Histochemical demonstration of two mercury pools in trout tissues: Mercury in kidney and liver after...

ECOTOXICOLOGY AND ENVIRONMENTAL SAFETY 12,267-282 (1986)

Histochemical Demonstration of Two Mercury Pools in Trout Tissues: Mercury in Kidney and Liver

after Mercuric Chloride Exposure

ERIK BAATRUP, MOGENS GISSEL NIELSEN, AND GORM DANSCHER

Institute ofAnatomy B, (NeurobioIogy) and Institute of Zoology and Zoophysiology, University ofAarhus, DK-8000 Aarhus C, Denmark

Received June 13. I986

Juvenile rainbow trout (Sabno gairdneri) were exposed to 100 ppb mercury (as HgClr) in the water for 14 days. Concentrations of mercury in water and fish organs were monitored using radiolabelcd mercury. Tissues from kidney and liver were fixed, and sections were developed by autometallography, a method whereby accumulations of mercury sulfides and/or mercury selenides are silver amplified. In the kidney, mercury was found within lysosomes and extracellularly in the basal lamina of proximal tubules. In the liver, mercury was found within lysosomes of the hepatocytes. Additional groups of mercury-exposed trout were subjected to selenium (as NarSeO,), administered intraperitoneally 2 hr before fixation. Following this treatment, additional mercury could be visualized in the kidney circulatory system, including glomeruli, and in the nucleus and endoplasmic reticulum of liver cells. It is suggested that the mercury visualized prior to selenium treatment represents inorganic mercury, while additional mercury visualized after selenium administration represents an organic form. 0 1986 Academic Press, Inc.

INTRODUCTION

Fish accumulate mercury as a result of an efficient uptake and a slow rate of elimination (Olson et al., 1973; Rouhtula and Miettinen, 1975). The concentration of mercury attained by a specific tissue depends on the mercurial in question, its concentration in water or food, time of exposure, and the rate of metabolism or elimination from the tissue (Hannerz, 1968; Matida et al., 197 1; McKim et al., 1976; Olson et al., 1978).

Mercury is highly toxic to fish, inducing morphological (McKim et al., 1976; Gill and Pant, 1985) and physiological defects (Hawryshyn et al., 1982; Menezes and Qasim, 1984) with consequent behavioral abnormalities (Matida et al., 1971; Rodgers and Beamish, 1982). In order to understand where and how mercury ex- presses its toxic effects it is necessary to determine where mercury accumulates within the fish.

The distribution of mercury among organs has been studied in a number of fish species (Hannerz, 1968; Backstrom, 1969; Matida et al., 197 1). This has provided valuable information on the retention and elimination of mercury in fish organs under various conditions. Most of these investigations have applied quantitative methods (reviewed by Drabaek and Carlsen, 1984), which can detect minute amounts of mercury within a tissue sample, but do not allow to localize mercury deposits within an organ and its cells.

The subcellular distribution of mercury has been approached by measuring labeled mercury in centrifugal fractions of disrupted tissue cells (Olson et al., 1978; Passino and Kramer, 1982). Assuming pure fractions and intact organelles, despite the rough

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268 BAATRUP, GISSEL NIELSEN, AND DANSCHER

FIG. 1. Principles of autometallography (physical development). Accumulations of mercury sulfide and/ or mercury selenide can act as electrodes that conduct electrons from reducing molecules (Hq) to silver ions if both units adhere to their surfaces. As the reduced silver ions have the same catalytic capability, a silver amplification of the original mercury will result.

treatment, this method allows mercury bound to the nucleus to be distinguished from that in various cytoplasmic compartments. Autoradiography was used by Backstrom (1969) to show the distribution of various mercury compounds among fish organs. Still, autoradiography is unacceptable at higher resolution work because of the relatively energetic beta radiation from the *03Hg isotope.

