Green tea polyphenols alleviate di-(2-ethylhexyl)phthalate-induced liver injury in mice

BACKGROUND Di(2-ethylhexyl)phthalate(DEHP)is a common plasticizer known to cause liver injury.Green tea is reported to exert therapeutic effects on heavy metal exposureinduced organ damage.However,limited studies have examined the therapeutic effects of green tea polyphenols(GTPs)on DEHP-induced liver damage.AIM To evaluate the molecular mechanism underlying the therapeutic effects of GTPs on DEHP-induced liver damage.METHODS C57BL/6J mice were divided into the following five groups:Control,model[DEHP(1500 mg/kg bodyweight)],treatment[DEHP(1500 mg/kg bodyweight)+GTP(70 mg/kg bodyweight),oil,and GTP(70 mg/kg bodyweight)]groups.After 8 wk,the liver function,blood lipid profile,and liver histopathology were examined.Differentially expressed micro RNAs(miRNAs)and mRNAs in the liver tissues were examined using high-throughput sequencing.Additionally,functional enrichment analysis and immune infiltration prediction were performed.The miRNA-mRNA regulatory axis was elucidated using the starBase database.Protein expression was evaluated using immunohistochemistry.RESULTS GTPs alleviated DHEP-induced liver dysfunction,blood lipid dysregulation,fatty liver disease,liver fibrosis,and mitochondrial and endoplasmic reticulum lesions in mice.The infiltration of macrophages,mast cells,and natural killer cells varied between the model and treatment groups.mmu-miR-141-3p(a differentially expressed miRNA),Zcchc24(a differentially expressed mRNA),and Zcchc24(a differentially expressed protein)constituted the miRNA-mRNA-protein regulatory axis involved in mediating the therapeutic effects of GTPs on DEHP-induced liver damage in mice.CONCLUSION This study demonstrated that GTPs mitigate DEHP-induced liver dysfunction,blood lipid dysregulation,fatty liver disease,and partial liver fibrosis,and regulate immune cell infiltration.Additionally,an important miRNAmRNA-protein molecular regulatory axis involved in mediating the therapeutic effects of GTPs on DEHP-induced liver damage was elucidated.

Glycerin Monostearate Aggravates Male Reproductive Toxicity Caused by Di(2-ethylhexyl)Phthalate in Rats

Human beings are increasingly exposed to phthalates,which are a group of chemicals used to make plastics more flexible and harder to break,and simultaneously ingesting abundant food emulsifiers via daily diet.The purpose of this study was to investigate the effect of the food emulsifier glycerin monostearate(GMS)on male reproductive toxicity caused by di(2-ethylhexyl)phthalate(DEHP,one of the phthalates)and explore the underlying mechanism.Thirty male Sprague-Dawley rats were randomly divided into control group,DEHP group and DEHP+GMS group.Rats in the DEHP group and DEHP+GMS group were orally administered with 200 mg/kg/d DEHP with or without 20 mg/kg/d GMS.After 30 days of continuous intervention,it was found that the serum testosterone level was significantly lowered in DEHP group and DEHP+GMS group than that in control group(P<0.01).The serum testosterone level and the relative testis weight were significantly decreased in the DEHP+GMS group as compared with those in the DEHP group and control group(P<0.05).More spermatids were observed to be shed off in DEHP+GMS group than in DEHP group.The expression levels of cell cycle checkpoint kinase 1(Chkl),cell division cycle gene 2(Cdc2),and cyclin-dependent kinase 2(CDK2)were down-regulated in DEHP group,and this tendency was more significant in DEHP+GMS group(P<0.05 or P<0.01).There was no significant difference in the P-glycoprotein(P-gp)expression between DEHP group and control group.However,P-gp was markedly down-regulated in DEHP+GMS group(P<O.Ol).The results indicated that the food emulsifier GMS aggravated the toxicity of DEHP on male reproduction by inhibiting the cell cycle of testicular cells and the expression of P-gp in testis tissues.

