Suppression by phthalates of the calcium signaling of human nicotinic acetylcholine receptors in human neuroblastoma SH-SY5Y cells

Phthalates are widely used in industry and cause public concern since they have genomic estrogenic-like effects via estrogen receptors. We previously found that some phthalates have nongenomic effects, exerting inhibitory effects on the functional activities of nicotinic acetylcholine receptors (nAChRs) in bovine chromaffin cells. In this study, we investigated the effects of eight phthalates on the calcium signaling of human nAChR by using human neuroblastoma SH-SY5Y cells. All eight phthalates, with different potency, have inhibitory roles on the calcium signaling coupled with human nAChR, but not muscarinic acetylcholine receptors (mAChRs). For inhibition of human nAChR, the strongest to weakest potencies were observed as di-n-pentyl phthalate (DPP) → butyl benzyl phthalate (BBP) → di-n-butyl phthalate (DBP) → dicyclohexyl phthalate (DCHP) → di-n-hexyl phthalate (DHP) → di-(2-ethyl hexyl) phthalate (DEHP) → di-n-propyl phthalate (DPrP) → diethyl phthalate (DEP). The potencies of phthalates were associated with their structures such that the most effective ones had dialkyl group carbon numbers of C4 or C5, with shorter or longer numbers resulting in decreased potency. At as low as 0.1 μM, DPP, DBP, BBP, DCHP and DHP significantly inhibited the calcium signaling of human nAChR. The IC₅₀ of phthalates on human nAChR, ranging from 0.32 to 7.96 μM, were 10–50 lower than those for bovine nAChR. We suggest that some phthalates effectively inhibit the calcium signaling of human nAChR, and these nongenomic effects are cause for concern.     

Effects and toxicity of phthalate esters to hemocytes of giant freshwater prawn,Macrobrachium rosenbergii

Phthalate esters (PAEs) have been considered as environmental pollutants and have been subject to control in the United States of America and Japan. The aim of this study was to investigate the effects and toxicity of eight PAEs to hemocytes and the defense functions of giant freshwater prawn (Macrobrachium rosenbergii), including hemocytic adhesion, pseudopodia formation, phenoloxidase (PO) activity, and superoxide anion (O(2)(-)) production, by means of in vitro exposure experiments. After hemocytes were treated separately with eight PAEs at concentrations of 100 microg/ml, the results showed that two PAEs (dipropyl phthalate, DPrP and diethyl phthalate, DEP) increased cells with pseudopodia formation, but decreased adhesive cells; reduction in the percentages of both pseudopodia formation and adhesive cells were detected in the dihexyl phthalate (DHP) and diphenyl phthalate (DPP) experiment groups; and di-(2-ethyl hexyl) phthalate (DEHP) decreased pseudopodia formation, but did not affect the adhesion. In addition, both PO activity and O(2)(-) production were decreased after hemocytes were treated with five PAEs (benzyl butyl phthalate (BBP), di-n-butyl phthalate (DBP), DEP, DHP and DPrP), respectively. At the same time, microscopy showed that both DPrP and DHP altered morphology of the cell nucleus and led to the presence of vacuoles in cytosol of hemocytes. Using the annexin assay, and after analysis of DNA fragmentation and transmission electron microscopy (TEM), it was found that hemocytes exposed to DHP and DPrP for more than 10 min would primarily die via apoptosis, the fatality correlates with increasing treatment time; and hemocytes treated with either BBP, dicyclohexyl phthalate (DCP), DEP or DPP would primarily die via necrosis. According to these results, we suggest that all eight PAEs examined could damage hemocytes and further influence the defense mechanism of prawns. This study reveals an important precaution for prawn cultivation.     

