Evaluation of a predictive in vitro Leydig cell assay for anti-androgenicity of phthalate esters in the rat

An in vitro assay using the rat Leydig cell line R2C was evaluated for its ability to quantitatively predict inhibition of testosterone synthesis. Results obtained for endocrine active phthalates (MEHP, MBP), and inactive phthalates (MMP and MEP) were highly consistent with in vivo results based on tissue and media concentrations. Statistically significant inhibition of testosterone synthesis (p < 0.05, 1-way ANOVA) was observed at 1 μM MBP and 3 μM MEHP, while MEP and MMP did not affect inhibition of testosterone synthesis until much higher concentrations (?100 μM). Concentrations causing 50% inhibition of testosterone synthesis for MBP and MEHP (3 and 6 μM respectively), were similar to in vivo values (3 and 7 μM). The R2C assay was used to determine the relative potency of 14 structurally diverse monoesters and oxidative metabolites of MEHP. Monoesters with alkyl chains 4–5 carbons in length had the highest potency for testosterone inhibition, while 0–2 carbon alkyl chains were least potent. Phase I metabolism did not completely inactivate MEHP, underscoring the need for metabolism data when interpreting in vitro pharmacodynamic data. This steroid inhibition assay provides a predictive in vitro alternative to expensive and timeconsuming developmental rat studies for phthalate-induced antiandrogenicity.     

Human urinary phthalate metabolites level and main semen parameters,sperm chromatin structure,sperm aneuploidy and reproductive hormones

The aim of the study was to assess the association of phthalate metabolites levels in urine with semen parameters (sperm concentration, motility, morphology, CASA parameters), sperm chromatin structure, sperm aneuploidy and reproductive hormones. The study population consisted of 269 men who were attending an infertility clinic and had normal semen concentration (20–300 mln/ml) or slight oligozoospermia (15–20 mln/ml). Participants were interviewed and provided a semen sample. The phthalate metabolites were analysed in the urine using a procedure based on the LC–MS/MS method. Urinary phthalate metabolites levels were significantly associated with a decrease in sperm motility (5OH MEHP, MEHP, MINP), CASA parameters (MBP), testosterone level (MEHP) and an increase sperm DNA damage (MBP) and sperm aneuploidy (MBzP, MBP, MEHP, MEP). In view of the importance of human reproductive health and the widespread usage of phthalates, it is important to further investigate these correlations.     

Kinetics of selected di-n-butyl phthalate metabolites and fetal testosterone following repeated and single administration in pregnant rats

Human exposure to phthalic acid diesters occurs through a variety of pathways as a result of their widespread use in consumer products and plastics. Repeated doses of di-n-butyl phthalate (DBP) from gestation day (GD) 12 to 19 disrupt testosterone synthesis and male sexual development in the fetal rat. Currently little is known about the disposition of DBP metabolites, such as monobutyl phthalate (MBP) and its glucuronide conjugate (MBP-G), during gestation after repeated exposure to DBP. In order to gain a better understanding of the effect of repeated dosing on maternal and fetal metabolism and distribution, pregnant Sprague–Dawley rats were given a single dose of 500 mg/kg DBP on GD 19 or daily doses of 50, 100, and 500 mg/(kg day) from GD 12 to 19 via corn oil gavage. Dose–response evaluation revealed a non-linear increase in maternal and fetal plasma concentrations of MBP. Maternal and fetal MBP levels were slightly lower in animals after 8 days of dosing at 500 mg/(kg day). Fetal plasma MBP levels closely followed maternal plasma, while the appearance and elimination of MBP-G in fetal plasma were significantly delayed. MBP-G accumulated over time in the amniotic fluid. Inhibition of testosterone was rapid in fetal testes when exposed to DBP (500 mg/(kg day)) on GD 19. Within 24 h, the level of inhibition in the fetus was similar between animals exposed to a single or multiple daily doses of 500 mg/(kg day). Examination of testosterone time-course data indicates a rapid recovery to normal levels within 24 h post-dosing at DBP doses of 50 and 100 mg/(kg day), with a rebound to higher than normal concentrations at later time-points. MBP kinetics in fetal testes allows direct comparison of active metabolite concentrations and testosterone response in the fetal testes.     

