Epidermal CYP2 family cytochromes P450

Toxicology and Applied Pharmacology 195 (2004) 278 – 287 www.elsevier.com/locate/ytaap

Review

Epidermal CYP2 family cytochromes P450 Liping Du, a Susan M.G. Hoffman, b and Diane S. Keeney a,c,d,* a

Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN 37232, USA b Department of Zoology, Miami University, Oxford, OH 45056, USA c VA Tennessee Valley Healthcare System, Nashville, TN 37212, USA d Department of Medicine/Dermatology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA Received 14 August 2003; accepted 16 September 2003

Abstract Skin is the largest and most accessible drug-metabolizing organ. In mammals, it is the competent barrier that protects against exposure to harmful stimuli in the environment and in the systemic circulation. Skin expresses many cytochromes P450 that have critical roles in exogenous and endogenous substrate metabolism. Here, we review evidence for epidermal expression of genes from the large CYP2 gene family, many of which are expressed preferentially in extrahepatic tissues or specifically in epithelia at the environmental interface. At least 13 CYP2 genes (CYP2A6, 2A7, 2B6, 2C9, 2C18, 2C19, 2D6, 2E1, 2J2, 2R1, 2S1, 2U1, and 2W1) are expressed in skin from at least some human individuals, and the majority of these genes are expressed in epidermis or cultured keratinocytes. Where epidermal expression has been localized in situ by hybridization or immunocytochemistry, CYP2 transcripts and proteins are most often expressed in differentiated keratinocytes comprising the outer (suprabasal) cell layers of the epidermis and skin appendages. The tissue-specific transcriptional regulation of CYP2 genes in the epidermis, and in other epithelia that interface with the environment, suggests important roles for at least some CYP2 gene products in the production and disposition of molecules affecting competency of the epidermal barrier. Published by Elsevier Inc. Keywords: Cytochromes P450; CYP; Keratinocyte; Epidermis; Skin

Introduction Enzymes of the cytochrome P450 superfamily have numerous important roles in exogenous and endogenous substrate metabolism (e.g., therapeutic drugs, xenobiotics, fatty acids, eicosanoids, sterols, steroids, vitamins A and D) (Nebert and Russell, 2002). It is now known that many cytochromes P450 (CYP gene products) are expressed in cutaneous tissues and are therefore relevant to dermatotoxicology and human health (Mukhtar, 1992). This review is limited to discussing genes from the large CYP2 gene family, many of which are preferentially expressed in extrahepatic tissues, with an emphasis on the CYP2 genes specifically expressed in epithelial tissues at the environmental interface. Tissue-specific

* Corresponding author. Department of Medicine/Dermatology and Biochemistry, Vanderbilt University, School of Medicine, 607 Light Hall (0146), Nashville, TN 37232-0146. Fax: +1-615-322-4349. E-mail address: [email protected] (D.S. Keeney). 0041-008X/$ - see front matter. Published by Elsevier Inc. doi:10.1016/j.taap.2003.09.020

transcriptional regulation at these sites suggests important roles for at least some CYP2 gene products in epithelial barrier functions in skin and in other organ systems such as the nasal, respiratory, oral, and digestive systems (Ding and Kaminsky, 2003; Ladd et al., 2003; Smith et al., 2003). Historically, cutaneous cytochromes P450 were studied from the perspective of polycyclic aromatic hydrocarbon (PAH) metabolism. Cutaneous PAH metabolism has been a critical component of the multistage model of mouse skin carcinogenesis, the origins of which predate the discovery of cytochromes P450 (in the late 1950s) by at least three decades (reviewed by Conney, 2003; DiGiovanni, 1992; Mukhtar, 1992). Since CYP1 family enzymes are efficient PAH hydroxylases and metabolically activate procarcinogens leading to skin tumors (Ahmad et al., 1996), these have been by far the most extensively studied cytochromes P450 in cutaneous tissues. The activities of cutaneous cytochromes P450 that metabolize foreign compounds were studied extensively throughout the 1980s (reviewed by Bickers, 1991; Hotch-

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kiss, 1992; Kao and Carver, 1991; Merk et al., 1996). At the same time, the multiplicity and diversity of the CYP gene superfamily were being discovered by molecular cloning approaches (Nebert et al., 1991). Two seminal observations from this period were that the epidermis is the major site of drug metabolism in skin (Bickers et al., 1982), and that within the epidermis, activities are greatest in the more differentiated keratinocytes (Reiners et al., 1991). In the 1990s, these advances led to the identification of specific CYP genes expressed in skin, the products of which were likely responsible for the cutaneous drug metabolism reported previously (Mukhtar, 1992). Important advances in the present decade include the identification of cytochromes P450 involved in endogenous substrate metabolism and information on their roles in epithelial differentiation and in vivo functions. Future progress will likely include the identification of cytochromes P450 involved in detoxification and epithelial barrier repair after injury, in response to specific physical, chemical, and biological agents. The CYP2 gene family, which encodes a significant fraction of all cytochromes P450 in vertebrates, encompasses a large number of genes in humans that are classified into 13 subfamilies (Nelson, 2003). The CYP2 subfamilies that contain one or a few member loci generally encode orthologous genes in human and rodent species (CYP2E, 2F, 2G, 2R, 2S, 2T, 2U, 2W). Other CYP2 subfamilies, however, have undergone relatively recent expansions in some mammalian lineages, resulting in variable numbers of loci per species whose orthologous relationships are obscure (CYP2A, 2B, 2C, 2D, 2J). Here we review data reported on expression of specific CYP2 genes in human epidermis, based on nucleic acid and immunocytochemical studies. Results obtained using mouse or rat epidermis are included where they complement or fill voids in the literature. With a few exceptions, relatively little is known about CYP2 genes in other mammalian species (Nelson, 2003). A comprehensive picture of all CYP loci in a given species requires nearly complete genomic sequencing and is therefore currently available only for humans and mice (Nelson et al., 2004).

