Aryl hydrocarbon receptor-dependent induction of apoptosis by 2,3,7,8-tetrachlorodibenzo-p-dioxin in cerebellar granule cells from mouse

Authors


Address correspondence and reprint requests to Jaime M. Merino, Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias, Universidad de Extremadura, 06071-Badajoz, Spain. E-mail: jmmerino@unex.es

Abstract

J. Neurochem. (2011) 10.1111/j.1471-4159.2011.07291.x

Abstract

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a prototypical environmental contaminant with neurotoxic properties that alters neurodevelopment and behavior. TCDD is a ligand of the aryl hydrocarbon receptor (AhR), which is a key signaling molecule to fully understand the toxic and carcinogenic properties of dioxin. Much effort is underway to unravel the molecular mechanisms and the signaling pathways involved in TCDD-induced neurotoxicity, and to define its molecular targets in neurons. We have used cerebellar granule cells (CGC) from wild-type (AhR+/+) and AhR-null (AhR−/−) mice to characterize the cell death that takes place in neurons after TCDD toxicity. TCDD induced cell death in CGC cultures from wild-type mice with an EC50 of 127 ± 21 nM. On the contrary, when CGC neurons from AhR-null mice were treated with TCDD no significant cell death was observed. The role of AhR in TCDD-induced death was further assessed by using the antagonists resveratrol and α-naphtoflavone, which readily protected against TCDD toxicity in AhR+/+ CGC cultures. AhR+/+ CGC cultures treated with TCDD showed nuclear fragmentation, DNA laddering, and increased caspase 3 activity, similarly to what was found by the use of staurosporine, a well-established inducer of apoptosis. Finally, the AhR pathway was active in CGC because TCDD could induce the expression of the target gene cytochrome P450 1A2 in AhR+/+ CGC cultures. All together these results support the hypothesis that TCDD toxicity in CGC neurons involves the AhR and that it takes place mainly through an apoptotic process. AhR could be then considered a novel target in neurotoxicity and neurodegeneration whose down-modulation could block certain xenobiotic-related adverse effects in CNS.

Abbreviations used
AhR

aryl hydrocarbon receptor

ARNT

Ah receptor nuclear translocator

DAPI

4′,6-diamidino-2-phenylindole

MTT

3-[4,5-dimethyl thiazol-2-yl]-2,5-diphenyl-tetrazolium

NGF

nerve growth factor

PBS

phosphate buffer solution

ROS

reactive oxygen species

TCDD

2,3,7,8-tetrachlorodibenzo-p-dioxin

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is one of the most potent xenobiotic agent known and exposure to this chemical results in toxicity and cancer (Cole et al. 2003; Bock and Köhle 2006). The aryl hydrocarbon receptor (AhR) mediates the teratogenic and carcinogenic effects of TCDD which binds this receptor as a high affinity exogenous ligand. An extensive amount of work supports that AhR is the principal intracellular molecule implicated in the regulation of the toxic and carcinogenic properties of TCDD (Denison and Nagy 2003; Mimura and Fujii-Kuriyama 2003). AhR is a ligand-activated transcription factor that belongs to a large family of proteins called basic helix-loop-helix that share in common conserved sequences for DNA binding and for protein dimerization (Burbach et al. 1992; Whitlock 1999). After ligand activation, AhR translocates into the nucleus and dimerizes with the Ah receptor nuclear translocator (ARNT) (Reyes et al. 1992) to finally promote the expression of target genes, such as cytochromes P450 and phase II enzymes (Hankinson 1995; Rivera et al. 2002; Nebert et al. 2004).

Although it is assumed that most TCDD-induced toxicological effects are mediated via binding to AhR, many aspects are still to be defined. Thus, the studies directed to unravel the molecular mechanisms of TCDD toxicity are of great relevance. Interestingly, the neurotoxic effects of xenobiotics have been less profoundly studied as compared with their carcinogenic activity.

The pattern of AhR and ARNT expression in the rat brain has been studied previously and it was found that both proteins are widely distributed throughout the brain and brainstem (Kainu et al. 1995; Petersen et al. 2000). Similarly, in mouse embryos, AhR and ARNT are present in neuroepithelium and neural tube (Abbott and Probst 1995; Abbott et al. 1995).