Effect of Mono-2-ethyhexyl Phthalate on DNA Methylation in Human Prostate Cancer LNCaP Cells

Objective To evaluate whether mono（2‐ethylhexyl） phthalate（MEHP） affects genomic DNA methylation and the methylation status of some specific genes such as patched gene（PTCH） and smoothened gene（SMO） in LNCaP cells. Methods LNCaP cells were treated with MEHP（0, 1, 5, 10, and 25 μmol/L） for 3 days. An ELISA assay was preformed to detect genomic methylation, including 5‐methylcytosine（5‐mC） and 5‐hydroxymethylcytosine（5‐hmC） content. A pyrosequencing assay was applied to assess DNA methylation in PTCH and SMO gene promoters. The correlation between DNA methylation and gene expression was assessed. Results The proportion of cytosines with 5‐mC methylation in LNCaP cells was significantly decreased by MEHP（1, 5, 10, and 25 μmol/L） in a dose‐dependent manner（P 〈 0.01）. For genes in the Hedgehog pathway, there was no significant MEHP concentration‐dependent difference in the DNA methylation of PTCH and SMO. Conclusion MEHP might affect the progression of prostate cancer through its effect on global DNA methylation.

Urine Metabonomic Analysis of Interventions Effect of Soy Isoflavones on Rats Exposed to Di-(2-ethylhexyl) Phthalate

Objective Di-(2-ethylhexyl) phthalate(DEHP) is a ubiquitous environmental contaminant.As an endocrine disruptor,it seriously threatens human health and ecological environmental safety.This study examines the impact of intervention with soybean isoflavones(SIF) on DEHP-induced toxicity using a metabonomics approach.Methods Rats were randomly divided into control(H),SIF-treated(A,86 mg/kg body weight),DEHP-treated(B,68 mg/kg),and SIF plus DEHP-treated(D) groups.Rats were given SIF and DEHP daily through diet and gavage,respectively.After 30 d of treatment,rat urine was tested using UPLC/MS with multivariate analysis.Metabolic changes were also evaluated using biochemical assays.Results Metabolomics analyses revealed that p-cresol glucuronide,methyl hippuric acid,N1-methyl-2-pyridone-5-carboxamide,lysophosphatidycholine [18:2(9 Z,12 Z)] {lyso PC [18:2(9 Z,12 Z)]},lyso PC(16:0),xanthosine,undecanedioic acid,and N6-acetyl-l-lysine were present at significantly different levels in control and treatment groups.Conclusion SIF supplementation partially protects rats from DEHP-induced metabolic abnormalities by regulating fatty acid metabolism,antioxidant defense system,amino acid metabolism,and is also involved in the protection of mitochondria.

Impairment of Di(2-Ethylhexyl) Phthalate on Cellular Immunity in Kunming Mice

Immunity is crucial to the health of animals and it can determine their survival and fitness. Di(2-ethylhexyl) phthalate (DEHP) is widely used as a plasticizer and hence is the most abundant phthalate in the environment. Exposure to DEHP is of great concern for human health. In the present study, we tested the hypothesis that exposure to DEHP would suppress T cell-mediated immunity in mice. Twenty adult male Kunming mice were randomly assigned into the control (n = 10) and the DEHP treatment (n = 10) groups. Both groups have free access to food and water, while the mice in the latter group drank DEHP solution (2000 mg/L) for 42 days. T cell-mediated immunity assessed by phytohaemagglutinin (PHA) response was depressed in the DEHP treated mice compared with the controls, however, wet thymus and spleen mass, white blood cells were not influenced by DEHP treatment. Taken together, different immunological parameters responded differently to DEHP treatment in Kunming mice.