CYP2A subfamily There are three complete CYP2A genes in humans— CYP2A6, CYP2A7, and CYP2A13—and a pseudogene (CYP2A18P) that is split into two fragments by an insertion. All four loci are part of a gene cluster on chromosome 19q13.2 (Hoffman et al., 2001; Nelson, 2003). Only the CYP2A6 and CYP2A7 genes are expressed in skin. The CYP2A13 gene is expressed and has been studied extensively in other epithelial tissues (Ding and Kaminsky, 2003), but no information is available for skin. The CYP2A7 gene is expressed in

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human skin fibroblasts, but there is yet no evidence for expression in keratinocytes. Dermal fibroblasts express both wild-type and alternatively spliced CYP2A7, which lacks exon 2, but neither of these transcripts resulted in the production of catalytically active CYP2A7 when expressed heterologously (Ding et al., 1995). The alternatively spliced CYP2A7 transcript might encode a defective enzyme, but it is not yet clear whether the wild-type CYP2A7 transcript encodes a defective enzyme or if the substrate for this enzyme has just not yet been identified. CYP2A6 transcripts were detected in five of nine human skin samples analyzed by quantitative ribonuclease protection assay, but not in proliferating, subconfluent cultures of epidermal keratinocytes or in the keratinocytederived HaCaT cell line (Janmohamed et al., 2001). CYP2A7 and CYP2A13 transcripts were not studied. These results suggest that CYP2A6 transcripts are produced in the dermis, not the epidermis. Alternatively, epidermal expression might be limited to terminally differentiated keratinocytes that were not represented in the proliferating cell cultures. Similar results were obtained for proliferating keratinocyte cultures, using reverse transcriptase PCR (RT-PCR). Expression levels of the CYP2A6 and CYP2A7 genes were below the detection limit, while that for CYP2A13 was not studied (Saeki et al., 2002). In a more recent study of 27 individual (male) human skin biopsies, cutaneous CYP2A6 transcript levels were also below the detection limits of a quantitative, real-time RT-PCR assay (Yengi et al., 2003). CYP2A7 and CYP2A13 transcripts were not studied. Since full-thickness skin punch biopsies (dermis and epidermis) were analyzed, expression in dermal fibroblasts, for example, should have been detected. Apparently, cutaneous CYP2A6 gene expression differed in the population studied by Yengi et al. (2003) versus the subset of individuals studied by Janmohamed et al. (2001). Interindividual variations in cutaneous CYP gene expression are common in humans and might be explained by gender differences among donors and other genetic or environmental factors affecting the populations sampled in these studies. There are no immunological or functional data for CYP2A proteins in human skin. A few physiological substrates and natural compounds are metabolized by CYP2A6, in addition to a large number of drugs and synthetic chemicals. Fewer substrates have been identified for CYP2A13 and none for CYP2A7 (Table 1). There are at least three CYP2A genes in both the mouse and rat, none of which is a clear ortholog of any human CYP2A gene (Nelson et al., 2004; Wang et al., 2003). Cutaneous expression of CYP2A genes has not been reported in rodent species, except for one RT-PCR study in which CYP2A1 transcripts were detected in rat skin and shown to be upregulated by treatment with h-naphthoflavone (Mukhtar, 1992).

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Table 1 Examples of known substrates metabolized by human CYP2 enzymesa,b

CYP2B subfamily

Enzyme

Physiological substrates

Natural compounds

CYP2A6

all trans-retinoic acid, arachidonic acid, progesterone

aflatoxin B1, benzophenone, coumarin, diallyl disulfide, nicotine, NV-nitrosonornicotine, psoralen coumarin coumarin, nicotine, ochratoxin A

