Research Article Assessment of Styrene Oxide Neurotoxicity Using In Vitro Auditory Cortex Networks

International Scholarly Research Network ISRN Otolaryngology Volume 211, Article ID 2484, 8 pages doi:1.542/211/2484 Research Article Assessment of Styrene Oxide Neurotoxicity Using In Vitro Auditory Cortex
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International Scholarly Research Network ISRN Otolaryngology Volume 211, Article ID 2484, 8 pages doi:1.542/211/2484 Research Article Assessment of Styrene Oxide Neurotoxicity Using In Vitro Auditory Cortex Networks Kamakshi V. Gopal, 1, 2 Calvin Wu, 1, 2, 3 Ernest J. Moore, 1, 2 and Guenter W. Gross 2, 3 1 Department of Speech and Hearing Sciences, University of North Texas, P.O. Box 351, Denton, TX , USA 2 Center for Network Neuroscience, University of North Texas, P.O. Box 351, Denton, TX , USA 3 Department of Biological Sciences, University of North Texas, P.O. Box 351, Denton, TX , USA Correspondence should be addressed to Kamakshi V. Gopal, Received 24 May 211; Accepted 6 July 211 Academic Editor: A. Bath Copyright 211 Kamakshi V. Gopal et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Styrene oxide (SO) (C 8 H 8 O), the major metabolite of styrene (C 6 H 5 CH=CH 2 ), is widely used in industrial applications. Styrene and SO are neurotoxic and cause damaging effects on the auditory system. However, little is known about their concentrationdependent electrophysiological and morphological effects. We used spontaneouslyactive auditorycortex networks (ACNs) growing on microelectrode arrays (MEA) to characterize neurotoxic effects of SO. Acute application of.1 to 3. mm SO showed concentration-dependent inhibition of spike activity with no noticeable morphological changes. The spike rate IC 5 (concentration inducing 5% inhibition) was 511 ± 6 µm(n = 1). Subchronic (5 hr)single applications of.5 mm SO also showed 5% activity reduction with no overt changes in morphology. The results imply that electrophysiological toxicity precedes cytotoxicity. Fivehour exposures to 2 mm SO revealed neuronal death, irreversible activity loss, and pronounced glial swelling. Paradoxical protection by 4 µm bicuculline suggests binding of SO to GABA receptors. 1. Introduction Styrene is a colorless chemical solvent with an aromatic odor. It is extensively used in industries that manufacture polymers, plastics, and resins. Styrene enters the human body through several routes, especially the respiratory system. More than 8% of the inhaled styrene undergoes bioactivation to styrene oxide (SO) by cytochrome P45 monooxygenases [1]. SO is widely used also in industries as a diluent for epoxy resins and as a chemical intermediate in the manufacturing of cosmetics, agricultural chemicals, and surface coatings. A review of the literature indicates that there is a considerable amount of evidence that styrene and SO are neurotoxic although the precise mechanisms are unclear and quantitative data are lacking [2]. Exposure of low levels of styrene and its metabolites (including SO) may cause irritation of skin, eyes, and mucus membranes, but there is evidence that high doses can lead to neurological disorders [1].The permissible styrene exposure limit set by the Occupational Safety and Health Administration (OSHA) and the threshold limit value recommended by the American Conference of Industrial Hygienists is 5 ppm (approximately 416 µm) for long-term exposure and 1 ppm (approximately 85 µm) for shortterm exposure [3]. There has been no permissible exposure limits set for SO although SO is the most active metabolite formed from styrene. Many of the adverse effects of styrene have been attributed to the accumulation of SO [4]. Otoneurologic tests on industrial workers with long-term styrene exposure at levels below 25 ppm (approximately µm) have revealed problems with the vestibular and auditory systems [5]. Other central nervous system problems induced by styrene exposure include vigilance, memory, vision, visuomotor performance, perceptual speed, and central auditory functions [5 7]. Styrene and SO are shown to impede the functioning of various neurotransmitters in the brain including dopamine and serotonin although uncertainties exist about the exact nature of the hindrance caused by these compounds [3, 8, 9]. The cytotoxicity from styrene and SO exposure is 2 ISRN Otolaryngology thought to be similar to oxidative stress-induced conditions caused by oxidizing protein thiols [4]. Primary cerebellar granule neurons and human neuroblastoma cells exposed to SO (.3 to 1 mm) induced apoptosis, which can be triggered by oxidative stress [1, 1 12]. There is also evidence that exposure of primary striatal neurons to SO induces synaptic impairments [2], which might be the reflection of morphological alteration of the neuronal cytoskeleton. Furthermore, these data supported the hypothesis of reactive oxygen species initiating the events of SO cytotoxicity. SO is a proven animal carcinogen and is classified as a possible human carcinogen (group 2B) by