Gadolinium induces macrophage apoptosis.

Gadolinium (Gd) suppresses reticuloendothelial functions in vivo by unknown mechanisms. In vitro exposure of rat alveolar macrophages to GdCl3.6H20 caused cell death, as measured by trypan blue permeability, in both dose- and time-dependent fashions. Even a 10-min exposure to Gd caused significant cell death by 24 h. The morphology of Gd-treated cells, pyknosis and karyorrhexis prior to loss of membrane integrity, suggested apoptosis. Upon flow cytometric examination, Gd-treated propidium iodide-excluding cells demonstrated light scatter changes characteristic of apoptotic cells (decreased forward and increased right angle scatter). Gel electrophoresis of DNA from Gd-treated macrophages clearly showed the ladder pattern unique to apoptotic cells. Electron-dense structures containing Gd were observed via electron spectroscopic imaging within phagosomes and also within nuclei (associated with condensed chromatin). Gadolinium, endocytosed by macrophages and distributed to nuclei, causes apoptosis of macrophages in vitro.

Contribution of osmium tetroxide to the image quality and detectability of iron in cells studied by electron spectroscopic imaging and electron energy loss spectroscopy.

The detection of elemental distributions within ultrastructural cellular components presents a number of challenges. There are many technical questions that need to be resolved including optimal fixation protocols. Another is the impact of heavy metals, such as osmium tetroxide (OsO4), on the detectability of other elements when OsO4 is used in chemical fixation protocols for biological samples. OsO4 was examined by varying its concentrations from 0% to 1% and time of fixation from 5 to 30 minutes with hamster alveolar macrophages. The morphological quality of cellular images observed and the detectability of iron using electron spectroscopic imaging (ESI) and electron energy loss spectroscopy (EELS) were evaluated. One percent OsO4 for 30 minutes in the chemical fixation protocol enhances the quality of the ESI and does not interfere with the ESI or EELS signal of iron. Positive results from both methods indicate the presence of the specific element. The loss of 59Fe during the chemical fixation procedure was also studied. Less than 10% was lost during the primary fixation step, but minimal losses occurred through dehydration, embedding, and sectioning. Careful technical assessment of the presence of an element as well as factors which might interfere with its detection is an important step in the application of any analytical microscopic technique.

Endocytosis of ultrafine particles by A549 cells.

Alveolar epithelium's capacity to ingest inhaled ultrafine particles is not well characterized. The objectives of this study were to use an in vitro model of type II lung epithelium and evaluate the cells' ability to take up ultrafine particles (titanium dioxide [TiO(2)], 50 nm diameter). The human epithelial cell line A549 was grown on aclar substrates and exposed to 40 microg/ml TiO(2) particles for 3, 6, and 24 h before imaging with energy-filtering transmission electron microscopy. Elemental mapping and electron energy loss spectroscopy were used to colocalize Ti/O with electron-dense particles. Particle endocytosis was compared in A549 cells with and without pretreatment with cytochalasin D (cyto D) (2 microg/ml). After 3 h of TiO(2) exposure, cells internalized aggregates of the ultrafine particles which were observed in cytosolic, membrane-bound vacuoles. After 24 h of exposure there were considerably more intracellular aggregates of membrane-bound particles, and aggregated particles were also enmeshed in loosely and tightly packed lamellar bodies. Throughout 24 h of exposure a preponderance of particles remained associated with the free surface of the cells and were not internalized. The majority of membrane-bound vacuoles contained aggregates of particles and only occasionally did they contain as few as two or three particles, despite the use of several different approaches to assure the possibility for individual particles to be ingested and detected. There was morphologic evidence of microfilament disturbance, but no evidence of a decrease in internalized particles in cells pretreated with cyto D. Thus, this model of type II epithelium is able to internalize aggregates of ultrafine particles.

Ultrastructural localization of a fluorinated bile salt in hepatocytes.

BACKGROUND: Interactions of bile salts with hepatocellular organelles are critical for the formation of bile, yet these interactions remain poorly characterized. We present a novel approach for visualizing bile salts at the ultrastructural level within hepatocytes, using a unique fluorinated bile salt conjugate and electron energy loss spectroscopy. EXPERIMENTAL DESIGN: Isolated rat hepatocytes were incubated for 5 and 20 minutes with the 2-fluoro-beta-alanine (FBAL) N-acyl amidate conjugate of cholic acid (C-FBAL, 50 microM). FBAL is a byproduct of hepatic 5-fluorouracil catabolism, and when conjugated to cholic acid is excreted into bile in a manner similar to the naturally occurring N-acyl amidates of bile salts. Cells were subjected to rapid cryofixation and automated freeze-drying followed by vapor-phase fixation using the LifeCell system, thus avoiding exposure to the leaching action of liquid fixatives. After resin infiltration, the cellular distribution of fluorine was determined in ultrathin sections with a Zeiss CEM902 electron microscope equipped for electron energy loss spectroscopy. RESULTS: Fluorine was detected primarily in association with intracellular membranes, particularly membranes of the endoplasmic reticulum (p < 0.05 at 20 minutes by morphometric analysis). Fluorine also was detected in association with membranes of the Golgi apparatus. The fluorine signal was confirmed by serial spectra of cell regions containing these organelles (p < 0.01), but was not detectable in the free cytosol, mitochondria or extracellular medium, nor in hepatocytes not exposed to C-FBAL. CONCLUSIONS: We conclude that cryofixation and freeze-dry processing followed by electron microscopy with electron energy loss spectroscopy is a valuable technique for examining intracellular processing of bile salts. Our results suggest that bile salts localize to the membranes, but not lumena, of organelles during hepatocyte exposure to bile salts, calling into question the proposed role for vesicular transport of bile salts within hepatocytes.

Deposition and phagocytosis of inhaled particles in the gas exchange region of the duck, Anas platyrhynchos.

Little is known about the fate of inhaled aerosol particles in birds; even the anatomical location of phagocytic cells within the lungs has yet to be clearly demonstrated. We exposed 2 anesthetized, spontaneously breathing ducks to a non-toxic iron oxide aerosol (aerodynamic mass mean diameter = 0.18 micron; 460 mg/m3) for 1.75 h and 2 awake, resting ducks to less concentrated aerosol (38 mg/m3) for 6 h on two consecutive days. All 4 ducks were sacrificed 24 h after the end of the last exposure. Their lungs, as well as the lungs from a control duck not exposed to the aerosol, were fixed in situ by insufflation of osmium tetroxide vapor or by intravascular perfusion. Then samples of the gas exchange region were examined with a transmission electron microscope. We found iron oxide particles: trapped within the trilaminar substance that is unique to avian lungs and coats the atria and infundibula; within epithelial cells of the atria and initial portions of the infundibula; and within interstitial macrophages. Only occasionally, small amounts of particles were found in the air capillaries. We conclude that both epithelial cells and interstitial macrophages can phagocytize particles in avian lungs, and that there is some convective transport of aerosol to the atria and the initial portions of the infundibula.