Effects of an environmentally relevant PCB-mixture on immune function, clinical chemistry, and thyroid hormone levels in adult female B6C3F1 mice.

Polychlorinated biphenyls (PCBs) have been assessed for immunotoxicity; however, humans and wildlife are exposed to multiple PCBs environmentally. Therefore, the aim of this study was to examine the effects of a complex 37 PCB congener mixture identified in blubber specific to dolphins residing in the estuarine waters of Charleston, South Carolina. Immunotoxicity was determined in adult female B6C3F1 mice by assessing lymphocyte proliferation, splenic and thymic immunophenotypes, and IgM production. Mice were exposed via oral gavage to the PCB-mixture (0, 1.8, 3.6, 7.1, or 14.3 mg/kg/day) for 28 days to yield a targeted total administered dose (TAD) 0, 50, 100, 200, or 400 mg/kg. Significant increased liver weight occurred at the highest treatment. IgM production was suppressed compared to control for all treatments. Numbers of thymic CD4+/CD8+, CD4-/CD8-, and CD4+/CD8- cells were not altered, but numbers of thymic CD4-/CD8+ cells were significantly increased in the highest treatment. Lymphocyte proliferation was not markedly affected by any treatment. The numbers of splenic CD4/CD8 T-cells or MHCII+ cells were not significantly changed. Humoral immunity using the plaque-forming cell assay for determining the specific IgM antibody-forming cell response appeared to be the most sensitive endpoint affected. As the lowest concentration tested resulted in decreased IgM production and total and free thyroxine (T4) serum levels a NOAEL was not identified. The calculated ED50 for suppression of IgM production was 2.4 mg/kg/day.

Comparative Innate and Adaptive Immune Responses in Atlantic Bottlenose Dolphins (Tursiops truncatus) With Viral, Bacterial, and Fungal Infections.

Free-ranging Atlantic bottlenose dolphins (n = 360) from two southeastern U.S. estuarine sites were given comprehensive health examinations between 2003 and 2015 as part of a multi-disciplinary research project focused on individual and population health. The study sites (and sample sizes) included the Indian River Lagoon (IRL), Florida, USA (n = 246) and Charleston harbor and associated rivers (CHS), South Carolina, USA (n = 114). Results of a suite of clinicoimmunopathologic tests revealed that both populations have a high prevalence of infectious and neoplastic disease and a variety of abnormalities of their innate and adaptive immune systems. Subclinical infections with cetacean morbillivirus and Chlamydiaceae were detected serologically. Clinical evidence of orogenital papillomatosis was supported by the detection of a new strain of dolphin papillomavirus and herpesvirus by molecular pathology. Dolphins with cutaneous lobomycosis/lacaziasis were subsequently shown to be infected with a novel, uncultivated strain of Paracoccidioides brasiliensis, now established as the etiologic agent of this enigmatic disease in dolphins. In this review, innate and adaptive immunologic responses are compared between healthy dolphins and those with clinical and/or immunopathologic evidence of infection with these specific viral, bacterial, and fungal pathogens. A wide range of immunologic host responses was associated with each pathogen, reflecting the dynamic and complex interplay between the innate, humoral, and cell-mediated immune systems in the dolphin. Collectively, these studies document the comparative innate and adaptive immune responses to various types of infectious diseases in free-ranging Atlantic bottlenose dolphins. Evaluation of the type, pattern, and degree of immunologic response to these pathogens provides novel insight on disease immunopathogenesis in this species and as a comparative model. Importantly, the data suggest that in some cases infection may be associated with subclinical immunopathologic perturbations that could impact overall individual and population health.

Chronic debilitation in stranded loggerhead sea turtles (Caretta caretta) in the southeastern United States: Morphometrics and clinicopathological findings.