One CYP2B gene (CYP2B6) and one pseudogene (CYP2B7P) are found in humans, and both loci localize to chromosome 19q13.2 (Hoffman et al., 2001). The CYP2B7P pseudogene is expressed in human lung tissue (Czerwinski et al., 1994), but known sequences contain a premature stop codon, so products arising from this locus presumably would be catalytically inert. Variant and defective CYP2B transcripts, as well as apparently functional ones, were previously characterized in human liver (Miles et al., 1988; Yamano et al., 1989). It is not always clear which of the two genes produced these transcripts. CYP2B6 transcripts were detected in all three of the human skin samples analyzed by Janmohamed et al. (2001), in quantitative ribonuclease protection assays. Epidermal expression was confirmed by the presence of CYP2B6 transcripts in proliferating cultures of human epidermal keratinocytes and HaCaT cells. Epidermal expression also was detected in the RT-PCR study by Baron et al. (2001) but not in that reported by Saeki et al. (2002). Both of these studies analyzed RNA from proliferating (subconfluent) human keratinocyte cultures. These results are consistent with the high degree of interindividual variation in cutaneous CYP2B6 gene expression reported by Yengi et al. (2003). In their realtime RT-PCR analyses, which included full-thickness skin punch biopsies from 27 individuals, CYP2B6 transcripts levels ranged from undetectable to values that were among the highest measured for the 10 expressed CYP genes in their studies. As in human lung, human epidermis contains variant and defective CYP2B transcripts, identified by sequencing cDNAs generated by RT-PCR (P.A. Ladd and D.S. Keeney, unpublished data). Since the CYP2B6 and CYP2B7P genes share extensive sequence identity, likely, the oligonucleotide primers used in all of these studies amplified transcripts arising from both loci. The different cDNA products would be indistinguishable based on predicted molecular sizes. There is no known function for the variant and defective CYP2B transcripts expressed in skin and other human tissues. Results of Western blot analyses and immunocytochemical studies provide further evidence that the CYP2B6 gene is expressed in epidermis, in at least some human individuals. Anti-CYP2B6 interacted with microsomal proteins from proliferating human keratinocyte cultures (Baron et al., 2001). The immunoreactivity was localized to the suprabasal cell layers of human foreskin epidermis (Baron et al., 2001) and to the epidermis, sebaceous glands and hair follicles of adult skin (Baron et al., 1983). If CYP2B6 transcripts are expressed in differentiated suprabasal keratinocytes in situ, then the expression detected in proliferating cultures is likely explained by the ability of cell contact inhibition to induce the differentiation of human keratinocytes grown in monolayer cultures. It is not known whether CYP2B6 functions in endogenous or exogenous metabolism

CYP2A13 CYP2B6

CYP2C8

CYP2C9

CYP2C18 CYP2C19

all trans-retinoic acid, 17h-estradiol, estrone, testosterone 9-cis- and all trans-retinoic acid, retinol, arachidonic and linoleic acids, androstenedione, 17h-estradiol 9-cis- and all trans-retinoic acid, arachidonic and linoleic acids, 5a-androstane-3a,17h-diol, 17h-estradiol, testosterone

all trans-retinoic acid, progesterone 9-cis-retinal, arachidonic and linoleic acids, melatonin, 17h-estradiol, progesterone, testosterone

CYP2D6

all trans-retinal, progesterone, testosterone, tryptamine, 5-methoxytryptamine

CYP2E1

all trans-retinoic acid, fatty acids, 17h-estradiol, estrone, phosphatidylcholine, uroporphyrinogen, prostaglandin H2 none identified arachidonic and linoleic acids, testosterone vitamin D

CYP2F1c CYP2J2 CYP2R1 (Cheng et al., 2003) CYP2S1 (Smith et al., 2003) CYP2U1 (Helvig et al., 2003)

capsaicin, (+, )-limonene, nicotine

capsaicin, diallyl disulfide, galangin, genistein 4V-methyl ether, kaempferide, (+, )-limonene, nicotine, ochratoxin A, tamarixetin (+, )-limonene capsaicin, diallyl disulfide, genistein 4V-methyl ether, (+, )-limonene, nicotine, ochratoxin A, tetrahydrocannabinol aflatoxin B1, capsaicin, curcumin, diallyl disulfide, emetine, genistein 4V-methyl ether, ibogaine, nicotine, ochratoxin A, sparteine aflatoxin B1, capsaicin, diallyl disulfide, diallyl sulfide, genistein, genistein 4V-methyl ether, methyleugenol, nicotine none identified

all trans-retinoic acid

fatty acids

a Rendic (2002) compiled metabolism data for cytochrome P450 enzymes in the CYP2A, 2B, 2C, 2D, 2E, 2F, and 2J subfamilies, through 2001; updated information is found at [world wide web site] http:// www.gentest.com. b Drugs and other synthetic substrates, too numerous to list here, are summarized by Rendic (2002). c Known substrates include foreign compounds (Rendic, 2002).

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in the epidermis. The CYP2B6 gene is expressed in other extrahepatic tissues as well as in the liver, and the encoded enzyme is active toward several physiological and natural compounds, in addition to a large number of xenobiotics (Ekins and Wrighton, 1999; Table 1). The mouse and rat have five and six functional CYP2B genes, respectively, but none appear to be orthologous to the human CYP2B6 gene (Nelson et al., 2004; Wang et al., 2003). In the mouse, cutaneous CYP2Bs were studied initially using antibodies raised against the rat proteins CYP2B1/2B2. Anti-CYP2B1/2B2 interacted with microsomal proteins from mouse skin and liver (Jugert et al., 1994). Topical application of dexamethasone induced the levels of immunoreactivity detected on Western blots, but ethanol and coal tar extracts had no effect. In tissue sections, the anti-CYP2B1/2B2 immunoreactivity localized to keratinocytes in the suprabasal cell layers of interfollicular epidermis and in the hair follicles of mouse skin (Jugert et al., 1994). This result is consistent with the suprabasal localization of proteins that interacted with anti-CYP2B6 in human foreskin epidermis (Baron et al., 2001). The antiCYP2B1/2B2 used in these studies likely interacted with mouse CYP2B19. Cyp2b19 is the major Cyp2b gene expressed in mouse epidermis, and it is a specific gene marker for terminally differentiated keratinocytes. By comparison, relatively low expression levels of the predominantly hepatic Cyp2b genes (e.g., Cyp2b10) were detected by RT-PCR (Keeney et al., 1998a). Transcripts encoding the newly discovered Cyp2b23 gene (Wang et al., 2003) were not detected in epidermis from CD-1 outbred mice by RT-PCR, although low expression levels cannot be ruled out (M. Neis and D.S. Keeney, unpublished data). In situ hybridization studies showed that the Cyp2b19 gene is transcriptionally activated in fetal mouse skin when suprabasal keratinocytes first appear during skin morphogenesis ( c embryonic day 15.5) and that CYP2B19 transcripts co-localize to the same cell layers that express profilaggrin. In the neonate and throughout postnatal development, CYP2B19 transcripts were localized to differentiated keratinocytes in the granular cell layer of interfollicular epidermis, the inner cell layer of hair follicle inner root sheaths, and the sebocytes in sebaceous glands. CYP2B19 is an arachidonic acid epoxygenase. In addition to epoxyeicosatrienoic (EET) acids, it produces lesser amounts of hydroxyeicosatetraenoic acids (Keeney et al., 1998a). The levels of CYP2B19 immunoreactivity detected by Western blotting increased during differentiation in vitro. The CYP2B19 product 14,15-EET increased transglutaminase enzyme activities and the number of cornified cells in differentiating cultures derived from human and mouse epidermis (Ladd et al., 2003). While the mechanisms of action have not yet been elucidated, these results suggest physiological roles for cytochrome P450-derived EETs in regulating epithelial cornification.

