Effects of environmental chemicals on human reproductive health and sex hormone levels have been reported for several years, but compared to other environmental chemicals, such as heavy metals, PCBs, triclosan, Phthalate, the links between polycyclic aromatic hydrocarbons (PAHs) and sex hormone levels have not been studied widely. Therefore, our purpose of research was to study the associations between urinary PAH metabolites and serum total testosterone (T) levels among men. The data was obtained from the independent cross-section wave (2011–2012) of the National Health and Nutrition Examination Survey (NHANES), including demographic, socioeconomic, dietary, health-related questions, examinations and laboratory test. All analyses were performed by R software (version 3.2.3), including one-way analysis of variance, multivariable linear regression, stratified analysis and heterogeneity test. Of 1102 American adults aged 20 and above included in the statistical analysis, we found that urinary 3-hydroxyfluorene and 2-hydroxyfluorene were significantly positively associated with serum T levels (β = 40.62, 95%CI = 21.78–59.46, P = 2.56 × 10−5; β = 35.17, 95%CI = 13.18–57.15, P = 1.75 × 10−3, respectively). The associations between urinary PAH metabolites and serum T levels signified a major public health problem over the world. Prospective work is needed to investigate the potential long-term health consequences of these findings.

•BPA is slightly correlated with semen quality in Chinese males independently.•BPA is significantly negatively correlated with sperm count in obese men.•There is an interaction between BPA and obesity on sperm count in a mouse model.•Interaction related metabolites play key role in fatty acid oxidation and TCA cycle.•Increased oxidative stress is associated with male reproductive dysfunction.Both bisphenol A (BPA) and obesity affect male reproductive system. However, whether there is an interaction between them remains poorly understood. The aim of the present study was to evaluate the interaction between BPA exposure and obesity on semen quality and elucidate the mechanism in humans and animals. We firstly analyzed the interaction on semen volume, sperm count per ejaculate, sperm concentration and sperm motility in 357 men, and found that urinary BPA concentration was significantly correlated with sperm count per ejaculate in obese men (β = − 34.62; 95% CI: − 60.75, − 8.48; P = 0.01). Then we validated the interaction using lean and obese mice with administration of BPA. Significant interactions between BPA exposure and obesity on sperm count and sperm concentration was observed in mice. Finally, we conducted metabolomics analyses to identify metabolites related to the interaction. Metabolites related to the interaction, including capric acid, dodecanoic acid, l-palmitoylcarnitine, niacinamide, etc., are known to play critical roles in fatty acid oxidation and tricarboxylic acid cycle indicating increased oxidative stress associated with male reproductive dysfunction. Thus, our study finds an interaction between BPA exposure and obesity on sperm count and reveals potential metabolic mechanisms. It emphasizes the importance to study interactions between endocrine disrupting chemicals and obesity, and opens avenues for the possible use of animal models in identifying the interactions.

•Exposure to 4-n-OP has an adverse health effect on male reproductive function.•Y-hg O3* has a protective effect on male reproductive function.•Y-hg O3* makes individuals more adaptive to the adverse effect of 4-n-OP.Certain genetic background (mainly Y chromosome haplogroups, Y-hg) may modify the susceptibility of certain environmental exposure to some diseases. Compared with respective main effects of genetic background or environmental exposure, interactions between them reflect more realistic combined effects on the susceptibility to a disease. To identify the interactions on spermatogenic impairment, we performed Y chromosome haplotyping and measurement of 9 urinary phenols concentrations in 774 infertile males and 520 healthy controls in a Han Chinese population, and likelihood ratio tests were used to examine the interactions between Y-hgs and phenols. Originally, we observed that Y-hg C and Y-hg F* might modify the susceptibility to male infertility with urinary 4-n-octylphenol (4-n-OP) level (Pinter = 0.005 and 0.019, respectively). Subsequently, based on our results, two panels were tested to identify the possible protective sub-branches of Y-hg F* to 4-n-OP exposure, and Y-hg O3* was uncovered to interact with 4-n-OP (Pinter = 0.019). In conclusion, while 4-n-OP shows an adverse effect on spermatogenesis, Y-hg O3* makes individuals more adaptive to such an effect for maintaining basic reproductive capacity.

