Journal of Clinical Pathology, 1978, 31, 893-896

Identification of thorium dioxide in human liver cellsby electron microscopic x-ray microanalysisARNE 0DEGAARD, EGIL M. OPHUS, AND AILEEN M. LARSEN

From the Department ofPathology, Trondheim Regional Hospital, 7000 Trondheim, Norway

SUMMARY Thirty-two years after injection of thorium dioxide (Thorotrast) for diagnostic x-ray

studies in a female patient deposits were found by light microscopy in the liver macrophages(Kupffer cells). They were shown by electron microscopy to be located inside secondary lyso-somes, and by autoradiography and x-ray microanalysis they were identified as thorium.

The aim of the present study was to undertake anultrastructural examination of liver cells obtainedby biopsy from a 47-year-old woman who in 1945had received an unknown amount of thoriumdioxide (Thorotrast) intravenously for diagnosticx-ray purposes before the removal of a braintumour. The deposits of Thorotrast in the liver andspleen were discovered incidentally during x-rayexamination of the kidney for an intercurrent dis-ease. The biopsy was taken specifically for mor-phological study of the liver. The intracellularpresence and localisation of aggregates of thoriumdioxide particles was studied using light microscopicautoradiography and electron microscopic x-raymicroanalysis. The latter technique has proved avaluable tool for the identification of elements natu-rally occurring in biological samples as well as inexogenously introduced elements. To our knowledge,this is the first study demonstrating thoriumdioxide (Thorotrast) in liver cells by x-ray micro-analysis.

Material and methods

Tissue from needle biopsy of the liver was fixed im-mediately in 4% glutaraldehyde in 0-1 M cacodylatebuffer at pH 7-2. One-half of the tissue was preparedfor electron microscopy while the other was trans-ferred to buffered formalin and processed by routinehistological methods.

Paraffin-embedded sections, 5 Jum thick, weredeparaffinised, dehydrated, and coated with Kodaknuclear track emulsion NTB 2 for autoradiography.The emulsion-coated slides were stored in darknessat 40C. After four weeks they were developed andstained with haematoxylin and eosin.

Received for publication 13 February 1978

The material for electron microscopic studieswas transferred to a mixture with a final concentra-tion of 2% glutaraldehyde and 2% osmium tetroxidebuffered to pH 7-2 by 0-1 M cacodylate. After onehour the tissue was washed in buffer and postosmi-fication was carried out in 2% OS04 for one hour(Franke et al., 1969). Subsequently the tissue wasdehydrated and embedded in Epon 812. All sub-stances used for preparation were of high chemicalpurity with an insignificant amount of thorium. Sec-tions approximately 60 nm thick, judged from inter-ference colours, were cut with glass knives, mountedon copper grids, and examined in a Zeiss EM-9S-2electron microscope at 60 keV. Some sections wereexamined without prior staining, others afterstaining with uranyl acetate and lead citrate (Rey-nolds, 1963). For electron microscopic x-raymicroanalysis unstained sections, about 150 nmthick, were mounted on copper grids and analysedin a Transmission Electron Microscope (Phillips300) equipped with an energy dispersive x-rayanalysis system (EDAX). The electron microscopewas operated at 100 kV with a specimen tilt of 42degrees. Analysing time was 55 seconds.

Results

MORPHOLOGICAL FINDINGS

Light microscopy (Fig. 1)The hepatic architecture was preserved. A variableamount of granular material was observed in thesinusoidal macrophages (Kupffer cells).

Electron microscopy (Figs 2 and 3)In the ultrastuctural study one or more electron-dense cytoplasmic inclusions were found withineach liver macrophage. The study of 60 nm thickstained sections revealed that the inclusions lay

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Arne Odegaard, Egil M. Ophus, and Ailleen M. Larsen

Fig. 1 Liver biopsy specimenwith preserved architecture andcollection of Thorotrast-containingmacrophages (Haematoxylinand eosin x 450)

Fig. 2 Electron micrographshowing a residual body con-taining electron-dense Thorotrastparticles in liver macrophage.Uranyl acetate and lead citratex 24 000

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inside single-membrane bound cell organelles,identified as residual bodies (Fig. 2). Some of thesebodies were completely filled with the dense material.In some instances electron-dense inclusions could bedemonstrated inside autophagic vacuoles (Fig. 3).