The histochemical method of autometallography is the most sensitive method for visualizing mercury within an organ and its cells. When bound to sulfide or selenide as few as 10 mercury atoms can be made visible at both light and electron microscopic levels (Danscher and Moller-Madsen, 1985). As shown in Fig. 1, autometallography (physical development-see Discussion) is based on a “photographic” process, in which accumulations of mercury sulfide or mercury selenide molecules in the tissues act as catalysts, i.e., electron carriers. Silver ions adhering to the surface of the catalyst receive electrons from likewise adhering reducing molecules, and are thus trans- formed into metallic silver atoms which stick firmly to the catalyst. Because the newly formed silver atoms are catalytic themselves, the process will continue, and layer upon layer of silver will encapsulate the original catalyst. The precise localization of mercury in the tissue can then be seen and photographed at desired magnifications.

In the present study we used autometallography to visualize mercury deposits in kidney and liver tissues from trout exposed to a sublethal concentration of mercuric

VISUALIZATION OF MERCURY IN TROUT TISSUES 269

AIR WATER

RG. 2. Schematic outline of the polyethylene aquaria used in the experiments. Waste was removed by opening the lower valve, A. A constant water level was maintained through water replacements by conduct- ing the water across valve B and the overflow device.

chloride (HgC12). Two autometallography methods have been used. In one procedure mercury bound to endogenous sulfide was made visible by silver amplification. In the other, selenium was used to precipitate additional mercury as HgSe, which could then be amplified by silver. The study is the initial step in a continuing effort to elucidate the histological and ultrastructural locations of mercury in trout tissues.

MATERIALS AND METHODS

Juvenile rainbow trout (Salvo guirdneri) were obtained from a private hatchery and brought to the aquarium facilities in the Department of Zoology, University of Aarhus. The fish were held at 13°C and a 12-hr photoperiod in a 700~liter aquarium filled with chlorine-free tap water. The water was constantly recirculated through a cotton filter and aerated. The fish were fed daily on a commercial trout fodder (Silver Cup, Denmark) with amounts equivalent to l-2% of their body weight. During the 2-month acclimation period the fish increased their weight from about 0.4 to 2 g.

All experiments were carried out in circular, polyethylene aquaria (Fig. 2) containing 4 liters of water. Each aquarium had a funnel-shaped bottom with an outlet at the lowest point so that feces and uneaten food could be removed by opening the valve. This arrangement made it possible to maintain clean water in the aquaria with very iittle replacement and without disturbing the fish. An overflow device was used for total water replacements. The water was aerated to oxygen saturation (10.4 mg 02/liter); the pH was 7.8, and total hardness was 2.7 mmol/liter.

Mercury and Selenium Exposures

In experiments involving mercury, the water contained 100 ppb Hg (as HgC&) including 10 &i/liter 203Hg (as 203HgC12). The concentration of mercury in the water was controlled daily by measuring the radioactivity. Samples of water (5 ml) were taken from the aquaria and their radioactivities were compared with those of the initial samples. Proportional amounts of HgC12 and 203HgC12 were then added in order to bring the concentration of mercury back to the initial 100 ppb. Pilot experiments showed that the mercury concentration decreased up to 20% after 24 hr and


in some cases up to 50% after 48 hr. Apart from the mercury absorbed by the fish a substantial part was lost to the polyethylene wall; probably due to microbiological growth, since a rather radioactive coating was easily removed. Every third day the fish were transferred to clean aquaria with freshly made solutions.

The uptake of mercury by the whole fish (“whole body”) and nine selected organs was determined for five fish by measuring the uptake of *03Hg. After 14 days of mercury exposure and a 2-hr clearing period in clean tap water, the fish were anesthetised in a l-m&f ethyl m-aminobenzoate solution and weighed. The radioactivity of each fish was measured in a 2 X 1 i in. NaI well-crystal connected to a one-channel an- alyzer, and the overall amount of mercury taken up by the fish was calculated by comparing the counting rate with that of a “standard fish” with known *03Hg content. Tissue samples from the posterior kidney, the liver, the intestines, the gills, and the muscle were then removed together with the brain, the spleen, the gall bladder, and the stomach. After the samples were weighed their radioactivities were measured in the same crystal as described above, and the counting rates were compared with a standard in order to calculate the uptake of mercury.

