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Mitochondria Are the Main Target Organelle for Trivalent Monomethylarsonous Acid (MMAIII)-Induced Cytotoxicity Hua Naranmandura,*,† Shi Xu,† Takashi Sawata,‡ Wen Hui Hao,† Huan Liu,† Na Bu,† Yasumitsu Ogra,§ Yi Jia Lou,† and Noriyuki Suzuki‡ †
Department of Pharmacology and Toxicology, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China Graduate School of Pharmaceutical Sciences, Chiba University, Chuo, Chiba 260-8675, Japan § Laboratory of Chemical Toxicology and Environmental Health, Showa Pharmaceutical University, Machida, Tokyo 194-8543, Japan ‡
bS Supporting Information ABSTRACT: Excessive generation of reactive oxygen species (ROS) is considered to play an important role in arsenic-induced carcinogenicity in the liver, lungs, and urinary bladder. However, little is known about the mechanism of ROS-based carcinogenicity, including where the ROS are generated, and which arsenic species are the most effective ROS inducers. In order to better understand the mechanism of arsenic toxicity, rat liver RLC-16 cells were exposed to arsenite (iAsIII) and its intermediate metabolites [i.e., monomethylarsonous acid (MMAIII) and dimethylarsinous acid (DMAIII)]. MMAIII (IC50 = 1 μM) was found to be the most toxic form, followed by DMAIII (IC50 = 2 μM) and iAsIII (IC50 = 18 μM). Following exposure to MMAIII, ROS were found to be generated primarily in the mitochondria. DMAIII exposure resulted in ROS generation in other organelles, while no ROS generation was seen following exposures to low levels of iAsIII. This suggests the mechanisms of induction of ROS are different among the three arsenicals. The effects of iAsIII, MMAIII, and DMAIII on activities of complexes IIV in the electron transport chain (ETC) of rat liver submitochondrial particles and on the stimulation of ROS production in intact mitochondria were also studied. Activities of complexes II and IV were significantly inhibited by MMAIII, but only the activity of complexes II was inhibited by DMAIII. Incubation with iAsIII had no inhibitory effects on any of the four complexes. Generation of ROS in intact mitochondria was significantly increased following incubation with MMAIII, while low levels of ROS generation were observed following incubation with DMAIII. ROS was not produced in mitochondria following exposure to iAsIII. The mechanism underlying cell death is different among AsIII, MMAIII, and DMAIII, with mitochondria being one of the primary target organelles for MMAIII-induced cytotoxicity.
’ INTRODUCTION Exposure to arsenic is associated with cancer of the skin, liver, lungs, and urinary bladder.1,2 In mammals, ingested inorganic arsenite (iAsIII) or inorganic arsenate (iAsV) can be biotransformed through a series of enzymatically mediated methylation reactions, producing methylated trivalent and pentavalent metabolites,3 and the final metabolites that are excreted in the urine are mostly mono- and dimethylated pentavalent species.3,4 However, it is not known which of these forms (inorganic arsenic or its metabolites) is responsible for arsenic’s carcinogenicity, and the mechanism is not well understood. A few studies have indicated that the arsenic-induced cancer associated with chronic exposure to inorganic arsenic may be mediated by its intermediate metabolites such as monomethylarsonous acid (MMAIII) and dimethylarsinous acid (DMAIII).57 In addition, the formation of these products may result in the generation of reactive oxygen species (ROS) and free radicals that could exert carcinogenic effects.8 In fact, recent evidence suggests that an increase in oxidative stress in cells treated with arsenicals may be the molecular mechanism behind arsenic-induced carcinogenesis.9,10 r 2011 American Chemical Society
