefore investigated whether the LDL receptor was present on exponentially growing SF 767 cells. We report here our initial results indicating relatively high levels of LDL-receptors per cell (over 200,000) from the SF-767 line.