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Investigative Treatment Division, National Cancer Center Research Institute East, Kashiwa-shi, Chiba, 277-8577, Japan [M. T., S. N., T. O., H. E.]; Chemical Exposure and Health Effects Research Team, Regional Environmental Research Group, National Institute for Environmental Studies, Tsukuba, 305-0053, Japan [H. S.]; and Internal Medicine, Saiseikai Central Hospital, Tokyo, 108-0073, Japan [M. T., H. N.]
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Introduction |
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CYPs,3 together with P450 reductase, play an important role in detoxification of toxic xenobiotics into inactive metabolites (2) . These enzymes are expressed mainly in the liver, the major organ responsible for clearance of most xenobiotics. These enzymes are also present extrahepatically in epithelial cells of the gastrointestinal tract, particularly in the small intestine, possibly for local detoxification of dietary xenobiotics (3) . However, certain CYPs such as CYP1A, CYP3A, CYP2D, CYP2E, and others are known to cause metabolic activation of chemical carcinogens to highly reactive electrophilic intermediates, resulting in mutagenicity. The majority of carcinogenic chemicals do not produce their biological effect per se, but require metabolic activation before they can interact with cellular macromolecules.
The CYP-reductase activation system of procarcinogens in the alimentary tract has been well defined in the small and large intestines and esophagus. However, little attention has been paid to the stomach because CYPs are expressed less in the normal gastric mucosa than in the small intestine and gastric epithelium exhibits a secretary, rather than absorptive, function. The normal gastric mucosa is protected against exposure to chemical agents by a mucus barrier (4) . This is consistent with the results of animal experiments in which several carcinogens readily produced squamous tumors of the rat forestomach, but experimental production of adenocarcinomas in the glandular portion has generally required the direct intramural injection of carcinogens (5) . In addition, agents used in these animal models of gastric carcinogenesis do not require metabolic activation (6) . This coincides with the evidence that CYP genes are expressed less in gastric mucosa than in the liver (7) . In the carcinogenic process of intestinal-type of human gastric cancer, intestinal metaplasia is always present before the cancer arises. Mucosa with intestinal metaplasia has an increased ability to transport lipids from the lumen into the lamina propria (8) . Many carcinogens, polycyclic aromatic hydrocarbons, various nitrosamines, and heterocyclic amines, are lipophilic. Thus, these carcinogens may be easily absorbed from the epithelium of intestinal metaplasia and activated by CYPs within the glands of intestinal metaplasia if CYPs and P450 reductase are coexpressed and are functional. The present study reports the presence of a CYP-reductase carcinogen activation system in gastric mucosa with intestinal metaplasia.
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Materials and Methods |
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Immunohistochemistry.
Specimens obtained surgically were fixed in 10% formalin and embedded in paraffin. Deparaffinized and rehydrated sections were prepared routinely. To examine localization of P450 reductase, the sections were incubated at room temperature for 30 min in 0.01 M PBS containing 1% trypsin and 1% CaCl2. To examine CYP1A1 and CYP1A2, sections were microwaved (750W) for 20 min at 95°C in 0.01 M citrate buffer (pH 6.0). Then, blocking for endogenous peroxidase activity and nonspecific immunoreaction was performed using the DAKO LSAB kit. The sections were reacted with the primary antibody, P450 reductase (1:250; Chemicon International, Inc.), CYP1A1 (1:1,000; a kind gift from Dr. Imaoka, Department of Chemical Biology, Osaka City University Medical School, Japan), and CYP1A2 (1:10,000; Chemicon International, Inc.). Immunoreactivity was visualized using an avidin-biotin immunoperoxidase technique (DAKO LASB kit). The peroxidase label was developed with 3,3'-diaminobenzidine, tetrahydrochloride (Dojindo, Kumamoto, Japan), and 0.3% H2O2 dissolved in 50 mM Tris-HCl (pH 7.6). After washing with 0.01 M PBS, the sections were counterstained with hematoxylin. The same procedure, but without primary antibodies, was carried as a negative control.
NADPH-Diaphorase Staining.
Tissue Section.
