Several pharmaceuticals impaired harbor seal lymphocytes (Phoca vitulina) in vitro

In human and veterinary therapeutics, approximately 3000 different drugs are used and prescribed in high quantities.1 The main substance classes are steroidal hormones, antimicrobials and pharmaceuticals and their metabolites.1,2 These compounds could become problematic in the environment, because >80% of the drugs pass the body without being biotransformed, and furthermore their elimination in sewage treatment plants is incomplete.1,3 Additionally, the compounds are constructed for a specific intended effect on living beings, and therefore show intrinsic physico-chemical behavior.4 Since the compounds are chemically different and peer-reviewed ecotoxicological data is available for less than 1% of human pharmaceuticals, the effects on most trophic levels of aquatic life are hard to predict for a single substance or a mix of various ones.5-8 The purpose of this study was to identify the impact of pharmaceuticals commonly found in surface waters on immune cells of harbor seals (Phoca vitulina) in vitro. Peripheral blood mononuclear cells (PBMCs) from captive seals and a seal B lymphoma cell line (11B7501)9 were exposed to selected single substances. Compounds of interest were analgesics (ibuprofen, naproxen), psychoactive substances (carbamazepine, paroxetine), antibiotics (erythromycin, sulfamethoxazole, trimethoprim), cholesterol lowering compounds (gemfibrozil), steroidal hormones (ethynyl estradiol) and caffeine.


Introduction
In human and veterinary therapeutics, approximately 3000 different drugs are used and prescribed in high quantities. 1 The main substance classes are steroidal hormones, antimicrobials and pharmaceuticals and their metabolites. 1,2 These compounds could become problematic in the environment, because >80% of the drugs pass the body without being biotransformed, and furthermore their elimination in sewage treatment plants is incomplete. 1,3 Additionally, the compounds are constructed for a specific intended effect on living beings, and therefore show intrinsic physico-chemical behavior. 4 Since the compounds are chemically different and peer-reviewed ecotoxicological data is available for less than 1% of human pharmaceuticals, the effects on most trophic levels of aquatic life are hard to predict for a single substance or a mix of various ones. [5][6][7][8] The purpose of this study was to identify the impact of pharmaceuticals commonly found in surface waters on immune cells of harbor seals (Phoca vitulina) in vitro. Peripheral blood mononuclear cells (PBMCs) from captive seals and a seal B lymphoma cell line (11B7501) 9 were exposed to selected single substances. Compounds of interest were analgesics (ibuprofen, naproxen), psychoactive substances (carbamazepine, paroxetine), antibiotics (erythromycin, sulfamethoxazole, trimethoprim), cholesterol lowering compounds (gemfibrozil), steroidal hormones (ethynyl estradiol) and caffeine.

Materials and Methods
Sampling and isolation of peripheral blood mononuclear cells and cell culture Whole blood samples (7-15 mL) were collect-ed from the extradural intervertebral vein of four female harbor seal adults in captivity (Aquarium du Québec, Quebec, Canada). Blood was kept in heparin tubes at RT for 6 h after sampling until separation with Lympholyte-Mammal (Cedarlane, Burlington, Canada). The PBMCs were resuspended in completed RPMI-1640 and kept at 4°C overnight until the start of the incubation with xenobiotics.

Preparation of xenobiotics
Chemicals (all Sigma-Aldrich) were dissolved in dimethyl sulfoxide (DMSO), ethanol or water. The final concentration of DMSO or ethanol in the samples never exceeded 0.1%.

In vitro exposures
For the in vitro exposures, cells were adjusted to the test specific concentration. In case of the lymphoblastic proliferation 200 µL of cells (1.25x10 6 cells/mL) were incubated with 2 µL of pharmaceutical product. In case of phagocytosis and cell cycle and apoptosis, 500 µL of cells (1.0x10 6 cells/mL and 0.5x10 6 cells/mL, respectively) were incubated with 5 µL of pharmaceutical product. Viability assays were set up parallel to each of the three assays as an additional control.
For the experiments with PBMCs, concentrations of pharmaceuticals were based on environmentally relevant values found in municipal effluents. The concentration 1x refers to the maximum concentration detected in the environment (Table 1), as cited. 10,11 Further concentrations tested were: 0x, 0.01x, 0.1x, 10x, 100x (Table 1).
In experiments with the 11B7501 cell line, concentrations of pharmaceuticals were increased to be able to observe a possible negative impact of the single substance on the harbor seal immune cells (Table 1).

