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Tissue Uptake of Bismuth from Shotgun Pellets
Environmental Research Section A 82, 258}262 (2000) doi:10.1006/enrs.1999.4016, available online at http://www.idealibrary.com on
Tissue Uptake of Bismuth from Shotgun Pellets Roger Pamphlett,*,1 Gorm Danscher,- J+rgen Rungby,- and Meredin Stoltenberg*Department of Pathology, University of Sydney, NSW 2006, Australia; and -Department of Neurobiology, Institute of Anatomy, University of Aarhus, DK-8000, Aarhus C, Denmark Received May 10, 1999
pellets on the environment, and in particular on animals wounded by these pellets, however, is not known. This is potentially important, since the popularity of recreational hunting has meant that a number of wild animals and birds are wounded by shotgun pellets but survive for prolonged periods of time. From human clinical experience it is known that bismuth-related toxicity is not directly related to the dose or duration of bismuth exposure (MartinBouyer et al., 1980). We were interested therefore to see whether bismuth could be released from shotgun pellets and enter the tissues of animals. Until recently it was dif7cult to localize small amounts of bismuth within tissues. However, when mice were injected with large doses of bismuth subnitrate intraperitoneally and their tissues examined with the technique of autometallography (AMG), bismuth was claimed to be detected in sections of their tissues (Ross et al., 1994). AMG is a histochemical technique that can detect mercury, gold, silver, and zinc in tissues because these metals are surrounded with a collar of reduced silver molecules (Danscher et al., 1997). Proton induced X-ray microanalysis (PIXE) has demonstrated that the AMG grains in sections from bismuth-exposed animals encase bismuth and sulfur (Danscher et al., in preparation). Having con7rmed the speci7city of the AMG bismuth technique, we used this method to look for bismuth that could be released from shotgun pellets that had been inserted into mice. In addition, because the pellets contained a small amount of tin, we also ensured that tin could not give rise to AMG silver grains.
Shotgun pellets containing bismuth have been suggested to be less environmentally toxic than those containing other metals. We sought to And if bismuth from shotgun pellets embedded within an animal enters the tissues of that animal. Five bismuth-containing shotgun pellets were placed intraperitoneally into adult mice. Four or 9 weeks later the tissue distribution of bismuth was examined histologically using silver lactate autometallography. Bismuth was seen in the nervous system of the mice, either in cells with processes outside the nervous system or in cells not protected by the blood-brain barrier. Bismuth was also seen in the kidney, liver, spleen, and lung. The amount of bismuth within tissues varied widely between animals at both time intervals. Bismuth from shotgun pellets enters the tissues of mice, with some mice taking up more bismuth than others. Some animals wounded with bismuth pellets are therefore likely to accumulate large amounts of potentially toxic bismuth in their tissues. ( 2000 Academic Press Key Words: bismuth; toxicity; heavy metal; shotgun pellets; autometallography.
INTRODUCTION
Because of environmental concerns, the use of lead in shotgun pellets has been forbidden in many countries, including Denmark, so interest has arisen in 7nding different materials for pellets. Iron pellets have been recommended from an environmental point of view, but these have poor ballistic qualities because of their lightness, and iron embedded in tree trunks can damage forestry equipment. Bismuth pellets have gained popularity because of their good ballistic qualities and have been in use in the United States for a number of years. The impact of these
MATERIALS AND METHODS
Animals Six-week-old BALB/c mice were used in all experiments. Animals were kept four to a cage in a room with a 12-h light/dark cycle and a temperature of
1
To whom correspondence should be addressed. Fax: int#(612) 9531-3429. E-mail: [email protected]. 258 0013-9351/00 $35.00 Copyright ( 2000 by Academic Press All rights of reproduction in any form reserved.
