Yan-Qiu Liang

Long-term exposure to environmentally relevant concentrations of progesterone and norgestrel affects sex differentiation in zebrafish (Danio rerio)

The aim of this study was to investigate the effects of progestins on the sex differentiation of zebrafish by measuring the sex ratio and transcriptions of genes related to sex differentiation (Amh, Dmrt1, Figa, Sox9a and Sox9b genes) as well as sex hormone levels and transcriptional expression profiles along the hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituitary-adrenal (HPA) axes in juvenile zebrafish. Exposure of zebrafish to 4, 33, 63ngL(-1) progesterone (P4) or 4, 34, 77ngL(-1) norgestrel (NGT) started at 20 days post fertilization (dpf) and ended at 60 dpf. The results showed that exposure to P4 caused a significant increase in proportion of females as well as significant down-regulation of Amh gene and up-regulation of Figa at a concentration of 63ngL(-1). However, the shift in the sex ratio toward males was observed following exposure to 34 and 77ngL(-1) NGT, which came along with the significant induction of Dmrt1 gene and inhibition of Figa gene. The sex hormones in exposed fish were measured with estrone being detected only in the fish exposed to the highest P4 concentration; whereas estradiol and androstenedione were detected only in the fish of the control and lowest NGT concentration. Furthermore, the increase in females was associated with the significant up-regulation of several key genes controlling the synthesis of sex hormones (i.e., Cyp17, Cyp19a1a and Hsd3b) following exposure to 63ngL(-1) P4 whereas the significant down-regulation of Cyp11a1, Cyp17, Cyp19a1a and Hsd3b genes was observed in the male-biased populations caused by 34 and 77ngL(-1) NGT. The overall results imply that both P4 and NGT could significantly affect sex differentiation in zebrafish, and that changes may be reflected by altered sex hormone levels and transcriptional expression profiles of genes related to synthesis of sex hormones.

Expression patterns of metallothionein, cytochrome P450 1A and vitellogenin genes in western mosquitofish (Gambusia affinis) in response to heavy metals

The aim of this study was to evaluate the effects of three metals (Zn, Cd and Pb) on hepatic metallothionein (MT), cytochrome P450 1A (CYP1A) and vitellogenin (Vtg) mRNA expression in the liver of adult female mosquitofish (Gambusia affinis) after 1, 3 or 8d. Both concentration-response and time-course effects of hepatic MT, CYP1A and Vtg at the transcription level were determined by quantitative real-time PCR. The results from this study showed that Zn, Cd and Pb could significantly induced MT, CYP1A and Vtg mRNA expression levels in mosquitofish. In general, this study demonstrated that heavy metals modulate MT, CYP1A and Vtg mRNA expression levels in a metal-, concentration- or time-dependent manner.

A time‐course transcriptional kinetics of the hypothalamic–pituitary–gonadal and hypothalamic–pituitary–adrenal axes in zebrafish eleutheroembryos after exposure to norgestrel

The objective of the present study was to investigate the effects of norgestrel on the hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituitary-adrenal (HPA) axes in zebrafish eleutheroembryos. Zebrafish embryos were exposed to different concentrations of norgestrel (0u2009ngu2009L(-1) , 5u2009ngu2009L(-1) , 50u2009ngu2009L(-1) , and 100u2009ngu2009L(-1) ) for 144u2009h post fertilization (hpf), and the transcriptional profiles of the HPG and HPA axes were examined every day. Norgestrel modulated the expression of Pgr and Vtg1 messenger (m)RNAs mainly at 96 hpf for all treatment groups. In addition, norgestrel strongly altered the expression of Cyp11a1 mRNA above 5u2009ngu2009L(-1) (significant upregulation from 48 hpf to 120 hpf and significant downregulation for 144 hpf). Norgestrel treatment could significantly induce expression of Cyp19a1a, Cyp11b, Gnrh2, Gnrh3, and Lhb mRNAs but inhibit transcripts of Hsd11b2 and Crh genes above 5u2009ngu2009L(-1) at different time points. The transcriptional expression levels of Esr1, Ar, Star, Hsd17b3, Fshb, and Pomc were also mediated by 5u2009ngu2009L(-1) norgestrel or higher during different exposure periods. Taken together, the overall results imply that the transcriptional changes in zebrafish eleutheroembryos may pose a potential effect on embryonic development, in particular in the brain and gonadogenesis.