Chronically debilitated loggerhead sea turtles (Caretta caretta) (DT) are characterized by emaciation, lethargy, and heavy barnacle coverage. Although histopathological findings associated with this condition have been reported, only limited data is available on health variables with clinical application. The objectives of this study were to 1) to compare morphometrics, clinicopathological variables, and immune functions of DTs to a group of apparently healthy loggerhead turtles to better understand the pathophysiology of the condition and 2) to assess health parameters in live debilitated turtles as they recovered during rehabilitation in order to identify potential prognostic indicators. We examined and sampled 43 DTs stranded from North Carolina to Florida for 47 health variables using standardized protocols to further characterize the condition. DTs were grouped into categories of severity of the condition, and those that survived were sampled at four time points through rehabilitation. All groups and time points were compared among DTs and to clinically healthy loggerhead turtles. Compared to healthy turtles, DTs had significantly lower body condition index, packed cell volume (PCV), total white blood cell (WBC) count, lymphocytes, glucose (Glc), total protein, all protein fractions as determined by electrophoresis, calcium (Ca), phosphorus (P), Ca:P ratio, potassium (K), lymphocyte proliferation, and greater heterophil toxicity and left-shifting, uric acid (UA), aspartate aminotransferase, creatine kinase, lysozyme, and respiratory burst. From admission to recovery, hematology and plasma chemistry data improved as expected. The most informative prognostic indicators, as determined by correlations with a novel severity indicator (based on survival times), were plastron concavity, P, albumin, total solids, UA, lymphocyte proliferation, WBC, K, Glc, Ca:P, and PCV. The results of this study document the wide range and extent of morphometric and metabolic derangements in chronically debilitated turtles. Monitoring morphometrics and clinicopathological variables of these animals is essential for diagnosis, treatment, and prognosis during rehabilitation.

In vitro evaluation of the effects of perfluorooctanesulfonic acid (PFOS) and perfluorooctanoic acid (PFOA) on IL-2 production in human T-cells.

Perfluorinated compounds, such as perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA), have been shown to alter various immune functions suggesting they are immunotoxic. This study assessed the effects of PFOS and PFOA on interleukin (IL)-2 production in the human Jurkat T-cell line and PFOS in healthy human primary T cells. Jurkat cells were stimulated with phytohemagglutinin (PHA)/phorbol myristate acetate (PMA), anti CD-3/anti CD-28, or anti CD-3, and dosed with 0, 0.05, 0.1, 0.5, 1, 5, 10, 50, 75, or 100 µg ml(-1) PFOS or 0, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5, or 10 µg ml(-1) PFOA. Jurkat cells stimulated with PHA/PMA or anti CD-3 exhibited decreased IL-2 production beginning at 50 µg PFOS ml(-1) and 5 µg PFOS ml(-1) respectively, but stimulation with anti-CD3/anti-CD28 resulted in no changes compared with the control. Addition of the PPAR-alpha antagonist GW6471 to PFOS-dosed cells stimulated with PHA/PMA resulted in decreases in IL-2 production starting at 50 µg PFOS ml(-1), which suggests PFOS affected T-cell IL-2 production via PPAR-alpha-independent mechanisms. Exposure to PFOA, PFOA + GW6471, or PFOS + PFOA in Jurkat cells resulted in no significant differences in IL-2 production. In vitro dosing studies using healthy primary human CD4+ T cells were consistent with the Jurkat results. These data demonstrated that PFOA did not impact IL-2 production, but PFOS suppressed IL-2 production in both a human cell line and human primary cells at dose levels within the high end of the human exposure range. A decrease in IL-2 production is characteristic of autoimmune diseases such as systemic lupus erythematosus and should be further investigated.

In vitro exposure of DE-71, a penta-PBDE mixture, on immune endpoints in bottlenose dolphins (Tursiops truncatus) and B6C3F1 mice.

Polybrominated diphenyl ethers (PBDEs) are an emerging contaminant of concern with low level exposures demonstrating toxicity in laboratory animals and wildlife, although immunotoxicity studies have been limited. Bottlenose dolphin peripheral blood leukocytes (PBLs) and mouse splenocytes were exposed to environmentally relevant DE-71 (a penta-PBDE mixture) concentrations (0-50 µg ml(-1) ) in vitro. Natural killer (NK) cell activity and lymphocyte (B and T cell) proliferation were evaluated using the parallelogram approach for risk assessment. This study aimed to substantiate results from field studies with dolphins, assess the sensitivities between the mouse model and dolphins, and to evaluate risk using the parallelogram approach. In mouse cells, NK cell activity increased at in vitro doses 0.05, 0.5 and 25 µg DE-71 ml(-1) , whereas proliferation was not modulated. In dolphin cells, NK cell activity and lymphocyte proliferation was not altered after in vitro exposure. In vitro exposure of dolphin PBLs to DE-71 showed similar results to correlative field studies; NK cell activity in mice was more sensitive to in vitro exposure than dolphins, and the parallelogram approach showed correlation with all three endpoints to predict risk in bottlenose dolphins.

