The Effects of Di(2-Ethylhexyl)Phthalate Exposure and Selenium Nutrition on Sertoli Cell Vimentin Structure and Germ-Cell Apoptosis in Rat Testis

This study aimed to investigate the effects of di(2-ethylhexyl)phthalate (DEHP) on Sertoli-cell vimentin filaments and germ-cell apoptosis in testes of pubertal rats at different selenium (Se) status. Se deficiency was produced in 3-weeks old Sprague-Dawley rats by feeding them ≤0.05 Se mg/kg diet for 5 weeks, Se supplementation group was on 1 mg Se/kg diet, and DEHP was applied at 1000 mg/kg dose by gavage during the last 10 days of the feeding period. The diet with excess Se did not cause any appreciable alteration in vimentin staining and apoptosis of germ cells, but Se deficiency caused a mild decrease in the intensity of vimentin immunoreactivity and enhanced germ-cell apoptosis significantly (approximately 3-fold, p <0.0033). DEHP exposure caused disruption and collapse of vimentin filaments and significantly induced apoptotic death of germ cells (approximately 8-fold, p <0.0033). In DEHP-exposed Se-deficient animals, compared with the control, collapse of vimentin filaments was more prominent; there was serious damage to the seminiferous epithelium; and a high increment (approximately 25-fold, p <0.0033) in apoptotic germ cells was observed. Thus, Se deficiency exacerbated the toxicity of DEHP on Sertoli cells and spermatogenesis, whereas Se supplementation provided protection. These results put forward the critical role of Se in the modulation of redox status of testicular cells and emphasize the importance of Se status for reproductive health.     

Phthalates Impair Germ Cell Development in the Human Fetal Testis in Vitro without Change in Testosterone Production

Background

Several studies have described an increasing frequency of male reproductive disorders, which may have a common origin in fetal life and which are hypothesized to be caused by endocrine disruptors. Phthalate esters represent a class of environmental endocrine-active chemicals known to disrupt development of the male reproductive tract by decreasing testosterone production in the fetal rat.

Objectives

Using the organ culture system we developed previously, we investigated the effects on the development of human fetal testis of one phthalate—mono-2-ethylhexyl phthalate (MEHP)—an industrial chemical found in many products, which has been incriminated as a disruptor of male reproductive function.

Methods

Human fetal testes were recovered during the first trimester (7–12 weeks) of gestation, a critical period for testicular differentiation, and cultured for 3 days with or without MEHP in basal conditions or stimulated with luteinizing hormone (LH).

Results

Whatever the dose, MEHP treatment had no effect on basal or LH-stimulated testosterone produced by the human fetal testis in vitro, although testosterone production can be modulated in our culture system. MEHP (10⁻⁴ M) did not affect proliferation or apoptosis of Sertoli cells, but it reduced the mRNA expression of anti-Müllerian hormone. MEHP (10⁻⁴ M) reduced the number of germ cells by increasing their apoptosis, measured by the detection of caspase-3–positive germ cells, without modification of their proliferation.

Conclusions

This is the first experimental demonstration that phthalates alter the development of the germ cell lineage in humans. However, in contrast to results observed in the rat, phthalates did not affect steroidogenesis.     

Time- and Dose-Related Effects of Di-(2-ethylhexyl) Phthalate and Its Main Metabolites on the Function of the Rat Fetal Testis in Vitro

Background

Endocrine-disrupting effects of phthalates are understood primarily from in utero exposures within the fetal rat testis. Nevertheless, their path of action, dose–response character, and cellular target(s) within the fetal testis are not known.

Objectives

In this study we investigated the effects of di-(2-ethylhexyl) phthalate (DEHP), mono-(2-ethylhexyl) phthalate (MEHP), and several of their metabolites on the development of organo-cultured testes from rat fetus.

Methods

We removed testes from 14.5-day-old rat fetuses and cultured them for 1–3 days with or without DEHP, MEHP, and the metabolites.