259
BISMUTH FROM SHOTGUN PELLETS
RESULTS
21}22@C. Mice were given a continuous supply of Altromin No. 1324 rat and mouse diet and tap water. Pellet Analysis The average weight of 10 shotgun pellets (Eley 12) was 0.17g (SD 0.01 g). Proton-induced X-ray emission, performed at the Danish Environmental Research Institute, Roskilde, showed that the pellets contained 91% bismuth and 9% tin. Pellet Placement Eight mice were anesthetized with halothane and 7ve alcohol-cleaned pellets were inserted into the peritoneal cavity though a 0.5-cm incision in the anterior abdominal wall. Four mice had sham operations with no insertion of pellets. Incisions were closed with two Clay Adams stainless-steel autoclips (Beckton Dickinson), which were removed 7 days later. Preparation and Staining of Tissue At 4 or 9 weeks after pellet implantation, four mice were anesthetized with halothane and perfused through the left ventricle of the heart with 50 ml of 3% glutaraldehyde in phosphate buffer. The right lung, liver, right kidney, spleen, four lumbar posterior root ganglia, brain, and cervical and lumbar spinal cord were removed and post7xed in glutaraldehyde for 48 h. After 7xation, blocks were immersed in 30% sucrose overnight, mounted on chucks with Tissue Tek OCT Compound (Sakura), and frozen with carbon dioxide. Serial 30-lm sections were cut in a cryostat and placed in physical developer comprising 60 ml of 50% gum arabic, 10 ml citrate buffer, 30 ml of 5.6 g/100 ml hydroquinone, and 0.5 ml of 17 g/100 ml of silver lactate and kept in the dark at 26@C for 60 min (Danscher and M+ller-Madsen, 1985). Sections were counterstained with toluidine blue. Black granules represent encased bismuth sul7de molecules (BiAMG). The nervous system distribution of BiAMG was mapped with the aid of a mouse brain atlas (Franklin and Paxinos, 1997).
The animals showed no ill effects from the operation or from the intraabdominal pellets and gained weight, moved, ate, and drank normally. Bismuth Uptake No BiAMG was seen in the tissues of any of the sham-operated mice. BiAMG was seen in the organs of all mice with intraperitoneal pellets. Within the affected cells, BiAMG was present in the cytoplasm of the cell bodies, but not in the nuclei. At both survival intervals the tissue distribution of bismuth was similar. However, the amount of BiAMG within cells varied widely between animals, in both the 4and 9-week interval groups. Cerebrum. BiAMG was present within neurons in the supraoptic, paraventricular (Fig. 1), suprachiasmatic, and arcuate nuclei. The median eminence adjacent to the arcuate nuclei stained positively. The subfornicial organ stained strongly, with BiAMG extending into the white matter of the supraadjacent hippocampal commissure. The vascular organ of the lamina terminalis stained moderately. No staining was seen in cells in the cerebral cortex, basal ganglia, or thalamus. Cerebellum. No BiAMG was seen in the cerebellar cortex or deep nuclei. Brain stem. BiAMG was found in neurons of the trochlear, oculomotor, mesencephalic trigeminal, motor trigeminal, abducens, facial, ambiguus, and
Testing the AMG Method for Tin Four mice were injected intraperitoneally with stannous chloride at doses of 1, 2, 4, or 8 lg/g. One week later, the animals were perfused as above, and 30-lm frozen sections of lumbar spinal cord were stained with AMG.
FIG. 1. Coronal section of the cerebrum showing BiAMG staining in the supraoptic (7lled arrow) and paraventricular (open arrow) nuclei, at 9 weeks after pellet implantation. opt, optic tract. AMG and toluidine blue. Magni7cation, ]65.
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FIG. 2. In the medulla oblongata, both the hypoglossal nuclei (12) and the area postrema (AP) stain positively for bismuth at 9 weeks after pellet implantation. Patchy staining is present in the ependyma of the cerebral aqueduct (arrow), but this was also seen in controls. AMG and toluidine blue. Magni7cation, ]90.
FIG. 3. Black granules of silver-enhanced bismuth (BiAMG) are present in the cytoplasm of large motor neurons in the lumbar spinal cord, at 4 weeks after pellet implantation. AMG and toluidine blue. Magni7cation, ]1250.
hypoglossal nuclei (Fig. 2). The area postrema also stained positively for BiAMG (Fig. 2).
