Elsevier

Journal of Proteomics

Volume 75, Issue 8, 18 April 2012, Pages 2417-2430
Journal of Proteomics

Insight into the neuroproteomics effects of the food-contaminant non-dioxin like polychlorinated biphenyls

https://doi.org/10.1016/j.jprot.2012.02.023Get rights and content

Abstract

Recent studies showed that food-contaminant non-dioxin-like polychlorinated biphenyls (NDL-PCBs) congeners (PCB52, PCB138, PCB180) have neurotoxic potential, but the cellular and molecular mechanisms underlying neuronal damage are not entirely known. The aim of this study was to assess whether in-vitro exposure to NDL-PCBs may alter the proteome profile of primary cerebellar neurons in order to expand our knowledge on NDL-PCBs neurotoxicity. Comparison of proteome from unexposed and exposed rat cerebellar neurons was performed using state-of-the-art label-free semi-quantitative mass-spectrometry method. We observed significant changes in the abundance of several proteins, that fall into two main classes: (i) novel targets for both PCB138 and 180, mediating the dysregulation of CREB pathways and ubiquitin–proteasome system; (ii) different congeners-specific targets (alpha-actinin-1 for PCB138; microtubule-associated-protein-2 for PCB180) that might lead to similar deleterious consequences on neurons cytoskeleton organization. Interference of the PCB congeners with synaptic formation was supported by the increased expression of pre- and post-synaptic proteins quantified by western blot and immunocytochemistry. Expression alteration of synaptic markers was confirmed in the cerebellum of rats developmentally exposed to these congeners, suggesting an adaptive response to neurodevelopmental toxicity on brain structures. As such, our work is expected to lead to new insights into the mechanisms of NDL-PCBs neurotoxicity.

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.

References (48)

  • M.D. Minana et al.

    Nicotine prevents glutamate-induced proteolysis of the microtubule-associated protein MAP-2 and glutamate neurotoxicity in primary cultures of cerebellar neurons

    Neuropharmacology

    (1998)
  • W. Thangnipon et al.

    Observations on rat cerebellar cells in vitro: influence of substratum, potassium concentration and relationship between neurones and astrocytes

    Brain Res

    (1983)
  • R.D. Brinton et al.

    Progesterone receptors: form and function in brain

    Front Neuroendocrinol

    (2008)
  • M.M. Hoskison et al.

    Microtubule disruption, not calpain-dependent loss of MAP2, contributes to enduring NMDA-induced dendritic dysfunction in acute hippocampal slices

    Exp Neurol

    (2006)
  • C.A. Meacham et al.

    Accumulation of methylmercury or polychlorinated biphenyls in in vitro models of rat neuronal tissue

    Toxicol Appl Pharmacol

    (2005)
  • M. Stenberg et al.

    Multivariate toxicity profiles and QSAR modeling of non-dioxin-like PCBs–an investigation of in vitro screening data from ultra-pure congeners

    Chemosphere

    (2011)
  • A. Fontan-Lozano et al.

    Lack of DREAM protein enhances learning and memory and slows brain aging

    Curr Biol

    (2009)
  • T. Nakagawa et al.

    The dynamic turnover and functional roles of alpha-actinin in dendritic spines

    Neuropharmacology

    (2004)
  • C.A. Boyle et al.

    Birth defects and disabilities: a public health issue for the 21st century

    Am J Public Health

    (2005)
  • T. Schettler

    Toxic threats to neurologic development of children

    Environ Health Perspect

    (2001)
  • European Food Safety Agency

    Opinion of the scientific panel on contaminants in the food chain on a request from the commission related to the presence of non dioxin-like polychlorinated biphenyls (PCB) in feed and food

    EFSA J

    (2005)
  • J.L. Jacobson et al.

    Intellectual impairment in children exposed to polychlorinated biphenyls in utero

    N Engl J Med

    (1996)
  • B. Piedrafita et al.

    Developmental exposure to polychlorinated biphenyls PCB153 or PCB126 impairs learning ability in young but not in adult rats

    Eur J Neurosci

    (2008)
  • O. Faroon et al.

    Effects of polychlorinated biphenyls on the nervous system

    Toxicol Ind Health

    (2001)
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