Human cytochromes P45011β (CYP11B1) and P450aldo (CYP11B2) are monooxygenases that synthesize cortisol through steroid 11β-hydroxylation and aldosterone through a three-step process comprising 11β-hydroxylation and two 18-hydroxylations, respectively. CYP11B1 also catalyzes 18-monohydroxylation and 11β,18-dihydroxylation. To study the molecular basis of such catalytic divergence of the two enzymes, we examined a CYP11B1 mutant (Mt-CYP11B1) with amino acid replacements on the distal surface by determining the catalytic activities and crystal structure in the metyrapone-bound form at 1.4 Å resolution. Mt-CY11B1 retained both 11β-hydroxylase and 18-hydroxylase activities of the wild type (Wt-CYP11B1) but lacked 11β,18-dihydroxylase activity. Comparisons of the crystal structure of Mt-CYP11B1 to those of Wt-CYP11B1 and CYP11B2 that were already reported show that the mutation reduced the innermost space putatively surrounding the C3 side of substrate 11-deoxycorticosterone (DOC) bound to Wt-CYP11B1, while the corresponding space in CYP11B2 is enlarged markedly and accessible to bulk water through a channel. Molecular dynamics simulations of their DOC-bound forms supported the above findings and revealed that the enlarged space of CYP11B2 had a hydrogen bonding network involving water molecules that position DOC. Thus, upon positioning 11β-hydroxysteroid for 18-hydroxylation in their substrate-binding sites, steric hindrance could occur more strongly in Mt-CYP11B1 than in Wt-CYP11B1 but less in CYP11B2. Our investigation employing Mt-CYP11B1 sheds light on the divergence in structure and function between CYP11B1 and CYP11B2 and suggests that CYP11B1 with spatially-restricted substrate-binding site serves as 11β-hydroxylase, while CYP11B2 with spatially-extended substrate-binding site successively processes additional 18-hydroxylations to produce aldosterone.

Context: Mutations in cytochrome P450 oxidoreductase (POR) cause a form of congenital adrenal hyperplasia (CAH). We report a novel R550W mutation in POR identified in a 46,XX patient with signs of aromatase deficiency. Objective: Analysis of aromatase deficiency from the R550W mutation in POR. Design, setting, and patient: Both the child and the mother had signs of virilization. Ultrasound revealed the presence of uterus and ovaries. No defects in CYP19A1 were found, but further analysis with a targeted Disorders of Sexual Development NGS panel (DSDSeq.V1, 111 genes) on a NextSeq (Illumina) platform in Madrid and Barcelona, Spain, revealed compound heterozygous mutations c.73_74delCT/p.L25FfsTer93 and c.1648C > T/p.R550W in POR. Wild-type and R550W POR were produced as recombinant proteins and tested with multiple cytochrome P450 enzymes at University Children's Hospital, Bern, Switzerland. Main outcome measure and Results: POR-R550W showed 41% of the WT activity in cytochrome c and 7.7% activity for reduction of MTT. Assays of CYP19A1 showed a severe loss of activity, and CYP17A1 as well as CYP21A2 activities were also lost by more than 95%. Loss of CYP2C9, CYP2C19, and CYP3A4 activities was observed for the R550W-POR. Predicted adverse effect on aromatase activity as well as a reduction in binding of NADPH was confirmed. Conclusions: Pathological effects due to POR-R550W were identified, expanding the knowledge of molecular pathways associated with aromatase deficiency. Screening of the POR gene may provide a diagnosis in CAH without defects in genes for steroid metabolizing enzymes.

