High throughput (RNA-seq) screening for biomarkers of azinphos methyl (AZM) exposure in Rhinella arenarum

MARDIROSIAN M.; CESCHIN D.G.; PIRES N; LASCANO C; VENTURINO A

Natal

Congreso; 9th Congress of Toxicology in Developing Countries and XIX Congress of Brazilian Society of Toxicology; 2015

Soc. Bras. Toxicol. - IUTOX

ntroduction: Azinphos methyl (AZM) is the main organophosphate pesticide applied in Río Negro-Neuquénvalley in Argentina. In this sense, it is necessary to control the levels ofresidues and environmental impact of pesticides. Traditional ecotoxicologicalbiomarkers like reduced glutathione (GSH) levels and the activities ofGlutathione S transferase (GST) and Catalase (CAT) are commonly used forbiomonitoring, however they may fail for sensitivity and unspecifically respondto oxidative stress. Native species is a preferred resource for its ecologicalsignificance. In this sense, we have evaluated pesticide effects on common toadR. arenarum larvae. In this work, we screenfor molecular biomarkers using RNA-Seq technology and compare the response withtraditional biomarkers, in order to develop new specific and sensitivebiomarkers.Objective: todevelop new early, specific, sensitive biomarkers to AZM exposure..Materials andMethods: R. arenarum embryos were obtained by in vitro fertilization. Larvae (complete operculum (CO) + 11 days)were exposed to sublethal concentration of AZM (0.5 mg/L) [96h-LC50 10 mg/L)].Samples were taken at 6h and 24h to evaluate GSH levels and the activities ofGST and CAT. At the same time, samples were collected for RNApurification and massive sequencing. Results anddiscussion: We could detect changes onlyin GSH content but not in GST or CAT activities at 6h and 24h exposures to AZM.Regarding to  the transcriptomic profile,we could determine up- and down-regulated genes compared with control for both6h and 24h of exposure. After gene enrichment analyses, overexpression of detoxificationand oxidative stress pathways was found. Moreover, for Gene Ontologyclassification there were hits for Biological Process category such as celldifferentiation, developmental growth, catabolic and hormone metabolic process,cell proliferation and cell cycle.Conclusion: here we show that transcriptomic analysis is a goodtool to pick up gene candidates to be used as biomarkers. After gene expressionanalysis, we could detect changes in the level of several genes, even at earlytime of exposition (6h), while classical biomarkers did not show modificationcompared with control. Thus, after verification, some genes could be selectedto develop more specific and sensitive biomarkers for biomonitoring pesticide impact.