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In the rat, anti-CYP2B1/CYP2B2 interacted with proteins in skin microsomes that migrated slightly faster than those in liver microsomes (Pham et al., 1989; Zhu et al., 2002). These Western blot data might be explained by interactions of the antibody with CYP2B12 and CYP2B15, because CYP2B12 and CYP2B15 are the major CYP2B genes expressed in rat skin (Friedberg et al., 1992; Keeney et al., 1998a, 1998b). In situ hybridization studies showed that the CYP2B12 gene is expressed predominantly in sebocytes, the terminally differentiated keratinocytes in sebaceous glands. Like mouse CYP2B19, rat CYP2B12 is an arachidonic acid epoxygenase (Keeney et al., 1998b). The patterns of CYP2B15 gene expression in rat skin are the same as those described for Cyp2b19 in mouse skin, but CYP2B15 enzyme activities have not yet been characterized. Expression of the predominantly hepatic CYP2B1 gene was detected in rat skin by RT-PCR and was upregulated by treatment with h-naphthoflavone (Mukhtar, 1992). It is not understood why mouse skin expresses a single major CYP2B enzyme (CYP2B19), although two (CYP2B12 and CYP2B15) are expressed in rat skin. Mechanisms regulating the transcription of these keratinocyte-specific genes are not known except that expression of all three genes is activated during late differentiation.

CYP2C subfamily Four CYP2C genes (CYP2C8, CYP2C9, CYP2C18, and CYP2C19) are found in humans on chromosome 10q23.31 – 24.33 (Nelson, 2003). There are also eight human CYP2C pseudogenes, all of which are clearly nonfunctional except CYP2C9-de1b, which may serve as an alternative first exon for the CYP2C9 gene (Warner et al., 2001). The structures of these genes make them particularly prone to recombination, conversion, and transplicing events. Many CYP2C transcripts have hybrid sequences derived from two different genes (Finta and Zaphiropoulos, 2000), making the identification of transcripts tenuous at best. Some of the splice variants involving CYP2C18, CYP2C19, and CYP2C8 were characterized in human epidermis (Finta and Zaphiropoulos, 2000; Zaphiropoulos, 1999). These findings should be considered when interpreting results of RT-PCR studies, in the absence of DNA sequence analyses. CYP2C9, CYP2C18, and CYP2C19 transcripts were detected in the 27 individual human skin samples analyzed by quantitative real-time RT-PCR (Yengi et al., 2003). CYP2C18 transcripts were the most abundant, although those encoding CYP2C8 were below the detection limits of this assay. Since this study analyzed full-thickness skin punch biopsies, dermal and epidermal expression cannot be differentiated. However, epidermal expression in at least some humans was demonstrated in the RT-PCR study of Saeki et al. (2002). CYP2C transcripts were detected in proliferating epidermal keratinocytes from five of six

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individuals analyzed, but the oligonucleotide primers used could not distinguish which CYP2C genes were expressed, and DNA sequence data were not reported. Gonzalez et al. (2001) also studied CYP2C19 gene expression in proliferating human epidermal keratinocytes using RT-PCR. CYP2C19 transcripts were detected in clofibrate-treated, but not in control, monolayer cell cultures. Other CYP2C genes were not analyzed. At the genomic level, CYP2C19 alleles were studied in 327 psoriatic patients. Heterozygosity at this locus was associated with increased risk of late onset psoriasis and was protective against psoriatic arthritis (Richter-Hintz et al., 2003). The significance of these correlations is difficult to assess—there are neither immunological data for epidermal CYP2C proteins nor have their physiological functions been defined. An obvious possibility, however, is a role in fatty acid metabolism and eicosanoid formation. Human CYP2C genes are expressed in many other extrahepatic tissues as well as in the liver (Klose et al., 1999). CYP2C proteins metabolize a diverse array of physiological (e.g., fatty acids) and natural compounds, and a very large number of drugs and synthetic chemicals (Table 1). The CYP2C gene subfamily has expanded significantly in the mouse (16 genes) and the rat (at least seven genes). None of these genes can be clearly identified as orthologous to any of the four human genes (Nelson et al., 2004). In mouse tissues, antibodies raised against CYP2C38 interacted with microsomal proteins from skin that were similar in size to those detected in several other extrahepatic tissues. Results of immunocytochemical studies corroborated these Western blot data. The immunoreactivity was localized to the epidermis, hair follicles, and sebaceous glands (Tsao et al., 2001). Since the immunopositive cell layers within these cutaneous structures were not specified, it is not possible to evaluate the stage of differentiation of the CYP2C-expressing cells. In the same study, CYP2C40 transcripts were detected in mouse skin by RT-PCR. It was suggested that this enzyme, which produces mainly 16-hydroxyeicosatetraenoic acids from arachidonate, was responsible for the immunoreactivity observed in situ. In Western blot analyses of rat tissues, a CYP2C13-specific peptide antibody interacted with microsomal proteins from skin that were similar in size to those from liver (Zhu et al., 2002). However, no interactions were observed using anti-CYP2C12 with rat skin microsomes, even though this peptide antibody interacted with proteins in liver microsomes in the same study.

