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Glufosinate-ammonium (GLA) is a widely used herbicide with emerging concern over its neural and reproductive toxicity. To uncover potential effects of GLA on male reproductive health in mammals, adult male C57BL/6J mice were administered 0.2 mg/kg·d GLA for 5 weeks. After examination on fertility, testis histology and semen quality in the GLA group, we performed deep sequencing to identify repressive epigenetic marks including DNA methylation and histone modifications (H3K27me3 and H3K9me3), together with mRNA transcript levels in sperm. Then, we integrated multi-omics sequencing data to comprehensively explore GLA-induced epigenetic and transcriptomic alterations. We found no significant difference either on fertility, testis histology or semen quality-related indicators. As for epigenome, the protein level of H3K27me3 was significantly increased in GLA sperm. Next generation sequencing showed alterations of these epigenetic marks and extensive transcription inhibition in sperm. These differential repressive marks were mainly distributed at intergenic regions and introns. According to results by Gene Ontology enrichment analysis, both differentially methylated and expressed genes were mainly enriched in pathways related to synapse organization. Subtle differences in genomic imprinting were also observed between the two groups. These results suggested that GLA predominantly impaired sperm epigenome and transcriptome in mice, with little effect on fertility, testis histology or semen quality. Further studies on human sperm using similar strategies need to be conducted for a better understanding of the male reproductive toxicity of GLA.

Surface water is essential for human health and ecological diversity, but some endocrine disrupting chemicals are detectable. Both thyroid receptor (TR) and androgen receptor (AR) agonistic/antagonistic potencies in grade II surface water in East China were investigated using reporter gene assays. While none of the water exhibited agonistic potency, significant AR and TR antagonistic potencies were detectable. TR antagonistic equivalents (TR-AntEQ) and AR antagonistic equivalents (AR-AntEQ) ranged from 3.6 to 76.1 μg dibutyl phthalate/L and from 2.3 to 242.6 μg flutamide/L, respectively. The TR and AR antagonistic potencies in the Yangtze River watershed were highlighted, with equivalents greater than the lowest observable effect concentration (LOEC) of dibutyl phthalate and flutamide, respectively. Phthalate esters (PAEs) being the most abundant explained most of the TR antagonistic potency, contributing more than 65% of the TR-AntEQ and diisobutyl phthalate (DiBP) was the major contributor. In most surface waters studied, PAEs contributed little of the AR-AntEQ, but the frequently detected octylphenol, nonylphenol and benzo[a]pyrene might be responsible.

In this study, the metabolic responses of mice after 30 days of exposure to benalaxyl were assessed using NMR-based untargeted metabolomics and LC-MS-based targeted profiling of 20 amino acids. Urinary 1H NMR analyses revealed alterations in energy metabolism, lipid metabolism, vitamin B metabolism, the urea cycle and amino acid metabolism, and targeted analyses indicated that the serum levels of asparagine, histidine, lysine and aspartic acid were significantly altered. Additionally, significant oxidative stress was observed in the liver and kidney, although no apparent histopathological injury was observed. The tissue distribution indicated a significant stereoselectivity in the brain, where (-)-R-benalaxyl was enriched. These data provide a comprehensive picture of the subacute toxic effects of benalaxyl in mice. The results of this study suggested that, for a toxicity evaluation, metabolomics analysis is much more sensitive than traditional toxicological methods. The results also highlight the combined use of untargeted and targeted metabolomics approaches in evaluating the health risks of xenobiotics.

Perfluorooctanoic acid (PFOA), a persistent perfluorinated compound, is distributed widely in wildlife and humans. Recent studies showed that PFOA is a suspected endocrine disruptor. But the results are somewhat contradictory and the mechanisms are unclear. In this study, we investigated the endocrine-related effects of PFOA using a series of assays. The lower dose effect of PFOA on development and endocrine-related gene expression were assessed in a short-term zebrafish assay in vivo. To clarify the mechanism of PFOA, in vitro assays were performed. We tested the hormone receptor activities of ER, AR, and TR against PFOA using reporter gene assays. The hormone levels of estradiol (E2) and testosterone (T), the expression of major steroidogenic genes and the key steroidogenic gene regulator steroidogenic factors 1 (SF-1) were measured after PFOA exposure in H295R steroidogenesis assay. Exposure of zebrafish embryo to PFOA resulted in higher expression of esr1, hhex and pax. PFOA is able to interfere with hormone receptor ER and TR. In H295R cells, PFOA could increase the E2 production and decrease the T production, altered the expression of major steroidogenic genes and regulator SF-1. The current findings indicated the potential endocrine-related effects of PFOA and provided novel information for human risk assessment.

