Putative Role Of Oxidative Damage On Ochratoxin A Induced Cell Cycle Arrest In Vitro

Mycotoxins are the secondary metabolites produced by different fungithat contaminate a large variety of grains and feedstuffs in the world, whichcan cause several health problems. Zanhuang County is one of the highincidence areas of gastric cancer in north China with an annual gastric cancermortality being77.67/100,000/year. Our previous study showed thatmycotoxin Ochratoxin A (OTA) in wheat samples reached to2.41μg/kg inthis area, which was significantly higher than that of provisional tolerableweekly intake allocated by the Joint FAO/WHO Expert Committee on FoodAdditives (JECFA).OTA is a mycotoxin considered of concern for human health. It is producedby a number of Aspergillus and Penicillium fungal species known to colonizea range of food commodities including cereals, wine, spices, dried fruits, grapejuice, as well as animal products. When ingested as a food contaminant, OTAis a persistent toxin with a blood half-life of35days following a single oraldose. Epidemiological studies have indicated that OTA might contribute to theetiology of some sporadic diseases such as the Balkan endemic nephropathy,chronic interstitial nephropathy, and urothelial tumors. Under experimentalconditions, OTA had a diverse range of toxicological effects, includingnephrotoxicity, teratogenicity, immunotoxicity, neurotoxicity andhepatotoxicity. Therefore, OTA was classified as a possible human carcinogen(group2B) by the International Agency for Research on Cancer.It has been generally accepted that the induced cell cycle arrest andapoptosis is the important bioeffects of many carcinogenic mycotoxins. Ourprevious study showed that OTA could induce G2phase arrest and apoptosisin immortalized human gastric epithelial cells (GES-1). We also found that theactivation of ERK, p38pathways and ATM pathway were involved in OTA-induced G2arrest. However, the detailed molecular mechanism of howOTA trigerring cell cycle arrest through ATM and MAPK signaling is stillunknown.It looks mostly accepted that oxidative damage is a critical event in theinitiation and development of carcinoma. Mutations and/or acquired defectsbrought about by DNA damage are thought to underlie the development andprogression of cancer. Oxidative stress is elicited by reactive oxygen species(ROS) generation. Oxidative stress can trigger cell damage by oxidizingbiomolecules including that of lipids, proteins and DNA, and modify theirbiological functions that ultimately cause cell cycle arrest and cell apoptosis.Mitochondria are the major source and at the same time are targets of ROS;this 'vicious cycle' leads to an accumulation of damages to several moleculesincluding the mitochondrial DNA (mtDNA). Several studies have shown thatoxidative DNA damage plays an important role in the toxins-induced cellcycle arrest. Amongst the mechanisms of OTA carcinogenic, oxidative stresshas been highlighted as one of the most probable by JECFA. A number ofstudies have demonstrated that OTA could result in oxidative stress associatedwith the production of ROS in different cells through various direct andindirect mechanisms. Thus, OTA-induced oxidative DNA damage mightcontributes to OTA-induced cells cycle arrest, which associated with thedevelopment of gastric cancer.Thus based on our previous study, the current study first evaluated theeffects of OTA on ROS production and DNA damage in GES-1cells,as wellas the role of oxidative stress in OTA-induced G2phase arrest through ATMand MAPK pathways in GES-1cells. Furthermore, the effects of OTAexposure on mitochondria damage in GES-1cells were investigated.The microenvironment, especially the immune system is playing animportant role in the development and metachoresis of carcinoma. Resentstudys indicated that mycotoxins could lead to immunosuppressive effects,which may be associated with an increased susceptibility to tumors.Finally, we explored the putative toxicological effects and related mechanism of OTA on human peripheral blood mononuclear cells (hPBMC).Our study may provide new data to elucidate its possible