An Estimation of the Daily Intake of Di(2-ethlhexyl) Phthalate(DEHP) among Workers in Flavoring Factories

Objective To estimate the daily intake of DEHP among workers in flavoring factories. Methods 71 workers in two flavoring manufacturers, 27 administrators in those factories and 31 laboratory technicians in a research institute were recruited and assigned to exposure group, control group 1 and control group 2 respectively. Their urinary DEHP metabolites, mono（2-ethylhexyl） phthalate （MEHP）, mono-（2-ethyl-5-hydroxyhexyl） phthalate （MEHHP）, mono-（2-ethyl-5-oxohexyl） phthalate （MEOHP）, were detected by isotope dilution-ultra performance liquid chromatography-tandem mass spectrometry（UPLC-MS/MS）. The urinary metabolites concentrations were converted into DEHP intake levels using two pharmacokinetic models： the urine creatinine-excretion （UCE） one and the urine volume （UV） one. Results No significant differences were found among the three groups. Based on the urinary concentrations of Z3MEHP, we got a median daily DEHP intake of 3.22 or 1.85 μg/kg body-weight/day applying the UV or UCE models respectively. Depending on the UV model, three subjects （2.34%） exceeded the RfD value given by US EPA and the P50 of estimate daily DEHP intakes accounted for 16.10% of the RfD value. No subjects exceeded the limitation depending on the UCE model. Conclusion The workers in flavoring factories were not supposed to be the high DEHP exposure ones and their exposure level remained at a low risk.

Reducing children’s exposure to di(2-ethylhexyl)phthalate in homes and kindergartens in China:Impact on lifetime cancer risks and burden of disease

Exposure to di(2-ethylhexyl)phthalate(DEHP)in the indoor environment has been linked with significant health risks for Chinese children.Multi-phase DEHP concentrations in Chinese residences and kindergartens were estimated using a mass balance model based on the current baseline condition and control strategies(i.e.,increasing ventilation rate,reducing area of sources,using mechanical ventilation systems,and using portable air cleaners).The health benefits of each control strategy were quantified as the reduction in lifetime cancer risks(LCR)and burden of disease(BoD).In the current situation,the mean LCR and disability-adjusted life years(DALY)number attributable to indoor DEHP exposure for Chinese children were around 6.0×10^(−6) and 155 thousand,respectively.The mean LCR and DALY might be reduced by 25%-54%and 16%-40%,respectively,by increasing air exchange rates by 100%,reducing the use of source materials by two-thirds or deploying commercial air cleaners in naturally ventilated buildings.Meanwhile,avoidable DALYs could result in a reduction of mean economic losses of 2.2-5.3 billion RMB.Mechanical ventilation systems with filtration units may not be helpful for reducing children’s health risks.House-specific and tailor-made control measures are critical in lowering indoor exposure to DEHP to promote sustainable buildings and children’s health in China.

Maslinic acid supplementation prevents di(2-ethylhexyl)phthalate-induced apoptosis via PRDX6 in peritubular myoid cells of Chinese forest musk deer

Chinese forest musk deer(FMD),an endangered species,have exhibited low reproductive rates even in captivity due to stress conditions.Investigation revealed the presence of di(2-ethylhexyl)phthalate(DEHP),an environmental endocrine disruptor,in the serum and skin of captive FMDs.Feeding FMDs with maslinic acid(MA)has been observed to alleviate the stress response and improve reproductive rates,although the precise molecular mechanisms remain unclear.Therefore,this study aims to investigate the molecular mechanisms underlying the alleviation of DEHP-induced oxidative stress and cell apoptosis in primary peritubular myoid cells(PMCs)through MA intake.Primary PMCs were isolated and exposed to DEHP in vitro.The results demonstrated that DEHP significantly suppressed antioxidant levels and promoted cell apoptosis in primary PMCs.Moreover,interfering with the expression of PRDX6 was found to induce excessive reactive oxygen species(ROS)production and cell apoptosis in primary PMCs.Supplementation with MA significantly upregulated the expression of PRDX6,thereby attenuating DEHP-induced oxidative stress and cell apoptosis in primary PMCs.These findings provide a theoretical foundation for mitigating stress levels and enhancing reproductive capacity of in captive FMDs.