Results

DEHP (10⁻⁵ M) produced a proandrogenic effect after 3 days of culture, whereas MEHP disrupted testis morphology and function. Leydig cells were the first affected by MEHP, with a number of them being inappropriately located within some seminiferous tubules. Additionally, we found a time- and dose-dependent reduction of testosterone. By 48 hr, gonocyte proliferation had decreased, whereas apoptosis increased. Sertoli cell number was unaffected, although some cells appeared vacuolated, and production of anti-Müllerian hormone decreased in a time- and dose-dependent manner. The derived metabolite mono-(2-ethyl-5-hydroxyhexyl) phthalate was the only one to cause deleterious effects to the rat fetal testis in vitro.

Conclusion

We hope that this in vitro method will facilitate the study of different phthalate esters and other endocrine disruptors for direct testicular effects.     

Germ Cell Adenylyl Cyclase Activity in the Human Undescended Testis and the Effect of HCG-Treatment

In the present study we have examined the soluble Mn²⁺ dependent, germ cell specific adenylyl cyclase (AC) activity and germ cell morphology in testicular biopsies from twenty-three boys, aged 2—17 years, with undescended testes and three adult men with normally descended testes. Eight of the boys were treated with hCG immediately before surgery. Germ cell AC-activity (3.2—3.4 pmoles cAMP/mg protein/min) was found in the adult testes with normal germ cell morphology. Furthermore, low but significant AC-activity (0.2—0.8 pmoles cAMP/mg protein/min) was noted in biopsies containing only primary spermatocytes. There was no Mn^2- dependent AC-activity in Sertoli cell only testes or in testes where only spermatogonia were observed. Treatment with hCG did not influence germ cell maturation or soluble Mn²⁺ dependent AC-activity.     

器官形成期高温暴露对大鼠胚胎生长发育的影响

目的探讨高温暴露对器官形成期胚胎生长发育的影响。方法将15只健康成年未生育清洁级的SD孕鼠随机分为对照组、高温暴露30min组、高温暴露60min组,每组5只。孕鼠于妊娠第8～10天分别暴露于(39.5±0.5)℃的高温环境中。在妊娠第11天检测孕鼠血清中乳酸脱氢酶(LDH)和肌酸激酶(CK)活力及胚胎数、头长和畸形率以及胎鼠脑蛋白质与HSP70含量。结果与对照组比较,高温暴露60min组孕鼠血清中LDH和CK活力均升高,差异有统计学意义(P0.05)。而高温暴露30min组和对照组孕鼠血清中LDH和CK活力间比较,差异均无统计学意义。随着高温暴露时间的延长,胎鼠头长和脑蛋白质含量呈下降趋势,畸形率和HSP70含量呈上升趋势;但各高温暴露组胎鼠胎鼠头长、畸形率、HSP70含量和脑蛋白质含量间比较,差异均无统计学意义。结论器官形成期孕鼠高温暴露对胎鼠的生长发育产生不良影响。     

Assessment of Adult Human Exposure to Phthalate Esters in the Urban Centre of Paris (France)

Human exposure to phthalates was assessed through digestive and respiratory intakes. Six phthalates (DMP, DEP, DnBP, BBP, DEHP, DnOP) were investigated in drinking water, in current foodstuff and in ambient air. Digestive intake was prevailing (92 %) with a major contribution of food (95.5 %). Phthalate intake from water was mainly due to bottled water (60 %) in spite of the minor volume absorbed daily. From the respiratory tract, it was dominated by DEP: 30.3 ng kg^?1 bw day^?1 and the part played by indoor air prevailed. Total intake were as ng kg^?1 bw day^?1, for DEHP: 1458, DnBP: 191.8, BBP: 164.3, DEP: 107.7, DMP: 79.1.     

Transdermal Uptake of Diethyl Phthalate and Di(n-butyl) Phthalate Directly from Air: Experimental Verification

Background

Fundamental considerations indicate that, for certain phthalate esters, dermal absorption from air is an uptake pathway that is comparable to or greater than inhalation. Yet this pathway has not been experimentally evaluated and has been largely overlooked when assessing uptake of phthalate esters.

Objectives

This study investigated transdermal uptake, directly from air, of diethyl phthalate (DEP) and di(n-butyl) phthalate (DnBP) in humans.

