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Hepatic phase I and phase II enzymes play an important role in contaminant metabolism in mammals. Knowledge of these enzymes is essential since their presence and activity determines the potential biological effects of contaminant exposure. In this study activities of hepatic phase I enzymes (cytochrome P450 (CYP)) and phase II enzymes (UDP glucuronosyl transferase (UDPGT) and glutathione S -transferase (GST)) in Ringed seals ( Phoca hispida ) were assessed. In addition, CYP enzymes were characterized using catalytic activities, selective inhibitors, mRNA analyses, as well as Western blotting. Both UDPGT and GST activities were present, indicating that these seals may form the reactive methylsulfonated PCB metabolites. The results from the CYP characterization showed ethoxyresorufin- O -deethylation (EROD) and caffeine demethylation activity, while the pentoxyresorufin- O -depenthylation activity was low. The activity towards testosterone resulted in several hydroxy-metabolites. Based on these activity studies the presence of CYP1A, CYP3A, but not CYP2B was insinuated. The inhibition of EROD and caffeine demethylation by α -naphthoflavone but not by furafylline suggested that in this seal species only one CYP1A enzyme was present. This was supported by the results from the mRNA measurements and Western blots. Only one mRNA band cross-hybridized with human CYP1A cDNA probes at the rat CYP1A1 position, while also one protein band, cross reacting with anti-rat CYP1A, was detected. The selective inhibition of the formation of the testosterone 2 β - and 6 β -hydroxy metabolites by ketoconazole supported the suggestion that the formation of these metabolites was mediated by CYP3A. The mRNA measurements and the results from the Western blots confirmed these results. The Northern blots showed cross hybridization with human CYP3A cDNA, while in the Western blots one protein band cross-reacting with anti-rat CYP3A was detected. No cross hybridization with rat CYP2B1/2 cDNA was observed. However, the Western blots showed a band cross-reacting with anti-rat CYP2B, suggesting the presence of a CYP2B-like protein. In conclusion, this study has shown that Ringed seal liver contains multiple forms of CYP as well as phase II enzymes, showing different catalytic activities, i.e. EROD, caffeine- N -demethylation, and testosterone hydroxylation at different positions. Only one CYP1A isoform seemed to be present as well as a CYP3A-like isoform. Although the catalytic activities and mRNA analyses did not indicate the presence of a CYP2B-like protein in Ringed seals, the Western blots suggested the presence of a CYP2B-like enzyme. However, its functional significance remains unclear. |
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