reduce silver ions to metallic silver on their surfaces if placed in an autometallographic developer (Danscher et al., 1997). As a result, AMG grains will be created exactly where the clusters are created in vivo or in vitro. Recently, bismuth has been added to the list of metallic ions that can be visualized with this technique (Ross et al., 1994; Danscher et al., 1997). Since the shotgun pellets contained a minor amount of tin, it was important to know whether tin could create catalytic sul7de or selenide clusters that could be silver-enhanced by AMG. We therefore injected some animals with stannous chloride alone,
Spinal cord. A large amount of BiAMG was seen in the cell bodies of large motor neurons in the anterior horn of the cervical and lumbar spinal cord (Fig. 3). More staining was seen in lumbar than in cervical motor neurons. Posterior root ganglia. About 40% of large neuron cell bodies in the spinal ganglia contained BiAMG. Other nervous system cells. A patchy uptake of bismuth was seen in the leptomeninges. Silver staining was seen in the ependyma, but this was present equally in control animals. The choroid plexus showed no signi7cant BiAMG staining. General organs. In nonnervous tissue, BiAMG was seen in cell bodies of renal tubular cells (Fig. 4), macrophages in the lung, and dendritic cells in the liver and spleen. Tin Uptake No AMG silver deposits were seen in the lumbar motor neurons of any animals injected intraperitoneally with stannous chloride. DISCUSSION
AMG for Bismuth Nanoclusters of metallic gold, silver, mercury, and zinc ions, combined with sul7de or selenide ions, can
FIG. 4. In the kidney, heavy BiAMG staining is seen in the renal tubules, while the glomeruli (G) are free of bismuth, at 9 weeks after pellet implantation. AMG and toluidine blue. Magni7cation, ]400.
BISMUTH FROM SHOTGUN PELLETS
since tin ions arising from this salt are likely to be similar chemically to the tin arising from the pellets. Lumbar spinal motor neutrons, one of the 7rst cells in the body to take up xenobiotics (Rungby and Danscher, 1983; Danscher et al., 1997; Pamphlett and Coote, 1997), showed no grains after development. Although bismuth was not detected directly in the granules by elemental analysis, we concluded on the balance of probabilities that it is bismuth sul7de or selenide, originating from the intraperitoneal bismuth shotgun pellets, that is being demonstrated by AMG in the tissues of these animals. Nervous System Distribution of Xenobiotics The nervous system distribution of bismuth, with a predominant involvement of motor neurons, parallels that of other xenobiotics such as mercury vapor (M+ller}Madsen, 1992), inorganic mercury (Danscher and M+ller-Madsen, 1985; M+ller}Madsen, 1990), and silver (Rungby and Danscher, 1983). The overall distribution of bismuth is also very similar to that of horseeradish peroxidase injected intravenously into mice (Broadwell and Brightman, 1976). The striking uptake by motor neurons of all of these substances had led to the suggestion that, while most neurons are protected by the blood-brain barrier, these xenobiotics enter distal motor axons at the neuromuscular junction and travel to the cell body by retrograde axonal transport (Arvidson, 1989). This suggestion has been supported by experiments showing retrograde transport of metals injected directly into striated muscle (Arvidson, 1992; Schi+nning, 1993). On the other hand, it is possible that some neurons with very high metabolic activity take up exogenous metals from the blood stream, since after high doses of bismuth large neurons that do not have processes outside the central nervous system can be shown to contain the metal (Ross et al., 1996). With the present model of low-dose xenobiotic uptake, it appears that two pathways are operative. The 7rst is the retrograde axonal route, with the xenobiotic entering the cell bodies of alpha motor neurons (axons terminating in striated muscle), mesencephalic trigeminal neurons (axons terminating in muscle spindles), and supraoptic, paraventricular, suprachiasmatic, and arcuate nuclei (axons terminating in circumventricular organs) (Broadwell and Brightman, 1976). The second pathway is the blood-borne route, with the xenobiotic entering cells not protected by the blood-brain barrier such as the circumventricular organs and the posterior root ganglia. At exposure to very high doses of
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a xenobiotic, a third route appears to be operative, leading to selective uptake by large neurons with processes that are con7ned to the central nervous system (Ross et al., 1996). These neurons were, however, not affected by the doses of bismuth arising from shotgun pellets in this experiment. We saw a wide variation in bismuth uptake from the shotgun pellets between animals. This variation has been seen both in mice given intraperitoneal doses of bismuth subnitrate (where it may be due to varying uptake of the water-insoluble suspension) (Ross et al., 1996) and in humans in whom plasma concentrations of bismuth are not proportional to the dose given (Koch et al., 1996). The cause for the variability in cellular bismuth in our mice is not clear. Potential Damage from Bismuth In humans, the form of toxicity from bismuth depends on the type of bismuth compound. Neurotoxic effects are associated with more insoluble compounds and kidney disorders with the more soluble compounds (Slikkerveer and de Wolff, 1989). The best-documented neurotoxicity was from an epidemic of bismuth encephalopathy in France (MartinBouyer et al., 1978). In this epidemic, symptomatic patients had a wide range of bismuth blood concentrations, from 10 to 4600 lg/L, suggesting a large interindividual susceptibility to bismuth (MartinBouyer et al., 1978). Disturbances of bone mineralization were also noted in patients with bismuth encephalopathy, and another osteoarthropathy has been seen in patients treated with bismuth for syphilis (Slikkerveer and de Wolff, 1989). Animal experiments have shown that bismuth distorts the inner mitochondrial membrane and reduces the activity of heme-synthesizing enzymes (Woods and Fowler, 1987). There is therefore compelling evidence to suggest that exposure to bismuth should be kept to a minimum. A large amount of bismuth was apparent in the renal tubules of our mice. Rodents are not alone in taking up bismuth into their kidneys, since after a bismuth-containing shot was embedded in the breast muscles of ducks, increased concentrations of bismuth were detected in the kidney and liver 1 year later (Sanderson et al., 1998). Animal experimentation has shown that bismuth binds to proteins of low molecular weight in the kidney (Szymanska and Piotrowski, 1980). Soluble bismuth compounds can poison the proximal renal tubules (Slikkerveer and de Wolff, 1989) and reversible renal failure in humans has been seen after intoxication with colloidal bismuth subcitrate (Hudson and Mowat, 1989).
