Jamie C. DeWitt

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.

Perfluorooctanoic acid-induced immunomodulation in adult C57BL/6J or C57BL/6N female mice.

Background Perfluorooctanoic acid (PFOA), an environmentally persistent compound of regulatory concern, has been reported to reduce antibody responses in mice at a single dose. Objective The aim of this study was to evaluate PFOA effects on humoral and cellular immunity using standard assays for assessing immune function, and to derive dose–response data. Methods C57BL/6J mice received 0 or 30 mg PFOA/kg/day for 10 days; half of the exposed groups were switched to vehicle and half continued on PFOA for five days. C57BL/6N mice received 0–30 mg/kg/day of PFOA in drinking water for 15 days. Mice were immunized with sheep red blood cells or sensitized to bovine serum albumin in Freund’s complete adjuvant on day 10 of exposure; immune responses were determined 1 day post-exposure. Results We found that 30 mg PFOA/kg/day given for 10 or 15 days reduced IgM synthesis; serum collected 1 day postexposure contained 8.4 × 104 or 2.7 × 105 ng PFOA/mL, respectively. IgM synthesis was suppressed at exposures ≥ 3.75 mg PFOA/kg/day in a dose-dependent manner, and IgG titers were elevated at 3.75 and 7.5 mg PFOA/kg/day. Serum PFOA at 3.75 mg/kg/day was 7.4 × 104 ng/mL 1 day postexposure, or 150-fold greater than the levels reported in individuals living near a PFOA production site. Using a second-degree polynomial model, we calculated a benchmark dose of 3 mg/kg/day, with a lower bound (95% confidence limit) of 1.75 mg/kg/day. Cell-mediated function was not affected. Conclusions IgM antibodies were suppressed after PFOA exposure. The margin of exposure for reduced IgM antibody synthesis was approximately 150 for highly exposed human populations.

Breaking Patterns of Environmentally Influenced Disease for Health Risk Reduction: Immune Perspectives

Background Diseases rarely, if ever, occur in isolation. Instead, most represent part of a more complex web or “pattern” of conditions that are connected via underlying biological mechanisms and processes, emerge across a lifetime, and have been identified with the aid of large medical databases. Objective We have described how an understanding of patterns of disease may be used to develop new strategies for reducing the prevalence and risk of major immune-based illnesses and diseases influenced by environmental stimuli. Findings Examples of recently defined patterns of diseases that begin in childhood include not only metabolic syndrome, with its characteristics of inflammatory dysregulation, but also allergic, autoimmune, recurrent infection, and other inflammatory patterns of disease. The recent identification of major immune-based disease patterns beginning in childhood suggests that the immune system may play an even more important role in determining health status and health care needs across a lifetime than was previously understood. Conclusions Focusing on patterns of disease, as opposed to individual conditions, offers two important venues for environmental health risk reduction. First, prevention of developmental immunotoxicity and pediatric immune dysfunction can be used to act against multiple diseases. Second, pattern-based treatment of entryway diseases can be tailored with the aim of disrupting the entire disease pattern and reducing the risk of later-life illnesses connected to underlying immune dysfunction. Disease-pattern–based evaluation, prevention, and treatment will require a change from the current approach for both immune safety testing and pediatric disease management.

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.

External heart deformities in passerine birds exposed to environmental mixtures of polychlorinated biphenyls during development

Necropsy-observable cardiac deformities were evaluated from 283 nestling passerines collected from one reference site and five polychlorinated biphenyl (PCB)-contaminated sites around Bloomington and Bedford, Indiana, USA. Hearts were weighed and assessed on relative scales in three dimensions (height, length, and width) and for externally visible deformities. Heart weights normalized to body weight (heart somatic index) were decreased significantly at the more contaminated sites in both house wren (Troglodytes aedon) and tree swallow (Tachycineta bicolor). Heart somatic indices significantly correlated with log PCB concentrations in Carolina chickadee (Parus carolinesis) and tree swallow and with log 2,3,7,8-tetrachlorodibenzo-p-dioxin toxic equivalent values in tree swallow alone. Ventricular length was increased significantly in eastern bluebirds (Sialia sialis) and decreased significantly in Carolina chickadee and tree swallow from contaminated sites versus the reference site. Heart length regressed significantly against the log PCB concentrations (Carolina chickadee and tree swallow) or the square of the PCB concentrations (red-winged blackbird [Agelaius phoeniceus]) in a sibling bird. The deformities that were observed most at the contaminated sites included abnormal tips (pointed, rounded, or flattened), center rolls, macro- and microsurface roughness, ventricular indentations on the ventral or dorsal surface, lateral ventricular notches, visibly thin ventricular walls, and changes in overall heart shape. A pooled heart deformity index regressed significantly against the logged contaminant concentrations for all species except red-winged blackbird. These results indicate that developmental changes in heart morphometrics and shape abnormalities are quantifiable and may be sensitive and useful indicators of PCB-related developmental impacts across many avian species.

Immune Responses in Sprague–Dawley Rats Exposed to Dibutyltin Dichloride in Drinking Water as Adults

Organotins are used commercially as agricultural pesticides, antifouling agents, and stabilizers for polyvinyl chloride (PVC) pipe. Mono- and di-substituted methyl and butyltins, used in PVC pipe production, are of concern as they leach from supply pipes into drinking water and have been reported to cause multisystem toxicity, including immunotoxicity. As part of an ongoing study to evaluate immunotoxic effects of organotins, we assessed immune function in adult Sprague–Dawley (CD) rats after exposure to dibutyltin dichloride (DBTC). Individually-housed adult male and female CD rats were given drinking water containing 0, 10, or 25 mg DBTC/L (final concentration) in 0.5% Alkamuls for 28 days. Water bottles were changed and water consumption was monitored twice weekly and body weights (BW) were recorded weekly. Delayed-type hypersensitivity (DTH), primary and secondary antibody responses to sheep red blood cells, and natural killer (NK) cell activity were evaluated in separate groups of treated and control animals on day 29 of exposure. Water consumption was significantly decreased in both sexes at 25 mg DBTC/L. BW, immune organ weights, the DTH response, and NK cell activity did not vary by dose. Different results for antibody responses in male rats were obtained in two experimental replicates. In the first replicate, IgG was elevated at the highest dose whereas in the second replicate, IgM was suppressed. However, as these effects occurred at the high dose of 25 mg DBTC/L, which is a concentration a million times higher than levels of DBTC reported in drinking water, our data suggest that DBTC is unlikely to cause immunotoxicity at concentrations found in drinking water supplies.

