Carboxilesterase como biomarcador de hipoxia em peixes

Abstract

When the esterases acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and carboxylesterase (CarbE) hydrolyze phosphate esters their active sites undergo inhibition by phosphorylation. Therefore, such phosphorylation may protect living beings against the spreading of organophosphates xenobiotics into their bodies, since these enzymes are capable of collecting organophosphate pesticide molecules stoichiometrically. Terrestrial organisms live in an environmental with more oxygen than aquatics organisms. In water, once the level of oxygen reaches 2.6 mg/mL, the environment is a hypoxic one. This occurrence affects aquatic ecosystem, as many organisms cannot adapt to low oxygen availability. We have been studying fish exposed to hypoxia and hyperoxia in order to understand better the biochemistry of enzymes capable of picking up organophosphates when fish are exposed to changes in the physical-chemistry of their habitats. Two groups of at least six pacus (Piaractus mesopotamicus), six goldfishes (Carassius auratus auratus), six tilapias (Oreochromis niloticus niloticus), six piavussus (Leporinus macrocephalus), six oscars (Astronotus ocellatus) or six carps (Cyprinus carpio carpio) were acclimated in two aquariums of 250 L. In separate aquaria, at least three assay animals of each species suffered hypoxia by decreasing the concentration of oxygen up to 0.5 mg/mL by bubbling nitrogen in the water. Once these animals reached hypoxia they were maintained at 0.5 mg/mL of oxygen for 6, 8, 24 or no more than 42 hours. Three control fish of each species were kept in normoxia (4.5 to 7 mg/mL of oxygen). Then, 3.5 mL of blood were withdrawn, fishes were sacrificed and their liver removed. After clotting, blood was centrifuged to remove the supernatant serum, which was used as a sample for assaying esterase activities. Livers were stored at -70 °C and, at the time of the enzyme assays, homogenized and centrifuged to obtain the cytosolic and microsomal fractions. Esterase activities were assayed using a spectrophotometer with the substrates acetylthiocholine, butyrylthiocholine or p-nitrophenylacetate. The serum and liver activities with p-nitrophenylacetate fell in all specimens subjected to hypoxia. Typically, in pacu kept for 42 h below 1.0 mg of oxygen per liter this activity dropped about 50%. The time required for the drop on enzyme activities varied from species to species.