The estrogenic activities of source water from Yangtze River, Huaihe River, Taihu Lake and groundwater in Yangtze River Delta in the dry and wet season were determined by use of reporter gene assays based on African green monkey kidney (CV-1) cell lines. Higher estrogenic activities were observed in the dry season, and the estrogenic potentials in water samples from Taihu Lake were greater than other river basins. None of the samples from groundwater showed estrogen receptor (ER) agonist activity. The 17β-Estradiol (E2) equivalents (EEQs) of water samples in the dry season ranged from 9.41 × 10 −1 to 1.20 × 10 1 ng E2 L −1 . In the wet season, EEQs of all the water samples were below the detection limit as 9.00 × 10 −1 ng E2 L −1 except for one sample from Huaihe River. The highest contribution of E2 was detected in Yangtze River as 99% of estrogenic activity. Nonylphenol (NP, 100% detection rate) and octylphenol (OP, 100% detection rate) might also be responsible for the estrogenic activities in water sources. Potential health risk induced by the estrogenic chemicals in source water may be posed to the residents through water drinking.

The enantiomerization and enantioselective metabolism of benalaxyl in mice after a single gavage administration were investigated in the present study. The pharmacokinetic result indicated that elimination of (−)-R-benalaxyl in plasma was slightly faster with the t1/2 of 26.65 h and 28.88 h for (−)-R- and (+)-S-benalaxyl, respectively. Consistent with this, elimination in tissues and excretion were also enantioselective, with (+)-S-benalaxyl enriched in all tissues, urine and feces. And formation of the metabolites also exhibited an enantioselective manner. Both benalaxyl and most of its metabolites exhibited a potent excretion to feces and urine. In addition, significant enantiomerization of benalaxyl enantiomers was observed in plasma, with a larger extent from R to S than S to R. Thus, this difference in enantiomerization may be one of the reasons to explain the enantioselective enrichment of (+)-S-benalaxyl in mice observed in this study. Data from this study proves that besides metabolic enzymes, enantiomerization could be another important factor that contributes to the enantioselective metabolism of a chiral pesticide. Thus, research at enantiomeric level is necessary for efficient risk assessment of chiral pesticides.

Cypermethrin, a type II pyrethroid insecticide, is widely used throughout the world in agriculture, forestry, horticulture and homes. Though the neurotoxicity of cypermethrin has been thoroughly studied in adult rodents, little is so far available regarding the developmental toxicity of cypermethrin to fish in early life stages. To explore the potential developmental toxicity of cypermethrin, 4-h post-fertilization (hpf) zebrafish embryos were exposed to various concentrations of cypermethrin (0, 25, 50, 100, 200 and 400 μg L⁻¹) until 96 h. Among a suite of morphological abnormalities, the unique phenotype curvature was observed at concentrations as low as 25 μg L⁻¹. Studies revealed that 400 μg L⁻¹ cypermethrin significantly increased malondialdehyde production. In addition, activity of antioxidative enzymes including superoxide dismutase and catalase were significantly induced in zebrafish larvae in a concentration-dependent manner. To further investigate the toxic effects of cypermethrin on fish, acridine orange (AO) staining was performed at 400 μg L⁻¹ cypermethrin and the result showed notable signs of apoptosis mainly in the nervous system. Cypermethrin also down-regulated ogg1 and increased p53 gene expression as well as the caspase-3 activity. Our results demonstrate that cypermethrin was able to induce oxidative stress and produce apoptosis through the involvement of caspases in zebrafish embryos. In this study, we investigated the developmental toxicity of cypermethrin using zebrafish embryos, which could be helpful in fully understanding the potential mechanisms of cypermethrin exposure during embryogenesis and also suggested that zebrafish could serve as an ideal model for studying developmental toxicity of environmental contaminants.

