Expression of (CYP) in Human Nonpigmented Ciliary Epithelial Cells: Induction of CYP1B1 Expression by TCDD

Marjo Volotinen,1,2 Jukka Ma¨enpa¨a¨,2 Esko Kankuri,1 Olli Oksala,2 Olavi Pelkonen,3 Miki Nakajima,4 Tsuyoshi Yokoi,4 and Jukka Hakkola3

PURPOSE. Cytochrome P450 (CYP) enzymes metabolize endog- ytochrome P450 (CYP) enzymes are a superfamily of enous compounds such as steroid hormones, fatty acids, and Cheme-containing proteins important in oxidative metabo- xenobiotics, including drugs and carcinogens. Expression of lism.1,2 CYP enzymes are responsible for the metabolism of CYP enzymes in ocular tissues is poorly known. However, endogenous substances such as steroid hormones and fatty mutations in the CYP1B1 have been linked to congenital acids and of xenobiotics including drugs and carcinogens.1 glaucoma. The aim of the present study was to investigate the Fifty-seven cytochrome P450 have been identified in the expression and regulation of cytochrome P450 enzymes in a , whereas the CYP1, CYP2, and CYP3 families human nonpigmented ciliary epithelial cell line. contribute primarily to the metabolism of drugs.2 CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 are the most impor- METHODS. Expression of mRNAs for major xenobiotic metabo- tant CYPs responsible for the metabolism of most drugs.1 lizing CYPs in families 1–3 and regulatory factors involved in CYP enzymes are most highly expressed in the liver. How- the induction of CYPs was studied using reverse transcriptase– ever, they are known to be present in many other tissues, such polymerase chain reaction. For induction studies, the cells as in small intestine, lung, and kidney.3 The expression and were treated with dexamethasone or 2,3,7,8-tetrachlorodi- regulation of CYP enzymes in ocular tissues is poorly known. benzo-p-dioxin (TCDD) for 24 hours. RNA and immunoblotting High expression of CYP1B1 mRNA has been reported in the analysis were used to study CYP induction. Transcriptional human eye in the nonpigmented epithelium of the ciliary body regulation of CYP1B1 gene was studied by transient transfec- and in the iris.4 Very low levels of other CYP expression have tion of reporter gene constructs. recently been detected in human ocular tissues.5 RESULTS. mRNAs of CYP1A1, CYP1B1, and CYP2D6 and of the Several antiglaucoma medicines and many other ophthalmic regulatory factors aryl hydrocarbon receptor (AHR), aryl hy- drugs used topically are substrates, inhibitors, or inducers of drocarbon receptor nuclear translocator, and glucocorticoid CYP enzymes. For example, the anti-inflammatory and analge- receptor were expressed in the human nonpigmented ciliary sic agent diclofenac is a substrate of CYP2C9.6 Dexamethasone epithelial cell line. CYP1B1 mRNA was strongly and dose de- (DEX) is a CYP3A4 substrate and a potent inducer of CYP3A4 pendently induced by TCDD. CYP1B1 protein was detected activity.7–9 One widely used glaucoma medicine, the ␤-adren- only after TCDD treatment of the human nonpigmented ciliary ergic blocking agent timolol, is a CYP2D6 substrate, and the epithelial cells. CYP1B1 promoter was activated by TCDD. The antibiotic chloramphenicol is an inhibitor of CYP3A4.10,11 