Elsevier

Toxicology in Vitro

Volume 23, Issue 3, April 2009, Pages 378-385
Toxicology in Vitro

The in vitro effects of selenomethionine on methylmercury-induced neurotoxicity

https://doi.org/10.1016/j.tiv.2008.12.024Get rights and content

Abstract

Selenium (Se) has been reported to reduce the severity of MeHg-induced neurological deficits. Therefore, we investigated whether 24 h. preincubation or 50 min. coincubation with selenomethionine (SeMet) was effective in reducing methylmercury (MeHg)-induced cytotoxicity in C6-glioma and B35-neuronal cell lines. As indicators of cytotoxicity, reduced glutathione (GSH), reactive oxygen species (ROS) and mitochondrial activity (MTT) was assessed. Measurement of GSH with the fluorescent indicator MCB-monochlorobimane indicated that in SeMet preincubated C6 cells, MeHg treatment resulted in a significant (p < 0.001) decrease in GSH levels as compared to coincubation group. Treatment with SeMet did not induce any significant changes in MTT activity in either of the cell lines as compared to the MeHg group. However, the amount of MeHg-induced ROS was significantly reduced (p < 0.001) after SeMet preincubation in both the cell lines. The intracellular Se content was measured with high resolution-inductively coupled plasma mass spectrometry (HR-ICPMS). In both the cell lines the intracellular Se levels increased after pre- and coincubation with 20 and 50 μM SeMet. However, the preincubation group exhibited increased Se content in both the cell lines and varied (p < 0.001) from coincubation group. These differences in the Se content were maintained after 10 μM MeHg treatment for 50 min. In C6-gliomas, the cell associated-MeHg measurements using 14C-labeled MeHg indicated a significant increase (p < 0.001) in MeHg content in preincubated cells as compared to coincubated cells. These findings provide experimental evidence that preincubation with SeMet increases Se content in cells and prevents against increased MeHg-induced ROS generation.

Introduction

Methylmercury (MeHg) is a hazardous environmental pollutant, which bioaccumulates (Inza et al., 1998, Hammerschmidt et al., 2002) and biomagnifies in the aquatic food chain, thereby affecting human health (Lebel et al., 1998, Myers et al., 2000, Clarkson et al., 2003). Considerable attention in the scientific and health policy fora is focused on the question of whether MeHg intake from a diet high in fish is associated with aberrant central nervous system (CNS) function. Studies from New Zealand (Kjellstrom et al., 1986, Kjellstrom et al., 1989), Canada (Keown-Eyssen et al., 1983) and the Faroe Islands (Grandjean et al., 1997) suggest that during pregnancy, maternal exposure to MeHg via fish consumption is associated with neurological deficits in their offspring. Notably, however, this outcome has not been replicated in the Seychelles (Myers et al., 1995, Myers et al., 1997, Davidson et al., 1998). The reasons for the discrepancy in these outcomes have yet to be identified (CENR, OSTP and The White house, 1998).

Selenium (Se) is an essential trace element. Seafood species known to accumulate significant amounts of Se are potentially a good dietary source of this element (World Health Organisation, 1987, USDA, 2005). Previous studies suggest that the majority of Se in fish is in the organic form, selenomethionine (SeMet) (Akesson and Srikumar, 1994, Quijano et al., 2000). Moreover, SeMet is more bioavailable than inorganic forms (Daniels, 1996). Se is a major component of the tetrameric selenoenzyme, glutathione peroxidase (GPx), which catalyzes the reduction of organic hydroperoxides (Flohe, 1988). At least 13 different selenoproteins are known and their physiological functions include at least five GPx isoforms, selenoprotein P, three iodothyronine deiodinases, three thioredoxin reductases and selenophosphate synthetase (Allan et al., 1999). The intake of Se-rich fish diet has been shown to be highly correlated with GPx activity as well as Se protein-P plasma levels (Bergmann et al., 1998, Hagmar et al., 1998).

