Institute of Aquaculture, University of Stirling, FK9 4LA, Stirling, UK.
Environ Sci Pollut Res Int. 2010 May;17(4):917-33. doi: 10.1007/s11356-009-0282-6. Epub 2010 Feb 6.
BACKGROUND, AIM, AND SCOPE: Pharmaceuticals are emerging pollutants widely used in everyday urban activities which can be detected in surface, ground, and drinking waters. Their presence is derived from consumption of medicines, disposal of expired medications, release of treated and untreated urban effluents, and from the pharmaceutical industry. Their growing use has become an alarming environmental problem which potentially will become dangerous in the future. However, there is still a lack of knowledge about long-term effects in non-target organisms as well as for human health. Toxicity testing has indicated a relatively low acute toxicity to fish species, but no information is available on possible sublethal effects. This study provides data on the physiological pathways involved in the exposure of Atlantic salmon as representative test species to three pharmaceutical compounds found in ground, surface, and drinking waters based on the evaluation of the xenobiotic-induced impairment resulting in the activation and silencing of specific genes.
Individuals of Atlantic salmon (Salmo salar) parr were exposed during 5 days to environmentally relevant concentrations of three representative pharmaceutical compounds with high consumption rates: the analgesic acetaminophen (54.77+/-34.67 microg L(-1)), the anticonvulsant carbamazepine (7.85+/-0.13 microg L(-1)), and the beta-blocker atenolol (11.08+/-7.98 microg L(-1)). Five immature males were selected for transcriptome analysis in brain tissues by means of a 17k salmon cDNA microarray. For this purpose, mRNA was isolated and reverse-transcribed into cDNA which was labeled with fluorescent dyes and hybridized against a common pool to the arrays. Lists of significantly up- and down-regulated candidate genes were submitted to KEGG (Kyoto Encyclopedia of Genes and Genomes) in order to analyze for induced pathways and to evaluate the usefulness of this method in cases of not completely annotated test organisms.
Exposure during 5 days to environmentally relevant concentrations of the selected pharmaceutical compounds acetaminophen, carbamazepine, and atenolol produced differences in the expression of 659, 700, and 480 candidate genes, respectively. KEGG annotation numbers (KO annotations) were obtained for between 26.57% and 33.33% of these differently expressed genes per treatment in comparison to non-exposure conditions. Pathways that showed to be induced did not always follow previously reported targets or metabolic routes for the employed treatments; however, several other pathways have been found (four or more features) to be significantly induced.
Energy-related pathways have been altered under exposure in all the selected treatments, indicating a possible energy budget leakage due to additional processes resulting from the exposure to environmental contaminants. Observed induction of pathways may indicate additional processes involved in the mode of action of the selected pharmaceuticals which may not have been detected with conventional methods like quantitative PCR in which only suspected features are analyzed punctually for effects. The employment of novel high-throughput screening techniques in combination with global pathway analysis methods, even if the organism is not completely annotated, allows the examination of a much broader range of candidates for potential effects of exposure at the gene level.
The continuously growing number of annotations of representative species relevant for environmental quality testing is facilitating pathway analysis processes for not completely annotated organisms. KEGG has shown to be a useful tool for the analysis of induced pathways from data generated by microarray techniques with the selected pharmaceutical contaminants acetaminophen, carbamazepine, and atenolol, but further studies have to be carried out in order to determine if a similar expression pattern in terms of fold change quantity and pathways is observed after long-term exposure. Together with the information obtained in this study, it will then be possible to evaluate the potential risk that the continuous release of these compounds may have on the environment and ecosystem functioning.
背景、目的和范围：药品是一种在日常城市活动中广泛使用的新兴污染物，可在地表水、地下水和饮用水中检测到。它们的存在来源于药品的使用、过期药物的处理、处理和未处理的城市污水的排放以及制药工业。它们的使用量不断增加，已成为一个令人担忧的环境问题，未来可能会变得更加危险。然而，人们对非目标生物以及对人类健康的长期影响仍知之甚少。毒性测试表明，这些药物对鱼类物种具有相对较低的急性毒性，但目前尚不清楚可能存在的亚致死效应。本研究提供了有关大西洋鲑暴露于三种在地表水、地下水和饮用水中发现的药物化合物的生理途径的信息，这些药物化合物的浓度与环境相关，评估了由此导致的特定基因激活和沉默的外源性物质诱导损伤，以此作为代表性测试物种。
将大西洋鲑（Salmo salar）幼鱼暴露于 5 天，使其接触三种具有高消耗率的代表性药物化合物的环境相关浓度：止痛药对乙酰氨基酚（54.77+/-34.67 µg/L）、抗惊厥药卡马西平（7.85+/-0.13 µg/L）和β-阻断剂阿替洛尔（11.08+/-7.98 µg/L）。选择 5 条未成熟的雄性鱼进行脑组织转录组分析，采用 17k 鲑鱼 cDNA 微阵列。为此，将 mRNA 分离并逆转录成 cDNA，然后用荧光染料标记并与共同池杂交到阵列上。提交显著上调和下调的候选基因列表到 KEGG（京都基因和基因组百科全书），以分析诱导途径，并评估在不完全注释测试生物体的情况下使用这种方法的有用性。
在 5 天内接触选定的药物化合物对乙酰氨基酚、卡马西平和阿替洛尔的环境相关浓度，分别导致 659、700 和 480 个候选基因的表达差异。与非暴露条件相比，每种处理下，这些差异表达基因中有 26.57%至 33.33%获得了 KEGG 注释编号（KO 注释）。所观察到的途径诱导并不总是遵循先前报道的治疗方法的目标或代谢途径；然而，已经发现了其他几个途径（具有四个或更多特征）被显著诱导。
所有选定的处理中，与能量相关的途径都发生了改变，这表明由于暴露于环境污染物而导致额外的过程，可能导致能量预算泄漏。观察到的途径诱导可能表明所选择的药物的作用方式涉及其他可能未被常规方法（如定量 PCR）检测到的额外过程，这些方法仅针对可疑特征进行有针对性的分析。即使生物体未完全注释，采用新型高通量筛选技术与全局途径分析方法相结合，也可以在基因水平上检查更多潜在暴露效应的候选者。
与环境质量测试相关的代表性物种的注释数量不断增加，这为不完全注释的生物体的途径分析过程提供了便利。KEGG 已被证明是一种有用的工具，可用于分析由微阵列技术产生的数据中的诱导途径，这些数据来自选定的药物污染物对乙酰氨基酚、卡马西平和阿替洛尔，但需要进一步研究以确定在长期暴露后是否观察到类似的表达模式，包括数量和途径。结合本研究获得的信息，然后可以评估这些化合物的持续释放可能对环境和生态系统功能造成的潜在风险。
