Keywords

9.1 Introduction and Background

From the fertilization of the egg, through gestation and during the first years after birth, the brain is subjected to a continuously development and disturbances during susceptible periods can induce many different types of negative alterations. Transfer of xenobiotics occurs from the mother to the fetus through the umbilical cord, via mother’s milk to the newborn and via direct inhalation and ingestion to the newborn and toddler. Knowledge about the exposure situation in fetuses, newborns, toddlers in addition to adults is therefore important in order to predict toxic effects. Exposure to environmental contaminants have been suspected as agents for an increased prevalence of attention deficit hyperactivity disorder (ADHD) and susceptibility of dementia disorders, such as Parkinson’s disease (Barkley 1998; Brown et al. 2005; Hardell et al. 2002; Hoffman et al. 2010; Lai et al. 2002; Rice 2000; Schettler 2001). It has been hypothesized that environmental contaminants can affect cognitive functions, such as learning and behavior, and motor skills (Grandjean and Landrigan 2006; Grandjean et al. 2014; Mariussen and Fonnum 2006). Human exposures to PFCs are reviewed in Part I of this book, but it is worth repeating that certain tissues and fluids are of extra interest concerning the possible developmental neurotoxic effects of PFCs. Generally it can be said that the concentrations of PFCs are significantly higher in maternal serum than in umbilical cord serum (Apelberg et al. 2007; Inoue et al. 2004; Midasch et al. 2007; Monroy et al. 2008). Unlike PFOS, PFOA appears to cross the placental barrier unhindered (Midasch et al. 2007). Only a few studies have analyzed the levels of PFCs in brain tissue. The PFC found in the highest concentration in brain tissue is PFOS, followed by PFOA, which probably reflects their historical use, persistency and accumulation potential, and rate of elimination (Mariussen 2012). In general the levels of the PFCs are lower in the brain than in other organs, such as the liver and kidney, and even in developmentally exposed laboratory animals (Kawamoto et al. 2011; Mariussen 2012; Sato et al. 2009). Both the adult and developing brain are protected by the so-called blood brain barrier (BBB) reducing the access of both exogenous and endogenous compounds into the brain (Ek et al. 2012). The properties of PFCs as surfactants may, however, modulate the membrane fluidity of the cells (Levitt and Liss 1986) and there are in vitro studies implicating that PFOS can increase the permeability of the blood brain barrier. Wang and co-workers showed that PFOS induced disassembly of endothelial tight junctions in brain endothelial cells (Wang et al. 2011) and later it was also observed that PFOS reduced mRNA expression of cellular adhesion markers in neuronal cells (Choi et al. 2013). Newborns, toddlers and children are the most exposed part of the population, on a body weight basis, since they tend to inhale and ingest more than the adult population (Trudel et al. 2008). Furthermore, it is a well-known fact that many environmental pollutants end up in the mother’s milk, exposing the nursing neonate to a cocktail of chemicals, including several of the PFCs, such as PFOS, PFOA, PFHxS, PFBA, PFHxA, PFHpA and PFNA (Antignac et al. 2013; Mondal et al. 2014; Sundstrom et al. 2011). The concentrations of the PFCs in breast milk tend to be highest in mothers who is nursing for the first time compared to mothers who have previously nursed (Tao et al. 2008). One study have compared concentrations of PFOS between the adult and juvenile brain showing a higher relative concentration in brain of the rat fetuses compared with the brains from their mothers (Chang et al. 2009), indicating that the BBB of the fetus has increased permeability of PFOS. Bearing in mind that the PFCs are only one group of toxicants reaching potential targets in the brain it is of importance to both screen the extent of exposure and to evaluate their hazardous potential.

9.2 Epidemiological Studies

It is always hard to study toxicological effects in humans especially when it comes to reproductive and developmental effects. Despite that, effort has been put into epidemiological studies to investigate if there are connections or correlations between levels/concentrations of PFCs in maternal serum, umbilical cord serum and birth weight, size and other markers of development in humans. This is summarized in Chap. 14, but some of the effects seen in these epidemiological studies indicate that PFCs can contribute to developmental neurotoxic effects in human. In 2007, reports came from Maryland, U.S., that both PFOS and PFOA concentrations in umbilical cord serum were negatively associated with birth weight and head circumference. When looking at maternal concentrations of PFOS and PFOA in relation to motor and mental developmental in children, it can be seen that children from mothers with high PFOS concentration are slightly delayed in time of sitting without support (Fei et al. 2008) and also self-reported birth defects were associated with high PFOA exposures (Stein et al. 2009). The same research group used the same cohort to look into the correlation between the serum levels of PFCs in children and teenagers and the prevalence of ADHD, and found indications that high levels of PFCs, especially PFOA and possibly also PFHxS could be factors behind the induction of ADHD (Stein and Savitz 2011). Higher risk of ADHD in relation to PFC levels has also been proposed by other researchers and in that study it was not only the usual suspects mentioned, but also PFNA (Hoffman et al. 2010).

