Mercury immune toxicity in harbour seals: links to in vitro toxicity.

BACKGROUND: Mercury is known to bioaccumulate and to magnify in marine mammals, which is a cause of great concern in terms of their general health. In particular, the immune system is known to be susceptible to long-term mercury exposure. The aims of the present study were (1) to determine the mercury level in the blood of free-ranging harbour seals from the North Sea and (2) to examine the link between methylmercury in vitro exposure and immune functions using seal and human mitogen-stimulated peripheral blood mononuclear cells (T-lymphocytes). METHODS: Total mercury was analysed in the blood of 22 harbour seals. Peripheral blood mononuclear cells were isolated from seals (n = 11) and from humans (n = 9). Stimulated lymphocytes of both species were exposed to functional tests (proliferation, metabolic activity, radioactive precursor incorporation) under increasing doses of methylmercury (0.1 to 10 microM). The expression of cytokines (IL-2, IL-4 and TGF-beta) was investigated in seal lymphocytes by RT-PCR and by real time quantitative PCR (n = 5) at methylmercury concentrations of 0.2 and 1 microM. Finally, proteomics analysis was attempted on human lymphocytes (cytoplasmic fraction) in order to identify biochemical pathways of toxicity at concentration of 1 microM (n = 3). RESULTS: The results showed that the number of seal lymphocytes, viability, metabolic activity, DNA and RNA synthesis were reduced in vitro, suggesting deleterious effects of methylmercury concentrations naturally encountered in free-ranging seals. Similar results were found for human lymphocytes. Functional tests showed that a 1 microM concentration was the critical concentration above which lymphocyte activity, proliferation and survival were compromised. The expression of IL-2 and TGF-beta mRNA was weaker in exposed seal lymphocytes compared to control cells (0.2 and 1 microM). Proteomics showed some variation in the protein expression profile (e.g. vimentin). CONCLUSION: Our results suggest that seal and human PBMCs react in a comparable way to MeHg in vitro exposure with, however, larger inter-individual variations. MeHg could be an additional cofactor in the immunosuppressive pollutant cocktail generally described in the blood of seals and this therefore raises the possibility of additional additive effects in the marine mammal immune system.

Neurotoxicity and immunotoxicity assessment in CBA/J mice with chronic Toxoplasma gondii infection and single-dose exposure to methylmercury.

Toxoplasma gondii is a protozoan parasite that localizes in the brain where it can cause life-threatening disease. Methylmercury (MeHg) is a well-documented neurotoxicant that accumulates in the brain. We investigated end points associated with immunotoxicity and neurotoxicity in mice exposed to MeHg during a chronic T. gondii infection. Two groups of 6-week-old, female CBA/J mice were either fed 25 T. gondii tissue cysts of the ME-49 strain or given vehicle. Six weeks later, half of the mice in each group were orally gavaged with a single dose of 20 mg/kg body weight of MeHg, creating four groups of mice (vehicle control, T. gondii, MeHg, and T. gondii/MeHg). Mice were sacrificed 7 days post MeHg exposure. MeHg exposure caused a significant decrease in mouse body weight. MeHg administration resulted in an increase of splenic cellularity and spleen-to-body weight ratios. MeHg had no significant effect on the percentages of CD4(+), CD8(+), or non-T-cell subpopulations in the spleen. MeHg dosed mice demonstrated an increase in absolute numbers of splenic CD4(+), CD8(+), or non-T cells when compared to mice in control and T. gondii-infected groups. Thymic CD4(+)CD8(+) T-cell subpopulations were decreased (p <.05) by MeHg with or without a concurrent T. gondii infection. There was a significant (p <.05) increase in brain tissue cyst counts within the group exposed to both MeHg and T. gondii (16 +/- 4, mean +/- SE, n = 7) versus T. gondii alone (4 +/- 1, n = 8). Histopathological examination demonstrated encephalitis, gliosis, and meningitis in brains from mice infected with T. gondii. These data indicate that exposure to both MeHg and T. gondii has synergistic effects, with effects of MeHg especially on the immune system.

Low-level exposure to methylmercury modifies muscarinic cholinergic receptor binding characteristics in rat brain and lymphocytes: physiologic implications and new opportunities in biologic monitoring.

