Blood was sampled from 33 harbour seals caught along the German coast or kept in captivity (Seal Station, Friedrichskoog, Germany) between 1997 and 2006 (Table 1). The seals were physically restrained. Health status was determined by physical examination, using routine haematological analysis (blood counts) and serum chemistry (detailed protocol described previously 50).

Blood was drawn from the extradural venous sinus into sterile evacuated blood collection Monovette^® tubes (serum tubes for Hg analysis, EDTA tubes for RT-PCR and heparinised tubes for functional tests and proteomics analysis). Blood tubes were kept at -20°C until Hg analysis or were processed within 18 hours for cell cultures.

Buffy coats from healthy 30–60 year old male humans (n = 9) were integrated into this study for the PBMC culture (Croix Rouge de Belgique).

The measurement of the concentration of T-Hg in blood is generally a good surrogate for the concentration of MeHg in blood in human populations with a high fish consumption 1216. T-Hg was analysed in full blood from 22 harbour seals by cold vapour atomic absorption spectrometry on a Perkin-Elmer Coleman Mas-50 Mercury Analyser (wavelength 253.7 nm). The freeze-dried samples were subjected to microwave-assisted digestion with nitric acid and H₂O₂, as described previously for blood and other tissues 5152. Quality control measurements for T-Hg included replicate analysis resulting in coefficients of variation < 10% and analysis of certified material (DORM-1, NRC, Canada). The Hg absolute detection limit was 10 ng, corresponding to 0.13 μg.g^-1 fresh weight (fw) for an average of 1.5 g of sample analysed. All samples were above the detection limit.

The expression of the housekeeping gene glyceraldehyde-3-phosphate (GAPDH), of interleukins-IL-2 and IL-4 and of the transforming growth factor (TGF)-β was investigated in vitro in control and contaminated seal PBMCs (n = 6, Table 1) using RT-PCR and real time PCR, as described elsewhere 535556. For a quantitative analysis of mRNA expression, a comparison between samples can be made by relating gene expression levels to housekeeping gene expression, the latter regarded as being unaffected by exposure conditions. After the incubation period (72 h), total RNA was isolated from exposed and control PBMCs (RNeasy^® Mini Kit, Qiagen Sciences, Maryland, USA) following the manufacturer's recommendations. After DNase-treatment (DNA-free™, Ambion kit), RNA was reverse transcribed with murine reverse transcriptase (RNA PCR Core Kit™, Applied Biosystems, Weiterstadt, Germany) and the resulting cDNA served as a template for PCR following the manufacturer's protocol and using Thermocycler MX4000™ (Stratagene Europe). For real-time quantification, the Brilliant SYBRGreen QPCR Master mix (Stratagen Europe) was used 56. This contained SYBRGreen I as a fluorescence dye, dNTPs, MgCl₂ and a hot start Taq DNA polymerase. Primers for the PCR were designed using DNAstarTM software (GATC Biotech, Konstanz, Germany). Primers for the detection of IL-2 and IL-4 were selected from conserved nucleotide sequences of grey seals (Halichoerus grypus) and dogs (Canis familiaris) respectively (GenBank accession numbers AF072871, AF187322, AF104245). Primer sequences for the amplification of GAPDH and TGF-β were selected from previously published sequences 53 (Table 2).

The fluorescence response was monitored in a linear fashion, as the PCR product was generated over a range of PCR cycles. For each cytokine, a standard curve was prepared using a dilution series from 10⁹ to 10² copies. The PCR started with an initial step at 95°C for 10 min, followed by 40 cycles with denaturation at 95°C for 1 min, annealing temperature for 30 sec and elongation at 72°C for 1 min. The fluorescence was measured at the end of the annealing and at the end of the dissociation program at a wavelength of 530 nm. In order to exclude measurement of non specific PCR products and primer dimers, and to determine true amplification, each PCR was followed by a dissociation program for 1 min at 95°C, followed by 41 cycles during which the temperature was increased in each cycle, starting at 55°C and ending at 95°C. Only PCR reactions with one well-defined peak were used for analysis. All reactions were performed in duplicate and two separate PCR reactions were performed. GAPDH was used as the control gene. In order to calculate cytokine expression, GAPDH was used as the calibration compound. The cytokine expression index (CI) was calculated as follows:

CI = Number of cytokine copies/Number of GAPDH copies

The viability and number of PHA-stimulated PBMCs were determined microscopically by trypan blue exclusion assay and a haemocytometer before and after MeHg exposure.

After 72 hours of MeHg exposure (0.1, 0.2, 0.5, 1, 1.5, 5, 10 μM), the metabolic activity of the cells was quantified by a colorimetric microtitre plate MTS assay 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium, inner salt (MTS, Promega), according to the manufacturer's instructions. 10 μl of prepared MTS solutions were added to the 100 μl culture medium containing the cells. MTS is chemically reduced by cells into formazan, which is soluble in culture medium. After 2 h in the dark and in the incubator, the measurement of the absorbance of the formazan was carried out on a spectrophotometer (Powerwave X, Bio-Tek) at 492 nm. Each condition was produced in 5 wells.

