Various deep, compact, sedimentary formations have been studied in recent years as potential host rock for a repository for high-level, long-lived radioactive waste. Considering that microbial activities may influence radionuclide chemistry and migration in such environments, we investigated the potential presence of microorganisms in the Opalinus Clay formation, from unperturbed sediment samples (i.e., not affected by gallery excavation and borehole drilling) recovered under aseptic conditions in the Mont Terri Underground Rock Laboratory (Switzerland). A combination of molecular biology techniques and a cultivation-based approach suggested the presence of a few sparse autochthonous microbial cells in the Opalinus Clay. For the first time, ribosomal RNA (rRNA) genes were sequenced from enrichment cultures from such samples. The results suggested that at least two of the bacterial strains isolated were likely unknown species of the Sphingomonas and Alicyclobacillus genera, as their fully-sequenced 16S-rRNA genes shared less than 97% similarity with validly published sequences. Early genetic divergence occurring after physical isolation of bacterial ancestors in the geosphere by the sedimentation process or following later geological events may have resulted in the generation of particular taxa in the subsurface.

Opalinus Clay is a candidate host rock for a high-level radioactive waste repository in Switzerland. Microbial metabolism and its by-products could affect the physical and (geo)chemical conditions in such a repository. This study investigated the occurrence of indigenous microbes, their community size and -structure in an Opalinus Clay core from the Mont Terri Underground Research Laboratory, Switzerland, drilled with aseptic techniques. Core sub-samples were distributed to five laboratories, where they were analysed with microscopy, culture-and molecular biology techniques. Evidence supporting a viable microbial community in Opalinus Clay included: five positive culture results (including for sulphate-reducing bacteria, SRB) out of 20 culture attempts; extraction of 64 ng phospholipid fatty acids (PLFA) per g clay, suggesting the presence of viable cells; detection of PLFA biomarkers for anaerobic Gram-negative bacteria and SRB; and the presence of sufficient nutrients to support growth of indigenous and non-indigenous microorganisms for two months. Evidence against a thriving microbial community in Opalinus Clay included: 15 negative culture results out of 20 attempts; lack of cells by application of microscope techniques ( phase contrast, AODC, CARD-FISH); consistent failure to extract PCR-amplifiable DNA from the core; the presence of 14 times higher amounts of lipids indicative of cell debris than those indicative of viable cells (PFLA); very small pore sizes; and very low water content. The combined results of this study (and evidence from other studies in comparable environments) suggest that unperturbed Opalinus Clay appears to contain only a small viable microbial community, which is probably metabolically almost inactive ( dormant), due to space and water restrictions. However, any disturbances that would provide space, water and nutrients, as would be the case during repository excavation and construction, could revive the dormant organisms, unless the host rock was kept intact.

Amongst all the fission fragments, actinides and activation products coming from the nuclear energy processes, a dozen radionuclides are a hazard for Man and environment, due to their half-life and toxicity. For the last 3.5-4 billion years (Ga), microorganisms have been present on Earth in all sorts of surroundings, even the most hostile ones, characterized by extreme pH, temperature and/or radioactivity. To grow and multiply, they developed enzymatically induced oxido-reduction reactions by coupling reduction of metals/metalloids with oxidation of organic matter, which, in turn, may change soluble forms of radionuclides or stable elements into non-soluble forms. When this happens, microorganisms contribute to fix the radionuclides and prevent dissemination.

Washed cell suspensions of the anaerobic hyperthermophilic archaea Thermococcus pacificus and Thermoproteus uzoniensis and the anaerobic thermophilic gram-positive bacteria Thermoterrabacterium ferrireducens and Tepidibacter thalassicus reduced technetium [ 99 Tc(VII)], supplied as soluble pertechnetate with molecular hydrogen as an electron donor, forming highly insoluble Tc(IV)-containing grayish-black precipitate. Apart from molecular hydrogen, T. ferrireducens reduced Tc(VII) with lactate, glycerol, and yeast extract as electron donors, and T. thalassicus reduced it with peptone. Scanning electron microscopy and X-ray microanalysis of cell suspensions of T. ferrireducens showed the presence of Tc-containing particles attached to the surfaces of non-lysed cells. This is the first report on the reduction in Tc(VII) by thermophilic microorganisms of the domain Bacteria and by archaea of the phylum Euryarchaeota .