With respect to the neurotoxic effects of TCDD, experiments in rhesus monkeys showed that TCDD induced changes in behavior (Schantz et al. 1992) and in long-term cognitive deficits (Schantz and Bowman 1989), both during development and in adulthood. An important set of studies analyzed TCDD effects in humans after gestational exposure, and demonstrated major alterations in neurodevelopment (Rogan and Gladen 1992; Jacobson and Jacobson 1996, 1997a,b). Also, it has been demonstrated that TCDD alters normal rat brain development and produces cognitive disability and motor dysfunction (Legare et al. 2000; Nayyar et al. 2002).

The mechanisms of TCDD-induced neurotoxicity have been recently studied in vitro using cerebellar granule cells and cortical neurons. These studies link mainly TCDD toxicity to the glutamate receptor of the NMDA subtype. Such linkage is proposed to take place through an increase in the intracellular calcium concentration, and because of the high permeability of NMDA receptors to this cation, it was also suggested that neuronal death takes place as a result of a massive generation of reactive oxygen species (ROS) (Kim and Yang 2005). In cortical neurons, TCDD activation of AhR induces gene expression and calcium entry that can be blocked by dizolcipine (MK-801), a selective blocker of the NMDA receptor (Lin et al. 2008). Remarkably, TCDD enhances NMDA excitotoxicity whereas AhR knockdown reduces NMDA excitotoxicity and NMDA-dependent calcium entry (Lin et al. 2009). From gestational TCDD exposure experiments, it was concluded that the induction of cortical deficits were a consequence of lower expression of NMDA and AMPA receptor subunits in rat brain (Hood et al. 2006). Together, although these data are very useful to understand TCDD toxicity on neurons, the mechanisms of TCDD-induced neurotoxicity, the signaling pathways involved and the identity of its molecular targets are still to be defined.

AhR activation by dioxin promotes gene expression. Typical target genes are members of the cytochrome P450 (CYP450) superfamily of monooxigenases such as CYP1A1, 1A2 and 1B1 (Nebert et al. 2004), which, in fact, are implicated not only in xenobiotic but also in endobiotic metabolism. Interestingly, our understanding of CYP1 enzymes has changed as different evidences have been accumulated. The detoxification function of CYP1 enzymes establishes a balance between beneficial, xenobiotic elimination-related and detrimental effects since its metabolic products can cause toxicity and cancer. Overall, CYP1 enzymes are generally considered more protective than destructive during the environmental insult (Nebert et al. 2004).

However, these issues have not been explored in the nervous system. We have previously shown that nitric oxide has a role in P450 induction in cerebellar granule cells and suggested that neurodegeneration could diminish the induction of specific P450s and impair the metabolism of toxins in the CNS (Mulero-Navarro et al. 2003). In addition, other studies have reported that granule cells neurogenesis can be disrupted by TCDD exposure (Williamson et al. 2005). Thus, the study of TCDD toxicity in cerebellar granule cells would be of high interest to understand the mechanisms of dioxin neurotoxicity.

In a previous study, we have also described the effects of TCDD toxicity on nerve growth factor (NGF)-differentiated PC12 cells (Sánchez-Martín et al. 2010a). The results indicated that TCDD induces cell death through an apoptotic process, but the role of AhR in such dioxin toxicity was not prominent. Although pheochromocytoma (PC12) cells can be differentiated to neuronal-like cells by the addition of NGF, and they have been widely used for neurobiology research, important phenotypic differences exist with respect to primary neurons. In this work, we study TCDD toxicity in primary cerebellar granule cells from wild-type and AhR-null mice aiming to determine the role of AhR in TCDD toxicity. We conclude that TCDD toxicity in cerebellar granule cells involves the AhR and that it takes place mainly through an apoptotic process.

Methods

Animals

The AhR −/− cells used in this study were obtained from AhR-null mice that were generated by homologous recombination in mouse embryonic stem cells as previously described (Fernández-Salguero et al. 1995).

