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. 1999 Jun;1(2):145–153. doi: 10.1038/sj.neo.7900017

Cytochromes P450 are Expressed in Proliferating Cells in Barrett's Metaplasia

Steven J Hughes *, Mark A Morse †, Christopher M Weghorst †, Hyesook Kim ‡, Paul B Watkins §, F Peter Guengerich , Mark B Orringer *, David G Beer *
PMCID: PMC1508133  PMID: 10933049

Abstract

The expression of cytochromes P450 (CYP) in Barrett's esophagus and esophageal squamous mucosa was investigated. Esophagectomy specimens from 23 patients were examined for CYP expression of CYP1A2, CYP3A4, CYP2C9/10, and CYP2E1 by immunohistochemical analysis, and the expression of CYP1A1, CYP3A4, CYP1B1, CYP2E1, and CYP2C9/10 in these tissues was further confirmed by reverse transcription polymerase chain reaction. Immunohistochemical analysis of esophageal squamous mucosa (n = 12) showed expression of CYP1A2, CYP3A4, CYP2E1, and CYP2C9/10 proteins, but it was noted that cells within the basal proliferative zone did not express CYPs. Immunohistochemical analysis of Barrett's esophagus (n = 13) showed expression of CYP1A2, CYP3A4, CYP2E1, and CYP2C9/10 that was prominent in the basal glandular regions, which are areas containing a high percentage of actively proliferating cells. Immunohistochemical staining for both proliferating cell nuclear antigen and the CYPs further supported the colocalization of CYP expression to areas of active cell proliferation in Barrett's esophagus, whereas in the esophageal squamous epithelium, CYP expression is limited to cells that are not proliferating. RT-PCR with amplification product sequence analysis confirmed CYP1A1, CYP3A4, CYP1B1, CYP2E1, and CYP2C9/10 mRNA expression in Barrett's esophagus. These data suggest that the potential ability of cells in Barrett's esophagus to both activate carcinogens and proliferate may be important risk factors affecting carcinogenesis in this metaplastic tissue.

Keywords: malignancy, esophagus, carcinogen, carcinogenesis, PCNA

Introduction

Esophageal adenocarcinoma, which carries a very poor prognosis, is now one of the 15 most prevalent cancers in the United States [1–3]. Barrett's esophagus is a metaplastic columnar epithelium, with three histological subtypes [2] (fundic, junctional, and intestinal) that replaces the normal squamous epithelium of the esophagus in 12% of patients with gastroesophageal reflux disease [4–6]. Barrett's esophagus is associated with an estimated 40-fold increased risk for the development of esophageal adenocarcinoma [2]. It is the intestinal-type Barrett's esophagus that has been shown to be most closely associated with adenocarcinoma development [3,7–10]. We have demonstrated that Barrett's esophagus and esophageal adenocarcinomas express the intestinal-specific, brush-border enzymes sucrase isomaltase [11] and aminopeptidase N [12], providing strong evidence that intestinal-type Barrett's esophagus is not only histologically, but also biochemically similar to the small intestine. However, unlike the small intestine, which has a very low incidence of adenocarcinoma, Barrett's esophagus is at high risk for development into adenocarcinoma, although the reasons for this are unknown.

One possible reason for the difference in the incidence of cancer between the small intestine and Barrett's esophagus may lie in the expression of enzymes that can activate carcinogens. The cytochromes P450 (CYP) are responsible for the metabolism and detoxification of many xenobiotics and endogenous compounds via oxidation [13–16], and these enzymes also may oxidize procarcinogens to reactive electrophilic intermediates capable of inducing mutations. Evidence also exists that CYP electrophilic metabolites can serve as tumor promoters [17,18]. Aflatoxin, nitrosamines such as N'-nitrosonornicotine, and aromatic hydrocarbons such as benzo[a]pyrene are examples of well-studied carcinogens present in tobacco and the diet that are activated by specific forms of CYP enzymes [19–22]. These carcinogens have been specifically shown to induce cancers of the esophagus in rodents [23], and tobacco and alcohol use have been shown to be risk factors for the development of esophageal squamous carcinoma and possibly adenocarcinoma [24,25].