A large number of organophosphorus (OP) pesticides are used globally, and the effects of OP pesticides on human health have attracted attention. However, methods focusing on the determination of various OP pesticides in human blood serum are still scarce, and the exposure level of various OP pesticides in humans remains largely unknown. We have developed a method for the quantification of 20 OP pesticides in human blood serum simultaneously. The determination was performed using solid-phase extraction with a small blood serum sample volume (200 μL) and gas chromatographic analysis with triple quadrupole mass spectrometry (GC-MS/MS). Among the 20 pesticides, limits of detection ranged from 0.01 to 2.70 ng mL−1; limits of quantitation ranged from 0.05 to 9.00 ng mL−1; linearity ranged from 0.02 to 135.00 ng mL−1; recoveries ranged from 90 to 120%; intra-day and inter-day relative standard deviations were mostly less than 15% at 2.70 and 27.00 ng mL−1 in the spiked blood serum samples. This method was further used for the determination of OP pesticides in 160 human blood serum samples, which generated the first-hand data regarding the OP pesticide exposure profile in Chinese adults without occupational exposure.

•We evaluated the results of reanalysis with three variable modifications.•Ubiquitinated peptides could be co-enriched with acetylated peptides.•Phosphorylated peptides are randomly eluted with acetylated peptides.•About 500 proteins contain more than one type of PTM in human sperm.Sperm is an ideal model for studying post-translational modifications since its transcriptional and translational activities are nearly silent. Thus, sperm functions are mainly regulated at the protein level, especially by means of post-translational modifications. Published proteomic datasets may contain valuable undiscovered information. In this study, we reanalyzed the raw data from previous acetylproteome study on human capacitated sperm to include two additional modifications: phosphorylation and ubiquitination. We successfully identified approximately 500 proteins with multiple types of modifications. Compared with recently developed serial enrichment strategy for multiple modifications, reanalysis of single modification enriched data provides a direct and efficient alternative approach. These results greatly expand our knowledge of protein modifications in human sperm.

The purpose of this study was to determine the relationship between hypomethylation of HOXA10 gene’s promoter and high expression in malignant ovarian tissues, and to confirm the level of hypomethylation in ovarian cell lines.We performed the methylation status of 29 samples from ovarian carcinomas and 16 from normal tissues by methylation-specific polymerase chain reaction (MSP). Then, we evaluated the expression of mRNA and protein of HOXA10 in all samples to work out the relationship between the methylation status of HOXA10 and its expression in transcriptional and translational levels. We then confirmed our present study usingSKOV3 and HEY ovarian cancer cell lines treated with the demethylating agent 5-aza-2′-deoxycytidine (5-aza-dC) to detect whether the expression of HoxA10 in the two cell lines was altered.Increased expression of HOXA10 was detected in almost all ovarian carcinomas (p < 0.05). Promoter hypomethylation was found in (17 of 29) 58.62% ovarian cancers and (4 of 16) 25% normal ovaries (p < 0.05). The HOXA10 expression is higher when the status of HOXA10 gene promoter is hypomethylated than in methylated tissues (p < 0.05). After 5-aza-dC treatment, the expression level of HOXA10 mRNA transcript was increased in the two cell lines.Our results indicate that promoter hypomethylation is an important mechanism for high expression of HOXA10 in human ovarian cancer and may be a potential prognostic factor in ovarian cancer.

Most of the general populations are exposed to polycyclic aromatic hydrocarbons (PAHs) at different levels. A limited number of studies have suggested that PAHs exposure may be associated with semen quality. To examine the association of four PAH metabolites, 1-hydroxynapthalene (1-N), 2-hydroxynapthalene (2-N), 1-hydroxypyrene (1-OHP) and 2-hydroxyfluorene (2-OHF) with altered semen quality, 542 subjects were recruited through the clinic following strict eligibility screening. Using LC-MS/MS, individual exposures were measured as spot urinary concentrations of PAH metabolites adjusted by creatinine (CR). Semen quality was assessed by semen volume, sperm concentration, sperm number per ejaculum, and sperm motility. First, we confirmed the variability of PAH metabolites in human urine. Our results showed that the median CR-adjusted concentrations of 1-N, 2-N, 1-OHP, 2-OHF were 2.35, 4.05, 1.14, and 2.89 μg/g of CR, respectively. Significant P-values for trend were found that men with higher 1-OHP (assessed as quintiles) were more likely to have below-reference sperm concentration and sperm number per ejaculum. These results indicate that PAHs exposure might be related to altered human semen quality.

A significant proportion of male infertility is accompanied by an abnormal semen analysis, azoospermia or severe oligozoospermia, which is generally assumed to be the result of spermatogenic failure. The genetic contribution in the process of spermatogenesis, particularly the role of the Y chromosome in determination of semen quality, is still obscure. In order to explore the relationship between Y chromosome haplogroup and spermatogenic failure, we collected 285 idiopathic infertile males with azoo-/oligozoospermia and 515 fertile men, adopted 12 binary markers and recruited the subjects (cases and controls) in the same region to test whether there is a possible susceptibility of certain Y haplogroups to spermatogenic failure in the Han Chinese population. The results indicated that the prevalences of hg K* in the control and the case population were 0.78% (4/515) and 2.80% (8/285), respectively. The difference between the frequencies of the hg K* in the infertile males and the normal control population was significant [odds ratio (OR) = 3.69; 95% confidence interval (CI) = 1.10–12.36] (P = 0.028). However, in the other haplogroups no significant differences were found. In conclusion, Y haplogroup-K* might bear a risk factor of male infertility, and the individuals in the haplogroup need to be further examined.