epigeneticmechanism of OTA hazard bioeffects and carcinogenicity. Our findings in thisreport provide new insights in the possible carcinogenic mechanism of OTAexposure in human gastric cancer.PartⅠOchratoxin A induced Oxidative stress involved in G2arrest inGES-1cells in vitroObjective: Based on our previous study, the current study systematicallyevaluated the role of ROS production, DNA damage, as well as oxidativestress-mediated ERK, p38and ATM activation on OTA-induced G2phasearrest in GES-1cells.Methods: GES-1cells were treated with5,10and20μM OTA orpre-treated with4mM NAC plus10μM OTA for24h.1ROS were detectedby staining the cells with DHE or DCFH-DA.2Total SOD activity wasdetermined by a SOD detection kits.38-OHdG was assayed by HPLC-ECD.We detected the generation of γ-H2AX foci by immunofluorescence stainingand assessed the expression of γ-H2AX using Western blot.4The percentageof cells in each phase of the cell cycle was determined using flow cytometry.5Effects of OTA on expression of Cdc25C, Cdc2, cyclin B1, ERK, p38andATM were analyzed by Western blot.6The effect of OTA on theCdc2-cyclinB1complex was detected by immunoprecipitation.Results:1.1Effect of OTA exposure on intracellular ROS levelThe results demonstrated that both of DCF and DHE mean fluorescenceintensity (MFI) were notably increased after OTA exposure for24h (P<0.05).Pretreatment of GES-1cells with NAC, a well-established antioxidant, greatlyinhibited OTA-induced increase in MFI of DCF and DHE (P<0.05). Inconclusion, OTA caused increases in intracellular steady-state levels of ROS(i.e., superoxide and hydroperoxides) in GES-1cells.1.2Effect of OTA exposure on SOD activitySOD activity was significantly increased in the OTA-exposed groups (P< 0.05). OTA-induced the increase of SOD was prevented by NAC pretreatment.1.3Effects of OTA exposure on oxidative DNA damageTo further determine OTA induced oxidative DNA damage in GES-1cells,we determined the levels of8-OHdG, as a sensitive marker of oxidative DNAdamage, in OTA treated cells. The results showed the levels of8-OHdG inOTA treatment groups were significantly higher than that of control group(P<0.05), which suggested that OTA could induce oxidative DNA damage inGES-1cells.Among different types of DNA damage, double-DNA breaks are arguablyone of the most deleterious lesions. γ-H2AX is a reliable and exquisitelysensitive marker for this lesion. We observed that OTA could induce theaccumulation of γ-H2AX foci in nucleus after20μM OTA treatment byimmunofluorescence staining. In addition, Western blot results showed thatOTA could significantly increase the expression of γ-H2AX in GES-1cells(P<0.05). It was in consistent with the result of immunofluorescence staining,further confirmed that OTA exposure causes double-DNA breaks in GES-1cells.To further confirm that OTA-induced oxidative stress trigerred the DNAdamage, NAC was applied prior to treatment with10μM OTA in GES-1cells.The result showed that pre-treatment with NAC resulted in a significantreduction in the expression of γ-H2AX (P<0.05).Taken together, these results confirmed that OTA could induce oxidativeDNA damage in GES-1cells.1.4Antioxidants NAC blocked OTA-induced activation of ATMHere, to dissect the role of oxidative stress in OTA-induced activation ofATM, GES-1cells were pre-incubated with NAC. Western blot result showedthat NAC resulted in a significant reduction in OTA-induced the up-regulationof ATM phosphorylation (P<0.05).1.5Antioxidants NAC blocked OTA-induced phosphorylation of ERKand p38MAPKOur previous study demonstrated that ERK and p38MAPK signaling pathways were involved in the regulation of OTA-induced G2arrest in GES-1cells. To investigate whether oxidative stress in response to OTA regulatedERK and p38MAPK activation, GES-1cells were pre-treated with NAC. Wefound that NAC markedly reduced OTA-induced ERK and p38MAPKphosphorylation (P<0.05). The results showed that OTA-induced oxidativedamage activated ERK and p38MAPK pathway in GES-1cells.1.6Antioxidants NAC abolishes OTA-induced G2arrest in GES-1cellsFor evaluate whether ROS-induced DNA damage may contribute toOTA-induced