BackgroundCytochrome P450 oxidoreductase (POR) deficiency (PORD) is a form of congenital adrenal hyperplasia (CAH) and results in loss of steroid and drug metabolizing cytochrome P450 activities. Mutations in POR cause mild to severe forms of CAH with/without bone malformation. We report a novel POR Arg550Trp mutation identified in a 46, XX patient with signs of CYP19A1 deficiency. Child and mother had signs of virilization. Mother had elevated T on the fifth day that returned to normal in the fourth month. The daughter was born with fused labioscrotal folds. Ultrasound revealed the presence of uterus and ovaries. Sequencing of the CYP19A1 gene did not reveal any defects, and further analysis revealed compound heterozygous mutations c.70_71delTC/p.Leu25PhefsTer93 and c.1648C>T/p.Arg550Trp in POR. At eight years the adrenal function of the patient is normal except for slightly elevated 17‐OH‐prog.MethodsDNA was analyzed with a targeted Disorders of Sexual Development NGS panel (DSDSeq.V1, 111 genes) on a NextSeq (Illumina) platform. We analyzed the ability of POR wild‐type (WT) and Arg550Trp to reduce ferricyanide, MTT, cytochrome c and activity towards the drug and steroid metabolizing cytochromes P450. The POR WT and Arg550Trp variants were expressed and produced as recombinant proteins in bacteria and combined with recombinant P450 proteins and small molecule substrates for enzyme assays.ResultsWe found severe effects of Arg550Trp mutation on activities with different substrates. Arg550Trp showed 41% of the WT activity in cytochrome c and only 7.7% activity towards the reduction of MTT. A 2.75 fold increase in Michaelis constant (Km) was observed in ferricyanide reduction assays compared to WT POR. Severe effect on NADPH binding by R550W mutation was observed using both cytochrome c and MTT as substrates with variable NADPH concentration. Assays of CYP19A1 activity showed loss of >90% activities using androstenedione as substrate.ConclusionThe mutation Arg550Trp is located in the NADPH binding region of POR. Computational analysis predicted instability in the NADPH binding region of POR by R550W mutation, Computationally predicted adverse effect on aromatase activity as well as a reduction in binding of NADPH was confirmed by experiments using recombinant proteins. These results suggest a pathological effect of POR R550W and a diagnosis of PORD in the patient with p.Arg550Trp/p.Leu25PhefsTer93 in POR.Support or Funding InformationSupported by grants to Amit V Pandey from the Swiss National Science Foundation, and Novartis Foundation for Medical Biological Research and to Mónica Fernández‐Cancio and Sara Benito from Instituto de Salud Carlos III, Madrid, Spain.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cytochromes P450 located in the endoplasmic reticulum require NADPH cytochrome P450 oxidoreductase (POR) for their catalytic activities. Mutations in POR cause multiple disorders in humans related to the biosynthesis of steroid hormones and also affect drug-metabolizing cytochrome P450 activities. Electron transfer in POR occurs from NADH to FAD to FMN, and the flexible hinge region in POR is essential for domain movements to bring the FAD and FMN close together for electron transfer. We tested the effect of variations in the hinge region of POR to check if the effects would be similar across all redox partners or there will be differences in activities. Here we are reporting the effects of a POR genetic variant P284T located in the hinge region of POR that is necessary for the domain movements and internal electron transfer between co-factors. Human wild-type and P284T mutant of POR and cytochrome P450 proteins were expressed in bacteria, purified, and reconstituted for enzyme assays. We found that for the P284T variant of POR, the cytochrome c reduction activity was reduced to 47% of the WT and MTT reduction was reduced to only 15% of the WT. No impact on ferricyanide reduction activity was observed, indicating intact direct electron transfer from FAD to ferricyanide, but a severe loss of CYP19A1 (aromatase) activity was observed (9% of WT). In the assays of drug-metabolizing cytochrome P450 enzymes, the P284T variant of POR showed 26% activity for CYP2C9, 44% activity for CYP2C19, 23% activity for CYP3A4, and 44% activity in CYP3A5 assays compared to the WT POR. These results indicate a severe effect on several cytochrome P450 activities due to the P284T variation in POR, which suggests a negative impact on both the steroid as well as drug metabolism in the individuals carrying this variation. The negative impact of P284T mutation in the hinge region of POR seems to be due to disruption of FAD to FMN electron transfer. These results further emphasize the importance of hinge region in POR for protein flexibility and electron transfer within POR as well as the interaction of POR with different redox partners.