major drug-metabolizing enzymes CYP1A2, CYP2Cs, and However, scant information is available regarding whether the CYP3As were not detected in these cells, and dexamethasone major drug-metabolizing CYPs are expressed in ocular tissues. treatment had no effect on CYP expression. The presence and activity of CYP enzymes may significantly affect the efficacy and safety of ophthalmic drugs. CONCLUSIONS. TCDD potently induces CYP1B1 mRNA in human Among the xenobiotic-metabolizing CYP enzymes in families nonpigmented ciliary epithelial cells, suggesting the involve- CYP1 to CYP3, CYP1B1 is the only member directly linked to a ment of an AHR-mediated pathway in the regulation of ciliary disease. CYP1B1 has been found to be a causative disease gene for CYP1B1 expression. (Invest Ophthalmol Vis Sci. 2009;50: primary congenital glaucoma and a modifier gene, or rarely a 3099–3105) DOI:10.1167/iovs.08-2790 causative gene, in primary open angle glaucoma.12 Furthermore, CYP1B1 null mice exhibit abnormalities in their ocular drainage structure and trabecular meshwork, which are similar to those in 12 From the 1Institute of Biomedicine, Pharmacology, University of patients with primary congenital glaucoma. Helsinki, Helsinki, Finland; 2Santen Oy, Tampere, Finland; 3Depart- An important characteristic of many xenobiotic-metabolizing ment of Pharmacology and Toxicology, University of Oulu, Oulu, CYP enzymes is adjustment to chemical environment by induc- Finland; and 4Drug Metabolism and Toxicology, Faculty of Pharmaceu- tion. This induction is mediated primarily by intracellular xenobi- tical Sciences, Kanazawa University, Kanazawa, Japan. otic-sensing receptors, including pregnane X receptor (PXR), con- Supported by Santen Oy, the Academy of Finland (Contracts stitutive androstane receptor (CAR), and aryl hydrocarbon 110591 and 125216), and the Sigrid Juse´lius Foundation. Submitted for publication August 29, 2008; revised February 13, receptor (AHR). AHR functions as a heterodimer with aryl hydro- 2009; accepted May 14, 2009. carbon receptor nuclear translocator (ARNT). Furthermore, glu- Disclosure: M. Volotinen, Santen Oy (E, F); J. Ma¨enpa¨a¨, Santen cocorticoid receptor (GR) may also be involved in some induction Oy (E, F); E. Kankuri, None; O. Oksala, Santen Oy (E, F); O. phenomena.13 DEX induces the expression of CYP3A6 in Pelkonen, None; M. Nakajima, None; T. Yokoi, None; J. Hakkola, the rabbit lacrimal gland.14 Almost nothing is known regarding None the regulation of CYP enzymes in the human eye. The publication costs of this article were defrayed in part by page The aim of the present study was to investigate the charge payment. This article must therefore be marked “advertise- ment” in accordance with 18 U.S.C. §1734 solely to indicate this fact. expression and regulation of CYP enzymes in a human Corresponding author: Jukka Hakkola, Department of Pharmacol- nonpigmented ciliary epithelial cell line. We show that few ogy and Toxicology, University of Oulu, POB 5000 (Aapistie 5B), 90014 CYP forms are expressed in this cell line. More important, University of Oulu, Oulu, Finland; jukka.hakkola@oulu.fi. we show for the first time that CYP1B1 is inducible in a