Cerebellar granule cell cultures

Cerebellar granule cells (CGC) cultures were prepared as previously described (Mulero-Navarro et al. 2003). Briefly, CGC cultures were isolated from 3-day-old C57BL/6 mouse pups (Mus musculus). Cerebella were dissected, the meninges carefully removed and the tissue finely minced and washed two times in Hank′s balanced salt solution containing 0.3% bovine serum albumin and 20 mM HEPES. Tissue was digested at 37°C for 15 min with gentle agitation in Hank′s balanced salt solution –bovine serum albumin–HEPES containing 0.025% trypsin. After digestion, trypsin was inactivated by addition of three volumes of complete medium: minimal essential medium containing 10% fetal bovine serum, 25 mM d(+)-glucose, 0.1 μg/mL apoptransferrin, 2 mM l-glutamine, 2 μg/mL insulin, 20 mM KCl, 100 μg/mL gentamicine sulphate and 20 mM HEPES. Tissue was then dissociated by several passes through a siliconized Pasteur pipette. Cells were centrifuged for 7 min at 200 g and pellets were resuspended in complete medium. CGC were plated at 3.5 × 106 cells in 35 mm or 7 × 105 cells in 24-well poly d-lysine coated dishes and maintained at 37°C in a 5% carbon dioxide atmosphere. To eliminate proliferating cells, cultures were treated two days after plating with 10 μM Ara-C as mitotic inhibitor. Treatment with Ara-C did not affect CGC viability under the experimental conditions used. Cells were maintained in the same culture medium for the extent of the experiment. Addition of 2.7 mM d(+)-glucose and sterile water every 2 days is necessary to maintain a carbon source for neurons and to compensate evaporation, respectively. The experiments involving animals were performed in compliance with the guidelines established by the Animal Care and Use Committee of the University of Extremadura.

TCDD toxicity assays

To analyze TCDD toxicity, we used CGCs cultured in 24-well poly-d-lysine coated plates. Neurons were incubated with different TCDD concentrations ranging from 1 to 500 nM at 37°C in culture medium. The medium was removed after 30 min incubation with dioxin and fresh culture medium was added. Then, cultures were returned to the incubator (37°C/5% CO2), and cell death was assessed 18–24 h later using the trypan blue exclusion assay. The effect of AhR antagonists was assayed using TCDD in presence of resveratrol or α-naphthoflavone. The EC50 values for TCDD toxicity were calculated using the logistic dose response curve from the OriginTM software. These values state the 50% of cell death exerted by TCDD in CGCs.

Cell viability determination by trypan blue exclusion and MTT reduction assays

Cell death was blindly assessed using the trypan blue exclusion assay as previously described (Valera et al. 2008). Neurons were incubated with trypan blue dye at 0.4% (w/v) and a minimum of 500 cells were counted in 4–5 microscopic fields per plate separating non-stained cells (live cells) and blue stained cells with swollen soma (dead cells) and using an Axioplan ZEISS microscope. A minimum of two plates per condition were used in three separate cultures.

Alternatively, cell viability were analyzed by using the 3-[4,5-dimethyl thiazol-2-yl]-2,5-diphenyl-tetrazolium (MTT) reduction assay as previously described by Mulero-Navarro et al. (2003). CGCs cultures were incubated at 37°C for 1 h with 150 μg/mL MTT in basic saline solution (137 mM NaCl, 3.5 mM KCl, 0.4 mM KH2PO4, 0.33 mM Na2HPO4·7 H2O, 5 mM N-Tris(hydroxymethyl)methyl-2-aminoethanesulfonicd acid pH 7.4, 10 mM d-glucose). The mitochondrial dehydrogenase activity of live cells generates formazan as a precipitate that can be dissolved in dimethyl sulfoxide (Sigma, St Louis, MO, USA) and measured using spectroscopy with the difference in absorbance at 490 and 650 nm. Data are shown as percentage of control cultures which are taken as 100%.

Nuclear morphology analysis by DAPI labeling

To evaluate nuclear morphology 4′,6-diamidino-2-phenylindole (DAPI) labeling was carried out in CGCs cultured in 35-mm poly-d-lysine coated dishes. After TCDD treatment at 250 nM for 30 min, CGCs were incubated for 5 min at 37°C with 2 μM DAPI (Sigma) in phosphate buffer solution (PBS) (137 mM NaCl, 2.7 mM KCl, 1.47 mM KH2PO4 and 4.3 mM Na2HPO4·7 H2O). CGCs were washed twice with PBS buffer to eliminate the excess of the dye. DAPI fluorescence was visualized using an Axioplan ZEISS microscope with excitation and emission wavelengths of 350 and 460 nm, respectively.

