Department of Aquaculture, Szent István University, Páter Károly u. 1., Gödöllő, H-2100, Hungary.
Environ Sci Pollut Res Int. 2018 May;25(15):14426-14436. doi: 10.1007/s11356-018-1613-2. Epub 2018 Mar 10.
The effect of seven heavy metals on the motility parameter of zebrafish sperm was tested in order to develop an in vitro toxicological test system as an alternative to live animal testing. In vitro test systems are currently preferred in ecotoxicology due to their practical and ethical advantages and fish sperm can be a suitable model. A number of studies had been carried out previously on this topic, but the described methods had not been standardized in numerous aspects (donor species, measured endpoint, etc.). In this study, heavy metals (mercury, arsenic, chromium, zinc, nickel, copper, cadmium) were used as reference toxicants with known toxicity to develop a standardized fish sperm in vitro assay. The tested concentrations were determined based on preliminary range finding tests. The endpoints were progressive motility (PMOT, %), curvilinear velocity (VCL, μm/s), and linearity (LIN, %) measured by a computer-assisted sperm analysis (CASA) system. According to our results, PMOT was the most sensitive of the three investigated parameters: dose-response curves were observed for each metal at relatively low concentrations. VCL values were less sensitive: higher concentrations were needed to observe changes. Of the three parameters, LIN was the least affected: dose-response relationship was observed only in the case of mercury (e.g., lowest observed effect concentration (LOEC) of Hg at 120 min: 1 mg/L for PMOT, 2.5 mg/L for VCL, 5 mg/L for LIN; LOEC of Cu at 120 min: 1 mg/L for PMOT, 5 mg/L for VCL, any for LIN). The order of toxicity as determined by PMOT was as follows: Hg > As > Cd > Cu > Zn > Cr > Ni. In conclusion, we found that PMOT of zebrafish sperm was an accurate and fast bioindicator of heavy metal load. Sperm analysis can be adopted to estimate the possible toxic effects of various chemicals in vitro. Future investigations should concentrate on the applicability of this assay to other contaminants (e.g., organic pollutants).
为了开发一种替代活体动物测试的体外毒理学测试系统，我们测试了七种重金属对斑马鱼精子运动参数的影响。由于其实用性和伦理优势，体外测试系统目前在生态毒理学中受到青睐，而鱼类精子可以作为一种合适的模型。此前已经有许多关于这个主题的研究，但在许多方面（供体物种、测量终点等），所描述的方法尚未标准化。在这项研究中，我们使用汞、砷、铬、锌、镍、铜、镉等重金属作为参考毒物，开发了一种标准化的鱼类精子体外分析。测试浓度是根据初步的范围发现测试确定的。终点是通过计算机辅助精子分析（CASA）系统测量的直线运动（PMOT，%）、曲线速度（VCL，μm/s）和直线性（LIN，%）。根据我们的结果，PMOT 是三个调查参数中最敏感的：在相对较低的浓度下，观察到每种金属的剂量-反应曲线。VCL 值不太敏感：需要更高的浓度才能观察到变化。在这三个参数中，LIN 受影响最小：仅在汞的情况下观察到剂量-反应关系（例如，PMOT 的最低观察到的效应浓度（LOEC）为 120 分钟时的 1 毫克/升，VCL 为 2.5 毫克/升，LIN 为 5 毫克/升；Hg 的 120 分钟时的 LOEC 为 1 毫克/升，VCL 为 5 毫克/升，LIN 为任何浓度）。PMOT 确定的毒性顺序如下：Hg>As>Cd>Cu>Zn>Cr>Ni。总之，我们发现斑马鱼精子的 PMOT 是重金属负荷的一种准确、快速的生物指标。精子分析可用于体外估计各种化学物质的可能毒性影响。未来的研究应集中在该测定法对其他污染物（如有机污染物）的适用性上。
