Mager Edward M, Wintz Henri, Vulpe Chris D, Brix Kevin V, Grosell Martin
Division of Marine Biology and Fisheries, University of Miami, Rosenstiel School of Marine and Atmospheric Science, Miami, FL 33149, USA.
Aquat Toxicol. 2008 May 1;87(3):200-9. doi: 10.1016/j.aquatox.2008.02.001. Epub 2008 Feb 12.
Establishment of water quality criteria (WQC), intended to protect aquatic life, continues to rely principally on water hardness (i.e. Ca(2+)) for lead (Pb) despite growing evidence that other chemical parameters also strongly influence toxicity. To more clearly define the water chemistry parameters mediating Pb toxicity, we evaluated the effects of hardness as CaSO(4) and dissolved organic carbon (DOC) as humic acid during chronic (150 days) exposures to the fathead minnow. Measured Pb concentrations ranged from 157+/-5 nM (33+/-1 microg/L) Pb in base water to 177+/-7 (37+/-1 microg/L) and 187+/-7 nM (39+/-1 microg/L) Pb in CaSO(4)- or HA-supplemented water, respectively. Fish were collected at 2, 4, 10, 30, 63, 90 and 150 days of exposure. Traditional toxicological endpoints were examined alongside gene transcription analyses to help clarify the underlying mechanisms of Pb toxicity and to identify candidate molecular markers that might ultimately serve as robust indicators of exposure and effect. Addition of CaSO(4) did not prevent whole body Pb accumulation whereas DOC afforded strong protection (about half the amount accumulated by fish in base water) suggesting that current, hardness-based WQC are likely inaccurate for predicting chronic Pb effects in aquatic systems. Custom-made microarrays were co-hybridized with base water samples+/-Pb up to the 30 days time point. Quantitative PCR was employed to verify gene transcription responses and to extend analysis to the CaSO(4) and HA treatments and the 150 days time point. Identification of four genes by microarray analysis revealed clear Pb-induced responses over time: glucose-6-phosphate dehydrogenase, glutathione-S-transferase, ferritin and beta-globin. Results obtained by qPCR were in strong agreement with microarray data by regression analysis (r(2)=0.82, slope=1.28). The associated pathways implicated herein for these genes provide further evidence supporting roles for anemia and neurological disorders in chronic Pb toxicity. Effects of water chemistry on Pb accumulation and gene transcription responses were in close parallel, though alterations in ionoregulatory and morphological endpoints were not observed. Whereas DOC was protective against Pb accumulation and mRNA expression changes, Ca(2+) was not. Additionally, several hypothesis-driven genes (ECaC, DMT-1, and ALA-D) were examined by qPCR but revealed either no change or small Pb-induced responses lacking any clear influence attributable to water chemistry. These findings should help pave the way toward development of a new chronic Pb BLM and a Pb-responsive gene transcript profile for fathead minnows, both of which would greatly aid future environmental monitoring and regulatory strategies for Pb.
旨在保护水生生物的水质标准（WQC）的制定，尽管越来越多的证据表明其他化学参数也对毒性有强烈影响，但目前在铅（Pb）方面仍主要依赖水硬度（即Ca(2+)）。为了更清楚地界定介导铅毒性的水化学参数，我们在对黑头呆鱼进行慢性（150天）暴露期间，评估了以CaSO(4)形式存在的硬度和以腐殖酸形式存在的溶解有机碳（DOC）的影响。测得的铅浓度范围从基础水中的157±5 nM（33±1微克/升）铅，到添加CaSO(4)或腐殖酸的水中分别为177±7（37±1微克/升）和187±7 nM（39±1微克/升）铅。在暴露的第2、4、10、30、63、90和150天收集鱼类。除了基因转录分析外，还检测了传统的毒理学终点，以帮助阐明铅毒性的潜在机制，并识别可能最终作为暴露和效应可靠指标的候选分子标记。添加CaSO(4)并不能阻止全身铅的积累，而DOC则提供了强有力的保护（约为基础水中鱼类积累量的一半），这表明当前基于硬度的WQC可能无法准确预测水生系统中铅的慢性影响。定制的微阵列与基础水样±铅共同杂交，直至30天时间点。采用定量PCR来验证基因转录反应，并将分析扩展到CaSO(4)和腐殖酸处理以及150天时间点。通过微阵列分析鉴定出四个基因，揭示了随着时间推移铅诱导的明显反应：葡萄糖-6-磷酸脱氢酶、谷胱甘肽-S-转移酶、铁蛋白和β-珠蛋白。通过定量PCR获得的结果与微阵列数据经回归分析后高度一致（r(2)=0.82，斜率=1.28）。本文中涉及这些基因的相关途径为贫血和神经紊乱在慢性铅毒性中的作用提供了进一步证据。水化学对铅积累和基因转录反应的影响密切平行，尽管未观察到离子调节和形态学终点的变化。虽然DOC对铅积累和mRNA表达变化具有保护作用，但Ca(2+)则不然。此外，通过定量PCR检测了几个基于假设的基因（ECaC、DMT-1和ALA-D），但结果显示要么没有变化，要么铅诱导的反应很小，且没有任何明显的水化学影响。这些发现应该有助于为开发新的慢性铅生物配体模型和黑头呆鱼的铅反应基因转录谱铺平道路，这两者都将极大地有助于未来对铅的环境监测和监管策略。
