Abstract Cocaine has a short half-life of only about an hour but its effects, predominantly on the central nervous system (CNS), are fairly long-lasting. Of all cells within the CNS, astrocytes may be the first to display cocaine toxicity owing to their relative abundance in the brain. Cocaine entry could trigger several early response changes that adversely affect their survival, and inhibiting these changes could conversely increase their rate of survival. In order to identify these changes and the minimal concentrations of cocaine that can elicit them in vitro, rat C6 astroglia-like cells were treated with cocaine (2–4 mM for 1h) and assayed for alterations in gross cell morphology, cytoplasmic vacuolation, viability, reactive oxygen species (ROS) generation, glutathione (GSH) levels, cell membrane integrity, F-actin cytoskeleton, and histone methylation. We report here that all of the above identified features are significantly altered by cocaine, and may collectively represent the key pathology underlying acute toxicity-mediated death of astroglia-like cells. Pretreatment of the cells with the clinically available antioxidant N-acetyl cysteine (NAC, 5 mM for 30 min) inhibited these changes during subsequent application of cocaine and mitigated cocaine-induced toxicity. Despite repeated cocaine exposure, NAC pretreated cells remained highly viable and post NAC treatment also increased viability of cocaine treated cells to a smaller yet significant level. We show further that this alleviation by NAC is mediated through an increase in GSH levels in the cells. These findings, coupled with the fact that astrocytes maintain neuronal integrity, suggest that compounds which target and mitigate these early toxic changes in astrocytes could have a potentially broad therapeutic role in cocaine-induced CNS damage.

Citation: Badisa RB, Kumar SS, Mazzio E, Haughbrook RD, Allen JR, Davidson MW, et al. (2015) N-Acetyl Cysteine Mitigates the Acute Effects of Cocaine-Induced Toxicity in Astroglia-Like Cells. PLoS ONE 10(1): e0114285. https://doi.org/10.1371/journal.pone.0114285 Academic Editor: Ryan K. Bachtell, University of Colorado, UNITED STATES Received: June 18, 2014; Accepted: November 3, 2014; Published: January 24, 2015 Copyright: © 2015 Badisa et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited Data Availability: All relevant data are within the paper. Funding: This study was supported by the National Institute on Minority Health and Health Disparities of the National Institutes of Health under award number G12MD007582 of the USA. Competing interests: The authors declare that no competing interests exist.

Introduction Cocaine is an addictive and widely abused psychostimulant that can evade the protection of the blood brain barrier (BBB) to enter the brain and compromise its normal functioning. Cocaine's effects on biochemical processes in the CNS is an area of active research, and how these cocaine-induced changes impact neurons and astrocytes is not well understood. Although acute exposure to cocaine has been shown to alter gene expression [1], it is the changed cell biochemistry that appears to underlie many of the clinical symptoms. Identification of early biochemical symptoms such as vacuolation and changes in mitochondrial membrane potential may offer clues about underlying mechanisms and therapeutic avenues. While the long-term/chronic effects of cocaine, including post-translational modifications such as acetylation, methylation [2, 3], phosphorylation have been well established in the literature, early precipitating events that lead to these chronic changes following acute exposure are much less understood. Furthermore, cocaine's ability to interfere with normal signaling pathways in neurons [4] has narrowed the focus of research within CNS to neurons, despite evidence that astrocytes–cells that provide both physical and chemical support to neurons [5] and maintain the integrity of the BBB [6]–are also vulnerable. The present study is geared towards unraveling the acute morphological and epigenetic changes in astrocytes upon exposure to cocaine. Incorporating data from our previous studies that focused on the chronic effects of cocaine [7, 8] and considering that astrocytes outnumber neurons in most brain regions [9], we postulate that toxic effects of cocaine manifest in astrocytes prior to any neuronal damage. Cocaine's entry into the brain through the BBB, known for its astroglial interaction [10, 11], may also expose astrocytes to cocaine sooner and for longer periods than any other cell-type in the CNS thereby enhancing their vulnerability to cocaine-induced toxicity. Because neurons depend on astrocytes for survival [12, 13], loss of astrocytes due to cocaine toxicity could ultimately lead to loss of neurons / neuronal function [14]–a circumstance that could possibly be avoided in the initial stages of cocaine addiction by protecting astrocytes from the acute effects of cocaine-induced toxicity. This study tests the hypothesis that inhibition of the acute effects of cocaine in astrocytes increases their survival. The objectives of the present study are to identify various early response changes associated with acute exposure of astroglia-like cells to physiologically-relevant doses of cocaine in vitro; to determine the minimal doses that compromise their viability; and to investigate the role of NAC in mitigating cocaine-induced toxicity in these cells and determining its mode of action. To this end, we used a CNS derived rat C6 astroglia-like cell line (CCL-107) which is astrocytic in origin and unlike other CNS cell lines, exhibits a high degree of similarity with human astrocytes in its gene expression [15] and enzymes [16]. Studies have also shown that this cell line contains undifferentiated glial cells [17] that release glial cell line-derived neurotrophic factors similar to astrocytes [18]. Taken together, these properties demonstrate that C6 cell cultures behave like an astroglia-like cell line. In the past, C6 cells have also been used extensively for in vitro drug abuse research [7, 8, 19–22] and in the study of astrocytic function [23–30].

