The aim of this study was to evaluate the effects of low concentrations of DEHP and MEHP on steroidogenesis in a murine Leydig tumor cell line (MLTC-1) in vitro. The result of flow cytometry analysis revealed that the proportion of apoptotic cells was significantly increased after the exposure to DEHP. All three genes (P450scc, P450c17, and 3βHSD) under study showed an increased expression following exposure to DEHP or MEHP, although some insignificant inhibitory effects appeared in the 10 μmol/L treatment group as compared with the controls. It was also found that compared with the controls. It was also found that DEHP or MEHP stimulated INSL3 mRNA and protein especially in the 0.001 μmol/L treatment group. Testosterone secretions were stimulated after the exposure to DEHP or MEHP. Alternations of steroidogenic enzymes and INSL3 in MLTC-1 cells might be involved in the biphasic effects of DEHP/MEHP on androgen production.
Animals ; Apoptosis ; drug effects ; Cell Line, Tumor ; Diethylhexyl Phthalate ; analogs & derivatives ; toxicity ; Leydig Cell Tumor ; metabolism ; pathology ; Mice ; Steroids ; biosynthesis

OBJECTIVETo study the association between phthalate esters (PAEs) metabolites in maternal urine and 11beta-hydroxysteroid dehydrogenase type 2 (11β-HSD2 ) enzyme activity, explore the possible mechanism of PAEs effect on fetal development.

METHODSAll of 33 cases of intrauterine growth retardation (IUGR) newborn were selected by random sampling in 2012. And 33 cases of normal control newborn were enrolled, use high performance liquid chromatography-tandem mass spectrometry method was used to detect 4 kinds of phthalate esters (PAEs) metabolites in maternal urine: mono-n-butyl phthalate ester (MBP), mono (2-ethylhexyl) phthalate (MEHP), mono (2-ethyl-5-hydroxyhexyl) phthalate (MEHHP), mono (2-ethyl-5-oxohexyl) phthalate (MEOHP) and three kinds of cortisol corticosterone metabolites, tetrahydrocortisol (THF), allo-tetrahydrocortisol (allo-THF), tetrahydrocortisone (THE), and analyze the association between phthalate esters (PAEs) metabolites in maternal urine and 11β-HSD2 enzyme activity.

RESULTSMBP, MEHP, MEHHP, MEOHP metabolites can be detected in 98% (65 cases) , 89% (59 cases), 91% (60 cases), 91% (60 cases) of all 66 maternal urine samples, respectively. The median concentrations of test material in case group were 31.20 ng/ml for MBP, 24.61 ng/ml for MEHHP, 11.72 ng/ml for MEOHP and 48.67 ng/ml for SumDEHP which were significantly higher than those of the control group (were 17.32, 12.03, 5.68 and 28.64 ng/ml); 11β-HSD2 activity in case group ((THF+allo-THF)/THE = (0.79 ± 0.09) ng/ml) was significantly lower than that of the control group((THF+allo-THF)/THE = (0.58 ± 0.04) ng/ml); PAEs metabolites MBP (β' = 1.12), MEHHP(β' = 1.14), MEOHP(β' = 1.10), SumDEHP(β' = 1.08) in baby boy mother's urine was reversely correlated to 11β-HSD2 activity.

CONCLUSIONSPAEs could affect fetal development by inhibit 11β-HSD2 activity.

11-beta-Hydroxysteroid Dehydrogenase Type 2 ; Chromatography, Liquid ; Diethylhexyl Phthalate ; analogs & derivatives ; Fetal Development ; Humans ; Infant, Newborn ; Male ; Mass Spectrometry ; Phthalic Acids ; Tetrahydrocortisol ; analogs & derivatives ; Tetrahydrocortisone

OBJECTIVETo explore the influences of di-(2-ethylhexyl)phthalate (DEHP) and its principle metabolite mono(2-ethylhexyl)phthalate (MEHP) on spermatogenic cell apoptosis in young male Wistar rats.

METHODSNinety-eight 2-week-old male Wistar rats were randomly divided into 14 equal groups to receive daily intragastric administration of 0.2 ml/kg normal saline for 3 weeks (normal control), 100 mg/kg cyclophosphamide (CTX) for 1 week (positive control), 100, 200, and 300 mg/kg DEHP or MEHP for 1 week, or 100 mg/kg DEHP or MEHP for 1, 2, and 3 weeks. After the treatments, the pathological changes of the testicular tissues were examined, spermatogenic cell apoptosis was detected, and serum sex hormones levels were measured using TUNEL assay or radioimmunoassays.

