Yubang Wang

Effects of permethrin, cypermethrin and 3-phenoxybenzoic acid on rat sperm motility in vitro evaluated with computer-assisted sperm analysis

Pyrethroid pesticides, produced and used worldwide, have been reported to impair male reproductive function by reducing sperm count and sperm motility. They are divided into two types: type I pyrethroids including permethrin, etc. and type II pyrethroids including cypermethrin, fenvalerate, cyfluthrin, etc. Our previous study showed that fenvalerate and cypermethrin could reduce sperm motility in vitro. However, it is not clear whether permethrin and 3-phenoxybenzoic acid (3-PBA, the major metabolite of pyrethroids) affect sperm motility directly or indirectly by affecting spermatogenesis via interaction with androgens and/or their receptors. In this study, rat sperm suspensions were treated respectively with permethrin, cypermethrin and 3-PBA, at various concentrations (0, 1, 4, 16, or 64mmol/L) for various times (1, 2, or 4h). The motility parameters of sperm were analyzed with a computer-assisted sperm analysis (CASA) system. The differential effects of permethrin and cypermethrin on sperm motility patterns in vitro were also compared. Our study revealed that permethrin and cypermethrin could reduce sperm motility in vitro in a concentration- and time-dependent manner. Marked differences between the two pyrethroids were not found in this study. Moreover, 3-PBA did not reduce sperm motility directly at all concentrations and treatment periods. These results provide further evidence that permethrin and cypermethrin can directly affect mature rat sperm motility.

Decreased androgen receptor expression may contribute to spermatogenesis failure in rats exposed to low concentration of bisphenol A

To investigate the effects of a low bisphenol A (BPA) concentration on male reproduction, adult rats were administered a concentration of BPA that was less than the no observable adverse effect level (0.0005-5 mg/kg/bw) for 8 weeks. General toxicity, reproductive hormones, and spermatogenesis were then determined. The expression of genes related to hormone synthesis and spermatogenesis was also analyzed. These BPA concentrations generated no general toxicity and no significant changes on serum hormones. However, the testicular testosterone, hormone synthesis-related genes StAR and Cyp450scc increased, whereas 3β-HSD, 17β-HSD, and Cyp450arom decreased. Additionally, BPA significantly decreased the epithelial height and round spermatids in seminiferous tubules, sperm count, androgen receptor expression, and the expression of the spermatogenesis-related genes outer dense fiber protein 1 (ODF1) and transition protein 1. Our results indicate that a low BPA concentration can induce spermatogenesis disorders mainly through decreasing androgen receptor expression. The present results may bring attention to the risk of environmental BPA exposure.

Arsenic Induces Functional Re-Expression of Estrogen Receptor α by Demethylation of DNA in Estrogen Receptor-Negative Human Breast Cancer

Estrogen receptor α (ERα) is a marker predictive for response of breast cancers to endocrine therapy. About 30% of breast cancers, however, are hormone- independent because of lack of ERα expression. New strategies are needed for re-expression of ERα and sensitization of ER-negative breast cancer cells to selective ER modulators. The present report shows that arsenic trioxide induces reactivated ERα, providing a target for therapy with ER antagonists. Exposure of ER-negative breast cancer cells to arsenic trioxide leads to re-expression of ERα mRNA and functional ERα protein in in vitro and in vivo. Luciferase reporter gene assays and 3-(4,5-dimethylthiazol-2-yl)- 5-(3-carboxymethoxyphenyl)- 2-(4-sulfophenyl)- 2H-tetrazolium (MTS) assays show that, upon exposure to arsenic trioxide, formerly unresponsive, ER-negative MDA-MB-231 breast cancer cells become responsive to ER antagonists, 4-hydroxytamoxifen and ICI 182,780. Furthermore, methylation- specific PCR and bisulfite-sequencing PCR assays show that arsenic trioxide induces partial demethylation of the ERα promoter. A methyl donor, S-adenosylmethionine (SAM), reduces the degree of arsenic trioxide-induced re-expression of ERα and demethylation. Moreover, Western blot and ChIP assays show that arsenic trioxide represses expression of DNMT1 and DNMT3a along with partial dissociation of DNMT1 from the ERα promoter. Thus, arsenic trioxide exhibits a previously undefined function which induces re-expression ERα in ER-negative breast cancer cells through demethylation of the ERα promoter. These findings could provide important information regarding the application of therapeutic agents targeting epigenetic changes in breast cancers and potential implication of arsenic trioxide as a new drug for the treatment of ER–negative human breast cancer.