Biological impact of phthalates

Esters of phthalic acid are chemical agents used to improve the plasticity of industrial polymers. Their ubiquitous use in multiple commercial products results in extensive exposure to humans and the environment. This study investigated cytotoxicity, endocrine disruption, effects mediated via AhR, lipid peroxidation and effects on expression of enzymes of xenobiotic metabolism caused by di-(2-ethy hexyl) phthalate (DEHP), diethyl phthalate (DEP), dibutyl phthalate (DBP) and benzyl butyl phthalate (BBP) in developing fish embryos. Oxidative stress was identified as the critical mechanism of toxicity (CMTA) in the case of DEHP and DEP, while the efficient removal of DBP and BBP by phase 1 enzymes resulted in lesser toxicity. DEHP and DEP did not mimic estradiol (E₂) in transactivation studies, but at concentrations of 10 mg/L synthesis of sex steroid hormones was affected. Exposure to 10 mg BBP/L resulted in weak transactivation of the estrogen receptor (ER). All phthalates exhibited weak potency as agonists of the aryl hydrocarbon receptor (AhR). The order of potency of the 4 phthalates studied was; DEHP > DEP > BBP >> DBP. The study highlights the need for simultaneous assessment of: (1) multiple cellular targets affected by phthalates and (2) phthalate mixtures to account for additive effects when multiple phthalates modulate the same pathway. Such cumulative assessment of multiple biological parameters is more realistic, and offers the possibility of more accurately identifying the CMTA.     

Structure-activity relationships of phthalates in inhibition of human placental 3β-hydroxysteroid dehydrogenase 1 and aromatase

Phthalates are associated with preterm delivery. However, the mechanism is unclear. Progesterone formed by 3β-hydroxysteroid dehydrogenase 1 (HSD3B1) and estradiol by aromatase (CYP19A1) in placenta are critical for maintaining pregnancy. In this study, we compared structure-activity relationships (SAR) of 14 phthalates varied in carbon atoms in alcohol moiety to inhibit human HSD3B1 in COS1 and CYP19A1 in JEG-3 cells. There were responses in that only diphthalates with 4–7 carbon atoms were competitive HSD3B1 inhibitors and diphthalates with 6 carbon atoms were CYP19A1 inhibitors. IC₅₀s of dipentyl (DPP), bis(2-butoxyethyl) (BBOP), dicyclohexyl (DCHP), dibutyl (DBP), and diheptyl phthalate (DHP) were 50.12, 32.41, 31.42, 9.69, and 4.87 μM for HSD3B1, respectively. DCHP and BBOP inhibited CYP19A1, with IC₅₀s of 64.70 and 56.47 μM. DPP, BBOP, DCHP, DBP, and DHP inhibited progesterone production in JEG-3 cells. In conclusion, our results indicate that there is clear SAR for phthalates in inhibition of HSD3B1 and CYP19A1.     

Phthalates inhibit tamoxifen-induced apoptosis in MCF-7 human breast cancer cells

Environmental estrogens represent a class of compounds that can mimic the function or activity of the endogenous estrogen 17 -estradiol (E2). Phthalates including butyl benzyl phthalate (BBP), di(n-butyl) phthalate (DBP), and di(2-ethylhexyl) phthalate (DEHP) are used as plasticizers, and also widely used in food wraps and cosmetic formulations. Phthalates have been shown to mimic estrogen and are capable of binding to the estrogen receptor (ER). It has been demonstrated that estrogen promotes drug resistance to tamoxifen (TAM) in breast cancer. In order to further evaluate the potential role of the phthalates as environmental estrogens, the effect of phthalates was investigated on TAM-induced apoptosis in MCF-7 human breast cancer cells. Our results show that phthalates, BBP (100 M), DBP (10 M), and DEHP (10 M), significantly increased cell proliferation in MCF-7, but not in MDA-MB-231 cells. In addition, BBP, DBP, and DEHP mimicked estrogen in the inhibition of TAM-induced apoptosis in MCF-7 cells. Our data suggest that the inhibitory effect of phthalates on TAM-induced apoptosis involves an increase in intracellular Bcl-2 to Bax ratio. Given that the phthalates are widely used in cosmetics mainly for women, our findings that revealed the promoting effect of BBP, DBP, and DEHP on chemotherapeutic drug resistance to TAM in breast cancer may be of biological relevance.     

