N-acetyl-L-cysteine amide protects retinal pigment epithelium against methamphetamine-induced oxidative stress

Joshua W. Carey, Shakila Tobwala, Xinsheng Zhang, Atrayee Banerjee, Nuran Ercal, Eylem Y. Pinarci, Humeyra Karacal

Department of Chemistry, Missouri University of Science & Technology, Rolla, USA.
Department of Ophthalmology, Baskent University School of Medicine, Ankara, Turkey.
Department of Ophthalmology, Washington University in St. Louis, St. Louis, USA.

DOI: 10.4236/jbpc.2012.32012                

Abstract

Methamphetamine (METH), a highly addictive drug used worldwide, induces oxidative stress in various animal organs. Recent animal studies indicate that methamphetamine also induces oxidative stress in the retina, which is an embryonic extension of the forebrain. The aim of this study, therefore, was to evaluate the protecttive effects of N-acetylcysteine amide (NACA) against oxidative stress induced by METH in retinal pigment epithelium (RPE) cells. Our studies showed that NACA protected against METH-induced oxidative stress in retinal pigment epithelial cells. Although METH significantly decreased glutathione (GSH) levels and increased reactive oxygen species (ROS) and malondialdehyde (MDA) levels, these returned to control levels with NACA treatment. Overall observations indicated that NACA protected RPE cells against oxidative cell damage and death by inhibiting lipid peroxidation, scavenging ROS, increasing levels of intracellular GSH, and maintaining the antioxidant enzyme activity and the integrity of the bloodretinal barrier (BRB). The effectiveness of NACA should be further evaluated to determine its potential for the treatment of numerous retinal diseases caused by oxidative stress.

Keywords

Methamphetamine; Oxidative Stress; Antioxidant; Reactive Oxygen Species; N-Acetylcysteine Amide; Blood-Retina Barrier

1. INTRODUCTION

Methamphetamine (METH), a highly addictive psychostimulant, is abused by approximately 35 million people worldwide [1]. A dramatic increase in METH-related emergency department visits is alarming, with >50% involving young adults between the ages of 18 - 34 years [2]. The relative ease of METH’s availability, coupled with its toxicity, has resulted in increased numbers of associated medical complications and fatalities [2,3].

Oxidative stress is believed to play a crucial role in METH-induced toxicity and is supported by several studies which reported decreased glutathione (GSH) levels, reduced levels and activities of antioxidant enzymes, and increased lipid peroxidation and protein carbonylation, all hallmarks of oxidative stress [4-8]. In the case of the brain, it is believed that, initially, METH causes a massive release of dopamine by inhibiting monoamine oxidase activity and dopamine uptake [9]. With higher doses, however, it causes dopamine depletion by degenerating dopaminergic terminals, damaging dopaminergic neurons, and decreasing dopamine transporter numbers [10,11]. Dopamine then reacts with molecular oxygen to form reactive oxygen species (ROS), such as hydrogen peroxide, superoxide, and hydroxyl free radicals, resulting in a condition known as oxidative stress [11] and causes neuronal death by apoptosis [12].

Oxidative stress acts as a propagating force in the pathogenesis of many ocular disorders, including cataracts, glaucoma, diabetic retinopathy, HIV-related retinopathy, and age-related macular degeneration (AMD) [13-15]. Oxidative stress possibly contributes to the death of retinal cells and degeneration of the macula [16-20]. Free radical formation in a developing retina has been reported to be induced by xenobiotics, e.g., environmental pollutants, pharmacological substances, and alcohol [21].

