Developmental toxicity in white leghorn chickens following in ovo exposure to perfluorooctane sulfonate (PFOS)
Introduction
Perfluorinated alkyl acids (PFAAs), have been manufactured for over 50 years and are currently used throughout industry as stain and water repellents, in floor waxes, as coatings on paper food containers, as firefighting foams, flame retardants, denture cleaners, carpet spot cleaners, pharmaceuticals, and as pesticides [1]. Due to the environmental persistence of these compounds, the U.S. Interagency Testing Committee (ITC) has identified 50 perfluorinated compounds as chemicals of interest [2]. Two of the major classes of concern are the perfluorinated carboxylates and the perfluorinated sulfonates, which together are referred to as PFAAs. They are typically nonvolatile, with relatively high molecular weights, and surface-active properties related to the attached carboxylic acid or sulfonic acid moiety. Due to the high energy of the carbon–fluorine ionic bond, many perfluorinated compounds are resistant to hydrolysis, photolysis, biodegradation and metabolism; therefore, some of these compounds are very stable in the environment [3]. Environmental exposure is evident as compounds from these perfluorinated classes such as perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) are found in blood samples both from wildlife and humans [2], [3], [4], [5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16]. Concentrations in eggs have been reported in various avian species with levels in Caspian tern eggs from the Great Lakes area (max 3.4 μg/kg egg) being among the highest [11], [13], [17], [18], [19], [20].
General toxicological effects of PFAAs indicate that they are peroxisome proliferator-activated receptor (PPAR)-α agonists that casue peroxisomal proliferation, hepatomegaly, body weight loss associated with a wasting syndrome peroxisomal fatty acid β-oxidation, decreases in serum cholesterol, and increases in serum alanine amino transferase (ALT) [21], [22], [23], [24], [25], [26], [27], [28], [29], [30], [31]. More recent studies suggest PFOS and PFOA disrupt endocrine function. PFOS decreases aromatase activity with corresponding increases in testosterone and dihydrotestosterone and plasma 11-ketotestosterone and testosterone, respectively, depending on the species [32], [33]. PFOS also decreases uterine weight in females [34], [35]. Alternatively, PFOA increases aromatase activity and plasma estradiol levels and induces Leydig cell adenomas [36], [37].
Emerging data suggest that the immune system is a sensitive target for PFAAs. Studies indicate that PFOA suppresses antibody production, causes thymic and splenic atrophy, and alters T-cell populations [38], [39], [40], [41], [42]. Perfluorodecanoic acid (PFDA) suppresses antibody production and perfluorononanoic acid (PFNA) alters interleukin (IL)-4 and interferon (IFN)-γ production [43], [44]. PFOS suppresses both T-dependent and T-independent antibody production [45]. In addition, PFOS causes increased ex vivo T-cell IL-4 production (stimulated with phorbol myristate acetate) and B-cell IL-6 production (stimulated with either anti-CD40 or lipopolysaccharide), and modulates serum levels of IL-6 and tumor necrosis factor (TNF)-α [34], [45], [46]. Moreover, liver levels of PFOS have been correlated with infectious disease states in sea otters [47].
Several studies have demonstrated that embryonic exposure to PFOS causes developmental and reproductive effects including increased incidence of prenatal mortality, low birth weights, structural defects, and developmental delays [18], [27], [31], [48], [49], [50], [51], [52]. Specifically, PFOS causes high pup mortality, chick mortality, altered thyroid hormone levels in F1 mouse offspring, and in combination with maternal restraint stress decreases performance in an open-field test [18], [49], [53]. Only one study has assessed the integrity of the immune system following exposure to PFOS during development. Keil et al. [54] showed that mice exposed in utero exhibited long-term effects in both male and female offspring. Functional immune suppression of NK cell activity (males and females) and sheep red blood cell (SRBC)-specific IgM production (males only) were observed at 8 weeks of age, but not at 4 weeks of age.