9.3 Animal Studies (In Vivo and Ex Vivo Studies)

Despite few studies it is plausible to believe that PFCs have toxic effects in humans, even though there are limited methods of measuring both exposure and effects. Instead animal studies are used to investigate neurotoxicity and there is a number of different experimental methods that can measure a variety of endpoints from several different exposure paradigms, with different doses, in several different species. Results generated from animal studies can be used to extrapolate and predict human toxicity and are therefore of great importance. Generally, it can be said that PFC neurotoxicity (mainly PFOS and PFOA) have been studied in all types of animals including fishes, birds and mammals, but the vast majority of studies have been done on rodents (Table 9.1). Usually, in order to exert an effect a compound has to be present in the target organ. In the section about the toxicokinetics of PFCs it was described that these compounds can reach the brain, both during development and in adults, which indicates that neurotoxic effects may arise. There are several known neurotoxic effects of PFCs and here we will look into some of them, starting with neuropsychiatric and neuromotoric effects. Effects that can be linked to the above mentioned epidemiological findings. An overview of studies on neurobehavioral effects of different PFCs are, in addition, presented in Table 9.1.

Table 9.1 Summary of neurobehavioral studies of PFCs* on rodents, birds and fish
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PFOS exposure in mice during the gestational period (6 mg/kg bw/day) delayed a couple of landmarks of neuromotor maturation, such as decreased resistance to backward pull on postnatal days 10 and 11 and decreased climb ability and forelimb strength on postnatal day 11. These effects were transient and not seen later during the postnatal period (Fuentes et al. 2007b). In a more recent study dams were exposed to different doses (0.1, 0.3, and 1.0 mg/kg bw/day) of PFOS from gestational day 0 through postnatal day 20. PFOS treatments had no effect during the postnatal period when looking at the auditory startle response and learning and memory in a swim maze. However, locomotor activity increased in PFOS treated animals (0.3 and 1.0 mg/kg bw/day) on postnatal day 17, which ultimately leads to the inability of the animals to habituate to the novel test environment (Butenhoff et al. 2009a).

