Approved Abstracts

Life after death? Exploring biochemical and molecular changes following organismal death in green sea turtles, Chelonia mydas (Linnaeus, 1758)



Author(s): Righetti, B.P.H; Lima, D.; Dias, V.H.V.; Mattos, J.J.; Vilas-Boas, L.O.B.; Alves, T.C.; Almeida, E.A.; Luchmann, K.H.; Bainy, A.C.D.;
Presenter: Afonso C Dias Bainy

Green sea turtles, Chelonia mydas, have been included in biomonitoring efforts, given their status as an endangered species. Many studies, however, rely on samples from stranded animals, raising the question of how the death affect the biochemical and molecular parameters (biomarkers). The goal of this study was to investigate post mortem fluctuations in the antioxidant response, metabolism of carbohydrates and nucleic acids in liver of C. mydas. Liver samples of euthanized turtles (n = 4) were collected immediately after death (t = 0) and at various time intervals (1, 2, 3, 4, 5, 6 and 24 hours post mortem), frozen in liquid nitrogen and stored at -80°C. The turtles were collected during the sampling efforts of the Projeto de Monitoramento de Praias da Bacia de Santos Beach Monitoring Project – (PMP-BS) (“Beach Monitoring Project of Santos Basin”), carried out by PETROBRAS at the Santos Basin to satisfy environmental constraint of the federal environmental licensing of PETROBRAS’ activities of production and outflow of oil and natural gas in Santos Basin, conducted by IBAMA. The PMP-BS’ objective is to assess the possible impacts of oil and natural gas production and outflow activities on birds, turtles and marine mammals, through monitoring of beaches and veterinary care for live animals and necropsy of animals found dead. The PMP-BS is carried out from Laguna (Santa Catarina state) to Saquarema (Rio de Janeiro state), being divided into 15 sections. The activity of antioxidant enzymes (CAT, GPx, GR and G6PDH), glycogen concentration and RNA integrity (RNA IQ) was evaluated. Antioxidant activity varied greatly between organisms, presenting the following ranges: CAT – 7.6 to 36.9 mU.mgprt-1, GPx – 183.0 to 712.8 mU.mgprt-1, GR – 14.6 to 46.7 mU.mgprt-1 and G6PDH – < DL to 15.6 mU.mgprt-1. Glycogen concentration varied from 771.4 to 1597.4 µg.mL-1 and RNA IQ values ranged from 7.8 to 9.5. Given the high inter-individual variability of baseline values (t = 0), data for each interval were normalized by the value at t = 0 of each animal (i.e. value of t = 0 equals 1). Comparison between intervals indicated that the activities of CAT, GR and GPx were stable until 24 h post mortem (CAT: H = 6.3, p = 0.5, GR: H = 9.7, p = 0.2, GPx: H = 9.9, p = 0.19). G6PDH activity, however, tended to peak at 5 h post mortem remaining stable after that. Despite the observed pattern, post mortem changes were not statistically significant when considering all intervals (H = 11.2, p = 0.13). However, median G6PDH activity values were significantly higher at 5 and 24 h post mortem when compared to t = 0 (p = 0.02 for both). Similarly, glycogen concentration tended to decrease until 4 h post mortem and remained stable afterwards, despite such fluctuations not being statistically significant (H = 5.7, p = 0.5). Also, RNA IQ values were stable among individuals and post mortem intervals, suggesting that RNA integrity did not change within the 24 hours post mortem period. Collectively, our results suggest that changes in biochemical and molecular parameters following organismal death are not immediate, and some enzymatic activities may remain constant up to 24 hours after death. However, some post mortem fluctuations were observed, suggesting that the rate and pattern of change following organismal death may be parameter-specific. The next step of our research will include the analysis of transcript levels of carbonic anhydrase and pyruvate carboxylase genes, to better comprehend the changes in carbohydrate metabolism following death, and the determination of RNA/DNA ratio, a physiological index of activity and synthetic capacity of cells.

Keywords: Sea turtles; Chelonia mydas; Biomarkers

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