Mono(2-ethylhexyl)phthalate and mono-n-butyl phthalate activation of peroxisome proliferator activated-receptors alpha and gamma in breast

The phthalates di(2-ethylhexyl)phthalate (DEHP) and di-n-butyl phthalate (DBP) are environmental contaminants with significant human exposures. Both compounds are known reproductive toxins in rodents and DEHP also induces rodent hepatocarcinogenesis in a process believed to be mediated via the peroxisome proliferator-activated receptor alpha (PPARalpha). DEHP and DBP are metabolised to their respective monoesters, mono-(2-ethylhexyl)phthalate (MEHP) and mono-n-butyl phthalate (MBP), which are the active metabolites. MEHP also activates another member of the PPAR subfamily, PPARgamma. The effects of PPARalpha and PPARgamma activation in human breast cells appears to be opposing; PPARalpha activators in breast cells cause an increase in proliferation, while PPARgamma activation in breast cells is associated with differentiation and an inhibition of cell proliferation. Further to this the activation of the PPARs is cell and ligand specific, suggesting the importance of examining the effect of MEHP and MBP on the activation of PPARalpha, PPARbeta and PPARgamma in human breast. We used the common model of human breast cancer MCF-7 and examined the ability of MEHP and MBP to activate human PPARs in this system. The ability of MBP and MEHP to block PPAR responses was also assessed. We found that both human PPARalpha and PPARgamma were activated by MEHP whereas MEHP could not activate PPARbeta. MBP was unable to activate any PPAR isoforms in this breast model, despite being a weak peroxisome proliferator in liver, although MBP was an antagonist for both PPARgamma and PPARbeta. Our results suggest that the toxicological consequences of MEHP in the breast could be complex given the opposing effects of PPARalpha and PPARgamma in human breast cells.     

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.     

Detection of phthalate metabolites in human saliva

In assessment of exposure to environmental contaminants, the use of unconventional matrices is becoming an increasingly important area of research. Saliva is one of the most promising alternative matrices because its collection is easy, noninvasive, and inexpensive. In this study, we measured the salivary concentrations of 14 phthalate metabolites in 39 anonymous adult volunteers using isotope-dilution, automated solid phase extraction-high performance liquid chromatography-tandem mass spectrometry. Seven phthalate metabolites were detected at the concentrations ranging from below the limit of detection (<1 ng/mL) to 10.6 ng/mL for phthalic acid, 3.1 ng/mL for monomethyl phthalate (MMP), 91.4 ng/mL for monoethyl phthalate (MEP), 65.8 ng/mL for mono-n-butyl phthalate (MBP), 17.9 ng/mL for mono-iso-butyl phthalate, 353.6 ng/mL for monobenzyl phthalate, and 6.8 ng/mL for mono-2-ethylhexyl phthalate (MEHP). The frequency of detection was highest for MBP (85%) and lowest for MMP (8%). The median salivary MBP level in this group of adults was higher than the median serum MBP level in another non-occupationally exposed human adult population in the United States, whereas, the median salivary levels of MEP and MEHP were lower than the corresponding median serum levels. The frequency of detection and the salivary levels of each phthalate monoester in this study population were lower than the frequency of detection and urinary level of the same monoester in the general US population. Although urine is preferred for exposure assessment to non-persistent chemicals such as phthalates, the similar levels in serum and saliva suggest that saliva could be used as a surrogate matrix for measuring the bioavailable dose of phthalates in biomonitoring studies.     

Testicular atrophy induced by phthalic acid monoesters: Effects of zinc and testosterone concentrations

Induction of testicular atrophy by monoesters of phthalic acid were compared in male Wistar rats. Dietary administration of monobutyl (MBP), mono-iso-butyl (MIBP) and mono-2-ethylhexyl phthalate (MEHP) induced severe atrophy of the testes. Furthermore, high testosterone concentration in the testes was found in MBP- and MIBP-treated rats, whereas low zinc concentration was found in MBP-, MIBP- and MEHP-treated rats. On the other hand, in monooctyl phthalate (MOP)-treated rats, testosterone and zinc concentrations in the testes were not changed. These were similar to the results with their diesters.     