Investigative Ophthalmology & Visual Science, July 2009, Vol. 50, No. 7 Copyright © Association for Research in Vision and Ophthalmology 3099

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ciliary cell line by an AHR receptor ligand, 2,3,7,8-tetrachlo- Reverse Transcriptase–Polymerase rodibenzo-p-dioxin (TCDD). Chain Reaction Each PCR reaction contained 1 ␮L cDNA (of a total 15 ␮L), 2.5 ␮Lof10ϫ ␮ METHODS PCR reaction buffer (Finnzymes, Espoo, Finland), 0.5 L dNTP (10 mM) reaction mixture (Finnzymes), 1 ␮L sense and antisense primer (10 ␮M), Human Ciliary Epithelial Cells 0.5 ␮L DNA polymerase (DyNAzyme II, 2 U/mL; Finnzymes), and water to a final volume of 25 ␮L. The primers (Sigma-Genosys, Cambridge, UK) The nonpigmented human ciliary cell line, developed from primary used in the study are presented in Table 1. The CYP2C8–19 primers 15,16 cultures of nonpigmented human ciliary epithelium, was a gener- detect all CYP2C8, CYP2C9, CYP2C18, and CYP2C19. ous gift from Miguel Coca-Prados (Yale University, New Haven, CT). PCR cycles were conducted as follows: 1 minute at 94°C, then 35 This cell line has many characteristics of normal nonpigmented human cycles for 10 seconds at 94°C, 10 seconds at annealing temperature, 30 ciliary epithelium. The nonpigmented human ciliary cells (passages seconds at 72°C, and a final extension of 5 minutes at 72°C. The 5–10) were cultured in Dulbecco modified Eagle medium (Sigma- annealing temperature used for each primer pair is shown in Table 1. Aldrich, Steinheim, Germany) supplemented with 10% (vol/vol) fetal Every series of PCR reactions contained a negative control to exclude bovine serum (Invitrogen, Paisley, Scotland, UK) and 1% penicillin- contamination and a human liver cDNA sample as positive control. streptomycin. Cells were cultured in standard conditions at 37°C, 5% After PCR amplification, 8 ␮L reaction mixture was electrophoresed CO2, and saturated humidity. into 0.5% agarose gel and stained with ethidium bromide. All PCR reactions were repeated at least twice. Induction Studies Nearly confluent cells were treated with DEX (Sigma-Aldrich) and CYP2D6 Sequencing TCDD (National Cancer Institute Chemical Carcinogen Repository, Agarose gel was prepared with low melting point agar (Agarose Prep; Bethesda, MD) for 24 hours in serum-free Dulbecco modified Eagle Amersham Biosciences AB, Uppsala, Sweden), and the CYP2D6 RT-PCR medium. Inducers were dissolved in dimethyl sulfoxide (DMSO). Con- products were run on the gel. The three most prominent bands were centrations used in screening studies (100 nM for DEX, 10 nM for extracted from the gel with an extraction kit (Qiaquick Gel Extraction Kit; TCDD) were selected based on our previous studies showing that Qiagen GmbH, Hilden, Germany) according to the manufacturer’s proto- these concentrations are sufficient to cause strong GR- and AHR- col. Purified DNA products were then subjected to direct sequencing mediated inductions.17,18 Control cells were incubated with an equal (Biocenter Oulu DNA Sequencing Core Facility, Oulu, Finland). volume of DMSO for 24 hours. After 24-hour exposure, the cells were collected and used for RNA or protein extraction. Northern Blot Analysis Five micrograms of the total RNA was electrophoretically resolved and Extraction of RNA and cDNA Synthesis transferred onto a nylon membrane (Hybond-Nϩ; Amersham Bio- Nearly confluent cells were washed twice with phosphate-buffered sciences, Little Chalfont, UK). RNA was fixed by ultraviolet light cross- ␣32 saline and were collected into a small volume of lysis buffer for RNA linking, and the membrane was hybridized with [ P] dCTP-labeled extraction. Total RNA was extracted from cultured ciliary epithelial probes. CYP1A1 and CYP1B1 cDNA probes were prepared as previ- 19 cells with reagent (TRI reagent; Sigma-Aldrich) according to the man- ously described ; the 18S probe was kindly provided by Heikki Rus- ufacturer’s protocol. The purity and concentration of the isolated RNA koaho (University of Oulu, Finland). was measured spectrophotometrically, and mRNA was stored at Ϫ70°C until cDNA synthesis. Immunoblot Analysis Complementary DNA (cDNA) was synthesized with a cDNA syn- Cultured cells were washed and collected into phosphate-buffered saline. thesis kit (First-Strand; Amersham Biosciences, Buckinghamshire, UK). The cells were broken by sonication and centrifuged. Sixty micrograms of One microgram RNA was used in each synthesis. Negative control was the 10,000g supernatant protein was subjected to SDS-PAGE (7.5% % prepared by making a cDNA synthesis reaction without RNA. The polyacrylamide). CYP1B1 supersomes (0.1 ␮g; BD Biosciences, San Jose, cDNAs and the negative control were stored at Ϫ20°C until PCR CA) were used as recombinant control. Proteins were transferred electro- amplifications were performed. phoretically to a polyvinylidene difluoride transfer membrane (Immo-

TABLE 1. Description of the Primers Used for PCR Analysis and Annealing Temperature for Each Primer

