This article was downloaded by: [Blanca Laffon] On: 17 July 2012, At: 04:23 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK Journal of Toxicology and Environmental Health, Part A: Current Issues Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/uteh20 Alterations in Metabolism-Related Genes Induced in SHSY5Y Cells by Okadaic Acid Exposure Vanessa Valdiglesias a b , Juan Fernández-Tajes b , Carla Costa c , Josefina Méndez b , Eduardo Pásaro a & Blanca Laffon a a Toxicology Unit, Department of Psychobiology, University of A Coruña, A Coruña, Spain b Department of Cell and Molecular Biology, University of A Coruña, A Coruña, Spain c Environmental Health Department, National Institute of Health Dr. Ricardo Jorge, Porto, Portugal Version of record first published: 12 Jul 2012 To cite this article: Vanessa Valdiglesias, Juan Fernández-Tajes, Carla Costa, Josefina Méndez, Eduardo Pásaro & Blanca Laffon (2012): Alterations in Metabolism-Related Genes Induced in SHSY5Y Cells by Okadaic Acid Exposure, Journal of Toxicology and Environmental Health, Part A: Current Issues, 75:13-15, 844-856 To link to this article: http://dx.doi.org/10.1080/15287394.2012.690703 PLEASE SCROLL DOWN FOR ARTICLE Full terms and conditions of use: http://www.tandfonline.com/page/terms-and-conditions This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. The publisher does not give any warranty express or implied or make any representation that the contents will be complete or accurate or up to date. The accuracy of any instructions, formulae, and drug doses should be independently verified with primary sources. The publisher shall not be liable for any loss, actions, claims, proceedings, demand, or costs or damages whatsoever or howsoever caused arising directly or indirectly in connection with or arising out of the use of this material.
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This article was downloaded by: [Blanca Laffon]On: 17 July 2012, At: 04:23Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House,37-41 Mortimer Street, London W1T 3JH, UK
Journal of Toxicology and Environmental Health, PartA: Current IssuesPublication details, including instructions for authors and subscription information:http://www.tandfonline.com/loi/uteh20
Alterations in Metabolism-Related Genes Induced inSHSY5Y Cells by Okadaic Acid ExposureVanessa Valdiglesias a b , Juan Fernández-Tajes b , Carla Costa c , Josefina Méndez b ,Eduardo Pásaro a & Blanca Laffon aa Toxicology Unit, Department of Psychobiology, University of A Coruña, A Coruña, Spainb Department of Cell and Molecular Biology, University of A Coruña, A Coruña, Spainc Environmental Health Department, National Institute of Health Dr. Ricardo Jorge, Porto,Portugal
Version of record first published: 12 Jul 2012
To cite this article: Vanessa Valdiglesias, Juan Fernández-Tajes, Carla Costa, Josefina Méndez, Eduardo Pásaro & BlancaLaffon (2012): Alterations in Metabolism-Related Genes Induced in SHSY5Y Cells by Okadaic Acid Exposure, Journal ofToxicology and Environmental Health, Part A: Current Issues, 75:13-15, 844-856
To link to this article: http://dx.doi.org/10.1080/15287394.2012.690703
PLEASE SCROLL DOWN FOR ARTICLE
Full terms and conditions of use: http://www.tandfonline.com/page/terms-and-conditions
This article may be used for research, teaching, and private study purposes. Any substantial or systematicreproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form toanyone is expressly forbidden.
The publisher does not give any warranty express or implied or make any representation that the contentswill be complete or accurate or up to date. The accuracy of any instructions, formulae, and drug doses shouldbe independently verified with primary sources. The publisher shall not be liable for any loss, actions, claims,proceedings, demand, or costs or damages whatsoever or howsoever caused arising directly or indirectly inconnection with or arising out of the use of this material.