DNA fragmentation analysis

DNA fragmentation was analyzed as described by Martín-Romero et al. (2002). Cell lysis was performed in 0.3 mL of a solution containing 5 mM Tris (pH 7.5), 20 mM EDTA and 0.5% Triton X-100 for 1 h at 4°C. Nuclei were precipitated by centrifugation at 10 000 g for 15 min. To obtain the DNA, supernatants were treated with 200 μg/mL proteinase K for 30 min at 60°C and the DNA was purified by sequential extraction with phenol, phenol–chloroform (1 : 1) and chloroform and precipitated in ethanol-sodium acetate solution (overnight at −20°C). Samples were then centrifuged for 30 min at 15 000 g at 4°C, pellets were washed with 80% ethanol and centrifuged again under the same conditions. Finally, DNA samples were resuspended in TE buffer or water and incubated with 30 μg/mL of RNase for 30 min at 37°C. Samples were run in 1% agarose gels and visualized by ethidium bromide staining and photographed.

Measurement of caspase 3 activity

Caspase 3 activity was measured as previously described (Sánchez-Martín et al. 2010b). The caspase 3 substrate used was N-Acetyl-Asp-Glu-Val-Asp-7-amido-4-methylcoumarin (Sigma). The fluorescence was measured with an spectrofluorimeter Shimadzu RF-5301-PC using excitation and emission wavelengths of 360 and 460 nm, respectively. Data were calculated as fluorescence units/μg protein. Data are showed as percentage relative to activity measured in control cultures.

Measurement of intracellular reactive oxygen species

To determine the ROS generation after TCDD incubation, CGCs were cultured in 35-mm poly-d-lysine coated dishes. The fluorescent dye dichlorofluorescein diacetate was added at 1 μM to the culture medium and cells were incubated for 15 min at 37°C (Valera et al. 2002; Mulero-Navarro et al. 2003). The excess of the dye is eliminated washing twice with PBS. Dichlorofluorescein diacetate fluorescence was visualized using an Axioplan ZEISS microscope with excitation and emission wavelengths of 494 and 525 nm, respectively. Fluorescence intensity was measured using the ImageJ software.

Reverse transcription and real-time PCR

Total RNA was isolation from CGCs, reverse transcription and real-time PCR was performed as previously described (Sánchez-Martín et al. 2010a) to quantify the expression levels of the CYP1A2 mRNA. Primers for CYP1A2 were 5′-ATGTCACCTCAGGGAATGC-3′ (forward) and 5′-GATGAGACCGCCATTGTCTT-3′ (reverse). Primers for GAPDH mRNA (to normalize expression levels) were 5′-TGAAGCAGCCATCTGAGCG-3′ (forward) and 5′-GCAAGCTGCAAGAGTCCCAG-3′ (reverse). Raw data was analyzed using the 2ΔΔCt method.

Cell lysates and western immunoblot analysis

Western blot were performed as previously indicated (Sánchez-Martín et al. 2010a). Antibodies are primary rabbit anti-AhR (Thermo Scientific, Fremont, CA, USA) and anti-β-actin (Sigma) and secondary donkey anti-rabbit (ABR). Molecular weights for the mouse AhR and β-actin proteins are 95 and 42 KDa, respectively. Quantification of band intensity was evaluated using the ImageJ software.

Statistical analysis of the data

Statistical analyses were carried out using one-way anova tests followed by post hoc Tukey tests using Prism 5.0 software (GraphPad). Significance was set at p < 0.05. Data shown are mean ± SD.

Results

AhR-dependent TCDD toxicity in cerebellar granule cells

To analyze the role of the aryl hydrocarbon receptor in TCDD neurotoxicity, we prepared cerebellar granule cells from wild-type (AhR+/+) and AhR-null (AhR−/−) mice. First, we measured AhR protein expression by western blot in AhR+/+ and AhR−/− CGC cultures. The results are shown in Fig. 1a. We confirmed that while AhR+/+ cultures expressed the Ah receptor, AhR−/− cultures did not. Thus, AhR+/+ and AhR−/− CGC primary cultures represent an adequate model to study AhR dependency in TCDD-induced neurotoxicity.

Figure 1.

