ABSTRACT
The common toad Rhinella arenarum is widely distributed in Argentina, where it is utilised as an autochthonous model in ecotoxicological research and environmental toxicology. However, the lack of a reference genome makes molecular assays and gene expression studies difficult to carry out on this non-model species. To address this issue, we performed a genome-wide transcriptome analysis on R. arenarum larvae through massive RNA sequencing, followed by de novo assembly, annotation, and gene prediction. We obtained 57,407 well-annotated transcripts representing 99.4% of transcriptome completeness (available at http://rhinella.uncoma.edu.ar). We also defined a set of 52,800 high-confidence lncRNA transcripts and demonstrated the reliability of the transcriptome data to perform phylogenetic analysis. Our comprehensive transcriptome analysis of R. arenarum represents a valuable resource to perform functional genomic studies and to identify potential molecular biomarkers in ecotoxicological research.
Subject(s)
Bufonidae/genetics , Genome/genetics , Transcriptome/genetics , Animals , Argentina , Base Sequence , Female , Gene Expression Profiling , High-Throughput Nucleotide Sequencing , Male , Molecular Sequence Annotation/methods , RNA, Long Noncoding/genetics , Sequence Analysis, RNAABSTRACT
Arsenic, a natural element of ecological relevance, is one of the most toxic elements present in various regions of the world. It can be found in natural water sources throughout Argentina in concentrations between 0.01 and 15mgL-1. The Argentinean autochthonous toad Rhinella arenarum was selected to study the molecular mechanisms involved in the effects and response to the chronic As exposure along its embryonic and larval development. We evaluated the effects on MAPK signal transduction pathway and transcription factors c-FOS and c-JUN, and the regulation of the expression at protein levels of different antioxidant enzymes. Our results indicated that As is modulating the MAPK pathway, increasing MEK and ERK levels both in the nuclear and post-nuclear fraction along the embryonic development and mainly at the beginning of the larval stage. Through this pathway, As can upregulate transcription factors like c-FOS and c-JUN, impacting the antioxidant response of the exposed embryos and larvae through antioxidant enzymes and recycling of GSH. Arsenic triggered specifically the synthesis of antioxidant enzymes in exposed R. arenarum embryo and larvae. In particular, the expression levels of SOD, CAT and GST enzymes analyzed by Western blot showed a similar behavior to their enzymatic activities in our previous work. This fact suggests that not only the synthesis of these antioxidant enzymes but also their rapid degradation after inactivation would be regulated in response to ROS levels. Antioxidant enzymes may show dual responses of induction and inactivation followed by degradation depending on the levels of oxidative stress and impact on ROS targets when the exposure is sustained in time and intensity. We also performed a probability of exceedence analysis including our previous results to visualize a progression of the response in time and also established the best early-responding biomarkers at the lowest As concentrations. As a conclusion, the molecular biomarkers such as the MAPKs MEK and ERK and transcription factors c-FOS and c-JUN are early induced in the response of developing toad embryos exposed to very low As concentrations in water. The advantage of counting with molecular biomarkers early responding to low concentrations of As in a chronic exposure is that they may anticipate the irreversible damage at later developmental stages due to the constant oxidative challenge.
Subject(s)
Arsenic/toxicity , Biomarkers/metabolism , Bufo arenarum/embryology , Bufo arenarum/genetics , Embryonic Development/drug effects , Environmental Exposure/analysis , Animals , Antioxidants/metabolism , Cell Nucleus/drug effects , Cell Nucleus/metabolism , Embryo, Nonmammalian/drug effects , Embryo, Nonmammalian/enzymology , Extracellular Signal-Regulated MAP Kinases/metabolism , Glutathione Transferase/metabolism , Larva/drug effects , Mitogen-Activated Protein Kinase Kinases/metabolism , Principal Component Analysis , Probability , Signal Transduction/drug effects , Transcription Factors/metabolism , Water Pollutants, Chemical/toxicityABSTRACT
The Argentinean autochthonous toad Rhinella arenarum was selected to study the chronic toxicity of arsenic (As) and the biochemical responses elicited by exposure to As in water during embryonic and larval development. Significant decreases in the total reactive antioxidant potential and in catalase activity were observed in individuals exposed chronically to sublethal concentrations of As, which is indicative of an oxidative stress situation. However, an antioxidant response was elicited during chronic exposure to As, as evidenced by the increase in endogenous reduced glutathione content and glutathione-related enzymatic activities such as glutathione S-transferase (GST) and glutathione reductase. This protective response might prevent a deeper decline in the antioxidant system and further oxidative damage. Alternatively, it might be linked to As conjugation with reduced glutathione for its excretion. Considering the sustained increase in GST activity and the decrease in the total antioxidant reactive potential observed, the authors propose them as good candidates to be used as biomarkers during As exposure. Interestingly, glutathione reductase activity was inhibited at a very low concentration of As considered safe for aquatic life. Environ Toxicol Chem 2017;36:1614-1621. © 2016 SETAC.
