Perfluorooctane sulfonate (PFOS) is a widespread and persistent chemical in the environment. We investigated the endocrine‐disrupting effects of PFOS using a combination of in vitro and in vivo assays. Reporter gene assays were used to detect receptor‐mediated (anti‐)estrogenic, (anti‐)androgenic, and (anti‐)thyroid hormone activities. The effect of PFOS on steroidogenesis was assessed both at hormone levels in the supernatant and at expression levels of hormone‐induced genes in the H295R cell. A zebrafish‐based short‐term screening method was developed to detect the effect of PFOS on endocrine function in vivo. The results indicate that PFOS can act as an estrogen receptor agonist and thyroid hormone receptor antagonist. Exposure to PFOS decreased supernatant testosterone (T), increased estradiol (E2) concentrations in H295R cell medium and altered the expression of several genes involved in steroidogenesis. In addition, PFOS increased early thyroid development gene (hhex and pax8) expression in a concentration‐dependent manner, decreased steroidogenic enzyme gene (CYP17, CYP19a, CYP19b) expression, and changed the expression pattern of estrogen receptor production genes (esr1, esr2b) after 500 µg/L PFOS treatment in zebrafish embryos. These results indicate that PFOS has the ability to act as an endocrine disruptor both in vitro and in vivo by disrupting the function of nuclear hormone receptors, interfering with steroidogenesis, and altering the expression of endocrine‐related genes in zebrafish embryo. Environ. Toxicol. Chem. 2013;32:353–360. © 2012 SETAC

Pyrethroid insecticides, the most commonly used insecticides worldwide, are suspected endocrine-disrupting chemicals. But their interactions with hormone receptors are still unclear. The present study intended to evaluate and compare the hormone receptor (estrogen receptor [ER], androgen receptor [AR], and thyroid hormone receptor [TR]) activities of nine pyrethroids (cycloprothrin, cyfluthrin, cyhalothrin, cypermethrin, deltamethrin, etofenprox, fenvalerate, permethrin, and tetramethrin) and their metabolites (3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropne carboxylic acid [DCCA] and 3-phenoxybenzoic acid [3-PBA]) using receptor-mediated luciferase reporter gene assays. Of the 11 compounds tested, four showed very weak ER agonistic activities and six displayed antiestrogenic effects, among which cyhalothrin and DCCA possessed the most potent estrogenic and antiestrogenic activity respectively. Antagonistic effects to AR were found in 7 compounds, with cyfluthrin and deltamethrin exhibiting stronger AR antagonistic capacity. In the TR assay, all of tested chemicals except DCCA showed antagonistic effects. In this study, we provided evidence that a variety of pyrethroids and their metabolites might disrupt the function of multiple nuclear hormone receptors and thus have the potentials to affect the endocrine and the reproductive systems in humans.

Abnormal germline DNA methylation in males has been proposed as a possible mechanism compromising spermatogenesis of some men currently diagnosed with idiopathic infertility. Previous studies have been focused on imprinted genes with DNA methylation in poor quality human sperms. However, recent but limited data have revealed that sperm methylation abnormalities may involve large numbers of genes or shown that genes that are not imprinted are also affected. Using the methylation-specific polymerase chain reaction and bisulfite sequencing method, we examined methylation patterns of the promoter of methylenetetrahydrofolate reductase (MTHFR) gene (NG_013351: 1538–1719) in sperm DNA obtained from 94 idiopathic infertile men and 54 normal fertile controls. Subjects with idiopathic infertility were further divided into groups of normozoospermia and oligozoospermia. Overall, 45% (41/94) of idiopathic infertile males had MTHFR hypermethylation (both hemimethylation and full methylation), compared with 15% of fertile controls (P<0.05). Subjects with higher methylation level of MTHFR were more likely to have idiopathic male infertility (P-value for trend = 0.0007). Comparing the two groups of idiopathic infertile subjects with different sperm concentrations, a higher methylation pattern was found in the group with oligozoospermia. Hypermethylation of the promoter of MTHFR gene in sperms is associated with idiopathic male infertility. The functional relevance of hypermathylation of MTHFR to male fertility warrants further investigation.

MicroRNAs (miRNAs) are small non-coding RNAs (18–25 nt), playing important regulatory roles via interaction with cellular messenger RNAs. The altered expression of miRNAs in specific tissues has been associated with diseases such as cancer and diabetes. We examined the presence of two selected miRNAs (miR-19b and let-7a) in human seminal plasma from fertile men and idiopathic infertile patients with oligozoospermia and non-obstructive azoospermia (NOA) using quantitative real-time PCR. We detected miRNAs in the seminal plasma of humans. The levels of miRNAs in the seminal plasma were reproducible in repeat samples from the same individuals. In addition, we examined the expression patterns of two selected miRNAs in 96 idiopathic infertile males (48 oligozoospermia and 48 NOA) and 48 fertile controls. Another 48 individuals of each group were used for verification. Our data showed that the expression levels of these two miRNAs in the seminal plasma significantly increased in idiopathic infertile males with NOA compared with fertile controls, whereas the expression levels were similar between idiopathic infertile males with oligozoospermia and fertile controls. In conclusion our results indicate that the expression of miR-19b and let-7a in the seminal plasma are reproducible and stable. Aberrant over-expression levels of miR-19b and let-7a may be an indicator of spermatogenic failure.

Aims:To determine sperm nuclear DNA integrity and to investigate the relation between fenvalerate (FE) exposure and spermatozoa DNA damage.Methods:Sperm DNA fragmentation was detected by a modified alkaline single cell gel electrophoresis (Comet) assay and a terminal deoxynucleotidyl transferase mediated dUTP nick end labelling (TUNEL) assay. The olive tail moment (OTM) and percentage tail DNA were measured by the Comet assay, and cell positive percentage was measured by the TUNEL assay for DNA damage evaluation.Results:The DNA integrity of spermatozoa of external and internal control groups were both significantly greater than that of the FE exposed group. The median value of tail DNA percentage in the exposure group was 11.30, which was significantly higher than 5.60 in the internal control group and 5.10 in the external control group. The median value of OTM was 3.80 in the exposure group, significantly higher than 1.50 in the internal control group and 2.00 in the external control group. Mean cell positive was 31.2% in the exposure group, significantly higher than 17.4% in the internal control and 19.6% in the external control groups. Cell positive (%) was significantly correlated with tail DNA percentage and with OTM of whole subjects (n = 63).Conclusions:Results showed that occupational FE exposure is associated with an increase in sperm DNA damage. A combination of the Comet and TUNEL assays would offer more comprehensive information for a better understanding of sperm DNA damage, and the biological significance of sperm DNA damage in sperm function and male infertility.