Specimens were immediately fixed for 8 h with 4% paraformaldehyde in 0.1 M PB (pH 7.4) and embedded in OTC mounting medium (Tissue-Tek; Miles, Inc., Elkhart, IN) before storing at -80°C. Cryostat sections (7 µm) were cut, mounted on a slide glass, and air-dried. Sections were incubated in 0.01 M PBS containing 0.1% Triton X-100 for 12 h at 4°C. After washing with 0.01 M PBS, sections were incubated in 0.01 M PBS containing 0.1% Triton X-100, 0.5 mg/ml ß-NADPH (Oriental Yeast Co., Ltd., Tokyo, Japan), and 0.1 mg/ml nitroblue tetrazolium (Sigma Chemical Co., St. Louis, MO) for 1 h at 37°C (9)
. The reaction was stopped in 0.01 M PBS, and sections were photographed under bright-field illumination. To confirm specificity to P450 reductase activity, sections were incubated in reaction buffer containing 25 µM DPI (Sigma Chemical Co.), a potent P450 reductase inhibitor.
Whole Mount.
Specimens obtained surgically were pinned on a board and immediately fixed for 4 h with 4% paraformaldehyde in 0.1 M PB (pH 7.4) and washed with 0.01 M PBS. Then, specimens were incubated in 0.01 M PBS containing 0.1% Triton X-100, 0.5 mg/ml ß-NADPH, and 0.1 mg/ml nitroblue tetrazolium for 5 min at 37°C. The reaction was stopped in 0.01 M PBS.
RNA Isolation and RT-PCR.
Total RNA was isolated from gastric specimens obtained endoscopically or surgically. First-strand cDNA was synthesized, and PCR was performed as described elsewhere (10)
. To detect CYP1A1, CYP1A2, CYP1B1, CYP3A4, CYP2D6, CYP2E1, sucrase, trehalase, and ß-actin mRNAs, 0.3 µl of first-strand cDNA was amplified by PCR with the respective set of oligonucleotide primers (Table 1)
. Denaturation, annealing, and elongation in the PCR were carried out at 94°C, 55°C, and 72°C for 30 s, 1 min, and 2 min, respectively, for: 35 cycles for trehalase; 30 cycles for CYP1A1, CYP1A2, CYP1B1, CYP3A4, CYP2D6, CYP2E1, and sucrase; and 28 cycles for ß-actin. The specificity and quantity of the amplified DNA fragments were determined by Southern blot hybridization, with specific internal oligonucleotide (Table 1)
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Immunoblotting.
The microsomal pellet was resuspended with radioimmunoprecipitation assay buffer [50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.1% SDS, 1% NP40, and 0.5% deoxycholic acid sodium salt]. SDS-PAGE was routinely performed with 10% acrylamide gels. The microsomal proteins (50 µg) were electrophoretically transferred to the Immobilon membranes (Millipore, Bedford, MA). The membranes were blocked with 5% skim milk and 2% BSA in 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 0.1% Tween 20 at 4°C for 12 h. The membranes were incubated with primary antibodies [CYP1A1 (1:10000) and CYP1A2 (1:5000); both purchased from Chemicon International, Inc.] at room temperature for 2 h. After washing, the membranes were incubated with the secondary antibodies associated with primary antibodies at room temperature for 1 h. The immunoreactive bands were detected using an enhanced chemiluminescence Western blotting detection kit (Amersham Life Science Ltd., Buckinghamshire, England).
Mutation Assay.
The microsomal pellet was resuspended in homogenizing buffer, and protein concentration was determined by the Bradford method using BSA as a standard. The microsomal protein was mixed with a cofactor set (Oriental Yeast Co., Ltd.), and glucose-6-phosphate dehydrogenase (Sigma Chemical Co.) was added at 1 unit/plate as microsomal mixture (12)
. Top agar ( 2 ml) containing 0.01 M histidine and avidine was added in the following order: 0.1 ml of Salmonella typhimurium bacteria TA98; 0.75 µg of PhIP, 5 µg of benzo(a)pyrene, 0.5 µg of IQ, and 0.5 µg of MeIQx and 0.5 ml of microsomal mixture. The mixture was incubated for 20 min at 37°C before pouring to the plates. Revertant numbers were counted after a 48-h incubation at 37°C.