•Our study used NGS to sequence of complete human mtDNA genomes in blood cells.•We found that the variants C16179T and A12361G were associated with increased risk of abnormal semen parameter.•We studied the mtDNA haplogroup distribution of case and control groups, the genetic backgrounds may not affect our results.Mitochondrial DNA (mtDNA) is believed to be both the source and target of reactive oxygen species (ROS), and mtDNA genetic alterations have been reported to be associated with molecular defects in the oxidative phosphorylation (OXPHOS) system. In order to investigate the potentially susceptible mtDNA genetic variants to oligoasthenospermia, we conducted a two-stage study in 921 idiopathic infertile men with oligoasthenospermia and 766 healthy controls using comprehensive molecular analysis. In the screen stage, we used next generation sequencing (NGS) in 233 cases and 233 controls to screen oligoasthenospermia susceptible mitochondrial genetic variants. In total, seven variants (C5601T, T12338C, A12361G, G13928C, A15235G, C16179T and G16291A) were screened to be potentially associated with idiopathic oligoasthenospermia. In the validation stage, we replicated these variants in 688 cases and 533 healthy controls using SNPscan. Our results demonstrated that the genetic alteration of C16179T was associated with idiopathic male infertility (odds ratio (OR) 3.10, 95% CI 1.41–6.79) (p = 3.10 × 10− 3). To elucidate the exact role of the genetic variants in spermatogenesis, two main sperm parameters (sperm count and motility) were taken into account. We found that C16179T was associated with both low sperm count and motility, with ORs of 4.18 (95% CI 1.86–9.40) (p = 1.90 × 10− 4) and 3.17 (95% CI 1.40–7.16) (p = 3.50 × 10− 3), respectively. Additionally, A12361G was found to be associated with low sperm count, with an OR of 3.30 (95% CI 1.36–8.04) (p = 5.50 × 10− 3). These results indicated that C16179T influenced both the process of spermatogenesis and sperm motility, while A12361G may just only participate in the process of spermatogenesis. Further investigation in larger populations and functional characterizations are needed to validate our findings.

•We examined relations between exposure to PEs and idiopathic male infertility.•We determined urinary concentrations of various PEs.•DAI, GEN and SEC were associated with male infertility.•DAI, GEN and SEC were associated with infertility with abnormal semen quality.Phytoestrogens (PEs) are naturally occurring chemical constituents of certain plants. The internal PE exposures, mainly from diet, vary among different populations and in different regions due to various eating habits. To investigate the potential relationship between urinary PE levels and idiopathic male infertility and semen quality in Chinese adult males, 608 idiopathic infertile men and 469 fertile controls were recruited by eligibility screening procedures. Individual exposure to PEs was measured using UPLC–MS/MS as spot urinary concentrations of 6 PEs (daidzein, DAI; equol, EQU; genistein, GEN; naringenin, NAR; coumestrol, COU; and secoisolariciresinol, SEC), which were adjusted with urinary creatinine (CR). Semen quality was assessed by sperm concentration, number per ejaculum and motility. We found that exposures to DAI, GEN and SEC were significantly associated with idiopathic male infertility (P-value for trend = 0.036; 0.002; and 0.0001, respectively), while these exposures had stronger association with infertile subjects with at least one abnormal semen parameter than those with all normal semen parameters. Exposures to DAI, GEN and SEC were also related to idiopathic male infertility with abnormal sperm concentration, number per ejaculum and motility (P-value for trend < 0.05), while these exposures had stronger association with the infertile men with abnormal sperm number per ejaculum. These findings provide the evidence that PE exposures are related to male reproductive function and raise a public health concern because that exposure to PEs is ubiquitous in China.

