Final Report Summary - ERA4PHARM (Environmental risk assessment of four pharmaceutical compounds employing genomic tools and the seabream, Sparus aurata)
This summary presents research highlights and conclusions of the project titled ERA4PHARM.
Environmental stressors exert their toxic effects on exposed non-target organisms by producing an impairment of normal cellular functions which, depending on the duration and concentration of the exposure may lead to physiological damage. One group of compounds found in increasing concentrations in the environment are pharmaceutics used in human and veterinary medicine. The concern over pharmaceuticals has emerged recently and they are now possible candidates for their introduction into the Water Framework Directive list of priority substances (2000/60/EC). Once these compounds have been consumed, the original compound or its metabolites are excreted and reach sewage systems and receiving water bodies where they may exert effects on non-target organisms either through the effects of the original compounds, or by unknown effects of metabolites. Concentrations in the range of ng/L to µg/L levels have been found in waste water treatment influents and effluents and may thus enter surface waters. In order to evaluate the effects and the environmental impact of pharmaceutical waste that is present in aquatic environments, approximately one-year-old individuals of gilthead sea bream were exposed to environmentally relevant concentrations of selected representative pharmaceutical compounds. These compounds included the analgesic (pain reliever) and antipyretic (fever reducer), acetaminophen (APAP), the anti depressive and antiepileptic drug, carbamazepine (CBZ) and the beta-blocher atenolol (AT). Effects were evaluated at the level of altered gene (transcriptomics) and protein (proteomics) expression in different target tissues (liver and brain) of the exposed organisms by means of complementary deoxyribonucleic acid (cDNA) microarray and two-dimensional (2D) differential gel electrophoresis (DIGE) techniques.
The proposed objectives of this project were to:
1. evaluate the xenobiotic-induced impairment resulting in the activation and silencing of specific genes in the gilthead sea bream, Sparus aurata, due to exposure to selected representative pharmaceuticals
2. elucidate the underlying molecular mechanisms of higher level damage and relate effect concentrations for different endpoints (molecular, cellular, organ, organism)
3. develop biomarkers of pharmaceutical contamination
4. use the obtained data to review existing extrapolation techniques in environmental risk assessment and
5. carry out a risk assessment exercise in the Bay of Cadiz for the selected compounds.
Research highlights
Most consistent outcomes were:
1. exposure to the selected pharmaceutical compounds, APAP, CBZ and AT, at environmentally relevant concentrations resulted in a number of differentially expressed transcripts in the brain of the gilthead sea bream, Sparus aurata. The treatment with the greatest number of differentially expressed genes was CBZ with 612 features, compared with 411 for APAP and 7 for AT
2. some of the differently expressed features were common between the treatments and other were treatment specific, being these possible candidates for the development of specific biomarkers
3. gene ontology (go) term and pathway enrichment analysis revealed that several processes were affected by the treatments. After APAP treatment, the most significantly enriched process is related with epithelium development and morphogenesis, as well as those related with transcription and ribonucleic acid (RNA) metabolic processes
4. after CBZ treatment, processes related to glycosylation and glycoproteins, as well as RNA metabolic processes and DNA dependent transcription were altered. Enriched molecular functioning was related with development, steroid dehydrogenase activity and transcriptionas well as monovalent inorganic cation transmembrane transporter activity. Due to its low exposure concentration, AT did not produce a significant enrichment in any GO term nor metabolic pathway
5. Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis for the differentially expressed features showed one enriched pathway for each, APAP and CBZ treatment compared with control animals: the lysosome and the androgen and estrogen metabolism, respectively.
Conclusions
Compared to control organisms (exposed to clean sea water), organisms exposed to environmentally relevant concentrations of APAP, CBZ and AT produce a transcriptomic response with a significant number of differently expressed features. Important relationships and similarities between effects of consumption / exposure in humans and rodents of/to these compounds and our test species have been detected in our study, indicating a high degree of conservation of the genetic targets of these drugs at different organisational levels. CBZ has been previously suspected to have endocrine disrupting activity. We identified a number of candidate genes in the brain that are estrogen responsive after CBZ treatment. Thus, our findings of its effects on androgen and estrogen metabolism pathway genes of the sea bream support these findings (Hampel et al., submitted to PlosONE).