The expression of specific forms of CYP proteins in Barrett's esophagus has not been described. CYP proteins are expressed by cells in the villi of small intestine [26,27] and importantly, localization of CYP proteins within the gut has demonstrated that they are limited to the more well differentiated epithelial cells of the villi, away from areas of active cell proliferation [26,28,29]. In the present studies, we examined whether Barrett's esophagus expresses CYP enzymes and if the pattern of expression differs from that present in the normal intestine or esophageal squamous epithelium. The expression of six different CYP enzymes was determined in normal esophageal squamous mucosa and Barrett's esophagus by using immunohistochemical analysis and reverse transcription polymerase chain reaction (RT-PCR). The specific relationship in these tissues between the proliferative compartment and the areas expressing CYP enzymes was determined using serial sections to compare areas expressing CYPs to areas expressing proliferating cell nuclear antigen (PCNA).

Materials and Methods

Patients

After obtaining written consent, tissue was obtained from 23 patients (21 men, 2 women; 22 whites, 1 African-American) who underwent esophagectomy at the University of Michigan Medical Center from 1988 to 1995. High-grade dysplasia was determined to be present by the final pathology report of the surgical specimens from 11 patients (48%). There were 18 (78%) specimens that contained an associated adenocarcinoma and 5 (22%) specimens without tumors. The patients' medical records were examined retrospectively. Twenty (87%) were smokers, 18 (78%) drank alcohol, and 17 (74%) of patients both smoke and drank alcohol. Eight of the patients (35%) were prescribed omeprazole, and nine (39%) were prescribed histamine antagonists. None of the patients reported the use of other medications known to affect the expression of CYPs. Severe gastroesophageal reflux symptoms were present in 20 (87%). None of the patients had undergone radiation or chemotherapy.

Tissue

Specimens of normal esophagus and Barrett's esophagus were obtained immediately after esophagectomy and placed in Hank's balanced salt solution (Sigma Chemical Co, St. Louis, MO) on ice for transportation to the laboratory. They were then quick frozen in liquid nitrogen and maintained at -70°C until time of assay. Tissues for immunohistochemical analysis were immediately fixed for 24 hours in 10% formalin at room temperature, dehydrated in a series of alcohols, and then processed in paraffin wax.

Antibodies

All anti-CYP antibodies were produced by Great Lakes Environmental and Biomedical Research (GLEBR, Romeo, MI) by immunization of rabbits with 200 µg of human recombinant CYP proteins expressed in Escherichia coli [30], as has been previously described [30–33]. The murine anti-PCNA (Dako, Carpinteria, CA) has been previously used in the analysis of Barrett's esophagus [34].

Conformation of Antibody Specificity

Purified recombinant human CYP proteins (0.1 µg per lane) expressed in E coli, and total protein (10 µg per lane) extract from human liver microsomes. Microsomes were prepared from liver samples obtained from Tennessee Donor Services, Nashville, TN, as previously described [35]. The liver microsomes, previously shown to express multiple CYP isoforms [36], were size-fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nylon membranes. The membranes were probed individually with a 1:1000 dilution of each of the polyclonal antibodies. Immunoreactivity was detected with an alkaline phosphatase-conjugated, donkey anti-rabbit IgG secondary antibody (Jackson ImmunoResearch Laboratory, Inc, West Grove, PA) and developed with a substrate mixture containing 5-bromo-4-chloro-3-indolyl phosphate, p-toluidine, and nitroblue tetrazolium.