CYP2D subfamily One CYP2D gene (CYP2D6) and two pseudogenes (CYP2D7AP, CYP2D8P) are found in humans. These loci localize to chromosome 22q13.2 (Nelson, 2003). CYP2D6 transcripts were detected by real-time RT-PCR in full-

thickness skin biopsies from 27 human individuals (Yengi et al., 2003). The levels of CYP2D6 transcripts were among the highest measured in this study, out of 10 expressed CYP genes. Saeki et al. (2002) studied CYP2D6 gene expression in different skin cell types by RT-PCR. CYP2D6 transcripts were below detection limits in proliferating subconfluent cultures of epidermal keratinocytes, but they were detectable in dermal fibroblasts. These results suggest that like CYP2A6 and CYP2A7, the dermis expresses the CYP2D6 transcripts. However, it is also possible that terminally differentiated keratinocytes that were not represented in the proliferating cultures do express CYP2D6 transcripts. Immunological data are not available to corroborate these gene expression data. In patients having basal cell carcinoma, homozygosity for wild-type CYP2D6 alleles was associated with the clinical presentation of multiple lesions (Ramachandran et al., 1999). The significance of these correlations is difficult to evaluate because physiological roles for CYP2D6 have not been studied in skin. This enzyme is active toward several physiological substrates and natural compounds, and a vast array of foreign compounds (Table 1). Like the CYP2C subfamily, the CYP2D subfamily has undergone an independent expansion in murine rodents, with seven and nine genes in the rat and mouse, respectively, and many pseudogenes (Nelson et al., 2004). None of these genes is a true ortholog of the human CYP2D6 gene. Little is known of CYP2D gene expression or CYP2D enzyme activities in rodent skin except for one Western blot study in the rat. A CYP2D1-specific peptide antibody interacted with proteins in skin microsomes that were similar in size to those detected in liver microsomes (Zhu et al., 2002). In the same study, another peptide antibody that was specific for CYP2D4 interacted with proteins in skin, but not in liver, microsomes.

CYP2E subfamily Human, mouse, and rat each have a single CYP2E gene, and these genes are considered to be mutually orthologous (Nelson et al., 2004). The human CYP2E1 gene localizes to chromosome 10q26.3 (Nelson, 2003). Three CYP2E1 transcripts (1.8, 2.6, and 4 kb) were detected in human tissues by Northern blot analyses (Botto et al., 1994). Liver expresses mainly 1.8-kb transcripts, although skin expresses lower levels of the 2.6- and 4-kb transcripts. Since there is a single CYP2E1 gene in humans, all three transcripts must arise from this locus. Results of hybridization and PCR studies suggest that expression of the 4-kb transcript involves a putative, upstream site of transcription initiation. The 4- and 2.3kb transcripts contain 3V-noncoding sequences from the CYP2E1 gene that are not present in the 2.6-kb transcript, suggesting alternative polyadenylation sites (Botto et al., 1994).

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The human CYP2E1 gene is expressed constitutively in proliferating keratinocyte cultures and in freshly isolated epidermal cells, as determined by RT-PCR assays (Baron et al., 2001; Gonzalez et al., 2001; Saeki et al., 2002). Cutaneous CYP2E1 transcripts were also detected in fullthickness skin biopsies (Yengi et al., 2003). Epidermal CYP2E1 gene expression has been corroborated by Western blot analyses and immunocytochemical studies. AntiCYP2E1 interacted with microsomal proteins from proliferating keratinocyte cultures, and the immunoreactivity was preferentially localized to the suprabasal cell layers of human foreskin epidermis (Baron et al., 2001). These results are similar to those obtained for CYP2B6. As discussed previously, if CYP2E1 transcripts are expressed in differentiated suprabasal keratinocytes in situ, the immunoreactive CYP2E1 detected in proliferating monolayer cultures is likely explained by the presence of differentiating keratinocytes in the cell contact-inhibited (late subconfluent) cultures. While functions for epidermal CYP2E1 are not known, several physiological and natural substrates and a very large number of foreign compounds are metabolized by CYP2E1 (Table 1). Like human skin, mouse skin expresses CYP2E1 transcripts constitutively (Sampol et al., 1997). AntiCYP2E1 interacted with microsomal proteins from mouse skin, detected by Western blotting. The levels of immunoreactivity in skin increased after dexamethasone or salicylic acid were applied topically or administered intraperitoneally, but these treatments had no effects on the levels of hepatic anti-CYP2E1 immunoreactivity (Jugert et al., 1994; Sampol et al., 1997). In the rat, a CYP2E1-specific peptide antibody interacted with proteins in skin microsomes (Zhu et al., 2002). Primary rat keratinocyte cultures were also analyzed, and the results confirmed that the expression was epidermal. The levels of anti-CYP2E1 immunoreactivity in keratinocyte microsomes increased as the cultures became cell contact-inhibited and began to differentiate. Similar to human epidermis, these Western blot data suggest that epidermal CYP2E1 in the rat is likely expressed in the differentiating suprabasal cell layers (Zhu et al., 2002).

