biological-samples-preparation-for-speciation-at-cryogenic

Biological Samples Preparation for Speciation at Cryogenic Temperature using High-Resolution  X-Ray Absorption Spectroscopy

The study of elements with X-ray absorption spectroscopy (XAS) is of particular interest when studying the role of metals in biological systems. Sample ...

cell-culture-on-silicon-nitride-membranes-cryopreparation-for

Cell Culture on Silicon Nitride Membranes and Cryopreparation for Synchrotron X-ray Fluorescence Nano-analysis

Very little is known about the distribution of metal ions at the subcellular level. However, those chemical elements have essential regulatory functions ...

INSERM UA7, Synchrotron Radiation for Biomedicine (STROBE)

ESRF, The European Synchrotron

Université Grenoble Alpes

Bisphosphonates, potent inhibitors of bone resorption, have been used clinically to correct the continued loss of bone mass in osteoporosis and in other conditions. However, there has been some concern that long-term treatment with these compounds, as well as more recently developed drugs, may also decrease the rate of bone formation. Bisphosphonates, which are strongly bound to hydroxyapatite crystals, may alter the structure and reactivity of the crystals, interfere with new crystal nucleation and growth, as well as alter the short-range order of newly formed crystals. We have investigated the chemistry and structure of the solid calcium-phosphate mineral phase of lumbar vertebrae of ovariectomized, 6.5-month-old rats treated with bisphosphonates for 1 year after onset of osteopenia. Appropriate control groups were used for comparison. The techniques used to assess the mineral phase were chemical analyses, Fourier transform-infrared (FT-IR) and FT-Raman spectroscopy, FT-IR microspectroscopy, and phosphorus-31 magic-angle-sample spinning nuclear magnetic resonance spectroscopy ((31)P MAS NMR). The (31)P MAS NMR spectra of trabecular bone of lumbar vertebrae of control, ovariectomized, and treated animals were similar. However, there were several significant differences in the results obtained by FT-IR spectroscopy of the whole tissue samples, FT-IR microspectroscopy of sections of bone, and chemical analyses. For example, whereas chemical analyses demonstrated that the CO(3) content of the mineral phase of the ovariectomized animals was decreased compared with controls, FT-IR microspectroscopy of bone sections showed no changes in the relative CO(3) content, but some changes in the environment of the CO(3) groups. However, chemical analyses of the crystals, combined with data from all three spectroscopic methods and with data from serum analysis, did indicate small changes in the mineral phase after ovariectomy, corrected after treatment with bisphosphonates. In any event, the chemical and structural data in the present studies demonstrate that the bisphosphonate, tiludronate, does not significantly alter the mineral components of bone after 1 year of treatment during the course of which bone loss was reversed.

Characterization of the trabecular rat bone mineral: effect of ovariectomy and bisphosphonate treatment.

Very little is known about the sub-cellular distribution of metal ions in cells. Some metals such as zinc, copper and iron are essential and play an important role in the cell metabolism. Dysfunctions in this delicate housekeeping may be at the origin of major diseases. There is also a prevalent use of metals in a wide range of diagnostic agents and drugs for the diagnosis or treatment of a variety of disorders. This is becoming more and more of a concern in the field of nanomedicine with the increasing development and use of nanoparticles, which are suspected of causing adverse effects on cells and organ tissues. Synchrotron-based X-ray and Fourier-transformed infrared microspectroscopies are developing into well-suited sub-micrometer analytical tools for addressing new problems when studying the role of metals in biology. As a complementary tool to optical and electron microscopes, developments and studies have demonstrated the unique capabilities of multi-keV microscopy: namely, an ultra-low detection limit, large penetration depth, chemical sensitivity and three-dimensional imaging capabilities. More recently, the capabilities have been extended towards sub-100nm lateral resolutions, thus enabling sub-cellular chemical imaging. Possibilities offered by these techniques in the biomedical field are described through examples of applications performed at the ESRF synchrotron-based microspectroscopy platform (ID21 and ID22 beamlines).

Biomedical applications of the ESRF synchrotron-based microspectroscopy platform.

Synchrotron-based x-ray fluorescence microscopy, immunofluorescence, and Western blotting were used to investigate changes in copper (Cu) and Cu-associated pathways in the vulnerable substantia nigra (SN) and locus coeruleus (LC) and in nondegenerating brain regions in cases of Parkinson's disease (PD) and appropriate healthy and disease controls. In PD and incidental Lewy body disease, levels of Cu and Cu transporter protein 1, were significantly reduced in surviving neurons in the SN and LC. Specific activity of the cuproprotein superoxide dismutase 1 was unchanged in the SN in PD but was enhanced in the parkinsonian anterior cingulate cortex, a region with α-synuclein pathology, normal Cu, and limited cell loss. These data suggest that regions affected by α-synuclein pathology may display enhanced vulnerability and cell loss if Cu-dependent protective mechanisms are compromised. Additional investigation of copper pathology in PD may identify novel targets for the development of protective therapies for this disorder.