THE EXTRACTED COMPLEX FORMED BY MONO (2-ETHYLHEXYL)-2-ETHYLHEXYL PHOSPHATE WITH DIVALENT Mn

In order to clarify the mechanism and the complex formed in the extraction of divalent Mn by alkylphosphonic acid monoester, the solid complex has been prepared by mono (2-ethylhexyl)-2-ethylhexyl phosphate, HEH(EH)P, HA with divalent Mn. The elemental analysis, magnetic susceptibility and thermogravimetry have been determined for the solid complex of HEH(EH)P-Mn(II) and the infrared spectrum has been carried out in comparison to the extractant HEH(EH)P. The extracted compound has also been studied by electronic and electron spin resonance spectroscopy in octane solvent and solid state at room temperature. On the basis of the measurements, it is concluded that the structure of the solid polymeric species MnA_2 is in a tetrahedral arrangement.

Aquatic toxicity of di (2-eihylhexyl) phthalate to duckweeds

This study is concerned with the effects of di （2-ethylhexyl） phthalate （DEHP） on two kinds of duckweeds （Spirodela polyrhiza and Lemna minor）.The results indicate that DEHP has aquatic toxicity to Spirodela polyrhiza at 0.4 mg/L and to Lemna minor at over 0.1 mg/L by changing their physiologic-biochemical characteristics.The contents of duckweed chlorophyll and soluble protein decrease with increasing DEHP concentration after 7 d of exposure.DEHP shows the stimulating role in catalase （CAT） and superoxide dismutase （SOD） systems at relative low levels.At 0.01 mg/L and 0.005 mg/L,SOD activities of Spirodela polyrhiza and Lemna minor reach their peak values respectively,while CAT activity reaches its maximum value at 0.05 mg/L and 0.01 mg/L.When DEHP levels are too high,the protection enzyme system would be destroyed and plant growth is inhibited.The analysis of malondialdehyde （MDA） and Fourier transform infrared spectroscopy manifest that DEHP could affect the tested duckweeds by destroying its cell membranes,and Spirodela polyrhiza is more resistant to DEHP exposure than Lemna minor.

m6A甲基化对邻苯二甲酸单(2-乙基己基)酯诱导小鼠睾丸间质细胞自噬的作用

目的 探讨邻苯二甲酸单(2-乙基己基)酯[mono(2-ethylhexyl)phthalate,MEHP]致小鼠睾丸间质(TM-3)细胞自噬中m6A甲基化的作用。方法 将对数生长期的TM-3细胞暴露于浓度梯度(0、5、10、20、40、80μmol·L^(-1))的m6A甲基化抑制剂3-脱氮腺苷(3-Deazaadenosine,DAA)24 h,CCK-8法检测细胞活力,确定3-脱氮腺苷的染毒剂量后,梯度稀释为对照组(0μmol·L^(-1))、MEHP低剂量组(200μmol·L^(-1))、MEHP中剂量组(400μmol·L^(-1))、MEHP高剂量组(800μmol·L^(-1)),将TM-3细胞分别暴露于0、200、400、800μmol·L^(-1)MEHP,40μmol·L^(-1)DAA+400μmol·L^(-1)MEHP和40μmol·L^(-1)DAA染毒组24 h,光镜下观察TM-3细胞形态。CCK-8法检测细胞存活率;m6A RNA甲基化定量检测试剂盒检测m6A修饰水平;丹酰尸胺(MDC)荧光染色检测自噬形成情况;Western blot检测LC3-Ⅱ、LC3-Ⅱ/LC3-Ⅰ和Beclin-1的蛋白表达水平。结果 与对照组相比,DAA浓度为5、10、20、40μmol·L^(-1)时,细胞活力均无明显变化(P均>0.05);DAA浓度为80μmol·L^(-1)时,细胞活力下降(P<0.01);各MEHP染毒组TM-3细胞活力和m6A甲基化水平均降低(P均<0.01),细胞空隙变大,圆形细胞增多,贴壁细胞的数量明显减少;DAA组m6A甲基化水平较对照组降低(P<0.01);与MEHP中剂量组相比,DAA+MEHP组细胞活力和m6A甲基化水平均下降(P均<0.01),细胞发生皱缩,细胞间隔稍有增大。与对照组相比,各MEHP染毒组、DAA+MEHP组和DAA处理组LC3-Ⅱ的蛋白表达水平均升高(P均<0.05),MEHP低剂量组MDC荧光信号强度、LC3-Ⅱ/LC3-Ⅰ和Beclin-1的蛋白表达量均无明显变化(P均>0.05);MEHP中、高剂量组和DAA+MEHP组荧光强度、LC3-Ⅱ/LC3-Ⅰ和Beclin-1的表达量均升高(P均<0.01);DAA组荧光强度和Beclin-1的表达水平均升高(P均<0.01),LC3-Ⅱ/LC3-Ⅰ无明显变化(P均>0.05)。DAA+MEHP组荧光强度和LC3-Ⅱ/LC3-Ⅰ较MEHP中剂量组呈上升趋势(P<0.05)。结论 MEHP可能通过降低RNA甲基化m6A修饰的水平来诱导TM-3细胞发生自噬。

