Disrupted Iron Storage in Dental Fluorosis.

Enamel formation and quality are dependent on environmental conditions, including exposure to fluoride, which is a widespread natural element. Fluoride is routinely used to prevent caries. However, when absorbed in excess, fluoride may also lead to altered enamel structural properties associated with enamel gene expression modulations. As iron plays a determinant role in enamel quality, the aim of our study was to evaluate the iron metabolism in dental epithelial cells and forming enamel of mice exposed to fluoride, as well as its putative relation with enamel mechanical properties. Iron storage was investigated in dental epithelial cells with Perl's blue staining and secondary ion mass spectrometry imaging. Iron was mainly stored by maturation-stage ameloblasts involved in terminal enamel mineralization. Iron storage was drastically reduced by fluoride. Among the proteins involved in iron metabolism, ferritin heavy chain (Fth), in charge of iron storage, appeared as the preferential target of fluoride according to quantitative real-time polymerase chain reaction, Western blotting, and immunohistochemistry analyses. Fluorotic enamel presented a decreased quantity of iron oxides attested by electron spin resonance technique, altered mechanical properties measured by nanoindentation, and ultrastructural defects analyzed by scanning electron microscopy and energy dispersive x-ray spectroscopy. The in vivo functional role of Fth was illustrated with Fth+/- mice, which incorporated less iron into their dental epithelium and exhibited poor enamel quality. These data demonstrate that exposure to excessive fluoride decreases ameloblast iron storage, which contributes to the defective structural and mechanical properties in rodent fluorotic enamel. They raise the question of fluoride's effects on iron storage in other cells and organs that may contribute to its effects on population health.

Mammalian cells loaded with platinum-containing molecules are sensitized to fast atomic ions.

PURPOSE: This work investigates whether a synergy in cell death induction exists in combining atomic ions irradiation and addition of platinum salts. Such a synergy could be of interest in view of new cancer therapy protocol based on atomic ions--hadrontherapy--with the addition of radiosensitizing agents containing high-Z atoms. The experiment consists in irradiating by fast ions cultured cells previously exposed to dichloroterpyridine Platinum (PtTC) and analyzing cell survival by a colony-forming assay. MATERIALS AND METHODS: Chinese Hamster Ovary (CHO) cells were incubated for six hours in medium containing 350 microM PtTC, and then irradiated by fast ions C(6+) and He(2+), with Linear Energy Transfer (LET) within range 2-70 keV/microm. In some experiments, dimethyl sulfoxide (DMSO) was added to investigate the role of free radicals. The intracellular localization of platinum was determined by Nano Secondary Ion Mass Spectroscopy (Nano-SIMS). RESULTS: For all LET examined, cell death rate is largely enhanced when irradiating in presence of PtTC. At fixed irradiation dose, cell death rate increases with increasing LET, while the platinum relative effect is larger at low LET. CONCLUSION: This finding suggests that hadrontherapy or protontherapy therapeutic index could be improved by combining irradiation procedure with concomitant chemotherapy protocols using platinum salts.

Kinetics of intracolloidal iodine in thyroid of iodine-deficient or equilibrated newborn rats. Direct imaging using secondary ion mass spectrometry.

The most significant impact of the Chernobyl accident is the increased incidence of thyroid cancers among children. In order to accurately estimate the radiation dose provided by radioiodines, it is important to examine how the distribution of newly incorporated iodine varies with time and if this distribution varies according to the iodine status. The kinetic distribution of intra colloidal newly organified iodine in the rat immature thyroid was recorded and analysed using the ionic nanoprobe NanoSims50. Our observations imply that in case of radioiodine contamination, the energy deposits vary (i) with time, (ii) from one follicle to another, and (iii) from one cell to another inside the same follicle regardless the iodine status. The kinetic heterogeneity of iodine distribution must be take in account in thyroid dose evaluation.

