Hypothesis-driven dragging of transcriptomic data to analyze proven targeted pathways in Rhinella arenarum larvae exposed to organophosphorus pesticides.

Transcriptional analysis of the network of transcription regulators and target pathways in exposed organisms may be a hard task when their genome remains unknown. The development of hundreds of qPCR assays, including primer design and normalization of the results with the appropriate housekeeping genes, seems an unreachable task. Alternatively, we took advantage of a whole transcriptome study on Rhinella arenarum larvae exposed to the organophosphorus pesticides azinphos-methyl and chlorpyrifos to evaluate the transcriptional effects on a priori selected groups of genes. This approach allowed us to evaluate the effects on hypothesis-selected pathways such as target esterases, detoxifying enzymes, polyamine metabolism and signaling, and regulatory pathways modulating them. We could then compare the responses at the transcriptional level with previously described effects at the enzymatic or metabolic levels to obtain global insight into toxicity-response mechanisms. The effects of both pesticides on the transcript levels of these pathways could be considered moderate, while chlorpyrifos-induced responses were more potent and earlier than those elicited by azinphos-methyl. Finally, we inferred a prevailing downregulation effect of pesticides on signaling pathways and transcription factor transcripts encoding products that modulate/control the polyamine and antioxidant response pathways. We also tested and selected potential housekeeping genes based on those reported for other species. These results allow us to conduct future confirmatory studies on pesticide modulation of gene expression in toad larvae.

Toxicity of the insecticide chlorpyrifos to the South American toad Rhinella arenarum at larval developmental stage.

Chlorpyrifos (CPF) is an insecticide widely used for pest control in the fruit-productive region of North Patagonia, Argentina, where it is found in superficial waters. The aim of this study was to establish the toxic effects of CPF in Rhinella arenarum toad larvae as a potentially exposed species. We determined the 96 h-LC50 (1.46 ± 0.27 mg/L), the LOEC (0.81 mg/L, LC10) and NOEC (0.43 mg/L, LC1) for CPF lethality as endpoint. We also analyzed biochemical biomarkers in larvae exposed to sublethal CPF concentrations. The IC50 for cholinesterase was 0.113 ± 0.026 mg/L, one order of magnitude lower than the LC50. Carboxylesterase activity was inhibited, buffering OP toxicity on cholinesterase. Reduced glutathione increased after 24h as an antioxidant response, and decreased at 96 h together with catalase activity, due to oxidative stress. These biochemical effects suggest that environmentally relevant CPF concentrations pose a threat to R. arenarum larvae progression.

Antioxidant responses to azinphos methyl and carbaryl during the embryonic development of the toad Rhinella (Bufo) arenarum Hensel.

Amphibian embryos are naturally exposed to prooxidant conditions throughout their development. Environmental exposure to contaminants may affect their capacity to respond to challenging conditions, to progress in a normal ontogenesis, and finally to survive and succeed in completing metamorphosis. We studied the effects of the exposure to two anticholinesterase agents, the carbamate carbaryl and the organophosphate azinphos methyl, on the antioxidant defenses of developing embryos of the toad Rhinella (Bufo) arenarum. Reduced glutathione (GSH) levels were increased early by carbaryl, but were decreased by both pesticides at the end of embryonic development. The GSH-dependent enzymes glutathione reductase and glutathione peroxidases showed oscillating activity patterns that could be attributed to an induction of activity in response to oxidative stress and inactivation by excess of reactive oxygen species. Glutathione-S-transferases, which may participate in the conjugation of lipid peroxide products in addition to pesticide detoxification, showed an increase of activity at the beginning and at the end of development. Catalase also showed variations in the activity suggesting, successively, induction and inactivation in response to pesticide exposure-induced oxidative stress. Superoxide dismutase activity was increased by carbaryl and transiently decreased by azinphos methyl exposure. Judging from the depletion in GSH levels and glutathione reductase inhibition at the end of embryonic development, the oxidative stress caused by azinphos methyl seemed to be greater than that caused by carbaryl, which might be in turn related with a higher number of developmental alterations caused by the organophosphate. GSH content is a good biomarker of oxidative stress in the developing embryos exposed to pesticides. The antioxidant enzymes are in turn revealing the balance between their protective capacity and the oxidative damage to the enzyme molecules, decreasing their activity.