Long-term exposure of Mytilus galloprovincialis to Diclofenac, Ibuprofen and Ketoprofen: insights into bioavailbility, biomarkers and transcriptomic changes

Pharmaceutical compounds are emerging contaminants in aquatic environment due to their massive use (human and veterinary medicines, agriculture and aquaculture) and a limited removal by waste water treatment plants (WWTPs). In this work, a representative determination of ecotoxicological potential of Ketoprofen s was studied in the sensitive bioindicator marine organism M. Galloprovincialis. Mussels were exposed, under regulated laboratory conditions, to Ketoprofen (KET) a, dosed alone at the realistic environmental concentration of 2.5µg/L for 14, 30 and 60 days. Gene expression analyses of Mytilus galloprovincialis exposed to KET have been performed trough a DNA microarray platform. Mussels Mytilus galloprovincialis (5 ± 1 cm shell length) were obtained from a local farm (Numana, Ancona) and acclimatized for 10 days to laboratory conditions with aerated seawater, at 18 ± 1 °C, 37 ? salinity, pH 7.5 ± 0.5 and oxygen saturation >94%. Mussels were distributed into three 17 L aquarium and exposed at 2.5µg/L to ketoprofen (KET) for 14, 30 and 60 days . All treatments were compared to control (CTRL) containing 0.00001% of methanol. Water was changed every other day and concentration of molecules were restored. Gene transcription analyses of 4 digestive glands pools (four pools for each treatment composed by 3 digestive glands; CTRL, KET) were performed using a 8X60K Agilent oligo-DNA microarray platform GPL18667. Microarrays were synthesized in situ using the Agilent non-contact ink-jet technology including default positive and negative controls. Total RNA was isolated using Extract-all (Eurobio) procedure. RNA quality and integrity was controlled on the Agilent bioanalyzer using RNA nanochips and Agilent RNA 6000 nanoreagents (Agilent Technologies, Waldbronn, Germany). RNA concentrations were measured at 260 nm using a ND-1000 spectrophotometer (Nanodrop Technologies) using the conversion factor 1 OD = 40 mg/mL total RNA. Samples were stored at -80°C until further use. Gene expression profiling was performed using an Mytilus galloprovincialis oligo-DNA microarray of 59,971 probes based on single-colour detection (Cyanine-3 only). Microarrays were scanned with Agilent scanner G2565BA at a resolution of 2 microns; all slides were scanned twice at two different sensitivity settings (XDRHi 100% and XDRLo 10%); the scanner software created a unique ID for each pair of XDR scans and saved it to both scan image files. FeatureExtraction v10.7.3.1 used XDR ID to link the pairs of scans together automatically when extracting data. The signal left after all the FE processing steps have been completed is ProcessedSignal that contains the Multiplicatively Detrended, Background-Subtracted Signal.

药物化合物因被大量使用（涵盖人类用药、兽药、农业及水产养殖用药）且污水处理厂（waste water treatment plants, WWTPs）对其去除效率有限，已成为水生环境中的新型污染物。本研究针对酮洛芬（Ketoprofen）的生态毒理潜力开展代表性测定，选用敏感海洋生物指示物种——盖贻贝（Mytilus galloprovincialis，常缩写为M. galloprovincialis）作为实验对象。实验在标准化实验室条件下开展，将贻贝暴露于单独给药的酮洛芬（KET）环境中，给药浓度为符合实际环境水平的2.5μg/L，暴露时长分别为14天、30天与60天。本研究通过DNA微阵列（DNA microarray）平台对暴露于酮洛芬的盖贻贝进行了基因表达分析。实验所用的盖贻贝（Mytilus galloprovincialis）壳长为5±1 cm，购自意大利安科纳省努马纳的本地养殖场。实验前将贻贝在充气海水环境中驯化10天，驯化条件为：温度18±1℃、盐度37‰、pH 7.5±0.5、氧饱和度>94%。将贻贝分配至3个17L的水族箱中，分别暴露于2.5μg/L的酮洛芬（KET）环境中，暴露时长为14天、30天与60天。所有处理组均以添加0.00001%甲醇的空白对照组（CTRL）作为参照。每两天更换一次养殖水体，并补充恢复目标药物浓度。本研究通过8×60K安捷伦（Agilent）寡核苷酸DNA微阵列平台GPL18667，对4组消化腺混合样本（每个处理组含4组混合样本，每组由3个消化腺组成；涵盖空白对照组与酮洛芬处理组）开展基因转录分析。微阵列采用安捷伦非接触式喷墨技术原位合成，内置默认的阳性与阴性对照。总RNA的提取采用Extract-all（Eurobio公司）试剂盒流程。使用RNA纳米芯片与安捷伦RNA 6000纳米试剂（安捷伦科技公司，德国瓦尔德布龙），在安捷伦生物分析仪上检测RNA的质量与完整性。采用ND-1000分光光度计（Nanodrop科技公司）在260nm波长下检测RNA浓度，转换系数为1 OD = 40 mg/mL总RNA。所有样本均保存于-80℃环境中，待后续实验使用。基因表达谱分析采用包含59971个探针的盖贻贝寡核苷酸DNA微阵列，仅采用单色荧光检测（仅Cyanine-3荧光标记）。使用安捷伦G2565BA扫描仪以2微米的分辨率扫描微阵列玻片；所有玻片均采用两种不同灵敏度设置（XDRHi 100%与XDRLo 10%）分别扫描一次；扫描仪软件为每一组XDR扫描对生成唯一ID，并将该ID保存至两张扫描图像文件中。FeatureExtraction v10.7.3.1软件在提取数据时，可通过XDR ID自动将成对的扫描结果进行关联。经过FE所有处理步骤后得到的信号为ProcessedSignal，该信号包含经乘法去趋势化、背景扣除后的信号值。