In experiments designed to label tissue mercury by selenium, some mercury exposed fish as well as nonexposed fish were treated intravitally with selenium (as Na2- Se03). The fish were anesthetised and 1 .O mg Se/kg fish was injected intraperitoneally. The fish were then returned to the aquarium for 2 hr.

Experiments

Four aquaria (I-IV), each containing five fish, made up the experimental series. The aquaria were set up at the same time and were treated identically except for mercury and selenium treatments. The fish in aquaria I and II were exposed to mercuric chloride while those in aquaria III and IV were kept in tap water. After 14 days the aquaria were divided into the following combinations: (I) mercury, (II) mercury + selenium, (III) selenium, (IV) control.

Histological Procedures

Fixation and embedding. After mercury and/or selenium treatments the fish were anesthetised. The body cavity was opened ventrally by a median incision and flushed with fixative, containing 2% glutaraldehyde and 1% paraformaldehyde in 0.08 M phosphate buffer (pH 7.2). Tissue blocks less than 1 mm3 were removed from the posterior kidney and the liver and immersed in the fixative. Alternatively, kidneys and livers were fixed by perfusion through one of the posterior cardinal veins. Tissue blocks were then cut out and immersed in the fixative. After 2 hr at room temperature the fixation continued at 4°C overnight. The tissue blocks were then rinsed in the buffer, dehydrated, and embedded in Epon 8 12. Three-micrometer sections were cut and fastened to glass slides on a heating plate at 45°C for 2 hr. Control material from untreated trout was always included.

Development. The mercury in the tissue was silver amplified according to the auto- metallographic method of Danscher and MBller-Madsen (1985). Prior to development the slides were dipped in 0.5% gelatin to coat the sections. The developer was made up immediately before use by mixing (1) 60 ml gum arabic (from lumps of crude acacia tree resin); (2) 10 ml buffer (0.80 Msoclium citrate in 1.27 Mcitric acid); (3) 15 ml reducing agent (0.5 1 M hydroquinone); (4) 15 ml silver ion supply (0.03 M


FIG. 3. Histogram showing mean concentrations (+ SD) of mercury attained by five fish (whole body) and nine tissues. The fish were exposed to 100 ppb Hg, including 5 &i *03HgJiter for 14 days. The intes tines, stomachs, and gall bladders were emptied before measuring the uptake of *“Hg.

silver lactate). The slides were placed in jars filled with the developer and kept in a water bath at 26°C for 45 min in darkness.

After development the sections were washed in running tap water at 40°C for 10 min. The slides were then immersed in an ordinary film fixative for 10 set and rinsed in distilled water. Finally, the slides were dipped in Farmer’s solution (9 parts of 10% sodium thiosulfate plus 1 part of 10% potassium ferricyanide) for 10 set and rinsed in distilled water. Further details on the procedure are given in Danscher and M@ller- Madsen ( 1985).

Preparation for electron microscopy. Following silver amplification, the sections were stained with toluidine blue and examined in the light microscope. Selected sections were covered with a drop of liquid Epon and a blank Epon block placed on top. After polymerization, the block and attached section were removed from the slide by a brief heating on a 90°C heating plate. Thin sections for electron microscopy were cut and counterstained with lead citrate and uranyl acetate.

