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 cr ude oil. Microsomes were prepared from the midgut gland (hepatopancreas), e xamined by gel electrophoresis and Western blotting and assayed for B(a)P m onooxygenase activity In early experiments there was evidence of protein de gradation and results were inconsistent and inconclusive. However, when ste ps were taken to minimize this in subsequent experiments, including the inc lusion of four protease inhibitors in the homogenization buffer, there was consistent evidence for an increase of proteins of estimated molecular weig ht 45-60 kDa, and particularly of a distinct band at c. 48 kDa, following e xposure 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 s uggest that one or more forms of cytochrome P450 may be induced by PAHs in these species. However, Western blotting using antibodies raised to vertebr ate P450s, and representing four different gene families, failed to recogni ze any proteins in either the PAH-treated samples or in the controls. The i solation and characterization of induced protein, and the production of ant ibodies may provide the basis for a biomarker assay to measure a response t o environmental PAHs in crabs.