Effect of dibutyl phthalate on expression of connexin 43 and testosterone production of leydig cells in adult rats

To investigate the adverse effect of dibutyl phthalate (DBP) on Leydig cells and its mechanism related to gap junction, Leydig cells isolated from adult rats were treated with 0.1% dimethylsulfoxide (DMSO), 50 mg/L DBP, 50 mg/L DBP + 10 μM prostaglandin E2 (PGE2) and 40 μM flutamide respectively. Radioimmunoassay, semi-quantitative RT-PCR, immunofluorescence and Western blot were applied to determine the expression of testosterone and Connexin 43 (Cx43) in Leydig cells. The expression of testosterone and Cx43 were both decreased in DBP group (P < 0.05). While Cx43 was up-regulated after administered to PGE2, there was no significant change in testosterone. However, testosterone was down-regulated with a significant decrease of Cx43 in flutamide group. The results indicated that the inhibitory effect of DBP on testosterone production was not through the down-regulation of Cx43. On the contrary, the change of testosterone can influence the expression of Cx43 in Leydig cells.     

Disposition of diiosononyl phthalate and its effects on sexual development of the male fetus following repeated dosing in pregnant rats

Pregnant Sprague-Dawley rats received 50, 250, and 500 mg/kg/day diisononyl phthalate (DiNP) from GD 12 to 19 via corn oil gavage to study the dose response for effects on fetal male rat sexual development as well as metabolite disposition in the dam and fetus. Monoisononyl phthalate (MiNP), mono(carboxy-isooctyl) phthalate (MCiOP), mono(hydroxyl-isononyl) phthalate (MHiNP), mono(oxo-isononyl) phthalate (MOiNP), and monoisononyl phthalate glucuronide (MiNP-G) were found in all measured tissues. MCiOP was the major metabolite, followed in decreasing order by MiNP, MHiNP, MOiNP, and MiNP-G. Percentage of dose absorbed decreased at 750 mg/kg/day. Testosterone concentration in the fetal testes was reduced at 250 and 750 mg/kg/day. Multinucleated germ cells were increased in the testes of rats at 250 and 750 mg/kg/day. The no observed effect level (NOEL) for this study was 50 mg/kg/day based on increased MNGs and reduced testes testosterone concentration in the fetal rat.     

Short term exposure to di-n-butyl phthalate (DBP) disrupts ovarian function in young CD-1 mice

Di-n-butyl phthalate (DBP) is present in many beauty and medical products. Human exposure estimates range from 0.007–0.01 mg/kg/day in the general population and up to 0.233 mg/kg/day in patients taking DBP-coated medications. Levels of phthalates tend to be higher in women, thus, evaluating ovarian effects of DBP exposure is of great importance. Mice were given corn oil (vehicle) or DBP at 0.01, 0.1, and 1000 mg/kg/day (high dose) for 10 days to test whether DBP causes ovarian toxicity. Estrous cyclicity, steroidogenesis, ovarian morphology, and apoptosis and steroidogenesis gene expression were evaluated. DBP exposure decreased serum E₂ at all doses, while 0.1DBP increased FSH, decreased antral follicle numbers, and increased mRNA encoding pro-apoptotic genes (Bax, Bad, Bid). Interestingly, mRNAs encoding the steroidogenic enzymes Hsd17b1, Cyp17a1 and Cyp19a1 were increased in all DBP-treated groups. These novel findings show that DBP can disrupt ovarian function in mice at doses relevant to humans.     

Medications as a potential source of exposure to phthalates among women of childbearing age

ObjectiveTo evaluate the association between the use of medications potentially containing phthalates and urinary concentrations of specific phthalate metabolites around conception.MethodsWomen enrolled in the Environment and Reproductive Health project from 2006 to 2009 completed questionnaires about the use of medications and provided multiple urine samples before and after conception. We compared the mean urinary concentration of phthalate metabolites between users of phthalate containing medications and a matched unexposed control group.ResultsOne woman used Asacol^® (mesalamine), which utilizes dibutyl phthalate (DBP) as a delayed release coating material, and had a mean urinary concentration of the main DBP metabolite 200 times higher than the controls (8176 μg/L vs. 37.5 μg/L). The three users of stool softeners had a higher concentration of the main diethyl phthalate (DEP) metabolite (8636 μg/L vs. 714.2 μg/L). Neither the three additional Prilosec^® (omeprazole) users nor one cyclobenzaprine user had higher urinary concentration than controls.ConclusionSelected medications may be important sources of DBP and DEP exposures around conception.     

