Molecular Mechanisms Of Immature Testicular Injuery Due To Ferroptosis Caused By DEHP Exposure

Objectives:To confirm the mechanisms of immature testicular injury due to Diethylhexyl phthalate（DEHP）exposure and the toxicological effects of Mono-（2-ethylhexyl）phthalate（MEHP,a major bio-metabolite of DEHP）exposure on Leydig and Sertoli cells,RNA sequencing and bioinformatics analysis were adopted.Methods:Part I:Male neonatal mice were selected at postnatal day（PND）21.Since we aimed to mimic the prepubertal testicular injury induced by DEHP exposure,all male mice were randomly divided into four groups and treated by oral gavage from PND22 to PND35 with corn oil,or DEHP at a dose of 100 mg/kg/day,250 mg/kg/day,or 500 mg/kg/day.Firstly,the organ coefficients of testis were calculated and histopathology of each mouse were observed using H&E sections to confirm the reproductive toxicity of DEHP on male prepubertal mice.Secondly,the total RNA of mouse testes were extracted and used for RNA sequencing,and the molecular mechanisms of testicular injuries caused by DEHP exposure were investigated using bioinformatics analysis.Part II:Cell lines of Leydig（TM3）and Sertoli（TM4）cells were treated with different concentrations of MEHP:（1）Cell viability and death were examined to evaluate the toxic effect of MEHP on Leydig and Sertoli cells.（2）RNA sequencing and bioinformatics analysis were adopted to investigate the toxicological effects of MEHP exposure on testicular cells.（3）Transmission electron microscopy was used to observe the mitochondrial morphology.Cellular ROS,Fe²⁺,and MDA levels were detected.The expression patterns of ferroptotic molecules at the m RNA and protein levels were also evaluated.At the meantime,a ferroptosis inhibitor（Ferrostatin-1）was used to rescue the MEHP-induced death of Leydig and Sertoli cells.（4）The expression patterns of molecules regarding the HIF-1 signaling pathway were verified at the m RNA and protein levels.Using the cellular immunofluorescence and chromatin immunoprecipitation,we identified that HIF-1α,as a transcriptional factor,could regulate the transcription of Hmox1 gene which was a key molecule in the ferroptosis pathway.（5）CRISPR/Cas9 technique was used to knock out the Hif-1αgene in Leydig and Sertoli cells,and cellular ferroptosis was also evaluated to confirm that HIF-1αplayed an important role in MEHP-induced ferroptosis.Results:Part I:Prepubertal DEHP exposure led to immature testicular injury in mice,which was mainly manifested as organ coefficient reduction and seminiferous tubule abnormality.RNA-seq and bioinformatics analysis revealed that DEHP exposure led to ferroptosis and activation of HIF-1signaling pathway.At the meantime,testicular ferrous iron and MDA levels were elevated.The ferroptotic proteins were also differentially expressed indicating the presence of ferroptosis in testes.Immunofluorescence of testicular section indicated that the level of HIF-1αwas increased in seminiferous tubules and interstitial cells.Part II:MEHP exposure compromised viability of Leydig and Sertoli cells.Bioinformatics analysis revealed that MEHP exposure led to ferroptosis and activation of HIF-1 signaling pathway.Meanwhile,glutathione pathway was also activated and DNA replication was inhibited.The subsequent experiments also verified that:（1）MEHP exposure resulted in abnormal mitochondrial morphology with fewer mitochondrial cristae and denser outer membranes.The levels of cellular ROS,ferrous iron and MDA were also elevated leading to ferroptosis in testicular cells;（2）Ferrostatin-1 reversed ferroptosis induced by MEHP exposure;（3）MEHP exposure caused HIF-1αaccumulation and stabilization,resulted in HIF-1αtranslocation into the nucleus,and induced HIF-1α/Hmox1 binding in Leydig and Sertoli cells;（4）Hif-1αknockout rescued MEHP-induced ferroptosis in Leydig and Sertoli cells.Conclusions:Prepubertal DEHP exposure causes damage to immature testes in mice.HIF-1α/HO-1 signaling pathway mediated ferroptosis is involved in the MEHP-induced injury of Leydig cells and Sertoli cells.