Context: Mutations in Cytochrome P450 oxidoreductase (POR) cause a form of congenital adrenal hyperplasia (CAH). We are reporting a novel R550W mutation in POR identified in a 46, XX patient with signs of aromatase deficiency. Objective: Analysis of aromatase deficiency from R550W mutation in POR. Design, Setting, and Patient: Both the child and the mother had signs of virilization. Ultrasound revealed the presence of uterus and ovaries. No defects in CYP19A1 were found, but further analysis with a targeted Disorders of Sexual Development NGS panel (DSDSeq.V1, 111 genes) on a NextSeq (Illumina) platform in Madrid and Barcelona, Spain, revealed compound heterozygous mutations c.73_74delCT/p.L25FfsTer93 and c.1648C>T/p.R550W in POR. WT and R550W POR were produced as recombinant proteins and tested with multiple cytochrome P450 enzymes at University Children’s Hospital, Bern, Switzerland. Main Outcome Measure and Results: R550W POR showed 41% of the WT activity in cytochrome c and 7.7% activity for reduction of MTT. Assays of CYP19A1 showed a severe loss of activity and CYP17A1, as well as CYP21A2 activities, were also lost by more than 95%. Loss of CYP2C9, CYP2C19, and CYP3A4 activities was observed for the R550W-POR. Predicted adverse effect on aromatase activity as well as a reduction in binding of NADPH was confirmed. Conclusions: Pathological effects due to POR R550W were identified, expanding the knowledge of molecular pathways associated with aromatase deficiency. Screening of the POR gene may provide a diagnosis in CAH without defects in genes for steroid metabolizing enzymes.

The vitamin D-3 25-hydroxylase (CYP27A1), 25-hydroxyvitamin D-3 1alpha-hydroxylase (CYP27B1) and 1alpha, 25-dihydroxyvitamin D-3 24-hydroxylase (CYP24A1) are members of the cytochrome P450 superfamily, and key enzymes of vitamin D-3 metabolism. Using the heterologous expression in E. coli, enzymatic properties of the P450s were recently investigated in detail. Upon analyses of the metabolites of vitamin D-3 by the reconstituted system, CYP27A1 surprisingly produced at least seven forms of minor metabolites including 1alpha, 25( OH)(2)D-3 in addition to the major metabolite 25( OH)D-3. These results indicated that human CYP27A1 catalyzes multiple reactions involved in the vitamin D-3 metabolism. In contrast, CYP27B1 only catalyzes the hydroxylation at C-1alpha position of 25(OH) D-3 and 24R, 25(OH)(2)D-3. Enzymatic studies on substrate specificity of CYP27B1 suggest that the 1alpha-hydroxylase activity of CYP27B1 requires the presence of 25-hydroxyl group of vitamin D-3 and is enhanced by 24-hydroxyl group while the presence of 23-hydroxyl group greatly reduced the activity. Eight types of missense mutations in the CYP27B1 gene found in vitamin D-dependent rickets type I (VDDR-I) patients completely abolished the 1alpha-hydroxylase activity. A three-dimensional model of CYP27B1 structure simulated on the basis of the crystal structure of rabbit CYP2C5 support s the experimental data from mutagenesis study of CYP27B1 that the mutated amino acid residues may be involved in protein folding, heme-propionate binding or activation of molecular oxygen. CYP24A1 expressed in E. coli showed a remarkable metabolic processes of 25(OH) D-3 and 1alpha, 25( OH)(2)D-3. Rat CYP24A1 catalyzed six sequential monooxygenation reactions that convert 1alpha, 25(OH)(2)D-3 into calcitroic acid, a known final metabolite of C-24 oxidation pathway. In addition to the C-24 oxidation pathway, human CYP24A1 catalyzed also C-23 oxidation pathway to produce 1alpha, 25(OH)(2)D-3- 26,23-lactone. Surprisingly, more than 70 % of the vitamin D metabolites observed in a living body were found to be the products formed by the activities of CYP27A1, CYP27B1 and CYP24A1. The species-based difference was also observed in the metabolism of vitamin D analogs by CYP24A1, suggesting that the recombinant system for human CYP24A1 may be of great use for the prediction of the metabolism of vitamin D analogs in humans.

