Phase I and phase II enzyme activities in Ringed seals (Phoca hispida): characterization of hepatic cytochrome P450 by activity patterns, inhibition studies, mRNA analyses, and western blotting

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 contam inant exposure. In this study activities of hepatic phase I enzymes (cytoch rome 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 activ ities, selective inhibitors, mRNA analyses, as well as Western blotting. Bo th UDPGT and GST activities were present, indicating that these seals may f orm 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 activi ty was low. The activity towards testosterone resulted in several hydroxy-m etabolites. Based on these activity studies the presence of CYP1A, CYP3A, b ut not C-YP2B was insinuated. The inhibition of EROD and caffeine demethyla tion by alpha-naphthoflavone but not by furafylline suggested that in this seal species only one CYP1A enzyme was present. This was supported by the r esults from the mRNA measurements and Western blots. Only one mRNA band cro ss-hybridized with human CYP1A cDNA probes at the fat CYP1A1 position, whil e also one protein band, cross reacting with anti-rat CYP1A, was detected. The selective inhibition of the formation of the testosterone 2 beta- and 6 beta-hydroxy metabolites by ketoconazole supported the suggestion that the formation of these metabolites was mediated by CYP3A. The mRNA measurement s and the results from the Western blots confirmed these results The Northe rn blots showed cross hybridization with human CYP3A cDNA, while in the Wes tern 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 t hat Ringed seal liver contains multiple forms of CYP as well as phase II en zymes, showing different catalytic activities, i.e. EROD, caffeine-N-demeth ylation, and testosterone hydroxylation at different positions. Only one CY P1A 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 pres ence of a CYP2B-like enzyme. However, its functional significance remains u nclear. (C) 1998 Elsevier Science B.V. All rights reserved.