GASTROENTEROLOGY 1992;103:1509-1516

Induction of Cytochrome P45OIA Genes (CYPZA) by Omeprazole in the Human Alimentary Tract W. MICHAEL

MCDONNELL,

JAMES

M. SCHEIMAN,

and PETER

G. TRABER

Division of Gastroenterology, Department of Internal Medicine, University of Michigan School of Medicine and The Ann Arbor Veterans Administration Medical Center, Ann Arbor, Michigan

Cytochrome P45O enzymes are capable of converting procarcinogens into either active mutagens or inactive metabolites. Because the distribution of these enzymes may be important for tissue susceptibility to procarcinogens, the expression and induction of CYPZA genes in the human alimentary tract were investigated. Endoscopic biopsy specimens were obtained from buccal mucosa, esophagus, gastric body, antrum, duodenum, and colon of 6 healthy volunteers before and 1 week after taking 20 mg of omeprazole daily. Tissue specimens were analyzed for the presence of CYPlAl and lA2 transcripts using hybridization methods and the polymerase chain reaction. P450-dependent enzymatic activity was assessed by deethylation of ethoxyresorufin. CYPlAl messenger RNA (mRNA) and ethoxyresorufin activity were present constitutively in the duodenum of each volunteer. Omeprazole (20 mg/day for 1 week) induced CYPlAl mRNA and enzymatic activity in 5 of 6 volunteers. The one individual who did not initially respond had a marked increase in both mRNA and enzymatic activity after receiving 60 mg of omeprazole daily for 1 week. After treatment with omeprazole, two individuals had low levels of CYPlAl mRNA in several other alimentary tissues as well as low levels of CYPlA2 mRNA in the duodenum. The expression and induction by a pharmaceutical agent of CYPZA genes may have implications for intestinal metabolism of ingested xenobiotics including procarcinogens. he cytochrome P450 gene superfamily (CYP)* encodes for multiple P45O enzymes that metabolize a broad range of endogenous and exogenous substrates.’ These microsomal enzymes convert environ-

T

* Cytochromes P450 in this report are named according to the recent recommendations for the standardization of P450 gene nomenclature.’ Italicized CYP for cytochrome P450 refers to the gene or cDNA and nonitalicized CYP refers to the P450 messenger RNA or protein.

mental organic compounds (xenobiotics) to either stable metabolites or to intermediate compounds that undergo additional metabolism by other enzyme systems. Some compounds are converted to highly reactive intermediates that covalently bind to cellular macromo1ecules.2 The majority of known procarcinogens are converted by P450-dependent reactions into electrophiles that covalently bind DNA. The alimentary tract is exposed to a large variety of dietary xenobiotics.3 How epithelial cells lining the alimentary tract metabolize these compounds may be important for tissue susceptibility to specific environmental procarcinogens and may have an affect on the systemic absorption of xenobiotics including drugs. In this regard, it is of interest that there is wide variability between alimentary tissues in the propensity to develop carcinoma. Cytochrome P4501Al (CYPZAZ) and IA2 (CYP1A2), the two known members of the CYPZA gene family, activate and/or inactivate a broad range of procarcinogens including polyaromatic hydrocabons (e.g., benzo[a]pyrene),4 arylamines,’ and aflatoxin Bl.’ CYPlAl is inducible by many of its substrates as well as xenobiotics found in cruciferous vegetables such as flavones and indoles. CYPZA gene products have not been conclusively identified in the human alimentary tract. Aryl hydrocarbon hydroxylase activity, one of the known enzymatic reactions of CYPlA enzymes, has been measured in small intestine by several investigators.7f8 However, identification of CYPlA proteins in human small intestine using antibodies has yielded conflicting results.g*‘o Furthermore, studies in rat have shown that enzymatic activity for the same P450 may differ depending on the tissue in which it is expressed.*’ The aims of this study were to determine if CYPlA genes are expressed in human alimentary tissue and to determine if levels are induced by a pharmacological agent. We chose to use omeprazole, a H+/K+aden0 1992 by the American Gastroenterological 0016-5085/92/$3.00