Methods

In a series of experiments, six human participants were exposed for 6 hr in a chamber containing deliberately elevated air concentrations of DEP and DnBP. The participants either wore a hood and breathed air with phthalate concentrations substantially below those in the chamber or did not wear a hood and breathed chamber air. All urinations were collected from initiation of exposure until 54 hr later. Metabolites of DEP and DnBP were measured in these samples and extrapolated to parent phthalate intakes, corrected for background and hood air exposures.

Results

For DEP, the median dermal uptake directly from air was 4.0 μg/(μg/m³ in air) compared with an inhalation intake of 3.8 μg/(μg/m³ in air). For DnBP, the median dermal uptake from air was 3.1 μg/(μg/m³ in air) compared with an inhalation intake of 3.9 μg/(μg/m³ in air).

Conclusions

This study shows that dermal uptake directly from air can be a meaningful exposure pathway for DEP and DnBP. For other semivolatile organic compounds (SVOCs) whose molecular weight and lipid/air partition coefficient are in the appropriate range, direct absorption from air is also anticipated to be significant.

Citation

Weschler CJ, Bekö G, Koch HM, Salthammer T, Schripp T, Toftum J, Clausen G. 2015. Transdermal uptake of diethyl phthalate and di(n-butyl) phthalate directly from air: experimental verification. Environ Health Perspect 123:928–934; http://dx.doi.org/10.1289/ehp.1409151     

非编码RNA与哺乳动物生殖细胞生长发育

基因转录后调控是表观遗传学的主要内容,是指通过DNA层面的修饰使原来拥有相同基因型的不同组织或细胞呈现出不同的表型和功能。非编码RNA作为此表观遗传调控的主要执行者之一,能够在转录水平调控蛋白质丰度,在生殖系统中更是发挥了基础调节者的作用。广义的非编码RNA包含了rRNA、tRNA、微小RNA（miRNA）、小干扰RNA（siRNA）、Piwi蛋白相互作用的RNA（piRNA）和长链非编码RNA（lncRNA）等。由于rRNA、tRNA已被人们较为熟知,文章主要选取miRNA、siRNA、piRNA等非编码RNA,针对其近期的研究进展,阐述该类非编码RNA在哺乳动物生殖细胞中的表达以及对生殖细胞发生、成熟的影响及作用机制。与此同时,还对另一类非编码RNA——lncRNA做基本的介绍。     

A Controlled Challenge Study on Di(2-ethylhexyl) Phthalate (DEHP) in House Dust and the Immune Response in Human Nasal Mucosa of Allergic Subjects

Background

Few studies have yet addressed the effects of di(2-ethylhexyl) phthalate (DEHP) in house dust on human nasal mucosa.

Objectives

We investigated the effects of house dust containing DEHP on nasal mucosa of healthy and house dust mite (HDM)–allergic subjects in a short-term exposure setting.

Methods

We challenged 16 healthy and 16 HDM-allergic subjects for 3 hr with house dust at a concentration of 300 μg/m³ containing either low (0.41 mg/g) or high (2.09 mg/g) levels of DEHP. Exposure to filtered air served as control. After exposure, we measured proteins and performed a DNA microarray analysis.

Results

Nasal exposure to house dust with low or high DEHP had no effect on symptom scores. Healthy subjects had almost no response to inhaled dust, but HDM-allergic subjects showed varied responses: DEHP_low house dust increased eosinophil cationic protein, granulocyte-colony–stimulating factor (G-CSF), interleukin (IL)-5, and IL-6, whereas DEHP_high house dust decreased G-CSF and IL-6. Furthermore, in healthy subjects, DEHP concentration resulted in 10 differentially expressed genes, whereas 16 genes were differentially expressed in HDM-allergic subjects, among them anti-Müllerian hormone, which was significantly up-regulated after exposure to DEHP_high house dust compared with exposure to DEHP_low house dust, and fibroblast growth factor 9, IL-6, and transforming growth factor-β1, which were down-regulated.

Conclusions

Short-term exposure to house dust with high concentrations of DEHP has attenuating effects on human nasal immune response in HDM-allergic subjects, concerning both gene expression and cytokines.     

