(7.16)--2019 Hepatic and renal tissue da环境与健康环境与健康.pdf

Contents lists available at ScienceDirectFood and Chemical Toxicologyjournal homepage: communicationHepatic and renal tissue damage in Balb/c mice exposed to diisodecylphthalate:The role of oxidative stress pathwaysYingying Chena,b,Chongyao Lia,b,Peng Songa,b,Biao Yana,Xu Yanga,Yang Wua,Ping Maa,aLaboratory of Environment-Immunological and Neurological Diseases,School of Basic Medical Sciences,Hubei University of Science and Technology,Xianning,437100,ChinabSchool of Pharmacy,Hubei University of Science and Technology,Xianning,437100,ChinaA R T I C L E I N F OKeywords:Diisononyl phthalateOxidative stressHepaticRenalA B S T R A C TDiisononyl phthalate(DIDP)is commonly used as a plasticizer in industrial and consumer products,however,itstoxicity remains unclear.This study investigated the possible involvement of oxidative stress in DIDP-inducedliver and kidney toxicity.Liver function and kidney function,tissue lesions,oxidative stress biomarkers,in-flammatory mediators and apoptosis factors were investigated in this study.The results showed that oral ex-posure to DIDP induced a marked increase in lever of alanine aminotransferase(ALT),aspartate amino-transferase(AST),urinary nitrogen(UREA)and creatinine(CREA),decrease in albumin(ALB)level,as well ascausing hepatic and renal histopathological change.Investigation of the role of oxidative stress pathwaysshowed that DBP exposure could lead to a significant increase in levels of reactive oxygen species(ROS),malondialdehyde(MDA),8-hydroxy-2-deoxyguanosine(8-OHdG),interleukin-1(IL-1),tumor necrosis factor-(TNF-)and nuclear factor-B(NF-B),while a decrease in glutathione(GSH)levels were observed.Administration of vitamin E to DIDP-treated mice restored these biochemical parameters to within normal levels,and resulted in less damage to livers and kidneys.Overall,these results suggest that the oxidative stress pathwayis involved in DIDP-induced toxicity.1.IntroductionDiisodecyl phthalate(DIDP)is commonly used as a plasticizer,withmany applications in industrial and consumer products,including butnot limited to food wrap,building materials and toys(Morse,2011;Saravanabhavan and Murray,2012).DIDP molecules are easily emittedfrom materials since they are not tightly bound to the polymer matrix(Halden,2010).A previous study reported that the concentration ofDIDP in dust is 73mg/kg,in soil is 0.007 mg/kg,and in indoor air is2.8 ng/m3(Wormuth et al.,2006).Some studies have already shownthat products containing DIDP can result in human exposure,primarilyvia dermal,oral and inhalation routes,with oral contact being the mainexposure route for humans,particularly for children(Bradley et al.,2013;Giovanoulis et al.,2018).It is therefore important to study anypotential toxic effects of DIDP in vivo.DIDP is not known to affect the reproductive system nor to affectdevelopment(Chen et al.,2014;Patyna et al.,2006).However,thepotential for DIDP to affect the liver and kidney has received con-siderable critical attention,since DIDP predominantly distributes tothese organs after oral ingestion(Saravanabhavan and Murray,2012).Cho et al.reported that higher doses of DIDP can reduce the weigh andlongevity of rats,and can result in the enlargement of theirs livers andkidneys(Cho et al.,2008).There is emerging evidence that exposure toDIDP may result in a higher number of hepatocellular adenomas in themale rasH2 mice(Cho et al.,2011).To date,the mechanisms behindDIDP-induced liver and kidney damage remains poorly understood.Previous studies have reported that the toxicity induced by DIDPmay be attributed to the enhanced production of reactive oxygen spe-cies(ROS),which in turn results in oxidative stress(Qin et al.,2018;Shen