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In the lungs of our mice, bismuth was seen in macrophages, and in the liver and spleen it was present in dendritic cells. Other xenobiotics and in particular inorganic mercury have also been noted to enter cells of the immune system (Christensen, 1996). Of interest, bismuth may inhibit migration of macrophages after it is phagocytosed (Soutar and Coghill, 1986), so damage to the immune system of animals exposed to bismuth is a possibility. CONCLUSION
In conclusion, bismuth arising from shotgun pellets can be detected in the nervous system and other organs of mice. The long-term effects of bismuth on animals that harbor bismuth-containing shotgun pellets are not known. However, given the present knowledge on the toxicity of bismuth compounds, if recreational shooting of wild animals is to continue on a large scale it seems prudent to look for pellet material that is more inert as regards the tissue uptake of its components. ACKNOWLEDGMENTS We thank Dorete Jensen and Thorkild Nielsen for skilled technical assistance. Kas re Kemp of the Danish Environmental Research Institute performed the PIXE analysis.
REFERENCES Arvidson, B. (1989). Retrograde axonal transport of metals. J. Trace Elem. Exp. Med. 2, 343}347. Arvidson, B. (1992). Inorganic mercury is transported from muscular nerve terminals to spinal and brainstem motoneurons. Muscle Nerve 15, 1089}1094. Broadwell, R. D., and Brightman, M. W. (1976). Entry of peroxidase into neurons of the central and peripheral nervous systems from extracerebral and cerebral blood. J. Comp. Neurol. 166, 257}284. Christensen, M. M. (1996). Histochemical localization of autometallographically detectable mercury in tissues of the immune system from mice exposed to mercuric chloride. Histochem. J. 28, 217}225. Danscher, G., Jensen, K. B., Kraft, J., and Stoltenberg, M. (1997). Autometallographic silver enhancement of submicroscopic metal containing catalytic crystallites;a histochemical tool for detection of gold, silver, bismuth, mercury and zinc. Cell Vision 4, 375}386. Danscher, G., and M+ller-Madsen, B. (1985). Silver ampli7cation of mercury sul7de and selenide: A histochemical method for light and electron microscopic localization of mercury in tissue. J. Histochem. Cytochem. 33, 219}228.