RESULTSCTX, DEHP, and MEHP all caused shrinkage, development retardation and quantitative reduction of spermatogenic cells with and mitochondrial swelling vacuolar changes. The damage of spermatogenic cells increased significantly with the increment of DEHP and MEHP doses and exposure time. Both DEHP and MEHP treatments resulted in significantly increased cell apoptosis index (AI) in close correlation with the exposure doses and duration (P<0.01). DEHP and MEHP treatments also significantly increased serum levels of follicle stimulating hormone and luteinizing hormone and decreased testosterone levels in a dose- and time-dependent manner (P<0.05).

CONCLUSIONDEHP and MEHP can induce obvious apoptosis of spermatogenic cells in young male rats with a dose- and time-dependent effect.

Animals ; Apoptosis ; drug effects ; Diethylhexyl Phthalate ; analogs & derivatives ; toxicity ; Dose-Response Relationship, Drug ; Environmental Exposure ; Male ; Rats ; Rats, Wistar ; Spermatozoa ; cytology ; drug effects

OBJECTIVE: Prenatal phthalate exposure has been associated with placental inflammatory factors and infant allergic rhinitis (AR). However, the results are inconclusive. We designed a population-based cohort study to examine the effects of placental inflammatory biomarkers on the sex-dependent associations between maternal phthalate exposure and infant AR. METHODS: A total of 2,348 pregnant women from Ma'anshan, Anhui Province, China, who were screened before antenatal visits and met the inclusion criteria, were included in the present study. We assessed AR in their offspring aged 36 months with a questionnaire. Quantitative PCR was performed to measure placental inflammatory factor mRNAs. The independent samples t-test and multivariable logistic regression were used to determine the associations between infant AR and maternal phthalates. RESULTS: Childhood AR may be related to education and family monthly income ( P = 0.01). The phthalate metabolites, mono (2-ethylhexyl) phthalate (MEHP), mono (2-ethyl-5-hydroxyl) phthalate (MEHHP), in pregnant women were associated with a significantly increased risk for infant AR in males [ P < 0.05; odds ratio ( OR): 1.285; 95% confidence interval ( CI): 1.037-1.591, and OR: 1.232, 95% CI: 1.008-1.507, respectively], but not females. Additionally, irritably-increased expression levels of HO-1 and IL-4 were associated with AR in male infants ( OR: 1.175; 95% CI: 1.038-1.329 and OR: 1.181; 95% CI: 1.056-1.322, respectively). The association between maternal urinary MEHHP and placental HO-1 was marginally significant according to mediation analysis. CONCLUSION The associations of maternal MEHHP and MEOHP levels with fetal AR in males were significant. Placental HO-1 was a fractional mediator in the associations between MEHHP and AR. Thus, the placenta should be further investigated as a potential mediator of maternal exposure-induced disease risk in children.
Biomarkers ; Child, Preschool ; Cohort Studies ; Diethylhexyl Phthalate/analogs & derivatives* ; Female ; Humans ; Interleukin-4/pharmacology* ; Male ; Maternal Exposure/adverse effects* ; Phthalic Acids/adverse effects* ; Placenta ; Pregnancy ; Rhinitis, Allergic/epidemiology*