Immunohistochemistry

Formalin-fixed, paraffin-embedded specimens were sectioned at 5 µm and placed on poly-l-lysine-treated slides. The slides were then deparaffinized in two changes of xylene (20 minutes each) and then rehydrated down an alcohol gradient. Endogenous peroxidase activity was quenched with 2 changes (20 min each) of 0.3% hydrogen peroxide in phosphate-buffered saline (PBS). For immunostaining with the anti-CYP3A4, anti-CYP1A2, anti-CYP2C9/10, and anti-CYP2E1 antibodies, nonspecific binding was blocked with undiluted goat serum (30 minutes); for anti-PCNA immunostaining, nonspecific binding was blocked with 1:20 horse serum in PBS-1% bovine serum albumin (BSA) (30 minutes). Incubations of primary antibodies were performed at 4°C overnight at the following dilutions in PBS-1% BSA: 1) Anti-CYP1A2 and anti-CYP3A4 antibody, 1:200 dilution; 2) anti-CYP2E1 antibody, 1:300 dilution; 3) anti-CYP2C9/10 antibody, 1:100 dilution; and 4) anti-PCNA antibody, 1:100 dilution. A section of tissue on each slide received only PBS-BSA and no primary antibody and served to identify potential nonspecific immunostaining. Immunoreactivity was developed with the species-appropriate Vectastain detection kit (Vector, Burlingame, CA) per the manufacturers recommendations. The reactions with the chromogens 3,3′-diaminobenzidine (Sigma Chemical Co) or aminoethylcarbazole (Sigma Chemical Co) at 0.1 mg/mL dissolved in PBS were monitored by using light microscopy and quenched in distilled water, and then the sections then lightly counterstained with Harris modified hematoxylin. The slides were then dehydrated up an alcohol gradient and permanently mounted before examination under light microscopy by three independent observers. The amount of immunoreactivity in each sample was assessed and given a number as follows: 0 if no staining was present or 1, 2, or 3 based upon intensity of staining. The localization of staining in the tissues was also recorded.

Reverse Transcription Polymerase Chain Reaction

The expression of CYP1A1, CYP3A4, CYP1B1, CYP2E1, and CYP2C mRNAs was determined by PCR by using reverse transcribed total RNA, as previously described [11]. Ten micrograms of total RNA was reverse transcribed into cDNA using random hexamer primers (125 pmol/reaction, Promega, Madison, WI) in a solution containing 50 mmol/L Tris HCl (pH 8.3), 50 mmol/L KCl, 8 mmol/L MgCl2, 10 mmol/L dithiothreitol, and 1 mmol/L each of dATP, dCTP, dGTP, and dTTP in a total volume of 25 µL. The reaction mixture was heated to 65°C for 5 minutes, cooled to 41°C, and avian myeloblastosis virus reverse transcriptase was added (7 units per reaction, Boehringer Mannheim, Indianapolis, IN). The reaction was allowed to proceed for 1 hour at 41°C.

After first-strand cDNA generation, PCR amplification was done with 5 mL of RT-PCR product added to a mixture of 10 mmol/L Tris HCl; pH 8.3, 50 mmol/L KCl; 2.5 mmol/L MgCl2 (or 1.5 mmol/L for CYP1B1 and CYP3A4 primer sets); 0.2 mmol/L each of dATP, dGTP, dCTP, and TTP; 0.25 pmol/L of each primer; and 0.625 units of Taq DNA polymerase, in a final reaction volume of 25 µL. The reaction mixture was initially heated to 95°C for 30 seconds before 35 thermal cycles with each cycle consisting of 95°C for 15 seconds, 60°C for 30 seconds, and 72°C for 30 seconds, followed by an incubation for 7 minutes at 72°C. The primer sets for CYP enzyme are as follows:

All primer pairs produce amplification products that span introns to control for potential DNA contamination. The amplification of ubiquitin served as a control for mRNA integrity [11]. The PCR amplification products were analyzed on 1.8% agarose gels containing ethidium bromide. The PCR amplification products were purified with Microcon-100 (Amicon, Beverly, MA) centrifugal concentrators, and sequence analysis was performed with an ABI Prism 377 DNA Sequencer (PE Applied Biosystems, Foster City, CA).

Western Blot Analysis

Total tissue protein was extracted in a buffer containing Nonidet P-40 as previously described [39]. One hundred micrograms of total protein extract was fractionated by 12% SDS-PAGE and transferred to nylon membranes. The isoforms of CYP protein were detected with the anti-CYP1A2, anti-CYP3A4, anti-CYP2C9/10, and anti-CYP2E1 antibodies at 1:1000 dilutions, followed by incubation with a peroxidase-conjugated secondary antibody at a 1:10000 dilution (Sigma). Enhanced chemiluminescence (Pierce, Rockford, IL) and Hyperfilm (Amersham, Arlington Heights, IL) were used for detection. Loading of total protein was evaluated by commassie blue staining of acrylamide gels.