Franklin, B. J., and Paxinos, G. (1997). ‘‘The Mouse Brain in Stereotaxic Coordinates.’’ Academic Press, San Diego. Hudson, M., and Mowat, N. A. (1989). Reversible toxicity in poisoning with colloidal bismuth subcitrate. Br. Med. J. 299, 159. Koch, K. M., Davis, I. M., Gooding, A. E., and Yin, Y. (1996). Pharmacokinetics of bismuth and ranitidine following single doses of ranitidine bismuth citrate. Br. J. Clin. Pharmacol. 42, 201}205. Martin-Bouyer, G., Barin, C., Beugnet, A., Cordier, J., and Guerbois, H. (1978). Intoxications par les sels de bismuth administreH s par voie orale. Gastroenterol. Clin. Biol. 2, 349}356. Martin-Bouyer, G., Foulon, B., Guerbois, H., and Barin, C. (1980). Epidemiological aspects of the encephalopathies resulting from oral administration of bismuth. Therapie 35, 307}313. M+ller-Madsen, B. (1990). Localization of mercury in CNS of the rat. II. Intraperitoneal injection of methylmercuric chloride (CH HgCl) and mercuric chloride (HgCl2). Toxicol. Appl. Phar3 macol. 103, 303}323. M+ller-Madsen, B. (1992). Localization of mercury in CNS of the rat. V. Inhalation exposure to metallic mercury. Arch. Toxicol. 66, 79}89. Pamphlett, R., and Coote, P. (1997). Entry of low doses of mercury vapour into the nervous system. Neurotoxicology 19, 39}48. Ross, J. F., Broadwell, R. D., Poston, M. R., and Lawhorn, G. T. (1994). Highest brain bismuth levels and neuropathology are adjacent to fenestrated blood vessels in mouse brain after intraperitoneal dosing of bismuth subnitrate. Toxicol. Appl. Pharmacol. 124, 191}200. Ross, J. F., Switzer, R. C., Poston, M. R., and Lawhorn, G. T. (1996). Distribution of bismuth in the brain after intraperitoneal dosing of bismuth subnitrate in mice: Implications for routes of entry of xenobiotic metals into the brain. Brain Res. 725, 137}154. Rungby, J., and Danscher, G. (1983). Localization of exogenous silver in brain and spinal cord of silver exposed rats. Acta Neuropathol. 60, 92}98. Sanderson, G. C., Anderson, W. L., Foley, G. L., Havera, S. P., Skowron, L. M., Brawn, J. W., Taylor, G. D., and Seets, J. W. (1998). Effects of lead, iron, and bismuth alloy shot embedded in the breast muscles of game-farm mallards. J. Wildl. Dis. 34, 688}697. Schi+nning, J. D. (1993). Retrograde axonal transport of mercury in rat sciatic nerve. Toxicol. Appl. Pharmacol. 121, 43}49. Slikkerveer, A., and de Wolff, F. A. (1989). Pharmacokinetics and toxicity of bismuth compounds. Med. Toxicol. Adverse Drug Exp. 4, 303}323. Soutar, R. L., and Coghill, S. B. (1986). Interaction of tripotassium dicitrato bismuthate with macrophages in the rat and in vitro. Gastroenterology 91, 84}93. Szymanska, J. A., and Piotrowski, J. K. (1980). Studies to identify the low molecular weight bismuth-binding proteins in rat kidney. Biochem. Pharmacol. 29, 2913}2918. Woods, J. S., and Fowler, B. A. (1987). Alteration of mitochondrial structure and heme biosynthetic parameters in liver and kidney cells by bismuth. Toxicol. Appl. Pharmacol. 90, 274}283.
Tissue Uptake of Bismuth from Shotgun Pellets Roger Pamphlett,*,1 Gorm Danscher,- J+rgen Rungby,- and Meredin Stoltenberg*Department of Pathology, University of Sydney, NSW 2006, Australia; and -Department of Neurobiology, Institute of Anatomy, University of Aarhus, DK-8000, Aarhus C, Denmark Received May 10, 1999
pellets on the environment, and in particular on animals wounded by these pellets, however, is not known. This is potentially important, since the popularity of recreational hunting has meant that a number of wild animals and birds are wounded by shotgun pellets but survive for prolonged periods of time. From human clinical experience it is known that bismuth-related toxicity is not directly related to the dose or duration of bismuth exposure (MartinBouyer et al., 1980). We were interested therefore to see whether bismuth could be released from shotgun pellets and enter the tissues of animals. Until recently it was dif7cult to localize small amounts of bismuth within tissues. However, when mice were injected with large doses of bismuth subnitrate intraperitoneally and their tissues examined with the technique of autometallography (AMG), bismuth was claimed to be detected in sections of their tissues (Ross et al., 1994). AMG is a histochemical technique that can detect mercury, gold, silver, and zinc in tissues because these metals are surrounded with a collar of reduced silver molecules (Danscher et al., 1997). Proton induced X-ray microanalysis (PIXE) has demonstrated that the AMG grains in sections from bismuth-exposed animals encase bismuth and sulfur (Danscher et al., in preparation). Having con7rmed the speci7city of the AMG bismuth technique, we used this method to look for bismuth that could be released from shotgun pellets that had been inserted into mice. In addition, because the pellets contained a small amount of tin, we also ensured that tin could not give rise to AMG silver grains.
