Study On Thiol-redox Microenvironment In Platinum-containing Drug Induced Nephrotoxicity

Cisplatin is one of the wildly used antineoplastic drugs and exhibits effective therapeutic efficiency in testicular cancer,ovarian cancer,non-small cell lung cancer and solid tumor of head and neck.However,the severe nephrotoxicity limits its clinical implementation.Thus it is meaningful to clarify the mechanism of cisplatin-induced nephrotoxicity and seek for renal protective drugs.Thioredoxin system is one of the most important antioxidant system.In mammalian cells,it participates in maintaining the balance of redox environment together with GSH system.By transforming electrons to ribonucleotide reductase,thioredoxin and GSH participate in DNA replication and reparation.As a platinum-containing antitumor drug,cisplatin can bind with selenocysteine to inhibit the activity of thioredoxin reductase（TXNRD）,and also interact with cysteine in glutathione（GSH）to reduce the content of GSH,which triggers imbalance of redox microenvironment in the cells.In order to investigate whether cisplatin-induced nephrotoxicity is related to its inhibition on TXNRD and to find viable renal protective drugs,we designed experiments in vitro and in vivo,and compared the inhibitory effects of three platinum drugs on TXNRD.The specific research as follows:1.In vitro inhibition on TXNRD by different platinum drugsThree platinum-containing anticancer drugs（cisplatin,carboplatin and oxaliplatin）were selected for study of WT-TXNRD1 inhibition.Afterwards,mutant TXNRD1（GCUG→GCCG in C terminal active site）was selected for study of action site.The result demonstrated that three platinum containing drugs could inhibit WT-TXNRD1 activity,and cisplatin exhibited the strongest inhibitory effect with IC50 of 35±1.4 n M.Carboplatin and oxaliplatin were 28.8±1.1μM and 3.23±0.15μM respectively..However,the mutant TXNRD1 could not be inhibited,which indicated that the inhibitory effect was selenocysteine dependent.Furthermore,the H₂O₂ production was measured with no increase,indicating that platinum containing drugs could not promote ROS accumulation by TXNRD1.We subsequently detected the glutathione reductase activity,the result demonstrated that GR activity would not be inhibited by platinum containing drugs.2.In vivo inhibition on TXNRD and antioxidant regulation by platinum drugsC57 mice were chosen for model of nephrotoxicity,and were intraperitoneally injected with the three platinum drugs.6 hr and 72 hr after injection,all mice were sacrificed for biological analysis.The result demonstrated that only cisplatin increased serum CREA and BUN level,and HE staining exhibited significant tissue damage.Then,TXNRD activity in different tissues were investigated,and the m RNA levels and protein expression levels were detected.All the results indicated that cisplatin could inhibit TXNRD activity in kidney 6 hr after injection,inhibition rate was 54.25%,for oxaliplatin and carboplatin,it was 27%and 10.37%.After 72 hr of injection,TXNRD in cisplatin treated group was upregulated.The Nrf2 mediated antioxidant regulation was activated and further upregulating TXNRD1 protein level.Moreover,the result demonstrated that the regulation of antioxidant was in a hierarchic manner.Txnrd1 and Homx1 were significantly upregulated,in comparison,Nqo1 and Grx1were lower upregulated.However,Txnrd2,Prxs and GPxs have no significant change,indicated that TXNRD1 is important in cisplatin-induced nephrotoxicity.3.Cisplatin induced renal redox imbalance is time dependentBy investigating cisplatin-induced oxidative stress in different time points,we found that cisplatin induced renal redox imbalance was a time dependent process.Total thiol level and protein glutathionylation was dynamically changed.By detecting m RNA level of antioxidant,we found that Txnrd1 and Homx1 were upregulated 6 hr after treatment,in same time,TXNRD was inhibited.Subsequently,Nqo1 and Grx1 were upregulated.The m RNA level of Gclc,Gclm and Ggt,indicated that GSH is important in cisplatin nephrotoxicity,because GSH synthetase was upregulated and GSH degrading enzyme was downregulated.Txnrd2 and Txn2 have no change in any time course,indicated that the redox environment of mitochondrial matrix was not affected.So that,TXNRD1 inhibition induced antioxidant regulation was fastest and most robust which may become the principal factor of cisplatin nephrotoxicity.Cisplatin treatment also induced TXN1 oxidation,and was not reverse even TXNRD activity was increased,this might induce ASK1 mediated apoptosis.4.glrx2 knockout in cisplatin induced nephrotoxicityProtein glutathionylation is a significant manifestation in cisplatin induced nephrotoxicity.Grx2is a key factor in regulation of protein glutathionylation,particularly in mitochondria.So we investigated glrx2 knockout in cisplatin induced nephrotoxicity.The result demonstrated that glrx2knockout upregulated glutathionylation level but did not increase serum CRAE,BUN,AST and ALT level,indicated that Glrx2 knock out will not affect cisplatin induced nephrotoxicity or hepatotoxicity.Furthermore,TXNRD,GPx and Grx activity and GSH content were not affected.5.Selenomethionine and selenite attenuated cisplatin induced nephrotoxicityTheoretically,selenium supplement may able to attenuate cisplatin induced nephrotoxicity because cisplatin inhibits TXNRD activity by interacting with its selenocysteine.By supplementing selenium in drinking water,mice were pre-administrated with selenomethionine and selenite for 11days,and then were i.p.injected with cisplatin.The preliminary result indicated that selenomethionine and selenite were able todecrease CREA and BUN level,indicated selenium supplementation could attenuate ciaplatin induced nephrotoxicity,and HE staining demonsatrated the same conclusion.Furthermore,selenomethionine and selenite increased GSH content and affected TXNRD and GPx regulation.Selenium supplementation could reduce cisplatin-induced protein glutathionylation,indicated selenomethionine and selenite can attenuate cisplatin induced nephrotoxicity by affecting tenal redox environment.However,the potential mechanism needs further investigation.In conclusion,our study demonstrated that cisplatin could inhibit TXNRD activity,and interupting intracellular redox imbalance.But the antioxidant system could not reverse oxidative stress,so activated ASK1 induced cell death.On the other hand,pre-administration of selenomethionine and selenite could alleviate cisplatin induced nephrotoxicity,but the potential mechanism needs further investigation.Our studies demonstrate the mechanism of cisplatin induced nephrotoxicity targeting thiol redox systems.Furthermore,two potential compounds in renal protection were found,which may provide a new approach for the clinical application of cisplatin detoxification.