Widespread use of phenols has led to ubiquitous exposure to phenols. In experimental animals, phenols increased resorptions, reduced live litter size and fetal body weights. However, there are limited epidemiological evidences of the relationships between exposure to phenols and pregnancy outcomes. We evaluated the associations between parental urinary levels of various phenols and spontaneous abortion in a Chinese population residing in the middle and lower reaches of the Yangtze River. A case-control study was conducted that included 70 case couples with medically unexplained spontaneous abortion and 180 control couples who did not have a history of spontaneous abortion and had at least one living child. Both parental urinary phenols were measured by ultra-high performance liquid chromatography-tandem mass spectrometry including bisphenol A (BPA), benzophenone-3 (BP-3), 2,3,4-trichlorophenol (2,3,4-TCP), pentachlorophenol (PCP), 4-n-octylphenol (4-n-OP) and 4-n-nonylphenol (4-n-NP). Compared with the low exposure group, there was an increased risk of spontaneous abortion with high paternal urinary PCP concentration [odds ratio (OR)=2.09, 95% Confidence Interval (CI), 1.05-4.14], and maternal exposure to 4-n-OP and alkylphenol(s) also significantly increased the risk of spontaneous abortion (OR=2.21, 95% CI, 1.02-4.80; OR=2.81, 95% CI, 1.39-5.65, respectively). Our study firstly provides the evidence that paternal PCP exposure, maternal 4-n-OP and alkylphenol(s) exposure are associated with spontaneous abortion in humans.

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.

Many pesticides possess hormonal activities and have been classified as endocrine disrupting chemicals. Synthetic pyrethroids are one kind of the most common pesticides used in the world. In the present study, we explored the association between serum reproductive hormone levels and urinary creatine (CR) adjusted concentration of 3-phenoxybenzoic acid (3-PBA), a general metabolite of pyrethroids, in Chinese adult men. The study subjects (n=212) were from the affiliated hospitals of Nanjing Medical University. By using GC-MS, urinary 3-PBA level of each subject was measured and adjusted by urinary CR. Blood samples were collected for measuring the serum levels of reproductive hormones, including follicle stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E2), testosterone (T) and prolactin (PRL). All the subjects had detectable levels of 3-PBA in their urine samples. The median concentration of 3-PBA was 0.815 microg g (-1)of CR. The results showed that there was positive associations between the levels of serum LH and 3-PBA (p=0.013) but negative associations between E2 and 3-PBA level (p=0.022), and the adjusting p-value was 0.044 for LH and E2, which suggested that pyrethroids are capable of disrupting the male endocrine function. In adult men, urinary 3-PBA levels were associated with increased LH and reduced E2 levels. On a population level, these reductions show potential public health importance because of widespread exposure to these pesticides.

Alkylphenols (APs) are widely used as important industrial materials and have attracted lots of attention because of their potential estrogenic activities. In this study, we developed human estrogen receptor alpha (hERalpha) and rat estrogen receptor alpha (rERalpha) mediated reporter gene assays and compared the estrogenic activity of APs and related chemicals based on the two ERalpha. Human breast cancer cell line MCF-7 was co-transfected with Gal4-fused hERalpha and corresponding reporter plasmid; African green monkey kidney cell line CV-1 was co-transfected with rERalpha and reporter gene. Both assays showed acceptable response to natural estrogen 17beta-estradiol (E2) with EC50 of 0.16 nM and 4.7 nM. Then the estrogenic activity of 4-APs, 4-phenylphenol and bisphenol-A were evaluated and compared with the effects of E2. The data suggested that test APs and related chemicals possessed weakly estrogenic activity and the activity of test APs increased with the increase of substituent size. This structure-activity relationship helped to infer the activity of chemicals with similar feature. Furthermore, test APs showed similar effect on the function of hERalpha and rERalpha. This consistency helped to extrapolate in vivo rodent data to human being when performing risk assessment of endocrine disruptors.

Many pesticides possess hormonal activity and have thus been classified as endocrine disruptors. Pyrethroids are commonly used pesticides worldwide, but little has been done to characterize their antiandrogenic activity potential. We tested three frequently encountered pyrethroids (fenvalerate, cypermethrin, permethrin) and their metabolite 3-phenoxybenzoic acid (3-PBA) for antiandrogenic and androgenic activity using a human androgen receptor (AR) mediated luciferase reporter gene assay in CV-1 African green monkey kidney cell. The assay displayed appropriate response to the known AR agonist 5alpha-dihydrotestosterone and AR antagonist nilutamide and flutamide. At 0.1mM, all the three tested pyrethroids significantly suppressed the luciferase expression. Further, their metabolite 3-PBA also showed antagonist activity. None of the test chemicals showed androgenic activity. Through the antiandrogenic pathways, exposure to certain pyrethroids may contribute to the damage of reproductive system. In conclusion, pyrethroid pesticides can act as antiandrogen in vitro, and metabolizing to 3-PBA cannot eliminate the antagonist activity. This result provides useful information for risk assessment of pyrethroid pesticides.