Styrene 7,8-oxide induces caspase activation and regular DNA fragmentation in neuronal cells

Brain Research 933 (2002) 12–22 www.elsevier.com / locate / bres

Research report

Styrene 7,8-oxide induces caspase activation and regular DNA fragmentation in neuronal cells Elisabetta Dare´ a , Roshan Tofighi a , Maria Vittoria Vettori b , Takashi Momoi c , Diana Poli b , Takaomi C. Saido d , Antonio Mutti b , Sandra Ceccatelli a , * a

The National Institute of Environmental Medicine, Division of Toxicology and Neurotoxicology, Karolinska Institutet, Box 210, S-171 77 Stockholm, Sweden b Laboratory of Industrial Toxicology, Istituto di Clinica Medica e di Nefrologia, Universita´ di Parma, Via A. Gramsci, 14, I-43100 Parma, Italy c Division of Development and Differentiation, National Institute of Neuroscience, NCNP, Kodaira, Tokyo, Japan d Laboratory for Proteolytic Neuroscience, RIXEN Brain Science Institute, 2 -1 Hirosawa, Waho-shi, Saitama, 351 -0198, Japan Accepted 22 November 2001

Abstract Neurobehavioral changes have been described in workers occupationally exposed to styrene vapors. Alterations of neurotransmitters and loss of neurons have been observed in brains of styrene-exposed rats. However, the mechanisms of neuronal damage are not yet clearly understood. We have characterized the cellular alterations induced by the main reactive intermediate of styrene metabolism, styrene 7,8-oxide (SO) in the human neuroblastoma SK-N-MC cell line and primary culture of rat cerebellar granule cells (CGC). SK-N-MC cells exposed to SO (0.3–1 mM) displayed apoptotic morphology, together with chromatin condensation and DNA cleavage into high molecular weight fragments of regular size. These features were accompanied by the activation of class II caspases, as detected with the DEVD assay, by following the cleavage of the caspase-substrate poly (ADP-ribose) polymerase (PARP) and by detection of the active fragment of caspase-3. Pre-incubation of the cells with the caspase inhibitor z-VAD-fmk reduced the cellular damage induced by SO, suggesting that caspases play an important role in SO toxicity. Increased proteolysis by class II caspases was detected also in primary culture of CGC exposed to SO. In addition, the presence of the 150-kDa cleavage product of a-fodrin suggests a possible activation of calpains in SK-N-MC cells. Moreover, SO did not affect the level of expression of the p53 protein, even though it is known to cause DNA damage. The identified intracellular pathways affected by SO exposure provides end-points that can be used in future studies for the evaluation of the neurotoxic effect of styrene in vivo.  2002 Elsevier Science B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Neurotoxicity Keywords: Styrene oxide; Neuronal apoptosis; DNA fragmentation; Caspase activity; Calpains

1. Introduction Styrene monomer remains one of the most important chemicals used world-wide in several applications, including polyester resins, plastics, latex paints and coatings, and synthetic rubbers. In humans, more than 80% of inhaled styrene is taken up and undergoes bioactivation to styrene 7,8-oxide (SO) by cytochrome P450 mono-oxygenases, mainly by the isoforms 2E1 and 2B6 [7,34]. SO acts as a

*Corresponding author. Tel.: 146-8-728-7586; fax: 146-8-329-041. E-mail address: [email protected] (S. Ceccatelli).

DNA-alkylating agent causing single-strand breaks to occur [1]. SO is a proven animal carcinogen and is classified as a possible human carcinogen (group 2B) by IARC [16]. There is consistent evidence indicating that styrene exposure is associated with neurological disorders [17]. Neurotoxic effects that have been related to occupational exposure to styrene include electroencephalographic alterations, peripheral neuropathy with decreased nerve conduction velocities, and sensory involvement, mainly ototoxicity and acquired dyschromatopsia [9,10,12,49]. Neurobehavioral effects are characterized by reduced vigilance and attention, resulting in impaired performance in several

0006-8993 / 02 / $ – see front matter  2002 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 02 )02274-6

E. Dare´ et al. / Brain Research 933 (2002) 12 – 22

neurobehavioral tasks, including reaction times, learning and memory tests [30]. Styrene is known to alter neurotransmission in the brain [15,20]. Decreased levels of dopamine in rabbit brain tissue and impairment of the dopamine transport in rat striatal synaptic vesicles have been reported [4,28,29], suggesting a possible dopamine-mediated effect of styrene neurotoxicity. In rats sub-chronically exposed to styrene vapors, a reduced density of retinal amacrine cells associated with dopamine depletion confirmed the vulnerability of dopaminergic systems to styrene toxicity, providing some insights on the possible mechanism of loss in chromatic discrimination recorded among workers exposed to styrene [46]. Increased levels of prolactin in styreneexposed workers have also been interpreted as an impaired tubero-infundibular dopaminergic modulation of pituitary secretion [2,32]. Moreover, neuronal loss has been observed in sensory-, motor cortex, and hippocampus of rats chronically exposed to styrene [38]. Studies performed with non-neuronal cell models have indicated that the metabolite SO can induce DNA fragmentation, sister chromatid exchanges, inhibition of cell growth and activation of DNA repair mechanisms [1]. In addition to these effects, decrease in intracellular glutathione (GSH) and depletion of adenosine triphosphate (ATP) have also been demonstrated after exposure to SO [8,18]. Similarly to styrene, SO can cross the blood / brain barrier. SO and styrene have been shown to be acutely cytotoxic for primary cultures of motor and sensory neurons [21]. However, at present scant information is available on the mechanisms of SO-induced neuronal damage. Exposure to toxic chemicals often induces cell death through the apoptotic program, which assures ordered dismantling of the cells [19,37]. Hallmarks of apoptosis are cell shrinkage, plasma membrane integrity, chromatin condensation, specific patterns of DNA fragmentation and proteolytic cleavage. Caspases, a family of cysteine-proteases cleaving after aspartate residues, have been found implicated in apoptosis [44]. To clarify intracellular events involved in SO neuronal cell death we have used the human neuroblastoma SK-NMC cell line and primary cultures of rat cerebellar granule cells (CGC) as in vitro models. Cells exposed to SO were analyzed according to the typical parameters of apoptosis to find end-points related to cellular damage. In addition, to evaluate the persistence of SO during the exposure, the concentration of SO in aliquots of the medium taken at different time points was measured.