Association

1510 MCDONNELL ET AL.

osine triphosphatase inhibitor, as the inducing agent. This drug was recently shown to induce CYPIA genes in human hepatocytes,” although these findings have been controversia1.13*14 We used Northern blot hybridization, quantitative dot blots, and RNA polymerase chain reaction (PCR) to identify CYPlA transcripts in endoscopic biopsy specimens throughout the alimentary tracts of normal volunteers before and after administration of omeprazole. The ethoxyresorufin deethylase assay, a measure of CYPlA enzyme activity, was performed on duodenal samples and correlated with the presence of CYPlAl transcripts. Taken together, our results show that CYPZAZ is constitutively expressed in duodenum and inducible by omeprazole. Other tissues have variable expression of CYPlAl after omeprazole treatment. CYPlA2 was expressed in low levels in duodenum of two individuals after omeprazole. These results may have implications for the metabolic capacity of the alimentary tract mucosa. Materials and Methods Subjects Six healthy volunteers, 3 men and 3 women, between the ages of 25 and 35 years were studied. All were nonsmokers who abstained from caffeinated beverages, tobacco products, and medications 3 days before each endoscopy. After an overnight fast, upper endoscopy was performed and 2-mm pinch biopsy samples4-5 were obtained from the buccal mucosa, esophagus, gastric body, gastric antrum, and duodenum distal to the ampulla of Vater for RNA analysis. An additional four biopsy samples were taken from the antrum and duodenum for analysis of enzymatic activity. A flexible sigmoidoscopy was also performed with biopsy samples obtained from the sigmoid colon. The volunteers then took omeprazole, 20 mg/day, for 7 days and underwent repeat endoscopy. Volunteers were midazolam for sedated with up to 10 mg of intravenous endoscopy. The protocol and consent form were approved by The University of Michigan Institutional Review Board.

RNA Extraction, Northern Blot, and Quantitative Slot Blot Analysis RNA was extracted according to the method of Chomczynski and Sacchi.” Four biopsy specimens from each tissue were placed in 750 pL of 4-mol/L guanidinium isothiocyanate, 25-mmol/L sodium citrate (pH 7.0), O.lmol/L 2-mercaptoethanol, and 0.5% sarcosyl and then were immediately placed in liquid nitrogen for later processing. Total RNA of 30-100 pg per sample was obtained ratio of >1.8. Ten pg of total RNA were with a A,,/A,,, separated in 2.2-mol/L formaldehyde, 1% SeaKem agarose gels (FMC Biochemical, Rockland, ME), transferred to Hybond-N membranes (Amersham Co., Arlington Heights, IL) by positive pressure (Posiblot, Stratagene, La Jolla, CA), and crosslinked to the membrane with 0.12 J UV radiation

GASTROENTEROLOGY Vol. 103,No. 5

(Stratalinker, Stratagene). The membranes were prehybridized, hybridized at 65“C, and washed as previously described.‘” Quantitative slot blots were performed as previously described” with prehybridization, hybridization, and washing the same as for northern blots. Escherichia Coli transfer RNA (tRNA) was used on each blot to control for nonspecific hybridization. Autoradiography was performed with Kodak X-Omat film (Eastman Kodak, Rochester, NY) between two intensifying screens at -7O’C. Slot blots were quantitated with a computer-assisted densitometer (Eastern Microscope Co., Raleigh, NC) and expressed as optical density (OD). Molecular probes used for analysis included a human CYPlAZ complementary DNA (cDNA), phIAl, kindly provided by Dr. Frank Gonzalez (National Institutes of Health, Bethesda, MD)” and a human p-actin. cDNA, pHFA-1, kindly provided by Dr. Craig Thompson (University of Michigan). phIA includes almost the entire length of the CYPlAl cDNA and after digestion with Eco Rl yields a 0.8 kb portion of the 5’end of the cDNA.‘* The j3-actin cDNA probe was used to ensure equivalent loading of RNA into each of the lanes. The cDNAs were isolated from their plasmid vectors by gel purification and radiolabeled with [32P]d adenosine triphosphate (Amersham) using a random-primed oligonucleotide-labeling method as previously described.16 Unincorporated nucleotides were removed from the labeled DNA with a size exclusion resin column (NucTrap Push Column, Stratagene).

