Department of Bioanalytical Ecotoxicology, UFZ - Helmholtz Centre for Environmental Research, 04318 Leipzig, Germany.
BMC Biol. 2013 Jun 17;11:69. doi: 10.1186/1741-7007-11-69.
In mammals, ABCB1 constitutes a cellular "first line of defense" against a wide array of chemicals and drugs conferring cellular multidrug or multixenobiotic resistance (MDR/MXR). We tested the hypothesis that an ABCB1 ortholog serves as protection for the sensitive developmental processes in zebrafish embryos against adverse compounds dissolved in the water.
Indication for ABCB1-type efflux counteracting the accumulation of chemicals in zebrafish embryos comes from experiments with fluorescent and toxic transporter substrates and inhibitors. With inhibitors present, levels of fluorescent dyes in embryo tissue and sensitivity of embryos to toxic substrates were generally elevated. We verified two predicted sequences from zebrafish, previously annotated as abcb1, by cloning; our synteny analyses, however, identified them as abcb4 and abcb5, respectively. The abcb1 gene is absent in the zebrafish genome and we explored whether instead Abcb4 and/or Abcb5 show toxicant defense properties. Quantitative real-time polymerase chain reaction (qPCR) analyses showed the presence of transcripts of both genes throughout the first 48 hours of zebrafish development. Similar to transporter inhibitors, morpholino knock-down of Abcb4 increased accumulation of fluorescent substrates in embryo tissue and sensitivity of embryos toward toxic compounds. In contrast, morpholino knock-down of Abcb5 did not exert this effect. ATPase assays with recombinant protein obtained with the baculovirus expression system confirmed that dye and toxic compounds act as substrates of zebrafish Abcb4 and inhibitors block its function. The compounds tested comprised model substrates of human ABCB1, namely the fluorescent dyes rhodamine B and calcein-am and the toxic compounds vinblastine, vincristine and doxorubicin; cyclosporin A, PSC833, MK571 and verapamil were applied as inhibitors. Additionally, tests were performed with ecotoxicologically relevant compounds: phenanthrene (a polycyclic aromatic hydrocarbon) and galaxolide and tonalide (two polycyclic musks).
We show that zebrafish Abcb4 is a cellular toxicant transporter and provides protection of embryos against toxic chemicals dissolved in the water. Zebrafish Abcb4 thus is functionally similar to mammalian ABCB1, but differs from mammalian ABCB4, which is not involved in cellular resistance to chemicals but specifically transports phospholipids in the liver. Our data have important implications: Abcb4 could affect bioavailability - and thus toxicologic and pharmacologic potency - of chemicals to zebrafish embryos and inhibition of Abcb4 therefore causes chemosensitization, that is, enhanced sensitivity of embryos to toxicants. These aspects should be considered in (eco)toxicologic and pharmacologic chemical screens with the zebrafish embryo, a major vertebrate model.
在哺乳动物中，ABCB1 构成了细胞抵御多种化学物质和药物的第一道防线，这些物质和药物使细胞具有多药或多外源性物质耐药性（MDR/MXR）。我们检验了这样一个假设，即 ABCB1 直系同源物在斑马鱼胚胎的敏感发育过程中充当保护者，使其免受水中溶解的有害物质的侵害。
用荧光和毒性转运体底物和抑制剂进行的实验表明，ABCB1 型外排泵可阻止化学物质在斑马鱼胚胎中的积累。有抑制剂存在时，胚胎组织中荧光染料的水平和胚胎对毒性底物的敏感性通常会升高。我们通过克隆验证了斑马鱼的两个先前被注释为 abcb1 的预测序列；然而，我们的基因座分析将它们分别鉴定为 abcb4 和 abcb5。abcb1 基因在斑马鱼基因组中缺失，我们探讨了 Abcb4 和/或 Abcb5 是否具有抗毒物的特性。实时定量聚合酶链反应（qPCR）分析显示，在斑马鱼发育的前 48 小时内，这两个基因的转录本都存在。与转运体抑制剂类似，Abcb4 的 morpholino 敲低增加了荧光底物在胚胎组织中的积累，并使胚胎对毒性化合物的敏感性增加。相比之下，Abcb5 的 morpholino 敲低没有产生这种效果。用杆状病毒表达系统获得的重组蛋白进行的 ATP 酶测定证实，染料和毒性化合物是斑马鱼 Abcb4 的底物，而抑制剂则阻断了其功能。测试的化合物包括人 ABCB1 的模型底物，即荧光染料罗丹明 B 和钙黄绿素 AM，以及毒性化合物长春新碱、长春碱和阿霉素；环孢菌素 A、PSC833、MK571 和维拉帕米被用作抑制剂。此外，还进行了与生态毒理学相关的化合物的测试：蒽（一种多环芳烃）以及麝香酮和酮麝香。
我们表明，斑马鱼 Abcb4 是一种细胞毒物转运体，为胚胎提供了对水中溶解的有毒化学物质的保护。因此，斑马鱼 Abcb4 在功能上类似于哺乳动物 ABCB1，但与不参与细胞对化学物质的耐药性但专门在肝脏中运输磷脂的哺乳动物 ABCB4 不同。我们的数据具有重要意义：Abcb4 可能会影响化学物质对斑马鱼胚胎的生物利用度-从而影响其毒理学和药理学效力-并且 Abcb4 的抑制会导致化学增敏，即胚胎对毒物的敏感性增强。在使用斑马鱼胚胎作为主要的脊椎动物模型进行（生态）毒理学和药理学化学筛选时，应考虑这些方面。