G2arrest through MAPK and ATM pathway, we pre-treatedGES-1cells with NAC to examine the role of OTA on G2arrest in GES-1cells.The result showed that pre-treatment with NAC was associated with areduction of cells arresting at the G2/M cell cycle phase (P<0.05). In addition,Western bolt results showed that OTA caused a significant down-regulation ofG2/M phase related proteins (Cdc25C/p-Cdc25C, Cdc2/p-Cdc2and cyclinB1)and the cyclinB1-Cdc2complex in GES-1cells, which were abolished byNAC (P<0.05).Taken together, the results indicated that OTA-induced oxidative damageregulated G2arrest in GES-1cells through ERK and p38MAPK and ATMsignaling pathway.PartⅡThe effects of OTA exposure on mitochondria damage in GES-1cellsObjective: To explore the effect of OTA on mitochondria DNA andmitochondrial function in GES-1cells.Methods:1MnSOD activity was determined by a SOD detection kits.28-OHdG was assayed by HPLC-ECD.3Mitochondrial genes encodedproteins mRNA expression was determined by Real-time quantitative PCR.4The activity of respiratory chain complex Ⅰ were assayed using adetection kits.5Mitochondrial respiratory function was measuredpolarographically at25℃using a Clark-type oxygen electrode.6The level ofmitochondrial membrane potential was determined using amitochondria-sensitive dye Rhodamine123by flow cytometry.7To quantify the OTA-induced apoptotic death of GES-1cells, Annexin V and PI stainingwas performed by flow cytometry.8The espression of MnSOD, OGG1, Bax,Bcl-2, Bcl-xL, cytochrome c and caspase-9were measured by Western blotanalysis.Results:2.1Effect of OTA on MnSOD in GES-1cellsMnSOD represents the first line of cell defence againstmitochondria-derived ROS. We measured directly the activity of the enzymein OTA-exposured cells and found OTA (5,10and20μM) induced significantincreases in MnSOD activity (115.26±9.83,162.63±13.18,285.04±3.10U/mgprotein vs64.73±4.42U/mg protein, P<0.05). Western blot analysis alsorevealed that OTA treatment for24h caused a significant increase in MnSODexpression in GES-1cells (P<0.05). Thus, the elevated of MnSOD activity inOTA groups might point to an adaptive reaction to oxidative stress.2.2Effect of OTA on8-OHdG in mtDNAConcerning oxidative damage to mtDNA, namely8-OHdG, significantincreases were observed in OTA groups compared with control group(P<0.05).2.3Effect of OTA on the expression of mitochondrial OGG1protein8-oxoguanine glycosylase1(OGG1), a key base excision repair enzyme,plays a key role in the removal of8-OHdG adducts. Western blot analysis,compared to control group, showed that OTA led to strong depression of theamount of mitochondrial OGG1protein in OTA-treated groups (P<0.05). Thisdata suggested that OTA inhibited the mitochondrial base excision repair.2.4Effect of OTA on mitochondrial gene expressionReal-time PCR studies detected mRNA transcription corresponding to13mitochondria genes encoded protein in complex Ⅰ, Ⅲ, Ⅳ, and Ⅴ ofmitochondrial respiratory chain. We observed significant increases intranscription level of ND1, ND2, ND3, ND4L, ND5, ND6and ATP6in20μMOTA group (Fold change＞1.5).2.5Effects of OTA on the activity of mitochondrial respiratory chain complex I.The enzymatic activity related to complex I was performed onmitochondrial fraction prepared from GES-1cells. Our findings documented asignificant decrease in the specific activity of complex I following5,10and20μM OTA treatment for24h (P<0.05).2.6Effect of OTA on mitochondrial respiratory functionOTA induced a significant decrease in state3respiration rate in5,10and20μM OTA groups compared with control group (P<0.05). Meanwhile,mitochondrial RCR values were significantly decline in10and20μM OTAgroups (P<0.05), suggesting OTA impairs mitochondrial respiratory function.2.7Effect of OTA on apoptosis of GES-1cellsOxidative stress, mitochondrial damage and disrupted mitochondrialrespiration have been found to promote cell death, functional failure, anddegeneration. Thus, we investigated whether mitochondrial damage isinvolved in apoptosis caused by OTA in GES-1cells.2.7.1Effect of OTA on mitochondrial membrane potential (ΔΨm)Mitochondrial dysfunction characterized