Contact details: Dr Miriam Hampel, postdoctoral fellow
Instituto de Ciencias Marinas de Andalucia CSIC Spain
E-mail address: miriam.hampel@icman.csic.es
Environmental stressors exert their toxic effects on exposed non-target organisms by producing an impairment of normal cellular functions which, depending on the duration and concentration of the exposure may lead to physiological damage. One group of compounds found in increasing concentrations in the environment are pharmaceutics used in human and veterinary medicine. The concern over pharmaceuticals has emerged recently and they are now possible candidates for their introduction into the Water Framework Directive list of priority substances (2000/60/EC). Once these compounds have been consumed, the original compound or its metabolites are excreted and reach sewage systems and receiving water bodies where they may exert effects on non-target organisms either through the effects of the original compounds, or by unknown effects of metabolites. Concentrations in the range of ng/L to µg/L levels have been found in waste water treatment influents and effluents and may thus enter surface waters. In order to evaluate the effects and the environmental impact of pharmaceutical waste that is present in aquatic environments, approximately one-year-old individuals of gilthead sea bream were exposed to environmentally relevant concentrations of selected representative pharmaceutical compounds. These compounds included the analgesic (pain reliever) and antipyretic (fever reducer), acetaminophen (APAP), the anti depressive and antiepileptic drug, carbamazepine (CBZ) and the beta-blocher atenolol (AT). Effects were evaluated at the level of altered gene (transcriptomics) and protein (proteomics) expression in different target tissues (liver and brain) of the exposed organisms by means of complementary deoxyribonucleic acid (cDNA) microarray and two-dimensional (2D) differential gel electrophoresis (DIGE) techniques.
The proposed objectives of this project were to:
1. evaluate the xenobiotic-induced impairment resulting in the activation and silencing of specific genes in the gilthead sea bream, Sparus aurata, due to exposure to selected representative pharmaceuticals
2. elucidate the underlying molecular mechanisms of higher level damage and relate effect concentrations for different endpoints (molecular, cellular, organ, organism)
3. develop biomarkers of pharmaceutical contamination
4. use the obtained data to review existing extrapolation techniques in environmental risk assessment and
5. carry out a risk assessment exercise in the Bay of Cadiz for the selected compounds.
Research highlights
Most consistent outcomes were:
1. exposure to the selected pharmaceutical compounds, APAP, CBZ and AT, at environmentally relevant concentrations resulted in a number of differentially expressed transcripts in the brain of the gilthead sea bream, Sparus aurata. The treatment with the greatest number of differentially expressed genes was CBZ with 612 features, compared with 411 for APAP and 7 for AT
2. some of the differently expressed features were common between the treatments and other were treatment specific, being these possible candidates for the development of specific biomarkers
3. gene ontology (go) term and pathway enrichment analysis revealed that several processes were affected by the treatments. After APAP treatment, the most significantly enriched process is related with epithelium development and morphogenesis, as well as those related with transcription and ribonucleic acid (RNA) metabolic processes
4. after CBZ treatment, processes related to glycosylation and glycoproteins, as well as RNA metabolic processes and DNA dependent transcription were altered. Enriched molecular functioning was related with development, steroid dehydrogenase activity and transcriptionas well as monovalent inorganic cation transmembrane transporter activity. Due to its low exposure concentration, AT did not produce a significant enrichment in any GO term nor metabolic pathway
5. Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis for the differentially expressed features showed one enriched pathway for each, APAP and CBZ treatment compared with control animals: the lysosome and the androgen and estrogen metabolism, respectively.
Conclusions
Compared to control organisms (exposed to clean sea water), organisms exposed to environmentally relevant concentrations of APAP, CBZ and AT produce a transcriptomic response with a significant number of differently expressed features. Important relationships and similarities between effects of consumption / exposure in humans and rodents of/to these compounds and our test species have been detected in our study, indicating a high degree of conservation of the genetic targets of these drugs at different organisational levels. CBZ has been previously suspected to have endocrine disrupting activity. We identified a number of candidate genes in the brain that are estrogen responsive after CBZ treatment. Thus, our findings of its effects on androgen and estrogen metabolism pathway genes of the sea bream support these findings (Hampel et al., submitted to PlosONE).
Contact details: Dr Miriam Hampel, postdoctoral fellow
Instituto de Ciencias Marinas de Andalucia CSIC Spain
E-mail address: miriam.hampel@icman.csic.es