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Results |
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. However, foveolar epithelium without intestinal metaplasia in the fundic gland showed no immunoreactivity to the antibody. NADPH-diaphorase staining was performed in tissue sections and whole mount tissue. NADPH-diaphorase activity was detected in neural cells, neural fibers, vascular endothelial cells, and infiltrating cells, particularly macrophages and plasma cells. Epithelial cells with intestinal metaplasia also showed strong activity (Fig. 1C)
. In NADPH-diaphorase staining, coincubation with DMSO, a solvent of DPI, did not affect NADPH-diaphorase activities (Fig. 1D)
. These reactivities were completely inhibited by cotreatment with DPI (Fig. 1E)
. The distribution of intestinal metaplasia was also clearly detected by whole mount NADPH-diaphorase staining (Fig. 1F)
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. The proper number of PCR cycles was selected as the conditions under which the radioactivity of the PCR product increased in proportion to the amount of mRNA. Sucrase was used as a marker of intestinal metaplasia. Trehalase was used to determine the subtype of intestinal metaplasia. When sucrase mRNA expression was observed, the specimen was assumed to be associated with intestinal metaplasia. When trehalase mRNA was detected in sucrase-positive specimens, intestinal metaplasia was classified as complete type. Trehalase-negative and sucrase-positive specimens were classified as incomplete type of intestinal metaplasia. Expression of CYP1A2, CYP3A4, and CYP2D6 mRNA in sucrase-positive specimens was higher than in sucrase-negative specimens. In contrast, the level of CYP2E1 in sucrase-negative specimens was higher than that in sucrase-positive specimens.
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. Immunoreactivity to CYP1A1 was detected in the epithelium of intestinal metaplasia, and its distribution was the same as that of P450 reductase (Fig. 2C)
. However, CYP1A2 protein was detected mainly in pyloric gland cells near the intestinal metaplastic gland (Fig. 2, D and E)
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Mutation Assay.
We examined the functional activity of CYPs by measuring activation of mutagens by the microsomal fraction. We tested four carcinogens: benzo(a)pyrene, PhIP, IQ, and MeIQx using microsomes prepared from 20 specimens of nine resected stomachs. Benzo(a)pyrene and IQ were metabolically activated by microsomes of intestinal metaplasia (Fig. 3, A and B)
, whereas PhIP and MeIQx were not activated (data not shown).
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Discussion |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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Intestinal metaplasia of the stomach is characterized by morphological similarity to the intestine and Paneth cell, goblet cell and characteristics of absorbing mucosa, striated boarder, and brush boarder. In addition, these tissues are characterized by expression of sucrase, trehalase, alkalinephosphatase, amino peptidases, and sialylated and sulfated mucins. Several CYP species were found to be expressed in the small and large intestines, suggesting that CYP is also a biochemical marker of the intestine. However, little is known about the expression of CYP in intestinal metaplasia (14) .
A broad range of procarcinogens including heterocyclic amines, polyaromatic hydrocarbons, arylamines, aflatoxin B1, and nitrosamines are activated by CYPs (15) . CYP1A plays important roles in the activation of procarcinogens. PhIP, IQ, and MeIQx, representative carcinogens related to human dietary lifestyle, undergo mutagenic activation via P450-mediated N-hydroxylation. It is now generally accepted that the isoforms showing the highest catalytic activity toward them are CYP1A1 and CYP1A2. In this study, we showed activation of IQ by microsomal fraction of intestinal metaplasia. Although we also examined activation of PhIP and MeIQx, results were not clear. One possible reason for this is the requirement of secondary metabolizing enzymes for efficient activation. In this study, we used microsomal protein instead of S-9 fraction because excess amounts of S-9 fraction prepared from the alimentary tract are reported to, and were found to, decrease the number of revertant colonies (16) .4 Because microsome fraction contains less secondary metabolizing enzymes that might be necessary for activation, we used YG1024, a strain of TA 98 harboring acetyltransferase gene, a secondary activating enzyme (17) . But no clear activation was observed.4 Another possibility is that although we concentrated CYP from intestinal metaplasia by purifying microsomes, the amount of CYP used in the Ames test might be far below optimum. In some experiments, we tried to increase the amount of CYP by increasing the amount of gastric mucosa; but, once excess amounts of microsomal fraction were used, the number of revertant colonies decreased by some unknown reasons. This might be also explained why we could not obtain high activity even toward B(a)P and IQ in the present study.