by a loss of transmembranepotential is one of the earliest intracellular events leading to cell damage. Inthis study we evaluated mitochondrial ΔΨm as an indicator of mitochondrialhealth in cells treated with OTA at different concentrations for24h. OTAexposure significantly decreased Rhodamine123MFI compared to the control(P<0.05). In order to ascertain whether ROS were involved in the alteration ofΔΨm, the effects of OTA on ΔΨm were evaluated in presence or absence ofNAC. The result showed that NAC treatment counteracted the effect of OTA.2.7.2NAC protected OTA induced apoptosis of GES-1cellsTo investigate the possible role of ROS in OTA-induced apoptosis, theeffects of specific modifiers of ROS on apoptosis were determined. Theapoptotic rate in NAC pretreatment with OTA group was5.56±1.80%significantly lower than that in only OTA treatment group12.67±2.12%(P<0.05).2.7.3Effects of OTA on the regulatory factors of mitochondrial pathway, with NAC pretreatmentTo investigate the mitochondrial apoptotic events involved in OTA-inducedapoptosis, we first analyzed the changes in the levels of pro-apoptotic proteinsBax and anti-apoptotic proteins Bcl-2and Bcl-xL. Immunoblot analysisshowed that treatment of GES-1cells with OTA increased Bax protein levels.In contrast, OTA decreased Bcl-2and Bcl-xLlevels.To elucidate whether the release of cytochrome c from mitochondria wasinvolved in OTA-induced apoptosis, mitochondria and cytosolic fractionswere prepared from GES-1cells. We found the release of cytochrome c intocytosol was detected relative to gradual decrease in mitochondrial cytochromec.Caspase-9is activated in response to cytochrome c. Proteolytic cleavage ofprocaspase-9observed in OTA-treated cells.Bax increase, Bcl-2and Bcl-xLreduction in response to OTA were blockedby NAC pretreatment. It was also found that NAC has abrogated the OTAinduced cytochrome c release and caspase-9activation.All the results indicated that OTA induced the execution of apoptosisthrough activation of the mitochondrial pathway and ROS were probablyinvolved in OTA-induced apoptosis in GES-1cells.PartⅢ Ochratoxin A induces oxidative DNA damage and G1phase arrestin human peripheral blood mononuclear cells in vitroObjective: To explore the putative toxicological effects and relatedmechanism of OTA on hPBMC.Methods:1The level of intracellular ROS (e.g. superoxide andhydroperoxides) was estimated by oxidations of DCFH-DA and DHE. Themean fluorescence intensity (MFI) was detected by a FACS flow cytometer.2The intracellular content of glutathione (GSH) was assessed using a reducedGSH assay kit.3To investigate the possibility of types of DNA damage, weperformed a comprehensive analysis of OTA-induced DNA damage.8-OHdGwas assayed by HPLC-ECD. Alkaline Comet Assay was performed todetermine whether OTA induce DNA damage. After that, the property of the stand breaks was further analyzed by the detection of γ-H2AX protein byWestern blot.4Flow cytometry was used to analyze cell cycle and Westernbolt measured the expression of cyclinD1and CDK4protein.5The cells werestained with PI and analyzed by flow cytometry. Apoptosis was quantified asthe percentage of cells containing hypodiploid amounts of DNA (SubG1peak).In addition, cells were analyzed for apoptotic nuclei fluorescence staining withHoechst33258.6hPBMC were pre-treated with4mM NAC for1h, awell-established antioxidant, followed by20μM OTA for24h. Intracellularlevels of ROS and GSH, DNA damage, cell cycle and apoptosis inOTA-induced cells were assessed as previously described.Results:3.1OTA induced increased steady state levels of superoxide andhydroperoxides in hPBMCThe state levels of intracellular superoxide and hydroperoxides weremeasured in hPBMC after treated with different concentrations of OTA for24h using FCM assay. The mean fluorescence intensity of DCF was significantlyincreased with5,10and20μM OTA treatment (P<0.05). We also found thatOTA increased the MFI of DHE in hPBMC (P<0.05).To further confirm that OTA induces the increased ROS generation inhPBMC, NAC was applied as a blocker for the increased ROS. The data fromthe flow cytometry analysis