DNA, RNA and protein synthesis were determined by measuring the amount of incorporated radioactive precursors using a scintillation counter. 2 × 10⁵ cells were seeded in 200 μl culture medium. During the last 24 h of culture, 1 μCi.ml^-1 of methyl-[³H] thymidine (specific activity 20–40 Ci.mmole^-1), [³H] uridine (specific activity 20–40 Ci.mmole^-1) or [³H] L-leucine (specific activity 40–60 Ci.mmole^-1, MP Biomedicals) was added to each well and cellular incorporation was determined. Cells were harvested on a filter (Multiscreen HTS, Millipore) with a millipore aspiration system, as described by the manufacturer's instructions. The following steps were carried out with an automatic dispenser (Precision Power 2000): 100 μl PBS, 120 μl trypsin 10 min, 100 μl PBS, 100 μl ethanol 20 min, 3× 100 μl ethanol. The plates were dried overnight, and the filters were collected in vials (Puncher Millipore). 400 μl sodium hypochlorite (1:10 v:v) were then added. After 30 min agitation, 4 ml scintillating liquid (Ready Safe, Beckman) were added and quantification of the retained radioactivity by the cells was made by liquid scintillation in a counter (LS 6500 Scintillation system, Beckman). Results from triplicates (means of counts per minute ± standard deviations) are expressed in percentages (control taken to be 100%).

Preliminary proteomics analysis (the study of the proteome) was carried out on human PBMCs. We focus this part of the study on cytoplasmic fraction (soluble proteins). Cells were treated with 1 μM MeHgCl in the presence of PHA for 72 h. Quantitative analysis was carried out on 2D DIGE gels in two steps. First, individuals were treated separately (n = 3) in order to discover interindividual variability and secondly, individuals were pooled in order to obtain sufficient amount of proteins to run the gel.

Student's t-test was used for comparing control and MeHg-treated cells.

T-Hg levels in full blood varied from 0.04 to 0.56 μg.g^-1 fw (43 to 611 μg Hg.L^-1) with a mean concentration of 0.16 μg.g^-1 fw (Table 3). T-Hg concentrations were found to be similar between males and females (Student's t-test, p > 0.5) and individuals were therefore regrouped. T-Hg concentration in blood was found to be significantly correlated to the body mass (r_p = 0.59 and p < 0.001) and length of the seals (r_p = 0.60 and p < 0.001) (Figure 1).

The PHA-induced proliferative responses of PBMCs collected from seals and humans are shown in Figure 2. A significant decrease in the percentage of lymphocyte proliferation was found at MeHg concentrations of 5 and 10 μM in both seals and humans (Figure 3). Cell mortality was found to be moderate at 1 and 1.5 μM of MeHg.

A decrease in DNA, RNA and protein synthesis was observed even at low MeHg concentrations (0.2 and 1 μM), whereas inhibition of synthesis was clearly noticed at 10 μM (Figure 4). DNA, RNA and protein synthesis showed a similar profile but nucleic acid synthesis seemed more inhibited compared to proteins. After 48 h, the concentrations inhibiting 50% of DNA and RNA synthesis of the two species were found to be around 1 μM and 1.5 μM, while the 50% protein synthesis inhibiting concentration was found to be around 1.5 μM.

The MTS assay (water soluble tetrazolium salts) reflects cell proliferation and the metabolic activity of the mitogen-stimulated lymphocytes. Metabolic activity was significantly reduced compared to the controls at around 1 μM both for human and seal cells (Figure 5).

In this set of experiments, seal PBMCs were cultured in the presence of non-cytotoxic doses of MeHg (0.2 and 1 μM) in order to examine mRNA expression. GAPDH, IL-2, IL-4 and TGF-β mRNA were successfully detected in both controlled and exposed PBMCs (Figures 6, 7, 8). As a general feature, change in mRNA expression occurred after exposure: the number of copies decreased at 0.2 μM, except for IL-4. The large dispersion of data between seals (coefficient of variation ≤ 68%) indicated high grade inter-individual variability in cytokine mRNA responses. Cytokine indexes were strikingly low for IL-2 and TGF-β even at 0.2 μM (Figures 6 and 7), while an increasing trend was observed for IL-4 (Figure 8).

Most of the proteins had an Ip of between 3 and 10. A Mowse score was calculated for each identification, which was considered as significant above 54 (Additional file 1). Low doses of MeHg (1 μM) were found to affect cytoplasmic protein expression. Proteins are generally less expressed in treated gels. Volume ratio (expression of protein in treated gel compared to control) was positive (over-expression) for spot 1142 and spot 1452 but negative (lower-expression) for spots 1319 (vimentin), 897 and 923 (Table 2, Figures 9 and 10). These results showed that the identified proteins are involved in many cellular functions such as cell proliferation (SYW), the building of the cytoskeleton (VIME), protein degradation (PRS10), melatonin biosynthesis and the creation of transduction pathways (GBLP, AN32A) (Additional file 1).

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 7374. The proteome of T-lymphocytes is well known 757677. 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 78. 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 7980. The earliest detectable event following MeHg exposure is a change in the level of mitochondria 81. 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. 8182. A previous study observed the expression and activation of different caspases after 16 h of treatment with MeHg 82. Caspases are cysteine proteases that are essential for executing apoptosis and for degrading vimentin 8384. 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.