Results

Conformation of Antibody CYP Isoform Specificity

Western blot analysis with polyclonal anti-CYP antibodies was used to probe membranes containing 0.1 µg of recombinant human CYP1A1, CYP1A2, CYP3A4, CYP2C9/10, CYP2E1, CYP2D6, and three individual human liver microsomal protein preparations (Figure 1). These analyses showed isoform specific bands of approximately 50 kDa for each antibody. The anti-CYP3A4 antibody also reacted with an approximately 40 kDa protein that was apparent in the purified CYP3A4 protein preparation. This may represent a contaminant or a truncated form of the CYP3A4 protein. The anti-CYP2C9/10 antibody cross-reacted slightly with CYP1A1. None of the antibodies significantly cross-reacted with other proteins present in human liver microsomes.

Figure 1.

Figure 1

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SDS-PAGE of purified recombinant human CYP proteins expressed in E coli and human liver microsome protein extracts known to contain high concentrations of human CYP [36]. Western blot analysis of this membrane using the anti-CYP3A4 antibody, anti-CYP1A2 antibody, anti-CYP2C10 antibody, and anti-CYP2E1 antibody demonstrates isoform specificity for each of these antibodies. The anti-CYP2C10 antibody slightly cross-reacts with CYP1A1 protein. A second band of approximately 40 kDa is present in the lane containing recombinant CYP3A4, and this band also is recognized by the anti-CYP3A4 antibody. None of the anti-CYP antibodies cross-react with other proteins expressed in human liver microsomes.

Identification of Areas of Active Cell Proliferation within Esophageal Squamous Mucosa and Barrett's Esophagus

Immunohistochemistry with the anti-PCNA antibody on sections of esophageal squamous mucosa and Barrett's esophagus specimens showed nuclear staining in 100% of the specimens (n = 14) examined (Figure 2A, B). In Barrett's esophagus, intense nuclear staining was observed in a high percentage of cells within the basal glandular regions, but a few cells within the villi also stained positively for PCNA expression. Squamous epithelium showed intense nuclear staining within a high percentage of cells within the basal, proliferative cell layer, with no staining detected in cell nuclei superficial to this. These patterns of PCNA expression in esophageal squamous mucosa and Barrett's esophagus have been previously described [34] and demonstrate the areas of active cell proliferation within these tissues.

Figure 2.

Figure 2

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Photomicrographs of sections of esophageal squamous mucosa (A) and intestinal-type Barrett's metaplasia (B) after immunohistochemical staining with an anti-PCNA antibody showing nuclear staining of cells within these tissues that are actively proliferating (arrows). In the esophageal squamous mucosa, cell proliferation is limited to the most basal cell regions. In Barrett's metaplasia, cell proliferation is primarily located in the basal glandular regions of the tissue and some of the cells constituting lower portions of the villi (v). (C) A section of terminal ileum following immunohistochemical staining with an anti-CYP3A4 antibody demonstrates cytoplasmic expression of CYP3A4 along the tips of the villi (v) and not in the crypts (c), which are the areas of active cell proliferation (arrows) within the small intestine. (D) Esophageal squamous mucosa expresses CYP3A4, and, similar to the small intestine, cells within the basal areas of active proliferation (arrows) do not express CYP3A4. In contrast, (E) an area of esophagus with intestinal-type Barrett's metaplasia shows expression of CYP3A4 in the basal glandular region, which also show a high percentage of actively proliferating cells (arrows) by positive immunostaining with the anti-PCNA antibody in a near-serial section (F). Mitotic figures are also visible (m). The esophageal squamous epithelium (s), which flanks this intestinal metaplasia, however, does not express CYP3A4 in areas of active cell proliferation (arrow). (G) Immunohistochemical analysis of esophageal squamous mucosa and (H) intestinal-type Barrett's metaplasia with an anti-CYP1A2 antibody showing a similar pattern of expression of the CYP1A2 isotype with intense staining of the glandular regions (g) of the intestinal-type metaplasia but not in the proliferative zone of the squamous mucosa. Arrows denote areas of active cell proliferation. (Original magnifications x 100).