Generally speaking, trivalent arsenicals are more cytotoxic and genotoxic than their pentavalent counterparts.11,12 In addition, trivalent arsenicals are able to induce oxidative DNA damage in animals as well as a greater frequency of chromosomal aberrations in cells.1315 Previous studies from our laboratory suggest that iAsIII methylation occurs in the liver, and the trivalent arsenicals are bound to proteins throughout the methylation process (iAsIII f MMAIII f DMAIII).16 The liver is also one of the identified target organ for arsenic toxicity and carcinogenicity, but it remains unclear if the cytotoxcity of these trivalent arenicals, or the ROS production associated with their formation may be responsible for this toxic effect. Kitchin et al. has indicated that the generation of ROS is likely to play an important role in the early stages of arsenic carcinogenesis, as well as risk factor for cancer development in target organs in humans.17 In other studies, high levels of products of DNA oxidation, such as oxidation8-oxo-20 -deoxyguanosine Received: March 4, 2011 Published: June 07, 2011 1094
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Chemical Research in Toxicology (8-oxodG), were detected in arsenic-induced human skin cancer in China, as well as in human urine after exposure to cadmium and arsenic.18,19 Similar results were also obtained from rats after long-term exposure to DMAV, with the generation of 8-OHdG being increased in the liver and bladder.20,21 Thus, understanding of the mechanism of arsenic induced ROS generation is important for better understanding the mechanism of carcinogenicity and could also aid in the development of strategies to counteract these effects. Mitochondria are known to be one of the major generators of ROS in cells. Dysfunction of electron transfer through the mitochondrial respiratory chain may result in increased ROS formation, including the formation of the superoxide anion radical (O2•), hydrogen peroxide (H2O2), and the hydroxyl radical (OH•).22,23 Moreover, complexes I and III in the electron transport chain (ETC) are thought to be the main leak sites for ROS production, as some of the electrons passing through the mitochondrial respiratory chain (i.e., complex I and III) leak out to molecular oxygen (O2) to form superoxide radicals and then dismutate to H2O2.24 The present study explores the mechanism of toxicity and localization of ROS generated by iAsIII and its intermediate metabolites such as MMAIII and DMAIII in rat liver RLC-16 cells. Although these trivalent arsenicals are highly toxic to RLC-16 cells, the induction mechanism of cell death seems to vary with species. The majority of ROS were generated in mitochondria following exposure to MMAIII, while other organelles produced lower amounts of ROS following exposure to DMAIII. No ROS were detected in cells following exposure to low levels iAsIII. This study also investigated how the complexes of the electron transfer chain (ETC) within the mitochondria were inhibited by AsIII, MMAIII, and DMAIII. Activities of complex II and IV were strongly inhibited by exposure to MMAIII, but only the activity of complexes II was inhibited by exposure to DMAIII. No inhibitory effects were observed following exposure to iAsIII, suggesting ROS are generated mainly through the inhibition of complex IV due to exposure to MMAIII.
’ MATERIAL AND METHODS Reagents. All reagents were of analytical grade or better. Milli-Q water (Millipore) was used throughout. Trizma HCl, Trizma Base, cytochrome c oxidase assay kit, rotenone, malonic acid, antimycin A, diphenyleneiodonium chloride, 2,6-dichloroindophenol sodium salt hydrate (DCPIP), phenazene methosulfate, decylubiquinone, 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), and 20 ,70 -dichlorofluorescin diacetate (DCFH-DA) were purchased from Sigma (St. Louis, MO, USA). Nitric acid, hydrogen peroxide (30%), cytochrome c (Horse Heart), L-glutamic acid, L-()-malic acid, L-cysteine, succinic acid, sodium hydrosulfite, 10% formalin neutral buffer solution, potassium cyanide, sodium arsenite (iAsIII), and dimethylarsinic acid [(CH3)2AsO(OH)] (DMAV) were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Monomethylarsonic acid (MMAV) was obtained from Tri Chemicals (Yamanashi, Japan). Fluorescein was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). The ICPMS arsenic standard solution (1,000 μg/mL) was purchased from SPEX CentiPrep (Metuchen, NJ, USA). Stock solutions of all arsenic compounds (10 mmol/L) were prepared from their respective compounds and were stored in the dark at 4 °C. The stock solutions were diluted daily as necessary. Preparation of Monomethylarsonous Acid (MMAIII) and Dimethylarsinous Acid (DMAIII). MMAIII and DMAIII were prepared
