Estimation of plasma levels of bisphenol-A & phthalates in fertile & infertile women by gas chromatography-mass spectrometry

Background & objectives: Bisphenol-A (BPA) and phthalates are utilized widely in consumer products. Due to their ubiquitous presence in the environment, a concern is expressed worldwide about their possible effect on human reproductive health. This study was conducted to compare the internal exposure of BPA and phthalates (using their metabolites as biomarkers) in plasma samples of infertile and fertile women. Methods: A sensitive gas chromatographic-mass spectrometric (GC-MS) method was developed to simultaneously quantify BPA and four phthalate monoester metabolites [namely mono-methyl phthalate (MMP), mono-benzyl phthalate (MBzP), mono-2-ethylhexyl phthalate (MEHP) and mono-(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP)] in human plasma. The method was validated using charcoal-stripped human plasma. Activated charcoal was also utilized to reduce contamination from reagents. The method was designed to account for and/or eliminate background contamination from all sources. Results: The limit of quantification for the method was 5 ng/ml for MMP and MBzP, while 1 ng/ml for BPA, MEHP and MEHHP, respectively. The precision and accuracy were well within the acceptable range. BPA was detectable in 77 per cent of plasma samples of infertile women and 29 per cent of fertile women. All the four phthalate metabolites were detected in plasma samples of both fertile and infertile women. Interpretation & conclusions: A GC-MS was developed and validated to estimate the BPA and four phthalate monoester metabolites in human plasma. It was utilised to analyse the plasma samples from fertile and infertile women. The infertile women showed significantly higher plasma concentrations of MBzP, BPA and MEHHP as compared to fertile women. The levels of MMP and MEHP were not significantly different between the two groups. Further studies need to be done to confirm these preliminary findings.

Effect of mono-2-ethylhexyl phthalate on proliferation and migration of neural stem cells / 中国药理学与毒理学杂志

OBJECTIVE To investigate the effect of mono-2-ethylhexyl phthalate(MEHP) on proliferation of primary neural stem cells(NSCs)of rats and NE-4C cells of mice and on the migration of NE-4C cells and the mechanism. METHODS NE-4C or NSCs were treated with MEHP 1,10,100 and 1000 μmol · L-1 for 72 h,respectively. The cytotoxicity was estimated with the cell counting kit-8 (CCK-8). Cell proliferation was analyzed by EdU assay. The mRNA expression levels of the glucocorticoid receptor(GR),signal transducer and activator of transcription 3(Stat3)and sex determining region Y (SRY)-box 2(Sox2) were detected by qRT-PCR. The protein expression levels of total GR,GRβ, Sox2,Stat3 and p-Stat3 were measured by Western blotting. RESULTS Cell viability of NE-4C cells and NSCs at MEHP 1000μmol·L-1 was significantly decreased,which was 70.3%and 40.0%of the control group, respectively. EdU assay showed that MEHP 100 μmol · L-1 decreased NE-4C cells and NSCs by 74.8%and 12.0%(P<0.05)compared with control. The effect of MEHP on the cell migration of NE-4C was evidenced by the fact that the migration was obviously reduced to (63.4±2.0)%(P<0.05)after treatment with MEHP 100μmol · L-1 for 72 h. The mRNA expression levels associated with proliferation and migration in NE-4C of GR,Stat3 and Sox2 in MEHP 100 μmol · L-1 group were down-regulated to 49.8%,26.0% and 14.0%of control(P<0.05). At MEHP 100μmol · L-1,mRNA of GR, Stat3 and Sox2 in NSCs declined to 10.0%,14.0% and 15.3% of normal control. Western blotting results revealed that protein expressions of GR,GRβ,Sox2 and p-Stat3 were remarkably inhibited by MEHP 100 μmol · L-1 in that the relative expression of NE-4C was 0.92 ± 0.17,0.87 ± 0.35,0.81 ± 0.22 and 0.62 ± 0.24(P<0.05). The corresponding protein expression in NSCs was 0.82 ± 0.20,0.56 ± 0.12,0.84 ± 0.36 and 0.53 ± 0.20(P<0.05)when the cells were treated with MEHP 100μmol · L-1 for 72 h. CONCLUSION MEHP can inhibit the proliferation and migration of NE-4C cells and NSCs possibly by decreasing Stat3 and Sox2 that are mediated by GRβ.

An ultrastructural study on cytotoxic effects of mono(2-ethylhexyl) phthalate (MEHP) on testes in Shiba goat in vitro

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.

Age-dependent Effect of Metabolism and Testicular Toxicity to di(2-ethylhexyl) Phthalate / 대한산업의학회지

OBJECTIVES: The purpose of this study was to evaluate the age-dependent response of testicular toxicity and the mechanism of di(2-ethylhexyl) phthalate (DEHP) induced testicular toxicity. METHODS: DEHP was administered orally in doses of 0, 1.0 and 2.0 g/kg/day, for 7 days, to 3, 6 and 9 week-old Sprague-Dawley rats. Testicular weight and sperm head counts, plasma level of DEHP, mono(2-ethylhexyl) phthalate (MEHP) and testicular lipid peroxidation were measured. Histopathological changes in the testis were observed. RESULTS: Reductions in weight gains, and relative testis weights, were observed in the 3 week-old rats in a dose-dependent manner, but not in the 6 and 9 week-old rats, compared to those of the control rats. Sperm head counts were decreased in the 6 week-old rats exposed to 2.0 g/kg/day, but not in the 9 week-old rats. Testicular atrophy and significant size reduction of the seminiferous tubule were observed in a dose-dependent manner in the 3 week-old rats. The plasma concentrations of MEHP were higher than those of DEHP, with these levels being most elevated in the younger rats. Lipid peroxidation, following exposed to DEHP, was increased in a dose-dependent manner in the 3 week-old, but with no changes in the 6 and 9 week-old rats. CONCLUSIONS: Our results suggest the age related difference observed in the testicular response to the oral administration of DEHP may be due to the metabolism, and that oxidative stress may be related to the mechanism of DEHP induced testicular toxicity.

Age-dependent Effect of Metabolism and Testicular Toxicity to di(2-ethylhexyl) Phthalate / 대한산업의학회지

OBJECTIVES: The purpose of this study was to evaluate the age-dependent response of testicular toxicity and the mechanism of di(2-ethylhexyl) phthalate (DEHP) induced testicular toxicity. METHODS: DEHP was administered orally in doses of 0, 1.0 and 2.0 g/kg/day, for 7 days, to 3, 6 and 9 week-old Sprague-Dawley rats. Testicular weight and sperm head counts, plasma level of DEHP, mono(2-ethylhexyl) phthalate (MEHP) and testicular lipid peroxidation were measured. Histopathological changes in the testis were observed. RESULTS: Reductions in weight gains, and relative testis weights, were observed in the 3 week-old rats in a dose-dependent manner, but not in the 6 and 9 week-old rats, compared to those of the control rats. Sperm head counts were decreased in the 6 week-old rats exposed to 2.0 g/kg/day, but not in the 9 week-old rats. Testicular atrophy and significant size reduction of the seminiferous tubule were observed in a dose-dependent manner in the 3 week-old rats. The plasma concentrations of MEHP were higher than those of DEHP, with these levels being most elevated in the younger rats. Lipid peroxidation, following exposed to DEHP, was increased in a dose-dependent manner in the 3 week-old, but with no changes in the 6 and 9 week-old rats. CONCLUSIONS: Our results suggest the age related difference observed in the testicular response to the oral administration of DEHP may be due to the metabolism, and that oxidative stress may be related to the mechanism of DEHP induced testicular toxicity.