PERSISTENT ORGANIC POLLUTANTS (POPs) AND IMMUNEFUNCTION IN US ATLANTIC COAST HARBOR SEALS(Phoca vitulina concolor )Susan D. Shaw 1, Diane Brenner1, Christie A. Mahaffey1, Sylvain De Guise2, Christopher R.
Perkins3, George C. Clark4, Michael S. Denison5, and Gordon T. Waring6
1

Marine Environmental Research Institute, Center for Marine Studies, PO Box 1652, Blue Hill,
Maine 04614 USA
2
Department of Pathobiology, University of Connecticut, Storrs, CT 06269 USA
3
Environmental Research Institute, U. of Connecticut, Storrs, CT 06269 USA
4
Xenobiotic Detection Systems, Inc., 1601 E. Geer St. Suite S, Durham, NC 27704 USA
5University of California, Davis, Davis, CA 95616 USA
6NOAA Fisheries Northeast Fisheries Science Center, Woods Hole, MA 02543 USA

Introduction
Polyhalogenated aromatic hydrocarbons (PHAHs), particularly the dioxin-like polychlorinatedbiphenyls (PCBs), dioxins and furans (PCDD/Fs) are potent immunotoxins that biomagnify inaquatic food chains and are associated with disease susceptibility and immune-disrupting effects inmarine mammals and humans1,2. In 1979-1980, a large-scale outbreak of type A influenza virus
resulted in the deaths of over 500 harbor seals (Phoca vitulina concolor) inhabiting the US
Atlantic coast3. An immunomodulatory role of environmental chemicals was not investigated;
however, studies conducted in the 1970s4 reported that levels of PHAHs in blubber of these seals
were comparable to those of seals in polluted areas of Europe. Recent data5 indicate that PHAH
levels have decreased since that time, but remain in concentration ranges known to be associatedwith impaired immune function in the species6. The sensitivity of harbor seals to the effects ofPHAH exposure was previously demonstrated by a semi-field study in which numerous immune
functions were suppressed in seals fed PHAH-contaminated fish from the Baltic Sea7. Similarly, afield study found associations between reduced immune responses of harbor seal pups andincreasing blubber levels of dioxin-like PCBs following low-level perinatal exposure8. Theseobservations support the hypothesis that dioxin-like PHAHs may play a role in the viral epizootics
occurring among harbor seals, by impairing normal immune resilience to pathogens. The
mechanisms of these effects are not yet clear, but are likely to involve interactions with the arylhydrocarbon receptor (AhR) signaling pathway9. Here we report analysis of the relationshipbetween body burdens of dioxin-like PHAHs and immune functions in free-ranging harbor seals
from the US Atlantic coast.
Methods and Materials
Peripheral blood samples were collected from 29 free-ranging harbor seals captured during March
2001 in Chatham Bay, Massachusetts and April in Penobscot Bay, Maine. Seal blood samples
were drawn from the extradural vein through a 20-gauge needle into four 10 ml heparinizedvacuum tubes, placed on ice packs, and shipped overnight for next day analysis. Lymphocyte
Proliferation. Peripheral blood mononuclear cells (PBMC) were isolated from the seal blood
samples and lymphocytes (2 x 105) were cultured in flat-bottom 96 well tissue culture plates in theabsence or presence of sub-optimal and optimal concentrations of Concanavalin A (Con A),

Organohalogen Compounds, Volumes 60-65, Dioxin 2003 Boston, MAphytohemagglutinin (PHA), and lipopolysaccharide (LPS). Cells were cultured for 66 hours with
5-bromo-2'-deoxyuridine (BrdU), a thymidine analog, added during the last 16 hours. The Cell
Proliferation ELISA, BrdU (colorimetric) assay was used to quantify proliferation as theincorporation of BrdU. Results were expressed as a stimulation index (SI), the proliferation ratioof stimulated to unstimulated cells. Natural killer (NK) cell activity. Lymphocytes wereincubated for 150 minutes in round-bottom 96-well plates at 37°C with 5% CO2 with labeled K562 target cells at effector:target cells ratio of 100:1, 50:1, and 25:1. Target cell mortality (% cellslysed) was evaluated using propidium iodide and two color flow cytometry. The fluorescence ofapproximately 1000 target cells was read with a FACScan flow cytometer using the Cell Quest
software. CALUX Analysis. PHAHs were extracted from seal plasma samples using acid-silica
and XCARB as described10. The ability of each extract to induce Ah receptor-dependent gene
expression was determined for each of the using the H1L6.1c3 CALUX cell bioassay (Xenobiotics
Detection Systems, Inc., Durham, NC) and luciferase activity measured10, 11. The relative potencyof each sample was determined by analysis of a dose (dilution) response for luciferase induction
using a four-parameter Hill plot analysis of the dose curves, the EC50 values directly compared toa TCDD standard curve and the relative induction equivalents (CALUX-IEQs) for each sample
calculated. CALUX-TEQ values are reported as pg/g lw.
Results and Discussion
Regression analysis showed a significant positive linear relationship between CALUX-TEQ
values in the adult harbor seals (n=11) and lymphoproliferative responses to the T cell mitogen
PHA (estimate = 0.039, SE = 0.011, F = 11.76, p = 0.008, r=0.753, r2 = 0.566, r2adj = 0.518,Figure 1) and the B cell mitogen LPS (estimate = 0.008, SE = 0.003, F = 8.68, p=0.016, r=0.701,
r2=0.491, r2adj = 0.434, Figure 2). The PHA and LPS-induced responses in the adult seals wererelated only to the independent factor CALUX-TEQ and not to the factors sex or condition index.
CALUX-TEQ values in adult seals were not significantly correlated with lymphoproliferative
responses to the mitogen Con A or to NK cell activity. There were no significant relationships
between CALUX-TEQ and mitogen responses or NK activity in juvenile seals.
10LPS Stimulation Index

