Effect Of Ferroptosis On Testicular Injury Induced By TDCIPP In Adolescent Male Mice

Tri(1,3-dichloro-2-propyl)phosphate(TDCIPP)is a novel environmental endocrine disruptor.With the global prohibition of brominated flame retardants,TDCIPP has rapidly become a replacement product due to its excellent flame-retardant performance,low smoke,and low halogen characteristics.Studies have shown that TDCIPP can exert carcinogenic,neurotoxic,and developmental toxic effects through multiple mechanisms.However,the specific mechanism by which TDCIPP causes damage to the male reproductive system is currently unclear.Ferroptosis,as a new type of cell death,has been demonstrated to be related to male reproductive damage.In this study,animal and in vitro cell models were used to investigate the reproductive damage caused by TDCIPP in adolescent male mice,as well as the role and mechanism of ferroptosis in this process of damage.Part 1.Ferroptosis is involved in testicular injury induced by TDCIPP in male adolescent miceObjective: To investigate the role of ferroptosis in testicular injury of male mice induced by TDCIPP.Methods: Thirty healthy three-week-old male C57BL/6 mice,weighing 13±2g,were selected and acclimated for one week.The mice were randomly divided into five groups: control group,low-dose group,medium-dose group,high-dose group,and ferroptosis inhibitor group,with six mice in each group.The low,medium,and high-dose groups were treated with 5 mg/(kg·d),25 mg/(kg·d),and 125 mg/(kg·d)TDCIPP by gavage for 28 days,while the control group was given an equal amount of corn oil by gavage for 28 days.Body weight was recorded daily.The ferroptosis inhibitor group was treated with 125 mg/(kg·d)TDCIPP by gavage for 28 days,and30 mg/kg of deferoxamine mesylate(DFO)physiological saline solution was injected into the abdominal cavity three times a week.After treatment,the mice were euthanized,and their epididymides were separated for sperm counting.H&E staining was used to observe sperm morphology and calculate the sperm deformity rate.The testes were collected to calculate the testis organ coefficient.H&E staining was used to observe the morphological changes of mouse testicular tissues.The tissue iron detection kit was used to measure the iron ion content in testicular tissues.The ROS assay kit was used to measure the level of reactive oxygen species(ROS)in testicular tissues,and the MDA assay kit was used to measure the MDA content in mouse testicular tissues.The JC-1 assay kit was used to measure the mitochondrial membrane potential in mouse testicular cells.Western blot was used to detect the expression levels of ferroptosis-related proteins GPX4 and COX2 in testicular tissues.Results: Compared with the control group,the weight of mice in the high-dose group decreased significantly from day 21,and the difference was statistically significant(P < 0.05).The arrangement of spermatogenic cells in the testicular tissues of the medium and high-dose groups was disordered,presenting a vacuolar structure,and the number of sperm in the epididymis was significantly reduced,while the sperm deformity rate was significantly increased(P < 0.05).The iron ion content and MDA content in mouse testicular tissues were significantly increased(P < 0.001),and the mitochondrial membrane potential of mouse testicular cells was significantly decreased(P < 0.01).The ferroptosis-related proteins GPX4 levels were significantly decreased(P < 0.05),while COX2 levels were significantly increased(P < 0.01).Compared with high-dose group group,spermatogenic cells in ferroptosis inhibitor group were closely arranged and normal,and ROS and Fe contents in testicular tissue were significantly decreased(P < 0.01);GPX4 protein expression was significantly increased while COX2 protein expression was significantly decreased(P < 0.05).Conclusion: Ferroptosis is involved in testicular tissue damage in male mice induced by TDCIPP during puberty.Part 2.The role of ferroptosis on cytotoxic damage of GC-2 spd induced by TDCIPP exposureObjective: To explore the effect of TDCIPP exposure on GC-2 spd cells,and further explore the role of iron death in the damage of GC-2 spd cells caused by TDCIPP exposure.Methods: The CCK-8 assay was used to determine the effect of TDCIPP on GC-2 spd cell viability and to determine the toxic dose.Based on the results of the CCK-8 assay,GC-2 spd cells were treated with different concentrations of TDCIPP,and the levels of lipid reactive oxygen species(ROS)in GC-2 spd cells were detected using the Liperfluo fluorescent probe.The iron ion content in GC-2 spd cells was measured using a cell iron detection kit.The levels of ROS and malondialdehyde(MDA)in GC-2 spd cells were measured using ROS and MDA detection kits,respectively.The expression levels of the ferroptosis-related proteins HO-1,GPX4,and COX2,as well as the iron autophagy-related proteins NCOA4,ATG5,and LC3 in GC-2 spd cells were detected using Western blotting.Results: The CCK-8 assay showed that compared with the control group,TDCIPP exposure significantly reduced cell survival rate(P < 0.05).The IC50 of TDCIPP was calculated as 77.00 μM [95%CI 72.66,81.52],and subsequent experiments were conducted using TDCIPP concentrations of 0,15,30,and 60 μM.Compared with the control group,the levels of lipid ROS,iron ions,MDA,and ROS in GC-2 spd cells in the 30 and 60 μM TDCIPP-treated groups were significantly higher,and the differences were statistically significant(P < 0.05).Western blotting showed that compared with the control group,the expression levels of GPX4 protein in GC-2 spd cells were significantly decreased,while the expression levels of HO-1and COX2 proteins were significantly increased in the 30 and 60 μM TDCIPP-treated groups(P < 0.05).In addition,compared with the control group,the expression levels of NCOA4 and ATG5 proteins in GC-2 spd cells were significantly increased in the30 and 60 μM TDCIPP-treated groups(P < 0.01),and the LC3Ⅱ/β-actin and LC3Ⅱ/LC3Ⅰ ratios in GC-2 spd cells were significantly increased in the 15,30,and60 μM TDCIPP-treated groups(P < 0.05).Conclusion: TDCIPP can cause iron metabolism disorder in GC-2 spd cells,and thus induce ferroptosis.This process may be related to abnormal ferritinophagy induced by TDCIPP.