Clinicoimmunopathologic findings in Atlantic bottlenose dolphins Tursiops truncatus with positive Chlamydiaceae antibody titers.

Sera from free-ranging Atlantic bottlenose dolphins Tursiops truncatus inhabiting the Indian River Lagoon (IRL), Florida, and coastal waters of Charleston (CHS), South Carolina, USA, were tested for antibodies to Chlamydiaceae as part of a multidisciplinary study of individual and population health. A suite of clinicoimmunopathologic variables was evaluated in Chlamydiaceae-seropositive dolphins (n = 43) and seronegative healthy dolphins (n = 83). Fibrinogen, lactate dehydrogenase, amylase, and absolute numbers of neutrophils, lymphocytes, and basophils were significantly higher, and serum bicarbonate, total alpha globulin, and alpha-2 globulin were significantly lower in dolphins with positive Chlamydiaceae titers compared with seronegative healthy dolphins. Several differences in markers of innate and adaptive immunity were also found. Concanavalin A-induced T lymphocyte proliferation, lipopolysaccharide-induced B lymphocyte proliferation, and granulocytic phagocytosis were significantly lower, and absolute numbers of mature CD 21 B lymphocytes, natural killer cell activity and lysozyme concentration were significantly higher in dolphins with positive Chlamydiaceae antibody titers compared to seronegative healthy dolphins. Additionally, dolphins with positive Chlamydiaceae antibody titers had significant increases in ELISA antibody titers to Erysipelothrix rhusiopathiae. These data suggest that Chlamydiaceae infection may produce subclinical clinicoimmunopathologic perturbations that impact health. Any potential subclinical health impacts are important for the IRL and CHS dolphin populations, as past studies have indicated that both dolphin populations are affected by other complex infectious and neoplastic diseases, often associated with immunologic perturbations and anthropogenic contaminants.

In vitro PFOS exposure on immune endpoints in bottlenose dolphins (Tursiops truncatus) and mice.

Previous studies in our lab have shown that perfluorooctane sulfonate (PFOS) modulates immune function in mice and correlates with many immune parameters in bottlenose dolphins (Tursiops truncatus). In this study, bottlenose dolphin peripheral blood leukocytes (PBLs) and adult female B6C3F1 mouse splenocytes were exposed to environmentally relevant PFOS concentrations (0-5 µg ml(-1)) in vitro; and natural killer (NK) cell activity and lymphocyte proliferation (T and B cell) were assessed using the parallelogram approach for risk assessment. The objectives were: to corroborate results from the correlative studies in bottlenose dolphins with in vitro PFOS exposures; to evaluate the sensitivity of the mouse model as compared with bottlenose dolphins; and to assess risk using the parallelogram approach. In mouse cells, NK cell activity was decreased at in vitro doses of 0.01, 0.5, 0.1, 0.5 and 1 µg PFOS ml(-1) and increased at 5 µg ml(-1). Additionally, B cell proliferation was not altered, but T cell proliferation was decreased at all in vitro PFOS exposures. In dolphin cells, NK cell activity and T cell proliferation were not altered by in vitro PFOS exposure, but B cell proliferation exhibited a positive association in relation to PFOS dose. Overall, the data indicates that: the in vitro exposures of bottlenose dolphin PBLs exhibited results similar to reported correlative fields studies; that mice were generally more sensitive (for these selected endpoints) than were dolphins; and that the parallelogram approach could be used two-thirds of the time to predict the effects in bottlenose dolphins.

DNA strand breaks (comet assay) in blood lymphocytes from wild bottlenose dolphins.