The nucleotide-excision repair (NER) system is crucial for the removal of bulky DNA adducts during spermatogenesis. Dysfunction of its repair capacity is likely related to the increased susceptibility to DNA damage. In this study, four polymorphisms in NER pathway (XPA(–4) G/A, ERCC1 C8092A, XPD Lys751Gln and XPF Ser835Ser) were selected to evaluate their potential impact on sperm DNA damage and male infertility. Genotypes were determined by PCR-restriction fragment length polymorphism. Sperm DNA damage was evaluated by TdT-mediated dUDP nick-end labelling assay. A case-only study of 620 infertile men found a significant association between XPA(–4) G/A polymorphism and sperm DNA damage. Individuals with the XPA(–4) A allele showed more sperm DNA damage and lower sperm concentration than G allele carriers. Further analysis, including 620 patients and 385 controls, revealed a 1.52-fold risk (95% CI 1.08–2.02) of developing male infertility in the XPA(–4) AA carriers compared with noncarriers. Luciferase assay verified that the promoter with the XPA(–4) A allele had a lower transcriptional activity than that with the G allele. These data provide the first evidence that –4 G/A polymorphism in XPA promoter alters its transcriptional activity and, thus, might contribute to sperm DNA damage and male infertility.Sperm DNA integrity is essential for the accurate transmission of genetic information. To our knowledge, few studies have elucidated the effect of DNA repair gene single-nucleotide polymorphisms on sperm DNA integrity, although the DNA repair system is indispensable in maintaining genetic stability and normal spermatogenesis. In this original study, we evaluated the potential impact of the polymorphisms in the nucleotide-excision repair pathway on the risk of sperm DNA damage based on 620 infertile patients and 385 controls, and provided the first evidence that –4 G/A polymorphism in the promoter for the xeroderma pigmentosum group A gene altered its transcriptional activity, which might contribute to sperm DNA damage and male infertility.

Polycyclic aromatic hydrocarbons (PAH) are ubiquitous global pollutants. Limited studies suggested that PAH may interfere with thyroid function in animals, but little is known about humans. A population of 480 Chinese males was recruited. Using LC–MS/MS, four urinary metabolites of PAH including 1-hydroxynaphthalene (1-N), 2-hydroxynaphthalene (2-N), 1-hydroxypyrene (1-P) and 2-hydroxyfluorene (2-F) were measured in spot urinary samples, which were adjusted by urinary creatinine (CR). Blood samples were collected for measuring serum levels of thyroid hormones including total thyroxine (TT4), free triiodothyronine (FT3), free thyroxine (FT4) and thyroid-stimulating hormone (TSH). The median CR-adjusted urine PAH concentrations of 1-N, 2-N, 1-P, 2-F were 2.306, 4.047, 1.155 and 2.899 μg g−1 of CR, respectively. Significant p-values for trend were found for men with higher 2-F tertiles and were more likely to possess high-reference TSH levels. In addition, the multivariate linear regression models showed significant positive correlations for TSH levels with increased CR-adjusted 2-F concentration. No significant associations were found between other thyroid hormones levels and PAH metabolite concentrations. These results indicated that PAH exposure might be related to altered male thyroid hormone levels, but further study is needed to confirm these observed findings.

•Men with GSTM1-null and GSTT1-present genotypes are susceptible to infertility.•Men with GSTM1-null are more susceptible to infertility when exposed to 4-n-OP.•Men with GSTM1-null and GSTT1-present are most susceptible when exposed to 4-n-OP.•Metabolomics analysis helps explain such susceptibilities.•Changed metabolites are involved in TCA-cycle which is related to male infertility.Infertility affects about 17% couples, and males contribute to half of the cases. Compared with independent effects of genetic and environmental factors, interactions between them help in the understanding of the susceptibility to male infertility. Thus, we genotyped 25 polymorphisms, measured 16 urinary chemical concentrations and explored interactions between gene-gene and gene-environment in 1039 Han Chinese using metabolomic analysis. We first observed that GSTT1 might interact with GSTM1 (Pinter = 6.33 × 10− 8). Furthermore, an interaction between GSTM1 and 4-n-octylphenol (4-n-OP) was identified (Pinter = 7.00 × 10− 3), as well as a 2-order interaction among GSTT1, GSTM1 and 4-n-OP (Pinter = 0.04). Subjects with GSTT1-present and GSTM1-null genotypes were susceptible to male infertility when exposed to 4-n-OP (OR = 14.05, 95% CI = 4.78–60.20, P = 2.34 × 10− 5). Most metabolites identified were involved in the tricarboxylic acid cycle. In conclusion, it is a novel study of the interaction on male infertility from the aspect of metabolomics.

Caspase-8 (CASP8) plays a key role in apoptosis. We examined by genotyping whether the −652 six-nucleotide insertion-deletion (6N ins/del) polymorphism in the CASP8 promoter region was associated with prostate cancer risk in a hospital-based case-control study of 406 Chinese prostate cancer patients and 408 age-matched cancerfree controls. Additionally, 23 prostate cancer tissues were analyzed for CASP8 mRNA expression. We found a significantly decreased prostate cancer risk for the 6N ins/del genotype [adjusted odds ratio (OR)=0.68; 95% confidence interval (CI)=0.51-0.92] and del/del genotype (OR=0.34; 95% CI=0.19-0.63) compared with the ins/ins genotype. The 6N del allele was associated dose-dependently with decreased prostate cancer risk (Ptrend = 0.001). RT-PCR showed that individuals with the 6N del allele had lower CASP8 mRNA levels than those with the ins/ins genotype (P = 0.024). These findings suggested that the CASP8-652 6N ins/del polymorphism may affect the susceptibility to prostate cancer and reduce prostate cancer risk among Chinese men.