In vitro phototoxicity of glycoconjugated porphyrins and chlorins in colorectal adenocarcinoma (HT29) and retinoblastoma (Y79) cell lines.

BACKGROUND: Retinoblastoma is the most common malignant intraocular tumor in children. The current treatment gives a good vital prognostic but there are several drawbacks to the arsenal of "classical antitumoral" therapies. Photodynamic therapy (PDT) could be an exciting non-toxic and non-mutagenic alternative protocol. METHOD: In this paper, we report about the screening of the in vitro photocytotoxicity of hydrophenylporphyrins and chlorins and their glycoconjugated derivatives in a human retinoblastoma cell line (Y79) and for comparison in a colorectal adenocarcinoma cell line (HT29). RESULTS: Despite lower photodynamic activity than that observed for hydroxylated photosensitizers, in particular Foscan(®) glycoconjugated derivatives display phototoxicity (IC50 2.4-0.05µM ±10%) against Y79 cells with examples of significant intrinsic cytotoxicity. Amongst them the triglucosyl porphyrin 10 is highly photocytotoxic (IC50 0.9µM ±10%) but is fully devoid of cytotoxicity (IC50>15µM). The photoactivity is highly modulated by the presence of a diethyleneglycol spacer between the chromophore and the glycoside (compounds 14-17, IC50 0.5, 0.6, 0.05 and 0.35µM ±10%) and by the anomeric configuration of the sugar (compound 15 and 17, IC50 0.6 and 0.05µM ±10% respectively). One of the main problems for the use of Foscan(®) is its poor solubility which might be improved by glycoconjugation. Moreover Foscan has been shown to induce necrosis after PDT leading to a possible ulceration of surrounding tissues unsuitable for a conservative treatment. A preferential mitochondrial subcellular localization which has been previously reported for some glycoconjugated photosensitizers could enhance the contribution of apoptosis process. CONCLUSION: Tri-α-O-galactosyl porphyrin 16 is a better candidate than Foscan(®) for a clinical application of PDT for a conservative therapy of retinoblastoma.

pH dependence of 5-fluorouracil uptake observed by in vivo 31P and 19F nuclear magnetic resonance spectroscopy.

Multinuclear nuclear magnetic resonance spectroscopy was used to follow the metabolism and kinetics of 5-fluorouracil (5FU) after i.v. administration at a dose of 100 mg/kg on Wistar rats. 31P spectra allow one to determine both the energetic status and the pH of the tissues under investigation, while serial 19F spectra reveal the drug clearance. Analyses of 5FU kinetics show that the half-life of 5FU elimination is about 35 min in tissue with a pH of 7.3. However, this half-life increases 2.5-fold when the local pH decreases below 6.9. Thus, acidification seems to induce a local retention of 5FU, which tends to prove the existence of active transport. This retention of the drug may have significant clinical implications for assessing and improving chemotherapy alone or in combination.

Follow-up by 31P NMR spectroscopy of the energy metabolism of malignant tumor in rats during treatment.

The energy metabolism of tumors in rats was investigated by in vivo 31P-NMR spectroscopy. The effects of radiotherapy, chemotherapy or radiotherapy combined with 5-fluorouracil (5-FU) chemotherapy were evaluated by observing the changes of these spectra in chemically induced subcutaneous fibrosarcoma in rats. Two milligrams of DMBA in solution in olive oil were administered subcutaneously in the flank of 20 Wistar rats and 17 fibrosarcoma occurred. 31P NMR spectra were recorded with a Brüker Medspec 30/47 spectrometer using a surface coil positioned over the tumor. We did not observe significant changes in the spectra during tumor growth. Radiotherapy and 5-FU chemotherapy alone did not induce major changes in the 31P spectra. But the situation was completely different for animals receiving the therapeutic combination. A clear increase in the ratio of inorganic phosphate to total phosphorus signal was observed 48 h after the first irradiation session. The pH shifted concurrently to the acidic range. No effect on tumor regression was observed in the rats from the chemotherapy group, while regression was less than 50% in rats treated by irradiation only, and at least 80% in the combined group.