by reducing monomethylarsonic acid (MMAV) and dimethylarsinic acid
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(DMAV), respectively, with 5 mol equiv of L-cysteine in distilled water at 90 °C for 1 h. The identities of the trivalent forms were confirmed by comparison to their iodide forms using gel filtration HPLCICP MS.25
Isolation of Subcellular Fractions from Rat Livers and Cells and the Preparation of Submitochondrial Particles. All animal experiments were carried out according to the “Principles of Laboratory Animal Care” (NIH version, revised 1996) and the Guidelines of the Animal Investigation Committee, Showa Pharmaceutical University, Japan. Six week old male Wistar rats were purchased from Clea Japan (Tokyo). The rats were housed in a humidity-controlled room, maintained at 2225 °C with a 12 h lightdark cycle. The animals were fed a commercial diet (MF; Clea Japan) and tap water ad libitum. Following a one-week acclimatization period, rats at 7 weeks of age (body weight, 180220 g) were used for experiments. Whole liver perfusion was performed following the method developed in our laboratory. Briefly, rats under sodium pentobarbital anesthesia were dissected to expose the heart, and then 0.2 mL of heparin was injected into the left ventricle. The remaining blood was perfused through the portal vein by a roller pump with phosphate buffered saline (PBS) at a flow rate of 60 mL/min. Blood-free liver (or cells) was minced in ice-cold homogenization buffer A (230 mM Mannitol, 70 mM sucrose, 10 mM Tris-HCl, 1 mM EDTA 3 2Na, and 0. 5% bovine serum albumin (BSA), pH 7.4) by using a Dounce homogenizer to make 20% (w/v) liver (or cells) homogenates and then kept on ice for 5 min to remove unbroken cells and connective tissues. After removing the unbroken cells, the supernatant was centrifuged twice at 700g for 10 min at 4 °C to obtain the crude cell nuclear fractions. Cytoplasmic and nuclear marker proteins (i.e., GAPDH and p84, respectively) were used to determine the purification of nuclear fractions. Mitochondria were isolated by subjecting the supernatant to centrifugation at 12,000g for 10 min at 4 °C to obtain the pellet. The pellet was washed twice and resuspended in isolation buffer B (230 mM Mannitol, 70 mM sucrose, 10 mM Tris-HCl, and 1 mM EDTA 3 2Na) at a protein concentration of 10 mg/mL. Mitochondria activity was determined by the cytochrome c oxidase (CCO) assay kit (Sigma, USA). Moreover, the pooled supernatant was further centrifuged at 105,000g for 10 min at 4 °C to obtain the pellet fraction as the crude microsome. Submitochondrial particles were prepared from frozen and thawed mitochondria (1020 mg proteins/mL) and disrupted by sonication for three 15 s periods with 30 s intervals at an output of 40 W using an ultrasonic homogenizer (Bio rupter UCD-200, Cosmo BioCo., Ltd., Tokyo, Japan) and then centrifuged at 10,000g for 10 min (4 °C). The supernatant was decanted and centrifuged at 105,000g for 30 min. The pellet (i.e., submitochondrial particles) was washed once and resuspended in isolation buffer B, and the protein concentration was determined according to Lowry’s method using BSA as the standard. Culture of RLC-16 Cells. Rat liver cell line RLC-16 was obtained from the Riken Cell Bank (Tsukuba, Japan). Cells were seeded at a density of 1.0 106 in a 10 cm dish and were maintained in low glucose Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% fetal bovine serum (FBS), 100 U/mL of penicillin, and 100 mg/mL of streptomycin, at 37 °C under a 5% CO2 atmosphere.
