Elsevier

Neurotoxicology and Teratology

Volume 81, September–October 2020, 106907
Neurotoxicology and Teratology

Full length article
Single and mixture per- and polyfluoroalkyl substances accumulate in developing Northern leopard frog brains and produce complex neurotransmission alterations

https://doi.org/10.1016/j.ntt.2020.106907Get rights and content

Highlights

  • PFAS neurotoxicity was tested in developing Northern leopard frogs.

  • PFOS, or a PFAS mixture produced time- and dose-dependent brain accumulation.

  • Developmental PFAS exposure changes neurotransmitters, especially acetylcholine.

  • PFAS exposure needs to be further evaluated for long-term neurological effects.

Abstract

Per- and polyfluoroalkyl substances (PFAS) are present in water and >99% of human serum. They are found in brains of wildlife; however, little is known about effects on the developing brain. To determine the effects of PFAS on brain and cardiac innervation, we conducted an outdoor mesocosm experiment with Northern leopard frog larvae (Rana pipiens) exposed to control, 10 ppb perfluorooctane sulfonate (PFOS), or a PFAS mixture totaling 10 ppb that mimicked aqueous film forming foam-impacted surface water (4 ppb PFOS, 3 ppb perfluorohexane sulfonate, 1.25 ppb perfluorooctanoate, 1.25 ppb perfluorohexanoate, and 0.5 ppb perfluoro-n-pentanoate). Water was spiked with PFAS and 25 larvae (Gosner stage (GS) 25) added to each mesocosm (n = 4 mesocosms per treatment). After 30 days, we harvested eight brains per mesocosm and remaining larvae developed to GS 46 (i.e. metamorphosis) before brains and hearts were collected. Weight, length, GS, and time to metamorphosis were recorded. Brain concentrations of all five PFAS were quantified using LC/MS/MS. Dopamine and metabolites, serotonin and its metabolite, norepinephrine, γ-aminobutyric acid, and glutamate were quantified using High Performance Liquid Chromatography with electrochemical detection while acetylcholine and acetylcholinesterase activity were quantified with the Invitrogen Amplex Red Acetylcholine Assay. PFOS accumulated in the brain time- and dose-dependently. After 30 days, the mixture decreased serotonin while both PFAS treatments decreased glutamate. Interestingly, acetylcholine increased in PFAS treatments at GS 46. This research shows that developmental environmentally relevant exposure to PFAS changes neurotransmitters, especially acetylcholine.

Introduction

Per- and polyfluoroalkyl substances (PFAS) are synthetic compounds that have been used since the 1940s in many different household applications, such as nonstick cookware, stain repellents, water repellents, beauty products, food packaging, and aqueous film forming foam (AFFF) firefighting foam (Chen et al., 2014; Moody et al., 2003; Olsen et al., 2007; Onishchenko et al., 2011; Stahl et al., 2012). Some longer chain PFAS started being phased out in the United States in 2009, but are still found throughout the world while short chain PFAS are still in use (Moody et al., 2003). Use of AFFF foams on miltary bases can contaminate surface waters with mixtures of PFAS including perfluorooctane sulfonate (PFOS), perfluorooctanoate (PFOA), perfluorohexane sulfonate (PFHxS), perfluorohexanoate (PFHxA), and perfluoro-n-pentanoate (PFPeA), among others (Anderson et al., 2016). PFAS have been shown to accumulate in humans and animals, leading to decreased birth weight-to-height ratios and decreased viability (Olsen et al., 2009). The main organs that accumulate PFAS tend to be liver and blood, but studies in various model systems have found that accumulation and distribution differ widely, with the blood half-life of PFOS in humans being 4.8 years, between 84 and 200 days in cynomolgus monkeys, 24–83 days for rats, and 30–38 days for mice (Olsen et al., 2007; Johnson et al., 1984; Pizzurro et al., 2019; Seacat et al., 2002). However, other PFAS have different accumulation rates. For example, perfluorobutane sulfonate (PFBS) and PFOA do not accumulate to the same amount as PFOS, even when exposure levels are the same (Chen et al., 2018a; Chen et al., 2018b). Furthermore, given the broader diversity of applications associated with PFAS usage, humans and wildlife are exposed to PFAS mixtures rather than individual PFAS (Anderson et al., 2016). However, few studies have exposed animals to specific mixtures to determine potential changes in toxicity and whether effects are additive, antagonistic, or synergistic.