Shotgun pellets containing bismuth have been suggested to be less environmentally toxic than those containing other metals. We sought to And if bismuth from shotgun pellets embedded within an animal enters the tissues of that animal. Five bismuth-containing shotgun pellets were placed intraperitoneally into adult mice. Four or 9 weeks later the tissue distribution of bismuth was examined histologically using silver lactate autometallography. Bismuth was seen in the nervous system of the mice, either in cells with processes outside the nervous system or in cells not protected by the blood-brain barrier. Bismuth was also seen in the kidney, liver, spleen, and lung. The amount of bismuth within tissues varied widely between animals at both time intervals. Bismuth from shotgun pellets enters the tissues of mice, with some mice taking up more bismuth than others. Some animals wounded with bismuth pellets are therefore likely to accumulate large amounts of potentially toxic bismuth in their tissues. ( 2000 Academic Press Key Words: bismuth; toxicity; heavy metal; shotgun pellets; autometallography.
INTRODUCTION
Because of environmental concerns, the use of lead in shotgun pellets has been forbidden in many countries, including Denmark, so interest has arisen in 7nding different materials for pellets. Iron pellets have been recommended from an environmental point of view, but these have poor ballistic qualities because of their lightness, and iron embedded in tree trunks can damage forestry equipment. Bismuth pellets have gained popularity because of their good ballistic qualities and have been in use in the United States for a number of years. The impact of these
MATERIALS AND METHODS
Animals Six-week-old BALB/c mice were used in all experiments. Animals were kept four to a cage in a room with a 12-h light/dark cycle and a temperature of
1
To whom correspondence should be addressed. Fax: int#(612) 9531-3429. E-mail: [email protected]. 258 0013-9351/00 $35.00 Copyright ( 2000 by Academic Press All rights of reproduction in any form reserved.
259
BISMUTH FROM SHOTGUN PELLETS
RESULTS
21}22@C. Mice were given a continuous supply of Altromin No. 1324 rat and mouse diet and tap water. Pellet Analysis The average weight of 10 shotgun pellets (Eley 12) was 0.17g (SD 0.01 g). Proton-induced X-ray emission, performed at the Danish Environmental Research Institute, Roskilde, showed that the pellets contained 91% bismuth and 9% tin. Pellet Placement Eight mice were anesthetized with halothane and 7ve alcohol-cleaned pellets were inserted into the peritoneal cavity though a 0.5-cm incision in the anterior abdominal wall. Four mice had sham operations with no insertion of pellets. Incisions were closed with two Clay Adams stainless-steel autoclips (Beckton Dickinson), which were removed 7 days later. Preparation and Staining of Tissue At 4 or 9 weeks after pellet implantation, four mice were anesthetized with halothane and perfused through the left ventricle of the heart with 50 ml of 3% glutaraldehyde in phosphate buffer. The right lung, liver, right kidney, spleen, four lumbar posterior root ganglia, brain, and cervical and lumbar spinal cord were removed and post7xed in glutaraldehyde for 48 h. After 7xation, blocks were immersed in 30% sucrose overnight, mounted on chucks with Tissue Tek OCT Compound (Sakura), and frozen with carbon dioxide. Serial 30-lm sections were cut in a cryostat and placed in physical developer comprising 60 ml of 50% gum arabic, 10 ml citrate buffer, 30 ml of 5.6 g/100 ml hydroquinone, and 0.5 ml of 17 g/100 ml of silver lactate and kept in the dark at 26@C for 60 min (Danscher and M+ller-Madsen, 1985). Sections were counterstained with toluidine blue. Black granules represent encased bismuth sul7de molecules (BiAMG). The nervous system distribution of BiAMG was mapped with the aid of a mouse brain atlas (Franklin and Paxinos, 1997).
The animals showed no ill effects from the operation or from the intraabdominal pellets and gained weight, moved, ate, and drank normally. Bismuth Uptake No BiAMG was seen in the tissues of any of the sham-operated mice. BiAMG was seen in the organs of all mice with intraperitoneal pellets. Within the affected cells, BiAMG was present in the cytoplasm of the cell bodies, but not in the nuclei. At both survival intervals the tissue distribution of bismuth was similar. However, the amount of BiAMG within cells varied widely between animals, in both the 4and 9-week interval groups. Cerebrum. BiAMG was present within neurons in the supraoptic, paraventricular (Fig. 1), suprachiasmatic, and arcuate nuclei. The median eminence adjacent to the arcuate nuclei stained positively. The subfornicial organ stained strongly, with BiAMG extending into the white matter of the supraadjacent hippocampal commissure. The vascular organ of the lamina terminalis stained moderately. No staining was seen in cells in the cerebral cortex, basal ganglia, or thalamus. Cerebellum. No BiAMG was seen in the cerebellar cortex or deep nuclei. Brain stem. BiAMG was found in neurons of the trochlear, oculomotor, mesencephalic trigeminal, motor trigeminal, abducens, facial, ambiguus, and
Testing the AMG Method for Tin Four mice were injected intraperitoneally with stannous chloride at doses of 1, 2, 4, or 8 lg/g. One week later, the animals were perfused as above, and 30-lm frozen sections of lumbar spinal cord were stained with AMG.