 Concentration dependence of TCDD toxicity in cerebellar granule cells. (a) Protein expression for AhR in wild-type and AhR-null mice cerebellar granule cells. Immunoblotting was carried out as described in the Methods section. Representative western blot for AhR expression is shown for AhR+/+ and AhR−/− CGC cultures. Molecular weights for the mouse AhR and β-actin proteins are 95 and 42 KDa, respectively. (b) Neurons from wild-type (•) and AhR-null (○) mice were incubated with different TCDD concentrations (1, 10, 100, 250, and 500 nM) for 30 min. Cell death was determined 18–24 h later by the trypan blue exclusion assay. The EC50 values calculated were 127 ± 21 nM (AhR+/+ neurons) and > 500 nM (AhR−/− neurons). EC50 values were calculated using the logistic dose response curve from OriginTM software. The experiments were carried out, at least, in six different cultures. Data shown are mean ± SD (n ≥ 12). *p < 0.05 (different from AhR−/−).

To characterize TCDD toxicity in cultured neurons, CGCs from AhR+/+ and AhR−/− mice were exposed to dioxin. We incubated CGCs in presence of TCDD concentrations ranging from 10 to 500 nM for 30 min. Cultures were returned to the incubator for additional 18–24 h and cell death was measured using the trypan blue exclusion assay as described in the Methods section. We also analyzed cell death using the MTT reduction assay (data not shown) and both experimental approaches similarly addressed the effects of TCDD on CGCs viability.

TCDD induced a loss in viability in wild-type CGC cultures with an EC50 value of 127 ± 21 nM (Fig. 1b), a concentration lower to that obtained in NGF-differentiated PC12 cells (218 ± 24 nM) (Sánchez-Martín et al. 2010a). The main difference between these two set of experiments is the incubation time: 30 min for primary cerebellar neurons and 90 min for differentiated PC12 cells. Thus, cell type affects TCDD sensitivity, being higher in primary cultures as compared with established cell lines. In agreement with these values in CGCs, the EC50 obtained for rat primary hippocampal neurons is 140 ± 10 nM after 30 min of TCDD incubation (Sánchez-Martín et al. 2010a).

The use of cultured neurons from AhR-null mice allowed us to establish the role of the dioxin receptor in TCDD toxicity. As shown in Fig. 1b, the incubation of AhR−/− CGCs with TCDD up to a concentration of 500 nM did not induce any significant loss of cell viability. Thus, these results suggest that TCDD toxicity in CGCs requires the aryl hydrocarbon receptor (AhR). We then decided to determine if blocking this receptor could also block TCDD toxicity. For this, we assayed the effects of two known AhR antagonists: resveratrol and α-naphthoflavone. CGCs were pre-incubated for 1 h with resveratrol (10 μM) or α-naphthoflavone (50 μM), followed by 30 min co-incubation with 250 nM TCDD. As shown in Fig. 2, α-naphthoflavone significantly protected whereas resveratrol fully protected against TCDD toxicity. This result further supports the tenet that TCDD-induced cell death in CGCs implies the AhR signaling pathway.

Figure 2.

 Effects of resveratrol and α-naphthoflavone on TCDD-induced toxicity in cerebellar granule cells. AhR+/+ CGC cultures were incubated with 250 nM TCDD for 30 min in absence or presence of each antagonist. Resveratrol (10 μM) or α-naphthoflavone (50 μM) were added for 1 h prior to incubation with 250 nM TCDD for additional 30 min. Cell death was determined 18–24 h later by the trypan blue exclusion assay. The experiments were carried out, at least, in three different cultures. Data shown are mean ± SD (n ≥ 6). *p < 0.05 (different from TCDD).

