Guo-Yong Huang

Expression and characterization analysis of type 2 metallothionein from grey mangrove species (Avicennia marina) in response to metal stress.

Metallothioneins (MTs) are a family of low-molecular-weight cysteine-rich proteins and are thought to play possible roles in metal metabolism or detoxification. To evaluate the roles of metallothioneins in metal homeostasis or tolerance in Avicennia marina, a real-time quantitative PCR protocol was developed to directly evaluate the expression of AmMT2 mRNA, when A. marina seedlings were exposed to different concentrations of zinc (Zn), copper (Cu) or lead (Pb) for 3 and 7d. Real-time quantitative PCR results indicated that the regulation of AmMT2 mRNA expression by Zn, Cu and Pb was strongly dependent on concentration and time of exposure. A significant increase in the transcripts of AmMT2 gene was also found in response to Zn, Cu and Pb, at least under some experimental conditions. When AmMT2 was overexpressed in Escherichia coli BL21 as a carboxy-terminal extension of glutathione-S-transferase (GST), the transgenic bacteria showed an increased tolerance to Zn, Cu, Pb and Cd exposure as compared to control strains. Moreover, GST-AmMT2 was purified from E. coli cells grown in the presence of 400 microM Zn, Cu, Pb or Cd. The purified GST-AmMT2 fusion protein could bind higher levels of all four metals than GST alone. Taken together, these data support the hypothesis that AmMT2 may be involved in processes of metal homeostasis or tolerance in A. marina.

Physiological and biochemical responses in the leaves of two mangrove plant seedlings (Kandelia candel and Bruguiera gymnorrhiza) exposed to multiple heavy metals

The accumulation of heavy metals and their effect on photosynthetic pigments, proline, glutathione (GSH) and phytochelatins (PCs-SH) were studied in the leaves of two mangrove plants seedlings (Kandelia candel and Bruguiera gymnorrhiza) grown for 30 days in the nutrient solution containing four different concentrations of Cd(2+), Pb(2+) and Hg(2+) (T(1), T(2), T(3) and T(4)). An increase in Cd, Pb and Hg content was found in the leaves of both species exposed to multiple heavy metal stress, whereas higher heavy metal levels (>T(1)) led to a remarkable breakdown of chlorophyll in the leaves of both species. The content of proline, GSH and PCs-SH in the leaves of both species exhibited a significant increase in response to heavy metal stress, at least under most of experimental conditions. Increased contents of proline, GSH and PCs-SH in metal-treated plants suggest that metal tolerance in both K. candel and B. gymnorrhiza might be associated to the efficiency of these antioxidants. Moreover, proline, GSH and PCs-SH in K. candel may play more important role in ameliorating the effect of heavy metal toxicity than those in B. gymnorrhiza.

Tissue-specific bioaccumulation of human and veterinary antibiotics in bile, plasma, liver and muscle tissues of wild fish from a highly urbanized region

We investigated the bioaccumulation of antibiotics in bile, plasma, liver and muscle tissues of wild fish from four rivers in the Pearl River Delta region. In total, 12 antibiotics were present in at least one type of fish tissues from nine wild fish species in the four rivers. The mean values of log bioaccumulation factors (log BAFs) for the detected antibiotics in fish bile, plasma, liver, and muscle tissues were at the range of 2.06-4.08, 1.85-3.47, 1.41-3.51, and 0.48-2.70, respectively. As the digestion tissues, fish bile, plasma, and liver showed strong bioaccumulation ability for some antibiotics, indicating a different bioaccumulation pattern from hydrophobic organic contaminants. Human health risk assessment based on potential fish consumption indicates that these antibiotics do not appear to pose an appreciable risk to human health. To the best of our knowledge, this is first report of bioaccumulation patterns of antibiotics in wild fish bile and plasma.

Expression analysis of type 2 metallothionein gene in mangrove species (Bruguiera gymnorrhiza) under heavy metal stress

In this paper, we aimed to assess the roles of metallothioneins (MTs) in heavy metal tolerance by analyzing the expression level of BgMT2 in leaves of Bruguiera gymnorrhiza in response to heavy metals. Eight-month-old B. gymnorrhiza seedlings were exposed to different concentrations of zinc (Zn), copper (Cu) or lead (Pb) for 1, 3 and 7 d. A Real-time quantitative PCR protocol was developed to directly evaluate the expression of BgMT2, using 18S rRNA as a reference gene. Real-time quantitative PCR analysis demonstrated BgMT2 mRNA expression was regulated by Zn, Cu and Pb, but the regulation pattern was different for the three metals tested. Significant increase in the transcript level of BgMT2 was also found in response to Zn, Cu and Pb in some experimental conditions. Our results confirm that BgMT2 gene is involved in the regulation of Zn, Cu and Pb in B. gymnorrhiza leaves.