Effects of Fenvalerate and Cypermethrin on Rat Sperm Motility Patterns In Vitro as Measured by Computer-Assisted Sperm Analysis

Fenvalerate and cypermethrin were reported to impair male reproductive function, inducing significant reductions in epididymal sperm count. Further, fenvalerate was shown to reduce sperm motility. However, it is not clear whether fenvalerate and cypermethrin might impact sperm motility directly or indirectly by affecting spermatogenesis via interaction with androgens or their receptors. In this study, sperm suspensions were treated with fenvalerate and cypermethrin, respectively, at various concentrations (0, 1, 4, 16, or 64 μmol/L) for various times (1, 2, or 4 h). The motility parameters of sperm treated with these two insecticides were analyzed with a computer-assisted sperm analysis (CASA) system. The differential effects of fenvalerate and cypermethrin on rat sperm motility patterns in vitro were also compared. Our study revealed that fenvalerate and cypermethrin reduced sperm motility in vitro in a concentration- and time-dependent manner. Cypermethrin exerted a greater effect on sperm motility in comparison to fenvalerate. These results provided evidence that fenvalerate and cypermethrin directly influence mature rat sperm motility.

Genetic variants in antioxidant genes are associated with sperm DNA damage and risk of male infertility in a Chinese population

To test the hypothesis that polymorphisms in antioxidant genes are more susceptible to sperm DNA damage and male infertility, we examined 11 single-nucleotide polymorphisms from six antioxidant genes (GPX1, CAT, PON1, NQO1, SOD2/MnSOD, and SOD3) in 580 infertility cases and 580 controls from a Chinese population-based case-control study (NJMU Infertility Study). Genotypes were determined using the OpenArray platform. Sperm DNA fragmentation was detected using the Tdt-mediated dUTP nick-end labeling assay, and the level of 8-hydroxydeoxyguanosine (8-OHdG) in sperm DNA was measured using immunofluorescence. The adjusted odds ratio and 95% confidence interval (CI) were estimated using unconditional logistic regression. The results indicated that the PON1 Arg192Glu (rs662) and SOD2 Val16Ala (rs4880) variant genotypes were associated with a significantly higher risk of male infertility. In addition, subjects carrying variant genotypes of both loci had a twofold (95% CI, 1.42-2.90) increase in the risk of male infertility, indicating a significant gene-gene interaction between these two loci (P for multiplicative interaction=0.045). Moreover, linear regression analysis showed that individuals carrying the PON1 Arg192Glu (rs662) or SOD2 Val16Ala (rs4880) variants have significantly higher levels of sperm DNA fragmentation and 8-OHdG. These data suggest that genetic variations in antioxidant genes may contribute to oxidative sperm DNA damage and male infertility.

Neurotoxicity of Perfluorooctane Sulfonate to Hippocampal Cells in Adult Mice

Perfluorooctane sulfonate (PFOS) is a ubiquitous pollutant and found in the environment and in biota. The neurotoxicity of PFOS has received much concern among its various toxic effects when given during developing period of brain. However, little is known about the neurotoxic effects and potential mechanisms of PFOS in the mature brain. Our study demonstrated the neurotoxicity and the potential mechanisms of PFOS in the hippocampus of adult mice for the first time. The impairments of spatial learning and memory were observed by water maze studies after exposure to PFOS for three months. Significant apoptosis was found in hippocampal cells after PFOS exposure, accompanied with a increase of glutamate in the hippocampus and decreases of dopamine (DA) and 3,4-dihydrophenylacetic acid (DOPAC) in Caudate Putamen in the 10.75 mg/kg PFOS group. By two-dimensional fluorescence difference in gel electrophoresis (2D-DIGE) analysis, seven related proteins in the hippocampus that responded to PFOS exposure were identified, among which, Mib1 protein (an E3 ubiquitin-protein ligase), Herc5 (hect domain and RLD 5 isoform 2) and Tyro3 (TYRO3 protein tyrosine kinase 3) were found down-regulated, while Sdha (Succinate dehydrogenase flavoprotein subunit), Gzma (Isoform HF1 of Granzyme A precursor), Plau (Urokinase-type plasminogen activator precursor) and Lig4 (DNA ligase 4) were found up-regulated in the 10.75 mg/kg PFOS-treated group compare with control group. Furthermore, we also found that (i) increased expression of caspase-3 protein and decreased expression of Bcl-2, Bcl-XL and survivin proteins, (ii) the increased glutamate release in the hippocampus. All these might contribute to the dysfunction of hippocampus which finally account for the impairments of spatial learning and memory in adult mice.