RESULTS

The amounts of mercury incorporated by the whole fish (“whole body”) and nine selected organs were determined by measuring the uptake of 203Hg. The mean concentrations from five fish which had been exposed to 100 ppb Hg and 10 &i/liter 203Hg for 14 days are shown in Fig. 3. Of the nine organs, the kidney showed by far the highest accumulation of mercury (42.3 ppm) followed by the gills (2 1 .O ppm), the spleen (10.5 ppm), and the liver (8.6 ppm). Gall bladders, when empty, contained 5.6 ppm of mercury, whereas gall bladders filled with bile contained 60 ppm mercury.

Location of Suljide Associated Mercury

Kidney tissue. The light microscopic appearance of the posterior kidney from an untreated fish is shown in Fig. 4. The circular or oblong arrangements of cells are

FIG. 4. Light micrograph from the kidney of an untreated (control) fish. The various nephron segments, including renal corpuscles (RC), proximal segments (PS), distal segments (DS), and collecting tubules (CT) are embedded in an abundant interstitial tissue. Pigment cells (PC) are numerous in the trout kidney. This section was subjected to autometallography, but showed no silver precipitates. Scale bar 20 pm.

FIG. 5. After exposure to 100 ppb Hg for 14 days and subsequent autometallography, numerous black grains were displayed in kidney sections. The grams, showing the localization of mercury deposits, are mainly situated in the epithelia and basal lamina (BL) of proximal segments (PS), but may be found also in other segments and in interstitial cells (arrows). (GL) glomerulus of renal corpuscle. Scale bar 20 pm.

272


nephrons cut at different angles. Several of the various nephron segments can be found within a random section (Fig. 4). A detailed description of the nephron segments and their histological characteristics is given in, e. g., Hickman and Trump (1969) and Trump et al. (1975). The nephrons are embedded in an abundant inter& tial tissue, containing conspicuous pigment cells with dense accumulations of dark granules (Fig. 4).

Silver amplification revealed clear differences between the kidney tissues of the control and the mercury-treated fish. Most conspicuous were the accumulations of dark grains within the nephron epithelium of the mercury-treated fish. In the toluidine-stained sections, the black or brown deposits were easily distinguished from the blue-staining cell organelles. The highest density of grains was found in the epithelium of certain proximal tubules, where they formed a dark layer in the supranuclear region (Fig. 5). Grains were also seen along the tubule periphery in connection with the basal lamina (Fig. 5). Extensively vacuolized cells were often seen in epithelia with a high density of precipitates.

Silver grains were found also in other nephron segments, but not as numerous as in the proximal tubules, and more scattered within the epithelium. Only the collecting tubules were devoid of grains. Silver precipitates in the interstitial tissue were difficult to distinguish from the pigment granules, although their presence became evident when comparing developed and nondeveloped sections. Unspecific silver precipitates were not observed, and tissue from control fish not treated with mercury showed no silver grains at all (Fig. 4).

At the ultrastructural level the grains were seen to consist of accumulations of smaller silver precipitates with a predominant circular outline. Intracellularly, the precipitates were almost exclusively situated within lysosomes (Fig. 6), the only ex- ception being a few grains near the apical surface (Fig. 7). Lysosomes labeled with silver precipitates were especially numerous in proximal tubule cells, where the highest concentration of precipitates within individual lysosomes was also found.

Cell injuries were often seen in epithelia with heavily loaded cells. The damages ranged from swollen tubular cells to advanced necrosis. Lysosomes with irregular outlines and disrupted membranes, or completely disintegrated lysosomes were com- mon (Fig. 9). Among the various interstitial cell types silver precipitates were found in the lysosomes of macrophages and what appeared to be leukocytes at various im- mature stages. Severe injuries were not observed in these cells although the density of precipitates was as high as in the proximal tubule cells.

Extracellularly, silver grains were found in the basal lamina surrounding the nephron (Fig. 6), including that of the capsular epithelium. The distribution and density of precipitates along the basal lamina clearly reflected the amount of silver grains in apposing tubular cells. Macrophages with silver labeled lysosomes were often seen close to the basal lamina (Fig. 8). In the tubular lumen precipitates were occasionally observed on the surface of microvilli.