Annealing Temperature (Gene Sense Primer Sequence (5؅ 3 3؅) Antisense Primer Sequence (5؅ 3 3؅) (°C

CYP1A1 TCACAGACAGCCTGATTGAG GATGGGTTGACCCATAGCTT 58 CYP1A2 TGGCTTCTACATCCCCAAGAAAT TTCATGGTCAGCCCGTAGAT 57 CYP1B1 AACGTACCGGCCACTATCAC ACGACCTGATCCAATTCTGC 55 CYP2A6 GTGTGGACATGATGCCGT AGGACTTGAGGCGGAAGT 55 CYP2B6 CCATACACAGAGGCAGTCAT GGTGTCAGATCGATGTCTTC 58 CYP2C8-19 GCTAAAGTCCAGGAAGAGATGA TCCTGCTGAGAAAGGCATGAAGT 58 CYP2D6 GGAGATCGACGACGTGATAG ACCAGGAAAGCAAAGACACC 62 CYP2E1 AGCACAACTCTGAGATATGG ATAGTCACTGTACTTGAACT 58 CYP3A4 CCAAGCTATGCTCTTCACCG TCAGGCTCCACTTACGGTGC 58 CYP3A5 TGTCCAGCAGAAACTGCAAA TTGAAGAAGTCCTTGCGTGTC 57 CYP3A7 CTATGATACTGTCTACAGT TCAGGCTCCACTTACGGTCT 55 AHR GGACTTGGGTCCAGTCTAATGCAC AGCCAGGAGGGAACTAGGATTGAG 56 ARNT CCCTAGTCTCACCAATCGTGGATC GTAGCTGTTGCTCTGATCTCCCAG 56 CAR AGATGAGCTGAGGAACTGTG CAGCATCTAAGCACTTCTGC 55 GR GGAGTTTTTCTTCTGGGTCCC GAGAGCTTACATCTGGTCTC 55 PXR AGCTGGAACCATGCTGACTT TCCTTCTTCATGCCGCTCTC 55

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bilon; Millipore, Bedford, MA) in a semidry transfer cell (Trans-Blot SD; Bio-Rad Laboratories Inc., Hercules, CA) at 15 V for 30 minutes. The membrane was blocked in 5% milk in Tris-buffered saline/Tween 20 buffer overnight. The sheet was then incubated for 1 hour with an antibody raised against a human CYP1B1 pentapeptide20 (1:1000 dilution) and for 1 hour with a secondary peroxidase-conjugated goat anti–rabbit antibody (Sigma-Aldrich, St. Louis, MO) (1:25,000 dilution). After washing, the immunoreactive bands were visualized with chemiluminescent per- oxidase substrate (Sigma-Aldrich).

Plasmids and Transfection Assay Preparation and site-directed mutagenesis of the CYP1B1 5Ј-luciferase reporter constructs have been described previously.21 pGL3-basic plas- mids contained the human CYP1B1 gene 5Ј-flanking region from Ϫ2299 to ϩ25, from Ϫ910 to ϩ25, and from Ϫ852 to ϩ25. Plasmids Ϫ910/XRE3 mt and Ϫ910/XRE2 mt have mutations in AHR-binding sites. Ciliary epithelial cells were seeded into a 24-well plate and cultured for 24 hours before transient transfection using reagent (Tfx- 20; Promega, Madison, WI) according to the manufacturer’s protocol. Cells in each well were transfected with 0.5 ␮g CYP1B1/luc reporter gene plasmid and 0.1 ␮g control reporter plasmid pRL-TK (Promega) in 200 ␮L medium (Opti-MEM I; Invitrogen). Twenty-four hours after transfection, the cells were treated with 100 nM TCDD of vehicle (DMSO) only. After 24-hour treatment, luciferase activities were mea- sured by the Dual-Luciferase Reporter Assay System (Promega).

