To our knowledge, this is the first study to assess the effects of PFCs on mRNA expression in cultured avian neuronal cells. Specific molecular endpoints were responsive to PFC exposure in CEN and HGEN cells, helping to elucidate the possible mechanisms of action of PFCs in the avian brain. To ensure that transcriptional changes were related to mRNA expression and not cell viability, the mRNA data reported are from treatment groups that had no significant effect on cell viability. The highest PFC concentration used for mRNA expression analysis was 10μM. T3 was administered at maximum concentrations of 30 and 300nM in CEN and HGEN cells, respectively.
The optimal incubation period required to observe mRNA expression effects was determined in HGEN cells exposed to T3 for 3, 6, 12, 18, 24, and 36 h. The minimum time required to demonstrate significant mRNA expression changes in the TH-responsive genes was 24 h as no alterations were observed at 3, 6, 12, or 18 h. T3 upregulated TR-α and TR-β mRNA expression (at 24 and 36 h), which indicated that primary cultures of CEN and HGEN cells responded to treatment with the natural ligand. During chicken development, maximal concentrations of TRs and high T3 levels are detected in the avian brain at midincubation (McNabb, 2007
), the developmental stage assessed in this study. PFC effects were compared with those of T3, which was included as a positive control for TH pathway activation in this study.
Exposure to T3 and several PFCAs (PFHxA, PFHpA, and PFNA) induced D2 mRNA expression in CEN cells. D2 converts T4 to its active form, T3, via 5′ outer ring deiodination (Bianco and Kim, 2006
). Increased D2 mRNA levels following T3 administration could be partially explained by the developmental time period at which the neuronal cells were harvested (i.e., day 11 embryos). TH concentrations and D2 mRNA levels are elevated at day 11 and, in fact, D2 mRNA increases up to 14-fold from day 10 to day 17 (Gereben et al., 2004
). This may be a means to increase T3 bioavailability in preparation for pipping, hatching, and growth at later stages (McNabb, 2007
). The concordance of D2 induction following T3, PFHxA, PFHpA, and PFNA treatment suggests a similar mode of action.
PFHxA, PFHxS, and PFBS induced D3 mRNA expression twofold to fivefold in CEN cells. D3 catalyzes inner ring 5′-deiodination, which degrades both T4 and T3 to their inactive derivatives, reverse T3 (rT3) and 3,3′-diiodothyronine (T2), respectively (Hernandez, 2005
). D3 is found primarily in the cerebral cortex (Hernandez, 2005
), the brain region from which neuronal cells were harvested in this experiment. Elevated D3 mRNA levels would be expected when TH concentrations are over abundant as this would provide a mechanism to eliminate excess TH and maintain homeostasis. Hernandez (2005)
suggested that D3 upregulation may occur as a means to protect the brain from elevated TH levels.
TTR mRNA expression was significantly downregulated following PFHxS exposure at concentrations as low as 0.1μM; at 1μM TTR mRNA levels were diminished by ~10-fold. T3 also decreased TTR mRNA levels indicating a potential shared mechanism of action. Previous studies in our laboratory measured TTR mRNA expression following T3 administration in primary cultures of CEN (36 h exposure as opposed to 24 h in this study) and HGEN cells (24 h exposure). As in the present study, T3 decreased TTR expression in CEN (Crump, unpublished results) and HGEN cells (twofold to fivefold; Crump et al., 2008c
). TTR, a major TH-binding protein in the bloodstream of birds, is synthesized by the choroid plexus and transports T4 into the central nervous system where it is converted to its more active T3 form by deiodination (review Schreiber, 2002
; review McNabb, 2007
). A reduction of TTR expression could result in decreased TH availability to target tissues, which could affect TH-dependent processes.