1Toxicology Unit, Department of Psychobiology, University of A Coruña, A Coruña, Spain2Department of Cell and Molecular Biology, University of A Coruña, A Coruña, Spain3Environmental Health Department, National Institute of Health Dr. Ricardo Jorge, Porto, Portugal
Okadaic acid (OA) is a widely distributed marine toxin produced by several phytoplank-tonic species and responsible for diarrheic shellfish poisoning in humans. At the molecularlevel OA is a specific inhibitor of several types of serine/threonine protein phosphatases.Due to this enzymic inhibition, OA was reported to induce numerous alterations in rele-vant cellular physiological processes, including several metabolic pathways such as glucoseuptake, lipolysis and glycolysis, heme metabolism, and glycogen and protein synthesis.In order to further understand the underlying mechanisms involved in OA-induced effectson cellular metabolism, the expression levels of six genes related to different catabolicand anabolic metabolism-related processes were analyzed by real-time polymerase chainreaction. Specifically, the expression patterns of GAPDH, TOMM5, SLC25A4, COII, QARS,and RGS5 genes were determined in SHSY5Y human neuroblastoma cells exposed to OAfor 3, 24, or 48 h. All these genes showed alterations in their expression levels afterat least one of the OA treatments tested. These alterations provide a basis to under-stand the mechanisms underlying the previously described OA-induced effects on differentmetabolic processes, mainly regarding glucose and mitochondrial metabolism. However,other OA-induced affected genes can not be ruled out, and further studies are required tomore comprehensively characterize in the mechanisms of OA-induced interaction on cellmetabolism.
Okadaic acid (OA) is a lypophilic marinetoxin produced by several phytoplanktonicspecies and responsible for diarrheic shellfishpoisoning in humans. Okadaic acid is usuallyaccumulated by several molluscs, mainlybivalves such as mussels, scallops, oysters, orclams, as well as by fish, by eating phyto-plankton containing OA. This toxin is widelydistributed globally but is especially abundantin Europe and Japan (FAO 2004). In lastdecades, concern regarding its adverse effects
This work was funded by a grant from the Spanish Ministry of Science and Innovation (PSI2010-15115). V. Valdiglesias was sup-ported by a fellowship from the University of A Coruña. The authors thank the Genomics Service from INIBIC (Complejo HospitalarioUniversitario A Coruña) for providing their facilities. Funding from the New INDIGO program (NanoLINEN – 045-036-073 project,PIM2010ENI-00632) is gratefully acknowledged.
Address correspondence to Vanessa Valdiglesias, Toxicology Unit, University of A Coruña, Edificio de Servicios Centrales deInvestigación, Campus Elviña s/n, 15071-A Coruña, Spain. E-mail: [email protected]
on humans has been increasing especially afterproofs of its activity as tumor promoter in ani-mals and cell death inducer in several systemswere provided (reviewed in Gehringer, 2004).
OA is known to be a potent inhibitorof serine/threonine protein phosphatases (PP)type 1 and 2A. However, Louzao et al. (2005)reported that OA decreased not only PP1 andPP2A activities, but also to a lesser degreeactivities of PP4, PP5, and PP2B. Therefore,OA has the potential to disturb the tightly
METABOLISM-RELATED GENES ALTERED BY OKADAIC ACID 845
regulated equilibrium of phosphorylation anddephosphorylation status, which is importantfor developing relevant physiological processesin the cell, including cell cycle regulation andmodulation of intracellular protein metabolism(Ehlers et al. 2011).