Bisphenol A Alters n-6 Fatty Acid Composition and Decreases Antioxidant Enzyme Levels in Rat Testes: A LC-QTOF-Based Metabolomics Study

Background Male reproductive toxicity induced by exposure to bisphenol A (BPA) has been widely reported. The testes have proven to be a major target organ of BPA toxicity, so studying testicular metabolite variation holds promise for the discovery of mechanisms linked to the toxic effects of BPA on reproduction. Methodology/Principal Findings Male Sprague-Dawley rats were orally administered doses of BPA at the levels of 0, 50 mg/kg/d for 8 weeks. We used an unbiased liquid chromatography-quadrupole time-of-flight (LC-QTOF)-based metabolomics approach to discover, identify, and analyze the variation of testicular metabolites. Two n-6 fatty acids, linoleic acid (LA) and arachidonic acid (AA) were identified as potential testicular biomarkers. Decreased levels of LA and increased levels of AA as well as AA/LA ratio were observed in the testes of the exposed group. According to these suggestions, testicular antioxidant enzyme levels were detected. Testicular superoxide dismutase (SOD) declined significantly in the exposed group compared with that in the non-exposed group, and the glutathione peroxidase (GSH-Px) as well as catalase (CAT) also showed a decreasing trend in BPA treated group. Conclusions/Significance BPA caused testicular n-6 fatty acid composition variation and decreased antioxidant enzyme levels. This study emphasizes that metabolomics brings the promise of biomarkers identification for the discovery of mechanisms underlying reproductive toxicity.

Metabolomic Analysis Reveals Metabolic Changes Caused By Bisphenol A in Rats

Bisphenol A (BPA) is a widely used material known to cause adverse effects in humans and other mammals. To date, little is known about the global metabolomic alterations caused by BPA using urinalysis. Sprague-Dawley rats were orally administrated BPA at the levels of 0, 0.5 μg/kg/day and 50 mg/kg/day covering a low dose and a reference dose for 8 weeks. We conducted a capillary electrophoresis in tandem with electrospray ionization time-of-flight mass spectrometry based nontargeted metabolomic analysis using rat urine. To verify the metabolic alteration at both low and high doses, reverse transcription-polymerase chain reaction (RT-PCR) and western blotting were further conducted to analyze hepatic expression of methionine adenosyltransferase Iα (Mat1a) and methionine adenosyltransferase IIα (Mat2a). Hepatic S-adenosylmethionine (SAMe) was also analyzed. A total of 199 metabolites were profiled. Statistical analysis and pathway mapping indicated that the most significant metabolic perturbations induced by BPA were the increased biotin and riboflavin excretion, increased synthesis of methylated products, elevated purine nucleotide catabolism, and increased flux through the choline metabolism pathway. We found significantly higher mRNA and protein levels of Mat1a and Mat2a, and significantly higher SAMe levels in rat liver at both low and high doses. These two genes encode critical isoenzymes that catalyze the formation of SAMe, the principal biological methyl donor involved in the choline metabolism. In conclusion, an elevated choline metabolism is underlying the mechanism of highly methylated environment and related metabolic alterations caused by BPA. The data of BPA-elevated accepted biomarkers of injury indicate that BPA induces DNA methylation damage and broad protein degradation, and the increased deleterious metabolites in choline pathway may also be involved in the toxicity of BPA.