The comet assay was carried out on blood lymphocytes from a large number of wild dolphins (71 from Indian River Lagoon, FL, USA; 51 from Charleston Harbor, SC, USA) and provides a baseline study of DNA strand breaks in wild dolphin populations. There were no significant differences in the comet assay (% DNA in tail) results between the different age and sex categories. Significant difference in DNA strand breaks were found between Charleston Harbor dolphins (median--17.4% DNA in tail) and Indian River Lagoon dolphins (median--14.0% DNA in tail). A strong correlation found between T-cell proliferation and DNA strand breaks in dolphin lymphocytes suggests that dolphins with a high numbers of DNA strand breaks have a decreased ability to respond to infection. Higher concentrations of genotoxic agents in Charleston Harbor compared with Indian River lagoon may have been one of the causes of higher DNA strand breaks in these dolphins.

Associations between perfluoroalkyl compounds and immune and clinical chemistry parameters in highly exposed bottlenose dolphins (Tursiops truncatus).

Perfluoroalkyl compounds (PFCs) are ubiquitous, persistent chemical contaminants found in the environment, wildlife, and humans. Despite the widespread occurrence of PFCs, little is known about the impact these contaminants have on the health of wildlife populations. The authors investigated the relationship between PFCs (including ∑perfluorocarboxylates, ∑perfluoroalkyl sulfonates, perfluorooctane sulfonate, perfluorooctanoic acid, and perfluorodecanoic acid) and the clinocopathologic and immune parameters in a highly exposed population (n = 79) of Atlantic bottlenose dolphins (mean ∑PFCs = 1970 ng/ml; range 574-8670 ng/ml) sampled from 2003 to 2005 near Charleston, South Carolina, USA. Age-adjusted linear regression models showed statistically significant positive associations between exposure to one or more of the PFC totals and/or individual analytes and the following immunological parameters: absolute numbers of CD2+ T cells, CD4+ helper T cells, CD19+ immature B cells, CD21+ mature B cells, CD2/CD21 ratio, MHCII+ cells, B cell proliferation, serum IgG1, granulocytic, and monocytic phagocytosis. Several PFC analyte groups were also positively associated with serum alanine aminotransferase, gamma-glutamyltransferase, creatinine, phosphorus, amylase, and anion gap and negatively associated with cholesterol levels, creatinine phosphokinase, eosinophils, and monocytes. Based on these relationships, the authors suggest that the PFC concentrations found in Charleston dolphins may have effects on immune, hematopoietic, kidney, and liver function. The results contribute to the emerging data on PFC health effects in this first study to describe associations between PFCs and health parameters in dolphins.

Developmental immunotoxicity (DIT): assays for evaluating effects of exogenous agents on development of the immune system.

Developmental immunotoxicity (DIT) occurs when exposure to environmental risk factors prior to adulthood, including chemical, biological, physical, or physiological factors, alters immune system development. DIT may elicit suppression, hyperactivation, or misregulation of immune responses and may present clinically as decreased resistance to pathogens, allergic and autoimmune diseases, and inflammatory diseases. Immunotoxicity testing guidelines established by the Environmental Protection Agency for adult animals (OPPTS 8703.7800) require functional tests and immunophenotyping that are suitable for detecting immunomodulation, especially immunosuppression. However, evaluating immune function in offspring that are not fully immunocompetent yields results that are challenging to interpret. Therefore, this unit will describe an optimum exposure scenario, reference two assays (immunophenotyping and histopathology) appropriate for detecting immunomodulation in weaning-age offspring, and reference four assays (immunophenotyping, histopathology, T cell-dependent antibody responses, and delayed-type hypersensitivity) appropriate for detecting immunomodulation in immunocompetent offspring. The protocol also will reference other assays (natural killer cell and cytotoxic T lymphocyte) with potential utility for assessing DIT.

Immune function in female B(6)C(3)F(1) mice is modulated by DE-71, a commercial polybrominated diphenyl ether mixture.