Indeed, OA was shown to affect both dif-ferent catabolic and anabolic metabolic path-ways in intact cells (Leira et al. 2002; Louzaoet al. 2005). Regarding the catabolic processes,OA was found to stimulate glucose trans-port in rat adipocytes (Rampal et al. 1995),increase glucose uptake in primary hepato-cytes (Espiña et al. 2010), and enhance lipol-ysis (Louzao et al. 2005) and glycolysis (Leiraet al. 2002; Tanti et al. 1991) in several celltypes. As for anabolism, OA was reported toalter heme metabolism of human hepatic celllines (Cable et al. 2002) and inhibit the fattyacid synthesis in adipocytes (Louzao et al.2005), glycogen synthesis in cultured rat hep-atocytes (Pugazhenthi et al. 1993), and pro-tein synthesis in different cell types (Matíaset al. 1996; 1999; Redpath and Proud 1989;Waschulewski et al. 1996). However, it was alsoshown to increase gluconeogenesis in hepato-cytes (Louzao et al. 2005). The predominantOA effects on metabolic processes may beexplained by its activity as a PP inhibitor sincevarious enzymes involved in those pathways,such as glycogen synthase or glycogen phos-phorylase, are modulated by phosphorylationor dephosphorylation on serine and/or thre-onine residues (Denton 1986). Specifically,PP1 and PP2A are closely involved in glucoseand glycogen metabolism (Hubbard and Cohen1989; Traoré et al. 2003; Ugi et al. 2004).Further, the existence of OA binding to proteinsother than phosphatases, previously demon-strated in several marine organisms (Schröderet al. 2006; Sugiyama et al. 2007), cannot beruled out in humans.
Toxicokinetic studies in mice concludedthat OA administered by gavage is wellabsorbed by the gastrointestinal tract (GIT) andrapidly distributed in animals (Ito et al. 2002;Matias et al. 1999), even crossing the placen-tal barrier of pregnant mice (Matias and Creppy1996). Several in vivo studies also reported that
OA produced lesions in the liver, small intes-tine, and forestomach after oral administration(Le Hégarat et al. 2006; Tubaro et al. 2004).Further, OA was detected in mouse liver 2 wkafter its administration and excreted in faecesduring at least 4 wk (Ito et al. 2002). All thesestudies suggest that OA may represent a poten-tial risk for the humans, especially for regularshellfish consumers.
Thus, on the basis of the previouslyreported OA-induced effects on differentmetabolic pathways, the aim of the cur-rent study was to elucidate the underly-ing mechanisms associated with these effects.SHSY5Y neuroblastoma cells were treatedwith OA for 3, 24, or 48 h. In total,six genes involved in different metabolism-related (both catabolic and anabolic) processes(GAPDH, TOMM5, SLC25A4, COII, QARS, andRGS5) were selected from suppression subtrac-tive hybridization (SSH) forward and reverselibraries previously performed under this expo-sure conditions (Valdiglesias et al. 2012), andtheir expression levels were analyzed by real-time polymerase chain reaction (PCR).
METHODS
ChemicalsOkadaic acid (OA) (95%, CAS num-
ber 78111-17-8) was purchased from Sigma-Aldrich Co. (Madrid, Spain) and dissolved indimethyl sulfoxide (DMSO) prior to use. Allcompounds used to prepare the SHSY5Y cul-ture media together with the TRIZOL reagentwere purchased from Invitrogen (Barcelona,Spain).
Cell Culture and OA TreatmentOn the basis of previous studies reporting
neurotoxic effects of OA (Ferrero-Gutiérrezet al. 2008; Túnez et al. 2010; Valdiglesiaset al. 2011a; 2011b; 2011c), human neu-roblastoma SHSY5Y cell line was selectedto undertake this study. SHSY5Y cells wereobtained from the European Collection ofCell Cultures and cultured in nutrient mixture
EMEM/F12 (1:1) medium with 1% nonessen-tial amino acids, 1% antibiotic and antimycoticsolution, and supplemented with 10% heat-inactivated fetal bovine serum, all fromInvitrogen (Barcelona, Spain). The cells wereincubated in a humidified atmosphere with 5%CO2 at 37◦C. For OA treatments, cells wereexposed to OA (100 nM) or control (DMSO) at1% of final volume for 3, 24, or 48 h.
Total RNA Isolation and cDNA SynthesisAfter OA treatments, total RNA was isolated
from SHSY5Y cells with TRIZOL reagent follow-ing the manufacturer’s instructions, and thendissolved in nuclease-free water. RNA qualitywas checked by a NANODROP 1000 spec-trophotometer (Thermo Scientific, Madrid,Spain) to confirm integrity of RNA preparations.cDNA synthesis was subsequently performedas previously described (Valdiglesias et al.2012).