Cytochromes P450 proteins are responsible for the metabolism of many steroid hormones as well as drugs and xenobiotics. All cytochromes P450s in the endoplasmic reticulum rely on P450 oxidoreductase (POR) for their catalytic activities. Previously we and others have shown that mutations in POR cause metabolic disorders of steroid hormone biosynthesis and also affect certain drug metabolizing P450 activities. Human POR has distinct subdomains, which bind flavin molecules and interact with redox partners. We studied the mutations identified in flavin mononucleotide (FMN) binding domain of POR that interacts with partner proteins. We found that mutations A115V, T142A located close to the FMN binding site had reduced flavin content compared to wild type POR and lost almost all activity to metabolize androstenedione via CYP19A1 and also showed reduced CYP3A4 activity. The variant A284L identified from normal subjects also had severe loss of both CYP19A1 and CYP3A4 activities, indicating this to be a potentially disease causing mutation. The mutation Q153R initially identified in a patient with disordered steroidogenesis showed remarkably increased activities of both CYP19A1 and CYP3A4 without any significant change in flavin content, indicating improved protein-protein interactions between POR Q153R and some P450 proteins. Such improved activities could be exploited for biotechnological applications to improve product yields. These results indicate that effects of mutations on activities of individual cytochromes P450 can be variable and a detailed analysis of each variant with different partner proteins is necessary to accurately determine the genotype-phenotype correlations of POR variants. Support or Funding Information This work was supported by a grant from the Swiss National Science Foundation (31003A-134926) to Amit V Pandey. The estrogen biosynthesis process requires CYP19A1 activity that is dependent on POR. The deficiency in POR can alter the enzymatic reactions of many cytochrome P450 enzymes and these changes can be variable based on structural properties of the mutation and nature of the redox partner (Pandey, A.V., and Flück, C.E. NADPH P450 oxidoreductase: structure, function, and pathology of diseases. Pharmacol Ther 2013, 138: 229–254). The estrogen biosynthesis process requires CYP19A1 activity that is dependent on POR. The deficiency in POR can alter the enzymatic reactions of many cytochrome P450 enzymes and these changes can be variable based on structural properties of the mutation and nature of the redox partner (Pandey, A.V., and Flück, C.E. NADPH P450 oxidoreductase: structure, function, and pathology of diseases. Pharmacol Ther 2013, 138: 229–254).

All cytochromes P450s in the endoplasmic reticulum rely on P450 oxidoreductase (POR) for their catalytic activities. Mutations in POR cause metabolic disorders of steroid hormone biosynthesis and affect certain drug metabolizing P450 activities. We studied mutations A115V, T142A, Q153R identified in the flavin mononucleotide (FMN) binding domain of POR that interacts with partner proteins and P284L located in the hinge region that is required for flexibility and domain movements in POR. Human wild-type (WT) and mutant POR as well as CYP3A4 and CYP19A1 proteins in recombinant form were expressed in bacteria, and purified proteins were reconstituted in liposomes for enzyme kinetic assays. Quality of POR protein was checked by cytochrome c reduction assay as well as flavin content measurements. We found that proteins carrying mutations A115V, T142A located close to the FMN binding site had reduced flavin content compared to WT POR and lost almost all activity to metabolize androstenedione via CYP19A1 and showed reduced CYP3A4 activity. The variant P284L identified from apparently normal subjects also had severe loss of both CYP19A1 and CYP3A4 activities, indicating this to be a potentially disease causing mutation. The mutation Q153R initially identified in a patient with disordered steroidogenesis showed remarkably increased activities of both CYP19A1 and CYP3A4 without any significant change in flavin content, indicating improved protein-protein interactions between POR Q153R and some P450 proteins. These results indicate that effects of mutations on activities of individual cytochromes P450 can be variable and a detailed analysis of each variant with different partner proteins is necessary to accurately determine the genotype-phenotype correlations of POR variants.

Human mitochondrial CYP11B1 catalyzes a one-step regio- and stereoselective 11β-hydroxylation of 11-deoxycortisol yielding cortisol which constitutes not only the major human stress hormone but also represents a commercially relevant therapeutic drug due to its anti-inflammatory and immunosuppressive properties. Moreover, it is an important intermediate in the industrial production of synthetic pharmaceutical glucocorticoids. CYP11B1 thus offers a great potential for biotechnological application in large-scale synthesis of cortisol. Because of its nature as external monooxygenase, CYP11B1-dependent steroid hydroxylation requires reducing equivalents which are provided from NADPH via a redox chain, consisting of adrenodoxin reductase (AdR) and adrenodoxin (Adx).

The occurrence of lung cancer is associated with smoking, which exposes smokers to a series of carcinogenic chemicals. CYP (cytochrome P450) usually metabolizes carcinogens to their inactive derivatives, but occasionally convert the chemicals to more potent carcinogens. In addition to the metabolism of carcinogenic compounds, CYP also participates in the activation and/or inactivation of anti-carcinogenic agents, suggesting that the local CYP expression in lung cancer and surrounding tissues could be an important determinant of efficacy of anticancer drugs. Furthermore, CYP19 (aromatase), estrogen synthase P450, expressed in more than 80 percent of non-small cell lung cancers. It suggests an association between estrogens and cancer development, which makes aromatase an attractive therapeutic target for the treatment of lung cancer. 1alpha,25-Dihydroxyvitamin D3 has an inhibitory effect on the proliferation of cancer tissues, and is converted to its inactive 24-hydroxylated derivatives by CYP24, which is frequently expressed in lung cancer tissues. Therefore, understanding the CYP expression in tumor tissues is important in developing better therapies for lung cancer, and may lead us to standardized, tailor-made therapies for individuals.

