Cytochrome P450 and dependent activities in unexposed and PAH-exposed terrestrial annelids

The cytochrome P450 system of the oligochaetes Eisenia f. fetida (tiger worm) and Enchytraeus crypticus (pot worm) was analysed using ethoxy-, pentoxy- and benzoxyresorufin as substrates for monooxygenase activity. Whole body microsomes of the earthworm E.f. fetida displayed PentROD activity in the range from 0.26 to 1.05 pmol mg protein-1 min-1 and BenzROD activity in the range from 0.14 to 0.30 pmol mg protein-1 min-1. Exposure of the animals for up to four weeks to 100 mg fluoranthene or benzo[a]pyrene kg-1 soil (dry weight) did not induce significant changes in the activity of these monooxygenases. In E. crypticus EROD activity was in the range from 2.10 to 6.18 pmol mg protein-1 min-1 and PentROD activity in the range from 1.75 to 4.78 pmol mg protein-1 min-1. Short-term exposure to BaP by feeding reduced the EROD activity significantly by 45%, but did not effect PentROD activity. After long-term (8 weeks) exposure to BaP in the agar-agar medium EROD activity was not changed but PentROD had decreased to zero. In both species cytochrome P420 and NADPH-cytochrome C reductase activity were present. In E.f. fetida microsomes are associated with the giant haemoglobin. Both can be separated by gel filtration on a Sepharose B2 column or by hydrophobic interaction chromatography after solubilisation with cholate. NADPH-cytochrome C reductase elutes together with haemoglobin. Cytochrome P420 is eluted with Emulgen 911 and can be further purified by ion exchange chromatography using HA-Ultrogel. By SDS-PAGE of the purified microsomal proteins three protein bands are visualised in the range of cytochrome P450 displaying an apparent molecular mass of 54, 56 and 58 kDa. Only the 54-kDa protein interacts weakly with perch (Perca fluviatilis) CYP1A antibodies, while two proteins with an apparent molecular mass of 65 and 71 kDa give a strong antibody signal.