•Lower miR-135a indicates poor progression-free and overall survival of EOC patients.•MiR-135a regulated HOXA10 expression by targeting its 3′-UTR in EOC cell lines.•MiR-135a/HOXA10/p53 pathway mediates EOC cell proliferation, apoptosis, and adhesion.The activation of homeobox A10 (HOXA10) has been proved to be an important event in epithelial ovarian carcinogenesis, yet its regulation in epithelial ovarian cancer (EOC) is still not fully understood. Here, we aimed to reveal the mechanism that a predicted target miRNA regulates HOXA10 expression and the association of its expression with progression of EOC. Here, by using computer-assisted algorithms from PicTar, TargetScan, and miRBase, we identified that the predicted target miRNA of HOXA10 was miR-135a. MiR-135a expression in EOC tissues and controls was measured with quantitative RT-PCR. The role of miR-135a and HOXA10 in the growth and survival of several EOC cell lines was determined with several in vitro approaches. We found that miR-135a expression was downregulated in an EOC patient cohort. Also, patients with low miR-135a expression had shorter overall survival and progression-free survival durations than those with high expression. Functional analysis of three EOC-derived cell lines (SKOV-3, HEY, and OVCAR-3) demonstrated that miR-135a directly regulated HOXA10 expression by targeting its 3′-UTR. Inhibition of HOXA10 expression with miR-135a mimics and HOXA10 siRNA consistently resulted in cell apoptosis with concomitant enhancement of caspase-3, increase of p53 expression and reduction of Bcl-2 expression, and also suppressed cell growth and adhesion. These findings suggest that ubiquitous loss of miR-135a expression is a critical mechanism for the overexpression of HOXA10 in EOC cells, which is implicated in epithelial ovarian carcinogenesis. Furthermore, miR-135a may be predictive of EOC prognosis.

ObjectiveNanoparticles are becoming an important method of targeted drug delivery. To evaluate the importance of folate-conjugated human serum albumin (HSA) magnetic nanoparticles (Folate-CDDP/HSA MNP), we prepared drug-loaded Folate-CDDP/HSA MNPs and characterized their features.MethodsFirst, folate was conjugated with HSA under the effect of a condensing agent, and the conjugating rate was evaluated by a colorimetric method using 2, 4, 6 – trinitrobenzene sulfonic acid. Second, under N2 gas, Fe3O4 magnetic nanomaterials were prepared and characterized by using transmission electron microscopy (TEM), SEM-EDS and X-ray diffraction (XRD). Finally, Folate-CDDP/HSA MNP was prepared by using a solvent evaporation technique. TEM was used to observe particle morphology. The particle size and distribution of the prepared complexes were determined by a Laser particle size analyzer. Drug loading volume and drug release were investigated by a high performance liquid chromatography method (HPLC) in vitro.ResultsWe successfully prepared folate-conjugated HSA and its conjugating rate was 27.26 μg/mg. Under TEM, Fe3O4 magnetic nanoparticles were highly electron density and had an even size distribution in the range of 10-20 nm. It was confirmed by SEM-EDS and XRD that Fe3O4 magnetic nanoparticles had been successfully prepared. Under TEM, drug-loaded magnetic nanoparticles were observed, which had a round shape, similar uniform size and smooth surface. Their average size was 79 nm which was determined by laser scattering, and they exhibited magnetic responsiveness. Encapsulation efficiency was 89.75% and effective drug loading was calculated to be 15.25%. The release results in vitro showed that the half release time (t1/2) of cisplatin in cisplatin Solution and Folate-CDDP/HSA MNP was 65 min and 24 h respectively, which indicated that microspheres had an obvious effect of sustained-release. Conclusion: Folate-CDDP/HSA MNPs were prepared successfully. The preparation process and related characteristics data provided a foundation for further study, including the mechanism of the nanoparticles distribution in vivo and their intake by tumor cells.

Observations in several western and Asiatic countries point toward a decline in semen quality which may be associated with environmental exposures. To investigate the effect of environmental exposure to pyrethroids on sperm DNA integrity and semen quality, 240 men were recruited from an infertility clinic through the clinic following strict eligibility screening. Urinary 3-phenoxybenzoic acid (3-PBA) concentration, semen quality, and sperm DNA integrity were evaluated. After adjustment for potential confounders, a significant inverse correlation was observed between the urinary 3-PBA level and the sperm concentration (β = −0.27, 95%CI: −0.41 to −0.12, P < 0.001). Moreover, we also found a significant positive correlation between urinary 3-PBA level and sperm DNA fragmentation (β = 0.27, 95%CI: 0.15–0.39, P < 0.001). Our results suggest that non-occupational environmental pyrethroids exposure may have a negative impact on sperm DNA integrity and semen quality in Chinese males.