The behavioral effects of PFC exposure, such as negative impact on memory, learning, and motor functions, may involve effects on several neurochemical targets. A major challenge is to link the behavioral effects to changes in the nervous tissue, which often probably result from effects on several neurochemical targets. In some of the studies of which neurobehavioral effects of PFCs have been elucidated, efforts have been performed to reveal neurochemical effects ex vivo (Table 9.2). In a series of studies it has been shown that different PFCs may affect spontaneous behavior and cognitive functions after administration of a single dose at specific time points (Johansson et al. 2008, 2009; Viberg et al. 2013). Here the spontaneous behavior, locomotion (horizontal movement), rearing (vertical movement) and total activity, was measured for an hour. In the beginning of the 60-min test period the activity was decreased in animals exposed to PFOS, PFOA, and PFHxS, but in the end these animals had not habituated to the novel environment and the activity was higher than in the control animals. This type of behavior was observed both in 2 and 4 months old animals and these behavioral effects were persistent and actually worsened with age. A fourth perfluorinated compound, PFDA (perfluorodecanoic acid) had no effects on adult behavior. So not all PFC have the potency to induce behavioral and cognitive disturbances. When looking at the neurochemical targets it was showed that mice exposed to single doses of PFOS (11.3 mg/kg), PFOA (8.7 mg/kg) and PFHxS (6.1 mg/kg) 10 days after birth, had increased levels of the proteins CaMKII, synaptophysin and tau in hippocampus 1 day after the exposure (Johansson et al. 2009; Lee and Viberg 2013). It was, in addition, shown that PFOA and PFOS induced increased levels of synaptophysin in the mice cerebral cortex (Johansson et al. 2009). The effects on CaMKII by PFOS, PFOA and PFHxS are supported by changes in the gene expression of calcium-dependent signaling molecules in rat hippocampus after perinatal PFOS exposure. The expression of calcium-related signaling molecules, which are critical to the function of the central nervous system, such as N-methyl-D-aspartate receptors, calmodulin, Ca(2+)/calmodulin-dependent kinase II alpha and cAMP-response element-binding, were increased in the PFOS exposure group on postnatal day 1 (PND 1). In some cases these changes lasted for only a short period in postnatal life, but calmodulin and the N-methyl-D-aspartate receptor subtype-2B were still reduced on postnatal day 35 (Liu et al. 2010a). Furthermore these proteins are involved in neuronal growth, synaptogenesis and mediation of neurotransmitter release and indicate that the exposure of PFCs may influence the development of the juvenile mouse brain related to cognitive functions. Synaptophysin, which is a synaptic vesicle associated protein, has for example also been shown to be involved in modulation of cognitive functions such as learning and memory, and novelty exploration (Schmitt et al. 2009). A similar study was performed by (Zeng et al. 2011a, b) who exposed pregnant rats daily from GD2 to GD21 for 0.1, 0.6 and 2.0 mg PFOS/kg/day. The levels of synaptophysin and synapsin in hippocampus were analyzed at PND 0 and 21 showing a reduction in the levels in hippocampus, and an increase in the levels of synaptophysin and a decrease in the levels of synapsin in the cerebral cortex. Synapsin are synaptic vesicle associated proteins involved in the regulation of neurotransmitter release, and in the study by Zeng et al. (2011a) it was also claimed that PFOS induced morphological changes in the synaptic structure and reduced numbers of synaptic vesicles. The discrepancy from the findings by Johansson et al. (2009), who observed an increase in the levels of synaptophysin in the mice brain, may be due to the different administration procedure. The juvenile rats in the study by Zeng et al. (2011a, b) were exposed chronically during pregnancy and the juvenile mice in the study by Johansson et al. (2009) were administered a single oral acute dose 10 days after birth. These studies, therefore, indicate that the PFCs might influence synaptic plasticity, which may have consequences for neuronal development.

Table 9.2 Summary of ex vivo studies of PFCs*
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When the neonatally animals, which showed effects on cognitive function after exposure to PFOS, PFOA, and PFHxS, were challenged with nicotine in adulthood, their response was changed compared to normal animals.

Control animals became significantly hyperactive by the adult nicotine injection, while the neonatally exposed animals reacted totally opposite with very little activity, displaying a clear hypoactivity. This indicates that PFC could affect the cholinergic transmitter system during the neonatal brain development, because the cholinergic system is involved in many physiological functions, including cognitive capacity (Johansson et al. 2008; Viberg et al. 2013). Other studies support that the cholinergic system could be a target for developmental PFC exposure. For example choline acetyltransferase, a very important enzyme in the cholinergic system of mammals, is involved in the recycling of the neurotransmitter acetylcholine by joining of Acetyl-CoA and choline to reform acetylcholine. In utero exposure to 3 mg PFOS/kg bw/day, during the gestational period, in rats resulted in decreased activity of choline acetyltransferase in prefrontal cortex at different postnatal ages (Lau et al. 2003). Interestingly these effects on cognitive function, behavior and motor activity, are similar to developmental neurotoxicological effects seen after gestational or neonatal exposure to other persistent organic pollutants, such as PCBs and PBDEs (Eriksson 1998; Eriksson et al. 2001; Viberg et al. 2003a, b). In addition, the mechanistic background to these disturbances are also similar, meaning that they seem to affect the same types of proteins and the same transmitter systems (Eriksson 1998; Viberg et al. 2002, 2007).