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.

The effect of multiple heavy metals on ascorbate, glutathione and related enzymes in two mangrove plant seedlings [Kandelia candel and Bruguiera gymnorrhiza]

The effect of multiple heavy metals on ascorbate, glutathione and related enzymes in two mangrove plant seedlings (Kandelia candel and Bruguiera gymnorrhiza) In this study, the effect of multiple heavy metal stress on ascorbate (AsA), glutathione (GSH) and related enzymes was investigated in the leaves, stems and roots of Kandelia candel and Bruguiera gymnorrhiza. Mangrove seedlings were treated with five different concentrations of a heavy metal mixture (Cd2+, Pb2+ and Hg2+). Antioxidants in both the species were analyzed after one month. AsA, GSH, ascorbate peroxidase (APX, EC 1.11.1.11) and glutathione reductase (GR, EC 1.6.4.2) exhibited a similar trend with initial increase and subsequent decrease in response to heavy metal stress. At the highest metal concentration, a significant decrease of AsA and GR was observed in K. candel and B. gymnorrhiza. Glutathione peroxidase (GPX, EC 1.11.1.9) in the leaves, stems and roots of K. candel reached their respective maximal values at the highest metal concentration, whereas GPX activity in roots and stems of B. gymnorrhiza was similar to the controls at higher metal concentrations. Our results demonstrate that AsA, GSH, APX, GR and GPX in K. candel may play more important roles in defending against reactive oxygen species (ROS) than those in B. gymnorrhiza.

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.

Contamination profiles of perfluoroalkyl substances in five typical rivers of the Pearl River Delta region, South China

A survey on contamination profiles of eighteen perfluoroalkyl substances (PFASs) was performed via high performance liquid chromatography-tandem mass spectrometry for surface water and sediments from five typical rivers of the Pearl River Delta region, South China in summer and winter in 2012. The total concentrations of the PFASs in the water phase of the five rivers ranged from 0.14 to 346.72 ng L(-1). The PFAS concentrations in the water phase were correlated positively to some selected water quality parameters such as chemical oxygen demand (COD) (0.7913) and conductivity (0.5642). The monitoring results for the water samples showed significant seasonal variations, while those for the sediment samples showed no obvious seasonal variations. Among the selected 18 PFASs, perfluorooctane sulfonic acid (PFOS) was the dominant PFAS compound both in water and sediment for two seasons with its maximum concentration of 320.5 ng L(-1) in water and 11.4 ng g(-1) dry weight (dw) in sediment, followed by perfluorooctanoic acid (PFOA) with its maximum concentration of 26.48 ng L(-1) in water and 0.99 ng g(-1) dw in sediment. PFOS and PFOA were found at relatively higher concentrations in the Shima River and Danshui River than in the other three rivers (Xizhijiang River, Dongjiang River and Shahe River). The principal component analysis for the PFASs concentrations in water and sediment separated the sampling sites into two groups: rural and agricultural area, and urban and industrial area, suggesting the PFASs in the riverine environment were mainly originated from industrial and urban activities in the region.

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 (0 ng L(-1) , 5 ng L(-1) , 50 ng L(-1) , and 100 ng L(-1) ) for 144 h 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 5 ng L(-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 5 ng L(-1) at different time points. The transcriptional expression levels of Esr1, Ar, Star, Hsd17b3, Fshb, and Pomc were also mediated by 5 ng L(-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.

Regulation of reproduction- and biomarker-related gene expression by sex steroids in the livers and ovaries of adult female western mosquitofish (Gambusia affinis)

To assess the adverse toxicological effects of steroid hormones on western mosquitofish (Gambusia affinis), 180 adult females were exposed to individual or binary combinations of progesterone (1μg/L), testosterone (1μg/L) and 17β-estradiol (1μg/L) for eight days. The expression patterns of vitellogenin, estrogen receptor, androgen receptor, metallothionein, and cytochrome P450 1A genes in mosquitofish varied according to tissue as well as the specificity of steroids. Treatment by progesterone or testosterone alone inhibited target gene expression in the livers. The expression levels of both vitellogenin A and vitellogenin B mRNAs were up-regulated by17β-estradiol, and a parallel induction of estrogen receptor α mRNA expression was also observed in the livers. In addition, 17β-estradiol treatment alone suppressed androgen receptor α, metallothionein and cytochrome P450 1A mRNA expression in the livers. In general, multiple hormone treatments had different effects on target gene expression compared with corresponding hormone alone. The results demonstrate that steroid hormones cause multiple biological responses including the expression of vitellogenin, estrogen receptor and androgen receptor mRNA in the hormone signaling pathways and the expression of metallothionein and cytochrome P450 1A mRNA in the xenobiotic signaling pathway.