Lead toxicity in Hermissenda crassicornis embryos and cultured neurons.
Lead toxicity in Hermissenda crassicornis embryos and cultured neurons.
复制标题
Hermissenda crassicornis 胚胎和培养神经元中的铅毒性。
DOI：
10.1086/bblv187n2p251
复制
发表时间：
1994
期刊：
影响因子：
--
通讯作者：
Kuzirian,AM
中科院分区：
文献类型：
--
作者：
Tamse,CT;Smith,PJ;Aloulou,A;Epstein,HT;Kuzirian,AM
铅中毒可导致哺乳动物和其他脊椎动物出现形态异常、行为缺陷和学习障碍（1，2）。这项研究旨在利用Hermissenda作为重金属毒性的非哺乳动物生物医学和毒理学研究模型，特别是铅，及其对这种海洋软体动物广泛研究的学习和行为模式的影响（3）。我们报告了铅如何影响Hermissenda胚胎发育和培养神经元生长的基线研究的第一个结果。从Hermissenda Resource Facility，Marine Biological Laboratory（MBL），伍兹霍尔，马萨诸塞州收集新产的卵块，并进行铅的静态慢性毒性试验。将卵块切成5-mm股，并单独分配到含有天然海水的50-ml锥形管中。每条链中的平均胚胎数为1000个。采用三种海水方案：不含EDTA的Lillie建筑物环境海水（LSW）; 74 pA 4）EDTA（LESW），其常规用于Hermissenda培养;和环境海洋资源中心，MBL海水（MSW）。使用以下总醋酸铅（PbAc）浓度：0.05、0.1、0.5、1.0、5.0、7.0、10.0 mg/l（分别为0.09、0.18、0.9、1.8、9、12.7、18 mg/l），加上阴性对照（不添加铅或EDTA）和阳性对照（不添加铅但添加EDTA）。重复每个试验浓度，并随机分配。将试管在12 ℃下孵育，并在铅暴露6天后记录每次处理50只动物的胚胎发育。对于长期体外神经元暴露于PbAc，遵循为Hermissenda开发的细胞培养技术（4）。将Hermissenda的中枢神经系统（CNS）解剖，在12 ℃下在蛋白酶中消化，孵育过夜，并用1.8%牛血清白蛋白的人工海水（ASW）洗涤两次，然后进行最后的ASW洗涤。将CNS切割成四个区域：左和右胸膜神经节和左和右足神经节。然后将神经元细胞解离，转移到含有3 ml补充有无机盐的Leibovitz L-15培养基（5）的聚赖氨酸包被的培养皿（35 × 10 mm）中。然后将培养皿置于12 ℃培养箱中，直到24小时后检查生长形态，此后每天检查。将培养7-14天的神经元暴露于PbAc，所述神经元具有良好的神经炎形态和稳定的生长。将1 M PbAc储备溶液直接加入L-15培养基中，以获得0.5、2.0和4.0 μ g/ml的总浓度（分别为0.9、3.6、7.2 μ l）。铅暴露的神经元密切观察前2-4小时的形态学变化，此后每24小时。细胞在L-15培养基-PbAc溶液中孵育3-10天，慢性毒性试验清楚地表明，Hermissenda胚胎对铅敏感，必须常规地向LSW中添加EDTA以使其正常和同步发育。这意味着从Lilhe大楼向海水中加入少量EDTA（0.74@）足以螯合海水中存在的未知重金属。在获得更多关于LSW中明显污染物的信息之前，将使用MSW进行涉及铅研究的未来测试。观察到的毒性效应与铅浓度的增加成正比。随着铅浓度的增加（0.5-1.0毫克/升），胚胎发育的时间进程延长，并以早期发育阶段为主。在较高的浓度（5.0-10.0毫克/升）加入到环境LSW，发展被逮捕的桑椹胚。
Morphological abnormalities, behavioral deficits, and learning disabilities can be induced in mammals and other vertebrates by lead poisoning (1, 2). This study seeks to exploit Hermissenda as a non-mammalian biomedical and toxicological research model of heavy metal toxicity, particularly lead, and its effects on the extensively studied learning and behavioral patterns of this marine mollusc (3). We report the first results of a baseline study on how lead affects the development of Hermissenda embryos and the growth of cultured neurons. Newly laid egg masses were collected from the Hermissenda Resource Facility, Marine Biological Laboratory (MBL), Woods Hole, Massachusetts, and processed for static, chronic toxicity tests for lead. The egg mass was cut into 5-mm strands and distributed individually into 50-ml conical tubes containing natural seawater. The average number of embryos in each strand was 1000. Three seawater regimes were used: ambient Lillie building seawater without EDTA (LSW); Lillie seawater with 0.25 mg/l (O. 74 pA4) EDTA (LESW), which we routinely use for Hermissenda culture; and ambient Marine Resources Center, MBL seawater (MSW). The following total lead acetate (PbAc) concentrations were used: 0.05, 0.1, 0.5, 1.0, 5.0, 7.0, 10.0 mg/1 (0.09, 0.18, 0.9, 1.8, 9, 12.7, 18 &f, respectively), plus negative (no lead or EDTA added) and positive (no lead but with EDTA added) controls. Each test concentration was replicated, and the distribution was randomized. The tubes were incubated at 12 C and the embryonic development of 50 animals per treatment was recorded after 6 days lead exposure. For the chronic in vitro neuronal exposure to PbAc, cell culture techniques developed for Hermissenda were followed (4). The central nervous system (CNS) of Hermissenda was dissected, digested in protease at 12” C, incubated overnight, and washed twice with 1.8% bovine serum albumin in artificial seawater (ASW), followed by a final ASW wash. The CNS was cut into four regions: left and right cerebropleural ganglia and left and right pedal ganglia. Neuronal cells were then dissociated, transferred to a poly+ lysine-coated culture dish (35 X 10 mm) containing 3 ml of Leibovitz L-15 medium (5) supplemented with inorganic salts. Dishes were then left in a 12 C incubator until examined 24 h later, and daily thereafter, for growth morphology. Neurons 7-14 days into culture, with well-established neuritic morphology and stable growth, were exposed to PbAc. A 1 M stock solution of PbAc was added directly to the L-15 medium to obtain total concentrations of 0.5, 2.0, and 4.0 CLg/ml (0.9, 3.6, 7.2 fl, respectively). Lead-exposed neurons were observed closely for the first 2-4 h for morphologic changes, and every 24 h thereafter. Cells were incubated in the L-15 medium-PbAc solution for 3-10 days.The chronic toxicity tests clearly indicate that Hermissenda embryos are sensitive to lead, and that EDTA must be added routinely to LSW for their normal and synchronous development. This implies that the small amount of EDTA (0.74@) added to seawater from the Lilhe building was sufficient to chelate an unknown heavy metal present in the seawater. Future tests involving lead studies will be conducted using MSW until more information is available about the apparent contaminant in LSW. Toxicity effects observed were directly proportional to increasing concentrations of lead. The time course for embryonic development was lengthened and dominated by earlier developmental stages as the lead concentrations increased (0.5-1.0 mg/l). At higher concentrations (5.0-10.0 mg/l) added to ambient LSW, development was arrested at the morula …