Visualization of the glomerular endothelial glycocalyx by electron microscopy using cationic colloidal thorium dioxide.

Biological material itself appears with poor contrast in electron microscopy (EM), due to its composition mostly of light elements. Classical staining agents such as osmium tetroxide, uranyl acetate, and lead citrate preserve and/or stain cellular structures such as membranes, cytoplasm, and organelles well for EM. However, extracellular polymeric substances (EPS) show no or only poor contrast with these staining agents. The endothelial glycocalyx in blood vessels consists mainly of proteoglycans. It can be visualized by EM only by additional staining with heavy metal ions such as copper (Alcian blue, cupromeronic blue), ruthenium (ruthenium red), or lanthanum. Best results are achieved by combined perfusion of fixative and stain. Cationic hydrous thorium dioxide colloids (named here cThO2) trace acidic groups in EPS. We describe here the use of cThO2 to visualize the glomerular endothelial glycocalyx in the mouse kidney. cThO2 shows high electron density and binds to a continuous layer of up to a few hundred nanometers thickness on the glomerular endothelium, as well as on epithelia in other blood vessels in perfused animals. The observed staining pattern gives rise to periodic densities, with a spacing varying between 50 and 200 nm, depending on the overall layer thickness, which varies between below 50 up to 300 nm. Due to high electron density of the used cThO2 particles, the introduced method allows distinct imaging and precise fine structural analysis of the endothelial glycocalyx.

Uptake of colloidal thorium dioxide by mast cells.

Mast cells from the peritoneal cavity of the rat were obtained at various times following in situ injection of a colloidal thorium dioxide preparation (Thorotrast). They were prepared for electron microscopy by aldehyde fixation, osmium tetroxide postfixation, and embedding in Epon. Thorotrast was rapidly taken up by mast cells through enhanced or newly elicited surface specializations. It was confined at first to large vesicles which moved to the Golgi area. Subsequently, in a matter of a few hours only, it became associated with progressively more mature granules, including "fully" mature ones. In addition to demonstrating a further phagocytic or pinocytotic activity of mast cells, the findings suggest that mast cell granules share a common membranous investment, and that substances from the tissue environment may theoretically percolate over and interact with the granules. Mast cell function could thus be served primarily by absorptive rather than secretory processes.

Internalization of macromolecules from the medium in Suctoria.

Takophrya infusionum like all other Suctoria lacks an oral cavity. Its feeding apparatus consists of tentacles, long narrow tubes through which the contents of the living prey are ingested. For normal growth, reproduction, and longevity of clones, Tokophrya needs supplements deriving from the medium in addition to living prey. Since Tokophrya lacks a mouth, these supplements can reach the cytoplasm only through the complex structure of the cortex, which is composed of a three-membraned pellicle and a dense epiplasm. In addition, external to the cortex, an extraneous coat covers the whole organism. Only the outer pellicular plasma membrane is continuous; the other two and the epiplasm are interrupted by the outer plasma membrane which invaginates at intervals forming the so-called pits. The invaginated plasma membrane dips down into the cytoplasm where it extends to form a saccule. Experiments with cationized ferritin and Thorotrast provide evidence that internalization of these macromolecules takes place through the pits by pinocytosis. The membrane of the saccules of the pits forms invaginations which pinch off giving rise to small, flattened vesicles containing the tracers. The tracers were never found free in the cytoplasm but exclusively in the flat vesicles. These vesicles are thus the vehicles transporting macromolecules from the medium to the cytoplasm. The saccules of the pits are the natural loci of pinocytosis and together with the flattened vesicles perform an important function in Suctoria, supplying the organisms with macromolecules from the medium.

Permeability of the ovarian follicle of Aedes aegypti mosquitoes.

The passage of tracers of various molecular weights into resting and vitellogenic ovarian follicles of Aedes aegypti mosquitoes was studied ultrastructurally. The outermost layer of the follicular sheath (the basement lamina) is a coarse mechanical filter. It is freely permeable to particles with molecular weights ranging from 12,000 to 500,000 (i.e. cytochrome c, peroxidase, hemoglobin, catalase, ferritin, immunoglobulin (IgG)-peroxidase, iron dextran and Thorotrast) that have dimensions less than 110 A. Molecules as large as carbon (300-500 A) are totally excluded. Whereas proteins and polysaccharide tracers permeate the basement lamina with apparent ease, certain inert particles (e.g. Thorotrast, Fellows-Testager Div., Fellows Mfg. Co., Inc., Detroit, Mich.) penetrate more slowly. With respect to the tracers tested, resting follicles are as permeable as vitellogenic follicles. The follicle epithelium of resting or vitellogenic follicles is penetrated by narrow intercellular channels. Our observations suggest that these spaces are lined with mucopolysaccharide material. After permeating the basement lamina, exogenous tracers fill these channels, while the bulk of material accumulates in the perioocytic space. Within 3 hr after imbibing blood, the pinocytotic mechanism of the oocyte is greatly augmented. Pinocytosis is not selective with regard to material in the perioocytic space, since double tracer studies show that exogenous compounds are not separated, but are incorporated into the same pinocytotic vesicle. During later stages of vitellogenesis, 36-48 hr after the blood-meal, the pinocytotic mechanism of the oocyte is diminished. Simultaneously, the intercellular channels become occluded by desmosomes, and the vitelline membrane plaques separate the oocyte and follicle epithelium.