Immunohistochemical Analysis of CYP1A2, CYP2C9/10, CYP2E1, and CYP3A4 Expression

Immunohistochemical analysis using an anti-CYP3A4 antibody showed expression of CYP3A4 protein in a specimen of terminal ileum mucosa, 100% of specimens of intestinal-type Barrett's esophagus (n = 13), and 100% of specimens of squamous epithelium (n = 12) examined. The localization and pattern of immunostaining, however, differed among these types of epithelium (Figures 2C, D and E). In the terminal ileum, epithelial cells along the tips of the villi expressed CYP3A4 protein, but cells within the region of active cell proliferation (crypts) did not. The detection of CYP3A4 with this antibody is consistent with the known distribution of CYP3A4 in this tissue [31]. Similarly, the esophageal squamous mucosa stained homogeneously for CYP3A4 protein expression with the exception of cells constituting the proliferative zone along the basement membrane, which do not express the CYP3A4 protein. In contrast, Barrett's metaplastic columnar epithelium expressed CYP3A4 protein throughout the tissue including the basal, glandular regions that are areas of active cell proliferation [34]. In fact, these glandular regions of the metaplastic epithelium appeared to stain more intensely than the villi in 53% of the samples examined. The separation of CYP3A4 expression and PCNA expression found in esophageal squamous mucosa differed significantly from the colocalization of CYP3A4 expression and PCNA expression observed in intestinal-type Barrett's metaplasia. Near-serial sections of a region containing both types of epithelium and stained for either CYP3A4 or PCNA expression (Figure 2E,F), clearly demonstrate the expression of CYP3A4 in proliferating cells in Barrett's mucosa but not in the esophageal squamous epithelium. No differences were observed in either the intensity or the localization of CYP3A4 staining between specimens from patients based on prior use of tobacco, ethanol, and/or histamine antagonists/omeprazole. Immunohistochemical analysis of the esophageal tissues with the anti-CYP1A2 (Figure 2G,H), anti-CYP2E1, and anti-CYP2C9/10 (Figure 3) antibodies showed identical results to those obtained with the anti-CYP3A4 antibody. Thus, like epithelial cells from the small intestine and esophageal squamous mucosa, intestinal-type Barrett's metaplastic cells express multiple isoforms of CYP. Importantly, this expression of CYPs in Barrett's esophagus colocalizes to areas of active cell proliferation, thus differing from the normal tissues examined in which the regions of active cell proliferation did not express CYPs.

Figure 3.

Figure 3

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Photomicrographs of esophageal tissues immunostained with an anti-CYP2E1 antibody showing expression of CYP2E1 in the superficial squamous cells (s) and not in the proliferative zone (arrows) of esophageal squamous epithelium (A). However, in Barrett's columnar metaplasia (B), CYP2E1 is primarily expressed by cells constituting the glandular regions (g), which are areas of active cell proliferation (arrows). A similar pattern of expression of CYP2C9/10 in esophageal squamous epithelium (C) and Barrett's metaplasia (D) tissues is shown by immunostaining with an anti-CYP2C9/10 antibody. (Original magnifications x 100).

RT-PCR Analysis of CYP1A1, CYP1B1, CYP2E1, and CYP2C9/10 mRNA Expression

To confirm the expression of CYPs by Barrett's esophagus, the expression of CYP1A1, CYP3A4, CYP1B1, CYP2E1, and CYP2C9/10 mRNA was analyzed with RT-PCR. Amplification with specific primers for CYP1A1 produced the expected 237-bp band in all nine samples of Barrett's esophagus examined (Figure 4). The CYP1A1 RT-PCR product was also detected in some normal squamous epithelium as expected and weakly in the gastric mucosa samples [26,37]. The integrity of the mRNA was confirmed in each sample by RT-PCR of ubiquitin, which was identified as a 219 bp product. The amplification products obtained with the CYP1A1, CYP1B1, CYP2E1, and CYP3A4 primer pairs shared 100% homology with the predicted sequences (Genbank) of these CYP isoforms. Sequence analysis of the amplification product obtained with the CYP2C primer pair identified small regions of degenerative sequence, suggesting amplification of the isoforms from the CYP2C family in addition to CYP2C9/10 including CYP2C18, CYP2C19, and CYP2C8. Results of amplifications in normal esophagus and the Barrett's mucosa from the same patients using primers specific for the coding sequences of these CYP isoenzymes are summarized in Table 1.

Figure 4.

Figure 4

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Examination of the expression of mRNA for the CYP1A1 gene by using RT-PCR in specimens of normal esophageal squamous mucosa (N), normal gastric mucosa (G), and Barrett's esophagus mucosa (B) from the same patients. Primers for the human CYP1A1 gene were used, which produce an expected 237-bp product. The integrity of the mRNA is shown by the ability to amplify a 219-bp ubiquitin control gene by RT-PCR.