The sorption of technetium by powdered and polished mineral stibnite Sb2S3 has been investigated in simulated and natural underground waters from the Meuse/Haute–Marne region (France). The sorption by powdered stibnite has been found to be complete under both aerobic and anaerobic conditions in batch experiments. The sorption rate is higher in the absence of oxygen than under aerobic condition. Increasing the temperature from 30 °C to 60 °C results in a rise of the sorption rate by 9.1 and 27 times under anaerobic and aerobic conditions, respectively. The observed differences in sorption kinetics in the presence and in absence of oxygen are explained by the interaction of oxygen with sulfide ion in aerobic conditions and by the reduction of technetium(VII) by iron(II) and by other impurities present in natural water and in the mineral, and by the subsequent sorption of Tc(IV) on stibnite under anaerobic conditions. The sorption on a polished mineral surface resulted in the formation of a technetium film, probably Tc2S7, with a thickness of 1−3 μg Tc/cm2 at pH 3−6 and 4−12 μg Tc/cm2 at 9−12. The simultaneous formation of stibnite colloids with adsorbed technetium occurs at pH 9−12. The study of the technetium film on the mineral by proton induced X-ray emission analysis showed it to be at least one order of magnitude thinner on the SiO2 impurities than on the main Sb2S3 component and the iron impurities.

References 1. Finlay EMIl, Gaskell SJ. Determination of testosterone in plasma from men by gas chromatography/mass spectrometry with high-resolution selected-ion monitoring and metastable peak monitoring. Clin Chem 27, 1165-1170 (1981). 2. Gaskell SJ, Collins CJ, Thorne CC, Groom GV. External quality assessment of assays for cortisol in plasma: Use of target data obtained by gas chromatography/mass spectrometry. Clin Chem 29, 862-867 (1983). 3. Nocke-Finck L, Rohie G, Siekmann L, Breuer H. Collaborative surveys for steroidhormones in the Federal Republic of Germany. In Quality Control in Clinical Endocrinology, DW Wilson, SJ Gaskell, K Kemp, Eds., Alpha Omega Publishing, Cardifi U.K., 1981,pp 175-182. 4. Lantto 0, BjOrkhem I, Blomstrand R, Kallner A. Interlaboratoiy evaluation of four RIA kits for determination of plasma cortisol,

A high resolution inductively coupled plasma mass spectrometric (HR-ICP-MS) method for the determination of plutonium isotopes, Am and the 240Pu/239Pu isotope ratio utilising modification of Pu-02-RC Plutonium in Soil Samples, Pu-03-RC Plutonium in Soil Residue—Total Dissolution Method, Pu-11-RC Plutonium Purification—Ion Exchange Technique, Pu-12-RC Plutonium and/or Americium in Soil or Sediments, HASL-300 was developed. Total plutonium concentrations (239+240Pu) measured in environmental samples by this HR-ICP-MS method were in good agreement with recommended data obtained from a-spectrometry. It was achieved the decreasing of the time to analyze the samples over than 33%.

Leaves of plants have the ability to accumulate the long-lived fission product (99)Tc. In the present work, an attempt was made to separate and characterize technetium species formed in maize grown on soil contaminated with Tc(VII)O(4)(-) solution. Data obtained from selective extraction, a Phosphorimager and liquid scintillation were employed.

It is shown for the first time that haloalkaliphilic bacteria, isolated from soda-lake environments were capable of reducing Tc(VII)O4− to the Tc(V), Tc(IV) and Tc(III) at pH 10 in carbonate medium, whereas no reduction took place without bacteria or in the presence of dead biomass. After 34 h of incubation, 55% remained as Tc(VII), 36% was found as Tc(IV) and 8% as Tc(V) and after 2 months 80% of the technetium was reduced. Technetium has a toxic effect on bacteria. Reduction of TcO4− was drastically decreased at concentration above 1.5 mM. The microbial reduction has been suggested as a potential mechanism for the removal of Tc from contaminated environments or waste streams.

