Evaluation Of The Intervention Effect Of Zinc On Reproductive Toxicity Induced By Phthalate Esters In Male Rats Basing On Detection Of Target Compounds

Background:Environmental endocrine disruptors（EEDs）is a kind of common environmentally harmful substances,through a variety of ways enriched in human and animal bodies,interfering aspects of synthesis,secretion,release,transport,binding,effect,clearance and so on to simulate or antagonize endogenous hormones which resulted in an increase in the incidence of reproductive dysfunction,malformation of reproductive system and malignant tumor.Among them,phthalates（PAEs）as a plasticizer,widely used in plastics and chemical industry,have become one of the most common environmental pollutants in the world.Human beings are inevitably exposed to long-term low-dose MIXPs（6 priority controlled PAEs mixed by equal toxicity）.Therefore,it has a significance in public health to find out potential risks caused by PAEs as soon as possible and protective factors for reproductive toxicity induced by PAEs.The intervention effect of protective factors on the reproductive system should be dynamically monitored.In our prophase research,we found that long-term exposure to low-dose MIXPs lead to male reproductive toxicity which mainly showed that changes in sex hormone levels,pathological changes of testicular tissue,changes in the expression level of testosterone synthesis protein and so on.The low-dose MIXPs as exposure dose was obtained by conversion with the species coefficient between human and rat according to actual exposure level of PAEs in the residents in China.Moreover,17 exposure biomarkers of MIXPs were screened out in male rat urine samples in which5 exposure biomarkers（androstenedione,testosterone,dehydroepiandrosterone,dihydrotestosterone and estrone）were related to metabolic pathways of androgen and male reproductive toxicity.Whether these 5 exposure biomarkers could be serviced as indirect index of toxic effects to sensitively represent early effects of reproductive toxicity in male rats exposed to PAEs?We found that zinc enrichment could improve reproductive toxicity in male rats exposed to MIXPs which showed the improvement of body weight,male reproductive organs’absolute and relative weight,serum testosterone,luteinizing hormone（LH）,follicle-stimulating hormone（FSH）,pathological structure of testicular tissue and expression level of testosterone synthesis protein and so on.Whether these 5 exposure biomarkers could be applied to the evaluation of intervention effect of protective factors such as zinc on male reproductive toxicity induced by PAEs if these 5exposure biomarkers could be serviced as indirect index of toxic effects?And on this basis,studies of this paper are as follows:Objective:This study basing on target analysis made quantitative analysis of 5 exposure biomarkers（androstenedione,testosterone,dehydroepiandrosterone,dihydrotestosterone and estrone）screened by non-target metabolomics which were closely related with function of reproductive system.The objectives of this study are to investigate the 5 exposure biomarkers whether could be serviced as indirect index of toxic effects to represent reproductive toxicity in male rats exposed to PAEs and whether could be serviced as indicators of intervention effects for the evaluation of male reproductive toxicity.Methods:（1）Animal experiment:50 male Sprague-Dawley（SD）rats（180²20 g）were selected and randomly divided into five groups（n=10）:A:NC;B:ZD;C:MIXPs;D:MIXPs-ZD;E:MIXPs-ZE.A:Rats in normal control rats were fed with regular feed and intragastric administration of 5%CMC-Na same volume one time every day;B:Rats in zinc deficiency control group were fed with zinc deficiency feed;C:Rats in MIXPs exposure group were fed with regular feed and intragastric administration of 160 mg/kg/d MIXPs;D:Rats in zinc deficiency-MIXPs exposure group were fed with zinc deficiency feed and intragastric administration of 160 mg/kg/d PAEs;E:Rats in zinc enrichment-MIXPs exposure group were fed with zinc deficiency feed and intragastric administration of 160 mg/kg/d PAEs and 13 mg/kg/d ZnSO₄.All rats were administered continuously by gavage for 90 days.24 hours’urine of all rats were collected on day 0 and 30,60and 90 days respectively.