TCDD induces apoptosis in cerebellar granule cells

Cell death can take place through different mechanisms. To discriminate the cell death process (e.g. necrosis or apoptosis) induced by TCDD in CGCs, we analyzed both the presence of chromatin condensation and the appearance of nuclear fragmentation by fluorescence microscopy using the DNA-binding fluorescent dye DAPI. Control CGC cultures, in absence of TCDD, showed nuclei with homogeneous chromatin distribution, as indicated by a low intensity DAPI labeling (Fig. 3a, control). This result was observed in both AhR+/+ and AhR−/− CGC cultures. After incubation of wild-type CGC cultures with 250 nM TCDD (a concentration two times the EC50) for 30 min, chromatin condensation was observed as revealed by an intense DAPI labeling 24 h later (Fig. 3a, TCDD); some cells showed a typical pattern of nuclear fragmentation of apoptotic nuclei. A quantification of apoptotic cells (considered positive for nuclear fragmentation) is shown in Fig. 3b. Almost 40% of the TCDD-treated CGCs had fragmented nuclei as compared with control cultures, which only reached 4–5% of fragmentation. As a positive control, CGCs were also treated with 30 nM staurosporine (Weil et al. 1996) for 24 h, and a characteristic nuclear fragmentation was observed (Fig. 3a, staurosporine) with a percentage of positive CGCs (40%) that was similar to that observed with TCDD (Fig. 3b). However, the incubation of AhR−/− CGCs with TCDD did not induce any significant chromatin condensation or nuclear fragmentation (Fig. 3a and b, TCDD), whereas staurosporine produced a characteristic pattern of apoptotic cell death (Fig. 3a and b, staurosporine).

Figure 3.

 TCDD induces nuclear fragmentation in cerebellar granule cells. (a) Chromatin condensation and nuclear fragmentation were analyzed by fluorescence microscopy using the DNA-binding fluorescent dye DAPI as described in the Methods section. AhR+/+ and AhR−/− CGC cultures were treated with 250 nM TCDD for 30 min and the pattern of DAPI labeling analyzed 24 h later. Staurosporine (incubation time of 24 h) was used as a positive control of nuclear fragmentation. Arrows indicate apoptotic fragmented nuclei. The experiments were carried out, at least, in three different cultures. (b) Percentage of apoptotic cells (positive for nuclear fragmentation) after TCDD incubation and staurosporine treatment using the data from panel a. Data shown are mean ± SD (n ≥ 6), and at least 500 cells were counted per experimental condition. Scale bar 20 μm.

To further analyze the induction of nuclear fragmentation in AhR+/+ CGCs, DNA laddering was studied in absence and in presence of TCDD. Incubation of AhR+/+ CGCs with TCDD for 30 min produced a DNA laddering that was not observed in control, untreated neurons (Fig. 4). Again, as a control, staurosporine generated a characteristic DNA laddering of an apoptotic process.

Figure 4.

 TCDD induces DNA laddering in cerebellar granule cells. DNA fragmentation was analyzed in agarose gels as described in the Methods section. AhR+/+ CGC cultures were treated with 250 nM TCDD for 30 min and DNA fragmentation was visualized after 24 h of incubation without TCDD. Staurosporine (incubation time of 24 h) was used as a positive control of DNA laddering. The experiments were carried out, at least, in three different cultures.

When apoptosis takes place, the activation of caspases is a widely accepted marker of this cell death mechanism. Thus, we have measured caspase 3 activity following TCDD incubation and the results are shown in Fig. 5. Incubation with 250 nM TCDD for 30 min produced a significant 2-3 fold increase in caspase 3 activation in AhR+/+ CGCs that continued for 1 h, to decrease to control levels 6–24 h after TCDD treatment. Incubation with staurosporine also provoked a large increase (4 to 5 times) in caspase 3 activity in CGCs (Fig. 5). In agreement with the data obtained in TCDD toxicity and DAPI labeling experiments, incubation of AhR−/− CGCs with TCDD did not produce any increase in caspase 3 activity after 30 min TCDD incubation or after 24 h post-treatment. Staurosporine provoked a twofold increase in caspase 3 activation in AhR−/− CGCs, indicating that these cells do have the potential to develop an apoptotic response (Fig. 5).

Figure 5.

 TCDD increases caspase 3 activity in cerebellar granule cells. Caspase 3 activity was measured as described in the Methods section. AhR+/+ and AhR−/− CGC cultures were treated with 250 nM TCDD for 30 min and caspase 3 activity was measured immediately after TCDD incubation and after 1, 6 or 24 h of additional incubation without TCDD. Staurosporine (incubation time of 24 h) was used as a positive control of caspase 3 activity. The experiments were carried out, at least, in three different cultures. Data shown are mean ± SD (n ≥ 6). *p < 0.05 (different from control).