RESULTS Detection of CYP mRNA Expression in Human Nonpigmented Ciliary Epithelial Cell Line Expression of several CYP genes in families CYP1–3 was stud- ied by an RT-PCR method. The presence of a band of the correct size in agarose gel was regarded as evidence for . mRNAs of CYP1A1 and CYP1B1 were detected in the human nonpigmented ciliary epithelial cell line and the human liver (Fig. 1). mRNAs of CYP1A2, CYP2A6, CYP2B6, CYP2Cs, CYP2E1, CYP3A4, CYP3A5, and CYP3A7 were not detected in the ciliary epithelial cell samples, but all were present in the control liver sample. RT-PCR for CYP2D6 mRNA produced a single band of cor- rect size for the liver sample, as expected. In contrast, in the ciliary epithelial cell sample, four separate bands were de- tected (Fig. 2A). Of these, band number four was of expected size, but the three other products were larger. To establish the identity of these PCR products, the three most prominent FIGURE 1. Detection of cytochrome P450 mRNAs in the human non- bands (bands 1, 2, 4) were extracted from the gel and se- pigmented ciliary epithelial cell line by RT-PCR. Liver RNA was used as quenced. CYP2D6 sense and antisense primers were designed positive control. Ciliary, nonpigmented ciliary epithelial cell line. to target sequences at exons 7 and 9, respectively. Band 4, of similar size as the liver band, represented correctly sliced CYP2D6 mRNA. Band 1 contained the introns between exons AHR-mediated regulatory pathways could be functional in the 7 to 8 and 8 to 9, and band 2 contained the intron between current human nonpigmented ciliary epithelial cell line, exons 7 to 8 but not between exons 8 to 9. Band 3 was not whereas the CAR and PXR pathways were not. sequenced, but its size suggested that it might have contained a product with the intron between exons 8 to 9 but not Induction of CYPs in Human Nonpigmented between exons 7 to 8 (Fig. 2B). Ciliary Epithelial Cell Line Detection of CYP-Regulating Receptor mRNA Given that the RT-PCR results suggested that GR- and AHR- Expression in Human Nonpigmented Ciliary mediated regulatory pathways might have been functional in the cell line studied, we next treated the cells with DEX and Epithelial Cell Line TCDD, which are prototypic ligands for GR and AHR, respec- Expression of mRNAs for major regulators of CYP induction, tively. Expression of all studied CYP forms was then screened including the nuclear receptors CAR, PXR, and GR, and for with conventional RT-PCR. Although the qualitative RT-PCR basic helix-loop-helix factors AHR and ARNT was studied by an method used was not designed to produce true quantitative RT-PCR method. All these mRNAs could be detected in the data, it could be used to detect substantial differences in mRNA liver, as expected. In the ciliary epithelial cell sample GR, AHR, amounts or for the detection of mRNAs detectable only after and ARNT were readily detected, whereas CAR and PXR were treatment with an inducing agent. Expression of CYP1A1 and not present (Fig. 3). These findings suggested that the GR- and CYP1B1 mRNAs was strongly induced by 24-hour treatment

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FIGURE 2. (A) Detection of cyto- chrome P450 CYP2D6 mRNA in the human nonpigmented ciliary epithe- lial cell line by RT-PCR. Four different PCR products amplified from the cil- iary epithelial cells are numbered. Liver was used as positive control. (B) Schematic presentation of the splicing variants detected in the hu- man nonpigmented ciliary epithelial cell line. Bands 1, 2, and 4 in (A) were extracted from the gel and se- quenced. Band 3 was not sequenced, and the variant 3 is hypothetical, based on size of the band.

