[CANCER RESEARCH 60, 3440 –3444, July 1, 2000] Cytochrome P450 1B1 (CYP1B1) Pharmacogenetics: Association of Polymorphisms with Functional Differences in Estrogen Hydroxylation Activity 1 Imad H. Hanna, Sheila Dawling, Nady Roodi, F. Peter Guengerich, and Fritz F. Parl 2 Departments of Biochemistry [I. H. H., F. P. G.] and Pathology[S. D., N. R., F. F. P.], Vanderbilt University Medical Center, Nashville, Tennessee 37232 ABSTRACT Activation of 17-estradiol (E2) through the formation of catechol estrogen metabolites, 2-OH-E2 and 4-OH-E2, and the C-16 hydroxyla- tion product, 16-OH-E2, has been postulated to be a factor in mammary carcinogenesis. Cytochrome P450 1B1 (CYP1B1) exceeds other P450 en- zymes in both estrogen hydroxylation activity and expression level in breast tissue. To determine whether inherited variants of CYP1B1 differ from wild-type CYP1B1 in estrogen hydroxylase activity, we expressed recombinant wild-type and five polymorphic variants of CYP1B1: variant 1 (codon 48Arg3Gly), variant 2 (codon 119Ala3Ser), variant 3 (codon 432Val3Leu), variant 4 (codon453Asn3 Ser), variant 5 (48Gly, 119Ser, 432Leu, 453Ser). The His-tagged proteins were purified by nickel-nitrilo- triacetic acid (Ni-NTA) chromatography and analyzed by electrophoresis and spectrophotometry. We performed assays of E2 hydroxylation activ- ity and quantitated production of 2-OH-E2, 4-OH-E2, and 16-OH-E2 by gas chromatography/mass spectrometry. Wild-type CYP1B1 formed 4-OH-E2 as main product (K m , 40  8 M; k cat 4.4  0.4, min -1 ; k cat /K m , 110 mM -1 min -1 ), followed by 2-OH-E2 (K m , 34  4 M; k cat , 1.9  0.1 min -1 ; k cat /K m , 55 mM -1 min -1 ) and 16-OH-E2 (K m , 39  5.7 M; k cat , 0.30  0.02 min -1 ; k cat /K m , 7.6 mM -1 min -1 ). The CYP1B1 variants also formed 4-OH-E2 as the main product but displayed 2.4- to 3.4-fold higher catalytic efficiencies k cat /K m than the wild-type enzyme, ranging from 270 mM -1 min -1 for variant 4, to 370 mM -1 min -1 for variant 2. The variant enzymes also exceeded wild-type CYP1B1 with respect to 2- and 16- hydroxylation activity. Thus, inherited alterations in CYP1B1 estrogen hydroxylation activity may be associated with significant changes in es- trogen metabolism and, thereby, may possibly explain interindividual differences in breast cancer risk associated with estrogen-mediated car- cinogenicity. INTRODUCTION Metabolic activation of E2 3 has been postulated to be a factor in mammary carcinogenesis. E2 is metabolized via two major pathways: formation of catechol estrogens, the 2-OH and 4-OH derivatives; and C-16 hydroxylation. Two enzymes, CYP1A1 and CYP1B1, are responsible for the hydroxylation to the 2-OH and 4-OH catechol estrogens (i.e., 2-OH-E2 and 4-OH-E2). The 2-OH and 4-OH catechol estrogens are oxidized to semiquinones and quinones. The latter are reactive electrophilic metabolites and are capable of forming DNA adducts (1, 2). Further DNA damage results from quinone-semiqui- none redox cycling, generated by enzymatic reduction of catechol estrogen quinones to semiquinones and subsequent auto-oxidation back to quinones (3– 6). C-16 hydroxylation has also been suggested to be involved in breast carcinogenesis (7, 8). Although other cytochrome P450 enzymes, such as CYP1A2 and CYP3A4, are involved in hepatic and extrahepatic estrogen hydroxy- lation, CYP1A1 and CYP1B1 display the highest levels of expression in breast tissue (9, 10). In turn, CYP1B1 exceeds CYP1A1 in its catalytic efficiency as an E2 hydroxylase and differs from CYP1A1 in its principal site of catalysis (11–13). CYP1B1 has its primary activity at the C-4 position of E2, whereas CYP1A1 has its primary activity at the C-2 position in preference to 4-hydroxylation. The 4-hydroxyla- tion activity of CYP1B1 has received particular attention because of