Quantitative PCRSix metabolism-related genes were cho-
sen from suppression subtractive hybridization(SSH) forward and reverse libraries previouslyperformed following the same OA exposureconditions as described earlier (Valdiglesiaset al. 2012), for their specific analysis byreal-time PCR: GAPDH, TOMM5, SLC25A4,COII, QARS, and RGS5 (Table 1). These geneswere classified as associated with metabolismaccording to the definition of Gene Ontology
(GO) (http://www.geneontology.org), related tothe aspects of biological and molecular func-tion. Oligonucleotide primers were designedbased on the EST sequences determined forcandidate differentially expressed genes usingthe web tool Universal ProbeLibrary (Table 2).Quantitative PCR was run in triplicate usingLightCycler SYBR green I master kit (Roche)and the LightCycler 480 real-time PCR detec-tion system (Roche). The PCR conditions were95◦C for 10 s, 60◦C for 10 s, and 72◦C for5 s, for 45 cycles, and final extension of 5 min.A subsequent melting-temperature curve of theamplicon was performed. Prior to running sam-ples, efficiency of target amplification was opti-mized for each of the 6 primer pairs by assaying4 primer concentrations (200, 150, 100, and50 nM). LightCycler software 1.5.0 (Roche) wasused for computing the number of amplifica-tion steps required to reach the threshold cyclenumber (Ct). Constant Ct values were observedat a 100-nM final primer concentration foreach of the primer pairs. Software qBasePlus(Hellemans et al. 2007) was used to calculaterelative expression values of each gene. TPR,ACTB, and NM23A were shown to be reli-able reference genes in SHSY5Y (Valdiglesiaset al. 2012) and, thus, used for normalizing theexpression levels. The genes obtained at 3 hof exposure were normalized against both TPRand ACTB, those obtained at 24 h of expo-sure were normalized against both ACTB andNM23A, and finally the ones obtained at 48 hof exposure were normalized against both TPRand NM23A.
TABLE 1. Genes Evaluated in This Study and Metabolic-Related Processes in Which They Are Involved
Genesymbol
Genebanknumber Gene name/description Location Related-metabolic process
Statistical AnalysisStatistical comparisons between groups
were performed using Student’s t-test.Significance was set at p < .05. SPSS forWindows statistical package (version 18.0) wasused for the statistical analysis. Experimentaldata were expressed as mean ± standard error.
RESULTS AND DISCUSSION
OA is the main representative diarrheicshellfish poisoning toxin. The consumption ofOA-contaminated shellfish by humans resultsin gastrointestinal-tract (GIT) distress, nausea,vomiting, and abdominal pain (Tubaro et al.2008). Nevertheless, beyond its role as a diar-rheic toxin, OA was also reported to induceseveral molecular and cellular effects, includingDNA damage, cell cycle alterations, embry-otoxicity, and DNA repair modulations (Ehlerset al. 2010; Le Hégarat et al. 2006; Matiaset al. 1999; Traoré et al. 2001; Valdiglesias et al.2010; 2011a; 2011b; 2011c).
Previously Valdiglesias et al. (2012) per-formed SSH to determine differential geneexpression in SHSY5Y cells exposed to OAfor 3, 24, or 48 h and noted a signifi-cant percentage of altered genes were relatedto electron transport chain and metabolism(between 18% and 26%, depending on the
time exposure). Specifically, among the genesaltered at 3 h of OA treatment, 10% wereinvolved in metabolic processes and 16%related to electron transport chain and redoxhomeostasis. The percentages obtained at24 and 48 h were 13% for both processes inthe first case, and 9%, also for both processes,in the second. On this basis, and taking intoaccount other previous studies in which OAwas found to alter relevant metabolic routes,mainly glycolysis, protein biosynthesis, andoxidative phosphorylation (Ferrero-Gutiérrezet al. 2008; Lago et al. 2005; Leira et al.2002; Matías et al. 1999; Tanti et al. 1991),the possible alterations in expression levelsof relevant genes involved in cell metabolismand electron transport chain and redoxhomeostasis were analyzed in OA-treatedSHSY5Y cells by means of real-time PCR. Sixgenes were selected for the study: GAPDH(glyceraldehyde-3-phosphate dehydrogenase),TOMM5 (translocase of outer mitochondrialmembrane 5), SLC25A4 (solute carrier family25, member 4), COII (cytochrome c oxidasesubunit II), QARS (glutaminyl-tRNA synthetase),and RGS5 (regulator of G-protein signaling 5).All of them showed altered expression patternsfollowing OA treatment (Figures 1–6).