Table 1.

RT-PCR Analysis of CYP mRNA Expression.

Number of Samples With RT-PCR Product
Tissues CYP1A1 CYP3A4 CYP1B1 CYP2E1 CYP2C
Esophageal squamous 5/7 7/7 7/7 7/7 6/7
Barrett's esophagus 7/7 7/7 6/7 7/7 7/7
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Western Blot Analysis of CYP1A2, CYP2C9/10, CYP2E1, and CYP3A4 Protein

Western blot analysis of total tissue protein extracts from normal squamous esophagus and Barrett's esophagus was performed to further confirm CYP protein expression in these tissues (Figure 5). CYP proteins of appropriate relative molecular mass (Mr) were detected in both normal squamous and Barrett's esophagus specimens by using the anti-CYP1A2, anti-CYP3A4, anti-CYP2C9/10, and anti-CYP2E1 antibodies. The identification of immunoreactive proteins of the expected Mr indicates the staining observed in the immunohistochemical studies is specific for CYP proteins.

Figure 5.

Figure 5

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Western blot analysis of total protein extracts from normal squamous and Barrett's esophagus showing CYP1A2, CYP3A4, CYP2C9/10, and CYP2E1 proteins in these tissues. Purified recombinant CYP isoforms served as positive controls. The bacterial source of these recombinant proteins results in subtle migration differences compared with the proteins from tissue extracts.

Discussion

The major risk factor for development of adenocarcinoma of the esophagus is the presence of the intestinal-type Barrett's esophagus. Why this subtype of Barrett's esophagus is at high risk to develop dysplasia and progress to invasive adenocarcinoma is unknown. This study demonstrates that Barrett's esophagus expresses multiple CYP isoenzymes that are necessary to activate a number of potential carcinogens. This is the first description of expression of CYP enzymes in this important metaplastic mucosa.

We found that Barrett's esophagus has a different cellular distribution pattern of CYP enzyme expression, compared with either the small intestine or squamous esophageal mucosa. In Barrett's esophagus, the most intense staining was present within cells in basal glandular structures that are areas of active cell proliferation. The expression of CYP1A2, CYP3A4, and CYP2E1 isoenzymes, three forms known to activate carcinogens in cells that are actively proliferating [14,15] (the CYP2C9/10 enzyme is not known to activate carcinogens [15]), may be a critical difference between normal tissues that express CYPs and Barrett's esophagus. In normal tissues of the gut, the cells that express CYP1A2 and CYP3A4 enzymes and are thus capable of carcinogen activation are confined within the nonproliferating, well-differentiated regions. These cells are also regularly shed into the esophageal or intestinal lumen, and therefore cells that may sustain DNA damage by CYPs-activated carcinogens, may not pose a carcinogenic threat [28,29]. In contrast, the metaplastic cells within the basal, glandular regions of Barrett's esophagus that can both actively proliferate and express CYP1A2, CYP3A4, and CYP2E1 may be more likely to sustain genetic damage from activated carcinogens and progress towards dysplasia and adenocarcinoma.

The expression of CYPs by Barrett's esophagus was expected. The inducible and constitutive expression of CYP isoenzymes in the normal gut likely serves a vital protective role from ingested toxins [28,29,41], and the expression of CYPs in Barrett's esophagus likely serves a similar role. The distal esophagus of patients with Barrett's esophagus is chronically bathed by enteric contents, increasing exposure time to ingested toxins and possibly inducing the expression of CYPs. In addition, the pharmacological treatment of symptoms from gastroesophageal reflux with omeprazole and/or histamine antagonists may induce CYP expression in Barrett's esophagus, as 74% of the patients in this study were taking one of these drugs. Finally, tobacco use is known to induce CYP1A1 expression in the lungs [42], and 87% of the patients in this study smoked; alcohol is known to induce CYP2E1 expression, and alcohol was consumed regularly by 78% of these patients. However, constitutive expression of these CYP isoforms was identified in specimens of normal squamous and Barrett's esophagus from patients who denied alcohol, tobacco, and histamine antagonists/omeprazole exposure.