Methylmercury (MeHg) affects several parameters of cholinergic function. These alterations are thought to play a role in MeHg neurotoxicity. In vitro experiments have indicated that MeHg acts as a strong competitive inhibitor of radioligand binding to muscarinic cholinergic receptors (mAChRs) in rat brain. Furthermore, rat brain mAChRs share several pharmacologic characteristics of similar receptors present on lymphocytes. Using the muscarinic antagonist [(3)H]quinuclidinyl benzilate (QNB) to label receptors, we investigated the in vivo interactions of MeHg with rat brain mAChRs. We also investigated whether MeHg-induced central mAChR changes are reflected by similar alterations in splenic lymphocytes. Exposure to low doses of MeHg--0.5 or 2 mg/kg/day in drinking water--for 16 days significantly increased (20-44% of control) mAChRs density (B(max)) in the hippocampus and cerebellum without affecting receptor affinity (K(d)). The effect of MeHg did not occur immediately; it was not apparent until 2 weeks after the termination of treatment. No significant changes in [(3)H]QNB binding were observed in the cerebral cortex. In splenic lymphocytes, mAChR density was remarkably increased (95-198% of control) by day 14 of MeHg exposure and remained enhanced 14 days after the cessation of treatment. These results suggest up-regulation of mAChRs in selected brain regions (hippocampus and cerebellum) after prolonged low-level ingestion of MeHg in rats. These cerebral effects are delayed in onset and are preceded by a marked increase in density of mAChRs on lymphocytes. In chronic MeHg exposure, peripheral lymphocytes may represent a sensitive target for the interaction of MeHg with mAChRs and, therefore, may be predictive indicators of later adaptive response involving cerebral mAChRs. Additionally, the effect of MeHg on lymphocyte mAChRs in vivo indicates that this receptor system should be investigated further as a possible target for MeHg immunotoxicity.

Methyl mercury-induced autoimmunity in mice.

Female SJL/N, A.SW, B10.S (H-2s), BALB/C, DBA/2 (H-2d), A.TL and B10. TL (H-2t1) mice were treated with sc injections of 1.0 mg CH3HgCl/kg body weight every third day for 4 weeks. Controls were given sterile, isotonic NaCl. CH3HgCl (MeHg) induced in SJL, A.SW and B10.S mice antinucleolar antibodies (ANoA) targeting the nucleolar 34-kDa protein fibrillarin. The susceptibility to develop ANoA in response to MeHg was linked to the mouse major histocompatibility complex (H-2), since H-2s but not H-2t1 mice sharing background (non-H-2) genes developed ANoA. However, the background genes decided the strength of the ANoA response in the susceptible H-2s mice, and the ANoA titer was in the order: A.SW > SJL > B10.S. Although MeHg as well as inorganic mercury induced ANoA, the two forms of mercury differed both quantitatively and qualitatively in their effect on the immune system. MeHg induced in H-2s mice a weaker general (polyclonal) and specific (ANoA) B-cell response than HgCl2, probably due to weaker activation of Th2 cells with lower IL-4 production, as indicated by the minimal increase in serum IgE. The A. TL strain with a susceptible genetic background, but a H-2 haplotype resistant to HgCl2, responded to MeHg with a modest polyclonal B-cell response dominated by Th1-associated Ig isotypes. H-2s mice treated with MeHg showed in contrast to HgCl2-treated mice no systemic immune-complex (IC) deposits, which may be due to the weaker immune activation after MeHg treatment. The increase in serum IgE concentration and ANoA titer 2-6 weeks after stopping treatment with MeHg is identical to reactions during the first 2-3 weeks of HgCl2 treatment. Therefore, demethylation of MeHg probably increased the concentration of inorganic mercury in the body sufficiently to reactivate the immune system. This reactivation indicated that genetically susceptible mice are not resistant to challenge with mercury, making them distinctly different from rats.

Exposure to methyl mercury results in serum autoantibodies to neurotypic and gliotypic proteins.

Environmental exposure to methyl mercury (MeHg) continues to pose a threat to humans, making early detection of neurotoxic effects a pressing concern. An enzyme-linked immunosorbent assay (ELISA) to measure serum autoantibodies (Ig) to neurotypic and gliotypic proteins [neurofilament triplet (NF68; NF160; NF200), myelin basic protein (MBP) and glial fibrillary acid protein (GFAP)] as markers of subclinical neurotoxicity was developed and tested in Fisher 344 rats exposed orally to 16 or 32 ppm MeHg. Both levels of MeHg resulted in serum Ig to all 5 proteins, not normally seen in controls. For anti-NFs and anti-GFAP, IgM isotype predominated significantly (p < 0.05) over IgG.Ig for MBP were of the IgG isotype, IgM were not detected. Significant differences (p < 0.05) between 16 and 32 ppm MeHg in levels of anti-NF 68 and GFAP, IgM, were evident at 7 days of exposure, but not at 14 days. Anti-NF 160, IgM, was significantly (p < 0.01) elevated in rats exposed to 32 ppm vs 16 ppm at 14 days. However, at both dose levels anti-NF 68 titers were the most elevated of the three NF proteins (p < 0.0001). For anti-NF 200 and anti-MBP it was the IgG isotype that was significantly (p < 0.01) elevated in the 32 ppm group at 7 days. GFAP levels as a marker of neurotoxicity were determined in the cortex, hippocampus and cerebellum. Exposure to 32ppm MeHg resulted in decreased (p < 0.05) levels in the cortex at 14 days. Both levels of MeHg resulted in increased GFAP in the cerebellum at 14 days. This study suggests that assay of autoantibodies against nervous system proteins may provide a means of assessing the early neurotoxic effects of environmental MeHg exposure.