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Neurotoxicity of Dental Amalgam is Mediated by Zinc
1 Dept. of Biomedical Sciences and Correspondence: *corresponding author, Doug.Lobner{at}marquette.edu
The use of dental amalgam is controversial largely because it contains mercury. We tested whether amalgam caused toxicity in neuronal cultures and whether that toxicity was caused by mercury. In this study, we used cortical cell cultures to show for the first time that amalgam causes nerve cell toxicity in culture. However, the toxicity was not blocked by the mercury chelator, 2,3-dimercaptopropane-1-sulphonate (DMPS), but was blocked by the metal chelator, calcium disodium ethylenediaminetetraacetate (CaEDTA). DMPS was an effective mercury chelator in this system, since it blocked mercury toxicity. Of the components that comprise amalgam (mercury, zinc, tin, copper, and silver), only zinc neurotoxicity was blocked by CaEDTA. These results indicate that amalgam is toxic to nerve cells in culture by releasing zinc. While zinc is known to be neurotoxic, ingestion of zinc is not a major concern because zinc levels in the body are tightly regulated.
Key Words: amalgam • neurotoxicity • zinc • mercury
The use of dental amalgam has come under attack for potentially causing systemic health problems (Dodes, 2001). Principal among the concerns is that the release of mercury from amalgam may cause neurological disorders (Clarkson, 1989). Amalgam restorations do release mercury vapors into the mouth (Patterson et al., 1985). However, whether mercury exposure from amalgam is high enough to have a significant impact on total body mercury exposure, given the ubiquitous nature of mercury in the environment, is considered unlikely (Wahl, 2001). The most convincing evidence that amalgam restorations do not cause neurological deficits comes from the "Nun Study". In this study, it was found that the number and surface area of dental amalgams were not correlated with lower performance on 8 different tests of cognitive function (Saxe et al., 1995). There is evidence that exposure to mercury vapor from the handling of dental amalgam may cause neurological deficits in dentists and dental assistants. One such study, conducted in Singapore, found a strong correlation between the level of mercury exposure and poor performance on motor and cognitive tests in dentists (Ngim et al., 1992). Other studies have also indicated a correlation between exposure, and body burden of mercury, with neurobehavioral effects in dental workers (Shapiro et al., 1982; Uzzel and Oler, 1986; Echeverria et al., 1995, 1998), although the adverse effects were typically at a pre-clinical level and were likely associated with poor mercury-handling procedures. The focus on potential toxicity of dental amalgam has been on the presence of mercury. However, amalgam contains other substances that may be neurotoxic. The exact composition of amalgam varies with manufacturer. We chose to study the nerve cell toxicity of Dispersalloy, which is the world‘s most widely used amalgam. It contains a combination of mercury (Hg), zinc (Zn), tin (Sn), copper (Cu), and silver (Ag). Each of these metals can be neurotoxic. The purpose of this study was, first, to test if amalgam was toxic to nerve cells in culture, and, second, to determine what component of amalgam was responsible for the toxicity.
Materials Timed pregnant Swiss Webster mice were obtained from Charles River Laboratories (Wilmington, DE, USA). Amalgam (Dispersalloy) was obtained from Dentsply (Milford, DE, USA). All other chemicals were obtained from Sigma (St. Louis, MO, USA).
Cortical Cell Cultures A messing gun was used to make standard-sized rods of amalgam (0.010 ± 0.002 g each). The freshly prepared amalgam was placed on tissue culture inserts suspended above the cultured cells. In this way, the amalgam was in contact with the media bathing the cultures, but not in direct contact with the cells. This system avoids possible cell damage due to physical disruption of the cells.
Cell Death Assessment
Statistical Analysis
Fig. 1  is a photomicrograph of cultures taken through the culture insert, on which was placed a rod of amalgam, showing that amalgam exposure induced widespread neuronal death after 24 hrs. Quantifying the cell death by the LDH assay indicated that about 50% of the cells were dead (Fig. 2A ). Addition of the mercury chelator, 2,3-dimercaptopropane-1-sulphonate (DMPS), to the bathing media had no effect on cell death, while the metal chelator, ethylenediaminetetraacetate (CaEDTA), greatly attenuated death (Fig. 2A). The cultures tested were mixed neuronal and glial cultures. Trypan blue staining indicated that death of not only neurons but also some glial cells occurred. The treatment with CaEDTA was profoundly protective against death for both neurons and glia (data not shown). We next tested whether DMPS does in fact block mercury toxicity. Addition of 5 µM HgCl2 induced death of approximately 50% of the cells. The toxicity was blocked by DMPS, while CaEDTA had no effect (Fig. 2B). Analysis of the data indicates that amalgam toxicity was not mediated by release of mercury. The other components found in amalgam are zinc, tin, copper, and silver. Each of these metals was toxic, although even a high concentration of tin (200 µM) caused only about 15% cell death. Addition of CaEDTA blocked the toxicity of only zinc (Fig. 3).
