Effects Of N-monoacetyl-p-phenylenediamine On Ovary In Mice

ObjectiveP-phenylenediamine（PPD）is the main constituent of permanent hair dye,and is widely used in the dye,photographic,and rubber industries,the main route of human exposure to PPD is skin contact.N-acetyltransferases-1 in the epidermis converts PDD into MAPPD,resulting in systemic exposure to MAPPD.Studies have shown that PPD is harmful to humans,while PPD metabolites MAPPD play an effect in the body;however,the effects of MAPPD internal exposure on female reproduction have not been reported.This study established a model of MAPPD toxicity in mice to explore the effects of MAPPD on female reproduction and related mechanisms,and provide a theoretical basis for exploring the toxic effects of metabolites and related mechanisms for future research.Methods1.In vivo experiments:（1）The KM mouse MAPPD exposure model was established.Sixty female Kunming mice（8–10-weeks-old,28–30g）were randomly assigned to three groups of 20 mice each:one control group（methylcellulose）and two MAPPD treated groups（100 and 300 mg/kg）.They were dosed once daily by gastric intubation for 28 days.At the end of the MAPPD exposure period,six mice in each group（female:male=2:1）were pair-housed.The time of pregnancy was defined as gestation day（GD）0.5,when a vaginal plug was observed.The uterus was collected on GD3.5 days,and the remaining mice received mouse ovarian tissue at the same stage of the emotional cycle.（2）At GD 3.5,the uterus of the mice was removed,and 2m L normal saline was injected into the uterine horn to flush the uterine cavity.The normal saline was collected in a clean plate,the morphology and structure of the early embryos were observed under a microscope.The inner cell masses,zona pellucida and blastocyst cavity were observed in the normal blastocyst.The mouse ovarian coefficient was counted.（3）After Hematoxylin and eosin（H&E）staining,ovarian morphology was observed,all follicles were counted.The levels of estrogen and progesterone in the serum and ovary tissue were quantified using ELISA kits;FSH,LH,and T levels in the serum were also measured.We also used ELISA to assess the levels of factors involved in oxidative stress,including ROS,MDA,SOD,CAT,and GPX.The changes of FSHR and LHR in mouse ovarian tissue were detected by immunohistochemistry and PCR.（4）The changes of apoptosis in ovarian tissue were detected by TUNEL,and the changes of BAX and BCL-2 in ovarian tissue were detected by Western blot.2.In vitro experiments:（1）MAPPD exposure model of human ovarian granulosa cell line（KGN）was established.KGN cells were cultured with MAPPD of 0,100and 300μg/m L.（2）Changes in cell activity were detected by CCK8.Detection of intracellular changes of reactive oxygen species using ROS kit.（3）The m RNA expression levels of FSHR,LHR,P450arom,P450acc,and St AR were detected using q RT-PCR.（4）The changes of apoptosis were detected by flow cytometry.Western blot were used to detect changes in protein levels in BAX,BCL-2and MAPK family members ERK,JNK,and P38.Results1.In vivo experiments:Compared with the control group,the number of abnormal blastocysts increased.The number of antral follicles decreased,the levels of E₂ and P₄ decreased in ovarian tissue.The serum levels of E₂,P₄,LH,and T decreased,and FSH increased.The expression of follicle-stimulating hormone receptor（FSHR）and luteinizing hormone receptor（LHR）was significantly down-regulated and the level of oxidative stress was significantly increased.The apoptosis level in ovarian granulosa cells increased significantly,the expression of BAX protein in ovarian tissue increased,but the expression of BCL-2 decreased.2.In vitro experiments:Compared with the control group,the level of ROS in the MAPPD treatment group increased in a dose-dependent manner.The m RNA levels of FSHR,LHR,and aromatase increased in the MAPPD treatment group.The results of flow cytometry showed that the level of apoptosis increased significantly.P-ERK/ERK expression level increased significantly.ConclusionMAPPD exposure can reduce the level of serum and ovarian hormone secretion in mice,and cause abnormal follicle and blastocyst development.MAPPD exposure induces apoptosis of ovarian granulosa cells by activating oxidative stress and ERK pathway.