Polybrominated diphenyl ethers (PBDEs) are an important class of flame-retardants that are environmentally persistent and bioaccumulative. Toxicity of these compounds has become a concern because detectable levels of PBDEs are present in humans and wildlife and they are structurally similar to polychlorinated biphenyls (PCBs). This study examined the effects of the commercial penta-BDE mixture, DE-71, in adult female B(6)C(3)F(1) mice on hematology, serum clinical chemistry, thyroid hormones, tissue histology, and several immunotoxicity end-points (lymphocyte proliferation, NK cell activity, splenic immunophenotypes, and SRBC-specific-IgM production). Mice were exposed via oral gavage for 28 days to achieve total administered doses (TAD) of 0, 0.5, 5, 50, or 100 mg/kg. No changes in histology, clinical chemistry, body or organ weights were observed. Serum total T3 and T4 levels were not altered by any of the DE-71 treatments. Peripheral blood monocyte numbers were decreased by the 0.5, 5, and 50 mg/kg treatments, but not by the 100 mg/kg TAD concentration. Compared to controls, mitogen-stimulated T- and B-cell proliferation was increased by the 100 mg/kg TAD concentration (ED(50) = 60 mg/kg TAD [2.14 mg/kg/day] and 58 mg/kg TAD [2.57 mg/kg/day], respectively). NK cell activity was decreased compared to controls by the 100 mg/kg TAD concentration (ED(50) = 20 mg/kg TAD [0.7 mg/kg/day]). No alterations were noted in thymic T-cell populations or in SRBC-specific-IgM production. Numbers of CD19(+)CD21(-), CD19(+)CD21(+), CD4(+)CD8(-), CD4(-)CD8(+), CD4(-)CD8(-), and MHC-II(+) cells in the spleen were not affected. However, the numbers of splenic CD4(+)CD8(+) cells were decreased compared to the controls by 0.5, 5, and 100 mg/kg TAD. This study provides an assessment of the systemic toxicity and immunotoxicity of DE-71, and indicates that immune parameters are modulated at exposure concentrations lower than previously reported.

Immunotoxicity of perfluorinated compounds: recent developments.

Perfluorinated compounds (PFCs) are environmentally widespread, persistent, and bioaccumulative chemicals with multiple toxicities reported in experimental models and wildlife, including immunomodulation. The two most commonly detected compounds, which also generally occur in the highest concentrations in environmentally exposed organisms, are perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS). PFOA and PFOS have been reported to alter inflammatory responses, production of cytokines, and adaptive and innate immune responses in rodent models, avian models, reptilian models, and mammalian and nonmammalian wildlife. Mounting evidence suggests that immune effects in laboratory animal models occur at serum concentrations below, within the reported range, or just above those reported for highly exposed humans and wildlife. Thus, the risk of immune effects for humans and wildlife exposed to PFCs cannot be discounted, especially when bioaccumulation and exposure to multiple PFCs are considered. This review contains brief descriptions of current and recently published work exploring immunomodulation by PFOA, PFOS, and other PFCs in rodent models, alternative laboratory models, and wildlife.

Current status of developmental immunotoxicity: early-life patterns and testing.

Developmental immunotoxicity (DIT) occurs when exposure to environmental risk factors prior to adulthood, including chemical, biological, physical, or physiological factors, alters immune system development. DIT may elicit suppression, hyperactivation, or misregulation of immune responses and therefore may present clinically as decreased resistance to pathogens, allergic and autoimmune diseases, and inflammatory diseases. When evaluating DIT in an animal model, specific endpoints are assessed, which can reveal the potential for a risk factor to alter immune system development. However, linking DIT evaluation in an animal model with clinical realities observed in human populations requires that DIT testing regimens evaluate critical windows in immune system development. In addition, pathways leading to DIT may not be apparent without the stressors that induce aberrant and detectable responses. This review contains brief descriptions of recently published work that addresses disease patterns associated with DIT and solutions for altering such patterns of disease. We also comment on gaps between DIT testing in animal models and the clinical manifestation of immune-based diseases in children that can be filled by a better understanding of critical windows in immune system development and DIT testing that includes multiple functional assays.

Effects of environmentally-relevant levels of perfluorooctane sulfonate on clinical parameters and immunological functions in B6C3F1 mice.