To further complete the analysis of the cell death process induced by TCDD in CGCs, we measured ROS generation by using a dichlorofluorescein dye that fluoresces after intense oxidation. TCDD treatment of AhR+/+ CGCs induced a significant level of ROS generation (Fig. 6a and b). The quantification of fluorescence positive cells indicated that 75–80% of CGCs generated significant ROS levels after TCDD incubation. On the contrary, we could not observe ROS generation in cultured AhR−/− CGCs (Fig. 6a and b). A positive test of ROS generation was carried out by incubation of both cell types with 250 μM zinc for 6 h; it was observed that zinc readily increased ROS content in both wild-type and AhR-null neurons (Fig. 6a and b). These results suggest that ROS generation could play an important role in TCDD-dependent apoptosis in cerebellar granule cells.

Figure 6.

 TCDD induces reactive oxygen species generation in cerebellar granule cells. (a) AhR+/+ and AhR−/− CGC cultures were treated with 250 μM TCDD for 30 min and ROS generation was monitored by using a dichlorofluorescein dye as described in the Methods section. Zinc was added as a positive control of ROS generation. CGCs were incubated with 250 μM zinc for 6 h. The experiments were carried out, at least, in three different cultures. (b) Percentage of fluorescent cells after TCDD and zinc incubation using the data from panel a. Data shown are mean ± SD (n ≥ 6), and at least 500 cells were counted per experimental condition. Scale bar 40 μm.

TCDD induces CYP1A2 expression in cerebellar granule cells

It is well established that TCDD induces the expression of cytochrome P450 (CYP) enzymes with detoxifying activities (Hankinson 1995). In addition, the mechanisms of TCDD-induced environmental toxicity or cancer have been related to CYP gene expression induced by dioxin through AhR-dependent transcriptional activity (Nebert et al. 1996, 2004). Thus, we examined the mRNA expression of cytochrome P450 1A2 (CYP1A2), which is expressed in the brain, in CGCs. Figure 7 shows that TCDD incubation of AhR+/+ CGCs at 250 nM concentration for 30 min induced the expression of CYP1A2 above the low levels of control, untreated CGCs. CYP1A2 expression reached its maximum 6 h after TCDD incubation to decrease to basal TCDD-induced levels 24 h after treatment.

Figure 7.

 TCDD induces the mRNA expression of CYP1A2 in cerebellar granule cells. AhR+/+ CGC cultures were treated with 250 nM TCDD for 30 min and CYP1A2 expression measured by real time RT-PCR as described in the Methods section. CYP1A2 expression was analyzed after TCDD incubation and after 6 or 24 h of additional incubation without TCDD. The experiments were carried out, at least, in three different cultures. Data shown are mean ± SD (n ≥ 6). *p < 0.05 (different from control).

Discussion

2,3,7,8-Tetrachlorodibenzo-p-dioxin is a prototypical environmental contaminant with highly toxic effects. Its accumulation in the organs of the rat has been analyzed (Pohjanvirta et al. 1990) and the results indicate that the liver is the major site of accumulation, although TCDD can be also detected in other organs, the brain among them. The neurotoxic properties of TCDD have been also analyzed during the last three decades. Its effects in neurodevelopment and behavior are of high interest, and the studies performed have been mostly focused on its detrimental action in development and behavior of animals and humans that were exposed to this toxin during the gestational period (Schantz and Bowman 1989; Rogan and Gladen 1992; Schantz et al. 1992; Jacobson and Jacobson 1996, 1997a; b). Recent studies have been performed to analyze the effect of fetal exposure to TCDD on male reproductive system of the rat (Bell et al. 2007a,b). Interestingly, acute versus chronic dosing of TCDD has been shown to modulate the potency of dioxin in inducing adverse effects (Bell et al. 2010). Thus, a single exposure to TCDD during the prenatal period alters the myelination and gliogenic potential of the mature CNS (Fernández et al. 2010). However, the mechanisms of TCDD-induced neurotoxicity, the signaling pathways involved and the molecular targets in neurons are still to be defined.

To know the molecular mechanisms of TCDD toxicity, different studies have used cultured neurons, for example, cerebellar granule cells and cortical neurons. Those studies have linked TCDD with protein kinase C activity through a mechanism translocating PKC from the cytosol to the plasma membrane via the receptor for activated C kinase-1 (Lee et al. 2007). Glutamate receptors of the N-methyl-d-aspartate subtype have been also implicated by increasing the intracellular calcium concentration (Kim and Yang 2005; Lin et al. 2008). An increase in [Ca2+]i was also observed in neuroblastoma cells after TCDD exposure (Sul et al. 2009) whereas calcium signaling enhances AhR-mediated gene expression and increases dioxin neurotoxicity in cortical neurons (Lin et al. 2008). Consistently, TCDD enhances NMDA excitotoxicity whereas AhR knockdown by siRNA reduces NMDA excitotoxicity (Lin et al. 2009). The role of AhR in TCDD toxicity through its interaction with glutamate receptors is an interesting hypothesis to explain the molecular mechanisms of TCDD toxicity in neuronal cells, although further studies need to be made to fully understand the process.

