环境毒理.docx

1、环境毒理Thyroid endocrine disrupting activity of pentachlorophenol: an in vitro and in vivo assaAbstractThe objective of the present study was to evaluate thyroid endocrine disrupting activity of pentachlorophenol (PCP) by using an in vitro and in vivo assay. In the in vitro assay, rat pituitary GH3 cel

2、ls were exposed to 0, 0.1, 0.3, and 1.0 M PCP. We selected deiodinase 1 (Dio1) gene transcription as the sensitive and specific thyroid hormone-responsive gene to indicate thyroid endocrine disruption. The results showed that PCP exposure alone could significantly down-regulated Dio 1 gene transcrip

3、tions and thus has the potential thyroid endocrine disrupting activity; while co-exposure PCP with physiological levels of triiodothyronine (T3), the Dio 1 gene transcriptions induced by T3 were markedly down-regulated by PCP, indicating antagnostic activity of PCP in vitro. In the in vivo assay, ze

4、brafish embryos were exposed to 0, 1, 3, and 10 g/L of PCP until 14 days post-fertilization (dpf). PCP exposure resulted in decreased of thyroxine (T4) levels, but elevated contents of whole-body T3. Gene transcriptions were further measured in the hypothalamic-pituitary-thyroid (HPT) axis. PCP expo

5、sure significantly up-regulated gene transcriptions, including thyroid-stimulating hormone (TSH), sodium/iodide symporter (NIS), thyroglobulin (TG), Dio1 and Dio2, thyroid hormone receptors (TR and TR), and uridinediphosphate-glucuronosyl-transferase (UGT1ab). PCP exposure did not influence the gene

6、 transcription of transthyretin (TTR). The results indicate that PCP has the potential as thyroid endocrine disruptor both in vitro and in vivo and further confirmed the reliability of the developing zebrafish larvae as models for assessment of the thyroid endocrine disruption of chemicals. Keywords

7、: Pentachlorophenol; T-Screen assay; Zebrafish larvae; Thyroid endocrine disruption; Hypothalamic-pituitary-thyroid (HPT) axis 1. IntroductionPentachlorophenol (PCP) was mainly used as pesticide and wood preservative worldwide. Due to its widespread use for these purposes, PCP has become a ubiquitou

8、s environmental contaminant (reviewed by Zheng et al., 2012). Although some countries have banned or control the use of PCP, it is still used as a wood preservative, and detected in the aquatic environment, wild animals and human samples (Farhadi et al., 2009; Zheng et al., 2011; Zheng et al., 2012)

9、. China has restricted to use PCP in 1997, however, PCP was used as a molluscide to control the re-emergence of schistosomiasis in some areas (Tan and Zhang, 2008). Because of the large amount of use of PCP, there is widespread environmental contamination. PCP has been detected in surface waters in

10、China, up to micromolar (g/L) concentrations (Zheng et al., 2000; Gao et al., 2008; Han et al., 2009). These data indicated that PCP usage induced obvious PCP pollution in the local water environment and may cause adverse effects on aquatic organisms and public health. The impact of environmental ch

11、emicals on thyroid endocrine system have received great attention in recent years, especially because thyroid hormones (THs) are of special importance in fetal development, as development of the brain is dependent on normal levels of thyroid hormones (reviewed by Boas et al., 2012). Endocrine disrup

12、ting chemicals can have a direct impact on thyroid hormone (TH) synthesis, transport, binding, catabolism and clearance of circulating THs (Kloas and Lutz, 2006). Limited information showed that PCP may have potential to disrupt thyroid endocrine functions. For example, PCP has 3,3,5-triiodothyronin

13、e (T3)-antagonist activity in vitro and in vivo assays in Xenopus laevis (Sugiyama et al., 2005). Developmental exposure of PCP to rats caused lower total thyroxine (T4) concentrations in plasma (Kawaguchi et al., 2008). In humans, the negative association between maternal plasma PCP levels and cord

14、 plasma free thyroxine (fT4) concentrations in neonates was reported (Dallaire et al., 2009). Given the important roles of THs in the growth and development of fetuses, the potential thyroid endocrine disruption of PCP has raised great concern over its adverse environmental health risks. In the eval

15、uating TH endocrine disruption, an in vitro model using the rat pituitary tumor cell line GH3 (T-Screen) has been developed. GH3 cells can synthesize and secret growth hormone (GH) and prolactin (PRL) and T3 can induce the two hormones production as well as gene transcriptions (Spindler et al., 1982

16、; Stanley, 1988). This T-Screen is based on the T3 dependent cell growth, mediated by specific, high-affinity thyroid receptor (TR), binding THs to thyroid hormone responsive elements (TREs) and ultimately leading to gene expression. Thus the T-Screen assay can be used for in vitro detection of agon

17、istic and antagonistic properties of compounds at the level of the TR in the absence and in the presence of T3 (Gutleb et al., 2005; Schriks et al., 2006). In addition, gene transcriptions can be measured based on modulation of basal GH and PRL secretion in GH3 cells is related to the regulation of