Glucose MetabolismGlucose uptake is one of the princi-
pal phenomena related to metabolic activ-ity since glucose is a fundamental sourceof energy for all eukaryotic cells (Leiraet al. 2002). In rat adipocytes (Lawrenceet al. 1990) and mice skeletal muscle (Tanti
GAPDH
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Control OA (100 nM)
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FIGURE 1. GAPDH gene expression after OA exposure for 3, 24,and 48 h. Bars represent mean standard error. Asterisk indicatessignificant difference from control, p < .05.
FIGURE 2. TOMM5 gene expression after OA exposure for 3, 24,and 48 h. Bars represent mean standard error. Asterisk indicatessignificant difference from control, p < .05.
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SLC25A4
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Control OA (100 nM)
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FIGURE 3. SLC25A4 gene expression after OA exposure for3, 24, and 48 h. Bars represent mean standard error. Asteriskindicates significant difference from control, p < .05.
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COII
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Control OA (100 nM)
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FIGURE 4. COII gene expression after OA exposure for 3, 24,and 48 h. Bars represent mean standard error. Asterisk indicatessignificant difference from control, p < .05.
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QARS
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Control OA (100 nM)
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FIGURE 5. QARS gene expression after OA exposure for 3, 24,and 48 h. Bars represent mean standard error. Asterisk indicatessignificant difference from control, p < .05.
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RGS5
0
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FIGURE 6. RGS5 gene expression after OA exposure for 3, 24,and 48 h. Bars represent mean standard error. Asterisk indicatessignificant difference from control, p < .05.
et al. 1991), OA was shown to mimicinsulin by stimulating 2-deoxyglucose uptake,[3H]glucose incorporation into lipids, and gly-colysis. The OA insulin-like effects may beattributed to its role as PP inhibitor compoundsince insulin, like OA, is also thought to exertits effects on cellular function through thephosphorylation or dephosphorylation of spe-cific regulatory substrates (Corvera et al. 1991).
The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase is encoded byGAPDH gene. This enzyme catalyzes the con-version of glyceraldehyde 3-phosphate into D-glycerate-1,3-bisphosphate, the sixth reactionin glycolysis, in two coupled steps (Laschet et al.2004). In the present study a statistically signif-icant underexpression of this gene was foundat 3 h of OA treatment, but an overexpressionwas observed at 24 h, and no effects at 48 h,suggesting that OA deregulates GAPDH expres-sion in the short term (within at least the first24 h), but then stabilizes and returns to basallevels (Figure 1). These results differ from thosereported by You and Bird (1995), who stud-ied the expression of different genes, mainlyrelated to cell cycle regulation and progressionin HeLa S3 cells, and oberved that GAPDHmRNA levels were not markedly affected by OAtreatment for 24 h.
Espiña et al. (2010) examined the effectsof OA on the metabolic rate and glucoseuptake in different cell types. OA decreasedthe metabolic rate of Clone 9 rat hepatocytesand human BE(2)-M17 neuroblastoma cells.In addition, OA reduced the metabolic activityof primary cultured hepatocytes and increased
METABOLISM-RELATED GENES ALTERED BY OKADAIC ACID 849
glucose uptake in these cells. Other studiesutilizing 24 h of OA exposure demonstratedthat this OA enhanced glucose uptake in manymammalian cells (Leira et al. 2002; Robinsonet al. 1993; Tanti et al. 1991). The rise in intra-cellular glucose levels found in these studiesmay be due to a glycogen synthesis inhibitionor an increase in the externalization rate ofGLUT4, a glucose transporter as suggested byPugazhenthi et al. (1993) and Rampal et al.(1995). The results obtained in our study mayhelp to elucidate the promotion in glucoseuptake found in previous studies, since GAPDHis involved in one of the most important glycol-ysis reactions. Consequently, increased levels ofthis enzyme may lead to stimulating glucosemetabolism and therefore enhancing demandfor this nutrient.
