In vitro biotransformation rates of surfactants in carp and rainbow trout liver subcellular fractions
The aim of the present work was to investigate, using in vitro approaches, the fish capabilities to metabolize the surfactants which may be present in surrounding water. The study of surfactants biotransformation rates has been undertaken using carp liver subcellular systems incubated with radio-labeled substances to investigate the disappearance of the parent compound and to quantify the amount of metabolites formed. For comparison, the biotransformation rates of these surfactants was also studied in rainbow trout microsomes.
Methodological aspects, including the preparation of the subcellular systems (homogenates, S9 fractions and microsomes) from common carp (Cyprinus carpio) liver and the
characterization of these fractions by protein concentration, esterase activity and Ethoxycoumarine O-deethylase activity (ECOD) were described in the Materials & Methods
section. The possible effects of surfactants on xenobiotic metabolizing enzymes was investigated, using ECOD activity as a test.
Immature common carp (Cyprinus carpio) and rainbow trout (Oncorhynchus mykiss) weighing 200-300g were maintained at 22-26°C and 16-18°C, respectively. After the sacrifice of the animals, the livers were immediately removed and frozen in liquid nitrogen then stored at –80°C until use. Homogenates, S9 and microsomes were prepared and protein concentration was determined for each sample. Metabolic capability of the carp subcellular fractions was determined measuring ethoxycoumarin O-deethylase (ECOD) and esterase activities. For rainbow trout, microsomes were the only subcellular fraction tested.
The metabolism of two anionic surfactants, i.e. sodium 2 dodecyl benzene sulfonate (C12-LAS) and diethylene glycol monotetradecyl sulfate (C14-E2S) and two non ionic surfactants, i.e. octaethylene glycol monotridecyl ether (C13EO8), and octaethylene glycol monohexadecyl ether (C16EO8) was investigated using radio-labelled molecules incubated with the subcellular liver fractions. The remaining parent compound as well as the resulting metabolites were analyzed by radio-TLC.
The incidence of substrate concentrations and of incubation times on metabolism was tested for each compound.
All the in vitro systems were found to be able to metabolize xenobiotics, however, significant differences were found between surfactants and between fish species. The biotransformation rates of C12-LAS in carp liver subcellular fractions were 5-8 fold higher than for the other surfactants. Trout microsomes were more efficient than carp microsomes for the biotransformation of C13EO8, C16EO8, and C14-E2S, whereas for C12-LAS a higher metabolic metabolic rate was observed with carp microsomes .