Materials and Methods Chemicals All chemicals used were of analytical grade. RPMI 1640, fetal bovine serum (FBS), penicillin/streptomycin sulfate, amphotericin B, phosphate-buffered saline (PBS) and L-glutamine were obtained from Media Tech (Herndon, VA, USA). Cocaine (Ecgonine methyl ester benzoate) hydrochloride, crystal violet, dichlorodihydrofluorescin diacetate dye (H 2 DCFDA), L-glutaraldehyde, 0.5 M EDTA (ethylene diamine tetraacetic acid) solution, 5,5-dithiobis-2-nitrobenzoic acid (DTNB), nicotinamide adenosine dinucleotide phosphate (NADPH), 5-sulfosalicylic acid, NAC and trypan blue were obtained from Sigma-Aldrich (St. Louis, MO, USA) and used according to various protocols. Cell Culture The CNS derived rat C6 astroglia-like cell line (CCL-107) was purchased from the American Type Culture Collection (Rockville, MD, USA) and maintained as a monolayer culture as described before [7]. Immunocytochemistry of Glial Fibrillary Acidic Protein We assayed for the presence of glial fibrillary acidic protein (GFAP), an important marker protein expressed abundantly only in astrocytes, in C6 cells. Cells were cultured in 96-well plates (5x104/well) over night following which they were fixed in 4% paraformaldehyde, permeabilized in 0.1% triton X 100 in PBS and incubated with rabbit-anti-rat GFAP 1° antibody (ab7260) (Abcam, Cambridge, MA) for 2 h at room temp. Samples were washed in PBS and subsequently incubated with goat anti-rabbit Alexa Fluor 488 conjugate for 2 h at room temp. These were counterstained for nuclei with propidium iodide (PI) and photomicrographed using an inverted microscope with a 40x objective, CCD camera. Data was acquired using ToupTek View (TouTek Photonics Co, Zhejiang, China). Treatment with Cocaine and NAC Cocaine hydrochloride stock (1M) and working stock solutions (80–160 mM) were prepared in PBS as described previously [7]. Cytotoxic studies were performed in polystyrene, flat bottom 96-well (0.32 cm2) microtiter plates (BD Labware, NJ, USA). Cells were seeded at a starting density of 2x104 cells per well in a total volume of 195 μl of complete RPMI 1640 growth medium with 10% FBS and allowed to adhere overnight in an incubator (37°C, 5% CO 2 ). Cocaine was added to the medium under sterile conditions using minimal volumes of the working stock solutions (5 μl/well) to achieve final concentrations of 2, 3 and 4 mM without disrupting pH. Untreated cells received equal volumes of PBS (5 μl/well) and served as vehicle controls. Treated samples were interspersed with controls in different wells of the same 96-well microtiter culture plates. Treatment with cocaine was carried out in an incubator (37°C; 5% CO 2 ) and lasted for 1h to mimic its biological half-life in the body [31, 32] and the fact that a single-dose of cocaine in addicts wears off after an hour for typical amounts and routes of intake (National Institute of Drug Abuse, Research Report Series, March 2010, Bethesda, Maryland, USA). In a subset of experiments, cells were pretreated with 5 mM NAC [33] for 30 min prior to cocaine exposure, while in another set of experiments, 5 mM NAC (30 min) was added to cells post 1h cocaine exposure. Cell viability and GSH levels were assayed at the end of an hour-long incubation using methods outlined below. Assessment of Morphology and Vacuolation To evaluate gross morphological changes including vacuolation, cells were stained with crystal violet (0.1%) and observed under an inverted phase contrast IX-70 Olympus microscope (Ontario, NY, USA) with a 40x objective. Photomicrographs were taken using an ocular video-camera system (MD35 Electronic eyepiece, Zhejiang JinCheng Scientific & Technology Co., Ltd, HangZhou, China) running C-Imaging System Software (Compix Inc. Cranberry Township, PA, USA). Assessment of Cell Viability Cell viability was assessed by the crystal violet dye uptake assay, described previously [34]. Briefly, at the end of the 1h treatment, 100 μl of 0.25% glutaraldehyde was added to each well and the cultures incubated for 30 min to fix cells to the polystyrene surface of culture plates. The plates were then gently washed three times and air-dried. Following staining with 0.1% aqueous crystal violet dye (15 min), the plates were washed and air dried again. The dye in each well was extracted with 100 μl of 50 mM sodium phosphate mono basic solution containing 50% ethyl alcohol. Optical density (OD) measurements of incorporated dye in viable cells were obtained at 540 nm using a microplate reader (Bio-Tek Instruments Inc, Wincoski, VT, USA). Cell viability was re-confirmed using 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethonyphenol)-2-(4-sulfophenyl)-2H tetrazolium (MTS) Cell Titer 96 AQueous One Solution Reagent kit (Promega, Madison, WI, USA) and titer plates were read for dye color in a micro plate reader at 490 nm. Measurements of Intracellular ROS