Exposure to the four- and six-carbon PFSAs induced RC3 mRNA expression up to 2- and 11-fold, respectively. Similarly, T3 administration increased RC3 mRNA expression up to ~30-fold. Iniguez et al. (1993)
reported that TH altered RC3 mRNA expression in brain regions of rodents, including the cerebral cortex, striatum, and hippocampus. Furthermore, a commercial mixture of polychlorinated biphenyls, Aroclor 1254 (A1254), induced RC3 mRNA expression in the cortex of rodents (Gauger et al., 2004
). The transcriptional response of RC3 following exposure to T3, PFCs, and other anthropogenic chemicals makes it a useful marker to examine disturbances in TH homeostasis. RC3 is a calmodulin-binding PKC substrate that is involved in the Ca2+
signal transduction system and implicated in events that lead to long-term potentiation (Gerendasy, 1999
; Iniguez et al., 1993
). Changes in RC3 expression could have functional consequences in synaptic plasticity, associative learning, and memory (Iniguez et al., 1993
PFHxA treatment induced MBP mRNA expression in CEN cells (at 3 and 10μM). MBP is essential in the process of nerve myelination, which facilitates the conduction of nerve impulses. MBP contains a thyroid-responsive element in its promoter region, and in vitro
binding studies demonstrated that both TH and TRs are required to positively regulate MBP expression (Farsetti et al., 1991
). T3 administration did not alter MBP mRNA expression in this study, which may have been due to differences in experimental designs (e.g., T3 concentrations, experimental approach [binding study vs. gene expression study], and species differences).
Although many significant changes were observed in TH-responsive genes following PFC exposure in CEN cells, this was not the case in PFC-treated HGEN cells. The mRNA levels of three TH-responsive genes (D2, Oct-1, and RC3) were examined in HGEN cells treated with short-chained PFCs because, based on the results in CEN cells, short-chained PFCs were more transcriptionally active. TR-α and TR-β mRNA levels were also measured in HGEN cells following T3 treatment and were induced at 300nM. A previous study by Crump et al. (2008c)
also reported an induction of TR-α and TR-β in HGEN cells, albeit at lower T3 concentrations. Although differences were observed between the Crump et al. (2008c)
study and the present study, the important finding was that HGEN cells from both studies were responsive to a TR agonist. Variability in response of HGEN cells to T3 between the two studies, conducted with herring gull eggs collected 2 years apart, could be attributed to factors including: (1) maternal influences (e.g., variability in hormone and contaminant deposition to the developing embryo during ovogenesis), (ii) interannual variation in colony dynamics (e.g., stress, influence of predators, weather conditions), (iii) genetic variability—especially compared with chicken embryos that come from a managed flock, and (iv) variation in the stage of embryonic development at which neurons were obtained—ideally, midincubation (i.e., day 14)—due to incubation prior to collection. The use of a wild avian species for such an in vitro
screening approach poses several challenges not present in a domestic species; however, it is important to generate data for species exposed naturally to environmental contaminants.
It was determined that exposure to short-chained PFCs elicited variable transcriptional responses when comparing primary cultures of herring gull and chicken neuronal cells (refer to ). Differences in the transcriptional responses between species may be related to variable levels of baseline contaminant exposure and/or unknown factors that determine species-specific differences in sensitivity. For example, the chicken embryos were obtained from a managed breeding facility where the exposure to environmental contaminants is minimal. Herring gull eggs were collected from Chantry Island, a Great Lakes colony that is used as a reference (i.e., clean) site; however, detectable levels of a wide range of chemical contaminants are found in egg homogenates (Gauthier et al. 2009
; Gebbink et al. 2009
; Jeremyn-Gee et al. 2005
). ∑PFC concentrations in herring gull eggs collected from Chantry Island were 192 ng/g ww (Gebbink et al., 2009
). In order to put these environmentally relevant levels in the context of in vitro
concentrations (micromolar) used in this study, calculations were made based on the weight of the neuronal cells in each well and the administered PFC concentration. The maximum possible uptake rate into neuronal cells was 100%, and based on this, the lowest effective in vitro
concentration (3μM for Oct-1 mRNA expression) would be approximately three orders of magnitude greater than levels detected in herring gull eggs collected from Chantry Island (~340 μg/g vs. 0.192 μg/g ww). It is important to note that actual PFC concentrations in neuronal cells were not determined, and this is a key consideration for future in vitro
studies in order to determine if the 100% uptake rate assumption, that forms the basis of the comparative calculation, is accurate.
