Study Of Ochratoxin A On Mitochondrial Oxidative Stress Injury In Human Esophageal Epithelium Het-1A Cells

Ochratoxin A(OTA)is a mycotoxin produced by some strains of Aspergillus and Penicillium.This mycotoxin is very common in nature and widely exists in food,beverage,feed and animal tissues.Studies have showed that some biological effects such as hepatotoxicity,neurotoxicity,immunotoxicity and carcinogenicity have been caused by OTA.Due to its natural origin,universal existence and high chemical stability,OTA can be produced and exist for a long time in the process of food processing,storage or transportation.It is inevitable that humans will take more or less OTA every day.The previous study of our research team showed that OTA is the dominant toxin in the most common diet-based contamination mycotoxins among residents in the high incidence area of esophageal cancer and gastric cancer in Hebei Province.Reactive oxygen species(ROS)is a general term of highly bioactive oxygen-containing compounds produced in the process of intracellular aerobic metabolism.It can act as a second messenger to participate in signal transduction and initiate a variety of cellular biological effects.However,when the ROS caused by various exogenous or endogenous factors exceeds the body’s ability to remove them,the ROS level will rise,causing damage to biological macromolecules such as DNA,proteins and lipids in cells,namely oxidative stress.It has been reported that OTA can induce oxidative stress damage in human liver cells and kidney cells.Our previous study found that OTA treatment on human gastric mucosal epithelium(GES-1)cells can cause the increase of ROS and oxidative stress DNA damage.Once oxidative stress occurs in cells,DNA damage may lead to a series of cascading reactions such as death,repair,gene mutation,and even canceration.However,the role of oxidative stress in the injury of esophageal epithelial cells induced by OTA still remains unclear.Mitochondria are the most important organelles in cells,which not only provide energy for cell life activities,but also play an important role in the regulation of cell apoptosis.Mitochondria are not only the main source of ROS,but also the main attack target of ROS.They are easily affected by oxidative stress,leading to structural and functional abnormalities,including the decrease of mitochondrial membrane potential,the change of respiratory chain complex activity,and the decrease of oxidative phosphorylation.Mitochondrial dysfunction can be involved in the activation of caspases cascade reaction and the mitochondrial apoptosis pathways marked by Bcl-2family members(Bcl-2 and Bcl-x L,etc.),as well as Bax and Bad,etc.,and can also cause abnormal energy metabolism pathways by inhibiting the expression of key enzymes of oxidative phosphorylation.Recent studies have found that some carcinogenic substances,including mycotoxins,can induce mitochondrial damage through oxidative stress,leading to cell apoptosis or abnormal glucose metabolism pathway: patulin can induce apoptosis in HEK293 cell by regulating ROS mediated mitochondrial dysfunction and caspase signaling pathway;when aflatoxin B1 induces steatosis in mouse hepatocytes,oxidative stress mediated mitochondrial function impairment and abnormal glucose metabolism pathways including glycolysis and tricarboxylic acid cycle occur.Our previous study found that OTA can induce apoptosis of Het-1A cells.However,it is not clear whether the occurrence of OTA is related to mitochondrial oxidative stress injury.In the meanwhile,the effect of OTA on glucose metabolism in Het-1A cells and the relationship between mitochondrial oxidative stress injury still need to be further studied.Therefore,from the perspective of mitochondria,the target of ROS action,this research further studied the toxic damage effects of OTA on the function,apoptosis and glucose metabolism of mitochondria of human esophageal epithelial cells Het-1A.Objective: The damage effects of OTA on Het-1A cells were observed from several aspects of intracellular ROS production,mitochondrial damage,glucose metabolism and apoptosis.Methods:1.Cell culture and treatmentThe cells were routinely cultured in DMEM supplemented with 100U/ml penicillin,100U/ml streptomycin,and 10% fetal bovine serum(FBS).Het-1A cells in logarithmic growth phase were randomly divided into four groups.The cells were then treated with solvent(methanol)alone or with various