In contrast to the mercury-treated fish, silver precipitates were not observed in the kidney cells of control fish.

Liver tissue. In the light microscope only few silver grains were seen in developed sections from livers of mercury-exposed trouts (Fig. 10). Those found were situated in the cytoplasm of the hepatocytes.

Ultrastructural observations revealed that the grains in liver tissue also originated in lysosomal accumulations of silver precipitates (Fig. 11). Within the hepatocytes,

FIG. 6. Electron micrograph showing a proximal tubule cell from a mercury exposed fish. Intracellularly, the silver amplified mercury deposits are located almost exclusively within lysosomes (LY). Precipitates are seen also along the basal lamina (BL). (TL) tubular lumen. Scale bar 1 pm.

FIG. 7. Visualized mercury was occasionally found in the apical region of proximal tubule cells. Whether these precipitates were related to, e.g., endocytic vesicles was not established. (MV) microvillus; (LY) lyso- some; (TL) tubular lumen. Scale bar 0.5 pm.

FIG. 8. Macrophages with silver labeled lysosomes (LY) often apposed the proximal tubules. (TE) tubular epithelium. Scale bar 2 pm.

274


FIG. 9. Electron micrograph showing irregular and disintegrated lysosomes within a proximal tubule cell. Such cell injuries cohere with a high mercury content. Scale bar 1 pm.

the silver labeled lysosomes were commonly situated in the region facing a bile canaliculus.

Tissue from control fish did not contain silver precipitates.

Location of Selenium Associated Mercury

Kidney tissue, From light microscopy on kidney tissue, it appeared that the general distribution of silver grains in the mercury-treated fish was unchanged following selenium treatment. Except for the collecting tubules, silver precipitates were found in all nephron segments, with the highest density constantly seen in the epithelia of proximal tubules (Fig. 12). However, following selenium treatment additional mercury deposits were visualized in glomerular endothelium and mesangial cells (Fig. 12). Further, selenium induced silver amplification of mercury deposits in erythrocytes (Fig. 12).

Electron microscopy indicated that the silver precipitates were still confined to the lysosomes in tubular and interstitial cells after selenium treatment, i.e., no additional subcellular locations of mercury deposits were observed in these cells. In the kidney circulatory system, it appeared that selenium induced visualization of mercury in glomerular cells and in the endothelium and surrounding muscle cells of smaller arteries as well (Fig. 13). In endothelium and mesangial cells mercury deposits were seen within lysosomes, but most precipitates were located in the cell periphery, bound to the filaments of the terminal web. In the smooth muscle cells mercury deposits were mainly seen in specific granules (Fig. 13). The presence of silver labeled erythrocytes was confirmed in the electron microscope. The precipitates were found in both the nucleus and in the hemoglobin filled portion (Fig. 14).

276 BAATRUP, GlSSEL NIELSEN, AND DANSCHER

FIG. 10. Light micrograph from the liver of a fish treated with HgClz. Following autometallography scattered accumulations of silver grains (arrows) can be seen around bile canalicuh (BC). (BV) blood vessel: (ET) erythrocytes. Scale bar 10 pm.

FIG. 11. Micrograph demonstrating the lysosomal (LY) localization of visualized mercury in trout liver cells. Labeled lysosomes were usually found near a bile canaliculus (BC). Scale bar 1 rm.

FIG. 12. Micrograph from the kidney of a trout treated with selenium after mercury exposure. The general distribution of silver precipitates was not changed by selenium; the highest density was constantly seen in the epithelia of proximal segments (PS). However, selenium induced visualization of mercury in glomeruli, including the erythrocytes (inset). Scale bar 20 pm, 10 pm (inset).

FIG. 13. Electron micrograph showing the wall of an artery in the trout kidney. Following selenium treatment mercury deposits (arrows) were displayed in the cytoplasm of the endothelial cells (EC) and in granules of surrounding smooth muscle cells (SM). Scale bar 1 pm.

