Effects of organochlorine contaminants on loggerhead sea turtle immunity: comparison of a correlative field study and in vitro exposure experiments

Abstract

Several laboratory and field studies indicate that organochlorine contaminants (OCs), such as polychlorinated biphenyls (PCBs) and pesticides, modulate immune responses in rodents, wildlife, and humans. In the present study we examined the effects of OCs on immunity in free-ranging loggerhead sea turtles (Caretta caretta). Mitogen-induced lymphocyte proliferation responses, lysozyme activity, and OC concentrations were measured from blood samples. Mitogens chosen in the lymphocyte proliferation assay were phytohemagglutinin (PHA) and concanavalin A (ConA) for T-lymphocyte stimulation, and lipopolysaccharide (LPS) and phorbol 12,13-dibutyrate (PDB) for B-lymphocyte stimulation. Lysozyme activity was significantly and negatively correlated with whole-blood concentrations of 4,4 -dichlorodiphenyldichloroethylene (4,4 -DDE) and the sum of chlordanes. Lymphocyte proliferation responses stimulated by PHA, LPS, and PDB were significantly and positively correlated with concentrations of the sum of PCBs measured in whole blood. LPS- and PDB-induced proliferation were also significantly and positively correlated with 4,4 -DDE blood concentrations. These correlative observations in free-ranging turtles suggest that current, chronic exposure to OCs may suppress innate immunity and enhance certain lymphocyte functions of loggerhead sea turtles. To further test this hypothesis, lymphocyte proliferation was measured after in vitro exposure of peripheral blood leukocytes from 16 turtles to Aroclor 1254 (0-13.5 microg/mL) or 4,4 -DDE (0-13.4 microg/mL). Both contaminants increased PHA- and PDB-induced proliferation at concentrations below those that affected cell viability. Moreover, the concentrations that enhanced PDB-induced proliferation in vitro were similar to concentrations measured in turtles with the highest proliferative responses. The similarities between the in vitro experiments and the correlative field study suggest that OC exposure modulates immunity in loggerhead turtles.

Figure 1

Scatterplots of plasma lysozyme activity versus concentrations of 4,4′-DDE (A; r_S = −0.310, p = 0.038) and ∑chlordanes (B; r_S = −0.368, p = 0.013) measured in the blood of loggerhead sea turtles. Linear trend lines demonstrate the negative relationships determined using Spearman rank correlations.

Figure 2

Scatterplots of ∑PCB blood concentrations versus loggerhead sea turtle lymphocyte proliferation (SI) responses stimulated for 5 days with 5 μg/mL PHA (A; r_S = 0.596, p = 0.012) and 0.8 μg/mL PDB (B; r_S = 0.564, p = 0.018). SI = cpm of mitogen-stimulated cells/cpm of unstimulated cells. Linear trend lines demonstrate the positive relationships determined using Spearman rank correlations.

Figure 3

Scatterplots of 4,4′-DDE blood concentrations versus loggerhead sea turtle lymphocyte proliferation (SI) responses stimulated for 5 days with 5 μg/mL PHA (A; r_S = 0.431; p = 0.084) and 0.8 μg/mL PDB (B; r_S = 0.507; p = 0.038). SI = cpm of mitogen-stimulated cells/cpm of unstimulated cells. Linear trend lines demonstrate the positive relationships determined using Spearman rank correlations.

Figure 4

The effect of a 5-day in vitro exposure to Aroclor 1254 on loggerhead sea turtle lymphocyte proliferation (SI) responses stimulated by 5 μg/mL PHA (A) and 0.2 μg/mL PDB (B). Data are shown as mean ± SE of the percentage of the SI measured in the control (no DMSO or Aroclor 1254) for each turtle. Sample sizes are 8 or 16 depending on the treatment group. The x-axis crosses the y-axis at the percentage of the control value for the wells receiving only DMSO. The mean ± SE SI for the DMSO controls in the PHA and PDB experiments was 96.6 ± 15.0 and 172 ± 39, respectively. Vertical dashed lines indicate the range of ∑PCB concentrations measured in the blood of 17 loggerhead sea turtles used in the correlative field study. *Significantly different from the DMSO control (ANOVA with log-transformed data, Dunnet’s multiple comparison test; p < 0.05).

Figure 5

The effect of a 5-day in vitro exposure to 4,4′-DDE on loggerhead sea turtle lymphocyte proliferation (SI) responses stimulated by 5 μg/mL PHA (A) and 0.2 μg/mL PDB (B). Data are shown as mean ± SE of the percentage of the SI measured in the control (no DMSO or 4,4′-DDE) for each turtle. Sample sizes are 8 or 16 depending on the treatment group. The x-axis crosses the y-axis at the percentage of the control value for the wells receiving only DMSO. The mean ± SE SI for the DMSO controls in the PHA and PDB experiments was 96.6 ± 15.0 and 172 ± 39, respectively. Vertical dashed lines indicate the range of 4,4′-DDE concentrations measured in the blood of 17 loggerhead sea turtles used in the correlative field study. *Significantly different from the DMSO control (ANOVA with log-transformed data, Dunnet’s multiple comparison test; p < 0.05).