Meredin Stoltenberg

Kupffer cells are central in the removal of nanoparticles from the organism

BackgroundThe study aims at revealing the fate of nanoparticles administered intravenously and intraperitoneally to adult female mice, some of which were pregnant. Gold nanoparticles were chosen as a model because these particles have been found to be chemically inert and at the same time are easily traced by autometallography (AMG) at both ultrastructural and light microscopic levels.ResultsGold nanoparticles were injected intravenously (IV) or intraperitoneally (IP) and traced after 1, 4 or 24 hours. For IV injections 2 and 40 nm particles were used; for IP injections 40 nm particles only. The injected nanoparticles were found in macrophages only, and at moderate exposure primarily in the Kupffer cells in the liver. IV injections resulted in a rapid accumulation/clustering of nanoparticles in these liver macrophages, while the uptake in spleen macrophages was moderate. IP injections were followed by a delayed uptake in the liver and included a moderate uptake in macrophages located in mesenteric lymph nodes, spleen and small intestine. Ultrastructurally, the AMG silver enhanced nanocrystals were found in lysosome-like organelles of the Kupffer cells and other macrophages wherever located.Accumulations of gold nanoparticles were not found in any other organs analysed, i.e. kidneys, brain, lungs, adrenals, ovaries, placenta, and fetal liver, and the control animals were all void of AMG staining.ConclusionOur results suggest that: (1) inert gold nanoparticles do not penetrate cell membranes by non-endocytotic mechanisms, but are rather taken up by endocytosis; (2) gold nanoparticles, independent of size, are taken up primarily by Kupffer cells in the liver and secondarily by macrophages in other places; (3) gold nanoparticles do not seem to penetrate the placenta barrier; (4) the blood-brain barrier seems to protect the central nervous system from gold nanoparticles; (5) 2 nanometer gold particles seem to be removed not only by endocytosis by macrophages, and we hypothesize that part of these tiny nanoparticles are released into the urine as a result of simple filtration in the renal glomeruli.

Protracted elimination of gold nanoparticles from mouse liver

The present study aims at revealing the fate of 40-nm gold nanoparticles after intravenous injections. The gold nanoparticles were traced histochemically with light and transmission electron microscopy using autometallographic (AMG) staining, and the gold content in the liver was determined with inductively coupled plasma mass spectrometry (ICP-MS). Gold nanoparticles were identified in almost all Kupffer cells one day after the injection, but the fraction of gold-loaded cells gradually decreased to about one fifth after 6 months. Transmission electron microscopic analysis showed that the gold nanoparticles had accumulated inside the vesicular lysosome/endosome-like structures of the macrophages. At day 1, about 4.5 per thousand of the area of the liver sections was AMG-stained, after 1 month it had decreased to 0.7 per thousand, and thereafter no further significant reduction was recorded. Because ICP-MS only showed a 9% fall in the gold content over the observed 6 months, the AMG finding of a significant reduction in the stained area of the liver sections and number of macrophages loaded with gold nanoparticles reveals that over time an increasing part of the total amount of gold nanoparticles in the liver is contained in fewer macrophages accumulated in growing clusters.

Increased amount of zinc in the hippocampus and amygdala of Alzheimer's diseased brains: A proton-induced X-ray emission spectroscopic analysis of cryostat sections from autopsy material

Zinc has been implicated as a contributing cause of the neuropathology of Alzheimers disease (AD), but consensus on the zinc content of AD brains has not yet been established. In the present study, multi-element PIXE was used to measure zinc in cryostat sections of brain tissue from AD patients and from normal control subjects. Compared to their age-matched controls, the AD patients showed an increase in zinc in the hippocampal and amygdalar regions. The instrumental PIXE assays do not show whether the zinc changes are due to altered zinc in the boutons of Zinc-ENriched (ZEN) neurons, i.e., zinc ions in synaptic vesicles, or to changes in the amount of zinc tightly bound to macromolecules. We hypothesise that the increased zinc level is caused by an increase in the amount of ZEN terminals. Such an increase could be the result of a sprout of ZEN terminals in diseased areas of the brain.

The role of metallothionein in oncogenesis and cancer prognosis.