PCR DNA was synthesized from 3 pg total RNA using random hexamer primers and AMV reverse transcriptase as previously described. *’ Oligonucleotide primers were designed to amplify specific sequences of CYPlAl, CYPlA2 and proubiquitin, which were 237 base pair (bp), 205 bp, and 219 bp in size, respectively.20*21 The proubiquitin primers span almost the entire ubiquitin carboxyterminal precursor sequence22: CYPlAl: 5’ primer 5’ ACT GCT TAG CCT AGT CAA CC 3’, 3’primer 5’CTT CTC CTG ACA GTG CTC AA 3’, CYPlA2: 5’primer 5’ATC CTT CGC TAC CTG CCT AA 3’, 3’primer 5’TGA CAA TCT TCT CTT GTG GG 3’, nested probe 5’GTC CTG ATA GTG CTC CTG GA 3’; proubiquitin: 5’primer 5’TCT TAC ACC ACT CCC AAG AAG 3’, 3’primer 5’ CTT GTC TTC TGG TTT GTT GAA 3’. Each segment of cDNA to be amplified spanned an intron so that any contaminating genomic DNA amplified would produce a significantly larger sequence than the desired cDNA. Ubiquitin primers were used as a positive control for the reverse transcription reaction and the PCR reaction. Amplification was performed on a programmable heating block (Perkin-Elmer Cetus, Norwalk, CT) using a cycle of 94°C for 1 minute, 55°C for 2 minutes, and 72°C for 3 minutes for 35 cycles followed by a lo-minute extension period at 72°C. Amplification products were separated on an agarose gel stained with ethidium bromide and some were transferred to Hybond-N membrane and probed with a 32P end-labeled, internal oligonucleotide as previously described.lg

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CYPlA GENE EXPKESSION 1511

Enzyme Assay Four pinch biopsy specimens from the antrum and duodenum of each volunteer were placed in 200 pL O.l-mol/L 4-[2-hydroxyethyll-l-piperazineethanesulfonic acid and then placed in liquid nitrogen for later processing. The samples were homogenized in a 2-mL dounce homogenizer and then sonicated for 10 seconds. The 7-ethoxyresorufin deethylase (ERD) assay was modified after the method of Pohl and FoutsZ3 The reaction was performed by adding 100 pL of homogenate to 2 mL of reaction mixture [O.l-moI/L 4-[Z-hydroxyethyll-l-piperazineethanesulfonic acid pH 7.8,1.5pmol/L ethoxyresorufin (Pierce Chemical Co., Rockford, IL), 5 mmol/L glucose-6phosphate (Sigma Chemical Co., St. Louis, MO), 1 U/mL glucose-6-phosphate dehydrogenase (Sigma), 5 mmol/L MgSO, (Sigma), 0.6 nmol/ml NADPH (Sigma)] in a cuvette with a stir bar in the chamber of a spectrofluorimeter (1681 Spectrometer, SPEX Industries, Edison, NJ) maintained at 3i”C. The fluorescent activity was continuously monitored for 10 minutes on a graphic display. A standard curve was determined from known dilutions of resorufin (Pierce). Total DNA of the tissue homogenate was determined using the method of Labarca and Paigen.24 Ethoxyresorufin deethylase values were normalized to total DNA and expressed as fmol resorufin produced - min-’ - pg-’ DNA. Statistical

Methods

The data obtained before and after omeprazole treatment was compared using the Wilcoxon paired-sample test (one tailed) and the coefficient of determination, r2. Results

Volunteers served as their own control with initial specimens representing constitutive expression and those obtained after omeprazole treatment representing induced expression. We were successful in obtaining high quality, undegraded messenger RNA (mRNA) from endoscopic pinch biopsies (260 nm/280 nm > 1.8, intact ribosomal RNA on denaturing gel electrophoresis). This allowed detailed evaluation of CYPlA gene expression in the alimentary tract by three complimentary methods. Northern analysis examined the qualitative presence of CYPlAl and CYPlA2 on a size gradient gel. Slot blots were used to quantitate the amount of hybridization found by northern analysis. PCR of reverse transcribed mRNA was able to confirm the presence or absence of CYPlA forms in alimentary tissue using a highly sensitive technique. Northern blots using the CYPlAl cDNA as a probe revealed a single, 2.8-kilobase (kb) RNA band in the duodenum of all volunteers as represented by a single volunteer in Figure 1. This band matches the reported 2.8 kb size of CYPlAl mRNA,” whereas CYPlA2 is 3.3 kb in size.” After omeprazole treat-