Mitochondrial MetabolismMetabolic intermediates generated during
catabolic metabolism are transported into themitochondria to be converted into energyvia oxidative phosphorylation through electrontransport chain (Yang et al. 2010). Electrontransport chain is located at the mitochondrialinner membrane and composed of a numberof protein complex. Alterations in this chainlead to induction of oxidative phosphorylationenzymes, increased reactive oxygen speciesgeneration, and excessive mtDNA damage(Subramaniam et al. 2008). Mitochondrionis involved in several critical cell functions,and if mitochondria processes are disrupted,not only energy production but also cell-specific products needed for normal cellfunctioning are affected (Hendrickson et al.2010). TOMM5, SLC25A4, and COII genesencode mitochondrial proteins directly relatedto energy metabolism.
Tight coordination between the nucleusand mitochondria is required for maintenanceof mitochondrial function and includes bothanterograde (nucleus to mitochondria) andretrograde (mitochondria to nucleus) signals(Budzinska et al. 2009). The translocase of themitochondrial outer membrane (TOM) com-plex is a multimeric complex that mediates
recognition of preproteins and their transferacross the outer membrane, via a general trans-port pore, becoming the entry gate for the vastmajority of precursor proteins that are importedinto the mitochondria (Schmitt et al. 2005).TOMM5 gene encodes the mitochondrial outermembrane protein TOM5, which is associ-ated with the protein-conducting channel andseems to be crucial for protein import to allmitochondrial subcompartments (Schmitt et al.2005). In yeast, TOM5 appears to be requiredfor maintaining the stability of the TOM com-plex at high temperatures. Subsequently, this isa prerequisite for functionality of pre-proteinimport of the mitochondrial outer membraneprotein translocation machinery (Schmitt et al.2005). In our study a significant decrease in theTOMM5 expression levels was found in cellsexposed to OA for 3 or 24 h; however, nomarked differences with regard to the controlcells were observed at 48 h of OA treatment(Figure 2). Han et al. (2003) suggested thatthe TOM complex may transport the superox-ide radical O2◦− across the mitochondrial outermembrane, and thus changes in TOM proteinsexpression levels in mitochondria coincide withchanges of O2◦− release from mitochondria,leading to an imbalance in the intracellularredox homeostasis (Budzinska et al. 2009).
The mitochondrial carrier family, or thesolute carrier family 25 (SLC25), comprises alarge group of proteins that transport a variety ofsubstrates across the inner mitochondrial mem-brane (Pebay-Peyroula et al. 2003). Becauseof their multitask nature, these proteins areinvolved in several aspects of cell metabolism,as well as in cell survival/death processes, andare related to several pathologies, includingcancer and neurodegenerative diseases (Lenaet al. 2010). All of the characterized SLC25,with the exception of SLC25A17, are local-ized in the inner mitochondrial membrane andtherefore are reported to be mitochondrial car-riers (Visser et al. 2002). SLC25A4 gene encod-ing mitochondrial adenine nucleotide translo-cator type 1 (ANT1), or ADP/ATP translo-cator, is the most abundant protein in themitochondrion (Park et al. 2011) and catalyzesthe last step of oxidative phosphorylation: the
exchange of ATP generated in mitochondria byATP synthase with the ADP produced in thecytosol by most energy-consuming reactions(Palmieri 2004). In humans, four different ANTisoforms were identified. ANT1 is more abun-dant in differentiated tissues and predominantlyexpressed in heart and skeletal muscle (Lenaet al. 2010). In the current study, increasedSLC25A4 expression levels were found at 3 and24 h of OA treatment but not 48 h (Figure 3).Buck et al. (2003) also observed an increase ofANT1 levels in astrocytes in response to cen-tral nervous system (CNS) injury, and postulatedthat the rise in mitochondrial ATP transloca-tor mobilizes mitochondrial ATP stores to ele-vate cytosolic ATP levels, thereby respondingto an increased bioenergetic demand. Severalstudies reported that ANT1 overexpressioninduces apoptosis in a variety of immortalizedfibroblasts and tumor cell lines, not includingglioma cells (Jang et al. 2008; Zamora et al.2004). These authors attributed the proapop-totic effect of ANT1 overexpression to its func-tion in modulating mitochondrial permeabilitytransition pore opening. However, it seems tobe dependent on the cell type, since in astro-cytes a high expression level of ANT1 does notplay a proapoptotic role (Lena et al. 2010).Our previous results in SHSY5Y cells showedthat 100 nM OA treatments for 3 h did notinduce apoptosis in the absence of an externalmetabolic activation system (Valdiglesias et al.2011b). Thus the currently observed overex-pression of SLC25A4 is not related to apoptosisinduction in the short term. The ability of OAto induce apoptosis in SHSY5Y cells after longerOA exposure remains to be investigated.