the International Agency for Research on Cancer [13]. Johnson et al., 6, reviewed nine studies that examined the relationship between occupational exposure to styrene and hearing loss [14]. They found that in seven of the nine studies, there was an association between styrene exposure and hearing loss. Occupational exposure to styrene levels of 4 5 ppm for more than 1 years showed elevated hearing thresholds at frequencies up to 15 Hz [15]. However, at lower concentrations of styrene (below 2 ppm), no association between exposure and hearing deficit was found. Chen et al., 8, reported styrene-induced cochlear injury prior to functional loss in an animal model [16]. Exposure to styrene in the presence of industrial noise is shown to have a synergistic effect on the hearing loss incurred by animals and humans [17 19]. Styrene exposure in combination with noise levels within recommended limits has an effect on the auditory system [2]. Chen and Henderson, 9, have suggested that individual exposure to noise or styrene may cause stress, temporary alteration, or nonlethal injury to cochlear hair cells, but the combination exposure of noise and styrene strengthens the stress on the hair cells, leading to cell death [21]. Neurophysiologic testing of brain dysfunction demonstrates reaction time deficiencies in people exposed to styrene [22]. Studies have also shown that styrene has adverse effects on the performance of the central auditory system, including temporal processing skills [14, 23 25]. The European Directive (EU 3) has specified that risk assessment should include interactions of noise and work-related ototoxic substances such as styrene [26]. NIOSH has recommended establishment of exposure limits for ototoxic chemicals in the presence and absence of noise [27]. Styrene ranks at the top of the list along with toluene as a potentially ototoxic solvent that is widely used in industrial settings [28]. There is, however, a limited understanding of the effects of styrene or its major metabolite SO on the central auditory system. This study was undertaken to assess the toxicity of SO using an in vitro model of auditory cortex networks (ACNs) growing on multielectrode arrays (MEAs). The objective was to characterize electrophysiological (functional) toxicity and cellular toxicity for acute and subchronic SO exposures and determine if functional toxicity preceded cellular toxicity. Acute neurotoxicity was assessed by serial additions of the SO to mature cultures (21 div or older) that were spontaneously active. The criterion time point selected was 3 minutes at each concentration. For subchronic neurotoxicity assessment, mature cultures that were spontaneously active were exposed to a single concentration of SO for five hours. The concentration-response relationship of SO was compared to exposure levels of styrene seen in industrial settings, since no such levels are currently available for SO exposure. 2. Materials and Methods 2.1. Cell Culture. The cell culture techniques using ACNs have been published earlier [29 31]. This study was approved by the University of North Texas Institutional Animal Care and Use Committee. Briefly, auditory cortices were dissected from E16-E17 Balb-C/ICR mouse embryos, and were subjected to the standard culturing procedures. The dissociated neurons were seeded at a density of approximately 1, cells/mm 2 on MEAs with substrate-integrated microelectrodes [29]. The networks were maintained in the incubatorsat37 C in a 1% CO 2 atmosphere and fed twice a week using half-medium changes of Dulbecco s modified minimum essential medium (DMEM) supplemented with 5% horse serum. On the day of the experiment, the original medium was completely replaced with serum-free DMEM (stock medium) Microelectrode Arrays (MEA). Previous publications have described in detail the procedures adopted in the inhouse MEA fabrication [32 34]. Briefly, the MEAs were photoetched from commercially available indium-tin oxide (ITO) plates (Applied Films Corp., Boulder, Colo, USA) to generate 5 cm 2 and 1 mm thick plates with 32 amplifier contact strips on either side. The contact strips terminated in the center of the plate in a.8 mm.8 mm recording matrix consisting of 64 recording sites (electrodes). The electrode terminals were either arranged in 4 rows and 16 columns or in 8 rows and 8 columns and conductors measured 1 Å in thickness and 8 µm in width. The processed ITO plates were spin insulated with methyltrimethoxysilane resin and deinsulated at the conductor tips with single laser shots. The exposed metal sites were then gold plated to lower the electrode impedance at 1 khz to approximately.8 mohms Electrophysiological Recording. The MEA recording techniques used in the study have also been described in detail in previous publications [29, 32].The matrix region was treated with poly-d-lysine and laminin to support adhesion of dissociated cells. For electrophysiologic recording, only ACNs that were at least three weeks old in vitro were used. By three weeks after seeding, the