Viability assay
After the respective exposure time, viability of cells was evaluated using 4 µL of a 100 μg/mL of propidium iodide (PI) solution (Sigma-Aldrich) to 500 µL of cell suspension. A FACSCalibur (Becton Dickinson, San Jose, CA, USA) with an air-cooled argon laser providing an excitation at 488 nm was used. For each sample 5 000 events were acquired at a fluorescence emission of 620 nm (FL3). The cell population was electronically gated in a FSC/SSC dot plot and the fluorescence frequency distribution histogram was obtained using FL3. The percentage of dead cells was determined using a marker. Data collection and analysis were performed with the CellQuest Pro software (Version 4.0.1). The results were expressed in percentage of viable cells.

Phagocytosis assay
Phagocytosis was measured using carboxylate coated fluorescent latex beads (Polysciences Inc.; 100 beads:1 cell). After 24 h exposure to single substances of pharmaceuticals, cells and beads were incubated for 1.5 h at 37°C to allow attachment and phagocytosis. To remove most free beads after incubation, the suspension was centrifuged on a gradient of 3% BSA (MP Biomedicals) prepared in completed medium. The fluorescence emission was collected at 520 nm (FL1). For each sample 10,000 events were acquired. Using a FSC/SSC dot plot, the adequate population was electronically gated and the fluorescence frequency distribution histogram was obtained using FL1. Data was expressed as percentage of cells with phagocytic activity (cells that engulfed>one bead) and phagocytotic efficiency (cells that engulfed>three beads). The lymphocyte population was gated as a negative control. The raw data was expressed as counts per minute and was then converted in percent proliferation of control.

Cell cycle and apoptosis assay
The DNA content of each cell was measured using PI. After fixation of the cells with 70% EtOH following the 24 h exposure and 72 h in RPMI, cells were washed and then resuspended in a PBS solution containing PI (50 μg/mL) and RNAse (100 μg/mL) (all Sigma-Aldrich). PI also binds to double stranded regions of RNA, necessitating treatment with nucleases. 12 Using the FSC/ SSC dot plot, the cell population was electronically gated and the fluorescence frequency distribution histogram using FL2 (585 nm) was obtained. In a second dot plot, the gated lymphocyte population is expressed in FL2-A/FL2-W. Doublets (two G 1 /G 0 cells attached to each other, which seem to have the same DNA content as one cell in the G 2 /M phase) are discriminated using a second gate. The population lymphocytes minus doublets was analyzed in FL3-A, and apoptosis as well as the phases of the cell cycle were gated independently. For each sample 5000 events were acquired. The results were expressed in percentage of cells in different stages of the cell cycle plus apoptotic events.

Statistical analyses
Differences between controls and treated groups were evaluated by one-way ANOVA followed by Tukey's Multiple Comparison post test. The calculations were performed using GraphPad Prism 5 for Windows (GraphPad Software). The level of significance was set at P≤0.05.

Results and Discussion
The experiments conducted with PBMCs exposed to concentrations of pharmaceuticals similar to those found in surface showed no significant impairment of cellular function in the phagocytosis or lymphoblastic proliferation assay (data not shown).
In the experiments with the 11B7501 cell line, three compounds influenced the B lymphocytes at high concentrations. While the phagocytosis experiment showed no significant effect with any compound (data not shown), the lymphoblastic proliferation with LPS was modulated in the case of naproxen (>25 µg/mL**) and carbamazepine (>50 µg/mL*) ( Figure 1A). The cell cycle and apoptosis assay revealed a significant increase in apoptotic events at 50 µg/mL for ethynylestradiol, while the percentage of cells in the G2/M phase decreased ( Figure 1B). For carbamazepine, the percentage of apoptotic events slightly decreased at 50 µg/mL, while the percentage of cells in the G0/G1 phase increased ( Figure  1B).
The absence of an effect in the lower doses of pharmaceutical compounds does not neces-  sarily conclude to the harmlessness of current environmentally relevant levels of pharmaceuticals towards the immune system of marine vertebrates. In contrast, many studies on fish already showed bioaccumulation in various tissues and a significant change in immune parameters, behavior and distribution that were caused by environmentally relevant levels of pharmaceuticals. [13][14][15][16] With fish accumulating pharmaceutical products and seals being piscivorous, they will likely be exposed to higher concentrations through nutrition, opposed to those concentrations found in the water column. Therefore, the true environmentally relevant concentrations for the exposures of marine mammals towards pharmaceuticals still have to be assessed.

Article
Our study shows that more sensitive methods are needed to evaluate the impact of pharmaceuticals on the immune system of marine mammals. Risk assessment in this area is necessary, because not only seals but also humans consume fish. If pharmaceuticals in the environment cause a possible reduction in certain functional activities of the immune system of large vertebrates that may alter the host's resistance to pathogens, the knowledge of these effects would therefore most certainly have implications on the treatment of municipal wastewaters in the future.