2. Materials and methods

2.1. Chemicals All chemicals used were of analytical grade. Styrene

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oxide (SO) was purchased from Sigma–Aldrich (Stockholm, Sweden). SO stock solutions were prepared in DMSO, whereas further dilutions were made in water. Trypan blue (T.B.), propidium iodide (P.I.), staurosporine, fetal calf serum (FCS), cytosine arabinoside and poly-Llysine (MW 300 000) were purchased from Sigma (St Louis, MO, USA). All other chemicals for cell culture were supplied by Life Technologies (Life Technologies, Gibco BRL, Grand Island, NY, USA). The caspase substrate DEVD-MCA [Ac-Asp-Glu-Val-Asp-a-(4-methylcoumaryl-7-amide)] and the caspase inhibitor z-VAD-fmk [Z-Val-Ala-Asp(Ome)-FMK] were purchased from Peptide Institute (Osaka, Japan). The Calpain Inhibitor I (C.I. I) was obtained from Roche (Bromma, Sweden).

2.2. Antibodies 2.2.1. Primary antibodies Rabbit polyclonal antibody against the large fragment of PARP (89 kDa) (New England Biolabs Inc, Beverly, MA, USA), working dilution 1 / 2000; rabbit polyclonal antibody against p11 / p20 fragments of caspase-3 and procaspase-3 (Santa Cruz Biotechnology Inc, SDS, Sweden), working dilution 1 / 500; rabbit polyclonal antibody against the active fragment of caspase-3, p17 [22,45], working dilution 1 / 200; mouse monoclonal antibody to a-fodrin (Affiniti, Mamhead Exeter, UK), working dilution 1 / 1000; rabbit polyclonal antibody to the 150-kDa fragment of a-fodrin produced specifically by calpain cleavage [39], working dilution 1 / 400; and mouse monoclonal antibody to p53 protein (DO-1) (Novocastra Laboratories Ltd, Newcastle upon Tyne, UK), working dilution 1 / 1000. 2.2.2. Secondary antibodies Goat anti rabbit IgG, peroxidase conjugated (Pierce, Rockford, IL, USA), working dilution 1 / 10 000; goat anti mouse IgG, horseradish peroxidase conjugated (Pierce, Rockford, IL, USA), working dilution 1 / 10 000; and FITC-conjugated secondary antibody (Amersham, Little Chalfont, Bucks, UK), working dilution 1 / 40. For the immunoblotting, the antibodies were diluted in a solution containing 1% BSA in HSBT (0.05% Tween-20, 500 mM NaCl, 50 mM Tris–HCl, pH 7.5), except for the p53 antibody, which was diluted in 5% non-fat dry milk in HSBT. 2.3. Cell culture, exposure to toxic agents and harvesting of cells The human neuroblastoma SK-N-MC clonal cell line was purchased from the American Type Culture Collection (Manassas, VA, USA). Cells were routinely seeded at the density of 40 000 cells / cm 2 in CO 2 -independent medium supplemented with 10% fetal calf serum (FCS) (Sigma, St Louis, MO, USA), 4 mM L-glutamine, 100 U / ml penicillin and 100 mg / ml streptomycin. The cell culture flasks were

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locked and the cells were incubated at 37 8C for 24 h before the treatments. Cerebellar granule cells (CGC) were prepared from rat pups on postnatal day 7 as previously described [6,41]. Pregnant Sprague–Dawley rats (B&K, Stockholm, Sweden) were housed singly and checked twice daily. Animals were kept in air-conditioned quarters, with a controlled photoperiod (14 h light, 10 h darkness) and free access to food and tap water. Procedures used in animal experimentation comply with the Karolinska Institute’s regulations for the care and use of laboratory animals. In brief, the cerebella from rat pups were dissected, minced with a McIlwain tissue chopper (Histo-Lab, Gothenburg, Sweden), dissociated with trypsin and seeded on dishes (or glass coverslips for microscopic analysis) coated with poly-L-lysine at a density of 500 000 cells / cm 2 . Cells were maintained in basal Eagle’s medium supplemented with 10% FCS, 25 mM potassium chloride and 0.5% (v / v) penicillin–streptomycin. To prevent growth of glial cells 10 mM cytosine arabinoside was added to the cultures 40 h after seeding. The cells were left for 7 days in culture to differentiate before exposure to SO. The range of 0.3–1 mM was chosen to mimic the human occupational exposure [29,31,40,46]. SO was directly added to the conditioned medium. The concentration of SO in the medium never reached 1%. During the exposure to SO, the bottles and dishes with the cells were kept in a sealed exposure chamber at 37 8C. The caspase inhibitor z-VAD-fmk or the calpain inhibitor C.I. I was added to the medium 30 min before SO and left in the culture for the entire exposure period. For harvesting, floating cells collected from the medium by centrifugation were pooled together with cells detached by scraping or with trypsin.