neurons develop shallow, three-dimensional networks with neuronal cell bodies generally situatedontop,andneuralprocessessituatedaboveandbelow the glial carpet [35]. Moreover, neuronal networks grown in this manner remain spontaneously active and pharmacologically responsive for more than 6 months [32]. On the day of the experiment, the cultures were maintained at 37 ±.5 C on an inverted microscope stage in a special recording chamber [32]. The recording chamber allows for network maintenance in a constant bath of 1 to 2 ml medium and is well suited for rapid medium changes and short-term (24 hours or less) pharmacological studies. The ph was stabilized at 7.4 by passing a stream of ISRN Otolaryngology 3 humidified 1% CO 2 in air through a cap fitted with a heated ITO window to prevent condensation, which permitted microscopic observations. Osmolarity was maintained at 3 to 32 mosm by adding water at a rate of 65 µl/hr via a syringe pump. Neuronal activity was recorded with a twostage, 64 channel amplification and signal processing system (Plexon Inc., Dallas) with a total system gain set at 1 k. Channels were assigned to 64 digital signal processors with a 4 khz sampling rate. Waveshapes representing single active units were discriminated via template matching. Up to four different templates per physical channel could be discriminated in real time and assigned to separate logical channels. To follow the behavior of the network before, during, and after test compound application, data were displayed as mean network activity per minute. Channels with best signal-to-noise ratios were selected for further monitoring Experimental Protocol. Following the assembly on the recording chamber, the ACNs were allowed to stabilize in fresh medium consisting of stock DMEM without serum. The spontaneous activity was generally recorded for a minimum of 3 minutes and was termed reference activity. Following this period, addition of SO (Sigma-Aldrich, St Louis, Mo, USA) was undertaken, while the ongoing activity was continuously recorded. SO stock solution was prepared in dimethyl sulfoxide (DMSO), since SO is only slightly soluble in water. The maximum DMSO volume in the bath did not exceed 4% of the total bath volume. Control studies were done with DMSO alone to account for its independent effects on the ACNs. For acute experiments, SO dissolved in DMSO was added to the bath with a micropipette to achieve final concentrations ranging from 1 to 3 µm, mimicking the styrene levels of 12 ppm to 36 ppm in occupational exposures in vivo [2]. The network activity was continuously monitored for about 3 minutes prior to the addition of the next dosage to allow for network stabilization. For subchronic experiments, ACNs were exposed to a onetime application of.5 or 2 mm SO, and the activity was monitored continuously for five hours before a complete wash (replacement of the medium with fresh stock DMEM) was undertaken. The cells were monitored optically for morphological changes during acute and subchronic experiments. Cell stress was determined by observations of vesiculation, swelling, obscuration of nucleus, and phase brightness. In a subset of experiments, bicuculline (4 µm) was added prior to addition of SO Data Analysis. To establish SO-induced changes in the ACNs, mean spike rates were obtained for each concentration level. To quantify the changes induced by SO, the reference activity was compared to network activity at each concentration of SO, and a normalized percent change was obtained. Independent samples t-test was used to identify if the reduction in spike activity seen in ACNs exposed to SO was significantly more than the reduction of spike activity in ACNs exposed to DMSO only. A criterion alpha level of.5 was adopted for the comparison. All computations were conducted using the Statistical Package for the Social REF Percent DMSO µm 15 µm (b1) (b2) WASH Figure 1: Effects of DMSO on ACN spontaneous activity and morphology. A gradual decrease in mean spikes per min from 26 units. The sudden augmentation of activity seen during the addition of the test compound is due to mixing of the compound in the bath medium. Horizontal bars represent quasistable states used or quantification of activity changes. Neurons and glia in referencemedium(b1)andafterminin4%dmso(b2).no overt changes in neuronal morphology (phase bright cells) can be identified. Sciences (SPSS) software. It is important to note that single cells are not reliable indicators of pharmacological responses. Population responses are more fault tolerant and provide representative dose-response functions [36]. 