Pinkas and co-workers exposed chicks prenatally to single doses of PFOS and PFOA at incubation day 0 for 5 and 10 mg/kg (Pinkas et al. 2010). The chicks were subjected to behavioral testing at hatching day 1 and showed impaired imprinting behavior. An ex vivo examination of the brains showed that the PFOS exposed birds had an overall reduction in the levels of different cytosolic PKC isoforms (PKC -α, -β, -γ), whereas PFOA induced an overall increase in the levels of cytosolic PKC. No effects on membrane bound PKC were found. According to the authors, translocation of cytosolic PKC to the membrane is required for imprinting and plays a role in the transfer of cholinergic input involved in learning and memory. Different PKC isoforms have previously been postulated as possible targets following both adult and developmental exposure to halogenated aromatic hydrocarbons, such as the PCBs (Kodavanti et al. 1994, 1998; Yang et al. 2003). The doses used in the experiment by Pinkas et al. (2010) lead, however to high mortality. Between 30 and 50 % of the exposed eggs did not develop embryos indicating that the doses used were detrimental to the chicks leading to other substantial non-neurotoxic effects. Additional studies have been done by Sean Kennedy’s research group, who exposed chickens in ovo to high doses of PFHxS and PFHxA. They saw that PFHxS induced increases in mRNA levels of neurogranin in cerebral cortex (Cassone et al. 2012). Neurogranin is expressed solely in central nervous system, particularly in dendrites, and is a calmodulin-binding protein, participating in the protein kinase C signaling pathway. PFHxA on the other hand did not have an effect on the mRNA levels of neurogranin.

PFCs can affect the nervous system of mammals and birds, but other studies have also shown that fish are susceptible to PFC exposure during their development. PFOS and PFOA are the most studied (Shi et al. 2009; Spulber et al. 2014; Zhang et al. 2011), and there is one particular interesting study out showing that water exposure to several different PFCs, in zebrafish, can cause behavioral disturbances in locomotor activity. Among the PFCs inducing behavioral disturbances were TFAA, PFNA, PFBS and PFOS. When looking at the structure of the PFCs, PFCs with longer carbon chain length and with attached sulfonic groups showed larger potential to affect locomotor behavior in zebrafish larvae (Ulhaq et al. 2013).

9.4 Cell Cultures (In Vitro Studies)

In order to look into the developmental neurotoxicity of PFCs and to get a better understanding of the potential mechanisms behind the neurotoxic effects, in vitro experiments have been conducted and investigated such as on cell differentiation and synaptic plasticity (Table 9.3). It has been shown that PFOSA and PFOS (50–250 μM) promote differentiation of the PC12 cell into the cholinergic phenotype at the expense of the dopaminergic phenotype (Slotkin et al. 2008). At the highest concentration, the effect of PFOSA (100 μM) switched and promoted differentiation into the dopaminergic phenotype. No mechanisms for the effects were postulated, but it was suggested that the induction of oxidative stress could be a factor. PFOSA was shown to induce lipid peroxidation and was also the most cytotoxic compound. Wan Ibrahim and co-workers examined the effects of low PFOS concentrations (12.5–100 nM) on differentiation of neural stem cell (Wan Ibrahim et al. 2013). It was shown that 100 nM PFOS reduced cell viability, whereas the lower concentrations increased neuronal differentiation, as shown as a lower numbers of proliferating cells and a higher number of neurite bearing cells. The effect was attributed to PPARγ activation.

Table 9.3 Summary of in vitro studies of PFCs*
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One strategy to evaluate neurochemical targets of PFCs has been to exploit their effects on cell viability, which may indicate a neurotoxic potential of the compounds. Reduced cell viability may be a response of a range of cellular processes triggered by the toxic agents, such as oxidative stress, disruption of the calcium homeostasis, and effects on neurotransmission and signaling. These are cellular processes that are important for neuronal development and survival. Cerebellar granule cells (CGCs) have been a convenient model to evaluate the neurotoxic potential and mechanisms of effect of a range of environmental toxicants, such as polychlorinated biphenyls, brominated flame retardants as well as PFCs. In two studies by Lee and co-workers, CGCs were exposed to PFOS (3 and 30 μM) and PFHxS (≥100 μM). It was observed induction of apoptosis as shown by increased caspase-3 activity and induced DNA-fragmentation (Lee et al. 2012, 2014b). The PFOS induced apoptosis was connected to activation of different subtypes of protein kinase C (PKC-α, PKC-βII and PKC-ε). The effect of PFOS and PFOA on PKC-translocation was also observed by Pinkas et al. (2010) on developmentally exposed chicks as described in the previous section. PKC is involved in a range of processes in the brain such as cognitive functions, learning and memory and several of studies have shown that other environmental toxicants, such as PCBs, dioxins and brominated flame retardants also influence PKC activity (Kodavanti et al. 1994). This may have implications on the risk of being exposed to mixtures of contaminants which have effects on similar targets. A later study by Lee and co-workers showed that the PFOS activation of PKC was followed by activation of the ERK-pathway (Lee et al. 2013), which is one of the mitogen-activated protein kinases (MAPKs). Also PFHxS was shown to induce ERK (Lee et al. 2014b). By inhibiting the ERK-pathway, the PFOS and PFHxS induced apoptosis was blocked. The ERK-pathway has also been shown activated in cerebellar granule cells by tetrabromobisphenol A (TBBPA), which is a brominated flame retardant and possibly by hydroxyl-PCBs, which are metabolites of PCBs (Dreiem et al. 2009), and recently by PFHxS in PC12 cells (Lee et al. 2014a).