Assessing the relevance of in vitro measures of phthalate inhibition of steroidogenesis for in vivo response

Human phthalate exposure occurs as mixtures of diesters with varying activity towards testosterone-dependent development. Dibutyl (DBP), diethylhexyl (DEHP) and butylbenzyl (BBP) phthalate disrupt sexual development in the fetal rat. Dimethyl (DMP) and diethyl (DEP) phthalate do not. These differences in potency may result from differential delivery of the monophthalates to the testes or from variation in the abilities of the compounds to alter steroidogenesis. We tested five phthalates in pregnant rats (500 mg/kg-d, GD12–19) and analyzed the fetal testes for corresponding monoesters (MMP, MEP, MBP, MEHP, MBeP). Testes MMP and MEP levels were 2–40-fold higher than the active monoesters, MBP and MEHP. BBP exposure led to low concentrations of MBeP, but similar MBP levels to DBP. An in vitro MA-10 cell assay measured the direct effect of monophthalates on testosterone production. MEHP inhibited LH-stimulated testosterone production at 1 μM. RT-PCR confirmed down-regulation of genes associated with cholesterol transport and steroid synthesis and metabolism by MEHP. Five additional phthalates were tested for testosterone inhibition. MBP and mono-n-octyl phthalate were similar to MEHP; MMP, MEP and MBeP were poor inhibitors of testosterone production. Based on these results, differences in the phthalates’ ability to interfere with sexual development in vivo appears to be more associated with differential potency for testosterone inhibition than differences in tissue doses.     

Estimated exposure to phthalates in cosmetics and risk assessment

Some phthalates such as di(2-ethylhexyl) phthalate (DEHP) and dibutyl phthalate (DBP) and their metabolites are suspected of producing teratogenic or endocrine-disrupting effects. To predict possible human exposure to phthalates in cosmetics, the levels of DEHP, diethyl phthalate (DEP), DBP, and butylbenzyl phthalate (BBP) were determined by high-performance liquid chromatography (HPLC) in 102 branded hair sprays, perfumes, deodorants, and nail polishes. DBP was detected in 19 of the 21 nail polishes and in 11 of the 42 perfumes, and DEP was detected in 24 of the 42 perfumes and 2 of the 8 deodorants. Median exposure levels to phthalates in cosmetics by dermal absorption were estimated to be 0.0006 g/kg body weight (bw)/d for DEHP, 0.6 g/kg bw/d for DEP, and 0.103 g/kg bw/d for DBP. Furthermore, if phthalates in cosmetics were assumed to be absorbed exclusively via 100% inhalation, the median daily exposure levels to phthalates in cosmetics were estimated to be 0.026 g/kg bw/d for DEHP, 81.471 g/kg bw/d for DEP, and 22.917 g/kg bw/d for DBP, which are far lower than the regulation levels set buy the Scientific Committee on Toxicity, Ecotoxicity, and the Environment (CSTEE) (37 g/kg bw/d, DEHP), Agency for Toxic Substances and Disease Registry (ATSDR) (7000 g/kg bw/d, DEP), and International Programme on Chemical Safety (IPCS) (66 g/kg bw/d, DBP), respectively. Based on these data, hazard indices (HI, daily exposure level/regulation level) were calculated to be 0.0007 for DEHP, 0.012 for DEP, and 0.347 for DBP. These data suggest that estimated exposure to-phthalates in the cosmetics mentioned are relatively small. However, total exposure levels from several sources may be greater and require further investigation.     

Phthalates residues in plastic bottled waters

This study was conducted to determine the presence of phthalates in 10 different brands of bottled water available in Saudi markets and stored under different conditions. Dimethyl phthalate (DMP), diethylphthalate (DEP), di-n-butyl phthalate (DBP), benzyl butyl phthalate (BBP) and diethyl hexyl phthalate (DEHP) were measured by headspace solid-phase microextraction followed by gas chromatography- mass spectrometer detector. Most of these phthalates were detected in the selected bottled water sample that might be either leached from the plastic packaging materials or contamination during bottling processes. Bottled waters stored at 4°C contained higher levels of DMP, DEP, BBP and DEHP than those stored at room temperature and outdoors. On the other hand, the levels of DMP, DEP and BBP were significantly lower in bottled waters stored at room temperature than those outdoor. It seems that temperature and sunlight play a role in the degradation of phthalates within time. The levels of BBP were the highest at 4°C storage (4.592 ± 3.081 μg/l; range: 1.194-21.128 μg/l) and approximately 76% of the bottled waters had BBP above the limit of quantification (LOQ) of 0.994 μg/l. Apart from DEHP (< 6 μg/l), there are not current legislations for other phthalates. Regardless of storage conditions, all our samples did not exceed the maximum established limit of DEHP. Although, the levels of phthalates in tested bottled waters were low, one should not dismiss that these chemicals may cause endocrine disruption through several mechanisms, especially to potentially vulnerable populations such as infants and pregnant women. Saudi Arabia ranks 12 in bottled water consumption (88 L per capita in 2004) among the 71 reported countries. With this high consumption, a quality assurance scheme for residue monitoring in water is quite important. Although, one cannot avoid phthalates contamination in bottled waters due to manufacturing process but at least special care should be taken regarding their storage conditions.     

