重金属暴露可以改变秀丽线虫的脂肪积累

1、重金属暴露可以改变秀丽线虫的脂肪积累         11-03-25 13:21:00     作者：武秋立,杜敏,王大勇    编辑：studa20【摘要】  目的:研究重金属暴露对秀丽线虫脂肪积累的急性中毒效应,鉴定重金属暴露秀丽线虫中发生表达模式改变的与脂肪酸代谢相关的基因。方法:银、镉、铬、铜、汞、锰和锌作为测试重金属,Nile Red荧光探针染色分析重金属暴露线虫肠道的脂肪酸含量变化,并测定重金属暴露线虫中脂肪酸代

2、谢相关基因的转录水平变化。结果:在所分析重金属中,高浓度银、铬和铜暴露可以显著诱导线虫肠道脂肪积累的增加,而高浓度镉暴露可以显著降低线虫肠道脂肪的积累。而且,与对照组相比,高浓度的银和铬暴露不明显影响基因pod2、gei7、lbp8、gpd35、fat6和fat7的表达水平显著升高,lbp1、acs2和ech1的表达水平显著降低。结论:高浓度银、铬、铜和镉暴露可以导致异常的秀丽线虫肠道的脂肪积累变化,其中高浓度银和铬暴露导致线虫脂肪积累增加的主要原因可能归因于暴露动物体内脂肪酸去饱和与线粒体氧化代谢途径以及脂肪酸结合能力的异常。 【关键词】  脂肪积累; 金属暴露; 代谢途径; Ni

3、le Red染色; 秀丽线虫Bioassays, usually used in monitoring scheme in toxicological studies, can be explored to monitor the acute toxicity of aquatic effluents according to the guideline set by regulatory authority. Physical and chemical methods can also be used for such monitoring by employing sophisticate

4、d equipment. However, considering the facts that field samples often contain unidentified components, and referencing of all toxicants may not be available, whole sample analysis with bioindicator animals was proposed by the U.S. Environment Protection Agency to circumvent such a limitation1. A free

5、living nematode, Caenorhabditis elegans, a nonparasitic bacterial feeder that lives in soil interstitial, satisfies all the criteria for bioindicator2. Because of its convenient handling in the laboratory, its sensitivity to different kinds of stresses, and its abundance in soil ecosystems, C. elega

6、ns is widely used in ecotoxicological studies using different exposure media, including soil and water3-4.The ability of an organism to regulate the production, storage, and release of energy is crucial for health and survival, and a major source of energy is stored as fat, which is required for the

7、 life cycle of organisms5. Fat storage regulation is a fundamental process, and abnormalities in fat storage will underline important human diseases6. The alarming worldwide increase in obesity has intensified the research to identify genes that control the differentiation and function of fatstoring

8、 tissues, and to examine environmental clues that induce the severe abnormalities in fat accumulation5. Previous studies have suggested that the lipid metabolism can be altered in acute and chronic lead(Pb) exposed humans and animals7, and cadmium(Cd) exposure can result in the impaired lipid storag

9、e in European eel8. Model organisms are a powerful resource for the discovery of genes and environmental clues critical to human health and disease. So far, it has been proven that the C. elegans is an excellent model to analyze the mechanisms of fat storage5. Worms have homologs of many mammalian l

10、ipogenic and lipolytic enzymes, and genes of encompassing a wide range of components of the mammalian fat regulation cascade5,9.Thus, the current literature regarding C. elegans provides insight concerning the relative sensitivity of the fat storage endpoint. In the present study, the toxicological

11、effects of metal exposure on fat storage were examined in nematodes. Silver(Ag), Cd, chromium(Cr), copper(Cu), mercury(Hg), manganese(Mn), and zinc(Zn) were chosen as test metals both because of the availability of toxicological data in the literature regarding nematodes and other organisms and beca

12、use of their abundance in the environment. The aims of the present study were to evaluate the acute toxicity of metal exposure on fat storage of nematodes, and to identify the sensitive genes required for the metabolism of fatty acid in metal exposed nematodes.1 Materials and methods1.1 ChemicalsThe

13、 metal concentrations used in this study were selected as previously described10-13. Three concentration levels of AgNO3, CdCl2, CrCl2, CuSO4, HgCl2, MnCl2, and ZnCl2 solutions were used in the current study, and they were 2.5, 75, and 200 mol·L-1, respectively. All the chemicals were obtained