concentrations of OTA for 24 h.2.Western blotAfter treated with OTA for 24 h,the expression of SOD2,Rad51,γ-H2 AX,PDHK1,IDH1,OGDH and apoptosis-related proteins at protein level in GES-1 cells was determined by Western blot.3.Real time-PCRAfter treatment,the total RNA of Het-1A was extracted using TRIzol agent,the RNA was reversed transcribed into c DNA with an RT reagent kit,and PCR amplification was then carried out using a SYBR Premix Ex Taq kit.The relative fold induction was quantified using the 2-ΔΔCt method4.Detection of cell viabilityThe cells were incubated with different doses of OTA in serum containing medium for 24 h.Then,cell counting kit-8(CCK-8)method was used to detect cell viability according to the assay kit.5.Isolation of mitochondriaHet-1A cells were collected and sedimented,and the cell suspension was then transferred to an appropriately sized glass homogenizer and homogenized approximately 10-30 times.Then,the cell homogenates were centrifuged twice to obtain the cytosolic and mitochondrial proteins.6.Cell ROS was detected by flow cytometryAfter 24 h of OTA treatment,cells in each group were collected,washed with PBS,and then DCFH-DA probe was added and incubated in dark for 30 min to detect the changes of ROS in cells.7.Detection of SOD activityThe treated cells were collected,SOD sample preparation solution was added to lyse the cells,the supernatant was taken as the sample to be tested,processed according to the requirements of the kit,and the absorbance of the cells was tested with the enzyme label instrument at last.8.Detection of GSH contentsHet-1A cells were collected and centrifuged,and the sediment was collected and washed with ice-cold PBS and then resuspended in protein removal reagent M solution.After two rapid freezing-thawing cycles using liquid nitrogen and a 37°C water bath,the mixture was incubated in an ice bath and centrifuged.The absorbance of the sample(412nm)was detected with a microplate reader.9.ImmunofluorescenceAfter culturing with different concentrations of OTA for 24 h on glass coverslips,the Het-1A cells were fixed with ice-cold acetone,permeabilized with Triton X-100(0.5%)and blocked with 5% normal goat serum.γ-H2 AX protein was labeled using rabbit anti-γ-H2AX(1:200).Alexa 594-conjugated anti-rabbit Ig G at a concentration of 1:1,000 was used as the secondary antibody.Cell nuclei were stained with DAPI.Fluorescent images were examined with a laser-scanning confocal microscope.10.Detection of mitochondrial membrane potential(Δψm)Het-1A cells were seeded into a six-well plate and treated with OTA for24 h,washed twice with PBS and incubated with JC-1 probe(5μM)at 37°C in a dark incubator for 30 min.After washing with PBS,the Δψm was detected by fluorescence microscopy.11.Detection of respiratory chain complex I activityAfter treating with different doses of OTA for 24 h,the Het-1A cells were collected and homogenized on ice.After centrifugation twice,extraction solution was add into the precipitate.Then following ultrasonic treatment,the absorbance at 340 nm was measured by microplate reader,and the activity of respiratory chain complex I was calculated according to the instructions of the kit.12.Glucose and lactic acid measurementCulture medium was collected after treated with OTA for 24 h.The glucose and lactate concentrations within the medium were determined by a glucose assay kit and a lactate assay kit,respectively.Values were normalized to the corresponding protein concentration levels of cells.13.Detection of lipid oxidation(MDA)After exposure for 24 h,Het-1A cells were lysed using cell lysis buffer,and the samples(0.1ml)were then mixed with MDA working solution(0.2ml)and heated for 15 minutes.A microplate reader was used to measure the absorbance(532nm)when the samples were cooled to room temperature.The protein concentration was measured using the BCA Protein Assay Kit.14.Statistical analysisAll data were analyzed by SPSS statistics17.0 statistical software,and the results were expressed by X ± SD.The differences of measurement data among groups were analyzed by one-way ANOVA.Statistical significance was defined as P < 0.05.Results:1.Effects of OTA on the viability of Het-1A cellsAfter treatment with different concentrations of OTA(2.5,5 or 10 μM)for 24 h,Het-1A cells were observed under a microscope.The results showed that the cell numbers in the 5 and 10μM OTA groups were both decreased compared with those in the control