呋喃妥因肠溶片中邻苯二甲酸酯类物质的定性分析及定量检测

目的建立呋喃妥因肠溶片中邻苯二甲酸酯类物质定性分析及定量检测的方法。方法采用气质联用色谱法进行定性分析。采用高效液相色谱法对筛查出的5种邻苯二甲酸酯类物质进行定量检测,色谱柱为Phenomenex C18色谱柱(250mm×4.6mm,5μm),以乙腈-水(90∶10)为流动相,流速1.0mL·min-1,检测波长为224nm,柱温为30℃。结果对用气质联用色谱法筛查出的5种邻苯二甲酸酯类物质,建立了高效液相色谱法测定方法。DMP,DEP,DBP,DPP和BBP质量浓度在1²50μg·mL-1范围内线性关系良好,回收率在87%250μg·mL-1范围内线性关系良好,回收率在87%¹07%范围内。结论该法简便、快速、准确,可用于呋喃妥因肠溶片中邻苯二甲酸酯类化合物的定性分析及定量检测。     

Phthalates in Belgian cow’s milk and the role of feed and other contamination pathways at farm level

This study investigated the occurrence of dimethyl phthalate (DMP), diethyl phthalate (DEP), diisobutyl phthalate (DiBP), di-n-butyl phthalate (DnBP), benzylbutyl phthalate (BBP), di(2-ethylhexyl) phthalate (DEHP), dicyclohexyl phthalate (DCHP) and di-n-octyl phthalate (DnOP) in raw cow’s milk and feed from Belgian farms in order to determine their most relevant contamination pathways in milk. Measurable levels of DMP, DEP, DnBP, DCHP and DnOP were found in various feed samples, although they were not observed in milk. A plausible explanation for this is that they are rapidly metabolised in cows. DEHP and in a smaller degree also DiBP and BBP levels in milk seemed to vary across seasons and farms. DiBP and BBP levels were lower in summer than in winter milk, which was in contrast with what was observed for DEHP. This is possibly due to another feed composition during summer and winter. Comparing BBP and DEHP concentrations in manually with those in mechanically obtained milk revealed that, besides environmental contamination via feed ingestion, contact materials used during the mechanical milking process is another important contamination pathway. Concentrations observed in this study confirm the decreasing trend of DEHP in European cow’s milk owing to the substitution of DEHP by other plasticisers.     

A comparative study on antioxidant status combined with integrated biomarker response in Carassius auratus fish exposed to nine phthalates

Laboratory experiments were performed to determine the antioxidant responses to nine phthalates (PAEs) in the liver of the goldfish Carassius auratus. The fish were injected with 10 mg/kg body weight of each PAE for 1 day and 4, 8, and 15 days. The potential biotoxicity of the PAEs were examined using the antioxidase and lipid peroxide indices. We determined that the superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and malondialdehyde (MDA) levels displayed different trends following prolonged treatment, suggesting that metabolism generated either less toxic or more active substances. Based on the intensity of enzymes inhibition, MDA content, and the calculated integrated biomarker response (IBR), the toxicity order was determined as follows: dibutyl phthalate (DBP) > diethyl phthalate (DEP) > diisodecyl phthalate (DIDP) > diphenyl phthalate (DPP) > butyl benzyl phthalate (BBP) > diallyl phthalate (DAP) > dicyclohexyl phthalate (DCHP) > dimethyl phthalate (DMP) > di(2‐ethylhexyl) phthalate (DEHP). In particular, DBP, which exhibited significant inhibition of enzyme activity and the greatest decrease in MDA content, may be a highly toxic contaminant. Furthermore, our results suggest that the IBR may be a general marker of pollution. © 2014 Wiley Periodicals, Inc. Environ Toxicol 30: 1125–1134, 2015.     