Cytotoxic effects of mono-（2-ethylhexyl） phthalate on human embryonic stem cells

Background Mono-（2-ethylhexyl） phthalate （MEHP）, the metabolite of di-（2-ethylhexyl） phthalate （DEHP）, was suspected to be toxic to human embryos. This study contributes to investigating its toxic effects by an embryonic stem cell test （EST） based on two human embryonic stem cell （hESCs） lines. Methods CH1 established in our own lab and H1, a federally registered cell line were two human embryonic stem cell lines used in this test. Four endpoint measurements were performed consisting of cell viability, proliferation ability, apoptosis as well as changes of gene expression patterns after spontaneous differentiation were determined. For measuring effects on the first three endpoints, the cells were treated with various concentrations of MEHP dissolved in dimethyl sulfoxide （DMSO） and only with DMSO which served as control and harvested after 5 days. For measuring effects during spontaneous differentiation, the RNA of embryoid bodies （EBs） formed after 8 days＇ MEHP exposure was collected and changes in differentiation specific gene expression patterns were analyzed by quantitative real time RT-PCR. Results As a result the viability and proliferation ability of both cell lines decreased significantly at 1000 μmol/L MEHP, while there was no effect on apoptosis or cell morphology. In addition MEHP also changed the gene expression pattern in the EBs of both cell lines. Conclusion MEHP in a high dose was cytotoxic and affected the development of hESCs, which indicates its embryo toxicitv in human embrvos.