Hormonal effects of tetrabromobisphenol A using a combination of in vitro and in vivo assays

The aim of this study was to investigate the hormonal effects of tetrabromobisphenol A (TBBPA) in vitro on recombinant yeasts and in vivo on mosquitofish (Gambusia affinis). The in vitro bioassays for (anti-)androgenic activities showed that TBBPA had a weak androgenic activity in vitro with recombinant yeast systems carrying human androgen receptor (hAR). In the in vivo bioassays, the gene expression patterns of vitellogenin (Vtg), estrogen receptors (ERα and ERβ), and androgen receptors (ARα and ARβ) in adult males and juveniles after exposure to TBBPA for 60 days were evaluated. Significant up-regulation of Vtg, ERα, and ERβ mRNAs was observed in the liver after exposure to 500 nM of TBBPA. In the testis, the lowest concentration of TBBPA (50 nM) markedly induced Vtg, ERβ, and ARβ mRNA expression, but the same concentration significantly inhibited ARα mRNA expression. In addition, in juveniles, 100 nM of TBBPA significantly up-regulated the expression of Vtg, ERβ, and ARα mRNAs. However, TBPPA did not cause histological alterations in the liver and testis of adult male mosquitofish. The results from this present study suggest that TBBPA could display low but multiple hormonal activities despite its low toxicity to mosquitofish.

Feminization and masculinization of western mosquitofish ( Gambusia affinis ) observed in rivers impacted by municipal wastewaters

Municipal wastewaters have been known to contain various estrogens and androgens. Little is known about the joint action of these chemicals from wastewaters on fishes in the aquatic environment. The objectives of this study were to investigate the estrogenic and/or androgenic effects in wild mosquitofish (Gambusia affinis) of two effluent-impacted rivers in South China by determining morphological changes and hepatic mRNA expression levels of relevant genes such as vitellogenin (Vtg), estrogen receptor (ERα) and androgen receptors (ARα and ARβ), and to assess the linkages of those morphological changes and hepatic mRNA expression levels to the chemical concentrations measured by in vitro bioassays and chemical analysis. The results showed a significant induction of Vtg and ERα mRNA in the livers of the males and a gonopodium-like anal fin in the females collected at the majority of sites. Redundancy analysis and Pearson correlation analysis showed that the chemical concentrations obtained by in vitro bioassays and chemical analysis had significant correlations with some of the endpoints for the estrogenic and/or androgenic effects in mosquitofish. The findings from this study indicate that the estrogens and androgens present in the two rivers could cause the observed estrogenic and androgenic effects in mosquitofish.

Progesterone and norgestrel alter transcriptional expression of genes along the hypothalamic-pituitary-thyroid axis in zebrafish embryos-larvae.

The aim of this study was to investigate the effects of progestins on the hypothalamic-pituitary-thyroid (HPT) axis in the early stage of zebrafish. Zebrafish embryos were exposed to progesterone (P4) or norgestrel (NGT) at 5, 50 and 100 ng L(-1) for 144 h post fertilization (hpf), and the transcriptional levels of target genes along the hypothalamic-pituitary-thyroid axis were determined daily. The results showed that P4 had only minor effects on the mRNA expression of thyroglobulin (Tg), iodothyronine deiodinase type Ι (Dio1) and thyroid hormone receptor β (Thrb) genes. Similarly, the effects of NGT on transcripts of thyrotropin-releasing hormone (Trh), Dio1, iodothyronine deiodinase type II (Dio2) and thyroid hormone receptor α (Thra) genes were generally low. In addition, NGT resulted in some alterations of Tg and Thrb transcripts at different time points. However, a strong induction of Nis mRNA by P4 and NGT was observed in zebrafish embryos-larvae. The overall results showed that besides Nis no effects on the hypothalamic-pituitary-thyroid (HPT) axis are observed following exposure to P4 and NGT, which imply that both P4 and NGT have potential effects on the thyroid endocrine system by inducing transcript of Nis gene during the early stage of zebrafish.

The effects of progesterone on transcriptional expression profiles of genes associated with hypothalamic–pituitary–gonadal and hypothalamic–pituitary–adrenal axes during the early development of zebrafish (Danio rerio)