The antiapoptotic, antioxidant, proliferative, and angiogenic effects of metallothionein (MT)-I+II has resulted in increased focus on their role in oncogenesis, tumor progression, therapy response, and patient prognosis. Studies have reported increased expression of MT-I+II mRNA and protein in various human cancers; such as breast, kidney, lung, nasopharynx, ovary, prostate, salivary gland, testes, urinary bladder, cervical, endometrial, skin carcinoma, melanoma, acute lymphoblastic leukemia (ALL), and pancreatic cancers, where MT-I+II expression is sometimes correlated to higher tumor grade/stage, chemotherapy/radiation resistance, and poor prognosis. However, MT-I+II are downregulated in other types of tumors (e.g. hepatocellular, gastric, colorectal, central nervous system (CNS), and thyroid cancers) where MT-I+II is either inversely correlated or unrelated to mortality. Large discrepancies exist between different tumor types, and no distinct and reliable association exists between MT-I+II expression in tumor tissues and prognosis and therapy resistance. Furthermore, a parallel has been drawn between MT-I+II expression as a potential marker for prognosis, and MT-I+IIs role as oncogenic factors, without any direct evidence supporting such a parallel. This review aims at discussing the role of MT-I+II both as a prognostic marker for survival and therapy response, as well as for the hypothesized role of MT-I+II as causal oncogenes.

Biodistribution of gold nanoparticles in mouse lung following intratracheal instillation

BackgroundThe fate of gold nanoparticles, 2, 40 and 100 nm, administered intratracheally to adult female mice was examined. The nanoparticles were traced by autometallography (AMG) at both ultrastructural and light microscopic levels. Also, the gold content was quantified by inductively coupled plasma mass spectrometry (ICP-MS) and neutron activation analysis (NAA). The liver is the major site of deposition of circulating gold nanoparticles. Therefore the degree of translocation was determined by the hepatic deposition of gold. Mice were instilled with 5 intratracheal doses of gold nanoparticles distributed over a period of 3 weeks and were killed 24 h after the last dose. One group of mice were given a single intratracheal dose and were killed after 1 h.ResultsThe instilled nanoparticles were found in lung macrophages already 1 h after a single instillation. In mice instilled treated repeatedly during 3 weeks, the load was substantial. Ultrastructurally, AMG silver enhanced gold nanoparticles were found in lysosome-/endosome-like organelles of the macrophages and analysis with AMG, ICP-MS and NAA of the liver revealed an almost total lack of translocation of nanoparticles. In mice given repeated instillations of 2 nm gold nanoparticles, 1.4‰ (by ICP-MS) to 1.9‰ (by NAA) of the instilled gold was detected in the liver. With the 40 nm gold, no gold was detected in the liver (detection level 2 ng, 0.1‰) except for one mouse in which 3‰ of the instilled gold was found in the liver. No gold was detected in any liver of mice instilled with 100 nm gold (detection level 2 ng, 0.1‰) except in a single animal with 0.39‰ of the dose in the liver.ConclusionWe found that that: (1) inert gold nanoparticles, administered intratracheally are phagocytosed by lung macrophages; (2) only a tiny fraction of the gold particles is translocated into systemic circulation. (3) The translocation rate was greatest with the 2 nm gold particles.

AMYLOID PLAQUES ARISE FROM ZINC-ENRICHED CORTICAL LAYERS IN APP/PS1 TRANSGENIC MICE AND ARE PARADOXICALLY ENLARGED WITH DIETARY ZINC DEFICIENCY

The ZnT3 zinc transporter is uniquely expressed in cortical glutamatergic synapses where it organizes zinc release into the synaptic cleft and mediates beta-amyloid deposition in transgenic mice. We studied the association of zinc in plaques in relation to cytoarchitectural zinc localization in the APP/PS1 transgenic mouse model of Alzheimers disease. The effects of low dietary zinc for 3 months upon brain pathology were also studied. We determined that synaptic zinc distribution within cortical layers is paralleled by amyloid burden, which is heaviest for both in layers 2-3 and 5. ZnT3 immunoreactivity is prominent in dystrophic neurites within amyloid plaques. Low dietary zinc caused a significant 25% increase in total plaque volume in Alzheimers mice using stereological measures. The level of oxidized proteins in brain tissue did not changed in animals on a zinc-deficient diet compared with controls. No obvious changes were observed in the autometallographic pattern of zinc-enriched terminals in the neocortex or in the expression levels of zinc transporters, zinc importers or metallothioneins. A small decrease in plasma zinc induced by the low-zinc diet was consistent with the subclinical zinc deficiency that is common in older human populations. While the mechanism remains uncertain, our findings indicate that subclinical zinc deficiency may be a risk factor for Alzheimers pathology.