A

4

2.8 kb

B

C --mm-Buccal

IC

IC Esoph

IC B&Y

IC Antrum

IC Duod

I Colon

c

I

Duod

Figure 1. Northern blot analysis of alimentary tissues for expression of CYPlAl and lA2 mRNA.Total RNA was extracted from tissues of each volunteer before (control-C) and after omeprazole treatment (induced-L)as described in Methods. (A) This is a representative blot of RNA obtained from one volunteer. Ten micrograms of total RNA was separated in a 1% agarose gel, transferred to a nylon membrane and hybridized with a 3ZP-labeled CYPlAl cDNA as described in Materials and Methods. The film was exposed for 96 hours. (B) The blot depicted in (A) was washed to remove the CYPlAl probe and rehybridized with a 32P-labeled human p-actin cDNA as described in Materials and Methods. The purpose of probing the membrane with an actin cDNA is to ensure equal loading of the lanes with RNA. The film was exposed for 24 hours. (C) The volunteer who initially did not respond to 20 mg of omeprazole was later restudied using 60 mg of omeprazole as shown here. The top blot was probed with the CYPlAl cDNA and exposed for 46 hours and then washed and exposed to human fl-actin cDNA and exposed for 10 hours. A faint band was seen in the control lane after exposure to CYPZAZ cDNA that was not apparent after being photographed.

ment (20 mg/day) five of six volunteers had increased CYPlAl mRNA in duodenum. CYPlAl mRNA from one volunteer did not increase after treatment with omeprazole. Slot blots were used to quantitate the amount of induction by omeprazole (Figure 2). Five of the volunteers showed induction ranging from 2- to Is-fold with an average of s-fold induction. The volunteer who did not induce was further studied by repeating the study protocol using 60 mg of omeprazole per day. Repeat northern blot showed a dramatic increase in CYPlAl mRNA at the higher dose of omeprazole (Figure 1). Enzyme activity was measured in the antrum and duodenum using an assay specific for CYP1A1.26 Although mono-oxygenase enzymatic activity has been measured from human small intestinal biopsy specimens previously, this has only been with the use of large biopsy capsules used blindly.7,27 We developed a technique for doing the ethoxyresorufin deethylase assay on endoscopic pinch biopsy specimens using a kinetic fluorometric assay. This technique insured that the small amount of activity measured was significant by documenting linearity over time (Figure 3A). In the five volunteers who showed

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GASTROENTEROLOGY

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Control

Omeprazole

Figure 2. Quantification of duodenal CYPlAl mRNA before and after treatment with omeprazole, 20 mg/day. Quantitative slot blots were performed using 0.5,1.0, and 2.0 pg of total RNA from each tissue as described in Materials and Methods. The blots were hybridized sequentially with a “P-labeled CYPlAl cDNA and then a 32P-labeled human (3-actin cDNA. All duodenal samples were run on the same blot to avoid interblot variation. The optical density of each band was determined with a densitometer, and the optical density per pg RNA was determined using the 3 samples. The data are expressed as a ratio of ODm,,,/pg RNA over OD,,.,&pg RNA. Statistical analysis was performed using the Wilcoxon paired sample test (P < 0.02).

induction of CYPlAl mRNA, ERD constitutive activity ranged from 45 to 152 fmol . min-’ - ug-’ DNA before induction and from 90-926 fmol. min-’ - pg-’ DNA after omeprazole; this represented a mean in-