Progesterone (P4) has been reported in surface water, and it may have adverse effects on aquatic organisms. This study provided the transcriptional effects of P4 during the early development of zebrafish. Zebrafish embryos were exposed for 144 h post fertilization (hpf) to 0, 6, 45 and 90 ng L(-1) P4, and transcriptional expression profiles of the hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituitary-adrenal (HPA) axes were assessed every day. For the receptor signaling pathways, P4 significantly induced the transcript of Pgr gene above 45 ng L(-1) at 72 and 144 hpf, but inhibited its transcript above 6 ng L(-1) at 96 and 120 hpf. A significant up-regulation of Vtg1 mRNA was observed at 6 ng L(-1) P4 or higher at 24, 96 and 144 hpf. For the steroidogenic pathways, the transcriptional expression of Cyp11a1 and Hsd17b3 mRNAs was mediated by 6 ng L(-1) P4 or higher according to different exposure time points. In addition, P4 resulted in a significant induction of Cyp19a1a and Cyp11b mRNA expression while it caused a significant inhibition of Hsd11b2 mRNA expression above 6 ng L(-1). For the other target genes related to hypothalamic and pituitary hormones, P4 mainly modulated the transcripts of Gnrh2, Fshb and Lhb genes at 6 ng L(-1) or higher. The overall results from the present study indicate that P4 at environmentally relevant concentrations could cause the potential effects on zebrafish reproductive and adrenal endocrine systems by interfering with the HPG and HPA axes.

Effects of steroid hormones on reproduction- and detoxification-related gene expression in adult male mosquitofish, Gambusia affinis

The molecular mechanisms that mediate fish reproduction and detoxification in response to steroid hormones were studied by using adult male western mosquitofish (Gambusia affinis) as sentinel species. The expression patterns of three vitellogenins (VtgA, VtgB and VtgC), two estrogen receptors (ERα and ERβ), two androgen receptors (ARα and ARβ), metallothionein (MT) and cytochrome P450 1A (CYP1A) in the liver and testis of adult male mosquitofish were assessed through exposure treatments with progesterone (P), testosterone (T) and 17β-estradiol (E2), alone and in combination for eight days. The results showed that expression patterns of Vtg subtype, ER subtype, AR subtype, MT and CYP1A genes in male mosquitofish varied according to tissue and specific hormone stress. Vtg subtype mRNA expression was induced in the liver in E2-added treatments, and an up-regulation of ERα mRNA expression was also observed. In addition, hormone treatments increased three Vtg subtype mRNA expression levels in the testis, at least to some extent. All hormone treatments significantly inhibited ERα, ERβ and ARβ mRNA expression in the testis. Some of hormone treatments could affect MT and CYP1A gene expression in mosquitofish. In general, multiple hormone treatments showed different effects on target gene expression compared with corresponding hormone alone. The results from the present study provided valuable information on the toxicological effects of steroid hormones in mosquitofish.

CYP1A/Regucalcin Gene Expression and Edema Formation in Zebrafish Embryos Exposed to 2,3,7,8-Tetrachlorodibenzo- p-dioxin

In this study, zebrafish eggs were exposed to a relatively low concentration (50xa0pg/mL) of 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) for 72xa0h and then transferred to vehicle/TCDD-free water for the remainder of the experiments. Mortality, heart rates, edema severity, CYP1A, and regucalcin gene expressions were investigated to study TCDD-induced toxicity in zebrafish during the early life stage. Results indicated that the 50xa0pg/mL TCDD caused severe and visible developmental toxicity. Further research of the long term and low concentration of TCDD exposure is required.

Multi-biomarker responses as indication of contaminant effects in Gambusia affinis from impacted rivers by municipal effluents

This study investigated toxic effects in mosquitofish from two urban rivers of South China impacted by municipal effluents by using multiple biomarkers including fish morphology, biochemical indicators and transcriptional responses, and explored potential cause-effect relationship with a list of chemicals (metals, polycyclic aromatic hydrocarbons (PAHs) and pesticides). The results showed significant alterations in metallothionein (MT) protein and mRNA expression in mosquitofish collected from the two rivers and a strong association between MT protein and mRNA expression levels and heavy metals in the river water. Both ethoxyresorufin-O-deethylase (EROD) activity and cytochromes P450 1A (CYP1A) mRNA expression were significantly enhanced in mosquitofish at most sampling sites. There existed a strong correlation between EROD activity and CYP1A mRNA expression levels, but no clear correlations between these responses and PAHs in the river water possibly because of the presence of many other agonists of the aryl hydrocarbon receptor in the two rivers. Significant acetylcholinesterase (AChE) inhibition was observed in mosquitofish brain samples. The pesticides in the two rivers showed an influence on the AChE activity, which was also found to be significantly negatively correlated to fipronil concentrations. Moreover, the result also indicates that metals and pesticides present in the two rivers might cause the observed estrogenic and androgenic effects in mosquitofish. The findings from this study clearly showed morphological, biochemical and transcriptional responses in mosquitofish due to chemical contamination of the two urban rivers. This multi-biomarker approach using mosquitofish can be applied to evaluate contamination of riverine environments.