Monobutyl Phthalate Inhibits Steroidogenesis by Downregulating Steroidogenic Acute Regulatory Protein Expression in Mouse Leydig Tumor Cells (MLTC-1)

Di-n-butyl phthalate (DBP) and its active metabolite, monobutyl phthalate (MBP), display no binding affinity for the androgen receptor, yet exert antiandrogenic effects by altering steroid biosynthesis. However, the mechanisms underlying this observed effect are not known. The purpose of this study was to determine the site of MBP action on steroidogenesis in vitro using mouse Leydig tumor cells (MLTC-1). Various concentrations of MBP (0, 50, 100, 200, 400, or 800 μmol/L) were added to the medium for 24 h followed by stimulation with some compounds such as human chorionic gonadotrophin (hCG), cholera toxin (CT), cAMP analog 8-Br-cAMP, 22(R)-hydroxycholesterol (22R-HC), and pregnenolone. Data showed that MBP inhibited the increases in progesterone production induced by hCG and CT. In contrast, the levels of intracellular cAMP remained unaltered. In addition, 8-Br-cAMP-stimulated progesterone production was also suppressed by MBP. These results suggested that the site in the steroid biosynthesis pathway affected by MBP occurs downstream of PKA activation in MLTC-1 cells. Moreover, incubation with 22R-HC and pregnenolone as progesterone precursors for P-450 side-chain cleavage enzyme (P450scc) and 3β-hydroxysteroid dehydrogenase (3βHSD) respectively resulted in no marked change in progesterone production, indicating that MBP did not influence P450scc and 3βHSD but did exert an effect on cholesterol transportation into mitochondria, the rate-limiting step. These results were supported by the downregulated StAR expression seen with MBP administration, as StAR is a key factor in this process. Data indicate that MBP interfered with steroid hormone production by affecting StAR expression in MLTC-1 cells.

Low-dose monobutyl phthalate stimulates steroidogenesis through steroidogenic acute regulatory protein regulated by SF-1, GATA-4 and C/EBP-beta in mouse Leydig tumor cells

BackgroundThe ubiquitous use of dibutyl phthalate (DBP), one of the most widely used plasticizers, results in extensive exposure to humans and the environment. DBP and its major metabolite, monobutyl phthalate (MBP), may alter steroid biosynthesis and their exposure may lead to damage to male reproductive function. Low-doses of DBP/MBP may result in increased steroidogenesis in vitro and in vivo. However, the mechanisms of possible effects of low-dose MBP on steroidogenesis remain unclear. The aim of present study was to elaborate the role of transcription factors and steroidogenic acute regulatory protein in low-dose MBP-induced distruption of steroidogenesis in mouse Leydig tumor cells (MLTC-1 cells).MethodsIn the present study, MLTC-1 cells were cultured in RPMI 1640 medium supplemented with 2 g/L sodium bicarbonate. Progesterone level was examined by I125-pregesterone Coat-A-Count radioimmunoassay (RIA) kits. mRNA and protein levels were assessed by reverse transcription-polymerase chain reaction (RT-PCR) and western blot, respectively. DNA-binding of several transcription factors was examined by electrophoretic mobility shift assay (EMSA).ResultsIn this study, various doses of MBP (0, 10(−9), 10(−8), 10(−7), or 10(−6) M) were added to the medium followed by stimulation of MLTC-1 cells with human chorionic gonadotrophin (hCG). The results showed that MBP increased progesterone production and steroidogenic acute regulatory protein (StAR) mRNA and protein levels. However, the protein levels of cytochrome P450scc and 3 beta-hydroxy-steroid dehydrogenase (3 beta-HSD) were unchanged after MBP treatment. EMSA assay showed that DNA-binding of steroidogenic factors 1(SF-1), GATA-4 and CCAAT/enhancer binding protein-beta (C/EBP-beta) was increased in a dose-dependent manner after MBP exposure. Western blot tests were next employed and confirmed that the protein levels of SF-1, GATA-4 and C/EBP-beta were also increased. Additionally, western blot tests confirmed the expression of DAX-1, negative factor of SF-1, was dose-dependently down regulated after MBP exposure, which further confirmed the role of SF-1 in MBP-stimulated steroid biosynthesis.ConclusionsIn conclusion, we firstly delineated the regulation of StAR by transcription factors including SF-1, GATA-4 and C/EBP-beta maybe critical mechanism involved in low-dose MBP-stimulated steroidogenesis.