Complementary advantages of fluorescence and SIMS microscopies in the study of cellular localization of two new antitumor drugs.

Low light level fluorescence microscopy studies have been carried out on MCF7-P human mammary tumor cells to localize the intracellular distribution of two new anticancer drugs, Pazelliptine and Intoplicine, which are currently under clinical evaluation. These two molecules are thought to act at the nuclear level, through DNA topoisomerase interactions. Because fluorescence of these compounds appears strongly quenched by intercalation in double strand DNA, secondary ion mass spectrometry (SIMS) imaging was used to check the presence of the drugs in the nuclear compartment. In spite of chemical structure similitudes, pazelliptine and intoplicine appear to be distributed in quite different ways within the cells. Incubation for 1 and 24 hours also allowed us to bring to light strong differences in the distribution kinetics. Pazelliptine quickly enters into the nucleoli but is no longer present in the nucleus after 24 hours incubation. Intoplicine was not detected by fluorescence in the nucleus, however SIMS microscopy allowed us to show its accumulation within this cellular compartment as a function of time of exposure. This study shows the complementarity of fluorescence and SIMS microscopies.

Energy deposition patterns in an amputated human lower leg heated with a miniannular phased array.

The energy deposition patterns in both alcohol-fixed and unfixed amputated human lower legs produced by a miniannular phased array (MAPA) applicator have been determined. The nontumor bearing portions of four human legs, amputated for therapeutic purposes, were heated within the MAPA. Experimental measurements of the time rate of temperature rise at many locations inside the leg (between 125 and 150) were transformed to specific absorption rate (SAR) values at each point. A simple model was developed which predicts the axial variations in SAR inside the heated limb based upon quantitative details of the leg's geometry obtained from computerized tomography scans. The axial location of the region of maximum energy deposition was predicted by the model with a precision of approximately 1 to 2 cm. Significant time rate of temperature rise was measured inside the cortical portion of the tibia, while the temperature rise in the cancellous (marrow) portion of the tibia was negligible. The alcohol fixation process appears to have no significant effect on the energy deposition patterns within the various leg tissues.

Experimental characterization of the miniannular phased array as a hyperthermia applicator.

A series of experiments has been carried out in order to characterize a miniannular phased array applicator prior to possible clinical implementation. The energy deposition patterns over the frequency range of 100 to 200 MHz were determined in several human limb models of different complexities by measuring the electric field strength patterns. The point of maximum energy deposition within a homogeneous, muscle-equivalent cylindrical phantom positioned coaxially within the MAPA was found to be at the center of the applicator. The energy deposition patterns seem to be more uniform at the lower frequencies. Inclusion of a cylindrical bone-equivalent phantom positioned coaxially with this muscle-equivalent phantom does not seem to significantly alter the energy deposition patterns in the muscle-equivalent region. For more realistically shaped, homogeneous muscle-equivalent limb models, the resulting energy deposition patterns appear to be confined mostly to the intended treatment region. However, the point of maximum energy deposition was not at the middle of the applicator as with the cylindrical model, but shifted towards a smaller cross-sectional region. This shift in location of the point of maximum energy deposition varies with the location of the MAPA on the limb. A secondary region of high-field strength was also observed at the ankle for a MAPA centered about the knee. In this study, the energy deposition patterns appear to be significantly dependent on the shape of the model. Therefore, this factor must be taken into consideration for the proper prediction and control of the heating patterns resulting from the use of this type of applicator for clinical hyperthermia treatment.

Human leg heating using a mini-annular phased array.

