SUBCELLULAR METAL DISTRIBUTION AND METALLOTHIONEINMEDIATED METAL DETOXIFICATION IN GUIANA DOLPHINS FROM SOUTHEASTERN BRAZIL

The Guiana dolphin (Sotalia guianensis) is a top-chain predator with a long life
expectancy and is considered an indicator of environmental contamination1. However, detoxification and homeostasis mechanisms in these animals are still poorly understood. Thus, the aim of the present study was to investigate metallothionein metal detoxification pathways in Sotalia guianensis liver (n=21) and kidneys (n=8) from incidentally caught specimens and carcasses found beached long the coastal area of the state of Rio de Janeiro. Metals and metalloids (Al, Cd, Co, Cu, Pb, Fe, Hg, Ni, Ag, Se and Zn) were determined by ICP-MS. Quality control was performed by the analysis of a certified reference material (DORM-4). Method reproducibility was evaluated using the coefficient of variation of the replicates, which was always less than 10%. Limits of quantification (LOQ) were from below 0.01 mg kg-1 for Ag, Cd, Co and Pb, up to 2.6 mg kg-1 for Fe. Total elemental concentrations in these organs were < 0.1 mg kg-1 only for Cd and Co and > 50 mg kg-1 for Fe and Zn. All others were in between these values. Liver and kidney samples were submitted to subcellular fractionation, by homogenizing 50 mg with a Tris-HCl buffer at room temperature for 3 min followed by centrifugation. The supernatant (cytosol) was then separated from the pellets (insoluble fraction - IN),
heated at 70 oC for 10 min and centrifuged in order to separate the precipitate containing the thermolabile high molecular weight proteins (TL) from the thermostable supernatant (TS) containing metallothioneins (MT). Al, Cu and Zn were present in all fractions in both liver and kidney, indicating homeostasis mechanisms involving MT. Co, Cd, Hg, Ag, Ni and Se were present in both organs only in the IN, most over 90% of the total concentrations, and below the LOQs in the TL and TS fractions, indicating that MT play no role in the detoxification/homeostasis of these elements. Pb and Fe in liver, on the other hand, were observed in all fractions in liver, linked to MT detoxification, while in kidneys they were present only in the IN and TL fractions, and below the LOQs in the TS fraction, indicating inter-organ differences in MT mechanisms of action.