Electron microscopic x-ray microanalysis (Figs 4and 5)In the semithick unstained sections prepared forx-ray microanalysis little cellular detail and nomembranes could be identified. However, the

electron dense inclusions were easily located (Fig. 4).Figure 5 shows the x-ray spectrum from micro-analysis of the heavily loaded cells shown in Figure4. The spectrum shows prominent thorium peaks(M-line at 3-06, La at 12-96, L 81 at 16-19, L/,2 at15-62, and L; at 18-92 keV, respectively). Noneof these peaks was present in the spectrum from anadjacent cell without electron-dense inclusions.The other peaks present in Fig. 5 represent elements(P, Cl, Cr, Ni, Cu, Zn, Os) in the fixative, embeddingmaterial, and metal-containing parts of the ana-

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Identification of thorium dioxide in human liver cells by electron microscopic x-ray microanalysis

Fig. 3 Electron micrographshowing autophagic vacuolewith heterogenous granules.Liver macrophage. Uranylacetate and lead citrate x 14 000

Fig. 4 Semithick section(150 nm) of liver macrophageheavily loaded with inclusionsof Thorotrast. Unstained x 14 000

lysing instrument. These peaks were present in allthe x-ray spectra.

Autoradiography disclosed some short, straighttracks emanating in a spokelike fashion from thethorium inclusions, typical of those producedby alpha particles.The patient's present condition is excellent,

without signs of liver disease.

Discussion

The ultimate fate of phagocytosed material dependsnot only on the function of the lysosomal apparatus

but also on the nature of the ingested material.Digestible organic substances are broken down andmay enter the metabolism as simpler compounds.On the other hand, phagocytosed material resistantto the lysosomal enzymes is stored indefinitelyin residual bodies. Depending on the quantity andtype, such material may be injurious. An exampleof a potentially hazardous material is radioactiveThorotrast particles. Thorotrast, a colloidal solutionof thorium dioxide, was used widely some 30 yearsago as a contrast medium for diagnostic X-raypurposes. Thorotrast contains the radioactiveisotope, thorium-232, which is a nearly pure alpha

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896 Arne Odegaard, Egil M. Ophus, and Aileen M. Larsen

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Fig. 5 X-ray spectrum frommicroanalysis of liver

- -. _ macrophage (Fig. 4) showingcharacteristic thorium peaks(Th). Non-labelled peaks

lTh represent various otherelements from the preparationprocedure and the instrument

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particle emitter. Its biological half-life has beencalculated to be over 400 years (Hursh et al., 1957).

There is a voluminous literature on Thorotrastand the liver. The induction of liver cancer inindividuals with Thorotrast deposits has gainedparticular attention (Smoron and Battifora, 1972;Kiely et al., 1973).

In these studies the presence of Thorotrast has beenproved by light microscopic autoradiography andby morphological studies with the electron micro-scope. However, only analytical electron microscopycan determine in situ the exact elementary composi-tion of the deposits.

In this study we identify and demonstrate thesub-cellular localisation of Thorotrast particlesinjected more than 30 years ago. The Thorotrastparticles were mainly located in liver macrophages(Kupffer cells). The liver tissue appeared otherwiseundamaged. Electron microscopic examinationshowed that the particles were retained insideresidual bodies. With the analytical electron micro-scope it was possible to identify thorium-containingdeposits.A major limiting factor in x-ray microanalysis is

the possible loss and redistribution of elementsduring conventional preparation techniques (Morganet al., 1975). Fixation and embedding of the tissuemay further introduce new elements into thesubstrate to be analysed. Nevertheless, whenfirmly bound elements are investigated, the routinepreparation for transmission electron microscopy

may be adequate (Yarom et al., 1976). As shownin this study, thorium deposits in the liver areeasily detected by this method.

References

Franke, W. W., Krien, S., and Brown, R. M., Jr. (1969).Simultaneous glutaraldehyde-osmium tetroxide fixationwith postosmication. Histochemie, 19, 162-164.

Hursh, J. B., Stedman, L. T., Looney, W. B., andColodzin, M. (1957). Excretion of thorium andthorium daughters after Thorotrast administration.Acta Radiologica, 47, 481-498.

Kiely, J. M., Titus, J. L., and Orvis, A. L. (1973). Thoro-trast-induced hepatoma presenting as hyperpara-thyroidism. Cancer, 31, 1312-1214.

Morgan, A. J., Davies, T. W., and Erasmus, D. A.(1975). Changes in the concentration and distributionof elements during electron microscopic preparativeprocedure. Micron, 6, 11-23.

Reynolds, E. S. (1963). The use of lead citrate at highpH as an electron opaque stain for electron microscopy.Journal of Cell Biology, 17, 208-213.

Smoron, G. L., and Battifora, H. A. (1972). Thorotrast-induced hepatoma. Cancer, 30, 1252-1259.

Yarom, R., Peters, P. D., and Hall, T. A. (1976). Pre-paration of tissues and cells for x-ray microanalysis.Sixth European Congress on Electron Microscopy,2, 232-234.

Requests for reprints to: Dr Arne 0degaard, Departmentof Pathology, The Norwegian Radium Hospital, Monte-bello, Oslo 3, Norway.

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