Margie M. Peden-Adams

Suppression of Humoral Immunity in Mice following Exposure to Perfluorooctane Sulfonate

Adult male and female B6C3F1 mice were exposed to perfluorooctane sulfonate (PFOS) daily via gavage for 28 days (0, 0.005, 0.05, 0.1, 0.5, 1, or 5 mg/kg total administered dose [TAD]). Following exposure, various immune parameters were assessed and serum PFOS concentrations were determined. Lymphocyte proliferation was not altered in either gender. Natural killer cell activity was increased compared with control at 0.5, 1, and 5 mg/kg TAD in male mice but was not altered in female mice. At these treatment levels, splenic T-cell immunophenotypes were minimally altered in females, but all T-cell subpopulations were significantly modulated in males beginning at 0.1 mg/kg TAD. The sheep red blood cell (SRBC) plaque-forming cell (PFC) response was suppressed in male mice beginning at 0.05 mg/kg TAD and in females at 0.5 mg/kg TAD. Serum trinitrophenyl (TNP)-specific IgM titers were also decreased by PFOS after TNP-LPS (TNP conjugated to lipopolysacharide) challenge suggesting that the humoral immune effects may be attributed to the B-cell rather than T-cell because both T-dependent (SRBC) and T-independent (TI) (TNP-LPS) antigens result in suppressed IgM production. Based on the PFC response, the low observed effect level (LOEL) for males was 0.05 mg/kg TAD (ED(50) = 0.021 mg/kg TAD) and for females was 0.5 mg/kg TAD (ED(50) = 0.59 mg/kg TAD). Measured PFOS serum concentrations at these dose levels were 91.5 +/- 22.2 ng/g and 666 +/- 108 ng/g (mean +/- SD), respectively. The male LOEL serum level was approximately 14-fold lower than reported mean blood levels from occupationally exposed humans and fell in the upper range of concentrations reported for the general population. Overall, this study provides a profile of PFOS immunotoxicity showing effects at levels reported in humans and identifies the B-cells as a potential target.

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.

Immunotoxicity of perfluorooctanoic acid and perfluorooctane sulfonate and the role of peroxisome proliferator-activated receptor alpha.

Perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) are environmentally widespread and persistent chemicals with multiple toxicities reported in experimental animals and humans. These compounds can trigger biological activity by activating the alpha isotype of peroxisome proliferator-activated receptors (PPARs), ligand-activated transcription factors that regulate gene expression; however, some biological effects may occur independently of the receptor. Activation of the peroxisome proliferator-activated receptor alpha (PPARα) modulates lipid and glucose homeostasis, cell proliferation and differentiation, and inflammation. Reported immunomodulation in experimental animals exposed to PFOA and PFOS has included altered inflammatory responses, production of cytokines and other proteins, reduced lymphoid organ weights, and altered antibody synthesis. Mounting experimental animal evidence suggests PPARα independence of some immune effects. This evidence originates primarily from studies with PPARα knockout models exposed to PFOA that demonstrate hepatic peroxisome proliferation, reduced lymphoid organ weights, and altered antibody synthesis. As human PPARα expression is significantly less than that of rodents, potential PPARα independence indicates that future research must explore mechanisms of action of these compounds, including PPARα -dependent and -independent pathways. This multiauthored review contains brief descriptions of current and recently published work exploring immunomodulation by PFOA and PFOS, as well as a short overview of other PPARα ligands of therapeutic and environmental interest.

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

Several laboratory and field studies indicate that organochlorine contaminants (OCs), such as poly-chlorinated 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 μg/mL) or 4,4′-DDE (0–13.4 μg/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.

Gestational Exposure to Perfluorooctane Sulfonate Suppresses Immune Function in B6C3F1 Mice

Perfluorinated alkyl acids (PFAAs) are used in a multitude of applications and are categorized as high-production volume chemicals produced in quantities exceeding 10,000 lbs/year. As a result, widespread exposure has been documented in adults, children, and infants. It is generally accepted that children are more sensitive to the effects of xenobiotic exposures during fetal and postnatal periods of development; therefore, considerable efforts are required to investigate the potential impact of a model PFAA, perfluorooctane sulfonate (PFOS) on childrens immunological health. Using the pairing of female C57BL/6N mice with male C3H/HeJ, developmental immunotoxicity was evaluated in B6C3F1 pups following oral maternal exposure to PFOS on gestations days 1-17. Exposure levels included 0.1, 1, and 5 mg/kg/day PFOS. Natural killer (NK) cell activity, SRBC IgM plaque assay, CD4/8 lymphocytic subpopulations, nitrite production in peritoneal macrophages, and body/organ weights were evaluated at 4 and 8 weeks of age in F1 pups. No significant dose-responsive changes in maternal or pup body weights, flow cytometry, or macrophage function were observed, yet hepatomegaly was indicated in F1 male pups at 4 weeks of age. Functional deficits were not evident until 8 weeks of age when NK cell function and IgM production were significantly decreased. When compared with females, male pups were more sensitive to the effects of PFOS thereby establishing a no observed adverse effect level and low observed adverse effect level of 0.1 and 1.0 mg/kg/day (males only) following maternal PFOS exposure level, respectively. This study establishes that the developing immune system is sensitive to the effects of PFOS and results in functional deficits in innate and humoral immunity detectable at adulthood.