Copper pathology in vulnerable brain regions in Parkinson's disease.

Manganese-enhanced magnetic resonance imaging (MEMRI) is a powerful tool for in vivo tract tracing or functional imaging of the central nervous system. However Mn(2+) may be toxic at high levels. In this study, we addressed the impact of Mn(2+) on mouse hippocampal neurons (HN) and neuron-like N2a cells in culture, using several approaches. Both HN and N2a cells not exposed to exogenous MnCl2 were shown by synchrotron X-ray fluorescence to contain 5 mg/g Mn. Concentrations of Mn(2+) leading to 50% lethality (LC50) after 24 h of incubation were much higher for N2a cells (863 mM) than for HN (90 mM). The distribution of Mn(2+) in both cell types exposed to Mn(2+) concentrations below LC50 was perinuclear whereas that in cells exposed to concentrations above LC50 was more diffuse, suggesting an overloading of cell storage/detoxification capacity. In addition, Mn(2+) had a cell-type and dose-dependent impact on the total amount of intracellular P, Ca, Fe and Zn measured by synchrotron X-ray fluorescence. For HN neurons, immunofluorescence studies revealed that concentrations of Mn(2+) below LC50 shortened neuritic length and decreased mitochondria velocity after 24 h of incubation. Similar concentrations of Mn(2+) also facilitated the opening of the mitochondrial permeability transition pore in isolated mitochondria from rat brains. The sensitivity of primary HN to Mn(2+) demonstrated here supports their use as a relevant model to study Mn(2+) -induced neurotoxicity.

Impact of manganese on primary hippocampal neurons from rodents.

A series of tamoxifen-like metallocifens of the group-8 metals (Fe, Ru, and Os) has strong antiproliferative activity on the triple-negative breast cancer cells (MDA-MB-231). To shed light on the mechanism of action of these molecules, synchrotron radiation X-ray fluorescence nanoimaging studies were performed on cells exposed to osmocenyl-tamoxifen (Oc-OH-Tam) to disclose its intracellular distribution. High-resolution mapping of the lipophilic Oc-OH-Tam in cells revealed its preferential accumulation in the endomembrane system. This is consistent with the ability of the amino nitrogen chain of the compounds to be protonated at physiological pH and responsible for electrostatic interactions between Oc-OH-Tam and membranes. A comprehensive scenario is proposed that provides new insight into the cellular behavior and activation of Oc-OH-Tam and advances the understanding of its mechanism of action.

Intracellular Localization of an Osmocenyl-Tamoxifen Derivative in Breast Cancer Cells Revealed by Synchrotron Radiation X-ray Fluorescence Nanoimaging.

The speciation of highly-diluted elements by X-ray absorption spectroscopy in a diverse range of materials is extremely challenging, especially in biological matrices such as articular cartilage. Here we show that using a high energy resolution fluorescence detected X-ray absorption spectroscopy (HERFD-XAS) technique coupled to an array of crystal analyzers, selenium speciation down to 400 ppb (μg kg) within articular cartilage can be demonstrated. This is a major advance in the speciation of highly-diluted elements through X-ray absorption spectroscopy and opens new possibilities to study the metabolic role of selenium and other elements in biological samples.

ArticleTotal : 2	Year
Publication title Cited by 3	2022
Publication title Cited by 16	2019

Article	Year
Characterization of the trabecular rat bone mineral: effect of ovariectomy and bisphosphonate treatment. Bone\| PubMed ID: 10719276	2000
Biomedical applications of the ESRF synchrotron-based microspectroscopy platform. Journal of structural biology\| PubMed ID: 22182732	2012
Copper pathology in vulnerable brain regions in Parkinson's disease. Neurobiology of aging\| PubMed ID: 24176624	2014
Impact of manganese on primary hippocampal neurons from rodents. Hippocampus\| PubMed ID: 24500839	2014
Intracellular Localization of an Osmocenyl-Tamoxifen Derivative in Breast Cancer Cells Revealed by Synchrotron Radiation X-ray Fluorescence Nanoimaging. Angewandte Chemie (International ed. in English)\| PubMed ID: 30663197	2019
Sub-ppm level high energy resolution fluorescence detected X-ray absorption spectroscopy of selenium in articular cartilage. The Analyst\| PubMed ID: 30939183	2019

Sylvain Bohic

Biography

JoVE Journal Publications

Other Publications