OBJECTIVES: In most DEHP exposure assessment studies, single spot urine sample was used. It could not compare the exposure level among studies. Therefore, we are going to represent the necessity of selection of proper sampling time of spot urine for assessing the environmental DEHP exposure, and the association urinary DEHP metabolites with steroid hormones. METHODS: We collected urine and plasma from 25 men. The urine sampling times were at the end of the shift (post-shift) and the next morning before the beginning of the shift (pre-shift). Three metabolites of DEHP {mono(2-ethylhexyl) phthalate [MEHP], mono-(2-ethyl-5-hydroxyhexyl)phthalate [MEHHP], and mono(2-ethyl-5-oxohexyl)phthalate [MEOHP]} in urine were analyzed by HPLC/MS/MS. Plasma luteinzing hormone, follicle stimulating hormone, testosterone, and 17beta-estradiol were measured at pre-shift using a ELISA kit. A log-transformed creatinine-adjusted urinary MEHP, MEHHP, and MEOHP concentration were compared between the post- and pre-shift. The Pearson's correlation was calculated to assess the relationships between log-transformed urinary MEHP concentrations in pre-shift urine and hormone levels. RESULTS: The three urinary metabolite concentrations at post-shift were significantly higher than the concentrations in the pre-shift (p<0.0001). The plasma hormones were not significantly correlated with log-transformed creatinine - adjusted DEHP metabolites. CONCLUSIONS: To assess the environmental DEHP exposure, it is necessary to select the urine sampling time according to the study object. There were no correlation between the concentration of urinary DEHP metabolites and serum hormone levels.
Adult ; Diethylhexyl Phthalate/analogs & derivatives/pharmacokinetics/*urine ; Estradiol/blood ; Follicle Stimulating Hormone/blood ; Humans ; *Laboratories, Dental ; Luteinizing Hormone/blood ; Male ; Middle Aged ; Occupational Exposure/*analysis ; Phthalic Acids/urine ; Specimen Handling/*methods ; Testosterone/blood ; Time Factors

OBJECTIVEThe purposes of our study were to investigate the association between maternal urinary phthalate metabolites and the levels of inhibin B (INHB) and insulin-like factor 3 (INSL3) in the cord blood in a Chinese pregnant population.

METHODSMaternal urine samples in the third trimester of pregnancy of 69 participants were collected and stored, and the samples of cord blood (10 ml) were collected at delivery between June 2011 and September 2012 in a comprehensive hospital of gynecology and obstetrics in Tianjin, China.Four phthalate metabolites, monomethyl phthalate (MMP), monoethyl phthalate (MEP), monobutyl phthalate (MBP), and mono-2-ethylhexyl phthalate (MEHP) were measured in the urine samples using liquid chromatography-tandem mass spectrometry. The levels of INHB, INSL3 in the cord blood were tested by ELISA. Associations of phthalate exposure with INHB and INSL3 levels were determined by spearman correlation and multiple regression model analysis.

RESULTSThe median concentrations of observed metabolites in descending order were 49.74 µg/L for MMP, 24.96 µg/L for MEHP, 19.52 µg/L for MEP and 17.73 µg/L for MBP. The median concentrations of INHB and INSL3 were 89.09 and 106.21 ng/L.Significant negative associations between INHB and MMP(β' = -0.252), MEP(β' = -0.363) or the sum value (∑PAEs) (β' = -0.346) were found by the multiple regression model analysis. For INSL3, only the sum value (β' = -0.313) was inversely significantly associated with the levels of INSL3 in the cord blood.

CONCLUSIONSMaternal urinary phthalate metabolites were associated with INHB and INSL3 in the cord blood in a Chinese population.

Adult ; Diethylhexyl Phthalate ; analogs & derivatives ; urine ; Female ; Fetal Blood ; chemistry ; Humans ; Infant, Newborn ; Inhibin-beta Subunits ; blood ; Insulin ; blood ; Male ; Maternal Exposure ; Phthalic Acids ; urine ; Pregnancy ; Proteins ; Testicular Hormones ; blood ; Young Adult

OBJECTIVETo investigate the effects of mono(2-ethylhexyl) phthalate(MEHP), the primary metabolite of di(2-ethylhexyl) phthalate (DEHP), on testosterone biosynthesis in Leydig cells cultured from the Sprague Dawley rat testis.

METHODSBased on the primary Leydig cell culture model, MEHP exposure groups involved control (0 micromol/L), 62.5, 125, 250, 500 and 1000 micromol/L. We observed mitochondria activity with the MTT method, measured the testosterone level with RIA and determined steroidogenesis acute regulatory protein (StAR) mRNA expression with RT-PCR.

RESULTSAfter Leydig cells were exposed to MEHP for 24 hours, the activity of mitochondria enhanced evidently at 250 micromol/L and then declined markedly at 1000 micromol/L compared with the control group (P < 0.01). The testosterone level showed an increasing tendency in both basal and hCG-stimulated states with statistical significance at 250 and 500 micromol/L compared with the control group (P < 0.01). However, the expression of StAR mRNA appeared unchanged at 62.5, 125 or 250 micromol/L, but exhibited a decreasing tendency at 500 and 1000 micromol/L (P < 0.01).