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crease of 3.1-fold (Figure 3B). The observed increase in ERD activity correlated with the increase in mRNA (r2 = 0.74, P < 0.05). The volunteer that did not show induction of CYPlAl mRNA did not have detectable levels of ERD activity. However, when this volunteer was restudied with 60 mg of omeprazole daily there was a twofold increase in ERD activity from 89 to 213 fmol - min-l - pg-’ DNA after treatment with omeprazole. ERD activity was not detected in any gastric antrum specimen that correlated with the lack of CYPlAl mRNA. PCR was used to confirm the identity of transcripts detected in duodenum by northern analysis. Furthermore, the great sensitivity of this method was used to determine if low levels of transcripts were present in other tissues that may be below the level of detectability by northern hybridization. CYPlAl mRNA was confirmed to be expressed in the duodenum of each volunteer before and after omeprazole treatment. In two volunteers, CYPlAl mRNA was identified only in the duodenum. In the remaining four volunteers, CYPlAl mRNA was also found in buccal mucosa after treatment with omeprazole. In two volunteers, CYPlAl appeared to be induced by omeprazole in all alimentary tissues studied (Figure 4). In contrast to CYPlAl, CYPlA2 mRNA was not present constitutively in any tissue studied by PCR. CYPlA2 was detected in the duodenum by PCR after omeprazole in two volunteers; they were the same volunteers who showed CYPlAl mRNA induction throughout the alimentary tract. Because the 205-bp CYPlA2 band on the ethidium bromide gel was quite light, this gel was transferred to a nylon membrane and probed with an end-labeled nested oligonucleotide probe for further identification (Figure 5).

350 1

300Z

250-

Duodenum-omeprazolc

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$ 2 200 1 0

E 8 ‘B

150-

loo-

Duodenum- coouol

50--

.A 0

I

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Seconds

Control

Omeprazole

Figure 3. (A)Graph of kinetic ERD assay of duodenal homogenate showing resort&n formation over time from a single volunteer before and after omeprazole treatment. (B) Ethoxyresorufin deethylase activity in mucosal homogenates from duodenum before and after treatment with omeprazole, 20 pg/day (Wilcoxon paired sample test P < 0.02).

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A 237 bp m

B 219 bp c

* Cl m_----Ladder

Bucd

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CYPlA GENE EXPRESSION

Cl

EwNws

Cl

@W

CICI

Cl

AntrumDuodsnum

Colon

Figure 4. PCR analysis of CYPlAl expression in the alimentary tract. RNA was extracted from each tissue and cDNA synthesized as described in Materials and Methods. (A)PCR was performed using primers designed to amplify a 237 bp portion of CYPlAl mRNA. PCR products were analyzed on a 3% NuSieve, 1% SeaKem agarose gel, stained with ethidium bromide and photographed under UV light. Products were identified in all tissues induced with omeprazole and in the duodenum of control tissue. DNA fragments were used as size markers in the first lane marked “ladder”. The CYPlAl product is between DNA size markers 220bp and 298 bp. (B)A 219 bp portion of proubiquitin mRNA was amplified in a parallel reaction as a positive control to ensure proper reverse transcription of mRNA. The proubiquitin product was detected in each tissue sample and is seen adjacent to the 229 bp DNA size marker.

Discussion We showed that CYPlAl mRNA is expressed constitutively in the duodenum of man. A standard dose of omeprazole administered for 1 week resulted in induction of CYPlAl mRNA and enzymatic activity in five of six volunteers. The one nonresponder had a marked increase in both CYPlAl mRNA and enzymatic activity after increasing the dose of omeprazole threefold to 60 mg/day. This represents the first definitive demonstration of CYPlAZ gene expression in the human alimentary tract and induction by a pharmaceutical agent. The parallel increase in enzymatic activity with CYPlAl mRNA indicates that the mRNA was likely translated into a functional protein and that the necessary coenzyme, NADPH cytochrome P450 reductase, is also expressed in duodenum. Although CYPlA2 may have some affinity for ethoxyresorufin as assessed in rat,‘” CYPlA2 was not present in the majority of our human volunteers. Therefore, ethoxyresorufin is an excellent representative substrate for the evaluation of CYPlAl activity in intestine. The direct demonstration of CYPlAl apoprotein by immunoblot analysis was not attempted because this method is not feasible using such small tissue samples. Furthermore, antibodies are not available to the human CYPlAl protein; antibodies to orthologous proteins from other species may lack sufficient specificity for the