The third possible explanation for the absence of activation of PhIP and MeIQx is described. In the present study, we detected CYP1A2 mRNA in intestinal metaplasia tissue and trace amounts even in normal gastric mucosa. It has long been believed that the expression of CYP1A2 is largely restricted to the liver, with only trace amounts ever detected in extrahepatic tissues. In this study, significantly higher CYP1A2 mRNA expression was observed in intestinal metaplasia over normal gastric mucosa, and CYP1A2 protein was clearly detected by Western blot analysis. However, we have to be cautious to conclude that this protein is truly CYP1A2 because the antibody used in the present study might recognize CYP1B1, a recently identified extrahepatic CYP1 family protein. CYP1B1 mRNA was also expressed in gastric mucosa with intestinal metaplasia. Therefore, it is not easy to determine whether the immunohistochemically detected CYP is 1A2 or 1B1 or both. Considering knowledge of catalytic potential of these two isozymes to activate heterocyclic amines (18) , 1B1 might be dominant in the gastric mucosa with intestinal metaplasia. It is very difficult to differentiate these proteins by presently available antibodies.
Intestinal metaplasia is classified into incomplete and complete type. Incomplete type intestinal metaplasia is more closely associated with gastric cancer than complete type. However, NADPH-diaphorase activity is more strongly enhanced in the epithelium of complete type intestinal metaplasia than incomplete type.5 This apparent inverse correlation between P450 reductase activity and cancer proneness may be due to expression of phase II enzymes, such as glutathione S-transferases, N-acetyltransferase, or epoxide hydorase, which play a critical role in detoxification of activated carcinogens. Earlier studies showed that these enzymes were as highly expressed in the small intestine as in the liver, but at low levels in the gastric mucosa and large intestine (19) . Complete-type intestinal metaplasia has the structural and biochemical characteristics of the small intestine, whereas, incomplete-type intestinal metaplasia is similar to the epithelium of the large intestine. Thus, a balance between CYP-reductase system and phase II enzymes needs to be carefully considered in relation to carcinogenic potential between complete type and incomplete type. In addition, intestinal metaplasia has a similar structure to the intestine, but lower lipid clearance, which means slower clearance of activated carcinogens may contribute to carcinogenesis.
RT-PCR analysis revealed expression of several kinds of CYPs in gastric mucosa with intestinal metaplasia, compared with the mucosa without intestinal metaplasia. Over the last decade, cancer susceptibility has been widely studied by assessing phenotypes in metabolic activation of xenobiotics or genotype of CYP and phase II enzyme genes, and these genetic determinants have a high impact on cancer susceptibility (20) . However, this study suggested the possibility that pathological changes might have some impact on carcinogen activation, in addition to genetic determinants. Therefore, it is important to identify expression profiles of CYPs in each pathological condition related to carcinogenesis, and our findings provide the first step toward this end.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by grants from the Ministry of Health and Welfare for the 2nd-term Comprehensive 10-year Strategy for Cancer Control, and a Grant-in-Aid for Cancer Research and a Grant-in-Aid for Scientific Research (C) from the Ministry of Education, Science and Culture of Japan. 
2 To whom requests for reprints should be addressed, at Investigative Treatment Division, National Cancer Center Research Institute East, 6-5-1, Kashiwanoha, Kashiwa-shi, Chiba, 277-8577, Japan. Phone: 81-471-33-1111, ext. 5101; Fax: 81-471-34-6859.
3 The abbreviations used are: CYP, cytochrome P450 monooxygenases; PhIP, 2-amino-1-meyhkl-6-phenylimidazo[4,5-b]pyridine; IQ, 2-amino-3-methylimidazo[4,5-f]quinoline; MeIQx, 2-amino-3, 8-dimethylimidazo[4,5-f]quinoxaline; PB, phosphate buffer; RT-PCR, reverse transcription-PCR; iNOS, inducible nitric oxide synthase; DPI, diphenyleneiodonium chloride.
4 M. Tatemichi et al., unpublished data.
5 S. Nomura et al., unpublished data.
Received 5/14/99. Accepted 7/ 2/99.
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, antrum; 
, corpus.