Reactive oxygen species (ROS) is a collective term for short lived, highly reactive compounds, often including oxygen radicals and non-radical products of oxygen. The brain is especially vulnerable to oxidative damage, partly because of its high oxygen demand, corresponding to about 20 % of the basal oxygen consumption. The membrane lipids of the nerve cells are rich in polyunsaturated fatty acids which are sensitive to attack from ROS. Nerve cells have often a large surface area making them more exposed to attack from ROS. ROS are typically generated as by-products in cellular metabolism, from toxic agents, inflammations and diseases (Halliwell and Guttenridge 1999). Oxidative stress refers to the consequence of a mismatch between the production of ROS and the ability of the cell to defend itself against them. In the study by (Lee et al. 2012) it was shown that PFOS induced production of reactive oxygen species (ROS), and the detrimental effect of PFOS, both the PKC-activation and apoptosis, was blocked by pretreatment of N-acetylcysteine (NAC). NAC is used as a scavenger of ROS-products. Reistad and co-workers exposed CGCs to four different PFCs to evaluate their potential to affect cell viability and induce ROS-formation (Reistad et al. 2013). The effect of the PFCs varied of which PFOSA and FTOH 8:2 were considerably more cytotoxic than PFOS and PFOA. PFOSA and FTOH 8:2 had EC50-values of 13 and 15 μM respectively, whereas PFOS had an EC50-value of 61 μM. PFOA did not induce cell death at concentrations up to 100 μM. Similar to these studies Slotkin et al. (2008) showed that PFOSA was the most potent in reducing the cell viability of PC12 cells followed by PFOS, and PFOA did not induce loss of cell viability. An interesting observation in the study by Reistad et al. (2013) was the lack of correlation between cytotoxicity and ROS formation. PFOS and PFOA were equally potent ROS inducers, with EC-50 concentrations of 27 μM and 25 μM respectively, but PFOA did not induce cell death. PFOSA induced ROS with an EC50-concentration of 57 μM whereas FTOH had no effect, but they were equally cytotoxic. For PFOS Lee et al. (2012) found a correlation between ROS formation and apoptosis in cerebellar granule cells, whereas this appeared not to be the case in PFHxS induced apoptosis (Lee et al. 2014b). Slotkin et al. (2008) found a correlation between cell viability and lipid peroxidation in PC12 cells exposed to PFOSA and PFOS. Similar correlation has also been found for PCBs, OH-PCBs and TBBPA (Dreiem et al. 2009; Mariussen et al. 2002; Reistad et al. 2007). Cellular damage should probably ultimately lead to increased ROS formation. A possible explanation for the lack of correlation between cell death and ROS formation may be the selectivity of the method used to identify ROS and the time after exposure when the endpoints were measured. In addition, the PFCs also differ with respect to their physical-chemical properties such as water solubility, which probably will reflect their ability to reach targets in cells.