Cytochrome c oxidase (CO), which takespart in the mitochondrial respiratory chain,is a large transmembrane protein complexwhere subunits are encoded by both nuclearand mitochondrial genes (Poyton and McEwen1996). Pathogenic mutations affecting thisenzyme frequently result in severe, often fatal,energy metabolism dysfunction (Pecina et al.2004). The mammalian CO is composed of13 subunits; the three largest (I, II, and III)are encoded by mitochondrial genes and formthe catalytic core of the enzyme (Pecina et al.
2004). COII gene (also called MTCOII andCOXII) encodes the subunit II of human CO,which transfers the electrons from cytochromec, via its binuclear copper A center, to thebimetallic center of the catalytic subunit I.In this study, a significant decrease in the COIIexpression levels was found at 24 h, but nomarked differences with regard to control cellswere observed at 3 or 48 h of OA treatment(Figure 4). In agreement with these results, Chinet al. (2000) also found altered expression pat-terns in several genes involved in the energymetabolism of OA-treated glioma cells.
OA was previously reported to decreasemitochondrial membrane potential (Kamatet al. 2011; Lago et al. 2005) and increasedoxidative stress in both in vivo (Túnez et al.2003), and in vitro systems (Ferrero-Gutiérrezet al. 2008; Valdiglesias et al. 2011a). Further,Montilla-López et al. (2002) found that expo-sure of cells to 50 nM OA for 2 h diminishedthe activities of cellular glutathione transferase,glutathione reductase and catalase. All thesealterations produce oxidative stress related todevelopment of different pathologies. Theseeffects may be partly explained by alterationsin gene expression found in the current study.Alterations in the electron transport chain usu-ally lead to fall of the mitochondrial membranepotential and production of reactive oxygenspecies (ROS) (Echtay 2007). Modifications inmitochondrial membrane potential were notedto be critical effectors of apoptosis in a varietyof cells (Tatton and Chalmers-Redman 1998).Therefore, the OA-induced apoptosis foundin a number of previous studies (Gehringer2004; Messner et al. 2001; Valdiglesias et al.2011b) might be mediated by the currentlyreported alterations in the expression of capitalgenes involved in the electron transport chain.Further, these effects may also be involved inoxidative stress and DNA oxidative damagenoted previously (Ferrero-Gutiérrez et al. 2008;Túnez et al. 2003; Valdiglesias et al. 2011a).
Protein SynthesisProtein biosynthesis is the anabolic pro-
METABOLISM-RELATED GENES ALTERED BY OKADAIC ACID 851
this process, the enzymes aminoacyl-tRNAsynthetases translate the genetic code by cat-alyzing the specific pairing of amino acidsand tRNAs (Ibba and Söll 2000). At leastone synthetase exists in the cytoplasm foreach amino acid. Glutaminyl-tRNA synthetase(GlnRS) appears to be the largest of the knownmammalian synthetases, and is part of a largemultienzyme complex, which includes at least8 of 20 cytoplasmic mammalian aminoacyl-tRNA synthetases (Rodríguez-Hernández et al.2010). QARS gene (Q, one-letter symbolfor glutamine; ARS, aminoacyl-tRNA syn-thetase) encodes the GlnRS in humans. In thisstudy, QARS expression levels were found todecrease significantly at all OA incubation times(Figure 3). This fall was especially prominentapproximately fivefold reduction after 24 h ofexposure.