human protein and, when used to detect human CYPlA proteins, have yielded conflicting resultsg*” Variability of CYPlAZ expression among individuals in control samples was present and probably represents numerous factors including differences in daily exposure to xenobiotics and genetic differences in the regulation of gene expression. Variability in constitutive expression of CYPZA has been previously described.‘3*2* Baseline variability in constitutive expression stresses the importance of each person acting as their own control in this study. Use of PCR to amplify cDNA synthesized from RNA is an extremely sensitive method for identifying rare RNA transcripts. “The specificity of the reaction can be manipulated so that only single transcripts are amplified.lg Using this method, we confirmed that the mRNA identified in duodenum by northern analysis was CYPlA. Furthermore, CYPlAl mRNA was identified in buccal mucosa of four volunteers after treatment with omeprazole. In two of these four individuals CYPlAl was inducible by omeprazole in every alimentary tissue tested. The physiological significance of finding these transcripts only by PCR when mRNA is not detectable by northern analysis is uncertain.2g However, it is possible that expression of CYPlAl in a small subpopulation of mucosal cells could have a metabolic effect in those individual cells. Furthermore, it is possible that omeprazole is a relatively weak inducer and that more potent inducers may have a greater effect. We showed that treatment of rats with a strong inducer, P-naphthoflavone, results in marked induction of CYPlAl mRNA and enzymatic activity throughout the alimentary tract.30 An intriguing finding of the PCR analysis was that CYPlA2 mRNA was identified in the duodenum of two individuals following treatment with omeprazole. This is considered a liver-specific P&O and had not previously been identified in extrahepatic tissue

c

I

c

I

I

c

c

I

c

I

c

I

----m

Buccal

Esophagus

Body

Antrum

Duodenum

Colon

Figure 5. PCR analysis of CYPlA2 expression in the alimentary tract from the same volunteer used in Figure 4. PCR of alimentary tissue RNA was performed as described in Figure 4, using CYPlA2 primers designed to amplify a 205 bp portion CYPlA2. The gel was then transferred to a nylon membrane and probed with a “P end-labeled oligonucleotide designed to hybridize to the amplified CYPlA2 PCR fragment. CYP lA2 amplified transcript was identified in the duodenum after treatment with omeprazole in two volunteers. The control for this PCR reaction is shown in Figure 4B.

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of rat10*31s32or human.” CYPlAZ is expressed in mouse small intestine and co1on.33 We recently reported that CYPlA2 mRNA is inducible in stomach and duodenum of the rat.30 As with the low level expression of CYPZAl in tissues other than duodenum, the physiological significance of CYPlA2 mRNA detected by PCR in the duodenum is unknown. A study conducted in the whole organism cannot assess whether induction of CYPlA genes by a xenobiotic is due to the parent compound, a metabolite, or to a secondary effect of the agent. Omeprazole inhibits parietal cell acid secretion, which results in stimulation of gastrin release from antral G cells and elevated serum gastrin levels. It was suggested in earlier studies that pentagastrin may induce expression of aryl hydrocarbon hydroxylase (CYPlA activity) in rat co1on.34 However, a carefully performed study using constant infusion of pentagastrin failed to show any effect on aryl hydrocarbon hydroxylase activity in the rat.35 Furthermore, CYPlA mRNA and enzymatic activity were induced in cultured human hepatocytes when exposed to omeprazole.12 Therefore, it seems more likely that induction of CYPlAZ was due to either the parent compound or to a metabolite. The induction of CYPlA is mediated by binding of xenobiotics to the Ah (aromatic hydrocarbon) receptor36 and, therefore, induction of CYPlAl is considered to be a marker for the presence of the Ah receptor.37 The Ah receptor is a cytosolic protein which acts in a similar fashion to proteins of the steroid receptor family.36 After binding of a ligand in the cytoplasm, the complex is translocated to the nucleus where it activates transcription of the CYPZAl gene via binding to DNA regulatory elements.36 Although transcription was not directly measured in this study, increased transcription is the mechanism of CYPlAl induction in liver of several species3**3g and in the rat small intestine.30 The significance of finding CYPlAl expression and induction in human tissues is controversial. Classically the induction of CYPZA expression as manifested by AHH activity is looked upon as being detrimental primarily because of work on procarcinogens such as benzo[a]pyrene which are not only strong inducers of CYPZA but are also metabolized by CYPlA to carcinogenic forms. Recently, the toxicity of 2,3,7,8 Tetrachlorodibenzo-p-dioxin (TCDD), the dioxin found in Agent Orange, was defined by its ability to bind the Ah receptor and induce CYPZAZ37; it was stated that manifestations of toxicity resulted from that initial event. It is generally agreed that CYPlAl activates procarcinogens to reactive compounds that are genotoxic and carcinogenic. Carcin-