MicroRNA biogenesis genes have been confirmed involved in lots of diseases. This study evaluated the role of genetic variants in microRNA biogenesis genes in semen quality and idiopathic male infertility. Seven single-nucleotide polymorphisms (SNP) of DICER1 (rs13078, rs1057035 and rs12323635) and DROSHA (rs10719, rs2291109, rs17409893 and rs642321) were determined by TaqMan probes and SNPstream in 667 eligible infertile men and 419 fertile controls. Semen quality analysis was performed by computer-assisted sperm analysis. It was found that genetic variants of rs12323635 was associated with idiopathic male infertility. Additionally, in strategy analysis, the rs12323635 C allele might decrease the risk of oligozoospermia (OR 0.42, 95% CI 0.26–0.66; P = 0.0002). The rs642321 TT genotype may have a higher risk of oligozoospermia (OR 2.38, 95% CI 1.34–4.25; P = 0.003). These significant differences were retained after Bonferroni correction. The results showed that variants of DICER1 and DROSHA may modify the risk of abnormal semen parameters, which could result in male infertility.MicroRNA have been confirmed involved in lots of diseases. To our knowledge, few studies have elucidated the role of genetic variants in microRNA biogenesis genes in semen quality and idiopathic male infertility, although microRNA is indispensable in normal spermatogenesis. In this original study, we evaluated the potential impact of the polymorphisms in microRNA biogenesis genes on the risk of abnormal semen quality based on 667 infertile patients and 419 controls, and provided the first evidence that polymorphism in rs12323635 in DICER1 may modify the risk of abnormal semen parameters, which could result in male infertility.

Polybrominated diphenyl ethers (PBDEs) are commonly used as flame retardants in textiles, plastics and electronics and represent a group of persistent environmental contaminants. They have been found to accumulate in human and marine mammals. Previous studies have shown that PBDEs have endocrine-disrupting properties and reproductive toxicity. However, the mechanisms under the reproductive disruptions are still not well understood. In this study, we explored the effects of 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47) on progesterone biosynthesis and possible mechanisms in mouse Leydig tumor cells (mLTC-1). Our results showed that BDE-47 could reduce progesterone production and decrease the intracellular cAMP level induced by hCG or forskolin. These suggested that BDE-47 decreasing progesterone production in mLTC-1 cells may be associated with the decline of intracellular cAMP level. Moreover, our data also indicated that the site G protein in cAMP-PKA pathway may be involved in this process. Furthermore, the addition of cAMP analog, 8-Br-cAMP, could not reverse the decrease of progesterone biosynthesis, indicating that a post-cAMP site (or sites) might be involved into the BDE-47-decreased progesterone production. In addition, we found BDE-47 reduced the activity of P450 side chain cleavage enzyme (P450scc), which was companied with the decline of P450scc mRNA and protein level in mLTC-1 cells. Put all together, these results suggested that progesterone synthesis decrease induced by BDE-47 may be associated with attenuation of cAMP generation and reduction of P450scc activity.

Dibutyl phthalate (DBP) had been widely used and its exposure in children has been thought to be one of the reasons causing a trend of advanced pubertal timing in girls. Puberty starts from hypothalamic gonadotropin-releasing hormone release which is controlled by many factors including neurotransmitter kisspeptin and its receptor GPR54. These neural organization or reorganization happens in hypothalamus during neonatal or prepubertal period which may be two target windows of DBP exposure. The present study was designed to determine: (1) the difference between the effects of neonatal and prepubertal DBP exposure on female pubertal timing; (2) whether kisspeptin/GPR54 expression in hypothalamus would respond to neonatal and prepubertal DBP exposure differently. Female Sprague-Dawley rats were exposed by subcutaneous injection of 0.5, 5 and 50 mg/kg DBP during Postnatal day (P)1–5 (neonatal) or P26–30 (prepubertal). Physiological data demonstrated that both neonatal and prepubertal DBP exposure could advance pubertal timing significantly accompanied by irregular estrous cycles but only a little gonadal impairment. Exposure-period-related difference was found significant with prepubertal exposure groups having longer estrous cycle duration, heavier at vaginal opening and having higher serum estradiol level compared with neonatal exposure groups. Molecular data showed an up-regulated trend in kisspeptin mRNA and immunoreactivity levels of hypothalamic area arcuate but a down-regulation in GPR54 mRNA expression after P1–5 DBP treatment. In P26–30 groups, kisspeptin mRNA and immunoreactivity levels tended to be lower after DBP treatment. These results demonstrated small dose of DBP could induce earlier pubertal timing in females and both neonatal and prepubertal periods were critical windows for DBP exposure.Download full-size image