邻苯二甲酸二乙基己酯及代谢产物邻苯二酸-单-2-乙基己酯对雄性幼鼠生精细胞凋亡的影响

目的了解环境内分泌干扰因子邻苯二甲酸二乙基已酯(DEHP)及代谢产物邻苯二酸-单-2-乙基己酯(MEHP)对雄性幼鼠生精细胞凋亡有否影响。方法生后2周龄的Wistar雄性幼鼠98只,随机分为14组,7只/组。随机选择1组行生理盐水(0.9%NS 0.2 ml.kg-.1d-1)灌胃喂养3周,作为正常对照组(NC组);再选1组行环磷酰胺(CTX 100 mg·kg-1·d-1)灌胃喂养1周,作为阳性对照组(PC组);余各组分别用DEHP、MEHP按低剂量(100 mg·kg-1·d-1)、中剂量(200 mg·kg-1·d-1)、高剂量(300 mg·kg-1·d-1)灌胃喂养1周;以及分别按低剂量(100 mg·kg-1·d-1)灌胃喂养1、2、3周分组,建立动物模型。观察不同时期、不同剂量下睾丸生精细胞的形态学变化,并应用TUNEL法检查睾丸生精细胞凋亡情况,放射免疫法检测血清性激素(卵泡刺激素FSH、黄体生成素LH以及睾酮T)水平。结果(1)睾丸生精细胞的变化:用药组生精细胞萎缩、发育落后、数量少,线粒体肿胀、空泡化等;在喂养1周的不同剂量组内,随剂量增加生精细胞损害加重,在低剂量不同喂养时间组内,随喂养时间的延长生精细胞损害亦加重;(2)睾丸组织中生精细胞凋亡:与NC组比较,用药组生精细胞凋亡显著增多(P<0.01);在喂养1周的不同剂量组内、以及低剂量不同喂养时间组内生精细胞凋亡间比较,差异均有统计学意义(P<0.05);(3)血清性激素水平:与NC组比较,用药组FSH、LH水平升高,T水平下降(P<0.05);在喂养1周的不同剂量组内、以及低剂量不同喂养时间组内性激素水平间比较,差异也有统计学意义(P<0.05)。结论 DEHP及代谢产物MEHP对幼鼠生精细胞凋亡有明显影响,且呈时间-量效依赖效应。

长期暴露的邻苯二甲酸二(2-乙基己基)酯在雌性大鼠体内的代谢及对肝脏的影响

目的:研究邻苯二甲酸二(2-乙基己基)酯(DEHP)长期暴露后,雌性大鼠体内的代谢情况及对肝脏的影响。方法:将Wistar雌性大鼠64只按体质量随机分为4组,分别为对照(玉米油)组和150、300、600 mg/mL DEHP染毒组,每组16只。采用灌胃方式进行染毒,每天灌胃1次,每次按2.5μL/g给予,连续灌胃6个月。期间观察大鼠一般生长状况,分别在染毒后3个月和6个月两个时间点采集大鼠尿液。6个月后处死各组大鼠,摘取大鼠的肝、脾和肾称取质量,计算脏体比值;并制备肝组织切片,采用HE染色法观察肝组织形态变化。对采集的2个时间点大鼠尿液,采用高效液相色谱的方法检测尿中DEHP及其代谢物邻苯二甲酸单(乙基己基)酯(MEHP)的含量。结果:与对照组相比,各剂量染毒组大鼠一般生长情况无明显差异,但肝脏的脏体比值增加,HE染色显示300和600 mg/mL DEHP染毒组大鼠肝脏组织轻微病理改变。与对照组相比,3个月时,300和600 mg/mL DEHP组随着染毒剂量的增加,大鼠尿液中DEHP和MEHP含量均明显增加,差异均有统计学意义(P均<0.05)。6个月时,600 mg/mL DEHP染毒组大鼠尿液中DEHP和MEHP含量高于其他3组,差异有统计学意义(P<0.05)。结论:雌性大鼠长期暴露DEHP,体内DEHP和MEHP的残留和蓄积会增加,超过正常的代谢能力,可能会对肝脏造成一定程度的损伤。