GASTROENTEROLOGY Vol. 103, No. 5

ogen-induced DNA adduct formation is increased in tissues, including small intestine, that have aryl hydrocarbon hydroxylase activity.40 In apprarent contrast to this information, multiple studies have shown that pretreatment of animals with nongenotoxic inducers of CYPlAl results in decreased tumor incidence in some tissues.41*42 We have shown constitutive and inducible expression of CYPlAl in human small intestine, a tissue that has a very low incidence of primary carcinoma. This suggests that the presence of this enzyme may not be, in and of itself, detrimental to small intestinal mucosa. It is possible that CYPlA itself may be important for the observed resistance to the development of cancer; however, there are many other potential mechanisms for this resistance. Some investigators have expressed concern over the fact that omeprazole induces CYPZA genes in human tissue.43 The initial report stimulated controversy as to the validity of the findings as well as the interpretation of potential detrimental effects of induction of CYPlAl and lA2 in human liver. Because omeprazole is not genotoxic44 and has a short halflife compared with environmental toxins such as TCDD, it may be viewed in the same light as other nongenotoxic inducers of CYPlA such as cruciferous vegetables. However, with the expanding use of this drug, more information is needed on whether it directly binds to the Ah receptor as well as which enzymes are induced, Furthermore, the biological effect of Ah receptor activation on individual tissues with regards to carcinogen metabolism and subsequent mutagenesis requires investigation, For now, the effect of omeprazole on induction of CYPlAl in duodenal mucosa warrants a reminder that omeprazole and other pharmacological P&O inducers should be used when clearly indicated and for a defined duration. References 1. Nebert DW, Nelson DR, Coon MJ, Estabrook

RW, Feyereisen R, Fujii-Kuriyama Y, Gonzalez FJ, Guengerich FP, Gunsalus IC, Johnson EF, Loper JC, Sato R, Waterman MR, Waxman DJ. The P45O superfamily: Update on new sequences, gene mapping, and recommended nomenclature. DNA Cell Biol 1991;10:1-14. P450 enzymes in chemi2. Guengerich FP. Roles of cytochrome cal carcinogenesis and cancer chemotherapy. Cancer Res 1968;48:2946-2954. 3. Ames BN, Profet M, Gold LS. Dietary pesticides (99.99% all natural). Proc Nat1 Acad Sci USA 1990;87:7777-7781. 4. Conney AH. Induction of microsomal enzymes by foreign chemicals and carcinogenesis by polycyclic aromatic hydrocarbons: G. H. A. Clowes memorial lecture. Cancer Res 1982;42:4875-4917. 5. Trottier Y, Waithe WI, Anderson A. Rat liver cytochrome P450IA2 synthesized by transfected COS-1 cells efficiently