3. Results The ACNs used in this study ranged in age from 21 to 42 days in vitro, with a mean age of 31 ± 6.6 days.this study evaluated acute (3 minute exposure) and semichronic effects (five hour exposure) of SO on electrophysiological activity and morphological aspects of ACNs. Independent effects of DMSO were first evaluated for reference purposes followed by investigations of the effects of SO dissolved in DMSO. Further, the effects of SO on ACNs exposed to 4 µm bicuculline (a competitive antagonist of GABA A receptors) were also examined Acute Effects of DMSO on ACNs. Sequential application of DMSO induced a concentration-dependent inhibition of network spike activity. Figure 1 depicts the responses from an ACN that was subjected to DMSO at concentrations ranging from.1% to 4% followed by a complete medium change. The average spike rate decreased as a function of DMSO concentration. To identify morphological changes, neurons and neuronal processes in the matrix area were monitored throughout the experiment. No significant morphological changes were identified between reference (no DMSO) and 4% DMSO (Figure 1). 4 ISRN Otolaryngology 6 5 REF SO concentration (mm) WASH µm 2 µm Percent inhibition SO (microm) (b1) (b2) Figure 2: Acute effects of SO on ACN spontaneous activity and morphology. A stepwise dose-dependent inhibition of mean spike rate per min (15 units). The activity was partially reversible with a single wash. Neurons and glia in reference medium (b1) and after 2 min in 3. mm SO (b2). No overt changes in morphology can be identified. The round black circles seen in the figures are gold-plated electrodes. Table 1: Percent average spike rate reduction with acute application of DMSO and SO (maximum DMSO = 3%). Concentrations Percent activity decrease Percent difference SO DMSO DMSO only SO in DMSO (SO effect) (mm) (%) (n = 3) (n = 1) ± ± ± ± ± ± ± ± ± ± ± ± Acute Effects of SO. With cumulative application of SO (dissolved in DMSO), a pronounced concentration-dependent spike rate inhibition was noticed. Figure 2 shows a typical ACN response to SO addition: gradual stepwise reduction in average spikes as a function of concentration. At 3. mm SO, more than 9% of the spiking activity was lost. When the culture was subjected to a complete wash, there was partial (38%) recovery of the activity. The spike rate IC 5 (concentration inducing 5% inhibition) occurred at approximately 5 µm. Morphological analysis of the neurons monitored throughout the experiment showed no noticeable changes in cell morphology (Figure 2). Table 1 shows the average reduction of mean spike activity in ACNs exposed to DMSO alone (N = 3 experiments), and to SO dissolved in DMSO (N = 1 experiments). Figure 3: Concentration-response curve for SO. Percent inhibition of network spike rate per concentration of SO was calculated for each network using the specific reference activity of that network and averaged. The spike rate IC 5 mean ± SE is 511 ± 6.1 µmso. A significantly greater reduction in activity was obtained for ACNs exposed to SO compared to DMSO alone. This difference was significant at the.5 level (t-test, P =.3). With a single wash following 3 mm SO application, the activity recovered to only to 28.9 ± 8.9% of the original reference level. This shows that the inhibitory effects of SO remain even after a full medium change Concentration-Response Characteristics. The concentration-response curve obtained from 1 ACNs (total of 23 neurons) exposed to acute sequential application of SO is shown in Figure 3. ACNs displayed inhibitory monotonic responses with sequential addition of SO. The IC 5 value, which represents the mean concentration at which spike rates were inhibited 5% of their original level, was 511±6.1 µm Subchronic (Five-Hour Exposure) Functional and Cellular Effects of SO on ACNs. Since the permissible exposure limit set by OSHA for styrene is 5 ppm (approximately 416 µm), which is close to our IC 5 concentration of 511 µm, we investigated the effects of a single dose of SO application at a concentration of.5 mm on ACNs for a period of five hours (to represent a subchronic condition). Additionally, for comparison purposes, we evaluated the effects of a one-time 2. mm sub-chronic exposure. The changes were monitored electrophysiologically as well as morphologically for five hours. Figure 4 depicts the effects of a one-time (nonsequential) application of.5 mm SO on an ACN resulting in an immediate cessation of activity. A rapid spontaneous recovery followed, leading to an eventual activity stabilization at approximately 4% of the original reference activity. A complete wash did not facilitate any recovery in this experiment. Morphologically, the neurons appeared to be normal and healthy throughout the duration of the experiment (Figure 4). ISRN Otolaryngology 35 REF 5 SO.5 mm 3 WASH Ref SO 2 mm Wash (b1) µm (b1) 2 µm 15 µm (b2) (b2) 2 µm (c1) (c2) 2 µm 2 µm (c1) 15 µm (c2) 15 µm (c) (c) Figure 4: Subchronic effects of.5 mm SO (in.5% DMSO) on ACN spontaneous activity and morphology. Mean spike rate per min from 33 units show initial cessation of activity followed by recovery of activity that stabilized to about 4% of the original level at the end of five hours. A complete wash did not lead to any further recovery. and (c) Neurons and glia in reference medium (1) and after 5 hrs in.5 mm SO (2). Only minor changes in morphology can be identif
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