Hexachloronaphthalene Induces Mitochondrial-Dependent Neurotoxicity via a Mechanism of Enhanced Production of Reactive Oxygen Species

Abstract

Hexachloronaphthalene (PCN67) is one of the most toxic among polychlorinated naphthalenes. Despite the known high bioaccumulation and persistence of PCN67 in the environment, it is still unclear to what extent exposure to these substances may interfere with normal neuronal physiology and lead to neurotoxicity. Therefore, the primary goal of this study was to assess the effect of PCN67 in neuronal in vitro models. Neuronal death was assessed upon PCN67 treatment using differentiated PC12 cells and primary hippocampal neurons. At 72 h postexposure, cell viability assays showed an IC₅₀ value of 0.35 μg/ml and dose-dependent damage of neurites and concomitant downregulation of neurofilaments L and M. Moreover, we found that younger primary neurons (DIV4) were much more sensitive to PCN67 toxicity than mature cultures (DIV14). Our comprehensive analysis indicated that the application of PCN67 at the IC₅₀ concentration caused necrosis, which was reflected by an increase in LDH release, HMGB1 protein export to the cytosol, nuclear swelling, and loss of homeostatic control of energy balance. The blockage of mitochondrial calcium uniporter partially rescued the cell viability, loss of mitochondrial membrane potential (ΔΨ _m), and the overproduction of reactive oxygen species, suggesting that the underlying mechanism of neurotoxicity involved mitochondrial calcium accumulation. Increased lipid peroxidation as a consequence of oxidative stress was additionally seen for 0.1 μg/ml of PCN67, while this concentration did not affect ΔΨ _m and plasma membrane permeability. Our results show for the first time that neuronal mitochondria act as a target for PCN67 and indicate that exposure to this drug may result in neuron loss via mitochondrial-dependent mechanisms.

Figure 1

Dose- and time-dependent effect of PCN67 on differentiated PC12 cells. The cells were treated with PCN67 concentration ranging from 0.001 μg/ml to 25 μg/ml for 24 h (a), 48 h (b), and 72 h (c). The half-maximal inhibitory concentration (IC₅₀) was determined based on viability data obtained following 72 h treatment using nonlinear regression analysis (d). Cell viability in vehicle-treated cells was taken as 100%. ^∗P < 0.05 and ^∗∗∗P < 0.001.

Figure 2

The effect of PCN67 on PC12 cell differentiation. (a) Morphology of control and PCN67-treated differentiated PC12 cells photographed under an inverted phase microscope with a CCD camera. Scale bar 10 μm. (b) Quantification of an average length of neurites. The cell protrusion was counted as neurite when its length was at least twice of cell diameter. (c) Quantification of an average number of neurite-forming cells. Cells with at least one visible neurite were considered. (d) The expression of neurofilament M (NF-M) and (e) neurofilament L (NF-L) was assessed by real-time PCR 72 h after PCN67 treatment. The expression level in vehicle-treated cells was taken as 1. ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001.

Figure 3

Toxicity of PCN67 depends on the maturation stage of primary hippocampal neurons. (a) The grayscale images of neuronal network photographed using an inverted phase microscope with a CCD camera. Scale bar 10 μm. (b) Neurons transfected with GFP plasmid cultured in defined media in the presence or absence of KCl (40 mM). KCl was added together with PCN67, and the images were taken 2 days later. (c) Quantification of axon length at DIV4 and (d) at DIV14. The longest neurite was measured. ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001.

Figure 4

PCN67 treatment induces characteristics of necrotic death in differentiated PC12 cells. (a) Representative dot plots of necrotic/apoptotic cell distribution following 72 h of PCN67 treatment. Cells were stained with Annexin V and propidium iodide and analyzed with flow cytometry. (b) Quantification of viable (V), early apoptotic (EA), late apoptotic (LA), and necrotic (N) cells in a population. (c) Determination of plasma membrane integrity measured by the release of lactate dehydrogenase (LDH) 72 h following PCN67 treatment. (d) Western blot-based quantification of HMGB1 protein in cytosolic fraction collected from 72 h-treated cells. The results are presented as arbitrary units following normalization to the GAPDH level used as a marker of a cytosolic fraction. Histone H3, a protein marker of a nuclear fraction, was used to determine the purity of fractionation. (e) Average measurement of the nuclear diameter of differentiated PC12 cells after 72 h of PCN67 treatment. (f) The changes in the ATP level following 72 h of PCN67 treatment. The results were normalized to the protein level and are expressed as nmoles/mg. ^∗P < 0.05 and ^∗∗∗P < 0.001.

Figure 5

PCN67 induces intracellular calcium rise and plasma membrane depolarization. (a) Differentiated PC12 cells were treated with PCN67 for 72 h in the presence of intracellular calcium indicator—GcAMP3 and (b) propidium iodide (7.5 μM). The cells were placed into an environmental chamber with controlled temperature and CO₂ concentration, and the fluorescence changes were recorded every 4 h using an Axio Observer 7 Marianas™ Microscope equipped with 63x objective. The fluorescence of single cells was processed as ΔF/F₀ after background subtraction. (c) Representative images of cells stained with propidium iodide (7.5 μM) following treatment with PCN67 (IC₅₀) for 72 h in the presence or absence of calcium in the culture media. Scale bar 10 μm. (d) Chelation of intracellular calcium partially protected from cell death. BAPTA-AM (5 μM) was added to the culture 48 h after PCN67 treatment, and the viability was determined the day after. Vehicle-treated cells were taken as 100%. (e) Plasma membrane potential was measured 72 h following PCN67 treatment using DiSBAC₂ (1 μM). Cells were loaded with a dye for 30 min, and the fluorescence was analyzed using flow cytometry. ^∗P < 0.05 and ^∗∗∗P < 0.001.

Figure 6

The role of mitochondria in PCN67-induced toxicity. (a) For measuring mitochondrial membrane potential, differentiated PC12 cells were cultured in the presence of PCN67 for 72 h and then loaded with TMRE (25 nM) for 30 min. TMRE-loaded cells were analyzed for fluorescence intensity using flow cytometry. Individual data points are shown. (b) Partial prevention of PCN67-induced death by Ru360. Ru360 was added 30 min before PCN67 treatment, and the viability was measured 72 h after. (c) Viability assessment of cells cultured in the presence of glucose (25 mM) or galactose (10 mM) supplemented RPMI for 5 days and then treated with PCN67 for 24 h. (d) PCN67-induced cell death is not inhibited by cyclosporine A (CsA). Differentiated PC12 cells were cotreated with PCN67 and CsA, and the viability was assessed 72 h later. ^∗P < 0.05 and ^∗∗∗P < 0.001.

Figure 7

The effect of PCN67 on oxidative and nitrative stress. (a) Differentiated PC12 cells were treated with vehicle and PCN67 for 72 h. ROS generation was assessed with DCFH-DA (10 μM) using the excitation and emission filters set at 488 nm and 525 nm. Individual data points are shown. (b) The oxidative damage of lipids by reactive oxygen species was assessed by measuring thiobarbituric acid reactive substances (TBARS), and the results are normalized to the protein level. Individual data points are shown. (c) The level of NO was determined using Griess reagent, and the absorbance readings at 540 nm were measured by a microplate reader. ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001.