PHA Stimulation Index

40302010086420-100 100 200 300 400 500 600 700
CALUX-TEQ (pg/g lw)

Fig. 1. Relationships between PHA responseand CALUX-TEQs (± 95% confidenceinterval) in adult harbor seals

0 100 200 300 400 500 600 700
CALUX-TEQ (pg/g lw)

Fig. 2. Relationship between LPS response
and CALUX-TEQs (± 95% confidenceinterval) in adult harbor seals

Organohalogen Compounds, Volumes 60-65, Dioxin 2003 Boston, MAThus, the CALUX-TEQ explained 57% and 49% of the variation in proliferative responses ofharbor seal lymphocytes to the mitogens PHA and LPS, respectively. To our knowledge, apositive relationship between CALUX-TEQ values and mitogen-induced responses of peripheral
lymphocytes of free-ranging phocid seals has not been previously documented. Recently, apositive correlation between PCBs in blubber and PHA and LPS-induced lymphocyte proliferation
was reported in an experiment using captive harbor seal weanling pups (Levin, pers. comm.).
Segre and coworkers12 recently reported a significant positive correlation between PCB bodyburdens and mitogen responses of thymocytes in white-footed mice (Peromyscus leucopus).
Alterations in immune function can have devastating effects on the survival of an organism,
particularly during critical phases of development. Whereas chemical-induced immune
suppression may result in increased susceptibility, incidence, and severity of infectious diseases,
conversely, immune enhancement may result in the loss of regulation within the immune systemand can lead to adverse outcomes including as autoimmune disease, anergy (inability to mountantigen-specific immune responses), and cancer9. Because PHAHs can interfere with cellsignaling pathways via interactions with the AhR, they may elicit a broad spectrum ofimmunologic and toxic responses1,9. For example, the significantly enhanced mitogenic responses
observed in the harbor seals in this study might be explained by the presence of PCB-targetedregulatory cells such as CD4+CD25+ cells which act via contact or soluble factors to inhibit or
down-regulate proliferating cells13. Alternatively, the production of cytokines, in particular IL-2
and the display of the IL-2 receptor on the surface of the activated T cells may be increased byexposure to PCBs14, resulting in enhanced proliferation.
The CALUX-TEQ values detected in free-ranging US Atlantic coast harbor seals reflect relatively
high burdens of dioxin-like compounds (Figure 3), although the relative contribution of individual
PHAHs to the overall CALUX response remains to be determined. There were no correlations
between the CALUX-TEQs and age, gender or condition of the animals. The levels of dioxin-likecompounds in these seals (mean TEQ 235 ± 155 and 137 ± 94 pg/g lw for adults and juveniles,
respectively) are comparable to or higher than those recently reported in Baltic ringed seals(Phoca hispida baltica) and gray seals (Halichoerus grypus) using a CALUX bioassay on liver
extracts 15.
As a tool for measuring the toxic potency of dioxin-like compounds, the CALUX bioassay has
several advantages, including its applicability to diverse tissues and its fast, relatively inexpensive,straightforward procedures. In the present study, logistical constraints prevented invasive
sampling and thus correlations between the CALUX response and blubber burdens of dioxin-likecompounds could not be examined. However, validation studies10,16 have revealed a goodcorrelation between the activity in these assays and TCDD/HAH concentrations in environmental
and biologic samples as determined by GC/MS.
Although possible links between contaminant burdens and health status are not clear, the CALUXTEQ values in adult harbor seals from the US Atlantic coast exceed the estimated threshold valueof 160 pg TEQ/g lw in blubber for adverse effects on immune function in the species 6. Dioxinlike PHAHs causing either inhibition of immunoregulatory cells or increased synthesis ofcytokines may explain the observed positive correlation between lymphocyte mitogenesis andCALUX-TEQ burdens. While this relationship is not evidence per se of a direct cause-effectassociation, the results indicate that exposure to environmentally relevant levels of dioxin-likecompounds may modulate immune responses in free-ranging harbor seals.

Organohalogen Compounds, Volumes 60-65, Dioxin 2003 Boston, MA

350--- Proposed

CALUX-TEQ pg/g lw

300

threshold for effects
on immune functionin harbor seals

250
200
150
100
500Juvenile

AdultFig. 3. CALUX-TEQ values (± 95% confidence interval)
in juvenile and adult harbor seals
Acknowledgements
The authors wish to acknowledge Christopher Taylor and the Northeast Fisheries Science Center,
Protected Species Branch, Woods Hole, MA, for providing harbor seal blood samples.
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