Oxygen-dependent metabolism of organic xenobiotics in marine fish and mussel
Studies were undertaken to examine the processes of oxygen-dependent organic xenobiotic metabolism in fish and mussel. The antioxidant enzymes superoxide dismutase (EC 22.214.171.124 ), catalase (EC 126.96.36.199), selenium-dependent glutathione peroxidase (SeGPX; EC 188.8.131.52) glutathione reducate (EC 184.108.40.206), DT Diaphorase (quinone oxidoreductase; EC 220.127.116.11), total GPX activity (sum of SeGPX and Se-independent GPX activities) and levels of cytochrome P450 (CYP) were determined in 13 500g supernatants of embryos, 3- day and 11-day post-hatch larvae of turbot Scophthalmus maximus. The early life stages of S. maximus demonstrated the potential to detoxify reactive oxygen species (ROS), organic peroxides and redox cycling compounds. The in vitro capacity of larval 13 500g supernatants to generate ROS was quantified and basal rates were stimulated upto 3 fold by redox cycling compounds. Exposure to benzo[a]pyrene (BaP) increased CYP1A protein levels and 7-ethoxyresorufin O-deethylase (EROD) activity in larval and juvenile life stages. BaP was metabolised by 4-day post-hatch larvae to phenols (54%), dihydrodiols (32%) and diones (quinones 14%) and DNA adducts were formed when juvenile S. maximus were exposed to 25 ppb BaP. EROD and antioxidant enzyme activities were measured in feral sardine Sardina pilchardus larvae from the north coast of Spain. Western analysis of microsomes and partially purified cytochrome P450 (CYP) from digestive gland of mussel Mytilus edulis was undertaken using polyclonal antibodies to hepatic perch Perca fluviatilis CYP1A, rainbow trout Oncorhynchus mykiss CYP3A and rat CYP2B, CYP2E and CYP4A isoforms. The apparent molecular weights in kD (mean ± range or SD; n = 2-4) for partially purified CYP were 42.5 ± 0.5 and 48.1 ± 0.3 (2 bands, anti-CYPlA); 67.4 ± 0.7, 52.8 ± 0.6 and 44.5 ± 2.5 (3 bands, anti-CYP3A); 52.8 ± 0.7, 48.1 ± 1.1 and 43 .9 ± 1.1 (3 bands, anti-CYP2B); 52.7 ± 0.8 and 47.2 ± 0.2 (2 bands, anti-CYP2E); 50.9 ± 0.3 and 44.1 ± 0.2 kD (2 bands, anti-CYP4A). Digestive gland microsomes of indigenous M galloprovincialis from a polluted compared to a clean field site showed higher levels of bands recognised by anti-CYP1A, anti-CYP2E and anti-CYP4A but not anti-CYP2B and anti-CYP3A (P < 0.05), indicative of independent regulation of different CYP forms. Three weeks after transplantation from a clean to a polluted site, levels of only the CYPlA-immunopositive protein were determined to be higher (63 %) than levels for the clean site (P < 0.05), indicating that the anti-CYP I A antibody showed greater specificity for a contaminant-inducible CYP form than the other antibodies. Overall, the apparent molecular weight and field studies indicate at least five different digestive gland CYP forms. Hepatic flavin-dependent monooxygenase (FMO; EC 18.104.22.168) dimethylamine N-oxidase activity was characterised in S. maximus. The presence of FMO I and FM02 were indicated by immunorecognition of single bands (c55 kD) using anti-pig liver FMOI and anti-rabbit lung FM02 antibodies. Tissue specific differential expression and activity were determined in the euryhaline flounder Platichthys flesus and a correlation between salinity and branchial FMO activity but not cytochrome P450 content was observed and indicated a potential osmoregulatory role for FMO.