showed the increased mean fluorescenceintensities of DCF and DHE in20μM OTA-treated hPBMC were significantlyattenuated by NAC pre-treatment (P<0.05). All these results showed thatincreased oxidation of DHE and DCFH suggesting increases in steady statelevels of superoxide and hydroperoxides in hPBMC by OTA.3.2OTA decreased intracellular GSH in hPBMCWe further measured the intracellular content of glutathione (GSH) inhPBMC after OTA exposure for24h. The results showed that a significantdecrease of intracellular reduced GSH content in OTA-treated in hPBMCcould be found (P<0.05). We also found that NAC effectively blocked thedecrease in GSH induced by OTA (P<0.05). 3.3OTA induced DNA damage in hPBMCWe further evaluated whether the accumulated ROS in hPBMC by OTAtreatment could induce DNA damage.8-OHdG has been established as animportant biomarker of oxidative DNA damage. HPLC-ECD results indicatedthat the level of8-OHdG was significantly higher in OTA treatment groupsthan that in control group (P<0.05).The comet assay was performed under alkaline conditions for the detectionof a broad spectrum of DNA lesions. Treatment with OTA caused a significantincrease in%Tail DNA, Tail length and Olive tail moment (P<0.05). Next, wedetected γ-H2AX protein expression, a maker of DNA double-strand breaks.Treatment of hPBMC with5,10and20μM OTA resulted in the up-regulationof γ-H2AX protein in a dose-effect manner (r=0.998, P<0.05). Pre-treatmentwith antioxidant reagent NAC resulted in a significant reduction in DNAdamage as well as γ-H2AX protein expression in20μM OTA-treated group(P<0.05). All these results confirmed that OTA-induced ROS contributed tooxidative DNA damage in hPBMC.3.4OTA induced cell cycle arrested at G1phase in hPBMCAs we know that DNA damage is often accompanied by arrest in cell cycle,so we detected cell cycle arrest in hPBMC using flow cytometry analysis. Incomparison with the control group, the proportion of cells in G1phase wasaccumulated markedly after treated with10and20μM OTA for24h (P<0.05).To estimate the molecular mechanism accounting for cell cycle arrest in G1phase, G1-associated regulatory proteins (CDK4and cyclinD1) were furtherexamined. Western blot analysis demonstrated that cyclinD1and CDK4protein expression were both markedly decreased in hPBMC treated withdifferent concentrations of OTA (P<0.05).To determine whether ROS-induced DNA damage may contribute toOTA-induced G1arrest, hPBMC were pre-treated with the antioxidant NAC.We observed NAC inhibited OTA-induced G1arrest, which demonstrated thatOTA induced G1phase arrest is in part mediated through ROS-accumulationoxidative DNA damage (P<0.05). 3.5OTA induced apoptosis in hPBMCIn order to quantify the extent of apoptosis, the content of DNA in cells wasmeasured by flow cytometry. We found hPBMC exposed to5,10and20μMOTA for24h showed significant increase in cells in SubG1phase (P<0.05). Aremarkable feature of apoptosis is the condensation and fragmentation ofnuclear chromatin, which can be observed under fluorescence microscopeafter staining with Hoechst33258. Within24h of treatment with20μM OTA,hPBMC exhibited significant morphological changes and chromosomalcondensation, which was indicative of apoptotic cell. Furthermore,pre-treatment with NAC partly protected OTA-induced apoptosis in hPBMC(P<0.05).Conclusions:1OTA induced oxidative stress damage in GES-1cells.2OTA-induced oxidative damage regulated G2arrest in GES-1cellsthrough ERK and p38MAPK and ATM signaling pathway.3OTA induced oxidative mtDNA damage, inhibited the mitochondrial baseexcision repair and impaired mitochondrial function, which acted as thetrigger in OTA induced oxidative stress in GES-1cells.4Mitochondrial damage is involved in apoptosis caused by OTA in GES-1cells.5OTA-induced oxidative DNA damage caused G1phase arrest andapoptosis in hPBMC, which indicate that oxidative stress is involved inOTA-induced human immunotoxicity.6OTA-induced oxidative damage mediated cell cycle arrest and apoptosis ingastric epithelium cells and hPBMC, which might contribute to a possiblecarcinogenic mechanism of OTA exposure in human gastric cancer.