Summary of the Overall Significant mRNA Expression Changes in CEN and HGEN Cells Following Exposure to T3 and Several PFCs
T3- and PFHpA-treated HGEN cells induced RC3 mRNA expression up to twofold and sixfold, respectively. This was similar to the transcriptional response observed in CEN cells treated with short-chained PFCs (PFBS and PFHxS). Similar transcriptional responses in RC3 following exposure to T3 and PFCs in the neuronal cells of two different avian species make this gene a useful marker to examine disturbances in TH homeostasis.
Oct-1 mRNA expression was induced following exposure to PFBS, PFHxA, and PFHxS in HGEN cells but was invariable in CEN cells. Oct-1, a DNA-binding protein, has been associated with DNA replication and the transcription of histone 2B genes, small nuclear RNAs, and immunoglobulins (Fletcher et al., 1987
; Mittal et al., 1996
; Sive and Roeder, 1986
; Verrijzer et al., 1990
). Oct-1 may be important in regulating the growth of eukaryotic cells because reduced expression was associated with cell cycle arrest and morphological differentiation (Lakin et al., 1995
). Oct-1 is considered to be a TH-responsive gene for several reasons. Dowling et al. (2000)
reported that a single T4 administration increased Oct-1 mRNA in the cortex of rat pups exposed at gestation day 16 (athyroid state). Furthermore, Oct-1 expression was regulated by T3 from fetal life until adulthood in rodents. Not only has Oct-1 demonstrated TH responsiveness but also it has shown responsiveness following exposure to environmental contaminants; A1254 induced Oct-1 expression in the cortex of rodents (Gauger et al., 2004
This study reported significant gene expression changes (up to 12-fold) in PFC-treated avian neuronal cells. It was determined that PFCs differ substantially in their effects on mRNA expression in avian neuronal cells. The six-carbon PFHxA and PFHxS were the most transcriptionally active compounds in CEN cells, altering the expression of three TH-responsive genes. The results suggest that PFBS, PFHxS, and PFHxA may share similar modes of action with T3 given the similar patterns in transcriptional responses. These findings are important given the persistence of newer, replacement PFCs in tissues, eggs, and blood of avian species worldwide. Short-chained PFCs are being manufactured as replacement alternatives to PFOS and PFOA and the concentrations of short-chained PFCs are expected to rise due to increasing usage and production.
Studies that have assessed PFC chain length effects have reported that the degree of fluorination, carbon chain length, and functional group are key factors that influence PFC effects (e.g., binding potency and neural functions) (Liao et al., 2009
; Upham et al., 1998
; Weiss et al., 2009
). Long-chained PFCs bind more strongly to protein than short-chained PFCs (Jones et al., 2003
), which may have caused these compounds to bind to the protein in the medium, the walls of the plastic wells, or to nonspecific proteins on the outside of neuronal cells rendering them less available for uptake into neuronal cells. Therefore, the augmented transcriptional response elicited by short-chained PFCs in this study may be due to their bioavailability. In other words, they could have entered neuronal cells more readily due to their lower binding affinities to extracellular proteins.
The TH-responsive genes appear to be useful endpoints for in vitro screening of effects of PFCs in primary cultures of avian neuronal cells. This technique permits an initial assessment of PFC effects on various avian species and provides more insight into the mechanisms of action of these contaminants. It is important to validate these findings in ovo to determine how predictive the in vitro approach is in terms of identifying whole animal effects. In addition, future work should examine alterations of the proteins encoded by these transcripts. This study has contributed to our understanding of PFC effects on the TH pathway and has not only provided evidence that the brain may be a target organ for PFC effects but that these contaminants have the potential to alter TH homeostasis in birds.