In the first part of a series of studies to account for perfluorooctane sulfonate (PFOS)-induced sheep red blood cell (SRBC)-specific immunoglobulin M (IgM) antibody suppression in mice, a survey of clinical and immunotoxicological endpoints was examined. Adult female B6C3F1 mice were exposed orally for 28 days to a total administered dose (TAD) of 0, 0.1, 0.5, 1, or 5 mg PFOS/kg. Uterus wet weight was significantly decreased compared with control at the 5 mg/kg dose. No indications of wasting syndrome, malnutrition, alteration of thyroid homeostasis, or signs of overt toxicity were observed. Numbers of splenic CD19+/CD21⁻, CD19+/CD21+, B220+/CD40+, CD4+/CD154⁻, CD4+/CD154+, and MHC-II+ cells were not altered. Additionally, ex vivo interleukin-4 (IL-4), IL-5, and IL-6 production by in vitro anti-CD3- or phorbol myristate acetate-stimulated CD4+ T-cells was not affected. Ex vivo IL-6 production by B-cells was significantly increased by in vitro stimulation with either anti-CD40 or lipopolysaccharide. Increased IL-6 production by B-cells was the most sensitive endpoint assessed resulting in alterations at the lowest dose tested (0.1 mg/kg TAD) following anti-CD40 stimulation. Further studies are required to characterize effects on inflammatory markers such as IL-6 at environmentally relevant concentrations of PFOS and to determine the key events associated with PFOS-induced IgM suppression to address potential human health risks.

Effects of perfluorooctane sulfonate (PFOS) exposure on markers of inflammation in female B6C3F1 mice.

Perfluorooctane sulfonate (PFOS; 1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8,8-heptadecafluoro-1-octanesulfonic acid) has been reported to alter humoral immune functions, but inflammatory processes following PFOS exposure have not been fully characterized. Therefore, the current study, assessed TNF-α and IL-6 concentrations in serum and peritoneal lavage fluid, numbers of splenoctyes expressing intracellular TNF-α, IL-6, IL-10 or IL-1, and ex vivo TNF-α and IL-6 production by peritoneal macrophages following either in vivo or in vitro LPS exposure. Adult female B6C3F1 mice were exposed orally for 28 days to 0, 1, 3, or 300 mg PFOS/kg total administered dose [TAD] (e.g., 0, 0.0331, 0.0993 or 9.93 mg/kg/day). Body and spleen masses were significantly reduced in the highest PFOS treatment group compared to the control group, whereas liver mass was significantly increased. Serum TNF-α levels were significantly decreased following exposure to 1 mg PFOS/kg TAD as compared to controls, while serum IL-6 levels were increased. IL-6 concentrations in peritoneal lavage fluid decreased with increasing dose. PFOS treatment did not alter numbers of splenocytes expressing intracellular levels of TNF-α, IL-10 or IL-1. Numbers of splenocytes expressing intracellular levels of IL-6 were significantly decreased in the 3 mg/kg treatment as compared to controls. Overall, these data suggest that PFOS exposure can alter some inflammatory processes, which could potentially lead to misdirected inflammatory responses.

Clinicoimmunopathologic findings in Atlantic bottlenose dolphins Tursiops truncatus with positive cetacean morbillivirus antibody titers.

Sera from free-ranging Atlantic bottlenose dolphins Tursiops truncatus inhabiting the Indian River Lagoon (IRL), Florida were tested for antibodies to cetacean morbilliviruses from 2003 to 2007 as part of a multidisciplinary study of individual and population health. A suite of clinicoimmunopathologic variables were evaluated in morbillivirus-seropositive dolphins (n = 14) and seronegative healthy dolphins (n = 49). Several important differences were found. Serum alkaline phosphatase, creatine phosphokinase, chloride, albumin and albumin/globulin ratios were significantly lower in seropositive dolphins. Innate immunity appeared to be upregulated with significant increases in lysozyme concentration and marginally significant increases in monocytic phagocytosis. Adaptive immunity was also impacted in dolphins with positive morbillivirus antibody titers. Mitogen-induced T lymphocyte proliferation responses were significantly reduced in dolphins with positive morbillivirus antibody titers, and marginally significant decreases were found for absolute numbers of CD4+ lymphocytes. The findings suggest impairment of cell-mediated adaptive immunity, similar to the immunologic pattern reported with acute morbillivirus infection in other species. In contrast, dolphins with positive morbillivirus antibody titers appeared to have at least a partially upregulated humoral immune response with significantly higher levels of gamma globulins than healthy dolphins, which may represent an antibody response to morbillivirus infection or other pathogens. These data suggest that subclinical dolphin morbillivirus infection in IRL dolphins may produce clinicoimmunopathologic perturbations that impact overall health.