We have previously showed that TCDD induces apoptosis in NGF-differentiated PC12 cells (Sánchez-Martín et al. 2010a), albeit the contribution of AhR to such process appeared to be minor. Interestingly, the role of TCDD as an inducer of apoptosis is a matter of discussion with paradoxical effects in non-neural cells suggesting that TCDD binding makes AhR competent to interact with different signaling pathways (reviewed in Puga et al. 2009). The effect of TCDD on apoptosis is a very important and contradictory issue because dioxin acts as a tumor promoter in several cell types after binding to AhR, possibly by inhibition of apoptosis (Schwarz et al. 2000), while it has been demonstrated to induce apoptosis in thymocytes (McConkey et al. 1988; Kurl et al. 1993). This work shows that AhR has an important role in TCDD-induced apoptosis in cerebellar granule cells as cultured neurons from AhR knockout mouse did not show any significant cell death after acute TCDD exposure. In addition, antagonists of the Ah receptor resveratrol and α-naphtoflavone fully protected against apoptosis induction in AhR+/+ CGC cultures.

The characterization of TCDD toxicity in CGCs as an apoptotic process is supported by nuclear fragmentation, increased caspase 3 activity and DNA laddering. Furthermore, the effects induced by TCDD are qualitatively similar to that exerted by staurosporine, a prototypical molecule of apoptosis induction (Weil et al. 1996). Together, these data demonstrate that cerebellar granule cells suffer apoptosis after TCDD treatment. In support to our hypothesis, a significant ROS generation after TCDD incubation was measured in our CGC cultures which also agrees with previous studies using CGCs (Kim and Yang 2005) or hepatocytes (Aly and Doménech 2009).

Cytochrome P450 expression was found in rat brain and members of the CYP1 family have been located in the cerebellum (Warner et al. 1988). We have previously shown that CYP1A2 induction by benzo-[a]-pyrene is affected by maturation of mouse cerebellar granule cells (Mulero-Navarro et al. 2003). The down-regulation of CYP1A2 was related in that study to an increase in nitric oxide that accumulated as a result of the activity of the neuronal nitric oxide synthase. Thus, we proposed that neurodegeneration could diminish the induction of specific CYP450s, impairing the metabolism of toxic chemicals in the CNS (Mulero-Navarro et al. 2003). The role of the Ah receptor-mediated induction of CYP1 enzymes in environmental toxicity and cancer has been previously discussed (Nebert et al. 2004). Although CYP1 enzymes were first thought to be beneficial because of their detoxification activity (Hankinson 1995), additional experimental evidences revealed a detrimental effect of CYP1 enzymes in TCDD-induced toxicity and cancer through a mechanism linking their increased gene expression with enhanced AhR transcriptional activity (Nebert et al. 1996, 2004). The results obtained in the present work suggest the possibility of a link between TCDD toxicity, apoptotic neuronal cell death and CYP1A2 induction. Additional experiments need to be performed to asses this point.

In conclusion, the results presented in this paper show that TCDD-induced cell death in cerebellar granule cells takes place through an apoptotic process. AhR plays a critical role in this process of TCDD-induced toxicity. Taken together, these findings suggest that induction of apoptosis plays an important role in TCDD-induced neurotoxicity.

Acknowledgements

This work was supported by a Grant from the Junta de Extremadura, Spain (PRI07A019 to J.M. Merino) and from the Red Temática de Investigación Cooperativa en Cáncer (RTICC) (RD 06/020/1016, Fondo de Investigaciones Sanitarias (FIS), Carlos III Institute, Spanish Ministry of Health, to P.M.F.-S.). F.J. Sánchez-Martín was the recipient of a predoctoral fellowship from the Junta de Extremadura (Spain). All Spanish funding is co-sponsored by the European Union FEDER program.

Conflict of interests

The authors declare that there are no conflicts of interests.

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