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Biological Oxidations and P450 Reactions☆

https://doi.org/10.1006/abbi.1997.0339Get rights and content

Abstract

Orthologs of a previously identified CYP1B subfamily designated CYP1B1, which are constitutively expressed in mammary, uterine, and embryonic cells, have previously been functionally linked to 7,12-dimethylbenz[a]anthracene (DMBA) metabolism. A chimeric construct of mouse CYP1B1 in which the 20 NH2-terminal amino acids have been replaced by eight residues from human CYP17 has been expressed inEscherichia coli.This recombinant mouse CYP1B1 (recCYP1B1m) exhibited DMBA metabolism accurately reproducing the characteristic product distribution and specific activity of 3.4 nmol/nmol P450/min seen in C3H10T1/2 cells from which this cDNA has been cloned. The high proportion of 10,11- and 3,4-dihydrodiols and near absence of 5,6-dihyrodiol- and 7-hydroxy-DMBA metabolites are seen only in rodent microsomes where CYP1B1 is highly expressed. This distribution of products from recCYP1B1mwas highly dependent on addition of epoxide hydrolase, particularly the ratio of 3,4-dihydrodiol to 4-phenol metabolites. These characteristics in addition to inhibition by antibodies raised to recCYP1B1mestablish that the CYP1B1 cDNA indeed encodes the P450 responsible for polycyclic aromatic hydrocarbon (PAH) metabolism from C3H10T1/2 cells. DMBA metabolites from cDNA-expressed human CYP1B1 (recCYP1B1h) however, exhibited a different regioselectivity toward DMBA resembling human CYP1A1 catalyzed DMBA metabolism. Reconstitution of recCYP1B1mwith different concentrations of NADPH-P450 reductase indicated a high affinity interaction with an apparentKmof 3 nm. Large PAH such as benz[a]pyrene, benz[e]pyrene, benz[a]anthracene, DMBA, 3-methylcholanthrene, and 1-ethynylpyrene bound to recCYP1B1mwith high affinity (Kd0.08 to 0.22 μM) concomitant with substantial spectral shifts (40% low to high spin state change). Smaller PAHs like pyrene, phenanthrene, and naphthalene neither produced spectral changes nor inhibited the spectral change caused by benz[a]pyrene. Among tested steroids, progesterone bound weakly to recCYP1B1m(Kd> 20 μm) with a comparable spectral shift and was a weak inhibitor of DMBA metabolism, but was not metabolized. While 17β-estradiol is a substrate for human CYP1B1 we have found no evidence for binding to mouse CYP1B1. This data establishes CYP1B1 as an important contributor to activation of PAHs, particularly in extra hepatic tissues that are susceptible to cancer where CYP1B1 in contrast to CYP1A1 is constitutively expressed.

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    ☆

    This work has been presented at the International Congress of Toxicology-VII Seattle, Washington (1995), and the Annual Meeting of the Society of Toxicology, Anaheim, California, 1996.

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    To whom correspondence should be addressed at the Department of Pharmacology, Medical Science Center, University of Wisconsin, 1300 University Avenue, Madison WI 53706. Fax: (608) 262-1257. E-mail: [email protected].

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