with 10 nM TCDD but not by a similar exposure to 100 nM ing elements; several have been shown to bind AHR in vitro.21 DEX (Fig. 4). Neither TCDD nor DEX induced expression of Previous studies in several cell lines suggested that the region any other CYP forms studied. from Ϫ910 to Ϫ852 containing two AHR-binding elements (XRE2 and XRE3) plays an important role in the AHR-mediated Induction of CYP1B1 mRNA by TCDD regulation of CYP1B1.21 However, in our present study, 5Ј Ϫ To further establish the induction of CYP1 forms by TCDD in deletion of the CYP1B1 promoter to 910 strongly reduced human nonpigmented ciliary epithelial cells by an accurate quan- TCDD induction, indicating that in the human ciliary epithelial titative method, we carried out a dose-response experiment and cell line, the more upstream AHR sites play the major role in Ј detected the mRNAs with Northern blot analysis. The level of TCDD induction. The 5 deletions of the promoter or muta- CYP1B1 mRNA was increased statistically significantly and dose- tions of the two XRE sites had no major effect on the consti- dependently by 1 to 100 nM TCDD (Fig. 5). The CYP1A1 mRNA tutive activity of the CYP1B1 promoter in the human ciliary could not be detected by Northern blot analysis in any of the epithelial cell line studied. samples, indicating that the expression level of CYP1A1 was much lower than that of CYP1B1 in this cell line. DISCUSSION Induction of CYP1B1 Protein Expression by TCDD Few reports have been published on the expression pattern of xenobiotic metabolizing CYP enzymes in ocular tissues or in Human nonpigmented ciliary epithelial cells were treated with different ocular cell types. Furthermore, almost nothing is 10nM TCDD or vehicle (DMSO) only for 24 hours. The pres- known of the regulation of CYP genes in the human eye. In the ence of CYP1B1 protein was analyzed by immunoblotting with present study, we characterized the expression profile of the human anti–CYP1B1 antibody. A band similar in size to the major CYP forms in families CYP1–3 in a nonpigmented ciliary recombinant CYP1B1 was detected in the TCDD-treated sam- epithelial cell line. Additionally, we elucidated the presence ple, but no clear protein expression was detected in the un- and functionality of the main regulatory pathways involved in treated cells (Fig. 6). the induction of CYP enzymes by chemical compounds. Transcriptional Regulation of the CYP1B1 Gene CYP1A1 and CYP1B1 were found to be expressed in the nonpigmented ciliary epithelial cell line. Both are well known by TCDD for their ability to metabolize a number of carcinogens, such as To further study regulatory mechanisms of CYP1B1 induction polycyclic aromatic hydrocarbons.22 CYP1A1 and CYP1B1 are by TCDD in human ciliary cells, we transfected CYP1B1 5Ј- only weakly expressed in the human liver but, in contrast, can luciferase reporter constructs into the human ciliary epithelial be found in a number of other tissues (e.g., breast).22–25 In cell line. TCDD induced CYP1B1 Ϫ2299/ϩ25 promoter frag- ocular tissues, CYP1B1 has previously been detected at mRNA ment–regulated luciferase activity 5.7-fold, indicating transcrip- level in the iris, ciliary body, and nonpigmented ciliary epithe- tional regulation of the CYP1B1 gene by TCDD (Fig. 7). The lial line and at lower levels in the cornea, retinal-pigment current promoter fragment contained eight putative AHR-bind- epithelium and retina.26,27 Doshi et al.,27 using an immunolo-

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FIGURE 3. Detection of glucocorticoid receptor (GR), constitutive an- drostane receptor (CAR), pregnane X receptor (PXR), aryl hydrocar- bon receptor (AHR), and aryl hydrocarbon receptor nuclear transloca- tor (ARNT) mRNAs in the human nonpigmented ciliary epithelial cell line by RT-PCR. Liver was used as positive control.