CYP2F and CYP2G subfamilies Two CYP2F and two CYP2G loci are found in humans, only one of which, CYP2F1, is functional. All four loci are within the CYP2 gene cluster on human chromosome 19q13.2 (Hoffman et al., 2001). In humans, CYP2F1P and CYP2G1P are deleted pseudogenes. The human CYP2G2P pseudogene has one or two premature stop codons in all known sequences, but functional alleles may exist in human populations. It is orthologous to the functional CYP2G1 genes in the mouse and rat, which are expressed primarily in nasal mucosa (Sheng et al., 2000).

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Fig. 1. Transcripts encoding the CYP2 genes CYP2J2, CYP2R1, CYP2W1, and CYP2U1 were detected in human epidermal keratinocyte cultures differentiated for 6 days; CYP2F1 transcripts were not detected. Poly(A)+ mRNAs were isolated from cell cultures differentiated in DMEM/F12 media as described by Sando et al. (1996); 20 ng of template (+) or water ( ) was reacted with reverse transcriptase, and the cDNAs were amplified by PCR using gene-specific oligonucleotides. The reaction products were size fractionated on agarose gels and visualized by ethidium bromide; identities were confirmed by DNA sequence analyses and expected product sizes (2F1, 344 bp; 2J2, 336 bp; 2R1, 307 bp; 2W1, 385 bp; 2U1, 451 bp). A 100-bp molecular ladder is shown.

The human CYP2F1 gene is expressed primarily in lung epithelium, and it is orthologous to the single functional CYP2F genes in the mouse, rat, goat, and gorilla (Chen et al., 2002; Wang et al., 1998). Thus far, CYP2F1 transcripts have not been detected in the epidermis or in cultured keratinocytes (Fig. 1). Of 11 human CYP2 gene subfamilies that contain functional loci, CYP2F is the only one in which there is no evidence for dermal or epidermal expression. CYP2F1 is active toward pneumotoxins and tobacco-specific nitrosamines and may have roles in detoxification in epithelia of the nasal and respiratory systems (Ding and Kaminsky, 2003; Table 1).

CYP2J subfamily One CYP2J gene (CYP2J2) is presently recognized in humans and localizes to chromosome 1p32.1 (Nelson, 2003). Differentiating human epidermal keratinocytes express CYP2J2 transcripts in vitro (Fig. 1). These results were corroborated by the interactions of anti-CYP2J2 with proteins in the keratinocyte cell lysates, detected by Western blotting (D.C. Zeldin and D.S. Keeney, unpublished data). While CYP2J2 has not been studied further in skin, the CYP2J2 gene is expressed in many extrahepatic tissues, including most of the major organ systems. The activities of CYP2J2 toward fatty acids have been studied intensively (Scarborough et al., 1999), but no information is available concerning roles for this enzyme in eicosanoid formation in the epidermis. In addition to fatty acids, CYP2J2 is active toward some foreign compounds (Table 1). The mouse has

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eight Cyp2j genes, but none is orthologous to the human CYP2J2 gene (Nelson et al., 2004). The expression of these mouse genes is not fully characterized, but at least some of them are expressed in various epithelial tissues (Xie et al., 2000).

CYP2R, CYP2U, and CYP2W subfamilies One CYP2R gene (CYP2R1), one CYP2U gene (CYP2U1), and one CYP2W gene (CYP2W1) are presently recognized in humans. These loci localize to chromosomes 11p15.1, 4q23, and 7p22.3, respectively (Nelson, 2003). Apparent orthologs of the human CYP2R1, CYP2U1, and CYP2W1 genes are found in the mouse (Nelson et al., 2004). Transcripts encoding all three of these recently discovered CYP2 genes were detected in human epidermal keratinocytes differentiated in vitro (Fig. 1). CYP2R1 has been studied most intensively in the mouse. The Cyp2r1 gene is expressed in mouse skin and several other tissues, but highest expression levels are found in testis and liver, as determined by real-time RT-PCR (Cheng et al., 2003). The human and mouse CYP2R1 genes were cloned and expressed heterologously. Both genes encode microsomal enzymes that have 25-hydroxylase activities toward vitamin D (Cheng et al., 2003). These data suggest that CYP2R1 has important roles in endogenous substrate metabolism. This enzyme might be involved in regulating epithelial differentiation, because the 1,25-dihydroxy metabolite of vitamin D is a pro-differentiating factor in the epidermis (Bikle and Pillai, 1993). Less information exists for CYP2U1 and CYP2W1. Recently, it was reported that the CYP2U1 gene is expressed in thymus, heart, and brain (Helvig et al., 2003; Karlgren and Ingelman-Sundberg, 2003). The only expression data for skin is that shown for differentiating human keratinocytes (Fig. 1). Recombinant CYP2U1 expressed in sf9 insect cells is active toward medium and long chain fatty acids, and it generates mainly 19- and 20-hydroxyeicosatetraenoic acids from arachidonate (Helvig et al., 2003). It is feasible that CYP2U1 functions in endogenous fatty acid metabolism in the epidermis. The only available data for CYP2W1 gene expression are those in Fig. 1 for human keratinocytes, and the activities of CYP2W1 are unknown.

