RESUMEN
New approach methodologies (NAM), including omics and in vitro approaches, are contributing to the implementation of 3R (reduction, refinement and replacement) strategies in regulatory science and risk assessment. In this study, we present an integrative transcriptomics and proteomics analysis workflow for the validation and revision of complex fish genomes and demonstrate how proteogenomics expression matrices can be used to support multi-level omics data integration in non-model species in vivo and in vitro. Using Atlantic salmon as an example, we constructed proteogenomic databases from publicly available transcriptomic data and in-house generated RNA-Seq and LC-MS/MS data. Our analysis identified â¼80,000 peptides, providing direct evidence of translation for over 40,000 RefSeq structures. The data also highlighted 183 co-located peptide groups that supported a single transcript each, and in each case, either corrected a previous annotation, supported Ensembl annotations not present in RefSeq, or identified novel previously unannotated genes. Proteogenomics data-derived expression matrices revealed distinct profiles for the different tissue types analyzed. Focusing on proteins involved in defense against xenobiotics, we detected distinct expression patterns across different salmon tissues and observed homology in the expression of chemical defense proteins between in vivo and in vitro liver systems. Our study demonstrates the potential of proteogenomic analyses in extending our understanding of complex fish genomes and provides an advanced bioinformatic toolkit to support the further development of NAMs and their application in regulatory science and (eco)toxicological studies of non-model species.
Asunto(s)
Proteogenómica , Animales , Proteogenómica/métodos , Anotación de Secuencia Molecular , Cromatografía Liquida , Espectrometría de Masas en Tándem , Proteómica/métodos , Péptidos/análisis , Péptidos/genética , Péptidos/metabolismoRESUMEN
A novel 3D Atlantic salmon co-culture model was developed using primary hepatocytes and kidney epithelial cells isolated from the same fish. Mono and co-cultures of primary hepatocytes and kidney epithelial cells were exposed for 48 h to glyphosate (5, 50 and 500 µM). For comparison, cells were also exposed to chlorpyrifos, benzo(a)pyrene and cadmium. Cell staining, cell viability assessments, RT-qPCR and global metabolomic profiling were used to examine the toxicological effects on liver and renal function and to compare responses in 3D and 2D cultures. The 3D hepatocyte cell culture was considered superior to the 2D culture due to the ATP binding cassette subfamily B member 1 (Abcb1) response and was thus used further in co-culture with kidney cells. Metabolomic analysis of co-cultured cells showed that glyphosate exposure (500 µM) altered lipid metabolism in both hepatocytes and kidney cells. Elevated levels of several types of PUFAs and long-chain fatty acids were observed in exposed hepatocytes, owing to increased uptake and phospholipid remodelling. Glyphosate suppressed the expression of estrogen receptor 1 (Esr1) and vitellogenin (Vtg) and altered histidine metabolism in exposed hepatocytes. Increased levels of cholesterol and downregulation of clusterin (Clu) suggest that glyphosate treatment affected membrane stability in Atlantic salmon kidney cells. This study demonstrates the usefulness of applying 3D co-culture models in risk assessment.
Asunto(s)
Salmo salar , Animales , Células Cultivadas , Técnicas de Cocultivo , Glicina/análogos & derivados , Hepatocitos/metabolismo , Riñón/fisiología , GlifosatoRESUMEN
Exposure to contaminants can lead to accumulation of lipids in the liver. This study aimed to examine whether eicosapentaenoic acid (EPA) and arachidonic acid (ARA) supplementation can protect fish cells against the negative impact of chlorpyrifos (CPF). Atlantic salmon hepatocytes were exposed to either 100⯵M CPF, 200⯵M EPA, 200⯵M ARA, or combinations of these for 48â¯h, and endpoints included lipid droplet formation, gene expression, and global metabolomic analysis. The results showed that polyunsaturated fatty acid (PUFA) supplementation modified the cell lipid composition, reduced uptake of CPF and increased the cellular number and size of lipid droplets. CPF exposure induced the transcription of ppara and fabp3, and reduced the levels of several PUFAs, and lead to accumulation of monoacylglycerols (MAGs) in the cells. Supplementation of EPA or ARA did not prevent CPF-induced accumulation of MAGs and only to a limited degree rescued the response on other lipids. CPF exposure further reduced energy metabolism, a response partly restored by PUFA supplementation. Reduced levels of glutathione indicated oxidative stress; an effect not ameliorated by the PUFAs. Altogether, this study shows that PUFA supplementation only modestly protects Atlantic salmon hepatocytes against the negative impact of CPF.
Asunto(s)
Ácido Araquidónico/farmacología , Cloropirifos/toxicidad , Ácido Eicosapentaenoico/análogos & derivados , Hepatocitos/efectos de los fármacos , Insecticidas/toxicidad , Metabolismo de los Lípidos/efectos de los fármacos , Animales , Células Cultivadas , Ácido Eicosapentaenoico/farmacología , Glutatión/metabolismo , Hepatocitos/metabolismo , Masculino , Estrés Oxidativo , Salmo salarRESUMEN
Elevated levels of chlorpyrifosmethyl have been detected in plant-based Atlantic salmon feeds. To evaluate the potential negative effects of long-term and continuous dietary exposure to chlorpyrifosmethyl in fish, we fed juvenile Atlantic salmon three concentrations (0.1, 1.0 and 8.0â¯mg/kg) of the pesticide for about two months. Brain and liver tissues were collected after 30 and 67â¯days of exposure. Homogenized brain tissue was examined for effects on acetylcholinesterase, and brain and liver tissue from fish exposed to 8.0â¯mg/kg were used for transcriptional analysis (RNA-seq). The results showed a transient accumulation of chlorpyrifosmethyl in the brain with lower levels after 67â¯days of exposure compared to after 30â¯days of exposure. In contrast, the liver showed a time-dependent accumulation pattern. No effect on acetylcholinesterase activity, the primary target of chlorpyrifosmethyl, was seen in the brain. However, after 30â¯days of exposure, 98 significantly differentially expressed genes (DEGs) were found in brain tissue and 239 DEGs in liver tissue. After 67â¯days of exposure, two and 258 DEGs were found in brain and liver tissue, respectively. Continuous dietary exposure of chlorpyrifosmethyl most profoundly affected mechanisms associated with protein degradation and lipid metabolism in both brain and liver. Specific for the brain, many of the significant DEGs encode proteins involved in neuron function. In conclusion, this study shows that chlorpyrifosmethyl affects the transcription of genes involved in neurological function in Atlantic salmon brain, even at exposure concentrations below the threshold for systemic toxicity as seen from brain acetylcholinesterase inhibition.