Aldosterone, the most important human mineralocorticoid, is involved in the regulation of the blood pressure and has been reported to play a key role in the formation of arterial hypertension, heart failure and myocardial fibrosis. Aldosterone synthase (CYP11B2) catalyzes the biosynthesis of aldosterone by successive 11β- and 18-hydroxylation followed by an 18-oxidation of 11-deoxycorticosterone and thus comprises an important drug target. For more than 20 years, all attempts to purify recombinant human CYP11B2 in significant amounts for detailed analysis failed due to its hydrophobic nature as a membrane protein. Here, we present the successful expression of the protein in E. coli yielding approx. 90 nmol/l culture, its purification and detailed enzymatic characterization. Biochemical analyses have been performed using in vitro conversion assays which revelead a V(max) of 238±8 nmol products/nmol hCYP11B2/min and a K(m) of 103±8 μM 11-deoxycorticosterone. Furthermore, binding analyses indicated a very loose binding of the first intermediate of the reaction, corticosterone with a K(d) value of 115±6 μM whereas for 11-deoxycorticosterone a K(d) of 1.34±0.13 μM was estimated. Upon substrate conversion of 11-deoxycorticosterone, new intermediates have been identified as 19- and 18-hydroxylated products not described before for the human enzyme. To understand the differences in substrate conversion, we constructed a new homology model based on the 3D structure of CYP11A1, performed docking studies and calculated the activation energy for hydrogen abstraction of the different ligands. The data demonstrated that the 11β-hydroxylation requires much less abstraction energy than hydroxylation at C18 and C19. However, the C18 and C19 hydroxylated products might be of clinical importance. Finally, purified CYP11B2 represents a suitable tool for the investigation of potential inhibitors of this protein for the development of novel drugs against hypertension and heart failure as was shown using ketoconazole.

Steroid 21-hydroxylase (cytochrome P450 21A2, CYP21A2) deficiency accounts for ∼95% of individuals with congenital adrenal hyperplasia, a common autosomal recessive metabolic disorder of adrenal steroidogenesis. The effects of amino acid mutations on CYP21A2 activity lead to impairment of the synthesis of cortisol and aldosterone and the excessive production of androgens. In order to understand the structural and molecular basis of this group of diseases, the bovine CYP21A2 crystal structure complexed with the substrate 17-hydroxyprogesterone (17OHP) was determined to 3.0 Å resolution. An intriguing result from this structure is that there are two molecules of 17OHP bound to the enzyme, the distal one being located at the entrance of the substrate access channel and the proximal one bound in the active site. The substrate binding features locate the key substrate recognition residues not only around the heme but also along the substrate access channel. In addition, orientation of the skeleton of the proximal molecule is toward the interior of the enzyme away from the substrate access channel. The 17OHP complex of CYP21A2 provides a good relationship between the crystal structure, clinical data, and genetic mutants documented in the literature, thereby enhancing our understanding of congenital adrenal hyperplasia. In addition, the location of certain CYP21A2 mutations provides general understanding of structure/function relationships in P450s.

Small compounds that activate the insulin-dependent signaling pathway have potential therapeutic applications in controlling insulin-independent diabetes mellitus. In this study, we investigated whether soybean isoflavones could induce the expression of SHARP-2, a downstream component of insulin-dependent signaling pathway, associated with the regulation of blood glucose. One such compound called genistein, rapidly and temporarily induced SHARP-2 mRNA levels in a dose-dependent manner in rat H4IIE hepatoma cells. This induction process was rapidly stimulated by a protein kinase C (PKC) activator and blocked by a PKC inhibitor, suggesting that SHARP-2 may be induced via PKC activation. Upon Western blot analysis, genistein showed a stimulation of PKC phosphorylation. Therefore, we concluded that genistein might transcriptionally induce SHARP-2 through the activation of PKC in H4IIE cells. Our results suggest that genistein might be a useful dietary supplement to control insulin-independent diabetes mellitus by inducing the SHARP-2 expression via a bypass of the insulin-dependent signaling pathway.