Complex contaminant exposure in cetaceans: a comparative E-Screen analysis of bottlenose dolphin blubber and mixtures of four persistent organic pollutants.

Cetaceans are federally protected species that are prone to accumulate complex mixtures of persistent organic pollutants (POPs), which individually may exert estrogenic or antiestrogenic effects. In the present study it was assessed whether contaminant mixtures harbored by cetaceans are estrogenic or antiestrogenic using a comparative approach. Interactions of antiestrogenic and estrogenic compounds were first investigated with the E-Screen assay using a mixture of four POPs (dichlorodiphenyldichloroethylene [4,4'-DDE], trans-nonachlor, and polychlorinated biphenyls [PCBs] 138 180) prevalent in cetacean blubber. Estrogenic/antiestrogenic activity was determined for the individual compounds and their binary, tertiary, and quaternary combinations. Significantly different responses were observed for the various POP mixtures, including enhanced estrogenic and antiestrogenic effects and antagonistic interactions. These results were then compared to the concentrations and estrogenic/antiestrogenic activity of contaminant mixtures isolated directly from the blubber of 15 bottlenose dolphins (Tursiops truncatus) collected from five U.S. Atlantic and Gulf of Mexico locations. The lowest observed effect concentrations (LOECs) determined for 4,4'-DDE (20 µmol/L), PCB 138 (20 µmol/L), PCB 180 (21 µmol/L), and trans-nonachlor (3 µmol/L) in the E-Screen were greater than estimated dolphin blood concentrations. Although estimated blood concentrations were below the LOECs, significant estrogenic activity was detected in diluted dolphin blubber from Cape May, NJ and Bermuda. Positive correlations between blubber estrogenicity and select POP concentrations (ΣDDTs, ΣPBDEs, ΣHCB, Σestrogenic PCBs, Σestrogenic POPs) were also observed. Collectively, these results suggest that select bottlenose dolphin populations may be exposed to contaminants that act in concert to exert estrogenic effects at biologically relevant concentrations. These observations do not necessarily provide direct evidence of endocrine disruption; however, they may indicate an environmental source of xenoestrogenic exposure warranting future research.

Comparison of mercury burdens in chronically debilitated and healthy loggerhead sea turtles (Caretta caretta).

An increase in the incidence of debilitated loggerhead sea turtle (Caretta caretta) strandings in the southeastern United States has been observed in recent years. These turtles are characterized by emaciation and heavy burdens of external and internal parasites, and bacterial infections, but the underlying cause of their condition is unknown. To investigate further the causes of these strandings, a health assessment was performed on stranded, debilitated loggerhead turtles, and contaminant concentrations in various tissues were compared to those from healthy turtles. This portion of the study investigated the potential role of mercury (Hg) toxicity in the debilitated condition described above. Hematocrit, total protein, albumin, globulin, glucose, calcium, lymphocyte counts, heterophil:lymphocyte ratios, aspartate aminotransferase, uric acid, sodium, and chloride were altered in debilitated loggerheads relative to healthy animals. However, none of the aforementioned health indicators correlated with Hg concentrations in either red blood cells (RBCs) or plasma. The Hg concentration in RBCs was 129+/-72 (mean+/-standard deviation) times higher than in plasma, causing a significant dilution of Hg in whole blood due to extreme anemia. Mercury concentrations in RBCs (73.7+/-21.2 ng/g) and scutes (455+/-57 ng/g) from debilitated turtles were similar to or lower than those reported for healthy animals, indicating no elevation in Hg exposure before and during the progression of this condition. These findings suggest that Hg toxicity does not play a role in the debilitated loggerhead condition observed in the southeastern United States.