邻苯二甲酸(2-乙基己基)酯低剂量暴露对雄性小鼠生殖发育的影响

目的观察邻苯二甲酸(2-乙基己基)酯(DEHP)低剂量暴露对雄性小鼠的生殖发育毒性作用。方法将清洁级ICR小鼠随机分为4个DEHP染毒组(1/120LD50,1/60LD50,1/30LD50,1/15LD50,即250、500、1000、2000mg/kg组),阳性对照组(环磷酰胺,40mg/kg)和阴性对照组(玉米油);DEHP染毒组和阴性对照组灌胃染毒30d,阳性对照组灌胃5d,每天1次。采用一般生殖毒性试验和显性致死突变试验的方法,研究DEHP低剂量暴露对雄性小鼠生殖细胞的损伤作用和生育能力的影响。结果一般生殖毒性试验结果显示,低剂量DEHP染毒雄性小鼠睾丸、附睾重量降低(r睾丸=-0.578,P<0.01;r附睾=-0.661,P<0.01),精子总数减少(r=-0.608,P<0.01),精子畸形率增高(r=0.836,P<0.01),精子活动度降低(P<0.05)。显性致死突变试验研究结果显示,250mg/kgDEHP染毒组小鼠受孕率降低,但与阴性对照组比较,差异无统计学意义(P>0.05);DEHP染毒组子代小鼠平均着床数减少(r=-0.892,P<0.01),与阴性对照组比较,差异均具有统计学意义(P<0.01);平均早期死亡胚胎数随染毒剂量增加而增加(r=0.996,P<0.01),250mg/kg染毒组与阴性对照组比较,差异无统计学意义(P>0.05)。结论DEHP低剂量暴露对雄性小鼠具有生殖毒性作用,可降低小鼠受孕率,使子代小鼠平均着床数减少,平均早期死亡胚胎数增加。     

不同烹调油样品中邻苯二甲酸二丁酯与邻苯二甲酸二（2-乙基己基）酯的分析

[目的]了解不同烹调油样品中邻苯二甲酸二丁酯（di-n—butyl phthalate,DBP）与邻苯二甲酸二（2-乙基己基）酯[di（2-ethylhexyl）phthalate,DEHP]的污染情况。[方法]采用气相色谱-质谱联用仪的选择离子检测（SIR）方式,测定取自居民家中未加热食用油、烹调剩油和厨房抽油烟机内油盒中的烹调油烟冷凝物,食堂中未加热食用油、烹调剩油和烹调油烟冷凝物;小摊贩所用的未加热食用油及其锅内剩油;快餐店未加热过的固体起酥油及烹调剩油等样品中DBP与DEHP的含量。[结果]9户居民家庭厨房和食堂不同烹调油样品中DBP和DEHP均为阳性,且烹调油烟冷凝物中的含量明显高于未加热食用油和烹调剩油中的含量;DBP和DEHP含量：前者分别为2．29～597．01μg／g和38．96～4774．72μg／g;后者分别为3．22～42．81μg／g和15．61～112．64μg／g。小摊贩和快餐店不同烹调油样品中,仅1例炸面食剩油检出DBP和DEHP。[结论]大部分烹调油样品被DBP、DEHP污染,且烹调油烟冷凝物中的含量大大增加。     

Pre-pubertal Di(2-ethylhexyl) Phthalate (DEHP) Exposure of Young Boars Did Not Affect Sperm In vitro Penetration Capacity of Homologous Oocytes Post-puberty