2.4. Trypan blue exclusion test Cells were harvested with trypsin as described in the previous section, and then an aliquot of the cell suspension was mixed with an equal volume of 0.4% Trypan blue in PBS. Cells were scored at the phase contrast microscope using a Neubauer improved counting chamber. Cells with damaged cell membrane stained blue (dead cells), while cells with plasma membrane integrity prevented the dye entry remaining unstained (healthy cells and apoptotic cells). The experiments were performed in triplicate and repeated twice.

2.5. Nuclear staining with propidium iodide Cells were grown on coverslips inside the exposure chamber, treated with SO, then fixed in ice-cold methanol / water (8 / 25v / v), at 220 8C for 30 min. Each coverslip was then washed with PBS. After washing with PBS, the coverslips were stained with P.I. (2.5 mg / ml in PBS) for 5 min, and mounted onto glass slides with PBS / glycerol

(1:9, v / v) containing 0.1% (w / v) phenylenediamine. Stained cells were analyzed with the fluorescence microscope (Olympus BX60). The smaller size, irregular shape and higher intensity of chromatin stained with P.I. identified apoptotic nuclei. Images of the nuclei were obtained with the C-4742-95-10sc digital camera (Hamamatsu Photonics Norden AB, Solna, Sweden).

2.6. Detection of high molecular weight ( HMW)-DNA fragments by pulse field gel electrophoresis ( FIGE) To monitor the formation of large size DNA fragments, field inversion gel electrophoresis (FIGE) was performed according to a method previously described [50]. Briefly, cells were harvested with trypsin, centrifuged at 1503g for 5 min and washed with PBS. Equal amounts of cells per sample (1310 6 ) were immobilized into agarose plugs and subjected to proteinase K digestion prior to loading into agarose gels. Two sets of pulse markers DNA were used for determination of molecular weights: (i) chromosomes from Saccharomyces cerevisiae (225–2200 kbp); and (ii) a mixture of l-DNA, l-Hind III fragments, and l-DNA concatemers (0.1–200 kbp) purchased from Sigma (St Louis, MO). The gels were stained with ethidium bromide (50 mg / l) to visualize the DNA and photographed on a 305-nm UV-transilluminator with Polaroid 665 positive / negative films.

2.7. Measurements of caspase II-like activity ( DEVDase assay) Caspases are divided into three classes, based on the substrate-specificity. A fluorogenic assay which evaluates cleavage of the substrate DEVD can be used to evaluate the activity of class II caspases (2, 3 and 7), which are considered the main executioner caspases [13,14,36,44]. Cells were harvested by scraping. The capability of cell extracts to cleave off the substrate DEVD-MCA, leading to the release of free 4-methyl-coumaryl-7-amide (excitation 355 nm, emission 460 nm), was monitored at 37 8C using a Fluoroskan II (Labsystem AB, Stockholm, Sweden), as previously described [13]. Fluorescent units were converted to pmoles of 4-methyl-coumaryl-7-amide released using a standard curve generated with 4-methyl-coumaryl7-amide and subsequently related to protein content. The measurements were performed in triplicate and the experiments were repeated two times.

2.8. Protein extraction, SDS–PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) and immunoblotting SK-N-MC cells were harvested with trypsin, centrifuged and washed with PBS. The cells were sonicated in a solution containing 1 mM Pefablock (Boehringer Mannheim, Bromma, Sweden), 10 mM EDTA and 2 mM DTT

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in PBS. Proteins were quantified with the micro-BCA protein assay reagent kit (Pierce, Rockford, IL, USA). Protein extracts were mixed with gel loading buffer (final concentration: 0.4% SDS, 4% glycerol, 1% b-mercaptoethanol, 12.5 mM Tris–HCl, pH 6.8) and boiled for 5 min. The proteins were separated by SDS–PAGE in Tris– glycine buffer, pH 8.3 [23]. Equivalent amounts of proteins (25 mg) were loaded in the wells. The proteins were then electroblotted onto nitrocellulose membranes at 80 V for 1–2 h using as transfer buffer 20% (v / v) methanol, 186 mM glycine, 26 mM Tris, pH 8.3. The blots were blocked with a solution containing 5% non-fat dry milk, 1% BSA in HSB (500 mM NaCl, 50 mM Tris–HCl, pH 7.5), at 4 8C overnight. The membranes were then incubated with a primary antibody at room temperature for 1 h. The incubation with the p53 antibody was performed at 4 8C for 15 h. After washing three times for 15 min with HSBT, the blots were incubated with a secondary antibody at room temperature for 1 h. They were then washed again three times as described above, rinsed twice for 5 min with 150 mM NaCl, 50 mM Tris, pH 7.5, incubated with ECL reagents for chemiluminescence (Amersham, Little Chalfont, Bucks, UK) and exposed to X-ray autoradiography films (Fuji, Japan).

2.9. Immunocytochemistry SK-N-MC cells grown on coverslips were fixed with 4% paraformaldehyde for 1 h at 4 8C. The coverslips were incubated with a polyclonal antibody against the active fragment of caspase-3, p17 diluted 1 / 200 in PBS supplemented with 0.3% Triton X-100 and 0.5% BSA, in a humid chamber at 4 8C overnight. The cells were then rinsed with PBS, and incubated with a FITC-conjugated secondary antibody diluted in PBS containing 0.3% Triton X-100 for 30 min at room temperature. After three rinses with PBS, coverslips were mounted in glycerol–PBS containing 0.1% phenylenediamine. The cells were then examined with an Olympus BX60 fluorescence microscope. Images were collected with the C4742-95-10sc digital camera.