A crucial factor for normal functioning cells is maintenance of the intracellular Ca2+-homeostasis. Ca2+ is an important second messenger in the cells. However, a sustained increase of the intracellular level of Ca2+ may induce formation of reactive oxygen species (ROS) followed by cellular injury. Calcium is also crucial for neurotransmitter release. Upon stimulation of a neuron the transmitter molecules are released from the nerve terminal into the synapse by a Ca2+ dependent process. In a study by Harada and co-workers it was shown that 30 μM of PFOS had a modulating effect on ion currents in rat cerebellar Purkinje cells leading towards a hyperpolarized state (Harada et al. 2006). The effect involved voltage gated Ca2+, Na+ and K+ channels. In a later study, Liao and co-workers also showed that PFOS increases K+ currents at doses over 10 μM towards a hyperpolarized direction in hippocampal neurons (Liao et al. 2009b) similar as observed by Harada et al. (2006) on cerebellar Purkinje cells. Effects of PFOS on nervous ion currents were also found by Liao, who showed increased Ca2+ currents recorded in the CA1 region of hippocampal slices and in cultured hippocampal neurons (Liao et al. 2008). It was also shown that PFOS inhibits neurite growth and synaptogenesis in cultured neurons. The effects could be blocked by the L-type voltage gated Ca2+ channel blocker nifedipine. These findings indicate that PFOS may facilitate influx of calcium leading to an increased susceptibility of calcium related effects, which ultimately may lead to reduced cell viability or impairment of cellular growth. A structure activity study showed that the effect on the calcium currents increased with the carbon chain length of the tail moiety of the PFCs, and that the effects of the carboxylated compounds were less pronounced than the sulfonates (Liao et al. 2009a). The effects of PFOS and PFOA on the Ca2+-homeostasis in hippocampal neurons have been elucidated in more detail by Liu and co-workers, showing that the PFCs affect several calcium dependent processes (Liu et al. 2011). The sulfonated PFOS (30 μM) was more potent than the carboxylated PFOA (100 μM) to induce elevated intracellular concentrations of Ca2+. The increase intracellular Ca2+ appeared to be of both extracellular origin involving voltage gated Ca2+ channels, as shown by Liao et al. (2008), and intracellular origin such as activation of ryanodine receptors and inositol phosphate-3 (IP3)-receptors. The disturbance of the Ca2+-homeostasis was followed by an increase in oxidative stress, as measured with DCF, and an increased expression of calcineurin, which is a Ca2+ activated protein phosphatase. Another interesting finding by Liao et al. (2009a) was that a low concentration of PFOS (1 μM) increased inward glutamate currents whereas higher concentrations of PFOS (10 and 100 μM) dose-dependently reduced the inward glutamate currents. Glutamate is the quantitatively the most important excitatoric neurotransmitter in the brain. Glutamate is an excitotoxin so a prolonged stimulation of glutamatergic receptors in the brain may cause a sustained elevation of the intracellular Ca2+ level in the neuron, which can mobilize Ca2+- dependent processes, leading to inflammation, ROS production, and ultimately cause cell death (Fonnum 1998). Hippocampus, which is mainly glutamatergic, is one of the major brain areas concerned with the acquirement of memory, and only minor damage to this area is sufficient to produce memory disturbances (Bliss and Collingridge 1993; Fonnum et al. 1995; Milner 1972; Victor et al. 1961).

Cytotoxicity and oxidative stress may also be induced as a consequence of inflammatory responses, such as immune responses. In prenatally PFOS exposed rats it was observed an increase inflammatory response in the juvenile rat brains as shown by increased levels of the astrocyte markers fibrillary acidic protein and S100 Ca2+-binding protein B in hippocampus and cortex (Zeng et al. 2011b). It was also found an increase in the mRNA levels of proinflammatory cytokines, such as interleukin 1β, tumour necrosis factor α, AP-1, NF-kappa-B and CREB. Changes in the mRNA levels as a response on an exposure may not necessarily imply changes or harmful effects on a higher protein or cellular level. There is, however, previously been shown that PFOS and PFOA enhance inflammatory responses of macrophages to lipopolysaccharide in mice, indicating that PFCs may be implicated in stress responses related to the immune system (Qazi et al. 2009).

9.5 Summary and Conclusion

There is no doubt that PFCs can induce developmental neurotoxic effects, since research in humans, animals and cell cultures all point in the same direction. Functional effects in animals, such as impaired behavior and cognitive functions, have also been investigated to elucidate the mechanisms behind the effects. Disturbances in the processes of synaptogenesis, dendritic outgrowth and ontogeny of neurotransmitter systems all look as plausible mechanisms and apoptosis, specific proteins, signaling molecules, calcium homeostasis as well as oxidative stress can be the molecular reasons behind the disturbances of these processes. It is important to remember, though, that the real world is much more complicated than exposure to one single compound at the time. Therefore, effects of PFCs in combination and/or in combination with other environmental pollutants need to be investigated. At the moment it is hard to find good examples of neurotoxic effects after combination exposure to PFCs or PFCs and other types of chemicals, but one study shows that combined exposure to low doses of PFOA and the polybrominated diphenyl ether PBDE 209, during the neonatal period, can interact and exacerbate adult functional neurobehavioral effects, compared to the single compounds alone (Johansson 2009).