Di(2-ethylhexyl) phthalate (DEHP), a plastic softener used in polyvinylchloride (PVC) products (e.g., plastic bags and medical equipment), has been reported to have toxic effects on animal reproduction and is considered an environmental hazard based, mostly, on rodent studies. However, the doses used in these studies are often considerably higher than that presumed in human exposure. In the present study we used young boars as model animals to assess the effects of pre-pubertal DEHP exposure on the ability of spermatozoa to penetrate homologous oocytes in vitro. Eight pairs of cross-bred male boar siblings were used. One brother in each pair became, at random, the test animal exposed to DEHP per os, three times a week, from 3 to 7 weeks of age while the other acted as the control, i.e., placebo-exposed. Semen was collected and frozen between 8 and 9 months of age and stored until spermatozoa were evaluated for their ability to in vitro penetrate in vitro-matured homologous oocytes post-thaw. Both the penetration rate and the number of spermatozoa per oocyte were considered within expected ranges for frozen boar semen of good quality. Penetration rate did not significantly differ (p > 0.05) between the groups with DEHP-exposed: 50%; control: 59%, which could be owing to a large variation between boars, and between replicates. The number of spermatozoa in the ooplasm was low and similar (p > 0.05) between the groups with DEHP-exposed: 1.5 and the control: 1.7. Under the conditions of the present experiment, pre-pubertal exposure to DEHP does not seem to cause a deleterious effect on the in vitro fertilizing ability of frozen spermatozoa post-puberty.     

Dispersion and Ecological Risk Assessment of Di (2-Ethylhexyl) Phthalate (DEHP) in the Surface Waters of Thailand

Previous environmental monitoring indicated that di (2-ethylhexyl) phthalate (DEHP) was released into the environment. This study collected both water and sediment samples from the source area, deltas of major rivers discharging into the Gulf of Thailand, and 2 tourist beaches from February 2007–August 2009. The analyses showed that the highest DEHP concentrations were 8.64 μg l⁻¹ and 14.51 μg g⁻¹ for water and suspended sediment samples, respectively. Nevertheless, the monitoring results indicated that the dispersion of DEHP was limited to the source area. The ecological risk assessment indicated that the ecological risk did not exceed the acceptable level for the current degree of contaminations.     

大鼠孕期及哺乳期邻苯二甲酸二(2-乙基己基)酯暴露致雄性仔鼠脑组织病理学改变

目的观察邻苯二甲酸二(2-乙基已基)酯(DEHP)宫内及哺乳期暴露对子代雄性大鼠脑组织发育的影响。方法采用围生期毒性试验的研究方法,将清洁级SD大鼠随机分为阴性对照组(玉米油)和4个DEHP染毒组(125、250、500、1000mg/kg),采用宫内及哺乳期经口灌胃的方式染毒。记录比较孕鼠孕0、20d(GD0、GD20)、仔鼠出生后21d(PND21)母鼠体重及体重增长量、雄性仔鼠不同发育阶段体重、脑脏器系数;观察雄性仔鼠不同发育阶段脑组织病理学及超微病理学改变。结果母鼠染毒期间,体重及体重增长量不同程度降低(P0.05)。不同发育阶段雄性仔鼠体重显示,0～500mg/kg组随染毒剂量增加而增加,1000mg/kg组体重均明显低于相同发育阶段对照组仔鼠体重,仅PND21仔鼠体重500mg/kg组与对照组比较,差异有统计学意义(P0.05)。PND21雄性仔鼠500mg/kg组脑脏器系数低于对照组,1000mg/kg组高于对照组,差异有统计学意义(P0.05)。各发育阶段海马区锥体细胞和大脑皮质神经元细胞表现为不同程度的核固缩变性,500、1000mg/kg组表现较为严重。电镜结果显示各发育阶段海马区锥体细胞核不同程度固缩、线粒体灶性损伤,粗面内质网不同程度损伤,突触增多。结论 DEHP宫内及孕期暴露可引起雄性仔鼠脑组织病理形态改变,可影响雄性仔鼠脑组织生长。     

Effects of Dicyclohexyl Phthalate Exposure on PXR Activation and Lipid Homeostasis in Mice