FIG. 1. Coronal section of the cerebrum showing BiAMG staining in the supraoptic (7lled arrow) and paraventricular (open arrow) nuclei, at 9 weeks after pellet implantation. opt, optic tract. AMG and toluidine blue. Magni7cation, ]65.
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FIG. 2. In the medulla oblongata, both the hypoglossal nuclei (12) and the area postrema (AP) stain positively for bismuth at 9 weeks after pellet implantation. Patchy staining is present in the ependyma of the cerebral aqueduct (arrow), but this was also seen in controls. AMG and toluidine blue. Magni7cation, ]90.
FIG. 3. Black granules of silver-enhanced bismuth (BiAMG) are present in the cytoplasm of large motor neurons in the lumbar spinal cord, at 4 weeks after pellet implantation. AMG and toluidine blue. Magni7cation, ]1250.
hypoglossal nuclei (Fig. 2). The area postrema also stained positively for BiAMG (Fig. 2).
reduce silver ions to metallic silver on their surfaces if placed in an autometallographic developer (Danscher et al., 1997). As a result, AMG grains will be created exactly where the clusters are created in vivo or in vitro. Recently, bismuth has been added to the list of metallic ions that can be visualized with this technique (Ross et al., 1994; Danscher et al., 1997). Since the shotgun pellets contained a minor amount of tin, it was important to know whether tin could create catalytic sul7de or selenide clusters that could be silver-enhanced by AMG. We therefore injected some animals with stannous chloride alone,
Spinal cord. A large amount of BiAMG was seen in the cell bodies of large motor neurons in the anterior horn of the cervical and lumbar spinal cord (Fig. 3). More staining was seen in lumbar than in cervical motor neurons. Posterior root ganglia. About 40% of large neuron cell bodies in the spinal ganglia contained BiAMG. Other nervous system cells. A patchy uptake of bismuth was seen in the leptomeninges. Silver staining was seen in the ependyma, but this was present equally in control animals. The choroid plexus showed no signi7cant BiAMG staining. General organs. In nonnervous tissue, BiAMG was seen in cell bodies of renal tubular cells (Fig. 4), macrophages in the lung, and dendritic cells in the liver and spleen. Tin Uptake No AMG silver deposits were seen in the lumbar motor neurons of any animals injected intraperitoneally with stannous chloride. DISCUSSION
AMG for Bismuth Nanoclusters of metallic gold, silver, mercury, and zinc ions, combined with sul7de or selenide ions, can
FIG. 4. In the kidney, heavy BiAMG staining is seen in the renal tubules, while the glomeruli (G) are free of bismuth, at 9 weeks after pellet implantation. AMG and toluidine blue. Magni7cation, ]400.