An association between amalgam and neurotoxicity has been suggested because mercury is neurotoxic (Clarkson, 1989) and amalgam contains mercury. However, this is the first study to show directly that amalgam is toxic to nerve cells in culture. Previous studies have primarily focused on amalgam toxicity to fibroblasts (Kawahara et al., 1975; Kaga et al., 1988; Osorio et al., 1998; Keiser et al., 2000) and have quantified the cell injury by measuring mitochondrial function (Osorio et al., 1998; Keiser et al., 2000), counting cell numbers (Kawahara et al., 1975), or measuring a zone of inhibition of cell growth (Kaga et al., 1998). When these assays are used on dividing cells, such as fibroblasts, the assays provide a measure of inhibition of cell replication, not necessarily a direct measure of cell death. That a substance inhibits cell replication is important information, but it is information slightly different from that provided by a direct assay of cell death. The cultures used in this study contain non-dividing neurons and a confluent glial cell layer that is contact-inhibited and not proliferating. The assay used measures the release of the cytosolic enzyme lactate dehydrogenase, which can leave only cells whose membrane has been disrupted. This assay has been shown to be an accurate measure of cell death (Koh and Choi, 1987; Lobner, 2000). The idea that zinc release from amalgam is toxic is not new. Zinc-containing amalgams have been shown to be more toxic than non-zinc-containing amalgams (Kaga et al., 1988). However, this is the first report to show that chelating zinc could block the cytotoxicity of amalgam. It is not clear why CaEDTA did not block the toxicity of metals other than zinc, since CaEDTA binds with high affinity to the other metals tested (Bell, 1977). However, binding does not always lead to diminished toxic effects of metals (Koh et al., 1996). The amalgam used in the present studies was Dispersalloy, the composition of which is: 50% mercury, 35% silver, 9% tin, 6% copper, and 0.5% zinc. Considering the low percentage of zinc, it may seem surprising that zinc was the primary toxic substance released. However, zinc has been shown to be the main corrosion product released from amalgam (Brune, 1981). There is enough zinc in the amalgam to be responsible for the toxicity to nerve cells in culture. The average piece of amalgam placed in the culture media weighed 0.010 ± 0.002 g, of which 0.5% was zinc. The culture medium in each pit was 625 µL. Therefore, approximately 1/25th of the zinc present in the amalgam would have to be released into the bathing medium to achieve a toxic concentration of 50 µM. Further experiments to measure the concentration of each of the metals released from amalgam will be required to confirm that zinc is the toxic component. Zinc is known to be neurotoxic (Gaskin et al., 1978; Lobner et al., 2000). Release of zinc from synaptic vesicles has been shown to contribute to neuronal death induced by global ischemia in rats (Koh et al., 1996). However, zinc levels in the brain are tightly regulated, and zinc has been used for the treatment of Wilson‘s disease for many years without adverse effects (Brewer et al., 1998). The situation with mercury is much different. The amount of mercury exposure determines the amount of mercury in the brain (Hursh et al., 1976) and the degree of neurological deficit (Grandjean et al., 1999). Therefore, while the present study shows that amalgam is toxic to nerve cells in culture, it also indicates that this toxicity is not a major concern. The study does not address the issue of whether long-term exposure to amalgam may produce mercury-mediated neurological deficits. It does, however, indicate that the interpretation of cytotoxicity of complex materials requires determination of which substance in that material is responsible for the toxicity.
This investigation was supported by NIH/NIA grant AG16708. The authors are grateful to Julie Hjelmhaug for her technical assistance. Received for publication February 20, 2002. Revision received August 30, 2002. Accepted for publication November 26, 2002.
Journal of Dental Research, Vol. 82, No. 3,
243-246 (2003)
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