The ability of bacteria to reduce pertechnetate in alkaline conditions was investigated using halophilic bacteria isolated from soda-lakes environments. Anaerobic halophilic bacteria were able to reduce as much as 0.25 mM pertechnetate, whereas no reduction took place without bacteria or in the presence of heat-killed bacteria. The results obtained showed reduction of Tc(VII)O4 − to the Tc(V) and Tc(IV) at pH 10 in the carbonate-bicarbonate medium. About 57% of the total technetium was determined to be Tc(IV), 1–3% as a Tc(V) and 17–20% as a Tc(VII) after 1–3 days of incubation with bacteria. The microbial reduction of Tc(VII) in alkaline conditions has been suggested as a potential mechanism for the removal of Tc from contaminated environments or waste streams.

Neuroblastoma is a severe cancer form in early childhood. N-myc amplification is causally involved in the progression of the disease and associated with advanced stages of malignancy. Available metals, including iron, copper and zinc, may have some role in promoting tumor cell growth. In previous studies, we showed that a relationship exists between intracellular trace metal concentrations of cultured neuroblasts and the N-myc oncogene amplification rate. We already published preliminary results on determinations of trace metal contents in tumors xenografts developed after injection of cells from the same cell lines in athymic nude mice. In order to confirm our previous results, Fe, Cu and Zn concentrations have been measured in two other human neuroblastoma cell lines characterized by different degrees of N-myc amplification as well as in the corresponding tumors. One of them is a non-amplified cell line transfected with N-myc gene. This complete study allows a direct comparison of intracellular trace metal contents in vitro and in vivo and confirms the relationship already established between Fe, Cu and Zn contents and the degree of N-myc oncogene amplification in neuroblastoma.

Iron is an essential micronutrient required for cell division and growth. Incorporation of iron into cells is achieved by endocytosis of transferrin. Then, iron may be stored in ferritin and hemosiderin. Increased intracellular iron concentrations may promote malignant cell growth. Patients with advanced-stage neuroblastoma (NB) show abnormally high levels of serum ferritin, very likely synthesized and secreted by the tumor in vivo and consistent with a frequent accumulation of iron in ferritin in NB tumor tissues. In a previous study, we showed that there is no iron accumulation in cultured neuroblasts, and intracellular iron concentrations proved to be especially low. Bio-Normalizer® (BioN) is a nutritional supplement sold to be an anti-oxidant and metal-chelator. In the present study, we tested cell viability and measured iron concentrations in neuroblasts treated or not with BioN. We found that BioN, probably thanks to papain, presents an anti-proliferative effect on NB cultured cells. Besides, preliminary results tend to prove that this natural anti-oxidant and iron-chelator could present interesting effects on trace metal concentrations in neuroblasts. Such a compound may be useful in treatment of this pathology.

The retina as well as other tissues needs iron to survive, but modifications in iron metabolism have also been suggested to contribute to cerebral neurodegenerative diseases. Our study was intended to investigate iron distribution in the retina of normal rats and Royal College of Surgeons (RCS) rats affected by hereditary degeneration of the photoreceptors at different developmental stages (35, 45 and 55 days after birth).Iron (Fe) distribution was determined by proton induced X-ray emission (PIXE) microanalysis on retinal sections and compared to other tissues (cornea, liver, spleen) and to other elements (Cu, Zn, Ca). Elemental concentrations were determined in different retinal layers especially the photoreceptors, which are progressively altered and disappear in the RCS rats.Iron is unevenly distributed throughout the rat retina. The highest concentration is observed in the choroid and the retinal pigmented epithelium and in the inner segments of photoreceptors. Iron content is lower in the outer segments but still significant. It increases during both the development and the disease at the level of the segments. This last localised iron increase can result in an overproduction of free radicals and be correlated with the photoreceptor cell loss. The distributions of other elements (Cu, Zn, Ca) revealed interesting temporal progressions. (C) 2001 Elsevier Science B.V. All rights reserved.

N-myc oncogene amplification is one of the most established prognostic factors in neuroblastoma (NB), a young children solid tumor. Amounts of ferritin, an iron storage protein, are abnormally increased in serum of patients with advanced stage disease. N-myc amplified NB cells can synthesize zinc metalloenzymes allowing tumor invasion and metastases formation. The aim of this study was to find a relationship between N-myc amplification and trace metals in human neuroblasts. Coupling PIXE and RBS techniques, nuclear microprobe allowed to analyze elemental distributions and to determine trace metal concentrations within cultured neuroblasts characterized by various degrees of N-myc amplification. They were compared to trace metal distributions and concentrations in tumor xenograft models of human NB, after injection of cells from the same lines in athymic nude mice. Our data allowed to establish a relation between trace metal contents and mechanisms of NB oncogenesis, amplified cell lines representing more aggressive phenotypes of the disease. They should be confirmed by analysis of cultured neuroblasts and tumors issued from a non-amplified cell line transfected with the N-myc oncogene.