Unlike mammalian studies, avian toxicity studies with PFAAs are limited and somewhat varied in scope. Studies in mallards and quail assessing body weight, organ weight, and mortality following a 5-day PFOS exposure via feed report an LD50 in juvenile mallards (Anas platyrhynchus) of 150 and 61 mg/(kg day) in bobwhite quail (Colinus virginianus) [55]. In ovo exposures with white leghorn chickens (Gallus gallus) indicate a lowest observed adverse effect level (LOAEL) of 0.1 mg PFOS/kg egg wt (based on decreased hatch rates) and an LD50 of 4.9 mg PFOS/kg egg wt [18]. Only one study has assessed sublethal health effects in birds. Hoff et al. [56] noted increased serum ALT, and decreased serum cholesterol and triglyceride levels in both the great tit (Parus major) and blue tit (Parus caeruleus) in relation to hepatic PFOS levels. Cwinn et al. [57] showed that embryonic hepatocytes from white leghorn chickens respond to PPAR-α agonism following PFOS exposure leading to increased mRNA for malic enzyme, peroxisomal acyl-CoA oxidase, liver fatty acid binding protein, and enoly-Coenzyme A but do not exhibit an increase in PPARα mRNA. Other gene expression studies in chicken hepatocytes indicate differing gene expression patterns following PFOS or PFOA exposure [58].
No avian studies to date have, however, assessed alterations in immune function or sublethal toxicity following embryonic exposure to PFOS. PFOS is the predominate PFAA found in both human and wildlife blood samples [3], [7], [8], [14], [16]. The developing embryo and immune system are often sensitive targets for xenobiotics, and recent studies suggest that immune function may be highly susceptible to effects of PFOS [45], [54], [59], [60], [61], [62], [63], [64]. Therefore, the current study utilized traditional measures of avian immunotoxicity [65], [66] to assess developmental effects of embryonic exposure using the white leghorn chicken following air sac injection on embryonic day 0 (ED0). In addition, typical measures of developmental and reproductive toxicity (i.e., hatch rates, deformities, survival, limb measurements, length measurements, and weights) along with brain asymmetry, hematology, clinical chemistry, and serum PFOS concentrations were assessed to determine potential health effects of egg concentrations documented in the literature and to identify what those injection concentrations would translate to in serum levels at 14 days of age.
Section snippets
Chemicals, antibodies, and supplies
Unless otherwise specified, all chemicals and mitogens were purchased from Sigma (St. Louis, MO). Perfluorooctane sulfonic acid (PFOS) potassium salt (stated purity >98%) used for animal treatments was obtained from Fluka (via Sigma, CAS No. 2795-39-3). Perfluorooctane sulfonic acid potassium salt (PFOS; 98%) for analytical chemistry determinations was obtained from Alfa Aesar (Ward Hill, MA). 13C labeled sodium PFOS and perfluorooctanoic acid (PFOA) were from Wellington Laboratory (Guelph,
Hatch rates and deformities
Neither hatching success nor egg viability was altered by PFOS treatment (Table 1). In the 2.5 mg/kg egg wt treatment four chicks out of 52 exhibited deformities (1 with cranial edema, 2 with abdominal cavities not formed correctly, 1 with exposed brain, beak not formed, and right eye not formed). However, in the 5 mg/kg egg wt treatment only two out of 52 chicks exhibited deformities (1 with crossbeak, 1 with cranial edema and missing leg and wing).
Body and organ mass, immune organ cellularity, and developmental measures
Body, brain, bursa and thymus mass were not
Discussion
This is the first study to detail developmental and immune modulation following PFOS exposure in an avian species. To survey for potential clinical health effects following PFOS exposures, hematology and clinical chemistry were included along with organ and body mass, and brain asymmetry measurements. In ovo exposure had little effect on hematological parameters, but caused statistically significant changes in liver clinical chemistry parameters such as ALT and LDH. ALT in birds is not as
Conflict of interest statement
The authors have no conflict of interest to declare.
Acknowledgments
The authors thank Carol Foster, Ann Miller, Jan Young, Ron Gosset, and Norman Ellis for their assistance. The authors also thank the following reviewers for their critical review of the manuscript: Dr. Jennifer Keller and Mr. Jeff Mollenhauer. The research described in this paper has not been subject to the U.S. Environmental Protection Agency (EPA) peer and administrative review and therefore may not necessarily reflect the views of the EPA; nor does the mention of trade names or commercial
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