Background: Exposure to plastic-associated endocrine disrupting chemicals (EDCs) has been associated with an increased risk of cardiovascular disease (CVD) in humans. However, the underlying mechanisms for this association are unclear. Many EDCs have been shown to function as ligands of the nuclear receptor pregnane X receptor (PXR), which functions as xenobiotic sensor but also has pro-atherogenic effects in vivo.Objective: We sought to investigate the contribution of PXR to the adverse effects dicyclohexyl phthalate (DCHP), a widely used phthalate plasticizer, on lipid homeostasis and CVD risk factors.Methods: Cell-based assays, primary organoid cultures, and PXR conditional knockout and PXR-humanized mouse models were used to investigate the impact of DCHP exposure on PXR activation and lipid homeostasis in vitro and in vivo. Targeted lipidomics were performed to measure circulating ceramides, novel predictors for CVD.Results: DCHP was identified as a potent PXR-selective agonist that led to higher plasma cholesterol levels in wild-type mice. DCHP was then demonstrated to activate intestinal PXR to elicit hyperlipidemia by using tissue-specific PXR-deficient mice. Interestingly, DCHP exposure also led to higher circulating ceramides in a PXR-dependent manner. DCHP-mediated PXR activation stimulated the expression of intestinal genes mediating lipogenesis and ceramide synthesis. Given that PXR exhibits considerable species-specific differences in receptor pharmacology, PXR-humanized mice were also used to replicate these findings.Discussion: Although the adverse health effects of several well-known phthalates have attracted considerable attention, little is known about the potential impact of DCHP on human health. Our studies demonstrate that DCHP activated PXR to induce hypercholesterolemia and ceramide production in mice. These results indicate a potentially important role of PXR in contributing to the deleterious effects of plastic-associated EDCs on cardiovascular health in humans. Testing PXR activation should be considered for risk assessment of phthalates and other EDCs. https://doi.org/10.1289/EHP9262     

睾丸热压内裤研制及对精子发生影响的临床研究

目的:研制适用于人用睾丸热压内裤(THU),并观察其对精子发生的影响。方法:恒温控制器、定时器、交流电变压装置及热压内裤的设计和研制,9例已育成年健康男性志愿者,每周穿THU2次,每次穿50min,3个月为1个实验周期。在实验前、中、后分别取精液和血液,以穿THU前的相关指标作自身对照,实验期分别于1个月、2个月和3个月观察,恢复期分别于1个月、2个月、3个月和6个月观察随访。结果:恒温控制器温度43℃±0.5℃,定时器0～60min,输入电压220V,输出电压12V,穿THU2个月后,精液常规中精子密度和精子活动率明显下降,而畸形精子比率明显升高;3个月后,精子密度和精子活动率进一步明显下降,9例受试者精子密度均下降到20×106/ml以下,3例<5×106/ml,其中有1例2×106/ml;在恢复期2个月、3个月和6个月后,上述各项指标又恢复到实验前水平。血清T、FSH、LH实验前后无明显改变。结论:THU对精子发生抑制效果较为明显,可考虑与男性激素合用抑制精子发生而达到避孕目的。     

孕期二月桂酸二丁基锡暴露对子代大鼠睾丸酶活力和生精功能的影响

目的 探讨孕期接触二月桂酸二丁基锡(DBTD)对Wistar大鼠子代雄性鼠性成熟后睾丸酶活力和生精功能的影响.方法 以0、10、20和30 mg/kg剂量的DBTD溶液对妊娠第12天的Wistar孕鼠连续灌胃染毒至第20天,仔鼠出生后第70天,每组随机抽取10只雄性大鼠,称重并处死,测定睾丸脏器系数、附睾精子数,并采用分光光度法测定睾丸酶活力.结果 各剂量组孕鼠染毒期间体重增加量、产仔数、仔鼠雌雄比例及子代雄性大鼠体重与对照组比较,差异均无统计学意义(P＞0.05);30 mg/kg组睾丸重量和睾丸脏器系数高于对照组,差异有统计学意义(P＜0.01,P＜0.05);20和30 mg/kg组精子数高于对照组,差异有统计学意义(P＜0.05,P＜0.01).各剂量组琥珀酸脱氢酶(SDH)活力和一氧化氮合酶(NOS)活力与对照组比较,差异无统计学意义(P＞0.05);10和20 mg/kg组酸性磷酸酶(ACP)活力、10 mg/kg组乳酸脱氢酶(LDH)活力、30 mg/kg组一氧化氮(N0)含量均高于对照组,差异有统计学意义(P＜0.05).结论 在本实验染毒时间和剂量范围内,DBTD对子代雄性大鼠睾丸发育和精子形成有促进作用,而对睾丸酶活力无直接影响.