FIG. 14. Silver precipitates were displayed in erythrocytes after administration of selenium. Note that precipitates are seen also in the nucleus (white arrows). Scale bar 1 pm.

211


Liver tissue. The histological appearance of liver tissue from mercury exposed fish was quite different after selenium treatment. Numerous silver grains were displayed in the cytoplasm of hepatocytes, predominantly in the region facing a bile canaliculus (Fig. 15). In addition, each nucleus contained one large precipitate surrounded by several smaller (Fig. 15). As in the case of kidney tissue silver precipitates showed in erythrocytes after administration of selenium.

Electron microscopy conf&med the increased stainability of liver tissue after selenium treatment. The highest density of cytoplasmic silver precipitates was still seen in the lysosomes, including autophagic vacuoles (Fig. 16), but unlike livers not treated with selenium, precipitates were also found outside lysosomes. These precipitates were always associated with the rough endoplasmic reticulum. Moreover, silver precipitates inside the nucleus constituted an increase in visualized mercury. The large nuclear grain observed in the light microscope proved to be accumulations of smaller precipitates in the nucleolus. As shown in Fig. 16 the grains were restricted to the dense material of the nucleolus. Scattered precipitates were also seen elsewhere in the nucleus. Extracellularly, precipitates were occasionally observed within the canalicu- lar lumen (Fig. 16).

Developed liver sections treated with selenium, but not mercury were observed to have no trace of silver grains at either light or ultrastructural levels.

DISCUSSION

Radiometric Data

Fish may absorb mercury from the water across the respiratory surfaces (Olson et al., 1973) and/or from the consumed food within the digestive tract (Phillips and Buhler, 1978). We administered the mercuric chloride by adding it to the water, since inorganic mercury is poorly absorbed from the intestine (Nagashima et al., 1984, Boudon and Ribeyre, 1985). We assume that most mercury was absorbed as Hg*+, but it is likely that some was converted by microorganisms or mucosal reactions (Jer- neliiv and Lann, 197 1; Rudd et al., 1980) and absorbed by the fish as organic mercury (e.g., CH3Hg+).

Our estimates of total mercury uptake by the fish and the distribution of mercury among the analyzed organs parallel previous data on fish (Matida et al., 197 1; Weis- hart, 1973). By far the highest concentration of mercury was found in the kidney, which is a well-known target organ of inorganic mercury (Gritzka and Trump, 1968; Weisbart, 1973). The high mercury content in gall bladders tiled with bile agrees with findings in mammals (Norseth and Clarkson, 197 l), and supports that the liver also in fish is important in the excretion of mercury (Olson et al., 1973).

The quantitative analysis of mercury in the tissues was, however, primarily included as a reference to the histochemical demonstration. By treating the fish used for quantitative measurements under the same conditions as those used for microscopy, we were able to compare the relationship between visualized mercury and the amount ofmercury actually taken up by the fish.

Histochemical Localization

The present autometallography method has been worked out for demonstrating metals and metal containing molecules, including mercury in mammalian tissues (Danscher, 1984; Danscher and Moller-Madsen, 1985; M@ller-Madsen and Dan-


FlG. 15. The number of silver precipitates increased considerably in liver cells when the fish was post- treated with selenium. Besides an increased stainability of cytoplasmic mercury (CM) (compare with Fig. lo), selenium also displayed mercury bound in the nuclei (arrows). Also, erythrocytes (ET) were labeled. Scale bar 10 pm.