METH abuse is implicated in a number of serious ocular pathologies, including corneal ulceration, retinal vasculitis, episcleritis, scleritis, panophthalmitis, endophthalmitis, and retinopathy [22-24]. It has been reported that METH induces oxidative stress in the retina and adversely affects the dopaminergic system of the rat retina [25], particularly during central nervous system (CNS) development. Prudencio et al. [26] demonstrated that METH altered retinal plasma membrane integrity, esterase activity, and/or pH in rat retina homogenates. Another study reported exacerbation of damaging effects of kainic acid on the retina by METH [27]. Under physiological conditions, high levels of antioxidant enzymes and small antioxidant molecules, particularly glutathione (GSH), in the Muller (glial) cells protects retinal pigment epithelium (RPE) cells against oxidative stress. GSH in the Muller cells is depleted under oxidative stress and, since GSH cannot be transported directly into the cells, the need arises for permeable compounds that can increase intracellular GSH levels. One such compound, known to increase intracellular GSH levels in cells, is the low molecular weight thiol antioxidant, N-acetylcysteine amide (NACA). Previous work by our group has demonstrated that NACA restored the levels of GSH, and scavenged the ROS produced in human brain endothelial cells, upon treatment with METH [4].

Even though the adverse effects of METH on the brain are linked to oxidative stress, little is known about its effect on the retina. Considering the ability of NACA to protect cells from oxidative stress [28-30], the effectiveness of this antioxidant was evaluated as a treatment option for METH-induced oxidative damage to RPE cells. Understanding NACA’s protective role against METHinduced oxidative damage to RPE cells would help develop NACA as a potential therapeutic agent for treating RPE cells against numerous oxidative stress-related ocular diseases.

2. MATERIALS AND METHODS

2.1. Materials

The retinal pigment epithelial cell line, ARPE-19, was purchased from the American Type Culture Collection (ATCC # CRL-2302) (Manassas, VA), and the N-acetylcysteine amide (NACA) from Dr. Glenn Goldstein of David Pharmaceuticals in New York, NY. HPLC chemicals were obtained from Fisher Scientific (Pittsburgh, PA). Cell culture reagents were purchased from ATCC in Manassas, VA, and Calcein AM was bought from Biotium, Inc. (Hayward, CA). The National Institute on Drug Abuse (NIDA) provided methamphetamine, while other chemicals were obtained from Sigma-Aldrich (St. Louis, MO), unless otherwise stated.

2.2. ARPE-19 Cell Culture and Treatment

The ARPE-19 cells were cultured in a one-to-one ratio of DMEM: F-12 culture medium, and supplemented with 10% (v/v) FBS, 100 U/mL of penicillin, and 100 μg/mL of streptomycin. Cells were maintained in a 37˚C incubator and supplied with 95% air and 5% CO₂. Cells between passage numbers 26 and 32 were used for experiments.

2.3. Determination of Cell Viability

The ARPE-19 cells were seeded in a 96-well tissue culture plate, at a density of approximately 1.25 × 10⁴ cells/well, for a day. The media was then discarded and the cells were treated with various concentrations of METH and NACA in serum-free media for 24 hours and 2 hours, respectively. Protective effects of NACA were studied by pre-incubating cells with NACA for 2 hours, followed by treatment with 500 µM of METH for 24 hours. After 24 hours of METH treatment, the medium was discarded and a Calcein AM assay KIT (Biotium, Inc. CA) was used to determine cell viability relative to the control group [31]. The cells were then washed three times with PBS, and 100 µL of 2.0 M Calcein AM in PBS were added to each well for 30 minutes at 37˚C. The fluorescence was measured with an excitation wavelength at 485 nm and an emission wavelength of 530 nm, using a microplate reader (FLUOstar, BMG Labtechnologies, Durham, NC, USA).

2.4. Intracellular ROS Measurement

Intracellular ROS generation was measured using a well-characterized probe, 2’, 7’-dichlorofluorescin diacetate (DCFH-DA) [32]. ARPE-19 cells were seeded at a density of 1.25 × 10⁴ cells/well in a 96-well plate. DCFH-DA is hydrolyzed by esterases to dichlorofluorescin (DCFH), which was trapped within the cell. This nonfluorescent molecule is then oxidized to fluorescent dichlorofluorescin (DCF) by the action of cellular oxidants. In groups with NACA pretreatment, media containing 1 mM NACA was added and incubated for 2 hours. Once pretreated, the cells were washed twice with PBS and incubated with a solution of 50 μM DCFH-DA in phenol red free media for 30 minutes. This was followed by washing the cells twice with PBS, and the respective groups were then dosed either with 500 µM METH or plain media for 4 hours and then fluorescence was determined at 485 nm excitation and 520 nm emission, using a microplate reader (FLUOstar, BMG Labtechnologies, Durham, NC, USA).