Mono-2-ethyhexyl phthalate advancing the progression of prostate cancer through activating the hedgehog pathway in LNCaP cells

Hedgehog (Hh) pathway plays a critical role in the progression of prostate cancer (PCa), the most commonly diagnosed non-cutaneous cancer in male adults. Studies showed that di-n-butyl phthalate (DBP) could interference with the Hh pathway. Di-2-ethylhexyl phthalate (DEHP), the congener of DBP, is the major plasticizer used in plastic materials that are inevitably exposed by patients with PCa. The aim of this in vitro study was to investigate whether mono-2-ethyhexyl phthalate (MEHP, the active metabolite of DEHP) could activate the Hh pathway of LNCaP cells. Results showed that the expression of the critical gene of Hh pathway PTCH and androgen-regulated gene KLK3 was significantly decreased on 3, 6 and 9 days with Hh pathway inhibitor cyclopamine's treatment. MEHP notably up-regulated the expression of PTCH with a dose–response relationship in the presence of cyclopamine, which indicate that MEHP might target on the downstream components of Hh pathway and advance the progression of PCa through activating the Hh pathway.     

A dose response study to assess effects after dietary administration of diisononyl phthalate (DINP) in gestation and lactation on male rat sexual development

Male rat sexual development was evaluated after dietary administration of 0, 760, 3800, 11,400 ppm diisononyl phthalate (DiNP) and 7600 ppm dibutyl phthalate (DBP) from gestation day (GD) 12 to postnatal day (PND) 14. Maternal weight was reduced on GD 20, PND 2 and 14 at 11,400 ppm DiNP. Pup weight was reduced on PND 2 and 14 at 11,400 and 3800 ppm DiNP. DBP induced multinucleated germ cells (MNGs) and Leydig cell aggregates (LCAs) in PND 2 testes. 7600 ppm DBP reduced anogenital distance (AGD) on PND 2 and 14, and increased nipple retention and reproductive tract malformations on PND 49. DiNP induced MNGs (3800 ppm) and LCAs (11,400 ppm) on PND 2, and reduced AGD (11,400 ppm) on PND 14. DiNP did not alter AGD, nipple retention or reproductive tract malformations on PND 49. Global endpoint analysis showed no evidence of a rat “phthalate syndrome” on PND 49 with DiNP administration.     

Role of PPARalpha in mediating the effects of phthalates and metabolites in the liver

Phthalate esters belong to a large class of compounds known as peroxisome proliferators (PP). PP include chemicals that activate different subtypes of the peroxisome proliferator-activated receptor (PPAR) family. The ability of phthalate esters and their metabolites to activate responses through different PPAR subtypes is not fully characterized. We investigated the ability of two phthalate esters di-(2-ethylhexyl) phthalate (DEHP) and di-n-butyl phthalate (DBP) and selected metabolites to activate PPAR (alpha, beta/delta, gamma) using a transient transfection assay. The monoester of DEHP, mono-(2-ethylhexyl) phthalate (MEHP) activated all three subtypes of PPAR, but preferentially activated PPARalpha. A second metabolite of DEHP, 2-ethylhexanoic acid (2-EHXA) was a weaker activator of all three subtypes. DBP, but not the primary metabolite mono-n-butyl phthalate weakly activated all three PPAR subtypes. MEHP and DBP but not DEHP and MBP interacted directly with human PPARalpha and PPARgamma as determined by scintillation proximity assays. Both DEHP and DBP activated expression of PP-inducible gene products in wild-type but not PPARalpha-null mice suggesting that both of these phthalates exert their effects by activation of PPARalpha in vivo. The preferential activation of PPARalpha by phthalate ester metabolites suggests that these phthalates mediate their toxic effects in rodent liver in a manner indistinguishable from other PP.     