Zinc-specific Autometallographic In Vivo Selenium Methods: Tracing of Zinc-enriched (ZEN) Terminals, ZEN Pathways, and Pools of Zinc Ions in a Multitude of Other ZEN Cells

In vivo-applied sodium selenide or sodium selenite causes the appearance of zinc-selenium nanocrystals in places where free or loosely bound zinc ions are present. These nanocrystals can in turn be silver enhanced by autometallographic (AMG) development. The selenium method was introduced in 1982 as a tool for zinc-ion tracing, e.g., in vesicular compartments such as synaptic vesicles of zinc-enriched (ZEN) terminals in the central nervous system, and for visualization of zinc ions in ZEN secretory vesicles of, e.g., somatotrophic cells in the pituitary, zymogene granules in pancreatic acinar cells, beta-cells of the islets of Langerhans, Paneth cells of the crypts of Lieberkühn, secretory cells of the tubuloacinar glands of prostate, epithelium of parts of ductus epididymidis, and osteoblasts. If sodium selenide/selenite is injected into brain, spinal cord, spinal nerves containing sympathetic axons, or intraperitoneally, retrograde axonal transport of zinc-selenium nanocrystals takes place in ZEN neurons, resulting in accumulation of zinc-selenium nanocrystals in lysosomes of the neuronal somata. The technique is, therefore, also a highly specific tool for tracing ZEN pathways. The present review includes an update of the 1982 paper and presents evidence that only zinc ions are traced with the AMG selenium techniques if the protocols are followed to the letter.

Abundant expression of zinc transporters in the amyloid plaques of Alzheimer's disease brain.

The pathological key features of Alzheimers disease (AD) are beta-amyloid peptide (Abeta)-containing senile plaques (SP) and neurofibrillary tangles. Previous studies have suggested that an extracellular elevation of the zinc concentration can initiate the deposition of Abeta and lead to the formation of SP. In the present study, we present data showing a correlation between zinc ions, zinc transporters (ZNTs) and AD, using immersion autometallography (AMG) and double immunofluorescence for the ZNTs and Abeta. We found that all the ZNTs tested (ZNT1, 3, 4, 5, 6, 7) were extensively present in the Abeta-positive plaques in the cortex of human AD brains, and the density of autometallographic silver enhanced zinc-sulphur nanoparticles were much higher in the plaques than in the surrounding zinc enriched (ZEN) terminals. Moreover, we found an abundant expression of ZNT3 and autometallographic grains in the amyloid angiopathic vessels. The subcellular localization of ZNTs and zinc ions were not detected, due to the limited tissue preservation in the present study. In conclusion, our data provided significant morphological evidence of zinc ions and ZNTs being actively involved in the pathological processes that lead to plaque formation.

SLC30A3 Responds to Glucose- and Zinc Variations in ß-Cells and Is Critical for Insulin Production and In Vivo Glucose-Metabolism During ß-Cell Stress

Background Ion transporters of the Slc30A- (ZnT-) family regulate zinc fluxes into sub-cellular compartments. β-cells depend on zinc for both insulin crystallization and regulation of cell mass. Methodology/Principal Findings This study examined: the effect of glucose and zinc chelation on ZnT gene and protein levels and apoptosis in β-cells and pancreatic islets, the effects of ZnT-3 knock-down on insulin secretion in a β-cell line and ZnT-3 knock-out on glucose metabolism in mice during streptozotocin-induced β-cell stress. In INS-1E cells 2 mM glucose down-regulated ZnT-3 and up-regulated ZnT-5 expression relative to 5 mM. 16 mM glucose increased ZnT-3 and decreased ZnT-8 expression. Zinc chelation by DEDTC lowered INS-1E insulin content and insulin expression. Furthermore, zinc depletion increased ZnT-3- and decreased ZnT-8 gene expression whereas the amount of ZnT-3 protein in the cells was decreased. Zinc depletion and high glucose induced apoptosis and necrosis in INS-1E cells. The most responsive zinc transporter, ZnT-3, was investigated further; by immunohistochemistry and western blotting ZnT-3 was demonstrated in INS-1E cells. 44% knock-down of ZnT-3 by siRNA transfection in INS-1E cells decreased insulin expression and secretion. Streptozotocin-treated mice had higher glucose levels after ZnT-3 knock-out, particularly in overt diabetic animals. Conclusion/Significance Zinc transporting proteins in β-cells respond to variations in glucose and zinc levels. ZnT-3, which is pivotal in the development of cellular changes as also seen in type 2 diabetes (e.g. amyloidosis in Alzheimers disease) but not previously described in β-cells, is present in this cell type, up-regulated by glucose in a concentration dependent manner and up-regulated by zinc depletion which by contrast decreased ZnT-3 protein levels. Knock-down of the ZnT-3 gene lowers insulin secretion in vitro and affects in vivo glucose metabolism after streptozotocin treatment.

Ultrastructural localization of zinc ions in the rat prostate : An autometallographic study

The prostate contains high amounts of free zinc ions which are excreted into the seminal fluid. The extra‐ and intracellular distribution of zinc ions using the highly specific autometallographical (AMG) method is described.