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. Materials and methods 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 µg L⁻¹), the anticonvulsant carbamazepine (7.85 ± 0.13 µg L⁻¹), and the beta-blocker atenolol (11.08 ± 7.98 µg 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. Results 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. Discussion 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. Conclusions 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.

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. Materials and methods: 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±34.67μg L-1), the anticonvulsant carbamazepine (7.85±0.13μg L-1), and the beta-blocker atenolol (11.08±7.98μg 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. Results: 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. Discussion: 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. Conclusions: 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.

7 páginas, 3 figuras, 4 tablas. | 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. | Discussion: 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. | Conclusions: 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. | Miriam Hampel was funded in the framework of the FP6 Marie Curie Action: Intra European Fellowships (MEIF-CT-2006-39691, acronym SALMONPHARM) | Peer reviewed