Cocaine-induced ROS release was measured with H 2 DCFDA dye in 96-well plates. Prior to cocaine exposure, 10 μM of the dye was loaded into cells [35] for 30 min and washed. Cells were subsequently treated with phenol red free media before exposure to cocaine. Plates were read using a micro plate fluorometer model 7620, Version 5.02, Cambridge Technology, Inc., (Watertown, MA, USA) with the excitation and emission filters set at 485 and 530 nm, respectively. Assessment of Total Cellular GSH Levels Total cellular GSH was assayed according to [36]. Briefly, following cocaine treatment, cells were fixed with 0.25% glutaraldehyde for 30 min followed by gentle washing and air drying. Cells were then deproteinized with 2% 5-sulfosalicylic acid (10 μl/well) for 30 min at 37°C followed by incubation with 90 μl of a reaction mixture containing (in mM): 0.416 sodium EDTA, 0.416 NADPH, 0.835 DTNB and 0.083 sodium phosphate buffer (pH 7.5) for 30 min at 37°C. Absorbance of color was measured on a micro plate reader at 412 nm. Plasma Membrane Integrity Assay Cell membrane integrity following exposure to various concentrations of cocaine was determined by measuring lactose dehydrogenase (LDH) release with CytoTox 96 non-radioactive assay kit (Promega) as per kit instructions provided by the manufacturer. Color intensity was measured using a micro plate reader at 490 nm. Fluorescence Microscopy for Nuclear and F-Actin Staining Briefly, following cocaine treatment, cells were fixed in 4% paraformaldehyde for 15 min and subsequently permeabilized in 0.25% triton X 100 in PBS for 15 min. Stock solutions for fluorescent dyes (Life Technologies, Carlsbad, CA, USA) were made in ethanol (5 mg/ml) and diluted in HBSS before being added to cells. Final concentrations for PI (excitation: 535 nm, emission 617 nm) and Alexa Fluor 488 Phalloidin (excitation: 488 nm; emission: 520 nm) were 5 μg/ml and 6.6 μM respectively. Images show Alexa Fluor 488 Phalloidin and PI nuclear counterstain to corroborate changes in cytoskeleton F-actin tertiary structure. Samples were analyzed photographically using XDY-1 inverted fluorescent microscope with a 40x objective, CCD camera and data acquired using ToupTek View (TouTek Photonics Co, Zhejiang, China). Fluorescence intensity was acquired using Image J software, National Institutes of Health (NIH), Bethesda, Maryland, USA. Histone H3-K27 Methylation Assay Global histone H3-K27 methylation assay kit (Epigentek, Farmingdale, NY, USA) was used to measure H3-K27 methylation in the cells. Briefly, cells (starting density: 1.5x106) were seeded in 100 mm sterile culture dishes per 10 ml of complete DMEM containing 5.5 mM glucose and incubated overnight. They were exposed to cocaine (1h) on the following day and harvested by trypsinization. Histone extraction and methylation detection were done as per the protocol supplied by the manufacturer (Epigentek). OD measurements were obtained from a micro plate reader at 450 nm. The amount of methylation (ng/mg protein) in H3 histone was determined by the following equation: (OD/slope) × 1000. Slope was determined from the standard curve of the positive control (H3-K27) supplied with the kit. Statistical Analysis Experimental results are presented as the mean ± standard error of the mean (S.E.M.). The data were analyzed for significance by one-way ANOVA and then compared using Dunnett's multiple comparison tests in GraphPad Prism Software, version 5 (San Diego, CA, USA). The lethal concentration of cocaine (mM) needed to kill 50% of cells (LC 50 ), was determined as described earlier [37].

Conclusion We identified several early response changes such as alterations in mitochondrial membrane potential [7], mitochondrial respiratory status [8], vacuolation (Fig. 2B), ROS production (Fig. 3B), cellular GSH levels (Fig. 3C), F-actin cytoskeleton (Fig. 4), and histone methylation (Fig. 7B) in C6 astroglia-like cells following acute exposure to cocaine. Our data provide compelling evidence to support the hypothesis that inhibition of the early response changes by NAC enhances cell survival through increased GSH levels. Compounds which support GSH synthesis could therefore mitigate toxicity of early response events in cells exposed to cocaine [22]. The recapitulation of cocaine-induced changes observed in our model (e.g. vacuolization [58]) lends credibility to its use for studying cocaine induced toxicity in vivo.

Acknowledgments The authors thank Dr. Sandra Suther for critically reading the manuscript and for suggestions.

Author Contributions Wrote the paper: RBB SSK. Conceived, designed and performed most of the research: RBB. Provided overall support and advice and served as the person-in-charge of the project: CBG. Conducted fluorescent microscopy studies: EM JRA MWD. Performed the methylation study: RDH. Helped interpret the data and write portions of the paper: CAF.