title: Characterization of ethoxyresorufin O-deethylase activity (EROD) in oyster Crassostrea brasiliana.
abstract: Cytochrome P450 family 1 (CYP1) is involved in polycyclic aromatic hydrocarbons (PAHs) biotransformation. PAHs can induce CYP1 protein expression and enzyme activity, the latter being usually quantified as 7-ethoxyresorufin O-deethylase activity (EROD). The aim of this study was to characterize EROD activity in the bivalve mollusk Crassostrea brasiliana. EROD activity was evaluated in cytosolic and microsomal fractions of gills, digestive gland and mantle of C. brasiliana. No EROD activity was detected in mantle, but it was present in microsomal fraction of gills and digestive gland with NADPH as coenzyme. Optima temperature and pH for EROD assay were 30°C and 7.4, respectively. EROD apparent Km(Kmapp) was 4.32μM for gills and 5.56μM for digestive gland. EROD Vmaxwas 337.3fmol·min-1·mg of protein-1in gills and 297.7fmol·min-1·mg of protein-1in digestive gland. Compared to other bivalves, a higher Kmappand a lower Vmaxwas found in oyster which may suggest that oyster CYP1-like enzyme has lower affinity for substrate 7-ethoxyresorufin (7-ER) than those species. CYP1 inhibitor ellipticine (ELP) inhibited EROD activity in all tested concentrations in both tissues. The higher ELP concentration, 100μM, inhibited 78% of EROD activity in gills and 47% in digestive gland. The CYP1 inhibitors α-naphthoflavone and furafylline did not inhibited EROD activity in microsomes of both tissues. In conclusion, EROD activity can be used to determine CYP1-like activity in oysters and possibly a CYP1A1/A2-like enzyme is responsible for this catalysis.
authors: Laboratory of Biomarkers of Aquatic Contamination and Immunochemistry, LABCAI, Federal University of Santa Catarina, UFSC, Florianópolis, Santa Catarina, Brazil.
authors: Marilia N
authors: MN
authors: Siebert
authors: Aquaculture Pathology Research Center, NEPAQ, Federal University of Santa Catarina, UFSC, Florianópolis, Brazil.
authors: Jacó J
authors: JJ
authors: Mattos
authors: Laboratory of Biomarkers of Aquatic Contamination and Immunochemistry, LABCAI, Federal University of Santa Catarina, UFSC, Florianópolis, Santa Catarina, Brazil.
authors: Clei E
authors: CE
authors: Piazza
authors: Laboratory of Biomarkers of Aquatic Contamination and Immunochemistry, LABCAI, Federal University of Santa Catarina, UFSC, Florianópolis, Santa Catarina, Brazil.
authors: Daína
authors: D
authors: de Lima
authors: Laboratory of Marine Mollusks, LMM, Federal University of Santa Catarina, UFSC, Florianópolis, Brazil.
authors: Carlos Henrique A M
authors: CH
authors: Gomes
authors: Laboratory of Marine Mollusks, LMM, Federal University of Santa Catarina, UFSC, Florianópolis, Brazil.
authors: Cláudio M R
authors: CM
authors: de Melo
authors: Laboratory of Biomarkers of Aquatic Contamination and Immunochemistry, LABCAI, Federal University of Santa Catarina, UFSC, Florianópolis, Santa Catarina, Brazil. Electronic address: afonso.bainy@ufsc.br.
authors: Afonso C D
authors: AC
authors: Bainy
mesh_terms: D000818:Animals; D049896:Crassostrea / Q000201:enzymology* / Q000378:metabolism; D019363:Cytochrome P-450 CYP1A1 / Q000378:metabolism*; D005880:Gills / Q000166:cytology; D006863:Hydrogen-Ion Concentration; D008861:Microsomes / Q000378:metabolism; D010078:Oxazines / Q000378:metabolism; D013696:Temperature
chemical_list: D010078:Oxazines; C014180:resorufin; D019363:Cytochrome P-450 CYP1A1
keywords: Biotransformation; Cytochrome P450; EROD; Mollusk; Oyster; Phase I