Purfication and Characterization of Two Forms of 2, 3, 4, 7, 8-Pentachlorodibenzofuran-Inducible Cytochrome P-450 in Hamster Liver1

Two forms of cytochrome P-450 (P-450) from liver microsomes of hamsters treated with 2,3,4,7,8-pentachlorodibenzofuran (PenCDF), which possesses the potent acute toxicity and 3-methylcholanthrene (MC)-type inducing ability of liver microsomal monooxygenases in animals, were purified and characterized. These P-450 forms, designated as hamster P-450H and hamster P-450L, had the molecular masses of 52 and 50 kDa, respectively, and showed the absorption maximum of CO-reduced difference spectra at 446 nm. The absolute spectra of their oxidized forms indicated that hamster P-450H was in high-spin state and hamster P-450L was in low-spin state. A part of PenCDF injected into hamster was tightly bound to purified hamster P-450H at a ratio of 0. 107 nmol PenCDF/nmol P-450. In a reconstituted system, both hamster P-450H and hamster P-450L showed relatively low catalytic activities for 3-hydroxylation of benzo[α]pyrene and O-deethylations of both 7-ethoxyresorufin and 7-ethoxycoumarin, while they both catalyzed lα- and 2α-hydroxylations of testosterone effectively to a similar extent. Addition of cytochrome b5 to a reconstituted system accelerated the formation of 7α-hydroxytestosterone 5. 3-fold with hamster P-450L and 2. 2-fold with hamster P-450H. In addition, hamster P-450H catalyzed estradiol 2-hydroxylation at a high rate but hamster P-450L did not. Immunochemical studies using antiserum to each P-450 form revealed that hamster P-450H and hamster P-450L differ from each other and comprise about 61 and 31% of the total P-450 in PenCDF-treated microsomes, respectively, indicating that these are PenCDF-inducible and major forms of P-450 in PenCDF-treated hamsters. Similarly to PenCDF, inducers such as MC, 3, 4, 5, 3', 4' -pentachlorobiphenyl, and isosafrole also preferentially induced hamster P-450H rather than hamster P-450L, but β-naphthoflavone preferentially increased hamster P-450L. Phenobarbital, pregnenolone 16α-carbonitrile and ethanol did not affect the contents of these forms at all. Analyses of NH2-terminal amino acid sequences demonstrated that hamster P-450H and hamster P-450L correspond to rat P-450d and rat P-450a, respectively.