Insight into the neuroproteomics effects of the food-contaminant non-dioxin like polychlorinated biphenyls
Graphical abstract
Highlights
► Food-contaminant PCBs alter the proteome profile of rat cerebellar neurons. ► A state-of-the-art label-free semi-quantitative mass-spectrometry approach was used. ► Major targets include proteins involved in CREB, ubiquitin, cytoskeleton pathways. ► PCB congeners interfere with synaptic proteins both in vitro and in vivo. ► Implication of the findings to PCB neurodevelopmental toxicity.
Introduction
There is a growing concern about the apparent increased incidence of neurodevelopmental disorders [1], [2]. Besides genetic and socioeconomic aspects, environmental factors, including industrial chemicals entering the food-chain, may represent possible causes for neurological disabilities. Among the contaminants abundantly found in food sources, there are the polychlorinated biphenyls (PCBs). Although the production and use of PCBs has been banned in most industrialized countries since the 1970s, PCBs persist in the environment, making them a permanent risk to human health [3].
Depending on their structural characteristics and toxicological effects, PCBs can be divided in two groups: coplanar dioxin-like PCBs (DL-PCBs) and non coplanar non-dioxin-like PCB (NDL-PCBs). The DL-PCBs have been extensively studied and their toxic and biological effects are mainly associated with binding and activation of the aryl hydrocarbon receptor (AhR) transduction pathway. The NDL-PCBs have generally been considered less toxic because of their ortho-substituted chlorines, which impair interaction with the AhR [4]. However, both groups of PCBs have been associated with neurotoxic effects and neurodevelopmental deficits in humans and animal models, although different PCBs congeners might act through shared or different pathways [5], [6], [7], [8], [9], [10], [11], [12], [13], [14].
Evidence suggests that low NDL-PCBs doses, such as those similar to background contamination in food, can cause subtle effects when exposure is prolonged over time. The need of a better understanding of NDL-PCB toxicity has been raised by the European Food Safety Authority, EFSA [3], [15].
In vivo and in vitro studies have shown that NDL-PCBs have neurotoxic potential by interfering with intracellular signalling and Ca2+ homeostasis (for review see [16]). Moreover they inhibit the uptake of dopamine, serotonin, glutamate and GABA in synaptosomes and synaptic vesicles isolated from rat brain [17], [18]. Additionally, low-chlorinated NDL-PCBs have been shown to potentiate the human GABAA receptor, with detrimental consequences on the main inhibitory feedback mechanism for learning and memory as well as motor activity [19], [20]. Recently, non-cytotoxic concentrations of PCB153 and 180 have been reported to interfere with neuronal differentiation of embryonic neural stem cells [21]. It has been shown that long-term exposure of primary cultures of cerebellar neurons to food-relevant NDL-PCBs such as PCB52, 138 and 180 impairs the glutamate-nitric oxide (NO)-cGMP pathway which contributes to the modulation of some learning abilities [22]. Each of the congeners affected the pathway differently, at more than one step with different potency and, for some steps, in opposite ways [23].
All these alterations can possibly underlie the observed neurobehavioral effects in vivo, which include changes in motor activity, learning, memory and attention [10], [24].
Knowledge of the neural functions implies knowledge of the functions of the proteins involved in those processes. Recent advances in proteomic technology provide the opportunity to investigate simultaneously multiple components of protein pathways and cascades in neurons, thereby increasing our understanding of the complex mechanisms underlying PCB-induced neurotoxicity.
Therefore, the objective of this study was to detect changes in the proteome profile of cerebellar neurons after exposure to PCB52, 138 and 180 by using a large-scale proteomic approach and a label-free semi-quantitative mass spectrometry method. In combination with network protein analysis tools, we sought to identify cellular targets that might mediate the different mechanism of neurotoxicity exerted by the different NDL-PCB congeners. We focused on PCB52, 138, 180 congeners, the most abundant in food and human blood and milk [3], because of our interest in gaining a more holistic perspectives of the primary neurons’ response to these contaminants, that was partially characterized by members of this group in a previous work [23].
Section snippets
Chemicals
PCB52 (2,2′,5,5′-tetrachlorobiphenyl), PCB138 (2,2′,3,4,4′5 ′-hexachlorobiphenyl) and PCB180 (2,2′,3,4,4′,5,5′-heptachlorobiphenyl), were obtained from Chiron (Trondheim; Norway) and purified at the Environmental Chemistry Department, Umea University (Sweden) to 99,9999% purity (ATHON European project (FOOD-CT_2006-022923 ATHON). Briefly, PCDD/Fs and DL-PCBs impurities in the NDL-PCBs was removed by applying a fractionation on active carbon as described previously [25]. Purification efficiency
Proteomic profiling reveals congener-specific patterns of changes in rat primary neurons exposed to PCB52, PCB138 and PCB180
To investigate the PCB congeners effect on the proteome of rat cerebellar neurons we used a 1-DE gel approach for protein pre-fractionation integrated into a typical LC-MS/MS workflow for protein identification. A simple and convenient label-free approach (spectral counting) was then used for relative protein abundance quantification.
Overall, our proteomic analysis identified 677 proteins in the total lysate of untreated and treated neurons (3 replicates/group) meeting the identification
Discussion
The exposure of primary cultures of rat cerebellar neurons to PCBs congeners 180, 138 and 52 significantly altered the cell protein expression profile.
Proteomic profiling reveals congener-specific patterns of changes. PCB52 resulted to be less potent than PCB138 and PCB180 in terms of number of proteins, the concentration of which was significantly modulated by the congeners. The concentration of PCB52 required to induce a significant effect on neurons proteome is 10 to 100-fold higher than
Conclusions
In this study, we have highlighted (i) the importance of differential proteome profile as contributor to NDL-PCB congener-specific sensitivity in rat primary neurons; (ii) the identification of novel and plausible mediators integral to major NDL-PCBs neurotoxicity pathways (e.g. CREB pathway, ubiquitin–proteasome system, synaptic proteins and synaptic plasticity) conferring congeners sensitivity and (iii) the identification of several novel PCB congeners-specific proteins (e.g. CSEN, ACTN1,
Acknowledgements
This work was carried out with the financial support from the European Commission (FOOD-CT-2006-022923 ATHON). We would like to thank Dr. Patrick Andersson and Mia Stenberg (Environmental Chemistry Department, Umea University, Sweden) for purifying NDL-PCBs.
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