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(W/EH076) Determination of glutathione S-transferases isoforms from Ruditapes decussatus under PB and pp’DDE. Hoarau, P.1, Gnassia-Barelli, M.1, Roméo, M.1, Girard, J.P.1, 1 ABSTRACT- Glutathione S-transferase (GST) activity, considered as an exposure biomarker of organochlorine compounds, was measured in clams Ruditapes decussatus exposed to phenobarbital (PB) and pp'DDE. Two substrates CDNB and ETHA were used to measure GST activity. The GST-CDNB activity was significantly higher 36 % and 96 % for PB and pp'DDE. GST purification by affinity chroma-tography was then performed in control and treated clams, using two columns : a glutathione (GSH) agarose and an S-hexyl GSH agarose, separately eluted with GSH (0.2 and 5 mM). Four fractions were obtained. GST/CDNB activities determined in the purified fractions were increased only in pp'DDE-treated mol-lusks. A strong GST activity was noted with ETHA in fractions purified through GSH column for PB treatment and through S-hexyl agarose column for both pollutants. Bands were more or less intense according to the contaminant. Our method had permitted to show differential impact of the two tested compounds, using SDS-PAGE and 2D electrophoresis technique, we purified 11 isoforms of GST. Molecular weights ranging from 22 to 24 kDa. Four isoforms were cho-sen, implicated in the detoxication system, to determine their kinetic parameters (Km and Vmax) for CDNB and GSH and their induction as a function of con-tamination. Key words: Gluthatione S-transferases, clam, phenobarbital, 2,2-bis-(p-chlorophenyl)-1,1-dichloroethylene |