Dibutyl phthalate (DBP) had been widely used and its exposure in children has been thought to be one of the reasons causing a trend of advanced pubertal timing in girls. Puberty starts from hypothalamic gonadotropin-releasing hormone release which is controlled by many factors including neurotransmitter kisspeptin and its receptor GPR54. These neural organization or reorganization happens in hypothalamus during neonatal or prepubertal period which may be two target windows of DBP exposure. The present study was designed to determine: (1) the difference between the effects of neonatal and prepubertal DBP exposure on female pubertal timing; (2) whether kisspeptin/GPR54 expression in hypothalamus would respond to neonatal and prepubertal DBP exposure differently. Female Sprague-Dawley rats were exposed by subcutaneous injection of 0.5, 5 and 50 mg/kg DBP during Postnatal day (P)1–5 (neonatal) or P26–30 (prepubertal). Physiological data demonstrated that both neonatal and prepubertal DBP exposure could advance pubertal timing significantly accompanied by irregular estrous cycles but only a little gonadal impairment. Exposure-period-related difference was found significant with prepubertal exposure groups having longer estrous cycle duration, heavier at vaginal opening and having higher serum estradiol level compared with neonatal exposure groups. Molecular data showed an up-regulated trend in kisspeptin mRNA and immunoreactivity levels of hypothalamic area arcuate but a down-regulation in GPR54 mRNA expression after P1–5 DBP treatment. In P26–30 groups, kisspeptin mRNA and immunoreactivity levels tended to be lower after DBP treatment. These results demonstrated small dose of DBP could induce earlier pubertal timing in females and both neonatal and prepubertal periods were critical windows for DBP exposure.Download full-size image

Fenvalerate is a widely used synthetic pyrethroid insecticide and is known to impede the male reproductive function. However, the mechanisms remain to be elucidated. In this study, mouse Leydig tumor cells (MLTC-1) were used to investigate the effects of fenvalerate on progesterone production. Fenvalerate treatment inhibited progesterone secretion induced by human chorionic gonadotropin (hCG), cholera toxin (CT) or forskolin and decreased cAMP levels induced by hCG, but not by CT or forskolin, which suggested a repaired site on the upstream components of G protein or G protein per se by fenvalerate in the cAMP-mediated signal pathway. Furthermore, the addition of cAMP analog, 8-Br-cAMP, could not reverse fenvalerate-suppressed progesterone synthesis, indicating that fenvalerate interfered with the downstream molecules of cAMP. In addition, fenvalerate decreased steroidogenic acute regulatory protein (StAR) mRNA and protein levels, and also profoundly inhibited the activity of P450 side chain cleavage enzyme (P450scc) which was consistent with the decreased expression of P450scc mRNA and protein in MLTC-1 cells. These results suggested that fenvalerate might inhibit progesterone production by attenuating cAMP generation and inhibiting StAR expression and P450scc activity.

Effects of pesticides on the function of thyroid have attracted lots of attention because thyroid hormones (THs) play a major role in mammalian brain development. In order to screen for compounds that acted on the thyroid hormone receptor (TR) signaling pathway, we transiently transfected the vector pGal4-L-TRβ1 (Gal4 DBD fused to hTRβ1 LBD) and Gal4-responsive luciferase reporter pUAS-tk-Luc into HepG2 cell, developing a reporter gene assay which showed good response to triiodothyronine (T3) and thyroxine (T4) with the median effective concentration (EC50) of 0.46 and 25.53 nM, respectively. Bisphenol A exhibited weak anti-thyroid hormone activity with median inhibitory concentration (IC50) value of 6.45 × 10−5 M. The assay showed acceptable repeatability to T3 with intra coefficient of variability (CV) of 5.9% and inter CV of 11.7%. Carbaryl, 1-naphthol (1-NAP) and 2-naphthol (2-NAP) were tested for their agonist and antagonist activities. As a result, we found that all the three related chemicals possessed TR antagonist activity and none of them showed the agonist activity. These results further indicated that TR might be the targets of industrial chemicals. And this assay provided a useful tool for investigating the effects of environment chemicals on thyoid function.