18、GH and PRL gene transcription (Tamura et al., 2000). Furthermore, GH3 cells possess both deiodinase 1 (Dio 1) and 2 (Dio 2) activity (St. Germain, 1985; Mori et al., 2007), and are responsive to T3 (Bauer et al., 1997). Both the deiodinases are important role in maintaining local T3 levels in the br

19、ain and pituitary in vivo. T-Screen assay has proven to successfully predict the effects of some thyroid hormone disrupting chemicals, such as plasticizers, alkylphenols, pesticides, polychlorinated biphenyls (PCBs), and brominated flame-retardants, personal care products (Ghisari and Bonefeld-Jorge

20、nsen, 2005; Hamers et al., 2006; Schriks et al., 2006; Hansen et al., 2009; Taxvig et al., 2011; Hinther et al., 2011). In fish, the thyroid endocrine system is controlled primarily by the hypothalamicpituitarythyroid (HPT) axis, which is responsible for regulating thyroid hormone dynamics by coordi

21、nating their synthesis, secretion, transport and metabolism (reviewed by Carr and Patio, 2011). Recently, an in vivo model for testing endocrine disruption of THs was developed in developing zebrafish larvae (Yu et al., 2010). This assay indicated that the HPT axis can be evaluated to determine thyr

22、oid endocrine disruption of TH endocrine disruptors, e.g., PBDEs and also to some extent on potential mechanisms of action (Yu et al., 2010; Chen et al., 2012). However, the thyroid endocrine disruption of PCP and environmental risk in fish is not well-known. The objectives of the present study were

23、 to investigate the thyroid endocrine disrupting activity of PCP by means of in vitro and in vivo assay. In the in vitro study, the TH-endocrine disrupting activity was assessed using the T-Screen assay. TH-responsive gene transcriptions were examined upon PCP exposure; We further examined the impac

24、t of PCP on THs levels and gene transcriptions in the HPT axis and the potential mechanisms of thyroid endocrine disruption in the developing zebrafish larvae.2. Materials and methods2.1. ChemicalsPentachlorophenol (PCP) (99%, CAS No. 87-86-5) was purchased from Sigma-Aldrich (St. Louis, MO, USA). I

25、t was dissolved in dimethyl sulfoxide (DMSO), and stored at 4 C. 4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT) was purchased from Duchefa (the Netherlands). All other chemicals used in the present study were of analytical grade. 2.2. Cell cultureThe rat pituitary GH3 cells were obta

26、ined from the Cell Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (Beijing, China). The cells were cultured in phenol-red freeDMEM/F12 medium (Sigma, St. Louis, MO, USA), supplemented with 10% fetal calf serum (FCS, Gibco), 2.5% 100 U/mL penicillin, and 100 g/

27、mL of streptomycin. The cells were maintained at 37C in an atmosphere of 5% CO2. 2.3. Cell viability assay and chemicals exposureCell viability was measured by the colorimetric monitoring conversion of MTT to formazan. Briey, the GH3 cells were seeded into 96-well culture plates (Falcon, Franklin La

28、kes, NJ, USA) at a density of 5 104 cells/well. After 24 h of culture, the cultured medium was removed and serum-starved culture medium was added for another 24 h. PCP was diluted with serum-free culture medium (0, 0.1, 0.3, and 1.0 M) and was added to the wells for 44 h. Then a 20 L of MTT solution

29、 (5 mg/mL MTT in PBS) was added to each well and the plates were incubated for additional 4 h. The culture medium was removed and 150 l of DMSO added to each well. After incubation for another 20 min, the extent to which the MTT had reduced to a formazan product was determined using a microplate rea

30、der (M2, Molecular Device, Union City, CA, USA) at 490 nm. The cell viability was expressed as a percentage of the cell survival rate compared with the control (Yu et al., 2008). There were six replicates and each treatment contained a blank and solvent control (0.1% DMSO). For thyroid endocrine dis

31、ruption assay, the GH3 cells with a density of 2 105 per well were seeded into 12-well plates for 24 h. Then the culture medium was removed and the cells were rinsed with serum-free medium. The cells were then exposed to PCP (0.1, 0.3, and 1.0 M) in serum- and phenol red-free DMEM/F12 for 48 h. Ther

32、e were three replicate for each treatment and control. 2.4. Zebrash maintenance and embryo exposureAdult zebrash (Danio rerio) (AB strain) maintenance and embryo exposure to PCP were carried out following the method described previously (Yu et al., 2010). Briey, embryos that developed normally and r

33、eached the blastula stage (2 hours post-fertilization, hpf) were selected for experiments. Approximately 400 embryos were randomly distributed into glass beakers containing 500 mL of PCP solution (0, 1, 3, and 10 g/L or equal to 0.0037, 0.011, 0.033 M). There were 3 replicates in each exposure group and control group for gene