Tilton Fred, La Du Jane K, Vue Meng, Alzarban Noor, Tanguay Robert L
Department of Environmental and Molecular Toxicology, Environmental Health Sciences Center and the Marine and Freshwater Biomedical Sciences Center, Oregon State University, Corvallis, OR 97331, USA.
Toxicol Appl Pharmacol. 2006 Oct 1;216(1):55-68. doi: 10.1016/j.taap.2006.04.014. Epub 2006 May 16.
We previously determined that the dithiocarbamate pesticide sodium metam (NaM) and its active ingredient methylisothiocyanate (MITC) were developmentally toxic causing notochord distortions in the zebrafish. In this study, developing zebrafish were exposed to isothiocyanates (ITCs), dithiocarbamates (DTCs) and several degradation products to determine the teratogenic relationship of these chemical classes at the molecular level. All dithiocarbamates tested elicited notochord distortions with notochord NOELs from <4 to 40 ppb, while none of the ITCs caused notochord distortions with the exception of MITC. Carbon disulfide (CS(2)), a common DTC degradate, also caused distortions at concentrations >200 times the DTCs. Whole mount in situ hybridization of developmental markers for collagen (collagen2a1), muscle (myoD), and body axis formation (no tail) was perturbed well after cessation of treatment with pyrolidine-DTC (PDTC), dimethyl-DTC (DMDTC), NaM, MITC, and CS(2). Therefore, distinct albeit related chemical classes share a common toxic effect on zebrafish notochord development. To test the responsiveness of the distortion to metal perturbation, five metal chelators and 2 metals were studied. The membrane permeable copper chelator neocuproine (NCu) was found to cause notochord distortions similar to DTC-related molecules. DMDTC and NCu treated animals were protected with copper, and collagen 2a1 and no tail gene expression patterns were identical to controls in these animals. PDTC, NaM, MITC, and CS(2) were not responsive to copper indicating that the chelation of metals is not the primary means by which these molecules elicit their developmental toxicity. Embryos treated with DMDTC, NaM, and NCu were rescued by adding triciaine (MS-222) which abolishes the spontaneous muscle contractions that begin at 18 hpf. In these animals, only collagen 2a1 expression showed a similar pattern to the other notochord distorting molecules. This indicates that the perturbation of no tail expression is in response to the muscle contractions distorting the notochord, while collagen 2a1 is associated with the impact of these molecules on much earlier developmental processes.
我们之前已确定二硫代氨基甲酸盐农药威百亩钠（NaM）及其活性成分甲基异硫氰酸盐（MITC）具有发育毒性，可导致斑马鱼脊索畸变。在本研究中，将发育中的斑马鱼暴露于异硫氰酸盐（ITCs）、二硫代氨基甲酸盐（DTCs）及几种降解产物中，以在分子水平确定这些化学类别之间的致畸关系。所有测试的二硫代氨基甲酸盐均引发了脊索畸变，其脊索未观察到有害作用水平（NOELs）为<4至40 ppb，而除MITC外，没有一种ITC会导致脊索畸变。二硫化碳（CS₂）是一种常见的DTC降解产物，在浓度高于DTCs 200倍时也会导致畸变。在用吡咯烷 - DTC（PDTC）、二甲基 - DTC（DMDTC）、NaM、MITC和CS₂处理停止后很久，用于检测胶原蛋白（collagen2a1）、肌肉（myoD）和体轴形成（no tail）的发育标记物的全胚胎原位杂交仍受到干扰。因此，不同但相关的化学类别对斑马鱼脊索发育具有共同的毒性作用。为了测试畸变对金属干扰的反应性，研究了五种金属螯合剂和两种金属。发现可透过细胞膜的铜螯合剂新铜试剂（NCu）会导致与DTC相关分子类似的脊索畸变。用铜对DMDTC和NCu处理过的动物进行保护后，这些动物中胶原蛋白2a1和no tail基因的表达模式与对照相同。PDTC、NaM、MITC和CS₂对铜无反应，这表明金属螯合不是这些分子引发其发育毒性的主要方式。通过添加三卡因（MS - 222）可挽救用DMDTC、NaM和NCu处理过的胚胎，三卡因可消除在受精后18小时开始出现的自发肌肉收缩。在这些动物中，只有胶原蛋白2a1的表达显示出与其他导致脊索畸变的分子类似的模式。这表明no tail表达的干扰是对扭曲脊索的肌肉收缩的反应，而胶原蛋白2a1与这些分子对更早发育过程的影响有关。