Human aromatase (CYP19, P450arom) is responsible for the conversion of androgens to estrogens. In addition to the estrogen biosynthesis in gonads and adrenals in a classical endocrine manner, this enzyme is widely expressed in various tissues and locally regulates the estrogen level or estrogen/androgen ratio in an intracrine manner. Since estrogen biosynthesis is involved in many essential biological events in humans, aromatase is an attractive target for investigations in basic biomedical and pharmacological sciences. Aromatase is a membrane protein localized on the endoplasmic reticulum, and its instability and hydrophobic nature has hampered the investigation of this important biological system. To investigate the structure function relationship of human aromatase by obtaining quantities of the purified enzyme, we have developed the expression system of human aromatase in E. coli. The human aromatase has two major forms from its genetic polymorphism, arginine (264R) and cysteine (2640) at the amino acid position 264. Although there is only one amino acid difference between the two forms of aromatase, the 264C form was expressed in E. coli with the cold stress response induced by chloramphenicol while the 264R form was expressed by the coexpression of heat shock proteins GroES/GroEL. In the case of human aromatase, a clear protocol is important to determine the expression levels by the reduced CO-difference spectrum. Therefore, the expression methods for the two forms of human aromatase as well as a method for the determination of the reduced CO-difference spectrum will be described.

The first systematic study of the complete cytochrome P450 complement (CYPome) of Sorangium cellulosum So ce56, which is a producer of important secondary metabolites and has the largest bacterial genome sequenced to date, is presented. We describe the bioinformatic analysis of the So ce56 cytochrome P450 complement consisting of 21 putative P450 genes. Because fatty acids play a pivotal role during the complex life cycle of myxobacteria, we focused our studies on the characterization of fatty acid hydroxylases. Three novel potential fatty acid hydroxylases (CYP109D1, CYP264A1, and CYP266A1) were used for detailed characterization. One of them, CYP109D1 was able to perform subterminal hydroxylation of saturated fatty acids with the support of two autologous and one heterologous electron transfer system(s). The kinetic parameters for the product hydroxylation were derived.

Human body might be exposed to acetaldehyde from smoking or occupational environment, which is known to be associated with cancer through the formation of DNA adducts, in particular, N2-ethylidene-2'- deoxyguanosine (N2-ethylidene-dG). Aldehyde dehydrogenase 2 (ALDH2) is the major enzyme that contribute to the detoxification of acetaldehyde in human body. In this study, wild type (Aldh2+/+) and Aldh2KO (Aldh2-/-) mice were exposed to the air containing 0, 125, 500 ppm acetaldehyde for 2 weeks. After inhalation, levels of N2- ethylidene-dG in the chromosomal DNA were analyzed by liquid chromatography tandem mass spectrometry (LC/MS/MS). N2-ethylidene-dG levels in livers of Aldh2-/- mice were always lower than those of Aldh2+/+ mice, suggesting that Aldh2 deficiency might cause the induction of acetaldehyde metabolizing enzymes in the liver such as P450s. The differences between Aldh2-/- and Aldh2+/+ mice were greater in the order of nasal epithelium > lung > dorsal skin, suggesting that nasal epithelium and lung are the major target sites for acetaldehyde. Acetaldehyde inhalation may cause a high risk in nasal epithelium and lung cancers for individuals with inactive ALDH2.

Human aromatase (CYP19) responsible for the conversion of androgens to estrogens is expressed not only in gonads and adrenals but also in many other tissues, including normal lungs and lung cancers. To investigate the involvement of CYP19 in lung cancer development, purified CYP19 protein and antibody are required. In this study, we have developed an efficient expression method of human aromatase in E. coli (>1000 nmol/L culture). The protein purified from E. coli was used to raise an antibody against the human CYP19 in rabbits. The resulting antibody showed a high titer judged by ELISA, which allowed us to determine the expression of CYP19 in non-small cell lung cancer (NSCLC). Of 78 NSCLC specimens from Japanese patients, 50 (64%) NSCLC aberrantly expressed CYP19. This CYP19 expression in NSCLC was independent of any clinical and pathological parameters as well as the expression of other P450s, except tumor stage. The results suggest that the aromatase inhibitors might be useful for the management of non-small cell lung cancer in postmenopausal women.