Biological oxidations and P450 reactions. Recombinant mouse CYP1B1 expressed in Escherichia coli exhibits selective binding by polycyclic hydrocarbons and metabolism which parallels C3H10T1/2 cell microsomes, but differs from human recombinant CYP1B1.
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 A chimeric construct of mouse CYP1B1 in which the 20 NH2-terminal amino acids have been replaced by eight residues from human CYP17 has been expressed in Escherichia coli.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 recCYP1B1m was 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 recCYP1B1m establish 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 recCYP1B1m with different concentrations of NADPH-P450 reductase indicated a high affinity interaction with an apparent Km of 3 nM. Large PAH such as benz[a]pyrene, benz[e]pyrene, benz[a]anthracene, DMBA, 3-methylcholanthrene, and 1-ethynylpyrene bound to recCYP1B1m with high affinity (Kd 0.08 to 0.22 microM) 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 microM) with a comparable spectral shift and was a weak inhibitor of DMBA metabolism, but was not metabolized. While 17beta-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.
Savas
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Department of Pharmacology, Medical Science Center, 1300 University Avenue, Madison, Wisconsin 53706, USA.
Carstens
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Jefcoate
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D015127:9,10-Dimethyl-1,2-benzanthracene / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D065633:Cytochrome P-450 CYP1B1; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D004851:Epoxide Hydrolases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D051379:Mice; D008861:Microsomes / Q000201:enzymology; D008969:Molecular Sequence Data; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism; D000072317:Polychlorinated Dibenzodioxins / Q000378:metabolism; D011083:Polycyclic Compounds / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013045:Species Specificity; D013053:Spectrophotometry; D013379:Substrate Specificity
D000072317:Polychlorinated Dibenzodioxins; D011083:Polycyclic Compounds; D011993:Recombinant Fusion Proteins; D015127:9,10-Dimethyl-1,2-benzanthracene; D003577:Cytochrome P-450 Enzyme System; D001189:Aryl Hydrocarbon Hydroxylases; C585496:CYP1B1 protein, human; C585498:Cyp1b1 protein, mouse; D065633:Cytochrome P-450 CYP1B1; D009251:NADPH-Ferrihemoprotein Reductase; D004851:Epoxide Hydrolases