In our previous study (Valdiglesias et al.2012), a considerable percent of genes forwhich expression was altered after OA treat-ment were related to transcription and trans-lation processes. Specifically, among the genesaltered at 3 h of OA treatment, 10% wereinvolved in transcription and the same percent-age was related to translation; these percent-ages were 4 and 15%, respectively, at 24 h, and6 and 14%, respectively, at 48 h. Therefore, theinhibition of the protein synthesis induced byexposure to OA found in several studies (Matíaset al. 1996; 1999; Redpath and Proud 1989;Waschulewski et al. 1996) might be explained,at least in part, by alterations in the expressionpatterns of genes related to transcription andtranslation processes, such as QARS.
Regulation of G-Protein SignalingG proteins act as signal transducers and
transmit chemical signals from many hormones,neurotransmitters, and other signalling factors.Thus, G proteins are involved in control of fun-damental life processes in the cells by regulatingimportant parts of the cell machinery, suchas metabolic enzymes. Regulators of G-proteinsignaling (RGS) proteins are a large family ofmammalian proteins that function as GTPase-activating proteins (GAP) for the heterotrimeric
G proteins (Hendriks-Balk et al. 2008). Basedon the homologies within the RGS domain,the RGS proteins may be arbitrarily categorizedinto several distinct subfamilies (Willars 2006).Regulator of G-protein signaling 5 (RGS5) isone member of B/R4 family. RGS5 found to behighly expressed in vascular pericytes of bothhepatocellular carcinoma and renal cell carci-noma (Chen et al. 2004; Furuya et al. 2004),in non-small-cell lung cancer (NSCLC) (Huanget al. 2011), and in ovarian angiogenesis andin granulation tissue of cutaneous wounds (Jinet al. 2009). RGS5 expression level was previ-ously proposed to be used an important prog-nostic factor for gastric carcinoma (Wang et al.2010), for clear cell renal carcinoma (Boss et al.2007; Yao et al. 2008), and as a meaningful pre-dictive biomarker of tumor cell differentiationand metastasis in non-small-cell lung cancer(NSCLC) (Huang et al. 2011).
In our study, no marked OA effects onRGS5 gene expression were observed at 3 h.However, significant decreases in the levels ofthis gene were obtained at both 24 and 48 h(Figure 6). Hamzah et al. (2008) previouslyreported loss of RGS5 results in pericyte matu-ration, vascular normalization, and consequentmarked reductions in tumor hypoxia and ves-sel leakiness. Campbell et al. (2008) showedthat RGS5, together with RGS2, may influ-ence the severity of schizophrenia symptoms,and suggested that RGS proteins are associatedwith an aspect of illness severity that influencespatients’ capacity to respond to antipsychoticdrug treatment. Further, since RGS proteinsare involved in protein translocation and vesi-cles transport processes, RGS5 underexpressionfound in this study may help to understand theresults obtained by Waschulewski et al. (1996),who found that OA interfered in the secretionof newly synthesized proteins and exocytosisprocess in rats.
In summary, the expression patterns ofdifferent metabolism-related genes, specificallyGAPDH, TOMM5, SLC25A4, COII, QARS,and RGS5, were found to be altered by OAexposure. These alterations provide a basisto understand the molecular mechanismsunderlying the effects of this marine toxin on
different metabolic processes, mainly regardingglucose and mitochondrial metabolisms, pre-viously observed in other in vitro and in vivostudies. However, other OA-affected genescannot be ruled out, and further investigationsare required to characterize in depth themechanisms underlying OA interaction withcell metabolism.
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