group(P < 0.05).The CCK-8 results showed that cell viability was also decreased by OTA(5 and 10μM)in Het-1A cells(P < 0.05).2.The effect of OTA on oxidative stress in Het-1A cellsFlow cytometry showed that After 24 h of treatment with 2.5,5 and 10μM OTA,the ROS levels in Het-1A cells were significantly elevated compared with those in the control group(P < 0.05).Similar to the results of ROS generation,the MDA content in Het-1A cells treated with 5 and 10μM OTA was also significantly increased(P < 0.05).3 Effect of OTA on antioxidant enzymes in Het-1A cellsThe SOD activity in Het-1A cells exposed to OTA was significantly increased(P < 0.05).PCR and western blot assays demonstrated that the m RNA and protein levels of SOD showed similar increasing trends as the activity.However,the changes were not statistically significant(P < 0.05).Conversely,an increase in GSH content was observed in cells treated with various doses of OTA compared with the control group(P < 0.05).4.Effect of OTA on DNA damage in Het-1A cellsWestern blot assays showed that when Het-1A cells were treated with OTA(0-10 μM)for 24 h,H2AX phosphorylation could indeed be induced(P <0.05).This result was confirmed by immunofluorescence staining,which detected the accumulation of γ-H2 AX foci in the nucleus of the OTA(10 μM)treatment group.which confirmed that at the same time as DNA damage was induced by OTA,the m RNA and protein levels of Rad51 were significantly downregulated(P < 0.05).These results further verified the genotoxic effect of OTA.5.Influence of OTA on mitochondrial function of Het-1AJC-1 is often used for the detection of the Δψm.In healthy cells with a normal Δψm,JC-1 dye forms red fluorescent J-aggregates.However,a lack of mitochondrial Δψm leads to a decrease in J-aggregate formation,expressed as green fluorescent J-monomers.Our results showed that in the control group,JC-1 mainly existed in the cytoplasm in the form of J-aggregates(red fluorescent),while in the 10 μM OTA treatment group,the J-aggregates(red fluorescent)decreased and the J-monomers(green fluorescent)obviously increased,indicating the effect of reducing Δψm on Het-1A cells by OTA.Meanwhile,activities of respiratory chain complex I(RCC I)in Het-1A cells treated with OTA were significantly decreased in the 5 and 10 μM groups compared with the control group,further proving the injury action of OTA on mitochondria.6.Effect of OTA on apoptosis of Het-1A cellsFlow cytometry showed that the percentages of apoptotic cells in the 5and 10 μM OTA groups were significantly increased compared with that in the control group(P < 0.05).western blot assay demonstrated that compared with the control group,the expression levels of Bcl-2,Bcl-xl,caspase-3 and caspase-9 were significantly decreased in the OTA exposure groups,while the expression levels of Bax,cleaved caspase-3,cleaved caspase-9 and cytochrome c were significantly elevated(P < 0.05),which emphasized the occurrence of apoptosis.7.Effect of OTA on glycolysis of Het-1A cellsThe influence of OTA on glucose metabolism in Het-1A cells was assessed using a glucose assay kit.The results demonstrated that compared with control cells,10 μM OTA-treated Het-1A cells exhibited significantly increased glucose consumption(P < 0.05).Then,we tested lactate production with a lactate assay kit.The results showed enhanced lactate production in Het-1A cells treated with 10 μM OTA(P < 0.05).The above findings demonstrated that OTA might be involved in the glucose metabolism reprogramming process in Het-1A cells.8.Effects of OTA on enzymes related to glucose metabolism in Het-1A cellsThe expression profiles of IDH1 and OGDH were further examined to evaluate the effect of OTA on the TCA cycle.Compared with the levels in the control group,the expression levels of IDH1 and OGDH were significantly weakened at both the protein and m RNA levels after OTA exposure in Het-1A cells.The results demonstrated that OTA inhibited the oxidative phosphorylation pathway of Het-1A cells by downregulating the expression of TCA cycle-associated enzymes.Conclusion:1.Ochratoxin A(OTA)induces oxidative stress DNA damage in human esophageal mucosal epithelial Het-1A cells.2.Mitochondrial dysfunction mediated by oxidative stress injury is involved in OTA-induced apoptosis.3.OTA induced mitochondrial injury and dysexpression of glucose metabolism-related enzymes led to the transformation of glucose metabolism into glycolysis in Het-1A cells.