Neurobehavioral teratogenicity of perfluorinated alkyls in an avian model

Abstract

Perfluorinated alkyls are widely-used agents that accumulate in ecosystems and organisms because of their slow rate of degradation. There is increasing concern that these agents may be developmental neurotoxicants and the present study was designed to develop an avian model for the neurobehavioral teratogenicity of perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS). Fertilized chicken eggs were injected with 5 or 10 mg/kg of either compound on incubation day 0. On the day of hatching, imprinting behavior was impaired by both compounds. We then explored underlying mechanisms involving the targeting of protein kinase C (PKC) isoforms (α, β, γ) in the intermedial part of the hyperstriatum ventrale, the region most closely associated with imprinting. With PFOA exposure, cytosolic PKC concentrations were significantly elevated for all three isoforms; despite the overall increase in PKC expression, membrane-associated PKC was unaffected, indicating a defect in PKC translocation. In contrast, PFOS exposure evoked a significant decrease in cytosolic PKC, primarily for the β and γ isoforms, but again without a corresponding change in membrane-associated enzyme; this likely partial, compensatory increases in translocation to offset the net PKC deficiency. Our studies indicate that perfluorinated alkyls are indeed developmental neurotoxicants that affect posthatch cognitive performance but that the underlying synaptic mechanisms may differ substantially among the various members of this class of compounds, setting the stage for disparate outcomes later in life.

Introduction

Perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) are prominent members of the family of perfluorinated alkyls (PFAs). These compounds are widely used for manufacturing non-stick coatings and stain repellents in fabrics, as well as in food-packaging and lubricants. PFAs are very poorly biodegradable and are accumulating in humans and ecosystems worldwide [17], extending even to animals in remote locations [10]. Thus, despite recent efforts to reduce or phase out PFA production [9], these agents will persist in the environment [20], [26].

Parallel to PFA bioaccumulation, toxicological studies in adult rats given high doses have shown damage to several internal organs, including the liver, kidneys and heart [43], preceded by substantial pathological changes in gene expression patterns [12]; parallel findings have been reported in zebra fish [29] and chicks [42]. In developing rodents, prenatal exposure to perfluorooctane sulfonate (PFOS) compromises survival rates and delays general growth and development [35]. In chicks, prenatal exposure to PFOS or perfluorooctanoic acid (PFOA) elicits reductions in hatchability, interference with pigmentation and pathological changes in the liver [24], [38]. In our earlier work, we pointed out that the biochemical characteristics that underlie plumage pigmentation in the chick are also critical for nervous system development [38], suggesting that the PFAs might be developmental neurotoxicants, a prediction we confirmed with in vitro models of neuronal development [30].

Studies in rodents similarly point to behavioral deficits in adulthood after neonatal exposure to PFOA or PFOS [16], reflecting an adverse impact on functioning of critical neurochemical events required for synaptic development and function [15]. Nevertheless, rodent models have inherent methodological shortcomings from confounding maternal factors, notably maternal care and mother–offspring interaction [27], [32], and the so-called “litter effect,” [33], where animals within a given litter are not independent of each other. Obviously, in the chick model, these limitations do not apply, since there is no maternal interaction and every individual represents an independent sample; further, because the chick is more mature at hatching than are newborn rats or mice, cognitive behavior, in the form of imprinting performance, can be evaluated immediately, prior to any potential impact of prenatal treatment on feeding or other indirect contributors to behavioral abnormalities. A newborn chick tends to follow the first object it sees after hatching [21] and can thus be imprinted upon an artificial object, which then becomes a suitable subject for studying the effect of prenatal treatments on imprinting behavior [5], [8], [31]. We have already demonstrated how this parallels hippocampus-related visuospatial cognitive performance in rodents [39], involving in the chick the left intermedial part of the hyperstriatum ventrale (IMHV) [5], [13]; specifically, cholinergic innervation in the left IMHV controls the chicks' perception of the imprinting stimulus [22], [36]. Given the postulated involvement of cholinergic deficits in the long-term neurobehavioral effects of neonatal PFOA and PFOS exposure in rodents [16], in the present study we evaluated whether these agents have an adverse effect on imprinting performance in the chick model.

In addition to behavioral assessments, we also evaluated the impact of PFOA and PFOS exposure on the concentration of protein kinase C (PKC) isoforms, which play an integral mechanistic role in transducing cholinergic input involved in learning and memory. Specifically, the translocation of PKC from the cytoplasm to the membrane is required for imprinting [3], [4], [25], [37]. In our studies, we obtained the brain samples immediately after the imprinting test so as to examine the ability of the imprinting stimulus to evoke PKC translocation. Indeed, in earlier work with other neurotoxicants known to target cholinergic function, we confirmed the direct relationship between adverse effects on PKC translocation and imprinting performance [14]. Here, we identified different patterns of effects of PFOA and PFOS on PKC-related mechanisms in association with impaired imprinting behavior.