Relationship of Blood Mercury Levels to Health Parameters in the Loggerhead Sea Turtle (Caretta caretta)

Background Mercury is a pervasive environmental pollutant whose toxic effects have not been studied in sea turtles in spite of their threatened status and evidence of immunosuppression in diseased populations. Objectives In the present study we investigate mercury toxicity in loggerhead sea turtles (Caretta caretta) by examining trends between blood mercury concentrations and various health parameters. Methods Blood was collected from free-ranging turtles, and correlations between blood mercury concentrations and plasma chemistries, complete blood counts, lysozyme, and lymphocyte proliferation were examined. Lymphocytes were also harvested from free-ranging turtles and exposed in vitro to methylmercury to assess proliferative responses. Results Blood mercury concentrations were positively correlated with hematocrit and creatine phosphokinase activity, and negatively correlated with lymphocyte cell counts and aspartate amino-transferase. Ex vivo negative correlations between blood mercury concentrations and B-cell proliferation were observed in 2001 and 2003 under optimal assay conditions. In vitro exposure of peripheral blood leukocytes to methylmercury resulted in suppression of proliferative responses for B cells (0.1 μg/g and 0.35 μg/g) and T cells (0.7 μg/g). Conclusions The positive correlation between blood mercury concentration and hematocrit reflects the higher affinity of mercury species for erythrocytes than plasma, and demonstrates the importance of measuring hematocrit when analyzing whole blood for mercury. In vitro immunosuppression occurred at methylmercury concentrations that correspond to approximately 5% of the individuals captured in the wild. This observation and the negative correlation found ex vivo between mercury and lymphocyte numbers and mercury and B-cell proliferative responses suggests that subtle negative impacts of mercury on sea turtle immune function are possible at concentrations observed in the wild.

Immune dysfunction in Atlantic bottlenose dolphins (Tursiops truncatus) with lobomycosis

Lobomycosis (Lacaziosis) occurs only in humans and dolphins under natural conditions. We evaluated the immune status of eight dolphins with lobomycosis and 40 healthy dolphins from the Indian River Lagoon (IRL), Florida. Lobomycosis cases had multiple abnormalities in their immunologic parameters when compared to healthy dolphins. The absolute number of circulating lymphocytes and serum albumin concentration were reduced (P<0.05) while the segmented neutrophils, alpha 1, total beta, total gamma and total globulins were increased (P<0.05). Although innate immunity was relatively intact and phagocytosis and natural killer cell activity were not affected, the plasma lysozyme concentrations were elevated in dolphins with lobomycosis (P<0.05). Adaptive immunity was depressed with statistically significant decreases found in the absolute numbers of CD4(+) helper T cells and CD19(+) and CD21(+) B cells. The ratios of CD2(+) T cells to CD4(+) cells and CD2(+) to CD21(+) cells were increased (P=0.05 and P<0.05, respectively) and the numbers of lymphocytes expressing MHC class II molecules was decreased in dolphins with lobomycosis (P<0.05). Lymphocyte proliferation was reduced in response to stimulation with lipopolysaccharide and concanavalin A (P<0.05). Antibody titers to Erysipelas rhusiopathiae, a common marine micro-organism, were decreased (P<0.05). In summary, dolphins with lobomycosis exhibit significant impairment in adaptive immunity.

Suppression of Humoral Immunity Following Exposure to the Perfluorinated Insecticide Sulfluramid

Perfluorinated hydrocarbons have been manufactured for over 40 yr and have numerous applications in industry. This group of compounds has recently generated much interest, as some of these compounds such as perfluorooctane sulfonate (PFOS) and perfluoroctanic acid (PFOA) are persistent in the environment and detectable in blood samples of both wildlife and humans. Studies show that these perfluorinated compounds induce peroxisomal proliferation, induce hepatomegaly, alter steroidogenesis, and decrease body weight, accompanied by a wasting syndrome; however, effects on immune function have not been addressed at length. This study examined sulfluramid, a perfluorinated pesticide that is currently available in the marketplace and is a representative member of this class of chemicals. Adult female B6C3F1 mice were exposed via gavage to either an oil carrier control or sulfluramid for 14 d (1, 3, 10, or 30 mg/kg/d) or 28 d (0.3, 1, 3, or 10 mg/kg/d). Although responses were normal in natural killer cell activity and lymphocyte proliferation, dose-responsive suppression was noted in the plaque forming cell (PFC) response at exposure levels as low as 3 mg/kg/d in the 14-d exposure and 0.3 mg/kg/d for 28 d. Dose-responsive increases in liver mass were observed following treatment with 1, 3, 10, or 30 mg/kg/d for 14 d and 0.3, 1, 3, or 10 mg/kg/d for 28 d. A significant reduction in body weight was observed at the highest dose level in each study. Novel findings in this study indicate that sulfluramid suppresses immunoglobulin (Ig) M production. Additional immunotoxicity studies are required to understand potential mechanisms of suppression and determine potential health risks associated with exposure to perfluorinated hydrocarbons.

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.