In the present study, T-Hg concentrations in the blood of harbour seals were found to vary widely, correlated to length and body mass of the seals (Figure 1). The observed correlation reflects daily Hg intake and thus, the amount of fish ingested, which differs according the body mass of animals. Adult harbour seals eat 5% to 6% of their body weight per day, up to 7 kg for big individuals [59, 60]. T-Hg levels measured in the blood of harbour seals caught in the North Sea are higher than those previously described in North Atlantic or in Arctic regions (Table 3) [61–63]. Interestingly, the level of T-Hg in the blood of harbour seals from the North Sea is not lower than that encountered in other seal species 30 years ago. Similarly, Hg levels have not decreased in Arctic biota despite the recent reductions in emissions in North America and Western Europe [4–6].
A commonly used reference interval for human beings is 0.6 – 59 μg.L-1[64, 65]. Clear signs of Hg toxicity develop in most individuals only at some point much higher than the upper reference limit. The Environmental Protection Agency (USA) has recommended that blood Hg levels should not be higher than 5.8 μg.L-1, at least for the more sensitive individuals such as pregnant women . A study of a human cohort with high fish consumption in the Faroe Islands found a median cord blood concentration of 24 μg.L-1 . Obviously, the harbour seals we studied from the North Sea displayed higher concentrations (mean Hg concentration = 172 μg.L-1, around 1 μM). Knowing that Hg is mainly under a methylated form in the blood of marine mammals (up to 90%), questions arise regarding the potential biochemical effects of these Hg levels on harbour seals and human immune cells. To tentatively answer this question, a set of in vitro experiments were carried out on T-lymphocytes using low MeHg exposure (around 1 μM).
We exposed harbour seal and human lymphocytes in vitro to MeHg and we examined the effects on cell-mediated immunity: cell mortality, synthesis of DNA, RNA and protein and metabolic activity. Cell mortality was reduced in the interval 0.1–1 μM (Figure 3) both for human and seal PHA-stimulated PBMCs. Up to 90% of cells exposed to 1 – 1.5 μM of MeHg remained alive. However, a clear suppressive effect of MeHg on DNA, RNA and protein synthesis in seal and human PBMCs was found to be present, even at low concentrations. Protein synthesis seemed less affected, probably due to a longer response time (Figure 4). Proliferation and metabolic activity reflected by MTS assay confirmed that 1 μM was a critical concentration with significantly reduced in vitro activity of human and seal PBMCs relative to controls (Figure 5). No striking difference appeared between human and seal in vitro resistance to MeHg. At higher concentrations, 5–10 μM, 75% of cells remained alive, but metabolic activity dropped to very low levels.
Cytokine expression also decreased following MeHg exposure: IL-2 and TGF-β seemed highly sensitive to in vitro MeHg exposure in regard to their dramatic decrease in gene expression at 0.2 μM and 1 μM compared to controls (Figures 6, 7, 8). However, large inter-individual variability has previously been observed and cytokine expression in seal PBMCs has been found to vary widely, depending also on the duration of MeHg exposure . A decrease in PBMC IL-2 expression has also been documented in harbour seals following in vitro exposure to PHA and PCB , raising the possibility of additive effects of MeHg and other organic pollutants.
IL-2 has a prime role in immune response, as it is responsible for T-cell clonal expansion after antigen recognition. IL-2 also increases synthesis of other T-cell cytokines, promotes the proliferation and differentiation of NK cells, and acts as a growth factor and stimulus for B-cell antibody synthesis. TGF-β is often considered as an anti-inflammatory cytokine [67, 68]. A variety of murine models have provided evidence that eliminating TGF-β or disrupting its downstream signalling cascade leads to inflammatory disease .
In contrast, in the present study, the cytokine index of IL-4 showed an increasing trend from control to 1 μM (Figure 8). IL-4 is known to induce the differentiation of naive helper T cells (Th0 cells) into Th2 cells. Similarly, Devos et al. found that inorganic mercury (HgCl2) and MeHg was capable of increasing IL-4 production in Con A-stimulated human PBMC in vitro . They also observed that IL-4 production occurred in a dose-dependent fashion, although MeHg was found to be much more potent than inorganic mercury in inducing IL-4 production . Furthermore, they observed MeHg induced IL-4 production at a similar range of concentrations to those found in our own investigation (0.1–0.5 uM) .
These preliminary in vitro results suggest that MeHg could induce a differentiation of naïve cells (increase of IL-4), while T-lymphocyte clonal expansion is inhibited (decrease of IL-2); a decrease in TGF-β suggests an increase in inflammatory response and would require further investigation such as through a polynuclear cell model. Our findings are consistent with those of previous researchers working with human and rodent systems and support a hypothesis of contaminant-altered lymphocyte function mediated (at least in part) by the disruption of cytokine production (TH2 cells) [34, 37, 71, 72]. Whether this phenomenon has clinical relevance in marine mammal populations remains to be determined.
Study of the proteome
Proteomics facilitates the identification of new biomarkers of chemical exposure and studies of mechanisms by which protein modification contribute to the adverse effects of environmental exposure [73, 74]. The proteome of T-lymphocytes is well known [75–77]. However, the expression of proteins in MeHg-exposed T-lymphocytes has not yet been described. Our results showed that identified proteins are involved in many cellular functions such as cell proliferation (SYW), the building of the cytoskeleton (VIME), protein degradation (PRS10), melatonin biosynthesis (ASML) and the creation of transduction pathways (GBLP, AN32A). Human lymphoid cells are an important physiological source of melatonin, which could be involved in the regulation of the human immune system . As a general feature, many spots are underexpressed in exposed gels, reflecting the inhibition of protein synthesis linked to MeHg toxicity. As for cytokine mRNA expression, high variability between individuals was evidenced here contrasting with functional tests displaying weaker inter-individual variations. This feature raises several issues within the framework of individual susceptibility to pollutants.
Some of the proteins identified here are in agreement with previous research describing MeHg toxicity mechanisms leading to cell death. MeHg exposure is known to induce a rapid and sustained increase in intracellular calcium levels [79, 80]. The earliest detectable event following MeHg exposure is a change in the level of mitochondria . Exposure of T-Cells to MeHg chloride has been found to cause a decrease in the overall size of mitochondria and changes in the structure of the cristae, leading finally to apoptosis. [81, 82]. A previous study observed the expression and activation of different caspases after 16 h of treatment with MeHg . Caspases are cysteine proteases that are essential for executing apoptosis and for degrading vimentin [83, 84]. The lower vimentin expression found here in exposed MeHg lymphocytes agrees with this caspase activation.
In this study, we found that exposure for 72 hours in vitro to 1 μM of MeHg affected not only cell proliferation but also many cellular functions such as the building of the cytoskeleton, melatonin biosynthesis, the creation of signal transduction pathways and signal transcription. This variability in affected cellular functions suggests various toxicity pathways, depending on duration of exposure, MeHg concentration, cell type and individual susceptibility. This study shows the potential of using a proteomics approach in deciphering the intracellular changes in cells exposed in vitro to MeHg.