Mono-(2-ethylhexyl) phthalate (MEHP) affects intercellular junctions of Sertoli cell: A potential role of oxidative stress

We analyzed the potential role of oxidative stress induced by mono (2-ethylhexyl) phthalate (MEHP) in adherent cell junction protein expression of prepubertal rat Sertoli cells (SC) in vitro. Five-day SC cultures were treated with MEHP (200 μM) for 24 h and compared to cells in basal conditions. Western blot and immunofluorescent (IF) analyses showed that MEHP induced increase of N-cadherin and catenin expression, modifying its distribution. Concomitantly, Cx-43 expression decreased significantly and delocalization of the IF signal for tight junction proteins (occludin, claudin-11 and ZO-1) occurred. Indicative of oxidative stress, MEHP induced in SC an increase of lipoperoxides, a decrease in glutathione (GSH) levels and a concomitant increase in Glutathione S-Transferases (GST) activity. Antioxidant N-acetyl-cysteine (1 mM) treatment prevented GSH decrease and N-cadherin and α-catenin up-regulation induced by MEHP. Our data suggest that oxidative stress signaling is a mechanism involved in adherent cell junctions disruption induced by MEHP in SC cultures.     

Phthalate and di-(2-ethylhexyl) adipate (DEHA) intake by German infants based on the results of a duplicate diet study and biomonitoring data (INES 2)

Phthalates as well as di-(2-ethylhexyl) adipate (DEHA) are used as plasticizers in diverse applications and are of toxicological concern.The study was conducted with a study population of 25 German subjects aged between 15 and 21 months. Overall, 16 phthalates and DEHA were measured by gas chromatography–mass spectrometry in a total of 171 duplicate diet samples collected over 7 consecutive days, and 20 phthalate metabolites were analyzed in the urine samples collected over 7 consecutive days using a liquid chromatography–tandem mass spectrometry method.The median “high” daily dietary intake based on 95th percentiles was 4.66 μg/kg b.w. for di-2-ethylhexyl phthalate (DEHP), 1.03 μg/kg b.w. for di-isobutyl phthalate (DiBP), and 0.70 μg/kg b.w. for di-n-butyl phthalate (DnBP), and 1.0 μg/kg b.w. for DEHA. The “high” daily total intake from biomonitoring data was 4.9 μg/kg b.w. for DEHP, 2.2 μg/kg b.w. for DnBP, 3.9 μg/kg b.w. for DiBP, and 2.6 μg/kg b.w. for di-isononyl phthalate.The comparison of the two intake estimates indicates that the dominant intake source of DEHP was food ingestion, whereas other sources considerably contributed to the total intake of other phthalates. Using our “high” intake scenario, none of the analyzed phthalates reached the recommended tolerable daily intake levels.     

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.     

Co-treatment of mouse antral follicles with 17β-estradiol interferes with mono-2-ethylhexyl phthalate (MEHP)-induced atresia and altered apoptosis gene expression

Mono-2-ethyhexyl phthalate (MEHP) is a metabolite of a plasticizer found in many consumer products. MEHP inhibits mouse ovarian follicle growth by reducing 17β-estradiol (E₂) production. Yet, whether MEHP causes follicle death (atresia) is unclear. We hypothesized that MEHP causes atresia by altering apoptosis gene expression, and that E₂ co-treatment blocks these effects. Follicles were exposed to MEHP (0.36–36 μM) ± E₂ for 48–96 h to determine the effect of MEHP ± E₂ on atresia and gene expression. MEHP increased atresia, but this effect was blocked by co-treatment with E₂. MEHP increased the expression of the pro-apoptotic gene Aifm1, but decreased that of the pro-apoptotic gene Bok and the anti-apoptotic gene Bcl2l10. E₂ interfered with MEHP-induced changes in Aifm1 and Bcl2l10. Our findings suggest that decreased E₂ levels are required for MEHP-induced follicle atresia and that Aifm1, Bok, and Bcl2l10 are involved in this process.