FIG. 16. Electron micrograph showing the subcellular distribution of visualized mercury deposits following selenium treatment. Precipitates were still numerous in the lysosomes (LY), but showed also on the endoplasmic reticulum (arrows), and occasionally in the lumen of a bile canaliculus (BC). Nuclear deposits were predominantly situated in the nucleolus (NU), bound to the electrondense material (inset). Scale bar 1 ,um, 1 pm (inset).


scher, 1986). It has been established that mercury bound to endogenous sulfide makes out catalytic centers for the reduction of silver ions to metallic silver (Timm et al., 1966; Danscher and MBller-Madsen, 1985). The method has not previously been used on fish tissues, but the fact that silver grains appeared only in animals treated with mercury, and not in the controls, strongly suggests that the catalytic centers were created by Hg-S molecules.

Intracellularly, mercury was found predominantly in the lysosomes of kidney and liver cells. Extracellularly, mercury was seen to accumulate in the basal lamina of kidney proximal segments. These results are consistent with the distribution of mercury in the proximal tubules of the rat (Miura et al., 198 1). These authors used a histochemical gold impregnation method to demonstrate mercury accumulations after administration of HgC12.

The cell injuries observed in the present study were closely correlated to the burden of mercury deposits, and agree with previous microscopic studies on the toxic effects of mercury in fish (Trump et al., 1975) and rat (Gritzka and Trump, 1968).

The stainability of kidney and liver tissues did not parallel the uptake of *03Hg. From the quantitative measurements, the density of silver grains in liver sections was expected to be about one-fifth of the density in kidney sections (8.6 ppm/42.3 ppm). However, even without counting silver grains in the liver sections, it was evident that this organ contained less stainable mercury than expected. This result suggests that a smaller fraction of the total mercury formed catalytic centers with sulfur in liver than in kidney. It is believed that each catalytic center consists of crosslinked mercury and sulfur atoms, involving 10 or perhaps fewer mercury atoms (Danscher and Moller- Madsen, 1985). A lower level of available sulfur in liver than in kidney might account for the relatively fewer silver precipitates in the liver sections. A reduced number of catalytic centers in liver tissue would also result if some of the mercury was present in a monovalent (CHsHg+), organic form, incapable of crosslinking the sulfide ions. This latter interpretation implies that only inorganic mercury (Hg2’) was demonstrated without prior selenium treatment.

It is well known that mercury has a high affinity for selenium in organic tissues (Magos and Webb, 1980) and form HgSe complexes, which can be made visible by autometallography (Danscher, 1982). In the present study, selenium induced visualization of mercury at new locations in both kidney and liver tissues, indicating the presence of a separate mercury pool. The following three observations seem to sup- port the theory of the selenium-mercury pool being organic mercury.

(1) In the kidney, selenium displayed additional mercury only in the circulatory system, while the stainability of the renal tubules was unaltered. These findings agree with previous reports of the high affinity of kidneys in fish and rats for inorganic mercury (McKim et al., 1976; Norseth and Clarkson, 197 1).

(2) In the liver cells, selenium treatment caused visualization of additional mercury deposits in the nucleus and the endoplasmic reticulum. This observation is consistent with the subcellular distribution of organic mercury in the rat liver, found by Norseth and Brendeford (197 1).

(3) Mercury accumulations were demonstrated in erythrocytes only after the administration of selenium. It has been shown that inorganic mercury binds almost exclusively to the plasma fraction in fish blood, while organic mercurials bind to the erythrocytes (Olson et al., 1973).


Further studies have been undertaken to confirm whether the selenium-mercury pool represents organic mercury, and, if so, whether it represents all or only a fraction of the organic tissue mercury.

ACKNOWLEDGMENTS

The authors acknowledge the technical assistance of Mrs. Herdis B. Andersen, Ms. Bertha Beth, Mrs. Karin Wiedemann, Mrs. Kirsten Baatrup, Mr. Bjame Krunderup, and Mr. Albert Meier. We also thank Mrs. Else Rasmussen and Dr. Christopher J. Frederickson for comments on the manuscript. The work was supported by The Faculty of Science, University of Aarhus, and Aarhus University Research Foundation.

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Histochemical demonstration of two mercury pools in trout tissues: Mercury in kidney and liver after...

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