et al.,2017).At high concentrations,ROS can be notable med-iators of damage to cell macromolecules,such as lipids,membranes,proteins and nucleic acids(Valko et al.,2006;Ma et al.,2013;Fenget al.,2013a).These alterations probably transform cell function,andeventually lead to tissue lesions and cell apoptosis(Majhi et al.,2011;Feng et al.,2013b,2015).Injuries occur in various organs,includingthe liver and kidney,and toxic reactions are known to be involved ininflammation,fatty liver disease,and diabetic kidney disease(Barceloset al.,2017;Paradies et al.,2014;Jha et al.,2016).However,there ishttps:/doi.org/10.1016/j.fct.2019.110600Received 23 February 2019;Received in revised form 16 June 2019;Accepted 19 June 2019Corresponding author.School of Basic Medical Science,Hubei University of Science and Technology,Xianning,437100,China.Corresponding author.School of Basic Medical Science,Hubei University of Science and Technology,Xianning,437100,China.E-mail addresses:(Y.Wu),(P.Ma).Food and Chemical Toxicology 132(2019)110600Available online 20 June 20190278-6915/2019 Elsevier Ltd.All rights reserved.Tvery little research into the possible involvement of oxidative stress inDIDP-induced liver and kidney damage.We hypothesized that DIDP exposure could lead to oxidative da-mage in hepatic and renal tissues.In this study,Balb/c mice were orallyexposed to DIDP for 14 consecutive days.Liver function was assessed byexamining levels of alanine aminotransferase(ALT),aspartate amino-transferase(AST)and albumin(ALB).Kidney function was evaluated bymeasuring urinary nitrogen(UREA)and creatinine(CREA)levels.Hepatic and renal tissue lesions were observed using Hematoxylin-eosin(H&E)staining.Oxidative stress was indicated by ROS,glutathione(GSH),malondialdehyde(MDA)and 8-hydroxy-2-deoxyguanosine(8-OH-dG)levels.Inflammatory mediators were determined by measuringthe levels of interleukin-1(IL-1),tumor necrosis factor-(TNF-)and Nuclear factor-B(NF-B).Cell apoptosis was evaluated by ex-amining levels of cysteine-aspartic acid protease 3(caspase-3),and byobservation after Hoechst 33258 staining.Additionally,we evaluatedthe antioxidant effect of administering Vitamin E,by looking at thelevels of oxidative stress.Such changes may have implications for thecare of people accidently exposed to DIDP.2.Materials and methods2.1.Animal care and ethics statement5-6 week-old SPF male Balb/c mice were purchased from the HubeiProvince Experimental Animal Center(Wuhan,China)All mice werehoused in pathogen-free cages at 2426C and 12h light-dark cyclewith 55%75%humidity.A commercial diet and filtered water wasprovided ad libitum.Mice were quarantined for at least 7 days prior tocommencing the study.All animal experiments were conducted in ac-cordance with the National Institutes of Health Guide for the Care andUse of Laboratory Animals,and were approved by the Office ofScientific Research Management of Hubei University of Science andTechnology(Xianning,China)with a Certificate approval ID:HBUST-IACUC-2018-001.2.2.Reagents and kitsDIDP(99%),27-dichlordihydrofluorescein(DCFH-DA),2-thio-barbituric acid(TBA),hematoxylin and eosin,Hoechst 33258 andVitamin E were purchased from Sigma-Aldrich(St.Louis,MO,USA).Tween-80 was purchased from Amresco(Solon,OH,USA).MouseELISA kits for 8-OHdG,TNF-,IL-1 and caspase-3 were purchasedfrom eBioscience(San Diego,CA,USA).A colorimetric kit for glu-tathione(GSH)and a Folin-phenol reagent kit were purchased fromJiancheng Bioengineering Institute(Nanjing,China).Colorimetric kitsfor alanine aminotransferase(ALT),aspartate aminotransferase(AST),albumin(ALB),urinary nitrogen(UREA)and creatinine(CREA)werepurchased from iCubio Biomedical Technology(Shenzhen,China).Allother chemicals were of the highest grade commercially