The expression of CYPs by Barrett's esophagus may provide a selective advantage. Cells expressing CYPs may be more resistant to damage and death in the hostile environment created by chronic reflux of enteric contents. For this epithelium to regenerate after ulceration, these same cells would need to be capable of proliferation. This mechanism could underlie some of the selection factors that lead to the replacement of squamous cells by Barrett's esophagus after ulceration, and may explain why there is expression of CYPs by Barrett's epithelium in cells within deep glandular structures that actively proliferate.

The presence of CYPs in Barrett's esophagus suggests a potential role for CYP-dependent activation of carcinogens in the development of adenocarcinoma of the esophagus. An approximately twofold increased risk of adenocarcinoma of the esophagus associated with tobacco use [24,25], and at least a subgroup of p53 mutations detected in esophageal adenocarcinoma, may implicate CYP-activated carcinogens [43]. Mutational analysis of the p53 gene in esophageal adenocarcinoma showed G:C to T:A transversions in 20% of the adenocarcinomas examined [43]. Importantly, mutation and/or loss of wild-type p53 allele is a frequent and early event in the development of esophageal adenocarcinomas [44,45]. These findings suggest that CYP-activated carcinogens may be involved in the pathogenesis of at least a subset of Barrett's esophagus adenocarcinomas.

Our study confirms the previous findings of CYP1A1, CYP1A2, and CYP3A4 expression in normal human esophagus [26,40] and further describes apparently constitutive expression of CYP2C9/10 and CYP2E1 in normal esophageal squamous epithelium and Barrett's esophagus. Previous studies have used immunohistochemistry or in situ hybridization to localize CYP expression within the esophagus and the small intestine and demonstrate expression primarily in the well-differentiated cells closer to the luminal surface (tips of villi in the small intestine, flattened squamous cells of the esophagus) [26–29]. Our findings also confirm these previous studies in esophageal squamous mucosa.

This study identified CYP protein expression by immunohistochemistry and Western blot analysis and examined CYP mRNA expression by RT-PCR. Confirmation of CYP expression by bioassay showing specific catalytic activity would further support expression of these CYP isoforms, however, the quantity of tissue necessary for these assays precludes their use on human surgical specimens.

In summary, we have demonstrated the expression of CYP isoenzymes in Barrett's epithelium. Cells within Barrett's esophagus that are actively proliferating express CYP1A2 and CYP3A4, differing from the small intestine and normal esophageal mucosa, which lack expression of these isoenzymes in proliferating cells. This potential ability to activate procarcinogens within cells that actively proliferate may be one reason Barrett's esophagus is prone to the development of dysplasia and adenocarcinoma.

CYP Primers Product (bp) Reference
1A1 Forward: 5′-TCACAGACAGCCTCATTCAC-3′ 432 [37]
Reverse: 5′-GATGGGTTGACCCATAGCTT-3′
1B1 Forward: 5′-ACCCCCAGTCTCAATCTCAAC-3′ 313 [38]
Reverse: 5′-CGTTCGGGCTGAGGCTGGTGC-3′
2C Forward: 5′-GCTAAAGTCCAGGAAGAGATTGA-3′ 332 [37]
Reverse: 5′-TCCTGCTGAGAAAGGCATGAAGT-3′
2E1 Forward: 5′-AGCACAACTCTGAGATATGG-3′ 365 [37]
Reverse: 5′-ATAGTCACTGTACTTGAACT-3′
3A4 Forward: 5′-AACCTGGCTTCTCCTGGCTGTCAG-3′ 353 [37]
Reverse: 5′-ATCTCTTCCATTCTTCATCCTCAGCT-3′
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Acknowledgements

The authors wish to acknowledge the expert technical assistance of Mr. Ted A. Skolarus. This work was supported in part by grant CN-171 from the American Cancer Society and the Roy Weber Research Endowment (D.G.B.), NIH contract ES 42002 (H.K.), NIH grants ES 00267 and CA 44353 (F.P.G.), NIH Training Grant CA 09672, and the Frederick A. Coller Surgical Society (S.J.H.)

Abbreviations

BSA

bovine serum albumin

CYP

cytochrome P450

DTT

dithiothreitol

PBS

phosphate-buffered saline

PCNA

proliferating cell nuclear antigen

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