（2）Establishment and evaluation of analytical method for target compounds:LC-MS/MS was applied to the detection of androstenedione,testosterone,dehydroepiandrosterone and dihydrotestosterone in rat urine.The chromatographic separation was performed on an Acquity UPLC BEH C18（2.1 mm×100 mm,1.7μm）column at 30°C with the injection volume of 5μL.The flow rate was 0.4 mL/min.The mobile phase consisted of solvent A（0.1%formic acid-water）and solvent B（acetonitrile）and nonlinear gradient elution was used.The ESI mode of androstenedione,testosterone,dehydroepiandrosterone and dihydrotestosterone was in the positive ion mode.In this scanning mode,the parent ion of the target object was firstly selected,and then the product scan was performed to determine the qualitative ion pair and collision energy.HPLC-DAD was applied to the detection of estrone in rat urine.The chromatographic separation was performed on the Diamonsil C18 column（250 mm×4.6 mm,5μm）at 30°C with the injection volume of 20μL.The mobile phase was solvent A（0.1%formic acid-water）and solvent B（acetonitrile）with solvent A maintaining at 30%and isocratic and gradient elution was used.The flow rate was 1.0 mL/min and estrone was determined under the wavelength of 230 nm.Results:（1）The detection limits（LOD）of five target compounds were 0.02 ng/mL for androstenedione,0.01 ng/mL for testosterone,0.15 ng/mL for dehydroepiandrosterone,0.12 ng/mL for dihydrotestosterone and 1 ng/mL for estrone and the linearity ranges were respectively 0.06-100ng/mL for androstenedione,0.03¹00 ng/mL for testosterone,0.5¹00 ng/mL for dehydroepiandrosterone,0.4¹00 ng/mL for dihydrotestosterone and 3¹00 ng/mL for estrone.Satisfactory spiked recoveries of five exposure biomarkers were 88.2%¹10.0%（RSD<15.8%,n=6）at three concentration levels in rat urine samples with good accuracy and the precision.Compared with existing methods,the amount of organic solvent used in our new method was decreased.Compared with existing methods,the accuracy of androstenedione,testosterone and dihydrotestosterone were better.Home-developed PA6 nanofibers mat-based solid phase extraction was used for the determination of estrone in rat urine samples with simple procedure.The consumption of extraction medium and organic solvent were low.The recovery of this new method was 88.2¹10.0%,which met the requirements in actual samples.（2）Detection of urine samples:The levels of target compounds in rat urine samples were corrected by creatinine.The levels of androstenedione,testosterone,dehydroepiandrosterone and dihydrotestosterone in rat urine samples in MIXPs group were higher than those in control group and significantly higher than those in control group in the 60^th day（P<0.05）.However,the change of estrone opposite to the change of the four exposure biomarkers in urine was in decline and levels of estrone in rat urine samples were significantly decreased since 30^th day（P<0.05）.The levels of five exposure biomarkers in urine were significantly decreased in rats fed with zinc deficiency feed for 90 days compared with those in control group（P<0.05）.The changes of levels and timeliness of five exposure biomarkers in MIXPs-ZD group was same with the changes in MIXPs group.The levels of five exposure biomarkers in urine were all improved in MIXPs-ZE group for 90 days compared with those in MIXPs group and MIXPs-ZD group（P<0.05）which tend to levels of five exposure biomarkers in control group.Conclusions:（1）C18-based solid phase extraction applied to high performance liquid chromatography-tandem mass spectrometry（LC-MS/MS）and PA6 nanofiber-solid phase extraction applied to high performance liquid chromatography（HPLC）method were established sensitively and accurately for the detection of five exposure biomarkers（androstenedione,testosterone,and dehydroepiandrosterone,dihydrotestosterone and estrone）in rat urine samples.（2）Five exposure biomarkers in urine can be serviced as indirect index of male reproductive toxicity induced by PAEs and early indicators of treatment effect of zinc intervention on male reproductive toxicity induced by PAEs to enable non-invasive and dynamic monitoring of occurrence and development of male reproductive toxicity induced by PAEs and provide objective foundations for the prediction and warning of the risk of toxic effects induced by PAEs,early discovery and intervention and evaluation of intervening measures and effects.Convenient urine sampling was contributed to the non-invasive and dynamic monitoring of male reproductive toxicity induced by PAEs.The detection of five target compounds at the time was also improve the reliability of monitoring for toxicity.