2.10. Measurement of the SO concentration in the conditioned culture medium The actual concentration of volatile chemicals in exposure medium results from the interaction among a number of complex phenomena, including the reactivity of the test substance with components of the medium, its partition coefficients, and its relative volatility versus that of the solvent. In our experiments, cells were exposed to the nominal concentration of 0.3 mM SO in closed bottles at 37 8C, as described above. The actual concentration of SO was measured in the medium of cells after exposure for 0, 8, 16, and 24 h. Experiments were performed in duplicate and two samples were collected from flasks at each time

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point. Aliquots of conditioned medium (1 ml) were transferred into 4-ml vials containing 0.5 g NaCl, a stirring bar, and 2 ml of a 100 ppm deuterate styrene solution (Sigma, St Louis, MO, USA). The vials were sealed immediately with Teflon-lined septa and hole caps (Supelco, Bellafonte, PA, USA). The samples were stored at 280 8C until analysis. Headspace-SPME studies were carried out using a 85 mm Polyacrylate fiber (Supelco, Bellafonte, PA, USA). Extraction procedures were performed with an autosampler at 70 8C for 20 min stirring. Before the analysis the samples were thawed and analyzed after 10 min at room temperature. After sampling, the fiber was immediately inserted into the gas chromatograph (GC) injection port for thermal desorption (5 min at 280 8C). GC–MS analysis was carried out on a Hewlett Packard HP 6890 GC coupled to a HP 5973 Mass Selective Detector (Palo Alto, CA, USA). The analytes were separated on a HP-5MS column (30 m30.25 mm, 0.25 mm film) (Palo Alto, CA, USA). Hydrogen was used as carrier gas. The temperature program was: 100 8C hold 5 min, 10 8C / min to 120 8C, hold 1 min. The temperatures of injector and MS detector were both 280 8C. MS acquisition was performed in SIM. The percentage decrease in SO concentration was calculated from the ratio between the peak area of the internal standard (deuterate styrene) and the peak area of styrene oxide. Values were obtained from the arithmetical average of four different measurements and the associated error was the propagation of the average standard deviation on the experimental data. The fit was performed with the program Origin 6.0 (Microcal, Northampton, MA, USA) using the Levenberg–Marquardt algorithm.

3. Results

3.1. Evaluation of cell membrane permeability by Trypan blue staining A time- and dose-dependent cytotoxicity was detected in SK-N-MC cells exposed to 0.1–1 mM SO using Trypan blue staining. SK-N-MC cells exposed to 0.3–1 mM SO displayed a significant increase in permeability to Trypan blue already after 8 h, indicating damaged plasma membrane (Fig. 1A). Furthermore, SO induced a significant decrease in the total number of cells at all of the doses tested (Fig. 1B).

3.2. Morphological alterations induced by SO SK-N-MC cells exposed to SO for 24 h displayed abnormal morphology characterized by cell shrinkage (Fig. 2). Moreover, nuclear staining of fixed cells with propidium iodide revealed the presence of chromatin condensation and convolution of the nuclear outline, as well as

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Fig. 1. SO-induced alterations of membrane permeability and total cell number. SK-N-MC cells exposed to SO were detached from the surface, then aliquots of the cell suspension were stained with Trypan blue and counted at the microscope. (A) Percentage of cells impermeable to Trypan blue. (B) Total cell number. Values are means6S.E.M. of three independent determinations. Statistical analysis of the values obtained at each time point was performed with ANOVA followed by Tukey–Kramer multiple comparison test. *Significantly different from control (P,0.05). **Significantly different from control (P,0.01). [ Significantly different from control (P,0.0001).

nuclear fragmentation (Fig. 2). These morphological features indicated the occurrence of apoptosis.

3.3. Detection of DNA fragmentation by FIGE During apoptosis the DNA is typically cleaved by endonucleases into fragments of regular sizes [47,48]. Exposure to 0.3–1 mM SO for 16 h induced generation of high molecular weight (HMW) DNA fragments of 700-, 300- and 50-kDa in SK-N-MC cells (Fig. 3). The pattern of DNA fragmentation was similar to that generated by

staurosporine, an inhibitor of protein kinase C that has been shown to induce neuronal apoptosis [35]. However, no increase in DNA fragmentation was detectable after incubating the cells with 0.1 mM SO for either 16 or 24 h (Fig. 3).

3.4. Analysis of caspase activation Activation of caspases is a typical feature of apoptosis, resulting in cleavage of a wide variety of intracellular polypeptides [43]. The enzyme poly (ADP-ribose) poly-

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Fig. 2. Morphological features of SK-N-MC cells exposed to SO. Cell shrinkage was observed with the phase contrast microscope after incubation with 0.3 mM SO for 20 h (top panels). The nuclei of SO-treated cells stained with P.I. displayed chromatin condensation (bottom panels).

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merase (PARP) is a target for caspase proteolysis. The fragment of 89 kDa produced by specific cleavage of PARP by caspases can be used as a useful marker for evaluating caspase activation. This fragment was hardly detectable in control cells by immunoblotting, whereas its level increased in protein extracts obtained after exposure of SK-N-MC cells to 0.3–1 mM SO for 16 h (Fig. 4A), or to 0.05–0.1 mM SO for 20 h (Fig. 4B). To further confirm the activation of caspases by SO, we measured the caspase activity in extracts obtained from SK-N-MC cells. The analysis revealed that 0.05–1 mM SO induced a significant increase in DEVDase activity compared to control, with the strongest activation observed after exposure to 0.3 mM SO for 20 h (Fig. 5A). To verify whether this pathway was relevant in primary neurons, the caspase activity was also measured in primary culture of rat CGC exposed to SO. As shown in Fig. 5B, exposure to 1 mM SO for 16–24 h induced a significant increase in DEVDase activity in CGC. Pro-caspase 3 (32 kDa) cleavage generates the active p17 / p20 fragment. Cells positively stained for p17 were detected by immunocytochemistry after exposure to SO for 16 h (Fig. 6A). Immunoblotting of protein extracts obtained from cells exposed to 0.3–1 mM SO for 16 h confirmed the cleavage of pro-caspase 3 into activated caspase 3 (Fig. 6C). Pre-incubation of SK-N-MC cells with the pan-caspase inhibitor z-VAD-fmk (20 mM) 30 min prior to exposure to SO significantly reduced the percentage of Trypan blue permeable-cells induced by either 0.1 or 1 mM SO (Fig. 7). Thus, inhibition of caspases protected the cells, suggesting that the proteolysis due to caspases might be crucial in the process of cell death induced by SO.