calization method, also suggested that CYP1B1 protein is present in the fetal and adult nonpigmented ciliary epithelium. CYP1B1 may have many functions in human ocular tissues, and it has been linked especially to various forms of glaucoma. Congenital glaucoma has been reported to be associated with FIGURE 5. Effect of TCDD on the expression of CYP1B1 mRNA in the chromosomal abnormalities in at least 17 different auto- human nonpigmented ciliary epithelial cell line. Cells were treated somes.28 In particular, homozygous mutations in the CYP1B1 with different concentrations of TCDD or vehicle (DMSO) only for 24 gene, which codes CYP1B1 and is located on hours, after which the RNA was extracted. (A) Five micrograms of total 2p22-p21, have been linked to congenital glaucoma.29 RNA was electrophoresed, blotted, and hybridized with human In agreement with previous studies, we could detect a rela- CYP1B1 and rat 18S probes. (B) Densitometric quantification of tively high level of CYP1B1 mRNA expression in the nonpig- CYP1B1 mRNA blot. Values are normalized against 18S control levels, and mean Ϯ range of two or three normalized samples are compared mented ciliary epithelial line. More important, we showed for the with those of untreated cells (0 nM TCDD). Difference from untreated first time the strong induction of CYP1B1 mRNA and protein cells ***P Ͻ 0.001 and **P Ͻ 0.01 (one-way ANOVA followed by LSD (and, to a lesser extent, CYP1A1 mRNA) by TCDD in an ocular post hoc test). cell type. TCDD induction of CYP1B1 is mediated by AHR, and AHR may also play a role in constitutive expression of CYP1B1.30,31 Previous studies in MCF-7 (breast carcinoma), HepG2 (hepatocellular carcinoma), LS-180 (colon carcinoma), and OMC-3 (ovarian carcinoma) cell lines suggested that the region from Ϫ910 to Ϫ852 containing two AHR binding elements (XRE2 and XRE3) plays an important role in AHR-mediated regu- lation of CYP1B1 in these cell lines.21 In contrast, our current findings indicate that in the nonpigmented ciliary epithelial cells

FIGURE 6. Effect of TCDD on the expression of CYP1B1 protein in the FIGURE 4. Effect of TCDD and DEX on the expression of CYP1A1 and human nonpigmented ciliary epithelial cell line. Cells were treated with CYP1B1 in the human nonpigmented ciliary epithelial cell line. Cells were 10 nM TCDD or vehicle (DMSO) only for 24 hours. Sixty micrograms of treated with 10 nM TCDD, 100 nM DEX, or vehicle (DMSO) for 24 hours, the 10,000g supernatant protein was subjected to SDS-PAGE. Proteins after which the RNA was extracted and CYP1A1 and CYP1B1 mRNA were transferred to PVDF membrane and stained with CYP1B1 antibody. expression was detected by an RT-PCR. Liver was used as positive control. REC, 0.1 ␮g CYP1B1 supersome used as recombinant control.

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FIGURE 7. Effect of TCDD on transcriptional regulation of CYP1B1. A series of CYP1B1 5Ј-luciferase reporter constructs, together with pRL-TK control plasmid, was transfected into the human nonpigmented ciliary epithelial cell line. Cells were treated with 100 nM TCDD or vehicle (DMSO) for 24 hours, after which they were harvested and luciferase activities were measured. The activities produced by the studied CYP1B1 promoter constructs were normalized against the cotransfected control plasmid (pRL-TK) activ- ities. Values represent mean Ϯ SD of four individual samples. Differences between TCDD-treated samples and DMSO-treated samples. **P Ͻ 0.01 (Student’s t-test).

