Few reports have been published on the expression pattern of xenobiotic metabolizing CYP enzymes in ocular tissues or in different ocular cell types. Furthermore, almost nothing is known of the regulation of CYP genes in the human eye. In the present study, we characterized the expression profile of the major CYP forms in families CYP1–3 in a nonpigmented ciliary epithelial cell line. Additionally, we elucidated the presence and functionality of the main regulatory pathways involved in the induction of CYP enzymes by chemical compounds.
CYP1A1 and CYP1B1 were found to be expressed in the nonpigmented ciliary epithelial cell line. Both are well known for their ability to metabolize a number of carcinogens, such as polycyclic aromatic hydrocarbons.
22 CYP1A1 and CYP1B1 are only weakly expressed in the human liver but, in contrast, can be found in a number of other tissues (e.g., breast).
22 23 24 25 In ocular tissues, CYP1B1 has previously been detected at mRNA level in the iris, ciliary body, and nonpigmented ciliary epithelial line and at lower levels in the cornea, retinal-pigment epithelium and retina.
26 27 Doshi et al.,
27 using an immunolocalization 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 chromosomal abnormalities in at least 17 different autosomes.
28 In particular, homozygous mutations in the
CYP1B1 gene, which codes CYP1B1 and is located on chromosome 2p22-p21, have been linked to congenital glaucoma.
29
In agreement with previous studies, we could detect a relatively high level of CYP1B1 mRNA expression in the nonpigmented ciliary epithelial line. More important, we showed for the first time the strong induction of CYP1B1 mRNA and protein (and, to a lesser extent, CYP1A1 mRNA) by TCDD in an ocular 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 −910 to −852 containing two AHR binding elements (XRE2 and XRE3) plays an important role in AHR-mediated regulation of
CYP1B1 in these cell lines.
21 In contrast, our current findings indicate that in the nonpigmented ciliary epithelial cells further upstream, AHR binding sites play the predominant role. This suggests cell type specific regulation of
CYP1B1 promoter.
The role of CYP1B1 in the developing eye and in congenital glaucoma is not fully understood.
32 However, it has been suggested that CYP1B1 participates in the development of the iridocorneal angle.
33 In addition,
CYP1B1 has been identified as a modifier gene in primary open-angle glaucoma and rarely as a causative gene in this disorder.
12 Substrates of CYP1B1 include endogenous compounds such as steroids, retinoic acids, and melatonin.
12 Interindividual variability in the metabolism of these or other as yet unidentified substrates may contribute to the pathophysiology of various types of glaucoma. Recently, CYP1B1 was reported to decrease oxidative stress and to promote angiogenesis in retinal endothelial cells.
34 Whether this mechanism could be relevant to iridocorneal angle development remains to be shown. An ability to increase the CYP1B1 expression level could potentially represent an interesting novel treatment strategy. However, use of AHR activation, even locally, is probably limited by toxic effects. The AHR receptor may play an important physiological role during development,
35 and a number of endogenous ligands have been identified for AHR.
36 Whether the AHR-mediated regulation of CYP1B1 plays a significant role during fetal development and contributes to iridocorneal angle development remains 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.
Most CYP forms involved in the metabolism of pharmaceuticals, such as CYP1A2, CYP2Cs, and CYP3A4, were not expressed in the nonpigmented ciliary epithelial cells. This is in line with a recent study in which low levels of CYP mRNAs were observed in the human iris-ciliary body.
5 CYP2D6 was an exception, and some mRNA expression could be detected for this enzyme. CYP2D6 has been reported to be involved in the metabolism of approximately 20% to 30% of all commonly used drugs.
37 38 Recently timolol, which is used to lower intraocular pressure, has been shown to be metabolized mainly by CYP2D6.
11 However, only very low concentrations of timolol metabolites were detected in the human aqueous humor in a few patients after a single dose of ophthalmic timolol.
39 It seems evident that a negligible amount of CYP2D6 protein is expressed in ocular tissues. In the present study, we detected several different splice variants of CYP2D6 in the nonpigmented ciliary epithelial cell line, but not in the liver sample. Previously, Huang et al.
40 41 reported extensive alternative slicing of CYP2D6 in lung and breast tissues. The very low amount of correctly spliced CYP2D6 mRNA may explain the lack of significant CYP2D6 enzyme expression in the eye.
Previously, CYP3A expression has been shown be inducible in the rabbit lacrimal gland by topical treatment with DEX.
14 In the liver, DEX is known to induce CYP3A4 mainly through PXR, which in turn is regulated by GR.
42 However, in fetal hepatocytes and in the lung, DEX appears to regulate members of the CYP3A subfamily directly.
17 43 In the present study, GR, but not PXR, was found to be expressed in the nonpigmented ciliary epithelial cell line. However, DEX did not induce the expression of CYP3A enzymes, possibly because of the lack of PXR or other transcription factors necessary for CYP3A induction or in consequence of repression of
CYP3A genes in this cell type by epigenetic mechanisms.
In conclusion, most drug-metabolizing CYPs were not expressed in the human nonpigmented ciliary epithelial cells. CYP1A1, CYP1B1, and CYP2D6 mRNAs as well as mRNAs for regulatory factors GR, AHR, and ARNT were detected. TCDD induced CYP1B1 expression by transcriptional mechanism, suggesting that an AHR-mediated pathway may play an important role in the regulation of ciliary CYP1B1 expression. AHR activation may thus affect developmental functions of CYP1B1 in the human eye. The human nonpigmented ciliary epithelial cell line used here will offer a useful model to study the regulation of CYP1B1 in the ciliary cell type.
The authors thank Ritva Tauriainen and Päivi Tyni for excellent technical assistance, Miguel Coca-Prados for the nonpigmented human ciliary cell line, and Robert J. Edwards (Section on Experimental Medicine and Toxicology, Imperial College London, London, UK) for the anti–CYP1B1 antibody.