3.5. Evaluation of cleavage of the cytoskeletal protein a -fodrin The cytoskeletal protein a-fodrin is a substrate for both caspases and calpains, which cleave it into distinct

Fig. 3. Analysis of HMW DNA-fragments by FIGE. The picture shows the DNA stained with ethidium bromide after agarose gel separation. Lane 1, control, 16 h; lane 2, 0.25 mM staurosporine, 16 h; lane 3, 0.1 mM SO, 16 h; lane 4, 0.3 mM SO, 16 h; lane 5, 0.5 mM SO, 16 h; lane 6, 1 mM SO, 16 h; lane 7, molecular weight markers, chromosomes from Saccharomyces cerevisiae; lane 8, control, 24 h; lane 9, 0.25 mM staurosporine, 24 h; lane 10, 0.1 mM SO, 24 h; and lane 11, 0.3 mM SO, 24 h.

Fig. 4. Caspase-mediated PARP cleavage in SK-N-MC cells exposed to SO. The 89-kDa PARP fragment produced by caspase cleavage was detected by immunoblotting using protein extract obtained from cells exposed to SO for either 16 h (A) or 20 h (B).

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Fig. 5. Exposure to SO increased caspase 3-like activity in neuronal cells. The activity was measured in cell extracts obtained either from SK-N-MC cells (A) or from CGC (B), using the fluorogenic DEVDase assay. Values are the means6S.E.M. of three determinations. Statistical analysis was performed with one-way ANOVA. For each dose, values obtained at different time points were compared to non-exposed control using the Dunnet multiple comparison test. *Significantly different from nontreated control (P,0.01).

proteolytic fragments of 120 and 150 kDa, respectively [35]. Analysis by Western blotting of SK-N-MC cells revealed that 0.3–1 mM SO for 16 h induced an increase in both breakdown products (Fig. 8A). Activation of calpain was confirmed using a polyclonal antibody specific for the 150-kDa fragment [35,39] generated by calpain cleavage (Fig. 8B). As expected, a lower level of the 150-kDa fragment was found in cells incubated with SO in the presence of 20 mM Calpain Inhibitor I. The analysis of a-fodrin cleavage, besides confirming SO-mediated activation of caspases, suggested that calpains also contribute to the proteolysis of cellular components.

3.6. Evaluation of p53 expression by immunoblotting The p53 transcription factor, which is involved in the regulation of DNA repair, is enhanced by DNA damage.

Fig. 6. Detection of active caspase-3 in SK-N-MC cells exposed to SO. The presence of the active subunit of caspase 3 was evaluated in cells exposed to 0.3 mM SO for 16 h (A) and in non-exposed control cells (B) using immunocytochemistry, and in cell extracts by Western blotting (panel C).

Increased expression of the p53 protein can induce cell cycle arrest or, alternatively, it can trigger apoptosis [27]. Since SO is known to cause DNA alterations, we have looked at the expression of p53 during the time that precedes apoptosis, that is when caspases are not yet activated. As shown in Fig. 8C, SK-N-MC cells exposed to 0.3–1 mM SO for 3–8 h did not display relevant changes in the p53 protein level, compared to control cells. The expression of p53 was also unchanged after exposure to 0.3 mM SO for 16 h, when hallmarks of apoptosis, such as DNA fragmentation and caspase activation, were already detectable.

3.7. Measurement of the SO concentration in the conditioned medium The nominal concentration of 0.3 mM was selected for this analysis, since it caused all the typical features of apoptosis, including the highest caspase activity. The actual concentration measured in the medium decreased progressively with time, reaching 11% of the initial

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Fig. 7. The caspase inhibitor z-VAD-fmk protected SK-N-MC cells from SO toxicity. The caspase inhibitor z-VAD-fmk was added to the medium 30 min before SO and left in the culture for the entire time. Cells were harvested, stained with Trypan blue and counted with the microscope. Statistical analysis was performed with ANOVA followed by Tukey–Kramer multiple comparison test. **Significantly different from control (P,0.001). [ Significantly different from 0.1 mM SO (P,0.01). *Significantly different from 1 mM SO (P,0.001).

Fig. 8. Western blot analysis of a-fodrin and p53 in SK-N-MC cells exposed to SO. Distinct proteolytic cleavage of a-fodrin into the 120- and 150-kDa break down products was observed by immunoblotting after exposure to SO for 16 h (A). The 150-kDa fragment produced by calpain cleavage, detected with a specific antibody [39], increased in cells exposed to SO (B). The presence of calpain inhibitor I (C.I. I) reduced a-fodrin proteolysis executed by calpain (B). The expression of the p53 protein was found unchanged both before and after caspase activation (C).

Fig. 9. Time-dependent decrease in SO concentration measured in the conditioned medium during the exposure of SK-N-MC cells. Cells were treated with the initial concentration of 0.3 mM SO, then SO was measured in aliquots of the medium at different time points. Values are means6S.D. of two determinations. The fitting function is a single exponential decay ( y 5 A 1 e 2kx). The calculated half-life of SO in this experimental model is 6.5 h.

administered dose after 24 h (Table 1). As shown in Fig. 9, the fit function was a single exponential decay. Even in the absence of cells, the half-life of SO in the medium was 6.5 h. The area under the curve corresponding to the first 24 h of exposure accounted for 0.109 mM, corresponding to 36% of the administered dose.