邻苯二甲酸二(2-乙基己)酯(DEHP)和邻苯二甲酸单乙基己基酯(MEHP)长期暴露对海洋青鳉(Oryzias melastigma)内分泌干扰效应的评价

为探讨邻苯二甲酸二(2-乙基己)酯(DEHP)和邻苯二甲酸单乙基己基酯(MEHP)长期暴露对海水生物的内分泌干扰效应及机制,将孵化后1周的海洋青鳉(Oryzias melastigma)分别暴露于DEHP(0.1 mg·L-1和0.5 mg·L-1)和MEHP(0.1 mg·L-1和0.5 mg·L-1)6个月。结果显示:DEHP显著增加了雌性和雄性海洋青鳉的肝指数,而MEHP只在高剂量时显著增加雄性青鳉的肝指数。对于雌性青鳉,DEHP暴露后肝脏雌激素相关基因ERα、ERβ、ERγ、VTG1、VTG2、ChgH和ChgL的表达水平显著上调,而对于雄性青鳉,DEHP暴露后,只有肝脏ERβ的表达水平显著上调。相比之下,MEHP暴露对雌性和雄性青鳉肝VTG和Chg基因表达无显著影响。DEHP激活了雌性和雄性青鳉的肝过氧化物增殖激活受体PPARα和PPARγ,而MEHP只在低剂量时上调了雄性青鳉PPARγ的表达。在雌性和雄性青鳉体内,VTG和Chg的表达与ERα和ERγ的表达显著相关,并且ER与PPAR也显著相关。研究表明,DEHP长期暴露可通过激活肝性激素受体调控肝雌激素响应基因(VTG和Chg)和过氧化物增殖激活受体(PPARα和PPARγ)的表达而对海洋青鳉产生内分泌干扰效应,并且显示出性别特异性。MEHP对海洋青鳉的内分泌干扰效应弱于DEHP。

S_2O_8^(2-)/ZrO_2催化合成邻苯二甲酸单月桂醇酯

用固体超强酸S2O82-/ZrO2催化合成了邻苯二甲酸单月桂醇酯。讨论了催化剂制备条件对其活性的影响,考察了影响酯收率的因素。结果表明,用0.75mol/L过硫酸铵溶液浸渍Zr(OH)4且在600℃下焙烧3h制备的固体酸催化活性最高。当原料邻苯二甲酸与月桂醇的摩尔比在1∶1~1.4范围内,催化剂S2O82-/ZrO2用量为0.02g/mL,90℃下反应4h,酯收率可达87%以上。

Primary neuronal-astrocytic co-culture platform for neurotoxicity assessment of di-(2-ethylhexyl) phthalate

Plastics such as polyvinyl chlorides （PVC） are widely used in many indoor constructed environments; however, their unbound chemicals, such as di-（2-ethylhexyl） phthalates （DEHP）, can leach into the surrounding environment. This study focused on DEHP＇s effect on the central nervous system by determining the precise DEHP content in mice brain tissue after exposure to the chemical, to evaluate the specific exposure range. Primary neuronal-astrocyte co-culture systems were used as in vitro models for chemical hazard identification of DEHP. Oxidative stress was hypothesized as a probable mechanism involved, and therefore the total reactive oxygen species （ROS） concentration was determined as a biomarker of oxidative stress. In addition, NeuriteTracer, a neurite tracing plugin with ImageJ, was used to develop an assay for neurotoxicity to provide quantitative measurements of neurological parameters, such as neuronal number, neuron count and neurite length, all of which could indicate neurotoxic effects. The results showed that with 1 nmol/L DEHP exposure, there was a significant increase in ROS concentrations, indicating that the neuronal-astrocyte cultures were injured due to exposure to DEHP. In response, astrocyte proliferation （gliosis） was initiated, serving as a mechanism to maintain a homeostatic environment for neurons and protect neurons from toxic chemicals. There is a need to assess the cumulative effects of DEHP in animals to evaluate the possible uotake and effects on the human neuronal system from exoosure to DEHP in the indoor environment.

Plastic Additives Decrease Agrin-Induced Acetylcholine Receptor Clusters and Myotube Formation in C2C12 Skeletal Muscle Cell Culture

