Toxicokinetics And Reproductive Toxicity From Low-dose Long-term Exposure To 4-Nonylphenol In Female Rats

4-nonylphenol?4-NP?,as a typical endocrine disrupting chemicals?EDCs?,is ubiquitous in the environment.It could enter into the body through food-chain and bioaccumulation so as to influence the endocrine and reproductive system.Dietary exposure is the main route which is latent and persistent.The current researches mainly focus on the single oral or high dose exposure.The lowest dose was 10 mg/kg and the exposure only lasted 14 days.It could not describe the real situation accordingly.In addition,male reproductive toxicity was paid more attention while the female was ignored.It is important to understand the toxicokinetics in serum from long-term exposure to 4-NP and explore the female reproductive toxicity further.In this study,the female Sprague Dawley?SD?rats were used to explore the changes of 4-NP concentration at different times in serum after low-dose long-term exposure to 4-NP.The PKSolver in MS EXCEL was used to calculate the toxicokinetics parameters.In addition,the influence on hormones and proteins related to energy metabolism were also evaluated to explore the female reproductive toxicity arising from low-dose long-term exposure to 4-NP.This research was composed of two parts.??Toxicokinetics from low-dose long-term exposure to 4-NP80 SD rats were randomly divided into 4 groups.The dose for each group were 0,50?g/kg,500?g/kg and 10 mg/kg,respectively.At day 0?pre-dose?and 1,2,4,6,8,10,13,17 week?post-dose?,the serun were collected for analysis.In addition,at 2,4,13,17 week,5 rats from each group were executed and the uterus,ovary were collected for analysis.All samples were stored at-80°C.After the incubation with?-glucuronidase/sulfatase,the C18 solid-phase extraction cartridges were used to extract 4-NP from the serum samples,following by the determination by ultra-high performance liquid chromatography-tandem mass spectrometry?UPLC-MS/MS?.Based on the PKSolver in MS EXCEL,the toxicokinetics parameters were calculated.The changes in serum fitted to two compartment model better.The 4-NP in serum reached peak concentration at about 0.93-,1.14-,1.26 week for low-,middle-,high dose group,respectively.The corresponding peak concentration(C_max)were 2.58-,2.73-,and 2.74 ng/mL.The mean residence time were 5.28-,6.98-,7.03 week.After 10 week,the serum concentration of 4-NP all decreased to the level under 0.5 ng/mL.As the dose increased,the rate of elimination from central compartment(k₁₀)and from central compartment to peripheral compartment(k₁₂)decreased.The k₁₀ were0.62-,0.48-,0.32 week^-1.The k₁₂ were 0.38-,0.31-,0.18 week^-1.Also,the half-life in phase of distribution(t_1/2?)increased.The t_1/2?were 0.64-,0.81-,1.20 week.The parameters related to central compartment were associated with the dose changes.However,the transport rate from peripheral compartment to central compartment(k₂₁)were 0.21-,0.17-,0.18 week^-1.The half-life in phase of elimination(t_1/2?)were 5.74-,7.55-,6.93 week.In the low-and middle dose groups,the values of k₂₁ decreased and those of t_1/2?increased as the dose increased.Also,the value of t_1/2?decreased compared with that from middle dose group.The values of k₂₁ and k₁₂ were equal which indicated the balance was reached between central and peripheral compartments and the clearance increased in the peripheral compartment.The area under curve?AUC?and C_max increased limitedly as the dose increased.The bioavailability of 4-NP reached saturation.The total clearance of central compartment?CL?and peripheral compartment?CL₂?along with the apparent volume of distribution of central compartment?V?and peripheral compartment?V₂?all increased in proportion as the dose increased,it showed that the total clearance and distribution increased as the dose increased.The low-dose long-term exposure to 4-NP affected the serum metabolism and the peak concentration increased as the dose increased.Compared with the current research on single exposure,the half of lives in serum were longer.The steady state was delayed along with the mean residence time.Under low-dose long-term exposure,the influence on rat serum from 4-NP lasted longer.??Reproductive toxicity from low-dose long-term exposure to 4-NPThe hormones could reflect the endocrine situation.The GnRH level in rat serum increased as the exposure continued till 5 week.After 5 week,the GnRH concentration in middle-,high dose group decreased while that in low dose group continued to increase.The effect on GnRH from 4-NP could be regarded as‘stimulation at low dose,prohibition at high dose'and the appearance was more easily noticed at the endpoint.This could also be noticed in phytoestrogens.LH was secreted by pituitary and influenced by GnRH.In the low-dose group,the level of LH increased continuously,while in middle-and high dose groups,the LH would decrease after 10 week,which was corresponding to the trend of GnRH.The FSH in low-dose group did not change apparntly while that in middle-and high dose groups presented the same tendency as LH.E₂was secreted by ovary and decreased constantly in experimental groups compared with that in control group.Thus,under the low-dose long-term exposure,the target of 4-NP in female rat was ovary and gradually affected hypothalamus and pituitary as the dose increased so as to disturb the balance of regulating shaft.From the pre-research,low-dose long-term exposure to 4-NP could alter energy homeostasis in the reproductive system of female rats.In this study,the energy metabolism related proteins in rat uterus were detected through Western Blot from the view of glucose metabolism and?-oxidation.The expression of PI3K,p-AKT1,PKM2 and c-Myc increased and that of AMPK,CPT1 and PPARg decreased.Thus,the 4-NP long-term exposure could activate PI3K/AKT signaling pathway.Then,the PKM2 protein were preferred by the alternative splicing,triggering aerobic glycolysis and lowering the availability of glucose.In addition,the decrease of CPT1 and PPARg proteins showed that the fatty acids?-oxidation was restrained,thus disrupting the energy metabolism in reproductive system.The low-dose long-term exposure to 4-NP could affect the endocrine and energy metabolism in female rat.At the low dose exposure,the target of 4-NP in female rat was ovary and gradually affected hypothalamus and pituitary as dose increased to disturb the balance of regulating shaft,resulting in the disfunction of ovary.In addition,the glucose metabolism in uterus changed from oxidative phosphorylation to aerobic glycolysis.The?-oxidation of fatty acids were inhibited and the tricarboxylic acid cycle was damaged.Therefore,the energy metabolism in uterus was interrupted.