CYP2S subfamily One CYP2S gene (CYP2S1) is found in humans, within the chromosome 19q13.2 CYP2 gene cluster (Hoffman et al., 2001). It has clear orthologs in both the mouse and rat (Nelson et al., 2004). Hybridization studies of Rylander et al. (2001) demonstrated that the CYP2S1 gene is highly expressed in epithelial tissues exposed to the environment, especially in the respiratory and digestive systems, but skin was not studied. Since the tissue distribution of CYP2S1 was similar to that of CYP2A13 and CYP2F1, a common

function in xenobiotic metabolism was suggested for CYP2S1 enzyme activities. These expression data were corroborated by the study of Smith et al. (2003), in which real-time RT-PCR was used to analyze RNA from different human tissues. CYP2S1 transcript levels in extrahepatic tissues were generally greater than those in liver, and digestive tract tissues contained the highest levels. In the same study, CYP2S1 gene expression was studied in fullthickness skin punch biopsies from 26 patients with psoriasis and from unaffected individuals. Like many other CYP2 genes, interindividual variation in the levels of constitutive CYP2S1 gene expression was pronounced. The levels of CYP2S1 and NADPH-cytochrome P450 reductase transcripts were greater in lesional versus non-lesional skin in these patients. These RT-PCR analyses of skin biopsy material could not distinguish whether the CYP2S1-expressing cells were dermal or epidermal constituents. Using a CYP2S1-specific antibody, immunocytochemical data clearly showed that CYP2S1 localizes to the outermost, differentiated cell layers of human epidermis (Smith et al., 2003). In both human and mouse cell lines, dioxin induces CYP2S1 gene expression (Rivera et al., 2002). Analogous to CYP1A genes, the dioxin-induced transcriptional activation required both aryl hydrocarbon receptor and aryl hydrocarbon nuclear translocator proteins in mouse hepatoma (Hepa1c1c7) cell lines (Rivera et al., 2002). The human CYP2S1 gene promoter region contains two xenobiotic response elements, identical to those in CYP1 genes, and many retinoic acid receptor consensus half-site sequences (Smith et al., 2003). To obtain evidence that these response elements are functional, crude coal tar and retinoic acid were applied topically. These treatments induced the levels of CYP2S1 transcripts in some but not all patients and increased the levels of anti-CYP2S1 immunoreactivity in skin biopsy sections, as detected by a CYP2S1-specific peptide antibody. Cutaneous CYP2S1 gene expression also was induced by ultraviolet irradiation in this study. CYP2S1 expressed in E. coli is active toward all trans-retinoic acid, and it generates mainly 4-hydroxy and 5,6-epoxy retinoic acid (Smith et al., 2003). Since CYP2S1 transcript levels were an order of magnitude greater than those of CYP26, even after retinoic acid induction, CYP2S1 activities would potentially have greater impact on cutaneous retinoic acid metabolism. Since retinoic acid inhibits terminal differentiation in the epidermis (Bikle and Pillai, 1993), CYP2S1 may have important roles in mechanisms regulating epithelial differentiation and barrier functions.

CYP2T subfamily Two CYP2T pseudogenes (CYP2T2P, CYP2T3P) are found in humans and localize to the human chromosome 19q13.2 CYP2 gene cluster (Hoffman et al., 2001). It is not known whether these pseudogenes are transcribed in the epidermis or any other tissue. Like CYP2G2P, known

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CYP2T gene sequences contain premature stop codons, but it is possible that functional alleles will be found in future population studies. Orthologous and apparently functional genes are known in the mouse (Cyp2t4) and rat (CYP2T1), but their expression has not been characterized (Hoffman et al., 2001).

Summary Here we have reviewed evidence that at least 13 CYP2 genes (CYP2A6, 2A7, 2B6, 2C9, 2C18, 2C19, 2D6, 2E1, 2J2, 2R1, 2S1, 2U1, and 2W1) are expressed in skin from at least some human individuals and that the majority of these genes is expressed in the epidermis or in cultured keratinocytes. Molecular and cellular studies during the last two decades have corroborated two seminal observations established in studies during the early 1980s. These are that the epidermis is the major site of drug metabolism in skin (Bickers et al., 1982) and that cytochrome P450 activities within the epidermis are greatest in differentiating keratinocytes (Reiners et al., 1991). Results of these early studies are the foundation for our current knowledge of cutaneous cytochromes P450. While it is not always possible to discern dermal versus epidermal expression in more recent studies, where epidermal expression has been localized in situ by hybridization or immunocytochemistry, CYP2 transcripts and proteins are most often expressed in differentiated keratinocytes comprising the outer (suprabasal) cell layers of the epidermis and skin appendages, in humans and rodents. Statistically, 11 of the 13 human CYP2 gene subfamilies contain functional member loci (CYP2A, 2B, 2C, 2D, 2E, 2F, 2J, 2R, 2S, 2U, 2W) (Table 2). Functionality of the CYP2W1 locus is inferred from genomic analyses

Table 2 List of human CYP2 gene subfamilies—summary of those containing functional member loci and evidence for cutaneous versus epidermal expression CYP2 gene subfamilies

