Oxidative Stress Biomarkers in Erythrocytes of Captive Pre-Juvenile Loggerhead Turtles Following Acute Exposure to Methylmercury

Abstract

This study describes the use of erythrocytes (RBCs) of loggerhead turtles as in vitro models for evaluating their toxicity to methylmercury. Blood samples of loggerhead turtles that were born in the Colombian Caribbean were used. The LC50 of RBCs to methylmercury was determined at 96 h using methylmercury concentrations of 0.5–100 mg L−1. Next, the viability of the RBCs and the activity of the enzymes superoxide dismutase (SOD), glutathione S-transferase (GST), and lipid peroxidation by malondialdehyde (MDA) at 6 and 12 h of exposure to acute concentrations of 0, 1, and 5 mg L−1 were evaluated. The LC50 for loggerhead turtle RBCs was 8.32 mg L−1. The cell viability bioassay of RBCs exposed for 12 h only showed 100% cell viability. Increasing in vitro MeHg concentrations caused a corresponding increase in MDA concentration as well as decreases in the activities of SOD and GST. The RBCs represent an excellent model for ecotoxicological studies and SOD, GST, and MDA are biomarkers of environmental pollution and oxidative stress in loggerhead turtles. This was the first study conducted on loggerhead turtle where the response of RBCs to MeHg-induced oxidative stress is evaluated.

Transcriptional Analyses of Acute Exposure to Methylmercury on Erythrocytes of Loggerhead Sea Turtle

“…Peripheral blood was collected from five captive, pre-juvenile loggerhead turtles at the El Rodadero Aquarium in Santa Marta (11°1301.09″ N, 74°14013.75″ W). The details of blood sample collection and RBC isolation have been described previously [ 32 ]. In brief, blood samples were extracted from the dorsal region of the cervical sinus.…”

“…In a previous experiment, we used the same RBC samples [ 32 ] as in this study to determine the activities of the Cu/Zinc-SOD, Mn-SOD, GST enzymes, and the amount of MDA produced. To verify the correlation between the relative expression (FPKM) of the GST, Cu/Zinc-SOD, Mn-SOD, and Tbxas1 genes and the enzymatic activities [ 32 ], we used Pearson’s multiple correlation analysis using the StatR v.1.8 statistical packages designed for Rwizard v.4.3 [ 54 ] The mapped read count measures the relative expression level of the genes and statistical methods are then applied to test the significance of the differences between groups [ 55 ]. This approach of using FPKM as a measure of relative expression has been used previously [ 29 , 56 , 57 , 58 ].…”

“…In previous studies, we demonstrated that an increase in the enzymatic activity of Glutathione S transferase and superoxide dismutase, as well as the production of malonaldehyde, was revealed when loggerhead turtle RBCs were exposed to 0, 1, and 5 mg L −1 of MeHg for 12 h. The observed response shows the increase in reactive oxygen species in RBCs [ 32 ]. In this study, we set out to perform an analysis of the global transcriptional profiles in RBCs of the loggerhead turtle, Caretta caretta , to identify cellular pathways or biological processes affected by MeHg while preliminarily elucidating the molecular mechanisms of toxicity used by this xenobiotic against this marine turtle.…”

“…These marine chelonians are in population decline due to anthropic and environmental influences, appearing on the red list of threatened species of the International Union for Conservation of Nature (IUCN, 2019-2) as critically endangered ( , accessed on 13 January 2021). For this reason, it is important to propose alternative study methodologies that do not compromise the life of these animals that are instrumental for the health of marine ecosystems [ 32 ]. Recently, cell cultures of different tissues of sea turtles have been established to allow research in toxicology [ 33 ].…”

“…These cells are being used in in vitro tests, producing a frame of reference on the initial response, with the effects of different xenobiotics evaluated [ 33 , 34 , 35 , 36 , 37 ]. The nucleated RBCs of sea turtles represent a model for in vivo ecotoxicology research to reveal the effects of Hg and other heavy metals and environmental pollutants [ 32 ].…”