BISMUTH FROM SHOTGUN PELLETS
since tin ions arising from this salt are likely to be similar chemically to the tin arising from the pellets. Lumbar spinal motor neutrons, one of the 7rst cells in the body to take up xenobiotics (Rungby and Danscher, 1983; Danscher et al., 1997; Pamphlett and Coote, 1997), showed no grains after development. Although bismuth was not detected directly in the granules by elemental analysis, we concluded on the balance of probabilities that it is bismuth sul7de or selenide, originating from the intraperitoneal bismuth shotgun pellets, that is being demonstrated by AMG in the tissues of these animals. Nervous System Distribution of Xenobiotics The nervous system distribution of bismuth, with a predominant involvement of motor neurons, parallels that of other xenobiotics such as mercury vapor (M+ller}Madsen, 1992), inorganic mercury (Danscher and M+ller-Madsen, 1985; M+ller}Madsen, 1990), and silver (Rungby and Danscher, 1983). The overall distribution of bismuth is also very similar to that of horseeradish peroxidase injected intravenously into mice (Broadwell and Brightman, 1976). The striking uptake by motor neurons of all of these substances had led to the suggestion that, while most neurons are protected by the blood-brain barrier, these xenobiotics enter distal motor axons at the neuromuscular junction and travel to the cell body by retrograde axonal transport (Arvidson, 1989). This suggestion has been supported by experiments showing retrograde transport of metals injected directly into striated muscle (Arvidson, 1992; Schi+nning, 1993). On the other hand, it is possible that some neurons with very high metabolic activity take up exogenous metals from the blood stream, since after high doses of bismuth large neurons that do not have processes outside the central nervous system can be shown to contain the metal (Ross et al., 1996). With the present model of low-dose xenobiotic uptake, it appears that two pathways are operative. The 7rst is the retrograde axonal route, with the xenobiotic entering the cell bodies of alpha motor neurons (axons terminating in striated muscle), mesencephalic trigeminal neurons (axons terminating in muscle spindles), and supraoptic, paraventricular, suprachiasmatic, and arcuate nuclei (axons terminating in circumventricular organs) (Broadwell and Brightman, 1976). The second pathway is the blood-borne route, with the xenobiotic entering cells not protected by the blood-brain barrier such as the circumventricular organs and the posterior root ganglia. At exposure to very high doses of
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a xenobiotic, a third route appears to be operative, leading to selective uptake by large neurons with processes that are con7ned to the central nervous system (Ross et al., 1996). These neurons were, however, not affected by the doses of bismuth arising from shotgun pellets in this experiment. We saw a wide variation in bismuth uptake from the shotgun pellets between animals. This variation has been seen both in mice given intraperitoneal doses of bismuth subnitrate (where it may be due to varying uptake of the water-insoluble suspension) (Ross et al., 1996) and in humans in whom plasma concentrations of bismuth are not proportional to the dose given (Koch et al., 1996). The cause for the variability in cellular bismuth in our mice is not clear. Potential Damage from Bismuth In humans, the form of toxicity from bismuth depends on the type of bismuth compound. Neurotoxic effects are associated with more insoluble compounds and kidney disorders with the more soluble compounds (Slikkerveer and de Wolff, 1989). The best-documented neurotoxicity was from an epidemic of bismuth encephalopathy in France (MartinBouyer et al., 1978). In this epidemic, symptomatic patients had a wide range of bismuth blood concentrations, from 10 to 4600 lg/L, suggesting a large interindividual susceptibility to bismuth (MartinBouyer et al., 1978). Disturbances of bone mineralization were also noted in patients with bismuth encephalopathy, and another osteoarthropathy has been seen in patients treated with bismuth for syphilis (Slikkerveer and de Wolff, 1989). Animal experiments have shown that bismuth distorts the inner mitochondrial membrane and reduces the activity of heme-synthesizing enzymes (Woods and Fowler, 1987). There is therefore compelling evidence to suggest that exposure to bismuth should be kept to a minimum. A large amount of bismuth was apparent in the renal tubules of our mice. Rodents are not alone in taking up bismuth into their kidneys, since after a bismuth-containing shot was embedded in the breast muscles of ducks, increased concentrations of bismuth were detected in the kidney and liver 1 year later (Sanderson et al., 1998). Animal experimentation has shown that bismuth binds to proteins of low molecular weight in the kidney (Szymanska and Piotrowski, 1980). Soluble bismuth compounds can poison the proximal renal tubules (Slikkerveer and de Wolff, 1989) and reversible renal failure in humans has been seen after intoxication with colloidal bismuth subcitrate (Hudson and Mowat, 1989).
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In the lungs of our mice, bismuth was seen in macrophages, and in the liver and spleen it was present in dendritic cells. Other xenobiotics and in particular inorganic mercury have also been noted to enter cells of the immune system (Christensen, 1996). Of interest, bismuth may inhibit migration of macrophages after it is phagocytosed (Soutar and Coghill, 1986), so damage to the immune system of animals exposed to bismuth is a possibility. CONCLUSION
In conclusion, bismuth arising from shotgun pellets can be detected in the nervous system and other organs of mice. The long-term effects of bismuth on animals that harbor bismuth-containing shotgun pellets are not known. However, given the present knowledge on the toxicity of bismuth compounds, if recreational shooting of wild animals is to continue on a large scale it seems prudent to look for pellet material that is more inert as regards the tissue uptake of its components. ACKNOWLEDGMENTS We thank Dorete Jensen and Thorkild Nielsen for skilled technical assistance. Kas re Kemp of the Danish Environmental Research Institute performed the PIXE analysis.
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