N-myc oncogene amplification is a powerful predictor of aggressive behavior of neuroblastoma (NB), the most common solid tumor of the early childhood. Since N-myc overexpression – subsequent to amplification – determines a phenotype of invasiveness and metastatic spreading, it is assumed that N-myc amplified neuroblasts synthesize zinc metalloenzymes leading to tumor invasion and formation of metastases. In order to test a possible relation between N-myc oncogene amplification and trace metal contents in human NB cells, Fe, Cu and Zn concentrations have been measured by nuclear microprobe analysis in three human neuroblastoma cell lines with various degrees of N-myc amplification. Elemental determinations show uniform distribution of trace metals within the cells, but variations of intracellular trace metal concentrations with respect to the degree of N-myc amplification are highly dependent on the nature of the element. Zinc concentration is higher in both N-myc amplified cell lines (IMR-32 and IGR-N-91) than in the non-amplified cells (SK–N–SH). In contrast, intracellular iron content is particularly low in N-myc amplified cell lines. Moreover, copper concentrations showed an increase with the degree of N-myc amplification. These results indicate that a relationship exists between intracellular trace metals and N-myc oncogene amplification. They further suggest that trace metals very probably play a determinant role in mechanisms of the neuroblastoma invasiveness.

The effect of extracellular adhesion factors, and of distinct culture substrata, on trace element content of epithelial cancer cells was investigated using nuclear microprobe analysis. Essential trace metal concentrations in human carcinoma cells cultured either onto tissue culture plastic dishes, onto microanalysis supports made of thin Formvar surfaces coated or not with gelatin gel, or onto polycarbonate films, were compared. Abnormally high copper concentrations were noticed specifically in cisplatin-resistant epithelial cancer cells cultured onto Formvar or polycarbonate surfaces with or without reconstituted extracellular matrix. Manganese, iron and zinc concentrations remained unchanged in these cells. These results indicate that (1) metal uptake of cells cultured onto substrata used for X-ray microanalysis can be different to that of cells cultured onto usual plastic dishes, (2) cisplatin-resistant epithelial cancer cells specifically accumulate copper when cultured onto thin Formvar or polycarbonate films, and could be used as in vitro models for copper overload studies. (C) 1999 Elsevier Science B.V. All rights reserved.

Several types of hereditary retinal degeneration with progressive alteration of photoreceptors exist in men and animals. Recent immunohistochemical results have shown strong degradation of transferrin, the protein responsible for iron transport, in retinas of rats with hereditary retinal degeneration. Freeze-dried thin sections of rat retinas from different stages of the disease, and respective coeval control sections, have been analyzed using nuclear microprobe. In this first part of the study, the rat retinas at post-natal stages of 35 and 45 days have been analyzed. The sample preparation and the post-irradiation staining to determine precisely the retinal layers involved are described. Preliminary results of element distributions (K, Ca, Fe) in the rat retina layers are discussed. A very high content of calcium in the choriocapillaris of dystrophic rat retinas was observed. Preliminary results on iron distribution in the rat retina layers are presented.

In this study, we performed nuclear microprobe analysis on cultured human ovarian cancer cells exposed to pharmacological concentrations of 4'-iodo-4'-deoxydoxorubicin (IDX), an anthracycline anticancer drug. We observed that iodine and iron cellular distributions were strongly correlated, suggesting intracellular iron chelation by the anthracycline. The average cellular iron concentration did not change during drug exposure, but the cellular distribution of iron was modified following the preferential nuclear localization of iodine, as determined by single cell microanalysis. These results are important for understanding the cellular pharmacology of anthracyclines. They suggest that iron cellular delocalization and its subsequent nuclear accumulation may participate to the overall cytotoxicity of IDX, and more generally to anthracycline antitumor activity. (C) 1997 Elsevier Science B.V.