Phenobarbital, drug metabolism, and human cancer.

To investigate the possible influence of anticonvulsant treatment on cancer risk, a nested case-control study of 104 lung cancers, 18 bladder cancers, and 322 cancer-free controls was conducted. The background for the study was previous observations among 8004 epileptics in Denmark with a significantly high risk for lung cancer and a significantly low risk for bladder cancer. Cigarette smoking appears to explain the lung cancer excess but not the low risk for bladder cancer, another tobacco-related disease. Information was abstracted on 94 and 95% of the cases and controls, respectively. Lung cancer was not associated with any anticonvulsant drug, but bladder cancer was inversely related to use of phenobarbital (PB). The apparent protective effect of PB was further evaluated in a study of rats given 4-aminobiphenyl (ABP), a bladder carcinogen. The levels of 4-aminobiphenyl adducts in hemoglobin and in bladder and liver DNA were significantly lower in rats given PB prior to 4-aminobiphenyl, compared to controls. These studies suggest that PB may induce drug-metabolizing enzymes of the liver that deactivate bladder carcinogens found in cigarette smoke and provide clues to the role of activation and detoxification of carcinogens in humans.

Translocation of Thorotrast in the body.

To investigate whether there is a permanent translocation of Thorotrast in the body, the liver of male Lewis rats was removed 4 weeks after injection of Thorotrast (300 microliters) and replaced by a donor liver. In half of the animals the spleen was removed as well. Measuring the 232Th content in the donor liver at different times after implantation demonstrated a permanent transport of 232Th into the surrogate organ. After 231 days a 232Th depot of about 1.1 mg was found, representing about 3% of the total body burden. The additional removal of the spleen resulted in a significantly lower transport of 232Th into the implanted liver. Histological examinations of the donated livers revealed increasing local concentration of Thorotrast granules, leading to the development of conglomerates. A comparable translocation of Thorotrast was verified in two humans who required liver transplantation more than 40 years after Thorotrast injection.

Systemic deposits of thorium in thorotrast patients with particular reference to sites of minor storage.

It is well established that injected Thorotrast is deposited in the liver, spleen, bone marrow, and lymph nodes, but accumulations in organs with lower macrophage activity have previously been given little attention. In this work, neutron activation analysis has been used to investigate concentrations of thorium in autopsy samples taken at sites of major and minor deposition in 24 Thorotrast patients. In the latter category, the highest values were found in the testis [40 x 10(-6) g/g(wet)], followed by those in the adrenal gland, gallbladder, lung, and pancreas. The resulting alpha-particle dose rates (mGy/year) are tentatively estimated to be 8.5 to the testis, 5.5 to the gallbladder, and 5.3 to the lung. These results may be relevant to the residual excess mortality among Thorotrast patients after diseases of the principal organs of deposition have been excluded; they also support previous indications that thorium deposited in pulmonary tissues is responsible for an important component of the total dose to the lung. In another context, our data may bear on the connection, postulated elsewhere, between exposure to alpha-particle emitters and elevated incidence of leukemia in the children of workers engaged in the reprocessing of nuclear fuel.

[Metastasising carcinoma of the renal pelvis after Thorotrast pyelography]. / Metastasierendes Nierenbeckenkarzinom nach Thorotrast-Pyelographie

A carcinoma of the renal pelvis was diagnosed in a female patient 42 years after a retrograde Thorotrast pyelogram. The patient died as a result of the carcinoma at the age of 60. The literature is reviewed. As a result of this case, the need is stressed for early recognition of Thorotrast depositis in the kidney. In this case it was demonstrated by autoradiography. Appropriate treatment is discussed.

Managing metabolic model deficiencies for routine radiation protection purposes at a thorium dioxide facility.

At a metallurgical facility using thorium dioxide feed materials, a number of conventional direct- and indirect-bioassay methods were potentially suitable monitoring methodologies when industry-standard dose assessment methodologies were based upon ICRP recommendations issued in 1959. When the ICRP recommended a different dose assessment methodology in 1977, the intake limits for thorium dropped significantly, and conventional bioassay methods for routine monitoring were rendered ineffective. While determining the technical basis for its air monitoring program, the facility noted that the radioactive materials used as feed to the process were highly insoluble. Therefore, their solubility in simulated lung fluids was determined. The results of this determination indicated that the facility's materials, if inhaled, dissolve in the lung at a half-time that is significantly longer than that contained in international consensus standards for Y-class compounds. They also indicated that the secondary dose limits, if adjusted to eliminate the dose contribution from translocated radionuclides, should be higher, and that a derived air concentration based upon more recent international recommendations is more appropriate for use in the facility's routine monitoring program.