Nearly all fish contain detectable amounts of MeHg (Bulato et al., 2007). The major dietary route of adult human exposure to MeHg is via the ingestion of seafood and for infants via maternal milk (Manfroi et al., 2004). SeMet has also been detected in human milk (Michalke and Schramel, 1997) and shown to prevent the decline in plasma Se in nursing mothers. It was observed that marine mammals could accumulate exceptionally high concentrations of Hg and Se compounds without obvious intoxication symptoms (Koeman et al., 1973). It was later reported that toxic effects of organic and inorganic Hg could be prevented by Se compounds (Chen et al., 1974, Lindh and Johansson, 1987). Hence, when exposure to a toxicant as well as an essential nutrient occurs from the same food source, their interactions may influence the net neurobehavioral outcome (Budtz-Jørgensen et al., 2007). Therefore, the presence of Se as a confounding factor could be responsible for different outcomes in studies evaluating the effect of MeHg from seafood diet. Se was recognized as a possible confounder in the Faroe Islands study and was measured in cord tissue, but failed to meet the inclusion criterion for statistical analysis (CENR, OSTP and The White house, 1998).

Although interactions between Hg and Se have been sporadically addressed, the mechanisms have yet to be fully understood (Cuvin-Aralar and Furness, 1991). Se has been reported to bind to MeHg (Magos et al., 1979) and influence its uptake (Frisk et al., 2001). A major mechanism of MeHg-induced neurotoxicity is via the generation of oxidative stress (Ali et al., 1992, Yee and Choi, 1996, Sarafian, 1999, Sanfeliu et al., 2001). It reflects a marked imbalance between ROS and their removal by antioxidant systems. Overproduction of ROS can damage major classes of biological macromolecules, such as nucleic acid, proteins, lipids and carbohydrates. On the other hand Se protects against ROS acting as a scavager, thereby reducing lipid, protein and DNA oxidation (Xiong et al., 2007). SeMet has also been reported to prevent the decline of GPx activity (Michalke and Schramel, 1997). The ability of SeMet to modify MeHg-induced ROS has not been investigated before. Moreover, there is little information regarding the protective roles of SeMet against MeHg toxicity, especially with respect to the ability of SeMet on to attenuate MeHg uptake in neuronal cells. The innovative aspect of this study resides in the monitoring of both concurrent and separate exposures to MeHg and SeMet, thus mimicking physiological exposure conditions. The study design emphasizes mechanisms of MeHg and Se interactions, contributing to the understanding of the risk/benefit binomium resulting from exposure to MeHg and Se.

Section snippets

Materials

24-well plastic tissue culture plates were purchased from Falcon (Becton Dickinson Labware, USA). Fetal bovine serum (Cat. No. A15-151) was purchased from PAA Laboratories, Pasching (Austria). The medium for culturing C6-gliomas (F12 Kaighn’s nutrient mixture, Cat. No. 21127) was purchased from Invitrogen (Norway). The DMEM media (Cat. No. E15-810) for culturing B35 cells was purchased from PAA Laboratories, Pasching (Austria). SeMet (Cat. No. S3132), MCB (Cat. No. 69899), MTT (Cat. No. M2128)

Modification of Se content by treatment with SeMet

The Se content in the C6 and B35 cell lines (Table 1) was analyzed with HR-ICPMS. Treatment with SeMet resulted in a significant (p < 0.001) concentration-dependent cellular uptake of Se in both the cell lines. However for both the cell lines, the amount of Se in preincubated cells was significantly higher (p < 0.001) than coincubated cells. After MeHg exposure in the 50 μM SeMet preincubated cell lines, there was a significant 13% (C6) and 9% (B35) reduction (p < 0.001) in cellular content of Se. For

Discussion

Se, an essential dietary trace element known to accumulate in plants and seafood species (USDA, 2005) can attenuate MeHg toxicity (Morris and Levander, 1970, Parizek and Ostadalova, 1967). Since potential Se–Hg interaction would have important public health implications, additional investigation is warranted. The innovative aspect of this study is to correlate intracellular MeHg and Se content and decipher potential mechanisms associated with Se’s protective effects in concurrent or separate

Funding

This work was funded by the scholarship grant from The Research Council of Norway (Grant no. 173389/110). MA was supported by Public Health Service Grant ES07331 from the National Institute of Health (to MA).

The authors also wish to thank the project collaborators Anne-Katrine Lundebye (NIFES, Norway) and Christer Hogstrand (King’s College London, UK) for their cooperation and valuable guidance.

Conflict of Interest Statement

None declared.

Acknowledgments

The authors gratefully acknowledge the technical assistance from Bente Urfjell and Sunniva Hoel (NTNU, Norway). The authors are also indebted to Chris Glover (SCION, New Zealand) for his guidance.

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