the fact that the 2-OH and 4-OH catechol estrogens differ in carcino- genicity. Treatment with 4-OH-E2, but not 2-OH-E2, induced renal cancer in Syrian hamster (14, 15). Analysis of renal DNA demon- strated that 4-OH-E2 significantly increased 8-hydroxydeox- yguanosine levels, whereas 2-OH-E2 did not cause oxidative DNA damage (16). Similarly, 4-OH-E2 induced DNA single-strand breaks whereas 2-OH-E2 had a negligible effect (17). Comparison of the corresponding catechol estrogen quinones showed that E2–3,4- quinone produced two to three orders of magnitude higher levels of depurinating adducts than E2–2,3-quinone (18). In addition to the induction of renal cancer in the hamster model, 4-OH-E2 is capable of inducing uterine adenocarcinoma, a hormonally related cancer, in mice. Administration of E2, 2-OH-E2, and 4-OH-E2 induced endo- metrial carcinomas in 7, 12, and 66%, respectively, of treated CD-1 mice (19). Finally, examination of microsomal E2 hydroxylation in human breast cancer showed significantly higher 4-OH-E2/2-OH-E2 ratios in tumor tissue than in adjacent normal breast tissue (20). All of these findings support a causative role of 4-OH catechol estrogens in carcinogenesis and implicate CYP1B1 as a key player in the process. Mutations and polymorphisms have both been identified in the CYP1B1 gene. Primary congenital glaucoma, a rare autosomal recessive eye disorder, has been linked to homozygous frameshift and missense mutations in affected Turkish and Saudi Arabian families (21–23). Six polymorphisms of the CYP1B1 gene have been described in the Anglo- American population, of which four result in amino acid substitutions (Table 1; Refs. 23, 24). We described two of these amino acid substitu- tions in exon 3, which encodes the heme-binding domain: codon 432Val3Leu and codon 453Asn3Ser (24). Stoilov et al. (23) described the other two amino acid substitutions in codons 48Arg3Gly and 119Ala3Ser in exon 2. Polymorphisms are inherited and, therefore, dictate exposure levels to metabolites for life. Thus, inherited alterations in the activity of CYP1B1 hold the potential to define differences in estrogen metabolism and, thereby, possibly explain interindividual dif- ferences in breast cancer risk associated with estrogen-mediated carcino- genesis. However, to support this hypothesis, formal proof is needed that these inherited enzyme variants are indeed associated with significant changes in estrogen metabolism. In the present study, we determined whether the polymorphic variants of CYP1B1 differ from wild-type CYP1B1 in 2-, 4-, and 16-estradiol hydroxylation activities. MATERIALS AND METHODS Construction of CYP1B1 Bacterial Expression Plasmid. To facilitate expression and purification of CYP1B1, the hydrophobic NH 2 -terminal 25 amino acids were replaced by six histidine residues. This was accomplished by designing primers to contain BamHI and KpnI sites, respectively, at their 5' ends to allow amplification of wild-type and polymorphic CYP1B1 cDNA. The amplification reaction was carried out with 1 g of cDNA in a 100-l volume containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl 2 ,5 l DMSO, 200 M each of the four deoxyribonucleotides, native Pfu DNA Received 12/14/99; accepted 4/26/00. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported in part by NIH F32 CA79162 (I. H. H.), R35 CA44353 (F. P. G.), and P30 ES00267 (F. F. P., F. P. G.). 2 To whom requests for reprints should be addressed, at Department of Pathology, TVC 4918, Vanderbilt University Medical Center, Nashville, TN 37232. Phone: (615) 343-9117; Fax: (615) 343-9563; E-mail: fritz.parl@mcmail.vanderbilt.edu. 3 The abbreviations used are: E2, 17-estradiol; CYP1B1, cytochrome P450 1B1; CYP1A1, cytochrome P450 1A1; TMS, trimethylsilyl; Ni-NTA, nickel-nitrilotriacetic acid. 3440