Induction of a putative monooxygenase of crabs (Carcinus spp.) by polycyclic aromatic hydrocarbons

As part of a programme to develop biomarker assays for polycyclic aromatic hydrocarbons (PAHs) in marine invertebrates, two species of crabs, Carcinus maenas and Carcinus aestuarii were exposed to benzo(a)pyrene (B(a)P) or crude oil. Microsomes were prepared from the midgut gland (hepatopancreas), examined by gel electrophoresis and Western blotting and assayed for B(a)P monooxygenase activity. In early experiments there was evidence of protein degradation and results were inconsistent and inconclusive. However, when steps were taken to minimize this in subsequent experiments, including the inclusion of four protease inhibitors in the homogenization buffer, there was consistent evidence for an increase of proteins of estimated molecular weight 45-60 kDa, and particularly of a distinct band at c. 48 kDa, following exposure to PAH at levels down to 0.1 ppm in ambient water. In C. aestuarii the increase in this band was found to coincide with an 8-12-fold increase of B(a)P monooxygenase activity in midgut gland microsomes. These results suggest that one or more forms of cytochrome P450 may be induced by PAHs in these species. However, Western blotting using antibodies raised to vertebrate P450s, and representing four different gene families, failed to recognize any proteins in either the PAH-treated samples or in the controls. The isolation and characterization of induced protein, and the production of antibodies may provide the basis for a biomarker assay to measure a response to environmental PAHs in crabs.