Organohalogen contaminants in blood of Kemp's ridley (Lepidochelys kempii) and green sea turtles (Chelonia mydas) from the Gulf of Mexico.

The threat that exposure to organohalogen (OH) contaminants poses to endangered populations of Kemp's ridley (Lepidochelys kempii) and green sea turtles (Chelonia mydas) is not well understood, partly because few OH data are available. Blood samples from live juvenile and sub-adult L. kempii (n = 46) and C. mydas (n = 9) from the Gulf of Mexico and from L. kempii from the southeastern US coast (n = 3) were extracted using microwave-assisted extraction, and analyzed by large volume injection gas chromatography-mass spectrometry for 85 polychlorinated biphenyls (PCBs), 25 organochlorine pesticides (OCPs) and 27 polybrominated diphenyl ethers (PBDEs). Plasma chemistries, hematology and immune responses were also assessed. Concentrations of SigmaPCBs (geometric mean, range: 3190 pg g(-1), 227-21590 pg g(-1) blood), SigmaDDTs (geometric mean, range: 541 pg g(-1), 161-4310 pg g(-1) blood) and OCPs in L. kempii from the Gulf were comparable to those reported in L. kempii from the Atlantic. SigmaPBDEs were detected in all samples (geometric mean, range: 146 pg g(-1), 19.5-1450 pg g(-1) blood), with PBDE 47, 99, 100, 153 and 154 being the predominant congeners. SigmaPCBs, SigmaDDTs and Sigmachlordanes were one order of magnitude lower in green turtles, and SigmaPBDE concentrations were lower by half due to trophic level differences. L. kempii from the southeast USA had higher percentages of highly chlorinated PCBs indicating exposure to Aroclor 1268. Blood urea nitrogen was positively correlated to Sigmachlordanes, and SigmaPCBs were inversely correlated to creatine phosphokinase in L. kempii. These data help establish baseline contaminant concentrations in live L. kempii and C. mydas.

Assessment of trichloroethylene (TCE) exposure in murine strains genetically-prone and non-prone to develop autoimmune disease.

There is increasing laboratory and epidemiologic evidence relating exposure to trichloroethylene (TCE) with autoimmune disease including scleroderma and lupus. New Zealand Black/New Zealand White (NZBWF1) and B6C3F1 mice were exposed to TCE (0, 1, 400 or 14,000 ppb) via drinking water for 27 or 30 weeks, respectively. NZBWF1 mice spontaneously develop autoimmune disease while B6C3F1 mice, a standard strain used in immunotoxicology testing, are not genetically prone to develop autoimmune disease. During the TCE exposure period, serum levels of total IgG, and autoantibodies (anti-ssDNA, -dsDNA, and -glomerular antigen [GA]) were monitored. At the termination of the study, renal pathology, natural killer (NK) cell activity, total IgG levels, autoantibody production, T-cell activation, and lymphocytic proliferative responses were evaluated. TCE did not alter NK cell activity, or T- and B-cell proliferation in either strain. Numbers of activated T-cells (CD4+/CD44+) were increased in the B6C3F1 mice but not in the NZBWF1 mice. Renal pathology, as indicated by renal score, was significantly increased in the B6C3F1, but not in the NZBWF1 mice. Serum levels of autoantibodies to dsDNA and ssDNA were increased at more time points in B6C3F1, as compared to the NZBWF1 mice. Anti-GA autoantibodies were increased by TCE treatment in early stages of the study in NZBWF1 mice, but by 23 weeks of age, control levels were comparable to those of TCE-exposed animals. Serum levels anti-GA autoantibodies in B6C3F1 were not affected by TCE exposure. Overall, these data suggest that TCE did not contribute to the progression of autoimmune disease in autoimmune-prone mice during the period of 11-36 weeks of age, but rather lead to increased expression of markers associated with autoimmune disease in a non-genetically prone mouse strain.