available.2.3.Chemical exposure and experimental groupsThe acceptable daily intake(ADI)of DIDP is considered to be0.15 mg/kg/d,a figure proposed by the United States ConsumerProducts Safety Commission in 2010(Kransler et al.,2013).Based onthis,we chose DIDP exposure concentrations of 0.15,1.5,15,150 mg/kg/d for our experiment.Animals were divided randomly into sevengroups of eight mice each,and treated for 14 consecutive days as fol-lows:Saline group;0.15 mg/kg/d DIDP group(DIDP 0.15);1.5 mg/kg/d DIDP group(DIDP 1.5);15 mg/kg/d DIDP group(DIDP 15);150 mg/kg/d DIDP group(DIDP 150);150 mg/kg/d DIDP+100 mg/kg/d Vi-tamins E group(DIDP150+Vit E100);100 mg/kg/d Vitamins E group(Vit E100).Different concentrations of DIDP were prepared in Tween-80(1:1 v/v)and diluted with sterile saline for oral administration.Theconcentration of Tween 80 used in our experiments has been shown inprevious in vivo pharmacological experiments to be inert,and to haveno toxic side effects on the organism(Dimitrov et al.,2011).Therefore,the negative control group was given only saline.The VitE dose levelwas selected based on daily VitE ingestion in normal human life.Thedose of VitE administered was chosen to be 100 mg/kg/d according toYousef et al.(2006).DIDP and VitE were administered via gavage at thesame time every day,After 14 days,all mice were anesthetized in-traperitoneally with pentobarbital sodium(100 mg/kg bw).Serumsamples were then extracted from heart blood by centrifugation(3000 rpmat 4C for 15min)and stored at 70C.Livers and kidneyswere removed for tissue sectioning and for the preparation of ahomogenate.2.4.Liver and kidney functionThe serum levels of ALT,AST,and ALB for liver function,and UREAand CREA for kidney function,were determined by using appropriatekits according to the manufacturers instructions.The OD values wereobtained using an automatic biochemical analyzer iMagic-V7(iCubioBiomedical Technology,Shenzhen,China).2.5.Hematoxylin-eosin stainingThe hepatic and renal tissues were collected and fixed in 10%par-aformaldehyde solution for 24hat room temperature,after which theywere embedded in paraffin and sectioned into 5m slices forHematoxylin-eosin staining.Each section was observed using a DP73microscope(Olympus,Tokyo,Japan).Tissue sections were examinedqualitatively by two experienced pathologists in a blinded fashion.2.6.Preparation of tissue homogenatesThe hepatic and renal tissues were placed in 10mL/g ice-cold 1 PBS(pH 7.5)and homogenized using a glass homogenizer.This homogenatewas centrifuged at 9800g for 10 minat 4C,and the supernatant col-lected and kept frozen at 70 C until needed.The protein concentra-tion of the supernatant was determined using a Folin-phenol assay.2.7.ROS,GSH and MDA assayROS levels in the supernatant were determined by using a generalROS indicator,DCFH-DA,as previously described(Lebel et al.,1992).The supernatant was diluted 50-fold with PBS(pH=7.5),then 100 Lof the dilution was transferred to a 96-well microplate,and 100L of10molL-1 DCFH-DA fluorescent dye was added.This was incubated inthe dark at 37C for 30min.Fluorescence intensity was measured at anexcitation wavelength of 488 nm and an emission wavelength of525nm using a fluorescence reader(Hide Chameleon V,Hidex,Fin-land).GSH is a major scavenger of ROS in tissues.The GSH concentrationin the supernatant was measured using a kit in strict accordance withthe manufacturers instructions.Samples were analyzed using a mi-croplate reader to measure absorbance at 405nm.GSH levels werecalculated according to the formula:GSH(molg-1 prot)=(measureOD405 blank OD405)/(standard OD405-blank OD405)standardconcentration sampledilutionfactor/homogenateproteincon-centration.MDA is a typical biomarker for evaluating lipid peroxidation