The energy deposition pattern within an isolated human leg heated with a mini-annular phased array (MAPA) hyperthermia applicator has been determined. The non-tumor-bearing lower portion of a human leg amputated at the hip due to the presence of a large tumor in the thigh was "fixed" in a 50% ethanol in 0.9% saline solution. Subsequent to this fixation process, the leg was rehydrated in 0.9% saline and heated four times using a MAPA operating at 122 MHz. Specific absorption rates and electric field strengths were calculated from the rates of change of temperature with time measured at 143 different anatomical locations within the leg. When the leg was coaxial with the MAPA and the MAPA was axially positioned midway between the knee and the ankle, the points of maximum heating were skewed away from the center of the MAPA, towards the ankle of the leg and along the central axis of the MAPA. Significant temperature rise was measured inside the bone and the fat as well as inside the muscle of the leg. Bone heating was reduced when the leg was shifted away from the MAPA axis.

[Microwave hyperthermia: influence of blood flow and thermoregulation processes (author's transl)]. / Hyperthermie micro-ondes: influence du flux sanguin et des phénomènes thermorégulateurs.

Various problems are encountered during production of local, deep-seated microwave hyperthermia, involving the technology of the irradiating system, and the complexity of heat transfer in living tissues. Before starting investigations on patients, preliminary studies were conducted on different models. Taking the influence of the blood flow into account, the thermal effects of microwaves were simulated on a numerical model, and a perfused phantom. These studies were completed by investigations in animals. The analysis of findings demonstrates that, in given conditions of irradiation, the temperature distribution is strongly dependent on blood flow. This means that the phantom models are only useful to evaluate the influence of the irradiation parameters and to develop and compare the generator-applicator systems, and that accurate planning of therapeutic trials requires in vivo studies on animals as well as on patients.

Cyanobacterial diversity and activity in modern conical microbialites.

Modern conical microbialites are similar to some ancient conical stromatolites, but growth, behavior and diversity of cyanobacteria in modern conical microbialites remain poorly characterized. Here, we analyze the diversity of cyanobacterial 16S rRNA gene sequences in conical microbialites from 14 ponds fed by four thermal sources in Yellowstone National Park and compare cyanobacterial activity in the tips of cones and in the surrounding topographic lows (mats), respectively, by high-resolution mapping of labeled carbon. Cones and adjacent mats contain similar 16S rRNA gene sequences from genetically distinct clusters of filamentous, non-heterocystous cyanobacteria from Subsection III and unicellular cyanobacteria from Subsection I. These sequences vary among different ponds and between two sampling years, suggesting that coniform mats through time and space contain a number of cyanobacteria capable of vertical aggregation, filamentous cyanobacteria incapable of initiating cone formation and unicellular cyanobacteria. Unicellular cyanobacteria are more diverse in topographic lows, where some of these organisms respond to nutrient pulses more rapidly than thin filamentous cyanobacteria. The densest active cyanobacteria are found below the upper 50 µm of the cone tip, whereas cyanobacterial cells in mats are less dense, and are more commonly degraded or encrusted by silica. These spatial differences in cellular activity and density within macroscopic coniform mats imply a strong role for diffusion limitation in the development and the persistence of the conical shape. Similar mechanisms may have controlled the growth, morphology and persistence of small coniform stromatolites in shallow, quiet environments throughout geologic history.

Study of the localization of iron, ferritin, and hemosiderin in Alzheimer's disease hippocampus by analytical microscopy at the subcellular level.

Previous studies of the structure of core nanocrystals of ferritin (Ft) in the brains of patients with Alzheimer's disease (AD) have shown differences in the mineral compound in comparison with physiological Ft. Both Ft cores have a polyphasic composition but whereas the major phase in physiological Ft is hexagonal ferric iron oxide (ferrihydrite), the major phases in brain AD Ft are two cubic mixed ferric-ferrous iron oxides (magnetite and wüstite). One of these (wüstite) is similar to what is detected in hemosiderin (Hm) cores in primary hemochromatosis (Quintana, C., Cowley, J.M, Marhic, C., 2004. Electron nanodiffraction and high resolution electron microscopy studies of the structure and composition of physiological and pathological ferritin. J. Struct. Biol. 147, 166-178). We have studied, herein, the distribution of iron, Ft, and Hm in sections of AD hippocampus using analytical microscopy. Iron present in Ft cores was directly mapped in a nanoSIMS microscope and the iron distribution has been correlated with the constituent elements N, P, and S. Ft and Hm cores were visualized at an ultrastructural level in an analytical transmission electron microscope. In senile plaques, Ft was observed in the coronal region associated with a non-beta-amyloid component and in the periphery of plaques, together with Hm, in sulfur-rich dense bodies of dystrophic neurites. Hm was also found in lysosomes and siderosomes of glial cells. Ft was observed in the cytoplasm and nucleus of oligodendrocytes. Ft was particularly abundant in myelinated axons in association with oligodendrocyte processes. These findings provide new arguments to support the hypothesis of a dysfunction of Ft (with eventual degradation to Hm) in AD resulting in an increase of toxic brain ferrous ions that may contribute to the production of free radicals that induce both cellular oxidative stress and aged-related myelin breakdown associated with cognitive decline and AD (Bartzokis, G., 2004. Age-related myelin breakdown: a developmental model of cognitive decline and Alzheimer's disease. Neurobiol. Aging 25, 5-18).

Active microwave tomographic imaging of isolated, perfused animal organs.

The relative transparency of biological materials to high-frequency electromagnetic waves has encouraged the development of new systems for imaging. This report describes experiments of microwave tomography conducted on a prototype. The object to be analyzed is submerged in water and is illuminated by a plane wave. The total electric field is analyzed by a microwave camera. The recorded data are then processed numerically in order to reconstruct the image that corresponds to the distribution of equivalent currents in a defined plane of a section. Experiments have been conducted on isolated kidneys with and without perfusion. The influence of the perfusing solution temperature has also been studied. These experiments show the potential of this system, especially through the correlation between microwave images and the biological structures. They also confirm previous results concerning spatial resolution and depth of exploration. Finally, the results demonstrate the influence of temperature and support the applicability of this imaging system in non-invasive thermometry, especially for clinical hyperthermia.

13C-NMR studies of Corynebacterium melassecola metabolic pathways.

Coryneform bacteria are widely used to produce amino acids, in particularly glutamic acid, by fermentation. To study the metabolic fate of glucose as the carbon source, we developed a method to analyze intracellular extracts by NMR and HPLC. The intracellular metabolites represent the metabolic state of the cells. Glutamic acid was the major metabolic intermediate found in the extracts and its 13C isotopic enrichment reflected that of pyruvic acid. Thus, it was possible to determine the respective contributions of the two major glucose catabolic pathways during the exponential growth phase; glycolysis (55%) and the pentose phosphate pathway (45%). Absolute glutamate 13C enrichments resulting from the incorporation of [1-13C]glucose were determined to quantify the contribution of several metabolic pathways such as anaplerotic pathways (61%; phosphoenolpyruvate carboxylase, pyruvate carboxylase, malic enzyme), a single turn (32%) or multiple turns of the Krebs cycle and the glyoxylate shunt, to oxaloacetate synthesis. A previously described model was adapted to C. melassecola for these calculations. The Krebs cycle was active, whereas the glyoxylate shunt was inactive in exponentially growing cells of C. melassecola with glucose as the sole carbon source. The contributions of anaplerotic enzymes and pyruvate dehydrogenase to replenishing the Krebs' cycle were determined to be 38% and 62%, respectively.

Carbon-flux distribution in the central metabolic pathways of Corynebacterium glutamicum during growth on fructose.

Growth of Corynebacterium glutamicum on fructose was significantly less than that obtained on glucose, despite similar rates of substrate uptake. This was in part due to the production of overflow metabolites (dihydroxyacetone and lactate) but also to the increased production of CO2 during growth on fructose. These differences in carbon-metabolite accumulation are indicative of a different pattern of carbon-flux distribution through the central metabolic pathways. Growth on glucose has been previously shown to involve a high flux (> 50% of total glucose consumption) via the pentose pathway to generate anabolic reducing equivalents. NMR analysis of carbon-isotope distribution patterns of the glutamate pool after growth on 1-13C- or 6-13C-enriched fructose indicates that the contribution of the pentose pathway is significantly diminished during exponential growth on fructose with glycolysis being the predominant pathway (80% of total fructose consumption). The increased flux through glycolysis during growth on fructose is associated with an increased NADH/NAD+ ratio susceptible to inhibit both glyceraldehyde-3-phosphate dehydrogenase and pyruvate dehydrogenase, and provoking the overflow of metabolites derived from the substrates of these two enzymes. The biomass yield observed experimentally is higher than can be estimated from the apparent quantity of NADPH associated with the pentose pathway and the flux through isocitrate dehydrogenase, suggesting an additional reaction yielding NADPH. This may involve a modified tricarboxylic acid cycle involving malic enzyme, expressed to significantly higher levels during growth on fructose than on glucose, and a pyruvate carboxylating anaplerotic enzyme.

Direct in vivo observation of 5-fluorouracil release from a prodrug in human tumors heterotransplanted in nude mice: a magnetic resonance study.

A glucuro-conjugated carbamate derivative of 5-fluorouracil (5-FU), originally designed as a prodrug for antibody-directed enzyme prodrug therapy (ADEPT) application, has been used for direct in vivo observation of in situ 5-FU generation in two human colon tumors heterotransplanted in nude mice. Because of the very fast elimination of glucuro-conjugated drugs, this observation required intratumoral injection. These tumors, when becoming necrotic, are rich enough in beta-glucuronidase to allow (19)F magnetic resonance spectroscopy monitoring, at the tumor level, of both prodrug elimination and 5-FU liberation without preliminary treatment by a specifically targeted enzyme conjugate. Convenient tumors have been selected by magnetic resonance imaging (MRI) on the basis of a correlative study between MRI and conventional histology. This contribution is the first report evidencing such a direct intra-tumoral conversion of a glucuro-conjugated prodrug into the expected active drug. This method, which should allow overall estimation of the beta-glucuronidase content of tumors, might also be helpful for selecting tumors as specific targets for non-toxic glucuro-conjugated prodrugs without prior treatment with a fusion protein.

Towards a transgenic mouse model of sickle cell disease: hemoglobin SAD.

In order to obtain a transgenic mouse model of sickle cell disease, we have synthesized a novel human beta-globin gene, beta SAD, designed to increase the polymerization of the transgenic human hemoglobin S (Hb S) in vivo. beta SAD (beta S-Antilles-D Punjab) includes the beta 6Val substitution of the beta S chain, as well as two other mutations, Antilles (beta 23Ile) and D Punjab (beta 121Gln) each of which promotes the polymerization of Hb S in human. The beta SAD gene and the human alpha 2-globin gene, each linked to the beta-globin locus control region (LCR) were co-introduced into the mouse germ line. In one of the five transgenic lines obtained, SAD-1, red blood cells contained 19% human Hb SAD (alpha 2 human 1 beta 2SAD) and mouse-human hybrids in addition to mouse hemoglobin. Adult SAD-1 transgenic mice were not anemic but had some abnormal features of erythrocytes and slightly enlarged spleens. Their erythrocytes displayed sickling upon deoxygenation in vitro. SAD-1 neonates were anemic and many did not survive. In order to generate adult mice with a more severe sickle cell syndrome, crosses between the SAD progeny and homozygous for beta-thalassemic mice were performed. Hemoglobin SAD was increased to 26% in beta-thal/SAD-1 mice which exhibited: (i) abnormal erythrocytes with regard to shape and density; (ii) an enlarged spleen and a high reticulocyte count indicating an increased erythropoiesis; (iii) mortality upon hypoxia; (iv) polymerization of hemolysate similar to that obtained in human homozygous sickle cell disease; and (v) anemia and mortality during development.