further upstream, AHR binding sites play the predominant role. Previously, CYP3A expression has been shown be inducible This suggests cell type specific regulation of CYP1B1 promoter. in the rabbit lacrimal gland by topical treatment with DEX.14 In The role of CYP1B1 in the developing eye and in congenital the liver, DEX is known to induce CYP3A4 mainly through glaucoma is not fully understood.32 However, it has been sug- PXR, which in turn is regulated by GR.42 However, in fetal gested that CYP1B1 participates in the development of the irido- hepatocytes and in the lung, DEX appears to regulate members corneal angle.33 In addition, CYP1B1 has been identified as a of the CYP3A subfamily directly.17,43 In the present study, GR, modifier gene in primary open-angle glaucoma and rarely as a but not PXR, was found to be expressed in the nonpigmented causative gene in this disorder.12 Substrates of CYP1B1 include ciliary epithelial cell line. However, DEX did not induce the endogenous compounds such as steroids, retinoic acids, and mel- expression of CYP3A enzymes, possibly because of the lack of atonin.12 Interindividual variability in the metabolism of these or PXR or other transcription factors necessary for CYP3A induc- other as yet unidentified substrates may contribute to the patho- tion or in consequence of repression of CYP3A genes in this physiology of various types of glaucoma. Recently, CYP1B1 was cell type by epigenetic mechanisms. reported to decrease oxidative stress and to promote angiogenesis In conclusion, most drug-metabolizing CYPs were not ex- in retinal endothelial cells.34 Whether this mechanism could be pressed in the human nonpigmented ciliary epithelial cells. relevant to iridocorneal angle development remains to be shown. CYP1A1, CYP1B1, and CYP2D6 mRNAs as well as mRNAs for An ability to increase the CYP1B1 expression level could poten- regulatory factors GR, AHR, and ARNT were detected. TCDD tially represent an interesting novel treatment strategy. However, induced CYP1B1 expression by transcriptional mechanism, use of AHR activation, even locally, is probably limited by toxic suggesting that an AHR-mediated pathway may play an impor- effects. The AHR receptor may play an important physiological tant role in the regulation of ciliary CYP1B1 expression. AHR role during development,35 and a number of endogenous ligands activation may thus affect developmental functions of CYP1B1 have been identified for AHR.36 Whether the AHR-mediated reg- in the human eye. The human nonpigmented ciliary epithelial ulation of CYP1B1 plays a significant role during fetal develop- cell line used here will offer a useful model to study the ment and contributes to iridocorneal angle development remains regulation of CYP1B1 in the ciliary cell type. an important subject for future studies. Furthermore, exposure to exogenous AHR ligands such as environmental contaminants may disturb physiological regulation of CYP1B1 in the human eye. Acknowledgments Most CYP forms involved in the metabolism of pharmaceu- ticals, such as CYP1A2, CYP2Cs, and CYP3A4, were not ex- The authors thank Ritva Tauriainen and Pa¨ivi Tyni for excellent tech- pressed in the nonpigmented ciliary epithelial cells. This is in nical assistance, Miguel Coca-Prados for the nonpigmented human line with a recent study in which low levels of CYP mRNAs ciliary cell line, and Robert J. Edwards (Section on Experimental Med- were observed in the human iris-ciliary body.5 CYP2D6 was an icine and Toxicology, Imperial College London, London, UK) for the exception, and some mRNA expression could be detected for anti–CYP1B1 antibody. this enzyme. CYP2D6 has been reported to be involved in the metabolism of approximately 20% to 30% of all commonly used References drugs.37,38 Recently timolol, which is used to lower intraocular pressure, has been shown to be metabolized mainly by 1. Sikka R, Magauran B, Ulrich A, Shannon M. Bench to bedside: CYP2D6.11 However, only very low concentrations of timolol pharmacogenomics, adverse drug reactions, and the cytochrome metabolites were detected in the human aqueous humor in a P450 system. Acad Emerg Med. 2005;12(12):1227–1235. few patients after a single dose of ophthalmic timolol.39 It 2. Wilkinson GR. and variability among patients in drug response. N Engl J Med. 2005;352(21):2211–2221. seems evident that a negligible amount of CYP2D6 protein is 3. Attar M, Shen J, Ling K-HJ, Tang-Liu D. Ophthalmic drug delivery expressed in ocular tissues. In the present study, we detected considerations at the cellular level: drug-metabolising enzymes and several different splice variants of CYP2D6 in the nonpig- transporters. Expert Opin Drug Deliv. 2005;2(5):891–908. mented ciliary epithelial cell line, but not in the liver sample. 40,41 4. Coca-Prados M, Escribano J. New perspectives in aqueous humor Previously, Huang et al. reported extensive alternative secretion and in glaucoma: the ciliary body as a multifunctional slicing of CYP2D6 in lung and breast tissues. The very low neuroendocrine gland. Prog Retin Eye Res. 2007;26(3):239–262. amount of correctly spliced CYP2D6 mRNA may explain the 5. Zhang T, Xiang CD, Gale D, Carreiro S, Wu E, Zhang E. Drug lack of significant CYP2D6 enzyme expression in the eye. transporter and CYP P450 mRNA expression in human ocular

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