Exposure to fenvalerate has been shown to be associated with decreased steroid hormone production by mouse Leydig tumor cells (MLTC-1) in our previous study and the interference with cAMP–PKA pathway cannot explain this inhibitory effect completely. In this study, the same cell line was used to investigate the potential involvement of insulin-like growth factor I (IGF-I) signaling pathway in the downregulation of steroidogenesis by fenvalerate. Results showed that fenvalerate treatment decreased IGF-I secretion significantly which was consistent with the reduced expression of IGF-I mRNA. Then inhibitors of the two downstream pathways of IGF-I were added to the medium. The addition of LY294002 (inhibitor of phosphatidylinositol (PI)-3-kinase) did not alter the declining trend of progesterone production with increasing dosages of fenvalerate treatment while the addition of UO126 (inhibitor of extracellular signal-regulated kinases 1/2 (ERK1/2)) markedly attenuated this trend, which strongly indicated the possible involvement of pathway ERK1/2. In addition, phosphorylation of ERK1/2 was also suppressed by fenvalerate. The results suggest that the mechanism by which fenvalerate decreased steroid hormone production might involve the impairment of IGF-I signal pathway by attenuating the IGF-I production and ERK1/2 phosphorylation.

Fenvalerate is a widely used synthetic pyrethroid insecticide and is reported to disrupt reproductive function in humans and animals. However, little is known about its influence on follicular development. In this study, rat preantral follicles were primary cultured to investigate the effects of fenvalerate on follicular survival rate, morphological change, steroid hormone levels and steroidogenesis related gene mRNA expression. Follicles were cultured with 0, 1, 5 and 25 μmol/L fenvalerate for 72 h. And then the morphous was assessed by conventional light microscopy, steroid hormones were measured by RIA, and the expressions of steroidogenic acute regulatory protein (StAR) and cytochrome P450 side-chain cleavage enzyme (P450scc) were monitored by real-time quantitative PCR analysis. Results showed that fenvalerate inhibited the augmentation of follicular diameters but did not have detectable effects on follicular survival rates. The level of steroid hormones, such as progesterone, testosterone and estradiol, was inhibited. The inhibition might be due to the decreased expression levels of StAR and P450scc. These results suggested that fenvalerate restrained the follicular growth, and inhibited steroidogenesis by reducing StAR and P450scc gene expression, which might further contribute to the fenvalerate-induced reproductive dysfunction.

Compared with increasing evidence suggesting that fenvalerate is neurotoxic to adults, further information regarding developmental toxicity of this compound attracts more attention. In this study, we used zebrafish as an environmental monitoring model to further explore the potential toxicity of fenvalerate. Our results demonstrated that larvae exposed to fenvalerate for 24–96 h displayed obvious morphological abnormalities, and the LC50 concentrations were 131.95 μg/L (LC50-24 h), 107.18 μg/L (LC50-48 h), 21.76 μg/L (LC50-72 h), and 6.25 μg/L (LC50-96 h). To further investigate the effects of fenvalerate on embryos and larvae, acridine orange staining was performed at a 50 μg/L concentration. Staining showed notable signs of apoptosis mainly in the brain. Further studies revealed that fenvalerate induced alterations in SOD activity in larvae were concentration dependent and also related to the length of exposure. Fenvalerate also down-regulated the expression of ogg1 and dlx2 genes in a concentration dependent manner, which indicated that the oxidative-DNA repair system as well as neurogenesis were impaired. In this study, we investigated the toxicity of fenvalerate using zebrafish, that provided new evidence of observable brain impairment during embryogenesis due to fenvalerate exposure and discussed their implications for the development of fenvalerate induced neurotoxicity.

Phthalates are widely used in the plastic industry and food packaging, imparting softness and flexibility to normally rigid plastic medical devices and children's toys. Even though phthalates display low general toxicity, there is increasing concern on the effects of endocrine system induced by some of phthalate compounds. The hormone activity of dibutyl phthalate (DBP), mono-n-butyl phthalate (MBP) and di-2-ethylhexyl phthalate (DEHP) were assessed using the luciferase reporter gene assays. The results showed that DBP, MBP and DEHP, not only exhibited potent antiandrogenic activity, with IC50 value of 1.05 × 10−6, 1.22 × 10−7 M and exceeding 1 × 10−4 M respectively, but also showed the androgenic activity with EC50 value of 6.17 × 10−6, 1.13 × 10−5 M and exceeding 1 × 10−4 M. We also found that all the three related chemicals possessed thyroid receptor (TR) antagonist activity with IC50 of 1.31 × 10−5, 2.77 × 10−6 M and exceeding 1 × 10−4 M respectively, and none showed TR agonist activity. These results indicate that TR might be the targets of industrial chemicals. In the ER mediate reporter gene assay, three chemicals showed no agonistic activity except for DBP, which appeared weakly estrogenic at the concentration of 1.0 × 10−4 M. Together, the findings demonstrate that the three phthalates could simultaneously disrupt the function of two or more hormonal receptors. Therefore, these phthalates should be considered in risk assessments for human health.