Functional loci

Cutaneous expression

Epidermal expression

Member loci expressed

CYP2A

+

+

CYP2B CYP2C

+ +

+ +

+ +

CYP2D CYP2E CYP2F CYP2G CYP2J CYP2R CYP2S CYP2T CYP2U CYP2W

+ + +

+ +

+

CYP2A6, CYP2A7 CYP2B6 CYP2C9, 2C18, 2C19 CYP2D6 CYP2E1

+ + +

+ + +

+ + +

CYP2J2 CYP2R1 CYP2S1

+ +

+ +

+ +

CYP2U1 CYP2W1

285

because no other information is available. The CYP2G and CYP2T gene subfamilies contain only nonfunctional loci, although some functional alleles may exist in human populations. Cutaneous expression has been reported for 10 of the 11 subfamilies that contain functional loci (Table 2). Thus far, there is neither evidence that the CYP2F1 gene is expressed in human skin, nor is there evidence that CYP2A or CYP2D genes are expressed in the epidermis even though transcripts were detected in full-thickness (dermis and epidermal) skin biopsy material. In future studies, it would not be surprising to find that epidermal keratinocytes express CYP2F, CYP2A, and CYP2D genes and more CYP2 member loci than listed in Table 2. The current state of knowledge is still fragmentary because the main source of expression data are derived from RTPCR amplification of RNA templates. The major template sources are either skin biopsies (dermal and epidermal components) or proliferating keratinocytes in monolayer culture. Hence, the identity and differentiation state of the CYP-expressing cells are generally unknown (e.g., keratinocyte, dermal fibroblast, melanocyte, Langerhan). These types of information could be established by future in situ hybridization studies, because this technology provides three-dimensional gene expression data in tissue sections, and it is quite sensitive at detecting gene expression in minority cell types. Valuable new information would also be learned from in vitro differentiation studies, because many CYP2 genes are likely induced during terminal differentiation. Gene expression data should be interpreted cautiously because transcript levels do not necessarily reflect the cellular levels of proteins or enzyme activities. In human skin, CYP gene expression levels vary remarkably among individuals due to genetic and environmental factors (e.g., gender, medical conditions, and drug usage). The literature also reflects inherent experimental variability due in part to limitations in sampling sizes and methods, with respect to human populations. Variability can also be due to differences in anatomic sites sampled, and the sensitivity and specificity of different analytical methods. The specificity of PCR primers toward functional and nonfunctional loci is relevant as there are many examples of expressed CYP pseudogenes. Another important consideration is that gene expression patterns in keratinocyte cultures, especially differentiating cell cultures, are highly dependent on culture conditions and may differ from natural patterns in the epidermis. For major advances in future studies, it will be important to carry out comparative studies under in vivo and in vitro conditions, to identify the CYP-expressing cell types, and to correlate the levels of CYP message, protein, and enzyme activities with each other and with the differentiation state of the CYP-expressing cells. The proteomes of basal, spinous, and granular keratinocytes are expected to differ remarkably and to reflect known differences in the functions of these epidermal cells. If it is known which CYP genes are expressed in which epithelial cell types, correlations of these data with

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cell type-specific functions will be predictive of physiological functions for the respective cytochrome P450 activities. It will also be instructive to compare CYP gene profiles in stratified squamous epithelia having fundamentally different terminal differentiation programs. For example, in future studies, it may be found that different sets of CYP proteins are preferentially expressed in keratinizing (e.g., CYP2B19, 2B15, 2B12) versus non-keratinizing or mucosal (e.g., CYP2A6, 2A13, 2F1) epithelia. Other CYP proteins may be expressed in both (e.g., CYP2J2, 2S1, 2R1) or none (CYP2A7?) of these epithelial types. Conceptually, such studies should reveal fundamental differences in how the barrier properties of different epithelia are regulated and in how the expression and functions of cytochromes P450 are regulated at these sites. In summary, at this juncture, there are insufficient data to conclude which CYP2 proteins are most relevant to epidermal functions during skin development, homeostasis, or exposure to hostile environments that can lead to injury, infection, and disease. By now, most of the human and mouse CYP genes have been sequenced and identified, even though much genomic analysis remains to be done. Recent advances in these genome projects have greatly facilitated the major tasks ahead, which are to identify endogenous substrates and physiological functions for epidermal CYP gene products, and to identify those products having important roles in detoxification and in responses to specific environmental insults. Successful approaches to these problems will provide insight into mechanisms regulating epithelial CYP gene expression and CYP enzyme activities. Intracellular signaling pathways will be identified in which CYP proteins have fundamental roles in the formation and disposition of biologically activated metabolites affecting the differentiation and functions of epithelia (e.g., CYP2R1, CYP2S1, and the keratinocyte-specific CYP2Bs in rodent skin). Because the epidermis and other stratified squamous epithelia directly interface the environment, they are primary sites of disease, including cancer. Undoubtedly, CYP2 proteins will be found to have roles in the production and disposition of molecules affecting the competency of these epithelial barriers. Acknowledgments The authors thank Patricia Ladd and Dr. Mark Neis for technical assistance with keratinocyte cultures and PCR analyses. Grant support for D.S.K.: Department of Veterans Affairs and NIH AR47357. Support and resources from the NIH P30 grants AR41943 and ES00267 facilitated this work. S.M.G.H. was supported in part by NIH R15 GM55951. References Ahmad, N., Agarwal, R., Mukhtar, H., 1996. Cytochrome P-450-dependent drug metabolism in skin. Clin. Dermatol. 14, 407 – 415.

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