Table 1 Time-dependent decrease in SO concentration in the conditioned medium Time of exposure (h)

SO concentration (% of nominal value)

4. Discussion

0 8 16 24

10066 42.7263.7 21.5561.61 11.4761.07

Although previous studies have shown that SO is cytotoxic to neuronal cells in culture [21], the intracellular mechanisms leading to cell death have not been clarified. Here we demonstrate that neuronal cells exposed to nominal concentration of SO ranging from 0.05 to 1 mM die through apoptotic pathways. Our data indicate that actual doses are much lower than nominal levels. SO concentrations measured in the medium decreased over

SK-N-MC cells were exposed to 0.3 mM SO by adding the stock solution to the conditioned medium, then the actual SO concentration was measured in aliquots of the medium collected at different time points. Values are means6S.D. of the values obtained from three independent cultures.

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time, with the area under the curve during the first 24 h accounting for 0.109 mM or 36% of the administered dose (0.3 mM). Such critical levels (lowest effective concentrations) are consistent with the hypothesis of styreneinduced neurotoxic effects after long-term low level exposure in vivo both in occupationally exposed workers and experimental animals. SK-N-MC cells exposed to SO exhibited morphological signs, such as cell shrinkage and chromatin condensation, which are characteristic of apoptosis. SO also induced cleavage of DNA into HMW-fragments of regular size, an apoptotic feature that had been previously observed in human blood exposed to SO in vitro [26,33]. Our data confirm that DNA is a target for SO toxicity. The presence of fragments with defined molecular weights (700-, 300and 50-kbp) in SO-exposed SK-N-MC cells suggests that DNA cleavage is achieved through non-random cuts due to activation of specific endonucleases [47]. SO is known to alkylate DNA, causing single-strand breaks to occur. Moreover, SO binds to SH groups, determining glutathione depletion and thereby sensitizing cells to oxidative stress, as shown by increased levels of lipid peroxidation [3,8,18]. Thus, additional DNA damage could arise from DNA oxidation secondary to glutathione depletion. SK-N-MC cells exposed to SO exhibited activation of caspases, which is often associated with apoptotic cell death. Following caspase activation, specific caspase-activated DNases (CAD) can execute DNA fragmentation [11,33]. However, at concentrations of SO50.1 mM, no DNA fragmentation was detectable by FIGE, in spite of the increase in caspase activity shown by the specific proteolysis of PARP and by the DEVDase assay. It is possible that HMW-DNA fragments were present at levels below the detection limit of FIGE. Alternatively, DNA fragmentation might have been delayed. Activation of caspases emerges as a key mechanism in the process of neuronal cell death induced by SO in SK-N-MC cells. The importance of this finding is strengthened by the observation that also increases caspase activity in primary cultures of CGC. Thus, caspase activity appears as a relevant marker for SO neurotoxicity. Furthermore, SK-N-MC cells could be protected, at least partially, by pre-incubation with the caspase inhibitor z-VAD-fmk, suggesting that activation of caspases is a critical event in the cascade leading to cellular damage. Apoptosis requires ATP [25]. Since previous studies have indicated that 1 mM SO induces a decrease in ATP in PC12 cells, it is possible to speculate that at this dose of SO, depletion of ATP also occurred in SK-N-MC. This is consistent with the finding that at the highest dose we have observed more necrotic cells and less caspase activity. The products of a-fodrin proteolysis were increased in SK-N-MC cells exposed to SO. The cleavage of certain cytoskeletal proteins, such as a-fodrin, can be executed by both caspases and calpains and may contribute to the apoptotic morphology of the cell [5]. Calpains are calcium-

regulated proteases that have been found implicated in the process of cell death [5,42]. Based on previous studies showing SO-induced intracellular Ca 21 -overload [8], and on the specific pattern of a-fodrin cleavage observed in SK-N-MC cells exposed to SO, it is tempting to speculate that activation of calpains, proteases regulated by Ca 21 , occurs in our experimental model. A recent report on gene expression induced by SO in cultured human lymphocytes has suggested that SO can cause up-regulation of p53-mRNA [24]. An increased level of p53 could be the signal that induces either cell cycle arrest or entry into apoptosis after DNA damage [27]. In our experimental model the occurrence of apoptosis does not seem to follow a p53-dependent pathway, since the expression of the p53 protein was not modified after exposure to SO. In conclusion, we have shown that clonal SK-N-MC cells exposed to SO undergo apoptotic cell death with activation of caspases and DNA fragmentation at specific sites. Activation of class II caspases was also found in primary cultured CGC exposed to SO, suggesting that this family of proteases might play an important role in cell death induced by the main reactive metabolite of styrene. The mechanism-based end-points identified in our in vitro models might be useful for future in vivo studies.

Acknowledgements This study was supported by the QLK4-1999-01356 grant from the European Commission and the Swedish National Board for Laboratory Animals (CFN). The authors are grateful to Dr B. Zhivotovsky for technical help in setting up the analysis of DNA by FIGE.