Subject(s)
Antioxidants/metabolism , Arsenic/toxicity , Bufo arenarum/embryology , Oxidative Stress/drug effects , Animals , Arsenic/administration & dosage , Biomarkers/blood , Bufo arenarum/growth & development , Catalase/metabolism , Drug Administration Schedule , Environmental Pollutants/toxicity , Glutathione/metabolism , Glutathione Reductase/metabolism , Glutathione Transferase/metabolism , Larva/drug effects , Oxidative Stress/physiologyABSTRACT
Arsenic (As), a natural element of ecological relevance, is found in natural water sources throughout Argentina in concentrations between 0.01 mg/L and 15 mg/L. The autochthonous toad Rhinella arenarum was selected to study the acute toxicity of As and the biochemical responses elicited by the exposure to As in water during its embryonic development. The median lethal concentration (LC50) value averaged 24.3 mg/L As and remained constant along the embryonic development. However, As toxicity drastically decreased when embryos were exposed from heartbeat-stage on day 4 of development, suggesting the onset of detoxification mechanisms. Given the environmental concentrations of As in Argentina, there is a probability of exceeding lethal levels at 1% of sites. Arsenic at sublethal concentrations caused a significant decrease in the total antioxidant potential but generated an increase in endogenous glutathione (GSH) content and glutathione S-transferase (GST) activity. This protective response might prevent a deeper decline in the antioxidant system and further oxidative damage. Alternatively, it might be linked to As conjugation with GSH for its excretion. The authors conclude that toad embryos are more sensitive to As during early developmental stages and that relatively high concentrations of this toxic element are required to elicit mortality, but oxidative stress may be an adverse effect at sublethal concentrations.
Subject(s)
Arsenic/toxicity , Bufo arenarum/metabolism , Embryo, Nonmammalian/metabolism , Oxidative Stress/drug effects , Animals , Antioxidants/analysis , Argentina , Arsenic/analysis , Arsenites/toxicity , Embryonic Development/drug effects , Glutathione/analysis , Glutathione Transferase/analysis , Risk Assessment , Sodium Compounds/toxicityABSTRACT
Los plaguicidas organofosforados (OP) son masivamente aplicados en el Alto Valle de Río Negro y Neuquén, afectando al ecosistema. Utilizamos un modelo embrionario de anfibios (Rhinella arenarum) para estudiar mecanismos por los cuales OP como metilazinfos (MA) y clorpirifos (CP) podrían provocar teratogénesis. Los embriones fueron desarrollados en diferentes concentraciones de MA o CP hasta opérculo completo (OC), analizando: malformaciones, histología, glutatión reducido (GSH) y enzimas antioxidantes, poliaminas, actividad de ornitina-decarboxilasa (ODC) y proteínaquinasa-C (PKC). Ambos OP provocaron un incremento tiempo/concentración-dependiente de malformaciones, llegando a 100% de teratogénesis en estadios avanzados y a las mayores concentraciones, incluyendo: exogastrulación, curvaturas de aleta caudal, acortamiento axial, edema, y atrofia branquial. Se evidenció una condición de estrés oxidativo creciente: las enzimas GSH-dependientes (S-transferasa (GST), peroxidasa y reductasa) fueron inducidas tempranamente a bajas concentraciones, pero inhibidas en elestadio de OC a altas concentraciones, junto con una caída significativa de GSH (62%) para MA. MA incrementó significativamente (18X) la actividad de ODC en OC, aumentando los niveles de putrescina (60%) pero disminuyendo espermidina (56%) y espermina (100%); CP disminuyó en estadios tempranos la actividad de ODC y niveles de poliaminas. La disminución de poliaminas podría deberse al incremento de degradación por poliamino-oxidasa, contribuyendo al estrés oxidativo inducido por OP. Esto causaría la disminución de GSH, y la activación de PKC en OC (55%), que participaría en el control positivo de GST y ODC. Finalmente, el estrés oxidativo y la disminución en los niveles de poliaminas podrían ser causantes de alteraciones del desarrollo embrionario.