Common additives in plastics such as bisphenol A (BPA) or phthalates like di-(2-ethylhexyl) phthalate (DEHP) are environmental estrogens that have been shown to be endocrine disruptors in some experimental animal models. This project used the C2C12 cell culture model to examine how exposure to BPA or DEHP affects two aspects of skeletal muscle development, the fusion of myoblasts into myotubes and agrin-induced clustering of acetylcholine receptors (AChRs). During myotube formation AChRs cluster spontaneously. Treatment with motor neuron derived agrin increases the frequency of AChR clusters through an agrin signaling pathway that also clusters other postsynaptic components of the neuromuscular synapse. For this project C2C12 cells were exposed to BPA or DEHP while myoblasts fused into myotubes. After exposure to 10 μM BPA or 100 μM DEHP the frequency of agrin-induced AChR clusters decreased. In addition, myotube formation decreased as a higher percentage of nuclei remained in myoblasts. Furthermore, BPA or DEHP reduced the amount of the myogenic regulatory factor myogenin. This suggests that BPA and DEHP decrease AChR clustering by reducing myogenin. Moreover, plastic additives like BPA and DEHP may pose a risk for skeletal muscle development in humans.

肌细胞增强因子2A介导邻苯二甲酸单(2-乙基己基)酯影响睾丸间质细胞甾体激素mRNA表达水平

目的探讨肌细胞增强因子2A(MEF2A)在邻苯二甲酸单(2-乙基己基)酯(MEHP)抑制睾丸间质细胞甾体激素合成的作用。方法体外培养细胞mLTC-1,取对数生长期的细胞用于实验。设DMSO溶媒对照组(0μmol/L)、MEHP处理组(200、400、800μmol/L),作用24h,加hCG0.1U/mL作用4h后收集细胞培养液用于检测孕酮;提取细胞总RNA用于检测类固醇激素合成关键酶(StAR/P450scc/3βHSD)及MEF2A的mRNA表达水平。结果MEHP作用24h后,细胞培养液中孕酮(PROG)随染毒剂量增高呈下降趋势,其中800μmol/L组与对照组比较,差异有统计学意义(P<0.01)。随着MEHP染毒剂量的增高,StAR mRNA逐渐降低,与对照组比较,差异有统计学意义(P<0.01)。P450sccmRNA和3βHSD mRNA表达水平在400μmol/L和800μmol/L组与对照组比较差异均有统计学意义(P<0.05或P<0.01)。不同实验组MEF2A mRNA均比对照组低(P均<0.01)。结论MEHP对雄性的生殖毒性机制可能通过抑制MEF2A mRNA的表达,干扰孕酮合成关键酶,从而影响睾酮活性。

邻苯二甲酸单乙基己基酯暴露对肝脏脂质代谢的影响及调控机制

邻苯二甲酸二(2-乙基己基)酯(Di(2-ethylhexyl)phthalate,DEHP)是一种重要的内分泌干扰物,对神经系统、生殖系统、内分泌系统等都具有特定的损伤作用.DEHP被机体摄入后,在肝脏和肠道中被消化酶代谢为邻苯二甲酸单(2-乙基己基)酯(mono-(2-ethylhexyl)phthalate,MEHP)以发挥毒性作用.本研究采用体外HepG2暴露结合mRNA芯片以及生信分析的方法,探讨MEHP暴露对HepG2细胞脂质代谢的影响.首先,采用油红O染色法观察细胞内脂滴蓄积情况,发现MEHP暴露能够以浓度依赖性的方式增加HepG2细胞内脂质蓄积.为了探究其可能的机制,采用Agilent(人)表达谱芯片分析并筛选差异基因,共筛选出93个差异表达基因,其中上调基因57个,下调基因36个.在此基础上,通过DAVID在线数据库对差异表达基因进行基因功能富集分析,发现其主要参与细胞脂质代谢过程(cellular lipid metabolic process)、跨膜运输(transmembrane transport)、跨膜转运的调控(regulation of transmembrane transport)等生物学过程.随后,通过qRT-PCR法对关键基因的mRNA表达水平进行验证.最后,通过GEPIA在线验证上述基因在肝细胞癌(LIHC)中的表达.综上,MEHP暴露能够影响细胞脂质代谢通路中的基因FADS6、MVD、SC5D、PLA2G4E在HepG2细胞中的表达,在一定程度上促进了肝脏细胞中的脂肪蓄积,从而使肝细胞内脂质代谢紊乱.