2.5. Experimental Design for Oxidative Stress Parameters

Parameters, including GSH, MDA, and activities of glutathione peroxidase and catalase, were measured after the cells were treated, as described below. After seeding the cells, the flasks were divided into the following four groups: 1) control; 2) NACA-only; 3) METH-only; and 4) METH + NACA. In groups with NACA pretreatment, media containing 1 mM NACA was added and incubated for 2 hours. After pretreatment, the media in the control and NACA-only groups were replaced with plain media, while both of the remaining two groups received media containing METH for 24 hours. The cell pellets obtained were then further processed for appropriate assays.

2.5.1. Determination of Glutathione (GSH)

The levels of GSH in the tissues were determined by RP-HPLC, according to the method developed in our laboratory [33]. The HPLC system (Thermo Electron Corporation) consisted of a Finnigan Spectra System vacuum membrane degasser (model SCM1000), a gradient pump (model P2000), autosampler (model AS3000), and a fluorescence detector (model FL3000) with l_ex = 330 nm and l_em = 376 nm. The HPLC column used was a Reliasil ODS-1 C18 column (5 µm packing material) with 250 mm × 4.6 mm i.d. (Column Engineering, Ontario, CA). The mobile phase (70% acetonitrile and 30% water) was adjusted to a pH of 2 with acetic acid and o-phosphoric acid. The N-(1-pyrenyl)-maleimide (NPM) derivatives of GSH were eluted from the column isocratically at a flow rate of 1 mL/min. The cell samples were homogenized in a serine borate buffer (100 mM Tris HCl, 10 mM borate, 5 mM serine, 1 mM diethylenetriaminepentaacetic acid), centrifuged, and 50 µL of the supernatant were added to 230 µL of HPLC grade water and 750 µL of NPM (1 mM in acetonitrile). The resulting solution was incubated at room temperature for 5 minutes, and the reaction was stopped by adding 10 µL of 2 N HCl. The samples were then filtered through a 0.45 µm filter and injected into the HPLC system.

2.5.2. Determination of Malondialdehyde (MDA)

The MDA levels were determined according to the method described by Draper et al. [34]. Briefly, 550 µL of 5% tricholoroacetic acid (TCA) and 100 µL of 500 ppm butylated hydroxytoluene (BHT) in methanol were added to 350 µL of the cell homogenates, and boiled for 30 minutes in a water bath. After cooling on ice, the mixtures were centrifuged, and the supernatant collected was mixed 1:1 with saturated thiobarbituric acid (TBA). The mixture was again heated in a water bath for 30 minutes, followed by cooling on ice. 500 µL of the mixture was extracted with 1 mL of nbutanol and centrifuged to facilitate the separation of phases. The resulting organic layers were first filtered through 0.45 µm filters and then injected into the HPLC system (Shimadzu, US), which consisted of a pump (model LC-6A), a Rheodyne injection valve and a fluorescence detector (model RF 535). The column was a 100 mm × 4.6 mm i.d. C18 column (3 µm packing material, Astec, Bellefonte, PA). The mobile phase used contained 69.4% sodium phosphate buffer, 30% acetonitrile, and 0.6% tetrahydrofuran. The fluorescent product was monitored at l_ex = 515 nm and l_em = 550 nm. The concentrations of the TBA-MDA complex in the mixture was determined by using the calibration curve obtained from a 1,1,3,3,-tetraethoxypropane standard solution. This method differs from commonly used TBARS assay in that it specifically determines TBA-MDA adducts by HPLC.