Thorotrast distribution in monkey bone marrow at early and late times after injection.

Thorotrast, a 25% colloidal suspension of 232ThO2, was formerly used as a radiographic contrast medium. Although epidemiological studies have shown that alpha particles emitted from 232Th and its decay products incorporated in the bone marrow cause leukemia, the use of these data for alpha particle induced leukemogenesis risk estimation has been criticized mainly for inhomogeneity of Thorotrast distribution. Four monkeys were injected with Thorotrast to investigate the degree of inhomogeneity in the thorium content of different bone marrow sites and the cellular localization of Thorotrast. Two were injected via an artery and two via a vein and sacrificed either at 1 wk or 3 to 4 y after injection. Microscopic, solid state autoradiography and back scatter electron imaging methods were applied to several bone sites to determine the degree of inhomogeneity. Quantification was performed using x-ray fluorescence for trabecular bone and bone marrow and neutron activation analysis for compact bones. At 1 wk Thorotrast was found to be distributed evenly in the red marrow; by 3 and 4 y conglomerates were seen which were restricted to macrophages. The monkey was found to be a good model for humans. The choice of injection route did not noticeably affect the Thorotrast distribution in bones of the skeletal system. Considering the even distribution of Thorotrast within the red bone marrow at early times after its injection, the inevitable diffusion of thorium progeny from the particles, the mobility of bone marrow macrophages, and the well established correction factor of self-absorption within conglomerates, these results suggest that data derived from Thorotrast patients are useful for risk estimation of alpha particle induced leukemia.

Recent results of the German Thorotrast study--dose relevant physical and biological properties of Thorotrast equivalent colloids.

As a prerequisite for quantifying the non-radiation effect of Thorotrast, nonradioactive and radioactive aquasols with identical physicochemical properties and with biophysical behavior comparable to that of Thorotrast were developed and produced for a second long-term animal experiment. Comparative investigations with hafnium and zirconium (zirconotrast) dioxide aquasols showed the latter to be most appropriate considering the size of both the dispersoids and the aggregates in the liver tissue. The average particle diameters of ThO2, ZrO2 and HfO2 proved to be values of 9.3 nm, 15 nm and 45 nm, respectively. The size of the aggregates shows a slight dependence on the applied amount but no dependence on the duration of body burden of the colloid. The aggregate diameters in the 600 microliters group turned out to be 9.6 microns, 14.4 microns and 5.3 microns, respectively. Radioactive zirconotrast was prepared by radiochemical incorporation of 230Th and 228Th at dose rates which produce accumulated doses in the liver of rats equivalent to those of commercial 230Th enriched Thorotrast after 1.5 yr. Five different colloids were prepared with alpha-energy emission rates increased by factors of 1, 2.5, 5, 10 and 25 compared to Thorotrast and then injected into rats.

Locoregional late effects of paravascular thorotrast deposits: results of the german thorotrast study.

PURPOSE: The aim of this study was to assess late effects of long-term exposure to alpha irradiation caused by paravascular Thorotrast deposits. SUBJECTS AND METHODS: 899 patients, who had received the radioactive contrast medium Thorotrast for angiography in the 1930s and 1940s, and 662 controls were followed-up since 1968 every two years by standardized clinical and laboratory examinations. Initially, X-ray plain films of the thorax, upper abdomen and the former injection site were performed. In selected patients the sites of paravascular Thorotrast deposits were evaluated by ultrasonography, CT and MRI. RESULTS: Paravascular Thorotrast deposits were detected in 245 patients. Clinical symptoms related to deposits appeared 10 to 30 years after Thorotrast administration. The severity of symptoms depended on the location and extension of granulomas and were mainly caused by fibrosis, nerve paralysis and vascular changes. Four malignant tumors adjacent to granulomas were observed (one soft tissue sarcoma in the groin, two squamous cell carcinomas of the parotid gland and one lymphoepithelial carcinoma of the nasopharynx). MRI including MRA allowed an accurate determination of tissue damage, whereas the utility of US and CT was restricted due to strong sound attenuation and streak artefacts caused by the high X-ray absorption of Thorotrast. DISCUSSION AND CONCLUSION: Locoregional late effects of paravascular Thorotrast deposits mainly comprise radiation induced, fibrotic tissue destruction. The incidence of malignant tumors, in particular sarcomas, adjacent to deposits, however, is much lower than initially expected.