Developmental Exposure to 1.0 or 2.5 mg/kg of Dibutyltin Dichloride Does Not Impair Immune Function in Sprague-Dawley Rats

Organotins are used commercially as pesticides, antifouling agents, and stabilizers for polyvinyl chloride (PVC) pipe. Mono-and di-substituted butyltins, used in PVC pipe production, are of concern to the United States EPA, they leach from supply pipes into drinking water and are reported multisystem toxicants. We assessed immune function in Sprague–Dawley rats after developmental dibutyltin dichloride (DBTC) exposure. Pregnant rats were given drinking water containing 0, 10, or 25 mg/L of DBTC (final concentration) in 0.5% Alkamuls from gestational Day (GD) 6 through weaning of pups (37 days total). Approximate doses to dams: 1 and 2.5 mg DBTC/kg body weight (BW) during gestation, or 2.0 and 4.4 mg DBTC/kg BW while nursing. Litters were sexed, weighed, and culled to 4 males and 4 females per dam on postnatal Day (PND) 2. Beginning on PND3, litters of half of the dams per dose were gavaged with 0, 1.0, or 2.5 mg DBTC/kg BW 3X/week for 10 doses (maternal + direct treatment); remaining litters were exposed indirectly via lactation (maternal treatment). BW of litters exposed to 2.5 mg DBTC/kg BW was 10–20% lower (p ≤ 0.05) relative to other groups from PND14 (males) or PND17 (females) through PND37. Delayed-type hypersensitivity (DTH), antibody synthesis, and natural killer (NK) cell activity were evaluated in immunologically mature offspring (N = 6/sex/group). DTH responses and antibody synthesis did not differ by dose, sex, or exposure. NK cell activity in the 10 mg DBTC/L maternal only group was greater in male offspring than in female. In female offspring from the maternal + direct group, cytotoxicity increased by dose at the 25:1 effector:target cell ratio. Our data suggest that developmental immunotoxicity from DBTC-tainted drinking water is unlikely as the concentrations we used were several orders of magnitude higher than concentrations expected to leach from PVC pipes.

Serum Supplementation Modulates the Effects of Dibutyltin on Human Natural Killer Cell Function

Natural killer (NK) cells are a subset of lymphocytes capable of killing tumor cells, virally infected cells and antibody-coated cells. Dibutyltin (DBT) dichloride is an organotin used as a stabilizer in polyvinylchloride (PVC) plastics and as a deworming product in poultry. DBT may leach from PVC water supply pipes and therefore poses a potential risk to human health. We previously reported diminished NK cells lysis of tumor cells following exposure to DBT in serum-free cell culture medium. However, under in vivo conditions, circulating cells will be exposed to DBT in the presence of 100% plasma; thus we investigated whether serum supplementation and incubation time modulates DBT effects on NK cell killing and the accumulation of DBT in freshly isolated NK cells, to determine whether a serum-free model accurately predicts possible effects of DBT on human NK cells under in vivo conditions. Lytic function was decreased by approximately 35% at an intracellular DBT (DBTi) concentration of 200 microM and nearly complete loss of lytic function was observed at DBTi above 300 microM for one h. However, an intracellular concentration of 50 microM DBT, achieved over 24 h of exposure in 50% serum, reduced lytic function by 50%. Thus, conditions that reflect prolonged contact with circulating DBT, in the presence of serum, suggest that NK cell activity is decreased at lower DBTi. These data indicate that the model is useful in predicting potential human effects of relatively low DBTi concentrations.

An Organotin Mixture Found in Polyvinyl Chloride (PVC) Pipe is not Immunotoxic to Adult Sprague-Dawley Rats

Organotin compounds used in polyvinyl chloride (PVC) pipe production are of concern to the U.S. Environmental Protection Agency (EPA) because they leach from supply pipes into drinking water and are reported multisystem toxicants. Immune function was assessed in male Sprague-Dawley rats exposed to the mixture of organotins used in PVC pipe production. Although several of these organotins are reported immunotoxicants, their immunotoxicity as a mixture when given by drinking water has not been evaluated. Adult male rats were given drinking water for 28 d containing a mixture of dibutyltin dichloride (DBTC), dimethyltin dichloride (DMTC), monobutyltin trichloride (MBT), and monomethyltin trichloride (MMT) in a 2:2:1:1 ratio, respectively, at 3 different concentrations (5:5:2.5:2.5, 10:10:5:5, or 20:20:10:10 mg organotin/L), MMT alone (20 or 40 mg MMT/L), or plain water as a control. Delayed-type hypersensitivity, antibody synthesis, and natural killer cell cytotoxicity were evaluated in separate endpoint groups (n = 8/dose; 24/endpoint) immediately after exposure ended. The evaluated immune functions were not affected by the mixture or by MMT alone. Our data suggest that immunotoxicity is unlikely to result from the concentration of organotins present in drinking water delivered via PVC pipes, as the concentrations used were several orders of magnitude higher than those expected to leach from PVC pipes.