Effect of N-Acetylcysteine (NAC) on the Viability of RLC-16 Cells. To investigate the effect of N-acetyl-L-cysteine (NAC) on RLC-
16 cells, the cells were cultured in a 96-well culture dish in the presence or absence of 2 mM NAC for 24 h and then exposed to various concentrations of arsenic compounds (i.e., iAsIII, MMAIII, and DMAIII) for 24 h. Then, 10 μL of an MTT solution was added to each well (at the final concentration of 0.5 mg/mL), and the plates were incubated for an additional 3 h at 37 °C. Afterward, the cell cultures were washed with PBS, and 150 μL of DMSO was added to each well. Cell viability was measured as the absorbance at 570 nm with a microplate reader and expressed as a percentage of the control level (n = 5). 1095
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MTT Assay for Cellular Viability. RLC-16 cells were seeded at a density of 2 104 cells/100 μL/well in 96-well microtiter plates (Promega Corporation). Twenty-four hours postseeding, the cultures were washed twice with PBS and then exposed to various concentrations of trivalent arsenic compounds for 24 h. Then, 10 μL of an MTT solution was added to each well (at the final concentration of 0.5 mg/mL), and the plates were incubated for an additional 3 h at 37 °C. Afterward, cell cultures were washed with PBS, and 150 μL of DMSO was added to each well. Cell viability was measured as the absorbance at 570 nm with a microplate reader and expressed as a percentage of the control level. Arsenic Measurements. Cells were seeded at a density of 1.0 106 cells in a 10 cm culture dish (n = 3) and then exposed to arsenic compounds in fresh medium. After exposure, the cell monolayer was washed twice with PBS. The cells were collected and then suspended in 300 μL of a 100 mM ammonium acetate solution (pH 6.5 at 25 °C; dissolved oxygen was purged by bubbling with nitrogen gas). The suspended cells were disrupted on ice with an ultrasonic homogenizer (Biorupter UCD-200, Cosmo Bio Co., Ltd., Tokyo, Japan) operating at 200 W and 20 kHz for 30 s. This was done three times with intervals of 45 s, followed by centrifugation at 105,000g for 1 h at 4 °C to yield the supernatant (soluble fraction) and insoluble sediment fractions (i.e., precipitates). The concentrations of arsenic in the supernatant and insoluble fractions of cells were determined with an Agilent 7500ce ICPMS (Agilent Technologies, Tokyo, Japan) equipped with an octopole reaction system (ORS) with an He flow of 3.0 mL/min to prevent molecular interference by 40Ar35Cl+ (signal at m/z 75). Prior to analysis, liver samples were wet-ashed with a mixture of concentrated nitric acid and 30% H2O2 (1:1, v/v) at 150 °C for 2 days. Removing Unbound Arsenic Compounds in Supernatants of Cells. The supernatants (500 μL) were dialyzed two times for 6 h each in a Slide-A-Lyzer Dialysis Cassette against 500 mL of 50 mM ammonium acetate buffer (pH 7.4) at 4 °C to remove the unbound arsenicals (i.e., free arsenicals) according to the method reported elsewhere.16 The cell supernatants and the protein-bound arsenic in the remaining dialyzed supernatant were analyzed using ICPMS following wet-digesting with a mixture of concentrated nitric acid and 30% H2O2 (1:1, v/v) at 150 °C for 2 days. Concentration of free arsenic was calculated as supernatant minus dialyzed supernatant, while the concentration of arsenic bound to proteins was calculated as total arsenic minus unbound free arsenic.
Preparation of Decylubiquinol and Reduced Cytochrome c.
The preparation of decylubiquinol was carried out according to Fisher’s method26 with slight modifications. Briefly, 100 μL of 500 mmol/L decylubiquinone was diluted using absolute ethanol to a concentration of 25 mmol/L, followed by the addition of 6 mL of a solution consisting of 0.1 mol/L potassium phosphate buffer, pH 7.4, and 0.25 mol/L sucrose. Then, 1 mL of cyclohexane and 0.1 g of solid sodium dithionite were added to the solution. The resulting mixture was shaken until colorless. The upper layer of cyclohexane was decanted into a separate vial. The aqueous solution was extracted twice with a 1 mL portion of cyclohexane twice, with each organic phase being decanted off and then combined with the initial organic layer. The combined organic phase was evaporated under vacuum until a light-yellow syrup remained in the bottom of the tube. This syrup was dissolved in 900 μL of absolute ethanol and 100 μL of 0.1 mol/L HCl, and stored in aliquots at 80 °C. Reduced cytochrome c was prepared by reducing the cytochrome c with ascorbic acid in 10 mM potassium phosphate buffer (pH 7.0). After reduction, the ascorbic acid was removed by dialysis with the Slide-ALyzer Dialysis cassette (cutoff molecular weight, MW,