CONCLUSIONME- HP directly affected the activity of mitochondria and testosterone biosynthesis of the Leydig cells in vitro. StAR, the regulator of cholesterol transport into mitochondria, might not be responsible for the increase of testosterone biosynthesis induced by MEHP.

Animals ; Cells, Cultured ; Diethylhexyl Phthalate ; analogs & derivatives ; pharmacology ; Dose-Response Relationship, Drug ; Leydig Cells ; drug effects ; metabolism ; Male ; Phosphoproteins ; biosynthesis ; genetics ; RNA, Messenger ; genetics ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction ; Testosterone ; biosynthesis

OBJECTIVETo study the constituents from roots of Euphorbia hylonoma.

METHODColumn chromatographic techniques were used for isolation and purification of the chemical constituents and their structures were identified by spectral analysis (IR, 1H-NMR, 13C-NMR, 2D-NMR and MS).

RESULTSix compounds were isolated and elucidated as nonane (1), bis (2-ethylhexyl) phthalate (2), euphol (3), beta-sitosterol (4), acalyphol (5) and daucosterol (6) respectively.

CONCLUSIONCompounds 1, 2, 3, 5 and 6 were isolated from the plant for the first time.

Alkanes ; chemistry ; isolation & purification ; Diethylhexyl Phthalate ; chemistry ; isolation & purification ; Euphorbia ; chemistry ; Lanosterol ; analogs & derivatives ; chemistry ; isolation & purification ; Plant Roots ; chemistry ; Plants, Medicinal ; chemistry ; Sitosterols ; chemistry ; isolation & purification ; Triterpenes ; chemistry ; isolation & purification

BACKGROUNDMono-(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.

METHODSCH1 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.

RESULTSAs 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.

CONCLUSIONMEHP in a high dose was cytotoxic and affected the development of hESCs, which indicates its embryo toxicity in human embryos.

Apoptosis ; drug effects ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; Cells, Cultured ; Diethylhexyl Phthalate ; analogs & derivatives ; toxicity ; Dose-Response Relationship, Drug ; Embryonic Stem Cells ; drug effects ; pathology ; Humans

In this study, the effects of mono(2-ethylhexyl) phthalate (MEHP), one of metabolites of di(2-ethylhexyl) phthalate, on immature Shiba goat testes in vitro were examined. The testes of 2-month-old Shiba goats were cut into smaller pieces, and seeded in medium. At 1, 3, 6 and 9 hr after administration of MEHP at various concentrations (0, 100 nmol ml-1, 1 nmol ml-1, and 1 x 10-3 nmol ml-1, respectively), the specimens were obtained for light and transmission electron microscopic observations. As a result, at 1 hr after exposure to MEHP, the vacuolization and nuclear membrane rupture appeared in Sertoli cells. Such alterations tended to gradually increase in number in timeand dose-dependent manners. Moreover, by MEHP treatment, apoptotic spermatogenic cells (characterized with chromatin condensation, cytoplasm shrinkage without membrane rupture, still functioning cell organelles, and packed cell contents in membrane-bounded bodies), apoptotic Sertoli cells (characterized with nuclear membrane lysis, nuclear condensation), necrotic spermatogenic cells (characterized with swollen and ruptured mitochondria, plasma membrane lysis, spilt cell contents, and chromatin clumps), and necrotic Sertoli cells (characterized with marginated chromatins along the nuclear membrane, ruptured vesicles within the MNB, some swollen and ruptured cell organelles, e.g. mitochondria) could be identified. Conclusively, ultrastructurally the treatment with MEHP at low concentration tends to lead spermatogenic and Sertoli cells to apoptosis, whereas that at high concentration tends to lead spermatogenic and Sertoli cells to necrosis. Thus, the testicular tissue culture is advantageous for screening testicular toxicity of chemicals.
Animals ; Apoptosis/drug effects/physiology ; Diethylhexyl Phthalate/*analogs&derivatives/*toxicity ; Goat Diseases/*chemically induced/pathology ; Goats ; Male ; Microscopy, Electron, Transmission/veterinary ; Necrosis ; Sertoli Cells/ultrastructure ; Spermatozoa/drug effects/pathology/ultrastructure ; Testicular Diseases/*chemically induced/pathology ; Testis/*drug effects/metabolism/pathology/ultrastructure ; Vacuoles/physiology/ultrastructure