Shoulin Wang

Assessing Hormone Receptor Activities of Pyrethroid Insecticides and Their Metabolites in Reporter Gene Assays

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.

Idiopathic Male Infertility Is Strongly Associated with Aberrant Promoter Methylation of Methylenetetrahydrofolate Reductase (MTHFR)

Background 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. Methodology/Principal Findings 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. Conclusions 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.

The b2/b3 subdeletion shows higher risk of spermatogenic failure and higher frequency of complete AZFc deletion than the gr/gr subdeletion in a Chinese population

Microdeletions in the azoospermia factor (AZF) regions on the long arm of the human Y chromosome are known to be associated with spermatogenic failure. Although AZFc is recurrently deleted in azoospermic or oligozoospermic males, no definitive conclusion has been reached for the contribution of different partial AZFc deletions to spermatogenic failure. To further investigate the roles of partial deletions in spermatogenic failure and the relationship between the complete and partial AZFc deletions, we performed deletion typing and Y chromosome haplogrouping in 756 idiopathic infertile Han-Chinese and 391 healthy Han-Chinese. We found that both the b2/b3 partial deletion and the DAZ3/4+CDY1a deletion pattern were associated with spermatogenic failure. We also confirmed that two previously reported fixations, the b2/b3 deletion in haplogroup N1 and the gr/gr deletion in haplogroup Q1. Remarkably, the frequency of the complete AZFc deletion in haplogroup N1 was significantly higher than that in the haplogroup Q1. These results suggest that the b2/b3 partial deletion was associated with a higher risk of complete AZFc deletion compared with the gr/gr partial deletion. Compared with the gr/gr deletion, the b2/b3 deletion presents a shorter distance among recombination targets and longer recombination substrates, which may be responsible for the increased incidence of subsequent recombination events that can lead to the complete AZFc deletion in this Chinese study population. The susceptibility of the b2/b3 partial deletion to the complete AZFc deletion deserves further investigation in larger and diverse populations, especially those with a relatively high frequency of b2/b3 and gr/gr partial deletions.

Developmental toxicity of cypermethrin in embryo-larval stages of zebrafish

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.

The relationship of 3-PBA pyrethroids metabolite and male reproductive hormones among non-occupational exposure males

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.

Sertoli cell is a potential target for perfluorooctane sulfonate-induced reproductive dysfunction in male mice.

Perfluorooctane sulfonate (PFOS) is associated with male reproductive disorders, but its targets and mechanisms are poorly understood. We used in vitro and in vivo models to explore the roles of Sertoli cells and the blood-testis barrier (BTB) in PFOS-induced male reproductive dysfunction. First, we used primary Sertoli cell to estimate PFOS-induced cytotoxicity, junction proteins expression, and the changes of barrier function. ICR mice were then administered PFOS (0.25-50mg/kg/day) for 4 weeks. Sperm count, ultrastructure and permeability of the Sertoli cell-based BTB, and testicular PFOS were estimated. Furthermore, the expression and localization of proteins related to junctions between Sertoli cells and mitogen-activated protein kinase (MAPK) signaling pathway were evaluated. Apparent decreases in sperm count were found. PFOS significantly increased vacuolization in Sertoli cells in seminiferous tubules and BTB ultrastructural disassembly, which subsequently increased BTB permeability and testicular PFOS levels, which was confirmed by in vitro results that PFOS decreased transepithelial electrical resistance between Sertoli cells. Additionally, PFOS decreased the expression of junction proteins in Sertoli cells, which was further confirmed by in vivo results that PFOS decreased or dislocated junction proteins (i.e., ZO-1, occludin, claudin-11, and connexin-43) and increased proteins related to the MAPK signaling pathway (i.e., Erk and p38), whereas basal ectoplasmic specialization proteins did not change. The results were confirmed by SB203580, a p38 MAPK selective inhibitor. Sertoli cells appear to be a new cellular target for PFOS. Together with disruption of BTB integrity and function, these cells play an important role in PFOS-induced male reproductive toxicity.

Effects of non-occupational environmental exposure to pyrethroids on semen quality and sperm DNA integrity in Chinese men

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.

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.

The Repressive Effect of NF-κB on p53 by Mot-2 Is Involved in Human Keratinocyte Transformation Induced by Low Levels of Arsenite

Inorganic arsenic is a ubiquitous environmental contaminant associated with an increased risk of skin hyperkeratosis and cancer. Although several hypotheses that relate to arsenic-induced carcinogenesis have been suggested, the mechanism of action remains obscure. In the present study, molecular mechanisms underlying the inactivation of p53 function and the genomic instability in malignant transformation of the human keratinocyte cell line, HaCaT, induced by low levels of arsenic were investigated. Our results show that long-term exposure of HaCaT cells to sodium arsenite (1.0 microM) increases their proliferation, causes DNA double-strand breaks, and induce anchorage-independent growth. In arsenite-exposed cells, the levels of phospho-p53, p21, and mdm2 increase at early times after exposure. The levels, however, decrease with longer times. Interaction of the promoter of mot-2 (a p53 inhibitor) with nuclear factor kappaB (NF-kappaB) was established by Southwestern and Western blot assays. Blockage of NF-kappaB prevents the increases of arsenite-induced mot-2 levels, and knockdown of mot-2 facilitates the nuclear translocation of p53, indicating that, in HaCaT cells exposed to arsenite, NF-kappaB inhibits p53 function by mot-2. Moreover, inactivation of NF-kappaB facilitated p53-mediated DNA repair and prevented arsenite-induced malignant transformation. Together, the results suggest that the repressive effect of NF-kappaB on p53 by mot-2 leads to genomic instability, which is involved in arsenite-induced malignant transformation of human keratinocytes.

Cytochrome P450 2A13 mediates aflatoxin B1-induced cytotoxicity and apoptosis in human bronchial epithelial cells.

Cytochrome P450 (CYP) 2A13 is mainly expressed in the respiratory system and has the ability to metabolize aflatoxin B(1) (AFB(1)). However, the role of CYP2A13-mediated AFB(1) metabolism and its consequences in human lung epithelial cell is not clear. Therefore, the objectives of this study were to investigate the significance of CYP2A13 in AFB(1)-induced cytotoxicity, DNA adducts, and apoptosis. To achieve these objectives, CYP2A13 was stably over-expressed in immortalized human bronchial epithelial BEAS-2B cells (B-2A13) and its significance in AFB(1)-induced cytotoxicity, DNA adducts, and apoptosis was compared to cells with stably expression of CYP1A2 (B-1A2), the predominant AFB(1) metabolizing enzyme in liver, as well as CYP2A6 (B-2A6) as controls. AFB(1) induced B-2A13 cytotoxicity and apoptosis in a dose- and time-dependent manner. The cytotoxic and apoptotic effects of AFB(1) were significantly remarkable in B-2A13 cells than those of B-1A2 and B-2A6 cells. The increased expression of pro-apoptotic proteins, such as C-PARP, C-caspase-3, and Bax, and decreased expression of anti-apoptotic proteins, such as caspase-3, Bcl-2, and p-Bad further confirmed the data of AFB(1)-induced cytotoxicity and apoptosis. Furthermore, increased DNA adduct was observed in B-2A13 after AFB(1) treatment as compared to B-1A2 cells and B-2A6 cells. Finally, treatment with nicotine, a competitor of AFB(1), and 8-methoxypsoralen (8-MOP), an inhibitor of CYP enzyme, further confirm the critical role of CYP2A13 in AFB(1)-induced cytotoxicity and apoptosis. Collectively, these findings suggest adverse effects of AFB(1) in respiratory diseases mediated by CYP2A13.