Mortensen Anne S, Arukwe Augustine
Department of Biology, Norwegian University of Science and Technology (NTNU), Høgskoleringen 5, 7491 Trondheim, Norway.
Aquat Toxicol. 2006 Aug 12;79(1):99-103. doi: 10.1016/j.aquatox.2006.05.009. Epub 2006 Jun 3.
Dimethyl sulfoxide (DMSO) has been frequently used as carrier solvent in toxicological experiments where the most compelling DMSO attributes are its exceptionally low toxicity and environmental impact. We were inspired by recent and consistent observations that ethanol and DMSO modulate endocrine-disruptor biomarker responses in both in vitro and in vivo studies in our laboratory, to take a critical evaluation of these effects. Quantitative (real-time) polymerase chain reaction (PCR) method with specific primer pairs was used in this study to measure DMSO-induced time-dependent modulation of estrogen receptor (ER) isoforms, vitellogenin (Vtg) and zona radiata-protein (Zr-protein) gene expression patterns in primary culture of salmon hepatocytes. In addition, immunochemical analysis, using indirect enzyme linked immunosorbent assay (ELISA) with monoclonal (Vtg) and polyclonal (Zr-proteins) antibodies was used to detect and measure Vtg and Zr-proteins secreted in culture media. Salmon hepatocytes were isolated by a two-step collagenase perfusion method and exposed to 0.1% or 10 microL/L of DMSO after 48 h pre-culture. Cells were harvested at 12, 24, 48 and 72 h after exposure and analysed for ERalpha, ERbeta, Vtg and Zr-protein gene expression using real-time PCR method. Media samples were collected at similar time-intervals for protein analysis. Our data show that DMSO-induced significant increase in ERalpha, ERbeta, Vtg and Zr-protein genes in a time-dependent manner. Indirect ELISA analysis showed a time-specific effect of DMSO. The use of DMSO as carrier solvent in fish endocrine disruption studies should be re-evaluated. We recommend more investigation, using other endocrine-disruptor biomarkers in order to validate the suitability of common carrier solvents used in toxicology with the aim of setting new maximum allowable concentrations. In particular, given the high sensitivity of genomic approaches in toxicology, these results may have serious consequences for the interpretation of biomarker responses.
二甲基亚砜（DMSO）在毒理学实验中常被用作载体溶剂，其最突出的特性是毒性极低且对环境影响小。我们受到近期在实验室进行的体外和体内研究中乙醇和DMSO调节内分泌干扰物生物标志物反应的一致观察结果的启发，对这些影响进行了批判性评估。本研究采用定量（实时）聚合酶链反应（PCR）方法，使用特异性引物对来测量DMSO诱导的鲑鱼肝细胞原代培养物中雌激素受体（ER）亚型、卵黄蛋白原（Vtg）和放射带蛋白（Zr蛋白）基因表达模式的时间依赖性调节。此外，使用间接酶联免疫吸附测定（ELISA），结合单克隆（Vtg）和多克隆（Zr蛋白）抗体进行免疫化学分析，以检测和测量培养基中分泌的Vtg和Zr蛋白。通过两步胶原酶灌注法分离鲑鱼肝细胞，并在预培养48小时后暴露于0.1%或10微升/升的DMSO中。在暴露后12、24、48和72小时收获细胞，并使用实时PCR方法分析ERα、ERβ、Vtg和Zr蛋白基因表达。在相似的时间间隔收集培养基样本进行蛋白质分析。我们的数据表明，DMSO以时间依赖性方式显著增加ERα、ERβ、Vtg和Zr蛋白基因的表达。间接ELISA分析显示DMSO具有时间特异性效应。在鱼类内分泌干扰研究中使用DMSO作为载体溶剂应重新评估。我们建议进行更多研究，使用其他内分泌干扰物生物标志物，以验证毒理学中常用载体溶剂的适用性，目的是设定新的最大允许浓度。特别是，鉴于基因组方法在毒理学中的高敏感性，这些结果可能对生物标志物反应的解释产生严重影响。