14、from SigmaAldrich(St. Louis, MO, USA).1.2 StrainsAll nematodes used were wildtype Bristol strain N2, originally obtained from the Caenorhabditis Genetics Center(CGC). They were maintained on nematode growth medium(NGM) plates seeded with Escherichia coli OP50 at 20 as described14. Gravid nematodes w

15、ere washed off the plates into centrifuge tubes and were lysed with a bleaching mixture(0.45 mol·L-1 NaOH, 2% HOCl). Age synchronous populations of N2(L4larvae stage) were obtained by the collection as described15. The L4larvae stage nematodes were washed with doubledistilled water twice, follo

16、wed by washing with modified K medium once(50 mmol·L-1 NaCl, 30 mmol·L-1 KCl, 10 mmol·L-1 NaOAc, pH 5.5)16. Exposures were performed in 12well sterile tissue culture plates. All exposures were 24 h, and were carried out in 20 incubator in the presence of food.1.3 Nile Red stainingThe

17、method was performed as described17. Nile Red(5Hbenzophenoxazine5one, 9diethylamino) powder(N1142 Molecular Probes) was dissolved in acetone at 500 g·ml-1, diluted in phosphate buffered saline(PBS) and added on top of NGM plates already seeded with OP50 bacteria, to a final concentration of 0.0

18、5 g·ml-1. Nematodes were then grown on plates with Nile Red as eggs, and photographed with epifluorescence(rhodamine channel). Their staining phenotypes were assessed prior to starvation at the young adult stages. Fat content was monitored and evaluated using ImageJ Software(NIH Image) by deter

19、mining average intensity in each animals intestine. More than 30 nematodes were counted for the statistical analysis.1.4 Analysis of gene expression in metal exposed nematodesPoly(A)+RNA of nematodes was prepared and analyzed as described18-19. The frozen nematodes pellets were harvested from 1 lite

20、r of mixed stage liquid cultures. A total of 1 g of poly(A)+RNA per lane was separated by electrophoresis on a 1.2% agarose gel containing 2 mol·L-1 formaldehyde and 0.02 mol·L-1 MOPS, and transferred onto a nylon membrane by a mild alkaline transfer method. Northern hybridizations of tran

21、sferred poly(A)+RNA were performed using PCR product probes labeled with32PdCTP at 65 overnight. Genespecific primers were designed for gpd3(gpd3f, 5ATGACCAAGCCAAGTGTC3; gpd3r, 5TAGGCCTTGGTAGCAATG3), for W05G11.6(W05G11.6f, 5GAGTGTCGTTCTCTGCG3; W05G11.6r, 5CCTGGCCACAAGAACTTG3), for C03H5.4(C03H5.4f,

22、 5GCCAGAAACTCTGAATAC3; C03H5.4r, 5CTCAACATCTTCTTCTGC3), for C07E3.9(C07E3.9f, 5TTCTAGTGTTTCTAGCCG3; C07E3.9r, 5CAATGTTGGAAATGTTTC3), for acs2 (acs2f, 5CCTCGCTCTACACTCTCG3; acs2r, 5GACGACATGAGCATCAGC3), for ech1(ech1f, 5GATTCTTGACGTTGTCCG3; ech1r, 5ACAGATTCGTACACTCCG3), for gei7(gei7f, 5CTATTGCACACTG

23、AGCCA3; gei7r, 5GGCTCCAGAGTTCATAGC3), for fat5(fat5f, 5GAGATCCGACAAATGCAG3; fat5r, 5CGAACTTCTTGCACTGTC3), for fat6(fat6f, 5CTCTTCACTTCGCTGCAG3; fat6r, 5GGCCGATAATCTCATCAC3), for fat7(fat7f, 5GCATTGCCAAGAAGATTG3; fat7r, 5CAAGACCAAGAACAGCTG3), for pod2 (pod2f, 5GGAGCAGTTCATTCACTC3; pod2r, 5GTCGAGCACATCGTATTC3), for lbp1 (lbp1f, 5TCGCTCTTCTCCTGGTTC3; lbp1r, 5GTCGTTTGTAGAATCGGC3), and for lbp8 (lbp8f, 5ATGGTTTCCATGAAAGAG3; lbp8r, 5TCTAATTTTGAAAG