Analysis of phthalates in food products and packaging materials sold on the Belgian market

Phthalates are organic lipophilic compounds that are principally used as plasticiser to increase the flexibility of plastic polymers. Other applications are a.o. the use of phthalates in printing inks and lacquers. Human exposure to phthalates mainly occurs via food ingestion and can induce adverse health effects. In this study, the presence of eight phthalate compounds – dimethyl phthalate (DMP), diethyl phthalate (DEP), diisobutyl phthalate (DiBP), di-n-butyl phthalate (DnBP), benzylbutyl phthalate (BBP), di(2-ethylhexyl) phthalate (DEHP), dicyclohexyl phthalate (DCHP) and di-n-octyl phthalate (DnOP) – was investigated in 400 food products, divided over eleven groups, and packages sold on the Belgian market. For this purpose, suitable extraction techniques were developed and validated for four different matrices, namely high-fat foods, low-fat food products, aqueous-based beverages and packaging materials. The instrumental analysis was performed by means of gas chromatography–low resolution-mass spectrometry with electron impact ionisation (GC–EI–MS). A wide variety of phthalate concentrations was observed in the different groups. DEHP was found in the highest concentration in almost every group. Moreover, DEHP was the most abundant phthalate compound, followed by DiBP, DnBP and BBP. This survey is part of the PHTAL project, which is the first project that discusses phthalate contamination on the Belgian food market.     

Perinatal exposure to the phthalates DEHP, BBP, and DINP, but not DEP, DMP, or DOTP, alters sexual differentiation of the male rat.

In mammals, exposure to antiandrogenic chemicals during sexual differentiation can produce malformations of the reproductive tract. Perinatal administration of AR antagonists like vinclozolin and procymidone or chemicals like di(2-ethylhexyl) phthalate (DEHP) that inhibit fetal testicular testosterone production demasculinize the males such that they display reduced anogenital distance (AGD), retained nipples, cleft phallus with hypospadias, undescended testes, a vaginal pouch, epididymal agenesis, and small to absent sex accessory glands as adults. In addition to DEHP, di-n-butyl (DBP) also has been shown to display antiandrogenic activity and induce malformations in male rats. In the current investigation, we examined several phthalate esters to determine if they altered sexual differentiation in an antiandrogenic manner. We hypothesized that the phthalate esters that altered testis function in the pubertal male rat would also alter testis function in the fetal male and produce malformations of androgen-dependent tissues. In this regard, we expected that benzyl butyl (BBP) and diethylhexyl (DEHP) phthalate would alter sexual differentiation, while dioctyl tere- (DOTP or DEHT), diethyl (DEP), and dimethyl (DMP) phthalate would not. We expected that the phthalate mixture diisononyl phthalate (DINP) would be weakly active due to the presence of some phthalates with a 6-7 ester group. DEHP, BBP, DINP, DEP, DMP, or DOTP were administered orally to the dam at 0.75 g/kg from gestational day (GD) 14 to postnatal day (PND) 3. None of the treatments induced overt maternal toxicity or reduced litter sizes. While only DEHP treatment reduced maternal weight gain during the entire dosing period by about 15 g, both DEHP and DINP reduced pregnancy weight gain to GD 21 by 24 g and 14 g, respectively. DEHP and BBP treatments reduced pup weight at birth (15%). Male (but not female) pups from the DEHP and BBP groups displayed shortened AGDs (about 30%) and reduced testis weights (about 35%). As infants, males in the DEHP, BBP, and DINP groups displayed femalelike areolas/nipples (87, 70, and 22% (p < 0.01), respectively, versus 0% in other groups). All three of the phthalate treatments that induced areolas also induced a significant incidence of reproductive malformations. The percentages of males with malformations were 82% (p < 0.0001) for DEHP, 84% (p < 0.0001) for BBP, and 7.7% (p < 0.04) in the DINP group. In summary, DEHP, BBP, and DINP all altered sexual differentiation, whereas DOTP, DEP, and DMP were ineffective at this dose. Whereas DEHP and BBP were of equivalent potency, DINP was about an order of magnitude less active.     