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activate food-derived promutagens. DNA Cell Biol 1991;lO: 33-39. 6. Crespi CL, Steimal DT, Aoyama T, Gelboin HV, Gonzalez FJ. Stable expression of human cytochrome P450IA2 cDNA in a human lymphoblastoid cell line: role of the enzyme in the metabolic activation of aflatoxin Bl.Mol Carcinog 1990;3:5-8. 7. Stahlberg MR. Hietanen E, Maki M. Mucosal biotransformation rates in the small intestine of children. Gut 1988;29:10581063. 8. Wattenberg LW. Carcinogen-detoxifying mechanisms in the gastrointestinal tract. Gastroenterology 1966;51:932-935. 9. Peters WM, Kremers PG. Cytochromes P-450 in the intestinal mucosa of man. Biochem Pharmacol1989;38:1535-1538. 10. dewaziers I, Cugnenc PH, Yang CS, Leroux JP, Beaune PH. Cytochrome P450 isoenzymes, epoxide hydrolase and glutathione transferases in rat and human hepatic and extrahepatic tissues. J Pharmacol Exp Ther 1990;253:387-394. 11.Sesardic D, Cole KJ, Edwards RJ, Davies DS, Thomas PE, Levin W, Boobis AR. The inducibility and catalytic activity of cytochromes P45Oc (P450IAl) and P45Od (P450IA2) in rat tissues. Biochem Pharmacol 1990;39:499-506. 12. Diaz D, Fabre I, Daujat M, Saint Aubert B, Bories P, Michel H, Maurel P. Omeprazole is an aryl hydrocarbon-like inducer of human hepatic cytochrome P450. Gastroenterology 1990;99: 737-747. 13. Wrighton SA, Watkins PW. Nonuniform distribution of cytochrome P450IA2 in liver. Gastroenterology 1991;100:14871488. 14. Andersson T, Bergstrand R, Cederberg C, Eriksson S, LagerStrom P, Skanberg I. Omeprazole treatment does not affect the metabolism of caffeine. Gastroenterology 1991;101:943-947. 15. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987;162:156-159. 16. Traber PG. Regulation of sucrase-isomaltase gene expression along the crypt-villus axis of rat small intestine. Biochem Biophys Res Commun 1990;173:765-773. 17. Traber PG, Chianale J, Florence R, Kim K, Wojcik E, Gumucio JJ. Expression of cytochrome P45Ob and P45Oe genes in small intestinal mucosa of rats following treatment with phenobarbital, polyhalogenated biphenyls, and organochlorine pesticides. J Biol Chem 1988;263:9449-9455. 18. Jaiswal AK, Gonzalez FJ, Nebert DW. Human dioxin-inducible cytochrome Pl-450: complementary DNA and amino acid sequence. Science 1985;228:80-83. 19.Traber PG, Wang W, McDonnell M, Gumucio J. P450IIB gene expression in rat small intestine: Cloning of intestinal P450IIBl mRNA using the polymerase chain reaction and transcriptional regulation of induction. Mol Pharmacol 1990:37:810-819. 20. Jaiswal AK, Gonzalez FJ, Nebert DW. Human PI-450 gene sequence and correlation of mRNA with genetic differences in benzo[a]pyrene metabolism. Nucleic Acids Res 1985;13: 4503-4520. 21. Ikeya K, Jaiswal AK, Owens RA, Jones JE, Nebert DW, Kimura S. Human CYPlA2: sequence, gene structure, comparison with the mouse and rat orthologous gene, and differences in liver lA2 expression. Mol Endocrinol 1989;3:1399-1408. 22. Lund PK, Moats-Staats BM, Simmons JG, Hoyt E, D’Ecoles AJ, Martin F, Van Wyk JJ. Nucleotide sequence analysis of a cDNA encoding human ubiquitin reveals that ubiquitin is synthesized as a precursor. J Biol Chem 1985;260:7609-7613. 23. Pohl RJ, Fouts JR. A rapid method for the metabolism of 7-ethoxyresorufin by microsomal subcellular fractions. Anal Biothem 1980;107:150-155.

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Received February 12, 1992. Accepted May 27,1992. Address requests for reprints to: Peter G. Traber, M.D., 600 Clinical Research Building, 422 Curie Boulevard, Philadelphia, PA 19104-6144.

GASTROENTEROLOGY

Vol. 103, No. 5

Supported by National Institutes of Health grant DK41393 (P,G.T.), a Veterans Administration Research Associate Award and Merit Review (P.G.T.), a Veterans Administration Associate Investigator Award (W.M.M.), and a grant from the University of Michigan Cancer Center’s Institutional Grant from the American Cancer Society (W.M.M.). The authors thank Paul Watkins, Joseph Kolars, and Daryl Daugherty for their helpful comments. They also acknowledge the support of the Clinical Research Center at the University of Michigan.