Organophosphate (OP) pesticides are widely applied in the region of Alto Valle de Río Negro y Neuquén, affecting the ecosystem. We use an amphibian embryonic model (Rhinella arenarum) in order to assess the mechanisms by which the OP pesticides azinphos methyl (AM) and chlorpyrifos (CP) could cause teratogenesis. The embryos were developed in different concentrations of AM or CP until they reached the stage of complete operculum (CO). We analyzed malformations, histology, reduced gluthatione content (GSH) and activity of antioxidant enzymes, polyamine content, ornithine decarboxilase (ODC) and protein kinase C (PKC) activities. Both OP pesticides caused a time-and dose-dependent increase in the number of malformations, reaching 100% teratogenesis in late embryonic development at the highest OP concentrations used. Malformations assessed include exogastrulation, caudalfin curvature, axial shortening, edema, and gill atrophy. Increasing evidence of oxidative stress was observed: GSH dependent enzymes (S- transferase, GST; peroxidase and reductase) were early induced in embryos exposed to low concentrations of the OP pesticides, but their activities were inhibited in the stage of CO at high concentrations of OP. These changes were accompanied by a significant decrease in GSH content (62%) in embryos exposed to AM. Besides, AM significantly increased (18X) ODC activity in the stage of CO, along with putrescine levels (60% of increase) but spermidine and spermine levels were significantly decreased (56% and 100%, respectively). The OP pesticide CP caused and early decrease in ODC activity and polyamine levels.The decrease in polyamine levels could be due to an increase in their degradation by polyamine oxidase, contributing to the oxidative stress induced by OP. This, in turn, would cause the decline in GSH levels and the activation of PKC in the embryonic stage of CO (55%), which is involved in the positive feedback of GST and ODC...
Subject(s)
Animals , Amphibians/embryology , Bufo bufo , Insecticides, Organophosphate/adverse effects , Biomarkers , Oxidative Stress , PolyaminesABSTRACT
Los plaguicidas organofosforados (OP) son masivamente aplicados en el Alto Valle de Río Negro y Neuquén, afectando al ecosistema. Utilizamos un modelo embrionario de anfibios (Rhinella arenarum) para estudiar mecanismos por los cuales OP como metilazinfos (MA) y clorpirifos (CP) podrían provocar teratogénesis. Los embriones fueron desarrollados en diferentes concentraciones de MA o CP hasta opérculo completo (OC), analizando: malformaciones, histología, glutatión reducido (GSH) y enzimas antioxidantes, poliaminas, actividad de ornitina-decarboxilasa (ODC) y proteínaquinasa-C (PKC). Ambos OP provocaron un incremento tiempo/concentración-dependiente de malformaciones, llegando a 100% de teratogénesis en estadios avanzados y a las mayores concentraciones, incluyendo: exogastrulación, curvaturas de aleta caudal, acortamiento axial, edema, y atrofia branquial. Se evidenció una condición de estrés oxidativo creciente: las enzimas GSH-dependientes (S-transferasa (GST), peroxidasa y reductasa) fueron inducidas tempranamente a bajas concentraciones, pero inhibidas en elestadio de OC a altas concentraciones, junto con una caída significativa de GSH (62%) para MA. MA incrementó significativamente (18X) la actividad de ODC en OC, aumentando los niveles de putrescina (60%) pero disminuyendo espermidina (56%) y espermina (100%); CP disminuyó en estadios tempranos la actividad de ODC y niveles de poliaminas. La disminución de poliaminas podría deberse al incremento de degradación por poliamino-oxidasa, contribuyendo al estrés oxidativo inducido por OP. Esto causaría la disminución de GSH, y la activación de PKC en OC (55%), que participaría en el control positivo de GST y ODC. Finalmente, el estrés oxidativo y la disminución en los niveles de poliaminas podrían ser causantes de alteraciones del desarrollo embrionario.(AU)
Organophosphate (OP) pesticides are widely applied in the region of Alto Valle de Río Negro y Neuquén, affecting the ecosystem. We use an amphibian embryonic model (Rhinella arenarum) in order to assess the mechanisms by which the OP pesticides azinphos methyl (AM) and chlorpyrifos (CP) could cause teratogenesis. The embryos were developed in different concentrations of AM or CP until they reached the stage of complete operculum (CO). We analyzed malformations, histology, reduced gluthatione content (GSH) and activity of antioxidant enzymes, polyamine content, ornithine decarboxilase (ODC) and protein kinase C (PKC) activities. Both OP pesticides caused a time-and dose-dependent increase in the number of malformations, reaching 100% teratogenesis in late embryonic development at the highest OP concentrations used. Malformations assessed include exogastrulation, caudalfin curvature, axial shortening, edema, and gill atrophy. Increasing evidence of oxidative stress was observed: GSH dependent enzymes (S- transferase, GST; peroxidase and reductase) were early induced in embryos exposed to low concentrations of the OP pesticides, but their activities were inhibited in the stage of CO at high concentrations of OP. These changes were accompanied by a significant decrease in GSH content (62%) in embryos exposed to AM. Besides, AM significantly increased (18X) ODC activity in the stage of CO, along with putrescine levels (60% of increase) but spermidine and spermine levels were significantly decreased (56% and 100%, respectively). The OP pesticide CP caused and early decrease in ODC activity and polyamine levels.The decrease in polyamine levels could be due to an increase in their degradation by polyamine oxidase, contributing to the oxidative stress induced by OP. This, in turn, would cause the decline in GSH levels and the activation of PKC in the embryonic stage of CO (55%), which is involved in the positive feedback of GST and ODC... (AU)