Hershberger assay for antiandrogenic effects of phthalates

The antiandrogenic effects of seven phthalates, di(2-ethylhexyl) phthalate (DEHP), dibutyl phthalate (DBP), butyl benzyl phthalate (BBP), di-isononyl phthalate (DINP), di-isodecyl phthalate (DIDP), di-n-heptyl phthalate (DnHP), and mono-2-ethyhexyl phthalate (MEHP), were investigated by Hershberger assay in castrated male SD rats. An androgen agonist, testosterone (0.4 mg/kg/d), was administered for 10 consecutive days by subcutaneous (s.c.) injection as a positive control. Additionally, 20, 100, or 500 mg/kg body weight (bw)/d of 6 phthalates (DEHP, DBP, BBP, DINP, DIDP, or DnHP) or 10, 50, or 250 mg/kg bw/d of MEHP, the primary metabolite of DEHP, were also administered orally in combination with testosterone (0.4 mg/kg/d, s.c.) for 10 consecutive days, respectively. In the testosterone-treated groups, glans penis, seminal vesicles, ventral prostate, and levator ani/bulbocavernosus muscles (LABC) weights were found to be significantly increased. Ventral prostate weights were significantly decreased in animals treated with DEHP or DBP at doses of 20 mg/kg bw/d or above, 500 mg/kg bw/d DIDP, and 250 mg/kg bw/d MEHP. Seminal vesicles weights were also significantly decreased by DEHP at > 100 mg/kg bw/d, DINP at > 20 mg/kg bw/d, DIDP at 500 mg/kg bw/d, or MEHP at 50 or 250 mg/kg bw/d, respectively. In addition, LABC weights were decreased by DEHP at 500 mg/kg bw/d, DINP at 500 mg/kg bw/d, and MEHP at 50 or 100 mg/kg bw/d. These data suggest that some phthalates possess antiandrogenic activity, and that multiple cross-talk between androgen, estrogen, and steroid hormone receptors occurs.     

Effect of cooking at home on the levels of eight phthalates in foods

Food products can be contaminated with toxic compounds via the environment. Another possibility of food contamination is that toxicants are generated in foods or that chemicals migrate from food contact materials into foods during processing. In this study, the effect of cooking at home on the levels of phthalates – world’s most used group of plasticisers – in various food types (starchy products, vegetables and meat and fish) was examined. Eight compounds were considered, namely dimethyl phthalate (DMP), diethyl phthalate (DEP), diisobutyl phthalate (DiBP), di-n-butyl phthalate (DnBP), benzylbutyl phthalate (BBP), di(2-ethylhexyl) phthalate (DEHP), dicyclohexyl phthalate (DCHP) and di-n-octyl phthalate (DnOP). Food products were analysed before as well as after cooking (boiling, steaming, (deep-)frying or grilling). In general, phthalate concentrations in foods declined after cooking, except in vegetables, where almost no effect was seen. Several factors influenced the degree of this decline (e.g. weight difference, fat uptake, etc.). Of all phthalates, DEHP, DiBP and BBP were affected the most. In conclusion, cooking at home definitely affected phthalate concentrations in foods and thus needs to be considered in order to correctly assess humans’ dietary exposure to these contaminants.     

Effects of Phthalates on the Human Corneal Endothelial Cell Line B4G12

Phthalates are industrial chemicals used in many cosmetics. We evaluated an in vitro model for eye irritancy testing using the human corneal endothelial cell line B4G12. Cell proliferation and toxicity were assessed after exposing to di-n-butyl phthalate (DBP), benzyl butyl phthalate (BBP), di-2-ethylhexyl phthalate (DEHP), diisodecyl phthalate (DIDP), di-n-octyl phthalate (DnOP), and di-isononyl phthalate (DINP). Gene expression and secretion of inflammatory cytokines were evaluated after exposure to DBP. Decreased cell proliferation was observed for the phthalates DBP, BBP, and DEHP, and cell toxicity was observed for DBP and BBP. Upon DBP exposure at nontoxic concentrations, a significant increased gene expression and cytokine cell secretion were observed for interleukin-1β (IL-1β) and IL-8, and also an increased IL-6 secretion was observed. In conclusion, the human corneal endothelial cell line B4G12 may be a potential model for inflammatory eye irritancy testing of phthalates.     