injury.MDA concentration in the brain supernatant was determined using thethiobarbituric acid(TBA)method(Draper and Hadley,1990).A 0.5mLsample was added to 2mL of 0.6%TBA solution and allowed to react inboiling water for 15min.After cooling with cold water,the mixtureswere centrifuged at 9800g for 10 minat 4C,and the supernatantcollected to measure absorbance at 532,600 and 450nm.MDA levelswereobtainedaccordingtotheformula:MDA(molg-1Y.Chen,et al.Food and Chemical Toxicology 132(2019)1106002prot)=6.45(OD532-OD600)-0.56 OD450/homogenate proteinconcentration.2.8.ELISA assayLevels of 8-OH-dG,IL-1,TNF-and caspase-3 in liver and kidneysupernatants were measured using ELISA kits.All procedures wereconducted according to the manufacturers instructions.Concentrationswere determined in duplicate for each sample.The sensitivities of thekits were 8pg/mL for TNF-,80pg/mL for IL-1,and 1pg/mL for NF-B and caspase-3.2.9.ImmunohistochemistrySections of tissue were incubated with 3%hydrogen peroxide(H2O2)and blocked using an appropriate normal serum of endogenousperoxides.The sections were then boiled in sodium citrate(0.01mol/L,pH 9.0)for antigen retrieval to unmask the antigen epitopes,permea-bilized with 0.2%Triton X-100 for 10min,and blocked with 5%bovineserum albumin(BSA)in phosphate buffer saline(PBS,PH=7.4)for30 minat room temperature.Sections were then incubated at 4Covernight with the monoclonal primary antibodies:mouse antiNFB-antibodies(Abcam,Cambridge,USA).Slides were washed with PBS,incubated with secondary antibodies for 30min at 37C and detectedwith a rabbit IgG peroxidase conjugated streptavidin-biotin complex(SABC-POD)kit,followed by incubation with a diaminobenzidine(DAB)kit.Immunostained sections were viewed under a DP73 micro-scope.The staining intensity was determined as the average opticaldensityusingImage-ProPlus6.0software(MediaCybernetics,Bethesda,MD,USA).A non-stained region was selected and set as thebackground.All tissue sections were examined qualitatively by twoexperienced pathologists in a blinded fashion.2.10.Hoechst 33258 stainingHoechst 33258 staining was performed to capture apoptotic in-duction of cells.Tissue sections were washed 3 times with PBS,thenstained with Hoechst 33258 solution and kept in the dark for 5minatroom temperature.Sections were again washed 3 times with PBS andimmediately imaged using a DP73 fluorescence microscope(Olympus,Tokyo,Japan).2.11.Statistical analysisData are presented as the mean standard deviation(SD).A one-way ANOVA followed by an LSD t-test was used to compare the dif-ferences between groups.A p value 0.05 was considered sig-nificantly different.Data analyses were carried out using IBM SPSSAdvanced Statistics 20(IBM,Armonk,NY,USA).Statistical graphs weregenerated using GraphPad Prism 6.0(GraphPad,San Diego,CA,USA).3.Results3.1.DIDP treatment resulted in liver and kidney dysfunctionWhen compared with the saline group,the DIDP150 group de-monstrated an increase in ALT content(p 0.05)(Fig.1A),and theDIDP15 and DIDP150 groups showed a decrease in AST content(p 0.05)(Fig.1C).A decrease in ALB was found in the serum fromthe DIDP150 group(p 0.01)(Fig.1E).The ALT levels of theDIDP150+VitE100 group were significantly lower than the DIDP150group(p 0.05)(Fig.1B and D).Administering vitamin E alongsideDIDP reversed the decrease in the levels of ALB that were seen in theDIDP150 group(p 0.05)(Fig.1F).An increase in CREA was found in the serum of the DIDP15 group(p 0.05)(Fig.1G).The level of UREA in the serum from theDIDP150 group was significantly higher than that in the saline group(p 0.01)(Fig.1I).Vitamin E administered to mice exposed to DIDPsignificantly attenuated the levels of CREA and UREA compared withlevels in the DIDP150 group(CREA:p 0.05,UREA: