Evaluation of endocrine disrupting activity of plasticizers in polyvinyl chloride tubes by estrogen receptor alpha binding assay

Polyvinyl chloride (PVC) tubing is an indispensable medical material for extracorporeal circulation therapy. However, di(2-ethylhexyl)phthalate (DEHP), a suspected endocrine disruptor, can be eluted from PVC, suggesting that an alternative material that does not contain DEHP is needed for clinical applications. First, we evaluated the endocrine disrupting risks of the plasticizers contained in PVC tubes by investigating their binding affinities for the human estrogen receptor alpha (ERα). Our results revealed that, while DEHP has some binding affinity for ERα, neither epoxidized soybean oil nor tris(2-ethylhexyl)trimellitate (an alternative to DEHP) has any affinity for ERα. Second, we evaluated the endocrine disrupting risks of a tube made of newly developed plasticizer-free (PF) materials. We confirmed the presence of DEHP and detected several unidentified substances in plasma stored within the PVC tube. This plasma's competitive binding affinity for ERα was significantly higher than that of control plasma (P < 0.01). In contrast, the profile of plasma stored in the PF tube was similar to that of the control, both in terms of high-performance liquid chromatography chromatograms and competitive binding capacity for ERα, suggesting that the PF tube is biocompatible and is useful for reducing the elution of substances capable of binding to ERα. Presented in part at the 42nd Congress of the Japanese Society for Artificial Organs, October 5–7, 2004, Tokyo, Japan     

Non-linearities in the pharmacokinetics of di-(2-ethylhexyl) phthalate and metabolites in male rats

The disposition of the plasticizer di-(2-ethylhexyl) phthalate (DEHP) and four of its major metabolites was studied in male rats given single infusions of a DEHP emulsion in doses of 5, 50 or 500 mg DEHP/kg body weight. Plasma concentrations of DEHP and metabolites were followed for 24 h after the start of the infusion. The kinetics of the primary metabolite mono-(2-ethylhexyl) phthalate (MEHP) was studied separately.The concentrations of DEHP in plasma were at all times considerably higher than those of MEHP, and the concentrations of MEHP were much higher than those of the other investigated metabolites. In animals given 500 mg DEHP/kg, the areas under the plasma concentration-time curves (AUCs) of the other investigated metabolites were at most 15% of that of MEHP. Parallel decreases in the plasma concentrations of DEHP, MEHP and the and (-1) oxidized metabolites indicated that the elimination of DEHP was the rate-limiting step in the disposition of the metabolites. This was partly supported by the observation that the clearance of MEHP was higher than that of DEHP. Nonlinear increases in the AUCs of DEHP and MEHP indicated saturation in the formation as well as the elimination of the potentially toxic metabolite MEHP.     

固相微萃取技术测定塑料浸取液中邻苯二甲酸二辛酯

目的　探讨塑料浸取液中邻苯二甲酸二辛酯 (DEHP)的测定方法。方法　采用聚硅氧烷和富勒烯聚二甲基硅氧烷混合固定相自制萃取头 ,利用顶空固相微萃取与气相色谱联用技术 (HS -SPME -GC)分析塑料浸取液中邻苯二甲酸二辛酯 ,研究萃取时间、热解吸时间、溶液的酸碱度和离子强度等因素对方法灵敏度的影响 ,并与商用聚二甲基硅氧烷 (PDMS)萃取头比较。结果　该萃取头的萃取选择性优于PDMS萃取头 ,方法具有良好的线性范围(0 .4～ 5 0 0 μg·L-1) ,最低检出限为 0 .15 μg·L-1,相对标准偏差为 4.9% (n =6 )。结论　固相微萃取技术分析水样中邻苯二甲酸二辛酯具有高效、灵敏、操作简单、无需溶剂等优点。     

Participation of the Fas-Signaling System in the Initiation of Germ Cell Apoptosis in Young Rat Testes after Exposure to Mono-(2-Ethylhexyl) Phthalate

The Fas-signaling system is composed of the interacting proteins Fas (CD95/APO-1) and Fas ligand (FasL, CD95L, APO-1L) and is proposed to act in the testis as a paracrine signaling mechanism by which FasL-expressing Sertoli cells initiate apoptosis of Fas-bearing germ cells. Here we describe alterations in the expression of Fas and FasL in the testis after the intimate physical association between Sertoli cells and germ cells is disrupted by exposure to the Sertoli cell toxicant mono-2-(ethylhexyl) phthalate (MEHP). Young, 28-day-old Fisher rats were treated with MEHP (2 g/kg po) and killed 0, 3, 6, and 12 h after exposure. Immunohistochemical analyses revealed a significant increase in the numbers of Fas-positive germ cells as well as increases in the expression of Sertoli cell FasL. Western blot analysis demonstrated a time-dependent increase in the production of the soluble form of FasL after MEHP exposure and suggests that it may participate in triggering apoptosis in germ cells that have lost their intimate association with the Sertoli cells. Measurement of Fas in cytosolic and membrane fractions of testis homogenates by Western blot analysis revealed a significant shift of Fas expression into the membrane fraction after MEHP exposure. Taken together, these observations indicate that the Fas-mediated paracrine signaling mechanism participates in triggering apoptosis of germ cells despite the loss of their close physical association with Sertoli cells. A working model is presented to explain the involvement of the Fas-system in stimulating germ cell apoptosis after MEHP exposure.     

Effects of Diethyl Phthalate and Other Plasticizers on Laurate Hydroxylation in Rat Liver Microsomes

Diethyl phthalate (DEP) is used in pharmaceutical coatings, cosmetics, and plastic films to wrap foods. There is a health concern associated with the exposure to certain phthalate esters because they belong to a class of compounds referred to as peroxisome proliferators which have been shown to increase the incidence of liver tumors when administered to rats. In this study, we have compared DEP to four other commonly used plasticizers, 2-diethylhexyl phthalate (DEHP), dibutyl phthalate (DBP), 2-diethylhexyl adipate (DEHA), and acetyltributyl citrate (ATBC), for their ability to induce the cytochrome P450-mediated fatty acid -hydroxylation system, which is one of the initial cellular responses when animals are treated with peroxisome proliferators. The administration of DEHP, DBP, and DEHA to rats increased the specific activity of laurate 12-hydroxylase from 2.8 ± 1.1 in control rats to 30.3 ± 11.6, 14.5 ± 4.1, and 9.7 ± 1.9 nmol 12-hydroxylaurate formed/min/nmol P450, respectively. In contrast, laurate 12-hydroxylase activity in DEP-and ATBC-treated rats were 4.4 ± 1.2 and 4.4 ± 1.0 nmol 12-hydroxylaurate formed/min/nmol P450, respectively. In addition, whereas DEHP increased peroxisomal palmitoyl-CoA oxidation 6-fold, DEP increased this activity only 1.3-fold. Two protein bands, at 51 and 52 kDa, were found to increase 6- to 12-fold in microsomes of DEHP-, DBP-, and DEHA-treated rats, but these bands were increased only 2-fold in DEP- or ATBC-treated rats.     

Altered gene expression during rat Wolffian duct development following di(n-butyl) phthalate exposure.

Di(n-butyl) phthalate (DBP) is a common plasticizer and solvent that disrupts androgen-dependent male reproductive development in rats. In utero exposure to 500 mg/kg/day DBP on gestation days (GD) 12 to 21 decreases androgen biosynthetic enzymes, resulting in decreased fetal testicular testosterone levels. One consequence of prenatal DBP exposure is malformed epididymides in adult rats. Reduced fetal testosterone levels may be responsible for the malformation, since testosterone is required for Wolffian duct stabilization and their development into epididymides. Currently, little is understood about the molecular mechanisms of Wolffian duct differentiation. The objective of this study was to identify changes in gene expression associated with altered morphology of the proximal Wolffian duct following in utero exposure to DBP. Pregnant Crl:CD(R) (SD) rats were gavaged with corn oil vehicle or 500 mg/kg/day DBP from GD 12 to GD 19 or 21. There were only small morphological differences between control and DBP-exposed Wolffian ducts on GD 19. On GD 21, 89% of male fetuses in the DBP dose group showed marked underdevelopment of Wolffian ducts, characterized by decreased coiling. RNA was isolated from Wolffian ducts on GD 19 and 21. Together with empirical information, cDNA microarrays were used to help identify candidate genes that could be associated with the morphological changes observed on GD 21. These candidate genes were analyzed by real-time RT-PCR. Changes in mRNA expression were observed in genes within the insulin-like growth factor (IGF) pathway, the matrix metalloproteinase (MMP) family, the extracellular matrix, and in other developmentally conserved signaling pathways. On GD 19, immunolocalization of IGF-1 receptor protein demonstrated an increase in cytoplasmic expression in the mesenchymal and epithelial cells. There was also a variable decrease in androgen receptor protein in ductal epithelial cells on GD 19. This study provides insight into the effects of antiandrogens on the molecular mechanisms involved in Wolffian duct development. The altered morphology and changes in gene expression following DBP exposure are suggestive of altered paracrine interactions between ductal epithelial cells and the surrounding mesenchyme during Wolffian duct differentiation due to lowered testosterone production.     

邻苯二甲酸二丁酯致大鼠隐睾睾丸和附睾病理组织学改变研究

目的：邻苯二甲酸二丁酯（DBP）孕晚期染毒诱导子代雄鼠隐睾的发生和探讨隐睾睾丸、附睾病理组织学的改变。方法：孕鼠40只，随机分组，在怀孕14-18天期间，每天分别灌胃给予大豆油（A组）、DBP100mg／kg（B组）、500mg／kg（C组）、800mg／kg（D组）。出生70天后观察雄仔鼠隐睾发生率及隐睾睾丸、附睾病理组织学改变。结果：C、D组雄仔鼠隐睾发生率为10．8％和63．5％；隐睾睾丸、附睾的脏器系数显著减轻；睾丸生精上皮萎缩，生精细胞层减少甚至消失，曲精小管体积减小；同时附睾管腔中精子缺如。电镜下隐睾睾丸中出现异常的支持细胞。结论：睾丸是DBP主要作用的靶器官；中、高剂量DBP孕晚期染毒可致雄仔鼠隐睾的发生、睾丸生精上皮的损害，从而影响其生育能力。     

邻苯二甲酸二环己酯人工抗原的制备研究

目的合成邻苯二甲酸二环己酯半抗原衍生物和合格的人工全抗原,探讨其制备方法。方法通过保留环己基结构和在芳环上引入氨基取代基合成了一种邻苯二甲酸二环己酯半抗原衍生物4-氨基邻苯二甲酸二环己酯,产物经通过1HNMR、IR和UV确证了结构。半抗原衍生物再通过重氮化反应与BSA偶联。结果得到的半抗原衍生物4-硝基邻苯二甲酸二环己酯的2个紫外吸收峰分别为:λ1=214nm,λ2=256nm;4-氨基邻苯二甲酸二环己酯的2个紫外吸收峰分别为:λ1=226nm,λ2=288nm。人工全抗原4-氨基邻苯二甲酸二环己酯-BSA,经荧光光谱(λex=307nm,λem=468nm)、体系颜色变化和免疫实验确证偶联成功。抗原结合比为19∶1。人工全抗原通过免疫动物得到了效价高、特异性好、纯度也较高的抗体。结论通过保留环己基结构和在芳环上引入氨基的设计思路可以合成邻苯二甲酸二环己酯的人工全抗原。     

邻苯二甲酸二丁酯对雌性果蝇生殖能力的双重作用

目的:研究邻苯二甲酸二丁酯(DBP)对雌性果蝇生殖能力的影响。方法:将未交配雌性果蝇随机分为5组,分别给予含DBP0.1%(A组)、0.4%(B组)、1.6%(C组)和6.4%(D组)的培养基喂饲,并以0浓度为对照组(E组),在染毒10d和20d时,分别取存活的实验雌蝇进行交配试验和生育试验。结果:各染毒组雌蝇1h交配率随DBP浓度升高而降低,D组雌蝇的1h交配率和生育率明显低于E组(P<0.05或P<0.01);染毒10d后C组的平均生育数和平均产卵期均明显高于E组(P<0.05),且在连续产卵10d后仍保持较高的生育水平;染毒20d后D组的平均生育数和平均产卵期均明显低于E组(P<0.01)。结论:DBP对雌蝇生殖能力有双重作用,较低浓度可提高雌性果蝇生育力,而较高浓度可使其生育能力降低。     

Effects of di(2‐ethylhexyl)phthalate on testicular oxidant/antioxidant status in selenium‐deficient and selenium‐supplemented rats

Di(ethylhexyl)phthalate (DEHP), the most widely used plasticizer, was investigated to determine whether an oxidative stress process was one of the underlying mechanisms for its testicular toxicity potential. To evaluate the effects of selenium (Se), status on the toxicity of DEHP was further objective of this study, as Se is known to play a critical role in testis and in the modulation of intracellular redox equilibrium. Se deficiency was produced in 3‐weeks‐old Sprague–Dawley rats feeding them ≤0.05 mg Se /kg diet for 5 weeks, and Se‐supplementation group was on 1 mg Se/kg diet. DEHP‐treated groups received 1000 mg/kg dose by gavage during the last 10 days of the feeding period. Activities of antioxidant selenoenzymes [glutathione peroxidase 1 (GPx1), glutathione peroxidase 4 (GPx4), thioredoxin reductase (TrxR)], catalase (CAT), superoxide dismutase (SOD), and glutathione S‐transferase (GST); concentrations of reduced glutathione (GSH), oxidized glutathione (GSSG), and thus the GSH/GSSG redox ratio; and thiobarbituric acid reactive substance (TBARS) levels were measured. DEHP was found to induce oxidative stress in rat testis, as evidenced by significant decrease in GSH/GSSG redox ratio (>10‐fold) and marked increase in TBARS levels, and its effects were more pronounced in Se‐deficient rats with ～18.5‐fold decrease in GSH/GSSG redox ratio and a significant decrease in GPx4 activity, whereas Se supplementation was protective by providing substantial elevation of redox ratio and reducing the lipid peroxidation. These findings emphasized the critical role of Se as an effective redox regulator and the importance of Se status in protecting testicular tissue from the oxidant stressor activity of DEHP. © 2011 Wiley Periodicals, Inc. Environ Toxicol 29: 98–107, 2014.