References [1] R. Barale, The genetic toxicology of styrene and styrene oxide, Mutat. Res. 257 (1991) 107–126. [2] A. Bergamaschi, A. Smargiassi, A. Mutti, S. Cavazzini, M.V. Vettori, R. Alinovi, I. Franchini, D. Mergler, Peripheral markers of catecholaminergic dysfunction and symptoms of neurotoxicity among styrene-exposed workers, Int. Arch. Occup. Environ. Health 69 (1997) 209–214. [3] M. Byfalt Nordqvist, A. Lof, S. Osterman-Golkar, S.A. Walles, Covalent binding of styrene and styrene-7,8-oxide to plasma proteins, hemoglobin and DNA in the mouse, Chem. Biol. Interact. 55 (1985) 63–73. [4] S.K. Chakrabarti, Styrene and styrene oxide affect the transport of dopamine in purified rat striatal synaptic vesicles, Biochem. Biophys. Res. Commun. 255 (1999) 70–74. [5] S.L. Chan, M.P. Mattson, Caspase and calpain substrates: roles in synaptic plasticity and cell death, J. Neurosci. Res. 58 (1999) 167–190. [6] E. Dare, M.E. Gotz, B. Zhivotovsky, L. Manzo, S. Ceccatelli, Antioxidants J811 and 17beta-estradiol protect cerebellar granule cells from methylmercury-induced apoptotic cell death, J. Neurosci. Res. 62 (2000) 557–565.

E. Dare´ et al. / Brain Research 933 (2002) 12 – 22 [7] L.P. Delbressine, P.J. Van Bladeren, F.L. Smeets, F. Seutter-Berlage, Stereoselective oxidation of styrene to styrene oxide in rats as measured by mercapturic acid excretion, Xenobiotica 11 (1981) 589–594. [8] J.M. Dypbukt, L.G. Costa, L. Manzo, S. Orrenius, P. Nicotera, Cytotoxic and genotoxic effects of styrene-7,8-oxide in neuroadrenergic Pc 12 cells, Carcinogenesis 13 (1992) 417–424. [9] C. Edling, H. Anundi, G. Johanson, K. Nilsson, Increase in neuropsychiatric symptoms after occupational exposure to low levels of styrene, Br. J. Ind. Med. 50 (1993) 843–850. [10] T. Eguchi, R. Kishi, I. Harabuchi, J. Yuasa, Y. Arata, Y. Katakura, H. Miyake, Impaired colour discrimination among workers exposed to styrene: relevance of a urinary metabolite, Occup. Environ. Med. 52 (1995) 534–538. [11] M. Enari, H. Sakahira, H. Yokoyama, K. Okawa, A. Iwamatsu, S. Nagata, A caspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD, Nature 391 (1998) 43–50. [12] F. Gobba, F. Cavalleri, D. Bontadi, P. Torri, R. Dainese, Peripheral neuropathy in styrene-exposed workers, Scand. J. Work Environ. Health 21 (1995) 517–520. [13] A.M. Gorman, U.A. Hirt, S. Orrenius, S. Ceccatelli, Dexamethasone pre-treatment interferes with apoptotic death in glioma cells, Neuroscience 96 (2000) 417–425. [14] D.R. Green, J.C. Reed, Mitochondria and apoptosis, Science 281 (1998) 1309–1312. [15] R. Husain, S.P. Srivastava, M. Mushtaq, P.K. Seth, Effect of styrene on levels of serotonin, noradrenaline, dopamine and activity of acetyl cholinesterase and monoamine oxidase in rat brain, Toxicol. Lett. 7 (1980) 47–50. [16] IARC, Styrene-7,8-oxide, IARC Monogr. Eval. Carcinog. Risks Hum. 60 (1994) 321–346. ´ [17] D. Jegaden, J.F. Simon, M. Habault, B. Legoux, P. Galopin, Study of the neurobehavioural toxicity of styrene at low levels of exposure, Int. Arch. Occup. Environ. Health 64 (1993) 527–531. [18] T. Katoh, K. Higashi, N. Inoue, Sub-chronic effects of styrene and styrene oxide on lipid peroxidation and the metabolism of glutathione in rat liver and brain, J. Toxicol. Sci. 14 (1989) 1–9. [19] J.F. Kerr, A.H. Wyllie, A.R. Currie, Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics, Br. J. Cancer 26 (1972) 239–257. [20] R. Kishi, Y. Katakura, T. Ikeda, B.Q. Chen, H. Miyake, Neurochemical effects in rats following gestational exposure to styrene, Toxicol. Lett. 63 (1992) 141–146. [21] J. Kohn, S. Minotti, H. Durham, Assessment of the neurotoxicity of styrene, styrene oxide, and styrene glycol in primary cultures of motor and sensory neurons, Toxicol. Lett. 75 (1995) 29–37. [22] Y. Kouroku, K. Urase, E. Fujita, K. Isahara, Y. Ohsawa, Y. Uchiyama, M.Y. Momoi, T. Momoi, Detection of activated Caspase3 by a cleavage site-directed antiserum during naturally occurring DRG neurons apoptosis, Biochem. Biophys. Res. Commun. 247 (1998) 780–784. [23] U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227 (1970) 680–685. [24] B. Laffon, E. Pasaro, J. Mendez, Effects of styrene-7,8-oxide over p53, p21, bcl-2 and bax expression in human lymphocyte cultures, Mutagenesis 16 (2001) 127–132. [25] M. Leist, B. Single, A.F. Castoldi, S. Kuhnle, P. Nicotera, Intracellular adenosine triphosphate (ATP) concentration: a switch in the decision between apoptosis and necrosis, J. Exp. Med. 185 (1997) 1481–1486. [26] B. Marczynski, M. Peel, X. Baur, Changes in high molecular weight DNA fragmentation following human blood exposure to styrene-7,8oxide, Toxicology 120 (1997) 111–117. [27] P. May, E. May, Twenty years of p53 research: structural and functional aspects of the p53 protein, Oncogene 18 (1999) 7621– 7636. [28] A. Mutti, M. Falzoi, A. Romanelli, M.C. Bocchi, C. Ferroni, I.