PFAS have been found in the brain, indicating that they cross the blood brain barrier and are potentially neurotoxic (Eggers Pedersen et al., 2015; Greaves and Letcher, 2013). Previous research in mice and rats have conflicting evidence as to whether PFOS causes decreased motor function after developmental exposure (Onishchenko et al., 2011; Johansson et al., 2009; Johansson et al., 2008). In vitro studies have shown that PFOS exposure causes a rat neuroblastoma cell line, PC12, to preferentially differentiate to cholinergic neurons at the expense of dopaminergic neurons (Slotkin et al., 2008). PFOS has also been shown to cause dopaminergic neurotoxicity and decreased motor function in Ceanorhabditis elegans (Sammi et al., 2019). In polar bears, various PFAS have been correlated with increased monoamine oxidase activity, muscarinic acetylcholine receptor density, and decreased acetylcholinesterase (AChE) activity (Eggers Pedersen et al., 2015). Female marine medaka exposed to PFBS also had increased acetylcholine (ACh) and choline levels, while males did not (Chen et al., 2018b). Our laboratory also previously reported that PFOS and PFOA decreased whole brain dopamine (DA) and significantly increased DA turnover in Northern leopard frogs, Rana pipiens, at 100–1000 μg/L (Foguth et al., 2019).

The brain is also key to innervation of the heart, which is regulated by norepinephrine (NE) for sympathetic innervation and ACh for the parasympathetic system. PFAS have been implicated in improper development of the heart, such as increased apoptosis in rats exposed to PFOS in utero and decreased expression of cardiac-specific genes in mouse embryonic stem cells during differentiation (Cheng et al., 2013; Zeng et al., 2015). Furthermore, there are correlations between serum PFAS levels and cardiovascular disease, including congestive heart failure, coronary heart disease, heart attacks, and strokes (Huang et al., 2018). PFOS also caused apoptosis of cells while differentiating into cardiomyocytes in rats exposed in utero through 21 days after birth and in cultured mouse embryonic stem cells (Cheng et al., 2013; Zeng et al., 2015). Interestingly, the effect of PFAS on the development of the sympathetic and parasympathetic nervous systems have not been tested, which could also be causing heart problems not seen early in development.

In this study, we used Northern leopard frogs as a model for evaluating neurotoxicity resulting from exposure to PFOS or a PFAS mixture. Northern leopard frogs are a suitable model species, especially for neurotoxicity, because they express neuromelanin, a product of DA oxidation that has been implicated in toxicity due to accumulation and later release of neurotoxic compounds, leading to a large exposure all at once (Karlsson et al., 2009; Kemali and Gioffre, 1985; Lindquist et al., 1988; Zecca et al., 2006). While most rodent models do not produce neuromelanin, Northern leopard frogs do, even as tadpoles (Kemali and Gioffre, 1985; Lindquist et al., 1988). This could indicate enhanced sensitivity to environmental exposures compared to laboratory studies with rodents and other animals lacking neuromelanin. Furthermore, frogs have large broods of eggs that are easily manipulated compared to developmental studies in rodents (Sachs and Buchholz, 2017; Scheenen et al., 2009). Their eggs are also free living, so it is possible to expose eggs at early life stages directly, with known concentrations, rather than exposing in utero. Building on our prior publication on neurotoxic effects (neurotransmitter changes) of PFOA and PFOS individually at relatively high exposure levels in frogs (Foguth et al., 2019), the goals of this study were to assess effects under more environmentally relevant exposures (lower concentrations and for a longer duration) including PFAS mixtures which more commonly occur in nature. We also quantified the effects of PFAS on innervation of the heart, which has not been previously studied.

Section snippets

Animals, exposure, survival, and measurement of PFAS levels

All animal studies were approved by the Purdue Animal Care and Use Committee and collection was under IN Scientific Purposes License 19–343 issued to Tyler Hoskins (TDH). On April 1, 2019, six partial Northern leopard frog egg masses were collected from an ephemeral wetland at the Purdue Wildlife Area (40.452275, −87.054853). This wetland hosts a diverse community of native amphibians, lies within a protected area, and has no known history of PFAS contamination. Eggs were grown in outdoor tanks

PFAS in water and brain

Measured water concentrations were slightly above nominals for the majority of chemicals tested (Table 1). As expected for such widespread contaminants, we detected background PFAS in some controls and the PFOS-only mesocosms, but concentrations were low relative to exposure concentrations and < LLOQ in most cases (Table 1, Table S2). Among PFAS examined in brain, only PFOS showed clear evidence of bioaccumulation. PFOS and the PFAS mixture treatments increased brain accumulation for PFOS in a

Discussion

PFAS exposure is a major public health concern. In this study, we exposed Northern leopard frogs, a sentinel species, to environmentally relevant concentrations of PFAS, including a mixture reflective of AFFF impacted sites to assess potential neurotoxicity. We showed brain accumulation and complex neurotransmission changes in the brain. Taken together with previous findings from our group and others, the literature strongly suggest that the nervous system is an important target organ affected

Funding

Ralph W. and Grace M. Showalter Research Trust (to J.R.C.). Strategic Environmental Research and Development Program (ER-2626) (to M.S.·S, J.T.H., and L.S.L.). We are grateful to the Digital Environment for Enabling Data Driven Science (DEEDS) team for facilitating quality assurance and quality control. The DEEDS project is supported by the National Science Foundation Office of Advanced Cyberinfrastructure (OAC) under grant number 1724728 (PI: AC Catlin).

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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