2.5.3. Measurement of Catalase (CAT) Activity

Catalase activity was measured according to the method described by Aebi [35]. Briefly, it was measured spectrophotometrically at a wavelength of 240 nm, in the supernatant of the cell homogenate, following the exponential disappearance of hydrogen peroxide (H₂O₂, 10 mM). The catalase activity was calculated from the equation A₆₀ = A_initiale^–kt, where k represents the rate constant, A_initial is the initial absorbance and A₆₀ is the absorbance after 60 seconds have passed.

2.5.4. Measurement of Glutathione Peroxidase Activity

Glutathione peroxidase (GPx) activity was determined using a glutathione peroxidase colorimetric assay kit purchased from Oxis Research (Foster City, CA). The cell samples were homogenized in 50 mM of phosphate buffer (pH 7.4), containing 1.0 mM EDTA, and then centrifuged at 7500 × g for 10 minutes. The resulting supernatant was collected to be used for the assay while the remaining debris was discarded. In brief, the assay buffer, supernatant, and NADPH reagent (containing glutathione reductase, GSH, and NADPH) were placed in a cuvette and the reaction initiated by the addition of t-butylhydroperoxide (tBHP). The decrease in absorbance at 340 nm was recorded for 3 minutes and the change in A₃₄₀/min from the initial linear portion of the curve was used to calculate the GPx enzyme activity. The GPx activity was calculated using the extinction coefficient of NADPH (6220 M^–1·cm^–1) and expressed as U/mg of protein.

2.5.5. Dextran Permeability Study and Trans-Endothelial Electrical Resistance (TEER) Measurement

ARPE-19 cells were seeded at a density of 1.5 × 10⁵ cells/well onto collagen-coated inserts with a pore size of 0.4 µm and allowed to form a monolayer. Trans-endothelial electric resistance (TEER) measurement by an EVOM voltohmmeter (World Precision Instrument, Sarasota, FL, USA) assessed the tightness of the ARPE monolayer [4]. The cell monolayer was then treated as described in Experimental Design. After this, the media was replaced with 150 µL of fresh medium. The insert containing the cell monolayer was then transferred to a fresh plate containing 500 µL of serum-free medium. The TEER reading was recorded immediately and TEER values were calculated as: Resistance × 0.32 cm² (insert surface area). Thus, resistance is proportional to the effective membrane.

2.6. Determination of Protein

Protein levels of the cell samples were measured by the Bradford method [36] using bovine serum albumin as standard.

2.7. Statistical Analysis

Statistical significance was calculated using an unpaired two-tailed student’s t test in which “*” represents a p-value ≤ 0.05 when compared to the control group, and “**” represents a p-value ≤ 0.05 when compared to the METH group. All reported values were represented as mean ± S.D. of triplets.

3. RESULTS

3.1. Protection from METH Toxicity

Cell viability was measured as a function of METH and NACA dose to determine the optimum concentrations for both METH and NACA, respectively. Figures 1(a) and (b) revealed that a concentration of 500 µM METH and 1 mM NACA are the minimum toxic doses of METH and NACA, for ARPE-19 cells and, therefore, these doses were selected for further experiments. Figure 2 shows that pretreatment with 1.0 mM of NACA successfully protected the ARPE-19 cells from METHinduced death, thereby increasing viability to ~92% of that of control levels.

3.2. Intracellular ROS Measurements

After treatment with 500 µM of METH, ROS production was found to have increased. METH (500 µM) induced an increase in the DCF fluorescence by approximately 30%, as compared to that of the control (Figure 3), while pretreatment with 1 mM NACA (2 hours prior to exposure) completely erased this increase. NACA alone did not significantly alter DCF fluorescence compared to that of the control.

3.3. Intracellular Glutathione

Figure 4 shows the effect of METH on cellular GSH levels in ARPE-19 cells in the presence and absence of NACA. A 24-hour exposure to METH resulted in an increase in cellular oxidative stress. Treatment with 500 µM of METH altered the GSH level to 80% of that of the control. A 500 µM treatment of METH, with a 1 mM pretreatment of NACA, was significantly different from that of the METH group alone, with p ≤ 0.05 and a GSH concentration of 98% of that of the control group. The

Conflicts of Interest

The authors declare no conflicts of interest.

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