[29]

[30]

[31]

[32]

[33] [34]

[35]

[36]

[37]

[38]

[39]

[40]

[41]

[42] [43]

[44] [45]

[46]

[47]

21

Franchini, Brain dopamine as a target for solvent toxicity: effects of some monocyclic aromatic hydrocarbons, Toxicology 49 (1988) 77–82. A. Mutti, M. Falzoi, A. Romanelli, I. Franchini, Regional alterations of brain catecholamines by styrene exposure in rabbits, Arch. Toxicol. 55 (1984) 173–177. A. Mutti, A. Mazzucchi, P. Rustichelli, G. Frigeri, G. Arfini, I. Franchini, Exposure–effect and exposure–response relationships between occupational exposure to styrene and neuropsychological functions, Am. J. Ind. Med. 5 (1984) 275–286. A. Mutti, A. Romanelli, M. Falzoi, S. Lucertini, I. Franchini, Styrene metabolism and striatal dopamine depletion in rabbits, Arch. Toxicol. 8 (1985) 447–450. A. Mutti, P.P. Vescovi, M. Falzoi, G. Arfini, G. Valenti, I. Franchini, Neuroendocrine effects of styrene on occupationally exposed workers, Scand. J. Work Environ. Health 10 (1984) 225–228. S. Nagata, Apoptotic DNA fragmentation, Exp. Cell Res. 256 (2000) 12–18. T. Nakajima, E. Elovaara, F.J. Gonzalez, H.V. Gelboin, H. Raunio, O. Pelkonen, H. Vainio, T. Aoyama, Styrene metabolism by cDNAexpressed human hepatic and pulmonary cytochromes P450, Chem. Res. Toxicol. 7 (1994) 891–896. R. Nath, K.J. Raser, D. Stafford, I. Hajimohammadreza, A. Posner, H. Allen, R.V. Talanian, P. Yuen, R.B. Gilbertsen, K.K. Wang, Non-erythroid alpha-spectrin breakdown by calpain and interleukin 1 b-converting-enzyme-like protease(s) in apoptotic cells: contributory roles of both protease families in neuronal apoptosis, Biochem. J. 319 (1996) 683–690. D.W. Nicholson, A. Ali, N.A. Thornberry, J.P. Vaillancourt, C.K. Ding, M. Gallant, Y. Gareau, P.R. Griffin, M. Labelle, Y.A. Lazebnik, Identification and inhibition of the ICE / CED-3 protease necessary for mammalian apoptosis, Nature 376 (1995) 37–43. J.D. Robertson, S. Orrenius, Molecular mechanisms of apoptosis induced by cytotoxic chemicals, Crit. Rev. Toxicol. 30 (2000) 609–627. L.E. Rosengren, K.G. Haglid, Long term neurotoxicity of styrene. A quantitative study of glial fibrillary acidic protein (GFA) and S-100, Br. J. Ind. Med. 46 (1989) 316–320. T.C. Saido, M. Yokota, S. Nagao, I. Yamaura, E. Tani, T. Tsuchiya, K. Suzuki, S. Kawashima, Spatial resolution of fodrin proteolysis in postischemic brain, J. Biol. Chem. 268 (1993) 25239–25243. H. Savolainen, P. Pfaffli, Accumulation of styrene monomer and neurochemical effects of long-term inhalation exposure in rats, Scand. J. Work Environ. Health 4 (1978) 78–83. A. Schousboe, E. Meier, J. Drejer, L. Hertz, Preparation of primary cultures of mouse (rat) cerebellar granule cells, in: A. Shahar, J. de Vellis, A. Vernadakis, B. Haber (Eds.), A Dissection and Tissue Culture Manual of the Nervous System, Wiley–Liss, New York, 1989, pp. 203–206. H. Sorimachi, S. Ishiura, K. Suzuki, Structure and physiological function of calpains, Biochem. J. 328 (1997) 721–732. C. Stroh, K. Schulze-Osthoff’, Death by a thousand cuts: an ever increasing list of caspase substrates, Cell Death Differ. 5 (1998) 997–1000. N.A. Thornberry, Y. Lazebnik, Caspases: enemies within, Science 281 (1998) 1312–1316. K. Urase, E. Fujita, Y. Miho, Y. Kouroku, T. Mukasa, Y. Yagi, M.Y. Momoi, T. Momoi, Detection of activated caspase-3 (CPP32) in the vertebrate nervous system during development by a cleavage sitedirected antiserum, Brain Res. Dev. Brain Res. 111 (1998) 77–87. M.V. Vettori, D. Corradi, T. Coccini, A. Carta, S. Cavazzini, L. Manzo, A. Mutti, Styrene-induced changes in amacrine retinal cells: an experimental study in the rat, Neurotoxicology 21 (2000) 607– 614. A.H. Wyllie, Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation, Nature 284 (1980) 555–556.

22

E. Dare´ et al. / Brain Research 933 (2002) 12 – 22

[48] A.H. Wyllie, J.F. Kerr, A.R. Currie, Cell death: the significance of apoptosis, Int. Rev. Cytol. 68 (1980) 251–306. [49] J. Yuasa, R. Kishi, T. Eguchi, I. Harabuchi, Y. Arata, Y. Katakura, T. Imai, H. Matsumoto, H. Yokoyama, H. Miyake, Study of urinary mandelic acid concentration and peripheral nerve conduction among styrene workers, Am. J. Ind. Med. 30 (1996) 41–47.

[50] B. Zhivotovsky, D. Wade, A. Gahm, S. Orrenius, P. Nicotera, Formation of 50 kbp chromatin fragments in isolated liver nuclei is mediated by protease and endonuclease activation, FEBS Lett. 351 (1994) 150–154.