Dermal toxicity elicited by phthalates: Evaluation of skin absorption,immunohistology, and functional proteomics

The toxicity of phthalates is an important concern in the fields of environmental health and toxicology. Dermal exposure via skin care products, soil, and dust is a main route for phthalate delivery. We had explored the effect of topically-applied phthalates on skin absorption and toxicity. Immunohistology, functional proteomics, and Western blotting were employed as methodologies for validating phthalate toxicity. Among 5 phthalates tested, di(2-ethylhexyl)phthalate (DEHP) showed the highest skin reservoir. Only diethyl phthalate (DEP) and dibutyl phthalate (DBP) could penetrate across skin. Strat-M^® membrane could be used as permeation barrier for predicting phthalate penetration through skin. The accumulation of DEHP in hair follicles was ∼15 nmol/cm², which was significantly greater than DBP and DEP. DBP induced apoptosis of keratinocytes and fibroblasts via caspase-3 activation. This result was confirmed by downregulation of 14-3-3 and immunohistology of TUNEL. On the other hand, the HSP60 overexpression and immunostaining of COX-2 suggested inflammatory response induced by DEP and DEHP. The proteomic profiling verified the role of calcium homeostasis on skin inflammation. Some proteins investigated in this study can be sensitive biomarkers for dermal toxicity of phthalates. These included HSPs, 14-3-3, and cytokeratin. This work provided novel platforms for examining phthalate toxicity on skin.     

Reproductive effects of four phthalic acid esters in the mouse

These studies compared the reproductive toxicity of four phthalates by a continuous breeding protocol. Mice were given diets with diethyl phthalate (DEP) (0.0, 0.25, 1.25, or 2.5%), di-n-butyl phthalate (DBP) (0.0, 0.03, 0.3, or 1.0%), di-n-hexyl phthalate (DHP) (0.0, 0.3, 0.6, or 1.2%), or di(2-ethylhexyl) phthalate (DEHP) (0.0, 0.01, 0.1, or 0.3%). Both male and female CD-1 mice were dosed for 7 days prior to and during a 98-day cohabitation period. Reproductive function was evaluated during the cohabitation period by measuring the numbers of litters per pair and of live pups per litter, pup weight, and offspring survival. There was no apparent effect on reproductive function in the animals exposed to DEP, despite significant effects on body weight gain and liver weight. DBP exposure resulted in a reduction in the numbers of litters per pair and of live pups per litter and in the proportion of pups born alive at the 1.0% amount, but not at lower dose levels. A crossover mating trial demonstrated that female mice, but not males, were affected by DBP, as shown by significant decreases in the percentage of fertile pairs, the number of live pups per litter, the proportion of pups born alive, and live pup weight. DHP in the diet resulted in dose-related adverse effects on the numbers of litters per pair and of live pups per litter and proportion of pups born alive at 0.3, 0.6, and 1.2% DHP in the diet. A crossover mating study demonstrated that both sexes were affected. DEHP (at 0.1 and 0.3%) caused dose-dependent decreases in fertility and in the number and the proportion of pups born alive. A crossover mating trial showed that both sexes were affected by exposure to DEHP. These data demonstrate the ability of the continuous breeding protocol to discriminate the qualitative and quantitative reproductive effects of the more and less active congeners as well as the large differences in reproductive toxicity attributable to subtle changes in the alkyl substitution of phthalate esters.     

Human monitoring of phthalates and risk assessment

Some phthalates, such as di(2-ethylhexyl) phthalate (DEHP) and dibutyl phthalate (DBP), and their metabolites are suspected of producing teratogenic and endocrino-disrupting effects. In this study, urinary levels of phthalates (DEHP, DBP, diethyl phthalate (DEP), butylbenzyl phthalate BBP), and monoethylhexyl phthalate (MEHP, a major metabolite of DEHP) were measured by high performance liquid chromatography (HPLC) in human populations (women [hospital visitors], n = 150, and children, n = 150). Daily exposure level of DEHP in children was estimated to be 12.4 microg/kg body weight/d (male 9.9 microg/kg body weight/d, female 17.8 microg/kg body weight/d), but, in women was estimated to be 41.7 microg/kg body weight/d, which exceeded the tolerable daily intake (TDI, 37 microg/kg body weight/day) level established by the European Union (EU) Scientific Committee for Toxicity, Ecotoxicity, and the Environment (SCTEE) based on reproductive toxicity. Based on these data, hazard indices (HIs) were calculated to be 1.12 (41.7/37 TDI) for women and 0.33 (12.4/37 TDI) for children, respectively. These data suggest that Koreans (women and children) were exposed to significant levels of phthalates, which should be reduced to as low a level as technologically feasible to protect Koreans from the exposure to toxic phthalates.